WORLD HOSPITAL DIRECTORY
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WORLD HOSPITAL DIRECTORY is the one and only largest database of hospitals around the world. There are over 35,000 plus records of hospitals across globe

WORLD HOSPITAL DIRECTORY has the World's largest online database of general, multispeciality, eye, dental, children, maternity, cardiac care, orthopedic, nephrology, neurology, diabetic, psychiatric, cancer, ENT, hospice, Rehabilitation, alternate medicine, veterinary hospitals across globe. Discover the complete list of hospitals available in North America, South America, Europe, Asia, Australia, New Zealand, rest of the world and online.


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1CEMIC
CEMIC
Category: General Hospitals
Argentina
South America, America
2Croydon Hospital (Tablelands HSD)
Croydon Hospital (Tablelands HSD)
Category: Mulispeciality Hospitals
Australia
Australia and New Zealand, Oceanic
3Regionaal Hospital Sint-Maria
Regionaal Hospital Sint-Maria
Category: Mulispeciality Hospitals
Belgium
Western Europe, Europe
4Bairro Passo D'areia Farmaconte Dist. Produtos Hospitalares Ltda.
Bairro Passo D'areia Farmaconte Dist. Produtos Hospitalares Ltda.
Category: Mulispeciality Hospitals
Brazil
South America, America
5BCIT - Technology Centre, Health Technology Research Group
BCIT - Technology Centre, Health Technology Research Group
Category: General Hospitals
Canada
North America, America

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Protect Yourself Against the Flu Vaccine!
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Increasing Patient Care and Reducing Liability in Seven Simple Steps
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New Surgical Treatment Options for Hernias
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The sun is not hurried by early risers


Time-lapse in the IVF-lab: how should we assess potential benefit?
<p>Time-lapse imaging of embryos has been widely introduced to fertility laboratories worldwide with the aim of identifying the best quality embryos to transfer that will ultimately improve IVF success rates. In this opinion paper, we explore the lack of evidence of benefit of this novel intervention, analyse the methodological flaws of current studies, offer ideal study designs that assess the various features of time-lapse imaging, and discuss forthcoming studies. In particular, we emphasize the ethical aspects of hastily adopting a costly technology without current high level evidence of improved live birth rates, safety and cost effectiveness.</p>


Individual fertility assessment and pro-fertility counselling; should this be offered to women and men of reproductive age?
<p>During the 1970s new contraceptive options developed and legal abortions became accessible. Family planning clinics targeting young women and men provided advice and assistance on contraception. Today, delayed childbearing, low total fertility rates and increasing use of social oocyte freezing create a need for pro-fertility initiatives. Three years ago we established a new separate unit: The Fertility Assessment and Counselling (FAC) clinic. The FAC clinic offers free individual counselling based on a clinical assessment including measurement of serum anti-M&uuml;llerian hormone and ovarian and pelvic sonography in women, sperm analysis in men, and a review of reproductive risk factors in both sexes. The FAC clinic includes a research programme with the goal to improve prediction and protection of fertility. Our first proposition is that clinics for individual assessment and counselling need to be established, as there is a strong unmet demand among women and men to obtain: (i) knowledge of fertility status, (ii) knowledge of reproductive lifespan (women) and (iii) pro-fertility advice. Addressing these issues is often more challenging than treating infertile patients. Therefore, we propose that fertility assessment and counselling should be developed by specialists in reproductive medicine. There are two main areas of concern: As our current knowledge on reproductive risk factors is primarily based on data from infertile patients, the first concern is how precisely we are able to forecast future reproductive problems. Predictive parameters from infertile couples, such as duration of infertility, are not applicable, diagnostic factors like tubal patency are unavailable and other parameters may be unsuitable when applied to the general population. Therefore, strict validation of reproductive forecasting in women and men from the general population is crucial. The second main concern is that we may turn clients into patients. Screening including reproductive forecasting may induce unnecessary anxiety through false positive predictions and may even result in overtreatment in contrast to the intended preventive concept. False negative findings may create false reassurance and result in postponement of conceptions.</p>


Putting 'family' back in family planning
<p>Family planning visits are designed to help women build families in a manner most compatible with their life goals. Women's knowledge regarding age-related fertility is suboptimal, and first wanted pregnancies are now occurring at older ages. Here we review the issue of diminishing chances of a pregnancy occurring in women over 30 years of age. A debate arises over whether to perform a standard fertility assessment at an age when, for example, oocyte freezing is still practical and feasible, knowing that the proven predictors in subfertile couples may be less informative, or even inappropriate, in women without complaints about fertility. Studies have demonstrated that if women knew that their fertility was diminishing, they might alter life plans, including having children sooner or considering oocyte preservation. Therefore, we argue that physicians need to make an effort to evaluate a woman's childbearing priorities, though not necessarily their fertility, during the initial family planning visit.</p>


The type of culture medium and the duration of in vitro culture do not influence birthweight of ART singletons
<sec><st>STUDY QUESTION</st> <p>Does the type of <I>in vitro</I> culture medium or the duration of <I>in vitro</I> culture influence singleton birthweight after IVF/ICSI treatment?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>In a comparison of two culture media, neither the medium nor the duration of culture (Day 3 versus Day 5 blastocyst transfer) had any effect on mean singleton birthweight.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Previous studies indicated that <I>in vitro</I> culture of human embryos may affect birthweight of live born singletons. Both the type of culture medium and the duration of culture may be implicated. However, these studies are small and report conflicting results.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A large retrospective analysis was performed including all singleton live births after transferring fresh Day 3 or Day 5 embryos. IVF and ICSI cycles performed between April 2004 and December 2009 at a tertiary care centre were included for analysis.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>A total of 2098 singleton live births resulting from singleton pregnancies were included for analysis. Two different sequential embryo culture media were concurrently used in an alternating way: Medicult (<I>n</I> = 1388) and Vitrolife (<I>n</I> = 710). Maternal age, maternal and paternal BMI, maternal parity, maternal smoking, main cause of infertility, cycle rank, stimulation protocol, method of fertilization (IVF or ICSI), time in culture and number of embryos transferred were taken into account. Embryo transfers were performed either on Day 3 (<I>n</I> = 1234) or on Day 5 (<I>n</I> = 864). Singleton birthweight was the primary outcome parameter. Gestational age and gender of the newborn were accounted for in the multiple regression analysis.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>No significant differences in mean singleton birthweight were observed between the two culture media: Medicult 3222 g (&plusmn;15 SE) and Vitrolife 3251 g (&plusmn;21 SE) (<I>P</I> = 0.264). The mean singleton birthweight was not different between Day 3 embryo transfers (3219 &plusmn; 16 g) and Day 5 blastocyst transfers (3250 &plusmn; 19 g; <I>P</I> = 0.209). Multiple regression analysis controlling for potential maternal, paternal, treatment and newborn confounders confirmed the non-significant differences in mean singleton birthweight between the two culture media. Likewise, the adjusted mean singleton birthweight was not different according to the duration of <I>in vitro</I> culture (<I>P</I> = 0.521).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The conclusions are limited by its retrospective design; however, the two different sequential culture systems were used in an alternating way during the same time period. Pregnancy-associated factors possibly influencing birthweight (such as diabetes, hypertension, pre-eclampsia) were not included in the analysis.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This large retrospective study does not support earlier concerns that both the type of culture medium and the duration of embryo culture influence singleton birthweight. However, a continuous surveillance of human embryo culture procedures (medium type, culture duration and other culture conditions) should remain a priority within assisted reproduction technology.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>None.</p> </sec>


Which set of embryo variables is most predictive for live birth? A prospective study in 6252 single embryo transfers to construct an embryo score for the ranking and selection of embryos
<sec><st>STUDY QUESTION</st> <p>Which embryo score variables are most powerful for predicting live birth after single embryo transfer (SET) at the early cleavage stage?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>This large prospective study of visual embryo scoring variables shows that blastomere number (BL), the proportion of mononucleated blastomeres (NU) and the degree of fragmentation (FR) have independent prognostic power to predict live birth.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Other studies suggest prognostic power, at least univariately and for implantation potential, for all five variables. A previous study from the same centre on double embryo transfers with implantation as the end-point resulted in the integrated morphology cleavage (IMC) score, which incorporates BL, NU and EQ.</p> </sec> <sec><st>STUDY DESIGN, SIZE AND DURATION</st> <p>A prospective cohort study of IVF/ICSI SET on Day 2 (<I>n</I> = 6252) during a 6-year period (2006&ndash;2012). The five variables (BL NU, FR, EQ and symmetry of cleavage (SY)) were scored in 3- to 5-step scales and subsequently related to clinical pregnancy and LBR.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>A total of 4304 women undergoing IVF/ICSI in a university-affiliated private fertility clinic were included. Generalized estimating equation models evaluated live birth (yes/no) as primary outcome using the embryo variables as predictors. Odds ratios with 95% confidence intervals and <I>P</I>-values were presented for each predictor. The C statistic (i.e. area under receiver operating characteristic curve) was calculated for each model. Model calibration was assessed with the Hosmer&ndash;Lemeshow test. A shrinkage method was applied to remove bias in c statistics due to over-fitting.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>LBR was 27.1% (1693/6252). BL, NU, FR and EQ were univariately highly significantly associated with LBR. In a multivariate model, BL, NU and FR were independently significant, with c statistic 0.579 (age-adjusted c statistic 0.637). EQ did not retain significance in the multivariate model. Prediction model calibration was good for both pregnancy and live birth. We present a ranking tree with combinations of values of the BL, NU and FR embryo variables for optimal selection of the embryo/s to transfer, providing a revised IMC score. The five embryo variables had similar effects over all age groups.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Limitations of the present study are those inherent for real-time visual scoring, including risks of inter-observer variation and the hazards of fixed time-point scoring procedures in a dynamic process. The study is restricted to Day-2 transfers.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>To our knowledge this is the largest prospective, SET study performed with the explicit aim of constructing an evidence-based embryo score for the ranking and selection of early cleavage stage embryos. In line with previous research, our data suggest that the symmetry of cleavage variable may be omitted when scoring embryos in the early cleavage stage. We suggest that, following validation in other populations, the revised IMC score may be used when international standards for embryo scoring are discussed.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST</st> <p>Carl von Linn&eacute; Clinic, Uppsala and the Department of Women's and Children's Health and the Family Planning Fund in Uppsala, Uppsala University, Uppsala, Sweden financed this study. There are no competing interests to declare.</p> </sec>


Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification
<sec><st>STUDY QUESTION</st> <p>Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The use of more &lsquo;steps&rsquo; of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem&ndash;Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (<I>P</I> &lt; 0.001), more spherical cellular morphology (<I>P</I> &lt; 0.001), increased cytoplasmic lipid retention in vitrified and warmed bovine oocytes (<I>P</I> &lt; 0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (<I>P</I> &lt; 0.05) and improved developmental competence of vitrified and warmed murine zygotes (<I>P</I> &lt; 0.05) than CPA exposure using the current clinically used manual pipetting method.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>It is necessary to design the microfluidic device to be more user-friendly for widespread use.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest.</p> </sec>


Repair of congenital 'disconnected uterus': a new female genital anomaly?
<p>Congenital uterine anomaly is a female genital disorder caused by developmental anomaly of the M&uuml;llerian ducts. In this report, we present a case of repair of congenital &lsquo;disconnected uterus&rsquo; between the cervix and the body of the uterus. The case did not correspond to the consensus classifications that have been proposed for congenital uterine anomaly. The patient was a young woman whose chief complaints were not having first menstruation and experiencing monthly severe lower abdominal pain. Magnetic resonance imaging showed that the uterine body was separated from the uterine cervix. Uteroplasty was conducted to anastomose the separated uterus. Periodic menstruation started 1 month after surgery and abdominal pain was improved. Performance of uteroplasty in this case was extremely significant and greatly improved the quality of life of the patient.</p>


Protein oxidative stress markers in peritoneal fluids of women with deep infiltrating endometriosis are increased
<sec><st>STUDY QUESTION</st> <p>Are protein oxidative stress markers [thiols, advanced oxidation protein products (AOPP), protein carbonyls and nitrates/nitrites] in perioperative peritoneal fluid higher in women with histologically proven endometriosis when compared with endometriosis-free controls?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Protein oxidative stress markers are significantly increased in peritoneal fluids from women with deep infiltrating endometriosis with intestinal involvement when compared with endometriosis-free controls.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Endometriosis is a common gynaecologic condition characterized by an important inflammatory process. Various source of evidence support the role of oxidative stress in the development of endometriosis.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>We conducted a prospective laboratory study in a tertiary-care university hospital between January 2011 and December 2012, and included 235 non-pregnant women, younger than 42 year old, undergoing surgery for a benign gynaecological condition.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>After complete surgical exploration of the abdomino-pelvic cavity, 150 women with histologically proven endometriosis and 85 endometriosis-free controls women were enrolled. Women with endometriosis were staged according to a surgical classification in three different phenotypes of endometriosis: superficial peritoneal endometriosis (SUP), ovarian endometrioma (OMA) and deeply infiltrating endometriosis (DIE). Perioperative peritoneal fluids samples were obtained from all study participants. Thiols, AOPP, protein carbonyls and nitrates/nitrites were assayed in all peritoneal samples.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Concentrations of peritoneal AOPP were significantly higher in endometriosis patients than in the control group (median, 128.9 &micro;mol/l; range, 0.3&ndash;1180.1 versus median, 77.8 &micro;mol/l; range, 0.8&ndash;616.1; <I>P</I> &lt; 0.001). In a similar manner concentrations of peritoneal nitrates/nitrites were higher in endometriosis patients than in the control group (median, 24.8 &micro;mol/l; range, 1.6&ndash;681.6 versus median, 18.5 &micro;mol/l; range, 1.6&ndash;184.5; <I>P</I> &lt; 0.05). According to the surgical classification, peritoneal fluids protein AOPP and nitrates/nitrites were significantly increased only in DIE samples when compared with controls (<I>P</I> &lt; 0.001 and <I>P</I> &lt; 0.05; respectively), whereas the others forms of endometriosis (SUP and OMA) showed non-statistically significant increases. We found positive correlations between peritoneal fluids AOPP concentrations, nitrites/nitrates levels and the total number of intestinal DIE lesions (<I>r</I> = 0.464; <I>P</I> &lt; 0.001 and <I>r</I> = 0.366; <I>P</I> = 0.007; respectively).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Inclusion of only surgical patients may constitute a possible selection bias. In fact, our control group involved women who underwent surgery for benign gynaecological conditions. This specificity of our control group may lead to biases stemming from the fact that some of these conditions, such as fibroids, ovarian cysts or tubal infertility, might be associated with altered peritoneal proteins oxidative stress markers.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>We demonstrate the existence of a significantly increased protein oxidative stress status in peritoneal fluid from women with endometriosis especially in cases of DIE with intestinal involvement. This study opens the way to future more mechanistics studies to determine the exact role of protein oxidative stress in the pathogenesis of endometriosis. Even if an association does not establish proof of cause and effect, these intrinsic biochemical characteristics of endometriosis may lead to the evaluation of therapeutic approaches targeting oxidative imbalance.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No funding was used for this study. The authors have no conflict of interest.</p> </sec>


Differential effects of tumor necrosis factor-{alpha} on matrix metalloproteinase-2 expression in human myometrial and uterine leiomyoma smooth muscle cells
<sec><st>STUDY QUESTION</st> <p>Does tumor necrosis factor-&alpha; (TNF-&alpha;) differentially regulate matrix metalloproteinase-2 (MMP-2) expression in leiomyomas compared with normal myometrium?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>TNF-&alpha; up-regulates MMP-2 expression and stimulates cell migration through the activation of extracellular signal-regulated kinase (ERK) signaling pathway in leiomyoma smooth muscle cells (SMCs), but not in normal myometrial SMCs.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Uterine leiomyoma, the benign smooth muscle cell tumor, is the single most common indication for hysterectomy. High expression of MMPs or TNF-&alpha; has been reported in uterine leiomyomas; however, the molecular mechanism underlying these observations remains unknown.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Samples were obtained between 2009 and 2013 from 12 women of reproductive age at the proliferative phase of the menstrual cycle by hysterectomy. Leiomyomas and matched normal myometrium from each woman were analyzed <I>in vitro</I>.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Western blot, RT&ndash;qPCR and a wound-healing assay were used to investigate the effects of TNF-&alpha; on MMP-2 expression and intracellular signal transduction in cultured SMCs from leiomyomas and matched myometrium.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Western blot and RT&ndash;qPCR analyses using tissues from clinical patients showed that the levels of MMP-2 protein (<I>P</I> = 0.008) and mRNA (<I>P</I> = 0.009) were significantly higher in uterine leiomyomas compared with their matched myometrium. Treatment with TNF-&alpha; significantly up-regulated the protein (<I>P</I> = 0.039) and mRNA (<I>P</I> = 0.037) levels of MMP-2 in cultured leiomyoma SMCs but not in matched myometrial SMCs. The extracellular signal-regulated kinase (ERK) and nuclear factor-kappa B (NF-B) pathways were activated by TNF-&alpha; in leiomyoma SMCs. Specific inhibitors of the ERK or NF-B pathway (PD98059 or Bay11-7082) suppressed TNF-&alpha;-induced MMP-2 expression in leiomyoma SMCs. The wound-healing assay revealed that TNF-&alpha; promoted the migration of cultured leiomyoma SMCs (<I>P</I> = 0.036); however, PD98059 compromised the cell migration triggered by TNF-&alpha;.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This study is descriptive and although we observed clear differential regulation of MMP-2 by TNF-&alpha; at mRNA and protein levels in leiomyoma, future studies are needed to identify why the difference in TNF-&alpha; response exists between human leiomyoma tissue and normal myometrium. Including some of the experiments such as transfection studies for TNF-&alpha; and MMP-2 promoter mapping could have added more insight as to why this difference exists. In addition, further studies <I>in vivo</I> are needed to verify the results obtained from primary cultured SMCs.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Considering the positive effect of TNF-&alpha; on leiomyoma SMC migration, strategies targeting TNF-&alpha;, in parallel with the production of more specific inhibitors of MMPs, may provide alternative therapeutic approaches for the treatment of leiomyoma.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was partially supported by grants from the Program for New Century Excellent Talents in University (NCET-12-0282), National Natural Science Foundation of China (81371620) and Tianjin Natural Science Foundation (12JCZDJC24900). The authors have no conflicts of interest to declare.</p> </sec>


Overtreatment in couples with unexplained infertility
<sec><st>STUDY QUESTION</st> <p>What is the percentage of overtreatment, i.e. fertility treatment started too early, in couples with unexplained infertility who were eligible for tailored expectant management?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Overtreatment occurred in 36% of couples with unexplained infertility who were eligible for an expectant management of at least 6 months.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Prognostic models in reproductive medicine can help to identify infertile couples that would benefit from fertility treatment. In couples with unexplained infertility with a good chance of natural conception within 1 year, based on the Hunault prediction model, an expectant management of 6&ndash;12 months, as recommended in international fertility guidelines, prevents unnecessary treatment.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A retrospective cohort study in 25 participating clinics, with follow-up of all couples who were seen for infertility in 2011&ndash;2012.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>In all, 9818 couples were seen for infertility in the participating clinics. Couples were eligible to participate if they were diagnosed with unexplained infertility and had a good prognosis of natural conception (&gt;30%) within 1 year based on the Hunault prediction model. Data to assess overtreatment were collected from medical records. Multilevel regression analyses were performed to investigate associations of overtreatment with patient and clinic characteristics.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Five hundred and forty-four couples eligible for expectant management were included in this study. Among these, overtreatment, i.e. starting medically assisted reproduction within 6 months, occurred in 36%. The underlying quality indicators showed that in 34% no prognosis was calculated and that in 42% expectant management was not recommended. Finally, 16% of the couples for whom a correct recommendation of expectant management for at least 6 months was made, started treatment within 6 months anyway. Overtreatment was associated with childlessness, higher female age and a longer duration of infertility. No associations between overtreatment and clinic characteristics were found.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The response rate was low compared with other fertility studies. Evaluation of possible selection bias showed that responders had a higher socio-economic status than non-responders.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our findings show that developing and publishing guideline recommendations on tailored expectant management (TEM) is not enough and that overtreatment still occurs frequently. Future research should focus on tailored efforts to implement guideline recommendations on TEM.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>Supported by Netherlands Organisation for Health Research and Development (ZonMW). ZonMW had no role in designing the study, data collection, analysis and interpretation of data or writing of the report. Competing interests: none.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p><A HREF="www.trialregister.nl">www.trialregister.nl</A> NTR3405.</p> </sec>


Can you ever collect too many oocytes?
<sec><st>STUDY QUESTION</st> <p>Does the chance of pregnancy keep improving with increasing number of oocytes, or can you collect too many?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Clinical pregnancy (CP) and live birth (LB) rates per embryo transfer varied from 10.2 and 9.2% following one oocyte collected to 37.7 and 31.3% when &gt;16 oocytes were collected. Regression modelling indicated success rates increased or at least stayed the same with number of oocytes collected.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>It has been suggested that if &gt;15 oocytes are collected, the success rate for fresh embryo transfers decreases. As this is counterintuitive, as more oocytes should result in more embryos, with a better choice of quality embryos, we decided to analyse the recent experience in a busy IVF unit.</p> </sec> <sec><st>STUDY DESIGN, SIZE DURATION</st> <p>A retrospective analysis of clinical pregnancy and live birth outcome, with respect to number of oocytes collected at Monash IVF for the 2-year period between August 2010 and July 2012, where patients under the age of 45 years underwent a fresh embryo transfer. This included 7697 stimulated cycles for IVF and ICSI.</p> </sec> <sec><st>PARTICIPANT/MATERIALS, SETTING, METHODS</st> <p>Statistical analysis involved data tables and graphs comparing oocyte number with outcome. Results of women who had their first oocyte collection with an embryo transfer within the reference period were analysed by logistic regression analysis including other covariates that might influence pregnancy outcome. Analysis was also carried out of all the 7679 oocyte collections undertaken, resulting in fresh embryo transfers by generalized estimating equations to allow for the within subject correlation in outcomes for repeated treatments.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The number of oocytes collected varied from 1 to 48. Clinical pregnancy and live birth rates per embryo transfer varied from 10.2 and 9.2% when only one oocyte was collected to 37.7 and 31.3% when &gt;16 oocytes were collected. Regression modelling indicated success rates increased or at least stayed the same or with the number of oocytes collected. The percentage of women with embryos cryopreserved increased from under 20% with &lt;4 oocytes collected to over 70% with &gt;16 oocytes collected. There was a slight increase (from 18 to 22%) in oocyte immaturity and a more marked increase (from 0 to 3%) in cancelling fresh transfers to prevent Ovarian Hyperstimulation Syndrome (OHSS) with increase in number of oocytes collected above 16. The results of this study suggest that you cannot collect too many oocytes as both clinical pregnancy and live birth rates do not decrease with high numbers of oocytes collected. However, once &gt;15 oocytes are collected, everything gets quite uncertain.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>As the data become sparse above 15 oocytes, we could not demonstrate a significant increase in pregnancy rates above this number. Larger studies would be required to answer the question whether there is a plateau, or rates continue to increase. The negative of aggressive stimulation to produce many oocytes is that the risk of OHSS increases, and this is the most serious complication of ovarian stimulation.</p> </sec> <sec><st>STUDY FUNDING/COMPLETING OF INTEREST(S)</st> <p>No funding was required. There is no conflict of interest, except that G.K., V.M. and C.M. are shareholders in Monash IVF Pty Ltd.</p> </sec>


In vitro maturation as an alternative to standard in vitro fertilization for patients diagnosed with polycystic ovaries: a comparative analysis of fresh, frozen and cumulative cycle outcomes
<sec><st>STUDY QUESTION</st> <p>Is <I>in vitro</I> maturation (IVM) as successful as standard <I>in vitro</I> fertilization (IVF) for the treatment of patients with polycystic ovaries (PCO) in terms of fresh, frozen and cumulative pregnancy outcomes?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>There was no difference in clinical pregnancy rates in fresh or frozen embryo transfer (FET) cycles between the two treatment groups however, the IVM group showed a lower clinical pregnancy rate cumulatively. There was significantly fewer live births resulting from IVM treatment for both fresh and cumulative cycle outcomes however, there was no difference in live birth rates resulting from FETs between IVM and IVF treatment.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>IVM is well recognized as the only treatment option to eliminate completely the incidence of ovarian hyperstimulation syndrome. However, historically IVM has been less successful than standard IVF in terms of clinical pregnancy, implantation and live birth rates.</p> </sec> <sec><st>STUDY DESIGN, SIZE, AND DURATION</st> <p>This paper represents a retrospective case&ndash;control study. The study involved 121 participants who underwent 178 treatment cycles. Cycles were completed between March 2007 and December 2012. All fresh cycles and subsequent FET cycles were included in the analysis to calculate cumulative outcomes.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, AND METHODS</st> <p>All participants were prospectively diagnosed with PCO morphology or polycystic ovarian syndrome (PCOS) and underwent either IVM or standard IVF treatment. Their treatment outcomes were analysed with regard to embryological data, and the rate of biochemical pregnancy, clinical pregnancy and live birth, in addition maternal and neonatal outcomes were assessed. Fifty-six patients underwent 80 cycles of IVM treatment and 65 patients underwent 98 cycles of standard IVF treatment.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>For fresh cycles, the differences in the biochemical pregnancy, clinical pregnancy or miscarriage rates between the two treatment groups were not statistically significant. The IVM group showed significantly lower live birth rates in fresh cycles in comparison to standard IVF treatment (18.8 versus 31.0%, <I>P</I> = 0.021). For frozen embryo transfer (FET) cycles the differences in biochemical pregnancy, clinical pregnancy, live birth or miscarriage rates between the two treatments groups were not statistically significant. The cumulative biochemical pregnancy (67.5 versus 83.7%, <I>P</I> = 0.018), clinical pregnancy (51.3 versus 65.3%, <I>P</I> = 0.021) and live birth rates (41.3 versus 55.1%, <I>P</I> = 0.005) were significantly lower in the IVM group in comparison to the standard IVF treatment group. There was no overall difference in the cumulative miscarriage rates between the two treatment groups. There was no difference between treatment methods with regard to the neonatal outcomes, and the IVM group had a significantly lower rate of ovarian hyperstimulation syndrome (0 versus 7.1%, <I>P</I> &lt; 0.001).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This was an observational study and further randomized clinical trials are required to clarify the difference in outcomes between standard IVF and IVM for patients with PCO/PCOS.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This is the first study to compare IVM with standard IVF in PCO/PCOS patients using blastocyst development and single embryo transfer. Furthermore, it is the first study to show the results of fresh, frozen and cumulative treatment cycle outcomes between the two groups. Our results show similar success rates to those reported from other groups, particularly in relation to the incidence of miscarriage in fresh IVM cycles and improved success from FET cycles. Maternal and neonatal outcomes are consistent with the limited literature available.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was supported by the Women's and Infant's Research Foundation of Western Australia. Professor Hart is Medical Director of Fertility Specialists of Western Australia (FSWA) and a shareholder Western IVF. He has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. T.H. is a consultant with FSWA and a shareholder in Western IVF. She has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. The other authors have no competing interests.</p> </sec>


Treatment with Ca2+ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study
<sec><st>STUDY QUESTION</st> <p>Does calcium ionophore treatment (A23187, calcimycin) improve embryo development and outcome in patients with a history of developmental problems/arrest?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Application of A23187 leads to increased rates of cleavage to 2-cell stage, blastocyst formation and clinical pregnancy/live birth.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Studies on lower animals indicate that changes in intracellular free calcium trigger and regulate the events of cell division. In humans, calcium fluctuations were detected with a peak shortly before cell division. Interestingly, these calcium oscillations disappeared in arrested embryos. Mitotic division blocked with a Ca<sup>2+</sup> chelator could be restored by means of ionophores in an animal model.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This prospective, multicenter (five Austrian centers), uncontrolled intervention study (duration 1 year) includes 57 patients who provided informed consent.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Inclusion criteria were complete embryo developmental arrest in a previous cycle (no transfer), complete developmental delay (no morula/blastocyst on Day 5), or reduced blastocyst formation on Day 5 (&le;15%). Severe male factor patients and patients with &lt;30% fertilization rate after ICSI were excluded because these would be routine indications for ionophore usage. The total of the 57 immediately preceding cycles in the same patients constituted the control cycles/control group. In the treatment cycles, all metaphase II-oocytes were exposed to a commercially available ready-to-use ionophore for 15 min immediately after ICSI. After a three-step washing procedure, <I>in vitro</I> culture was performed as in the control cycles, up to blastocyst stage when achievable.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Fertilization rate did not differ (75.4 versus 73.2%); however, further cleavage to 2-cell stage was significantly higher (<I>P</I> &lt; 0.001) in the ionophore group (98.5%) when compared with the control cycles (91.9%). In addition, significantly more (<I>P</I> &lt; 0.05) blastocysts formed on Day 5 in the study compared with the control group (47.6 versus 5.5%, respectively) and this was associated with a significant increase (<I>P</I> &lt; 0.01) in the rates of implantation (44.4 versus 12.5%), clinical pregnancy (45.1 versus 12.8%) and live birth (45.1 versus 12.8%). All babies born at the time of writing (22/28) were healthy.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The frequency of patients showing embryo developmental problems was expected to be low; therefore, a multicenter approach was chosen in order to increase sample size. In one-third of the cycles, the clinician or patient requested a change of stimulation protocol; however, this did not influence the developmental rate of embryos.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This is the first evidence that developmental incompetence of embryos is an additional indication for ionophore treatment. The present approach is exclusively for overcoming cleavage arrest.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No funding received. T.E. reports fees from Gynemed, outside the submitted work. All co-authors have no interest to declare.</p> </sec>


Mental distress and personality in women undergoing GnRH agonist versus GnRH antagonist protocols for assisted reproductive technology
<sec><st>STUDY QUESTION</st> <p>Do mental distress and mood fluctuations in women undergoing GnRH agonist and GnRH antagonist protocols for assisted reproductive technology (ART) differ depending on protocol and the personality trait, neuroticism?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>ART treatment did not induce elevated levels of mental distress in either GnRH antagonist or agonist protocols but neuroticism was positively associated with increased mental distress, independent of protocols.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>ART treatment may increase mental distress by mechanisms linked to sex hormone fluctuations. General psychological characteristics, such as personality traits indexing negative emotionality, e.g. neuroticism, are likely to affect mental distress during ART treatment.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A total of 83 women undergoing their first ART cycle were consecutively randomized 1:1 to GnRH antagonist (<I>n</I> = 42) or GnRH agonist (<I>n</I> = 41) protocol. The study population was a subgroup of a larger ongoing Danish clinical randomized trial and was established as an add-on in the period 2010&ndash;2012.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Women in the GnRH antagonist protocol received daily injections with recombinant follicle-stimulating hormone, Puregon<sup>&reg;</sup> and subcutaneous injections with GnRH antagonist, Orgalutran<sup>&reg;</sup>. Women in the GnRH agonist protocol received nasal administration of the GnRH agonist, Synarela<sup>&reg;</sup> and subcutaneous injections with FSH, Puregon<sup>&reg;</sup>. The study design did not allow for a blinding procedure. All women self-reported the Profile of Mood States, the Perceived Stress Scale, the Symptom Checklist-92-Revised, and the Major Depression Inventory questionnaires, at baseline, at ART cycle day 35, on the day of oocyte pick-up, and on the day of hCG testing. Also, a series of Profile of Mood States were reported daily during pharmacological treatment to monitor mood fluctuations. The personality trait Neuroticism was assessed at baseline by the self-reported NEO-PI-R questionnaire.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>ART did not induce within- or between-protocol changes in any of the applied measures of mental distress. However, the GnRH antagonist protocol was associated with more pronounced median mood fluctuations during the stimulation phase (antagonist, 11.0 SD, [IQR = 21.1&ndash;6.1]; agonist, 8.9 SD, [IQR = 11.3&ndash;5.7], <I>P</I> = 0.025). This association became non-significant after applying a Bonferroni&ndash;Holm correction. Neuroticism was highly positively associated with increased levels of mental distress throughout treatment independent of protocols (all <I>P</I>-values &lt;0.006), and cross-sectional analysis revealed that women with high or low Neuroticism scores at baseline showed a significant trend towards lower chances of a positive pregnancy test (<I>P</I>-value =0.028).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Information on prognostic factors such as preceding length of infertility, number of retrieved oocytes and number of prior insemination treatments was not accounted for in the analyses. The stratification of protocols by age in the subgroups of women included in this study was suboptimal. Women with prior or current use of antidepressant medication were excluded from our study.</p> </sec> <sec><st>WIDER IMPLICATIONS</st> <p>Our results imply that mental distress emerging during ART treatment is not causally linked to hypogonadism <I>per se</I> or to the choice of protocol. Rather, our data highlight the potential importance of (i) rapid increases in ovarian steroids and (ii) addressing personality traits indexing negative emotionality, i.e. Neuroticism, in women undergoing ART treatment, to optimize both emotional adjustment and, possibly, the chances of obtaining pregnancy.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The Danish Research Council for Independent Research and MSD, Denmark kindly supported the study. The authors declare no competing financial interests.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>EudraCT &ndash; 2008-005452-24.</p> </sec>


Expectations and experiences of gamete donors and donor-conceived adults searching for genetic relatives using DNA linking through a voluntary register
<sec><st>STUDY QUESTION</st> <p>What are the experiences of donor-conceived adults and donors who are searching for a genetic link through the use of a DNA-based voluntary register service?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Donor-conceived adults and donors held positive beliefs about their search and although some concerns in relation to finding a genetically linked relative were reported, these were not a barrier to searching.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Research with donor-conceived people has consistently identified their interest in learning about&mdash;and in some cases making contact with&mdash;their donor and other genetic relatives. However, donor-conceived individuals or donors rarely have the opportunity to act on these desires.</p> </sec> <sec><st>STUDY DESIGN, SIZE, AND DURATION</st> <p>A questionnaire was administered for online completion using Bristol Online Surveys. The survey was live for 3 months and responses were collected anonymously.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, AND METHODS</st> <p>The survey was completed by 65 donor-conceived adults, 21 sperm donors and 5 oocyte donors who had registered with a DNA-based voluntary contact register in the UK. The questionnaire included socio-demographic questions, questions specifically developed for the purposes of this study and the standardized Aspects of Identity Questionnaire (AIQ).</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Motivations for searching for genetic relatives were varied, with the most common reasons being curiosity and passing on information. Overall, participants who were already linked and those awaiting a link were positive about being linked and valued access to a DNA-based register. Collective identity (reflecting self-defining feelings of continuity and uniqueness), as assessed by the AIQ, was significantly lower for donor-conceived adults when compared with the donor groups (<I>P</I> &lt; 0.05), but not significantly different between linked/not linked or length of time since disclosure of donor conception (all <I>P</I>s &gt; 0.05) for donor-conceived adults.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Participants were members of a UK DNA-based registry which is unique. It was therefore not possible to determine how representative participants were of those who did not register for the service, those in other countries or of those who do not seek information exchange or contact.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This is the first survey exploring the experiences of donor-conceived adults and donors using a DNA-based voluntary register to seek information about and contact with genetic relatives and the first to measure aspects of identity using standardized measures. Findings provide valuable information about patterns of expectations and experiences of searching through DNA linking, identity and of having contact in the context of donor conception that will inform future research, practice and policy development.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTERESTS</st> <p>No funding was obtained for this study. The authors have no competing interests to declare except for M.C. who was national adviser to UKDL from 2003&ndash;2013.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


Human embryos from overweight and obese women display phenotypic and metabolic abnormalities
<sec><st>STUDY QUESTION</st> <p>Is the developmental timing and metabolic regulation disrupted in embryos from overweight or obese women?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Oocytes from overweight or obese women are smaller than those from women of healthy weight, yet post-fertilization they reach the morula stage faster and, as blastocysts, show reduced glucose consumption and elevated endogenous triglyceride levels.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Female overweight and obesity is associated with infertility. Moreover, being overweight or obese around conception may have significant consequences for the unborn child, since there are widely acknowledged links between events occurring during early development and the incidence of a number of adult disorders.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>We have performed a retrospective, observational analysis of oocyte size and the subsequent developmental kinetics of 218 oocytes from 29 consecutive women attending for ICSI treatment and have related time to reach key developmental stages to maternal bodyweight. In addition, we have measured non-invasively the metabolic activity of 150 IVF/ICSI embryos from a further 29 consecutive women who donated their surplus embryos to research, and have related the data retrospectively to their body mass index (BMI).</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>In a clinical IVF setting, we compared oocyte morphology and developmental kinetics of supernumerary embryos collected from overweight and obese women, with a BMI in excess of 25 kg/m<sup>2</sup> to those from women of healthy weight. A Primovision Time-Lapse system was used to measure developmental kinetics and the non-invasive COnsumption/RElese of glucose, pyruvate, amino acids and lactate were measured on spent droplets of culture medium. Total triglyceride levels within individual embryos were also determined.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Human oocytes from women presenting for fertility treatment with a BMI exceeding 25 kg/m<sup>2</sup> are smaller (<I>R</I><sup>2</sup> = &ndash;0.45; <I>P</I> = 0.001) and therefore less likely to complete development post-fertilization (<I>P</I> &lt; 0.001). Those embryos that do develop reach the morula stage faster than embryos from women of a BMI &lt; 25 kg/m<sup>2</sup> (&lt;0.001) and the resulting blastocysts contain fewer cells notably in the trophectoderm (<I>P</I> = 0.01). The resulting blastocysts also have reduced glucose consumption (<I>R</I><sup>2</sup> = &ndash;0.61; <I>P</I> = 0.001), modified amino acid metabolism and increased levels of endogenous triglyceride (<I>t</I> = 4.11, <I>P</I> &lt; 0.001). Our data further indicate that these differences are independent of male BMI.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Although statistical power has been achieved, this is a retrospective study and relatively small due to the scarcity of human embryos available for research. Consequently, subanalysis of overweight and obese was not possible based on the sample size. The analysis has been performed on supernumerary embryos, originating from a single IVF unit and not selected for use in treatment. Thus, it was not possible to speculate how representative the findings would be of the better quality embryos transferred or frozen for each patient.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The data indicate that a high BMI of women at conception is associated with distinct phenotypic changes in the embryo during the preimplantation period, highlighting the importance of prepregnancy body weight in optimizing the chances of fertility and safeguarding maternal and offspring health. These changes to the metabolic fingerprint of human embryos which are most likely a legacy of the ovarian conditions under which the oocyte has matured may reduce the chances of conception for overweight women and provide good evidence that the metabolic profile of the early embryo is set by sub-optimal conditions around the time of conception. The observed changes could indicate long-term implications for the health of the offspring of overweight and obese women.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by the Hull IVF Unit Charitable Trust and the Hull York Medical School. There are no conflict of interests.</p> </sec>


Overlap of proteomics biomarkers between women with pre-eclampsia and PCOS: a systematic review and biomarker database integration
<sec><st>STUDY QUESTION</st> <p>Do any proteomic biomarkers previously identified for pre-eclampsia (PE) overlap with those identified in women with polycystic ovary syndrome (PCOS).</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Five previously identified proteomic biomarkers were found to be common in women with PE and PCOS when compared with controls.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Various studies have indicated an association between PCOS and PE; however, the pathophysiological mechanisms supporting this association are not known.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A systematic review and update of our PCOS proteomic biomarker database was performed, along with a parallel review of PE biomarkers. The study included papers from 1980 to December 2013.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>In all the studies analysed, there were a total of 1423 patients and controls. The number of proteomic biomarkers that were catalogued for PE was 192.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Five proteomic biomarkers were shown to be differentially expressed in women with PE and PCOS when compared with controls: transferrin, fibrinogen &alpha;, &beta; and chain variants, kininogen-1, annexin 2 and peroxiredoxin 2. In PE, the biomarkers were identified in serum, plasma and placenta and in PCOS, the biomarkers were identified in serum, follicular fluid, and ovarian and omental biopsies.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The techniques employed to detect proteomics have limited ability in identifying proteins that are of low abundance, some of which may have a diagnostic potential. The sample sizes and number of biomarkers identified from these studies do not exclude the risk of false positives, a limitation of all biomarker studies. The biomarkers common to PE and PCOS were identified from proteomic analyses of different tissues.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This data amalgamation of the proteomic studies in PE and in PCOS, for the first time, discovered a panel of five biomarkers for PE which are common to women with PCOS, including transferrin, fibrinogen &alpha;, &beta; and chain variants, kininogen-1, annexin 2 and peroxiredoxin 2. If validated, these biomarkers could provide a useful framework for the knowledge infrastructure in this area. To accomplish this goal, a well co-ordinated multidisciplinary collaboration of clinicians, basic scientists and mathematicians is vital.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No financial support was obtained for this project. There are no conflicts of interest.</p> </sec>


Inhibitor of apoptosis proteins (IAPs) may be effective therapeutic targets for treating endometriosis
<sec><st>STUDY QUESTION</st> <p>What is the role of the inhibitor of apoptosis proteins (IAPs) in human endometriotic tissues and a mouse model of endometriosis?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Four IAP proteins were expressed in endometriotic tissue indicating IAPs may be a key factor in the pathogenesis and progression of endometriosis.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Overexpression of IAPs protects against a number of proapoptotic stimuli. IAPs (c-IAP1, c-IAP2, XIAP and Survivin) are expressed in human ectopic endometrial stromal cells (ESCs) from ovarian endometriomas.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Forty-eight women with or without ovarian endometrioma are included in this study. BALB/c mice (<I>n</I> = 24) were used for the mouse endometriosis model. Mice with surgically induced endometriosis were treated with an IAP antagonist (BV6) for 4 weeks.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Human ectopic endometrial tissues from chocolate cysts and eutopic endometrial tissue were collected. ESCs were enzymatically isolated from these tissues. ESC proliferation was examined by 5-bromo-2'-deoxyuridine&mdash;enzyme-linked immunosorbent assay. IAPs expression in tissue derived from eutopic endometria and chocolate cysts was evaluated using real-time RT&ndash;PCR and immunohistochemistry. A homologous mouse endometriosis model was established by transplanting donor mouse uterine tissue into the abdominal cavities of recipient mice. After treating the mice with BV6 (i.p. 10 mg/ml), the extent of endometriosis-like lesions in mice was measured and proliferative activity assessed by Ki67 staining. All experiments were repeated a minimum of three times.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>IAP (c-IAP1, c-IAP2, XIAP and Survivin) mRNA and protein in human ectopic endometrial tissues were expressed at higher levels than in eutopic endometrial tissues (<I>P</I> &lt; 0.05). All four IAPs proteins were expressed in mouse endometriosis-like implants. BV6 inhibited BrdU incorporation of human ESCs (<I>P</I> &lt; 0.05 versus control). BV6 also decreased the total number, weight, surface area and Ki67 positive cells in the endometriosis-like lesions in the mice (<I>P</I> &lt; 0.05 versus control).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue only, not human pathological endometriotic tissue. Furthermore, the effects of BV6 on human ESCs and mouse endometriosis-like lesions may differ between the species.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our data support the hypothesis that IAPs are involved in the development of endometriosis, and therefore an inhibitor of IAPs has potential as a novel treatment for endometriosis.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was supported by KAKENHI (Japan Society for the Promotion of Science, Grant-in-Aid: to F.T.; 21592098 and to T.H.; 24659731) and Yamaguchi Endocrine Research Foundation. The authors have no conflicts of interest to disclose.</p> </sec>


NLRP7 and KHDC3L, the two maternal-effect proteins responsible for recurrent hydatidiform moles, co-localize to the oocyte cytoskeleton
<sec><st>STUDY QUESTION</st> <p>What is the subcellular localization in human oocytes and preimplantation embryos, of the two maternal-effect proteins, NLRP7 and KHDC3L, responsible for recurrent hydatidiform moles (RHMs)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>NLRP7 and KHDC3L localize to the oocyte cytoskeleton and are polar and absent from the cell-to-cell contact region in early preimplantation embryos.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>NLRP7 and KHDC3L expression has been described at the RNA level in some stages of human oocytes and preimplantation embryos and at the protein level by immunohistochemistry in human and bovine ovaries. NLRP7 and KHDC3L co-localize to the microtubule organizing center and/or the Golgi apparatus in human hematopoietic cells.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A total of 164 spare human oocytes and embryos from patients undergoing <I>in vitro</I> fertilization were used.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Oocytes and early cleavage-stage embryos were fixed, immunostained with NLRP7 and/or KHDC3L antibodies, and analyzed using high-resolution confocal immunofluorescence and electron microscopies.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>NLRP7 and KHDC3L localize to the cytoskeleton and are predominant at the cortical region in growing oocytes. After the first cellular division, these two maternal-effect proteins become asymmetrically confined to the outer cortical region and excluded from the cell-to-cell contact region until the blastocyst stage where NLRP7 and KHDC3L homogeneously redistribute to the cytoplasm and the nucleus, respectively.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>We could not analyze fresh human oocytes and embryos. The analyzed materials were donated by patients undergoing assisted reproductive technologies and released for research 1&ndash;3 days after their collection and the transfer of embryos to the patients.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our study is the first comprehensive and high-resolution localization of the only two known maternal-effect proteins, NLRP7 and KHDC3L, in human oocytes and preimplantation embryos. Our data contribute to a better understanding of the roles of these two proteins in the integrity of the oocytes, post-zygotic divisions, and cell-lineage differentiation.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was supported by the Canadian Institute of Health Research (86546 to R.S.); E.A. was supported by fellowships from the Research Institute of the McGill University Health Centre and a CREATE award from the R&eacute;seau Qu&eacute;b&eacute;cois en Reproduction. All authors declare no conflict of interest.</p> </sec>


Ovarian response prediction in GnRH antagonist treatment for IVF using anti-Mullerian hormone
<sec><st>STUDY QUESTION</st> <p>What is the clinical value of anti-M&uuml;llerian hormone (AMH) for the prediction of high or low ovarian response in controlled ovarian stimulation for IVF using GnRH antagonist treatment?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>AMH as a single test has substantial accuracy for ovarian response prediction in GnRH antagonist treatment for IVF, with a higher accuracy for predicting a high response than for low response.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The role of AMH and other patient characteristics in ovarian response prediction has been studied extensively in long GnRH agonist protocols; however, little information is available regarding the clinical value in GnRH antagonists.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This is an observational (retrospective) substudy as part of an ongoing cohort study. A total of 487 patients scheduled for IVF/ICSI between 2006 and 2011 were included in the study.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Patients with a regular cycle who underwent their first IVF/ICSI cycle with GnRH antagonist treatment while receiving a starting dose of 150 or 225 IU recombinant FSH were included in the study. Patients were divided into three subgroups according to the following ovarian response categories: high (&gt;15 oocytes or cycle cancellation), normal (4&ndash;15 oocytes) and low (&lt;4 oocytes or cycle cancellation). Serum samples collected prior to IVF treatment were used to determine serum AMH levels.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>According to the predefined ovarian response categories, 58 patients were classified as high, 326 as normal and 101 as low responders, and the ongoing pregnancy rates did not differ among groups (19.0, 22.1 and 16.8%, respectively, <I>P</I> = 0.9). For the prediction of high response, AMH had an area under the receiver-operating characteristic curve (AUC) of 0.87. Both female age and BMI had lower accuracy (AUC 0.66 and 0.58, respectively). For low response prediction, again AMH had a better accuracy (AUC 0.79) than female age and BMI (AUC 0.59 and 0.56, respectively). In a multivariate model, including the factors age, AMH, BMI, smoking, type and duration of subfertility, only BMI added some predictive value to AMH for both high and low response prediction. Clinical test characteristics demonstrated that using a specificity of ~90%, the detection rate of AMH for high and low response, corresponding with a test cut-off of 4.5 and 0.8 &micro;g/l, was ~60 and ~45%, respectively.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The impact of the antral follicle count (AFC) on ovarian response prediction in GnRH antagonists was not assessed; however, previously studies demonstrated that for GnRH antagonists, AMH has a better accuracy than AFC.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The current study demonstrates that AMH is an adequate predictor for both high and low response in GnRH antagonist cycles, showing a similar accuracy to GnRH agonists, as reported previously. The optimization and individualization of GnRH antagonist protocols may be improved by using an AMH-tailored approach.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by the Academic Institutional Resources of the Department of Reproductive Medicine of the UMC Utrecht. O.H., M.J.C.E, E.W.G.L and H.L.T. have nothing to declare. N.S.M. has received fees and/or grant support from the following companies (in alphabetic order): Anecova, Ferring, Informa, Merck Serono and MSD. B.C.J.M.F. has received fees and/or grant support from the following companies (in alphabetic order); Childhealth, CVON, Ferring, Ova-Science, PregLem, Roche and Watson laboratories. F.J.B. has received fees and/or grant support from the following companies (in alphabetic order); Merck Serono and MSD.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p><A HREF="www.clinicaltrials.gov">www.clinicaltrials.gov</A>, Protocol ID 13-109.</p> </sec>


Highly purified hMG versus recombinant FSH plus recombinant LH in intrauterine insemination cycles in women >=35 years: a RCT
<sec><st>STUDY QUESTION</st> <p>Is the treatment with recombinant FSH (rFSH) <I>plus</I> recombinant LH (rLH) more effective than highly purified (HP)-hMG in terms of ongoing pregnancy rate (PR) in women &ge;35 years of age undergoing intrauterine insemination (IUI) cycles?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The ongoing PR was not significantly different in women treated with rFSH <I>plus</I> rLH or with HP-hMG.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Although previous studies have shown beneficial effects of the addition of LH activity to FSH, in terms of PR in patients aged over 34 years having ovulation induction, no studies have compared two different gonadotrophin preparations containing LH activity in women &ge;35 years of age in IUI cycles.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A single-centre RCT was performed between May 2012 and September 2013 with 579 women &ge;35 years of age undergoing IUI cycles. The patients were randomly assigned to one of the two groups, rFSH in combination with rLH group or HP-hMG (Meropur) group, by giving them a code number from a computer generated randomization list, in order of enrolment. The randomization visit took place on the first day of ovarian stimulation.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Five hundred and seventy-nine patients with unexplained infertility or mild male factor undergoing IUI cycles were recruited in a university hospital setting. All women were enrolled in this study only for one cycle of treatment. Five hundred and seventy-nine cycles were included in the final analysis. Two hundred and ninety patients were treated with rFSH in combination with rLH and 289 patients were treated with HP-hMG. The ovarian stimulation cycle started on the third day of the menstrual cycle and the starting gonadotrophin doses used were 150 IU/day of rFSH <I>plus</I> 150 IU/day of rLH or 150 IU/day of HP-hMG. The drug dose was adjusted according to the individual follicular response. A single IUI per cycle was performed 34&ndash;36 h after hCG injection.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The main outcome measures were ongoing PR and number of interrupted cycles for high risk of ovarian hyperstimulation syndrome (OHSS). Ongoing pregnancy rates were 48/290 (17.3%) in the recombinant group versus 35/289 (12.2%) in the HP-hMG group [(odds ratio (OR) 1.50, 95% CI 0.94&ndash;2.41, <I>P</I> = 0.09]. The number of interrupted cycles for high risk of OHSS was 13/290 (4.5%) in the rFSH <I>plus</I> rLH group and 2/289 (0.7%) in the HP-hMG group (OR 6.73, 95% CI 1.51&ndash;30.12, <I>P</I> = 0.013).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>One of the limitations of this study was the early closure and the ongoing PR could be overestimated. Both patient and gynaecologist were informed of the assigned treatment.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our results demonstrated the lack of differences in terms of ongoing PR between recombinant product and HP-hMG, in women &ge;35 years undergoing controlled ovarian stimulation for IUI cycles. HP-hMG was safer than recombinant gonadotrophin concerning the risk of OHSS.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>None.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>NCT01604044.</p> </sec>


Male experiences of unintended pregnancy: characteristics and prevalence
<sec><st>STUDY QUESTION</st> <p>What are the characteristics and circumstances of pregnancies men report as unintended in France?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Pregnancies reported as unintended were most prevalent among young men with insecure financial situations, less stable relationships and inconsistent use of contraception or false assumptions about their partner's use of contraception.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Efforts to involve men in family planning have increased over the last decade; however, little is known about factors associated with men's pregnancy intentions and associated contraceptive behaviours.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>The data presented in this study were drawn from the nationally representative <I>FECOND</I> study, a population-based survey conducted in France in 2010. The sample comprised 8675 individuals (3373 men), aged 15&ndash;49 years, who responded to a telephone interview about socio-demographics and topics related to sexual and reproductive health. The total refusal rate was 20%.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>This study included 2997 men, of whom 664 reported 893 recent pregnancies (in the 5 years preceding the survey). Multivariate Poisson's regression with population-averaged marginal effects was applied to assess the individual and contextual factors associated with men's intentions for recent pregnancies. The contraceptive circumstances leading to the unintended pregnancies were also assessed.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Of all heterosexually active men, 5% reported they had experienced an unintended pregnancy with a partner in the last 5 years. A total of 20% of recent pregnancies reported by men were qualified to be unintended, of which 45% ended in induced abortion. Of pregnancies following a previous unintended pregnancy, 68% were themselves unintended. Among all heterosexually active men, recent experience of an unintended pregnancy was related to age, mother's education, age at first sex, parity, contraceptive method history, lifetime number of female partners and the relationship situation at the time of survey. Recent unintended pregnancies were also related to pregnancy order and to the financial and professional situation at the time of conception. The majority of unintended pregnancies occurred when men or their partners were using contraceptives; 58% of contraceptive users considered that the pregnancy was due to inconsistent use and 39% considered that it resulted from method failure. Half of the non-users who reported an unintended pregnancy thought that their partner was using a contraceptive method. The relative risk of non-use of a contraceptive method during the month of conception of a recent unintended pregnancy was higher among those without a high school degree (IRR = 2.9, CI 1.6, 5.2) and higher among men for whom the pregnancy interfered with education (IRR = 1.8, CI 1.0, 3.1) or work (IRR = 1.9, CI 1.1, 3.6).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>From the perspective of men, the unintended pregnancy rates may be underestimated due to a combination of underreporting of abortion and post-rationalization of birth intentions. Our use of a dichotomous measure of unintended pregnancy is unlikely to fully capture the multidimensional construct of pregnancy intentions.</p> </sec> <sec><st>WIDER IMPLICATION OF THE FINDINGS</st> <p>These results call for gender-inclusive family planning programmes, which fully engage men as active participants in their own rights.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The FECOND study was supported by a grant from the French Ministry of Health, a grant from the French National Agency of Research (#ANR-08-BLAN-0286-01; PIs N.B., C.M.), and funding from National Institute of Health and Medical Research (INSERM) and the National Institute for Demographic Research (INED). None of the authors have competing interests.</p> </sec>


Cervical conization doubles the risk of preterm and very preterm birth in assisted reproductive technology twin pregnancies
<sec><st>STUDY QUESTION</st> <p>Does cervical conization add an additional risk of preterm birth (PTB) in assisted reproduction technology (ART) singleton and twin pregnancies?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Cervical conization doubles the risk of preterm and very PTB in ART twin pregnancies.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>ART and cervical conization are both risk factors for PTB.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>In this national population-based controlled cohort study, we included all ART singletons and twin deliveries from 1995 to 2009 in Denmark by cross-linkage of maternal and child data from the National IVF register and the Medical Birth register. Furthermore, control groups of naturally conceived (NC) singletons and twins were extracted. Cervical diagnoses were obtained from the Danish Pathology register. Cervical conization included both cold knife cone and LEEP (loop electrosurgical excision procedure) but not cervical biopsies. The main outcomes measures were PTB (PTB &le; 37 + 0 gestational weeks), very preterm birth (VPTB &le; 32 + 0 gestational weeks) and preterm premature rupture of membranes (PPROM).</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>In all 16 923 ART singletons and 4829 ART twin deliveries were included. A random sample of NC singletons, 2-fold the size of the ART singleton group matched by date and year of birth (<I>n</I> = 33 835) and all NC twin deliveries (<I>n</I> = 15 112), was also extracted. Multiple logistic regression analyses were performed to adjust for the following confounders: maternal age, parity, year of child birth and sex of child.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Cervical morbidity (dysplasia and conization) was more often observed in ART pregnancies (6.2% of ART singletons and 5.4% ART twins) than in NC pregnancies (4.2% for NC singletons and 4.5% for NC twins), both for singletons and twins. In ART singleton deliveries, the PTB rate was 13.1 versus 8.2% in women with and without conization, respectively, with an adjusted odds ratio (aOR) of 1.56 [95% confidence interval (CI) 1.21&ndash;2.01]. In ART twin deliveries, the prevalence of PTB was 58.2 versus 41.3% in women with and without conization, respectively, with an aOR 1.94 (95% CI 1.36&ndash;2.77), and the risk of VPTB was also doubled. Furthermore, previous dysplasia (without conization) increased the risk of VPTB in ART twins (aOR 1.74, 95% CI 1.04&ndash;2.94). Cervical dysplasia did not increase the risk of any of the other adverse outcomes in ART singletons or twins. The risk of PPROM was increased in both in ART and NC singleton deliveries with conization versus no conization; however, this increased risk of PPROM after conization was not observed in either ART or NC twin pregnancies.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>We were not able to adjust for the height of the cervical cone or the severity of the cervical intraepithelial neoplasia (CIN) or the time window between diagnosis of CIN and ART treatment. The finding on an increased risk of VPTB in ART twin pregnancies after dysplasia without conization may be random as we found no other increased risk after dysplasia alone either in singletons or in twins.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>After ART and prior conization, 58% of twin pregnancies versus 13% of ART singleton pregnancies result in PTB. There is a doubled risk of preterm delivery in ART twins with conization versus ART twins with no prior conization. Single-embryo transfer should always be recommended in women with prior conization irrespective of female age, embryo quality and prior number of ART attempts.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No external funding was achieved for this project.</p> </sec>


Pregnancy outcome and long-term follow-up after in vitro fertilization in women with renal transplantation
<sec><st>STUDY QUESTION</st> <p>What is the child morbidity after IVF in women who have received a kidney transplant?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Overall, obstetric outcome and morbidity in children of women who had undergone renal transplantation and IVF treatment were favourable.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>There are several studies of the obstetric outcome in women with spontaneous conception after solid organ transplantation as well as studies of obstetric outcome after IVF in general. There are only a few case reports of women with kidney or pancreas-kidney transplantation and deliveries after IVF treatment.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A population-based retrospective register study was carried out in Sweden including all women with solid organ transplantation and deliveries after IVF; however, only women with kidney transplants were recruited. It also included information on all singleton deliveries after kidney transplantation and spontaneous conception between 1973 and 2012.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING AND METHODS</st> <p>We cross-linked the IVF registers with the Medical Birth Register, the Patient Register and the Cause of Death Register. Study group 1 consisted of women with kidney transplantation and deliveries after IVF treatment. Study group 2 consisted of women with kidney transplantation and singleton deliveries after spontaneous conception. Group 3 (control group to singletons in study group 1) consisted of women without organ transplantation and with singleton deliveries after IVF, matched for maternal age, parity and date of birth. Group 4 (control group to study group 2) consisted of women without organ transplantation and with singleton deliveries after spontaneous conception, matched for maternal age, parity and year of birth.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Seven singletons and one set of twins were born after organ transplantation and IVF. All women in this group had renal transplants. Among singletons, two (28.6%) were preterm births (PTB), one (14.3%) had very low birthweight (VLBW) (672 g) and one (14.3%) was small for gestational age (SGA). Two infants had minor birth defects. One woman developed pre-eclampsia (14.3%). Mean age at follow-up of the children was 9.7 years (SD 4.2). Two children were diagnosed with hyperactivity disorders. There were 199 singletons born after renal transplantation and spontaneous conception. The rates of pre-eclampsia (23.6%), PTB (48.5%), LBW (43.7%) and SGA (21.2%) were significantly higher in pregnancies of women with renal transplants who had conceived spontaneously than in pregnancies where there was no transplantation and conception was spontaneous. Neonatal morbidity, early neonatal and infant mortality were all significantly higher. No increase in birth defects was seen. Mean age at follow-up of the children was 14.7 years (SD 9.4). Acute bronchitis, systemic lupus erythematosus and hyperactivity disorders were more common in children delivered to women with renal transplantation than in children delivered to women with no transplanted organs. Otherwise, long-term child morbidity was comparable.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The women who had received renal transplants and who had given birth after IVF were a small group and may represent a selected group of comparatively healthy women.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The results are important to transplant recipients with infertility problems. Neonatal outcomes after maternal renal transplantation and spontaneous conception were in agreement with the literature. Long-term follow-up of this group of children has long been asked for and findings are included in this report.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTERESTS</st> <p>No conflict of interest was reported. The study was supported by grants from Swedish Association of Local Authorities and Regions and by grants from the University of Gothenburg/Sahlgrenska University hospital (LUA/ALF 70940).</p> </sec>


Time to second abortion or continued pregnancy following a first abortion: a retrospective cohort study
<sec><st>STUDY QUESTION</st> <p>What proportions of women have a second abortion or continued pregnancy within 12&ndash;46 months of a first abortion?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Estimated return rates for a second abortion were 5, 10.9 and 19.8% at 12, 24 and 46-months, respectively, and rates of continued pregnancy were 5.6, 12.9 and 24.3% at the same intervals.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Studies attempting to identify women at risk for &lsquo;repeat abortion&rsquo; for intervention purposes have described a range of demographic and behavioural characteristics associated with presentation for more than one abortion, but few have taken timing of abortions into account.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Retrospective cohort study involving women presenting for a first abortion at a public hospital abortion clinic in New Zealand (2007&ndash;2010).</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Electronically stored records were analysed for women discharged from a public hospital abortion clinic in New Zealand. Outcome measures were the proportion of women having a second abortion or continued pregnancy within 24 months of a first abortion, and characteristics associated with shorter time to subsequent pregnancy. Cox proportional hazards modelling was used to detect factors associated with time to a second abortion or continued pregnancy, and Kaplan&ndash;Meier survival analyses were used to estimate time to one of these two pregnancy outcomes.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>A total of 6767 women had a first abortion between 2007 and 2010. Some data were missing for 11 women so were excluded from the cohort and analyses. Return rates for a second abortion estimated from survival analyses were 5, 10.9 and 19.8% at 12, 24 and 46 months, respectively. Estimated rates of continued pregnancies were 5.6, 12.9 and 24.3% at 12, 24 and 46 months, respectively. Younger age, non-European ethnicity and greater parity were significantly associated with shorter time to a second abortion and to a subsequent continued pregnancy (<I>P</I> &lt; 0.01 for all factor <I>P</I>-values). Hazard ratios (HR) for a second abortion were highest among those aged 16&ndash;19 years (HR 1.6, 95% confidence interval (CI) 1.3&ndash;1.9, Reference 20&ndash;24), of Pacific Island (HR 1.35, 95% CI 1.1&ndash;1.7) or Maori ethnicity (HR 1.26, 95% CI 1.1&ndash;1.5, Reference New Zealand European), and with 1 (HR 1.41, 95% CI 1.1&ndash;1.7) or 2 (HR 1.41, 95% CI 1.1&ndash;1.9, Reference nulliparous) children at the time of the first abortion. Both pregnancy outcomes were observed among 120 women (1.8%), with 60% of these women having a second abortion before the continued pregnancy.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This study was limited to analysis of routinely collected clinical and demographic data for women presenting for abortion over a 4-year period. Conclusions could not be drawn about a wider range of personal and situational factors influencing pregnancy and pregnancy outcomes. Data were drawn from only one clinic but characteristics of the study sample were broadly representative of those reported nationally. Loss to follow-up for women seeking a second abortion elsewhere in the country cannot be ruled out and would serve to underestimate return rates reported here.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>To date, the most effective public health measure known to reduce abortion return rates within 24 months is the initiation of long-acting reversible contraception (LARC) at the time of an abortion. The high proportion of women seeking a second abortion &lt;4 years after a first abortion (20%) could be significantly reduced by use of LARC, as could unintended pregnancies that are continued soon after a first abortion, particularly among teenaged and young women. Barrier-free access to a range of LARC methods should be prioritized to prevent unintended and mistimed pregnancies.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>Funded by a Lottery Health Research Grant and a University of Otago Research Grant. The authors have no competing interests.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


Risk for borderline ovarian tumours after exposure to fertility drugs: results of a population-based cohort study
<sec><st>STUDY QUESTION</st> <p>Do fertility drugs increase the risk for borderline ovarian tumours, overall and according to histological subtype?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The use of any fertility drug did not increase the overall risk for borderline ovarian tumours, but an increased risk for serous borderline ovarian tumours was observed after the use of progesterone.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Many epidemiological studies have addressed the connection between fertility drugs use and risk for ovarian cancer; most have found no strong association. Fewer studies have assessed the association between use of fertility drugs and risk for borderline ovarian tumours, and the results are inconsistent.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A retrospective case&ndash;cohort study was designed with data from a cohort of 96 545 Danish women with fertility problems referred to all Danish fertility clinics in the period 1963&ndash;2006. All women were followed for first occurrence of a borderline ovarian tumour from the initial date of infertility evaluation until a date of migration, date of death or 31 December 2006, whichever occurred first. The median length of follow-up was 11.3 years.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Included in the analyses were 142 women with borderline ovarian tumours (cases) and 1328 randomly selected sub-cohort members identified in the cohort during the follow-up through 2006. Cases were identified by linkage to the Danish Cancer Register and the Danish Register of Pathology by use of personal identification numbers. To obtain information on use of fertility drugs, hospital files and medical records of infertility-associated visits to all Danish fertility clinics were collected and supplemented with information from the Danish IVF register. We used case&ndash;cohort techniques to calculate rate ratios (RRs) and corresponding 95% confidence intervals (CIs) for borderline ovarian tumours, overall and according to histological subtype, associated with the use of any fertility drug or five specific groups of fertility drugs: clomiphene citrate, gonadotrophins (human menopausal gonadotrophins and follicle-stimulating hormone), gonadotrophin-releasing hormone analogues, human chorionic gonadotrophins and progesterone.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Analyses within the cohort showed that the overall risk for borderline ovarian tumours was not associated with the use of any fertility drug (RR 1.00; 95% CI 0.67&ndash;1.51) or of gonadotrophins (RR 1.32; 95% CI 0.81&ndash;2.14), clomiphene citrate (RR 0.96; 95% CI 0.64&ndash;1.44), human chorionic gonadotrophins (RR 0.91; 95% CI 0.61&ndash;1.36) or gonadotrophin-releasing hormone analogues (RR 1.10; 95% CI 0.66&ndash;1.81). Furthermore, no associations were observed between the risk for borderline ovarian tumours and these groups of fertility drugs according to the number of cycles of use, length of follow-up or parity. In contrast, use of progesterone increased the risk for borderline ovarian tumours, particularly serous tumours, for which statistically significantly increased risks were observed with any use of progesterone (RR 1.82; 95% CI 1.03&ndash;3.24), among women treated with &ge;4 cycles of progesterone (RR 2.63; 95% CI 1.04&ndash;6.64) and for all women followed up for &ge;4 years after their first treatment with progesterone.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Although we tried to minimize the effects of the underlying infertility, the severity of infertility might have affected our risk estimates, as women with more severe fertility problems may receive more treatment. The results from the subgroup analyses, e.g. the findings of an elevated risk for borderline ovarian tumours associated with increased time since first use of progesterone and with increased number of treatment cycles, should be interpreted with caution as these analyses are based on a limited number of women with borderline ovarian tumours.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Although this study, which is the largest to date, provides reassuring evidence that there is no strong link between the use of fertility drugs and risk for borderline ovarian tumours, the novel observation of an increased risk for serous tumours after use of progesterone should be investigated in large epidemiological studies. The results of the present study provide valuable knowledge for clinicians and other health care personnel involved in the diagnosis and treatment of fertility problems.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No conflict of interest was reported. S.M.B. was supported by a research scholarship from the Danish Cancer Society.</p> </sec>


No evidence for mutations in NLRP7, NLRP2 or KHDC3L in women with unexplained recurrent pregnancy loss or infertility
<sec><st>STUDY QUESTION</st> <p>Are mutations in <I>NLRP2/7 (NACHT, LRR and PYD domains-containing protein 2/7)</I> or <I>KHDC3L (KH Domain Containing 3 Like)</I> associated with recurrent pregnancy loss (RPL) or infertility?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>We found no evidence for mutations in <I>NLRP2/7</I> or <I>KHDC3L</I> in unexplained RPL or infertility.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Mutations in <I>NLRP7</I> and <I>KHDC3L</I> are known to cause biparental hydatidiform moles (BiHMs), a rare form of pregnancy loss. <I>NLRP2</I>, while not associated with the BiHM pathology, is known to cause recurrent Beckwith Weidemann Syndrome (BWS).</p> </sec> <sec><st>STUDY DESIGN, SIZE, AND DURATION</st> <p>Ninety-four patients with well characterized, unexplained infertility were recruited over a 9-year period from three IVF clinics in Sweden. Blood samples from 24 patients with 3 or more consecutive miscarriages of unknown etiology were provided by the Recurrent Miscarriage Clinic at St Mary's Hospital, London, UK.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Patients were recruited into both cohorts following extensive clinical studies. Genomic DNA was isolated from peripheral blood and subject to Sanger sequencing of <I>NLRP2</I>, <I>NLRP7</I> and <I>KHDC3L</I>. Sequence electropherograms were analyzed by Sequencher v5.0 software and variants compared with those observed in the 1000 Genomes, single nucleotide polymorphism database (dbSNP) and HapMap databases. Functional effects of non-synonymous variants were predicted using Polyphen-2 and sorting intolerant from tolerant (SIFT).</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>No disease-causing mutations were identified in NLRP2, NLRP7 and KHDC3L in our cohorts of unexplained infertility and RPL.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Due to the limited patient size, it is difficult to conclude if the low frequency single nucleotide polymorphisms observed in the present study are causative of the phenotype. The design of the present study therefore is only capable of detecting highly penetrant mutations.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The present study supports the hypothesis that mutations in <I>NLRP7</I> and <I>KHDC3L</I> are specific for the BiHM phenotype and do not play a role in other adverse reproductive outcomes. Furthermore, to date, mutations in <I>NLRP2</I> have only been associated with the imprinting disorder BWS in offspring and there is no evidence for a role in molar pregnancies, RPL or unexplained infertility.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by the following sources: Estonian Ministry of Education and Research (Grant SF0180044s09), Enterprise Estonia (Grant EU30020); Mentored Resident research project (Department of Obstetrics and Gynecology, Baylor College of Medicine); Imperial NIHR Biomedical Research Centre; Grant Number C06RR029965 from the National Center for Research Resources (NCCR; NIH). No competing interests declared.</p> </sec>


Association between endometriosis and the interleukin 1A (IL1A) locus
<sec><st>STUDY QUESTION</st> <p>Are single-nucleotide polymorphisms (SNPs) at the interleukin 1A (<I>IL1A</I>) gene locus associated with endometriosis risk?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>We found evidence for strong association between <I>IL1A</I> SNPs and endometriosis risk.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Genetic factors contribute substantially to the complex aetiology of endometriosis and the disease has an estimated heritability of ~51%. We, and others, have conducted genome-wide association (GWA) studies for endometriosis, which identified a total of nine independent risk loci. Recently, two small Japanese studies reported eight SNPs (rs6542095, rs11677416, rs3783550, rs3783525, rs3783553, rs2856836, rs1304037 and rs17561) at the <I>IL1A</I> gene locus as suggestively associated with endometriosis risk. There is also evidence of a link between inflammation and endometriosis.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>We sought to further investigate the eight <I>IL1A</I> SNPs for association with endometriosis using an independent sample of 3908 endometriosis cases and 8568 controls of European and Japanese ancestry. The study was conducted between October 2013 and July 2014.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>By leveraging GWA data from our previous multi-ethnic GWA meta-analysis for endometriosis, we imputed variants in the <I>IL1A</I> region, using a recent 1000 Genomes reference panel. After combining summary statistics for the eight SNPs from our European and Japanese imputed data with the published results, a fixed-effect meta-analysis was performed. An additional meta-analysis restricted to endometriosis cases with moderate-to-severe (revised American Fertility Society stage 3 or 4) disease versus controls was also performed.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>All eight <I>IL1A</I> SNPs successfully replicated at <I>P</I> &lt; 0.014 in the European imputed data with concordant direction and similar size to the effects reported in the original Japanese studies. Of these, three SNPs (rs6542095, rs3783550 and rs3783525) also showed association with endometriosis at a nominal <I>P</I> &lt; 0.05 in our independent Japanese sample. Fixed-effect meta-analysis of the eight SNPs for moderate-to-severe endometriosis produced a genome-wide significant association for rs6542095 (odds ratio = 1.21; 95% confidence interval = 1.13&ndash;1.29; <I>P</I> = 3.43 <FONT FACE="arial,helvetica">x</FONT> 10<sup>&ndash;8</sup>).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The meta-analysis for moderate-to-severe endometriosis included results of moderate-to-severe endometriosis cases from our European data sets and all endometriosis cases from the Japanese data sets, as disease stage information was not available for endometriosis cases in the Japanese data sets.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>SNP rs6542095 is located ~2.3 kb downstream of the <I>IL1A</I> gene and ~6.9 kb upstream of cytoskeleton-associated protein 2-like (<I>CKAP2L</I>) gene. The <I>IL1A</I> gene encodes the IL1a protein, a member of the interleukin 1 cytokine family which is involved in various immune responses and inflammatory processes. These results provide important replication in an independent Japanese sample and, for the first time, association of the <I>IL1A</I> locus in endometriosis patients of European ancestry. SNPs within the <I>IL1A</I> locus may regulate other genes, but if <I>IL1A</I> is the target, our results provide supporting evidence for a link between inflammatory responses and the pathogenesis of endometriosis.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.</p> </sec>


Doxycycline alters the expression of inflammatory and immune-related cytokines and chemokines in human endometrial cells: implications in irregular uterine bleeding


Antral follicle count might be underestimated in the presence of an ovarian endometrioma


Reply: Antral follicle count might be underestimated in the presence of an ovarian endometrioma


Endometriosis of the retrocervical septum is proposed to replace the anatomically incorrect term endometriosis of the rectovaginal septum
<p>We propose that the term retrocervical septum be added to the medical lexicon to designate the anatomic location of endometriosis of the septum that separates the vagina and posterior vaginal fornix from the rectovaginal pouch of Douglas. Use of the terms retrocervical septum and endometriosis of the retrocervical septum would correct the century-long misuse of the anatomically incorrect term, endometriosis of the rectovaginal septum.</p>


Ovarian reserve screening: a scientific and ethical analysis
<p>Ovarian reserve (OR) screening of the general population is generally not advocated as it has not been conclusively established to reflect immediate potential for natural conception, while it may also potentially create anxiety for women. However, in this paper, we argue in support of screening of the general population for diminished OR. First, OR tests such as measurements of anti-M&uuml;llerian hormone and antral follicle count are predictive of the chances of IVF conception, and therefore predict a woman's total fertility potential (i.e. chances of natural and IVF-related conceptions). Since the requirement for assisted conception increases with age, this is an important point. Secondly, women identified as having low OR are at increased risk of early loss of fertility potential in the longer term, limiting their reproductive life span and the size of their family if they delay conception. Thirdly, women often disregard generic advice to avoid delaying conception beyond 30 years of age, yet studies suggest that personalized risk assessment tools such as OR testing can actually increase an individual's motivation for positive change. A poor OR screening test result is more likely to convince a woman to bring forward her plans for natural conception, or alternatively explore oocyte vitrification, at a stage when these approaches still have reasonable prospects of success. Finally, we believe that women have a right, based on the ethical concept of autonomy, to be made aware of OR screening, so that they themselves can determine if OR testing is useful in assisting them with reproductive life planning.</p>


Practical problems in the posthumous retrieval of sperm
<p>This communication discusses the practical problems that arise during the collection and processing of sperm that have been retrieved posthumously. It is based on a small group, namely the last six men from whom we carried out posthumous retrieval. The reason for each retrieval, the method of that retrieval, the assessment of the sperm retrieved, the subsequent viability of the sperm and their storage method are discussed. The many ethical and legal problems that arise both before and after posthumous sperm retrieval are huge in their complexity. Therefore, they will not be discussed here and this communication will be limited to the practical aspects of posthumous sperm retrieval. The purpose of this communication is to make some suggestions that will facilitate such collections. The whole subject of posthumous sperm collection is gaining increasing clinical importance and has begun to interest the media as demonstrated by the recent national coverage in an Australian newspaper.</p>


ESX1 mRNA expression in seminal fluid is an indicator of residual spermatogenesis in non-obstructive azoospermic men
<sec><st>STUDY QUESTION</st> <p>Is the presence <I>of ESX1</I> mRNA in seminal fluid (SF) an indicator of residual spermatogenesis in men with non-obstructive azoospermic (NOA)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p><I>ESX1</I> mRNA in SF is a suitable molecular marker for predicting the presence of residual spermatogenesis in testis.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p><I>ESX1</I> is an X-linked homeobox gene whose expression in testis is restricted to germ cells. We previously reported, in the testicular biopsies from azoospermic men, a positive correlation between the presence of <I>ESX1</I> mRNA and residual spermatogenesis.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>We investigated <I>ESX1</I> mRNA expression in 70 testicular fragments (TF) and 56 (SF) of 70 NOA men. As controls, we analyzed 8 TF from men with obstructive azoospermic (OA) and 9 SF from normozoospermic men. For all patients we considered the histological classification of testis biopsies and the recovery of spermatozoa by surgical procedures.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Relative <I>ESX1</I> mRNA expression was evaluated by quantitative RT&ndash;PCR using the Ct method. The results were compared with the recovery of spermatozoa at surgery.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>In TF from NOA patients we found that: (i) <I>ESX1</I> mRNA level was significantly decreased as the severity of spermatogenic defects increased (<I>P</I> &lt; 0.0001, one-way analysis of variance); (ii) the presence of <I>ESX1</I> mRNA can predict the success of sperm retrieval (sensitivity: 80%). In SF from NOA patients we found that: (i) <I>ESX1</I> mRNA was present in 78.5% of NOA men; (ii) the presence of <I>ESX1</I> mRNA could predict the success of sperm retrieval (sensitivity: 84%).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Spermatozoa were recovered at surgery in 5 out of 12 patients whose SF was negative for <I>ESX1</I> mRNA expression. We think that discrepancies between molecular and clinical results could be reduced by analyzing more than one ejaculate from each man.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The data confirm that the <I>ESX1</I> transcript in the semen of men with NOA is a suitable molecular marker for predicting the presence of residual foci of spermatogenesis in the testis. The implication of these results is that some patients &lsquo;with azoospermia&rsquo;, although having a severe impairment of spermatogenesis, could still maintain residual foci of spermatogenesis in limited areas of the testes, not always recovered by surgery.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was supported by the Fondazione IRCCS Ca&rsquo; Granda, Ospedale Maggiore Policlinico: Ricerca Corrente [grant number RC2014/519-02] to M.M. and from ASM onlus 2010&ndash;2011 to M.M. The authors declare that they have no conflict of interest.</p> </sec>


The influence of IVF/ICSI treatment on human embryonic growth trajectories
<sec><st>STUDY QUESTION</st> <p>Is <I>in vitro</I> fertilization treatment with or without intracytoplasmatic sperm injection (IVF/ICSI) associated with changes in first and second trimester embryonic and fetal growth trajectories and birthweight in singleton pregnancies?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Embryonic and fetal growth trajectories and birthweight are not significantly different between pregnancies conceived with IVF/ICSI treatment and spontaneously conceived pregnancies with reliable pregnancy dating.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>IVF/ICSI treatment has been associated with increased risks of preterm birth, fetal growth restriction and low birthweight. Decreased first-trimester crown-rump length (CRL) in the general population has been inversely associated with the same adverse pregnancy outcomes.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>In a prospective periconception birth cohort study conducted in a tertiary centre, 146 singleton pregnancies with reliable pregnancy dating and nonmalformed live borns were investigated, comprised of 88 spontaneous and 58 IVF/ICSI pregnancies.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Serial 3D ultrasound scans were performed from 6 to 12 weeks of gestation. As estimates of embryonic growth, CRL and embryonic volume (EV) were measured using the I-Space virtual reality system. General characteristics were obtained from self-administered questionnaires at enrolment. Fetal growth parameters at 20 weeks and birthweight were obtained from medical records. To assess associations between IVF/ICSI and embryonic growth trajectories, estimated fetal weight and birthweight, stepwise linear mixed model analyses and linear regression analyses were performed using square root transformed CRL and fourth root transformed EV.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>In 146 pregnancies, 934 ultrasound scans were performed of which 849 (90.9%) CRLs and 549 (58.8%) EVs could be measured. Embryonic growth trajectories were comparable between IVF/ICSI pregnancies and spontaneously conceived pregnancies (CRL: <I>&beta;</I><SUB>IVF/ICSI</SUB> = 0.10mm; <I>P</I> = 0.10; EV: <I>&beta;</I><SUB>IVF/ICSI</SUB> = 0.03<sup>4</sup>cm&sup3;; <I>P</I> = 0.13). Estimated fetal weight and birthweight were also comparable between both groups (<I>&beta;</I><SUB>IVF/ICSI</SUB> = 6 g; <I>P</I> = 0.36 and <I>&beta;</I><SUB>IVF/ICSI</SUB> = 80 g; <I>P</I> = 0.24, respectively).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Variations in embryonic growth trajectories of spontaneously conceived pregnancies with reliable pregnancy dating may partially be a result of less precise pregnancy dating and differences in endometrium receptivity compared with IVF/ICSI pregnancies.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The absence of a significant difference in embryonic and fetal growth trajectories suggests safety of IVF/ICSI treatment with regard to early embryonic growth. However, further research is warranted to ascertain the influence of IVF/ICSI treatments in a larger study population, and to estimate the impact of the underlying causes of the subfertility and other periconceptional exposures on human embryonic and fetal growth trajectories.</p> </sec> <sec><st>FUNDING STATEMENT</st> <p>This study was supported by the Department of Obstetrics and Gynaecology of the Erasmus MC, University Medical Centre.</p> </sec> <sec><st>CONFLICT OF INTEREST</st> <p>No competing interests are declared.</p> </sec>


Maternal KIR haplotype influences live birth rate after double embryo transfer in IVF cycles in patients with recurrent miscarriages and implantation failure
<sec><st>STUDY QUESTION</st> <p>In patients with recurrent miscarriages (RM) or recurrent implantation failure (RIF), does the maternal killer immunoglobulin-like receptor (KIR) haplotype have an impact on live birth rates per cycle after embryo transfer with the patient's own or donated oocytes?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>After double embryo transfer (DET) in patients with the maternal KIR AA haplotype, a significantly increased early miscarriage rate was observed when the patient's own oocytes were used, and a significantly decreased live birth rate per cycle after embryo transfer was observed when donated oocytes were used.</p> </sec> <sec><st>WHAT IS ALREADY KNOWN</st> <p>Interactions between fetal HLA-C and maternal KIR influence placentation during human pregnancy. There is an increased risk of RM, pre-eclampsia or fetal growth restriction in mothers with the KIR AA haplotype when the fetus has more HLA-C2 genes than the mother.</p> </sec> <sec><st>STUDY DESIGN, SIZE AND DURATION</st> <p>Between 2010 and 2014, we performed a retrospective study that included 291 women, with RM or RIF, who had a total of 1304 assisted reproductive cycles.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Pregnancy, miscarriage and live birth rates per cycle after single or DET, categorized by the origin of the oocytes and the presence of maternal KIR haplotypes, were studied. KIR haplotype regions were defined by the presence of the following KIR genes: Cen-A/2DL3; Tel-A/3DL1 and 2DS4; Cen-B/2DL2 and 2DS2; as well as Tel-B/2DS1 and 3DS1.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Higher rates of early miscarriage per cycle after DET with the patient's own oocytes in mothers with the KIR AA haplotype (22.8%) followed by those with the KIR AB haplotype (16.7%) compared with mothers with the KIR BB haplotype (11.1%) were observed (<I>P</I> = 0.03). Significantly decreased live birth rates per cycle were observed after DET of donated oocytes in mothers with the KIR AA haplotype (7.5%) compared with those with the KIR AB (26.4%) and KIR BB (21.5%) haplotypes (<I>P</I> = 0.006). No statistically significant differences were observed for pregnancy, miscarriage and live birth rates per cycle among those with maternal KIR AA, AB and BB haplotypes after single embryo transfer (SET) with the patient's own or donated oocytes. The large number of cases studied strengthens the results and provides sufficient power to the statistical analysis.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>During the IVF procedure, DET induces the expression of more than one paternal HLA-C and the oocyte-derived maternal HLA-C in the oocyte-donation cycles probably behaves like paternal HLA-C. Because this was a retrospective study, we did not have data about the HLA-C of the parent, donor, chorionic villi, or infant, which is a limitation because we cannot show differences according to paternal or oocyte donor HLA-C1 and HLA-C2.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>These new insights could have an impact on the selection of SET in patients with RM or RIF, and a KIR AA haplotype. Also, it may help in oocyte and/or sperm donor selection by HLA-C in patients with RM or RIF and a KIR AA haplotype.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No funding was received for this study. The authors have no conflicts of interest to declare.</p> </sec>


First trimester trophoblast and placental bed vascular volume measurements in IVF or IVF/ICSI pregnancies
<sec><st>STUDY QUESTION</st> <p>Are first trimester trophoblast volume (TV) and placental bed vascular volume (PBVV) different in IVF or IVF/ICSI pregnancies in comparison with spontaneously conceived pregnancies?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Any possible abnormal placentation in IVF or IVF/ICSI pregnancies in comparison with spontaneously conceived pregnancies is not detected by a difference in PBVV or TV at an early gestational age (GA).</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Assisted reproductive technology pregnancies have been associated with an increased risk of placenta-related adverse pregnancy outcomes. It is unclear whether these effects originate from infertility or from the technique itself.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>We performed a retrospective cohort study in which 154 pregnant patients qualified for participation.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Out of 154 pregnant patients, 84 conceived spontaneously and 70 conceived after IVF or IVF/ICSI. We determined the TV at 10 weeks GA by Virtual Organ Computer-aided AnaLysis measuring application and the PBVV at 12 weeks GA by the virtual reality operating system of BARCO I-Space in both subgroups. The investigators were blinded to the mode of conception during the measurements. Analysis was limited to singleton pregnancies with only one sac ever detectable.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>There were no differences in TV (mean 42.7, SD 15.9 versus mean 41.2, SD 13.9, <I>P</I> = 0.70) and PBVV (mean 27.6, SD 16.9 versus mean 24.8, SD 19.9, <I>P</I> = 0.20) between IVF or IVF/ICSI pregnancies and spontaneously conceived pregnancies. There was a significant correlation between TV and PBVV (<I>r<SUB>s</SUB></I> = 0.283, <I>P</I> = 0.004).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The limitations of the present study concern the small size of the study groups.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>IVF or IVF/ICSI does not seem to be associated with abnormal placentation.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was financially supported by the Erasmus Trustfonds, the Meindert de Hoop foundation and the Fonds NutsOhra. No competing interests are declared.</p> </sec>


Proposed guidelines on the nomenclature and annotation of dynamic human embryo monitoring by a time-lapse user group
<sec><st>STUDY QUESTION</st> <p>Can the approach to, and terminology for, time-lapse monitoring of preimplantation embryo development be uniformly defined in order to improve the utilization and impact of this novel technology?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The adoption of the proposed guidelines for defining annotation practice and universal nomenclature would help unify time-lapse monitoring practice, allow validation of published embryo selection algorithms and facilitate progress in this field.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>An increasing quantity of publications and communications relating to time-lapse imaging of <I>in vitro</I> embryo development have demonstrated the added clinical value of morphokinetic data for embryo selection. Several articles have identified similar embryo selection or de-selection variables but have termed them differently. An evidence-based consensus document exists for static embryo grading and selection but, to date, no such reference document is available for time-lapse methodology or dynamic embryo grading and selection.</p> </sec> <sec><st>STUDY DESIGN, SIZE AND DURATION</st> <p>A series of meetings were held between September 2011 and May 2014 involving time-lapse users from seven different European centres. The group reached consensus on commonly identified and novel time-lapse variables.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Definitions, calculated variables and additional annotations for the dynamic monitoring of human preimplantation development were all documented.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Guidelines are proposed for a standard methodology and terminology for the of use time-lapse monitoring of preimplantation embryo development.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The time-lapse variables considered by this group may not be exhaustive. This is a relatively new clinical technology and it is likely that new variables will be introduced in time, requiring revised guidelines. A different group of users from those participating in this process may have yielded subtly different terms or definitions for some of the morphokinetic variables discussed. Due to the technical processes involved in time-lapse monitoring, and acquisition of images at varied intervals through limited focal planes, this technology does not currently allow continuous monitoring such that the entire process of preimplantation embryo development may be visualized.</p> </sec> <sec><st>WIDER IMPLICATIONS</st> <p>This is the first time that a group of experienced time-lapse users has systematically evaluated current evidence and theoretical aspects of morphokinetic monitoring to propose guidelines for a standard methodology and terminology of its use and study, and its clinical application in IVF. The adoption of a more uniform approach to the terminology and definitions of morphokinetic variables within this developing field of clinical embryology would allow practitioners to benefit from improved interpretation of data and the sharing of best practice and experience, which could impact positively and more swiftly on patient treatment outcome.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>There was no specific funding for the preparation of these proposed guidelines. Meetings were held opportunistically during scientific conferences and using online communication tools.</p> <p>H.N.C. is a scientific consultant for ESCO, supplier of Miri TL. I.E.A. is a minor shareholder in Unisense Fertilitech, supplier of the EmbryoScope. Full disclosures of all participants are presented herein. The remaining authors have no conflict of interest.</p> </sec>


Cell-free DNA in human follicular fluid as a biomarker of embryo quality
<sec><st>STUDY QUESTION</st> <p>Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for <I>in vitro</I> fertilization (IVF) outcomes?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (<I>n</I> = 26) or ICSI (<I>n</I> = 17). CfDNA level was quantified in each individual follicular fluid sample.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean &plusmn; SD, 1.62 &plusmn; 2.08 ng/&micro;l). CfDNA level was significantly higher in small follicles (8&ndash;12 mm in diameter) than in large ones (&gt;18 mm) (mean &plusmn; SD, 2.54 &plusmn; 0.78 ng/&micro;l versus 0.71 &plusmn; 0.44 ng/&micro;l, respectively, <I>P</I> = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (<I>r</I> = &ndash;0.34; <I>P</I> = 0.003). A weak significant negative correlation between DNA integrity and 17&beta;-estradiol level in follicular fluid samples at oocyte collection day was observed (<I>r</I> = &ndash;0.26; <I>P</I> = 0.008). CfDNA level in follicular fluid samples corresponding to top quality embryos was significantly lower than in follicular fluid samples related to poor quality embryos (<I>P</I> = 0.022). Similarly, cfDNA level was also significantly lower in follicular fluid samples related to embryos with low fragmentation rate (&le;25%) than with high fragmentation rate (&gt;25%) (<I>P</I> = 0.02).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>A larger study should be conducted in order to establish the predictive value of cfDNA level for embryo quality and to investigate whether follicular fluid cfDNA levels are correlated with embryo implantation rates and pregnancy outcomes. Moreover, the role of follicular fluid cfDNA on embryo quality should be studied to determine whether high cfDNA concentration in follicular fluid is only a consequence or also a cause of follicular dysfunction.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>CfDNA evaluation in individual follicular fluid samples might represent an innovative biomarker of embryo quality to use as a supplemental tool to predict embryo quality during IVF.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was partially supported by the University Hospital of Montpellier and Ferring Pharmaceuticals. The authors of the study have no competing interests to report.</p> </sec>


Laser-assisted zona pellucida thinning does not facilitate hatching and may disrupt the in vitro hatching process: a morphokinetic study in the mouse
<sec><st>STUDY QUESTION</st> <p>Does laser-assisted zona thinning of cleavage stage mouse embryos facilitate hatching <I>in vitro</I>?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>No, unlike laser zona opening, zona thinning does not facilitate embryo hatching.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Artificial opening of the zona pellucida facilitates hatching of mouse and human embryos. Laser-assisted zona thinning has also been used for the purpose of assisted hatching of human embryos but it has not been properly investigated in an animal model; thinning methods have produced inconsistent clinical results.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Time-lapse microscopy was used to study the hatching process in the mouse after zona opening and zona thinning; a control group of embryos was not zona-manipulated but exposed to the same laser energy.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Eight-cell CB6F1/J mouse embryos were pooled and allocated to three groups (<I>n</I> = 56 per group): A control group of embryos that were exposed to a dose of laser energy focused outside the zona pellucida (zona intact); one experimental group of embryos in which the zona pellucida was opened by complete ablation using the same total number of pulses as the control group; a second experimental group of embryos in which the zona pellucida was thinned to establish a smooth lased area using the same number of pulses as used in the other two groups. The width of the zona opening was 25 &mu;m and width of the thinned area was 35 &mu;m. Development was monitored by time-lapse microscopy. Overall treatment differences for continuous variables were analyzed by analysis of variance and pairwise comparisons using the Student <I>t</I>-test allowing for unequal variances, while for categorical data, a standard chi-squared test was utilized for all pairwise comparisons.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The frequency of complete hatching was 33.9% in the control group, 94.4% after zona opening, and 39.3% after zona thinning (overall group comparison, <I>P</I> &lt; 0.0001). Overall, 60.7% of the zona-thinned embryos did not complete the hatching process and remained trapped within the zona; when they did hatch, they did not necessarily hatch from the zona-thinned area. Hatching in about one-third of the zona-intact embryos began with breaches at multiple sites by small groups of cells. Likewise, 53.6% of zona-thinned embryos had multiple breaches, always involving an area outside the thinned zone. Zona opening decreased multiple breaching and led to blastocyst escape an average of 14 h earlier than zona-thinned embryos and 5.5 h before control embryos (<I>P</I> = 0.0003).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The experiments presented here were limited to <I>in vitro</I> experiments performed in the mouse. Whether human embryos would behave the same way under similar circumstances is unknown. We postulate that zona thinning is not beneficial in human embryos.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The experiments demonstrate that zona thinning is not equivalent to zona opening for assisted hatching. The study provides reason for systematic reviews of assisted hatching trials to take the method of assisted hatching into consideration and not combine the results of zona thinning and zona opening procedures.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>Institutional funds were used for the study. No competing interests are declared.</p> </sec>


Smoking in infertile women with polycystic ovary syndrome: baseline validation of self-report and effects on phenotype
<sec><st>STUDY QUESTION</st> <p>Do women with polycystic ovary syndrome (PCOS) seeking fertility treatment report smoking accurately and does participation in infertility treatment alter smoking?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Self-report of smoking in infertile women with PCOS is accurate (based on serum cotinine levels) and smoking is unlikely to change over time with infertility treatment.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Women with PCOS have high rates of smoking and it is associated with worse insulin resistance and metabolic dysfunction.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Secondary study of smoking history from a large randomized controlled trial of infertility treatments in women with PCOS (<I>N</I> = 626) including a nested case&ndash;control study (<I>N</I> = 148) of serum cotinine levels within this cohort to validate self-report of smoking.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Women with PCOS, age 18&ndash;40, seeking fertility who participated in a multi-center clinical trial testing first-line ovulation induction agents conducted at academic health centers in the USA.</p> </sec> <sec><st>MAIN RESULT(S) AND THE ROLE OF CHANCE</st> <p>Overall, self-report of smoking in the nested case&ndash;control study agreed well with smoking status as determined by measure of serum cotinine levels, at 90% or better for each of the groups at baseline (98% of never smokers had cotinine levels &lt;15 ng/ml compared with 90% of past smokers and 6% of current smokers). There were minor changes in smoking status as determined by serum cotinine levels over time, with the greatest change found in the smoking groups (past or current smokers). In the larger cohort, hirsutism scores at baseline were lower in the never smokers compared with past smokers. Total testosterone levels at baseline were also lower in the never smokers compared with current smokers. At end of study follow-up insulin levels and homeostatic index of insulin resistance increased in the current smokers (<I>P</I> &lt; 0.01 for both) compared with baseline and with non-smokers. The chance for ovulation was not associated with smoking status, but live birth rates were increased (non-significantly) in never or past smokers.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The limitations include the selection bias involved in our nested case&ndash;control study, the possibility of misclassifying exposure to second hand smoke as smoking and our failure to capture self-reported changes in smoking status after enrollment in the trial.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Because self-report of smoking is accurate, further testing of smoking status is not necessary in women with PCOS. Because smoking status is unlikely to change during infertility treatment, extra attention should be focused on smoking cessation in current or recent smokers who seek or who are receiving infertility treatment.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>Sponsored by the <I>Eugene Kennedy Shriver</I> National Institute of Child Health and Human Development of the U.S. National Institutes of Health.</p> </sec> <sec><st>CLINICAL TRIAL REGISTRATION NUMBERS</st> <p>ClinicalTrials.gov numbers, NCT00068861 and NCT00719186.</p> </sec>


A randomized double blind comparison of atosiban in patients undergoing IVF treatment
<sec><st>STUDY QUESTION</st> <p>Does atosiban (oxytocin/vasopressin V1A receptor antagonist), given around embryo transfer improve the live birth rate of women undergoing IVF treatment?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The use of atosiban around embryo transfer did not improve the live birth rate in a general population of IVF patients.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Uterine contractions in IVF cycles were significantly increased following ovarian stimulation and women with frequent uterine contractions had a lower pregnancy rates. A few observational studies suggested that the use of atosiban around embryo transfer resulted in higher pregnancy rates in women with repeated implantation failure (RIF). A non-randomized trial of IVF patients also reported higher implantation and clinical pregnancy rates after the use of atosiban.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This multi-centre randomized double blind study recruited 800 general subfertile women undergoing IVF treatment between November 2011 and March 2013. Subjects were randomized into the atosiban (<I>n</I> = 400) and placebo (<I>n</I> = 400) groups according to a computer-generated randomization list.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Subjects were recruited and randomized in the three IVF units in Guangzhou, Hong Kong and Ho Chi Minh City. Women in the atosiban group received i.v. atosiban 30 min before embryo transfer with a bolus dose of 6.75 mg, and the infusion was continued at 18 mg/h for ~1 h. The dose of atosiban was then reduced to 6 mg/h continued for another 2 h. Those in the placebo group received i.v. normal saline only. The primary outcome measure was the live birth rate.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>There was no significant difference in the live birth rate between the atosiban and placebo groups (39.8 versus 38.0%, <I>P</I> = 0.612, rate ratio 1.051, 95% confidence interval: 0.884&ndash;1.251). No significant differences were found between the two groups in the positive pregnancy test, clinical pregnancy, ongoing pregnancy, miscarriage, multiple pregnancy, ectopic pregnancy rates and implantation rate per woman. Similar results were found between the groups at different IVF centres, with a repeated cycle, presence of uterine fibroids or a serum estradiol level on the day of hCG above the median level.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Limitations include the transfer of early cleavage embryos, no measurement of uterine contractions, no documentation of adenomyosis and incomplete tracking of congenital abnormalities in newborns.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This randomized double blind study demonstrated that the use of atosiban given around embryo transfer did not improve the live birth rate in a general population of IVF patients; therefore atosiban should be given only in the context of clinical research.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>Centres in Hong Kong and Vietnam received research funding from Ferring, which was not involved in study design, execution, data analysis and manuscript preparation. There are no conflicts of interest.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>ClinicalTrials.gov Identifier: NCT01501214.</p> </sec>


Patient-centred quality of care in an IVF programme evaluated by men and women
<sec><st>STUDY QUESTION</st> <p>Do men and women value the same aspects of quality of care during IVF treatment when measuring rates of importance by the validated instrument, quality from the patient's perspective of <I>in vitro</I> fertilization (QPP-IVF)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Women valued most aspects of care as significantly more important than their partner although men and women evaluated the importance of the different care factors in a similar pattern.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>A few validated tools measuring patient-centred quality of care during IVF have been developed. Few studies of gender differences concerning experiences of patient-centred quality of care have been reported in the literature to date.</p> </sec> <sec><st>STUDY DESIGN, SIZE AND DURATION</st> <p>A two-centre study was conducted between September 2011 and May 2012. Heterosexual couples (<I>n</I> = 497) undergoing IVF were invited to complete a questionnaire before receiving the result of the pregnancy test.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>In all, 363 women and 292 men evaluated quality of care by answering the QPP-IVF questionnaire. The measurements consisted of two kinds of evaluations: the rating of the importance of various aspects of treatment (subjective importance) and the rating of perceived quality of care (perceived reality). Comparisons between men and women on importance ratings and perceived reality ratings were performed both on factor (subscale) and single item levels by intra-couple analyses and corrected for age. A stepwise multiple logistic regression analysis was performed in order to select baseline variables independently predicting evaluation at factor level.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The response rate was 67.5%, with 363 women (74.2%) and 292 men (60.6%) completing the study. Both the woman and man responded in 251 couples. Women rated the different care aspects as significantly more important than their partner in all factors except the factor, &lsquo;Responsibility/continuity&rsquo;. Both genders gave the factors, &lsquo;Medical care&rsquo; and &lsquo;Information after treatment&rsquo;, the highest scores. At item level women rated the majority of items as significantly more important than men. Perceived reality for the majority of factors and items was similarly rated by men and women in the couples. For women, receiving embryo transfer, short duration of infertility, IVF as a method and number of previous cycles were independently correlated to the highest score of importance of certain factors.</p> </sec> <sec><st>LIMITATIONS, REASON FOR CAUTION</st> <p>The lower response rate of men compared with women (60.6 versus 74.2%, respectively) might have influenced the results through selection bias. Only patients who had adequate fluency in the Swedish language participated.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This study is an important contribution in comparing the needs of men and women undergoing IVF treatments. The QPP-IVF instrument is a suitable instrument for revealing important care aspects identified by both men and women and a useful tool for stimulating patient-centred quality improvements within and between clinics.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST</st> <p>The study was supported by the LUA/ALF agreement at Sahlgrenska University Hospital, Gothenburg, Sweden, and by Hjalmar Svensson's Research Foundation. None of the authors declared any conflict of interests.</p> </sec>


'Will I be able to have a baby?' Results from online focus group discussions with childhood cancer survivors in Sweden
<sec><st>STUDY QUESTION</st> <p>What do adolescent and young adult survivors of childhood cancer think about the risk of being infertile?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The potential infertility, as well as the experience of having had cancer, affects well-being, intimate relationships and the desire to have children in the future.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Many childhood cancer survivors want to have children and worry about possible infertility.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>For this qualitative study with a cross-sectional design, data were collected through 39 online focus group discussions during 2013.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Cancer survivors previously treated for selected diagnoses were identified from The Swedish Childhood Cancer Register (16&ndash;24 years old at inclusion, <unl>&ge;</unl>5 years after diagnosis) and approached regarding study participation. Online focus group discussions of mixed sex (<I>n</I> = 133) were performed on a chat platform in real time. Texts from the group discussions were analysed using qualitative content analysis.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The analysis resulted in the main category <I>Is it possible to have a baby?</I> including five generic categories: <I>Risk of infertility affects well-being</I>, <I>Dealing with possible infertility</I>, <I>Disclosure of possible infertility is a challenge</I>, <I>Issues related to heredity</I> and <I>Parenthood may be affected</I>. The risk of infertility was described as having a negative impact on well-being and intimate relationships. Furthermore, the participants described hesitation about becoming a parent due to perceived or anticipated physical and psychological consequences of having had cancer.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Given the sensitive topic of the study, the response rate (36%) is considered acceptable. The sample included participants who varied with regard to received fertility-related information, current fertility status and concerns related to the risk of being infertile.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The results may be transferred to similar contexts with other groups of patients of childbearing age and a risk of impaired fertility due to disease. The findings imply that achieving parenthood, whether or not with biological children, is an area that needs to be addressed by health care services.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was financially supported by The Cancer Research Foundations of Radiumhemmet, The Swedish Childhood Cancer Foundation and the Doctoral School in Health Care Science, Karolinska Institutet. The authors report no conflicts of interest.</p> </sec>


Clarifying the benefits of the positive reappraisal coping intervention for women waiting for the outcome of IVF
<sec><st>STUDY QUESTION</st> <p>Does the use of a positive reappraisal coping intervention (PRCI) alone following IVF embryo transfer influence anxiety, the depression and treatment outcome when compared with its use combined with monitoring emotions, monitoring emotions alone or no intervention?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Woman using the PRCI alone had significantly lower anxiety levels at Day 10 of the waiting period and 6 weeks after the start of the waiting period but also a significantly higher clinical pregnancy rate compared with the other three groups.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The waiting period, which follows embryo transfer after IVF/ICSI is very stressful. The use of the PRCI together with a daily monitoring form increases positive emotions but appears not to reduce anxiety. The impact of using the PRCI without daily recording of emotions may be more beneficial.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Following completion of recruitment to a recently published 3-arm randomized controlled trial (RCT) of the use of the PRCI in the post-embryo transfer waiting period, a further 110 participants were recruited to study the impact of the PRCI in clinical practice without concurrent emotional monitoring. Data collection took place between May 2012 and December 2012. Outcomes were compared with those generated by a RCT of the PRCI with daily emotional monitoring, daily emotional monitoring only or routine care.</p> </sec> <sec><st>PARTICIPANT, MATERIALS, SETTING, METHODS</st> <p>To capture the impact of the PRCI on this further group, questionnaires were completed at three time points: just before the waiting period (Time 1: preintervention), on Day 10 of the 14-day waiting period (Time 2: waiting period intervention) and 6 weeks after the start of the waiting period (Time 3: post-intervention). Data generated were compared with the data from the RCT. To compare the impact over time on anxiety and depression, a repeated multilevel linear model design was used.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Nighty-eight of the 110 women who were recruited received the PRCI intervention without daily monitoring (PRCI-comparison group). After correcting for known confounding factors, compared with women in all three groups of the original RCT, women in the PRCI-comparison group had a significantly lower anxiety at Time 2 (<I>n</I> = 83) and Time 3 (<I>n</I> = 70) but not significantly lower depression levels. Women in the PRCI-comparison group had a significantly higher clinical pregnancy rate (39.8%, <I>P</I> = 0.033) but there were no significant differences in clinical pregnancies with fetal heartbeat (<I>P</I> = 0.10).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>A limitation of this study is that the additional study group was not randomized to the intervention, and may therefore be subject to selection bias. The study was also done subsequent to the other three groups.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This simple low cost self-help coping intervention can be offered to women during the waiting period in an IVF/ICSI treatment. A further RCT comparing PRCI only to a non-intervention group is necessary to confirm these findings.</p> </sec> <sec><st>STUDY FUNDING/COMPETENT INTEREST(S)</st> <p>The Women and Baby Division of the University Medical Centre Utrecht funded the study. The authors have no conflicting interest(s).</p> </sec>


Damaging legacy: maternal cigarette smoking has long-term consequences for male offspring fertility
<sec><st>STUDY QUESTION</st> <p>What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of <I>in utero</I> exposure on offspring fertility is limited.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult).</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (<I>P</I> &lt; 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (<I>P</I> &lt; 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (<I>P</I> &lt; 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (<I>P</I> &lt; 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (<I>P</I> &lt; 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (<I>P</I> &lt; 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (<I>P</I> &lt; 0.01) reduction in litter size.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.</p> </sec>


Expression of neurokinin B/NK3 receptor and kisspeptin/KISS1 receptor in human granulosa cells
<sec><st>STUDY QUESTION</st> <p>Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus&ndash;oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT&ndash;PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca<sup>2+</sup> levels, [Ca<sup>2+</sup>]<SUB>i</SUB>, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured.</p> </sec> <sec><st>MAIN OUTCOME AND THE ROLE OF CHANCE</st> <p>NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca<sup>2+</sup>]<SUB>i</SUB> in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca<sup>2+</sup>]<SUB>i</SUB>. However, the [Ca<sup>2+</sup>]<SUB>i</SUB> response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells.</p> </sec> <sec><st>STUDY FUNDING/ COMPETING INTEREST(S)</st> <p>This work was supported by grants from Ministerio de Econom&iacute;a y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andaluc&iacute;a (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare.</p> </sec>


Inhibition of dual specificity phosphatase-2 by hypoxia promotes interleukin-8-mediated angiogenesis in endometriosis
<sec><st>STUDY QUESTION</st> <p>How does hypoxia-mediated down-regulation of dual specificity phosphatase-2 (DUSP2) promote endometriotic lesion development?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Inhibition of DUSP2 by hypoxia enhances endometriotic lesion growth via promoting interleukin-8 (IL-8)-dependent angiogenesis.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Angiogenesis is a prerequisite for the development of endometriosis. DUSP2 is down-regulated in endometriotic stromal cells in a hypoxia inducible factor-1&alpha;-dependent manner. Down-regulation of DUSP2 contributes to the pathological process of endometriosis.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A laboratory study recruiting 20 patients of reproductive age with endometriosis and normal menstrual cycles, and an autoimplant-induced mouse model of endometriosis using 13 mice in a 28-day treatment.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>IL-8 mRNA levels were assayed in endometrial stromal cells maintained in normoxic or hypoxic (1% O<SUB>2</SUB>) conditions, with or without DUSP2 knockdown. Promoter activity and chromatin immunoprecipitation (ChIP) assays were conducted to characterize the regulation of IL-8 by DUSP2. Conditioned media from cells maintained in normoxic or hypoxic conditions, and cells with/without DUSP2 knockdown were collected to investigate the angiogenic capacity using an <I>in vitro</I> tube formation assay. Reparixin, an IL-8 receptor blocker, was administered to investigate the role of IL-8 in hypoxia-mediated angiogenesis and the development of endometriotic-like lesions in an autotransplanted mouse model.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>IL-8 mRNA was increased by both hypoxia and DUSP2 knockdown in endometrial stromal cells in an extracellular signal-regulated protein kinase-dependent manner (<I>P</I> &lt; 0.05 versus control). Promoter activity and ChIP assays demonstrated that expression of IL-8 was regulated by CCAAT/enhancer binding protein &alpha; (<I>P</I> &lt; 0.05 versus control). Furthermore, conditioned media collected from hypoxia-exposed or DUSP2 knockdown endometrial stromal cells promoted tube formation, which was abolished by co-treatment with reparixin (<I>P</I> &lt; 0.05 versus control). Results from the autotransplanted mouse model demonstrated that number of blood vessels and size of endometriotic-like lesions were markedly reduced in recipient mice treated with reparixin (<I>P</I> &lt; 0.05 versus control).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This study was conducted in primary human cell cultures and a mouse model, therefore may not fully reflect the situation <I>in vivo</I>.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This is the first study to highlight the potential application of an IL-8 receptor blocker as a therapeutic target to treat endometriosis. This study demonstrates IL-8 as a key angiogenic factor regulated by hypoxia/DUSP2, which suggests an alternative mechanism through which hypoxia may promote angiogenesis.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by the National Science Council of Taiwan (NSC101-2314-B-006-043-MY2). The author declares that there is no conflict of interest.</p> </sec>


Young women with polycystic ovary syndrome have raised levels of circulating annexin V-positive platelet microparticles
<sec><st>STUDY QUESTION</st> <p>Are circulating microparticles (MPs) altered in young women with polycystic ovary syndrome (PCOS)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Women with PCOS have elevated concentrations of circulating platelet-derived MPs, which exhibit increased annexin V binding and altered microRNA (miR) profiles compared with healthy volunteers.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Some studies have shown that cardiovascular risk is increased in young women with PCOS but the mechanisms by which this occurs are uncertain. Circulating MPs are elevated in patients with cardiovascular disease but the characteristics of MPs in patients with PCOS are unclear.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Case&ndash;control study comprising 17 women with PCOS (mean &plusmn; SD; age 31 &plusmn; 7 years, BMI 29 &plusmn; 6 kg/m<sup>2</sup>) and 18 healthy volunteers (age 31 &plusmn; 6 years, BMI 30 &plusmn; 6 kg/m<sup>2</sup>).</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>The study was conducted in a University hospital. Nanoparticle tracking analysis (NTA) and flow cytometry (CD41 platelet, CD11b monocyte, CD144 endothelial) were used to determine MP size, concentration, cellular origin and annexin V positivity (reflecting phosphatidylserine exposure). Fatty acid analysis was performed by gas chromatography and MP miR expression profiles were compared by microarray.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>PCOS subjects showed increased MP concentrations compared with healthy volunteers (mean &plusmn; SD; 11.5 &plusmn; 5 <FONT FACE="arial,helvetica">x</FONT> 10<sup>12</sup>/ml versus 10.0 &plusmn; 4 <FONT FACE="arial,helvetica">x</FONT> 10<sup>12</sup>/ml, respectively; <I>P</I> = 0.03), which correlated with the homeostasis model of insulin resistance (<I>r</I> = 0.53, <I>P</I> = 0.03). This difference was predominantly seen in MPs whose size was in the small exosomal range (&lt;150 nm in diameter, <I>P</I>&lt; 0.05)<I>.</I> PCOS patients showed a greater percentage of annexin V<sup>+</sup> MPs compared with healthy volunteers (84 &plusmn; 18 versus 74 &plusmn; 24%, respectively, <I>P</I> = 0.05) but the cellular origin of MPs, which were predominantly platelet-derived (PCOS: 99 &plusmn; 0.9%; controls: 99 &plusmn; 2.5%), did not differ. MP fatty acid concentration and composition was similar between groups but 16 miRs were differentially expressed (<I>P</I> &lt; 0.05).</p> </sec> <sec><st>LIMITATIONS, REASON FOR CAUTION</st> <p>Patients with PCOS were classified by the Rotterdam criteria, which describes a less severe metabolic phenotype than other definitions of the syndrome. Our findings may thus not be generalizable to all patients with PCOS. MicroRNA expression analysis was only undertaken in an exploratory subset of the overall study population hence, validation of our findings in a larger cohort is mandatory. Furthermore, miR levels were unaltered for the highly expressed miRs and it is unclear whether differences in the lowly expressed miRs carries pathological relevance.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This study suggests that women with PCOS have an altered MP profile but further studies are needed to confirm this, to explore the mechanisms by which these alterations develop and to establish whether therapies that improve insulin sensitivity are able to reduce circulating MP concentrations.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was funded by grants from the Wales Heart Research Institute and Mrs John Nixon Scholarship. The authors have no conflicts of interest to declare.</p> </sec>


Evidence for gonadotrophin secretory and steroidogenic abnormalities in brothers of women with polycystic ovary syndrome
<sec><st>STUDY QUESTION</st> <p>Are there abnormalities in gonadotrophin secretion, adrenal steroidogenesis and/or testicular steroidogenesis in brothers of women with polycystic ovary syndrome (PCOS)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Brothers of women with PCOS have increased gonadotrophin responses to gonadotrophin releasing hormone (GnRH) agonist stimulation and alterations in adrenal and gonadal steroidogenesis.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>PCOS is a complex genetic disease. Male as well as female first-degree relatives have reproductive features of the syndrome. We previously reported that brothers of affected women have elevated circulating dehydroepiandrosterone sulfate levels.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This was a case&ndash;control study performed in 29 non-Hispanic white brothers of 22 women with PCOS and 18 control men.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>PCOS brothers and control men were of comparable age, weight and ethnicity. Adrenocorticotrophic hormone (ACTH) and GnRH agonist stimulation tests were performed. Gonadotrophin responses to GnRH agonist as well as changes in precursor-product steroid pairs (delta, ) across steroidogenic pathways in response to ACTH and GnRH agonist were examined.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Basal total (T) levels did not differ, but dehydroepiandrosterone (DHEA) levels (0.13 &plusmn; 0.08 brothers versus 0.22 &plusmn; 0.09 controls, nmol/l, <I>P</I> = 0.03) were lower in brothers compared with control men. ACTH-stimulated 17-hydroxypregnenolone (17Preg)/17-hydroxyprogesterone (17Prog) (7.8 &plusmn; 24.2 brothers versus 18.9 &plusmn; 21.3 controls, <I>P</I> = 0.04) and DHEA/androstenedione (AD) (0.10 &plusmn; 0.05 brothers versus 0.14 &plusmn; 0.08 controls, <I>P</I> = 0.04) were lower in brothers than in the controls. GnRH agonist-stimulated 17Prog/AD (0.28 &plusmn; 8.47 brothers versus 4.79 &plusmn; 10.28 controls, <I>P</I> = 0.003) was decreased and luteinizing hormone (38.6 &plusmn; 20.6 brothers versus 26.0 &plusmn; 9.8 controls, IU/l, <I>P</I> = 0.02), follicle-stimulating hormone (10.2 &plusmn; 7.5 brothers versus 4.8 &plusmn; 4.1 controls, IU/l <I>P</I> = 0.002), AD (1.7 &plusmn; 1.4 brothers versus 0.9 &plusmn; 1.5 controls, nmol/l, <I>P</I> = 0.02) and AD/T (0.16 &plusmn; 0.14 brothers versus 0.08 &plusmn; 0.12 controls, <I>P</I> = 0.005) responses were increased in brothers compared with controls.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The modest sample size may have limited our ability to observe other possible differences in steroidogenesis between PCOS brothers and control men.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Decreased ACTH-stimulated 17Preg/17Prog and DHEA/AD responses suggested increased adrenal 3&beta;-hydroxysteroid dehydrogenase activity in the brothers. Decreased 17Prog/AD and increased AD/T responses to GnRH agonist stimulation suggested increased gonadal 17,20-lyase and decreased gonadal 17&beta;-hydroxysteroid dehydrogenase activity in the brothers. Increased LH and FSH responses to GnRH agonist stimulation suggested neuroendocrine alterations in the regulation of gonadotrophin secretion similar to those in their proband sisters. These changes in PCOS brothers may reflect the impact of PCOS susceptibility genes and/or programming effects of the intrauterine environment.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This research was supported by P50 HD044405 (A.D.), K12 HD055884 (L.C.T.), U54 HD034449 (A.D., R.S.L.) from the National Institute of Child Health and Development. Some hormone assays were performed at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core that is supported by U54 HD28934 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Partial support for some of the clinical studies was provided by UL1 RR025741 and UL1 TR000150 (Northwestern University Clinical and Translational Sciences Institute) from the National Center for Research Resources, National Institutes of Health, which is now the National Center for Advancing Translational Sciences. The authors have no conflict of interest to declare.</p> </sec>


Increased time to pregnancy is associated with less optimal neurological condition in 4-year-old singletons, in vitro fertilization itself is not
<sec><st>STUDY QUESTION</st> <p>Does ovarian hyperstimulation, the <I>in vitro</I> procedures required for <I>in vitro</I> fertilization (IVF)/ intracytoplasmic sperm injection or the combination of both, affect the neurological outcome of 4-year-old singletons?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Ovarian hyperstimulation, the <I>in vitro</I> procedure and the combination of both, were not associated with the worse neurological outcome in 4-year-old singletons.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Assisted reproduction techniques (ARTs) are not associated with neurological dysfunction during the first post-natal years; however, effects on the long-term neurological outcome are still inconclusive. An increased time to pregnancy (TTP, a proxy for the severity of subfertility) has been associated with a less optimal neurological condition at age 2. The present study focuses on the neurodevelopmental outcome of 4-year-old ART-offspring.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Longitudinal, prospective follow-up study.</p> </sec> <sec><st>PARTICIPANTS, SETTING, METHODS</st> <p>Four-year-old singletons born to subfertile parents (subfertile group, <I>n</I> = 195), including singletons born after controlled ovarian hyperstimulation IVF (COH-IVF, <I>n</I> = 63), modified natural cycle IVF (MNC-IVF, <I>n</I> = 53) and natural conception (Sub-NC, <I>n</I> = 79). Data on underlying cause of subfertility and TTP were present. In addition, we assessed newly recruited 4-year-old singletons born to fertile parents after natural conception (reference group, <I>n</I> = 98). Neurological development was evaluated with the neurological examination according to Hempel, resulting in a neurological optimality score (NOS), a fluency score and the occurrence of the clinically relevant form of minor neurological dysfunction (complex MND). The primary outcome was the fluency score, as fluency of movements is easily reduced by subtle brain dysfunction. Data were analysed with univariable and multivariable regression analyses, in which special attention was paid to sex differences in the neurological outcome.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The fluency score, NOS and the prevalence of complex MND were similar in COH-IVF, MNC-IVF and Sub-NC children. The neurological condition of children born to subfertile parents was similar to that of children of fertile parents and was independent of the underlying cause of subfertility. No statistically significant associations were found between TTP and the fluency score and NOS. However, a positive correlation was found between TTP and the prevalence of complex MND (TTP in years, adjusted odds ratio [OR] [95% confidence interval, CI]: 1.207 [1.038 to 1.404], <I>P</I> = 0.014); a correlation which could be attributed to girls, in whom an evident positive correlation was present (adjusted OR [95% CI]: 1.542 [1.161 to 2.047], <I>P</I> = 0.003). A similar association was absent in boys.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The prospective design of our study and small post-natal attrition rate (9.3%) reduced potential selection bias based on the child's development or health. The assessors were blind to the mode of conception, except for the group of children born to fertile parents, which was newly recruited. The study lacks sufficient power to conclude firmly that increased TTP is associated with a higher prevalence of complex MND.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our study suggests that the severity of subfertility, rather than its simple presence or components of IVF treatment, affects the neurological outcome. Moreover, girls may be neurologically more vulnerable for the effect of severity of subfertility. The finding that the severity of subfertility may be the decisive factor rather than the presence of a history of subfertility <I>per se</I> corroborates previous reports. Our results cannot be generalized to multiples, as we studied singletons only.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was financially supported by the University Medical Center Groningen, grant number: 754510, the Junior Scientific Masterclass, the Postgraduate School Behavioural and Cognitive Neurosciences and the Cornelia Foundation, Groningen, The Netherlands. The authors have no conflicts of interest to declare.</p> </sec>


Risk of placenta praevia is linked to endometrial thickness in a retrospective cohort study of 4537 singleton assisted reproduction technology births
<sec><st>STUDY QUESTION</st> <p>Is endometrial thickness measured prior to embryo transfer associated with placenta praevia?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Following IVF, the risk of placenta praevia is increased 4-fold in women with an endometrial thickness of &gt;12 mm compared with women with an endometrial thickness of &lt;9 mm.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Placenta praevia is a serious complication of pregnancy with adverse maternal and neonatal outcomes. Placenta praevia is 2- to 6-fold more likely to occur following IVF treatment but it remains unknown what factors contribute to that increased risk.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Retrospective cohort study involving 4007 women who had 4537 singleton assisted reproduction technology (ART) births occurring between January 2006 and June 2012 with no loss to follow-up. The primary outcome measure was the diagnosis of placenta praevia, made by the treating obstetrician on a transvaginal ultrasound in the third trimester.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Women who had singleton births following single embryo transfer performed at Monash IVF in Melbourne, Australia were included. Of the 4537 cycles leading to a singleton ART birth, 2951 were stimulated cycles with fresh embryo transfers; 355 were hormone replacement therapy frozen embryo transfers and 1231 were natural cycles with frozen embryo transfers. The dataset was analysed using binary logistic general estimating equations to calculate odds ratios for placenta praevia adjusted (aOR) for known confounders.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The study groups did not differ significantly in age, BMI and aetiologies of infertility prior to IVF treatment. When compared with stimulated cycles, placenta praevia was less common in women undergoing natural cycles with frozen embryo transfers (OR 0.44, 95% confidence interval (CI) 0.27&ndash;0.70, <I>P</I> &lt; 0.01) but hormone replacement therapy frozen embryo transfer cycles were not associated with a lower risk (OR 0.89, 95% CI 0.48&ndash;1.63). After adjusting for confounders, smoking (aOR 2.58, 95% CI 1.07&ndash;6.24, <I>P</I> = 0.04, endometriosis (aOR 2.01, 95% CI 1.21&ndash;3.33, <I>P</I> &lt; 0.01) and endometrial thickness remained statistically significant as independent risk factors for placenta praevia. Compared with women with an endometrial thickness of &lt;9 mm, women with an endometrial thickness of 9&ndash;12 mm had an aOR of 2.02 (95% CI 1.12&ndash;3.65, <I>P</I> = 0.02) and women with an endometrial thickness &gt;12 mm had an aOR of 3.74 (95% CI 1.90&ndash;7.34, <I>P</I> &lt; 0.01). These differences remained statistically significant after performing a sensitivity analysis limited to women with no previous births.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The study is retrospective in nature, not all confounders may have been accounted for and details on previous intrauterine surgery, a known risk factor, were not available. In addition, ultrasound assessments were carried out by several highly trained operators measuring the endometrial thickness, the main independent variable, in a two-dimensional plane and some inter-observer variability may therefore be present.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The findings of a higher risk of placenta praevia in patients with endometriosis and in those that smoke are in agreement with the current literature on natural conception. There have so far been no reports of an association between endometrial thickness and placenta praevia after ART. This novel finding warrants further study to elucidate the underlying cause of the association and to assess how to minimize harm to IVF patients and their offspring. The fact that the observed increased risk is not linked to the type of embryo transfer (fresh/frozen) but to the type of endometrial preparation, suggests that the risk of placenta praevia in ART can be reduced by considering an elective frozen embryo transfer in a natural cycle, especially given the growing evidence that this strategy also provides a number of other maternal and neonatal benefits.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No funding was required for this study. L.R. has a minority shareholding in Monash IVF and has received unconditional research and educational grants from MSD<sup>&reg;</sup>, Merck-Serono<sup>&reg;</sup> and Ferring<sup>&reg;</sup>. L.R. serves on an advisory board for MSD<sup>&reg;</sup> and Ferring<sup>&reg;</sup>.</p> </sec>


Clinical outcomes following cryopreservation of blastocysts by vitrification or slow freezing: a population-based cohort study
<sec><st>STUDY QUESTION</st> <p>What are the clinical efficacy and perinatal outcomes following transfer of vitrified blastocysts compared with transfer of fresh or of slow frozen blastocysts?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Compared with slow frozen blastocysts, vitrified blastocysts resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes at population level.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Although vitrification has been reported to be associated with significantly increased post-thaw survival rates compared with slow freezing, there has been a lack of general consensus over which method of cryopreservation (vitrification versus slow freezing) is most appropriate for blastocysts.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A population-based cohort of autologous fresh and initiated thaw cycles (a cycle where embryos were thawed with intention to transfer) performed between January 2009 and December 2011 in Australia and New Zealand was evaluated retrospectively. A total of 46 890 fresh blastocyst transfer cycles, 12 852 initiated slow frozen blastocyst thaw cycles and 20 887 initiated vitrified blastocyst warming cycles were included in the data analysis.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Pairwise comparisons were made between the vitrified blastocyst group and slow frozen or fresh blastocyst group. A Chi-square test was used for categorical variables and <I>t</I>-test was used for continuous variables. Cox regression was used to examine the pregnancy outcomes (clinical pregnancy rate, miscarriage rate and live delivery rate) and perinatal outcomes (preterm delivery, low birthweight births, small for gestational age (SGA) births, large for gestational age (LGA) births and perinatal mortality) following transfer of fresh, slow frozen and vitrified blastocysts.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The 46 890 fresh blastocyst transfers, 11 644 slow frozen blastocyst transfers and 19 978 vitrified blastocyst transfers resulted in 16 845, 2766 and 6537 clinical pregnancies, which led to 13 049, 2065 and 4955 live deliveries, respectively. Compared with slow frozen blastocyst transfer cycles, vitrified blastocyst transfer cycles resulted in a significantly higher clinical pregnancy rate (adjusted relative risk (ARR): 1.47, 95% confidence intervals (CI): 1.39&ndash;1.55) and live delivery rate (ARR: 1.41, 95% CI: 1.34&ndash;1.49). Compared with singletons born after transfer of fresh blastocysts, singletons born after transfer of vitrified blastocysts were at 14% less risk of being born preterm (ARR: 0.86, 95% CI: 0.77&ndash;0.96), 33% less risk of being low birthweight (ARR: 0.67, 95% CI: 0.58&ndash;0.78) and 40% less risk of being SGA (ARR: 0.60, 95% CI: 0.53&ndash;0.68).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>A limitation of this population-based study is the lack of information available on clinic-specific cryopreservation protocols and processes for slow freezing-thaw and vitrification-warm of blastocysts and the potential impact on outcomes.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This study presents population-based evidence on clinical efficacy and perinatal outcomes associated with transfer of fresh, slow frozen and vitrified blastocysts. Vitrified blastocyst transfer resulted in significantly higher clinical pregnancy and live delivery rates with similar perinatal outcomes compared with slow frozen blastocyst transfer. Comparably better perinatal outcomes were reported for singletons born after transfer of vitrified blastocysts than singletons born after transfer of fresh blastocysts. Elective vitrification could be considered as an alternative embryo transfer strategy to achieve better perinatal outcomes following Assisted Reproduction Technology (ART) treatment.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No specific funding was obtained. The authors have no conflicts of interest to declare.</p> </sec>


Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles
<sec><st>STUDY QUESTION</st> <p>Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Extensive application of NGS in clinical preimplantation genetic screening (PGS) cycles demonstrates that this methodology is reliable, allowing identification and transfer of euploid embryos resulting in ongoing pregnancies.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The effectiveness of PGS is dependent upon the biology of the early embryo and the limitations of the technology. Fluorescence <I>in situ</I> hybridization, used to test for a few chromosomes, has largely been superseded by microarray techniques that test all 24 chromosomes. Array comparative genomic hybridization (array-CGH) has been demonstrated to be an accurate PGS method and has become the <I>de facto</I> gold standard, but new techniques, such as NGS, continue to emerge.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>The study consisted of a prospective trial involving a double blind parallel evaluation, with both NGS and array-CGH techniques, of 192 blastocysts obtained from 55 consecutive clinical PGS cycles undertaken during the period of September to October 2013. Consistency of NGS-based aneuploidy detection was assessed by matching the results obtained with array-CGH-based diagnoses. Primary outcome measure was accuracy of the chromosomal analysis; secondary outcome measures were clinical outcomes.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTINGS, METHODS</st> <p>Fifty-five patients (median age 39.3 years, range 32&ndash;46) undergoing PGS were enrolled in the study. All embryos were cultured to blastocyst stage; trophectoderm biopsy was performed on Day 5 of development or Day 6/7 for slower growing embryos. The method involved whole genome amplification followed by both NGS and array-CGH. The MiSeq<sup>&reg;</sup> control software, real-time analysis and reporter performed on-board primary and secondary bioinformatics analysis. Copy number variation analysis was accomplished with BlueFuse Multi software.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>A total of 192 blastocysts were blindly evaluated with the NGS-based protocol. Paired comparison between NGS and array-CGH from individual embryos showed concordant results in 191/192 (99.5%) of the blastocysts tested. In total 4608 chromosomes were assessed, 211 (4.6%) of which carried a copy number imbalance. NGS specificity for aneuploidy calling (consistency of chromosome copy number assignment) was 99.98% (4333/4334; 95% confidence interval [95% CI]: 99.87&ndash;100) with a sensitivity of 100% (211/211, 95% CI: 99.25&ndash;100). Despite one discordant result, NGS specificity and sensitivity for aneuploid embryo calling (24-chromosome diagnosis consistency) were both 100% since the discordant sample presented several other aneuploidies. Clinical application of the NGS-based approach revealed 74/192 (38.5%) euploid blastocysts. Following transfer of 50 embryos in 47 women, 34 women had positive hCG levels: 30 pregnancies continued, confirmed by at least one fetal sac and heart beat (63.8% clinical pregnancy rate/embryo transfer), 3 were biochemical and 1 miscarried. A total of 32 embryos implanted and led to the presence of a fetal sac (64.0% implantation rate). All pregnancies went to term resulting in the birth of 31 healthy babies.</p> </sec> <sec><st>LIMITATION, REASON FOR CAUTION</st> <p>Although clinical results reported high pregnancy outcomes following transfer of screened embryos, further data and broad-based clinical application are required to better define the role of NGS in PGS. Before recommending widespread application, a randomized controlled trial confirming its clinical effectiveness is advisable.</p> </sec> <sec><st>WIDER IMPLICATION OF THE FINDING</st> <p>This is the first study reporting extensive application of NGS-based comprehensive aneuploidy screening on embryos at blastocyst stage in a clinical setting versus array-CGH as test of reference. NGS has demonstrated a reliable methodology, with the potential to improve chromosomal diagnosis on embryos especially in terms of high-throughput, automation and ability to detect aneuploidy. NGS methodology may represent a valuable alternative to the other comprehensive aneuploidy screening techniques currently available.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>No external funding was sought for this study. Drs F.K. and C.-E.M. are full-time employees of Illumina, Inc., which provided NGS library and sequencing reagents for the study. All other authors have no conflicts to declare.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


Discordant sex in monozygotic XXY/XX twins: a case report
<p>We report a case of discordant phenotypic sex in monozygotic twins mosaic 47,XXY/46,XX: monozygotic heterokaryotypic twins. The twins presented with cognitive and comprehension delay, behavioural and language disorders, all symptoms frequently reported in Klinefelter syndrome. Molecular zygosity analysis with several markers confirmed that the twins are in effect monozygotic (MZ). Array comparative genomic hybridization found no evidence for the implication of copy number variation in the phenotypes. Ultrasound scans of the reproductive organs revealed no abnormalities. Endocrine tests showed a low testosterone level in Twin 1 (male phenotype) and a low gonadotrophin level in Twin 2 (female phenotype) which, combined with the results from ultrasound examination, provided useful information for potentially predicting the future fertility potential of the twins. Blood karyotypes revealed the presence of a normal 46,XX cell line and an aneuplo&iuml;d 47,XXY cell line in both patients. Examination of the chromosome constitutions of various tissues such as blood, buccal smear and urinary sediment not surprisingly showed different proportions for the 46,XX and 47,XXY cell lines, which most likely explains the discordant phenotypic sex and mild Klinefelter features. The most plausible underlying biological mechanism is a post-zygotic loss of the Y chromosome in an initially 47,XXY zygote. This would result in an embryo with both 46,XX and 47,XXY cells lines which could subsequently divide into two monozygotic embryos through a twinning process. The two cell lines would then be distributed differently between tissues which could result in phenotypic discordances in the twins. These observations emphasize the importance of regular paediatric evaluations to determine the optimal timing for fertility preservation measures and to detect new Klinefelter features which could appear throughout childhood in the two subjects.</p>


Altered gene expression in human placentas after IVF/ICSI
<sec><st>STUDY QUESTION</st> <p>Is gene expression in placental tissue of IVF/ICSI patients altered when compared with a spontaneously conceived group, and are these alterations due to loss of imprinting (LOI) in the case of imprinted genes?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>An altered imprinted gene expression of <I>H19</I> and Pleckstrin homology-like domain family A member 2 (<I>PHLDA2</I>), which was not due to LOI, was observed in human placentas after IVF/ICSI and several biological pathways were significantly overrepresented and mostly up-regulated.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Genomic imprinting plays an important role in placental biology and in placental adaptive responses triggered by external stimuli. Changes in placental development and function can have dramatic effects on the fetus and its ability to cope with the intrauterine environment. An increased frequency of placenta-related problems as well as an adverse perinatal outcome is seen in IVF/ICSI derived pregnancies, but the role of placental epigenetic deregulation is not clear yet.</p> </sec> <sec><st>STUDY DESIGN AND PARTICIPANTS</st> <p>In this prospective cohort study, a total of 115 IVF/ICSI and 138 control couples were included during pregnancy. After applying several exclusion criteria (i.e. preterm birth or stillbirth, no placental samples, pregnancy complications or birth defects), respectively, 81 and 105 placentas from IVF/ICSI and control pregnancies remained for analysis. Saliva samples were collected from both parents.</p> </sec> <sec><st>METHODS</st> <p>We quantitatively analysed the mRNA expression of several growth-related imprinted genes [<I>H19</I>, insulin-like growth factor 2 (IGF2), PHLDA2, cyclin-dependent kinase inhibitor 1C (CDKN1C), mesoderm-specific transcript homolog (MEST) isoform &alpha; and &beta; by quantitative PCR] after standardization against three housekeeping genes [Succinate dehydrogenase A (SDHA), YWHAZ and TATA-binding protein (TBP)]. A quantitative allele-specific expression analysis of the differentially expressed imprinted genes was performed to investigate LOI, independent of the mechanism of imprinting. Furthermore, a microarray analysis was carried out (<I>n</I> = 10 in each group) to investigate the expression of non-imprinted genes as well.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Both <I>H19</I> and <I>PHLDA2</I> showed a significant change, respectively, a 1.3-fold (<I>P</I> = 0.033) and 1.5-fold (<I>P</I> = 0.002) increase in mRNA expression in the IVF/ICSI versus control group. However, we found no indication that there is an increased frequency of LOI in IVF/ICSI placental samples. Genome-wide mRNA expression revealed 13 significantly overrepresented biological pathways involved in metabolism, immune response, transmembrane signalling and cell cycle control, which were mostly up-regulated in the IVF/ICSI placental samples.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Only a subset of samples was found to be fully informative, which unavoidably led to lower sample numbers for our LOI analysis. Our study cannot distinguish whether the reported differences in the IVF/ICSI group are exclusively attributable to the IVF/ICSI technique itself or to the underlying subfertility of the patients.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Whether these placental adaptations observed in pregnancies conceived by IVF/ICSI might be connected to an adverse perinatal outcome after IVF remains unknown. However, it is possible that these differences affect fetal development and long-term patterns of gene expression, as well as maternal gestational physiology.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>Partly funded by an unrestricted research grant by Organon BV (now MSD BV) and GROW School for Oncology and Developmental Biology without any role in study design, data collection and analysis or preparation of the manuscript. No conflict of interests to declare.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Dutch Trial Registry (NTR) number 1298.</p> </sec>


A single nucleotide polymorphism of bone morphogenic protein-15 is not associated with ovarian reserve or response to ovarian stimulation
<sec><st>STUDY QUESTION</st> <p>Is there any effect of the -9C&gt;G variant in the bone morphogenic protein-15 (<I>BMP15</I>) gene on ovarian response and/or current markers of ovarian reserve in patients undergoing <I>in vitro</I> fertilization (IVF) treatment?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>No significant associations of <I>BMP15</I> genotypes with ovarian response (number of oocytes retrieved) and/or markers of ovarian reserve were detected in our cohort of women undergoing IVF treatment.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>There is evidence that genetic variation influences patients' response to ovarian stimulation therapy. BMP15 plays a role in the recruitment of primordial follicles. Therefore, variation in <I>BMP15</I> could predict ovarian reserve and response to ovarian stimulation. Two previous studies have determined a significant correlation between the <I>BMP15</I> -9C&gt;G variant and over-response to ovarian stimulation. No studies to date have correlated this variant with ovarian reserve markers.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>In this prospective observational study, we genotyped the <I>BMP15</I> -9C&gt;G single nucleotide polymorphism in 239 unrelated women undergoing their first cycle of controlled ovarian stimulation for IVF and ICSI (intra-cytoplasmic sperm injection) using gonadotrophins at a tertiary referral centre for reproductive medicine between March 2009 and August 2010.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTINGS, METHODS</st> <p>Baseline pelvic ultrasound and blood tests were taken on Days 2&ndash;3 of the cycle for assessment of baseline hormones and for DNA extraction. Genotypes were determined using TaqMan allelic discrimination assay. Regression analysis was performed to assess the effect of the <I>BMP15</I> genotype on the ovarian reserve markers, serum anti-M&uuml;llerian hormone (s-AMH), follicle stimulating hormone (s-FSH) and antral follicle count (AFC), with adjustment for age and body mass index (BMI), and on the primary outcomes of response (number of oocytes retrieved and gonadotrophin dose) with adjustment for age, BMI and treatment received.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>There was no evidence of any statistically significant (<I>P</I> &lt; 0.05) difference in basal s-FSH, s-AMH and AFC between individuals with different <I>BMP15</I> genotypes. The number of oocytes retrieved and gonadotrophin dose used were also comparable between the individuals with different genotypes.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>A larger sample size would be required in order to determine if the <I>BMP15</I> genotype has a small effect on ovarian reserve or response.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>When considering the development of integrative clinical algorithms for individual FSH doses, our analysis suggests that the genotyping of <I>BMP15</I> -9C&gt;G does not provide additional useful information as a predictor of ovarian reserve or response to ovarian stimulation.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was funded by the Manchester Biomedical Research Centre. The authors have no competing interests to declare.</p> </sec>


A novel MKRN3 missense mutation causing familial precocious puberty
<p>Central precocious puberty may be familial in about a quarter of the idiopathic cases. However, little is known about the genetic causes responsible for the disorder. In this report we describe a family with central precocious puberty associated with a mutation in the <I>makorin RING-finger protein 3 (MKRN3)</I> gene. A novel missense mutation (p.H420Q) in the imprinted MKRN3 gene was identified in the four affected siblings, in their unaffected father and in his affected mother<I>.</I> An <I>in silico</I> mutant <I>MKRN3</I> model predicts that the mutation p.H420Q leads to reduced zinc binding and, subsequently, impaired RNA binding. These findings support the fundamental role of the <I>MKRN3</I> protein in determining pubertal timing.</p>


Genetic variations associated with the effect of testicular cancer treatment on gonadal hormones
<sec><st>STUDY QUESTION</st> <p>Do genetic variations in the testosterone pathway genes modify the effect of treatment on the levels of testosterone and LH in long-term testicular cancer (TC) survivors (TCSs)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Variations in LH receptor (LHR) and in 5&alpha;-reductase II (SRD5A2) genes may modify the effect of TC treatment on testosterone levels, whereas genetic variations in the androgen receptor (AR) may modify the effect on LH levels.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>TCSs experience variable degrees of long-term reduction in gonadal function after treatment. This variability can in part be explained by treatment intensity, but may also be due to individual variations in genes involved in the function and metabolism of reproductive hormones.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Cross-sectional study on testosterone and LH levels in 637 Norwegian TCSs in relation to genetic variants and TC treatment.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>The single nucleotide polymorphisms <I>LHR Asn291Ser</I> (rs12470652) and <I>Ser312Asn</I> (rs2293275), as well as <I>SRD5A2 Ala49Thr</I> (rs9282858) and <I>Val89Leu</I> (rs523349) were analyzed by allele-specific PCR. The insertion polymorphism <I>LHR InsLQ</I> (rs4539842) was analyzed by sequencing. The numbers of <I>AR CAG</I> and <I>GGN</I> repeats were determined by capillary electrophoresis. Blood samples were collected 5&ndash;21 years after diagnosis (median 11 years) and serum total testosterone and LH were analyzed by commercial immunoassays. The TCSs were divided into four groups according to their treatment; surgery only, radiotherapy and chemotherapy with &le;850 or &gt;850 mg of cisplatin. Polymorphisms presenting <I>P</I> &lt; 0.1 for the interaction term with treatment in an initial two-way analysis of covariance (ANCOVA) were investigated further in two consecutive one-way ANCOVA analyses to elucidate the interaction between treatment and genotype.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>For the whole group of TCSs, there were no significant differences between the hormone levels in homozygotes for the wild type and carriers of at least one polymorphic allele for the investigated polymorphisms. Three of the polymorphisms showed signs of interaction with treatment, i.e. <I>LHR InsLQ</I>, <I>SRD5A2 A49T</I> and the AR CAG repeat. Follow-up analyses revealed three situations where only one of the genotypes of the polymorphism where associated with significantly different hormone levels after surgery compared with after additional cytotoxic treatment: For <I>LHR InsLQ</I>, only the wild-type allele was associated with lower testosterone levels after cisplatin &gt; 850 mg compared with after surgery (24% lower, <I>P</I> &lt; 0.001). For <I>SRD5A2 A49T</I>, testosterone levels were lower after radiotherapy compared with after surgery, but only for the heterozygotes for the polymorphism (39% lower, <I>P</I> = 0.001). In comparison, the testosterone levels were just slightly lower after radiotherapy (6% lower, <I>P</I> = 0.039) or cisplatin &le; 850 mg (7% lower, <I>P</I> = 0.041), compared with surgery, independent of genotypes. For <I>AR CAG</I>, only the reference length of CAG = 21&ndash;22 had significantly higher LH levels after cisplatin &le; 850 mg compared with after surgery (70% higher, <I>P</I> &lt; 0.001). Independent of genotypes, however, LH levels after cisplatin &le; 850 mg were only 26% higher than after surgery (<I>P</I> = 0.005).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Unadjusted <I>P</I>-values are presented. For analysis involving genotypes, the level of statistical significance was adjusted for the total number of polymorphisms tested, <I>n</I> = 7, i.e. to <I>P</I> &lt; 0.007 (0.5/7). The rather weak associations indicate that additional polymorphisms are involved in the modulation.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>To our knowledge, this is the first study supporting the notion that polymorphisms may explain at least some of the inter-individual differences in endocrine response to TC treatment. Our findings suggest that individuals with certain genotypes may be more vulnerable to certain treatments. Knowledge on genetic predisposition concerning treatment-related endocrine gonadotoxicity to different treatment regimens may help tailoring TC therapy when possible.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was supported by the Research Council of Norway (Grant No. 160619). There were no competing interests.</p> </sec>


Endometrioma excision and ovarian reserve; do assessments by antral follicle count and anti-Mullerian hormone yield contradictory results?


Reply: Endometrioma excision and ovarian reserve: do assessments by antral follicle count and anti-Mullerian hormone yield contradictory results?


Endometrial scratching for women with repeated implantation failure


Reply: Endometrial scratching for women with repeated implantation failure


A nod is as good as a wink to a blind horse


Sins of the fathers: sperm DNA damage in the context of assisted reproduction


Sperm vacuoles cannot help to differentiate fertile men from infertile men with normal sperm parameter values
<sec><st>STUDY QUESTION</st> <p>Can the assessment of sperm vacuoles at high magnification contribute to the explanation of idiopathic infertility?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The characteristics of sperm head vacuoles (number, area, position) are no different between fertile controls and patients with unexplained infertility.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Until now, the assessment of sperm head vacuoles has been focused on a therapeutic goal in the intracytoplasmic morphologically selected sperm injection (IMSI) procedure, but it could be pertinent as a new diagnostic tool for the evaluation of male fertility.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This diagnostic test study with blind assessment included a population of 50 fertile men and 51 men with idiopathic infertility. They were selected from September 2011 to May 2013.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Fertile men were within couples who had a spontaneous pregnancy in the last 2 years. Infertile men were within couples who had unexplained infertility and were consulting in our centre. After analysis of conventional sperm parameters, we investigated the number, position and area of sperm head vacuoles at high magnification (<FONT FACE="arial,helvetica">x</FONT>6000) with interference contrast using an image analysis software. We also carried out a nuclear status analysis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL), sperm chromatin structure assay (SCSA) and aniline blue staining.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Concerning the vacuoles data, we did not find any significant difference between the two populations. We found no significant correlation between the vacuolar parameters (mean number of vacuoles, relative vacuole area and percentage of spermatozoa with large vacuoles) and either conventional semen parameters, male age or the data from the aniline blue staining, SCSA assay and TUNEL assay.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Despite the fact all of the vacuole parameters values were identical in fertile and infertile men, we cannot totally exclude that a very small cause of unexplained infertilities could be related to an excess of sperm vacuoles.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>In line with its widely debated use as a therapeutic tool, sperm vacuole assessment for diagnostic purposes does not seem useful.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was funded by a grant from Association pour la Recherche sur les Traitements de la St&eacute;rilit&eacute;. There are no competing interests to declare.</p> </sec>


Neutralization of ASC improves sperm motility in men with spinal cord injury
<sec><st>STUDY QUESTION</st> <p>Does neutralization of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) improve sperm motility in men with spinal cord injury (SCI)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Neutralization of ASC improves sperm motility in men with SCI.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Semen of men with SCI contains normal sperm concentrations but abnormally low sperm motility. Inflammatory cytokines, activated via the inflammasome complex, are contributory. A key component of the inflammasome is ASC.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This prospective study included semen samples collected from 32 men with SCI.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>At a major university medical center, untreated semen was compared with semen treated with anti-ASC polyclonal antibody. Semen treated with IgG was used as a control.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Addition of anti-ASC polyclonal antibody to semen significantly increased mean sperm motility from 11.5% (95% CI, 6.3&ndash;16.7) to 18.3% (95% CI, 11.8&ndash;24.8). Improvements were most pronounced in the subgroup whose starting motility ranged between 6 and 40%. In this subgroup, the mean sperm motility improved from 13.3% (95% CI, 9.3&ndash;17.3) to 23.9% (95% CI, 14.7&ndash;23.0). Sperm motility did not improve after treatment with IgG.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This study is limited by the small sample size as this is a rare population.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Blockade of the inflammasome via treatment with anti-ASC improved sperm motility in men with SCI. In doing so, this treatment significantly increased their total motile sperm count. This is the first study to demonstrate that interference with the inflammasome improves sperm motility in men with SCI. This treatment has potential as a therapeutic intervention.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by the Craig H. Neilsen Foundation, Grant # 224598, the University of Miami Miller School of Medicine and the Miami Project to Cure Paralysis, Miami, FL, USA. R.W.K. and J.P.d.R.V. hold a patent for the treatment of inflammation after central nervous system injury using antibodies against inflammasome proteins. The other authors have no conflicts of interest to declare.</p> </sec>


Morphological alterations in protamine-deficient spermatozoa
<sec><st>STUDY QUESTION</st> <p>How are protamine deficiencies associated with sperm head morphology in subfertile men?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The prevalence of morphological variations and large nuclear vacuoles was slightly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>A protamine deficiency was previously reported to be associated with an abnormal sperm morphology; however, how they are related to each other remains unclear. This is further confounded by a number of protamine-deficient spermatozoa having a normal head morphology.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This is a cross-sectional study, including 36 men diagnosed with male factor infertility or participating in an assisted reproduction program. To assess sperm head morphology, this study analyzed 2400 spermatozoa with a protamine deficiency and 2400 spermatozoa with a normal protamine status. An additional 21 men were analyzed to examine DNA fragmentation and its relationship with protamine deficiencies and sperm head morphologies.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>The morphology of the sperm head was evaluated based on its shape, size and nuclear vacuoles at a magnification of &gt;6000<FONT FACE="arial,helvetica">x</FONT>. Using elliptic Fourier analysis, the shape was summarized into four numeric variables. The protamine status was evaluated with chromomycin A<SUB>3</SUB> (CMA<SUB>3</SUB>). Sperm head size, vacuoles and shape were compared between protamine-deficient and non-deficient spermatozoa. DNA fragmentation was evaluated with the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay. The percentages of protamine-deficient spermatozoa and DNA fragmentation were compared between spermatozoa with morphologically normal heads and those with abnormal heads.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Variations in head size (<I>P</I> &lt; 0.0001) and shape (<I>P</I> &lt; 0.0001) were significantly higher, with narrower (<I>P</I> &lt; 0.001), more fan-shaped (<I>P</I> &lt; 0.01) and more square-shaped forms (<I>P</I> &lt; 0.001) in protamine-deficient spermatozoa than in non-deficient spermatozoa; however, the distribution of morphological variations markedly overlapped. Protamine deficiencies were more frequently observed in spermatozoa with large nuclear vacuoles than in those without them (32.0 &plusmn; 3.1 versus 39.4 &plusmn; 2.9%, <I>P</I> &lt; 0.001). The percentage of protamine-deficient spermatozoa was significantly lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (25.4 &plusmn; 2.6 versus 38.0 &plusmn; 2.5%, <I>P</I> &lt; 0.001). The percentage of DNA fragmentation was significantly higher in protamine-deficient spermatozoa than in non-deficient spermatozoa (11.3 &plusmn; 2.1 versus 1.6 &plusmn; 0.6%, <I>P</I> &lt; 0.001), and was lower in spermatozoa with a normal head morphology than in those with an abnormal head morphology (2.6 &plusmn; 0.7 versus 6.4 &plusmn; 0.2%, <I>P</I> &lt; 0.001).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>We were unable to discriminate the kind of protamines or quantify the extent of the protamine deficiency in spermatozoa using the CMA<SUB>3</SUB> staining method.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This study provided a novel insight into how abnormal protamination affects sperm head morphology as well as the relationship between sperm head morphology and its own molecular integrity. Our results will contribute to a deeper understanding of the benefits and limitations of the morphological selection of spermatozoa for ICSI.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was supported by a JSPS Grant-in-Aid for the Encouragement of Scientists (25931009, 26931010). All authors have no conflicts of interest to disclose.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>N/A.</p> </sec>


Autoantibodies against protein disulfide isomerase ER-60 are a diagnostic marker for low-grade testicular inflammation
<sec><st>STUDY QUESTION</st> <p>Is there a non-invasive biomarker for the diagnosis of testicular inflammatory lesions?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>In sera from infertile azoospermic patients with histologically confirmed low-grade testicular inflammation, significantly elevated titers of autoantibodies against disulfide isomerase family A, member 3 (ER-60) were found.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Infection and inflammation of the genital tract are supposed to be responsible for up to 15% of cases among infertile males. However, specific seminal or serological markers are not available to assess subacute or chronic inflammatory conditions in the testis.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This study consisted of the identification of autoantibodies for testicular antigens in sera of patients with low-grade testicular inflammation, validation of candidates, development of an ELISA for the most promising target antigen and measurement of autoantibodies titers in healthy normozoospermic men (<I>n</I> = 20); male blood donors (<I>n</I> = 14); men with impaired semen quality without (<I>n</I> = 14) or with (<I>n</I> = 26) symptoms of genital tract infection/inflammation; azoospermic men with histologically confirmed testicular inflammatory lesions (<I>n</I> = 16); men after pharmacotherapy of genital tract infection/inflammation (<I>n</I> = 15) and men with acute epididymo-orchitis (<I>n</I> = 30).</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Proteins in lysates of normal testicular tissue were separated by high-resolution 2D gel electrophoresis and probed with sera of 13 patients with histologically confirmed chronic testicular inflammation. There were 14 proteins that immunoreacted with a majority of these sera and could be identified by mass spectrometry. Of these 14 proteins, disulfide isomerase family A, member 3 (ER-60), transferrin and chaperonin containing TCP1 complex, subunit 5 (epsilon) (CCT5) were considered as specific. Since ER-60 reacted with 92% of patient sera, an ER-60-autoantibody ELISA was developed.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The newly established ELISA detected significantly elevated titers of autoantibodies against ER-60 in the sera from infertile men with histologically confirmed chronic testicular inflammation (median 8.6; <I>P</I> &lt; 0.01) compared with the control groups. Moreover, elevated levels of anti-ER-60 titers were detected in patients suffering from acute epididymo-orchitis (median 3.3; <I>P</I> &lt; 0.05) as compared with healthy normozoospermic men (median 2.13; <I>P</I> &lt; 0.001), male blood donors with unknown fertility status (median 2.72; <I>P</I> &lt; 0.01), patients with impaired semen quality but no infection/inflammation (median 2.59; <I>P</I> &lt; 0.001) and patients with symptoms of genital tract infections and/or inflammation (median 2.18; <I>P</I> &lt; 0.001). Significantly lower levels of anti-ER-60 antibodies were measured in sera from patients after application of anti-inflammatory pharmacotherapy (median 1.9; <I>P</I> &lt; 0.01) compared with those with histologically confirmed chronic testicular inflammation. The cut-off value of the assay was set to 6.6 U/ml based on a calculated sensitivity of 100% and a specificity of 81.2%.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The results obtained in this study showed statistically significant elevated titers of ER-60 antibodies in sera from patients with histologically confirmed testicular inflammatory lesions and from a few patients with acute epididymo-orchitis. However, the number of serum samples tested was limited. Severe testicular damage seen in azoospermic patients could represent a bias towards ER-60 reactivity, while the assay does not allow for different etiologies of the lesions to be distinguished. Due to ethical reasons, the prevalence of testicular inflammatory lesions among controls and non-azoospermic men cannot be studied at the histological level.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Measurement of ER-60 autoantibody titers in serum could be a novel non-invasive marker for the diagnosis of asymptomatic testicular inflammation causing male fertility disturbances.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was supported by a grant of the Deutsche Forschungsgemeinschaft (ME 1323/4-4) and the Translational Science Fund (Wirtschafts-und Strukturbank Hessen&mdash;WI Bank). M.F., A.P., W.W., H.-C.S. and A.M. are supported by the LOEWE focus group &lsquo;MIBIE&rsquo; (Male infertility during infection and inflammation). The ER-60 ELISA is protected by a patent to the Justus-Liebig-University of Giessen with A.M. and M.F. as inventors (patent no. DE 10 2008 053 503). T.Z. as employee of the DRG Company was responsible for the ELISA development.</p> </sec>


Controlled ovarian hyperstimulation leads to high progesterone and estradiol levels during early pregnancy
<sec><st>STUDY QUESTION</st> <p>Are there differences in estrogen and progesterone secretion in singleton pregnancies, up to Week 11, between spontaneous pregnancies, after controlled ovarian hyperstimulation and fresh embryo transfer (COH + ET) and after frozen embryo transfer in a spontaneous cycle (FET)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Serum progesterone and estradiol (E<SUB>2</SUB>) concentrations after COH + ET were higher in early pregnancy, lasting up to Week 7&ndash;8, than FET and spontaneous pregnancies, while hormone levels after FET did not differ from spontaneous pregnancies.</p> </sec> <sec><st>WHAT IS ALREADY KNOWN</st> <p>The risk of adverse perinatal outcomes after COH + ET seems to be increased when compared with spontaneous pregnancies. One of the reasons suggested for this is related to ovarian hyperstimulation.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This was a prospective cohort study consisting of three different groups of pregnant women which were followed-up weekly until Week 11 of their pregnancies. The spontaneous pregnancy group consisted of 41 women, the COH + ET group consisted of 39 and the FET group consisted of 30 women.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Women in the control group with spontaneous conception were recruited from local prenatal clinics. Women in the COH + ET and FET groups were recruited from the Reproductive Unit of Oulu University Hospital. At each visit, a three-dimensional ultrasonography was performed to examine the ovarian volumes and vascularization. A blood sample was drawn to analyse progesterone and E<SUB>2</SUB> levels. The pregnancy outcome was included in the analysis.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>At pregnancy Week 5, the serum progesterone levels were higher after the COH + ET (median 312, inter-quartile range 183&ndash;480 nmol/l), when compared with the spontaneous (63, 52&ndash;80 nmol/l; <I>P</I> &lt; 0.001) and FET (74, 48&ndash;96 nmol/l; <I>P</I> &lt; 0.001) pregnancies. At Week 11, the <I>P</I> (189, 124&ndash;260 nmol/l) was still higher in the COH + ET group (FET 101, 78&ndash;120 nmol/l, <I>P</I> &lt; 0.001; spontaneous 115, 80&ndash;139 nmol/l, <I>P</I> &lt; 0.01) than the other two groups. The E<SUB>2</SUB> levels at Week 5 were also significantly higher after COH + ET (4.1, 2.2&ndash;6.6 nmol/l) than in the spontaneous pregnancies (1.1, 0.7&ndash;1.6 nmol/l, <I>P</I> &lt; 0.001) or after FET (0.7, 0.6&ndash;0.9 nmol/l, <I>P</I> &lt; 0.001). The volume of the ovaries and the intraovarian vasculature in the COH + ET group were significantly higher when compared with the other two groups (<I>P</I> &lt; 0.001). The birthweight was negatively correlated with the serum <I>P</I> (R &ndash;0.340, <I>P</I> &lt; 0.01) and E<SUB>2</SUB> (<I>R</I>= &ndash;0.275, <I>P</I> &lt; 0.05) in pregnancy Weeks 5&ndash;8. In the multivariate analysis evaluating the factors affecting birthweight of the newborn, the significant factors were the length of gestation, maternal height and progesterone or E<SUB>2</SUB> secretion during Weeks 5&ndash;8.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Because of the low number of patients in this study, larger cohort studies are required to confirm the findings.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The findings here indicate that COH-induced increased luteal activity should be evaluated by measuring steroid levels or the ovarian size or vascularity, rather than number of oocytes retrieved. If unphysiologically high steroid activity during pregnancy after COH contributes to the risk of adverse perinatal outcomes after fresh embryo transfer, milder stimulation protocols or even freezing of all of the embryos should be considered.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTERESTS</st> <p>This study was supported by a research grant from the Academy of Finland. The authors declare no conflicts of interest.</p> </sec>


Paternal influence of sperm DNA integrity on early embryonic development
<sec><st>STUDY QUESTION</st> <p>Does sperm DNA damage affect early embryonic development?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Increased sperm DNA damage adversely affects embryo quality starting at Day 2 of early embryonic development and continuing after embryo transfer, resulting in reduced implantation rates and pregnancy outcomes.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Abnormalities in the sperm DNA in the form of single and double strand breaks can be assessed by an alkaline Comet assay. Some prior studies have shown a strong paternal effect of sperm DNA damage on IVF outcome, including reduced fertilization, reduced embryo quality and cleavage rates, reduced numbers of embryos developing into blastocysts, increased percentage of embryos undergoing developmental arrest, and reduced implantation and pregnancy rates.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A cross-sectional study of 215 men from infertile couples undergoing assisted reproduction techniques at the University of Utah Center for Reproductive Medicine.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Sperm from men undergoing ART were analyzed for DNA damage using an alkaline Comet assay and classified into three groups: &lsquo;low damage&rsquo; (0&ndash;30%), &lsquo;intermediate damage&rsquo; (31&ndash;70%) and &lsquo;high damage&rsquo; (71&ndash;100%). The cause of couples' infertility was categorized into one of the three types (male, female or unexplained). Each embryo was categorized as &lsquo;good&rsquo;, &lsquo;fair&rsquo; or &lsquo;poor&rsquo; quality, based on the number and grade of blastomeres. The influence of sperm DNA damage on early embryonic development was observed and classified into four stages: peri-fertilization effect (fertilization rate), early paternal effect (embryonic days 1&ndash;2), late paternal effect (embryonic days 3&ndash;5) and implantation stage effect.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The paternal effect of sperm DNA damage was observed at each stage of early embryonic development. The peri-fertilization effect was higher in oocytes from patients with female infertility (20.85%) compared with male (8.22%; <I>P</I> &lt; 0.001) and unexplained (7.30%; <I>P</I> &lt; 0.001) infertility factors. In both the early and late paternal effect stages, the low DNA damage group had a higher percentage of good quality embryos (<I>P</I> &lt; 0.05) and lower percentage of poor quality embryos (<I>P</I> &lt; 0.05) compared with the high DNA damage group. Implantation was lower in the high DNA damage (33.33%) compared with intermediate DNA damage (55.26%; <I>P</I> &lt; 0.001) and low DNA damage (65.00%; <I>P</I> &lt; 0.001) groups. The implantation rate was higher following blastocyst transfer (58.33%), when compared with early stage blastocyst (53.85%; <I>P</I> = 0.554) and cavitating morula transfers (34.40%; <I>P</I> &lt; 0.001). Implantation was higher when the female partner age was &le;35 years when compared with &gt;35 year age group (52.75 versus 35.44%; <I>P</I> = 0.008).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>A potential limitation of this study is that it is cross-sectional. Generally in such studies more than one variable could affect the outcome. Analyzing sperm is one part of the equation but a number of environmental and female factors also have the potential to influence embryo development and implantation. Furthermore, the selection of morphologically normal and physiologically motile sperm may result in isolation of sperm with reduced DNA damage. Therefore, selecting the best available sperm for ICSI may lead to experimental bias, as the selected sperm do not represent the overall sperm population in which the DNA damage is measured. Similar studies on selected sperm and with a larger sample size are now required.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The paternal influence of damaged chromatin is more prominent after zygotic transcriptional activation. A prolonged paternal effect on the developing embryo may be due to the active repair mechanism present in oocytes that tends to overcome the damaged paternal chromatin. The probability of eliminating an embryo fertilized by a sperm with damaged DNA is higher at the blastocyst stage than the cleavage stage; therefore blastocyst transfer could be recommended for better implantation success. Finally, we recommend ICSI treatment for patients with a higher percentage of sperm with DNA damage as well as additional studies with a larger sample size aimed at assessing DNA damage analysis as a diagnostic tool for IVF.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was supported by the University of Utah internal funds. The authors declare no competing interests.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>N/A.</p> </sec>


Nuclear magnetic resonance metabolomic profiling of Day 3 and 5 embryo culture medium does not predict pregnancy outcome in good prognosis patients: a prospective cohort study on single transferred embryos
<sec><st>STUDY QUESTION</st> <p>Does the metabolomic profile, obtained with nuclear magnetic resonance (NMR), of spent culture media from human embryos correlate with reproductive potential in a cohort of good prognosis patients?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>In a large cohort of single transferred blastocysts from a homogeneous group of good prognosis patients, we find a high degree of individual variation in the metabolome that, however, has no relation to pregnancy outcome.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Differences among various specific metabolites have been linked to reproductive potential. Although results from retrospective near infrared (NIR) spectroscopy analyses of spent culture medias from transferred embryos were promising, randomized controlled trials were unable to demonstrate that NIR analysis improved pregnancy rates. Therefore, a more detailed investigation of the relation between embryo metabolism and reproductive potential is required. NMR is a powerful technique that provides detailed structural and dynamic information.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>A prospective cohort study was conducted at the Fertility Clinic, Aarhus University Hospital between February 2011 and July 2012. Infertile patients aged &lt;38 years without endometriosis were offered participation and their embryos were included if greater than or equal to eight oocytes were retrieved. In total, 161 infertile patients were included in the cohort.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Spent culture media was collected on Days 3 and 5 after oocyte retrieval from 148 single transferred embryos. NMR spectra were obtained from 12 &micro;l of spent media. Data were quantitatively analysed using multivariate analysis with respect to pregnancy outcome, defined as a live fetus by ultrasound in gestational Week 8, along with patient and treatment related variables such as embryo score, age, BMI, fertilization method and cause of infertility.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>A total of 148 cycles were included in the analysis [embryo transfer cancelled (<I>n</I> = 12), no media collected (<I>n</I> = 1)]. Clinical pregnancy was confirmed in 47 patients (32%). We obtained high quality NMR spectra for 141 Day 3 and 137 Day 5 samples. Our spectra show a high degree of individual variation. Multivariate data analysis was performed on spectral data with several different pre-processing combinations, i.e. binning, alignment, normalization and scaling in the attempt to develop a valid prediction model. Different strategies of multivariate analysis showed, however, no correlation between the NMR profiles and pregnancy outcome, patient or treatment characteristics. No model could therefore be developed for prediction of pregnancy outcome. We conclude that within this group of good prognosis patients, large-scale metabolic variations between embryos detected with NMR have no apparent association with pregnancy outcome.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Although this study is the largest we know of using NMR to investigate metabolomic profiles of single-transferred embryos, there may be differences that would be detected with a larger study. When analysing such a small sample volume, even small variations in the amount of media and dilution may introduce a large uncertainty in the results.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our study questions the usefulness of the entire metabolome for embryo selection, which should direct the search for viability markers in the culture media towards individual components.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTERESTS</st> <p>Funding was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation. Research at the Fertility Clinic, Aarhus Universtity Hospital is supported by an unrestricted grant from MSD and Ferring. The authors declare no competing interest.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>NCT01139268.</p> </sec>


Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis
<sec><st>STUDY QUESTION</st> <p>Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography&ndash;tandem mass spectrometry (LC&ndash;MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC&ndash;MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Using advanced mass spectrometry and high confidence criteria for accepting proteins (<I>P</I> &lt; 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92&ndash;97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp &amp; Dohme Corp and Ferring. The authors declare no conflicts of interest.</p> </sec>


Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method
<sec><st>STUDY QUESTION</st> <p>Can the equilibration steps prior to embryo vitrification be automated?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>We have developed the &lsquo;Gavi&rsquo; system which automatically performs equilibration steps before closed system vitrification on up to four embryos at a time and gives <I>in vitro</I> outcomes equivalent to the manual Cryotop method.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Embryo cryopreservation is an essential component of a successful assisted reproduction clinic, with vitrification providing excellent embryo survival and pregnancy outcomes. However, vitrification is a manual, labour-intensive and highly skilled procedure, and results can vary between embryologists and clinics. A closed system whereby the embryo does not come in direct contact with liquid nitrogen is preferred by many clinics and is a regulatory requirement in some countries.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>The Gavi system, an automation instrument with a novel closed system device, was used to equilibrate embryos prior to vitrification. Outcomes for embryos automatically processed with the Gavi system were compared with those processed with the manual Cryotop method and with fresh (non-vitrified) controls.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>The efficacy of the Gavi system (Alpha model) was assessed for mouse (Quackenbush Swiss and F1 C57BL/6J x CBA) zygotes, cleavage stage embryos and blastocysts, and for donated human vitrified-warmed blastocysts. The main outcomes assessed included recovery, survival and <I>in vitro</I> embryo development after vitrification-warming. Cooling and warming rates were measured using a thermocouple probe.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Mouse embryos vitrified after processing with the automated Gavi system achieved equivalent <I>in vitro</I> outcomes to that of Cryotop controls. For example, for mouse blastocysts both the Gavi system (<I>n</I> = 176) and manual Cryotop method (<I>n</I> = 172) gave a 99% recovery rate, of which 54 and 50%, respectively, progressed to fully hatched blastocysts 48 h after warming. The outcomes for human blastocysts processed with the Gavi system (<I>n</I> = 23) were also equivalent to Cryotop controls (<I>n</I> = 13) including 100% recovery for both groups, of which 17 and 15%, respectively, progressed to fully hatched blastocysts 48 h after warming. The cooling and warming rates achieved with the Gavi system were 14 136&deg;C/min and 11 239&deg;C/min, respectively.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Testing of the Gavi system described here was limited to <I>in vitro</I> development of embryos from two mouse strains and a limited number of human embryos. Validation of Gavi system advanced production models is now required to confirm the success of semi-automated vitrification, including clinical evaluation of pregnancy outcomes from the transfer of Gavi vitrified-warmed human embryos.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The Gavi system has the potential to revolutionize and standardize vitrification of embryos and oocytes. The success of the Gavi system shows that it is possible to semi-automate complicated labour-intensive ART methods and processes, and opens up the possibility for further improvements in clinical outcomes and efficiencies in the ART clinic.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by Genea Ltd. S.B., N.M.T., T.T.P., S.J.M., M.C.B. and T.S. are shareholders of Genea Ltd. E.V., C.H., C.L., S.R.L. and S.M.D. are shareholders of Planet Innovation Pty Ltd. The remaining authors are employees of either Genea Ltd. or Planet Innovation Pty Ltd.</p> </sec>


Effect of intracervical anesthesia on pain associated with the insertion of the levonorgestrel-releasing intrauterine system in women without previous vaginal delivery: a RCT
<sec><st>STUDY QUESTION</st> <p>Is the pain associated with levonorgestrel-releasing intrauterine system (LNG-IUS) insertion reduced by intracervical anesthesia in women without previous vaginal birth?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Intracervical anesthesia was not associated with reduced pain in women without previous vaginal birth.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The pain associated with the insertion of intrauterine contraceptives (IUCs) is a limiting factor for the use of these contraceptives by some women. No prophylactic pharmacological intervention has proven efficacy in relieving pain during or after the insertion of IUCs. However, previous studies included women with previous vaginal delivery, and injectable intracervical anesthesia was not evaluated in any of these studies.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This was a randomized, open, parallel-group clinical trial that evaluated 100 women without previous vaginal delivery who wished to use the LNG-IUS for the first time. These women were evaluated immediately after LNG-IUS insertion and then 2 h and 6 h later.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>The 100 women were randomized into two groups: (i) use of a non-steroidal anti-inflammatory drug (NSAID) (ibuprofen, 400 mg) 1 h prior to LNG-IUS insertion; or (ii) 2% lidocaine intracervical injection 5 min prior to LNG-IUS insertion. The women were evaluated immediately after LNG-IUS insertion and then 2 h and 6 h after insertion. Two pain scales were used (the visual analogue scale and the facial pain scale) in addition to assessing the ease of insertion (as rated by the provider) and the level of discomfort during the procedure (as rated by the patient). Multivariate logistic regression was performed to analyze the predictors associated with moderate/severe pain.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The pain and discomfort associated with LNG-IUS insertion, and the ease of insertion of the LNG-IUS did not differ between the groups. Nulliparity was more associated with moderate/severe pain [adjusted odds ratio (OR): 3.1 (95% confidence interval (CI): 1.3&ndash;7.80]. Injectable intracervical anesthesia use reduced the risk of moderate/severe pain by 40% [adjusted OR: 0.6 (95% CI: 0.2&ndash;1.4)]. The difference between the mean pain score in the intracervical anesthesia group and the NSAID group was &lt;10%; thus, the effect size of the intervention was not significant.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Intracervical anesthesia was compared with an oral medication in this study. Intracervical injection of a saline solution or even a dry needling as the placebo for a double-blind study could be a more adequate control; however, this approach was not a protocol approved by the institutional review board. Considering that the majority of the insertions were easy (&gt;80% in both groups), the results may not be extrapolated to difficult insertions with moderate/severe pain where local anesthesia may have a role.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The findings can be generalized to most insertions in nulliparous women or in those without a previous vaginal delivery. There is currently no evidence to recommend the routine use of prophylactic intracervical anesthesia prior to LNG-IUS insertion; there is no evidence that this treatment reduces insertion-related pain.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>RAF and CSV give occasional lectures for Bayer Healthcare. This study received funding from the National Institute of Hormones and Women's Health, National Council for Scientific and Technological Development (CNPq).</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>NCT02155166.</p> </sec>


Intra-uterine microbial colonization and occurrence of endometritis in women with endometriosis
<sec><st>STUDY QUESTION</st> <p>Is there any risk of intra-uterine bacterial colonization and concurrent occurrence of endometritis in women with endometriosis?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>An increase in intra-uterine microbial colonization and concurrent endometritis occurred in women with endometriosis that was further increased after GnRH agonist (GnRHa) treatment.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Higher bacterial contamination of menstrual blood and increased endotoxin level in menstrual and peritoneal fluids have been found in women with endometriosis than in control women. However, information on intra-uterine microbial colonization across the phases of the menstrual cycle and possible occurrence of endometritis in women with endometriosis is still lacking.</p> </sec> <sec><st>STUDY DESIGN, SIZE AND DURATION</st> <p>This is a case-controlled study with prospective collection of vaginal smears/endometrial samples from women with and without endometriosis and retrospective evaluation.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Vaginal smears and endometrial smears were collected from 73 women with endometriosis and 55 control women. Twenty of the women with endometriosis and 19 controls had received GnRHa therapy for a period of 4&ndash;6 months. Vaginal pH was measured by intra-vaginal insertion of a pH paper strip. The bacterial vaginosis (BV) score was analyzed by Gram-staining of vaginal smears and based on a modified Nugent-BV scoring system. A panel of bacteria was analyzed by culture of endometrial samples from women treated with GnRHa or not treated. Immunohistochemcial analysis was performed using antibody against Syndecan-1 (CD138) and myeloperoxidase in endometrial biopsy specimens from women with and without endometriosis.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>A significant shifting of intra-vaginal pH to &ge;4.5 was observed in women with endometriosis compared with control women (79.3 versus 58.4%, <I>P</I> &lt; 0.03). Compared with untreated women, use of GnRHa therapy also shifted vaginal pH to &ge;4.5 in both control women (<I>P</I> = 0.004) and in women with endometriosis (<I>P</I> = 0.03). A higher risk of increasing intermediate flora (total score, 4&ndash;6) (<I>P</I> = 0.05) was observed in women with endometriosis who had GnRHa treatment versus untreated women. The number of colony forming units (CFU/ml) of <I>Gardnerella, &alpha;-Streptococcus, Enterococci and Escherichia coli</I> was significantly higher in endometrial samples from women with endometriosis than control women (<I>P</I> &lt; 0.05 for each bacteria). GnRHa-treated women also showed significantly higher colony formation for some of these bacteria in endometrial samples than in untreated women (<I>Gardnerella</I> and <I>E. coli</I> for controls<I>; Gardnerella, Enterococci</I> and <I>E. coli</I> for women with endometriosis, <I>P</I> &lt; 0.05 for all). Although there was no significant difference in the occurrence of acute endometritis between women with and without endometriosis, both GnRHa-treated controls and women with endometriosis had a significantly higher occurrence of acute endometritis (<I>P</I> = 0.003 for controls, <I>P</I> = 0.001 for endometriosis versus untreated women). Multiple analysis of covariance analysis revealed that an intra-vaginal pH of &ge;4.5 (<I>P</I> = 0.03) and use of GnRHa (<I>P</I> = 0.04) were potential factors that were significantly and independently associated with intra-uterine microbial colonization and occurrence of endometritis in women with endometriosis. These findings indicated the occurrence of sub-clinical uterine infection and endometritis in women with endometriosis after GnRHa treatment.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>We cannot exclude the introduction of bias from unknown previous treatment with immunosuppressing or anti-microbial agents. We have studied a limited range of bacterial species and used only culture-based methods. More sensitive molecular approaches would further delineate the similarities/differences between the vaginal cavity and uterine environment.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Our current findings may have epidemiological and biological implications and help in understanding the pathogenesis of endometriosis and related disease burden. The worsening of intra-uterine microbial colonization and higher occurrence of endometritis in women with endometriosis who were treated with GnRHa identifies some future therapeutic avenues for the management, as well as prevention of recurrence, of endometriosis. Further studies are needed to examine intra-uterine colonization of a broad range of common bacteria as well as different viruses and their role in the occurrence of endometritis.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan. There is no conflict of interest related to this study.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


A population-based case-control study of urinary bisphenol A concentrations and risk of endometriosis
<sec><st>STUDY QUESTION</st> <p>Is bisphenol A (BPA) exposure associated with the risk of endometriosis, an estrogen-driven disease of women of reproductive age?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Our study suggests that increased urinary BPA is associated with an increased risk of non-ovarian pelvic endometriosis, but not ovarian endometriosis.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>BPA, a high-volume chemical used in the polymer industry, has been the focus of public and scientific concern given its demonstrated estrogenic effects <I>in vivo</I> and <I>in vitro</I> and widespread human exposure. Prior studies of BPA and endometriosis have yielded inconsistent results and were limited by the participant sampling framework, small sample size or use of serum (which has very low/transient concentrations) instead of urine to measure BPA concentrations.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>We used data from the Women's Risk of Endometriosis study, a population-based case&ndash;control study of endometriosis, conducted among female enrollees of a large healthcare system in the US Pacific Northwest. Cases were women with incident, surgically confirmed endometriosis diagnosed between 1996 and 2001 and controls were women randomly selected from the defined population that gave rise to the cases, without a current or prior diagnosis of endometriosis.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTINGS, METHODS</st> <p>Total urinary BPA concentrations were measured in 143 cases and 287 population-based controls using single, spot urine samples collected after disease diagnosis in cases. Total urinary BPA concentration (free and conjugated species) was quantified using a high-performance liquid chromatography-mass spectrometry method. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression, adjusting for urinary creatinine concentrations, age and reference year. We also evaluated the association by disease subtypes, ovarian and non-ovarian pelvic endometriosis, that may be etiologically distinct.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>We did not observe a statistically significant association between total urinary BPA concentrations and endometriosis overall. We did observe statistically significant positive associations when evaluating total urinary BPA concentrations in relation to non-ovarian pelvic endometriosis (second versus lowest quartile: OR 3.0; 95% CI: 1.2, 7.3; third versus lowest quartile: OR 3.0; 95% CI: 1.1, 7.6), but not in relation to ovarian endometriosis.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Given the short elimination half-life of BPA, our study was limited by the timing of collection of the single urine sample, that occurred after case diagnosis. Thus, our BPA measurements may not accurately represent the participants' levels during the etiologically relevant time period for endometriosis development. In addition, since it was not feasible in this population-based study to surgically confirm the absence of disease, it is possible that some controls may have had undiagnosed endometriosis.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>By using population-based data, it is more likely that the controls represented the underlying frequency of BPA exposure in contrast to prior studies that used for comparison control women undergoing surgical evaluation, where the indication for surgery may be associated with BPA exposure. The significant associations observed in this study suggest that BPA may affect the normal dynamic structural changes of hormonally responsive endometrial tissue during the menstrual cycle, promoting the establishment and persistence of refluxed endometrial tissue in cases with non-ovarian pelvic endometriosis. Further research is warranted to confirm our novel findings in endometriosis subtypes that may be etiologically distinct.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTERESTS</st> <p>This work was supported by the National Institutes of Health, National Institute of Environmental Health Sciences (grant number R03 ES019976), the <I>Eunice Kennedy Shriver</I> National Institute of Child Health and Human Development (grant number R01 HD033792); US Environmental Protection Agency, Science to Achieve Results (STAR) (grant number R82943-01-0) and National Institute of Nursing Research (grant number F31NR013092) to KU for training support. This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institute of Child Health and Human Development, National Institute of Environmental Health Sciences, National Institute of Nursing Research or the National Institutes of Health. The authors have no actual or potential competing financial interests.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


A first-in-human study of PDC31 (prostaglandin F2{alpha} receptor inhibitor) in primary dysmenorrhea
<sec><st>STUDY QUESTION</st> <p>What is the safe and pharmacodynamically active dose range for PDC31 (prostaglandin F<SUB>2&alpha;</SUB> receptor inhibitor) in patients with primary dysmenorrhea (PD)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The 1 mg/kg/h dose of PDC31 appears to be safe and potentially effective in reducing intrauterine pressure (IUP) and pain associated with excessive uterine contractility when given as a 3-h infusion in patients with PD.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>PDC31 has previously been shown to reduce the duration and strength of PGF<SUB>2&alpha;</SUB>-induced contractions in human uterine myometrial strip models and to delay delivery in animal models of preterm labor.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This was a prospective, multi-center, dose-escalating first-in-human Phase I study conducted from March 2011 to June 2012. A total of 24 women with PD were enrolled and treated with one of five doses (0.01, 0.05, 0.15, 0.3, 0.5 and 1 mg/kg/h) of PDC31 given as a 3-h infusion. Patients were observed for a further 24 h.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>This study was conducted at four hospitals in Europe in non-pregnant, menstruating women with PD. Women with PD (<I>n</I> = 24) received PDC31 infused over 3 h within 8&ndash;10 h of the onset of menstruation. IUP and pain monitoring through the visual analog scale (VAS) was assessed prior to, during and following the infusion. Patients were observed for dose-limiting toxicities and other adverse events. Pharmacokinetic samples were also taken to profile the drug.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>A 3-h infusion of PDC31 was safe up to and including doses of 1 mg/kg/h. Most adverse events were mild (<I>n</I> = 15; 83.3%) and not considered associated with PDC31 (<I>n</I> = 14; 77.8%). PDC31 infusion decreased uterine activity based on IUP and pain (VAS) scores. IUP was decreased by 23% over all dose levels, reaching a minimum at 135&ndash;150 min. There appeared to be a dose-dependent effect on IUP, with the high dose group (1 mg/kg/h) showing the largest decrease in IUP. There was a statistically significant linear dose&ndash;effect and concentration&ndash;effect relationship for several IUP parameters over the evaluation period of 60&ndash;180 min. A dose differentiating effect on pain was seen with the two highest doses. PDC31 demonstrated uncomplicated, linear pharmacokinetics with a terminal half-life of ~2 h.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This was a first-in-human study and exposure to PDC31 was limited for safety reasons. As such, pharmacodynamic parameters were assessed at a two-sided Type I error of 20%, an appropriate level for the exploratory nature of this study without a placebo control arm. This limited the chance of false positive findings to one in five.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Like PD, preterm labor is associated with prostaglandin-mediated uterine contractions; therefore, the findings of this study support further development of PDC31 as a treatment for both PD and preterm labor.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was funded by PDC Biotech GmbH, Vienna, Austria. B.B., R.M.L., L.W., R.J.S., K.J.B. and C.F.S. received reimbursement for the conduct of this study from PDC Biotech GmbH. W.H., M.S. and R.P.S. are paid consultants for PDC Biotech GmbH. P.G. is a paid consultant and shareholder of PDC Biotech GmbH.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>NCT01250587 at <A HREF="www.clinicaltrials.gov">www.clinicaltrials.gov</A>.</p> </sec>


The effect of endometrial injury on ongoing pregnancy rate in unselected subfertile women undergoing in vitro fertilization: a randomized controlled trial
<sec><st>STUDY QUESTION</st> <p>Does endometrial injury in the cycle preceding ovarian stimulation for <I>in vitro</I> fertilization (IVF) improve the ongoing pregnancy rate in unselected subfertile women?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Endometrial injury induced by endometrial aspiration in the preceding cycle does not improve the ongoing pregnancy rate in unselected subfertile women undergoing IVF.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Implantation failure remains one of the major limiting factors for IVF success. Mechanical endometrial injury in the cycle preceding ovarian stimulation of IVF treatment has been shown to improve implantation and pregnancy rates in women with repeated implantation failures. There is limited data on unselected subfertile women, especially those undergoing their first IVF treatment.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This randomized controlled trial recruited 300 unselected subfertile women scheduled for IVF/ICSI treatment between March 2011 and August 2013. Subjects were randomized into endometrial aspiration (EA) (<I>n</I> = 150) and non-EA (<I>n</I> = 150) groups according to a computer-generated randomization list.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Subjects were recruited and randomized in the assisted reproductive unit at the University of Hong Kong. In the preceding cycle, women in the EA group underwent endometrial aspiration using a Pipelle catheter in mid-luteal phase. All women were treated with a cycle of IVF/ICSI. Pregnancy outcomes were compared.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>There were no significant differences in baseline or cycle characteristics between the groups. There were 209 subjects (69.7%) who were undergoing their first IVF cycle and 91 (30.3%) subjects who had repeated cycles. There was no significant difference in ongoing pregnancy rates [26.7% (40/150) versus 32.0% (48/150); RR 0.833 (95% CI 0.585&ndash;1.187), <I>P</I> = 0.375] in the EA and non-EA groups. The implantation rates [32.8% (67/204) versus 29.7% (68/229); RR 1.080 (95% CI 0.804&ndash;1.450), <I>P</I> = 0.120], clinical pregnancy rates [34.0% (51/150) versus 38.0 (57/150); RR 0.895 (95% CI 0.661&ndash;1.211), <I>P</I> = 0.548], miscarriage rates [30.3% (17/56) versus 18.6% (11/59), RR 1.628 (95% CI 0.838&ndash;3.164), <I>P</I> = 0.150] and multiple pregnancy rates [31.3% (16/51) versus 19.3% (11/57), RR 1.626 (95% CI 0.833&ndash;3.172), <I>P</I> = 0.154] were all comparable between the EA and non-EA groups. Subgroup analysis in women having first embryo transfer (<I>n</I> = 209) also demonstrated no significant difference in ongoing pregnancy rates, but for women undergoing repeated cycles (<I>n</I> = 91), the on-going pregnancy rate was significantly lower in the EA group than in the non-EA group.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The study aimed at assessing an unselected population of subfertile women by recruiting consecutive women attending our fertility clinic. However, since the majority of the recruited women (69.7%) were having their first IVF treatments, the results may not be generalizable to all women undergoing IVF.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Previous RCTs and meta-analyses have suggested improved pregnancy rates after pretreatment endometrial injury in women with repeated implantation failure. A recent RCT also showed increased pregnancy rates in unselected subfertile women after endometrial injury, although that study was terminated early and thus underpowered. Our study showed with adequate power that no significant improvement in pregnancy rates was observed after endometrial injury in unselected women undergoing IVF treatment.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was supported by the Small Project Funding 201309176012 of the Committee on Research and Conference Grants, University of Hong Kong. The authors have nothing to disclose.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>HKCTR-1646 and NCT 01977976.</p> </sec>


How long should we continue clomiphene citrate in anovulatory women?
<sec><st>STUDY QUESTION</st> <p>What is the effectiveness of continued treatment with clomiphene citrate (CC) in women with World Health Organization (WHO) type II anovulation who have had at least six ovulatory cycles with CC but did not conceive?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>When women continued CC after six treatment cycles, the cumulative incidence rate of the ongoing pregnancy rate was 54% (95% CI 37&ndash;78%) for cycles 7&ndash;12.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>If women with WHO type II anovulation fail to conceive with CC within six ovulatory cycles, guidelines advise switching to gonadotrophins, which have a high risk of multiple gestation and are expensive. It is however not clear what success rate could be achieved by continued treatment with CC.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>We performed a retrospective cohort study of women with WHO II anovulation who visited the fertility clinics of five hospitals in the Netherlands between 1994 and 2010. We included women treated with CC who had had at least six ovulatory cycles without successful conception (<I>n</I> = 114) after which CC was continued using dosages varying from 50 to 150 mg per day for 5 days.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Follow-up was a total of 12 treatment cycles. Primary outcome was the cumulative incidence rate of an ongoing pregnancy at the end of treatment.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>We recruited 114 women that had ovulated on CC for at least six cycles but had not conceived. Of these 114 women, 35 (31%) had an ongoing pregnancy resulting in a cumulative incidence rate of an ongoing pregnancy of 54% after 7&ndash;12 treatment cycles with CC.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Limitations of our study are its retrospective approach.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Randomized trials comparing continued treatment with CC with the relatively established second line treatment with gonadotrophins are justified. In the meantime, we suggest to only begin this less convenient and more expensive treatment for women who do not conceive after 12 ovulatory cycles with CC.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>None.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


Parent psychological adjustment, donor conception and disclosure: a follow-up over 10 years
<sec><st>STUDY QUESTION</st> <p>What is the relationship between parent psychological adjustment, type of gamete donation (donor insemination, egg donation) and parents' disclosure of their use of donated gametes to their children.</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Disclosure of donor origins to the child was not always associated with optimal levels of psychological adjustment, especially for fathers in donor insemination families.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Cross-sectional analyses have found mothers and fathers who conceived a child using donated sperm or eggs to be psychologically well-adjusted, with few differences emerging between parents in gamete donation families and parents in families in which parents conceived naturally. The relationship between mothers' and fathers' psychological well-being, type of gamete donation (donor insemination, egg donation) and parents' disclosure decisions has not yet been examined.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>In this follow-up study, data were obtained from mothers and fathers in donor insemination and egg donation families at 5 time points; when the children in the families were aged 1, 2, 3, 7 and 10. In the first phase of the study, 50 donor insemination families and 51 egg donation families with a 1-year-old child participated. By age 10, the study included 34 families with a child conceived by donor insemination and 30 families with a child conceived by egg donation, representing 68 and 58% of the original sample, respectively.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Families were recruited through nine fertility clinics in the UK. Standardized questionnaires assessing depression, stress and anxiety were administered to mothers and fathers in donor insemination and egg donation families.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Mothers and fathers in both donor insemination and egg donation families were found to be psychologically well-adjusted; for the vast majority of parents' levels of depression, anxiety and parenting stress were found to be within the normal range at all 5 time points. Disclosure of the child's donor origins to the child was not always associated with optimal levels of parental psychological adjustment. For example, disclosure was associated with lower levels of psychological well-being for certain groups in particular (such as fathers in donor insemination families), at certain times (when children are in middle childhood and have a more sophisticated understanding of their donor origins).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Owing to small sample sizes, the value of this study lies not in its generalizability, but in its potential to point future research in new directions.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Donor insemination and egg donation families are a heterogeneous group, and future research should endeavour to obtain data from fathers as well as mothers. Support and guidance in terms of disclosure and family functioning might be most beneficial for parents (and especially fathers) in donor insemination families, particularly as the child grows older. The more that is known about the process of disclosure over time, from the perspective of the different members of the family, the better supported parents and their children can be.</p> </sec> <sec><st>STUDY FUNDING COMPETING INTEREST(S)</st> <p>The project described was supported by grant number RO1HD051621 from the National Institute of Child Health and Human Development. The content is solely the responsibility of the authors and does not represent the official views of the National Institute of Child Health and Human Development or the National Institutes of Health. The authors have no conflict of interest to declare.</p> </sec>


Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture
<sec><st>STUDY QUESTION</st> <p>Can human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Germ cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1&ndash;2 weeks) but not late (&gt; 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Testicular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Immunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured <I>in vitro</I> for 1&ndash;2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (<I>UTF1</I>, <I>FGFR3</I>, <I>ZBTB16</I>, <I>GPR125, DAZL, GATA4</I> and <I>VIM</I>) and flow cytometry (CD9 and SSEA4).</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p><I>UTF1</I>, <I>FGFR3, DAZL and ZBTB16</I> qRT&ndash;PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, <I>GPR125</I> mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1&ndash;2 weeks), their expression steadily declined over time <I>in vitro</I>. After 6 weeks in culture only somatic cells were detected.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Different groups attempting SSC culture have utilized different sources of human testes and minor differences in the preparation and maintenance of the testicular cell cultures. Differences in outcome may be explained by genetic background of the source tissue or technical differences.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The ability to propagate human SSCs <I>in vitro</I> is a prerequisite for proposed autologous transplantation therapy aimed at restoring fertility to men who have been treated for childhood cancer. By applying the assays validated here it will be possible to quantitatively compare human SSC culture conditions. The eventual development of conditions for long-term propagation of human SSCs <I>in vitro</I> will greatly facilitate learning about the basic biology of these cells and in turn the ability to use human SSCs in therapy.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The experiments presented in this manuscript were funded by a Project Development Team within the ICTSI <grant-sponsor id="cs1" refid="cn1">NIH/NCRR</grant-sponsor><?release-delay 12|0?> Grant Number <grant-num id="cn1" refid="cs1">TR000006</grant-num>. The authors declare no competing interests.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


Transgenerational impaired male fertility with an Igf2 epigenetic defect in the rat are induced by the endocrine disruptor p,p'-DDE
<sec><st>STUDY QUESTION</st> <p>What are the epigenetic mechanisms underlying the transgenerational effect of <I>p</I>,<I>p</I>'-DDE on male fertility?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Impaired male fertility with an <I>Igf2</I> epigenetic defect is transgenerationally inherited upon exposure of <I>p</I>,<I>p</I>'-DDE.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p><I>p</I>,<I>p</I>'-Dichlorodiphenoxydichloroethylene (<I>p</I>,<I>p</I>'-DDE) is one of the primary metabolite products of the ancestral organochlorine pesticide dichlorodiphenoxytrichloroethane. As it is a known anti-androgen endocrine disruptor, it could cause harmful effects on the male reproductive system.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Pregnant rats (F0) were administered with <I>p</I>,<I>p</I>'-DDE or corn oil at the critical time of testis development, i.e. from gestation days 8 to 15. Male and female rats of the F1 generation were mated with each other to produce F2 progeny. To reveal whether the transgenerational phenotype is produced by the maternal or paternal line, F3 progeny were generated by intercrossing control (C) and treated (DDE) males and females of the F2 generation according to the following groups: (i) C-C, (ii) DDE-DDE, (iii) DDE-C and (iv) C-DDE.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Mature sperm and testes were collected from male offspring of the F1&ndash;F3 generations for the examination of male fertility parameters, i.e. sperm count and motility, testis histology and apoptosis. Expression of the imprinted genes, <I>H19</I> and <I>Igf2</I>, was detected by real-time PCR. <I>Igf2</I> DMR2 methylation was analyzed by bisulfite genomic sequencing.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Upon exposure of <I>p</I>,<I>p</I>'-DDE, the male F1 generation showed impaired male fertility and altered imprinted gene expression caused by <I>Igf2</I> DMR2 hypomethylation. These defects were transferred to the F3 generation through the male germline.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>This study has examined the effect of <I>p</I>,<I>p</I>'-DDE only on the sperm number and motility and the possible mechanism of <I>Igf2</I> DMR2 methylation <I>in vivo</I> and thus has some limitations. Further investigation is necessary to focus on the epigenetic effects of <I>p</I>,<I>p</I>'-DDE at the genome level and to include a more detailed semen quality analysis including sperm morphology assessment.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Impaired male fertility with epigenetic alterations is transgenerationally inherited after environmental exposure of <I>p</I>,<I>p</I>'-DDE, posing significant implications in the etiology of male infertility.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The present research was supported by National Natural Science Fund for Young Scholar (81102161), the Natural Science Fund of Zhejiang Province (LY14H260004) and funding from the Health Department of Zhejiang Province (201475777). No competing interests are declared.</p> </sec>


Lipid profiles and ovarian reserve status: a longitudinal study
<sec><st>STUDY QUESTION</st> <p>Is there any association between ovarian reserve status and lipid profile changes?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Women with lower ovarian reserve might be susceptible to higher cardiovascular risks, especially lipid disturbances, even during their reproductive life span.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>The risk of developing cardiovascular disease (CVD) in women increases after menopause, but the association between ovarian reserve status and CVD is not known.</p> </sec> <sec><st>STUDY DESIGN, SIZE AND DURATION</st> <p>This longitudinal study was conducted on 1015 participants of Tehran Lipid and Glucose Study, an ongoing population based cohort study with 12 years follow-up.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>There were 1015 women who were aged 20&ndash;50 years and met our eligibility criteria. Their ovarian reserve status was identified according to their age-specific AMH levels, calculated using the exponential&ndash;normal 3-parameter model. At the time of recruitment, 268, 233, 256 and 258 subjects were in the first, second, third and fourth quartiles of age-specific AMH, respectively. The cardiovascular risk factors of these groups were compared.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Anthropometric measurements, lipid profiles and mean systolic and diastolic blood pressures in the first and fourth AMH quartiles did not differ at the initiation of the study. Total cholesterol (TC) net changes per year were incremental in the first AMH quartile but not in the fourth quartile (<I>P</I> &lt; 0.001). According to the generalized estimating equation (GEE), after adjustment for age, BMI, time interaction and menopause status, the changes across time in TC, LDL and HDL were varied according to the age-specific AMH status.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>A potential limitation is that development of cardiovascular risk is a major long-term event that needs decades of follow-up from birth to death; hence further studies with longer follow-up times are needed.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>We provide the insight that women with lower ovarian reserve might be susceptible to developing cardiovascular risk factors, particularly lipid disturbances, even during their reproductive life span.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences. The authors report no conflict of interest.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>Not applicable.</p> </sec>


The ovarian response to controlled stimulation in IVF cycles may be predictive of the age at menopause
<sec><st>STUDY QUESTION</st> <p>Can the number of oocytes retrieved in IVF cycles be predictive of the age at menopause?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>The number of retrieved oocytes can be used as an indirect assessment of the extent of ovarian reserve to provide information on the duration of the reproductive life span in women of different ages.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Menopause is determined by the exhaustion of the ovarian follicular pool. Ovarian reserve is the main factor influencing ovarian response in IVF cycles. As a consequence the response to ovarian stimulation with the administration of gonadotrophins in IVF treatment may be informative about the age at menopause.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>In the present cross-sectional study, participants were 1585 infertile women from an IVF clinic and 2635 menopausal women from a more general population.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>For all infertile women, the response to ovarian stimulation with gonadotrophins was recorded. For menopausal women, relevant demographic characteristics were available for the analysis.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>A cubic function described the relationship between mean numbers of oocytes and age, with all terms being statistically significant. From the estimated residual distribution of the actual number of oocytes about this mean, a distribution of the age when there would be no oocytes retrieved following ovarian stimulation was derived. This was compared with the distribution of the age at menopause from the menopausal women, showing that menopause occurred about a year later.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The retrieved oocyte data were from infertile women, while the menopausal ages were from a more general population.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>In the present study, we have shown some similarity between the distributions of the age when no retrieved oocytes can be expected after ovarian stimulation and the age at menopause. For a given age, the lower the ovarian reserve, the lower the number of retrieved oocytes would be and the earlier the age that menopause would occur.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was supported by a grant from the Italian Ministry of Health (GR-2009-1580036). There are no conflicts of interest.</p> </sec>


Using cluster analysis to identify a homogeneous subpopulation of women with polycystic ovarian morphology in a population of non-hyperandrogenic women with regular menstrual cycles
<sec><st>STUDY QUESTION</st> <p>Can cluster analysis can be used to identify a homogeneous subpopulation of women with polycystic ovarian morphology (PCOM) within a very large population of control women in a non-subjective way?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Identification and exclusion of the cluster corresponding to women with PCOM from controls improved the diagnostic power of serum anti-M&uuml;llerian hormone (AMH) level and follicle number per ovary (FNPO) in discriminating between women with or without polycystic ovary syndrome (PCOS).</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>There is disagreement as to whether women with PCOM should be excluded from the control population when establishing FNPO and AMH diagnostic thresholds for the definition of PCOS and how to identify such women. It has been demonstrated that cluster analysis can detect women with PCOM within the control population through a set of classifying variables among which the most relevant was AMH. The adequacy of this approach has not been confirmed in other clinical settings.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This was a retrospective study using clinical and laboratory data derived from the computerized database. The data were collected from March 2011 to May 2013.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>The study included 893 patients referred for routine infertility evaluation and treatment. The patients were divided into three groups: (i) the control group (<I>n</I> = 621) included women with regular menstrual cycles and no signs of hyperandrogenism (HA), (ii) the full-blown PCOS group (<I>n</I> = 95) consisted of women who were diagnosed as having PCOS based on the presence of both HA and oligo/amenorrhoea (OA), (iii) the mild PCOS group included women with only two items of the Rotterdam classification, i.e. PCOM at ultrasonography according to the FNPO threshold of 12 or more and either OA (<I>n</I> = 110) or HA (<I>n</I> = 67).</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>After exclusion of women with PCOM from the controls, the AMH threshold of 28 pmol/l with specificity 97.5% and sensitivity 84.2% [area under the curve (AUC) 0.948 (95% confidence interval (CI) 0.915&ndash;0.982)] and FNPO threshold of 12 with specificity 92.5% and sensitivity 83.2% [AUC 0.940 (95% CI 0.909&ndash;0.971)] for identifying PCOS were derived from the receiver operating characteristic curve analysis. The AMH threshold of 28 pmol/l had the same specificity for discriminating the mild and the full-blown PCOS phenotypes from controls.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>There was no selection bias other than being evaluated for infertility treatment, however, this could also be considered as a limitation of the study as these women may not necessarily be a representative sample of the general population. The study demonstrated that serum AMH has intrinsically a very high potency to detect women with PCOM within a control group. However, the AMH threshold is specific for this method and clinical setting.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>AMH threshold value for the definition of PCOM is method specific and cannot be universally applied. This study confirmed the results of previous studies that cluster analysis can identify women with PCOM within the very large control population without using a predefined diagnostic threshold for FNPO, and that AMH can detect women with PCOM with a high specificity. The exclusion of PCOM women from the controls by using a cluster analysis should be considered when establishing reference intervals and decision threshold values for various parameters used to characterize PCOS.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The authors have no funding<b>/</b>competing interest(s) to declare.</p> </sec>


Short-term changes in hormonal profiles after laparoscopic ovarian laser evaporation compared with diagnostic laparoscopy for PCOS
<sec><st>STUDY QUESTION</st> <p>Which reproductive endocrine changes are attributed exclusively to laparoscopic ovarian drilling in polycystic ovarian syndrome (PCOS)?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Laser evaporation-specific endocrine effects were the prevention of an immediate increase in inhibin B and a sustained decrease in testosterone, androstenedione and anti-M&uuml;llarian hormone (AMH).</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>All ovarian drilling procedures result in reproductive endocrine changes. It is not known which of these changes are the result of ovarian drilling and which are related to the surgery <I>per se</I>.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This prospective controlled study was performed at an outpatient academic fertility clinic. Between 2007 and 2010, a total of 21 oligo- or amenorrheic PCOS patients were included.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Included were oligo- or amenorrheic PCOS patients with all three of the Rotterdam criteria and luteinizing hormone (LH) &gt;6.5 U/l. All PCOS patients had an indication for diagnostic surgery due to subfertility. There were 12 PCOS patients who chose to undergo ovarian laser evaporation (CO<SUB>2</SUB> laser, 25 W, 20 times/ovary) and 9 PCOS who chose a diagnostic laparoscopy only (controls). Reproductive endocrinology was measured before, and until 5 days after, surgery, and four gonadotrophin-releasing hormone (GnRH) &lsquo;double pulse&rsquo; tests were included. The main outcome measures were changes in reproductive endocrinology and pituitary sensitivity/priming to GnRH after laser evaporation compared with diagnostic laparoscopy only.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>In the first hours after surgery, both groups showed an increase in LH, follicle stimulating hormone, estrogen and a decrease in testosterone, androstenedione, AMH and insulin growth factor-1 (<I>P</I> &lt; 0.05). Inhibin B increased in the laparoscopy only group (<I>P</I> &lt; 0.05). In the first days after surgery, testosterone, androstenedione and AMH remained at lower than baseline levels exclusively in the laser group (<I>P</I> &lt; 0.05). Pituitary sensitivity/priming to GnRH was not altered after either laser evaporation or laparoscopy only.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The limitations of this study are the short follow-up period and the relatively small groups.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>The strength of this study is the integrally measured endocrine profiles in combination with an optimal control group of PCOS patients undergoing diagnostic laparoscopy only. Interestingly, most of the immediate endocrine changes after laser evaporation could be related to the surgical context and not to the ovarian drilling procedure itself.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The study was funded by the Foundation of Scientific Research in Obstetrics and Gynaecology and the study medication, Lutrelef, was donated by Ferring, The Netherlands, Hoofdorphe There were no conflicts of interests mentioned by the authors.</p> </sec>


Successive time to pregnancy among women experiencing pregnancy loss
<sec><st>STUDY QUESTION</st> <p>Is time to pregnancy (TTP) similar across successive pregnancy attempts among women experiencing pregnancy loss?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>TTP after a loss may be longer compared with TTP before a loss.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Two pregnancy cohort studies have reported that TTP is similar across pregnancy attempts in fertile women. However, this has not been investigated among women experiencing pregnancy losses.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>Data for this analysis come from the Longitudinal Investigation of Fertility and the Environment Study, a population-based, preconception cohort of couples attempting pregnancy. During 2005&ndash;2009, recruitment was targeted to 16 counties in Michigan and Texas with reported exposures to persistent environmental chemicals. A total of 501 couples were recruited and followed for up to 12 months of pregnancy attempts allowing for continued participation of women with pregnancy losses until censoring.</p> </sec> <sec><st>PARTICIPANTS, SETTING, METHODS</st> <p>We assessed TTP among 70 couples recruited upon discontinuing contraception for purposes of becoming pregnant and experiencing &ge;1 prospectively observed pregnancy losses during 12 months of trying. There were 61 couples who contributed two pregnancy attempts and 9 who contributed three. Women were instructed in the use of urine-based home fertility monitors to time intercourse relative to ovulation and recorded their bleeding patterns in daily journals. TTP was defined as the number of menstrual cycles taken to achieve pregnancy. Women were also instructed in the use of home digital pregnancy tests and asked to begin pregnancy testing on the day of expected menses. Women recorded the results of their pregnancy tests in a daily journal with a single positive pregnancy test result indicating an hCG-confirmed pregnancy. Pregnancy losses were ascertained from a subsequent recorded negative pregnancy test or clinically confirmed loss. We estimated fecundability odds ratios (FORs) comparing subsequent to first TTP using discrete Cox models with robust standard errors, accounting for cycles off contraception before study entry and adjusting for maternal age, body mass index, reproductive history and time-varying cigarette, alcohol and caffeine usage while trying.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>The mean female age was 30.3 &plusmn; 4.3 years; 21% had a prior pregnancy loss before study entry. Of the second and third attempts, 59 and 43%, respectively, were longer compared with the first attempt. FORs &lt;1 suggest reduced fecundability or a longer TTP when comparing the second with the first attempt (0.42, 95% confidence interval (CI): 0.28, 0.65), and similarly for the third relative to the first attempt (0.64, 95% CI: 0.18, 2.36). TTP in the second attempt was a median of 1 cycle longer (interquartile range: 0, 3 cycles) compared with TTP in the first attempt.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>As this is the first study to investigate successive TTP exclusively among women experiencing pregnancy loss, our findings await corroboration since most losses occurred early in gestation. As such, the generalizability of our findings for all pregnancy losses awaits further research. We also had limited power to detect a reduction in fecundability for the third compared with first pregnancy attempt.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Unlike fertile women, TTP in women experiencing early pregnancy losses may trend towards longer subsequent attempts. If the findings are corroborated, women experiencing losses may benefit from counselling regarding trying times.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTERESTS</st> <p>This research was supported by the Intramural Research Program of the <I>Eunice Kennedy Shriver</I> National Institute of Child Health and Human Development (contracts N01-HD-3-3355, N01-HD-3-3356 and NOH-HD-3-3358). K.J.S. was supported by an Intramural Research Training Award from the <I>Eunice Kennedy Shriver</I> National Institute of Child Health and Human Development, Division of Intramural Population Health Research. The authors have no conflicts of interest to declare.</p> </sec>


Does a Caesarean section increase the time to a second live birth? A register-based cohort study
<sec><st>STUDY QUESTION</st> <p>Does a primary Caesarean section influence the rate of, and time to, subsequent live birth compared with vaginal delivery?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Caesarean section was associated with a reduction in the rate of subsequent live birth, particularly among elective and maternal-requested Caesareans indicating maternal choice plays a role.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>Several studies have examined the relationship between Caesarean section and subsequent birth rate with conflicting results primarily due to poor epidemiological methods.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This Danish population register-based cohort study covered the period from 1982 to 2010 (<I>N</I> = 832 996).</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>All women with index live births were followed until their subsequent live birth or censored (maternal death, emigration or study end) using Cox regression models.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>In all 577 830 (69%) women had a subsequent live birth. Women with any type of Caesarean had a reduced rate of subsequent live birth (hazard ratio [HR] 0.86, 95% confidence intervals [CI] 0.85, 0.87) compared with spontaneous vaginal delivery. This effect was consistent when analyses were stratified by type of Caesarean: emergency (HR 0.87, 95% CI 0.86, 0.88), elective (HR 0.83, 95% CI 0.82, 0.84) and maternal-requested (HR 0.61, 95% CI 0.57, 0.66) and in the extensive sub-analyses performed.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Lack of biological data to measure a woman's fertility is a major limitation of the current study. Unmeasured confounding and limited availability of data (maternal BMI, smoking, access to fertility services and maternal-requested Caesarean section) as well as changes in maternity care over time may also influence the findings.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This is the largest study to date and shows that Caesarean section is most likely not causally related to a reduction in fertility. Maternal choice to delay or avoid childbirth is the most plausible explanation. Our findings are generalizable to other middle- to high-income countries; however, cross country variations in Caesarean section rates and social or cultural differences are acknowledged.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>Funding was provided by the National Perinatal Epidemiology Centre, Cork, Ireland and conducted as part of the Health Research Board PhD Scholars programme in Health Services Research (Grant No. PHD/2007/16). L.C.K. is a Science Foundation Ireland Principal Investigator (08/IN.1/B2083) and the Director of the SFI funded Centre, INFANT (12/RC/2272). The authors have no competing interests to declare.</p> </sec>


Mode of delivery and subsequent fertility
<sec><st>STUDY QUESTION</st> <p>When compared with vaginal delivery, is Cesarean delivery associated with reduced childbearing, a prolonged inter-birth interval or infertility?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Women whose first delivery was by Cesarean section were not significantly different from those who delivered vaginally with respect to subsequent deliveries, inter-birth interval or infertility after delivery.</p> </sec> <sec><st>WHAT IS ALREADY KNOWN</st> <p>Some studies have suggested that delivery by Cesarean section reduces subsequent fertility, while others have reported no association.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This was a planned secondary analysis of the Mothers' Outcomes After Delivery study, a longitudinal cohort study. This analysis included 956 women with 1835 deliveries, who completed a study questionnaire at 6&ndash;11 years (median [interquartile range]: 8.1 [7.1, 9.8]) after their first delivery.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Exclusion criteria regarding the first birth were: maternal age &lt;15 or &gt;50 years, delivery at &lt;37 weeks gestation, placenta previa, multiple gestation, known fetal congenital abnormality, stillbirth, prior myomectomy and abruption. Of the 956 women included, the first delivery was by Cesarean section for 534 women and by vaginal birth for 422 women. Infertility was self-reported. To compare maternal characteristics by mode of first delivery, <I>P</I>-values were calculated using Fisher's exact test or Pearson's <I></I><sup>2</sup> test for categorical variables and a Kruskall&ndash;Wallis test for continuous variables. We also considered whether, across all deliveries to date, a prior Cesarean is associated with decreased fertility. In this analysis, self-reported infertility after each delivery (across all participants) was considered as a function of one or more prior Cesarean births, using generalized estimating equations to control for within-woman correlation.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>No differences were observed between the Cesarean and vaginal groups (for first delivery) with respect to infertility after their most recent delivery (7 versus 6%, <I>P</I> = 0.597), the interval between their first and second births (30.8 versus 30.6 months, <I>P</I> = 0.872), or multiparity (75 versus 76%, <I>P</I> = 0.650). Across all births, a history of Cesarean delivery was not significantly associated with infertility (odds ratio [OR], 0.90; 95% confidence interval [CI], 0.64&ndash;1.26). Women who reported infertility prior to their first delivery were significantly more likely to report infertility after each subsequent delivery (OR, 5.16; 95% CI, 3.60&ndash;7.39).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>Due to the use of self-reported infertility, the fertility status of some participants may be misclassified. Also, the small sample size may result in insufficient power to detect small differences between groups. Finally, a relatively high proportion of our participants were over age 35 at the time of first delivery (26%) and highly educated (37% with graduate degrees), which may indicate that our population may not be generalizable.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>While some prior studies have shown decreased family size among women who deliver by Cesarean, our results suggest that the rate of infertility is not different after Cesarean compared with vaginal birth. Our findings should be reassuring to women who deliver by Cesarean section.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This study was funded by the US National Institutes of Health (NIH, R01-HD056275). No competing interests are declared.</p> </sec> <sec><st>TRIAL REGISTRATION NUMBER</st> <p>N/A.</p> </sec>


Paternal physical and sedentary activities in relation to semen quality and reproductive outcomes among couples from a fertility center
<sec><st>STUDY QUESTION</st> <p>Is paternal physical activity associated with semen quality parameters and with outcomes of infertility treatment?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Among men presenting for infertility treatment, weightlifting and outdoor activities were associated with higher sperm concentrations but not with greater reproductive success.</p> </sec> <sec><st>WHAT IS ALREADY KNOWN</st> <p>Higher physical activity is related to better semen quality but no studies to date have investigated whether it predicts greater reproductive success.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>The Environment and Reproductive Health (EARTH) Study is an on-going prospective cohort study which enrolls subfertile couples presenting at Massachusetts General Hospital (2005&ndash;2013). In total, 231 men provided 433 semen samples and 163 couples underwent 421 IVF or intrauterine insemination cycles.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Leisure time spent in physical and sedentary activities over the past year was self-reported using a validated questionnaire. We used mixed models to analyze the association of physical and sedentary activities with semen quality and with clinical pregnancy and live birth rates.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Men in this cohort engaged in a median of 3.2 h/week of moderate-to-vigorous activities. Men in the highest quartile of moderate-to-vigorous activity had 43% (95% confidence interval (CI) 9, 87%) higher sperm concentrations than men in the lowest quartile (<I>P</I>-trend = 0.04). Men in the highest category of outdoor activity (&ge;1.5 h/week) and weightlifting (&ge;2 h/week) had 42% (95% CI 10, 84%) and 25% (95% CI &ndash;10, 74%) higher sperm concentrations, respectively, compared with men in the lowest category (0 h/week) (<I>P</I>-trend = 0.04 and 0.02). Conversely, men who reported bicycling &ge;1.5 h/week had 34% (95% CI 4, 55%) lower sperm concentrations compared with men who reported no bicycling (<I>P</I>-trend = 0.05). Paternal physical and sedentary activities were not related to clinical pregnancy or live birth rates following infertility treatment.</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>The generalizability of the findings on live birth rates to populations not undergoing infertility treatment is limited.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>Certain types of physical activity, specifically weightlifting and outdoor activities, may improve semen quality but may not lead to improved success of infertility treatments. Further research is needed in other non-clinical populations.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>The authors are supported by NIH grants R01-ES009718, ES000002, P30-DK046200, T32-DK007703-16 and ES022955 T32-HD060454. None of the authors has any conflicts of interest to declare.</p> </sec>


The phenotype of an IVF child is associated with peri-conception measures of follicular characteristics and embryo quality
<sec><st>STUDY QUESTION</st> <p>Are childhood measures of phenotype associated with peri-conception parental, IVF treatment and/or embryonic characteristics of IVF children?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Birthweight, childhood body mass index (BMI) and height of pre-pubertal IVF children were strongly associated with peri-conception factors, including follicular and embryonic characteristics.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>A growing number of studies have identified a range of phenotypic differences between IVF and naturally conceived pre-pubertal children; for example, birthweights are lower following a fresh compared with a thawed embryo transfer.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This retrospective cohort study included IVF children (<I>n</I> = 96) born at term (&gt;37 weeks) after a singleton pregnancy from the transfer of either fresh or thawed embryos in New Zealand. Between March 2004 and November 2008, these children were subjected to clinical assessment before puberty.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Clinical assessment provided anthropometric measures of children aged 3.5&ndash;11 years old. Peri-conception factors (<I>n</I> = 36) derived retrospectively from parental, treatment, laboratory and embryonic variables (<I>n</I> = 69) were analysed using multiple stepwise regression with respect to standard deviation scores (SDSs) of the birthweight, mid-parental corrected BMI and height of the IVF children. Data from children conceived from fresh (<I>n</I> = 60) or thawed (<I>n</I> = 36) embryos, that met inclusion criteria and had high-quality data with &gt;90% completeness, were analysed.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>Embryo treatment at transfer was identified as a predictor of birthweight with thawed embryos resulting in heavier birthweights than fresh embryos [<I>P</I> = 0.02, 95% confidence interval (CI) fresh minus thawed: &ndash;1.047 to &ndash;0.006]. Birthweight SDS was positively associated with mid-parental corrected BMI SDS (<I>P</I> = 0.003, slope 0.339 &plusmn; 0.100). Four factors were related (<I>P</I> &lt; 0.05) to mid-parental corrected height SDS. In particular, child height was inversely associated with the diameter of lead follicles at oocyte retrieval (<I>P</I> &lt; 0.0001, slope &ndash;0.144 &plusmn; 0.040) and with the quality score of embryos at transfer (<I>P</I> = 0.0008, slope &ndash;0.425 &plusmn; 0.157), and directly associated with the number of follicles retrieved (<I>P</I> = 0.05, slope 1.011 &plusmn; 0.497). Child height was also positively associated with the transfer of a fresh as opposed to thawed embryo (<I>P</I> &lt; 0.001, 95% CI 0.275&ndash;0.750).</p> </sec> <sec><st>LIMITATIONS, REASONS FOR CAUTION</st> <p>More than one embryo was transferred in most cycles so mean development and quality data were used. The large number of variables measured was on a relatively small sample size. Large cohorts from multiple clinics using a variety of treatment protocols and embryology methods are needed to confirm the associations identified and ultimately to test these factors as possible predictors of phenotype.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE FINDINGS</st> <p>This is the first study to directly associate peri-conception measures of IVF treatment with a pre-pubertal child's phenotype. Demonstration that peri-conception measures relate to a pre-pubertal child's phenotype extends the range of factors that may influence growth and development. These findings, if corroborated by larger studies, would provide invaluable information for practitioners, who may want to consider the impact of ovarian stimulation protocols as well as the quality of the embryo transferred on a child's growth and development, in addition to their impact on pregnancy rate.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST(S)</st> <p>This work was supported by grants from the National Research Centre of Growth and Development New Zealand (grant 3682065) and the Australasian Paediatric Endocrine Group (APEG; grant 3621994), as well as a fellowship from Fertility Associates New Zealand awarded to M.P.G. In terms of competing interest, J.C.P is a shareholder of Fertility Associates. M.P.G. currently holds the position of Merck Serono Lecturer in Reproductive Biology. W.S.C. and P.L.H. have also received grants and non-financial support from Novo Nordisk, as well as personal fees from Pfizer that are unrelated to the current study. The other authors have no conflict of interest to declare.</p> </sec>


Severe teenage acne and risk of endometriosis
<sec><st>STUDY QUESTION</st> <p>Is there a relationship between severe teenage acne and endometriosis?</p> </sec> <sec><st>SUMMARY ANSWER</st> <p>Endometriosis is positively associated with severe teenage acne.</p> </sec> <sec><st>WHAT IS KNOWN ALREADY</st> <p>No studies have specifically explored a possible association between severe acne in adolescence and risk of endometriosis.</p> </sec> <sec><st>STUDY DESIGN, SIZE, DURATION</st> <p>This prospective cohort study used data collected from 88 623 female nurses from September 1989 to June 2009 as part of the Nurses' Health Study II (NHS II) cohort.</p> </sec> <sec><st>PARTICIPANTS/MATERIALS, SETTING, METHODS</st> <p>Regression models were used to calculate hazard ratios (HRs) and confidence intervals (CIs) for endometriosis among women with and without severe teenage acne. Multivariate models were adjusted for established risk factors of endometriosis.</p> </sec> <sec><st>MAIN RESULTS AND THE ROLE OF CHANCE</st> <p>A total of 4 382 laparoscopically confirmed endometriosis cases were documented during 1 132 272 woman-years of follow-up. Compared with women without a history of severe teenage acne, women who had severe teenage acne had a 20% increased risk of endometriosis (HR = 1.20, 95% CI: 1.08&ndash;1.32). The association was not affected by adjusting for use of tetracycline or isotretinoin.</p> </sec> <sec><st>LIMITATIONS AND REASONS FOR CAUTION</st> <p>The HR is likely to be underestimated since we only included endometriosis cases confirmed by laparoscopy. Although geographically diverse, the NHS II cohort is primarily Caucasian, which may limit generalization to more ethnically diverse populations.</p> </sec> <sec><st>WIDER IMPLICATIONS OF THE STUDY</st> <p>The results of this study suggest that severe teenage acne is associated with an increased risk of endometriosis. As a visible and non-invasive clinical indicator, severe teenage acne may be useful for early detection of endometriosis. We bring this counter-intuitive association to the attention of clinicians for the benefit of the patient and an early diagnosis of endometriosis.</p> </sec> <sec><st>STUDY FUNDING/COMPETING INTEREST</st> <p>This study was funded by research grant CA176726 from the National Institute of Health. M.K. is supported by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (#PIOF-GA-2011-302078). The funding agencies had no role in the design of the study, in the analysis and interpretation of the data, in the writing of the report or in the decision to submit the paper for publication.</p> </sec>


Implication of the liberal use of ART in Nordic countries: should stricter guidelines be created to prevent unnecessary stillbirth and preterm delivery?


Reply: Implication of the liberal use of ART in Nordic countries: should stricter guidelines be created to prevent unnecessary stillbirth and preterm delivery?