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Este mes en... Human Reproduction

Sumarios de Revistas

Este mes en... Human Reproduction:

  • Equipoise and the RCT

    Clinical decisions in reproductive medicine are often made in uncertainty. To reduce uncertainty and to improve clinical decision-making, RCTs are increasingly called upon. A key concept underpinning the ethics of RCTs is equipoise. Here, we aimed to dissect the basic reasoning behind the concept of equipoise and we proposed a line of thinking delineating under which conditions it is ethical to design and execute an RCT. This might prevent a priori negative trials, reduce research waste and aid in the design of meaningful ones. It is these trials that will provide insight on how to safely and effectively assist subfertile couples.

  • The Rotterdam criteria for polycystic ovary syndrome: evidence-based criteria?

    The Rotterdam criteria for polycystic ovary syndrome (PCOS) are used by a wide range of medical professionals and researchers. However, the development of these criteria was based on expert meetings and not on evidence-based treatment guidance. Over the last decade, the Rotterdam criteria have been useful in guiding research, and a number of clinical studies on PCOS have been published consequently. We plead to revisit the Rotterdam criteria based on the available evidence in prognostic studies and randomized controlled trials. In this opinion paper, we provide arguments of the strengths and limitations of the Rotterdam criteria in guiding treatment selections and predicting prognoses in women with infertility. While the Rotterdam criteria have shown their advantages in predicting reproductive prognosis, the next step is to evaluate whether they can guide treatment choices in infertility as well as other health aspects of the syndrome. Based on available data in clinical studies, we should be able to determine whether the Rotterdam criteria are evidence-based criteria.

  • Factors associated with seminal impairment in HIV-infected men under antiretroviral therapy
    STUDY QUESTION

    How do semen parameters of HIV-infected men under antiretroviral treatment compare with WHO parameters of normal semen, and what factors are associated with any differences?

    SUMMARY ANSWER

    Semen quality in most HIV-infected patients under antiretroviral treatment exceeds WHO limits, but the number falling below them is higher than would be expected in a healthy population. Exposure to efavirenz has a significant association with dysmotility.

    WHAT IS KNOWN ALREADY

    Dysmotility is the most frequently described sperm alteration related to HIV infection, and it has recently been linked to treatment with non-nucleoside reverse transcriptase inhibitors, particularly to efavirenz.

    STUDY DESIGN, SIZE, DURATION

    Prospective cohort study. Between March 2002 and December 2013, 139 HIV-infected men undergoing antiretroviral therapy were enrolled.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Participants were male members of serodiscordant couples who attended a clinic for sexually transmitted infections (STIs) in Madrid and requested reproductive counselling. Sociodemographic, behavioural and clinical data were collected. CD4+ lymphocyte count, HIV viral load, serology/viral load of hepatitis B and C viruses, syphilis serology and other STIs diagnosis were performed. Semen parameters were assessed through standard sperm analysis and were compared with WHO 2010 reference values. Factors associated with impaired semen parameters were evaluated by bivariate and multivariate analysis.

    MAIN RESULTS AND THE ROLE OF CHANCE

    The median values of all assessed semen parameters were within a normal range, but in up to 19% of HIV-positive males, at least one parameter of semen quality was below the normal range. A significant association between treatment with efavirenz and the presence of dysmotility was detected in the multivariate analysis.

    LIMITATIONS, REASONS FOR CAUTION

    Our results cannot demonstrate a causal relationship between exposure to efavirenz and impaired motility. We do not have a real comparison group as the WHO cohort is international and may not reflect local variations in semen characteristics. Subjects who requested reproductive counselling might not be representative of HIV-positive men in general.

    WIDER IMPLICATIONS OF THE FINDINGS

    Since efavirenz is still widely used in current therapeutic regimens may be affecting fertility in seropositive men who desire procreation.

    STUDY FUNDING/COMPETING INTEREST(S)

    No external funding was used for this study. The authors have no conflict of interest to declare.

  • Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation
    STUDY QUESTION

    Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm?

    SUMMARY ANSWER

    No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared.

    WHAT IS KNOWN ALREADY

    Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation.

    STUDY DESIGN, SIZE, DURATION

    This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996–1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 μg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods.

    LARGE SCALE DATA

    Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781.

    LIMITATIONS, REASONS FOR CAUTION

    This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated.

    WIDER IMPLICATIONS OF THE FINDINGS

    Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 μg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome.

    STUDY FUNDING/COMPETING INTERESTS

    This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

  • Pregnancy and live birth rates after microsurgical vasoepididymostomy for azoospermic patients with epididymal obstruction
    STUDY QUESTION

    Can microsurgical vasoepididymostomy (MVE) be an effective treatment for azoospermic men with epididymal obstruction?

    SUMMARY ANSWER

    MVE is an effective treatment for epididymal obstruction, with overall patency and live birth rates of 76.3% and 34.8%, respectively.

    WHAT IS KNOWN ALREADY

    We showed that MVE was an effective treatment for non-vasectomized patients with epididymal obstruction and prior failed sperm retrieval for ICSI. ICSI is the preferred treatment for obstructive azoospermia in some reproductive centers. Some small studies documented that MVE could achieve high patency and pregnancy rates.

    STUDY DESIGN, SIZE, DURATION

    This retrospective study was designed to investigate the natural pregnancy and live birth rates after MVE and to identify possible predictors of pregnancy. From January 2011 to July 2013, 241 patients underwent MVE for epididymal obstruction in our andrology center.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    All patients underwent scrotal exploration and MVE. Semen was analyzed every 3 months postoperatively until pregnancy was achieved. Patency, pregnancy and live birth rates were evaluated. Preoperative and intraoperative data were compared between patent and non-patent groups to identify factors affecting the patency rate. Predictors of pregnancy were identified by univariate and multivariate analyses with Cox regression models.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Data from 198 males (82.2%) were analyzed. The mean (±SD) age of males and female partners was 31.0 ± 5.8 and 28.4 ± 4.4 years, respectively. Sperm was present in the ejaculate of 151 patients (76.3%) postoperatively. Patency rates were increased for patients with bilateral anastomosis, distant anastomosis and motile sperm in epididymal fluid. Overall, 81/198 males (40.9%) reported pregnancy in partners and 73 newborns were delivered. The overall live birth rate was 34.8%. Male age (hazard ratio (HR) [95% CI] 0.407 [0.203–0.816], P = 0.011), sperm concentration (HR [95% CI] 4.988 [2.777–8.957], P < 0.001) and forward motility (HR [95% CI] 1.751 [1.042–2.945], P = 0.035) were predictors of pregnancy.

    LIMITATIONS, REASONS FOR CAUTION

    A randomized control trial comparing pregnancy rates, live birth rates, risks and medical costs of MVE and IVF/ICSI is needed. The sample size of females >35 years old was small, so we could not determine whether female age was a predictor of pregnancy.

    WIDER IMPLICATIONS OF THE FINDINGS

    MVE is an effective therapy for azoospermic patients with epididymal obstruction. Sperm concentration and forward motility may predict pregnancy after the procedure. Microsurgical reconstruction could be a first choice for epididymal obstruction.

    STUDY FUNDING/COMPETING INTEREST(S)

    No external funding was received. The authors have no competing interests.

    TRIAL REGISTRATION NUMBER

    Not applicable.

  • Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa
    STUDY QUESTION

    Is diethylstilbestrol (DES), a prototypical endocrine-disrupting chemical (EDC), able to induce physiological changes in human spermatozoa and affect progesterone actions?

    SUMMARY ANSWER

    DES promoted Ca2+ flux into human spermatozoa by activating the cation channel of sperm (CatSper) and suppressed progesterone-induced Ca2+ signaling, tyrosine phosphorylation and sperm functions.

    WHAT IS KNOWN ALREADY

    DES significantly impairs the male reproductive system both in fetal and postnatal exposure. Although various EDCs affect human spermatozoa in a non-genomic manner, the effect of DES on human spermatozoa remains unknown.

    STUDY DESIGN, SIZE, DURATION

    Sperm samples from normozoospermic donors were exposed in vitro to a range of DES concentrations with or without progesterone at 37°C in a 5% CO2 incubator to mimic the putative exposure to this toxicant in seminal plasma and the female reproductive tract fluids. The incubation time varied according to the experimental protocols. All experiments were repeated at least five times using different individual sperm samples.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a multimode plate reader following sperm loading with Ca2+ indicator Fluo-4 AM, and the whole-cell patch-clamp technique was performed to record CatSper and alkalinization-activated sperm K+ channel (KSper) currents. Sperm viability and motility parameters were assessed by an eosin–nigrosin staining kit and a computer-assisted semen analysis system, respectively. The ability of sperm to penetrate into viscous media was examined by penetration into 1% methylcellulose. The sperm acrosome reaction was measured using chlortetracycline staining. The level of tyrosine phosphorylation was determined by western blot assay.

    MAIN RESULTS AND THE ROLE OF CHANCE

    DES exposure rapidly increased human sperm [Ca2+]i dose dependently and even at an environmentally relevant concentration (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and mainly mediated by CatSper. Although DES did not affect sperm viability, motility, penetration into viscous media, tyrosine phosphorylation or the acrosome reaction, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1–100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous media and acrosome reaction.

    LARGE SCALE DATA

    N/A.

    LIMITATIONS, REASONS FOR CAUTION

    Although DES has been shown to disturb progesterone actions on human spermatozoa, this study was performed in vitro, and caution must be taken when extrapolating the results in practical applications.

    WIDER IMPLICATIONS OF THE FINDINGS

    The present study revealed that DES interfered with progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation, ultimately inhibited progesterone-induced human sperm functions and, thereby, might impair sperm fertility. The non-genomic manner in which DES disturbs progesterone actions may be a potential mechanism for some estrogenic endocrine disruptors to affect human sperm function.

    STUDY FUNDING/COMPETING INTEREST(S)

    National Natural Science Foundation of China (No. 31400996); Natural Science Foundation of Jiangxi, China (No. 20161BAB204167 and No. 20142BAB215050); open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07) to T. Luo; National Natural Science Foundation of China (No. 81300539) to L.P. Zheng. The authors have no conflicts of interest to declare.

  • Previous miscarriages and GLI2 are associated with anorectal malformations in offspring
    STUDY QUESTION

    Are anorectal malformations (ARMs) associated with previous miscarriages or single nucleotide polymorphisms (SNPs) in the Bone Morphogenetic Protein 4 (BMP4) and GLI family zinc finger 2 (GLI2) genes?

    SUMMARY ANSWER

    The SNP rs3738880 in GLI2 and miscarriages were associated with ARM, especially in patients with multiple congenital anomalies (MCA).

    WHAT IS KNOWN ALREADY

    ARM are one of the most common birth defects of the gastrointestinal tract. The etiology is likely to be multifactorial, involving both environmental and genetic factors. SNPs in BMP4 and GLI2 genes were associated with ARM in non-Caucasian populations. During a patient information day, several mothers of ARM patients reported their concerns about previous miscarriages.

    STUDY DESIGN, SIZE, DURATION

    A case-control study was performed among 427 ARM patients and 663 population-based controls.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    We examined the associations of ARM with SNPs in GLI2 and BMP4 using DNA samples of the children and associations with previous miscarriages using parental questionnaires. In addition, gene–gene and gene–environment interaction analyses were performed.

    MAIN RESULTS AND THE ROLE OF CHANCE

    The SNP rs3738880 in GLI2 was associated with ARM, especially in patients with MCA (homozygous GG-genotype: odds ratio (OR): 2.1; 95% CI: 1.2, 3.7). We identified previous miscarriages as a new risk factor for ARM, especially when occurring in the pregnancy directly preceding the index pregnancy and in patients with MCA (OR: 2.1; 95% CI: 1.3, 3.5). No association with rs17563 in BMP4, nor gene–gene or gene–environment interactions were found.

    LIMITATIONS, REASONS FOR CAUTION

    The possibility of recall errors for previous miscarriage, but we expect these errors to be limited, as a miscarriage is a major life event. In addition, potential misclassification regarding miscarriages and stillbirth, but sensitivity analyses showed that this did not influence our results.

    WIDER IMPLICATIONS OF THE FINDINGS

    This study showed associations of ARM with rs3738880 in GLI2 and with previous miscarriages. Both associations were stronger in patients with MCA, showing the importance of stratifying the analyses by patients with isolated ARM or MCA.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was funded by the Radboudumc. The authors have no conflict of interest to disclose.

  • Inter-observer and intra-observer agreement between embryologists during selection of a single Day 5 embryo for transfer: a multicenter study
    STUDY QUESTION

    What is the inter-observer and intra-observer agreement between embryologists when selecting a single Day 5 embryo for transfer?

    SUMMARY ANSWER

    The inter-observer and intra-observer agreement between embryologists when selecting a single Day 5 embryo for transfer was generally good, although not optimal, even among experienced embryologists.

    WHAT IS KNOWN ALREADY

    Previous research on the morphological assessment of early stage (two pronuclei to Day 3) embryos has shown varying levels of inter-observer and intra-observer agreement. However, single blastocyst transfer is now becoming increasingly popular and there are no published data that assess inter-observer and intra-observer agreement when selecting a single embryo for Day 5 transfer.

    STUDY DESIGN, SIZE, DURATION

    This was a prospective study involving 10 embryologists working at five different IVF clinics within a single organization between July 2013 and November 2015.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    The top 10 embryologists were selected based on their yearly Quality Assurance Program scores for blastocyst grading and were asked to morphologically grade all Day 5 embryos and choose a single embryo for transfer in a survey of 100 cases using 2D images. A total of 1000 decisions were therefore assessed. For each case, Day 5 images were shown, followed by a Day 3 and Day 5 image of the same embryo. Subgroup analyses were also performed based on the following characteristics of embryologists: the level of clinical embryology experience in the laboratory; amount of research experience; number of days per week spent grading embryos. The agreement between these embryologists and the one that scored the embryos on the actual day of transfer was also evaluated. Inter-observer and intra-observer variability was assessed using the kappa coefficient to evaluate the extent of agreement.

    MAIN RESULTS AND THE ROLE OF CHANCE

    This study showed that all 10 embryologists agreed on the embryo chosen for transfer in 50 out of 100 cases. In 93 out of 100 cases, at least 6 out of the 10 embryologists agreed. The inter-observer and intra-observer agreement among embryologists when selecting a single Day 5 embryo for transfer was generally good as assessed by the kappa scores (kappa = 0.734, 95% CI: 0.665–0.791 and 0.759, 95% CI: 0.622–0.833, respectively). The subgroup analyses did not substantially alter the inter-observer and intra-observer agreement among embryologists. The agreement when Day 3 images were included alongside Day 5 images of the same embryos resulted in a change of mind at least three times by each embryologist (on average for <10% of cases) and resulted in a small decrease in inter-observer and intra-observer agreement between embryologists (kappa = 0.676, 95% CI: 0.617–0.724 and 0.752, 95% CI: 0.656–808, respectively).

    The assessment of the inter-observer agreement with regard to morphological grading of Day 5 embryos showed only a fair-to-moderate agreement, which was observed across all subgroup analyses. The highest overall kappa coefficient was seen for the grading of the developmental stage of an embryo (0.513; 95% CI: 0.492–0.538). The findings were similar when the individual embryologists were compared with the embryologist who made the morphological assessments of the available embryos on the actual day of transfer.

    LIMITATIONS, REASONS FOR CAUTION

    All embryologists had already completed their training and were working under one organization with similar policies between the five clinics. Therefore, the inter-observer agreement might not be as high between embryologists working in clinics with different policies or with different levels of training.

    WIDER IMPLICATIONS OF THE FINDINGS

    The generally good, although not optimal uniformity between participating embryologists when selecting a Day 5 embryo for transfer, as well as, the surprisingly low agreement when morphologically grading Day 5 embryos could be improved, potentially resulting in increased pregnancy rates. Future studies need to be directed toward technologies that can help achieve this.

    STUDY FUNDING/COMPETING INTEREST(S)

    None declared.

    TRIAL REGISTRATION NUMBER

    Not applicable.

  • World Endometriosis Society consensus on the classification of endometriosis
    STUDY QUESTION

    What is the global consensus on the classification of endometriosis that considers the views of women with endometriosis?

    SUMMARY ANSWER

    We have produced an international consensus statement on the classification of endometriosis through systematic appraisal of evidence and a consensus process that included representatives of national and international, medical and non-medical societies, patient organizations, and companies with an interest in endometriosis.

    WHAT IS KNOWN ALREADY

    Classification systems of endometriosis, developed by several professional organizations, traditionally have been based on lesion appearance, pelvic adhesions, and anatomic location of disease. One system predicts fertility outcome and none predicts pelvic pain, response to medications, disease recurrence, risks for associated disorders, quality of life measures, and other endpoints important to women and health care providers for guiding appropriate therapeutic options and prognosis.

    STUDY DESIGN, SIZE, DURATION

    A consensus meeting, in conjunction with pre- and post-meeting processes, was undertaken.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    A consensus meeting was held on 30 April 2014 in conjunction with the World Endometriosis Society's 12th World Congress on Endometriosis. Rigorous pre- and post-meeting processes, involving 55 representatives of 29 national and international, medical and non-medical organizations from a range of disciplines, led to this consensus statement.

    MAIN RESULTS AND THE ROLE OF CHANCE

    A total of 28 consensus statements were made. Of all, 10 statements had unanimous consensus, however none of the statements was made without expression of a caveat about the strength of the statement or the statement itself. Two statements did not achieve majority consensus. The statements covered women's priorities, aspects of classification, impact of low resources, as well as all the major classification systems for endometriosis. Until better classification systems are developed, we propose a classification toolbox (that includes the revised American Society for Reproductive Medicine and, where appropriate, the Enzian and Endometriosis Fertility Index staging systems), that may be used by all surgeons in each case of surgery undertaken for women with endometriosis. We also propose wider use of the World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project surgical and clinical data collection tools for research to improve classification of endometriosis in the future, of particular relevance when surgery is not undertaken.

    LIMITATIONS, REASONS FOR CAUTION

    This consensus process differed from that of formal guideline development, although based on the same available evidence. A different group of international experts from those participating in this process may have yielded subtly different consensus statements.

    WIDER IMPLICATIONS OF THE FINDINGS

    This is the first time that a large, global, consortium–representing 29 major stake-holding organizations, from 19 countries – has convened to systematically evaluate the best available evidence on the classification of endometriosis and reach consensus. In addition to 21 international medical organizations and companies, representatives from eight national endometriosis organizations were involved, including lay support groups, thus generating and including input from women who suffer from endometriosis in an endeavour to keep uppermost the goal of optimizing quality of life for women with endometriosis.

    STUDY FUNDING/COMPETING INTEREST(S)

    The World Endometriosis Society convened and hosted the consensus meeting. Financial support for participants to attend the meeting was provided by the organizations that they represented. There was no other specific funding for this consensus process. Mauricio Abrao is an advisor to Bayer Pharma, and a consultant to AbbVie and AstraZeneca; G David Adamson is the Owner of Advanced Reproductive Care Inc and Ziva and a consultant to Bayer Pharma, Ferring, and AbbVie; Deborah Bush has received travel grants from Fisher & Paykel Healthcare and Bayer Pharmaceuticals; Linda Giudice is a consultant to AbbVie, Juniper Pharmaceutical, and NextGen Jane, holds research grant from the NIH, is site PI on a clinical trial sponsored by Bayer, and is a shareholder in Merck and Pfizer; Lone Hummelshoj is an unpaid consultant to AbbVie; Neil Johnson has received conference expenses from Bayer Pharma, Merck-Serono, and MSD, research funding from AbbVie, and is a consultant to Vifor Pharma and Guerbet; Jörg Keckstein has received a travel grant from AbbVie; Ludwig Kiesel is a consultant to Bayer Pharma, AbbVie, AstraZeneca, Gedeon Richter, and Shionogi, and holds a research grant from Bayer Pharma; Luk Rombauts is an advisor to MSD, Merck Serono, and Ferring, and a shareholder in Monash IVF. The following have declared that they have nothing to disclose: Kathy Sharpe Timms; Rulla Tamimi; Hugh Taylor.

    TRIAL REGISTRATION NUMBER

    N/A

  • Total circulating microparticle levels are increased in patients with deep infiltrating endometriosis
    STUDY QUESTION

    Are the levels of total circulating cell-derived microparticles (cMPs) and circulating tissue factor-containing microparticles (cMP-TF) increased in patients with endometriosis?

    SUMMARY ANSWER

    The levels of total cMP, but not cMP-TF, were higher in patients with endometriosis, and these were attributed to higher levels in patients with deep infiltrating endometriosis (DIE).

    WHAT IS KNOWN ALREADY

    Previous studies have reported elevated levels of total cMP in inflammatory conditions as well as higher levels of other inflammatory biomarkers in endometriosis. Increased expression of tissue factor (a transmembrane receptor for Factor VII/VIIa) in eutopic and ectopic endometrium from patients with endometriosis has been described. There is no previous data regarding total cMP and cMP-TF levels in patients with endometriosis.

    STUDY DESIGN, SIZE, DURATION

    A prospective case–control study including two groups of patients was carried out. The E group included 65 patients with surgically confirmed endometriosis (37 with DIE lesions) and the C group comprises 33 women without surgical findings of any form of endometriosis. Patients and controls were recruited during the same 10-month period. Controls were the next patient without endometriosis undergoing surgery, after including two patients with endometriosis.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Venous blood samples for total cMP and cMP-TF determinations were obtained at the time of surgery, before anesthesia at a tertiary care center. To assess total cMP, an ELISA functional assay was used and cMP-TF activity in plasma was measured using an ELISA kit.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Total cMP levels in plasma were higher in the E group compared with the C group (P < 0.0001). The subanalysis of endometriosis patients with DIE or with ovarian endometriomas without DIE showed that total cMP levels were higher in the DIE group (P = 0.001). There were no statistically significant differences in cMP-TF levels among the groups analyzed.

    LIMITATIONS, REASONS FOR CAUTION

    This is a preliminary study in which the sample size was arbitrarily decided, albeit in keeping with previous studies analyzing cMP in other inflammatory diseases and other biomarkers in endometriosis. The control group included patients with other pathologies as well as healthy controls, and blood samples were taken at different phases of the cycle.

    WIDER IMPLICATIONS OF THE FINDINGS

    Elevated total cMP levels in DIE patients may reflect an inflammatory and/or procoagulant systemic status in these patients. Further studies are warranted to confirm our findings and to assess the role of cMP levels in the pathophysiology of DIE.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was supported in part by a grant from FIS-PI11/01560 and FIS-PI11/00977 within the ‘Plan Nacional de I + D + I’ and co-funded by the ‘ISCIII-Subdirección General de Evaluación’ and ‘Fondo Europeo de Desarrollo Regional (FEDER)’ and by the grant ‘Premi Fi de Residència Emili Letang 2015’ from the Hospital Clínic of Barcelona. The authors have no competing interests to disclose.

  • mRNA levels of low-density lipoprotein receptors are overexpressed in the foci of deep bowel endometriosis
    STUDY QUESTION

    Is mRNA expression of LDL receptors altered in deep bowel endometriotic foci?

    SUMMARY ANSWER

    mRNA expression of LDL receptors is up-regulated in deep bowel endometriotic foci of patients with endometriosis.

    WHAT IS KNOWN ALREADY

    Several studies have demonstrated the overexpression of low-density lipoprotein receptors in various tumour cell lines and endometriosis has similar aspects to cancer, mainly concerning the pathogenesis of both diseases. This is the first study we know of to investigate lipoprotein receptors expression in deep endometriosis with bowel involvement.

    STUDY DESIGN, SIZE, DURATION

    During 2014–2015, an exploratory case-control study was conducted with 39 patients, including 20 women with a histological diagnosis of deep endometriosis compromising the bowel and 19 women without endometriosis who underwent laparoscopic tubal ligation.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Peripheral blood samples were collected on the day of surgery for lipid profile analysis, and samples of endometrial tissue and of bowel endometriotic lesions were also collected. The tissue samples were sent for histopathological analysis and for LDL-R and LRP-1 gene expression screening using quantitative real-time PCR.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Patients with deep endometriosis had lower LDL-cholesterol than patients without the disease (119 ± 23 versus 156 ± 35; P = 0.001). Gene expression analysis of LDL receptors revealed that LDL-R was more highly expressed in endometriotic lesions when compared to the endometrium of the same patient but not more than in the endometrium of women without endometriosis (0.027 ± 0.022 versus 0.012 ± 0.009 versus 0.019 ± 0.01, respectively; P < 0.001). LRP-1 was more highly expressed in endometriotic lesions, both when compared with the endometrium of the same patient and when compared with the endometrium of patients without the disease (0.307 ± 0.207 versus 0.089 ± 0.076 and versus 0.126 ± 0.072, respectively; P < 0.001). The study also showed that LDL-R gene expression in the endometrium of women with endometriosis was higher during the secretory phase of the menstrual cycle (P = 0.001). LRP-1 gene expression was increased during the secretory phase in the endometrium of women without the disease (P = 0.008).

    LIMITATIONS, REASONS FOR CAUTION

    In the endometriotic lesions, the presence of fibrosis is substantial, restricting access to the stromal and glandular components of the lesion. Despite that, we found that LDL receptor mRNA was overexpressed. Future studies may perform laser microdissection to isolate the area of interest in the target tissue, excluding fibrosis contamination.

    WIDER IMPLICATIONS OF THE FINDINGS

    This study supports the feasibility of LDL-R targeted therapy in the treatment of deep endometriosis.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was supported by Fundacão de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2011/17245-0). The authors have no conflicts of interest to declare.

  • The natural history of endometrial polyps
    STUDY QUESTION

    What is the natural history of endometrial polyps in women who are managed expectantly?

    SUMMARY ANSWER

    The growth rates of expectantly managed polyps vary considerably and cannot be accurately predicted.

    WHAT IS KNOWN ALREADY

    The majority of polyps detected on ultrasound are treated surgically, and therefore little is known about their natural history. Some polyps have been reported to regress spontaneously without the need for treatment; however, the factors predictive of regression are unknown.

    STUDY DESIGN, SIZE, DURATION

    This was a retrospective cohort study conducted at the Department of Gynaecology, University College London Hospitals. We searched our ultrasound clinic database between July 1997 and September 2015, to identify women aged 18 years or older with endometrial polyps that were managed expectantly for ≥6 months. All women attended for a minimum of two ultrasound scans.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    A single expert operator performed all ultrasound scans. Those with <6-month follow-up and those who were taking hormonal contraception, HRT or tamoxifen were excluded from the study. The mean diameter of each polyp was calculated from the measurements in three perpendicular planes. The polyp growth rate was expressed as annual percentage change in the mean diameter. Non-parametric tests and the Fisher's exact test were used to compare differences in polyp mean diameters and growth rates between women of different demographic characteristics. To correct for multiple significance testing, we used the Bonferroni method, giving the level of probability at which findings were considered significant as P < 0.0029 (as 17 tests were undertaken).

    MAIN RESULTS AND THE ROLE OF CHANCE

    We included 112 women with endometrial polyps, which were expectantly managed over a median period of 22.5 months (range, 6–136). The annual endometrial polyp growth rate varied with a median of 1.0% (interquartile range, –6.5 to 14.3). There was no association between women's demographic characteristics or polyps’ morphology and their growth rates. Eleven out of 75 (15% (95% CI, 6.9%–23.1%)) women who initially did not have abnormal uterine bleeding subsequently developed abnormal bleeding during the follow-up period. Polyp growth rate was not associated with the subsequent development of abnormal uterine bleeding (P = 0.397). Seven out of 112 (6.3% (95% CI, 1.8%–10.8%)) women had complete regression of their polyps without treatment during a median follow-up period of 28 months (range, 9–56). Spontaneous regression appeared to occur more frequently in premenopausal women (P = 0.016) and in those who presented with abnormal uterine bleeding at diagnosis (P = 0.004); however, the differences did not reach statistical significance after correction for multiple comparisons.

    LIMITATIONS, REASONS FOR CAUTION

    This study was retrospective and therefore may be prone to selection and information biases. The lack of histological confirmation on all ultrasound diagnoses may also be considered as a limitation.

    WIDER IMPLICATIONS OF THE FINDINGS

    Women should be advised that the growth pattern of an individual polyp cannot be accurately predicted; however, a small proportion of polyps do regress spontaneously. There was no correlation between polyps’ growth rate and the subsequent development of abnormal uterine bleeding. In view of that, routine monitoring of asymptomatic polyps by ultrasound is not helpful and encouraging women to report clinical symptoms is more useful in deciding whether treatment is required. In contrast to previous studies, we found that polyps may regress more frequently in premenopausal women and in those who presented with abnormal uterine bleeding; a larger sample size would give us greater power to detect a difference in these subgroups of women.

    STUDY FUNDING/COMPETING INTERESTS

    No study funding was received and no competing interests are present.

    TRIAL REGISTRATION NUMBER

    N/A

  • Natural conception: repeated predictions over time
    STUDY QUESTION

    How can we predict chances of natural conception at various time points in couples diagnosed with unexplained subfertility?

    SUMMARY ANSWER

    We developed a dynamic prediction model that can make repeated predictions over time for couples with unexplained subfertility that underwent a fertility workup at a fertility clinic.

    WHAT IS KNOWN ALREADY

    The most frequently used prediction model for natural conception (the ‘Hunault model’) estimates the probability of natural conception only once per couple, that is, after completion of the fertility workup. This model cannot be used for a second or third time for couples who wish to know their renewed chances after a certain period of expectant management.

    STUDY DESIGN, SIZE, DURATION

    A prospective cohort studying the long-term follow-up of subfertile couples included in 38 centres in the Netherlands between January 2002 and February 2004. Couples with bilateral tubal occlusion, anovulation or a total motile sperm count <1 x 106 were excluded.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    The primary endpoint was time to natural conception, leading to an ongoing pregnancy. Follow-up time was censored at the start of treatment or at the last date of contact. In developing the new dynamic prediction model, we used the same predictors as the Hunault model, i.e. female age, duration of subfertility, female subfertility being primary or secondary, sperm motility and referral status. The performance of the model was evaluated in terms of calibration and discrimination. Additionally, we assessed the utility of the model in terms of the variability of the calculated predictions.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Of the 4999 couples in the cohort, 1053 (21%) women reached a natural conception leading to an ongoing pregnancy within a mean follow-up of 8 months (5th and 95th percentile: 1–21). Our newly developed dynamic prediction model estimated the median probability of conceiving in the first year after the completion of the fertility workup at 27%. For couples not yet pregnant after half a year, after one year and after one and a half years of expectant management, the median probability of conceiving over the next year was estimated at 20, 15 and 13%, respectively. The model performed fair in an internal validation. The prediction ranges were sufficiently broad to aid in counselling couples for at least two years after their fertility workup.

    LIMITATIONS, REASONS FOR CAUTION

    The dynamic prediction model needs to be validated in an external population.

    WIDER IMPLICATIONS OF THE FINDINGS

    This dynamic prediction model allows reassessment of natural conception chances after various periods of unsuccessful expectant management. This gives valuable information to counsel couples with unexplained subfertility that are seen for a fertility workup.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was facilitated by grant 945/12/002 from ZonMW, The Netherlands Organization for Health Research and Development, The Hague, The Netherlands. No competing interests.

  • Effect of pretreatment with oral contraceptives and progestins on IVF outcomes in women with polycystic ovary syndrome
    STUDY QUESTION

    Do oral contraceptives (OCs) and progestins impact live birth rate of IVF when used for cycle scheduling in women with polycystic ovary syndrome (PCOS)?

    SUMMARY ANSWER

    OCs used for scheduling IVF cycle were associated with lowered rates of pregnancy and live birth after fresh embryo transfer, whereas progestins used for this purpose yield higher rates of pregnancy and live birth than OCs.

    WHAT IS KNOWN ALREADY

    Due to oligo-menorrhea in PCOS, OCs and progestin are extensively used to schedule the start of an IVF cycle in women with PCOS. Little is known about the effect of such pretreatments on outcomes, especially, the rate of live birth.

    STUDY DESIGN, SIZE, DURATION

    This was a nested cohort study and secondary analysis of a multicenter randomized trial, which was designed to compare live birth rate after fresh embryo transfer vs frozen embryo transfer (FET) in women with PCOS (Frefro-PCOS). A total of 1508 women were enrolled from 14 centers between June 2013 and May 2014.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    At the discretion of local investigators, subjects were instructed to wait for spontaneous menses (Control group, n = 323), or were prescribed progestins (P group, n = 283) or OCs (OCs group, n = 902) to induce menstruation prior to the start of ovarian stimulation. GnRH antagonist protocol was initiated at Day 2 or 3 of induced or spontaneous menses cycle. The rates of pregnancy, pregnancy loss and live birth after either fresh embryo transfer or FET were compared among these three groups.

    MAIN RESULTS AND THE ROLE OF CHANCE

    With fresh embryo transfer, women with OC-induced menses had lower rates of clinical pregnancy (48.8% vs 63.6%, relative rate (RR): 0.77, 95% CI: 0.66–0.89) and live birth (36.1% vs 48.1%, RR: 0.75, 95% CI: 0.61–0.92) than women with spontaneous menses. With freeze-all and deferred FET, women with OC-induced menses had a similar pregnancy rate but a higher pregnancy loss rate (27.7% vs 13.0%, RR: 2.13, 95% CI: 1.28–3.52) after FET than women with spontaneous menses. The live birth rate after FET in women with OC-induced menses, progestin-induced menses and spontaneous menses was 49.4%, 50.7% and 60.2%, respectively (P = 0.06). Progestin-induced menses was associated with similar rates of pregnancy, pregnancy loss and live birth after transfer of either fresh or frozen embryos compared with spontaneous menses. Multivariate logistic regression analysis showed that OCs used for menses induction was associated with lower rate of live birth.

    LIMITATIONS, REASONS FOR CAUTION

    The methods for menses induction were not assigned randomly, thus selection bias was highly likely because of the study design and significant differences that were observed in the baseline characteristics of the women in the different groups. The mean BMI in this study population was relatively normal; the applicability of this result to obese PCOS women needs to be evaluated in further study.

    WIDER IMPLICATIONS OF THE FINDINGS

    Our results suggest that either waiting for a spontaneous menses or using progestin is a better option than using OCs to induce menses in women with PCOS prior to ovarian stimulation using GnRH antagonist protocol for IVF. Further randomized controlled studies are needed to confirm our findings.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was funded by National Basic Research Program of China (973 Program) (2012CB944700), the State Key Program of National Natural Science Foundation of China (81430029), National Natural Science Foundation of China (81471428) and Thousand Talents Program (Drs Legro and Zhang H). Dr Legro reports receiving consulting fees from Euroscreen, Kindex, Bayer and Millendo Pharmaceuticals and research funding from Ferring. Others report no disclosures.

    TRIAL REGISTRATION NUMBER

    Frefro-PCOS was registered at Clinicaltrials.gov: NCT01841528.

  • The slow growing embryo and premature progesterone elevation: compounding factors for embryo-endometrial asynchrony
    STUDY QUESTION

    Is there an association of progesterone (P4) on the day of trigger with live birth in autologous ART transfer cycles on day 5 versus day 6?

    SUMMARY ANSWER

    P4 had a greater negative effect on live birth in day 6 fresh transfers compared to day 5 fresh transfers.

    WHAT IS KNOWN ALREADY

    Premature P4 elevation is associated with lower live birth rates in fresh autologous ART cycles, likely due to worsened endometrial-embryo asynchrony. Few studies have evaluated whether the effect of an elevated P4 on the day of trigger is different on live birth rates with a day 5 compared to a day 6 embryo transfer.

    STUDY DESIGN SIZE, DURATION

    This was a retrospective cohort study with autologous IVF cycles with fresh embryo transfers on day 5 and day 6 from 2011 to 2014. A total of 4120 day 5 and 230 day 6 fresh autologous embryo transfers were included. The primary outcome was live birth, defined as a live born baby at 24 weeks gestation or later.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Patients from a large private ART practice were included. Analysis was performed with generalized estimating equations (GEE) modeling and receiver operating characteristic (ROC) curves.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Day 6 transfers were less likely to have good quality embryos (73% versus 83%, P < 0.001) but the cohorts had similar rates of blastocyst stage transfer (92% versus 91%, P = 0.92). Live birth was less likely in fresh day 6 versus day 5 embryo transfers (34% versus 46%, P = 0.01) even when controlling for embryo confounders. In adjusted GEE models, the effect of P4 as a continuous variable on live birth was more pronounced on day 6 (P < 0.001). Similarly, the effect of P4 > 1.5 ng/ml on day of trigger was more pronounced on day 6 than day 5 (P < 0.001). Day 6 live birth rates were 8% lower than day 5 when P4 was in the normal range (P = 0.04), but became 17% lower when P4 was > 1.5 ng/ml (P < 0.01). ROC curves for P4 predicting live birth demonstrated a greater AUC in day 6 transfers (AUC 0.59, 95% CI 0.51–0.66) than day 5 (AUC 0.54, 95% CI 0.52–0.55). Interaction testing of P4 x day of embryo transfer was highly significant (P < 0.001), further suggesting that the effect of P4 was more pronounced on day 6 embryo transfer. In fresh oocyte retrieval cycles with elevated P4, a subsequent 760 frozen–thaw transfers did not demonstrate a difference between embryos that were frozen after blastulation on day 5 versus 6.

    LIMITATIONS REASONS FOR CAUTION

    Limitations include the retrospective design and the inability to control for certain confounding variables, such as thaw survival rates between day 5 and day 6 blastocysts. Also, the data set lacks the known ploidy status of the embryos and the progesterone assay is not currently optimized to discriminate between patients with a P4 of 1.5 versus 1.8 ng/ml.

    WIDER IMPLICATIONS OF THE FINDINGS

    This study suggests further endometrial-embryo asynchrony when a slow growing embryo is combined with an advanced endometrium, ultimately leading to decreased live births. This suggests that premature elevated P4 may be a factor in the lower live birth rates in day 6 fresh embryo transfers. Further studies are needed to evaluate if a frozen embryo transfer cycle can ameliorate the effect of elevated P4 on the day of trigger among these slower growing embryos that reach blastocyst staging on day 6.

    STUDY FUNDING/COMPETING INTEREST(S)

    No external funding was received for this study. There are no conflicts of interest to declare.

    TRIAL REGISTRATION NUMBER

    Not applicable.

  • There is no evidence that the time from egg retrieval to embryo transfer affects live birth rates in a freeze-all strategy
    STUDY QUESTION

    Does the time from ovum pick-up (OPU) to frozen embryo transfer (FET) affect reproductive outcomes in a freeze-all strategy?

    SUMMARY ANSWER

    Our study did not detect statistically significant differences between first and subsequent cycles, clinically relevant differences are not ruled out and further and larger studies are required.

    WHAT IS KNOWN ALREADY

    Following controlled ovarian hyperstimulation (COH) delaying FET until the endometrium has returned to an optimal pre-stimulation state may have a significant emotional impact on patients, which adds to the stress and anxiety accompanying a standard IVF cycle. Currently there is no agreement on the best time to perform a FET after a freeze-all cycle in order to maximize reproductive outcomes for the patient.

    STUDY DESIGN, SIZE, DURATION

    Retrospective cohort study of 512 freeze-all cycles, performed between January 2012 and December 2014. COH was performed by either a GnRH antagonist (n = 397) or a long GnRH agonist protocol (n = 115). Ovulation was triggered using either a GnRH agonist (n = 258) or hCG (n = 254). Endometrial preparation was performed in an artificial cycle by either oral (n = 238) or transdermal (n = 274) oestrogen. Differences were considered significant if P < 0.05.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Reproductive outcomes between FETs which took place either within the first menstrual cycle following OPU (Cycle 1; n = 263) or afterwards (Cycle ≥2; n = 249) were compared. Student's t-test for independent samples, Mann–Whitney U-test and Chi-square analysis were used where appropriate. A multivariable logistic regression analysis was performed adjusting for maternal age, drug used for ovulation trigger, number of retrieved oocytes, number of embryos obtained, day of embryonic development at transfer, number of embryos transferred and type of endometrial preparation. Differences were considered significant if P < 0.05.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Live birth rate (LBR) was significantly higher in FET performed during Cycle 1 vs Cycle ≥2 (37.6% vs 27.3%, respectively; P = 0.01) before adjusting for confounding factors. We found no difference for biochemical pregnancy (49.8% vs 43.8%; P = 0.17), clinical pregnancy (44.1% vs 36.1%; P = 0.07) or pregnancy loss (11.8% vs 16.1%; P = 0.16). A multivariable analysis found no impact of timing of elective FET on LBR (odds ratio, OR 0.73; 95% CI 0.49–1.08). The impact remained not significant after adjusting for number of retrieved oocytes, drug used for ovulation trigger (hCG vs GnRH agonist) and reason for cryopreservation. The factors that significantly affected LBR were: maternal age in both age categories (women between 35 and 40 years vs women below 35 years, OR 0.63, 95% CI 0.4–0.95; and women over 40 years vs women below 35 years, OR 0.34, 95% CI 0.2–0.7), day of embryonic development at transfer (day +4 vs +3; OR 1.7, 95% CI 1.1–2.8) and number of transferred embryos (OR 2.2, 95% CI 1.4–3.3) and oestrogen used for endometrial preparation (transdermal vs oral; OR 0.62, 95% CI 0.4–0.9).

    LIMITATIONS REASONS FOR CAUTION

    The main limitation of our study is its retrospective nature. Although we adjusted our statistical analysis for a number of known and suspected confounders, we cannot exclude the possibility of residual confounding factors.

    WIDER IMPLICATIONS OF THE FINDINGS

    According to our results, clinicians might not need to wait more than one menstrual cycle before performing FET. This allows us to reduce unnecessary delays in FET, without compromising reproductive outcomes.

    STUDY FUNDING/COMPETING INTEREST(S)

    No funding was sought for this study. Authors declare no competing interests.

    TRIAL REGISTRATION NUMBER

    NA.

  • COMPI Fertility Problem Stress Scales is a brief, valid and reliable tool for assessing stress in patients seeking treatment
    STUDY QUESTION

    Are the Copenhagen Multi-Centre Psychosocial Infertility research program Fertility Problem Stress Scales (COMPI-FPSS) a reliable and valid measure across gender and culture?

    SUMMARY ANSWER

    The COMPI-FPSS is a valid and reliable measure, presenting excellent or good fit in the majority of the analyzed countries, and demonstrating full invariance across genders and partial invariance across cultures.

    WHAT IS KNOWN ALREADY

    Cross-cultural and gender validation is needed to consider a measure as standard care within fertility. The present study is the first attempting to establish comparability of fertility-related stress across genders and countries.

    STUDY DESIGN SIZE, DURATION

    Cross-sectional study. First, we tested the structure of the COMPI-FPSS. Then, reliability and validity (convergent and discriminant) were examined for the final model. Finally, measurement invariance both across genders and cultures was tested.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Our final sample had 3923 fertility patients (1691 men and 2232 women) recruited in clinical settings from seven different countries: Denmark, China, Croatia, Germany, Greece, Hungary and Sweden. Participants had a mean age of 34 years and the majority (84%) were childless.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Findings confirmed the original three-factor structure of the COMPI-FPSS, although suggesting a shortened measurement model using less items that fitted the data better than the full version model. While data from the Chinese and Croatian subsamples did not fit, all other counties presented good fit (2/df ≤ 5.4; comparative fit index ≥ 0.94; root-mean-square error of approximation ≤ 0.07; modified expected cross-validation index ≤ 0.77). In general, reliability, convergent validity, and discriminant validity were observed in all subscales from each country (composite reliability ≥ 0.63; average variance extracted ≥ 0.38; squared correlation ≥ 0.13). Full invariance was established across genders, and partial invariance was demonstrated across countries.

    LIMITATIONS REASONS FOR CAUTION

    Generalizability regarding the validation of the COMPI-FPSS cannot be made regarding infertile individuals not seeking treatment, or non-European patients. This study did not investigate predictive validity, and hence the capability of this instrument in detecting changes in fertility-specific adjustment over time and predicting the psychological impact needs to be established in future research.

    WIDER IMPLICATIONS OF THE FINDINGS

    Besides extending knowledge on the psychometric properties of one of the most used fertility stress questionnaire, this study demonstrates both research and clinical usefulness of the COMPI-FPSS.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was supported by European Union Funds (FEDER/COMPETE—Operational Competitiveness Program, and by national funds (FCT—Portuguese Foundation for Science and Technology) under the projects PTDC/MHC-PSC/4195/2012 and SFRH/BPD/85789/2012). There are no conflicts of interest to declare.

    TRIAL REGISTRATION NUMBER

    N/A

  • Transgender men's experiences of fertility preservation: a qualitative study
    STUDY QUESTION

    How do transgender men experience fertility preservation (FP) by cryopreservation of oocytes?

    SUMMARY ANSWER

    The procedures required prior to oocyte cryopreservation, such as hormonal ovarian stimulation and transvaginal ultrasound (TVS), have a negative impact on gender dysphoria as they are closely linked to the men's female assigned sex at birth, which is incompatible with their current status.

    WHAT IS KNOWN ALREADY

    Transgender persons often have high dissatisfaction with assigned sex-specific body features, such as the genital organs and androgen/oestrogen-responsive features. Thus, undergoing FP that requires genital-specific examinations, aimed at obtaining oocytes to cryopreserve, could be distressing. As no previous studies have investigated transgender men's experiences of FP involving cryopreservation of oocytes, little is known about their experience of the procedures.

    STUDY DESIGN, SIZE, DURATION

    This is a prospective study among adult transgender men referred for FP between March 2014 and December 2015. Individual in-depth qualitative interviews were conducted shortly after FP treatment. The interviews lasted between 62 and 111 min (mean 81 min) and were digitally recorded and transcribed verbatim.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Participants were recruited on their first visit to the assisted reproduction clinic for reproductive counseling. There were 15 men, scheduled for FP, who chose to participate in the study (age 19–35); none had given birth and eight had a partner. Data were analyzed by thematic content analysis.

    MAIN RESULTS AND THE ROLE OF CHANCE

    The analysis resulted in three main categories: the journey to FP, reactions to the FP proceedings and strategies for coping. The referral for FP was an important part of the assessment and diagnosis and sometimes lined with frustrating waits and doubts. The reaction to the FP proceedings revealed that the genital examinations and the physical changes associated with discontinuation of testosterone or hormonal stimulation treatment triggered gender incongruence and dysphoria. However, for some, the negative expectations were not met. The participants used several coping strategies in order to manage the procedure, such as focusing on their reasons for undergoing FP, reaching out to friends and family for support and the cognitive approaches of not hating their body or using non-gendered names for their body parts. The results demonstrate the importance of contextual sensitivity during FP procedures.

    LIMITATIONS, REASONS FOR CAUTION

    The authors have strived to be reflective about their pre-understanding of the phenomenon. The majority of the participants resided in large urban areas; it is possible that transgender men living in rural areas have different experiences.

    WIDER IMPLICATIONS OF THE FINDINGS

    As the results are based on qualitative data from 15 transgender men, the results cannot readily be generalized to larger populations. However, the results are suggested to be applicable to other transgender men who want to undergo FP by cryopreservation of oocytes. The results show that transgender men's experience of FP places may elicit gender incongruence and gender dysphoria. However, health care personnel can alleviate distress by using a gender-neutral language and the preferred pronoun. Also, reassuringly, the men also have coping strategies of how to handle the situation. This knowledge is important to ensure adequate professional support for patients with gender dysphoria during FP.

    STUDY FUNDING/COMPETING INTEREST(S)

    Swedish Society of Medicine, Stockholm County Council and Karolinska Institutet (to K.A.R.-W.).

    TRIAL REGISTRATION NUMBER

    N/A.

  • The effect of expressive writing intervention for infertile couples: a randomized controlled trial
    STUDY QUESTION

    Is expressive writing intervention (EWI) efficacious in reducing distress and improving pregnancy rates for couples going through ART treatment?

    SUMMARY ANSWER

    Compared to controls, EWI statistically significantly reduced depressive symptoms but not anxiety and infertility-related distress.

    WHAT IS KNOWN ALREADY

    ART treatment is considered stressful. So far, various psychological interventions have been tested for their potential in reducing infertility-related distress and the results are generally positive. It remains unclear whether EWI, a brief and potentially cost-effective intervention, could be advantageous.

    STUDY DESIGN SIZE, DURATION

    Between November 2010 and July 2012, a total of 295 participants (163 women, 132 men) were randomly allocated to EWI or a neutral writing control group.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Participants were couples undergoing IVF/ICSI treatment. Single women and couples with Preimplantation Genetic Diagnosis or acute change of procedure from insemination to IVF, were excluded. EWI participants participated in three 20-min home-based writing exercises focusing on emotional disclosure in relation to infertility/fertility treatment (two sessions) and benefit finding (one session). Controls wrote non-emotionally in three 20-min sessions about their daily activities. The participants completed questionnaires at the beginning of treatment (t1), prior to the pregnancy test (t2), and 3 months later (t3). In total, 26.8% (79/295) were lost to follow-up. Mixed linear models were chosen to compare the two groups over time for psychological outcomes (depression, anxiety and infertility-related distress), and a Chi2 test was employed in order to examine group differences in pregnancy rates

    MAIN RESULTS AND THE ROLE OF CHANCE

    One hundred and fifty-three participants received EWI (women = 83; men = 70) and 142 participants were allocated to the neutral writing control group (women = 83; men = 62). Both women and partners in the EWI group exhibited greater reductions in depressive symptoms compared with controls (P = 0.049; [CI 95%: –0.04; –0.01] Cohen's d = 0.27). The effect of EWI on anxiety did not reach statistical significance. Overall infertility-related distress increased marginally for the partners in the EWI group compared to the partners in the control group (P = 0.06; Cohen's d = 0.17). However, in relation to the personal subdomain, the increase was statistically significant (P = 0.01; Cohen's d = 0.24). EWI had no statistically significant effect on pregnancy rates with 42/83 (50.6%) achieving pregnancy in the EWI group compared with 40/80 (49.4%) in the control group (RR = 0.99 [CI 95% = 0.725, 1.341]; P = 0.94).

    LIMITATIONS, REASONS FOR CAUTION

    The results for depressive symptoms corresponded to a small effect size and the remaining results failed to reach statistical significance. This could be due to sample characteristics leading to a possible floor-effect, as we did not exclude participants with low levels of emotional distress at baseline. Furthermore, men showed increased infertility-related distress over time.

    WIDER IMPLICATIONS OF THE FINDINGS

    EWI is a potentially cost-effective and easy to implement home-based intervention, and even small effects may be relevant. When faced with infertility, EWI could thus be a relevant tool for alleviating depressive symptoms by allowing the expression of feelings about infertility that may be perceived as socially unacceptable. However, the implications do not seem to be applicable for men, who presented with increased infertility-related distress over time.

    STUDY FUNDING/COMPETING INTEREST(S)

    The present study was supported by research grants from Merck Sharpe and Dohme and The Danish Agency for Science Technology and Innovation as part of a publicly funded PhD. The funding bodies had no influence on the data collection, analysis or conclusions of the study. None of the authors have any conflicts of interest to declare.

    TRIAL REGISTRATION NUMBER

    Clinicaltrials.gov, trial no. NCT01187095.

    TRIAL REGISTRATION DATE

    7th September 2010

    DATE OF FIRST PATIENT'S ENROLMENT

    23rd November 2010

  • Perceived challenges of working in a fertility clinic: a qualitative analysis of work stressors and difficulties working with patients
    STUDY QUESTION

    What are some of the challenges of working in a fertility clinic?

    SUMMARY ANSWER

    The most frequently mentioned challenges were workload (e.g. high time pressure) and patient-related sources (e.g. unrealistic expectations).

    WHAT IS KNOWN ALREADY

    One study showed a too high workload, worry about handling human material and low success rates were main stressors in fertility clinics.

    STUDY DESIGN, SIZE, DURATION

    An online open-ended survey inviting participants to respond to seven questions was distributed to 5902 members of the European Society for Human Reproduction and Embryology (ESHRE, October 2010). Questions asked participants to describe the top three factors that made (i) their work stressful (hereafter ‘Work stressors’) and (ii) working with patients difficult (hereafter ‘Perceived sources of difficulties’), and (iii) to choose from these factors which top three issues they would be willing to attend a workshop to resolve (hereafter ‘Workshops’). A qualitative content analysis using inductive coding for each question was used to extract meaningful themes from the text replies, at three levels of increasing abstraction (lower and higher categories, general themes).

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    The final sample comprised 526 respondents (8.9% participation rate). Respondents were predominantly clinicians (41.3%, n = 216) or embryologists (35.5%, n = 186) from European countries (73.0%, n = 386).

    MAIN RESULTS AND THE ROLE OF CHANCE

    The number of text replies generated for each question was 1421, 1208 and 907 for the ‘Work Stressors’, ‘Perceived sources of difficulties’ and ‘Workshop’ questions, respectively. The most often reported higher-order categories of Work Stressors were ‘Time and Workload’ (61.6%, e.g. time pressure), ‘Organisation, Team and management issues’ (60.4%, e.g. team conflicts) and ‘Job content and work environment’ (50.3%, e.g. burdensome administration). For ‘Perceived sources of difficulties’ these were ‘Patient-related sources’ (66.7%, e.g. unrealistic expectations), ‘Communication and Counselling with patients’ (33.7%, e.g. strained information giving) and ‘Misinformation and lack of knowledge’ (27.8%, e.g. Dr Google). Finally, the topics participants would be willing to address in Workshops were ‘Communicating and Counselling with Patients’ (24.9%), ‘Dealing with Patient-related sources’ (19.6%) and ‘Clinical topics’ (19.6%). Three general themes emerged. First, a theme of ‘time and time trade-offs’ expressed the oft-mentioned need to trade-off time spent on one activity (e.g. managing patient demands) against another activity (e.g. clinical workload, administration) with stress level dependent on the efficacy of trading-off. Second, the theme of ‘multifactorial causes’ of challenging patient interactions that embodied the many sources of difficulties working with patients. What staff would be willing to address in workshops was indicated by the final general theme of ‘a little of everything’, which linked to the need for multiple workshops addressing the multifactorial nature of challenges in fertility clinics.

    LIMITATIONS, REASONS FOR CAUTION

    Only about 10% of members receiving the survey participated. The work was limited to the stressful and difficult aspects of working in fertility clinics, which may give a more negative impression than if questions about the rewards and benefits had also been included.

    WIDER IMPLICATIONS OF THE FINDINGS

    The nature of stressors and difficulties of working in a fertility clinic are consistent with models of occupational stress and patient complexity. Specialized psychologists, management consultants and other occupational experts could assist fertility teams in overcoming many of the challenges. More research is required on the effect of encountered work stressors and perceived sources of difficulties in working with patients on staff and patient outcomes.

    STUDY FUNDING/COMPETING INTEREST(S)

    None declared.

  • Bisphenol-A exposure and gene expression in human luteinized membrana granulosa cells in vitro
    STUDY QUESTION

    Does bisphenol-A (BPA) affect gene expression in human membrana granulosa cells (MGC)?

    SUMMARY ANSWER

    In vitro, short exposure to supra-physiological concentrations of BPA alters human MGC gene expression.

    WHAT IS KNOWN ALREADY

    Exposure to BPA may interfere with reproductive endocrine signaling. In vitro studies, mostly in animal models, have shown an inverse correlation between exposure to BPA and follicular growth, meiosis, and steroid hormone production in granulosa cells.

    STUDY DESIGN, SIZE, DURATION

    Primary cultures of MGC obtained from 24 patients undergoing IVF (for PGD, male factor infertility or unexplained infertility) were exposed to various concentrations of BPA (0, 0.02, 0.2, 2 or 20 µg/ml) for 48 h.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    The study was conducted in a university-affiliated hospital. Microarray analysis was used to identify genes exhibiting expression changes following BPA exposure. Genes significantly altered were identified based on changes greater than 2-fold relative to the control group (not treated by BPA) and a Student's t-test P-value <0.05. Statistical significance was adjusted for multiple comparisons using the Benjamini–Hochberg method. Alterations in the expression of genes that are involved in the enriched functional annotations altered by BPA at the concentration of 20 µg/ml were confirmed by real-time PCR.

    MAIN RESULTS AND THE ROLE OF CHANCE

    A distinct pattern of gene expression was observed in primary cultures of MGC exposed to the highest BPA concentration compared with untreated cells. We identified 652 genes that exhibited at least 2-fold differences in expression after BPA exposure (all P < 0.05 versus untreated). These genes were significantly enriched for annotations related to cell cycle progression, segregation of chromosomes, steroid metabolism, apoptosis, lipid synthesis, oocyte maturation and chromosomal alignment. No significant changes in gene expression were found at the lower doses of BPA most relevant to human exposure.

    LARGE SCALE DATA

    N/A.

    LIMITATIONS, REASONS FOR CAUTION

    Human exposure to BPA in vivo occurs over long periods of time. In this in vitro model, cells were exposed to the chemical for 48 h only. Thus, the effects of BPA on the human follicle might be underestimated.

    WIDER IMPLICATIONS OF THE FINDINGS

    As BPA exposure is ubiquitous, understanding the effects of the chemical on the ovary, specifically in women of reproductive age, has public health significance. The clinical evidence to date points to an association between BPA exposure and impaired IVF outcome, although not all studies have shown negative effects. Our study adds valuable mechanistic information showing that exposure to BPA alters granulosa cell gene expression at high and supra-physiological doses.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was supported by grant number 1936/12 from the ISF. The authors have nothing to disclose.

  • Performance of mass spectrometry steroid profiling for diagnosis of polycystic ovary syndrome
    STUDY QUESTION

    How well does multi-analyte steroid mass spectrometry (MS) profiling classify women with and without polycystic ovary syndrome (PCOS)?

    SUMMARY ANSWER

    Our liquid chromatography MS (LC–MS) steroid profiling only minimally improves discrimination of women with and without PCOS compared with a direct testosterone immunoassay (T_IA) and the free androgen index (FAI).

    WHAT IS KNOWN ALREADY

    Blood testosterone measured by direct (non-extraction) immunoassay overlaps between women with and without PCOS. Multi-analyte MS provides greater specificity and accuracy for steroid measurement so might improve the classification.

    STUDY DESIGN, SIZE, DURATION

    An observational, cross-sectional study of women with PCOS (n = 152) defined by Rotterdam criteria and matched non-PCOS (n = 45) control women was conducted.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Serum steroid profiles of testosterone (T), dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), androstenedione (A4), estradiol (E2), estrone (E1), 17 hydroxy progesterone (17OHP4), progesterone (P4) and cortisol were measured by LC–MS; T_IA and sex hormone binding globulin were measured by immunoassay; and FAI, calculated free testosterone (cFT) and total androgen index (TAI) were calculated. Classification was based on logistic regression with corresponding univariate and multivariate C-statistics.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Serum testosterone by immunoassay demonstrated levels more than 100% higher than that measured by LC–MS. Compared with the controls, women with PCOS had higher serum T, DHEA, A4, TAI, T_IA, cFT, FAI and E2 but not serum DHT, E1, P4, 17OHP4 or cortisol. Univariate C-statistics were highest for FAI (0.89) and T_IA (0.82) compared with other androgens (T [0.72], DHT [0.40]), pro-androgens (A4 [0.74], DHEA[0.71]) or derivatives (cFT [0.75], TAI [0.60]). For all multivariate models, the overall correct predictions (81–86%) featured high sensitivity (92–96%) but low specificity (28–43%). and substituting LC–MS steroid measurements for T_IA and FAI produced only minimal improvements in classification.

    LIMITATIONS REASONS FOR CAUTION

    The study cohort is limited in size and only unconjugated steroids were measured.

    WIDER IMPLICATIONS OF THE FINDINGS

    Multi-analyte steroid profiling of unconjugated circulating steroids provides only limited improvement on direct T_IA in classifying women with and without PCOS.

    STUDY FUNDING/COMPETING INTEREST(S)

    None.

    TRIAL REGISTRATION NUMBER

    N/A.

  • Overweight and obese but not normal weight women with PCOS are at increased risk of Type 2 diabetes mellitus--a prospective, population-based cohort study
    STUDY QUESTION

    What are the respective roles of polycystic ovary syndrome (PCOS), long-term weight gain and obesity for the development of prediabetes or Type 2 diabetes mellitus (T2DM) by age 46 years?

    SUMMARY ANSWER

    The risk of T2DM in women with PCOS is mainly due to overweight and obesity, although these two factors have a synergistic effect on the development of T2DM.

    WHAT IS KNOWN ALREADY

    PCOS is associated with an increased risk of prediabetes and T2DM. However, the respective roles of PCOS per se and BMI for the development of T2DM have remained unclear.

    STUDY DESIGN, SIZE, DURATION

    In a prospective, general population-based follow-up birth cohort 1966 (n = 5889), postal questionnaires were sent at ages 14 (95% answered), 31 (80% answered) and 46 years (72% answered). Questions about oligoamenorrhoea and hirsutism were asked at age 31 years, and a question about PCOS diagnosis at 46 years. Clinical examination and blood sampling were performed at 31 years in 3127 women, and at 46 years in 3280 women. A 2-h oral glucose tolerance test (OGTT) was performed at 46 years of age in 2780 women.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Women reporting both oligoamenorrhoea and hirsutism at age 31 years and/or diagnosis of PCOS by 46 years were considered as women with PCOS (n = 279). Women without any symptoms at 31 years and without PCOS diagnosis by 46 years were considered as controls (n = 1577). The level of glucose metabolism was classified according to the results of the OGTT and previous information of glucose metabolism status from the national drug and hospital discharge registers.

    MAIN RESULTS AND THE ROLE OF CHANCE

    PCOS per se significantly increased the risk of T2DM in overweight/obese (BMI ≥ 25.0 kg/m2) women with PCOS when compared to overweight/obese controls (odds ratio: 2.45, 95% CI: 1.28–4.67). Normal weight women with PCOS did not present with an increased risk of prediabetes or T2DM. The increase in weight between ages 14, 31 and 46 years was significantly greater in women with PCOS developing T2DM than in women with PCOS and normal glucose tolerance, with the most significant increase occurring in early adulthood (between 14 and 31 years: median with [25%; 75% quartiles]: 27.25 kg [20.43; 34.78] versus 13.80 kg [8.55; 20.20], P < 0.001).

    LIMITATIONS, REASONS FOR CAUTION

    The diagnosis of PCOS was based on self-reporting, and the questionnaire at 46 years did not distinguish between polycystic ovaries only in ultrasonography and the syndrome. Ovarian ultrasonography was not available to aid the diagnosis of PCOS.

    WIDER IMPLICATIONS OF THE FINDINGS

    These results emphasize weight management already during adolescence and early adulthood to prevent the development of T2DM in women with PCOS, as the period between 14 and 31 years seems to be a crucial time-window during which the women with PCOS who are destined to develop T2DM by 46 years of age experience a dramatic weight gain. Furthermore, our results support the view that, particularly in times of limited sources of healthcare systems, OGTT screening should be targeted to overweight/obese women with PCOS rather than to all women with PCOS.

    STUDY FUNDING/COMPETING INTEREST(S)

    Finnish Medical Foundation; North Ostrobothnia Regional Fund; Academy of Finland (project grants 104781, 120315, 129269, 1114194, 24300796, Center of Excellence in Complex Disease Genetics and SALVE); Sigrid Juselius Foundation; Biocenter Oulu; University Hospital Oulu and University of Oulu (75617); Medical Research Center Oulu; National Institute for Health Research (UK); National Heart, Lung, and Blood Institute (grant 5R01HL087679-02) through the STAMPEED program (1RL1MH083268-01); National Institute of Health/National Institute of Mental Health (5R01MH63706:02); ENGAGE project and grant agreement HEALTH-F4-2007–201413; EU FP7 EurHEALTHAgeing-277849 European Commission and Medical Research Council, UK (G0500539, G0600705, G1002319, PrevMetSyn/SALVE) and Medical Research Center, Centenary Early Career Award. The authors have no conflicts of interests.

    TRIAL REGISTRATION NUMBER

    N/A.

  • Pre-term birth and low birth weight following preimplantation genetic diagnosis: analysis of 88 010 singleton live births following PGD and IVF cycles
    STUDY QUESTION

    Is PGD associated with the risk of adverse perinatal outcomes such as pre-term birth (PTB) and low birth weight (LBW)?

    SUMMARY ANSWER

    There was no increase in the risk of adverse perinatal outcomes of PTB, and LBW following PGD compared with autologous IVF.

    WHAT IS KNOWN ALREADY

    Pregnancies resulting from ART are associated with a higher risk of pregnancy complications compared with spontaneously conceived pregnancies. The possible reason of adverse obstetric outcomes following ART has been attributed to the underlying infertility itself and embryo specific epigenetic modifications due to the IVF techniques. It is of interest whether interventions such as embryo biopsy as performed in PGD affect perinatal outcomes.

    STUDY DESIGN, SIZE, DURATION

    Anonymous data were obtained from the Human Fertilization and Embryology Authority (HFEA), the statutory regulator of ART in the UK. The HFEA has collected data prospectively on all ART performed in the UK since 1991. Data from 1996 to 2011 involving a total of 88 010 singleton live births were analysed including 87 571 following autologous stimulated IVF ± ICSI and 439 following PGD cycles.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Data on all women undergoing either a stimulated fresh IVF ± ICSI treatment cycle or a PGD cycle during the period from 1996 to 2011 were analysed to compare perinatal outcomes of PTB and LBW among singleton live births. Logistic regression analysis was performed adjusting for female age category, year of treatment, previous IVF cycles, infertility diagnosis, number of oocytes retrieved, whether IVF or ICSI was used and day of embryo transfer.

    MAIN RESULTS AND THE ROLE OF CHANCE

    There was no increase in the risk of PTB and LBW following PGD versus autologous stimulated IVF ± ICSI treatment, unadjusted odds of PTB (odds ratio (OR) 0.68, 95% CI: 0.46–0.99) and LBW (OR 0.56, 95% CI: 0.37–0.85). After adjusting for the potential confounders, there was again no increase in the risk of the adverse perinatal outcomes following PGD: PTB (adjusted odds ratio (aOR) 0.66, 95% CI: 0.45–0.98) and LBW (aOR 0.58, 95% CI: 0.38–0.88).

    LIMITATIONS, REASONS FOR CAUTION

    Although the analysis was adjusted for a number of important confounders, the data set had no information on confounders such as smoking, body mass index and the medical history of women during pregnancy to allow adjustment. There was no information on the stage of embryo at biopsy, whether blastomere or trophectoderm biopsy.

    WIDER IMPLICATIONS FOR THE FINDINGS

    The demonstration that PGD is not associated with higher risk of PTB and LBW provides reassurance towards its current expanding application.

    STUDY FUNDING/COMPETING INTEREST(S)

    No funding was obtained. There are no competing interests to declare.

  • Reproductive hormones of ICSI-conceived young adult men: the first results
    STUDY QUESTION

    Are reproductive hormone levels (FSH, LH, inhibin B and testosterone) in male offspring conceived by ICSI because of male infertility comparable with those from peers born after spontaneous conception?

    SUMMARY ANSWER

    In this cohort of 54 young men conceived by ICSI because of male-factor infertility, mean and median reproductive hormone levels were found to be comparable with results from spontaneously conceived peers, but ICSI-conceived men were more likely to have low inhibin B (<10th percentile) and high FSH (>90th percentile) levels.

    WHAT IS KNOWN ALREADY

    Since the worldwide oldest ICSI offspring have recently reached young adulthood, their reproductive health can now be investigated. This typically involves semen analysis and a hormonal profiling including the measurement of FSH, LH, inhibin B and testosterone. Circulating levels of FSH and inhibin B are generally known as markers of the exocrine function of the testis, i.e. spermatogenesis, while LH and testosterone reflect its endocrine function. We have previously observed a normal pubertal development and comparable levels of inhibin B and testosterone among pubertal ICSI boys when compared to spontaneously conceived peers. However, at present, information on the gonadal function of ICSI offspring in adulthood is still lacking.

    STUDY DESIGN, SIZE, DURATION

    This study, conducted between March 2013 and April 2016 at the UZ Brussel, is part of a larger follow-up project focusing on reproductive and metabolic health of young adults between 18 and 22 years and conceived after ICSI because of male infertility. The ICSI men are part of a longitudinally followed cohort while the spontaneously conceived controls were recruited cross-sectionally.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Results of a single fasting blood sample from 54 young adult ICSI men were compared to that of 57 spontaneously conceived peers. Reproductive hormone analysis involved FSH, LH, testosterone and inhibin B measurement. Furthermore, the association between their reproductive hormones and their sperm parameters was examined. Data were analyzed by multiple linear and logistic regression adjusted for covariates.

    MAIN RESULTS AND THE ROLE OF CHANCE

    ICSI men had comparable mean levels of FSH, LH, testosterone and inhibin B in comparison to spontaneously conceived counterparts, even after adjustment for confounders, such as age, BMI and season. Young ICSI-conceived men were more likely to have inhibin B levels below the 10th percentile (<125.2 ng/l; Adjusted Odds Ratio (AOR) 4.0; 95% CI: 0.9–18.4; P = 0.07) compared with spontaneously conceived peers and were more likely to have FSH levels above the 90th percentile (>5.5 IU/L; AOR 3.3; 95% CI: 0.9–11.9; P = 0.06) compared with spontaneously conceived peers, but neither difference reached statistical significance. FSH, LH and inhibin B, but not testosterone, levels were significantly associated with sperm concentration and total sperm count.

    LIMITATIONS, REASONS FOR CAUTION

    The main limitation is the small study population. Furthermore, the results of this study should be interpreted according to the background of the participants: all subjects in our study group were conceived by ICSI because of severe male infertility and hence the results cannot be generalized to all ICSI offspring because the indications for performing ICSI have since been widened.

    WIDER IMPLICATIONS OF THE FINDINGS

    These first results in a small group of ICSI men show reassuring reproductive hormonal levels. However, larger studies are required to confirm our results. Since inhibin B and FSH are consistently correlated with semen characteristics, we would suggest that the reproductive status of young adults conceived by ICSI is explored with a hormonal assessment given its easier acceptance compared to semen sampling.

    STUDY FUNDING/COMPETING INTEREST(S)

    This study was supported by Methusalem grants and by grants from Wetenschappelijk Fonds Willy Gepts, all issued by the Vrije Universiteit Brussel (VUB). A grant from the Belgian Society for Pediatric Endocrinology and Diabetology was received for this project. All co-authors, except M.B. and H.T., declare no conflict of interest. M.B. has received consultancy fees from MSD, Serono Symposia and Merck. The Universitair Ziekenhuis Brussel (UZ Brussel) and the Centre for Medical Genetics have received several educational grants from IBSA, Ferring, Organon, Shering-Plough, Merck for establishing the database for follow-up research and organizing the data collection. The institution of HT receives research grants from the ’Research Fund of Flanders’ (FWO), an unconditional grant from Ferring for research on testicular stem cells and research grants from Ferring, Merck, MSD, Roche, Besins, Goodlife and Cook for several research projects in female infertility. H.T. has received consultancy fees from Finox, Abbott and ObsEva for research projects in female infertility.

    TRIAL REGISTRATION NUMBER

    N/A.

  • Academic performance in adolescents born after ART--a nationwide registry-based cohort study
    STUDY QUESTION

    Is academic performance in adolescents aged 15–16 years and conceived after ART, measured as test scores in ninth grade, comparable to that for spontaneously conceived (SC) adolescents?

    SUMMARY ANSWER

    ART singletons had a significantly lower mean test score in the adjusted analysis when compared with SC singletons, yet the differences were small and probably not of clinical relevance.

    WHAT IS KNOWN ALREADY

    Previous studies have shown similar intelligence quotient (IQ) levels in ART and SC children, but only a few have been on adolescents. Academic performance measured with standardized national tests has not previously been explored in a complete national cohort of adolescents conceived after ART.

    STUDY DESIGN, SIZE, DURATION

    A Danish national registry-based cohort including all 4766 ART adolescents (n = 2836 singletons and n = 1930 twins) born in 1995–1998 were compared with two SC control cohorts: a randomly selected singleton population (n = 5660) and all twins (n = 7064) born from 1995 to 1998 in Denmark. Nine children who died during the follow-up period were excluded from the study.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Mean test scores on a 7-point-marking scale from –3 to 12 were compared, and adjustments were made for relevant reproductive and socio-demographic covariates including occupational and educational level of the parents.

    MAIN RESULTS AND THE ROLE OF CHANCE

    The crude mean test score was higher in both ART singletons and ART twins compared with SC adolescents. The crude mean differences were +0.41 (95% CI 0.30–0.53) and +0.45 (95% CI 0.28–0.62) between ART and SC singletons and between ART and SC twins, respectively. However, the adjusted mean overall test score was significantly lower for ART singletons compared with SC singletons (adjusted mean difference –0.15 (95% CI –0.29–(–0.02))). For comparison, the adjusted mean difference was +2.05 (95% CI 1.82–2.28) between the highest and the lowest parental educational level, suggesting that the effect of ART is weak compared with the conventional predictors. The adjusted analyses showed significantly lower mean test scores in mathematics and physics/chemistry for ART singletons compared with SC singletons. Comparing ART twins with SC twins yielded no difference in academic performance in the adjusted analyses. Similar crude and adjusted overall mean test scores were found when comparing ART singletons and ART twins.

    LIMITATIONS, REASONS FOR CAUTION

    Missing data on educational test scores occurred in 6.6% of adolescents aged 15–16 years for the birth cohorts 1995–1997, where all of the children according to their age should have passed the ninth grade exam at the time of data retrieval. As sensitivity analyses yielded no significant difference in the adjusted risk of having missing test scores between any of the groups, it is unlikely that this should bias our results. Adjustment for body mass index and smoking during pregnancy was not possible.

    WIDER IMPLICATIONS OF THE FINDINGS

    As our results are based on national data, our findings can be applied to other populations. The findings of this paper suggest that a possible small negative effect of parental subfertility or ART treatment is counterbalanced by the higher educational level in the ART parents.

    STUDY FUNDING/COMPETING INTEREST(S)

    The Danish Medical Association in Copenhagen (KMS) funded this study with a scholarship grant. None of the authors had any competing interests.

    TRIAL REGISTRATION NO. (STATISTICS DENMARK)

    704676.

  • Novel mutations and structural deletions in TUBB8: expanding mutational and phenotypic spectrum of patients with arrest in oocyte maturation, fertilization or early embryonic development
    STUDY QUESTION

    Are there any new type of mutations and novel phenotypes in patients with arrest in oocyte maturation, fertilization or early embryonic development having tubulin beta eight class VIII (TUBB8) mutations?

    SUMMARY ANSWER

    We identified new types of mutations in TUBB8 associated with maturation, fertilization and developmental arrest.

    WHAT IS KNOWN ALREADY

    We previously found heterozygous mutations and a homozygous frameshift/internal seven amino acid deletion in TUBB8 that are responsible for oocyte maturation arrest.

    STUDY DESIGN, SIZE, DURATION

    We recruited 10 new primary infertility patients from 9 families from December 2015 to May 2016, most of which exhibited failures in oocyte maturation.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    Ten primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA samples from the affected individuals, their family members and healthy controls were extracted from peripheral blood. TUBB8 in the DNA samples were sequenced by Sanger sequencing. TUBB8 sequence was then aligned by CodonCode software to identify rare variants. ExAC database was used to search frequency of corresponding mutations. In silico analysis of mutations was used by Polyphen and PROVEAN. Phenotypes of oocytes and embryos were evaluated by light microscopy, polarization microscopy or immunolabeling.

    MAIN RESULTS AND THE ROLE OF CHANCE

    Besides several novel heterozygous missense mutations, we also identified other new types of genetic variants, including homozygous mutations and a de novo compound heterozygous mutation. We also found a patient with a homozygous deletion of the whole TUBB8 gene, which is the first reported case of a large structural variation in this gene. In addition, we found different mutations in TUBB8 that could result in variability in oocyte/embryo phenotypes, including oocyte maturation arrest, first polar body (PB1) oocytes that cannot be fertilized, and PB1 oocytes that can be fertilized but arrest at an early embryonic stage.

    LIMITATIONS, REASONS FOR CAUTION

    The exact molecular mechanism has not been analyzed and should be further investigated in the future. In addition, immunostaining of more oocytes with mutations and checking spindle status of oocytes with mutations non-invasively by polarization microscopy needs to be done in order to determine exact stage of PB1 oocytes and the functional differences of these mutations.

    WIDER IMPLICATIONS OF THE FINDINGS

    The results not only emphasize the important role of TUBB8 in oocyte maturation, fertilization and early embryonic development but they also provide a basis for determining the genetic variations in TUBB8 as a potential additional criterion for evaluating the quality of patients’ functional PB1 oocytes.

    STUDY FUNDING/COMPETING INTEREST(S)

    National Key R&D Program of China (2016YFC1000600); Basic Research Program of China (2015CB943300); National Natural Science Foundation of China (81270747 and 81571501). No competing interests declared.

  • Pseudoautosomal abnormalities in terminal AZFb+c deletions are associated with isochromosomes Yp and may lead to abnormal growth and neuropsychiatric function
    STUDY QUESTION

    Are copy number variations (CNVs) in the pseudoautosomal regions (PARs) frequent in subjects with Y-chromosome microdeletions and can they lead to abnormal stature and/or neuropsychiatric disorders?

    SUMMARY ANSWER

    Only subjects diagnosed with azoospermia factor (AZF)b+c deletions spanning to the end of the Y chromosome (i.e. terminal deletions) harbor Y isochromosomes and/or cells 45,X that lead to pseudoautosomal gene CNVs, which were associated with abnormal stature and/or neuropsychiatric disorders.

    WHAT IS KNOWN ALREADY

    The microdeletions in the long arm of the Y chromosome (Yq) that include the loss of one to three AZF regions, referred to as Yq microdeletions, constitute the most important known etiological factor for primary spermatogenic failure. Recently, controversy has arisen about whether Yq microdeletions are associated with gain or loss of PAR genes, which are implicated in skeletal development and neuropsychiatric function.

    STUDY DESIGN, SIZE, DURATION

    We studied a cohort of 42 Chilean patients with complete AZF deletions (4 AZFa, 4 AZFb, 23 AZFc, 11 AZFb+c) from a university medical center, diagnosed over a period of 15 years. The subjects underwent complete medical examinations with special attention to their stature and neuropsychiatric function.

    PARTICIPANTS/MATERIALS, SETTING, METHODS

    All subjects were characterized for Yq breakpoints by PCR, and for CNVs in PARs by multiplex ligation-dependent probe amplification (MLPA), followed by qPCR analysis for genes in PAR1 (SHOX and ZBED1), PAR2 (IL9R) and two single copy genes (SRY and DDX3Y, respectively located in Yp11.3 and AZFa). In addition, karyotypes revision and fluorescence in situ hybridization (FISH) for SRY and centromeric probes for X (DXZ1) and Y (DYZ3) chromosomes were performed in males affected with CNVs.

    MAIN RESULTS AND THE ROLE OF CHANCE

    We did not detect CNVs in any of the 35 AZF-deleted men with interstitial deletions (AZFa, AZFb, AZFc or AZFb+c). However, six of the seven patients with terminal AZFb+c deletions showed CNVs: two patients showed a loss and four patients showed a gain of PAR1 genes, with the expected loss of VAMP-7 in PAR2. In these patients, the Yq breakpoints localized to the palindromes P8, P5 or P4. In the four cases with gain of PAR1, qPCR analysis showed duplicated signals for SRY and DDX3Y and one copy of IL9R, indicating isodicentric Yp chromosomes [idic(Y)] with breakpoint in Yq11.22. The two patients who had loss of PAR1, as shown by MLPA, had an additional reduction for SRY and DDX3Y, as shown by qPCR, associated with a high proportion of 45,X cells, as determined by FISH and karyotype. In agreement with the karyotype analysis, we detected DYZ3++ and DYZ3+ cells by FISH in the six patients, confirming idic(Y) and revealing additional monocentric Y chromosome [i(Y)]. Five patients had a history of major depressive disorders or bipolar disorder, and three had language impairment, whereas two patients showed severe short stature (Z score: –2.75 and –2.62), while a man with bipolar disorder was very tall (Z score: +2.56).

    LARGE SCALE DATA

    N/A.

    LIMITATIONS, REASONS FOR CAUTION

    The number of males studied with Y-chromosome microdeletions and normozoospermic controls with normal karyotypes may not be enough to rule out an association between AZF deletions and PAR abnormalities. The prevalence of Y isochromosomes and/or 45,X cells detected in peripheral blood does not necessarily reflect the variations of PAR genes in target tissues.

    WIDER IMPLICATIONS OF THE FINDINGS

    This study shows that CNVs in PARs were present exclusively in patients with terminal AZFb+c deletions associated with the presence of Y isochromosomes and 45,X cells, and may lead to neuropsychiatric and growth disorders. In contrast, we show that men with interstitial Yq microdeletions with normal karyotypes do not have an increased risk of PAR abnormalities and of phenotypical consequences. Moreover, our results highlight the importance of performing molecular studies, which are not considered in the usual screening for patients with Yq microdeletions.

    STUDY FUNDING/COMPETING INTEREST(S)

    This work was supported by the National Fund for Scientific and Technological Development of Chile (FONDECYT), grant no. 1120176 (A.C.). The authors declare that no conflicting interests exist.

  • PGS for recurrent pregnancy loss: still an open question
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