The metabolic health of young men conceived using intracytoplasmic sperm injection.

STUDY QUESTION: Is the metabolic health of men conceived using ICSI different to that of IVF and spontaneously conceived (SC) men? SUMMARY ANSWER: ICSI-conceived men aged 18-24 years, compared with SC controls, showed differences in some metabolic parameters including higher resting diastolic blood pressure (BP) and homeostasis model assessment for insulin resistance (HOMA-IR) scores, although the metabolic parameters of ICSI- and IVF-conceived singleton men were more comparable. WHAT IS KNOWN ALREADY: Some studies suggest that IVF-conceived offspring may have poorer cardiovascular and metabolic profiles than SC children. Few studies have examined the metabolic health of ICSI-conceived offspring. STUDY DESIGN, SIZE, DURATION: This cohort study compared the metabolic health of ICSI-conceived men to IVF-conceived and SC controls who were derived from prior cohorts. Participants included 121 ICSI-conceived men (including 100 singletons), 74 IVF-conceived controls (all singletons) and 688 SC controls (including 662 singletons). PARTICIPANTS/MATERIALS, SETTING, METHODS: Resting systolic and diastolic BP (measured using an automated sphygmomanometer), height, weight, BMI, body surface area and fasting serum metabolic markers including fasting insulin, glucose, total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol, triglycerides, highly sensitive C-reactive protein (hsCRP) and HOMA-IR were compared between groups. Data were analysed using multivariable linear regression adjusted for various covariates including age and education level. MAIN RESULTS AND THE ROLE OF CHANCE: After adjusting for covariates, compared to 688 SC controls, 121 ICSI-conceived men had higher diastolic BP (ß 4.9, 95% CI 1.1-8.7), lower fasting glucose (ß -0.7, 95% CI -0.9 to -0.5), higher fasting insulin (ratio 2.2, 95% CI 1.6-3.0), higher HOMA-IR (ratio 1.9, 95% CI 1.4-2.6), higher HDLC (ß 0.2, 95% CI 0.07-0.3) and lower hsCRP (ratio 0.4, 95% CI 0.2-0.7) levels. Compared to 74 IVF-conceived singletons, only glucose differed in the ICSI-conceived singleton men (ß -0.4, 95% CI -0.7 to -0.1). No differences were seen in the paternal infertility subgroups. LIMITATIONS, REASONS FOR CAUTION: The recruitment rate of ICSI-conceived men in this study was low and potential for recruitment bias exists. The ICSI-conceived men, the IVF-conceived men and SC controls were from different cohorts with different birth years and different geographical locations. Assessment of study groups and controls was not contemporaneous, and the measurements differed for some outcomes (BP, insulin, glucose, lipids and hsCRP). WIDER IMPLICATIONS OF THE FINDINGS: These observations require confirmation in a larger study with a focus on potential mechanisms. Further efforts to identify whether health differences are due to parental characteristics and/or factors related to the ICSI procedure are also necessary. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by an Australian National Health and Medical Research Council Partnership Grant (NHMRC APP1140706) and was partially funded by the Monash IVF Research and Education Foundation. S.R.C. was supported through an Australian Government Research Training Program Scholarship. R.J.H. is supported by an NHMRC project grant (634457), and J.H. and R.I.M. have been supported by the NHMRC as Senior and Principal Research Fellows respectively (J.H. fellowship number: 1021252; R.I.M. fellowship number: 1022327). L.R. is a minority shareholder and the Group Medical Director for Monash IVF Group, and reports personal fees from Monash IVF Group and Ferring Australia, honoraria from Ferring Australia and travel fees from Merck Serono and MSD and Guerbet; R.J.H. is the Medical Director of Fertility Specialists of Western Australia and has equity in Western IVF; R.I.M. is a consultant for and shareholder of Monash IVF Group and S.R.C. reports personal fees from Besins Healthcare and nonfinancial support from Merck outside of the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Health and fertility of ICSI-conceived young men: study protocol.

STUDY QUESTIONS: What are the long-term health and reproductive outcomes for young men conceived using ICSI whose fathers had spermatogenic failure (STF)? Are there epigenetic consequences of ICSI conception? WHAT IS KNOWN ALREADY: Currently, little is known about the health of ICSI-conceived adults, and in particular the health and reproductive potential of ICSI-conceived men whose fathers had STF. Only one group to date has assessed semen parameters and reproductive hormones in ICSI-conceived men and suggested higher rates of impaired semen quality compared to spontaneously conceived (SC) peers. Metabolic parameters in this same cohort of men were mostly comparable. No study has yet evaluated other aspects of adult health. STUDY DESIGN SIZE DURATION: This cohort study aims to evaluate the general health and development (aim 1), fertility and metabolic parameters (aim 2) and epigenetic signatures (aim 3) of ICSI-conceived sons whose fathers had STF (ICSI study group). There are three age-matched control groups: ICSI-conceived sons whose fathers had obstructive azoospermia (OAZ) and who will be recruited in this study, as well as IVF sons and SC sons, recruited from other studies. Of 1112 ICSI parents including fathers with STF and OAZ, 78% (n = 867) of mothers and 74% (n = 823) of fathers were traced and contacted. Recruitment of ICSI sons started in March 2017 and will finish in July 2020. Based on preliminary participation rates, we estimate the following sample size will be achieved for the ICSI study group: mothers n = 275, fathers n = 225, sons n = 115. Per aim, the sample sizes of OAZ-ICSI (estimated), IVF and SC controls are: Aim 1-OAZ-ICSI: 28 (maternal surveys)/12 (son surveys), IVF: 352 (maternal surveys)/244 (son surveys), SC: 428 (maternal surveys)/255 (son surveys); Aim 2-OAZ-ICSI: 12, IVF: 72 (metabolic data), SC: 391 (metabolic data)/365 (reproductive data); Aim 3-OAZ-ICSI: 12, IVF: 71, SC: 292. PARTICIPANTS/MATERIALS SETTING METHODS: Eligible parents are those who underwent ICSI at one of two major infertility treatment centres in Victoria, Australia and gave birth to one or more males between January 1994 and January 2000. Eligible sons are those aged 18 years or older, whose fathers had STF or OAZ, and whose parents allow researchers to approach sons. IVF and SC controls are age-matched men derived from previous studies, some from the same source population. Participating ICSI parents and sons complete a questionnaire, the latter also undergoing a clinical assessment. Outcome measures include validated survey questions, physical examination (testicular volumes, BMI and resting blood pressure), reproductive hormones (testosterone, sex hormone-binding globulin, FSH, LH), serum metabolic parameters (fasting glucose, insulin, lipid profile, highly sensitive C-reactive protein) and semen analysis. For epigenetic and future genetic analyses, ICSI sons provide specimens of blood, saliva, sperm and seminal fluid while their parents provide a saliva sample. The primary outcomes of interest are the number of mother-reported hospitalisations of the son; son-reported quality of life; prevalence of moderate-severe oligozoospermia (sperm concentration <5 million/ml) and DNA methylation profile. For each outcome, differences between the ICSI study group and each control group will be investigated using multivariable linear and logistic regression for continuous and binary outcomes, respectively. Results will be presented as adjusted odds ratios and 95% CIs. STUDY FUNDING/COMPETING INTERESTS: This study is funded by an Australian National Health and Medical Research Council Partnership Grant (NHMRC APP1140706) and was partially funded by the Monash IVF Research and Education Foundation. L.R. is a minority shareholder and the Group Medical Director for Monash IVF Group, and reports personal fees from Monash IVF group and Ferring Australia, honoraria from Ferring Australia, and travel fees from Merck Serono, MSD and Guerbet; R.J.H. is the Medical Director of Fertility Specialists of Western Australia and has equity in Western IVF; R.I.M. is a consultant for and a shareholder of Monash IVF Group and S.R.C. reports personal fees from Besins Healthcare and non-financial support from Merck outside of the submitted work. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable. TRIAL REGISTRATION DATE: Not applicable. DATE OF FIRST PATIENT'S ENROLMENT: Not applicable.

A core outcome set for future endometriosis research: an international consensus development study.

OBJECTIVE: To develop a core outcome set for endometriosis. DESIGN: Consensus development study. SETTING: International. POPULATION: One hundred and sixteen healthcare professionals, 31 researchers and 206 patient representatives. METHODS: Modified Delphi method and modified nominal group technique. RESULTS: The final core outcome set includes three core outcomes for trials evaluating potential treatments for pain and other symptoms associated with endometriosis: overall pain; improvement in the most troublesome symptom; and quality of life. In addition, eight core outcomes for trials evaluating potential treatments for infertility associated with endometriosis were identified: viable intrauterine pregnancy confirmed by ultrasound; pregnancy loss, including ectopic pregnancy, miscarriage, stillbirth and termination of pregnancy; live birth; time to pregnancy leading to live birth; gestational age at delivery; birthweight; neonatal mortality; and major congenital abnormalities. Two core outcomes applicable to all trials were also identified: adverse events and patient satisfaction with treatment. CONCLUSIONS: Using robust consensus science methods, healthcare professionals, researchers and women with endometriosis have developed a core outcome set to standardise outcome selection, collection and reporting across future randomised controlled trials and systematic reviews evaluating potential treatments for endometriosis. TWEETABLE ABSTRACT: @coreoutcomes for future #endometriosis research have been developed @jamesmnduffy.

Evidence summaries and recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome: assessment and treatment of infertility.

STUDY QUESTION: What is the recommended assessment and management of infertile women with polycystic ovary syndrome (PCOS), based on the best available evidence, clinical expertize and consumer preference? SUMMARY ANSWER: International evidence-based guidelines, including 44 recommendations and practice points, addressed prioritized questions to promote consistent, evidence-based care and improve the experience and health outcomes of infertile women with PCOS. WHAT IS KNOWN ALREADY: Previous guidelines on PCOS lacked rigorous evidence-based processes, failed to engage consumer and multidisciplinary perspectives or were outdated. The assessment and management of infertile women with PCOS are inconsistent. The needs of women with PCOS are not being adequately met and evidence practice gaps persist. PARTICIPANTS/MATERIALS SETTING METHODS: Governance included a six continent international advisory and a project board, a multidisciplinary international guideline development group (GDG), consumer and translation committees. Extensive health professional and consumer engagement informed the guideline scope and priorities. The engaged international society-nominated panel included endocrinology, gynaecology, reproductive endocrinology, obstetrics, public health and other experts, alongside consumers, project management, evidence synthesis and translation experts. Thirty-seven societies and organizations covering 71 countries engaged in the process. Extensive online communication and two face-to-face meetings over 15 months addressed 19 prioritized clinical questions involving nine evidence-based reviews and 10 narrative reviews. Evidence-based recommendations (EBRs) were formulated prior to consensus voting within the guideline panel. STUDY DESIGN SIZE DURATION: International evidence-based guideline development engaged professional societies and consumer organizations with multidisciplinary experts and women with PCOS directly involved at all stages. A (AGREE) II-compliant processes were followed, with extensive evidence synthesis. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) framework was applied across evidence quality, desirable and undesirable consequences, feasibility, acceptability, cost, implementation and ultimately recommendation strength. The guideline was peer-reviewed by special interest groups across our partner and collaborating societies and consumer organizations, was independently assessed against AGREE II criteria and underwent methodological review. This guideline was approved by all members of the GDG and has been approved by the NHMRC. MAIN RESULTS AND THE ROLE OF CHANCE: The quality of evidence (QOE) for the EBRs in the assessment and management of infertility in PCOS included very low (n = 1), low (n = 9) and moderate (n = 4) quality with no EBRs based on high-quality evidence. The guideline provides 14 EBRs, 10 clinical consensus recommendations (CCRs) and 20 clinical practice points on the assessment and management of infertility in PCOS. Key changes in this guideline include emphasizing evidence-based fertility therapy, including cheaper and safer fertility management. LIMITATIONS REASONS FOR CAUTION: Overall evidence is generally of low to moderate quality, requiring significantly greater research in this neglected, yet common condition. Regional health systems vary and a process for adaptation of this guideline is provided. WIDER IMPLICATIONS OF THE FINDINGS: The international guideline for the assessment and management of infertility in PCOS provides clinicians with clear advice on best practice based on the best available evidence, expert multidisciplinary input and consumer preferences. Research recommendations have been generated and a comprehensive multifaceted dissemination and translation program supports the guideline with an integrated evaluation program. STUDY FUNDING/COMPETING INTERESTS: The guideline was primarily funded by the Australian National Health and Medical Research Council of Australia (NHMRC) supported by a partnership with ESHRE and the American Society for Reproductive Medicine (ASRM). GDG members did not receive payment. Travel expenses were covered by the sponsoring organizations. Disclosures of conflicts of interest were declared at the outset and updated throughout the guideline process, aligned with NHMRC guideline processes. Dr Costello has declared shares in Virtus Health and past sponsorship from Merck Serono for conference presentations. Prof. Norman has declared a minor shareholder interest in the IVF unit Fertility SA, travel support from Merck and grants from Ferring. Prof. Norman also has scientific advisory board duties for Ferring. The remaining authors have no conflicts of interest to declare.This article was not externally peer-reviewed by Human Reproduction Open.

Germ cell arrest associated with aSETX mutation in ataxia oculomotor apraxia type 2.

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar atrophy, peripheral neuropathy and oculomotor apraxia. It is caused by mutations in the SETX gene that encodes senataxin, a ubiquitously expressed protein that mediates processes, including transcription, transcription termination, DNA repair, RNA processing, DNA-RNA hybrid (R-loop) elimination and telomere stability. In mice, senataxin is essential for male germ cell development and fertility through its role in meiotic recombination and sex chromosome inactivation. AOA2 is associated with hypogonadism in women, but there are no reports of hypogonadism or infertility in men. We describe the first case of human male infertility caused by germ cell arrest in a man with AOA2. Our patient has a homozygous mutation in the SETX gene (NC_000009.11:g.135158775dup), which results in a frameshift and premature protein termination (NM_015046.6:c.6422dup, p.[Ser2142Glufs*23]). In accordance with the murine phenotype, testis histology revealed disrupted seminiferous tubules with spermatogonia and primary spermatocytes, but absent spermatids. Collectively, these data support an essential role of senataxin in human spermatogenesis, and provide a compelling case that men with AOA2 should be counselled at diagnosis about the possibility of infertility.

Survey of Reproductive Experiences and Outcomes of Cancer Survivors Who Stored Reproductive Material Before Treatment.

STUDY QUESTION: What are the reproductive experiences and outcomes of people who store reproductive material before cancer treatment? SUMMARY ANSWER: Of respondents who had tried to achieve pregnancy since completing cancer treatment almost all had succeeded, in most cases through natural conception. WHAT IS KNOWN ALREADY: People of reproductive age who are diagnosed with cancer can cryopreserve reproductive material to guard against the adverse effects on fertility of gonadotoxic treatment. Little is known about the reproductive outcomes of people who undergo fertility preservation before cancer treatment. STUDY DESIGN, SIZE, DURATION: Cross-sectional survey. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women and men who had stored reproductive material before cancer treatment at two private and one public fertility clinics up to June 2014 and were at least 18 years old at the time were identified from medical records and invited to complete an anonymous questionnaire about their reproductive experiences. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 870 potential respondents 302 (171 female and 131 male) returned completed questionnaires yielding a response rate of 34.5% (39.5% and 29.7% for female and male respondents, respectively). Current age was similar for women and men (37.2 years) but men had been diagnosed with cancer significantly earlier in life than women (28.2 versus 30.3 years, P = 0.03). Almost two-thirds of respondents wished to have a child or another child in the future, some of whom knew that they were unable to. One in ten respondents was a parent before the cancer diagnosis and around one-third had had a child since diagnosis or was pregnant (or a partner in pregnancy) at the time of the survey. Of those who had tried to conceive since completing cancer treatment (N = 119) 84% (79% of women and 90% of men) had had a child or were pregnant (or a partner in pregnancy). Most of the pregnancies since the diagnosis of cancer occurred after natural conception (58/100, 58%). Of the 22 women (13% of all women) and 35 men (27% of all men) who had used their stored reproductive material four women (18%) and 28 men (80%) had had a child or were pregnant or a partner in pregnancy at the time of completing the survey. The most commonly stated reason for not using the stored material was not being ready to try for a baby. LIMITATIONS, REASON FOR CAUTION: The relatively low response rate, particularly among men, means that participation bias may have influenced the findings. As type of cancer was self-reported and we did not ask questions about respondents' cancer treatments, it is not possible to link reproductive outcomes to type of cancer or cancer treatment. Also, there is no way of comparing the sample with the populations they were drawn from as data on reproductive outcomes of people who store reproductive material before cancer treatment are not collected routinely. This might have led to over- or underestimates of the reproductive experiences and outcomes reported in this paper. WIDER IMPLICATIONS OF THE FINDINGS: The findings add to the limited evidence about the reproductive outcomes of this growing group of people and can be used to inform the advice given to those contemplating fertility preservation in the context of cancer. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the National Health and Medical Research Council (APP1042347). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Risk of ectopic pregnancy is linked to endometrial thickness in a retrospective cohort study of 8120 assisted reproduction technology cycles.

STUDY QUESTION: Is endometrial combined thickness (ECT) measured prior to embryo transfer (ET) associated with ectopic pregnancy (EP)? SUMMARY ANSWER: Following IVF, the risk of EP is 4-fold increased in women with an ECT of <9 mm compared with women with an ECT of >12 mm. WHAT IS KNOWN ALREADY: Known risk factors for EP include tubal damage, maternal cigarette smoking and endometriosis. EP is also more common following IVF but the underlying causes for this remain unclear. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study restricted to all IVF cycles leading to a pregnancy (ßhCG > 50 IU/l) between January 2006 and December 2014. A total of 6465 patients achieved a pregnancy in 8120 cycles. Cycles using preimplantation genetic screening or donor oocytes were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: This cohort consists of 6465 patients achieving a pregnancy in 6920 stimulated cycles with fresh embryo transfers (STIM ET) and 1200 hormone replacement therapy frozen embryo transfers (HRT-FET) cycles at a private IVF unit (Monash IVF, Melbourne, Australia). ECT was the primary independent variable of interest; the primary outcome was a diagnosis of EP. The dataset was analysed using binary logistic general estimating equations (SPSS v22.0) to calculate odds ratio (OR) for EP adjusted for known confounders (aOR). There was no loss to follow-up in the dataset. MAIN RESULTS AND THE ROLE OF CHANCE: The study groups did not differ significantly prior to IVF treatment. After adjusting for confounders, ECT remained statistically significant as an independent risk factor for EP. Compared with women with an ECT of <9 mm, women with an ECT of 9-12 mm had an aOR of 0.44 (95% CI 0.29-0.69, P < 0.01) and women with an ECT > 12 mm had an aOR of 0.27 (95% CI 0.10-0.77, P = 0.01). These differences remained statistically significant after performing a sensitivity analysis excluding HRT-FET, smokers and patients with tubal infertility. LIMITATIONS, REASONS FOR CAUTION: The study design is retrospective, and it is possible that not all confounders have been accounted for. Measurement of ECT was performed by highly trained sonographers, but some inconsistency between individuals may be present. WIDER IMPLICATIONS OF THE FINDINGS: Our group has previously demonstrated an increased risk of placenta praevia with increased ECT. These new findings suggest that the directionality of the uterine peristalsis waves matters more than their frequency or amplitude. Combining the data from both studies we now hypothesize that increased ECT is a marker for increased fundus-to-cervix uterine peristalsis, explaining both the increased placenta praevia risk and the lower EP risk. Further prospective studies are required to confirm these observations.

Human and Pathogen Factors Associated with Chlamydia trachomatis-Related Infertility in Women.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen worldwide. Infection can result in serious reproductive pathologies, including pelvic inflammatory disease, ectopic pregnancy, and infertility, in women. However, the processes that result in these reproductive pathologies have not been well defined. Here we review the evidence for the human disease burden of these chlamydial reproductive pathologies. We then review human-based evidence that links Chlamydia with reproductive pathologies in women. We present data supporting the idea that host, immunological, epidemiological, and pathogen factors may all contribute to the development of infertility. Specifically, we review the existing evidence that host and pathogen genotypes, host hormone status, age of sexual debut, sexual behavior, coinfections, and repeat infections are all likely to be contributory factors in development of infertility. Pathogen factors such as infectious burden, treatment failure, and tissue tropisms or ascension capacity are also potential contributory factors. We present four possible processes of pathology development and how these processes are supported by the published data. We highlight the limitations of the evidence and propose future studies that could improve our understanding of how chlamydial infertility in women occurs and possible future interventions to reduce this disease burden.

Live birth rates after combined adjuvant therapy in IVF-ICSI cycles: a matched case-control study.

The effectiveness of combined co-treatment with aspirin, doxycycline, prednisolone, with or without oestradiol patches, was investigated on live birth (LBR) rates after fresh and frozen embryo transfers (FET) in IVF and intracytoplasmic sperm injection cycles. Cases (n = 485) and controls (n = 485) were extensively matched in a one-to-one ratio on nine physical and clinical parameters: maternal age, body mass index, smoking status, stimulation cycle number, cumulative dose of FSH, stimulation protocol, insemination method, day of embryo transfer and number of embryos transferred. No significant differences were found in fresh cycles between cases and controls for the pregnancy outcomes analysed, but fewer surplus embryos were available for freezing in the combined adjuvant group. In FET cycles, LBR was lower in the treatment group (OR: 0.49, 95% CI 0.25 to 0.95). The lower LBR in FET cycles seemed to be clustered in patients receiving combined adjuvant treatment without luteal oestradiol (OR 0.37, 95% CI 0.17 to 0.80). No difference was found in LBR between cases and controls when stratified according to the number of previous cycles (<3 or ≥3). There is no benefit of this combined adjuvant strategy in fresh IVF cycles, and possible harm when used in frozen cycles.

Risk of placenta praevia is linked to endometrial thickness in a retrospective cohort study of 4537 singleton assisted reproduction technology births.

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

Incidence and zygosity of twin births following transfers using a single fresh or frozen embryo.

STUDY QUESTION: Are all twin births following single embryo transfer (SET) monozygotic? SUMMARY ANSWER: Between 1 in 10 and in 1 in 5 twins born after SET are the result of a concurrent natural conception. WHAT IS KNOWN ALREADY: The twinning rate after SET is higher than following natural conception. Most studies of twins following SET have incorrectly assumed monozygosity or have not been able to assess the zygosity. STUDY DESIGN, SIZE, DURATION: This study is a retrospective cohort study assessing the gender discordance of all live born twins following fresh or frozen SET. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 4701 patients in a large private IVF unit who gave birth following SET with a fresh or frozen embryo with complete follow-up. Of 137 viable twins at the 7-week ultrasound, 109 were delivered as twins. Gender discordance and Weinberg's differential rule were used to estimate dizygosity. Twin rates were compared for fresh and frozen transfers by insemination method and transfer day. MAIN RESULTS AND THE ROLE OF CHANCE: The overall live twin birth rate was 2.3% (109/4701). Based on the 7-week scan, 2 of the twins were monochorionic monoamniotic, 62 were monochorionic diamniotic and 45 were dichorionic diamniotic. There were a total of 12 gender discordant twins (11%), 7 from the Day 2/3 transfers and 5 from Day 5 transfers. Nine of the 12 discordant twins were from natural cycle frozen embryo transfers, the remaining 3 were from fresh cycles. LIMITATIONS, REASONS FOR CAUTION; To assess gender discordance only live born twins were studied. DNA fingerprinting of twins is a more accurate way to assess zygosity than measuring gender discordance. Same sex twins in this study are not necessarily monozygotic and the dizygotic rate in this study may therefore be higher. This rate was estimated using Weinberg's differential rule. WIDER IMPLICATIONS OF THE FINDINGS: As many as 1 in 5 twins born after SET may be the result of a concurrent natural conception. Couples therefore need to be counselled regarding the relative benefits and risks of intercourse in assisted reproduction technology cycles where spontaneous conception is possible. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: Not applicable.

Preimplantation human blastocysts release factors that differentially alter human endometrial epithelial cell adhesion and gene expression relative to IVF success.

STUDY QUESTION: Do human blastocysts which subsequently implant release factors that regulate endometrial epithelial cell gene expression and adhesion to facilitate endometrial receptivity? SUMMARY ANSWER: Blastocysts which subsequently implanted released factors that altered endometrial epithelial gene expression and facilitated endometrial adhesion while blastocysts that failed to implant did not. WHAT IS KNOWN ALREADY: Human preimplantation blastocysts are thought to interact with the endometrium to facilitate implantation. Very little is known of the mechanisms by which this occurs and to our knowledge there is no information on whether human blastocysts facilitate blastocyst attachment to the endometrium. STUDY DESIGN, SIZE, DURATION: We used blastocyst-conditioned medium (BCM) from blastocysts that implanted (n = 28) and blastocysts that did not implant (n = 28) following IVF. Primary human endometrial epithelial cells (HEECs) (n = 3 experiments) were treated with BCM and the effect on gene expression and adhesion to trophoblast cells determined. We compared the protein production of selected genes in the endometrium of women with normal fertility (n = 40) and infertility (n = 6) during the receptive phase. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used real-time RT-PCR arrays containing 84 genes associated with the epithelial to mesenchymal transition. We validated selected genes by real-time RT-PCR (n = 3) and immunohistochemistry in the human endometrium (n = 46). Adhesion assays were performed using HEECs and a trophoblast cell line (n = 3). MAIN RESULTS AND THE ROLE OF CHANCE: Blastocysts that implanted released factors that differentially altered mRNA levels for six genes (>1.5 fold) compared with blastocysts that did not implant. A cohort of genes was validated at the protein level: SPARC and Jagged1 were down-regulated (P < 0.01), while SNAI2 and TGF-B1 were up-regulated (P < 0.05) by implanted compared with non-implanted BCM. Jagged-1 (P < 0.05) and Snai-2 protein (P < 0.01) showed cyclical changes in the endometrium across the cycle, and Jagged-1 staining differed in women with normal fertility versus infertility (only) (P < 0.01). HEEC adhesion to a trophoblast cell line was increased after treatment with implanted BCM compared with untreated control (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and it would be beneficial to validate our findings using a physiological model, such as mouse. WIDER IMPLICATIONS OF THE FINDINGS: This new strategy has identified novel pathways that may be important for human preimplantation blastocyst-endometrial interactions and opens the possibility of examining and manipulating specific pathways to improve implantation and pregnancy success. STUDY FUNDING/COMPETING INTEREST: This study was supported by the National Health and Medical Research Council of Australia (Fellowship support #550905, #611827) and project grants by Monash IVF, Australia. There are no conflicts of interest to be declared.

Uterine lavage or aspirate: which view of the intrauterine environment?

Fluid within the uterine cavity provides the microenvironment for preimplantation blastocyst development and early implantation. Analysis of uterine fluid sampled by aspiration or lavage provides a view of this microenvironment but the similarity or otherwise of the sample components is not known. This study compared proteins in aspirates versus lavage samples taken sequentially from the same women, using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), multiplex cytokine assays, and an activity assay for proprotein convertase 6. Both lavage and aspiration enabled analysis of uterine fluid components, but they provided substantially different protein profiles. Although there were many similarities in overall protein profiles and most specific proteins examined were detected in both fluids, these were neither qualitatively nor quantitatively comparable within each participant. A likely explanation is that lavage samples the entire uterine cavity including washing the endometrial surface (glycocalyx), whereas aspiration sampling is very local.

Analysis of fertility-related soluble mediators in human uterine fluid identifies VEGF as a key regulator of embryo implantation.

Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the midsecretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen contains important mediators that modulate the endometrium and support the conceptus during implantation. This study aimed first to identify the growth factor and cytokine profile of human uterine fluid from fertile women during the midproliferative (MP; nonreceptive) and MS (receptive) phases of the cycle, and from women with unexplained infertility during the MS phase. The second aim was to determine important functions of endometrial secretions for embryo implantation. Analysis of uterine fluid using quantitative Luminex assays revealed the presence of over 30 cytokines and growth factors, of which eight [platelet-derived growth factor-AA, TNF-B, soluble IL-2 receptor-A, Fms-like tyrosine kinase 3 ligand, soluble CD40 ligand, IL-7, interferon-A2, and chemokine (C-X-C motif) ligand 1-3] were previously unknown in human uterine fluid. Comparison of the fertile MP, MS, and infertile MS cohorts revealed vascular endothelial growth factor (VEGF) levels are significantly reduced in uterine fluid during the MS phase in women with unexplained infertility compared with fertile women. Functional studies demonstrated that culturing mouse embryos with either MS-phase uterine fluid from fertile women or recombinant human VEGF significantly enhanced blastocyst outgrowth. Furthermore, treatment of human endometrial epithelial cells with uterine fluid or recombinant human VEGF-A significantly increased endometrial epithelial cell adhesion. Taken together, our data support the concept that endometrial secretions, including VEGF, play important roles during implantation. Identifying the soluble mediators in human uterine fluid and their actions during implantation provides insight into interactions essential for establishing pregnancy, fertility markers, and infertility treatment options.

Preimplantation genetic screening outcomes are associated with culture conditions.

BACKGROUND: Although preimplantation genetic screening (PGS) is widely offered, there are contradictory reports on the clinical merit of this procedure. Any gain from embryo selection following aneuploidy screening must significantly outweigh the impact of the procedure. Variability of technical expertise in embryo biopsy, blastomere fixation, fluorescence in situ hybridization analysis, along with suboptimal laboratory quality control and inappropriate patient selection may impact PGS outcomes. METHODS: To investigate such effects, a total of 1508 stimulated in vitro fertilization (IVF) cycles were retrospectively analysed. During 2004, a significant change was made to the embryo culture media used. Clinical outcomes from cycles with PGS were compared prior to and after the change in media and compared with matched controls not utilizing PGS during the same period. RESULTS: Clinical PGS success rates were found to improve following the media change. For patients aged less than 40, clinical outcomes following PGS were significantly lower than those without PGS prior to the change, but became equivalent after the change. For patients >or=40 years and

Highly specific and sensitive rise in Days 14-17 pro-alphaC inhibin with clinical pregnancy after frozen embryo transfer with ovulatory cycles.

BACKGROUND: Pro-alphaC inhibins are luteal derived analytes peaking in the maternal serum as early as Day 16 after conception. We set out to verify a previous post hoc analysis which suggested that pro-alphaC levels measured this early are extremely sensitive in predicting clinical pregnancy success after unstimulated IVF with ovulatory cycles. METHODS: Prospective observational study of 246 women undergoing frozen embryo transfer with ovulatory cycles. Serum pro-alphaC and beta-HCG levels at 14-17 days after conception were measured by enzyme-linked immunosorbent assay and grouped according to whether a clinical pregnancy occurred (demonstrable cardiac activity at > or =6 weeks' gestation). RESULTS: Of 34 (13.8%) women who achieved a clinical pregnancy, median (25th-75th centile) Days 14-17 pro-alphaC levels were 995 pg/ml (758-1463), 6- to 7-fold higher than levels observed in the remainder who did not fall pregnant (112.8 pg/ml (104-121); P < 0.0001). At a fixed 95% specificity, pro-alphaC was 100% sensitive in predicting clinical pregnancy. The best specificities achieved at 100% sensitivity were; 94.8% for pro-alphaC, 96.7% for beta-HCG and 98.1% when both analytes were combined. CONCLUSIONS: Clinical pregnancy is always associated with a release of luteal derived pro-alphaC 14-17 days after conception. Pro-alphaC may play a possible biological role and be a useful clinical biomarker of luteal health.

GnRH antagonists in ovarian stimulation for IVF.

The present review describes, on the basis of the currently available evidence, the consensus reached by a group of experts on the use of gonadotropin-releasing hormone (GnRH) antagonists in ovarian stimulation for IVF. The single or multiple low-dose administration of GnRH antagonist during the late-follicular phase effectively prevents a premature rise in serum luteinizing hormone (LH) levels in most women. Although controversy remains, most comparative studies suggest a slight, not significant reduction in the probability of pregnancy after IVF using GnRH antagonist versus GnRH agonist co-treatment. Published meta-analyses suggest that this slight difference in pregnancy rates is not attributed to chance. Further studies applying varying treatment regimens and outcome measures are required. Data are not in favour of a need to modify the starting dose of gonadotropins. Data are not in favour of increasing gonadotropin dose at GnRH antagonist initiation. The addition of LH from the initiation of ovarian stimulation or from GnRH antagonist administration does not appear to be necessary. Replacement of human chorionic gonadotropin (HCG) by GnRH agonist for triggering final oocyte maturation is associated with a lower probability of pregnancy. The optimal timing for HCG administration needs to be explored further. GnRH antagonist initiation on day 6 of stimulation appears to be superior to flexible initiation by a follicle of 14-16 mm, although earlier GnRH antagonist administration is worth further evaluation. Luteal phase supplementation in GnRH antagonist protocols remains mandatory in IVF. Effects of GnRH antagonist co-treatment on the incidence of ovarian hyperstimulation syndrome remains uncertain, although a trend is present in favour of the GnRH antagonists. The role of GnRH antagonists in ovarian stimulation for IVF appears to be promising, although many questions regarding preferred dose regimens and effects on clinical outcomes remain.

Ultrasound prediction of risk of spontaneous miscarriage in live embryos from assisted conceptions.

OBJECTIVE: An accurate method to predict subsequent miscarriage in live embryos has not yet been established. This pilot study aimed to determine the most discriminatory ultrasound-based model for predicting spontaneous miscarriage after embryonic life was first detected in assisted conceptions. A method for estimating individual risk of miscarriage was developed. METHODS: This was a prospective cross-sectional survey of 322 live singleton embryos in women from an assisted reproductive technology program. Mean sac diameter (MSD), crown-rump length (CRL), embryonic heart rate (EHR), maternal age and gestational age at the first transvaginal scan detecting embryonic life (between 42 and 62 days) were observed. These variables were included in a multivariate model for predicting spontaneous miscarriage occurring prior to 20 weeks. MSD, CRL and MSD minus CRL were assessed in univariate logistic regression analyses. The global diagnostic accuracy of each model was compared directly using receiver-operating characteristics (ROC) curves. RESULTS: The multivariate model demonstrated the best ROC curve for predicting miscarriage (ROC area 0.87; 95% CI, 0.80-0.95). The separate univariate analyses had less diagnostic accuracy. In particular, MSD - CRL had a significantly smaller ROC area (0.65) than did the multivariate model (P < 0.01). CONCLUSIONS: The most discriminatory test for predicting spontaneous miscarriage in live embryos was a multivariate model, which allows estimation of individual risk levels.

Diagnostic accuracy of transvaginal hydrolaparoscopy in infertility.

OBJECTIVE: To determine whether the accuracy of transvaginal hydrolaparoscopy is comparable to that of standard laparoscopy for the diagnosis of infertility. DESIGN: Prospective, comparative study. SETTING: General hospital in Belgium. PATIENT(S): Ten infertile patients without obvious pelvic pathology. INTERVENTION(S): Two gynecologists independently performed transvaginal hydrolaparoscopy and standard laparoscopy and reported the observations in a confidential manner to a third person. MAIN OUTCOME MEASURE(S): Endometriosis, adhesions, and other pelvic pathology related to infertility. RESULT(S): Minimal or mild endometriosis was diagnosed in seven patients and sequelae of pelvic inflammatory disease in one patient at both procedures. The interobserver agreement for tuboovarian adhesions was 95% at transvaginal hydrolaparoscopy and 74% at standard laparoscopy. Ovarian adhesions were detected in 63% at transvaginal hydrolaparoscopy and in 37% at standard laparoscopy. CONCLUSION(S): Transvaginal hydrolaparoscopy is comparable in accuracy to standard laparoscopy for the diagnosis of adhesions and endometriosis in infertile patients without obvious pathology.