Clinical outcomes of uninterrupted embryo culture with or without time-lapse-based embryo selection versus interrupted standard culture (SelecTIMO): a three-armed, multicentre, double-blind, randomised controlled trial.

BACKGROUND: Time-lapse monitoring is increasingly used in fertility laboratories to culture and select embryos for transfer. This method is offered to couples with the promise of improving pregnancy chances, even though there is currently insufficient evidence for superior clinical results. We aimed to evaluate whether a potential improvement by time-lapse monitoring is caused by the time-lapse-based embryo selection method itself or the uninterrupted culture environment that is part of the system. METHODS: In this three-armed, multicentre, double-blind, randomised controlled trial, couples undergoing in-vitro fertilisation or intracytoplasmic sperm injection were recruited from 15 fertility clinics in the Netherlands and randomly assigned using a web-based, computerised randomisation service to one of three groups. Couples and physicians were masked to treatment group, but embryologists and laboratory technicians could not be. The time-lapse early embryo viability assessment (EEVA; TLE) group received embryo selection based on the EEVA time-lapse selection method and uninterrupted culture. The time-lapse routine (TLR) group received routine embryo selection and uninterrupted culture. The control group received routine embryo selection and interrupted culture. The co-primary endpoints were the cumulative ongoing pregnancy rate within 12 months in all women and the ongoing pregnancy rate after fresh single embryo transfer in a good prognosis population. Analysis was by intention to treat. This trial is registered on the ICTRP Search Portal, NTR5423, and is closed to new participants. FINDINGS: 1731 couples were randomly assigned between June 15, 2017, and March 31, 2020 (577 to the TLE group, 579 to the TLR group, and 575 to the control group). The 12-month cumulative ongoing pregnancy rate did not differ significantly between the three groups: 50·8% (293 of 577) in the TLE group, 50·9% (295 of 579) in the TLR group, and 49·4% (284 of 575) in the control group (p=0·85). The ongoing pregnancy rates after fresh single embryo transfer in a good prognosis population were 38·2% (125 of 327) in the TLE group, 36·8% (119 of 323) in the TLR group, and 37·8% (123 of 325) in the control group (p=0·90). Ten serious adverse events were reported (five TLE, four TLR, and one in the control group), which were not related to study procedures. INTERPRETATION: Neither time-lapse-based embryo selection using the EEVA test nor uninterrupted culture conditions in a time-lapse incubator improved clinical outcomes compared with routine methods. Widespread application of time-lapse monitoring for fertility treatments with the promise of improved results should be questioned. FUNDING: Health Care Efficiency Research programme from Netherlands Organisation for Health Research and Development and Merck.

Economic evaluation of endometrial scratching before the second IVF/ICSI treatment: a cost-effectiveness analysis of a randomized controlled trial (SCRaTCH trial).

STUDY QUESTION: Is a single endometrial scratch prior to the second fresh IVF/ICSI treatment cost-effective compared to no scratch, when evaluated over a 12-month follow-up period? SUMMARY ANSWER: The incremental cost-effectiveness ratio (ICER) for an endometrial scratch was €6524 per additional live birth, but due to uncertainty regarding the increase in live birth rate this has to be interpreted with caution. WHAT IS KNOWN ALREADY: Endometrial scratching is thought to improve the chances of success in couples with previously failed embryo implantation in IVF/ICSI treatment. It has been widely implemented in daily practice, despite the lack of conclusive evidence of its effectiveness and without investigating whether scratching allows for a cost-effective method to reduce the number of IVF/ICSI cycles needed to achieve a live birth. STUDY DESIGN, SIZE, DURATION: This economic evaluation is based on a multicentre randomized controlled trial carried out in the Netherlands (SCRaTCH trial) that compared a single scratch prior to the second IVF/ICSI treatment with no scratch in couples with a failed full first IVF/ICSI cycle. Follow-up was 12 months after randomization.Economic evaluation was performed from a healthcare and societal perspective by taking both direct medical costs and lost productivity costs into account. It was performed for the primary outcome of biochemical pregnancy leading to live birth after 12 months of follow-up as well as the secondary outcome of live birth after the second fresh IVF/ICSI treatment (i.e. the first after randomization). To allow for worldwide interpretation of the data, cost level scenario analysis and sensitivity analysis was performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: From January 2016 until July 2018, 933 women with a failed first IVF/ICSI cycle were included in the trial. Data on treatment and pregnancy were recorded up until 12 months after randomization, and the resulting live birth outcomes (even if after 12 months) were also recorded.Total costs were calculated for the second fresh IVF/ICSI treatment and for the full 12 month period for each participant. We included costs of all treatments, medication, complications and lost productivity costs. Cost-effectiveness analysis was carried out by calculating ICERs for scratch compared to control. Bootstrap resampling was used to estimate the uncertainty around cost and effect differences and ICERs. In the sensitivity and scenario analyses, various unit costs for a single scratch were introduced, amongst them, unit costs as they apply for the United Kingdom (UK). MAIN RESULTS AND THE ROLE OF CHANCE: More live births occurred in the scratch group, but this also came with increased costs over a 12-month period. The estimated chance of a live birth after 12 months of follow-up was 44.1% in the scratch group compared to 39.3% in the control group (risk difference 4.8%, 95% CI -1.6% to +11.2%). The mean costs were on average €283 (95% CI: -€299 to €810) higher in the scratch group so that the point average ICER was €5846 per additional live birth. The ICER estimate was surrounded with a high level of uncertainty, as indicated by the fact that the cost-effectiveness acceptability curve (CEAC) showed that there is an 80% chance that endometrial scratching is cost-effective if society is willing to pay ∼€17 500 for each additional live birth. LIMITATIONS, REASONS FOR CAUTION: There was a high uncertainty surrounding the effects, mainly in the clinical effect, i.e. the difference in the chance of live birth, which meant that a single straightforward conclusion could not be ascertained as for now. WIDER IMPLICATIONS OF THE FINDINGS: This is the first formal cost-effectiveness analysis of endometrial scratching in women undergoing IVF/ICSI treatment. The results presented in this manuscript cannot provide a clear-cut expenditure for one additional birth, but they do allow for estimating costs per additional live birth in different scenarios once the clinical effectiveness of scratching is known. As the SCRaTCH trial was the only trial with a follow-up of 12 months, it allows for the most complete estimation of costs to date. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by ZonMW, the Dutch organization for funding healthcare research. A.E.P.C., F.J.M.B., E.R.G. and C.B. L. reported having received fees or grants during, but outside of, this trial. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NL5193/NTR 5342).

Birthweight and other perinatal outcomes of singletons conceived after assisted reproduction compared to natural conceived singletons in couples with unexplained subfertility: follow-up of two randomized clinical trials.

STUDY QUESTION: Does assisted reproduction, such as ovarian stimulation and/or laboratory procedures, have impact on perinatal outcomes of singleton live births compared to natural conception in couples with unexplained subfertility? SUMMARY ANSWER: Compared to natural conception, singletons born after intrauterine insemination with ovarian stimulation (IUI-OS) had a lower birthweight, while singletons born after IVF had comparable birthweights, in couples with unexplained subfertility. WHAT IS KNOWN ALREADY: Singletons conceived by assisted reproduction have different perinatal outcomes such as low birthweight and a higher risk of premature birth than naturally conceived singletons. This might be due to the assisted reproduction, such as laboratory procedures or the ovarian stimulation, or to an intrinsic factor in couples with subfertility. STUDY DESIGN, SIZE, DURATION: We performed a prospective cohort study using the follow-up data of two randomized clinical trials performed in couples with unexplained subfertility. We evaluated perinatal outcomes of 472 live birth singletons conceived after assisted reproduction or after natural conception within the time horizon of the studies. PARTICIPANTS/MATERIALS, SETTING, METHODS: To assess the possible impact of ovarian stimulation we compared the singletons conceived after IUI with FSH or clomiphene citrate (CC) and IVF in a modified natural cycle (IVF-MNC) or standard IVF with single embryo transfer (IVF-SET) to naturally conceived singletons in the same cohorts. To further look into the possible effect of the laboratory procedures, we put both IUI and IVF groups together into IUI-OS and IVF and compared both to singletons born after natural conception. We only included singletons conceived after fresh embryo transfers. The main outcome was birthweight presented as absolute weight in grams and gestational age- and gender-adjusted percentiles. We calculated differences in birthweight using regression analyses adjusted for maternal age, BMI, smoking, parity, duration of subfertility and child gender. MAIN RESULTS AND THE ROLE OF CHANCE: In total, there were 472 live birth singletons. Of the 472 singleton pregnancies, 209 were conceived after IUI-OS (136 with FSH and 73 with CC as ovarian stimulation), 138 after IVF (50 after IVF-MNC and 88 after IVF-SET) and 125 were conceived naturally.Singletons conceived following IUI-FSH and IUI-CC both had lower birthweights compared to naturally conceived singletons (adjusted difference IUI-FSH -156.3 g, 95% CI -287.9 to -24.7; IUI-CC -160.3 g, 95% CI -316.7 to -3.8). When we compared IVF-MNC and IVF-SET to naturally conceived singletons, no significant difference was found (adjusted difference IVF-MNC 75.8 g, 95% CI -102.0 to 253.7; IVF-SET -10.6 g, 95% CI -159.2 to 138.1). The mean birthweight percentile was only significantly lower in the IUI-FSH group (-7.0 percentile, 95% CI -13.9 to -0.2). The IUI-CC and IVF-SET group had a lower mean percentile and the IVF-MNC group a higher mean percentile, but these groups were not significant different compared to the naturally conceived group (IUI-CC -5.1 percentile, 95% CI -13.3 to 3.0; IVF-MNC 4.4 percentile, 95% CI -4.9 to 13.6; IVF-SET -1.3 percentile, 95% CI -9.1 to 6.4).Looking at the laboratory process that took place, singletons conceived following IUI-OS had lower birthweights than naturally conceived singletons (adjusted difference -157.7 g, 95% CI -277.4 to -38.0). The IVF group had comparable birthweights with the naturally conceived group (adjusted difference 20.9 g, 95% CI -110.8 to 152.6). The mean birthweight percentile was significantly lower in the IUI-OS group compared to the natural group (-6.4 percentile, 95% CI -12.6 to -0.1). The IVF group was comparable (0.7 percentile, 95% CI -6.1 to 7.6). LIMITATIONS, REASONS FOR CAUTION: The results are limited by the number of cases. The data were collected prospectively alongside the randomized controlled trials, but analyzed as treated. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest IUI in a stimulated cycle may have a negative impact on the birthweight of the child and possibly on pre-eclampsia. Further research should look into the effect of different methods of ovarian stimulation on placenta pathology and pre-eclampsia in couples with unexplained subfertility using naturally conceived singletons in the unexplained population as a reference. STUDY FUNDING/COMPETING INTEREST(S): Both initial trials were supported by a grant from ZonMW, the Dutch Organization for Health Research and Development (INeS 120620027, SUPER 80-83600-98-10192). The INeS study also had a grant from Zorgverzekeraars Nederland, the Dutch association of healthcare insurers (09-003). B.W.J.M. is supported by an NHMRC investigator Grant (GNT1176437) and reports consultancy for ObsEva, Merck Merck KGaA, Guerbet and iGenomix, outside the submitted work. A.H. reports grants from Ferring Pharmaceutical company (the Netherlands), outside the submitted work. F.J.M.B. receives monetary compensation as a member of the external advisory board for Merck Serono (the Netherlands), Ferring Pharmaceutics BV (the Netherlands) and Gedeon Richter (Belgium), he receives personal fees from educational activities for Ferring BV (the Netherlands) and for advisory and consultancy work for Roche and he receives research support grants from Merck Serono and Ferring Pharmaceutics BV, outside the submitted work. The remaining authors have nothing to disclose. TRIAL REGISTRATION NUMBER: INeS study Trial NL915 (NTR939); SUPER Trial NL3895 (NTR4057).

Effect of parental and ART treatment characteristics on perinatal outcomes.

STUDY QUESTION: Do parental characteristics and treatment with ART affect perinatal outcomes in singleton pregnancies? SUMMARY ANSWER: Both parental and ART treatment characteristics affect perinatal outcomes in singleton pregnancies. WHAT IS KNOWN ALREADY: Previous studies have shown that singleton pregnancies resulting from ART are at risk of preterm birth. ART children are lighter at birth after correction for duration of gestation and at increased risk of congenital abnormalities compared to naturally conceived children. This association is confounded by parental characteristics that are also known to affect perinatal outcomes. It is unclear to which extent parental and ART treatment characteristics independently affect perinatal outcomes. STUDY DESIGN, SIZE, DURATION: All IVF clinics in the Netherlands (n = 13) were requested to provide data on all ART treatment cycles (IVF, ICSI and frozen-thawed embryo transfers (FET)), performed between 1 January 2000, and 1 January 2011, which resulted in a pregnancy. Using probabilistic data-linkage, these data (n = 36 683) were linked to the Dutch Perinatal Registry (Perined), which includes all children born in the Netherlands in the same time period (n = 2 548 977). PARTICIPANTS/MATERIALS, SETTING, METHODS: Analyses were limited to singleton pregnancies that resulted from IVF, ICSI or FET cycles. Multivariable models for linear and logistic regression were fitted including parental characteristics as well as ART treatment characteristics. Analyses were performed separately for fresh cycles and for fresh and FET cycles combined. We assessed the impact on the following perinatal outcomes: birth weight, preterm birth below 37 or 32 weeks of gestation, congenital malformations and perinatal mortality. MAIN RESULTS AND THE ROLE OF CHANCE: The perinatal outcomes of 31 184 out of the 36 683 ART treatment cycles leading to a pregnancy were retrieved through linkage with the Perined (85% linkage). Of those, 23 671 concerned singleton pregnancies resulting from IVF, ICSI or FET. Birth weight was independently associated with both parental and ART treatment characteristics. Characteristics associated with lower birth weight included maternal hypertensive disease, non-Dutch maternal ethnicity, nulliparity, increasing duration of subfertility, hCG for luteal phase support (compared to progesterone), shorter embryo culture duration, increasing number of oocytes retrieved and fresh embryo transfer. The parental characteristic with the greatest effect size on birth weight was maternal diabetes (adjusted difference 283 g, 95% CI 228-338). FET was the ART treatment characteristic with the greatest effect size on birth weight (adjusted difference 100 g, 95% CI 84-117) compared to fresh embryo transfer. Preterm birth was more common among mothers of South-Asian ethnicity. Preterm birth was less common among multiparous women and women with 'male factor' as treatment indication (compared to 'tubal factor'). LIMITATIONS, REASONS FOR CAUTION: Due to the retrospective nature of our study, we cannot prove causality. Further limitations of our study were the inability to adjust for mothers giving birth more than once in our dataset, missing values for several variables and limited information on parental lifestyle and general health. WIDER IMPLICATIONS OF THE FINDINGS: Multiple parental and ART treatment characteristics affect perinatal outcomes, with birth weight being influenced by the widest range of factors. This highlights the importance of assessing both parental and ART treatment characteristics in studies that focus on the health of ART-offspring, with the purpose of modifying these factors where possible. Our results further support the hypothesis that the embryo is sensitive to its early environment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Foreest Medical School, Alkmaar, the Netherlands (grants: FIO 1307 and FIO 1505). B.W.M. reports grants from NHMRC and consultancy for ObsEva, Merck KGaA, iGenomics and Guerbet. F.B. reports research support grants from Merck Serono and personal fees from Merck Serono. A.C. reports travel support from Ferring BV. and Theramex BV. and personal fees from UpToDate (Hyperthecosis), all outside the remit of the current work. The remaining authors report no conflict of interests. TRIAL REGISTRATION NUMBER: N/A.

Endometrial scratching in women with one failed IVF/ICSI cycle-outcomes of a randomised controlled trial (SCRaTCH).

STUDY QUESTION: Does endometrial scratching in women with one failed IVF/ICSI treatment affect the chance of a live birth of the subsequent fresh IVF/ICSI cycle? SUMMARY ANSWER: In this study, 4.6% more live births were observed in the scratch group, with a likely certainty range between -0.7% and +9.9%. WHAT IS KNOWN ALREADY: Since the first suggestion that endometrial scratching might improve embryo implantation during IVF/ICSI, many clinical trials have been conducted. However, due to limitations in sample size and study quality, it remains unclear whether endometrial scratching improves IVF/ICSI outcomes. STUDY DESIGN, SIZE, DURATION: The SCRaTCH trial was a non-blinded randomised controlled trial in women with one unsuccessful IVF/ICSI cycle and assessed whether a single endometrial scratch using an endometrial biopsy catheter would lead to a higher live birth rate after the subsequent IVF/ICSI treatment compared to no scratch. The study took place in 8 academic and 24 general hospitals. Participants were randomised between January 2016 and July 2018 by a web-based randomisation programme. Secondary outcomes included cumulative 12-month ongoing pregnancy leading to live birth rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with one previous failed IVF/ICSI treatment and planning a second fresh IVF/ICSI treatment were eligible. In total, 933 participants out of 1065 eligibles were included (participation rate 88%). MAIN RESULTS AND THE ROLE OF CHANCE: After the fresh transfer, 4.6% more live births were observed in the scratch compared to control group (110/465 versus 88/461, respectively, risk ratio (RR) 1.24 [95% CI 0.96-1.59]). These data are consistent with a true difference of between -0.7% and +9.9% (95% CI), indicating that while the largest proportion of the 95% CI is positive, scratching could have no or even a small negative effect. Biochemical pregnancy loss and miscarriage rate did not differ between the two groups: in the scratch group 27/153 biochemical pregnancy losses and 14/126 miscarriages occurred, while this was 19/130 and 17/111 for the control group (RR 1.21 (95% CI 0.71-2.07) and RR 0.73 (95% CI 0.38-1.40), respectively). After 12 months of follow-up, 5.1% more live births were observed in the scratch group (202/467 versus 178/466), of which the true difference most likely lies between -1.2% and +11.4% (95% CI). LIMITATIONS, REASONS FOR CAUTION: This study was not blinded. Knowledge of allocation may have been an incentive for participants allocated to the scratch group to continue treatment in situations where they may otherwise have cancelled or stopped. In addition, this study was powered to detect a difference in live birth rate of 9%. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study are an incentive for further assessment of the efficacy and clinical implications of endometrial scratching. If a true effect exists, it may be smaller than previously anticipated or may be limited to specific groups of women undergoing IVF/ICSI. Studying this will require larger sample sizes, which will be provided by the ongoing international individual participant data-analysis (PROSPERO CRD42017079120). At present, endometrial scratching should not be performed outside of clinical trials. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by ZonMW, the Dutch organisation for funding healthcare research. J.S.E. Laven reports grants and personal fees from AnshLabs (Webster, Tx, USA), Ferring (Hoofddorp, The Netherlands) and Ministry of Health (CIBG, The Hague, The Netherlands) outside the submitted work. A.E.P. Cantineau reports 'other' from Ferring BV, personal fees from Up to date Hyperthecosis, 'other' from Theramex BV, outside the submitted work. E.R. Groenewoud reports grants from Titus Health Care during the conduct of the study. A.M. van Heusden reports personal fees from Merck Serono, personal fees from Ferring, personal fees from Goodlife, outside the submitted work. F.J.M. Broekmans reports personal fees as Member of the external advisory board for Ferring BV, The Netherlands, personal fees as Member of the external advisory board for Merck Serono, The Netherlands, personal fees as Member of the external advisory for Gedeon Richter, Belgium, personal fees from Educational activities for Ferring BV, The Netherlands, grants from Research support grant Merck Serono, grants from Research support grant Ferring, personal fees from Advisory and consultancy work Roche, outside the submitted work. C.B. Lambalk reports grants from Ferring, grants from Merck, grants from Guerbet, outside the submitted work. TRIAL REGISTRATION NUMBER: Registered in the Netherlands Trial Register (NL5193/NTR 5342). TRIAL REGISTRATION DATE: 31 July 2015. DATE OF FIRST PATIENT'S ENROLMENT: 26 January 2016.

The vaginal microbiome as a predictor for outcome of in vitro fertilization with or without intracytoplasmic sperm injection: a prospective study.

STUDY QUESTION: Is the presence or absence of certain vaginal bacteria associated with failure or success to become pregnant after an in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (IVF-ICSI) treatment? SUMMARY ANSWER: Microbiome profiling with the use of interspace profiling (IS-pro) technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. WHAT IS KNOWN ALREADY: Live-birth rates for an IVF or IVF-ICSI treatment vary between 25 and 35% per cycle and it is difficult to predict who will or will not get pregnant after embryo transfer (ET). Recently, it was suggested that the composition of the vaginal microbiota prior to treatment might predict pregnancy outcome. Analysis of the vaginal microbiome prior to treatment might, therefore, offer an opportunity to improve the success rate of IVF or IVF-ICSI. STUDY DESIGN, SIZE, DURATION: In a prospective cohort study, 303 women (age, 20-42 years) undergoing IVF or IVF-ICSI treatment in the Netherlands were included between June 2015 and March 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Study subjects provided a vaginal sample before the start of the IVF or IVF-ICSI procedure. The vaginal microbiota composition was determined using the IS-pro technique. IS-pro is a eubacterial technique based on the detection and categorization of the length of the 16S-23S rRNA gene interspace region. Microbiome profiles were assigned to community state types based on the dominant bacterial species. The predictive accuracy of the microbiome profiles for IVF and IVF-ICSI outcome of fresh ET was evaluated by a combined prediction model based on a small number of bacterial species. From this cohort, a model was built to predict outcome of fertility treatment. This model was externally validated in a cohort of 50 women who were undergoing IVF or IVF-ICSI treatment between March 2018 and May 2018 in the Dutch division of the MVZ VivaNeo Kinderwunschzentrum Düsseldorf, Germany. MAIN RESULTS AND THE ROLE OF CHANCE: In total, the vaginal microbiota of 192 women who underwent a fresh ET could be analysed. Women with a low percentage of Lactobacillus in their vaginal sample were less likely to have a successful embryo implantation. The prediction model identified a subgroup of women (17.7%, n = 34) who had a low chance to become pregnant following fresh ET. This failure was correctly predicted in 32 out of 34 women based on the vaginal microbiota composition, resulting in a predictive accuracy of 94% (sensitivity, 26%; specificity, 97%). Additionally, the degree of dominance of Lactobacillus crispatus was an important factor in predicting pregnancy. Women who had a favourable profile as well as <60% L. crispatus had a high chance of pregnancy: more than half of these women (50 out of 95) became pregnant. In the external validation cohort, none of the women who had a negative prediction (low chance of pregnancy) became pregnant. LIMITATIONS, REASONS FOR CAUTION: Because our study uses a well-defined study population, the results will be limited to the IVF or IVF-ICSI population. Whether these results can be extrapolated to the general population trying to achieve pregnancy without ART cannot be determined from these data. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that vaginal microbiome profiling using the IS-pro technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. Knowledge of their vaginal microbiota may enable couples to make a more balanced decision regarding timing and continuation of their IVF or IVF-ICSI treatment cycles. STUDY FUNDING/COMPETING INTEREST(S): This study was financed by NGI Pre-Seed 2014-2016, RedMedTech Discovery Fund 2014-2017, STW Valorisation grant 1 2014-2015, STW Take-off early phase trajectory 2015-2016 and Eurostars VALBIOME grant (reference number: 8884). The employer of W.J.S.S.C. has in collaboration with ARTPred acquired a MIND subsidy to cover part of the costs of this collaboration project. The following grants are received but not used to finance this study: grants from Innovatie Prestatie Contract, MIT Haalbaarheid, other from Dutch R&D tax credit WBSO, RedMedTech Discovery Fund, (J.D.d.J.). Grants from Ferring (J.S.E.L., K.F., C.B.L. and J.M.J.S.S.), Merck Serono (K.F. and C.B.L.), Dutch Heart Foundation (J.S.E.L.), Metagenics Inc. (J.S.E.L.), GoodLife (K.F.), Guerbet (C.B.L.). R.K. is employed by ARTPred B.V. during her PhD at Erasmus Medical Centre (MC). S.A.M. has a 100% University appointment. I.S.P.H.M.S., S.A.M. and A.E.B. are co-owners of IS-Diagnostics Ltd. J.D.d.J. is co-owner of ARTPred B.V., from which he reports personal fees. P.H.M.S. reports non-financial support from ARTPred B.V. P.H.M.S., J.D.d.J. and A.E.B. have obtained patents `Microbial population analysis' (9506109) and `Microbial population analysis' (20170159108), both licenced to ARTPred B.V. J.D.d.J. and A.E.B. report patent applications `Method and kit for predicting the outcome of an assisted reproductive technology procedure' (392EPP0) and patent `Method and kit for altering the outcome of an assisted reproductive technology procedure' by ARTPred. W.J.S.S.C. received personal consultancy and educational fees from Goodlife Fertility B.V. J.S.E.L. reports personal consultancy fees from ARTPred B.V., Titus Health B.V., Danone, Euroscreen and Roche during the conduct of the study. J.S.E.L. and N.G.M.B. are co-applicants on an Erasmus MC patent (New method and kit for prediction success of in vitro fertilization) licenced to ARTPred B.V. F.J.M.B. reports personal fees from Advisory Board Ferring, Advisory Board Merck Serono, Advisory Board Gedeon Richter and personal fees from Educational activities for Ferring, outside the submitted work. K.F. reports personal fees from Ferring (commercial sponsor) and personal fees from GoodLife (commercial sponsor). C.B.L. received speakers' fee from Ferring. J.M.J.S.S. reports personal fees and other from Merck Serono and personal fees from Ferring, unrelated to the submitted paper. The other authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: ISRCTN83157250. Registered 17 August 2018. Retrospectively registered.

Predicting the cumulative chance of live birth over multiple complete cycles of in vitro fertilization: an external validation study.

STUDY QUESTION: Are the published pre-treatment and post-treatment McLernon models, predicting cumulative live birth rates (LBR) over multiple complete IVF cycles, valid in a different context? SUMMARY ANSWER: With minor recalibration of the pre-treatment model, both McLernon models accurately predict cumulative LBR in a different geographical context and a more recent time period. WHAT IS KNOWN ALREADY: Previous IVF prediction models have estimated the chance of a live birth after a single fresh embryo transfer, thereby excluding the important contribution of embryo cryopreservation and subsequent IVF cycles to cumulative LBR. In contrast, the recently developed McLernon models predict the cumulative chance of a live birth over multiple complete IVF cycles at two certain time points: (i) before initiating treatment using baseline characteristics (pre-treatment model) and (ii) after the first IVF cycle adding treatment related information to update predictions (post-treatment model). Before implementation of these models in clinical practice, their predictive performance needs to be validated in an independent cohort. STUDY DESIGN, SIZE, DURATION: External validation study in an independent prospective cohort of 1515 Dutch women who participated in the OPTIMIST study (NTR2657) and underwent their first IVF treatment between 2011 and 2014. Participants underwent a total of 2881 complete treatment cycles, with a complete cycle defined as all fresh and frozen thawed embryo transfers resulting from one episode of ovarian stimulation. The follow up duration was 18 months after inclusion, and the primary outcome was ongoing pregnancy leading to live birth. PARTICIPANTS/MATERIALS, SETTING, METHODS: Model performance was externally validated up to three complete treatment cycles, using the linear predictor as described by McLernon et al. to calculate the probability of a live birth. Discrimination was expressed by the c-statistic and calibration was depicted graphically in a calibration plot. In contrast to the original model development cohort, anti-Müllerian hormone (AMH), antral follicle count (AFC) and body weight were available in the OPTIMIST cohort, and evaluated as potential additional predictors for model improvement. MAIN RESULTS AND THE ROLE OF CHANCE: Applying the McLernon models to the OPTIMIST cohort, the c-statistic of the pre-treatment model was 0.62 (95% CI: 0.59-0.64) and of the post-treatment model 0.71 (95% CI: 0.69-0.74). The calibration plot of the pre-treatment model indicated a slight overestimation of the cumulative LBR. To improve calibration, the pre-treatment model was recalibrated by subtracting 0.35 from the intercept. The post-treatment model calibration plot revealed accurate cumulative LBR predictions. After addition of AMH, AFC and body weight to the McLernon models, the c-statistic of the updated pre-treatment model improved slightly to 0.66 (95% CI: 0.64-0.68), and of the updated post-treatment model remained at the previous level of 0.71 (95% CI: 0.69-0.73). Using the recalibrated pre-treatment model, a woman aged 30 years with 2 years of primary infertility who starts ICSI treatment for male factor infertility has a chance of 40% of a live birth from the first complete cycle, increasing to 72% over three complete cycles. If this woman weighs 70 kg, has an AMH of 1.5 ng/mL and an AFC of 10 measured at the beginning of her treatment, the updated pre-treatment model revises the estimated chance of a live birth to 30% in the first complete cycle and 59% over three complete cycles. If this woman then has five retrieved oocytes, no embryos cryopreserved and a single fresh cleavage stage embryo transfer in her first ICSI cycle, the post-treatment model estimates the chances of a live birth at 28 and 58%, respectively. LIMITATIONS, REASONS FOR CAUTION: Two randomized controlled trials (RCT) evaluating the effectiveness of gonadotropin dose individualization on basis of the AFC were nested within the OPTIMIST study. The strict dosing regimens, the RCT in- and exclusion criteria and the limited follow up time of 18 months might have influenced model performance in this independent cohort. Also, consistent with the original model development study, external validation was performed using the optimistic assumption that the cumulative LBR in couples who discontinue treatment without a live birth would have been equal to that of those who continue treatment. WIDER IMPLICATIONS OF THE FINDINGS: After national recalibration to account for geographical differences in IVF treatment, the McLernon prediction models can be introduced as new counselling tools in clinical practice to inform patients and to complement clinical reasoning. These models are the first to offer an objective and personalized estimate of the cumulative probability of a live birth over multiple complete IVF cycles. STUDY FUNDING/COMPETING INTEREST(S): No external funds were obtained for this study. M.J.C.E., D.J.M. and S.B. have nothing to disclose. J.A.L, S.C.O, T.C.v.T. and H.LT. received an unrestricted personal grant from Merck BV. B.W.M. is supported by a NHMRC Practitioner Fellowship (GNT1082548) and reports consultancy for ObsEva, Merck and Guerbet. F.J.M.B. receives monetary compensation as a member of the external advisory board for Merck BV (the Netherlands) and Ferring pharmaceutics BV (the Netherlands), for consultancy work for Gedeon Richter (Belgium) and Roche Diagnostics on automated AMH assay development, and for a research cooperation with Ansh Labs (USA). TRIAL REGISTRATION NUMBER: Not applicable.

Follicle stimulating hormone versus clomiphene citrate in intrauterine insemination for unexplained subfertility: a randomized controlled trial.

STUDY QUESTION: Is FSH or clomiphene citrate (CC) the most effective stimulation regimen in terms of ongoing pregnancies in couples with unexplained subfertility undergoing IUI with adherence to strict cancellation criteria as a measure to reduce the number of multiple pregnancies? SUMMARY ANSWER: In IUI with adherence to strict cancellation criteria, ovarian stimulation with FSH is not superior to CC in terms of the cumulative ongoing pregnancy rate, and yields a similar, low multiple pregnancy rate. WHAT IS ALREADY KNOWN: FSH has been shown to result in higher pregnancy rates compared to CC, but at the cost of high multiple pregnancy rates. To reduce the risk of multiple pregnancy, new ovarian stimulation regimens have been suggested, these include strict cancellation criteria to limit the number of dominant follicles per cycle i.e. withholding insemination when more than three dominant follicles develop. With such a strategy, it is unclear whether the ovarian stimulation should be done with FSH or with CC. STUDY DESIGN, SIZE, DURATION: We performed an open-label multicenter randomized superiority controlled trial in the Netherlands (NTR 4057). PARTICIPANTS/MATERIALS, SETTING, METHODS: We randomized couples diagnosed with unexplained subfertility and scheduled for a maximum of four cycles of IUI with ovarian stimulation with 75 IU FSH or 100 mg CC. Cycles were cancelled when more then three dominant follicles developed. The primary outcome was cumulative ongoing pregnancy rate. Multiple pregnancy was a secondary outcome. We analysed the data on intention to treat basis. We calculated relative risks and absolute risk difference with 95% CI. MAIN RESULTS AND THE ROLE OF CHANCE: Between July 2013 and March 2016, we allocated 369 women to ovarian stimulation with FSH and 369 women to ovarian stimulation with CC. A total of 113 women (31%) had an ongoing pregnancy following ovarian stimulation with FSH and 97 women (26%) had an ongoing pregnancy following ovarian stimulation with CC (RR = 1.16, 95% CI: 0.93-1.47, ARD = 0.04, 95% CI: -0.02 to 0.11). Five women (1.4%) had a multiple pregnancy following ovarian stimulation with FSH and eight women (2.2%) had a multiple pregnancy following ovarian stimulation with CC (RR = 0.63, 95% CI: 0.21-1.89, ARD = -0.01, 95% CI: -0.03 to 0.01). LIMITATIONS, REASONS FOR CAUTION: We were not able to blind this study due to the nature of the interventions. We consider it unlikely that this has introduced performance bias, since pregnancy outcomes are objective outcome measures. WIDER IMPLICATIONS OF THE FINDINGS: We revealed that adherence to strict cancellation criteria is a successful solution to reduce the number of multiple pregnancies in IUI. To decide whether ovarian stimulation with FSH or with CC should be the regimen of choice, costs and patients' preferences should be taken into account. STUDY FUNDING/COMPETING INTEREST(S): This trial received funding from the Dutch Organization for Health Research and Development (ZonMw). Prof. Dr B.W.J. Mol is supported by a NHMRC Practitioner Fellowship (GNT1082548). B.W.M. reports consultancy for Merck, ObsEva and Guerbet. The other authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: Nederlands Trial Register NTR4057. TRIAL REGISTRATION DATE: 1 July 2013. DATE OF FIRST PATIENT'S ENROLMENT: The first patient was randomized at 27 August 2013.

Does endometrial scratching increase the rate of spontaneous conception in couples with unexplained infertility and a good prognosis (Hunault > 30%)? Study protocol of the SCRaTCH-OFO trial: a randomized controlled trial.

BACKGROUND: In the Netherlands, couples with unexplained infertility and a good prognosis to conceive spontaneously (i.e. Hunault > 30%) are advised to perform timed intercourse for at least another 6 months. If couples fail to conceive within this period, they will usually start assisted reproductive technology (ART). However, treatment of unexplained infertility by ART is empirical and can involve significant burdens. Intentional endometrial injury, also called 'endometrial scratching', has been proposed to positively affect the chance of embryo implantation in patients undergoing in vitro fertilization (IVF). It might also be beneficial for couples with unexplained infertility as defective endometrial receptivity may play a role in these women. The primary aim of this study is to determine whether endometrial scratching increases live birth rates in women with unexplained infertility. METHOD: A multicentre randomized controlled trial will be conducted in Dutch academic and non-academic hospitals starting from November 2017. A total of 792 women with unexplained infertility and a good prognosis for spontaneous conception < 12 months (Hunault > 30%) will be included, of whom half will undergo endometrial scratching in the luteal phase of the natural cycle. The women in the control group will not undergo endometrial scratching. According to Dutch guidelines, both groups will subsequently perform timed intercourse for at least 6 months. The primary endpoint is cumulative live birth rate. Secondary endpoints are clinical and ongoing pregnancy rate; miscarriage rate; biochemical pregnancy loss; multiple pregnancy rate; time to pregnancy; progression to intrauterine insemination (IUI) or IVF; pregnancy complications; complications of endometrial scratching; costs and endometrial tissue parameters associated with reproductive success or failure. The follow-up duration is 12 months. DISCUSSION: Several small studies show a possible beneficial effect of endometrial scratching in women with unexplained infertility trying to conceive naturally or through IUI. However, the quality of this evidence is very low, making it unclear whether these women will truly benefit from this procedure. The SCRaTCH-OFO trial aims to investigate the effect of endometrial scratching on live birth rate in women with unexplained infertility and a good prognosis for spontaneous conception < 12 months. TRIAL REGISTRATION: NTR6687 , registered August 31st, 2017. PROTOCOL VERSION: Version 2.6, November 14th, 2018.

Unraveling the associations of age and menopause with cardiovascular risk factors in a large population-based study.

BACKGROUND: Although the association between menopause and cardiovascular disease (CVD) risk has been studied extensively, the simultaneous role of chronological aging herein remains underexposed. This study aims to disentangle the relationships of menopausal status and chronological aging with CVD risk factors in the largest study population to date. METHODS: In this cross-sectional study, CVD risk factors were compared between women with a different menopausal status within the same yearly age strata. The study population comprised female participants of the baseline visit of the population-based LifeLines Cohort Study. A total of 63,466 women, aged between 18 and 65 years, was included. Of them, 39,379 women were considered to be premenopausal, 8669 were perimenopausal, 14,514 were naturally postmenopausal, and 904 were surgically postmenopausal. RESULTS: Compared to postmenopausal women aged 45 years, average total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) were 0.5 and 0.4 mmol/L higher, respectively, in postmenopausal women aged 50. Systolic and diastolic blood pressure levels were 4 and 1 mmHg higher, respectively. At all ages between 46 and 55 years, and after adjustment for confounders, naturally postmenopausal women had 0.2 to 0.4 mmol/L higher TC and 0.1 to 0.3 mmol/L higher LDL-c levels compared to premenopausal women in the same age range. Systolic blood pressure levels were up to 4 mmHg lower in naturally post- compared to premenopausal women at all ages between 29 and 52 years. Body mass index levels were up to 3.2 kg/m2 higher in women with surgical menopause compared to all other women between the ages 32 and 52 years. All aforementioned results were statistically significant. CONCLUSIONS: Chronological age and menopausal status are both independently associated with CVD risk factors. Based on the comparatively smaller observed differences associated with menopausal status than with chronological aging, the significance of a more unfavorable lipid profile in a later reproductive stage may be less obvious than previously thought.

Implementing targeted expectant management in fertility care using prognostic modelling: a cluster randomized trial with a multifaceted strategy.

STUDY QUESTION: What is the effectiveness of a multifaceted implementation strategy compared to usual care on improving the adherence to guideline recommendations on expectant management for couples with unexplained infertility? SUMMARY ANSWER: The multifaceted implementation strategy did not significantly increase adherence to guideline recommendations on expectant management compared to care as usual. WHAT IS KNOWN ALREADY: Intrauterine insemination (IUI) with or without ovarian hyperstimulation has no beneficial effect compared to no treatment for 6 months after the fertility work-up for couples with unexplained infertility and a good prognosis of natural conception. Therefore, various professionals and policy makers have advocated the use of prognostic profiles and expectant management in guideline recommendations. STUDY DESIGN, SIZE, DURATION: A cluster randomized controlled trial in 25 clinics in the Netherlands was conducted between March 2013 and May 2014. Clinics were randomized between the implementation strategy (intervention, n = 13) and care as usual (control, n = 12). The effect of the implementation strategy was evaluated by comparing baseline and effect measurement data. Data collection was retrospective and obtained from medical record research and a patient questionnaire. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 544 couples were included at baseline and 485 at the effect measurement (247 intervention group/238 control group). MAIN RESULTS AND THE ROLE OF CHANCE: Guideline adherence increased from 49 to 69% (OR 2.66; 95% CI 1.45-4.89) in the intervention group, and from 49 to 61% (OR 2.03; 95% CI 1.38-3.00) in the control group. Multilevel analysis with case-mix adjustment showed that the difference of 8% was not statistically significant (OR 1.31; 95% CI 0.67-2.59). The ongoing pregnancy rate within six months after fertility work-up did not significantly differ between intervention and control group (25% versus 27%: OR 0.72; 95% CI 0.40-1.27). LIMITATIONS REASONS FOR CAUTION: There is a possible selection bias, couples included in the study had a higher socio-economic status than non-responders. How this affects guideline adherence is unclear. Furthermore, when powering for this study we did not take into account the unexpected improvement of adherence in the control group. WIDER IMPLICATIONS OF THE FINDINGS: Generalization of our results to other countries with recommendations on expectant management might be questionable because barriers for expectant management can be very different in other countries. Furthermore, due to a large variation in improved adherence rate in the intervention group it will be interesting to further analyse the process of implementation in each clinic with a process evaluation on professionals and couples' exposure to and experiences with the strategy. STUDY FUNDING/COMPETING INTEREST(S): Supported by Netherlands Organisation for Health Research and Development (ZonMW, project number 171203005). No competing interests. TRIAL REGISTRATION NUMBER: Dutch trial Register, www.trialregister.nl NTR3405. TRIAL REGISTRATION DATE: 19 April 2012. DATE OF FIRST PATIENT'S ENROLMENT: 10 July 2012.

Endometrial scratching in women with implantation failure after a first IVF/ICSI cycle; does it lead to a higher live birth rate? The SCRaTCH study: a randomized controlled trial (NTR 5342).

BACKGROUND: Success rates of assisted reproductive techniques (ART) are approximately 30%, with the most important limiting factor being embryo implantation. Mechanical endometrial injury, also called 'scratching', has been proposed to positively affect the chance of implantation after embryo transfer, but the currently available evidence is not yet conclusive. The primary aim of this study is to determine the effect of endometrial scratching prior to a second fresh in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle on live birth rates in women with a failed first IVF/ICSI cycle. METHOD: Multicenter randomized controlled trial in Dutch academic and non-academic hospitals. A total of 900 women will be included of whom half will undergo an endometrial scratch in the luteal phase of the cycle prior to controlled ovarian hyperstimulation using an endometrial biopsy catheter. The primary endpoint is the live birth rate after the 2nd fresh IVF/ICSI cycle. Secondary endpoints are costs, cumulative live birth rate (after the full 2nd IVF/ICSI cycle and over 12 months of follow-up); clinical and ongoing pregnancy rate; multiple pregnancy rate; miscarriage rate and endometrial tissue parameters associated with implantation failure. DISCUSSION: Multiple studies have been performed to investigate the effect of endometrial scratching on live birth rates in women undergoing IVF/ICSI cycles. Due to heterogeneity in both the method and population being scratched, it remains unclear which group of women will benefit from the procedure. The SCRaTCH trial proposed here aims to investigate the effect of endometrial scratching prior to controlled ovarian hyperstimulation in a large group of women undergoing a second IVF/ICSI cycle. TRIAL REGISTRATION: NTR 5342 , registered July 31st, 2015. PROTOCOL VERSION: Version 4.10, January 4th, 2017.

Anti-Müllerian hormone does not predict time to pregnancy: results of a prospective cohort study.

In order to study whether ovarian reserve tests (ORTs) can predict time to ongoing pregnancy, we conducted a prospective cohort study in a cohort of healthy pregnancy planners. A total of 102 pregnancy planners were followed for 1 year, or until ongoing pregnancy occurred, after cessation of contraceptives). A baseline measurement of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH) and antral follicle count (AFC) was conducted. At the end of follow-up, a semen analysis was performed and chlamydia antibody titres were assessed. A univariate prediction model demonstrated age and the AFC to be significantly capable of predicting time to pregnancy (hazard ratio 0.92, 95% CI 0.87-0.98, p = 0.01; 1.04, 95% CI 1.01-1.07, p = 0.02 respectively). In the multivariate model, however, correcting for female age, we found no predictive effect of AMH, basal FSH or the AFC for time to ongoing pregnancy (hazard ratios 1.43, 95% CI 0.84-2.46, p = 0.36; 0.96, 95% CI 0.86-1.06, p = 0.43; 1.03, 95% CI 1.00-1.07, p = 0.08, respectively). This was confirmed by the low C-statistic. We therefore concluded that baseline AMH, AFC or FSH levels do not predict time to ongoing pregnancy in a cohort of healthy pregnancy planners. These results limit the usability of these ORTs in the assessment of current fertility.

BRCA1 mutation carriers have a lower number of mature oocytes after ovarian stimulation for IVF/PGD.

PURPOSE: The aim of this study was to determine whether BRCA1/2 mutation carriers produce fewer mature oocytes after ovarian stimulation for in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD), in comparison to a PGD control group. METHODS: A retrospective, international, multicenter cohort study was performed on data of first PGD cycles performed between January 2006 and September 2015. Data were extracted from medical files. The study was performed in one PGD center and three affiliated IVF centers in the Netherlands and one PGD center in Belgium. Exposed couples underwent PGD because of a pathogenic BRCA1/2 mutation, controls for other monogenic conditions. Only couples treated in a long gonadotropin-releasing hormone (GnRH) agonist-suppressive protocol, stimulated with at least 150 IU follicle stimulating hormone (FSH), were included. Women suspected to have a diminished ovarian reserve status due to chemotherapy, auto-immune disorders, or genetic conditions (other than BRCA1/2 mutations) were excluded. A total of 106 BRCA1/2 mutation carriers underwent PGD in this period, of which 43 (20 BRCA1 and 23 BRCA2 mutation carriers) met the inclusion criteria. They were compared to 174 controls selected by frequency matching. RESULTS: Thirty-eight BRCA1/2 mutation carriers (18 BRCA1 and 20 BRCA2 mutation carriers) and 154 controls proceeded to oocyte pickup. The median number of mature oocytes was 7.0 (interquartile range (IQR) 4.0-9.0) in the BRCA group as a whole, 6.5 (IQR 4.0-8.0) in BRCA1 mutation carriers, 7.5 (IQR 5.5-9.0) in BRCA2 mutation carriers, and 8.0 (IQR 6.0-11.0) in controls. Multiple linear regression analysis with the number of mature oocytes as a dependent variable and adjustment for treatment center, female age, female body mass index (BMI), type of gonadotropin used, and the total dose of gonadotropins administered revealed a significantly lower yield of mature oocytes in the BRCA group as compared to controls (p = 0.04). This finding could be fully accounted for by the BRCA1 subgroup (BRCA1 mutation carriers versus controls p = 0.02, BRCA2 mutation carriers versus controls p = 0.50). CONCLUSIONS: Ovarian response to stimulation, expressed as the number of mature oocytes, was reduced in BRCA1 but not in BRCA2 mutation carriers. Although oocyte yield was in correspondence to a normal response in all subgroups, this finding points to a possible negative influence of the BRCA1 gene on ovarian reserve.

Back to the basics of ovarian aging: a population-based study on longitudinal anti-Müllerian hormone decline.

BACKGROUND: Anti-Müllerian hormone (AMH) is currently used as an ovarian reserve marker for individualized fertility counseling, but very little is known of individual AMH decline in women. This study assessed whether the decline trajectory of AMH is uniform for all women, and whether baseline age-specific AMH levels remain consistently high or low during this trajectory. METHODS: A total of 3326 female participants from the population-based Doetinchem Cohort Study were followed with five visits over a 20-year period. Baseline age was 40 ± 10 years with a range of 20-59 years. AMH was measured in 12,929 stored plasma samples using the picoAMH assay (AnshLabs). Decline trajectories of AMH were studied with both chronological age and reproductive age, i.e., time to menopause. Multivariable linear mixed effects models characterized the individual AMH decline trajectories. RESULTS: The overall rate of AMH decline accelerated after 40 years of age. Mixed models with varying age-specific AMH levels and decline rates provided the significantly best fit to the data, indicating that the fall in AMH levels over time does not follow a fixed pattern for individual women. AMH levels remained consistent along individual trajectories of age, with an intraclass correlation coefficient (ICC) of 0.87. The ICC of 0.32 for AMH trajectories with time to menopause expressed the large variation in AMH levels at a given time before the menopause. The differences between low and high age-specific AMH levels remained distinguishable, but became increasingly smaller with increasing chronological and reproductive age. CONCLUSIONS: This is the first study to characterize individual AMH decline over a long time period and broad age range. The varying AMH decline rates do not support the premise of a uniform AMH decline trajectory. Although age-specific AMH levels remain consistently high or low with increasing age, the converging trajectories and variance of AMH levels at a given time before menopause shed doubt on the added value of AMH to represent individualized reproductive age.

Association between vascular health and ovarian ageing in type 1 diabetes mellitus.

STUDY QUESTION: Is vascular health associated with ovarian reserve status using type 1 diabetes mellitus (DM-1) as a model for vascular compromise? SUMMARY ANSWER: No conclusive evidence for an association between vascular health and ovarian ageing was found in women with DM-1. WHAT IS KNOWN ALREADY: The mechanism behind advanced ovarian ageing has not yet been elucidated. We hypothesize that vascular impairment precedes ovarian ageing. DM-1 is hallmarked by premature vascular complications that may consequently play a role in the rate of primordial follicle decline. STUDY DESIGN, SIZE, DURATION: A cross-sectional, patient-control study was performed in 150 premenopausal, regular cycling women with DM-1, as well as a reference population of 177 healthy, fertile women. PARTICIPANTS/MATERIALS, SETTING, METHODS: In a single-study visit, an inventory of both ovarian reserve and vascular status was carried out in the DM-1 group. A transvaginal ultrasound to calculate the antral follicle count (AFC) and blood sampling for anti-Müllerian hormone (AMH), lipids, C-reactive protein and HbA1c measurements were performed. Furthermore, vascular screening including measurements of blood pressure, flow-mediated dilation, peripheral arterial tonometry, pulse wave velocity, pulse wave analysis and intima-media thickness was carried out. The relative decrease in serum AMH levels in women with DM-1 compared with healthy references was investigated. MAIN RESULTS AND THE ROLE OF CHANCE: Systolic blood pressure was negatively correlated with both serum AMH (P= 0.006) and AFC (P= 0.004) in the DM-1 group. A non-linear relationship between HDL-cholesterol and serum AMH was found (P= 0.0001). No associations were detected between other vascular risk factors or vascular function tests and serum AMH or AFC in women with DM-1. With regard to the comparison of AMH levels between women with and without DM-1, mean AMH levels were 2.5 ± 1.9 ng/ml and 3.0 ± 2.8 ng/ml, respectively. After adjustment for confounders the difference in AMH levels between both groups appeared non-significant (fold change: 0.92, 95% confidence interval: 0.68-1.23). LIMITATIONS, REASON FOR CAUTION: The use of different AMH assays and the cross-sectional design may limit the interpretation of this study. WIDER IMPLICATIONS OF THE FINDINGS: The lack of evident association between vascular health and ovarian ageing may be the result of an insufficient vascular compromise in the relatively young, DM-1 group. STUDY FUNDING/COMPETING INTERESTS: No external funding was received for conducting or publishing this study. F.Y., W.S., A.F., F.L.J.V., M.J.C.E. and H.W.d.V. have nothing to disclose. F.J.M.B. has received fees and grant support from the following companies: Ferring, Gedeon Richter, Merck Serono, Medical Specialties Distributors and Roche. TRIAL REGISTRATION NUMBER: Not applicable.

Does anti-Müllerian hormone predict menopause in the general population? Results of a prospective ongoing cohort study.

STUDY QUESTION: Do ovarian reserve tests (ORTs) predict age at natural menopause (ANM) in a cohort of healthy women with a regular menstrual cycle? SUMMARY ANSWER: Of the ORTs researched, anti-Müllerian hormone (AMH) alone predicts age at menopause. However, its predictive value decreased with increasing age of the woman, prediction intervals were broad and extreme ages at menopause could not be predicted. WHAT IS KNOWN ALREADY: A fixed interval is hypothesized to exist between ANM and age at loss of natural fertility. Therefore, if it is possible to predict ANM, one could identify women destined for early menopause and thus at higher risk for age-related subfertility. Of ORTs researched in the prediction of ANM, AMH is the most promising one. STUDY DESIGN, STUDY SIZE AND DURATION: A long-term, extended follow-up study was conducted, results of the first follow-up round were previously published. Two hundred and sixty-five normo-ovulatory women (21-46 years) were included between 1992 and 2001, 49 women (18.5%) could not be reached in the current follow-up round. PARTICIPANTS, SETTING, METHODS: Two hundred and sixty-five healthy normo-ovulatory women were included, recruited in an Academic hospital. We measured baseline AMH, follicle-stimulating hormone and the antral follicle count (AFC). At follow-up (2009 and 2013), menopausal status was determined via questionnaires. Cox regression analysis calculated time to menopause (TTM) using age and ORT. A check of (non-) proportionality of the predictive effect of AMH was performed. A Weibull survival model was used in order to predict individual ANM. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 155 women were available for analyses. Eighty-one women (37.5%) had become post-menopausal during follow-up. Univariable Cox regression analysis demonstrated age and ORTs to be significantly correlated with TTM. Multivariable Cox regression analysis, adjusting for baseline age and smoking; however, demonstrated AMH alone to be an independent predictor of TTM (Hazard Ratio 0.70, 95% Confidence Interval 0.56-0.86, P-value <0.001). A (non-)proportionality analysis of AMH over time demonstrated AMH's predictive effect to decline over time. LIMITATIONS, REASON FOR CAUTION: The observed predictive effect of AMH became less strong with increasing age of the woman. Individual AMH-based age at menopause predictions did not cover the full range of menopausal ages, but did reduce the variation around the predicted ANM from 20 to 10.1 years. WIDER IMPLICATIONS OF THE FINDINGS: Age-specific AMH levels are predictive for ANM. Unlike in our previous publication however, a declining AMH effect with increasing age was observed. This declining AMH effect is in line with recent long-term follow-up data published by others. Moreover, the accompanying predictive inaccuracy observed in individual age at menopause predictions based on AMH, makes this marker currently unsuitable for use in clinical practice. STUDY FUNDING/COMPETING INTERESTS: No external funds were used for this study. M.D., M.J.C.E, S.L.B., G.J.S. and I.A.J.R. have nothing to declare. J.S.E.L. has received fees and grant support from the following companies (in alphabetical order): Ferring, Merck-Serono, MSD, Organon, Serono and Schering Plough. F.J.M.B. receives monetary compensation: member of the external advisory board for Merck Serono, the Netherlands; consultancy work for Gedeon Richter, Belgium; educational activities for Ferring BV, the Netherlands; strategic cooperation with Roche on automated AMH assay development.

The association of low ovarian reserve with cardiovascular disease risk: a cross-sectional population-based study.

STUDY QUESTION: Is there a relationship between serum anti-Müllerian hormone (AMH) level and cardiovascular disease (CVD) risk in premenopausal women? SUMMARY ANSWER: There are indications that premenopausal women with very low ovarian reserve may have an unfavorable CVD risk profile. WHAT IS KNOWN ALREADY: Age at menopause is frequently linked to CVD occurrence. AMH is produced by ovarian antral follicles and provides a measure of remaining ovarian reserve Literature on whether AMH is related to CVD risk is still scarce and heterogeneous. STUDY DESIGN, SIZE, DURATION: Cross-sectional study in 2338 women (age range of 20-57 years) from the general population, participating in the Doetinchem Cohort Study between 1993 and 1997. PARTICIPANTS/MATERIALS, SETTING, METHODS: CVD risk was compared between 2338 premenopausal women in different AMH level-categories, with adjustment for confounders. CVD risk was assessed through levels of systolic and diastolic blood pressure, total cholesterol, high-density lipoprotein cholesterol and glucose, in addition to a summed score of CVD risk factors. Among other factors, analyses were corrected for smoking, oral contraceptive use and BMI. MAIN RESULTS AND THE ROLE OF CHANCE: The relationship of serum AMH levels with CVD risk factor outcomes was nonlinear. Women with AMH levels <0.16 µg/l had 0.11 (95% confidence intervals (CIs) 0.01; 0.21) more metabolic risk factors compared with women with AMH levels ≥0.16 µg/l. There was no association of individual risk factor levels with AMH levels, besides a tendency towards lower total cholesterol levels of 0.11 mmol/l (95% CI -0.23; 0.01) in women with AMH levels <0.002 µg/l compared with women with AMH levels ≥0.16 µg/l. Although not statistically significant, these effect sizes were larger in women below 40 years of age. LIMITATIONS, REASONS FOR CAUTION: Causality and temporality of the studied association cannot be addressed here. Moreover, the clinical and statistical significance of the results of this exploratory study should be interpreted with caution due to the absence of adjustment for multiple statistical testing. WIDER IMPLICATIONS OF THE FINDINGS: This population-based study supports previous findings that premenopausal women with very low AMH levels may have an increased CVD risk. It lays the groundwork for future research to focus on this group of women. Longitudinal studies with more sensitive AMH assays may furthermore help better understand the implications of these results. STUDY FUNDING/COMPETING INTEREST: No financial support was received for this research or manuscript. The Doetinchem Cohort Study is conducted and funded by the Dutch National Institute for Public Health and the Environment F.J.M.B. has received fees and grant support from Merck Serono, Gedeon Richter, Ferring BV and Roche. TRIAL REGISTRATION NUMBER: N/A.

Effectiveness of lifestyle intervention in subgroups of obese infertile women: a subgroup analysis of a RCT.

STUDY QUESTION: Do age, ovulatory status, severity of obesity and body fat distribution affect the effectiveness of lifestyle intervention in obese infertile women? SUMMARY ANSWER: We did not identify a subgroup in which lifestyle intervention increased the healthy live birth rate however it did increase the natural conception rate in anovulatory obese infertile women. WHAT IS KNOWN ALREADY: Obese women are at increased risk of infertility and are less likely to conceive after infertility treatment. We previously demonstrated that a 6-month lifestyle intervention preceding infertility treatment did not increase the rate of healthy live births (vaginal live birth of a healthy singleton at term) within 24 months of follow-up as compared to prompt infertility treatment in obese infertile women. Natural conceptions occurred more frequently in women who received a 6-month lifestyle intervention preceding infertility treatment. STUDY DESIGN, SIZE, DURATION: This is a secondary analysis of a multicentre RCT (randomized controlled trial), the LIFEstyle study. Between 2009 and 2012, 577 obese infertile women were randomly assigned to a 6-month lifestyle intervention followed by infertility treatment (intervention group) or to prompt infertility treatment (control group). Subgroups were predefined in the study protocol, based on frequently used cut-off values in the literature: age (≥36 or <36 years), ovulatory status (anovulatory or ovulatory), BMI (≥35 or <35 kg/m2) and waist-hip (WH) ratio (≥0.8 or <0.8). PARTICIPANTS/MATERIALS, SETTING, METHODS: Data of 564 (98%) randomized women who completed follow-up were analyzed. We studied the effect of the intervention program in various subgroups on healthy live birth rate within 24 months, as well as the rate of overall live births (live births independent of gestational age, mode of delivery and health) and natural conceptions within 24 months. Live birth rates included pregnancies resulting from both treatment dependent and natural conceptions. Logistic regression models with randomization group, subgroup and the interaction between randomization group and subgroup were used. Significant interaction was defined as a P-value <0.1. MAIN RESULTS AND THE ROLE OF CHANCE: Neither maternal age, ovulatory status nor BMI had an impact on the healthy live birth rate within 24 months, nor did they influence the overall live birth rate within 24 months after randomization. WH ratio showed a significant interaction with the effect of lifestyle intervention on healthy live birth rate (P = 0.05), resulting in a lower healthy live birth rate in women with a WH ratio <0.8. WH ratio had no interaction regarding overall live birth rate (P = 0.27) or natural conception rate (P = 0.38). In anovulatory women, the effect of lifestyle intervention resulted in more natural conceptions compared to ovulatory women (P-value for interaction = 0.02). There was no interaction between other subgroups and the effect of the intervention on the rate of natural conception. LIMITATIONS, REASONS FOR CAUTION: Since this was a subgroup analysis of a RCT and sample size determination of the trial was based on the primary outcome of the study, the study was not powered for analyses of all subgroups. WIDER IMPLICATIONS OF THE FINDINGS: Our finding that lifestyle intervention leads to increased natural conception in anovulatory obese women could be used in the counselling of these women, but requires further research using an appropriately powered study in order to confirm this result. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a grant from ZonMw, the Dutch Organisation for Health Research and Development (50-50110-96-518). The Department of Obstetrics and Gynaecology of the UMCG received an unrestricted educational grant from Ferring pharmaceuticals BV, The Netherlands. Ben Mol is a consultant for ObsEva, Geneva. Annemieke Hoek received a speaker's fee for a postgraduate education from MSD pharmaceutical company, outside the submitted work. TRIAL REGISTRATION NUMBER: The LIFEstyle study was registered at the Dutch trial registry (NTR 1530).