Aneuploidy in oocytes from women of advanced maternal age: analysis of the causal meiotic errors and impact on embryo development.

STUDY QUESTION: In oocytes of advanced maternal age (AMA) women, what are the mechanisms leading to aneuploidy and what is the association of aneuploidy with embryo development? SUMMARY ANSWER: Known chromosome segregation errors such as precocious separation of sister chromatids explained 90.4% of abnormal chromosome copy numbers in polar bodies (PBs), underlying impaired embryo development. WHAT IS KNOWN ALREADY: Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years) and can affect over half of oocytes in this age group. This underlies the rationale for PB biopsy as a form of early preimplantation genetic testing for aneuploidy (PGT-A), as performed in the 'ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis' (ESTEEM) randomized controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), Meiosis II (MII) non-disjunction (ND), and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. STUDY DESIGN, SIZE, DURATION: Data were sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridization (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs (PGT-A group), and on embryo morphology in a subset of embryos, up to Day 6 post-insemination, from both the intervention (PB biopsy and PGT-A) and control groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (two pronuclei) in the PB biopsy group and 1082 in the control group were used for statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 461/693 PB pairs showed abnormal segregation in 1162/10 810 chromosomes. The main observed abnormal segregations were compatible with PSSC in Meiosis I (MI) (n = 568/1162; 48.9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%), and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9.6%), we observed a chromosome copy number in the first PB (PB1) and second PB (PB2) that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 days post-insemination compared to the control group, indicating that PB biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day, aneuploidy was associated with a decrease in embryo quality on Day 3 (adjusted odds ratio (aOR) 0.62, 95% CI 0.43-0.90), Day 4 (aOR 0.15, 95% CI 0.06-0.39), and Day 5 (aOR 0.28, 95% CI 0.14-0.58). LIMITATIONS, REASON FOR CAUTION: RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. WIDER IMPLICATIONS OF THE FINDINGS: Our finding of frequent unexplained chromosome copy numbers in PBs indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by mis-segregation of chromosomes during MI and MII. STUDY FUNDING/COMPETING INTEREST(S): Data were mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of PBs. None of the authors have competing interests. TRIAL REGISTRATION NUMBER: Data were mined from the ESTEEM study (ClinicalTrials.gov Identifier NCT01532284).

The HERA (Hyper-response Risk Assessment) Delphi consensus for the management of hyper-responders in in vitro fertilization.

PURPOSE: To provide agreed-upon guidelines on the management of a hyper-responsive patient undergoing ovarian stimulation (OS) METHODS: A literature search was performed regarding the management of hyper-response to OS for assisted reproductive technology. A scientific committee consisting of 4 experts discussed, amended, and selected the final statements. A priori, it was decided that consensus would be reached when ≥66% of the participants agreed, and ≤3 rounds would be used to obtain this consensus. A total of 28/31 experts responded (selected for global coverage), anonymous to each other. RESULTS: A total of 26/28 statements reached consensus. The most relevant are summarized here. The target number of oocytes to be collected in a stimulation cycle for IVF in an anticipated hyper-responder is 15-19 (89.3% consensus). For a potential hyper-responder, it is preferable to achieve a hyper-response and freeze all than aim for a fresh transfer (71.4% consensus). GnRH agonists should be avoided for pituitary suppression in anticipated hyper-responders performing IVF (96.4% consensus). The preferred starting dose in the first IVF stimulation cycle of an anticipated hyper-responder of average weight is 150 IU/day (82.1% consensus). ICoasting in order to decrease the risk of OHSS should not be used (89.7% consensus). Metformin should be added before/during ovarian stimulation to anticipated hyper-responders only if the patient has PCOS and is insulin resistant (82.1% consensus). In the case of a hyper-response, a dopaminergic agent should be used only if hCG will be used as a trigger (including dual/double trigger) with or without a fresh transfer (67.9% consensus). After using a GnRH agonist trigger due to a perceived risk of OHSS, luteal phase rescue with hCG and an attempt of a fresh transfer is discouraged regardless of the number of oocytes collected (72.4% consensus). The choice of the FET protocol is not influenced by the fact that the patient is a hyper-responder (82.8% consensus). In the cases of freeze all due to OHSS risk, a FET cycle can be performed in the immediate first menstrual cycle (92.9% consensus). CONCLUSION: These guidelines for the management of hyper-response can be useful for tailoring patient care and for harmonizing future research.

The HERA (Hyper-response Risk Assessment) Delphi consensus definition of hyper-responders for in-vitro fertilization.

PURPOSE: To provide an agreed upon definition of hyper-response for women undergoing ovarian stimulation (OS)? METHODS: A literature search was performed regarding hyper-response to ovarian stimulation for assisted reproductive technology. A scientific committee consisting of 5 experts discussed, amended, and selected the final statements in the questionnaire for the first round of the Delphi consensus. The questionnaire was distributed to 31 experts, 22 of whom responded (with representation selected for global coverage), each anonymous to the others. A priori, it was decided that consensus would be reached when ≥ 66% of the participants agreed and ≤ 3 rounds would be used to obtain this consensus. RESULTS: 17/18 statements reached consensus. The most relevant are summarized here. (I) Definition of a hyper-response: Collection of ≥ 15 oocytes is characterized as a hyper-response (72.7% agreement). OHSS is not relevant for the definition of hyper-response if the number of collected oocytes is above a threshold (≥ 15) (77.3% agreement). The most important factor in defining a hyper-response during stimulation is the number of follicles ≥ 10 mm in mean diameter (86.4% agreement). (II) Risk factors for hyper-response: AMH values (95.5% agreement), AFC (95.5% agreement), patient's age (77.3% agreement) but not ovarian volume (72.7% agreement). In a patient without previous ovarian stimulation, the most important risk factor for a hyper-response is the antral follicular count (AFC) (68.2% agreement). In a patient without previous ovarian stimulation, when AMH and AFC are discordant, one suggesting a hyper-response and the other not, AFC is the more reliable marker (68.2% agreement). The lowest serum AMH value that would place one at risk for a hyper-response is ≥ 2 ng/ml (14.3 pmol/L) (72.7% agreement). The lowest AFC that would place one at risk for a hyper-response is ≥ 18 (81.8% agreement). Women with polycystic ovarian syndrome (PCOS) as per Rotterdam criteria are at a higher risk of hyper-response than women without PCOS with equivalent follicle counts and gonadotropin doses during ovarian stimulation for IVF (86.4% agreement). No consensus was reached regarding the number of growing follicles ≥ 10 mm that would define a hyper-response. CONCLUSION: The definition of hyper-response and its risk factors can be useful for harmonizing research, improving understanding of the subject, and tailoring patient care.

Is a freeze-all policy the optimal solution to circumvent the effect of late follicular elevated progesterone? A multicentric matched-control retrospective study analysing cumulative live birth rate in 942 non-elective freeze-all cycles.

STUDY QUESTION: Is late follicular elevated progesterone (LFEP) in the fresh cycle hindering cumulative live birth rates (CLBRs) when a freeze only strategy is applied? SUMMARY ANSWER: LFEP in the fresh cycle does not affect the CLBR of the frozen transfers in a freeze only approach, nor the embryo freezing rate. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which has been demonstrated to have a deleterious effect on IVF outcomes. While there is robust evidence that this elevation produces impaired endometrial receptivity, the impact on embryo quality remains a matter of debate. In particular, previous studies have shown that LFEP is associated with a hindered CLBR. However, most clinical insight on the effect of progesterone on embryo quality in terms of CLBRs have focused on embryo transfers performed after the fresh transfer, thus excluding the first embryo of the cohort. To be really informative on the possible detrimental effects of LFEP, evidence should be derived from freeze-all cycles where no fresh embryo transfer is performed in the presence of progesterone elevation, and the entire cohort of embryos is cryopreserved. STUDY DESIGN, SIZE, DURATION: This was a matched case-control, multicentre (three centres), retrospective analysis including all GnRH antagonist ICSI cycles in which a freeze all (FA) policy of embryos on day 3/5/6 of embryonic development was applied between 2012 and 2018. A total of 942 patients (471 cases with elevated P and 471 matched controls with normal P values) were included in the analysis. Each patient was included only once. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sample was divided according to the following P levels on the day of ovulation triggering: <1.50 ng/ml and ≥1.50 ng/ml. The matching of the controls was performed according to age (±1 year) and number of oocytes retrieved (±10%). The main outcome was CLBR defined as a live-born delivery after 24 weeks of gestation. MAIN RESULTS AND THE ROLE OF CHANCE: The baseline characteristics of the two groups were similar. Estradiol levels on the day of trigger were significantly higher in the elevated P group. There was no significant difference in terms of fertilisation rate between the two groups. The elevated P group had significantly more cleavage stage frozen embryos compared to the normal P group while the total number of cryopreserved blastocyst stage embryos was the same. The CLBR did not differ between the two study groups (29.3% and 28.2% in the normal versus LFEP respectively, P = 0.773), also following confounder adjustment using multivariable GEE regression analysis (accounting for age at oocyte retrieval, total dose of FSH, progesterone levels on the day of ovulation trigger, day of freezing, at least one top-quality embryo transferred and number of previous IVF cycles, as the independent variables). LIMITATIONS, REASONS FOR CAUTION: This is a multicentre observational study based on a retrospective data analysis. Better extrapolation of the results could be validated by performing a prospective analysis. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study demonstrating that LFEP in the fresh cycle does not hinder CLBR of the subsequent frozen cycles in a FA approach. Thus, a FA strategy circumvents the issue of elevated P in the late follicular phase. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. Throughout the study period and manuscript preparation, authors were supported by departmental funds from: Centre for Reproductive Medicine, Brussels, Belgium; Infertility Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Centro Scienze Natalità, San Raffaele Scientific Institute, Milan, Italy; and IVI-RMA, Lisbon, Portugal. E.S. has competing interests with Ferring, Merck-Serono, Theramex and Gedeon-Richter outside the submitted work. E.P. reports grants from Ferring, grants and personal fees from Merck-Serono, grants and personal fees from MSD and grants from IBSA outside the submitted work. All the other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Parameters of poor prognosis in preimplantation genetic testing for monogenic disorders.

STUDY QUESTION: What is the likelihood of success of a single cycle of preimplantation genetic testing for monogenic disorders (PGT-M), measured as the cumulative live birth rate (CLBR) and based on various patient demographics? SUMMARY ANSWER: For all women aged ≤40 years, the CLBR was at least 10% when the number of oocytes was ≥7 (range 10-30%) or was at least 5% when the number of oocytes was ≥3 (range 5-17%). WHAT IS KNOWN ALREADY: The number of oocytes is significantly associated with the number of embryos for genetic testing and the clinical outcome in PGT-M. Embryos diagnosed as affected or embryos that remain without diagnosis cannot be used for embryo transfer. The size of the group of embryos non-suitable for transfer varies between 25% and 81%, depending on the indication. Thus, PGT-M is more likely to be more severely impacted by suboptimal ovarian response, poor fertilization and suboptimal embryo development than conventional IVF/ICSI schemes without PGT. STUDY DESIGN, SIZE, DURATION: This was a single-centre retrospective comparative cohort study, of cycles between January 2011 and December 2015. A total number of 2265 PGT-M cycles were compared to 2833 conventional ICSI cycles. The principal aim of our study was the identification of the parameters of poor CLBR in couples undergoing PGT-M using multiplex short tandem repeat (STR) markers on blastomere biopsy DNA. The secondary aim was to compare the parameters of poor CLBR of the PGT-M population to those of couples undergoing ICSI without PGT. PARTICIPANTS/MATERIALS, SETTING, METHODS: The baseline characteristics of the PGT-M group were compared to the conventional ICSI group. A multiple regression analysis was applied to account for the following potential confounding factors: female age, number of previous ART cycles, number of oocytes/suitable embryos for transfer and dosage of gonadotrophins used for ovarian stimulation. MAIN RESULTS AND THE ROLE OF CHANCE: The PGT-M group was younger (female age 32.0 vs 34.5 years), had a higher number of previous ART cycles (1.1 vs 0.9 cycles) and used more gonadotrophins (2367 vs 1984 IU). Per cycle, the PGT-M group had more retrieved oocytes (11.8 vs 8.3 oocytes), fewer suitable embryos for transfer (1.7 vs 2.8 embryos) and a lower CLBR (29.4% vs 35.0%). Multiple regression analysis showed that the CLBR in the PGT-M group was significantly influenced by female age, the number of previous ART cycles, the number of oocytes and the dose of ovarian stimulation. In both groups, the predicted CLBR increased with increasing numbers of oocytes and suitable embryos. At least two retrieved oocytes or one embryo per single PGT-M cycle could confer an estimated CLBR above 10%. By assessing female age and the number of retrieved oocytes together, it was shown that for all women aged ≤40 years, the predicted CLBR per single PGT-M cycle was ≥10% when the number of oocytes was ≥7 or was ≥5% when the number of oocytes was ≥3. LIMITATIONS, REASONS FOR CAUTION: Despite the large sample size, the findings are confined by limited confounder adjustment and the lack of specific PGT-M comparators. WIDER IMPLICATIONS OF THE FINDINGS: This study aimed to describe the likelihood of success of PGT-M treatment, measured as CLBR, based on various patient demographics. In a PGT-M program, couples need to be informed of the prognosis more specifically when it is futile. The table of predicted CLBRs presented in this study is a useful tool in counselling PGT-M couples for making reproductive choices. STUDY FUNDING/COMPETING INTEREST(S): No funding was required and there are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Early pregnancy loss in patients with polycystic ovary syndrome after IVM versus standard ovarian stimulation for IVF/ICSI.

STUDY QUESTION: Is the incidence of early pregnancy loss (EPL) in patients with polycystic ovary syndrome (PCOS) higher after IVM of oocytes than after ovarian stimulation (OS) for IVF/ICSI? SUMMARY ANSWER: Women with PCOS who are pregnant after fresh embryo transfer have a higher probability of EPL following IVM, but after frozen embryo transfer (FET), no significant difference in the incidence of EPL was observed following IVM compared to OS. WHAT IS KNOWN ALREADY: There is conflicting evidence in the current literature with regard to the risk of EPL after IVM of oocytes when compared with OS. Because of the limited sample size in previous studies, the use of different IVM systems and the possible bias introduced by patient characteristics and treatment type, firm conclusions cannot be drawn. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study evaluating 800 women, with a diagnosis of infertility and PCOS as defined by Rotterdam criteria, who had a first positive pregnancy test after fresh or FET following IVM or OS between January 2010 and December 2017 in a tertiary care academic medical centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pregnancies after non-hCG triggered IVM following a short course of highly purified human menopausal gonadotropin were compared with those after conventional OS. The primary outcome was EPL, defined as a spontaneous pregnancy loss before 10 weeks of gestation. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 329 patients with a positive pregnancy test after IVM and 471 patients with a positive pregnancy test after OS were included. Women who were pregnant after IVM were younger (28.6 ± 3.4 years vs 29.3 ± 3.6 years, P = 0.005) and had higher serum anti-Mullerian hormone levels (11.5 ± 8.1 ng/ml vs 7.2 ± 4.1 ng/ml, P < 0.001) compared to those who were pregnant after OS. The distribution of PCOS phenotypes was significantly different among women in the IVM group compared to those in the OS group and women who were pregnant after OS had previously suffered EPL more often (28% vs 17.6%, P = 0.003). EPL was significantly higher after fresh embryo transfer following IVM compared to OS (57/122 (46.7%) vs 53/305 (17.4%), P < 0.001), while the results were comparable after FET (63/207 (30.4%) vs 60/166 (36.1%), respectively, P = 0.24). In the multivariate logistic regression analysis evaluating fresh embryo transfer cycles, IVM was the only independent factor (adjusted odds ratio (aOR) 4.24, 95% CI 2.44-7.37, P < 0.001)) significantly associated with increased odds of EPL. On the other hand, when the same model was applied to FET cycles, the type of treatment (IVM vs OS) was not significantly associated with EPL (aOR 0.73, 95% CI 0.43-1.25, P = 0.25). LIMITATIONS, REASONS FOR CAUTION: The current data are limited by the retrospective nature of the study and the potential of bias due to unmeasured confounders. WIDER IMPLICATIONS OF THE FINDINGS: The increased risk of EPL after fresh embryo transfer following IVM may point towards inadequate endometrial development in IVM cycles. Adopting a freeze-all strategy after IVM seems more appropriate. Future studies are needed to ascertain the underlying cause of this observation. STUDY FUNDING/COMPETING INTEREST(S): The Clinical IVM research has been supported by research grants from Cook Medical and Besins Healthcare. All authors declared no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Follicular-phase endometrial scratching: a truncated randomized controlled trial.

STUDY QUESTION: Does intentional endometrial injury (scratching) during the follicular phase of ovarian stimulation (OS) increase the clinical pregnancy rate (CPR) in ART? SUMMARY ANSWER: CPR did not vary between the endometrial injury and the control group, but the trial was underpowered due to early termination because of a higher clinical miscarriage rate observed in the endometrial injury arm after a prespecified interim analysis. WHAT IS KNOWN ALREADY: Intentional endometrial injury has been put forward as an inexpensive clinical tool capable of enhancing endometrial receptivity. However, despite its widespread use, the benefit of endometrial scratching remains controversial, with several recent randomized controlled trials (RCTs) being unable to confirm its added value. So far, most research has focused on endometrial scratching during the luteal phase of the cycle preceding the one with embryo transfer (ET), while only a few studies investigated in-cycle injury during the follicular phase of OS. Also, the persistence of a scratch effect in subsequent treatment cycles remains unclear and possible harms have been insufficiently studied. STUDY DESIGN, SIZE, DURATION: This RCT was performed in a tertiary hospital setting between 3 April 2014 and 8 October 2017. A total of 200 women (100 per study arm) undergoing IVF/ICSI in a GnRH antagonist suppressed cycle followed by fresh ET were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were randomized with a 1:1 allocation ratio to either undergo a pipelle endometrial biopsy between Days 6 and 8 of OS or to be in the control group.The primary outcome was CPR. Secondary outcomes included biochemical pregnancy rate, live birth rate (LBR), early pregnancy loss (biochemical pregnancy losses and clinical miscarriages), excessive procedure pain/bleeding and cumulative reproductive outcomes within 6 months of the study cycle. MAIN RESULTS AND THE ROLE OF CHANCE: The RCT was stopped prematurely by the trial team after the second prespecified interim analysis raised safety concerns, namely a higher clinical miscarriage rate in the intervention group. The intention-to-treat CPR was similar between the biopsy and the control arm (respectively, 44 versus 40%, P = 0.61, risk difference = 3.6 with 95% confidence interval = -10.1;17.3), as was the LBR (respectively, 32 versus 36%, P = 0.52). The incidence of a biochemical pregnancy loss was comparable between both groups (10% in the intervention group versus 15% in the control, P = 0.49), but clinical miscarriages occurred significantly more frequent in the biopsy group (25% versus 8%, P = 0.032). In the intervention group, 3% of the patients experienced excessive procedure pain and 5% bleeding. The cumulative LBR taking into account all conceptions (spontaneous or following ART) within 6 months of randomization was not significantly different between the biopsy and the control group (54% versus 60%, respectively, P = 0.43). LIMITATIONS, REASONS FOR CAUTION: The trial was stopped prematurely due to safety concerns after the inclusion of 200 of the required 360 patients. Not reaching the predefined sample size implies that definite conclusions on the outcome parameters cannot be drawn. Furthermore, the pragmatic design of the study may have limited the detection of specific subgroups of women who may benefit from endometrial scratching. WIDER IMPLICATIONS OF THE FINDINGS: Intentional endometrial injury during the follicular phase of OS warrants further attention in future research, as it may be harmful. These findings should be taken in consideration together with the growing evidence from other RCTs that scratching may not be beneficial. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by 'Fonds Wetenschappelijk Onderzoek' (FWO, Flanders, Belgium, 11M9415N, 1524417N). None of the authors have a conflict of interest to declare with regard to this study.

To trigger or not to trigger ovulation in a natural cycle for frozen embryo transfer: a randomized controlled trial.

STUDY QUESTION: Is the clinical pregnancy rate (CPR) following a frozen embryo transfer (FET) in a natural cycle (NC) higher after spontaneous ovulation than after triggered ovulation [natural cycle frozen embryo transfer (NC-FET) versus modified NC-FET]? SUMMARY ANSWER: The CPR did not vary significantly between the two FET preparation protocols. WHAT IS KNOWN ALREADY: Although the use of FET is continuously increasing, the most optimal endometrial preparation protocol is still under debate. For transfer in the NC specifically, conflicting results have been reported in terms of the outcome following spontaneous or triggered ovulation. STUDY DESIGN, SIZE, DURATION: In a tertiary hospital setting, subjects were randomized with a 1:1 allocation into two groups between January 2014 and January 2019. Patients in group A underwent an NC-FET, while in group B, a modified NC-FET was performed with a subcutaneous hCG injection to trigger ovulation. In neither group was additional luteal phase support administered. All embryos were vitrified-warmed on Day 3 and transferred on Day 4 of embryonic development. The primary outcome was CPR at 7 weeks. All patients were followed further until 10 weeks of gestation when the ongoing pregnancy rate (OPR) was defined by the observation of foetal cardiac activity on ultrasound scan. Other secondary outcomes included biochemical pregnancy rate, early pregnancy loss and the number of visits, blood samples and ultrasonographic examinations prior to FET. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 260 patients (130 per study arm) were randomized, of whom 12 withdrew consent after study arm allocation. A total of 3 women conceived spontaneously before initiating the study cycle and 16 did not start for personal or medical reasons. Of the 229 actually commencing monitoring for the study FET cycle, 7 patients needed to be switched to a hormonal replacement treatment protocol due to the absence of follicular development, 12 had no embryo available for transfer after warming and 37 had a spontaneous LH surge before the ovulation trigger could be administered, although they were allocated to group B. Given the above, an intention-to-treat (ITT) analysis was performed taking into account 248 patients (125 in group A and 123 in group B), as well as a per protocol (PP) analysis on a subset of 173 patients (110 in group A and 63 in group B). MAIN RESULTS AND THE ROLE OF CHANCE: Demographic features were evenly distributed between the study groups, as were the relevant fresh and frozen ET cycle characteristics. According to the ITT analysis, the CPR and OPR in group A (33.6% and 27.2%, respectively) and group B (29.3% and 24.4%, respectively) did not vary significantly [relative risk (RR) 0.87, 95% CI (0.60;1.26), P = 0.46 and RR 0.90, 95% CI (0.59;1.37), P = 0.61, respectively]. Biochemical pregnancy rate and early pregnancy loss were also found to be not statistically significantly different between the groups. In contrast, more clinic visits and blood samplings for cycle monitoring were required in the NC-FET group (4.05 ± 1.39) compared with the modified NC-FET group (3.03 ± 1.16, P = <0.001), while the number of ultrasound scans performed were comparable (1.70 ± 0.88 in group A versus 1.62 ± 1.04 in group B). The additional PP analysis was in line with the ITT results: CPR in group A was 36.4% versus 38.1% in group B [RR 1.05, 95% CI (0.70;1.56), P = 0.82]. LIMITATIONS, REASONS FOR CAUTION: The results are limited by the high drop-out rate for the PP analysis in the modified NC-FET group as more than one-third of the subjects allocated to this group ovulated spontaneously before ovulation triggering. Nonetheless, this issue is inherent to routine clinical practice and is an important observation of an event that can only be avoided by performing a very extensive monitoring that limits the practical advantages associated with modified NC-FET. Furthermore, although this is the largest randomized controlled trial (RCT) investigating this specific research question so far, a higher sample size would allow smaller differences in clinical outcome to be detected, since currently they may be left undetected. WIDER IMPLICATIONS OF THE FINDINGS: This RCT adds new high-quality evidence to the existing controversial literature concerning the performance of NC-FET versus modified NC-FET. Based on our results showing no statistically significant differences in clinical outcomes between the protocols, the treatment choice may be made according to the patient's and treating physician's preferences. However, the modified NC-FET strategy reduces the need for hormonal monitoring and may therefore be considered a more patient-friendly and potentially cost-effective approach. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was available for this study. None of the authors have a conflict of interest to declare with regard to this study. TRIAL REGISTRATION NUMBER: NCT02145819. TRIAL REGISTRATION DATE: 8 January 2014. DATE OF FIRST PATIENT'S ENROLMENT: 21 January 2014.

The proliferative phase endometrium in IVF/ICSI: an in-cycle molecular analysis predictive of the outcome following fresh embryo transfer.

STUDY QUESTION: Does an early proliferative phase endometrial biopsy harvested during ovarian stimulation harbour information predictive of the outcome following fresh embryo transfer (ET) in that same cycle? SUMMARY ANSWER: Transcriptome analysis of the whole-tissue endometrium did not reveal significant differential gene expression (DGE) in relation to the outcome; however, the secretome profile of isolated, cultured and in vitro decidualized endometrial stromal cells (EnSCs) varied significantly between patients who had a live birth compared to those with an implantation failure following fresh ET in the same cycle as the biopsy. WHAT IS KNOWN ALREADY: In the majority of endometrial receptivity research protocols, biopsies are harvested during the window of implantation (WOI). This, however, precludes ET in that same cycle, which is preferable as the endometrium has been shown to adapt over time. Endometrial biopsies taken during ovarian stimulation have been reported not to harm the chances of implantation, and in such biopsies DGE has been observed between women who achieve pregnancy versus those who do not. The impact of the endometrial proliferative phase on human embryo implantation remains unclear, but deserves further attention, especially since in luteal phase endometrial biopsies, a transcriptional signature predictive for repeated implantation failure has been associated with reduced cell proliferation, possibly indicating proliferative phase involvement. Isolation, culture and in vitro decidualization (IVD) of EnSCs is a frequently applied basic research technique to assess endometrial functioning, and a disordered EnSC secretome has previously been linked with failed implantation. STUDY DESIGN, SIZE, DURATION: This study was nested in a randomized controlled trial (RCT) investigating the effect of endometrial scratching during the early follicular phase of ovarian stimulation on clinical pregnancy rates after IVF/ICSI. Of the 96 endometrial biopsies available, after eliminating those without fresh ET and after extensive matching in order to minimize the risk of potential confounding, 18 samples were retained to study two clinical groups: nine biopsies of patients with a live birth versus nine biopsies of patients with an implantation failure, both following fresh ET performed in the same cycle as the biopsy. We studied the proliferative endometrium by analysing its transcriptome and by isolating, culturing and decidualizing EnSCs in vitro. We applied this latter technique for the first time on proliferative endometrial biopsies obtained during ovarian stimulation for in-cycle outcome prediction, in an attempt to overcome inter-cycle variability. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA-sequencing was performed for 18 individual whole-tissue endometrial biopsies on an Illumina HiSeq1500 machine. DGE was analysed three times using different approaches (DESeq2, EdgeR and the Wilcoxon rank-sum test, all in R). EnSC isolation and IVD was performed (for 2 and 4 days) for a subset of nine samples, after which media from undifferentiated and decidualized cultures were harvested, stored at -80°C and later assayed for 45 cytokines using a multiplex suspension bead immunoassay. The analysis was performed by partial least squares regression modelling. MAIN RESULTS AND THE ROLE OF CHANCE: After correction for multiple hypothesis testing, DGE analysis revealed no significant differences between endometrial samples from patients who had a live birth and those with an implantation failure following fresh ET. However secretome analysis after EnSC isolation and culture, showed two distinct clusters that clearly corresponded to the two clinical groups. Upon IVD, the secretome profiles shifted from that of undifferentiated cells but the difference between the two clinical groups remained yet were muted, suggesting convergence of cytokine profiles after decidualization. LIMITATIONS, REASONS FOR CAUTION: Caution is warranted due to the limited sample size of the study and the in vitro nature of the EnSC experiment. Validation on a larger scale is necessary, however, hard to fulfil given the very limited availability of in-cycle proliferative endometrial biopsies outside a RCT setting. WIDER IMPLICATIONS OF THE FINDINGS: These data support the hypothesis that the endometrium should be assessed not only during the WOI and that certain endometrial dysfunctionalities can probably be detected early in a cycle by making use of the proliferative phase. This insight opens new horizons for the development of endometrial tests, whether diagnostic or predictive of IVF/ICSI treatment outcome. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Fonds Wetenschappelijk Onderzoek (FWO, Flanders, Belgium, 11M9415N, 1 524 417N), Wetenschappelijk Fonds Willy Gepts (WFWG G160, Universitair Ziekenhuis Brussel, Belgium) and the National Medicine Research Council (NMRC/CG/M003/2017, Singapore). There are no conflicts of interests. TRIAL REGISTRATION NUMBER: NCT02061228.

Do we need to measure progesterone in oocyte donation cycles? A retrospective analysis evaluating cumulative live birth rates and embryo quality.

STUDY QUESTION: Does late follicular-phase elevated serum progesterone (LFEP) during ovarian stimulation for oocyte donation have an impact on embryo quality (EQ) and cumulative live birth rate (CLBR)? SUMMARY ANSWER: LFEP does not have an influence on EQ nor CLBR in oocyte donation cycles. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which, when elevated during the follicular phase, has been demonstrated to have a deleterious effect in autologous fresh IVF outcomes. While there is robust evidence that this elevation results in impaired endometrial receptivity, the impact on EQ remains a matter of debate. The oocyte donation model is an excellent tool to assess the effects of LFEP on EQ from those on endometrium receptivity separately. Previous studies in oocyte donation cycles investigating the influence of elevated P on pregnancy outcomes in oocyte recipients showed conflicting results. STUDY DESIGN, SIZE, DURATION: This is a retrospective analysis including all GnRH antagonist down-regulated cycles for fresh oocyte donation taking place in a tertiary referral university hospital between 2010 and 2017. A total of 397 fresh donor-recipient cycles were included. Each donor was included only once in the analysis and could be associated to a single recipient. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sample was stratified according to serum P levels of ≤1.5 and >1.5 ng/mL on the day of ovulation triggering. The primary endpoint of the study was the top-quality embryo rate on Day 3, and the secondary outcome measure was CLBR defined as a live-born delivery beyond 24 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: Three hundred ninety-seven fresh oocyte donation cycles were included in the analysis, of which 314 (79%) had a serum P ≤ 1.5 ng/mL and 83 (20.9%) had a serum P > 1.5 ng/mL. The average age of the oocyte donors was 31.4 ± 4.7 and 29.9 ± 4.5 years, respectively, for normal and elevated P (P = 0.017). The mean number of oocytes retrieved was significantly higher in the elevated P group with 16.6 ± 10.6 vs 11.5 ± 6.9 in the P ≤ 1.5 group (P < 0.001).In parallel, the total number of embryos on Day 3, as well as the number of good-quality embryos at this stage, was significantly higher in the elevated P group (6.6 ± 5.6 vs 4.15 ± 3.5 and 8.7 ± 6.3 vs 6.1 ± 4.4; respectively, P < 0.001). However, maturation and fertilization rates did not vary significantly between the two study groups and neither did the top- and good-quality embryo rate and the embryo utilization rate, all evaluated on Day 3 (P = 0.384, P = 0.405 and P = 0.645, respectively). A multivariable regression analysis accounting for P groups, age of the donor, number of retrieved oocytes and top-quality embryo rate as potential confounders showed that LFEP negatively influenced neither the top-quality embryo rate nor the CLBR. LIMITATIONS, REASONS FOR CAUTION: This is an observational study based on a retrospective data analysis. Better extrapolation of the results could be validated by performing a prospective trial. Furthermore, this study was focused on oocyte donation cycles and hence the results cannot be generalized to the entire infertile population. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study providing evidence that LFEP does not influence CLBR and is adding strong evidence to the existing literature that LFEP does not harm EQ in oocyte donation programs. STUDY FUNDING/COMPETING INTERESTS: Not applicable.

Predicting suboptimal oocyte yield following GnRH agonist trigger by measuring serum LH at the start of ovarian stimulation.

STUDY QUESTION: Are the LH levels at the start of ovarian stimulation predictive of suboptimal oocyte yield from GnRH agonist triggering in GnRH antagonist down-regulated cycles? SUMMARY ANSWER: LH levels at the start of ovarian stimulation are an independent predictor of suboptimal oocyte yield following a GnRH agonist trigger. WHAT IS KNOWN ALREADY: A GnRH agonist ovulation trigger may result in an inadequate oocyte yield in a small subset of patients. This failure can range from empty follicle syndrome to the retrieval of much fewer oocytes than expected. Suboptimal response to a GnRH agonist trigger has been defined as the presence of circulating LH levels <15 IU/l 12 h after triggering. It has been shown that patients with immeasurable LH levels on trigger day have an up to 25% risk of suboptimal response. STUDY DESIGN, SIZE, DURATION: In this retrospective cohort study, all patients (n = 3334) who received GnRH agonist triggering (using Triptoreline 0.2 mg) for final oocyte maturation undergoing a GnRH antagonist cycle in our centre from 2011 to 2017 were included. The primary outcome of the study was oocyte yield, defined as the ratio between the total number of collected oocytes and the number of follicles with a mean diameter >10 mm prior to GnRH agonist trigger. PARTICIPANTS/MATERIALS, SETTING, METHODS: The endocrine profile of all patients was studied at initiation as well as at the end of ovarian stimulation. In order to evaluate whether LH levels, not only at the end but also at the start, of ovarian stimulation predicted oocyte yield, we performed multivariable regression analysis adjusting for the following confounding factors: female age, body mass index, oral contraceptives before treatment, basal and trigger day estradiol levels, starting FSH levels, use of highly purified human menopausal gonadotrophin and total gonadotropin dose. Suboptimal response to GnRH agonist trigger was defined as <10th percentile of oocyte yield. MAIN RESULTS AND THE ROLE OF CHANCE: The average age was 31.9 years, and the mean oocyte yield was 89%. The suboptimal response to GnRH agonist trigger cut-off (<10th percentile) was 45%, which was exhibited by 340 patients. Following confounder adjustment, multivariable regression analysis showed that LH levels at the initiation of ovarian stimulation remained an independent predictor of suboptimal response even in the multivariable model (adjusted OR 0.920, 95% CI 0.871-0.971). Patients with immeasurable LH levels at the start of stimulation (<0.1 IU/l) had a 45.2% risk of suboptimal response, while the risk decreased with increasing basal LH levels; baseline circulating LH <0.5 IU/L, <2 IU/L and <5 IU/L were associated with a 39.1%, 25.2% and 13.6% risk, respectively. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study is its retrospective design. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest study of GnRH agonist trigger cycles only, since most of the previous research on the predictive value of basal LH levels was performed in dual trigger cycles. LH values should be measured prior to start of ovarian stimulation. In cases where they are immeasurable, suboptimal response to GnRH agonist trigger can be anticipated, and an individualized approach is warranted. STUDY FUNDING/COMPETING INTEREST(S): There was no funding and no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Obstetric and neonatal outcome of ART in patients with polycystic ovary syndrome: IVM of oocytes versus controlled ovarian stimulation.

STUDY QUESTION: Does IVM of immature oocytes retrieved from small antral follicles in women with polycystic ovary syndrome (PCOS) have an impact on obstetric and neonatal outcomes compared to controlled ovarian stimulation (COS)? SUMMARY ANSWER: Obstetric and neonatal outcomes after IVM appear to be similar to those after COS. WHAT IS KNOW ALREADY: Women with PCOS have an increased risk of adverse pregnancy outcomes and congenital malformations in their offspring. For patients with PCOS who require IVF, IVM of germinal vesicle (GV)-stage oocytes retrieved from antral follicles has been adopted as a mild approach ART, with improved pregnancy rates over the last two decades. Although reports of obstetrical and neonatal outcomes after IVM have been reassuring, the limited sample sizes in previous studies preclude firm conclusions, and further study is warranted. STUDY DESIGN, SIZE, DURATION: This is a retrospective observational study analysing obstetric and neonatal data from 1036 clinical pregnancies in unique patients with PCOS who conceived following a cycle of IVM or COS between January 2010 and December 2016 in a tertiary reproductive centre. In total, 393 singleton pregnancies with a gestational age beyond 20 weeks were included. A phenotypic approach was used for the diagnosis of PCOS. Pregnancies following oocyte donation, standard IVF (as opposed to ICSI) or preimplantation genetic testing and pregnancies requiring testicular biopsy in the male partners were excluded. PARTICIPANTS/MATERIALS,SETTING, METHODS: Pregnancy outcomes were analysed in women with PCOS phenotype A, C or D, as defined by different combinations of the Rotterdam criteria. Data from 164 pregnancies beyond 20 weeks after IVM were compared with those from 229 pregnancies after COS. Pregnancies in the IVM group were obtained after minimal ovarian stimulation and IVF with ICSI of transvaginally collected GV oocytes that had reached the metaphase II stage in vitro after 28 to 40 h of culture. No hCG trigger was administered before oocyte retrieval. Outcome measures were analysed or reported in singleton pregnancies only and included adverse obstetric events and neonatal health parameters, in particular birthweight, prematurity, small-for-gestational age, large-for-gestational age, perinatal death and major/minor malformation rates. The incidence of hypertensive disorders of pregnancy (HDP) and birthweight was analysed by multiple linear and logistic regression, adjusted for relevant treatment variables and maternal characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: The IVM and the COS groups differed significantly (P < 0.001) for maternal circulating AMH levels and PCOS phenotype distribution, with more of the PCOS phenotype A in the IVM group. Pregnant women in the IVM group were younger than pregnant women in the COS group (P = 0.05). With regard to obstetric complications in singleton pregnancies, in the unadjusted analysis, mothers of infants in the IVM group more often had HDP (29/164 (17.9%) vs 22/229 (9.6%), P = 0.02) compared with mothers in the COS group. Singletons born after IVM and COS had a similar birthweight standard deviation score (SDS) (0.51 ± 0.94 after IVM vs 0.33 ± 1.05 after COS, P = 0.19). Preterm birth rate (32-36.9 weeks) and early preterm birth rate (<32 weeks) were also similar in both groups. The total malformation rate was 4.1% in singletons after IVM and 2.4% in singletons after COS. Multivariate linear regression analysis accounting for relevant confounders demonstrated that parity was the only independent predictive factor (P = 0.04) for birthweight SDS. Multivariate logistic regression analysis showed that BMI, parity and type of ART (IVM as opposed to COS) were significantly correlated with the incidence of HDP. Only patients with the PCOS phenotype A showed a tendency towards a higher risk of HDP in those who underwent IVM compared to those who had COS. LIMITATIONS, REASONS FOR CAUTION: The study is limited by its retrospective nature and loss to follow-up of a subset of children with no information regarding congenital malformations. Furthermore, the paediatricians who assessed the children after birth were not blinded for the type of ART procedure. WIDER IMPLICATIONS OF THE FINDINGS: This study provides further evidence that, compared to COS, IVM of oocytes derived from small antral follicles does not adversely affect the neonatal health of the offspring of patients with PCOS. The observed increased risk of HDP in patients with PCOS phenotype A following IVM treatment warrants further scrutiny. STUDY FUNDING/COMPETING INTEREST(S): Translational IVM research at Universitair Ziekenhuis Brussel (UZ Brussel) and Vrije Universiteit Brussel (VUB) has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13) and the Belgian Foundation Against Cancer (HOPE project, Dossier C69). Clinical IVM research was supported by research grants from Cook Medical and Besins Healthcare. M.D.V. reports honoraria for lectures from Cook Medical and Besins Healthcare outside the submitted work. S.S.R. reports honoraria for lectures by MSD and Besins and research grants by MSD, Ferring and Merck Serono outside of the submitted work. C.B. reports personal fees from Merck-Serono, Ferring, IBSA, Finox, MSD and Abbott outside the submitted work. H.T. reports grants from Merck, MSD, Goodlife, Cook, Roche, Besins, Ferring, Mithra (now Allergan) and the Research Fund of Flanders (FWO) and consultancy fees from Finox, Abbott, Obseva and Ovascience outside the submitted work. The other authors have nothing to disclose.

Impact of late-follicular phase elevated serum progesterone on cumulative live birth rates: is there a deleterious effect on embryo quality?

STUDY QUESTION: Is elevated late-follicular phase progesterone (EP) associated with a deleterious impact on embryo quality (EQ) and cumulative live birth rates (LBRs)? SUMMARY ANSWER: EP was associated with a decrease in embryo utilization and cumulative LBRs. WHAT IS KNOWN ALREADY: Ovarian stimulation promotes the production of progesterone (P) which adversely affects IVF pregnancy outcomes. However, evidence regarding a potential association between EP an EQ is lacking. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of all GnRH antagonist down-regulated ICSI cycles followed by a fresh embryo transfer (ET) between 2010 and 2015 was performed. The sample was stratified according to the following P levels on the day of ovulation triggering: ≤0.50, 0.51-1.49 and ≥1.50 ng/ml. The primary outcomes were embryo utilization rates (number of embryos transferred or cryopreserved) and cumulative LBR, defined as the occurrence of the first live-birth after either the fresh or one of the subsequent frozen ET. PARTICIPANTS/MATERIALS, SETTING, METHODS: Overall, 3400 cycles were included in the analysis, using multivariable regression to account for potential confounding. MAIN RESULTS AND THE ROLE OF CHANCE: Female age and the number of oocytes retrieved increased significantly with increasing serum P values. Utilization rates decreased linearly as P increased for Day 3 embryos (72.3, 63.0 and 45.4%, respectively), while for Day 5 embryos only the EP group was associated with a significant decrease (48.8, 47.8 and 38.8%, respectively). EP was also associated with decreased fresh and cumulative LBRs. LIMITATIONS REASONS FOR CAUTION: The main limitations of this study were its retrospective nature and the fact that it was restricted to GnRH antagonist cycles. WIDER IMPLICATIONS OF THE FINDINGS: These results raise the question whether EP may also be associated with a decrease in cumulative pregnancy outcomes by increasing embryo wastage. Further studies may evaluate the potential benefit of additional measures besides the freeze-all strategy to avoid this issue, such as lowering the stimulation dose or applying a step-down protocol. STUDY FUNDING/COMPETING INTEREST(S): None.

Frozen embryo transfer: a review on the optimal endometrial preparation and timing.

STUDY QUESTION: What is the optimal endometrial preparation protocol for a frozen embryo transfer (FET)? SUMMARY ANSWER: Although the optimal endometrial preparation protocol for FET needs further research and is yet to be determined, we propose a standardized timing strategy based on the current available evidence which could assist in the harmonization and comparability of clinic practice and future trials. WHAT IS KNOWN ALREADY: Amid a continuous increase in the number of FET cycles, determining the optimal endometrial preparation protocol has become paramount to maximize ART success. In current daily practice, different FET preparation methods and timing strategies are used. STUDY DESIGN, SIZE, DURATION: This is a review of the current literature on FET preparation methods, with special attention to the timing of the embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Literature on the topic was retrieved in PubMed and references from relevant articles were investigated until June 2017. MAIN RESULTS AND THE ROLE OF CHANCE: The number of high quality randomized controlled trials (RCTs) is scarce and, hence, the evidence for the best protocol for FET is poor. Future research should compare both the pregnancy and neonatal outcomes between HRT and true natural cycle (NC) FET. In terms of embryo transfer timing, we propose to start progesterone intake on the theoretical day of oocyte retrieval in HRT and to perform blastocyst transfer at hCG + 7 or LH + 6 in modified or true NC, respectively. LIMITATIONS REASONS FOR CAUTION: As only a few high quality RCTs on the optimal preparation for FET are available in the existing literature, no definitive conclusion for benefit of one protocol over the other can be drawn so far. WIDER IMPLICATIONS OF THE FINDINGS: Caution when using HRT for FET is warranted since the rate of early pregnancy loss is alarmingly high in some reports. STUDY FUNDING/COMPETING INTEREST(S): S.M. is funded by the Research Fund of Flanders (FWO). H.T. and C.B. report grants from Merck, Goodlife, Besins and Abbott during the conduct of the study. TRIAL REGISTRATION NUMBER: Not applicable.

Vitrified-warmed blastocyst transfer on the 5th or 7th day of progesterone supplementation in an artificial cycle: a randomised controlled trial.

Prospective studies comparing different durations of progesterone supplementation before transfer of vitrified-warmed blastocysts in an artificial cycle are lacking. However, in oocyte donation programmes, the sporadic available evidence demonstrates considerable differences in clinical pregnancy rates according to the duration of progesterone administration. This randomised controlled trial (RCT), included 303 patients undergoing a frozen-thawed embryo transfer (FET) of one or two vitrified-warmed blastocyst(s) in an artificial cycle. Randomisation was performed when the endometrial thickness reached ≥7 mm after oestrogen supplementation. One hundred and fifty two patients in group A received 7 d of vaginal micronised progesterone tablets and 151 patients in group B received 5 d of micronised vaginal progesterone before FET. No differences were seen in clinical pregnancy rate between both groups: 42/152 (27.6%) in group A versus 49/151 (32.5%) in group B. Although no statistically significant difference was observed in clinical pregnancy rates, our study was powered to detect an absolute difference of 16%. In this regard, we cannot exclude that smaller, clinically relevant differences might exist and our study did not have the power to detect this. Patients were also not blinded for the intervention, causing a potential bias.

Do ARTs affect the incidence of monozygotic twinning?

STUDY QUESTION: Does the manipulation of gametes or embryos during ARTs increase the risk for monozygotic twinning (MZT)? SUMMARY ANSWER: Frozen embryo transfer (ET) is associated with a lower MZT rate, while blastocyst culture is associated with an increased risk of monozygotic pregnancy. WHAT IS KNOWN ALREADY: Monozygotic twins have a higher risk for perinatal complications. Although an increased incidence of monozygotic pregnancies after ART has been previously reported, data regarding the possible impact of different laboratory procedures are conflicting. STUDY DESIGN, SIZE, DURATION: All clinical pregnancies after single ET carried out in our centre between 2004 and 2013 (n = 6096) were retrospectively analysed for the incidence of MZT. The effect of different laboratory procedures on the incidence of MZT was evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: The following ART risk factors were assessed: maternal age, type of ET (fresh versus frozen), zona pellucida (ZP) manipulation (specifically, ICSI, embryo biopsy and assisted hatching), use of donor oocytes, embryo stage at time of ET (cleavage, compaction, early or advanced blastocyst) and culture media. MAIN RESULTS AND THE ROLE OF CHANCE: The overall MZT rate was 2.2% (136/6096). Frozen ET was associated with a significant reduction in MZT incidence (adjusted odds ratio (aOR) 0.48, 95% CI 0.29-0.80), while blastocyst transfer (early or advanced blastocyst) was associated with a significant increase in MZT risk (aOR 2.70, 95% CI 1.36-5.34; aOR 2.05, 95% CI 1.29-3.26, respectively). No significant differences were found between the MZT and singleton (non-MZT) groups regarding maternal age, the use of different ZP manipulation techniques, not type of culture media used. LIMITATION, REASONS FOR CAUTION: This study is limited by its retrospective nature and the fact that monozygosity was not confirmed by genetic testing. Furthermore, since monozygotic pregnancy is a rare event, other ART parameters that may influence its incidence could not be assessed during our analysis. WIDER IMPLICATION OF THE FINDINGS: Our findings warrant future studies designed to investigate the association between specific ART procedures and MZT, namely the potential risk of blastocyst transfer to increase MZT. STUDY FUNDING/COMPETING INTERESTS: No external funding was used for this study. There are no conflicts of interest.

Cyclin E1 plays a key role in balancing between totipotency and differentiation in human embryonic cells.

STUDY HYPOTHESIS: We aimed to investigate if Cyclin E1 (CCNE1) plays a role in human embryogenesis, in particular during the early developmental stages characterized by a short cell cycle. STUDY FINDING: CCNE1 is expressed in plenipotent human embryonic cells and plays a critical role during hESC derivation via the naïve state and, potentially, normal embryo development. WHAT IS KNOWN ALREADY: A short cell cycle due to a truncated G1 phase has been associated with the high developmental capacity of embryonic cells. CCNE1 is a critical G1/S transition regulator. CCNE1 overexpression can cause shortening of the cell cycle and it is constitutively expressed in mouse embryonic stem cells and cancer cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated expression of CCNE1 in human preimplantation embryo development and embryonic stem cells (hESC). Functional studies included CCNE1 overexpression in hESC and CCNE1 downregulation in the outgrowths formed by plated human blastocysts. Analysis was performed by immunocytochemistry and quantitative real-time PCR. Mann-Whitney statistical test was applied. MAIN RESULTS AND THE ROLE OF CHANCE: The CCNE1 protein was ubiquitously and constitutively expressed in the plenipotent cells of the embryo from the 4-cell stage up to and including the full blastocyst. During blastocyst expansion, CCNE1 was downregulated in the trophectoderm (TE) cells. CCNE1 shortly co-localized with NANOG in the inner cell mass (ICM) of expanding blastocysts, mimicking the situation in naïve hESC. In the ICM of expanded blastocysts, which corresponds with primed hESC, CCNE1 defined a subpopulation of cells different from NANOG/POU5F1-expressing pluripotent epiblast (EPI) cells and GATA4/SOX17-expressing primitive endoderm (PrE) cells. This CCNE1-positive cell population was associated with visceral endoderm based on transthyretin expression and marked the third cell lineage within the ICM, besides EPI and PrE, which had never been described before. We also investigated the role of CCNE1 by plating expanded blastocysts for hESC derivation. As a result, all the cells including TE cells re-gained CCNE1 and, consequently, NANOG expression, resembling the phenotype of naïve hESC. The inhibition of CCNE1 expression with siRNA blocked proliferation and caused degeneration of those plated cells. LIMITATIONS, REASONS FOR CAUTION: The study is based on a limited number of good-quality human embryos donated to research. WIDER IMPLICATIONS OF THE FINDINGS: Our study sheds light on the processes underlying the high developmental potential of early human embryonic cells. The CCNE1-positive plenipotent cell type corresponds with a phenotype that enables early human embryos to recover after fragmentation, cryodamage or (single cell) biopsy on day 3 for preimplantation genetic diagnosis. Knowledge on the expression and function of genes responsible for this flexibility will help us to better understand the undifferentiated state in stem cell biology and might enable us to improve technologies in assisted reproduction. LARGE SCALE DATA: NA STUDY FUNDING AND COMPETING INTERESTS: This research is supported by grants from the Fund for Scientific Research - Flanders (FWO-Vlaanderen), the Methusalem (METH) of the VUB and Scientific Research Fond Willy Gepts of UZ Brussel. There are no competing interests.

Live birth rates after IVF are reduced by both low and high progesterone levels on the day of human chorionic gonadotrophin administration.

STUDY QUESTION: Are low serum progesterone levels on the day of human chorionic gonadotrophin (hCG) administration detrimental for live birth delivery rates during in vitro fertilization (IVF)? SUMMARY ANSWER: Progesterone levels ≤0.5 ng/ml on the day of hCG administration hinder live birth rates. WHAT IS KNOWN ALREADY: Fundamental research has shown that the presence of late follicular phase progesterone is essential for follicular development, ovulation and endometrial receptivity. However, previous studies in patients undergoing ovarian stimulation have only assessed if progesterone levels in the higher range are detrimental for pregnancy or not. That said, information on the effect of the full range of late follicular progesterone on IVF outcomes is still lacking. STUDY DESIGN, SIZE, DURATION: This was a retrospective, single-centre cohort study with 2723 cycles performed in patients aged between 19 and 36 and undergoing controlled ovarian stimulation between January 2006 and March 2012 for their first or second attempt of IVF followed by a fresh embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent ovarian stimulation using a gonadotrophin-releasing hormone (GnRH) antagonist for pituitary down-regulation. Final oocyte maturation was triggered with hCG 36 h before oocyte retrieval. On the day of hCG administration, serum progesterone evaluation was performed. Live birth delivery rates were compared amongst various ordinal and regular progesterone intervals (≤0.50, 0.50-0.75, 0.75-1.00, 1.00-1.25, 1.25-1.50, >1.50 ng/ml) using logistic regression. MAIN RESULTS AND THE ROLE OF CHANCE: The average age of our sample was 30.5 years. Almost 82% of all embryo transfers were of a single embryo and 51.8% were performed with a Day 5 embryo. The average value (±standard deviation) of progesterone on the day of hCG administration was 1.02 ± 0.50 ng/ml and the live birth rate was 23.4%. The live birth rates (according to the above-described ordinal serum progesterone intervals) were 17.1, 25.1, 26.7, 25.5, 21.9 and 16.6%, respectively. The live birth rates were significantly lower in patients with both low (≤0.5 ng/ml) and high (>1.5 ng/ml) late follicular progesterone levels (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: The main limitation of our study was its retrospective nature. Furthermore, our study was restricted to patients under GnRH antagonist pituitary suppression and requires confirmation in a GnRH agonist setting. WIDER IMPLICATIONS OF THE FINDINGS: This study comprehensively assessed the relationship between live birth delivery rates and progesterone levels on the day of hCG administration during ovarian stimulation for IVF. Clinically relevant lower (≤0.5 ng/ml) and higher (>1.5 ng/ml) progesterone level limits were determined. STUDY FUNDING/COMPETING INTERESTS: No funding was received for this study and the authors have no conflicts of interest to declare.