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RESEARCH QUESTION: Does additional supplementation with oral dydrogesterone improve reproductive outcomes in patients with low serum progesterone concentrations on the day of frozen embryo transfer (FET) after artificial (HRT) endometrial preparation? DESIGN: Retrospective, single-centre cohort study including 694 unique patients performing single blastocyst transfer in an HRT cycle. For luteal phase support, intravaginal micronized vaginal progesterone (MVP, 400 mg twice daily) was administered. Serum progesterone concentrations were assessed prior to FET and outco-mes were compared among patients with normal serum progesterone (≥8.8 ng/ml) continuing the routine protocol and patients with low serum progesterone (

OBJECTIVE: To study the relationship between the steroid concentration in the endometrium, in serum, and the gene expression level of steroid-metabolizing enzymes in the context of endometrial receptivity in in vitro fertilization (IVF) patients.DESIGN: Case-control study of 40 IVF patients recruited in the SCRaTCH study (NTR5342), a randomized controlled trial investigating pregnancy outcome after "endometrial scratching." Endometrial biopsies and serum were obtained from patients with a first failed IVF cycle randomized to the endometrial scratch in the midluteal phase of the natural cycle before the next fresh embryo transfer during the second IVF cycle.SETTING: University hopsital.PATIENTS: Twenty women with clinical pregnancy were compared with 20 women who did not conceive after fresh embryo transfer. Cases and controls were matched for primary vs. secondary infertility, embryo quality, and age.INTERVENTION: None.MAIN OUTCOME MEASURE(S): Steroid concentrations in endometrial tissue homogenates and serum were measured with liquid chromatography-mass spectrometry. The endometrial transcriptome was profiled by RNA-sequencing, followed by principal component analysis and differential expression analysis. False discovery rate-adjusted and log-fold change >|0.5| were selected as the threshold for differentially expressed genes.RESULT(S): Estrogen levels were comparable in both serum (n = 16) and endometrium (n = 40). Androgens and 17-hydroxyprogesterone were higher in serum than that in endometrium. Although steroid levels did not vary between pregnant and nonpregnant groups, subgroup analysis of primary women with infertility showed a significantly lower estrone concentration and estrone:androstenedione ratio in serum of the pregnant group (n = 5) compared with the nonpregnant group (n = 2). Expression of 34 out of 46 genes encoding the enzymes controlling the local steroid metabolism was detected, and estrogen receptor β gene was differentially expressed between pregnant and nonpregnant women. When only the primary infertile group was considered, 28 genes were differentially expressed between pregnant and nonpregnant women, including HSD11B2, that catalyzes the conversion of cortisol into cortisone.CONCLUSION(S): Steroidomic and transcriptomic analyses show that steroid concentrations are regulated by the local metabolism in the endometrium. Although no differences were found in endometrial steroid concentration in the pregnant and nonpregnant IVF patients, primary women with infertility showed deviations in steroid levels and gene expression, indicating that a more homogeneous patient group is required to uncover the exact role of steroid metabolism in endometrial receptivity.CLINICAL TRIAL REGISTRATION NUMBER: The study was registered in the Dutch trial registry (www.trialregister.nl), registration number NL5193/NTR5342, available at https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6687. The date of registration is July 31, 2015. The first enrollment is on January 1, 2016.

Research question Do cumulative live birth rates (CLBR) differ between polycystic ovary syndrome (PCOS) phenotypes when a freeze-all strategy is used to prevent ovarian hyperstimulation syndrome (OHSS) after ovarian stimulation (OS)? Design This is a single-centre, retrospective cohort study including 422 women with PCOS as defined by Rotterdam criteria or with PCO-like ovarian morphology-only (PCOM) in whom, between January 2015 and December 2019, a freeze-all strategy was applied after GnRH agonist triggering in the context of hyper-response defined as ≥18 follicles of ≥11 mm in their first or second IVF/ICSI cycle in a tertiary referral hospital. The primary outcome was CLBR resulting from the transfer of all cryopreserved embryos from the same IVF/ICSI cycle. Analysis was performed by multivariate logistic regression analysis adjusting for confounders. Results In total, 91/422 (21.6%) patients had PCOS phenotype A (hyperandrogenism + ovulation disorder + PCOM = HOP), 33/422 (7.8%) had phenotype C (hyperandrogenism + PCOM = HP), 161/422 (38.2%) had phenotype D (ovulation disorder + PCOM = OP) and 137/422 (32.5%) had PCOM. Unadjusted CLBR was similar among the groups (69.2%, 69.7%, 79.5% and 67.9%, respectively; p= 0.11). The multivariate logistic regression model adjusting for age, BMI, number of oocytes, fertilization rate and embryo stage at vitrification confirmed that the PCOS/PCOM phenotype did not have any impact on CLBR [OR 0.72, CI 0.24-2.14 (phenotype C); OR 1.55, CI 0.71-3.36 (phenotype D); OR 0.84, CI 0.39-1.83 (PCOM); p= 0.2, with phenotype A as reference]. Conclusions In subfertile women with PCOS eligible for ART, hyper-response after OS confers excellent cumulative live birth rates of ∼70%, irrespective of the phenotype, when a freeze-all strategy is used. This contrasts with the unfavourable clinical outcomes that have previously been observed in hyperandrogenic PCOS women who underwent mild OS targeting normal ovarian response and fresh embryo transfer. The data should however be interpreted with caution as the retrospective nature of the study holds the possibility of unmeasured confounding factors. The results cannot be generalized to all ART cycles in women with polycystic ovaries as they pertain to those cycles in which hyper-response is observed after OS.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal–cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

Chronic endometritis is a poorly understood infectious or inflammatory process, potentially disrupting the correct implantation of a human embryo (Puente et al., 2020). The exact prevalence is a subject of discussion and ranges across the available literature from 2% to almost 60%, with a higher suspicion of the condition being present in women with recurrent early pregnancy loss and recurrent implantation failure (Puente et al., 2020). The impact of chronic endometritis on reproductive outcomes following IVF remains questionable given the lack of proper data convincingly showing an improvement after diagnosis and treatment. This article aims to provide the reader with a critical appraisal of current diagnostic methods, treatments and patient populations to be tested for chronic endometritis.

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endometrial microbiome is of particular interest given the potential impact on the embryo implantation. To date, all endometrial microbiome studies have applied a metagenomics approach. A sequencing-based technique, however, has its limitations, more specifically in adequately exploring low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were included after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial microbiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab®-assisted culturomics to investigate the low biomass endometrial microbiome at a species level.

PURPOSE: To investigate whether treatment with commercially available ready-to-use A23187 ionophore (GM508-CultActive) improves embryo development outcome in patients with a history of embryo developmental problems. METHODS: This is a uni-center prospective study in which sibling oocytes of patients with embryos of poor quality on day 5 in the previous cycle were treated or not with CultActive. RESULTS: Two hundred forty-seven metaphase II (MII) oocytes from 19 cycles performed between 2016 and 2019 were included in the study. After ICSI, the sibling oocytes were assigned to the treatment group or to the control group, following an electronically generated randomization list. A number of 122 MII were treated with CultActive and 125 MII had no treatment and were assigned to the control group. No difference in fertilization rate (p = 0.255) or in the capacity of embryos to reach good quality on day 5 (p = 0.197) was observed between the two groups. The utilization rates defined as the number of embryos transferred or cryopreserved per mature oocyte (p = 0.438) or per fertilized oocytes (p = 0.299) were not significantly different between the treated group and the control group. CONCLUSION: The results of the current study do not support the use of CultActive in cases with embryo developmental problems.

Research question Is there an association between ovarian response and perinatal outcomes? Design A retrospective, single-centre cohort study including all women undergoing their first ovarian stimulation cycle in a gonadotrophin releasing hormone antagonist protocol, with a fresh embryo transfer that resulted in a singleton live birth from January 2009 to December 2015. Patients were categorized into four groups according to the number of oocytes retrieved: one to three (category 1), four to nine (category 2), 10–15 (category 3), or over 15 oocytes (category 4). Results The overall number of patients analysed was 964. No relevant statistical difference was found among neonatal outcomes across the four ovarian response categories. Neonatal weight (in grams) was comparable between all groups (3222 ± 607 versus 3254 ± 537 versus 3235 ± 575 versus 3200 ± 622; P = 0.85, in categories 1, 2, 3 and 4, respectively). No statistically significant differences were found among the ovarian response categories for birth weight z-scores (taking into account neonatal sex and delivery term). The incidence of pre-term birth and low birth weight was comparable across the different ovarian response groups (P = 0.127 and P = 0.19, respectively). Finally, the occurrence of adverse obstetric outcomes did not differ among the ovarian response categories. Multivariate regression analysis revealed that the number of oocytes was not associated with neonatal birth weight. Conclusions No association was found between ovarian response and adverse perinatal outcomes in antagonist IVF and intracytoplasmic sperm injection cycles. Future, larger scale and prospectively designed investigations are needed to validate these results.

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 = 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.

RESEARCH QUESTION: What are the effects of the oxytocin receptor (OTR) antagonist nolasiban on uterine contractions, endometrial perfusion and endometrial mRNA expression? DESIGN: Randomized, double-blind, parallel-group, mechanism-of-action study with nolasiban. Forty-five healthy, pre-menopausal women were treated with placebo, 900 mg or 1800 mg nolasiban on the day corresponding to blastocyst transfer. Ultrasonographic uterine contraction frequency and endometrial perfusion were assessed, and endometrial biopsies analysed by next-generation sequencing. RESULTS: Both doses of nolasiban showed decreased contraction frequency and increased endometrial perfusion depending on the time point assessed. At 1800 mg, 10 endometrial genes (DPP4, CNTNAP3, CNTN4, CXCL12, TNXB, CTSE, OLFM4, KRT5, KRT6A, IDO2) were significantly differentially expressed (adjusted P < 0.05). Of these, OLFM4, DPP4 and CXCL12 were regulated in the same direction as genes involved in implantation during the window of implantation. In addition, three genes (DPP4, CXCL12 and IDO2) were associated with decidualization and endometrial receptivity. CONCLUSIONS: These data expand our knowledge of the mechanism of action of nolasiban in increasing pregnancy rates after embryo transfer. The results suggest more marked effects of nolasiban 1800 mg compared with the 900 mg dose, supporting testing at higher doses in IVF patients.

Study question What is the impact of seven days versus fourteen days’ estrogen (E2) priming on the clinical outcome of frozen-embryo-transfer in artificially prepared endometrium (FET-HRT) cycles? Summary answer No significant difference in clinical/ongoing pregnancy rate was observed when comparing 7 versus 14 days of estrogen priming before starting progesterone (P) supplementation. What is known already One (effective) method for endometrial preparation prior to frozen embryo transfer is hormone replacement therapy (HRT), a sequential regimen with E2 and P, which aims to mimic the endocrine exposure of the endometrium in a physiological cycle. The average duration of E2 supplementation is generally 12–14 days, however, this protocol has been arbitrarily chosen whereas, the optimal duration of E2 implementation remains unknown. Study design, size, duration This is a single-center, randomized, controlled, open-label pilot study. All FET-HRT cycles were performed in a tertiary centre between October 2018 and December 2020. Overall, 150 patients were randomized of whom 132 were included in the analysis after screening failure and drop-out. Participants/materials, setting, methods The included patients were randomized into one of 2 groups; group A (7 days of E2 prior to P supplementation) and group B (14 days of E2 prior to P supplementation). Both groups received blastocyst stage embryos for transfer on the 6th day of vaginal P administration. Pregnancy was assessed by an hCG blood test 12 days after FET and clinical pregnancy was confirmed by transvaginal ultrasound at 7 weeks of gestation. Main results and the role of chance Following the exclusion of drop-outs and screening failures, 132 patients were finally included both in group A (69 patients) or group B (63 patients). Demographic characteristics for both groups were comparable. The positive pregnancy rate was 46.4% and 53.9%, (p 0.462) for group A and group B, respectively. With regard to the clinical pregnancy rate at 7 weeks, no statistically significant difference was observed (36.2% vs 36.5% for group A and group B, respectively, p = 0.499). The secondary outcomes of the study (biochemical pregnancy, miscarriage and live birth rate) were also comparable between the two arms for both PP and ITT analysis. Multivariable logistic regression showed that the HRT scheme is not associated with pregnancy rate, however, the P value on the day of ET is significantly associated with the pregnancy outcome. Limitations, reasons for caution This study was designed as a proof of principle trial with a limited study population and therefore underpowered to determine the superiority of one intervention over another. Instead, the purpose of the present study was to explore trends in outcome differences and to allow us to safely design larger RCTs. Wider implications of the findings: The results of this study give the confidence to perform larger-scale RCTs to confirm whether a FET-HRT can be performed safely in a shorter time frame, thus, reducing the TTP, while maintaining comparable pregnancy and live birth rates. Trial registration number NCT03930706

Study question Do cumulative live birth rates (CLBR) differ between PCOS phenotypes when a freeze-all strategy is used to prevent OHSS after ovarian stimulation (OS)? Summary answer When conventional-dose OS resulted in high response, a CLBR of ∼ 70% was observed after “freeze-all” in women with PCOS, irrespective of their phenotype. What is known already Previous observational studies have shown that CLBR in women with PCOS who undergo assisted reproductive technologies (ART) may depend on their phenotype. When OS was performed with caution to avoid ovarian hyperresponse, CLBR was lower in women with a hyperandrogenic PCOS phenotype. However, when women with PCOS do exhibit hyperresponse and a freeze-all strategy is used, the impact of the PCOS phenotype on the clinical outcome of the ART cycle is unclear. Study design, size, duration This is a single-centre, retrospective cohort study including 422 women with polycystic ovary syndrome (PCOS) as defined by Rotterdam criteria or PCO-like ovarian morphology-only (PCOM) in whom a freeze-all strategy was applied after GnRH agonist triggering in the context of hyperresponse defined as ³19 follicles of ³11mm in their first or second IVF-ICSI cycle between January 2015 and December 2019 in a tertiary referral hospital. Participants/materials, setting, methods PCOS phenotype was based on hyperandrogenism (H), ovulatory dysfunction (O) and PCO-like ovarian morphology (P). Ovarian stimulation was performed with rFSH or HPhMG, adjusted to BMI. The primary outcome was cumulative live birth rate (CLBR) resulting from the transfer of all cryopreserved embryos from the same IVF-ICSI cycle. Patient and cycle characteristics and laboratory and clinical data were analysed. Data were analysed by multivariate logistic regression adjusting for covariates. Main results and the role of chance In total, 91/422 (21.6%) patients had PCOS phenotype A (HOP); 33 (7.8%) had phenotype C (HP), 161/422 (38.2%) had phenotype D (OP) and 137/422 (32.5%) had PCOM (n = 137). BMI, AMH and AFC were significantly different between phenotype groups (p Limitations, reasons for caution These data should be interpreted with caution as the retrospective nature of the study holds the possibility of unmeasured confounding factors. The results cannot be generalised to all ART cycles in women with polycystic ovaries as they pertain to those cycles where OS leads to hyperresponse. Wider implications of the findings In subfertile women with PCOS eligible for ART, hyperresponse after OS confers excellent cumulative live birth rates when a freeze-all strategy is used, eliminating unfavourable clinical outcomes that had previously been observed in hyperandrogenic PCOS women after mild OS targeting normal ovarian response and fresh embryo transfer. Trial registration number not applicable

Study question How does the blood pharmacokinetic (PK) profile of OD/MVP differ after the first and last administration dose when used as LPS for fresh embryo transfer? Summary answer: The PK profile differed strongly between both LPS administration strategies with a more rapid absorption, metabolism and clearance of OD in comparison with MVP. What is known already Adequate LPS is crucial to achieve a successful pregnancy following ovarian stimulation (OS) and fresh embryo transfer. OD has been proven to be non-inferior compared to MVP in two phase III clinical trials. Additionally, a combined individual participant data and aggregate data meta-analysis showed an odds ratio in favor of OD for live birth. Little information is available on the PK of LPS strategies, leaving an important field unexplored. Individualization of LPS has recently gained more interest and insight into the PK of progestogens is essential to correctly interpret the potential impact of circulating hormone levels on reproductive outcomes. Study design, size, duration Twenty oocyte donors underwent two OS cycles followed by one week of LPS (OD or MVP) in a randomized, cross-over, double blind, double dummy fashion. As both dydrogesterone (D) and 20αdihydrodydrogesterone (DHD) are progestogenic, D, DHD and progesterone (P) plasma levels were established using a validated liquid chromatography tandem mass spectrometry assay in each cycle, on the 1st (single dose PK) and 8th day (multiple dose PK) of LPS (9 and 12 harvesting time-points, respectively). Participants/materials, setting, methods All oocyte donors were Main results and the role of chance The mean (±SD) age of the subjects was 27.4 (± 3.8) years and BMI was 24.0 (±3.2) kg/m2. The mean (±SD) number of oocytes retrieved was 19.7 ±10. No adverse events were reported during the intake of the study medication. The PK results are best estimates as sampling was reduced compared to a formal PK study. Following the intake of the first dose of OD, the observed maximal plasma concentrations (Cmax) for D and DHD were 2.9 and 77 ng/ml (single dose). The Cmax for D and DHD was reached after 1.5 and 1.6 hours (=Tmax), respectively. On the 8th day of LPS the first administration of that day gave rise to a Cmax of 3.6 and 88 ng/ml for D and DHD (multiple dose). For both, the observed Tmax was 1.5 hours. Following the intake of the first dose of MVP, the Cmax for P was 16 ng/mL with a Tmax of 4.2 hours. On the 8th day of LPS the first administration of that day showed a Cmax for P of 21 ng/mL with a Tmax of 7.3 hours. Although low, the role of chance could be influenced by the relatively low sampling numbers and frequency. Limitations, reasons for caution Peripheral concentrations do not necessarily reflect the steroidogenic effect on endometrial progesterone receptors. Extrapolation to clinical practice is therefore difficult, however, molecular analyses of endometrial tissue harvested within this study protocol are underway to investigate further pharmacodynamics and the progestogenic impact on endometrial receptivity during the embryo implantation period. Wider implications of the findings: This is the first study comparing OD/MVP pharmacokinetics in IVF/ICSI. Results suggest administration frequency to be as important as dose, definitely for OD, showing a rapid absorption/clearance. More studies are needed to investigate blood levels in relation to time of LPS administration, especially in (artificially prepared) FET and LPS individualization. Trial registration number EUDRACT 2018–000105–23

Study question Does a 3-day pretreatment course with a GnRH antagonist in the early follicular phase increase the number of oocytes in a GnRH antagonist stimulation protocol? Summary answer The administration of 3 days of GnRH antagonist before starting ovarian stimulation in a GnRH antagonist protocol increases the number of COCs (Cumulus-Oocyte-Complexes). What is known already The GnRH antagonist protocol is characterized by higher gonadotropin and E2 serum levels at the start of ovarian stimulation (OS), compared with a long pituitary down regulation protocol. The unsuppressed FSH level at the start of a GnRH antagonist cycle allows the initial growth of follicles before addition of exogenous FSH, which may result in asynchrony of the follicular cohort. Menstrual administration of a GnRH antagonist can inhibit follicle growth and improve homogeneity of recruitable follicles. Previous studies showed a trend toward higher numbers of COCs and improved maturation and fertilization rates of retrieved oocytes. Study design, size, duration Retrospective single center crossover study, including consecutive women enrolled in an IVF program in a university hospital from January 2011 to December 2020. All women underwent one standard GnRH antagonist stimulation cycle (“standard cycle”) and one GnRH antagonist stimulation cycle preceded by early administration of GnRH antagonist for 3 days (“pretreatment cycle”). Women with basal progesterone levels >1.5ng/ml, and women undergoing oocyte freezing, oocyte donation or PGT were excluded. In total, 427 patients were included. Participants/materials, setting, methods Women were included when the pretreatment cycle occurred within a time interval of Main results and the role of chance The average female age was 35.1 ± 4.7 years. Indications for fertility treatment included unexplained infertility (34.3%), male-factor infertility (33.3%), age (16.9%), PCOS (8.2%) and endometriosis (2.6%). All cycles were divided into two groups: group 1 (standard, 427 cycles) and group 2 (pretreatment, 427 cycles). The mean duration of stimulation was similar in both groups (10.3 vs 10.3 days, p = 0.2). The starting dose of gonadotropin (196.8 vs 234.9IU, p Limitations, reasons for caution Despite the large dataset, the presence of biases related to the retrospective study design cannot be excluded. Besides, the impact of GnRH pretreatment on pregnancy rate cannot be assessed because of the crossover design. Wider implications of the findings: A 3-day course of GnRH antagonist pretreatment increases the number of COCs obtained after OS. Furthermore, since the initiation of OS in a GnRH antagonist protocol relies on the occurrence of spontaneous menses, addition of three days of GnRH antagonist pretreatment may enhance scheduling flexibility without reducing efficacy. Trial registration number Not applicable

Study question Does the dose or type of gonadotropin affect the reproductive outcomes of poor responders undergoing MNC-IVF? Summary answer Neither the type nor the dose of gonadotropins affects the reproductive outcomes of poor responders undergoing MNC-IVF. What is known already Poor ovarian response (POR) to ovarian stimulation remains a major therapeutic challenge in routine IVF practice, because of the association with low live birth rates and high cancellation rates. Although high doses of gonadotropins are traditionally used to stimulate the ovaries in women with predicted POR, MNC-IVF has been proposed as a mild-approach alternative in this population. Typically, the MNC protocol includes GnRH-antagonists to avoid premature ovulation and gonadotropin add-back stimulation at the late follicular phase. However, evidence is sparse, and there is no consensus regarding a specific dose or type of gonadotropins in this mild stimulation protocol. Study design, size, duration This is a retrospective cohort study including patients attending a tertiary referral University Hospital from 1st January 2017 until 1st March 2020. Participants/materials, setting, methods All women who underwent MNC-IVF in our center were included. Gonadotropins [recombinant FSH (rFSH), urinary FSH (uFSH) or highly purified human menopausal gonadotrophin (hp-hMG)] were started when a follicle with a mean diameter of 12–14 mm was observed on ultrasound scan, followed by GnRH antagonists (0.25mg/day) from the next day onwards. Mature oocytes were inseminated using ICSI. Main results and the role of chance In total, 484 patients undergoing 1398 cycles were included. Mean (SD) age and serum AMH were 38.2 (3.7) years and 0.46 (0.78) ng/ml, respectively. The daily dose of gonadotropins was either Limitations, reasons for caution The main limitation is the retrospective design of our study, with an inherent risk of bias. Wider implications of the findings: This is the first and largest study evaluating MNC-IVF protocol modalities. Our data demonstrate that any type of gonadotropin can be used and there is no benefit from daily doses beyond 75IU. Trial registration number N/A

Study question When repeated cycles of OS for planned oocyte cryopreservation using a standard GnRH antagonist protocol are required, can OS protocol modifications improve oocyte yield? Summary answer Compared to repeating a standard GnRH antagonist protocol, switching to a long GnRH agonist protocol for POC results in a higher number of cryopreserved oocytes. What is known already The total number of cryopreserved oocytes is a key parameter of POC programs because of its association with livebirth. A substantial proportion of women embarking on POC will undergo repeated cycles of OS to reach their desired target number of vitrified oocytes. According to recent guidelines, the GnRH antagonist protocol with GnRH agonist triggering is considered the first choice protocol for POC, because of its safety profile and convenience. However, in women with normal ovarian reserve, the long GnRH agonist protocol results in a higher number of oocytes retrieved. Evidence regarding the optimal protocol for POC is limited. Study design, size, duration This is a single-centre, retrospective cohort study including 283 women who had a first cycle for POC using a standard GnRH antagonist protocol and who requested a second OS cycle to increase their total number of vitrified oocytes for later use. The choice of protocol for the second cycle was left at the discretion of the reproductive medicine specialist. All OS cycles took place between January 2009 and December 2019 in a tertiary referral hospital. Participants/materials, setting, methods After ovarian reserve testing, the first cycle OS was performed using rFSH or HPhMG in a GnRH antagonist protocol. For the second cycle, a GnRH antagonist protocol with or without antagonist pretreatment, or a long GnRH agonist protocol was prescribed. The primary outcome was the number of mature oocytes (MII) vitrified per cycle. Cycle characteristics were compared. Data were assessed by generalized estimating equation (GEE) regression analysis adjusting for covariates. Main results and the role of chance In total, 226 (79.9%) women had a GnRH antagonist protocol and 57 (20.1%) had a long GnRH agonist protocol in their second OS cycle for POC. Overall, mean age was 36.6±2.4 years. The median (CI) number of mature oocytes vitrified after the second OS cycle was significantly higher than that after the first cycle [8 (5–11) vs. 7 (4–10), p Similarly, of 174 women who underwent their first OS cycle with a standard GnRH antagonist protocol, 133 women (76.4%) had the same protocol for their second cycle and 41 women (23.6%) an additional three-day course of GnRH antagonist pretreatment. According to GEE multivariate regression, this protocol modification did not result in more mature oocytes available for vitrification (coefficient –0.25, CI –1.86–1.36, p-value = 0.76). Limitations, reasons for caution These data should be interpreted with caution because of the retrospective design and limited sample. Although more oocytes were obtained with a long GnRH agonist protocol we have no data on livebirth in women returning to use their oocytes to support the choice for a specific OS protocol for POC. Wider implications of the findings: Although oocyte yield in the context of POC is an important parameter that may be modulated by the choice of OS protocol, the ultimate outcome measure of a successful POC program is livebirth after oocyte vitrification. Future research of oocyte parameters reflecting oocyte quality is paramount. Trial registration number Not applicable

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 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 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 ( 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.

Assisted reproductive technology (ART) has vastly improved over the last 40 years, from a frequently unsuccessful and complicated procedure requiring hospital admission and routine laparoscopy to a fairly simple outpatient technique with relatively high success rates. However, it is important to stress that ART is not without risk and medical complications may still occur. The incidence of most of these ART-related complications is associated with how women undergo ovarian stimulation. For this reason, physicians should be aware that a carefully thought-out ovarian stimulation protocol and cycle monitoring are of paramount importance to maximise the success of the treatment while avoiding potentially life-threating complications to occur in this frequently otherwise healthy patient population. This review discusses the rationale and evolution of ovarian stimulation strategies over the years and the current developments towards finding a balance between the retrieval of a sufficient number of oocytes and ART-related complication prevention.

BACKGROUND: The existence of an extensive microbiome in and on the human body has increasingly dominated the scientific literature during the last decade. A shift from culture-dependent to culture-independent identification of microbes has occurred since the emergence of next-generation sequencing (NGS) techniques, whole genome shotgun and metagenomic sequencing. These sequencing analyses have revealed the presence of a rich diversity of microbes in most exposed surfaces of the human body, such as throughout the reproductive tract. The results of microbiota analyses are influenced by the technical specifications of the applied methods of analyses. Therefore, it is difficult to correctly compare and interpret the results of different studies of the same anatomical niche. OBJECTIVES AND RATIONALE: The aim of this narrative review is to provide an overview of the currently used techniques and the reported microbiota compositions in the different anatomical parts of the female and male reproductive tracts since the introduction of NGS in 2005. This is crucial to understand and determine the interactions and roles of the different microbes necessary for successful reproduction. SEARCH METHODS: A search in Embase, Medline Ovid, Web of science, Cochrane and Google scholar was conducted. The search was limited to English language and studies published between January 2005 and April 2018. Included articles needed to be original microbiome research related to the reproductive tracts. OUTCOMES: The review provides an extensive up-to-date overview of current microbiome research in the field of human reproductive medicine. The possibility of drawing general conclusions is limited due to diversity in the execution of analytical steps in microbiome research, such as local protocols, sampling methods, primers used, sequencing techniques and bioinformatic pipelines, making it difficult to compare and interpret results of the available studies. Although some microbiota are associated with reproductive success and a good pregnancy outcome, it is still unknown whether a causal link exists. More research is needed to further explore the possible clinical implications and therapeutic interventions. WIDER IMPLICATIONS: For the field of reproductive medicine, determination of what is a favourable reproductive tract microbiome will provide insight into the mechanisms of both unsuccessful and successful human reproduction. To increase pregnancy chances with live birth and to reduce reproduction-related health costs, future research could focus on postponing treatment or conception in case of the presence of unfavourable microbiota and on the development of therapeutic interventions, such as microbial therapeutics and lifestyle adaptations.

BACKGROUND: Non-elective freeze-all policy has been increasingly utilized in assisted reproductive treatment, but the optimal timing of frozen-thawed embryo transfer (FET) after controlled ovarian stimulation (COS) remains to be investigated. METHODS: This retrospective cohort study included 2,998 patients who underwent their first FETs after the first COS cycles using the non-elective freeze-all strategy from Jan 2013 to Dec 2016 at a tertiary-care academic medical center. Patients were divided into the “immediate” group in which FET took place within the first menstrual cycle after oocyte retrieval, and the “delayed” group where FET started after one or more menstrual cycles following COS. RESULTS: The mean interval between oocyte retrieval and FET was 33.3±5.8 days in the immediate group (n=280; 9.3%) and 91.3±19.4 days in the delayed group (n=2,718; 90.7%). Cycles with delayed FET had a significantly lower live birth rate than those with immediate FET before [1,246/2,718 (45.8%) vs. 156/280 (55.7%); P=0.002] and after propensity score matching (PSM) [123/280 (43.9%) vs. 156/280 (55.7%); P=0.005]. When controlling for a number of confounding factors by multivariable logistic regression analysis, the risk remained significant with the adjusted odds ratio (aOR) [95% confidence interval (CI)] of 0.69 (0.53–0.90) and 0.60 (0.42–0.85) before and after matching, respectively. CONCLUSIONS: Performing FET immediately within the first menstrual cycle following COS was associated with a higher chance to achieve live birth compared with delaying FET to subsequent cycles in a non-elective freeze-all policy. However, further randomized controlled trials are still needed to confirm this conclusion.

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 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.

RESEARCH QUESTION: What are the reproductive outcomes of women aged 43 years and older undergoing IVF and intracytoplasmic sperm injection (ICSI) treatment using their own eggs. DESIGN: Retrospective study of 833 woman aged 43 years or older undergoing their first IVF and ICSI cycle using autologous oocytes at a tertiary referral hospital between January 1995 and December 2019. Live birth rate (LBR) after 24 weeks' gestation was the primary outcome. RESULTS: Ninety-five out of 833 (11.4%) had a positive HCG, whereas 59 (62.1% per positive HCG) had a miscarriage before 12 weeks' gestation and 36 (4.3%) live births were achieved. Analysis by age showed that the number of cumulus-oocyte complexes retrieved was significantly different between the four age groups: 43 years (5 [3-9]); 44 years (5 [2-7]); 45 years (3 [2-8)]); ≥45 years (2.5 [2-6]); P < 0.01; the number of metaphase II oocytes, however, was similar. Positive HCG rates remained low: 43 years (78/580 [13.4%]); 44 years (14/192 [7.3%]); 45 years (1/39[2.6%]; and ≥46 years (2/22 [9.1%]); P = 0.03, as did LBR: 43 years (28 [4.8%]); 44 (7 [3.6%]); 45 years (0 [0%]); and ≥46 years (1 [4.5%]); P = 0.5. Multivariate regression analysis revealed that only number of metaphase II was significantly associated with LBR, when age was considered as a continuous (OR 1.08, 96% CI 1.004 to 1.16) or categorical variable (OR 1.08, 95% CI 1.005 to 1.16). CONCLUSION: The chances of achieving a live birth in patients aged 43 years and older undergoing IVF/ICSI with their own gametes are low, even in cases of patients with a relatively 'normal' ovarian reserve for their age.

Research question Does clinical performance of personalized embryo transfer (PET) guided by endometrial receptivity analysis (ERA) differ from frozen embryo transfer (FET) or fresh embryo transfer in infertile patients undergoing IVF? Design Multicentre, open-label randomized controlled trial; 458 patients aged 37 years or younger undergoing IVF with blastocyst transfer at first appointment were randomized to PET guided by ERA, FET or fresh embryo transfer in 16 reproductive clinics. Results Clinical outcomes by intention-to-treat analysis were comparable, but cumulative pregnancy rate was significantly higher in the PET (93.6%) compared with FET (79.7%) (P = 0.0005) and fresh embryo transfer groups (80.7%) (P = 0.0013). Analysis per protocol demonstrates that live birth rates at first embryo transfer were 56.2% in PET versus 42.4% in FET (P = 0.09), and 45.7% in fresh embryo transfer groups (P = 0.17). Cumulative live birth rates after 12 months were 71.2% in PET versus 55.4% in FET (P = 0.04), and 48.9% in fresh embryo transfer (P = 0.003). Pregnancy rates at the first embryo transfer in PET, FET and fresh embryo transfer arms were 72.5% versus 54.3% (P = 0.01) and 58.5% (P = 0.05), respectively. Implantation rates at first embryo transfer were 57.3% versus 43.2% (P = 0.03), and 38.6% (P = 0.004), respectively. Obstetrical outcomes, type of delivery and neonatal outcomes were similar in all groups. Conclusions Despite 50% of patients dropping out compared with 30% initially planned, per protocol analysis demonstrates statistically significant improvement in pregnancy, implantation and cumulative live birth rates in PET compared with FET and fresh embryo transfer arms, indicating the potential utility of PET guided by the ERA test at the first appointment.

STUDY QUESTION Does the phenotype of patients with polycystic ovary syndrome (PCOS) affect clinical outcomes of ART following in-vitro oocyte maturation? SUMMARY ANSWER Cumulative live birth rates (CLBRs) after IVM were significantly different between distinct PCOS phenotypes, with the highest CLBR observed in patients with phenotype A/HOP (= hyperandrogenism + ovulatory disorder + polycystic ovaries), while IVM in patients with phenotype C/HP (hyperandrogenism + polycystic ovaries) or D/OP (ovulatory disorder + polycystic ovaries) resulted in lower CLBRs (OR 0.26 (CI 0.06–1.05) and OR 0.47 (CI 0.25–0.88), respectively, P = 0.03). WHAT IS KNOWN ALREADY CLBRs in women with hyperandrogenic PCOS phenotypes (A/HOP and C/HP) have been reported to be lower after ovarian stimulation (OS) and ART when compared to CLBR in women with a normo-androgenic PCOS phenotype (D/OP) and non-PCOS patients with a PCO-like ovarian morphology (PCOM). Whether there is an influence of the different PCOS phenotypes on success rates of IVM has been unknown. STUDY DESIGN, SIZE, DURATION This was a single-centre, retrospective cohort study including 320 unique PCOS patients performing their first IVM cycle between April 2014 and January 2018 in a tertiary referral hospital. PARTICIPANTS/MATERIALS, SETTING, METHODS Baseline patient characteristics and IVM treatment cycle data were collected. The clinical outcomes following the first IVM embryo transfer were retrieved, including the CLBR defined as the number of deliveries with at least one live birth resulting from one IVM cycle and all appended cycles in which fresh or frozen embryos were transferred until a live birth occurred or until all embryos were used. The latter was considered as the primary outcome. A multivariate regression model was developed to identify prognostic factors for CLBR and test the impact of the patient’s PCOS phenotype. MAIN RESULTS AND THE ROLE OF CHANCE Half of the patients presented with a hyperandrogenic PCOS phenotype (n = 140 A/HOP and n = 20 C/HP vs. n = 160 D/OP). BMI was significantly different between phenotype groups (27.4 ± 5.4 kg/m2 for A/HOP, 27.1 ± 5.4 kg/m2 for C/HP and 23.3 ± 4.4 kg/m2 for D/OP, P LIMITATIONS, REASONS FOR CAUTION These data should be interpreted with caution as the retrospective nature of the study holds the possibility of unmeasured confounding factors and misassignment of the PCOS phenotype. Moreover, the sample size for phenotype C/HP was too small to draw conclusions for this subgroup of patients. WIDER IMPLICATIONS OF THE FINDINGS Caucasian infertile patients with a PCOS phenotype A/HOP who undergo IVM achieved a higher CLBR than their counterparts with C/HP and D/OP. This is in strong contrast with previously reported outcomes following OS where women with PCOS and hyperandrogenism (A/HOP and C/HP) performed significantly worse. For PCOS patients who require ART, the strategy of OS followed by an elective freeze-all strategy remains to be compared with IVM in a prospective fashion; however, the current data provide support for IVM as a valid treatment option, especially in the most severe PCOS phenotypes (A/HOP). Our data suggest that proper patient selection is of utmost importance in an IVM programme. 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.

Does intentional endometrial injury (i.e. endometrial scratching) during ART enhance pregnancy rates?We propose a randomized controlled clinical trial in women performing ART in which the intervention group will undergo an additional endometrial biopsy during exogenous ovarian stimulation.Although endometrial receptivity has been extensively studied, the mechanisms behind the implantation of an embryo remain largely a mystery. Intentional endometrial injury has been put forward by many researchers as an inexpensive clinical tool capable of enhancing endometrial receptivity. However, despite its widespread use, the benefit of endometrial scratching is still a contentious and unresolved issue.Pragmatic two-arm randomized, single-centre, controlled open-label trial in women undergoing exogenous gonadotropin ovarian stimulation for ART followed by a fresh embryo transfer in a gonadotropin-releasing hormone antagonist suppressed cycle. The trial will include 360 women in total with a 1:1 allocation ratio and an expected total duration of up to 45 months.Subjects in the intervention group will undergo an endometrial biopsy during the follicular phase, on the sixth to eighth day of exogenous stimulation. Furthermore, nested within this clinical trial, we will also evaluate whether the transcriptomic signatures of the material collected during the biopsy may accurately distinguish women who become pregnant from those who do not. These endometrial transcriptomic signatures will be assessed both immediately after the biopsy and followingOur primary objective is to assess the effect of endometrial injury during exogenous gonadotropin ovarian stimulation on clinical pregnancy rates after ART. Secondary efficacy and safety outcomes include: live-birth delivery after 24 weeks, the endometrial transcriptomic profile among women in the intervention group, short-term safety (e.g. procedure intolerance due to pain, post-procedure bleeding) and long-term safety (e.g. cancelled transfers, miscarriage) outcomes.Owing to its pragmatic design, this study may have limited power to determine one or more of our secondary outcomes and whether there are specific subgroups of women who may benefit significantly from performing endometrial scratching and endometrial transcriptomic profiling.Despite the weak biological plausibility, heterogeneity in the existing randomized controlled trials and lack of evaluation of any potential risks associated with endometrial scratching, this procedure is still widely applied in current clinical practice. This clinical trial aims to pragmatically assess the potential benefits and harms of the generalized use of this strategy.this study has received a grant from the Research Foundation-Flanders (FWO, 1524417N). This organization has no further role in the study, namely with regards to protocol development, study conduction and evaluation of results.NCT02061228.10 February 2014.3 April 2014.2.0.

STUDY QUESTION Does the freeze-all strategy in high-responders increase pregnancy rates and improve safety outcomes when compared with GnRH agonist triggering followed by low-dose hCG intensified luteal support with a fresh embryo transfer? SUMMARY ANSWER Pregnancy rates after either fresh embryo transfer with intensified luteal phase support using low-dose hCG or the freeze-all strategy did not vary significantly; however, moderate-to-severe ovarian hyperstimulation syndrome (OHSS) occurred more frequently in the women who attempted a fresh embryo transfer. WHAT IS KNOWN ALREADY Two strategies following GnRH agonist triggering (the freeze-all approach and a fresh embryo transfer attempt using a low-dose of hCG for intensified luteal phase support) are safer alternatives when compared with conventional hCG triggering with similar pregnancy outcomes. However, these two strategies have never been compared head-to-head in an unrestricted predicted hyper-responder population. STUDY DESIGN, SIZE, DURATION This study included women with an excessive response to ovarian stimulation (≥18 follicles measuring ≥11 mm) undergoing IVF/ICSI in a GnRH antagonist suppressed cycle between 2014 and 2017. Our primary outcome was clinical pregnancy at 7 weeks after the first embryo transfer. Secondary outcomes included live birth and the development of moderate-to-severe OHSS. PARTICIPANTS/MATERIALS, SETTING, METHODS Following GnRH agonist triggering, women were randomized either to cryopreserve all good-quality embryos followed by a frozen embryo transfer in an subsequent artificial cycle or to perform a fresh embryo transfer with intensified luteal phase support (1500 IU hCG on the day of oocyte retrieval, plus oral estradiol 2 mg two times a day, plus 200 mg of micronized vaginal progesterone three times a day). MAIN RESULTS AND THE ROLE OF CHANCE A total of 212 patients (106 in each arm) were recruited in the study, with three patients (one in the fresh embryo transfer group and two in the freeze-all group) later withdrawing their consent to participate in the study. One patient in the freeze-all group became pregnant naturally (clinical pregnancy diagnosed 38 days after randomization) prior to the first frozen embryo transfer. The study arms did not vary significantly in terms of the number of oocytes retrieved and embryos produced/transferred. The intention to treat clinical pregnancy and live birth rates (with the latter excluding four cases lost to follow-up: one in the fresh transfer and three in the freeze-all arms, respectively) after the first embryo transfer did not vary significantly among the fresh embryo transfer and freeze-all study arms: 51/105 (48.6%) versus 57/104 (54.8%) and 41/104 (39.4%) versus 42/101 (41.6%), respectively (relative risk for clinical pregnancy 1.13, 95% CI 0.87–1.47; P = 0.41). However, moderate-to-severe OHSS occurred solely in the group that received low-dose hCG (9/105, 8.6%, 95% CI 3.2% to 13.9% vs 0/104, 95% CI 0 to 3.7, P LIMITATIONS, REASONS FOR CAUTION The sample size calculation was based on a 19% absolute difference in terms of clinical pregnancy rates, therefore smaller differences, as observed in the trial, cannot be reliably excluded as non-significant. WIDER IMPLICATIONS OF THE FINDINGS This study offers the first comparative analysis of two common strategies applied to women performing IVF/ICSI with a high risk to develop OHSS. While pregnancy rates did not vary significantly, a fresh embryo transfer with intensified luteal phase support may still not avoid the risk of moderate-to-severe OHSS and serious consideration should be made before recommending it as a routine first-line treatment. Future trials may allow us to confirm these findings. STUDY FUNDING/COMPETING INTEREST(S) The authors have no conflicts of interest to disclose. No external funding was obtained for this study. TRIAL REGISTRATION NUMBER ClinicalTrials.gov identifier NCT02148393. TRIAL REGISTRATION DATE 28 May 2014 DATE OF FIRST PATIENT’S ENROLMENT 30 May 2014

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.

Resume But de l’etude Dans la neoplasie mammaire, l’envahissement ganglionnaire est un facteur de mauvais pronostic. En cas de carcinome invasif, le curage axillaire est indispensable car il conditionne les traitements adjuvants mais presente malheureusement une morbidite non negligeable, occasionnant notamment paresthesie et lymphœdeme dans 10 a 20 %. L’objectif de cette etude etait d’evaluer la valeur predictive positive et negative de l’imagerie axillaire (echographie et resonance magnetique) de la reponse a la chimiotherapie neoadjuvante chez les patientes atteintes d’une neoplasie mammaire avec metastase ganglionnaire confirmee par analyse anatomopathologique au bilan initial. Le second objectif etait d’etudier la possibilite de reduire la morbidite en evitant une chirurgie axillaire chez les patientes presentant une reponse complete a l’imagerie (N0) apres la chimiotherapie neoadjuvante. Materiels et methodes Il s’agit d’une etude retrospective incluant les patientes prises en charge au centre hospitalier de Wallonie picarde par chimiotherapie neoadjuvante de janvier 2010 a decembre 2016, qui etaient atteintes d’une neoplasie mammaire invasive avec envahissement ganglionnaire, confirme par cytoponction, et bilan d’extension a distance negatif. Elles ont d’abord beneficie d’une chimiotherapie neoadjuvante avec suivi par imagerie par resonance magnetique et/ou echographie, ensuite d’une chirurgie incluant un curage axillaire et enfin une radiotherapie mammaire et ganglionnaire (avec ou sans hormonotherapie et herceptine selon les caracteristiques immunohistochimiques). Resultats Soixante-dix-huit patients presentant les criteres d’inclusion ont ete pris en charge (un homme et 77 femmes), dont deux avaient un cancer mammaire bilateral. L’âge variait entre 30 et 93 ans. Trente et un ont presente une reponse axillaire complete a l’imagerie de fin de chimiotherapie (soit 38,8 %), dont 15 presentaient un envahissement retrouve a l’histologie (soit 48,4 %). Sur les 49 patients dont l’examen radiologique en fin de chimiotherapie neoadjuvante decrivait une persistance de l’envahissement ganglionnaire (soit 61,2 %), 17 ne presentaient pas d’infiltration tumorale a l’histologie (soit 34,7 %). Une valeur predictive positive de 65,3 % et une valeur predictive negative de 51,6 % sont rapportees. Conclusion L’imagerie axillaire n’est pas fiable dans l’evaluation de l’envahissement neoplasique axillaire apres chimiotherapie neoadjuvante. Chez les patientes ayant un envahissement ganglionnaire metastatique au bilan initial et beneficiant d’une chimiotherapie neoadjuvante, la chirurgie axillaire en fin de traitement reste actuellement recommandee malgre sa morbidite.

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.

Objective To study the difference in live birth rate (LBR) between frozen-warmed blastocyst transfer (FET) on the 6th or the 7th day of progesterone administration in artificially prepared cycles. Design Retrospective cohort study. Setting Tertiary university-based referral hospital. Patient(s) Patients who underwent FET between December 2015 and December 2017 in a hormone replacement therapy cycle (HRT). Intervention(s) Group A included all eligible patients who underwent transfer of a vitrified-warmed blastocyst on the 6th day of progesterone administration; group B included patients who underwent blastocyst transfer on the 7th day of progesterone. The artificial HRT protocol in this study consisted of estrogen administration at a dose of 2 mg twice daily for 7 days followed by 2 mg three times daily for 6 days and micronized vaginal progesterone 200 mg three times daily from an adequately considered endometrial thickness onward. Main Outcome Measure(s) Live birth rate. Results The study included 619 patients, 346 in group A and 273 in group B. The LBRs were comparable between both groups (36.6% for group A and group B), even after adjustment for confounding factors (adjusted odds ratio 1.073, 95% confidence interval 0.740–1.556). Subgroup analysis revealed significantly higher miscarriage rates for day 6 blastocysts transferred on the 6th day of progesterone supplementation compared with transfer on the 7th day of progesterone supplementation (50.0% versus 21.4%, respectively). Additionally, there was a tendency toward a higher LBR when the 7-day progesterone supplementation protocol was used for transfer of a day 6 blastocyst (21.5% and 35.5% for group A and group B, respectively). Conclusion Warmed blastocyst transfer on the 6th compared with the 7th day of progesterone administration in an HRT cycle results in similar LBR. Subgroup analysis of day 6 blastocysts showed significantly higher miscarriage rates when FET was performed on the 6th day of progesterone administration.

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.

STUDY QUESTIONDoes 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 ANSWERLFEP does not have an influence on EQ nor CLBR in oocyte donation cycles.WHAT IS KNOWN ALREADYOvarian 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, DURATIONThis 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, METHODSThe 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 CHANCEThree 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 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 LIMITATIONS, REASONS FOR CAUTIONThis 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 FINDINGSThis 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 INTERESTSNot applicable.

Study question What is the impact on clinical pregnancy rates when vitrified cleavage stage Day 3 embryos, warmed and cultured overnight to Day 4, are transferred on the 3rd or 5th day of progesterone administration in an artificial cycle? Summary answer Clinical pregnancy rates are similar when transferring a vitrified-warmed cleavage stage Day 3 embryo after overnight culture on the 3rd or 5th day of progesterone administration. What is known already In artificially prepared cycles, progesterone supplementation is generally started 3 days before embryo transfer, although the optimal length of exposure to progesterone before frozen embryo transfer (FET) has not been established. However, in a natural cycle, serum progesterone levels start to rise before ovulation, due to the LH-stimulated production by the peripheral granulosa cells. Hence, it could be postulated that progesterone supplementation before embryo transfer in an artificial cycle should start earlier or even later. Study design, size, duration Prospective, randomized controlled trial, encompassing 300 patients who had embryos frozen on Day 3 and who underwent FET in an artificial cycle. Between 1 November 2012 and 31 December 2014, 300 patients were allocated to one of two groups as soon as endometrial thickness reached ≥7 mm on ultrasound after estrogen supplementation. A computer-generated randomization list was used, not concealed to the physicians. Each patient was enrolled into the study only once. FET was performed on the fifth day of progesterone supplementation in Group A, whereas in Group B, FET was performed on the third day of vaginal micronized progesterone administration. Embryos were thawed the day before transfer and after overnight culture, one or two Day 4 embryos were transferred. Participants/materials, setting, methods One hundred and fifty patients in Group A received 5 days of vaginal micronized progesterone tablets and one hundred and fifty patients in Group B received 3 days of micronized progesterone vaginally before FET. In Group A, 13 patients did not have an embryo transfer, compared with 12 patients in Group B. Main results and the role of chance Clinical pregnancy rates did not differ significantly between both groups (37/137 (27.0%) in Group A versus 26/138 (18.8%) in Group B (OR 1.6 (CI 0.9-2.82), ITALIC! P = 0.11). However, early pregnancy loss was significantly higher in Group B (32/58 (55.2%)) compared with Group A (21/58 (36.2%)) (OR 0.46 (CI 0.22-0.97), ITALIC! P = 0.04). Limitations, reasons for caution Although no statistically significant difference was seen in the primary outcome, the study may have been underpowered to detect smaller differences. The study was also not blinded and patients were aware of the exact duration of progesterone supplementation. Wider implications of the findings This is the first randomized controlled trial to show that duration of progesterone administration in an artificially prepared FET cycle may modulate cycle outcome and that too short progesterone supplementation might be deleterious. Study funding/competing interests No external finance was involved in this study. All authors declare to have no conflict of interest with regard to this trial. Trial registration number The trial was registered at clinicaltrials.gov (NCT01940653). Trial registration date 9 September 2013. Date of first patient's enrollment 1 November 2012.

Research question Progesterone overproduction during ovarian stimulation is associated with lower live birth rates (LBR) after fresh embryo transfer. Therefore, circulating P concentrations on the day of HCG administration are frequently measured in clinical practice and followed by an elective cryopreservation strategy whenever late-follicular elevated P (LFEP) occurs. A recent study concluded that the duration of LFEP >1.00 ng/mL prior to HCG administration may also affect clinical pregnancy rates. The objective of this current study was to assess whether this hypothesis was reproducible using LBR as the primary outcome. Design Retrospective analysis including women undergoing IVF/ICSI between 2010-2015. LBR were compared among different P elevation duration subgroups (0, 1 or >1 day) using two LFEP thresholds (>1.00 ng/mL and >1.50 ng/mL). Results The duration of LFEP >1.00 ng/mL was not associated with a significant decrease in LBR according to whether the patient had LFEP lasting for 0, 1 or >1 days (29.9%, 30.3% and 26.3%, respectively). Conversely, when using >1.50 ng/mL as the LFEP threshold, LBR decreased significantly (30.3% 20.4% and 20.5%, respectively). However, the relative frequency of having LFEP >1.50 ng/mL for >1 day was exceedingly rare (1.9%) and the additional benefit of evaluating LFEP beyond the day of HCG triggering no longer remained statistically significant after confounder-adjustment with multivariable regression analysis. Conclusion These results suggest a lack of benefit in measuring serum P in the days preceding HCG administration, since LBR in women with LFEP >1 day do not vary significantly from those with LFEP detected only on the day of HCG administration.

Study question Does intentional endometrial injury (i.e. endometrial scratching) during ART enhance pregnancy rates? Summary answer We propose a randomized controlled clinical trial in women performing ART in which the intervention group will undergo an additional endometrial biopsy during exogenous ovarian stimulation. What is known already Although endometrial receptivity has been extensively studied, the mechanisms behind the implantation of an embryo remain largely a mystery. Intentional endometrial injury has been put forward by many researchers as an inexpensive clinical tool capable of enhancing endometrial receptivity. However, despite its widespread use, the benefit of endometrial scratching is still a contentious and unresolved issue. Study design size duration Pragmatic two-arm randomized, single-centre, controlled open-label trial in women undergoing exogenous gonadotropin ovarian stimulation for ART followed by a fresh embryo transfer in a gonadotropin-releasing hormone antagonist suppressed cycle. The trial will include 360 women in total with a 1:1 allocation ratio and an expected total duration of up to 45 months. Participants/materials setting methods Subjects in the intervention group will undergo an endometrial biopsy during the follicular phase, on the sixth to eighth day of exogenous stimulation. Furthermore, nested within this clinical trial, we will also evaluate whether the transcriptomic signatures of the material collected during the biopsy may accurately distinguish women who become pregnant from those who do not. These endometrial transcriptomic signatures will be assessed both immediately after the biopsy and following in-vitro decidualization. Main results and the role of chance Our primary objective is to assess the effect of endometrial injury during exogenous gonadotropin ovarian stimulation on clinical pregnancy rates after ART. Secondary efficacy and safety outcomes include: live-birth delivery after 24 weeks, the endometrial transcriptomic profile among women in the intervention group, short-term safety (e.g. procedure intolerance due to pain, post-procedure bleeding) and long-term safety (e.g. cancelled transfers, miscarriage) outcomes. Limitations reasons for caution Owing to its pragmatic design, this study may have limited power to determine one or more of our secondary outcomes and whether there are specific subgroups of women who may benefit significantly from performing endometrial scratching and endometrial transcriptomic profiling. Wider implications of the findings Despite the weak biological plausibility, heterogeneity in the existing randomized controlled trials and lack of evaluation of any potential risks associated with endometrial scratching, this procedure is still widely applied in current clinical practice. This clinical trial aims to pragmatically assess the potential benefits and harms of the generalized use of this strategy. Study funding/competing interests this study has received a grant from the Research Foundation-Flanders (FWO, 1524417N). This organization has no further role in the study, namely with regards to protocol development, study conduction and evaluation of results. Trial registration number NCT02061228. Trial registration date 10 February 2014. Date of first patient’s enrolment 3 April 2014. Protocol version 2.0.

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.

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.

The early 1980s saw the first cases of live births after the transfer of autologous frozen embryos be reported [1, 2]. These preliminary reports were met with reasonable resistance by the scientific community owing to the limited efficacy of earlier cryopreservation methods and concerns regarding the overall safety of embryo cryopreservation. This uncertainty effectively relayed embryo cryopreservation at its genesis to the status of an “adjuvant method” for cycles in which the number of embryos produced was deemed too excessive for simultaneous replacement during the fresh embryo transfer (ET) attempt. However, following the advent of more efficient cryopreservation strategies [3] (i.e. vitrification) and reassuring safety data [4, 5], the use of embryo cryopreservation has progressively increased, currently accounting for up to one third of all children born after assisted reproductive technologies (ART) in the United States [6]. Furthermore, cryopreservation has now become an indispensable tool in everyday clinical practice, providing the necessary means to assure the safe storage of embryos while minimizing the many risks associated to the multiple pregnancies [7]. For this reason, an increasing amount of scientific societies and governments have encouraged [8] or even enforced [9, 10] elective single ET policies to ART clinics, progressively changing the benchmark of ART from pregnancy rates to Birth Emphasizing a Successful Singleton at Term (BESST) [11]. These considerations have set the stage for a new stance on embryo cryopreservation in modern-day medicine, which is no longer viewed as a simple adjuvant of fresh ET [12], a mindset that has transpired across to oocyte donation and/or embryo cryopreservation programs as well. In parallel, an increasing number of women are currently opting to electively cryopreserve oocytes in anticipation of age-related gamete exhaustion. Since the first live birth following the transfer of a cryopreserved oocyte occurred already in 1986 [13], physicians have the possibility to extrapolate from the abundance of data already existent from both fresh/frozen oocyte donation and embryo cryopreservation programs in order to determine the best ET strategy for women thawing electively cryopreserved oocytes. In this chapter, we give a detailed explanation on how the preparation of the uterus for the transfer of embryos deriving from cryopreserved oocytes is generally performed, followed by a brief overview of which methods one may consider to optimize the safety and pregnancy/neonatal outcomes of these treatment cycles.

STUDY QUESTION: Is natural cycle frozen-thawed embryo transfer (NC-FET) associated with better clinical pregnancy rates (CPR) when compared to modified natural cycle frozen-thawed embryo transfer (mNC-FET)? SUMMARY ANSWER: NC-FET is associated with a higher CPR compared to mNC-FET. WHAT IS KNOWN ALREADY: There is conflicting evidence regarding the impact of hCG triggering on clinical outcomes after frozen-thawed embryo transfer (FET), and information on the effect of luteal phase support (LPS) is lacking. STUDY DESIGN, SIZE, DURATION: This retrospective study included all (n = 2353) consecutive cycles with FET of vitrified cleavage and blastocyst stage embryos warmed between January 2010 and April 2015 in a tertiary centre. The FET cycles were grouped by type as follows: NC (n = 501), NC + LPS (n = 828) or mNC + LPS (n = 1024). Artificial cycles were excluded from the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed mixed-effect multilevel multivariable regression analysis to account for the clustering of FETs using embryos derived from the same patient and/or ovarian stimulation cycle. Adjustment for the following potential confounders was also performed: female age at oocyte retrieval, number of oocytes retrieved, fresh cycle pregnancy outcome, embryo transfer rank, number of embryos transferred, embryo stage and grade and endometrial thickness. Bonferroni adjustment for multiple comparisons was performed whenever indicated. MAIN RESULTS AND THE ROLE OF CHANCE: The unadjusted CPR per cycle was significantly higher in the NC-FET group (46.9%) when compared with the mNC-FET + LPS groups (29.7%, P < 0.001) but not the NC-FET + LPS group (39.9%, P = 0.069). The lower clinical performance of mNC-FET + LPS remained significant even after adjusting for potential confounders [adjusted odds ratio (95% CI) compared to the NC-FET groups: 2.18 (1.64-2.90) and 1.67 (1.31-2.12) for the NC-FET and NC-FET + LPS groups, respectively]. A sensitivity analysis restricting the sample only to the first FET performed by the couple in our centre was also performed. The predicted CPR in this multivariable logistic regression model remained significantly higher in the NC-FET (53.9%) and NC-FET + LPS (44.9%) groups when compared to mNC-FET + LPS (34.2%, all Bonferroni-adjusted pairwise comparisons with P ≤ 0.01). LIMITATIONS, REASONS FOR CAUTION: The interpretation of the findings of this study is limited by the retrospective nature of the analysis and the potential for unmeasured confounding. WIDER IMPLICATIONS OF THE FINDINGS: The use of mNC-FET, using hCG triggering or progesterone supplementation, should be reconsidered in light of the potential negative effect on pregnancy outcome. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: N/A.

Study question Does the timing of the first frozen embryo transfer (FET) after gonadotropin-releasing hormone (GnRH) agonist triggering with the elective cryopreservation of all embryos affect pregnancy outcome? Summary answer FETs performed immediately after a freeze-all cycle did not vary significantly from delayed FETs in terms of pregnancy rates. What is known already As interest in, and use of, the freeze-all strategy expands in the field of reproductive medicine, the optimal timing to perform the subsequent FET has become increasingly important. Thus far, all clinical trials evaluating the efficacy of the segmentation strategy have opted to electively defer the first FET for at least one menstrual cycle. However, this mere empirical approach may cause unnecessary distress to infertile patients eager to conceive as soon as possible. Study design, size and duration This retrospective cohort study included the first FET cycle of all women who underwent a freeze-all protocol between October 2010 and October 2015 in two reproductive medicine centres (in Belgium and Vietnam, respectively). Participants/materials, setting and methods A total of 333 FET cycles were included in the analysis. Following the freeze-all cycle, the preparation of the endometrium consisted of the sequential administration of oestradiol valerate and micronized vaginal progesterone. The start of the FET was classified as either immediate (following the GnRH agonist withdrawal bleeding) or delayed (by at least one menstrual cycle). Clinical pregnancy rate (CPR) was the main outcome of our study. Main results and the role of chance Women in the immediate FET group were slightly younger on average (30.9 ± 4.1 versus 31.8 ± 4.3, P = 0.045) on the date of oocyte retrieval. Moreover, women in the immediate FET group received a blastocyst transfer more frequently (53.4% versus 41.6%, P = 0.038) and had fewer embryos transferred on average compared to the delayed FET group (1.8 ± 0.8 versus 2.0 ± 0.8, P = 0.013). CPR/FET was marginally significantly higher in the immediate FET group in our crude analysis (52.9% after immediate FET versus 41.6% after delayed FET, P = 0.046). In order to assess if CPR/FET remained unaltered after adjusting for measured confounding, we performed mixed-effects multivariable regression analysis. Timing of the FET no longer affected significantly the CPR of the first FET in the adjusted analysis (adjusted odds ratio (aOR): 0.62, 95% CI: 0.38-1.00; predicted CPR of 52.5% for immediate FET versus 41.8% for delayed FET). Limitations, reasons for caution The results are limited by the retrospective design and the potential for unmeasured confounding. Furthermore, we only evaluated the effect of the FET timing of the first FET on CPRs in artificially supplemented cycles and, thus, the results should not be extrapolated to live birth rates or natural-cycle FETs. Wider implications of the findings This study offers a simple but potentially relevant measure to increase patient satisfaction and adherence in couples who seek to become pregnant both safely and as soon as possible. Study funding/competing interests No funding was received for this study. The authors have no conflicts of interest to declare.