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Study question Do Gonadotropin-releasing hormone agonists (GnRHa) triggering improves oocyte maturation, clinical outcomes, and safety compared to human chorionic gonadotropin (hCG) triggering during controlled ovarian stimulation with an antagonist protocol? Summary answer The final triggering using GnRHa allows a higher number of retrieved and mature oocytes to be obtained with comparable clinical outcomes and lower OHSS risk. What is known already GnRHa represent an alternative to hCG for ovulation triggering after controlled ovarian stimulation with an antagonist protocol for IVF/ICSI. GnRHa triggering is thought to be more physiological due to the endogenous surges in LH and FSH levels. However, the benefit of GnRHa over hCG triggering on oocyte maturation remains controversial. Study design, size, duration A systematic review and meta-analysis of randomised controlled clinical trials. Searches were conducted from 01 January 1990 to 15 April 2020 on MEDLINE, EMBASE, the Cochrane Library, ClinicalTrials.gov and EudraCT, using the following search terms: ‘GnRH agonist’, ‘hCG’, ‘triggering’. Two independent reviewers carried out the study selection, the bias assessment using the RoB2 tool, and the data extraction according to Cochrane methods. Participants/materials, setting, methods The primary outcomes were the total number of retrieved oocytes and the number of mature oocytes. The main secondary outcomes were the number of embryos obtained, the clinical pregnancy rate, the early pregnancy loss rate, the live birth rate, and the incidence of ovarian hyperstimulation syndrome (OHSS). Random-effects meta-analysis was performed followed by prespecified sensitivity and subgroup analyses. Main results and the role of chance A total of 29 randomised controlled trials were included. The mean number of retrieved oocytes [difference in means (95% CI) 0.99 (0.21, 1.78); p = 0.01; n = 26] and of mature oocytes [0.68 (0.04, 1.33); p = 0.04; n = 12] were statistically significantly higher after GnRHa than after hCG triggering. A similar difference was observed for the number of embryos [0.94 (0.19, 1.68); p = 0.01; n = 10]. No differences in the clinical pregnancy rate [risk ratio 1.01 (0.90, 1.14); p = 0.83; n = 23], early pregnancy loss [1.27 (0.94, 1.71); p = 0.13; n = 16], and live birth rate [1.00 (0.77, 1.29); p = 0.97; n = 6] were noted. GnRHa was associated with a lower incidence of OHSS [odds ratio 0.25 (0.08, 0.74); p = 0.012; n = 20]. Limitations, reasons for caution The validity of meta-analysis results depends mainly on the quality and the number of the published studies available. Wider implications of the findings: In light of its safety and effectiveness, GnRHa should be the new standard for triggering in antagonist cycles, with dual triggering with hCG when the risk of OHSS is low and a fresh embryo transfer approach is used. Trial registration number NA

Study question Is there any association between stage of embryo at transfer based on extended in vitro culture and gestational trophoblastic disease risk during assisted reproduction? Summary answer No significant association between stages of embryo transfer from zygote stage to blastocyst stage was found after analysing 540376 cycles of IVF and ICSI. What is known already Gestational trophoblastic disease (GTD), commonly referred to as molar pregnancy, results from abnormal proliferation of the trophoblast with a reported incidence of ∼1 in 700 in the UK. Despite technological advances such as ICSI, PGT and selection of normally fertilised (2PN) embryos, there are reported cases of GTD following assisted reproduction. Blastocyst transfer is associated with higher pregnancy and live birth rates but evidence is lacking whether extended embryo culture to blastocyst stage influences implantation of an abnormal embryo or abnormal trophoblastic proliferation leading to GTD. Study design, size, duration A retrospective study was carried out using Human Fertilisation and Embryology Authority (HFEA) anonymised register data from 1999 to 2016. HFEA holds the longest running register for fertility treatment data in the world and is the national database for fertility treatment data in UK. A total of 540376 fresh IVF or ICSI assisted reproduction cycles were analysed. Participants/materials, setting, methods There were 1033588 treatment cycles during the study period but only 540376 cycles met the inclusion criteria of fresh IVF or ICSI. Cycles with incomplete data, frozen embryo transfers, donor treatment or surrogacy were excluded. A subgroup analysis of those with primary subfertility was performed after excluding subjects with secondary infertility in order to exclude an effect of a previous molar pregnancy. Multivariate regression analysis was used to adjust for possible confounders. Main results and the role of chance 78 molar pregnancies were reported in the original sample giving a prevalence of 4/10000 live births (78/228461), much lower than the prevalence given with natural pregnancies. Prevalence of molar pregnancy amongst the study population after meeting exclusion criteria was 4 /10000 livebirths (53/156683). Incidence of molar pregnancy was not statistically different between treatment type (0.0001 vs 0.00009). Significantly higher incidence of GTD was seen in the 40 to 42 age category compared to 18-34 category(OR 1.86(95% CI 8.7-3.75)),in par with known higher GTD risk in women above 40 in the general population. Of interesting note, although the incidence of molar pregnancy was significantly lower in women undergoing assisted reproduction increased risk with advancing age is not totally eliminated with treatment. There was no significant association between the occurrence of molar pregnancy with the type and cause for infertility and number of embryos transferred. Crude (1.06 (95% CI 0.852-1.31)) and adjusted odds ratios (1.07 (95% CI (0.857-1.32)) did not show any association between day of embryo transfer and molar pregnancy even after adjusting for age and excluding secondary infertility. Selection of blastocyst stage embryo after extended culture did not alter the likelihood of having a GTD compared to cleavage stage embryo. Limitations, reasons for caution The retrospective analysis of anonymised HFEA data limited adjustments for confounders such as smoking, previous history of GTD, ethnicity etc that predispose to GTD. Caution needs to be exercised for under-reporting of GTD to HFEA and lack of information on type of GTD identified. Wider implications of the findings Though GTD cannot be prevented by IVF/ICSI, the incidence is significantly low and extended culture is not associated with higher risk of abnormal trophoblastic proliferation or GTD occurrence with IVF/ ICSI treatment. These findings would aid informed implications counselling and reassurance of patients during assisted reproduction treatments. Trial registration number not applicable

Study question Is the incidence of miscarriage rates increased in frozen embryo transfer (ET) compared with fresh ET? Summary answer The frozen ET is not associated with an increased risk of miscarriage in comparison with fresh ET. What is known already Nowadays, the freeze-all policy has gained great popularity because it minimizes OHSS and it seems to improve the reproductive outcomes. This increase was possible thanks to progress in vitrification procedures. Nevertheless, some recent evidence suggests more caution on the wide use of elective frozen ET in a general IVF population for its possible effect on some pregnancy-related complications. Endometrial preparation schemes for frozen ET include natural cycles or, more frequently, artificial cycles in which the endometrium is prepared with exogenous steroids. In this last case, the corpus luteum is absent, but its demise could be correlated with some pregnancy-related complications. Study design, size, duration A meta-analysis, based on PubMed, Cochrane CENTRAL, EMBASE, was conducted to estimate and compare the miscarriage rate (MR) in frozen versus fresh ET. Following PICOS, inclusion criteria were: Population, patients undergoing IVF/ICSI; Intervention, frozen ET; Comparison, fresh ET; Outcome, MR (primary one); clinical pregnancy rate (CPR), ongoing pregnancy rate (OPR) and live birth rates (LBR) (secondary ones). Study design: RCT. Participants/materials, setting, methods Electronic and manual search, conducted from 1990 to 2020, yielded 269 studies. Two researchers reviewed the studies independently, excluding 247 and 14 studies after the first and second screening. The outcome data from the 8 included studies were combined using a Mantel–Haenszel model and applying the random effects models. The dichotomous data results of each study were expressed as risk ratio (RR) with 95% confidence intervals (CI). Heterogeneity was evaluated using the I2 statistic. Main results and the role of chance The eight studies comprise 6,934 participants, 3,450 undergoing frozen ET and 3,484 fresh ET (Ferraretti et al., 1999; Shapiro et al., 2011a; Shapiro et al.; 2011b; Chen et al., 2016; Shi et al., 2018; Le et al., 2018; Wei et al., 2019; Stormlund et al., 2020). Preliminary analyses excluded interaction between covariates. RR of miscarriage ranged from 0.29 (Chen et al., 2016) to 3.24 (Ferraretti et al., 1999). The level of heterogeneity (I2) was 48% and it was considered as moderate. MR resulted not significantly different in frozen [279/1,601 pregnancies] compared with fresh ET [308/1,458 pregnancies] (RR = 0.727, 95%CI=0.43–1.25, p = 0.25). By analysing the 5 trials in which the endometrium was prepared with steroids (Ferraretti et al., 1999; Shapiro et al., 2011a; Shapiro et al., 2011b; Chen et al., 2016; Le et al., 2018), MR resulted not significantly different in frozen [126/720] versus fresh ET [160/682] (RR = 0.605, 95%CI 0.22–1.66; p = 0.33; I2=43%). For the secondary outcomes, CPR (RR = 1.14; 95%CI=0.97–1.35; 0.56 vs 0.52 for frozen and fresh ET, respectively), OPR (RR = 1.12; 95%CI=0.95–1.33; 0.49 vs 0.46 for frozen and fresh ET, respectively) and LBR (RR = 0.93; 95%CI=0.60–1.44; 0.49 vs 0.47 for frozen and fresh ET, respectively) were not significantly different between the two groups. Limitations, reasons for caution The MR was not the primary outcome of the included RCTs. This could have determined a sample size not calibrated for the analysis of the primary outcome of the present meta-analysis. Wider implications of the findings: This meta-analysis indicates that MR was similar between frozen and fresh ET. The endometrial preparation, in artificial cycles without corpus luteum, does not seem to influence the good course of pregnancy. Based on these results, the choice of a fresh ET should not derive by the presumed reduction of MR. Trial registration number Not applicable

Study question The purpose of this systematic review is to calculate dropout-rates of IVF/ICSI treatment by analysing the published cumulative live birth rates of IVF/ICSI treatment. Summary answer One out of three patients stop their treatment after their first IVF/ICSI cycle and dropout-rates tend to increase per consecutive cycle. What is known already Cumulative live birth rates (CLBRs) have created the possibility to present realistic probabilities of having a live birth after IVF/ICSI treatment. However, it is noted that a significant percentage of the patients stop their treatment before having a child (“dropout”). Possible reasons and predicting factors for dropout of treatment are already extensively investigated. However, only a few studies try to report about the incidence of dropout. Publications on CLBRs of large numbers of patients allow the extraction of dropout-rates. These rates will provide insight in the extent of the problem and could be used as a reference for interventional studies. Study design, size, duration Four databases (PubMed, The Cochrane Library, EMBASE, DoKS) were systematically searched from 1992 to December 2020. Search terms referred to “cumulative live birth” AND “ART/IVF/ICSI”. No restrictions were made on the type or language of publication. Studies were included if they reported absolute numbers of patients and live births per consecutive complete IVF/ICSI cycle or per consecutive embryo transfer cycle, starting from the first IVF/ICSI cycle for each patient. Participants/materials, setting, methods Dropout-rates per cycle were calculated in two manners: “intrinsic dropout-rate” with all patients that started the particular IVF/ICSI cycle in the denominator, and “potential dropout-rate” with all patients who did not achieve a live birth after IVF/ICSI (and potentially could have started a consecutive cycle) in the denominator. Dropout-rates were analysed for consecutive complete cycles and consecutive embryo transfer cycles, because these two manners are used in reporting CLBRs, often related to the reimbursement policy. Main results and the role of chance This review included 29 studies and almost 800,000 patients from different countries and registries. Regarding the patients who started their first IVF/ICSI cycle, trying to conceive their first child by IVF/ICSI, intrinsic dropout-rate was 33% (weighted average) after the first complete cycle, meaning they did not return for their second oocyte retrieval cycle. After the first embryo transfer cycle, intrinsic dropout-rate was 27% (weighted average), meaning those patients did not return for their next frozen-thawed embryo transfer cycle or for the next oocyte retrieval cycle. Regarding the patients who did not achieve a live birth after the first complete cycle, potential dropout-rate was 48% (weighted average), and 37% (weighted average) after the first embryo transfer cycle. Both potential and intrinsic dropout-rates for both consecutive complete and embryo transfer cycles tended to increase with cycle number. One study on second IVF/ICSI conceived children showed a potential dropout-rate after the first complete cycle of 29%. From studies on women >40 years of age, the potential dropout-rate after the first complete cycle was 45% (weighted average) and from studies with the uses of testicular sperm extraction, the potential dropout-rate after the first complete cycle was 34% (weighted average). Limitations, reasons for caution Our analysis was hampered by the different ways of reporting on CLBRs (complete cycles versus embryo transfer cycles), informative censoring, patients changing clinics and spontaneous pregnancies. Dropout-rates were potentially overestimated given that spontaneous pregnancies were not taken into account. Wider implications of the findings The extent of dropout in IVF/ICSI treatment is substantial and has an important impact on its effectiveness. Therefore, it is a challenge for fertility centers to try to keep patients longer on board, by taking into account the patients’ preferences and managing their expectations. Trial registration number PROSPERO Registration number: CRD42020223512

Study question Can frozen embryo transfer (FET) be offered immediately after a stimulated IVF/ICSI cycle without compromising live birth rate (LBR)? Summary answer FET in the menstrual cycle immediately following the stimulated IVF/ICSI cycle was associated with a slightly higher LBR compared to standard postponed FET. What is known already It is standard clinical practice to postpone FET for at least one menstrual cycle following a failed fresh transfer or a freeze-all cycle. This practice is thought to minimize any possible residual negative effect of ovarian stimulation, with excessive steroid levels and multiple corpora lutea, on the resumption of a normal ovulatory cycle and receptivity of the endometrium. Even so, elective deferral of FET is an empirical strategy based on suggestions rather than solid scientific evidence and may unnecessarily delay time to pregnancy, causing frustration and decreased quality of life to couples. Study design, size, duration Systematic review and meta-analysis according to PRISMA guidelines. Original studies on subfertile women aged 18-46 with any indication for treatment with IVF/ICSI investigating the timing of FET after IVF/ICSI were included. Intervention was defined as FET in the menstrual cycle immediately following the stimulated IVF/ICSI cycle. Comparator was defined as FET in the second or subsequent menstrual cycle following IVF/ICSI. Risk of bias was assessed using Robins-I and quality of evidence using GRADE. Participants/materials, setting, methods PubMed (MEDLINE) and EMBASE databases were searched for MeSH and Emtree terms, as well as text words related to timing of FET, up to March 2020. There were no limitations regarding year of publication or duration of follow-up but to English language. The primary outcome was LBR. Secondary outcomes were implantation rate, pregnancy rate, clinical pregnancy rate (CPR), time-to-pregnancy, miscarriage rate (MR), cycle cancellation rate and patient wellbeing. Main results and the role of chance Out of 4124 search results, 15 studies were included in the review. Studies reporting adjusted odds ratios (aOR) for LBR, CPR and MR were included in meta-analyses. All studies (n = 15) were retrospective cohort studies involving a total of 6,304 immediate FET cycles and 13,851 postponed FET cycles including 8,019 matched controls. Twelve studies of very low to moderate quality reported no difference in LBR with immediate versus postponed FET. Two studies of moderate quality reported a statistically significant increase in LBR with immediate FET and one small study of very low quality reported better LBR with postponed FET. Trends in rates of secondary outcomes followed trends in LBR regarding timing of FET. The meta-analyses showed a significant advantage of immediate FET (n = 2,076) compared to postponed FET (n = 3,833), with a pooled aOR of 1.20 (95% CI 1.01-1.44) for LBR and a pooled aOR of 1.22 (95% CI 1.07-1.39) for CPR. Limitations, reasons for caution Limitations include the retrospective design and heterogeneity of studies included, limiting comparison and pooling of data. With little transparency regarding cancellation rates, the risk of selection bias is apparent. Further, confounding by embryo quality is a limitation. Small sample sizes are a limitation to subgroup meta-analyses. Wider implications of the findings The standard clinical practice of postponing FET for at least one menstrual cycle following a failed fresh transfer or a freeze-all cycle may not be best clinical practice. Randomized controlled trials including data on cancellation rates are highly needed to provide high grade evidence regarding clinical practice and patient counseling. Trial registration number not applicable

Study question Does time between ejaculation and processing, and time between processing and insemination/injection affect fertilization rate (FR) and ongoing pregnancy rate (OPR) in IVF/ICSI treatments? Summary answer Increasing time between processing and insemination significantly decreased the OPR after IVF. FRs after IVF/ICSI and OPR after ICSI were not affected by different time-intervals. What is known already The choice for IVF or ICSI depends on semen quality, however, this doesn’t affect the outcome of IVF/ICSI treatments (Mariappen et al 2018). After ejaculation, the percentage of motile spermatozoa decreases progressively at a rate of about 10%/hour (Makler 1979). According to the ESHRE-guideline, semen should be processed within 1 hour after ejaculation. In our laboratory, a validation was performed that confirmed a decrease in sperm motility after ejaculation. During incubation at 37 °C after processing, the sample remained stable in incubation medium (unpublished data). Therefore, we analyzed the effect of handling time and incubation time with regard to IVF/ICSI outcomes. Study design, size, duration This retrospective data analysis examines the effect of time between ejaculation and processing using density-gradient centrifugation (handling time) and time between processing and insemination (IVF)/injection (ICSI) (incubation time) on the FR and OPR, irrespective of the initial semen quality. A total of 1488 oocyte pickups (844 IVF, 644 ICSI) were included from 1060 patients undergoing fertility treatment between 2017 and 2019. Oocyte pickups without oocytes, with oocyte vitrification, or with donor oocytes were excluded. Participants/materials, setting, methods Anonymized data were obtained from the laboratory database ProMISe. Handling time and incubation time of the semen incubated at 37 °C and 5% CO2 were analyzed in relation to the occurrence of TFF (Total Fertilization Failure), FR and OPR. Linear and logistic regression was performed in SPSS version 25. In case of significant association, the data were adjusted for potential confounders, such as woman’s age, semen quality before and after preparation, and number of oocytes. Main results and the role of chance This study shows that increasing the incubation time of the semen significantly reduced the OPR per ET in IVF treatments (from 30,8% within 3,5 hours to 24,1% after 6 hours) even after adjusting for the potential confouders. However, the OPR in ICSI treatments was not significantly affected by the incubation time (rather, there was an opposite trend). Also, the handling time of the semen did not significant effect the FR per OPU and the OPR per ET in IVF/ICSI treatments. The overall percentage of TFF was 3,5% and did not differ significantly between the IVF and ICSI treatments. Both handling time and incubation time did not have a significant effect on the occurrence of TFF. An explanation for the decrease in OPR in IVF treatments may be that increasing the incubation time at 37 °C reduces the sperm quality as the capacitation reaction takes place too early, energy levels are reduced, DNA damage increases, or vacuoles arise in the sperm heads (Thijssen et al 2014, Jackson et al 2010, Peer et al 2007). Incubation at room temperature and reduction of the insemination time may improve OPR. Limitations, reasons for caution Retrospective study limitations (bias), no data on DNA fragmentation, incubation of semen only at 37 °C after preparation. Wider implications of the findings: Although it is recommended to produce semen at the IVF-department, our results show that an exception can be made, when production of a semen sample in a clinical setting is stressful, with no negative effect on the outcome. Furthermore, incubation-time at room temperature may have a positive effect on OPR. Trial registration number Not applicable

Study question What is the influence of time on the vaginal microbiome-based prediction of IVF/ICSI success? Summary answer Time influences the vaginal microbiome-based prediction of IVF/ICSI success. What is known already The association between the microbiome of the lower female reproductive tract and subfertility is discussed extensively suggesting its importance for fertility and fertility treatment. Using a modified next generation sequencing technique, an assay of the vaginal microbiome that predicts the pregnancy chances before starting the IVF/ICSI procedure has been developed and validated (1) displaying profiles associated with a low, medium and high chance of implantation. The vaginal microbiome is already known to change over time (2). However, it remains unclear to what extent spontaneous improvement from a low score can occur and over what time period. Study design, size, duration To investigate the spontaneous reversal capacity and associated time period of a low score microbiome profile in IVF-ICSI patients, an observational prospective cohort study of 77 women was performed using the ReceptIVFity assay. Women with medium or high profiles were encouraged to proceed with their ART treatment, whereas women with a low profile were suggested to delay the treatment for 1 month until a subsequent swab was taken with a maximum of 4 repeats. Participants/materials, setting, methods The study was carried out in a University based single center setting. Ethical approval was obtained (6259R MPG§23b). Patients between 24 and 41 years of age were included when eligible for their first, second or third IVF or IVF-ICSI attempt. Exclusion criteria were: antibiotic treatment in the 3 months prior to the test, women who have started with hormone treatment in the last 2 months in the context of ovarian stimulation, or downregulation of endometriosis. Main results and the role of chance Of the 77 patients included, 53 had a high or medium profile and proceeded with their treatment. 24 had a low profile and were supposed to delay the treatment in favor of a subsequent test. The low profile patients were followed up as indicated in the study description. Unfortunately, 11 of the 24 low score patients dropped out of the study. This relatively high number can only in parts be explained by unswayable medical reasons as no fertilization or embryo arrest but a comparable number of patients dropped out most likely due to Corona restrictions or Corona-related anxiety reasons. In the low score group, 1 month after the initial test, 12 patients repeated the swab; 4 remained low (33,33%), whereas 8 shifted to the medium or high (66,67%) groups. After 2 months, 4 patients had another test; 1 remained low (25%), 3 shifted to medium and high (75%). Therewith, in two months’ time 91,7% shifted from low to a better (medium/high) profile. So far, only 1 patient of the initial lows remained low for 5 months. The 12 shifters had a clinical pregnancy rate of 40% after the first embryo transfer after changing the microbiome profile from low to medium/high. Limitations, reasons for caution The results described were generated from a smaller group than intended initially due to a relative high dropout rate for no medical reasons. Wider implications of the findings: Patients suffering from infertility have a clinical benefit from performing a ReceptIVFity test before ART treatment and to delay treatment, when the result is low, since the spontaneous conversion time to a better profile, and therewith a higher pregnancy chance, occurred within 2 month in almost all patients. Trial registration number 2018124928

Study question Does an elevated SDF (>15%) increase the odds of adverse maternal and neonatal outcomes in autologous oocyte IVF-ICSI cycles from unselected couples? Summary answer No adverse effects of high SDF on obstetric and neonatal outcomes have been found in couples with sperm fragmentation undergoing IVF-ICSI cycles with own eggs. What is known already Sperm chromatin integrity assessment has been implemented as an additional tool in the clinical evaluation of sperm quality in infertile patients undergoing an assisted reproduction treatment. All of the published reports to date appraise its effect on clinical outcomes, and how it impacts embryo quality and the pregnancy chances after IVF and ICSI cycles. Sperm DNA integrity has also been hypothesized to affect offspring health but not many studies have reported in humans if an elevated SDF raises the risks of obstetric, delivery and neonatal outcomes. Study design, size, duration Multicentric retrospective cohort study of all IVF-ICSI cycles using autologous oocytes between January 2000-March 2019 at Spain IVIRMA clinics of couples with a SDF test on their ejaculated semen. The sperm fragmentation index was measured in all men with TUNEL assay. The database included 228 couples which had a delivery with at least a newborn. Subjects were divided into two study groups according to their level of SDF: ≤15% (low SDF) or > 15% (high SDF). Participants/materials, setting, methods Patients with missed information on maternal and neonatal outcomes were not counted for the analysis. The obstetric outcomes were gestational age, gestational diabetes, preeclampsia (hypertension with proteinuria after 20 weeks of gestation) and type of delivery. Neonatal outcomes were sex, birth weight, length, head circumference, Apgar score at 1, 5, 10 minutes, and neonatal intensive care unit (NICU) admission. Student’s t-test and Fisher’s test were used for statistical analysis. A p-value Main results and the role of chance Maternal age mean was 37.4 years (95%CI 36.9-38.0) in ≤ 15%SDF group and 37.2 years (95%CI 36.1-38.4) in > 15%SDF group (p = 0.8). Similar gestational age was found, 41.8 weeks (95%CI 41.3-42.2) in ≤ 15%SDF and 41.3 weeks (95%CI 40.4-42.3) in > 15%SDF. Gestational diabetes incidence was higher in > 15%SDF compared to ≤ 15%SDF group (3.5% versus 1.7% (OR = 2.0 (95%CI 0.03-39.8), p = 0.5). Equally, the incidence of preeclampsia was 3.6% in patients with high SDF versus 1.7% in couples with low SDF, OR = 2.1 (95%CI 0.03-41.3), p = 0.5. Type of delivery frequency was in the ≤15%SDF group 61.9% vaginal and 38.1% cesarean, while in the >15%SDF group 62.1% vaginal and 37.9% cesarean (OR = 1.0 (95%CI 0.4-2.6), p = 1.0). The overall proportion of singleton pregnancies was 87.2% (95%CI 82.4-91.2) and twins 12.8% (95%CI 8.8-17.6). There were no statistically differences between groups in the rate of delivery of twins and in the sex ratio of the newborns. When comparing the newborns of ≤ 15%SDF with >15%SDF group, the average of weight was 3011.7g (95%CI 2912.2-3111.2) versus 2986.4g (95%CI 2753.1-3219.7), of length was 49.2cm (95%CI 48.3-50.0) versus 49.5cm (95%CI 49.2-49.9), of head circumference was 34.9cm (95%CI 34.6-35.2) versus 34.3cm (95%CI 33.4-35.2). No statistically differences were observed for Apgar punctuation and for NICU admission. Limitations, reasons for caution Due to the retrospective nature of the study we have missing data from the lack of follow-up of many patients after the confirmation of the ongoing pregnancy. Although pregnancies of couples with elevated SDF have a higher incidence of gestational diabetes and preeclampsia, the sample size evaluated is a limitation. Wider implications of the findings This is one of the first reports to evaluate the relationship between paternal DNA damage and obstetric risks and neonatal health in couples with high SDF who underwent IVF-ICSI in our centers. Despite SDF did not jeopardize the maternal and neonatal outcomes, more studies are needed to confirm this conclusion. Trial registration number NA

Study question Is reproductive success measured as CLBR per inseminated oocyte, per embryo transfer and per embryo transferred affected by paternal age in autologous IVF-ICSI cycles? Summary answer The number of embryo transfers and embryos transferred until live birth, but not the number of inseminated oocytes, were significantly different among the age groups. What is known already In recent years, there has been an increase of the average paternal age at which the first child is conceived. Therefore, there is a growing interest on the study of the impact of male age on the reproductive outcomes in assisted reproduction cycles (ART). Several studies have shown negative effects of advanced paternal age on semen parameters, embryo aneuploidy, miscarriage, male infertility,.... However, other studies have found no association between them. Hence, the impact of paternal age on reproductive outcomes still remains uncertain, leading to a need of more research on this topic, which this study tries to address. Study design, size, duration This retrospective observational multicentric cohort study has included autologous IVF-ICSI treatments (n = 6295) performed to couples with etiology of male infertility (non-normozoospermic) in Spain IVIRMA clinics between January 2008 and March 2020 using patients’ own sperm sample. Paternal age ranged from 20 to 75 years. The study population was categorized in 5 groups following the criterion of homogenizing the number of observations between groups: 20–34 (A), 34–37 (B), 37–39 (C), 39–42 (D) and 42–75 (E) years. Participants/materials, setting, methods Considering that male age could be a factor affecting reproductive outcomes, we evaluated men with different age that performed an autologous IVF-ICSI treatment with their own semen, etiology of male infertility and known age. Data was exported in order to obtain the clinical database and Kaplan-Meier was used for data analysis. P Main results and the role of chance This study considered approximately 2976 patients and 4385 embryo transfers. The CLBR per inseminated oocyte showed no significant difference between the study groups: A (6.43%, 48.24%, 81.38%), B (5.74%, 52.14%, 82.87%), C (6.14%, 49.83%, 83.69%), D (5.89%, 53.60%, 81.16%) and E (6.61%, 47, 52%, 77.85%) for 4, 13 and 21/22 inseminated oocytes, respectively. In terms of CLBR per embryo transfer, the results obtained for each of the age groups were: A (31.81%, 71.89%, 87.63%), B (28.89%, 67.87%, 82.63%), C (27.10%, 68.87%, 88.17%), D (23.45%, 64.63%, 100.00%) and E (22.88%, 55.48%, 63.31%) for 1, 4 and 7 embryo transfers, respectively. There were statistically significant differences in the CLBR per embryo transfer between the studied age groups (p Limitations, reasons for caution The retrospective nature of this study leads to biases derived from the clinical practice and to the presence of missing data (limiting sample size). Moreover, this study considered autologous cycles, therefore the results were not adjusted for female factors. Wider implications of the findings: Our study showed that reproductive outcomes measured by CLBR per embryo transfer and embryo transferred until live birth were significantly different between the paternal age groups in autologous IVF-ICSI cycles of infertile males. Hence, paternal age could be affecting reproductive outcomes and it should be considered for improving infertility counselling. Trial registration number NA

Study question Does GCSF by intrauterine route leads to better result in the treatment of thin endometrium as compared to GCSF by the subcutaneous route, in IVF-ICSI Cycles? Summary answer Yes, GCSF by intrauterine route leads to better result in the treatment of thin endometrium as compared to subcutaneous-GCSF, in ART Cycles? What is known already GCSF, is a member of the colony stimulating factor family of cytokines and growth factors. GCSF receptors are expressed in high concentration on dominant follicle, particularly at preovulatory stage.The endometrium also shows an increased expression of these receptors. GCSF concentration rises in the follicular fluid at the same time. Serum levels of GCSF are found to be in direct correlation with levels of GCSF in follicular fluid. Serum levels increase progressively from the day the embryo-transfer to the day of implantation. GCSF has been found to be beneficial in patients with thin endometrium and recurrent implantation failure. Study design, size, duration This was a RCT conducted between 2018–2019. 30 patients with thin endometrium were enrolled in each group. In either group, GCSF was given if endometrium was less than 7mm on day 14, maximum of two doses were administered. Patients undergoing frozen embryo transfer were recruited in the study, after meeting the inclusion and exclusion criteria. Primary outcome measured was increase in endometrium thickness and the secondary outcome was the clinical pregnancy rate and abortion-rate. Participants/materials, setting, methods 60 patients with thin endometrium were randomly divided into two groups. Group A: Inj. GCSF (300 mcg/1 ml) subcutaneously on Day 14 onwards alternate days for two doses. Group B: Inj. GCSF (300 mcg/1 ml) instilled slowly into the uterine cavity using an intrauterine insemination (IUI) catheter under USG guidance. Endometrial thickness was assessed after 48 h. If endometrial thickness was found to be Main results and the role of chance In the subcutaneous group (group-A) the mean endometrial thickness before GCSF injection was 5.8 ± 0.6 mm and, after injection it increased to 6.9 ± 0.4 mm. Similarly, in the intrauterine group (group-B) the mean endometrial thickness before GCSF was 5.9 ± 0.7 which increased to a mean of 7.9 ± 0.5 after GCSF instillation. The difference between endometrial thickness before and after intrauterine infusion of GCSF was more than that in the subcutaneous group. In group-A, 08 patients conceived out of 30 patients ( clinical pregnancy rate 26.6%) and in group B 11 conceived out of 30 patients in whom GCSF was instilled intrauterine (pregnancy rate 36.6%). Thus, there was a difference in the clinical pregnancy rate in the two groups, the intrauterine group yielding a higher clinical pregnancy rate, but it was not statistically significant. Because of the thin endometrium, we found an abortion rate of 25% (2/8) in the subcutaneous-GCSF group, and an abortion rate of 18% (2/11) in the intrauterine GCSF group. Limitations, reasons for caution There are few potential limitations because of the small sample size. Confounders such as obesity, smoking and alcohol intake, presence of adenomyosis and endometriosis, were not taken into consideration. Though prevalence of obesity is usually low in Indian women. Habits of smoking and alcohol are exceedingly uncommon in Indian women. Wider implications of the findings: Use of GCSF plays an important role in management of patients of thin endometrium undergoing embryo transfer. It is an easily available and economical preparation in developing countries and the intrauterine instillation of GCSF can be easily practiced in an ART unit with good results in resistant thin endometrium patients. Trial registration number Not applicable

Study question After one failed IVF/ICSI cycle, does the endometrial microbiota composition differ between women who will or will not reach a live birth within 12 months? Summary answer The endometrial microbiota composition did not significantly differ in women with one failed IVF/ICSI cycle with or without live birth, but statistical power was low. What is known already Evidence for the presence of an indigenous endometrial microbiome is mounting, and its composition may be associated with implantation success. However, a ‘core’ endometrial microbiome has not yet been defined, and its role in embryo implantation is still poorly understood. Further investigation of this topic may allow improvement and personalisation of clinical care for infertile couples. Endometrial microbiome analysis in infertile women has not yet been performed using transcervically obtained endometrial tissue. Using endometrial tissue instead of swabs or fluid may increase the bacterial DNA yield and therefore the precision of microbiome analyses. Study design, size, duration Endometrial tissue was obtained from a cohort of 141 infertile women undergoing endometrial scratching within a randomised controlled trial (RCT) (SCRaTCH trial, NL5193/NTR5342). Briefly, women aged 18-44 years with failed implantation after one full IVF/ICSI cycle and planning a subsequent IVF/ICSI cycle, were eligible. Participants were followed-up until 12 months after randomisation, with the primary outcome being live birth, defined as the delivery of at least one live foetus after 24 weeks of gestation. Participants/materials, setting, methods Endometrial tissue was obtained with an endometrial biopsy catheter in the midluteal phase of a natural cycle preceding subsequent IVF/ICSI, snap-frozen and stored at -80 °C until use. Total DNA was isolated from these biopsies, followed by 16S rRNA sequencing (V3-V4 region) to determine the endometrial microbiota composition. Positive (mock communities) and negative controls (DNA extraction and PCRs) were included. QIIME2 and DADA2 were used for the data analysis, followed by statistical analysis in R studio. Main results and the role of chance During the 12-month follow-up, 61/141 women (43.3%) reached a live birth. While endometrial microbiota profiles of all 141 women were analysed, only samples with ≥100 reads were included in the analysis, resulting in a total of 46 samples (32.6%) that were included in the analysis, which consisted of samples from 25 women who did not have and 21 women who did have a live birth within 12 months. The median number of reads per sample was not significantly different between the two groups (respectively 2,317 (IQR 651-19,031) and 1,335 (IQR 296-3,180), p = 0.29 by Mann-Whitney test). The endometrial microbiota detected, were bacterial genera frequently reported within the vaginal microbiota (e.g. Lactobacillus, Atopobium and Gardnerella). A clear dominance of Lactobacillus (relative abundance 55-100%, n = 22) or an unclassified bacterium genus (relative abundance 52-76%, n = 18) was observed in the majority of the samples; however, this dominance was not associated with the outcome of live birth. In addition, the samples dominated by Lactobacillus genera were mostly dominated by one species of Lactobacillus each (L. crispatus, L. iners, L. gasseri or L. jensenii). Limitations, reasons for caution The low biomass and the low ratio of bacterial versus human DNA in endometrial tissue were limiting factors in endometrial microbiota analysis. Furthermore, tissue was obtained transcervically, and contamination with vaginal/cervical microbiota could therefore have occurred. In the SCRaTCH trial no vaginal swabs were taken to serve as internal controls. Wider implications of the findings Future endometrial microbiota studies should consider the use of samples with a lower proportion of human DNA to maximize bacterial DNA yield. Furthermore, for endometrial microbiota research, sampling devices avoiding cervicovaginal contamination are desirable and may be developed in the future. Trial registration number SCRaTCH trial, NL5193/NTR5342

Study question Does the endometrial synthesis and inactivation of 17β- estradiol differ between receptive and non-receptive endometrium in women undergoing IVF/ICSI? Summary answer The synthesis and inactivation of 17β-estradiol is similar in the endometrium of women who did and did not achieve a clinical pregnancy through IVF/ICSI. What is known already Implantation failure of high-quality embryos is a main concern in IVF/ICSI treatment. Blood sex-steroid concentrations do not reflect their corresponding concentrations in endometrial tissue. This is in line with the concept that blood steroids (and precursors) are locally converted to bioactive metabolites and vice versa, by expressing steroid-metabolising enzymes, such as 17β-hydroxy steroid dehydrogenase (17β-HSD). Studies indicate that alterations in intracrinology might modulate endometrial receptivity. We hypothesize that the local 17β-HSD activity during the window of implantation (WOI) differs between pregnant and non-pregnant IVF/ICSI patients. Study design, size, duration Case-control study of 40 patients that were recruited in the SCRaTCH study (NL5193/NTR5342), a randomised trial exploring whether ‘endometrial scratching’ in patients with a previous IVF/ICSI cycle failure affects pregnancy outcome in a subsequent IVF/ICSI cycle. For the present investigation, 20 endometrial biopsies from women who achieved clinical pregnancy after fresh embryo transfer (ET) were compared with 20 endometrial biopsies of women that did not conceive after fresh ET. Participants/materials, setting, methods Endometrial biopsies and serum were obtained at LH + 5-8 days (urinary test) in a natural cycle, prior to the fresh ET cycle. Cases (negative pregnancy test, n = 20) and controls (clinically pregnant, n = 20) were matched for primary vs. secondary infertility, embryo quality and age. Reduction of estrone to 17β-estradiol (synthesizing 17β-HSDs) and oxidation of 17β-estradiol to estrone (inactivating 17β-HSDs) were determined by high-performance liquid chromatography (HPLC). Results were compared with the Wilcoxon-Mann-Whitney Rank Sum Test. Main results and the role of chance Activity of 17β-HSDs responsible for the reduction of estrone to 17β-estradiol (mainly 17β-HSD type 1) was detected in all samples and ranged from 55 to 1864 pmol 17β-estradiol formed/mg protein/24 h. The values obtained from pregnant women (median: 1054) were not significantly different to those obtained from non-pregnant women (median: 997), p = 0.97. The activity of enzymes responsible for the oxidation of 17β-­ estradiol (mainly 17β-HSD type 2) into the less active estrone ranged from 32 to 1731 estrone formed/mg protein/24 h. The values obtained from pregnant women (median: 737) were not significantly different to those obtained from non-pregnant women (median: 624), p = 0.90. The ratio of 17β-HSD type 1:17β-HSD type 2 had a median of 1.63 in the pregnant woman compared to 1.95 in the group of non-pregnant woman (p = 0.57). Limitations, reasons for caution The study is pilot in nature and study population is small. Primary and secondary infertility patients were analysed together. A chance phenomenon could have occurred as included women were included after their first IVF/ICSI cycle, hence not every included study person met the criteria for repeated implantation failure (RIF). Wider implications of the findings 17β-estradiol metabolism takes place during the WOI, controlling the final 17β-estradiol level. Although the present investigation did not show differences between pregnant and non-pregnant women, it remains important to explore if estrogen balance deviations, e.g. in other cycle phases plays a role in clinical conditions such as primary infertility/RIF. Trial registration number NL5193/NTR5342

Study question Is the incidence of ectopic pregnancy (EP) increased in fresh compared with frozen embryo transfer (ET)? Summary answer The fresh ET is not associated with an increase of the incidence of EP in comparison with frozen ET. What is known already EP represents the first cause of mortality in the first trimester of pregnancy. Assisted reproductive technologies (ART) are associated with an increased EP risk. The reasons of this effect are inadequately explained and may be associated with variables patients-related as tubal diseases and ART-related as the number of embryos transferred, the depth of insertion of the catheter tip during ET and the supraphysiological estradiol levels during ovarian stimulation affecting endometrial receptivity and tubal function. The role of this last factor seems to be corroborated by higher incidence of EP in fresh versus frozen ET, as highlighted by some retrospective studies. Study design, size, duration A meta-analysis, based on PubMed, Cochrane CENTRAL, EMBASE, was conducted to estimate and compare the EP rate in fresh versus frozen-thawed ET. Following PICOS, inclusion criteria were: Population, patients undergoing IVF/ICSI; Intervention, fresh ET; Comparison, frozen/thawed ET; Outcome, EP (primary one), clinical/ongoing pregnancy and live birth rates (secondary ones). Study design: RCT. Participants/materials, setting, methods Electronic and manual search, conducted from 1990 to 2020, yielded 269 studies. Two researchers reviewed the studies independently, excluding 247 and 16 studies after the first and second screening. The outcome data from the 6 included studies were combined using a Mantel–Haenszel model and applying the random effects models. The dichotomous data results of each study were expressed as risk ratio (RR) with 95% confidence intervals (CI). Heterogeneity was evaluated using the I2 statistic. Main results and the role of chance The six studies included in the present review comprise in total 6,675 participants, 3,320 undergoing frozen ET and 3,355 fresh ET (Ferraretti et al., 1999; Chen et al., 2016; Le et al., 2018; Shi et al., 2018; Wei et al., 2019; Stormlund et al., 2020). Preliminary analyses excluded interaction between covariates, defining intervention/control groups and outcome. In particular, the incidence of tubal infertility was comparable between the two groups. Risk ratio of EP ranged from 0.03 (Ferraretti et al., 1999) to 2.77 (Shi et al., 2018). The level of heterogeneity (I2) between studies was 42% and it was considered as moderate. EP incidence resulted not significantly different in fresh ET [56/1,703 pregnancies] compared with frozen ET [44/1,799 pregnancies] (RR = 0.450, 95% CI 0.13–1.81, p = 0.29; I2=42%). Analysis of the secondary outcomes was conducted on 5 studies; results showed that clinical pregnancy rate was not significantly different between fresh (0.52) and frozen ET (0.56) (RR = 1.11; 95% CI = 0.93–1.34); at the same manner, the ongoing pregnancy rate (RR = 1.11; 95%CI=0.91–1.37; 0.46 vs 0.49 for fresh and frozen, respectively) and the live birth rate (RR = 0.93; 95%CI=0.60–1.44; 0.47 vs 0.49 for fresh and frozen, respectively) resulted not significantly different between the two groups. Limitations, reasons for caution The EP incidence was not the primary outcome of the included RCTs. This could have determined a sample size not calibrated for the analysis of the primary outcome of the present meta-analysis. Wider implications of the findings: This meta-analysis indicates that EP incidence is similar between fresh and frozen ET. The possible role on EP risk of the supraphysiological estradiol levels during ovarian stimulation should be reconsidered. Based on these results, the choice of a frozen ET should not derive by the presumed reduction of EP risk. Trial registration number Not applicable

Study question Is the outcome of IVF/ICSI cycles done with continuous (single step ) embryo culture system different from that with sequential embryo culture system ? Summary answer Yes the outcome of IVF / ICSI cycles done with continuous (single step ) embryo-culture system is better than that with sequential embryo-culture system . What is known already Embryo culture media are important factors in IVF, which can significantly influence the clinical outcome of IVF/ICSI cycles. However it is not clear which formulation is most optimal and whether sequential or continuous media (single step) should be favored. Sequential media complies with embryo demands based on developmental stage , taking into account metabolic changes embryos undergo in-vivo, while moving from the oviduct to the uterus. The embryos in the early cleavage stage prefer to use pyruvate to produce energy, whereas once development nears the blastocyst stage , the embryos start using glucose in the process of glycolysis . Study design, size, duration A prospective RCT was carried out at our centre between 2018–2019 and IVF-ICSI patients meeting inclusion criteria (at least six normal MII - Oocytes) were included in this study. The aim of study was to compare blastocyst formation rates after embryo-culture in two different culture media systems. 436 metaphase II Oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media or single-step media. Participants/materials, setting, methods In this prospective trial with sibling oocytes, 436 metaphase II oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media ( n = 218 MII oocytes) or a single medium ( n = 218 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Main results and the role of chance Blastocyst formation rates on day 5 were significantly higher following culture in single step media 60.55% (132 / 218 ) as compared to sequential media 34.86% ( 76 / 218) . The percentage of good quality blastocysts was also significantly higher in single step media. In conclusion, culture in single step media was associated with higher blastocyst formation rates compared to sequential media , suggesting that the single medium may provide better support to the developing embryo. The proportion of poor quality embryos was significantly higher in the sequential media group. Results indicate that embryo culture in continuous media could be as efficient as embryo culture in sequential media. A significant difference observed was the proportion of poor quality embryos on day 5 , which was significantly higher when the embryos were cultured in sequential media. Our results suggest that the type of embryo culture media can influence the quality of embryos both at the cleavage stage and blastocyst stage. The use of continuous embryo culture media does not seem to cause an adverse effect; in fact, their use can lower the workload in busy IVF labs and lower the stress that embryos are exposed to during handling. Limitations, reasons for caution Although single-step-medium for extended culture has practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the embryo-culture to days 5/6. Further studies comparing these two media systems in well-designed trials should be performed. Wider implications of the findings: When employing sequential media for embryo culture , it is necessary to transfer the embryos from one medium to another ( cleavage stage medium to blastocyst stage medium) which increases stress related embryo damage . Therefore, single-step media is beneficial as the embryos can develop undisturbed till blastocyst stage. Trial registration number Not applicable

Study question Are the ovarian reserve parameters and the outcome of ovarian stimulation for IVF / ICSI influenced by ethnicity? Summary answer Ethnicity influences ovarian reserve parameters and the outcome of ovarian stimulation for IVF / ICSI What is known already Infertility affects couples worldwide and due to a lack of a standardized reporting system, the real number, especially in developing countries, might be underestimated. The etiology of infertility may differ around the world and is often subjected not only to social, cultural and religious peculiarities, but also to different genetic influences. Published data suggest that ethnicity influences the ovarian reserve as well as the outcome of Assisted-Reproductive-Techniques (ART)-treatments. Key players of a successful ART outcome are the ovarian reserve and consequently the number of oocytes retrieved. Until today, the impact of ethnical differences is not sufficiently addressed in research. Study design, size, duration Prospective observational study, performed in 10 infertility centers worldwide (Europe (4 centers), Middle East North Africa (MENA) region (2 centers), Iran (2 centers), South America (1 center), India (1 center)) between May 2019 and September 2020, evaluating ovarian reserve and outcome parameters of ovarian stimulation treatments for IVF/ICSI. The study was approved by the Ethical Committee of each participating center. Participants/materials, setting, methods Couples with primary / secondary infertility and an indication for an IVF/ICSI treatment were included into this study. Besides anamnestic data regarding the history of the infertility and self-reported ethnicity (Arab, Caucasian, Hispanic, Ohters, Persian and South Asian), data obtained during the basic fertility assessment on the ovarian reserve parameters (Antral follicle count (AFC) and Anti-Muellerian-Hormone (AMH)) as well as stimulation parameters from the ovarian stimulation treatment were collected and analyzed. Main results and the role of chance This study comprised 1032 couples with the following distribution of the ethnicities: Arab 21.5%, Caucasian 15.9%, Hispanic 5%, Others 1.2%, Persian 33.4%, and South Asian 23%. The unadjusted means, SD and 95%CI (Confidence Interval) of AMH (ng/ml) for the groups were 3.33±0.19 [2.95–3.71]; 2.03±0.25 [1.55–2.52]; 2.43±0.74 [0.97–3.88]; 2.76±0.96 [0.88–4.64]; 3.10±0.16 [2.79–3.41]; 3.62±0.19 [3.25–3.98], for AFC 15.52±0.53 [14.49–16.55]; 12.00±0.67 [10.69–13.31]; 12.69±1.08 [10.57–14.81]; 15.11±2.60 [10.01–20.21]; 13.58±0.42 [12.75–14.41]; 13.49±0.51 [12.49–14.48] and for the number of retrieved oocytes (rCOC) 14.08±0.61 [12.88–15.27]; 9.84±0.71 [8.44–11.24]; 7.94±1.26 [5.48–10.41]; 9.92±2.62 [4.78–15.05]; 10.83±0.49 [9.87–11.79]; 17.06±0.59 [15.90–18.21], respectively. Univariate analysis of AMH, AFC and rCOC with the ethnicities revealed highly statistically significant differences for AMH and rCOC (p As age is a major confounder for the ovarian reserve, multivariate analyses were performed. After adjusting for age, AMH was significantly different between Arab-Persian, Arab-South Asian and Arab-Caucasian (p Limitations, reasons for caution Limitations of the study are an unequal number of included patients per ethnicity and that the data for the ovarian reserve parameters and the stimulation outcome were not available for all of the included patients. Wider implications of the findings: Counselling of couples with infertility have to take, besides all other factors, also the ethnicity of the couple into account as ethnicity influences the ovarian reserve parameters as well as the number of retrieved oocytes in ovarian stimulation cycles for IVF/ICSI. Trial registration number ClinicalTrials.gov Identifier: NCT03927417

Study question To investigate differences in In Vitro Fertilization (IVF)/Intracytoplasmic Sperm Injection (ICSI) outcomes between endometriosis women who do or don’t have a concomitant autoimmune disease. Summary answer Despite a higher oocyte yield, a trend for reduction in clinical pregnancy rates was observed in the autoimmunity group compared to women without concomitant autoimmunity. What is known already Endometriosis is an inflammatory chronic gynaecological disorder with a known detrimental impact on fertility. Endometriosis pathogenesis is still unclear. It has been postulated a role of both innate and adaptive immune system. The coexistence of endometriosis and autoimmunity is a well-documented occurrence Some recent findings have revealed an increased risk to have concomitant autoimmune disease in women with endometriosis, but no study has so far investigated whether this association could affect IVF/ICSI outcomes. Indeed, autoimmune phenomena, including proinflammatory cytokines and auto-antibody production, may result in diminished quality of oocytes/embryos with lower pregnancy rates among these patients. Study design, size, duration This was a retrospective observational study carried out at the Fertility Unit of IRCSS San Raffaele Hospital (Milan). We reviewed medical patients’ notes of women with a confirmed diagnosis of endometriosis who referred to our Fertility Unit from October 2018 to January 2021. Participants/materials, setting, methods Out of 1441 patients undergoing IVF/ICSI, 98 women had surgical/histopathological diagnosis of endometriosis. 25 of them had a clinical and/or serological diagnosis of autoimmunity. Autoimmunity was assessed by clinical data (blood tests for auto-antibodies or rheumatological records) obtained from the electronic patient files stored in the database of our Fertility Centre. Clinical pregnancy was defined as the presence of at least one intrauterine gestational sac with a viable embryo at week 6 after transfer. Main results and the role of chance 25/98 (25.5%) endometriosis women with a concomitant autoimmune disease (cases) were compared with 73/98 (74.5%) endometriosis patients without autoimmunity (controls). The mean age was 37.36±3.63 and 36.93±3.79 (p=.623) in cases and controls respectively. The mean number of oocytes retrieved was higher in cases (5.78±4.07) than in controls (3.82±2.69;p=.041); similarly, cases showed an higher number of embryos (2.13±1.93 vs. 1.19±1.37;p=.041) and blastocysts (1.89±2.02 vs. 0.85±1.61;p=.041) obtained. A total of 47 fresh embryo transfer (ET) were performed. Considering all the endometriosis patients, the clinical pregnancy rate (CPR) per cycle was 34.0% (16/47); when stratifying for the presence of autoimmunity the CPR was 23.1% (3/13) in cases, and 38.2% (13/34) in controls (p=.494). Limitations, reasons for caution This is a retrospective study based on data extraction from electronic records of our Fertility Centre. The sample size is limited and some information about past medical history could be missed. Results should be interpreted with caution until validated by future research providing more standardized data collection. Wider implications of the findings: Despite significantly higher numbers of oocytes retrieved and embryos/blastocysts formed, the presence of concomitant autoimmune disease in patients with endometriosis may impair pregnancy rates. Whether this finding is confirmed and whether it could be due to a defect in embryo/blastocysts quality or in endometrial receptivity deserves further studies. Trial registration number Not applicable

Study question Does presence of endometrioma has worse IVF/ICSI outcome than endometriosis per se? What about the impact of cystectomy of endometrioma on IVF/ICSI outcomes? Summary answer IVF/ICSI outcome of patients with endometrioma is comparable than with endometriosis. Cystectomy for endometrioma did not alter IVF/ICSI outcomes if ovarian reserve is comparable. What is known already Previous studies revealed women with endometrioma undergoing IVF/ICSI had similar reproductive outcomes compared with those without. Most of the comparisons are between women with endometrioma and women without endometriosis. However, endometrioma per se, different from endometriosis may have specific impact on IVF/ICSI outcomes. There is now molecular, histological and morphological evidence to suggest endometrioma is detrimental to the ovaries. Studies comparing IVF/ICSI outcomes between women with endometrioma and women with endometriosis are few. Cystectomy of endometrioma may worse ovarian reserve, and subsequently adversely affect IVF/ICSI outcomes. But there are possible complications associated with the persistence of endometrioma during IVF/ICSI. Study design, size, duration Retrospective analysis of 2153 IVF/ICSI cases during Jan/01/2014 to Dec/31/2018 in VGHTC. We included women who received ART due to endometriosis(n = 208). Exclusion criteria including patients >40 years-old, simulation day < 5 days, severe male factor, uterine factor (including adenomyosis) and immunological factors. Patients whose embryos were not completely transferred back or who received embryo transfer from different OPU cycles are excluded. We followed up these patients till 2020/6. The primary outcome is cumulative LBR Participants/materials, setting, methods For first analysis, we divided 208 cases to patients with endometrioma during IVF/ICSI(n = 89), and patients only diagnosed of endometriosis (n = 119). Second analysis on the effect of cystectomy of endometrioma on IVF/ICSI outcomes. Patients with endometrioma (n = 89) during IVF/ICSI were further divided to patients with primary endometrioma (n = 70) and patients with recurrent endometrioma (n = 19, ever received cystectomy for endometrioma). Another group is patients without endometrioma during IVF/ICSI, but ever received cystectomy before (n = 40) Main results and the role of chance For the first analysis, age, BMI and AMH were comparable in endometrioma (n = 89) and endometriosis group(n = 119). The usage gonadotropin dose was significantly higher in the endometrioma group (FSH 3619IU vs 3471IU, p = 0.001. LH 1224 IU vs 941 IU, p = 0.009). The Blastocyst formation rate is lower in the endometrioma group (49.4% vs. 57.7% p = 0.005). The OPU number, LBR and cumulative LBR were comparable in both groups (10.3 vs 12.4 p = 0.131, 33.3% vs 37%, p = 0.687, 49.4% vs 60.5%, endometrioma vs endometriosis). For the second analysis, when comparing cystectomy before IVF/ICSI group with primary endometrioma group, cystectomy group were younger (32.8 vs 34.8 p = 0.006). AMH level were comparable. The BC formation rate was significantly higher in the cystectomy group (61.5% vs 50.4% 0= 0.007). The LBR and cumulative LBR were comparable in both groups (43.5% vs 28.1%, 60% vs 48% in cystectomy vs primary endometrioma group). As for the recurrent endometrioma group, the age and AMH level were comparable with cystectomy group, but the usage gonadotropin dose was significantly higher than other two groups. The BC formation rate was also lower than cystectomy group (47.8% vs 61.5% p = 0.042). The LBR and cumulative LBR were comparable with other two groups (55.6%, 57.9%). Limitations, reasons for caution This is a retrospective study, and the sample size is limit. We did not analysis the size of endometrioma nor the unilateral or bilateral endometrioma. Wider implications of the findings Cystectomy for endometrioma must be carefully selected since it did not alter IVF/ICSI outcome only if the ovarian reserve is not affected. Recurrent endometriomas do not have a worse impact on IVF/ICSI outcome than primary endometrioma. If there is recurrent endometrioma, IVF/ICSI may be the first priority. Trial registration number not applicable

Study question Is there any association between stage of embryo at transfer based on extended in vitro culture and gestational trophoblastic disease risk during assisted reproduction? Summary answer No significant association between stages of embryo transfer from zygote stage to blastocyst stage was found after analysing 540376 cycles of IVF and ICSI. What is known already Gestational trophoblastic disease (GTD), commonly referred to as molar pregnancy, results from abnormal proliferation of the trophoblast with a reported incidence of ∼1 in 700 in the UK. Despite technological advances such as ICSI, PGT and selection of normally fertilised (2PN) embryos, there are reported cases of GTD following assisted reproduction. Blastocyst transfer is associated with higher pregnancy and live birth rates but evidence is lacking whether extended embryo culture to blastocyst stage influences implantation of an abnormal embryo or abnormal trophoblastic proliferation leading to GTD. Study design, size, duration A retrospective study was carried out using Human Fertilisation and Embryology Authority (HFEA) anonymised register data from 1999 to 2016. HFEA holds the longest running register for fertility treatment data in the world and is the national database for fertility treatment data in UK. A total of 540376 fresh IVF or ICSI assisted reproduction cycles were analysed. Participants/materials, setting, methods There were 1033588 treatment cycles during the study period but only 540376 cycles met the inclusion criteria of fresh IVF or ICSI. Cycles with incomplete data, frozen embryo transfers, donor treatment or surrogacy were excluded. A subgroup analysis of those with primary subfertility was performed after excluding subjects with secondary infertility in order to exclude an effect of a previous molar pregnancy. Multivariate regression analysis was used to adjust for possible confounders. Main results and the role of chance 78 molar pregnancies were reported in the original sample giving a prevalence of 4/10000 live births (78/228461), much lower than the prevalence given with natural pregnancies. Prevalence of molar pregnancy amongst the study population after meeting exclusion criteria was 4 /10000 livebirths (53/156683). Incidence of molar pregnancy was not statistically different between treatment type (0.0001 vs 0.00009). Significantly higher incidence of GTD was seen in the 40 to 42 age category compared to 18–34 category(OR 1.86(95% CI 8.7–3.75)),in par with known higher GTD risk in women above 40 in the general population. Of interesting note, although the incidence of molar pregnancy was significantly lower in women undergoing assisted reproduction increased risk with advancing age is not totally eliminated with treatment. There was no significant association between the occurrence of molar pregnancy with the type and cause for infertility and number of embryos transferred. Crude (1.06 (95% CI 0.852–1.31)) and adjusted odds ratios (1.07 (95% CI (0.857–1.32)) did not show any association between day of embryo transfer and molar pregnancy even after adjusting for age and excluding secondary infertility. Selection of blastocyst stage embryo after extended culture did not alter the likelihood of having a GTD compared to cleavage stage embryo. Limitations, reasons for caution The retrospective analysis of anonymised HFEA data limited adjustments for confounders such as smoking, previous history of GTD, ethnicity etc that predispose to GTD. Caution needs to be exercised for under-reporting of GTD to HFEA and lack of information on type of GTD identified. Wider implications of the findings: Though GTD cannot be prevented by IVF/ICSI, the incidence is significantly low and extended culture is not associated with higher risk of abnormal trophoblastic proliferation or GTD occurrence with IVF/ ICSI treatment. These findings would aid informed implications counselling and reassurance of patients during assisted reproduction treatments. Trial registration number Not applicable

Study question To compare the effect of sperm preparation methods on the DFI of semen sample for patients undergoing ICSI. Summary answer On comparing the results, microfluidic sperm sorting yielded sperms with significantly less DFI as compared to density gradient method of sperm preparation. What is known already The DNA integrity of the sperm plays an important role to ensure formation of good quality embryos with increased potential of fertilization, growth and ultimately implantation.. Centrifugation has shown to add stress to the sperm and leading to DNA damage, therefore there is a need to develop techniques of sperm preparation which help in retrieving as many sperms with intact DNA from the unprocessed sample as possible. Microfludic is fluid dynamic based technique of sperm preparation. in this study, we evaluated if microfluidic sperm sorter can recover motile sperm with better DNA integrity compared to density gradient preparation method. Study design, size, duration Prospective randomized study conducted in 80 patients undergoing IVF-ICSI with normal semen parameters (based WHO criteria 2010). DFI was done using Sperm Chromatin Dispersion (SCD) test in split semen samples prepared by microfluidic sperm sorter and density gradient method. Sperm morphology and motility were also recorded and evaluated based on the WHO 2010 criteria. Participants/materials, setting, methods Semen parameters of the sample were assessed by microscopic examination. DFI of each unprocessed sample was carried out using SCD test, following that the sample was split and sperm preparation was done using microfluidic sperm sorter and density gradient. the recovered sperm were tested for DFI and the results were compared. Main results and the role of chance Mean DFI in unprocessed semen samples was 23%. the analysis of split semen samples post preparation showed that the DFI was significantly reduced with the use of microfluidic sperm sorter (mean DFI 0.6%) as compared to density gradient (mean DFI 9%). Limitations, reasons for caution A major limitation of the microfluidic sperm sorter is the use sperm concentration and motility of the semen sample. In oligospermic and asthenospermic samples, density gradient is the preferred method of preparation. Lack of data showing improvement in clinical outcomes with reduced DFI is also a major limitation. Wider implications of the findings: Microfluidics has shown to significantly reduce the DFI of the semen sample, it requires no extra equipment and cost and is relatively easy to pick up. Density gradient method of sperm preparation continues to be the preferred method due to its versatility and recovery of good quality sperm. Trial registration number Not applicable

STUDY QUESTION Are perinatal outcomes different between singleton live births conceived from fresh blastocyst transfer and those following the transfer of fresh cleavage-stage embryos? SUMMARY ANSWER Fresh blastocyst transfer does not increase risks of preterm birth (PTB), low/high birth weight or congenital anomaly and does not alter the sex ratio at birth or prejudice the chance of having a healthy baby. WHAT IS KNOWN ALREADY Extended embryo culture is currently considered the best option for embryo selection, but concerns have been raised about increased risks of preterm delivery and large-for-gestational-age (LGA) babies. STUDY DESIGN, SIZE, DURATION We conducted a retrospective cohort study based on data from the Human Fertilisation and Embryology Authority (HFEA) anonymised and cycle-based dataset in the UK between 1999 and 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS Baseline characteristics were compared between in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) blastocyst-stage and cleavage-stage embryo transfer cycles using the χ2 test for categorical/dichotomised covariates and the Mann–Whitney test for continuous covariates. Statistical significance was set at MAIN RESULTS AND THE ROLE OF CHANCE Of a total of 67 147 IVF/ICSI cycles, 11 152 involved blastocyst-stage embryo(s) and 55 995 involved cleavage-stage embryo(s). The two groups were comparable with regards to the risk of PTB (aRR, 1.00; 99.5% CI, 0.79–1.25), very-preterm birth (VPTB) (aRR, 1.00; 99.5% CI, 0.63–1.54), very-low birth weight (VLBW) (aRR, 0.84; 99.5% CI, 0.53–1.34), low birth weight (LBW) (aRR, 0.92; 99.5% CI, 0.73–1.16), high birth weight (HBW) (aRR, 0.94; 99.5% CI, 0.75–1.18) and very-high birth weight (VHBW) (aRR, 1.05; 99.5% CI, 0.66–1.65). The risk of congenital anomaly was 16% higher in the blastocyst-stage group than in the cleavage-stage group, but this was not statistically significant (aRR, 1.16; 99.5% CI, 0.90–1.49). The chance of having a healthy baby (born at term, with a normal birth weight and no congenital anomalies) was not altered by extended culture (aRR, 1.00; 99.5% CI, 0.93–1.07). Extended culture was associated with a marginal increase in the chance having a male baby in the main cycle-based analysis (aRR, 1.04; 99.5% CI, 1.01–1.09) but not in the sub-group analysis of women undergoing their first cycle of treatment (aRR, 1.04; 95% CI, 1.00–1.08). In the sub-group analysis, the risk of congenital anomalies was significantly higher after blastocyst-stage embryo transfer (aRR, 1.42; 95% CI, 1.12–1.81). LIMITATIONS, REASONS FOR CAUTION This study is limited by the use of observational data and inability to adjust for key confounders, such as maternal smoking status and body mass index (BMI), which were not recorded in the HFEA dataset. As the main analysis was cycle-based and we were unable to link cycles within women undergoing more than one IVF/ICSI cycle, we undertook a sub-group analysis on women undergoing their first treatment cycle. WIDER IMPLICATIONS OF THE FINDINGS Our findings should reassure women undergoing blastocyst-stage embryo transfer. For the first time, we have shown that babies born after blastocyst transfer have a similar chance of being healthy as those born after cleavage-stage embryos transfer. STUDY FUNDING/COMPETING INTEREST(S) The research activity of Dr Nicola Marconi was funded by the scholarship ‘A. Griffini-J. Miglierina’, Fondazione Comunitaria del Varesotto, Provincia di Varese, Italy. The authors do not have any competing interests to disclose. TRIAL REGISTRATION NUMBER N/A

All agree that in hindsight the rapid adoption of preimplantation genetic screening (PGS) using cleavage stage biopsy and fluorescence in situ hybridization (FISH) in routine clinical practice without proper evaluation of (cost-)effectiveness basically resulted in couples paying more money for a less effective treatment. Now, almost 20 years later, we are on the verge of a new era of PGS. But have things really changed or are we simply going back to the future?

Contains fulltext : 53223.pdf (Publisher’s version ) (Closed access) BACKGROUND: The aim of this study was to gain more insight into long-term psychological adjustment to IVF in women. METHODS: In a prospective cohort study, 298 women entering their first IVF treatment cycle (including ICSI) completed standardized psychological questionnaires before the start of the treatment, just after the last treatment cycle, and 6 months and 3-5 years after the last treatment cycle. The main outcome measures were state anxiety, depression and mode of adaptation to unsuccessful IVF. RESULTS: Anxiety and depression were found at follow-up to return to baseline levels following treatment not resulting in a live birth, after an initial increase during treatment. On the contrary, treatment resulting in a live birth was found to lead to a more positive long-term emotional status. Women who focused on new life goals as a mode of adaptation 3-5 years after IVF without a live birth showed lower levels of anxiety and depression compared with those who persisted in their attempts to get pregnant. CONCLUSIONS: Treatment that resulted in a live birth led to more positive emotional adjustment. In addition, most women who did not give birth to a child after treatment adjusted well psychologically. Positive adjustment was related to developing new life goals rather than persisting in attempts to get pregnant. Helping women to change life goals after abandoning treatment might have beneficial effects on the adaptation process.

Udgivelsesdato: May 12 BACKGROUND: In a Danish population-based cohort study assessing the risk of cerebral palsy in children bornafter IVF, we made some interesting observations regarding ‘vanishing co-embryos’. METHODS andRESULTS: All live-born children born in Denmark from 1 January 1995 to 31 December 2000 were included inthis analysis. The children conceived by IVF/ICSI (9444) were identified through the IVF Register, the childrenconceived without IVF/ICSI (395 025) were identified through The Danish Medical Birth Register. Main outcomemeasure was the incidence of cerebral palsy. Within the IVF/ICSI children we found indications of an increasedrisk of cerebral palsy in those children resulting from pregnancies, where the number of embryos transferred washigher than the number of children born. CONCLUSIONS: The association between vanishing embryo syndromeand incidence of cerebral palsy following IVF requires further investigation in larger, adequately powered, studies.