On the reproductive capabilities of aneuploid human preimplantation embryos.

In IVF cycles, the application of aneuploidy testing at the blastocyst stage is quickly growing, and the latest reports estimate almost half of cycles in the US undergo preimplantation genetic testing for aneuploidies (PGT-A). Following PGT-A cycles, understanding the predictive value of an aneuploidy result is paramount for making informed decisions about the embryo's fate and utilization. Compelling evidence from non-selection trials strongly supports that embryos diagnosed with a uniform whole-chromosome aneuploidy very rarely result in the live birth of a healthy baby, while their transfer exposes women to significant risks of miscarriage and chromosomally abnormal pregnancy. On the other hand, embryos displaying low range mosaicism for whole chromosomes have shown reproductive capabilities somewhat equivalent to uniformly euploid embryos, and they have comparable clinical outcomes and gestational risks. Therefore, given their clearly distinct biological origin and clinical consequences, careful differentiation between uniform and mosaic aneuploidy is critical in both the clinical setting when counseling individuals and in the research setting when presenting aneuploidy studies in human embryology. Here, we focus on the evidence gathered so far on PGT-A diagnostic predictive values and reproductive outcomes observed across the broad spectrum of whole-chromosome aneuploidies detected at the blastocyst stage to obtain evidence-based conclusions on the clinical management of aneuploid embryos in the quickly growing PGT-A clinical setting.

Assisted reproductive technology use and outcomes in childhood cancer survivors.

BACKGROUND: Treatment exposures for childhood cancer reduce ovarian reserve. However, the success of assisted reproductive technology (ART) among female survivors is not well established. METHODS: Five-year survivors of childhood cancer in the Childhood Cancer Survivor Study were linked to the Society for Assisted Reproductive Technology Clinic Outcome Reporting System, which captures national ART outcomes. The authors assessed the live birth rate, the relative risk (RR) with 95% confidence intervals (95% CIs), and associations with treatment exposure using generalized estimating equations to account for multiple ovarian stimulations per individual. Siblings from a random sample of survivors were recruited to serve as a comparison group. RESULTS: Among 9885 female survivors, 137 (1.4%; median age at diagnosis, 10 years [range, 0-20 years]; median years of follow-up after age 18 years, 11 years [range, 2-11 years]) underwent 224 ovarian stimulations using autologous or donor eggs and/or gestational carriers (157 autologous ovarian stimulation cycles, 67 donor ovarian stimulation cycles). In siblings, 33 (1.4%) underwent 51 autologous or donor ovarian stimulations. Of those who used embryos from autologous eggs without using gestational carriers, 97 survivors underwent 155 stimulations, resulting in 49 live births, for a 31.6% chance of live birth per ovarian stimulation (vs. 38.3% for siblings; p = .39) and a 43.9% chance of live birth per transfer (vs. 50.0%; p = .33). Prior treatment with cranial radiation therapy (RR, 0.44; 95% CI, 0.20-0.97) and pelvic radiation therapy (RR, 0.33; 95% CI, 0.15-0.73) resulted in a reduced chance of live birth compared with siblings. The likelihood of live birth after ART treatment in survivors was not affected by alkylator exposure (cyclophosphamide-equivalent dose, ≥8000 mg/m2 vs. none; RR, 1.04; 95% CI, 0.52-2.05). CONCLUSIONS: Childhood cancer survivors are as likely to undergo treatment using ART as sibling controls. The success of ART treatment was not reduced after alkylator exposure. The results from the current study provide needed guidance on the use of ART in this population.

SARS-CoV-2 infection is detrimental to pregnancy outcomes after embryo transfer in IVF/ICSI: a prospective cohort study.

BACKGROUND: To explore whether SARS-CoV-2 infection affects the pregnancy outcomes of assisted reproductive techniques (ART). METHODS: A prospective cohort study recruited patients for embryo transfer from December 01, 2022, to December 31, 2022. All patients were closely followed up for SARS-CoV-2 infection after embryo transfer. The SARS-CoV-2 "diagnosed group" was defined as RNA or antigen-positive. The SARS-CoV-2 "suspected infection group" was defined as having apparent SARS-CoV-2 symptoms without an RNA or antigen test, while the "uninfected group" was defined as having a negative SARS-CoV-2 RNA or antigen test and no SARS-CoV-2 symptoms. RESULTS: A total of 1330 patients participated in the study, 687 of whom were in the SARS-CoV-2 diagnosed group, 219 in the suspected infection group, and 424 in the uninfected group. There was no significant difference in basic characteristics among the three groups. The clinical pregnancy rate was 68% in the SARS-CoV-2 diagnosed group, 63% in the uninfected group, and 51% in the suspected infection group (P < 0.001). The ongoing pregnancy rate was 58% in the SARS-CoV-2 diagnosed group, 53% in the uninfected group, and 45% in the suspected infection group (P < 0.001). Upon analyzing the factors influencing clinical pregnancy, it was found that suspected infection (odds ratio [OR] 0.618, 95% CI 0.444-0.862, P = 0.005) and the short time (≤ 22 days) between embryo transfer and SARS-CoV-2 infection (OR 3.76, 95% CI 1.92-8.24, P < 0.001) were not conducive to clinical pregnancy. In addition, the concurrent presence of fever and dizziness/headache SARS-CoV-2 symptoms (OR 0.715, 95% CI 0.526-0.972, P = 0.032) decreased the clinical pregnancy rate. However, vaccination administered 2-3 times (OR 1.804, 95% CI 1.332-2.444, P < 0.001) was associated with an improvement in clinical pregnancy rate. CONCLUSIONS: This prospective cohort study shows that SARS-CoV-2 infection in a short period of time after embryo transfer is not conducive to clinical pregnancy. Reproductive physicians should advise patients to avoid SARS-CoV-2 infection shortly after embryo transfer. Meanwhile, women should be encouraged to vaccinate at least 2-3 times before embryo transfer or pregnancy.

Pretreatment prediction for IVF outcomes: generalized applicable model or centre-specific model?

STUDY QUESTION: What was the performance of different pretreatment prediction models for IVF, which were developed based on UK/US population (McLernon 2016 model, Luke model, Dhillon model, and McLernon 2022 model), in wider populations? SUMMARY ANSWER: For a patient in China, the published pretreatment prediction models based on the UK/US population provide similar discriminatory power with reasonable AUCs and underestimated predictions. WHAT IS KNOWN ALREADY: Several pretreatment prediction models for IVF allow patients and clinicians to estimate the cumulative probability of live birth in a cycle before the treatment, but they are mostly based on the population of Europe or the USA, and their performance and applicability in the countries and regions beyond these regions are largely unknown. STUDY DESIGN, SIZE, DURATION: A total of 26 382 Chinese patients underwent oocyte pick-up cycles between January 2013 and December 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: UK/US model performance was externally validated according to the coefficients and intercepts they provided. Centre-specific models were established with XGboost, Lasso, and generalized linear model algorithms. Discriminatory power and calibration of the models were compared as the forms of the AUC of the Receiver Operator Characteristic and calibration curves. MAIN RESULTS AND THE ROLE OF CHANCE: The AUCs for McLernon 2016 model, Luke model, Dhillon model, and McLernon 2022 model were 0.69 (95% CI 0.68-0.69), 0.67 (95% CI 0.67-0.68), 0.69 (95% CI 0.68-0.69), and 0.67 (95% CI 0.67-0.68), respectively. The centre-specific yielded an AUC of 0.71 (95% CI 0.71-0.72) with key predictors including age, duration of infertility, and endocrine parameters. All external models suggested underestimation. Among the external models, the rescaled McLernon 2022 model demonstrated the best calibration (Slope 1.12, intercept 0.06). LIMITATIONS, REASONS FOR CAUTION: The study is limited by its single-centre design and may not be representative elsewhere. Only per-complete cycle validation was carried out to provide a similar framework to compare different models in the sample population. Newer predictors, such as AMH, were not used. WIDER IMPLICATIONS OF THE FINDINGS: Existing pretreatment prediction models for IVF may be used to provide useful discriminatory power in populations different from those on which they were developed. However, models based on newer more relevant datasets may provide better calibrations. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China [grant number 22176159], the Xiamen Medical Advantage Subspecialty Construction Project [grant number 2018296], and the Special Fund for Clinical and Scientific Research of Chinese Medical Association [grant number 18010360765]. TRIAL REGISTRATION NUMBER: N/A.

Unexplained infertility and age-related infertility: indistinguishable diagnostic entities but different IVF prognosis.

STUDY QUESTION: Is IVF indicated for couples with age-related infertility? SUMMARY ANSWER: IVF may be of doubtful utility for age-related infertility. WHAT IS KNOWN ALREADY: A diagnosis of unexplained infertility is drawn when the diagnostic work-up fails to identify any patent cause. Although typically managed uniformly, unexplained infertility is likely to comprise a wide range of conditions, including age-related infertility (at least in older women). Unfortunately, no validated tests for the identification of age-related infertility exist and these women are typically treated as unexplained cases. However, homologous ART may be less effective for these women because these techniques may be unable to treat the detrimental effects of ageing on oocyte competence. STUDY DESIGN, SIZE, DURATION: Women aged 18-42 years who underwent IVF procedures between January 2014 and December 2021 were selected retrospectively. In the first part of the study, we aimed to assess whether the proportion of women with unexplained infertility (i.e. without patent causes of infertility) increased with age. In the second part of the study, women with unexplained infertility were matched 1:1 by age, study period, and duration of infertility, to those with a patent cause of infertility. If our hypothesis is valid, the first part of the study should highlight an increase in the proportion of unexplained infertility with age. Moreover, in the second part of the study, one should observe a sharper decrease in the rate of IVF success of the procedure with age in women with an unremarkable work-up compared to those with a definite cause of infertility. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women were included if: they had been trying to conceive for more than 2 years, they had retrieved more than three oocytes, and had not undergone previous IVF attempts. We exclude couples with severe male factor (criptozoospermia), chronic anovulation, untreated hydrosalpinx, or intracavitary diseases. The first part of the study aimed at investigating the relative proportion of unexplained infertility with age. The outcome of the second part was the distribution of the live births between unexplained versus explained infertility, in women younger or older than 35 years. Only the results of the first IVF cycle were considered (including both fresh and frozen cycles). The live birth rate corresponded to the cumulative chance of a live birth per oocyte retrieval. MAIN RESULTS AND THE ROLE OF CHANCE: One thousand five hundred and thirty-five women were selected for the first part of the study; 742 of them had unexplained infertility (48%). The frequency of this diagnosis was lower among women aged <35 years (40%) compared to those ≥35 years (52%) (P < 0.001). A clear gradient emerged when considering smaller intervals of age (P < 0.001). A total of 1134 women (567 unexplained cases and 567 explained cases) were selected for the second part of the study. Baseline variables were comparable between women with unexplained and explained infertility. Among women younger than 35 years (n = 229 unexplained cases and 229 explained cases), 108 live births were observed in women with unexplained infertility (47%) and 88 in those with explained infertility (38%). In comparison, among women older than 35 years, the live births occurred in 90 (27%) and 114 (34%) couples, respectively (P = 0.03). The adjusted odds ratio (OR) for a live birth in older women with unexplained infertility was 0.63 (95% CI: 0.43-0.94). In other words, the effectiveness of IVF in older women with unexplained infertility is reduced by an additional 37% when compared to women of similar age with a patent cause of infertility. Moreover, when considering smaller intervals of age, a gradient of the adverse effect of age on the distribution of live births between unexplained and explained infertility emerged (P = 0.003). Overall, these results support the hypothesis that IVF may be of modest benefit in women with age-related infertility. The decline in IVF success is sharper in women with unexplained infertility compared to those with explained infertility, indirectly suggesting that IVF cannot effectively treat age-related infertility. LIMITATIONS, REASONS FOR CAUTION: We postulated that the greater decline in IVF success with age in the unexplained group could be related to the concomitant increase in the proportion of women with age-related infertility. However, even if this is theoretically logical, the unavailability of validated tools to diagnose age-related infertility makes our inference speculative. We cannot exclude that the prevalence of other unknown causes of infertility that cannot also be effectively overcome with IVF could increase with age. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that IVF may be of modest utility for treating age-related infertility. Offering this procedure to older women with an unremarkable infertility work-up may be questioned. However, the diagnosis of age-related infertility remains challenging and identifying a biomarker that could reliably diagnose age-related infertility is a priority. STUDY FUNDING/COMPETING INTEREST(S): The study was partially funded by the Italian Ministry of Health-current research IRCCS and by a specific grant supported by Ferring. ES declares receiving honoraria for lectures at meetings from IBSA and Gedeon-Richter and he also handles private grants of research from Ferring, IBSA, Theramex, and Gedeon-Richter. All the other authors do not have any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Protocol change improves live birth and recurrent cycle cancellation rates after a previous IVF cycle cancellation: an analysis of 13 000 autologous cycles reported to SART CORS.

STUDY QUESTION: After an IVF cycle cancellation, does changing the stimulation protocol affect the odds of live birth and recurrent cancellation in the subsequent cycle? SUMMARY ANSWER: After IVF cycle cancellation, compared to those who repeated the same stimulation protocol, those who changed their protocol had higher odds of live birth and lower odds of recurrent cycle cancellation. WHAT IS KNOWN ALREADY: There is limited data addressing the effect of changing the stimulation protocol after an IVF cycle is cancelled during initial stimulation. The odds of live birth outcomes are not known so far in studies addressing the effect of changing the protocol. STUDY DESIGN, SIZE, DURATION: Retrospective Cohort Study using the 2014-2017 Society for Assisted Reproductive Technology Clinic Outcome Reporting System (SART CORS) database. PARTICIPANTS/MATERIALS, SETTING, METHODS: The data included 13 135 patients with a first autologous IVF cycle that resulted in a cycle cancellation and was followed by a second autologous cycle within the study period. We excluded fertility preservation cycles, supernumerary cycle attempts after the second IVF cycle attempt, and cycles with more than one stimulation protocol documented per cycle start. Patients who received the same protocol for both cycles (n = 6434) were compared to those who changed their protocol in the second cycle (n = 6701). Multivariable logistic regression analyses were performed to estimate the adjusted odds of live birth and recurrent cancellation. MAIN RESULTS AND THE ROLE OF CHANCE: Changing the protocol in the second cycle resulted 14% lower odds of recurrent cycle cancellation (P = 0.01) and 17% higher odds of live birth after fresh transfers (P = 0.04). When stratifying the data by specific combinations of protocol change (agonist flare, agonist suppression, antagonist), there was an increase in live birth when switching from antagonist to agonist suppression (odds ratio (OR) = 1.36, P = 0.03) and from agonist suppression to antagonist (OR = 1.73, P = 0.01) compared to those who repeated their same stimulation protocol. Specifically in poor responders, outcomes were worse when using the agonist flare protocol and significantly improved with the agonist suppression protocol. LIMITATIONS, REASONS FOR CAUTION: Comparison of response to stimulation between first and second cycles cannot be made in this study because the index IVF cycle was cancelled during ovarian stimulation, and thus there is no reportable outcome data for that cycle. Additionally, SART only tracks the three stimulation protocols addressed in this study and does not have data on more contemporary protocols that are used in poor responders thus limiting the generalizability of our findings. WIDER IMPLICATIONS OF THE FINDINGS: Using the SART CORS database, which includes >90% of all reported IVF cycles in the USA, provides generalizability to the demographically diverse IVF populations found here. In agreement with prior studies assessing change in IVF protocols, the agonist flare protocol seems to result in worse IVF outcomes, and based on our results, we believe that there is no role for the agonist flare protocol in patients with a prior poor response to stimulation. STUDY FUNDING/COMPETING INTEREST(S): None declared. TRIAL REGISTRATION NUMBER: N/A.

What do women undergoing in vitro fertilization (IVF) understand about their chance of IVF success?

STUDY QUESTION: How well informed are Australian women who undergo IVF about their chances of having a baby? SUMMARY ANSWER: Only one in four women estimated their individual chance of success with IVF accurately, with most women overestimating their chance. WHAT IS KNOWN ALREADY: Limited knowledge about infertility and infertility treatment in the general population is well-documented. The few studies that have investigated patients' knowledge about the chance of IVF success suggest that while IVF patients are aware of average success rates, they tend to be unrealistic about their own chance of success. STUDY DESIGN, SIZE, DURATION: We conducted an anonymous online survey of 217 women who had started IVF since 2018 in Australia. The survey was advertised on social media, enabling women from across Australia to participate. Responses were collected in June 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: The survey included questions on demographic characteristics and IVF history. It asked what participants thought their chance of having a baby from one IVF treatment cycle was, how they rated their knowledge about chance of success, and about their experience of receiving IVF-related information. Participants' estimations of their chance of success were compared with their chance as calculated by the Society for Assisted Reproductive Technology's (SART) online calculator. Responses to a free-text question about what information women wished they had been given when they started treatment were analysed thematically. MAIN RESULTS AND THE ROLE OF CHANCE: Only about a quarter (58/217, 27%) of participants accurately estimated their chance of having a baby within 20% relative to their SART calculated chance, with more than half (118/217, 54%) overestimating their chance. Ninety percent of women indicated that their preferred source of treatment information was a consultation with their doctor, despite less than half (44%) reporting that doctors explained the probability of having a baby with IVF well (mean 5.9/10). In free-text responses, many women also reported that they wished they had been given more realistic information about IVF and their chance of success. LIMITATIONS, REASONS FOR CAUTION: The dissemination method precludes calculation of response rate, and it is not possible to know if participants are representative of all women undergoing IVF. Additionally, we only surveyed women undergoing IVF, while those who decided not to have IVF were not included. Therefore, women who overestimated their chance may have been overrepresented. There is also inherent imprecision in the way understanding of chance of success was estimated. The potential impact of recall bias could neither be quantified nor excluded. It is difficult to determine to what extent women's lack of understanding of what is possible with IVF is due to poor information-provision by clinicians and the clinic, and how much can be explained by optimism bias. WIDER IMPLICATIONS OF THE FINDINGS: The finding of poor understanding of personal chance of success amongst women undergoing IVF in Australia requires further investigation to determine potential reasons for this. The findings can be used by clinics to develop strategies for improvement in the information-provision process to ensure that women can make informed decisions about their fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This study received no external funding. S.L. is supported by a NHMRC Investigator Grant (APP1195189). R.W. is supported by a NHMRC Investigator Grant (APP2009767). B.W.M. is supported by a NHMRC Investigator Grant (GNT1176437). B.W.M. reports consultancy for Merck and ObsEva and has received research funding and travel funding from Merck. The other authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Oral dydrogesterone versus micronized vaginal progesterone for luteal phase support: a double-blind crossover study investigating pharmacokinetics and impact on the endometrium.

STUDY QUESTION: How do plasma progesterone (P) and dydrogesterone (D) concentrations together with endometrial histology, transcriptomic signatures, and immune cell composition differ when oral dydrogesterone (O-DYD) or micronized vaginal progesterone (MVP) is used for luteal phase support (LPS)? SUMMARY ANSWER: Although after O-DYD intake, even at steady-state, plasma D and 20αdihydrodydrogesterone (DHD) concentrations spiked in comparison to P concentrations, a similar endometrial signature was observed by histological and transcriptomic analysis of the endometrium. WHAT IS KNOWN ALREADY: O-DYD for LPS has been proven to be noninferior compared to MVP in two phase III randomized controlled trials. Additionally, a combined individual participant data and aggregate data meta-analysis indicated that a higher pregnancy rate and live birth rate may be obtained in women receiving O-DYD versus MVP for LPS in fresh IVF/ICSI cycles. Little data are available on the pharmacokinetic (PK) profiles of O-DYD versus MVP and their potential molecular differences at the level of the reproductive organs, particularly at the endometrial level. STUDY DESIGN, SIZE, DURATION: Thirty oocyte donors were planned to undergo two ovarian stimulation (OS) cycles with dual triggering (1.000 IU hCG + 0.2 mg triptorelin), each followed by 1 week of LPS: O-DYD or MVP, in a randomized, cross-over, double-blind, double-dummy fashion. On both the first and eighth days of LPS, serial blood samples upon first dosing were harvested for plasma D, DHD, and P concentration analyses. On Day 8 of LPS, an endometrial biopsy was collected for histologic examination, transcriptomics, and immune cell analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: All oocyte donors were <35 years old, had regular menstrual cycles, no intrauterine contraceptive device, anti-Müllerian hormone within normal range and a BMI ≤29 kg/m2. OS was performed on a GnRH antagonist protocol followed by dual triggering (1.000 IU hCG + 0.2 mg triptorelin) as soon as ≥3 follicles of 20 mm were present. Following oocyte retrieval, subjects initiated LPS consisting of MVP 200 mg or O-DYD 10 mg, both three times daily. D, DHD, and P plasma levels were measured using liquid chromatography-tandem mass spectrometry. Histological assessment was carried out using the Noyes criteria. Endometrial RNA-sequencing was performed for individual biopsies and differential gene expression was analyzed. Endometrial single-cell suspensions were created followed by flow cytometry for immune cell typing. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 21 women completed the entire study protocol. Subjects and stimulation characteristics were found to be similar between groups. Following the first dose of O-DYD, the average observed maximal plasma concentrations (Cmax) for D and DHD were 2.9 and 77 ng/ml, respectively. The Cmax for D and DHD was reached after 1.5 and 1.6 h (=Tmax), respectively. On the eighth 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, respectively. For both, the observed Tmax was 1.5 h. Following the first dose of MVP, the Cmax for P was 16 ng/ml with a Tmax of 4.2 h. On the eighth day of LPS, the first administration of that day showed a Cmax for P of 21 ng/ml with a Tmax of 7.3 h. All 42 biopsies showed endometrium in the secretory phase. The mean cycle day was 23.9 (±1.2) in the O-DYD group versus 24.0 (±1.3) in the MVP group. RNA-sequencing did not reveal significantly differentially expressed genes between samples of both study groups. The average Euclidean distance between samples following O-DYD was significantly lower than following MVP (respectively 12.1 versus 18.8, Mann-Whitney P = 6.98e-14). Immune cell profiling showed a decrease of CD3 T-cell, γδ T-cell, and B-cell frequencies after MVP treatment compared to O-DYD, while the frequency of natural killer (NK) cells was significantly increased. LIMITATIONS, REASONS FOR CAUTION: The main reason for caution is the small sample size, given the basic research nature of the project. The plasma concentrations are best estimates as this was not a formal PK study. Whole tissue bulk RNA-sequencing has been performed not correcting for bias caused by different tissue compositions across biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study comparing O-DYD/MVP, head-to-head, in a randomized design on a molecular level in IVF/ICSI. Plasma serum concentrations suggest that administration frequency is important, in addition to dose, specifically for O-DYD showing a rapid clearance. The molecular endometrial data are overall comparable and thus support the previously reported noninferior reproductive outcomes for O-DYD as compared to MVP. Further research is needed to explore the smaller intersample distance following O-DYD and the subtle changes detected in endometrial immune cells. STUDY FUNDING/COMPETING INTEREST(S): Not related to this work, C.Bl. has received honoraria for lectures, presentations, manuscript writing, educational events, or scientific advice from Abbott, Ferring, Organon, Cooper Surgical, Gedeon-Richter, IBSA, and Merck. H.T. has received honoraria for lectures, presentations, manuscript writing, educational events, or scientific advice from Abbott, Ferring, Cooper Surgical, Gedeon-Richter, Cook, and Goodlife. S.M. has received honoraria for lectures, presentations, educational events, or scientific advice from Abbott, Cooper Surgical, Gedeon-Richter, IBSA, and Merck and Oxolife. G.G. has received honoraria for lectures, presentations, educational events, or scientific advice from Merck, MSD, Organon, Ferring, Theramex, Gedeon-Richter, Abbott, Biosilu, ReprodWissen, Obseva, PregLem, Guerbet, Cooper, Igyxos, and OxoLife. S.V.-S. is listed as inventor on two patents (WO2019115755A1 and WO2022073973A1), which are not related to this work. TRIAL REGISTRATION NUMBER: EUDRACT 2018-000105-23.

Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

Intra-patient analysis of individual weight gain or loss between IVF cycles: cycle now and transfer later.

STUDY QUESTION: What is the impact of clinically significant weight change on outcomes related to IVF cycle performance? SUMMARY ANSWER: While individual weight loss did not significantly impact ovarian response to stimulation or other cycle outcome parameters in our study, some positive associations were found for individual weight gain. WHAT IS KNOWN ALREADY: The role of weight-change in patients undergoing IVF has been largely studied by comparing weight loss in different cohorts of patients stratified by a static BMI. Specifically, obesity has been extensively studied in relation to its negative effects on assisted or unassisted conception outcomes and ovulatory function. Previous research has shown conflicting results, while BMI, which is commonly used as a marker of obesity, may not accurately reflect the underlying factors affecting fertility in obese patients. STUDY DESIGN, SIZE, DURATION: This study utilized a retrospective within-patient repeated measurement analysis design to assess the impact of weight change on IVF outcomes in cycles where all embryos were cryopreserved at the blastocyst stage for transfer at a later date. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at an academically affiliated fertility center. The data included 961 women who underwent at least two IVF cycles between December 2014 and June 2020, with documented short-term weight gain (n = 607) or weight loss (n = 354) within 1 year from their initial IVF cycle. Multivariable generalized estimating equations (GEE) and generalized linear mixed models (GLMM) were employed to assess associations between weight change and outcomes across cycles. MAIN RESULTS AND THE ROLE OF CHANCE: The multivariable models indicated that weight loss did not show any significant associations with the numbers of oocytes retrieved, or mature oocytes, the fertilization rate or the blastulation rate. However, weight gain demonstrated a minor positive association with the number of oocytes retrieved in both GEE models (coefficient: 0.01, 95% CI: 0.00-0.01) and GLMM models (0.01, 95% CI: 0.01-0.00). There was also a potential increase in the fertilization rate with weight gain, as indicated by a positive coefficient in both GEE models (coefficient: 0.01, 95% CI: 0.00-0.02) and GLMM models (coefficient: 0.01, 95% CI: 0.00-0.01). However, the association between weight gain and the embryo blastulation rate was not statistically significant in any model. LIMITATIONS, REASONS FOR CAUTION: This study focused on cycle performance parameters instead of reproductive outcomes, which restricted our ability to evaluate the impact of weight change on cumulative live birth rates. Additionally, the study did not account for variables such as stimulation protocols, potentially introducing confounding factors and limiting the generalizability of the results. WIDER IMPLICATIONS OF THE FINDINGS: Although obesity is associated with adverse obstetrical risks, there is less evidence of adverse reproductive outcomes in IVF cycles. We therefore recommend that an IVF cycle should not be delayed due to weight, so that the patient is not adversely affected by increasing age. The IVF cycle should aim to freeze all embryos, so that embryo transfer can then occur after weight loss, so as to limit the recognized obstetrical risks. STUDY FUNDING/COMPETING INTEREST(S): The study was not funded and there were no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Fertility clinics have a duty of care towards patients who do not have children with treatment.

In medically assisted reproduction (MAR) success has mostly been measured in terms of achieving (healthy) livebirths. We argue this focus is too narrow and that success should be measured in terms of alleviating patient suffering caused by an unfulfilled child wish. The major implication is that clinics must better tailored care to effectively support patients who do not have child(ren) with treatment. First, we argue that clinics have a duty of care towards patients for whom MAR does not result in children because this is a common treatment outcome, because treatment is burdensome and creates new losses for patients, and because the field has the necessary expertise to provide support and it is part of patient-centred care. Then, we examine concerns about the adequacy of addressing the possibility that treatment may end without children, namely, that this may hinder patients' hope and put them off doing treatment, and that it may be perceived as a sign of clinical incompetence, as well as concerns about the required skill set. We end with a set of research-informed recommendations to promote healthy adjustment to ending fertility treatment without children. These focus on the need to reconceptualize 'success' and 'failure' in MAR, to promote open discussion about the possibility of treatment not resulting in children and encourage patients to develop 'plan(s) B', to support patients who end treatment without children, and to create the organizational structures needed to support clinics and healthcare professionals in this endeavour.

A modified flexible GnRH antagonist protocol using antagonist early cessation and a gonadotropin step-down approach improves live birth rates in fresh cycles: a randomized controlled trial.

STUDY QUESTION: Can pregnancy outcomes following fresh elective single embryo transfer (eSET) in gonadotropin-releasing hormone (GnRH) antagonist protocols increase using a gonadotropin (Gn) step-down approach with cessation of GnRH antagonist on the day of hCG administration (hCG day) in patients with normal ovarian response? SUMMARY ANSWER: The modified GnRH antagonist protocol using the Gn step-down approach and cessation of GnRH antagonist on the hCG day is effective in improving live birth rates (LBRs) per fresh eSET cycle. WHAT IS KNOWN ALREADY: Currently, there is no consensus on optimal GnRH antagonist regimens. Studies have shown that fresh GnRH antagonist cycles result in poorer pregnancy outcomes than the long GnRH agonist (GnRHa) protocol. Endometrial receptivity is a key factor that contributes to this phenomenon. STUDY DESIGN, SIZE, DURATION: An open label randomized controlled trial (RCT) was performed between November 2021 and August 2022. There were 546 patients allocated to either the modified GnRH antagonist or the conventional antagonist protocol at a 1:1 ratio. PARTICIPANTS/MATERIALS, SETTING, METHODS: Both IVF and ICSI cycles were included, and the sperm samples used were either fresh or frozen from the partner, or from frozen donor ejaculates. The primary outcome was the LBRs per fresh SET cycle. Secondary outcomes included rates of implantation, clinical and ongoing pregnancy, miscarriage, and ovarian hyperstimulation syndrome (OHSS), as well as clinical outcomes of ovarian stimulation. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline demographic features were not significantly different between the two ovarian stimulation groups. However, in the intention-to-treat (ITT) population, the LBRs in the modified antagonist group were significantly higher than in the conventional group (38.1% [104/273] vs. 27.5% [75/273], relative risk 1.39 [95% CI, 1.09-1.77], P = 0.008). Using a per-protocol (PP) analysis which included all the patients who received an embryo transfer, the LBRs in the modified antagonist group were also significantly higher than in the conventional group (48.6% [103/212] vs. 36.8% [74/201], relative risk 1.32 [95% CI, 1.05-1.66], P = 0.016). The modified antagonist group achieved significantly higher implantation rates, and clinical and ongoing pregnancy rates than the conventional group in both the ITT and PP analyses (P < 0.05). The two groups did not show significant differences between the number of oocytes retrieved or mature oocytes, two-pronuclear zygote (2PN) rates, the number of embryos obtained, blastocyst progression and good-quality embryo rates, early miscarriage rates, or OHSS incidence rates (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: A limitation of our study was that the subjects were not blinded to the treatment allocation in the RCT trial. Only women under 40 years of age who had a good prognosis were included in the analysis. Therefore, use of the modified antagonist protocol in older patients with a low ovarian reserve remains to be investigated. In addition, the sample size for Day 5 elective SET was small, so larger trials will be required to strengthen these findings. WIDER IMPLICATIONS OF THE FINDINGS: The modified GnRH antagonist protocol using the Gn step-down approach and cessation of GnRH antagonist on hCG day improved the LBRs per fresh eSET cycle in normal responders. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by grant 2022YFC2702503 from the National Key Research & Development Program of China and grant 2021140 from the Beijing Health Promotion Association. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: The RCT was registered in the Chinese Clinical Trial Registry; Study Number: ChiCTR2100053453. TRIAL REGISTRATION DATE: 21 November 2021. DATE OF FIRST PATIENT'S ENROLLMENT: 23 November 2021.

Developmental outcomes of children born through ICSI versus conventional IVF (cIVF) in couples with non-male factor infertility.

STUDY QUESTION: In non-male factor infertile couples, are there any differences in the developmental outcomes between children born through ICSI and conventional IVF (cIVF)? SUMMARY ANSWER: In this preliminary study, ICSI and cIVF seem to have a comparable effect on developmental outcomes after 12 months in children born to non-male factor infertile couples. WHAT IS KNOWN ALREADY: ICSI, an invasive technique, has raised concerns about potential developmental abnormalities in children. Limited data are available regarding the developmental outcomes of ICSI-conceived infants born to non-male factor infertile couples. STUDY DESIGN, SIZE, DURATION: This prospective cohort study involved a follow-up of all children aged 12 months or older who were born from pregnancies resulting from either ICSI or cIVF as part of a previous randomized controlled trial (RCT) (NCT03428919). PARTICIPANTS/MATERIALS, SETTING, METHODS: In the original RCT, 1064 women were randomly assigned to the ICSI or cIVF groups (532 women for each group). Follow-up was conducted with 155 couples (195 children) in the ICSI group and 141 couples (185 children) in the cIVF group. The Vietnamese version of the Ages & Stages Third Edition Questionnaires (ASQ-3) and the Development Red Flags questionnaires were completed by the participants. A total of 141 (90.1%) women (177 children) in the ICSI group and 113 (80.1%) women (145 children) in the cIVF group returned fully completed questionnaires. The primary outcomes were the developmental outcomes based on responses to the ASQ-3 and the Red Flags questionnaire. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age of children at follow-up was 19.5 ± 5.0 months in the ICSI group and 19.3 ± 5.5 months in the cIVF group. The mean height and weight of children in both groups were similar. The overall proportion of children with any abnormal ASQ-3 score did not differ significantly between the ICSI and cIVF groups (16.9% vs 13.1%, P = 0.34). The proportion of children with Red Flag signs was also comparable between the two groups (6.2% vs 9.2%, P = 0.36, ICSI vs cIVF, respectively). LIMITATIONS, REASONS FOR CAUTION: Despite a reasonably high follow-up response rate, there is a potential risk of sampling bias, and overall, the number of children with developmental abnormalities was very small. The study relied solely on questionnaires as screening tools, rather than incorporating additional behavioral observations or physical developmental tests; this may have affected the statistical power and the significance of between-group comparisons. WIDER IMPLICATIONS OF THE FINDINGS: The current findings contribute to the existing evidence and support the comparative safety of ICSI and cIVF regarding early childhood development. However, more extensive and prolonged follow-up data for these children are needed to draw definitive conclusions. STUDY FUNDING/COMPETING INTEREST(S): No external funding was received for this study, and no authors reported conflicting interests. TRIAL REGISTRATION NUMBER: NCT04866524 (clinicaltrials.gov).

Survey on ART and IUI: legislation, regulation, funding, and registries in European countries-an update.

STUDY QUESTION: How are ART and IUI regulated, funded, and registered in European countries, and how has the situation changed since 2018? SUMMARY ANSWER: Of the 43 countries performing ART and IUI in Europe, and participating in the survey, specific legislation exists in only 39 countries, public funding varies across and sometimes within countries (and is lacking or minimal in four countries), and national registries are in place in 33 countries; only a small number of changes were identified, most of them in the direction of improving accessibility, through increased public financial support and/or opening access to additional subgroups. WHAT IS KNOWN ALREADY: The annual reports of the European IVF-Monitoring Consortium (EIM) clearly show the existence of different approaches across Europe regarding accessibility to and efficacy of ART and IUI treatments. In a previous survey, some coherent information was gathered about how those techniques were regulated, funded, and registered in European countries, showing that diversity is the paradigm in this medical field. STUDY DESIGN, SIZE, DURATION: A survey was designed using the SurveyMonkey tool consisting of 90 questions covering several domains (legal, funding, and registry) and considering specific details on the situation of third-party donations. New questions widened the scope of the previous survey. Answers refer to the situation of countries on 31 December 2022. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: All members of the EIM were invited to participate. The received answers were checked and initial responders were asked to address unclear answers and to provide any additional information considered relevant. Tables resulting from the consolidated data were then sent to members of the Committee of National Representatives of ESHRE, requesting a second check. Conflicting information was clarified by direct contact. MAIN RESULTS AND THE ROLE OF CHANCE: Information was received from 43 out of the 45 European countries where ART and IUI are performed. There were 39 countries with specific legislation on ART, and artificial insemination was considered an ART technique in 33 of them. Accessibility is limited to infertile couples only in 8 of the 43 countries. In 5 countries, ART and IUI are permitted also for treatments of single women and all same sex couples, while a total of 33 offer treatment to single women and 19 offer treatment to female couples. Use of donated sperm is allowed in all except 2 countries, oocyte donation is allowed in 38, simultaneous donation of sperm and oocyte is allowed in 32, and embryo donation is allowed in 29 countries. Preimplantation genetic testing (PGT)-M/SR (for monogenetic disorders, structural rearrangements) is not allowed in 3 countries and PGT-A (for aneuploidy) is not allowed in 10; surrogacy is accepted in 15 countries. Except for marital/sexual situation, female age is the most frequently reported limiting criterion for legal access to ART: minimal age is usually set at 18 years and the maximum ranges from 42 to 54 with some countries not using numeric definition. Male maximum age is set in very few countries. Where third-party donors are permitted, age is frequently a limiting criterion (male maximum age ranging from 35 to 50; female maximum age from 30 to 37). Other legal restrictions in third-party donation are the number of children born from the same donor (or, in some countries, the number of families with children from the same donor) and, in 12 countries, there is a maximum number of oocyte donations. How countries deal with the anonymity is diverse: strict anonymity, anonymity just for the recipients (not for children when reaching legal adulthood age), a mixed system (anonymous and non-anonymous donations), and strict non-anonymity. Inquiring about donors' genetic screening showed that most countries have enforced either mandatory or scientific recommendations that exclude the most prevalent genetic diseases, although, again, diversity is evident. Reimbursement/compensation systems exist in more than 30 European countries, with around 10 describing clearly defined maximum amounts considered acceptable. Public funding systems are extremely variable. One country provides no financial assistance to ART/IUI patients and three offer only minimal support. Limits to the provision of funding are defined in the others i.e. age (female maximum age is the most used), existence of previous children, BMI, maximum number of treatments publicly supported, and techniques not entitled for funding. In a few countries reimbursement is linked to a clinical policy. The definitions of the type of expenses covered within an IVF/ICSI cycle, up to which limit, and the proportion of out-of-pocket costs for patients are also extremely dissimilar. National registries of ART are in place in 33 out of the 43 countries contributing to the survey and a registry of donors exists in 19 of them. When comparing with the results of the previous survey, the main changes are: (i) an extension of the beneficiaries of ART techniques (and IUI), evident in nine countries; (ii) public financial support exists now in Albania and Armenia; (iii) in Luxembourg, the only ART centre expanded its on-site activities; (iv) donor-conceived children are entitled to know the donor identity in six countries more than in 2018; and (v) four more countries have set a maximum number of oocyte donations. LIMITATIONS, REASONS FOR CAUTION: Although the responses were provided by well-informed and committed individuals and submitted to double checking, no formal validation by official bodies was in place. Therefore, possible inaccuracies cannot be excluded. The results presented are a cross-section in time, and ART and IUI frameworks within European countries undergo continuous modification. Finally, some domains of ART activity were deliberately left out of the scope of this survey. WIDER IMPLICATIONS OF THE FINDINGS: Our results offer a detailed updated view of the ART and IUI situation in European countries. It provides extensive answers to many relevant questions related to ART usage at the national level and could be used by institutions and policymakers at both national and European levels. STUDY FUNDING/COMPETING INTEREST(S): The study has no external funding, and all costs were covered by ESHRE. There were no competing interests.

Applying a simplified economic evaluation approach to evaluate infertility treatments in clinical practice.

IVF is the backbone of infertility treatment, but due to its costs, it is not affordable for everyone. The cost of IVF is further escalated by interventions added to the routine treatment, which are claimed to boost pregnancy rates, so-called add-ons. Consequently, it is critical to offset the increased costs of an intervention against a potentially higher benefit. Here, we propose using a simplified framework considering the cost of a standard IVF procedure to create one live-born baby as a benchmark for the cost-effectiveness of other fertility treatments, add-ons inclusive. This framework is a simplified approach to a formal economic evaluation, enabling a rapid assessment of cost effectiveness in clinical settings. For a 30-year-old woman, assuming a 44.6% cumulative live birth rate and a cost of $12 000 per complete cycle, the cost to create one live-born baby would be ∼$27 000 (i.e. willingness to pay). Under this concept, the decision whether to accept or reject a new treatment depends from an economic perspective on the incremental cost per additional live birth from the new treatment/add-on, with the $27 000 per live-born baby as a reference threshold. This threshold can vary with women's age, and other factors such as the economic perspective and risk of side effects can play a role. If a new add-on or treatment costs >$27 000 per live birth, it might be more rational to invest in a new IVF cycle rather than spending on the add-on. With the increasing number of novel technologies in IVF and the lack of a rapid approach to evaluate their cost-effectiveness, this simplified framework will help with a more objective assessment of the cost-effectiveness of infertility treatments, including add-ons.

Circadian serum progesterone variations on the day of frozen embryo transfer in a modified natural cycle protocol.

STUDY QUESTION: Is there a circadian variation of serum progesterone (P) on the day of frozen embryo transfer (FET) in a modified natural cycle (mNC)? SUMMARY ANSWER: There is a statistically significant diurnal variation of serum P on the day of a FET in an mNC protocol. WHAT IS KNOWN ALREADY: In recent years, the proportion of FET cycles has increased dramatically. To further optimize pregnancy outcomes after FET, recent studies have focused on serum luteal P levels in both natural and artificially prepared FET cycles. Despite the different cut-off values proposed to define low serum P in the NC, it is generally accepted that lower serum P values (<10 ng/ml) around the day of FET are associated with negative reproductive outcomes. However, a single serum P measurement is not reliable given that P levels are prone to diurnal fluctuations and are impacted by patients' characteristics. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was conducted in a single university-affiliated fertility center, including 22 patients performing a single blastocyst mNC-FET from August 2022 to August 2023. Serum P levels were measured on the day of transfer at 08:00h, 12:00h, 16:00h, and 20:00h. Differences between P levels were compared using the Wilcoxon signed-rank test. The sample size was calculated to detect a difference of 15% between the first and last P measurements with a 5% false-positive rate and a 95% CI. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a normal BMI, between 18 and 40 years old, without uterine diseases were eligible. Patients utilizing donated oocytes were excluded. The mNC-FET protocol involved monitoring the normal ovarian cycle and triggering ovulation with an injection of 250 µg of choriogonadotropin alfa when a pre-ovulatory follicle (16-20 mm diameter) was visualized. The blastocyst was transferred seven days later. The patients were not supplemented with exogenous P at any time before the day of the FET. MAIN RESULTS AND THE ROLE OF CHANCE: The mean age and BMI of the study population were 33.6 ± 3.8 years and 22.7 ± 1.8 kg/m2, respectively. Mean P values at 08:00h, 12:00h, 16:00h, and 20:00h were 14.6 ± 4.5, 14.7 ± 4.1, 12.9 ± 3.5, and 14.6 ± 4.3 ng/ml, respectively. The mean P levels at 16:00h were significantly lower compared to all other time points (P < 0.05: P = 0.007 between P at 8:00h and 16:00h; P = 0.003 between P at 12:00h and 16:00h; P = 0.007 between P at 16:00h and 20:00h). No statistically significant difference was observed between P values at the other time points (P > 0.05: P = 0.88 between P at 8:00h and 12:00h; P = 0.96 between P at 8:00h and 20:00h; P = 0.83 between P at 12:00h and 20:00h). LIMITATIONS, REASONS FOR CAUTION: The study's limitations include the small sample size that may cause a bias when the results are extrapolated to a larger subfertile population undergoing mNC-FET. Ideally, larger prospective trials including a more heterogeneous patient population would be necessary to validate our findings. WIDER IMPLICATIONS OF THE FINDINGS: The current study demonstrates the existence of a diurnal fluctuation of serum P on the day of mNC-FET highlighting the importance of a standardized time point for its measurement. This is especially important for considering clinical actions, such as additional exogenous P supplementation, when encountering P values lower than 10 ng/ml on the day of FET. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained for the study. The authors have no conflicts of interest to declare regarding the content of the study. TRIAL REGISTRATION NUMBER: NCT05511272.

Disposables used cumulatively in routine IVF procedures could display toxicity.

STUDY QUESTION: Is there a cumulative toxicity of disposables used in IVF procedures? SUMMARY ANSWER: A toxicity may be detected when consumables are used cumulatively, while no toxicity is detected when the same consumables are used and tested individually. WHAT IS KNOWN ALREADY: Many components of items used in IVF laboratories may impair human embryonic development. Consequently, it is necessary to screen all reagents and materials which could be in contact with gametes and embryos. Toxicity tests, such as the mouse embryo assay and the human sperm motility assay (HSMA), are used by manufacturers as quality control tools to demonstrate the safety of their products. This evaluation is currently individually performed for each single consumable. However, during an IVF cycle, several devices are used sequentially, potentially creating a cumulative exposure to chemical contaminants, which could not be detected for individually tested consumables. STUDY DESIGN, SIZE, DURATION: The objective of this observational study conducted from March 2021 to October 2022 was to evaluate with the HSMA methodology if there was a cumulative toxicity when several disposables are sequentially used. Fourteen categories of consumables currently used in routine IVF procedures were studied, which included devices used for sperm and oocyte collection (cups, condoms, and oocyte aspiration needles), manipulation (flasks, tubes, tips, pipettes, embryo transfer catheters, syringes, and gloves), culture (dishes), and storage (straws). PARTICIPANTS/MATERIALS, SETTING, METHODS: After obtaining patient consent, the surplus semen assessed as having normal parameters according to the World Health Organization 2010 criteria were used to perform the HSMAs. First, each consumable was tested individually. Then, associations of three, four, and five consumables, previously validated as non-toxic when tested individually, were analyzed. HSMAs were conducted three times to ensure reproducibility, with a defined toxicity threshold of a sperm motility index (SMI) below 0.85 in at least two of three tests. MAIN RESULTS AND THE ROLE OF CHANCE: Thirty-six references of disposables were first individually tested across 53 lots. Forty-nine (92%) demonstrated compliance. However, four (8%) devices revealed toxicity: one lot of 1 ml syringes, two lots of sperm cups, and one lot of 25 cm2 flasks. These four references were excluded from the IVF routine procedures. A total of 48 combinations of consumables were assessed, involving 41 lots from 32 references that were previously individually tested. Among the evaluated combinations, 17 out of 48 (35%) associations exhibited toxicity with a SMI below 0.85 for two of the three tests (n = 8) or all the three tests (n = 9). Notably, three out of 17 (18%) of the three-consumable associations, five out of 16 (31%) of the four-consumable associations, and nine out of 15 (60%) of the five-consumable associations were found not compliant. The toxicity did not originate from a single consumable, because only consumables that were individually pre-validated as non-toxic were included in the combinations, but the toxicity had a cumulative origin. The risk of cumulative toxicity increased with the number of consumables included in the association (Cochran-Mantel-Haenszel statistic, P = 0.013). LIMITATIONS, REASONS FOR CAUTION: The high proportion of non-compliant combinations of disposables can be attributed directly to the extreme rigorous extraction conditions employed during the tests, which could deviate from the conditions encountered in routine clinical use. Also, the methodology employed in the HSMAs (e.g. toxicity extraction duration, sperm concentrations, and protein supplementation of the medium) can influence the sensitivity of the tests. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the significance of performing toxicity testing on devices before introducing them into clinical practice. Disposables should be tested individually to detect immediate toxicities and also in combination. Our results advocate rationalizing the number of consumables used in each IVF procedure and re-evaluating the use of glass consumables. STUDY FUNDING/COMPETING INTEREST(S): This study received fundings from GCS Ramsay Santé pour l'Enseignement et la Recherche (Paris, France) and the Centre de Biologie Médicale BIOGROUP (Le Chesnay-Rocquencourt, France). The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the reported research. TRIAL REGISTRATION NUMBER: N/A.

BlastAssist: a deep learning pipeline to measure interpretable features of human embryos.

STUDY QUESTION: Can the BlastAssist deep learning pipeline perform comparably to or outperform human experts and embryologists at measuring interpretable, clinically relevant features of human embryos in IVF? SUMMARY ANSWER: The BlastAssist pipeline can measure a comprehensive set of interpretable features of human embryos and either outperform or perform comparably to embryologists and human experts in measuring these features. WHAT IS KNOWN ALREADY: Some studies have applied deep learning and developed 'black-box' algorithms to predict embryo viability directly from microscope images and videos but these lack interpretability and generalizability. Other studies have developed deep learning networks to measure individual features of embryos but fail to conduct careful comparisons to embryologists' performance, which are fundamental to demonstrate the network's effectiveness. STUDY DESIGN, SIZE, DURATION: We applied the BlastAssist pipeline to 67 043 973 images (32 939 embryos) recorded in the IVF lab from 2012 to 2017 in Tel Aviv Sourasky Medical Center. We first compared the pipeline measurements of individual images/embryos to manual measurements by human experts for sets of features, including: (i) fertilization status (n = 207 embryos), (ii) cell symmetry (n = 109 embryos), (iii) degree of fragmentation (n = 6664 images), and (iv) developmental timing (n = 21 036 images). We then conducted detailed comparisons between pipeline outputs and annotations made by embryologists during routine treatments for features, including: (i) fertilization status (n = 18 922 embryos), (ii) pronuclei (PN) fade time (n = 13 781 embryos), (iii) degree of fragmentation on Day 2 (n = 11 582 embryos), and (iv) time of blastulation (n = 3266 embryos). In addition, we compared the pipeline outputs to the implantation results of 723 single embryo transfer (SET) cycles, and to the live birth results of 3421 embryos transferred in 1801 cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: In addition to EmbryoScope™ image data, manual embryo grading and annotations, and electronic health record (EHR) data on treatment outcomes were also included. We integrated the deep learning networks we developed for individual features to construct the BlastAssist pipeline. Pearson's χ2 test was used to evaluate the statistical independence of individual features and implantation success. Bayesian statistics was used to evaluate the association of the probability of an embryo resulting in live birth to BlastAssist inputs. MAIN RESULTS AND THE ROLE OF CHANCE: The BlastAssist pipeline integrates five deep learning networks and measures comprehensive, interpretable, and quantitative features in clinical IVF. The pipeline performs similarly or better than manual measurements. For fertilization status, the network performs with very good parameters of specificity and sensitivity (area under the receiver operating characteristics (AUROC) 0.84-0.94). For symmetry score, the pipeline performs comparably to the human expert at both 2-cell (r = 0.71 ± 0.06) and 4-cell stages (r = 0.77 ± 0.07). For degree of fragmentation, the pipeline (acc = 69.4%) slightly under-performs compared to human experts (acc = 73.8%). For developmental timing, the pipeline (acc = 90.0%) performs similarly to human experts (acc = 91.4%). There is also strong agreement between pipeline outputs and annotations made by embryologists during routine treatments. For fertilization status, the pipeline and embryologists strongly agree (acc = 79.6%), and there is strong correlation between the two measurements (r = 0.683). For degree of fragmentation, the pipeline and embryologists mostly agree (acc = 55.4%), and there is also strong correlation between the two measurements (r = 0.648). For both PN fade time (r = 0.787) and time of blastulation (r = 0.887), there's strong correlation between the pipeline and embryologists. For SET cycles, 2-cell time (P < 0.01) and 2-cell symmetry (P < 0.03) are significantly correlated with implantation success rate, while other features showed correlations with implantation success without statistical significance. In addition, 2-cell time (P < 5 × 10-11), PN fade time (P < 5 × 10-10), degree of fragmentation on Day 3 (P < 5 × 10-4), and 2-cell symmetry (P < 5 × 10-3) showed statistically significant correlation with the probability of the transferred embryo resulting in live birth. LIMITATIONS, REASONS FOR CAUTION: We have not tested the BlastAssist pipeline on data from other clinics or other time-lapse microscopy (TLM) systems. The association study we conducted with live birth results do not take into account confounding variables, which will be necessary to construct an embryo selection algorithm. Randomized controlled trials (RCT) will be necessary to determine whether the pipeline can improve success rates in clinical IVF. WIDER IMPLICATIONS OF THE FINDINGS: BlastAssist provides a comprehensive and holistic means of evaluating human embryos. Instead of using a black-box algorithm, BlastAssist outputs meaningful measurements of embryos that can be interpreted and corroborated by embryologists, which is crucial in clinical decision making. Furthermore, the unprecedentedly large dataset generated by BlastAssist measurements can be used as a powerful resource for further research in human embryology and IVF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Harvard Quantitative Biology Initiative, the NSF-Simons Center for Mathematical and Statistical Analysis of Biology at Harvard (award number 1764269), the National Institute of Heath (award number R01HD104969), the Perelson Fund, and the Sagol fund for embryos and stem cells as part of the Sagol Network. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Granulosa cell metabolism at ovulation correlates with oocyte competence and is disrupted by obesity and aging.

STUDY QUESTION: Is oocyte developmental competence associated with changes in granulosa cell (GC) metabolism? SUMMARY ANSWER: GC metabolism is regulated by the LH surge, altered by obesity and reproductive aging, and, in women, specific metabolic profiles are associated with failed fertilization versus increased blastocyst development. WHAT IS KNOWN ALREADY: The cellular environment in which an oocyte matures is critical to its future developmental competence. Metabolism is emerging as a potentially important factor; however, relative energy production profiles between GCs and cumulus cells and their use of differential substrates under normal in vivo ovulatory conditions are not well understood. STUDY DESIGN, SIZE, DURATION: This study identified metabolic and substrate utilization profiles within ovarian cells in response to the LH surge, using mouse models and GCs of women undergoing gonadotropin-induced oocyte aspiration followed by IVF/ICSI. PARTICIPANTS/MATERIALS, SETTING, METHODS: To comprehensively assess follicular energy metabolism, we used real-time metabolic analysis (Seahorse XFe96) to map energy metabolism dynamics (mitochondrial respiration, glycolysis, and fatty acid oxidation) in mouse GCs and cumulus-oocyte complexes (COCs) across a detailed time course in the lead up to ovulation. In parallel, the metabolic profile of GCs was measured in a cohort of 85 women undergoing IVF/ICSI (n = 21 with normal ovarian function; n = 64 with ovarian infertility) and correlated with clinical parameters and cycle outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: Our study reveals dynamic changes in GC energy metabolism in response to ovulatory LH, with mitochondrial respiration and glycolysis differentially affected by obesity versus aging, in both mice and women. High respiration in GCs is associated with failed fertilization (P < 0.05) in a subset of women, while glycolytic reserve and mitochondrial ATP production are correlated with on-time development at Day 3 (P < 0.05) and blastocyst formation (P < 0.01) respectively. These data provide new insights into the cellular mechanisms of infertility, by uncovering significant associations between metabolism within the ovarian follicle and oocyte developmental competence. LIMITATIONS, REASONS FOR CAUTION: A larger prospective study is needed before the metabolic markers that were positively and negatively associated with oocyte quality can be used clinically to predict embryo outcomes. WIDER IMPLICATIONS OF THE FINDINGS: This study offers new insights into the importance of GC metabolism for subsequent embryonic development and highlights the potential for therapeutic strategies focused on optimizing mitochondrial metabolism to support embryonic development. STUDY FUNDING/COMPETING INTEREST(S): National Health and Medical Research Council (Australia). The authors have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

IVF and risk of Type 1 diabetes mellitus: a population-based nested case-control study.

STUDY QUESTION: Is the mode of conception (natural, subfertility and non-IVF, and IVF) associated with the risk of Type 1 diabetes mellitus among offspring? SUMMARY ANSWER: The risk of Type 1 diabetes in offspring does not differ among natural, subfertility and non-IVF, and IVF conceptions. WHAT IS KNOWN ALREADY: Evidence has shown that children born through IVF have an increased risk of impaired metabolic function. STUDY DESIGN, SIZE, DURATION: A population-based, nested case-control study was carried out, including 769 children with and 3110 children without Type 1 diabetes mellitus within the prospective cohort of 2 228 073 eligible parent-child triads between 1 January 2004 and 31 December 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using registry data from Taiwan, the mode of conception was divided into three categories: natural conception, subfertility, and non-IVF (indicating infertility diagnosis but no IVF-facilitated conception), and IVF conception. The diagnosis of Type 1 diabetes mellitus was determined according to the International Classification of Diseases, 9th or 10th Revision, Clinical Modification. Each case was matched to four controls randomly selected after matching for child age and sex, residential township, and calendar date of Type 1 diabetes mellitus occurrence. MAIN RESULTS AND THE ROLE OF CHANCE: Based on 14.3 million person-years of follow-up (median, 10 years), the incidence rates of Type 1 diabetes were 5.33, 5.61, and 4.74 per 100 000 person-years for natural, subfertility and non-IVF, and IVF conceptions, respectively. Compared with natural conception, no significant differences in the risk of Type 1 diabetes were observed for subfertility and non-IVF conception (adjusted odds ratio, 1.04 [95% CI, 0.85-1.27]) and IVF conception (adjusted odds ratio, 1.00 [95% CI, 0.50-2.03]). In addition, there were no significant differences in the risk of Type 1 diabetes according to infertility source (male/female/both) and embryo type (fresh/frozen). LIMITATIONS, REASONS FOR CAUTION: Although the population-level data from Taiwanese registries was used, a limited number of exposed cases was included. We showed risk of Type 1 diabetes was not associated with infertility source or embryo type; however, caution with interpretation is required owing to the limited number of exposed events after the stratification. The exclusion criterion regarding parents' history of diabetes mellitus was only applicable after 1997, and this might have caused residual confounding. WIDER IMPLICATIONS OF THE FINDINGS: It has been reported that children born to parents who conceived through IVF had worse metabolic profiles than those who conceived naturally. Considering the findings of the present and previous studies, poor metabolic profiles may not be sufficient to develop Type 1 diabetes mellitus during childhood. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from Shin Kong Wu Ho-Su Memorial Hospital (No. 109GB006-1). The funders had no role in considering the study design or in the collection, analysis, interpretation of data, writing of the report, or decision to submit the article for publication. The authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: N/A.