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Study Question: Can sperm donation increase live birth rates following ICSI in advanced maternal age (AMA) patients?Summary Answer: Sperm donation increases the live birth rate in AMA ICSI cycles.What Is Known Already: In ICSI practice, sperm donation has been predominantly applied to overcome male infertility. The involvement of paternal age and lower sperm quality in the severe reduction in fertility observed in AMA patients remains to be clarified.Study Design, Size, Duration: Retrospective multicenter cohort study including data generated between 2015 and 2019 from 755 ICSI cycles achieving a fresh embryo transfer, of which 337 were first homologous cycles (normozoospermic partner sperm and homologous oocytes) and 418 were first sperm donation cycles (donor sperm and homologous oocytes). The association of sperm origin (partner vs donor) with live birth was assessed by multivariate analysis in non-AMA (<37 years, n = 278) and AMA (≥37 years, n = 477) patients, separately, including in the model all variables previously found to be associated with live birth in a univariate analysis (number of MII oocytes recovered, number of embryos transferred, and maternal age). ICSI outcomes were compared between sperm donation and homologous cycles in overall, non-AMA and AMA patients.Participants/materials, Setting, Methods: The study was conducted in three fertility clinics and included 755 Caucasian patients aged 24-42 years undergoing their first homologous or sperm donation ICSI cycle achieving a fresh embryo transfer.Main Results and the Role Of Chance: The multivariate analysis revealed that sperm donation was positively associated with the likelihood of a live birth independently of all other variables tested in AMA (P = 0.02), but not in non-AMA patients. Live birth, delivery, and miscarriage rates differed substantially between sperm donation and homologous AMA cycles; live birth and delivery rates were 70-75% higher (25.4% vs 14.5% and 22.5% vs 13.5%, respectively; P < 0.01), while miscarriage occurrence was less than half (18.0% vs 39.5%; P < 0.01) in sperm donation compared to homologous AMA cycles.Limitations, Reasons For Caution: This study is limited by its retrospective nature, differences in patients profiles between sperm donation and homologous-control groups and varying proportion of donor cycles between fertility centers, although these variations have been controlled for in the statistical analysis.Wider Implications Of the Findings: The findings suggest that sperm donation increases live birth rates while reducing miscarriage occurrence in AMA patients, and thus may be a valid strategy to improve ICSI outcomes in this growing and challenging patient group.Study Funding/competing Interest(s): N/A.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Can focused application of time-lapse microscopy (TLM) lead to a more detailed map of the morphokinetics of human fertilization, revealing novel or neglected aspects of this process?Summary Answer: Intensive harnessing of TLM reveals novel or previously poorly characterised phenomena of fertilization, such as a cytoplasmic wave (CW) preceding pronuclear formation and kinetics of pronuclear chromatin polarization, thereby suggesting novel non-invasive biomarkers of embryo quality.What Is Known Already: In recent years, human preimplantation development has been the object of TLM studies with the intent to develop morphokinetic algorithms able to predict blastocyst formation and implantation. Regardless, our appreciation of the morphokinetics of fertilization remains rather scarce, currently including only times of polar body II (PBII) emission, pronuclear appearance and fading, and first cleavage. This is not consistent with the complexity and importance of this process, calling for further TLM studies aimed at describing previously unrecognized or undetected morphokinetic events and identifying novel developmental biomarkers.Study Design, Size, Duration: The study involved a retrospective observation by TLM of the fertilization process in 500 oocytes utilized in consecutive ICSI cycles carried out in 2016. A maximum of five fertilized oocytes per patients were included in the analysis to reduce possible patient-specific biases. Oocytes of patients with different diagnoses of infertility where included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded.Participants/materials, Settings, Methods: Microinjected oocytes where assessed by a combined TLM-culture system (Embryoscope). Oocytes that were not amenable to TLM assessment, due to excess of residual corona cells or inadequate orientation for the observation of PBII emission, were not analysed. We identified and monitored 28 parameters relevant to meiotic resumption, pronuclear dynamics, chromatin organization, and cytoplasmic/cortical modifications. Times (T) were expressed as mean ± SD hours post-insemination (p.i.) and analysed, where appropriate, by Paired T Student or Fisher's exact tests.Main Results and Role Of Chance: PBII emission was occasionally followed (4.3% of cases) by the transient appearance of a protrusion of the cell surface, the fertilization cone (FC), probably resulting from interaction of the male chromatin with the oocyte cortex. Pronuclear formation was always preceded by a radial CW originating from the initial position of the male pronucleus (PN) and extending towards the oocyte periphery. The appearance of the CW followed a precise sequence, occurring always 2-3 h after PBII emission and shortly before PN appearance. Male and female PN appeared virtually simultaneously at approximately 6.2 h p.i. However, while the female PN always formed cortically and near the site of emission of the PBII, the initial position of the male PN was cortical, intermediate, or central (15.2%, 31.2% and 53.6%, respectively). PN juxtaposition involved rapid and straight movement of the female PN towards the male PN. In addition, the initial position of male PN formation was predictive of the position of PN juxtaposition. It was also observed that nucleolar precursor bodies (NPBs) aligned along the juxtaposition area and this happened considerably earlier for the female PN (8.2 ± 2.6 vs.11.2 ± 4.1 h, P = 0.0001). Although it occurred rarely, displacement of juxtaposed PN to the cortex was strongly associated (P < 0.0001) with direct cleavage into three blastomeres at the first cell division. The times of PN breakdown and first cleavage showed a very consistent trend, occurring earlier or progressively later depending on whether initial male PN positioning was central, intermediate or cortical, respectively. Finally, time intervals between discrete fertilization events were strongly associated with embryo quality on Day 3. For example, longer intervals between disappearance of the cytoplasmic halo and PN breakdown were highly predictive of reduced blastomere number and increased fragmentation (P = 0.0001).Large Scale Data: N/A.Limitations, Reason For Caution: Some of the morphokinetic parameters assessed in this study may require better definition to reduce inter-operator annotation variability.Wider Implications Of the Findings: To our knowledge, overall, these data represent the most detailed morphokinetic description of human fertilization. Many of the illustrated parameters are novel and may be amenable to further elaboration into algorithms able to predict embryo quality, as suggested by the findings presented in this study.Study Funding/competing Interests: None. [ABSTRACT FROM AUTHOR]

Study Question: What is the clinical efficacy of an oocyte donation program based on the transportation of frozen semen and embryos between two countries?Summary Answer: The transnational oocyte donation program is efficient and reliable and it could provide a first-line strategy to overcome the lack of donors in some countries.What Is Known Already: While there is increasing need for donated oocytes, in many countries the availability of donors is still insufficient to cover the therapeutic demands, and patients are referred abroad for treatment. Since embryo cryopreservation is reliable and efficient, we propose a strategy based on frozen embryos instead of frozen oocytes to satisfy the increasing demand for cross border oocyte donation.Study Design, Size, Duration: This is a retrospective cohort study including 630 patients treated from December 2015 to July 2017.Participants/materials, Setting, Methods: Infertile women were treated with elective vitrified-thawed embryo shipping and embryo transfer (ET) between two IVF clinics, one in Spain and one in Italy.Main Results and the Role Of Chance: A total of 2617 embryos were created for the 630 patients and the survival rate after warming was 98.5%. After the first ET the live birth rate (LBR) was 30.6%. In 476 patients (75.5%), embryos were transferred at the cleavage stage (Day 2 or 3) and the LBR was 29.2%. Vitrified blastocysts were available for 154 patients (24.5%) and the LBR was 35%. Among patients who did not achieve a pregnancy after the first frozen ET (FET), 92.5% had at least one frozen embryo for successive procedures. 213 patients underwent a second FET. The LBR at the second FET was 30%. The cumulative LBR at the end of the observation period was 39.3%.Limitations, Reasons For Caution: The study design was retrospective. A direct comparison with vitrified oocyte donors cycle and subsequent fresh ET would have permitted to compare this strategy versus the current standard based on vitrified gametes.Wider Implications Of the Findings: The LBR found in our study is more than acceptable and seems to be higher than what reported with vitrified oocytes. The transnational fresh oocyte donation program may have several advantages over the shipment of vitrified oocytes: similarly to the fresh oocyte donation program it allows for personalized care in oocyte recipient, which is provided by assigning a flexible number of oocytes, and at the same time it maintains the benefit of a frozen ART program permitting scheduling flexibility. The TOD program is efficient and may be proposed as a first-line strategy for distance and inter-countries oocyte donation programs.Study Funding, Competing Interest(s): None.Trial Registration Number: NA. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Which add-ons are safe and effective to be used in ART treatment? SUMMARY ANSWER Forty-two recommendations were formulated on the use of add-ons in the diagnosis of fertility problems, the IVF laboratory and clinical management of IVF treatment. WHAT IS KNOWN ALREADY The innovative nature of ART combined with the extremely high motivation of the patients has opened the door to the wide application of what has become known as 'add-ons' in reproductive medicine. These supplementary options are available to patients in addition to standard fertility procedures, typically incurring an additional cost. A diverse array of supplementary options is made available, encompassing tests, drugs, equipment, complementary or alternative therapies, laboratory procedures, and surgical interventions. These options share the common aim of stating to enhance pregnancy or live birth rates, mitigate the risk of miscarriage, or expedite the time to achieving pregnancy. STUDY DESIGN, SIZE, DURATION ESHRE aimed to develop clinically relevant and evidence-based recommendations focusing on the safety and efficacy of add-ons currently used in fertility procedures in order to improve the quality of care for patients with infertility. PARTICIPANTS/MATERIALS, SETTING, METHODS ESHRE appointed a European multidisciplinary working group consisting of practising clinicians, embryologists, and researchers who have demonstrated leadership and expertise in the care and research of infertility. Patient representatives were included in the working group. To ensure that the guidelines are evidence-based, the literature identified from a systematic search was reviewed and critically appraised. In the absence of any clear scientific evidence, recommendations were based on the professional experience and consensus of the working group. The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by 46 independent international reviewers. A total of 272 comments were received and incorporated where relevant. MAIN RESULTS AND THE ROLE OF CHANCE The multidisciplinary working group formulated 42 recommendations in three sections; diagnosis and diagnostic tests, laboratory tests and interventions, and clinical management. LIMITATIONS, REASONS FOR CAUTION Of the 42 recommendations, none could be based on high-quality evidence and only four could be based on moderate-quality evidence, implicating that 95% of the recommendations are supported only by low-quality randomized controlled trials, observational data, professional experience, or consensus of the development group. WIDER IMPLICATIONS OF THE FINDINGS These guidelines offer valuable direction for healthcare professionals who are responsible for the care of patients undergoing ART treatment for infertility. Their purpose is to promote safe and effective ART treatment, enabling patients to make informed decisions based on realistic expectations. The guidelines aim to ensure that patients are fully informed about the various treatment options available to them and the likelihood of any additional treatment or test to improve the chance of achieving a live birth. STUDY FUNDING/COMPETING INTEREST(S) All costs relating to the development process were covered from ESHRE funds. There was no external funding of the development process or manuscript production. K.L. reports speakers fees from Merck and was part of a research study by Vitrolife (unpaid). T.E. reports consulting fees from Gynemed, speakers fees from Gynemed and is part of the scientific advisory board of Hamilton Thorne. N.P.P. reports grants from Merck Serono, Ferring Pharmaceutical, Theramex, Gedeon Richter, Organon, Roche, IBSA and Besins Healthcare, speakers fees from Merck Serono, Ferring Pharmaceutical, Theramex, Gedeon Richter, Organon, Roche, IBSA and Besins Healthcare. S.R.H. declares being managing director of Fertility Europe, a not-for-profit organization receiving financial support from ESHRE. I.S. is a scientific advisor for and has stock options from Alife Health, is co-founder of IVFvision LTD (unpaid) and received speakers' fee from the 2023 ART Young Leader Prestige workshop in China. A.P. reports grants from Gedeon Richter, Ferring Pharmaceuticals and Merck A/S, consulting fees from Preglem, Novo Nordisk, Ferring Pharmaceuticals, Gedeon Richter, Cryos and Merck A/S, speakers fees from Gedeon Richter, Ferring Pharmaceuticals, Merck A/S, Theramex and Organon, travel fees from Gedeon Richter. The other authors disclosed no conflicts of interest. DISCLAIMER This Good Practice Recommendations (GPRs) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or bedeemedinclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results.Theydo not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What clinical and laboratory differences emerge from parallel direct comparison of embryos reaching the blastocyst stage between Days 4, 5, 6, and 7 (Days 4–7)? SUMMARY ANSWER Increasing times to blastocyst formation are associated with a worse clinical outcome and perturbations in developmental patterns appear as early as the fertilization stage. WHAT IS KNOWN ALREADY Previous evidence indicates that later times to blastocyst development are associated with a worse clinical outcome. However, the vast majority of these data concern Day 5 and Day 6 blastocysts, while Day 4 and Day 7 blastocysts remain less thoroughly investigated. In addition, studies comparing in parallel the developmental patterns and trajectories of Day 4–7 blastocysts are lacking. This leaves unanswered the question of when and how differences among such embryos emerge. Acquisition of such knowledge would significantly contribute to understanding the relative impact of intrinsic and extrinsic causes of embryo developmental kinetics and competence. STUDY DESIGN, SIZE, DURATION This retrospective study involved time-lapse technology (TLT) monitoring of Day 4 (N = 70), Day 5 (N = 6147), Day 6 (N = 3243), and Day 7 (N = 149) blastocysts generated in 9450 ICSI cycles. Oocyte retrievals were carried out after clomiphene citrate-based minimal ovarian stimulation, between January 2020 and April 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS Couples included in the study presented with different diagnoses, mainly male factor and unexplained infertility. Cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Microinjected oocytes were assessed by a combined TLT-culture system. Day 4–7 blastocyst groups were compared in terms of morphokinetics (pronuclear dynamics, cleavage patterns and timings, and embryo quality) and clinical outcome. Clinically usable blastocysts were cryopreserved and transferred in single vitrified-warmed blastocyst transfers (SVBT). MAIN RESULTS AND THE ROLE OF CHANCE From 19 846 microinjected oocytes, 17 144 zygotes (86.4%) were obtained. Overall, the blastocyst development rate was 56.0%. Rates of blastocysts formation on Days 4, 5, 6, and 7 were 0.7%, 64.0%, 33.8%, and 1.6%, respectively. The average expanded blastocyst development times were 98.4 ± 0.4, 112.4 ± 0.1, 131.6 ± 0.1, and 151.2 ± 0.5 h in the Day 4–7 groups, respectively. Female age was positively associated with longer times to blastocyst development. Rates of both inner cell mass (ICM) and trophectoderm (TE) morphological grade A blastocysts were negatively associated with the day of blastocyst development (P  < 0.0001). The differences in development times and intervals increased progressively until blastocyst expansion (P  < 0.0001 for all development times). Strikingly, such differences were already markedly evident as early as the time of pronuclear fading (tPNf) (20.6 ± 0.3, 22.5 ± 0.0, 24.0 ± 0.0, 25.5 ± 0.3; Days 4–7, respectively; P  < 0.0001). Rates of cleavage anomalies (tri-/multi-chotomous mitosis or rapid cleavage) occurring at the first or second/third division cycles were also positively associated with longer times to blastocyst development. Implantation, ongoing pregnancy, and live birth rates were progressively reduced with increasing blastocyst development times (P  < 0.0001), even after stratification for maternal age. When controlled for female age, male age, number of previous embryo transfer cycles, morphological grade of the ICM and TE, and progesterone supplementation, the probabilities of implantation, clinical, and ongoing pregnancy and live birth were significantly decreased in Day 6 blastocysts in comparison to Day 5 blastocysts. Follow-up data on birth length, weight, and malformations were comparable among the four blastocyst groups. LIMITATIONS, REASONS FOR CAUTION The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation. WIDER IMPLICATIONS OF THE FINDINGS This study extends previous data on the relation between time of blastocyst formation and clinical outcome. It also indicates that differences in developmental times and patterns of Day 4–7 blastocysts occur as early as the fertilization stage, possibly dictated by intrinsic gamete-derived factors. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the participating institutions. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Which patients might benefit from insemination of delayed-matured oocytes? SUMMARY ANSWER Delayed-matured oocytes had a ≥50% contribution to the available cohort of biopsied blastocysts in patients with advanced maternal age, low maturation, and/or low fertilization rates. WHAT IS KNOWN ALREADY Retrieved immature oocytes that progress to the MII stage in vitro could increase the number of embryos available during ICSI cycles. However, these delayed-matured oocytes are associated with lower fertilization rates and compromised embryo quality. Data on the ploidy of these embryos are controversial, but studies failed to compare euploidy rates of embryos derived from delayed-matured oocytes to patients' own immediate mature sibling oocytes. This strategy efficiently allows to identify the patient population that would benefit from this approach. STUDY DESIGN, SIZE, DURATION This observational study was performed between January 2019 and June 2021 including a total of 5449 cumulus oocytes complexes from 469 ovarian stimulation cycles, from which 3455 inseminated matured oocytes from ICSI (n = 2911) and IVF (n = 544) were considered as the sibling controls (MII-D0) to the delayed-matured oocytes (MII-D1) (n = 910). Euploidy rates were assessed between delayed-matured (MII-D1) and mature sibling oocytes (MII-D0) in relation to patients' clinical characteristics such as BMI, AMH, age, sperm origin, and the laboratory outcomes, maturation, fertilization, and blastocyst utilization rates. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 390 patients undergoing IVF/ICSI, who had at least one metaphase I (MI) or germinal-vesicle (GV) oocyte on the day of oocyte collection (Day 0), which matured in 20–28 h after denudation were included. MI and GV oocytes that matured overnight were inseminated on the following day (Day 1, MII-D1) by ICSI. Only cycles planned for preimplantation genetic testing for aneuploidy using fresh own oocytes were included. MAIN RESULTS AND THE ROLE OF CHANCE Fertilization (FR) and blastocyst utilization rates were significantly higher for MII-D0 compared to delayed-matured oocytes (MII-D1) (69.5% versus 55.9%, P  < 0.001; and 59.5% versus 18.5%, P  < 0.001, respectively). However, no significant difference was observed in the rate of euploid embryos between MII-D0 and MII-D1 (46.3% versus 39.0%, P  = 0.163). For evaluation of the benefit of inseminating MI/GV oocytes on D1 per cycle in relation to the total number of biopsied embryos, cycles were split into three groups based on the proportion of MII-D1 embryos that were biopsied in that cycle (0%, 1–50%, and ≥50%). The results demonstrate that patients who had ≥50% contribution of delayed-matured oocytes to the available cohort of biopsied embryos were those of advanced maternal age (mean age 37.7 years), <10 oocytes retrieved presenting <34% maturation rate, and <60% fertilization rate. Every MII oocyte injected next day significantly increased the chances of obtaining a euploid embryo [odds ratio (OR) = 1.83, CI: 1.50–2.24, P  < 0.001] among MII-D1. The odds of enhanced euploidy were slightly higher among the MII-D1-GV matured group (OR = 1.78, CI: 1.42–2.22, P  < 0.001) than the MII-D1-MI matured group (OR = 1.54, CI: 1.25–1.89, P  < 0.001). Inseminating at least eight MII-D1 would have >50% probability of getting a euploid embryo among the MII-D1 group. LIMITATIONS, REASONS FOR CAUTION ICSI of MII-D1 was performed with the fresh or frozen ejaculates or testicular samples from the previous day. The exact timing of polar body extrusion of delayed-matured MI/GV was not identified. Furthermore, the time point of the final oocyte maturation to MII for the immature oocytes and for the oocytes inseminated by IVF could not be identified. WIDER IMPLICATIONS OF THE FINDINGS The results of this study might provide guidance to the IVF laboratories for targeting the patient's population who would benefit from MII-D1 ICSI without adhering to unnecessary costs and workload. STUDY FUNDING/COMPETING INTEREST(S) No external funding was received for this study. There are no conflicts of interest to be declared for any of the authors. There are no patents, products in development, or marketed products to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Human embryos are very frequently affected by maternally inherited aneuploidies, which in the vast majority of cases determine developmental failure at pre- or post-implantation stages. However, recent evidence, generated by the alliance between diverse technologies now routinely employed in the IVF laboratory, has revealed a broader, more complex scenario. Aberrant patterns occurring at the cellular or molecular level can impact at multiple stages of the trajectory of development to blastocyst. In this context, fertilization is an extremely delicate phase, as it marks the transition between gametic and embryonic life. Centrosomes, essential for mitosis, are assembled ex novo from components of both parents. Very large and initially distant nuclei (the pronuclei) are brought together and positioned centrally. The overall cell arrangement is converted from being asymmetric to symmetric. The maternal and paternal chromosome sets, initially separate and scattered within their respective pronuclei, become clustered where the pronuclei juxtapose, to facilitate their assembly in the mitotic spindle. The meiotic spindle is replaced by a segregation machinery that may form as a transient or persistent dual mitotic spindle. Maternal proteins assist the decay of maternal mRNAs to allow the translation of newly synthesized zygotic transcripts. The diversity and complexity of these events, regulated in a precise temporal order and occurring in narrow time windows, make fertilization a highly error-prone process. As a consequence, at the first mitotic division, cellular or genomic integrity may be lost, with fatal consequences for embryonic development. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Is a commercially available embryo assessment algorithm for early embryo evaluation based on the automatic annotation of morphokinetic timings a useful tool for embryo selection in IVF cycles? SUMMARY ANSWER The classification provided by the algorithm was shown to be significantly predictive, especially when combined with conventional morphological evaluation, for development to blastocyst, implantation, and live birth, but not for euploidy. WHAT IS KNOWN ALREADY The gold standard for embryo selection is still morphological evaluation conducted by embryologists. Since the introduction of time-lapse technology to embryo culture, many algorithms for embryo selection have been developed based on embryo morphokinetics, providing complementary information to morphological evaluation. However, manual annotations of developmental events and application of algorithms can be time-consuming and subjective processes. The introduction of automation to morphokinetic annotations is a promising approach that can potentially reduce subjectivity in the embryo selection process and improve the workflow in IVF laboratories. STUDY DESIGN, SIZE, DURATION This observational, retrospective cohort study was performed in a single IVF clinic between 2018 and 2021 and included 3736 embryos from oocyte donation cycles (423 cycles) and 1291 embryos from autologous cycles with preimplantation genetic testing for aneuploidies (PGT-A, 185 cycles). Embryos were classified on Day 3 with a score from 1 (best) to 5 (worst) by the automatic embryo assessment algorithm. The performance of the embryo classification model for blastocyst development, implantation, live birth, and euploidy prediction was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS All embryos were monitored by a time-lapse system with an automatic cell-tracking and embryo assessment software during culture. The embryo assessment algorithm was applied on Day 3, resulting in embryo classification from 1 to 5 (from highest to lowest developmental potential) depending on four parameters: P2 (t3–t2), P3 (t4–t3), oocyte age, and number of cells. There were 959 embryos selected for transfer on Day 5 or 6 based on conventional morphological evaluation. The blastocyst development, implantation, live birth, and euploidy rates (for embryos subjected to PGT-A) were compared between the different scores. The correlation of the algorithm scoring with the occurrence of those outcomes was quantified by generalized estimating equations (GEEs). Finally, the performance of the GEE model using the embryo assessment algorithm as the predictor was compared to that using conventional morphological evaluation, as well as to a model using a combination of both classification systems. MAIN RESULTS AND THE ROLE OF CHANCE The blastocyst rate was higher with lower the scores generated by the embryo assessment algorithm. A GEE model confirmed the positive association between lower embryo score and higher odds of blastulation (odds ratio (OR) (1 vs 5 score) = 15.849; P  <   0.001). This association was consistent in both oocyte donation and autologous embryos subjected to PGT-A. The automatic embryo classification results were also statistically associated with implantation and live birth. The OR of Score 1 vs 5 was 2.920 (95% CI 1.440–5.925; P  =   0.003; E  =   2.81) for implantation and 3.317 (95% CI 1.615–6.814; P  =   0.001; E  =   3.04) for live birth. However, this association was not found in embryos subjected to PGT-A. The highest performance was achieved when combining the automatic embryo scoring and traditional morphological classification (AUC for implantation potential = 0.629; AUC for live-birth potential = 0.636). Again, no association was found between the embryo classification and euploidy status in embryos subjected to PGT-A (OR (1 vs 5) = 0.755 (95% CI 0.255–0.981); P  =   0.489; E  =   1.57). LIMITATIONS, REASONS FOR CAUTION The retrospective nature of this study may be a reason for caution, although the large sample size reinforced the ability of the model for embryo selection. WIDER IMPLICATIONS OF THE FINDINGS Time-lapse technology with automated embryo assessment can be used together with conventional morphological evaluation to increase the accuracy of embryo selection process and improve the success rates of assisted reproduction cycles. To our knowledge, this is the largest embryo dataset analysed with this embryo assessment algorithm. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by Agencia Valenciana de Innovació and European Social Fund (ACIF/2019/264 and CIBEFP/2021/13). In the last 5 years, M.M. received speaker fees from Vitrolife, Merck, Ferring, Gideon Richter, Angelini, and Theramex, and B.A.-R. received speaker fees from Merck. The remaining authors have no competing interests to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Are the early pregnancy outcomes of IVF pregnancies conceived with donor sperm different to those conceived with partner sperm? SUMMARY ANSWER Pregnancies conceived with donor sperm have a lower odds of early pregnancy loss and ectopic pregnancy compared to pregnancies conceived with partner sperm. WHAT IS KNOWN ALREADY The number of cycles using donor sperm has risen significantly in recent years. Adverse early pregnancy outcomes have a negative impact on women and their partners. The evidence available to date regarding early pregnancy outcomes for pregnancies conceived with IVF donor sperm is limited by low numbers and lower-quality studies. STUDY DESIGN, SIZE, DURATION This is a retrospective cohort study of 1 376 454 cycles conceived with either donor or partner sperm between 1991 and 2016 as recorded in the Human Fertilisation and Embryology Authority (HFEA) Register. PARTICIPANTS/MATERIALS, SETTING, METHODS The HFEA has recorded data on all fertility treatments carried out in the UK from 1991 onwards, and it publishes this data in an anonymized form. This study assessed the outcomes of all pregnancies conceived with donor sperm and compared them to those conceived with partner sperm among IVF cycles recorded in the HFEA anonymized dataset from 1991 to 2016. Cycles that included intrauterine insemination, donor oocytes, preimplantation genetic testing, oocyte thaw cycles and alternative fertility treatments were excluded. The outcomes of interest were biochemical pregnancy, miscarriage, ectopic pregnancy, stillbirth and live birth. Logistic regression was used to adjust for confounding factors including age of the female partner, cause of infertility, history of previous pregnancy, fresh or frozen cycle, IVF or ICSI, number of embryos transferred, and year of treatment. Results are reported as adjusted odds ratios (aOR) and 95% CIs. MAIN RESULTS AND THE ROLE OF CHANCE This study found reductions in the odds of biochemical pregnancy (aOR 0.82, 95% CI 0.78–0.86), miscarriage (aOR 0.93, 95% CI 0.89–0.97), and ectopic pregnancy (aOR 0.77, 95% CI 0.66–0.90) among pregnancies as a result of the use of donor sperm as opposed to partner sperm. LIMITATIONS, REASONS FOR CAUTION This study is retrospective and limited by the constraints of routinely collected data. No data were available for maternal characteristics such as BMI, smoking and partner age, which could all be potential confounders. Clustering of multiple pregnancies within women could not be accounted for as the data are reported only at the cycle level with no maternal identifiers. WIDER IMPLICATIONS OF THE FINDINGS This study has demonstrated that there are no increased risks of adverse pregnancy outcome with donor sperm pregnancies. The reduction in miscarriage in pregnancies using donor sperm suggests that sperm could have a role in miscarriage, as the selection process for being accepted as donor is stringent. STUDY FUNDING/COMPETING INTEREST(S) No external funding was sought for this study. C.A. has received funding from Ferring to attend a UK meeting for trainees in reproductive Medicine. A.M. has received funding from Ferring, Cook, Merck Serono, Geodon Ritcher, and Pharmasure for speaking at, or attending, meetings relating to reproductive medicine. She has also participated in a Ferring advisory board. S.B. has received grants from Tenovus and the UK Medical Research Council. She has also been supported with a Medical Research Scotland PhD studentship. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? SUMMARY ANSWER Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. WHAT IS KNOWN ALREADY In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. STUDY DESIGN, SIZE, DURATION A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (<35, 35–37, 38–40, 41–42, and >42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers—Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)—involved in blastocyst morphogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41–42 and >42-year groups (P  <   0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the >42-year group than those in the <35-year group (P  =   0.0022 and P  <   0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41–42 and >42-year groups (P  =   0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P  =   0.0027 and P  <   0.0122, respectively). Developmental speed at cleavage stage, incidence of irregularity of first cleavage, type and duration of blastomere movement, and number of multinucleated cells were comparable among age groups. Delayed embryonic compaction and an increased number of extruded blastomeres were observed in the >42-year group (P  =   0.0002 and P  =   0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41–42 and >42-year groups (P  < 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the >42-year group (P  <   0.0001 for both). LIMITATIONS, REASONS FOR CAUTION At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. WIDER IMPLICATIONS OF THE FINDINGS The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response?Summary Answer: A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women.What Is Known Already: Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART.Study Design, Size, Duration: hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021.Participants/materials, Setting, Methods: hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well.Main Results and the Role Of Chance: hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders.Limitations, Reasons For Caution: Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage.Wider Implications Of the Findings: This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction.Study Funding/competing Interest(s): This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does culture in a high relative humidity atmosphere improve clinical outcomes when using a time-lapse integrated incubator and single-step culture medium? SUMMARY ANSWER Using an integrated time-lapse system and single-step culture medium, culture in a high relative humidity atmosphere increases the likelihood of embryos, especially those subjected to preimplantation genetic testing for aneuploidies, to achieve a pregnancy compared to those cultured in dry conditions. WHAT IS KNOWN ALREADY The use of a humid atmosphere inside incubators can reduce changes in culture media osmolality, which has been reported to have a significant effect on embryo quality and morphokinetics. Studies assessing the effect of humid culture (HC) in clinical outcomes are, however, scarce and inconclusive, mostly due to a high variability in culture conditions and reduced sample size. STUDY DESIGN, SIZE, DURATION Retrospective cohort study performed over 1627 ICSI cycles performed during 3 consecutive years in which embryo cohorts were cultured in a time-lapse incubator with three dry and three humidified chambers, and using single-step culture medium. Clinical outcomes were compared between treatments in which embryo cohorts were cultured in either humid (n = 833) or dry (n = 794) conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS The study includes autologous treatments, with (N = 492) and without (N = 372) preimplantation genetic testing for aneuploidies (PGT-A) and ovum donation treatments (N = 763), performed in three university-affiliated private IVF centres. Stimulation, oocyte pickup and fertilization were performed according to the standard procedures of the clinic. All embryo cohorts were cultured in the same model of time-lapse incubator, distributed to either a dry or humidified chamber, while the rest of the culture variables remained equal. The population was weighted by the inverse probability of treatment to control for all measured confounders. The association between HC and the main outcome was assessed by logistic regression over the weighted population. The E- value was reported as a way of considering for unmeasured confounders. Differences in embryo development and other secondary outcomes between the study groups were assessed by Pearson Chi-squared test, ANOVA test and Kaplan–Meier survival analysis. MAIN RESULTS AND THE ROLE OF CHANCE An univariable logistic regression analysis, weighted by the inverse probability of treatment, determined that embryos cultured in humid conditions are more likely to achieve a clinical pregnancy than those cultured in dry conditions (odds ratio (OR) = 1.236 (95% CI 1.009–1.515), P  =   0.041, E  = 1.460). Through stratification, it was determined that said effect is dependent on the type of treatment: no improvement in clinical pregnancy was present in ovum donation or autologous treatments, but a statistically significant positive effect was present in treatments with preimplantation genetic testing (OR = 1.699 (95% CI 1.084–2.663), P =  0.021, E  =   1.930). Said increase does not relate with an improvement in later outcomes. Differences were also found in variables related to embryo developmental morphokinetics. LIMITATIONS, REASONS FOR CAUTION The retrospective nature of the study makes it susceptible to some bias linked to the characteristics of the treatments. To lessen the effect of possible biases, cases were weighted by the inverse probability of treatment prior to the evaluation of the outcome, as means to assess for measured confounders. In addition, the E -value of the weighted OR was calculated as a sensitivity analysis for unmeasured confounders. A randomized prospective study could be performed for further assessing the effect of humid conditions in clinical outcome. WIDER IMPLICATIONS OF THE FINDINGS These results support that embryo culture under conditions of high relative humidity contributes to optimize clinical results in undisturbed culture in a time-lapse incubator with single-step medium. To our knowledge, this is the largest study on the matter and the first performing a propensity score-based analysis. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the "Centro para el Desarrollo Tecnologico Industrial" from the Spanish Ministry of Science, Innovation, and Universities (CDTI-20170310) and Generalitat Valenciana and European Social Fund (ACIF/2019/264). None of the authors have any competing interest to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can rapamycin improve the developmental competence of human oocytes during the IVM process? SUMMARY ANSWER Rapamycin at 10 nM could markedly improve the developmental competence of human oocytes undergoing IVM. WHAT IS KNOWN ALREADY Embryos derived from oocytes that mature in vitro have lower developmental competence than sibling embryos derived from oocytes matured in vivo. Rapamycin was shown to effectively improve IVM outcomes in mammalian oocytes; however, its effects on IVM of human oocytes have not been investigated. STUDY DESIGN, SIZE, DURATION In 2021, donated immature oocytes (n = 202) from 80 infertile couples receiving ICSI were included in a control group, and 156 oocytes from 72 couples were included in a rapamycin group. The oocytes underwent IVM with 10 nM rapamycin or without (control) rapamycin, followed by insemination by ICSI and embryo culture. PARTICIPANTS/MATERIALS, SETTING, METHODS The germinal vesicle breakdown (GVBD), maturation, normal fertilization, high-quality embryo (HQE) and blastocyst formation rates were calculated to evaluate the developmental competence of IVM oocytes, and fluorescence staining was used to assess DNA damage levels of oocytes in both groups. Whole-genome amplification and DNA sequencing were performed to analyze chromosome euploidy in embryos derived from the rapamycin group. MAIN RESULTS AND THE ROLE OF CHANCE The baseline characteristics of patients who donated oocytes for the two experimental groups were similar. In the control group, GVBD happened in 135 (66.8%) oocytes, and the maturation rate reached 52.5% at 24 h and 63.4% at 48 h. In the rapamycin group, 143 (91.7%) oocytes underwent GVBD, and the maturation rate reached 60.3% at 24 h and 82.7% at 48 h. Following ICSI, more HQEs were obtained in the rapamycin group versus control (34.2% versus 22.1%, respectively, P  =   0.040), although with comparable fertilization rates in the two groups. In addition, the levels of histone γH2AX in oocytes cultured with 10 nM rapamycin were markedly decreased, compared with those in the control group (0.3 ± 0.0 versus 0.6 ± 0.1, respectively, P  =   0.048). Embryos with normal karyotype could be obtained from oocytes cultured with rapamycin. LIMITATIONS, REASONS FOR CAUTION Our preliminary results indicated that the addition of rapamycin during human oocyte IVM did not cause extra aneuploidy. However, this safety evaluation of rapamycin treatment was based on limited samples and more data are needed before possible application in the clinic. WIDER IMPLICATIONS OF THE FINDINGS In the current study, 10 nM rapamycin was applied in the IVM process of human oocytes for the first time and showed positive effects, providing new insights for potentially improving IVM outcomes in the clinic. There were subtle differences between the results presented here on human oocytes and our previous studies on mouse oocytes, indicating the necessity of more research on human samples. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the research grants from National Key Research and Development Project (2018YFC1002103) and Health Commission of Hubei Province scientific research project (WJ2021M110). All authors declared no conflicts of interest. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does culture in a high relative humidity atmosphere improve clinical outcomes when using a time-lapse integrated incubator and single-step culture medium?Summary Answer: Using an integrated time-lapse system and single-step culture medium, culture in a high relative humidity atmosphere increases the likelihood of embryos, especially those subjected to preimplantation genetic testing for aneuploidies, to achieve a pregnancy compared to those cultured in dry conditions.What Is Known Already: The use of a humid atmosphere inside incubators can reduce changes in culture media osmolality, which has been reported to have a significant effect on embryo quality and morphokinetics. Studies assessing the effect of humid culture (HC) in clinical outcomes are, however, scarce and inconclusive, mostly due to a high variability in culture conditions and reduced sample size.Study Design, Size, Duration: Retrospective cohort study performed over 1627 ICSI cycles performed during 3 consecutive years in which embryo cohorts were cultured in a time-lapse incubator with three dry and three humidified chambers, and using single-step culture medium. Clinical outcomes were compared between treatments in which embryo cohorts were cultured in either humid (n = 833) or dry (n = 794) conditions.Participants/materials, Setting, Methods: The study includes autologous treatments, with (N = 492) and without (N = 372) preimplantation genetic testing for aneuploidies (PGT-A) and ovum donation treatments (N = 763), performed in three university-affiliated private IVF centres. Stimulation, oocyte pickup and fertilization were performed according to the standard procedures of the clinic. All embryo cohorts were cultured in the same model of time-lapse incubator, distributed to either a dry or humidified chamber, while the rest of the culture variables remained equal. The population was weighted by the inverse probability of treatment to control for all measured confounders. The association between HC and the main outcome was assessed by logistic regression over the weighted population. The E-value was reported as a way of considering for unmeasured confounders. Differences in embryo development and other secondary outcomes between the study groups were assessed by Pearson Chi-squared test, ANOVA test and Kaplan-Meier survival analysis.Main Results and the Role Of Chance: An univariable logistic regression analysis, weighted by the inverse probability of treatment, determined that embryos cultured in humid conditions are more likely to achieve a clinical pregnancy than those cultured in dry conditions (odds ratio (OR) = 1.236 (95% CI 1.009-1.515), P = 0.041, E = 1.460). Through stratification, it was determined that said effect is dependent on the type of treatment: no improvement in clinical pregnancy was present in ovum donation or autologous treatments, but a statistically significant positive effect was present in treatments with preimplantation genetic testing (OR = 1.699 (95% CI 1.084-2.663), P = 0.021, E = 1.930). Said increase does not relate with an improvement in later outcomes. Differences were also found in variables related to embryo developmental morphokinetics.Limitations, Reasons For Caution: The retrospective nature of the study makes it susceptible to some bias linked to the characteristics of the treatments. To lessen the effect of possible biases, cases were weighted by the inverse probability of treatment prior to the evaluation of the outcome, as means to assess for measured confounders. In addition, the E-value of the weighted OR was calculated as a sensitivity analysis for unmeasured confounders. A randomized prospective study could be performed for further assessing the effect of humid conditions in clinical outcome.Wider Implications Of the Findings: These results support that embryo culture under conditions of high relative humidity contributes to optimize clinical results in undisturbed culture in a time-lapse incubator with single-step medium. To our knowledge, this is the largest study on the matter and the first performing a propensity score-based analysis.Study Funding/competing Interest(s): This work was supported by the ''Centro para el Desarrollo Tecnologico Industrial'' from the Spanish Ministry of Science, Innovation, and Universities (CDTI-20170310) and Generalitat Valenciana and European Social Fund (ACIF/2019/264). None of the authors have any competing interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is there any difference in developmental outcomes in children born after capacitation IVM (CAPA IVM) compared with conventional IVF?Summary Answer: Overall development up to 24 months of age was comparable in children born after CAPA IVM compared with IVF.What Is Known Already: IVM has been shown to be a feasible alternative to conventional IVF in women with a high antral follicle count (AFC). In addition to live birth rate, childhood development is also a relevant metric to compare between the two approaches to ART and there are currently no data on this.Study Design, Size, Duration: This study was a follow-up of babies born to women who participated in a randomized controlled trial comparing IVM with a pre-maturation step (CAPA IVM) and IVF. Developmental assessments were performed on 231 children over 24 months of follow-up.Participants/materials, Setting, Methods: Participants in the randomized controlled trial had an indication for ART and a high AFC (≥24 follicles in both ovaries). They were randomized to undergo one cycle of either IVM (n = 273) or IVF (n = 273). Of these, 96 women and 118 women, respectively, had live births. Seventy-six women (94 children, 79.2%) and 104 women (137 children, 88.1%), respectively, completed Ages & Stages Third Edition Questionnaire assessment (ASQ-3), and underwent evaluation of Developmental Red Flags at 6, 12 and 24 months of age.Main Results and the Role Of Chance: Baseline characteristics of participants in the follow-up study between the IVM and IVF groups were comparable. Overall, there were no significant differences in ASQ-3 scores at 6, 12 and 24 months between children born after IVM or IVF. The proportion of children with developmental red flags was low and did not differ between the two groups. Slightly, but significantly, lower ASQ-3 problem solving and personal-social scores in twins from the IVM versus IVF group at 6 months were still within the normal range and had caught up to the IVF group in the 12- and 24-month assessments. The number of children confirmed to have abnormal mental and/or motor development after specialist assessment was four in the IVM group and two in the IVF group (relative risk 2.91, 95% CI 0.54-15.6; P = 0.23).Limitations, Reasons For Caution: This study is an open-label follow-up of participants in a randomized controlled trial, and not all original trial subjects took part in the follow-up. The self-selected nature of the follow-up population could have introduced bias, and the sample size may have been insufficient to detect significant between-group differences in developmental outcomes.Wider Implications Of the Findings: Based on the current findings at 2 years of follow-up, there does not appear to be any significant concern about the effects of IVM on childhood development. These data add to the evidence available to physicians when considering different approaches to fertility treatment, but require validation in larger studies.Study Funding/competing Interest(s): This work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number FWO.106-YS.2017.02. L.N.V. has received speaker and conference fees from Merck, grant, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring; T.M.H. has received speaker fees from Merck, Merck Sharp and Dohme, and Ferring; R.J.N. has receives grant funding from the National Health and Medical Research Council (NHMRC) of Australia; B.W.M. has acted as a paid consultant to Merck, ObsEva and Guerbet and is the recipient of grant money from an NHMRC Investigator Grant; J.E.J.S. reports lecture fees from Ferring Pharmaceuticals, Biomérieux and Besins Female Healthcare, grants from Fund for Research Flanders (FWO) and is co-inventor on granted patents on CAPA-IVM methodology in the USA (US10392601B2) and Europe (EP3234112B1); T.D.P., M.H.N.N., N.A.N., T.T.L., V.T.T.T., N.T.N., H.L.T.H. and X.T.H.L. have no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years, and no other relationships or activities that could appear to have influenced the submitted work.Trial Registration Number: NCT04296357 (www.clinicaltrials.gov).Trial Registration Date: 5 March 2020.Date Of First Patient’s Enrolment: 7 March 2020. [ABSTRACT FROM AUTHOR]

Reproductive immunology has grown in importance in recent years and has even developed into a discipline of its own within the field of reproductive medicine. Many aspects of reproductive failure such as repeated implantation failure or recurrent miscarriages are, meanwhile, seen as a consequence of aberrant expression of immunological factors. This is reflected by the increasing number of tests for assessing and quantifying different immune cell types as well as by a wide range of immune therapies offered to a clientele consisting of desperate patients requesting additional 'IVF tools': first, what is still usually disregarded is the enormous plasticity and fluctuation of most immune cells in the genital tract; second, their still poorly characterized functions in the endometrial cycle: further, their partially unknown role in embryo implantation and in establishing a pregnancy; and third, the fact that one of the fundamental hypotheses of reproductive immunology—of note—the Medawar concept or 'Medawar's Paradox' of semi-allogeneic graft embryo, is partially based on an erroneous assumption, i.e. the immunologic rejection and tolerance of an embryo. In the present opinion article, we comment on the diagnostic procedures and therapy approaches for chronic endometritis within the scope of reproductive medicine. [ABSTRACT FROM AUTHOR]

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography–high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration. [ABSTRACT FROM AUTHOR]

Study Question: Is the automatic embryo grading function of specific time-lapse systems clinically useful as a decision support tool for IVF laboratories?Summary Answer: Blastocyst grading according to the automatic scoring system is directly associated with the likelihood of implantation and live birth, at least in treatments without preimplantation genetic testing for aneuploidy (PGT-A).What Is Known Already: Several embryo selection algorithms have been described since the introduction of time-lapse technology in IVF laboratories, but no one algorithm has yet been sufficiently consolidated for universal use. Multicentric models based on automated grading systems offer promise for standardization of embryo selection.Study Design, Size, Duration: A retrospective cohort study was performed including 1678 patients who underwent IVF treatments between 2018 and 2020 and whose embryos (n = 12 468) were cultured in time-lapse systems.Participants/materials, Setting, Methods: After obtaining the required parameters (division time to 2, 3, 4 and 5 cells; time of blastocyst formation; inner cell mass quality; and trophectoderm quality), the automatic embryo score was calculated using the software included in the appropriate workstation. First, embryo score was compared with conventional morphological quality and the subsequent clinical outcomes of 1952 single blastocyst transfers. Second, we quantified the contribution of the automatic embryo score and conventional morphological grade to implantation and live birth outcome with multivariate logistic regression analysis in different patient populations.Main Results and the Role Of Chance: A higher embryo score was associated with a better clinical outcome of IVF treatment. The mean of the automatic embryo score varied significantly (P < 0.001) among embryos with different morphological categories, between euploid and aneuploid embryos, between embryos resulting in positive versus negative pregnancy, between implanted and non-implanted embryos, and between embryos resulting in positive and negative live birth. Embryo score was related to the odds of implantation and live birth in the oocyte donation program (odds ratio (OR)=1.29; 95% CI [1.19-1.39]; P < 0.001 for implantation and OR = 1.26; 95% CI [1.16-1.36]; P < 0.001 for live birth) and in conventional treatments with autologous oocytes (OR = 1.38; 95% CI [1.24-1.54]; P < 0.001 for implantation and OR = 1.47; 95% CI [1.30-1.65]; P < 0.001 for live birth). There was no significant association of embryo score with implantation or live birth in treatments involving PGT-A.Limitations, Reasons For Caution: This study is limited by its retrospective nature. Further prospective randomized trials are required to confirm the clinical impact of these findings. The single-center design should be taken into account when considering the universal application of the model.Wider Implications Of the Findings: Evidence of the clinical efficiency of automated embryo scoring for ranking embryos with different morphological grade and potential in order to achieve higher implantation and live birth rates may make it a decision support tool for embryologists when selecting blastocysts for embryo transfer.Study Funding/competing Interest(s): This research has been funded by a grant from the Ministry of Science, Innovation and Universities FIS (PI21/00283) awarded to M.M. There are no competing interests to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Background: Fertility loss during female ageing is associated with increasing basal FSH and decreasing anti-Müllerian hormone (AMH) concentrations, together with compromised oocyte quality, presumably due to increased oxidative stress (OS) and DNA damage, as well as reduced metabolic and meiotic competences. Basal FSH and AMH circulatory concentrations have been broadly utilized as IVF success predictors, regardless of fluctuations in prognostic accuracy; basal FSH and AMH perform better in pre-advanced maternal age (AMA: >35 years) and AMA patients, respectively. The relationships between FSH and AMH intrafollicular levels and IVF outcomes suggest, nevertheless, that both hormones regulate oocyte competence, supporting the hypothesis that changes in FSH/AMH levels cause, at least in part, oocyte quality degradation during ageing. To understand the reasons behind the fluctuations in FSH and AMH prognostic accuracies and to clarify their participation in mechanisms determining oocyte competence and age-related subfertility, a deeper knowledge of the regulation of FSH and AMH intrafollicular signalling during the female reproductive lifespan, and of their effects on the cumulus-oocyte complex, is required.Objective and Rationale: An extensive body of information on the regulation of FSH and AMH intrafollicular availability and signalling, as well as on the control of folliculogenesis and oocyte metabolism, has been accumulated. However, these datasets have been explored within the relatively narrow boundaries of their specific subjects. Given the aforementioned gaps in knowledge and their clinical relevance, herein we integrate clinical and basic data, within a wide biological perspective, aiming to shed light on (i) the reasons for the variability in the accuracy of serum FSH and AMH as fertility markers, and on (ii) the potential roles of these hormones in mechanisms regulating oocyte quality, particularly those associated with ageing.Search Methods: The PubMed database encompassing the period between 1960 and 2021 was searched. Principal search terms were FSH, FSH receptor, AMH, oocyte, maternal age, cumulus, transzonal projections (TZPs), actin, OS, redox, reactive oxygen species, mitochondria, DNA damage, DNA repair, aneuploidy, spindle, meiosis, gene expression, transcription, translation, oocyte secreted factors (OSFs), cAMP, cyclic guanosine monophosphate, natriuretic peptide C, growth differentiation factor 9, bone morphogenetic protein 15 and fibroblast growth factor.Outcomes: Our analysis suggests that variations in the accuracy of fertility prognosis reflect a modest association between circulatory AMH levels and oocyte quality as well as increasing basal FSH inter-cycle variability with age. In addition, the basic and clinical data articulated herein support the hypothesis that increased intrafollicular FSH levels, as maternal age advances, may override the physiological protective influences of AMH and OSFs against excessive FSH signalling in cumulus cells. This would result in the disruption of oocyte homeostasis via reduced TZP-mediated transfer of cumulus-derived molecules essential for meiotic competence, gene expression, redox activity and DNA repair.Wider Implications: In-depth data analysis, encompassing a wide biological perspective has revealed potential causative mechanisms of age-related subfertility triggered by alterations in FSH/AMH signalling during the female reproductive life. Insights from new mechanistic models arising from this analysis should contribute to advancing our comprehension of oocyte biology in humans and serve as a valuable reference for novel AMA subfertility treatments aimed at improving oocyte quality through the modulation of AMH/FSH action. [ABSTRACT FROM AUTHOR]

Study Question: Can non-invasive metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) detect variations in metabolic profiles between discarded human blastocysts?Summary Answer: FLIM revealed extensive variations in the metabolic state of discarded human blastocysts associated with blastocyst development over 36 h, the day after fertilization and blastocyst developmental stage, as well as metabolic heterogeneity within individual blastocysts.What Is Known Already: Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in ART to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state.Study Design, Size, Duration: We conducted a prospective observational study. We used 215 morphologically normal human embryos from 137 patients that were discarded and donated for research under an approved institutional review board protocol. These embryos were imaged using metabolic imaging via FLIM to measure the autofluorescence of two central coenzymes, nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD+), which are essential for cellular respiration and glycolysis.Participants/materials, Setting, Methods: Here, we used non-invasive FLIM to measure the metabolic state of human blastocysts. We first studied spatial patterns in the metabolic state within human blastocysts and the association of the metabolic state of the whole blastocysts with stage of expansion, day of development since fertilization and morphology. We explored the sensitivity of this technique in detecting metabolic variations between blastocysts from the same patient and between patients. Next, we explored whether FLIM can quantitatively measure metabolic changes through human blastocyst expansion and hatching via time-lapse imaging. For all test conditions, the level of significance was set at P < 0.05 after correction for multiple comparisons using Benjamini-Hochberg's false discovery rate.Main Results and the Role Of Chance: We found that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We found that metabolic variations between blastocyst are partially explained by both the time since fertilization and their developmental expansion stage (P < 0.05), but not their morphological grade. Substantial metabolic variations between blastocysts from the same patients remain, even after controlling for these factors. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida (P < 0.05). And finally, we observed that the metabolic state of human blastocysts continuously varies over time.Limitations, Reasons For Caution: Although we observed significant variations in metabolic parameters, our data are taken from human blastocysts that were discarded and donated for research and we do not know their clinical outcome. Moreover, the embryos used in this study are a mixture of aneuploid, euploid and embryos of unknown ploidy.Wider Implications Of the Findings: This work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism. These results further demonstrate that FLIM can provide biologically relevant information that may be valuable for the assessment of embryo quality.Study Funding/competing Interest(s): Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Oocyte in vitro maturation (IVM) involves the achievement of the process of oocyte maturation in vitro Clinically, it was introduced several decades ago as a potentially more patient-friendly and less expensive assisted reproductive technology (ART) approach, as immature oocytes collected from mid-antral follicles can be matured in vitro without prior gonadotrophin stimulation. However, IVM oocytes are developmentally less competent compared with oocytes matured in vivo, a fact that has encouraged the use of short FSH priming and/or hCG triggering in IVM cycles. These alternatives have generated much confusion about the definition and clinical outcome of IVM, also among ART specialists, especially because hCG triggering can support maturation in vivo even in follicles of 10-13 mm in diameter. In a recent manuscript, a team of IVM specialists propose that IVM should include any ART approach involving 'the collection of oocyte from small and intermediate sized follicles' even after hCG or GnRH triggering. It is more than predictable that other scientists and clinicians with an interest in IVM will object to such a definition, believing that semantically and operatively IVM should not be associated with pharmacological interventions aimed at promoting or achieving maturation in vivo, although partially. [ABSTRACT FROM AUTHOR]

Study Question: Using time-lapse data, can the current consensus for the timing of fertilisation assessment of oocytes, cultured in standard incubation, be optimised?Summary Answer: The optimum time to perform fertilisation assessment for oocytes cultured in standard incubation is 16.5 ± 0.5 h post-insemination (hpi), and the current consensus requires modification in order to minimise the chance of fertilisation being missed.What Is Known Already: Time-lapse incubation allows the embryologist to retrospectively review collated images of oocytes and embryos to capture important embryological observations that may otherwise be missed in standard incubation. According to expert consensus, the optimum time to perform the assessment of fertilisation is 17 ± 1 hpi.Study Design, Size, Duration: A retrospective, multicentre analysis utilised data obtained from 54 746 ICSI-derived embryos and 23 602 IVF-derived embryos cultured in time-lapse incubation between January 2011 and November 2019.Participants/materials, Setting, Methods: Using time-lapse imaging (TLI), the precise time of pronuclei appearance and disappearance was recorded, where applicable, and the number of oocytes with two pronuclei observable during 10 30-min intervals from 15 hpi to 20 hpi was determined.Main Results and the Role Of Chance: Between 15 and 17.5 hpi, the average number of oocytes exhibiting normal fertilisation, elicited as two pronuclei, was 98.19% with the highest proportion of oocytes having visible pronuclei at 16-16.5 hpi (98.32%). At 18-18.5 hpi, the number of visible pronuclei reduced to 95.53% and continued to fall to 87.02% at 19.5-20 hpi.Limitations, Reasons For Caution: The authors' expectation is that these findings are transferable to other settings, however it is possible that, with alternative culture media and incubation environments, calibration of this timing may be required. As data cannot readily be recorded for pronuclear appearance for IVF-derived embryos, it is not possible to determine the optimum time to perform the fertilisation assessment for IVF-derived embryos.Wider Implications Of the Findings: By fine-tuning the time at which fertilisation assessment takes place the accuracy of the assessment can be increased to maximise the number of fertilised oocytes identified, thereby increasing the number of usable embryos for the patient. Without TLI and following current consensus guidelines, over 11% (n = 3000) of oocytes would have been marked as unfertilised within this cohort. Further to this, depending on the time of a standard fertilisation assessment, up to 300 embryos which resulted in live births could have been categorised as unfertilised, as they presented no visible pronuclei at the conventional assessment time-point.Study Funding/competing Interest(s): A.C. is a minor shareholder in CARE Fertility Limited. Validated algorithmic time-lapse embryo selection is offered to patients at CARE Fertility at an additional charge as an adjuvant treatment option.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does LH protect mouse oocytes and female fertility from alkylating chemotherapy?Summary Answer: LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan.What Is Known Already: Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment.Study Design, Size, Duration: This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment.Participants/materials, Setting, Methods: In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis.Main Results and the Role Of Chance: LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties.Large Scale Data: N/A.Limitations, Reasons For Caution: This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation.Wider Implications Of the Findings: The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients.Study Funding/competing Interest(s): This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Study Question: What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them?Summary Answer: The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes.What Is Known Already: rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM.Study Design, Size, Duration: We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid.Participants/materials, Setting, Methods: Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-μM gallic acid on their maturation rate.Main Results and the Role Of Chance: The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%.Large Scale Data: Raw data from this study can be accessed through GSE158539.Limitations, Reasons For Caution: In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator.Wider Implications Of the Findings: This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes.Study Funding/competing Interest(s): This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]

Study Question: Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro?Summary Answer: In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes.What Is Known Already: Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes.Study Design, Size, Duration: Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture.Participants/materials, Setting, Methods: Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed.Main Results and the Role Of Chance: Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure.Limitations, Reasons For Caution: Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study.Wider Implications Of the Findings: Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women.Study Funding/competing Interest(s): NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is the microRNA (miRNA) expression pattern of cumulus oophorus cells (COCs) in women undergoing medically assisted reproduction (MAR) procedures differentially modulated according to patient age and gonadotropin treatment strategy?Summary Answer: Maternal age is an independent factor impacting miRNA expression in COCs while gonadotropin treatment may affect follicular miRNA expression and IVF efficacy.What Is Known Already: Epigenetic mechanisms in female infertility are complex and poorly studied. DNA methylation, histone modifications, miRNAs and nucleosome positioning influence cellular machinery through positive and negative feedback mechanisms either alone or interactively. miRNAs are important regulators during oogenesis, spermatogenesis and early embryogenesis, and are reported to play a role in regulating crosstalk between the oocyte and COCs. Although miRNome analysis has been performed in female human reproductive tissues (endometrium, myometrium, cervix and ovaries), epigenetic modifications in women with infertility have not been explored in detail. In addition, the impact of gonadotropin treatments during MAR on miRNA expression in COCs has not been fully investigated.Study Design, Size, Duration: This study was carried out in 53 COC samples obtained from mature metaphase II (MII) oocytes in 53 women undergoing MAR treatment. A total of 38 samples for assay development were pooled by maternal age and gonadotropin treatment into four predetermined subgroups: ≥36 years and recombinant human FSH (r-hFSH), n = 10; ≥36 years and r-hFSH+ recombinant human-luteinizing hormone (r-hLH), n = 10; ≤35 years and r-hFSH, n = 9; ≤35 years and r-hFSH+r-hLH, n = 9. miRNome profiles were determined and compared between subgroups. Expression of defined miRNAs was validated in the remaining fifteen samples, representative of each subgroup, by quantitative polymerase chain reaction (PCR).Participants/materials, Setting, Methods: COCs were processed for miRNA-enriched total RNA extraction and pooled in homogeneous subgroups to obtain a sufficient amount and quality of starting material to perform the analysis. Each pooled sample underwent miRNA profiling using PCR assay system to examine expression of 752 human miRNAs without pre-amplification. Data were analyzed using the delta-delta Ct method for relative quantitation and prediction of target genes (with at least four algorithms predicting the same miRNA-gene interaction pair (HIT)>4). The miRSystem database provided functional annotation enrichment (raw P-value <0.05) of co-expressed miRNAs.Main Results and the Role Of Chance: We found distinctive miRNA expression profiles in each subgroup correlating with age and MAR stimulation. In addition, a number of selective and co-expressed miRNAs were revealed by comparative analysis. A cluster of 37 miRNAs were commonly but differentially expressed in all four pools. Significant differences were observed in expression regulation of 37 miRNAs between age groups (≤35 or ≥36) in women receiving r-hFSH+r-hLH compared to those receiving r-hFSH alone. Higher concentrations and increased numbers of miRNAs were recorded in younger than in older patients, regardless of treatment. Functional and expression studies performed to retrieve common miRNome profiles revealed an enrichment of biological functions in oocyte growth and maturation, embryo development, steroidogenesis, ovarian hyperstimulation, apoptosis and cell survival, glucagon and lipid metabolism, and cell trafficking. The highest scored pathways of target genes of the 37 common miRNAs were associated with mitogen-activated protein kinase (MAPK) signaling pathways, G alpha signaling, transcription regulation, tight junctions, RNA polymerase I and III, and mitochondrial transcription. We identified a potential age- and MAR stimulation-dependent signature in the miRNA landscape of COCs.Limitations, Reasons For Caution: We cannot rule out the possibility that other unknown individual genetic or clinical factors may have interfered with the reported results. Since miRNA profiling was conducted with a predefined array of target probes, other miRNA molecules, potentially modulated by age and hormonal stimulation, may have been missed in this study.Wider Implications Of the Findings: miRNA expression in COCs is modulated by gonadotropin treatment and correlates strongly with age. A better understanding of the expression patterns and functions of miRNAs may lead to the development of novel therapeutics to treat ovarian dysfunction and improve fertility in older women.Study Funding/competing Interest: This study was funded by Merck KGaA, Darmstadt, Germany. All authors declared no competing interest, except SL and TD who are fully employed by Merck KGaA.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Background: IVM was implemented in medically assisted reproduction 25 years ago. IVM does not involve controlled ovarian stimulation (COS) and is mainly indicated in patients with a high risk of ovarian hyperstimulation syndrome, in particular in patients with polycystic ovary syndrome (PCOS); it is also an acknowledged option in fertility protection. However, the in-vitro culture of immature oocytes raises concerns over their developmental potential and the putative impact on children's health. Although an increasing number of studies on obstetric and neonatal outcomes of IVM children and their development have been published in recent years, study designs are difficult to compare, since IVM is used in women with various indications and IVM protocols do not follow the same standards.Objective and Rationale: The aim of this systematic review was to evaluate the current evidence from IVM children of an impact of in-vitro culture of immature oocytes. Primary outcome parameters were birthweight and children's development up to the age of 2 years. We also compared pregnancy pathologies and the outcome of IVM children and COS children in relation to maternal indications, in particular PCOS, and to the type of IVM protocols with or without ovulation trigger as the secondary outcome parameters. IVM is an accepted clinical option for many centres; however, a comprehensive analysis of the available data is needed to establish whether the use of human oocytes that are fully matured in vitro is safe for both children and their mothers.Search Methods: Google Scholar and PubMed were used for identifying peer-reviewed original articles and reviews through January 2020. A total of 191 studies were screened and 16 studies were included in the qualitative synthesis. Studies were stratified according to indications, the use of an ovulation trigger and multiplicity.Outcomes: Birthweights of IVM singletons and multiples were comparable to their respective COS controls: birthweights were also similar if the analysis was restricted to mothers with PCOS. IVM children had a comparable birthweight to COS children, irrespective of whether an ovulation trigger was used in IVM cycles or not. The frequency of gestational diabetes (GD) in singleton pregnancies was comparable between IVM and COS, regardless of infertility background. There was also no difference in GD frequency between IVM and COS, if an hCG ovulation trigger in IVM cycles was used or not. Hypertensive disorders in singleton pregnancies of women with PCOS were significantly more frequent after IVM compared to COS, in particular if IVM cycles were performed only with in-vitro matured oocytes. There was no difference in the preterm birth rate of singleton pregnancies between IVM and COS. Preterm birth rates were still similar if only women diagnosed with PCOS were compared and whether an ovulation trigger in IVM was used or not. The malformation rate in IVM children did not differ in COS children versus children after natural conception. At the age of 2 years, IVM singletons showed similar anthropometric and mental development compared to COS children or children from natural conception.Wider Implications: The higher incidence of hypertensive disorders in IVM pregnancies needs monitoring during pregnancy. Current data on the development of IVM children are encouraging, although the quality of many studies is limited and long-term data beyond 2 years are scarce. Further studies should be based on generally accepted IVM protocols. Studies on long-term outcomes beyond 2 years are needed to search for potential long-time sequelae of IVM. [ABSTRACT FROM AUTHOR]

Study Question: Is there any association between the appearance of smooth endoplasmic reticulum aggregates (SERa) in oocytes and ovarian stimulation, embryological, clinical and neonatal outcomes of ICSI and IVF cycles?Summary Answer: A suboptimal prolonged ovarian stimulation is detrimental to oocytes by inducing the occurrence of SERa, which reduces the reproductive potential of oocytes.What Is Known Already: Controlled ovarian stimulation recruits oocytes of different qualities. Based on current evidence, it was agreed that non-homogeneous cytoplasm may represent the normal variability among oocytes rather than a dysmorphism with developmental significance. The only exception is the appearance of SERa within the ooplasm. Owing to the lack of univocal evidence in this literature about the safety of injecting oocytes with SERa and the mechanism responsible for the occurrence of SERa, this topic is still a matter of debate.Study Design, Size, Duration: A retrospective, longitudinal cohort study performed at a tertiary level public infertility center. We included 1662 cycles (180 SERa+ and 1482 SERa-) from 1129 women (age: 20-44 years) who underwent IVF/ICSI treatments in 2012-2019. The SERa+ cycles had at least one SERa+ oocyte in the oocyte cohort. The SERa- cycles had morphologically unaffected oocytes.Participants/materials, Setting, Methods: We collected stimulation data and embryological, clinical, neonatal outcomes of SERa- and SERa+ cycles and oocytes.Main Results and the Role Of Chance: Overall, 347 out of 12 436 metaphase II oocytes (2.8%) were affected by SER. We performed only 12 transfers involving at least one SERa+ embryo. Stimulation length (P = 0.002), serum progesterone (P = 0.004) and follicle size (P = 0.046) at trigger, number of retrieved (P = 0.004) and metaphase II (P = 0.0001) oocytes were significantly higher in SERa+ than SERa- cycles. Fertilization rate was significantly (P < 0.0001) reduced in SERa+ cycles and oocytes compared to SERa- counterparts. Embryos of SERa+ cycles had a lower blastocyst formation rate compared to embryos of SERa- cycles (P = 0.059). Statistical analysis according to a generalized estimating equation model performed at patient level demonstrated that the duration of ovarian stimulation was predictive of SERa+ oocytes appearance. The clinical success of SERa+ cycles was lower than SERa- cycles, although no differences in neonatal birthweights or malformations were recorded in sibling unaffected oocytes of SERa+ cycles.Limitations, Reasons For Caution: Given that SERa+ oocytes were discarded in our center for years and transfers of embryos originating from affected oocytes were generally avoided, clinical outcomes of SERa+ cycles are largely attributable to the transfer of embryos derived from unaffected oocytes of SERa+ cycles and we did not have data about newborns from affected oocytes, since none of the transfers involving SERa+ embryos resulted in a progressive clinical pregnancy.Wider Implications Of the Findings: For the first time, we speculate that the late-follicular phase elevated serum progesterone caused by a suboptimal prolonged ovarian stimulation may be detrimental to the oocytes by inducing the occurrence of SERa, resulting in negative effects on their reproductive potential. This raises the question of whether some stimulation regimens could be worse than others and a change in stimulation protocol would reduce the possibility of producing oocytes with suboptimal maturation. In particular, our data highlight the importance of correct timing of the trigger in order to maximize oocyte collection, not only in terms of numerosity but also their reproductive potential.Study Funding/competing Interest(s): None.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Background: Mild ovarian stimulation has emerged as an alternative to conventional IVF with the advantages of being more patient-friendly and less expensive. Inadequate data on pregnancy outcomes and concerns about the cycle cancellation rate (CCR) have prevented mild, or low-dose, IVF from gaining wide acceptance.Objective and Rationale: To evaluate parallel-group randomised controlled trials (RCTs) on IVF where comparisons were made between a mild (≤150 IU daily dose) and conventional stimulation in terms of clinical outcomes and cost-effectiveness in patients described as poor, normal and non-polycystic ovary syndrome (PCOS) hyper-responders to IVF.Search Methods: Searches with no language restrictions were performed using Medline, Embase, Cochrane central, Pre-Medicine from January 1990 until April 2020, using pre-specified search terms. References of included studies were hand-searched as well as advance access articles to key journals. Only parallel-group RCTs that used ≤150 IU daily dose of gonadotrophin as mild-dose IVF (MD-IVF) and compared with a higher conventional dose (CD-IVF) were included. Studies were grouped under poor, normal or hyper-responders as described by the authors in their inclusion criteria. Women with PCOS were excluded in the hyper-responder group. The risk of bias was assessed as per Cochrane Handbook for the included studies. The quality of evidence (QoE) was assessed according to the GRADE system. PRISMA guidance was followed for review methodology.Outcomes: A total of 31 RCTs were included in the analysis: 15 in the poor, 14 in the normal and 2 in the hyper-responder group. Live birth rates (LBRs) per randomisation were similar following use of MD-IVF in poor (relative risk (RR) 0.91 (CI 0.68, 1.22)), normal (RR 0.88 (CI 0.69, 1.12)) and hyper-responders (RR 0.98 (CI 0.79, 1.22)) when compared to CD-IVF. QoE was moderate. Cumulative LBRs (5 RCTs, n = 2037) also were similar in all three patient types (RR 0.96 (CI 0.86 1.07) (moderate QoE). Risk of ovarian hyperstimulation syndrome was significantly less with MD-IVF than CD-IVF in both normal (RR 0.22 (CI 0.10, 0.50)) and hyper-responders (RR 0.47 (CI 0.31, 0.72)), with moderate QoE. The CCRs were comparable in poor (RR 1.33 (CI 0.96, 1.85)) and hyper-responders (RR 1.31 (CI 0.98, 1.77)) but increased with MD-IVF among normal responders (RR 2.08 (CI 1.38, 3.14)); all low to very low QoE. Although fewer oocytes were retrieved and fewer embryos created with MD-IVF, the proportion of high-grade embryos was similar in all three population types (low QoE). Compared to CD-IVF, MD-IVF was associated with less gonadotrophin use and lower cost.Wider Implications: This updated review provides reassurance on using MD-IVF not only for the LBR per cycle but also for the cumulative LBR, with moderate QoE. With risks identified with 'freeze-all' strategies, it may be time to recommend mild-dose ovarian stimulation for IVF for all categories of women i.e. hyper, poor and normal responders to IVF. [ABSTRACT FROM AUTHOR]

Ovarian tissue cryopreservation and banking provides a fertility preservation option for patients who cannot undergo oocyte retrieval; it is quickly becoming a critical component of assisted reproductive technology programs across the world. While the transplantation of cryopreserved ovarian tissue has resulted in over 130 live births, the field has ample room for technological improvements. Specifically, the functional timeline of grafted tissue and each patient's probability of achieving pregnancy is largely unpredictable due to patient-to-patient variability in ovarian reserve, lack of a reliable method for quantifying follicle numbers within tissue fragments, potential risk of reintroduction of cancer cells harbored in ovarian tissues, and an inability to control follicle activation rates. This review focuses on one of the most common physiological techniques used to study human ovarian tissue transplantation, xenotransplantation of human ovarian tissue to mice and endeavors to inform future studies by discussing the elements of the xenotransplantation model, challenges unique to the use of human ovarian tissue, and novel tissue engineering techniques currently under investigation. [ABSTRACT FROM AUTHOR]

Study Question: Do uterine arteries Doppler studies show different pulsatility index (UtA-PI) measurements in IVF/ICSI pregnancies with oocyte donation (OD) as compared to natural conceptions?Summary Answer: In IVF/ICSI pregnancies with OD, UtA-PI is reduced by an average of about 40% as compared to pregnancies with natural conception.What Is Known Already: OD pregnancies present worse pregnancy outcomes as compared to natural conception, particularly for increased incidence of pre-eclampsia (PE). Recent evidence shows that IVF/ICSI pregnancies with frozen blastocyst transfer also present higher prevalence of PE and 15% lower UtA-PI as compared to pregnancies after fresh blastocyst transfers.Study Design, Size, Duration: Prospective, longitudinal matched cohort study performed in the Fetal Medicine and Obstetric Departments of San Raffaele Hospital in Milan, between 2013 and 2018. The analysis is based on 584 Doppler observations collected from 296 women with different method of conception (OD n = 122; natural conception n = 174).Participants/materials, Setting, Methods: IVF/ICSI viable singleton pregnancies with OD and natural conception control pregnancies matched for BMI and smoking, performing repeated UtA-PI measurements at 11-34 weeks. Miscarriages, abnormalities, twins, significant maternal diseases and other types of ARTs were excluded. Log mean left-right UtA-PI was used for analysis with linear mixed model (LMM) and correction for significant confounders. Pregnancy outcome was also analyzed.Main Results and the Role Of Chance: Participants after OD were older and more frequently nulliparous (mean age: OD 43.4, 95% CI from 42.3 to 44.6; natural conception 35.1, 95% CI from 34.5 to 35.7; P-value < 0.001; nulliparous: OD 96.6%; natural conception 56.2%; P-value < 0.001). Mean pulsatility index was lower in OD (UtA-PI: natural conception 1.22; 95% CI from 1.11 to 1.28; OD 1.04; 95% CI from 0.96 to 1.12; P-value < 0.001). A significant effect of parity, gestational age (GA) modeled with a cubic polynomial and BMI was described in the LMM. The mean Log UtA-PI was on average 37% lower in OD as compared to natural conception pregnancies at LMM (P-value < 0.001). We also found a significant interaction between longitudinal UtA-PI Doppler and GA. Therefore, at 11 weeks' gestation the Log UtA-PI was 42% lower and, at 34 weeks, the differences reduced to 32%. GA at delivery and birth weight were statistically lower in OD group; however, birthweight centile was not statistically different. Preeclampsia was 11-fold more common in the OD group (0.6% and 6.6%, P-value = 0.003). No other significant difference in pregnancy outcome was shown in the study groups (gestational diabetes mellitus, small or large for GA).Limitations, Reasons For Caution: It was not possible to properly match for maternal age and to blind the assessment given the major differences between cohorts; however, we did not find significant within-groups effects related to maternal age. Future research is needed to reassess outcomes and correct them for maternal characteristics (e.g. cardiovascular function).Wider Implications Of the Findings: This finding reproduces our previous discovery of lower UtA-PI in frozen as compared to fresh blastocyst transfer. The vast majority of OD is obtained by the use of cryopreservation. We speculate that increased uterine perfusion may be the physiological response to compensate dysfunctions both in the mother and in the placenta.Study Funding/competing Interest(s): This is a non-funded study. The authors do not declare competing interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Does the time elapsed between oocyte pick-up (OPU) and denudation or injection affect the probability of achieving a live birth (LB) in ICSI cycles?Summary Answer: Prolonged oocyte culture before denudation (>4 h) was associated with an increase in clinical pregnancy (CP), LB and cumulative LB (CLB) rates when compared with earlier denudation timings.What Is Known Already: Oocyte maturation is a complex and dynamic process involving structural and biochemical modifications in the cell necessary to support fertilization and early embryo development. While meiotic competence is easily identifiable by the presence of an extruded first polar body, cytoplasmic maturation cannot be assessed microscopically. Culturing oocytes with their surrounding cumulus cells (CCs) prior to ICSI can enhance the completion of in vitro cytoplasmic maturation; conversely, prolonged culture may induce cell degeneration. The optimal culture intervals prior to oocyte denudation and/or injection have not yet been established and may prove relevant for the improvement of ICSI reproductive outcomes.Study Design, Size, Duration: This is a single-centre retrospective cohort analysis of 1378 ICSI cycles performed between January 2005 and October 2018. Data were categorized according to: (i) the time interval between OPU and denudation (<3 h, 3-4 h and ≥4 h), (ii) the time interval between denudation and ICSI (<1.5 h, 1.5-2 h, ≥2 h) and (iii) the time interval between OPU and ICSI (<5 h, 5-6 h and ≥6 h). The effect of these timings on fertilization, CP, LB and CLB rates were compared. The culture intervals between different procedures were dependent exclusively on laboratory workload.Participants/materials, Setting, Methods: ICSI cycles performed in women younger than 40 years old using autologous gametes with at least one metaphase II injected oocyte were included. The effect of oocyte culture duration prior to denudation and injection of the oocytes was compared using multivariable regression accounting for potential confounding variables.Main Results and the Role Of Chance: Fertilization and oocyte damage rate after ICSI was found to be independent of the time interval to denudation (<3 h, 3-4 h and ≥4 h) and/or injection (<5 h, 5-6 h and ≥6 h). Extending oocyte culture before denudation significantly improved CP (29.5%, 42.7% and 50.6%, respectively), LB (25.1%, 34.4% and 40.7%, respectively) and CLB rates (26.0%, 36.1% and 42.2%, respectively), particularly if the time interval was at least 4 h. Additionally, LB (31.7%, 35.8% and 27.4%, respectively) and CLB rates (34.2%, 36.6% and 27.7%, respectively) were also dependent on the time from OPU to injection.Limitations, Reasons For Caution: This study is limited by its retrospective nature and potential unmeasured confounding cannot be excluded. Furthermore, the effect of even shorter or longer periods of culture before denudation and/or injection were not evaluated and should not be extrapolated from these results.Wider Implications Of the Findings: Our findings propose new evidence of a previously unrecognized protective effect of the CCs-oocyte interactions in human ART, raising the question of a possible downstream effect in embryogenesis which significantly affects LB rates. Additionally, this is the first study to suggest a negative effect of further extending culture before ICSI on LB and CLB rates, thus potentially allowing for the narrowing of an optimal ICSI time interval. Simple strategies such as the establishment of more effective time frames to perform these procedures and adjusting laboratory practice may prove beneficial, ultimately improving ICSI reproductive outcomes.Study Funding/competing Interest(s): None.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Is the mitochondrial DNA (mtDNA) copy number of cumulus granulosa cells (CGCs) related to the maturation of oocyte cytoplasm?Summary Answer: Compared with the mtDNA copy number of CGCs from germinal vesicles (GV), CGCs from Metaphase I (MI) oocytes appear to have a lower mtDNA copy number.What Is Known Already: The growth and development of CGCs and oocyte are synchronised. The interaction between CGCs and the oocyte provides the appropriate balance of energy, which is necessary for mammalian oocyte development. Moreover, in the oocyte-cumulus complex (OCC), mature oocytes with higher mtDNA copy numbers tend to have corresponding CGCs with higher mtDNA copy numbers.Study Design, Size, Duration: This is a prospective study of 302 OCCs obtained from 70 women undergoing in vitro fertilisation with intracytoplasmic sperm injection (ICSI) at the Reproductive and Genetic Hospital of CITIC-Xiangya, between 24 February 2018 and 21 December 2019. The CGCs were divided into three groups (GV, MI and MII stages) based on the maturation status of their corresponding oocyte. The sample sizes (n = 302) of CGCs in the three stages were 63 (CGCGV), 70 (CGCMI) and 169 (CGCMII), respectively. Some of the samples (n = 257) was used to quantify the mtDNA copy number, while the rest (n = 45) were used to analyse the expression level of mitochondrial genes. Furthermore, we retrieved 82 immature oocytes from among the 257 OCCs used for mtDNA copy numbers, including 36 GV oocytes and 46 MI oocytes, for analysis of oocyte mtDNA.Participants/materials, Setting, Methods: We selected genes with high consistency of real-time PCR results to accurately measure the mtDNA copy number by testing the efficacy and the reproducibility in whole genome amplification (WGA) samples from a human embryonic stem cell line. The CGCs of each oocyte were individually isolated. The mtDNA copy number and gene expression of the CGCs were assessed using real-time PCR techniques. Mitochondrial DNA copy number of the corresponding immature oocytes was also evaluated.Main Results and the Role Of Chance: MT-ND1, MT-CO1 and β-globin genes were chosen for the assessment of mtDNA content, and mRNA expressions of MT-ND1, MT-CO1, PGC-1α and TFAM were also measured. The genome of 257 CGCs and 82 immature oocytes were amplified according to the multiple displacement amplification (MDA) protocol, and RNA was extracted from 45 CGCs. Compared with CGCGV, CGCMI had a significantly lower mtDNA copy number. In the MT-ND1 assay, the CGCGV: CGCMI was [270 ± 302]: [134 ± 201], P = 0.015. In the MT-CO1 assay, CGCGV: CGCMI was [205 ± 228]: [92 ± 112], P = 0.026. There was no statistical difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [270 ± 302]: [175 ± 223], P = 0.074. In the MT-CO1 assay, CGCGV: CGCMII was [205 ± 228]: [119 ± 192], P = 0.077. No statistical difference of mtDNA copy number was observed between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [134 ± 201]: [175 ± 223], P = 0.422. In the MT-CO1 assay, CGCMI: CGCMII was [92 ± 112]: [119 ± 192], P = 0.478. To verify the reliability of the above results, we further analysed the mtDNA copy number of CGCs of 14 patients with GV, MI and MII oocytes, and the results showed that the mtDNA copy number of CGCMI may be lower. The mtDNA copy number of CGCGV and CGCMI was statistically different in the MT-ND1 assay where CGCGV: CGCMI was [249 ± 173]: [118 ± 113], P = 0.016, but in the MT-CO1 assay, CGCGV: CGCMI was [208 ± 199]: [83 ± 98], P = 0.109. There was no significant difference in mtDNA between CGCGV and CGCMII. In the MT-ND1 assay, CGCGV: CGCMII was [249 ± 173]: [185 ± 200], P = 0.096. In the MT-CO1 assay, CGCGV: CGCMII was [208 ± 199]: [114 ± 139], P = 0.096. There was also no significant difference in mtDNA between CGCMI and CGCMII. In the MT-ND1 assay, CGCMI: CGCMII was [118 ± 113]: [185 ± 200], P = 0.198. In the MT-CO1 assay, CGCMI: CGCMII was [83 ± 98]: [114 ± 139], P = 0.470. Moreover, there were no statistical differences in the expression levels of MT-ND1, MT-CO1, PGC-1α and TFAM between CGCGV, CGCMI and CGCMII (P > 0.05).Large Scale Data: N/A.Limitations, Reasons For Caution: Due to the ethical issues, the study did not quantify the mtDNA content of MII oocytes. Thus, whether the change in mtDNA copy number in CGCs is related to the different developmental stages of oocytes has not been further confirmed. Moreover, the sample size was relatively small.Wider Implications Of the Findings: The mtDNA copy number of CGCs decreases from the GV phase to the MI phase and stays steady from the MI to MII stage. At different stages of oocyte maturation, the mtDNA of CGCs may undergo self-degradation and replication to meet the energy requirements of the corresponding oocyte and the maturation of the oocyte cytoplasm.Study Funding/competing Interest(s): Funding was provided by the National Key R&D Program of China (Grant 2018YFC1003100, to L.H.), the science and technology major project of the Ministry of Science and Technology of Hunan Province, China (grant 2017SK1030, to G.L.), the National Natural Science Foundation of China (grant 81873478, to L.H.), and Merck Serono China Research Fund for Fertility Experts (to L.H.). There is no conflict of interest. [ABSTRACT FROM AUTHOR]

Study Question: Which is the most suitable clinical strategy in egg donation IVF cycles conducted with imported donated vitrified oocytes?Summary Answer: The importation, and allocation, of at least eight vitrified eggs per couple during an egg donation cycle is associated with a high cumulative live birth delivery rate per cycle, as well as the confident adoption of a single blastocyst transfer strategy to minimize the risk of multiple pregnancies.What Is Known Already: IVF using donor eggs is commonly used worldwide to treat women who are unable to conceive with their own oocytes. In 2014, the Constitutional Court (n.162/2014) gave permission for gamete donation to be allowed for ART in Italy. Initially recommended as a therapeutic approach for premature ovarian insufficiency, the use of donated oocytes has become more and more common. In countries such as Italy, fresh oocyte donation is theoretically possible, but practically impossible due to the lack of donors. In fact, the Italian law does not allow reimbursement to the young women, who can only voluntarily donate their eggs. Therefore, Italian IVF centers have established several collaborations with international oocyte cryo-banks. The most popular workflow involves the importation of donated oocytes that have been vitrified. However, recent evidence has questioned the overall efficacy of such an approach. This is because detrimental effects arising from oocyte vitrification and warming might reduce the number of eggs available for insemination, with a consequential reduction in the achievable live birth rate per cycle.Study Design, Size, Duration: This was a longitudinal cohort study, conducted between October 2015 and December 2018 at two private IVF centers. Overall, 273 couples were treated (mean maternal age: 42.5 ± 3.5 years, range: 31-50 years; mean donor age: 25.7 ± 4.2, 20-35 years) with oocytes purchased from three different Spanish egg banks.Participants/materials, Setting, Methods: We performed an overall analysis, as well as several sub-analyses clustering the data according to the year of treatment (2015-2016, 2017 or 2018), the number of warmed (6, 7, 8 or 9) and surviving oocytes (≤4, 5, 6, 7, 8 or 9) and the cycle strategy adopted (cleavage stage embryo transfer and vitrification, cleavage stage embryo transfer and blastocyst vitrification, blastocyst stage embryo transfer and vitrification). This study aimed to create a workflow to maximize IVF efficacy, efficiency, and safety, during egg donation cycles with imported vitrified oocytes. The primary outcome was the cumulative live birth delivery rate among completed cycles (i.e. cycles where at least a delivery of a live birth was achieved, or no embryo was produced/left to transfer). All cycles, along with their embryological, obstetric and neonatal outcomes, were registered and inspected.Main Results and the Role Of Chance: The survival rate after warming was 86 ± 16%. When 6, 7, 8 and 9 oocytes were warmed, 94, 100, 72 and 70% of cycles were completed, resulting in 35, 44, 69 and 59% cumulative live birth delivery rates per completed cycle, respectively. When ≤4, 5, 6, 7, 8 and 9 oocytes survived, 98, 94, 85, 84, 66 and 68% of cycles were completed, resulting in 16, 46, 50, 61, 76 and 60% cumulative live birth delivery rates per completed cycle, respectively. When correcting for donor age, and oocyte bank, in a multivariate logistic regression analysis, warming eight to nine oocytes resulted in an odds ratio (OR) of 2.5 (95% CI: 1.07-6.03, P = 0.03) for the cumulative live birth delivery rate per completed cycle with respect to six to seven oocytes. Similarly, when seven to nine oocytes survived warming, the OR was 2.7 (95% CI: 1.28-5.71, P < 0.01) with respect to ≤6 oocytes. When cleavage stage embryos were transferred, a single embryo transfer strategy was adopted in 17% of cases (N = 28/162); the live birth delivery rate per transfer was 26% (n = 43/162), but among the pregnancies to term, 28% involved twins (n = 12/43). Conversely, when blastocysts were transferred, a single embryo transfer strategy was adopted in 96% of cases (n = 224/234) with a 30% live birth delivery rate per transfer (N = 70/234), and the pregnancies to term were all singleton (n = 70/70). During the study period, 125 babies were born from 113 patients. When comparing the obstetric outcomes for the cleavage and blastocyst stage transfer strategies, the only significant difference was the prevalence of low birthweight: 34 versus 5%, respectively (P < 0.01). However, several significant differences were identified when comparing singleton with twin pregnancies; in fact, the latter resulted in a generally lower birthweight (mean ± SD: 3048 ± 566 g versus 2271 ± 247 g, P < 0.01), a significantly shorter gestation (38 ± 2 versus 36 ± 2 weeks, P < 0.01), solely Caesarean sections (72 versus 100%, P = 0.02), a higher prevalence of low birthweight (8 versus 86%, P < 0.01), small newborns for gestational age (24 versus 57%, P = 0.02) and preterm births (25 versus 86%, P < 0.01).Limitations, Reasons For Caution: This retrospective study should now be confirmed across several IVF centers and with a greater sample size in order to improve the accuracy of the sub-analyses.Wider Implications Of the Findings: Single blastocyst transfer is the most suitable approach to achieve high success rates per procedure, thereby also limiting the obstetric complications that arise from twin pregnancies in oocyte donation programs. In this regard, the larger the cohort of imported donated vitrified oocytes, the more efficient the management of each cycle.Study Funding/competing Interest(s): None.Trial Registration Number: None. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What recommendations can be provided on the approach to and use of time-lapse technology (TLT) in an IVF laboratory? SUMMARY ANSWER The present ESHRE document provides 11 recommendations on how to introduce TLT in the IVF laboratory. WHAT IS KNOWN ALREADY Studies have been published on the use of TLT in clinical embryology. However, a systematic assessment of how to approach and introduce this technology is currently missing. STUDY DESIGN, SIZE, DURATION A working group of members of the Steering Committee of the ESHRE Special Interest Group in Embryology and selected ESHRE members was formed in order to write recommendations on the practical aspects of TLT for the IVF laboratory. PARTICIPANTS/MATERIALS, SETTING, METHODS The working group included 11 members of different nationalities with internationally recognized experience in clinical embryology and basic science embryology, in addition to TLT. This document is developed according to the manual for development of ESHRE recommendations for good practice. Where possible, the statements are supported by studies retrieved from a PUBMED literature search on 'time-lapse' and ART. MAIN RESULTS AND THE ROLE OF CHANCE A clear clinical benefit of the use of TLT, i.e. an increase in IVF success rates, remains to be proven. Meanwhile, TLT systems are being introduced in IVF laboratories. The working group listed 11 recommendations on what to do before introducing TLT in the lab. These statements include an assessment of the pros and cons of acquiring a TLT system, selection of relevant morphokinetic parameters, selection of an appropriate TLT system with technical and customer support, development of an internal checklist and education of staff. All these aspects are explained further here, based on the current literature and expert opinion. LIMITATIONS, REASONS FOR CAUTION Owing to the limited evidence available, recommendations are mostly based on clinical and technical expertise. The paper provides technical advice, but leaves any decision on whether or not to use TLT to the individual centres. WIDER IMPLICATIONS OF THE FINDINGS This document is expected to have a significant impact on future developments of clinical embryology, considering the increasing role and impact of TLT. STUDY FUNDING/COMPETING INTEREST(S) The meetings of the working group were funded by ESHRE. S.A. declares participation in the Nordic Embryology Academic Team with meetings sponsored by Gedeon Richter. T.E. declares to have organized workshops for Esco and receiving consulting fees from Ferring and Gynemed and speakers' fees from Esco and honorarium from Merck and MSD. T.F. received consulting fees from Vitrolife and Laboratoires Genévrier, speakers' fees from Merck Serono, Gedeon Richter, MSD and Ferring and research grants from Gedeon Richter and MSD. M.M. received sponsorship from Merck. M.M.E. received speakers' fees from Merck, Ferring and MSD. R.S. received a research grant from ESHRE. G.C. received speakers' fees from IBSA and Excemed. The other authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER N/A. DISCLAIMER This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation. ESHRE's GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care nor exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgment to each individual presentation, nor variations based on locality and facility type. Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring of any of the included technologies by ESHRE. †ESHRE Pages content is not externally peer reviewed. The manuscript has been approved by the Executive Committee of ESHRE. [ABSTRACT FROM AUTHOR]

Background: Despite great advances in assisted reproductive technology, poor ovarian response (POR) is still considered as one of the most challenging tasks in reproductive medicine.Objective and Rationale: The aim of this systemic review is to evaluate the role of different adjuvant treatment strategies on the probability of pregnancy achievement in poor responders undergoing IVF. Randomized controlled trials (RCTs) comparing 10 adjuvant treatments [testosterone, dehydroepiandrosterone (DHEA), letrozole, recombinant LH, recombinant hCG, oestradiol, clomiphene citrate, progesterone, growth hormone (GH) and coenzyme Q10 (CoQ10)] were included.Search Methods: Relevant studies published in the English language were comprehensively selected using PubMed, Embase and the Cochrane Central Register of Controlled Trials (CENTRAL) until 11 July 2018. We included studies that investigated various adjuvant agents, including androgen and androgen-modulating agents, oestrogen, progesterone, clomiphene citrate, GH and CoQ10, during IVF treatment and reported subsequent pregnancy outcomes. The administration of GnRH analogs and gonadotrophins without adjuvant treatment was set as the control. We measured study quality based on the methodology and categories listed in the Cochrane Collaboration Handbook. This review protocol was registered with PROSPERO (CRD42018086217).Outcomes: Of the 1124 studies initially identified, 46 trials reporting on 6312 women were included in this systematic review, while 19 trials defining POR using the Bologna criteria reporting 2677 women were included in the network meta-analysis. Compared with controls, DHEA and CoQ10 treatments resulted in a significantly higher chance of clinical pregnancy [odds ratio (OR) 2.46, 95% CI 1.16 to 5.23; 2.22, 1.08-4.58, respectively]. With regard to the number of retrieved oocytes, HCG, oestradiol and GH treatments had the highest number of oocytes retrieved [weighted mean difference (WMD) 2.08, 0.72 to 3.44; 2.02, 0.23 to 3.81; 1.72, 0.98 to 2.46, compared with controls, respectively]. With regard to the number of embryos transferred, testosterone and GH treatment led to the highest number of embryos transferred (WMD 0.72, 0.11 to 1.33; 0.67, 0.43 to 0.92; compared with controls, respectively). Moreover, GH resulted in the highest oestradiol level on the HCG day (WMD 797.63, 466.45 to 1128.81, compared with controls). Clomiphene citrate, letrozole and GH groups used the lowest dosages of gonadotrophins for ovarian stimulation (WMD 1760.00, -2890.55 to -629.45; -1110.17, -1753.37 to -466.96; -875.91, -1433.29 to -282.52; compared with controls, respectively). CoQ10 led to the lowest global cancelation rate (OR 0.33, 0.15 to 0.74, compared with controls).Wider Implications: For patients with POR, controlled ovarian stimulation protocols using adjuvant treatment with DHEA, CoQ10 and GH showed better clinical outcomes in terms of achieving pregnancy, and a lower dosage of gonadotrophin required for ovulation induction. Furthermore, high-level RCT studies using uniform standards for POR need to be incorporated into future meta-analyses. [ABSTRACT FROM AUTHOR]

Study Question: Do the ICSI-related procedural timings and operators affect the outcomes of an ART cycle?Summary Answer: The ICSI-related timings and operators do not associate with the mean blastulation rate per cohort of inseminated oocytes and the cumulative delivery rate per concluded cycle, except for a mild association between the times from induction of ovulation to oocyte denudation and the former outcome.What Is Known Already: In ART, specific timings, protocols and conditions must be complied with to preserve gamete developmental and reproductive competence during the required manipulations. ICSI represents a groundbreaking advancement that has been widely implemented. Nevertheless, the studies that examined the putative impact of ICSI-related procedural timings were mainly conducted in old-fashioned settings or in good prognosis patients. No report addressed issues like operators' skills and experience and uncertainties exist dealing with the effect of cumulus cells in the pre-incubation period in vitro before ICSI. However, all this information is crucial to efficiently plan the daily routine of an IVF lab, fill the existing gaps of knowledge and define proper key performance indicators.Study Design, Size, Duration: Observational study conducted at a private IVF clinic (January 2016 to January 2018). We included all consecutive ICSI procedures (n = 1084 infertile couples undergoing 1444 cycles with or without preimplantation genetic testing (PGT); mean ± SD maternal age: 38.1 ± 4.0 years) with fresh autologous oocytes (n = 7999 oocytes, 5.5 ± 3.2 per treatment) inseminated with fresh non-donor ejaculated sperm. All operators and critical procedural timings (induction of ovulation to oocyte denudation, denudation and ICSI) were automatically recorded through an electronic witnessing system. The primary outcome measure was the cumulative delivery rate among both non-PGT and PGT-concluded cycles (i.e. delivery achieved or no supernumerary cryopreserved blastocyst available). The secondary outcome measure was the mean blastulation rate per cohort of inseminated oocytes. All confounders were registered and included in generalized linear models and multivariate logistic regression analyses.Participants/materials, Setting, Methods: Fourteen and 12 operators were involved in denudation and ICSI procedures, respectively. Denudation was performed after 4.1 ± 1.2 h (2-7) of pre-incubation in vitro after oocyte retrieval, and ICSI was started immediately after. Beyond procedural timings and operators, all the putative confounders (patients' and cycles' characteristics) on the primary and/or secondary outcomes were systematically registered and included in the statistical analyses.Main Results and the Role Of Chance: The mean time from induction of ovulation to oocyte denudation was 39.3 ± 1.3 h. The mean procedural timings for denudation and ICSI were 8.1 ± 3.8 and 12.6 ± 6.4 min; both these variables were significantly dependent on the number of inseminated oocytes and the operators' skills and experience. The overall mean blastulation rate per cohort of inseminated oocytes was 34.0 ± 27.9%. This outcome was significantly associated with the time from induction of ovulation to oocyte denudation (mean blastulation rate stable in the time interval 38-42 h, but significantly higher for timings <38 h), maternal age (the mean blastulation rate drops especially beyond the age of 40 years) and categorized sperm concentration (highest mean blastulation rate for sperm concentrations ≥15 mil/ml and lowest for cryptozoospermic patients) through a generalized linear model that showed an adjusted r2 = 0.053 (P < 0.01). No association was found for denudation and ICSI timings and operators. Lastly, when adjusted for maternal age and number of inseminated oocytes, both ICSI-related procedural timings and operators did not associate with the cumulative delivery rate among both non-PGT- or PGT-concluded cycles.Limitations, Reasons For Caution: This is a single private IVF center study. Its reproducibility should be assessed in different laboratory conditions, with different protocols and in the hands of different operators. Moreover, specific studies are warranted to address the beneficial/detrimental effect of the other putative confounders under investigation (e.g. kind of ovulation trigger, culture media, incubator, etc.).Wider Implications Of the Findings: Proactive communication between the embryologists and the clinicians might contribute to a reasoned and more efficient organization of the daily workload and increase the mean blastulation rate, especially when poor prognosis couples (advanced maternal age, reduced sperm count and/or ovarian reserve) are treated.Study Funding/competing Interest(s): No funding. The authors declare no conflict of interest related to the present study. [ABSTRACT FROM AUTHOR]

Study Question: Does imprinted DNA methylation or imprinted gene expression differ between human blastocysts from conventional ovarian stimulation (COS) and an optimized two-step IVM method (CAPA-IVM) in age-matched polycystic ovary syndrome (PCOS) patients?Summary Answer: No significant differences in imprinted DNA methylation and gene expression were detected between COS and CAPA-IVM blastocysts.What Is Known Already: Animal models have revealed alterations in DNA methylation maintenance at imprinted germline differentially methylated regions (gDMRs) after use of ARTs. This effect increases as more ART interventions are applied to oocytes or embryos. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for patients. CAPA-IVM is an improved IVM system that includes a pre-maturation step (CAPA), followed by an IVM step, both in the presence of physiological compounds that promote oocyte developmental capacity.Study Design, Size, Duration: For DNA methylation analysis 20 CAPA-IVM blastocysts were compared to 12 COS blastocysts. For RNA-Seq analysis a separate set of 15 CAPA-IVM blastocysts were compared to 5 COS blastocysts.Participants/materials, Setting, Methods: COS embryos originated from 12 patients with PCOS (according to Rotterdam criteria) who underwent conventional ovarian stimulation. For CAPA-IVM 23 women were treated for 3-5 days with highly purified hMG (HP-hMG) and no hCG trigger was given before oocyte retrieval. Oocytes were first cultured in pre-maturation medium (CAPA for 24 h containing C-type natriuretic peptide), followed by an IVM step (30 h) in medium containing FSH and Amphiregulin. After ICSI, Day 5 or 6 embryos in both groups were vitrified and used for post-bisulphite adaptor tagging (PBAT) DNA methylation analysis or RNA-seq gene expression analysis of individual embryos. Data from specific genes and gDMRs were extracted from the PABT and RNA-seq datasets.Main Results and the Role Of Chance: CAPA-IVM blastocysts showed similar rates of methylation and gene expression at gDMRs compared to COS embryos. In addition, expression of major epigenetic regulators was similar between the groups.Limitations, Reasons For Caution: The embryos from the COS group were generated in a range of culture media. The CAPA-IVM embryos were all generated using the same sperm donor. The DNA methylation level of gDMRs in purely in vivo-derived human blastocysts is not known.Wider Implications Of the Findings: A follow-up of children born after CAPA-IVM is important as it is for other new ARTs, which are generally introduced into clinical practice without prior epigenetic safety studies on human blastocysts. CAPA-IVM opens new perspectives for patient-friendly ART in PCOS.Study Funding/competing Interest(s): IVM research at the Vrije Universiteit Brussel has been supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680), the Fund for Research Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen-FWO-AL 679 project, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69Ref Nr 2016-119) and the Vrije Universiteit Brussel (IOF Project 4R-ART Nr 2042). Work in G.K.'s laboratory is supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. The authors have no conflicts of interest. [ABSTRACT FROM AUTHOR]

Study Question: Can time-lapse imaging systems make it possible to identify novel early non-invasive biomarkers to predict live birth?Summary Answer: From mostly high-grade embryos, out of 35 morphometric, morphologic and morphokinetic variables, only pronuclei (PN) position at time of PN juxtaposition and the absence of multinucleated blastomeres at the 2-cell stage (MNB2cell), were potentially associated with live birth.What Is Known Already: Previous studies indicate that some kinetic markers may be predictive of blastocyst development and embryonic implantation. Certain teams have suggested including some of them in decisional algorithms for embryo transfers.Study Design, Size, Duration: Using a time-lapse incubator (EmbryoScope, Unisense FertiliTech), we retrospectively explored the associations between the morphometric, morphologic and morphokinetic parameters of oocytes, zygotes and embryos, and their associations with live birth. This study assessed 232 embryos from single embryo transfers after ICSI cycles performed between January 2014 and December 2017.Participants/materials, Setting, Methods: The morphometric, morphologic and morphokinetic parameters (18, 4 and 13, respectively) of oocytes, zygotes and early embryos were studied retrospectively. The associations between these parameters were examined using a Spearman's correlation, Mann-Whitney or chi-squared test as appropriate. We examined whether these parameters were associated with outcomes in univariate and multivariate logistic regression analyses.Main Results and the Role Of Chance: Central PN juxtaposition was associated with a 2-fold increase in the odds of live birth (OR = 2.20; 95% CI, [1.26-3.89]; P = 0.006), while the presence of MNB2cell was associated with half the odds of live birth (OR = 0.51; 95% CI, [0.27-0.95]; P = 0.035). These two parameters were independent of embryo kinetics. The 33 remaining parameters had no significant association with the capacity of transferred embryos to develop to term.Limitations, Reasons For Caution: Even though the population size was relatively small, our analyses were based on homogeneous cycles, i.e. young women whose transferred embryos were found to be high-grade according to conventional morphology evaluation. In addition, our conclusions were established from a specific, highly selected population, so other study populations, such as women in an older age bracket, may yield different results. Finally, because we assessed day 2/3 transfers, our findings cannot be generalized to embryos cultured up to the blastocyst stage.Wider Implications Of the Findings: It would be interesting to explore, prospectively, whether PN localisation is a relevant measure to predict embryo development when added into further algorithms and whether this parameter could be suitable for use in other IVF clinics. Further studies are needed, notably to explore the added value of timing evaluation in cohorts of embryos with low or intermediate morphology grade, as well as in other maternal populations (i.e. older women).Study Funding/competing Interest(s): No external funding was used for this study. P. Sagot received funding from the following commercial companies: Merck Serono, Finox Biotech, Ferring, MSD France SAS, Teva Sante ́ SAS, Allergan France, Gedeon Richter France, Effik S.A., Karl Storz Endoscopie France, GE Medical Systems SCS, Laboratoires Genevrier, H.A.C. Pharma and Ipsen.All the authors confirm that none of this funding was used to support the research in this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the journal policies on sharing data and materials. [ABSTRACT FROM AUTHOR]

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

Study Question: What is the effectiveness and safety of IVM compared with IVF for the treatment of women with high antral follicle count (AFC)?Summary Answer: In women with high AFC undergoing assisted reproductive technique (ART), IVM is an effective alternative compared with IVF, while it eliminates the risk of ovarian hyperstimulation syndrome (OHSS).What Is Known Already: IVM is postulated to be an alternative to conventional IVF to avoid OHSS. It has particular potential in women with high AFC who are known to be at increased risk of OHSS. To date, IVM and IVF have only been compared in small cohort studies.Study Design, Size and Duration: We performed a retrospective cohort study including 919 women, of whom 608 underwent IVM and 311 IVF. The treatments were conducted at IVFMD, My Duc Hospital, Ho Chi Minh, Vietnam, from July 2015 to December 2017.Participants/materials, Setting, Methods: We studied infertile women aged 18-38 years with an indication for ART and with an AFC ≥24. Women received either IVM or IVF treatment depending on patient's or physician's preference. In IVM cycles, women received 3 days of FSH 100 IU/day followed by hCG 10 000 IU. In IVF cycles, women underwent a gonadotropin-releasing hormone antagonist protocol and were triggered with hCG 6500 IU. Outcome measures were live birth rate (LBR) after first embryo transfer and cumulative LBR after one complete cycle, defined as the chance of having live birth after all fresh and frozen transfers of embryos derived from one IVM/IVF cycle. We also report on clinical pregnancy, miscarriage, multiple pregnancy and OHSS.Main Results and the Role Of Chance: Baseline characteristics including age and BMI were comparable between groups. In the IVM group (608 started cycles), there were 511 fresh and 167 frozen transfers. In the IVF group (311 started cycles), there were 209 fresh and 185 frozen transfers. The number of mature oocytes, embryos, good embryos and frozen embryos was significantly lower in the IVM compared with the IVF group. LBRs after the first transfer were 222/608 (36.5%) versus 127/311 (40.8%) (adjusted odds ratio [OR], 0.74; 95% confidence interval [CI], 0.42-1.30). Cumulative LBRs after one completed cycle were 239/608 (39.3%) versus 155/311 (49.8%) (adjusted OR, 0.52; 95% CI, 0.30-0.89). OHSS did not occur in the IVM group versus 11/311 (3.5%) in the IVF group.Limitations and Reasons For Caution: Our study is limited by its non-randomized design. Randomized clinical trials are required to precisely compare the outcomes after IVM versus IVF.Wider Implications Of the Findings: In infertile women with a high AFC, IVM is a feasible alternative to standard IVF that markedly reduces OHSS and is potentially more patient friendly and cost effective.Study Funding/competing Interest(s): No external funding was sought to support this work. B.W.M. is supported by a National Health Medical Research Council Practitioner Fellowship (GNT1082548). B.W.M. reports consultancy for ObsEva, Merck and Guerbet. All other authors have no conflicts of interest.Trial Registration Number: NA. [ABSTRACT FROM AUTHOR]

Study Question: Do culture conditions affect cytoplasmic maturation in denuded immature non-GV oocytes?Summary Answer: The maturation rate of denuded non-GV oocytes is not affected by culture media, but in vitro maturation seems to alter the mitochondrial membrane potential, endoplasmic reticulum (ER) and actin cytoskeleton compared with in vivo maturation.What Is Known Already: In vitro maturation of denuded immature non-GV oocytes benefits cycles with poor in vivo MII oocyte collection, but maturation levels of non-GV oocytes are only scored by polar body extrusion. Since oocyte maturation involves nuclear as well as cytoplasmic maturation for full meiotic competence, further knowledge is needed about cytoplasmic maturation in in vitro culture.Study Design, Size, Duration: This basic research study was carried out between January 2017 and September 2018.Participants/materials, Setting, Methods: A total of 339 denuded immature non-GV oocytes were cultured in SAGE 1-Step (177) or G-2 PLUS (162) for 6-8 h after retrieval, and 72 in vivo matured MII oocytes were used as controls. Cultured immature non-GV oocytes were scored for polar body extrusion and analysed for mitochondrial membrane potential (ΔΨm), ER clusters, cortical granules number and distribution, spindle morphology and actin cytoskeleton organization. The obtained parameter values were compared to in vivo matured MII oocyte parameter values.Main Results and the Role Of Chance: The maturation rates of oocytes cultured in G-2 PLUS and SAGE 1-Step were similar (65% vs 64.2%; P = 0.91). The differences observed in cortical granule density were not statistically significant. Also spindle morphometric parameters were mostly similar between in vitro and in vivo matured MII oocytes. However, the number of ER clusters, the ΔΨm and the cortical actin thickness showed significant differences between in vivo MII oocytes and denuded immature non-GV oocytes cultured in vitro until meiosis completion.Limitations, Reasons For Caution: Frozen-thawed oocytes together with fresh oocytes were used as controls. Due to technical limitations (fixation method and fluorochrome overlap), only one or two parameters could be studied per oocyte. Thus, a global view of the maturation status for each individual oocyte could not be obtained.Wider Implications Of the Findings: Characterization of in vitro matured oocytes at the cellular level will help us to understand the differences observed in the clinical outcomes reported with rescue IVM compared to in vivo MII oocytes and to improve the culture methods applied.Study Funding/competing Interest(s): This work was supported by intramural funding of Clinica Eugin and by the Torres Quevedo Program to A.F.-V. from the Spanish Ministry of Economy and Competitiveness. No competing interests are declared. [ABSTRACT FROM AUTHOR]

Study Question: Did the revised Alpha/ESHRE consensus (Vienna, 2017) bring a real answer on managing oocytes with aggregates of smooth endoplasmic reticulum (SERa)?Summary Answer: According to the currently available literature, a case by case approach on the time of injecting/inseminating SERa+ oocytes may be not helpful for embryologists making a decision, so we suggest fertilizing both SERa+ and SERa- oocytes and prioritizing embryos derived from SERa- oocytes.What Is Known Already?: In 2011, the Istanbul consensus recommended not to inject/inseminate SER+ oocytes due to adverse foetal outcomes reported in literature. At the end of 2017, a panel of experts reconsidered this recommendation and advised a case by case approach. Hence, with a lack of clear recommendations, in-vitro fertilization practitioners still have heterogeneous attitudes when managing SERa+ oocytes. In this context of controversy, an updated review could be helpful in (i) forming a common language for managing cases of SERa+ oocytes and (ii) offering the most ethical practice and best care for patients seeking infertility treatment or fertility preservation.Study Design, Size, Duration: This review (with a last literature search on 1 June 2018) evaluated the effect of the SER dysmorphism on embryological and neonatal outcomes.Participants/materials, Setting, Methods: Studies were considered for inclusion if they were prospective or retrospective cohort or case-control studies. Electronic searches of the Pubmed and Embase databases were done using the keyword combination: smooth endoplasmic reticulum, SER, oocyte and zygote. Abstracts and articles written in English and limited to humans were included.Main Results and the Role Of Chance: The search returned a total of 726 studies among which 21 met the inclusion criteria. The literature does not unanimously support a negative association between SERa and embryogenesis, implantation or assisted reproductive therapy outcomes. The reviewed studies reported 112 neonatal outcomes after transfers where at least one embryo originated from oocyte affected by SERa. They included 101 healthy babies, three live births with malformations, three neonatal deaths, one stillbirth and four medical interruptions of pregnancy. After transfer of embryos exclusively derived from SERa+ oocytes, a total of 48 healthy newborns were reported along with four babies with perinatal complications (including one ventricular septal defect), one stillbirth, one neonatal death and one pregnancy termination for multiple malformations.Limitations, Reasons For Caution: As with any review, this review was limited by the quality of the included studies especially in terms of possible methodological limitations, the limited sample size and the retrospective aspect of the studies. Among the 21 selected studies, seven were abstracts and two were case reports. Of the remaining 14 studies, only three were prospective. The tools used in identifying SERa+ oocytes may have varied from one study to another and a consequent misclassification cannot be excluded. Considering the poor resolution of light microscopy in detecting SER aggregates, we are not sure that apparently SERa- oocytes do not really exhibit such a dysmorphism if they were analysed under electronic microscopy or a time lapse system.Wider Implications Of the Findings: In the light of the existing data and the lack of a real link between fertilizing SERa+ oocytes and the occurrence of embryo aneuploidy/malformations, we think that discarding SERa+ oocytes may be not the most ethical approach even in patients with large cohorts on the day of oocyte retrieval. Avoiding the wastage of oocytes and embryos with respect to medical ethics remains a constant concern in daily IVF practice. Thus, we recommend that all mature oocytes could be fertilized and embryos originating from SERa- oocytes would be preferably transferred, even if they come from a cohort with SERa+ oocytes. The remaining embryos derived from SERa+ oocytes could be considered with a lower priority for transfer after obtaining consent from the couple if a strict follow-up of the pregnancy and the baby is performed.Study Funding/competing Interest(s): We have no conflict of interest to declare and no funding was received.Registration Number: N/A. [ABSTRACT FROM AUTHOR]