Background Abnormal pronuclear formation, including five pronuclei (5PN), is generally considered indicative of abnormal fertilization (e.g., polyspermy or failure of polar body extrusion) and leads to routine embryo discard in many IVF programs. However, emerging evidence suggests that a minority of embryos with atypical pronuclear patterns may develop into a blastocyst and present diploid constitution after genetic testing, challenging the assumption that abnormal pronuclear morphology invariably predicts non-viability. Case report We report a rare case in which a 5PN zygote developed into a morphologically high-quality blastocyst was confirmed euploid-diploid by PGT-A, transferred in a subsequent frozen embryo transfer cycle, and resulted in the live birth of a healthy child. Conclusion This case adds to the limited but growing evidence that embryos with atypical pronuclear presentation may retain reproductive potential when development on time-lapse appears coherent and validated genetic testing confirms diploidy and euploidy, supported by appropriate patient counseling and informed consent.

RESEARCH QUESTION:What are the reproductive outcomes of women who returned to a Brazilian fertility clinic to achieve motherhood after elective oocyte cryopreservation (EOC)? DESIGN:Retrospective single-centre study (n = 2073 women; n = 2431 EOC cycles) between January 2013 and December 2022. Women freezing oocytes for medical indications or infertility treatment were excluded. RESULTS:Mean age at freezing was 36.5 ± 2.8 years. The annual number of EOC cycles increased nearly 500% from 2013 to 2022. Of 1755 women with cryopreserved oocytes, 134 (7.6%) returned to the same clinic for oocyte warming after a mean interval of 4.1 ± 2.2 years; from this cohort, 69% achieved embryo transfer and 47.3% of those achieved a live birth. Older age at freezing was associated with lower odds of reaching embryo transfer (adjusted OR [aOR] 0.86, 95% CI 0.74 to 0.99, P = 0.037), whereas a higher cumulative number of vitrified mature oocytes increased those odds (aOR 1.11, 95% CI 1.03 to 1.20, P = 0.004). The number of oocytes warmed, post-warming survival, fertilization and blastocyst rate were independently associated with embryo transfer; no variable independently predicted live birth. Kaplan-Meier analysis showed cumulative utilization of 4.02% at 3 years and 9.37% at 5 years after freezing. Utilization differed according to cumulative number of vitrified mature oocytes, but not age at freezing. Among women who warmed oocytes, 27% pursued single motherhood. CONCLUSION:As elective oocyte cryopreservation increases, a greater number of women may return for future use. Counselling should emphasize freezing at younger ages, achieving an adequate number of vitrified mature oocytes, and the limitations of assisted reproductive technology at advanced maternal age.

The need to reduce the number of embryos transferred in assisted reproductive care to prevent multiple gestations has led to a stronger emphasis on selecting embryos with the highest morphological quality. Although this evaluation has traditionally been performed by trained embryologists, the increasing use of time-lapse incubators has introduced a greater volume of data and subjectivity in decision-making. Artificial intelligence (AI)-based tools can support embryologists by offering objective, standardized embryo assessments.In Brazil, like other countries, where imported embryo selection technologies may not account for local demographic and ethnic profiles, an AI model — Morphological Artificial Intelligence Assistance (MAIA) — was developed through a collaboration between a university and a private fertility clinic in São Paulo. The model was trained using 1,015 embryo images and prospectively tested in a clinical setting on 200 single embryo transfers. In clinical testing, MAIA achieved an overall accuracy of 66.5%. In elective embryo transfers, where there were more than one embryo eligible for transfer, MAIA achieved 70.1% accuracy for predicting clinical pregnancy. Designed with a user-friendly interface tailored by embryologists, MAIA provides real-time embryo evaluations to support decision-making in routine care.

To assess the impact of maternal age on the association between maternal basal FSH and aneuploidy. A retrospective study including data from 1749 blastocysts diagnosed as euploid or aneuploid by PGT-A (preimplantation genetic testing for aneuploidy). Aneuploidy incidence was compared between embryos from mothers with high vs. low basal FSH levels (above and below the group median, respectively) in total, pre-AMA (advanced maternal age; < 35 years, 198 embryos) and AMA (≥ 35 years, 1551 embryos) patient groups, separately. To control for the interference of potentially confounding variables, the association between aneuploidy and high basal FSH levels was assessed by multivariate logistic analysis in overall, pre-AMA and AMA patient groups. Overall, aneuploidy rate was 9

Abstract Study question Is live birth rate of autologous versus donor cycles similar after a euploid embryo transfer? Summary answer The live birth rate was significantly higher in autologous cycles after a euploid embryo transfer. What is known already Advanced maternal age (AMA) is a detrimental factor for patients to achieve a successful outcome in ART. Physiological ovarian aging negatively affects oocyte quality and quantity. Thus, the number of cycles with donated oocytes is increasing for AMA patients worldwide, with the perspective of surpass the oocyte factor and achieve a live birth in similar rates as younger patients. The goal of this study was to compare the live birth rates of autologous versus donor cycles after a euploid embryo transfer. Study design, size, duration Observational cohort study including all patients of IVF cycles with a euploid embryo transfer after PGT-A (NGS technology), according to medical referral between Jan/2016 and Dec/2020 (n = 3623 patients and 4025 cycles) in a private ART center. Participants/materials, setting, methods Reproductive outcomes between autologous and egg donation cycles were compared and data were analyzed in the follow subgroups: cycles with a fresh oocyte and fresh embryo transfer (FRESH), frozen embryo transfer (FET) and fresh embryo transfer from frozen oocytes (FOT). Positive pregnancy test (PPT), clinical pregnancy (CP), clinical miscarriage (CM) and live birth (LB) rates were compared. Kruskal-Wallis and Fisher test were applied for statistical analysis, significance was considered if p < 0,05. Main results and the role of chance In total, 3623 patients – 88.9% autologous and 11.1% with donated oocytes – were included. They performed 4025 ETs (3591/434) with 5313 biopsied blastocysts (4736/577) in a mean of 1,32/1,33 blastocysts per transfer in autologous/donated cycles respectively. The majority of patients using autologous and donated oocytes underwent a FET (97,2% and 63,1%), followed by FOT (1,5% and 34,7%) and FRESH (1,3% and 2,2%, respectively). Mean maternal age in autologous and donor cycles was 37,76±3,9yo and 43,47±4,26yo and oocyte donors were 24,99±3,86yo. In subgroups, autologous patients in FRESH were 38,33±2,69 yo, similar in FET (37,73±3,93 yo) and older in FOT, 39,25±3,93 yo (p = 0,03). Patients in donor cycles showed no difference in age. PPT and CP were similar in autologous and donated cycles (PPT: 59,2% vs 54,8%, p = 0.08; CP: 52,2% vs 48,2%, p = 0.11). In subgroup analysis, PPT and CP were higher in donated cycles vs autologous cycles for FOT (PPT: 50,7% vs 33,3%, p = 0.004; CP: 40,7% vs 20,8%, p = 0.01). Overall, CM were higher in donated cycles (15,5% vs 10,6%, p = 0.03), especially after FET (16,8% vs 10,6%, p = 0.025). LB rates were higher in autologous compared to donated cycles (37,8% vs 29,5%, p = 0.0007). There was no significant difference in LB rates in subgroup analysis. Limitations, reasons for caution This is a large cohort study; however, we cannot exclude potential bias due its retrospective nature. Including only euploid embryos, we excluded bias on ploidy status when comparing autologous versus donated oocytes cycles, yet the potential harmful of embryo biopsy may not be neglect. Wider implications of the findings Live birth rate was significantly higher in autologous cycles after a euploid embryo transfer, regardless of subgroups. AMA patients that choose to undergo an IVF treatment with donated oocytes should be aware that there are other important factors related to a successful reproductive outcome, including immunological adaptation and endometrial aging. Trial registration number Not applicable.

How is the performance of KIDScoreTM D5 version 3 for clinical pregnancy prediction in day 6 blastocysts? Differing on Day 5 performance, KIDScoreTM D5 algorithm does not predict clinical pregnancy for day 6 blastocysts according to score subgroup grades. Embryo selection is a crucial step for a successful outcome in assisted reproductive technologies (ART). In the past years, the use of software and algorithms developed with artificial intelligence (A.I.) tools brought new parameters to be considered by the embryologists in embryo decision at the ART lab. KIDScoreTM is a commercial software (Vitrolife®, Sweden) developed and validated with a large database of known clinical outcome embryos transferred on day 3 or day 5 of development. Yet, it is unknown the performance of KIDScore D5 v3 on day 6 blastocysts, which are widely used for embryo transfer. Large retrospective cohort study including sequential single embryo transfers with blastocysts submitted or not to trophoectoderm biopsy for PGT-A, cultured in the Embryoscope® Plus incubator (Vitrolife®) in a single private IVF center between Jan/2020 and Jul/2023. Positive or negative clinical pregnancy, CP (presence/absence fetal heartbeat and gestational sac) from 738 patients/771 blastocysts developed on day 5 or day 6 were considered. Biochemical pregnancy and miscarriage were excluded from this analysis. Day 5 and day 6 embryo transfer outcomes data were compared considering three KIDScoreTM subgroups, according to the following score intervals: subgroup 1: 1.0-3.9 (n = 102), subgroup 2: 4.0-6.9 (n = 273) and subgroup 3: 7.0-9.9 (n = 396 blastocysts). Euploid (n = 517) and non-biopsied embryos (n = 254) were also analyzed in the described subgroups. For the analysis, Mann-Whitney, Chi-square and Fisher tests were used properly for statistical analysis, values of p < 0.05 were considered significant. Maternal age were similar between positive and negative pregnancies in D5 and D6 transfers (D5:39,32±4,34 vs 39,02±4,47 and D6:38,75±3,99 vs 38,18±3,76, p = 0,35 and 0,28 respectively). In D5 transfers, CP rates significantly increased in higher score subgroups [subgroup 1:42.9% (6/14); subgroup 2:48.9% (86/176); subgroup 3:61.6% (236/383),p=0.01]. As expected, subgroup 3 showed a higher CP rate when compared to subgroup 1 + 2 (scores from 1.0 to 6.9,p=0,003). However, in D6 transfers, CP rates were similar between score subgroups 2 and 3 and lower in group 1 [subgroup 1:28.4% (25/88); subgroup 2:50.5% (49/87); subgroup 3:53.8% (7/13),p=0.005 and p = 0,001 between subgroup 1 and subgroups 2 + 3]. Similar results were seen in euploid and non-biopsied embryo transfers; in D5, CP rates were increased in higher scores subgroup (7.0-9.9) when compared to subgroups 1 + 2 (euploid:61,6% vs 42,9% p = 0,05, and non-biopsied:61,3% vs 42,6%,p=0,01 for positive and negative CP respectively in subgroup 3 vs subgroup 1 + 2). And in D6 embryo transfers CP rates were lower in subgroup 1 (1.0-3.9) and similar in subgroups 2 and 3 for both euploid and non-biopsied embryos (euploid:31,5% vs 49,4% p = 0,02, and non-biopsied:13,6% vs 56,5%,p=0,007 for positive and negative CP respectively in subgroup 1 vs subgroup 2 + 3, respectively). The KIDScoreTM D5 v3 algorithm is validated for blastocyst on day 5 of development; the results showed here recognizes the limitations of evaluate the potential of CP prediction on Day 6 blastocysts and the score were not considered for embryo selection decision during the time of study. The KIDScoreTM is effective to predict the potential of clinical pregnancy in blastocysts on day 5 of development, with higher rates on higher score subgroups, but not for blastocysts on day 6 of development, where the embryos had similar rates with score greater than 4.0. Not applicable.

Classical preimplantation embryo culture is performed in static fluid environments. Whether a dynamic fluid environment, like the fallopian tube, is beneficial for embryo development remains to be determined across mammalian species. Objectives of these proof-of-concept studies were to determine if controllable dynamic microfluidic culture would enhance preimplantation murine, bovine, and human embryo development compared to static culture. This prospective randomized controlled trial tested static versus controlled dynamic culture of preimplantation mouse (n = 397), bovine (n = 242), and human (n = 512) zygotes to blastocyst stages with outcome measures of embryo cleavage, cellular fragmentation, apoptosis, and blastocyst conversion rates. Dynamic culture of mouse and bovine zygotes with microfluidics significantly improved embryo development. Mouse placental imprinted gene expression was significantly different between embryos derived in vivo, by static culture, and by dynamic culture. Using human sibling zygotes, this dynamic microfluidic culture system increased the number of blastomeres per cleavage-stage embryo, reduced cellular fragmentation or apoptosis, improved blastocyst conversion rates, and enhanced blastocyst developmental stages. In conclusion, species-specific longitudinal studies demonstrated that dynamic microfluidic culture significantly improved embryo development, independent of culture media composition, temperature, and gaseous environment. These cellular indicators represent improved embryo development that can translate into higher pregnancy rates in transgenics, domestic livestock and endangered species and treating human infertility.

Natural conception in women after the age of 45 years is rare. The probability of successful pregnancy in this specific group of women after IVF and embryo transfer with autologous oocytes is also reduced. In addition, advanced maternal age is associated with an increased risk of aneuploidies and other associated complications during pregnancy. Generally, women who are over 44 years old are advised to receive IVF treatment with donated oocytes due to poor oocyte quality and low ovarian reserve. Although IVF outcomes in women of advanced age can be associated with the best prognosis when donated oocytes are used, IVF is not always well accepted by infertile couples. This is a case report of a woman who achieved a clinical pregnancy and live birth in her first attempt at IVF treatment with her own eggs and a euploid embryo at 48 years and 10 months, respectively, at the time of oocyte retrieval. This case demonstrates that limited attempts at assisted reproductive technology with older women's own eggs may be an option in specific cases.

Abstract Study question How is the performance of a Morphological Artificial Intelligence Assistant (MAIA) on embryo selection to clinical pregnancy prediction? Summary answer MAIA presented a sensibility of 79.8% and specificity of 72.9% to predict a clinical pregnancy and reached a performance of 64.7% on prospective validation test. What is known already Artificial intelligence (AI) tools have gained attention in assisted reproductive treatments. Algorithms for gametes and embryo classification and selection are commercially available in an exponential increasing race. Usually, those algorithms/software are developed based on data acquisition of several ART centers, from different countries and continents, which may overlap some but rarely all clinical and laboratory methodologies and protocols. In this way, the purpose to develop a software restricted to a single center may represent several advantages which is not highlighted at this moment. Here, we developed a full in-house software for embryo selection and prediction of clinical pregnancy. Study design, size, duration MAIA was developed through artificial neural networks technique and genetic algorithms using the input of morphological data from 1.008 sequential transferred blastocysts and known reproductive outcomes (clinical pregnancy, presence of gestational sac and heartbeat). Blastocysts images were obtained from IVF cycles based in a single ART center. All embryos were cultured in a time-lapse incubator (Embryoscope Plus, Vitrolife) and blastocyst digital image processing were analyzed considering 33 mathematic variables. Participants/materials, setting, methods Input data were randomized for training, validation and test (70, 15 and 15%, respectively). From 1008 images, 755 were used for effective AI learning, 174 were used for the blind test and 79 were excluded due to poor embryo visualization. For performance validation, the software was responsible for embryo selection in 34 elective single embryo transfers. The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was measured to obtain predictive power. Main results and the role of chance In the blind test (n = 174), 71 embryos were correctly classified as positive for clinical pregnancy (CP) – true positive – and 18 embryos were misclassified as negative for CP. In negative CP, 62 embryos were correctly classified as negative for CP – true negative – and 23 embryos were misclassified as positive for CP. This represented a sensibility of 79.8%, specificity of 72.9% and precision of 75.5% for CP. The AUC was 76.52%. In performance validation, 34 elective single embryo transfers, meaning there were from cycles with more than 1 viable blastocyst for selection, the chosen embryo was decided by MAIA. In those, 22 achieved a clinical pregnancy (64.7%), in which MAIA predicted correctly the CP in 19 (86.4%) – the software may predict that none of embryos available would achieve a CP. From 12 negative outcomes, MAIA predicted in 25% that none of the embryos available would resulted in CP. Overall prediction for positive and negative CP in performance validation was 64.7%. Limitations, reasons for caution MAIA software was built with the database from a single IVF center and its performance may be impacted if used in different ART services. The number of single embryo transfers used to validate the sofware performance is still low. Wider implications of the findings This is a new artificial intelligence software for embryo selection and clinical pregnancy prediction. Different from all available commercial software, MAIA was built in homogenous clinical and laboratory protocols, analyzing the database from a single IVF center, and were able to reach a performance of 64.7% on prospective validation test. Trial registration number Not applicable.