Foundation for Reproductive Medicine

Background Preimplantation genetic testing for aneuploidy (PGT-A) is used principally to identify embryos with whole chromosome aneuploidy, which are generally considered non-viable. Reports of healthy live births following transfer of embryos classified as fully aneuploid are therefore extraordinary and require rigorous verification. Methods This commentary evaluates how claims of live births from embryos diagnosed as fully aneuploid by PGT-A are generated, interpreted and reported. We examine prevailing evidentiary standards and highlight key sources of potential error, including lack of genetic identity confirmation, natural conception, laboratory or clerical error and misclassification of mosaic or segmental aneuploid embryos. Results To date, no published report has included DNA-based confirmation demonstrating that a healthy child originated from an embryo diagnosed as fully aneuploid. Failure to clearly distinguish whole chromosome aneuploidy from mosaic or segmental aneuploidy, which retain the potential to result in live birth, further complicates interpretation and risks overstating the implications of such reports. Conclusion In the absence of genetic identity confirmation, claims of live births from embryos classified as fully aneuploid cannot be considered evidentiary. As interest in this topic grows, rigorous genetic validation and standardised reporting should be required to ensure scientific credibility, support accurate patient counseling and guide responsible interpretation of these reports.

Abstract Study question How do frozen embryo transfer (FET) outcomes of preimplantation genetic testing (PGT)-tested embryos compare between those undergoing two versus a single freeze-thaw cycle(s)? Summary answer PGT-tested embryos subjected to two freeze-thaw cycles were noted to have poorer morphology, higher miscarriage rates, and lower live birth rates. What is known already For various clinical reasons, patients who initially froze untested embryos after controlled ovarian hyperstimulation may later thaw those embryos for PGT. However, the impact of two freeze-thaw cycles on embryo potential and FET outcomes remains unclear. Limited research exists on how repeated freeze-thaw cycles affects embryo quality, making patient counseling challenging. This study aims to clarify the effects of two freeze-thaw cycles on FET outcomes. Study design, size, duration A retrospective chart review analyzed FET cases with PGT-tested embryos at Weill Cornell Center for Reproductive Medicine (2020–2024). Among 4724 FETs, 4542 involved testing fresh embryos (T1), while 182 involved thawing frozen embryos for PGT, refreezing, and thawing again for FET (T2). Participants/materials, setting, methods Individuals aged 18–45 who underwent natural cycle or programmed FET with a PGT-tested embryo at a New York academic multicenter clinic were included. Reasons for embryo thawing for PGT were recorded. Blastocysts were categorized by final thaw morphology: excellent (≥3AA), good (3-6AB, 3-6BA, 1-2AA), average (3-6BB, 3-6AC, 3-6CA, 1-2AB, 1-2BA), and poor (1-6BC, 1-6CB, 1-6CC, 1-2BB). Descriptive statistics assessed population characteristics, and Fisher’s exact test compared FET outcomes between T1 and T2. Main results and the role of chance Between 2020 and 2024, 4542 FETs used PGT-tested embryos thawed once (T1). Additionally, 254 embryos underwent thawing for PGT, leading to 182 FETs (T2) of 183 euploid embryos among 127 individuals. Of 72 individuals who did not proceed with FET after thawing, 47 had no euploid embryos. The reasons for embryo thawing for PGT included prior pregnancy loss or failed implantation after untested transfers (n = 55), age-related aneuploidy screening or grouping from multiple retrieval cycles for PGT-A (n = 33), sex selection (n = 19), PGT-M (n = 13), prior termination for aneuploidy (n = 7), and rebiopsy (n = 2). Embryo morphology was poorer after a second thaw, with fewer “excellent” (T2: 0.5% vs. T1: 4.4%, p=.007) and “good” embryos (T2: 19.8% vs. 22.2%, p=.007). Live birth rates were lower in T2 (39.6% vs. 54.6%, p<.001), as were implantation rates per transfer (52.2% vs. 61.0%, p=.020) and per embryo (51.9% vs. 59.9%, p=.032). Miscarriage rates were higher (18.9% vs. 9.4%, p=.004). Within T2, individuals with a history of one or more pregnancy losses or failed implantation had comparable live birth and miscarriage rates to those with other reasons for undergoing a freeze-thaw cycle. Limitations, reasons for caution This single-center, retrospective study has limited generalizability and excludes rare cases of embryos not surviving the second thaw, necessitating larger multicenter studies for validation. Wider implications of the findings Multiple freeze-thaw cycles pose potential risks, including higher miscarriage rates and lower live birth rates. These findings emphasize the need for individualized counseling and treatment plans based on risk-benefit profiles. These findings also support the need for further investigation into laboratory freeze-thaw protocols to optimize embryo potential and outcomes. Trial registration number No

Abstract Study question What molecular mechanisms trigger aneuploid cell depletion in post-implantation mosaic embryos, enabling self-correction and pregnancy success? Summary answer Aneuploid cell depletion in day 9-11 mosaic embryos is driven by disrupted stress responses, pluripotency dysregulation and impaired energy expenditure, mediated by aneuploid-euploid cell competition. What is known already Embryo aneuploidy is a major cause of adverse pregnancy outcomes. However, mosaic embryos - especially those with low-level aneuploidy (<50%), have significantly higher developmental potential, resulting in ongoing pregnancy and live birth of normal babies. Although their implantation rates are lower than euploid embryos, multicenter data from over 3,500 mosaic embryo transfers show that only 1.2% of mosaicism persists during pregnancy and postnatally, suggesting a self-correction capacity in many mosaic embryos. Despite these promising outcomes, the use of mosaic embryos in IVF remains limited due to an incomplete understanding of the biological mechanisms underlying their developmental success. Study design, size, duration Donated preimplantation euploid, aneuploid and mosaic embryos from the CReATe Fertility Centre and Zouves Fertility Center (Veritas IRB protocol #16580) were cultured from day 5-6 to day 9-11. 3870 cells from 31 post-implantation embryos (aneuploid, n = 7; mosaic, n = 8; euploid, n = 16) were included for single-cell RNA sequencing (scRNA-seq) analysis. An iPSC-based mosaic model was established by co-culturing euploid and reversine-treated (aneuploid) naive iPSCs in a transwell system for 96 hours. Participants/materials, setting, methods Cultured embryos were dissociated into single cells and pooled for scRNA-seq (10x Chromium). Sequencing results were analyzed using Freemuxlet to identify embryo-of-origin, Seurat for clustering, and InferCNV to determine post-implantation cell ploidy. Lineage developmental trajectory was inferred from pseudotime analysis. Enriched genetic markers and pathways were identified through differential expression analyses. RT-qPCR and immunofluorescence assays were used to quantify gene and protein expression in iPSC models. Main results and the role of chance scRNA-seq data from 3870 embryonic cells revealed diverse post-implantation lineages, including epiblast, hypoblast, and multiple trophoblast subtypes. Transcriptomic and regulon analysis showed aneuploid cell survival was dependent on embryonic environments. In homogenous aneuploid embryos, we observed adaptive stress responses that support temporary survival and aneuploidy tolerance. This includes delayed differentiation across all lineages, upregulation of genes related to pluripotency (e.g. KLF4) and unfolded protein response (e.g. ATF4). In contrast, mosaic embryos exhibited complex cell competition dynamics, where aneuploid cells in mosaic embryos were driven toward pluripotency exit, evidenced by advanced progression along developmental trajectory and downregulated pluripotency genes. This shift was accompanied by disrupted homeostatic pathways, including glycolysis (e.g. MYC) and proteostasis (e.g. DDIT4), leading to elevated apoptotic activity (e.g. CASP8). Our iPSC-based mosaic model recapitulated differential fitness levels in mosaic embryos. In a mosaic environment, aneuploid iPSCs exhibited impaired viability and growth, along with significant downregulation of DDIT4 (log2FC=-3.16, p < 0.05), and KLF4 (log2FC=-0.54, p < 0.05). Enhanced autophagy was observed in euploid iPSCs, indicated by accumulation of LC3 complex, while aneuploid iPSCs displayed increased apoptotic signaling, marked by elevated Caspase-8 levels. Together, Our results pointed to a diminished survival advantage in the aneuploid cells of mosaic embryos, driving their selective depletion. Limitations, reasons for caution We compared the transcriptomic signatures of aneuploid cells in aneuploid and mosaic embryos. Expanding the sample size will improve generalizability of the findings. Furthermore, while the iPSC-based mosaic model mirrors key aspects of the mosaic environment, it may not fully recapitulate the complexity of in vivo embryonic development. Wider implications of the findings Our study unravels molecular mechanisms contributing to self-correction and pregnancy success of mosaic embryos. These results provide the foundation for interrogating developmental competence of mosaic embryos, potentially expanding the range of viable embryos suitable for IVF treatment and improving options for fertility preservation. Trial registration number No

This case report describes the strategy for reclassifying embryos initially diagnosed as segmental aneuploid through preimplantation genetic testing for aneuploidy (PGT-A), potentially rescuing those embryos for clinical use. A couple undergoing IVF, facing a lack of viable embryos, opted to re-biopsy a previously tested uniform (non-mosaic) segmental aneuploid embryo. The repeat PGT-A analysis produced a euploid result. Following thorough consultation with a genetic counselor and the treating physician, and after signing an informed consent detailing the associated risks, the couple proceeded with the embryo transfer. The pregnancy was confirmed, and prenatal testing by amniocentesis produced euploid results. This case highlights the potential of re-biopsy to more accurately assess embryos initially diagnosed as segmental aneuploid, helping to avoid the unnecessary discard of viable embryos while minimizing the clinical risks associated with constitutive segmental aneuploidies.

Lower birth rates and higher pregnancy loss are observed after childhood cancer. Studies specific to adolescent and young adult (AYA) cancer rarely have information on pregnancies that do not end in live birth, fertility preservation strategies, or detailed cancer treatment information to assess these risks. To address this gap, we examined pregnancy outcomes after cancer in a cohort of 30,020 AYAs with detailed clinical records. We identified 6021 survivors of AYA cancer matched to 23,999 AYAs without cancer enrolled in the Kaiser Permanente California health systems during 2004-2016. Of these, 950 survivors had ≥1 pregnancy during follow-up through 2018. Hazard ratios (HR) and 95% confidence intervals (CI) for pregnancy were estimated using multivariable subdistribution hazard models accounting for competing risks. Relative risks (RR) and 95% CIs for pregnancy loss were calculated with multivariable Poisson regression with generalized estimating equations. Pregnancy was less common after AYA breast (HR = 0.57; 95% CI: 0.50, 0.66) or gynecologic cancer (HR = 0.55; 95% CI: 0.42, 0.73) compared to AYAs without cancer. Among AYAs with cancer, some alkylating and platinum chemotherapy agents, but not gonadotoxic radiation, were associated with a lower likelihood of pregnancy. Use of assisted reproductive technologies was not common, and 69% of pregnancies after AYA cancer resulted in live birth. Pregnancy loss was not statistically significantly elevated for any cancer type. Among survivors, pregnancy loss was more common at older ages, with smoking during pregnancy, and among those who received cyclophosphamide. Our findings inform reproductive counseling and prenatal care for reproductive age cancer survivors.

Given the history of coercion in the provision of sterilization, it is important that patients who desire permanent contraception receive patient-centered care that respects their preferences. Recent literature examiningthe provision of postpartum permanent contraception has not explored whether patients who desire permanent contraception feel supported by their clinical teams. We interviewed patients from four medical centers around the United States with a documented desire for postpartum permanent contraception. We audio-recorded, transcribed, and analyzed interviews using rapid qualitative methods and thematic content analysis. Of the 81 patients interviewed, most (n = 64) felt supported by their clinician throughout their prenatal care and delivery hospitalization. Seventeen patients shared stories of feeling unsupported. Patients recalled feeling dismissed when they initiated conversations about permanent contraception. Additionally, they felt they had received incomplete counseling on permanent contraception and felt discouraged from getting the procedure during prenatal care encounters. During delivery hospitalization, patients discussed not being informed about barriers to permanent contraception as they surfaced, facing lack of prioritization of the procedure by clinicians, recognizing communications errors among the clinical team, and feeling pressured to undergo or not undergo permanent contraception. Most patients felt supported by their clinicians throughout permanent contraception decision-making and provision. Patients who did not feel supported reported a spectrum of experiences ranging from rushed appointments, perceived clinician bias in counseling, and communications barriers to the provision of permanent contraception after delivery. Patient-centered care that prioritizes patient experiences, values, and desires is necessary to ensure that all people can achieve their reproductive goals.

Abstract Study question Can a generalizable computer vision foundation model be trained using self-supervised learning on time-lapse embryo images to perform multiple clinically relevant downstream tasks? Summary answer We developed FEMI, a foundation model trained on eight million time-lapse images, to perform multiple clinical tasks, including blastocyst quality scoring, ploidy prediction, and segmentation. What is known already In vitro fertilization success critically depends on choosing viable embryos, a process hampered by current limited diagnostic tools, high costs, and ethical concerns. In recent years, various medical fields have increasingly explored foundation models using vision transformer architectures. These models, trained in a self-supervised manner on vast unlabeled image datasets, perform various clinically relevant tasks. Once trained on a massive unlabeled dataset, the encoder portion of the foundation model can be extracted and subsequently fine-tuned with a labeled dataset to perform various clinically relevant tasks like classification, regression, and segmentation. Study design, size, duration The foundation model was trained on 8 million Embryoscope® (E-SD) and Embryoscope+® (E+) images spanning clinics in the United States, Canada, and Europe. Datasets contained various additional information, such as ploidy status, blastocyst scores (numerical values from 3 to 14 based on Zhan et al. 2020), and segmentation masks (for trophectoderm [TE], inner cell mass [ICM], and zona pellucida [ZP]) that were used for training downstream tasks. Participants/materials, setting, methods A masked autoencoder architecture is trained on time-lapse images to create a foundation model (FEMI). The encoder from the autoencoder is extracted and fine-tuned on three image-based downstream tasks: ploidy prediction, blastocyst scoring, and embryo component segmentation. The performance of the fine-tuned foundation model is compared to VGG16 architectures, specifically trained for each downstream task. We evaluated performances using the area under the receiver-operating-characteristic (AUROC), mean absolute error (MAE), and mean intersection over union (mIoU). Main results and the role of chance The training dataset for the downstream tasks consisted of image data and labels, including PGT-A ploidy results (euploid or aneuploid), blastocyst scores (3-14), and segmentation masks. No clinical data like maternal age was used in order to assess FEMI’s learning capabilities from only time-lapse images against VGG16 model baselines. Both FEMI and baseline models used the same data splits for consistent comparisons. For ploidy prediction, models trained on a Weill Cornell Medicine (WCM) dataset were validated using WCM, Florida, and Spain data. FEMI achieved a 0.610 ± 0.004 AUROC, outperforming the baseline’s 0.590 ± 0.005. In blastocyst score prediction, FEMI attained a superior MAE of 0.099 ± 0.002 compared to the baseline’s 0.118 ± 0.001. For embryo segmentation (TE, ICM, ZP), a publicly available dataset from Simon Fraser University was split for training and validation. FEMI exceeded the baseline in mIoU for TE segmentation (0.738 ± 0.002 vs. 0.726 ± 0.002) and was comparable in ICM (0.832 ± 0.008 vs. 0.821 ± 0.011) and ZP segmentation (0.779 ± 0.002 vs. 0.778 ± 0.010). Limitations, reasons for caution The current version of FEMI is trained on a subset of our available datasets and can be improved through further training. Moreover, the downstream ploidy and blastocyst score models may be biased by errors in PGT results and the subjectivity of embryologists, respectively. Wider implications of the findings A generalizable foundational model for IVF time-lapse imaging aids clinicians in embryo selection by providing various clinical insights for better decision-making. FEMI, once fully trained, will be publicly accessible to the scientific community allowing for researchers to fine-tune FEMI on their own clinic-specific applications with their own image datasets. Trial registration number not applicable

Abstract Study question Is it possible to generate genotyped oocytes by utilizing accessible somatic cells in a somatic cell nuclear transfer model? Summary answer Genotyped oocytes generated through haploidization of endometrial somatic cells exhibited reliably completed embryo development and yielded healthy offspring. What is known already The generation of genotyped oocytes through neogametogensis or somatic cell nuclear transfer (SCNT) would be the ultimate solution to treat female infertility due to primary ovarian insufficiency or agonadism. Among the various approaches, somatic cell haploidization appears more feasible (Lee, et al 2022). Unfortunately, this approach has only been successful in a murine model using cumulus cells. However, this donor cell type is not ideal when translated into clinical setting, due to the absence of cumulus cells in patients who are void of oocytes, which arguably is the patient population that would potentially benefit the most from this technique. Study design, size, duration In a mouse SCNT model, endometrial stromal cells (EMSCs) were injected into enucleated oocytes. Cumulus cells (CCs) were used as another source of nuclear donor. Intact MII oocytes were used as control. Post-SCNT oocytes and controls were inseminated by spermatozoa from B6-EGFP mice to monitor paternal genome presence through embryo development and offspring. Embryos were cultured in a time-lapse microscope. To prove gamete competence, blastocysts were transferred into pseudo-pregnant recipients and offspring health was assessed. Participants/materials, setting, methods Endometrial stromal cells were cultured for 4 days and exposed to aphidicolin overnight to synchronize at G1 phase of cell cycle. Cumulus cells were collected after denudation. Oocytes from B6D2F1 mice were enucleated under polarized light microscope. SCNT was performed with CCs or EMSCs by lipofectamine-mediate fusion. Oocytes were inseminated with spermatozoa from GFP-positive transgenic mice. Embryos morphokinesis were assessed by a time-lapse system. Blastocysts were transferred into 2.5-dpc surrogate CD1 mice. Main results and the role of chance Control oocytes(n = 75) fertilized at 93.3%(70/75) and progressed to 2-cell stage at 93.3%(70/75), 8-cell at 93.3%(70/75) and blastocyst at 89.3%(67/75). The control embryos started syngamy at 14.4±1h, reaching the 4-cell stage at 37.3±3h and blastulation at 80.6±8h. A total of 484 oocytes were enucleated, 470(97.1%) survived and underwent SCNT using CCs(n = 85) or EMSCs(n = 385). Fusion rate was 95.5% for CC with a haploidization rate of 70.9%(56/79) that led to a fertilization rate of 64.3%(36/56). The CC cohort developed to 2-cell, 8-cell, and morula at 47.4%, 35.4%, and 22.8%, with a delay in development at syngamy at 19.0±8h(P<0.0001). In the EMSC cohort, fusion rate was 96.0%(361/376). Haploidization rate was 65.4%(236/361), comparable to the CC cohort(P = 0.35). Fertilization was lower in the EMSC cohort at 47.0%(P<0.01) compared to the CC cohort. Subsequently, EMSC cohort yielded similar cleavage rates to 2-cell stage at 30.7%, 8-cell stage at 29.4%, and morula stage at 28.5%. The blastocyst rate was higher in the EMSC cohort at 28.0%(P<0.05). The EMSC cohort yielded similar developmental timing comparing to controls from pronuclear appearance to 4-cell cleavage but a delay manifested afterward at 37.3±3h(P<0.0001). In the EMSC cohort, 42 blastocysts were transferred, yielding 3 healthy pups(2 male,1 female). Limitations, reasons for caution While we generated consistent haploidization and satisfactory fertilization, full pre-implantation development remained low. Even more limited was the live birth rate, which probably related to subtle factors such as an imprinting issue due to insufficient reprogramming of the somatic cell donor. Wider implications of the findings The SCNT model described to generate genotyped oocytes, although still with limited efficiency, represents a feasible reproductive option for women with agonadism and compromised ovarian reserve. This model is still a better approach to complete neogametogenesis. Trial registration number not applicable