Abstract STUDY QUESTION Do euploid (EU), trisomy 21 (T21), and monosomy 21 (M21) human embryos exhibit different patterns of secreted proteins and morphometric changes during extended in vitro culture, either with or without endometrial cell support? SUMMARY ANSWER Secreted protein profiles and morphometric parameters discriminate between euploid and aneuploid embryos during post-implantation in vitro culture with endometrial cell support. WHAT IS KNOWN ALREADY Extended in vitro culture systems with and without endometrial cells have enabled the study of early human post-implantation development and highlighted the importance of embryo–endometrial crosstalk during implantation. However, how embryo chromosomal status influences implantation dynamics and embryo–endometrial interactions remain poorly understood. STUDY DESIGN, SIZE, DURATION Laboratory-based study. Sixty-nine supernumerary human embryos donated for research at blastocyst stage (5 days post-fertilization, d.p.f.) (EU, n = 24; T21, n = 21; M21, n = 24) were used. PARTICIPANTS/MATERIALS, SETTING, METHODS Extended in vitro culture until 14 d.p.f. was performed using two systems: a cell-free culture medium optimized for post-implantation development, and a 3D culture based on cell-engineered receptive endometrial scaffold technology (CREST), providing epithelial and stromal cell support. Secreted levels of CALR (calreticulin), β-hCG, human placental lactogen (hPL), pregnancy-associated plasma protein A (PAPP-A), and placental growth factor (PlGF) were quantified at different developmental time points corresponding to 7, 10, 12, and 14 d.p.f. Embryo morphometric parameters, including area, degree of expansion, axis ratio, and circularity were also measured. MAIN RESULTS AND THE ROLE OF CHANCE Embryos cultured without endometrial support showed compromised viability beyond 10 d.p.f., with significantly reduced secretion of β-hCG, hPL, PAPP-A, PlGF, and CALR. The area of post-implanted EU and T21 embryos cultured in cell-free medium progressively increased up to 9 d.p.f., unlike M21 embryos. However, all showed no changes in blastocoel expansion and maintained a rounded morphology throughout all post-implantation culture. CREST systems extended viability up to 14 d.p.f. and enhanced secretion levels of all evaluated protein markers compared with embryos cultured in cell-free conditions. Among embryos cultured in CREST systems, M21 embryos exhibited diminished hPL, CALR, and PlGF secretion as early as 7 d.p.f. At 10 d.p.f., PAPP-A levels distinguished EU from both T21 and M21 embryos. Progressive increases in PAPP-A and PlGF were observed in EU embryos until 14 d.p.f., but not in T21 embryos. Morphometric analysis revealed that only EU and T21 embryos underwent implantation-like expansion, blastocoel collapse, and increased axis ratio. In contrast, M21 embryos retained a rounded morphology and failed to expand. LARGE SCALE DATA No OMIC technique was performed in this research. LIMITATIONS, REASONS FOR CAUTION One of the main limitations of this study is the number of embryos used. Although more than 10 embryos were included in each culture condition, biological variability may still have influenced the results. This limitation is related to the difficulty of obtaining pure chromosome 21 aneuploid embryos. In addition, while the CREST system successfully recapitulates key structural features of the endometrium, it lacks endothelial and immune cell components, which may influence embryo development and implantation dynamics. Finally, we selected a targeted set of secreted proteins based on their relevance to the main objective of the study; however, the use of broader secretomic approaches could have provided a more comprehensive characterization of embryo–endometrial interactions. The findings are limited to pure chromosome 21 aneuploidies; therefore, further studies including aneuploidies affecting other chromosomes are warranted to assess the generalizability of the results. WIDER IMPLICATIONS OF THE FINDINGS The obtained results demonstrate the usefulness of the CREST system to study implantation process. Besides, these findings support the potential of secreted proteins analysis in extended culture as a non-invasive strategy to improve embryo selection beyond conventional morphological assessment. In parallel, we establish the CREST model as a translational platform to identify novel reproductive competence biomarkers and evaluate interventions aimed at improving maternal–embryo communication, not only after implantation but also during the pre-implantation phase. FUNDING This research was funded by the Institute of Health Carlos III (ISCIII) (Spanish Ministry of Science and Innovation) through the FIS program granted to F.D. (PI20/00405, PI23/00860), co-funded by the European Union, the Generalitat Valenciana through the Grant Program for Consolidated Research Groups (CIAICO/2022/203 [F.D.]), Centro para el Desarrollo Tecnológico y la Innovación (CDTI; reference number IDI-20210916) (Spanish Ministry of Science and Innovation) granted to I.S.-R. and Fundación Merck Salud through XXXI Merck Research Grant on Fertility granted to F.D. A.P. and R.Y.-S. are funded by Generalitat Valenciana Grant for the hiring of support staff linked to a technology transfer project (CIAPOT/2022/18 [A.P.]) and training research (CIACIF/2021/051 [R.Y.-S.]). S.M.-A. is funded by Institute of Health Carlos III (ISCIII) (Spanish Ministry of Science and Innovation) through the contract of training research personnel into health (FI24/00068), co-funded by the European Union. M.A.M. was supported by the Stanford Dunlevie Maternal Fetal Medicine Center for Discovery, Innovation and Impact, the Duan Family Faculty Scholar in Maternal-Fetal Medicine, the FY25 ASRM Discovery & Innovation Grant from the American Society for Reproductive Medicine, and the California Institute for Regenerative Medicine (DISC0-17685). DISCLOSURES Matteo Molè, declares that he serves as an advisory board member and consultant for Opal Therapeutics (CA, USA) and Genie Fertility (UK). He is also a co-inventor of the patent ‘A Method of Generating an In Vitro Endometrium Assembloid’ (UK Patent No. BAB-C-P3371GBp). All other authors have no financial nor competing interest to declare. TRIAL REGISTRATION NUMBER N/A.
Abstract STUDY QUESTION Do premenarcheal girls have a high proportion of morphologically abnormal ovarian follicles? SUMMARY ANSWER A higher prevalence of follicles with an abnormal number of oocytes and nuclei was found in premenarcheal ovarian tissue compared with postmenarcheal tissue. WHAT IS KNOWN ALREADY Morphological aberrations of ovarian follicles, including multi-nuclear oocytes and multi-ovular follicles, have been documented across species, including humans. These specific morphological aberrations, together with other types of abnormal follicles, are more prevalent in the ovaries of children and decline in number with age. However, detailed studies of the prevalence of multi-nuclear and multi-ovular follicles in paediatric ovarian tissue are limited. STUDY DESIGN, SIZE, DURATION This was an observational study of the ovaries of children and adolescents who underwent ovarian cryopreservation prior to chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS The study recruited participants aged 0–18 years who underwent ovarian tissue cryopreservation at a single centre. Haematoxylin and eosin-stained slides of ovarian biopsies were scanned and analysed to determine follicle number and aberrations in morphology and the cortical area was measured. Abnormal follicles were defined as those with an abnormal number of oocytes or oocyte nuclei. Relationships with menarcheal status (premenarcheal; postmenarcheal) and age were assessed. MAIN RESULTS AND THE ROLE OF CHANCE A total of 149 participants’ ovarian samples were evaluated in the study, of whom 67 (45%) had follicles with abnormal numbers of oocytes or oocyte nuclei. Amongst 117 participants who had adequate follicle numbers, these abnormal follicle types accounted for 3.8% (902/23 891) of the total number of follicles and were significantly more common in premenarcheal girls (52/80; 65.0%) compared with postmenarcheal girls (15/37; 40.5%; P = 0.013). Ovular abnormalities (in 76.7% of abnormal follicles) were more common than nuclear abnormalities (in 23.3% of abnormal follicles; P < 0.001). Bi-ovular primordial follicles were the most common abnormality, comprising 54% of the total abnormal follicle population and were present in 41.9% of participants. LIMITATIONS, REASONS FOR CAUTION Small ovarian biopsies were analysed, which may not fully represent the whole ovary. There is also a limited number of participants in each menarcheal group. WIDER IMPLICATIONS OF THE FINDINGS Our study highlights that although follicle numbers are high in the prepubertal ovary, there was also a high proportion of follicles containing abnormal numbers of oocytes and oocyte nuclei. Further longitudinal and functional studies are recommended to determine whether this impacts fertility and the longevity of function following ovarian tissue grafting for restoration of fertility in these participants. Our work supports a separate multioocytic follicle class within the NICHD follicle classification, and we suggest expanding it to include a distinct multinuclear follicle category (oocyte contains more than one nucleus), as these likely represent different aberrations in oogenesis or folliculogenesis. This classification with clear definitions and nomenclature may improve consistency in reporting and facilitate future research. FUNDING This work was supported by an MRFF Grant (MRFAR000308) supporting the funding of coauthors M.J. and M.A. Y.J. is supported through a Dame Kate Campbell Fellowship, The University of Melbourne. Co-authors V.G.-L. and J.Y.M. are supported through National Institute of Health (NIH) Z1A HD009005. DISCLOSURES No conflict of interests are declared. TRIAL REGISTRATION NUMBER n/a.
Abstract STUDY QUESTION Does endometrial compaction, assessed by transvaginal ultrasound at two measurement time points, predict ongoing pregnancy in patients undergoing single euploid blastocyst transfer in a modified natural cycle frozen embryo transfer (mNC-FET)? SUMMARY ANSWER Endometrial compaction of ≥5% between the day of ovulation triggering and the day of embryo transfer was not associated with ongoing pregnancy rate or any secondary reproductive outcome in this prospective multicentre cohort. WHAT IS KNOWN ALREADY Endometrial compaction—a reduction in endometrial thickness (EMT) following progesterone-induced secretory transformation—has been proposed as a potential marker of endometrial receptivity in FET cycles. However, existing evidence is conflicting. Retrospective studies have reported positive associations between compaction and ongoing pregnancy in programmed cycles, while prospective data, including the only prior study restricted to euploid single FET, have not confirmed this relationship. Crucially, most studies have used transabdominal ultrasound for the transfer-day measurement, a recognized source of measurement error. No prospective study has examined compaction exclusively in mNC-FET using transvaginal ultrasound at both time points. STUDY DESIGN, SIZE, DURATION Prospective multicentre observational cohort study conducted between March 2023 and March 2025 across six assisted reproduction units of Instituto Bernabeu group (Spain). A total of 207 patients who met all inclusion criteria and completed the full protocol were analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS Women aged 18–50 years undergoing mNC-FET with a single euploid blastocyst (confirmed by preimplantation genetic testing for aneuploidy via next-generation sequencing) were included. EMT was measured transvaginally at T1 (day of hCG trigger) and T2 (day of embryo transfer). Compaction was defined as a ≥5% reduction in EMT from T1 to T2. Additional thresholds of ≥10% and ≥15% were evaluated as sensitivity analyses. The primary outcome was ongoing pregnancy, defined as foetal cardiac activity at 10 weeks of gestation. Multivariable logistic regression was performed, adjusting for age, previous live births, previous miscarriages, BMI, and oocyte origin. MAIN RESULTS AND THE ROLE OF CHANCE Of 207 patients, 58 (28.0%) met the compaction criterion (≥5%). The compaction group had a significantly greater T1 EMT (9.55 ± 1.54 mm vs 8.39 ± 1.14 mm, P < 0.001) and a lower T2 EMT (7.91 ± 1.19 mm vs 9.75 ± 1.55 mm, P < 0.001). Ongoing pregnancy rates were 50.0% in the compaction group versus 53.7% in the non-compaction group (P = 0.633). This difference was not significant on multivariable analysis (adjusted odds ratio 0.80, 95% CI 0.38–1.67, P = 0.546). The null association was consistent across all compaction thresholds (≥10%: P = 0.674; ≥15%: P = 0.928) and across all secondary outcomes, including clinical pregnancy rate (56.9% vs 59.1%, P = 0.777), biochemical pregnancy loss (10.8% vs 8.3%, P = 0.737), and early pregnancy loss rate (12.1% vs 9.1%, P = 0.734). The overall ongoing pregnancy rate was 52.7% (109/207). LIMITATIONS, REASONS FOR CAUTION The distribution between groups was asymmetric (28% compaction vs 72% non-compaction), deviating from the balanced allocation anticipated at protocol design, which may reduce statistical power. Although both measurements were transvaginal, a subset of T1 measurements from international patients were performed at external centres and validated remotely by Instituto Bernabeu’s physicians. Live birth data were only partially available at the time of analysis; however, ongoing pregnancy at 10 weeks is a validated and widely accepted surrogate for live birth in reproductive medicine trials. WIDER IMPLICATIONS OF THE FINDINGS These findings do not support the hypothesis that endometrial compaction is associated with improved reproductive outcomes in mNC-FET with euploid embryos. The consistent null result across multiple thresholds and outcomes, obtained with a fully standardized transvaginal ultrasound methodology, strengthens the current evidence base. Routine assessment of endometrial compaction with the aim of identifying patients with a favourable prognosis does not appear to be clinically justified in this setting; indeed, acting upon compaction status could lead to unnecessary cycle cancellations in patients who would otherwise achieve ongoing pregnancy. FUNDING This study was supported by a Doctoral Research Grant awarded by Miguel Hernandez Public University, Spain (Rectoral Resolution No. 04588/2021). The funding body had no role in the study design, data collection, analysis or interpretation of the data, preparation of the manuscript, or decision to submit the manuscript for publication. DISCLOSURES The authors declare no competing interests related to the present study. No personal payments, financial incentives, or commercial benefits were received by any author in relation to this work. TRIAL REGISTRATION NUMBER NCT05960370.
Abstract STUDY QUESTION What are the genetic causes of asthenoteratozoospermia in consanguineous families, and what is the specific role of cilia- and flagella-associated proteins (CFAP) 46 in mammalian spermiogenesis? SUMMARY ANSWER CFAP46 is a central apparatus protein essential for sperm flagellar assembly and head remodeling. WHAT IS KNOWN ALREADY Asthenoteratozoospermia is a genetically heterogeneous condition characterized by reduced sperm motility and abnormal morphology. While several CFAPs are known to support axonemal organization, the functional role of CFAP46 in mammals remained uncharacterized prior to this study. STUDY DESIGN, SIZE, DURATION We performed whole-genome or whole-exome sequencing on 13 consanguineous families affected by primary male infertility. Functional validation was conducted using three CRISPR/Cas9-engineered mouse models: a null allele, a FLAG-tagged knock-in, and a humanized patient-specific nonsense allele. PARTICIPANTS/MATERIALS, SETTING, METHODS Genomic DNA from affected individuals and fertile relatives was analyzed by NGS and validated via Sanger sequencing. Mouse phenotypes were characterized through sperm analysis, immunofluorescence, and electron microscopy. Protein interactions were identified via immunoprecipitation and mass spectrometry. Additionally, a gene-level burden analysis was applied to unresolved cases across 345 spermiogenesis-associated genes. MAIN RESULTS AND THE ROLE OF CHANCE We identified homozygous loss-of-function variants in CFAP46, CFAP57, SEPTIN4, and SPEM2 segregating with infertility. Cfap46-deficient males were infertile due to significantly decreased sperm count, motility, and severe axonemal disruptions. CFAP46 was found to form an in vivo complex with CFAP54, CFAP74, and CFAP221. In families without a monogenic cause, affected individuals harbored a significantly higher burden of rare deleterious variants (4–16 genes) compared to fertile controls. LARGE SCALE DATA Large-scale datasets and scripts are available via GitHub (https://github.com/sirusb/Nawaz_et_al_scripts). LIMITATIONS, REASONS FOR CAUTION While functional mouse data strongly supports the identified variants, the rare-variant burden analysis identifies associations that require further functional validation to confirm specific oligogenic interactions. WIDER IMPLICATIONS OF THE FINDINGS This study establishes CFAP46 as a regulator of mammalian spermiogenesis and suggests that, beyond monogenic defects, aggregated rare-variant burden may also contribute to male infertility. These findings refine the genetic basis of asthenoteratozoospermia, supporting precise molecular diagnosis, individualized prognostic counseling, and informed use of assisted reproduction (e.g. ICSI) in affected couples. FUNDING This work was supported by Sidra Medicine grant SDR400185 (to M.A.A.), the FERTILITY-IQ Study and Qatar National Research Fund (QNRF) grant NPRP12S-0318-190394 (to M.A.A. and K.F.), the Qatar Research, Development and Innovation (QRDI) grant GSRA12-I-0311-250014 (to M.A.Abd.), the Quaid-i-Azam University Research Project (URF-QAU) 2020-21 (to I.U.), Japan Society for the Promotion of Science KAKENHI grants JP24K12461 (to H.K.), JP23K18328 (to H.M.), and JP21H05033 and JP23K20043 (to M.I.), Japan Agency for Medical Research and Development (AMED) grants JP23jf0126001 and JP26gn0110011 (to M.I.), and JSRM Ferting Grant 2025 (to H.K.). Open access funding was provided by the Qatar National Library. DISCLOSURES The authors declare no competing interests.
Abstract Polycystic ovary syndrome (PCOS) has long been focused on the ovary and infertility aspects, rooted in the early observations by Stein and Leventhal (1935). The aggregated data, however, show that the syndrome has a wide comorbidity risk beyond fertility aspects involving several endocrine and metabolic derangements, yet without a true risk of ovarian cysts, benign or pathological, as expected with the old name. Moreover, with modern fertility care, women with the condition can reach cumulative childbirth rates comparable to those of unaffected women. The women may have spontaneous ovulations and pregnancies across the reproductive life course and, having high ovarian reserve, enter the menopause later. The endometrium and pregnancy complications have, however, been the blind spots in fertility care. Taking all this into account, in May 2026, a multi-step global consensus process culminating in a Lancet statement renamed the condition polyendocrine metabolic ovarian syndrome (PMOS), with transitional adoption already underway in current international guidance and consolidation into the next major international evidence-based guideline update scheduled for 2028. This opinion argues that the rename is not a cosmetic relabelling but a clinical mandate to widen fertility and pregnancy care, address fragmented care across primary, fertility, obstetric, and endocrine services, and acknowledge the endocrine, metabolic, psychological, and oncological burden. For Human Reproduction’s readership, the 2028 guideline update is the concrete deadline around which language, registry coding, trial protocols, education, and clinical practice can align.
