The PLCζ p.Arg385Gln (R385Q) variant was recently identified in infertile patients with recurrent fertilization failure and markedly reduced sperm PLCζ levels. However, whether this variant causes infertility through loss of catalytic function or protein insufficiency remains unknown. The structural and functional consequences of the R385Q variant were investigated using structural modelling, protein stability prediction, recombinant PLCζ enzymatic assays, and mouse oocyte microinjection followed by Ca2+ oscillation analysis. Structural modelling predicted that the R385Q substitution destabilizes the catalytic domain without disrupting the active site. Consistent with this prediction, recombinant PLCζR385Q exhibited phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolytic activity comparable to wild-type PLCζ. Likewise, microinjection of PLCζR385Q cRNA into mouse oocytes induced Ca2+ oscillations with frequencies and patterns indistinguishable from those elicited by wild-type PLCζ, demonstrating preserved catalytic and oocyte activation activity. These findings indicate that the R385Q variant is catalytically competent and does not cause infertility through loss of enzymatic function. Instead, together with our previous demonstration of markedly reduced PLCζ protein levels in patient sperm, the results support a protein insufficiency mechanism in which fertilization failure arises when PLCζ levels fall below the threshold required for physiological oocyte activation. This study expands the spectrum of pathogenic mechanisms underlying PLCζ-associated male infertility.
To investigate the association between maternal preconception body mass index (BMI) and offspring sex ratio among women undergoing intrauterine insemination (IUI) treatment. This retrospective cohort study analyzed 17,116 IUI cycles from 9581 women treated at a single large fertility center between 2015 and 2024. Maternal preconception BMI, measured at the start of each IUI cycle, was categorized into four groups: underweight (< 18.5 kg/m2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2), and obesity (≥ 30.0 kg/m2). The primary outcome was the proportion of male live births, with secondary sex ratio (SSR; male births per 100 female births) reported descriptively. Associations were estimated using generalized estimating equations to account for repeated cycles per patient, with adjustment for potential confounders using inverse probability weighting based on the generalized propensity score. Among 3224 live-born infants with documented sex, 1629 (50.5
To elucidate the genetic etiology of osteogenesis imperfecta (OI) in affected families and to characterize reproductive decision-making, reproductive interventions, and pregnancy outcomes of these families. Fifty-one families with clinically suspected OI underwent variant analysis by Sanger sequencing or next-generation sequencing (NGS) and clinical follow-up of reproductive options and pregnancy outcomes. Of the 51 enrolled families, 37 probands had mild phenotypes and 14 presented with severe phenotypes. Genetic analysis revealed that 46 probands (90.2
The aim of this study was to examine POC patient experience at multiple stages throughout the course of a treatment cycle. Email invitations were sent to 359 POC patients from November 2024 to August 2025 for this longitudinal, survey-based cohort study. Surveys were distributed at three different points throughout a POC treatment cycle: pre-cycle, mid-cycle and post-cycle. The survey questions related to the experiences of the participant in relation to eight core aspects of clinical treatment. Participants were asked to rate their experience (difficulty) and feelings on a scale of 1 to 10. Responses of first and subsequent cycle POC participants were compared for each aspect of treatment. A total of 101 participants were recruited (28
Assisted reproductive techniques (ART) are increasingly used worldwide. Concerns persist regarding the long-term cardiovascular health of ART-conceived offspring. To systematically review and meta-analyze cardiovascular, vascular, and echocardiographic outcomes in ART-conceived offspring compared with naturally conceived children. PubMed and Scopus were searched from database inception to January 2025. Reference lists of eligible articles were manually screened. Searches included terms related to assisted reproductive technologies, offspring, and cardiovascular outcomes. Only human studies published in English were included. Observational cohort, cross-sectional, and case–control studies evaluating cardiovascular outcomes in ART-conceived offspring with a naturally conceived comparison group were eligible. Studies without relevant outcomes, lacking a control group, or including populations with genetic conditions affecting cardiovascular health were excluded. Two reviewers independently screened studies, with disagreements resolved by a third investigator. Eighty-one studies met inclusion criteria. Data extraction was independently performed by two reviewers following PRISMA and MOOSE guidelines. Risk of bias and study quality were assessed using the Cambridge Quality Checklist. Meta-analyses were conducted using fixed- or random-effects models according to heterogeneity (I2 statistic). Meta-regression analyses explored the influence of parental and perinatal factors. Primary outcomes included vascular indices (carotid intima-media thickness [CIMT], flow-mediated dilation [FMD]), risk of congenital heart disease (CHD), echocardiographic measures of cardiac structure and function, and blood pressure. ART conception was associated with increased CIMT in singleton offspring (mean difference, 0.03 mm; 95
Polyendocrine metabolic ovarian syndrome (PMOS), an endocrine disorder with unknown aetiology is the leading cause of anovulatory infertility. N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, regulates multiple aspects of RNA metabolism and reproductive function. However, the contributions of m6A modification and its regulatory factors including microRNAs to the pathophysiology of PMOS remain poorly understood. We therefore investigated global m6A abundance, expression of key m6A regulatory genes, and selected experimentally validated m6A-associated microRNAs in granulosa cells (GCs) from women with PMOS. GCs were collected from 23 women with PMOS and 23 age- and BMI- matched controls undergoing in vitro fertilization. Global m6A modification was quantified by ELISA, whereas the m6A writer, reader, eraser (WRE) protein-coding genes and selected microRNAs were assessed by RT-qPCR. The total m6A levels were significantly increased in GCs from women with PMOS. Transcript encoding the core writer complex (METTL3, METTL14, WTAP, RBM15, VIRMA) and reader proteins (YTHDF1, YTHDF3, YTHDC1) were upregulated, whereas transcripts encoding the eraser proteins (FTO, ALKBH5) were downregulated. Elevated m6A, METTL3, METTL14 and YTHDF1 levels positively correlated with androgen excess, anti-Müllerian hormone and reduced oocyte fertilization rates. Among the experimentally validated m6A-associated microRNAs, miR-20b, whose maturation is regulated by METTL3, was downregulated, whereas miR-607, a validated regulator of FTO, was upregulated in PMOS. Coordinated alterations in global m6A abundance, m6A regulatory genes and the selected microRNAs indicate widespread epitranscriptomic dysregulation in GCs from women with PMOS, providing a foundation for future mechanistic and therapeutic studies.
Environmental microbiological monitoring is mandatory in IVF laboratories; however, procedure-specific risk assessment across the entire medically assisted reproduction workflow remains limited. This multicenter study, named “SMILE” (Study of Environmental Microbiological Monitoring in IVF Laboratories), aimed to assess settle plate positivity as a surrogate marker of environmental microbial contamination risk, in compliance with the guidelines of the European Directorate for the Quality of Medicines HealthCare (EDQM). Passive air monitoring using settle plates was performed during individual IVF laboratory procedures under different environmental cleanliness conditions. Nine IVF laboratories in Northern Italy prospectively collected environmental microbiological monitoring data between 2023 and 2025. Passive air monitoring using settle plates was performed during seven routine IVF procedures. Monitoring was conducted either under GMP Grade A laminar airflow or at the micromanipulation workstation in the validated GMP Grade D background environment (ICSI and embryo biopsy). A total of 2146 sentinel settle plates were exposed and incubated according to EDQM recommendations. Contamination probability was defined as the proportion of plates with ≥ 1 colony-forming unit (CFU), with exact 95
To study whether there is a marked interobserver discrepancy in the diagnosis of adenomyosis based on reviewing stored sonographic images of infertile patients? Three reproductive endocrinology and infertility specialists reviewed stored ultrasound images of 2134 patients attending tertiary IVF-centers. The physicians were blinded to each other as well as to the original diagnosis. The revised Morphological Uterus Sonographic Assessment (MUSA) criteria was used. The overall three-rater agreement for adenomyosis (Yes/No) was 62.3
Biallelic variants in FSIP2 have been associated with multiple morphological abnormalities of the sperm flagella (MMAF) and acrosomal defects. This study aimed to characterize the genetic, sperm phenotypic, and reproductive features of infertile men carrying homozygous FSIP2 variants, with particular attention to sperm nuclear ultrastructure and chromatin condensation. Two infertile men with severe sperm morphological abnormalities were enrolled. Whole-exome sequencing and Sanger sequencing were performed to identify candidate variants. Sperm morphology, FSIP2 expression, acrosomal status, nuclear ultrastructure, and chromatin condensation were evaluated using routine semen analysis, immunofluorescence staining, transmission electron microscopy, and chromomycin A3 (CMA3) staining. Intracytoplasmic sperm injection outcomes were reviewed. Two homozygous FSIP2 variants were identified in two men with primary infertility, including a frameshift variant, NM_173651.3: c.2519delA, p.(Asn840Metfs*43), and a missense variant, NM_173651.3: c.17798C > T, p.(Ser5933Phe). The missense variant, previously reported in a compound heterozygous context, was identified here in a homozygous state. Spermatozoa from both patients exhibited typical MMAF phenotypes and markedly reduced or absent FSIP2 signals. Acrosomal loss or abnormal acrosomal localization was also observed. Notably, spermatozoa from both patients showed prominent intranuclear vacuoles and increased CMA3 staining, suggesting defective chromatin condensation. After ICSI, both couples achieved fertilization, and one couple achieved a live birth. These findings suggest an expansion of the genotypic and phenotypic spectrum of FSIP2-associated male infertility. In the two cases studied, biallelic FSIP2 variants were associated with classical MMAF and acrosomal abnormalities, as well as with abnormal nuclear ultrastructure and increased CMA3 staining indicative of impaired chromatin condensation. However, further studies are needed to establish whether these nuclear features represent a direct consequence of FSIP2 deficiency.
Polycystic ovary syndrome (PCOS) and ovarian hyperstimulation syndrome (OHSS) are characterized by excessive follicular activity and profound alterations in the ovarian microenvironment. Kisspeptin and vascular endothelial growth factor (VEGF) play central roles in the regulation of folliculogenesis, angiogenesis, vascular permeability, and ovarian responsiveness. This study aimed to compare follicular fluid levels of Kisspeptin-1, Kisspeptin-54, and VEGF among women with PCOS, women who developed OHSS, and normoresponder controls, and to investigate their associations with established markers of ovarian response. In this retrospective cross-sectional study, 90 women undergoing IVF treatment were stratified into three groups: normoresponder controls (n = 30), women with PCOS (n = 30), and patients who developed OHSS (n = 30). Follicular fluid samples were collected at the time of oocyte retrieval. Concentrations of Kisspeptin-1, Kisspeptin-54, and VEGF were quantified using validated enzyme-linked immunosorbent assay (ELISA) methods. Clinical, hormonal, and embryological parameters were collected and analyzed using group comparisons, correlation analyses, and multivariable logistic regression models. Follicular fluid concentrations of Kisspeptin-1, Kisspeptin-54, and VEGF differed significantly among study groups (all p < 0.001), demonstrating a progressive increase from normoresponder controls to women with PCOS and those who developed OHSS. Kisspeptin-54 and VEGF exhibited strong positive correlations with established indicators of ovarian response, including AMH, AFC, trigger-day estradiol levels, total oocyte yield, MII oocyte count, and the number of good-quality embryos (r = 0.41–0.78, all p < 0.001). In multivariable logistic regression analyses, Kisspeptin-54 (OR = 1.031) and VEGF (OR = 1.008), together with AFC, AMH, trigger-day estradiol, and total oocyte count, remained significantly associated with OHSS occurrence (all p < 0.01). Follicular fluid levels of Kisspeptin-1, Kisspeptin-54, and VEGF are significantly elevated in ovarian hyperresponse conditions and exhibit strong associations with key indicators of ovarian stimulation outcomes. These findings suggest that Kisspeptin-54 and VEGF reflect the biological features of ovarian hyperresponsiveness and may serve as complementary biomarkers of the follicular microenvironment, rather than clinically actionable predictors of OHSS. Follicular fluid Kisspeptin-1, Kisspeptin-54, and VEGF levels increase progressively from normoresponder women to patients with PCOS and OHSS. The coordinated elevation of Kisspeptin-54 and VEGF parallels increasing ovarian responsiveness and is associated with established ovarian response parameters, supporting the concept of a follicular Kisspeptin–VEGF axis as a biological signature of the hyperresponsive ovarian microenvironment.
To characterize the occurrence, developmental competence, and ploidy outcomes of abnormally fertilized oocyte (AFO) subtypes in ICSI/PGT-A cycles, and to assess whether maternal age independently predicts abnormal fertilization. Prospective observational study including 1033 ICSI/PGT-A cycles (6373 inseminated oocytes). AFO occurrence was analyzed by binomial logistic regression and multivariable analysis. In 664 time-lapse cycles, pronuclear status was reassessed at 22–24 hpi (Check II) after routine evaluation at 16–18 hpi (Check I). Ploidy analysis was performed on 78 AFO-derived blastocysts by SNP genotyping using NGS technology. AFOs accounted for 6.8
Long non-coding RNAs (lncRNAs) are key regulators of transcriptional, epigenetic, and post-transcriptional processes involved in implantation, trophoblast function, and early placental development. Genetic polymorphisms within lncRNA loci may alter their expression or regulatory activity, potentially contributing to recurrent pregnancy loss (RPL). We aimed to evaluate the association between lncRNA gene polymorphisms and susceptibility to RPL. A systematic review and meta-analysis were conducted according to PRISMA and MOOSE guidelines and prospectively registered in PROSPERO (CRD420261298804). A systematic search was performed in PubMed, Embase, Scopus, and Web of Science from database inception to January 2026, without language or date restrictions. Two reviewers independently screened studies, extracted data, and assessed risk of bias using ROBINS-E. Meta-analyses were performed using fixed- or random-effects models according to heterogeneity. Hardy–Weinberg equilibrium testing, sensitivity analyses, subgroup analyses by ancestry, and certainty of evidence assessment using the GRADE framework were conducted. Sixteen case–control studies involving 11,088 participants were included, comprising 5169 women with RPL and 6454 controls. Eight polymorphisms across four lncRNA genes were quantitatively synthesized. Significant associations with increased RPL susceptibility were observed for HOTAIR rs4759314 (AG vs AA: OR, 2.75 [95
Maintaining physiological extracellular pH (pHe) is essential for oocyte viability and embryonic development, particularly during ICSI, when metaphase II oocytes have their intracellular pH (pHi) regulation mechanisms temporarily inactivated. HEPES- and MOPS-buffered handling media are routinely used in IVF laboratories, with decades of clinical use supporting their effectiveness and safety. Although concerns regarding their biological effects beyond pH buffering have occasionally emerged, most are derived from experimental settings and are not representative of clinical IVF procedures, more likely influenced by experimental conditions and media composition rather than by the zwitterions themselves. Nevertheless, while evidence from human oocytes remains extremely limited and preliminary, the improved embryological outcomes and transcriptomic profiles recently reported from bicarbonate-buffered handling media have renewed interest in exploring more physiological ICSI media formulations. While current evidence remains far from conclusive and insufficient to support immediate changes in clinical practice, emerging findings suggest that routine systems may not yet be fully optimized and could represent an opportunity for further improvement. Future evaluation of bicarbonate-buffered media for ICSI should include robust standardization of pH measurement methodologies together with optimization of handling conditions, including oil and dish characteristics, that influence pHe stability.
To develop a stage-structured, distribution-based prediction framework for in vitro fertilization (IVF) that generates full probability distributions at each treatment stage and enables conditional updating of downstream predictions when observed outcomes become known. We conducted an observational modeling study using de-identified UK Human Fertilisation and Embryology Authority (HFEA) registry data (2017–2018; up to 101,217 model-ready cycles after stage-specific filtering) to model egg retrieval, maturation, and fertilization. Egg retrieval was modeled using a zero-inflated negative binomial specification. Downstream transitions such as blastocyst formation, euploidy, vitrification survival, and live birth after euploid transfer were modeled using stage-specific logistic regressions calibrated to published cohorts and national registry summaries (429,507 additional observations). Predictive performance of HFEA-derived models was evaluated on held-out HFEA test sets using point-prediction accuracy, train-test gaps, prediction-interval coverage, and calibration across predicted outcome strata. The framework propagates full probability distributions across sequential IVF stages rather than point estimates. Held-out HFEA validation showed minimal train-test degradation ( R^2 gaps under 0.007), with modest expected-count accuracy for egg retrieval and stronger expected-count accuracy for maturity and fertilization. Egg-retrieval prediction intervals showed near-nominal coverage (50
The clinical application of sequencing guidelines often yields variants of uncertain significance (VUS), creating profound challenges in prenatal genetic counseling. This study demonstrates the clinical utility and real-world impact of dynamic, longitudinal variant re-classification on reproductive decision-making in a family affected by an atypical SEC24D-associated skeletal phenotype. Over a 3-year period (2023–2026), a multidisciplinary diagnostic workflow was employed, integrating trio whole-exome sequencing, protein structural modeling, prenatal ultrasound, fetal magnetic resonance imaging, post-mortem histopathology, and preimplantation genetic testing for monogenic disorders (PGT-M). Variant pathogenicity was sequentially curated according to the American College of Medical Genetics and Genomics (ACMG) framework. A homozygous SEC24D variant (c.1942G > C, p.Gly648Arg) was identified in a proband presenting with isolated craniofacial ossification defects without classic long-bone fractures. Over 3 years, the variant was dynamically re-classified from VUS to likely pathogenic (LP), back to VUS, and to LP again, driven by institutional peer-review debates regarding the conservative application of computational structural criteria versus clinical intuition. This diagnostic instability directly dictated a cascade of critical reproductive events: the termination of a second pregnancy demonstrating recurrent fetal cranial defects confirmed by amniocentesis and post-mortem examination, followed by the deployment of PGT-M within a diagnostic “gray zone.” Ultimately, the successful delivery of a healthy, non-carrier infant in June 2026 provided crucial family co-segregation data, successfully resolving the classification loop and securing a definitive likely pathogenic status. Diagnostic uncertainty in prenatal genomics is a persistent clinical reality rather than a transient evidence gap. Longitudinal variant reinterpretation combined with transparent shared decision-making is vital to safely navigate borderline classifications and optimize reproductive trajectories.
Similar live birth rates in frozen embryo transfer (FET) trials can be over-read as evidence that natural, modified natural, and programmed endometrial preparation protocols are biologically equivalent. This interpretation is not warranted when cancellation, delayed transfer, and rescue switching are built into care. Under these conditions, randomization compares starting strategies that unfold through managed clinical pathways, not uninterrupted protocols completed exactly as assigned. Using recent FET trials as practical examples, this Clinical Opinion argues for a narrower and more constructive interpretation: similar live birth supports pathway effectiveness only for the estimand that was actually analyzed. Clinicians should ask whether the reported effect reflects assignment to an initial strategy, completion of the first assigned protocol, or a hypothetical no-switch contrast. Cancellation and rescue switching should be treated as outcomes; pathway burden should be reported alongside live birth, and safety outcomes should use denominators aligned with the stage at which harms occur. These FET-specific reporting recommendations apply existing CONSORT, CONSORT Harms, and ICH E9(R1) principles to trials in which cancellation or switching can materially affect interpretation.
To assess the extent to which expanded carrier screening (ECS) panels capture the genetic conditions for which preimplantation genetic testing for monogenic diseases (PGT-M) is performed. A retrospective observational study conducted at a single university-affiliated PGT unit from 2015 to 2023. Women undergoing PGT-M for autosomal recessive or X-linked genetic conditions were included. Genetic conditions were cross-matched with five ECS panels (four commercial panels and one publicly funded ECS panel) and compared with a targeted, population-specific screening. Coverage was assessed at both the woman and gene levels using paired statistical comparisons. A total of 330 cases were included; seven women underwent PGT-M for two eligible conditions and were therefore counted twice. These cases involved 110 different genes with an uneven distribution: eight genes accounted for 53.2
To systematically evaluate the diagnostic accuracy and methodological quality of machine learning (ML) prediction models for pregnancy outcomes after assisted reproductive technology (ART). PubMed, Embase, the Cochrane Library, IEEE Xplore, MEDLINE, ClinicalTrials.gov, CNKI, Wanfang, and VIP were searched from inception to July 2026. Eligible studies developed or validated ML models to predict clinical pregnancy or live birth after ART. For studies reporting complete 2 × 2 contingency data, pooled sensitivity, specificity, diagnostic odds ratio (DOR), and summary receiver operating characteristic (SROC) curves were estimated using random-effects diagnostic meta-analysis. Risk of bias was assessed with PROBAST. Twenty studies were included in the systematic review, of which 14 contributed to the diagnostic meta-analysis. Overall risk of bias was low in 1 study (5.0
