The current study aimed to investigate whether cathepsin B (cath B) inhibition in cumulus cells of morphologically graded buffalo cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) could modulate apoptotic markers and enhance developmental potential. Cumulus cells from grade A and B buffalo COCs were collected at 0, 12, and 24h of IVM, with or without supplementation of the optimized cysteine protease inhibitor E-64. Cathepsin B and selected apoptotic regulators were quantified by qRT-PCR. Cathepsin B protein levels and apoptotic signals were assessed by immunostaining and TUNEL assay, respectively. Following 24h of IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were performed, and developmental competence was evaluated based on oocyte maturation, cleavage rate, blastocyst formation rate, and blastocyst quality. Expression of cath B and pro-apoptotic genes (BID, BCL2, BAX, caspase-3) was found higher along with lower expression of anti-apoptotic genes (BCL2 and XIAP) in the cumulus cells of B grade COCs. The addition of 10µM E-64 significantly (P < 0.05) reduced cath B and pro-apoptotic gene expression, lowered the BAX/BCL2 ratio, and decreased apoptosis signals in cumulus cells of both COC grades. E-64 supplementation improved blastocyst yield from grade A COCs and enhanced blastocyst quality in both grades, without affecting maturation and cleavage rates. E-64 supplementation during IVM modulates apoptotic markers in cumulus cells of different grades of buffalo COCs by inhibiting cath B, thereby enhancing the developmental competence and offering valuable insights for improving in vitro embryo production programs in this species.

To assess whether the endometrial receptivity analysis (ERA) captures receptivity changes attributed to endometrial aging and whether it may be useful for older patients undergoing fertility treatment. Retrospective cohort study of patients who underwent ERA testing at an academic center (01/2019-05/2024). The ERA inferred transcriptomic levels of canonical receptivity markers from biopsies obtained at standard timing. Demographic and treatment-related variables were analyzed by age group. The proportion of non-receptive ERA results (pre- and post-receptive combined) was compared using Fisher's exact test. Univariable and multivariable logistic regression assessed associations between predictors and non-receptive ERA. Of 210 patients, 205 were included. Age distribution was < 35 (n = 35, 17%), 35-37 (n = 58, 28.3%), 38-40 (n = 53, 25.8%), and ≥ 41 (n = 59, 28.8%). Overall, 166 (81.0%) ERAs were receptive, 33 (16.1%) pre-receptive, and 6 (2.9%) post-receptive. BMI, infertility diagnosis, and prior implantation or miscarriage history did not differ by age. Non-receptive ERA proportions were 20% (< 35), 17.2% (35-37), 17.0% (38-40), and 22.0% (≥ 41) (p = 0.52). In multivariable analysis adjusting for BMI and number of prior failed euploid transfers, age was not associated with non-receptive ERA (aOR 0.98, 95% CI 0.34-2.30, p = 0.97). Uterine age was not associated with increased odds of non-receptive ERA, suggesting that the test does not capture age-related changes in endometrial receptivity. Although endometrial aging is implicated in reduced embryo transfer success, the ERA should not be ordered solely on the basis of uterine age. The ERA may not reliably detect age-related endometrial differences in the window of implantation at a clinical level.

To compare dual triggering versus hCG-only triggering in poor responders undergoing controlled ovarian stimulation with a GnRH antagonist protocol in ART cycles. A systematic review and meta-analysis was performed. The primary outcome was the number of mature oocytes (MII). The number of oocytes retrieved, clinical pregnancy, and miscarriage rates were analyzed as secondary outcomes. Subgroup analyses were conducted according to the Bologna and POSEIDON classifications. Ten studies were included. Dual triggering significantly increased the number of retrieved and mature oocytes in patients classified according to the Bologna criteria, but not in those classified by the POSEIDON criteria. Age-related differences across studies appeared to influence the efficacy of dual triggering. A borderline improvement in clinical pregnancy rates was observed among Bologna-defined patients. Miscarriage rates did not differ significantly between the groups. Dual triggering appears to improve oocyte yield and maturity in Bologna-defined patients, but this effect is unlikely to apply uniformly across all phenotypes. Persistent heterogeneity in poor responder definitions limits the understanding of the benefits of dual triggering in this population. Well-designed prospective studies with rigorous phenotypic stratification are warranted to identify which patients are most likely to benefit from dual triggering.

Can sperm selection through cumulus cells improve embryo quality compared to conventional methods, and is its effectiveness influenced by parental age? This prospective clinical trial included 99 ICSI cycles from 95 couples. Sibling oocytes were randomly allocated at the oocyte level to either the study group (cumulus cell-mediated sperm selection after conventional density gradients centrifugation (DGC), 554 oocytes) or the control group (only sperm selection by DGC, 543 oocytes), using a dish designed to facilitate sperm interaction with cumulus cells. The inclusion criteria for this study were patients using their own oocytes, with a medical indication for ICSI, who had at least 6 mature oocytes (MII) in that cycle. For semen samples, inclusion required the ability to adjust the concentration to 10 million/mL. Exclusion criteria included the use of vitrified oocytes, donated oocytes, and semen samples obtained by testicular biopsy or aspiration. Embryo quality was assessed at the blastocyst stage on day 5 according to ASEBIR. A subanalysis evaluated the influence of parental age on outcomes. The study group showed a significantly higher proportion of good-quality day-5 blastocysts compared to controls (55.2% vs. 45.3%, p = 0.028). No statistically significant differences were observed in overall blastocyst formation or pregnancy rates, although favourable trends were noted. In an age-stratified analysis, a significant improvement in day-5 blastocyst quality among evaluable blastocysts was observed in women aged 40-45 (51.4% vs. 30.4%, p = 0.017), with a non-significant trend toward improved outcomes in men aged 40-53 (44.7% vs. 32.6%, p = 0.083). No differences were seen in younger age groups. Cumulus cell-mediated sperm selection after DGC using a specialized Oosafe® ICSI Dish with Sperm Selection Channels was associated with an increased proportion of good-quality day 5 blastocysts compared with conventional sperm preparation. While clinical outcomes did not differ significantly, these findings suggest a potential benefit in specific ART subpopulations, particularly those of advanced maternal age. Further adequately powered studies are required to confirm these observations and to determine their impact on clinical outcomes.

Oocyte maturation defects are one of the common clinical phenotypes contributing to recurrent IVF/ICSI failures and female infertility. We aimed to identify novel pathogenic variants responsible for oocyte maturation defects. Whole-exome sequencing and in silico analysis were used to identify variants in disease-causing genes in a cohort of 442 female infertility patients with oocyte maturation defects. In vitro functional studies were performed to validate the destructive effects and pathogenicity of the novel variants. We identified five novel PABPC1L variants (c.280C > A (p.Arg94Ser), c.283A > T (p.Lys95*), c.611A > G (p.Glu204Gly), c.583G > T (p.Val195Leu), and c.691C > T (p.Arg231Trp)) in two independent infertile families. These variants followed an autosomal recessive inheritance pattern. Western blotting analysis confirmed that all the novel variants led to the downregulation of PABPC1L protein expression in HEK293T cells. Immunofluorescence results showed that the nonsense variant c.283A > T (p.Lys95*) altered the cytoplasmic localization of PABPC1L in HeLa cells. In addition, three-dimensional molecular modeling and cycloheximide chase assay indicated that all four missense variants impair the stability of the PABPC1L protein. Our findings expand the mutational spectrum of the pathogenic gene PABPC1L, further highlight its essential function in female reproduction, and provide new support for the genetic diagnosis of female infertility.

Infertility affects 1 in 6 couples, yet male fertility assessments often rely on semen analysis alone, which has limited predictive value for treatment success. This study evaluates an epigenetic tool for assessing sperm quality, aiming to provide a more comprehensive view of male fertility and improve personalized treatment strategies. De-identified pregnancy outcomes from 537 couples treated at 10 US fertility clinics were analyzed. Partner ages, total motile sperm count, treatment type, and sperm epigenetic quality were considered to assess associations with fertility treatment outcomes. Men with abnormal sperm epigenetic profiles had significantly lower pregnancy success rates with intrauterine insemination (IUI) compared to those with normal profiles, despite similar sperm motility and concentration. In contrast, pregnancy rates did not differ between abnormal and normal profiles among couples undergoing in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI), suggesting that ICSI may overcome sperm epigenetic quality issues. This real-world analysis was limited by the availability of detailed clinical and phenotypic data, which may introduce potential confounders. Nevertheless, the findings highlight the clinical value of epigenetic sperm assessment as part of male fertility evaluation and support its potential to guide more effective, personalized fertility treatment pathways.

It is widely accepted that pursuing fertility treatment is a costly endeavor, however, there are limited reports on the indirect costs of pursuing treatment-including the impact on professional careers, and costs of time away from work or home required to pursue fertility care. A 36-question survey was developed in RedCap and distributed to patients at the Massachusetts General Hospital (MGH) Fertility Center, following exemption from the IRB. This study included English speaking patients, aged > 18 and < 50years old, undergoing IVF cycles between August 1 and December 31, 2024. Two hundred fifty-one survey invitations were sent through Epic Gateway patient messages with 54 unique responses (22% response rate) after trigger before oocyte retrieval. Self-reported impact of fertility care on professional lives including time away from work, additional financial burden, and workplace privacy. Distance and time spent travelled were calculated through GoogleMaps with cost estimates generated using IRS cost/mile and lost work hours. Forty-seven respondents (90%) reported missing work to attend appointments. Additionally, 31 (60%) patients reported attending appointments affected their privacy at work and 26 (50%) had to disclose the reason for missing work to their employer, of which 14 (54%) would have preferred not to disclose this information. Thirty-four (65%) participants reported missing professional obligations and 27 (52%) reported missing personal obligations to attend appointments. A total of 8 (15%) reported changing work responsibilities to accommodate fertility treatment. When accounting for travel time and potential work hours missed, an average of 260.15min/cycle and an average of $53.63/hour wage this amounts to $232.53 lost work hours/productivity. Total time accounted for an additional "cost" of $544.53 per IVF cycle. This pilot survey highlights the significant indirect costs of pursuing fertility care and identifies time away from work and workplace privacy as two of the two primary drivers. There is a clear need for additional research in the area of the indirect cost of fertility care.

The mammalian spermatozoon neck is a unique structure that functions during spermatid differentiation and spermatozoa swimming, and its contents are critical for post-fertilization embryogenesis. Mutations in proteins localizing to the neck connecting piece (the modified pericentriolar material) result in acephalic spermatozoa. In contrast, mutations in proteins localizing to the centriole often produce abnormal tail morphology. Acephalic spermatozoa can be categorized based on the exact location of the neck breakpoint. Here, we classify 24 proteins known to cause acephaly in human and mice spermatozoa into five different acephalic types, depending on where the breakpoint occurs. We also discuss other proteins found in the spermatozoonneck, which may result in spermatozoa acephaly. The relationship between the exact location of the neck's break and intracytoplasmic sperm injection (ICSI) outcomes is explored in the context of the spermatozoon centrosome's role. We conclude that to understand this relationship, future research should investigate DNA, phospholipase C zeta, and centriole functionality, in addition to the location of the acephalic breakpoint in the patient's sperm.

PGT utilizing SNP-based linkage analysis for monogenic defects associated with microdeletions/duplications has been the standard method for preventing disease inheritance. However, its high cost and time consumption limit its accessibility. This study introduces a novel platform, the GenoLab M DX gene sequencer, designed to leverage next-generation sequencing (NGS) technology, aiming to reduce costs and increase sequencing efficiency in preimplantation genetic testing (PGT) for microdeletions/duplications. Couples with microdeletion/duplication syndromes were recruited, and GenoLab was employed to detect copy number variations (CNVs) under 4Mb in their abnormal blastocysts, developmentally arrested embryos, and peripheral blood, assessing the diagnostic validity of GenoLab. Blastocysts and arrested embryos from eight couples were thawed and sequenced using GenoLab. Of 69 affected blastocyst samples, 49 (71.0%) exhibited submicroscopic abnormalities, with 94% concordance to prior biopsy findings. Among 15 arrested embryo samples, 6 (40%) showed submicroscopic abnormalities, while 3 (20%) were mosaic. Additionally, 138 developmentally arrested embryos, peripheral blood, and 1 abortive tissue were analyzed, achieving diagnostic accuracy exceeding 90%. Clinically, two couples with microdeletions/duplications had embryos successfully transferred, resulting in the birth of two healthy children. The GenoLab M DX sequencer is a reliable, rapid (< 2weeks), and cost-effective platform suitable for CNV analysis in monogenic diseases. It offers an innovative solution for cases involving de novo pathogenic variants or when family DNA samples are unavailable, representing a significant advancement in assisted reproduction.