Objective To determine whether trophectoderm (TE) biopsy performed for preimplantation genetic testing induces molecular, epigenetic, or developmental perturbations in preimplantation embryos and subsequent placental development. Design Comparative multi-omics investigation of human blastocysts combined with a murine embryo transfer model. Subjects Surplus cryopreserved human euploid blastocysts donated to research and murine blastocysts that were either subjected to TE biopsy or no biopsy. Exposure The impact of blastocyst-stage trophectoderm biopsy on transcriptomic, epigenomic, developmental, and/or placental outcomes. Main Outcome Measures Differential gene expression, imprinting methylation, whole-genome DNA methylation of inner cell mass (ICM) and TE, mitotic cell division mechanics, implantation rates, fetal and placental growth, placental methylation. Results Trophectoderm biopsy was not associated with biologically meaningful molecular or epigenetic alterations in human blastocysts. Gene expression, imprinting methylation, global DNA methylation, and DNA budding/shedding were comparable between biopsied and non-biopsied embryos. In a murine model, biopsy had no effect on implantation rates, fetal or placental growth, placental methylome, or imprinting methylation status. Conclusion A comprehensive multi-omics assessment of human blastocysts, supported by developmental and placental analyses in a murine model, found no evidence that trophectoderm biopsy adversely affects early embryonic molecular regulation or subsequent developmental outcomes. These findings support the use of TE biopsy as a minimally invasive micro-manipulation procedure.
OBJECTIVE:To investigate the potential pathogenic mechanisms of the liquid crystal monomer 4-ethyl-2-(4-(4-ethylbenzyl) phenoxy) benzonitrile (EBCN) in ovulatory dysfunction (composite phenotype including polycystic ovary syndrome [PCOS], anovulation, and primary ovarian insufficiency [POI]) through an integrative computational strategy that combines composite and subtype-specific Mendelian randomization. DESIGN:Integrative computational study combining network toxicology, Mendelian randomization, and molecular docking. SUBJECTS:For Mendelian randomization analysis, genetic data were derived from large cohorts of European ancestry, including plasma protein quantitative trait loci data and genome-wide association study summary statistics for a composite ovulatory dysfunction phenotype (FinnGen finngen_R12_E4_OVARDYS, 3,045 cases and 267,780 controls), which explicitly includes PCOS (E28.2), anovulatory infertility (N97.0), POI (E28.3), and other related diagnoses. Subtype-specific analyses were conducted for PCOS (finngen_R12_E4_PCOS, n_cases = 2,214) and POI (finngen_R12_E4_OVARFAIL, n_cases = 655). EXPOSURE:In silico exposure to EBCN for target prediction; genetically predicted plasma protein levels for causal inference. MAIN OUTCOME MEASURES:Identification of overlapping targets between EBCN and ovulatory dysfunction; functional enrichment pathways; causal effects of prioritized proteins on ovulatory dysfunction risk (odds ratios [OR] with 95% confidence intervals); binding affinities between EBCN and core targets (kcal/mol). RESULTS:Network toxicology identified 150 overlapping targets between EBCN and ovulatory dysfunction. Enrichment analyses revealed significant associations with cancer-related pathways, PI3K-Akt and MAPK signaling cascades, and processes involving inflammation, metabolism, and apoptosis. Mendelian randomization demonstrated that genetically elevated levels of JUN (OR 1.265), CYP3A4 (OR 1.192), FGFR3 (OR 1.182), MET (OR 1.128), and POLR1C (OR 1.190) were significantly associated with increased risk of the composite ovulatory dysfunction phenotype, whereas higher PSAP levels were protective (OR 0.931). Subtype-specific analyses revealed convergent signals (e.g., CYP3A4 in PCOS) and divergent signals (e.g., TYMS as a risk factor in PCOS but protective in POI). Molecular docking confirmed stable binding interactions between EBCN and these targets, with strongest affinities for CYP3A4 (-8.9 kcal/mol) and JUN (-7.7 kcal/mol). CONCLUSION:This integrative investigation identifies inflammatory regulators, xenobiotic-metabolizing enzymes, and cellular pathway proteins as potential mediators genetically associated with ovulatory dysfunction risk. However, whether EBCN directly affects these proteins and thereby contributes to ovulatory pathology remains to be experimentally validated. The findings provide prioritized targets and testable hypotheses for future experimental studies and environmental risk assessment.
OBJECTIVE:To understand the pathogenesis of polycystic ovary syndrome (PCOS), investigating the molecular mechanism of granulosa cell dysfunction is vital. DESIGN:On the basis of bioinformatics analysis, we analyzed the differentially expressed genes and their enrichment pathways between patients with PCOS and normal samples. We also analyzed the levels of CD14 and miR-296-3p in the patients with PCOS. Pearson correlation coefficient was used to analyze the correlation between CD14 expression and PCOS-related inflammatory factors. The CCK-8 assay, flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blotting were performed to explore the mechanism of CD14 modulation via miR-296-3p in KGN cells in vitro. SUBJECTS:The gene expression data of patients with PCOS and healthy individuals in the gene expression omnibus (GEO) database, as well as the experimental data in KGN cells. EXPOSURE:The KGN cells were subjected to the following interventions: transfection with miR-296-3p inhibitor to suppress miR-296-3p expression; transfection with small interfering ribonucleic acid (siRNA) targeting CD14 (si-CD14) to knock down CD14; co-transfection with si-CD14 and miR-296-3p inhibitor to evaluate the rescue effect. The detections included CCK-8, flow cytometry, qRT-PCR, and western blotting. MAIN OUTCOME MEASURES:The expression of CD14 in patients with PCOS and KGN cells, and its correlation with inflammatory factors; the expression of miR-296-3p in patients with PCOS, and its regulatory relationship with CD14; the effect of miR-296-3p/CD14 on the viability and apoptosis of KGN cells; the influence of miR-296-3p/CD14 on the nuclear factor kappa-B (NF-κB) pathway. RESULTS:CD14 was up-regulated in patients with PCOS and KGN cells, and the expression of CD14 was positively correlated with the expression of inflammatory factors (CAT, CCL2, ICAM1, IL10, MMP9, and TGFB1) related to PCOS. The miR-296-3p was the upstream regulatory factor of CD14, and miR-296-3p inhibitor could promote cell apoptosis and inhibit cell viability. On the contrary, knockdown of CD14 in KGN cells inhibited apoptosis and promoted cell viability. Importantly, KGN cells cotransfected with si-CD14 and miR-296-3p could reverse the effects of miR-296-3p inhibitor and si-CD14 on the biological behavior of KGN cells. Moreover, miR-296-3p/CD14 axis mediated activation of NF-κB pathway in KGN cells. CONCLUSIONS:Down-regulation of CD14 attenuated the miR-296-3p inhibitor-induced increase in apoptosis and decrease in cell viability by suppressing the NF-κB signaling pathway. Our findings contribute to further understanding of the pathogenesis of PCOS and provide potential biomarkers for PCOS.
OBJECTIVE:To investigate the protective effects of rosuvastatin against ovarian ischemia-reperfusion (I/R) injury in a rat model. DESIGN:Controlled experimental study using an induced ovarian torsion-detorsion model. SUBJECTS:Fifty adult female Wistar rats (aged 8-10 weeks, weighing 100-150 g). EXPOSURE:Intraperitoneal administration of rosuvastatin (5 mg/kg) 30 minutes before induction of ischemia in the designated treatment groups. MAIN OUTCOME MEASURES:Ovarian tissue levels of myeloperoxidase (MPO), malondialdehyde (MDA), total antioxidant capacity (TAC), and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT); mRNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-α (TNF-α), and caspase-3; and semiquantitative histopathological scoring of tissue damage. RESULTS:Ischemia and I/R significantly elevated MPO, MDA, VEGF, bFGF, TNF-α, and caspase-3 levels while worsening histological damage. Rosuvastatin pretreatment reduced MPO by 13%-16%, MDA by 9%, VEGF by 7%-14%, bFGF by 14%-37%, TNF-α by 34%-38%, and caspase-3 by 31%-39%; increased TAC by 20%; and markedly improved histopathological scores, including reduced follicular atresia. No significant changes were observed in SOD, GPx, or CAT activities. CONCLUSIONS:Rosuvastatin exerts protective effects against ovarian I/R by attenuating oxidative stress, inflammation, excessive angiogenesis, and apoptosis, independent of major antioxidant enzyme induction. These preclinical findings suggest potential adjunctive utility in limiting reperfusion injury after detorsion; however, protein-level validation, dose-response studies, postischemic administration, long-term fertility outcomes, and safety assessments are required before any clinical translation. TRIAL REGISTRATION:Not applicable. This study is an experimental animal study and was not registered as a clinical trial.
OBJECTIVE:To evaluate and select blastocysts with higher implantation potential using reactive oxygen species (ROS) levels and glutathione (GSH) regulated by arginine (Arg) with leucine (Leu). DESIGN:Experimental study. SUBJECTS:ICR mice. EXPOSURE:In vitro fertilization (IVF)-derived blastocysts were treated with Arg and Leu for 24 hours. A microgrid array was used for live-cell imaging of individual blastocysts, followed by embryo transfer to recipient females. MAIN OUTCOME MEASURES:Both ROS and GSH levels in individual blastocysts, selection of blastocysts at the ROS level, embryo transfer (ET), and implantation rates. RESULTS:For each in vitro fertilization cycle followed by ET, approximately 20 blastocysts were provided to measure levels of ROS and GSH. The ROS levels in individual blastocysts showed substantial variation, whereas the GSH levels were low and similar in each blastocyst. Therefore, the blastocysts were ranked and divided into two groups based on their ROS levels. The top six blastocysts were classified as the high-ROS group, and the bottom six as the low-ROS group. Blastocysts from the high- and low-ROS groups were transferred to one or the other uterine horns of the same female recipient mice, respectively. The implantation rate was significantly higher in the high-ROS group than in the low-ROS group. CONCLUSION:This study demonstrates that blastocysts with higher implantation potential can be selected on the basis of their ROS levels before ET. Increased ROS levels caused by the combination of Arg and Leu indicate a higher implantation potential in the blastocysts. This study also proposes a direct selection method for blastocysts using a live-cell imaging assay for ET.
OBJECTIVE:To develop a noninvasive, time-lapse imaging-based approach to stratify three-pronuclear zygotes by developmental competence and to identify embryos with higher potential for good-quality blastocyst development and live birth. DESIGN:Retrospective observational study. SUBJECTS:Zygotes exhibiting three pronuclei after intracytoplasmic sperm injection, collected from three fertility clinics. EXPOSURE:Time-lapse imaging assessment of second polar body (PB2) extrusion and quantitative measurement of pronuclear volumes. MAIN OUTCOME MEASURES:Discriminative performance of the ratio of combined female pronuclear volume to male pronuclear volume for classifying three-pronuclear zygotes on the basis of PB2 extrusion status (operational definition); blastocyst formation and good-quality blastocyst formation (≥3BB); and supportive posttransfer clinical outcomes (where available). RESULTS:A total of 222 three-pronuclear zygotes were analyzed, including 173 operationally classified as putative diploid and 49 as putative triploid according to PB2 extrusion status. The ratio of combined female to male pronuclear volume demonstrated strong discrimination between the PB2-extruded and PB2 extrusion-failed categories (area under the curve, 0.863), with a candidate cut-off of 1.189 yielding high sensitivity and specificity. The overall blastocyst formation rate was 40.1% (69/172) in the PB2-extruded and 32.7% (16/49) in the extrusion-failed group. The good-quality blastocyst formation rate was 23.3% (40/172) in the PB2-extruded and 10.2% (5/49) in the extrusion-failed group. After adjustment for maternal age, PB2 extrusion-based classification was not significantly associated with overall blastocyst formation (adjusted odds ratio [aOR], 1.44; 95% confidence interval [CI]: 0.73-2.86) but remained associated with higher odds of good-quality blastocyst formation (aOR, 2.82; 95% CI: 1.03-7.71). Posttransfer live birth outcomes were available for a subset of transferred embryos under site-specific transfer policies; seven live births were observed, and all live birth cases had volume ratios within 0.61-0.83. CONCLUSION:A time-lapse imaging-based framework integrating PB2 extrusion and pronuclear volumetrics feasibly provides a clinically actionable, noninvasive method to stratify three-pronuclear zygotes by developmental competence and may aid selection of embryos with developmental potential when genetic testing is unavailable or undesirable.
OBJECTIVE:To determine whether human blastocysts requiring an additional day of in vitro culture to achieve expansion exhibit differences in trophectoderm transcriptomic profiles associated with developmental and implantation-related processes. DESIGN:Prospective in vitro experimental cohort study including ribonucleic acid sequencing of mural trophectoderm biopsies from day 5 (D5) and day 6 (D6) (euploid) blastocysts. SUBJECTS:Twenty-one blastocysts donated by 16 couples undergoing intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A). Transcriptomic comparison focused on 21 blastocysts, classified as D5 (n = 13) vs. D6 (n = 8) embryos, and euploid D5 (n = 6) vs. euploid D6 (n = 4) embryos. EXPOSURE:Blastocyst developmental timing (D5 vs. D6) and embryo ploidy status. MAIN OUTCOME MEASURES:Identification of differentially expressed genes (DEGs) and deregulated molecular pathways between D5 and D6 (euploid) embryos, including functional enrichment analysis to explore their potential roles in embryo development and implantation. RESULTS:Global transcriptomic analysis revealed partial overlap between D5 and D6 blastocysts, indicating a largely shared gene expression landscape with underlying biological variability. Differential expression analysis identified 111 DEGs between D5 and D6 embryos, with functional enrichment analyses highlighting pathways related to translation, metabolism, and cellular organization. An exploratory euploid-only subanalysis yielded a larger number of genes meeting the selected differential expression thresholds, potentially reflecting reduced biological variability within the subset, together with enrichment of pathways associated with oxidative phosphorylation, ribosomal function, and energy metabolism, although these findings should be interpreted cautiously given the limited sample size. CONCLUSION:Human D5 blastocysts exhibit transcriptomic profiles enriched in pathways related to translation, metabolism, and cell adhesion compared with D6 blastocysts. However, these differences are subtle and occur within a largely shared global transcriptomic landscape, suggesting that they reflect variations in developmental timing rather than clearly distinct functional states. Although these findings are consistent with biological processes relevant to implantation, they should be interpreted within the broader context of embryo development, where additional molecular and cellular mechanisms also contribute to implantation competence.
OBJECTIVE:To study the potential pathogenic mechanisms of bisphenol A (BPA) in polycystic ovary syndrome (PCOS) using an integrative computational strategy. DESIGN:Integrative computational study combining network toxicology, Mendelian randomization (MR), and molecular docking. SUBJECTS:For MR analysis, genetic data were sourced from large European-ancestry cohorts, including plasma protein quantitative trait loci data and genome-wide association study summary statistics for PCOS (3,045 cases and 267,780 controls). EXPOSURE:In silico exposure to BPA for target prediction; genetically predicted plasma protein levels for causal inference. MAIN OUTCOME MEASURES:Identification of overlapping targets between BPA and PCOS; functional enrichment pathways; causal effects of prioritized proteins on PCOS risk (odds ratios with 95% confidence intervals); binding affinities between BPA and core targets (kcal/mol). RESULTS:Network toxicology identified 310 overlapping targets between BPA and PCOS. Enrichment analyses revealed significant involvement in endocrine signaling, inflammatory pathways (eg, IL-17), and cellular processes. MR demonstrated that genetically elevated levels of RET, CXCL8, HTR6, MMP1, MMP9, NTRK1, and TNNI2 were significantly associated with increased PCOS risk, whereas higher PSAP and SHBG levels were protective. Molecular docking confirmed stable binding between BPA and all nine key targets, with strongest affinity for SHBG (-8.4 kcal/mol), followed by NTRK1, TNNI2, and RET. CONCLUSION:This integrative investigation suggests that BPA may contribute to PCOS pathogenesis through multitarget interactions involving inflammatory mediators, endocrine regulators, and tissue remodeling proteins. The findings provide prioritized targets and mechanistic insights for future experimental validation and environmental risk assessment.
Objective To examine whether dynamic changes in circulating estradiol during ovarian stimulation are associated with the number and maturation status of oocytes recovered in cynomolgus macaques. Design Observational study based on retrospective analysis of ovarian stimulation cycles. Subjects Sixty-six ovarian stimulation cycles from adult female cynomolgus macaques (Macaca fascicularis) housed at the Primate Resources Center. Exposure Animals underwent a standardized gonadotropin-based ovarian stimulation protocol. Circulating estradiol concentrations were measured at multiple time points during the late follicular phase before oocyte recovery, and estradiol dynamics were defined as the difference between the maximum and minimum values observed across these measurements. Main Outcome Measures Total number of oocytes recovered per stimulation cycle, number of mature oocytes at the metaphase two stage, and proportional distribution of oocyte maturation stages. Results Dynamic changes in estradiol concentrations were positively associated with the total number of oocytes recovered per cycle (correlation coefficient 0.45). A similar but weaker association was observed with the number of mature oocytes recovered (correlation coefficient 0.36). In contrast, estradiol dynamics were not associated with the proportional distribution of oocyte maturation stages. Donor age and body weight were not significantly associated with oocyte recovery outcomes. Conclusion Dynamic changes in circulating estradiol during ovarian stimulation primarily reflect the quantitative dimension of ovarian response in cynomolgus macaques, with limited relevance for the maturation stage composition of recovered oocytes.
OBJECTIVE:To characterize the immune cell and hormonal profile of menstrual effluent compared with paired peripheral blood. DESIGN:This descriptive, cross-sectional study collected menstrual effluent and processed it to isolate cellular and soluble fractions; peripheral blood was processed in parallel. Steroid hormones were quantified using immunoassays, immune cell populations were assessed by multiparameter flow cytometry, and cytokines and chemokines were measured using multiplex assays. SUBJECTS:Menstrual effluent and peripheral blood were collected from 13 healthy participants (excluding those using NSAIDs or hormonal contraception) after informed consent. EXPOSURE:None MAIN OUTCOME MEASURES: The immune cell and hormonal profiles of menstrual effluent and peripheral blood were the main outcome measures. RESULTS:Progesterone and cortisol concentrations were significantly decreased in menstrual effluent compared with peripheral blood. Menstrual effluent exhibited distinct immune and cytokine profiles, including increased plasmacytoid and myeloid dendritic cells, neutrophils, natural killer (NK) cells, and memory CD8+ T cells, alongside decreased regulatory T cells, natural killer T (NKT) cells, and multiple B cell subsets. Cytokine analysis revealed multiple elevated cytokines in menstrual effluent, even after false discovery rate correction. CONCLUSIONS:Menstrual effluent is immunologically and hormonally distinct from peripheral blood, which may reflect inflammatory and tissue-repair activity. These findings contribute to the understanding of the normative baseline for menstrual effluent.
OBJECTIVE:To improve ovarian tissue transplantation effectiveness by limiting oxidative stress after transplantation, thus increasing follicle survival. DESIGN:Experimental laboratory-controlled study. Immunodeficient mice were transplanted with frozen-thawed human ovarian cortex pieces, and received 7 days of oral treatment. The grafts were recovered 7 days and 4 weeks after transplantation. SUBJECTS:A total of 2-4 ovarian tissue fragments per patient were donated by the patients (n = 26) undergoing ovarian tissue cryopreservation for fertility preservation and transplanted to immunodeficient mice. EXPOSURE:Xenotransplanted mice received 7 days of oral treatment with either 150 mg/kg N-acetylcysteine (NAC) (NAC 150), 300 mg/kg NAC (NAC 300), FERTILIX, or Control (vehicle). MAIN OUTCOME MEASURED:Follicle density 1 and 4 weeks after transplantation, malondialdehyde (MDA) immunohistochemistry assay and expression of antioxidant, mitochondrial, and apoptotic key genes. RESULTS:After 7 days of transplantation, ovarian xenografts showed no significant differences in antioxidant genes (HMOX and CATALASE), mitochondrial stress genes (TFAM and HSP10), oxidative stress gene (ALDH3A2), BAX/BCL2 ratio, or malondialdehyde-positive areas (Control: 7.54% ± 2.23%; NAC 150: 4.99% ± 1.23%; NAC 300: 5.91% ± 1.19%; and FERTILIX: 10.30% ± 2.63%). However, follicle densities (follicles/mm3, mean ± SEM) showed a modest, nonsignificant increase in the NAC 150 (13.2 ± 2.85) and FERTILIX (20 ± 7.79) groups compared with controls (8.5 ± 2.85) after 4 weeks of grafting. CONCLUSION:These findings suggest that orally administered FERTILIX and NAC have little to no efficacy in reducing oxidative stress or improving follicle survival in xenotransplanted human ovarian tissue.
OBJECTIVE:To assess deoxyribonucleic acid (DNA) methylation at imprinted and repetitive genomic regions in ejaculated and testicular sperm from men with oligospermia, azoospermia, and those undergoing vasectomy reversal (VR), compared with fertile controls. DESIGN:Observational case-control study. SUBJECTS:Samples were obtained from 68 men, including 29 infertile men (18 oligospermic [5-15 million/mL], 11 severely oligospermic [<5 million/mL]) and 20 fertile controls with confirmed natural conceptions. Testicular tissue was collected from 11 azoospermic men (7 obstructive azoospermia [OA], 4 nonobstructive azoospermia [NOA]) and 8 men with prior paternity undergoing VR. EXPOSURE:Sperm DNA methylation at four imprinted genes (H19, GTL2, MEST, and LIT1) and one repetitive element (LINE1). MAIN OUTCOME MEASURES:Methylation levels at CpG sites determined by bisulfite pyrosequencing. RESULTS:The H19 was significantly hypomethylated only in severe oligospermia compared with fertile controls, whereas other groups showed nonsignificant trends with overlapping distributions. In contrast, MEST was significantly hypermethylated in oligospermic, azoospermic, and VR groups compared with fertile controls. No significant differences were observed for GTL2, LIT1, or LINE1. CONCLUSION:The DNA methylation abnormalities in sperm are locus-specific and vary across infertility phenotypes. MEST alterations were consistent across groups, whereas H19 changes were limited to severe oligospermia. Similar methylation patterns in testicular sperm from azoospermic and VR groups suggest that epigenetic alterations may reflect the testicular environment or obstruction, or differences between testicular and ejaculated sperm.
Objective To study the association between self-rated health and in vitro fertilization outcomes in women with infertility undergoing embryo transfer. Design Prospective cohort study. Subjects A total of 800 women with infertility undergoing embryo transfer at a tertiary reproductive center in China. Exposure Self-rated health assessed across physical, psychological, social, and overall domains on the day of embryo transfer. Main Outcome Measures Clinical pregnancy, implantation rate, biochemical pregnancy, and early miscarriage. Results Self-rated health in any domain was not significantly associated with clinical pregnancy, implantation rate, biochemical pregnancy, or early miscarriage after adjustment for demographic and clinical confounders. Conclusion Self-rated health was not associated with in vitro fertilization outcomes, suggesting that treatment prognosis is primarily driven by biological and clinical factors.
Objective To study whether functional human spermatids can be generated from spermatogonial stem cells obtained from testicular biopsies of prepubertal boys newly diagnosed with cancer using a three-dimensional (3D) human testicular organoid system. Design Experimental laboratory study using a 3D human testicular organoid model incorporating Sertoli cells and endothelial cells to recreate the testes microenvironment. Subjects Testicular biopsy specimens obtained from prepubertal boys newly diagnosed with cancer before initiation of gonadotoxic cancer therapy for fertility preservation. Exposure Culture of prepubertal testicular tissue in a 3D human testicular organoid system with growth factors to support germ cell differentiation. Main Outcome Measures Generation of differentiated spermatids followed by verification of postmeiotic marker expression by immunofluorescence and assessment of functional competence through fertilization assays. Results Germ cells differentiated into round spermatid-like cells within our testicular organoid system. Immunofluorescence confirmed expression of markers consistent with postmeiotic germ cell differentiation. Functional competence was demonstrated by successful fertilization assays using mouse oocytes, indicating that the spermatid-like cells generated ex vivo could activate oocytes. Conclusion Spermatid-like cells can be generated from prepubertal testicular tissue in a three-dimensional organoid culture system, supporting the potential feasibility of fertility preservation for boys undergoing gonadotoxic cancer treatments.
OBJECTIVE:To evaluate whether hyaluronan-enriched transfer medium (HETM) is associated with improved live birth rates in biopsied euploid blastocyst transfers. DESIGN:Retrospective cohort study. SUBJECTS:A total of 1,221 single frozen euploid blastocyst transfers performed between January 2011 and December 2024. EXPOSURE:Embryo transfer using HETM compared with standard zwitterionic-buffered transfer medium. All embryos underwent trophectoderm biopsy. MAIN OUTCOME MEASURES:Live birth rate. Secondary outcomes included biochemical pregnancy and clinical pregnancy rates. RESULTS:Hyaluronan-enriched transfer medium was associated with significantly higher live birth rates compared with standard medium (59.1% vs. 43.2%; absolute difference 15.9%, 95% confidence interval 10.3%-21.5%; relative risk 1.37, 95% confidence interval 1.22-1.54). Clinical pregnancy and biochemical pregnancy rates were also significantly higher in the hyaluronan group. Sensitivity analysis restricted to first transfers per patient (n = 715) confirmed persistence of the live birth benefit (61.2% vs. 47.1%; absolute difference 14.1%, 95% confidence interval 6.9%-21.3%; relative risk 1.30, 95% confidence interval 1.13-1.49). Maternal age was comparable between groups. CONCLUSION:Use of HETM is associated with a clinically meaningful increase in live birth rates in biopsied euploid blastocyst transfers. Zona pellucida disruption created during trophectoderm biopsy may facilitate enhanced embryo-endometrial interaction, improving implantation efficiency. However, these results should be interpreted with caution, given the potential for unmeasured confounding variables and require validation in prospective randomized trials.
OBJECTIVE:To study the ameliorative effects of silymarin on hormonal balance, oxidative stress markers, sperm quality, and testicular histopathology in male mice exposed to cyclophosphamide (CP). DESIGN:This is a controlled experimental animal study. SUBJECTS:Eighty adult male mice were randomly assigned to 5 groups (N = 16 for each group) as untreated control, CP only, silymarin (100 mg/kg) + CP, silymarin (200 mg/kg) + CP, and silymarin only. EXPOSURE:Silymarin was given orally for 28 consecutive days, while a single intraperitoneal dose of CP (200 mg/kg) was administered on day 22 to induce testicular injury. MAIN OUTCOME MEASURES:Body and testicular weights were recorded at sacrifice, and serum concentrations of testosterone, luteinizing hormone, and follicle-stimulating hormone were quantified. Testicular tissues were analyzed for oxidative stress parameters (malondialdehyde, superoxide dismutase, catalase, and reduced glutathione), sperm characteristics including count, motility, morphology, and histopathological changes. RESULTS:Cyclophosphamide showed significantly decreased body and testicular weights, reduced testosterone levels, and impaired sperm parameters, accompanied by a rise in malondialdehyde and depletion of antioxidant enzymes along with severe histopathological changes. Co-administration of silymarin mitigated these adverse effects in a dose-dependent manner. High-dose silymarin markedly restored hormonal levels, sperm quality, antioxidant balance, and normal testicular architecture to near-control levels. Silymarin alone produced no detrimental effects. CONCLUSION:These results indicate that silymarin mitigates CP-induced testicular toxicity by enhancing antioxidant defenses, stabilizing hormonal function, and preserving spermatogenesis in male mice.
OBJECTIVE:To evaluate the association between per- and polyfluoroalkyl substances (PFAS) in follicular fluid (FF) and live birth after in vitro fertilization (IVF) and characterize the FF metabolome in relation to both. DESIGN:Retrospective cohort. SUBJECTS:Thirty-six women who underwent IVF treatment at 1 of the 3 centers in Eastern Massachusetts between 1999 and 2003. EXPOSURE:Twenty-four PFAS were measured in FF retrieved from the first follicle aspirated during the first treatment cycle. The 8 PFAS detected in >90% of samples were evaluated. MAIN OUTCOME MEASURES:We analyzed the FF metabolome using untargeted liquid chromatography high-resolution mass spectrometry. We used linear regression to estimate associations between FF PFAS and metabolic feature intensities, and logistic regression to estimate associations of FF PFAS and metabolites with live birth, adjusting for age and infertility type. We subsequently conducted pathway enrichment analyses and used a meet-in-the-middle approach to screen for overlapping metabolic pathways associated with FF PFAS and live birth. RESULTS:Higher FF perfluoroheptanesulfonic acid (PFHpS) was associated with lower odds of live birth (odds ratio = 0.42 [95% confidence interval: 0.15-0.97]). All other FF PFAS, except perfluorobutanesulfonic acid (PFBS), were also inversely associated with live birth, although confidence intervals included the null value. We evaluated 27,903 features detected in >25% of participant samples. For PFAS-feature associations, the top 5% of features for each PFAS were enriched for bile acid biosynthesis. Thirty-eight FF metabolic pathways enriched for features associated with live birth (yes/no) overlapped with pathways enriched for features associated with perfluorooctanoic acid (PFOA), PFHpS, or perfluorohexanesulfonic acid (PFHxS). These included pathways related to lipid (n = 11), carbohydrate (n = 9), and vitamin and cofactor metabolism (n = 6), among others. CONCLUSION:Higher FF PFAS concentrations were associated with lower odds of achieving live birth, although most 95% confidence intervals included the null. Metabolic pathways enriched for features associated with live birth overlapping with those associated with FF PFAS were related to lipid, carbohydrate, vitamin, and cofactor metabolism. Although our sample size was small, our findings align with other studies characterizing environmental exposures in reproductive organs and using the metabolome to assess their impact on fertility.
OBJECTIVE:To assess the prevalence of proliferative endometrial disorders in women with histologically confirmed adenomyosis and to evaluate the independent association between adenomyosis and each lesion type. DESIGN:Retrospective cohort study. SUBJECTS:Consecutive patients undergoing hysterectomy between January 2021 and August 2024, excluding those with histologically confirmed gynecological malignancies (n = 734). EXPOSURE:None. Histological and clinical records were reviewed to identify adenomyosis and coexisting endometrial disorders. MAIN OUTCOME MEASURES:Primary outcome was the prevalence of endometrial polyps, endometrial hyperplasia without atypia, and endometrial intraepithelial neoplasia in patients with histologically confirmed adenomyosis. Secondary outcome included the independent association between adenomyosis and each endometrial disorder. RESULTS:Adenomyosis was identified in 34.7% (255/734) of cases. Proliferative endometrial disorders were more prevalent among patients with adenomyosis (37.6% vs. 25.1%). In multivariable analysis, adenomyosis was independently associated with these conditions overall (aOR 1.87, 95% CI: 1.31, 2.65), including endometrial polyps (aOR 1.69, 95% CI: 1.03, 2.79), endometrial hyperplasia without atypia (aOR 2.07, 95% CI: 1.34, 3.21), and endometrial intraepithelial neoplasia (aOR 2.06, 95% CI: 1.16, 3.66). After adjustment for established clinical risk factors for endometrial cancer (obesity, menopause, nulliparity and abnormal uterine bleeding), adenomyosis remained in the predictive model for EIN. CONCLUSION:The present study identified an independent histopathological association between adenomyosis and proliferative endometrial disorders, including endometrial intraepithelial neoplasia, the lesion with the highest malignant potential. When coexisting with other risk factors, adenomyosis may contribute to a risk profile warranting targeted endometrial assessment.
OBJECTIVE:To evaluate the effects of letrozole alone and in combination with a gonadotropin-releasing hormone agonist on inflammatory cytokines, folliculogenesis, and hormonal profiles in a rat model of surgically induced peritoneal endometriosis. DESIGN:Prospective, controlled experimental laboratory study. SUBJECTS:Twenty-eight adult female Wistar albino rats randomly assigned to four groups (n = 7 each): sham, untreated endometriosis, letrozole, and letrozole plus gonadotropin-releasing hormone agonist. EXPOSURE:Endometriosis was induced by autologous uterine tissue transplantation onto the pelvic peritoneum. After a 28-day lesion development period, intraperitoneal letrozole or combined letrozole plus gonadotropin-releasing hormone agonist therapy was administered for 30 days. MAIN OUTCOME MEASURES:Peritoneal interleukin-6, vascular endothelial growth factor, and tumor necrosis factor-α concentrations; serum estradiol, CA-125, and antimüllerian hormone levels; and histological counts of ovarian follicle subtypes. RESULTS:Untreated endometriosis increased peritoneal interleukin-6, vascular endothelial growth factor, and tumor necrosis factor-α levels, as well as serum estradiol and CA-125 levels, reduced antimüllerian hormone concentrations, and disrupted folliculogenesis with higher atretic follicle counts compared with controls. Letrozole alone and combination therapy significantly reduced inflammatory cytokines, estradiol, and CA-125, and decreased atretic follicle numbers. Antimüllerian hormone levels and most follicular subtypes did not differ among treatment groups. No significant advantage of combination therapy over letrozole monotherapy was observed. CONCLUSION:Letrozole alone and combined therapy effectively attenuate inflammatory activity and reduce follicular atresia in experimental peritoneal endometriosis. No significant advantage of combination therapy over letrozole monotherapy was observed. However, direct comparisons between regimens should be interpreted with caution because of the different letrozole doses used across treatment arms and the inherent limitations of the experimental animal model.
