Zhen-Gao Sun

Per- and polyfluoroalkyl substances (PFAS) are a class of anthropogenic organic compounds ubiquitous in the environment. Growing evidence has underscored these associated health risks. Diminished ovarian reserve (DOR), which is characterized by a decline in oocyte quantity and quality, is a significant cause of female infertility. However, the role of PFAS in the pathogenesis of DOR remains unclear. This study integrated network toxicology, machine learning, molecular docking, and molecular dynamics (MD) simulations to investigate the mechanisms of PFAS on DOR, with subsequent validation using KGN cells. Multi-database screening identified 35 candidate targets. Gene Ontology and KEGG enrichment analyses revealed that PFAS perturb pathways governing cell proliferation and differentiation, immune-inflammatory responses, and hormone signaling. Furthermore, we identified three core targets by integrating the GEO database analysis, and machine learning. Single-gene GSEA elucidated multi-pathway regulatory mechanisms. Molecular docking demonstrated strong binding affinities between PFAS and these targets, while MD simulations confirmed the structural stability of the resulting complexes. PFAS exposure induces dose- and time-dependent cytotoxicity and oxidative stress in KGN cells in vitro. It activates apoptotic signaling via BAX upregulation and Bcl-2 downregulation and accelerates oxidative stress by suppressing glutathione S-transferase P1 (GSTP1) and peroxiredoxin 5 (PRDX5) expression. Additionally, PFAS impair ovarian reserve function by inhibiting the synthesis and secretion of anti-Müllerian hormone (AMH) and inhibin B (INHB). Notably, TGFBR1 upregulation suggests the activation of pro-fibrotic signaling markers, further compromising ovarian function. Our findings propose a potential mechanistic framework wherein PFAS may contribute to DOR progression by correlating with apoptosis, inducing oxidative stress, suppressing reserve markers, and activating fibrotic pathways. This study provides novel insights into the pathogenesis of PFAS-associated DOR.

Background: This study employs high-throughput targeted PFAS quantification analysis to analyze per- and polyfluoroalkyl substances (PFAS) metabolites in follicular fluid from women with Kidney Deficiency-Liver Stagnation Pattern poor ovarian response (POR) and IVF controls with normal ovarian reserve. Furthermore, we examine PFAS metabolite alterations in POR patients before and after intervention with kidney-tonifying and liver-soothing herbal medicine to suggest that PFAS compounds, particularly PFBA, are significant metabolic markers associated with the Kidney Deficiency-Liver Stagnation Pattern in POR. Methods: This study enrolled 93 patients undergoing in vitro fertilization and embryo transfer (IVF-ET). Participants were divided into three groups: (1) the non-herbal intervention group (POR-NON group, n =31); (2) the control group (POR-CON group, n =31); and (3) the herbal medicine intervention group (POR-TCM, n =31). And ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UHPLC-QTRAP MS) was used to detect metabolites in the three groups of samples. Results: Patients with Kidney Deficiency-Liver Stagnation Pattern POR showed significantly higher levels of PFBA in follicular fluid compared to IVF controls with normal ovarian reserve (P < 0.01). The level of PFBA in follicular fluid was significantly lower in patients with Kidney Deficiency-Liver Stagnation Pattern POR who received QZYST intervention (POR-TCM group) than in those who did not receive intervention (POR-NON group) (P < 0.05). QZYST intervention significantly reduced Kidney Deficiency-Liver Stagnation Pattern scores in POR patients compared to both baseline (P < 0.001) and the POR-NON group (P < 0.05). The POR-TCM group demonstrated significantly improved number of oocytes retrieved (P < 0.05) and two-pronuclei (2PN) zygotes (P < 0.05), along with significantly fewer cycles with no transferable embryos (P < 0.05) compared to the POR-NON group. Conclusions: Patients with Kidney Deficiency-Liver Stagnation Pattern POR exhibited significantly elevated levels of PFAS compounds in follicular fluid compared to IVF controls with normal ovarian reserve. Compared with the control group, patients who received QZYST intervention showed improvements in TCM syndrome manifestations and key clinical outcomes, and significantly lower PFBA levels were observed in the follicular fluid of the TCM intervention group than in those without intervention.

Objective: To evaluate the clinical efficacy of Xiaonang Yusi decoction (XNYSD, 消囊育嗣汤) in patients with phlegm-dampness type polycystic ovary syndrome (PCOS) undergoing in vitro fertilization and embryo transfer (IVF-ET), and to investigate its underlying mechanism by examining the arachidonic acid (AA) metabolic pathway. Methods: Women undergoing IVF-ET were divided into a treatment group (TCM treatment), a phlegm-dampness PCOS group, and a tubal factor control group ( n = 32 each); all received a GnRH antagonist protocol. The study evaluated IVF-ET laboratory and clinical outcomes, alongside changes in TCM syndrome scores. To elucidate the therapeutic mechanism, follicular fluid AA metabolites were quantified by targeted metabolomics. These data were integrated with a network pharmacology analysis to link the active ingredients of Xiaonang Yusi decoction, PCOS targets, and differential metabolites. A protein–protein interaction (PPI) network was then constructed (STRING) and analyzed for GO/KEGG enrichment (DAVID) to systematically clarify the treatment’s mode of action. Results: Clinical findings demonstrate that Traditional Chinese Medicine (TCM) intervention significantly ameliorates phlegm-dampness symptoms in PCOS patients, with marked improvements in embryo implantation rate (52.0% vs. 28.0%), clinical pregnancy rate (67.9% vs. 29.0%), and ongoing pregnancy rate (60.7% vs. 29.0%) (all p < 0.05), while showing no significant intergroup differences in early miscarriage rates ( p > 0.05). Metabolomic profiling revealed significantly elevated levels of four arachidonic acid metabolites, including 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), in follicular fluid of PCOS patients compared to controls ( p < 0.05), with TCM treatment effectively reducing 15(S)-HETE concentrations ( p < 0.05). Network pharmacological analysis suggests that Xiaonang Yusi Decoction may modulate PCOS pathophysiology through targeting 15(S)-HETE-mediated pathways, acting on core targets including ESR1 and SIRT1, and influencing critical signaling pathways such as cancer-related pathways, lipid metabolism, and PI3K-Akt signaling. Molecular docking results show favourable interactions, indicating the active compound can spontaneously bind and modulate multiple key targets. Conclusion: Xiaonang Yusi Decoction significantly improves TCM syndrome manifestations and IVF-ET outcomes in phlegm-dampness PCOS patients, enhancing embryo quality and pregnancy rates. The therapeutic effects appear mediated through regulation of arachidonic acid metabolites, particularly 15(S)-HETE, in follicular fluid. Network pharmacology analysis has preliminarily elucidated the underlying mechanisms, providing novel evidence for TCM-based PCOS treatment strategies.

Purpose: To evaluate whether diclofenac sodium administration reduces premature ovulation and improves in vitro fertilization (IVF) outcomes in patients with diminished ovarian reserve (DOR). Patients and Methods: Retrospective cohort study conducted at a single academic reproductive center from January 2022 to March 2025. We included women with DOR (anti-Müllerian hormone [AMH] < 1.1 ng/mL, antral follicle count [AFC] < 5) undergoing autologous IVF cycles with single dominant follicle development. A total of 1164 cycles from 616 patients were analyzed, comparing 382 cycles with diclofenac sodium 75mg daily from trigger day to oocyte retrieval versus 782 cycles without treatment. The primary endpoint was cycle cancellation due to premature ovulation. Secondary endpoints included oocyte retrieval success, fertilization rates, embryo outcomes, and clinical pregnancy rates. Generalized estimating equation (GEE) models with multivariable adjustment were used to account for correlation between multiple cycles from the same patient. A sensitivity analysis restricted to first cycles per patient (n=616) was performed using logistic regression to validate primary findings. Results: Diclofenac sodium significantly reduced cycle cancellation due to premature ovulation (7.3% vs 19.8%, adjusted odds ratio [OR] 0.39, 95% confidence interval [CI] 0.21– 0.73, P=0.003) and decreased the proportion of cycles with no oocytes retrieved (21.8% vs 27.1%, adjusted OR 0.54, 95% CI 0.34– 0.84, P=0.007). Normal fertilization rates were higher in the diclofenac sodium group (77.5% vs 69.8%, P=0.041), though this lost significance after adjustment (P=0.184). The proportion of cycles without viable embryos was comparable between groups (42.3% vs 41.8%, P=0.727). Among limited fresh embryo transfers (n=124), clinical pregnancy (19.2% vs 11.2%, P=0.847) and live birth rates (15.4% vs 8.2%, P=0.948) were similar. The sensitivity analysis using first cycles per patient (n=616) confirmed these findings, showing a 47% reduction in premature ovulation odds with diclofenac sodium (adjusted OR 0.53, 95% CI 0.29– 0.94, P=0.031). Conclusion: Diclofenac sodium is associated with reduced premature ovulation and improved oocyte retrieval success in DOR patients, though embryonic and pregnancy outcomes remain similar. These observational findings require validation through prospective randomized controlled trials with adequate power to assess cumulative reproductive outcomes.

Introduction: Effective ovarian stimulation and luteal phase support (LPS) are key factors for the success of in vitro fertilization and embryo transfer (IVF-ET) The present multicenter randomized trial evaluates whether adding Gushen Antai Pill (GSATP) to standard LPS improves the ongoing pregnancy rate (OPR) in women with normal ovarian reserve (NOR) undergoing fresh embryo transfer. Methods: This study is a multicenter, randomized, double-blind, placebo-controlled trial, taking place at 20 reproductive centers within public tertiary hospitals. We plan to enroll around 800 women who are expected to have a NOR. Participants are randomized 1:1 using a stratified block randomization method with block sizes of 4 and 6, stratified by age (< 35 vs ≥ 35 years). The primary endpoint is the OPR, with comprehensive safety evaluations also being performed. Data analysis will adhere to both intention-to-treat (ITT) and per-protocol (PP) principles to ensure robust statistical validity. Discussion: This randomized controlled trial (RCT) aims to evaluate the effectiveness and safety of combining LPS with GSATP to improve pregnancy outcomes in women with NOR who are undergoing fresh embryo transfer. By rigorously evaluating endpoints such as OPR and early pregnancy complications, this study seeks to establish evidence for GSATP as a potential adjuvant therapy in IVF-ET protocols. The findings may offer innovative therapeutic perspectives for integrating traditional Chinese medicine (TCM) into contemporary assisted reproductive technology, particularly in enhancing implantation conditions and reducing miscarriage risks during fresh cycles. Trial Registration: ClinicalTrials.gov, ID: NCT04872660, 05/04/2021.

Background and objective: Ovarian endometriosis (OE), characterized by endometriotic cysts, adversely affects ovarian function and comprises in vitro fertilization and embryo transfer (IVF-ET) outcomes. Acupoint application therapy (AAT), which integrates transdermal drug delivery with acupoint stimulation, may offer therapeutic benefits; however, its underlying mechanisms remain unclear. Methods: In this randomized trial, 81 IVF-ET patients were stratified into: a treatment group comprising OE patients (n=27) undergoing a gonadotropin hormone-releasing hormone (GnRH) antagonist protocol with medicated AAT, a placebo group consisting of OE patients (n=26) following an identical protocol but receiving a sham patch, and a control group, including patients with male-factor infertility (n=28) undergoing the standard protocol without additional interventions. Follicular fluid metabolomics (assessed by Ultra High-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS/MS) System)and IVF parameters were analyzed. Results: Significant intergroup differences were observed (one-way ANOVA, P < 0.05). The treatment group exhibited a shorter Gn duration (9.00 ± 0.68 days) compared to the placebo group (10.62 ± 2.43 days; P < 0.05), with a duration comparable to the male-factor control group (9.32 ± 1.89 days; NS). Similarly, the total Gn dose was lower in the treatment group (2112.50 ± 483.17 IU) than in the placebo group (2549.04 ± 677.44 IU; P < 0.05), and comparable to the control group (2105.89 ± 690.24 IU; NS). Regarding IVF outcomes, the treatment group yielded more oocytes retrieved (12.41 ± 7.27) than the placebo group (8.85 ± 7.89; P < 0.05), though fewer than the control group (15.25 ± 7.77). Fertilized oocytes were also higher in the treatment group (7.59 ± 4.58) compared to the placebo group (4.46 ± 3.40; P < 0.05), but fewer than in the control group (9.21 ± 4.82). The number of transferable embryos was comparable between the treatment group (3.33 ± 2.30) and the control group (3.43 ± 2.13; NS), and significantly higher than in the placebo group (1.69 ± 1.87; P < 0.05). Furthermore, the treatment group produced more high-quality embryos (3.04 ± 1.89) than the placebo group (1.35 ± 1.99; P < 0.05), and more than the control group (2.43 ± 1.95). No significant intergroup difference was found in fertilization rates (64.40% vs. 62.90% vs. 60.90%; NS). However, the high-quality embryo rate was significantly higher in the treatment group (47.02%) compared to both the placebo (31.15%; P < 0.05) and control (29.05%; P < 0.05) groups. Finally, the treatment group demonstrated a significantly greater reduction in peri-menstrual abdominal pain scores (Δ=−1.15 ± 0.36) compared to the placebo group (Δ=−0.35 ± 0.49; F = 6.84, P = 0.01). Metabolomic analysis revealed that the steroid hormone biosynthesis pathway was the most significantly disturbed in OE patients, characterized by markedly elevated levels of key intermediates such as 17α-hydroxyprogesterne. Critically, the levels of 17α-hydroxyprogesterone exhibited a significant negative correlation with oocyte yield (r = -0.286, p = 0.012), directly linking this metabolic dysregulation to impaired clinical outcome. Furthermore, oxidative stress metabolites showed a strong positive correlation with the luteinization marker 20α-hydroxy-4-pregnen-3-one (r = 0.43, p = 0.001), suggesting a potential interaction between oxidative stress and aberrant steroidogenesis. The AAT intervention effectively normalized this dysregulated steroidogenic profile, which underpinned the observed therapeutic benefits. Conclusion: AAT significantly alleviates peri-menstrual pain and enhances IVF outcomes in ovarian endometriosis patients, which may be attributed to the restoration of granulosa cell steroid metabolism, evidenced by normalized levels of 17α-hydroxyprogesterone and attenuation of oxidative stress. Clinical trial registration: https://www.chictr.org.cn/ , identifier ChiCTR2200057339.

Background: To evaluate the association between seasonal temperature variations and clinical outcomes of in vitro fertilization (IVF), aiming to provide theoretical foundations for optimizing protocol timing in reproductive medicine.Methods: This retrospective cohort study analyzed 2,551 first fresh IVF-embryo transfer (IVF-ET) cycles performed at a tertiary reproductive center of The First Affiliated Hospital of Shandong University of Traditional Chinese Medicine between January 2009 and January 2024. The study population comprised normo-ovulatory women aged < 35 years without uterine anomalies or severe male factor infertility (sperm concentration >1 × 106/mL). Cycles were stratified into four seasonal cohorts based on gonadotropin initiation dates: spring (March-May, n = 709), summer (June-August, n = 787), autumn (September-November, n = 640), and winter (December-February, n = 415). Primary outcomes included clinical pregnancy rate (CPR), live birth rate (LBR), miscarriage rate (MR), and Full-Term delivery rate (FTBR), analyzed through multivariable logistic regression models adjusting for mean daily temperature (°C), relative humidity (%), and daylight hours (h).Results: Compared with the winter control group, risk of miscarriage in cycles initiated in spring showed a statistically significant increase (95% CI 1.019, 2.846; P = 0.042). Although CPR showed no seasonal variation (spring: 54.30%, summer: 52.22%, autumn: 50.47%, winter: 50.36%; P = 0.464), the spring cohort exhibited a numerically higher Full-Term delivery rate (39.07 vs. 34.22%; P = 0.105). Sensitivity analysis using weighted analysis to balance sample sizes across groups revealed significantly higher full-term birth rates in spring compared to winter (P = 0.046) and the live birth rate in spring was also significantly higher than in winter (P = 0.029). For each unit increase in sunlight intensity on the trigger day, the probability of successful pregnancy decreases to approximately 0.978 times the original value (OR = 0.978 per lux-unit increase, 95% CI 0.960–0.997; P = 0.025).Conclusion: Seasonal microenvironmental factors during ovarian stimulation may modulate IVF success trajectories, suggesting potential benefits of climate-adaptive protocol personalization in temperate monsoon regions.Clinical trial registration: This is a retrospective case-control study.