Background
In recent years, IVF-ET has undergone significant advancements, offering hope to countless infertility patients. However, this technology also faces considerable challenges. During COS, a subset of patients exhibits suboptimal response to exogenous gonadotropins (Gn), characterized by: Limited follicular development; Low peak serum estradiol(E2) levels; High Gn dosage requirements; Reduced oocyte yield; Elevated cycle cancellation rate. These factors collectively contribute to diminished clinical pregnancy rates, defining a condition termed POR.
Epidemiological studies report a POR incidence of 5.6%–35.1% (1), accounting for approximately 9–26% of IVF indications (2). The 2011 Bologna Criteria established the first evidence-based framework for POR diagnosis and therapeutic strategies (3). In 2016, the Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) group introduced a novel stratification system, categorizing patients by age, ovarian reserve markers and previous stimulation response. This paradigm shift replaced the term “poor response” with “low prognosis” (POSEIDON Group 1–4), substantially reducing interpatient heterogeneity (4).
The etiology of POR remains unclear but may involve advanced maternal age, genetic predisposition, prior ovarian surgery, pelvic inflammatory disease, and environmental factors (5), with the latter emerging as a key research focus in recent years (6). Clarifying the pathogenesis of POR and developing targeted prevention and treatment strategies to obtain an optimal number of high-quality oocytes during COS, thereby improving IVF success rates in POR patients, represents a critical challenge in reproductive medicine.
Although traditional Chinese medicine (TCM) does not specifically mention the disease term “POR,” its clinical manifestations can generally be categorized under TCM syndromes such as “infertility,” “scanty menstruation,” “delayed menstruation,” “amenorrhea,” and “menopausal syndrome.” Clinically, POR patients most frequently present with a Kidney Deficiency-Liver Stagnation Pattern (7, 8). Professor Zhengao Sun, a leading TCM expert, proposes that POR pathogenesis is closely related to liver-kidney dysfunction, consistent with the TCM theory of “simultaneous liver-kidney disorders,” with Kidney Deficiency-Liver Stagnation Pattern being the core pathogenesis. The clinical application of QZYST, a kidney-tonifying and liver-regulating herbal formula that nourishes blood and activates blood circulation, has demonstrated efficacy in improving POR patients’ clinical symptoms, ovarian reserve function, and ovarian responsiveness.
PFAS, a class of widespread and persistent EEDs found in various consumer products, can accumulate in human tissues through multiple exposure routes, posing significant health risks including reproductive toxicity (9). Transport proteins such as serum albumin, solute carrier proteins, and ATP-binding cassette transporters facilitate PFAS distribution throughout the body, enabling their penetration through the follicular barrier and subsequent detection in female follicular fluid (10). Emerging evidence indicates these compounds disrupt steroidogenesis by altering the expression of key steroidogenic enzymes (11), thereby impairing follicular development, oocyte maturation, and ultimately leading to diminished ovarian reserve and compromised oocyte quality (12). While current evidence suggests a potential association between PFAS exposure and POR, this relationship remains to be conclusively established and warrants further investigation.
Follicular fluid, serving as the critical microenvironment for oocyte growth and development, exhibits metabolic profiles intrinsically linked to oocyte and granulosa cell quality (13). Consequently, investigating metabolite dynamics in follicular fluid holds significant diagnostic and therapeutic value for conditions like POR. The advent and rapid advancement of metabolomics technologies have enabled robust quantification of follicular fluid metabolites, providing insights into oocyte developmental potential (14) and offering transformative opportunities for elucidating POR pathogenesis and guiding clinical management. This study utilizes high-throughput targeted PFAS quantification analysis to identify differential PFAS metabolite profiles in follicular fluid between POR patients with Kidney Deficiency-Liver Stagnation Pattern and IVF controls with normal ovarian reserve, and analyze PFAS level alterations following intervention with kidney-tonifying and liver-regulating herbal medicine (QZYST), and investigate the correlation between follicular fluid PFAS compounds and the pathogenesis of Kidney Deficiency-Liver Stagnation Pattern POR, thereby advancing the mechanistic understanding of POR development.
Discussion
This study enrolled 93 women undergoing IVF-ET, who were assigned in a non-randomized, prospective, sequential comparative manner to the POR-NON (n = 31), POR-CON (n = 31), and POR-TCM (n = 31) groups. Using high-throughput targeted PFAS quantification analysis, we quantified six PFAS in follicular fluid to investigate their role in POR pathogenesis. While prior studies have examined PFAS exposure in relation to blood to follicle transfer efficiencies (16), embryo quality (17), and fertility outcomes (18)—or employed untargeted metabolomics to explore associations between follicular fluid endocrine disruptors and POR (19–21)—this work provides preliminary evidence for an association between follicular fluid PFAS compounds and the development of POR, suggesting they may play a role in the pathological microenvironment of the follicle and demonstrate the clinical efficacy of TCM intervention for PFAS-exposed POR patients.
Follicular fluid—an ultrafiltrate of plasma reflecting systemic metabolic dynamics—constitutes the critical microenvironment for oocyte development, where its compositional profile directly influences oocyte and granulosa cell quality (22). PFAS, as pervasive EEDs in industrial and consumer products, demonstrably impair endocrine and reproductive functions (23–25). In this study, targeted quantification of six PFAS congeners revealed PFOA as the predominant compound across all groups (POR-CON, POR-NON, POR-TCM), followed by PFOS, aligning with prior epidemiological evidence (17).
PFBA, a short-chain PFAS with high water solubility and low bioaccumulation potential (elimination half-life: 48–96 hours (26)), is increasingly used as an alternative to long-chain compounds like PFOA/PFOS. However, its widespread detection in drinking water sources raises toxicological concerns (27, 28). Our study revealed significantly elevated PFBA levels in follicular fluid of Kidney Deficiency-Liver Stagnation Pattern POR patients versus controls (P < 0.01). Experimental evidence indicates PFBA induces hepatotoxicity across species (rodents, zebrafish, humans) by disrupting lipid metabolism and gene expression via peroxisome proliferator-activated receptor alpha (PPARα) activation. Bjork et al. demonstrated 100 μM PFBA upregulated Acox, Cte/Acot1, and Cyp4a1/11 in rat hepatocytes, yet lost PPARα-stimulatory effects at 200 μM in both rat and human liver cells (29). Foreman et al. confirmed PPARα-dependent hepatomegaly in humanized PPARα mice (30). Zebrafish studies showed sublethal PFBA exposure caused metabolic disruption and lipid droplet accumulation despite unchanged PPAR, Fabp3, and crot expression (31). We thus propose that PFBA contributes to POR pathogenesis by suppressing aromatase expression via PPARα signaling (32), thereby impairing oocyte maturation, developmental competence, and quality.
PDA, a long-chain PFAS with a 10-carbon backbone, exhibits heightened toxicity proportional to chain length (33). Consequently, PDA poses greater environmental and human health risks than shorter-chain congeners. Although follicular fluid PDA levels showed no statistically significant difference between Kidney Deficiency-Liver Stagnation Pattern POR patients and controls (P > 0.05), a marginally elevated trend was observed (1.07 vs. 0.83 nmol/L). Experimental evidence confirms PDA activates PPARα, upregulating Cyp2B10 and 4A14 expression in murine liver to induce hepatotoxicity (34). In primary mouse renal cells, PDA elevates intracellular reactive oxygen species (ROS) while suppressing superoxide dismutase activity, exacerbating oxidative stress and triggering apoptosis (35). Porcine oocyte studies further demonstrate PDA disrupts calcium homeostasis, impairing gap junctional communication between cumulus cells and oocytes, thereby compromising developmental competence and assisted reproductive technology (ART) success rates (36). Deng et al. revealed PDA inhibits maturation-promoting factors, causing meiotic arrest at prophase I and spindle assembly checkpoint dysfunction—accelerating meiotic progression and aneuploidy risk. Concurrent mitochondrial defects and ROS accumulation induce oocyte apoptosis (37). We thus propose PDA contributes to Kidney Deficiency-Liver Stagnation Pattern POR pathogenesis through PPARα-mediated aromatase suppression, oxidative stress induction, disruption of granulosa-oocyte gap junctions, and interference with meiotic fidelity.
PFOS, a prototypical and extensively studied PFAS compound, showed marginally elevated levels in the follicular fluid of Kidney Deficiency-Liver Stagnation Pattern POR patients compared to controls (5.62 vs. 4.82 nmol/L), though this difference lacked statistical significance (P > 0.05). Accumulating evidence indicates PFOS induces hepatorenal damage via oxidative stress (38, 39) and reduces fertilization potential, as evidenced by negative dose-response relationships in meta-analyses (40). Crucially, PFOS impairs oocyte competence through three synergistic mechanisms: (1) Disrupting cytoskeletal assembly to cause spindle aberrations, chromosomal misalignment, and actin dysfunction; (2) Compromising mitochondrial dynamics to provoke oxidative stress, DNA damage, and apoptosis; (3) Altering cortical granule distribution to hinder sperm-oocyte fusion (41, 42). Given this mechanistic evidence and our observed trend of PFOS accumulation, we propose PFOS exposure contributes to POR pathogenesis in this TCM pattern.
PFNA, an unrestricted long-chain PFAS with a 9-carbon backbone widely used in firefighting foams, metal coatings, and imaging additives, exhibits heightened toxicity despite lower human blood concentrations than PFOA/PFOS. Our data revealed marginally elevated PFNA levels in the follicular fluid of Kidney Deficiency-Liver Stagnation Pattern POR patients versus controls (1.29 vs. 1.21 nmol/L, P > 0.05). Mechanistically, Jiao et al. demonstrated PFNA disrupts oocyte maturation by impairing germinal vesicle breakdown and polar body extrusion through dual pathways: (1) inducing DNA damage via ROS-triggered oxidative stress; (2) causing severe spindle defects and chromosomal misalignment in MII oocytes (43). We thus propose PFNA contributes to POR pathogenesis by compromising oocyte quality through sublethal mitochondrial stress and meiotic disorganization, ultimately diminishing ART success in affected patients.
PFOA, an 8-carbon PFAS with high persistence and bioaccumulation potential, enters humans via respiratory, digestive, and dermal routes, binding plasma albumin to disrupt endocrine and reproductive functions. Our study observed a marked elevation of follicular fluid PFOA in Kidney Deficiency-Liver Stagnation Pattern POR patients versus controls (28.2 vs. 19.1 nmol/L, P > 0.05). Experimental evidence reveals PFOA compromises female reproduction through multisystem toxicity: suppressing ovarian steroidogenesis to impair folliculogenesis (44), inducing mitochondrial dysfunction and γ-H2AX-marked DNA damage in oocytes with consequent meiotic defects (45), and triggering granulosa cell apoptosis via disrupted gap junctional intercellular communication (46). We therefore propose that elevated PFOA contributes to POR pathogenesis in this TCM pattern through tripartite mechanisms: dysregulating ovarian hormone synthesis, compromising oocyte metabolic integrity, and impairing granulosa-oocyte crosstalk.
PAUnA, an understudied PFAS congener with recognized human toxicity, showed a non-significant upward trend in the follicular fluid of Kidney Deficiency-Liver Stagnation Pattern POR patients versus controls (1.56 vs. 1.50 nmol/L, P > 0.05). Given its bioaccumulative potential, we hypothesize chronic PAUnA exposure may contribute to POR pathogenesis through progressive bodily accumulation, ultimately disrupting ovarian function and impairing oocyte developmental competence.
Current evidence on PFAS exposure and fertility outcomes remains inconsistent. A Chinese prospective cohort study found no significant associations between serum/follicular fluid PFAS levels and key ART endpoints—including oocyte maturation rate, fertilization rate, high-quality embryo rate, biochemical pregnancy, clinical pregnancy, or preclinical spontaneous abortion (47). McCoy et al. conversely reported reduced blastocyst conversion rates with follicular fluid PFNA and PFDA exposure, though no links to fertilization or pregnancy outcomes were observed (48). Notably, Ma et al. demonstrated maternal plasma PFOA inversely correlated with oocyte yield and 2PN embryos, while paternal PFOA levels reduced 2PN zygote formation (49). A Singaporean case-control study further revealed 5-10% decreased fecundability per quartile increase in preconception PFDA, PFOS, PFOA, and PFHpA, with PFAS mixtures reducing clinical pregnancy and live birth likelihood by 30-40% per quartile; however, PFHxS, PFNA, and PFHpS showed no associations (18). This heterogeneity may be attributable to limited sample sizes, demographic variability across study populations, and uncontrolled confounding factors.
While prior research predominantly focused on exposure-outcome relationships without addressing therapeutic interventions, this study pioneers the evaluation of QZYST efficacy specifically for POR patients presenting with Kidney Deficiency-Liver Stagnation Pattern. POR—a pathological condition characterized by suboptimal ovarian response to gonadotropin stimulation during IVF-ET—necessitates escalated Gn dosage and prolonged stimulation to achieve adequate follicular development on hCG day. This not only imposes significant financial burdens but also heightens risks of ovarian hyperstimulation syndrome (OHSS) and luteal phase deficiency, consequently increasing early pregnancy loss rates (21).
From a TCM perspective, Professor Sun Zhengao identifies POR as a disorder rooted in ovarian dysfunction attributable to concurrent kidney and liver system pathologies. The core pathogenesis involves Kidney essence depletion, Kidney Qi deficiency, and Liver Qi stagnation as pivotal contributing factors. Drawing on decades of clinical expertise, Professor Sun empirically developed QZYST to treat POR with Kidney Deficiency-Liver Stagnation Pattern. This formula aims to tonify the Kidney and regulate Liver Qi, thereby ameliorating clinical manifestations and enhancing reproductive outcomes. The intervention duration of 31–33 days was strategically designed to bridge the transition from the preceding cycle’s luteal phase to the current stimulation cycle. This ‘pretreatment’ or ‘priming’ window is consistent with TCM principles of preparing the ovarian microenvironment to enhance oocyte competence. Physiologically, this period covers the final stages of antral follicle recruitment, providing an adequate timeframe for QZYST to exert its antioxidant and metabolic regulatory effects. QZYST comprises 13 botanical drugs, each substantiated by extensive contemporary pharmacological research.
Rehmanniae Radix Praeparata contains bioactive constituents including iridoid glycosides, phenylethanoid glycosides, carbohydrates, and 5-hydroxymethylfurfural (5-HMF)—a Maillard reaction product formed during processing. Crucially, enhanced follicle-stimulating hormone receptor (FSHR) expression improves granulosa cell function, whereas FSHR silencing induces apoptosis and follicular atresia (50). Rehmanniae Radix Praeparata may elevate 5-HMF concentrations and upregulate FSHR levels, thereby promoting granulosa cell proliferation, folliculogenesis, and ultimately ovarian reserve parameters (51). Given that dysregulated free radical metabolism impairs follicular development, ovarian reserve, and oocyte quality, 5-HMF’s free radical scavenging capacity confers significant antioxidant effects (52). Collectively, Rehmanniae Radix Praeparata exhibits pharmacological properties that enhance granulosa cell viability, combat oxidative stress, and mitigate cellular senescence. Additionally, its immunomodulatory and anti-inflammatory actions may benefit POR patients with autoimmune comorbidities (51, 53).
Cornu Cervi Degelatinatum, primarily composed of calcium carbonate, calcium phosphate, colloids, nitrogen compounds, and multiple amino acids, demonstrates significant immunomodulatory effects. Both water-processed and vinegar-processed forms reduce pro-inflammatory cytokines (TNF-α, IL-6) while elevating anti-inflammatory cytokines (IL-10) in rat models of ulcerative colitis with Yang Deficiency of Spleen-Kidney Syndrome (54). It is postulated that this dual-processed material may enhance oocyte developmental competence by modulating immune-inflammatory responses within the ovarian microenvironment.
Cistanches Herba contains bioactive compounds including echinacoside, verbascoside, and salidroside, which collectively confer ovarian protection by suppressing granulosa cell apoptosis through modulation of Bcl-2 family-mediated mitochondrial pathways (55). Specifically, salidroside protects dihydrotestosterone (DHT)-induced human granulosa-like tumor cells (KGN) by activating the AMPK/Nrf2 axis to attenuate oxidative stress and apoptotic cascades (56). Furthermore, phenylethanoid glycosides from Cistanche deserticola demonstrate hepatoprotective efficacy via triple mechanisms: stimulating hepatocyte proliferation/regeneration, while suppressing inflammation, oxidative damage, and programmed cell death (57, 58).
Ecliptae Herba and Fructus Ligustri Lucidi collectively form Erzhi Pill (EZP), a classic TCM formula with established anti-aging properties. EZP mitigates pseudo-hypoxic microenvironments and inflammatory states by downregulating HIF1α to delay aging (59). Notably, ecliptasaponin A (from Ecliptae Herba) and specnuezhenide (from Fructus Ligustri Lucidi) upregulate ESR1 to protect against cyclophosphamide-induced damage in human KGN (60). Furthermore, EZP exerts hepatoprotective effects by inhibiting PI3K/Akt/Raptor/Rictor signaling to suppress hepatocyte apoptosis (61).
Curcumae Radix contains key bioactive constituents including curzerene, curcuminoids, and terpenoids. Curcuminoids enhance ovarian function through anti-inflammatory, anti-apoptotic, and antioxidant properties (62), notably by boosting antioxidant enzyme activity and activating the Nrf2-Keap1 pathway to increase cellular oxidative stress resistance and survival rates (63). In D-galactose-induced premature ovarian insufficiency mice, curcumin ameliorated oxidative damage and apoptosis, elevating SOD while reducing MDA levels and downregulating SOD2/CAT mRNA expression (64). This coincided with upregulated Nrf2 and HO-1 proteins—critical mediators of ROS scavenging (64). Additionally, Curcumae Radix exerts hepatoprotection in CCl4-induced liver injury by suppressing NF-κB activation, downregulating pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and upregulating anti-inflammatory IL-10 to mitigate inflammation and fibrosis (65).
Modern pharmacological studies confirm that Lycii Fructus (66, 67), Fructus Ligustri Lucidi (68), Fructus Mori (69), Rubi Fructus (70), Cuscutae Semen (71), Nelumbinis Plumula (72), and Rosae Laevigatae Fructus (73) collectively exhibit antioxidant and anti-aging properties, which mitigate oocyte senescence and reduce apoptosis. In TCM, these seven botanicals—unified as “Qizi” (Seven Seeds) due to shared Kidney-tonifying and Essence-nourishing functions—all belong to seed-derived drugs. Additionally, Rubi Fructus (74), Cuscutae Semen (75, 76), Nelumbinis Plumula (77), and Rosae Laevigatae Fructus (78) demonstrate hepatoprotective effects through distinct mechanisms.
Radix Glycyrrhizae contains bioactive triterpenoids and flavonoids that confer potent antioxidant and immunoenhancing effects, as substantiated by pharmacological studies (79, 80).
QZYST significantly ameliorated Kidney Deficiency-Liver Stagnation Pattern manifestations in POR women, evidenced by a marked reduction in total syndrome scores post-intervention (median [IQR]: 18 (15, 22) vs. 21 (18, 24); P < 0.001). Compared with the POR-NON group, QZYST-treated subjects demonstrated substantially lower syndrome scores (18 (15, 22) vs. 21 (19, 23); P < 0.05), confirming its therapeutic efficacy for this TCM pattern. QZYST treatment significantly improved key manifestations of Kidney Deficiency-Liver Stagnation Pattern (all P < 0.05), including: premenstrual irritability or chronic depression, soreness in lumbar and knees, dizziness and tinnitus, premenstrual breast distension, loss of libido and dark tongue body. Although symptomatic relief for scanty menstruation, darkish or dull menstrual blood, hot flashes with sweating, mental fatigue and weakness, insomnia with dreamfulness, hypochondriac distension/pain, clear profuse urine and deep thready or wiry pulse did not reach statistical significance (P > 0.05), amelioration trends were observed versus baseline. In the present study, the significant improvements in the number of oocytes retrieved and 2PN zygotes, coupled with a reduced rate of cycles with no transferable embryos, underscore the clinical utility of QZYST for POR patients. Although the clinical pregnancy and cumulative pregnancy rates showed only a modest numerical increase without statistical significance, this is likely attributable to the limited sample size, as binary pregnancy outcomes require larger cohorts to achieve sufficient statistical power. For POR patients—a population characterized by a low prognosis—the ability to obtain more oocytes and embryos is the fundamental prerequisite for any successful pregnancy (81). Our findings suggest that QZYST primarily acts by optimizing the follicular microenvironment and reducing the toxic impact of PFAS, thereby expanding the pool of available embryos for transfer.
Additionally, in the follicular fluid of patients with Kidney Deficiency-Liver Stagnation Pattern POR who received QZYST intervention (POR-TCM group), the level of PFBA was significantly lower than that in POR patients without Chinese herbal medicine intervention (POR-NON group), and the difference was statistically significant (P < 0.05). It is important to acknowledge that the observed association between reduced follicular PFBA levels and improved clinical outcomes in the POR-TCM group remains correlative. In the complex biological system of the ovary, QZYST likely exerts a synergistic effect by both enhancing the metabolic clearance of EEDs like PFBA and directly attenuating oxidative stress within granulosa cells. While our data suggest that the reduction of PFAS burden is a key metabolic feature of successful TCM intervention, further functional experiments—such as utilizing PFAS-exposed cell models or animal studies—are required to definitively establish a causal link and map the downstream molecular signaling pathways. However, although the levels of five other perfluoroalkyl substances—PDA, PFOS, PFNA, PFOA, and PAUnA—also showed a downward trend in the POR-TCM group, the differences were not statistically significant (P > 0.05). Therefore, we speculate that the improvement in TCM syndrome manifestations and clinical outcomes after QZYST intervention may be associated with changes in the levels of certain PFAS compounds in follicular fluid.