Per- and polyfluoroalkyl substances are associated with reduced cumulus cell MFN1 expression and lower oocyte maturation rates

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Seifer, Gizem Nur Sahin, Olga Chaplia, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9247718/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Purpose: To determine whether per- and polyfluoroalkyl substances (PFAS) are detectable in human follicular fluid (FF) and to evaluate their association with cumulus cell (CC) expression of Mitofusin-1 ( MFN1 ), a mitochondrial fusion gene linked to fertility, as well as selected assisted reproductive technology (ART) outcomes. Methods: This cross-sectional study included reproductive-aged women undergoing oocyte retrieval for IVF/ICSI. FF and CCs were collected at retrieval. Concentrations of 24 PFAS were measured in FF by liquid chromatography-mass spectrometry. MFN1 mRNA expression was measured in paired CCs by quantitative PCR. Associations between PFAS concentrations and MFN1 expression were assessed using Spearman correlation and tertile-based Kruskal-Wallis tests. Multivariable linear regression models were used to assess associations with ART outcomes. Results: Seventy FF samples from 57 women were analyzed, with perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) detected in all samples. Paired CCs were available for 38 FF samples. PFOS demonstrated a moderate inverse association with CC MFN1 expression (Spearman rank =-0.45, p=0.005), which persisted after adjustment for age (partial Spearman =-0.46, p=0.005). When PFOS was stratified into tertiles (low 2.77ng/mL), median MFN1 expression declined progressively with 4.56 arbitrary unit (AU) in the lowest tertile, 2.79AU in the middle tertile, and 1.46AU in the highest tertile (Kruskal Wallis H=9.84, p=0.007). FF PFOS levels were independently associated with oocyte maturation rate, with each 1ng/mL increase in PFOS associated with a 2.06% decrease in maturation (95% CI -3.20 to -0.92, p=0.0007). No significant associations were observed between FF PFAS levels and other ART outcomes or patient characteristics. Conclusion: PFAS are ubiquitously detectable in human FF. Higher FF PFOS levels are independently associated with lower MFN1 expression in CCs and reduced oocyte maturation rates, supporting a potential mitochondrial mechanism linking PFAS exposure to impaired reproductive function. Capsule : Higher follicular fluid PFAS concentrations are associated with reduced cumulus cell MFN1 expression and impaired oocyte maturation, linking endogenous PFAS exposure to mitochondrial dysfunction in human IVF cycles. per- and polyfluoroalkyl substances Mitofusin-1 (MFN1) mitochondria oocyte maturation follicular fluid ART Figures Figure 1 Introduction Per- and polyfluoroalkyl substances (PFAS) are a broad class of synthetic substances used since the 1950s in numerous products such as non-stick cookware, stain-resistant textiles, cosmetics and food contact materials [ 1 – 3 ]. Their carbon-fluorine backbones make them highly persistent and prone to bioaccumulation [ 1 , 4 , 5 ]. Among the PFAS, Perfluorooctanesulfonic Acid (PFOS) and Perfluorooctanoic Acid (PFOA) are notable for long human half-lives and strong anion driven binding to plasma proteins, especially albumin and, to a lesser extent, lipoproteins. The sulfonate head group of PFOS generally confers higher albumin affinity than PFOA, which often results in greater persistence [ 6 – 8 ]. Consistent with these properties, PFAS have been documented in multiple human tissues, including blood, urine, breast milk, and follicular fluid (FF) [ 1 , 9 – 15 ]. Several studies have detected PFAS in human FF, reported in cohorts from a number of countries, with PFOS and PFOA frequently the most abundant [ 13 , 14 , 16 , 17 ]. Given that these compounds are detectable within the ovarian micro-environment, there has been increasing interest on their potential impact on female reproduction. PFAS have repeatedly shown effects at the cellular level. In vitro, human granulosa cells (GCs) exposed to a PFAS mixture (PFOS/PFOA/PFHxS) demonstrated altered proliferation, hormone production and transcriptomic shifts [ 18 ]. GC work in mice has similarly shown that PFAS mixtures lower mitochondrial activity and membrane potential, reduce ATP and increase ROS, consistent with mitochondrial stress [ 19 ]. In placental/trophoblast models, PFOS specifically has been shown to reduce mtDNA copy number and down regulate mitochondrial fusions genes [ 20 ]. Similarly, in a zebrafish larvae, PFAS mixtures disrupt mitochondrial membrane potential and respiratory-chain gene expression, confirming PFAS can target mitochondrial function across species and cell types [ 21 ]. Building on in vitro evidence that PFAS can impair mitochondrial function, it is compelling to investigate whether endogenous PFAS levels (those actually present in human FF) also have effects on mitochondrial maintenance that supports follicular function. Mitochondrial integrity in GCs and cumulus cells (CCs) is governed not only by respiratory capacity but by mitochondrial dynamics. These dynamics consist of fusion, in which adjacent mitochondria join to share proteins, lipids and mtDNA, and fission, in which mitochondria divide to segregate damaged segments and facilitate turnover. Both together support organelle quality control [ 22 , 23 ]. Among the proteins that regulate outer mitochondrial membrane fusion, Mitofusin-1 is a key mediator, and is encoded by the nuclear gene MFN1 [ 24 , 25 ]. Prior work in mouse models demonstrated that loss of MFN1 critically impairs follicle development and fertility, indicating that adequate MFN1 expression is required for functional female reproduction [ 26 , 27 ]. On this basis, our aim was to determine if real-world PFAS concentrations in FF are sufficient to impact MFN1 expression in paired human CCs. Additionally, we examined whether PFAS levels correlated with IVF outcomes. Materials and Methods Sample collection and isolation The study participants included reproductive aged women, 18-46 years of age, undergoing oocyte retrieval for IVF, ICSI or oocyte cryopreservation at Yale Fertility Center (IRB No. 2000036885), at Yale School of Medicine, CT, USA. Exclusion criteria included women or their partners with known chromosomal abnormalities or exposure to prior chemotherapy. Ovarian stimulation protocols included standard long, short and flare protocols, and were chosen by the treating clinician. At retrieval, FF was aspirated from the first one to two large follicles per patient, with effort to obtain clear FF samples free of blood. Throughout the study, all samples were individually stored (not pooled), and the information regarding which follicle the oocyte, cumulus cells (CCs) and FF were derived from was recorded. From the collected samples, the CCs were mechanically denuded from the oocyte per lab protocol. The CCs were then placed in cryopreservation vials containing RNA later and frozen at -80C for later analysis. The FF was then centrifuged, and any granulosa cells, red blood cells or other cellular debris were removed. FF was frozen at -80C until further analysis. PFAS measurement by high-resolution liquid chromatography-mass spectrometry Twenty-four unique PFAS were quantified in FF by high-resolution liquid chromatography-mass spectrometry using an Agilent 1290 Infinity liquid chromatograph coupled to an Agilent 6546 quadrupole-time-of-flight mass spectrometer in negative electrospray ionization mode. Concentrations were reported as ng/mL. Samples were analyzed along with a six-point calibration curve from native PFAS standards at 0.01, 0.05, 0.1, 0.5, 1, 2 parts per billion. MFN1 measurement by quantitative reverse transcription polymerase chain reaction (qRT-PCR) RNA was isolated from CCs using the QIAGEN RNeasy kit according to manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using a manual reverse transcription (RT) protocol. For each sample, RT was performed in a total reaction volume of 20uL, consisting of 10uL RNA combined with 2 uL RT butter, 0.8 uL dNTP mix, 2uL of RT primer, 1 uL reverse transcriptase, 1 uL RNase inhibitor, and 3.2uL nuclease-free water. RT reactions were prepared in duplicate and then pooled for each sample. cDNA concentration was then measured by NanoDrop to allow normalization of template input for quantitative polymerase chain reaction (qPCR). QPCR was performed using SYBR Green chemistry on a Bio-Rad CFX Duet real-time PCR system. Each reaction was run in a total volume of 20uL using SYBR Green master mix, with the volume of cDNA and nuclease-free water adjusted for each reaction based on NanoDrop cDNA concentrations to ensure that each well received the same concentration of cDNA. Gene expression of MFN1 was quantified using gene-specific primers (MFN1 forward: 5’-GGT-GAA-TGA-GCG-GCT-TTC-CAA-G-3’; MFN1 reverse: 5’-TTC-TTC-ACC-AAG-AAA-TGC-AGG-C-3) and normalized to the reference gene GAPDH (GAPDH forward: 5’-GTC-TCC-TCT-GAC-TTC-AAC-AGC-G-3’; GAPDH reverse: 5’-ACC-ACC-CTG-TTG-CTG-TAG-CCA-A-3’). All reactions were performed in duplicate. To control for inter-plate variability, the same calibrator samples were included on each plate. MFN1 expression was calculated relative to GAPDH using the delta-delta cycle threshold method. Statistical Analysis To examine the relationship between FF PFAS concentrations and CC MFN1 expression, Spearman rank correlations were performed with age adjusted partial Spearman correlations. PFAS levels were additionally categorized into tertiles, and differences in MFN1 expression across groups was compared using the Kruskal-Wallis test. Baseline characteristics and IVF cycle parameters were compared across PFOS tertiles using one-way ANOVA for normally distributed variables and Kruskal-Wallis tests for non-normally distributed variables. Continuous variables are reported as mean +/- SEM or median (interquartile range), as appropriate. Using multiple linear regression models, the relationship between PFAS levels (PFOS and PFOA) and ART outcomes, including days of ovarian stimulation, total gonadotropin dose, number of oocytes retrieved, metaphase II (MII) rate (% of MII oocytes from total oocytes retrieved), number of two pronuclei (2PN) embryos, and blastocyst number and formation rate, were evaluated. Models were adjusted for potential patient level confounders such as age, body mass index (BMI), and anti-Mullerian hormone (AMH). Analyses were performed using GraphPad Prism (v10). Results PFAS in FF PFAS concentrations were measured in a total of 70 FF samples from 57 patients. Of the 24 PFAS measured, both PFOS and PFOA were detected in 100% of specimens, with means of 2.6ng/mL and 1.1ng/mL, respectively. Of the remaining PFAS measured, 20 other PFAS metabolites were detected in at least one FF sample (Table I). Follicular fluid PFOS and PFOA concentrations and cumulus cell MFN1 expression Paired CC material was available for 38 FF samples, which were analyzed for MFN1 expression correlated with FF PFOS and PFOA concentrations, given that these two PFAS were present in every FF sample. FF PFOS showed a moderate inverse association with MFN1 expression in CC (Spearman r = -0.45, p = 0.005). This relationship persisted after adjustment for age (partial Spearman = -0.46, p = 0005). When PFOS was stratified into tertiles (low 2.77ng/mL), median MFN1 expression declined progressively with higher PFOS concentration (Fig I), 4.56 arbitrary unit (AU) in the lowest tertile, 2.79AU in the middle tertile and 1.46AU in the highest tertile (Kruskal Wallis H 9.84, p = 0.007, Fig I). Baseline age and AMH were similar across PFOS tertiles. No significant differences were observed across PFOS tertiles for total gonadotropin dose, number of oocytes retrieved, MII rate, fertilization outcomes or blastocyst yield. BMI and days of ovarian stimulation differed between groups (Table II). PFOA demonstrated a weaker inverse trend with MFN1 (Spearman rank = -0.23, p = 0.16). When PFOA was stratified by tertiles (low 1.56ng/mL), the means fell progressively from the lowest to highest PFOA tertiles (11.19, 5.94, 3.99), however, comparing the medians (3.06, 2.33, 2.71) did not yield significant results (Kruskal Wallis H 2.13 p = 0.35). Follicular fluid PFOS and PFOA concentrations and ART outcomes FF PFOS and PFOA levels were examined in relation to patient characteristics and ART outcomes in the 57 patients included in the study (Table III). In multivariable linear regression models adjusted for age, BMI and AMH, FF PFOS concentrations were independently associated with maturation rate, with each 1ng/mL increase in PFOS associated with a 2.06% decrease in MII rate (CI -3.20 to -0.92, p = 0.0007). A similar trend was observed for FF PFOA, though it did not reach statistical significance (β = -7.043, CI -15.3 to 1.54, p = 0.1). FF PFAS levels were otherwise not associated with age, BMI or AMH in mutually adjusted models, nor were they associated with days of ovarian stimulation, total gonadotropin dose, number of oocytes retrieved, number of 2PN embryos, blast number, or blast formation rate. Discussion To our knowledge this is the first study to demonstrate that endogenously found PFAS concentrations in human FF are associated with cellular function changes, with higher FF PFOS levels linked to lower CC MFN1 expression, as well as reduced MII rates across rank-based, age-adjusted, and model-based analyses. Mechanistically, these observations align with experimental data showing that PFAS mixtures impair mitochondrial function by reducing ATP linked respiration, elevating ROS and down regulating biogenesis and fusion pathways [ 18 – 21 ]. Such shifts favor a dysfunctional mitochondrial network that can potentially compromise granulosa-oocyte metabolic coupling and oocyte competence. Because progression to the MII stage is an energy intensive process requiring tightly coordinated mitochondrial ATP production, impaired mitochondrial function is well documented to reduce maturation rates in both animal and human oocytes [ 28 – 31 ]. The current findings of parallel reductions in MFN1 expression and MII formation rate are thus biologically plausible and demonstrate a mechanism by which PFAS mixtures may induce a mitochondrial cellular effect. Furthermore, these data suggest that endogenously occurring PFAS concentrations are sufficient to produce measurable biological changes in human folliculogenesis. Although several FF studies did not find consistent associations with fertility outcomes, others do point towards potential reproductive risk. In China, a recent study found that higher FF PFOA was associated with greater odds of PCOS and higher androgens [ 32 ]. In an ART cohort from Sweden, FF PFAS were associated with lower embryo quality despite higher ovarian reserve in those patients, a pattern that is also compatible with PCOS-like physiology [ 17 ]. Another IVF study from China linked higher FF PFAS with fewer high quality embryos [ 33 ]. Complementing the FF literature, higher plasma PFAS concentrations were associated with reduced number of oocytes retrieved, 2PN zygotes and cleavage stage embryos [ 34 ]. In addition, our findings that higher PFOS levels are associated with reduced oocyte maturation rates strengthens the notion that ovarian PFAS exposure is clinically relevant, underscoring the need to further investigate the underlying disrupted cellular pathways and to determine how this may translate into impacting additional fertility outcomes. PFAS are present world-wide and found in industrialized nations. They are pervasive and persistent, with reported human FF concentrations varying across geographic regions. In China, one study measured 15 PFAS in FF and reported geometric means of PFOS at 4.8 ng/mL and PFOA at 4.6 ng/mL [ 16 ], with another Chinese study observing PFOA most abundant (median 5.6 ng/mL) followed by PFOS (median 4.3 ng/mL) [ 14 ]. In Australia, PFAS were detected in 100% of FF samples with mean levels of PFOS at 4.9 ng/mL and mean PFOA at 2.4 ng/mL [ 13 ]. By comparison, our cohort’s means were lower, with PFOS at 2.6 ng/mL and PFOA at 1.1 ng/mL. Differences between studies may reflect regional product use, legacy water contamination, population behaviors, sample measuring methods, pooling versus single-follicular samples, and participant factors such as BMI. Despite the differences, these studies underscore that PFAS exposure is a widespread world-wide concern. In our dataset, PFOA trended in the same direction as PFOS but with a smaller effect. This pattern is consistent with other studies, in which PFOS often affects cellular function at lower concentrations in several systems and exerts stronger mitochondrial inhibition than PFOA in side-by-side assays [ 35 , 36 ]. Several features may underlie this difference, PFOS has a sulfonate head group that is associated with stronger serum albumin binding and longer human half-life, which increase effective exposure in protein rich compartments like FF. Moreover, PFOS most consistently impacts mitochondrial bioenergetics across epithelia and reproductive cells [ 18 – 21 , 35 , 36 , 37 ]. Taken together, these considerations provide a plausible rational for why PFOS shows a clearer relationship with CC MFN1 expression and oocyte maturation rate in our cohort. Limitations of this study include the cross-sectional design, which precludes causal inference, use of mRNA rather than protein/function, low CC RNA concentrations limiting the number of gene panels, and a relatively small sample size. Replication in larger cohorts is warranted. Future work should test additional mitochondrial dynamics and quality control genes, connect molecular results to embryo competence, and assess fertility outcomes across regions. Conclusions In conclusion, PFAS are ubiquitously detectable in human FF, and higher PFOS is independently associated with lower MFN1 expression in paired cumulus cells. Coupled with the observed reduction in MII rate, these findings add human evidence of a mechanistic pathway that endogenous PFAS burdens in the real-world follicular microenvironment may compromise mitochondrial fusion pathways relevant to oocyte quality and ultimately fertility. Declarations -Funding – SREI/ASRM Young Investigators Award (MV) -Consent to participate – N/A (IRB for discard sample use) -Consent for publication – N/A -Ethical approval and its number – N/A -Data availability – N/A Author Contribution MV – funding acquisition, conceptualization, investigation, methodology, writing. 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Variable Low PFOS 2.77 (n=12) P value Age (years) 35.8 +/- 1.6 36.5 +/- 0.8 36.2 +/- 1 1.4 0.91 BMI (kg/m2) 26.1 +/- 1.6 30.4 +/- 1.8 23.7 +/- 1.3 0.02* AMH (ng/mL) 2.4 (1.5-4.7) 2.7 (0.9-3.5) 1.5 (0.7-2.3) 0.16 Total gonadotropin dose (IU) 3550 (1988-4500) 4200 (2813-4800) 4950 (3150-5288) 0.07 Days of stimulation 11.0 (9.5-11.5) 11.0 (10.0-12.0) 12.0 (11.3-13.0) 0.05* Oocytes retrieved (n) 11.0 (6.0-15.0) 11.0 (7.5-16.0) 10.5 (7.3-17.3) 0.90 MII rate (%) 75.0 (60.5-84.0) 81.5 (66.3-91.0) 60.0 (50.5-79.0) 0.27 Number of 2PN embryos (n) 5.0 (2.0 – 7.5) 6.0 (4.0-11.0) 6.0 (3.0-9.0) 0.47 Blastocysts (n) 1.0 (1.0-6.0) 4.0 (3.5 – 10.0) 3.0 (1.8-5.8) 0.09 PFOS (ng/mL) 1.3 (1.1-1.7) 2.3 (2.1-2.6) 3.8 (3.1-6.8) <0.0001**** Values are presented as mean +/- SEM or median (interquartile range). Comparisons across PFOS tertiles were performed using one-way ANOVA for normally distributed variables and Kruskal-Wallis test for non-normally distributed variables. *p >0.05, ****p<0.0001. MII = metaphase II, 2PN = two pronuclei. Table III. Multivariable linear regression analyses of follicular fluid PFAS concentrations in relation to patient characteristics and ART outcomes. Outcome PFOS β (95% CI) P value PFOA β (95% CI) P value Age (years) -0.17 (-0.54-0.21) 0.12 -0.05 (-0.12-0.01) 0.12 BMI (kg/m2) -0.09 (-0.36-0.17) 0.71 0.01 (-0.04-0.05) 0.92 AMH (ng/mL) -0.35(-1/06-0.36) 0.33 -0.07 (-0.18-0.05) 0.25 Total gonadotropin dose (IU) 48.66 (-7.87-105.20) 0.09 342.00 (-5.28-689.40) 0.06 Days of stimulation 0.08 (-0.01-0.17) 0.08 0.49 (-0.06-1.05) 0.08 Oocytes retrieved (n) 0.12 (-0.17-0.41) 0.41 0.49 (-1.32-2.29) 0.59 MII rate (%) -2.06 (-3.2 – 0.9) 0.0007*** -7.04 (-15.63 – 1.54) 0.11 2PN embryos (n) 0.07 (-0.14-0.28) 0.51 0.23 (-1.08-1.53) 0.73 Blastocysts (n) 0.02 (-0.15-0.19) 0.83 0.17 (-0.89-1.24) 0.75 Blastocyst formation rate (%) -0.28 (-1.82-1.25) 0.71 -0.89 (-10.40-8.61) 0.85 Results from multivariable linear regression models (n=57). All models were adjusted for age, body mass index (BMI), and anti-Mullerian hormone (AMH). β coefficients represent the estimated change in the outcome variable per 1ng/mL increase in FF PFOS or PFOA concentrations. ***p<0.001. MII = metaphase II, 2PN = two pronuclei. Additional Declarations No competing interests reported. Supplementary Files TableI.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 30 Apr, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers invited by journal 06 Apr, 2026 Editor assigned by journal 31 Mar, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 27 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9247718","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620530981,"identity":"35251ecd-b0b4-48bd-ad54-fc0f44e89c89","order_by":0,"name":"Michelle Volovsky","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie2QsUrEQBBARwKJxXK2E4SLPyDMshCvyMdsEK6ysJIUkksVm8P67i8Ef2DDgtcs2OawSXWVReyuEHEPPEHMbS24r9iZgXnMzAJ4PH+RAEKA68kuPVL2IYgqgG5XK5dCCPseAmaDdCnwS0HpVs7vgk3XE44BL5V6q0tB61feywLGo1YOKqkOL/iCUABOZbOsdUovVwKlAREfVFh4ygjzihnSzKjMKrasIX9wKe+Esy+lzGhtxDb/gJlTsedLiOZWKYKUWpbaoSDp8C1pPCfkdVTLZlFoEZvpzUQ+IV+ablh51hvcFllyEgSN/bqS36/0Y9vfZsloNTzlm3CfnKljsgHd7T9Iqmh4IY/H4/m3fAJ/yl/0KiOTOAAAAABJRU5ErkJggg==","orcid":"","institution":"Yale University","correspondingAuthor":true,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Volovsky","suffix":""},{"id":620530982,"identity":"b8a5909c-0f40-4a09-b84e-45e3fb2728ce","order_by":1,"name":"David B. Seifer","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"B.","lastName":"Seifer","suffix":""},{"id":620530984,"identity":"3b4d7beb-cc35-460a-8263-40cc3552a138","order_by":2,"name":"Gizem Nur Sahin","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Gizem","middleName":"Nur","lastName":"Sahin","suffix":""},{"id":620530986,"identity":"4899e13e-6da1-4387-80dc-72de3ed3fab5","order_by":3,"name":"Olga Chaplia","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"","lastName":"Chaplia","suffix":""},{"id":620530988,"identity":"66b34d8b-f25a-4a5b-9bb3-081dcf4a7f24","order_by":4,"name":"Stephanie M Nichols-Burns","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"M","lastName":"Nichols-Burns","suffix":""},{"id":620530989,"identity":"39ea7120-e335-445e-af8b-38ecb2ac3a3b","order_by":5,"name":"Cihan Halicigil","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Cihan","middleName":"","lastName":"Halicigil","suffix":""},{"id":620530990,"identity":"bf40518d-a694-4356-9132-df20a4912421","order_by":6,"name":"Emre Seli","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Emre","middleName":"","lastName":"Seli","suffix":""}],"badges":[],"createdAt":"2026-03-27 18:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9247718/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9247718/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106702844,"identity":"9da9d2a4-678a-4ab1-9068-6570681649bd","added_by":"auto","created_at":"2026-04-12 07:36:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCumulus cell \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMFN1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA expression stratified by follicular fluid PFOS concentration tertiles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBars represent median MFN1 expression levels in arbitrary units (AU). Expression decreased progressively across increasing PFOS tertiles low (\u0026lt;1.83ng/mL, 4.56AU), moderate (1.83-2.77ng/mL, 2.79AU) and high (\u0026gt;2.77ng/mL, 1.46AU), Kruskal-Wallis H 9.84, p = 0.007).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9247718/v1/692469ac9cfdac6e6589c95f.png"},{"id":106728209,"identity":"603f15a6-211e-4629-b928-aee77652219c","added_by":"auto","created_at":"2026-04-12 18:42:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":962694,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9247718/v1/655bfe64-c2d6-42c3-8d1b-84b01816fdad.pdf"},{"id":106702845,"identity":"694eba08-3bd8-4cd9-9971-16a6920e0ba3","added_by":"auto","created_at":"2026-04-12 07:36:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18739,"visible":true,"origin":"","legend":"","description":"","filename":"TableI.docx","url":"https://assets-eu.researchsquare.com/files/rs-9247718/v1/cc0cc2ea540539856f75062f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePer- and polyfluoroalkyl substances are associated with reduced cumulus cell MFN1 expression and lower oocyte maturation rates\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePer- and polyfluoroalkyl substances (PFAS) are a broad class of synthetic substances used since the 1950s in numerous products such as non-stick cookware, stain-resistant textiles, cosmetics and food contact materials [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Their carbon-fluorine backbones make them highly persistent and prone to bioaccumulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among the PFAS, Perfluorooctanesulfonic Acid (PFOS) and Perfluorooctanoic Acid (PFOA) are notable for long human half-lives and strong anion driven binding to plasma proteins, especially albumin and, to a lesser extent, lipoproteins. The sulfonate head group of PFOS generally confers higher albumin affinity than PFOA, which often results in greater persistence [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Consistent with these properties, PFAS have been documented in multiple human tissues, including blood, urine, breast milk, and follicular fluid (FF) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Several studies have detected PFAS in human FF, reported in cohorts from a number of countries, with PFOS and PFOA frequently the most abundant [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven that these compounds are detectable within the ovarian micro-environment, there has been increasing interest on their potential impact on female reproduction. PFAS have repeatedly shown effects at the cellular level. In vitro, human granulosa cells (GCs) exposed to a PFAS mixture (PFOS/PFOA/PFHxS) demonstrated altered proliferation, hormone production and transcriptomic shifts [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. GC work in mice has similarly shown that PFAS mixtures lower mitochondrial activity and membrane potential, reduce ATP and increase ROS, consistent with mitochondrial stress [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In placental/trophoblast models, PFOS specifically has been shown to reduce mtDNA copy number and down regulate mitochondrial fusions genes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Similarly, in a zebrafish larvae, PFAS mixtures disrupt mitochondrial membrane potential and respiratory-chain gene expression, confirming PFAS can target mitochondrial function across species and cell types [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBuilding on in vitro evidence that PFAS can impair mitochondrial function, it is compelling to investigate whether endogenous PFAS levels (those actually present in human FF) also have effects on mitochondrial maintenance that supports follicular function. Mitochondrial integrity in GCs and cumulus cells (CCs) is governed not only by respiratory capacity but by mitochondrial dynamics. These dynamics consist of fusion, in which adjacent mitochondria join to share proteins, lipids and mtDNA, and fission, in which mitochondria divide to segregate damaged segments and facilitate turnover. Both together support organelle quality control [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Among the proteins that regulate outer mitochondrial membrane fusion, Mitofusin-1 is a key mediator, and is encoded by the nuclear gene \u003cem\u003eMFN1\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Prior work in mouse models demonstrated that loss of MFN1 critically impairs follicle development and fertility, indicating that adequate \u003cem\u003eMFN1\u003c/em\u003e expression is required for functional female reproduction [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. On this basis, our aim was to determine if real-world PFAS concentrations in FF are sufficient to impact \u003cem\u003eMFN1\u003c/em\u003e expression in paired human CCs. Additionally, we examined whether PFAS levels correlated with IVF outcomes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection and isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study participants included reproductive aged women, 18-46 years of age, undergoing oocyte retrieval for IVF, ICSI or oocyte cryopreservation at Yale Fertility Center (IRB No. 2000036885), at Yale School of Medicine, CT, USA. Exclusion criteria included women or their partners with known chromosomal abnormalities or exposure to prior chemotherapy. Ovarian stimulation protocols included standard long, short and flare protocols, and were chosen by the treating clinician. At retrieval, FF was aspirated from the first one to two large follicles per patient, with effort to obtain clear FF samples free of blood. Throughout the study, all samples were individually stored (not pooled), and the information regarding which follicle the oocyte, cumulus cells (CCs) and FF were derived from was recorded.\u003c/p\u003e\n\n\u003cp\u003eFrom the collected samples, the CCs were mechanically denuded from the oocyte per lab protocol. The CCs were then placed in cryopreservation vials containing RNA later and frozen at -80C for later analysis. The FF was then centrifuged, and any granulosa cells, red blood cells or other cellular debris were removed. FF was frozen at -80C until further analysis. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePFAS measurement by high-resolution liquid chromatography-mass spectrometry \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four unique PFAS were quantified in FF by high-resolution liquid chromatography-mass spectrometry using an Agilent 1290 Infinity liquid chromatograph coupled to an Agilent 6546 quadrupole-time-of-flight mass spectrometer in negative electrospray ionization mode. Concentrations were reported as ng/mL. Samples were analyzed along with a six-point calibration curve from native PFAS standards at 0.01, 0.05, 0.1, 0.5, 1, 2 parts per billion. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMFN1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e measurement by quantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eRNA was isolated from CCs using the QIAGEN RNeasy kit according to manufacturer\u0026rsquo;s protocol. Complementary DNA (cDNA) was synthesized using a manual reverse transcription (RT) protocol. For each sample, RT was performed in a total reaction volume of 20uL, consisting of 10uL RNA combined with 2 uL RT butter, 0.8 uL dNTP mix, 2uL of RT primer, 1 uL reverse transcriptase, 1 uL RNase inhibitor, and 3.2uL nuclease-free water. RT reactions were prepared in duplicate and then pooled for each sample. cDNA concentration was then measured by NanoDrop to allow normalization of template input for quantitative polymerase chain reaction (qPCR). \u003c/p\u003e\n\n\u003cp\u003eQPCR was performed using SYBR Green chemistry on a Bio-Rad CFX Duet real-time PCR system. Each reaction was run in a total volume of 20uL using SYBR Green master mix, with the volume of cDNA and nuclease-free water adjusted for each reaction based on NanoDrop cDNA concentrations to ensure that each well received the same concentration of cDNA. Gene expression of \u003cem\u003eMFN1\u003c/em\u003e was quantified using gene-specific primers (MFN1 forward: 5\u0026rsquo;-GGT-GAA-TGA-GCG-GCT-TTC-CAA-G-3\u0026rsquo;; MFN1 reverse: 5\u0026rsquo;-TTC-TTC-ACC-AAG-AAA-TGC-AGG-C-3) and normalized to the reference gene \u003cem\u003eGAPDH\u003c/em\u003e (GAPDH forward: 5\u0026rsquo;-GTC-TCC-TCT-GAC-TTC-AAC-AGC-G-3\u0026rsquo;; GAPDH reverse: 5\u0026rsquo;-ACC-ACC-CTG-TTG-CTG-TAG-CCA-A-3\u0026rsquo;). All reactions were performed in duplicate. To control for inter-plate variability, the same calibrator samples were included on each plate. \u003cem\u003eMFN1\u003c/em\u003e expression was calculated relative to \u003cem\u003eGAPDH\u003c/em\u003e using the delta-delta cycle threshold method. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the relationship between FF PFAS concentrations and CC \u003cem\u003eMFN1\u003c/em\u003e expression, Spearman rank correlations were performed with age adjusted partial Spearman correlations. PFAS levels were additionally categorized into tertiles, and differences in \u003cem\u003eMFN1\u003c/em\u003e expression across groups was compared using the Kruskal-Wallis test. Baseline characteristics and IVF cycle parameters were compared across PFOS tertiles using one-way ANOVA for normally distributed variables and Kruskal-Wallis tests for non-normally distributed variables. Continuous variables are reported as mean +/- SEM or median (interquartile range), as appropriate. \u003c/p\u003e\n\n\u003cp\u003eUsing multiple linear regression models, the relationship between PFAS levels (PFOS and PFOA) and ART outcomes, including days of ovarian stimulation, total gonadotropin dose, number of oocytes retrieved, metaphase II (MII) rate (% of MII oocytes from total oocytes retrieved), number of two pronuclei (2PN) embryos, and blastocyst number and formation rate, were evaluated. Models were adjusted for potential patient level confounders such as age, body mass index (BMI), and anti-Mullerian hormone (AMH). Analyses were performed using GraphPad Prism (v10). \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePFAS in FF\u003c/h2\u003e \u003cp\u003ePFAS concentrations were measured in a total of 70 FF samples from 57 patients. Of the 24 PFAS measured, both PFOS and PFOA were detected in 100% of specimens, with means of 2.6ng/mL and 1.1ng/mL, respectively. Of the remaining PFAS measured, 20 other PFAS metabolites were detected in at least one FF sample (Table I).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFollicular fluid PFOS and PFOA concentrations and cumulus cell\u003c/b\u003e \u003cb\u003eMFN1\u003c/b\u003e \u003cb\u003eexpression\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePaired CC material was available for 38 FF samples, which were analyzed for \u003cem\u003eMFN1\u003c/em\u003e expression correlated with FF PFOS and PFOA concentrations, given that these two PFAS were present in every FF sample.\u003c/p\u003e \u003cp\u003eFF PFOS showed a moderate inverse association with \u003cem\u003eMFN1\u003c/em\u003e expression in CC (Spearman r = -0.45, p\u0026thinsp;=\u0026thinsp;0.005). This relationship persisted after adjustment for age (partial Spearman = -0.46, p\u0026thinsp;=\u0026thinsp;0005). When PFOS was stratified into tertiles (low\u0026thinsp;\u0026lt;\u0026thinsp;1.83, middle 1.83\u0026ndash;2.77, high\u0026thinsp;\u0026gt;\u0026thinsp;2.77ng/mL), median \u003cem\u003eMFN1\u003c/em\u003e expression declined progressively with higher PFOS concentration (Fig I), 4.56 arbitrary unit (AU) in the lowest tertile, 2.79AU in the middle tertile and 1.46AU in the highest tertile (Kruskal Wallis H 9.84, p\u0026thinsp;=\u0026thinsp;0.007, Fig I).\u003c/p\u003e \u003cp\u003eBaseline age and AMH were similar across PFOS tertiles. No significant differences were observed across PFOS tertiles for total gonadotropin dose, number of oocytes retrieved, MII rate, fertilization outcomes or blastocyst yield. BMI and days of ovarian stimulation differed between groups (Table II).\u003c/p\u003e \u003cp\u003ePFOA demonstrated a weaker inverse trend with \u003cem\u003eMFN1\u003c/em\u003e (Spearman rank = -0.23, p\u0026thinsp;=\u0026thinsp;0.16). When PFOA was stratified by tertiles (low\u0026thinsp;\u0026lt;\u0026thinsp;0.77, middle 0.78\u0026ndash;1.54, high\u0026thinsp;\u0026gt;\u0026thinsp;1.56ng/mL), the means fell progressively from the lowest to highest PFOA tertiles (11.19, 5.94, 3.99), however, comparing the medians (3.06, 2.33, 2.71) did not yield significant results (Kruskal Wallis H 2.13 p\u0026thinsp;=\u0026thinsp;0.35).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFollicular fluid PFOS and PFOA concentrations and ART outcomes\u003c/h2\u003e \u003cp\u003eFF PFOS and PFOA levels were examined in relation to patient characteristics and ART outcomes in the 57 patients included in the study (Table III). In multivariable linear regression models adjusted for age, BMI and AMH, FF PFOS concentrations were independently associated with maturation rate, with each 1ng/mL increase in PFOS associated with a 2.06% decrease in MII rate (CI -3.20 to -0.92, p\u0026thinsp;=\u0026thinsp;0.0007). A similar trend was observed for FF PFOA, though it did not reach statistical significance (β = -7.043, CI -15.3 to 1.54, p\u0026thinsp;=\u0026thinsp;0.1). FF PFAS levels were otherwise not associated with age, BMI or AMH in mutually adjusted models, nor were they associated with days of ovarian stimulation, total gonadotropin dose, number of oocytes retrieved, number of 2PN embryos, blast number, or blast formation rate.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo our knowledge this is the first study to demonstrate that endogenously found PFAS concentrations in human FF are associated with cellular function changes, with higher FF PFOS levels linked to lower CC \u003cem\u003eMFN1\u003c/em\u003e expression, as well as reduced MII rates across rank-based, age-adjusted, and model-based analyses. Mechanistically, these observations align with experimental data showing that PFAS mixtures impair mitochondrial function by reducing ATP linked respiration, elevating ROS and down regulating biogenesis and fusion pathways [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Such shifts favor a dysfunctional mitochondrial network that can potentially compromise granulosa-oocyte metabolic coupling and oocyte competence. Because progression to the MII stage is an energy intensive process requiring tightly coordinated mitochondrial ATP production, impaired mitochondrial function is well documented to reduce maturation rates in both animal and human oocytes [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The current findings of parallel reductions in \u003cem\u003eMFN1\u003c/em\u003e expression and MII formation rate are thus biologically plausible and demonstrate a mechanism by which PFAS mixtures may induce a mitochondrial cellular effect. Furthermore, these data suggest that endogenously occurring PFAS concentrations are sufficient to produce measurable biological changes in human folliculogenesis.\u003c/p\u003e \u003cp\u003eAlthough several FF studies did not find consistent associations with fertility outcomes, others do point towards potential reproductive risk. In China, a recent study found that higher FF PFOA was associated with greater odds of PCOS and higher androgens [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In an ART cohort from Sweden, FF PFAS were associated with lower embryo quality despite higher ovarian reserve in those patients, a pattern that is also compatible with PCOS-like physiology [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Another IVF study from China linked higher FF PFAS with fewer high quality embryos [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Complementing the FF literature, higher plasma PFAS concentrations were associated with reduced number of oocytes retrieved, 2PN zygotes and cleavage stage embryos [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In addition, our findings that higher PFOS levels are associated with reduced oocyte maturation rates strengthens the notion that ovarian PFAS exposure is clinically relevant, underscoring the need to further investigate the underlying disrupted cellular pathways and to determine how this may translate into impacting additional fertility outcomes.\u003c/p\u003e \u003cp\u003ePFAS are present world-wide and found in industrialized nations. They are pervasive and persistent, with reported human FF concentrations varying across geographic regions. In China, one study measured 15 PFAS in FF and reported geometric means of PFOS at 4.8 ng/mL and PFOA at 4.6 ng/mL [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], with another Chinese study observing PFOA most abundant (median 5.6 ng/mL) followed by PFOS (median 4.3 ng/mL) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In Australia, PFAS were detected in 100% of FF samples with mean levels of PFOS at 4.9 ng/mL and mean PFOA at 2.4 ng/mL [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. By comparison, our cohort\u0026rsquo;s means were lower, with PFOS at 2.6 ng/mL and PFOA at 1.1 ng/mL. Differences between studies may reflect regional product use, legacy water contamination, population behaviors, sample measuring methods, pooling versus single-follicular samples, and participant factors such as BMI. Despite the differences, these studies underscore that PFAS exposure is a widespread world-wide concern.\u003c/p\u003e \u003cp\u003eIn our dataset, PFOA trended in the same direction as PFOS but with a smaller effect. This pattern is consistent with other studies, in which PFOS often affects cellular function at lower concentrations in several systems and exerts stronger mitochondrial inhibition than PFOA in side-by-side assays [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Several features may underlie this difference, PFOS has a sulfonate head group that is associated with stronger serum albumin binding and longer human half-life, which increase effective exposure in protein rich compartments like FF. Moreover, PFOS most consistently impacts mitochondrial bioenergetics across epithelia and reproductive cells [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Taken together, these considerations provide a plausible rational for why PFOS shows a clearer relationship with CC \u003cem\u003eMFN1\u003c/em\u003e expression and oocyte maturation rate in our cohort.\u003c/p\u003e \u003cp\u003eLimitations of this study include the cross-sectional design, which precludes causal inference, use of mRNA rather than protein/function, low CC RNA concentrations limiting the number of gene panels, and a relatively small sample size. Replication in larger cohorts is warranted. Future work should test additional mitochondrial dynamics and quality control genes, connect molecular results to embryo competence, and assess fertility outcomes across regions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, PFAS are ubiquitously detectable in human FF, and higher PFOS is independently associated with lower \u003cem\u003eMFN1\u003c/em\u003e expression in paired cumulus cells. Coupled with the observed reduction in MII rate, these findings add human evidence of a mechanistic pathway that endogenous PFAS burdens in the real-world follicular microenvironment may compromise mitochondrial fusion pathways relevant to oocyte quality and ultimately fertility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e-Funding \u0026ndash; SREI/ASRM Young Investigators Award (MV)\u003c/p\u003e\n\u003cp\u003e-Consent to participate \u0026ndash; N/A (IRB for discard sample use)\u003c/p\u003e\n\u003cp\u003e-Consent for publication \u0026ndash; N/A\u003c/p\u003e\n\u003cp\u003e-Ethical approval and its number \u0026ndash; N/A\u003c/p\u003e\n\u003cp\u003e-Data availability \u0026ndash; N/A\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eMV \u0026ndash; funding acquisition, conceptualization, investigation, methodology, writing. DS \u0026ndash; conceptualization, supervision, review \u0026amp; editing. GS, OC, SN, CH \u0026ndash; investigation, sample acquisition and processing. ES \u0026ndash; supervision, resources, writing, review \u0026amp; editing.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eKrystal Pollitt PhD and Sheng Liu at the Pollitt Lab, Yale School of Public Health\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSunderland EM, Hu XC, Dassuncao C, Tokranov AK, Wagner CC, Allen JG. A review of the pathways of human exposure to poly-and perfluoroalkyl substances (PFASs) and present understanding of health effects. Journal of exposure science \u0026amp; environmental epidemiology. 2019 Mar;29(2):131-47.\u003c/li\u003e\n \u003cli\u003eDeWitt JC, editor. Toxicological effects of perfluoroalkyl and polyfluoroalkyl substances. Cham: Springer International Publishing; 2015 Apr 14.\u003c/li\u003e\n \u003cli\u003eGaines LG. Historical and current usage of per‐and polyfluoroalkyl substances (PFAS): A literature review. 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Follicular fluid and blood levels of persistent organic pollutants and reproductive outcomes among women undergoing assisted reproductive technologies. Environmental Research. 2022 May 15;208:112626.\u003c/li\u003e\n \u003cli\u003eClark KL, Shukla M, George JW, Gustin S, Rowley MJ, Davis JS. An environmentally relevant mixture of per-and polyfluoroalkyl substances (PFAS) impacts proliferation, steroid hormone synthesis, and gene transcription in primary human granulosa cells. Toxicological Sciences. 2024 Jul;200(1):57-69.\u003c/li\u003e\n \u003cli\u003eTatarczuch A, Gogola-Mruk J, Kotarska K, Polański Z, Ptak A. Mitochondrial activity and steroid secretion in mouse ovarian granulosa cells are suppressed by a PFAS mixture. Toxicology. 2025 Mar 1;512:154083.\u003c/li\u003e\n \u003cli\u003eHofmann A, Mishra JS, Yadav P, Dangudubiyyam SV, Blesson CS, Kumar S. PFOS impairs mitochondrial biogenesis and dynamics and reduces oxygen consumption in human trophoblasts. Journal of environmental science and public health. 2023 Oct 10;7(4):164.\u003c/li\u003e\n \u003cli\u003eLiu Y, Liu S, Huang J, Liu Y, Wang Q, Chen J, Sun L, Tu W. Mitochondrial dysfunction in metabolic disorders induced by per-and polyfluoroalkyl substance mixtures in zebrafish larvae. Environment international. 2023 Jun 1;176:107977.\u003c/li\u003e\n \u003cli\u003eChan DC. Mitochondrial dynamics and its involvement in disease. Annual review of pathology: mechanisms of disease. 2020 Jan 24;15(1):235-59\u003c/li\u003e\n \u003cli\u003eWestermann B. Mitochondrial fusion and fission in cell life and death. Nature reviews Molecular cell biology. 2010 Dec;11(12):872-84.\u003c/li\u003e\n \u003cli\u003eChen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development. The Journal of cell biology. 2003 Jan 20;160(2):189-200.\u003c/li\u003e\n \u003cli\u003eCao YL, Meng S, Chen Y, Feng JX, Gu DD, Yu B, Li YJ, Yang JY, Liao S, Chan DC, Gao S. MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion. Nature. 2017 Feb 16;542(7641):372-6.\u003c/li\u003e\n \u003cli\u003eCozzolino M, Ergun Y, Seli E. Targeted deletion of mitofusin 1 and mitofusin 2 causes female infertility and loss of follicular reserve. Reproductive Sciences. 2023 Feb;30(2):560-8.\u003c/li\u003e\n \u003cli\u003eZhang M, Bener MB, Jiang Z, Wang T, Esencan E, Scott III R, Horvath T, Seli E. Mitofusin 1 is required for female fertility and to maintain ovarian follicular reserve. Cell Death \u0026amp; Disease. 2019 Jul 22;10(8):560.\u003c/li\u003e\n \u003cli\u003eBabayev E, Seli E. Oocyte mitochondrial function and reproduction. Current Opinion in Obstetrics and Gynecology. 2015 Jun 1;27(3):175-81.\u003c/li\u003e\n \u003cli\u003eKirillova A, Smitz JE, Sukhikh GT, Mazunin I. The role of mitochondria in oocyte maturation. Cells. 2021 Sep 19;10(9):2484.\u003c/li\u003e\n \u003cli\u003eYu Y, Dumollard R, Rossbach A, Lai FA, Swann K. Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation. Journal of cellular physiology. 2010 Sep;224(3):672-80.\u003c/li\u003e\n \u003cli\u003eVan Blerkom J. Mitochondrial function in the human oocyte and embryo and their role in developmental competence. Mitochondrion. 2011 Sep 1;11(5):797-813.\u003c/li\u003e\n \u003cli\u003eLi S, Li G, Lin Y, Sun F, Zheng L, Yu Y, Xu H. Association between perfluoroalkyl substances in follicular fluid and polycystic ovary syndrome in infertile women. Toxics. 2024 Jan 26;12(2):104.\u003c/li\u003e\n \u003cli\u003eZeng XW, Bloom MS, Wei F, Liu L, Qin J, Xue L, Wang S, Huang G, Teng M, He B, Mao X. Perfluoroalkyl acids in follicular fluid and embryo quality during IVF: a prospective IVF cohort in China. Environmental health perspectives. 2023 Feb 1;131(2):027002.\u003c/li\u003e\n \u003cli\u003eShen J, Mao Y, Zhang H, Lou H, Zhang L, Moreira JP, Jin F. Exposure of women undergoing in-vitro fertilization to per-and polyfluoroalkyl substances: Evidence on negative effects on fertilization and high-quality embryos. Environmental Pollution. 2024 Oct 15;359:124474.\u003c/li\u003e\n \u003cli\u003eWallace KB, Kissling GE, Melnick RL, Blystone CR. Structure\u0026ndash;activity relationships for perfluoroalkane-induced in vitro interference with rat liver mitochondrial respiration. Toxicology letters. 2013 Oct 9;222(3):257-64.\u003c/li\u003e\n \u003cli\u003eTukker AM, Bouwman LM, van Kleef RG, Hendriks HS, Legler J, Westerink RH. Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) acutely affect human \u0026alpha;1\u0026beta;2\u0026gamma;2L GABAA receptor and spontaneous neuronal network function in vitro. Scientific reports. 2020 Mar 24;10(1):5311.\u003c/li\u003e\n \u003cli\u003eZhao Y, Zhao H, Xu H, An P, Ma B, Lu H, Zhou Q, Li X, Xiong Y. Perfluorooctane sulfonate exposure induces preeclampsia-like syndromes by damaging trophoblast mitochondria in pregnant mice. Ecotoxicology and Environmental Safety. 2022 Dec 1;247:114256.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable I is available in the Supplementary Files section.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable II\u003c/strong\u003e. Baseline characteristics and IVF cycle parameters between PFOS concentration tertile groups.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow PFOS \u0026lt;1.83 (n=13)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eModerate PFOS 1.83-2.77 (n=13)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh PFOS \u0026gt;2.77 (n=12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e35.8 +/- 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e36.5 +/- 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e36.2 +/- 1 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI (kg/m2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e26.1 +/- 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e30.4 +/- 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e23.7 +/- 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.02*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAMH (ng/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e2.4 (1.5-4.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e2.7 (0.9-3.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e1.5 (0.7-2.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal gonadotropin dose (IU)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e3550 (1988-4500)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e4200 (2813-4800)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e4950 (3150-5288)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDays of stimulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e11.0 (9.5-11.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e11.0 (10.0-12.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e12.0 (11.3-13.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.05*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOocytes retrieved (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e11.0 (6.0-15.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e11.0 (7.5-16.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e10.5 (7.3-17.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMII rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e75.0 (60.5-84.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e81.5 (66.3-91.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e60.0 (50.5-79.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of 2PN embryos (n)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e5.0 (2.0 \u0026ndash; 7.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e6.0 (4.0-11.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e6.0 (3.0-9.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlastocysts (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e1.0 (1.0-6.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e4.0 (3.5 \u0026ndash; 10.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e3.0 (1.8-5.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePFOS (ng/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 127px;\"\u003e\n \u003cp\u003e1.3 (1.1-1.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e2.3 (2.1-2.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003e3.8 (3.1-6.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001****\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues are presented as mean +/- SEM or median (interquartile range). Comparisons across PFOS tertiles were performed using one-way ANOVA for normally distributed variables and Kruskal-Wallis test for non-normally distributed variables. *p \u0026gt;0.05, ****p\u0026lt;0.0001. MII = metaphase II, 2PN = two pronuclei.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable III.\u003c/strong\u003e Multivariable linear regression analyses of follicular fluid PFAS concentrations in relation to patient characteristics and ART outcomes.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOutcome\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePFOS \u0026beta; (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePFOA \u0026beta; (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-0.17 (-0.54-0.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-0.05 (-0.12-0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBMI (kg/m2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-0.09 (-0.36-0.17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.01 (-0.04-0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAMH (ng/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-0.35(-1/06-0.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-0.07 (-0.18-0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal gonadotropin dose (IU)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e48.66 (-7.87-105.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e342.00 (-5.28-689.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDays of stimulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.08 (-0.01-0.17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.49 (-0.06-1.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOocytes retrieved (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.12 (-0.17-0.41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.49 (-1.32-2.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMII rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-2.06 (-3.2 \u0026ndash; 0.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0007***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-7.04 (-15.63 \u0026ndash; 1.54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2PN embryos (n)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.07 (-0.14-0.28)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.23 (-1.08-1.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlastocysts (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.02 (-0.15-0.19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.17 (-0.89-1.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlastocyst formation rate (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-0.28 (-1.82-1.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e-0.89 (-10.40-8.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults from multivariable linear regression models (n=57). All models were adjusted for age, body mass index (BMI), and anti-Mullerian hormone (AMH). \u0026beta; coefficients represent the estimated change in the outcome variable per 1ng/mL increase in FF PFOS or PFOA concentrations. ***p\u0026lt;0.001. MII = metaphase II, 2PN = two pronuclei.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"per- and polyfluoroalkyl substances, Mitofusin-1 (MFN1), mitochondria, oocyte maturation, follicular fluid, ART","lastPublishedDoi":"10.21203/rs.3.rs-9247718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9247718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose: \u0026nbsp;\u003c/strong\u003eTo determine whether per- and polyfluoroalkyl substances (PFAS) are detectable in human follicular fluid (FF) and to evaluate their association with cumulus cell (CC) expression of Mitofusin-1 (\u003cem\u003eMFN1\u003c/em\u003e), a mitochondrial fusion gene linked to fertility, as well as selected assisted reproductive technology (ART) outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThis cross-sectional study included reproductive-aged women undergoing oocyte retrieval for IVF/ICSI.\u003cstrong\u003e \u003c/strong\u003eFF and CCs were collected at retrieval. Concentrations of 24 PFAS were measured in FF by liquid chromatography-mass spectrometry. \u003cem\u003eMFN1\u003c/em\u003e mRNA expression was measured in paired CCs by quantitative PCR. Associations between PFAS concentrations and \u003cem\u003eMFN1\u003c/em\u003eexpression were assessed using Spearman correlation and tertile-based Kruskal-Wallis tests. Multivariable linear regression models were used to assess associations with ART outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eSeventy FF samples from 57 women were analyzed, with perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) detected in all samples. Paired CCs were available for 38 FF samples. PFOS demonstrated a moderate inverse association with CC \u003cem\u003eMFN1\u003c/em\u003e expression (Spearman rank =-0.45, p=0.005), which persisted after adjustment for age (partial Spearman =-0.46, p=0.005). When PFOS was stratified into tertiles (low \u0026lt;1.83, middle 1.83-2.77, high \u0026gt;2.77ng/mL), median \u003cem\u003eMFN1\u003c/em\u003e expression declined progressively with 4.56 arbitrary unit (AU) in the lowest tertile, 2.79AU in the middle tertile, and 1.46AU in the highest tertile (Kruskal Wallis H=9.84, p=0.007). FF PFOS levels were independently associated with oocyte maturation rate, with each 1ng/mL increase in PFOS associated with a 2.06% decrease in maturation (95% CI -3.20 to -0.92, p=0.0007). No significant associations were observed between FF PFAS levels and other ART outcomes or patient characteristics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003ePFAS are ubiquitously detectable in human FF. Higher FF PFOS levels are independently associated with lower \u003cem\u003eMFN1\u003c/em\u003eexpression in CCs and reduced oocyte maturation rates, supporting a potential mitochondrial mechanism linking PFAS exposure to impaired reproductive function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCapsule\u003c/strong\u003e: Higher follicular fluid PFAS concentrations are associated with reduced cumulus cell \u003cem\u003eMFN1\u003c/em\u003e expression and impaired oocyte maturation, linking endogenous PFAS exposure to mitochondrial dysfunction in human IVF cycles.\u003c/p\u003e","manuscriptTitle":"Per- and polyfluoroalkyl substances are associated with reduced cumulus cell MFN1 expression and lower oocyte maturation rates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-12 07:36:34","doi":"10.21203/rs.3.rs-9247718/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"264965542846902917187999166001509959987","date":"2026-04-30T07:01:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197969640235097638770873939758656490193","date":"2026-04-30T06:46:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-06T06:43:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T01:40:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T01:39:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Assisted Reproduction and Genetics","date":"2026-03-27T18:38:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"journal-of-assisted-reproduction-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Journal of Assisted Reproduction and Genetics","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7b0ade08-30dd-45e3-bafe-bdbabe6eec4b","owner":[],"postedDate":"April 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"264965542846902917187999166001509959987","date":"2026-04-30T07:01:30+00:00","index":37,"fulltext":""},{"type":"reviewerAgreed","content":"197969640235097638770873939758656490193","date":"2026-04-30T06:46:59+00:00","index":36,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-12T07:36:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-12 07:36:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9247718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9247718","identity":"rs-9247718","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}