Lipid dysregulation in mouse ovarian antral follicles after oral exposure to a human relevant mixture of three phthalates.

Dibutyl phthalate (DBP, CAS no. 84-74-2, Cat no. 524980, 99% purity), di-2-ethylhexyl phthalate (DEHP, CAS no. 117-81-7, Cat no. D201154, ≥99.5% purity), and benzyl butyl phthalate (BBP, CAS no. 85-68-7, Cat no. 308501, 98% purity) were obtained from Sigma-Aldrich (St Louis, MO). Tocopherol-stripped corn oil (vehicle for DBP, DEHP, and BBP) was obtained from Dyets, Inc. (Bethlehem, PA). Buffered Formalde-Fresh (Cat no. SF93-4), Hematoxylin (Cat no. 7211), and Bluing Reagent (Cat no. 7301) were obtained from Fisher Scientific (Waltham, MA). Oil Red O stain (Cat no. 1277A) was obtained from Newcomers Supply (Middleton, WI). Aqueous mounting media (Cat no. HK099-5K) was obtained from Biogenex (Fremont, CA). Animal use and experiments were conducted according to the guidelines stated in the United States Public Health Service’s Guide for the Care and Use of Laboratory Animals 45 and approved by the University of Arizona Institutional Animal Care and Use Committee. Adult female CD-1 mice, 60 days old to mimic women of reproductive age, were purchased from Charles River Laboratories (Charles River, CA) and housed two per cage in single use BPA-free Innovive Disposable Rodent Caging at the University of Arizona Animal Care Facility. Temperature was maintained at 22 ± 1 ° C, food and water were provided ad libitum , and animals were subjected to 14L:10D cycles. After arrival at the animal facility, mice were allowed to acclimate for at least 24 h before handling. Estrous stage and weight gain were monitored daily for 15 days prior and throughout oral dosing. Animals were euthanized in the estrous stage of diestrus by decapitation under isoflurane anesthesia at least two hours after receiving their final oral dose. Following euthanasia both ovaries were dissected and cleaned for whole ovary fixation or mechanical isolation of fresh follicles as applicable. The liver was dissected from each animal and snap frozen in liquid nitrogen. Trunk blood was collected from each mouse and the serum separated. Serum and snap frozen tissues were stored at −80 o C until subsequent gene expression, triglyceride, and LC-MS/MS lipid analyses as applicable. The estrous cycle of each animal was monitored by vaginal cytology for 15 days prior to inclusion in the study. Vaginal smears were collected and analyzed using methods based on techniques 46 , 47 , 27 and criteria 48 previously described. Specifically, cyclicity data from each animal was evaluated for normalcy prior to dosing. The cyclicity pattern of an animal was considered abnormal using criteria previously described 30 . Briefly, cycles were marked as abnormal if the animal showed no cycles (no change in stage), extended estrus (>4 days), and/or extended diestrus (>10 days) during the evaluation period. No abnormal cycles were observed; thus, all animals were randomly distributed to control or treatment groups and used downstream in the study. The parent compounds, DBP, DEHP, and BBP produce three major metabolites in vivo: monobutyl phthalate (MBP), mono-2-ethylhexyl phthalate (MEHP), and monobenzyl phthalate (MBzP), respectively. These metabolites have been detected in human follicular fluid at a median concentration (ng/mL) of 1.68, 2.2, and 0.84, respectively 20 . Thus, the relative quantity of each phthalate metabolite in follicular fluid was used to set the parent mixture at 36% DBP, 52% DEHP, and 12% BBP. A stock solution (1 g) of the phthalate mixture was created by weight and then diluted in tocopherol-stripped corn oil to achieve the experimental dose. At the end of the pre- dosing evaluation period, normally cycling mice (~75 days old; equivalent to reproductively aged adult women) were randomly assigned to receive tocopherol-stripped corn oil (vehicle) or the phthalate mixture dissolved in vehicle at 32 μg/kg/day. This dose was selected to resemble estimates of cumulative daily exposure to the three phthalates as reported individually in humans in the general population and match our previous work showing dysregulated proteins involved in lipid metabolism in antral follicles 30 . The selection of this dose, while environmentally relevant and informed by epidemiological studies, only partially models human exposure due to unknown mixture toxicokinetics and species differences. Animals were weighed and dosed orally via pipet feeding 29 , 27 , 24 , 28 . To avoid bias introduced by hormonal status, the dosing scheme was developed so that estrous stage matching was achieved. Specifically, mice were dosed for 10-15 consecutive days until they reached the stage of diestrus when they were euthanized and their tissues collected as previously described 30 . The minimum 10-day dosing regimen permits exposure to vehicle or the phthalate mixture over the course of late antral follicle development in the mouse as described by Hirshfield 49 for type 6 through 8 follicles in Pedersen and Peters’ follicular classification criteria 50 . Ovaries from each mouse were excised, trimmed of fat and placed in alpha-minimal essential media (MEMα; Life Technologies #12571-048) for follicle isolation as previously described 30 , 47 , 51 , 52 , 53 . Briefly, antral follicles (≥250 μm diameter) were mechanically isolated from each ovary in MEMα using watchmaker forceps (Dumont No.5, 11254-20). For each mouse, isolated antral follicles were combined per ovary in separate tubes, immediately snap frozen in liquid nitrogen, and stored −80 o C until subsequent gene expression, triglyceride, and LC-MS/MS lipid analyses as applicable. Due to their small size, isolated antral follicles are limited to use in a single experimental endpoint; therefore, distinct subsets of follicle samples from the same study were used for each downstream analysis. Starting material quantity was comparable for each analysis based on follicle number between treatments and the quantification of internal controls. Lipid metabolites were isolated from antral follicles (51-177 follicles), liver tissue (~20mg), or serum (50μl) from each mouse (n=5 mice/treatment) via a single-phase extraction method and separated using reversed-phase chromatography with methods as previously described 54 . Briefly, targeted lipidomic analysis was performed using an Agilent 1200 HPLC Tandem Thermo Quantum Ultra triple quadrupole mass spectrometer (Agilent Technologies) to quantify levels of major molecular species. Free fatty acids (FFA) were extracted separately utilizing methods adapted from Okahashi et al 55 and mass transitions adapted from Huynh et al 56 . Acylcarnitine (AC) analysis was achieved utilizing mass transitions adapted from Giesbertz et al 57 . Phospholipid (PC, PE) and lysophospholipid (LPC, LPE) analysis was achieved utilizing mass transitions adapted from Huynh et al 56 . Ceramide (Cer) analysis was achieved utilizing mass transitions adapted from Bielawski et al 58 . Data was processed through Thermo LCQuan 2.7 software. Antral follicle data were normalized by total lipid phosphate (Pi) present in the organic phase of the Bligh and Dyer extraction 59 detected by phosphomolybdate assay 60 . Liver and serum data were normalized by weight and volume, respectively. As described in Miller et al ., (2024) vehicle and phthalate mixture treated antral follicle proteome profiles demonstrated significant differences in protein abundance between treatments 30 . Proteome data from that study (ProteomeXchange, dataset identifier PXD047061) were analyzed to evaluate protein abundance for lipid metabolism pathways of interest including lipid and fatty acid metabolic process. Protein abundance values were compared between antral follicle samples (n=4/treatment) from mice exposed to oil and 32 μg/kg/day (PHT32) of the phthalate mixture. Abundance of proteins of interest within the PHT32 group were normalized to values measured in the oil group for analysis. Snap frozen samples (antral follicles from one ovary per mouse (n=7 mice/treatment; 24-69 follicles) or liver (n=7-9 mice/treatment)) were subjected to RNA extraction with DNAse treatment using Qiagen RNeasy Micro Kit (Qiagen Valencia, California). RNA concentrations were determined at 260nm using a Take3 Microvolume plate on a Synergy H1m microplate reader (Biotek, Winooski, Vermont). RNA samples were reverse transcribed to cDNA using iScript cDNA synthesis kits (Bio-Rad, Hercules, California). Quantitative polymerase chain reaction (qPCR) was performed on each sample in triplicate using 1μL of each primer (5μM), 5μL of Ssofast EvaGreen Supermix (Bio-Rad, Hercules, California), 2μL of nuclease free water, and 1μL of cDNA (10ng). Reaction controls included no template, no primer, and no reverse transcriptase samples. Each reaction was performed in triplicate using manufacturer’s suggested protocol. Primers used for genes involved in lipid metabolic pathways and housekeeping genes were verified on PrimerBlast software for transcript specificity and amplification size, purchased from Integrated DNA Technologies, and de novo designed primers were validated using agarose gel electrophoresis ( Table 1 ). The reference genes: B2m and Hmbs for liver 61 , and Tbp, Actb, Ppia, and Hprt1 for follicles 62 were confirmed to not be differentially expressed between treatments before analysis of the genes of interest. Data were analyzed using the ΔΔCt model for relative quantification and normalized to the average expression of the two most stable reference genes. One ovary per mouse (n=4 mice/treatment) was frozen in optimal cutting temperature compound (OCT, Tissue-Tek) and dry ice and stored at −80°C until sectioning. Sections (8μm thick) were mounted on slides and fixed in 10% neutral buffered formalin. Fixed slides were stained with oil red o and counterstained with hematoxylin, mounted with an aqueous gelatin-based media, and cover slipped. Slides were scanned at 40x using a Leica Aperio AT2 Scanner at the Comparative Pathology Core Laboratory, University of Arizona Cancer Center. Images were analyzed by Qupath: Open-source software for digital pathology image analysis 63 to quantify percent positive staining by oil red o. Whole ovary and antral follicles within the ovaries were hand traced and a non-biased color threshold was created to classify positive staining. All image analyses were performed blinded to treatment groups. Triglyceride content was determined from antral follicle samples (21-69 follicles) and liver samples (~25mg) from mice (n=4 mice/treatment), using a Triglyceride-Glo Assay (#J3160, Promega) following the protocol provided by the manufacturer. Briefly, samples were sonicated for 10 seconds at 50 amplitude twice in 1X-phosphate buffered solution (PBS), diluted with buffer included in kit, and ran in duplicate per the manufacturer recommendations. The concentration was calculated based on concurrently generated standard curve and normalized to free glycerol present in samples. For serum samples (n=3 mice/treatment; 10μl), triglyceride content was determined using Point Triglyceride (GPO) Liquid Reagent Set (#T7532, MedTest) following the protocol provided by the manufacturer. Briefly, serum samples were diluted in the enzymatic reagent provided and then concentrations were calculated based on the provided standard. For univariate tests with normal outcomes, n=5 animals per group allow detection of a mean difference of 2.0 standard deviations with significance level 0.05 and 80% power. This is typical of many mouse experiments where treatments exhibit a relatively strong effect. In previous protein profiling experiments, it was observed that about 80% of signals exhibit a coefficient of variation CV<0.40. Using this threshold and results from the statistics literature linking detectable fold change to CV 64 , the sample sizes used in this study permit detection of significant fold change differences in omics experiments with 0.05 significance and 80% power. Data were compared between treatments using GraphPad Prism Software (version 10.4.0). Data were subjected to normality and homogeneity of variance tests prior to analysis. ROUT testing (5%) was performed when extreme values were observed to identify statistically significant outliers for removal. One outlier was removed from the lipidomic analysis. Additionally, outliers identified in control samples were removed from average prior to normalization for gene, protein, or triglyceride data. Comparisons between oil and PHT32 groups were performed using unpaired two-sided t tests or equivalent non-parametric tests when applicable. All data are represented in bars showing the mean ± standard deviation of the mean (SD) overlaid with individual data points corresponding to each animal. Statistical significance was assigned when p ≤ 0.05.

To identify whether fatty acid profile changes occur in antral follicles in response to phthalate exposure, antral follicle samples (n=4-5 mice/treatment) from control (oil) and phthalate mixture (PHT32) treated mice were subjected to free fatty acid profiling via LC-MS/MS ( Figure 1 ). Fifteen free fatty acids were detected in the profile, two: palmitic acid (C16:0) and stearic acid (C18:0) were saturated while the remaining thirteen (C16:1, C18:1, C18:2, C18:3, C20:2, C20:3, C20:4, C20:5, C22:2, C22:3, C22:4, C22:5, C22:6) were unsaturated. The saturated fatty acids made up the vast majority (~80%) of the total quantity of free fatty acids detected in the antral follicles from both treatments. Interestingly, the quantity (μM) of total free fatty acids in the antral follicles following phthalate exposure significantly increased (p = 0.029; Figure 1A ). This change was accompanied by a significant increase in saturated fatty acids (p = 0.033), while total unsaturated fatty acids remained unchanged ( Figure 1A ). The increase in total free saturated fatty acids in the antral follicles from phthalate exposed mice appeared to be driven by a significant increase in stearic acid (C18:0), though there was also a trending increase in palmitic acid (C16:0; p = 0.019 & 0.063, respectively; Figure 1B & Supplemental Table 1 ). Of the thirteen free unsaturated fatty acids, C22:3 (docosapentaenoic acid, DPA), had a significant increase following exposure to PHT32, while the others were unchanged (p = 0.042; Figure 1B & Supplemental Table 1 ). A previously published antral follicle proteome profile 30 was probed to evaluate protein abundance changes of key FFA synthesis enzymes. Acetyl-CoA carboxylase 1 (ACACA) and fatty acid synthase (FAS, Fasn ) were both detected in the proteome, while very long chain fatty acid elongase 6 (ELOV6) and acyl-coA desaturase 1 (ACOD1, Scd1 ) were not. The protein abundance of ACACA was unchanged; however, the abundance of FAS was significantly increased in the antral follicles from the mice exposed to PHT32 (p = 0.017; Figure 2A – B ). To further understand the impact of phthalates on fatty acid homeostasis, qPCR analysis was used to determine transcript levels for genes involved in the fatty acid synthesis pathway in antral follicle samples (n=7 mice/treatment) from oil- and PHT32-treated mice. As with protein, there was no change in mRNA levels for Acaca, but a significant increase in Fasn transcript levels was observed (p = 0.029; Figure 2C – D ). Although not detected in the proteome profiles, mRNA for Elovl6 was significantly decreased (p =0.039; Figure 2E ), while that for Scd1 was unchanged in antral follicles treated with the phthalate mixture ( Figure 2F ). The distinctive feature of the antral follicle is the antrum, which is filled with fluid containing a variety of molecules that are not only derived from the follicular cells but also from the systemic circulation 65 . Therefore, it is critical to also evaluate the influence of phthalate exposure on systemic lipid production. Thus, lipid profiling like that done for antral follicles was performed with liver and serum samples from oil and PHT32-treated mice using LC/MS-MS (n=5 mice/treatment). The total quantity of free, saturated, and unsaturated free fatty acids was unchanged in the liver and serum in response to phthalate mixture exposure ( Figure 3A – B ). None of the fifteen individual free fatty acids detected in the liver were significantly altered ( Figure 3C ; Supplemental Table 2 ), however of the seventeen detected in the serum, palmitoleic acid (C16:1), was significantly increased (p = 0.0265; Figure 3D & Supplemental Table 3 ). Liver samples from oil and PHT32 mice were also subjected to qPCR analysis (n=7-8 mice/treatment) to investigate transcript levels of enzymes involved in the fatty acid synthesis pathway. No significant change was observed in hepatic transcript levels for Acaca, Fasn, Elovl6, or Scd1 ( Table 2 ). To assess phthalate effects on antral follicle beta oxidation, levels of acylcarnitines and expression profiles of key enzymes in the acylcarnitine shuttle pathway were evaluated in antral follicles. Antral follicle samples (n=4-5 mice/treatment) from oil or PHT32-treated mice were subjected to acylcarnitine profiling by LC-MS/MS ( Figure 4 ). A total of 26 individual acylcarnitine (AC) and hydroxyacylcarnitine (AC-OH) species were detected, of which 11 were saturated and 15 were unsaturated. Relative to global levels in vehicle controls, antral follicle samples from PHT32 mice showed a total increase in unsaturated AC (p = 0.043; Figure 3A ). Interestingly, when evaluating AC species individually, increases in both saturated and unsaturated species were observed ( Figure 4B & Supplemental Table 4 ). The quantity of 6 out of 11 individual saturated AC and AC-OH species (AC6:0, AC8:0, AC14:0, AC14:0-OH, AC16:0-OH, AC18:0-OH) was significantly increased (p ≤ 0.05; Figure 4B & Supplemental Table 4 ). A similar pattern was observed in the individual unsaturated AC and AC-OH species with 11 out of 15 species showing a significant increase (AC8:1, AC12:1, AC14:1, AC18:2, AC18:3, AC20:3, AC22:5, AC22:6, AC14:1-OH, AC18:1-OH, AC18:2-OH; p ≤ 0.05; Figure 4B & Supplemental Table 4 ) in follicles from phthalate-treated mice. Interestingly, the unsaturated species, AC16:1 and AC16:1-OH, were not significantly changed by exposure to the phthalate mixture. The protein abundance of four key enzymes in the acylcarnitine shuttle pathway, acyl-coenzyme A synthetase (ACSM3), carnitine o-palmitoyltransferase 1 (CPT1), mitochondrial carnitine/acylcarnitine carrier protein (MCAT, Slc25a20 ), and carnitine o-palmitoyltransferase 2 (CPT2), were evaluated using our previously published proteome profile 30 . The abundance of ACSM3, CPT1, and MCAT were unchanged by phthalate exposure; however, the abundance of CPT2 was significantly decreased (p = 0.038; Figure 5A – D ). These enzymes were further evaluated at the mRNA level by subjecting antral follicle samples (n=7 mice/treatment) from oil and PHT32- treated mice to qPCR analysis. Transcript levels for Acsm3, Cpt1a, Cpt1b, Slc25a20, and Cpt2 were not significantly changed by exposure to PHT32 ( Figure 5E – H ). Lipid profiling via LC/MS-MS (n=5 mice/treatment) was performed with liver and serum samples from oil and PHT32-treated mice to evaluate the influence of phthalate exposure on systemic acylcarnitine (AC) levels. Total AC, as well as individual saturated and unsaturated AC species in the liver and serum samples were unchanged following PHT32 exposure ( Figure 6A – B ). However, of the 26 individual AC species detected in the liver samples, four saturated species, C4:0, C6:0, C8:0, and C10:0 were significantly increased (p ≤ 0.05; Figure 6C ; Supplementary Table 5 ). Additionally, the quantity of one unsaturated AC species, C20:3 in the serum was significantly increased following phthalate exposure (p = 0.0342; Figure 6D ; Supplementary Table 6 ). Liver samples from oil and PHT32-treated mice were also subjected to qPCR analysis (n=7-9 mice/treatment) to investigate transcript levels of enzymes involved in the acylcarnitine pathway. No significant change was observed in hepatic transcript levels for Acsm3, Cpt1a/b, or Cpt2 , however a significant decrease in the expressions of Slc25a20 (p = 0.0445; Table 2 ) was observed. To evaluate lipid storage within the cells of the ovary and antral follicle, one ovary per mouse (n=4 mice/treatment) was subjected to fresh frozen fixation and Oil Red O staining, a marker for neutral lipid droplet formation ( Figure 7A – F ). The percent positive staining in the whole ovary and in the antral follicles was analyzed using QuPath Software. The percent positive staining in both the whole ovary and antral follicle population were unchanged by phthalate mixture exposure ( Figure 7C & F ). To further evaluate the lipid storage, antral follicles (n=4 mice/treatment) were subjected to triglyceride measurements using a luminescence-based assay. The triglyceride content (μM) was significantly decreased in the antral follicles from mice exposed to PHT32 (p = 0.027; Figure 7G ). In addition, the systemic impacts of PHT32 exposure were evaluated via liver and serum triglyceride content. Triglyceride measurements (n=3 mice/treatment) revealed a significant increase in triglyceride species in the liver of PHT32-treated mice which was also accompanied by a trending increase in the serum (p = 0.045 & 0.089 respectively; Figure 7H & I ). The protein abundance changes of three key enzymes involved in the hydrolysis of triglycerides to free fatty acids, patatin-like phospholipase domain-containing protein 2 (PLPL2, Pnpla2 ), hormone-sensitive lipase (LIPS, Lipe ), and monoglyceride lipase (MGLL), were evaluated using our previously published proteome profile 30 . When using all the samples, the abundance of PLPL2 was unchanged by phthalate exposure ( Supplemental Figure 1 ), however when two extreme values (one oil and one PHT32) were removed from the analysis, it was observed that the protein abundance of PLPL2 was significantly increased (p = 0.0032; Figure 8A ). The protein abundance of LIPS was significantly decreased (p = 0.0163; Figure 8B ), while MGLL remained unchanged in the antral follicle samples from the mice treated with the phthalate mixture ( Figure 8C ). The transcript levels for the genes encoding these enzymes were not changed between treatment groups ( Figure 8D – F ). Additionally, hepatic transcript levels of these lipid hydrolysis genes were evaluated through qPCR analysis of liver samples from oil and PHT32 mice (n=7-9 mice/treatment). No significant change was observed in hepatic transcript levels for Pnpla2, Lipe, or Mgll ( Table 2 ). To identify changes associated with phthalate exposure on lipids important in providing structural support within antral follicles, antral follicle samples from control and PHT32 groups (n=4-5 mice/treatment) were subjected to phospholipid, lysophospholipid, and ceramide profiling by LC-MS/MS. Exposure to PHT32 did not alter the quantity of total phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC), and ceramide (Cer) species in antral follicles ( Supplementary Tables 7 – 11 ). Interestingly, there was a significant increase in 6 of the 8 individual LPC species (p ≤ 0.05; Supplementary Table 7 ). Phthalate exposure did not induce any significant changes on the 6 individual LPE species ( Supplementary Table 8 ) or the 17 individual PE species detected in the antral follicle samples ( Supplementary Table 9 ). Additionally, PHT32 exposure led to significant changes in some individual PC and Cer species including significant increases in the quantity of PC 40:5 and Cer d18:1/14:0 (p = 0.012 & 0.026; Supplementary Table 10 – 11 ), and decreases in dhCer d18:0/16:0 and dhCer d18:0/24:0 (p = 0.025 & 0.030; Supplementary Table 11 ). Finally, the effects of phthalate exposure on lipids important in providing cellular structural support systemically were investigated by subjecting liver and serum samples from control and PHT32 groups (n=4-5 mice/treatment) to phospholipid and lysophospholipid profiling by LC-MS/MS. Exposure to PHT32 did not alter the quantity of total PE, PC, LPE, or LPC species in the liver samples ( Supplementary Table 12 – 15 ). Interestingly, PHT32 exposure led to a significant decrease in the total quantity of PE species found in the serum (p = 0.0175), accompanied by a decrease in one individual species PE 32:1 (p = 0.0478; Supplementary Table 16 ). However, the other structural support lipids PC, LPE, and LPC were unchanged by phthalate exposure ( Supplementary Table 17 – 19 ).

This study evaluated lipid, protein, and gene expression changes within antral follicles and identified significant phthalate-induced changes in the quantity of free fatty acids, acylcarnitines, and triglycerides as well as gene and protein expression changes associated with fatty acid synthesis, acylcarnitine shuttling for beta oxidation, and lipid storage in antral follicle samples. The results obtained imply that there is an associated systemic influence at play given the observed differences in response between antral follicles, liver and serum lipid profiles. These findings strongly suggest that exposure to this environmentally relevant mixture of DBP, DEHP, and BBP leads to changes consistent with increased fatty acid synthesis, decreased beta oxidation, decreased triglyceride abundance, and increased lysophosphatidylcholines within antral follicles ( Figure 9 ). Finally, this work reveals key mechanistic events that may become useful in explaining previously reported associations between phthalate burden, antral follicle function, and lipid metabolic dysfunction. Targeted lipidomic profiling was performed on antral follicles to help address gaps in mechanistic understanding of in vivo phthalate exposure on ovarian antral follicle lipid metabolism. Other lipidomic profiling studies have highlighted the importance of lipid metabolism in ovarian function in the context of ovarian cancer 66 , high fat diet 67 , PCOS 68 , and tributylin exposure 69 . The discovery of dysregulated intrafollicular fatty acid, acylcarnitine, triglycerides, and lysophosphatidylcholines in phthalate-treated mice are unique and contribute significantly to understanding the associations between phthalate exposure and adverse antral follicle outcomes in women. These observations are supported by previous toxicoproteomic findings showing aberrant abundance of lipid metabolism proteins in mouse antral follicles 30 and a recent study reporting lipid-related transcriptional changes in isolated primary granulosa cells 70 . Homeostatic fatty acid synthesis, beta oxidation, and lipid storage serve in concert to maintain antral follicle and oocyte health and function. Therefore, these proteomic, lipidomic and transcriptomic studies provide strong evidence for disrupted lipid metabolic processes as a significant key event worth evaluating further for fit in the adverse outcome pathway for phthalate effects on the ovary. In this study, mRNA, protein, and lipid data suggest that phthalate mixture exposure may increase intrafollicular FFA concentrations by stimulating de novo synthesis. Fatty acid synthesis requires the conversion of acetyl-CoA into malonyl-CoA via ACACA, sequential addition of two-carbon units to a growing fatty acid chain by FAS ( Fasn ) to produce palmitate (16:0), extension of 16:0 by ELOV6 into stearate (18:0), and desaturation into oleate (18:1) by ACOD1 ( Scd1 ). Present findings show that exposure to the three-phthalate mixture increases follicular Fasn mRNA, FAS protein, and accumulation of saturated FFAs (mainly stearate). Lipid metabolism is essential for oocyte and cumulus cell function 71 and dysregulated levels of FFAs are associated with poor cumulus-oocyte-complex (COC) morphology, reduced ovarian reserve, and IVF outcomes 72 , 73 in women. Given that increased phthalate burden has been associated with reduced ovarian reserve 25 , 74 , 75 , 23 and poor IVF outcomes 76 in women, it is possible that increased intrafollicular FFA accumulation could be a key event to be prioritized in the study of phthalates. Future studies will be needed to identify the molecular initiating event leading to enhanced Fasn expression from among known transcriptional and post-translational regulators of the enzyme. Fatty acid synthesis and oxidation are reciprocally regulated; thus, changes in markers of beta oxidation were also observed in response to phthalate mixture exposure. The protein and lipid profiling results in this study suggest that phthalate mixture exposure may increase intrafollicular acylcarnitine concentration by shutting down the carnitine shuttle system and inhibiting beta oxidation in antral follicles. In preparation for oxidation, fatty acids are activated to fatty acyl-CoA derivatives by ACSM and then transported into the mitochondrial matrix via the carnitine shuttle system comprised of CPT1A/B, MCAT ( Slc25a20 ), and CPT2. Due to its role in the last step of the shuttle system, CPT2 deficiency is known to cause acylcarnitine accumulation, impaired oxidation, and dysfunction in cells highly reliant on beta oxidation 77 . In this study, exposure to the phthalate mixture decreased the abundance of CPT2 protein in the antral follicle, thus suggesting inhibition of the last carnitine shuttle step prior to beta oxidation. Because CPT2 abundance is reduced, acylcarnitine conversion back to an acyl CoA and carnitine may be impaired thus leading to accumulation of acylcarnitine molecules. Interestingly, the phthalate mixture did not affect transcript levels for Cpt2 suggesting that transcriptional regulation alone may not entirely explain the decrease in CPT2 protein and that transcription-independent regulation should be evaluated in future studies. Different populations of antral follicles were used in the proteome and transcript analyses; therefore, it is possible that sampling may have had an effect. However, strong functional agreement among mRNA, protein, and lipid assays conducted across different antral follicle samples in this study and previous work 30 provide significant evidence against sampling bias. Finally, given that beta oxidation is essential for mouse oocyte developmental competence and early embryo development 78 , 71 and women with high phthalate burden show poor assisted reproduction outcomes 76 and early pregnancy loss rates 10 , future work in this area should determine whether a key event in phthalate toxicity includes not only increased fatty synthesis via high FAS, but also blocked carnitine shuttle function and impaired oxidation via low CPT2. A decrease in antral follicle triglyceride content was observed following phthalate exposure, indicating that triglyceride homeostasis may be impaired. Fatty acids are stored as triglycerides for later use in energy production and signaling pathways. Triglycerides are hydrolyzed to produce three fatty acids and glycerol in a three-step process. First, the triglyceride is converted into a diglyceride (DG) by PLPL2 ( Pnpla2 ), then to a monoglyceride (MG) by LIPS, ( Lipe ), and finally to FFAs and glycerol by MGLL. Findings from this study show that exposure to the phthalate mixture led to reduced antral follicle triglyceride content with alterations in two triglyceride hydrolysis proteins, PLPL2 and LIPS, when extreme/outlier values were removed from analysis. These results suggest that the impairment in antral follicle triglyceride content may be through altered lipase action; however, future experiments measuring specific states of the acylglycerides (i.e., tri-, di-, mono-) will be required. Triglyceride accumulation typically occurs within adipocytes and DEHP has been shown to individually decrease triglyceride accumulation in human adipocytes 79 . Besides systemic contribution and local storage 80 , the ovaries may also receive triglycerides from periovarian adipose tissue (POAT) 81 , which should be considered in future studies as its collection and characterization was not part of the antral follicle isolation protocol used in this study. While the categories of phospholipids, ceramides, and lysophospholipids were not extensively affected, several individual lysophosphatidylcholines (LPCs) were significantly increased following exposure to the phthalate mixture. LPCs in follicular fluid have been shown to be important for follicular development and a potential predictor of ovarian sensitivity in women 82 . Elevated LPCs have been shown to induce apoptosis and autophagy in mouse ovarian granulosa cells through oxidative stress pathways 132 . Increased oxidative stress has been described for phthalates in the ovary 83 , 84 , 21 thus, increased follicular LPC in the study gives insight to a potential molecular link between phthalate exposure effects on lipid homeostasis and follicular death pathways. Interestingly, urinary phthalate metabolites in women have been associated with increased plasma LPC levels, likely through enhanced phospholipase activity 85 ; therefore, future work is essential and should determine how phthalate-induced LPC levels in mouse antral follicle are linked to phospholipase activity, oxidative stress pathways, and ultimately cellular death. While relatively fewer in quantity, some significant effects on serum and liver FFA, acylcarnitine, and triglyceride profiles were observed in response to phthalate mixture exposure. Interestingly, four individual acylcarnitine species were significantly altered in the liver in the same direction as observed in the follicle samples, indicating that a related systemic effect on short and medium chain species may be occurring. Additionally, C20:3 AC was significantly elevated in the serum and antral follicles of phthalate-treated mice in our study. Observations made in this study are supported by reported associations and/or dysfunction in acylcarnitine homeostasis with phthalates, tributylin, and bisphenol exposures in other tissues 86 , 87 , 88 , 89 , 90 . Finally, the increased triglyceride content, at first glance, appears to contradict work published by others reporting phthalate impacts on hepatic and adipocyte lipid metabolism, but it is critical to acknowledge that high dose, single phthalate exposure studies are not directly comparable to those reported here. Under homeostatic conditions, the liver stores low amounts of triglycerides with the majority being packaged into lipoprotein for transport to other tissue including muscle, fat, and the ovary and antral follicle 91 , 92 . Results from this study suggest that phthalates cause an imbalance in this process possibly favoring accumulation, which could lead to pathologies like nonalcoholic liver disease 91 . Antral follicles utilize circulating lipoproteins as a source for cholesterol for steroid hormones production 92 thus an increased retention of triglycerides in liver and serum may result in aberrant antral follicle uptake. Additional studies will be needed to determine the impact of phthalates on antral follicle lipid uptake, di- and monoglyceride pools, lipase activity, and lipoprotein secretion and profiles. Notably, results showed related, but not overwhelming, changes in lipid profiles peripherally to the ovary provide support for hypotheses that favor direct intraovarian responses to phthalates. While this study provides novel insights into the impacts of phthalate mixture exposures on antral follicle lipid homeostasis, it is important to highlight its limitations. First, while the use of an experimental rodent model is advantageous by providing the strength of a simplified, well known and controllable mammalian system with relevance to humans, it is limited in its ability to fully recapitulate the complexity of human physiology (i.e., non-conserved aspects of lipid metabolism) and interactions with the environment (e.g., toxicokinetics and co-exposures unique to humans). Although the toxicokinetics of individual phthalates have been described in humans and rodents, the same level of multi-species understanding is not currently available for mixture exposures. In the absence of mixture data from humans to perfectly align the phthalate composition and dose selections for animal studies, this study used individual phthalate information to fill this gap and, thus, this limitation must be accounted for when interpreting results. Second, using distinct samples for mRNA and protein analyses is standard practice when evaluating toxicodynamic outcomes in small tissues like mouse ovarian follicles. While this approach might be perceived as a limitation due to known discrepancies between transcript and protein levels, our results, alongside Miller et al. (2024) 30 , demonstrate strong functional agreement among mRNA, protein, and lipid assays conducted across different antral follicle samples. Finally, this study focused on elucidating the impacts of phthalates on the lipid profile of antral follicles and identified strong candidates for the mechanism of action; however, future studies that incorporate a thorough characterization of the mixture including chemical composition analysis of stock solutions, complete serum and tissue-level toxicokinetic profiling, and ovarian compartment exposure verification will further the translational significance of these findings.

In conclusion, this study shows that exposure to a human relevant mixture of DEHP, DBP, and BBP may disrupt antral follicle lipid metabolism in mice as evidenced by alterations in gene and protein abundance and lipid profiles. These findings suggest that phthalate exposure may lead to increased free fatty acid synthesis and decrease beta oxidation in mouse antral follicles by targeting FAS and CPT2 expression, respectively. These observations are relevant to risk assessment and, with additional research, have potential to be useful in the assembly of adverse outcome pathways for phthalates in the ovary. Additionally, these findings have potential relevance to reproductive medicine because elevated follicular fluid LPCs correlate with antral follicle count and ovarian sensitivity in women and, in bovine, stearic acid correlates with reduced numbers of fully mature oocytes 93 , impaired oocyte maturation, fertilization rates, and poor quality embryos 72 . Finally, this work highlights the need to fully understand homeostatic ovarian lipid metabolism and its susceptibility to chemical exposures.

Phthalates are found in everyday products including personal care products, medical equipment, the coating of some pharmaceuticals, and in food packaging 1 . Although new congeners are currently in use, metabolites of dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), and di-2-ethylhexyl phthalate (DEHP) are still detected in human biofluids 2 , 3 . Daily chronic exposure to DBP, BBP, and DEHP in the general population has been estimated via human monitoring studies to range between 7-10 μg/kg/day, 2 μg/kg/day, and 0.1-18.9 μg/kg/day, respectively 4 , 5 , 6 , 7 , 8 . In women, urinary phthalate metabolite levels are associated with several negative reproductive outcomes including early menopause 9 , increased pregnancy loss 10 , and reproductive diseases such as polycystic ovarian syndrome (PCOS) 11 and endometriosis 12 . Urinary phthalate metabolites are also associated with an increased risk for metabolic syndrome and obesity 13 , 14 , which in turn are associated with infertility via negative impacts on female fecundity and medically-assisted reproduction success 15 , 16 . Considering the global prevalence of infertility is 17.5% 17 and in 2022 alone approximately 100,000 infants in the US were conceived with the assistance of artificial reproductive technology 18 , it is critical to elucidate the mechanisms responsible for associations between phthalates and poor reproductive and metabolic health outcomes in women. Phthalate metabolites are known to reach the ovary as demonstrated by their detection in the ovarian functional units known as ovarian follicles 19 – 24 . Ovarian follicles house the oocyte and range in developmental stage from quiescent primordial follicles to mature functional antral follicles. Antral follicles, characterized by the presence of a fluid-filled cavity known as the antrum, sustain steroidogenesis and are capable of ovulation. The follicular fluid within the antrum is derived from follicular cell secretions and the systemic circulation. Studies in women seeking medically assisted reproduction have reported associations between follicular fluid phthalate metabolite concentrations and low ovarian follicle count and low intraovarian hormone concentrations 25 , 20 , 26 . The underlying mechanisms explaining the associations observed in human studies still remain to be fully elucidated. Due to the multiorgan complexity of reproductive toxicity and challenges associated with primate research, the phenotypes associated with exposure to single phthalate and phthalate mixtures have been replicated in animal models in various studies including our own 27 , 28 , 24 , 29 , 30 , 31 , 32 . Specifically, mice provide a feasible and controllable whole-body mammalian model of human reproductive system responses to toxicants that in vitro and in silico models cannot yet replicate. In vivo exposure to environmentally relevant concentrations of a mixture of DBP, BBP, and DEHP has been shown to significantly alter the antral follicle proteome 30 . Specifically, a dose that mimics general population human exposure in mice significantly changed the abundance of follicular proteins involved in lipid and fatty acid metabolic functions 30 . Lipid metabolism includes the synthesis and catabolism of lipids for structural, signaling, and energy functions. It is well known that lipid metabolism is maintained in a delicate balance in which synthesis, storage, and oxidation of fatty acids are carefully regulated, and significant deviations in these processes lead to metabolic dysfunction. Importantly, fatty acid metabolism is critical to follicular cell and oocyte functions. Particularly in oocytes, alterations in fatty acid oxidation result in impaired maturation and development 33 , 34 . High saturated free fatty acids cause reduced cell viability, increased apoptosis, and disrupted steroidogenesis in oocytes 35 , 36 , bovine and mouse cumulus oocyte complexes (COCs) 37 , 38 , 39 , 40 , and bovine granulosa and theca cells 41 , respectively. Therefore, significant disruptions to lipid metabolism in antral follicles can cause reproductive disease and, in the case of phthalates, help explain the poor medically assisted reproductive performance observed in women with high urinary burden of phthalate exposure markers. While there is evidence in laboratory rodents that phthalates, particularly DEHP, influence fatty acid production and oxidation, triglyceride hydrolysis and lipid accumulation 42 , 43 , 44 , no studies to date have characterized the impacts of epidemiologically relevant phthalate exposures at low doses and including multiple phthalates. Based on this need and previous work showing alterations in antral follicle proteins involved in lipid metabolism, the goal of this study was to investigate the consequences of oral phthalate exposure on antral follicle lipid and systemic metabolism processes (i.e., fatty acid synthesis, beta oxidation, and lipid storage pathways). To achieve this goal, various lipid profiles, mRNA and protein expression of key enzymes, and lipid content were analyzed in isolated antral follicles, liver, and serum samples from adult female mice orally exposed to vehicle control or a human relevant mixture of DBP, BBP, and DEHP.