Phenol biomarker concentrations in human ovarian follicular fluid and the associations with in-vitro fertilization outcomes.

The 143 women included in this analysis had a median (IQR) age and BMI of 34.0 (32.0–38.0) years and 22.6 (20.9–24.9) kg/m 2 , respectively ( Table 1 ). Women were predominantly Caucasian (83%), and 78% had never smoked. Detection rates of the different phenols are shown in Table 2 and varied from high (79–95%) for MPB, PPB and BPS to moderate (44–65%) for BPA, BP3 and TCS to low (1–34%) for 2,4DCP, 2,5DCP, EPB, BPB, BPF, TCC ( Table 2 ). Phenols with a low detection rate were not considered for further analysis. Correlations among FF phenol biomarkers were noted between MPB and PPB ( r =0.78), BPA and BPS ( r =0.25), BP3 and TCS ( r =0.18), as well as BP3 and BPA ( r =0.15) ( Supplemental Table 1 ). Correlations among FF and urine concentrations for the studied phenol biomarkers were strong for TCS ( r =0.88) and BP3 ( r =0.86), moderate for MPB ( r =0.64) and PBP ( r =0.63), and weak for BPS ( r =0.21) and BPA ( r =0.12) ( Supplemental Table 2 ). Table 5 depicts the FF phenol concentrations over time, demonstrating that average BPA concentrations decreased over time (0.27ng/nl to 0.19ng/ml ( p =0.001)) whereas BPS concentrations increased (0.89ng/ml to 1.85ng/ml ( p =0.001)) from 2007 to 2015. No other trends were observed for the FF phenol biomarker concentrations studied. While most of the examined FF phenol biomarker concentrations were not related to early IVF outcomes, including endometrial thickness, total and mature (MII) oocyte yield, and fertilization rates, we observed significant associations between PPB and endometrial thickness, and between BP3 and MII oocyte yield ( Table 6 ). Specifically, women in the highest, middle, and lowest tertile of FF PPB biomarker concentrations had a mean adjusted endometrial thickness in millimeters (95% CI) of 9.85 (9.28, 10.4), 10.5 (9.81, 11.1) and 10.9 (10.3, 11.5), respectively. Also, women in the highest, middle, and lowest tertile of FF BP3 biomarker concentrations had a mean adjusted MII oocyte yield (95% CI) of 10.5 (9.4, 11.7), 10.3 (9.0, 11.8) and 8.7 (7.5, 10.0), respectively. However, these did not translate into any effects on pregnancy outcomes, as we did not find any relationship between any of the phenols investigated in the FF with probabilities of implantation, clinical pregnancy and live birth ( Table 7 ).

Study participants were women enrolled in the Environment and Reproductive Health (EARTH) Study, a prospective cohort established since 2004 to evaluate environmental, dietary and lifestyle factors that may impact reproductive and pregnancy outcomes among couples seeking fertility treatment ( Messerlian et al. 2018 ). Women aged 18–45 years were eligible to participate and approximately 60% of those contacted by the research study staff ultimately enrolled, as previously described (Minguez-Alarcon et al 2019). The current analysis includes 143 women who completed one IVF cycle between 2009 and 2015 at the Massachusetts General Hospital (MGH) Fertility Center and had available FF and urine biomarker data. Accounting for clinical indications and factors such as age and infertility diagnosis, women underwent one of the following ovarian stimulation protocols: a ) luteal-phase gonadotropin-releasing hormone (GnRH) agonist (low-, regular-, or high-dose leuprolide acetate; Lupron), b ) follicular-phase GnRH-agonist/Flare stimulation, and c ) GnRH-antagonist. Ovulation trigger was achieved with human chorionic gonadotropin (hCG). Women were followed from study entry throughout their fertility care and treatment outcomes including, pregnancy, labor and delivery. At entry, the participant’s date of birth was collected, and weight and height were measured by trained study staff. In addition, research staff administered socio-demographic, lifestyle, and medical history questionnaires to participants. Study participants provided additional information at study entry by completing a more comprehensive questionnaire on family, medical, reproductive and occupational history, product use, smoking history, and physical activity. Exclusion criteria for this analysis included couples with male factor infertility and donor gamete cycles. The study was approved by the Human Subject Committees of the Harvard T.H. Chan School of Public Health, MGH, and the Centers for Disease Control and Prevention (CDC). Participants signed an informed consent after the study procedures were explained by trained research staff. Women enrolled in the EARTH study undergoing an oocyte retrieval provided a urine sample on the day of their procedure as previously described (Liao et al. 2012). In addition, FF from the first three follicles aspirated with a 16 G needle during the oocyte retrieval was collected. The physician performing the oocyte retrieval was asked to aspirate only one follicle per specimen collection tube, which was prepared by trained study staff with 1 ml of flushing media, for each of these first 3 follicles. After the FF was thoroughly examined by the fertility center embryologist for any oocytes, the FF was poured into a 15ml Falcon tube labeled Trap #1, #2 and #3 for each follicle aspirated. The embryologists noted the time of the FF collection, and the tubes were capped and placed on ice until they were processed by the experienced EARTH study research staff. The research staff member made three 1ml aliquots from the FF supernatant per follicle aspirated and stored the specimens in 2ml cryovials at −20 °C initially and ultimately at −80 °C. From the pellet of the specimen, the research staff made two 100 μl aliquots which were then stored in 500 μl cryovials in liquid nitrogen. Follicular fluid specimens from 143 women undergoing their first IVF cycle at MGH Fertility Center and enrolled in the EARTH study were sent to the NSF International LLC laboratory in Ann Arbor, Michigan for analysis. NSF developed a sensitive Multiple Reaction Monitoring (MRM) negative mode method for measuring the following twelve phenols: 2,4-dichloro-phenol (2,4DCP), 2,5-dichloro-phenol (2,5DCP), methyl-paraben (MPB), ethyl-paraben (EPB), propyl-paraben (PPB), butyl-paraben (BPB), bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), benzophenone-3 (BP3), triclosan (TCS) and triclorocarban (TCC). The method was developed to simulate the CDC (Centers for Disease Control and Prevention) method “Bisphenol A and Parabens in Urine Method Laboratory Procedure 6301.01.” The NSF method was evaluated against similar acceptance criteria established within the CDC method. The validated analyte calibration curve correlation coefficient (R 2 ) range was 0.995–1.000. The method accuracy (% nominal concentration) and precision (%RSD) were determined through six replicate analyses of analytes spiked at three different concentrations in human FF across validation runs on three separate days (n =18), which reflects both the intra-day and inter-day variability of the assay. The accuracy (% nominal concentration) range across all analytes was 102–114% with precision (%RSD) range for the FF quality control samples across all analytes being 2.7–7.2%. Using a Thermo Scientific Accela Autosampler, samples were pre-concentrated by on-line solid phase extraction (SPE) interfaced with a Thermo Scientific Transend TXII system utilizing a Cyclone-P extraction column. The analytes were then separated and focused using a Dionex UltiMate 3000 UHPLC system with reversed-phase chromatography using a Waters XBridge C18 analytical column. All chemicals of interest were detected by atmospheric pressure chemical ionization (APCI)–MS/MS using a Thermo Scientific Quantiva Mass Spectrometer. Optima LC/MS methanol and Optima LC/MS water were purchased from Fisher Chemical (Fair Lawn, NJ USA). Control human FF (pooled) was donated from The University of Michigan (Ann Arbor, MI, U.S.A.). 2,4DCP, 2,5DCP, BPA, BPS, BPF, BPB, EPB, PBP, TCC, 4-Methylumbelliferyl glucuronide, β-glucuronidase from helix pomatia, ammonium acetate and acetic acid were purchased from Sigma-Aldrich (St.Louis, MO, U.S.A.). BP3 was purchased from AccuStandard (New Haven, CT, U.S.A). MPB was purchased from Fluka (Ronkonkoma, NY, U.S.A.). TCS was purchased from USP Reference Standards (Rockville, MD, U.S.A.). Isotopically labeled analogues of the analytes, 2,4-Dichlorphenol- 13 C6, 2,5-Dichlorphenol- 13 C 6 , Bisphenol-A- 13 C 12 , Bisphenol-S- 13 C 12 , Bisphenol-F- 13 C 12 , Butylparaben- 13 C 6 , Ethylparaben- 13 C 6 , Propylparaben- 13 C 6 , Methylparaben- 13 C 6 , Oxobenzone- 13 C 6 , Triclocaraban- 13 C 6, Triclosan- 13 C 12 , and 2,3,4-Methyl- 13 C 4 -methylumbelliferone were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). 4-methylumbelliferyl sulfate was purchased from Marker Gene Technologies (Eugene, OR, U.S.A.). All standards and internal standards used in the method have purity greater than 98%. All other reagents used in the method have purity greater than 99%. XBridge C18 analytical column (3.0 × 50mm x 5μm) was purchased from Waters Corp. (Milford, MA, U.S.A.). Cyclone-P extraction column (0.5 × 50mm) was purchased from Thermo Scientific (Franklin, MA, U.S.A.). The study samples sent were randomly chosen from oocyte retrievals performed at MGH Fertility Center after September 2009, at which time there was a change in the FF collection protocol. The collection protocol called for the samples from each patient to not be pooled and all aliquots were centrifuged. Non-bloody supernatant of FF from one follicle per patient with a minimum of 400 μl per the NFS lab’s requirements was sent for analysis. Samples were thawed and vortexed to ensure homogeneity. All standards and reagents were allowed to reach room temperature before sample preparation. All samples, method blanks, follicular fluid matrix blanks, calibration standards, and Quality Control (QC) samples were prepared with the assistance of a Hamilton MICROLAB STARlet Liquid Handling System. 50 μL β-glucuronidase was added to all vials. 200 μL of the FF samples was then added to the sample vials while 200 μL of water was added to the method blanks and calibration standards. Afterwards, 200 μL of control human FF was added to the QC samples and matrix blanks and 100 μL of internal standard solution was added to all vials. All vials were incubated at 37 °C for 2 hours. After incubation, 200 μL of methanol was added to the FF samples, method blanks, and the control follicular fluid matrix blanks. 200 μL of the standard solution was then added to the respective calibration standards and the QC samples. Finally, 1500 μL of water was added to all samples. PTFE/Silicone pre-split caps were screwed onto the vials and all samples vortexed. The women also provided a spot urine sample on the day of oocyte retrieval. The urine was collected in a sterile polypropylene specimen cup. Specific gravity, which was used to correct phenol concentrations for urine dilution, was measured at room temperature using a handheld refractometer (National Instrument Company, Inc., Baltimore, MD, USA) calibrated with deionized water before each measurement. The samples were then shipped to the CDC on dry ice and were analyzed as previously described with online solid-phase extraction coupled with isotope dilution-high-performance liquid chromatography-tandem mass spectrometry for urinary concentrations of phenols ( Silva et al. 2007 , Ye et al. 2005 ) Demographic and reproductive characteristics were presented using median ± interquartile ranges (IQRs) or counts (%). The FF phenol concentrations received from the lab were corrected for the above-mentioned dilution with 1 ml of flushing media. The adjustment was made as follows: Corrected Phenol Concentration = Lab-resulted Phenol Concentration x Specimen Total Volume / Specimen Total Volume – 1 Distribution of FF phenol concentrations were reported as mean (standard deviation) and percentiles. Detection rates of the various phenols were calculated as percentages using the different limits of detection (LODs). Follicular fluid phenol concentrations were natural log-transformed to more closely approximate a normal distribution. Spearman correlations were used to summarize the relationship between the different phenol concentrations in FF, and the different phenol concentrations between FF and urine. Multivariable generalized linear models were used to estimate the differences in FF phenol concentrations across the years (normal distribution and identity link). Women’s biomarker phenol concentrations were then categorized into tertiles or two groups based on the detection rate for each biomarker, with the lowest tertile considered as the reference group. Generalized linear models were used to investigate the associations between FF concentrations of phenols and IVF outcomes, adjusting for age, BMI, infertility diagnosis and year of sample collection. A Poisson distribution and log link function were specified for oocyte counts, and a binomial distribution and logit link function were specified for fertilization rates, and other clinical outcomes (implantation, clinical pregnancy, and live birth). To allow for better interpretation of the results they were back-transformed and population marginal means ( Searle et al. 1980 ) were presented. Statistical analyses were performed with SAS (version 9.4; SAS Institute Inc., Cary, NC, USA).

We quantified phenol biomarker concentrations in FF samples among 143 women attending the Fertility Center in Boston (MA) and evaluated correlation with urine samples and associations with reproductive outcomes. We identified the presence of various phenols in human ovarian FF and demonstrated correlation of some phenol biomarkers in FF and urine. We also identified a decrease in the presence of BPA in the FF and a concurrent increase in BPS which could be explained by the recent temporal trend in using BPA substitutes. While a couple of FF phenol concentrations were associated with intermediate reproductive outcomes, these associations did not translate into any effects on probabilities of clinical pregnancy or live birth. Given the increasing rates of infertility in Western countries and the scarce literature on phenol biomarkers measured in the target organ, additional studies are warranted. Phenols are a category of short-lived EDCs which are widely used in the manufacture of consumer products. Data from both animal and human studies show that the endocrine activities of phenols may lead to adverse reproductive outcomes ( Qiu et al, 2019 , Mansur et al. 2017, Karwacka et al. 2019 ). However, most published human studies concentrate on the association of urinary phenol concentrations with reproductive outcomes ( Souter et al. 2013 , Smith et al. 2013 , Mínguez-Alarcón et al 2019, Hua et al. 2017 , Ehrlich et al. 2012 , Mínguez-Alarcón et al 2016 ). Urinary concentrations are mostly used as a biomarker of exposure but may not represent exposure at the target organ, such as the ovary and ultimately the oocyte. As the FF is the matrix in direct contact with the female gamete, one can hypothesize that the presence of EDCs in the FF may have an effect on the oocyte, and by extension, on fertility. Martinez et al . showed that urinary concentrations of select phenol metabolites are correlated with altered extracellular vesicle-miRNAs expression in FF, providing some insight regarding the potential molecular mechanism underlying the reported adverse associations of phenol exposure on female fertility ( Martinez et al. 2019 ). Mansur et al . showed that when cumulus cells surrounding the oocyte were exposed to increasing BPA, gene expression associated with apoptosis was altered, implying ovarian toxicity and providing another important molecular mechanism of BPA action on human reproduction (Mansur et al. 2017). Furthermore, animal studies have suggested an impact of BPA directly on the oocyte affecting meiosis ( Shin et al. 2019 ) as well as disturbed steroidogenesis ( Zhang et al. 2018 ). However, only three studies have attempted to evaluate whether BPA can be detected in human FF with contradicting conclusions. In a study conducted in Japan, Ikezuki et al . detected BPA in FF, supporting the importance of further studies to evaluate the potential effect this phenol may have on human reproduction ( Ikezuki et al. 2002 ). In contrast, Krotz et al. argued that the accumulation of BPA in the microenvironment of the human oocyte is non-detectable and thus further research is not useful, with a major criticism of the study being that only five samples were analyzed ( Krotz et al. 2012 ). Most recently, Kim et al. published a prospective cohort study investigating BPA concentrations in multiple human matrices, including urine, plasma, FF and semen. This group noted that BPA concentrations in these bodily fluids were not significantly associated with outcomes in couples undergoing IVF ( Kim et al. 2019 ). Our findings were similar to Kim et al. for BPA. It is important to note that our study differs from these studies in terms of study design, population, number of EDCs evaluated and lab methods. In our study, we confirmed the detection of various EDCs of the phenol group, some with high detection rates: MPB (95%), BPS (81%) and PBP (79%), others with moderate detection rates: BP3 (65%), BPA (48%) and TCS (44%), and others with low detection rates: 2,4DCP (32%), 2,5DCP (1%), EPB (34%), BPB (28%), BPF (1%), TCC (1%). The strength of our study lies in the number of samples examined and the various phenols measured. However, a limitation of our study is the generalizability as the study population was limited to infertile women seeking fertility treatments. Nevertheless, the collection of FF is typically only performed in women undergoing an oocyte retrieval for fertility treatments. With BPA being under intense scrutiny as an environmental reproductive toxicant by regulatory agencies in many countries, including the United States, its use has been restricted in specific products such as infant feeding bottles. In response, manufacturers have introduced substitute chemicals including BPS, one of the most used BPA replacements. Little is known about the safety of this compound and close analog of BPA, with studies suggesting that BPS is also an EDC ( Mathew et al. 2014 ) and can be detected in approximately 80% of human urine samples (Liao et al. 2012). Interestingly, BPS is found to be more resistant to environmental degradation than BPA ( Ike et al. 2006 ). At the time our study was conducted, there were no studies evaluating the presence of BPS in FF and the potential reproductive impact. In our study, we show that BPS is detected in human ovarian FF, with a higher detection rate than that of BPA (81% vs 48%, respectively). In addition, we show that the presence of BPA in FF is decreasing over time in contrast with BPS, which is increasing. This phenomenon could be explained by the increasing tendency to replace BPA with BPS. The health outcomes of this substitution are still unknown and under investigation. However, in our study, no significant associations were found for BPA and BPS in FF with early IVF outcomes (peak E 2 levels, endometrial thickness, total and MII oocyte yield, fertilization rates) or pregnancy outcomes (implantation, clinical pregnancy and live birth rates) as depicted in tables 6 and 7 . This contradicts findings in the literature suggesting an association of urinary BPA and adverse IVF outcomes including decreased antral follicle counts, peak E 2 levels, number of total and MII oocytes retrieved, number of fertilized oocytes, embryo quality, pregnancy rates and neonatal outcomes ( Minguez-Alarcon & Gaskins 2017 , Souter et al. 2013 , Karwacka et al. 2019 , Ehrlich et al. 2012 , Mok-Lin et al. 2010 , Mustieles et al. 2018 ). This discrepancy in results between phenols measured in urine verses FF is difficult to explain, especially given that several of these previous publications used urinary phenol concentrations measured in the same EARTH study cohort that the current analysis was performed in. An additional fact to consider for the present analysis is that during the sample collection years of our study, patients undergoing oocyte retrievals were instructed to have nothing by mouth (NPO) after 10pm the night prior to their procedure. This is important to consider as bisphenols, which have a known short half-life, are excreted through the urine and a major route of human exposure is via ingestion. Nowadays, with the introduction of the Enhanced Recovery after Surgery (ERAS) protocol in gynecology ( Grant et al. 2019 ) this limitation can be overcome. In support of our findings, it is important to mention that the recent case-control study by Tian T et al , focusing on decreasing ovarian reserve (DOR) and conducted after the completion of our study, did not show an association between BPS FF concentrations and DOR ( Tian et al. 2023 ). Regarding the two statistically significant findings of associations found between FF phenol concentrations and early IVF outcomes, one could argue that the clinical relevance is minimal, if any. The endometrial thickness difference between the first and third tertile of PPB FF concentration was 1.05mm thinner in the higher concentration group. Although studies have shown a decreasing probability of implantation and ongoing pregnancy with thinner endometrial thickness at the time of an embryo transfer, the absolute endometrial thickness findings were all above 9 mm in our study, which is above the commonly used 6mm endometrial thickness cut-off used to proceed with a transfer ( Jacobs et al. 2022 ) and likely limits the clinical significance of this finding. The MII oocyte yield difference between the first and third tertile of BP3 FF concentration was an additional 1.8 MII oocytes retrieved in the higher concentration group. This finding is overall reassuring and consistent with the outcome that the FF phenol concentrations investigated in this study did not clinically affect early IVF or pregnancy outcomes. However, while an increased oocyte yield may appear favorable, its clinical significance remains uncertain. The present study has some limitations. As already mentioned, and because of its design, it may not be possible to extrapolate the study findings to the general population of couples conceiving without medical intervention. However, these findings may be applicable to other women seeking infertility treatment. The study sample is limited as the quantification of phenol biomarkers in FF is technically complex and costly, and available funding was limited at the time of analysis. This may limit statistical power, however, our study represents one of the largest sample sizes to date investigating phenol biomarker concentrations in human ovarian follicular fluid in relation to IVF outcomes. In addition, misclassification of biomarker exposure based on concentrations from FF samples is possible because phenols are short-lived chemicals, and exposures are likely to be episodic in nature, potentially leading to attenuated associations. While we acknowledge that a single time-point measurement has inherent limitations for assessing short-lived chemical exposures, our use of FF as the biomatrix was intentional and based on its biological relevance to the outcome of interest. Non-persistent semi-volatile phenols are rapidly metabolized and excreted in urine, which is the most used and validated matrix for exposure assessment in environmental epidemiology. However, FF offers a unique opportunity to evaluate local exposure at the target tissue level, specifically the ovarian microenvironment where oocyte maturation occurs. Unlike serum or plasma, which reflect systemic exposure, FF directly bathes the developing oocyte and surrounding granulosa cells. Therefore, its use allows for the investigation of associations between biomarkers of exposure and reproductive endpoints that are intrinsically linked to the site of exposure. As highlighted by Hao et al. ( Hao et al, 2023 ), this matrix enhances the biological plausibility of observed associations. Lastly, the biomarkers were evaluated individually, not accounting for the potential effect of EDC mixtures. Strengths of our study also include its prospective design, which minimizes the possibility of reverse causation, the comprehensive adjustment of possible confounding variables, and the possibility to examine intermediate IVF outcomes given the selected group of women attending a fertility center. In summary and to the best of our knowledge, this is the largest prospective study that measured the presence of several phenols in human ovarian FF and found correlations between some phenol biomarkers in FF and urine. We also identified a decrease in the presence of BPA in the FF and a concurrent increase in BPS which could be explained by the recent temporal trend in using BPA substitutes. While a couple of FF phenol concentrations were associated with intermediate reproductive outcomes, these associations did not translate into any effects on probabilities of clinical pregnancy. Data on the relation between BPA and its substitutes, as well as other phenols, in FF and reproductive outcomes remain scarce and further studies with larger numbers in other study populations are needed to replicate our overall null findings, which should not be interpreted as evidence of safety solely based on the findings of this study. It is crucial for future studies to evaluate the role of EDC mixtures ( İnkaya & Barlas, 2022 , Tian et al. 2023 ) and ultimately attempt to establish biologic plausibility with the goal of evidence-based clinical recommendations for patients.

In recent years, the scientific community has become increasingly interested in the potential health effects of environmental exposures. Chemical substances known as endocrine disrupting chemicals (EDCs) have drawn great attention because of their potential impact on overall long-term health ( Rivera-Núñez et al. 2022 ) and specifically reproductive health ( Toft et al. 2004 , Diamanti-Kandarakis et al. 2009, Minguez-Alarcon & Gaskins 2017 , Hallberg et al. 2023 ). Phenols are a category of non-persistent EDCs found in a wide range of products, including plastics, personal care products, building materials, germicides, and disinfectants. Human exposure to phenols is widespread and can result from ingestion, inhalation or dermal contact. Many published studies have been suggested possible associations of phenols with reproductive outcomes ( Souter et al. 2013 , Smith et al. 2013 , Mínguez-Alarcón et al. 2019, Rattan et al. 2017 ), however the data remains inconsistent ( Minguez-Alarcon & Gaskins 2017 ). In addition, most studies have utilized serum, blood or urinary concentrations of phenols to explore associations with reproductive outcomes. Studies evaluating the presence of EDCs in human ovarian follicular fluid (FF), the liquid which fills the follicular antrum and surrounds the ovum, thus being in immediate contact with the female gametes, are relatively limited ( Ikezuki et al. 2002 , Krotz et al. 2012 , Kim et al. 2019 , Gokyer et al 2024 ). Evaluating EDCs in this matrix could provide a more direct understanding of the associations of these chemicals with oocyte development and thus aid in explaining associations with reproductive outcomes. The initial studies evaluating human ovarian FF concentration of phenols were limited in size and measured only one of the most common phenols, bisphenol-A (BPA). However, as of 2012 the Food and Drug Administration (FDA) implemented restrictions on the use of BPA. This led to the introduction of bisphenol-S (BPS), a BPA analog, as a replacement for BPA. Of note, BPS has limited labeling regulations and thus it is difficult for consumers to identify in products. Nevertheless, BPS has been detected in up to 81% of human urine samples, mostly as a result of thermal receipt exposure (Liao et al. 2012, Liao et al. 2012). BPA alternatives have since been identified in other reproductive matrices such as breast milk ( Nevoral et al. 2021 ). At the time of recruitment and analysis, no studies had evaluated the presence of BPS in FF. Since our study was completed, a few studies have been published confirming the presence of BPS in FF, all of which were limited in size with the largest studying 64 patients ( Amar et al. 2020 , Žalmanová et al. 2023 , Tian et al. 2023 ). In this study, we quantified multiple phenol biomarkers in human ovarian FF of a cohort of women undergoing fertility treatments. We also attempted to determine if the concentration of the phenols detected in the FF was correlated with the urinary concentrations of these phenols and evaluated trends in ovarian FF biomarkers concentrations over time. Lastly, we sought to evaluate potential associations of FF phenol biomarker concentrations with IVF outcomes.