**Background:** The menopausal transition is characterized by declining ovarian follicle count, reduced ovarian volume, and falling levels of anti-Müllerian hormone (AMH) and estradiol (E2). Phthalates are ubiquitous endocrine-disrupting chemicals that target the female reproductive system. Prior work in the Midlife Women's Health Study (MWHS) unexpectedly found positive associations between certain phthalate metabolites and AMH/E2 in midlife women. This study aimed to evaluate whether phthalates are associated with ovarian volume (a proxy for folliculogenesis) and whether ovarian volume mediates the positive phthalate–hormone associations.
**Methods:** The study included 614 women aged 45–54 from the MWHS cohort (Baltimore, MD, 2006–2015). Participants provided up to four urine and blood samples over four consecutive weeks. Nine phthalate metabolites (representing six parent phthalates: MBP, MiBP, MBzP, MCPP, MEP, and ∑DEHP) were quantified from pooled urine using isotope dilution HPLC-MS/MS. Serum AMH was measured from the first visit (LOD 0.2 ng/mL; 54% of women < LOD), and E2 was measured from all visits (LOD 9.714 pg/mL). Mean ovarian volume was calculated from transvaginal ultrasound measurements of both ovaries using the ellipsoid formula (length × height × width × 0.526). Multivariable linear regression (single-pollutant) and weighted quantile sum (WQS) regression (mixture analyses) evaluated associations with ovarian volume, adjusting for age, change in BMI since age 18, race/ethnicity, oral contraceptive use, parity, smoking, alcohol, income, marital status, and CES-D score. Cross-sectional mediation analysis tested whether ovarian volume mediated phthalate–hormone associations.
**Key Results:** Most women were non-Hispanic White (66%), premenopausal (67%), with median age 48 years (IQR: 46–50) and median ovarian volume 4.4 cm³ (IQR: 2.7–6.4). In single-pollutant models, a 10% increase in MCPP was associated with 0.44% larger ovarian volume (95% CI: −0.02%, 0.91%), and a 10% increase in MBzP with 0.62% larger volume (95% CI: 0.02%, 1.23%). In WQS mixture analysis, a 10% increase in all biomarkers was associated with 2.89% larger ovarian volume (95% CI: 0.27%, 5.59%), with MBzP (41% weight) and MCPP (35% weight) as major contributors. For AMH, a 10% increase in MCPP (OR: 1.02; 95% CI: 1.00, 1.03), MBzP (OR: 1.03; 95% CI: 1.01, 1.05), and MBP (OR: 1.04; 95% CI: 1.01, 1.07) was associated with higher odds of detectable AMH. For E2, a 10% increase in ∑DEHP (%Δ: 0.68; 95% CI: 0.04, 1.33), MCPP (%Δ: 0.46; 95% CI: 0.01, 0.91), MBP (%Δ: 0.87; 95% CI: 0.14, 1.60), and MiBP (%Δ: 0.89; 95% CI: 0.17, 1.62) was associated with higher E2. Mediation analysis showed that 16% of the MBzP–AMH association was mediated by ovarian volume (indirect effect OR: 1.004; 95% CI: 1.00, 1.01), and 23% of the MCPP–E2 association was mediated by ovarian volume (indirect effect %Δ: 0.11; 95% CI: −0.01, 0.22).
**Clinical Implications:** These findings suggest that phthalate exposure may be associated with larger ovarian volume and higher AMH/E2 in midlife women, contrary to patterns seen in younger women and animal models. The authors hypothesize that phthalates may accelerate folliculogenesis, rapidly depleting ovarian reserve and potentially contributing to earlier menopause. However, the cross-sectional design precludes causal inference, and the positive direction of associations conflicts with most prior literature. The study highlights a critical gap in understanding environmental exposures during the menopausal transition and underscores the need for prospective studies in this understudied population. Clinically, if confirmed, these findings could have implications for counseling perimenopausal women about environmental exposures and their potential impact on reproductive aging.