The estradiol-to-periovulatory follicle ratio (E2/N16) as a marker of follicular endocrine efficiency and embryological outcomes in ART cycles

Abstract

Background Controlled ovarian stimulation (COS) in assisted reproductive technology (ART) is traditionally assessed using quantitative parameters, particularly the number of retrieved oocytes. However, these measures may not fully capture the endocrine efficiency of the periovulatory follicular cohort. Objective To evaluate the association between the estradiol-to-periovulatory follicle ratio (E2/N16) and embryo yield, and to investigate its relationship with serum progesterone concentrations and ovarian stimulation characteristics. Methods This retrospective observational cohort study included 2,831 ART cycles performed between January 2022 and February 2025. E2/N16 was calculated by dividing serum estradiol concentration by the number of periovulatory follicles (≥16 mm) on the day of trigger. The primary outcome was viable embryo yield per cycle. E2/N16 was analyzed as a continuous variable and by quartiles. Multivariable models were adjusted for female age, body mass index, anti-Müllerian hormone, number of retrieved oocytes, total FSH dose, and trigger type. Restricted cubic splines with formal tests of non-linearity and sensitivity analyses were also performed. Results Viable embryo yield progressively decreased across increasing E2/N16 quartiles, from 2.09 to 1.77 embryos per cycle (p < 0.001). After multivariable adjustment, including trigger type, higher E2/N16 remained independently associated with lower embryo yield (β = −0.000218; 95% CI, −0.000427 to −0.000010; p = 0.040). The spline showed no statistically significant departure from linearity (P for non-linearity = 0.449), despite a modest initial rise followed by a decline. The association with serum progesterone was significantly non-linear (P for non-linearity = 0.009), with a U-shaped pattern. After exclusion of cycles with two or fewer periovulatory follicles, the association with embryo yield was attenuated but remained directionally consistent. Conclusions The estradiol-to-periovulatory follicle ratio (E2/N16) was independently associated with embryo yield and provided information complementary to conventional quantitative measures of ovarian response. Its relationship with progesterone was non-monotonic, indicating that E2/N16 should not be interpreted as a simple linear marker of ovarian steroidogenesis. Prospective studies are required to validate its clinical utility and define how it might contribute to the assessment of ovarian stimulation in ART.