Abstract Background In anovulatory hormone replacement therapy frozen embryo transfer (HRT-FET) cycles, reproductive success depends entirely on exogenous sex-steroid supplementation. Inadequate progesterone exposure remains a clinically relevant challenge, affecting up to one-third of patients receiving micronized vaginal progesterone (MVP) monotherapy. Combination regimens incorporating oral dydrogesterone (DYD) alongside MVP have been proposed to address this limitation. Critically, the absence of immunoassay cross-reactivity between DYD and progesterone enables simultaneous quantification of both progestins within a single patient – a methodological opportunity not yet exploited in outcome research. Using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS), we investigated the independent and joint associations of DYD, dihydrodydrogesterone (DHD), progesterone (P), and estradiol (E2) with clinical outcomes in a combination-progestin HRT-FET regimen, establishing a novel pharmacodynamic interaction framework for HRT-FET protocol optimization. Methods This nested analysis included 111 women undergoing anovulatory HRT-FET within a prospective multicenter cohort (NCT03507673). Patients received oral estradiol (2 mg tid) followed by MVP (400 mg bid) and DYD (10 mg tid) from days 13–15. Serum and plasma samples collected on the day of FET were analyzed using HPLC–MS/MS for DYD and DHD, and immunoassay for P and E2. Clinical pregnancy and live birth were assessed as primary outcomes. Stratified and interaction analyses were performed to explore combined hormone-level effects. Results Hormone concentrations showed broad interindividual variability with weak inter-analyte correlations (r ≤ 0.33), confirming pharmacodynamic independence of the two progestin pathways. No statistically significant independent association with live birth was observed for any single analyte. However, interaction analyses revealed consistent gradient patterns: live birth rates were highest when both DYD and P concentrations were elevated (67%) and lowest when both were in the lower range (27%). Analogous patterns were observed for DYD–E2 and P–E2 combinations, suggesting additive and substitutive pharmacodynamic interaction effects. Given the hypothesis-generating nature of this study, the sample size is appropriate for the exploratory interaction framework established here. Conclusions This study introduces simultaneous dual-progestin quantification as a methodological platform for pharmacodynamic interaction research in HRT-FET. Exploratory interaction patterns between both progestins and estradiol suggest clinically relevant additive effects, while patterns between the two progestins are compatible with potential substitutive dynamics that can only be evaluated within combination regimens without analytical cross-reactivity. These findings provide a mechanistic framework and generate hypotheses for adequately powered prospective studies investigating joint hormone exposure and reproductive outcomes. Trial registration NCT03507673.
Eggersmann et al. (Mon,) studied this question.