Pharmacodynamic Interaction Analysis of Dydrogesterone, Progesterone, and Estradiol in Combination-Progestin HRT Frozen Embryo Transfer: a prospective clinical cohort study

Pharmacodynamic Interaction Analysis of Dydrogesterone, Progesterone, and Estradiol in Combination-Progestin HRT Frozen Embryo Transfer: a prospective clinical cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pharmacodynamic Interaction Analysis of Dydrogesterone, Progesterone, and Estradiol in Combination-Progestin HRT Frozen Embryo Transfer: a prospective clinical cohort study Tanja K. Eggersmann, Noemi Hamala, Alexander R. Hiller, Marion Depenbusch, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8929363/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version 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 number: NCT03507673 frozen-thawed embryo transfer cycle luteal phase support dydrogesterone progesterone progestin Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Frozen-thawed embryo transfer (FET) cycles account for approximately 60% of all embryo transfers worldwide (Ory et al., 2022 ). FET can be performed using ovulatory or anovulatory protocols. In anovulatory protocols—also known as "hormone replacement therapy" (HRT), "programmed," or "artificial" cycles—follicular development, ovulation, and corpus luteum formation are suppressed by estradiol administration, while the implantation window is induced by progestin medication. As a result, implantation and early pregnancy rely entirely on exogenous sex-steroid support, as endogenous hormone production by the ovaries is absent. If pregnancy occurs, hormonal support must continue through the early first trimester to compensate for the lack of endogenous progesterone (Lutjen et al., 1984; Neumann et al., 2022 ). Concerns have been raised regarding anovulatory FET protocols (Magnusson et al., 2024 ; von Versen-Höynck & Griesinger, 2022 ). First, the absence of a corpus luteum has been linked to maternal and fetal risks (Asserhøj et al., 2021 ; Busnelli et al., 2022 ; Conrad et al., 2019 ; Ginström Ernstad et al., 2019 ; Hu et al., 2021 ; Moreno-Sepulveda et al., 2021 ; Roelens et al., 2022; Rosalik et al., 2021 ). Second, serum progesterone (P) levels vary widely depending on the route of administration, with low levels (< 8–10 ng/ml) after standard progestin mono-therapy with vaginal micronized progesterone (MVP) associated with reduced ongoing pregnancy and live birth rates (Cédrin-Durnerin et al., 2019 ; Labarta et al., 2017 , 2021 ; Maignien et al., 2022 ; Melo et al., 2021 ). A recent study confirmed this observation in an HRT-FET protocol using 10 mg oral dydrogesterone (DYD) three times daily ( ter-in-die; abbr.: tid) as a progestin mono-therapy. In this study, women in the lowest DYD quartile had significantly lower ongoing pregnancy rates (-22% absolute difference; 95% CI: -32 to -12, P < 0.0001) (Neumann et al., 2022 ) and a negative synergism of low DYD with low E2 levels was suggested. To optimize HRT protocols, two approaches have been proposed. First, following follicular suppression with estrogen and then MVP monotherapy, serum P levels can be measured in the early luteal phase to identify low-P patients, who then receive an additional progestin as a "rescue" intervention (du Boulet et al., 2022 ; Labarta et al., 2021 ; Mackens et al., 2023 ; Metello et al., 2024 ; Arik Alpcetin et al., 2025 ; Alsbjerg et al., 2023 ). Alternatively, combination protocols—simultaneous administration of two progestins for implantation and luteal support—have been explored in prospective (Vuong et al., 2021 ) and retrospective cohort studies (Vidal et al., 2023 ; Xu et al., 2021 ; Lawrenz et al., 2024 ; Zhu et al., 2023 ) respectively. Herein, we follow-up on our previous HRT-FET study utilizing 10mg oral DYD (tid) mono-therapy study (Neumann et al., 2022 ), now with a combination progestin protocol. The established lack of analytical interference of DYD and P in immunoassay (Neumann et al., 2020 ; Eggersmann et al., 2022 ) allows the simultaneous assessment of blood levels of both drugs and gives the unique opportunity to explore interaction effects, including possible synergism or functional substitution, which may inform future optimization of dosing strategies and compound selection. Materials and Methods Design and setting This prospective, observational, multicentre cohort study was conducted at three university-affiliated centres and one private reproductive medicine centre in Germany between February 2021 and August 2023. The study was embedded within an ongoing prospective platform trial (ClinicalTrials.gov identifier: NCT03507673), initiated on 14 April 2018. The platform trial investigates endocrine dynamics of the luteal phase and early pregnancy (Neumann et al., 2020 ; Neumann et al., 2022 ; Eggersmann et al., 2025 ), as well as the vaginal and endometrial microbiome (Depenbusch et al., 2024 ; Lüth et al., 2022 ), in women undergoing different frozen embryo transfer (FET) regimens. The present analysis represents a predefined observational cohort within the trial infrastructure. Study population and FET regimen The study cohort consists of female infertile patients undergoing a FET cycle following IVF or ICSI who had DYD (10mg per os tid; brand name Duphaston®, Abbott Biologicals B.V., Weesp, Netherlands) and micronized MVP suppositories (400mg per vaginam, two times a day, bis-in-die , abbr.: bid; brand name Cyclogest®, Gedeon Richter Pharma GmbH, Eschborn, Germany) as progestogenic drugs for endometrial transformation and support of early pregnancy. Patient inclusion was performed on day 13–15 of oral estradiol valerate (2mg per os , tid; brand name Progynova®, Jenapharm GmbH & Co. KG, Jena, Germany) intake in a programmed cycle. Exclusion criteria were evidence of ovulation on ultrasound prior to ET (defined as the presence of a follicle ≥ 14mm and/or P ≥ 1.0 ng/ml on endometrial preparation day 13–15. Patients with malformations of the uterus or endometrial abnormalities (on ultrasound or diagnosed by previous hysteroscopy) were also excluded. Serum and plasma samples were collected and stored on the day of the FET (together with vaginal and endometrial swabs for separate analyses on microbial colonisation) ( Fig. 1 ) . FET cycles were conducted after vitrification and warming using an open, manual system (Kitazato vitrification kit VT601, Gynemed, Lensahn, Germany, (Al-Hasani et al., 2007 )). FET was performed on days 3, 4, 5, or 6 of progestin administration, timed to match the embryo’s developmental stage: day 3 of DYD/MVP intake for a day 2 embryo, day 4 of DYD/MVP intake for a day 3 embryo, day 5 of DYD/MVP intake for a day 4 embryo, and day 6 of DYD/MVP intake for a day 5 embryo. Women who achieved pregnancy, continued E2/DYD/MVP intake into late first trimester, with exact timing per centre policy for anovulatory cycles. Sampling and outcomes FET was typically performed in the late morning. Before FET, blood samples (serum and plasma) were collected and stored at -80°C for later measurement of DYD, DHD, P, and E2. A vaginal swab and an aspiration of endometrial fluid were performed immediately before FET for microbiome analysis (separate analysis). A positive pregnancy test (i.e., serum hCG levels above the local center's reference range) 10–14 days after FET indicated implantation. Clinical pregnancy was defined by the presence of a fetal sac with a heartbeat on transvaginal sonography at GW 7 or later. After gestational week 7, pregnancy progression, adverse events, live birth, and child health were prospectively monitored through structured telephone interviews conducted by a study nurse or doctoral student. Pregnancy outcomes included maternal and fetal/neonate morbidity, termination of pregnancy, and fetal malformations assessed by prenatal diagnosis or at birth (separate analyses). Live birth, chosen as the primary outcome of interest for the present analysis, was defined as the delivery of a living infant at or beyond the point of viability. DYD and DHD measurement The concentration of dydrogesterone and dihydrodydrogesterone (DHD) was determined in 50 µL plasma samples using the High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (HPLC/MS/MS) method, which had been previously validated and adjusted to the concentration ranges of 0.050–10.000 ng/mL for DYD and 0.500–100.000 ng/mL for DHD in plasma. Dipotassium ethylenediaminetetraacetic acid (K2EDTA) was used as an anticoagulant. The standard calibration curves covered the ranges of 0.050 to 10.000 ng/mL for DYD and 0.500 to 100.000 ng/mL for DHD. Plasma samples were precipitated with a solution of internal standards dDYD and dDHD (dextrorotatory form of the molecule) in 80% Acetonitrile (ACN). The supernatant was analyzed using by HPLC/MS/MS by company QUINTA-ANALYTICA (Prague, Czech Republic). Estradiol and Progesterone measurement Estradiol in serum was measured using the Alinity i Estradiol assay. Intra-assay coefficient of variation (CV) values range from 2.2% to 7.2% across low, medium, and high control levels. Inter-assay CVs range from 2.6% to 7.7%. The assay's Limit of Detection (LoD), and Limit of Quantitation (LoQ) are 20 pg/mL, and 24 pg/mL, respectively. Progesterone in serum was measured using the ARCHITECT Progesterone assay, demonstrating intra-assay CVs ranging from 1.5% to 5.5%, and inter-assay CVs ≤ 6.2% for low controls, ≤ 2.9% for medium controls, and ≤ 3.9% for high controls. The LoD for progesterone is approximately 0.1 ng/mL. The assay reliably measures up to 40 ng/mL for progesterone and 1000 pg/mL for estradiol (Reliable maximal values). Trial objective and sample size In a previous study, a difference of -22% in the ongoing pregnancy/live birth rate was observed among women undergoing HRT-FET with oral DYD 10 mg (tid) monotherapy, comparing those below and above the 25th percentile of DYD plasma concentrations (odds ratio: 6.09) (Neumann et al., 2022 ). The present trial was designed to investigate a putative optimized HRT-CP-FET protocol, with the primary aim of refuting the presence of a similar difference in women with low DYD levels. With a sample size of 111 participants stratified at the 25th percentile, the power to detect the original effect (odds ratio: 6.09) at a one-sided significance level of 5% in an HRT-CP-FET protocol with DYD and additional MVP administration is approximately 80%. Statistical analysis All analyses were primarily descriptive and exploratory. Descriptive statistics, including mean, standard deviation (SD), confidence interval (CI), or median, percentiles, and range, as well as absolute numbers and relative proportions, were used to characterize patient demographics and outcomes. For hormonal values, empirical distribution functions were fitted to explore value distributions, and concentration data were log-transformed, when applicable, before further analysis. Spearman’s rank-order correlation coefficients were calculated to assess the strength and direction of monotonic associations between individual hormone concentrations. For all hormone values, the patient sample was then divided into below and above the 25th percentile (lower quartile) of concentrations at FET. Implantation, pregnancy rates, and live birth rates were described by the risk difference with 95% confidence intervals between hormone quarters. This was stratified by day of embryo transfer (days 2–3 versus 4–5) for computing P values. Live birth rates were color-coded in a scatter plot by hormone level terciles on the day of FET to explore potential interaction effects. A P-value of < 0.05 was considered statistically significant. All statistical analyses were performed using the software Jamovi (The jamovi project. (2022), Version 2.3.28, Sydney, Australia), R 4.0.3 (R Core Team. (2020), The R Foundation for Statistical Computing, Vienna, Austria) and G*Power 3 (version 3.1.9.7; Faul, F., et al., Behavior Research Methods , 41 , 1149–1160). Results Patient flow and demographics Between February 2021 and August 2023, 728 patients were included across four centres, of whom 111 underwent an embryo transfer in an HRT-CP-FET protocol, with plasma and serum samples collected, and follow-up to live birth. Supplementary Figure 1 shows inclusion and patient flow. Supplementary Table 1 shows patient demographics. In summary, patients were on average 32.4 years old, weighed 78.3 kg, 96.4% were Caucasian, 38.7% labelled regularly cycling, endometrial thickness of 9.21 mm (±1.72) and LH levels of 11.6 IU/L (±8.74) and Progesterone levels of 0.24 ng/ml (±0.20) on day of last monitoring before DYD initiation. FET was performed as single embryo transfer (sFET) in 77.5% of cases and as double embryo transfer (DET) or higher in 22.5% of cases. Clinical pregnancy and live birth rates per woman undergoing FET were 45.0% (95% CI: 36.1% to 54.3%) and 35.1% (95% CI: 26.9% to 44. 4%), respectively. Endocrine profile on day of FET and incidence of ovulation Empirical distributions of DYD, DHD, P, and E2 with lognormal or normal distribution functions overlayed are Supplementary Figure 2 . Hormone analyte levels were not associated with cycle progression ( Figure 2). Of note, 22/111 (19.8%) of patients were below a progesterone level of 8.90 ng/ml on day of FET previously reported as a predictive threshold for ongoing pregnancy or live birth in an HRT-FET protocol with MVP (Melo et al., 2021). DYD and DHD concentration distributions on the 3 rd to 6 th day of intake were consistent with previous findings (Neumann et al, 2022) ( Supplementary Table 2). Progesterone showed virtually no correlation with E2 (r P,E =-0.03), while DYD and E2 were also essentially uncorrelated (r DYD,E2 =0.09). Correlations of P with DYD and DHD were weak (r DYD,P =0.23; r DHD,P =0.33). Treatment outcome by hormonal levels on day of FET Among the 111 patients, 23 underwent embryo transfer on day 2/3 and 88 on day 4/5. The live birth rate was 13.0% (3/23) following day 2/3 transfers and 40.9% (36/88) following day 4/5 transfers (odds ratio 4.56, 95%-confidence interval 1.22 to 25.7, P value 0.014). Figure 3 illustrates non-significant risk differences with confidence intervals for implantation, clinical pregnancy, early and late pregnancy loss, and live birth in FET patients, categorized into subgroups with low vs. normal-high hormone levels by quartiles. Table S3 confirms no statistically significant associations of low vs. normal-high hormone levels by quartiles with these outcomes when stratifying for early (days 2–3) vs. later (days 4–5) FET. Figure 4 displays scatter plots stratified by (log-)terciles of hormone concentrations measured on the day of FET, overlaid with color-coded live birth rates for each hormone pair. The DYD–P plot indicates an additive pattern: live birth probability is lowest when both DYD and P are low (27%) and highest when both are high (67%). Notably, live birth rates are relatively high (62%) even when P is low, but DYD was elevated, suggesting that DYD may partially substitute for low progesterone exposure. A similar additive pattern was observed for DYD–E2 and a suggestion of synergy between P and E2. If higher levels of sex-steroids were associated with improved live birth probability, DYD might be a decisive contributor when hormonal exposures are discordant. Discussion In this prospective observational study of anovulatory HRT-FET cycles supported by a combination of oral dydrogesterone (DYD) and micronized vaginal progesterone (MVP), plasma DYD levels on the day of embryo transfer below 0.71 ng/dl were not much associated with pregnancy or live birth outcomes. Notably, DYD (and its active metabolite DHD) concentrations showed a distribution comparable to a previous 10mg DYD tid mono-therapy HRT-FET study (Neumann et al., 2022 ), in which DYD below 0.71 ng/dl was linked to reduced live birth rates. In contrast, our findings suggest that when DYD is combined with MVP, the predictive value of individual serum hormone levels—whether DYD, progesterone, or estradiol—appears limited. This may imply that routine measurement of only one of these hormones at the time of FET may be unnecessary for guiding clinical decision-making in an HRT-CP-FET protocol. These data thus corroborate previous observations showing that in HRT-CP-FET cycles with DYD and MVP, serum progesterone levels on the day of embryo transfer are not predictive of live birth. Specifically, Lawrenz et al. ( 2024 ) retrospectively analysed 560 HRT-FET cycles with euploid embryo transfer, in which luteal support consisted of 30 mg/day oral DYD and 300 mg/day MVP. Serum progesterone, but not DYD/DHD, was measured on the day of FET, and in patients with levels < 10 ng/ml, MVP dosing was initially increased. However, multivariable logistic regression adjusted for age, BMI, and embryo quality, as well as weighted analyses using inverse probability treatment weights, showed no significant association between serum progesterone and ongoing pregnancy when patients already receive DYD 10mg tid. Our findings not only support this conclusion but also suggest the mechanism of substitution: when progesterone levels are low, DYD levels appear to compensate, with DYD emerging as a potentially decisive contributor when hormonal exposures are discordant. The latter, however, needs confirmation in larger studies. A noteworthy observation in our dataset is the presence of several patients with extremely low serum progesterone levels despite administration of 2×400 mg MVP. As shown in Fig. 4 , five women had values at the lower detection limit of the assay (0.14 ng/mL), suggesting a potential subgroup with markedly impaired vaginal absorption or increased systemic clearance. This finding is concerning, particularly given that MVP monotherapy remains widely used in HRT-FET protocols (Ho et al.,2024). While non-compliance cannot be entirely excluded, it appears unlikely in this setting due to the typically high intrinsic motivation of women undergoing fertility treatment. Furthermore, comparable observations have also been reported in other studies using Cyclogest® at 2×400 mg/day. Based on the reported means and standard deviations—and assuming a normal distribution of serum progesterone—the estimated proportion of women with levels below 1 ng/mL can be estimated as 0.41% in Labarta et al. ( 2024 ; n = 663, mean = 14.5 ng/mL, SD = 5.1), 1.46% in Alsbjerg et al. ( 2023 ; n = 488, mean = 15.4 ng/mL, SD = 6.6), 0.26% in Herencia et al. ( 2023 ; n = 131, mean = 13.6 ng/mL, SD = 4.5), and 1.8% in Baldini et al. ( 2023 ; n = 281, mean = 14.0 ng/mL, SD = 6.2). Similar results can be estimated from other large observational studies using vaginal progesterone formulations such as Utrogestan®, Crinone®, and Endometrin® (summarized in Alsbjerg et al., 2025). Altogether, these findings highlight substantial interindividual variability in systemic progesterone exposure under MVP monotherapy and underscore the potential value of combination progestin strategies in HRT-FET protocols, allowing for substitution between different routes of administration. In our previous prospective study investigating DYD monotherapy in anovulatory HRT-FET cycles, we already observed a kind of interaction between serum E2 and DYD levels on the day of FET: elevated E2 levels appeared to mitigate the adverse association of low DYD with live birth rate, suggesting a substitution effect between these two hormones (depicted in Fig. 5 in Neumann et al., 2022 ). In the present study, with the addition of a second progestin (MVP) to the luteal support regimen, we now hypothesize a synergistic pattern between Progestins and E2. Live birth rates were consistently highest when both hormones of a given pair (DYD-E2, P-E2) were elevated and lowest when both were low. This observation, if further corroborated, has important ramifications for clinical practice and protocol optimization. Several studies have proposed the use of a single progesterone threshold on the day of FET as predictive of clinical pregnancy or live birth—for instance, Labarta et al. ( 2017 ) suggested a cut-off of 9.2 ng/ml, while other thresholds ranging from 8 to 11 ng/ml have been reported in the literature (see Melo et al., 2021 , for summary). However, such an approach may be overly simplistic for identifying patients with suboptimal sex-steroid support. Omitting potential interactions between estrogenic and progestogenic sex steroids assumes that each hormone exerts its effect in isolation, which may not reflect the complex endocrine milieu required for optimal endometrial receptivity and pregnancy maintenance. Single threshold-based assessments may indeed also distort smooth effects, misrepresent additive and substitution effects of different hormones and lead to over- and undertreatment in individual cases. This underscores the need for multivariable and interaction-sensitive models in evaluating hormone adequacy in HRT-FET protocols, particularly because all administered sex steroids in this setting are pharmacologic substitutes for endogenous production and display substantial inter-individual variability. Importantly, conventional logistic regression models—which rely on the logit link function—tend to model multiplicative effects on the odds scale and may obscure additive or substitutive relationships between covariates. When hormonal variables interact in a compensatory manner, such patterns may not be readily apparent in regression coefficients alone. Visual inspection of interaction plots, such as the hormone interaction matrices shown in Fig. 4 , can therefore provide valuable complementary insights into complex biological relationships and guide a more nuanced understanding. A novel and unexpected observation in this study is a potentially additive effect of a DYD and MVP combination, indicating that a higher exposure to both agents may confer a cumulative benefit. While our study was initially designed to explore how one progestin may partially compensate for low levels of the other—implying a substitution effect—an obvious opportunity arises for improving outcomes by optimizing overall progestin exposure, whether through increased dosing, alternative routes of administration, or individualized adjustments. This has obvious and potentially important implications for routine HRT-FET regimens, which have been suspected to carry a higher risk of pregnancy loss after implantation compared to natural cycles. Inadequate progestins exposure may be a contributing factor. Of note, such a phenomenon may not become apparent in pragmatic RCTs measuring mean effects at the population level (Ho et al., 2024 ) as this phenomenon may affect only a (too small) fragment of all women treated. Still, by leveraging the potential synergistic effect of dual-progestin strategies, protocols could be refined to ensure more consistent and potentially optimized exposure across patients. Of note, the sample size of this study is modest, which limits the statistical power for detecting small effect sizes and precludes more complex multivariable modelling adjusting for relevant predictors. This is partly due to the declining clinical use of HRT-FET protocols, which are no longer considered first-line in many centers for patients who can be treated within an ovulatory FET regimen (von Versen-Hoeynck & Griesinger, 2022), thereby restricting the number of eligible patients for prospective inclusion. While it was anticipated that approximately 200 patients would undergo HRT-FET within this prospective study encompassing also ovulatory FET cycles (Figure S1), only 111 observations have become available. Nonetheless, despite the limited sample size, potentially relevant patterns emerged in the descriptive analyses. Importantly, the weak pairwise correlations between DYD, P, and E2 (all rho < 0.3) proved advantageous in this exploratory setting. This independence reduces concerns of multicollinearity and allows for the meaningful visual separation of effects in cross-classification. In turn, this facilitates the interpretation of potential additive or substitution effects between hormone pairs—patterns that might be obscured in more collinear datasets. Hence, while the study is underpowered for more robust inferential testing, the independence of the hormonal variables lends credibility to the exploratory observation of effects and interaction effects. Conclusion In summary, this study confirms that serum progesterone levels on the day of FET are unlikely to predict reproductive outcomes in DYD + MVP-supported HRT-FET cycles, thereby challenging mono-progestin “screen-and-act” strategies based solely on progesterone thresholds. The observed interaction patterns—particularly between estradiol and progestins—indicate that single-hormone cut-off concepts may oversimplify the complex endocrine interplay underlying endometrial receptivity. By enabling simultaneous and analytically independent quantification of two progestins, this study establishes dual-progestin measurement as a novel methodological platform for pharmacodynamic interaction research in HRT-FET. The data suggest clinically relevant additive effects between progestins and estradiol, as well as potential substitutive dynamics between dydrogesterone and progesterone—phenomena that can only be meaningfully evaluated in combination regimens without analytical cross-reactivity. Together, these findings provide a mechanistic framework and generate hypotheses for adequately powered prospective studies investigating joint hormone exposure rather than isolated single-hormone thresholds in artificial FET protocols. Abbreviations ACN – Acetonitrile AI – Artificial Intelligence AMH – Anti-Müllerian Hormone bid – Bis in die (twice daily) BMI – Body Mass Index CI – Confidence Interval COCs – Cumulus–Oocyte Complexes CP – Combination Progestin CRF – Case Report Form CV – Coefficient of Variation DET – Double Embryo Transfer DHD – 20α-Dihydrodydrogesterone DOI – Digital Object Identifier DYD – Dydrogesterone E2 – Estradiol ELISA – Enzyme-Linked Immunosorbent Assay EMT – Endometrial Thickness ET – Embryo Transfer FET – Frozen-Thawed Embryo Transfer GnRH – Gonadotropin-Releasing Hormone hCG – Human Chorionic Gonadotropin HPLC-MS/MS – High-Performance Liquid Chromatography Tandem Mass Spectrometry HRT – Hormone Replacement Therapy HRT-CP-FET – Hormone Replacement Therapy Combination-Progestin Frozen Embryo Transfer ICSI – Intracytoplasmic Sperm Injection IFFS – International Federation of Fertility Societies IVF – In Vitro Fertilization IU – International Units K2EDTA – Dipotassium Ethylenediaminetetraacetic Acid LH – Luteinizing Hormone LoD – Limit of Detection LoQ – Limit of Quantitation MVP – Micronized Vaginal Progesterone ng/mL – Nanograms per Milliliter P – Progesterone pg/mL – Picograms per Milliliter PRISMA – Preferred Reporting Items for Systematic Reviews and Meta-Analyses RCT – Randomized Controlled Trial RD – Risk Difference REI – Reproductive Endocrinology and Infertility rho (r) – Spearman’s Rank Correlation Coefficient sFET – Single Frozen Embryo Transfer SET – Single Embryo Transfer SD – Standard Deviation tid – Ter in die (three times daily) VBL – Versorgungsanstalt des Bundes und der Länder WHO – World Health Organization Declarations Ethical approval: Institutional review board approval was granted (centers Luebeck & Kiel reference number 18-005; center Duesseldorf reference number 2022-1953; center Saarbruecken reference number 206/21) in accordance with the Declaration of Helsinki. Consent for publication: All patients provided written informed consent. Data availability: The analysis dataset and statistical code underlying this study are currently being curated and will be made publicly available on Zenodo and GitHub under the dataset title “HRT_CP_FET _Luebeck.” A DOI-linked Zenodo repository will be established to ensure long-term accessibility and transparency. In the meantime, the data are available from the corresponding author upon reasonable request. Competing interests: T.K.E. discloses honoraria from Ferring; travel support from Merck, Ferring, Theramex and Gedeon-Richter; and receipt of equipment/materials (to institution) from Arthrex and Besins Healthcare, outside the submitted work. N.H. has received travel support from Gedeon-Richter, Ferring and Merck, outside the submitted work. A.R.H. has received honoraria from Organon and travel support from Merck Serono, Gedeon Richter and Theramex, outside the submitted work. M.D. discloses support from Merck, outside the submitted work. P.E. discloses honoraria from Ferring, Theramex and Gedeon Richter; and travel support from Merck, Ferring, Theramex, Gedeon Richter and MSD, outside the submitted work. A.P.B discloses honoraria and travel support from Merck, Theramex, Gedeon Richter and Ferring; and participation on a data safety monitoring board or advisory board for Ferring and Merck, outside the submitted work. A.S-M, D.B-B., J.S.K, S.v O., W.J., S.T., R.V. declare no conflict of interest. G.G. discloses consulting fees and honoraria from Merck, Organon, Ferring, Theramex, Gedeon-Richter, Abbott, Biosilu, ReprodWissen, Obseva, PregLem, Guerbet, Cooper, Igyxos, OxoLife, and ReproNovo, outside the submitted work, and travel support from Merck, Organon, Ferring, Theramex, Gedeon-Richter, and Abbott, outside the submitted work. There are no conflicts relating directly to the submitted work. Funding: The trial was funded through institutional resources of the University Hospital of Schleswig-Holstein, Campus Lübeck. Expenses related to plasma and serum sample handling, storage, shipment, and the HPLC/MS/MS and ELISA analyses of DYD, DHD, estradiol, and progesterone were financially supported by Abbott Products Operations AG (Allschwil, Switzerland). The funding was provided in the form of a research grant to the Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital of Schleswig-Holstein, Campus Lübeck (no grant number applicable). Abbott Products Operations AG had no role in the study design, conduct, data collection, statistical analysis, data interpretation, manuscript preparation, or the decision to submit the article for publication. Authors’ contributions: Eggersmann T.K. : Conceptualization, Methodology, Study initiation, Conduct, Supervision, Data analysis, Data interpretation, Writing - review & editing; Hamala N. : Data acquisition from CRFs and patient files, Data analysis, Data interpretation, Writing - review & editing; Hiller A.R., Depenbusch M., Schultze-Mosgau A., Edimiris P., Baston-Büst D., Bielfeld A.P., Kruessel J-S., von Otte S., Junkers W., Tauchert S. : Investigation, Writing - review & editing; Vonthein R. : Data analysis, Data interpretation, Writing - original draft, Writing - review & editing; Griesinger G. : Study design, Conceptualization, Study initiation, Supervision, Data analysis, Data interpretation, Writing - original draft, Writing - review & editing. Acknowledgements: We sincerely thank Mrs. Andrea Knaak, study nurse at the Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital of Schleswig-Holstein, for her exceptional dedication in managing documentation, data entry, sample handling, and follow-up during pregnancy and neonatal period. References Ory S, Miller K, Horton M, et al. editors. International Federation of Fertility Societies’ Surveillance (IFFS) 2022: Global Trends in Reproductive Policy and Practice, 9th Edition. Glob Reprod Health. 2022;7(3):e58. 10.1097/GRH.0000000000000058 Lutjen P, Trounson A, Leeton J, Findlay J, Wood C, Renou P. The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature. 1984 Jan 12–18;307(5947):174-5. 10.1038/307174a0 . PMID: 6690997. Neumann K, Masuch A, Vonthein R, Depenbusch M, Schultze-Mosgau A, Eggersmann TK, Griesinger G. 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PMID: 36930501. Alsbjerg B, Humaidan P. What to expect from a 'standard vaginal progesterone regimen' in hormone replacement therapy frozen embryo transfer (HRT-FET) - a PRISMA review and meta-analysis. Reprod Biomed Online. 2025;50(5):104736. 10.1016/j.rbmo.2024.104736 . Epub 2024 Nov 26. PMID: 40132. Additional Declarations Competing interest reported. T.K.E. discloses honoraria from Ferring; travel support from Merck, Ferring, Theramex and Gedeon-Richter; and receipt of equipment/materials (to institution) from Arthrex and Besins Healthcare, outside the submitted work. N.H. has received travel support from Gedeon-Richter, Ferring and Merck, outside the submitted work. A.R.H. has received honoraria from Organon and travel support from Merck Serono, Gedeon Richter and Theramex, outside the submitted work. M.D. discloses support from Merck, outside the submitted work. P.E. discloses honoraria from Ferring, Theramex and Gedeon Richter; and travel support from Merck, Ferring, Theramex, Gedeon Richter and MSD, outside the submitted work. A.P.B discloses honoraria and travel support from Merck, Theramex, Gedeon Richter and Ferring; and participation on a data safety monitoring board or advisory board for Ferring and Merck, outside the submitted work. A.S-M, D.B-B., J.S.K, S.v O., W.J., S.T., R.V. declare no conflict of interest. G.G. discloses consulting fees and honoraria from Merck, Organon, Ferring, Theramex, Gedeon-Richter, Abbott, Biosilu, ReprodWissen, Obseva, PregLem, Guerbet, Cooper, Igyxos, OxoLife, and ReproNovo, outside the submitted work, and travel support from Merck, Organon, Ferring, Theramex, Gedeon-Richter, and Abbott, outside the submitted work. There are no conflicts relating directly to the submitted work. 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Hiller","email":"","orcid":"","institution":"Department of Reproductive Medicine and Gynecological Endocrinology, University Hospital of Schleswig-Holstein, Campus Luebeck, and Universitaeres Kinderwunschzentrum Luebeck und Manhagen","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"R.","lastName":"Hiller","suffix":""},{"id":600110961,"identity":"df0fda02-8b6a-4193-8399-d69a401a529f","order_by":3,"name":"Marion Depenbusch","email":"","orcid":"","institution":"Department of Reproductive Medicine and Gynecological Endocrinology, University Hospital of Schleswig-Holstein, Campus Luebeck, and Universitaeres Kinderwunschzentrum Luebeck und Manhagen","correspondingAuthor":false,"prefix":"","firstName":"Marion","middleName":"","lastName":"Depenbusch","suffix":""},{"id":600110962,"identity":"443c9b57-3c47-40f7-bc3f-550bbcd61d55","order_by":4,"name":"Askan Schultze-Mosgau","email":"","orcid":"","institution":"Department of Reproductive Medicine and Gynecological Endocrinology, University Hospital of Schleswig-Holstein, Campus Luebeck, and Universitaeres Kinderwunschzentrum Luebeck und Manhagen","correspondingAuthor":false,"prefix":"","firstName":"Askan","middleName":"","lastName":"Schultze-Mosgau","suffix":""},{"id":600110963,"identity":"8e800ecb-71ab-4100-94dc-e99e7499ae9c","order_by":5,"name":"Philippos Edimiris","email":"","orcid":"","institution":"Department of Obstetrics, Gynecology and REI (UniKiD), Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf","correspondingAuthor":false,"prefix":"","firstName":"Philippos","middleName":"","lastName":"Edimiris","suffix":""},{"id":600110964,"identity":"bd7bea47-0759-4cfd-822a-b8bd61f9ab3a","order_by":6,"name":"Dunja Baston-Buest","email":"","orcid":"","institution":"Department of Obstetrics, Gynecology and REI (UniKiD), Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf","correspondingAuthor":false,"prefix":"","firstName":"Dunja","middleName":"","lastName":"Baston-Buest","suffix":""},{"id":600110965,"identity":"f8e1db1b-57fa-4b08-8ca7-d66d366b3477","order_by":7,"name":"Alexandra P. Bielfeld","email":"","orcid":"","institution":"Department of Obstetrics, Gynecology and REI (UniKiD), Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"P.","lastName":"Bielfeld","suffix":""},{"id":600110966,"identity":"a5474d67-da09-464c-8c4d-f6fc9c41e891","order_by":8,"name":"Jan-Steffen Kruessel","email":"","orcid":"","institution":"Department of Obstetrics, Gynecology and REI (UniKiD), Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf","correspondingAuthor":false,"prefix":"","firstName":"Jan-Steffen","middleName":"","lastName":"Kruessel","suffix":""},{"id":600110967,"identity":"b8e62225-d770-4602-a149-4be8898d90d9","order_by":9,"name":"Sören von Otte","email":"","orcid":"","institution":"Department of Reproductive Medicine and Gynecological Endocrinology, University Hospital of Schleswig-Holstein, Campus Kiel, and Universitaeres Kinderwunschzentrum Kiel","correspondingAuthor":false,"prefix":"","firstName":"Sören","middleName":"","lastName":"von Otte","suffix":""},{"id":600110968,"identity":"b12dba15-2c58-47c5-ba72-f29114519e0e","order_by":10,"name":"Wiebe Junkers","email":"","orcid":"","institution":"Department of Reproductive Medicine and Gynecological Endocrinology, University Hospital of Schleswig-Holstein, Campus Kiel, and Universitaeres Kinderwunschzentrum Kiel","correspondingAuthor":false,"prefix":"","firstName":"Wiebe","middleName":"","lastName":"Junkers","suffix":""},{"id":600110969,"identity":"8249527a-ff12-4ba4-bf07-39d2472ebf95","order_by":11,"name":"Sascha Tauchert","email":"","orcid":"","institution":"Center for Reproductive Medicine IVF SAAR","correspondingAuthor":false,"prefix":"","firstName":"Sascha","middleName":"","lastName":"Tauchert","suffix":""},{"id":600110970,"identity":"42bb4015-45be-4333-be09-114e79b04e42","order_by":12,"name":"Reinhard Vonthein","email":"","orcid":"","institution":"Institute of Medical Biometry and Statistics, University of Luebeck and University Hospital of Schleswig-Holstein","correspondingAuthor":false,"prefix":"","firstName":"Reinhard","middleName":"","lastName":"Vonthein","suffix":""},{"id":600110971,"identity":"a01a3741-c62a-41ee-913e-efbcf8b29f72","order_by":13,"name":"Georg Griesinger","email":"data:image/png;base64,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","orcid":"","institution":"Department of Reproductive Medicine and Gynecological Endocrinology, University Hospital of Schleswig-Holstein, Campus Luebeck, and Universitaeres Kinderwunschzentrum Luebeck und Manhagen","correspondingAuthor":true,"prefix":"","firstName":"Georg","middleName":"","lastName":"Griesinger","suffix":""}],"badges":[],"createdAt":"2026-02-20 22:23:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8929363/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8929363/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104403432,"identity":"469538cf-65f2-4fd1-b577-a24041fd5203","added_by":"auto","created_at":"2026-03-11 12:18:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":69086,"visible":true,"origin":"","legend":"\u003cp\u003eHRT-CP-FET treatment protocol (abbr.: FET, frozen-thawed embryo transfer; EMT, endometrial thickness; HPLC/MS/MS, high performance liquid chromatography/tandem mass spectroscopy; ELISA, enzyme-linked-immunosorbent-assay).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8929363/v1/39df85faf60d3df39c9d2a98.png"},{"id":104175880,"identity":"1ae51840-832e-4d76-8b29-768e445667a9","added_by":"auto","created_at":"2026-03-08 16:33:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26634,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots of hormone values by day of FET (i.e. by increasing duration of DYD intake and thus cycle progression). (n=2 for DYD, n=6 for DHD, n=1 for P and n=4 for E2 have been censored at minimum or maximum reliable value; abbr.: P = progesterone; E2 = estradiol; DYD = dydrogesterone; DHD = 20α-dihydrodydrogesterone).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8929363/v1/2ad3e5e4d8b5f53282e219e6.png"},{"id":104175884,"identity":"da80c5e7-a7f0-417b-9f1c-295686c9798c","added_by":"auto","created_at":"2026-03-08 16:33:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64088,"visible":true,"origin":"","legend":"\u003cp\u003eForest plots with risk differences with confidence intervals for treatment outcome from implantation to live birth calculated per patient undergoing FET, in subgroups of low (≤25th percentile) versus normal-high (\u0026gt;25th percentile) hormone levels on day of FET.\u003c/p\u003e\n\u003cp\u003e(abbr.: A, DYD = dydrogesterone; B, DHD = 20α-dihydrodydrogesterone; C, Progesterone = progesterone; D, E2 = estradiol).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8929363/v1/32df18c6ca13600c9f75631b.png"},{"id":104175882,"identity":"1ca639dd-b7c3-4cac-9005-5cee92d88cee","added_by":"auto","created_at":"2026-03-08 16:33:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52248,"visible":true,"origin":"","legend":"\u003cp\u003eLive birth rates stratified by hormone concentration tertiles on the day of FET.\u003cbr\u003e\nScatterplots depict combinations (A-C) of dydrogesterone (DYD), progesterone, and estradiol (E2) levels measured on the day of frozen embryo transfer (FET). DYD concentrations were log-transformed. Each panel shows the observed live birth rates (blue numbers, in %) within the nine resulting strata. Filled green dots represent individual patients achieving live birth.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8929363/v1/dd61c9c35d10a72376adda56.png"},{"id":104408768,"identity":"c09a5274-28b5-4abc-b979-d3f3c783dcc7","added_by":"auto","created_at":"2026-03-11 12:43:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1058995,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8929363/v1/d595e9d3-c8f1-4506-8f47-973e4198153b.pdf"},{"id":104175881,"identity":"c91acc37-374a-45b9-bf4f-18758be4b2af","added_by":"auto","created_at":"2026-03-08 16:33:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":366856,"visible":true,"origin":"","legend":"","description":"","filename":"FigSTableS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8929363/v1/0c697b0e735297c28b81522c.docx"}],"financialInterests":"Competing interest reported. T.K.E. discloses honoraria from Ferring; travel support from Merck, Ferring, Theramex and Gedeon-Richter; and receipt of equipment/materials (to institution) from Arthrex and Besins Healthcare, outside the submitted work.\nN.H. has received travel support from Gedeon-Richter, Ferring and Merck, outside the submitted work.\nA.R.H. has received honoraria from Organon and travel support from Merck Serono, Gedeon Richter and Theramex, outside the submitted work.\nM.D. discloses support from Merck, outside the submitted work.\nP.E. discloses honoraria from Ferring, Theramex and Gedeon Richter; and travel support from Merck, Ferring, Theramex, Gedeon Richter and MSD, outside the submitted work.\nA.P.B discloses honoraria and travel support from Merck, Theramex, Gedeon Richter and Ferring; and participation on a data safety monitoring board or advisory board for Ferring and Merck, outside the submitted work.\nA.S-M, D.B-B., J.S.K, S.v O., W.J., S.T., R.V. declare no conflict of interest.\nG.G. discloses consulting fees and honoraria from Merck, Organon, Ferring, Theramex, Gedeon-Richter, Abbott, Biosilu, ReprodWissen, Obseva, PregLem, Guerbet, Cooper, Igyxos, OxoLife, and ReproNovo, outside the submitted work, and travel support from Merck, Organon, Ferring, Theramex, Gedeon-Richter, and Abbott, outside the submitted work.\nThere are no conflicts relating directly to the submitted work.","formattedTitle":"\u003cp\u003ePharmacodynamic Interaction Analysis of Dydrogesterone, Progesterone, and Estradiol in Combination-Progestin HRT Frozen Embryo Transfer: a prospective clinical cohort study\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eFrozen-thawed embryo transfer (FET) cycles account for approximately 60% of all embryo transfers worldwide (Ory et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). FET can be performed using ovulatory or anovulatory protocols. In anovulatory protocols\u0026mdash;also known as \"hormone replacement therapy\" (HRT), \"programmed,\" or \"artificial\" cycles\u0026mdash;follicular development, ovulation, and corpus luteum formation are suppressed by estradiol administration, while the implantation window is induced by progestin medication. As a result, implantation and early pregnancy rely entirely on exogenous sex-steroid support, as endogenous hormone production by the ovaries is absent. If pregnancy occurs, hormonal support must continue through the early first trimester to compensate for the lack of endogenous progesterone (Lutjen et al., 1984; Neumann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Concerns have been raised regarding anovulatory FET protocols (Magnusson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; von Versen-H\u0026ouml;ynck \u0026amp; Griesinger, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). First, the absence of a corpus luteum has been linked to maternal and fetal risks (Asserh\u0026oslash;j et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Busnelli et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Conrad et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ginstr\u0026ouml;m Ernstad et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Moreno-Sepulveda et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Roelens et al., 2022; Rosalik et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Second, serum progesterone (P) levels vary widely depending on the route of administration, with low levels (\u0026lt;\u0026thinsp;8\u0026ndash;10 ng/ml) after standard progestin mono-therapy with vaginal micronized progesterone (MVP) associated with reduced ongoing pregnancy and live birth rates (C\u0026eacute;drin-Durnerin et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Labarta et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Maignien et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Melo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A recent study confirmed this observation in an HRT-FET protocol using 10 mg oral dydrogesterone (DYD) three times daily (\u003cem\u003eter-in-die;\u003c/em\u003e abbr.: tid) as a progestin mono-therapy. In this study, women in the lowest DYD quartile had significantly lower ongoing pregnancy rates (-22% absolute difference; 95% CI: -32 to -12, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Neumann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and a negative synergism of low DYD with low E2 levels was suggested.\u003c/p\u003e \u003cp\u003eTo optimize HRT protocols, two approaches have been proposed. First, following follicular suppression with estrogen and then MVP monotherapy, serum P levels can be measured in the early luteal phase to identify low-P patients, who then receive an additional progestin as a \"rescue\" intervention (du Boulet et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Labarta et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mackens et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Metello et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Arik Alpcetin et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Alsbjerg et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Alternatively, combination protocols\u0026mdash;simultaneous administration of two progestins for implantation and luteal support\u0026mdash;have been explored in prospective (Vuong et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and retrospective cohort studies (Vidal et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lawrenz et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) respectively.\u003c/p\u003e \u003cp\u003eHerein, we follow-up on our previous HRT-FET study utilizing 10mg oral DYD (tid) mono-therapy study (Neumann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), now with a combination progestin protocol. The established lack of analytical interference of DYD and P in immunoassay (Neumann et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Eggersmann et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) allows the simultaneous assessment of blood levels of both drugs and gives the unique opportunity to explore interaction effects, including possible synergism or functional substitution, which may inform future optimization of dosing strategies and compound selection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDesign and setting\u003c/h2\u003e \u003cp\u003eThis prospective, observational, multicentre cohort study was conducted at three university-affiliated centres and one private reproductive medicine centre in Germany between February 2021 and August 2023. The study was embedded within an ongoing prospective platform trial (ClinicalTrials.gov identifier: NCT03507673), initiated on 14 April 2018. The platform trial investigates endocrine dynamics of the luteal phase and early pregnancy (Neumann et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Neumann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Eggersmann et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), as well as the vaginal and endometrial microbiome (Depenbusch et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; L\u0026uuml;th et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in women undergoing different frozen embryo transfer (FET) regimens. The present analysis represents a predefined observational cohort within the trial infrastructure.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy population and FET regimen\u003c/h3\u003e\n\u003cp\u003eThe study cohort consists of female infertile patients undergoing a FET cycle following IVF or ICSI who had DYD (10mg \u003cem\u003eper os\u003c/em\u003e tid; brand name Duphaston\u0026reg;, Abbott Biologicals B.V., Weesp, Netherlands) and micronized MVP suppositories (400mg per vaginam, two times a day, \u003cem\u003ebis-in-die\u003c/em\u003e, abbr.: bid; brand name Cyclogest\u0026reg;, Gedeon Richter Pharma GmbH, Eschborn, Germany) as progestogenic drugs for endometrial transformation and support of early pregnancy. Patient inclusion was performed on day 13\u0026ndash;15 of oral estradiol valerate (2mg \u003cem\u003eper os\u003c/em\u003e, tid; brand name Progynova\u0026reg;, Jenapharm GmbH \u0026amp; Co. KG, Jena, Germany) intake in a programmed cycle. Exclusion criteria were evidence of ovulation on ultrasound prior to ET (defined as the presence of a follicle \u0026ge;\u0026thinsp;14mm and/or P\u0026thinsp;\u0026ge;\u0026thinsp;1.0 ng/ml on endometrial preparation day 13\u0026ndash;15. Patients with malformations of the uterus or endometrial abnormalities (on ultrasound or diagnosed by previous hysteroscopy) were also excluded. Serum and plasma samples were collected and stored on the day of the FET (together with vaginal and endometrial swabs for separate analyses on microbial colonisation) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. FET cycles were conducted after vitrification and warming using an open, manual system (Kitazato vitrification kit VT601, Gynemed, Lensahn, Germany, (Al-Hasani et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e)). FET was performed on days 3, 4, 5, or 6 of progestin administration, timed to match the embryo\u0026rsquo;s developmental stage: day 3 of DYD/MVP intake for a day 2 embryo, day 4 of DYD/MVP intake for a day 3 embryo, day 5 of DYD/MVP intake for a day 4 embryo, and day 6 of DYD/MVP intake for a day 5 embryo. Women who achieved pregnancy, continued E2/DYD/MVP intake into late first trimester, with exact timing per centre policy for anovulatory cycles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSampling and outcomes\u003c/h3\u003e\n\u003cp\u003eFET was typically performed in the late morning. Before FET, blood samples (serum and plasma) were collected and stored at -80\u0026deg;C for later measurement of DYD, DHD, P, and E2. A vaginal swab and an aspiration of endometrial fluid were performed immediately before FET for microbiome analysis (separate analysis). A positive pregnancy test (i.e., serum hCG levels above the local center's reference range) 10\u0026ndash;14 days after FET indicated implantation. Clinical pregnancy was defined by the presence of a fetal sac with a heartbeat on transvaginal sonography at GW 7 or later. After gestational week 7, pregnancy progression, adverse events, live birth, and child health were prospectively monitored through structured telephone interviews conducted by a study nurse or doctoral student. Pregnancy outcomes included maternal and fetal/neonate morbidity, termination of pregnancy, and fetal malformations assessed by prenatal diagnosis or at birth (separate analyses). Live birth, chosen as the primary outcome of interest for the present analysis, was defined as the delivery of a living infant at or beyond the point of viability.\u003c/p\u003e\n\u003ch3\u003eDYD and DHD measurement\u003c/h3\u003e\n\u003cp\u003eThe concentration of dydrogesterone and dihydrodydrogesterone (DHD) was determined in 50 \u0026micro;L plasma samples using the High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (HPLC/MS/MS) method, which had been previously validated and adjusted to the concentration ranges of 0.050\u0026ndash;10.000 ng/mL for DYD and 0.500\u0026ndash;100.000 ng/mL for DHD in plasma. Dipotassium ethylenediaminetetraacetic acid (K2EDTA) was used as an anticoagulant. The standard calibration curves covered the ranges of 0.050 to 10.000 ng/mL for DYD and 0.500 to 100.000 ng/mL for DHD. Plasma samples were precipitated with a solution of internal standards dDYD and dDHD (dextrorotatory form of the molecule) in 80% Acetonitrile (ACN). The supernatant was analyzed using by HPLC/MS/MS by company QUINTA-ANALYTICA (Prague, Czech Republic).\u003c/p\u003e\n\u003ch3\u003eEstradiol and Progesterone measurement\u003c/h3\u003e\n\u003cp\u003eEstradiol in serum was measured using the Alinity i Estradiol assay. Intra-assay coefficient of variation (CV) values range from 2.2% to 7.2% across low, medium, and high control levels. Inter-assay CVs range from 2.6% to 7.7%. The assay's Limit of Detection (LoD), and Limit of Quantitation (LoQ) are 20 pg/mL, and 24 pg/mL, respectively. Progesterone in serum was measured using the ARCHITECT Progesterone assay, demonstrating intra-assay CVs ranging from 1.5% to 5.5%, and inter-assay CVs\u0026thinsp;\u0026le;\u0026thinsp;6.2% for low controls, \u0026le; 2.9% for medium controls, and \u0026le;\u0026thinsp;3.9% for high controls. The LoD for progesterone is approximately 0.1 ng/mL. The assay reliably measures up to 40 ng/mL for progesterone and 1000 pg/mL for estradiol (Reliable maximal values).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTrial objective and sample size\u003c/h2\u003e \u003cp\u003eIn a previous study, a difference of -22% in the ongoing pregnancy/live birth rate was observed among women undergoing HRT-FET with oral DYD 10 mg (tid) monotherapy, comparing those below and above the 25th percentile of DYD plasma concentrations (odds ratio: 6.09) (Neumann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The present trial was designed to investigate a putative optimized HRT-CP-FET protocol, with the primary aim of refuting the presence of a similar difference in women with low DYD levels. With a sample size of 111 participants stratified at the 25th percentile, the power to detect the original effect (odds ratio: 6.09) at a one-sided significance level of 5% in an HRT-CP-FET protocol with DYD and additional MVP administration is approximately 80%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were primarily descriptive and exploratory. Descriptive statistics, including mean, standard deviation (SD), confidence interval (CI), or median, percentiles, and range, as well as absolute numbers and relative proportions, were used to characterize patient demographics and outcomes. For hormonal values, empirical distribution functions were fitted to explore value distributions, and concentration data were log-transformed, when applicable, before further analysis. Spearman\u0026rsquo;s rank-order correlation coefficients were calculated to assess the strength and direction of monotonic associations between individual hormone concentrations. For all hormone values, the patient sample was then divided into below and above the 25th percentile (lower quartile) of concentrations at FET. Implantation, pregnancy rates, and live birth rates were described by the risk difference with 95% confidence intervals between hormone quarters. This was stratified by day of embryo transfer (days 2\u0026ndash;3 versus 4\u0026ndash;5) for computing P values. Live birth rates were color-coded in a scatter plot by hormone level terciles on the day of FET to explore potential interaction effects. A P-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were performed using the software Jamovi (The jamovi project. (2022), Version 2.3.28, Sydney, Australia), R 4.0.3 (R Core Team. (2020), The R Foundation for Statistical Computing, Vienna, Austria) and G*Power 3 (version 3.1.9.7; Faul, F., et al., \u003cem\u003eBehavior Research Methods\u003c/em\u003e, \u003cem\u003e41\u003c/em\u003e, 1149\u0026ndash;1160).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatient flow and demographics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetween February 2021 and August 2023, 728 patients were included across four centres, of whom 111 underwent an embryo transfer in an HRT-CP-FET protocol, with plasma and serum samples collected, and follow-up to live birth. \u003cstrong\u003e\u003cem\u003eSupplementary Figure 1\u003c/em\u003e\u003c/strong\u003e shows inclusion and patient flow. \u003cstrong\u003e\u003cem\u003eSupplementary Table 1\u003c/em\u003e\u003c/strong\u003e shows patient demographics. In summary, patients were on average 32.4 years old, weighed 78.3 kg, 96.4% were Caucasian, 38.7% labelled regularly cycling, endometrial thickness of 9.21 mm (±1.72) and LH levels of 11.6 IU/L (±8.74) and Progesterone levels of 0.24 ng/ml (±0.20) on day of last monitoring before DYD initiation. FET was performed as single embryo transfer (sFET) in 77.5% of cases and as double embryo transfer (DET) or higher in 22.5% of cases. Clinical pregnancy and live birth rates per woman undergoing FET were 45.0% (95% CI: 36.1% to 54.3%) and 35.1% (95% CI: 26.9% to 44. 4%), respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndocrine profile on day of FET and incidence of ovulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmpirical distributions of DYD, DHD, P, and E2 with lognormal or normal distribution functions overlayed are \u003cstrong\u003e\u003cem\u003eSupplementary\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eFigure 2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e Hormone analyte levels were not associated with cycle progression (\u003cstrong\u003e\u003cem\u003eFigure 2).\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eOf note, 22/111 (19.8%) of patients were below a progesterone level of 8.90 ng/ml on day of FET previously reported as a predictive threshold for ongoing pregnancy or live birth in an HRT-FET protocol with MVP (Melo et al., 2021). DYD and DHD concentration distributions on the 3\u003csup\u003erd\u003c/sup\u003e to 6\u003csup\u003eth\u003c/sup\u003e day of intake were consistent with previous findings (Neumann et al, 2022) (\u003cstrong\u003e\u003cem\u003eSupplementary Table 2).\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eProgesterone showed virtually no correlation with E2 (r\u003csub\u003eP,E\u003c/sub\u003e=-0.03), while DYD and E2 were also essentially uncorrelated (r\u003csub\u003eDYD,E2\u003c/sub\u003e=0.09). Correlations of P with DYD and DHD were weak (r\u003csub\u003eDYD,P\u003c/sub\u003e=0.23; r\u003csub\u003eDHD,P\u003c/sub\u003e=0.33).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment outcome by hormonal levels on day of FET\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 111 patients, 23 underwent embryo transfer on day 2/3 and 88 on day 4/5. The live birth rate was 13.0% (3/23) following day 2/3 transfers and 40.9% (36/88) following day 4/5 transfers (odds ratio 4.56, 95%-confidence interval 1.22 to 25.7, P value 0.014). \u003cstrong\u003e\u003cem\u003eFigure 3\u003c/em\u003e\u003c/strong\u003e illustrates non-significant risk differences with confidence intervals for implantation, clinical pregnancy, early and late pregnancy loss, and live birth in FET patients, categorized into subgroups with low vs. normal-high hormone levels by quartiles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable S3\u003c/em\u003e\u003c/strong\u003e confirms no statistically significant associations of low vs. normal-high hormone levels by quartiles with these outcomes when stratifying for early (days 2–3) vs. later (days 4–5) FET.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFigure 4\u003c/em\u003e\u003c/strong\u003e displays scatter plots stratified by (log-)terciles of hormone concentrations measured on the day of FET, overlaid with color-coded live birth rates for each hormone pair. The DYD–P plot indicates an additive pattern: live birth probability is lowest when both DYD and P are low (27%) and highest when both are high (67%). Notably, live birth rates are relatively high (62%) even when P is low, but DYD was elevated, suggesting that DYD may partially substitute for low progesterone exposure. A similar additive pattern was observed for DYD–E2 and a suggestion of synergy between P and E2. If higher levels of sex-steroids were associated with improved live birth probability, DYD might be a decisive contributor when hormonal exposures are discordant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this prospective observational study of anovulatory HRT-FET cycles supported by a combination of oral dydrogesterone (DYD) and micronized vaginal progesterone (MVP), plasma DYD levels on the day of embryo transfer below 0.71 ng/dl were not much associated with pregnancy or live birth outcomes. Notably, DYD (and its active metabolite DHD) concentrations showed a distribution comparable to a previous 10mg DYD tid mono-therapy HRT-FET study (Neumann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in which DYD below 0.71 ng/dl was linked to reduced live birth rates. In contrast, our findings suggest that when DYD is combined with MVP, the predictive value of individual serum hormone levels\u0026mdash;whether DYD, progesterone, or estradiol\u0026mdash;appears limited. This may imply that routine measurement of only one of these hormones at the time of FET may be unnecessary for guiding clinical decision-making in an HRT-CP-FET protocol.\u003c/p\u003e \u003cp\u003eThese data thus corroborate previous observations showing that in HRT-CP-FET cycles with DYD and MVP, serum progesterone levels on the day of embryo transfer are not predictive of live birth. Specifically, Lawrenz et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) retrospectively analysed 560 HRT-FET cycles with euploid embryo transfer, in which luteal support consisted of 30 mg/day oral DYD and 300 mg/day MVP. Serum progesterone, but not DYD/DHD, was measured on the day of FET, and in patients with levels\u0026thinsp;\u0026lt;\u0026thinsp;10 ng/ml, MVP dosing was initially increased. However, multivariable logistic regression adjusted for age, BMI, and embryo quality, as well as weighted analyses using inverse probability treatment weights, showed no significant association between serum progesterone and ongoing pregnancy when patients already receive DYD 10mg tid. Our findings not only support this conclusion but also suggest the mechanism of substitution: when progesterone levels are low, DYD levels appear to compensate, with DYD emerging as a potentially decisive contributor when hormonal exposures are discordant. The latter, however, needs confirmation in larger studies.\u003c/p\u003e \u003cp\u003eA noteworthy observation in our dataset is the presence of several patients with extremely low serum progesterone levels despite administration of 2\u0026times;400 mg MVP. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e, five women had values at the lower detection limit of the assay (0.14 ng/mL), suggesting a potential subgroup with markedly impaired vaginal absorption or increased systemic clearance. This finding is concerning, particularly given that MVP monotherapy remains widely used in HRT-FET protocols (Ho et al.,2024). While non-compliance cannot be entirely excluded, it appears unlikely in this setting due to the typically high intrinsic motivation of women undergoing fertility treatment. Furthermore, comparable observations have also been reported in other studies using Cyclogest\u0026reg; at 2\u0026times;400 mg/day. Based on the reported means and standard deviations\u0026mdash;and assuming a normal distribution of serum progesterone\u0026mdash;the estimated proportion of women with levels below 1 ng/mL can be estimated as 0.41% in Labarta et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; n\u0026thinsp;=\u0026thinsp;663, mean\u0026thinsp;=\u0026thinsp;14.5 ng/mL, SD\u0026thinsp;=\u0026thinsp;5.1), 1.46% in Alsbjerg et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; n\u0026thinsp;=\u0026thinsp;488, mean\u0026thinsp;=\u0026thinsp;15.4 ng/mL, SD\u0026thinsp;=\u0026thinsp;6.6), 0.26% in Herencia et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; n\u0026thinsp;=\u0026thinsp;131, mean\u0026thinsp;=\u0026thinsp;13.6 ng/mL, SD\u0026thinsp;=\u0026thinsp;4.5), and 1.8% in Baldini et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; n\u0026thinsp;=\u0026thinsp;281, mean\u0026thinsp;=\u0026thinsp;14.0 ng/mL, SD\u0026thinsp;=\u0026thinsp;6.2). Similar results can be estimated from other large observational studies using vaginal progesterone formulations such as Utrogestan\u0026reg;, Crinone\u0026reg;, and Endometrin\u0026reg; (summarized in Alsbjerg et al., 2025). Altogether, these findings highlight substantial interindividual variability in systemic progesterone exposure under MVP monotherapy and underscore the potential value of combination progestin strategies in HRT-FET protocols, allowing for substitution between different routes of administration.\u003c/p\u003e \u003cp\u003eIn our previous prospective study investigating DYD monotherapy in anovulatory HRT-FET cycles, we already observed a kind of interaction between serum E2 and DYD levels on the day of FET: elevated E2 levels appeared to mitigate the adverse association of low DYD with live birth rate, suggesting a substitution effect between these two hormones (depicted in Fig.\u0026nbsp;5 in Neumann et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, with the addition of a second progestin (MVP) to the luteal support regimen, we now hypothesize a synergistic pattern between Progestins and E2. Live birth rates were consistently highest when both hormones of a given pair (DYD-E2, P-E2) were elevated and lowest when both were low. This observation, if further corroborated, has important ramifications for clinical practice and protocol optimization. Several studies have proposed the use of a single progesterone threshold on the day of FET as predictive of clinical pregnancy or live birth\u0026mdash;for instance, Labarta et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) suggested a cut-off of 9.2 ng/ml, while other thresholds ranging from 8 to 11 ng/ml have been reported in the literature (see Melo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, for summary). However, such an approach may be overly simplistic for identifying patients with suboptimal sex-steroid support. Omitting potential interactions between estrogenic and progestogenic sex steroids assumes that each hormone exerts its effect in isolation, which may not reflect the complex endocrine milieu required for optimal endometrial receptivity and pregnancy maintenance. Single threshold-based assessments may indeed also distort smooth effects, misrepresent additive and substitution effects of different hormones and lead to over- and undertreatment in individual cases. This underscores the need for multivariable and interaction-sensitive models in evaluating hormone adequacy in HRT-FET protocols, particularly because all administered sex steroids in this setting are pharmacologic substitutes for endogenous production and display substantial inter-individual variability.\u003c/p\u003e \u003cp\u003eImportantly, conventional logistic regression models\u0026mdash;which rely on the logit link function\u0026mdash;tend to model multiplicative effects on the odds scale and may obscure additive or substitutive relationships between covariates. When hormonal variables interact in a compensatory manner, such patterns may not be readily apparent in regression coefficients alone. Visual inspection of interaction plots, such as the hormone interaction matrices shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e, can therefore provide valuable complementary insights into complex biological relationships and guide a more nuanced understanding.\u003c/p\u003e \u003cp\u003eA novel and unexpected observation in this study is a potentially additive effect of a DYD and MVP combination, indicating that a higher exposure to both agents may confer a cumulative benefit. While our study was initially designed to explore how one progestin may partially compensate for low levels of the other\u0026mdash;implying a substitution effect\u0026mdash;an obvious opportunity arises for improving outcomes by optimizing overall progestin exposure, whether through increased dosing, alternative routes of administration, or individualized adjustments. This has obvious and potentially important implications for routine HRT-FET regimens, which have been suspected to carry a higher risk of pregnancy loss after implantation compared to natural cycles. Inadequate progestins exposure may be a contributing factor. Of note, such a phenomenon may not become apparent in pragmatic RCTs measuring mean effects at the population level (Ho et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) as this phenomenon may affect only a (too small) fragment of all women treated. Still, by leveraging the potential synergistic effect of dual-progestin strategies, protocols could be refined to ensure more consistent and potentially optimized exposure across patients.\u003c/p\u003e \u003cp\u003eOf note, the sample size of this study is modest, which limits the statistical power for detecting small effect sizes and precludes more complex multivariable modelling adjusting for relevant predictors. This is partly due to the declining clinical use of HRT-FET protocols, which are no longer considered first-line in many centers for patients who can be treated within an ovulatory FET regimen (von Versen-Hoeynck \u0026amp; Griesinger, 2022), thereby restricting the number of eligible patients for prospective inclusion. While it was anticipated that approximately 200 patients would undergo HRT-FET within this prospective study encompassing also ovulatory FET cycles (Figure S1), only 111 observations have become available. Nonetheless, despite the limited sample size, potentially relevant patterns emerged in the descriptive analyses. Importantly, the weak pairwise correlations between DYD, P, and E2 (all rho\u0026thinsp;\u0026lt;\u0026thinsp;0.3) proved advantageous in this exploratory setting. This independence reduces concerns of multicollinearity and allows for the meaningful visual separation of effects in cross-classification. In turn, this facilitates the interpretation of potential additive or substitution effects between hormone pairs\u0026mdash;patterns that might be obscured in more collinear datasets. Hence, while the study is underpowered for more robust inferential testing, the independence of the hormonal variables lends credibility to the exploratory observation of effects and interaction effects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study confirms that serum progesterone levels on the day of FET are unlikely to predict reproductive outcomes in DYD\u0026thinsp;+\u0026thinsp;MVP-supported HRT-FET cycles, thereby challenging mono-progestin \u0026ldquo;screen-and-act\u0026rdquo; strategies based solely on progesterone thresholds. The observed interaction patterns\u0026mdash;particularly between estradiol and progestins\u0026mdash;indicate that single-hormone cut-off concepts may oversimplify the complex endocrine interplay underlying endometrial receptivity.\u003c/p\u003e \u003cp\u003eBy enabling simultaneous and analytically independent quantification of two progestins, this study establishes dual-progestin measurement as a novel methodological platform for pharmacodynamic interaction research in HRT-FET. The data suggest clinically relevant additive effects between progestins and estradiol, as well as potential substitutive dynamics between dydrogesterone and progesterone\u0026mdash;phenomena that can only be meaningfully evaluated in combination regimens without analytical cross-reactivity.\u003c/p\u003e \u003cp\u003eTogether, these findings provide a mechanistic framework and generate hypotheses for adequately powered prospective studies investigating joint hormone exposure rather than isolated single-hormone thresholds in artificial FET protocols.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACN \u0026ndash; Acetonitrile\u003c/p\u003e\n\u003cp\u003eAI \u0026ndash; Artificial Intelligence\u003c/p\u003e\n\u003cp\u003eAMH \u0026ndash; Anti-M\u0026uuml;llerian Hormone\u003c/p\u003e\n\u003cp\u003ebid \u0026ndash; Bis in die (twice daily)\u003c/p\u003e\n\u003cp\u003eBMI \u0026ndash; Body Mass Index\u003c/p\u003e\n\u003cp\u003eCI \u0026ndash; Confidence Interval\u003c/p\u003e\n\u003cp\u003eCOCs \u0026ndash; Cumulus\u0026ndash;Oocyte Complexes\u003c/p\u003e\n\u003cp\u003eCP \u0026ndash; Combination Progestin\u003c/p\u003e\n\u003cp\u003eCRF \u0026ndash; Case Report Form\u003c/p\u003e\n\u003cp\u003eCV \u0026ndash; Coefficient of Variation\u003c/p\u003e\n\u003cp\u003eDET \u0026ndash; Double Embryo Transfer\u003c/p\u003e\n\u003cp\u003eDHD \u0026ndash; 20\u0026alpha;-Dihydrodydrogesterone\u003c/p\u003e\n\u003cp\u003eDOI \u0026ndash; Digital Object Identifier\u003c/p\u003e\n\u003cp\u003eDYD \u0026ndash; Dydrogesterone\u003c/p\u003e\n\u003cp\u003eE2 \u0026ndash; Estradiol\u003c/p\u003e\n\u003cp\u003eELISA \u0026ndash; Enzyme-Linked Immunosorbent Assay\u003c/p\u003e\n\u003cp\u003eEMT \u0026ndash; Endometrial Thickness\u003c/p\u003e\n\u003cp\u003eET \u0026ndash; Embryo Transfer\u003c/p\u003e\n\u003cp\u003eFET \u0026ndash; Frozen-Thawed Embryo Transfer\u003c/p\u003e\n\u003cp\u003eGnRH \u0026ndash; Gonadotropin-Releasing Hormone\u003c/p\u003e\n\u003cp\u003ehCG \u0026ndash; Human Chorionic Gonadotropin\u003c/p\u003e\n\u003cp\u003eHPLC-MS/MS \u0026ndash; High-Performance Liquid Chromatography Tandem Mass Spectrometry\u003c/p\u003e\n\u003cp\u003eHRT \u0026ndash; Hormone Replacement Therapy\u003c/p\u003e\n\u003cp\u003eHRT-CP-FET \u0026ndash; Hormone Replacement Therapy Combination-Progestin Frozen Embryo Transfer\u003c/p\u003e\n\u003cp\u003eICSI \u0026ndash; Intracytoplasmic Sperm Injection\u003c/p\u003e\n\u003cp\u003eIFFS \u0026ndash; International Federation of Fertility Societies\u003c/p\u003e\n\u003cp\u003eIVF \u0026ndash; In Vitro Fertilization\u003c/p\u003e\n\u003cp\u003eIU \u0026ndash; International Units\u003c/p\u003e\n\u003cp\u003eK2EDTA \u0026ndash; Dipotassium Ethylenediaminetetraacetic Acid\u003c/p\u003e\n\u003cp\u003eLH \u0026ndash; Luteinizing Hormone\u003c/p\u003e\n\u003cp\u003eLoD \u0026ndash; Limit of Detection\u003c/p\u003e\n\u003cp\u003eLoQ \u0026ndash; Limit of Quantitation\u003c/p\u003e\n\u003cp\u003eMVP \u0026ndash; Micronized Vaginal Progesterone\u003c/p\u003e\n\u003cp\u003eng/mL \u0026ndash; Nanograms per Milliliter\u003c/p\u003e\n\u003cp\u003eP \u0026ndash; Progesterone\u003c/p\u003e\n\u003cp\u003epg/mL \u0026ndash; Picograms per Milliliter\u003c/p\u003e\n\u003cp\u003ePRISMA \u0026ndash; Preferred Reporting Items for Systematic Reviews and Meta-Analyses\u003c/p\u003e\n\u003cp\u003eRCT \u0026ndash; Randomized Controlled Trial\u003c/p\u003e\n\u003cp\u003eRD \u0026ndash; Risk Difference\u003c/p\u003e\n\u003cp\u003eREI \u0026ndash; Reproductive Endocrinology and Infertility\u003c/p\u003e\n\u003cp\u003erho (r) \u0026ndash; Spearman\u0026rsquo;s Rank Correlation Coefficient\u003c/p\u003e\n\u003cp\u003esFET \u0026ndash; Single Frozen Embryo Transfer\u003c/p\u003e\n\u003cp\u003eSET \u0026ndash; Single Embryo Transfer\u003c/p\u003e\n\u003cp\u003eSD \u0026ndash; Standard Deviation\u003c/p\u003e\n\u003cp\u003etid \u0026ndash; Ter in die (three times daily)\u003c/p\u003e\n\u003cp\u003eVBL \u0026ndash; Versorgungsanstalt des Bundes und der L\u0026auml;nder\u003c/p\u003e\n\u003cp\u003eWHO \u0026ndash; World Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInstitutional review board approval was granted (centers Luebeck \u0026amp; Kiel reference number 18-005; center Duesseldorf reference number 2022-1953; center Saarbruecken reference number 206/21) in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis dataset and statistical code underlying this study are currently being curated and will be made publicly available on Zenodo and GitHub under the dataset title \u0026ldquo;HRT_CP_FET _Luebeck.\u0026rdquo; A DOI-linked Zenodo repository will be established to ensure long-term accessibility and transparency. In the meantime, the data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.K.E. discloses honoraria from Ferring; travel support from Merck, Ferring, Theramex and Gedeon-Richter; and receipt of equipment/materials (to institution) from Arthrex and Besins Healthcare, outside the submitted work.\u003c/p\u003e\n\u003cp\u003eN.H. has received travel support from Gedeon-Richter, Ferring and Merck, outside the submitted work.\u003c/p\u003e\n\u003cp\u003eA.R.H. has received honoraria from Organon and travel support from Merck Serono, Gedeon Richter and Theramex, outside the submitted work.\u003c/p\u003e\n\u003cp\u003eM.D. discloses support from Merck, outside the submitted work.\u003c/p\u003e\n\u003cp\u003eP.E. discloses honoraria from Ferring, Theramex and Gedeon Richter; and travel support from Merck, Ferring, Theramex, Gedeon Richter and MSD, outside the submitted work.\u003c/p\u003e\n\u003cp\u003eA.P.B discloses honoraria and travel support from Merck, Theramex, Gedeon Richter and Ferring; and participation on a data safety monitoring board or advisory board for Ferring and Merck, outside the submitted work.\u003c/p\u003e\n\u003cp\u003eA.S-M, D.B-B., J.S.K, S.v O., W.J., S.T., R.V. declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eG.G. discloses consulting fees and honoraria from Merck, Organon, Ferring, Theramex, Gedeon-Richter, Abbott, Biosilu, ReprodWissen, Obseva, PregLem, Guerbet, Cooper, Igyxos, OxoLife, and ReproNovo, outside the submitted work, and travel support from Merck, Organon, Ferring, Theramex, Gedeon-Richter, and Abbott, outside the submitted work.\u003c/p\u003e\n\u003cp\u003eThere are no conflicts relating directly to the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial was funded through institutional resources of the University Hospital of Schleswig-Holstein, Campus L\u0026uuml;beck. Expenses related to plasma and serum sample handling, storage, shipment, and the HPLC/MS/MS and ELISA analyses of DYD, DHD, estradiol, and progesterone were financially supported by Abbott Products Operations AG (Allschwil, Switzerland). The funding was provided in the form of a research grant to the Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital of Schleswig-Holstein, Campus L\u0026uuml;beck (no grant number applicable). Abbott Products Operations AG had no role in the study design, conduct, data collection, statistical analysis, data interpretation, manuscript preparation, or the decision to submit the article for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEggersmann T.K.\u003c/strong\u003e: Conceptualization, Methodology, Study initiation, Conduct, Supervision, Data analysis, Data interpretation, Writing - review \u0026amp; editing; \u003cstrong\u003eHamala N.\u003c/strong\u003e: Data acquisition from CRFs and patient files, Data analysis, Data interpretation, Writing - review \u0026amp; editing; \u003cstrong\u003eHiller A.R., Depenbusch M., Schultze-Mosgau A., Edimiris P., Baston-B\u0026uuml;st D., Bielfeld A.P., Kruessel J-S., von Otte S., Junkers W., Tauchert S.\u003c/strong\u003e: Investigation, Writing - review \u0026amp; editing; \u003cstrong\u003eVonthein R.\u003c/strong\u003e: Data analysis, Data interpretation, Writing - original draft, Writing - review \u0026amp; editing; \u003cstrong\u003eGriesinger G.\u003c/strong\u003e: Study design, Conceptualization, Study initiation, Supervision, Data analysis, Data interpretation, Writing - original draft, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank Mrs. Andrea Knaak, study nurse at the Department of Gynecological Endocrinology and Reproductive Medicine, University Hospital of Schleswig-Holstein, for her exceptional dedication in managing documentation, data entry, sample handling, and follow-up during pregnancy and neonatal period.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOry S, Miller K, Horton M, et al. editors. 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Reprod Biomed Online. 2025;50(5):104736. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.rbmo.2024.104736\u003c/span\u003e\u003cspan address=\"10.1016/j.rbmo.2024.104736\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 2024 Nov 26. PMID: 40132.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"frozen-thawed embryo transfer cycle, luteal phase support, dydrogesterone, progesterone, progestin","lastPublishedDoi":"10.21203/rs.3.rs-8929363/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8929363/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn 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 \u0026ndash; 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.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis 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\u0026ndash;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.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHormone concentrations showed broad interindividual variability with weak inter-analyte correlations (r\u0026thinsp;\u0026le;\u0026thinsp;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\u0026ndash;E2 and P\u0026ndash;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.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis 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.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTrial registration number:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNCT03507673\u003c/p\u003e","manuscriptTitle":"Pharmacodynamic Interaction Analysis of Dydrogesterone, Progesterone, and Estradiol in Combination-Progestin HRT Frozen Embryo Transfer: a prospective clinical cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 16:33:25","doi":"10.21203/rs.3.rs-8929363/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T15:36:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T08:24:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T07:43:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220353888166272881909526398455005416285","date":"2026-03-16T06:27:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159098994583883577912555319017274647898","date":"2026-03-10T09:35:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-27T13:34:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T12:27:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T12:20:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2026-02-20T22:15:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6864423-aacf-4bbc-afea-1269ab290c7f","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T09:33:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 16:33:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8929363","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8929363","identity":"rs-8929363","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}