Live birth rates are unrelated to sex-steroid levels on ET day in a dydrogesterone-based 'programmed-ovulatory FET' protocol: a multi-centric prospective cohort study.

study questionWhat are the effects of three-times-a-day 10 mg oral dydrogesterone (DYD), initiated in the late follicular phase of natural menstrual cycles to induce endometrial receptivity for frozen-thawed embryo transfer (FET), on progesterone levels, indicative of ovulation on the day of FET, and how are levels of DYD, 20α-dihydrodydrogesterone (DHD), progesterone (P), and estradiol (E2) on the day of FET associated with clinical outcomes? SUMMARY ANSWER: Late follicular phase initiation of oral 30 mg DYD is compatible with progesterone levels indicative of ovulation in 98% of cases, and DYD, DHD, P, and E2 blood levels on the day of FET do not show a consistent relationship with live birth achievement, whether evaluated in isolation or interaction. WHAT IS KNOWN ALREADY: HRT regimens for FET have come under scrutiny due to: (i) the risk of insufficient progesterone exposure with conventional dosing schemes, and (ii) maternal and fetal risks associated with the iatrogenic absence of a corpus luteum. Oral DYD 10 mg three-times-a-day (tid) is considered unlikely to interfere with ovulation or corpus luteum formation and does not exhibit cross-reactivity with progesterone in ELISA. Therefore, it can be used to induce endometrial receptivity (i.e. to schedule the timing of FET in a natural cycle) and provide luteal phase support (LPS) while allowing ovulation to occur independently of the implantation window and enabling the monitoring of endogenous progesterone serum levels. STUDY DESIGN SIZE DURATION: Nested within a multi-centric, prospective, clinical cohort study (NCT03507673), 559 normally cycling women from the routine care population who underwent FET in a spontaneous menstrual cycle (12/2021-8/2023) had DYD, DHD, P, and E2 levels on day of FET measured by high-performance liquid chromatography/tandem mass spectroscopy (HPLC/MS/MS) or commercially available electrochemiluminescence immunoassay (ECLIA). PARTICIPANTS/MATERIALS SETTING

methodsStarting from cycle day 10, women underwent endocrine (E2, LH, and P) and sonographic monitoring until the following criteria were ideally met: a leading follicle ≥16 mm, endometrial thickness ≥6 mm, and E2 ≥ 180 pg/ml. At this point, physicians could initiate oral DYD 10 mg (tid) or, if there was no LH rise in serum, postpone initiation within 2 days of monitoring to align with patient and/or centre preferences (e.g. avoiding weekends). FET for Day 2-Day 5 embryos was performed on Day 3-6 of DYD intake. MAIN RESULTS AND THE ROLE OF CHANCE: DYD and DHD plasma levels on Day 3-6 of intake replicate previous findings. In FET with Day 4 or 5 embryos, serum P levels indicated ovulation in 98.1% (>1.5 ng/ml) and 95.4% (>3.0 ng/ml) of cases and levels were higher (ratio of geometric means 2.88, 95% CI 2.48-3.35) than in FET with Day 2 or 3 embryos. Progesterone levels on day of FET were unrelated (ratio of geometric means 1.13, 95% CI 0.95-1.34) to follicular size (≤16 mm vs >16 mm) and only slightly higher (ratio of geometric means 1.36, 95% CI 1.19-1.56) in cases of serum LH level ≥12.6 IU/l versus <12.6 IU/l on day of last monitoring (i.e. before DYD initiation). When analysed stratified for FET timing (Day 2/3 vs 4/5 embryos), risk differences (RD) for live birth in subjects were only marginally and not statistically significant different in patients with low (≤25th percentile) versus normal-high (>25th percentile) hormone levels (DYD RD -5.3%, 95% CI -14.0 to 3.2, LIMITATIONS REASONS FOR CAUTION: All outcomes were assessed only in subjects undergoing FET, not on intention-to-treat. Oral DYD 10 mg (tid), similar to other progestins given to induce endometrial receptivity or for LPS, will likely still interfere with the LH surge, ovulation, and luteal phase characteristics, warranting further investigations using a true natural cycle (NC) as the reference. WIDER IMPLICATIONS OF THE

findingsWe propose the term 'Programmed-Ovulatory (PO-) FET' for this protocol. The PO-FET protocol enables scheduling the window of implantation, allows ovulation and corpus luteum formation, provides double gestagenic support, and may eliminate the need for control measurements of sex steroids on the day of FET. It allows monitoring of corpus luteum activity during the luteal phase and early pregnancy, is injection-free, and has low drug costs. This protocol needs to be tested in RCTs against other ovulatory FET protocol options, such as the 'NC FET' or 'managed NC FET'. STUDY FUNDING/COMPETING INTERESTS: 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. 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 travel 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. TRIAL REGISTRATION NUMBER: NCT03507673.