Estradiol and progesterone levels in early pregnancy after modified natural, programmed, and gonadotrophin-stimulated frozen embryo transfer cycles: a randomized controlled trial.

Study Question: Are serum estradiol and progesterone levels higher in women treated with programmed cycle (PC) FET compared to women treated with a gonadotrophin-stimulated (gSC) FET or a modified natural cycle (mNC) FET during early pregnancy?

Summary Answer: Estradiol and progesterone levels are higher on the day of embryo transfer in women treated with PC compared to those treated with a mNC or gSC.

What Is Known Already: Various FET treatment regimens may affect the maternal hormone balance very differently in early pregnancy. Recent studies show that a PC is associated with increased risk of hypertensive pregnancy disorders, high birth weight, and postpartum hemorrhages. The underlying mechanism is not known but differences in hormone levels could play a role.

Study Design, Size, Duration: This was a parallel open-label randomized controlled drug trial conducted from April 2021 until December 2024. The target sample size was 100 cycles in each treatment arm for a power of 80%. A total of 305 cycles were included and 257 were included in the per-protocol analysis. Ovulatory women were randomized to either a PC or a mNC. Anovulatory women were randomized to a PC or a gSC. Blood samples were obtained at seven consecutive time points from the start of the cycle until gestational age (GA) 9 + 6 weeks. Obstetric and neonatal follow-up was completed through the patients' digital medical records. Primary outcomes were differences in serum estradiol and progesterone between patient groups and treatment regimens. Secondary outcomes were to compare the type of cycle with reproductive, obstetric, and neonatal outcomes between groups.

Participants/materials, Setting, Methods: The trial was conducted in the Fertility Clinic at Herlev University Hospital, Denmark. Participants were women aged 18-40 years with BMI ≤35 kg/m2. Only women using autologous frozen blastocysts were eligible and all women had a normal wet cervical wet smear within the last 3 years.

Main Results And The Role Of Chance: For ovulatory women, estradiol and progesterone levels were higher in PCs on the day of embryo transfer day compared to mNCs (estradiol: 0.86 nmol/l vs 0.54 nmol/l, adjusted P < 0.001, progesterone: 68.5 nmol/l vs 40.3 nmol/l, adjusted P < 0.001), and progesterone levels were also higher in PCs at GA 8 + 0 (87.5 nmol/l vs 61.2 nmol/l, adjusted P = 0.02). For anovulatory women, the same trend was observed when comparing PCs to gSCs. Serum estradiol was significantly higher on the day of embryo transfer day (0.96 nmol/l vs 0.54 nmol/l, adjusted P < 0.01), though no significant differences in progesterone were found. We found no differences in pregnancy rates, live birth rates, pregnancy loss rates, or cancelation rates. Ovulatory women treated with PCs had significantly larger children with an estimated treatment difference of 292 grams (P < 0.01) compared to those treated with mNCs. Furthermore, we observed a higher risk of postpartum hemorrhages in PCs compared to mNCs (22/40 (55%) vs 10/40 (25%), P = 0.01).

Limitations, Reasons For Caution: The target sample size of 100 cycles was not achieved in either of the anovulatory arms.

Wider Implications Of The Findings: This is the first randomized trial to investigate differences in maternal sex hormones between FET regimens. Although higher levels of estradiol and progesterone were demonstrated in PCs compared to mNCs and gSCs on the day of embryo transfer day, the significance of these findings is currently unclear. Further research is needed to verify these results and to examine possible effects on pregnancy and fetal development.

Study Funding/competing Interest(s): The study has received grants from Gedeon Richter Nordics AB (DK-2019-04, DK-2022-03, DK-2023-08, DK-2023-06, DK-2024-08) and a grant from the Gangsted-Rasmussen Foundation (ref. A39784). The study has also received a grant from the local research board at Herlev University Hospital. The funders of the trial had no role in the design and planning phase, data analysis, or interpretation of the results. Ongoing analyses of biobank samples have been funded by Merck A/S. M.S.S., H.U., and M.L.B. have no conflicts of interest. N.F.M. has, outside this work, received funding for congress registration from Gedeon Richter Nordics AB. P.F.S. has, outside this work, received grants from Merck A/S, Gedeon Richter Nordics AB, and Ferring Pharmaceuticals A/S. Outside this work, B.N. has received grants from Merck A/S, Gedeon Richter Nordics AB, and Ferring Pharmaceuticals A/S, personal fees from Ferring Pharmaceuticals A/S, travel support from Gedeon Richter Nordics AB, and participated in a data safety monitoring or advisory board for Ferring Pharmaceuticals A/S.

Trial Registration Number: 2020-001218-39 in EudraCT.

Trial Registration Date: 17 November 2020.

Date Of First Patient’s Enrolment: 20 April 2021.