Preparing and Timing of the Endometrium in Modified Natural Cycle Frozen-thawed Embryo Transfers

Preparing and Timing of the Endometrium in Modified Natural Cycle Frozen-thawed Embryo Transfers (mNC-FET) - a Randomized Controlled Multicenter Trial

Copenhagen University Hospital, Hvidovre, Regionshospitalet Horsens, Copenhagen University Hospital at Herlev, Aalborg University Hospital, Hillerod Hospital, Denmark, Zealand University Hospital, Regionshospitalet Viborg, Skive

The increasing use of FET emphasizes the importance of preparing and timing the endometrium in FET cycles, however there is no consensus on luteal phase progesterone supplementation in mNC-FET and the optimal day of blastocyst warming and transfer. The aim of this multicenter RCT is to assess the effect of progesterone supplementation in hCG-triggered mNC-FET and the effect of embryo thawing and transfer at hCG+6 or hCG+7 days, respectively. In total 604 patients will be included with n=151 in each of the four study arms. The primary outcome is live birth rate per transfer (LBR) and the goal is to show a 10% increase in LBR after progesterone supplementation and to assess whether blastocyst warming+transfer 6 days after hCG trigger is superior to 7 days after hCG trigger in mNC-FET.

Single embryo transfer and freezing of surplus embryos has lowered twin birth rates after in vitro fertilization (IVF) to a level of less than 5% in Denmark. However, several treatments with repeated frozen embryo transfers (FET) before a viable pregnancy is confirmed are burdensome to the patients. New freezing techniques has optimized the quality of the embryo transferred in FET cycles, but optimization of the endometrium in the luteal phase is still lacking behind. In a mNC-FET, which is the routine in many clinics, ovulation is induced with an hCG injection when the leading follicle is ≥17 mm. The hCG trigger is important for controlling the time of ovulation, but triggering an unhealthy follicle at an inappropriate time may cause luteal phase insufficiency and thus suboptimal function of the endometrium. Danish public fertility clinics are not routinely using progesterone supplementation in mNC-FET, but there may be a rationale to do so, and some implantations may be rescued. In this study we will compare live birth rates in mNC-FET with and without progesterone supplementation in the luteal phase, and further we will explore the optimal timing of blastocyst warming and transfer by comparing embryo transfer at hCG trigger +6 days versus +7 days. This is a superiority study with the aim to detect an increase in live birth rates of 10%. Hence, this adequately powered RCT may make a major contribution to knowledge on mNC-FET to the benefits of patients. We will include 604 patients divided 1:1 (302:302) in each arm +/- progesterone and these will further be divided 1:1 in blastocyst warming and transfer +6 and +7 days after hCG injection. The primary endpoint is live birth rate per transfer.

Randomized, controlled multicenter trial with inclusion of 604 mNC-FET cycles. We will include 604 patients divided 1:1 (302:302) in each arm +/- progesterone supplementation and these will further be divided 1:1 in blastocyst warming and transfer +6 and +7 days after hCG injection. Patients randomised to progesterone supplementation will start administering medicine four days after the ovulation trigger up til the day of the pregnancy test. If the pregnancy test is positive, medication will continue for 30 days more.

The study is a single blinded study; therefore, the study medication will be blinded for the treating doctors, but not for the patients, the non-treating doctors or the study nurses. Patients will only be seen by a treating doctor at the day of blastocyst transfer and at the day of the pregnancy scan. The participants will not take progesterone the morning of the blastocyst transfer, but immediately after to keep the treating doctors blinded. Patients will be instructed in not disclosing their study group to the treating doctor.

Four parallel groups of patients undergoing fertility treatment (modified natural cycle frozen embryo transfer) will be compared using/not using vaginal progesterone (Lutinus) and subject to blastocyst warming and transfer 6/7 days after hCG trigger.

Four parallel groups of patients undergoing fertility treatment (modified natural cycle frozen embryo transfer) will be compared using/not using vaginal progesterone (Lutinus) and subject to blastocyst warming and transfer 6/7 days after hCG trigger.

Four parallel groups of patients undergoing fertility treatment (modified natural cycle frozen embryo transfer) will be compared using/not using vaginal progesterone (Lutinus) and subject to blastocyst warming and transfer 6/7 days after hCG trigger.

Four parallel groups of patients undergoing fertility treatment (modified natural cycle frozen embryo transfer) will be compared using/not using vaginal progesterone (Lutinus) and subject to blastocyst warming and transfer 6/7 days after hCG trigger.

Comparison of live birth rates between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of chemical pregnancy rates between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of clinical pregnancy rates (ultrasound) between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or seven after hCG trigger.

Comparison of abortion rates between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

ALAT measured by blood sample to ensure normal liver parameters before administration of progesterone.

ALAT measured by blood sample to ensure normal liver parameters before administration of progesterone.

Comparison of AMH measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of estradiole measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of FSH measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of LH measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of progesterone measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison OH-progesterone measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of beta-hCG measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of TSH measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Measured at baseline, at ovulation trigger day (hCG+0), at transfer day (hCG+6/7), at hCG+11, at hCG+14 and at hCG+19.

Comparison of thyroglobulin antibodies measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of thyroid peroxidase antibodies measured by blood samples between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of obstetric complication rates between patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Comparison of neonatal complication rates for children of patients receiving and not receiving Lutinus, with blastocyst warming and transfer day 6 or 7 after hCG trigger.

Inclusion Criteria: Female age 18-41 years, regular menstrual cycle (23-35 days), vitrified blastocysts derived from 1.-3. IVF/ICSI cycle in a public hospital and undergoing single blastocyst transfer. Exclusion Criteria: - Previous participation in the study, uterine malformations, intrauterine polyps or submucosal myomas, breast feeding, oocyte donation, preimplantation genetic testing, blastocyst conceived with sperm from testicular sperm aspiration, HIV (woman), hepatitis B and C (woman), known luteal phase insufficiency or if patients are not fulfilling the inclusion criteria. Further exclusion criteria are the following contraindications to progesterone; allergy to the study medication, undiagnosed vaginal bleeding, current missed abortion or ectopic pregnancy, hepatic insufficiency or severe hepatic disease, genital or breast cancer, arterial or venous thromboembolism, thrombophlebitis or porphyria. For patients participating in the sub-study, thyroid disease is an exclusion criterion.

On request the study protocol and deidentified individual study data collected during the trial, including stored biobank samples, can be shared with research groups with relevant aims and a methodologically sound proposal. Approvals by necessary ethic committees and the Danish Data Protection Agency will be needed before sharing of data. All costs for data sharing will be covered by the party requesting the data. Data cannot be shared with groups working on research projects with the same aims, secondary aims or purposes. Further no data can be shared until 3 months after publication of first papers on the primary and secondary outcomes in this study. Biobank samples cannot be shared with research groups outside Denmark. Proposals of data sharing should be directed to anja.bisgaard.pinborg@regionh.dk. To gain access, data requestors will need to sign a data sharing agreement.

Data will be available from 3 months after publication of first papers on the primary and secondary outcomes in this study.

Approvals by necessary relevant ethic committees and the Danish Data Protection Agency will be needed before sharing of data. All costs for data sharing will be covered by the party requesting the data. Data cannot be shared with groups working on research projects with the same aims, secondary aims or purposes. Biobank samples cannot be shared with research groups outside Denmark.

Saupstad M, Freiesleben NC, Skouby SO, Andersen LF, Knudsen UB, Petersen KB, Husth M, Egeberg A, Petersen MR, Ziebe S, Andersen AN, Lossl K, Pinborg A. Preparation of the endometrium and timing of blastocyst transfer in modified natural cycle frozen-thawed embryo transfers (mNC-FET): a study protocol for a randomised controlled multicentre trial. BMJ Open. 2019 Dec 15;9(12):e031811. doi: 10.1136/bmjopen-2019-031811.