Study Design
A retrospective analysis was conducted among patients undergoing FET cycles from November 2015 to December 2021 in the Reproductive Center of Peking University Shenzhen Hospital. Medical records were reviewed. Patients aged between 20 and 42 years whose EMT ≦7 mm on the day of progesterone conversion in the present HRT cycle, or in the previous HRT cycle if MOS protocol was taken were included. Exclusion criteria included the followings: [1] corroborative chromosomal abnormality in either partner; [2] intrauterine conditions affecting the pregnancy outcomes of FET including uterine malformation, endometrial polyps, submucous myoma, intrauterine adhesions, endometrial tuberculosis, hydrosalpinx with reflux into uterine cavity which was observed by ultrasound; [3] cycles of spontaneous ovulation; [4] history of psychiatric disorders (anxiety, depression, and schizophrenia), coagulation disorders, infectious diseases (syphilis, human immunodeficiency virus infection, etc.), serious heart, kidney or liver disease. Finally, 52 FET cycles were included in the MOS group, while another 52 cycles were grouped into the HRT cycles.
Endometrial Preparation Protocol
In HRT protocol, patients were initially given oral estradiol twice a day with a total daily dosage of 4-6 mg (Progynova, Bayer, Leverkusen, Germany) from day 2-3 of the cycle. The dose was adjusted every 3-5 days according to the endometrium response. The maximal daily dose was 12 mg. When EMT failed to reach over 7 mm, patients were suggested to cancel the cycle and start a new FET cycle or accept embryo transfer despite of the thin endometrium. If they refused to terminate the cycle, transformation would be given.
Whether to receive MOS protocol depended on the willingness of the patients. Patients in the MOS group received intramuscular injections of human menopausal gonadotropin (Urofollitropin for Injection, Livzon, China) on Day 2-3 of the menstrual cycle. Two or more follicles were intended to be induced by the medicine. The starting dose (75 to 300 IU/Day) was determined by the clinician, based on the patients’ age, basal (Day 2–5) serum follicle stimulating hormone (FSH), anti-Mullerian hormone (AMH), antral follicle count (AFC) and the response to previous ovarian stimulation if available. On Day 6 or 7 of the menstrual cycle, an ultrasound and serum hormonal testing (P, E2, LH, FSH) were performed. Based on these results, the dose of gonadotropins was adjusted if needed.
The Luteal Phase Support (LPS)
The LPS with a daily dose of 90 mg of vaginal progesterone gel and 20 mg of oral dydrogesterone or 60 mg of progesterone injection (Xianju Pharma, China) was performed in the following. LPS continued for at least 8 weeks or till the HCG test result presented as negative.
Endometrial Thickness Measurement
On the day of progesterone administration, transvaginal ultrasound scanning (SSD-ALPHA7, ALOKA, HITACHI, Japan) with a 2D, 6.67MHz probe was performed by an expert gynecologist. Endometrial thickness was measured at the thickest part in the longitudinal section of the uterus and was noted in millimeters (mm) (16).
Embryo Thawing, Assessment and Transfer
The timing of the embryo thawing and transfer was calculated to achieve synchronization between the embryo and endometrium (17). The day of initiating progesterone was marked as Day 0. Cleavage-stage embryos were thawed on Day 4 and were transferred on the Day 5 after overnight culture, while blastocysts were thawed and transferred on Day 6. Cleavage-stage embryos were graded according to the SART grading system involving the evaluations of cell number, fragmentation, and symmetry (18). Blastocysts were morphologically scored according to Gardner’s three-part scoring system, which was based on blastocoele cavity, inner cell mass, and trophectodermal cells (19). Good embryo quality was defined as embryos of 7, 8 or 9 cells with ≤10% fragmentation, no multinucleation and with symmetric to slightly asymmetric blastomeres on thawing day (17). Good quality blastocysts were graded as ≥ 3BB on the thawing day (20).
Pregnancy Outcome Assessment
Serum HCG levels were measured 14 days after embryo transfer, and an HCG level of >30 mIU/mL indicated biochemical pregnancy. Clinical pregnancy was confirmed if the gestational sac was observed with ultrasonography at 4 weeks after embryo transfer, including ectopic pregnancy and miscarriage. Ongoing pregnancy was defined as at least a 12-week gestation age confirmed by an ultrasound scan. Live birth was defined as the delivery of one or more living infants at ≥28 weeks of gestation. The biochemical or clinical pregnancy rate was expressed as the number of cycles with biochemical or clinical pregnancy per 100 FET cycles. The ectopic pregnancy or miscarriage rate was expressed as the number of cycles with ectopic pregnancy or miscarriage per 100 clinical pregnancy cycles. The live birth rate was expressed as the number of cycles with live births per 100 FET cycles.
Statistical Analysis
The normality of continuous variables was tested by the Shapiro-Wilk test. Normally distributed data were described as mean (standard deviation, SD), otherwise, were presented as median (25th percentile to 75th percentile). Categorical variables were expressed as the number (n) and percentage (%). Student's t-test and Mann-Whitney U-test were used to compare continuous data, while the chi-squared test and Fisher's exact test were used for categorical data. Univariate logistic analysis was used to evaluate the effect on pregnancy outcomes for the variables with significant differences observed in the comparisons between the two groups. Multivariate logistic regression analysis was used to evaluate the association between different endometrial preparation protocols and pregnancy outcomes after adjusting for potential confounders including maternal age, infertility duration and type, body mass index, hysteroscopy before ET, the number type of embryos transferred, the stage and quality of embryos transferred. All statistical analyses were performed by Stata Statistical Soft-ware (Release 16, TX, USA). P value <0.05 was considered statistically significant.