Materials & Methods
The study was approved by the Ethics Committee of The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University (Approval number: 2023-K-205-01). We retrospectively collected all frozen blastocyst transfer cycles carried out between January 2020 and July 2022 at the reproductive center of The Second Affiliated Hospital of Wenzhou Medical University. Inclusion criteria: endometrial preparation protocol using HRT cycle with or without GnRH-a pretreatment. Exclusion criteria were: adenomyosis or endometriosis; PCOS; RIF; severe intrauterine adhesions, endometrial thickness was less than 6 mm; natural cycle IVF/ICSI; characteristic information incomplete; reproductive outcomes lost. Based on the criteria listed above, 4,091 cycles were included in this study. Cycles were grouped by maternal age: group A (<35 years old) and group B (≥35 years old). Each group was further subdivided into two sub-groups based on the endometrial preparation methods: the HRT group and the GnRHa-HRT group.

HRT group: Supplementation with estrogens was administered on day 2–5 of the menstrual cycle after the sex hormone test and ultrasonography showed no abnormalities. Endometrial thickness and morphology were regularly evaluated with vaginal ultrasonography. Monitor the levels of E2, progesterone, and LH, and adjust the drug usage and dosage when necessary. The timing of endometrial transformation was determined by the clinicians. If endometrial thickness and morphology were satisfactory and serum progesterone level <1.5 ng/ml, progesterone was added to achieve endometrial transformation. Blastocysts were transferred on the fifth day after transformation.

GnRHa-HRT group: 3.75mg long-acting GnRH agonist was injected on the 2nd–5th day of menstruation, and 28–30 days later, patients were examined for serum FSH, LH, and E2 levels, and an ultrasound was performed to evaluate endometrium. When pituitary downregulation reached standards: LH < 5U/L, FSH < 5U/L, E2 < 50 pg/ml, endometrial thickness < 5 mm, and the largest follicle diameter was ≤5–10 mm, and then the protocol was the same as for HRT.

Serum β -hCG levels were measured on day 10–14 after embryo transfer and transvaginal ultrasound was performed 2 weeks after the β -hCG test to diagnose pregnancy. Serum β -HCG >5 mIU/mL indicated HCG positive. Pregnancy outcomes included: biochemical pregnancy (a positive β -hCG level without visualization of an intrauterine gestational sac), clinical pregnancy (the presence of a gestational sac), miscarriage (a loss of pregnancy before 28 weeks of gestation), and singleton live birth (a live born infant after 28 weeks of gestation).

Primary observation indicator: live birth rate. Secondary observation indicators: clinical pregnancy rate and miscarriage rate.

Data were analyzed using Graphpad Prism (Graphpad Prism 9 software). Data with a skewed distribution are described as median (interquartile range (IQR)). Mann–Whitney test was performed for group comparison. The chi-square test or Fisher exact test was used for categorical variables. Multivariate logistic regression analysis was performed to explore the factors associated with live birth. The covariates included endometrial preparation protocol (with or without GnRH-a), maternal age, maternal body mass index (BMI), history of intrauterine adhesions surgery, and number of embryos transferred. P < 0.05 was considered statistically significant.