This prospective observational study was conducted at a tertiary care academic medical center in southwestern China. Women who received their first HRT-FET between November 2021 and July 2023 were invited to participate in this study. Based on previous ultrasound, hysterosalpingography, magnetic resonance imaging, or surgical records, patients with stage III and IV endometriosis, adenomyosis, hydrosalpinx, uterine fibroids, history of severe uterine adhesions, endometrial polyps, scarred uterus, or uterine malformations were excluded. Furthermore, patients with a history of recurrent miscarriage or repeated implantation failure (more than three occurrences) were excluded from the study. This study was approved by the Scientific Ethics Committee of Chengdu Women’s and Children’s Central Hospital (reference: B2021-7), and all enrolled patients provided informed consent.

For hormonal replacement therapy cycles, all patients received estradiol (Femoston red tablets; Abbott Biologicals B.V., Olst, NL) treatment at 4 mg orally, with the dosage regulated on a 7-day basis according to endometrial thickness (EMT). If the EMT was < 8 mm, the estradiol dosage was increased. When the EMT reached ≥ 8 mm, and the oral estradiol dosage reached ≥ 14 days, or the clinicians and patients recognized EMT as having a relatively maximum thickness, progesterone was administered to initiate endometrial transformation. The secretory transformation was initiated using fematon-yellow tablets (containing estradiol 2 mg and dydrogesterone 10 mg; Abbott Biologicals B.V.) twice daily, and 40 mg oil-based intramuscular progesterone injection (Zhejiang Xianju Pharmaceutical) once daily was administered from the day of transformation to the day of the pregnancy test.

Identity 3D power Doppler (3D PD-US, H60, Samsung, Seoul, South Korea) settings with a transvaginal probe of 4 − 9 MHz were used in patients for endometrial receptivity testing on the endometrial transformation day as well as on the day preceding embryo transfer at approximately 1 pm. The settings for this study were as follows: low frequency; signal magnifying, 50; pulse repetition frequency, 0.8 kHz; power Doppler map, 5; and power, 90%. The sector of interest was adjusted to cover the endometrial cavity in the longitudinal plane of the uterus. The color gain was adjusted to 80%±2% to optimize blood flow detection in the small vessels of endometrial and subendometrial areas.

The data produced by the PD-US measurement included EMT, endometrial patterns (classified as type A, trilinear endometrium; type Not-A, no trilinear endometrium), endometrial contraction, endometrial volume, blood flow patterns (type I, the vessels pass through the lateral hypo-echoic band of the endometrium but do not enter the hyper-echoic rim of the endometrium; type II, the vessels pass through the hyper-echoic rim of the endometrium but do not enter the endometrium; type III, the vessels enter the endometrium [8]), and endometrial blood flow branches (countable endometrial and subendometrial blood flow branches in a single plane) (Fig. 1). In this study, the subendometrial region was considered to be within 1 mm of the originally defined myometrial–endometrial contour [9]. The endometrial volume was drawn manually along the endometrial outline. Stored volumes were analyzed using VOCAL software of Three-dimensional (3D) Power Doppler. The results of the ultrasound evaluation did not affect the subsequent clinical procedures. The primary outcome measure was intrauterine clinical pregnancy (defined as the presence of a gestational sac in the uterus determined using ultrasonography).

Power Doppler ultrasound imaging of endometrial and subendometrial blood flow: type-branch. (A) type I - branch<5: the vessels pass through the lateral hypo-echoic band of the endometrium but do not enter the hyper-echoic rim of the endometrium, branch 1. (B) type II - branch<5: the vessels pass through the hyper-echoic rim of the endometrium but do not enter the endometrium, branch 4. (C) type III – branch <5: the vessels enter the endometrium, branch 4

The Kappa test was applied to evaluate the consistency of the endometrial blood flow pattern results, with a Kappa value of 0.778 (P = 0.001). The Kendall W test was used to evaluate the consistency of the endometrial blood flow branches, with the Kendall Wa being 0.979 (P = 0.001). An ROC curve was constructed to explore the cutoff value of EMBLB for Pregnancy outcome.

Statistical analyses were performed using the Statistical Program for Social Sciences (SPSS Version 26, IBM Corp, Armonk, NY, USA). Normal distribution was identified using the Shapiro-Wilk test. Data were statistically described in terms of mean ± standard deviation (± SD), median and range, or number of cases (frequencies) when appropriate normality of data was tested. A nonparametric Mann-Whitney U test was used to compare the non-normally distributed data. Other comparisons were performed using the chi-squared and Fisher’s Exact tests, where appropriate. Binary stepwise logistic regression was used to determine the independent predictive factor of clinical pregnancy, adjusting for patient age, antral follicles (AFC), number of embryos transferred, embryo transfer type, good quality embryos transfer, endometrial blood flow, and changes of blood flow at two time points. The results are presented as the odds ratio (OR) and 95% confidence interval (CI). The significance level for all analyses was set at P < 0.05.