All procedures in this study were approved by the Ethics Committee of Affiliated Women’s Hospital of Jiangnan University (2023-06-1213-65).

In this retrospective study, infertile women undergoing FET with NCs were included based on electronic medical records from 2012 to 2022.

Clinical characteristics such as maternal age at diagnosis, nationality, the years of infertility, body mass index (BMI), basal estradiol (bE2), basal follicle-stimulating hormone (bFSH) and basal luteinizing hormone (bLH), anti-Müllerian hormone (AMH), basal prolactin (bPRL), β-hCG levels, etc., were collected. Weight and height at time of diagnosis were measured by BMI calculated and classified according to the ethnicity-specific WHO classification for Asian/Indian women: underweight (< 18.4 kg/m2), normal weight (18.5–22.99 kg/m2), overweight (23–27.49 kg/m2), and obese (> 27.50 kg/m2) [7].

The study included the patients aged up to 35 years old and without major system disease. Patients with uterine malformation, uterine fibroids or adenomyosis, severely damaged endometrium and chromosomal abnormalities of either part of the couple were excluded. Patients with incomplete basic endocrine parameters and untimely β-hCG and E2 data were also excluded from the study. Additionally, patients with an endometrial thickness less than 8 mm were not included in the analysis. Those who required exogenous estrogen supplements were also excluded due to their slow endometrial growth without supplementation. Finally, a total of 1521 cycles with positive β-hCG values were collected, forming two groups: CE Group (n = 595) and B Group (n = 926).

Blood samples were collected on day 12 post-embryo transfer, followed by quantification of serum E2 and β-hCG levels. The CE Group was further divided into clinical pregnancy group (CE-CP Group, with gestational sac) and biochemical pregnancy group (CE-BP Group, without gestational sac), based on the cutoff value of β-hCG ≥ 5 mIU/mL [8]. Similarly, the B Group was divided into blastocyst-clinical pregnancy group (B-CP Group) and blastocyst-biochemical pregnancy group (B-BP Group). A flow diagram of the patient-selection process is presented in Fig. 1.

Detail criteria and selection of the cohort selection. FET: frozen-warmed embryo transfer; NCs: natural endometrial preparation cycles; CE: cleavage embryo; B: blastocyst; CE-CP: cleavage embryo to clinical pregnancy; CE-BP: cleavage embryo to biochemical pregnancy; B-CP: blastocyst to clinical pregnancy; B-BP: blastocyst to biochemical pregnancy

In the natural FET cycles, endometrial maturation is determined by endogenous estradiol and progesterone, which are generated by stimulating the growth of the endometrium and the development of follicles. Regularly menstruating women can use NCs to plan FET without the need for high dose exogenous hormone supplements. However, oral low-dose estrogen (Estradiol Valerate, Bayer, Germany) will be administered to certain patients whose endometrium thickness is consistently thin, particularly when the dominant follicle is up to 14 mm, in order to promote endometrial growth. Finally, FET will be carried out if the endometrial thickness is ≥ 8 mm. Otherwise, the cycles will be cancelled. Furthermore, for those who might not make enough progesterone during embryo transfer, progesterone treatment is carried out during natural cycles.

Following 12–14 days of menstruation, an ultrasound evaluation was done to track follicle growth and measure follicle size. When the follicle reaches a diameter of 16 mm, daily transvaginal ultrasound becomes necessary, and serum levels of LH, E2, and P can be detected until spontaneous ovulation. It is routine to do NCs-FET at the cleavage embryo and blastocyst on Days 0 + 3 and 0 + 5 days, respectively, taking into account that the day of ovulation is Day 0. For luteal phase support, oral progesterone tablets (Duphaston, Abbott, 20 mg) and vaginal progesterone gels (Crinone, Merck Serono, Germany, 90 mg qd) were used. Support for the luteal phase was continued until week 10, at when the β-hCG tests turned positive.

D3 cleavage embryos were divided into four groups based on their cell number, symmetry of blastomeres, and degree of fragmentation. Grade I included embryos with 7–9 cells, symmetrical blastomeres, and < 5% fragmentation; Grade II included embryos with 6 cells, symmetrical blastomeres, and < 5% fragmentation; Grade III included embryos with 4–5 cells, symmetrical blastomeres, and < 5% fragmentation; Grade IV included embryos with < 4 cells. We classified Grade I and Grade II as high-quality embryos.

Blastocyst morphology was assessed based on the Inner cell mass (ICM) and trophectoderm (TE) which were categorized according to the number and structure of cells. The appearance of tightly packed ICM/TE cells was defined as “A”; several grouped cells as “B”; a few loose cells as “C”. High-grade blastocysts were defined as AA, AB, BA or BB expanded or hatched blastocysts with high ICM/TE grading.

The levels of serum E2 and β-hCG were quantified using commercially available automated electrochemiluminescence immunoassays (DxI 800 Immunoassay System, Beckman Coulter, USA). All measurements were performed by skilled technicians in strict adherence to the manufacturer’s instructions.

Pregnancy outcomes were as follows: biochemical pregnancy, clinical pregnancy, ongoing pregnancy, and live birth [9]. Biochemical pregnancy [10] was defined as a serum β-hCG level ≥ 5 mIU/mL 12 days after embryo transfer, and that fails to progress to the point of ultrasound confirmation of gestational sac 28–35 days after embryo transfer. Clinical pregnancy was defined as an intrauterine/extra uterine gestational sac that was detected 28–35 days after embryo transfer by ultrasound.

IBM SPSS Statistics for Windows Version 27.0 (Armonk, NY: IBM Corp) software was used to analyze all the data. For analysis, we performed Kolmogorov-Smirnov test to determine whether variables were normally distributed or not, and then parametric continuous variables were expressed as mean values and standard deviation (̄x ± s), the difference was assessed using T-test. Where required, non-parametric variables were analyzed with the median (1st and 3rd quartiles) and compared using the Mann Whitney U test. A Chi-squared test was used to compare rates.

The receiver-operating characteristic (ROC) analysis was used to assess the predictive accuracy of the serum E2 and β-hCG concentrations. AUC measures the diagnostic accuracy of the test, with a greater area corresponding to a greater ability to discriminate between patients with clinical pregnancy or not. AUC < 0.5 indicated no predictive value, 0.5 ≤ AUC < 0.7 indicated a low predictive value, 0.7 ≤ AUC < 0.9 indicated a moderate prediction value and 0.9 ≤ AUC < 1 indicated a high predictive value.

Spearman’s rank correlation (rs) was used to examine the relationship between pregnancy outcome and early serum E2 and β-hCG levels. The Pearson’s test was used to assess the difference.

Logistic regression analyses were used to study the association between variables (day 12 serum E2 and β-hCG concentration) and pregnancy outcome. Hosmer-Lemeshow test was done to assess the model’s goodness-of-fit.

Statistical significance was set at a two-sided P < 0.05.