Human Ovary Granules KGN were obtained from Procell Life Science & Technology Co., Ltd (CL-0603, Wuhan, China). KGN cell-specific medium was sourced from Procell Life Science & Technology Co., Ltd (CL-0603, Wuhan, China). 25-hydroxyvitamin D3 (25 (OH) D3) was sourced from Sigma (17938-1MG, Darmstadt, Germany). The short tandem repeat (STR) typing of KGN cell line DNA showed that no cross contamination of human cells was found in the cell line.

Endometriosis patients in the First Hospital of Lanzhou University Reproductive Medicine Centre diagnosed between December 2021 and February 2022 were included in the study (75 cases). Patients with infertility due to fallopian tubes in the same period as the control group (219 cases). Inclusion criteria for the endometriosis group: women aged 21 to 38 years undergoing IVF assisted pregnancy; Patients with no history of hormone use within three months; Patients diagnosed with bilateral or unilateral ovarian endometriosis via laparoscopy, laparotomy, and took a biopsy and sent it for pathological examination, with a clear diagnosis [24]. According to the revised American Society of Reproductive Medicine (r-ASRM) classification [25], all patients with endometriosis were classified into stage I (micro): 1–5 points, stage II (mild): 6–15 points, stage III (medium): 16–40 points, and stage IV (severe): > 40 points. All patients with endometriosis included in our study were in stage II. Inclusion criteria for the control group: women aged 21–38 years undergoing IVF; Patients with no history of hormone use within three months; Patients with tubal obstruction (proximal, distal, unilateral, or bilateral) based on hysterosalpingography. Fresh day 3 blastocysts were transferred. To a greater extent, we controlled for confounding factors that affect vitamin D levels (season, stage, ethnic group, diet, medical conditions, kidney and digestive system diseases, etc.). Exclusion criteria included: patients with endometrial polyps, endometritis or endometrial cancer, polycystic ovarian syndrome (PCOS), hyperprolactinemia, hypothyroidism, androgen secreting tumor, Cushing's syndrome, congenital adrenal hyperplasia and diabetes; Patients with congenital abnormal uterine development, diseases that affect calcium or vitamin D anabolism; Patients with systemic conditions that are at high risk of pregnancy complications (hypertension, diabetes, coronary artery, kidney and liver diseases). The study was approved by the Clinical Research and Ethics Committee of the Lanzhou University Affiliated First Hospital, and informed written consent was obtained from all patients (Ethics number: LDYYSZLL2023-09).

ELISA assay was conducted as follows: 5 mL fasting venous blood was extracted via venipuncture, mixed upside-down, then left at room temperature for 2 h. The sample was centrifuged at 4000 r/min for 5 min, and the upper serum was frozen at -80 °C. FF was collected after egg extraction, centrifuged at 2000 r/min for 10 min, and the supernatant was frozen at -80 °C. Follicular fluid was collected during egg retrieval, and blood contaminated follicular fluid was not included. An enzyme-linked immunosorbent assay (ELISA) kit (Mei Jing Biotechnology Co., LTD., JM-04677H2, Jiangsu, China) was used to measure 25 (OH) D3 levels. The samples and standard product were reacted at 37 °C for 30 min. The samples were then washed and reacted with enzyme-labeled reagent for 30 min. Finally, the samples were washed, followed by addition of color developing agent A, B. A termination solution was used to stop the reaction. OD value was detected at 450 nm to calculate the actual concentration of the final sample.

Normal ovarian granule KGN cell line was used for CCK8 assay. The sample was cultured at 37 °C in 5% CO2 while cell growth was evaluated daily. The medium was changed regularly, and cell passage was performed after 2–3 days. Briefly, 1 × 105 KGN cells (per well) were inoculated in 96-well plates for 24 h, then treated with 25 (OH) D3 (1 nM, 10 nM, 100 nM, 1000 nM) for 0 h, 6 h, 12 h, 24 h. The medium was removed, then the sample was washed with PBS. CCK8 solution (10 μl; coolaber Technology Co., LTD., SK2060-500 T, Beijing, China) was added to each well, then incubated at 37 °C for 4 h. The absorbance was measured at 450 nm with an enzyme marker. Cell viability in each group was calculated as follows: cell viability = [OD (dosing) – OD (blank)] / [OD (no dosing) –OD (blank)] × 100%.

The cells were treated with 25 (OH) D3, then underwent trypsin hydrolysis (0.25% trypsin). The cells (1 × 106) were then washed with PBS, centrifuged at 1000 r/min for 5 min, and supernatant was discarded. DNA Staining solution (1 ml) and 10 ul Permeabilization solution were added to the cells (Linke Biological Co., LTD., CCS012, Hangzhou, China), mixed and incubated in the dark at room temperature for 30 min. Cell cycle was then detected via flow cytometry (BD, LSRFortessa, USA). The ratio of G0G1, S and G2M phase cells was also analyzed. Proliferous index (PI) was calculated as follows: PI = (S + G2M)/(G0G1 + S + G2M).

Granulosa cell expression of patients with ovarian endometriosis and tubal factor infertility was searched from the online website Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). GSE168214 dataset contains three pairs of endometriosis patients and tubal infertility patients with ovarian granulosa cell samples. Herein, the DEGs were screened at threshold | log2FC |≥ 1 and p < 0.05. The genes associated with 25 (OH) D3 anabolic metabolism were screened from Gene Set Enrichment Analysis (GSEA) (gsea-msigdb.org). Interactive genes were obtained from DEGs and vitamin D anabolism related genes. The DEGs were analyzed using Gene Ontology (GO) enrichment pathway to explore the functions and action pathways and understand the underlying molecular mechanisms.

RNA was extracted from cells using TRIzol (Coolaber, Beijing, China), RNA was reverse transcribed to prepare complementary DNA (cDNA). Quantitative real-time PCR was perfumed using fluorescence quantitative premix kit (TIANGEN, Beijing, China). After adding enzyme-free water, primer R, primer F, cDNA and Super Real PreMix Plus to the octet tube, centrifuge instantaneously and set the program and cycle. The mRNA expression levels of CDKN2D, PPARA, TGFB2 and THBD in the treatment group were analyzed using real-time PCR system. GAPDH was used as an internal reference gene. mRNA expression levels were calculated using the 2 − ΔΔCT method (Table 1).

KGN cell samples were lysed and centrifuged to extract protein. The lysate was added and sonicated on ice, centrifuged at 12000 r/min for 15 min and the supernatant was removed. The supernatant is the total protein solution. Protein concentration was measured using the Bicinchoninic Acid (BCA) method. The protein was denatured by adding loading buffer and boiling at 100 °C for 15 min, and stored at -80 °C in portions. The sample underwent electrophoresis after gluing at 80 V for 30 min, then at 120 V for 60 min. The sample was transferred to PVDF membrane, then sealed with 5% skim milk for 1 h. The sample was incubated with diluted primary antibody (1:1000, San Ying Biotechnology Co., LTD., 14318–1-AP, 10272–2-AP, 19999–1-AP, 15540–1-AP, 81115–1-RR, Wuhan, China) at 4℃ overnight. The sample was washed thrice the next day using TBST (10 min each wash), then incubated with secondary antibody (1:10000) at room temperature for 1 h. Color development was conducted for gel imaging analysis.

SPSS 22.0 and GraphPad Prism 9 were used for all statistical analyses. All experiments were performed in triplicates. Continuous variables were represented by mean ± standard deviation and compared using t-test. If normality was not met, Mann Whitney U test was used for comparison. Quantitative data that did not conform to normal distribution were expressed as median and quartile [M (P25, P75)]. Classification variables were expressed as percentages. Chi-square test was used for the comparison of qualitative data between groups. The influencing factors of pregnancy outcome in endometriosis patients were analyzed via Logistic regression. Pearson correlation analysis was also conducted. P < 0.05 was considered statistically significant difference.