Study subjects

In this study, a total of 1007 patients with chronic HBV infection were retrospectively recruited from eight counties/cities in Northeastern China, namely Changchun, Jilin, Yanji, Songyuan, Siping, Tonghua, Baishan, and Baicheng [16]. The chronic HBV-infected patients met the diagnostic criteria for chronic HBV infection according to clinical guidelines developed by the Chinese Medical Association (Guidelines for the prevention and treatment of chronic hepatitis B, 2019) [17]. The inclusion criteria for patients with chronic HBV infection were as follows: (1) age ≥ 18 years old; (2) serum HBsAg positive status for more than 6 months and serum HBV DNA quantitative positive status; (3) not receiving antiviral therapy at the time of enrollment. Of these patients, 951 were treatment-naïve patients with complete personal data and peripheral anticoagulated whole blood specimens who had not undergone liver biopsy, while 56 underwent percutaneous liver biopsy for a definitive diagnosis. The liver biopsies of these patients were obtained from the tissue specimen bank of China-Japan Union Hospital of Jilin University.

In addition, 1040 age-matched healthy individuals with a negative status of serum HBsAg and HBV DNA were included as controls. The following inclusion criteria were used for the healthy controls: (1) age ≥ 18 years old; (2) serum HBsAg negative status; (3) serum HBV DNA negative status.

The exclusion criteria were as follows: (1) chronic HCV infection combined with autoimmune liver disease, alcoholic liver disease, hepatic schistosomiasis, drug-related liver disease, genetic metabolic liver disease, or other liver diseases; (2) chronic HCV infection combined with liver cancer or other types of tumors; (3) recent use of steroid drugs and chronic HCV infection combined with pituitary tumors, polycystic ovary syndrome, or other diseases affecting hormone synthesis and secretion.

All the study subjects provided informed consent. This study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University (No. 2016- nsfc015) and was performed in compliance with the Declaration of Helsinki.

Clinical characteristics, laboratory tests, and virological characteristics

All the included patients were analyzed for determining their clinical characteristics, serum biochemical parameters, and serum virological parameters (i.e., HBV DNA quantification, hepatitis B serum markers, HBV genotype). Liver stiffness measurements (LSMs) were performed using transient ultrasound elastography (Fibroscan, Echosens, France) and expressed as E (kPa). The liver stiffness E values were graded according to the World Federation for Ultrasound in Medicine and Biology (WFUMB) 2015 guidelines [18]: no or mild fibrosis (corresponding to METAVIR stage F0–F1), E = 2.5–7.0 kPa; significant fibrosis (corresponding to METAVIR stage F2), E = 7.1–9.5 kPa; severe fibrosis (corresponding to METAVIR stage F3), E = 9.6–12.5 kPa; and cirrhosis (corresponding to METAVIR stage F4), E > 12.5 kPa.

Based on the approximate trend of sex hormone levels during reproductive aging, female patients were categorized into two groups: age ≤ 50 years old (i.e., reproductive women) and age > 50 years old (i.e., late menopausal women). Similarly, male patients were divided into two groups: age ≤ 50 years old and age > 50 years old [19, 20].

Single nucleotide polymorphism (SNP) analysis

The DNA extraction of blood samples was performed using the Blood Genomic DNA Rapid Extraction Kit (Shanghai Bioengineering Co., Shanghai, China). Primer Premier 5.0 software was utilized for designing SNP primers associated with AKR1C2, AKR1C3, HSD17B6, SRD5A1, SRD5A2, and ESR1. These primers were synthesized by Shanghai Bioengineering Co. Amplification of the SNP site sequences and the preparation of compatible Illumina sequencing libraries were accomplished through a two-step polymerase chain reaction (PCR). The resulting PCR products were then purified and recovered using AMPure XP magnetic beads. Equal amounts of individual PCR products were combined and sequenced using a HiSeq XTen sequencer (Illumina, San Diego, CA, USA). The key genes and primers are listed below.

AKR1C3 gene:

Forward primer: 5ʹ-CCCAGGTTCAATAGGAAAGAA-3ʹ, reverse primer: 5ʹ-ACCTTCACCCATGCACTTTC-3ʹ;

SNPs: rs2211623, rs2186174, rs2398203, rs2154306, rs4559587, rs12529, rs3763676, rs2096421.

AKR1C2 gene:

Forward primer: 5ʹ-CCGTCAAATTGGCAATAGAAGCC-3ʹ, reverse primer: 5ʹ-CAACTCTGGTCGATGGGAATTGCT-3ʹ;

SNPs: rs2801904, rs12414884.

HSD17B6 gene:

Forward primer: 5ʹ-TCTTTGTGGGAGAGTAGCTTC-3ʹ, reverse primer: 5ʹ-TTTGTCAGTCTCTTCCTTTCTCATC-3ʹ;

SNPs: rs4237805, rs898611.

SRD5A1 gene:

Forward primer: 5ʹ-GGTTTTGGCTTGTGGTTAACA-3ʹ, reverse primer: 5ʹ-CTCTTCAAATTTCCGGAGGTAC-3ʹ;

SNPs: rs248799, rs248800, rs6872996, rs3797177, rs1691053.

SRD5A2 gene:

Forward primer: 5ʹ-CGCGAAGTGATCCAGAAAC-3ʹ, reverse primer: 5ʹ-CAGGACCAGGTAGCCTGTG-3ʹ;

SNPs: rs2208532, rs12470143, rs523349, rs4952197, rs7594951, rs612224.

ESR1 gene:

Forward primer: 5ʹ-CUGUCUUCUGUUGUGGGAACA-3ʹ, reverse primer: 5ʹ-GGAGAAUGUUGAAACACAAUU-3ʹ;

SNPs: rs6909023, rs6920483, rs2077647, rs2234693, rs1062577, rs7753153, rs9340799.

Measurement of the METAVIR inflammatory fibrosis score and SRD5A2 protein expression level in liver tissue

Liver tissues from all the patients were fixed with formaldehyde, followed by routine dehydration and embedding in paraffin. Subsequently, the tissues were stained with hematoxylin–eosin (H&E) for pathological examination. The histopathological assessment of liver inflammation and fibrosis in chronic HBV-infected patients was conducted using the METAVIR scoring system as reported previously [21]. In this system, the inflammatory activity score A ranges from 0 to 3, while the fibrosis score (F) ranges from 0 to 4.

The primary antibody used in this study was SRD5A2 antibody (Anti-SRD5A2 antibody [EPR6281(B)]) obtained from Shanghai Uninvest Biotechnology Co. (Shanghai, China). The secondary antibody used was Goat-Anti-Rabbit IgG-HRP, diluted at a ratio of 1:10,000. Protein lysates were prepared from liver tissue using the Protein Extraction Kit (C510003-0050, Biotech), with the addition of broad-spectrum protease inhibitors and phosphatase inhibitors to the lysate. The protein concentration was determined using the BCA Protein Assay Kit (C503021-0500, Biotech). Subsequently, 20 μg of protein was mixed with the loading buffer and loaded into a 10–15% SDS–polyacrylamide gel electrophoresis system. The separated proteins were then electrotransferred onto a PVDF membrane. The resulting membrane was then incubated overnight at 4 ℃ with the primary antibody, followed by washing at room temperature for 2 h with the secondary antibody. Also, β-actin was used as an internal reference control to normalize the protein expression. The gray value of the bands was determined using Image J software, with the quantified value representing the ratio of the target protein to the internal reference protein.

Statistical analysis methods

Genetic equilibrium was assessed using the Hardy–Weinberg Equilibrium test (HWE) to determine the genotype frequencies, while the allele and genotype frequencies between groups were compared using the chi-square test. Count data were presented as frequencies and percentages, and statistical analysis was performed using either the chi-square test or Fisher's exact probability method. Measurement data were described by the mean and standard deviation (SD), and statistical analysis was performed using the t-test. Factor screening was carried out using logistic regression analysis, followed by sequential univariate and multivariate analyses. Variable screening was performed using the stepwise regression method. Differences were considered statistically significant at p < 0.05. Data analysis and graphical representation were conducted using SPSS 22.0 and GraphPad Prism 8 software.