Architect i2000

Serum HBsAg and HBeAg levels were measured by an Abbott Architect i2000 detection reagent (Abbott Diagnostics, Abbott Park, IL, USA); the HBsAg dynamic range was 0.05–250 U/ml. Samples with HBsAg levels >250 U/ml were diluted to 1:500–1:1000. The positive HBeAg levels were defined as >1 sample/cutoff (S/CO).

The parameters of liver function were measured using an automatic biochemical analyzer (7600-020, Hitachi, Japan). HBsAg, anti-HBs, HBeAg, and anti-HBe levels were measured using an Abbott Architect i2000 detection reagent (Abbott Diagnostics, Abbott Park, IL, USA). The dynamic range of HBsAg was 0.05–250 U/ml. Samples with HBsAg levels >250 U/ml were automatically retested at a 1:500 dilution. HBsAg <0.05 U/ml was defined as the disappearance of HBsAg. The serum HBV DNA level was quantitated with a Roche COBAS AmpliPrep/COBAS TaqMan 96 full automatic real-time fluorescence quantitative polymerase chain reaction detection reagent (with a lower limit of 20 U/ml) (Roche, Pleasanton, CA, USA).

Venous blood samples were obtained in the early morning (9–10 am) on the 3^rd to 5^th days of the menstrual cycle, which was spontaneous or induced by progesterone. Fasting plasma glucose levels were measured by photometric analysis (Abbott Architect C16000 AutoAnalyzer, Abbott Diagnostics, USA). Fasting serum insulin, follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, dehydroepiandrosterone sulfate (DHEAS), total testosterone, and thyroid stimulating hormone (TSH) levels were measured by chemiluminescent microparticle enzyme immunoassay (Abbott Architect i2000, Abbott Diagnostics, USA). Serum 17 hydroxyprogesterone (17-OHP) and free testosterone levels were specified by radioimmunoassay and high-sensitive C-reactive protein (hs-CRP) concentrations were measured by immunoturbidimetry (Abbott Architect C16000 AutoAnalyzer, Abbott Diagnostics, USA). The Homeostasis Model Assessment-Insulin Resistance (HOMA-IR) index was calculated as follows: $\text{HOMA-IR}=\text{Fasting\hspace{0.17em}plasma\hspace{0.17em}glucose\hspace{0.17em}}(\text{mmol}/\text{l})\times \text{Fasting\hspace{0.17em}serum\hspace{0.17em}insulin\hspace{0.17em}}(\text{mIU}/\text{ml})/22.5$

The parameters of liver and kidney functions, including ALT, aspartate aminotransferase, total bilirubin, albumin, creatinine, and blood urea nitrogen, were measured by Hitachi 7600 automatic biochemical analyzer (Hitachi 7600-11; Hitachi, Tokyo, Japan). Serum HBV DNA was quantitated using a Roche CobasAmpliPrep/CobasTaqMan 96 real-time fluorescence quantitative polymerase chain reaction detection reagent (with a lower limit of 20 U/ml; Roche, Pleasanton, CA, USA). The levels of HBsAg, anti-HBs, HBeAg, and anti-HBe were tested using an Abbott Architect i2000 detection reagent (Abbott Diagnostics, Abbott Park, IL, USA); the HBsAg dynamic range was 0.05–250.00 U/ml. Samples with HBsAg levels >250.00 U/ml were automatically re-tested at 1:500 dilution. HBsAg negative was defined as <0.05 U/ml.

Venipuncture was used to collect peripheral venous blood samples (5 mL) without anticoagulants between 7:00 and 9:00 am. The serum samples were centrifuged at 3000 rpm for 10 minutes and immediately separated, aliquoted, and stored at −80°C in a refrigerator until analysis. Blood tests were performed only once for each patient and control participant. The frozen samples were sent at the same time for analysis. Antinuclear antibody in serum was detected using an indirect immunofluorescence assay coated with HEp-2 cells (EUROIMMUN, Lubeck, Germany) using serial dilutions starting at 1:100. A fluorescence microscope was used to observe samples (EUROStarIII Plus, Lubeck, Germany). Antinuclear antibody profiles, which included anti-Jo-1, anti-nuclear ribonucleo-protein (nRNP), anti-Scl70, anti-Sm, anti-SS-A (R052), anti-SS-A, anti-SS-B, and anti-ds-DNA antibodies, were detected by line immunoassays (EURO Blot ONE, Lubeck, Germany). The sample was considered ANA positive if any of these antibodies were detected. The serum levels of anti-TPO and TG-Ab were measured with Abbott Architect I2000 (Abbott Diagnostics, Ireland). All analyses were performed following the instructions from the manufacturer, and recommended cut-off values were used. Borderline values were considered negative.