Study design and overview
This is a single-center, prospective observational study. The study were performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and all its protocols were approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine (approval number 2017[674]). Written informed consent was obtained from patients or their legal representatives prior to enrollment in the study.
Subjects
We consecutively recruited 229 patients with HBV-ACLF who underwent treatment with an artificial liver support system at the First Affiliated Hospital of Zhejiang University College of Medicine during the 14-month period between December 2018 and January 2020 by invitation. Of these, 69 were excluded from analysis (30 who were female, 28 who had other chronic liver disease, five with hepatocellular carcinoma, four who had undergone previous liver transplant, one who had undergone stem-cell transplantation, and one with pituitary tumor). During the study period, we recruited 151 age- and BMI-matched males with chronic hepatitis B but without liver failure from sample bank of major diseases in Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province as disease controls and 106 matched healthy controls without liver disease from the physical examination center. Exclusion criteria were: other diagnosed chronic liver disease (such as alcoholic liver disease, autoimmune liver disease, drug-induced liver injury, or other viral infection); hepatocellular carcinoma; previous transplant; hypogonadism; the presence of testicular, prostatic, or pituitary tumor; and use of hormone therapy (including estrogen, progesterone, testosterone compounds, or dehydroepiandrosterone supplements) or anticoagulation therapy such as warfarin.
Clinical diagnosis
The diagnostic criteria for HBV-ACLF followed the criteria of the Chinese Group on the Study of Severe Hepatitis B (COSSH) [3]: acute hepatic insult manifesting severe jaundice (total bilirubin [Tbil] ≥ 205 μmol/L) and coagulopathy (international normalized ratio [INR] ≥ 1.5) develops in patients with CHB and regardless of the presence of cirrhosis. We categorized subjects according to stage of HBV-ACLF; namely, early stage (1.5 ≤ INR < 1.9 without complications or extrahepatic organ failure), middle stage (1.9 ≤ INR < 2.6 with one complication and/or one extrahepatic organ failure), or end stage (INR ≥ 2.6 with two or more complications and/or extrahepatic organ failures).
Data collection
Clinical data was obtained from medical records relating to essential information (age, gender, weight, height), comorbidities (diabetes mellitus, hypertension), laboratory indexes (such as serum alanine aminotransferase [ALT], aspartate aminotransferase [AST], albumin, Tbil, INR, creatinine level, complete blood count), and complications (ascites, hepatic encephalopathy, infection, acute kidney injury, gastrointestinal hemorrhage). Severity scores (Model for End-stage Liver Disease [MELD] [14] and COSSH-ACLF [3] scores) were calculated using the most abnormal value for each organ system during hospitalization. The primary outcome, defined as death or transplantation within 90 days of first treatment of artificial liver support system, was analyzed through evaluation of medical records or by direct contact with the subjects or their families.
Laboratory analysis
Morning fasting blood samples were drawn from all subjects prior to treatment with the artificial liver support system and from controls at the time of recruitment. We analyzed the androgens using chemi-bioluminescent immunoassay. Serum TT, SHBG and DHEAS were measured on an Architect i4000 analyzer (Abbott Laboratories, Kallang Place, Singapore), cortisol was measured on an ADVIA Centaur XP (Siemens Healthcare Diagnostics Inc., Los Angeles, CA, USA) and androstenedione was measured on an Immulite 2000XPi (Siemens Healthcare Diagnostics Inc.). The FTI was calculated for each participant as (TT × 10)/SHBG [26]. The normal ranges for TT, SHBG, FTI, DHEAS, cortisol and androstenedione in males are 142.39–923.14 ng/dL, 17.1–77.6 nmol/L, 20.4–81.2%, 48.6-591.9 μg/dL, 5.27-22.45 μg/dL and 0.6-3.1 ng/mL, respectively.
Statistical analysis
All continuous variables are expressed as median, interquartile range (IQR; 25th and 75th percentiles), categorical data are presented as percentage and frequency. TT, FTI and SHBG levels were natural log-transformed and other androgen levels were square root-transformed. In univariate statistical comparisons, the Mann-Whitney non-parametric U test was used for continuous variables and the Kruskal-Wallis test to compare more than two groups. Categorical data were evaluated using a chi-squared test or Fisher’s exact test, as appropriate. Generalized linear models were used to predict an increase in stage of HBV-ACLF, ascites, and hepatic encephalopathy with decreasing or increasing androgen level. The relationships between androgen level and severity scores were examined in subjects with HBV-ACLF through multiple linear regression analysis. Kaplan-Meier estimation was used to evaluate the survival rates without transplant of groups with different testosterone levels. The log-rank test was used to compare mortality rates in terms of the composite outcome and in terms of death alone between groups with different testosterone levels. When analyzing mortality rates, subjects who underwent liver transplant were excluded. With regards to HBV-ACLF prognosis, the Cox proportional hazards model was fitted with a forward stepwise selection method (p-in: 0.05 and p-out: 0.01) to identify risk factors associated with the composite outcome of death or transplantation. All statistical analyses were carried out using SPSS version 19.0 (SPSS, Inc., Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.