Female Sex Hormones Pattern and Its Relation to Disease Severity and Treatment in Pre- and Postmenopausal Patients with Chronic Hepatitis C Virus (Genotype 4) Infection

Chronic hepatitis C (CHC) course revealed differences between men and women. Male gender and postmenopausal women are thought to be of the critical factors affecting HCV infection progression. The study aimed to assess female sex hormones and their relation to disease severity and treatment in HCV infected females. Subjects were divided to 2 groups: 44 CHC female patients and 44 controls. Both groups were classified to premenopausal and postmenopausal females. Serum estradiol (E2), progesterone (PRG), and total testosterone (TT) were assessed using chemiluminescent immunoassay. Our results showed that menopausal patients had significantly higher levels of estradiol, total testosterone, and progesterone compared to controls (P < 0.001). Reproductive aged patients had lower level of total testosterone compared to menopausal patients (P < 0.001). HCV infected females of reproductive age had higher level of progesterone compared to menopausal HCV infected females (P = 0.0014). Indicators of disease severity and treatment response were significantly worse in menopausal women compared to reproductive aged women (fibrosis: P < 0.001, activity: P = 0.045, and treatment: P < 0.001). We observed that lower estradiol level may be related to fibrosis severity in CHC females. Higher total testosterone and progesterone levels may be related to fibrosis severity and poor response to treatment in CHC menopausal females only.


Introduction
Hepatitis C is caused by HCV infection. It is estimated that about 3% of the world's population has been infected with HCV and 170 million are chronic carriers at risk of developing liver cirrhosis and/or liver cancer [1]. Prevalence of HCV in Egypt is 14.7% [2].
The clinical course of chronic hepatitis C revealed several differences between men and women. Male gender is thought to be one of the critical factors in the progression of HCV infection [3]. Besides, the development of hepatic fibrosis is faster in postmenopausal than in premenopausal women [4]. In addition, serum total testosterone is associated with increased risk of both advanced hepatic fibrosis and advanced hepatic inflammatory activity in HCV infected men [5].
Sex differences are believed to be one of the factors affecting response to therapy. Female sex correlated positively with sustained virological response (SVR) [6]. However, early menopause associated with lack of SVR among women with genotype 1 hepatitis C virus infection [7].

Type of Study and Selection of Patients.
Descriptive analytical study included 44 female patients, proven to have chronic hepatitis C infection [8] by polymerase chain reaction (PCR) and 44 control females, age matched to the patients, were selected from healthy blood donors, proven to be negative for HCV on hepatitis C antibody test.
Patients were recruited from Virology Center in Ismailia Fever Hospital, a referral center for treatment of HCV in Ismailia under the supervision of the Ministry of Health as part of the national project for combating viral hepatitis. Patients were subjected to thorough history taking, clinical examination, and routine workup including complete blood picture (CBC), serum transaminases (AST, ALT), total bilirubin, albumin, International Normalized Ratio (INR), alpha fetoprotein (AFP), and HCV quantitative PCR. Histopathological examination of histological activity and degree of hepatic fibrosis, of ultrasound guided percutaneous liver biopsy, was performed according to Metavir's score. Blood samples were included in the study between May 2013 and October 2013.

Serum Estradiol, Progesterone, and Total Testosterone
Assay. Serum estradiol, progesterone, and total testosterone were assessed using IMMULITE 1000 (Siemens, IMMULITE, catalog number LKE21 for estradiol, LKTW1 for total testosterone, and LKPW1 for progesterone), a solid-phase enzymelabeled, chemiluminescent immunoassay. Chemiluminescence generates electromagnetic radiation as light. The light intensity depends on a chemical reaction using a labeled enzyme and a chemiluminescent compound supplied as the enzyme substrate in excess to assure saturation kinetics. Light intensity is measured using stored master curves based on manufacturer's instructions.

Patients' Classification.
Patients were classified according to their reproductive status to 22 premenopausal and 22 postmenopausal females in each group (patients and controls). Further grouping of female patients according to (1) treatment (combined interferon and ribavirin therapy) response (nonresponders: patients who had detectable HCV RNA at weeks 12, 24, and 48; relapsers: patients who had detectable HCV RNA 24 weeks after stopping treatment; and responders: patients with sustained virological response (SVR) with undetectable HCV RNA six months after stopping treatment), (2) viral load (<600,000 and ≥600,000), and (3) fibrosis stage (≤F2 and >F2) was done to compare between groups.

Patients Characteristics.
Females included were nonsmokers, of reproductive age group (i.e., with regular menses, not pregnant or lactating), ≥18 years, and postmenopausal females (i.e., no menstrual period for 12 consecutive months).
CHC females having hepatocellular carcinoma (HCC), coinfection with human immunodeficiency virus (HIV), coinfection with hepatitis B virus (HBV), other causes of liver disease (alcoholic, autoimmune, and Wilson disease), previous liver transplant, hormone replacement therapy, hormonal contraception, and history of active substance or alcohol consumption were excluded.

Patients Consent.
All regulations adopted by the ethics committee in Faculty of Medicine, Suez Canal University, including patient consent were considered and approved.

Statistical Analysis.
Quantitative parametric variables were presented by mean and standard deviation (SD) and compared by Student's -test. Qualitative variables were compared by the chi-square test. In all tests, value was considered significant if <0.05.

Results
The demographic, clinical, biochemical, and histopathological data of the studied subjects are shown in Table 1. Similar distributions were noted for age, BMI, age at menarche, marital status, reproductive status and the presence of diabetes and/or hypertension in HCV female patients, at reproductive age and menopausal group, and their respective healthy controls. Among the baseline laboratory data platelets count and albumin levels were significantly lower ( = 0.007 and 0.03, resp.), while INR level was significantly higher ( = 0.005) in the menopausal patients compared to the reproductive aged patients. Menopausal women had worse indicators of disease severity (fibrosis: < 0.001; activity: = 0.045) and reduced response to treatment ( < 0.001) compared to reproductive aged women.
Hormonal levels in the studied subjects showed that estradiol level is higher in menopausal patients compared to their healthy controls ( < 0.001) (Table 1). Similarly, menopausal HCV infected females have significantly higher level of total testosterone than controls ( < 0.001). Total testosterone level is lower in reproductive aged HCV infected females than menopausal HCV infected females ( < 0.001). Progesterone level in HCV infected females of reproductive age group was higher than in menopausal HCV infected females ( = 0.0014).
The relation between fibrosis and hormonal levels in reproductive aged and menopausal patients is shown in Table 2. Estradiol level appeared to be higher in the group with fibrosis stage ≤F2 than the group of fibrosis stage >F2 ( = 0.005) for reproductive aged patients. In menopausal patients total testosterone and progesterone levels were significantly lower (TT: = 0.002; PRG: = 0.023), while estradiol level was significantly higher ( = 0.003) in group with fibrosis stage ≤F2 than the group of fibrosis stage >F2.
The relation between virological response and hormonal levels in the studied groups is shown in Table 3. While the majority of the reproductive aged women responded to HCV therapy, only 13.6% appeared to be nonresponders and 9.1% were relapsers. In menopausal patients, total testosterone and progesterone levels were higher in the nonresponder group than in the responder group (TT: < 0.001; PRG: < 0.001). No -test was done for reproductive aged patients as there were great differences in numbers of each group, which cannot be compared statistically. The study of menopausal patients included one patient who showed relapse in response to treatment with hormonal levels (E2 = 97.3, TT = 29.00, and PRG = 0.60). Table 4 showed that the comparative relationship of hormonal levels and viral load in both groups (reproductive aged and menopausal females) appeared with no statistically significant value in all variables. Table 5 showed the correlations between hormonal levels in reproductive aged and menopausal patients and the fibrosis stage, activity grade, and virological response. Table 6 showed the relation between estradiol level and fibrosis in reproductive aged and menopausal patients; lower estradiol level was associated with significant increase of fibrosis level in reproductive aged patients between 35 and International Journal of Chronic Diseases 3   International Journal of Chronic Diseases      and menopausal patients (Table 7) showed that estradiol level was significantly lower with nonresponders in menopausal patients between 47 and 55 years ( = 0.03).

Discussion
The findings of our study reveal that CHC infection is associated with elevated levels of estradiol, total testosterone, and progesterone in both pre-and postmenopausal females when International Journal of Chronic Diseases 5 compared to their healthy controls. This may be explained by the impaired metabolism of these hormones as a result of the defective liver function in chronic liver disease patients [9][10][11][12][13]. Menopausal HCV infected women had higher levels of estradiol, total testosterone, and progesterone when compared to their healthy controls ( < 0.001, <0.001, and <0.001, resp.). On the other hand reproductive aged women had statistically nonsignificant increased levels of these hormones when compared to their healthy controls ( < 0.28, <0.346, and <0.80, resp.). Menopausal Greek females with HCC females had also higher levels regarding estradiol and testosterone compared to their healthy controls [13]. To our knowledge this is the first study to evaluate progesterone level in women with HCV infection.
There were higher significant level of total testosterone and lower significant level of progesterone in menopausal women with CHC compared to females of reproductive age with CHC, and this was not the case for estradiol level that showed a slight, however, nonsignificant decrease ( = 0.093). On the other hand the comparison between the same groups (menopausal and reproductive aged females) in control women showed higher significant levels regarding estradiol and progesterone levels in reproductive age females ( < 0.001, <0.001, resp.) and nonsignificant difference in testosterone level between both groups.
HCV infection is associated with increased hormonal levels of estradiol, total testosterone, and progesterone in both groups reproductive aged and menopausal women; moreover estradiol and progesterone levels decreased with age, while total testosterone level increased with age in HCV group. In addition, although hormonal levels in reproductive aged group were higher than their healthy controls they are of statistically nonsignificant values and appeared to be minimally affected by hepatitis C, while menopausal group had hormonal levels high enough to be statistically significant in all variables compared to their healthy controls, providing evidence that this group was highly affected by hepatitis C.
Our study revealed that women of reproductive age had lower disease severity as measured by the liver necroinflammation activity and fibrosis compared to older menopausal women. The observed changes in the studied hormonal levels could be implicated in these findings. The high estradiol and progesterone levels and low total testosterone level in the reproductive aged patients may in part have protected against the development of severe liver injury. In contrast, menopausal women exhibited greater disease severity, probably due to the decline in estradiol and progesterone levels and the rise in total testosterone level that occurs with menopause development, supporting the concept that the progression of fibrosis in women is not on the same pace: mild during reproductive age and severe after menopause. Similar to our findings, Villa et al. 2012 conducted a study on four groups of women divided according to their reproductive stage (fully reproductive, premenopausal, early menopausal, and late menopausal) in addition to four age-matched groups of men serving as controls [14]. According to this study they found that liver fibrosis was advanced in the early menopausal HCV infected women compared to fully reproductive or premenopausal women. In addition, late menopausal women had higher liver fibrosis compared with younger women [14]. The favorable role of estrogens in liver disease has also been suggested by Shimizu et al., 2001, andMaffei et al., 2004, two case reports, in which different pathological conditions required prolonged estrogen treatment in two young men, one with hepatitis C and the other with nonalcoholic steatohepatitis. In the first case, the addition of estradiol reduced disease activity and maintained viral loads at lower levels than those observed before treatment. In the second case, it reversed hepatic steatosis and insulin resistance [15,16].
The total testosterone level was significantly higher in menopausal HCV infected females compared to reproductive aged HCV infected females. This finding is in agreement with the recently reported positive correlation between testosterone level and increased risk of both advanced hepatic fibrosis and advanced hepatic inflammatory activity in HCV infected men [5], in which higher unopposed level of testosterone related to higher severity of the disease and increased risk of HCC in men [25]. Other studies did not correlate high testosterone levels to hepatic fibrosis [26]. Potential explanations for the discordant findings of our study and those studies may include differences in underlying target populations, host factors, or difference in viral genotype.
Our findings of excess testosterone-associated risk in HCV positive females are consistent with the findings of increased liver cancer risk in males who abuse anabolic steroids, in androgen-treated Fanconi anemia patients [27], in women with hyperandrogenemia secondary to polycystic ovarian disease [28], and in HBV positive Taiwanese males with higher serum testosterone level [29][30][31].
To our knowledge no studies addressing the changes in progesterone level have been conducted yet. Our study showed that menopausal patients had poorer responses to combined therapy than reproductive aged patients (menopause versus reproductive age: < 0.001). Previous studies including genotype 1 (most common genotype in Japan and Italy) highlighted the same findings [7,32,33].
The relationship between hormonal parameters in menopausal women and virological response showed that higher total testosterone and progesterone levels were significantly associated with unfavorable outcomes ( < 0.001 and <0.001, resp.). Estradiol level appeared to be of no significant relationship. A number of previous studies suggested, however, a potential link to estrogen secretion [32,33]. Another nonconcordant study, the study of Furusyo  [34]. These inconsistent results may be explained by the difference in host genetic factors, viral genotype (Egypt common genotype 4 versus Japan common genotype 1), or ethnicity. Impairments in humoral, cellular, and innate immunity in the elderly and modulation of inflammatory factors, such as IL-6 and TNF-, at menopause may be another important factor which are responsible for decreasing successful responses to treatment in menopausal patients [7,35].
Our study revealed that there was a statistically nonsignificant difference regarding viral load values between the two groups. Besides, the comparative relationship of hormonal levels and viral load appeared to be of statistically nonsignificant value in all variables of both groups (menopausal and reproductive aged females). This finding is supported by one in vitro study that treated human hepatoma-derived cell line (Huh-7.5 cells) with E2 and PRG and found inhibition of HCV virion production (in case of E2 only) but not HCV RNA replication or HCV protein synthesis [36]. On the other hand, to our knowledge, no studies were found addressing its relation to total testosterone level.

Conclusion
The present study explored the possible involvement of female sex hormones in the pathogenesis and/or progression of liver disease in HCV infected Egyptian females. We observed that lower estradiol level is significantly related to fibrosis severity in CHC females, while higher total testosterone and progesterone levels are significantly related to fibrosis severity in CHC menopausal females only. Besides, higher total testosterone and progesterone levels are related to poorer treatment response in CHC menopausal females, while estradiol level is not. Additionally, the detected higher hormonal levels did not have significant relation to viral load in both groups. We suggest that screening of progesterone and total testosterone levels should selectively target females with high grade fibrosis. Our findings underscore the potential favorable effects of antitestosterone treatment to target HCV infected females. We assume that giving hormone replacement therapy to infected menopausal females would not have a beneficial effect regarding HCV course or treatment response.