Menopause is certainly not the first issue that comes to mind when talking about hepatitis C. However, the liver is much more sensitive to estrogen (and therefore to its deprivation) than it is usually believed. Changes induced by menopause have profound consequences not only on the classical target organs, such as the ovary, uterus, or breast, but also on the liver. The consequences are manifold: estrogen deprivation is associated with faster progression of hepatic fibrosis [1,2], lower sensitivity to antiviral therapy , and eventually to a high risk of developing hepatocellular carcinoma (HCC) [4,5]. It is therefore worthwhile to examine all the aspects of the relationship between estrogens and the liver in order to better understand these phenomena.
LIVER AS AN ENDOCRINE ORGAN
The liver is not a classical endocrine organ. However, it is certainly sensitive to the action of estrogens, to which it responds by increasing the synthesis and secretion of several glycoproteins, such as ceruloplasmin, corticosteroid-binding globulin, thyroid-binding globulin, and testosterone-estradiol–binding globulin. During pregnancy, there is a marked upregulation of several estrogen-dependent proteins (eg, ceruloplasmin), and this is an indirect demonstration of the sensitivity of the liver to estrogens. The molecular basis for this responsiveness is the presence in the liver of both alpha [6,7] and beta  estrogen receptors, which mediate the action of estrogens on the liver. This indirectly suggests that the fluctuations of hormonal balance throughout the course of the life span may exert more direct influences on apparently unrelated conditions, such as, for example, hepatitis and progression of chronic liver disease.
PROTECTIVE ROLE OF ESTROGENS ON THE LIVER
It is well known that estrogens are powerful antioxidants. In an experimental rat model of dimethylnitrosamine-induced fibrosis, Shimizu et al  showed that estradiol was capable of suppressing early apoptosis and development of hepatic fibrosis by inhibiting hepatic stellate cell proliferation and fibrogenesis. As estrogens and their antioxidant effect decline, lipogenesis and lipid peroxidative processes increase within the liver [2,10]. This may lead to enhanced production of reactive oxygen species (ROS), which constitutes a general feature of sustained inflammatory response and liver injury, once antioxidant mechanisms have been depleted. Estrogens are also thought to protect hepatocytes from oxidative damage, inflammatory cell injury, and cell death by suppression of activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B) activation and induction of Bcl-2 expression . Moreover, the persistence of the original insult (viral infection, alcohol abuse, or metal overload) causes prolonged activation of tissue repair mechanisms: the repair reaction becomes excessive and, instead of being effective in restoring a normal condition, leads to hepatic fibrosis.
Menopause and the liver
Traditionally, menopause is defined as the period of life in women when menstrual cycles definitively stop. The average age of menopause occurrence in Western countries is between 46 to 52 years. Physiologically, menopause represents functional ovarian exhaustion. It is divided into early (<5 years) and late (>5 years) menopause. As the pool of ovarian follicles becomes exhausted, there is a decrease of circulating estrogens. This determines a long series of consequences at the level of many different organs and tissues (bone, heart, brain, adipose tissue, etc) that are only partially counteracted by the use of hormone replacement therapy (HRT). The consequences at the hepatic level are less immediate to perceive, although present. Eventually, in menopausal women, there is a shift from a low-inflammatory state toward a proinflammatory state [3,12], with more rapid progression of fibrosis in post-menopausal women than that which occurs with younger, fertile females .
Mechanisms of progression of liver damage in menopause
In addition to the usual mechanisms of liver damage determined by either viruses or exogenous substances, menopause may have a synergic effect on liver damage. To further complicate the matter, menopause coincides with aging and aging per se is known to lead to the development of profound modifications and to aberrant body homeostasis. Indeed, though liver function does not deteriorate only because of age, nevertheless, several conditions, such as triglyceride levels, insulin resistance, nonalcoholic fatty liver diseases, increase with aging .
The hormonal changes occurring with menopause leads to an increased inflammatory status: there is a large body of evidence indicating that the decline in ovarian function is associated with spontaneous and marked increases in Th1 and Th17 cytokines (such as tumor necrosis factor-α [TNF]-α, interleukin (IL)-1β, and IL-6) [12,14]. In comparison, there is a profound imbalance of cytokines such as IL-10, which have anti-inflammatory properties . The final consequences of these changes are that, in a post-menopausal woman, there is evidence of profound change in the overall inflammatory status, from the balanced status during fertile age to a marked proinflammatory state soon before and immediately after menopause. Necroinflammatory status, as well as fibrosis [1,2] and steatosis , increase: in due course, there is also an increased risk of tumoral transformation . Estrogens, at physiological concentrations, were shown to inhibit the spontaneous secretion of these proinflammatory cytokines in whole blood cell cultures, as well as in vivo: treatment with estrogens transdermally in post-menopausal women was shown to decrease the spontaneous IL-6 production by peripheral blood mononuclear cells (PBMCs) after 12 months of therapy . HRT in vivo, however, must be initiated soon after the onset of menopause in order to be effective, otherwise positive effects may not be as remarkable .
Menopause and HCV
The relationship between menopause and hepatitis C is bidirectional, that is one influencing the other and vice versa.
Cieloszyk et al  showed, in a large cross-sectional study on hepatitis C virus (HCV) infection and its relationship to menopausal status and symptoms, that HCV is independently associated with natural menopause and with vasomotor symptoms. Women infected with HCV had an odds ratio (OR) of 1.68 post-menopausal at younger age than were uninfected women (median age of HCV-infected vs HCV-uninfected: 46 vs 47 years of age). The authors suggested that, in their patients, liver disease may alter the hypothalamic-pituitary axis, thus favoring cessation of menses. Additional risk factors present in this cohort (drug addiction, low social levels, human immunodeficiency virus [HIV] coinfection) could have contributed to an earlier onset of menopause; nevertheless, that HCV can anticipate the occurrence of menopause (and of the associated hepatic consequences) had not been shown before.
Di Martino and coworkers , who showed a few years ago that menopausal HCV-positive women had accelerated progression of fibrosis, suggested that HRT and pregnancies may have a beneficial effect on long-term progression of fibrosis. Codes et al  emphasized the relevance of metabolic features in the relationship between menopause and HCV: they suggested that the high severity of liver disease in menopause may be partly explained by the prevalence of features (increased intra-abdominal fat, atherogenic lipid profile, steatosis, insulin resistance) of the metabolic syndrome, which occurs in women during the transition period leading to menopause, and by the loss of the protective effect of estrogens.
The metabolic syndrome may depend on menopause, possibly due to a wrong diet behavior (leading to obesity), or to a specific genetic background (mostly associated with type 2 diabetes). Data also show that it may be due to HCV . HCV-infected women, therefore, face a risk of developing severe liver disease that is multiplied by 3 or 4 as a consequence of the intertwining of these factors.
Obesity per se may cause insulin resistance and hepatic steatosis leading to steatohepatitis and hepatic fibrosis, thus contributing to disease progression. In patients with HCV, the following are associated with inflammation: insulin resistance (mediated by both viral and host interactions), steatosis, progression of fibrosis, and reduced response to treatment. Charlton and coworkers  reviewed the mechanisms through which obesity is associated with less response to therapy: apart from causing insulin resistance and hepatic steatosis leading to steatohepatitis and hepatic fibrosis, these authors emphasized that obesity is also an inflammatory condition in which oxidative stress increases, through adipokine secretion (eg, leptin), which may modulate the immune response and have proinflammatory activity. Leptin also upregulates important proinflammatory cytokines (IL-1 beta, IL-6, IL-12, and TNF-α), reduces the activity of anti-inflammatory cytokines (IL-10) , and also appears to play a role in the activation of multiple signaling pathways in hepatic stellate cells, further contributing to intrahepatic inflammation and fibrogenesis.
The prevalence of overweight is rising worldwide, and weight gain in midlife is common among women. The effect of the menopausal transition (and therefore the circulating level of sex steroids) on weight and body fat distribution is not totally clear, but some studies suggest that it is associated with an accumulation of central fat, particularly intra-abdominal fat, which produces alterations in glucose utilization leading to insulin resistance . Furthermore, visceral fat contributes to increase the inflammatory burden as cytokines (especially IL-6) are produced in vitro 2 to 3 times more by visceral than by subcutaneous fat [19,20]. HRT has been shown, when given early during menopausal transition, to prevent the shift from subcutaneous to visceral fat .
HEPATITIS C AND AGE
Severity of disease, change in prognosis, and response to antiviral therapy
Chronic HCV infection is a disease of later life: HCV patients are constantly about a decade older than hepatitis B virus infected individuals and have either chronic hepatitis, cirrhosis, or HCC . Although the rate of fibrosis progression varies markedly from person to person, median time from infection to cirrhosis is roughly 30 years. Several studies have shown that cirrhosis is prevalent in men and in menopausal women and that the severity of fibrosis in chronic hepatitis C is significantly different between males and females: male sex is an independent risk factor for the severity of liver fibrosis. While females younger than 40 years of age seem to have mild disease and higher probability of spontaneous HCV clearance [2,22], females at approximately 50 years of age experience acceleration of the disease . Shimizu and Susumi  emphasized that cirrhosis, with the exception of autoimmune liver disease, is largely a disease of men and post-menopausal women and that mechanisms related to sex-linked differences may be based on biological factors, including estrogen-related female sex hormones, rather then simply gender differences in social environment and lifestyle.
Younger age (usually <40 years of age) has also often been identified as a predictor of sustained viral response (SVR) to antiviral therapy together with genotype, histological stage of liver disease, pretreatment viral load, weight-adjusted therapies, sex, and race [3,24-29]. So far, gender and age have been evaluated separately and only in a few studies was there an attempt at studying their possible relationship (Table 1). Hayashi et al  were among the first authors assessing the importance of age in relation to gender when evaluating response to conventional interferon α (IFNα) treatment alone. They concluded that women younger than 40 years of age had higher rates of SVR than did men (75 vs 33%) and that this advantage was lost after 40 years of age. They hypothesized that decreasing estrogen levels around the age of 40 could play a role in the worsening of response to IFNα. Sezaki et al , in a retrospective study, evaluated the influence of gender on treatment of genotype 1 chronic hepatitis C. They concluded that the response to combined therapy with pegylated (PEG) IFNα and ribavirin was poorer among females than among male HCV-infected patients aged 50 years or older (32 vs 63%) . More recently, Petta et al  showed in a series of 83 consecutive treatment-naive G1 chronic hepatitis C patients that female gender was associated with an OR of failing SVR of 0.132: 70% of women not achieving SVR were post-menopausal. It has to be emphasized, however, that in this study, females had several other associated risk factors, such as hepatic steatosis and other metabolic imbalances, all known factors associated with scarce response to IFN. In a recent study evaluating predictors of SVR for HCV genotype 1 patients enrolled in 2 phase III studies with PEG-IFNα-2a, Reddy and coworkers  showed that patients >50 years of age had poorer responses to combined therapy than did patients <50 years of age. Interestingly, the lower SVR rates in older patients primarily resulted from higher relapse rate.
||TABLE 1. Relationship between age and SVR
The importance of considering age in a patient initiating therapy for HCV is emphasized also by the recent study by Davis and coworkers , who developed a multicohort natural history model for predicting disease outcomes and benefits of therapy in HCV-infected patients. These authors showed that fibrosis progression was inversely related to age at infection, so cirrhosis and its complications were most common after the age of 60 years, regardless of age of infection: older patients who exhibit lower responses are indeed those most in need of an effective therapy.
Menopause and response to antiviral treatment
The data discussed earlier prompted the present authors to begin, more than 5 years ago, a comprehensive evaluation of the relationship between menopause, HCV infection, and response to antiviral treatment. On the whole, data was collected from a large cohort of 1000 male and female patients with chronic hepatitis C undergoing standard PEG-IFNα/ribavirin treatment and analyzed for predictors of SVR. Attention was focused especially on hormonal and metabolic features and the interplay with age . Menopause not only was correlated with more severe liver disease, with high necroinflammatory features, and with high rate of hepatic steatosis, but was also the strongest negative factor for SVR both in the whole female cohort and in the subgroup of HCV genotype 1 females (in whom HCV was the only independent factor for failure of antiviral therapy). A striking difference between fertile and menopausal females (which may help explain the significantly more severe disease in the latter) was the remarkable increase in the level of 2 cytokines (IL-6 and TNF-α) that cooperate in inducing the necroinflammatory damage in patients with HCV. Despite already being upregulated in female patients, these cytokines underwent a further and significant increase at the time of menopause, both at the circulating and hepatic level. Again, menopause may be considered causally involved in the strength of this upregulation, as the shift toward a different body fat composition, with increase in the visceral fat, strengthens the production of inflammatory cytokines [18,19]. This involvement of extrahepatic sites in contributing to inflammatory upregulation may be one of the reasons for the significantly higher SVR achieved, in this cohort, by PEG-IFNα-2b as compared with PEG-IFNα-2a. It has been suggested, in fact, that the different pharmacokinetics of the 2 PEG-IFNs, with the α-2b having smaller molecular weight and therefore being able to better reach extrahepatic tissues (eg, fat), may account for such a difference .
The most obvious, practical consequence of these results would be to foster use of HRT to increase SVR at levels reported for premenopausal women, as the data cited previously would suggest . Thus, more than 2 years ago, a prospective randomized trial of standard antiviral therapy combined with HRT was begun (EudraCT 2008-001260-36). It is too early to define whether this approach may indeed be useful in improving SVR. It is already evident, however, that the addition of HRT enhances tolerability of the combined antiviral therapy decreasing the risk of premature antiviral therapy termination.
Menopause certainly represents a seminal event in the life of a woman and, from many points of view, not an agreeable one. The possible interventions, in order to slow the inevitable, at the different levels, are many: diet, physical activity, and drugs counteracting the alterations of lipid and glucose metabolism. This holds true also for HCV-positive women entering menopause. However, for the latter ones, there is the additional burden of HCV infection and the negative influence of menopause on liver disease. Until effective measures to bypass these additional difficulties are available, the best strategy is to cure HCV-associated liver disease early, when the response is maximal and SVR high, as well as in difficult HCV genotypes (such as genotypes 1 or 4), disregarding the fact that liver disease in fertile women is mild. This favorable condition, in fact, will last only as long as women are exposed to estrogens.
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