Clinical impact of sexual dimorphism in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)

1 INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are leading cause of liver disease and liver transplantation (LT) worldwide.^{1, 2} NAFLD/NASH are sex-dimorphic diseases, with a general higher prevalence in men.³ However, protective effect observed in fertile women seems to be lost after menopause. Thus, consideration on sex and hormonal status related to age (puberty, menopause) is critical in defining risk factors, disease prevention and treatment of NAFLD.⁴

Despite many of the major risk factors for NAFLD, including metabolic syndrome (MS), type 2 diabetes mellitus (T2DM) and regional adiposity, are known to present clear and profound sex dimorphism, fewer publications describe sex differences in NAFLD, compared to other areas of medicine.

An appropriate attention to biological sex differences, age, hormonal status, but also gender differences, including dietary patterns, exercise and quality of life should be equally considered in the assessment of NAFLD.⁵ This consideration will lead to a better understanding of sex differences in NAFLD and NASH, and will aid in achieving sex-specific personalized treatments and therapies.^{6, 7}

In this review, we summarize the main current findings about the influence of sex and gender on epidemiology, pathogenesis, disease progression, indication for LT and treatments.

2 METHODS

A literature search was performed to identify studies regarding sex and gender influence in NAFLD/NASH, published up to March 2021. Relevant research and review articles were identified by searching the PubMed database, Ovid MEDLINE and Ovid EMBASE, limited to articles published in the English language but not date restricted.

Search terms included “fatty liver” OR “NAFLD” OR “NASH” OR “steatosis” AND “gender” OR “sex” OR “sex differences”. Additional searches were also made for each of the individual section of our review article. Selected articles referenced in these publications were also examined.

3 EPIDEMIOLOGY OF NAFLD/NASH

The incidence and prevalence of NAFLD are increasing dramatically in either Western or Eastern countries, reaching epidemic proportions.^{8, 9} The main reason for this dramatic increasing trend in NAFLD and NASH is the global increasing prevalence of obesity described by the World Health Organization.¹⁰ In longitudinal studies, mostly performed in Asia, the incidence of NAFLD ranges from 19 to 44.5/1000 person-years and is higher in males compared to females.^11-21 The global prevalence of NAFLD has been recently updated,^{1, 8, 9, 22} the estimated prevalence of NASH in Europe is 23.71% (95% CI 16.12%-33.45%).²³ In Italy, the Dionysos Study comprising 6,917 persons from the general population in two Northern areas found a 25% prevalence of fatty liver and was higher in males compared to females (40% vs 23%).^24-26 Considering the different components of metabolic syndrome, prevalence NAFLD is dramatically high in obese patients, reaching 98%²⁷ and in T2DM patients, 55.5% of cases, with the highest prevalence in studies from Europe (68%), whereas the prevalence of NASH is 37.3%.²⁸

Interestingly, children carry a high risk for NAFLD, having 8%-10% prevalence of NAFLD in the Italian population.²⁹ In a recent population-based study of young adults in UK, one in five young people had steatosis and one in 40 had fibrosis around the age of 24.³⁰ In the US, the prevalence of NAFLD in children and adolescents increased from 3.3 between 1988 and 1994 to 10.1% between 2005 and 2010. Similarly, the prevalence of NASH increased from 0.74% to 3.4% in the same period.^{30, 31}

The prevalence of NAFLD is higher in men than in women (Table 1, refs.^{11, 32-37}). In particular, the prevalence of men in NAFLD cohorts ranges between 4.3% and 42%, whereas the prevalence of women ranges between 1.6% and 24%. However, among each sex group, the wide difference in prevalence can be explained by the methods for definition of NAFLD, the gold standard being, among the non-invasive techniques, the magnetic resonance spectroscopy. The role of sex and reproductive status in the development and progression of NAFLD has been focused in a recent review by Ballestri et al.³⁸ One study from Japan¹⁵ reported that the prevalence of NAFLD in pre-menopausal women was lower (6%) than in men (24%) and in post-menopausal women (15%). Moreover, NAFLD was more prevalent in women receiving HRT than in pre-menopausal women. In Shanghai population, the prevalence of NAFLD was higher in males than females under the age of 50, but was lower in males than females among people older than 50 years.³⁹ Analogously, a study from Japan reported a higher prevalence of NAFLD in males than females, and showed an increasing prevalence of NAFLD during adulthood from young to middle age, and a reduction of prevalence after the age of 50-60.⁴⁰ In general, women are at reduced risk of NAFLD compared to men in fertile age, whereas after menopause women lose their protective effect and have a comparable prevalence of NAFLD as men.⁴¹ Post-menopausal women tend to increase body weight along with a shift in its distribution to an increase of visceral fat.⁴² Moreover, post-menopausal women on hormone replacement therapy (HRT) have been found to have a lower prevalence of NAFLD compared to post-menopausal women not taking HRT.⁴³ A double-blind randomized trial with HRT or placebo in post-menopausal women with T2DM and presumed NAFLD showed a significantly decreased levels of aminotransferase in the group treated with HRT compared to placebo.⁴⁴ These figures contrast with a paper in which the prevalence of NAFLD was explored in 1170 community-based adolescents in the Western Australian Pregnancy cohort.⁴⁵ Females compared with males had a significantly higher prevalence of NAFLD (16.3% vs 10.1%, P =.004). In this study, the sex differences in NAFLD prevalence were associated with significant differences in adipose distribution and metabolic parameters, including adipocytokine levels. Finally, a recent review summarizes the current knowledge on sex differences in NAFLD, identifies gaps and discusses important considerations for future research.³ Indeed, gender and sex hormones interact with numerous NAFLD factors including genetic variants, cytokines, stress, environmental factors and alter the risk profiles and phenotypes of NAFLD in individuals.³

4 PATHOGENESIS

NAFLD is a multi-factorial disease where a predisposed genetic background is under the negative influence of a number of environmental and lifestyle-related factors.⁶² In the presence of a positive calorie balance and unfavourable genetic pattern, free fatty acids (FAs) accumulate in ‘ectopic’ tissues, including the liver,⁶³ resulting in accumulation of fat droplets in hepatocytes. Excessive fat within the liver is associated with different degrees of lipotoxicity,⁶⁴ which triggers a multicellular response to damage, leading to the development of inflammation, fibrosis and eventually cirrhosis. This process is modulated by signals deriving from extrahepatic tissues, such as the adipose tissue, the intestine and possibly the skeletal muscle.^{64, 65} The differences in pathogenesis based on sex are summarized in Figure 1 and are discussed in details below.

5 END-STAGE LIVER DISEASE AND ACUTE-ON-CHRONIC LIVER FAILURE

The prevalence of cirrhosis among 492 biopsy-proven NASH patients was higher in women (57%) than in men (43%).⁹⁰ In a study on 144 patients with NAFLD, sex was not an independent risk factor for the progression of fibrosis, while, at multivariate analysis, older age (>45 years), obesity, presence of diabetes and AST/ALT ratio>1 were independent significant predictive factors of severe fibrosis.⁹¹

Acute-on-chronic liver failure (ACLF) is characterized by multiple organ failure with high short-term mortality. Despite ACLF incidence due to NASH is lower when compared to other aetiologies, it is the fastest growing liver disease aetiology among all ACLF hospitalizations, as demonstrated in a recent US population-based study.⁹² In this study, the authors detected an exponentially increased frequency of ACLF admissions for NASH aetiology with 12% of NASH-related ACLF in 2006-2008, 33% in 2009-2011 and 55% in 2012-2014. NASH-ACLF patients are more frequently women (60%) and older than in other aetiologies, with a more severe course characterized by higher mortality, more frequent need of dialysis and higher length of stay. Metabolic derangements such as obesity and diabetes might also play a confounding role in the pathophysiology, clinical course and prognosis of NASH patients with ACLF.⁹³

6 NASH AND HCC

In Europe, NASH is an increasingly common aetiology for HCC.⁹⁴ The yearly cumulative incidence of HCC is 2.6% in patients with NASH-related cirrhosis.⁹⁵ Irrespective of its aetiology, HCC predominantly affects males with an incidence four times higher in males than females.⁹⁶ The reasons for this sex disparity are complex and may be associated with a protective role of oestrogen on HCC development,⁹⁷ until menopause. For this reason, the median age for women is higher than that of men at the time of HCC diagnosis. Most studies showed that men are more likely to have larger tumours with increased rates of macrovascular invasion and extrahepatic spread, and are more likely to undergo transplantation than women.^{98, 99} The pathogenesis of HCC in the context of NAFLD is not completely clear; importantly 20% of NAFLD-HCC are diagnosed in the setting of non-cirrhotic liver.^100-102 Grade-based recommendations for surveillance in patients with NASH did not justify a systematic surveillance in NAFLD patients without cirrhosis, as a result of low currently observed incidence of HCC in this setting.¹⁰³ Older age, diabetes, advanced fibrosis and obesity are the main risk factors associated with HCC development in NAFLD patients, with or without cirrhosis.^104-106 In a US multicentre retrospective study,¹⁰⁷ the authors showed that there was significantly more non-cirrhotic HCC in women than in men. This may reflect the large number of NAFLD-related HCC in women in the cohort. This is very important from a surveillance point of view. Finally, they found that women had less-advanced HCC and had a greater overall survival, leading to different treatment options. Even when HCC is diagnosed at a potentially curable stage, LT or resection is not always feasible because of obesity and comorbidities. However, there have been excellent long-term outcomes in patients undergoing liver resection,¹⁰⁸ loco-regional therapies such as radiofrequency ablation, selective internal radiotherapy and transarterial chemoembolization can also be proposed.^109-113 Sex has a role also in HCC treatment, related to different tumour morphology at diagnosis, as men present larger HCC than women. Moreover, some studies showed that oestrogen can reduce the activation of stellate cell, making slower the liver fibrinogenesis.¹⁰⁷ Thus, women are less likely to have complications such as portal vein thrombosis and renal dysfunction, which may prevent HCC curative treatments. For both reasons, curative treatments for HCC are more common in women than men.^114-116 Sobotka et al¹¹⁵ confirmed that women are still more likely to undergo resection and also determined that women are more likely to undergo ablation because of more compensated liver disease, being NAFLD present similarly in the 2 groups.

In conclusions, women show frequently a more compensated liver disease compared to men, developing non-cirrhotic HCC-related NAFLD. Moreover, at diagnosis, women have smaller tumour than men, more amendable to curative HCC treatments.

7 NASH AS INDICATION FOR LIVER TRANSPLANTATION

NASH has become the leading indication for LT in the US¹¹⁷ and NASH/HCC is the most rapidly growing in the waiting list for LT.¹¹⁸ NASH was the second cause for LT globally in the last years, the leading cause for LT for women, the second leading cause for men and the leading cause by 2016 among Asian, Hispanic and non-Hispanic white females.¹¹⁹ In Nordic countries, NAFLD was the second most rapidly increasing indication for LT between 1994 and 2015, going from 2% in 1994-1995 to 6.2% in 2011-2015.¹²⁰ A recent Italian monocentric study highlighted that the proportion of NASH patients listed for LT significantly increased from 5% to 9.5% after the introduction of direct-acting antiviral agents for HCV, also showing a significant increase of those listed for HCC (from 2% to 5.9%).¹²¹

8 LIFESTYLE INTERVENTION IN NAFLD/NASH PATIENTS

The main strategy to treat NAFLD focuses on the control of underlying risk factors like diabetes, hyperlipidaemia, obesity and other comorbidities. Since no effective pharmacological treatment for NAFLD/NASH has been approved, the lifestyle changes, consisting of balanced diet and physical activity represent the cornerstone of intervention for NAFLD treatment.¹³²

Recent studies have reported that a 6- to 12-month widespread lifestyle modification, based on reduced energy intake and increased physical activity, leads to improvement in liver enzymes and metabolic parameters, and reduced steatosis and necroinflammation, including ballooning and fibrosi.^133-135 The most recent European and American guidelines recommend that lifestyle interventions should be included as part of the clinical care of all NAFLD patients, regardless of the stage of disease.^{136, 137}

Preclinical studies demonstrated in animal models that males and females react differently to caloric restriction and intermittent fasting,¹³⁸ but clinical human data are still deficient. Only few studies have explored whether sex differences affect weight loss after surgical and non-surgical treatments, achieving conflicting results.^{139, 140} In general, with lifestyle interventions, men lose more weight and present greater metabolic benefits compared to women.¹⁴¹ This higher metabolic effect could be partly explained by the fact that during weight loss, men lose mainly visceral adiposity compared with women, who principally lose subcutaneous adiposity.^{142, 143}

Similarly, in NAFLD patients, higher histological improvement was observed in men than women after weight loss. In fact, in male patients, a modest weight loss, between 7% and 10%, produces a significant histological improvement, while in women a more consistent weight loss (>10%) is necessary to obtain the same effect.¹⁴⁴

It is well known that inadequate physical activities and sedentary lifestyle are risk factors for NAFLD.¹⁴⁵ Considering this aspect, women are generally more sedentary and have a lower tendency to meet physical activity guidelines. However, they showed a stronger beneficial effect of increased physical activity against NAFLD compared to men. Indeed, the physiological response to exercise appears to be different in men and women because of the different composition of the muscle fibres and different lipid metabolism.^{66, 146} Women's muscle is characterized by a higher number of type I muscle fibres which has higher capabilities of lipid oxidation, a higher content of intramyocellular lipids and are more sensitive to insulin than men's muscle.¹⁴⁷

Some attention should be paid to post-menopausal women, in whom significant weight reduction achieved through dietary restrictions can induced negative effects on lean muscle and bone mass.¹⁴⁸ To prevent this side effect, an integrated approach consisting of dietary changes along with regular resistance training or aerobic exercises is mandatory.^{148, 149} However, only few studies have specifically assessed the role of physical activity in NAFLD post-menopausal women. The available data, however, confirm that physical activity and exercises effectively reduce liver enzymes in overweight post-menopausal women, probably because of the reduction in liver fat content and as the same time reduce cardiovascular risk factors.^{150, 151}

Thus, optimal lifestyle modifications may differ between men and women, or between pre- and post-menopausal women. However, this topic is still largely unexplored, so these important questions should be better addressed in future interventional clinical trials.

9 PHARMACOLOGICAL TREATMENT FOR NASH

Despite the high prevalence of NAFLD worldwide, currently there is no effective pharmacological treatment for NAFLD/NASH. The current therapies mainly focus on metabolic disorders associated with NAFLD.¹⁵²

Regardless the clear sexual dimorphism of NAFLD/NASH, very few studies on possible therapeutic strategies deal with sex differences or consider natural age-related hormone fluctuations as a disease-modifying factor. A recent Chinese study has found pioglitazone to be more efficient in reducing liver fat content in NAFLD female than in male diabetic patients.¹⁵³

A phase 2b study on MK-3655 (an insulin sensitizer) in pre-cirrhotic NASH is currently recruiting men and post-menopausal women (NCT04583423). Whether this patient selection will lead to the evaluation of sex-specific outcomes is not reported.

Most of the sex-specific studies on NAFLD therapy are targeted at sex hormones supplementation or inhibition; meanwhile, data on sex- and age-specific response to drugs in phase 3 trials are lacking (Table 2).

10 CONCLUSIONS AND FUTURE DIRECTIONS

Although sex differences exist in the prevalence, risk factors, fibrosis and clinical outcomes of NAFLD, our understanding of sex differences in NAFLD has remained significantly more limited so far, compared to other areas of medicine. To fill this gap, more accurate epidemiological and pathophysiological data obtained from larger cohort studies are needed.

The future direction in the management of NAFLD/NASH patients – regardless the gender – should be the early identification of NAFLD to prevent progression to fibrosis and NASH. Moreover, detailed program of education, especially in the young population at risk of develop overweight, obesity and liver steatosis should be developed.

Therefore, an appropriate consideration of sex, age, hormonal status and sociocultural background will lead to a better understanding of sex differences in NAFLD risk, and will aid in personalization of therapies. In addition, it is mandatory to stimulate the design of clinical studies aimed at sex and gender differences in NASH therapy to better characterize sex-specific therapeutic targets and responses.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.