Serum Testosterone Level Within Normal Range Is Positively Associated with Nonalcoholic Fatty Liver Disease in Premenopausal but Not Postmenopausal Women

Abstract

Background:

A positive relationship between testosterone level and nonalcoholic fatty liver disease (NAFLD) in women has been reported, but findings concerning the association are conflicting and inconclusive.

Materials and Methods:

We examined the association between testosterone level and the risk of NAFLD after stratification by menopausal status in 613 women (223 premenopausal women aged 21–52 years and 390 postmenopausal women aged 46–75 years). A diagnosis of fatty liver was based on abdominal ultrasonography. The odds ratios (ORs) and 95% confidence intervals (95% CIs) for NAFLD with 1 nmol/L increment in the log testosterone concentration were calculated after adjusting for confounding variables using multiple logistic regression analysis.

Results:

The prevalence of NAFLD was 19.2% among premenopausal women and 33.3% among postmenopausal women. After adjusting for age, regular exercise, type 2 diabetes, body mass index, mean arterial pressure, fasting plasma glucose, triglyceride, and high-density lipoprotein cholesterol, and testosterone levels, the OR (95% CI) for NAFLD was 2.79 (1.11–7.08) with 1 nmol/L increment of the log testosterone concentration in premenopausal women. However, these positive associations were not found in postmenopausal women after adjusting for the same covariables.

Conclusion:

Serum testosterone level was positively associated with NAFLD in premenopausal women but not in postmenopausal women. Our findings suggest that higher androgenic activity may be at least partly involved in the pathogenesis of NAFLD, particularly in premenopausal women.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is characterized by diffuse triglyceride accumulation in the liver not caused by excessive alcohol use or other causes of liver disease. This condition encompasses a spectrum of clinical syndromes ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) that can progress to advanced fibrosis, cirrhosis, and cirrhosis complicated by hepatocellular carcinoma.^1
–3 With the growing epidemic of obesity, NAFLD is one of the most prevalent causes of chronic liver disease, and NASH cirrhosis is expected to be the leading cause of liver-related death worldwide.^4
–6 While simple hepatic steatosis is more common among men, there is a higher rate of development of NASH from simple steatosis among women.^7,8 On the basis of these considerations, it is essential to understand the factors associated with NAFLD in women to prevent liver-related death due to NASH cirrhosis.

Accumulating evidence supports sex-specific relationships between androgen hormones and NAFLD. For men, testosterone deficiency has been consistently associated with an increased risk of NAFLD in cross-sectional and prospective studies.^9

–13 Contrary to the consistent relationships in men, epidemiological studies linking serum testosterone level with NAFLD found inconsistent and conflicting results in women. Some observational studies have reported positive associations in postmenopausal women¹³ and inverse associations in premenopausal women,¹⁴ whereas others have revealed no significant relationship.¹⁵ A recent meta-analysis showed that women with higher serum testosterone levels were more likely to have increased odds ratios (ORs) of NAFLD,⁹ but did not find a significant mean difference in serum testosterone concentration by NAFLD presence.

Although the reason for these discrepancies in the association between serum testosterone levels and NAFLD in women is not clear, the effects of testosterone on insulin resistance and metabolism may vary with menopausal status. We hypothesized that higher testosterone is associated with the presence of NAFLD according to menopausal status, since testosterone usually declines in postmenopausal women. Therefore, this study aimed to assess the association between serum testosterone level and NAFLD in premenopausal and postmenopausal women.

Materials and Methods

Study participants

We retrospectively reviewed the medical records of 716 women aged 20 years or older who underwent a medical examination at the Health Promotion Center of Gangnam Severance Hospital in Seoul, Korea, between November 2013 and July 2015. Natural menopause was defined as 12 consecutive months with no menstrual periods.

We excluded participants who met at least one of the following criteria according to a questionnaire regarding self-reported lifestyle, menstrual and medical history, and blood test results: those on current oral contraceptive medication, exogenous estrogen replacement therapy, or tamoxifen therapy; those with a history of induced menopause, such as bilateral oophorectomy or radiation- or drug-induced menopause; those with a history of hepatobiliary or renal disease; those with alcohol intake of 70 g/week or more, a positive test for hepatitis B antigens or hepatitis C antibodies, and those who did not fast for 12-hour before testing. We also excluded those with missing data. We also excluded women with an intermittent menstruation during the previous year or those with a higher than normal reference level of testosterone to rule out the possibility of polycystic ovarian syndrome or perimenopause. After applying the exclusion criteria, 613 women (223 premenopausal women aged 21–52 years and 390 postmenopausal women aged 46–75 years) were included in the final analysis.

The participants voluntarily attended the health promotion center regularly to undergo a health examination. Informed consent was obtained from each participant. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Yonsei University College of Medicine (Seoul, Korea).

Data collection

Each participant completed a questionnaire about self-reported lifestyle, menstrual, and medical history. Cigarette smoking, alcohol consumption, and physical activity were determined from the questionnaires on a weekly basis. Smoking status was categorized into nonsmoker, ex-smoker, and current smoker. Alcohol drinking was defined as consumption greater than or equal to twice per week. Participants were asked about the type and frequency of leisure-time physical activity on a weekly basis. Regular exercise was defined as exercise greater than or equal to three times per week. Menstrual history was also measured through a question “did you ever experience a lack of menstruation for 1 year or more?” with three alternatives: “Yes” “No, but there was intermittent menstruation during the previous year,” and “No, menstruation was normal.”

Medical examinations were performed by trained medical staff using a standardized procedure. Body mass and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, in light indoor clothing, without shoes. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m²). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in the patient's right arm with a standard mercury sphygmomanometer (Baumanometer; W.A. Baum Co., Inc., Copiague, NY). The mean arterial pressure was calculated using the equation (SBP +2 × DBP)/3.

All blood samples were obtained from the antecubital vein after a 12-hour overnight fast. Fasting plasma glucose, triglycerides, high-density lipoprotein (HDL)-cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured by enzymatic methods using AU5800, an automated chemistry analyzer (Beckman Coulter). Testosterone concentration was measured with eletrochemiluminescence immunoassay (ECLIA) using the Cobas e601 immunoanalyzer (Roche Diagnostics, Basel, Switzerland). The normal range of serum testosterone is 0.17–1.81 nmol/L in premenopausal women and 0.09–1.6 nmol/L in postmenopausal women.

Diagnosis of NAFLD

A diagnosis of fatty liver was based on abdominal ultrasonography with a 3.5-MHz transducer (HDI 5000; Philips, Bothell, WA). Ultrasonography was performed by two experienced radiologists who were unaware of the aims of the study and blinded to laboratory findings. We analyzed the linear weighted kappa statistics to analyze agreement between the two radiologists because the outcome encompasses ordinal scoring, and the kappa index (95% confidence interval [CI]) was 0.809 (0.7823–0.8676).

The presence of hepatic steatosis was determined according to the findings of high hepatorenal echo contrast, bright liver, or attenuation of ultrasound in a deep area of the liver. Hepatic steatosis was graded according to the criteria previously described¹⁶: mild, slight diffuse increase in bright homogeneous echoes in the liver parenchyma, with normal visualization of the diaphragm and portal and hepatic vein borders, and normal hepatorenal contrast; moderate, diffuse increase in bright echoes in the liver parenchyma, with slightly impaired visualization of the peripheral portal and hepatic vein borders; and severe, marked increase in bright echoes at a shallow depth, with deep attenuation and impaired visualization of the diaphragm and marked vascular blurring. Liver with any degree of hepatic steatosis was considered NAFLD in this study.

Statistical analysis

Normal distribution was evaluated by determination of skewness using the Kolmogorov–Smirnov test. The clinical characteristics of the study population according to the presence of NAFLD were expressed as mean (standard deviation), median (interquartile range [IQR]), or percentages and compared using the independent t-test or Wilcoxon rank-sum test for continuous variables and the chi-square test for categorical variables. Serum triglycerides, AST, ALT, and total testosterone levels have skewed distribution, so these variables were log transformed before multiple logistic regression analysis. The ORs and 95% CIs of log-transformed testosterone level for NAFLD were calculated after adjusting for confounding variables using multiple logistic regression analysis. All analyses were conducted using SAS statistical software (version 9.4; SAS Institute, Inc., Cary, NC). All statistical tests were two-sided, and statistical significance was determined at p < 0.05.

Results

Table 1 shows the clinical and biochemical characteristics of the participants in relationship to the presence of menopause and NAFLD. The overall prevalence of NAFLD was 28.1%, and it was significantly higher in postmenopausal women than premenopausal women (33.3% vs. 19.2%). The mean values of BMI, mean arterial pressure, fasting plasma glucose level, and leukocyte count and the median values of triglycerides, AST, ALT, and total testosterone levels were higher, while HDL-cholesterol level was lower in both pre- and postmenopausal women with NAFLD. The median (IQR) value of testosterone levels in postmenopausal women was significantly lower than in premenopausal women—0.50 (0.30–0.80) in postmenopausal women and 0.85 (0.60–1.20) in premenopausal women (p < 0.001; data not shown).

Table 1.

Clinical Characteristics of the Study Population by Menopausal Status and Nonalcoholic Fatty Liver Disease

	All women			Premenopausal women			Postmenopausal women
	NAFLD (−)	NAFLD (+)	p	NAFLD (−)	NAFLD (+)	p	NAFLD (−)	NAFLD (+)	p
N	440	173		180	43		260	130
Age (years)	52.1 ± 9.1	57.3 ± 7.0	<0.001	44.5 ± 6.6	48.8 ± 3.8	<0.001	57.6 ± 6.4	59.1 ± 6.1	0.009
BMI (kg/m²)	21.7 ± 2.6	24.7 ± 3.0	<0.001	21.2 ± 2.5	24.9 ± 3.2	<0.001	22.2 ± 2.5	24.7 ± 3.0	<0.001
MAP (mmHg)	87.8 ± 12.1	96.1 ± 13.0	<0.001	85.5 ± 11.8	92.1 ± 9.6	0.006	89.5 ± 12.0	97.0 ± 13.4	<0.001
FPG (mmol/L)	4.98 ± 0.61	5.58 ± 1.23	<0.001	4.86 ± 0.54	5.30 ± 0.69	0.048	5.06 ± 0.64	5.64 ± 1.31	<0.001
Triglycerides (mmol/L)	0.83 (0.64–1.11)	1.26 (0.89–1.79)	<0.001	0.80 (0.63–1.06)	1.15 (0.90–1.38)	<0.001	0.87 (0.67–1.21)	1.29 (0.89–1.83)	<0.001
HDL-cholesterol (mmol/L)	1.51 ± 0.34	1.31 ± 0.29	<0.001	1.54 ± 0.31	1.33 ± 0.25	<0.001	1.50 ± 0.36	1.30 ± 0.30	<0.001
AST (U/L)	19 (16–23)	22 (19–28)	<0.001	17 (15–20)	19 (16–24)	0.068	21 (18–24)	22 (20–30)	<0.001
ALT (U/L)	16 (12–20)	22 (18–34)	<0.001	14 (11–17)	18 (15–25)	<0.001	17 (14–22)	23 (18–36)	<0.001
Testosterone (nmol/L)	0.61 (0.40–1.01)	0.60 (0.42–0.98)	0.483	0.61 (0.32–1.12)	0.93 (0.69–1.19)	0.006	0.59 (0.38–0.87)	0.58 (0.36–0.89)	0.192
Current smoking (%)	4.4	3.0	0.798	6.6	3.3	0.907	3.0	3.5	0.965
Regular exercise (%)^a	40.4	38.0	0.639	31.1.	40.6	0.308	46.8	36.1	0.041
Type 2 diabetes (%)^b	1.9	8.7	<0.001	0.5	2.8	0.308	2.9	10.2	0.002

Data are expressed as mean ± standard deviation or median (IQR), or percentage. p Values were calculated using the independent two-sample t-test or the Wilcoxon rank-sum test for continuous variables or the chi-square test for categorical variables.

Regular exercise was defined as exercise at least three times per week.

Type 2 diabetes was defined as a current medication of antidiabetes or an FPG concentration ≥7.0 mmol/L.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; FPG, fasting plasma glucose; HDL, high-density lipoprotein; IQR, interquartile range; MAP, mean arterial pressure; NAFLD, nonalcoholic fatty liver disease.

Table 2 shows the results of multiple logistic regression analysis to assess the odds for predicting the presence of NAFLD with 1 nmol/L increment of log testosterone concentration according to menopausal status. After adjusting for age, regular exercise, type 2 diabetes, BMI, mean arterial pressure, fasting plasma glucose, triglyceride, HDL cholesterol, and testosterone levels, the OR (95% CI) for NAFLD was 2.79 (1.11–7.08) with 1 nmol/L increment of the log-testosterone concentration in premenopausal women. However, this positive association between testosterone concentration and prevalence risk of NAFLD was not found in postmenopausal women after adjusting for the same covariables.

Table 2.

Results of Multiple Logistic Regression Analysis to Assess Independent Relationships Between Log-Transformed Testosterone Levels for Nonalcoholic Fatty Liver Disease According to Menopausal Status

	All women	Premenopausal women	Postmenopausal women
Age (years)	1.03 (1.00–1.05)	1.17 (1.06–1.30)	0.98 (0.93–1.01)
Regular exercise (yes)	0.79 (0.50–1.23)	1.15 (0.44–2.99)	0.67 (0.40–1.14)
Type 2 diabetes (yes)	1.86 (0.58–5.93)	0.51 (0.11–24.63)	2.04 (0.58–7.09)
BMI (kg/m²)	1.35 (1.23–1.47)	1.50 (1.23–1.82)	1.35 (1.21–1.51)
MAP (mmHg)	1.01 (0.99–1.03)	1.00 (0.96–1.04)	1.01 (0.99–1.03)
FPG (mmol/L)	1.04 (1.02–1.06)	1.02 (0.97–1.07)	1.04 (1.02–1.06)
Triglyceride (mmol/L)^a	3.69 (2.10–6.51)	3.53 (1.05–11.80)	4.61 (2.34–9.11)
HDL-cholesterol (mmol/L)	0.99 (0.97–1.01)	0.97 (0.93–1.02)	1.01 (0.98–1.03)
Testosterone (nmol/L)^a	1.08 (0.77–1.51)	2.79 (1.11–7.08)	1.01 (0.70–1.47)

Multiple logistic regression analysis model included age, regular exercise, type 2 diabetes, BMI, MAP, FPG, triglyceride, HDL cholesterol, and testosterone levels.

Log-transformed values due to skewed distributions.

Discussion

In this cross-sectional study, we found that serum testosterone level was positively associated with NAFLD in premenopausal women after adjusting for potential confounding variables. Our findings are consistent with those of previous studies demonstrating that higher testosterone levels are closely associated with insulin resistance disorders, such as visceral adiposity,¹⁷ type 2 diabetes,¹⁸ metabolic syndrome,¹⁹ and NAFLD⁹ in women.

The results from previous studies concerning the association between testosterone level and NAFLD in women are conflicting and inconclusive. Using the dataset of the Coronary Artery Risk Development in Young Adults (CARDIA) cohort, Sarkar et al. reported a positive association between testosterone level and NAFLD in 1052 premenopausal women in the United States, which is consistent with the findings of our study.¹⁴ On the contrary, another observational study using the National Health and Nutrition Examination Survey in the United States, found that low total testosterone level was associated with suspected NAFLD in postmenopausal women and not in premenopausal women. However, the study defined NAFLD from the laboratory cutoff point of ALT levels and not on the basis of imaging studies, suggesting the possibility of misclassification bias.¹³ In a more recent meta-analysis, Jaruvongvanich et al. reported that the pooled OR of NAFLD in women was 1.40 (1.11–1.77) when summarizing three observational studies, but they failed to identify the pooled mean difference in the testosterone level of seven observational studies.⁹ Therefore, further longitudinal studies are warranted to establish the relationship between serum testosterone level and NAFLD by menopausal status.

The exact mechanisms underlying the menopause-specific relationship between testosterone level and NAFLD remains uncertain, but some plausible explanations can be offered. Although there is debate regarding whether a high circulating level of testosterone results from insulin resistance or is a driver of insulin resistance, a high testosterone level in women is associated with abdominal visceral fat mass, a decrease in skeletal muscle mass, and hepatic insulin resistance.²⁰ Furthermore, previous studies on spironolactone, an androgen receptor antagonist, have suggested that antiandrogen therapy could help prevent NAFLD in women. Spironolactone treatment improved visceral adiposity, insulin resistance, lipid profiles, and NASH histology in previous human and experimental studies.^21

–24 However, the metabolic effects of androgen hormones may be different according to menopausal status. During the menopausal transition period, there is a remarkable decrease in androgens along with changes in androgen receptors and sensitivity in the liver, which may attenuate the detrimental effect of androgens on metabolism in postmenopausal women.

There are several limitations to consider in the interpretation of these findings. First, this study had a cross-sectional design, suggesting that caution should be used in causal and temporal interpretations. Further longitudinal studies are required to establish causality between serum testosterone level and NAFLD by menopausal status. Second, because the study participants were confined to women who visited for health promotion screenings in a single hospital and appeared to be slightly healthier than most community-based cohorts, the study population may not be representative of the general population. Third, NAFLD was diagnosed based on abdominal ultrasonography scans and not liver biopsy, which is regarded as the gold standard for NAFLD diagnosis. Although abdominal ultrasonography is widely accepted as the diagnostic tool for NAFLD screening owing to its safety and accessibility, it has limited accuracy in detecting mild steatosis.^25
–27 Fourth, testosterone concentration was measured by ECLIA, not by liquid chromatography mass spectrometry (LC-MS). LC-MS method is considered the gold standard for measuring serum testosterone. However, testosterone measurement by the ECLIA method has been reported to have good agreement with the results obtained by the LC-MS method.²⁸ In addition, ECLIA has several advantages such as rapid turnaround time, high-throughput, and full automation. Finally, we could not measure dehydroepiandrosterone sulfate and sex hormone-binding globulin levels, which are important covariables to better understand the link between testosterone and NAFLD. Moreover, we did not collect measurements of waist circumference in our data and used BMI as a measure of obesity.

Despite these potential limitations, this study has several strengths. We separately assessed the association between serum testosterone and NAFLD in premenopausal and postmenopausal women and this may reveal the association between serum testosterone and NAFLD according to menopausal status. In addition, a wide range of confounding factors closely related to NAFLD, including exercise status, BMI, hepatic enzymes, and inflammatory markers were adjusted for in multiple logistic regression analyses. Moreover, our study excluded subjects with induced or secondary menopause and women with oral contraceptive use or undergoing estrogen replacement therapy to determine the true nature of testosterone and NAFLD in pre- and postmenopausal women. Induced menopause causes sudden onset of obesity and metabolic disturbances, followed by an abrupt decline in ovarian hormones, and tamoxifen therapy can induce secondary NAFLD, whereas estrogen replacement therapy can decrease hepatic steatosis.^29

–32

In conclusion, serum testosterone level was positively associated with NAFLD in premenopausal women, but not in postmenopausal women. Our findings suggest that higher androgenic activity may be at least partly involved in the pathogenesis of NAFLD, particularly in premenopausal women.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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