Abstract
Background and Aims
Men with cirrhosis frequently experience endocrine dysfunction which is worsened with use of spironolactone for ascites. Both decreased testosterone and increased estrogen levels have been incriminated for these effects.
Methods
A cross-sectional study was conducted on 45 men with cirrhosis. A comprehensive clinical assessment of cirrhosis severity and sex hormone profile [total testosterone (TT), free testosterone (FT), total estrogen, estrone (E1), estradiol (E2) levels, and ratios] were performed. The relationship between cirrhosis severity and spironolactone therapy (ST) with hormone levels was assessed.
Results
Forty-five men with cirrhosis (age 31–76 years, mean 60.3 ± 8.6) were evaluated, including 17 on ST and 28 not on (no history of spironolactone therapy). Low testosterone and elevated estrogen levels were present in 12 (26.7%) and 29 (65.9%) of the subjects, respectively. Elevated E1 and E2 levels were found in 26 (57.8%) and 19 (43.2%) of subjects, respectively. Cirrhosis severity and ST were not associated with T levels but were with elevated E levels and E/T ratios. Total estrogen, E1, and E2 levels and E1/TT and E1/FT ratios were higher in those with Child-Turcotte-Puch class B/C compared to those with class A cirrhosis (P < .05). E1 level was the only predictor of Child-Turcotte-Puch class B/C cirrhosis on multivariate analysis (P = .035). Subjects with Na-model for end-stage liver disease ≥15 had higher E1/TT and E1/FT ratios than those with Na-MELD <15 (P = .013 and P = .022, respectively). ST was associated with increased E1, E2 levels and E1/TT, E1/FT, E2/TT, and E2/FT ratios among subjects with Na-MELD <15 (P < .05).
Conclusion
Increased estrogen levels, especially E1, correlate with cirrhosis severity and ST.
Keywords: Androgens, Estrogens, Liver cirrhosis, Spironolactone
Introduction
Endocrine dysfunction is common in men with cirrhosis. Manifestations include sarcopenia and feminization with gynecomastia, erectile dysfunction, testicular atrophy, and sparse body hair which are frequently exacerbated by the administration of spironolactone for the treatment of ascites.1 The cause of these symptoms is frequently attributed to hypotestosteronism. Low testosterone (T) levels are present in many men with cirrhosis and can be secondary to hypothalamic/pituitary dysfunction and primary testicular failure in those with alcoholic cirrhosis from the toxic effects of ethanol.1
An alternative explanation for feminization in men with cirrhosis is high estrogen (E) levels. Most E in men originate from androstenedione produced by the adrenal gland which is secreted into the plasma and converted peripherally to either T by 17β-HSD type 5 or estrone (E1) by aromatase in adipose tissue.2 The healthy adult liver does not ordinarily express aromatase. However, cirrhosis and hepatocellular carcinoma are associated with increased expression,3,4 and its activity is further increased by factors frequently present in cirrhosis, including obesity, older age, hyperinsulinemia, elevated gonadotropin levels, and alcohol.5 There is a dynamic interconversion between E1 and estradiol (E2) by six 17β-hydroxysteroid dehydrogenase enzymes.6 Both E1 and E2 undergo sulfation and glucuronidation to form inactive hydrophilic compounds for excretion in the urine and bile, and an enterohepatic circulation influences E clearance via an estrogen-gut microbiome axis.7 Derangements of the gut microbiome can potentially affect the enterohepatic circulation. Shortening of intestinal transit time and reduction of the bacterial flora β-glucuronidase activity decreases serum E levels due to decreased enterohepatic recirculation. In cirrhosis, the opposite effect on E, especially E1, occurs due to delayed intestinal transit time and bacterial overgrowth.8,9 Finally, estrone 1 sulfate (E1S) serves as an E reservoir due to its low metabolic clearance rate, and it is reactivated to form an equilibrium with active E1.10 In cirrhosis this equilibrium shifts toward E1 due to low levels of sulfotransferase (STS) which converts E1 to E1S (Figure 1).11 Although there are conflicting reports, it has been reported that there is increased conversion of E1S to E1 due to inflammatory activation of NF-kB in chronic inflammatory liver diseases which induce STS hepatic gene expression and shunting of blood leading to increased peripheral conversion.12,13
Figure 1.
Adrenal production of androstenedione is converted peripherally to Estrone (E1) by aromatase in adipose tissue and into Testosterone by 17-beta hydroxysteroid dehydrogenase type 5 in intracrine tissue. Aromatase is not expressed in healthy adult liver, but it is expressed in cirrhosis and hepatocellular carcinoma and is increased by obesity, older age, hyperinsulinemia, elevated gonadotropin levels, alcohol. There is a dynamic interconversion by 7ß-hydroxysteroid dehydrogenase between E1 and Estradiol (E2). Both E1 and E2 undergo sulfation and glucuronidation to form inactive hydrophilic compounds in the liver for excretion in the urine and bile. Enterohepatic circulation: a) Estrogen clearance via an estrogen-gut microbiome axis b) Shortening of intestinal transit time and reduction of the bacteria flora ß-glucuronidase activity decreases serum E levels due to decreased enterohepatic recirculation c) E1 is increased in cirrhosis due to delayed intestinal transit time and bacterial overgrowth E1 Sulfate (E1S) serves as an estrogen reservoir due to its low metabolic clearance rate: a) Reactivated to form an equilibrium with active estrogens b) Equilibrium is altered in cirrhosis towards E1 due to low levels of sulfotransferase which converts E1 to E1S and induction of steroid sulfatase gene expression which converts E1S to E1 Estrogen sulfotransferase (SULT1E1) converts estradiol (E2) to estradiol sulfate (E2S), while steroid sulfatase (STS) converts E2S back to E2.
Most studies of hormone levels in men with cirrhosis have been incomplete in terms of the levels measured and correlation with severity of liver disease. Studies have assessed only T or E levels but not both, and those assessing E levels only reported levels of E1 or E2 but not both.14, 15, 16 In addition, it has recently been suggested that the E/T or E/free testosterone (FT) ratio is a more meaningful measure of hormonal balance of a man rather than T or E levels alone. Finally, the impact of spironolactone therapy (ST) which has been reported to affect both T and E levels was frequently not addressed.17,18 One study did measure all parameters discussed, but it only included men with low T levels.19 In this study, we assess the relationship of cirrhosis severity and ST in men with the complete hormone profile.
Methods
Subjects and Clinical Characteristics
Men with cirrhosis aged 18 years or older were prospectively enrolled. Diagnosis of cirrhosis was based on liver biopsy, radiographic findings, or endoscopic evidence of varices. Inclusion criteria consisted of either ST for at least 1 month prior to enrollment or no history of spironolactone therapy (NST). Exclusion criteria included inability to provide informed consent, history of transjugular portosystemic shunt, active infection, alcohol abuse or gastrointestinal bleeding within 3 months of enrollment, or severe co-existing chronic medical conditions, including cardiovascular or pulmonary co-morbidity, HIV infection, end-stage renal disease, and malignancy other than localized hepatocellular carcinoma. The study was approved by the Institutional Review Board of the Albert Einstein College of Medicine, and written informed consent was obtained from all subjects.
Medical records were reviewed for therapy with spironolactone and history of the presence and severity of ascites and hepatic encephalopathy (HE). Biochemical characteristics including complete blood count, serum electrolytes, renal and liver tests and international normalized ratio (INR) were recorded, and cirrhosis severity assessed by calculating Child-Turcotte-Puch (CTP) score and MELD and Na-MELD scores.
Hormone Levels and Ratios
Total testosterone (TT) levels were measured by chromatography/mass spectrometry (normal range: 250–1100 ng/dl), and FT levels were calculated by equilibrium dialysis using a modified Vermeulen equation (normal range: 35–155 pg/ml) (Quest Diagnostics, New York, USA). Total estrogen (TE) levels were measured by enzyme linked immunosorbent assay, and E1 and E2 levels were measured by chromatography/mass spectrometry (Quest Diagnostics, New York, USA). Estriol levels (Quest Diagnostics, New York, USA) and estetrol levels (Cayman Chemical Company, Michigan, USA), were measured by chromatography/mass spectrometry in 7 and 17 subjects, respectively. Sex hormone binding protein (SHBP) levels were measured by immunoassay in 16 subjects (Quest Diagnostics, New York, USA). Ratios of FT to TT (FT/TT), E1 to TT (E1/TT), E1 to FT (E1/FT), E2 to TT (E2/TT), and E2 to FT (E2/FT), the difference between the TE level and sum of E1 and E2 (E gap), and the proportion of the TE level accounted for by E1 and E2 ([E1+E2]/TE) were calculated for each subject.20
Relationship of Cirrhosis Severity With Hormone Levels and Ratios
Subjects were stratified based on CTP class (A, 5–6 points; B/C ≥ 7 points) and MELD and Na-MELD scores (<15 and ≥15). Parameters of cirrhosis severity were then correlated with the hormone levels and ratios and SHBP levels.
Relationship of Spironolactone Therapy Status With Cirrhosis Severity
Clinical and biochemical characteristics between the ST and NST subjects were compared. Subjects were stratified based on cirrhosis severity (CTP class A vs B/C) and MELD and Na-MELD scores (<15 vs ≥ 15), and parameters of cirrhosis severity between the ST and NST subgroups compared.
Relationship Between Spironolactone Therapy and Cirrhosis Severity With Hormone Levels and Ratios
Hormone levels and ratios in the ST subjects and NST subjects were compared. Levels were also compared in subjects stratified based on cirrhosis severity (CTP A vs B/C, Na-MELD <15 vs ≥ 15).
Univariate analysis between parameters of cirrhosis severity and ST with the hormone levels and ratios were performed. Logistic regression analysis was performed to identify independent factors associated with the various hormone levels. Variables which showed P < .1 in univariate analysis were included in a multivariable linear regression model. P < .05 was assessed as significant.
Statistical Analysis
The statistical program “SPSS for windows 22.0” was employed for analysis. For descriptive statistics of the data, mean, standard deviation, ratio, and frequency were used. The distribution of quantitative variables was checked with Kolmogorov-Smirnov test. Independent samples t test and Mann-Whitney U-test were used for variables distributed normally and non-normally, respectively. The chi-square test was used for the analysis of qualitative data. The correlation between quantitative variables distributed normally and non-normally was checked with Pearson correlation analysis and Spearman’s correlation analysis, respectively.
Results
Subjects and Clinical Characteristics
Forty-five subjects (age range 31–76 years, mean 60.27 ± 8.6) were studied (17 ST, 28 NST). The dose of spironolactone for those receiving the medication ranged from 25 mg to 200 mg (median 100 mg, mean 83.8 ± 44.1 mg), and the duration of use ranged from 1 month to 71 months (median 2 months, mean 11.2 ± 19.9). Twenty-eight participants (62.2%) had no ascites, 12 (26.7%) small volume or diuretic controlled, and 5 (11.1%) large volume ascites. Twenty-five participants (55.6%) had no symptoms of HE, 19 (42.2%) mild symptoms or were on HE therapy, and 1 (2.2%) had a history of overt HE. Details of the clinical assessment and biochemical characteristics are provided in Table 1. Median CTP score was 6 with a range from 5 to 11. Twenty-three participants (51.1%) were classified as CTP class A, and 22 (48.9%) as CTP class B/C cirrhosis. Mean MELD and Na-MELD scores were 10.9 ± 4.1 and 11.2 ± 4.4, respectively. Na-MELD score was <15 in 38 (84.4%), and ≥15 in 7 (15.6%).
Table 1.
Comparison of Biochemical, Clinical, and Hormonal Parameters and Ratios in the Patients According to Spironolactone Therapy Status
| Total n = 45 | Spironolactone therapy (S) n = 17 | No spironolactone therapy (NS) n = 28 | P | |
|---|---|---|---|---|
| Cirrhosis severity | ||||
| Na (mEq/l) | 138.33 ± 3.28 | 136.82 ± 3.13 | 139.25 ± 3.08 | .014 |
| BUN (mg/dL) | 13.38 ± 4.56 | 14.12 ± 4.97 | 12.93 ± 4.33 | .404a |
| Creatinine (mg/dL) | 0.88 ± 0.22 | 0.95 ± 0.24 | 0.83 ± 0.19 | .073 |
| WBC (K/ul) | 5.31 ± 1.98 | 4.91 ± 1.45 | 5.56 ± 2.24 | .291 |
| Hemoglobin (g/dL) | 12.91 ± 2.26 | 11.8 ± 2.07 | 13.58 ± 2.13 | .003a |
| Platelets (k/ul) | 112.84 ± 58.76 | 99.88 ± 46.65 | 120.71 ± 64.55 | .253 |
| T. protein (g/dL) | 7.28 ± 0.68 | 7.23 ± 0.79 | 7.3 ± 0.62 | .728 |
| Albumin (g/dL) | 3.77 ± 0.65 | 3.3 (0.45) | 4.2 (0.6) | < .001a |
| T. bilirubin (mg/dL) | 1.89 ± 2.35 | 1.91 ± 1.38 | 1.87 ± 2.8 | .133a |
| INR | 1.25 ± 0.27 | 1.38 ± 0.28 | 1.18 ± 0.25 | .001a |
| MELD score | 10.9 ± 4.1 | 12.65 ± 3.48 | 9.82 ± 4.06 | .003a |
| Na-MELD score | 11.2 ± 4.4 | 13.18 ± 4.08 | 10 ± 4.15 | .005a |
| CTP score | 6.84 ± 1.93 | 8.41 ± 1.73 | 5.89 ± 1.34 | < .001a |
| Ascites | ||||
| None | 28 (62.2) | 3 (17.6) | 25 (89.3) | < .001 |
| Present | 17 (37.8) | 14 (82.4) | 3 (10.7) | |
| Hepatic encephalopathy | ||||
| None | 25 (55.6) | 4 (23.5) | 21 (75) | .001 |
| Present | 20 (44.4) | 13 (76.5) | 7 (25) | |
| Endocrine status | ||||
| TT (ng/dL) | 510.62 ± 299.05 | 500.71 ± 335.87 | 516.64 ± 280.7 | .865 |
| % with low TT | 12 (26.7) | 5 (29.4) | 7 (25) | .743 |
| FT (pg/ml) | 46.24 ± 27.62 | 46.48 ± 33.28 | 46.09 ± 24.23 | .964 |
| % with low FT | 16 (35.6) | 6 (35.3) | 10 (35.7) | .977 |
| TE (pg/ml) | 259.37 ± 114.79 | 355.14 ± 130.66 | 211.67 ± 71.92 | < .001 |
| % with high TE | 29 (65.9) | 13 (76.5) | 16 (59.3) | .241 |
| E1 (pg/ml) | 84.96 ± 52.71 | 126.71 ± 48.57 | 59.61 ± 36.97 | < .001 |
| % with high E1 | 26 (57.8) | 16 (94.1) | 10 (35.7) | < .001 |
| E2 (pg/ml) | 41.75 ± 18.94 | 54.41 ± 20.18 | 33.78 ± 13.1 | < .001 |
| % with high E2 | 19 (43.2) | 13 (76.5) | 6 (22.2) | < .001 |
| E gap (pg/ml) | 132.82 ± 72.22 | 154.02 ± 89.63 | 118.97 ± 55.82 | .121 |
| SHBG (nmol/l) | 86 ± 22.93 | 86.29 ± 29.08 | 85.78 ± 18.74 | .967 |
| % high SHBGb | 15 (94) | 6 (85.7) | 9 (100) | .438 |
| FT/TT ratio | 0.0094 ± 0.0026 | 0.0095 ± 0.0029 | 0.0093 ± 0.0024 | .726 |
| E1/TT ratio | 0.054 ± 0.12 | 0.102 ± 0.17 | 0.026 ± 0.57 | .001a |
| E1/FT ratio | 6.31 ± 13.76 | 10.55 ± 16.76 | 3.74 ± 11.13 | .001a |
| E2/TT ratio | 0.022 ± 0.04 | 0.039 ± 0.06 | 0.011 ± 0.015 | .035a |
| E2/FT ratio | 2.52 ± 4.67 | 4.18 ± 6.23 | 1.48 ± 3.03 | .042 |
| E1+E2/TE ratio | 0.49 ± 0.16 | 0.56 ± 0.12 | 0.45 ± 0.17 | .034 |
Independent samples t test. Chi-square test. Bold values indicate statistical significance (P < .05)
Mean ± SD.
BUN- Blood urea nitrogen; WBC- White blood cells; TE- Total estrogen; E1- Estrone; E2- Estradiol; E Gap – difference between TE, and sum of E1 and E2; SHBG- Sex hormone binding globulin; E1/TT, ratio- Estrone/Total testosterone ratio; E1/FT, ratio- Estrone/Free testosterone ratio; E2/TT, ratio- Estradiol/Total testosterone ratio; E2/FT, ratio- Estradiol/Free testosterone ratio.
Mann-Whitney U test.
SHBG levels were evaluated in 16 participants.
Hormone Levels and Ratios
TT levels ranged from 20 to 1038 ng/dL (mean 510.62 ± 299.05 ng/dL) with 26.7% below the normal level for a man (250 ng/dL). FT levels ranged from 1.6 to 102.2 pg/ml (mean 46.24 ± 27.62 pg/ml) with 35.6% below the normal level for a man (35 pg/ml). TE levels ranged from 56 to 716.9 pg/ml (mean 259.37 ± 114.79 pg/ml) with 65.9% above the normal level for a man (190 pg/ml). E1 ranged from 10 to 253 (mean 84.96 ± 52.71 pg/ml) with 57.8% above the normal level for a man (68 pg/ml). E2 levels ranged from 5 to 99 pg/ml (41.75 ± 18.94 pg/ml) with 43.2% above the normal level for a man (40 pg/ml). Estriol levels and estetrol levels were undetectable in all. SHBP levels ranged from 39 to 123 nmol/l (mean 86 ± 22.93) with 94% above the normal level (50 nmol/l). Ratios of FT to TT (FT/TT), E1 to TT (E1/TT), E1 to FT (E1/FT), E2 to TT (E2/TT), and E2 to FT (E2/FT) are presented in Table 1. The difference between the TE level and sum of E1 and E2 (E gap) ranged from 17 to 421.9 pg/ml (mean 132.82 ± 72.22 pg/ml). The proportion of the TE level accounted for by E1 and E2 ([E1+E2]/TE) ranged from 0.15 to 0.82 (mean 0.49 ± 0.16).
Relationship of Cirrhosis Severity With Hormone Levels and Ratios
Correlations of parameters of cirrhosis severity with the hormone levels and ratios are presented in Table 2. TE levels were positively correlated with TBili, INR, MELD, Na-MELD, and CTP scores and negatively correlated with albumin levels (P < .05). E1 levels were positively correlated with TBili, INR, MELD, Na-MELD, and CTP scores, and negatively correlated with albumin, hemoglobin, and platelet levels (P < .05). E2 levels were positively correlated with INR, Na-MELD, and CTP scores and negatively correlated with albumin and platelet levels (P < .05). E1/FT ratio was negatively correlated with albumin, hemoglobin, and platelet levels, and positively correlated with INR levels, MELD, Na-MELD, and CTP scores (P < .05). E2/FT ratio was negatively correlated with albumin, hemoglobin, and platelet levels and positively correlated with INR, Na-MELD, and CTP scores (P < .05).
Table 2.
Correlation of the Various Hormones and the Ratios With the Parameters of Cirrhosis Severity in the Study Population
| Albumin (g/dL) |
Bilirubin (mg/dL) |
INR |
Hgb (g/dL) |
Platelets (k/ul) |
MELD score |
Na-MELD score |
CTP score |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| r | P | r | P | r | P | r | P | r | P | r | P | r | P | r | P | |
| TT (ng/dL) | 0.191 | .209a | 0.055 | .72 | −0.035 | .817 | 0.186 | .221 | 0.046 | .762 | −0.19 | .211 | −0.196 | .196 | −0.027 | .86 |
| FT (pg/ml) | 0.204 | .179a | 0.09 | .556 | 0.008 | .958 | 0.15 | .325 | 0.089 | .563 | −0.096 | .53 | −0.113 | .461 | −0.007 | .966 |
| TE (pg/ml) | −0.68 | < .001a | 0.388 | .009 | 0.66 | < .001 | −0.202 | .188 | −0.263 | .085 | 0.454 | .002 | 0.432 | .003 | 0.687 | < .001 |
| E1 (pg/ml) | −0.804 | < .001a | 0.507 | < .001 | 0.702 | < .001 | −0.412 | .005 | −0.386 | .009 | 0.628 | < .001 | 0.621 | < .001 | 0.794 | < .001 |
| E2 (pg/ml) | −0.619 | < .001a | 0.264 | .083 | 0.465 | .001 | −0.226 | .14 | −0.362 | .016 | 0.295 | .052 | 0.324 | .032 | 0.508 | < .001 |
| E1/FT ratio | −0.78 | < .001 | 0.285 | .058 | 0.532 | < .001 | −0.443 | < .001 | −0.389 | .008 | 0.538 | < .001 | 0.547 | < .001 | 0.553 | < .001 |
| E2/FT ratio | −0.601 | < .001 | 0.085 | .582 | 0.348 | .021 | −0.323 | .033 | −0.405 | .006 | 0.29 | .056 | 0.308 | .042 | 0.339 | .024 |
Bold values indicate statistical significant (P < .05).
E1- Estrone; E2- Estradiol; E1/FT, ratio- Estrone/Free testosterone ratio; E2/FT, ratio- Estradiol/Free testosterone ratio; TE- Total estrogen.
Pearson Correlation analysis, Spearman’s correlation analysis.
Hormone levels in subjects stratified by cirrhosis severity (CTP class A vs B/C and Na-MELD scores <15 vs ≥ 15) are presented in Table A1. TE, E1, E2 levels and the ratios of E1/TT, E1/FT, and (E1+E2)/TE were significantly lower in the subjects with CTP class A compared to CTP class B/C (P < .05). Testosterone levels and ratio were not significantly different between the CTP classes and Na MELD categories. The percentage of individuals with high E1 levels, and E1/TT and E1/FT ratios were also significantly lower in the subjects with Na-MELD <15 compared to those with Na-MELD ≥15 (P < .05).
Relationship of Spironolactone Therapy Status With Cirrhosis Severity
Comparison of the clinical and biochemical characteristics between the ST and NST subjects is presented in Table 1. Serum sodium, hemoglobin, and albumin levels were significantly lower in those on S, and INR levels, ammonia, MELD and Na-MELD levels and CTP scores significantly higher (P < .05). The percentage of individuals with ascites and HE was significantly higher in the S group compared to the NS group (P < .05) (Table 1).
Comparison of the clinical and biochemical characteristics between the ST and NST subjects stratified by CTP class and Na-MELD is presented in Table 3. Due to limited number in the ST group with CTP A, only analyses between ST and NST subjects with CTP class B/C were performed. Serum creatinine levels and the percentage of individuals with ascites were significantly higher in the ST group compared to the NST group (P < .05). Due to limited numbers in the Na-MELD ≥15 groups, only analyses between ST and NST subjects with Na-MELD <15 were performed. Hemoglobin and albumin levels were significantly lower, and INR and the percentage of individuals with ascites and HE were significantly higher in the ST group compared to the NST group (P < .05).
Table 3.
Comparison of Biochemical, Clinical, and Hormonal Parameters and Ratios in Cirrhotic Men With CTP Class B/C and Na-MELD <15 According to Spironolactone Therapy Status
| CTP class B/C S therapy n = 15 | CTP class B/C NS therapy n = 7 | P | Na-MELD scores <15 s therapy n = 12 | Na-MELD scores <15 NS therapy n = 26 | P | |
|---|---|---|---|---|---|---|
| Cirrhosis severity | ||||||
| Na (mEq/l) | 136.27 ± 2.79 | 138.14 ± 3.02 | .168 | 137.67 ± 2.87 | 139.42 ± 3.11 | .059a |
| BUN (mg/dL) | 13.53 ± 3.85 | 10.57 ± 3.6 | .102 | 13.92 ± 5.62 | 12.73 ± 4.43 | .538a |
| Creatinine (mg/dL) | 0.91 ± 0.19 | 0.7 ± 0.11 | .013 | 0.94 ± 0.27 | 0.84 ± 0.2 | .214a |
| WBC (K/ul) | 4.81 ± 1.32 | 5.03 ± 2.44 | .832 | 4.74 ± 1.52 | 5.84 ± 2.06 | .11 |
| Hemoglobin (g/dL) | 11.75 ± 2.16 | 13.13 ± 1.9 | .163 | 12.16 ± 1.94 | 13.82 ± 2.01 | .008a |
| Platelets (k/ul) | 100.67 ± 44.54 | 99.71 ± 52.9 | .965 | 101.17 ± 49.13 | 126.96 ± 62.69 | .217 |
| T. protein (g/dL) | 7.14 ± 0.76 | 7.06 ± 0.74 | .812 | 7.34 ± 0.87 | 7.35 ± 0.61 | .973 |
| Albumin (g/dL) | 3.21 ± 0.44 | 3.53 ± 0.61 | .19a | 3.38 ± 0.54 | 4.15 ± 0.45 | < .001 |
| T. bilirubin (mg/dL) | 2.06 ± 1.4 | 4.66 ± 4.69 | .056a | 1.42 ± 0.86 | 1.35 ± 1.23 | .321a |
| INR | 1.38 ± 0.29 | 1.43 ± 0.4 | .747 | 1.27 ± 0.17 | 1.12 ± 0.13 | .005a |
| Ascites | ||||||
| None | 1 (6.7%) | 5 (71.4%) | .004 | 3 (25) | 24 (92.3) | < .001 |
| Present | 14 (93.3%) | 2 (28.6%) | 9 (70) | 2 (7.7) | ||
| Hepatic encephalopathy | ||||||
| None | 3 (20%) | 3 (42.9%) | .334 | 2 (16.7%) | 21 (80.8%) | < .001 |
| Present | 12 (80%) | 4 (57.1%) | 10 (83.3%) | 5 (19.2%) | ||
| Endocrine status | ||||||
| TT (ng/dL) | 541.2 ± 330.93 | 502.86 ± 331.19 | .803 | 529.92 ± 374.62 | 549.19 ± 263.64 | .856 |
| % with low TT | 4 (26.7) | 2 (28.6) | 1 | 4 (33.3) | 5 (19.2) | .423 |
| FT (pg/ml) | 49.98 ± 33.23 | 42.99 ± 29.79 | .641 | 48.47 ± 37.89 | 49.03 ± 22.5 | .963 |
| % with low FT | 5 (33.3) | 3 (42.9) | 1 | 5 (41.7) | 8 (30.8) | .714 |
| TE (pg/ml) | 354.11 ± 125.17 | 266.14 ± 66.03 | .098 | 348.51 ± 149.57 | 203.59 ± 66.52 | < .001 |
| % with high TE | 12 (80) | 6 (85.7) | 1 | 9 (75) | 14 (56) | .306 |
| E1 (pg/ml) | 133.33 ± 47.43 | 102 ± 33.62 | .133 | 134.17 ± 51.66 | 53.08 ± 29.24 | < .001 |
| % with high E1 | 15 (100) | 6 (85.7) | .318 | 11 (91.7) | 8 (30.8) | < .001 |
| E2 (pg/ml) | 56.33 ± 20.44 | 39.43 ± 12.74 | .059 | 59.75 ± 20.6 | 33.88 ± 13.5 | < .001 |
| % with high E2 | 12 (80) | 2 (28.6) | .052 | 10 (83.3) | 6 (24) | .001 |
| E gap (pg/ml) | 164.44 ± 89.55 | 124.71 ± 63.4 | .306 | 154.59 ± 102.79 | 117.57 ± 55.13 | .166 |
| SHBG (nmol/l) | 90.83 ± 29 | 102.33 ± 17.21 | .554 | 92.33 ± 32.08 | 82 ± 15.96 | .479 |
| % high SHBG∗∗ | 5 (83.3) | 3 (100) | 1 | 3 (100) | 8 (100) | - |
| FT/TT ratio | 0.0096 ± 0.003 | 0.0083 ± 0.0027 | .363 | 0.0089 ± 0.0026 | 0.0094 ± 0.0023 | .563 |
| E1/TT ratio | 0.093 ± 0.18 | 0.068 ± 0.11 | .459a | 0.1 ± 0.17 | 0.012 ± 0.008 | < .001a |
| E1/FT ratio | 9.34 ± 16.29 | 11.38 ± 21.55 | .418a | 12.06 ± 18.79 | 1.32 ± 0.97 | < .001a |
| E2/TT ratio | 0.33 ± 0.56 | 0.02 ± 0.028 | .751a | 0.041 ± 0.62 | 0.008 ± 0.065 | .018a |
| E2/FT ratio | 3.4 ± 5.59 | 3.25 ± 5.77 | .698a | 4.94 ± 7.05 | 0.87 ± 0.68 | .014a |
| E1+E2/TE ratio | 0.55 ± 0.12 | 0.55 ± 0.16 | .939 | 0.58 ± 0.12 | 0.44 ± 0.17 | .015 |
Independent samples t test. Bold values indicate statistical significance (P < .05).
Mean ± SD.
BUN- Blood urea nitrogen; E1- Estrone; E2- Estradiol; E Gap – difference between TE, and sum of E1 and E2; SHBG- Sex hormone binding globulin; E1/FT, ratio- Estrone/Free testosterone ratio; E1/TT, ratio- Estrone/Total testosterone ratio; E2/FT, ratio- Estradiol/Free testosterone ratio; E2/TT, ratio- Estradiol/Total testosterone ratio; TE- Total estrogen; WBC- White blood cells.
Mann-Whitney U test.
Relationship Between Spironolactone Therapy and Cirrhosis Severity With Hormone Levels and Ratios
Testosterone and estrogen levels and ratios in the ST and NST subjects are presented in Table 1. There was no difference in the TT or percent of subjects with T level lower than the reference range between the ST and NST individuals. However, TE, E1, E2 levels, and E1/TT, E1/FT, E2/TT, and E2/FT, (E1+E2)/TE ratios were significantly higher in the ST compared to the NST subjects (P < .05).
Testosterone and estrogen levels and ratios in ST and NST subjects stratified by cirrhosis severity (CTP class A vs B/C and Na-MELD scores <15 vs ≥ 15) are presented in Table 3. There was no significant difference in the testosterone and estrogen levels and ratios between the S and NS groups in the subjects with CTP class B/C (P > .05). TE, E1, E2 levels, and E1/TT, E1/FT, E2/TT, E2/FT, (E1+E2)/TE ratios were significantly higher in the in the S compared to the NS group (P < .05) in the subjects with Na-MELD score <15 (P < .05).
Significant factors on univariate analysis between parameters of cirrhosis severity and ST with the hormone levels and ratios are presented in Tables 1 and 3. No variables were correlated with T or FT levels. Using variables with a statistical significance of P < .1 on univariate analysis (WBC, albumin, CTP score, Na-MELD score, and ST) in a multivariable linear regression model (age-adjusted), the only factor independently associated with E1 levels was an inverse relationship with albumin levels (P = .003). Using variables with a statistical significance of P < .1 on univariate analysis (WBC, albumin, CTP score, and ST) in a multivariable linear regression model (age-adjusted), the independent factors associated with E2 levels were an inverse relationship with WBC and albumin levels and spironolactone treatment (P < .05) (Table 4). Only E1 was independently associated with CTP class B/C (P = .035) in a multivariable regression model using variables with a statistical significance of P < .1 on univariate analysis (age, TE, E1, E2, FT/E1 ratio, and FT/E2 ratio).
Table 4.
Independent Predictors for E1 and E2 Levels by Multivariate Linear Regression Analysis
| Univariate analysis |
Multivariate analysis |
|||
|---|---|---|---|---|
| β | P | β | P | |
| E1 | ||||
| Age | −0.196 | .197 | −0.045 | .642 |
| WBC (k/ul) | −0.372 | .012 | −0.078 | .438 |
| Albumin (g/dL) | −0.804 | < .001 | −0.506 | .003 |
| CTP score | 0.742 | < .001 | 0.311 | .131 |
| Na-MELD score | 0.479 | .001 | −0.165 | .257 |
| Spironolactone therapy | 0.624 | < .001 | 0.185 | .126 |
| E2 | ||||
| Age | −0.062 | .688 | 0.08 | .513 |
| WBC (k/ul) | −0.484 | .001 | −0.295 | .029 |
| Albumin (g/dL) | −0.619 | < .001 | −0.433 | .041 |
| CTP score | 0.482 | .001 | −0.142 | .501 |
| Spironolactone therapy | 0.537 | < .001 | 0.332 | .032 |
Bold values indicate statistical significant (P < .05).
E1- Estrone; E2- Estradiol; WBC- White blood cells.
Discussion
The important findings in our study included significant increases in estrogen levels that correlated with cirrhosis severity and ST. Testosterone was low in only 25% of subjects and had no relationship with disease severity or ST. In contrast, estrogen levels, especially E1, were elevated in 57.8% of men, and levels increased in parallel with disease severity. Although E1/FT and E1/TT ratios were positively related to disease severity, the E1 level was the only predictor of CTP class B/C cirrhosis on multivariate analysis. ST was associated with increased E1 and E2 but not decreased testosterone levels among individuals with Na-MELD <15. In multivariate analysis, the decreasing albumin level emerged as the sole predictor of an increase in E1 level. Decreasing white blood cell counts, albumin levels, and spironolactone treatment were predictors of an increase in E2 level.
Our findings of increased E1 levels corroborate earlier studies that reported elevated levels in men with cirrhosis.13,21 The increase in E1 in patients with cirrhosis was first reported in the 1970s.22,23 Studies that simultaneously measured both E1 and E2 levels reported elevations in E1 more commonly than E2 in both compensated and decompensated cirrhosis.13,21 In a study of men with cirrhosis with and without hepatocellular carcinoma, E1 levels were significantly elevated in all, but E2 in only approximately 40%,24 and 2 other reports that also measured both E1 and E2 only reported only elevations in the E1 level.13,21 More recently, elevated E1 levels have been reported to correlate with disease severity in men with low T levels.19
Spironolactone is the mainstay for the treatment of ascites with its major metabolites exerting potent mineralocorticoid receptor antagonism.25 However, it is associated with significant endocrine side effects in men. When administered for congestive heart failure, 10% of men receiving spironolactone experienced gynecomastia or breast pain compared to 1% receiving placebo.26 In addition, it is utilized therapeutically in the treatment of androgen dependent dermatologic conditions and feminizing therapy.27
The adverse effect of spironolactone has historically been attributed to anti-androgenic activity.27 In vitro studies demonstrate that spironolactone is a weak inhibitor for 17β-HSD type 2 which is responsible for conversion of E2 to T.28 Spironolactone and its metabolites also competitively inhibit androgen receptor binding and weakly inhibit 17α-hydroxylase and 17–20 desmolase in testosterone biosynthesis.29,30 Finally, supratherapeutic doses in animal models are associated with decreased T levels, and there is evidence of increased T clearance.27 However, studies on the effect on T levels have yielded conflicting results, and there is limited clinical evidence to support a role in the effect of spironolactone on testosterone levels with only reports of small numbers of patients with conflicting results.31,32 There were no changes in T levels after administration of high doses to 6 healthy male volunteers27 Whereas Rose Li et al. reported decreased T levels in six hypertensive patients with gynecomastia undergoing ST compared to ten individuals not receiving the agent32 and its administration led to lower T level in 6 out of 16 men with hypertension,17 this effect was not confirmed in a study of six hypertensive men treated for 12 weeks.31
An alternative explanation for the effect of spironolactone is through an impact on estrogen metabolism. Spironolactone inhibits 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD type 2) which is responsible for converting E2 into testosterone.28 In addition, spironolactone increases the levels of free E2 by displacing it from SHBG.30 As with its clinical effect on T levels, there are inconsistent findings of its impact on E levels. Loriaux DL et al. reported no changes in E2 levels in five healthy men after two and 4 weeks of therapy.27 In contrast, Rose et al. reported increased peripheral conversion of T to E2 with higher E2 levels in 6 hypertensive men with gynecomastia treated with spironolactone compared to control subjects,32 and Miyatake A et al. reported increases in E1 and E2 levels in hypertensive men treated with ST.31
Painful gynecomastia is common in men with cirrhosis treated with spironolactone, and there is a reduction in painful gynecomastia after its substitution with eplerenone.18,33,34 As with the case in normal and hypertensive men, inconsistent alterations in T and E have been reported to occur in response to spironolactone in cirrhosis. Although there is indirect evidence that its effect is due to estrogenic activity due to an amelioration of symptoms with the co-administration of tamoxifen, reports of changes in E levels are inconsistent. Vaishnav et al. reported high E2 levels in 60 men with liver cirrhosis compared to 60 healthy controls,14 and Sinclair et al. reported that ST was associated with higher E1 levels but similar E2 levels.19
Our findings of a greater effect of spironolactone on E1 and relationship with severity of cirrhosis potentially explain the discrepancies of the various studies. E1 was elevated in 94.1% of individuals on spironolactone, whereas E2 was elevated in 76.5%. Advanced liver disease appears to have a greater impact on E1 than on E2, and the effect is readily apparent when subjects are stratified by cirrhosis severity. E1 was elevated in 100% of individuals on spironolactone with Na-MELD ≥15 whereas E2 was only elevated in 42.9% of individuals. The percentage of individuals with high E1 levels was significantly higher in the subjects with Na-MELD >15 compared to those with Na-MELD <15 (100% vs 50%). In contrast, there was no significant difference in E2 levels in the subjects with Na-MELD <15 versus ≥15. As a result, studies that only assessed E2 would have missed an effect of spironolactone on estrogen metabolism and a relationship with gynecomastia.17
An important question that our study raises is how the endocrine status of a cirrhotic man should be assessed. An understanding of the hormonal status requires more than just the assessment of the TT or TE level. The TE does not represent the sum of E1 and E2. Rather our findings indicate that there is a significant gap between the TE level as the sum of the individual measurements only accounts for 49% of the TE measurement. The composition and significance of the gap and its relation to the disease progression is unclear, but interestingly the E1+E2/TE ratio was significantly higher in the men with CTP class B/C compared to those with CTP class A and in those receiving spironolactone.
It has recently been suggested that the E/T ratio is a more meaningful measure of hormonal balance in a man rather than T or E levels alone. In a study on erectile dysfunction, the E2/T ratio was a more sensitive parameter of male hormonal balance than TT or E2 levels as assessed by International Index of Erectile Function questionnaire scores.35 In 2016, Wu et al. reported that sexual dysfunction in men was associated with E2 levels but not T levels and that the E2/T had greater significance.36
Our findings agree with previous reports of an altered E to T ratio in men with cirrhosis. Most studies have assessed only the relationship of E2 with T.14,37,38 The one study that evaluated both E1 and E2 ratios reported a greater difference for the E1/T ratio as was the case in our series.22 Our findings also support previous reports that the ratio is related to the severity of cirrhosis. In a survey of 95 men with low T levels, higher E1/TT and E2/TT ratios correlated with the disease severity.19 In our study, there was a strong correlation between the E1/FT and E1/TT ratios and disease severity. E1/TT (0.13 ± 0.18 vs 0.04 ± 0.104) and E1/FT (15.01 ± 21.79 vs 4.71 ± 11.45) ratios were significantly higher in the subjects with Na-MELD >15 compared to those with Na-MELD <15. However, this relationship was restricted to E1. There was no significant difference in E2/TT and E2/FT ratios in the subjects with Na-MELD <15 versus ≥15.
An important consideration that has not been fully studied in cirrhosis is alterations in hormone binding protein levels. Although calculation of the FT levels incorporates the effect of elevated SHBP levels, assessments of E1 and E2 levels do not include the impact of carrier proteins. The concentration of SHBG is the major factor regulating the distribution of sex steroids between the bound and free fractions. The predominance of T is bound to SHBP, and only FT is physiologically active. The important transport proteins for E1 and E2 are SBHP and albumin. As with T, both are less available to interact with their target tissues when bound to carrier proteins. E2 circulates with high affinity to SHBG, and only 2.2% is free or active. In contrast, E1 binds poorly to SHBG. Only 16% of E1 is bound to SHBG, and approximately 80% is bound to albumin.39,40 Finally, the binding affinity of SHBG is twice as high for T as for E2. Because SHBP levels are elevated and albumin levels decreased in cirrhosis, these changes have the potential to have additional effects on the balance between the free E1, free E2, and FT levels, and it has been suggested that SHBG is an estrogen amplifier for feminization symptoms in cirrhosis.41, 42, 43 Our finding that elevated E1 levels correlate with the various parameters of cirrhosis severity and decreasing albumin levels raises the possibility that increased estrogenic activity might be of even greater severity.
An important limitation in our study includes the relatively small number of participants, particularly for the categories of CTP class B/C and Na-MELD scores >15. We were also unable to evaluate the impact of dose and duration of S therapy on hormone levels due to the small sample size. Because of the limited number of subjects and variable spironolactone dose and duration, a correlation with the presence of gynecomastia was not performed. In addition, TE levels were measured with a different assay (enzyme linked immunosorbent assay) than E1 and E2 (chromatography/mass spectrometry). However, these are the assays that are employed by most clinical laboratories.
Our study highlights several important considerations when assessing the disturbances in sex hormones in men with cirrhosis. Our findings emphasize the importance of assessing both androgens and estrogens. The role of E1 in cirrhosis has not received wide attention, and it is frequently not included in the assessment. The derangements in E1 in men with cirrhosis identified by our current study highlights the importance of measuring this estrogen and questions the value of a TE measurement. An important question that remains to be answered is whether the endocrine disturbances participate in the pathophysiology and manifestations of cirrhosis or are only a result of a diseased liver. Feminization with gynecomastia is most likely a result of hyperestrogenemia, but there are other possible relationships. E1 has been reported to increase vascular endothelial nitric oxide synthesis which is a critical mediator of portal hypertension and the development of ascites and hepatorenal syndrome.44,45 Additional areas that could be affected by altered sex hormone levels include sarcopenia, anemia, diabetes mellitus, hepatic inflammation and the immune system, hepatocarcinogenesis, and overall feelings of fatigue and quality of life.46, 47, 48, 49, 50 Our study emphasizes the importance of including a comprehensive evaluation of the complete sex hormone profile that includes free hormone levels, liver function, and ST.
Acknowledgments
Authors’ Contributions
Asli Akin Belli: Data acquisition, data analysis, writing - original draft, visualization. Ben Flikshteyn: Study conceptualization and design, data acquisition, writing - original draft. Yonatan Ziv: Study conceptualization and design, data acquisition. Angela Lu: Data acquisition, visualization. Garrick Luo: Data acquisition. Nachum Lebovics: Data acquisition, writing - original draft. Samuel H. Sigal: Study conceptualization and design, funding acquisition, supervision, visualization, writing - final draft.
Footnotes
The Media Contact for NYU Grossman Long Island School of Medicine.
Rosemary Gomez, Phone: 516-663-2709, Email: Rosemary.Gomez@NYULangone.org
Conflicts of Interest: The authors disclose no conflicts.
Funding: Division of Hepatology, Department of Medicine, Montefiore Medical Center, Albert Einstein College of Medicine. Study sponsor had no role in study design; collection, analysis, or interpretation of data; writing of the report; or the decision to submit the paper for publication.
Ethical Statement: The study was approved by the Institutional Review Boards of the Albert Einstein College of Medicine, and written informed consent was obtained from all subjects (IRB Number: 2017–8060).
Data Transparency Statement: The dataset analyzed during the current study is not publicly available due to ethical restrictions and the absence of IRB approval for data sharing. Requests for access may be considered by the corresponding author, subject to institutional review and approval.
Reporting Guidelines: Helsinki Declaration.
Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.gastha.2025.100763.
Supplementary data
References
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