Non-alcoholic fatty liver disease in polycystic ovary syndrome women

To evaluate risk factors leading to non-alcoholic fatty liver disease (NAFLD) occurrence in polycystic ovarian syndrome (PCOS) women. A retrospective cohort study of a total of 586 women diagnosed with PCOS aged 13–35 years at the gynecology department at a university hospital was done to evaluate PCOS phenotype, metabolic syndrome (MetS) diagnosis, body composition, insulin sensitivity, sex hormones, lipid profile, liver function, and transient elastography (TE). In PCOS women with NAFLD compared to those without, MetS diagnosis (Hazard ratio [HR] 5.6, 95% Confidence interval [CI] 2.2–14.4, p < 0.01) and hyperandrogenism (HA) (HR 4.4, 95% CI 1.4–13.4, p = 0.01) were risk factors significantly associated with subsequent NAFLD occurrence, whereas 2-h insulin level in 75 g glucose tolerance test (GTT) (HR 1.2, 95% CI 0.5–2.5, p = 0.70) and body mass index (BMI) > 25 kg/m2 (HR 2.2, 95% CI 0.6–8.0, p = 0.24) was not. Among NAFLD patients who underwent TE, a higher number of MetS components indicated a worse degree of fibrosis and steatosis. MetS diagnosis and HA at PCOS diagnosis were risk factors associated with NAFLD, while 2-h insulin level in 75 g GTT and obesity were not. Although elevated aspartate aminotransferase levels were significant for NAFLD risk, liver enzyme elevations may not be present until late liver damage. Further prospective studies of PCOS women with MetS or HA are warranted to determine whether patients without liver enzyme elevations should undergo preemptive liver examinations.

The 75 g oral glucose tolerance testing (GTT) was routinely performed in all patients diagnosed with PCOS to evaluate insulin resistance. HOMA-IR was calculated as [fasting glucose (mg/dL) × basal insulin (µUI/mL)]/405. Patients were considered to have MetS if ≥ 3 of the following criteria, based on the criteria presented by the National Cholesterol Education Program/Adult Treatment Panel III (NCEP/ATP III) and adapted for patients with PCOS by the 2003 Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop, were satisfied: (1) abdominal obesity as waist circumference of > 88 cm, (2) elevated triglyceride level of ≥ 150 mg/dL, (3) low HDL cholesterol level of < 50 mg/dL, (4) elevated blood pressure of ≥ 130/ ≥ 85 mmHg, and (5) impaired fasting glucose level of 110-126 mg/dL and/or 2-h postprandial glucose level of 140-199 mg/dL 1 . Regrettably, because waist circumference was not routinely checked in our study, BMI was used as an alternative 24 . The cut-off for obesity in an Asian population was defined as BMI of ≥ 25 kg/m 225 . Those with BMI ≤ 20 kg/m 2 was defined as

Characteristics of NAFLD occurrence in PCOS patients. Baseline characteristics comparing PCOS
women with and without NAFLD are shown in Table 1. MetS diagnosis rate was significantly higher in women with NAFLD than in those without NAFLD. AST and ALT level elevation was also significantly correlated with NAFLD. Indices reflecting insulin resistance including 2-h 75 g GTT insulin, FGIR, HOMA-IR were markedly higher in NAFLD group. Of the 51 NAFLD patients, 43 (84.3%) had HA as opposed to 345 of 535 patients (64.5%) without NAFLD, which was significant (p = 0.01). Total testosterone and DHEA-S levels were not statistically significant. Mean BMI of our study population was 23.83 kg/m 2 , showing higher proportion of nonobese PCOS patients (n = 387, 66%) compared to obese PCOS patients (n = 199, 34%) with significant difference (p < 0.001). Proportion of obese PCOS was significantly higher in NAFLD compared to the non-NAFLD group (p < 0.001).
Baseline characteristics according to the age of PCOS diagnosis. Comparing adolescent and adult PCOS patients (Table 2), BMI was significantly higher in adolescents than in adults. Lipid profile was in the normal range for both groups. Indices reflecting insulin resistance seemed to be higher in the adolescent group. 2-h 75 g GTT insulin was more elevated in adolescents. HOMA-IR was 4.3 in adolescents and 2.7 in adults (p < 0.01). Establishing insulin resistance as HOMA-IR > 3.16 for adolescents 27 or > 2.5 for adults 28,29 and 2-h 75 g GTT insulin > 41 μU/mL 30 , both parameters showed a greater insulin resistance in adolescents compared to adults. FGIR showed conflicting results with 14.0 in adults which was higher than the 7.1 for adolescents (p < 0.01), with FGIR < 7 as abnormal for adolescents 31 and < 4.5 as abnormal for adults 32 . Both were not indicative of insulin resistance, although it was borderline for adolescents. The duration of time that lapsed after PCOS diagnosis to NAFLD diagnosis in adolescents compared to adults was not significant (35.6 months vs. 38.0 months, p = 0.48).

Relationship between MetS diagnosis and liver steatosis.
MetS diagnosis at the time of PCOS diagnosis was 62.7% in NAFLD patients and 10.5% in non-NAFLD patients (p < 0.01, Fig. 2). The proportion of NAFLD patients increased as the number of MetS components increased, which was not true for non-NAFLD patients (Table 1). Among NAFLD patients who underwent TE, the greater the number of MetS components, the worse was the degree of fibrosis and steatosis (Fig. 3). The mean stiffness scores on liver fibroscan were 328.9 dB/m, which suggests severe liver steatosis, and 7.7 kPa, which suggests significant liver fibrosis (Table 1). Univariate cox analysis was performed for risk factors associated with NAFLD occurrence in our PCOS population ( www.nature.com/scientificreports/ and impaired fasting glucose level, were statistically significant. As the number of MetS components increased from 1 to ≥ 3, the hazard ratio (HR) also increased from 4.2 to 53.8. Elevated AST and ALT levels were both linked to NAFLD. Insulin resistance, portrayed as 2-h 75 g GTT insulin ≥ 100 μU/mL, FGIR < 4.5, and HOMA-IR > 2.5 were all statistically significant. HA was significant, while age at PCOS diagnosis was not. Obesity, specified as BMI > 25, compared to the normal was significant compared to lean group which was not significant. Multivariate cox analyses of variables associated with NAFLD were presented in two models ( Table 4). The first model was according to MetS diagnosis, and the second model was according to the number of MetS components, which was not significant for a single component, but was significant for ≥ 3 components. AST level was significant in both models, while ALT level was not. Insulin resistance, represented as 2-h 75 g GTT insulin ≥ 100 μU/mL, was not significant in both models. Significance of HA also concurred in both models. BMI, both lean and obese groups were not significant compared to a reference BMI of 20-25 in both models. COX regression analysis of time from NAFLD diagnosis following PCOS diagnosis in months is shown in Fig. 4. The cumulative hazard function of NAFLD diagnosis following PCOS diagnosis was significantly higher when patients were diagnosed with MetS (Fig. 4a). It was also sequentially higher as the number of MetS components increased (Fig. 4b).  Table 5). The cox models showed that MetS diagnosis was a significant factor in consequent NAFLD development for adults, but not in adolescents. For elevated AST and ALT, there was contrasting results for the adolescent and adult group. In adolescents, both elevated AST and ALT was significant. In adults, only elevated AST was significant. 2-h 75 g GTT insulin ≥ 100 μU/mL was not a significant variable associated with NAFLD in both adolescents and adults. HA was significant in the adult group. It was not checked in the adolescent group as 100% of patients had HA. BMI was not significant for both adolescents and adults.

Discussion
Our study suggests that metabolic disturbances are intimately related to the pathophysiology and development of liver disease in women with PCOS. While the separate components of MetS were not critically connected to NAFLD, the greater the number of MetS components, the higher the prevalence of NAFLD diagnosis and more severe was the liver fibrosis and steatosis. In previous studies determining the relationship between NAFLD and MetS, features of metabolic disturbances were frequently detected in NAFLD, leading to the theory that NAFLD is the hepatic expression of MetS 6 . Even though MetS diagnosis and the number of MetS components showed significant risk increase of developing NAFLD, obesity and insulin resistance itself did not turn out to be a direct  33 . Although obese cases were dominant in the NAFLD group compared to the non-NAFLD group, still the BMI was relatively lower than other studies' population 25 . Generally, PCOS and NAFLD are representative metabolic diseases associated with insulin resistance 6 . The cut-offs of indices for insulin resistance and insulin sensitivity are controversial, especially in the non-diabetic population 5 . In the current study, we included 2-h 75 g GTT insulin, FGIR, and HOMA-IR. 2-h 75 g GTT insulin was the only index that may be applied commonly  www.nature.com/scientificreports/ in both adolescent and adult. Although traditional references suggest a cut-off of 100 μU/mL as grading severe insulin resistance, a few studies questioned whether ethnicity, race may affect insulin sensitivity and end organ response 34 . Therefore, our study presents similar results to suggest that MetS is associated with NAFLD in PCOS women, rather than obesity itself, especially in the lean BMI population.
As HA was a prerequisite in diagnosing PCOS in adolescents as opposed to being one of three requirements in young adults, it could be the decisive factor associated with NAFLD in lieu of age. Previously, liver fat accumulation was associated with increasing age 35 . This was not found to be true in our investigation. Because NAFLD can arise regardless of age, instead of waiting for outward manifestations of liver injury to present, identification of risk factors at the time of PCOS diagnosis can lead to preventative actions. We found that HA, independent of insulin resistance and obesity, was significantly associated with NAFLD occurrence in accordance with previous studies 4, 36 . This was consistent with a meta-analysis which showed that HA, was an independent factor associated with NAFLD in PCOS patients 18 .
In previous studies on women with PCOS, the prevalence of elevated liver enzyme levels was higher in women with PCOS than in controls 13 . The proportion of subjects with elevated ALT levels, which is more hepatocyte specific, was higher than that with elevated AST levels 13,15 . Interestingly, although both AST and ALT level elevations were associated with NAFLD in our univariate analysis, only AST was significant in our multivariate analysis. Up to 80% of all NAFLD patients have normal range ALT levels and it also decreases as liver fibrosis progresses to liver cirrhosis 37 , so the results of our study may actually be on par with the progression of NAFLD. Although elevated AST was significant in adolescents and elevated ALT was significant in adults, the actual serum AST/ ALT levels were not very divergent. The number of adolescent PCOS patients was very small, so this conflicting result may not have any clinical significance. Liver enzyme levels are not always elevated and can be normal in NAFLD patients [38][39][40][41] . Liver fat accumulation evaluation through TE or abdominal ultrasonography was not done if there were no elevations in serum AST/ALT levels. Because this is not a defining feature of liver injury in patients with PCOS, patients presenting with MetS and HA should consider liver evaluation. Most current PCOS Table 3. Univariate analysis of variables associated with NAFLD development in PCOS patients. The categorical variables were MetS component (reference: none), number of MetS components (reference: number 0), Age at PCOS diagnosis (reference: adolescent), Elevated AST (reference: normal AST level at PCOS diagnosis), Elevated ALT (reference: normal ALT level at PCOS diagnosis), 2-h 75 g GTT insulin (reference: < 100 µU/mL), FAI (reference < 4.5), HA (reference: none). ALT alanine aminotransferase, AMH anti-Müllerian hormone, AST aspartate transaminase, CI confidence interval, FAI free androgen index, FGIR fasting glucose insulin ratio, HA hyperandrogenism, HDL high-density lipoprotein, HOMA-IR homeostasis model assessment of insulin resistance, HR hazard ratio, HTN hypertension, GTT glucose tolerance test, LH luteinizing hormone, MetS metabolic syndrome, NAFLD non-alcoholic fatty liver disease, PCOS polycystic ovary syndrome, SHBG sex hormone binding globulin.    This study investigated NAFLD incidence in PCOS women and comprehensively examined all MetS components in relation to NAFLD. As liver biopsy was not performed in our study, the true prevalence of NAFLD and steatosis and fibrosis severity remain unknown. The limitations of this study stem from its retrospective design. Because not all patients had values available for all data items, the extent of potential relationships with NAFLD may have been diluted. A significantly higher number of adults were included in the study compared to adolescents, which could underestimate risk factors in the adolescent group. Except for incidentally found NAFLD, most of the patients were referred to the hepatology department for further examination due to liver enzyme abnormalities, and accordingly, there may have been selective bias in the diagnostic flow of NAFLD. Mild expressions of NAFLD without liver enzyme abnormality were not included, which contributes to the low prevalence of NAFLD in this study group. Waist circumference was not checked in most patients, so central obesity was not properly established. BMI was used as a substitute because studies published in Northeast Asian countries 25,43,44 and even a large cross-sectional study of U.S. and Spanish populations 45 showed BMI was correlated with metabolic impairment in lean PCOS patients. Further prospective studies of patients with MetS irregularities are warranted to determine whether patients without liver enzyme level elevations should undergo liver examinations.
In summary, our study results demonstrated that the diagnosis of MetS and HA were significant variables associated with NAFLD in women with PCOS. In light of the fact that MetS factors have a high correlation to NAFLD, patients presenting with metabolic disturbances at the time of PCOS diagnosis should be counseled to consider liver evaluation even if there are no outward manifestations of liver disease. Adding a cursory transabdominal ultrasound examination of the liver could be considered while doing a gynecologic ultrasound. PCOS manifests in women at reproductive age and evolves into metabolic problems with time. In the long run, NAFLD can have catastrophic liver-related mortalities if left untreated 46 . Earlier diagnosis can lead to more timely treatment. Although previous studies have shown the association between HA and MetS with NAFLD, we demonstrated in our study that adolescent PCOS patients, all of whom had HA, were susceptible to NAFLD when compounded with the diagnosis of MetS. Regardless of the age at which PCOS diagnosis was made, comorbidities can lead to an increased metabolic risk; thus, systematized investigation of NAFLD may be helpful.