Sex Differences in the Association between Liver Fibrosis and Clinical Outcomes in Acute Cardioembolic Stroke Patients with Nonvalvular Atrial Fibrillation


 Background: Liver cirrhosis is a confirmed risk factor for clinical outcomes of stroke patients. However, the contribution of liver fibrosis to cardioembolic stroke (CES) and its short-term outcomes are poorly understood. This study aimed to investigate the association between liver fibrosis and short-term clinical outcomes of acute CES patients, due to nonvalvular atrial fibrillation (NVAF), as well as the impacts of sex on the association. Methods: Using data of 522 patients with NVAF admitted within 48 hours after acute symptom of CES onset. We calculated Fibrosis-4 score (FIB-4) and defined liver fibrosis as: likely advanced fibrosis (FIB-4>3.25), indeterminate (FIB-4, 1.45-3.25), unlikely advanced fibrosis (FIB-4<1.45). We investigated the impact of liver fibrosis degree on stroke severity, major disability at discharge and all cause death at 90 days stratified by sex. Results: Among 522 acute CES patients with NVAF, the mean FIB-4 on admission reflected intermediate fibrosis with largely normal liver enzymes. After adjusting for all confounders, multivariate analyses revealed that likely advanced liver fibrosis was associated with severe stroke (OR=2.21, 95% CI: 1.04-3.54), major disability at discharge (OR=4.59, 95% CI: 1.88-11.18), and 90-days mortality (HR=1.25, 95% CI: 1.10-1.56). Further grouped by sex, these associations were stronger in males but not significant in females.Conclusions: In patients with largely normal liver enzyme, likely advanced liver fibrosis is associated with severe stroke, major disability and all cause death after acute CES due to NVAF, and the association unfolded more obvious in males, but not for females.


Introduction
Stroke is a common chronic disease, especially the ischemic stroke, which is the second leading cause of death and third leading cause of disability in adults worldwide.(1) Atrial brillation (AF) is a major modi able factor for stroke severity, associated with a more threefold mortality and disability rates than patients without AF. (2) Besides, the severity, treatments effectiveness, outcomes of stroke may differ from sex. (3,4) Except for sex, liver disease was associated with in-hospital mortality after ischemic stroke. (5) Several studies suggested that liver cirrhosis was not only associated with an increased risk of stroke, but an independent risk factor for poor prognosis of stoke patients. (5,6) Liver brosis, the commonly clinical feature of chronic liver disease, is often undetected because of no obvious clinical symptoms (7). Recent studies showed that there was a signi cant relationship between liver brosis and all-cause mortality in patients with cardiovascular disease, (8,9) intracerebral hemorrhage (10) as well as the ischemic stroke risk, but data are lacking regarding to the contribution of liver brosis on short-term outcomes of ischemic stroke. Therefore, we focused on the CES patients with NVAF, and investigated the association between liver brosis and CES outcomes among patients without overt liver disease. Furthermore, we also researched effect modi cation of sex on those associations.

Study Population
We performed a retrospective study using data from the First A liated Hospital of Xi'an JiaoTong University, a National Advanced Stroke Center which has both an acute stroke treatment center and a stroke rehabilitation center. Thus, all patients suffering acute CES admitted to the stroke center received consistent therapy in the acute and chronic phase during hospitalization. Patients were included in this study if they met the following requirements: (1) 18 years of age or older; (2) has a history of AF or presented symptom onset accompanied by AF; (3) admitted to the stroke center within 48 hours after onset; (4) without any limitation of physical activities before onset; (5) baseline assessment within 48 hours of CES, including symptom, function and imaging assessment; (6) assessment of CES recovery by cerebral imaging during hospitalization; (7) assessment of functional outcome at discharge and all cause death at 90 days after discharge; Exclusive criteria of this study: (1) missing data on liver chemistries, baseline stroke imaging, or important clinical covariates such as cardiac function, renal function, hospital treatment and CES severity; (2) history of viral hepatitis and liver cirrhosis; (3) alcohol use (men, >30 g/day; women, >20 g/day); (4) use of medications that causes liver damage: valproic acid, amiodarone, methotrexate, and tamoxifen; (11) (5) with poor cardiac function (EF 40%); (6) known malignancy and clinical signs of infection on admission; Over a 6-year period from January 2013 to June 2019, a total of 987 consecutive CES patients were admitted to the center for acute therapy and for further rehabilitation within 7 days of the CES onset. Of these, 522 CES patients with NVAF were included in the current study ( Figure 1). The protocol for the research project has been approved by the Ethics Committee of the First A liated Hospital of Xi'an Jiao Tong University (reference number, VL.0 2018-08-11) and that it conforms to the provisions of the Declaration of Helsinki.

De nition
We used a validated liver brosis indictor-FIB-4 score (12,13), which calculated as: and de ned liver brosis as FIB-4 >3.25, advanced brosis likely; 1.45-3.25, indeterminate level of brosis; FIB-4 <1.45, no advanced brosis likely. (14,15) CES was diagnosed according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. (16) The treatment for all CES patients was performed according to the American Heart Association/American Stroke Association Guideline. (17) The diagnosis of AF was based on at least 1 electrocardiogram obtained before or after admission.(18) Stroke severity was evaluated by the National Institutes of Health Stroke Scale (NIHSS) upon admission. NIHSS score of ≥8 was considered as severe stroke, (19) as well as the consciousness state of those patient assessed by Glasgow Coma Scale (GCS). (20) The modi ed Rankin Scale (mRS) was used to evaluate functional outcomes at discharge, and major disability was de ned as a mRS score of ≥3.(21) The primary outcomes were major disability at discharge. Our secondary outcomes were all-cause death at 90 days from discharge.

Demographics and CES Characteristics
We collected several demographics, clinical and analytical parameters. All patients included received 12-lead electrocardiogram, transthoracic echocardiography, carotid ultrasonography, and standard blood tests upon admission. The CHA2DS2-VASc score, and HAS-BLED score were measured at prespeci ed time points, as well as the treatment strategy in hospital and discharge (the use of antiplatelet and anticoagulant medication, intravenous thrombolysis, endovascular revascularization). Several biochemical parameters, including platelet count, liver chemistries (alanine aminotransferase, aspartate aminotransferase and albumin), N-terminal Pro-Btype Natriuretic Peptide (NT pro-BNP), and international normalized ratio (INR) were measured. The estimated glomerular ltration rate (eGFR) was calculated according to the Chronic Kidney Disease Epidemiology Collaboration equation. (22) Statistical Analysis Data are presented as means ± standard deviation or median and interquartile range for continuous variables and percentages for categorical variables. NT pro-BNP were lg-transformed to minimize skewness and treated as continuous variables. The population characteristics were described by FIB-4 score classi cation and strati ed by sex to explore the distribution of each interval. Kruskal Wallis test and/or chi-square test were used to compare differences among the three groups, as appropriate. Multivariate logistic regression analyses for stroke severity on admission and major disability at discharge and COX regression analysis for all-cause death at 90 days from discharge were performed after adjusting for general risk factors. Statistical signi cance was de ned as two-tailed P < 0.05. All analyses were conducted with SPSS 26.0 (SPSS, Inc., Chicago, IL, USA).

Characteristics of Study Population
The dataset of our study consisted of 522 CES patients with NVAF. Standard liver chemistry tests were generally in normal range of the study sample, with 11.7% of aspartate aminotransferase (AST)> 40 IU/L, 11.1% of alanine aminotransferase (ALT)>40 IU/L. Among these patients, 109, 280, 133 subjects were classi ed into unlikely advanced brosis group, indeterminate group, likely advanced brosis group, respectively. Patients in likely advanced brosis group had higher values for age, CHA2DS2-VASc score, HAS-BLED score and higher prevalence of smoking, diabetes melitus, hyperlipidemia, ischemic heart disease, P < 0.05 for both comparisons.
No difference was found in sex, BMI, hypertension, prior history of ischemic stroke/TIA, drug use before admission (anticoagulant, antiplatelet and lipid-lowering use) and treatment in hospital (intravenous thrombolysis and endovascular revascularization) among three groups ( Patients with likely advanced brosis group had signi cantly worse outcomes in hospital, such as higher NIHSS score on admission and mRS score at discharge, and they usually had a higher incidence of 90-days mortality, severe stroke, major disability compared with other 2 groups, P < 0.05 for both comparisons. (Table 1).

Stroke Severity and Short-term Outcomes of Study Population Strati ed by Sex
When strati ed by sex, the likely advanced brosis group had higher value for age and INR both in males and females, but beyond that, the male patients had higher CHA2DS2-VASc score, HAS-BLED score and higher proportion of smoker, hyperlipidemia, intravenous thrombolysis and endovascular revascularization in likely advanced brosis group. No differences were observed in sex, BMI, hypertension, prior history of ischemic stroke/TIA, drug use before admission among three groups both for males and females ( Table 2).
Both male and female patients in likely advanced brosis group had higher NIHSS score, mRS score, more severe stroke, and more major disability than their counterparts ( Table 2). For 90-days mortality, 8.0% male patients suffered death in likely advanced brosis group, which was higher than other 2 groups, but there was no signi cant difference among three groups in females (Table 2).

Discussion
In this study, we found that the higher FIB-4 score was independently associated with more severe strokes on admission, poor outcomes at discharge and a higher 90-days death risk among patients with CES due to NAVF.
More importantly, our study also suggests that sex can modify this associations, and the association become stronger in male patients, but not signi cant in females.
FIB-4 score is a validated non-invasive tool to assess live brosis in HIV and chronic hepatitis C virus coinfection, hepatitis C mono-infection and non-alcoholic fatty liver disease (NAFLD) populations. (12,13) NAFLD is the main cause of most clinically covert liver brosis, especially after excluding the in uence of alcohol consumption.(23) FIB-4 have been validated to have good accuracy in the identi cation of liver brosis in patients with NAFLD.(15) To our knowledge, there was no evidence to con rm the associations between subclinical liver disease and CES characteristics due to NVAF and clinical outcomes as well as the sex hybrid effect in previous studies. Liver brosis was associated with the risk of cardiovascular disease and mortality in the general population. (24) Remarkably, in NAFLD, cardiovascular disease is more commonly responsible for death than liver disease. (8) Although several previous studies have concluded that the severe abnormal liver enzyme may be signi cant risk factor for stroke and worse clinical outcomes of stroke patients with non-liver diseases, (5,25,26) however they only focused on the effects of nonspeci c liver enzyme index on stroke and patients with heavy alcohol consumption were not excluded, or they including all subgroups of stroke patients.
(5) In contrast, a novel association of severe stroke on admission, major disability at discharge and 90-days death after discharge for the recognized liver brosis indictor (FIB-4) was observed in the CES patients with NVAF of our study which of different pathogenesis from others. Moreover, this novel nding revealed that live brosis may represent more severe stroke on admission, worse functional outcomes at discharge and a higher 90-days mortality risk, without obvious clinical manifestations of liver disease, abnormal liver enzyme and con rmed liver disease, which share the same conclusion as those observation-studies that liver brosis could occur without special attention of many subjects, and almost 75% of subjects with liver brosis had normal liver chemistries levels. (23) To sum up, our study presented the evidence that FIB-4 index is independently associated to severe stroke, short-term outcome of CES due to NVAF.
Stroke risk, severity, reaction to endovascular therapy and outcomes could be differ from sex. Mounting studies generally suggested that older females suffered from more severe stroke, worse prognosis, and higher mortality than males. (3,4,27,28) We also observed the higher NIHSS score, mRS score and more death in women patients (Table S1). Therefore, when we grouped sex to eliminate the hybrid effect, we found that liver brosis became a stronger risk factor for server stroke, major disability and 90-days death in males, but not in females.
The results of the present study was not consistent with a previous study, which showed that sex would not affect the association between liver disease and stroke mortality among liver brosis population.(29) Maybe because the patients they researched almost did not suffer from AF, and the NIHSS score which represent stroke severity of those patients included in their study is lower than of the patients in our study (average NIHSS = 3.7 versus 8).
The possible mechanisms in the association between live brosis and poor prognosis of CES patients with NVAF may include metabolic pathways, immune-in ammatory and coagulopathy. (29)(30)(31) Liver involved in a variable degree in the acute ischemic stroke, and is responsible for the synthesis and metabolism of blood coagulation factors and brinolytic enzymes associated with pathophysiology of stroke.(30, 32) What's more, liver also can produced enzymes proportional to the injury size, to response signals from cerebral infarction. (32) But whether these mechanisms can also apply to explain our ndings referring to subclinical liver brosis remain uncertain and requires further studies to verify.
Our nding about sex can modify the relationship of liver brosis and the short-time outcomes of CES patients with NVAF, which share a different view of Baik's study, which suggested the association between liver brosis and cerebral infarction prognosis did not change with gender. (29) The disagreement might be related to discrepancies in several aspects between these two studies, including different study populations, durations of stroke after onset and timings to assess the outcomes. In Baik's study, (29) all types of acute stroke were investigated, and the patients they included with younger age, little atrial brillation, and milder function outcome at discharge. Moreover, their follow-up time was over 3 years. Several studies concluded that traditional cardiovascular risk factors varies by sex. (33)(34)(35) Women have speci c risk factors, including gestational hypertension and pre-eclampsia, gestational diabetes, and placental disorders such as intrauterine growth restriction and stillbirth. (36)(37)(38) It is worth noting that females remain an independent poor prognostic factor for CES due to AF. A prospective cohort study revealed the etiological stroke subtypes independently related to unfavorable outcomes at 90 days were CES in women, but large artery atherosclerosis in men. Their results suggested that AF affect females more than males in short-term outcome of stroke.(39) So in our study, AF maybe display an important role for the stroke outcomes of females, and liver brosis also presented sex difference related to the outcome of CES due to AF, which may be explained for the comorbidity affect patients differently classi ed by sex (39) and the sex differences in liver function, disease pathogenesis and metabolic genes for maintaining homeostasis in the liver. (40) But this hypothesis still need further studies to verify. This is the rst study showed that liver brosis is associated with stroke severity, outcomes due to NVAF, and sex can modify those relationships. It also con rmed the high burden of liver brosis in CES patients with NVAF.
Strengths of our study also include the aimed population with strict inclusion criteria, the availability of standardized outcome assessments, and the exclusion of patients with overt liver disease. In our study, almost 90% of subjects with liver brosis had normal liver chemistries levels, indicating even through the stroke patients with normal liver enzyme levels also need accept live brosis evaluation such as the FIB-4 sore, especially for male patients.
There were several limitations in the present study. First, it shares all the limitation of single-center, observational study, so the generalizability of our results may be limited. To minimize the biases caused by an observational study, our study included consecutive patients admitted during the study period thereby. Second, although our use of liver brosis indice-FIB-4 is consistent with other studies (41,42) to observe how liver brosis affect disease processes, we were unable to determine a causal relationship between liver brosis and clinical outcomes of CES patients with NVAF.

Conclusion
Among CES patients with NVAF, FIB-4was associated with admission stroke severity, major disability at discharge, and 90-days mortality, and this associations was more signi cant in males but not in females. Our work suggests that new risk assessment and therapeutic targets aimed at liver brosis, may bene t those who with poor outcomes of CES patients with NVAF.