Sex disparities in clinicopathological features and outcomes of patients with myeloperoxidase-ANCA-associated vasculitis: a retrospective study of 366 cases in a single Chinese center

There are a few studies that reported sex disparities in clinical features, pathological features and outcomes among ANCA-associated vasculitis (AAV) patients, but studies focusing on sex-specific differences of myeloperoxidase (MPO)-AAV patients are scarce. Therefore, the purpose of this study was to analyze sex differences in clinicopathological features and outcomes of MPO-AAV. Patients diagnosed with MPO-AAV in Xiangya Hospital from January 2010 to June 2021 were included in the study and separated into female and male groups. The differences in clinical manifestations, laboratory parameters, pathological features and prognosis between the two groups were retrospectively analyzed. Three hundred and sixty-six patients were included and divided into female group (n = 176) and male group (n = 190). The age of the male group was 62.41 ± 10.49 years, significantly higher than that of the female group (58.69 ± 16.39, p = 0.011). Compared with the female group, the male group had a shorter duration of disease, higher levels of hemoglobin, eosinophil count, proteinuria, serum C4, and lower levels of serum globulin, serum IgG and serum IgM (p < 0.05). No significant differences in kidney pathological features were observed between the two groups. During a median follow-up of 37.6 months, there was no significant difference in renal survival and patient survival between the two groups, but male patients had a worse composite outcome of renal and patient survival compared with the female patients (p = 0.044). This study found that male patients with MPO-AAV had a higher age of onset, shorter duration of disease, higher levels of hemoglobin, eosinophil count, proteinuria, serum C4, and lower levels of serum globulin, serum IgG and serum IgM. Male patients fared worse than female patients in terms of the composite outcome of renal and patient survival.


Introduction
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease, which leads to damage and dysfunction of multiple organs and tissues, most frequently presenting as renal, lung, ear, nose and throat (ENT) and nerve lesions [1].Its characteristics are inflammation and necrosis of small and medium-sized blood vessels and are associated with the presence of ANCAs in the serum [2].AAV contains three different types based on its clinical presentation, namely microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) and eosinophilic GPA (EGPA) [3].There are significant differences in heredity, epidemiological characteristics, organ involvement and prognosis between myeloperoxidase (MPO)-ANCA positive and proteinase 3 (PR3)-ANCA positive patients [4,5].
Advancements in diagnosis and treatment have changed AAV from a life-threatening disease to a controllable chronic disease.However, renal insufficiency remains a major challenge for clinicians, which can lead to end stage renal disease (ESRD) and even premature death [6].The main determinants leading to adverse outcomes were advanced age, baseline kidney function, renal histopathologic classification, Ting Meng and Peng Zhu authors have contributed equally to this work and share first authorship.
Yong Zhong Lead Contact.
Extended author information available on the last page of the article pulmonary hemorrhage, Birmingham vasculitis activity score (BVAS), ANCA titer and early infection, which are associated with ESRD and death [7][8][9][10].Recent studies have found that male AAV patients are more likely to develop ESRD [11], and being male is an independent all-cause mortality predictor [12].By contrast, Scott et al. did not observe a strong impact of gender on ESRD [13].The sex disparity of prognosis is still not clear among AAV patients.
Unlike other autoimmune diseases, the incidence of AAV has no significant gender predominance, but some sex-specific differences in disease manifestation have been investigated.Compared with male patients, female patients were older at diagnosis [13,14], had more joint and nervous system involvement [14], had a higher positive rate of muscle biopsy [15], more MPO-AAV [13], less PR3-ANCA [11], and more pANCA [16].There was less correlation among clinical features, laboratory parameters and histopathology in female patients [14].At present, there are no reports about the sex disparities in clinicopathological features and prognosis of MPO-AAV patients.Therefore, we systematically analyzed sex differences in clinical manifestations, pathological features and prognosis among MPO-AAV patients in a single Chinese center.

Study population
This retrospective study was approved by the Ethics Committee of Xiangya Hospital, Center South University in accordance with the 1964 Declaration of Helsinki.Written informed consent was not required for a retrospective study.(ID: 202108374).
AAV patients with newly diagnosed MPA or GPA from Xiangya Hospital, Center South University between January 2010 and June 2021 were retrospectively included in this study.Diagnosis was made according to the 2007 European Medicines Agency-EMA-algorithm and the 2012 revised Chapel Hill Consensus Nomenclature of Vasculitides [2,17].Inclusion criteria were AAV patients with MPO-ANCA serology positive at initial diagnosis; Exclusion criteria were patients diagnosed with secondary vasculitis or other types of vasculitis, including EGPA, PR3-AAV and anti-glomerular basement membrane (GBM) disease.

Data acquisition
We retrospectively collected the patient's demographic characteristics (age, sex), clinical presentation (duration of disease, BVAS [18], vasculitis type, organ involvement and comorbidities) and routine laboratory parameters (full blood examination, hemoglobin, serum albumin, serum globulin, serum creatinine (Scr), estimated glomerular filtration rate (eGFR), 24-h proteinuria, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), serum immunoglobulin, serum complement and ANCA serology) at the time of initial diagnosis obtained through the electronic medical records from Xiangya Hospital.We followed up the patients until they developed ESRD, or died or until November 2021 through telephone and/or WeChat.

Definition
ESRD was defined as eGFR < 15 mL/(min*1.73m 2 ) and requiring renal replacement therapy for more than 3 months or kidney transplantation [19].Duration of disease was defined as the duration from onset to the time of diagnosis.Renal survival was defined as the duration from onset to the time of developing ESRD.Patient survival was defined as the duration from onset to the time of death.Renal and patient survival was defined as the duration from onset to the time of developing ESRD or death.

Renal biopsy
A total of 151 patients received renal biopsy before receiving immunosuppressive therapy at the initial diagnosis of MPO-AAV.Two experienced pathologists scored the renal specimens independently and were blinded to the clinical data.Renal specimens were evaluated by direct immunofluorescence, light microscopy and electron microscopy [20].According to the Berden classification [10], which is based on the proportion of glomerular lesions, the patients were divided into focal, crescentic, mixed and sclerotic classes.Renal specimens were also evaluated similarly by the histological characteristics as described previously [21], including fibrinoid necrosis, Bowman's capsule rupture, periglomerular inflammation, granulomatous lesions, interstitial infiltrates and renal tubulointerstitial lesions.Interstitial infiltrates and renal tubulointerstitial lesions were scored semiquantitatively according to the extent of involvement, which can be divided into none (score 0, range of involvement = 0%), mild (score 1, range of involvement < 25%), moderate (score 2, range of involvement between 25-50%) and severe (score 3, range of involvement > 50%) [22,23].

Treatment protocol
The treatment of AAV was divided into two stages: induction treatment and maintenance treatment [24].All patients received glucocorticoid combined with CTX (0.5-0.75 g/ m 2 /months) or mycophenolate mofetil (MMF) (2 g/d) during the induction period, and then were switched to maintenance therapy after remission was achieved, which consisted of MMF (1.5 g-2 g/d) or azathioprine (AZA) (1.5-2 mg/kg/d) combined with low-dose prednisone (5-7.5 mg/d).Maintenance treatment lasted at least 18 months.All patients were treated with oral prednisone, the initial dose was 1 mg/ kg/d and gradually reduced according to the schedule as described previously [25].The dose of immunosuppressant was adjusted according to age, eGFR, adverse events and white blood cell count.According to the disease condition, some patients received high-dose methylprednisolone pulse therapy (5-10 mg/kg/d) and plasma exchange.

Statistical methods
The data types were divided into qualitative data and quantitative data.Qualitative data were presented as frequencies (percentage) and were compared by the Χ 2 test.Quantitative data were presented as meanplus standard deviation (SD) and compared by the Student's t test when they fitted a normal distribution, or they were presented as median with interquartile range (IQR) and compared by the Mann-Whitney U test or Kruskal-Wallis test when they were not normally distributed.Survival distribution was presented as Kaplan-Meier survival curves and compared by a log-rank test.All data analysis was performed using GraphPad Prism software (version 8) and SPSS Statistical software (version 25).If the two-sided p value < 0.05, the difference was considered to be statistically significant.

Sex differences among demographic characteristics and clinical features
A total of 366 patients with MPO-AAV were included in this study.According to gender, 176 female patients were included in the female group and 190 male patients were included in the male group.The main baseline demographic characteristics and clinical features of the two groups are summarized in Table 1.In this cohort, 176 (48.1%) were female, with a mean age at diagnosis of 58.69 ± 16.39 years.The average age of male patients was 62.41 ± 10.49 years, which was significantly higher than that of female patients (p = 0.011) (Table 1).Three hundred and sixteen (86.3%) patients were diagnosed with MPA.The median duration of disease in male patients was 50 days (IQR: 78-91), which was significantly shorter than that in female patients (p = 0.027) (Table 1).The initial mean BVAS was 17.02 ± 6.02.Three hundred and forty-four (94.0%) patients had renal involvement and 234 (63.9%) patients had lung involvement.

Sex differences among laboratory parameters
The main baseline laboratory parameters are shown in Table 1.In the total cohort, the median serum creatinine and eGFR was 306 μmol/L (IQR: 150-556) and 14.39 mL/ (min*1.73m 2 ) (IQR: 7.55-38.85),respectively.The serum creatinine of the male group was slightly higher than that of the female group, but there was no significant difference.The mean hemoglobin in the male group was significantly higher than that of the female group (p = 0.010) (Table 1).The median 24 h urinary protein in the male group was 1.31 g, which was higher than that in female group (p = 0.010) (Table 1).In addition, compared with the female group, the eosinophil count and serum C4 level were higher, and serum globulin, serum IgG and serum IgM levels were lower in male patients.There were no significant differences in other laboratory parameters between the two groups.

Sex differences among pathological features
A total of 151 patients with MPO-AAV received renal biopsy, including 67 female patients and 84 male patients.As shown in Table 2 No significant differences in glomerular injury classification were observed between the two groups (Table 2).There were no statistical differences in Bowman's capsule rupture, periglomerular inflammation, granulomatous lesions, interstitial infiltration and renal tubulointerstitial lesions score between the two groups (Table 2).

Renal survival and patient survival
During a median follow-up of 37.6 months, the overall 1-year and 5-year renal survival rates were 58.83 and 48.19% in the male group, and 67.18 and 59.41% in the female group, respectively (Table 3).There was no significant difference in renal survival between the two groups (p = 0.116) (Fig. 1).The overall 1-and 5-year survival rates were 81.21 and 62.44% in the male group, and 85.63 and 70.05% in the female group, respectively (Table 3).There was also no significant difference in patient survival between the two groups (p = 0.096) (Fig. 2).However we found in the first 3 years, male patients had a higher risk of progressing to ESRD or death compared with female patients.After adjusting for age and eGFR, the hazard ratio (HR) for ESRD in male patients versus female patients was 1.977 (95%CI, 1.429-2.737,p < 0.001), and the HR of males versus females for death was 1.392 (95%CI, 0.945-2.050,p = 0.094).We found that there was a significant difference between the two groups in the composite outcome of renal and patient survival (Fig. 3).The overall 1-year and 5-year renal and patient survival in the male group was 51.09 and 36.11%, which was significantly lower than that in the female group (61.05 and 48.65%), respectively (Table 3).Similarly, the HR of males versus females for renal patient survival was 1.698 (95%CI, 1.277-2.256,p < 0.001).

Discussion
In this retrospective study, we analyzed the sex differences in clinical manifestations, laboratory parameters, pathological features and outcome in patients with MPO-AAV.We found that male patients had a higher age of onset and  a shorter duration of disease than female patients.In addition, with regard to laboratory parameters, hemoglobin level, eosinophil count, proteinuria, serum C4 level were higher, while the level of serum globulin, serum IgG and serum IgM were lower in male patients.Previous studies have suggested that female patients are older than male patients with AAV [13,14].However we encountered inconsistent results, which firstly may be attributed to differences in the study population, latitudinal and longitudinal gradient, and ANCA specificity [26].Secondly, we observed that it is mainly because that there were ten patients in the female group but only one patient in the male group who were less than 18-years-old at the time of diagnosis, which is consistent with our previous findings that girls more commonly present with childhood AAV than boys [27].Neurological involvement was more  common in female patients in this study, which was consistent with a previous study [14], suggesting that there may be sex differences in neurological involvement.We found that proteinuria in male patients was significantly higher than in female patients.Proteinuria reflected the severity of disease and predicted poor renal and patient survival in a previous study [28].Similarly, our previous study suggested that hypoglobulin is a risk factor for the progression to ESRD in patients with MPO-AAV [29], suggesting that lower serum globulin in male patients may promote the progression to ESRD.A previous study suggested that low serum C3 levels in AAV patients is associated with adverse outcomes [30].Hakroush et al. reported that low serum C4 levels were associated with glomerulonephritis in ANCA-associated glomerulonephritis [31].
Here we found that male patients have higher serum C4 level than female patients, and the relationship between the sex difference of serum C4 level and prognosis needs further study.Although there were significant differences in other laboratory parameters such as hemoglobin, eosinophil count, serum globulin, serum IgG and serum IgM, these differences might be attributed to gender-fixed differences in the population [32][33][34], which may be related to hormone levels [32,35].We found that in terms of the composite outcome of renal and patient survival, male patients fared worse than female patients.After accounting for age and eGFR, male patients were more likely to progress to ESRD.Previous studies also suggested that the risk of progression to ESRD and death in male patients is significantly higher than those in female patients [11,12], which may be associated with baseline kidney function and histopathologic classification [10].Although there were no statistical differences in renal survival and patient survival in our study, renal and patient survival as a compound prognosis was worse in male patients.The reason may be due to the higher age of onset, and higher proteinuria in male patients, which are associated with adverse factors like ESRD and death [7,36,37].Finally, we included patients with MPO-AAV, who have a poorer renal function and a higher risk of progression to ESRD compared with other AAV types.Our 1-and 5-year renal survival rates were lower than those of previous AAV studies [11,13], which might narrow the sex difference in kidney survival.
To the best of our knowledge, this is the first study to report sex differences in clinicopathological features and prognosis in MPO-AAV patients.However, there are still some limitations.First of all, this was a retrospective study that we could not trace the exact cause of death in some patients but only assessed all-cause death.Secondly, this was a single-center study, which may have a selection bias.Further prospective, multicenter studies in the future are needed to verify the results of this study.

Conclusion
In conclusion, male patients with MPO-AAV had a higher age of onset, shorter duration of disease, with higher levels of hemoglobin, eosinophil count, proteinuria and serum C4, and lower levels of serum globulin, serum IgG and serum IgM.Male patients fared worse than female patients in the composite outcome in terms of renal and patient survival.
ards.The Ethic Committee of Xiangya Hospital approved the study protocol according to the Declaration of Helsinki (ID: 202108374).

Table 1
Baseline demographic characteristics and clinical features MPA microscopic ployangiitis, GPA granulomatosis with polyangiitis, BVAS Birmingham vasculitis activity score, ENT ear, nose and throat