The hidden interplay between sex and COVID-19 mortality: the role of cardiovascular calcification

Recent clinical and demographical studies on COVID-19 patients have demonstrated that men experience worse outcomes than women. However, in most cases, the data were not stratified according to gender, limiting the understanding of the real impact of gender on outcomes. This study aimed to evaluate the disaggregated in-hospital outcomes and explore the possible interactions between gender and cardiovascular calcifications. Data was derived from the sCORE-COVID-19 registry, an Italian multicentre registry that enrolled COVID-19 patients who had undergone a chest computer tomography scan on admission. A total of 1683 hospitalized patients (mean age 67±14 years) were included. Men had a higher prevalence of cardiovascular comorbidities, a higher pneumonia extension, more coronary calcifications (63% vs.50.9%, p<0.001), and a higher coronary calcium score (391±847 vs. 171±479 mm3, p<0.001). Men experienced a significantly higher mortality rate (24.4% vs. 17%, p=0.001), but the death event tended to occur earlier in women (15±7 vs. 8±7 days, p= 0.07). Non-survivors had a higher coronary, thoracic aorta, and aortic valve calcium score. Female sex, a known independent predictor of a favorable outcome in SARS-CoV2 infection, was not protective in women with a coronary calcification volume greater than 100 mm3. There were significant differences in cardiovascular comorbidities and vascular calcifications between men and women with SARS-CoV2 pneumonia. The differences in outcomes can be at least partially explained by the different cardiovascular profiles. However, women with poor outcomes had the same coronary calcific burden as men. The presumed favorable female sex bias in COVID-19 must therefore be reviewed in the context of comorbidities, especially cardiovascular ones.


Introduction
The SARS-CoV2 outbreak has had a significant impact on healthcare services and resource allocation. Accumulating evidence has shown that amongst COVID-19-infected patients, elevated mortality is observed in older patients and those with pre-existing comorbidities, including hypertension, diabetes mellitus, cardiovascular disease, chronic lung disease, and cancer [1][2][3].
The available sex-disaggregated data reveal that men experience a higher hospitalization rate, a more severe disease than women with a consequent higher overall case fatality ratio [1]. Epidemiological data on sex differences are still under investigation and need to be cleared of potential bias. A better knowledge of sex differences in incidence and mortality in the COVID-19 pandemic is the first step to analyze the biological patterns to define gender-specific prevention and treatment strategies.
The high cardiovascular risk profile of males and females has been proposed to explain the observed gender bias in outcomes.
Recent studies have demonstrated a complex interplay between comorbid cardiovascular disease, COVID-19 pathophysiology, and poor clinical outcomes. Coronary artery calcification (CAC) may therefore aid in risk stratification of COVID-19 patients.
Lung CT scans are obtained in COVID-19 patients to assess the extent of lung disease, monitor disease progression, and investigate the presence of disease complications.
The analysis of vascular calcifications (coronary, aortic, and valvular) can provide a rapid and quantifiable stratification of the cardiovascular profile. This imaging biomarker may provide further insight into the disease process from both pathophysiologic and clinical perspectives, performed in the context of a pandemic with limited health resources Considering that cardiovascular comorbidities represent a recognized risk factor for COVID-19 outcome, cardiovascular calcifications (calcium score) can be regarded as a surrogate radiological biomarker that can stratify the patient's cardiovascular risk [4][5][6].

Methods
The present study aims to describe gender differences in terms of clinical and radiological features and in-hospital outcomes of COVID-19 patients. The hypothesis that the different cardiovascular profile influences the different outcomes in men and women was explored through the comparison of cardiovascular calcifications.

Study cohort
The study cohort was derived from the multicentre, retrospective, and observational sCORE COVID-19 (calcium score for COVID-19 Risk Evaluation) registry, which has been described elsewhere [6]. Briefly, the sCORE COVID-19 registry included data from 16 Italian hospitals directly involved in the COVID-19 emergency during the study period (Mar 1-Apr 20, 2020). All consecutive patients with a positive qualitative polymerase chain reaction assay for SARS-CoV-2 and a non-contrast chest CT scan performed on admission to assess pneumonia severity were included. The study population was divided into two groups according to gender (male and female), and data were analyzed for each group [7]. The local ethics of each institution committee approved the study.

Data collection
Demographic characteristics, cardiovascular risk factors, comorbidities, and history of coronary artery disease were collected. Laboratory data included baseline admission values of hemoglobin, white blood cell count, creatinine, baseline high sensitivity troponin I (HS-TnI), lactate dehydrogenase (LDH), and C-reactive protein (CRP).

Chest ct scan analysis
Chest CT scans were sent to the central core lab (Experimental Imaging center, IRCCS, Ospedale San Raffaele, Milano). They were analyzed by three expert cardiothoracic radiologists blinded to patients' clinical data. Clinical and radiological data were integrated and analyzed by the coordinating center (Maria Cecilia Hospital, GVM Care & Research, Cotignola).
CT scans had been acquired with a standard nongated chest CT protocol, using multidetector scanners with at least 16 detector rows [6]. For lung parenchyma evaluation, CTs were reconstructed at each site with a sharp kernel and visualized at the core lab using a standard lung window (width 1400 HU; center −450 HU). Analysed lung parameters included (1) semi-quantitative pneumonia scoring [no pneumonia (0%); minimal pneumonia (1-25%); mild pneumonia (26-50%); moderate pneumonia (51-75%); and severe pneumonia (76-100%)] [8]. For calcium quantification, CTs were reconstructed at each site with a soft kernel, transferred to the core lab, reformatted at a standard slice thickness of 2.5 mm without overlap or gap, and visualized using a standard mediastinal window (width 350 HU; center 40 HU). Coronary artery calcifications were visually assessed (presence/absence and number of involved vessels) and quantitatively computed. Quantification of coronary artery (CAC) was performed both with Agatston calcium scoring (CS) [9] and calcium volume (CV) methodology [10], semi-automatically, on commercial software (IntelliSpace v 8.0, Philips, The Netherlands), as follows: vascular calcifications were automatically detected as a group of adjacent pixels with an area ≥ 1 mm 2 and a density above 130 HU. An experienced cardiothoracic radiologist labeled every calcification as belonging to coronary arteries (left main, left anterior descending, left circumflex, or right coronary artery). The interaction between cardiovascular calcifications and sex was investigated: the population was subdivided according to the coronary calcium score: absent of calcium score (CAC= 0), mild coronary calcifications (CAC 0-100), and moderate to severe coronary calcifications (CAC> 100).

Statistical analysis
The qualitative variables are expressed as a percentage, and the differences were tested with the chisquare test. Quantitative variables are expressed as mean and standard deviation. The Student t test was used to test the statistical difference of continuous variables, adjusted according to the distribution's possible normality.
The population was distinguished in the analysis based on biological sex (men vs. women).
The overall cohort was split based on gender and each gender further sub-categorized into survivors or non-survivors.
Continuous variables were compared with the Student t test adapted according to their distribution. Dedicated plots were constructed to compare the population's risk profile based on gender and outcome using the relative risk ratio. Cox regression was used using "time to death after admission" as a time variable. Statistical significance of age, sex, creatinine, white blood cells, LDH, and interstitial lung involvement percentage was calculated on the univariate Cox analysis.
The choice of the included variables was empirical, considering the variable's different domains and the preliminary data reported in the literature.
In addition to age (the most important demographic variable) and creatinine, white blood cell counts (expression of inflammation) and LDH (a marker of organ damage) were included in the model. Radiological lung injury was accounted for using the percentage of interstitial involvement categorized as greater/less than 50% of the lung parenchyma.
The univariate significance of these variables was included in a multivariate Cox regression model. The interaction of these variables with the calcium score and sex was explored by repeating the Cox multivariate in patients with mild coronary calcifications (0<CAC <100) and in those with moderate-severe coronary calcifications (CAC>100 mm 3 ).

Results
The study population included 1683 hospitalized patients with confirmed COVID-19 infection and admission chest CT. Table 1 shows the demographic characteristics and comorbidities of the overall population and two cohorts. Group A included 1131 men (67.2%) and group B 552 women (32.8%). Globally, the mean age was 67±14 years, with no statistically significant difference between the groups. In total, 910 patients (55%) had arterial hypertension, 319 (19.3%) diabetes mellitus, and 170 (11.4%) coronary artery disease with previous revascularization (percutaneous in 7.1% and surgical in 4.3%). Other prevalent comorbidities included chronic lung disease (166, 10%), chronic kidney disease, and active malignancy (86, 5.2%).
Non-survived men and women, compared to survived patients, had a higher calcium score in all the three analyzed districts (coronary, thoracic aorta, and aortic valve) (Fig. 1). Figure 2 shows in-hospital survival curves in male and female groups subdivided according to coronary calcification severity (absent, mild, and severe). Men and women with severe calcification results have the worst outcome. Cardiovascular calcifications had a mild-moderate sensitivity in predicting death with an AUC ranging between 0.67 and 0.72 based on the type of calcification considered. Coronary, thoracic aortic, and aortic valvular calcifications had slightly different sensitivity in predicting death between men and women.
Coronary and aortic calcifications were more sensitive in men, while aortic valvular calcifications were more significant in women (Fig. 3).
Age, female sex, creatinine, white blood cell count, LDH, interstitial lung involvement > 50%, and coronary calcium volume were significant predictors of the univariate Cox analysis.
The continuous variable with a higher HR (HR 1.52, 95% CI 1.42-1.62) was baseline creatinine. The categorical variable with a higher HR was interstitial lung involvement greater than 50%. Calcium volume was significant, with an HR of 1 (95% CI 1.0002-1.0004). The gender categorical variable was negatively associated with death with an HR of 0.66 (95% CI 0.52-0.84).
In multivariate analysis, the female sex maintained its negative predictivity for the death event (HR 0.65, 95% CI 0.50-0.92).
In patients with mild coronary calcifications, sex was significant (HR 0.68, 95% CI 0.49-0.96). In contrast, in patients with moderate-to-severe calcifications, female sex (but not age, creatinine, and LDH values) lost its statistical significance as a predictor of 30-day mortality (HR 0.89, CI 0.48-1.67).
Pulmonary radiological involvement also lost significance in patients with moderate to severe coronary calcifications (HR 1.54, CI 0.86-2.74).

Discussion
The main findings of this sCORE-COVID-19 registry sub-analysis are that women, compared to men, (1) had lower in-hospital mortality; (2) experienced a less pronounced inflammatory reaction with a lower pneumonia extension; and (3) had a lower cardiovascular risk profile with fewer coronary calcifications. This trend was even confirmed within the whole women group: indeed, non-survived women were older than the survived ones with a higher cardiovascular risk profile, including coronary calcifications. Our analysis is in line with the recent literature confirming that women are globally more protected from the COVID-19 adverse outcomes, having a more favorable clinical and radiological cardiovascular risk profile [8][9][10]. However, the protective effect of the female gender on mortality seems to be lost in women with moderate to severe coronary calcifications. Other possible reasons for sex disparities in COVID-19 outcome are the biological differences in sex chromosome genes and sex hormones that may contribute to the immune response's different regulation. Women are functional mosaics for X-lined genes [11,12].
The X chromosome contains a high density of immune-related genes; therefore, women generally mount more robust innate and adaptive immune responses than men. Genes encoded on X chromosomes, and sex hormones may explain the decreased fatality of COVID-19 in women.
The angiotensin-converting enzyme 2 gene is located on X chromosomes. Men, with a single X chromosome, may lack the alternative mechanism for cellular protection after exposure to SARS-CoV-2.
As an element of novelty, calcium score, diversified into coronary calcifications, aortic valve calcifications, and thoracic aortic calcifications, plays a role in COVID-19 mortality prediction.   The coronary calcium score is a well-recognized parameter that can identify heart disease and estimate cardiovascular events' risk. Nevertheless, calcium score provides additional information regarding total mortality risk beyond traditional risk factors [6,10].
Calcium score should be considered a measure of arterial aging reflecting several cardiovascular risk factors' cumulative effect.
Coronary calcium likely reveals a general biological weakness or a reduced microvascular or endothelial reserve, even more accurately than aging and other comorbidities. Cardiovascular calcifications appear to be an intergender parameter strongly associated with a poor outcome. Indeed, even if overall mortality in COVID-19 seems lower in women, the intergender gap is not confirmed in the subgroup of patients with moderate to severe CAC [13,14].
The analysis of cardiovascular calcifications allows to "photograph" the cardiovascular burden of every single patient at a precise moment. In contrast, cardiovascular anamnestic variables are often retrospective and may underestimate the cardiovascular burden, especially in women [7,15]. The present study's limitations are the retrospective analysis of the clinical and radiological data collected during the first pandemic wave peak. Therapeutical data have not been taken into account due to a standardized approach during the study period. Our study population included only COVID-19 patients who had undergone chest CT for lung assessment [16]. Therefore, these findings are potentially not reflective of all COVID-19 patients. However, it is relevant to underline that all consecutive patients by each participating center were enrolled. Moreover, our study population's clinical, laboratory, and outcome features were consistent with those previously reported in the literature for COVID-19 [17][18][19][20].

Limitations of the present study
In the differences in laboratory values (hemoglobin, creatinine, and white blood cells) and the values of cardiovascular calcifications, it is necessary to take into account that women have different basal values compared to men.
The comparison between survivors and deceased versus the overall population partially limits this bias. In our study, some risk factors are not available that could explain the different calcific burdens such as dyslipidemia or uric acid levels.
We have no information on chronic cardiovascular medications of these patients and therapies used in the treatment of COVID-19 disease beyond ventilation therapy.
The prophylactic use of heparin and steroids in SARS-CoV2 infection was not yet known at the time of the first wave and these data are not collected.
The outcomes of the present study concern the first region of the western world affected by the epidemic and certainly the outcomes are worse than those of other regions affected in the following weeks and compared to the other epidemic waves.

Conclusion
COVID-19-infected women were less hospitalized than men and had more favorable outcomes. The cardiovascular burden in terms of comorbidities and coronary, thoracic aorta, and aortic valve calcifications was less represented in women [7]. However, in the multivariate analysis model, the interaction between sex and mortality was not significant in the population with moderate-to-severe coronary calcifications. Our data suggest that the protective effect of the female sex is attenuated in women at increased cardiovascular risk. In a viral infection with multiple pleiotropic cardiovascular manifestations, the protective factor of sex is lost in women with coronary calcifications (calcium score volume > 100 mm 3 ).
The presumed favorable female sex bias in COVID-19 must therefore be reviewed in the context of comorbidities, especially cardiovascular ones.
Funding Open access funding provided by Università degli Studi di Ferrara within the CRUI-CARE Agreement.

Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.