The Role of Sex in the Risk of Mortality From COVID-19 Amongst Adult Patients: A Systematic Review

A worldwide outbreak of coronavirus disease 2019 (COVID-19), identified as being caused by the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), was classified as a Public Health Emergency of International Concern by the World Health Organisation (WHO) on January 30, 2020. Initial sex-disaggregated mortality data emerging from the Wuhan province of China identified male sex as a risk factor for increased COVID-19 mortality.   In this systematic review, we aimed to assess the role of sex in the risk of mortality from COVID-19 in adult patients through comparison of clinical markers and inflammatory indexes.  A systematic search was conducted on the following databases: PubMed, WHO COVID-19 database, Ovid MEDLINE, and Web of Science between the dates of June 15, 2020, and June 30, 2020. Key search terms used included: “sex”, “gender”, “SARS-COV-2”, “COVID” and “mortality”. We accepted the following types of studies concerning adult COVID-19 patients: retrospective cohort, observational cohort, case series, and applied research. Further studies were extracted from reference searching. The risk of bias was determined using the National Institutes of Health Quality Assessment Tool for Observational Cohort, Cross-Sectional Studies, and Case Series.  We identified a total of 16 studies published between January 2020 and June 2020 for analysis in this systematic review. Our study population consisted of 11 cohort studies, four case series, and one genetic study, including a total of 76,555 participants. Ten of the studies included in this review observed a higher risk of mortality among males compared to females, and eight of these studies found this risk to be statistically significant.   Sex-disaggregated COVID-19 mortality data identifies male patients with comorbidities as being at an increased risk of mortality worldwide. Further investigation revealed differences in immune response regulated by sex hormones, angiotensin-converting enzyme 2 (ACE2) expression, and health behaviours as contributing factors to increased risk of mortality from COVID-19 among males.   Nine out of the 16 studies included were conducted in China. In order to comprehensively assess sex-differences in the risk of mortality from COVID-19, more studies will need to be conducted worldwide. Sex-disaggregated COVID-19 data published in the medical literature is limited, however it has become evident that male sex is an important risk factor for mortality. Further exploration into the impact of sex on this pandemic is required in order to develop targeted therapies, as well as public health policies, and to prevent sex bias in treatment.


Introduction And Background
In December 2019, a rise in pneumonia cases of unknownaetiologywas seen in the Wuhan province of China. Approximately one month later, the coronavirus disease 19 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the pathogenic source of the disease [1].The virus is transmitted by talking, coughing, sneezing, aerosols and is now thought to be airborne [2].The rapidly spreading and contagious nature of the virus led to a Public Health Emergency of International Concern being declared by the World Health Organisation (WHO) as of January 30, 2020 [3].
Mild illness may present with symptoms such as fever, malaise, headache, muscle pain, dry cough, and sore throat. However, more severe disease may progress to Acute Respiratory Distress Syndrome (ARDS) and death [3].
As ofJune 30, 2020, England had 39,177 COVID-19 fatalities of which 56.9% were males and 43.1% were females [4].Further exploration into the impact of sex on this pandemic is required to develop targeted therapies and public health policies.Sex-disaggregated analysis of the COVID-19 outbreak isimportant,asmortalitydata from 49 countries indicatethat males have a higheroverallmortality rate than females [4].Although male and female susceptibility is the same, it has become evident that the male sex is an important risk factor for mortality [5].
In this systematic review we aimed to assess the role of sex in the risk of mortality from COVID-19 in adult patients through comparison of clinical markers and inflammatory indexes.

Search Strategy
A systematic search was conducted on the followingelectronicdatabases:PubMed, WHO COVID-19 database, Ovid MEDLINEandWeb of Sciencebetween the dates of June 15, 2020,and June 30, 2020.This systematic review was conducted in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelinesand hasbeenregistered on PROSPERO (identifier number: CRD42020196076) [6].

Selection Criteria
The preliminary search yielded1092papers.After removal of duplicates, 655abstracts and titles were screened for relevancy, through which598papers were excluded. The remaining57papers underwent full text screening. Anadditional25paperswere retrieved through screening ofrelevantreferences. A total of 82papers underwent full text screening. After applying inclusion and exclusion criteria, 16 papers were selected for analysis in this systematic review.
We accepted the following types of studies: retrospectivecohort, observationalcohort, case series, and applied research. Papers from any country concerning confirmed COVID-19 adult patients were accepted if they were written in English.Papers concerning paediatric (being under 18 years of age) and pregnant patients were excluded. Papers addressing other viral respiratory diseases, such assevere acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), were also excluded.
Our outcome of interest was mortality rate of males and females diagnosed with COVID-19. Indicators that we regarded as potentialreasonsfor the difference in mortality included: immunoglobulin G (IgG) antibody levels, immune cell levels,inflammatory indexes,receptor expression, hormone levels, behaviouralfactors, and disease severity.
Two reviewers screened the abstracts and titles retrievedfromthe searches. The third reviewer conducted a final check on the relevance of the papers against the inclusion and exclusion criteria. Data retrieved from the studies were collated into tabular form outlining the study design and main findings. Figure 1 shows the PRISMA flow diagram detailing the study identification, screening, and selection process.

Risk ofBias Assessment
Risk of bias was assessedusing theNational Institutes of Health (NIH) Quality Assessment Tool for Cohort,Cross-SectionalStudiesand Case series [7]. Two review authors assessed the risk of bias and this was reviewed by the third author.

Results
We identified a total of 16 studies published between January 2020 and June 2020 for analysis in this systematic review. Our study population consisted of 11 cohort studies, four case series and one genetic study, including a total of 76,555 participants.The characteristics and main outcomes of each study are summarised in Table 1

Sex and Mortality From COVID-19
Ten studiesfrom the database search observed higher risk of mortality amongst males compared to females. Eightstudiesfound male sex to be significantly associated with increased risk of mortality from COVID-19. Onestudyfound no significant association between male sex and mortality after adjusting for confounders. Figure 2 indicates whichstudies have observed an increased risk of mortality in males and those in which this association is significant.

FIGURE 2: Male sex and mortality from COVID-19
This figure shows which of the included studies observed an increased risk of mortality due to male sex, and which studies observed a significant association (p<0.05).   Were key potential confounding variables measured and adjusted statistically for their impact on the relationship between exposure(s) and outcome(s)?

Risk of Bias Assessment Results
Case Series: 1. Was the study question or objective clearly stated? 2. Was the study population clearly and fully described, including a case definition? 3. Were the cases consecutive? 4. Were the subjects comparable? 5. Was the intervention clearly described? 6. Were the outcome measures clearly defined, valid, reliable, and implemented consistently across all study participants? 7. Was the length of follow-up adequate? 8. Were the statistical methods well-described? 9. Were the results well-described?

Discussion
We undertook acomprehensivesearch of the literatureconcerningsex-disaggregated mortality from COVID-19 in order tosummarisethe potential underlying reasons for the disparity in mortality rate. We found that published evidence suggests the female sex has a protective role againstCOVID-19 mortality.Main reasons for this finding includethehigher levels of the circulating form of ACE2 in females,theimmunostimulatoryeffectof female sex hormones,more rapid clearance of pathogens by the female immune system and femalestending todisplay disease-preventingbehaviours.

Sex Differences in ACE2 Expression
Differences in angiotensin-Converting Enzyme 2 (ACE2) expression between sexes is thought to contribute tothe highermalemortality rate. ACE2 degrades Angiotensin II into Angiotensin 1-7, counteracting the Renin-Angiotensin-System (RAS) axis. This reduces the effects of the RAS axis which usually increases blood pressure, sympathetic tone, vasoconstriction, inflammation, and fibrosis. ACE2 also serves as the primary receptor for SARS-CoV-2 cellular invasion [23].The viral spike protein contains the S1 domain,which serves as a receptor-binding portion,and the S2 domain which facilitates cellular-viral fusion [24,25]. The high affinity of SARS-CoV-2 for the ACE2 receptor facilitates viral spread between person to person [26]. It is also thought that SARS-CoV-2 infection downregulates ACE2 expression, reducing its protective role and explaining the progression of patients into ARDS [23].
ACE2 is expressed on the PAR region of the X chromosome,which has a greater chance of escaping X chromosomal inactivation [27]. ACE2 is also upregulated byoestrogenleading todisparityin ACE2expressionin some organsbetween thesexes [27,28].The paradox of ACE2upregulationyet lower female mortality can be explained by a few theories. There are two types of ACE2: the membrane-bound, which provides the viral entry point, and the circulating which has a cardiovascular protective function. It is thought thatfemalesexpress more of the circulating ACE2 providing protection against disease progression into ARDS [29].
Vikseet al. suggest that the testes may serve as a reservoir for SARS-CoV-2, delaying viral clearance and increasing the likelihood of systemic tissue damage.The high levels of ACE2 expression and the immune-privileged nature of this organ concur with this theory [30].It is also thought that amino acid substitutions can influence viral S1-ACE2 interaction and viral infectivity. Due to hemizygosity in males, carrying a viral-boosting allelic variant of ACE2 may lead to increased susceptibility to severe disease [31,32].Li et al. propose that increased male mortality can be attributed to the increased likelihood of a cytokine storm in the lungs,accelerating progression into ARDS. They found a positive correlation between ACE2 expression and immune celllevels(Natural Killer (NK)cells, CD8+ cells) in male lung tissue whereas the opposite was found in females [16].

Role of Sex Hormones
Previous literature shows that differences in sex hormones impact the immune system and therefore may play a role inSARS-CoV-2 clearance.It is thought that testosterone (T) has both protective and adverse effects on mortality risk.
Low levels of T appear to be linked with increased susceptibility of respiratorydiseases [33].Rastrelliet al.demonstrate that low levels of Tand circulating free testosterone (cFT) are predictors for adverse outcomes and mortality from COVID-19 [19]. This concurs with existing literature in which an association between hypogonadism and proinflammatory cytokine levels isobserved [33,34].Severe infections are also associated with a reduction in numbers of CD4 + T cells, CD8 + T cells, B cells, and NK cells.The presence of androgen receptors (AR) on these cells suggests thatTis important in their function [34].
In addition to this,T plays a complex role in coagulation which could affect male mortality rate. Intravascular thrombosis and endothelialdysfunctioncomplicate COVID-19 prognosis.
Published evidenceindicates this occurs more frequently inmalesthan females [35]. T augments activation and aggregation of platelets by increasing platelet expression of thromboxane A2 receptors [36]. In contrast, a negative correlation between serum T levels and platelet reactivity has been discovered by an ex vivo study [37]. T enhancestheproduction of endothelial nitric oxide, a potent vasodilator and inhibitor of plateletrecruitment.Mean platelet volume, a biological indicator of platelet activation, is seen to be increased in hypogonadal males [38]. Therefore, it could behypothesisedthatTprotects males against new thrombotic events in COVID-19, an effect that is lost through hypogonadism [33].
T has a cardioprotective role and promotes myocardialhealth. Thus,maleswithhypogonadism are predisposed to increased cardiovascular risk from COVID-19.Tis vital in regulating glucose and maintainingfavourablelipid metabolism [35]. Furthermore, being a rapid onset vasodilator, Treducesblood pressure by blocking calcium channel opening.Males with cardiovascular diseases (CVD) tend to have lowserumT levels [39]. This further illustrates the importance of T in protecting against chronic CVD, as well as acute cardiac injury, which is typically associated with severe COVID-19 disease [17,33,40].
Although hypogonadism appears to be a risk factor for mortality, a contradictory 'Testosterone driven COVID-19' theory exists [28].Transmembrane Protease Serine 2 (TMPRSS2) cleaves the viral S protein at two sites allowing penetration changes on which viral entry into cells depends. It is thought that increased male mortality could be attributed to the androgen regulation of TMPRSS2. There is discourse in the literature as some papers find that there is no significant difference in TMPRSS2 expression in the lungs between the sexes [41].However, other papers find that males have significantly higher (P=0.029) expression of TMPRSS2 at the pulmonary level which may lead to viral progression and poorer outcomes [32].
Oestrogen(E) is thought to have a protective role against COVID-19 mortality in females. Lower female mortality could be attributed toEstimulating immune cell development, namely B cells, leading to humoral anti-viral responses.Ereceptors, present on various leukocytes induce pro-inflammatory cytokine production such as interleukin (IL)-12,tumor necrosis factor-alpha (TNF-alpha)and chemokine (C-C motif) ligand 2 (CCL2) [42]. The activated lymphocytes and alveolar macrophages increase type 1 and 2 interferon (IFN) production, reducing viral load.
Scotlandet al.suggest thatEmay also affect leukocyte function. They found that female mice have an increased number of resident T lymphocytes and that their tissue macrophages have a higher density of toll-like receptor (TLR), specifically TLR2 and TLR4; this allows rapid detection and elimination of pathogens [43].Channappanavaret al.also demonstrateE's protective role as they found oophorectomy or treating female mice with anERantagonist resulted in increased mortality from SARS-CoV-1, whereas gonadectomy did not affect mortality [44]. This finding may also be applicable to SARS-Cov-2, providing a potential explanation for higher male COVID-19 mortality.

Sex Differences in Immune Regulation
A study conducted byZenget al.highlights that females produce more serum (SARS-CoV-2) IgG in comparison to males in severe disease status [15]. TLR7, a pattern recognition receptor,is expressed on the X chromosome and can bypass X chromosomal inactivation [42]. Female X chromosomal homozygosity results in a greater gene dosage and expression of TLR7, allowing for stronger antigen detection [45]. TLR7 presenting plasmacytoid dendritic cells in females produce more type 1 IFN following ligand stimulationwhencompared to males [45]. In the presence of TLR7, type 1 IFN enhances B cell-mediated immunoglobulin secretion as well asproliferation [46].These biological processes provide an explanation for higher serum IgG in females.
Gene term enrichment analysis of the genesupregulated inaSARS-CoV-2 infection in human lung epithelium identify the 'cytokine-mediatedsignallingpathway' as the most significantly altered pathway [18].Qin et al.observe thatCOVID-19 diseaseseverity is positively correlated with inflammation [7].Following viral invasion of the lungs, aberrant release of inflammatory cytokines (soluble IL-2, IL-6, IL-8, IL-10) and proteins (LDH, ferritin,high-sensitivity CRP [hs-CRP]) damagethe alveolar epithelial cell barrier causingoedemaand hypoxialeading to ARDS [8,17].Inflammatory indexes within this cohort are significantly higher inmales. This is worth noting as mortality within this cohort was twice as likely in males and this could be as a result of the immunopathogenic damage caused by excess cytokine storms promoting acute lung injury [8].
Gene term enrichment analysis may provide further explanation for these sex-based differences in cytokine expression.SeveralDifferentially Expressed Genes (DEGs) identified upon SARS-CoV-2 infection of human lung epithelium are found to be modulated by sex hormones. Neutrophil chemotactic factor CXCL1 and dendritic cell chemotactic factor CCL20 are significant DEGs upregulated in SARS-CoV-2 infection.Bothfactors areregulated by AR,providing further evidence for the role of excess cytokine storms observed in malesincreasingmortality [18].
SARS-CoV-2 infection is known to result in significant lymphocytopenia, the extent of which differs betweenthesexes [8,10,11,14].Yang et al.observe lymphocytopenia in 80% of their most critically ill patients [11]. Qinet al.similarly note that male COVID-19 patients have a lower overall lymphocyte count compared to females when adjusting for age and comorbidity [8].
Additionally, previous research finds that femaleshave higher CD4 + T cell counts than agematched males, and after in vitro stimulation females produce higher numbers of activated CD4 + T cells [44].Thegreater reserve of CD4 + lymphocytes, combined withlowerrisk of lymphocytopenia in SARS-CoV-2 infection, may potentially decrease the risk of mortality from COVID-19 in females.

Sex Differences in Behaviour
Behavioural differences are also thought to contribute to the difference in COVID-19 mortality between sexes. Males tend to partake in higher-riskbehaviourssuch as smoking and drinking alcohol; the WHO reports that 40% ofmalesworldwide smoke compared to 9% of females [47].Additionally, it is thought that females are more likely to follow hygiene and preventative routines [48].
A study by Guan et al.finds an association between disease severity and smoking. Smokers make up a greater proportion of severe COVID-19 patients compared to non-severe patients (16.9% and 11.8%, respectively) [49]. However, it is worth noting that in this study sex is not taken into consideration.Bagoneet al. observe an association between cigarette smoke and increased heme oxygenase-1 induction (HO-1) of lung fibroblasts in mice. HO-1 is thought to have anti-viral and cytoprotective properties [50].This confounds the previous understanding of the relationship between smoking and SARS-CoV-2 infection. Therefore, further research and clarification are needed to determine the precise mechanisms of this relationship.

Strengths and limitations
Our review has several strengths.The search strategy we implemented contained a comprehensive list ofsearchwordsresulting inan in-depthanalysis ofavailableevidence. We adopted a pragmatic search strategy approach, appropriate for an ongoing pandemic setting, which aligned with PRISMA guidelines and WHO recommendations for rapidly reviewing evidence in the context of emergencies.Papers also underwent an extensive appraisal before being included in our review astwo reviewers assessed potential studies while athird reviewer verified this. Althoughthe risk of bias assessment showed most of the studies used had little or no bias, we found that disease severity was measured differently between studies. As a result, comparison of patients between studies must be done carefully.
There are severallimitations to our review. We limited our electronic database search due to the dynamic and rapidly evolving nature of the COVID-19 pandemic, therefore we included publications only in English betweenJanuary 2020 and June 2020. Our search strategywas also focusedsolely on sex and COVID-19 mortality; no comparisons were made regarding the role of sex in the SARS and MERS outbreaks. Additionally,nineout ofthe16studiesusedwere conducted in China. In order to comprehensively assess sex differences in the risk of mortality from COVID-19, more studies will need to be conducted worldwide.
Sex-disaggregated COVID-19 data published in medical literature is limited however it has become evident that the male sex is an important risk factor for mortality. Further exploration into the impact of sex on this pandemic is required in order to develop targeted therapies, as well as public health policies, and to prevent sex bias in treatment.

Conclusions
In conclusion, data emerging worldwide suggeststhat the male sex has a significant role in increasing risk of COVID-19 mortality amongst adult patients. This association may be explained by the findings that males tend to have lower serum IgG antibody generation, decreased CD4 + T cell reserves, and lower circulating ACE2 expression when compared to females. Male sex is also found to be associated with increased disease severity upon hospital admission, higher rates of ICU admission, and increased clinical markers such as lymphopenia and inflammatory indexes. Conversely, female sex is found to play a significant role in lowering risk of mortality from COVID-19.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.