High estradiol and low testosterone levels are associated with critical illness in male but not in female COVID-19 patients: a retrospective cohort study

ABSTRACT Male sex was repeatedly identified as a risk factor for death and intensive care admission. However, it is yet unclear whether sex hormones are associated with disease severity in COVID-19 patients. In this study, we analysed sex hormone levels (estradiol and testosterone) of male and female COVID-19 patients (n = 50) admitted to an intensive care unit (ICU) in comparison to control non-COVID-19 patients at the ICU (n = 42), non-COVID-19 patients with the most prevalent comorbidity (coronary heart diseases) present within the COVID-19 cohort (n = 39) and healthy individuals (n = 50). We detected significantly elevated estradiol levels in critically ill male COVID-19 patients compared to all control cohorts. Testosterone levels were significantly reduced in critically ill male COVID-19 patients compared to control cohorts. No statistically significant differences in sex hormone levels were detected in critically ill female COVID-19 patients, albeit similar trends towards elevated estradiol levels were observed. Linear regression analysis revealed that among a broad range of cytokines and chemokines analysed, IFN-γ levels are positively associated with estradiol levels in male and female COVID-19 patients. Furthermore, male COVID-19 patients with elevated estradiol levels were more likely to receive ECMO treatment. Thus, we herein identified that disturbance of sex hormone metabolism might present a hallmark in critically ill male COVID-19 patients.


Background
The current SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus Type 2) pandemic continues taking its toll on human health resulting in 4.1 million lives lost worldwide (as of 20 July 2021). The clinical spectrum of SARS-CoV-2 infection is broad, ranging from mild upper respiratory illnesses to severe primary pneumonia with respiratory failure, multiorgan failure and death [1]. Retrospective cohort studies revealed risk factors associated with disease severity and death. A study from Wuhan, China, which enrolled 191 inpatients on hospital admission since its first occurrence in China in December 2019, reported that older age and comorbidities, such as hypertension, diabetes and coronary heart diseases being among the top three, present poor prognostic markers at an early stage [1]. Another study from the UK linked 10,926 COVID-19-related deaths pseudonymously to primary care records of 17 million individuals, identifying being male, older age, diabetes, asthma, obesity as well as chronic heart diseases among the comorbidities associated with COVID-19 related death [2,3].
Thus, there is increasing evidence that being male constitutes a major risk factor associated with SARS-CoV-2 fatality. However, the underlying factors of sex disparity observed in COVID-19 remain unclear yet. We have recently shown using the golden hamster model, that SARS-CoV-2 infection attacks the reproductive organs and causes massive dysregulation of sex hormones in infected male and female animals [4]. Thus, we wanted to study whether the observed dysregulation in sex hormones upon SARS-CoV-2 infection is also present in COVID-19 patients and poses a risk factor for disease severity.
We herein compared sex hormone levels and cytokine responses in critically ill male and female COVID-19 patients to critically ill non-COVID-19 patients as well as to further control cohorts (healthy individuals and patients with coronary heart diseases due to the high prevalence in critically ill male COVID-19 patients). Analysis within the critically ill cohort was stratified by sex, age and Sequential Organ Failure Assessment (SOFA) scores.

Study design and participants
This retrospective cohort study includes the first 50 laboratory-confirmed (qRT-PCR) SARS-CoV-2 patients (39 males and 11 females) who were admitted to the Department of Intensive Care Medicine at the University Medical Center Hamburg-Eppendorf from 9 March to 29 April 2020 (herein referred as ICU . As a COVID-19 negative control cohort, we included 42 patients (27 males and 15 females) with laboratory-confirmed negative SARS-CoV-2 PCR who were admitted to the Department of Intensive Care Medicine at the University Medical Center Hamburg-Eppendorf as a control group (herein referred as ICU non-COVID- 19 ).
For controlled analysis, we recruited additional SARS-CoV-2 PCR-negative cohorts. We recruited patients with coronary heart diseases as one of the most prevalent comorbidities in our COVID-19 cohort (Institute for Pathology and Neuropathology, University Hospital Tübingen; 25 males, 14 females; herein referred as CHD cohort). We also recruited healthy donors (Institute for Transfusion Medicine, University Medical Center Hamburg-Eppendorf; 30 males, 20 females; herein referred as HC cohort).
A detailed description of the individual cohorts as well as respective inclusion and exclusion criteria are summarized in Supplementary Figure 1.

Setting
The University Medical Center Hamburg-Eppendorf is a tertiary care hospital with 1738 hospital beds. The Department of Intensive Care Medicine includes regularly 12 multidisciplinary ICUs with a total of 140 ICU beds. Since the first wave of SARS-CoV-2 pandemic in March 2020, a minimum of two and a maximum of four intensive care units treated simultaneously up to 40 patients suffering from COVID-19 or suspected COVID-19.

Data collection
The following demographic and clinical variables were collected retrospectively for the COVID-19 and non-COVID-19 cohort from the electronic patient data management system (PDMS) (ICM, Dräger, Lübeck, Germany): age, sex, body mass index, comorbidities, admission type and diagnosis, Acute Physiology and Chronic Health Score (APACHE II) and Simplified Acute Physiology Score II (SAPS II) on admission, Sequential Organ Failure Assessment Score (SOFA) within the first 24 h, classification of Acute Respiratory Distress Syndrome (ARDS) using the Berlin definition [5] and the need for mechanical ventilation and extracorporeal membrane oxygenation. Additionally, we recorded antiviral treatment, as well as supportive and experimental COVID-19 therapies for the COVID-19 cohort. Furthermore, we followed the course of the patients and recorded discharge or death.

Statistical analysis
Continuous variables are expressed as median with first to third quartile. Categorical variables are given as absolute and relative numbers. The distribution of data was visually interpreted using histograms. Variables were compared between groups (COVID-19 and non-COVID-19) with the Wilcoxon-Mann-Whitney U test, Student-T test and the Fisher's exact test as appropriate. All given p-values in the tables are of descriptive nature and not adjusted for multiple testing.
Statistical evaluation for quantitative data was performed with two-way-ANOVA using Holm-Šidák correction for multiple comparisons. For non-normal data unpaired Mann-Whitney or Kruskal-Wallis test ignoring any multiple comparisons were used. Statistical significance was defined as p < 0.05 (* p < 0.05, ** p < 0.01 and *** p < 0.001).
All statistical evaluations mentioned above were performed with SAS®, version 9.4 TS level 1M5 (SAS Institute Inc., Cary, NC, United States) and IBM® SPSS® Statistics 27. Graphical representation of the data was performed via GraphPad Prism 8 v. 8.4.2 (GraphPad Software, Inc.).

Results
Male COVID-19 patients present high estradiol and low testosterone levels on ICU admission A total of n = 292 SARS-CoV-2 PCR-positive patients were admitted to the Department of Intensive Care Medicine, at the University Medical Center Hamburg-Eppendorf from 9 March to 31 May 2021. Of these, n = 157 (54%) were patients secondarily admitted from referring intensive care units from all over northern Germany. In total, n = 194 (66%) of all COVID-19 patients were male and n = 98 (34%) of all COVID-19 patients were female. Thereof, n = 104 (36%) patients died; of which n = 71 (68%) were male and n = 33 (32%) were female. These data are in line with large epidemiological data analysis identifying being male as a high-risk factor for COVID-19 related death [2].
Critically ill COVID-19 patients were compared to the respective control group of critically ill non-COVID-19 patients with negative SARS-CoV-2 PCR (Table 1). All COVID-19 patients admitted to the ICU presented at least one comorbidity with obesity, arterial hypertension, coronary heart diseases and diabetes mellitus type II being among the most frequent (Table 1). Within the COVID-19 cohort, among all comorbidities present, coronary heart diseases (CHD) were slightly more prevalent in male COVID-19 patients (p = 0.6) (Table S1). A total of 39 (78%) patients met the ARDS criteria [5] in the COVID-19 group and 12 (28.6%) in the ICU non-COVID-19 group. There were no statistical relevant differences in the need for mechanical ventilation (invasive and non-invasive) in both groups (p = 0.856). In the ICU non-COVID-19 group, 14 (33.3%) patients had a suspected bacterial infection on admission to the ICU. CRP was significantly higher in COVID-19 patients (p < 0.001). In total, mortality within the COVID-19 cohort was 30% (n = 15) compared to 11.9% (n = 5) in the non-COVID-19 control group.
First, we wanted to assess whether sex hormone levels are altered within the critically ill male and female COVID-19 patients potentially providing a link to sexdependent disease severity. Since CHD was more prevalent in male COVID-19 patients compared to female COVID-19 patients (Table S1), we recruited an ageand sex-matched SARS-CoV-2 negative CHD control cohort (n = 39). As an additional healthy control group, we recruited age-and sex-matched SARS-CoV-2 negative male blood donors (HC) (n = 50). Furthermore, we recruited SARS-CoV-2 PCRnegative ICU patients (n = 42) as an additional control for critical illness independently of COVID-19 (herein referred to as ICU non-COVID-19). Critically ill male COVID-19 patients presented the highest estradiol levels compared to critically ill male non-COVID-19 patients (p = 0.0293) or male patients with CHD (p = 0.0004) or healthy males (p = 0.001) (Figure 1(a)). In line, comparing to published clinical reference values, estradiol levels in critically ill male COVID-19 patients were far above clinical references [6,7]. No significant difference in estradiol levels was detected within the control cohorts, which were within clinical reference values. Conversely, testosterone levels were lowest in critically ill male COVID-19 patients compared to critically ill male non-COVID-19 patients (p = 0.0250) or male patients with CHD (p = 0.0050) or healthy males (p < 0.0001) (Figure 1(b)). In accordance, testosterone levels of most critically ill male COVID-19 patients were below clinical reference values. No significant differences in testosterone levels were detected within the HC and CHD control cohorts. Critically ill female COVID-19 patients presented a trend towards elevated estradiol levels, albeit statistically not significant (Figure 1(c)). Testosterone levels were not significantly altered in critically ill female COVID-19 patients compared to female control cohorts (Figure 1(d)).
Collectively, these findings show that male COVID-19 patients present significantly increased estradiol and reduced testosterone levels compared to non-COVID-19 males. In contrast, female COVID-19 patients do not present significantly altered sex hormone levels.

Majority of critically ill male COVID-19 patients present secondary hypogonadism
To shed light on the origin of severe testosterone deficiency in male COVID-19 patients, we further analysed related hormones (Table 2). Free testosterone levels were reduced in 66.7% of male COVID-19 patients compared to reference values. Conversely, 54.5% of female COVID-19 patients presented elevated levels of free testosterone. Thus, changes in total testosterone levels correlate with levels of free bioavailable testosterone levels in the respective sex. We then measured levels of the sex hormone-binding globulin (SHBG), since the majority (98%) of total testosterone is bound to SHBG and only 2% is in its free, bioavailable form. Thus, in some cases, testosterone deficiencies might be masked by elevated SHBG levels. In 28.2% of male COVID-19 patients, SHBG levels were elevated, which might suggest masked testosterone deficiencies in some patients. Luteinizing hormone (LH) levels were elevated in 30.8% of male COVID-19 patients, while being within the normal range in all female patients. Interestingly, 7 out of the 28 male patients with low total testosterone levels presented elevated LH levels at the same time (data not shown), suggesting impairment of Leydig cell steroidogenesis in 25% of the male patients. Follicle- These findings suggest that in 25% of the male COVID-19 patients with low total testosterone levels, testosterone deficiency is likely of testicular origin. Thus, in 75% of male patients, the origin of testosterone deficiency is yet unclear.

Male COVID-19 patients present high estrone and low DHT levels on ICU admission
Testosterone is further metabolized to dihydrotestosterone by 5-α reductase. Dihydrotestosterone (DHT) also acts as an androgen and plays a key role in activating the transcription of various genes and activation of various immune cells similar to testosterone [8]. Thus, we wanted to assess whether alterations in testosterone levels detected in male COVID-19 patients are also reflected in its most potent metabolite. In male COVID-19 patients, dihydrotestosterone levels were reduced compared to HC males (p < 0.0001) (Supplementary Figure 2a). In line, a substantial proportion of plasma dihydrotestosterone levels in COVID-19 males was even below the lowest reference range, confirming dihydrotestosterone deficiency in men. In contrast, dihydrotestosterone levels were comparable and within clinical references in female COVID-19 and HC cohorts (p = 0.9568) (Supplementary Figure 2c). To assess whether the increase in estradiol levels can be attributed to a general increase in estrogens, we next measured estrone concentrations. Estrone levels in the plasma of COVID-19 males were higher than those measured in HC males (p < 0.0001) (Supplementary Figure 2b). Similarly, estrone levels were elevated in the plasma of female COVID-19 patients unlike HC females (p = 0.0009) (Supplementary Figure 2d).
Critically ill male COVID-19 patients present additionally elevated estrone levels accompanied by severely reduced DHT levels. Critically ill female COVID-19 patients present elevated estrone levels, while dihydrotestosterone levels remain unchanged.
Male COVID-19 patients with high estradiol levels are more likely to require ECMO treatment Next, we analyzed whether sex hormone levels correlate with an increased risk for severe disease outcome as assessed by the Sequential Organ Failure Assessment Score (SOFA) scores or the requirement for extracorporeal membrane oxygenation (ECMO) later during the patients' ICU stay. In critically ill male COVID-19 patients, estradiol levels increased with disease severity (Figure 2(a)) unlike in non-COVID-19 males (Figure 2(b)) (p = 0.0245 and p = 0.0273). In female COVID-19 patients, a trend towards higher estradiol levels with increasing SOFA scores was also observed (Figure 2(c)). Male COVID-19 patients with elevated estradiol levels were more likely to require ECMO treatment than those having estradiol levels within normal clinical references (p = 0.0307) (Figure 2(d)).  Within the female COVID-19 cohort, only one patient required ECMO treatment; thus, not allowing statistical analysis (Table 1). Testosterone levels did not show statistically significant changes in dependence of SOFA scores or patients requiring ECMO treatment (Supplementary Figure 3a-d). This might be due to the fact that most male patients presented low testosterone levels below clinical references [6,7]. These data show that critically ill male COVID-19 patients with elevated estradiol levels are more likely to require ECMO treatment.  (Figure 3(a-e)). In female COVID-19 patients, TNFα levels were higher in those with high SOFA scores compared to those with low SOFA scores (p = 0.0476) (Figure 3(f)). Albeit statistically not significant, IFN-γ showed a trend towards elevation with increasing SOFA scores (Figure 3(g)).
We then analyse whether changes in cytokine and chemokine responses in critically ill COVID-19 patients correlate with their respective sex hormone levels given that most immune cells possess androgen and estrogen receptors, using multivariable regression [8][9][10]. Among all 27 cytokine and chemokines assessed, only IFN-γ presented a significant correlation to estradiol in male and female COVID-19 patients (R 2 = 0.216, p = 0.0009; Figure 4). Multivariable analysis further revealed that testosterone levels are not associated with changes in IFN-γ levels (R 2 = 0.133, p = 0.3111; Supplementary Figure 4).
These findings suggest an association of estradiol levels with IFN-γ levels, which is in line with previous studies reporting on the estradiol-controlled transcription of IFN-γ due to the presence of an estrogen-responsive element (ERE) in its promoter region [11][12][13].

Discussion
In this study, we show that disturbance of sex hormone metabolism is associated with critical COVID-19 in men but not in women.
In critically ill men with COVID-19, we detected significantly elevated estradiol levels compared to control cohorts (HC, CHD and critically ill non-COVID- 19). Furthermore, testosterone levels were significantly reduced in males with COVID-19 compared to all control cohorts. Our findings were recently confirmed by two independent single-centre studies [14,15]. A study from Italy reported that men with COVID-19 have significantly reduced testosterone and significantly increased estradiol levels compared to healthy controls [15]. Therein, low testosterone levels in COVID-19 males were also associated with secondary hypogonadism and severe outcome in line with our findings reported here [15]. Another study from the US reported that lower testosterone concentrations during hospitalisation were associated with increased disease severity and inflammation in men with COVID-19 [14]. Estradiol levels were not significantly altered in the US study, where severely ill COVID-19 males were directly compared to severely ill non-COVID-19 males. However, it is important to define disease severity very precisely. In our study, we subdivided disease severity using SOFA scores. We found that men with COVID-19 presented a step-wise increase in estradiol levels with increasing SOFA scores unlike men without COVID-19. Furthermore, when comparing all cohorts, we identified estradiol to be a predictive marker for later ECMO requirement during the hospital stay in males with critical COVID-19.
Reduced testosterone levels were likely of testicular origin in 25% (primary hypogonadism) of all male COVID-19 cases in our study, suggesting that 75% can likely be attributed to hypothalamic-hypopituitary origin (secondary hypogonadism). These findings are further confirmed by the Italian single-centre study reporting that low testosterone levels in men with COVID-19 are associated with secondary hypogonadism [15]. The vast majority (95%) of testosterone is produced in Leydig cells of the testes depending on stimulation by luteinizing hormone (LH). Only small amounts (5%) are produced in the adrenal glands. Low levels of testosterone may either be of testicular origin (primary hypogonadism), of hypothalamic-pituitary origin (secondary hypogonadism) or a combination of both, with the latter predominantly found in the aging male population as late-onset hypogonadism [16,17]. Hypogonadism with and without elevated estradiol levels was reported before in patients with cardiovascular diseases as a risk factor for increased mortality in men [18][19][20]. Thus, it is tempting to speculate whether an initial comorbidity-driven hit, with respect to low testosterone levels, which are also reported in patients with obesity and type II diabetes [21,22] might put males at higher risk to develop severe COVID-19.
In women with COVID-19, we did not detect any significant alterations in testosterone or estradiol levels. However, some female COVID-19 patients had elevated testosterone levels albeit statistically not significant. It is important to note that all but one female COVID-19 patients were postmenopausal. However, the small female COVID-19 cohort size in our study is a potential limitation with respect to conclusions on female COVID-19 outcome. This variance in cohort size was due to that more men than women were admitted to the ICU, highlighting the importance of sex on critical COVID-19 outcome. Despite these limitations in the female COVID-19 cohort, others postulated an elevated COVID-19 risk for women with polycystic ovary syndrome, a condition characterized by increased androgen levels [23]. This highlights the need for further investigations to understand the impact of elevated testosterone levels in women in the context of COVID-19.
Comparative analysis of 27 different cytokines/chemokines in the male and female COVID-19 patients and subsequent regression analysis revealed that interferon-γ (IFN-γ) positively correlates with estradiol but not testosterone levels. This correlation is particularly interesting given that IFN-γ possesses an estrogenresponsive element (ERE) in its promoter region [11][12][13]. IFN-γ is a key activator of macrophages [13,24] and macrophage activation was repeatedly reported as a hallmark of COVID-19 severity [25]. Macrophages contain membrane-bound as well as nuclear androgen-and estrogen-receptors. Thus, it will be of high interest to dissect the role of sex hormones and IFN-γ in orchestrating, e.g. macrophage activation and cytokine storm in future studies as outlined in a hypothesis model (Supplementary Figure 5).
Collectively, our findings herein highlight that disturbance in circulating sex hormone levels is a hallmark of critical COVID-19 in males. Longitudinal hormone surveillance studies may be warranted during acute and recovery phases in COVID-19 patients to predict short-and potentially long-term risks [26][27][28][29].

Ethical approval and consent to participate
The study with laboratory-confirmed COVID-19 and non-COVID-19 ICU patients was reviewed and approved by the ethics committee at the Hamburg State Chamber of Physicians (registration no.: WF-053/20 and WF-073/21). The need for an informed consent for the ICU cohort, healthy blood donors (HC) and patients with coronary heart diseases (CHD) was waived by the ethics committee because data were retrieved anonymously and retrospectively from electronic health records. The study is registered under the number NCT04979091 (HAM-SEX-C19) on ClinicalTrials.gov. critical reading of the manuscript. GG conceived the study. SK, MS, DJ, KR, GdH and AN were responsible for COVID-19 patient management and recruitment. GG, SK, MS and BS (Berfin Schaumburg) designed and overviewed the study design. BS, HJ, MZ and SSB conducted the cytokine and chemokine assays. ZK, KK, TR and BS performed hormone assays. GG, BS, MS, AK and TB analysed data and developed the figures. LK, BetS (Bettina Schneider) and FS conducted ANOVA models and statistical tests. JG helped with data management and the analysis. JA, AM, TB, JH, HS, KK, TR critically reviewed the manuscript and were involved in the study design. GG and MS wrote the manuscript. All authors read and approved the final manuscript.

Disclosure statement
No potential conflict of interest was reported by the author (s).

Funding
This study was supported by a rapid response grant from the Federal Ministry of Health (Bundesministerium für Gesundheit, BMG) to GG. MS receives funding from the Medical Faculty of the University of Hamburg for clinical leave. The study funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.