Since the emergence of SARS-CoV-2 and the resulting COVID-19 pandemic, effective vaccines and therapeutic options, including antivirals and glucocorticoids, have significantly reduced mortality and rates of severe disease [1]. Nonetheless, infection remains common [2], and severe illness continues to pose a serious risk, especially among immunocompromised individuals and those with multiple comorbidities [3]. In this context, low-cost, widely accessible therapeutic alternatives may still offer clinical value. Transdermal estrogen may represent one such candidate.

Epidemiological studies have consistently shown sex-dependent differences in COVID-19 severity and fatality, with male patients more likely to develop severe illness, require hospitalization, and die than premenopausal female patients [4,5]. The mechanisms underlying this sex-specific difference are likely multifactorial and include immunological distinctions between male and female patients [6,7]. Among these factors, estrogens appear to play an important role.

Estrogen concentrations are higher during the reproductive years, and the relative protection observed in female patients appears to diminish after menopause [8,9]. Evidence from observational studies further suggests that this protection may partially be restored by estradiol therapy (odds ratio [OR] 0.33, 95 % CI 0.18-0.62; hazard ratio 0.29, 95% CI 0.11-0.76) [10].

Estrogens, and particularly 17β-estradiol, the most potent form, act through receptors on both innate and adaptive immune cells. Estrogen can modulate immune activity in both proinflammatory and anti-inflammatory directions [11], and female patients tend to show stronger innate and adaptive immune responses to pathogens than male patients [11,12]. Furthermore, nonimmunobiological mechanisms may also contribute to a protective role for estrogen. SARS-CoV-2 enters host cells via angiotensin-converting enzyme type 2 (ACE2) receptors, which are expressed across various tissues including the lung, vasculature, heart, kidney, and brain. ACE2 serves both as a cellular receptor for SARS-CoV-2 and as a key regulator of the renin-angiotensin system [13]. Under physiological conditions, ACE2 converts angiotensin II into the vasodilator angiotensin (1-7), which in turn regulates blood pressure [14]. However, SARS-CoV-2 infection downregulates ACE2 expression, thus increasing angiotensin II activity, which drives inflammation, vascular leakage, vasoconstriction, and pulmonary damage [15].

Preclinical studies further support a protective role for estrogen in COVID-19. Application of 17β-estradiol to Vero E6 monkey kidney cells reduced SARS-CoV-2 viral load [16] and attenuated ACE2 expression in human lung epithelial cells [17]. Mechanistically, estrogen modulates the innate immune system to suppress hyperinflammation and mitigate the cytokine storm by downregulating nuclear factor κ B (NF-κB) signaling [18,19]. Within the respiratory system, estrogen upregulates the Mas receptor, activating an anti-inflammatory and tissue-protective axis [20]. Furthermore, estrogen promotes endothelial nitric oxide synthase (eNOS) activity and subsequent nitric oxide production, which in turn improves microvascular endothelial function—a key issue in severe COVID-19 pathophysiology [21-23].

Despite these promising mechanistic and preclinical findings, clinical evidence to support benefit from estrogen therapy in COVID-19 remained limited [24,25], with no human efficacy data reported. We sought to address this issue and initiated a study to determine whether transdermal 17β-estradiol could reduce COVID-19 disease progression in such patients. However, the widespread COVID-19 vaccination program, declining COVID-19 disease severity, and changes to Qatar’s quarantine policy ultimately limited recruitment and prevented trial completion. Here, we present the study protocol, preliminary findings, and key operational lessons to inform future investigations of estrogen-based therapies for COVID-19 and other diseases caused by coronaviruses that use the ACE2 receptor to gain cellular entry.

Between February 21, 2022, and June 17, 2022, 1006 patients were screened. Of these, 760 patients were ineligible because of kidney disease (n=43, 5.7%), liver disease (n=15, 1.9%), participation in other clinical trials (n=180, 23.7%), inability to collect timely blood samples (n=244, 32.1%), presentation more than 48 hours after diagnosis (n=198, 26.1%), age <18 years (n=44, 5.8%), or a history of thromboembolic complications (n=36, 4.7%). Additionally, 203 patients declined to provide consent. Of the 43 patients initially eligible, 14 (32.6%) later withdrew consent, leaving 29 (67.4%) participants enrolled in the study (Figure 1).

The 29 enrolled participants were all men of Southeast Asian origin (from Bangladesh, India, Pakistan, Nepal, and Sri Lanka), reflecting the demographic profile of the eligible hospitalized population in Qatar during the recruitment period. Of the 29 enrolled participants, 13 (44.8%) were randomized to standard care and 16 (55.2%) to 17β-estradiol gel plus standard care. Mean age was similar between groups (mean 34.7, SD 8.66 years vs mean 36.3, SD 10.49 years, respectively; P=.67), with no statistically significant differences in baseline characteristics (Table 2). The small sample size precluded subgroup analysis. No adverse events, including thromboembolic complications, were reported among participants receiving the 17β-estradiol gel treatment during the 7-day treatment period.

Early termination of the trial, due to a rapidly evolving viral, public health, treatment, and vaccination landscape, meant that our study was underpowered to evaluate efficacy. Nevertheless, short-term transdermal 17β-estradiol administration was generally well tolerated in the enrolled participants, and no thromboembolic or other major adverse events were observed during the 28-day follow-up, including VTE or cardiovascular complications. Despite recruitment challenges, the study provides important operational lessons for the design and conduct of clinical trials during rapidly evolving infectious disease outbreaks.

Although preclinical studies have reported beneficial effects of 17β-estradiol in experimental models of COVID-19 [17,22], clinical evidence remains limited. Several trials investigating estrogen therapy for COVID-19 have been registered (NCT04359329, NCT04865029, NCT04539626, and NCT04801836), yet only 2 have reported results to date. Lovre et al [25] reported that a 5-day course of intramuscular estradiol combined with oral progesterone in 10 hospitalized patients with COVID-19 (5 men and 5 women) improved systemic inflammation. In contrast, Foidart et al [24] evaluated another form of estrogen, estetrol, a pregnancy-specific estrogen, in 87 patients with moderate COVID-19 and found that, although estetrol was well tolerated, it did not significantly improve clinical outcomes compared with those in 88 placebo-treated COVID-19 patients. Compared with these studies, our trial evaluated a different estrogen formulation and route of administration. Transdermal 17β-estradiol was selected because it provides continuous systemic delivery while avoiding first-pass hepatic metabolism [33]. Compared with oral estrogen therapy, transdermal administration has less effect on hepatic stimulation of coagulation factors and has been associated with a lower risk of VTE in other clinical settings [27]. However, the present study was not designed or powered to evaluate thromboembolic safety, and therefore no conclusions regarding the comparative risk of VTE can be drawn. Nonetheless, the practical experience gained from this trial provides valuable information to inform the design and conduct of future studies evaluating estrogen-based therapies and other host-directed interventions during infectious disease outbreaks.

Several lessons emerged from this study. First, investigational products and regulatory approvals should be secured as early as possible to minimize delays between protocol development and recruitment. Second, adaptive platform trial designs and methodologies that incorporate prespecified sample size re-estimation or expansion to additional sites may improve resilience to rapidly changing epidemiology. Third, future studies evaluating host-directed therapies such as transdermal 17β-estradiol would benefit from broader recruitment strategies and rapid trial implementation early in an outbreak, when disease incidence is highest, and recruitment targets are more likely to be achieved. Finally, multicenter or multinational recruitment should be considered to reduce dependence on local disease incidence and public health policy.

Of note, a low participation rate was observed among eligible patients. Although 246 patients fulfilled the eligibility criteria, only 43 (17.5%) initially consented to participate, and 14 (32.6%) subsequently withdrew. Selection bias may therefore have occurred, and the study population is not fully representative of the broader population of patients hospitalized with COVID-19.

A further limitation is that reasons for declining participation or withdrawing from the study were not systematically collected. Therefore, it was not possible to determine the factors influencing decisions to decline participation or withdraw from the study. Future clinical trials should prospectively collect reasons for nonparticipation, where feasible, to better understand barriers to recruitment and to inform strategies for improving enrollment.

The study population should also be interpreted within the demographic context of Qatar. All enrolled participants were male expatriate workers from South Asia. This reflects the demographic profile of Qatar, where the population in 2020 was approximately 2.8 million, 72% of whom were male and 83% were aged between 15 and 64 years [29]. Nevertheless, the resulting homogeneous study population limits the external validity of the findings. The observed safety profile cannot be assumed to apply to women, individuals from other ethnic backgrounds, older adults, patients with a higher risk of severe COVID-19, or those with comorbidities. Future multicenter studies recruiting more diverse populations will be important to determine whether the safety and potential efficacy of transdermal 17β-estradiol are consistent across different demographic and clinical groups.

Nevertheless, the biological rationale for investigating 17β-estradiol remains compelling. Sex-specific differences in COVID-19 outcomes have consistently been reported, with men experiencing higher rates of severe disease, intensive care admission, and mortality than women [5]. Estrogen has been shown to modulate innate and adaptive immune responses, reduce inflammatory cytokine production, and regulate ACE2 expression and activity, with the primary receptor used by SARS-CoV-2 for cellular entry [34]. Collectively, these mechanisms suggest that estrogen therapy may attenuate the dysregulated inflammatory responses associated with severe COVID-19. However, the optimal timing of treatment, route of administration, and patient population most likely to benefit remain to be determined. In addition, serial assessments of inflammatory or immunobiological biomarkers are essential to better characterize the biological effects and mechanisms of action of transdermal 17β-estradiol in the context of COVID-19. Finally, larger, adequately powered randomized controlled trials with longer follow-up are warranted to determine both the efficacy and safety of transdermal 17β-estradiol in patients with COVID-19, including its effects on uncommon but clinically important adverse events, such as VTE and cardiovascular complications. Such studies should incorporate mechanistic assessments to further elucidate the immunomodulatory effects of estrogen and identify the patient populations most likely to benefit. If efficacy and safety are confirmed, transdermal 17β-estradiol may have therapeutic potential not only in COVID-19 but also in other ACE2-dependent viral infections.