Comparison of Inflammatory Markers in Pre and Post-Menopausal Women During First and Second Waves of COVID-19

Introduction

The COVID-19 pandemic, caused by the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has emerged as an unprecedented global health crisis, disrupting societies, economies, and healthcare systems worldwide.¹ Since its initial outbreak in December 2019, the virus has continued to evolve, leading to the emergence of more contagious and potentially more virulent variants. Among the nations affected, India faced successive waves of COVID-19, with the devastating second wave striking in March 2021, characterized by a surge in cases and an unprecedented stress on healthcare infrastructure.²

While COVID-19 can impact individuals of all ages and backgrounds, certain populations, such as those with underlying health conditions, are at an increased risk of severe disease and adverse outcomes.³ Among these risk factors, age, gender, and hormonal status have garnered significant attention in recent research.

One intriguing aspect is the disproportionate prevalence of severe COVID-19 cases and the incidence of "long COVID" among women in their early 50s.⁴ This observation has raised compelling questions about the potential influence of hormonal changes, particularly the decline in ovarian steroid hormone production, on COVID-19 outcomes among women.

Menopause, a natural physiological process typically occurring around the age of 50, marks the cessation of menstruation and a decline in ovarian hormone production, primarily estrogen.⁵ Post-menopausal women often exhibit a higher prevalence of comorbidities, which may be attributed to a combination of factors, including aging, estrogen depletion, sedentary lifestyles, and increased susceptibility to certain health conditions⁶

In addition to these factors, the role of inflammatory markers has emerged as a key area of investigation in the context of COVID-19. Elevated levels of inflammatory markers, such as C-reactive protein (CRP), lactate dehydrogenase (LDH), D-dimer, and ferritin, have been associated with severe disease and poor outcomes in COVID-19 patients.⁷

In this context, understanding the potential impact of menopausal status on COVID-19 outcomes becomes crucial. The hormonal milieu of pre-menopausal and post-menopausal women differs significantly, which may contribute to variations in disease severity and immune responses during COVID-19 infection. Therefore, this research project aimed to address this gap in knowledge by comparing the profiles of various inflammatory markers in pre-menopausal and post-menopausal women during the first and second waves of the COVID-19 pandemic.

To provide a comprehensive context for this research, we have incorporated relevant findings from multiple studies. For instance, a study published in the Journal of Midlife Health in 2020 reported a higher mortality rate from COVID-19 among post-menopausal females, emphasizing the potential protective role of estrogen.⁸ Additionally, research published in Frontiers in Global Women Health in 2021 highlighted the association between post-menopausal status and more severe COVID-19 infections, particularly in women nearing the end of their reproductive years.⁹ Furthermore, a retrospective study published in Frontiers in Cellular and Infection Microbiology in 2021 found that post-menopausal women with COVID-19 had a significantly higher prevalence of comorbid conditions compared to pre-menopausal women.¹⁰ Lastly, a study in the Journal of Midlife Health in 2020 found that menopausal women with COVID-19 had a higher requirement for oxygen, ventilation, prolonged hospital stays, along with elevated levels of ferritin and residual symptoms.¹¹

Given these findings and the growing importance of understanding the interplay between hormonal status and COVID-19 outcomes, this research project aimed to investigate the severity of infection in pre-menopausal and post-menopausal women during the first and second waves of COVID-19. By comparing inflammatory marker profiles, including IL-6, ferritin, Erythrocyte Sedimentation Rate (ESR), C-Reactive Protein (CRP), Creatine Phosphokinase (CPK), Lactate Dehydrogenase (LDH), and D-dimer between these two groups, we can contribute valuable insights into the role of menopausal status in shaping COVID-19 outcomes among women.

Materials and Methods

Source of Data

The data for this study was sourced from Abhaya Hospital, located in Bengaluru, Karnataka. Abhaya Hospital served as the primary healthcare facility for the study, where patients with COVID-19 were diagnosed and treated.

Study Design

This research adopted a retrospective, observational study design. Data collection occurred over a defined period, specifically from March 2020 to September 2021. The study aimed to compare various inflammatory markers between pre-menopausal and post-menopausal women during the first and second waves of the COVID-19 pandemic.

Study Population

The study population consisted of individuals residing in Bengaluru, Karnataka, who met the inclusion and exclusion criteria outlined below.

Inclusion criteria: Participants were pre-menopausal and post-menopausal women.

1. Participants had tested positive for COVID-19 using Reverse Transcription Polymerase Chain Reaction (RTPCR) tests.
2. Participants were aged 12 years and above.

Exclusion criteria: Participants from other waves of COVID-19 pandemic.

Data Collection

The following marker levels were assessed in the study population.

• IL-6 levels

• Ferritin levels

• Erythrocyte Sedimentation Rate (ESR)

• C-Reactive Protein (CRP) levels

• Creatine Phosphokinase (CPK) levels

• Lactate Dehydrogenase (LDH) levels

• D-Dimer levels

The specimens for laboratory diagnosis were obtained through the following methods:

• Nasopharyngeal swab for COVID-19 diagnosis

• Serum for analyzing Ferritin, CRP, D-Dimer, LDH, and CPK levels

Laboratory Methods

The laboratory diagnosis of COVID-19 involved the use of RTPCR tests. For the other inflammatory markers, various laboratory methods were employed:

• CRP levels were determined using the latex agglutination method (Z3 CRP Hematology Analyzer, Zybio Inc, India).
• Ferritin levels were analyzed using immuno-turbidometry (INNOVANCE D-Dimer assay, Siemens Healthineers, India).
• D-Dimer levels were measured using automated turbidimetric immunoassay (VIDAS® Ferritin, bioMérieux Clinical Diagnostics).
• LDH levels were assessed using the Spectrophoto-metric method. (LDH P, Transasia Bio-Medicals)
• ESR was determined using the Westergren’s method (Microsed-System® Automated ESR Analyzer, LabX IVD).
• CPK levels were measured using the Spectrophotometric method (Creatine Kinase (CK) Activity Assay Kit, Elabscience).

Statistical Analysis

The collected data were subjected to statistical analysis using SPSS version 19 software. The results were presented as mean±SD, and statistical significance was defined as a P-value <0.05. The analysis aimed to identify significant differences in the inflammatory markers between pre-menopausal and post-menopausal women during the first and second waves of COVID-19.

Ethical Clearance

This study did not involve any interventions or investigations conducted on patients, human subjects, or animals. However, it received ethical clearance from the Padmashree Institutional Ethics Committee (PIEC/ MLT/ 20/2022 DATED 07TH JULY, 2022) of Padmashree Group of Institutions, ensuring compliance with ethical standards for data collection and analysis. The procedures followed were in accordance with the institutional ethical standards of the ethical committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000.

Results

In this study, we compared various inflammatory markers, including IL-6, Ferritin, Erythrocyte Sedimentation Rate (ESR), C-Reactive Protein (CRP), Creatine Phosphokinase (CPK), Lactate Dehydrogenase (LDH), and D-Dimer, between pre-menopausal and post-menopausal women during the first and second waves of COVID-19. The analysis revealed significant differences in several markers between the two groups.

The study compared various inflammatory markers, including IL-6, Ferritin, Erythrocyte Sedimentation Rate (ESR), C-Reactive Protein (CRP), Creatine Phosphokinase (CPK), Lactate Dehydrogenase (LDH), and D-Dimer, between pre-menopausal and post-menopausal women during the first and second waves of COVID-19. The analysis revealed significant differences in several markers between the two groups.

Median IL-6 levels were found to be significantly higher in post-menopausal women compared to pre-menopausal women during both waves of COVID-19 (P-value = 0.03). Similarly, median ferritin levels were significantly elevated in post-menopausal women compared to pre-menopausal women during both waves of COVID-19 (P-value = 0.034). While ESR showed a notable increase in post-menopausal women compared to pre-menopausal women, the difference was not statistically significant (P-value = 0.1). CRP levels were significantly higher in post-menopausal women compared to pre-menopausal women during both waves of COVID-19 (P-value = 0.004). However, CPK levels did not show a significant difference between the two groups (P-value = 0.07). Similarly, LDH levels also did not exhibit a statistically significant difference between pre-menopausal and post-menopausal women (P-value = 0.2). Additionally, D-Dimer levels were not significantly different between the two groups (P-value = 0.12) (Table 1).

Statistically significant increase in CRP, Ferritin, and IL-6 levels was observed among patients in the post-menopause group when compared with the premenopause group. However, other markers such as ESR, CPK, LDH, and D-Dimer did not show significant differences between the two groups. These findings suggest that there are differences in certain inflammatory markers between pre-menopausal and postmenopausal women during COVID-19 infection, which may have implications for the severity of COVID-19 outcomes in these groups.

Discussion

The findings of this retrospective, observational study shed light on the intricate relationship between menopausal status and COVID-19 outcomes, focusing on various inflammatory markers. Our analysis demonstrated significant differences in certain inflammatory markers between pre-menopausal and post-menopausal women during the first and second waves of the COVID-19 pandemic.

IL-6 emerged as a noteworthy marker in our study, with significantly higher levels observed in post-menopausal women during both waves of COVID-19. This finding aligns with previous research indicating that elevated IL-6 levels are associated with severe COVID-19 outcomes.¹ Interestingly, IL-6 has also been linked to estrogen regulation, suggesting that the decline in estrogen during menopause could contribute to the observed increase in IL-6 levels.⁸ These results highlight the potential role of IL-6 as a key player in the pathophysiology of COVID-19 in post-menopausal women.

Ferritin levels exhibited a significant increase in post-menopausal women compared to their pre-menopausal counterparts. Ferritin is a known marker of inflammation and iron storage, and elevated levels have been associated with poor COVID-19 outcomes.⁷ The observed rise in ferritin among post-menopausal women could be attributed to the multifaceted effects of estrogen, including its anti-inflammatory properties and regulation of iron metabolism.⁸ This finding emphasizes the need for further investigation into the link between ferritin, menopause, and COVID-19 severity.

While ESR showed a notable increase in post-menopausal women, the difference did not reach statistical significance. ESR is a non-sp ecific marker of inflammation, and its elevation can result from various underlying conditions.⁷ The trend observed in our study hints at a potential influence of menopausal status on ESR levels, but more extensive research is warranted to establish a concrete connection.

CRP, another crucial inflammatory marker, exhibited a significant increase in post-menopausal women during both waves of COVID-19. Elevated CRP levels have been consistently associated with severe COVID-19 and adverse outcomes.⁷ The connection between CRP and menopause may be multifaceted, as estrogen is known to modulate CRP production.⁸ Our findings underscore the importance of CRP in assessing COVID-19 severity, particularly in post-menopausal women.

CPK, LDH, and D-Dimer levels did not show significant differences between pre-menopausal and post-menopausal women during COVID-19 infection. While these markers are indicative of various aspects of COVID-19 pathophysiology, our results suggest that their levels may not be significantly influenced by menopausal status. However, it is essential to note that these markers remain valuable in the overall assessment of COVID-19 severity and prognosis.⁷

The observed variations in inflammatory markers between pre-menopausal and post-menopausal women have important clinical implications. Post-menopausal women, characterized by a decline in estrogen levels, appear to exhibit a pro-inflammatory profile, as evidenced by higher levels of IL-6, ferritin, and CRP. These markers have been linked to severe COVID-19 outcomes, indicating that post-menopausal women may face an increased risk of adverse effects.

Our study aligns with previous research reporting a higher prevalence of comorbid conditions among post-menopausal women with COVID-19.¹⁰ Comorbidities such as cardiovascular disease and diabetes have been associated with both menopause and COVID-19 severity, further highlighting the complex interplay between hormonal status and health outcomes.

The limitations of our study include a relatively small sample size and the focus on a single healthcare facility. A larger, multicenter study could provide more comprehensive insights into the relationship between menopausal status, inflammatory markers, and COVID-19 outcomes. Additionally, hormonal fluctuations within the pre-menopausal group were not considered in this analysis, which may be a potential avenue for future research.

Conclusion

Our study provides valuable insights into the association between menopausal status and inflammatory markers in the context of COVID-19. Post-menopausal women exhibited elevated levels of IL-6, ferritin, and CRP, suggesting a pro-inflammatory state that may contribute to the severity of COVID-19 outcomes. These findings underscore the need for personalized approaches to COVID-19 management, considering hormonal status as a potential factor influencing disease progression.

Further research is warranted to elucidate the precise mechanisms underlying these associations and to explore targeted interventions that could mitigate the heightened risk of severe COVID-19 among post-menopausal women. Understanding the interplay between menopause, inflammation, and COVID-19 may pave the way for improved clinical strategies and more tailored healthcare for women in this vulnerable demographic.

Conflict of Interest

The authors of the study declare that there are no conflicts of interest in this particular study.

Acknowledgements

The data for this study was collected from Abhaya Hospital, Dr. M H Mari Gowda Road, Wilson Garden, Bangalore.