key: cord-0984130-y67vbzb6
authors: Shah, Shilpa Bhuaptrai
title: COVID–19 and Progesterone: Part 2. Unraveling High Severity, Immunity Patterns, Immunity grading, Progesterone and its potential clinical use
date: 2021-08-08
journal: Endocr Metab Sci
DOI: 10.1016/j.endmts.2021.100110
sha: 2ff9acfcae36a9dc63ddf4da35fe207434c4e5b1
doc_id: 984130
cord_uid: y67vbzb6

Severely ill COVID–19(Corona Virus Disease of 2019) patients have a hyperinflammatory condition with a high concentration of pro-inflammatory cytokines termed the cytokine storm. This milieu is reported to cause acute lung injury, oxygen deprivation, multiorgan damage, critical illness, and often death. Post SARS–CoV–2(Severe Acute Respiratory Syndrome Coronavirus 2) infection, the fight between the invading virus and the host's immune system would either terminate in recovery, with eradication of the infection and regulation of the immune system; or there would be a continuation of immune attacks even after the virus has been cleared, leading to immune dysregulation and disease. This outcome is chiefly dependent on two factors: (1) the patient's immune response, and (2) sufficiency plus efficiency of the regulator(s). Concerning the first, the present research introduces a framework based on different types of immune responses to COVID–19 along with known disease examples,and how this relatesto varying clinical outcomes and treatment needs for COVID–19 patients. About the second factor of ‘regulator(s)’, part 1 of the manuscript described in depth the regulatory role of progesterone in COVID–19.The present study investigates the two immunity patterns and the status of the regulatory hormone progesterone with respect to the two established demographic risk factors for high-severity COVID–19: male sex, and old age. The study evaluates the status of progesterone as a credible participant of immune regulationand dysregulation. It duly relates the immunity patterns to clinical outcomes and evinces indications for clinical use of progesterone in COVID–19.It proposes a clear answer to the question: why are males and old patients most likely to have critical illness due to COVID–19? The study highlights clinical domains for the use of progesterone in COVID–19. Part 2 of this research introducing the concept of immunity patterns and immunity grading. These concepts herewith provided for the clinical course of COVID–19 also applies to other hyperinflammatory conditions. Possible clinical applications of progesterone to treat critically ill COVID–19 patients will open an avenue for hormonal treatments of infections and other immune-related diseases.

COVID-19 is an infectious disease characterized by varied scale, type and duration of adaptive immune responses to the SARS-CoV-2. The immune system is a chief determinant of pathogenesis due to SARS-CoV2 infection. When SARS-CoV-2 bypasses innate immune defenses, the adaptive immune system gets activated. It has two subdivisions: cell-mediated immunity T helper (Th) cells type 1 (Th1)and humoral immunity T helper (Th) cells type 2 (Th2). Cell-mediated immune responses involve destruction of SARS-CoV-2 infected cells by cytotoxic T cells and destruction of intracellular pathogens by Th1 cells. Th1 cells release proinflammatory cytokines inducing intracellular killing of SARS-CoV-2. Th1 cells also contribute to humoral immunity by inducing the production of opsonizing antibodies. Th2 cells initiate the humoral response by activating naive B cells to secrete IgM and inducing the production of other antibody isotypes. These specific antibodies help with opsonization, activation of the complement pathway, and phagocytosis of the SARS-CoV-2. Th2 cells resolve cell-mediated inflammation by secreting anti-inflammatory cytokines and regulating the Th1. Th1 and Th2 regulation are important for clearing SARS-CoV-2 infection and recovery from COVID-19 [1] . A SARS-CoV-2 infected individual's ability to achieve this regulation depends on three factors: viral load, immunity, and regulatory mechanisms. Part 1 of the manuscript is about the regulatory functions of progesterone. This part 2 investigates the role of individual immunity in COVID-19.

The present concept-based theoretical investigative research work was carried out in the city of Mumbai, Maharashtra, India from April 1, 2020 to December 31, 2020 based on the following stepwise methodology protocol.

The foundation of present research is the concept of immunity patterns. Different types of immunity patterns are outlined. This is followed by the presentation of disease conditions which are clinically well established examples representing each of the five different immunity patterns. This is followed up by establishing the roles of these immunity patterns for COVID-19 outcomes. The clinically supported concept of the immunity patterns and status of the regulator progesterone was evaluated in the COVID-19 high severity and low severity groups. To this end, the immunity grading method was established and introduced to investigate and explain the varying severity of COVID-19 in different age-sex groups.

Formative research questions based on theoretical investigations were carried out to find other biological factors that could affect progesterone levels and immunity patterns.This was a widely open question. The first step was to research for parameters that relate to high mortality in COVID- 19 . For example, one such parameter found was Vitamin D. For this factor research questions were (1) does Vitamin D relate to cell-mediated immunity? and (2) does vitamin D relate to progesterone? (3) Is there a connection among vitamin D, cell mediated immunity and progesterone? (4) What would be the outcome of these factors on immunity grading? In similar fashion the effect of these factors on COVID-19 outcomes was probed, attending to severity parameters irrespective of age or sex. All illustrations and tables are originally prepared.

In vivo, broadly there are three possible scenarios when cross-regulation of the cell-mediated and humoral immunity is active after challenge by an infectious agent: (1) Th1 and Th2 in balance, (2) low Th1 with raised Th2, and (3) raised Th1 with low Th2. When cross-regulation of cellmediated and humoral immunity is inactive, one more immunity pattern is possible (4) raised Th1 and raised Th2 (Fig. 1) . A fifth immunity pattern is immunodeficiency or immunosuppression. At the time of infection an individual may have any one of these immunity patterns to varying extents. The symptoms of disease depend on the kind of infection and replication of the infectious agent and the immune response pattern. Immune responses to SARS-CoV-2 are different in patients with severe and non-severe course [2] .

3.1.1 Immunity pattern 1: Th1and Th2 in balance. Th1/Th2 cells in a balanced state (Fig. 1A ) mount a moderate immune response taking moderate time for incubation as well as Th1/Th2 homeostasis. The incubation period is defined as the time between exposure to the pathogen and onset of signs and/or symptoms of clinically apparent disease. Asymptomatic patients have lower viral load [3] . COVID-19 patients with balanced Th1/Th2 typically are asymptomatic or present with mild symptoms depending on the viral load [4] .

3.1.2 Immunity pattern 2: Th1 low with Th2 high. Low Th1 with raised Th2 (Fig. 1B) is a suboptimal immunity status for killing the intracellular virus; and the clinical outcome of SARS-CoV-2 infection would depend on whether the patient's immune system is competent or impaired. In patients with lasting impairment of Th1 immunity post-infection, it would lead to an inability to clear the infection, the possibility of viral reactivations, and secondary infections because cell-mediated immunity is crucial for attacking the intracellular infection and more specifically for killing virus-infected cells [5] . With respect to SARS-CoV-2, Jiang et al. [6] and Amodio et al. [7] both noted that a longer incubation time may lead to a high rate of asymptomatic and sub-clinical infection among immunocompetent individuals. In immunocompetent patients, depending on the rising viral load and parallel induction of Th1 immunity, the patient may exhibit symptoms ranging from none to mild or moderate-as the infection and hence the disease progresses over a long incubation period. Depending upon the viral load, patients who initially had a low Th1 response could mount a stronger cell-mediated immune response later and more slowly. Interestingly, their start with low Th1 and high Th2 is an advantage, with better prospects for Th1/Th2 homeostasis. This is because when Th1 is initially low and then increases, Th2 which is initially high gets crossregulated [8] . After the rise of cell-mediated Th1 immunity, when the infection gets defeated and starts to clear, there would be the need for humoral Th2 immunity and regulatory mechanisms to normalize the elevated Th1 response. Thus higher Th2 humoral immunity initially, topped up with post-infection specific humoral immunity, is beneficial for recovery in COVID-19 patients with intact cross-regulation. . Lower ACE2 expression in the nasal epithelial cells of asthma and allergic rhinitis patients is reported as compared to healthy individuals [10] . A large, 2-site cohort study of patients positive for COVID-19 confirmed that atopy is significantly associated with less severe COVID-19 outcomes. It reports that the presence of atopy could be predictive of a decreased need for hospitalization for COVID-19 [11] . Atopic status protects against the most severe, often fatal consequences of SARS-CoV-2 infection. The protective effect of atopic status against severe lung disease is found to be evident throughout all age subsets in a mixed study group of atopic females and males [12] . 3.1.3 Immunity pattern 3. Th1 high with Th2 low. It has been proposed that an exception to the inverse relationship between incubation period and severity of the disease is unlikely for SARS-CoV-2 [13] . An inverse correlation between the length of the incubation period and the severity of the disease is reported for other coronaviruses [14] . Th1 dominance (Fig.1C ) contributes to the excessive inflammatory response and cytokine storm with tissue damage and organ injury along with the production of loads of debris from this protective yet destructive immune attack [15] . Further, SARS-CoV-2 acts on T cells with its superantigen [16] . Compared to a normal antigen-induced T-cell response where 0.0001-0.001% of the body's T-cells are activated, these superantigens are capable of activating up to 20% of the body's T-cells [17] . Activation of large numbers of T cells induced by the SARS-CoV-2 superantigens adds to the host-Th1 dominance [18] , and results in a massive release of cytokines and uncontrolled tissue damage, organ injury, and toxic shock [19] . In this situation the immune system, which had elevated Th1 prior to the infection, gets its Th1 manifold raised even more while responding to the SARS-CoV-2 infection. This accelerates and magnifies the cytokine storm and is considered the rootcause of pathogenic inflammation in COVID-19 [20] [21] [22] . These patients would have severe symptoms with a short incubation period either from the time of exposure to the virus or from the time of noticing the first symptoms. Plus, they would take a long time to achieve Th1/Th2 homeostasis post infection as Th1, which was already raised, is further amplified and so there will be poor and delayed cross-regulation of Th2. Hence, if patients with raised Th1 immunity got infected by SARS-CoV-2, they would be at risk of sufferingcritical COVID-19 illness. In some of these patients uncontrolled Th1 immune dysregulation may deteriorate their condition rapidly and prove fatal [23] . Remarkably, the occurrence of rheumatoid arthritisin atopic patients is found to reduce the atopy symptoms by 40 to 50% affirming that regulation of Th1 and Th2 cells strongly influences the inflammatory responses and disease outcome [27] .The observations about the above discussed two diseases,rheumatoid arthritis (Th1 predominance) and atopy (Th2 predominance), verify the above concept of immunity patterns for COVID-19. Further, infections are a common comorbidity in long-term atopic dermatitis [28] . Patients suffering from chronic atopic dermatitis show increase in Th1. This balance can vary during the chronic phase of atopic dermatitis [29] .

3.1.5 Immunity pattern 4: Th1high with Th2 high When the immune regulatory mechanisms fail, cell-mediated and humoral immunity do not cross-regulate each other effectively, resulting in immune disruption with both Th1 and Th2 being high (Fig.1D ) [30] . Earlier in this section, based on the immunity pattern concept for immunity pattern 2 (Th1 low and Th2 high) and immunity pattern 3 (Th1 high and Th2 low), the benefits of high Th2 and the risks of high Th1 for COVID-19 are explained in detail in the manuscript. In immunity pattern 4 with Th1 as well as Th2 high, the risk of high Th1 is applicable although Th2 is also raised due to pre-existing failure of cross-regulation. Patients with this immunity pattern are most likely to present severe symptoms with a short incubation period either from the time of exposure to the virus or from the time of noticing the first symptoms. Plus, they would take a long time to achieve Th1/Th2 homeostasis post infection as Th1, which was already raised, is further amplified and immune regulation and cross-regulation by Th2 both are unavailable. Hence,patients with immunity pattern 4 could get more critically ill than those with immunity pattern 3. In these patients,in absence of timely intervention with immune regulatory therapy, uncontrolled Th1 immune dysregulation may deteriorate their condition rapidly and prove fatal [23] . Disease example: Metabolic syndrome is a cluster of metabolic abnormalities associated with obesity, insulin resistance, dyslipidemia, and hypertension in which inflammation plays an important role. There is upregulation of both Th1 and Th2 cytokines in metabolic syndrome [31] . Patients with metabolic disorders like obesity, diabetes, cardiovascular and liver disease may face a higher risk of SARS-CoV-2 infection and COVID-19. These disorders greatly affect the course and prognosis of COVID-19 and are associated with significantly worse COVID-19 outcomes [32] .

A meta-analysis of main predictors of COVID-19 associated mortality rate in hospitalized patients found that diabetes mellitus is the best predictor of mortality rate in an age-and sexdependent manner [33] . Metabolic stress can cause pathologic activation of the immune system. Therefore, metabolic disorders including diabetes mellitus can manifest and progress as an inflammatory disorder, with inflammation producing severe consequences [34] . SARS-CoV-2 induced activation of the immune system could further aggravate these pre-existing inflammatory conditions.

Immunocompromised patients, including those with inborn errors of immunity, may be at increased risk for severe or prolonged infections with SARS-CoV-2 [35] .

Cancer and organ transplant patients are immunocompromised chiefly due to treatment. Compared to the general population, adult cancer and organ transplant patients with COVID-19 are found to suffer higher comorbidities, have higher levels of inflammatory markers at diagnosis, and higher rates of intensive care and hospital mortality [36] .

Inborn errors of immunity are genetic disorders with broad clinical manifestations, ranging from increased susceptibility to infections to significant immune dysregulation [37] . Primary immunodeficiency disorder (PID) refers to a large heterogeneous group of disorders that result from defects in immune system development and/or function. PIDs are broadly classified as disorders of adaptive immunity (i.e., T cell, B-cell or combined immunodeficiencies) or of innate immunity (e.g., phagocyte and complement disorders). Although the clinical manifestations of PIDs are highly variable, many disorders involve an increased susceptibility to infection. [38] . Young patients with primary immunodeficiency have a prevalence of Th2 but as they get older, due to alarmins, this balance can change to Th1 prevalence [39, 40] . Compared to the general population, adult patients with PID and symptomatic secondary immunodeficiency display grater morbidity and mortality from COVID-19 [41] .

Here it is important to mention that some orphan hereditary diseases present immunodeficiencies and hormone deregulation. For example, beta thalassemias are a group of hereditary blood disorders characterized by anomalies in the synthesis of the beta chains of hemoglobin resulting in variable phenotypes ranging from severe anemia to clinically asymptomatic individuals. Total annual incidence of symptomatic individuals is estimated at 1 in 100,000 throughout the world and 1 in 10,000 people in the European Union [42] . Immune competence is affected in betathalassemia and involves numerous quantitative and functional defects, involving T and B lymphocytes, immunoglobulin production, neutrophils and macrophages, chemotaxis and phagocytosis, as well as the complement system [43] . Iron excess may derange the immune balance in favor of the growth of infectious organism and the accumulation of iron in different organs leads to different clinical complications of iron overload. The anterior pituitary is particularly sensitive to iron overload which disrupts hormonal secretion resulting in hypogonadism [44].

In part 1 of the manuscript the regulatory role of progesterone for COVID-19 was corroborated.

To further substantiate applicability of both these parameters, it will be appropriate to appraise their status for the established COVID-19 high-severity groups.

3.2 (B) Immunity patterns and progesterone statusin COVID-19 high severity groups Two established high severity groups for COVID-19 are old age and male sex. Based on these two factors, it would be pertinent to explore (I) Effects of old age on the regulator progesterone, (II) Effects of old age on the immunity patterns, (III) Sex and levels of the regulator progesterone, (IV) Sex, immunity patterns and old age, and (V) Comprehensive presentation of the four age-sex groups, old males, young males, old females and young females, with respect to immunity patterns, status of the regulator progesterone, and different COVID-19 severity.

Progesteronedecreases significantly with age in both men and women [45] . Ageing, hormones and immunity are interconnected [46] .

3.2.2 (II) Effect of old age on the immunity patterns:

The respiratory system undergoes various hormonal, anatomical, physiological and immunological changes with age. Reduced levels of progesterone and estrogen could cause decreased muscular strength, decreased relaxation of bronchial smooth muscle, and increased compression of thoracic spine due to osteoporosis [47] . Age significantly determines the clinical features and prognosis of COVID-19. Elderly patients with COVID-19 are found to be more likely to progress to severe disease. Acute respiratory distress as graded by the Pneumonia Severity Index is higher in elderly than in young and middle-aged COVID-19 patients. Prognosis is worse in patients older than 60 years. Hospitalization, ICU admissions, and death increase with age. Mortality is higher in elderly than inyoung and middle-aged patients [48] [49] [50] [51] .

There are three main findings about effects of ageing on the T-cells.

(1) Immunosenescence: Immunosenescence refers to the gradual deterioration of the immune system brought on by advancing age [49] . Ferrando-Martínez S et al [53] reported that an important cause of elderly impaired responses to newly encountered pathogens is an age-related deregulation of T cell homeostasis and accumulation of age-associated defects in naive T cells.

(2) Defects in antiviral T cell responses and T cell regulation: MicroRNAs are single-stranded RNAs which are differentially expressed in viral acute respiratory infections. Theycan be responsible for high morbidity and mortality due to acute respiratory infections [51] . In old age, there is reduced microRNA-181a expression in T cells, which contributes to defective antiviral T cell responses; and there is increased production of microRNA-21 which favors Th1 effector cell generation [55] . Holcar M et al [56] have demonstrated a decrease in the percentage of total Tregulator cells and an increase in the percentage of total Teffector cells with age, and a consequent immense increase in the Teffector/Tregulator ratio.

(3) Increase in Th1: Aging is associated with increases in levels of circulating cytokines and proinflammatorymarkers; this phenomenon is called "inflamm-aging" [57] . The microenvironment in which T helper cells develop in older people may cause production of more cells committed to Th1 than that in younger subjects. On analysis of the effect of ageing on the responsiveness of human T helper cells to polyclonal stimuli, Sakata-Kaneko S et al [58] discovered that at the time of antigenic stimulation and subsequent induction of lymphokine secretion and cell adhesion molecule expression, the T helper subset in the peripheral blood of aged individuals is found to contain a greater proportion of cells committed to Th1type cells than that from young individuals [58] . These T cell related changes could contribute to dysregulated and damaging immune responses to SARS-CoV-2 in the elderly. Changes in sex steroids impact regulation of the Th1 and Th2 immune responses in old age, with implications for severe and fatal outcomes of infections [68] .It is found that women present strongerTh2 immunity [69] and men have stronger Th1 immunity [70] . In healthy men and women, the polarization of immune response into Th1/Th2 cytokines or T helper cell subsets is flexible.The ratio of these cells swiftly varies according to physiological demands and clinical conditions. Aging is associated with a loss of this flexibility due to alterations in the levels of sex hormones [71]. Yet even though bothsexes have declining sex hormones with ageing and increase in Th1 immunity, men and women have differentCOVID-19 outcomes. This is chiefly because increases of Th1 due to ageing, immunosenescence and hormonal changes do not affect the Th2 mediated humoral immune response. Tajima K et al [72] have proved that the levels of Th1-type cytokines in the bronchoalveolar lavage fluid of ovariectomized rats were significantly higher than those in sham rats, while the levels of Th2-type cytokines in both bronchoalveolar lavage fluid and serum were comparable between ovariectomized and sham rats.

These findings suggest that in the aged, though the immune responses would be in a Th1polarized manner while levels of sex hormones are declining,the basic humoral immune status is unchanged. This is more favorable for agedwomen than men suffering from COVID-19 because women, in general, have Th2 predominance which remains unaffected; while men, in general, have low Th2 which also remains unchanged. In consequence, aged women have higher Th2 than aged men. When equal numbers of severe COVID-19 female patients (average age 63.1 years) and severe COVID-19 male patients (average age 59.4 years) were analyzed for the concentration of serum SARS-CoV-2 IgG antibodies, the female patients had higher concentrations of serum SARS-CoV-2 IgG antibody than the male patients. This indicates that even after the age-related immunosenescence and increase in Th1, oldwomen have better humoral immunity than old men to SARS-CoV-2 [73] . Thus, despite similar age-related changeswith increased Th1 and low progesterone in both males and females, the general difference in Th1 and Th2 predominance means that chances of recovery from COVID-19 are higher in elderly women than in elderly men due to their stronger humoral immunity and antibody production. This is also evident from the betterefficacy of vaccines in older women than in older men. For example, when vaccine efficacy against influenza was measured using the hemagglutination inhibition assay, the antibody response titers were higher in women than in men at all ages. Thehigher antibody titers of older womenwere found to be associated with lower hospitalization and mortality rates compared to same-aged men [74, 75] . 

COVID-19 severity and mortality, from highest to lowest, are graded by age and sex as indicated in Table I : Old men> Old women > Young men > Young women. Table I . Age-Sex groups, Stages of Th1 escalation, Progesterone levels and COVID-19 severity. Old males:

In aged men infected with SARS-CoV-2, there would be three stages of Th1 escalation: (1) Generally pre-existing Th1 predominance in men (2) Rise in Th1 due to ageing with no crossregulation of Th2 due to Immunosenescence and hence the low Th2 status continues (Fig. 2) , and (3) Activation of the cell mediated Th1immune response to SARS-CoV-2 infection (Table 1) . This excessive Th1 activity with low Th2 would cause cytokine storm, tissue injury, organ damage, and severe disease with poor outcomes.It may prove fatal due to low levels of the regulator progesterone (inadequate regulatory actions), low Th2 (poor humoral immunity), and uncontrolled immune dysregulation.

Like old males, in old females too there would be age-related Th1 increase, but since in general women have Th2 orientation, there are only two stages of Th1 escalation in COVID-19 suffering older women: (1) Rise in Th1 immunity due to ageing, and (2) Activation of the cell-mediated Th1 immune response to SARS-CoV-2 infection (Table 1 ). This can lead to excessive Th1 activity with cytokine storm, tissue injury, organ damage, and severe disease. Menopause is an independent risk factor for female COVID-19 patients [76] . Even though at higher risk of immune dysregulation than younger women, aged postmenopausal COVID-19 women with low progesterone levels are presenting with less critical illness and are dying less of COVID-19 than equally old COVID-19 male patients. This is due to their lower baseline activity of Th1 than in the aged COVID-19 male patients, and higher Th2 humoral immunity, which neutralizes extracellular virus particles, thereby lessening the stimulation of Th1 cell-mediated immunity. Like old men, old women too have low progesterone levels and rise in Th1 due to ageing with missing cross-regulation of Th2. Hence, Th2 is unaffected and continues to be high (Fig. 2) . Their risk of severe illness and mortality is lower than old men but higher than that of young males. They both have two stages of Th1 escalation, but young men have higher progesterone than aged postmenopausal women [59,60].

In young men infected with SARS CoV-2, the rise in Th1 due to old age is not there. Hence, there would be two stages of Th1 escalation: (1) Generally preexisting Th1 predomination in men and (2) Activation of cell mediated Th1 immune response to SARS-CoV-2 infection. The young males have less severity and mortality due to COVID-19 than old males (Table 1) . Their progesterone levels are low but higher than those of aged men [59,60]; and they have one less stage of Th1 escalation. Although as explained further the young males have higher severity than young females.

In young females infected with SARS-CoV-2, generally there is Th2 predominance and the rise in Th1 due to old age is not there. Hence, there would be only one stage of Th1 escalation: which is activation of the cell-mediated Th1 immune response to SARS-CoV-2 infection (Table 1) . Plus, they also have higher levels of the regulator progesterone, and theoretically optimum chances of immune regulation. Initially there would be cross-regulation of Th2 followed by development of specific humoral immunity, immune homeostasis, and recovery. Thus young females have less severe illnessthan the other age-sex combinations.

The above evidence about low progesterone and raised Th1 in the high-severity groups substantiates and confirms applicability of the concept of immunity patterns and their regulator progesterone for COVID-19. The evidence indicates that highTh1 with decreased progesterone could lead to severe COVID-19 outcomes, and high risk of mortality (Fig. 3 ).

Low progesterone SARS COV-2 Figure 3 .Schematic illustration. SARS-CoV-2infection in a patient with high Th1 and low progesterone link to severe COVID-19.

While old age and male sex are the chief known risk factors for severe disease, there are other factors affecting progesterone and cell mediated immunity and hence immune regulation, and COVID-19 outcomes irrespective of age and sex. Table IIA enlists the factors affecting progesterone and/or cell-mediated immunity along with their reported COVID-19 mortality. Table IIB suggests the same list of factors as a check-list for possible risk of severity in COVID-19 patients.

Oxidative stress is associated with changes found in COVID-19 patients.It participatesin the cytokine storm, coagulopathy, and cell hypoxia [77] . Hypoxia results in decreased ATP synthesis and increased formation of reactive oxygen species while decreasing the activity of the normal cellular antioxidant system. The resulting oxidative stress initiates apoptosis, which contributes significantly to cell death observed in hypoxia [78] . Progesterone levels decrease in response to oxidative stress [79] . Various mechanisms, including the decline of sex hormones, link oxidative stress with ageing [80] . One of them is that the alveolar dead space increases with age, reducing arterial oxygen without impairing carbon dioxide elimination. Older adults have a diminished ventilatory response to hypoxia and hypercapnia [81] .

Progesterone is produced from cholesterol. Cytochrome P450 converts cholesterol into pregnenolone, which gets further converted into progesterone. Hence low cholesterol would directly relate to low progesterone [82] . Low cholesterol levels are associated with more severe disease in patients infected with SARS-CoV-2 [83] . Secondly, hypoxia, which is common COVID-19, decreases progesterone synthesis from cholesterol by attenuating cytochrome P450 production [84, 85] . 

The two hormones progesterone and vitamin D cooperate with each other for sequential and effective regulation of the immune system. Vitamin D plays an important role in modulating the immune response to infections. There is an established association between vitamin D deficiency and an increased risk for infections, and vitamin deficiency is associated with poorer outcomes from infectious diseases [86] . Connecting to the present study, both the immunity pattern concept and progesterone regulation can explain these associations of mortality with male sex and vitamin D deficiency. Thangamani S et al [90] have concluded that the progesterone and vitamin D nuclear hormone receptor ligands play important roles in regulating T cells. Progesterone is an inducer of the vitamin D receptor in T cells. This makes T cells highly sensitive to calcitriol, the active form of vitamin D. Increased expression of vitamin D receptors induced by progesterone allows highly sensitive regulation of T cells by vitamin D even when the vitamin D level issuboptimal. This regulatory pathway enhances the induction of regulatory T cells and suppresses Th1 cells. This results in effective regulation of T cells and helps to prevent adverse outcomes due to dysregulated immune responses. Males have low progesterone levels, and when their vitamin D is also deficient, it escalatesthe risk of an overshooting Th1 response, which can be described as the fourth stage of Th1 escalation [91] .

Average progesterone levels in women vary significantly at the inter-population and intrapopulation level as a function of age and acute metabolic energystatus related to energy intake, energy expenditure, or a combination of both. In addition to effects of acute stressors, baseline progesterone levels differ among populations. The causes of such chronic differences are not well understood, but it has been hypothesized that they may result from varying tempos of growth and maturation and, by implication, from diverse environmental conditions encountered during childhood and adolescence [92].

Use of progesterone in clinical practice for non-endocrine and non-gynecological disorders is well established. Progesterone is used in treatment of inflammatory diseases with Th1 dominance.

Disease example: Rheumatoid arthritis patients suffer from Th1-driven autoimmune inflammation [93, 94] . Disease activity of rheumatoid arthritis is characteristically regulated by hormones. Women with rheumatoid arthritis often go into remission during pregnancy [95] , and its outcomes get more severe with reproductive ageing. Women suffering from rheumatoid arthritis experience a slower physical decline before than after menopause [96] .Men older than 50 years suffering from rheumatoid arthritishave about twice the risk of osteoporosis compared to healthy controls [97] . Rheumatoid arthritis symptoms are related to changes in menstrual phases [98] . Plasma progesterone levels in the luteal phase are significantly lower in women suffering from rheumatoid arthritis than in the control group [99] . Progesterone is therapeutically used to treat rheumatoid arthritis patients. Progesterone receptors are widespread in human cells including osteoblasts and osteoclasts[100]. Intra-articular administration of progesterone reduces inflammation in arthritic joints [101], repairs cartilage, and regulates remodeling of affected bones [102,103].

As evident from the detailed research on the regulatory role of progesterone in both parts of this manuscript, progesterone could have therapeutic clinical applications for SARS-CoV-2 infection and COVID-19. Ideally,progesterone treatment of COVID-19 patientsshould be limited to treatment of the acute illness and recovery, irrespective of the patient's baseline plasma/serum levels of progesterone. It needs to be clearly understood that here the aimis to make best therapeutic use of the biological and regulatory effects of progesterone, and not hormone replacement therapyor corrections of hormones which is a different issue as well as a different application of the hormone.

As the majority of mildly or moderately symptomatic COVID-19 patients recover without complications of immune dysregulation, use of exogenous progesterone would be chiefly for those COVID-19 patients having severe symptoms with rapid worsening and critical illnessrelated toimmune dysregulation. As discussed before, theseverely affected patients are highly likely to be low on progesterone with high Th1 immunity,in part due to their sex (males) or their age (aged men and postmenopausal women). Ding 

If possible, measurement of serum/plasma progesterone level and Th1 cytokine panel levels will be useful for decision about the dose of progesterone, records, and follow-up. It is important to understand that while in routine applications, the pretreatment value of serum/plasma progesterone is interpreted with reference to the laboratory's normal limits, for COVID-19 severely and critically ill patients the targeted physiological reference range would be that of young females in their mid-luteal phase (reference range 12-32 ng/mL) [61] . Luteal phase is the Th1 regulating phase [104] . CRP is a routinely used marker of inflammation and infection in clinical practice. Gursoy AY et al [105] have reported that the progesterone rise during the luteal phase might have a subtle effect on CRP, which is significantly lower in the luteal phase than in the follicular phase. Thus, as young premenopausal females suffer the least severity and mortalityfrom COVID-19, they provide an indication of the range of progesterone target levels for therapy. It would be ideal to refer to the normal limits reported by the local laboratory which have been established for the local population.

Careful monitoring of symptoms is important. Administration of progesterone would be most promising at an early stage, before the patient gets critically illand beforeimmune dysregulation has caused critical complications.

Physiological data and clinical outcomes demonstrate that bioidentical progesterone is more efficacious than its synthetic and animal-derived counterparts [106] . It is important to note that micronized progesterone and pregnane derivatives are safe with respect to thrombosis, but not the norpregnane derivatives and Medroxyprogesterone acetate [107] . It is relevant to mention here that some synthetic progesterones are found to elevate alpha-1 antitrypsin concentrations in the blood. This could be protective as alpha-1 antitrypsin inhibits the TMPRSS2 involved in binding of SARS-CoV-2 to the host ACE2 [108,109].

The route of progesterone administration, dose, and duration of treatment would be the treating physician's decision based on the specifics of each case. The progesterone effect depends onconcentration as well as infusion mode. It is important to note that -progesterone has dose dependent effects on hypoxia and neurotransmission, with low pulsatile doses having a positive effect whereas continuous and/or high doses may have negative effects [110, 111] ‖. Unfer V et al [112] conducted detailed work on the use of progesterone in clinical practice and evaluated its efficacy in diverse indications using different routes of administration. The distribution and concentration of the hormone in the tissues could vary depending on the route of administration,affecting therapeutic outcomes. A single 100mg dose of progesterone was given orally or as intramuscular injection to women during the follicular phase of two consecutive menstrual cycles: After oral administration serum levels of progesterone increased rapidly to reach luteal phase values within 1-4 h;whereas following intramuscular injection too the progesterone levels increased rapidly but peaked 8 hours after administration to values 2.5 times higher than the luteal phase. Via both routes, the serum progesterone levels were found elevated for more than 12 hours [113].

Based on these findings, an appropriate dose for COVID-19 patientswould be either 100 mg via the oral routeor a lower dose for the intramuscular route to avoid the 2.5 times increment because the aim of administering the progesterone is regulation of theimmune responses, not excessive suppression of the Th1 response. Massive Th1 suppression or Th2 deviation would be risky as it could camouflage the disease symptoms and provide favorable conditions for replication of any remaining SARS-CoV-2 virus or new infections.

For example, pregnant womenhave high progesterone levels which increaseover the first to third trimesters (22.1 -225 ng/mL) [114] .Based on a systematic review and meta-analysis, Allotey J et al [115] have reported that pregnant women with COVID-19 are less likely to manifest symptoms of fever and myalgia than non-pregnant women of reproductive age,although they might be at increased risk of complications and admission to an intensive care unit. This is similar to findings of pregnant women infected with swine flu. H1N1 pathogenesis is similar to SARS-CoV-2 with heightened Th1 immunity. A study has reported the presence of antibodies to progesterone in pregnant women suffering from swine flu. It was related to high levels of this hormone during pregnancy whichcould deviate the immune system excessively towards Th2. Maternal Th2 immune status is protective for the fetus in order to avoid abortion, but it could lead to formation of auto-antibodies due to Th2 deviation and diminished Th1 resulting in impaired ability to fight the virus [116] .

Irrespective of the cause of immune dysregulation in COVID-19: be it due to heavy viral load, pre-existing immunity pattern with raised Th1,suboptimallevels of progesterone or other regulatory factors, the logical antidote to immune dysregulation is immunoregulation and not immunosuppression. Immunosuppression with corticosteroids is used in COVID-19 patients mainly for suppressing the inflammation. Efficacy of corticosteroid treatment depends on the stage of COVID-19 [117] and appropriate dose [118] . Administering it at an early stage of the SARS-CoV-2 infection in high doses is found to be harmful with excessive suppression of inflammatory responses leading to a virus-supporting milieu and also paving the way for mucormycosis [119] .Such clinical findings in some of the COVID-19 patients treated with corticosteoids indicate that an immune regulator like progesterone maybe a better option. Progesterone is one option to achieve optimal immunoregulation while avoiding excessive suppression of the Th1 response. Use of progesterone will also require clinical vigilance, especially about the right time of administrationwith regards to the stage of COVID-19 and appropriate dose. Disease example: COVID-19 outcomes are found to be worse in those inflammatory arthritis patients who are on glucocorticoids than in those who are on anti-cytokine therapy [120] .Most pathogenic fungi are intracellular infections and immunomodulating therapeutic agents that upregulate the immune response in the fight against fungal infections hold promise for enhancing the efficacy and safety of conventional antifungal therapy [121] . This is an indication that not immunosuppression but immunoregulation is the proper target to treat Th1 oriented COVID-19 patients with severe symptoms due to cytokine storm.

Suggested duration of treatment with progesterone for COVID-19 would be episodic, transient, only when needed, an emergency intervention to assist the patient to overcome the acute effects of immune dysregulation.

Blood pressure monitoring Progesterone treatment significantly reduces resting mean arterial pressure although it increases plasmavolumein humans [122] . There is no effect of progesterone treatment on adrenocorticotropic hormone, arginine vasopressin, renin, or heart rate responses to hypotension. It is suggested that a small increase in progesterone can reset resting mean arterial pressure and plasma volume, without altering reflex heart rate or endocrine responses to hypotension [123] . Like other vasodilators it could be administered early to acute heart failure patients with normal or high blood pressure at presentation,but is best avoided in critical patients with low blood pressure [124] .

Two interesting addendums in regard to the immunity patterns: First is the two-way effect of antibiotics. In patients with low Th1 and high Th2 immunity pattern, unless there is a bacterial infection present, antibiotics are best avoided to prevent lowering of the commensals and gut microbiota, as this could further lower the Th1 response and have a profound effect on the host immune system [125] . For the same reason, the use of antibiotics could be therapeutic for patients with high Th1 and low Th2 immunity patterns as lowering the microbiota lowers Th1 and susceptibility to cytokine storm [126] .Second, therapeutic drugs influence oxidative stress. It is interesting to note that the much-used drug hydroxychloroquine for COVID-19 downregulates oxidative stress [127] , which is also an effect of progesterone. Remarkably, it substantiates the present research as it is actually reported to shift the excessive cell-mediated immune responses to Th2, in a dose-dependent manner [128].

To conclude, the presented research indicates that recognizing the patient's immunity pattern and progesterone status is important for COVID-19.High Th1 and low progesterone predict severe outcomes of COVID-19. The study proposes reasons for the higher severity and mortality in males and the elderly. One stage lesser escalation of Th1combined with higherTh2 is the most likely reason for less severity and mortality in COVID-19 affected old women in comparison to old men. Younger women have the additional advantage of higher levels of the regulator progesterone, along with only one stage escalation of Th1as compared to older women as well as aged and young men.Part 1 of this research finds that progesterone could favor recovery by interfereing with SARS-CoV-2 infection and COVID-19 pathophysiology at multiple steps. It could be an important remedy itself or as an adjuvant to other antiviral drugs and the symptomsbased treatment protocol. It could be beneficial in COVID-19 patients with pathophysiological indications of immune dysregulation, severe symptoms, and critical illness. It may even have application for vaccination of those with low humoral immunity, to help them raise their antibody titers. The immunity pattern concept maybe widely applicable for other diseases, once the specific pattern for a disease is established.Understanding of immunity grading could be of use to evaluate immunity status for other disease conditions. A short summary of the present work would be to consider IPPR (Immunity pattern and progesterone regulation) for COVID-19 patients and to check for presence of the other enlisted parameters affecting cell-mediated immunity and/or progesterone as an alarmfor the possibility of severe or critical illness. Further studies based on the clinical applications of the present research in medical practice would be insightful.

AUTHOR'S CONTRIBUTIONS Conceptualization of the present research; reference work; published data collection; writing of the manuscript; validation of the concept; visualization and drawing of figures to explain the concepts; reviewing and editing the manuscript; and submission of the work to this journal. No medical writer or editor was involved in the creation of this manuscript.

No humans or animalsubjects are used for this study.

There is no unreleased data for this study. FUNDING:

The present study had no funding source.

There are no conflicts of interests for this study.

Author conveys sincere gratitude towards all her teachers. She thanks each of the authors for their referred work in the present manuscript. As this research is carried out during the SARS-CoV-2 pandemic lockdown period, author acknowledges her family for this work. She respectfully dedicates this manuscript to her parents Mrs. 

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Dear Respected Editor, Greetings! It is my pleasure to submit my original research work on "COVID-19 and Progesterone Part 2" to the

Please know that there are no conflicts of interest for this manuscript. With thanks and best wishes