Yoga, Meditation, Breathing Exercises, and Inflammatory Biomarkers with Possible Implications in COVID-19: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Introduction COVID-19, a multisystem disease, has implications for various immunity and infection biomarkers. Yoga (Y), meditation (M), and pranayama (P), and their combinations have shown positive changes on those biomarkers among other than COVID-19 patients and healthy people. So, we aimed to document the evidence of possible implication in a systematic way. Materials and Methods We screened 84 full texts, published in the last ten years, from three databases, from which only 44 met the eligibility criteria, and then extracted the data related to demographic characteristics, intervention, results, and strengths and limitations in two MS-Excel grids, and then presented them in tables and figures. Furthermore, we carried out meta-analysis including subgroup and sensitivity analysis using a random effects model of 11 RCTs and reported the mean difference, heterogeneity, and p value with 95% CI and presented them with forest and funnel plots and the tables. Results Twenty-five biomarkers of 4023 participants (range, 15–413) from 13 countries, healthy and clinical, from both sexes above 18 years, and from mainly clinical settings, were reported. YMP intervention, in solitary or in different possible combinations with varied durations among clinical and pregnant (range, 960–4800 minutes) and healthy (960–8400 minutes, excluding two studies of 20 minutes only) participants, was reported. It was revealed that 25 biomarkers, nine among the apparently healthy, 14 among the patients, and two among the pregnant, changed favourably (p < 0.05). Furthermore, either in meta- or subgroup-analysis, mean differences of IL-6 (−1.44 pg/ml) (95% CI) (−2.33, −0.55), (p = 0.002, I2 = 82%), Cortisol (−40.75 pg/ml) (95% CI) (−64.13, −17.38), (p = 0.0006, I2 = 87%), and TNF-α (−3.40 pg/ml) (95% CI) (−4.83, −1.98), (p < 0.0001, I2 = 79%) showed statistically significant changes. Nonetheless, considerable heterogeneity and publication bias were observed among the studies. Conclusion Although more than two dozens of biomarkers in individual studies showed favourable changes, only IL-6, Cortisol, and TNF-α produced significant combined results, even then with much less certainty. Further meta-analysis of biomarkers of COVID-19 patients is highly recommended. Registration: CRD42021283894.


Introduction
Yoga, meditation, and pranayama, or yogic breathing, have been practised worldwide since antiquity. Research, both here and elsewhere, has shown that practises like guided asanas, specific pranayama, and meditation can improve the body's immune system responses. To develop a holistic framework for individuals' health, the interrelationships between mind and body have been extensively studied in recent times [1][2][3][4][5]. With the advent of the pandemic, researchers and clinicians are in search of practises and therapeutics that can reduce the impact of COVID-19 on human health. In this line, recent reviews of complementary and alternative medicine (CAM) and traditional Chinese medicine (TCM) for COVID- 19 have provided preliminary evidence of effectiveness [6,7]. In addition, a recent review also underscored that the effects of natural compounds from Nigella sativa are promising [8]. It has been stated that as a result of COVID-19 disease, leukocytes, neutrophils, cytokine levels [IL-2R, IL-6, IL-8, and IL-10], and infection biomarkers like CRP, PCT, and ferritin are significantly increased [9,10]. Multiple studies have shown that yoga, meditation, and pranayama interventions are efficacious in regulating an array of biomarkers, including cytokines [4,5].
Further research in this area is ongoing since the disease is still uncertain in many aspects, especially regarding its long-term complications [10] and the outbreak of possible new mutants due to natural selection [11]. During the cytokine storm that occurs in COVID-19 syndrome, proinflammatory cytokines are released uncontrollably. A positive relationship between the severity of the viral infection and the mortality rate was observed following an uncontrolled and dysregulated secretion of inflammatory and proinflammatory cytokines [12]. Recent evidence also shows that systemic vasculitis and cytokine-mediated coagulation disorders act as the principal actors of multiorgan failure among COVID-19 patients with severe complications [10]. Assessing the effect of different biomarkers in COVID- 19 can help in the early diagnosis of the disease, confirming and classifying the disease severity, identifying the high-risk cohort, framing intensive care unit (ICU) admission criteria, rationalising therapy, assessing response to therapies, predicting outcomes, and framing criteria for discharge from the ICU and/or the hospital [9]. So, it is the need of the hour to assess and identify measures that can control the dysregulated secretion of cytokines and other inflammatory mediators with possible implications for COVID-19. Till date, multiple studies have shown that yoga, meditation, and pranayama interventions are efficacious in regulating an array of biomarkers, including cytokines [4,5]. However, these interventions and their outcomes presented over different settings and durations have shown mixed results. Considering the inconclusive evidence present, the heterogeneity in outcomes, and the duration of interventions given in the previous studies, we sought to systematically review the evidence of the effects of yoga, meditation, and pranayama interventions on COVID-19 related inflammatory biomarkers by including only randomized controlled trials (RCTs).

Materials and Methods
We conducted this review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.

Study Selection Criteria.
A systematic literature search was carried out after registering the review protocol in the international prospective register of systematic reviews (PROSPERO Regd. No. CRD42021283894). Peer-reviewed RCTs published during the last 10 years (2010-2021) were included. We sought to identify studies that used any version of yoga, meditation, and/or pranayama, intervened as solitary or in combination, among healthy and clinically ill individuals in adults (≥18 years), including pregnant women, to assess the effectiveness of biomarkers related to or influenced by COVID-19. We were also guided by our earlier scoping review on yoga, immunity, and COVID-19 for study selection [13]. e search was conducted mainly in three databases: e Cochrane Library, PubMed, and Google Scholar. Additionally, we also carried out a citation search. Keywords and MeSH terms were informed by previous literature searches. Six sets of search terms were used including terms related to (i) yoga, (ii) meditation, (iii) pranayama, (iv) intervention, (v) biomarker, and (vi) controlled trial (S1_ Search Strategy with Keywords and MeSH).

Data Extraction and Analysis.
Based on our previous scoping review of yoga, immunity, and COVID-19 [13], we developed and then extracted data using two standardized MS-Excel grids. e data extraction form were initially piloted (n = 2 studies) and then refined and finalised by all reviewers. e data were extracted by two reviewers (CA and ShS), and the third reviewer (KS) assessed when there was a discrepancy. Pre-post effectiveness of YMP on biomarkers was found using mean difference, difference in differences of means (∆1-∆2), and effect sizes (Cohen's d). However, we only reported p values along with the direction of effectiveness in interpreting the tests. e basic characteristics and detailed findings of the included studies are given in Tables 1 and 2, respectively. For data synthesis, we developed an Excel grid for the main outcomes (biomarkers). e evidence was synthesised into a narrative form. A descriptive analysis of the characteristics of study populations was carried out using range values and proportions.
For meta-analysis, we calculated the mean difference when at least three studies consistently reported the primary biomarkers. We produced forest plots, funnel plots, and risk of bias (ROB) assessment charts using the Cochrane risk of bias tool, from RevMan 5.4.1. e effect size of the meta-analysis was estimated through random effects, considering the post-pre mean change in biomarker concentration and standard deviation (SD). Studies that reported standard mean error (SME) were converted to SD, and for those not specifying SME or SD, either of the two was considered. Following the Cochrane guideline, we calculated the pooled SD from the given parameters, including an additional value of the correlation coefficient, assuming 0.8. Regarding three-armed studies, we pooled the mean and SD from the two experimental groups for the outcome measure.
Subgroup analysis was performed post hoc by stratifying the studies based on intervention type such as yoga, meditation, pranayama, or yoga only, or meditation only, and intervention duration in weeks and minutes. For this, we divided IL-6 into six subgroups, cortisol into two, and TNFa into three.  Evidence-Based Complementary and Alternative Medicine  (ii) Participants introduced to slow and deep breathing exercise from day 2 (till 21 days).      (ii) Observation was conducted only at baseline and after 3 months.
(iii) Patients with low-to-severe disease activity were included in the study. 17 Chen     adherence to the yoga and walking would be questionable.
(ii) Home-based yoga activity track report was difficult to maintain 28     (ii) HLW (healthy living workshop) included at least one HLW class (60 minutes long) per week for 10 weeks, addressing alcohol, nicotine, and caffeine; being a smart patient; brain diseases; cancer prevention; diabetes; nutrition (3 classes); germs, colds, and the flu; physical activity (2 classes); sleep; physical pain, prevalence and causes of depression; and protecting your heart (i) IL-6 levels reduced significantly in the intervention group compared to control.   YMP (i) Active intervention was carried out for 2 hours a day for 14 days, consisting physical postures, pranayama, interactive lecture, and ending with meditative relaxation, and then followed for next 10 weeks at home.
(ii) Dietician provided dietary intervention (DI) for both the groups (i) IL-6 and TBARS levels significantly decreased in the intervention group at two points of analysis.
(ii) TNF-α and SOD levels changed in groups (both within and between), but were insignificant.
(i) Both groups are homogenous.
(ii) Intention to treat analysis could not be carried out.
(ii) Home-based interventions have been suffered with noncompliance, which was often difficult to assess 42 Harkess et al., 2016, austrailia [46] YMP (i) An hour class of total 16 classes were offered twice weekly for 8 weeks.
(ii) Hatha yoga was taught (i) A moderate correlation (rho �0 .608, p < 0.01 between TNF and IL-6 was observed. (ii) e friedman and mixed between-within subjects ANOVA test represented nonsignificant changes in IL-6 or TNF and hsCRP levels, respectively.
(iii) Studied in a nonclinical population (i) e first study to explore DNA methylation.
(ii) A pilot study with small sample size and low power of the test 16 Evidence-Based Complementary and Alternative Medicine (iii) Participants attended at least 10 of the total 12 weeks and at least three times at home during the experiment period by watching a 40-minute DVD (i) e serum TBARS, and IL-12, TNF-α, IFN-c reduced significantly in the intervention group and within group comparison respectively.
(ii) e serum SOD levels decreased and cortisol levels increased significantly only in the control group.
(i) Many stress related biomarkers were studied.
(ii) e sample size was small, and the findings may not be generalized 44  full text. We removed 40 full-text articles that failed to meet the inclusion criteria, as well as 3 articles that were unavailable in full text. Finally, 44 articles were selected for the review (Figure 1).

Study Quality.
Of the 44, twenty-three RCTs were openlabel, followed by 19 single-blinded and 2 double-blinded trials. Among the 37 full RCTs, one was a crossover design. e comparator arms included 23 controls or wait-list or attention or observational controls, 11 usual or standard care, and 10 were given placebo or active control. Placebo interventions included health education (HE) with or without supportive therapy, counselling, exercise, rehabilitation, relaxation, healthy living workshop (HLW), dietary intervention, physical exercise, sleep hygiene education (SHE), and stress reduction. Risk of bias (ROB) of 11 studies included in meta-analyses, three were found to be low, one medium, and seven with high risk (Table 3).

Participants.
A total of 4023 participants were included in 44 trials, ranging from 15 to 413 participants. Most participants were patients, including those either from inpatient or out-patient clinics or hospital settings. Only a few studies (n = 4) were carried out in community and educational institutions. Almost all studies included both men and women aged 18 years or above, whereas some trials were exclusively conducted only on females (n = 13) [14, 20, 26, 27, 29-31, 46, 48, 51, 52, 54, 56] (Tables 1 and 2).
In a case report, a 55-year-old COVID-19 positive with comorbidities had his COVID-19 symptoms alleviated as well as his blood sugar level improved after treatment with ayurvedic medicine, yoga, dietary suggestions, lifestyle modifications, and allopathic medications [68]. Another ongoing study can strengthen the importance of yoga intervention in improving the health of COVID-19 patients [69].
Many studies have explored various immunity biomarkers in COVID-19 patients. T cells, an important component of lymphocytes responsible for robust immunity, are reduced significantly during severe infections, including COVID-19 [58,59]. Cytokines (IL-6, IL-10, IL-8, IL-2R, and so on) consist of various biomarkers produced by both innate and adaptive immune cells [70], which play a vital role in inflammation [71]. A fatal cytokine storm is usually observed in COVID-19 patients who are critically ill [72]. Cytokine storms lead to damaged tissues, resulting in thrombotic tendency and multiple-organ failure [73], suggesting cytokine control necessary. Both individual studies and systematic reviews indicate the importance of IL-6, reporting it to be an independent predictor of disease severity and survival in COVID-19 patients [74][75][76][77]. CRP, a prominent marker of systemic inflammation, was elevated in the majority of COVID-19 patients with severe illness compared with mild or nonsevere patients [78][79][80]. Cortisol regulation acts as an adaptive immunity, but extreme cortisol responses (relative adrenal insufficiency) are associated with a higher mortality rate [81][82][83]. Cortisol levels were high in COVID-19 positive patients compared to negative [84] and nonfatal [85] COVID-19 patients.
Although meta-analyses showed an overall decrease in stress biomarkers (IL-6, TNF-α, and cortisol), only TNF-α was significant. However, subgroup analysis showed TNF-α (8-12 wk, 3000-4800 minutes), IL-6 (yoga-meditationpranayama), and cortisol (6-12 wk, 1000-2000 minutes) statistically significant reductions favouring intervention. Our findings are in line with earlier conducted metaanalyses in which both IL-6 and TNF-α were reduced in the intervention group following the yoga and meditation [86,87]. e change was significant in one study reporting IL-6 [86], whereas there was an insignificant decrease in IL-6 and TNF-α [87], which could have been due to smaller number of studies (n � 2) included in the analysis. Recent reviews on the effects of phytosterols and the effect of an herb, Nigella sativa, have also been shown to reduce the cytokine storm; however, these are beyond the scope of this review and so, further suggested [8,88].
In the scarcity of RCTs reporting the effectiveness and efficacy of yoga, meditation, and pranayama in COVID-19 patients, our findings further add to the yoga-based interventions and their effects on the inflammatory biomarkers.
rough our findings, it may be possible to recommend treatment strategies to promote the health of both mild to moderate and severe cases, including symptomatic and asymptomatic COVID-19 patients.  Evidence-Based Complementary and Alternative Medicine Severe/critical/ICU/ dead e Indian Public Health Standards (IPHS), in its revised set of standards released in 2012, has integrated yoga as one of the components to be prescribed for AYUSH facilities [89]. Our findings strengthen the usefulness of yoga and its fraternity, which is in tandem with the recommendation of IPHS to implement yoga in primary and secondary health facilities. A minimum of an hour of intervention following a higher duration of yoga followed by pranayama and meditation in healthy and asymptomatic COVID-19 patients can have a positive impact on their health. As COVID-19 patients become more severe, pranayama should be practised for longer periods, followed by meditation and yoga, as various asanas in yoga might be complicated to perform in such patients. Our findings suggest that the intervention can have a long-term positive effect even if practised at home, but beyond 8 weeks, participants might feel unmotivated to continue. erefore, post-8-week refresher training might be granted as food for thought.
Despite this, the review has certain limitations. Because the studies and participants were from various settings and countries, significant heterogeneity was observed in the meta-analyses, which may cast doubt on the summary effects to some extent and should be approached with caution. Moreover, due to the low number of studies included in meta-analyses, it was unwise and so restricted to carry out quantitative analysis such as Egger's or Begg's tests for publication bias. In the systematic review, we included seven pilot and feasibility RCTs, which may have compromised the internal validity of the review. Furthermore, only a single study was respiratory-related (acute respiratory distress syndrome, ARDS) as a disease outcome; it should be extrapolated in COVID-19 patients with solicitude.

Conclusion
e systematic review of 44 studies revealed 25 immunity biomarkers, among healthy, clinical, and pregnant women, with possible implications for yoga-related interventions in COVID-19. Total doses of yoga, meditation, or pranayama ranged between 20 and 8400 minutes. However, a smaller dose of YMP was found to make significant changes among pregnant or clinical patients in comparison to healthier ones. Clearly, the combined action of YMP may be effective in lowering IL-6, cortisol, and TNF-α levels in patients, specifically with 2625, 3900, and 1500-minute interventions, respectively, spanned over 8-12 weeks. Also, the YMP package is warranted for pilot RCT among COVID-19 mild to moderate patients with standard care before reaching a firm conclusion. Furthermore, meta-analyses of biomarkers and phytosterols of COVID-19 patients are highly recommended.

Data Availability
Data extracted from the studies, of systematic reviews and meta-analysis, are provided by the corresponding author upon request.