Sperm a cell in distress: Yoga to the rescue

Causes and effects

The vulnerability of the sperm to damage due to oxidative insult is the result of sperm being transcriptionally inactive due to compaction, deficient in cytosolic antioxidants, a high content of PUFA content in cell membrane, and inadequate DNA damage detection and repair mechanisms (base excision repair mechanism).^{[3,4,7,15-18]} There are numerous lipid metabolites which are generated as a consequence of the oxidative attack by free radicals on the PUFA in the sperm membrane including lipid alkoxyl radicals, peroxyl radicals, and also various electrophiles, such as 4-hydroxynonenal (4HNE), malondialdehyde (MDA), and acrolein.^[15,16] These electrophillic aldehydes have the potential to form adducts with several proteins in spermatozoa affecting sperm function. Moazamian et al. showed the formation of an adduct formed with the flagellar axonemal protein, dynein heavy chain in their study, which highlighted the impact on sperm motility.^[15] Several proposed factors which induce seminal OS, include morphologically abnormal sperm, sedentary lifestyle, intake of nutritionally depleted food, infections, exposure to xenobiotics, psychological stress, high temperature, insecticides and pesticides, smoking, alcohol, obesity, mobile phone radiations, and advanced paternal age.^[7] This makes the spermatozoa susceptible to OS-induced damage to various biomolecules and to sperm mitochondrial and nuclear genome. The excess production of ROS by such pathways is detrimental for the sperm functions and acts by two mechanisms, (a) by producing lipid oxidation in the sperm plasma membrane of the sperm containing bountiful PUFAs making them susceptible to oxidative attack and (b) by affecting the sperm mitochondrial and nuclear DNA as well as RNA, thus further compromising embryonic development. The highly complex lipid peroxidation pathway, electrophilic aldehydes which further increases the production of OS.^[8,14,19] This is due to inherent potential of aldehydes to covalently bind to the nucleophilic centers of vulnerable proteins, in the mitochondrial electron transport chain (ETC) such as succinic acid dehydrogenase.^[8,20] The subsequent disruption of mitochondrial electron transport, leads to an electron efflux which become associated with oxygen, generating superoxide anion.^[8] This culminates in a self-perpetuating redox cycle and any factor predisposing to OS will lead to greater levels of oxidative damage, along with accelerated generation of mitochondrial ROS and a state of cell senescence, as these cells thus enter a truncated version of the intrinsic apoptotic cascade.^[5,21] One of the first functions affected by OS and lipid peroxidation is sperm motility.^[22,23] It has been proposed that oxidative damage to the axoneme and depletion of intracellular adenosine triphosphate (ATP) are responsible for asthenospermia which is one of the first manifestations of OS and lipid peroxidation. Shamsi et al. documented that OS and OS-induced mitochondrial dysfunction are associated with reduced ATP levels which impair development and differentiation of microtubular apparatus of sperm axoneme and results in asthenozoospermia.^[14]

The vulnerable spermatozoa are endowed by a repertoire of antioxidant scavenging mechanisms to safeguard from the ROS attack. Taking into account that spermatozoa are transcriptionally inactive and possess limited endogenous antioxidant defense mechanisms, the dependence on exogenous source of antioxidants has mushroomed among reproductive specialties.^[24,25] This fact has given rise to the adoption of different cocktails of enzymatic and non-enzymatic antioxidants which are currently used therapeutically. Most of the antioxidants used lack consistency and also the question that what defines the ideal redox balance remains unanswered.^[26,27] Adoption of a healthy yoga-based lifestyle (YBL) has not only seen to show improvement in decreasing ROS levels but also regulating levels of free radicals.

Nuclear genome

The high level of derangements in the nuclear chromatin in the male germ line is one of the hallmarks of male infertility. The compromise in the sperm genomic integrity has been found to be clinically associated with an array of adverse reproductive outcomes including impaired fertilization, aberrant embryogenesis, an increased rate of spontaneous miscarriage, implantation failure, congenital malformations, and significant morbidity in the offspring, including paediatric carcinomas.^[5,28] The detailed etiology of the defects in genomic integrity are still underexplored but they have been associated with efficiency of sperm chromatin compaction during the terminal stages of sperm differentiation in epididymis and oxidative insult on the spermatozoa.^[5,20,29,30] The guanine base in the sperm chromatin is more susceptible to the oxidative attack on the sperm due to its low oxidation potential. Due to the lack of adequate repair mechanisms in sperm, the impending attack results in accumulation of the mutagenic base adduct 8-hydroxy-2-deoxyguanosine (8-OHdG). 8-OHdG expression ahas been shown to correlate with DNA fragmentation on terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, chromatin integrity assays as well as in vivo fertility.^[28,31] 8-OHdG has the propensity to form a stable base pair with adenine, resulting in G:C to T:A transversion mutations during DNA replication. This has a highly mutagenic potential if it is able to escape from DNA repair.^[2-4,29] It thus becomes imperative that 8-OHdG lesions are removed from DNA-damaged spermatozoa to prevent it from affecting the early embryonic gene expression. OS and associated oxidative damage to both mitochondrial and nuclear DNA, shortening of telomeres, and accumulation of mitochondrial DNA (mtDNA) variations and highly mutagenic oxidized DNA adducts also predispose to an increased risk for cancer.^[2,3,19,32] The accumulation of the mutagenic base adducts may also lead to an increased risk of various gonadal and extragonadal tmalignancies later in life making male infertility an early marker of such late onset malignancies.^[33] OS is also associated with aberrant DNA methylation with global hypomethylation and associated loss of genomic stability may also increase risk for cancer.^[3,32] In an attempt to elucidate the paternal contributions in recurrent pregnancy loss (RPL), a study conducted in our laboratory estimated the OS and DNA integrity and significant difference was found between patients and healthy fertile controls. The odds of occurrence of RPL were estimated to be 4.23 time higher when ROS were >27.8 RLU/sec/million sperm (OR 4.23, 95% CI: 1.6–11.1), while it was 10.21 times higher when DFI was >30.7 (OR 10.21, 95% CI: 3.1–33.5). The study also assessed relative expression of the genes critical for early embryonic development.^[2] Thus, an intervention which results in decrease in ROS levels, improvement in sperm nuclear DNA integrity, increases in total antioxidant capacity (TAC), and also helps in maintenance of telomere length is ideal for reduction of idiopathic infertility, RPL, congenital malformation, and reduction of disease burden in children. [2,3,33] The lack of adequate detection and repair mechanisms of DNA damage in spermatozoa makes it susceptible to various oxidative insults and thus, it is better to adopt necessary changes to prevent the impending oxidative attack than only treating it later. Thus, it becomes prudent to minimize exposure causal factors inducing OS, and if diagnosed with high levels, then simple lifestyle intervention like yoga may normalize the levels.

Mitochondrial genome

Mitochondria are one of the potential endogenous source of ROS production in spermatozoa. Mitochondrion, present in sperm midpiece, is also the only organelle to have its own genome in the sperm. Despite their location, the mitochondria rely on the glycolytic pathway to meet their energy demands. This extrachromosomal genetic counterpart in the sperm has the capacity to synthesize proteins in a semi-autonomous manner. Human mtDNA circular dsDNA molecule (16569 bp). It is responsible for the coding for 2 rRNAs, 22 tRNAs, and 13 polypeptides.^[34-36] mtDNA only possesses the genes, which regulate OXPHOS pathway, and this reduction in genomic material warrants against any scope of error. The mtDNA is susceptible to oxidative attack as it lacks the protection by histones or DNA-binding proteins unlike the genomic DNA and has a very basic repair mechanism.^[4,35]

In the study conducted in our laboratory, mitochondrial health was assessed by calculation of mitochondrial copy number variation and transcripts associated with mitochondrial integrity: AMPK, IGF1R, PRC-1, TFAM, SIRT-1, TIMP-1, and CLOTHO.^[37] The study showed that there was a significant increase in mitochondrial copy number and increased expression of transcripts that maintain mitochondrial integrity after 12 weeks of intervention.^[37] Adoption of yoga-based lifestyle intervention as an integral part of our lifestyle may hold the key to increase mitochondrial copy number, increase the expression levels of transcripts that maintain mitochondrial integrity and its associated consequences on physical, mental, and reproductive health. Though the mitochondria present in sperm are not transmitted to the offspring, however, the mtDNA damage is associated with lower ATP production and higher free radical production which damages all biomolecules and both nuclear and mtDNA. It also results in production of sperm which are poorly differentiated and have impaired motility.^{[35,36,38,39]} Sperm mitochondria are prone to electron leakage, resulting in production of ROS and suppressing the sperm functions. A host of factors might trigger the production of ROS by mitochondria, (a) directly by interfering the passage of electrons to the electron transport chain, for example, polyunsaturated fatty acids,^[35] electromagnetic radiation^[40] and (b) indirectly by activation of intrinsic apoptotic pathway, for example, bisphenol A or parabens.^[41,42]

Impact on OS and ODD

Various studies previously conducted in our laboratory have reported a rapid significant decline in seminal OS even within just 10 days of regular practice of yoga and meditation.^{[54-56,66,67]} Dhawan et al., 2018a,b, reported a minimal non-significant improvement in the DNA fragmentation index measured to assess the ODD in male partners of couples with recurrent pregnancy loss and implantation failure in IVF cycles.^[55,56] While the significant improvement in ODD took 6 months as reported by decrease in levels of mutagenic base adduct 8-OHdG^[32,53,54] and an increase in seminal total antioxidant capacity (TAC) levels. [32,54] The decline in levels of this adduct is important as it can be a crucial causal factor of infertility, reproductive outcomes, and childhood disease burden.^{[31,32,51,55,56]} OS has shown to exert a biphasic response, as it is beneficial in low levels and deleterious in higher levels. Moderate levels of OS have been cited to be beneficial in maintenance of sperm telomere length in another study conducted in our laboratory whereas very high and low levels of seminal OS have been associated with shorter telomere length and decline in DNA integrity.^[18] Telomere length has been indicated as one of the most relevant indicators of sperm function. The sperm telomere length in male partners of females with RPL has been found to be significantly lower as compared to that seen in healthy fertile controls in the recent study from our laboratory (unpublished). The study also documented a significant positive increase in the relative sperm telomere length with the adoption of YBL intervention by the couples. It has also been cited to be a marker of psychological stress and aging.^[65,68,69] The integration of a healthy YBL intervention has seen to not only affect the telomere length but also another important marker of sperm biology, that is, the telomerase activity. Increase in telomerase activity along with decrease in ROS levels and increase in TAC levels were witnessed by a short-term (21 days) practice of YBLI. Yoga and meditation based lifestyle has proven to be therapeutic for sperm and the effects on sperm can be summarized in [Figure 1].^[54]

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Figure 1:

Effect of yoga and meditation based lifestyle intervention on the sperm.

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Yoga versus antioxidants

The therapeutic use of various enzymatic and nonenzymatic antioxidant formulations is being routinely adopted by specialists in reproductive medicine for alleviating the current burden of derangements due to OS and ODD.^[70] Different antioxidant formulations have been proposed to show improvement in oligoasthenospermia but therapeutic doses have seen to affect DNA damage in only a few studies.^[8,70] Yoga-based interventions have been significantly seen to impact ROS levels with a parallel increase in antioxidant levels (TAC levels) in as early as 10 days.^{[32,33,57,71-73]} They have been documented to regulate rather than simply scavenging reactive oxygen species. Lack of consensus, dosage schedules and indiscriminate use of antioxidants among practitioners causes very low levels of free radicals resulting in impaired sperm function and several redox sensitive metabolic reactions. YBLI has not only seen to mediate regulation of ROS levels but also cause an alleviation and reversal of various mechanisms causing stress. It has been shown to bring about positive changes by moderating OS-related gene expression^[74-80] immediately by parasympathetic activation, decrease in pro-oxidants,^[81] and increase in antioxidants (like melatonin).^[51,82]

Effect on gene expression

YBLI does not have short term effect only, but it has also shown long term effects by causing histone modifications.^[83] Spermatozoal transcripts, with a critical role in early embryogenesis showed normalization in the levels in male partners of couples with history of recurrent pregnancy loss and implantation failure by Dhawan et al., (2018) after adopting a brief YBLI for 21 days.^[55,56] The dysregulation in gene expression showed normalization toward that of healthy fertile controls with this brief intervention.^[55,56] In the study from our laboratory on primary open angle glaucoma patients, we found positive changes in gene expression pattern by using whole genome microarrays.^[61,66] An upregulation of genes involved in cellular repair and nerve growth maintenance and genes was observed in our patient cohort^[60,61,84] in the central as well as peripheral tissues. The multisystem effects observed by integration of yoga and meditation is thus ideal in management of infertility and reversing testicular aging. A pilot study conducted in our laboratory to assess the changes in the sperm epigenome of male infertility patients by next-generation sequencing documented alterations in the sperm methylome following yoga-based intervention for 21 days. This was seen as DNA methylation changes at 400 genes, out of which hypermethylation was seen in 147 genes and hypomethylation of 229 genes was found.^[85]