RESEARCH ARTICLE


https://doi.org/10.5005/jp-journals-10016-1318
International Journal of Infertility and Fetal Medicine
Volume 14 | Issue 3 | Year 2023

The Effects of Cigarette Smoke-induced Biophysical ROS on Spermatozoa Heads and Telomere Shortening in Infertile Smokers: A Prospective Study


Sridharan TB1, Kamini Rao2, Parameswari Ranganathan3

1Department of Industrial Biotechnology, Vellore Institute of Technology (VIT), Vellore, Tamil Nadu, India

2IIRRH-BACC, Bengaluru, Karnataka, India

3Department of Biotechnology, Thiruvalluvar University, Vellore, TamilNadu, India

Corresponding Author: Parameswari Ranganathan, Department of Biotechnology, Thiruvalluvar University, Vellore, Tamil Nadu, India, Phone: +91 4662202925, e-mail: eashvit@gmail.com

Received on: 16 May 2023; Accepted on: 22 June 2023; Published on: 25 November 2023

ABSTRACT

Background: Cigarette smoking is the leading source of oxidative species of free radicals, which causes an imbalance in the sperm’s homeostatic state during spermatogenesis. Telomere length is influenced by a variety of factors, including oxidative stress, obesity, infection, telomere uncapping, and autoimmune disorders.

Materials and methods: The study participants were divided into two groups—fertile nonsmokers (n = 85), who served as the control group, and infertile smokers (n = 85). The subjects chosen ranged in age from 26 to 39 years. After 48–72 hours of sexual abstinence by masturbation, semen samples were collected from each subject and allowed to stand at room temperature for 30 minutes for liquefaction. Following liquefaction, the samples were subjected to automated computer-assisted sperm analysis (CASA) to assess the quality of the sperm in accordance with World Health Organization (WHO) guidelines from 2010. The morphological characteristics and velocity parameters were evaluated using CASA, just like the major semen characteristics.

Results: The relative and absolute telomere length of sperm telomere loss was significantly affected by smoke toxicants in infertile subjects. Meanwhile, elevated 8-hydroxy-2’-deoxyguanosine (8-OhdG)/dG and reactive oxygen species (ROS) levels in infertile smokers were negatively correlated with absolute telomere length, and sperm morphology was directly related to damaged or telomere-shortened sperm. Furthermore, halo sperm acrosome status with reduced live sperm cells was discovered in infertile smokers.

Conclusion: Finally, our findings show that smoke toxicants directly or indirectly affect sperm cells by increasing oxidative species (ROS) and that sperm maturation, spermatogenesis, and sperm telomere shortening are all significant issues when sperm cells are subjected to oxidative stress, which causes deoxyribonucleic acid (DNA) integrity to be compromised.

Clinical significance: Male infertility, increased ROS, abortion, chromosomal abnormalities, defective sperm, and as a smoke toxicant apart from cancer, it also causes infertility in males.

How to cite this article: TB S, Rao K, Ranganathan P. The Effects of Cigarette Smoke-induced Biophysical ROS on Spermatozoa Heads and Telomere Shortening in Infertile Smokers: A Prospective Study. Int J Infertil Fetal Med 2023;14(3):109–116.

Source of support: Nil

Conflict of interest: Dr Kamini Rao is associated as Editor-in-Chief of this journal and this manuscript was subjected to this journal’s standard review procedures, with this peer review handled independently of the Editor-in-Chief and her research group.

Keywords: Acrosome, Cigarette smoking, Free radicals, Reactive oxygen species, Sperm telomere, 8-hydroxy-2’-deoxyguanosine

INTRODUCTION

Infertility is a couple’s inability to conceive a pregnancy after a year of regular unprotected sexual intercourse. Furthermore, “male infertility” refers to the physical inability to produce healthy sperm and achieve pregnancy with a healthy female after 1 year of abstinence.1 Male infertility is caused by a variety of factors.2 According to previous reports, among all the smoking and smoking-related health issues, there is an alarmingly high suspicion regarding male infertility.3 A total of 36.9% of the world’s population is addicted to tobacco and tobacco-related cigarette components. According to research, smoking causes nearly 40–50% of respiratory and cardiac problems.4 Meanwhile, the World Health Organization (WHO) reported that smoking is responsible for 15–30% of all reproductive problems.5,6 Cigarette smoking has now been identified as an attractive and intriguing habit in adolescents aged 15–19. Furthermore, the dominance of psychoactive drugs or the substantial presence of cigarette inhalation causes addiction.7 According to new research, cigarette smoking is not the only cause of the progression of various cancers and cardiovascular and pulmonary damage. It also plays a role in fertility issues in men who smoke more [as measured by the smoking index (SI)].8-10 Cigarette smoke contains over 2000 chemicals, including 40 toxic metal substances, and smokers are exposed to smoke.11 According to a recent study, cigarette smoking affects sperm motility, morphology, and homeostasis by causing damage to the sperm head and tail. This is a common misconception.12 Meanwhile, reproductive toxicants such as cadmium (Cd), lead, nicotine, cotinine, and cigarette smoke condition (CSC) have been identified.13,14 These toxins affect sperm cells either directly or indirectly by increasing oxidative species [reactive oxygen species (ROS)] during sperm production or sperm maturation.15,16 Telomeres are noncoding genes found in highly sealed regions of eukaryotic chromosomes that have been found to play a significant role in genome stabilization and protection.17 When a sperm cell is subjected to oxidative stress, telomere shortening arises as a strategy for losing deoxyribonucleic acid (DNA) integrity.18,19 Numerous factors, such as lifestyle, occupational, and socioeconomic status, generate free radicals, exacerbating the imbalance in sperm pathophysiology and resulting in sperm morphology and histological hindrance.1-3 According to Thilagavathi et al., a recent study, cigarette smoking causes oxidative stress, which causes sperm mitochondria to malfunction during spermatogenesis, resulting in short telomere length and infertility.4

The telomere length is critical for protein-mediated DNA repair mechanisms and apoptosis. The size of the telomere and its inadequacy in these proteins also normal apoptosis.5 Thus, telomeres play a significant role in chromosomal alignment and pairing, and the meiosis stage of germ cells is identified as a critical step.6 Also, telomere aids migration and spermatogenesis in male germ cells.7 Fertilization, synapsis, and cross-over depend on the V-shaped acrosome tip of the sperm head and the sperm telomere.8 Nevertheless, the acrosome cap damage was affected by various factors like oxidative stress,9 obesity,10 infection,11 telomeres uncapping and shortening, and autoimmune disorders.12 Cigarette smoking has become a popular pastime among adolescents and adults of reproductive age. CSC produces a large number of toxicants, which produce extreme free radicals that disrupt and damage sperm DNA through oxidative DNA damage by removing 8-hydroxy-2’-deoxyguanosine (8-OhdG), a sperm DNA damage marker (when seminal plasma releases a higher amount when compared to other toxicants, according to our previous study).13,14 As a result, the current research investigates the shortening of telomere length and acrosome loss caused by cigarette smoking caused by exposed metal hazards induced biophysical ROS status. A prospective study.

MATERIALS AND METHODS

Ethics

The Vellore Institute of Technology (VIT) institutional review board (IRB) approved this study (VIT/UHEC-3/NO.11) and set the study subjects with specific inclusion-exclusion criteria. Ages over 40, alcohol, chemotherapy, being on medication, sexually transmitted diseases-related diseases, and occupational and environmental exposure were all excluded. Similarly, study participants with a smoking history of at least 8 years (before enrolment in the study) were classified as smokers based on the SI unit. The human semen samples were collected with informed consent and maintained that their details were confidential.

Semen Collection and its Major Conventional Characteristics Examination by Computer-assisted Sperm Analysis (CASA) According to WHO (2010)

From January 2015 to January 2017, sperm samples were collected regularly from the International Institute of Reproductive Research in Health-Bangalore Assisted Conception Centre (IIRRH-BACC). Subjects had been experiencing infertility issues for at least 1 year. Although this study confirms that the infertile is a male partner with a healthy female partner, the study subjects were divided into two groups—group I, fertile nonsmokers (n = 85), who served as the control group, and group II, infertile smokers (n = 85). The subjects chosen were between the ages of 26 and 39. After 48–72 hours of sexual abstinence by masturbation, semen samples were collected from each subject and left at room temperature for 30 minutes to liquefy. Following liquefaction, the samples were subjected to automated CASA to determine the quality of the sperm (pH, volume, morphology, sperm count, total, and progressive motility) under WHO (2010)2 guidelines. Like the significant semen characteristics, the morphology and velocity parameters were assessed employing CASA following WHO guidelines.

Semen Separation

Following CASA, the semen samples were centrifuged at 1500 rpm for 30 minutes with Hank’s Balanced Salt Solution. The obtained sperm cells (pellet) were used to determine viability (HOST) acrosome status, while the remaining seminal plasma supernatant was used to determine individual trace elements and seminal ROS.

Seminal Calcium (Ca), Zinc (Zn), and Cd Estimation

Seminal plasma from semen was diluted 10-fold with double distilled water and measured for the trace elements Zn and Ca in study subjects using a flame atomic absorption spectrophotometer (PerkinElmer precisely Analyst 200) with a detection limit of 6.6 mg Zn/L, Zn chloride hydrate and 5.3 mg Ca/L, Ca chloride dihydrate, which are used as standards in this analysis. Approximately 1 mL of separated seminal fluid (supernatant) was double digested with an acid mixture of nitric acid (HNO3): perchloric acid at a 6:1 ratio and dissolved in 1 mL% HNO3, followed by Cd detection at 228.9 nm using a flame atomic absorption spectrophotometer (PerkinElmer precisely Analyst 200) with the respective standard (Cd chloride). The standard curve obtained will help determine the concentration of trace elements in seminal plasma.2

Biophysical ROS Estimation

Nitroblue tetrazolium (NBT) assay is used to measure the biophysical ROS level in fertile and infertile semen samples, which was established and standardized in our gene cloning and technology lab and includes the following steps briefly (under communicated):

  • Preparation of fertile and infertile samples along with respective working NBT standards.

  • Sperm cell lysis with lysis buffer followed by centrifugation at 500 gm.

  • Pellets washing with NBT solution followed by phosphate-buffered solution washing.

  • Stopped the reaction with the addition of 2 M potassium hydroxide and sodium hydroxide, measured final color development at 570–630 nm.

Sperm ROS was expressed in sperm ROS (×106).

Sperm Viability Analysis

Eosin-nigrosin sperm viability kit (L-7011) purchased from Molecular Probes, National Scientific Suppliers, Hi-Media, India. Live cell proportion was expressed in percentage.3

Acrosome Status

To know the smoke toxicant-induced acrosome damage was analyzed by acrosome halo assay in the infertile against control sperm cells was performed by the method of4 with few modifications for our lab conveniences. Briefly, the semen samples were diluted in phosphate buffered saline: D-glucose in the 1:20 ratio while equilibrated at room temperature for 30 minutes. After equilibration, 20 µL of diluted sperm cells were smeared gently on a gelatin-coated microscopic slide, evaporating the excess water. Then the slides were incubated at 37°C for 2 hours in a humid chamber, air-dried kept the drops under the microscope to check the percentage of acrosome halo in the sperm cell’s head. Acrosome halo in the sperm cells was expressed in percentage.

Sperm DNA Damage by 8-OHdG/dG Marker Analysis

For 8-OHdg oxidative damage marker analysis, the sperm DNA should be isolated first and then processed for 8-OHdG/dG marker analysis, so we followed the protocol from Selit et al.4 with few modifications due to our laboratory convenience. First, the semen/sperm DNA was isolated as per the procedure mentioned above against the 1 Kb DNA ladder to check the obtained isolated genomic DNA. The isolated DNA was allowed to review the sperm DNA integrity by the Gel Pre-Pro program analyzer. The remaining tris-ethylenediaminetetraacetic acid (EDTA) buffer dissolved isolated samples went for enzymatic DNA digestion with DNase I, nuclease P1, and alkaline phosphatase for 8-OH dg measurement through an high performance liquid chromatography system containing a Younglin make ACME 9000 gradient pump, C18 column with 250 × 4.6 mm of E95520, Kromasil syringe sample injector. The same Younglin packed 1050 ultraviolet detector (258 nm) connected with the electrode to monitor 8-OHdg. The mobile phase composition is 5% ethanol, 10 mM potassium chloride, 10 mM ammonium dihydrogen phosphate, and 1 mM EDTA with a pH of 4.8–5.0 (flow rate 1 mL/minute). With the standard dG and 8-OHdg, the unknown sample dG and 8-OHdg were calibrated and expressed in ng/mL.6

Sperm Telomere Length by Qualitative Polymerase Chain Reaction (PCR) Analysis

For telomere shortening analysis, the sperm DNA extraction done by the salting-out method, followed by quantitative real-time quantitative PCR, was used with 96-well plates filled with one nanogram of sperm DNA in PCR. The short telomere length was interpreted here in kbp of relative and absolute telomere length as per Darmishonnejad et al. and Rocca et al.6

Statistics

Results were represented here as mean ± standard error of the mean (SEM). IBM Statistical Package for the Social Sciences 21 statistical package and Graph pad Prism (version 6.0) were used for all statistical and correlation analyses. Two-way analysis of variance statistics (p < 0.0001; p < 0.001) were used for the significance of fertile and infertile subjects. The percentage of isolated sperm DNA and its integrity and abnormality was assessed by Gel Pro Program analyzer v 6.3.

RESULTS

Table 1 shows the primary conventional semen parameters and their calculated values, representing values as the mean and SEM. It was also measured employing age-influenced semen parameters and the duration of the smoking. Table 1 shows that compared to fertile subjects, the percentage of rapid progressive motility and normal sperm morphology was lower. Kruger’s strict morphology also confirmed it under WHO 2020 guidelines. Even though the infertile subject has a high sperm concentration, the cells are in a state of slow progressive motility due to a damaged head and tail, leading to the oligoasthenoteratospermic category, which is induced by cigarette smoking. Table 2 displays the velocity parameters of infertile subjects’ sperm cells. They were listed with their respective units. Curvilinear velocity (VCL) characterizes motility and mobility, and the individual’s fertility determines linearity. The current study demonstrates that infertile has a lower VCL and linearity than fertile ones.

Table 1: Statistical values of conventional semen parameters in fertile and infertile smokers and their comparison
Parameters Fertile (n= 85) Infertile Smokers (n= 85)
Age (years) 28 ± 4.62 34 ± 3.12
No. of cigarettes/day 12.93 ± 1.56
Duration of smoking (years) 12.49 ± 2.38
pH 7.411 ± 0.02 7.290 ± 0.03
Volume (mL) 2.839 ± 0.07 1.120 ± 0.39
Sperm count (millions/mL) 42.46 ± 0.74 8.20 ± 3.26
Motility% (total) 29.63 ± 5.74 7.20 ± 4.15
Motility% (rapid) 4.21 ± 0.40 2.31 ± 0.755
Motility% (slow) 25.30 ± 0.52 4.54 ± 2.24
% of morphology 37.31 ± 2.96 6.42 ± 2.06

Values are represented here as mean ± SEM

Table 2: Velocity parameters of sperm cells of infertile subjects
Velocity parameters Slow Medium Rapid Total
VCL (mm/s) 6.3 17.5 25.9 20.8
Straight line velocity (VSL) (mm/s) 2.3 6.0 10.8 7.4
Average path velocity (VAP) (mm/s) 4.0 11.0 16.5 13.0
Straightness (STR) (mm/s) 36.5 34.4 41.5 35.8
Straightness (STR) (%) 57.3 54.9 65.0 57.3
Wobble (WOB) (%) 63.6 62.5 63.9 62.5
Head displacement (ALH) (µm) 0.3 12 1.1

Values are represented here as mean ± SEM

Furthermore, velocity and linearity are essential in genetic inheritance and support sperm motility. The velocity parameters, on the other hand, influence the head and tail defects of normal morphology of semen characteristics. Furthermore, Table 3 stated that infertile subjects lose more head and tail and found midpiece defects and cytoplasmic droplets than fertile subjects. Following that, linearity was found to be negatively (p < 0.001 with R2 = −0.765) correlated with head and midpiece defects, and head defects (p < 0.001 with R = −0.0831) associated with linearity. Figure 1A–D shows that loss was also related to the number of cigarettes smoked per day (15–20/day), loss of sperm morphology (R2 = 0.914 and p < 0.0001), and loss of sperm counts (p < 0.001 and R2 = −0.894). It appears that linearity changes in velocity parameters influence the head and tail portions of sperm, resulting in rapid motility losses in infertile subjects compared to fertile subjects.

Table 3: Comparison of head, tail, midpiece, and cytoplasmic droplets in fertile and infertile subjects
Parameters Fertile (n= 85) Infertile (n = 85)
Cytoplasmic droplets (%) 2.83 ± 0.09 8.45 ± 0.319**
Granular debris (%) 0.85 ± 0.002 4.19 ± 0.220
Head defects (%) 12.92 ± 0.04 63.81 ± 0.697**
Head and midpiece defects (%) 2.93 ± 0.09 12.93 ± 0.184
Tail defects (%) 0.75 ± 0.0005 25.556 ± 0.230**

Values are represented here as mean ± SEM; **p < 0.05

Figs 1A to D: Sperm head, tail, and mid-piece defects and their correlation with semen parameters

Table 4 lists the essential trace elements Ca and Zn in seminal plasma. It demonstrates that Zn levels were significantly lower in infertile males than in fertile males and were negatively correlated with the number of cigarettes smoked (p < 0.001 and R = −0.835) when considering the age factor. At the same time, our previous study, Ranganathan et al.,8 clearly stated that Zn was influenced by Cd, was agreed with the present study, along with Ca also depleted by cigarette smoke. Table 4 also shows that infertile subjects had lower total seminal protein values than fertile subjects. Usually, lowering the protein level does not affect sperm characteristics. However, the current study finds that total protein concentrations in infertile subjects negatively correlate with seminal Cd (p < 0.001 and R = −0.0721). Furthermore, free radical elevation is higher in sterile sperm than in fertile (Table 4), and the free radical-influenced sperm visualized under the NBT stain is shown in Figure 2.

Table 4: Comparison of seminal trace elements, molecular marker vs smoke toxicant (Cd) in fertile and infertile subjects
Parameters Fertile (n= 85) Infertile (n= 85)
Seminal Zn (mg/mL) 2.43 ± 0.05* 0.08 ± 0.002**
Seminal Ca (mg/mL) 27.23 ± 1.34*** 9.19 ± o.89**
Seminal Cd (µg/L) 3.02 ± 0.24 9.82 ± 0.23***
Seminal protein (mg/mL) 22.51 ± 2.81** 8.32 ± 1.35**
Sperm ROS (× 106) 4.05 ± 0.005 25.35 ± 5.05***
8-OHdG/dG, (ng/mL) 0.672 ± 0.0003 9.45 ± 2.06**

Values are represented here mean ± SEM; **p < 0.05; furthermore, infertile smokers’ absolute telomere length (4.19 ± 0.71) was lower than in fertile nonsmokers’ (12.13 ± 0.91); infertile smokers’ relative telomere length was correlated (***p < 0.001 and 0.899) with the sperm DNA damage marker 8-OHdG/dG, confirming that sperm biophysical ROS influence affected total telomere length; finally, it reveals that smoking-induced ROS shortened total and relative telomere length in infertile smokers’ spermatozoa

Fig. 2: Nitroblue tetrazolium (NBT) assay stained infertile sperm cells

Figure 1A–D shows that the head, tail, and mid-piece damage influenced semen parameter changes in infertile smokers. It seems that sperm linearity and velocity parameters influence the head and tail portions of sperm, leading to more rapid motility losses in infertile subjects than in fertile subjects.

Cigarette smoke-exposed sperm cells of infertile subjects stained with NBT (light microscope 40×) and damaged cells were highlighted stained sperm cells.

Figure 3 shows the viable sperm cells with neat acrosome parts and the unviable or motile loss sperm cells with damaged acrosome parts (40×).

Figs 3A and B: Eosin-nigrosin stained sperm cells viability in fertile and infertile smoker’s subjects

Figure 3 depicts the live-cell population by eosin/nigrosin stain in fertile and infertile subjects; productive subjects had >50% of the neat live-cell population than infertile subjects. Figures 3B and C also show the eosin/nigrosin stained cells of fertile and infertile subjects and their live cells. A halo-spaced acrosome head was marked in a round space, and >60–80% of halo-spaced sperm cells were found in infertile sperm cells compared to fertile sperm cells, with <10% of halo-spaced sperm cells observed. Table 4 shows the results of a sperm DNA damage marker called 8-OHdG/dG used to measure cigarette smoke-induced sperm membrane damage. We discovered a higher level of 8-OHdG/dG in infertile smokers than in fertile smokers, and it was also positively correlated with seminal Cd and ROS levels. It was strongly suggested that cigarette smoke affects sperm DNA in infertile smokers. Finally, we wanted to confirm whether ROS-generated free radicals cause sperm telomere shortening, and the real-time PCR calculated telomere length result is shown in Figure 4, even though Figure 5 shows that the infertile have a relative telomere length of 0.68 ± 0.12 and the fertile has a relative telomere length of 1.24 ± 0.29.

Fig. 4: Acrosome status in infertile smokers

Figs 5A to C: Sperm telomere length and its comparison with 8-OHdG/dG

There is no halo space in marked sperm cells that shows the acrosome part of a sperm cell is damaged, and it concludes that sperm-egg fusion is a failure in conceive process.

Figures 5A and B show relative and absolute telomere length and its comparison in fertile and infertile subjects. Followed by a correlation was found with absolute telomere length vs 8-OHdG/dG, an oxidative damage marker, and it confirms that telomere shortening is due to biophysical ROS-triggered sperm membrane damage (8-OHdG/dG) in infertile smokers. Hence, cigarette smoking is also one of the reasons that damage the sperm tail of the telomere and causes infertility.

DISCUSSION

The purpose of this prospective study is to investigate the effects of cigarette smoking on fertility; increased seminal ROS, deteriorated sperm head, and acrosome changes with telomere shortening cause infertility. Most in vivo and in vitro findings confirm that ROS is a critical factor affecting spermatogenesis and steroidogenesis by interfering with the pathophysiology of homeostatic mechanisms.9-11 According to the literature, genotoxic, occupational, and environmental factors cause free radical ROS and impact fertility.13-40 Despite reports that cigarette smoking is the obvious factor that generates oxidative species by affecting spermatogenesis, physiology leads to poor motility and impaired morphology.16 Ranganathan et al.8 explained how free radicals from cigarette smoke and their released toxicants damaged, as well as acrosome cap tip and semenogelin, were discovered to have altered sperm integrity in our previous experiment. Traditional sperm parameters that determine male fertility include rapid motility, sperm count, and morphology tests. According to Ribas-Maynou et al.,24 age, radiation, and smoking increase oxidative stress affect the anti-ameliorative effect on telomeres, lead to sperm chromosome shortening, result in XY-linked recessive disorders during in vitro fertilization (IVF), or failure in the IVF procedure leads to unexplained infertility. According to Darmishonnejad et al.,7 age is not a predictive or influential factor in determining fertility for an individual, and it is dependent on personal habits that influence fertility. As a result of the current study, the results for semen parameters of rapid motility with poor morphology in infertile subjects were lower than in normal subjects. Following that, we looked at sperm velocity parameters as well as sperm head, tail, and granular defects. Chronic smoking generates a large amount of ROS, which reduces sperm curvilinear and straight-line velocity by >20–30%.15

Furthermore, sperm head, tail, and midpiece defects, as well as their percentage, were higher in infertile smokers than in fertile subjects. According to andrologists, the live cell population and quantity of sperm cells do not contribute to fertility if the sperm cell has morphological hindrance.14,16 In particular, the percentage of sperm cells and their live condition, without which we cannot declare the fertility ratio and the complete morphology and fast progressive movement, only confirm the subject fertility. As a result, these parameters of rapid movement, linearity, and VCL are required.

Meanwhile, the current study suggests that spermatozoa’s velocity and linearity parameters and head, tail, midpiece, and granular parts determine successful fertility. Toxins from smoking exert oxidative pressure on sperm DNA, causing acrosome cap damage and sperm membrane and integrity loss, according to research from the last three decades.17,18 Furthermore, many studies have found that free radicals are harmful to the pathophysiology of sperm cells, and smoking and its chemicals produce ROS, which disrupts normal fertility.19-22 The spermatozoa portion of the acrosome contains acrosin, which aids in the fusion of an egg’s gamete with the zona pellucida after ejaculation (capacitation process).23 Fertilization with a site is questionable if the acrosome portion of sperm is damaged during capacitation.

Furthermore, the specific interest in acrosome reaction cannot be treated because of homeostasis imbalance caused by ROS of oxidative stress.23-25 The acrosome portion of spermatozoa is sensitive and easily damaged, even at slightly higher temperatures. Mostafa describes how oxidative stress-induced free radicals from smoking cause chromosome abnormalities during spermatogenesis.26 Infertile smokers’ sperm also had halo-formed tubules, indicating poor acrosin formation and motility. According to findings, oxidative species produce ROS, a silent killer type that will remain silent until it takes over the sperm in infertile men rather than fertile men.17 According to the report, it is currently accepted that ROS interferes with fertility percentages by dominating sperm DNA integrity and damaging its DNA; in fact, it can modify the epigenetic profile of sperm cells.27 The current study discovered a high level of ROS in infertile smokers compared to fertile subjects. As a result, increased ROS in spermatozoon’s is expected to reduce the aerobic supply of oxygen to spermatogenesis, leading to immature sperm cells with slow progressive motility or telomere shortening. These sperm cells have an impact on men’s fertility in smokers. In young (30-year-old) smoker cancer patients admitted for treatment, they discovered the telomere, as mentioned above, shortening with offspring damage.

Sperm is a specially designed cell for fertilization, and its morphology is extremely sensitive to oxidative stress, which causes DNA damage.28 The risk is that a failure to fight oxidative species generates free electrons due to the defenseless function and silent nature of sperm biology design, leading to delayed recognition of ROS.29 Simply put, the quiet pack of sperm biology is inactive in terms of translation due to the absence or lack of cytosolic antioxidant enzymes (SOD, GPx) and nonenzyme antioxidants listed below (taurine, and vitamins C and E).30,31 In addition, polyunsaturated fatty acids and other lipid components are abundant in the sperm membrane. These lipids are peroxide-sensitive, resulting in increased free radicals during spermatogenesis and an effect on sperm DNA.32,33 So, sperm DNA damage in infertile subjects is not surprising because toxicants released by environmental and occupational factors are dangerous, and many studies have already reported sperm DNA damage. We have confirmed that 8-OHdG is a significant factor released in sperm DNA damage caused by smoking. Guz et al. said that 8-OHdG estimation confirmed that subjects with short DNA strands were highly infertile.34 As per the WHO manual, Vorilhon et al. demonstrated that 66% of men tested positive for sperm DNA damage by 8-OHdG.35 Simultaneously, 8-OHdG is a standard oxidative marker for sperm DNA damage, particularly in infertile smokers. The current study also confirmed ROS increased sperm membrane and DNA damage, as evidenced by 8-OHdG of sperm DNA damage. Meanwhile, 8-OHdG was negatively correlated with sperm morphology (p < 0.001), indicating that DNA strand damage occurs in infertile smokers.

However, in sperm, the telomere plays an essential role in maintaining chromosomal strength and its associated genomic integrity.36 Though various researchers and their research is focused on telomere length in tumorigeneses,37 cancer,38,39 age-related diseases,40 different receptors damaged signaling mechanism, cardiovascular diseases, heart failure, and reproductive biology.41 They discovered that telomere length damage causes embryo development loss via sperm-oocyte fusion after decades of research.37 The first study found that telomere length is unrelated to spermatozoa and its conventional parameters and DNA integrity. Turner and Hartshorne reported that telomere length is not required for male fertility; additionally, after fertilization occurs, the telomere length is reset during the embryo stage.42,43 However, in the current study, the causative agent of cigarette smoke increased metal toxicity, induced telomere shortening, and was critical. Telomere shortening may also be a significant cause of sperm DNA damage and sperm morphology against healthy male fertility, according to our findings. It was also influenced by increased biophysical ROS caused by metal toxicity caused by cigarette smoking. It also suggests that telomeres are linked to chromosomes and the nuclear membrane.

CONCLUSION

In conclusion, our study found that infertile smokers had shorter relative and absolute telomere lengths than fertile smokers due to the relatively high oxidative stress of biophysical ROS generated by chronic cigarette smoking. Nevertheless, our study confirms that smoke toxicants generate ROS that alter or damage sperm DNA integrity, as evidenced by a high elevation of the 8-OHdG/dG-oxidative damage marker, which is also one of the reasons for telomere shortening in infertile smokers.

CLinical Significance

Male infertility, increased ROS, abortion, chromosomal abnormalities, defective sperms, and as a smoke toxicant apart from cancer, it also causes infertility in males.

DECLARATIONS

Ethics Approval and Patient Consent to Participate

The VIT institutional review board (IRB) approved this study (VIT/UHEC-3/NO.11).

Patient Consent for Publication

Yes obtained and kept confidential.

Author Contributions

SB, supervision and validation; KAR, semen analysis and patient recruitment; PR, design, objective, analysis, results, and writing original article and proofreading.

Acknowledgments

The author (Parameswari Ranganathan) is very much thankful to the UGC DSKPDF (No. F.4-2/2006 (BSR)/BL/19-20/0246) fellowship. The authors are very much thankful to SBST, VIT Management for the instruments and infrastructure. Also, the authors are thankful to IIRRH-BACC, Bengaluru, Karnataka, India.

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