Oxidative DNA Damage in Male Germ Cells in Normozoospermic Infertile Men: A Case for Concern

Research Article

Austin J Reprod Med Infertil. 2015;2(3): 1017.

Oxidative DNA Damage in Male Germ Cells in Normozoospermic Infertile Men: A Case for Concern

Mishra SS¹, Kumar S¹, Singh G¹, Mohanty K¹, Vaid S¹, Malhotra N³, Singh N³, Kumar R² and Dada R¹*

1Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India

2Deptartment of Urology, All India Institute of Medical Sciences, New Delhi, India

3Deptartment of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi, India

*Corresponding author: Dada R, Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences (AIIMS), New Delhi-110029, India

Received: May 04, 2015; Accepted: June 15, 2015; Published: June 28, 2015


Background: Sperm DNA is highly susceptible to oxidative damage due to various endogenous/exogenous factors and deficient DNA damage detection and repair mechanisms. Telomeres are the favourable targets for oxidative damage. So, this study was planned to evaluate seminal oxidative stress, sperm DNA damage and sperm telomere length in normozoospermic infertile men.

Material and Method: The study included 30 infertile men and 30 controls. Reactive oxygen species (ROS) estimation was done by chemiluminesence method and 8-isoprostane (8-IP) and 8-hydroxy-2-deoxy-guanosine (8-OHdG) levels by ELISA. The average sperm telomere length was measured using quantitative Real Time PCR method. Sperm Chromatin Structure Assay (SCSA) was done by flow cytometry.

Results: Mean ROS (89.43±36.32 Vs. 15.04 ± 10.81 RLU/sec/million sperm; p=0.016) and 8-IP levels (698.7 ± 127.8 Vs. 278.02 ± 72.03 pg/ml; p=0.035) were significantly elevated in cases as compared to controls. 8−OHdG levels were higher in patients (25.33 ± 13.34 pg/ml) as compared to controls (15.34 ± 8.3 pg/ml) (p= 0.032). Mean DNA Fragmentation Index (DFI %) in patients was 40.31 ± 14.83, which was higher as compared to controls (24.43 ± 8.83) (p<0.0001). Mean telomere length was significantly (p=0.012) lower in patient group (0.737 ± 0.038) as compared to fertile control group (0.787 ± 0.054).

Conclusion: Normozoospermic infertile men may experience seminal oxidative stress, DNA damage and telomere shortening. Oxidative stress, loss of sperm DNA integrity, accumulation of mutagenic bases, telomere shortening may lead to genome hyper mutability and may be the underlying aetiology of male infertility. As majority of causes leading to oxidative stress are modifiable/ treatable (sedentary life style, excessive alcohol intake, smoking, excessive use of cell phone, acute or chronic infections, varicocele), management of oxidative stress may minimize free radical levels and subsequent oxidative damage to sperm DNA. Thus sperm with its limited antioxidant/DNA repair capacity may benefit by adopting a policy of prevention is better than cure.

Keywords: Oxidative stress; Sperm DNA damage; Telomere; Infertility


Infertility is a health problem affecting approximately 15% of all couples trying to conceive. It is now evident that in at least 50% of all cases, reduced semen quality is a factor contributing to the problem. In 20% of the couples, the main cause is solely male related, and in another 27%, both partners contribute to the problem. Male infertility can be the result of congenital or acquired urogenital abnormalities, infections of the genital tract, varicocele, endocrine disturbances, genetic or immunological factors. However, in at least 50% of the infertile men, no explanation to their reduced semen quality can be found. Recent studies have shown that alterations in the sperm molecular factors (paternal genome, mitochondrial DNA and transcripts) may be the underlying cause of infertility [1-3]. It is considered to be a complex lifestyle disease involving interplay of over 2000 genes and several lifestyle and environmental factors.

In past decade there has been a secular trend of decline in semen quality, concomitant with increase in male genitourinary abnormalities and testicular cancer. In recent years apart from an increase in number of cases with idiopathic azoospermia or oligozoospermia there has been a significant increase in numbers of cases with normal sperm parameters experiencing infertility and recurrent spontaneous abortions. Such cases with unexplained infertility need to be evaluated beyond the routine semen analysis as per WHO guidelines 1999. As about 1-3% of children each year are conceived via ART, the need for in depth analysis of sperm factors, such as free radical levels and assessment of integrity of sperm DNA is important. Use of sperm with oxidative DNA damage may impact health trajectory of child.

Studies have shown that sperm DNA integrity assessment can be applied in ART [4] in order to find the most effective treatment in a couple and counsel couple regarding management [5-7]. Semen quality is known to be influenced by a variety of lifestyle, environmental, and occupational factors. Although still much is unknown, the origin of sperm DNA damage is believed to be multifactorial where defects during spermatogenesis, abortive apoptosis and oxidative stress may be possible causes of a defective sperm DNA, but the damage is believed to be mainly oxidative [8].

Human sperm DNA is highly susceptible to oxidative damage. The most common types of DNA damage include single or double DNA strand breaks chemical modifications of DNA bases and inter and intra strand crosslinks. There are several factors that lead to sperm DNA damage. They include alterations in chromatin modelling during the process of spermiogenesis, abortive apoptosis, supra physiological levels of reactive oxygen species (ROS), and activation of caspases and endonucleases [7, 9]. Oxidative stress damages both mitochondrial and nuclear DNA. Mitochondrial DNA accumulates mutation and nuclear DNA shows fragmentation, denaturation and accumulation of highly mutagenic oxidized adducts like 8-hydroxy-2-deoxy guanosine (8-OHdG) [3]. Sperm cells are exposed to oxidative stress during transport and storage through the male reproductive tract and oxidative stress is believed to be major cause of DNA damage and sperm function [10]. Physiological levels of oxidative stress are essential for normal sperm function but levels of ROS exceeding the physiological limits cause sperm DNA damage. Advanced paternal age, storage in the epididymis, acute and chronic infections, chemotherapy, varicocele, sedentary life style and obesity are the risk factors associated with increased oxidative damage in sperm DNA. Unhealthy life style habits like smoking, alcohol consumption, sedentary and stressful lifestyle, occupational exposure to hazardous chemicals like lead, phthalates, organophosphorous compounds and environmental exposures to insecticides, pesticides, electromagnetic radiation, and high temperature exposure are other factors that increase ROS levels and consequently induce damage to the sperm DNA [11]. Presence of immature germ cells, morphologically abnormal spermatozoa and inflammatory cells in semen, varicocele are endogenous causes of excess free radical production [12]. The role of oxidative stress is now a well established aetiology of infertility and poor semen quality [11, 13, 14]. The important nucleic acid oxidation product includes 8-OHdG which is used as a oxidative DNA damage marker. 8-OHdG is a base adduct formed due to oxidation of guanine nucleotide found maximally in telomeric DNA in sperm nuclear periphery in the nucleohistone compartment. Telomeres are preferential targets of oxidative stress. Sperm with longer telomeres help to maintain critical telomere length during cleavage, which aids in maintaining species-specific telomere length in the newborn [15]. Studies on telomerase null mice have also shown that when these mice fertilized wild-type oocytes resulted in decreased fertilization and blastocyst formation, increased embryo fragmentation, and apoptosis [16]. Therefore, it may be possible that sperm with shortened telomere length may not respond to oocyte signals for pronucleus formation even if fertilization does occur. This may result in impaired cleavage, which may result in poor quality blastocyst, increased apoptosis, or failed implantation. Telomeric DNA is guanine rich and more susceptible to oxidative stress. If there is attrition in telomere or there is a double strand break in the ends of telomere, it recruits DNA repair enzymes or it activates DNA damage response pathways. Telomeres block unwanted DNA repair reactions and avoid detection by the DNA damage signaling pathway. So, telomeric damage or shortening may result in recognition of the chromosome ends as double strand breaks and recruitment of DNA damage response mechanisms [17]. Estimating the levels of 8-OHdG in the seminal plasma of the infertile patients is an efficient and reliable method to analyse oxidative damage to the sperm nucleus [18].

Recent studies from our lab have documented sperm DNA damage in infertile men with normal semen parameters and also in male partners of couples experiencing pre and post implantation losses [2]. Persistence of DNA damage may be due to limited DNA repair mechanisms Sperm a highly polarised, transcriptionally inert cell has only base excision repair (BER) but lacks APE and XRCC1. There may be altered or reduced expression of DNA damage detection and repair enzymes in infertile patients [14]. Kumar et al, 2015 documented that simple life style modification, adopting meditation and yoga practice for a minimum of 6 months can significantly reduce sperm DFI and levels of 8-OHdG. The group further documented that fathers of cases with non familial sporadic heritable retinoblastoma have elevated ROS levels and oxidative sperm DNA damage [19]. Majority of these men had advanced age (>35 yrs) and were smokers. As DNA damage is chiefly oxidative in nature and arises due to various life style factors, majority of which are modifiable, the integrity of sperm genome can be maintained by following a healthy lifestyle and this may have great impact on offspring health and can significantly reduce incidence of childhood morbidity and even children cancer.

Therefore in this study we have measured oxidative stress levels to understand aetiology of DNA damage, which was quantified by levels of ROS and 8 Isoprostane (8-IP) and oxidative DNA damage by estimating the levels of 8-OHdG. We have also measured telomere length in these normozoospermic infertile patients and controls to know the effect of oxidative stress on the telomere length. Since oxidative stress induced sperm DNA damage is chiefly produced by numerous endogenous and exogenous factors majority of which are modifiable, adoption of a healthy life style and prompt management of infections, inflammations and varicocele can minimize / prevent oxidative damage to sperm DNA.

Materials and Methods

The study was initiated after institutional ethical clearance and written informed consent from patient and controls. The female partners of all these cases were normal after complete clinical, gynecological, hormonal and radiological examination. Human ejaculates were obtained from 30 healthy volunteers of proven fertility from family planning OPD of Obstetrics and Gynecology department, and 30 male partners of couples experiencing primary infertility within age group of 18-45 years. A detailed family history was recorded in a pre-designed proforma. Cytogenetic analysis has been done for all cases to exclude cases with abnormal chromosome complement. All cases with recent history of fever, drug intake, any inflammatory disorders, and infections were excluded. Semen analysis was assessed by World Health Organization (1999) criteria. These patients after through clinical examination were referred from the Department of Gynecology and Obstetrics and Department of Urology, AIIMS, New Delhi. Statistical analysis was done using Student t test and Pearson correlation.

Semen analysis

Semen analysis was done twice at 2 weeks interval. Samples were collected after minimum of 48 hours and not longer than 7 days of sexual abstinence. The name of the patient, period of abstinence and time of collection were recorded on the form accompanying each semen analysis. Samples were collected in the privacy of room near the laboratory and were delivered to the laboratory within one hour after collection. The samples were obtained by masturbation and ejaculated into a clean, wide-mouthed glass or plastic container. The procedure for sample collection was explained to the patients and controls. Semen analysis was done as per WHO guidelines (1999).

ROS detection by chemiluminescence assay in neat semen

The ROS production in 400 μl of liquefied neat semen was measured after addition of 10 μl of 5 mM solution of luminol in DMSO (dimethylsulphoxide, Sigma Chemical Co.). A tube containing 10 μl of 5 mM luminol (5-amino-2,3-dihydro- 1,4-phthalazinedione, Sigma Chemical Co., St. Louis, MO, USA) solution in DMSO used as a blank. Chemiluminescence was measured in duplicate for 10 min using the Berthold detection luminometer (USA). Sample analysis was done along with blank, positive control (H2O2+PBS+Luminol) and negative control (PBS+luminol). Results were expressed in relative light units (RLU) per second and per 1 × 106 spermatozoa.

Sperm chromatin structure assay

Preparation of samples: The SCSA was performed according to the procedure described by Evenson et al, 2005 [20]. The aliquot from each ejaculate was thawed in a water bath at 37ºC for 30 seconds and diluted to a concentration of 2×106 sperm/mL in TNE buffer to a total of 200 mL in a falcon tube. Immediately, 0.4 mL of acid detergent solution (0.08 mol/L HCl, 0.15 mol/L NaCl, 0.1% v/v Triton X-100, pH 1.2) was added to the Falcon tube. After exactly 30 seconds, 1.2 mL of AO-staining solution (6 mg AO [chromatographicall purified: Polysciences, Inc. USA] per mL citrate buffer [0.037 mol/L citric acid, 0.126 mol/L Na2HPO4, 1.1 mmol/L EDTA disodium, 0.15 mol/L NaCl, pH 6.0]) was added. For every 6 test samples, 1 standard reference sample was analyzed to ensure instrument stability.

Flow cytometric measurements: The samples were analyzed using a FAC Scan flow cytometer (BD Biosciences), with an air cooled argon laser operated at 488 nm and a power of 15 mW. The green fluorescence (FL1) was collected through a 515-545 nm band pass filter, and the red fluorescence (FL3) was collected through a 650 nm long pass filter. The sheath/ sample was set on ‘‘low,’’ adjusted to a flow rate of 200 events/s when analyzing a sample containing 2 ×106 sperm/mL. Immediately after the addition of the AO staining solution, the sample was placed in the flow cytometer and run through the flow system. All the samples were assessed in duplicate at 1-month interval and the average was taken. After complete analysis of the sample, the X-mean (red fluorescence) and Y-mean (green fluorescence) values were recorded manually after selecting gate for sperm cells using FlowJo software (Oregon). Strict quality control was maintained throughout the experiment.

DFI Calculation: Post-acquisition DFI calculation was performed offline in the Flowjo software. The sperm cells are gated after excluding debris and high DNA stain ability (HDS) cells and mean values of red and green fluorescence were recorded manually. The DFI was then calculated by the formula, DFI = mean red fluorescence/ (mean red fluorescence + mean green fluorescence).

8 Isoprostane (8-IP) estimation: 8 Isoprostane levels were estimated by ELISA. The quantification was done by Cayman’s 8-IP EIA Kit. Protocol was followed as described by the manufacturer for the quantification.

8-Hydroxy-2-deoxy-Guanosine (8-OHdG) estimation: For this assay sperm DNA was isolated. Then it was digested by DNase1 and Alkaline phosphatase. The quantification of 8-OH-dG was achieved by using Cayman’s 8-OHdG EIA kit. Protocol was followed essentially as described by the manufacturer for the quantification of 8-OH-dG (Promega).

Telomere length estimation

Real time PCR: Sperm telomere length was determined from the sperm DNA by a quantitative real-time PCR-based method as described elsewhere [15]. Briefly, the relative mean telomere length was determined by comparing the value from absolute quantification of telomere DNA with a single copy reference gene, 36B4 (T/S ratio). These two assays were carried out as separate reactions on separate plates maintaining the sample positions between the two plates. Amplification signals were quantified by the standard curve method using a DNA template series (100 ng, 10 ng, 1 ng, 0.1 ng, 0.01 ng/microlitre) on every plate. All randomized DNA samples (20 ng) and standard dilution was processed as triplicates on 96-well plates using Bio-Rad CFX 96 (Her- cules, CA, USA). The purpose of the standard curve was to assess and compensate for inter-plate variations in PCR efficiency. Amplification of the telomeric repeat region was expressed relative to amplification of 36B4, a single copy gene (SCG) encoding acidic ribosomal phosphor protein located on chromosome 12. Real time kinetic quantitative PCR determines, for each sample well, the Ct, i.e., the fractional cycle number at which the well’s accumulating fluorescence crosses a set threshold that is several standard deviations above baseline fluorescence. A plot of Ct versus log (amount of input target DNA) is linear, allowing simple relative quantisation of unknowns in comparison to a standard curve derived from amplification, in the same plate, of serial dilutions of a known reference DNA sample. For this study, telomere (T) PCRs and SCG PCRs were always performed in separate 96-wellplates.

Statistical analysis: Pearson (χ2/Fisher’s exact test) was applied to make a comparison between two groups. P-values less than 0.05 were considered as significant. All tests were done using STATA 11.2 software for windows.


The sperm parameters like sperm count (p=0.026) and forward motility (p=0.0002) were significantly lower in infertile men compared to controls and no significant difference in the seminal volume and pH was observed between infertile men and controls. Out of 30 cases, 22 men (73.33%) had normal semen parameters as per WHO 1999 guidelines.

ROS levels

The seminal ROS level (RLU/sec/million sperm) were significantly higher (p=0.016) in infertile men (89.43±36.32 RLU/sec/million sperm) as compared to fertile controls (15.04 ± 10.81 RLU/sec/ million sperm). Out of 30 cases 17 cases (56.67%) had ROS > 22 RLU/ sec/million sperm and 13 cases (43.33%) had < 22 RLU/sec/million sperm, out of 30 controls 21 controls (70%) had ROS <22RLU/sec/ million sperm and 9 (30%) had > 22RLU/sec/million sperm (Figure 1).