Oxidative stress in seminal plasma and its relation to fertility potential of human male subjects

Pranjali Parikshit Muley; Parikshit Ashok Muley

doi:10.4103/jdmimsu.jdmimsu_110_20

ORIGINAL ARTICLE

Year : 2020 | Volume : 15 | Issue : 2 | Page : 172-175

Oxidative stress in seminal plasma and its relation to fertility potential of human male subjects

Pranjali Parikshit Muley, Parikshit Ashok Muley
Department of Physiology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi (Meghe), Wardha, Maharashtra, India

Date of Submission	13-Apr-2020
Date of Decision	22-Apr-2020
Date of Acceptance	27-Apr-2020
Date of Web Publication	21-Dec-2020

Correspondence Address:
Dr. Parikshit Ashok Muley
Plot No. 20, New Sneh Nagar, Wardha Road, Behind Laxmi-Narayan Hall, Nagpur - 440 015, Maharashtra
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jdmimsu.jdmimsu_110_20

Abstract

Background: There is growing evidence that damage to spermatozoa by reactive oxygen species (ROS) plays a key role in male infertility. Human spermatozoa are susceptible to ROS as their plasma membrane contains abundant polyunsaturated fatty acid which can undergo lipid peroxidation which may cause damage to the sperms. Lipid peroxidation of sperm membrane is an important mechanism of this ROS-induced sperm damage leading to infertility. The present study was undertaken using malondialdehyde (MDA) for assessing oxidative stress which is an end product of lipid peroxidation. Aim and Objectives: The aim of this study was to evaluate the oxidative stress by estimating MDA (marker of lipid peroxidation) in seminal plasma of different groups of individuals and to correlate the levels with different seminogram parameters. Materials and Methods: Semen samples were obtained and analyzed for routine seminogram parameters from 150 male partners between ages 20 and 58 years of infertile couples attending reproductive biology unit. The patients were grouped into abnormal ejaculate (asthenoteratozoospermic ORIGINAL ARTICLE, oligoasthenoteratozoospermic, and azoospermic) and normal ejaculate groups. Oxidative stress was measured spectrophotometrically by estimating MDA. Results: Higher MDA levels of seminal plasma were observed in the AT and azoospermic groups as compared to normozoospermics though the difference was not statistically different. A significant rise in MDA levels was observed in the oligoasthenoteratozoospermic group. Seminogram parameters were found to be negatively correlated with seminal MDA level. Conclusion: Increased seminal plasma lipid peroxidation is likely to have an association with poor semen quality. Hence, MDA may be used as one of the biomarkers for assessing oxidative stress on sperm.

Keywords: Lipid peroxidation, male infertility, malondialdehyde, oxidative stress, reactive oxygen species, seminal plasma

How to cite this article:
Muley PP, Muley PA. Oxidative stress in seminal plasma and its relation to fertility potential of human male subjects. J Datta Meghe Inst Med Sci Univ 2020;15:172-5

How to cite this URL:
Muley PP, Muley PA. Oxidative stress in seminal plasma and its relation to fertility potential of human male subjects. J Datta Meghe Inst Med Sci Univ [serial online] 2020 [cited 2021 Jan 26];15:172-5. Available from: http://www.journaldmims.com/text.asp?2020/15/2/172/304232

Introduction

Oxidative stress occurs when the balance between the production of reactive oxygen species (ROS) and antioxidant capacity of any system is distorted. ROS are highly reactive oxidizing agents belonging to class of free radicals containing one or more unpaired electrons which are continuously generated through various metabolic and pathologic processes.^[1]

Human spermatozoa are susceptible to ROS as their plasma membrane contains polyunsaturated fatty acid in abundance which can undergo lipid peroxidation after getting attacked by ROS which can grossly damage the sperms. Sperms also lack cytoplasm to generate preventive and repair mechanisms against ROS.^[2] Lipid peroxidation of sperm membrane is the mechanism of ROS-induced sperm damage which leads to infertility.^[3]

The exposure of human spermatozoa to ROS can cause oxidative stress which can result in peroxidative damage to the sperm plasma membrane and DNA damage to mitochondrial and nuclear genomes.^[4] One of the important markers of oxidative stress is malondialdehyde (MDA) which is an end product of lipid peroxidation. A high level of MDA represents a high level of lipid peroxidation rate which may cause changes in sperm and diminished fertility.^[5] Both spermatozoa and seminal plasma possess an antioxidant system capable of counteracting the harmful effects of ROS.^[6],[7]

The aim of the present study was to find the oxidative stress of seminal plasma in individuals with impaired fertility potential and to investigate its significance with different seminogram parameters.

Materials and Methods

The semen samples were obtained from 150 male individuals of infertile couples of age 20–58 years. Participants with hydrocoele, undescended testes, varicocele or any history of surgical intervention in the genitourinary tract, drug history like cancer chemotherapy, nitrofurantoin, niridazole, colchicine, or any hormonal preparation which may affect the spermatogenesis were excluded from the study after explaining the purpose of study, the procedure involved, and the confidentiality of data; the informed written consent was obtained from all the participants.

The participant was asked to observe 3 days of abstinence, and semen samples were taken on the 4^th day. Semen samples were collected by masturbation. After complete liquefaction at room temperature, each sample was tested.

Routine semen analysis

Routine semen analysis was done by SQA II C-P (Sperm Quality Analyzer) (Medical Electronic System Ltd., Israel) for sperm concentration (millions/ml), sperm motility, and sperm morphology. According to the World Health Organization (WHO) guideline, participants were grouped into three categories such as normozoospermic, oligozoospermic, asthenozoospermic, and azoospermic.^[8]

In this study, the samples collected from each group were as follows: normozoospermics – 80, asthenoteratozoospermics (AT) – 25, oligoasthenoteratozoospermics (OAT) – 26, and azoospermics – 19.

Estimation of malondialdehyde

After evaluation of physical parameters, 1 ml of semen sample was centrifuged at 3000 rotations/min for 10 min. Then, without disturbing pellet at the bottom, supernatant plasma was taken for estimation of MDA by trichloroacetic acid (TCA) and thiobarbituric acid (TBA) method (a modified procedure by Satoh).^[9]

Principle

The assay utilizes TBA and is based on acid-catalyzed decomposition of lipid hydroperoxides to MDA, which reacts with TBA to form a chromogen evaluated spectrophotometrically at 530 nm.

Reagents

TCA 20%, sulfuric acid (0.05 M), TBA (0.67%), and n-butyl alcohol (butanol) are used in this study.

Standard solution

MDA-bis-dimethyl acetal was dissolved in 0.05 M sulfuric acid to prepare 10 μM solution. This 10 μM solution was further diluted to obtain standard MDA of different concentrations such as 1 nmol/ml, 2 nmol/ml, 3 nmol/ml, 4 nmol/ml, and 5 nmol/ml.

Procedure

To 0.5 ml each of plasma and MDA standards, 2.5 ml of 20% TCA and 1 ml of TBA were added and vortexed. Then, the mixture was boiled in hot water bath for 30 min. The cold-water bath was used for cooling. After cooling, the resultant chromogen was extracted with 4 ml of n-butyl alcohol. The separation of organic phase was done by centrifugation at 3000 r. p. m. for 10 min.

The absorbance of butyl alcohol extract of standards and samples was measured in a spectrophotometer at 530 nm against butanol as a blank. The standard curve was plotted, and the concentration of total MDA in the sample was calculated and expressed as MDA in nmol/ml from the curve.

Statistical analysis

Statistical analysis of the data was carried out with SPSS, version 17 (Chicago, Illinois, USA). Data were reported as mean ± standard deviation. P < 0.05 was taken as statistically significant. Correlation between various parameters was evaluated using Spearman's rank correlation coefficients.

Ethical Approval

Ethical approval for this study (DMIMS(DU)/IEC/2019-20/2055.) was provided by the Ethical Committee of Datta Meghe Institute of Medical Sciences (Deemed to be University) on 30^th July 2019.

Results

The result of this study [Table 1 and [Figure 1] shows higher MDA levels of seminal plasma (in nmol/ml) in the AT (2.11 ± 0.66) and azoospermic (2.00 ± 0.81) groups as compared to normozoospermics (1.83 ± 0.56), but the difference was not statistically different. A significant rise in MDA levels was observed in the oligoasthenoteratozoospermic group (2.56 ± 0.80). [Figure 2], [Figure 3], [Figure 4] show a negative correlation between MDA and sperm parameters.

Figure 1: Malondialdehyde levels in seminal plasma of different groups (μmol/L) (Normo-normozoospermics, OAT oligoasthenoteratozoospermics, AT-asthenoteratozoospermics, and Azoo-azoospermics)

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Figure 2: Correlation between sperm concentration and seminal malondialdehyde

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Figure 3: Correlation between sperm motility and seminal malondialdehyde

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Figure 4: Correlation between sperm morphology and seminal malondialdehyde

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Table 1: Different physical parameters and malondialdehyde levels of semen in study group

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Discussion

In our present study, we obtained considerably elevated values of MDA which is an important marker of oxidative stress in the seminal plasma of the abnormal group (including oligoasthenoteratozoospermics, asthenoteratozoospermics, and azoospermics) than the normal group.

These findings were similar to the finding of Hsieh et al., Kobayashi et al., and Athayde et al.^[10],[11] Similarly, an elevated level of MDA was also noted in oligoasthenoteratozoospermia in a study conducted by Fraczek et al., 2001.^[12]

A prospective study demonstrated that men with high levels of ROS generation had less chance of initiating a pregnancy compared with low ROS.^[13] The ROS can cause an increase in DNA fragmentation, and pretreatment with antioxidants that dispose, scavenge, and suppress the formation of ROS can reduce DNA damage.^[14]

The presence of seminal oxidative stress in infertile males suggests its importance in pathophysiology via several mechanisms acting in synergism which can affect sperm characteristic and functional capacity.^[15] Human spermatozoa have an abundance of unsaturated lipid which is necessary to create the membrane fluidity required for the membrane fusion events of fertilization such as acrosomal exocytosis and sperm-oocyte fusion. Such a high abundance of unsaturated lipid makes spermatozoa vulnerable to oxidative injury.

Spermatozoa can become dysfunctional by lipid peroxidation as it leads to loss of membrane integrity which causes increased cell permeability, enzyme inactivation, and structural damage to DNA and cell death.Therefore, measurement of lipid peroxidation end products such as MDA and 4-hydroxyl generates a significant amount of information about the sperm population under study.

Such oxidative damage to spermatozoa membrane resulted in impairment of sperm concentration, sperm motility, and sperm morphology which is observed in results by a negative correlation between seminal MDA level and sperm concentration, motility, and morphology.

Decreased sperm count could be attributed to extended exposure of the seminiferous tubule to high level of ROS. ROS damage this tubule leading to testicular atrophy, decrease of gamete production, motility loss and DNA damage to matured sperm.^[13] The ROS affects the sperm motility due to damage to flagellum and axonemal structures of the tail of spermatozoa and causes sperm immobilization. Hence, it was revealed from our study that high lipid peroxidation is linked with overall poor sperm quality and affects the fertility of an individual.^{[14],[15],[16],[17],[18],[19],[20],[21],[22]}

Conclusion

The present study concluded that a high rate of lipid peroxidation is likely to have some association with poor semen quality. The identification of adequate molecular markers to diagnose a semen sample, in conjugation with the design of appropriate therapeutic strategies to combat defects and deficiencies, is decisive for resolving male factor problem. Among these, oxidative stress markers are relevant factors in male infertility, since they play a significant role in sperm physiology. A fine balance of these markers could modify the reproductive success. It is now observed that ROS-induced lipid peroxidation causes significant sperm membrane damage and influences sperm motility and morphology. It is quite probable that such deleterious effect may account for some cases of male infertility and evaluation of ROS and peroxidation parameters may be a part of infertile male workup in the near future. The negative correlation of sperm parameters with MDA indicates oxidative stress that adversely affects male fertility. Evaluation of MDA can be used for the diagnosis and prognosis of male infertility.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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