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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 25  |  Issue : 2  |  Page : 94-99

Assessment of seminal biomarker of lipid peroxidation among male partners of infertile couples at the university of ilorin teaching hospital, Nigeria


1 Department of Pathology, Federal Medical Centre, Abeokuta, Ogun State, Nigeria
2 Department of Chemical Pathology, University of Ilorin, Kwara State, Nigeria

Date of Web Publication19-Jul-2018

Correspondence Address:
Olatunbosun Waliu Oladosu
Department of Pathology, Federal Medical Centre, Idi-Aba, Abeokuta, Ogun State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/npmj.npmj_51_18

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  Abstract 

Background: Seminal fluid analysis (SFA) is the most important investigation of infertile males. It is however limited in revealing the aetiologies of the various spermatozoa abnormalities observed on microscopy. Increasing prevalence of male infertility and the challenges of diagnosis require biochemical investigations, such as assay of seminal malondialdehyde (MDA), a marker of sperm peroxidation and to support SFA in investigation of infertile males. Aims: The aim of this study was to determine the seminal level of biomarker of lipid peroxidation by measuring seminal MDA levels among infertile males in Ilorin. Settings and Design: This was a descriptive cross-sectional study. Subjects and Methods: One hundred and thirty infertile males served as patients and 50 fertile males as controls. Seminal MDA was assayed using ELISA method. Statistical Analysis Used: Statistical Package for the Social Sciences (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA) was used. Normally distributed data were expressed as mean ± standard deviation. Results: The mean age of the patients who were mainly civil servants was 38.6 ± 6.6 years compared to 37.0 ± 5.8 years among controls. Seminal MDA was significantly higher among infertile males in this study (P < 0.05) indicating higher degree of lipid peroxidation in their semen. Seminal MDA concentrations were found to be significantly higher among infertile males with a history of alcohol consumption (P < 0.05), cigarette smoking (P < 0.001) and sexually transmitted infections (P < 0.001) when compared to control with similar history. Conclusions: There is significant lipid peroxidation of spermatozoa among infertile males with lifestyle playing a significant role. Evaluation of infertile males should include assessment of seminal MDA. Prophylactic administration of antioxidants to this group of patients may be beneficial.

Keywords: Infertility, male, malondialdehyde, peroxidation, spermatozoa


How to cite this article:
Oladosu OW, Biliaminu SA, Abdulazeez IM, Nwadike VU, Yusuff JdO, Okesina AB. Assessment of seminal biomarker of lipid peroxidation among male partners of infertile couples at the university of ilorin teaching hospital, Nigeria. Niger Postgrad Med J 2018;25:94-9

How to cite this URL:
Oladosu OW, Biliaminu SA, Abdulazeez IM, Nwadike VU, Yusuff JdO, Okesina AB. Assessment of seminal biomarker of lipid peroxidation among male partners of infertile couples at the university of ilorin teaching hospital, Nigeria. Niger Postgrad Med J [serial online] 2018 [cited 2018 Dec 14];25:94-9. Available from: http://www.npmj.org/text.asp?2018/25/2/94/237087


  Introduction Top


Male infertility refers to a man's inability to achieve pregnancy in a fertile female after regular unprotected sexual intercourse for at least 1 year. Male factor accounts for 40%–50%[1] of infertility among couples, female factor also accounts for 40%–50%, while 10% of the causes of infertility are regarded as idiopathic. The prevalence of infertility among men globally is about 7%.[2] Male infertility is commonly due to deficiencies in semen quantity and quality and both characteristics are used as surrogate measure of male fecundity.

The activities of the spermatozoa are dependent on adenosine triphosphate generated from aerobic metabolism, with concurrent generation of free oxidised radicals including hydrogen peroxide and superoxide radicals.[3],[4] The peroxidation of lipid-rich spermatozoa by these oxidised free radicals causes damage to its membranes, manifesting as disorders of morphology, sperm count, viability and motility.[5] High concentration of reactive oxygen species (ROS), which are either due to excess production or due to reduced production of naturally occurring antioxidants, has been found in infertile males.[6] The degree of lipid peroxidation can be determined by assaying for malondialdehyde (MDA) in the semen.[7] MDA is a product of lipid peroxidation by ROS and has sensitivity, specificity, positive and negative prognostic values in semen as a marker of oxidative stress more than those of Vitamins C and E.[8] MDA is therefore regarded as a better biomarker of lipid peroxidation in the semen.[9]

The impairments of seminal antioxidants and lipid peroxidation status play important roles in the aetiopathogenesis and pathophysiology of male infertility and considered one of the major causes of male infertility.[10],[11],[12],[13] However, these ROS under physiological conditions are produced in small amount and are required for spermatozoa capacitation, acrosome reaction and fertilisation.[14] In situ ations where the ROS concentration is high enough to overwhelm the antioxidant system, either due to excessive production of oxidative stress or underproduction of antioxidants, it has deleterious effects on both the structural and functional integrity of the spermatozoa.

ROS are species that contain one or more unpaired electrons, singly occupying an atomic or molecular orbital. They are therefore unstable and highly reactive.[15] They interfere with normal sperm function by causing lipid peroxidation of the polyunsaturated fatty acid-rich spermatozoa membrane and DNA fragmentation of nucleic acids. These cause various impairments of the sperm cells, such as membrane damage, decreased motility, reduced survival, deranged metabolic functions and alterations in signal transduction mechanisms, which all affect fertility. Oxidative stress damage to spermatozoa also predisposes the semen to leucocyte infiltration and macrophage damage of the defective spermatozoa.[16] Defective sperm function has been found to be the single, most common and defined cause of human infertility. Therefore, evaluating the semen of all suspected cases of male infertility, for biomarkers of lipid peroxidation is now being advocated.[12] MDA is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells and form covalent protein adducts referred to as advanced lipoxidation end products, in analogy to advanced glycation end products.[17]

The objective of this study is to determine the semen level of MDA among male partners of infertile couples and controls. Ten to fifteen percent (10%–15%) of couples are considered infertile and male causes of infertility accounts for 40%–50% of infertility [1] among couples. Majority of the studies on infertility focused more on female infertility with little attention on male infertility, more so in the developing countries, including Nigeria. Seminal fluid analysis (SFA) is the most important investigation carried out on all suspected cases of male infertility. However, attention is paid mostly to microscopy component of SFA. Biochemical analysis of semen is often excluded despite its benefits.


  Subjects and Methods Top


Ethical approval for this study was obtained from the University of Ilorin Teaching Hospital Ethical Research Committee on 24 February 2016 with protocol number: NHREC/02/05/2010. The study was conducted at the Department of Chemical Pathology and Immunology, University of Ilorin Teaching Hospital, Ilorin, Kwara State, Northcentral, Nigeria. The hospital receives referrals of infertile male patients from Kwara State and other neighbouring states. The Hospital has a General Outpatient Department which serves as a referral point for infertile couples, Specialist Gynaecological and Urology clinics attending to female and male infertility cases, respectively, as well as an Assisted Reproductive Technology Unit (ARTU). The study population included male partners of infertile couples who were requested to carry out SFA at the microbiology laboratory in UITH and at the ARTU between 01 June 2016 and 01 February 2017. It was a descriptive cross-sectional study of consecutive infertile male patients. Sampling was conducted using the convenient recruitment method. One hundred and thirty consenting male partners of infertile couples were selected as the patients and fifty fertile males as controls.

The minimum sample size required for the study was estimated using the Fisher's formula:[18]

Given as:

n = The desired minimum sample size.

z = The standard normal deviation usually set at 1.96 which corresponds to 95% confidence interval.

p = The prevalence of male infertility in the target population from the previous study. This was estimated to be 8.45%.[19]

q = The proportion in the target population who do not have a particular characteristic, i.e.

q = 1 – P = 1–0.0845 = 0.9155,

d = Tolerable margin of error, an observed difference of 5% was taken as being significant.

Therefore,

Attrition rate of 10% was assumed (which is ≈ 12). Thus, minimum sample size was taken to be 130.

Patients with abnormalities such as cryptorchidism (undescended testes), atrophic testes and aspermia were excluded from this study. Written consent for inclusion in the study was obtained after explanation of the study and the procedure before samples were taken from the patients.

Semen samples were collected by masturbation into a sterile, wide-mouthed container, after at least 72 h (3–4 days) of sexual abstinence. Samples were allowed to liquefy at room temperature (20°C–25°C) for at least 45 min. After liquefaction, samples were centrifuged for 20 min at 3500 revolutions/min. The supernatants were collected and stored at −80°C (≤6 months) to avoid the loss of bioactivity and contamination.

The Elabscience MDA ELISA kits (Lot No.-AK0017APR18059 and Manufacturing date-18 April 2017) were used. It is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of MDA in biological sample like semen. The principle is based on competitive ELISA as the method. The microtiter plate provided in this kit has been precoated with MDA. During the reaction, MDA in the sample or standard competes with a fixed amount of MDA on the solid-phase supporter for sites on the Biotinylated Detection Ab specific to MDA. Excess conjugate and unbound sample or standard are washed from the plate and Avidin conjugated to horseradish peroxidase is added to each microplate well and incubated. Then, a TMB substrate solution was added to each well. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the colour change was measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of MDA in the samples was determined by comparing the optical density of the samples to the standard curve.

Statistical analysis was done with the Statistical Package for the Social Sciences (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA). Normally distributed data were expressed as mean ± standard deviation, while non-normally distributed data were expressed as median and interquartile range or transformed logarithmically and categorical variables were reported as percentages.


  Results Top


A total of 180 participants comprising of 130 male partners of infertile couples and 50 age-matched fertile males were recruited for this study. The mean age of the patients was 38.6 ± 6.6 years, while the mean age of controls was 37.0 ± 5.8 years. The patients and controls were mainly within the age group of 31–40 years, with 73 (56.2%) and 28 (56.0%), respectively [Figure 1]. The patients and controls were mainly civil servants consisting 63 (48.5%) and 28 (56%) of male partners of infertile couples and controls, respectively [Figure 2].
Figure 1: Age group distribution among male partners of infertile couples and controls

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Figure 2: Frequencies of occupation among male partners of infertile couples and controls

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Alcohol consumption, cigarette smoking and previous history of sexually transmitted infections (STIs) in patients and controls are shown in [Table 1]. No statistically significant differences observed between patients and controls.
Table 1: Relationship between alcohol use, cigarette smoking and sexually transmitted infections and infertility using Chi-square test test

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The mean seminal concentration of MDA in patients was 238.7 ± 2.4 ng/ml compared to 172.0 ± 1.8 ng/ml in control, and this was statistically significant (P< 0.05) as shown in [Table 2]. The mean seminal MDA among male partners of infertile couples, who consume alcohol, was significantly higher than among controls who consume alcohol (341.6 ± 2.3 ng/ml vs. 235.7 ± 1.8, P < 0.001). Furthermore, the mean seminal MDA concentration among male partners of infertile couples who consume alcohol was statistically significantly higher than the mean seminal MDA levels among infertile male non-alcoholics (341.6 ± 2.3 ng/ml vs. 217.4 ± 2.4 ng/ml, P < 0.05), as shown in [Table 3].
Table 2: Seminal level of malondialdehyde in male partners of infertile couples and controls using student t-test

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Table 3: Comparison of seminal malondialdehyde levels between social and clinical characteristics of patients and controls using Student's t-test

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The mean seminal MDA concentration among male partners of infertile couples who smoke was also significantly higher than among control smokers (506.8 ± 2.5 ng/ml vs. 148.4 ± 1.3 ng/ml, P < 0.001). The mean seminal MDA concentration among male partners of infertile couples who smoke was 506.8 ± 2.5 ng/ml and was significantly higher than 213.2 ± 2.3 ng/ml among non-smoking male partners of infertile couples (P< 0.001) [Table 3].

Furthermore, male partners of infertile couples with a history of STIs had mean seminal MDA concentration significantly higher than controls with history STIs (503.5 ± 1.9 ng/ml vs. 383.9 ± 1.1 ng/ml, P < 0.001). The mean serum MDA concentration among male partners of infertile couples with history of STIs was also significantly higher than the concentration among male partners of infertile couples with no history of STIs (503.5 ± 2.0 ng/ml vs. 213.4 ± 2.4 ng/ml, P < 0.001), as shown in [Table 3] below.


  Discussion Top


The mean ages of the patients and controls in this study were 38.6 ± 6.6 years and 37.0 ± 5.8 years, respectively, and no statistically significant difference between the mean ages in the two groups. This is similar to 35.7 years in a similar study in Northeastern Nigeria.[20] It was, however, lower than 43.7 ± 1.5 years in another similar study.[19] The relatively older mean age group in that study was attributed to delay in presentation by the patients at the health facility. It may also mean that there is now more awareness among patients about the benefits of early presentation at the hospitals.

Majority of the patients and controls (48.5% and 56%, respectively) were civil servants. This may be due to the study site being located at the state capital, where majority of the civil servants reside, which enhance accessibility to health-care services and the fact that educated patients tend to present more to hospitals than illiterates.

The mean seminal concentration of MDA among male partners of infertile couples in this study is significantly higher than among the fertile controls (P< 0.05). This is similar to previous studies carried out [13],[21],[22],[23] where MDA was found to be higher in the semen of infertile men compared to fertile men. Research has shown that there was a negative correlation between seminal MDA levels and the normal sperm motility and morphology,[24] so they suggested damaging effect of free radicals on sperm membrane integrity while another study also showed significant association between seminal MDA level and abnormal sperm morphology, decrease semen total antioxidant capacity and weak sperm motility.[25] Seminal MDA levels were also shown infertile males to be significantly lower than in asthenoteratospermic and oligoasthenoteratospermic patients in another study.[26]

Consumption of alcohol, cigarette smoking as well as STIs, three known sources of ROS, were found in this study to be higher among male partners of infertile couples than controls, though no statistically significant differences was observed. Various studies have implicated the trio in the aetiopathogenesis of male infertility.

The mean seminal MDA concentration among male partners of infertile couples who consumed alcohol was significantly higher than among male partners of infertile couples who are non-alcoholics (341.6 ± 2.3 ng/ml vs. 217.4 ± 2.4 ng/ml P < 0.05) in this study and the mean seminal MDA concentration was found to also be higher among male partners of infertile couples who are alcoholics than fertile male alcoholics with statistically significant difference in their means (341.6 ± 2.3 ng/ml vs. 235.7 ± 1.8, P < 0.05). This is similar to findings in a study which found that alcohol consumption correlated with high extent of lipid peroxidation and increased formation of MDA.[27] Another study also found that alcohol intake affects male infertility by reducing semen volume, increasing abnormality of sperm morphology and by causing leucocytospermia.[28] Cessation of alcohol intake, however, may cause a partial reversal of these parameters. A study also found that men with a history of heavy consumption of alcohol, >80 g/day, developed partial or complete spermatogenic arrest or Sertoli cell-only syndrome, manifesting as azoospermia and male infertility.[29]

Male partners of infertile couples who smoked cigarette had mean seminal MDA concentration (506.8 ± 2.5 ng/ml) significantly higher than both control male smokers (148.4 ± 1.3 ng/ml) and male partners of infertile couples non-smokers (213.2 ± 2.3 ng/ml), with P < 0.001, in both cases. This is similar to findings in other studies [30],[31] where seminal MDA levels were significantly elevated among male partners of infertile couples who smoked than among fertile control smokers and infertile non-smokers.[32] Other studies have also found increased levels of seminal oxidative stress among infertile male smokers compared to infertile and fertile non-smokers.[33] Smoking increases seminal ROS levels and decreases seminal antioxidants. The ROS is negatively correlated with the quality of sperm in semen and increases male infertility.

The mean seminal MDA concentration among male partners of infertile couples with a history of STIs was significantly higher than among controls with a history of STIs and male partners of infertile couples with no history of STIs (503.5 ± 1.9 vs. 385.9 ± 1.1 and 213.4 ± 2.4, respectively, with P < 0.001). This is consistent with a study [34] which showed that infections of the testis and epididymis are particularly detrimental to sperm cells because of prolong exposure to the adverse effects of ROS without adequate antioxidant protection. Infections of the male accessory organs have been shown to decrease sperm count while prostatitis causes sexual dysfunction and poor sperm parameters.[35],[36]


  Conclusions Top


This study has shown that seminal MDA is significantly elevated among male partners of infertile couples, indicating high level of peroxidation of polyunsaturated fatty acid-rich spermatozoa by ROS as shown by many other previous studies. It is therefore suggested that seminal MDA assay should be incorporated into the routine evaluation of male partners of infertile couples.

Antioxidant therapy, such as administration of Vitamins A, C and E as well as zinc, selenium and manganese should be administered as part of drug management of infertile males. More, however, still needs to be done to determine reference value of seminal MDA in healthy individuals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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