Egyptian Journal of Dermatology and Venerology

ORIGINAL ARTICLE
Year
: 2013  |  Volume : 33  |  Issue : 2  |  Page : 42--45

Evaluation of lipid peroxidation in cases of idiopathic male infertility: correlation with the hypo-osmotic swelling test


Hassan A Khodair1, Zakaria Mahran1, Osama Hashim1, Tarek Omran2,  
1 Department of Dermatology & Andrology, Al-Azhar University, Damietta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Damietta, Egypt

Correspondence Address:
Zakaria Mahran
Kerdasa, Giza
Egypt

Abstract

Background Lipid peroxidation of sperm plasma membranes has a deleterious effect on the semen quality, and measurement of malondialdehyde acid (MDA) is the most widely used method for assessing lipid peroxidation. High levels of seminal MDA represent increased lipid peroxidation rates and oxidative damage to the sperm plasma membranes, which may result in the impairment of the integrity and functioning of sperm plasma membranes and damage of both nuclear and mitochondrial DNA. Aim of the study The aim of the present study was to investigate the seminal plasma MDA concentration as a marker of lipid peroxidation and its correlation with the hypo-osmotic swelling test (HOST) scores as a predictor for the functional integrity of the sperm membrane in cases of idiopathic male infertility. Participants and methods A total of 93 men were included in this study: 68 men who had been attending to our andrology outpatient clinics with idiopathic infertility were selected as cases and 25 healthy fertile men were assigned as control group. Both groups were subjected to the following laboratory investigations: semen analysis, peroxidase test, HOST, and measurement of MDA in the seminal plasma. Results Our results showed that seminal plasma concentration of MDA was significantly higher in men with idiopathic infertility compared with the fertile control group ( P ≤ 0.001), and HOST was significantly lower in men with idiopathic infertility compared with the fertile control group ( P ≤ 0.001). In addition, the results showed a strong negative correlation between MDA concentration and HOST ( P ≤ 0.001). Conclusion Our results suggest that the evaluation of lipid peroxidation must be used in combination with the HOST as a diagnostic tool in men with idiopathic infertility in addition to standard semen analysis.



How to cite this article:
Khodair HA, Mahran Z, Hashim O, Omran T. Evaluation of lipid peroxidation in cases of idiopathic male infertility: correlation with the hypo-osmotic swelling test.Egypt J Dermatol Venerol 2013;33:42-45


How to cite this URL:
Khodair HA, Mahran Z, Hashim O, Omran T. Evaluation of lipid peroxidation in cases of idiopathic male infertility: correlation with the hypo-osmotic swelling test. Egypt J Dermatol Venerol [serial online] 2013 [cited 2022 Dec 10 ];33:42-45
Available from: http://www.ejdv.eg.net/text.asp?2013/33/2/42/123925


Full Text

 Introduction



Metabolism in human sperm produces various reactive oxygen species (ROS), which when present in excess possess potentially toxic effects on both sperm quality and function >sup>[1],[2],[3],[4]. However, small physiological levels of ROS are essential for the regulation of normal sperm functions such as sperm capacitation, acrosome reaction, and sperm-oocyte fusion [5] . Human ejaculate consists of different types of cells such as mature and immature spermatozoa, round cells, leukocytes, and epithelial cells. Of these, peroxidase-positive leukocytes and abnormal spermatozoa produce free radicals continuously [6],[7] . Seminal plasma contains enzymatic ROS scavengers such as superoxide dismutase, glutathione peroxidase, and catalase. These enzymes act as an antioxidant and inhibitor of lipid peroxidation [8-10]. The process of lipid peroxidation is the oxidative conversion of polyunsaturated fatty acids to products such as malondialdehyde acid (MDA). High levels of seminal MDA represent increased lipid peroxidation rates and oxidative damage to the sperm membranes [11] . MDA, an end product of lipid peroxidation, reacts with thiobarbituric acid (TBA) to form a colored substance. Measurement of MDA by TBA reactivity is the most widely used method for assessing lipid peroxidation [12] . The hypo-osmotic swelling test (HOST) was originally used as a sperm function test to evaluate the integrity of the sperm membrane [13],[14],[15] and as a vitality test alternative to dye exclusion when staining of spermatozoa is avoided [16] . A large population of apparently normal men have problem impregnating their partners even when their fertility status indicated by routine semen analysis is considered normal. These cases were classified as idiopathic infertility and represented about 60-75% of male infertility [14] .

 Participants and methods



This study was conducted during the period of June 2011 to June 2012 and included 93 men who had been attending to our andrology outpatient clinics at Damietta university hospital, Al-Azhar University. Sixty-eight men with idiopathic infertility, who had normal semen parameters with a duration of infertility of 2-7 years, were selected as cases and 25 healthy fertile men as control group. Both groups were comparable in terms of age with normal health. Patients taking medications or antioxidants and smokers were excluded. Patients suffering from varicocele and acute infections or leukocytospermia were excluded from the study because of their well-known high seminal ROS levels. The fertilizing capacity of the female partners of those infertile men including hormonal assay and folliculometry were assessed and were found functionally normal. Both groups were subjected to the following laboratory investigations: semen analysis including, peroxidase test and the HOST in addition to measurement of malondialdehyde content (MDA) in the seminal plasma.

 Laboratory evaluation



Semen collection and evaluation

All semen samples were produced by masturbation after 3-5 days of abstinence, collected in a sterile plastic container, and incubated at 37°C following collection. The following routine parameters were assessed according to the WHO laboratory manual. The specimens were analyzed for volume, pH, color, liquefaction, agglutination, sperm concentration, percentage and grade of motility, sperm morphology, sperm vitality, total sperm count, and total motile sperms. Results were considered normal when the semen volume was greater than 2 ml, sperm density was greater than 15 × 10 6 /ml, there was more than 32% forward progressive motility, and there were less than 85% abnormal forms including dead sperm [15],[16] .

Peroxidase test

Leukocytes were counted by the LeucoScreen method. Briefly, mix one drop of semen with one drop of working solution (leucoscreen stain and hydrogen peroxide), cover with a cover slip for 2 min, and then read the result at a magnification of ×400. Peroxidase-positive cells are stained yellow to brown, whereas other cells are stained pink [17] .

Hypo-osmotic swelling test evaluation

Live spermatozoa with normal membrane function show swelling of the cytoplasm and curling of the tail due to water influx when exposed to hypo-osmotic conditions. The incidence of greater than 60% swollen spermatozoa showing different degrees of swelling ranging from swelling only at the tip of the tail to the swollen area enveloping the curved tail of the sperm was considered as an index for a normal fertile sample [13] .

Measurement of malondialdehyde acid concentration

Lipid peroxidation was measured by determining the MDA production using the TBA method. After liquefaction, semen samples were centrifuged at 3500 rpm for 15 min to obtain seminal plasma. Then, 0.1 ml of seminal plasma was added to 0.75 ml of TBA reagent (0.8 g of 2-TBA dissolved in 80 ml of distilled water with 0.5 ml of NaOH). Perchloric acid (7%) was added to this mixture to adjust the pH to 7.4. This mixture was heated for 1 h at 95°C in a warm water bath. After cooling, the tube was centrifuged for 10 min at 4000 rpm and the absorbance of the supernatant was read at 535 nm using a spectrophotometer [12] . The results were expressed as nmol MDA/ml seminal plasma.

 Results



General characteristics of both fertile and idiopathic infertile groups

Mean age in idiopathic infertile and fertile control groups was 34.5 ± 3.5 (range 26-46 years) and 33.5 ± 2.5 (range 25-45 years), respectively, as shown in [Table 1].{Table 1}

Semen parameters studies

The study parameters were the color of the semen, coagulation, pH, semen volume, liquefaction, viscosity, sperm concentration, motility, grade of motility, sperm morphology, sperm vitality, total sperm count, and total motile sperm in both groups. The results (mean ± SD, P > 0.05) are given in [Table 2].{Table 2}

Peroxidase test

Mean white blood cells (10 6 /ml) in idiopathic infertile and fertile control groups were 0.05 ± 0.03 × 10 6 /ml and 0.06 ± 0.02 × 10 6 /ml (P > 0.05), respectively, as shown in [Table 3].{Table 3}

MDA

The results obtained in the present study showed that the seminal plasma concentration of MDA was significantly higher in men with idiopathic infertility compared with the fertile control group (29.67 ± 5.77 vs. 14.99 ± 5.60 nmol/ml, P ≤ 0.001), as shown in [Table 4].{Table 4}

HOST

The result was significantly lower in men with idiopathic infertility compared with the fertile control group (39.5 ± 3.7 vs. 71.0 ± 5.4%, P ≤ 0.001), as shown in [Table 5].{Table 5}

Correlation between MDA concentration and HOST

The results obtained in the present study showed a strong negative correlation between HOST and MDA concentration ( P ≤ 0.001), as shown in [Figure 1].{Figure 1}

 Discussion



Our study found no statistically significant differences in the data of semen parameters and white blood cells concentration (no leukocytospermia) between the idiopathic infertile men and fertile controls (P > 0.05). However, our results showed that the seminal MDA concentration was significantly higher in men with idiopathic infertility compared with the fertile control group ( P ≤ 0.001). Despite numerous studies explored a strong negative correlation between lipid peroxidation and semen parameters data, our study is the first to evaluate the MDA level in idiopathic infertile men. In previous studies, seminal MDA level significantly increased in patients with oligoasthenoteratozoospermia [18],[19],[20],[21]. Another studies found that the seminal MDA level significantly increased in patients with asthenozoospermia and oligoasthenoteratozoospermia [22-24].

Our study found that HOST was significantly lower in men with idiopathic infertility compared with the fertile control group ( P ≤ 0.001). Our study is the first to evaluate the correlation between lipid peroxidation, MDA level, and HOST. Our study found that elevated concentrations of seminal MDA were strongly and negatively associated with HOST (P ≤ 0.001). According to these results, high levels of seminal MDA represent increased lipid peroxidation rates and oxidative damage to the sperm plasma membranes, which may help to clarify the cause of impaired HOST in our idiopathic infertile men. Consequently, these findings suggest an explanation why patients with normal semen parameters experience idiopathic infertility. Our results support another studies in which an increased incidence of miscarriage was observed in couples with abnormal HOST results undergoing IVF procedures [25] . Our study explored that normal semen analysis does not guarantee the fertilization potential of the sperm, and studies have shown a significant overlap in the semen parameter values between fertile and infertile men.

 Conclusion



Lipid peroxidation of sperm plasma membrane has a deleterious effect on the sperm function and quality. Measurement of the MDA level in seminal plasma must be used as an important oxidative stress parameter in combination with HOST as a diagnostic tool in men with idiopathic infertility in addition to standard semen analysis.

 Acknowledgements



Conflicts of interest

None declared.

References

1Warren JS, Johnson Kja, Ward PA. Oxygen radicals in cell injury and cell death. Pathol Immunopathol Res 1987; 6 :301-315.
2De Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod 1995; 10 (Suppl 1):15-21.
3Padron OF, Brackett NL, Sharma RK, Lynne CM, Thomas AJ Jr, Agarwal A. Seminal reactive oxygen species and sperm motility and morphology in men with spinal cord injury. Fertil Steril 1997; 67 :1115-1120.
4Agarwal A, Ikemoto I, Loughlin KR. Relationship of sperm parameters with levels of reactive oxygen species in semen specimens. J Urol 1994; 152 :107-110.
5Sikka SC, Rajasekaran M, Hellstrom WJ. Role of oxidative stress and antioxidants in male infertility. J Androl 1995; 16:464-468.
6Aitken RJ, West KM. Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl 1990; 13 :433-451.
7Saleh RA, Agarwal A. Oxidative stress and male infertility: erom research bench to clinical practice. J Androl 2002; 23 (Suppl 6):737-752.
8Eskiocak S, Gozen AS, Yapar SB, Tavas F, Kilic AS, Eskiocak M. Glutathione and free Sulphydryl content of seminal plasma in healthy medical students during and after exam stress. Hum Reprod 2005; 20 (Suppl 9):2595-2600.
9Cummins JM, Jequier AM, Kan R. Molecular biology of human male infertility: links with aging, mitochondrial genetics and oxidative stress. Mol Reprod Dev 1994; 37 (Suppl 3):345-362.
10Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl 1987; 8 :338-348.
11Gomez E, Irvine DS, Aitken RJ. Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function. Int J Androl. 1998; 21 :81-94.
12Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med 1976; 5 (Suppl 2):212-216.
13Jeyendran RS, Van der Ven HH, Zaneveld LJ. The hypo-osmotic swelling test: an update. Arch Androl 1992; 29:105-116.
14Pasqualotto FF, Sharma RK, Kobayashi H, Nelson DR, Thomas AJ Jr, Agarwal A. Oxidative stress in normospermic men undergoing infertility evaluation. J Androl 2001; 22 :316-322.
15World Health Organization Who Laboratory Manual for the Examinatopn of Human Semen and Semen Cervical Mucus Interactions. 3rd ed. 1992;Cambridge, UK: Cambridge University Press; 35.
16World Health Organization Laboratory Manual for the Examination and Processing of Human Semen. 5th ed. 2010;Geneva: World Health Organization.
17Politch JA, Wolff H, Hill JA, Anderson DJ. Comparison of method to enumerate white blood cells in semen. Fertil Steril 1993; 60 :372-375.
18Hesham N, Moemen LA, Abu Elela MH Studying the levels of malondialdehyde and antioxidant parameters in normal and abnormal human seminal plasma. Aust J Basic Appl Sci 2008; 2 (Suppl 3):773-778.
19Hsieh YY, Chang CC. Lin CS Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility. Int J Biol Sci 2006; 2 (Suppl 1):23-29.
20Dandekar SP, Nadkarni GD, Kulkarni VS, Punekar S. Lipid peroxidation and antioxidant enzymes in male infertility. J Postgrad Med 2002; 48 :186-189.
21Fraczek M, Szkuntik D, Sanocka D, Kurpisz M. Peroxidation components of sperm lipid membranes in male infertility. Ginekol Pol 2001; 72 (Suppl 2):73-79.
22Tavilani H, Mohamed TG, Vaisi Al, Saeedeh SS, Hassan Z. Activity of antioxidant enzymes in seminal plasma and their relationship with lipid peroxidation of spermatozoa. 2008;Int Braz J Urol 34:485-491.
23Ben AF, Dammak I, Attia H, Hentati B, Ammar-Keskes L. Lipid peroxidation and antioxidant enzyme activities in infertile men: correlation with semen parameter. J Clin Lab Anal 2009; 23 (Suppl 2):99-104.
24Atig F, Raffa M, Ben Ali H, Abdelhamid K, Saad A, Ajina M. Altered antioxidant status and increased lipid per-oxidation in seminal plasma of tunisian infertile men. Int J Biol Sci 2012; 8 :139-149.
25Check JH, Stumpo L, Lurie D, Benfer K, Callan C. A comparative prospective study using matched samples to determine the influence of abnormal hypo-osmotic test scores of spermatozoa on subsequent fertilization and pregnancy rates following in vitro fertilization. Hum Reprod 1995; 10 :1197-1200.