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Seminal osteopontin relationship with semen variables in infertile men with varicocele

El-Haggar, Shawkia; Rashed, Lailab; Saleh, Neveen Y.a; Taymour, Maic; Mostafa, Taymoura

doi: 10.1097/01.XHA.0000436102.82137.23
Original articles
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Purpose To assess seminal plasma osteopontin (OPN) relationship with semen variables in fertile and infertile men with varicocele (Vx).

Patients and methods A total of 88 men were investigated, who were divided into the following groups: healthy fertile men without Vx, healthy fertile men with Vx, oligoasthenoteratozoospermic (OAT) infertile men without Vx, and OAT infertile men with Vx. They were subjected to assessment of history, clinical examination, semen analysis, and assessment of seminal OPN, malondialdehyde (MDA), and glutathione peroxidase (GPx).

Results Infertile men associated with Vx showed a significant increase in seminal OPN and MDA, and a significant decrease in seminal GPx compared with infertile men without Vx and fertile men with or without Vx. Infertile men without Vx showed a significant increase in seminal OPN and MDA, and a significant decrease in seminal GPx compared with fertile men with or without Vx. Fertile men with Vx showed a significant increase in seminal OPN and MDA, and a significant decrease in seminal GPx compared with fertile men without Vx. Seminal OPN showed a significant positive correlation with seminal MDA, significant negative correlations with sperm count, sperm motility, sperm normal forms, seminal GPx, and a nonsignificant correlation with age.

Conclusion Seminal OPN is significantly increased in infertile OAT men associated with Vx. Seminal OPN showed a positive correlation with seminal MDA and sperm abnormal forms and a significant negative correlation with sperm count, sperm motility, and seminal GPx.

Departments of aAndrology and Sexology

bMedical Biochemistry, Faculty of Medicine, Cairo University

cDepartment of Deramatology and Andrology, Egypt Air Hospital, Cairo, Egypt

Correspondence to Taymour Mostafa, MD, Department of Andrology and Sexology, Faculty of Medicine, Cairo University, 11562 Cairo, Egypt Tel: +20 100 515 0297; fax: +2 23654133;e-mail: taymour.mostafa@yahoo.com

Received September 1, 2013

Accepted September 4, 2013

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Introduction

Seminal plasma is composed of secretions from the male accessory sex glands and epididymis, which contains many organic and inorganic components that affect the sperm quality 1. The proteins secreted into the seminal plasma play an important role in sperm capacitation and fertilization and protect sperm from damage to maintain their longevity 2.

Osteopontin (OPN), also known as bone sialoprotein-I, is a glycoprotein that was first identified in 1986 in the osteoblasts as an important factor in bone remodeling, playing a role in anchoring osteoclasts to the mineral matrix of bones 3. Also, it is expressed in a range of immune cells, including macrophages, neutrophils, dendritic cells, and T and B cells, with varying kinetics acting as an immune modulator and an antiapoptotic factor. Several studies of its potential significance in mineralized tissues, vascular system, immune system, kidney, cellular transformation, and cancer have been carried out 4.

Varicocele (Vx), a varicosity of the pampiniform plexus, is considered to be the most treatable factor of male infertility, with an overall prevalence of 15% in postpubertal populations and a prevalence in infertility clinics approaching 40%. The effect of Vx on spermatogenesis in infertile men is often reflected by low sperm count, decreased sperm motility, increased sperm abnormal morphology, sperm DNA fragmentation, and apoptotic markers 5–7. Several studies have reported elevated levels of seminal oxidative stress (OS) in infertile men associated with Vx correlated with sperm dysfunction 8–10.

This study aimed to assess seminal plasma OPN relationship with semen variables in fertile and infertile men associated with Vx.

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Patients and methods

This study included 88 men who were consecutively recruited from the Andrology Department, University Hospital, after institutional review board approval and informed consent. All men had been married more than 2 years. They were divided into healthy fertile men without Vx (n=20), fertile men with Vx (n=20), infertile men without Vx (n=22), and infertile men with Vx (n=26). The Vx cases included were grade II and III. The exclusion criteria were as follows: genital trauma, radiation, smoking, leukocytospermia, gonadotoxin, abnormal karyotyping, drugs, or immunological factors.

All participants were subjected to assessment of history, clinical examination, and semen analysis. Vx diagnosis was made with the patient in the standing position in a temperature-controlled room by inspection and palpation with or without the Valsalva maneuver and using high-resolution color flow duplex Doppler ultrasonography. Semen samples were taken twice, 1 week apart, after 4–5 days of sexual abstinence. The samples were examined immediately after liquefaction according to WHO guidelines 11. Liquefied semen was centrifuged at 1200g for 10 min at 4°C; the supernatant seminal plasma was subjected to assessment of OPN, malondialdehyde (MDA), and glutathione peroxidase (GPx).

Seminal OPN was assayed using the human OPN immunoassay method (R&D Inc., Minneapolis, Minnesota, USA) 12.

Seminal MDA was analyzed using the thiobarbituric acid method. Briefly, 100 μl seminal plasma was added in 0.9 ml distilled water in a glass tube. To each tube, 0.5 ml of thiobarbituric acid reagent (0.67 g 2-thiobarbituric acid dissolved in 100 ml distilled water with 0.5 g NaOH and 100 ml glacial acetic acid) was added and then heated for 1 h in a boiling water bath. After cooling, each tube was centrifuged for 10 min at 4000g and the supernatant absorbance was read on a spectrophotometer at 534 nm 13.

Seminal GPx was determined using the Ransel Glutathione Peroxidase Kit (Randox Labs, Crumlin, Co Antrim, Ireland), where GPx catalyzes the oxidation of reduced glutathione with cumene hydroperoxide. In the presence of glutathione reductase and NADPH, oxidised glutathione is converted into reduced glutathione, with a concomitant oxidation of NADPH to NADP+. The decrease in the absorbance was measured at 340 nm at 37°C (pH 7.2) 14.

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Statistical analysis

Statistical package for social sciences program version 18 (SPSS Inc., Chicago, Illinois, USA) was used for statistical analysis. Numerical data were expressed as mean±SD and range. Comparisons were performed by the one-way analysis of variance test; the correlations between variables were assessed using Spearman’s test. P value of less than 0.05 was considered to indicate statistical significance.

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Results

Fertile men with Vx showed left-sided-Vx grade I in 13 cases and grade II in eight cases; oligoasthenoteratozoospermic (OAT) men with Vx had left-sided-Vx grade I in 13 cases and grade II in eight cases. OAT men associated with Vx showed a significant increase in seminal OPN and MDA, and a significant decrease in seminal GPx compared with OAT men without Vx and fertile men with or without Vx. OAT men without Vx showed a significant increase in seminal OPN and MDA and a significant decrease in seminal GPx compared with fertile men with or without Vx. Fertile men with Vx showed a significant increase in seminal OPN and MDA, and a significant decrease in seminal GPx compared with fertile men without Vx (Table 1).

Table 1

Table 1

Seminal OPN showed a significant positive correlation with seminal MDA (r=0.547, P=0.001) and sperm abnormal forms (r=0.604, P=0.001), a significant negative correlation with seminal GPx (r=−0.476, P=0.001), sperm count (r=−0.549, P=0.001), and sperm motility (r=−0.534, P=0.001), and a nonsignificant correlation with age (r=−0.205, P=0.129) (Figs 1 and 2).

Figure 1

Figure 1

Figure 2

Figure 2

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Discussion

OPN, a multifunctional protein, is considered to play a significant role in a variety of biological processes in bone resorption, immune cell activation, inhibiting vascular calcification, and extracellular matrix remodeling 15,16. Expression of OPN has been characteristically observed in different pathological conditions and pathophysiological responses 4. In female factor fertility, OPN, αvβ3 integrin complex, was proposed to distinguish receptive from nonreceptive endometrium and its apparent reduced expression, important in cell–cell adhesion, during the window of implantation in infertile women with isolated polycystic ovary 17,18.

In animals, Cancel et al.19 detected OPN in bull seminal plasma; Erikson et al. 20 reported OPN on the bovine sperm membrane, relating it to sperm function. Souza et al. 21 reported different isoforms in the seminal plasma and sperm membranes of dogs. Lin et al. 22 significantly associated seminal OPN with abnormal spermatozoa and decreased motility.

This is the first study to assess OPN in human seminal fluid. In the current study, seminal OPN, MDA was demonstrated to be significantly increased and seminal GPx was significantly decreased in the infertile men compared fertile participants. In addition, seminal OPN showed a significant negative correlation with sperm count, sperm motility, seminal GPx and a significant positive correlation with sperm abnormal forms, seminal MDA. Different studies reported insufficient antioxidant enzymes and increased seminal OS to be generally attributed to the risk of decreasing semen quality 23,24. Georgiadou et al.25 and Irita et al.4 reported an inverse relation between the presence of OPN and OS.

Infertile men with Vx showed a significant increase in seminal OPN compared with infertile men without Vx and in healthy fertile men compared with fertile men without Vx, indicating that Vx is an additional factor that decreases seminal OPN. A variety of inflammatory mediators and growth factors, including IL-1 and TNF-α, were shown to stimulate OPN transcription, often through activation of protein kinase C 26. Moretti et al.27 detected significantly higher levels of inflammatory mediators in Vx-associated cases, inducing an inflammatory effect, which could play a detrimental role in spermatogenesis, indicated by a decrease in sperm motility and the fertility index, concomitant with increased sperm immaturity.

Furthermore, different studies have reported increased seminal OS in Vx-associated cases 28,29. In addition, a significant increase in seminal OPN in infertile men with Vx could be associated with increases in different unstable, potentially toxic products such as superoxide anion, free radical activity, and inflammatory mediators prevalent in the semen of Vx-associated cases 5,30–32.

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Conclusion

Seminal OPN is significantly increased in infertile OAT men associated with Vx. Seminal OPN showed a positive correlation with seminal MDA and abnormal sperm forms and a significant negative correlation with sperm count, sperm motility, and seminal GPx.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

male infertility; osteopontin; oxidative stress; semen; varicocele

© 2013 Human Andrology