ORIGINAL ARTICLE

Seminal plasma oxytocin and oxidative stress levels in infertile men with varicocele T. Mostafa1, L. A. Rashed2, I. Osman1 & M. Marawan1 1 Department of Andrology and Sexology, Faculty of Medicine, Cairo University, Cairo, Egypt; 2 Department of Medical Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt

Keywords Male infertility—oxidative stress—oxytocin— semen—varicocele Correspondence Dr Taymour Mostafa, Department of Andrology and Sexology, Faculty of Medicine, Cairo University, Cairo 11562, Egypt. Tel.: +2 01005150297; E-mail: [email protected]

Accepted: December 29, 2013 doi: 10.1111/and.12248

Summary This study aimed to assess seminal plasma oxytocin (OT) and oxidative stress (OS) levels in infertile men with varicocele (Vx). A total of 131 men were divided into fertile men (n = 20), fertile men with Vx (n = 17), infertile men without Vx (n = 40) and infertile men with Vx (n = 54). OT, malondialdehyde (MDA) and glutathione peroxidase (GPx) were estimated in seminal plasma. Mean levels of seminal OT, MDA were significantly decreased, and the mean level of GPx was significantly increased in fertile men with/without Vx compared with infertile men with/without Vx. Mean levels of OT, MDA were increased, and mean level of GPx was significantly decreased in Vx grade III cases compared with Vx grades I, II cases and in bilateral Vx cases compared with unilateral Vx. There was significant negative correlation between seminal OT with sperm count, sperm motility, seminal GPx and significant positive correlation with sperm abnormal forms, seminal MDA. It is concluded that seminal OT is significantly decreased in fertile men with/without Vx compared with infertile men with/without Vx. Seminal OT demonstrated significant negative correlation with sperm count, sperm motility, seminal GPx and significant positive correlation with sperm abnormal forms, seminal MDA. Seminal OT is associated with Vx grade and its bilaterality.

Introduction Oxytocin (OT) is a neuropeptide that is synthesised in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei projecting to the posterior pituitary from which it is released into the blood stream. After its initial discovery as a uterus-contracting hormone in the 1950s, its facilitative effects on sexual and reproductive behaviours were rapidly raised both inside the scientific community and among the general public. In addition to being the ‘love hormone’ among the general public reflecting positive effects on affiliation and bonding, OT facilitates a wide range of processes and behaviour (Huffmeijer et al., 2013). In the brain, OT could act both as a neurotransmitter and as a neuromodulator. Also, OT is well known for its role in parturition, lactation, regulation of the hypothalamic–pituitary–adrenal axis, facilitating reproductive and maternal behaviour, infant attachment and social behaviour (MacDonald & MacDonald, 2010). In males, OT was demonstrated to be synthesised within the mammalian testis, epididymis, prostate, penis and the presence of its receptor through the reproductive © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5

tract supports a local important action (Thackare et al., 2006). OT also was shown to affect conversion of testosterone to DHT by modulating 5-a reductase, suppressing inflammation and regulating oxidant–antioxidant status (Assinder et al., 2004; Tas Hekimoglu et al., 2013). As well, OT is recognised to affect male reproduction where a burst of its release into the systematic circulation at orgasm stimulates contractions of the reproductive tract aiding sperm release (Thackare et al., 2006). Varicocele (Vx) is one of the well-known diagnosed causes of male infertility that has a prevalence of 15% in men evaluated for infertility (Hassan et al., 2011; El-Haggar et al., 2012). The effect of Vx on spermatogenesis in infertile men is often reflected by low sperm count, decreased sperm motility, increased sperm abnormal morphology and increased apoptotic indices (Sadek et al., 2011; Zalata et al., 2011; El-Kamshoushi et al., 2013; Tawadrous et al., 2013). Oxidative stress (OS) as a result of the imbalance between reactive oxygen species (ROS) and antioxidants in the semen gained momentum as it leads to sperm DNA damage and sperm deformity. In addition, OS 1

Seminal oxytocin – oxidative stress relationship

induces membrane and nucleus alterations, resulting in loss of motility, decline in sperm fertilising ability, abnormalities of pre-implantation development and miscarriages. Different studies demonstrated high levels of seminal OS in infertile men in general that is exaggerated if associated with Vx (Mostafa et al., 2001, 2006, 2009). This study aimed to assess seminal OT-OS levels in infertile men associated with Vx. Materials and methods This study consisted of 131 men, selected prospectively according to their semen parameters and fertility status after IRB approval from the Andrology Department and the University Hospital with matched age. Fertile men were fathering a pregnancy or a child within the last 12 months. They were divided into healthy fertile men (n = 20), fertile men with Vx (n = 17), infertile men without Vx (n = 40) and infertile men with Vx (n = 54). All participants were married for more than 2 years. Exclusion criteria were subclinical Vx, leucocytospermia, smoking, diabetes or antioxidant therapy. They were subjected to history taking, clinical examination and semen analysis. Clinical examination was carried out in a warm room at the standing position with/without Valsalva manoeuver. Vx was classified into grade I (palpable only during Valsalva manoeuver), grade II (palpable distension on standing upright) and grade III (visible through scrotal skin) (Chiou et al., 1997). Colour Doppler ultrasonography was conducted for assurance of Vx and its grade when one or more veins had a maximal diameter >3 mm with a retrograde flow at rest or under Valsalva manoeuver. The ejaculates were obtained after 4–5 days of sexual abstinence into sterile containers. More than one sample was provided 2 weeks apart. Semen was examined manually according to WHO guidelines (2010). The levels of malondialdehyde (MDA), glutathione peroxidase (GPx) and OT were estimated in seminal plasma. Seminal MDA was estimated using the photometric method described by Placer et al. (1966) and seminal GPx level was estimated using the method of Paglia & Valentine (1967) where seminal OT was estimated by enzyme linked immunosorbent assay (Enzo Life Sciences Inc., Farmingdale, NY, USA). Statistical analysis The statistics are presented as mean  SD. The results were analysed by SPSS version 18 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) for the continuous variables was analysed followed by Tukey’s HSD post hoc analysis for comparison where Pearson’s rank 2

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correlation coefficient was used to study the relationship between variables. P < 0.05 was considered statistically significant. Results The mean levels of seminal OT and MDA were significantly decreased, and the mean level of GPx was significantly increased in fertile men without Vx compared with fertile men with Vx. Also, the mean levels of seminal OT, MDA were significantly decreased, and the mean level of GPx was significantly increased in fertile men with/without Vx and infertile men without Vx compared with infertile men with Vx (Table 1). The mean levels of OT, MDA were increased, and the mean level of GPx was significantly decreased in infertile men with Vx grade III (n = 24) compared with Vx grade I and grade II cases (Table 2). The mean levels of OT, MDA were significantly increased in infertile men associated with bilateral Vx (n = 39) compared with infertile men associated with unilateral Vx (n = 17) (OT 91.9  24.3 versus 61.1  11.7 pg ml 1, MDA 3.06  1.4 versus 2.7  0.9 nmol ml 1, GPx 0.13  0.4 versus 0.29  0.08 U ml 1, respectively). There was significant negative correlation between seminal OT and sperm count (r = 0.406, P = 0.001), sperm motility (r = 0.457, P = 0.001), GPx (r = 0.572, P = 0.001) and significant positive correlation with sperm abnormal forms (r = 0.418, P = 0.001), MDA (r = 0.770, P = 0.001) (Figs 1–3). Discussion In males, the small peptide hormone OT is produced in similar quantities within the hypothalamo-pituitary magnocellular system as in the female, yet for the male little is known about its associated physiology. In the current study, the mean level of seminal OT was significantly decreased in fertile men without Vx compared with fertile men with Vx, in infertile men without Vx compared with fertile men with/without Vx and in infertile men with Vx compared with fertile men with/without Vx and infertile men without Vx. These relationships point to the intimate relation between seminal OT and decreased semen parameters associated with the male infertility status that is exaggerated if associated with Vx. Being a circulatory hormone, the majority of the available reports about OT relationship with male infertility are estimated in the peripheral circulation not in the semen. Previously, Ogawa et al. (1980) showed that there was no obvious correlation between plasma OT levels and sperm count, sperm motility or sperm abnormal forms. L€ u et al. (2010) demonstrated a significantly lower serum © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5

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Table 1 Comparison of different parameters in the investigated groups (mean  SD)

n Age (years) Semen volume (ml) Sperm count (106 ml 1) Sperm motility (%) Sperm abnormal forms (%) MDA (nmol ml 1)

Fertile men

Fertile men with Vx

OAT men without Vx

OAT men with Vx

20 30.15 2.5 79.15 56.5 33.0 1.57

17 33.4  5.4 2.4  1.5 78.8  23.9 55.0  6.2 37.6  8.1 2.3  0.7 P1 = 0.001*

40 30.9  5.1 3.1  1.2 11.6  2.75 17.8  8.7 78.0  19.2 2.71  0.9 P1 = 0.001* P2 = 0.01*

54 30.3  5.9 2.6  1.07 10.9  3.5 18.8  9.6 79.8  19.9 2.93  1.3 P1 = 0.001* P2 = 0.01* P3 = 0.03* 0.26  0.1 P1 = 0.001* P2 = 0.01* P3 = 0.03* 85.5  19.2 P1 = 0.001* P2 = 0.001* P3 = 0.01*

     

6.8 1.02 20.4 5.2 5.7 0.7

GPx (U ml 1)

0.49  0.05

0.37  0.09 P1 = 0.001*

0.31  0.09 P1 = 0.001* P2 = 0.022*

OT (pg ml 1)

61.1  11.7

70.2  11.2 P1 = 0.01*

76.7  17.8 P1 = 0.001* P2 = 0.01*

P1, Statistical difference compared with fertile men. P2, Statistical difference compared with fertile men with Vx. P3, Statistical difference compared with infertile men without Vx. *Significant. Table 2 Comparison of the seminal OT according to Vx grade in infertile men (mean  SD) Vx Grade I n MDA (nmol ml 1)

9 1.7  0.05

Vx Grade II 21 2.2  0.7 P1 = 0.001*

GPx (U ml 1)

0.33  0.05

0.30  0.1 P1 = 0.03*

OT (pg ml 1)

70.7  5.3

83.3  16.0 P1 = 0.01*

Vx Grade III 24 3.7 P1 P2 0.23 P1 P2 102.4 P1 P2

 1.1 = 0.001* = 0.001*  0.1 = 0.001* = 0.001*  15.03 = 0.001* = 0.001*

P1, Statistical difference compared with Vx grade I cases. P2, Statistical difference compared with Vx grade II cases. *Significant. Fig. 1 Significant negative correlation of seminal OT with sperm count (r = 0.406, P = 0.001).

content of OT in fertile men compared with oligozoospermic men, asthenozoospermic men and oligoasthenozoospermic men pointing to the association of OT with male infertility. Lui et al. (2010) showed that serum OT baseline concentrations in infertile groups were significantly higher than in fertile group and were not correlated with LH, FSH or testosterone hormonal levels. Goverde et al. (1998) studied the relationship between seminal OT by radioimmunoassay after extraction detecting no difference in fertile men, oligoasthenoteratozoospermic men or in vasectomised patients. They concluded that OT is not only derived from the testis; OT levels in © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–5

poor semen samples are not different from controls, and there was no relationship between OT seminal plasma levels and sperm characteristics. OT in the testis appears to have an autocrine/paracrine role modulating steroid metabolism, but it may in addition be involved in seminiferous tubules contractility (Ivell et al., 1997). The first indication of local OT synthesis within the male reproductive tract was obtained by Nicholson et al. (1984). Using radioimmunoassay and high-performance liquid chromatography, an OT-like peptide was detected in both the human and rat testis. 3

Seminal oxytocin – oxidative stress relationship

Fig. 2 Significant negative correlation of seminal OT with sperm motility (r = 0.457, P = 0.001).

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represent a neuroendocrine response contributing to the overall host response to infection by decreasing the pro-inflammatory response and OS. Deing et al. (2013) suggested that the OT system is a novel neuroendocrine mediator in human skin homoeostasis and clinically relevant to stressed skin conditions. In animals, OT injection in lactating buffaloes resulted in elevated OS by increasing the total homocysteine and ceruloplasmin oxidase activity and decreasing enzymatic activities of antioxidant enzymes (Iqbal et al., 2013). The mean level of seminal OT was significantly increased in men with Vx grade III compared with men with Vx grade II and in men with bilateral Vx cases compared with men with unilateral Vx. Different studies pointed to the increase of Vx deleterious effect(s) with its increased grade giving a significant difference between grade I and III of Vx. Vivas-Acevedo et al. (2010) showed that Vx grade could determine the extent of alteration to semen quality. Cocuzza et al. (2012) pointed that as Vx grade increased, seminal ROS increases and sperm concentration decreases. Mostafa et al. (2012) added that seminal OS is related to increased Vx grade in infertile OAT men associated with Vx. A point of limitation is to assess seminal OT levels after varicocelectomy and if these changes are correlated with semen parameters and/or pregnancy rates. It is concluded that seminal OT is significantly increased in infertile men with Vx compared with fertile men, fertile men with Vx and infertile men without Vx. Seminal OT demonstrated significant negative correlation with sperm count, sperm motility, seminal GPx and significant positive correlation with sperm abnormal forms, seminal MDA. Seminal OT in infertile men associated with Vx was associated with its grade and bilaterality.

Fig. 3 Significant positive correlation of seminal OT with sperm abnormal forms (r = 0.418, P = 0.001).

References Colocalisation of neurophysin with this OT-like peptide, together with the higher than plasma concentrations recorded, pointed towards local synthesis. In parallel, seminal OT demonstrated significant positive correlation with seminal MDA and significant negative correlation with seminal GPx. Of course, Vx is the most relevant condition in male infertility that is associated with increased reactive oxygen species with decreased antioxidant defences both at the local (seminal plasma) and systemic (blood plasma) levels (Abdel Aziz et al., 2010; Mostafa et al., 2012; Taha et al., 2012). Biyikli et al. (2006) showed that OT alleviates oxidant renal injury in pyelonephritic rats by its antioxidant actions and by preventing free radical damaging cascades that involves excessive infiltration of neutrophils. Oliveira-Pelegrin et al. (2013) demonstrated that OT secretion during sepsis may 4

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Seminal plasma oxytocin and oxidative stress levels in infertile men with varicocele.

This study aimed to assess seminal plasma oxytocin (OT) and oxidative stress (OS) levels in infertile men with varicocele (Vx). A total of 131 men wer...
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