Article Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility

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1 RBMOnline - Vol 14 No Reproductive BioMedicine Online; on web 13 December 2006 Article Relationship between male reproductive hormones, sperm DNA damage and markers of oxidative stress in infertility Dr Malathy Appasamy graduated in medicine from the TN Dr MGR Medical University in India in She has obtained her membership from the Royal College of Obstetricians and Gynaecologists (MRCOG), London. She is undergoing her post-graduate training in Obstetrics and Gynaecology in United Kingdom. Her speciality of interest is reproductive medicine. She is currently working towards an MD on the role of oxidative stress on male and female reproductive hormones in infertility and assisted conception in the Department of Obstetrics and Gynaecology at the University College, London. Dr Malathy Appasamy M Appasamy 1, S Muttukrishna 1, AR Pizzey 2, O Ozturk 3, NP Groome 4 P Serhal 3, E Jauniaux 1,5 1 Academic Department of Obstetrics and Gynaecology, Royal Free and University College London, UCL Campus, Chenies Mews, London, WC1E6HX; 2 Department of Haematology, UCL, London; 3 Assisted Conception Unit, UCL, London; 4 School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, United Kingdom 5 Correspondence: Tel: ; Fax: ; e.jauniaux@ucl.ac.uk Abstract This study investigated the relationship between male reproductive hormones and sperm DNA damage and markers of oxidative stress in men undergoing infertility evaluation for male factor (n = 66) and non-male factor (n = 63) infertility. Semen samples were analysed for DNA fragmentation index (DFI). Serum samples were analysed for FSH, inhibin B, anti- Müllerian hormone (AMH), testosterone and total antioxidant capacity (TAC). Serum inhibin B was significantly lower in the male factor group compared with the non-male factor group. Inhibin B showed a positive correlation with sperm concentration and motility, and serum AMH showed a positive correlation with sperm concentration and semen volume. DFI was 3-fold higher in the male factor group and showed a negative correlation with sperm motility. Blood plasma TAC was negatively related to sperm concentration. The results confirm that AMH and inhibin B are markers of Sertoli cell function. Sperm DNA damage is moderately increased in male factor infertility, and is negatively associated with sperm motility. A negative association between antioxidant activity and sperm concentration suggests that even minimal oxidative stress may influence sperm concentration. However, there was no significant relationship between hormone concentrations, sperm DNA damage and total antioxidant capacity, suggesting other mechanisms for sperm dysfunction. Keywords: AMH, inhibin B, male infertility, oxidative stress, sperm DNA damage Introduction Male infertility accounts for 25% of couples attending fertility clinics and for at least 50% of couples requiring assisted reproduction techniques (Hull et al., 1992; ESHRE, 2002). Spermatogenesis is initiated by the endocrine influence of gonadotrophins, e.g. FSH, that act on the Sertoli cells of the testis (Griswold, 1995). This results in the production of protein hormones such as inhibin B and anti-müllerian hormone (AMH) from the Sertoli cells (Risbridger et al., 1990). Inhibin B has been shown to mediate Sertoli germ cell interactions, thereby playing an important role in spermatogenesis (Risbridger et al., 1990). AMH concentration in the seminal plasma is significantly lower in infertile oligozoospermic men compared with healthy volunteers, and is related to sperm concentration and mean testicular volume (Fujisawa et al., 2002). The other male reproductive hormone that plays a role in spermatogenesis is testosterone, which is produced by the Leydig cells of the testis under the influence of LH (de Kretser et al., 1998). The ability of the spermatozoa to fertilize the ovum is largely dependent on the presence of compact chromatin, which protects them during transport through the male and female reproductive tract (Richthoff et al., 2002), and on intact epididymal function, Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 160 which determines maturation and sperm motility (Vernet et al., 2004). There are growing concerns that male factor infertility is increasing, but the underlying cause for sperm dysfunction is not yet well understood (Sakkas et al., 2003). In recent years, oxidative stress (OS) has been implicated as one of the underlying aetiologies in the mechanism leading to sperm dysfunction (Sharma and Agarwal, 1996; Pasqualotto et al., 2001) and DNA damage (Loft et al., 2003; Tesarik et al., 2006). OS occurs due to overproduction of reactive oxygen species (ROS) beyond cellular antioxidant scavenging capacity. It has been shown that human ejaculate is contaminated with potential sources of ROS (Aitken, 1995). This results in oxidative damage and loss of function of some spermatozoa in every ejaculate. Immature spermatozoa and seminal leukocytes have been identified to be the main sources of ROS in semen (Aitken and West., 1990; Gil-Guzman et al., 2001). Elevated concentrations of ROS induce lipid peroxidation, thereby affecting the sperm cell membrane and damage to intracellular proteins, and contributing indirectly to infertility (Sikka, 1996). Despite compelling evidence of the role of OS in male infertility, the efficacy of antioxidant treatment is still debatable (Agarwal et al., 2004). In view of the close proximity of Sertoli cells and germ cells, OS produced by the subsets of spermatozoa during the maturation process could affect the production or action of protein hormones from Sertoli cells, such as inhibin B and AMH, which play an important role in spermatogenesis. In addition, OS causes several forms of DNA damage such as chromatin cross-linking, chromosome deletion, DNA strand breaks and base oxidation (Agarwal and Said, 2003; Aitken et al., 2003) and mediates apoptosis (Said et al., 2004). OS is associated with increased numbers of immature spermatozoa (Gil-Guzman et al., 2001), which is negatively related to fertilization potential and pregnancy rates after IVF (Zorn et al., 2003) and in vivo (Aitken et al., 1991). Embryos derived from DNA damaged spermatozoa have lower potential to reach the blastocyst stage after ICSI (Henkel et al., 2003; Nasr-Esfahani et al., 2005). It is not known whether OS results in an increased number of immature spermatozoa by interfering with spermatogenesis via an indirect effect on the hormones produced from the Sertoli and Leydig cells that are in close proximity to the developing spermatozoa from the germ cells. There is controversial evidence that testosterone may act as a pro-oxidant or antioxidant (Peltola et al., 1996). In addition, a negative correlation has been found between sperm DNA damage (DNA fragmentation index; DFI) and oestradiol and between DFI and free testosterone in young fertile men (Elzanaty et al., 2002). However, there are currently no available data on the relationship between AMH, inhibin B, sperm DNA damage and markers of oxidative stress in the infertile population. The aim of this study was to investigate the relationship between serum AMH, inhibin B, testosterone, FSH and oxidative stress and sperm DNA in normal males with infertility and men with male factor infertility. Materials and methods In this prospective study, samples of semen and blood were collected from men undergoing evaluation and treatment for infertility over an 18 month period. Two groups were studied: a male factor infertility group (n = 66) and a non-male factor group (n = 63). The non-male factor group included men with normal semen parameters who had unexplained aetiology or female factor aetiology for infertility such as tubal damage, polycystic ovarian syndrome and endometriosis. Semen samples were obtained by masturbation after at least 48 h of abstinence and were allowed to liquefy for about 30 min at room temperature. The results of semen analysis performed by an embryologist blinded to the study were interpreted using World Health Organization criteria (World Health Organization, 1999). Specimens with a sperm count < /ml, sperm motility < 50% and percentage normal forms < 14% were considered to be positive for male factor infertility. An aliquot of semen was snap frozen in liquid nitrogen and stored at 80ºC for later analysis of sperm DNA damage (DFI) using sperm chromatin structure assay. Serum and plasma (heparinized) samples were obtained by centrifugation (10 min, 1500 g) within 1 h of sample collection, snap frozen in liquid nitrogen and stored at 80ºC for later analysis of hormones and total antioxidant capacity. The study was approved by the joint UCL/UCLH Committees on the Ethics of Human Research (Committee Alpha). Measurement of DNA fragmentation index (DFI) The sperm chromatin structure assay (SCSA) was performed according to the procedure described by Evenson et al. (1999). Briefly, samples stored at 80ºC were quickly thawed in a 37ºC water bath on the day of analysis and used immediately. The samples were diluted with TNE buffer containing 12.1 g Tris base, 3.7 g Na 2 EDTA.2H 2 O (ph 7.4) to obtain a cell concentration of /ml. The suspension was then treated with acid detergent buffer (ph 1.2) containing 0.1%Triton X-100, 0.15 mol/l NaCl and 0.08 mol/l HCl for 30 s by gentle shaking. Freshly prepared acridine orange (6 mg/ml) in phosphate citrate buffer, ph 6.0, was then added to stain the cells. After 3 min, the cells were analysed on an EPICS ELITE flow cytometer (Beckman-Coulter, High Wycombe, Bucks, UK; 488 nm light source). A total of at least 10,000 events were studied for each sample. Under 488nm excitation, cells stained with acridine orange (AO) exhibit metachromatic properties, so that AO intercalated with normal double-stranded DNA emits green fluorescence (labelled as native DNA in Figure 1a), while AO associated with denatured single-stranded DNA emits red fluorescence (labelled as fragmented DNA in Figure 1a). The extent of DNA denaturation was quantified and expressed as DNA fragmentation index (DFI) as described by Evenson et al. (2002). The results were expressed as αt %, ratio of red (fragmented DNA) to total fluorescence intensity (red plus green; fragmented plus native DNA as shown in Figure 1a, b). DFI >30% was considered as high sperm DNA damage and DFI between 15 and 30% was considered moderate DNA damage, according to Evenson et al. (1999). Measurement of total antioxidant capacity (TAC) When there is oxidative stress, there is excessive free radical formation. The antioxidants interact with the free radicals and neutralize the reactivity. Therefore, antioxidant capacity is

3 a b Fig gure 1. (a) Bivariate dot plot representing denatured or fragmented DNA on the x axis and native or normal DNA on the y axis. The peaks on the y axis and x axis represent the cell count exhibiting native (cell count; 4967) and fragmented DNA (cell count; 449), respectively. DNA fragmentation index (DFI: αt %) for this sample = R14/ R14 + R11 (8%, low DFI). (b) Bivariate dot plot representation of semen sample with high DNA fragmentation index ( αt % = 54%). x axis represents fragmented DNA (cell count: 4718) and y axis represents native DNA (cell count: 4021). 161

4 depleted when there is oxidative stress. The total antioxidant capacity measured in this study represents an integrated picture of oxidative stress (Said et al., 2003), and is expected to show an inverse relationship with oxidative stress. Total antioxidant capacity (TAC) was measured in blood plasma using the calorimetric micro plateassay (Product no: TA01; Oxford Biomedical Research Inc., Oxford, USA) according to the manufacturer s protocol. This assay quantitatively measures the total antioxidant capacity or antioxidant power within biological specimens, and encompasses the measurement of enzymes such as superoxide dismutase, catalase, glutathione peroxidase, larger molecules such as albumin, ferritin and ceruloplasmin as well as smaller molecules such as ascorbic acid, α-tocopherol, β-carotene, uric acid, bilirubin, glutathione and methionine. Hormone assays All serum samples were assayed in duplicate for FSH, inhibin B, AMH and free testosterone using ELISA (enzyme linked immunosorbent assay). AMH was measured using an in-house assay, as previously described (Al-Qahtani et al., 2005). The sensitivity of the assay was 0.09 ng/ml. The intraand inter-assay variations were <15%. Inhibin B was assayed using a commercially available kit (Oxford Bio Innovations- DSL, Oxford, UK). The sensitivity of the assay was 10 pg/ ml and the intra- and inter-assay variations were <10%. FSH was measured using a commercial ELISA (IBL Immuno- Biological Laboratories, Hamburg, Germany). The sensitivity of the assay was 1 miu/ml and the intra- and inter-assay variation were <7%. Serum free testosterone was measured using ELISA (IBL Immuno-Biological Laboratories). The sensitivity of the assay was 0.2 pmol/l and the intra- and inter-assay variation was <10%. Statistical analysis Statistical analysis was performed using SPSS v12.1 software (SPSS Inc., Chicago, IL, USA). Semen volume, serum FSH, inhibin B and AMH were normally distributed after log transformation. Sperm concentration, DFI and blood plasma TAC were not normally distributed. Non-parametric tests were performed to analyse these parameters. Independent samples t-test was used to compare the normally distributed parameters, namely age, semen parameters except sperm concentration, and male hormones in the two groups (male factor and nonmale factor group). Non-parametric Mann Whitney U-tests were performed for univariate comparison of sperm concentration, DFI and TAC in the two groups. Pearson s rank correlation coefficients were computed to compare the normally distributed continuous variables and Spearman s rank correlation coefficient was used for the non-parametric correlations. Data are presented as mean ± SEM. P-values < 0.05 are considered statistically significant. Results Male reproductive hormones, DFI and blood plasma TAC in the two groups Semen parameters in the two groups studied are summarized in Table 1. The age of the patients was similar between the male factor group (group 1) and the non-male factor group (group 2). As expected, sperm concentration, motility and progression score were significantly lower in group 1 compared with group 2 (P < 0.001). The hormone levels in the two groups are summarized in Table 1. Serum FSH was significantly higher in group 1 compared with group 2 (10.2 ± 2.6 miu/l versus 4.4 ± 0.4 miu/ml; P < 0.001) and serum inhibin B was significantly lower in group 1 compared with group 2 (99.7 ± 10.0 pg/ml versus ± 9.0 pg/ml; P < 0.05). Three patients (5%) in group 1 had DFI >30%, consistent with high DNA damage and nine patients (14%) had DFI between 15 and 30%, consistent with moderate DNA damage. All patients in group 2 had low DFI (<15%). DFI in group 1 (11.9 ± 3.0%) was significantly higher compared with group 2 (3.9 ± 1.0%; P < 0.05; Figure 2a). Blood plasma TAC was not significantly different between the two groups ( ± 43.8 mmol/l and ± mmol/l, in group 1 and 2 respectively; Figure 2b). Relationship between hormones, semen parameters, DFI and blood plasma TAC The age of the patient was negatively correlated with semen volume (r = 0.22, P < 0.05). There was a positive correlation between serum inhibin B and sperm concentration (r = 0.40, P < 0.001) and sperm motility (r = 0.36, P < 0.05). Serum AMH showed a positive correlation with sperm concentration (r = 0.46, P < 0.02) and semen volume (r = 0.30, P < 0.05). There was also a positive correlation between serum inhibin B and serum AMH (r = 0.50, P < 0.001) and between serum AMH and serum free testosterone (r = 0.25, P < 0.05). Serum FSH was negatively correlated with serum inhibin B (r = 0.37, P < 0.001), serum AMH (r = 0.24, P < 0.05) and sperm motility (r = 0.28, P < 0.05). There was no significant relationship between hormones and DFI. DFI showed a statistically significant negative correlation with sperm motility(r = 0.45, P < 0.001) and semen volume (r = 0.30, P < 0.05). Blood plasma TAC was positively correlated with the age of the patient (r = 0.25, P < 0.05) and negatively correlated with sperm concentration (r = 0.33, P < 0.05). There was no significant correlation between the hormones and blood plasma TAC. There was no significant relationship between DFI and TAC in blood plasma. 162

5 Table 1. Semen parameters, male reproductive hormones, DNA fragmentation index (DFI) and blood plasma total antioxidant capacity (TAC) in male factor (n = 66) and non-male factor infertility (n = 63). Parameter Male factor Non-male factor infertility (n = 66) infertility (n = 63) Age (years) 40.4 ± ± 0.6 Semen volume (ml) 3.2 ± ± 0.2 Sperm concentration ( 10 6 / ml) 12.7 ± 1.8 a 35.3 ± 1.7 a Total motility (%) 47.7 ± 3.2 b 62.6 ± 1.6 b Progression score (out of 4) 2.1 ± 0.1 c 2.4 ± 0.1 c FSH (miu/l) 10.2 ± 2.6 d 4.4 ±0.4 d Serum free testosterone (ng/ml) 3.5 ± ± 0.17 Serum inhibin B (pg/ml) 99.7 ± 10.0 e ± 9.0 e Serum AMH (ng/ml) 3.3 ± ± 0.3 DFI (%) 11.9 ± 3.0 f 3.9 ± 1.0 f TAC-blood plasma (mmol/l) ± ± Values are mean ± SEM; AMH = anti-müllerian hormone. a,b,c,d P < 0.001; e,f P < a b Figure 2. (a) Sperm DNA damage (DNA fragmentation index, DFI) in male factor (n = 66) and non-male factor infertility (n = 63). Data are presented as mean ± SEM. Asterisk (*) denotes P < (b) Blood plasma total antioxidant capacity (TAC) in male factor (n = 66) and non-male factor infertility (n = 63). Data are presented as mean ± SEM. 163

6 164 Discussion The formation of spermatozoa that could achieve fertilizing capacity depends on a complex interplay of testicular and posttesticular factors. Recent studies suggest that oxidative stress could result in sperm DNA damage (Saleh et al., 2003; Agarwal and Said, 2005) and that increased sperm DNA damage is associated with negative pregnancy outcome (Larson-Cook et al., 2003; Virro et al., 2004). It is apparent that normal sperm production and function requires the interaction between Sertoli/ Leydig cells and germ cells, as well as compact chromatin. In addition, intact epididymal function determines the maturation and motility of spermatozoa (Vernet et al., 2004), which is a good predictor of human male fertility in vivo and in vitro. One animal study has looked at the interactions between all the factors causing sperm dysfunction, namely male reproductive hormones, sperm DNA damage and antioxidant capacity (Kaur and Bansal, 2004). This study showed that mice on an antioxidant (selenium) deficient diet had poor semen parameters, low concentrations of FSH, LH and testosterone, deficient antioxidant enzymes and increased DNA damage. This suggests the role of oxidative stress as a common underlying mechanism affecting the interaction between the factors that influence sperm function. A recent human study demonstrated a negative association between serum free testosterone and oestradiol and DFI in fertile men, suggesting the possibility of oxidative stress as the underlying aetiology of sperm DNA damage as well as alteration in testicular hormones (Richthoff f et al., 2002). We have evaluated the interactions between factors that interfere with sperm function, namely hormones, sperm DNA damage and antioxidant activity, in men undergoing evaluation and treatment for infertility. Inhibin B and AMH, produced from testicular Sertoli cells may reflect Sertoli cell function and spermatogenesis (Trigo et al., 2004). AMH is inhibited by testosterone secreted by Leydig cells under the influence of LH. Therefore, alteration in testicular androgens could interfere with Sertoli cell function, thereby contributing to male infertility. Higher inhibin B concentrations are associated with increased sperm concentration and testicular volume (Klingmuller and Haidl, 1997). The study has further confirmed the role of inhibin B and AMH in spermatogenesis through a positive correlation with semen volume, sperm concentration and total motility. There was a good correlation between serum inhibin B and serum AMH, suggesting the common origin of these hormones from the Sertoli cells of the testis. Serum FSH is negatively related to serum inhibin B and serum AMH, confirming the negative feedback role of these Sertoli cell hormones on the pituitary gland. The findings showed that the majority of the patients with male factor infertility had low DFI (82%). However, there was a statistically significant increase in sperm DNA damage as assessed by SCSA in male factor infertility compared with the non-male factor group (P < 0.05) and was negatively related to sperm motility and semen volume. This is in agreement with previous studies (Evenson et al., 1999; Benchaib et al., 2003; Giwercman et al., 2003) that showed an association between increased sperm DNA damage in male factor infertility and a negative correlation with sperm motility. The study also showed a negative correlation between age of the patient and semen volume, suggesting the possibility of increased percentage of sperm DNA damage with increasing age. Plasma total antioxidant capacity, an indirect marker of oxidative stress, was negatively related to sperm concentration, suggesting that an increase in oxidative stress could result in an increase in sperm concentration. Previous studies have confirmed that a limited amount of oxidative stress is necessary for sperm function (Aitken et al., 1989) and stimulation of sperm capacitation, hyperactivation, acrosome reaction and oocyte fusion (de Lamirande et al., 1997). The study further suggests that some increase in oxidative stress resulting in reduction of total antioxidant capacity is associated with increased sperm concentration. Testosterone administration has been shown to increase oxidative stress in rabbit testis and that supplementation with vitamin E results in reduction of oxidative stress (Aydilek et al., 2004). There is controversial evidence from other animal studies suggesting that testosterone has an antioxidant effect (Peltola et al., 1996) and pro-oxidant properties. In this study, there was no statistically significant association between testosterone or the other male reproductive hormones and antioxidant activity. There was no significant relationship between these hormones and sperm DNA damage, suggesting other exogenous mechanisms for sperm DNA damage. OS is probably not the underlying mechanism causing alteration in male hormones, but more studies are needed to confirm this. There was no significant relationship between DFI and blood plasma TAC in this study, suggesting other mechanisms for sperm DNA damage. However, a previous study has reported that there is a significant negative relationship between sperm DNA damage as assessed by SCSA and oxidative stress, using ROS-TAC score as a measure of oxidative stress (Saleh et al., 2003). The discrepancy between the present observation and the previous study could be due to the different samples (blood plasma TAC in the present study as opposed to seminal plasma TAC in the previous study; seminal plasma TAC was not measured in the current study due to insufficient semen samples) and parameters measured as markers of OS that has to be confirmed in the future. It is also acknowledged that in the present study, most of the patients in the male factor group had low sperm DNA damage, and that could contribute to the lack of relationship. In conclusion, this study confirms that serum AMH and inhibin B are markers of Sertoli cell function. Sperm DNA damage is moderately increased in male factor infertility, and is negatively associated with sperm motility. A negative association between antioxidant activity and sperm concentration suggests that a limited amount of oxidative stress plays a role in influencing sperm concentration. However, there is no significant relationship between these hormones and sperm DNA damage or the total antioxidant capacity, suggesting other possible mechanisms for sperm dysfunction. This preliminary study should be followed up with further studies using fertile males as the control group. Acknowledgements We acknowledge the embryologists at the UCL assisted conception unit who carried out the semen analysis and the nurses in the assisted conception unit for their help in recruitment.

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