ASSOCIATION OF IMMUNOLOGICAL FACTOR IN MALE INFERTILITY WITH SEMINAL HORMONES

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1 Health and Population - Perspectives and Issues 36 (1&2), 57-65, 2013 ASSOCIATION OF IMMUNOLOGICAL FACTOR IN MALE INFERTILITY WITH SEMINAL HORMONES Kiran Rangari* and T. G. Shrivastav** ABSTRACT Male infertility is a multi-factorial disorder. With the progress in infertility management, newer technologies such as in-vitro fertilization (IVF) came into existence. It became more relevant to study the factors which might interfere the IVF process in any way. Immunological factor is one such factor, present in male or female genital tract fluid or in blood plasma; and they are found to be in interfering with the processes in IVF by inhibiting the steps in successful fertilization. Immunological factor is found to be present in approximately 10 per cent of the male patients and it generates anti-sperm antibodies either systemically in blood or locally in seminal plasma or cervical mucus. These antisperm antibodies can impair the fertilizing capacity of human spermatozoa, acting negatively on sperm motility and cervical mucus penetration and at the level of in-vitro gamete interaction. In the Andrology Out-patient Clinic of the National Institute of Health and Family Welfare, test for presence of anti-sperm antibodies is done in the patients with either abnormal post-coital test, auto-agglutination of sperm in ejaculates or in case of asthenozoospermia. The span of the current study is over 7 years and the immunological factor is tested in blood and semen of the male partners of the infertile couples. The researchers then tried to establish a relation of the presence of anti-sperm antibodies with that of hormonal levels present either in blood or semen such as testosterone, progesterone, estradiol and cortisol. The findings of the research reflect the presence of higher percentage of anti-sperm antibodies in the semen is associated with the altered seminal hormonal levels. Key words: Male infertility, Immunological infertility, Anti-sperm antibodies, Seminal hormones *Research Officer, kiranrangari@nihfw.org; ** Professor, tgsrivastava@nihfw. org, Department of Reproductive Bio-medicine, National Institute of Health and Family Welfare, Munirka, New Delhi

2 Male infertility contributes to about per cent of total infertility. The four factors which are related to the male fertilizing ability are (a) androgen deficiency, (b) anti-sperm antibodies, (c) testicular histology in men with idiopathic azoospermia and (d) genetic abnormalities. 1 Out of which, around 10 per cent infertility is due to the presence of anti-sperm antibodies present in blood plasma, or seminal plasma or attached to spermatozoa. Around 5 per cent of the infertile females have antibodies to spermatozoa in their circulation or in cervical mucus. The presence of sperm anti-bodies interfere with the process of fertilization invivo and in-vitro, and reduce the chances of pregnancy by about 18 per cent if present in males partners. 2-4 Earlier, this immunological factor was overlooked by the clinicians but with the progresses in ART, such as in-vitro fertilization (IVF), it becomes vital to rule out every factor which may interfere with the fertilization process since IVF is very critical, lengthy, and expensive that causes physical and mental agony to the patients if it fails. 5 In the infertile couples, repeated abnormal Post-Coital Test (PCT) during pre-ovulatory phase, reduction in sperm-motility or auto-agglutinates in semen suggest the presence of anti-sperm antibodies either in blood, cervical mucus or semen It remains unclear whether sperm antibodies are a chance finding or a primary cause of infertility or a sign of underlying defects in sperm membrane or sperm function. The immune system is a complex system and works with endocrine system. 11 So, it was hypothesized that if immune system goes wrong, the endocrine system may also get disturbed and may affect the steroidogenesis. As suggested by another researcher, 12 the immunological causes of decreasing fertility may depend on neuro-endocrinological relations. Earlier studies have shown that low levels of testosterone in vasectomized males with ASAB. 13,14 Recent studies also suggest a relationship between testicular steroid hormone levels with autoimmunity and sperm-antibodies which influence the motility of ejaculated spermatozoa. 15 Increased estradiol levels in serum and/or seminal plasma were also correlated with abnormal sperm functions such as damaged spermatozoa, infection of genital tract or loss of motility Progesterone regulates capacitation, hyperactivation, and acrosome reaction or priming of the cells to the subsequent action of zona pellucida proteins which is present in the semen but acts in the female genital tract or loss of motility Progesterone regulates capacitation, hyperactivation, and acrosome reaction. The first description of the hormone cortisol in human seminal plasma as estimated by Amersham Amerlite iuminescence immunoassay was / (31-94) ng/ml. This is 60 per cent of the level of cortisol in blood serum. 21 Detection of anti-sperm antibodies is a routine test in cases of abnormal PCT at NIHFW Andrology outpatient clinic. Data of 7 years from of detection of antisperm antibodies are for the research. The antibodies were detected in 58

3 the serum, semen or cervical mucus. The testosterone, estrogen progesterone and cortisol levels were estimated in randomly selected blood serum or semen samples from antibody positive and antibody negative samples. OBJECTIVES Objectives of this research are to: detect the percentage of immunological infertility in men attending the Andrology Out-patient Clinic of NIHFW, detect the percentage of agglutinating antibodies and immobilizing antibodies present in the blood serum and/or seminal plasma of the subjects, estimate the sex hormones such as testosterone, estradiol, progesterone and cortisol in the blood serum and/or seminal plasma of men with or without anti-sperm antibodies, and correlation of presence of agglutinating or immobilizing anti-sperm antibodies with that of altered serum or seminal sex hormone concentrations, such as testosterone, estradiol, progesterone and cortisol and its significance in male infertility. METHODOLOGY Subjects: Male partners of the infertile couples attending the Andrology Out-patient Clinic at NIHFW, New Delhi, in the age group of years were selected for this study. The study has been conducted over 7 years from January 2000 to June 2007, who has shown to have abnormal PCT. The total number of serum samples tested was 594 and seminal plasma samples were 591. Detection of Anti-sperm Antibodies: The serum and seminal plasma samples were incubated at 56 C for 30 minutes to inactivate the complement factor present in it. Agglutination test was carried out according to the method of Kibrick 22 and immobilization test is done according to Isojima et al. 23 Both the tests are done simultaneously. Hormone Estimation: Serum as well as seminal plasma levels of steroid hormones such as testosterone, progesterone, estradiol and cortisol were estimated. Testosterone was estimated by the RIA method of Abraham 24 and the progesterone and cortisol was estimated in serum and seminal plasma by the ELISA technique of Basu et al. 25 Estradiol was estimated in seminal plasma only. Statistical Analysis: Data were expressed as mean ± SEM and subjected to analysis adopting Students t-test. Differences were considered significant if the probability of their occurrence was <

4 FINDINGS Detection of Anti-sperm Antibodies: Of all the patients tested for immunological factor over January 2000-June 2007; the total number of serum samples tested were 594 and total number of semen samples tested were 591. Out of the 594 serum samples tested anti-sperm antibodies, 31 (5%) samples were found to be positive for agglutination test and 71 (11%) tested positive for immobilization test. Out of the 591 seminal plasma, only 18 (3%) samples were tested positive for agglutination test and 165 (28%) samples were found to be positive for immobilization test Table 1. TABLE 1 PRESENCE OF ANTI-SPERM ANTIBODIES IN THE BLOOD SERUM AND SEMINAL PLASMA SAMPLES Samples Assayed Antibody +ve Samples SAT SIV SAT SIV Blood (5%) 71 (11%) semen (3%) 165(28%) Steroid Hormone Levels Testosterone: No significant difference was found between the mean serum testosterone levels of men with (4.8 ±0.6 ng/ml) or without antibodies (4.6±0.4 ng/ml) whereas testosterone levels were found to be significantly lower ( p< 0.001) in antibody positive seminal plasma samples than the antibody negative samples (0.46±0.007ng/ml versus 0.99±0.06 ng/ml) Table 2. TABLE 2 TESTOSTERONE LEVELS IN ANTIBODY POSITIVE AND ANTIBODY NEGATIVE SERUM AND SEMEN SAMPLES Testosterone Levels in antibody positive samples (ng/ml) Testosterone levels in antibody negative samples (ng/ml) Blood 4.8±0.6 (n=20) 4.6±0.4 (n=17) Semen 0.46±0.07** (n=35) 0.99± 0.06 (n= 18) (**p<0.001) Estradiol: The seminal estradiol levels were significantly higher (p<0.005) in the antibody positive samples than in the antibody negative samples (99±24 versus 46±8.7 pg/ml) Table 3. 60

5 TABLE 3 ESTRADIOL LEVELS IN ANTIBODY POSITIVE AND ANTIBODY NEGATIVE SEMEN SAMPLES Estradiol levels in antibody positive samples )(pg/ml Semen 99±24*** (n=35) (***p<0.005) Estradiol levels in antibody )negative samples (pg/ml 46±8.7 (n= 18) Progesterone: Table 4 shows no significant difference in the mean progesterone levels in antibody positive and antibody negative serum samples (2.63±0.34ng/ ml and 3.02±0.24ng/mL). However, the mean seminal progesterone level was significantly higher (p<0.001) in antibody positive semen samples than in antibody negative semen samples (3.68±0.56 versus 2±0.02ng/mL). TABLE 4 PROGESTERONE LEVELS IN ANTIBODY POSITIVE AND ANTIBODY NEGATIVE SERUM AND SEMEN SAMPLES Progesterone Levels in antibody positive (ng/ml) Blood 2.63±0.34 (n=28) 2.82±0.24 (n=20) Semen 3.68±0.56 ** (n=40) 1.98±0.02 (n=34) (**p<0.001) Progesterone levels in antibody negative (ng/ml) Cortisol: The mean cortisol levels of antibody positive serum samples to that of antibody negative serum samples did not show any significant difference (245±23ng/mL versus 256±32ng/mL) whereas significantly reduced (p<0.05) cortisol levels were found in antibody positive seminal plasma (75±5.5ng/mL) than in antibody negative seminal plasma samples (95±6.7 ng/ml) Table 5. TABLE 5 CORTISOL LEVELS IN ANTIBODY POSITIVE AND ANTIBODY NEGATIVE SERUM AND SEMEN SAMPLES Cortisol levels in antibody positive (ng/ml) Blood 245±23 (n=18) 256±32 (n=20) Semen 75±5.5* (n=32) 95±6.7 (n=28) (*p<0.05) Cortisol levels in antibody negative (ng/ml) 61

6 DISCUSSION In the current study, the authors have detected the percentage of immunological infertility in men attending the Andrology Outpatient clinic to see the association of presence of anti-sperm antibodies with that of steroid hormones present in serum and seminal plasma. Antibodies were found to be more of local origin than of systemic origin. Activated Local immunity suggested a strong possibility of infection or mechanical causes. 26,27 The researchers found only 3 per cent of semen samples with agglutinating antibodies and 28 per cent semen samples with immobilizing antibodies. In circulation also, immobilizing antibodies were present in 11 per cent of the samples with higher concentration whereas only 5 per cent of the samples showed agglutinating type of antibodies. The data suggest that immobilizing antibodies which immobilizes the sperm in presence of complement making it non-functional may be associated with that of decreasing quality of semen with overall reduction in male fertility. 28 It is seen that steroid hormones such as testosterone and estrogen play a role in male fertility, and any alteration in their concentration leads to infertility in males. 19 Here again, it has been observed that antibody positive samples showed a higher level of estradiol and a lower level of testosterone in semen samples. Similarly, cortisol which is secreted by adrenal gland and known to be immunesuppressive hormone was found to be decreased in the antibody positive seminal plasma samples. The level of progesterone was also found to be elevated in the seminal plasma of antibody positive samples. The data is positively correlated with an earlier study of the current authors. 29 The immunological system works completely in accordance with the neurological and endocrinological system forming neuro-endocrino-immuno complex. 12 The factors such as stress may be responsible for triggering such a complex. 30 Further studies are desired to understand these relations. CONCLUSION From this study, it could be concluded that the higher percentage of immobilizing antisperm antibodies in the semen may be the cause of infertility in the couple and it could be related with the seminal hormonal levels. It would be interesting to study the seminal hormones in case of immunological male infertility. Acknowledgement: The study was supported by the National Institute of Health and Family Welfare, New Delhi, India. The authors acknowledge the support rendered by Prof. J.K. Das, Director, Prof. S.K. Sood, Dean of Studies and Prof. K. Kalaivani, Head, Department of Reproductive Biomedicine; during the course of the study. 62

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