The patterns and occupational distribution of hormonal abnormalities among men investigated for infertility in some centers in the southwest, Nigeria

Background and aim: Male factor infertility results from dysfunction at various levels of spermatogenesis, sex hormone abnormalities, and occupation or workplace exposure to toxins are involved. This study was designed to determine the frequency of occupational distribution of men who were evaluated for infertility, the patterns of hormonal abnormalities, and to associate hormonal abnormalities with occupational categories in some centers in Osun State, Nigeria. Methods: Semen and 5 mL of whole blood were collected from the infertile men (n=319) who were referred to the laboratories for fertility investigation after physical and medical examination. Semen analysis was performed microscopically according to the World Health Organization manual while serum gonadotrophin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and prolactin were determined by Enzyme-linked Immunosorbent assay technique using reagents supplied by Biorex Diagnostics Limited, Antrim, United Kingdom. The subjects were grouped based on semen characteristics. Results: More than half 164 (51.4%) of the subjects were within the age group of 31-40 years, followed by 70 (21.9%) in the age group of 21-30 years, while 67 (21.0%) was in the age group of 41-50 years. Some 133/319 (41.7%) of the subjects had normal sex hormone levels while 186/319 (58.3%) had abnormal hormone levels. The patterns of hormonal abnormalities observed were 96/186 (51.6%) normogonadotrophin-hypogonadism, 49/186 (26.3%) normogonadotrophin-hypergonadism, 14/186 (7.5%) elevated FSH levels, 15/186 (8.1%) elevated LH levels, 07/186 (3.8%) hypergonadotropic-hypergonadism, and 05/186 (2.7%) hyperprolactinemia. Artisans (OR 1.2252 95%CI 0.367-2.472), workers in chemical related industries (OR 1.667, 95%CI 0.594-4.676), and businessmen (OR 1.200, 95%CI 0.110-3.49) are more likely to be predisposed to hormonal abnormalities. Conclusion: The patterns of hormone abnormalities as well as their relative proportions are slightly different from those reported previously. Some occupations may predispose workers to hormonal disorder than the others. Relevance for patients: This is a cross-sectional study of males investigated for infertility; the contribution and patterns of hormonal abnormalities were evaluated. The possible association between workplace and infertility that may assist in the management of patients with male infertility was evaluated.


Introduction
Infertility is a disease of reproductive systems characterized by the inability of a sexually active, non-contraception couple to achieve pregnancy in 1 year after regular unprotected intercourse [1]. Globally, about 15% of all sexually active couples of reproductive age are affected by infertility and about 40-50% are attributed to abnormalities in males, called male factor infertility [2]. Infertility is not only a medical challenge; it also results in psychosocial problems such as marital disharmony, stigmatization, depression, and psychiatric challenges. Not until recently, Nigerian-sociocultural believed that infertility was attributed only to women. It is now clear that the problems are shared equally between males and females. There appears to be an increasing willingness of men nowadays than before to present for evaluation for infertility.
Male infertility ensues as a result of diminished bioavailability and dysfunctions at various levels along the hypothalamic pituitary-Gonadal axis (HPGA): Pre-testicular (damage at the hypothalamus or pituitary level), testicular (failure of the testis), post-testicular (normal testicular function but with obstruction or inflammation that leads to infertility) or a combination of these [3]. It was estimated that about 187 million couples worldwide or one out of every six couples of reproductive age are affected [4]. However, in sub-Saharan Africa, the prevalence of male infertility is higher and is currently reported to be between 30 and 50% [5][6][7][8].
The functionalities of the male reproductive system are largely dependent on hormones that are produced by endocrine glands. Abnormalities in hormone production and transportation challenges to the sites of action have been reported as one of the causes of male infertility and hormonal therapy has been used to correct the abnormality [9,10]. Furthermore, male factor fertility depends on viable production of normal quality, and quantity of healthy sperms in the testes, which, in turn, is dependent on adequate production and release of gonadotropin hormones by the pituitary gland at the base of the brain.
The etiological factors of male infertility are many and the contribution of endocrine abnormalities has been evaluated by several authors [7,8,[11][12][13][14]. Endocrine involvements in male infertility are increasingly identified and the patterns vary from one region of Nigeria to another probably due to differences in lifestyle behaviors such as nutrition [15,16]. Others have attributed this to differences in environmental pollutions as a result of crude oil exploration and exploitation [17,18]. These lifestyle habits are modifiable and evolving; therefore, it is important to periodically investigate hormonal patterns of male infertility.
Available evidence indicates that physical and chemical hazards in the workplace can impact negatively on male fertility, but the workers may be unaware of such challenges. The association of industrial workers with poor indices of semen among men underdoing fertility treatment has been reported. Exposure to occupational hazards can alter the sex drive of men and women, as well as impairment of fertilization potentials. Disruption of spermatogenesis may occur with occupational exposure to organic solvents which are commonly found in industrial companies [19].
It is imperative to have a recall of work-related toxins exposure when evaluating men for infertility.
This present study was designed to determine the frequency of occupational distribution of men who were evaluated for infertility, the patterns of hormonal abnormalities, and to associate hormonal abnormalities with occupational categories in some centers in Osun State, Nigeria.

Inclusion criteria
Thorough physical and medical examinations were conducted on the participants by the attending physicians. Only those who met the inclusion criteria were recruited in the study. They consist of males aged 21-60 years who were referred to the laboratory for semen analyses as part of their investigation for infertility, gave consent, without physical abnormalities or chronic illnesses were included in the study. Subjects without chronic clinical illnesses and had their babies within the last 1 year, whose seminal fluid concentrations were over 15 million sperm cells per milliliter according to the WHO criteria [20] were included and used as controls.

Exclusion criteria
After physical and clinical examinations, individuals with known pathological or congenital conditions such as severe hypertension, diabetes mellitus, sexually transmitted diseases, testicular varicocele, and genital warts were excluded from the study. Besides, individuals currently on antioxidant food supplements, smoke cigarettes, and consume alcohol were also excluded due to their high seminal reactive oxygen species levels and possibly low antioxidant activity which might lead to decreased motility and abnormal sperm morphology.

Sample size
The sample size (n) was calculated using an estimated prevalence of 40% from a previous study on male infertility in Nigeria (Emokpae et al., 2007) and sample size determination formula by Lwange and Lemeshow [21], N=Z 2 (1-P)P/d 2 . The calculated sample size was 369 which was increased to 400 for the purpose of this study. Some 81 respondents were later excluded because they admitted to smoking and consume alcohol and 319 subjects were eventually used in the study.

Specimen collection
A total of 319 male subjects evaluated for fertility were included in the study, and 100 age-matched healthy men who had fathered a child within the last 12 months were recruited as controls. Semi structured questionnaire was used to collect socio-demographic data of both infertile and control subjects. The questionnaire was administered by trained research assistants at the various centers. Thereafter, the subjects were taught on how to collect semen specimen after at least 3 days of sexual abstinence and brought to the laboratory immediately. The specimens were collected in a wide-mouth container without the use of condoms or spermicidal cream. Exactly 5 mL of blood specimen was obtained from the subjects in the morning for hormonal determination. The blood was allowed to clot and was centrifuged after clot retraction at 3000 rpm for 10 min. The serum was then kept frozen at −20°C until analyses were done. The semen analyses were done immediately the specimen was received in the laboratory.

Semen analysis
For each semen sample, the color, pH, viscosity, and liquefaction time were recorded. After, liquefaction, the sperm concentration was counted in million/mL using a Mackler counting chamber (semen Analysis Chamber, ISO 9001:2000) and was viewed under a binocular microscope (Olympus) at ×100. The motility, morphology, and progressive motility were also assessed microscopically according to the WHO recommendation [20]. An appropriate quality control measure was observed as recommended by the WHO. The semen assay was done on two different occasions at 3 months interval for those semen specimens that gave abnormal results and the mean was determined and used for the results.

Hormonal evaluations
The GnRH, FSH, LH, testosterone, and prolactin evaluations were done by Enzyme-linked-Immunosorbent Assay technique (ELISA) using reagents supplied obtained from Biorex Diagnostics Limited (ISO: 13485) Antrim, UK. The analyses were done by following the manufacturer's protocol and read with the aid of a microplate reader. The hormones analyzed include GnRH, FSH, LH, and testosterone. The assays were based on competitive ELISA technique.

Principle
The assay employs high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition in excess, of native antigen. On mixing monoclonal biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen reacts, to form an insoluble sandwich complex. Simultaneously, the complex is deposited to the well through the high-affinity reaction of streptavidin and biotinylated antibody. The antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration. Several different serum references of known antigen values were used to generate a dose response curve from which the antigen concentration of an unknown was calculated.

Statistical analysis
The data generated from the study were compared between the groups using unpaired Students-test and Chi-square. The statistical significance level was set at P<0.05. The statistical software SPSS version 21 (SPSS INC, Chicago, IL, USA) was used for the analysis.

Results
The findings from this study are presented in Tables 1-7. Table 1 shows the demographic characteristics of the studied participants. Most of them 164/319 (51.4%) belong to the age group 31-40    Table 2 indicates the lifestyle characteristics of the subjects; they were non-smokers, non-alcohol consumers and only 109/319 (34.2%) engage in regular exercise while majority 210/319 (65.8%) do not exercise regularly. Bivariate analysis of categorical data on exercise indicates a significant difference between those on regular exercise and those not exercising (P<0.001). Greater proportion of the respondents 311/319 (97.5%) do not use any form of contraceptive while 08/319 (2.5%) use one form of contraceptive.
There was no statistically significant association between the occupation of the test subjects and the subtype of infertility with (X) 2 (8)= 9.391 and P>0.05.

Discussion
Infertility is a major public health concern among couples, and occupational hazards may be the main cause of infertility in men. Several authors have reported that physical and chemical hazards in the workplace can have an adverse effect on male fertility [22]. Occupation and exposure to harmful environmental   contaminants are well known endocrine disruptors and are leading causes of infertility. Preventing adverse occupational effects on male infertility is important for health-care providers. Therefore, the provision of employee awareness, providing appropriate preventive measures when performing hazardous jobs would go a long way to preventing infertility. Furthermore, understanding the functions of the hypothalamic-pituitary-gonadal (HPG) axis is important in the investigation and management of male infertility. Despite the high control mechanisms involved in spermatogenesis, any derangement within the axis can result in the male reproductive failure. In this study, 58.3% of men investigated for infertility had hormonal abnormalities which is higher than 46.9% reported from Ilorin [14], 40% among azoospermia, 40% among oligozoospermia from Kano, respectively [7,12], and 7.3% reported from Maiduguri [23], but lower than 80.1% among males investigated for infertility reported from Enugu [8]. Male infertility of endocrine origin can manifest in several patterns. The observation of significantly lower FSH, testosterone, and higher LH levels aligns with the previous studies [24][25][26], but at variance with other authors [14,27]. The observed lower FSH and higher LH and testosterone levels may be attributed to testicular damage not involving the Leydig cells, but involves in androgen biosynthesis or extra-gonadal androgen synthesis [14].
In the same vein, five different patterns of hormonal profile abnormalities were reported among men investigated for infertility in Ilorin; (1.5% hypogonadotropic-hypogonadism, 11.5% hypergonadotropic-hypogonadism, 17.7% hypergonadotropicnormogonadism, 16.2% normogonadotropic-hypogonadism, and 53.1% normogonadotropic normogonadism) [14]. Six patterns of hormonal abnormalities were however observed in this study among infertile men with abnormal hormone levels, which are; 51.6% (96/186) normogonadotrophic-hypogonadism,  [8,23]. Such findings may be an indication of seminiferous tubular damage which may involve the Leydig cells. Individuals with this condition may benefit from sperm retrieval and in vitro cytoplasmic sperm injection techniques to achieve conception [14].
The observed isolated elevation of LH and FSH levels may be due to attempts to stimulate the Sertoli cells and Leydig cells, respectively, to produce androgen proteins and testosterone that is necessary for spermatogenesis. The high FSH level may also be an indication of decreased testicular function which can be attributed to altering feedback mechanism along the HPG axis.
The 2.7% hyperprolactinemia observed in this study is lower than 5.1% reported by Ozoemena et al. [8], 15.4% reported by Oladosu et al. [14], 11% by Masud et al. [28], and 12.2% by Soler-Fernadez et al. [29]. High serum prolactin level hurts spermatogenesis and steroidogenesis through its action on prolactin receptors on Sertoli cells and Leydig cells in the testes to cause hypogonadism by altering the pulsatile release of gonadotrophins [28]. Although hyperprolactinemia is not common among men with infertility, subjects with hyperprolactinemia may be investigated for thyroid dysfunction. About 4.7% of individuals were observed to have high levels of GnRH levels in this study. This is consistent with the previous study, where it was reported that acquired defects in GnRH release commonly referred to as the GnRH pulse generator [30]. It was commonly observed in women as a result of stress, excessive exercise, and eating disorders that impair reproductive potentials. Some authors have reported acute reversible changes in GnRH-induced gonadotrophin secretion among men with severe stress and illness [12].
Evidence linking work-place or occupational exposure to harmful effects on male infertility is on the increase. Infertile men might benefit from assessment of work-place contribution to their condition. The highest proportions of infertile men (68.5%) were artisans while the lowest 40.0% were farmers. The occupational distributions likely to be associated with abnormal hormone levels among participants are artisans (OR 1.25 95% CI 0.367-2.472), workers in chemical related industries (OR 1.667 95% CI 0.594-4.676), and businessmen (OR 1.200 95% CI 0.110-3.49). This is inconsistent with the previous study, where no association with occupation was observed among 1164 males treated for infertility. The authors reported no significant difference in the measured fertility indicators between occupational categories [22]. A significant decline in sperm quality and endocrine abnormalities among infertile men of different occupations has been reported [31].
In this study, overwhelming number of respondents (41.7%) had normal hormone levels. The cause of their infertility in this group is not immediately ascertained since the etiologies of male infertility are numerous and include genetic, physical abnormalities, injuries, drugs, infections of the genital tract, radiation, toxins, or unexplained [32]. The role lifestyle factors play in the etiology of infertility has been a subject of interest among researchers. Recently, some authors have provided evidence of a correlation between lifestyle behaviors and infertility in both men and women. The enumerated lifestyle factors include delayed childbearing due to pursuit of career or education, age of starting a family, eating of fat-rich diets, smoking, alcohol and caffeine consumption, exercise, risky sexual behaviors, drug misuse, anxiety/depression, cellular phones, and radiation among others [33]. In this study, majority of the respondents do not carry-out regular exercise and the difference in the number of respondents who carryout regular exercise and those who do not was statistically significant (P<0.001). Available evidence suggests that exercise may contribute to improvement in the quality of sperm parameters from 9.7% to 15.2% in men performing regular exercise of 1 h, at least 3 times a week [34]. Others have observed that moderate regular exercise may lead to the reduction of seminal plasma oxidative stress than rigorous regular exercise [35]. Regular moderate exercise was recommended and may improve fertility potential among infertile subjects [33].
The majority of the respondents (51.4%) were in the age group (31-40 years). This aligns with a previous study by Benbella et al. [36] among Moroccan subjects, where most of the infertile men were between the ages ranges of 35 and 40 years (34.8%). Variation in the age range and percentage could be due to population size differences.

Conclusion
This study has demonstrated the various patterns of endocrine abnormalities among males investigated for infertility. The patterns of hormonal abnormalities as well as their relative proportions are slightly different from those previously reported. Some occupations may predispose workers to hormonal disorders than the others.