HORMONAL PARAMETERS AND SEMEN MICROBIOLOGICAL PATTERN OF INFERTILE MALES: A COMPARATIVE CROSS-SECTIONAL STUDY IN BENIN CITY, SOUTH SOUTH NIGERIA

Abstract

Background

In many Nigerian studies, male factors were responsible for up to 50 to 70% of infertility.

Aim

To evaluate the contribution of endocrinopathy and bacteriospermia to male infertility in Benin.

Study design

A comparative cross-sectional study

Setting

Department of Obstetrics and Gynecology of Central Hospital, Benin City

Methodology

Male partners of 355 couples who presented with infertility over 8 months were studied. The data collected included socio-demographics, clinical features, hormonal assays, semen analysis, and semen culture. These were analyzed using Statistical Package for Social Sciences version 21.0, employing both univariate and bivariate analyses, and multivariate logistic regression to identify independent determinants of abnormal semen analysis.

Results

The mean age of the subjects was 34.98±4.67 years and mean duration of infertility was 4.5±2.17 years. A total of 59.7% patients were of low socioeconomic status. The participants had at least a primary school education, and 81.7% dwelt in the urban areas of the state. Secondary infertility was the commonest (82.3%); while 56.6% of the subjects were overweight and 14.9% obese. 66.5% of participants had abnormal semen parameters. Oligospermia was the commonest (22.8%) abnormality. The commonest hormonal disorders were abnormality of luteinizing hormone (79.1%) and follicle stimulating hormones (26.8%). There was positive semen culture in 162(43.6%) of participants, of which 135(83.3%) had abnormal semen analysis (p=0.000). Men with positive semen culture had more than four-fold odds of having abnormal semen analysis (OR=4.554; 95%CI=2.761–7.513).

Conclusion

This study has shown that male endocrinopathy and bacteriospermia contributed significantly to male infertility in our environment.

Introduction

The inability of a couple to procreate poses a medical problem with profound psychosocial and socio-cultural undertone. Infertility is a global problem affecting 10-20% of couples; with much higher prevalence reported in the developing countries1, 2. It afflicts about 20-45% of couples in Nigeria2, 3. Awareness of the contributions of the male gender to the burden of infertility is gradually increasing among Africans. In many Nigerian studies, male factors were responsible for up to 50 - 70% of infertility4, 5.

Evaluation of male reproductive failure poses a special challenge because of multiplicity of aetiological factors6. However, these factors ultimately affect the production or otherwise of functionally competent spermatozoa as normal semen parameter is a good predictor of normal male fertility potential7. But beyond routine seminal fluid analysis, ab-initio assessment of other male reproductive compartments has been advocated7. This includes the male hormonal status and assessment of semen for markers of infection-induced inflammatory process in the genital tract. A normal hormonal environment is required for optimal male reproductive functions8,9. Hormonal disorders in males are thought to be uncommon, but they are significant because they constitute a group of treatable causes of male infertility9,10.

Infection involving any segment of the male genital tracts can impair fertility. The pathophysiological mechanism of infection-induced infertility involves the inhibition of spermatogenesis, obstruction of the genital tubes, and disruption of the functional capacity of the spermatozoa6,11,12. Presence of bacterial organisms and pus cells in the semen suggests the possibility of ongoing genital infections.

The most widely used semen reference value is that defined by the World Health Organization13. In this system, semen is assessed in terms of number, motility and forms. There are limitations to this evaluation system14; it nevertheless remains a handy tool in assessing and predicting male infertility8. Evaluation of sperm function is a more advanced and sophisticated test, but they are not recommended for routine application15. The knowledge of how the male hormonal profile and semen microbiology affects the semen parameters is poorly documented in the African setting. Despite the high burden of infertility in Nigeria, not much effort has been made at tackling the problem9.

This study explored the relationship between the pattern of seminal fluid parameters, hormonal profile and semen microbiology among male partners of women with infertility in Benin City, Nigeria.

Patients and Methods

This was a comparative cross-sectional study conducted in the department of Obstetrics and Gynecology, Central Hospital Benin City, Nigeria. Consecutive male partners of women presenting with inability to conceive between January 2017 to August 2017 who after been counseled gave consent to participate in the study were enrolled. Men who objected to sperm collection, had chronic medical diseases, were on long term medications, or had been previously treated for infertility were excluded from the study. The sample size of 355 was determined using the formula by D.W. Taylor16>for cross sectional studies based on local infertility prevalence rate of 30%, and a 10% attrition rate.

Each participant was interviewed to extract basic information, after which physical examination was carried out. Thereafter, blood and semen were properly collected respectively for hormonal assay (follicle stimulating hormone [FSH], luteinizing hormone [LH], prolactin and testosterone) and semen analysis/culture.

To conduct a hormonal assay, 5mls of blood was collected into lithium heparin container, centrifuged at 2400 revolutions per minute for 15 minutes on a bench-top centrifuge. The plasma was separated and frozen at -20ºc, and allowed to thaw at room temperature before assay was carried out for testosterone, prolactin, LH, and FSH levels.

Two semen analyses were conducted on each participant two weeks apart. Those with consistent results using the 1999 WHO criteria13 were included in the study. Following instruction on methods of collection, semen samples were collected in the laboratory collection room by masturbation into a sterile wide-mouthed plastic container after a minimum of 3 to 5 days of ejaculatory sexual abstinence. The samples were submitted to laboratory within 15 minutes of collection.

Sperm motility test was conducted by placing a drop of the sperm cells suspension on a clean grease-free slide, covered with a cover slip and examined. A total of 100 spermatozoa were counted and the percentage of motility recorded. To determine the sperm count, a 1:10 dilution of the sperm suspension was made in physiological saline and a capillary tube was used in collecting a portion and charged into Neubauer haemocytometer. The cells in the appropriate ruled areas of the counting chamber were counted. Sperm viability test was conducted by mixing one drop (10 - 15µl) of 0.5% eosin with one drop of semen on a slide. After 2 minutes; the preparation was examined microscopically using x10 objective to focus the specimen and the x40 to count the percentage of viable and non-viable spermatozoa. In assessing sperm count and morphology, a thin smear of the liquefied well mixed semen was made on a clean grease-free slide and fixed with 95% ethanol for 5-10 minutes and then allowed to air dry. The smear was washed with sodium bicarbonate formation to remove any mucus that may be present. It was then rinsed several times, and stained with dilute carbolfuchsin (1 in 20) and allowed to stain for 3 minutes followed by washing with water. Dilute (1 in 20) loeflers methylene blue was used to counterstain the smear for 2 minutes. It was then washed with water, drained and allowed to air dry. Using the x100 objective, 100 cells were counted and the percentage showing normal morphology and the percentage that appears abnormal was recorded. Sample was also examined for appearance, liquefaction, viscosity, ph and cellular elements other than spermatozoa.

All the collected seminal fluid samples were subjected to culture and sensitivity. A loop full of the semen sample was inoculated into oven-dried chocolate agar, blood agar and Macconkay’s agar. The wire loop was flamed in between the streaking so as to get distinct colonies. Properly streaked plates were incubated at 37oc for 18-24 hours. Those with growth were subjected to identification for proper speciation. Those without growth were further incubated for another 24 hours. The identified pathogens were subjected to antibiotic sensitivity testing using Mueller-Hinton agar.

The study was approved by the ethics & research committee of Central Hospital, Benin.

Data management and analysis

Information was extracted using a data collection sheet designed for the purpose. The data were coded and analyzed using Statistical Package for Social Sciences (SPSS) version 21.0. This consisted of initial univariate and bivariates analyses, and then multivariate logistic regression analysis to identify the independent determinant of abnormal semen parameters in male infertility. Test of statistical significance was based on 95% confidence interval and p <0.05 using chi-square test with Fisher Exact correction where applicable.

Results

A total of 355 male subjects participated in the study. Table 1 shows the socio-demographic characteristics of the participants. The mean age of the subjects was 34.98±4.67 years. Majority (96.6%) were 25 years and above, and had been infertile for a mean duration of 4.5±2.17years. More than half (59.7%) were of low socioeconomic status, all participants had at least a primary school education, and most (81.7%) dwelt in the urban areas of the state. Secondary infertility was the commonest (82.3%). In all, 27.9% of the subjects had normal BMI (18.5-24.9kg/m2), the others were underweight (0.6%), overweight (56.6%), or obese (14.9%).

Table 1. Socio-demographic characteristics of male partners of women with infertility.

	N	%
Parameters
Age group (yrs)*
<25	12	3.4
25 – 35	170	47.9
>35	173	48.7
Duration of Infertility #
< 2 years	12	3.4
> 2 years	343	96.6
Social Status
Low	212	59.7
Middle	95	26.8
High	48	13.5
Educational Level
No formal educationPrimary	042	011.8
Secondary	174	49
Tertiary	139	39.2
Location of residence
Rural	65	18.3
Urban	290	81.7
BMI
Underweight	2	0.6
Normal weight	99	27.9
Overweight	201	56.6
Obesity	53	14.9
Subset of Infertility
Primary	63	17.7
Secondary	292	82.3
*=>Mean age = 34.98 (SD 4.67); Median age = 35.00; Range = 24-46. # =>Mean duration = 4.50 (SD 2.17); Median duration = 4.0; Range 1-12. §=> Mean duration = 4.52 (2.17); Median duration = 4.10; Range = 1-12

Abnormal semen parameters were observed in 236 (66.5%) patients. Fig. 1 shows the pattern of seminal fluid abnormalities. Oligospermia was the commonest (22.8%), and there were various degrees of single and combined abnormalities.

Fig. 1: Pattern of semen parameters among male partners of women with infertility.

Table 2-a shows the pattern of hormonal abnormalities among the study participants. Abnormalities of LH and FSH levels were the commonest hormonal disorders observed, being present in 79.1% and 26.8% of the subjects respectively. Testosterone level disorder was least common, found in 5.1%. Table 2-b shows the association between hormone patterns and semen analysis. Of the 95 participants with abnormal FSH level, 89.5% was associated with abnormal semen parameters while 10.5% had normal parameters; this association was statistically significant, showing a greater than six-fold chances of males with abnormal FSH levels having abnormal semen parameters (P=0.000; or 6.14, CI= 3.05 - 12.36). Of the 43 participants with abnormal prolactin level, 86% of them had abnormal semen parameters, with more than 3 folds odds of men with abnormal prolactin having abnormal semen parameters (p=0.004; or 3.50, CI-1.43-8.55). Abnormal testosterone level was associated with abnormal semen parameters in 94.4% of the 18 subjects affected, showing more than 9 folds odds of been associated with abnormal semen parameters (P=0.009; OR 9.16, CI 1.20 - 69.69). Although there were high proportions of subjects with abnormal LH level, 68.7% of these (representing the least among the four hormones tested) had abnormal semen parameters. Thus, the association between abnormal semen and abnormal LH was not statistically significant. Furthermore, upon multivariate logistic regression analysis of hormonal levels to male infertility (as shown in Table 2-c), abnormal FSH and Prolactin levels were found to be independent risk factors for abnormal semen parameters.

Table 2-a. Pattern of hormonal abnormality of the study participants.

	Abnormal		Normal
	N	%	N	%
FSH	95	26.8	260	72.2
LH	281	79.1	74	20.8
Prolactin	43	12.1	312	87.9
Testosterone	18	5.1	337	94.9

Table 2-b. Association between hormonal level and semen analysis.

	Semen Analysis
	Abnormal		Normal		P	OR	95% C.I for OR
	N	%	N	%
FSH
Abnormal	85	89.5	10	10.5	0.000	6.14	3.05 - 12.36
Normal	151	58.1	109	41.9
LH
Abnormal	193	68.7	88	31.3	0.086	1.581	0.93 - 2.68
Normal	43	58.1	31	41.9
Prolactin
Abnormal	37	86.0	6	14.0	0.004	3.5	1.43 - 8.55
Normal	199	63.8	113	36.2
Testosterone
Abnormal	17	94.4	1	5.6	0.009	9.16	1.20 - 69.69
Normal	219	65.0	118	35.0

Table 2-c. Multivariate logistic regression of hormonal level to male infertility.

	P-value	OR	95% C.I. for OR
FSH	0.00	0.19	0.09-0.38
LH	0.96	0.98	0.56-1.72
Prolactin	0.02	0.34	0.14-0.87
Testosterone	0.20	0.25	0.03-2.09

Table 3-a shows the microbiological pattern of organisms cultured in the semen of male partners of infertile females. Various bacterial organisms were isolated in 162 of the 355 participants, giving a positive semen culture rate of 43.6%. The commonest bacteria isolated were Staphylococcus aureus (89.9%), followed by Escherichia coli (25.3%), Klebsiella spp (7.4%) and Proteus spp (7.4%). Table 3-b shows the relationship between semen culture and abnormal seminalysis. Of the 162 men with positive bacteria semen culture, 135 (83.3%) had abnormal semen analysis parameters while 27 (16.7%) had normal semen analysis parameter. This difference was statistically significant (p=0.000). Men with positive bacteria semen culture had more than four folds odds of having abnormal semen parameters (OR= 4.554; 95%CI=2.761 – 7.513). Table 3-c shows the antibiotic sensitivity pattern of the isolates. Most of organisms were sensitive to ofloxacin (73.0%) and augmentin (54.1%).

Table 3-a. Microbiological pattern of the semen of the study participants.

	N	%
E. coli	41	25.3
Klebsiella	12	7.4
Proteus	12	7.4
Staph aureus	97	59.9

Table 3-b. Association between semen bacteria culture and semen analysis.

	Semen Analysis
	Abnormal236 (66.5%)		Normal119 (35.5%)		Total355 (100%)		χ 2	P-value	Odds Ratio	95% CI
	N	%	N	%	N	%
Semen Culture
Positive Culture	135	83.3	27	16.7	162	100	37.985	0.000	4.554	2.761 – 7.513
Negative Culture	101	52.3	92	47.7	193	100.

Table 3-c. Antibiotics sensitivity pattern of the semen of the study participants.

	Organisms				Total
	E. coli	Klebsiella	Proteus	Staph aureus	N	%
Ofloxacin	27	9	7	46	89	73.0
Augmentin	31	8	6	20	66	54.1
Genticin	2	2	3	5	13	10.7
Maxpan(Cefixime)	2	1	2	18	23	18.9
Erythromycin	0	0	2	3	5	4.1
Amoxicillin	0	0	2	2	4	3.3

Discussion

Male infertility is a common global reproductive health problem. Worldwide, semen quality has shown progressive decline over the last decades4. The contribution of hormonal abnormalities and positive semen culture to the decline in semen performances in this environment was explored in this study.

This study demonstrated at least one abnormality in semen parameters in more than two-third (66.5%) of the participants. Similar high prevalence of male factor infertility has been reported in other parts of Nigeria17^-19. This reinforces the fact that the male partners contribute in no little measure to the overall burden of infertility in our environment. Oligozoospermia alone (22.8%) was the most common semen disorder in this study, and general pattern of semen abnormalities is similar to the findings from other centers20-2323.

Normal hormonal milieu is a prerequisite for normal spermatogenesis24. The consequence of male reproductive endocrinopathy is male reproductive failure4,6. Endocrinopathy among male partners of infertile women had a high showing in this study. Emokpae et al25 reported 40% among infertile men in Kano. However, Geidam et al26 reported a lower overall incidence of 7.3% among men with abnormal semen parameters. Geidam also reported a comparable rate of prolactin disorder (12.5%), but much higher prevalence of FSH (66.7%) and testosterone (50%) abnormalities among infertile men26. Abnormal levels of FSH, prolactin and testosterone were demonstrated in this study to be significantly associated with abnormal semen parameters. This is consistent with findings by Akinloye et al27 in Ibadan, but differs from report by Ogbahon et al18 in Ilorin. Expectedly, abnormal testosterone level has the strongest association with abnormal semen parameters in this study (94.4%. p=0.009. OR=9. CI=61.20 - 69.69). This is because testosterone is an absolute requirement for the process of spermatogenesis and the maintenance of seminiferous tubular epithelium24.

On regression analysis, abnormal FSH and Prolactin levels were the only predictors of abnormal semen parameters. Suboptimal testicular activities will alter the normal feedback mechanism causing alteration in hypothalamic-pituitary responses and gonadotropin secretion24. While normal FSH levels does not guarantee optimal spermatogenesis, abnormal FSH level is commonly associated with impaired spermatogenesis6. Hyperprolactinemia has been thought to cause erectile dysfunctions, and ultimately male reproductive failures6. The role of prolactin in abnormal spermatogenesis is not clear, but may be in its ability to disrupt normal gonadotropin secretion24. In this study, abnormalities in luteinizing hormonal level did not show significant difference between subjects with normal and abnormal semen parameters.

Positive semen culture, especially involving pathogenic organisms, is a compelling presumption for reproductive tract infections28. In this study, bacterial organisms were isolated from more than two-fifth (43.6%) of the study population. Okonofua et al29 reported a similar rate of 45% among infertile males; this is however far higher than prevalence of 7% and 18% reported by Ekhaise et al30 in Benin and Ogunbajoin31 Ibadan respectively. Also in this study, men with positive bacteria semen culture had more than four folds odds of having abnormal semen parameters. The cause of the high prevalence of male reproductive tract infection in this study population is not clear. Male reproductive tract infection due to bacterial organisms can contribute to sub-fertility by inhibiting spermatogenesis and secretory function of the accessory glands. Infection can also induce canalicular system damage, testicular atrophy, obstructive azoospermia, direct spermatozoa damage, cause DNA fragmentation, and formation of antisperm antibodies11,12. Several studies have demonstrated higher positive culture rates in sub-fertile relative to fertile male populations4. Ekhaise et al30 reported a higher frequency of abnormal semen parameters in those with bacteriospermia. However, a study by Ombelet et al32 could not demonstrate such correlation. However, the presence of organism in the semen will affect the quality of the spermatozoa4

The organisms isolated in this study were Staphylococcus aureus, Escherichia coli, Klebsiella spp and Proteus spp. This is similar to findings by Onemu et al33 in Benin city, and Emma-Okon et al34 in Ile-Ife Nigeria. More virulent sexually transmitted infectious agents such as Chlamydia trachomatis and Nisserea gonorrhea were not isolated in this study. Most of organisms isolated were sensitive to ofloxacin in 73.0% of cases and augmentin in 54.1%. Onemu et al33also reported a similar sensitivity pattern to ofloxacin antibiotics. Although the benefit of antibiotic therapy in asymptomatic bacteriospermia is not yet established, treatment is recommended all the same4.

The limitations of this study include the fact that the culture method employed could not identify such infertility-associated organisms like Chlamydia trachomatis which could have been prevalent among the participants.

Conclusions

This study has shown that male endocrinopathy and bacteriospermia contributed significantly to male infertility in our environment. These pathologies should therefore be deliberately sort, and treated to improve treatment outcome of male infertility.