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Relationship Between Sperm Quality and Male Reproductive Hormones Among Male Partners with Fertility Complications: Attending CHUB

Background Infertility remains a highly prevalent global condition in the second decade of the new millennium. Reproductive hormones determine sperm quality as they initiate and maintain spermatogenesis. Hormonal imbalance can cause abnormal sperm quality that can be treated by hormonal replacement therapy. Objective To assess the relationship between sperm quality and male reproductive hormones among male partners with fertility complications attending CHUB. Methods The study was a descriptive cross-sectional, and a convenient sampling strategy was used to recruit subjects at CHUB. Sixty-two male subjects with fertility complications provided both semen and blood sample to analyze sperm quality and reproductive hormones. Descriptive statistics were used to analyze data. Results Both FSH and LH showed a strong negative correlation with sperm count which is more profound with FSH (r= -0.722) than LH (r= -0.545). Testosterone showed a strong positive correlation with sperm count (r= 0.712). FSH and LH showed a negative correlation with sperm motility which is more profound in FSH (r= 0.312) than LH (r= -0.302). Testosterone also showed a positive correlation with sperm motility (r= 0.360). Conclusion Our study found a correlation between sperm quality and male reproductive hormones. We further suggest other studies to investigate predictive power of male reproductive hormones. Rwanda J Med Health Sci 2020;3(3):315-328 _


Background
Infertility remains a highly prevalent global condition in the second decade of the new millennium, affecting about 186 million worldwide.
[1] For the several last decades, there is an increase of infertility and clinical presentation suggests the causes to be hormonal in nature.
[2] WHO defines the term infertility as a failure to conceive a child after frequent and unprotected sexual intercourse for 12 months. [3,4] Many scientists prefer the term sub fertility because half of couples conceive without intervention in the following 12-24 months.
[5] Reproductive hormones are important determinant of sperm quality as they initiate and maintain spermatogenesis. [6] Testosterone participate in spermatogenesis by acting on sertoli cells in seminiferous tubules and surrounding peritubular cells which increase sperm motility, and cause negative feedback to the LH and FSH. [7] Contrary, the levels of FSH and LH are inversely proportional to the sperm morphology, motility and concentration. [6] Globally, infertility is estimated to be 10 %-12% in couples. Female factors related to 88.9% of the cases while male factors related to 66% of the cases.
[8] The highest prevalence found in Africa and Central/East of Europe.
[9] The study done by Dhont et al (2011) about infertility in Rwanda showed that male factors contribute to 16%, female factors contribute 31% and combination of both male and female factors contribute to 50% while 3% was unexplained.
[10] Multiple factors like genetics, environment, dietary factors and infections might cause the infertility. [4] A study done in Nigeria found the prevalence of endocrine abnormalities of 7.3% in men investigated for infertility, hormonal abnormalities found in 22% of oligospermic males while 43% of hormonal abnormalities found in azoospermic males. [11] The sperm cells are formed by process of spermatogenesis. It starts with mitotic division of stem cells (spermatogonial stem cells) located near to the tubules' basement membrane that produces two types of cells: type A cells that replenish the stem cells, and type B cells that differentiate into spermatocytes. The primary spermatocyte divides by mitosis to produce two secondary spermatocytes; and then secondary spermatocyte divides by meiosis II into two equally haploid spermatids that transform into spermatozoa by spermiogenesis. Semen are formed by mixing of whitish fluid called seminal fluid produced by seminal vesicles and prostate gland with sperm during ejaculation. [12,13] Endocrine factors from brain highly regulate testicular functions, and hypothalamus-pituitary-testis circuit is core unit for maintaining endocrine balance and fertility. However, no research done to reveal the relationship between sperm quality and male reproductive hormones in Rwanda. There is more data on infertility in high-income countries, but in developing countries, more research on infertility are highly needed to generate information important for planning fertility interventions.(4) This research aims to assess the relationship between sperm quality and male reproductive hormones for evidenced-based management of male infertility.

Study design
This study was cross sectional study on the relationship between sperm quality and male reproductive hormones among male partners with fertility complications attended CHUB, in Huye district, Southern province, Rwanda from 18th June to 06th September 2019.

Participants recruitment
A study recruited 62 adult male patients attended Butare University Teaching Hospital presenting with fertility complications. Convenient sampling strategy was used to recruit participants.

Data collection
The participants requested not to have sexual intercourse or masturbate for 2-7 days prior to the sample collection. The blood sample was collected in red top tube with no additive, and the participants provided semen sample. The semen produced by having sexual intercourse with partner or masturbation in private room. The whole semen collected into plastic container and brought to the lab within 1 hour.

Laboratory testing
Semen were analyzed for count, motility and morphology according to WHO laboratory manual for the examination and processing of human semen, fifth edition. Semen incubated for 30 minutes at 37 o C. The sperms were counted using Neubauer counting chamber. The dilution of 1:20 was used and prepared by adding 50 μl of semen to the 950 μl diluent. The diluent was prepared adding 50 grams of sodium carbonate and 10 ml of 35% formalin to the distilled water to make 1000 ml. Zero sperm count were considered as azoospermia, less than 20x10 6 were considered as oligozoospermia, and at least 20x10 6 were considered as normal sperm count.
To examine the sperm morphology, semen specimens were stained with MGG and spermatozoa stained into dark blue purple hue. Normal sperm morphology for man was deliberated at normal morphology greater than or equal to 4% of total sperms morphology.
To examine motility, the semen was examined microscopically to estimate motility of sperm cells. 10 μl of semen was put on glass slide and covered with 22x22 mm coverslip. The slide was then examined microscopically using 40x objective lens. Motility was reported and graded as rapid progressive motility, slow progressive motility, non-progressive motility or immotility. Normal motility for man was deliberated at total motile greater than or equal to 40%, and progressive motility greater than or equal to 32% of total motility. [24] Testosterone, LH and FSH were tested using solid phase enzymelinked immunosorbent assay (ELISA) for quantitative determination of hormones in serum by following manufacturer's protocol. LH and FSH were reported in mlU/ml while testosterone was reported in ng/ml.

Data analysis
Collected data were analyzed using SPSS 25.0 to generate descriptive and inferential statistics. Data was firstly entered in Microsoft excel 2016 and imported in SPSS 25.0. Pearson's r was used to determine the correlation between semen quality and male reproductive hormones. Statistical significance was deliberated at P<0.05. The graphs were plotted using Microsoft excel 2016.

Ethical consideration
Data was collected after obtaining ethical clearance from University of Rwanda, College of Medicine and Health Sciences and data collection approval from the ethical committee of Butare University Teaching Hospital. Only patients who signed informed consent form were included in the study. Confidentiality maintained by using code numbers and data only accessed by authorized personnel.

Figure 2. Sperm motility
Sperm morphology were analyzed microscopically after staining with May Grunwald Giemsa. Abnormal sperm morphology in subjects were deliberated at normal sperm morphology less than 4% of total sperm seen. All subjects had no normal sperms greater than 4%; however, 19 (31%) were azoospermic with no any sperm in semen. The rest of subjects, 43 (69%) had normal sperm morphology.

Figure 4. Sperm quality categories
FSH and LH showed strong negative correlation with sperm count which is more profound with FSH (r= -0.722, and p<0.05) than LH (r= -0.545, and P<0.05). Testosterone showed strong positive correlation with sperm count (r= 0.712, and p<0.05).
FSH and LH showed a negative correlation with sperm motility which is more profound in FSH (r= -0.312, and p<0.05) than LH (r= -0.302, and p<0.05). Testosterone showed a positive correlation (r= 0.360, and p<0.05).
FSH have strong positive correlation with LH (r= 0.843, and p<0.05); while both FSH and LH showed a negative correlation with testosterone which is more profound in FSH (r= -0.648, *. Correlation is significant at the 0.05 level (2-tailed).

Figure 5. The mean levels of testosterone, LH and FSH in sperm quality categories
In azoospermic subjects, the mean level of gonadotropins were 19.20mIU/ml and 12.45mIU/ml for FSH and LH respectively. The mean level of testosterone was 3.83 ng/ml. In oligozoospermic subjects, the levels of FSH and LH were 17.53 mIU/ml and 9.71mIU/ml respectively, and testosterone was 4.19 ng/ml. In asthenozoospermic subjects, the levels of FSH and LH were 4.62mIU/ml and 4.27 respectively while the level of testosterone was 5.34 ng/ml. In normozoospermic subjects, the levels of FSH and LH were 6.43 mIU/ml and 6.55 mIU/ml while the level of testosterone was 5.46ng/ml.

Discussion
Male reproductive hormones initiate and maintain reproductive function, and they are associated with sperm quality.
[6] In this study, found strong negative correlation between sperm count and gonadotropins; FSH (r= -0.712, and p<0.05), and LH (r= -0.512, and p<0.05). Our study agreed with a study done by Ismael et al. (2017)  Other studies should be done to investigate effectiveness of hormonal replacement therapy in Rwanda.

Limitations
A study was done in one referral hospital in Rwanda located in Southern province (Butare University Teaching Hospital), and should not generalized to whole country.

Conclusion
Our study found correlation between sperm quality and male reproductive hormones. Gonadotropins negatively correlated with sperm count and sperm motility while testosterone showed positive correlation. Gonadotropins positively correlated with testosterone, and sperm count positively correlated with sperm motility. The previous studies had suggested use of reproductive hormones to predict semen quality in epidemiological studies, and in investigation of neuro-endocrine cause of infertility. However, an increase of endocrinopathy in infertile male rise the use of hormone replacement therapy. We recommend other study to investigate predictive power of male reproductive hormones measurement to predict response to hormonal replacement therapy.