Low Sperm Counts : Biophysical Profiles of Oligospermic Males in Sub-Saharan Africa

Introduction: Male infertility is a public health burden and a psychological dilemma in the life of the affected man. Subjects and Methods: A total of 911 men were studied retrospectively, from 2010 to 2015. Among these, 49.7% had normal sperm count, 39.3% were oligospermic and 12.0% were azoospermic. Azoospermic men were withdrawn from this study solely to investigate the seminal fluid parameters and the biophysical characteristics of oligospermic men in contrast to those with normal sperm count. Age was stratified into <30, 30 39.9, 40 49.9, 50 59.9 and ≥60 years; body mass index was categorized into underweight (<18.5), normal (18.5 24.9), overweight (25.0 29.9) and obese (≥30) and standard semen analysis was performed. Results: The means (±sd) of age and of BMI of the 802 subjects of the study were 42.7 (±7.0) years and 26.9 (3.9) kg/m respectively. There was no significant difference in the age or BMI of normal and oligospermic men. A total of 453 (56.5%) had normal sperm count while 349 (43.5%) were oligospermic. Compared to normal weight men, those overweight and those obese were, respectively, 1.11 (χ = 0.44, P-value = 0.51, OR = 1.11, 95% CI = 0.81, 1.54) and 1.56 times (χ = 4.50, P-value = 0.03, OR = 1.56, 95% CI = 1.03, 2.36) more likely to be oligospermic. The mean of normal oval head sperms was significantly higher (t = −7.31, P-value = 0.00001) in normal men (47.8 ± 8.9) than in oligospermic men (43.0 ± 10.7). Oligospermic men were over 4 times as likely to produce progressive sperm motility of <32% (χ = 70.90, P-value = 0.000001, OR = 4.24, 95% CI = 2.99, 6.02) than men with normal sperm count. Multivariate regression analysis shows negative but significant correlations between age and semen volume (coef. = −0.04, Std Err. = 0.01, t = −4.01, P-value = 0.0001, 95% CI: −0.06, −0.02) and between BMI and sperm count (coef. = −0.18, Std Err. = 0.06, t = −3.26, P-value = 0.001, 95% CI: −0.29, −0.07). Conclusion: Our findings suggest that overweight and obesity are How to cite this paper: Ajayi, A.B., Afolabi, B.M., Ajayi, V.D., Oyetunji, I. and Atiba, A. (2018) Low Sperm Counts: Biophysical Profiles of Oligospermic Males in Sub-Saharan Africa. Open Journal of Urology, 8, 228-247. https://doi.org/10.4236/oju.2018.88026 Received: June 5, 2018 Accepted: August 20, 2018 Published: August 23, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Infertility and problems of impaired fecundity have been a concern through ages [1].As a disease of the reproductive system, it is defined as failure to achieve clinical pregnancy after 12 months or more of regular unprotected sexual intercourse [2].Incidentally, the first study of the epidemiology of infertility was published in Scotland in 1866 by Matthews Duncan whose book, Fecundity, Fertility and Sterility, [3] was the first, and until recently the only, population-based study of infertility [4].It is regrettable that extremely few national data on the prevalence of infertility exists, especially in Africa where voluntary infertility is an anathema, though not in developed countries.A good example of voluntary infertility occurred in the US in the first 3 decades of the last century, though it became less common thereafter only to surface again in the 1960s [5].Globally, there has been a measure of difficulties in assessing the prevalence of infertility due to rarity of large-scale surveys on this subject [6] [7] except the US National Survey of Family Growth (NSFG) [8].
Infertility is estimated to affect as many as 186 million people worldwide.Although male infertility contributes to more than half of all cases of global childlessness, infertility remains a woman's social burden [9].In recent times, infertility has become a significant clinical problem affecting 8% -12% of couples worldwide [1].Male factor is responsible for about 40% -50% cases of infertility in couples who desire to have children and it is estimated that approximately 2% of men with male factor infertility have sub-optimal sperm parameters such as 1) low sperm concentration 2) poor sperm motility and 3) abnormal morphology [1].The high prevalence of infectious diseases in less developing countries may be responsible for high rate of infertility in these parts of the globe.Low "sperm concentration" or "sperm count" referred to as Oligospermia, is a male fertility issue defined as a low sperm concentration in the ejaculate (typically milliliters or mL).As defined by the World Health Organization (WHO) in 2000, oligospermia is low sperm countless than 15 million sperm/mL [10].The exact global prevalence of oligospermia is not known though studies have indicated regional variations in the mean sperm concentration among men in USA and France [11] [12] [13] [14].Also, geographic, ethnic, climatic and occupational factors have been suggested to be responsible for the regional differences in the sperm counts in men [13] [14] [15].Isolated studies show higher prevalence of oligospermia among Caucasians [16].The prevalence of oligospermia was 22.9% in Pakistan [17], 15.6% in Tanzania [18], 25.6% in South-west and Ile-Ife [19] and 35.9% in South-east Nigeria [20].A study in Ghana reported that "half of the respondents had very low sperm counts (oligospermia)" [21].The cause of oligospermia may be multifactorial, including chromosomal mutations-translocations, inversions, deletions, as well as structural and numerical abnormalities [22], microdeletions of AZF on the Y chromosome and CFTR mutations.It is estimated that more than 75% of infertile males have an underlying chromosomal error [23]; infections such as viral orchitis, caused by mumpsvirus, hepatitis and human immunodeficiency virus (HIV) infection; endocrine disorders affecting the hypothalamic-pituitary-gonadal axis such as hypogonadism, hypothyroidism, hyperprolactinemia (usually as a result of a pituitary prolactinoma), acromegaly and adrenal hyperplasia; iatrogenic causes, such as drugs which interfere with spermatogenesis, including chemotherapeutic agents, sulfasalazine, corticosteroids, estrogens, antimalarials, nicotine, cimetidine, ethanol, opioids, methotrexate and many other [24]; testicular and endocrine tumors, as well as trauma with subsequent orchidectomy [25].Clinical evaluation of male fertility begins with a detailed history and physical examination, which generally will provide valuable information to guide what additional laboratory investigations or imaging studies to complete the evaluation.The two main purposes of male infertility evaluation are 1) to identify any modifiable factors that can improve the fertility status of the man and 2) to identify any underlying serious condition(s) such as testis cancer, osteoporisis, endocrinological and genetic problems that present first as infertility.Semen analysis remains the single most useful and fundamental investigation with a sensitivity of 89.6%, that it can detect 9 out of 10 men with a genuine problem of male infertility [26].There is paucity of information on the biophysical characteristics of patients with oligospermia in sub-Saharan Africa which makes this the objective of this study among infertile males who present for Assisted Reproduction Technology (ART) in Nigeria.

Subjects and Methods
This has been described elsewhere [27].In summary, between 2010 and 2015, 916 men, aged between 23 and 73 years, consulted for infertility at the offices of Nordica Fertility Center (NFC) in three Nigerian cities-Lagos, Abuja and Asaba respectively.Among them, 607 (66.3%) were couples while (309, 33.7%) consulted alone.At first consultation, all the patients were counselled on the need for various laboratory analyses, especially seminal fluid analysis (SFA) for the male partners and other necessary investigations for each couple spouses to ascertain the exact cause(s) of infertility.In all, 907 (99.0%) of the 916 responded correctly to the counselling and produced semen for SFA.Various reasons were given by the remaining 9 men for not producing seminal fluid, such as 1) travelled out (4, 44.4%) 2) coincided with fasting period (2, 22.2%) and 3) absconded (2, 22.2%) and 4) specimen was discarded due to improper collection Open Journal of Urology (1, 11.1%).The data of all those who produced seminal fluid for analysis, in strict compliance with World Health Organization's (WHO) instruction for SFA [10] were analyzed.All the sampled patients consulted because their spouses or partners were not getting pregnant despite uninterrupted, regular and constant unprotected sexual intercourse.within a maximum of 3 -5 minutes' walk to according to WHO guidelines [22].

Subjects
The patient was counseled to abstain from sex for minimum/maximum duration of 2 -5 days and importance of abstinence time was explained.Patients were given clear and simple instructions on the following: 1) accuracy in the semen collection, 2) semen should be collected in a wide-mouth measuring cylinder that was provided, and 3) the importance of reporting any loss of semen sample during collection.For this semen collection process, each patient was provided with a medical record form where data such as name, age (or date of birth), days of abstinence, date and time of collection, if there was any loss in semen volume during collection (incomplete collection) and the time of commencement of seminal fluid analysis in the laboratory were appropriately filled in by the patient and the laboratory staff who collected the semen samples.
At the point of first contact each patient was weighed (kg) and had his height measured (cm) for BMI determination and each patient's medical, surgical, family and social history were taken, including marital status, occupation, smoking and alcohol use.Past histories of groin operation, sexually transmitted diseases and varicocelectomy were taken.Medical data of these patients were managed by retrieval, entered into Excel spreadsheet on a laptop computer, cleaned and analyzed.WHO specification was strictly followed in the analysis of the two semen samples collected by masturbation per patient.Prior to sample collection, the patients were counselled on the need to observe accuracy in semen collection into a wide-mouth measuring cylinder provided to each patient and any loss of semen sample during production and collection into the measuring cylinder should be reported.Thus, all patients were given a special wide-mouth calibrated cylinder in which to collect the seminal fluid; semen samples were collected, as sterile as possible, by the process of masturbation and submitted to the laboratory within 30 minutes of collection; and all patients were advised to not have sexual intercourse for a minimum of 2 days and a maximum of 5 days.In addition, each patient was given a specific record form where data demographic data, number of days of abstinence, date and time of collection, any loss in semen volume during collection and the time of commencement of SFA in the laboratory were appropriately filled in by the patient and the laboratory staff who collected the semen samples.
All semen samples were delivered to the laboratory on time and after liquefaction has taken place, the samples were analyzed for volume, total sperm concentration, motility, and morphology strictly according to WHO guidelines [10].
Sperm morphology is not reported in this paper.Semen volume was measured by directly from the meniscus of calibrated wide-mouth measuring cylinder given to each patient.Light microscope (×40 magnification) with eyepiece reticle having grid was used to determine 1) mean progressive motion-a) rapid forward, fast progressive motility, b) moderately forward, slow progressive motility, c) jerky non-progressive motility and d) immotile/no movement 2) sperm concentration-counting at least 200 spermatozoa and expressing these as 10 6 spermatozoa/ml, in a 3 -5 µL of semen sample transferred to the center of the Neubauer Chamber and 3) progressive motion-categorized into ≥32% or <32%.
Other parameters assessed in each semen sample were color, viscosity and liquefaction time.Age (years) was classified into <30, 30 -39.9, 40 -49.9,50 -59.9 and ≥60 and BMI (kg/m 2 ) into underweight (BMI < 18.5), normal weight (BMI 18.5 -24.9), overweight (BMI 25.0 -29.9) and obese (BMI ≥ 30) according to the conventional WHO classification [28].Patients who were not on any medication that would interfere with spermatogenesis, those not consuming herbal medication and who gave consent for their semen to be analyzed were included into the study.Patients initially screened out of this study were those who earlier gave a medical history of i) chronic liver disease, ii) renal failure, iii) HIV infection, iv) diabetes mellitus or v) tuberculosis of the genitourinary system, vi) urethral disease or those who gave surgical history of i) undescended testis or ii) operations on the testis.Further analysis of the data specifically focused on men with normal sperm count and those with oligospermia.
Data were analyzed using STATA 13; associations between Age, BMI and Age-for-BMI with normal sperm concentration and with oligospermia were assessed using chi-square, Odds ratio and 95% Confidence Interval.Spearman correlation and analysis of variance (ANOVA), and a multiple linear regression analysis were performed to determine correlation coefficients and significance of means of two variables.In addition, the relative risks for men having oligospermia, giving reference range of the WHO [10], were calculated.A P-value of ≤0.05 was taken as significant.Data were presented as Tables and bar charts.

Results
The total number of participants in this study was initially 911 among whom 453 (49.7%) had normal sperm count, 109 (12.0%) were azoospermic and 349 (38.3%) were oligospermic, according to WHO classification [10].For the purpose of this study, all the azoospermic patients were excluded, leaving 802 males whose means (±sd) of age (years) and BMI (kg/m 2 ) were 42.7 (7.0) and 26.9 (3.9) respectively, for further analysis.The main reason for the exclusion of the azoospermic patients was sole to study the biophysical characteristics of oligospermic patients relative to those with normal sperm count.Further, azoospermia from this same group of patients has been studied and reported earlier [27] 1).
Multivariate regression analysis in Table 6 shows that Age and BMI explained a significant 5.12% and 3.25% respectively of the differences observed in seminal fluid volume (R 2 = 0.0512, P-value = 0.0001) and in sperm count (R 2 = 0.0325, P-value = 0.0033) but an insignificant explanation of the variation observed in

Discussion
One of the intrinsic qualities of living beings, including humans, is the continuation of the genome of each entity and reproduction is essential in carrying out this task.Those who are not able to carry out this chore of the continuation of human genome through reproduction often may be subject of ridicule from close quarters-husband, wife, families, neighbors, others-resulting in isolation, dejection, abuse, stigmatization, deprived physical, mental and social, in some cases leading to physical trauma and even death.Some studies in with mild, moderate or severe oligospermia that this condition may be as a result abnormality in their chromosomes.Clinicians should then attempt to identify such abnormality through laboratory tests and correct it as much as possible.
This study analyzed the biophysical characteristics of infertile men who were given laboratory diagnosis of oligospermia and who sought for Assisted Reproduction Technology at the Nordica Fertility Center in Nigeria.The first key finding in this study is that majority of the patients were aged 40 -49 years, an older age range than the 30 years and above reported from Ghana [21] or the 35.8 years reported from Tanzania [18].This finding suggests that oligospermia may occur at older age in Nigeria than in other African countries where it occurs at a younger age.The fact that oligospermia possibly occurs in older age among Nigerians may be responsible for its large population with its consequences on infrastructures, education, housing and environmental degradation.
Another key finding is that a high proportion of these patients were overweight.Body Mass Index (BMI) of fertile and infertile men have been studied extensively.Some studies have shown that higher BMI is associated with impaired semen parameters, and others have shown improvement in semen parameters with weight loss [29].This study reports that of the 802 participants, 553 (69.1%) were overweight (408) and obese (145); figures that were much lower than the 72.8% combined overweight and obese patients in a similar study in New Zealand [29].Of the 408 men who were overweight in this study, 174 (42.6%) presented with oligospermia, similar to the 41% reported by Eisenberg et al. [30].Overall, age alone or BMI alone may not significantly contribute to seminal fluid volume as indicated in this study which agrees with what Schwartz [31] earlier reported.But when age was segregated, a significant difference was shown in the seminal fluid volume of normal and of oligospermic patients only at age 50 -59 years.Earlier studies did not separate the patients into normospermic, oligospermic and azoospermic patients in considering the effects of age on seminal fluid volume.
A relative high proportion of patients in this study, as in other studies [17] [32] [33] [34] [35], reported with oligospermia which predisposes them to higher risk of developing both testicular cancer and prostate cancer later in life [25].Therefore, oligospermia may be regarded as an accepted biomarker of imminentmale health problems.
At first glance, there was no significant difference in the mean liquefaction time (min) but when examined according to different categories of age and BMI; it became apparent that there were significant differences in liquefaction time at some different age groups and different BMI, especially in obese patients.Cui et al. [36] reported a significant longer semen liquefaction time in patients with Anti-sperm antibodies than in their control group, indicating a negative effect of ASA on liquefaction time.Longer liquefaction prevents timely availability of viable sperm cells for fertilization of accessible egg which may be problematic for patients with advanced age and those who are overweight.
In contrast to normal patients, a high proportion of patients with oligosper-mia presented with PM of <32%, especially in younger age group of <40 years and in obese patients, a situation that was reversed in older age group till <60 years and normal BMI.One of the consequences of low progressive sperm motility is inability of the sperm cells to swim forward in the female genital tract and reach the egg on time for fertilization to take place.It is possible that, as reported in a recent study [37], patients who presented with asthenozoospermia in our study also demonstrated high basal and stimulated reactive oxygen species (ROS) production with increased mitochondrial DNA (mtDNA) copy number but decreased mtDNA integrity associated with elevated ROS levels.Another study reported that aneuploidy is associated with low progressive sperm motility [38].This paper reports a mean of 32.1 (±9.1) concentration of small tapering head abnormal sperm cells among oligospermic patients compared to 29.9 (±7.4) of such cells among men with normal sperm count, with a highly significant statistical difference between the two.On the other hand, patients with oligospermia had a highly significant lower concentration of normal oval head sperms compared to those with normal sperm count.This finding aligns with the report from a Nigerian study which documented a statistically significant higher abnormal morphology (81% vs. 67%, P = 0.01) among oligospermic patients compared to those with normospermia [39].Complete non-motile sperm cells were rare in this study, occurring on only one patient aged 40 -49 years who was also overweight.This agrees with what Ortega et al., [40] reported in their study that complete as the nozoospermia occurred at a frequency of 1 of 5000 men.More than half of the oligospermic patients with poor mean progressive motion were aged 40 -49 years with BMI of 25 -29.9Very few (8.9%) proportion of oligospermic patients had sperms with good Mean Progressive Motion or Grade 4 movement, especially those aged 30 -39 years and with BMI of 25 -29.9 kg/m 2 .Progression refers to the forward movement of sperm and is recorded as: Grade 4: Fast and forward progression where sperm move in a straight direction.Several studies support the importance of the mitochondria in maintaining sperm quality and motility; in fact, as the nozoospermia [41] and oligoasthenozoospermia [42] have been reported in patients with typical mitochondrial diseases characterized by point mutations or multiple deletions in mtDNA.
Oligospermic patients had significant sperm cell defects compared to those with normal sperm count.The argument that reactive oxygen species (ROS) production is involved in the pathogenesis of sperm abnormalities may be valid also for oligospermia and defects in sperm morphology.A study reported high proportion of patients with severe asthenozoospermia who showed increased basal and stimulated ROS production and also demonstrated increased mitochondrial DNA (mtDNA) copy number but decreased mtDNA integrity and they were associated with elevated ROS levels [37].

Conclusion
In conclusion, biological and statistical data from the present research support the idea that oligospermia could be associated with transformation of sperm morphology, reduced progressive motility, mean progressive motion as well as with reduced liquefaction time, especially when biophysical properties of the patients such as age and Body Mass Index are segregated.Otherwise, merely reporting the observation without segregation of the two variables of age and BMI may give a false positive or a false negative.For example, though there appeared to be no significant difference in the liquefaction time (min.) between patients with normal sperm count and those with oligospermia, on segregation of BMI it was found that there was a significant difference in the liquefaction time of normal patients who were obese and oligospermic men who were obese.
2) Anthropometric data of patients were recorded at consultation, not volunteered by patients.
3) Up-to-date laboratory equipment was used for semen analysis and in almost all cases each analysis was on two consecutive samples.
4) Enough number of overweight and obese patients was available for the study.
5) The same study criteria were used for all the patients.

Weaknesses
1) This was a facility-based study limiting generalizability of the findings.A more robust study including the community is urgently needed.
2) Semen parameters may not be completely accurate measures to determine fertility.This study examined only oligospermia but not pregnancy success rates among oligospermic patients.Therefore, it is not certain how the findings relate to infertility per se in our setting.

Figure 1 .
Figure 1.Age distribution of normal and oligospermic patients who produced seminal fluid <1.5 ml.

Figure 2 .
Figure 2. BMI distribution of normal and oligospermic men who produced semen volume <1.5 ml.

Figure 3 .
Figure 3. Percent distribution of mean progressive motion in normal and in oligospermic men in the study.

Table 2 .
Means of semen volume, sperm concentration and liquefaction time by age and by BMI of normal and oligospermic males.

Table 3 .
Frequency distribution of Progressive Motility in normal and oligospermic patients.

Table 4 .
Frequency distribution of mean Progressive motion in normal and oligospermic patients.

Table 5 .
Sperm morphology in normal and oligospermic patients relative to age and BMI.