Priyadarsini Sunanda, Babita Panda, Chidananda Dash, Rabindra N. Padhy, Padmanav Routray

1Centre for Human Reproduction, IMS & SUM Hospital, Siksha ‘O’ Anusandhan University, K8-Kalinga Nagar, Bhubaneswar 751003, Odisha, India

2Centre Research Laboratory, IMS & SUM Hospital, Siksha ‘O’ Anusandhan University, K8-Kalinga Nagar, Bhubaneswar 751003, Odisha, India

3Aquaculture Production and Environment Division, Central Institute of Freshwater Aquaculture, Bhubaneswar-751002, Odisha, India

Effect of age and abstinence on semen quality: A retrospective study in a teaching hospital

Priyadarsini Sunanda1,2, Babita Panda1*, Chidananda Dash1, Rabindra N. Padhy2, Padmanav Routray3

1Centre for Human Reproduction, IMS & SUM Hospital, Siksha ‘O’ Anusandhan University, K8-Kalinga Nagar, Bhubaneswar 751003, Odisha, India

2Centre Research Laboratory, IMS & SUM Hospital, Siksha ‘O’ Anusandhan University, K8-Kalinga Nagar, Bhubaneswar 751003, Odisha, India

3Aquaculture Production and Environment Division, Central Institute of Freshwater Aquaculture, Bhubaneswar-751002, Odisha, India

ARTICLE INFO

Article history:

Received 19 February 2014

Received in revised form 24 February 2014

Accepted 26 February 2014

Available online 20 June 2014

Age

Objective: To elucidate the effect of age and sexual abstinence on semen quality (semen volume, total count, progressive motility, vitality and morphology). Methods: A total of 730 semen samples were analyzed. Subjects were grouped according to the age (20-29, 30-34, 35-39 and 40-50) and abstinence (2-3, 4-5 and 6-7). Semen parameters were evaluated following WHO standard criteria. Results: Analysis of 730 semen samples showed negative correlation of progressive motility (r=-0.131, P< 0.01), vitality (r=-0.173, P< 0.01), morphology (r=-0.324, P< 0.01) with age. With increase in age percentage of progressive motility, vitality and normal morphology in mean values declined after the age group of 35-39 to 40-50 years, but no change in volume and count were observed. Increase in abstinence with individual days significantly affected semen volume (H= 20.65, P< 0.001), count (H= 36.67, P<0.01), progressive motility (H= 13.53, P<0.05) and vitality (H= 15.33, P< 0.01). But, no effect was found on sperm morphology. Mann Whitney U test confirmed the changes in semen volume, total count and vitality in paired grouping from 2-7 days (P<0.05), but changes in sperm motility were observed after 5 days of abstinence in each paired group upto 7 days (P<0.05). Mean values of semen parameters among three abstinence groups (2-3, 4-5 and 6-7 days) also showed similar result. Conclusions: In the present study, age negatively affected progressive motility, vitality and morphology of human sperm. Semen samples showed intra varied results within WHO amended abstinence period.

1. Introduction

Infertility is the inability to contribute conception after 12 months of unprotected intercourse; this may be due to male, female or some unexplained factors. Males alone contribute 35% to 40 % of infertile cases[1, 2]. Pathogenesis of male infertility is multi-factorial[3] and any alteration to normal physiology of reproductive organs may affect sperm functions resulting in oligozoospermia (low sperm count), asthenozoospermia (loss of motility), teratozoospermia (abnormal morphology), azoospermia (absence of sperms in the ejaculate) and in severe form, oligoasthenoteratozoospermia (OAT) that causes problem for a successful/fruitful fertilization[2]. Apart from the most conventional causes, age and abstinence were found to have significant role in sperm physiology.

Males have the advantage over females that they can contribute to conception even after the age of 40 and upto an age beyond 40 years of sexual maturity[4]. However, in advanced ages, degenerative changes in germinal epithelium[5], decreased number of Leydig cells[6], and their functions affect spermatogenesis through decrease in testosterone level[7], starting at the age of 30 years[8]. In animal studies, it was found that aging of males affect the sperm quality and spermatogenesis[9]. An age related decrease in nitric oxide level has shown to be associated with erectile dysfunction in older age people. In addition,semen quality also affects the implantation loss to the fertilized embryo[10] and possible autosomal dominant disorders by wrongly selecting a sperm among the semen pool of aged males[11].

Testicular volume and histopathological changes were strongly associated with the age of males that result in alteration of sex hormone production affecting the sperm physiology. However, the threshold age of sperm production in males is yet to be defined[12, 13]. Further, in the changing life style scenario, delayed parenthood is an upcoming problem and targeted couples need an assisted reproduction procedure to have a child[14]. Some retrospective studies in USA, China and India showed age related effects in semen parameters following the WHO guideline[11, 15, 16]. Here, the varied representations are ambiguous to find the optimal age/s for the treatment of infertility.

Apart from age, abstinence plays an important role in the maintenance of sperm quality, recommended by World Health Organization (WHO) for assisted reproduction. In other words, evaluation of sperm count depends on both interval between the ejaculations and their frequency in hours[17]. There are reports of positive correlation of volume and total count with abstinence period[18-20]. Further, some studies also showed the inverse correlation of motility with abstinence period[21]. In between WHO specified period of sperm collection (2-7 days) there is a variation of sperm quality[22, 23], whereas, other studies showed no significant effect of abstinence[24]. Hence, there is an ambiguity of semen collection, quality determination and the rate of success in fertilization within the assigned period. As sperm gains motility following the addition of secretions from accessory glands (seminal fluid), the variations of semen parameters depend on the quantitative and qualitative function of respective reproductive organs. So, more studies can suggest the particular abstinence period to obtain better semen quality in different geographical regions.

In general, retrospective data implies the declined sperm counts through ages[25-27]. In India, diminished sperm quality were reported in Kolkata, Bombay, Bangalore, Jodhpur and some other places in South India[16, 28]. So, in the present study, semen parameters were analyzed in a clinical set up of Eastern India, from 2010-2013 with reference to paternal age and abstinence period.

2. Materials and methods

2.1. Subjects

Patients attending Infertility Unit of a tertiary healthcare centre and teaching hospital, for evaluation of infertile couples, were taken as subjects. Seminal fluid analysis was done for the male partners of 730 couples attending the Infertility unit from 2010 to 2013. Male partners having the complain of infertility between age 20-50 years and sexual abstinence 2-7 days were included in the study; patients (142/872) with diabetes, infectious diseases, hydrocele, hernia, varicocele, addiction to tobacco, alcohol and azoospermia cases were excluded.

2.2. Sample collection and semen analysis

Semen samples were collected by masturbation into widemouth plastic container, in a room close to the andrology laboratory. The patients were advised to keep the abstinence period around 2-7 days. Samples were analyzed within 30-60 min, after liquefaction at 37 ℃. Semen parameters like sperm count/mL, percentage of motile spermatozoa, percentage of normal spermatozoa were analyzed along with the presence of pus cells in semen according to the WHO standard criteria[2].

About 6-10 μL of semen was put on to a microscopic slide and sperm motility was analyzed under bright field microscope under 400× magnification. At least 200 sperms were counted, and the mean value from duplicate measurements was represented. Sperm counts were done by using Neubauer’s haemocytometer with requisite dilutions (1:2, 1:5, 1:20), as per the WHO manual, 2010[2]. Diluted semen samples were mixed properly and 10 μL of the sample was transferred to Neubauer’s chamber for counting of spermatoza. Replicate dilutions were prepared to get correct values. Sperm morphology was assessed in Papanicolau-stained smears (Haematoxylene, Orange-G and EA-50 stain) using light microscopy (Olympus India Ltd) under oil immersion at 1 000× magnification.

Vitality of sperms was estimated by the hypo-osmoticswelling (HOS) test that was performed by mixing equal volumes of semen and hypo-osmotic solution, prepared from 7.35 g sodium citrate and 13.5 g fructose in 1 000 mL distilled water. The mixture was incubated for 30 min at 37 ℃, from which an aliquot of 10 μL was immediately examined at the 40x magnification. The percentage of swollen (vital) sperm was assessed by counting a minimum of 200 spermatozoa.

2.3. Statistical analysis

Semen quality parameters were analyzed by the use ofaChi-square test,bPearson’s correlation andcKruskal-Wallis H test were applied, whenever needed. For each parameter where a statistically significant variation was found with respect to abstinence, pair-wise comparison was performed bydMann-Whitney U-test. For the same, Statistical programme for the Social Sciences (SPSS Inc., version 20.0) was used.

3. Results

Out of 730 subjects, 315 (43.2%) were found to be normozoospermic, 263 (36.02 %) oligoasthenozoospermic, 98 (13.4%), and 54 (7.4%) asthenozoospermic. Semen parameters showed different ranges like volume from 1.5-6.5 mL, total count 1-209 millions, Progressive motility 0-65 %, vitality 8%-88 % and percentage of normal sperms (morphology)between 5% to 37 %. Further, means of the semen parameters were 2.5 mL, 48.8 millions, 35.2 %, 75.6 %, 31.3 % respectively (Table 1).

In comparison to WHO reference values, 12.05 % of samples showed below standard criteria of the semen volume. Similarly for total count, progressive motility, vitality and morphology were 50.8%, 43.4%, 12.32% and 1.90 % respectively showed below WHO reference values (Table 2). Chi- square test was applied to compare the normal reference values among different age groups of 20-29, 30-34, 35-39, and 40-50 years except normal morphology, as subjects below <4 % reference values were very low. The difference in the subjects below standard criteria (WHO) among the age groups were found to be significant for progressive motility (χ2= 9.6,aP< 0.05) and vitality (χ2= 14.28,aP< 0.01) only.

Further, the mean values of semen parameters in different age groups were represented in Figure 1. Mean values of semen volume were almost equal in all age groups and for total sperm count it declined only after 40 years of age. With increase in age progressive motility, vitality and normal morphology found to decline slowly, that begin at the age group 35-39 years, but the best quality of semen was observed at the age group 30-34 years. Pearson’s correlation was applied to find out significant changes between age and semen parameters. Progressive motility (r=-0.131,bP< 0.01), vitality (r=-0.173,bP< 0.01) and normal morphology (r=-0.324,bP< 0.01) were found to be negatively correlated with increasing age (Figure 2). But significant changes were found for semen volume and total count.

With increasing sexual abstinence, volume of semenand total count showed increasing trend in mean values. Progressive motility was found to be same between 2-3 and 4-5 days, but a sudden decrease in motility was found after 5 days of abstinence (Figure 3). Kruskal-Wallis H test was applied to the semen parameters according to the individual days of sexual abstinence, where volume (H=20.65,cP< 0.001), total count (H= 36.67,cP< 0.001), progressive motility (H=13.53,cP< 0.05) and vitality (H=15.33,cP< 0.01) were found to be highly significant for abstinence of 2-7 days. No change was found for normal morphology of sperm with increasing abstinence period. Mann Whitney U test confirmed the changes in semen volume significantly in each paired grouping from 2 days upto 7 days (dP< 0.05) and total sperm count changed from 3 days of abstinence to 7 days (dP<0.05). However, vitality was observed to be different between 2 and 4 days, 3 and 5 days, 4 and 5 days, 4 and 6 days in paired groups of sexual abstinence period (dP<0.05). Significant changes in sperm motility were observed after 5 days of abstinence in each paired group upto 7 days (dP< 0.05).

Table 1Description of indices of semen quality.

Table 2Percentage of subjects below WHO reference values of semen parameters among different age groups.

Figure 1. Comparison of semen parameters in different age groups(Mean ± SE).

Figure 2. Scatter diagrams and linear regression lines of progressive motility (%), vitality and normal morphology (%) of sperm on age.

Figure 3. Comparison of semen parameters in different abstinence period (Mean ± SE ).

4. Discussion

Semen quality analysis is the preliminary step to know the fertility status of males for assisted reproduction (WHO, 2010). Researchers have differently analyzed age and abstinence to find their effect on fertility potential[16, 21]. Here, age significantly affected the sperm quality, viz; progressive motility, vitality and morphology. Similar results wereobtained in Chinese populations[15]. It was partly supported by another retrospective study, [29] where the decreasing trend was observed with viability, morphology and volume. Whereas, contradictory findings on Cordoba (Argentina) population and sub-fertile patients of Arabian Gulf University reported the age related decrease in all the semen parameters [30,31].

In the present study, means of the forward progressive motility decreased in older age groups. Similar result was obtained by Mukhopadhay et al. 2010[16] for motility only, but contradicted the trend of sperm concentration. As normal morphology of sperms is essential to gain motility during epididymal transit, it might be difficult to attain higher grade of motility, imperative for conception in older age males. There were reports of increased percentage of abnormal spermatozoa in older age groups than younger groups[32]. Further, detoriation of healthy germ cells in advanced age might be one of the reasons for loss of grade of motility[33]. Here, no significant change in semen volume was recorded, contradicting another study on older age patients undergoing IVF and ICSI, which showed no effect on semen parameters, rate of fertilization, miscarriages inadvanced age groups, except semen volume, and decreased rate of blastocyst embryo formation[10]. Further, animal studies demarcated histological changes in testes, anomaly in germ cells and seminiferous epithellium, preimplantation losses and increased frequency of spermatid mutations in the process of aging[34]. Damage of DNA was also found to be higher in aged males leading to spontaneous abortion[11].

Table 3A comparative study of age related changes in semen parameters.

Study of Zhu et al. 2011 showed the low reference value for motility (40.6%) and vitality (36.4%) in Chinese poulation. But in the present study, the percentage of subjects below standard reference values of WHO are very high in case of total count (50.8%) and progressive motility (43.4%). Good quality of semen was found in between the two initial age groups i.e., amid 20-34 years. Although age related changes in semen quality has been elucidated, in the present study semen parameters above the WHO certified criteria were very low, indicating declined sperm count and motility in the semen pool of 730 samples. As the whole process of spermatogenesis is regulated by hypothalamus-pituitary-Leydig cell endocrine axis and andogen dependent secretions, continuous exposure to environmental toxicants and today’s lifestyle, might be one of the factors that alter the normal number and function of the germ cells, Sertoli cells and Leydig cells prior to the relative changes in aging [35]. A comparative study analyzing the age related changes in semen parameters has been given in Table 3. So is the possible positive predictive factor of age for the management of male infertility during the treatment correlating the standard physiological conditions.

Semen volume and total sperm counts were found to be significantly correlated with increase in sexual abstinence from 2-7 days. This is supported partly by other studies [23, 24, 50] for positive correlation of volume, sperm concentration and total count with abstinence. Here, progressive motilityand vitality of sperms also declined after 5 days of abstinence. Similar result was too reported from elsewhere for oligozoospermic samples, contradicting the result of normozoospermic that all semen parameters show increasing trend with abstinence period[22], corroborated by Pellester et al. [20]. As sperms are deposited daily into the Epididymis, increase in total count is obvious with increasing abstinence. So, oligozoospermic cases are suggested by clinicians to maintain the abstinence period for a couple of weeks to get an acceptable sperm count to achieve pregnancy[51]. On the other hand, daily ejaculation in oligozoospermics having<10 million sperm count achieved fatherhood with a good sperm quality[52]. Another study on daily ejaculation at a gap of 24 hour, reported the decrease in semen volume and count to an abstinence period of 10 days, where sperm progressive motility was of good quality after 2nd day, but declined after 5 days of abstinence[53]. Similar results on motility and vitality was also observed here also. A comparative study analyzing the abstinence related changes in semen parameters has been given in Table 4. It is known that sperms remains immotile during epididymal transit and get motility after in touch with the accessory gland fluids. Epididymis contains some inhibitory factors like lactate which diffuse into the sperm and lowers the pH inhibiting the motility of sperm. Outside the epididymis, it is reversed by the influx of Na+and HCO3-from the fluid of accessory glands[55]. Exposure to these inhibitory factors in epididymis for a prolonged period might be affecting either on metabolic activity of sperms or the opening of ion channels that is responsible to regain motility outside Epididymis.

As daily sperm production in human is 100-500 millions [17], increase in total count and high level of semen for a prolonged period in the sperm ducts that store it, may affect the motility and viability of sperms creating an oxidative stress condition through inflammation of the prostate gland[56]. Apart from these increased exposure to pollutants, endocrine disrupters (pthalates, polychlorinated biphenyles, xenoestrogens, pesticides, etc.), consumption of junk food, alcohol and smoking, badly affects the semen quality either by disrupting hormonal control or by DNA damage though huge production of reactive oxygen species. Disregulation of sex hormones and accessory sex glands (seminal vesicles, prostate and Cowper’s glands) may affect the semen volume and potency of sperm for fertilization. These may be some of the possible reasons of the optimal activity of WHO amended semen parameters, in our 3 years retrospective data analysis.

In conclusion, present study predicted that aging negatively affect progressive motility, vitality and morphology of sperms and beyond 5 days of sexual abstinence sperm motility is reduced.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

SP is supported by a research fellowship from SOA University. Authors are thankful to Prof. DK Roy, Dean for consistent encouragement. Authors are also grateful to Er. Gopabandhu Kar, Managing Member, SOA University and Vice-Chancellor, SOA University for the financial support.

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ment heading

10.1016/S2305-0500(14)60017-8

*Corresponding author: Prof (Dr.) Babita Panda (Obst & Gyn), Head of the Unit, Centre for Human Reproduction, Institute of Medical Sciences & SUM Hospital, K8- Kalinga Nagar, Bhubaneswar-751003, Odisha, India.

E-mail: chrimsivf@gmail.com

Abstinence

Semen quality