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From the * Laboratory of Histology &
Embryology, Medical School of Sfax, Sfax, Tunisia; the
Unit of Biostatistics & Bioinformatics,
Centre of Biotechnology of Sfax, Sfax, Tunisia; the
Department of Gynaecology and the
Unit of Male Infertility Department of Urology,
Medical University of Sfax, Sfax, Tunisia.
| Correspondence to: Dr Nozha Chakroun Feki, Laboratory of Histology & Embryology, Medical School of Sfax, Route Majida Boulila, 3028, Sfax, Tunisia (e-mail: nozhafeki{at}yahoo.fr). |
| Received for publication June 10, 2008; accepted for publication January 29, 2009. |
Concerns about the worldwide decline in semen quality over the past 50
years are increasing. Western countries have shown a decline in semen quality.
However, in non-Western countries studies are sparse. We investigated trends
in semen parameters between 1996 and 2007 in the Sfax area of southern Tunisia
in a sample of 2940 men in infertile relationships. Age at semen collection,
duration of sexual abstinence, volume of seminal fluid, the sperm count,
percentages of motile and morphologically normal spermatozoa, and semen
leukocyte concentration were determined. Linear regression was used to examine
trends over time in sperm count, sperm motility, normal morphology, and semen
leukocyte concentration. Mean age and semen volume did not change between 1996
and 2007. Data adjusted for age and abstinence showed a decreasing trend in
sperm count and percentage of normal morphology over the last 12 years
(R2 = 0.71, P = .0004, and R2
= 0.87, P < .0001, respectively). There was no significant change
in sperm motility. However, semen leukocyte concentration increased
significantly over time (R2 = 0.38, P = .03).
These results coincide with the high prevalence of genital infectious diseases
in the Sfax area, suggesting that infection may be a potential contributing
factor in semen quality decline.
Key words: Semen quality, leukocyte, infertility, trend
Other less-studied factors, such as lifestyle issues and stress, have been associated with male reproductive malfunction. Moreover, it is suspected that infectious disease may affect reproductive function (Dieterle, 2008). Diagnosis of male genital infection remains difficult and the methods used for detection of these bacteria are not standardized. Although high prevalence of genital infection among men and women has been noticed in both industrialized and developing countries, the relationship between this factor and semen quality decline has never been investigated (LaMontagne et al, 2004; Gdoura et al, 2008).
Sfax is the biggest city of southern Tunisia with about 1 million inhabitants, and represents an important industrial and agricultural area. There are no available data for variations of semen parameters in different cities within this country or in other countries of North Africa.
The aim of this study was to investigate semen quality in men in infertile relationships who were under investigation for couple infertility and attended a clinic for routine semen analysis over a period of 12 years.
Materials and Methods
Subjects
In this retrospective study, we included men in infertile relationships who
attended the Laboratory of Histology and Embryology at the Sfax Medical School
for semen analysis between 1996 and 2007 (n = 2940). From the beginning (in
1995) and over the following 11 years (ie, 1996–2007) the recruitment of
men in infertile relationships at our laboratory was mainly from the
Department of Gynaecology and the Department of Urology of the Sfax hospital.
We considered only the first ejaculate if the patient had submitted more than
1 specimen during infertility investigation. Men with azoospermia and those
with spermatozoa only in the pellet were excluded. The mean (±SD) age
in the studied group was 36 ± 6.9 years (range 21–74).
The population consisted of both rural and urban men living and working in the area of Sfax. This study was approved by the Institutional Review Board of the Medical Faculty of Sfax.
Sample Collection
Semen samples were collected by masturbation into plastic containers, in
the laboratory, after a recorded abstinence time varying between 1 and 8
days.
Semen Analysis
Analysis was performed by a specially trained laboratory technician
according to the standardized methods recommended by the World Health
Organization (WHO) (1992). The
following semen parameters were analyzed: volume, sperm concentration, sperm
count, percentage of motile spermatozoa, and percentage of normal spermatozoa.
Samples were analyzed within 30 minutes to 1 hour to allow liquefaction. The
volume was determined by drawing up the entire sample into a 10-mL graduated
glass tube. For sperm concentration, diluted semen samples were mixed before
transferring a drop to the chamber of the hemocytometer. After about 5 minutes
in a moist chamber, to allow for the sedimentation of the cells, the
spermatozoa were counted under a light microscope at x4000
magnification. The number of sperm cells was counted in squares on the
diagonal of the chamber, and the mean value was calculated
(WHO, 1992). The sperm
concentration per mL of semen sample obtained was then multiplied by semen
volume to obtain the sperm count in the overall ejaculate.
To determine the percentage of motile spermatozoa, a 10-µL drop of gently mixed semen was placed on a heated glass slide (37°C) under a square cover glass (22 mm). The slide was placed on a heating stage (37°C) and observed at x4000 magnification. The percentage of motile spermatozoa was evaluated according to WHO guidelines (WHO, 1992).
Sperm morphology was assessed in Shoor-stained semen smears. All samples were examined using the same technique and by the same technician. Criteria and classification proposed by David et al (1975) were used for morphology assessment of all the samples during the 12 years. Internal quality control was performed regularly by the medical staff.
We differentiated leukocytes from germ cells by a histochemical method based on peroxidase staining recommended by the WHO (1992). For the assessment of leukocyte concentration per 1 mL of semen, stained samples were mixed before transferring a drop to the chamber of the hemocytometer. After sedimentation of the cells, the brown-colored cells (leukocytes) were counted under a light microscope at x4000 magnification in squares on the diagonal of the chamber, and the mean value was calculated.
Statistical Analysis
The data were analyzed using SPSS statistical computer software (13.0; SPSS
Inc, Chicago, Illinois) and Microsoft Office Excel. Linear regression was used
to examine trends over time in sperm density, motility, and normal morphology
and in semen leukocyte concentration. Because age and duration of abstinence
influence the characteristics of semen, we used a single multiple regression
model to assess the trend over time, adjusting for age and abstinence time.
The estimated slope parameter from this model represents the change over time
adjusted for age and abstinence time.
We analyzed the trend in semen parameters for infertile men with a normal
sperm concentration (
20 x 106/mL, n = 1835) to minimize
selection bias (Sripada 2007).
An analysis of variance (ANOVA) test was carried out to see if there were any overall differences in the means of sperm parameters, for each year of the period 1996 to 2007 (Table 1) and for age range (Table 2) and abstinence duration (Table 3).
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Results
Data on semen samples from a total of 2940 men were available for analysis. Age, volume, sperm count, sperm motility, and normal morphology had normal distributions. Semen leukocyte concentration did not have a normal distribution in our subject group of infertile men; the best transformation of the data that yielded normal distribution was obtained with logarithmic transformed values.
The means of age, sperm density, and percentages of motile and normal sperm for the studied population (n = 2940) were 36.0 years, 184.4 x 106/mL, 41.1%, and 22.3% respectively. The mean value for semen leukocyte concentration was 0.4 x 106/mL.
Descriptive statistics and ANOVA test results for unadjusted data for each of the sperm parameters for each year from 1996 to 2007 are listed in Table 1. There were significant differences in the average annual sperm density, motility, and normal morphology values across the 12-year period (Table 1). Of the 2940 men, 1835 had sperm concentrations greater than 20 x 106/mL (the normal sperm concentration according to WHO criteria; WHO, 1992). The mean age of the men (n = 1835) in the present study was 36 years and did not significantly change during the study. Sperm motility and morphology decreased with increasing age groups, whereas sperm concentration did not fluctuate among age groups (Table 2). Even if a time of abstinence from 3 to 5 days was recommended before ejaculation, only 71% of men respected this period, and the duration of abstinence varied between 1 and 8 days. Sperm motility, count, and morphology depended significantly on the number of abstaining days (Table 3).
The mean value (SD) for semen volume was 3.3 (1.6) mL and did not significantly change over the 12 years (Figure 1).
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The studies of trends in semen parameters in developing countries were sparse. There are great differences in environment, culture, lifestyle, and risk factors for infertility between African and Western countries. This is the first study of semen parameter trends of men in the north of Africa.
We found a decline in the sperm count and sperm morphology over a 12-year period among men in infertile relationships from the south of Tunisia, suggesting that the reported worldwide decline in semen quality is also real in certain areas of the African continent. This finding contrasted with the increase of semen leukocyte concentration, suggesting the implication of genital infection as a potential contributing factor in semen quality decline in the Sfax area.
Our results are in accordance with previous reports on a decline in semen quality in men in infertile relationships. Sripada et al (2007), using a similar design to our study, found a decrease in sperm concentration of men in infertile relationships from 75 x 106/mL in 1994 to 55 x 106/mL in 2005. Lackner et al (2005) reported the same trends in sperm concentration from 27 x 106/mL to 10 x 106/mL between 1986 and 2003. In contrast, Andolz et al (1999) found a statistically significant decline in semen volume and a nonsignificant increase in sperm concentration in a sample of 20 411 men in infertile relationships. One of the main weaknesses of the present study is the type of the recruitment of men; we included only men in infertile relationships, but to minimize selection bias, we excluded oligozoospermic samples having less than 20 x 106 spermatozoa per mL of semen (WHO, 1992). We did not have technical skews; indeed, only 1 technician carried out the analysis over such a long period. We did not introduce any newer method or equipment. In order to reduce intraindividual variations in semen samples, we used the first semen sample from each man. Variables such as sperm count, motility, and morphology were adjusted for the main confounding factors: age and abstinence. The other factors influencing semen quality, such as profession, education, smoking, or level of stress, were not considered in this study.
The main advantage of this study is the assessment of semen leukocyte concentration over the last 12 years, which showed an increase in this parameter coinciding with an increasing incidence of genital infection diseases in the Sfax area as reported by the microbiologists of the Medical School of Sfax in 2001 and 2008 (Gdoura et al, 2001, 2008). In fact, Chlamydia trachomatis, genital mycoplasmas, and genital ureaplasmas seem to be widespread among men in infertile relationships in southern Tunisia (43.3%, 14.4%, and 18.3%, respectively). In all, the prevalence of bacteriospermia in semen samples from men who underwent evaluation of fertility in the Sfax area was 56.9%. However, these authors failed to give evidence of the effect of these pathogens on semen parameters (Gdoura et al, 2001, 2008). In the current literature, there is an indication of an increase in the prevalence of genital infections among men and women (LaMontagne et al, 2004). In our area, this is likely related to the relative change in the sexual practices in young people.
High prevalence of sexually transmitted diseases in the developing countries of Africa was recorded recently (Ochsendorf, 2008).
Microorganisms might impair the male reproductive function in different ways. The inflammatory process in the genital tract may lead to deterioration of spermatogenesis and obstruction of the seminal tract (Keck et al, 1998), worsening characteristics of semen and sperm density. Activating leukocytes produce reactive oxygen species, which are known to impair DNA integrity (Agarwal et al, 2008).
A relationship between bacteriospermia and leukocytospermia at different cutoff levels in semen samples was rarely found (Punab et al, 2003; Lackner et al, 2006). This could be because of the technical difficulties in determining leukocyte concentration.
In our laboratory, we have used peroxidase staining since 1996 to quantify leukocytes in semen, which is a practical and reliable method recommended by WHO for determining leukocytes. This method differentiates only polymorphonuclear neutrophil granulocytes (PMNs) from leukocytes. Nevertheless, PMNs are primary antimicrobial effector cells of the innate immune system, and high levels of these cells may reflect marks of pathogen invasion.
Few data are available concerning trends of leukocyte concentration in infertile men. A trend toward lower numbers of leukocytes over 18 years in infertile men was reported by Lackner et al (2005). Unfortunately, their data are not suitable for comparison with ours; these authors did not identify and differentiate leukocytes from germ cells, resulting in high means of leukocyte concentration during their study.
Nevertheless, the presence and clinical significance of leukocytes in semen are still controversial (Tomlinson et al, 1993; Lackner et al, 2008). Elevated concentrations of leukocytes in semen have been associated with genital tract infection, poor semen quality, and in vitro fertilization failure (Wolff 1995).
Because Sfax is an important industrial and agricultural center, the hypothesis that the exposure to endocrine-disrupting chemicals in the area has contributed to the decline in sperm count cannot be ruled out. Exposure to environmental chemicals, including endocrine-disrupting compounds, is suspected in affecting male gonadal functions (Daston et al, 1997, McGlynn et al, 2008; Sikka and Wang, 2008). A generalized increase in gonadal dysfunction in various Western countries has been suggested by authors who have observed increases in the incidence of testicular cancer and cryptorchidism during the last 30 years (Boyle et al, 1987; Jackson, 1988; Carbone et al, 2006). In the north of Africa there are no reliable data that confirm similar trends.
Based on these considerations, prospective monitoring of sperm parameters should be carried out in the future to assess the changes in male gonadal function and to investigate contributory factors to observed declines.
In conclusion, this is the first reliable report about trends in the semen quality of Tunisian men in infertile relationships. We found a decline in sperm count and morphology associated with an increase of semen leukocytes over the last 12 years coinciding with a high incidence of genital infections in the area, especially Chlamydia trachomatis. The potential impact of a real decline in semen quality on human fertility remains doubtful; however, the increase on semen leukocyte and genital infectious diseases should be more alarming. Therefore, the introduction of preventive precautions should be considered as a priority among reproductive health concerns.
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