1 Fertility Center and 2 Department of Clinical Chemistry, Scanian Andrology Center, Lund University, Malmö University Hospital, SE 205 02 Malmö, Sweden
3 To whom correspondence should be addressed. Email: saad.elzanaty{at}kir.mas.lu.se
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: fructose/NAG/PSA/sexual abstinence period/sperm motility
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sperm motility is believed to be one of the most important parameters in evaluating the fertilizing ability of the ejaculated spermatozoa (Bongso et al., 1989; Eimers et al., 1994
; Donnelly et al., 1998
). Moreover, recent data have indicated that sperm motility characteristics obtained by computer-aided sperm analysing systems (CASAs) may also serve as predictive parameters of fertility (Larsen et al., 2000
; Hirano et al., 2001
).
Several factors have been shown to influence semen parameters, one of which is the length of sexual abstinence. Although there is general agreement that semen volume and sperm concentration increase with prolonged sexual abstinence (LeLannou et al., 1986; Blackwell and Zaneveld, 1992
; Pellestor et al., 1994
), the issue of sperm motility is still contradictory (Mortimer et al., 1982
; Sauer et al., 1988
; Check et al., 1991
; Magnus et al., 1991
; Blackwell and Zaneveld, 1992
; Pellestor et al., 1994
).
The results of seminal investigation play an important role for clinical decisions regarding the strategy for infertility treatment. Therefore, it is of a crucial importance to minimize the impact of variation in sample collection conditions on the final result of this test. According to the manual of the World Health Organization (WHO), which is the most accepted guideline for performing semen analysis, a 27 day abstinence period is recommended prior to semen analysis for infertility investigation (World Health Organization, 1999). However, in the recommendations by the European Society of Human Reproduction and Embryology (ESHRE) and the Nordic Association for Andrology (Kvist and Bjorndahl, 2002
), standardization of abstinence time to 34 days is strongly advised.
Apart from the lack of proper information about the correlation between the length of the sexual abstinence period and sperm motility, the possible mechanism of such an association is also unresolved. Epididymal and accessory sex gland secretions play a crucial role for proper sperm function (Mann, 1964; Tremblay et al., 1979
; Lilja, 1985
; Lilja et al., 1989
; Lee et al., 1989
; Kret and Milad, 1995
; Robert and Gagnon, 1996
). However, only one study addressed the issue of the length of sexual abstinence seen in relation to the epididymal and accessory sex gland secretions (Cooper et al., 1993
).
Therefore, the aim of this study was to determine the association between the length of sexual abstinence periods and the epididymal and accessory sex gland secretions and their relationship to sperm motility as assessed manually and by use of CASA.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Semen samples
The ejaculates were obtained by masturbation after 27 days of sexual abstinence (median 4 days). Only completely collected semen samples were included. For men delivering more than one sample during the study period, only the first ejaculate was included in the analysis.
Semen analysis
Thirty minutes after ejaculation, 450 µl of the ejaculate were taken off using a common air displacement pipette and mixed with 50 µl of benzamidine (0.1 mol/l) in order to stop the biochemical processes involved in liquefaction. The mixture was centrifuged for 20 min at 4500 g, and the seminal plasma was decanted and stored at 20°C until analysed for the activity of neutral -glucosidase (NAG), and the concentrations of prostate-specific antigen (PSA), zinc and fructose (see below). After liquefaction, within 1 h after ejaculation, the ejaculates were analysed for the following characteristics: semen volume, sperm concentration, sperm motility (the motility grade; a, b, c and d) and sperm morphology. All semen tests were performed according to the WHO semen manual (1999). Thereafter, CASA motility using the CRISMAS (Image House, Copenhagen, Denmark) system was performed as described before (Elzanaty et al., 2002
) with the following modification. Motility analysis was performed in 20 µl Microcell chambers (Leja, Oslo, Norway) instead of a 10 µl Makler chamber. The CASA motility was classified as follows; motile spermatozoa [curvilinear velocity (VCL)>25 µm/s], locally motile spermatozoa (VCL 525 µm/s) and immotile spermatozoa (VCL < 5 µm/s). CASA was performed in 375 of the 422 semen samples. The 47 samples with no CASA done did not differ from the remaining 375 regarding the length of the abstinence period.
Biochemical analysis
Biochemical markers of epididymal function (NAG), prostatic function (PSA and zinc) and seminal vesicles function (fructose) were assessed as described before (Elzanaty et al., 2002). Total
-glucosidase activity was first measured using a commercially available kit (Episcreen®; Fertipro, Gent, Belgium) according to the instructions given by the manufacturer; thereafter, the NAG activity was estimated by the use of the corresponding table provided by the manufacturer. The concentration of PSA in seminal plasma was determined with the PROSTATUSTM kit from (Wallac Oy, Turku, Finland). The concentration of zinc in seminal plasma was determined by a colorimetric method (Makino et al., 1982
). The concentration of fructose in seminal plasma was determined with a spectrophotographic method, essentially as described in Wetterauer and Heite (1976)
. Because of low semen volume, the biochemical markers were only measured in 401 of the 422 semen samples. The 21 samples with no biochemical analysis done did not differ from the remaining 401 regarding the length of the abstinence period.
Statistical methods
Statistical analysis was performed using the SPSS 11.0 software (SPSS Inc., Chicago, IL). The period of sexual abstinence was calculated (expressed as whole days) from the date and time of previous ejaculation, which was asked for in the questionnaire completed by the patients. The subjects were divided according to the length of sexual abstinence into three groups: G23 (23 days), G45 (45 days) and G67 (67 days). For each parameter, the variation between the three abstinence period groups was analysed primarily by means of KruskalWallis test. For each parameter where a statistically significant variation was found, pair-wise comparison was performed by MannWhitney U-test. No adjustment for seasonal variation in semen parameters was done. However, the sample deliveries and the length of the sexual abstinence period were evenly distributed throughout the study period. Finally, the proportion of men who fall below the WHO standards regarding semen volume, sperm concentration or percentage motile spermatozoa were compared between the different abstinence groups using Fisher's test. Morphology data were not included in this analysis since the WHO manual does not define any reference range for this parameter. A P-value <0.05 was considered as statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The time of abstinence and sperm parameters
Sperm concentration and total sperm count were significantly higher in G45 compared with G23 (P=0.010 and P<0.001, respectively) but were not different from G67. The percentage spermatozoa with tail defects was significantly higher in G67 when compared with G23 (P=0.011) and G45 (P=0.002) (Table I).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although in G45, as compared with the two other groups, a significantly higher proportion of men fulfilled the WHO criteria considering seminal volume, sperm concentration and percentage motile spermatozoa, the results of the current study do not allow us to pinpoint any subinterval within the range of 27 days as ideal from the sperm motility point of view, but rather to conclude that significant differences exist within this time frame.
Our finding of decreased sperm motility with a short abstinence period was in agreement with previous reports regarding men assessed for infertility (Mortimer et al., 1982) as well as normal men (Cooper et al., 1993
). In the study of Cooper et al., where the subjects were asked for multiple ejaculations for depletion of extra-gonadal reserves prior to the actual period of abstinence, the short length of the abstinence period was associated with a lower level of NAG activity and amount of zinc, which seems in accordance with our finding (Cooper et al., 1993
). Thus, it has been found that the final sperm function is dependent on epididymal transit where spermatozoa maturation occurs (Bedford, 1990
; Amann et al., 1993
). The transit time through the epididymis can be influenced by external factors such as sexual stimulus and ejaculatory frequency, inducing a sharp rise in intraluminal pressure, as a consequence of which the passage of spermatozoa is speeded up (Amir and Ortavant, 1968
; Tischner, 1972
) which might influence the maturity of spermatozoa. NAG is excreted mostly by the cauda epididymidis, and was found to correlate positively with the motility of spermatozoa (Viljoen et al., 1990
; Fourie et al., 1991
; Elzanaty et al., 2002
). Its exact physiological role is not yet known; however, it might be important to provide the spermatozoa with optimal levels of energy (Tremblay et al., 1979
).
Semenogelins are considered to be the main seminal coagulum proteins (Lundwall et al., 2002). PSA has been reported to be involved in degradation of semenogelins (Lilja et al., 1989
; Robert and Gagnon, 1996
), and previously found to correlate positively with sperm motility (Elzanaty et al., 2002
). Zinc represents another potentially important factor for the control of the proteolytic activity of PSA in seminal plasma. The concentration of this metal in seminal fluid (Zaneveld and Taubar, 1981
) was found to be at a level equal to one that has been shown to be inhibitory for the activity of the PSA (Watt et al., 1986
).
In vitro, fructose has been shown to be the main source for energy and metabolism of spermatozoa (Mann, 1964). Our study did not find a significant difference between the abstinence time groups regarding fructose. Cooper et al. (1993) observed a decreased amount of fructose with a short abstinence period in normal men and oligozoospermic patients.
Also, the present finding of decreased motility after a long abstinence period was in agreement with previous reports from men assessed for infertility (Mortimer et al., 1982) and from normal men (Magnus et al., 1991
; Pellestor et al., 1994
). Such motility impairment might be explained by the presence of an increased percentage of spermatozoa with a tail defect in that group. Blackwell and Zaneveld (1992)
and Pellestor et al. (1994)
observed a decreased percentage of spermatozoa with normal forms after a long abstinence period, a phenomenon that is suggested to be due to senescence of spermatozoa (Pellestor et al., 1994
). Sperm motility depends on morphological development of spermatozoa (Bedford, 1979
). Thus, a highly significant correlation was observed between the morphological characteristics of the tail and total motility as well as the progressive motility of spermatozoa (Haidl et al., 1987
). Moreover, light microscopic assessment of human spermatozoa in post-coital samples of cervical mucus revealed that the excluded spermatozoa were more likely to be those with tail defects (Mortimer et al., 1982
). However, several previous reports were not able to find any association between the period of abstinence and sperm motility characteristics (Sauer et al., 1988
; Check et al., 1991
; Blackwell and Zaneveld, 1992
).
Recent studies have concluded that some of the CASA parameters, including VCL, VSL, (VAP), ALH and LIN provide a reliable estimation of the fertilizing ability of human spermatozoa both in vivo and in vitro (Donnelly et al., 1998; Larsen et al., 2000
; Hirano et al., 2001
). Our study demonstrated higher ALH values in semen samples obtained after 45 days of abstinence as well as higher VSL and LIN values in samples obtained after 23 days, pointing to a possible association between the length of the abstinence period and not only the percentage of motile spermatozoa but also the qualitative characteristics of their movements.
Our study has some limitations, which need to be pointed out. It was not based on the background male population but on patients referred due to infertility problems of the couple. Therefore, our results may not be representative for men in the general population but, on the other hand, the cohort selected by us is probably representative for the type of men delivering semen samples as a part of infertility work-up. This study has a cross-sectional design. Thus, a potential confounding factor which, therefore, cannot be excluded is the possibility that the subgroups with differing times of abstinence are not equal regarding the general status of their reproductive function. We did have access to hormone levels from 25% of all subjects. Although this subgroup was not representative for the whole cohort, at least with regard to sperm concentration and motility, among those 107 men we could not find any correlation between their sexual hormone levels, the length of abstinence period or even age.
An additional factor which might affect ejaculate quality, i.e. the duration of pre-ejaculatory sexual arousal, was not estimated in our study. It has been found that the time taken to produce a specimen was positively correlated with sperm concentration but not with ejaculate volume (Pound et al., 2002).
In conclusion, our study demonstrated that among men delivering a semen sample as a part of an infertility investigation, significant differences in the number and percentage of motile sperm as well as in the motility characteristics exist, depending on whether the period of abstinence is 23, 45 or 67 days. This effect may be at least partly mediated through variation in secretions from the epididymis and prostatic gland as well as the morphology of the spermatozoa, particularly the percentage of tail defects.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amir D and Ortavant R (1968) The effect of collection frequency on duration of sperm transit in the rams epididymis. Ann Biol Anim Biochim Biophys 8, 195207.[ISI]
Bedford JM (1979) Evaluation of the sperm maturation and sperm storage functions of the epididymis. In Fawcett DW and Bedford JM (eds), The Spermatozoon. Urban and Schwarzenberg, Baltimore, pp. 721.
Bedford JM (1990) The mammalian spermatozoon, morphology, biochemistry and physiology. Reproduction in the male. In Lamming GE (ed.), Marshalls Pyhsiology of Reproduction, Vol. 2. Churchill Livingstone, London, pp. 379568.
Blackwell JM and Zaneveld LJD (1992) Effect of abstinence on sperm acrosin, hypoosmotic swelling, and other semen variables. Fertil Steril 58, 798802.[ISI][Medline]
Bongso TA, Ng SC, Mok H, Lim MN, Teo HL, Wong PC and Ratnam SS (1989) Effect of sperm motility on human in vitro fertilization. Arch Androl 22, 185190.[ISI][Medline]
Check JH, Epstein R and Long R (1991) Effect of time interval between ejaculations on semen parameters. Arch Androl 27, 9395.[ISI][Medline]
Cooper TG, Keck C, Oberdieck U and Nieschlag E (1993) Effects of multiple ejaculations after extended periods of sexual abstinence on total, motile and normal sperm numbers, as well as accessory gland secretions, from healthy normal and oligozoospermic men. Hum Reprod 8, 12511258.[Abstract]
Donnelly ET, Lewis SEM, McNally JA and Thompson W (1998) In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertil Steril 70, 305314.[CrossRef][ISI][Medline]
Eddy EM and O'Brien DA (1994) The spermatozoon. In Knobil E and Neill JD (eds), The Physiology of Reproduction, 2nd edn. Raven Press, New York, pp. 2977.
Eimers JM, Te Velde ER, Gerritse R, Vogelzang ET, Looman CW and Habbema JD (1994) The prediction of the chance to conceive in sub-fertile couples. Fertil Steril 61, 4452.[ISI][Medline]
Elzanaty S, Richthoff J, Malm J and Giwercman A (2002) The impact of epididymal and accessory sex gland function on sperm motility. Hum Reprod 17, 29042911.
Fourie MH, du Toit D, Bornman MS, Van der Merwe MP and Du Plessis DJ (1991) -Glucosidase, sperm ATP concentrations, and epididymal function. Arch Androl 26, 139141.[ISI][Medline]
Haidl G, Hartmann R and Hofmann N (1987) Morphological studies of spermatozoa in disorders of motility. Andrologia 19, 433447.[ISI][Medline]
Hirano Y, Shibahara H, Obara H, Suzuki T, Takamizawa S, Yamaguchi C, Tsunoda H and Sato I (2001) Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro. J Assist Reprod Genet 18, 213218.[ISI][Medline]
Kret B and Milad M (1995) New discriminatory level for glucosidase activity to diagnose epididymal obstruction or dysfunction. Arch Androl 35, 2933.[ISI][Medline]
Kvist U and Bjorndahl L (eds) (2002) ESHRE Monographs: Manual on Basic Semen Analysis. Oxford University Press, pp. 124.
Larsen L, Scheike T, Jensen TK, Bonde JP, Ernst E, Hjollund NH, Zhou Y, Skakkebæk NE and Giwercman A (2000) Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. Hum Reprod 15, 15621567.
Lee C, Keefer M, Zhao ZW, Kroes R, Berg L, Liu Xianxi and Sensibar J (1989) Demonstration of the role of prostate-specific antigen in semen liquefaction by two-dimensional electrophoresis. J Urol 10, 432438.
LeLannou D, Colleu D, Boujard D, le Couteux A, Lescoat D and Segalen J (1986) Effects of abstinence on maturity of human spermatozoa. Arch Androl 17, 3540.[ISI][Medline]
Lilja H (1985) A kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein. J Clin Invest 76, 18991903.[ISI][Medline]
Lilja H, Abrahamsson PA and Lundwall A (1989) Semenogelin, the predominant protein in human semen. Primary structure and identification of closely related protiens in the male accessory sex glands and on the spermatozoa. J Biol Chem 25, 18941900.
Lindholmer C (1974) The importance of seminal plasma for human sperm motility. Biol Reprod 10, 533542.[ISI][Medline]
Lundwall A, Bjartell A, Olsson AY and Malm J (2002) Semenogelin I and II, the predominant human seminal plasma proteins are also expressed in non-genital tissues. Mol Hum Reprod 8, 805810.
Magnus O, Tollefsrud A and Abyholm T (1991) Effects of varying the abstinence period in the same individuals on sperm quality. Arch Androl 26, 199203.[ISI][Medline]
Makino T, Saito M, Horiguchi D and Kina K (1982) A highly sensitive colorimetric determination of serum zinc using water-soluble pyridylazo dye. Clin Chim Acta 120, 127135.[CrossRef][ISI][Medline]
Mann T (1964) Fructose, polyols, and organic acids. In Nam T (ed.), The Biochemistry of Semen and of the Male Reproductive Tract. Methuen & Co. Ltd, Ltd, pp. 237264.
Mortimer D, Leslie EE, Kelly RW and Templeton AA (1982) Morphological selection of human spermatozoa in vivo and in vitro. J Reprod Fertil 64, 391399.[ISI][Medline]
Pellestor F, Girardet A and Andreo B (1994) Effect of long abstinence periods on human sperm quality. Int J Fertil 39, 278282.[ISI]
Pound M, Javed MH, Ruberto C, Shaikh MA and del Valle AP (2002) Duration of sexual arousal predicts semen parameters for masturbatory ejaculates. Physiol Behav 76, 685689.[CrossRef][ISI][Medline]
Robert M and Gagnon C (1996) Purification and characterization of the active precursor of a human sperm motility inhibitor secreted by the seminal vesicles: identity with semenogelin. Biol Reprod 55, 813821.[Abstract]
Sauer MV, Zeffer KB, Buster JE and Sokol RZ (1988) Effects of abstinence on sperm motility of normal men. Am J Obest Gynecol 158, 604607.
Tischner M (1972) The role of the vas deferens and the urethra in the transport of semen in ram. Acta Agraria Silvestre (Zootecnia) 12, 77.
Tremblay RR, Chapdelaine P and Mailhot J (1979) ,14 glucosidase activity in human semen: variations with number and motility of spermatozoa. Fertil Steril 31 31, 592593.[ISI][Medline]
Viljoen MH, Bornman MS, Van der Merwe MP and du Plessis DJ (1990) Alpha-glucosidase activity and sperm motility. Andrologia 22, 205208.[ISI][Medline]
Watt KWK, Lee PJ, Timkulu TM, Chan WP and Loor R (1986) Human prostate specific antigen: structural and functional similarity with serine protases. Proc Natl Acad Sci USA 83, 31663170.[Abstract]
Wetterauer U and Heite H-J (1976) Eine empfehlenswerte Methode zur gleichzeitigen enzymatischen Bestimmung von Citrat und Fruktose im Seminalplasma. Akt Dermatol 2, 239248.
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 4. Cambridge University Press.
Zaneveld LJD and Tauber PF (1981) Contribution of prostate fluid components to the ejaculate. In Murphy GP, Sandbery AA and Kan JP (eds), The Prostatic Cell: Structure and Function. Part A. Alan, R. Liss, New York, pp. 265277.
Submitted on March 23, 2004; resubmitted on August 3, 2004; accepted on October 4, 2004.
|