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From the * Reproductive Medicine Center, Scanian
Andrology Center, and the Department of
Clinical Chemistry, Lund University, Malmö University Hospital,
Malmö, Sweden
| Correspondence to: Dr Saad Elzanaty, Reproductive Medicine Center, Scanian Andrology Center, Lund University, Malmö University Hospital, SE 205 02 Malmö, Sweden (e-mail: saad.elzanaty{at}med.lu.se). |
| Received for publication February 3, 2007; accepted for publication May 29, 2007. |
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Abstract |
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-glucosidase [NAG]), prostate (prostate-specific antigen [PSA]
and zinc), and seminal vesicles (fructose). Three groups were defined
according to time from ejaculation to analysis: G
30
(24–30 minutes), G31–60 (31–60 minutes), and
G>60 (63–180 minutes). The proportion of progressively
motile sperm was significantly lower in G>60 than in
G30 (mean difference, 8.0%; 95% confidence interval [CI],
2.0%–13%) or G31–60 (mean difference, 6.0%; 95% CI,
1.0%–12%). The proportion of rapid progressive sperm motility was
significantly higher in G30 compared with
G31–60 (mean difference, 3.0%; 95% CI, 1.0%–5.0%) and
G>60 (mean difference, 6.0%; 95% CI, 1.0%–10%). Sperm
morphology and viability did not vary significantly between the groups.
However, PSA levels in G>60 were 29% and 31% significantly lower
than in G30 (95% CI, 3.0%–54%) and
G31–60 (95% CI, 7.0%–58%), respectively. Moreover, men
in G>60 had 29% and 17% significantly lower zinc compared with
those in G30 (95% CI, 4.0%–69%) and
G31–60 (95% CI, 4.0%–64%), respectively. Levels of NAG
and fructose did not differ significantly between the groups. There were
negative associations between the ejaculation-to-analysis interval and sperm
motility and levels of PSA and zinc. In male infertility assessments, semen
analysis should be performed within 60 minutes of ejaculation.
Key words: Biochemical markers, morphology, semen analysis, viability
The analysis of semen quality plays an important role in clinical decisions regarding the strategy for infertility treatment. Therefore, it is essential to minimize the impact of variation in sample delivery and analytic conditions on the outcome of this testing. In the World Health Organization (WHO) manual (1999), which is the most accepted guideline for semen analysis, it is recommended that assessment of semen in infertility investigations be performed within 60 minutes of ejaculation. However, both the European Society of Human Reproduction and Embryology (ESHRE) and the Nordic Association for Andrology (Kvist and Bjorndahl, 2002) strongly advise that semen analysis be done within 30 minutes of collection. In contrast to both those levels, Mortimer et al (1982) have postulated that analysis can done up to 3 hours after ejaculation, although it is preferable that it be achieved within 2 hours.
In addition to the very limited scientific knowledge about the effects of the ejaculation-to-analysis interval on sperm motility, information is also lacking about the mechanisms underlying that association. Therefore, the aim of the present study was to investigate sperm motility in relation to the impact of the time from ejaculation to analysis on markers of the functions of the epididymis and accessory sex glands.
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Materials and Methods |
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Semen Samples
The ejaculates were obtained by masturbation after 1–30 days (median,
4 days) of sexual abstinence. Only completely collected semen samples were
included. For men delivering more than 1 sample during the study period, only
the first ejaculate was included in the analysis. For each semen sample, the
time of delivery to the laboratory and the time of semen analysis were
recorded on the semen analysis form.
Semen Analysis
The semen samples were allowed to liquefy at 37°C. After liquefaction,
within 24–180 minutes of ejaculation, aliquots of the samples were
subsequently analyzed for semen volume, sperm concentration, motility (graded
as follows: a, rapid progressive motility; b, slow progressive motility; c,
local motility; or d, immotility), viability, and morphology. All these tests
were performed according to the WHO recommendations
(1999). Semen volumes were
measured by weighing the containers with and without semen using Sartorius
balances (Tillquist Analysis AB, Stockholm, Sweden). Sperm concentration was
assessed using positive displacement pipettes and improved Neubauer
hemocytometer. Sperm morphology was assessed after Papanicolaou staning, and
viability was assessed using eosin-nigrosin–stained smears using WHO
criteria. The analyses of ejacuiates were performed by 3 laboratory
assistants, and the interobserver coefficient of variation for motility
assessment was 8.5%. This laboratory participates in an external quality
control program organized by the Nordic Association of Andrology and
ESHRE.
For each sample, 450 µL of the remaining ejaculate was collected using a
common air displacement pipette and then mixed with 50 µL of benzamidine
(0.1 M) to stop the biochemical processes involved in liquefaction. The
mixture was centrifuged for 20 minutes at 4500 x g, and the
seminal plasma was decanted and stored at –20°C until analyzed for
neutral -glucosidase (NAG) activity and concentrations of
prostate-specific antigen (PSA), zinc, and fructose.
Biochemical Markers
Biochemical markers of function were assessed for the epididymis (NAG),
prostate (PSA and zinc), and seminal vesicles (fructose) as previously
described (Elzanaty et al,
2002). NAG was analyzed by first measuring total
-glucosidase activity using an Episcreen kit (Fertipro, Beernem,
Belgium) according to the instructions of the manufacturer and thereafter
estimating the NAG activity by use of the table included in the kit. The
concentrations of PSA, zinc, and fructose in seminal plasma were determined
using a PROSTATUS kit (Wallac Oy, Turku, Finland), a colorimetric method
(Makino et al, 1982), and a
spectrophotographic technique (essentially as described by
Wetterauer and Heite, 1976),
respectively.
Background Characteristics
The subjects included in the present study were 20–64 years of age
(median, 34 years). Ninety-five percent of the samples were analyzed within
24–60 minutes of ejaculation, and 5% were analyzed within 63–180
minutes. After subtracting the volume of semen required for routine analysis,
only 915 of the neat samples contained a sufficient amount of semen for
analysis of biochemical markers. Moreover, the biomarkers PSA, zinc, and
fructose were analyzed first, and thereafter only 504 of the 915 samples
contained enough semen for analysis of NAG. The proportions of semen samples
delivered in different seasons were as follows: 24.4% in spring
(March–May), 19.1% in summer (June–August), 29.3% in autumn
(September–November), and 27.2% in winter (December–February).
Samples found to have high viscoelasticity (n = 60) were excluded from the
analyses.
Statistical Methods
Statistical analysis was performed using SPSS 11.0 software (SPSS Inc,
Chicago, Ill). The normal distribution of residuals was determined using
normal probability plots, after which logarithmic transformations of total
activity of NAG and total amounts of PSA, zinc, and fructose were done to
ascertain the normal distribution of residuals. The remaining data were not
transformed. The subjects were divided into 3 groups according to the interval
from ejaculation to analysis: G30 (24–30 minutes),
G31–60 (31–60 minutes), and G>60
(63–180 minutes). Linear regression analysis models were applied to
investigate the effects of the ejaculation-to-analysis interval on sperm
motility, viability, and morphology and on amounts of NAG, PSA, zinc, and
fructose. As potential confounding factors, we considered the age of the
donors (years), the length of sexual abstinence (number of consecutive days),
and the season of semen collection (spring [March–May], summer
[June–August], autumn [September–November], and winter
[December–February]). P values below .05 were considered
statistically significant.
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Results |
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Sperm Motility, Morphology, and Viability
The proportion of rapid progressive sperm motility (grade a) was
significantly higher in the G30 compared with
G31–60 (mean difference, 3.0%; 95% confidence interval [CI],
1.0%–5.0%; P = .01) and G>60 (mean difference,
6.0%; 95% CI, 1.0%–10%; P = .02), but there was no significant
difference between the G31–60m and G>60 samples.
The proportion of progressive motility (grades a + b) was significantly lower
in G>60 compared with G30 (mean difference,
8.0%; 95% CI, 2.0%–13%; P = .01) and G31–60
(mean difference, 6.0%; 95% CI, 1.0%–12%; P = .02), whereas
there was no significant difference between G30 and
G31–60. The proportion of immotile spermatozoa (grade d) was
significantly higher in G>60 compared with G30
(mean difference, 8.0%; 95% CI, 3.0%–13%; P = .004) and
G31–60 (mean difference, 8.0%; 95% CI, 3.0%–13%;
P = .003), although there was no significant difference between
G30 and G31–60. Furthermore, the proportions
of spermatozoa exhibiting slow progressive motility (grade b) and local
motility (grade c) did not differ significantly between groups
(Table 2); nor did the
proportions of morphologically normal and viable cells
(Table 2).
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Markers of Epididymal and Accessory Sex Gland Function
PSA levels in the G>60 were 29% and 31% significantly lower
than those found in the G30 (95% CI, 3.0%–54%) and
G31–60 (95% CI, 7.0%–58%) samples, respectively. Also,
the zinc levels in G>60 were 29% and 17% significantly lower
than those in G30 (95% CI, 4.0%–69%) and
G31–60 (95% CI, 4.0%–64%), respectively. There were no
significant differences between G30 and G31–60
with regard to levels of PSA and zinc. Markers of epididymal function (NAG)
and seminal vesicle performance (fructose) did not differ significantly among
groups (Table 3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Sperm motility is considered to be one of the most important factors predicting the fertilizing ability of ejaculated spermatozoa both in vivo and in vitro (Bongso et al, 1989; Eimers et al, 1994; Donnelly et al, 1998). Notably, an earlier study indicated that a delay of up to 3 hours after ejaculation to the time to analysis had no significant adverse effect on the mobility of spermatozoa (Mortimer et al, 1982), whereas we observed significantly lower sperm movement when analysis was performed more than 1 hour after collection of semen samples.
Furthermore, during spermatogenesis, the locomotor apparatus of the spermatozoa is formed and becomes functional (Mohri and Ishjima, 1989), and considerable amounts of zinc are incorporated into the spermatids (Parizek et al, 1966). It has also been observed that the spermatozoa liberated from the rete testis and caput epididymis show only sluggish, nonprogressive movement (Cooper, 1986). The capacity for progressive motility is gained solely during maturation of the spermatozoa as they are transported through the epididymis (Haidl et al, 1994). In the course of that journey, the zinc content of the sperm is reduced by approximately 60% (Kaminska et al, 1987), which leads to the increased stabilization of the outer dense fiber (ODF) proteins that is induced when sulfhydryl groups are oxidized to form disulfide bridges (Calvin et al, 1973). In our study, prolonging the time from ejaculation to analysis to more than 1 hour was associated with significantly lower levels of zinc in the semen samples. A plausible explanation for that observation is that the binding of zinc to spermatozoa was augmented with increasing time from ejaculation to analysis, resulting in greater flexibility of the ODF proteins and consequently diminished motility of the spermatozoa. Perhaps future studies will confirm this assumption and, if so, they might also explain why the incorporation of zinc increases with time from ejaculation.
PSA is considered to be the primary proteolytic enzyme in seminal plasma, and it has been shown that this protein degrades the 2 major components of the semen coagulum (semenogelins I and II [SgI and II]; Lilja et al, 1989) into lower molecular weight fragments (Lilja, 1985; Robert and Gagnon, 1996) and thereby facilitates free movement of the spermatozoa (Malm et al, 2000). In the current study, we found that the levels of PSA decreased as the ejaculation-to-analysis time increased to more than 1 hour, which might be at least partially attributable to a modification in the antigenic epitopes. The biochemical mechanism behind such an alteration is not known, although it does seem to be of practical importance considering the time-related drop in PSA.
The fructose present in seminal plasma is believed to be the main source of energy for sperm metabolism and motility in vitro (Mann, 1964). Therefore, it is reasonable to assume that an increased interval between ejaculation and analysis will be associated with a decline in the levels of fructose. However, that notion is not supported by our results, possibly because there are other sources of energy present in seminal plasma, including glucose, which has been reported to constitute almost half of the sugar consumed by spermatozoa (Martikainen et al, 1980).
The morphologic development of spermatozoa is decisive for the motility of these gametes (Bedford, 1979). We found no significant difference in sperm morphology between the groups of semen samples investigated in our study, which agrees with earlier results reported by Mortimer and colleagues (1982). However, in contrast to the findings of Mortimer et al, we did not observe any effect of the time from ejaculation to analysis on the proportion of viable spermatozoa.
Our observations have obvious practical implications. Semen analysis is the cornerstone in male infertility assessment, and the information provided by such evaluation serves as a basis for the diagnosis and treatment of infertile men. Therefore, it is highly important to standardize semen investigation procedures to include shortening of the interval from collection to analysis of ejaculates, because that will improve the possibility of comparing the results of repeated analyses. Our study strongly suggests that investigation of semen be done within 60 minutes of ejaculation.
Our findings may also have therapeutic value. That conclusion is made in light of a study showing that intrauterine insemination performed with spermatozoa from semen samples processed more than 60 minutes after ejaculation resulted in no pregnancy, whereas the use of spermatozoa from semen processed within 30 and 60 minutes of ejaculation led to pregnancy rates of 29% and 13%, respectively (Yavas et al, 2004).
In conclusion, we found lower sperm motility with ejaculation-to-analysis time of more than 1 hour, and that finding was supported by measurements of PSA and zinc as markers of prostatic function. Further studies are needed to ascertain whether there is a connection between those 2 observations. In contrast, the interval from ejaculation to analysis had no apparent effect on sperm morphology or viability or on the markers of epididymal and seminal vesicle function (NAG and fructose). Our results also clearly indicate that semen analysis as a part of male infertility assessment should be done no longer than 1 hour after ejaculation.
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Acknowledgments |
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Footnotes |
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References |
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