FERTILITY AND STERILITY威

VOL. 82, NO. 1, JULY 2004

Copyright ©2004 American Society for Reproductive Medicine
Published by Elsevier Inc.
Printed on acid-free paper in U.S.A.

Influence of the abstinence period on

human sperm quality
Christopher De Jonge, Ph.D.,a Marie LaFromboise, B.S.,a Eugene Bosmans, Pharm.D.,b
Willem Ombelet, M.D., Ph.D.,b Annemie Cox,b and Martine Nijs, M.Sc.b
Genk Institute for Fertility Technology, Genk, Belgium, and Reproductive Medicine Center, University of
Minnesota, Minneapolis, Minnesota

Objective: To determine the influence of ejaculatory abstinence on within-subject semen parameters and
DNA fragmentation.
Design: Prospective study.
Setting: Private infertility institute and university-based research laboratory.
Patient(s): Sixteen consenting male volunteers undergoing infertility investigation.
Intervention(s): None.
Main Outcome Measure(s): Within-subject analysis of World Health Organization semen parameters and
sperm DNA fragmentation and chromatin packaging after 1, 3, 5, and 8 days’ abstinence.
Result(s): Of 16 men recruited, data for 11 men were included for statistical analysis because 5 men did not
strictly comply with abstinence criteria. Duration of abstinence had a statistically significant positive influence
on sperm concentration and semen volume. Abstinence had no statistically significant influence on pH,
viability, total and grade A motility, or morphology. The percentage of DNA fragmentation remained
unchanged relative to abstinence. The percentage of sperm with immature chromatin was statistically
significantly increased with 1 day of abstinence.
Conclusion(s): This is the first study to report on within-subject semen parameter, DNA fragmentation, and
chromatin packaging variations after specified target days of abstinence. Sperm numbers and semen volume
increased with duration of abstinence. Abstinence did not influence pH, viability, morphology, total or grade
A motility, or sperm DNA fragmentation. A short (24-hour) abstinence period negatively influenced chromatin
quality. (Fertil Steril威 2004;82:57– 65. ©2004 by American Society for Reproductive Medicine.)
Key Words: Abstinence, DNA fragmentation, chromatin, semen quality, IUI

The optimal ejaculatory abstinence time that abstinence period should be based on cop-
Received December 11,
2003; revised and
frame for assessing traditional semen parame- ulatory frequency, for instance, twice per week.
accepted March 2, 2004. ters, for example, volume, concentration, mo- In contrast, Freund (5) argued that sperm pro-
Presented at the Twin- tility, and morphology, as recommended by the duction is best measured after depletion of ep-
Meeting Alpha-Andrology World Health Organization (WHO) (1), is 2–7 ididymal reserve followed by a recuperation
2003, Antwerp, Belgium,
September 24 –27, 2003. days. However, the basis for this recommenda- period. Since these early studies, numerous
Reprint requests: tion is unclear because no supporting refer- other studies have attempted to clarify the im-
Christopher De Jonge, ences are provided. It can be concluded easily pact of abstinence interval on semen parame-
Ph.D., Reproductive
Medicine Center, 606 24th that establishing an evidence-based time frame ters.
Avenue South, Suite 500, for ejaculatory abstinence is beneficial because
Minneapolis, Minnesota Some reports investigating the influence of
(FAX: 612-627-4334; E-
it affords a better ability to compare interlabo-
male abstinence on semen parameters have
mail: [email protected]). ratory and intralaboratory semen analysis re-
a
evaluated extremes in abstinence duration,
Reproductive Medicine sults.
Center, University of such as ⱕ24 hours or ⱖ10 days. Oldereid et al.
Minnesota. There are a few benchmark articles that can (6) studied ejaculates from seven men who
b
Genk Institute for Fertility be said to have established current presump- produced samples every 8 hours and over a
Technology.
tions about ideal days of ejaculatory abstinence 2-day period. They found that sperm concen-
0015-0282/04/$30.00 for optimization of semen parameters (2–5). tration and total sperm number declined during
doi:10.1016/j.fertnstert.2004
03.014 For example, MacLeod and Gold (4) concluded the 2-day study period, but with no increase in

57
the proportion of immature forms. Matilsky et al. (7) eval- were found to positively correlate with abstinence duration.
uated semen characteristics from normozoospermic and oli- Acrosin remained stable initially but then decreased after 10
gozoospermic men who participated in an intrauterine in- days’ abstinence.
semination program. They found that men who produced A few studies have attempted to evaluate DNA packaging
samples after 4 days’ abstinence followed by 24 hours’ in relationship to ejaculatory abstinence. Oldereid et al. (6)
abstinence had, in general, significantly decreased semen found that DNA condensation was unaffected by frequent
volume, sperm concentration, total sperm count, and total ejaculation. Le Lannou et al. (13) determined that conden-
motile sperm count between the first and second specimens. sation of the DNA-protein complex was unaffected by 12
Other studies have relied on intervals or ranges in time hours or 7 days’ abstinence. Spano et al. (14), in an epide-
periods when investigating the influence of ejaculatory ab- miological study, found a significant positive correlation
stinence on semen parameters. Padova et al. (8) evaluated between days of abstinence and DNA fragmentation, the
semen samples from men who abstained for 2– 4 days, 5–7 latter measured by the sperm chromatin structure assay
days, and more than 7 days and found that only semen (SCSA). Those investigators attempted to analyze within-
volume increased with extended abstinence. Pellestor and subject variation, but because the required abstinence inter-
colleagues (9) analyzed ejaculates from six men abstaining vals were ranges of days as opposed to specified days, it was
for 2–18 days. They found that semen volume, concentra- more difficult to accurately detect true variation.
tion, and total sperm count positively correlated with in- The SCSA is a flow cytometric assay developed by Even-
creased abstinence. In contrast, motility and morphology son that has provided evidence that a relationship exists
decreased after 7 days of abstinence. between sperm chromatin structure and function (see, e.g.,
In general, the aforementioned studies evaluated semen Evenson and Jost [15]). The SCSA defines abnormal chro-
parameters in cohorts of men who abstained for generalized, matin structure as an increased susceptibility of sperm DNA
as compared with specifically designated, time periods. to acid-induced denaturation in situ. The SCSA parameters
More important, the above studies evaluated the between- of DNA fragmentation index (DFI) and high DNA stainabil-
subject variation in semen parameters as opposed to the ity (HDS) were found to be more consistent than were WHO
within-subject variation. Although studies that analyze be- semen analysis parameters that were evaluated in semen
tween-subject variation reveal important population data, samples from individual men over 8 months (16, 17). Also,
they do not, by virtue of their testing protocol, allow for the the SCSA has been reported to be only weakly correlated
detection of within-subject variation. with conventional semen parameters, and thus it provides
additional prognostic criteria for male infertility evaluations
Freund (5) concluded that “without repeated specimens, (14, 18, 19).
under controlled conditions, no estimate of the variation
among specimens within donors can be made. Without such Given the diversity in previously published reports of
an estimate of within-donor variation, no conclusive test of study designs used for assessing influences of ejaculatory
the significance of the differences among donors, among abstinence on semen parameters, we decided to evaluate
fertile and infertile groups, or among treated and control within-subject semen parameters, DNA integrity, and chro-
groups can be made.” This latter issue is clearly relevant. matin packaging variation in a group of men who were
collectively abstaining for 1, 3, 5, and 8 days.
A few studies have evaluated semen parameters in ejac-
ulates from men who collectively abstained for specifically
designated time periods. The benefit of doing so is that MATERIALS AND METHODS
within-subject variation in semen parameters in relationship Patient Demographics
to defined periods of abstinence can be analyzed. Sauer et al. Sixteen male volunteers undergoing infertility investiga-
(10) followed 10 fertile males over abstinence periods of 12 tion at the Genk Institute for Fertility Technology (Genk,
hours and 1, 3, and 5 days. Similar to the case with other Belgium) were recruited for participation in the study after
studies, they found that semen volume and concentration providing informed consent. This study had institutional
positively correlated with abstinence interval. In addition, review board approval. Average male age was 30 ⫾ 2.9
they found by computer-assisted sperm motion analysis that years (95% confidence interval, 28.7–31.9 years). None of
motility and sperm kinematics went unchanged in relation- the men reported fever or other illness during the 8 weeks
ship to abstinence. However, Magnus et al. (11) found that before and during the study; fever has been reported to
sperm progressive motility declined after 10 days’ absti- influence the percentage of fragmented DNA (20). Each
nence. subject was asked to deliver a fresh semen sample after
Blackwell and Zaneveld (12) analyzed ejaculates from abstinence periods of 1, 3, 5, and 8 days, covering a total of
men who abstained for 1, 2, 3, 4, 5, and 10 days. They 17 consecutive days. For 9 of 11 subjects, the 24-hour
evaluated traditional semen parameters and sperm acrosin. abstinence specimen collection followed either a 3- or 5-day
As for other studies, semen volume and sperm concentration abstinence period.

58 De Jonge et al. Ejaculatory abstinence and sperm quality Vol. 82, No. 1, July 2004
 Semen analyses were performed by a single experienced
laboratory technician and according to WHO (1) guidelines. TABLE 1
The following parameters were noted: semen volume, pH, Semen parameter descriptive statistics.
concentration, motility (total motility and grade A, B, C, and
D motility, as defined in the WHO guidelines [1] and mea- 95%
sured at 37°C). The number of round cells and white blood Confidence
Semen parameter Mean Range limits
cells were counted in the semen sample. Viability was de-
termined after eosin staining. Sperm morphology was scored Volume (mL) 3.3 0.8–9.7 2.7–3.8
by using strict criteria (21, 22). After completion of semen pH 7.5 7–8 7.5–7.6
analysis, aliquots from the samples were frozen in straws by Concentration (⫻ 106/mL) 55 1.4–245 37–73
Viability (%) 60 3–80 56–65
using the Sperm Freeze method (Fertipro, Beernem, Bel-
Motility (%) 54 0–76 48–59
gium). The sample straws were sent in a dry liquid nitrogen Grade A motility (%) 17 0–38 14–21
shipper to the Reproductive Medicine Center at the Univer- Morphology (%) 8 1–12 7–8
sity of Minnesota and were stored in liquid nitrogen until the DFI (%) 20.0 5.8–62.9 16.1–23.8
time of DNA/chromatin testing. HDS (%) 11.2 3.7–40.7 9.0–13.5
Note: Data are from 44 total observations of 11 men.
Sperm Chromatin Structure Assay De Jonge. Ejaculatory abstinence and sperm quality. Fertil Steril 2004.
Straws containing semen were removed from liquid ni-
trogen storage and placed in a 37°C water bath until the
entire specimen had just thawed. The sample was vortexed fluorescence emitted from individual sperm cells flowing at
briefly and immediately placed in crushed ice in preparation approximately 200 cells per second.
for SCSA testing. The procedure for SCSA testing for chro-
matin integrity exactly followed that of Evenson and Jost The SCSAsoft software calculates red/(red⫹green) fluo-
(23). rescence distribution (originally defined by Evenson et al.
[18] as ␣t) and associated parameters for each sample.
All chemicals were obtained from Fisher Scientific (Pitts- Sperm populations with normal chromatin structure have a
burgh, PA), except for acridine orange (AO), which was small mean ␣t (X␣t), standard deviation ␣t (SD␣t), and
obtained from Polysciences, Inc. (Warrington, PA). Two percentage of cells outside the main population, that is,
hundred microliters of sperm in Tris NaEDTA (TNE) buffer percentage of cells with denatured DNA (DFI, formerly
(1–2 ⫻ 106 sperm/mL) were treated for 30 seconds with 400 termed COMP␣t). The SCSA™ also identifies a sperm frac-
␮L of a pH 1.2 solution containing 0.1% Triton X-100, 0.15 tion with High DNA Stainability (HDS). HDS sperm are
M NaCl, and 0.08 N HCl. Triton X-100 permeabilizes sperm considered to be immature with unprocessed nuclear pro-
cell membranes, providing greater accessibility of AO to teins and/or poorly condensed chromatin.
DNA. The low-pH solution partially denatures DNA in
sperm with abnormal chromatin structure. Sperm with nor- Statistical Analysis
mal chromatin structure do not demonstrate DNA denatur- Semen parameter, percentage DFI, and percentage HDS
ation. After the 30-second acid treatment, 1.20 mL of AO data were tested for normality of distribution and equality of
staining buffer (6 ␮g of AO/mL, 37 mM citric acid, 126 mM variance. Log10 transform was applied when indicated. Data
Na2HPO4, 1 mM disodium ethylenediaminetetraacetic acid were analyzed by two-way randomized block ANOVA with
(EDTA), and 0.15 M NaCl, pH 6.0) was added to the cells Tukey multiple-comparison post hoc testing by StatsDirect
before analysis by flow cytometry. Acridine orange that statistical software, version 2.2.9 (StatsDirect Ltd, Cheshire,
intercalates into double-stranded DNA (native; normal) flu- UK).
oresces green (515–530 nm), whereas AO that associates
with single-stranded (denatured) DNA fluoresces red (ⱖ630
nm) when excited by a 488-nm light source (24). RESULTS
The extent of sperm DNA denaturation was quantified Of 16 men who participated in the study, 11 strictly
with a FACScan flow cytometer (Becton Dickinson, West- complied with abstinence requirements. The mean, range,
wood, MA) with a closed quartz flow cell and a 100-mW and 95% confidence limits of semen parameter data for the
argon ion laser operated at 35 mW of power that was population of men (n ⫽ 11) are provided in Table 1.
interfaced through a Becton Dickinson Power Acquisition As noted earlier, data for five men were excluded because
Control (BDPAC) to a Macintosh G4 computer running of noncompliance with study guidelines. However, when
CellQuest, version 3.2 (Becton Dickinson). Data for each data for those men were included with those of compliant
analyzed specimen was saved as an .fcs file, transferred to a participants (n ⫽ 11), the results were the same (n ⫽ 16).
personal computer, and analyzed by SCSAsoft software, Figure 1A–I contains graphs composed of semen parameter
version 1.07 (SCSA Diagnostics, Inc., Brookings, SD). The data for each man (n ⫽ 11) at the specified abstinence
CellQuest software measured the amount of red and green intervals.

FERTILITY & STERILITY威 59

 FIGURE 1

Sperm and semen parameters. In all panels, subject number corresponds to each study participant; mean is the calculated
mean value for all subjects at each abstinence time period; and colors indicate the following. Dark blue, day 1 (corresponds to
24 hours’ abstinence); red, day 3 (corresponds to 3 days’ abstinence); yellow, day 5 (corresponds to 5 days’ abstinence); light
blue, day 8 (corresponds to 8 days’ abstinence). (A) Volume (mL). (B) Sperm concentration (⫻ 106/mL). (C) pH. (D) Viability
(percentage). (E) Motility (percentage). (F) Grade A motility (percentage). (G) Morphology (percentage). (H) DFI (percentage). (I)
HDS (percentage).

De Jonge. Ejaculatory abstinence and sperm quality. Fertil Steril 2004.

Abstinence had a highly significant (P⬍.0001) influence Abstinence had a statistically significant (P⫽.02) positive
on semen volume (Fig. 1A). Semen volume was statistically influence on sperm concentration (Fig. 1B). Sperm concen-
significantly lower after 1 day’s abstinence in comparison tration was statistically significantly higher after abstinence
with specimen volume after abstinence intervals of 5 of 5 (P⫽.02) and 8 (P⫽.03) days in comparison with sperm
(P⬍.0001) and 8 (P⬍.0001) days. In addition, semen vol- concentration after 1 day’s abstinence. No statistically sig-
ume was statistically significantly greater after 5 days nificant (P⬎.05) influence of abstinence on sperm concen-
(P⫽.02) and 8 days (P⫽.04) of abstinence, in comparison tration was detected for any of the other comparisons. It has
with specimen volume on day 3. been reported that abstinence may differentially influence

60 De Jonge et al. Ejaculatory abstinence and sperm quality Vol. 82, No. 1, July 2004
 FIGURE 1 Continued.

normozoospermic and oligozoospermic men (7). Data anal- stainable sperm are considered to be immature, with unproc-
ysis revealed that there was no statistically significant essed nuclear proteins and/or poorly condensed chromatin.
(P⬎.05) influence of ejaculatory abstinence on sperm con- Abstinence had a significant (P⫽.03) influence on the per-
centration for either normozoospermic or oligozoospermic centage of HDS. A statistically significantly (P⫽.02) greater
men (data not shown). percentage of HDS was detected when the abstinence period
Duration of abstinence had no statistically significant was 1 day, in comparison with 5 days’ abstinence.
influence (P⬎.05) on pH, viability, sperm motility, or grade
A motility (Fig. 1C to F, respectively). In addition, absti- DISCUSSION
nence interval had no statistically significant (P⬎.05) effect A number of previous studies have investigated the in-
on sperm morphology as measured using strict criteria (Fig. fluences of ejaculatory abstinence on semen parameters.
1G). Last, the length of abstinence had no statistically sig- However, for many of those studies, conclusions were typ-
nificant (P⬎.05) influence on the percentage of sperm with ically based on single ejaculate data for individuals within a
fragmented DNA (DFI, Fig. 1H), as measured by using the population of men, that is, between-subject variations. Pop-
SCSA. ulation studies provide meaningful information; however,
In addition to DFI, the SCSA technique detects sperm that they fail to reveal variation in semen parameters within an
have high DNA stainability (HDS, Fig. 1I). Highly DNA- individual over specified time frames.

FERTILITY & STERILITY威 61

 FIGURE 1 Continued.

The present study represents one of only a few published after 3 days’ abstinence in comparison with the case with 1
reports that have evaluated the influence of defined periods days’ abstinence. These findings raise a question concerning
of ejaculatory abstinence on within-subject semen parame- daily sperm production. We have found that 5 and 8 days’
ters. Eleven men provided semen specimens for analysis abstinence produced the greatest semen volumes and sperm
according to WHO protocols (1) after strictly complying concentrations in comparison with 1 day’s abstinence.
with designated intervals of abstinence. Semen volume was
significantly increased after 5 and 8 days’ abstinence, rela- Total motile sperm count (data not shown) was found to
tive to the case with only 1 days’ abstinence. Further, semen be significantly higher after 5 and 8 days’ abstinence in
volume was significantly increased after 5 and 8 days’ ab- comparison with 1 days’ abstinence. Further, total motile
stinence, in comparison with ejaculate volume after 3 days’ count was marginally and significantly higher after 5 and 8
abstinence. The findings regarding semen volume are com- days’ abstinence, respectively, in comparison with the case
patible with data reported in similarly controlled studies (10, with 3 days’ abstinence. Collectively, our data support the
12) and also with population-based studies (7, 8, 13, 25–27). WHO 1999 (1) recommended abstinence periods of 2–7
Sperm concentration was significantly increased after days and specifically, the midpoint (day 5) of that range,
both 5 and 8 days’ abstinence in comparison with 1 day’s because it represents a time point at which within-individual
abstinence. However, sperm concentration was not different semen parameter variations are at a minimum.

62 De Jonge et al. Ejaculatory abstinence and sperm quality Vol. 82, No. 1, July 2004
 FIGURE 1 Continued.

FERTILITY & STERILITY威 63

 The composition of semen is complex and varied, not densed chromatin requires independent validation, it does
only between different men but, as the present study sug- cause attention to be drawn and caution to be given when
gests, from one ejaculate to another in the same man. After considering the use of sperm for therapeutic purposes after a
ejaculation, spermatozoa are exposed to the unique environ- short abstinence interval.
ment of the seminal plasma. Prostatic fluid contains both free
This study is the first to report on the within-subject
zinc and zinc bound to citrate. When sperm become exposed
influences of ejaculatory abstinence on DNA quality, defined
to prostatic fluid rich in zinc, they are able to retain their
as the percentage of DNA fragmentation and percentage of
endogenous chromatin zinc, and hence chromatin stability is
sperm with immature chromatin. By using a well-character-
less likely to be altered.
ized and well-controlled method for DNA fragmentation,
Seminal vesicle fluid contains proteins with high affinity that is, SCSA, it was determined that over the intervals used
for zinc. Bjorndahl and Kvist (28) found that spermatozoa for this study, abstinence does not influence the percentage
incubated in seminal vesicular fluid lose chromatin zinc and of sperm DNA fragmentation. Our within-subject abstinence
that spermatozoa expelled in vesicular fluid at ejaculation results are in contrast with those of Richthoff et al. (33), who
have lower zinc content in their chromatin. Our data support found that increased abstinence had a negative influence on
that after 1 day of abstinence, there is an alteration in semen DNA fragmentation in a between-subject study.
composition in the form of significantly lower volume, as
The finding in this study that DNA fragmentation was
well as an increase in immature chromatin in the spermato-
unaffected by abstinence applies only to the maximal absti-
zoa from these ejaculates.
nence period of 8 days. Longer periods of abstinence might
The nature of the semen volume decrease must arise from have an impact on the degree of sperm DNA fragmentation.
a decrease in prostatic and/or vesicular secretions. It is If subsequent studies confirm our within-subject abstinence
questionable whether an alteration in the prostatic to seminal data, then this finding should prove to be reassuring from the
vesicle volume ratio and/or a decrease in zinc leading to context of abstinence requirements for maximizing diagnos-
alteration in protamine content can occur in the short absti- tic semen analysis and for use of spermatozoa in assisted
nence time frame of 1 day and lead to chromatin instability. reproduction.
This is the subject of current investigation.
In conclusion, this simple study has revealed important
It has been shown that AO binding to DNA decreases as data regarding within-subject semen parameter variation rel-
human sperm transit through the epididymis from caput to ative to length of ejaculatory abstinence. The WHO-recom-
cauda, reflecting a sequential and progressive completion of mended (1) abstinence interval before diagnostic semen
nuclear maturation (29, 30). The increased percentage of analysis is supported, and with a focus on the midpoint of
spermatozoa with HDS detected after 24 hours’ abstinence that interval as being representative of the time that will
in the present study may relate to differences in epididymal provide optimization of individual semen parameters.
maturation. Specifically, the sperm population collected after
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