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Systematic Review
238
Revisiting The Relationship between The Ejaculatory
Abstinence Period and Semen Characteristics
Bashir M Ayad, M.Sc., Gerhard Van der Horst, Ph.D., Stefan S Du Plessis, Ph.D.*
Division of Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa
Abstract
Variation in the ejaculatory abstinence period suggested by different guidance bodies have resulted in a growing concern
among researchers and clinicians over what the precise period of ejaculatory abstinence ought to be for an optimal semen
sample. Several studies have thus been undertaken to examine the association between the length of sexual abstinence
and semen characteristics. Not all studies, however, have arrived at the same conclusions. This study aims to review all
existing literature published during the past few decades pertaining to the inuence of ejaculatory abstinence on semen
quality. For the purpose of this systematic review, all data related to sexual abstinence duration and seminal parameters
were re-analysed to homogenize the current data. Thorough PubMed, MEDLINE and Google Scholar, a literature search
was conducted using the keywords “sexual abstinence”, “ejaculatory abstinence”, “semen”, “spermatozoa”, “semen
analysis”, “sperm parameters”, “motility”, “reactive oxygen species (ROS)” and “DNA fragmentation”. After carefully
reviewing all the literature, 30 relevant papers, both written in English and published between January 1979 and Decem-
ber 2016, were included in this review. The weight of the evidence suggests that the decline in semen volume and sperm
concentration with shorter abstinence periods is accompanied by a substantial improvement in sperm motility charac-
teristics, especially progressive motility and velocity. Nevertheless, available data are insufcient to support denitive
conclusions regarding the inuence of the ejaculatory abstinence period on advanced semen parameters (ROS, DNA
fragmentation and seminal plasma antioxidant capacity) and pregnancy rates. In conclusion, taking all data into account,
shortening of the abstinence period may be benecial to sperm quality. Furthermore, we recommend that the current
guidelines regarding the prescribed abstinence period should be revisited.
Keywords: DNA Fragmentation, Semen Analysis, Sexual Abstinence, Spermatozoa, Sperm Motility
Citation: Ayad BM, Van der Horst G, Du Plessis SS. Revisiting the relationship between the ejaculatory abstinence period and semen characteristics. Int J Fertil
Steril. 2018; 11(4): 238-246. doi: 10.22074/ijfs.2018.5192.
Received: 30 Jan 2017, Accepted: 17 Mar 2017
*Corresponding Address: Division of Medical Physiology, Faculty of Med-
icine and Health Sciences, Stellenbosch University, Francie van Zijl Drive,
Tygerberg, 7505, South Africa
Email: ssdp@sun.ac.za Royan Institute
International Journal of Fertility and Sterility
Vol 11, No 4, Jan-Mar 2018, Pages: 238-246
Introduction
Infertility is the “failure to achieve a clinical pregnancy
after 12 months or more of regular unprotected sexual
intercourse” and is a condition estimated to affect about 15%
of all couples of reproductive age. Male factor infertility has
been found to be the sole contributor in approximately 20%
of all infertility cases and is partially implicated in another
30-40% (1). When the attributable causes of female infertility
have been eliminated and/or semen analysis results fail to
meet the World Health Organization (WHO) criteria, male
infertility is taken into consideration as the likely etiological
factor. Therefore, semen analysis still remains the established
cornerstone of the laboratory assessment of male infertility.
A considerable amount of variability has been shown to
exist in various semen characteristics within and among
individuals (2). These variations have been largely attributed
to several modiable intrinsic and extrinsic factors. These
factors include the length of sexual abstinence, ejaculation
frequency and method of collection. Other factors that have
the potential to inuence semen quality are general health
and lifestyle, infection, dysfunction of male sex glands,
urogenital surgery as well as therapeutic and environmental
exposures (3).
The WHO manuals for examining and processing human
semen provide a practical guide for standardizing semen
analysis. These manuals have been periodically published
and actively developed since its rst edition in 1980. The
WHO criteria for semen analysis have been adopted by most
human andrology and fertility laboratories around the world
for more than thirty years. The most recent guidelines of
WHO recommend that the minimum period of ejaculatory
abstinence prior to semen collection should not be less than
2 days and more than 7 days (4). The Nordic Association
for Andrology (NAFA) and the European Society of Human
Reproduction and Embryology (ESHRE) (5), however,
outline a narrower range of 3-4 days of abstinence. The basis
for these recommendations is nevertheless not supported
by sufcient scientic evidence and requires further
clarication.
In light of the differing ejaculatory abstinence periods
suggested by various regulatory bodies, a growing concern
has resulted over what the precise period of ejaculatory
Int J Fertil Steril, Vol 11, No 4, Jan-Mar 2018 239
Ayad et al.
abstinence ought to be for an optimal semen sample. This
has prompted several studies to examine the inuence of
abstinence periods on various semen parameters. The results
are, however, not conclusive. Interestingly, some studies
have even challenged the recommended guidelines in favour
of extremely shorter periods (i.e. <1 hour to 4 hours) due
to their advantageous effects on semen characteristics (6-9).
Studies on the association of abstinence length with semen
quality have examined a wide range of abstinence intervals.
Although numerous related articles have been published to
this date, a systematic review has not been undertaken. This
study therefore aims to review the existing scientic literature
over the past few decades pertaining to this association in
humans to evaluate the weight of evidence for the optimal
time period of ejaculatory abstinence.
Materials and Methods
For the purpose of this systematic review, all data related
to sexual abstinence duration and seminal parameters were
re-analysed to homogenize the current data. An extensive
review of the existing literature was performed in various
electronic databases, namely MEDLINE, PubMed and
Google Scholar by using the keywords “sexual abstinence”,
“ejaculatory abstinence”, “semen”, “spermatozoa”, “semen
analysis”, “sperm parameters”, “motility”, “reactive oxygen
species (ROS)” and “DNA fragmentation”. A total of 34
relevant articles were obtained, all of which were written in
English and published between January 1979 and December
2016. Four of these were excluded due to a lack of numerical
data. After careful review of the abstracts, these 30 studies
were included in the current review, of which 25 were
prospective and ve were retrospective. The majority of the
included studies had used donors recruited from the general
population, while twelve studies selected patients from
infertility clinics or assisted reproduction units. The seminal
parameters examined were semen pH, semen volume, sperm
concentration, sperm motility, sperm morphology, sperm
intracellular ROS and DNA fragmentation, and seminal
plasma antioxidant capacity. Here, ejaculatory abstinence
was classied into the time periods of ≤1 day, 2-3 days, 4-5
days, 6-7 days and ˃7 days.
Ejaculatory abstinence and conventional semen
parameters
The majority of the studies investigating the inuence
of ejaculatory abstinence on semen quality (Table 1) had
assessed the most conventional semen parameters (e.g.
volume, count and concentration, motility and morphology)
as described by WHO (4) with the latest version reporting a
reference range based on men with proven fertility.
Seminal pH
A slightly alkaline seminal uid is necessary to neutralize
the acidic environment of the vagina, which can negatively
impact sperm function (10). A substantial reduction in sperm
motility was reported in patients with semen pH less than the
WHO lower-bound threshold value of 7.2 (11), however, the
correlation between semen pH and sperm motility was not
statistically signicant (12). Only three studies considered
seminal pH as a parameter when investigating the relationship
between the abstinence period and semen quality (13-15).
Blackwell and Zaneveld (13) analysed semen samples from
ten men with abstinence periods of 1, 2, 3, 4, 5 and 10 days,
and found that seminal pH remained essentially unchanged.
In addition, De Jonge et al. (14) examined ejaculates from
11 men who had abstained for 1, 3, 5 and 8 days, and
reported no signicant changes in seminal pH across the
four abstinence periods. Similar results were also reported by
Agarwal et al. (15) who collected semen samples from seven
men each abstaining sequentially for 1, 2, 5, 7, 9 and 11
days, and observed that semen pH remained relatively stable
but declined signicantly after 11 days of abstinence. The
scarcity of studies examining seminal pH indicates that the
signicance of this semen marker has been underestimated.
Semen volume
According to the latest WHO guidelines, the lower-bound
reference value for semen volume is 1.5 ml. Accurate
measurement of the ejaculate volume is important as the
concentration of spermatozoa and non-sperm cells in the
ejaculate are based on the initial volume. Semen volume-
after the recommended standard period of abstinence-has
consequently been suggested to be an early indicator of low
semen quality even before identifying any abnormality in
concentration, motility and morphology of spermatozoa.
Semen volume has also been suggested to be a reliable
indicator of the secretory functions of the accessory glands,
particularly the seminal vesicles (4).
The relationship between the abstinence period and
semen volume was reported in twenty-four studies (Table
1). In all but one, there is robust and consistent evidence
for the signicant increase in semen volume with increase
in abstinence period (6, 8, 9, 13-32). In a retrospective
longitudinal study (22), the greatest overall mean of daily
increase in semen volume was observed at 11.9% per day
during the rst 4 days of abstinence. However, only one
study (33) failed to show any signicant change in semen
volume in both normozoospermic and asthenozoospermic
populations which is likely to be due to the small sample
size studied and the protracted period of the short abstinence.
Short abstinence-associated decreases in the ejaculate
volume may be attributed to insufciency of the accessory
sex glands to make an adequate contribution to the ejaculate
volume, particularly the seminal vesicles and the prostate
gland, which are the major contributors. The epithelial
tissues of these organs are targeted by androgen, which is
thought to regulate their mRNA production as well as the
synthesis of rough endoplasmic reticulum, thereby enhancing
the production of seminal plasma proteins (34). Improved
secretory capacity of the seminal vesicles and the prostate
gland has been associated with higher endogenous serum
testosterone levels in rats (35) and men (36). In addition,
higher testosterone serum levels have been reported following
a prolonged abstinence period compared with a shorter
Int J Fertil Steril, Vol 11, No 4, Jan-Mar 2018
240
Short Abstinence Improves Semen Quality
abstinence (37). Therefore, the potential stimulating effect
of testosterone on the major accessory glands associated
with long abstinence periods may contribute to the increased
semen volume after prolonged abstinence periods.
Sperm concentration and total count
Concentration of spermatozoa in semen, expressed
as millions per millilitre, is a critical indicator of semen
quality and a prognostic factor for fertility potential (38).
However, it is not recommended as an accurate measure
of spermatogenesis because it is inuenced by the volume
of secretions of the accessory sex glands in which the
concentrated epididymal spermatozoa are diluted in during
ejaculation (4). The total number of spermatozoa in the
ejaculate, expressed as millions per total ejaculate and
obtained by multiplying the sperm concentration by the
semen volume, is suggested to be a better parameter for the
evaluation of spermatogenic statuses (39). The lower-bound
threshold values of sperm concentration and total count
recommended by the WHO are 15×106 spermatozoa/mL and
39×106 spermatozoa/ejaculate respectively (4).
The inuence of the abstinence period on sperm
concentration was assessed in twenty-two of the studies
listed in Table 1. Of these, twenty (91%) reported a linear
increase in sperm concentration with increased abstinence
periods (8, 9, 13-16, 19, 21-26, 28-30, 32, 33, 40, 41). The
highest rise in the overall mean of sperm concentration
(14×106/mL) occurred when the abstinence period increased
from 2-3 days to 4-5 days (Table 2). Two studies found a
non-signicant mild increase in sperm concentration after
long abstinence compared with short abstinence periods
(18, 20). Eighteen studies (6, 9, 13, 15, 16, 18, 19, 21-24,
29-33, 41, 42) reported a signicant association between
long abstinence periods and increased total sperm count in
the ejaculate. The largest increase in the overall mean of
total sperm count was recorded when the abstinence period
extended from 6-7 days to ˃7 days.
During sexual inactivity an estimated 400 million
spermatozoa are reserved within the epididymis with the
majority stored in the cauda epididymis and lesser in the
caput and corpora with an average of 90 million in each
of these sections. The paired vas deferens with its ampulla
is estimated to contain about 75 million spermatozoa (39).
During the arousal phase, but prior to the emission phase, the
population of spermatozoa in the paired ampulla increases
dramatically as they move distally towards the urethra (43).
After particularly long periods of abstinence, the bulk of
the sperm population in the rst ejaculate mainly comprise
spermatozoa stored in the ampulla and vas deference, and
partly in the cauda epididymis. Consequent ejaculates in
quick successions are typically characterized by a lower
total count of spermatozoa as the residual spermatozoa are
ushed from the proximal cauda and corpus, and thereafter
from the caput (6), all of which contain much lower sperm
reserves (39). Despite these ndings, Bahadur et al. (8)
interestingly suggested that “combining the initial and
consecutive ejaculates allows for a potential shift of severe
and oligozoospermia patients towards the normospermia
range”. This approach may lead to a change in the treatment
strategies by possibly avoiding testicular biopsies.
The observed consistent positive correlation of sperm
concentration and total count with increasing abstinence
durations can be ascribed to daily sperm production, which
is determined to be approximately 130-270×106 per day (39).
The regulation of testicular functions and spermatogenesis
necessitates a complex combination of endocrine and paracrine
signals. Relatively higher levels of testosterone are essential
for the maintenance and proceeding of spermatogenesis.
Serum testosterone levels were shown to uctuate mainly
from the second to the fth day of abstinence, reaching a
peak (about 145% of the baseline) after the seventh day of
abstinence and remaining relatively constant even when the
abstinence period was prolonged (37).
Sperm motility and kinematics
Assessment of motility characteristics of ejaculated
spermatozoa has been shown to have the utmost importance
for the diagnosis of male fertility potential since it provides
vital information on the functional competence of the
spermatozoon. The percentage of motile spermatozoa in
the ejaculate provides an indication of epididymal sperm
maturation (44). However, progressive motility is required
for the spermatozoa to migrate through the harsh environment
of the female genital tract to reach the ovum. Motility is not
only necessary for sperm transit, but changes in agellar
motion also play an essential role at the site of fertilization.
The mechanical driving force generated by motility help the
sperm to propel through the outer layers of the cumulus-
oocyte complex (45). The lower-bound WHO threshold
values for the percentages of total motility and progressive
motility are 40 and 32% respectively (4).
Twelve studies examined the relationship between the
abstinence period and the total motile sperm (TMS) count
in the ejaculate. Eight of these (15, 23, 24, 26, 29, 40-42)
reported an increase in TMS count with increase in the
abstinence period, while the other four did not nd any
signicant effect of abstinence period on TMS (9, 22, 28,
33). The overall mean of TMS increased substantially as
the abstinence period increased from ≤ 1 to 3 days (Table
2). The mean TMS remained relatively stable between the
fourth and the seventh day, increased on the subsequent
days (>7) and declined gradually after day 9 to 10 of
abstinence (17, 19). The inuence of abstinence length on
the percentage of motile spermatozoa was investigated in
seventeen studies (6, 9, 14, 15, 17, 19, 21, 23, 24, 26-30,
32, 41, 42) (Table 1). We found little consensus among
the results of these studies. A slight or lack of association
between abstinence period and motile sperm percentage
was reported in eleven studies (9, 14, 15, 17, 21, 27, 28,
30, 32, 41, 42). In contrast, six studies (6, 19, 23, 24,
26, 29) reported a substantial decrease in the percentage
of motile spermatozoa with increasing abstinence; the
highest overall mean sperm motility percentage was
observed after ≤ 1 day of abstinence (Table 2).
Int J Fertil Steril, Vol 11, No 4, Jan-Mar 2018 241
Table 1: Absnence periods and semen characteriscs
Type of study Abstinence
periods
Subjects
Number of subjects/samples
Volume (mL)
Concentration (106/mL)
TSC (106/ejaculate)
TMS (106/ejaculate)
Motility (%)
Progressive motility (%)
Viability (%)
Normal morphology (%)
DNA fragmentation (%)
ROS
Prospective 4 hours and 3-5 days Volunteers 11 ↑ −− ↑ −− ↓ ↓ −− −− −− −−
Prospective 3 hours and 96 hours Normozoospermic 21 −− −− −− −− −− −− −− −− ↔ −−
Prospective 40 minutes and 2-7 days Oligozoospermic 73 ↑ ↑ −− −− −− ↓ −− ↓ −− −−
Prospective 2 hours and 3-4days Healthy 3 ↑ ↑ ↑ ↔ ↔ ↔ ↔ −− ↓ ↔
Prospective 1, 2, 3, 4, 5 and 10 days Volunteers 10 ↑ ↑ ↑ −− −− −− ↔ ↑ −− −−
Prospective 1, 3, 5, and 8 days Volunteers 11 ↑ ↑ −− −− ↔ −− ↔ ↔ ↔ −−
Prospective 1, 2, 5, 7, 9 and 11 days Normozoospermic 7 ↑ ↑ ↑ ↑ ↔ −− ↓ −− ↑ ↔
Prospective 1, 2, 3, 4, 5, 6 and 7 days Normal 36 ↑ ↑ ↑ −− −− −− −− −− −− −−
Retrospective ≤1, 2, 3, 4, 5, 6 and 7 days Suspected infertile 1801 ↑ −− −− −− ↔ −− ↔ ↔ −− −−
Prospective 8 hours and 3 days Volunteers 7 ↑ ↔ ↑ −− −− −− −− ↔ −− −−
Prospective 12 hours and 7 days Volunteers 10 ↑ ↑ ↑ −− ↓ −− −− ↔ −− −−
Prospective 2-4, 5-7 and ˃7 days Healthy men 195 ↑ ↔ −− −− −− −− −− −− −− −−
Prospective 2, 4, 7, 10, 15 and 18 days Volunteers 6 ↑ ↑ ↑ −− ↔ −− −− ↔ −− −−
Prospective ˂4, 4-6 and ˃6 days Healthy 27 ↑ ↑ ↑ ↔ −− −− −− ↔ −− −−
Prospective 2-3 and 4-7 days Non-azoospermic 422 ↑ ↑ ↑ ↑ ↓ ↓ −− ↓ −− −−
Retrospective 1, 2, 3, 4, 5, 6, 7, 8-10
and 11-14 days
Oligozoospermic 3506
samples
↑ ↑ ↑ ↑ ↓ −− −− ↓ −− −−
Retrospective 1, 2, 3, 4, 5, 6, 7, 8-10
and 11-14 days
Normozoospermic 5983
samples
↑ ↑ ↑ ↑ ↓ −− −− ↔ −− −−
Prospective 2, 3, 4 and 5 days Fertile 500 ↑ ↑ −− −− −− ↓ −− ↔ −− −−
Retrospective ≤2 and 3-7 days Undergoing IUI 372 ↑ ↑ −− ↑ ↓ −− ↓ −− −− −−
Prospective 1 and 4 days Undergoing ICSI 40 ↑ −− −− −− ↔ −− −− −− ↑ −−
Prospective 18-30 hours and 3-5 days Healthy 57 ↑ ↑ −− ↔ ↔ −− −− ↑ ↑ −−
Prospective 1, 2, 3, 4, 5, 6, 7, 8, 9
and 10 days
Normozoospermic 100
samples
↑ ↑ ↑ ↑ ↓ −− ↔ ↔ −− −−
Prospective 1 and 4 days Planning IUI 40 ↑ ↑ ↑ −− ↔ −− −− ↔ −− −−
Retrospective 2-3, 4-5 and 6-7 days Attending Infertility
Unit
730 ↑ −− ↑ −− −− ↓ ↓ ↔ −− −−
Prospective 1 and 3-4 days Healthy 6 ↑ ↑ ↑ −− ↔ ↔ ↔ ↔ ↔ ↔
Prospective 3, 6 and 10 days Normozoospermic 7 ↔ ↑ ↑ ↔ −− ↓ ↔ ↔ −− −−
Prospective 3, 6 and 10 days Asthenozoospermic 7 ↔ ↑ ↑ ↔ −− ↔ ↔ ↔ −− −−
Retrospective ≤3, 4-10 and ˃10 days Undergoing IUI 929 −− ↑ −− ↑ −− ↔ −− −− −− −−
Prospective 4 and 14 days Nonobstructive
azoospermic
50 −− ↑ ↑ ↑ ↔ −− −− −− −− −−
Prospective 1, 2, 4, 7, 10 and 14 days Healthy 4 −− −− ↑ ↑ ↔ ↔ ↔ ↔ −− −−
Prospective 1 and 3-4 days With high DNA
fragmentation
levels
35 −− −− −− −− −− −− −− −− ↑ −−
Prospective 1-10 days Healthy 36
samples
−− −− −− −− −− −− −− −− −− ↔
TSC; Total sperm count/ejaculate, TMS; Total motile sperm/ejaculate, ROS; Reactive oxygen species, ICSI; Intracytoplasmic sperm injection, IUI; Intrauterine insemination, ↑; Increase
signicantly with increasing abstinence period (P≤0.05), ↓; Decrease signicantly with increasing abstinence period (P≤0.05), ↔; Not signicantly different, and --; Not investigated.
Ayad et al.
Int J Fertil Steril, Vol 11, No 4, Jan-Mar 2018
242
Ten studies (6, 8, 9, 23, 25, 31-33, 40, 42) investigated
the relationship between ejaculatory abstinence and
progressive motility (Table 1). Five studies (6, 8, 23,
25, 31) reported a signicantly higher percentage of
progressively motile spermatozoa with shorter abstinence
periods, with the overall mean peak of progressive
motility observed after ≤1 day of abstinence (Table
2). Interestingly, shortening the abstinence interval to
about 30 minutes resulted in a signicant increase in the
percentage of fast progressive (type A) spermatozoa (8).
The results of Magnus et al. (33) were consistent with
those of the abovementioned studies where the progressive
motility of a normozoospermic population was found to
increase with decreasing abstinence time. However, they
analysed an asthenozoospermic population and found no
such association, corroborating the ndings of the other
relevant studies (9, 32, 40, 42).
Motility assessment in the majority of the studies was
performed manually using a light microscope and only
ve studies (23, 27-29, 42) used computer-aided sperm
analysis (CASA). Manual assessment of sperm motility
is subjective and is strongly associated with inter- and
intra-laboratory variation (46). The potential counting
and interpretation errors associated with the subjective
visual assessment of sperm motility have made automated
semen analyses an absolute necessity. CASA, in contrast
to subjective motility estimation, is certainly a powerful
approach for the objective assessment of sperm motion.
The most recent WHO guidelines on semen analysis
nevertheless indicate that the assessment of sperm
motility percentage using CASA may be unreliable due
to the potential misidentication of particulate debris
as immotile spermatozoa (4). This issue has recently
been addressed as modern CASA systems such as the
sperm class analyser (SCA6) are now equipped with
intelligent lters to accurately identify the spermatozoa
and eliminate the debris and other cells. The automatic
analysis of sperm motility by CASA instruments enables
the objective estimation of various parameters which
translate into certain kinematic measures of sperm
movement (47). The only study investigating the impact
of ejaculatory abstinence on sperm kinematics, among
other determinants of semen quality, had been conducted
by Elzanaty et al. (23). In this study, semen samples
collected from patients with a wide age range, undergoing
infertility assessment, were grouped into three categories
based on the abstinence period (i.e. 2-3 days, 4-5 days
and 6-7 days). Signicantly higher straight-line velocity
(VSL) and linearity (LIN) were found in the group
with the shortest abstinence period, while average path
velocity (VAP) and curvilinear velocity (VCL) were not
signicantly different among the three abstinence groups.
Variation in semen characteristics among individuals
may enhance the potential for observation bias (48) since
other factors besides ejaculatory abstinence may account
for the effects observed. However, collecting replicate
semen samples from the same individual is likely to be an
effective approach to controlling confounding factors. The
increase in semen volume and sperm concentration with
prolonged abstinence periods was almost consistently
accompanied by substantial deterioration in sperm
motility characteristics, especially progressive motility
and velocity. Although the exact mechanism as to how
ejaculatory abstinence may affect changes in semen
quality is unknown, a number of possibilities have been
suggested. For instance, reduction in the storage period
within the epididymis may minimize the exposure of
unejaculated spermatozoa to motility inhibitory factors
and enzymes released from the degenerating cells within
the same microenvironment (6). Furthermore, the sperm
Table 2: The overall mean values of basic semen parameters in relaon to dierent absnence periods calculated from values reported in relevant studies
referred to in Table 1
Semen parameter Day
≤1 2-3 4 -5 6-7 ˃7
Semen volume (mL) 2.198
n=15
2.72
n=13
3.251
n=18
3.773
n=13
4.229
n=14
Concentration (106/mL) 54.363
n=16
52.038
n=11
66.849
n=16
64.623
n=11
70.474
n=13
Total sperm count (106/ejaculate) 99.911
n=9
114.306
n=9
172.591
n=10
225.792
n=10
288.642
n=12
Total motile sperm (106/ejaculate) 36.56
n=8
49.618
n= 8
81.114
n=9
78.517
n=8
94.612
n=9
Motility (%) 56.03
n=15
44.813
n=11
52.044
n=15
41.277
n=12
43.325
n=13
Progressive motility (%) 57.083
n=6
54.533
n=3
53.887
n=6
49.15
n=2
-
-
Viability (%) 66.29
n=4
72.37
n=5
73.622
n=5
68.4
n=5
66.41
n=6
Normal morphology (%) 8.453
n=1
9.644
n=14
10.16
n=15
8.45
n=14
8.590
n=13
The average reported in each study contributed equally to the overall mean. Only studies reporng absolute values were included. All studies were
included in the calculaons (e.g. normozoospermic, oligozoospermic, volunteers, paents, etc.).
Short Abstinence Improves Semen Quality
Int J Fertil Steril, Vol 11, No 4, Jan-Mar 2018 243
reservoir capacity of the cauda epididymis is limited
(49), thus the substantial increase in sperm concentration
during prolonged ejaculatory abstinence may result in
the depletion of energy reserves and allow for senescent
spermatozoa to accumulate in the epididymis. The
relative contribution of these senescent spermatozoa
to the subsequent ejaculate impairs semen quality (27,
50). Extending the abstinence time may also enhance
susceptibility of unejaculated spermatozoa to recurrent
genital heat exposure, causing detrimental changes to
the membrane phospholipid architecture of epididymal
spermatozoa (51) and the functional properties of the
motor apparatus of the sperm agellum (52). Therefore,
reducing the abstinence period may minimize the
frequency and time span of heat exposure, thereby leading
to improved motility.
Sperm viability
Sperm viability is one of the parameters that is routinely
assessed in basic semen analysis, and is especially
recommended in samples where the percentage of motile
spermatozoa is less than about 40% (4). The viability
status of spermatozoa selected for intracytoplasmic sperm
injection (ICSI) has to be precisely examined since the
injection of a live spermatozoon is vital to the success of
the ICSI outcome (53). Furthermore, a direct correlation
has recently been identied between sperm viability
and the level of DNA fragmentation, showing that the
viability status may be a potential indicator of DNA
integrity of the ejaculated spermatozoa (54). The lower-
bound reference limit for sperm viability is estimated
to be 58% (4). The inuence of abstinence duration on
sperm viability was examined in eleven studies (9, 13-15,
17, 26, 29, 31-33, 42). This was done by using various
techniques including a dye exclusion assay (14, 15, 29,
33), the hypo-osmotic swelling test (13, 31, 42) and ow
cytometry (9, 32). Most of these studies reported slight
or no statistically signicant negative association between
sperm viability and abstinence period. The overall mean
percentage of viable spermatozoa peaked and remained
relatively unchanged between the second and the fth day
of abstinence, and declined thereafter (Table 2).
Sperm morphology
To be considered morphologically normal, the whole
spermatozoon and its three distinct areas, the head,
midpiece and the tail, must t with stringent criteria in
terms of their size and shape. The 5th centile lower-bound
reference limit for normal forms is 4% (4). It has also been
reported that morphologically abnormal spermatozoa,
with a special focus on the acrosomal region, have a lower
chance to bind to the zona pellucida (55). A correlation has
also been observed between sperm head abnormalities and
DNA integrity. Therefore, analysis of sperm morphology,
which may provide crucial evidence about semen quality,
is assessed by fairly simple and inexpensive methods
compared with expensive and elaborate assays such as
DNA fragmentation (56) and acrosome reaction (57). The
relationship between the abstinence duration and sperm
morphology was investigated in eighteen studies (8, 13,
14, 17-19, 21-25, 28-33, 42). All had assessed sperm
morphology manually via visual assessment except one
(29) which had used CASA.
Most of the studies (14 out of 18) reported no signicant
association between sperm morphology and the period of
abstinence. In contrast, one study reported signicantly
higher percentages of spermatozoa with tail defects
when the abstinence period was extended from 2-3 days
to 6-7 days. However, the overall proportion of normal
morphology did not differ between the two abstinence
groups (23). Furthermore, Levitas et al. (24) reported that
among mild to moderate oligozoospermic samples, the
highest percentage of normal morphology was reported
at ≤2 days of abstinence but this association was not
observed in a normozoospermic population. Bahadur et
al. (8) recently reported that an extremely short abstinence
period of 30 minutes could signicantly improve sperm
morphology among oligozoospermic men, all candidates
for intrauterine insemination (IUI) treatment. By contrast,
shortening the abstinence duration in normal individuals
from 3-5 days to only 18-30 hours resulted in a considerably
lower percentage of morphologically normal spermatozoa
(28). It may therefore be advantageous for patients with
oligozoospermia to abstain for shorter periods before sperm
collection in the process of fertility treatment. However,
it must be re-iterated that manual assessment of sperm
morphology is a subjective analysis with inter- and intra-
laboratory variation. This variability may be attributed to
several factors including the use of different xation and
staining techniques (58), differences in interpretation (59)
and technician expertise (60). Another important factor
that needs to be taken into consideration is that the WHO
guidelines and reference ranges have changed over the
years and may thus lead to differences in interpretation (4).
Ejaculatory abstinence and advanced semen parameters
Conventional semen parameters provide the essential
information on which clinicians base their preliminary
diagnosis (61). Approximately 25-40% of idiopathic
infertile males have been reported to have normal semen
proles (62). Therefore, a range of advanced sperm
quality parameters have been developed to circumvent the
limitations of the conventional semen analysis (63).
DNA fragmentation
Assessment of sperm DNA integrity, in addition to routine
semen analysis, provides further valuable information
about sperm quality as well as pregnancy outcomes (64,
65). It has been shown that high proportions of spermatozoa
with DNA fragmentation above 20% increase the risk
of infertility regardless of having normal basic semen
parameters (61). Eight studies (7, 9, 14, 15, 27, 28, 32, 66)
had investigated the relationship between the abstinence
period and sperm DNA fragmentation. Three studies (7, 14,
32) did not nd any effect while half of the studies (15, 27,
Ayad et al.
Int J Fertil Steril, Vol 11, No 4, Jan-Mar 2018
244
28, 66) showed an increase in sperm DNA fragmentation
rates with prolonged abstinence. Interestingly, the report
by Mayorga-Torres et al. (9) was to the contrary, showing
considerable increase in DNA fragmentation levels after an
extremely short abstinence periods of 2 hours compared
with the initial ejaculate that was collected after 3-4 days of
abstinence. The latter nding could be purely a result of the
extremely small and underrepresented sample size (n=3)
but still merits further investigation.
Reactive oxygen species production
Normal physiological levels of ROS are crucial for
maintaining various vital functions in spermatogenesis at
different maturational stages. These highly reactive species
can also act as essential mediators for signal transduction
involved in sperm capacitation, hyperactivation and
acrosome reaction (67). However, ROS levels must
be maintained within physiological ranges since ROS
overproduction or insufcient antioxidant defense can
result in a state of oxidative stress (68).
Three studies (9, 15, 32) examined the relationship
between the abstinence period and sperm intracellular ROS
production, while only one study examined the relationship
in terms of seminal ROS concentration (69). These studies
consistently reported no association of abstinence duration
with either intracellular ROS production or seminal ROS
levels. However, among the relevant studies a general
trend of reduction, albeit non-signicant, was observed in
intracellular ROS levels after short abstinence in comparison
with long abstinence. Interestingly, when four repeated
ejaculates were collected on the same day at 2 hour intervals,
a signicant reduction in intracellular ROS production was
observed in the fourth ejaculate compared with the initial
one obtained after 3 to 4 days of abstinence (9). During
their maturation and storage, spermatozoa are continuously
susceptible to oxidative damage induced by intracellular
and extracellular reactive species. Spermatozoa are highly
sensitive to ROS damage by lipid peroxidation due to their
membranes being highly rich in polyunsaturated fatty acids
(67). Therefore, the release of spermatozoa through more
frequent ejaculations may possibly minimize their adverse
effects on sperm quality (9).
Seminal plasma antioxidants
Spermatozoa have limited intracellular enzymatic
defense against oxidative stress, partly due to cytoplasmic
extrusion during spermatogenesis. This decient capacity
is effectively compensated for by a group of cellular
detoxifying enzymes with powerful antioxidant properties
including superoxide dismutase (SOD), catalase (CAT),
and glutathione peroxidises found within the seminal
plasma (68). Surprisingly, only one study had examined
the inuence of ejaculatory abstinence period on seminal
plasma antioxidants and lipid peroxidation of the sperm
membrane (30). By analysing ejaculates of forty men
undergoing IUI, Marshburn et al. (30) observed a
signicant improvement in the total antioxidant capacity
of seminal plasma after one day of abstinence compared
to four days. Lipid peroxidation of the sperm membrane
remained unchanged between the two abstinence periods.
They therefore suggested that short abstinence-related
increase of total antioxidant capacity in seminal plasma
could defend spermatozoa against oxidative stress through
a mechanism that is independent of lipid peroxidation.
Hitherto, there are no available data on the effect of the
abstinence period on acrosome reaction or any individual
antioxidants. With respect to the detoxyifying enzymes,
however, we have observed in our laboratory that a short
abstinence period of four hours led to a signicant increase
in SOD activity but did not change the activity of catalase
in seminal plasma (unpublished data).
Pregnancy rate
The conventional parameters of semen analysis provide
fundamental information for the initial diagnosis of male
infertility, but none is reliable enough to predict pregnancy
(38). Few studies had examined the inuence of ejaculatory
abstinence period on pregnancy rate, all of which had
recruited patients from infertility and assisted conception
clinics. For instance, among infertile couples undergoing
ovulation induction followed by IUI, the highest pregnancy
rate was observed for those with an abstinence period of ≤3
days, while a sharp decline in pregnancy rate was observed
for those with ≥10 days of abstinence. Interestingly,
the relationship between ejaculatory abstinence period
and pregnancy rate was independent of the variation
in conventional semen parameters (40). Another study,
examining a more general infertile population, revealed
that the highest IUI pregnancy rates were associated with
≤2 days of abstinence (26). Sánchez-Martín et al. (27)
reported that serial ejaculation every 24 hours for four
days with an ultimate abstinence of 12 hours, along with
sperm selection by density gradient centrifugation, could
signicantly improve pregnancy rate with ICSI. More
recently, Bahadur et al. (70) showed in a pilot study that
recurrent ejaculates successfully improved IUI pregnancy
rates. These ndings can be supported by the fact that
fertilization rates are directly related to sperm progressive
motility and inversely related to DNA fragmentation in vitro
(71) with both parameters generally found to be improved
with shorter abstinence periods. However, importantly,
large prospective randomized controlled trials are required
to validate that short abstinence periods improve pregnancy
and live birth rates, and may thus be recommended for
infertility treatments.
Conclusion
We conclude that in spite of the varied quality of existing
studies, the weight of evidence suggests that reducing the
ejaculatory abstinence period may positively inuence
semen quality based on a consistent trend towards an increase
in the percentage of motile, progressively motile and rapid
spermatozoa with shorter abstinence periods. However, the
small number of studies examining ROS production, DNA
fragmentation and seminal plasma antioxidant capacity
Short Abstinence Improves Semen Quality
Int J Fertil Steril, Vol 11, No 4, Jan-Mar 2018 245
limit any denitive conclusion regarding its effect on
advanced semen parameters. Further clinical trials with
sufcient number of subjects, and controlling for potential
confounders, may shed further light on this association.
We recommend that future studies incorporate CASA as
a more accurate and objective measurement tool as well
as utilize more sensitive measures of sperm function
such as sperm hyperactivity, sperm-zona binding ability,
acrosome reaction, and total and individual seminal plasma
antioxidants. It is, however, worth mentioning that even
after short abstinence periods of ≤1 day, the overall mean
values of the conventional semen parameters were always
above the lower-bound reference limits recommended by
WHO (fth version). Therefore, shortening the abstinence
period may be a potential strategy to improve sperm
quality. It is thus recommended that the current guidelines
regarding the prescribed abstinence period are revisited.
Acknowledgments
The authors wish to thank Miss Bongekile Skosana for
critical reading of the manuscript. We also thank the Harry
Crosley Foundation for their nancial support. The authors
declare no conicts of interest.
Author's Contributions
B.M.A.; Helped design the study, searched the data base,
analysed the results and wrote the manuscript. G.V.d.H.;
Helped with the study design and reviewed the nal version
of the manuscript. S.S.D.P.; Helped with the study design
and assisted with the writing of the manuscript.
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