Effects of long and short ejaculatory abstinence on sperm

parameters: a meta-analysis of randomized-controlled-trials

1 1
Arturo Lo Giudice , Maria Giovanna Asmundo , Sebastiano
1 2 3 4
C i m i n o , Andrea Cocci , M a r c o F a l c o n e , M a r c o C a p e c e , Ali S.
5 6 7
Abdelhameed , Paolo Capogrosso , Afonso Morgado , Georgios
8 4 9 *
T s a m p o u k a s , Celeste Manfredi , Giorgio Ivan Russo

1 2
University of Catania, Italy, University of Florence, Italy,
3 4
University of Turin, Italy, University of Naples Federico II, Italy,
5 6

l
King Saud University, Saudi Arabia, University of Varese, Italy,

a
7 8
University of Porto, Portugal, The Great Western Hospital, United

Kingdom,
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Department of Urology, University of Catania, Italy
Submitted to Journal:

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Frontiers in Endocrinology

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P r o v
Specialty Section:
Reproduction

ISSN:
1664-2392

Article type:
Review Article

Received on:
19 Jan 2024

Accepted on:
22 Mar 2024

Provisional PDF published on:

22 Mar 2024

Frontiers website link:

www.frontiersin.org

Citation:
Lo_giudice A, Asmundo M, Cimino S, Cocci A, Falcone M, Capece
M, Abdelhameed AS, Capogrosso P, Morgado A, Tsampoukas G,
Manfredi C and Russo G(2024) Effects of long and short ejaculatory
abstinence on sperm parameters: a meta-analysis of randomized-
controlled-trials. Front. Endocrinol. 15:1373426.
doi:10.3389/fendo.2024.1373426

Copyright statement:
© 2024 Lo_giudice, Asmundo, Cimino, Cocci, Falcone, Capece,
Abdelhameed, Capogrosso, Morgado, Tsampoukas, Manfredi and
Russo. This is an open-access article distributed under the terms
o f t h e C r e a t i v e C o m m o n s A t t r i b u t i o n L i c e n s e ( C C B Y ). T h e u s e ,
distribution and reproduction in other forums is permitted, provided
the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms.
This Provisional PDF corresponds to the article as it appeared upon acceptance, after
peer-review. Fully formatted PDF and full text (HTML) versions will be made available soon.

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 1 Effects of long and short ejaculatory abstinence on sperm parameters: a meta-analysis of

2 randomized-controlled-trials

4 ABSTRACT:

5 Purpose: To investigate whether ejaculatory abstinence would affect sperm parameters.

6 Methods: This analysis has been registered on PROSPERO (CRD42023472124). We performed a

7 search on Pubmed using the following text terms: ((“sperm parameters” OR “sperm analysis”

8 [Mesh]) AND (“sperm DNA fragmentation” OR “DNA fragmentation” [Mesh]) AND (“sexual

9 abstinence” [Mesh] OR “abstinence”)) and an advanced search in Scopus using the terms (“sperm

10 parameters” OR “sperm parameters” OR “DNA fragmentation”) AND (“abstinence”). Sperm

11 parameters that have been investigated were sperm volume, total sperm motility, progressive sperm

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motility, sperm concentration, sperm morphology and sperm DNA fragmentation (SDF). A two-day

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13 cut-off as a "short" or "long" abstinence period has been defined.

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Results: Thirteen studies published between 2013 and 2022 have been included in the meta-

analysis. In total, 2315 patients, ranging from 6 to 836 from each cohort, have been enrolled in the

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study. We showed that longer abstinence time was associated with greater sperm concentration

(mean difference [MD]: 8.19; p<0.01), sperm volume (MD: 0.96; p<0.01) and higher SDF (MD: 3.46;

p<0.01) but lower progressive sperm motility (MD: -1.83; p<0.01). Otherwise, a not statistically

19 significance difference was observed in patients with longer vs. shorter abstinence time regarding

20 total sperm motility (MD: -1.83; p=0.06). Meta-regression analysis showed that days of abstinence

21 were positively and linearly related to sperm concentration (slope: 3.74; p<0.01) and SDF (slope:

22 0.65; p=0.044).

23 Conclusions: According to our data, short ejaculatory abstinence is associated with better sperm

24 quality; indeed, higher percentage of progressive sperm motility and lower level of SDF were

25 reported in short abstinence cohort. Otherwise, long abstinence group reported higher sperm

26 concentration.

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27 Keywords: semen parameters; sperm parameters; ejaculatory abstinence; sexual abstinence; DNA

28 fragmentation

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30 1. Introduction

31 Despite variable and conflicting evidences on the decrease of male reproductive indices over the

32 past half century, male subfertility remains an area of concern with great academic, social and

33 financial interest worldwide [1]. Apart from the extensively researched and conventionally accepted

34 morphological, physiological, and genetic explanations of male infertility, the theory that the

35 spermatocyte is a "cell in crisis" because its genetical material is under danger from multiple

36 sources, is also a current theory [2]. In light of these theories and available data, recent studies

37 have sought to gain a deeper understanding of the underlying mechanisms of oxidative stress, which

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is the main root cause of sperm DNA fragmentation (SDF). However, it is unclear whether routine

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39 use of SDF is beneficial, other than in certain populations, such as those with recurrent miscarriages,

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modifiable risk lifestyle factors, and infertility [3]. Resolving practicalities concerning the

standardization of sperm collection may also help SDF role to be clarified and its utility to be

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maximized. Furthermore, improved sperm quality could result from improved standardization and

specification of the ideal time for sperm collection [4]. The physiology of ejaculation and the

individual quantitative and qualitative contribution of seminal vesicles, prostate gland and epididymis

45 after repeated ejaculations, and duration of ejaculation abstinence (EA) should be considered, as

46 abstinence may affect conventional sperm parameters such as volume and total sperm count in

47 both men with normospermia and dyspermia [5]. This has also been reported to be the case

48 regarding SDF, as length of abstinence was correlated positively with semen volume, sperm

49 concentration and total sperm count while SDF was significantly lower in shorter EA compared to a

50 recommended (3-7 days) in healthy donors [6]. In real-world application, these observations seem

51 to carry most clinical importance, as standardization of sperm collection may improve some of the

52 primary fertility endpoints. Borges et al. reported that EA of four days or less was associated to lower

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53 SDF, higher rates of fertilization and pregnancy comparing to longer ejaculatory abstinence in

54 couples undergoing assisted reproductive technology (ART) [7]. Despite accumulating evidences,

55 at the moment, the Worth Health Organization recommends a minimum two days and a maximum

56 of seven days of abstinence [8], a wide range that should be taken into account when interpreting

57 sperm quality. In this systematic review and meta-analysis, we aimed to evaluate the association

58 between ejaculation abstinence time and sperm quality in adult men undergoing male infertility work-

59 up from randomized clinical studies. By specifically focusing on RCTs, the paper aims to report a

60 higher level of evidence compared to observational or non-randomized studies.

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62 2. Methods

63 2.1 Systematic literature search

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65 This systematic review was conducted according to the Preferred Reporting Items for Systematic

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Reviews and Meta-analysis guidelines (PRISMA) [9] and it was registered on PROSPERO

(CRD42023472124), with the aim to answer the clinical question whether ejaculatory abstinence

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has an effect on sperm volume, sperm concentration, total motility, progressive motility, morphology

and SDF. In November 2022 we performed a search on major databases, and we collected data of

adult men undergoing male infertility evaluation due to couple infertility or healthy donors. We

71 performed a search in Pubmed, Scopus and Embase using the text terms ((“sperm parameters” OR

72 “sperm analysis” [Mesh]) AND (“sperm DNA fragmentation” OR “DNA fragmentation” [Mesh]) AND

73 (“sexual abstinence” [Mesh] OR “abstinence”)) and an advanced search in Scopus using the terms

74 (“semen parameters” OR “sperm parameters” OR “DNA fragmentation”) AND (“abstinence”). Finally,

75 we employed a snowball method to search for articles not identified in the first search. No time

76 limitations were applied. We included studies reporting: 1) the correlation between EA and sperm

77 DNA fragmentation, and 2) comparisons of SDF or sperm parameters across various intervals of

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 78 EA in both independent and paired groups. Studies involving non-adults, animals and retracted

79 publications were not included.

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81 2.2 Study selection

82 A total of 279, 1000 and 203 articles were selected from Pubmed, Scopus and Embase, respectively

83 (Suppl. Figure 1). Citation lists of selected studies were manually checked, and references reported

84 in included articles were screened to find more potentially pertinent papers. After duplicates and

85 only-abstracts have been removed, authors assessed eligibility and final inclusion in the meta-

86 analysis for 13 studies. Studies were reviewed by two independent reviewers (A.L.G. and G.T.);

87 differences in opinion were discussed in consultation with the last author (G.I.R.) who solved

88 discrepancies for further inclusion between the first two investigator. Studies with any design were

89 included.

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2.3 Risk of bias assessment

Before the extraction of the outcomes, two reviewers (M.G.A. and A.L.G.) assessed the risk of bias,

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concerning the following characteristics: Random sequence generation (selection bias), Allocation

concealment (selection bias), Blinding of outcome assessment (detection bias), Incomplete outcome

data (attrition bias), Selective reporting (reporting bias). To perform the risk of bias assessment, the

96 Cochrane risk of bias tool has been used; a third reviewer (G.I.R.) resolved disagreements between

97 reviewers’ judgements.

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99 2.4 Data extraction

100 The authors of the study defined a two-day cut-off as a "short" or "long" abstinence period. All

101 samples collected <2 days (2hrs to 2 days) were classified as short abstinence period while all

102 samples collected from day 3 upward were considered as long abstinence period. This period has

103 been arbitrarily defined on the basis of populations included and compared in the studies selected

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104 for meta-analysis. For each study, the difference between long and short abstinence interval was

105 calculated in order to obtain days of abstinence. In case that different interval time were reported in

106 the same study, data have been separately extracted and included in the forest plot.

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108 2.5 Statistical Analysis

109 Sperm parameters regarding semen volume, sperm concentration, total motility, progressive

110 motility, SDF and morphology have been reported as mean with SD. A meta-regression analysis

111 has been performed among the mean difference between long and short abstinence in populations;

112 the galbraith plot was used to identify potential outliers among the populations included; when

113 potential outliers were identified, a leave-one-out meta-analysis was subsequently performed.

114 Finally, in order to identify a linear relationship between the days of abstinence and the parameter

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under consideration, a linear regression was performed between each sperm parameter and the

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116 withdrawal time (reported as days between), the outcome was reported with a bubble plot. All

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analyses have been performed with the software Stata/SE 17.0 for Mac (Apple Silicon), StataCorp

4905 Lakeway Dr College Station, TX 77845 USA. All reported P values were based on two-sided

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tests and compared to a significance level of 5%.

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130 3 Results

131 3.1 Characteristics of the study

132 After removing 240 duplicates, 1265 records were screened, and 34 full-text articles were assessed

133 for eligibility. In the end, a total of 13 studies were included for the quantitative and qualitative

134 analysis [7, 10–21](Suppl. Figure 1). Baseline characteristics of the articles included are presented

135 in Suppl. Table 1. All the studies included in the meta-analysis are RCT enrolling patients from

136 infertility check-up visits or healthy volunteers. The final number of patients enrolled in the study (N)

137 was 2315, ranging from 6 to 836.

138

139 3.2 Analysis

140 We showed greater sperm volume (MD: 1; 95% CI 0.81-1.2; p<0.01) (Figure 1), sperm concentration

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(MD: 9.07; 95%CI 2.87-15.27; p<0.01) (Figure 1) and SDF (MD: 3.67; 95%CI 2.32-5.03; p<0.01)

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142 (Figure 2) in long abstinence group vs. short abstinence group.

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Moreover, we observed a not significant reduction of sperm progressive motility (MD: -1.34; p=0.1)

(Figure 3a) and total sperm motility (MD: -1.15; p=0.35) in patients with longer vs. shorter abstinence

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time (Figure 3b).

The meta-regression analysis revealed a positive association between days of abstinence and

sperm concentration (slope: 3.74; 95%CI 1.09-6.38; p<0.01) and SDF (slope: 0.65; 95%CI 0.018-

148 1.82; p=0.044); otherwise, the meta regression analysis did not highlight a statistically significant

149 relation between days of abstinence and volume (p= 0.24), progressive sperm motility (p= 0.11)

150 (Figure 4). Suppl. Figure 2 reports the risk of bias of included studies.

151 We used the Galbriath plot to test for heterogeneity among the included studies and identified some

152 potential outliers (Suppl. Figure 3); consequently, we performed a leave one out meta-analysis for

153 sperm concentration (Suppl. Figure 4), sperm volume (Suppl. Figure 5) and SDF (Suppl. Figure 6).

154 No changes in the statistical significance of the analysis were identified when a study was omitted.

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156 4 Discussion

157 In the present meta-analysis, we showed that days of ejaculatory abstinence significantly influenced

158 sperm quality. Moreover, a statistically significant association between longer ejaculatory abstinence

159 and sperm volume, sperm concentration and SDF has been showed. However, no relevant

160 association between days of abstinence and total sperm motility has been recorded.

161 Overall, according to WHO criteria a 2–7-day period of abstinence is recommended before collecting

162 ejaculate for appropriate semen analysis [22].

163 However, the ideal period of ejaculatory abstinence is still open to debate. Indeed, the worldwide

164 recommended period of abstinence is quite variable, and it is not plenty known how it could affect

165 the final sperm analysis results.

166 Short abstinence periods (1-2 days) may result in higher sperm motility and viability due to reduced

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sperm aging and decreased sperm DNA damage [23]. On the other hand, longer abstinence periods

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168 (5-7 days) may lead to higher sperm concentration but lower motility and viability due to increased

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sperm senescence and oxidative stress [24]. Therefore, a balance between abstinence duration and

sperm quality is crucial for optimizing fertility outcomes in infertile patients.

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Short abstinence periods may reduce oxidative stress levels and improve antioxidant capacity by

minimizing sperm exposure to reactive oxygen species (ROS) and lipid peroxidation. Conversely,

longer abstinence periods may exacerbate oxidative stress and deplete antioxidant reserves,

174 leading to sperm membrane damage and impaired fertility [25].

175 When the sample collection takes place after longer ejaculatory abstinence, the spermatozoa

176 remain for several days inside the epididymis. This undoubtedly leads to alterations which are

177 reflected in semen analysis results [26] .Moreover, it is important to underline at individual factors

178 may influence sperm parameters. In fact, lifestyle habits and anthropometric parameters can affect

179 sperm analysis [27] . An interesting study conducted by Dahan et al observed a relevant

180 improvement in SDF when comparing short abstinence group (3 hours) and long abstinence group

181 (3 days). Authors registered in 58 out of 112 patients, an improvement of 30% or more in SDF

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182 parameters in second sperm sample compared to the first one. Moreover, short abstinence group,

183 reported lower sperm volume and total sperm concentration with augmented sperm progressive

184 motility [28].

185 Since the abstinence time is fundamental to ensure both quality and quantity of spermatozoa

186 required to achieve natural and assisted pregnancy, recent studies had focused on the achievement

187 of the perfect sample [29]. Due to the increasing interest among artificial insemination, research had

188 suggested that shorter abstinence may be more appropriate in ART, than the conventional

189 abstinence recommended for routinary semen analysis [26].

190 An interesting meta-analysis presented by Calogero et al promoted very short abstinence period,

191 especially among patients affected by OAT. After a very brief period of abstinence (4 hours), the

192 authors reported improved sperm parameters in OAT-affected patients, including increased sperm

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concentration, total and progressive sperm motility, and decreased SDF level [30].

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194 Previous meta-analysis conducted on non-randomized clinical studies however have reported that

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short-term abstinence may be associated with limited improvements in semen quality in healthy men

but could be more beneficial for infertile men, especially within the first 4 days of abstinence [31].

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The positive association between days of abstinence and sperm concentration was substantially a

common finding in all the paper analyzed. This relationship may be ascribed to the presence of

stored sperm into the epididymis; it is therefore predictable a depleted sperm reserve and

200 consequent lower total sperm count in shorter abstinence groups [32]. Undoubtedly epididymis plays

201 a fundamental role on spermatozoa maturation, concentration, and survival; moreover, during

202 epididymal transit, spermatozoa acquire antioxidant enzymes [33].

203 Indeed, increasing evidence suggested that total antioxidant capacity (TAC) is considerably

204 improved after reduced ejaculatory abstinence and a possible link among short abstinence, TAC

205 and SDF had been suggested [34].

206 On the other side, longer ejaculatory abstinence may lead to sperm functional alterations that may

207 not be recognized by conventional sperm analysis; this hypothesis may explain the lower pregnancy

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208 and fertility rate in long abstinence, notwithstanding higher total sperm count and sperm

209 concentration [7]. Figure 5 shows the impact of identified mechanisms influencing sperm quality in

210 short abstinence time.

211 The strength of our study is to have explores and compares the effects of both long and short

212 ejaculatory abstinence periods on various sperm parameters. By specifically focusing on RCTs, the

213 paper ensures a higher level of evidence compared to observational studies or non-randomized

214 trials. This comparative analysis could provide valuable insights into the optimal abstinence duration

215 for improving sperm quality, which may have implications for fertility treatment and family planning.

216 In fact, our study almost investigated the impact of abstinence in healthy volunteer subject.

217 Before concluding we would like to highlight some limitations. Firstly, we did not assess the

218 pregnancy rate since it was not reported by the studies. Secondly, other markers of seminal

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oxidative stress markers were not reported in the studies and they may be influenced by the time of

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5 Conclusions

According to our data, short ejaculatory abstinence is associated with better sperm parameters;

indeed, higher percentage of progressive sperm motility and lower level of SDF were reported in

226 short abstinence cohort. Otherwise, long abstinence group reported higher sperm concentration.

227 These results should be considered especially when counseling patients during ART.

228 Instead of adopting a one-size-fits-all approach to abstinence duration, future practices may involve

229 personalized recommendations based on individual sperm quality and characteristics. Advanced

230 diagnostics and biomarkers could help assess sperm health, allowing clinicians to tailor the

231 abstinence period to maximize sperm quality for each donor.

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232 Future approach can also include artificial intelligence and machine learning algorithms that could

233 be employed to analyze large datasets of sperm quality parameters and donor characteristics to

234 identify patterns and optimize the timing of sperm donation relative to abstinence periods.

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236

237 Authors contribution:

238 Conceptualization, G.I.R.; methodology, G.I.R.; validation, all authors; formal analysis, G.T; M.G.A.;

239 G.I.R.; A.L.G.; investigation, G.T; M.G.A.; G.I.R.; A.L.G.; data curation, G.T; M.G.A.; G.I.R.; A.L.G.;

240 writing—original draft preparation, M.G.A. and G.T.; writing—review and editing, M.G.A., G.I.R.;

241 supervision, G.I.R.; project administration, G.I.R. All authors have read and agreed to the published

242 version of the manuscript.

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244 Acknowledgement: This review was registered on PROSPERO (CRD42023472124). Data can be

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requested to the corresponding author. Authors extend their sincere appreciation to Researchers

Supporting Project number (RSPD2023R750). King Saud University, Riyadh, Saudi Arabia.

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'Funding and/or Conflicts of interests/Competing interests: This research received no external

funding. The authors declare no conflict of interest.

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251 Ethical statement: Not applicable.

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255

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