Journal of

Clinical Medicine

Article
Does a Very Short Length of Abstinence Improve Assisted
Reproductive Technique Outcomes in Infertile Patients with
Severe Oligo-Asthenozoospermia?
Federica Barbagallo 1 , Aldo E. Calogero 1 , Rosita A. Condorelli 1 , Ashraf Farrag 2 , Emmanuele A. Jannini 3 ,
Sandro La Vignera 1 and Claudio Manna 2,4, *

1 Department of Clinical and Experimental Medicine, University of Catania, 95123 Catania, Italy;
federica.barbagallo11@gmail.com (F.B.); acaloger@unict.it (A.E.C.); rosita.condorelli@unict.it (R.A.C.);
sandrolavignera@unict.it (S.L.V.)
2 Biofertility IVF and Infertility Center, 00198 Rome, Italy; ashraf_farrag@yahoo.com
3 Department of Systems Medicine, University of Rome “Tor Vergata”, 00133 Rome, Italy; eajannini@gmail.com
4 Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, 00133 Rome, Italy
* Correspondence: claudiomanna55@gmail.com

Abstract: In recent years, a growing number of studies seem to support the beneficial effects of a very
short abstinence period on sperm parameters, especially in patients with oligo-asthenozoospermia
(OA). On this basis, the aim of this study was to evaluate the effects of a short period of absti-





 nence (1 h) on intracytoplasmic sperm injection (ICSI) outcomes in infertile patients with severe OA.
Citation: Barbagallo, F.; We performed a retrospective study on 313 ICSI cycles in which couples were divided into two dif-
Calogero, A.E.; Condorelli, R.A.; ferent groups based on sperm parameters of the male partners. Group 1 included normozoospermic
Farrag, A.; Jannini, E.A.; men or male partners with a mild OA (n = 223). Group 2 included male partners with severe OA
La Vignera, S.; Manna, C. Does a Very (n = 90). They were asked to provide a second consecutive ejaculation after 1 h from the first one.
Short Length of Abstinence Improve The best ejaculate was used to perform ICSI. We found a significant increase of total (p < 0.001) and
Assisted Reproductive Technique progressive motility (p < 0.001) in the second ejaculate of patients of Group 2 compared with those of
Outcomes in Infertile Patients with the first one. Spermatozoa of the second ejaculate were chosen for ICSI for all patients in Group 2.
Severe Oligo-Asthenozoospermia? J.
We found statistically significant improvement of clinical pregnancy rate (p = 0.001) and embryo
Clin. Med. 2021, 10, 4399. https://
quality (p = 0.003) in couples in Group 2 compared to those of Group 1. No statistically significant
doi.org/10.3390/jcm10194399
difference was found in fertilization, implantation, live birth delivery, and miscarriage rates between
the two groups. Therefore, a second semen sample collected after a very short time-interval in pa-
Academic Editor:
Marie-Madeleine Dolmans
tients with severe OA allowed us to obtain significantly higher clinical pregnancy rate with improved
embryo quality compared to normozoospermic men or patients with mild OA. Fertilization, implan-
Received: 1 August 2021 tation, live birth delivery, and miscarriage rates were similar between the two groups. The present
Accepted: 22 September 2021 study shows that a second consecutive ejaculate could represent a simple strategy to obtain better
Published: 26 September 2021 sperm parameters and assisted reproductive technology (ART) outcomes in infertile patients with
mild-severe OA.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in Keywords: ART; sperm parameters; ICSI; fertilization rate; pregnancy rate
published maps and institutional affil-
iations.

1. Introduction
In the second decade of the new millennium, infertility remains a global public health
Copyright: © 2021 by the authors. issue, affecting approximately 15% of all couples in the reproductive age in industrialized
Licensee MDPI, Basel, Switzerland. countries [1]. In recent decades, assisted reproductive technologies (ART) have spread
This article is an open access article
worldwide to help infertile couples to achieve pregnancy [2]. A proper diagnostic and
distributed under the terms and
therapeutic workup associated with adequate preparation of infertile couples before ART
conditions of the Creative Commons
is essential to obtain spermatozoa and oocytes of high quality and, in turn, to improve
Attribution (CC BY) license (https://
the outcomes of ART. The female factor has often been considered as the main factor
creativecommons.org/licenses/by/
responsible for the failure of ART cycles. However, a male factor is responsible for couple
4.0/).

J. Clin. Med. 2021, 10, 4399. https://doi.org/10.3390/jcm10194399 https://www.mdpi.com/journal/jcm

J. Clin. Med. 2021, 10, 4399 2 of 11

infertility in about half of the cases [1]. A breakthrough in this regard was the development
of intracytoplasmic sperm injection (ICSI) for infertile patients with severe oligoasthenoter-
atozoospermia (OAT) [3]. A better sperm quality enhances fertilization rate and embryo
quality following ICSI [4]. In fact, impairment of sperm parameters may affect fertilization
and cleavage rates and the quality of embryos [5]. Furthermore, it can be associated with
higher aneuploidy [6] and miscarriage rates [7]. In clinical practice, several antioxidants are
commonly used to improve male fertility before ART. Oral antioxidant treatment appears
to improve ICSI outcomes, especially in patients with sperm DNA damage, in whom
antioxidants reduce the percentage of damaged spermatozoa [8]. A position statement of
the Italian Society of Andrology and Sexual Medicine placed a high value on the adminis-
tration of antioxidants in patients with idiopathic infertility in the presence of documented
abnormal sperm parameters and altered sperm DNA fragmentation after an adequate
diagnostic procedure [9]. Therefore, a correct diagnostic and therapeutic management of
infertile patients before ART is essential to increase success rates, without simply leaving
ICSI to solve the severe male factor of infertility.
Due to the increasing number of infertile couples undergoing ART, several studies
have focused on identifying factors that can improve the outcome of these techniques.
Among the different factors that affect sperm quality, sexual abstinence is often overlooked,
although the length of sexual abstinence has been shown to influence sperm parameters.
The World Health Organization (WHO) recommends that semen should be collected for its
analysis after a minimum of 2 days and a maximum of 7 days of sexual abstinence [10];
however, the European Society of Human Reproduction and Embryology (ESHRE) advises
an abstinence period of 3–4 days only [11]. The basis for these recommendations is unclear
and the current indications on the abstinence length should be revisited [12]. Many years
ago, McLeod and Gold indicated that the abstinence period should be based on copulation
frequency [13]. They reported that a coital frequency less than three times per week may
result in delayed fertility by missing the ovulatory window and/or by impairing sperm
parameters [13].
Many studies have investigated the influence of the length of sexual abstinence on
sperm parameters, although the results are controversial. In fact, a longer abstinence period
seems to improve semen fluid volume and sperm count whereas the effects on motility,
morphology, and DNA fragmentation are contradictory [14]. However, these studies are
extremely variable both for the characteristics of men enrolled and for the length of sexual
abstinence [15].
A growing number of studies are focusing their attention on the possibility of using
a second ejaculate collected after a very short period of abstinence in infertile men, espe-
cially patients with oligo-asthenozoospermia (OA). We previously reported that a second
consecutive ejaculate (collected within 1 h from the first) resulted in better conventional
sperm parameters (motility and morphology) and a lower percentage of spermatozoa with
fragmented DNA in normozoospermic male partners of infertile couples and even more in
patients with OAT [16]. Our findings were in line with the most recent literature [4,17–25].
However, the optimal length of abstinence for couples undergoing ART cycles is
still debated. Short abstinence in terms of days [26,27] or hours [28] has improved the
pregnancy rate by intrauterine insemination. A recent meta-analysis described that a short
abstinence period (less than 4 days) was associated with higher implantation (p = 0.0001)
and pregnancy rates (p = 0.0006) compared to a period of abstinence of 4–7 days in ART
treatments [29].
Few studies have evaluated the effect of a very short period of abstinence on ART
outcomes. Sugyam et al. reported that the fertilization rate using spermatozoa collected
after 30–60 min (53.3%) was significantly higher than that from the first (28.9%, p < 0.05) [4].
A very recent study evaluated the effect of an abstinence period of 2 h on sperm parameters
and ART outcome in patients with severe OAT [25]. Their findings suggest that a very
short abstinence period in severe OAT allows the obtaining spermatozoa with better
J. Clin. Med. 2021, 10, 4399 3 of 11

quality, especially in terms of motility and, in turn, achievement of the same probability of
pregnancy compared to normozoospermic patients [25].
Moreover, an increase of the euploid blastocysts has been reported using ejaculates
obtained after very short abstinence (1 h) [15].
On this basis, this study aimed to evaluate the effects of a very short period of
abstinence (1 h) on intracytoplasmic sperm injection (ICSI) outcomes in infertile patients
with severe OA.

2. Materials and Methods

2.1. Sample Selection
This is a clinical study treating 313 infertile couples with ICSI which we have retro-
spectively evaluated. All 313 male partners had abstinence of 2–7 days, as suggested by the
WHO 2010 criteria. All semen samples were collected by masturbation within the fertility
center to minimize conditions that might alter sperm parameters.
All semen analyses were performed by the same experienced embryologist according
to the WHO 2010 criteria [10]. The evaluation of sperm motility was performed on a
10 µL drop on a glass slide with a 22 × 22 mm coverslip and heated stage at 37 ◦ C, with
a lens with a reticule. The slides were examined with a positive phase-contrast optics
at a magnification of 400×. We assessed 400 spermatozoa per replicate for an accurate
evaluation of motility. The assessment of morphology was done using Diff Quick stained
slides (Medion Diagnostics AG, Dudingen, Switzerland). Morphology was evaluated
at a magnification of 400× and 1000×. We prepared multiple slides to obtain at least
400 spermatozoa for a proper sperm morphological evaluation.
Couples were divided into two groups based on the sperm parameters of the male
partners. Group 1 (n = 223) included normozoospermic men or patients with mild OA,
whereas Group 2 (n = 90) included male partners with severe OA. The male partners of
Group 2 couples were asked to provide a second consecutive semen sample after explaining
to them the possibility of having better sperm parameters in the second ejaculate. Informed
consent was obtained from each patient. The second semen collection was obtained 1 h
after the first. Then, the second sample was analyzed and compared to the first for the
following parameters: concentration (mil/mL), total and progressive motility (%), and
morphology (%).
The ICSI procedure was performed with the best ejaculate and swim-up preparation
obtained according to the percentage of spermatozoa with progressive motility.

2.2. Controlled Ovarian Hyper Stimulation

Controlled ovarian hyper stimulation protocols were carried out using recombinant
follicle-stimulating hormone (FSH, Gonal-F, Merck Serono, Geneva, Switzerland) and
gonadotropin-releasing hormone (GnRH) antagonist according to the ovarian reserve.
Monitoring of the follicular development was performed by real-time ultrasound scans
from day 2 of treatment until the day of follicular aspiration guided by the patient’s
response to stimulation. When at least one ovarian follicle reached 18–20 mm diameter,
ICSI was performed 36–38 h after the administration of human chorionic gonadotropin
(hCG, Gonasi, 10,000 IU IBSA, Lodi, Italy).

2.3. Intracytoplasmic Sperm Injection Procedure

The first semen collection was obtained about 5–6 h before microinjection, scheduled
about 40 h after hCG administration. ICSI procedure was performed with spermatozoa
obtained by “swim-up” using the first or second ejaculate according to the male partner
sperm parameters as above reported. The “swim-up” technique was performed directly
from liquefied semen. To accomplish this, several semen aliquots were taken from each
sample and placed in tubes underneath an overlay of flushing medium (Origio Italia Srl,
Rome, Italy). Round-bottom tubes or four-well dishes were used to optimize the interface
surface area between the semen layer and the culture medium. The samples were left to
J. Clin. Med. 2021, 10, 4399 4 of 11

incubate at 37 ◦ C in an incubator for 30–45 min. Spermatozoa with the best motility and
ability to migrate were recovered.
The collected cumulus-enclosed oocytes were maintained in 500 µL multi dishes with
4 wells (Nunclon Surface, Roskilde, Denmark) in Continuous Single Culture™ Medium–
Complete (CSCM-C) (Irvine Scientific, FujiFilm, Tilburg, The Netherlands) under oil (oil
for embryo culture, Fuji Film, Europe) and maintained in the incubator for 2 h after their
retrieval. Then they were decumulated in hyaluronidase drops (Hyaluronidase Solution,
Fuji Film Europe Europe).
ICSI procedure was performed according to the standard technique. Embryo culture
was performed in a standard incubator at 37 ◦ C, 6% CO2 , 5% O2 in CSCM-C (Irvine
Scientific, FujiFilm, Tilburg, The Netherlands). Embryo transfer was performed after
2 days of culture. After 36–44 h in culture, all embryos were carefully examined with both
a dissecting and an inverted microscope. Embryo grading was carried out according to
the system proposed by Puissant and colleagues [30]. The number and size of blastomeres
as well as the presence or absence of anucleate fragments, were carefully recorded so that
embryos could be scored as follows: 4 = embryos with clear, regular blastomeres and either
no fragmentation or a maximum of five small anucleate fragments; 3 = embryos with few
or no fragments but with unequal blastomeres (>1/3 difference in size); 2 = embryos with
more fragments but over <1/3 of the embryonic surface; 1 = fragments over >1/3 of the
embryonic surface. Two points are added if the embryo had reached the 4-cell stage by
48 h after fertilization.
Then, we compared the ICSI outcomes of couples in which spermatozoa obtained
from ejaculate collected after 2–7 days of abstinence (as suggested by 2010 WHO criteria)
were used and couples in which a second ejaculate was obtained after a short period of
abstinence (1 h). The following primary outcomes were considered: fertilization rate (num-
ber of fertilized oocytes/number of oocytes inseminated), implantation rate (number of
gestational sacs/number of embryos transferred), clinical pregnancy rate (number of preg-
nancies with at least one fetal heartbeat/number of pick-up cycles with at least one oocyte
retrieved), live birth delivery rate (number of deliveries with at least 1 live birth/number
of pick-up with at least 1 oocyte retrieved), miscarriage rate (number of spontaneous
abortions/total number of pregnancies), and embryo quality. We also evaluated the type of
birth (natural or cesarean) and birth weight.

2.4. Ethical Approval

The study was conducted in the ART Biofertility IVF Center (Rome, Italy) on infertile
couples undergoing ICSI treatment. It was reviewed and approved by the Institutional
Review Board at the Biofertility IVF Centre who indicated that ethical approval was not
required for this study. Data collection followed the principles outlined in the Declaration
of Helsinki. All patients provided their informed consent, agreeing to supply their own
anonymous information for this and future studies.

2.5. Statistical Analysis

The Kolmogorov–Smirnov test was used to evaluate whether the data were normally
distributed. The Student’s t-test and the Mann–Whitney U test were used for continuous
variables. Dichotomous variables were analyzed by chi-square or Fisher’s exact test. A
p-value lower than 0.05 was considered statistically significant. The software SPSS 23.0 for
Windows (SPSS Inc., Chicago, IL, USA) was used.

3. Results
The age of the male partners was statistically significantly different between the two
groups (Group 1: 40.5 ± 6.4 vs. Group 2: 38.5 ± 6.7, p = 0.017). On the contrary, the age of
women in Groups 1 and 2 did not show any statistically significant difference. Likewise, no
statistically significant difference was found between antral follicle count (AFC) of women
in Groups 1 and 2. Moreover, the total dose of FSH administered to the female partners
J. Clin. Med. 2021, 10, 4399 5 of 11

for the controlled ovarian hyperstimulation was similar between the two groups. Table 1
shows the characteristics of female partners in the two groups. Table 2 shows the sperm
parameters in the two groups. Sperm concentration, total and progressive motility were
significantly higher in Group 1 than in Group 2. No statistically significant difference
was found between the percentage of spermatozoa with normal morphology between the
two groups.

Table 1. Comparison of female characteristics between Group 1 and Group 2.

Group 1 Group 2 p-Value

Age (years) a 38.1 ± 4.7 37.1 ± 5.5, 0.143
Antral follicle count a 11.2 ± 6.8 11.8 ± 7.4 0.68
Total FSH dose administered (IU) a 2731.7 ± 1515.7 2600.4 ± 1473.9 0.48
Causes of female infertility
• Recurrent pregnancy loss (%) 24 24 1
• Endometriosis (%) 29 24 0.7
• Pelvic (%) 22 12 0.3
• Tubal (%) 16 35 0.07
• Polycystic ovarian syndrome (%) 9 6 0.56
a Mean ± standard deviation. FSH = follicle stimulating hormone.

Table 2. Sperm parameters (mean ± SEM) of patients of Groups 1 (n = 223) and 2 (n = 90).

Group 1 Group 2 p-Value

Concentration (mil/mL) 41.1 ± 2.8 28.8 ± 3.4 0.005 *
Total motility (%) 45.4 ± 1.6 37.2 ± 2.3 0.035 *
Progressive motility (%) 25.3 ± 1.2 18.8 ± 1.0 0.024 *
Normal morphology (%) 13.4 ± 0.8 11 ± 1.1 0.07
* p < 0.05.

The comparison between the first and second semen samples in patients in Group 2
showed a significantly higher percentage of spermatozoa with total (p < 0.001) and progres-
sive (p < 0.001) motility in the second ejaculate than in the first (Table 3). The percentage
of morphologically normal spermatozoa was also higher in the second ejaculate, but the
difference did not, albeit only by a small margin, reach statistical significance compared to
Group 1 (p = 0.054). Therefore, spermatozoa from the second ejaculate were used for ICSI
for all 90 patients in Group 2.

Table 3. Sperm parameters (mean ± SEM) of the two ejaculates of the male partners in Group 2
couples (n = 90).

First Ejaculate Second Ejaculate p-Value

Concentration (mil/mL) 28.8 ± 3.4 31.6 ± 3.9 0.07
Total motility (%) 37.2 ± 2.3 47.7 ± 2.4 <0.0001 *
Progressive motility (%) 18.8 ± 1.7 25.9 ± 1.7 <0.0001 *
Normal morphology (%) 11 ± 1.1 11.2 ± 1.1 0.054
* p < 0.05.

The transfer was performed in all patients enrolled in this study. For patients of
Group 1, 586 embryos were transferred (2.7 ± 1.14), whereas 229 embryos (2.6 ± 1.2) were
transferred for patients of Group 2. There was no statistically significant difference in the
number of embryos transferred between the two groups (p = 0.5).
Fertilization, implantation, clinical pregnancy, live birth delivery, and miscarriage rates
are reported in Table 4. The clinical pregnancy rate of couples in Group 2 was significantly
 Group 1, 586 embryos were transferred (2.7 ± 1.14), whereas 229 embryos (2.6 ± 1.2) were
transferred for patients of Group 2. There was no statistically significant difference in the
number of embryos transferred between the two groups (p = 0.5).
Fertilization, implantation, clinical pregnancy, live birth delivery, and miscarriage
J. Clin. Med. 2021, 10, 4399 6 of 11
rates are reported in Table 4. The clinical pregnancy rate of couples in Group 2 was sig-
nificantly higher than that of Group 1 (p = 0.001). Interestingly, the former had also a sig-
nificantly higher percentage of embryos of grade I than couples in Group 1 (p = 0.003)
higher than
(Figure thatstatistically
1). No of Group 1 (p = 0.001). Interestingly,
significant the former
difference was found had also a significantly
in fertilization, implantation,
higher percentage of embryos of grade I than couples in Group
live birth delivery, and miscarriage rates between the two groups. 1 (p = 0.003) (Figure 1).
No statistically significant difference was found in fertilization, implantation, live birth
delivery,
Table and outcomes
4. ICSI miscarriage rates
of the between
couples the Group
of the two groups.
1 (n = 223) and 2 (n = 90).

Group
Table 4. ICSI outcomes of the couples of the Group 1 = 223) and 2 Group
1 (n (n = 90). 2 p-Value
Fertilization rate (%) 90%
Group 1 Group 292% p-Value 0.3
Implantation rate (%)
Fertilization rate (%)
15%
90% 92%
20% 0.3
0.09
Clinical pregnancy
Implantation rate (%)
rate (%) 20%
15% 20%
31% 0.09
0.001 *
Live birth delivery rate (%)
Clinical pregnancy rate (%)
18%
20% 31%
22% 0.001 *
0.4
Miscarriage rate (%) 16% 15% 0.9
Live birth delivery rate (%) 18% 22% 0.4
Birth weight (g) a 3154.5 ± 510.3 3007.5 ± 466.3 <0.05 *
Miscarriage rate (%) 16% 15% 0.9
Type of birth a
Birth weight (g) 3154.5 ± 510.3 3007.5 ± 466.3 <0.05 *
 Natural birth (n) 16 12
Type of birth
 Scheduled cesarean (n) 24 8
• Natural birth (n) 16 12
 • Unplanned cesarean
Scheduled cesarean (n) (n) 24
1 8
0
 • Twin Birth (%) (n)
Unplanned cesarean 1 29 0 35
• Twin Birth (%) 29 35
 Multiple Birth (%) 0 0
• Multiple Birth (%) 0 0
a
a
Mean ± standard deviation; * p < 0.05.
Mean ± standard deviation; * p < 0.05.

Figure 1. Embryo quality of couples in Groups 1 (n = 223) and 2 (n = 90).

Figure 1. Embryo quality of couples in Groups 1 (n = 223) and 2 (n = 90).

4. Discussion
4. Discussion
In recent decades, many authors have supported the beneficial effects of a very short
In recent decades, many authors have supported the beneficial effects of a very short
abstinence period on sperm parameters. We have previously reported that a second
abstinence
consecutiveperiod oncollected
ejaculate sperm parameters. We have
within 1 h resulted previously
in an reported
improvement thatmotility
of sperm a second con-
secutive ejaculate collected within 1 h resulted in an improvement of sperm
and morphology in normozoospermic male partners of infertile couples. The improvement motility and
morphology
was more marked in normozoospermic
in patients with OATmale
[16]. partners of infertile couples. The improvement
was more
On thismarked in patients
basis and with
according OAT
to the [16]. literature [4,18–25], we conducted this
recent
study to evaluate the effects of a very short period of abstinence (1 h) on ICSI outcomes
in infertile patients with severe OA. This subset of patients (Group 2) had significant
improvement of total (p < 0.001) and progressive motility (p < 0.001) in the second ejaculate
compared with those of the first (Table 3). Based on the increase of sperm progressive
J. Clin. Med. 2021, 10, 4399 7 of 11

motility, we chose to utilize spermatozoa of the second ejaculate in all 90 men of Group 2.
These results confirm our previous findings [16]. Most of the studies conducted on OAT
patients reported an increase of progressive motility in ejaculate collected after a very
short period of abstinence [18–25]; some of these studies also found an increase in normal
morphology [20,24,25] and sperm concentration [17,18,21,24,25].
Then, we compared ICSI outcomes of couples in Group 1 whose male partners col-
lected their seminal sample after a conventional period of abstinence (2–7 days) and couples
in Group 2 whose male partners were requested to provide a second ejaculate collected
after 1 h from the first. We found a statistically significant higher clinical pregnancy rate of
couples in Group 2 compared to Group 1 (p = 0.001), despite the worse seminal parame-
ters of the male partners. No statistically significant difference was found in fertilization,
implantation, live birth delivery, and miscarriage rates between the two groups. Thus, a
second semen sample collected after 1 h of sexual abstinence allowed the same results in
terms of fertilization, implantation, live birth delivery, and miscarriage rates of normo-
zoospermic patients or with mild OA, but increased clinical pregnancy rate, despite their
severe OA. A very small number of studies have focused on the effects of a very short
period of abstinence on ART outcomes. Barash et al. [31] conducted a study of 39 infertile
patients with OAT scheduled for in-vitro fertilization and embryo transfer (IVF-ET). They
reported increased fertilization (18.3 ± 25.8 vs. 29.6 ± 29.7; p < 0.05), cleavage (0.6 ± 1
vs. 1.9 ± 1.7; p < 0.05), and pregnancy (5/14 vs. 0/2; p < 0.05) rates when oocytes were
incubated with spermatozoa of an ejaculate collected 2 h after the first one, compared with
oocytes exposed to spermatozoa from ejaculate collected after 3 days of abstinence [31].
Sugyam et al. reported that the fertilization rate with a second ejaculate collected within
60 min was significantly higher than that from the first (53.3 vs. 28.9%, p < 0.05), whereas
cleavage rate and embryo quality were comparable [4]. More recently, Ciotti et al. found
that using seminal samples collected after an abstinence period of 2 h in severe OAT pa-
tients allowed the obtaining of similar rates of fertilization (p = 0.59), pregnancy (p = 0.34),
implantation (p = 0.23), and miscarriages (p = 0.60) to those achieved in normozoospermic
patients [25].
Interestingly, we also found a significantly higher percentage of embryos of grade I
in Group 2 compared with Group 1 (Figure 1). In a recent study, Scarselli et al. described
higher blastocyst euploid formation rates using ejaculates collected after an abstinence
length of one hour than by using ejaculates collected after the conventional length of
abstinence of 2–5 days (43.6% vs. 27.5%, p = 0.043) [15]. The authors hypothesized
that this finding may be ascribed to the higher percentage of spermatozoa with mature
chromatin in the second ejaculate. Specifically, they used the aniline blue (AB) test to
detect protamination and, in turn, chromatin integrity [15]. The chromatin compactness
is an important bio-functional sperm parameter for the evaluation of DNA maturity and
sperm damage. Staining with AB provides a specific positive reaction for lysine, the
main amino acid present in histones, whereas it is little represented in protamines [32].
The histone to protamine transition is a fundamental step in spermatogenesis to facilitate
chromatin compaction in the sperm head and, in turn, to prevent the paternal genome from
mutagenesis and damage [2]. In fact, DNA complexed with protamine is very stable and
resistant to enzymatic digestion. Defects in the histone replacement process may lead to
DNA damage and, consequently, to male infertility [2]. Scarselli et al. found a statistically
significantly higher percentage of pale-colored spermatozoa (a sign of mature chromatin)
in the second ejaculate compared to the first (54.0 ± 1.8% vs. 29.6 ± 2.3%, p < 0.005). A
lower percentage of spermatozoa with mature chromatin can alter zygote development
after ICSI and impair embryo quality [33].
Sperm DNA fragmentation (fDNA) is another bio-functional parameter increasingly
used [34] that may be associated with defective maturation, abortive apoptosis, and oxida-
tive stress (OS) [35]. A higher number of spermatozoa with fDNA in couples undergoing
ART is associated with worse outcomes [36]. Our previous study reported a decrease of
fDNA in both men with normozoospermia and patients with OAT after an abstinence
J. Clin. Med. 2021, 10, 4399 8 of 11

period of 1 h. A lower percentage of spermatozoa with fDNA was reported after recurrent
ejaculations every 24 h for four days with final abstinence of 12 h before ICSI [37].
Shen et al. found a decreased percentage of spermatozoa with fDNA, an increased total
antioxidant capacity (TAC), and higher sperm mitochondrial membrane potential (MMP)
in ejaculates from short (1–3 h) compared with long (3–7 days) length of abstinence [23].
MMP is a marker of sperm mitochondrial function that strictly correlates with sperm
motility [38].
In agreement with these findings, quantitative proteomic analysis has shown that
proteins overexpressed in spermatozoa of the second ejaculate are involved in specific
functions, such as sperm motility, capacitation, and antioxidant defense [15]. Human sper-
matozoa are rich in polyunsaturated fatty acids and, therefore, are very sensitive to damage
provoked by reactive oxygen species (ROS). It has been widely shown that an imbalance
between oxidative and antioxidant systems in the seminal plasma is detrimental for sperm
function and fertility outcome [39]. A short period of abstinence could reduce the time of
exposure of spermatozoa to the harmful effects of ROS in the cauda epididymis and, in turn,
may result in a “younger” population of spermatozoa [40]. Therefore, the improvement
in the sperm quality observed in the second ejaculate could be explained by a different
epididymal transit time. Interestingly, Johnson and Varner reported that the sperm transit
time through the epididymis was three times longer in patients with oligozoospermia than
in men with normozoospermia [41]. Thus, spermatozoa of patients with severe OA stay
in the genital tract for a prolonged time and, therefore, suffer more deeply the effects of
oxidative stress. This might explain the greater improvement of sperm quality after a very
short period of abstinence in patients with severe OA compared with normozoospermic
men [16]. During the epididymal transit, sperm maturation includes sperm surface modifi-
cations and changes of flagellar beating through which spermatozoa acquire their forward
motility and their fertilization capability [42]. In addition, epigenetic modifications occur
during the epididymal transit [43] and represent a crucial step during spermatogenesis,
sperm maturation, and fertilization process [44]. Shen et al. identified ten kinds of major
protein modifications to evaluate the difference in epigenetic modifications in ejaculates
after 1–3 h compared to 3–7 days [23]. Notably, sperm butyryl-lysine, propionyl-lysine, and
malonyl-lysine modifications were significantly decreased and trimethyl-lysine modifica-
tions increased significantly after lower abstinence length [23]. ART failure of couples with
male partner infertility could relate to epigenetic modifications found in blastocysts [45].
Epigenetic events in OAT patients directly impact embryogenesis and this could explain
the higher miscarriage rates for couples with OAT male partners compared to controls,
despite maternal age-match and a similar number of transferred euploid blastocyst [44].
Furthermore, changes in the seminal plasma composition could also have a role in the
improvement of sperm motility after a very short period of abstinence. A recent study
compared the seminal plasma metabolomics profile in two consecutive ejaculates collected
from normozoospermic men after long (4–7 days) followed by a short (2 h) ejaculation-
abstinence period. The results showed a lower absolute amount of all metabolites in the
second ejaculate. This may be related to the insufficient time available for the secretion
and accumulation of these metabolites by accessory sex glands including the epididymis.
However, the contemporary lower number of spermatozoa in the second ejaculate resulted
in increased absolute amounts of pyruvate and taurine per spermatozoa, together with
an improvement of sperm motility in these samples. Therefore, the authors speculated
that changes in the seminal plasma composition may influence spermatozoa motility and
kinematic parameters [45].

5. Limitations
The results of this study have been obtained in a single center. To corroborate these
findings, larger trials should be designed with prospective and randomized protocols.
Moreover, a limitation of our study is that the selection procedure may have influenced the
possible beneficial effect of shorter abstinence. A more suitable experimental model could
J. Clin. Med. 2021, 10, 4399 9 of 11

be to evaluate ART outcome (fertilization rate, embryo quality, pregnancy rate, and live
birth rate) using spermatozoa from the first and second ejaculate by splitting the oocytes
when they are retrieved in an adequate number.
Furthermore, another limit of our study is that, while full sexual intercourse has
been consistently found able to increase the circulating levels of testosterone in patients
recovering from erectile dysfunction [46,47], we do not have, currently, direct evidence that
this mechanism may play a role in the results found here. For this purpose, a dedicated
protocol is currently ongoing.

6. Conclusions
With the widespread use of ART to treat couple infertility, improved sperm quality
has become essential for better outcomes in ART cycles. In fact, by using better quality
spermatozoa, it should be possible to obtain a greater number of good quality embryos.
In recent years, a growing number of studies have supported beneficial effects on sperm
parameters when semen was collected after a short length of abstinence. Consequently, the
present study shows that a second ejaculate obtained after a very short time from the first
could represent a simple and useful strategy to obtain spermatozoa with better parameters
and to improve the results of ART in patients with abnormal sperm parameters and,
particularly, in those with severe OA. Better ICSI results could be achieved by this simple,
non-invasive, and inexpensive step. Certainly, the live birth rate is a multifactorial index
and other important factors must be considered to obtain the final result, including oocyte
quality and endometrial receptivity. However, the optimization of the abstinence length
together with an adequate diagnostic and therapeutic workup and proper preparation
of infertile couples before ART can contribute to an increase in their ART success rate.
Personalization of ART procedure should be regarded as the gold standard for optimization,
without leaving the solution to the severe male factor in infertility simply to the ICSI
technique. Finally, further studies are needed to investigate the underlying molecular
mechanisms and the role of epigenetic modifications in improving sperm quality after a
very short period of abstinence.

Author Contributions: Conceptualization, C.M. and A.E.C.; methodology, C.M.; formal analysis, F.B.
and S.L.V.; data curation, R.A.C., A.F.; writing—original draft preparation, F.B.; writing—review and
editing, F.B. and A.E.C. and C.M.; visualization, E.A.J.; supervision, A.E.C. and C.M. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki. It was reviewed and approved by the Institutional Review Board at
the Biofertility IVF Centre (Rome, Italy) who indicated that ethical approval was not required for
this study.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Conflicts of Interest: The authors declare no conflict of interest.

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