Effect of sperm DNA fragmentation on embryo development: clinical and biological aspects

Abstract

Objective:
The aim of this study was to investigate the effect of sperm DNA fragmentation on fertilization rate, embryo development (blastulation rate), and pregnancy outcomes for ICSI cycles performed in a cohort of couples using donor eggs and to assess the remaining embryos that were not transferred or frozen for apoptotic markers.

Methods:
Eighty-two women (egg recipients) were included in the study (2016) were included in the study. The recipients' mean age was 41.8±5.1 y/o (36-49), while the egg donors' mean age was 30.8±2.1 y/o (27-33). Even though donor egg cycles with frozen sperm samples are performed regularly in our center, 35 cycles were done using fresh sperm samples. The mean age of the males involved in the procedure was 40.1±5.2 y/o. Fertilization, blastulation, and pregnancy rates were assessed. The patients were divided into two groups, TUNEL <15% and ≥15%. In arrested embryos, ICC was performed to detect cleaved caspase-3, survivin, TUNEL, and DNA. The Student's t-test was used in between-group comparisons. The Mann-Whitney U-test was used to assess homogeneity. Pearson's correlation coefficient was also calculated. p<0.05 was considered statistically significant.

Results:
This study showed that there is a negative correlation (R=-0.5) between DNA fragmentation and blastulation rate. High levels of DNA fragmentation were associated with low blastulation and pregnancy rates (per transfer); however, fertilization rate was not affected. Samples with higher levels of DNA fragmentation were associated with higher levels of DNA fragmentation in blastomeres without activating the apoptotic pathway (9.1% vs. 15.9%) (p<0.05). Blastomeres from samples with high DNA fragmentation activated the apoptotic pathway in higher levels than samples with TUNEL <15% (16.4% vs. 21.9%) (p<0.05).

Conclusion:
Sperm DNA fragmentation was negatively correlated with blastulation and pregnancy rates even in good quality oocytes. High levels of DNA damage promote embryo arrest and induce the activation of the apoptotic pathway.

Full text

1
Received February 02, 2017
Accepted August 05, 2017
Original article
Eect of sperm DNA fragmentation on embryo development: clinical
and biological aspects
Cristian Alvarez Sedó1, Melina Bilinski1, Daniela Lorenzi1, Heydy Uriondo1, Felicitas Noblía1, Valeria Longobucco1,
Estefanía Ventimiglia Lagar1, Florencia Nodar1
1Centro de Estudios en Genética y Reproducción (CEGYR), Buenos Aires, Argentina
ABSTRACT
Objective: The aim of this study was to investigate
the eect of sperm DNA fragmentation on fertilization rate,
embryo development (blastulation rate), and pregnancy
outcomes for ICSI cycles performed in a cohort of couples
using donor eggs and to assess the remaining embryos
that were not transferred or frozen for apoptotic markers.
Methods: Eighty-two women (egg recipients) were
included in the study (2016). The recipients' mean age was
41.8±5.1 y/o (36-49), while the egg donors' mean age
was 30.8±2.1 y/o (27-33). Even though donor egg cycles
with frozen sperm samples are performed regularly in our
center, 35 cycles were done using fresh sperm samples.
The mean age of the males involved in the procedure was
40.1±5.2 y/o. Fertilization, blastulation, and pregnancy
rates were assessed. The patients were divided into two
groups, TUNEL <15% and ≥15%. In arrested embryos,
ICC was performed to detect cleaved caspase-3, survivin,
TUNEL, and DNA. The Student's t-test was used in between-
group comparisons. The Mann-Whitney U-test was used
to assess homogeneity. Pearson's correlation coecient
was also calculated. p<0.05 was considered statistically
signicant.
Results: This study showed that there is a negative
correlation (R=-0.5) between DNA fragmentation and
blastulation rate. High levels of DNA fragmentation were
associated with low blastulation and pregnancy rates (per
transfer); however, fertilization rate was not aected.
Samples with higher levels of DNA fragmentation were
associated with higher levels of DNA fragmentation in
blastomeres without activating the apoptotic pathway
(9.1% vs. 15.9%) (p<0.05). Blastomeres from samples
with high DNA fragmentation activated the apoptotic
pathway in higher levels than samples with TUNEL <15%
(16.4% vs. 21.9%) (p<0.05).
Conclusion: Sperm DNA fragmentation was negatively
correlated with blastulation and pregnancy rates even in
good quality oocytes. High levels of DNA damage promote
embryo arrest and induce the activation of the apoptotic
pathway.
Keywords: blastocyst, DNA fragmentation, blastulation
rate
JBRA Assisted Reproduction 2017;00(0):000-000
doi: 10.5935/1518-0557.20170061
INTRODUCTION
Male factor accounts for 30-40% of all cases of human
infertility. In the past, medical decisions on treating infertile
couples were based mostly on the results of conventional
semen analysis, assessing sperm concentration, motility,
and morphology in one or more semen samples.
In the early days of ART, severe male factor infertility
yielded frustrating results, in the form of poor fertilization
and pregnancy rates. With the preliminary reports on
ICSI (intracytoplasmic sperm injection) in the early
1990s, clinicians and embryologists believed a solution
had been found to all cases of male factor infertility.
However, unsuccessful treatments despite the use of
ICSI have indicated that other factors may be involved,
including sperm DNA fragmentation or sperm morphologic
damage undetected by the standard magnication used
in conventional ICSI. More recently, several number
of techniques designed to improve sperm selection
for conventional ICSI have demonstrated to increase
fertilization rates, enhance embryo quality after successful
fertilization, and optimize pregnancy rates.
Several studies have demonstrated the importance
of the stability of sperm nuclei and its correlation with
successful reproduction in animals and humans, and
the association of sperm nucleus damage with low
fertilization rates, poor embryo implantation, and
increased miscarriage rates (Aitken et al., 1998; Morris
et al., 2002; Bungum et al., 2004; Carrell et al., 2003;
Seli et al., 2004; Virro et al., 2004; Lewis & Aitken, 2005;
Aitken et al., 2009; Meseguer et al., 2008; Zini & Sigman,
2009; De Iuliis et al., 2009; Barratt et al., 2010; Sakkas
& Alvarez, 2010).
DNA damage may occur in the form of single or double
strand breaks, and both types can be analyzed and/
or quantied through dierent methods, including SCD
(Sperm chromatin dispersion), SCSA (Sperm Chromatin
Structure Assay) and TUNEL (terminal deoxynucleotidyl
transferase dUTP nick end labeling) (Evenson et al., 1980;
Gorczyca et al., 1993; Fernández et al., 2005; Chohan et
al., 2006). DNA damage may have a negative impact in IVF-
ICSI results (Evenson et al., 2002; Cordelli et al., 2005;
Sergerie et al., 2005; Greco et al., 2005; Boe-Hansen et
al., 2006; Makhlouf & Niederberger, 2006; Avendaño et
al., 2009a; Avendaño et al., 2009b; Góngora-Rodríguez
& Fontanilla-Ramírez, 2010; Borini et al., 2006; Ni et al.,
2014).
In the rst days of embryo culture, morphological
criteria alone are generally poor predictors of embryo
development and ability to achieve pregnancy (Guerif et
al., 2010; Nel-Themaat & Nagy, 2011). However, embryos
are still categorized and chosen for transfer based on
morphological and developmental scores (Alpha Scientists
in Reproductive Medicine and ESHRE Special Interest
Group of Embryology, 2011).
Higher levels of DNA fragmentation (SDF >30%) in
sperm cells have been associated with lower blastocyst
formation rates (Virro et al., 2004). Nasr-Esfahani et al.
(2005) reported that embryos derived from spermatozoa
with high levels of DNA damage are less likely to reach
later developmental or blastocyst stages. However, in
these studies the blastulation rates between the groups
with SDF >30% and SDF <30% in IVF cycles were not
dierent. The most likely explanation for this is “natural”
selection during IVF. In that sense, it seems that ICSI
results and embryo development (ICSI) are signicantly
aected by sperm quality.
Finally, regarding apoptosis, anomalies in cell death
control have been implicated as a cause or contributing

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JBRA Assist. Reprod. | v.00 | nº0 |
factor in a range of diseases, including cancer, autoimmunity,
and degenerative disorders. Cell death control involves
several proteins that promote or inhibit apoptosis and an
evolutionarily conserved multistep cascade. A number of
proteins, such as Bcl-2, Fas and Bax, aect processes
upstream of the cascade. Survivin, an apoptosis inhibitor,
may prolong cell survival by targeting terminal eector
caspase-3. Located at the end of the cascade, caspase-3
acts as both an initiator and executor of the apoptotic
process. Therefore, survivin and caspase-3 have received
signicant attention in the discussions on apoptosis (Li et
al., 2004).
This study looked into the eect of sperm DNA
fragmentation on fertilization rates, embryo development
(blastulation rate), and pregnancy outcomes of couples
using donor eggs oered ICSI cycles. The remaining
embryos that were not transferred or frozen were also
assessed for apoptotic markers.
MATERIALS AND METHODS
Population
This study included 82 egg recipients submitted to ART
procedures (2016). The recipient's mean age was 41.8±5.1
y/o (36-49) and the mean age of the oocyte donors was
30.8±2.1 y/o (27-33). Even though donor egg cycles
with frozen sperm samples are performed regularly in our
center, 35 cycles were done using fresh sperm samples.
The mean age of the males involved in the procedure was
40.1±5.2 y/o.
Egg donors had to comply with the following
requirements of the egg donation program: antral follicle
count >16, negative serology, psychological and genetic
counseling, and normal karyotype. A clinical geneticist
tested the patients for relevant family history.
Egg donor controlled ovarian stimulation
The donors were placed on a flexible GnRH antagonist
protocol for ovarian stimulation, with daily doses of
225-300 IU of a gonadotropin (Menopur®) (Ferring)
daily. When the leading follicle reached a diameter
of 14 mm, GnRH antagonist Orgalutran (MSD) was
administered daily until the day of Lupron® injection.
Once the leading follicle reached 17-18 mm in diameter
and estradiol levels were >+500 pg/ml, leuprolide
acetate (Lupron®, 2 mg) was administered 36 hours
prior to oocyte retrieval. Then the IVF procedure (ICSI)
was performed.
Endometrial preparation for ET
Seventy-ve embryo transfers (blastocyst) were
performed; patients received oral estradiol Valerate
(Ronfase®) 4 mg daily from day 2 of the menstrual cycle.
On day 10, an ultrasound examination was performed.
After ultrasound conrmation of endometrial thickness
>6mm and absence of ovarian activity, progesterone
(Utrogestan®) 600 mg daily was added for 5 days before
ET and up to 14 weeks afterwards when pregnancy was
conrmed.
The dose of Ronfase was increased to 6 mg daily if
endometrial thickness was less than 6mm. Ultrasound
examination was repeated within 7 days and cycles were
cancelled if the endometrium failed to reach the minimum
thickness.
Source of embryos
All remaining human embryos were donated for
research with the consent of the couples submitted to
ART procedures at CEGyR (Buenos Aires, Argentina). The
Internal Review Board and Ethics Committee of CEGyR
approved the procedures involving human embryos. None
of the presumed embryos donated for this project were
transferred to recipients after ICSI. If they had not been
donated to this research, these embryos would have been
discarded. All embryos were xed immediately 5 days after
oocyte fertilization was conrmed.
Chemicals and antibodies
All chemicals were obtained from Sigma Chemical Co.
(St. Louis, MO, USA), unless stated otherwise. Cleaved
caspase-3 (CC3) and Survivin (Surv) were detected using
anti-full-length human CC3 (rabbit monoclonal, dil: 1:100,
Cell Signaling, USA), and Surv (mouse monoclonal, dil:
1:100, Novus Biologicals, USA). Alexa Fluor secondary
antibodies were obtained from Molecular Probes
(Invitrogen, US). TUNEL (Roche, USA) assays were also
performed to detect DNA fragmentation. Hoechst 33258
(Sigma) was used for DNA staining.
Embryo processing
One hundred and eighty-seven embryos from 82
patients were collected after ICSI and studied. All embryos
were individually processed. The zona pellucida was slightly
dissolved by incubation in acidic Tyrode´s solution (Irvine
Scientic, US), and then the embryos were xed and
processed by immunocytochemistry (ICC) (see below).
Detection of cell damage and apoptosis in
embryos by immunohistochemistry
The human embryos were xed for 45 min in 2%
formaldehyde and washed with PBS + 0.1% Triton X-100
for an additional 45 min (method modied and based on
Messinger and Albertini, 1991). After xation and washing,
the samples were blocked for at least 1 h in PBS + 0.3%
bovine serum albumin (BSA) + 1% fetal calf serum prior
to incubation in humidied chambers with primary and
secondary antibodies overnight at 4°C and for 2 hours
at 37°C, respectively. The embryos were washed several
times with PBS-BSA. After washing, they were incubated
in TUNEL solution for 1 hour at 37°C. Finally, the embryos
were incubated with Hoechst 33258 for DNA detection.
Some images were obtained using an Olympus spectral
confocal microscope, with laser lines at 488-, 568- and
633 -nm wavelengths and then processed using Adobe
Photoshop C5; additional images were captured with an
Olympus BX40 Fluorescence Microscope. Negative controls
were run in the absence of primary antibodies. This assay
allowed us to determine the cytoplasmic activation of CC3
in the blastomeres of each of the embryos (apoptosis);
Surv activation meant that the cell performed a strategy
to stop the cell death; TUNEL (positive) meant that
DNA fragmentation (damage) had occurred, but nuclear
condensation and TUNEL were the nal evidence of cell
death by apoptosis (Figures 1 and 2).
Sperm analysis and DNA fragmentation
Semen analysis was performed accordingly to the
procedure established by the WHO (2010). After motile
sperm isolation, the samples were xed in 2% formaldehyde
in phosphate-buered saline solution (PBS; pH 7.4) for at
least 1 hour. Each sample was placed into one well of a
multiwell (4-mm diameter) Teon-printed slide (Electron
Microscopy Sciences) and allowed to settle. After 2-3
hours, each well was washed with 1X PBS (three times, 5
minutes each); the cells were then permeabilized with cold
methanol. Before incubation with TUNEL solution, each
well was washed again with 1X PBS. For each sample, one
extra well was incubated with DNAse (1 U/mL; Sigma)
for 30 minutes at 37°C as a positive control, and in
another well the TUNEL ''enzyme' 'solution was omitted

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Figure 1. In A, ICC for TUNEL and DNA staining fragmented DNA (1 and 2), non-fragmented DNA (3), and some blastomeres
without DNA (arrows). In B, Blastomeres without DNA damage and Surv (+) (*) or DNA fragmentation and Surv (-) (**).
Figure 2. Embryos with condensed DNA (arrowhead) and TUNEL (+) (arrow). Surv and CC3 show dierent positive
staining.
as a negative control. Then all samples were incubated in
TUNEL solution (Roche,) for 1 hour at 37°C. All samples
were nally washed with 1X PBS (three times, 5 minutes
each), and mounted in Vectashield H-1000 medium
(Vector Laboratories). 500 spermatozoa were counted by
uorescence microscopy. TUNEL staining was evaluated on
a uorescence microscope using a green lter (uorescein
isothiocyanate, 488 nm) (Figure 3).
Statistics
The Student's t-test was used for between-group
comparisons. The Mann-Whitney U-test was used to assess
homogeneity. Pearson's correlation coecient was also
calculated. p<0.05 was considered statistically signicant.
Statistical analyses were carried out on software program
MedCalc 12.5 (Belgium).
RESULTS
Table 1 shows sperm analysis data. Clinical outcomes
(ICSI results) in terms of fertilization, blastulation, and
pregnancy rates are shown in Table 2. The same data is
shown in Table 3 for sperm samples with high (≥15) or low
(<15%) levels of DNA fragmentation. The results revealed
the existence of a negative correlation (R=-0.5) between
DNA fragmentation and blastulation rates (Fig. 4), and
an association between high levels of DNA fragmentation
and low blastulation and pregnancy rates (per transfer);
however, fertilization rate was not aected.
On the other hand, when the remaining embryos
were analyzed, samples with higher levels of DNA
fragmentation were observed to induce higher levels of
DNA fragmentation in blastomeres without activating the

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Figure 3. TUNEL assay to assess sperm DNA fragmentation. Positive cells (*), negative cells (**).
Table 1. Results of sperm analysis performed according to the WHO guidelines (2010).
Sperm parameters (%) N=82
Volume (mL) 2.7±0.8
Sperm Concentration (mill/mL) 68.5±43.1
Sperm Morphology using Kruger’s strict criteria 6.5±3.8
Sperm Progressive Motility (a+b) 50.8±16.9
PMN 0.96±1.64
DNA fragmentation (TUNEL) 13.5±11.1
Table 2. Clinical outcomes for the studied population.
Clinical outcomes X±SD Min-Max
Female age 41.8±5.2 36-49
Donor age 30.8±2.1 27-33
Male age 40.1±5.2 33-60
Nº of oocytes assigned (MII) 8.7±2.1 4-14
Fertilization rate (%) 75.4±18.9 20-100
Blastulation rate (%) 51.8±26.3 0-100
Pregnancy rate (%)/transfer 50/75
67%

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Table 3. Clinical outcomes - Comparison between patients with TUNEL <15% vs. ≥15%.
DNA fragmentation
<15%
DNA fragmentation
≥15 % p
N54 28
Nº of oocytes assigned (MII) 8.9±2.3 8.5±1.8 0.75
Strict morphology (%) 6.5±3.1 5.9±4.2 0.40
DNA fragmentation (%) 7.6±3.8 24.9±11.5 0.001
Fertilization rate (%) 76.1±19.4 73.8±18.2 0.62
Blastulation rate (%) 59.2±22 37.5±28 0.003
Pregnancy rate (%)/transfer 38/51
74.5%
12/24
50% 0.06
Figure 4. Correlation analysis between DNA fragmentation and Blastulation rate (R=-0.5).
apoptosis pathway (9.1% vs. 15.9%) (p<0.05). Likewise,
blastomeres from samples with high DNA fragmentation
activated the apoptotic pathway in higher levels than
TUNEL <15% (16.4% vs. 21.9%) (p<0.05). The nal
expression of cell death, TUNEL (+), Surv (+), CC3 (+)
and DNA condensation, was increased in embryos coming
from sperm samples with high levels of DNA damage (3.0%
vs. 8.8%) (p<0.05). Finally, when TUNEL and CC3 were
positive and Surv was negative, there were no statistical
dierences between groups (Table 4).
DISCUSSION
Semen quality is usually expressed in terms of sperm
concentration, motility, and morphology (WHO, 2010).
Our group previously reported that these parameters,
specically morphology and motility, were closely related
to DNA alterations (Uriondo et al., 2011, Alvarez Sedó et
al., 2012). Though sperm DNA damage is not considered in
regular sperm analysis, the literature suggests that sperm
DNA damage produces relevant impact on male fertility.
Various theories have been put forward to explain sperm
DNA damage (apoptosis, chromatin remodeling, oxidative
damage) (Sakkas & Alvarez, 2010).
During the course of natural selection, eective
conception can only occur following the fertilization of
an oocyte by sperm with intact DNA. However, assisted
reproductive technologies have increased the possibility
of anomalous spermatozoa being used to fertilize oocytes
(Tavukçuoğlu et al., 2012). Sperm DNA fragmentation
is an important parameter of sperm quality that can be

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Table 4. Blastomere analysis - Comparison between patients with TUNEL <15% vs. ≥15%.
DNA fragmentation
<15%
DNA fragmentation
≥15 % p
Nº embryos 112 75
Nº of Blastomeres assessed 860 432
Blastomeres without DNA damage 458 (53.2%) 137 (31.7) 0.0001
Blastomeres with DNA damage
CC3 (-) Surv (-) 78 (9.1%) 69 (15.9%) 0.0004
Blastomeres with DNA damage
CC3 (+) Surv (-) 132 (15.3%) 76 (17.6%) 0.29
Blastomeres without DNA damage
CC3 (+) Surv (+) 25 (2.9%) 17 (3.9%) 0.45
Blastomeres with DNA damage
CC3 (+) Surv (+) 141 (16.4%) 95 (21.9%) 0.007
Blastomeres with DNA damage
CC3 (+) Surv (+) Nuclear condensation 26 (3.0%) 38 (8.8%) 0.0001
used to assess sperm nuclear integrity, which itself plays
an important role in fertilization and embryo development
(Bungum et al., 2007). The role of male factor infertility
on embryo development has gained attention since the
introduction of ICSI as a treatment option for patients with
very poor sperm characteristics. Our study demonstrated
that blastulation rates were associated with high levels of
DNA damage, suggesting a very early onset of paternal
eects on embryo development.
In the present study, sperm DNA damage was assessed
by TUNEL assay, performed on motile sperm prepared
with a swim-up procedure and used for ICSI. Our group
previously demonstrated that the predictive ability of
the sperm DNA integrity test, performed on raw sample,
diminished when spermatozoa were prepared using
techniques such as Swim-up or centrifugation density
gradient (Rougier et al., 2013, Alvarez Sedó et al., 2013).
However, in our population, several samples had high levels
of DNA fragmentation even after motile sperm isolation.
This preliminary data found no relationship between
sperm DNA damage and fertilization rates in ICSI
(p=0.62), but the blastulation rate was clearly diminished
when DNA damage was high (p<0.05). On the other
hand, patients with low DNA fragmentation (<15%) had
a clear tendency to attaining higher pregnancy rates
(p=0.06). These results may be accounted for by the fact
that high DNA fragmentation probably does not impede
fertilization, but prevents blastulation and/or successful
embryo development (Ahmadi & Ng, 1999). However, this
issue was mostly observed in ICSI patients; when IVF was
performed, these dierences were not evinced, probably
due to "natural" selection of sperm by the oocyte (Borini et
al., 2006). The amount of sperm DNA damage was related
to embryo development to the blastocyst stage, a time
when the embryonic genome is activated, transcriptional
activity has begun, and the paternal genome plays a
signicant role in embryo function toward implantation
(Seli et al., 2004).
This is the rst report that used an egg donation
model to assess the impact of DNA damage over clinical
and biological outcomes. Our biological results showed
that sperm DNA damage might promote blastomere DNA
fragmentation without the activation of the apoptotic
machinery, probably due the injection of sperm with
slight DNA damage that was able to advance during the
embryonic development. However, other mechanisms
might be at play in embryo arrest. On the other hand, most
of the blastomeres showed a complete apoptotic pattern
(TUNEL (+), CC3 (+) and Surv (+)), revealing that good
quality oocytes respond adequately to the induction of
apoptosis. However, this was observed largely in embryos
that came from samples with high levels of DNA damage,
conrming that sperm damage can cause further arrest in
embryonic development.
In conclusion, our data indicated that sperm DNA
fragmentation signicantly aected embryo blastulation and
implantation in ICSI patients who received donated eggs.
More specically, this study showed, for the rst time, that
sperm DNA fragmentation might compromise the progression
of embryo development, resulting in arrested embryos. This
study also underlined the better predictive value of DNA
fragmentation analysis versus traditional sperm parameter
evaluation in the assessment of ART outcomes. For this
reason, sperm DNA fragmentation should be considered
during the assessment of semen quality.
CONCLUSIONS
Sperm DNA fragmentation had a negative correlation
with blastulation and pregnancy rates even with good
quality oocytes. High DNA damage levels promoted
embryo arrest and induced the activation of the apoptotic
machinery.
CONFLICT OF INTERESTS
The authors had no conicts of interest to declare.
Corresponding author:
Cristian Alvarez Sedó
Centro de Estudios en Genética y Reproducción (CEGYR)
Buenos Aires, Argentina
E-mail address: calvarez@cegyr.com
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