Sperm DNA fragmentation induced by

cryopreservation: new insights and

effect of a natural extract from
Opuntia cus-indica
Mehrdad Meamar, Ph.D.,
a
Nassira Zribi, Ph.D.,
a
Marta Cambi,
a
Lara Tamburrino, Ph.D.,
a
Sara Marchiani, Ph.D.,
a
Erminio Filimberti, Ph.D.,
a
Maria Grazia Fino,
a
Annibale Biggeri, M.D.,
b
Yves Menezo, Ph.D.,
c
Gianni Forti, M.D.,
a
Elisabetta Baldi, Ph.D.,
a
and Monica Muratori, Ph.D.
a
a
Department of Clinical Physiopathology and
b
Department of Statistics, Center of Excellence DeNothe, University of
Florence, Florence, Italy; and
c
UNILABS, Clinique du Cotentin, Paris, France
Objective: To analyze the effect of cryopreservation on spermDNAfragmentation (SDF) in two cytometric spermpopulations, PI
brighter
and PI
dimmer
, and to test the effects of Opuntia cus-indica (OFI) extracts, which contain antioxidants and avanoids, and of resveratrol
on cryopreservation of human semen.
Design: In vitro prospective study.
Setting: Institutional study.
Patient(s): Twenty-one normozoospermic men undergoing semen analysis for couple infertility.
Intervention(s): Cryopreservation using the routine method in the presence of OFI extracts or resveratrol.
Main Outcome Measure(s): Measurment of SDF by TUNEL/PI ow cytometric method to evaluate sperm motility (by automated
motion analysis, CASA system) and viability (by eosin/nigrosin staining) in the two populations of sperm PI
br
and PI
dim
.
Result(s): Cryopreservation induced an increase of SDF only in the PI
br
sperm population. The increase was negatively dependent on
the basal values of SDF in the same population. Addition of OFI extracts and resveratrol to the cryopreservation medium slightly but
statistically signicantly reduced SDF in the PI
br
population without affecting the deleterious effect of cryopreservation on sperm
motion parameters or viability.
Conclusion(s): The increase of SDF in the PI
br
population, which is unrelated to semen quality, suggests that caution must be taken in
using cryopreserved semen, as morphologically normal and motile sperm may be damaged. The addition of substances with multifunc-
tional properties such as OFI extracts to cryopreservation medium is only slightly effective in preventing the dramatic effects on SDF.
(Fertil Steril

2012;-:--. 2012 by American Society for Reproductive Medicine.)

Key Words: Opuntia cus-indica, resveratrol, semen cryopreservation, sperm DNA fragmentation
S
perm cyropreservation may rep-
resent, for some men, the only
option to preserve their fertility
potential. Indeed, sperm cryopreserva-
tion is now widely used not only in
cases of malignancies or other condi-
tions requiring therapies that may be
harmful for testis function, but also in
cases of azoospermia or severe oligo-
zoospermia, as even a few sperm
retrieved from the testis or present in
semen can be cryopreserved for subse-
quent assisted reproduction techniques
(ART). In particular, as men with (se-
vere) oligozoospermia are considered
at risk of a further decrease of semen
quality, semen cryopreservation is
often recommended.
The deleterious effects of cryopres-
ervation on sperm function are well
known, with dramatic effects on viabil-
ity, motility, and chromatin integrity
(13). It is believed that most of these
deleterious effects are related to
reactive oxygen species (ROS)
generated during the procedure, and
several studies have reported the
generation of ROS during the
cryopreservation of sperm (46).
Spermatozoa are extremely sensitive
Received January 30, 2012; revised and accepted May 2, 2012.
M.M. has nothing to disclose. N.Z. has nothing to disclose. M.C. has nothing to disclose. L.T. has noth-
ing to disclose. S.M. has nothing to disclose. E.F. has nothing to disclose. M.G.F. has nothing to
disclose. A.B. has nothing to disclose. Y.M. has nothing to disclose. G.F. has nothing to disclose.
E.B. has nothing to disclose. M.M. has nothing to disclose.
Supported by Regione Toscana (to G.F.) and the Ministry of Education and Scientic Research, PRIN
Project (to E.B.).
Present address for M.M.: Yasouj University, Yasouj, Iran.
Reprint requests: Elisabetta Baldi, Ph.D., Department of Clinical Physiopathology, Section of Sexual
Medicine and Andrology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy
(E-mail: elisabetta.baldi@uni.it).
Fertility and Sterility Vol. -, No. -, -2012 0015-0282/$36.00
Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc.
doi:10.1016/j.fertnstert.2012.05.001
VOL. -NO. -/ -2012 1
ORIGINAL ARTICLE: ANDROLOGY
to ROS-induced damage; the elevated content of polyunsatu-
rated fatty acids in their membrane makes themsusceptible to
lipid peroxidation (7). Besides inducing alterations in the
motility and viability of sperm, ROS may induce DNA dam-
age, including sperm DNA fragmentation (SDF) and base
oxidation (7). In particular, the increase of DNA damage after
cryopreservation procedure represents a problem in cancer
patients; these men may have increased SDF even before
chemotherapy (8, 9), and the deleterious effects of the
cryopreservation procedure may increase it.
The increase of SDF after cryopreservation and thawing is
particularly relevant for the fertility status of these men, as
there is good evidence that a sperm with fragmented DNA
can be motile, apparently morphologically normal (10), and
can fertilize an oocyte, at least in animal models (11), thus
transmitting the genetic anomaly to the oocyte and the
embryo. In particular, we have recently demonstrated the
existence of two sperm populations characterized by different
levels of SDF as well as by differing relationships with
semen quality. These populations were named PI
brighter
(PI
br
)
and PI
dimmer
(PI
dim
), based on their avidity for the nuclear
probe propidium iodide (PI). The PI
dim
population, entirely
formed of dead (12) and DNA-fragmented spermatozoa, neg-
atively correlates with semen quality. The PI
br
population is
only partially fragmented, and the percentage of fragmented
sperm is independent of semen quality (13). The fragmented
PI
br
sperm may be motile, may show an apparently normal
morphology, may have a greater possibility than fragmented
PI
dim
sperm to participate in the process of fertilization,
especially in ART. Thus, the evaluation of the impact of cryo-
preservation procedures on the PI
br
population appears to be
important for future ART applications.
Despite the wide use of sperm cryopreservation and the
knowledge of its deleterious effects on sperm function, cryo-
preservation techniques have not improved in past few years.
Because ROS are believed to be involved in the decrease of
sperm quality during cryopreservation, attempts to reduce
the effects of oxidative stress during cryopreservation have
mainly focused on adding antioxidants to extension media
(1418) or using different cryoprotectants or techniques (19,
20). So far, few benecial effects on sperm motility,
viability, or DNA fragmentation have been reported. In fact,
standard cryopreservation procedures as suggested in the
latest edition of the World Health Organization (WHO)
manual for examination and processing of human semen
do not include the addition of antioxidants (21).
Prickly pear cactus, or Opuntia cus-indica (OPI), has
recentlyreceived a growinginterest inthe scientic community
because of its multifunctional properties (2224). Chemical
analysis has revealed that OFI extracts contain ascorbic acid,
polyphenols, carotenoids, taurine, and several types of
avanoids, in particular quercetin (22, 24). Furthermore, OFI
extracts have high free-radical scavenging and antioxidant ac-
tivity in vitro (22, 24). Recently, a study of an infertile couple
who had had several previous unsuccessful attempts in ART
reported an increase of sperm DNA condensation in the male
partner after he had been treated with an OFI extract (25).
We used terminal deoxynucleotidyl transferase dUTP
nick end labeling/propidium iodide (TUNEL/PI) to evaluate
DNA fragmentation in the PI
br
and PI
dim
sperm populations
after cryopreservation (13, 26). We then evaluated the SDF,
motility parameters, and viability of cryopreserved human
spermatozoa after supplementing the cryopreservation
medium with OFI extracts.
MATERIALS AND METHODS
Chemicals
The OFI extract was a generous gift of Prof. Marc Cohen (Pro-
crelys, Recherche et Traitement en Infertilite/AMP Natecia,
Lyon, France). The OFI extracts were prepared by crushing
the gs and drying the obtained grains at 40

C in the total ab-

sence of oxygen. The drying process of OFI extracts is a Nurilia
patent. At the end, the product is crushed into powder. To con-
trol the nal quality of the product, the aldehydes and qui-
none (oxidation products of alcohol and phenols) are
evaluated by liquid chromatography, according European
guidelines (CE no. 852/2004; CE no. 853/2004, and CE no.
2073/2005).
Test yolk cryopreservation medium was purchased from
Irvine Scientic. Human tubal uid (HTF) was purchased
from Celbio. The in situ cell death detection kit, uorescein,
was purchased from Roche Molecular Biochemicals. Propi-
dium iodide was obtained fromCalbiochem. The SpermVital-
stain was from Nidacon, and resveratrol and all the other
reagents were obtained from Sigma Aldrich.
Sample Collection
Semen samples were collected according to WHO criteria (27)
from a total of 21 patients, all normozoospermic by WHO cri-
teria (27), who were undergoing routine semen analysis for
couple infertility in the andrology laboratory of the Univer-
sity of Florence. Approval was obtained from the Hospital
Committee for Investigations in Humans (Prot. N. 2010/
0033789), and informed consent was obtained from the
men to use their remaining semen after routine semen
analysis.
Semen Analysis
Semen analysis was performed according to WHO guidelines
(27). Spermmorphology and motility were assessed by optical
microscopy, according to WHO criteria (27). Sperm viability
was evaluated by using Sperm Vitalstain according to the
manufacturer's instructions. The average sperm concentra-
tion was 96.3 87.0 10
6
/mL, the forward motility was
52.7 14.01, and the sperm normal morphology
6.42 5.66 (all mean standard deviation [SD], n 21).
Evaluation of Motion Parameters by CASA
We used computer-assisted semen analysis (CASA, Hamilton
Thorn Research) in fresh samples and after thawing to evalu-
ate sperm motility and hyperactivation parameters such as
average path velocity (VAP), curvilinear velocity (VCL),
straight-line velocity (VSL), beat-cross frequency (BCF), line-
arity (LIN VSL/VCL), and straightness (STR VSL/VAP).
The settings used during CASA procedure were: analysis
2 VOL. -NO. -/ -2012
ORIGINAL ARTICLE: ANDROLOGY
duration of 1 second (30 frames); minimum contrast: 80;
minimum size: 3; low-size and high-size gates: 0.7 and 2.6,
respectively; low intensity and 165 high-intensity gates:
0.34 and 1.40, respectively (28). A minimum of 100 cells
and four elds was analyzed for each aliquot. All analyses
were performed at 37

C.
Cryopresevation and Thawing of Semen Samples
After initial evaluations, each semen sample (n 15) was
divided into four aliquots. One aliquot (10 million sperm)
was used to determine the SDF infreshsamples. The remaining
three aliquots were used for cryopreservation in test yolk
buffer (TYB) with or without OFI extract (5 mg/mL nal
concentration, chosen on the basis of the oral daily male treat-
ment of 200 mg and the circulating levels) and/or resveratrol.
Resveratrol has been previously shown to reduce DNA
damage when added to cryopreservation medium at 10 mM
concentration (16), but it has been reported recently that
resveratrol is highly toxic for human spermatozoa at concen-
trations above 15 mM (29). To choose the correct concentra-
tion of resveratrol, preliminary studies were conducted that
added the antioxidant to freezing media at concentrations
of 10, 100, and 1,000 mM and evaluated the effect on DNA
fragmentation, motion parameters, and viability of spermato-
zoa after 3 days of storage. When added to cryopreservation
medium, resveratrol induced a slight, albeit signicant,
decrease of postthawing SDF levels with respect to control
at concentrations of 10 mM (mean SD: 76.2% 0.6 vs.
82.06% 3.29 control, P.03, n 3) and 100 mM
(mean SD: 79.5% 2.6 vs. 82.06% 3.29 control,
P.03, n 3) without statistically signicantly affecting
the motility or viability (not shown). Conversely, after thaw-
ing, sperm viability was statistically signicantly reduced by
1 mM resveratrol (mean SD: 18.3% 5.1 vs. 34% 5.3
control, P.01, n 3), conrming that the antioxidant
may be toxic for sperm at high concentrations (29). Based
on these results, in the next experiments, resveratrol was
used at the 15 mM concentration.
Resveratrol stock solution was made in dimethyl sulfox-
ide (DMSO), and the nal concentration of the solvent in TYB
was 0.1%. This concentration of DMSO did not demonstrate
toxic effects on sperm motility or viability of spermatozoa
(not shown). The OFI extract was prepared by grinding with
a pestle to obtain a ner powder, which was directly dissolved
in TYB after vortexing for some minutes followed by ltering
through gauze to eliminate any eventual cellulose residues
that may have been present in the powder.
Spermwere frozen in liquid nitrogen in a Taylor-Wharton
34HC tank (Taylor-Wharton) by a manually controlled freez-
ing procedure. Briey, samples were diluted 1:1 (vol:vol) by
dropwise addition of TYB (containing 0.1% DMSO as control)
with or without OFI extracts or resveratrol. After equilibration
at roomtemperature for 5 to 10 minutes, spermwere loaded in
500 mL straws. The straws were frozen via 8 minutes' exposure
to liquid nitrogen vapors and a nal plunge into liquid nitro-
gen, according to the Taylor-Wharton procedure. Thawing
was performed by transferring the straw at 37

C for 15
minutes. In another set of experiments, six semen samples
were incubated for 1 hour at 37

C with OFI extract (5 mg/

mL) or resveratrol (15 mM) before cryopreservation.
Evaluation of Sperm DNA Fragmentation (SDF)
We evaluated SDF by use of the TUNEL/PI assay recently set
up in our laboratory (26). Briey, 10 million spermatozoa
were xed in 4% paraformaldehyde and immediately pro-
cessed for TUNEL labeling. Sperm were centrifuged at
500 g for 10 minutes and washed twice with 200 mL of
phosphate-buffered saline (PBS) with 1% bovine serum albu-
min (BSA). Then the spermatozoa were permeabilized with
0.1% Triton X-100 in 100 mL of 0.1% sodium citrate for 2
minutes in ice. After washing two times, the labeling reaction
was performed by incubating sperm in 50 mL of labeling
solution (supplied with the In Situ Cell Death Detection Kit,
uorescein) containing the TdT enzyme for 1 hour at 37

C
in the dark. Finally, samples were washed twice, resuspended
in 500 mL of PBS, stained with 10 mL of PI (30 mg/mL in PBS),
and incubated in the dark for 10 minutes at roomtemperature.
For each test sample, a negative control (omitting TdT) and
a sample for uorescence compensation (labeled only with
TUNEL) were prepared. For each sample, 10,000 events were
recorded within the characteristic ame-shaped region in
the forward-light scatter/side-light scatter (FSC/SSC) dot
plot, which excludes debris and large cells (30, 31).
We determined SDF within the nucleated events (i.e., the
events labeled with PI) of the characteristic FSC/SSC region of
sperm(13). Green uorescence (of nucleotide conjugated with
uorescein) and red uorescence (of PI) were revealed,
respectively, by the FL-1 (515555 nm wavelength band)
and the FL-2 (563607 nm wavelength band) detectors of
a FACScan ow cytometer (Becton Dickinson) equipped
with a 15-mW argon-ion laser for excitation. We calculated
the percentage of DNA fragmented sperm within the
PI-positive events of the R1 region (13).
Statistical Analysis
Statistical analysis was performed using the program Origin,
version 6.1 (OriginLab). To identify differences in the mean,
a paired t-test was employed. P<.05 was considered statisti-
cally signicant. Relationships were identied by use of the
Pearson coefcient of correlation. To exclude regression to
the mean, we applied the Oldham test (32) and a linear mixed
effect model (33).
RESULTS
Effect of Cryopreservation on Sperm DNA
Fragmentation (SDF) in Presence or Absence of OFI
Extracts and Resveratrol
Cryopreservation induced an increase of DNA fragmentation
in the total and in the PI
br
sperm population, whereas no sta-
tistically signicant variations were observed in the PI
dim
population (Table 1). Figure 1A (left panel) shows variations
in the individual semen samples in the PI
br
spermpopulations.
As can be observed, most samples showa dramatic increase of
SDF in the PI
br
sperm. On average, the percentage increase of
PI
br
SDF following cryopreservation is 27.4. Collectively,
VOL. -NO. -/ -2012 3
Fertility and Sterility
these data demonstrate that the increase of SDF during
cryopreservation occurs in the PI
br
sperm population. A
highly statistically signicant negative correlation was
found between prefreeze and postthaw DNA fragmentation
in the PI
br
sperm populations (Pearson correlation coefcient
0.479, P>.03; Fig. 1B).
To exclude that the relationship was purely due to the re-
gression to the mean effect, we analyzed the data via Old-
ham's method (32). According to this test (P.04;
correlation between difference and mean values: 0.385;
90% condence limits: 0.018; 0.660), there is evidence of
change as a function of the initial value. Similar results
were obtained by linear-mixed effect models (33), where we
found evidence of a negative association of change with base-
line values. Overall, these results indicate that patients with
high SDF values in the PI
br
population of fresh samples may
experience almost no variation (or even a decrease, as we
found in 2 out of 21 samples) in their postthaw SDF levels.
Conversely, if the SDF levels in the fresh samples are low,
there is a high probability of a dramatic increase in the
TABLE 1
SpermDNA fragmentation in fresh samples (n [21) and after cryopreservation (3 days) in total, PI
br
and PI
dim
spermpopulation in the presence
or absence of Opuntia cus-indica (OFI) extract or resveratrol.
Sperm population Fresh Control OFI extract Resveratrol
Total 43.8 12.5 73.6 25.1
a
70.9 24.1 72.9 24.1
PI
br
29.9 9.8 60.9 20.7
b
55.6 20.1
c
55.8 20.2
d
PI
dim
13.9 10.2 12.6 8.9 15.2 8.4 16.1 9.1
Note: Values are mean standard deviation (SD).
a
P.003 versus fresh.
b
P.0007 versus fresh.
c
P.04 versus control.
d
P.009 versus control.
Meamar. Cryopreservation and sperm DNA fragmentation. Fertil Steril 2012.
FIGURE 1
Sperm DNA fragmentation (SDF) after cryopreservation in the presence or absence of Opuntia cus-indica (OFI) extracts and resveratrol. (A)
Individual SDF values in the PI
br
population before and after cryopreservation in basal conditions (left: control, n 21) and in the presence of
OFI extracts (center: n 15) and resveratrol (right: n 15). (B) Scatterplot and linear regression analysis of values before and after
cryopreservation in basal conditions with 95% condence interval.
Meamar. Cryopreservation and sperm DNA fragmentation. Fertil Steril 2012.
4 VOL. -NO. -/ -2012
ORIGINAL ARTICLE: ANDROLOGY
postthaw levels. In particular, in samples with pre-SDF PI
br
values below 30%, the average increase is of 45.9 4.48%
(mean SD, n 9); in patients with >30%, the average
increase is of 8.63 22% (mean SD, n 11, P.003).
The average postthawing values of DNAfragmentation in
total, PI
br
, and PI
dim
sperm populations in the presence or
absence of OFI extract or resveratrol are shown in Table 1.
In the presence of OFI extracts, the postthawing level of
DNA fragmentation in both total and PI
br
sperm populations
was statistically signicantly reduced with respect to control
values, and no effect was observed, as expected, on the PI
dim
population. A similar effect on SDF in the PI
br
sperm popula-
tion was observed in the presence of resveratrol. Figure 1A
(medium and right panels) shows the percentage of PI
br
DNA fragmentation in the individual sperm samples cryopre-
served without (C) or with (T) OFI extracts and resveratrol.
Effect of Resveratrol and OFI Extracts on Postthaw
Sperm Viability and Motility Parameters
Average motility parameters as evaluated by CASAand sperm
viability before and after cryopreservation in the presence or
absence of OFI extract and resveratrol are reported in Table 2.
As can be observed, statistically signicant variations were
present in all the parameters after cryopreservation (see
Table 2). Addition of resveratrol and OFI extracts to the cryo-
preservation medium did not result in any statistically signif-
icant ameliorative effect on sperm motion parameters or
viability (see Table 2).
Effect of Prefreezing Prolonged Incubation with
OFI Extracts and Resveratrol on Postthawing
Sperm Viability, Motility Parameters, and SDF
Overall, adding OFI extract and resveratrol to the cryopreserva-
tion medium did not result in statistically signicant
advantages in terms of sperm motility or viability with respect
to the control medium, demonstrating only a slight benecial
effect on sperm SDF. It is possible that addition of the two
antioxidants to TYB is not sufcient to prevent damage from
cryopreservation and that a longer incubation is needed to en-
sure penetration of the two antioxidants into the cells (15).
In the next set of experiments, OFI extracts (5 mg/mL
nal concentration) and resveratrol (15 mM) were directly
added to semen samples, which were incubated for 1 hour
at 37

C before the cryopreservation procedure. The results

of these experiments (n 6, not shown) demonstrated that
prolonged incubation with the two antioxidants did not result
in any benecial effect on the postthawing sperm parameters,
including SDF.
Relationship between Sperm DNA Fragmentation
and Sperm Motion Parameters in Fresh and
Cryopreserved Semen
No correlations were found among motion parameters or
viability and DNA fragmentation in the PI
br
or in the total
sperm population before the cryopreservation procedure, in
agreement with our previous studies demonstrating a lack
of correlation of this sperm population and semen quality
(13). Before cryopreservation, the PI
dim
population was found
to be negatively correlated to ALH (r 0.55, P.03, n 21)
and positively to BCF (r 0.66, P.006, n 21), STR
(r 0.59, P.02, n 21), and LIN (r 0.55, P.03,
n 21) (Fig. 2). After cryopreservation, the percentage of
SDF in the PI
br
population was found to negatively correlate
with VCL (r 0.49, P.02, n 21), whereas the PI
dim
pop-
ulation was statistically signicantly correlated with BCF
(r 0.55, P.03, n 21).
DISCUSSION
The deleterious effects of cryopreservation on sperm func-
tional parameters are well known (13). Our study
conrms the dramatic effect of cryopreservation on sperm
motion parameters (VAP, VSL, ALH, STR, and LIN),
viability, and SDF. The decrease in sperm motion
parameters and, in particular, of ALH may have a great
impact on subsequent application of rst-level ART using
cryopreserved semen. Freour et al. (34) recently demon-
strated that ALH is related to pregnancy in intrauterine
TABLE 2
Sperm motility parameters modications (CASA system) before and after cryopreservation of 15 semen samples in the absence or presence of
Opuntia cus-indica (OFI) extract and resveratrol.
Sperm After cryopreservation
P value Motion parameter Fresh sample Control OFI extract Resveratrol
VAP (mm ms) 57.9 6,4 27.9 29.7
a
23.8 23.3 24.1 27.7 .001
VSL (mm ms) 49.8 4.8 24.9 27.7
a
20.5 20.1 20.4 24.2 .003
VCL (mm ms) 82.2 11.5 65.3 71.1 38.08 39.6 33.2 3806
ALH (mm) 3.5 0.7 2.0 2.6
a
1.4 1.8 1.2 1.8 .03
BCF (Hz) 18.4 3.8 13.6 19.2 6.5 9.0 6.1 10.0
STR (%) 86.3 3.2 46.6 45.5
a
46.3 45.7 39.2 44.0 .005
LIN (%) 62.6 6.1 28.9 31.6
a
31.1 32.6 29.6 34.3 .001
Viability (%) 74.7 8.03 53.9 8.5
a
56.3 8.7 54.4 8.8 .00001
Note: Statistical signicance determined by Student's t-test. ALH amplitude of lateral head displacement; BCF beat-cross frequency; LIN linearity (VSL/VCL); STR straightness (VSL/VAP);
VAP average path velocity; VCL curvilinear velocity; VSL straight-line velocity.
a
Signicance vs. fresh sample at the P value shown in the last column.
Meamar. Cryopreservation and sperm DNA fragmentation. Fertil Steril 2012.
VOL. -NO. -/ -2012 5
Fertility and Sterility
insemination. For subsequent use of cryopreserved semen in
ART programs, the integrity of the genomic material is fun-
damentally important (35). In oncologic patients, who are
the main beneciaries of sperm cryopreservation programs,
the DNA damage from cryopreservation may be an addition
to the elevated basal sperm DNA damage found with certain
types of tumors (8, 9). Because in many cases cryopreserved
semen is used in second-level ART, high levels of SDF in-
crease the possibility that fragmented sperm will participate
in the fertilization process.
Several studies have ascribed the deleterious effects of
cryopreservation to oxidative stress generated during the pro-
cedure. Despite such evidence, studies evaluating the protec-
tive effects of antioxidants such as vitamin E (17), genistein
(14), or resveratrol (15) have been mostly disappointing,
showing minimal effects on sperm motion parameters or
DNA fragmentation. Our study evaluated whether the addi-
tion to cryopreservation medium of OFI extract, which con-
tains several types of antioxidants and is highly membrane
soluble (2224), would protect sperm from the deleterious
effects of cryopreservation. For comparison, we used the
antioxidant resveratrol, previously shown to have some
benecial effects during sperm cryopreservation when
added at 10 mM (15, 16). Our results demonstrated a slight
but statistically signicant protective effect of both
substances on SDF. In the case of resveratrol, the protective
effect was obtained at a much lower concentration than
used in previous studies (15, 16), and we demonstrated the
compound's toxic effect at concentrations higher than 1
mM, in agreement with previous results (29).
Collectively, our study and the results of other studies on
the addition of antioxidants or multifunctional substances
such as OFI extract to cryopreservation media (1517)
indicate that these strategies are not sufcient to completely
prevent damage from the cryopreservation process. It
cannot be excluded that, besides oxidative stress, other
mechanisms are responsible for cryopreservation damage,
such as an increase of apoptosis. An increase of caspase
activity (an index of apoptosis) has been shown to occur
during cryopreservation of human semen (14).
Another aim of our study was to employ a method (TU-
NEL/PI) recently set up in our laboratory (26) to determine
damage to DNA after cryopreservation procedures; this
method, which allows elimination of all the interferents pres-
ent in semen, results in a more precise evaluation of SDF, as
interferents lead to an underestimation in most of the samples
(13). The TUNEL/PI method allows distinguishing between the
PI
br
and PI
dim
spermpopulations (13), which are characterized
by properties such as differing levels of DNA fragmentation
and semen quality. The PI
dim
sperm are entirely DNA frag-
mented and are negatively related to semen quality. The
percentage of fragmented sperm in the PI
br
population varies,
and it is totally unrelated to semen parameters such as motil-
ity and morphology (13).
The increase of SDF after cryopreservation occurred in the
PI
br
population, but there was no change in the PI
dim
sperm.
This result was expected in view of the fact that PI
dim
sperm
are already entirely DNA fragmented, but we believe that
knowing of the percentage of SDF in the PI
br
sperm popula-
tion in frozen-thawed sperm is of interest for ART
FIGURE 2
S
D
F

(
P
I
d
i
m
)
p
r
e
-
f
r
e
e
z
e

l
e
v
e
l
s
ALH pre
2,0
2,5
3,0
3,5
4,0
4,5
5,0
R=-0.55,
STR pre
0 10 20 30 40 50
82
84
86
88
90
92
94
R=0.59,
S
D
F

(
P
I
d
i
m
)
p
r
e
-
f
r
e
e
z
e

l
e
v
e
l
s
BCF pre
8 10 12 14 16 18 20 22 24 26 28
0
10
20
30
40
50
R=0.66,
S
D
F

(
P
I
d
i
m
)
p
r
e
-
f
r
e
e
z
e

l
e
v
e
l
s
LIN pre
50 55 60 65 70 75 80
0
10
20
30
40
50
R=0.55,
S
D
F

(
P
I
d
i
m
)
p
r
e
-
f
r
e
e
z
e

l
e
v
e
l
s
Scatterplots and linear regression between sperm DNA fragmentation (PI
dim
population) and the sperm motility parameters of amplitude of lateral
head displacement (ALH: R 0.55, P.03), straightness (STR: R 0.59, P.02), beat-cross frequency (BCF: R 0.66, P.006), and linearity (LIN: R
0.55, P.03), as evaluated by CASA.
Meamar. Cryopreservation and sperm DNA fragmentation. Fertil Steril 2012.
6 VOL. -NO. -/ -2012
ORIGINAL ARTICLE: ANDROLOGY
applications with cryopreserved semen. Indeed, the lack of
correlation with semen quality in DNA fragmented PI
br
sperm
implies that DNA-fragmented spermatozoa in this population
may be motile and morphologically normal, giving them
a higher probability of participating in the fertilization
process. The increase of fragmentation in PI
br
sperm after
cryopreservation augments the possibility that a motile
DNA fragmented sperm with normal morphology could par-
ticipate in the process of fertilization (as it can be selected
for intracytoplasmic sperm injection, for instance), with
important consequences for the couple. Indeed, SDF has
been reported to have an impact on both natural and assisted
reproduction (35). In particular, in ART, the fertilization rate,
embryo quality, and, as pointed out by a recent meta-analysis
(36), the rate of miscarriages may be affected by SDF (35, 36).
Thus, clinicians should be aware of the increased probability
of these effects when cryopreserved semen is used in ART, and
the couples should be informed. After cryopreservation, the
percentage of SDF in PI
br
sperm was not related to motion
parameters except with VCL, a parameter related to
hyperactivation (37), which implies that thawed sperm with
characteristics of hyperactivation have a higher probability
of DNA integrity.
Although on average SDF dramatically increases after
cryopreservation, we found that the increase of SDF depends
in a negative manner on the level of SDF in fresh samples;
a sample with low PI
br
SDF in basal conditions will show
a more dramatic increase after thawing. These results have
important implications: low levels of SDF before cryopreser-
vation do not represent a favorable event per se, as these sam-
ples are more susceptible to increased DNA damage. Similar
results have been previously reported by Thomson et al.
(19). Overall, our results (19) indicate that the higher the
SDF in prefreeze samples, the lower the SDF after cryopreser-
vation. The decrease of SDF in patients with high basal levels
may be due to the loss of spermwith high DNAdamage during
the process of cryopreservation, as these spermatozoa may be
more fragile (19). The higher increase of SDF in semen
samples with lower basal levels is more difcult to explain.
However, although men included in our study were mostly
normospermic, they were all male partners of infertile cou-
ples, so it is possible that their sperm are characterized by
elevated chromatin instability or have other types of damage
that make them more susceptible to the deleterious effect of
cryopreservation on DNA.
Finally, we evaluated the relationship between SDF and
motility parameters as evaluated by the CASA system. We
found that prefreeze SDF in the PI
dim
population was nega-
tively associated with amplitude of lateral head (ALH) and
positively associated with beat-cross frequency (BCF),
straightness of the trajectory (STR), and linearity of the trajec-
tory (LIN). In line with our previous results (13), the percent-
age of fragmented sperm in the PI
br
population did not show
any statistically signicant relationship with sperm motion
parameters. To the best of our knowledge, a relationship
between CASA motility parameters and SDF has not been
described previously. Our results indicate the existence of
a negative relationship between the PI
dim
sperm population
and sperm hyperactivation parameters; indeed, ALH, which
is negatively related to PI
dim
sperm, increases as the percent-
age of hyperactivated sperm increases, but BCF, STR, and LIN,
which are negatively related to PI
dim
sperm, show an opposite
trend (37). Conversely, SDF in the PI
br
population appears to
be independent of sperm hyperactivation.
Our study provides newinsights on the deleterious effects
of cryopreservation on SDF, demonstrating that the damage
occurs in the PIbr sperm population which is unrelated to
semen quality. We also show that the addition of a cocktail
of antioxidants, such as OFI extract, has only a mildly bene-
cial effect on SDF after cryopreservation. The molecular
mechanisms in addition to oxidative stress that determine
the extent of sperm damage during cryopreservation proce-
dures need to be identied before we can develop new strat-
egies to prevent this damage.
Acknowledgments: The authors thank Dr. Angela Magini
(SOD Medicina della Sessualita e Andrologia, AOUC, Firenze)
for helpful advice.
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