JBRA Assist. Reprod. 2016;20 (1):17-22
ORIGINAL ARTICLE
doi: 10.5935/1518-0557.20160005
Assessment of sperm DNA in patients submitted the assisted reproduction technology procedures
1Clínica Endogin, Bauru/SP
2Department of Animal Reproduction, Veterinary Medicine and Animal Science School, São Paulo State University at Botucatu/SP
3Blood Center, School of Medicine of the São Paulo State University at Botucatu/SP
4Department of Urology, School of Medicine of the São Paulo State University at Botucatu/SP
CONFLICT OF INTERESTS
No conflict of interest have been declared.
ABSTRACT
Objective:
This study aimed to produce data on sperm quality while maintaining the
integrity of sperm DNA samples taken from patients submitted to in
vitro fertilization (IVF) procedures at our center, and determine
whether increased levels of histones were associated with sperm DNA
damage and decreased fertilization, cleavage, and pregnancy rates. Such
findings might shed light on the physiology and outcomes of pregnancy.
Methods:
Semen samples from 27 patients divided into two groups were analyzed.
The case group included individuals offered IVF; the control group had
subjects with normal spermograms. Sperm DNA structure was assessed
through phosphorylated histone H2AX analysis by flow cytometry.
Results:
The patients with altered sperm parameters had more histones in sperm
chromatin than the individuals with normal sperm parameters.
Conclusion:
Results indicated that increased levels of histone in sperm chromatin
do not affect embryo production, but affect the cleavage rate, embryo
quality, and might thus reduce pregnancy rates. The integrity of the
paternal genome is of paramount importance in the initiation and
maintenance of a viable pregnancy in patients treated with assisted
reproduction technology procedures. Further studies on sperm diagnostic
tests at a nuclear level might improve the treatment offered to
infertile couples.
Keywords: Sperm DNA, Histones, Pregnancy, Fertilization rate, ICSI.
INTRODUCTION
The Sperm DNA integrity is essential for the accurate transmission of
the genetic code, in addition to serving as an indicator of the quality
of spermatogenesis and male fertility (Smith et al., 2007).
Disturbances in the integrity of chromatin are characterized by the
presence of breakages in single or double strands of sperm DNA, which
lead to the formation of denatured segments (Rybar et al., 2004).
Increased susceptibility to denaturation is suggestive of chromatin
structure heterogeneity and has been linked to disturbances in
spermatogenesis, abnormal morphology (Enciso et al., 2011), decreased sperm concentration and motility (Benchaib et al., 2003), hampered embryonic development, and decreased fertility (Hallap, 2005).
Although many authors have reported high DNA fragmentation levels in
infertile patients with abnormal spermatozoa parameters (Aitken & De luliis, 2007; Henkel, 2007),
some patients with normal semen parameters and idiopathic infertility
have been shown to present higher levels of damaged sperm DNA (Saleh et al., 2003; Cohan et al., 2006).
More than 30% of these patients require longer times to establish a
normal pregnancy, more assisted reproduction cycles, and are at a
higher risk of experiencing a miscarriage (Giwercman et al., 2010).
Sperm DNA damage can occur within the testes, the epididymis or in
ejaculates. Its multifactorial etiology may include extrinsic and
intrinsic factors (Zini & Sigman, 2009; Sakkas & Alvarez, 2010).
Different hypotheses have been proposed to explain the origin of DNA
damage in mature sperm of infertile men, including abnormalities in
chromatin condensation, microdeletions, aneuploidies, chromosome
rearrangements, breakages in single or double strand DNA, apoptosis,
and oxidative stress (Gorczca et al., 1993; Manicardi et al., 1995; Sakkas et al., 1999; Barroso et al., 2000; Cho et al., 2001; Souza et al., 2010).
In addition to biological factors such as infection, sexually
transmitted diseases, obesity, diabetes, age, lifestyle factors
including smoking (Pacey, 2010) may also affect chromatin integrity.
Sperm chromatin is organized within a highly condensed and compressed
structure that significantly protects the DNA against damage while
making it promptly available to the ooplasm (Bjorndahl & Kvist, 2010).
Sperm chromatin undergoes a series of changes during spermatogenesis,
with the first important event being the hyperacetylation of histones
to facilitate the disassembly of the nucleosome. During this process,
sperm DNA is extensively complexed with transition proteins. At the end
of spermiogenesis these transition proteins are replaced by protamines,
leaving the DNA in human sperm chromatin a fraction in nucleohistones
and a fraction in nucleoprotamines, with 15% of the DNA bound to
histones and 85% bound to protamines (Bjorndahl & Kvist, 2010; Oliva, 2006).
Infertile men have increased histone to protamine ratios when compared
to fertile controls. This change in the ratio, which consists of
abnormal chromatin packaging, increases the susceptibility of sperm DNA
to external stress, due to the lower compaction of chromatin (Zini & Sigman, 2009; Ozmen et al., 2007).
In recent years, rapid advances in molecular biology have led to the
development of numerous tests to assess various aspects of DNA and
chromatin quality. The tests are divided into direct and indirect
assays. Direct assays include TUNEL, the neutral comet assay, and in
situ nick translation; indirect assays include the alkaline comet
assay, the sperm chromatin structure assay (SCSA), and the sperm
chromatin dispersion (SCD) test (Lewis et al., 2008).
These methods might detect cases of infertility missed in semen
analysis. Studies have shown that levels of DNA damage in mature
spermatozoa are significantly correlated with sperm fertilization
potential. DNA damage analysis may help predict the success of
pregnancy and birth produced by natural and assisted reproduction
technology (ART) procedures. Moreover, characterizing normal levels of
DNA damage in normal sperm might help prevent the use of samples with
high levels of DNA damage in ART procedures (Tesarik et al., 2004).
This study aimed to produce data on sperm quality while maintaining the
integrity of sperm DNA samples taken from patients submitted to in
vitro fertilization (IVF) procedures at our center, and determine
whether increased levels of histones were associated with sperm DNA
damage and decreased fertilization, cleavage, and pregnancy rates. Such
findings might shed light on the physiology and outcomes of pregnancy.
MATERIALS AND METHODS
Twenty-one couples were offered intracytoplasmic sperm injections
(ICSI) at the Endogin Clinic in Bauru, Brazil. Individuals who smoked,
drank alcohol, or had an infertile partner were excluded. Ejaculated
semen samples from six individuals with normal findings on physical
examination – normal volume, absence of varicocele, and history of
pregnancy with a partner without using ART procedures within the last
12 months – were used as controls. Only the samples and data of the
individuals who gave consent to participating in the study were used.
The patients referred to ICSI underwent controlled ovarian stimulation
along with suppression of the hypothalamic-pituitary axis. Pituitary
suppression was carried out between days 18 and 22 of the menstrual
cycle with a gonadotropin-releasing hormone analogue (Lupron TM Kit,
Abbott SA, Paris, France). Fourteen to twenty days later, the patients
were started on ovarian stimulation with follicle-stimulating hormone
(TM Gonal-F, Serono, Geneva, Switzerland) at dosages ranging between
150 and 300 IU depending on the patient’s age. Induction was followed
with serial transvaginal ultrasound scans. When the dominant follicle
reached 18 mm or at least two follicles reached 16 to 18 mm in
diameter, the patients were administered 250 ug of recombinant human
chorionic gonadotropin (rhCG) (Ovidrel, Serono, Geneva, Switzerland) to
enable final follicular maturation. Follicular aspiration was performed
approximately 34 to 36 hours after the administration of rhCG. The
recovered oocytes were denuded and incubated in the initial culture
medium (G1Plus, Vitrolife, Gothenburg, Sweden), covered with paraffin
oil (Ovoil, Vitrolife) previously equilibrated at pH 7.2-7.4 and kept
at 37°C.
Oocyte morphology was assessed and patients with most of their oocytes
showing a perivitelline space, extrusion of the first polar body (PB)
MII, and an intact oolemma were selected, administered microinjections,
and included in the case group.
Semen samples were obtained by masturbation after a period of three to
five days of abstinence from the day of oocyte aspiration. The
collected material was kept for 30 minutes at room temperature and
after liquefaction the samples were macroscopically assessed for color,
volume, viscosity, and liquefaction. Then microscopic analysis was
performed to determine sperm concentration and motility. Normal values
were defined as a concentration >15 million/ml or 39x106/ejaculate
and motility >32% (progressive motility) and >40% (total
motility). Morphology was considered normal when >4% according to
the Tygerberg criteria.
Portions of the semen samples were centrifuged at 300 g for seven
minutes and were then frozen. Seminal plasma was discarded and the
seminal pellet was washed twice in PBS at a pH of 7.4, resuspended at a
final concentration of 5x106/ml, and stored at -196oC for sperm
chromatin analysis. The same procedure was performed with the semen
samples of the control group.
Intracytoplasmic sperm injection (ICSI) was performed according to the standard technique described by Palermo et al., (1992). Embryo quality was assessed based on the modified morphological criteria described by Veeck (1991),
including items such as number and symmetry of blastomeres and degree
of fragmentation. Samples assigned to Grade I had no fragmentation and
equal blastomeres; samples on Grade II had <20% fragmentation and
equal blastomeres; samples on Grade III had <20% fragmentation and
unequal blastomeres; samples on Grade IV had >20% fragmentation and
unequal blastomeres; and samples on Grade V had > 50% fragmentation.
Three days after oocyte aspiration, the embryos were selected for
transfer. Fourteen days after embryo transfer the patients were tested
for beta hCG levels to verify whether embryo implantation was
successful. Ultrasound scans were performed in the 6th week of
pregnancy to confirm fetus viability. Clinical pregnancy was defined
when a gestational sac was detected in ultrasound scans. Clinically
pregnant individuals who lost their fetuses before the 12th week of
pregnancy were categorized as cases of miscarriage.
Tests to determine the presence of histones in sperm chromatin
A fraction of the semen samples was tested with the Apoptosis, DNA
Damage and Cell Proliferation Kit (BD Biosciences, catalog number
562253). Analysis by flow cytometry was performed according to the
manufacturer’s instructions. Sperm samples were thawed and diluted in
1ml buffer (1x106 sperm) and centrifuged at 250g for five minutes. The
supernatant was discarded and the pellet resuspended in 100 ul BD
Cytofix for fastening and Cytosperm (permeabilizing solution) and kept
at room temperature for 30 minutes. Then it was washed with 1 ml 1x BD
Perm / Wash Buffer, and the supernatant was again removed and the
pellet resuspended in 20ul BDPerm / Wash Buffer plus antibody Alexa
Fluor 647Mouse anti-H2AX (pS139.5μl / test). The samples were incubated
for 20 minutes at room temperature. After 60 minutes, the samples were
washed and resuspended in 1ml of 1 x BD Perm / Wash Buffer and
evaluated in the flow cytometer (FACScalibur, Becton Dickinson, San
Jose, California, USA). A minimum of 100,000 cells per sample were
assessed.
Statistical Analysis
Statistical
analysis was performed using software package GraphPad Prism Version
5.00 for Windows (GraphPad Software, San Diego California USA,
www.graphpad.com). The Kolmogorov-Smirnov test was used to verify the
normality and homogeneity of variance when the samples had parametric
data. The t-test was used the samples had non-parametric data. The
Mann-Whitney test or the Kruskal-Wallis test were performed followed by
Dunn’s test. Significance was attributed to events with a P<0.05. Fisher’s exact test was used to assess embryo quality and percentage of histones in relation to embryo quality.
RESULTS
The semen samples of 27 subjects were analyzed and divided into two
groups separating patients offered IVF and healthy controls with normal
spermograms. No statistically significant differences were found (Figure 1A) between case and control group subjects. A trend was observed (P
= 0.0808) in which IVF patients (n = 21; 3.9 ± 1.4) had a higher
incidence of histones in sperm DNA chromatin than controls (n = 6; 0:48
± 0:11). The impact of the level of histones in male fertility was
further analyzed in terms of positive and negative cases of clinical
pregnancy (Figure 1B). Again, no statistically significant difference was found in clinical pregnancy rates (P
= 0.2486). This may have occurred because one patient with high levels
of histones was positive for clinical pregnancy. Patients were also
assessed for semen quality before freezing (Figure 1C), but no statistically significant differences were found (P
= 0.3757). However, patients considered to have normal quality semen (n
= 13) had higher histone levels (5.4 ± 2.2) than controls (n = 6; 00:48
± 00:11), while individuals with altered semen quality had only a
slight increase in histone levels (n = 8; 1.8 ± 0.8).
Cleavage rate P = 0.0506 (Figure 2A)
and the percentage of histones in sperm chromatin influenced the
results. Patients with positive pregnancies (98.61 ± 1.38) had higher
cleavage rates than individuals negative for pregnancy (87.98 ± 4.0).
No statistically significance difference (P = 0.1245) was
observed in terms of the percentage of transferable embryos (64.57 ±
13.6 versus 57.6 ± 8.0, for individuals positive and negative for
pregnancy, respectively), as shown in Figure 2B.
Embryo quality was also assessed. As expected, clinical pregnancy rates
were higher among individuals with higher quality embryos (Grades I and
II) than in subjects with poorer quality embryos (Grades III and IV),
as shown in Figure 3A (P <0.001). The presence of histones in sperm chromatin significantly interfered with the quality of the transferred embryos (Figure 3B). Better quality embryos were produced from patients with lower sperm chromatin histone levels (P = 0.0123).
Figure 1: Representation of Whisker plots (min to max) of the data
obtained from the analysis of histones in the sperm chromatin of
patients undergoing in vitro fertilization procedures versus controls –
males whose partners have had spontaneous pregnancies within less than
12 months (A). Patients positive for pregnancy versus individuals
negative for pregnancy versus controls (B); Patients grouped according
to sperm quality into abnormal, normal, and controls (C).
Figure 2: Representation of Whisker plots (min to max) of the data
obtained from the analysis of histones in the sperm chromatin of
patients undergoing in vitro fertilization procedures grouped into
subjects positive for pregnancy versus individuals negative for
pregnancy in relation to cleavage rates (Fig A); and rate of
transferable embryos (Fig B).
Figure 3: Graphs representing the analysis of embryo quality versus
pregnancy using Fisher’s exact test (fig A). Thirty-two embryos of
Grades I and II (good quality) resulted in pregnancy, while 40 embryos
– 17 in Grades I and II and 23 in Grades III and IV (poor quality) –
did not result in pregnancy. Concerning the level of histones in sperm
chromatin, 22 transferred embryos (10 of good and 12 of poor quality)
had high histone levels (> 1%) and 50 (39 good and 11 poor quality
embryos) had low histone levels (<0.99%).
DISCUSSION
Oocytes fertilized by sperm with DNA alterations may significantly impair embryonic development and fetal health (Evenson et al., 2002), in addition to transmitting mutations that might affect the fertility of the next generation (Aitken & Krausz, 2001). Thus, chromatin integrity plays a vital role in the paternal contribution to the production of a healthy fetus.
This study compiled data on chromatin alterations that may compromise
the viability of sperm used in IVF. These changes are basically caused
by apoptosis during spermatogenesis, breaks during chromatin
remodeling, and fragmentation induced by reactive oxygen species (Tavalee et al., 2009; Sakkas & Alvarez, 2010).
In recent years, the evaluation of sperm DNA fragmentation was
recognized as a predictor of the likelihood of conception. The capacity
for natural conception decreases in sperm with abnormal chromatin (Spano et al., 2000; Sergerie et al., 2005). Henkell et al. (2004) and Simon et al. (2011)
found that patients treated repeatedly with conventional IVF
experienced negative effects on fertilization and blastocyst
development in the course of pregnancy. Reports on ICSI have published
contradictory results. Some groups found (statistically significant)
negative associations between the percentage of sperm with fragmented
DNA and fertilization rates with ICSI (Lopes et al., 1998; Simon et al., 2013),
while other authors did not observe differences in the fertilization
rate but reported negative effects upon pregnancy rates (Benchaib et al., 2003) or embryo quality (Muriel et al., 2006). Higher levels of DNA fragmentation have been described as a cause for low quality embryo implantation and miscarriage (Tesarik et al., 2004; Evenson et al., 2002; Borini et al., 2006). Conversely, some studies did not describe sperm DNA fragmentation as having a role in ICSI results (Bungum et al., 2004; Nicopoullos et al., 2008).
These authors looked into DNA fragmentation in the total population of
sperm (morphologically abnormal and normal), but during the ICSI
procedure, only sperm with good motility and morphology was selected
for injection. The inadvertent selection of sperm cells based solely on
normal motility and morphology without considering DNA integrity may
yield undesired outcomes in ICSI procedures, which might impair not
only embryonic development, but also the resulting offspring. Greco et al. (2005)
showed that microinjection of sperm with fragmentation above 15% as
analyzed using TUNEL resulted in a pregnancy rate of 5.6% versus 44.4%
when sperm DNA fragmentation was below 6%.
The results analyzed in
this study showed that even men with normal sperm counts may have high
levels of alteration in the compaction of chromatin, which means it
might take longer to have a successful pregnancy, thus increasing the
chances of having more ART cycles and the risk of having a miscarriage.
Tests showed that these alterations were not related to fertilization
rates, but to significant reductions in embryo quality and consequently
in the pregnancy rates attained with ICSI cycles.
Although contradictory, no associations were found between embryo
quality and sperm DNA damage measured by sperm chromatin structure
assay (SCSA) and TUNEL (Irvine et al., 2000; Larson et al., 2000; Benchaib et al. 2003; Huang et al., 2005; Benchaib et al., 2007; Frydman et al., 2008), despite the different test sensitivities and the types of DNA damage measured specifically in each test.
There is currently a threshold assigned to percent DNA fragmentation in
a sample above which pregnancy becomes unlikely. This threshold varies
between 12% (Duran et al., 2002) and 36.5% (Henkel et al., 2003), and has been described as 15% (Benchaib et al., 2007) and 20% (Sergerie et al., 2005).
Many are the possible reasons for the discrepant thresholds: different
study populations (intrauterine insemination (IUI), in vitro
fertilization (IVF) and/or intracytoplasmic sperm microinjection
(ICSI); type of sperm used (obtained after washing or selected); or the
type of count (operator or by flow cytometry).
Despite these observations, sperm with damaged DNA can preserve the potential for fertilization and produce viable embryos (Tesarik et al., 2004). While the oocyte might repair the damaged paternal DNA, this ability is limited and depends on the level of DNA damage (Fatehi et al., 2006). DNA damage may predispose the developing embryo to mutations and induce disease in the offspring (Aitken & Krausz, 2001).
Sperm DNA integrity is an important factor in the overall success of
ART procedures. Although several studies have demonstrated the
importance of assessing chromatin integrity and thresholds have been
set to assess fertility levels, this parameter has not been evaluated
routinely in andrology laboratories.
This is possibly due to the
complex protocols and expensive equipment – the likes of fluorescence
microscopes and flow cytometers – required to run such tests.
Successful ART procedures rely on many factors other than semen quality
and the ability of sperm to merge with the oolemma, such as the oocyte,
uterine receptivity, and maternal immune status.
CONCLUSIONS
Changes in
chromatin structure negatively affect the clinical outcomes of ICSI
procedures. Clinical evidence suggests that these changes may curtail
the effect of ART procedures even in men with normal sperm. The impact
of chromatin structure alterations on fertilization rates is still
controversial, but there is agreement over the negative effects it
produces on embryonic development and pregnancy rates.
The predictive value of a DNA fragmentation test consists of a sum of
factors, such as: 1) whether fragmentation occurred in single or
double-stranded DNA (single offers better prognosis); 2) percentage of
sperm with damaged DNA; higher percentages mean the likelihood of the
egg being fertilized by sperm with intact DNA is reduced; 3) degree of
DNA fragmentation in sperm (more intense fragmentation means DNA is
less likely to be repaired); 4) primary or secondary damage; 5) type of
test used (direct or indirect); 6) whether the damage affects encoding
sequences (relatively low likelihood, as more than 90% of the DNA
consists of non-encoding sequences); 7) the ability of the oocyte to
repair the damage; 8) ability of the embryo to repair the damage; 9)
number of oocytes in metaphase II; and 10) sample processing. It is
also important to remember that although the 2006 Practice Committee
Guidelines of the American Society for Reproductive Medicine (ASRM)
accepted that sperm DNA fragmentation is more common in infertile men
and that it may contribute to poor reproductive performance, there is
no proven role for routine testing of DNA integrity in the evaluation
of infertility, as current findings are enough only to suggest, rather
than confirm, an association between sperm DNA testing and ART
procedure outcomes.
The lack of an indication for routine testing was supported in a review published by Collins et al. (2008).
More studies are needed to verify the association of DNA damage test
results and the clinical outcomes of ART procedures. Oocyte quality may
also be a major determinant on the negative effects introduced by sperm
DNA damage.
REFERENCES
Aitken RJ, De luliis GN. Origins and consequences of DNA damage in male germ cells. Reprod BioMed Online 2007; 14: 727-33.
Medline Crossref
Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001; 122: 497-506.
Medline Crossref
Barroso
G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma
membrane translocation of phosphatidylserine and oxidative stress in
human spermatozoa. Hum Reprod 2000; 1338-44.
Medline Crossref
Benchaib
M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, Guérin JF. Sperm DNA
fragmentation decreases the pregnancy rate in an assisted reproductive
technique. Hum Reprod 2003; 18: 1023-8.
Medline Crossref
Benchaib
M, Lornage J, Mazoyer C, Lejeune H, Salle B, François Guerin J. Sperm
deoxyribonucleic acid fragmentation as a prognostic indicator of
assisted reproductive technology outcome. Fertil Steril 2007; 87:
93-100.
Medline Crossref
Bjorndahl L, Kvist U. Human sperm chromatin stabilization: a proposed model including zinc bridges. Mol Hum Reprod. 2010; 23-9.
Medline Crossref
Borini
A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamingni C, Coticchio G.
Sperm DNA fragmentation: paternal effect on early post-implantation
embryo development in ART. Hum Reprod. 2006; 21, 2876-81.
Medline Crossref
Bungum
M, Humaidan P, Spano M, Jepson K, Bungum L, Giwerchman A. The
predictive value of sperm chromatin structure assay (SCSA) parameters
for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod.
2004; 19: 1401-8.
Medline Crossref
Cho
C, Willis WD, Goulding EH, Jung-Ha, Choi YC, Hecht NB, Eddy EM.
Haploinsufficiency of protamine-1 or –2 causes infertility in mice. Nat
Genet 2001; 28: 82-6.
Medline Crossref
Cohan
KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT. Comparison of
cromatin assays for DNA fragmentation evaluation in human sperm. J
Androl 2006; 27:53-9.
Medline Crossref
Collins
JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict
pregnancy with in vitro fertilization? Fertil Steril. 2008; 823-31.
Medline Crossref
Duran
EH, Morshedi M, Taylor S, Oehninger S. Sperm DNA quality predicts
intrauterine insemination outcome: a prospective cohort study. Hum
Reprod 2002; 17: 3122-8.
Medline Crossref
Enciso
M, Iglesias M, Galán I, Sarasa J, Gosálvez A, Gosálvez J. The ability
of sperm selection techniques to remove single- or double –strand DNA
damage. Asian J Androl 2011; 13: 764-8.
Medline Crossref
Evenson
DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical
use for detecting sperm DNA fragmentation in male infertility and
comparisons with other techniques. J Androl 2002; 23: 25-43.
Medline Crossref
Fatehi
AN, Bevers MM, Schoevers E, Roelen BA, Colenbrander B, Gadella BM. DNA
damage in bovine sperm does not block fertilization and early embryonic
development but induces apoptosis after the first cleavages. J Androl
2006; 27: 176-88.
Medline Crossref
Frydman
N, Prisant N, Hesters L, Frydman R, Tachdjian G, Cohen-Bacrie P,
Fanchin R. Adequate ovarian follicular status does not prevent the
decrease in pregnancy rates associated with high sperm DNA
fragmantation. Fertil Steril 2008; 89: 92-7.
Medline Crossref
Giwercman
A, Lindstedt L, Larsson M, Bungum M, Spano M, Levine RJ, Rylander L.
Sperm chromatin structure assay as an independent predictor of
fertility in vivo: a case-control study. Int J Androl 2010; 33:221-7.
Medline Crossref
Gorczca
W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand
breaks and increased sensitivity of DNA in situ to denaturation in
abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp
Cell Res. 1993; 207: 202-5.
Medline Crossref
Greco
E, Scarcelli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Franco G,
Anniballo N, Mendoza C, Tesarik J. Efficiente treatment of infertility
due to sperm DNA damage by ICSI with testicular spermatozoa. Hum
Reprod. 2005; 20: 226-30.
Medline Crossref
Hallap
T. Assessment of sperm attributes of frozen-thawed AI doses from
Swedish and Estonian dairy bulls sires. Doctoral thesis (Divison of
comparative Reproduction, Obstetrics and Udder Health) – Department of
Clinical Sciences - Swedish University of Agricultural Sciences.
Uppsala. 2005.
Link
Henkel
R, Hajimohammad M, Stall T, Hoogendijk C, Mehnert C, Menkveld R, Gips
H, Schill WB, Kruger TF. Influence of deoxyribonucleic acid damage on
fertilization and pregnancy. Fert Steril. 2004; 81: 965-72.
Medline Crossref
Henkel
R, Kierspel E, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C,
Menkveld R, Schill WB, Kruger TF. DNA fragmentation of spermatozoa and
assisted reproduction technology. Reprod Biomed Online. 2003; 7: 477-84.
Medline Crossref
Huang
X, Halicta HD, Traganos F, Tanaka T, Kurose A, Darzynkiewicz Z.
Cytometric assessment of DNA damage in relation to cell cycle phase and
apoptosis. Cell Prolif 2005; 38: 223-43.
Medline Crossref
Irvine
DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity
in human spermatozoa: relationships with semen quality. J Androl. 2000;
21: 33-44.
Medline Crossref
Larson
KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin
structure assay parameters as predictors of failed pregnancy following
assisted reproductive techniques. Hum Reprod. 2000; 15: 1717-22.
Medline Crossref
Lewis SE, Agbaje I, Alvarez J. Sperm DNA tests as useful adjuncts to semen analysis. Syst Biol Reprod Med. 2008; 54:111-25.
Medline Crossref
Lopes
S, Jurisicova A, Casper RF. Gamete-specific DNA fragmentation in
unfertilized human oocytes after intracytoplasmatic sperm injection.
Hum Reprod. 1998; 13: 703-8.
Medline Crossref
Manicardi
GC, Bianchi PG, Pantano S, Azzoni P, Bizzaro D, Bianchi U, Sakkas D.
Presence of endogenous nicks in DNA of ejaculated human spermatozoa and
its relationship to chromomycin A3 accessibility. Biol Reprod. 1995;
52: 864-7.
Medline Crossref
Muriel
L, Garrido N, Fernandez JL, Remohi J, Pellicer A, de Los Santos MJ,
Meseguer M. Value of the sperm deoxyribonucleic acid fragmentation
level, as measured by the sperm chromatin dispersion test, in the
outcome of in vitro fertilization and intracytoplasmatic sperm
injection. Fertil Steril. 2006; 85: 371-83.
Medline Crossref
Nicopoullos
JD, Gilling-Smith C, Almeida PA, Homa S, Norman-Taylor JQ, Ramsay JW.
Sperm DNA fragmentation in subfertile men: the effect on the outcome of
intracytoplasmatic sperm injection and correlation with sperm
variables. BJU Int 2008; 101:1553-60.
Medline Crossref
Oliva R. Protamines and male infertility. Hum Reprod Update. 2006; 417-35.
Medline Crossref
Ozmen
B, Koutjaki N, Youssry M, Diedrich K, Al-Hasani S. DNA damage of human
spermatozoa in assisted reproduction: origins, diagnosis, impacts and
safety. Reprod Biomed Online. 2007; 14: 384-95.
Medline Crossref
Pacey AA. Environmental and lifestyle factors associated with sperm DNA damage. Hum Fertil (Camb). 2010; 13: 189-93.
Medline Crossref
Palermo
G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after
intracytoplasmic injection of single spermatozoon into an oocyte.
Lancet. 1992; 340:17-8.
Medline Crossref
Rybar
R, Faldicova L, Faldyna M, Machatkova M, Rubes J. Bull and boar sperm
DNA integrity evaluated by sperm chromatin structure assay in the Czech
Republic. Vet. Med. 2004; 49: 1-8.
Link
Sakkas
D, Alvarez, JG. Sperm DNA fragmentation mechanisms of origin, impact on
reproductive outcome, and analysis. Fertil Steril; 2010:93: 1027-36.
Medline Crossref
Sakkas
D, Mariethoz E, Manicardi GC, Bizzaro D, Bianchi PG, Bianchi U. Origin
of DNA damage in ejaculated human spermatozoa. Rev Reprod. 1999; 4:
31-7.
Medline Crossref
Saleh
RA, Agarwal A, Sharma RK, Said TM, Sikka SC, Thomas AJ. Evaluation of
nuclear DNA damage in spermatozoa from infertile men with varicocele.
Fertil Steril. 2003; 80: 1431-6.
Medline Crossref
Sergerie
M, Laforest G, Bujan L, Bissonnette F, Bleau G. Sperm DNA
fragmentation: threshold value in male fertility. Hum Reprod. 2005; 20:
3446-51.
Medline Crossref
Simon
L, Castilho J, Oliva R, Lewis SE. Relationships between human sperm
protamines, DNA damage and assisted reproduction outcomes. Reprod
Biomed Online 2011; 23:724-34.
Medline Crossref
Simon
L, Proutski I, Stevenson M, Jennings D, McManus J, Lutton D, Lewis SE.
Sperm DNA damage has a negative association with live-birth rates after
IVF. Reprod Biomed Online. 2013; 26:68-78.
Medline Crossref
Smith
R, Kaune H, Parodi D, Madariaga M, Morales I, Ríos R, Castro A. [Extent
of sperm DNA damage in spermatozoa from men examined for infertility.
Relationship with oxidative stress]. Rev Med Chil. 2007; 135: 279-86.
Medline Crossref
Souza AP, Santos JR, Santos TA. [The Significance of Sperm Chromatin Integrity in Male Infertility] Acta Urol 2010; 27:37-47.
Link
Spano
M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm
chromatin damage impairs human fertility. The Danish First Pregnancy
Planner Study Team. Fertil Steril 2000; 73: 43-50.
Medline Crossref
Tavalee
M, Razavi S, Nasr-Esfahani MH. Influence of sperm chromatin anomalies
on assisted reproductive technology outcome. Fertil Steril 2009; 91:
1119-26.
Medline Crossref
Tesarik
J, Greco E, Mendza C. Late, but no early, paternal effect on human
embryo development is related to sperm DNA fragmentation. Hum Reprod
2004; 19:611-5.
Medline Crossref
Veeck
L. Preembryo grading. In: Veek LL, ed. Atlas of human oocyte and early
conceptus. Vol. 2. Baltimore: Williams & Wilkins, 1991: 121-44.
Medline
Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J Androl 2009; 219-29.
Medline Crossref