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low fertilization after intracytoplasmic sperm injection
Accepted Manuscript
Evaluation of sperm telomere length in infertile men with failed fertilization after intra-cytoplasmic sperm injection Zahra Darmishonnejad , Marziyeh Tavalaee , Tayebeh Izadi , Somayeh Tanhaei , Mohammad Hossein Nasr-Esfahania PII: DOI: Reference:
S1472-6483(18)30651-5 https://doi.org/10.1016/j.rbmo.2018.12.022 RBMO 2084
To appear in:
Reproductive BioMedicine Online
Received date: Revised date: Accepted date:
7 June 2018 6 October 2018 10 December 2018
Please cite this article as: Zahra Darmishonnejad , Marziyeh Tavalaee , Tayebeh Izadi , Somayeh Tanhaei , Mohammad Hossein Nasr-Esfahania , Evaluation of sperm telomere length in infertile men with failed fertilization after intra-cytoplasmic sperm injection, Reproductive BioMedicine Online (2018), doi: https://doi.org/10.1016/j.rbmo.2018.12.022
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ACCEPTED MANUSCRIPT Evaluation of sperm telomere length in infertile men with failed fertilization after intracytoplasmic sperm injection
Zahra Darmishonnejada,b, Marziyeh Tavalaeea, Tayebeh Izadic, Somayeh Tanhaeic, Mohammad
a
ACECR Institute of Higher Education (Isfahan Branch)
b
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Hossein Nasr-Esfahaniad
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran c
d
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Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran Isfahan Fertility and Infertility Center, Isfahan, Iran
*
Corresponding author.
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CONTACT Mohammad Hossein Nasr-Esfahani [email protected] Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan
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Institute for Biotechnology, ACECR, Isfahan, Iran.
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ABSTRACT Research Question: Telomeres are non-coding, repetitive DNA sequences (TTAGGG repeats) that play an important role in maintaining genome integrity. Unlike in somatic cells, telomere length in sperm increases with men age and is considered as a molecular marker of sperm quality. Etiology of
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failed fertilization post- intracytoplasmic sperm injection (ICSI) technique is multifactorial; perhaps one of the reasons for this failure in these individuals is shortened sperm telomere length. Therefore, we aimed to assess sperm telomere length in addition to DNA damage, lipid peroxidation and
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protamine deficiency in infertile men with previously failed fertilization post-ICSI.
Design: Semen samples were obtained from infertile men with previous failed or reduced fertilization (n=10). Chromatin integrity (CMA3 staining and TUNEL assay), lipid peroxidation (Bodipy probe), and telomere length (Real time PCR) were compared with semen samples obtained
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from fertile individuals (n=10).
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Results: The results showed significantly higher mean of sperm DNA damage, lipid peroxidation and reduced telomere length in sperm of infertile men with previous failed or reduced fertilization
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compared to fertile individuals (p<0.05).
Conclusions: Low or failed fertilization rate could be related to oxidative stress resulting in short
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telomere length, and also increased sperm chromatin damage and lipid peroxidation. Based on literature, shortened telomere length may lead to detachment of chromosomes from nuclear
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membrane, the consequences of which are: defect in process of spermatogenesis, pronuclei formation, and delayed or arrested cell cycle post-ICSI. KEY MESSAGE Low or failed fertilization rate in infertile men with previously failed fertilization post-ICSI could be related to short telomere length in sperm due to a defect in spermatogenesis that results in failed pronuclei formation and delayed or arrested cell cycle post-ICSI.
ACCEPTED MANUSCRIPT Key words: sperm telomere length, protamine, oxidative stress, DNA fragmentation, ICSI, failed fertilization
Introduction Handling of human gametes outside the body is termed “assisted reproductive techniques” (ART).
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The application of these techniques has spread worldwide and is expanding day by day mainly due to increasing age of marriage and reduced quality of gametes. Intra-cytoplasmic sperm injection (ICSI) results in a high rate of fertilization and is one of the most commonly applied ART techniques, which has resulted in over 5million births globally (Palermo et al., 2012; Wiener-Megnazi et al., 2012). Notwithstanding the success of fertilization achieved by this technique, failed fertilization is
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reported in 1 to 5% of ICSI cycles. A study of literature suggests that severe male factor infertility is the main accountable factor that leads to failed oocyte activation and thereby failed fertilization (Flaherty et al., 1998; Swain et al., 2008; Kahyaoglu et al., 2014). In addition to the absence or low expression of phospholipase c zeta (PLCζ), which results in failed oocyte activation, loss of genomic integrity may also account for this dearth (Simon et al., 2010; Aghajanpour et al., 2011; Sanusi et al.,
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2015; Yelumalai et al., 2015; Javadian-Elyaderani et al., 2016; Tavalaee et al., 2016, 2018). Reduced sperm genomic integrity is related to several functional factors such as oxidative stress, apoptosis, and
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protamine deficiency, which can lead to DNA fragmentation (Nasr-Esfahani et al., 2008 and 2007; Bahreinian et al., 2015; Henkel et al., 2010; Utsuno et al., 2014). Recently, two authors have demonstrated significant negative correlations between reduced sperm telomere length and these three
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sperm functional factors (Rocca et al., 2016; Cariati et al., 2016). Among the aforementioned factors, imbalance in the oxidative state due to overproduction of reactive oxygen species (ROS) or reduced
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antioxidant capacity account for increased DNA fragmentation. In addition, aberrant histone/protamine and epigenetic anomalies render DNA sensitive to ROS and make DNA prone to fragmentation (Nasr-Esfahani et al., 2006; Tavalaee et al., 2008; Gharagozloo et al., 2011; Wright et
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al., 2014; Castillo et al., 2015). Another factor that affects chromosome and chromatin integrity is the length of DNA telomeres” (Cariati et al., 2016). Telomeres are DNA-protein complexes and single-stranded DNA, located at end of the
eukaryotic chromosome. They are heterochromatic structures of the non-coding DNA with the hexanucleutide sequence repeats 5'TTAGGG '3. In human somatic cells, telomeres have an average length of 5 to 10 kb while in germ cells telomeres are approximately 10 to 20 kb. Unlike somatic cells in which the length of telomeres decreases with cell divisions over time, in male germ cells the length of telomeres increases with age, emphasizing the importance of telomere in the maintenance of
ACCEPTED MANUSCRIPT genomic integrity and passage of intact genetic codes from one generation to the next (Thilagavathi et al., 2013a). The number of studies assessing the role of telomeres in human reproduction or ART outcomes are limited. The overall conclusion derived from these studies is that in semen samples with reduced sperm quality, and even in infertile men with normal semen parameters, the telomere length of sperm is reduced. Some of the studies show that this defect subsequently affects developmental competency of embryos (Rocca et al., 2016; Yang et al., 2015; Thilagavathi et al., 2013 b&c).
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However, to our knowledge there is no study that shows that shortened telomere length in sperm is another factor involved in human failed fertilization. Therefore in this study, in addition to assessing sperm protamine deficiency, DNA integrity and lipid peroxidation, we also assessed the telomere length in individuals with previous failed fertilization post-ICSI. Our results for the first time reveal that sperm telomere length is significantly reduced in men with previous failed fertilization post ICSI
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that prevalence of these infertile men in infertility in society is reported to be around 3%.
Materials and methods
This study was approved by the Ethical Board of Royan Institute
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(IR.ACECR.ROYAN.REC.1397.40). For this study, couples with previous low (< 20%) fertilization rate within the last 12 months (November 2010 to September 2014) were contacted and asked to
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participate in this study. Out of 28 infertile men that were contacted, only10 men accepted and/or were included to participate and provide a semen sample for analysis. Concomitant with the time that these individuals provided semen samples, 10 fertile men who requested pre-implantation genetic
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screening (PGS) for family balancing, were also accepted to participate in this study as the control group. All the individuals that participated in the study signed an informed consent form. Considering
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the low prevalence of failed fertilization (Kahyaoglu et al., 2014; Flaherty et al., 1998) post-ICSI (3%), and according to sample size formula, the minimum number of cases required was estimated to
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be 10.
Males aged over 50 years, and men with leukocytospermia (more than 1 million leukocytes in
1 ml of semen), azoospermia, Klinefelter syndrome and cancer patients were not included for this study. To reduce female factors, couples with fewer than 6 mature normal looking oocytes, polycystic ovarian diseases or endometriosis were also not included.
Semen samples collection
ACCEPTED MANUSCRIPT Semen samples were collected by masturbation into a sterile specimen container following 3– 5 days of sexual abstinence. These samples were maintained to liquefy for a period of 15–30 min at room temperature. Then, semen analysis was performed according to World Health Organization protocol (WHO, 2010). An aliquot of semen sample was used for assessment of sperm parameters. Sperm concentration and percentage of sperm motility was assessed by computer-aided sperm analysis (CASA) system (Video Test, Version Sperm 2.1©, Russia). The control of the system is carried out by a calibration test by a Neubauer chamber and microscopic ruler.
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For each sperm sample, 10 μl was placed on a sperm counter (Sperm Processor, Aurangabad, India) and sperm concentration and motility for each sample were analyzed and reported. In addition, sperm morphology was assessed by Diff-Quik staining (WHO, 2010) and analyzed by CASA system. A normal sperm has an oval head with an acrosome covering 40%–70% of the head area. Normal sperm have head length of 5-6 µm and diameter of 2.5-3.5 µm, with a single long tail. For each
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sample, 200 sperm were assessed and percentage of abnormal sperm morphology was reported.
The remaining sample was used for DNA extraction (Cawthon et al., 2002; O'Callaghan et al., 2011) and assessment of protamine deficiency (Chromomycin A3 staining) as a marker for sperm chromatin immaturity, DNA fragmentation (TUNEL assay), and sperm lipid peroxidation (bodipy
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probe) (Tavalaee et al., 2017; Aitken et al., 2007; Nasr-Esfahani et al., 2001).
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DNA extraction and telomere length assay by Quantitative Real-time PCR Semen samples were washed with phosphate-buffered saline (PBS) and, extraction of genomic DNA from washed sperm and peripheral blood leukocytes was performed using QIAamp
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DNA Mini Kit (QIAGEN, Milan, Italy) according to manufacturer’s recommendations. Sperm telomere length (STL) and leucocyte telomere length (LTL) were assessed using quantitative real-
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time polymerase chain reaction (qPCR) as previously described by Cawthon (Cawthon et al., 2002). Each sample was run in triplicate and telomere length was reported as relative and/or absolute value. The relative telomere length was calculated by the telomere to single-copy gene
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(T/S) ratio and expressed as [2^ (-ΔΔct)]. In regard to the assessment of absolute telomere length, two standard curves were created by serial dilutions of known amounts of oligomer standards (36B4 and telomere) in each reaction, and the data were expressed as kbp (O'Callaghan et al., 2011).
Assessment of sperm DNA fragmentation A droplet of washed sperm was smeared onto slides and fixed by freshly prepared 4% methanol-free paraformaldehyde for 25 minutes at room temperature. Next, slides were washed twice with PBS and then permeabilization of sperm was performed 0.2% Triton X-100 in PBS for 5
ACCEPTED MANUSCRIPT minutes. After equilibration of sperm with equilibration buffer for 7 minutes, a detection kit (Apoptosis Detection System Fluorescein, Promega, Germany) that contained nucleotide mix, rTdT and equilibration buffer was used and the process was continued according to the manufacturer's manual. Lastly, sperm were assessed using a fluorescence microscope (BX51, Tokyo, Japan) and for each sample, at least 500 sperm were counted. Sperm with green nuclei were considered as TUNEL positive (containing fragmented DNA) while sperm with red nuclei had intact
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DNA (Tavalaee et al., 2017).
Assessment of sperm protamine deficiency
Briefly, a droplet of washed sperm was fixed with Carnoy’s solution (methanol: glacial acetic acid, 3:1) and after 5 minutes, smears were prepared onto the slides. Then, slides were incubated with
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100 μl of Chromomycin A3 (CMA3) solution [0.25 mg/ml CMA3 in McIlvaine buffer (7 ml citric acid (0.1 M) 32.9 ml Na2HPO4.7 H2O (0.2 M), pH 7.0, containing 10 mM MgCl2)] for 20 minutes and washed twice with PBS. Samples were observed by using an Olympus fluorescence microscope (BX51, Tokyo, Japan) with the appropriate filters (460–470 nm) and at least 200 sperm cells were counted for each sample. Sperm with light yellow stain were considered as CMA3-positive
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content (Nasr-Esfahani et al., 2001).
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spermatozoa (protamine deficiency) while sperm with dark yellow stain had normal protamine
Assessment of sperm lipid peroxidation using BODIPY
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For sperm lipid peroxidation, BODIPY C11 loading BODIPYw 581/591 C11 (D3861, Molecular Probes) was used and added to 2 ×106 spermatozoa at a final concentration of 5 mM (30
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min at 37° C). Then, sperm were washed twice with PBS buffer and the percentage of lipid peroxidation or BODIPY positive spermatozoa was evaluated using a FACSCalibur flow
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cytometer (Becton Dickinson, San Jose, CA, USA) (Aitken et al., 2007).
Statistical analysis All statistical analyses were performed using SPSS software, version 22 (Chicago, IL, USA). The data was checked for normality with Shapiro-Wilk test. Then, an independent samples T test was used for comparison of mean values between groups. The data were expressed as mean ± standard error of mean (SEM). In addition, Pearson analysis was used to assess the correlations between
ACCEPTED MANUSCRIPT different parameters. P-values of less than 0.05 were considered as statistically significant. For this study, sample size was determined according to sample size formula:
n=
. In this formula, P was considered as percentage prevalence of failed fertilization
post-ICSI (0.03), ɑ and d were equal 0.05, and 0.1, respectively. Accordingly, the minimum number of cases in each group was considered to be 10.
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Results
In this study, semen samples were collected from 10 infertile men with previous failed or low fertilization and 10 fertile individuals with high fertilization. Sperm parameters and male age were compared between two groups (Table 1). Among these parameters, the mean of male age and semen volume were not significantly different between two groups. Mean of sperm concentration (38.14±8.9
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vs. 84.58±11.34), sperm count (86.27±10.07 vs. 314.27±65.7), and percentage of sperm motility
(44.00±6.01 vs. 58.71±2.8) were significantly lower in infertile men with failed or low fertilization compared to fertile individuals with high fertilization (p<0.05). In addition, percentage of abnormal sperm morphology was insignificantly higher in infertile men with failed or low fertilization than
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fertile individuals with high fertilization (p>0.05).
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Comparison of relative and absolute telomere length in sperm and leukocytes Telomere lengths in sperm and leukocytes were assessed by real-time PCR, and the data were
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analyzed via two approaches; relative and absolute method. Unlike leukocytes, the mean of relative telomere length in sperm was also significantly lower in infertile men with failed or low fertilization
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(0.74 ± 0.15) than fertile individuals (1.24 ± 0.18). Relative telomere length in leukocytes was not different between the two groups (Figure 1A). Similar to relative analysis, the mean of absolute telomere length in sperm was significantly lower in infertile men with failed or low fertilization (4.75
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± 0.84) compared to fertile individuals with high fertilization (8.80 ± 1.2) (Figure 1B).
Comparison of sperm lipid peroxidation and chromatin status Sperm lipid peroxidation, protamine deficiency and DNA fragmentation were assessed by bodipy probe, CMA3, and TUNEL staining, respectively (Figure 2). Mean percentage of lipid peroxidation (27.40 ± 4.30 vs.14.14 ±1.22; p=0.014), protamine deficiency (48.06 ±6.45 vs. 20.46
ACCEPTED MANUSCRIPT ±4.89; p=0.005), and DNA fragmentation (34.00 ± 5.43 vs. 12.65 ± 1.18; p=0.01) were significantly higher in infertile men with failed or low fertilization compared to fertile individuals (Figure 3).
Relationship between sperm parameters, telomere length, lipid peroxidation and chromatin status In this study, relationship between sperm parameters with absolute and relative telomere
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length in sperm and blood were assessed. Unlike sperm count, motility, and morphology, only sperm concentration showed a positive significant correlation with telomere length [absolute (r=0.586;
p=0.01) and relative (r=0.487; p=0.04)]. When relationship between lipid peroxidation and chromatin status were analyzed with telomere length in sperm and blood, there were negative significant
correlations between percentages of sperm lipid peroxidation with absolute and relative telomere
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length in sperm (Figure 4).
Significant negative correlations were also observed between sperm count with percentage of DNA damage (r= -0.547; p=0.028) and protamine deficiency (r= -0.477; p=0.04). Additionally, a negative significant correlation was observed between percent sperm motility and DNA damage (r=-0.815;
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p<0.001). We did not observe any significant correlations among other parameters (data not shown).
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Relationship between age (male age and maternal age at conception) with sperm parameters, lipid peroxidation, chromatin status and telomere length
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Negative significant correlations were observed between male age and percentage of lipid peroxidation (r=-0.530; p=0.029). With regard to maternal age at conception, there were negative
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significant correlations between absolute and relative telomere length in sperm with this parameter (r=
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-0.537; p=0.02, r= -0.515; p=0.02) respectively.
Relationship between fertilization rate and sperm telomere length, lipid peroxidation, and chromatin status Positive significant correlations were observed between fertilization rate with sperm telomere length, and negative significant correlations were also observed between fertilization rate with percentage of sperm lipid peroxidation, protamine deficiency, and DNA damage (Table 2).
ACCEPTED MANUSCRIPT Discussion In addition to numerous functions envisaged for telomeres in somatic cells, telomeres play important roles in both meiosis and early reproduction. In meiosis, telomeres ensure tethering of homologous chromosomes together and their attachment to the nuclear envelope. They also play important roles in synapsis and recombination events (Scherthan et al., 2007). Telomeres are among the first structures to contribute in the early event of male pronuclear formation after oocyte activation. This activity is mainly attributed to their attachment to the nuclear envelope (Scherthan et
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al., 2007; Zalenskaya et al., 2000). The telomeric part of the chromosome that attaches to the nuclear envelope has a nucleosomal organization rather than compaction via protamine (Zalenskaya et al., 2000).
The chromosome attaches to the nuclear envelope via two proteins, SUN1 and KASH5,
present on the inner and outer nuclear membrane, respectively. Attachment of chromosomes to the
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nuclear envelope is mediated via the histone rich region of telomere (Boateng et al., 2013; Link et al 2013, Ding etal., 2007; Morimoto et al., 2012). Therefore, a shortened telomere jeopardizes the proper attachment of chromosome to these proteins, the consequence of which would likely to be the higher rate of aneuploidy and failure of pronuclear formation (Garcia-Cruz et al., 2009). In this regard, there was significant association between sperm telomere length and fertilization rate and good quality
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embryos in IVF candidates (Yang et al., 2015). Despite these conclusions, other researchers believe that paternal telomere length is likely to be modified by recombination events in the oocyte through the blastocyst stage. Therefore, these authors concluded that there was no association between
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telomere length in human spermatozoa with sperm parameters and DNA integrity (Liu et al., 2002; Rodríguez et al., 2005; Liu and Li., 2010; Cariati et al., 2016; Turner and Hartshorne, 2013).
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Considering the importance of sperm telomere length in the initiation of pronuclear formation, we aimed to compare the length of sperm telomere in infertile men with previous low fertilization rate in
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comparison with fertile individuals with high fertilization rate. Several studies assessed only correlation of sperm telomere length with fertilization rate in infertile couple candidate for ICSI/ IVF/and or IUI. The advantage of this study is special selection of infertile couples with previous
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failed or low fertilization.
The results of this study revealed that in addition to lower sperm parameters in these individuals, both absolute and relative sperm telomere length were significantly shorter in infertile men with previous low fertilization rate than in fertile individuals. These results indicate that there might be an association between sperm telomere length and its ability to participate in pronuclear formation. These results are consistent with the proposed theory that sperm with shortened telomere length are unable to bind SUN1 and KASH5 on the nuclear membrane to induce nuclear decondensation and thereby pronuclear formation (Boateng et al., 2013; Link et al., 2013., Ding et al., 2007; Morimoto et
ACCEPTED MANUSCRIPT al., 2012). Some authors have suggested that shortened sperm telomere length is a compensatory mechanism by which oocytes ensure that development is initiated with a sperm with intact chromatin integrity as chromatin integrity is superimposed on telomere integrity, with telomere integrity more prone to DNA damage due to nucleosomal organization compared to protamine packed region of chromosomes (Derijck et al., 2008; Liu et al., 2002; Rodríguez et al., 2005; Liu and Li., 2010; Cariati et al., 2016; Turner and Hartshorne, 2013). The difference between the two groups is consistent with previous studies showing the association between fertilization rate and sperm telomere length while
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other studies did not observe such association (Yang et al., 2015; Turner et al., 2013). In this regard, a recently published paper by Kahyaoglu (2018) demonstrated that shortened sperm telomere length cannot effect fertilization and pregnancy outcome in ICSI cycles (Torra-Massana et al., 2018). These differences could be likely related to the cohort of individuals or couples recruited in their studies (IVF or ICSI candidates or idiopathic infertility) (Torra-Massana et al., 2018; Yang Q et al., 2015,). In this regard, Kurjanowicz et al., (2017) stated that type of analysis (comparative vs. correlative) in
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addition to patient characteristics like male and female age, BMI or oocyte quality, which affect
oocyte repair capacity (Yang et al 2016; Ferlin et al., 2013) account for the controversial differences observed between studies. In addition, they also stated significant differences for most measured parameters are observed, if the studies are comparative or if the number of samples are above 81 in the correlative studies and include individuals with normal semen parameters in their systemic review.
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This conclusion, therefore, appear to apply to samples that did not observed significant correlations. Our study was comparative and we observed differences for sperm telomere length between low
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versus high fertilization and hold the criteria stated by Kurjanowicz et al. (2017). It was interesting to note that we did not observe such a difference in leucocyte telomere length between the two groups, indicating that the observation is specific to sperm. Unlike shortened telomere length affecting the
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fertilization potential of sperm, recently Denomme et al (2017) demonstrated that alterations in the sperm histone-retained epigenome does not affect fertilization capacity but it may affect the
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developmental competency of resultant embryos (Denomme et al 2017). Therefore, normal length of sperm telomere can be considered as potential marker for competent fertilization. It is also important
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to note that Kurjanowicz et al. (2017) stated that method of DNA isolation which particularly reflects its effect on the telomere population assessed (Chromosomes vs. Chromosomes+ extrachromosomal telomere-specific sequences), could also affect the results. Isolation of DNA in current study was chromosomes+ extrachromosomal telomere-specific sequences which is less specific than chromosome DNA isolation. However, it is noticeable that we also presented two methods of sperm telomere analysis; absolute and relative. Since same procedure was applied for both group, we hope this may not undermine our results. To further investigate the relation between DNA integrity and sperm telomere length, we also compared the mean of sperm DNA fragmentation, chromatin compaction and lipid peroxidation as main inducer of DNA fragmentation between the two groups and our results revealed that these
ACCEPTED MANUSCRIPT parameters significantly were higher in infertile men compared to fertile individuals. These results may further correlate to the fact that sperm with short telomere may not be able to bind the nuclear envelope through SUN1 and KASH5 and such a sperm are unable to induce nuclear decondensation and, result in proper formation of pronuclei (Zalenskaya et al., 2000; Gineitis et al., 2000; Churikov et al., 2004; Reig-Viader et al., 2016).
Among the three aforementioned parameters only sperm lipid peroxidation and sperm
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concentration showed significant correlations with sperm telomere length. This is likely related to fact that the telomeric region has nucleosomes organization compared to protamine packed region of chromosomes which is less prone to DNA damage (Zalenskaya et al., 2000; Bahreinian et al., 2015). In addition, ROS damages to shelterin can induce cell cycle arrest via ATM/Chk1 (De Lange, 2009). In mouse, cell cycle arrest or delay was induced by hydrogen peroxide-treated epididymal
mouse spermatozoa which could be related to activation ATM/Chk1 through reduction of telomere
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length and inability of shelterin proteins to protect the telomere region and thereby the integrity of DNA (Wang et al., 2013). In addition, the significant negative associations observed between fertilization and the percentage of sperm DNA damage, lipid peroxidation and protamine deficiency also emphasize that these phenomena are highly related and may account for low or delayed fertilization observed in ICSI cycles.
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In conclusion, this result, for the first time, shows that one of the causes of failed fertilization after ICSI could be due to the reduction of sperm telomere length in the original sperm. Reduced sperm
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telomere length could be indirectly associated with an increase in the level of DNA fragmentation, deficiency of protamine and lipid peroxidation in these individuals. Therefore, reduction of sperm telomere length in infertile men with previous low or failed fertilization could be accompanied with a
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defect in pronuclei formation, and delayed or arrest in cell cycle post ICSI. One of the shortcomings of this study is the low number of cases due to the low prevalence of failed fertilization in infertile
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men, especially in centers practicing artificial oocyte activation. We also have not yet established a sequence of causality, though it seems likely that telomere length is an important starting point. More
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studies are needed for further verification of the proposed hypothesis.
Acknowledgement This study was supported by Royan Institute and we would like to express our gratitude to staff of Isfahan Fertility and Infertility for their full support. In addition, we also thank Dr. R.A. Lockshin for critical review of the manuscript.
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Link, J., Jahn, D., Schmitt, J., Göb, E., Baar, J., Ortega, S., Benavente, R., Alsheimer, M., 2013.The meiotic nuclear lamina regulates chromosome dynamics and promotes efficient homologous recombination in the mouse. PLoS Genet. 9, e1003261.
Liu, L., Blasco, M.A., Keefe, D.L., 2002.Requirement of functional telomeres for metaphase chromosome alignments and integrity of meiotic spindles. EMBO. Rep.3,230-4. Liu, J.P., Li, H., 2010.Telomerase in the ovary. Reproduction. 140,215-22.
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Morimoto, A., Shibuya, H., Zhu, X., Kim, J., Ishiguro, K., Han, M., Watanabe, Y., 2012.A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis. J. Cell.Biol. 198,165-72.
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Nasr-Esfahani, M.H., Naghshizadian, N., Imani, H., Razavi, S., Mardani, M., Kazemi, S., Shahvardi, H., 2006.Can sperm protamine deficiency induce sperm premature chromosomal condensation? Androl. 38,92-8.
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Nasr-Esfahani, M.H., Razavi, S., Mardani, M., Shirazi, R., Javanmardi, S., 2007.Effects of failed oocyte activation and sperm protamine deficiency on fertilization post-ICSI. Reprod. Biomed Online. 14,422-9.
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Nasr-Esfahani, M.H., Razavi, S., Mardani, M., 2001.Relation between different human sperm nuclear maturity tests and in vitro fertilization. J.Assist. Reprod. Genet. 18,219-25.
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Nasr-Esfahani, M.H., Razavi, S., Tavalaee, M., 2008.Failed fertilization after ICSI and spermiogenic defects. Fertil.Steril. 89,892-8. O'Callaghan, N.J., Fenech, M., 2011.A quantitative PCR method for measuring absolute telomere length. Biol Proced Online.13,3. Palermo, G.D., Neri, Q.V., Monahan, D., Kocent, J., Rosenwaks, Z.,2012. Development and current applications of assisted fertilization. Fertil Steril. 97,248-59. Reig-Viader, R., Garcia-Caldés, M., Ruiz-Herrera, A., 2016. Telomere homeostasis in mammalian germ cells: a review. Chromosoma. 125,337-51. Rocca, M.S., Speltra, E., Menegazzo, M., Garolla, A., Foresta, C., Ferlin, A., 2016.Sperm telomere length as a parameter of sperm quality in normozoospermic men. Hum. Reprod. 31,1158-63.
ACCEPTED MANUSCRIPT Rodríguez, S., Goyanes, V., Segrelles, E., Blasco, M., Gosálvez, J., Fernández, J.L., 2005. Critically short telomeres are associated with sperm DNA fragmentation. Fertil.Steril. 84,843-5. Sanusi, R., Yu. Y., Nomikos, M., Lai, F.A., Swann, K., 2015.Rescue of failed oocyte activation after ICSI in a mouse model of male factor infertility by recombinant phospholipase Cζ. Mol .Hum. Reprod. 21,783-91. Scherthan, H., 2007.Telomere attachment and clustering during meiosis. Cell Mol Life Sci. 64,117-24.
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Simon, L., Brunborg, G., Stevenson, M. Lutton, D., McManus, J., Lewis, S.E., 2010.Clinical significance of sperm DNA damage in assisted reproduction outcome. Hum. Reprod. 25,1594608. Swain, J. E., Pool, T. B., 2008. ART failure: oocyte contributions to unsuccessful fertilization. Hum. Reprod. 14, 431–446.
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Tavalaee, M., Nomikos, M., Lai, F.A., Nasr-Esfahani, M.H., 2018. Expression of sperm PLCζ and clinical outcomes of ICSI-AOA in men affected by globozoospermia due to DPY19L2 deletion. Reprod. Biomed Online. 36,348-355. Tavalaee, M., Nasr-Esfahani, M.H., 2016.Expression profile of PLCζ, PAWP, and TR-KIT in association with fertilization potential, embryo development, and pregnancy outcomes in globozoospermic candidates for intra-cytoplasmic sperminjection and artificial oocyte activation. Androl. 4,850-6.
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Tavalaee, M., Kiani-Esfahani, A., Nasr-Esfahani, M.H., 2017. Relationship between phospholipase C-zeta, semen parameters, and chromatin status. Syst Biol. Reprod. Med.63,259268.
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Thilagavathi, J., Venkatesh, S., Dada, R., 2013a.Telomere length in reproduction. Androl. 45,289-304. Thilagavathi, J., Kumar, M., Mishra, S.S., Venkatesh, S., Kumar, R., Dada, R., 2013b. Analysis of sperm telomere length in men with idiopathic infertility. Arch Gynecol Obstet. 287,803-7.
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Thilagavathi, J., Mishra, S.S., Kumar, M., Vemprala, K., Deka, D., Dhadwal, V., Dada, R., 2013c. Analysis of telomere length in couples experiencing idiopathic recurrent pregnancy loss. J. Assist. Reprod. Genet.30,793-8. Torra-Massana M., Barragán M., Bellu E., Oliva R., Rodríguez A., Vassena R., 2018. Sperm telomere length in donor samples is not related to ICSI outcome. J. Assist. Reprod. Genet. 35(4):649-657. Turner, S., Hartshorne, G.M., 2013.Telomere lengths in human pronuclei, oocytes and spermatozoa. Mol Hum. Reprod. 19,510-8. Utsuno, H., Miyamoto, T., Oka, K., Shiozawa, T., 2014.Morphological alterations in protaminedeficient spermatozoa. Hum. Reprod. 29,2374-81. Wang, B., Li, Z., Wang, C., Chen, M., Xiao, J., Wu, X., Xiao, W., Song, Y., Wang, X., 2013. Zygotic G2/M cell cycle arrest induced by ATM/Chk1 activation and DNA repair in mouse
ACCEPTED MANUSCRIPT embryos fertilized with hydrogen peroxide-treated epididymal mouse sperm. PLoS One. 8, e73987 Wiener-Megnazi, Z., Auslender, R., Dirnfeld, M.,2012. Advanced paternal age and reproductive outcome. Asian ,J. Androl. 14,69-76. World Health Organization., 2010.WHO Laboratory Manual for the Examination and Processing of Human Semen 5th edn. Cambridge, UK: Cambridge University Press.
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Wright, C., Milne, S., Leeson, H., 2014.Sperm DNA damage caused by oxidative stress: modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod. Biomed Online. 28,684-703. Yang, Q., Zhao, F., Dai, S., Zhang, N., Zhao, W., Bai, R., Sun, Y., 2015.Sperm telomere length is positively associated with the quality of early embryonic development. Hum. Reprod.30,1876-81.
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Yang Q., Zhao F., Hu L., Bai R., Zhang N., Yao G., Sun Y., 2016.Effect of paternal overweight or obesity on IVF treatment outcomes and the possible mechanisms involved. Sci. Rep. 14;6:29787. Yelumalai, S., Yeste, M., Jones, C., Amdani,S.N., Kashir, J., Mounce, G., Da Silva, S.J., Barratt, C.L., McVeigh, E., Coward, K., 2015. Total levels, localization patterns, and proportions of sperm exhibiting phospholipase C zeta are significantly correlated with fertilization rates after intracytoplasmic sperm injection. Fertil. Steril. 104,561-8. e4.
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Zalenskaya, I.A., Bradbury, E.M., Zalensky, A.O., 2000.Chromatin structure of telomere domain in human sperm. Biochem Biophys Res Commun. 279,213-8.
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Figure 1 - Comparison of absolute (A) and relative (B) sperm telomere length (STL) and leucocyte telomere length (LTL) between infertile men with failed fertilization and fertile
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individuals with high fertilization. * indicates P< 0.05.
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Figure 2 - Flow cytometric dot plot of sperm lipid peroxidation assessed by Bodipy in a fertile man with a high fertilization rate (A) and an infertile man
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with low fertilization rate (B). Figure C show fluorescence images of CMA3 staining for protamine deficiency (dark yellow stain is considered normal for protamine content while spermatozoa with light yellow stain are considered as protamine deficient). Figure 4D show fluorescence images of TUNEL positive sperm (red stain spermatozoa is considered as intact while spermatozoa with green stain are considered as containing fragmented DNA).
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60
P=0.005 50
P=0.01 P=0.01
30
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Percentage
40
20
0
Lipid peroxidation
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10
Protamine deficiency Fertile
DNA damage
Failed fertilization
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Figure 3 - Comparison of mean percentage of sperm lipid peroxidation, protamine
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deficiency, and DNA damage between infertile men with failed or low fertilization rate and
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fertile individuals with high fertilization.
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Figure 4 - Correlation between absolute (AB) and relative (R) sperm telomere
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length (S-TL) with sperm concentration (A) and sperm lipid peroxidation (B).
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Parameters
Failed fertilization
Fertile
Male age (Year)
38.1±4.17
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Table 1- Comparison of male age and sperm parameters between infertile men with failed fertilization (n=10) and fertile individuals with high fertilization (n=10).
Sperm concentration (106/ml)
38.14±8.9
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86.27±10.07
Sperm motility (%)
44.00±6.01
Semen volume (ml)
2.95±0.35
Abnormal sperm morphology (%)
97.10±0.65
40.11±3.14
0.249
84.58±11.34
0.005
314.27±65.7
0.002
58.71±2.8
0.049
3.83±0.7
0.260
96.7±0.5
0.551
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Sperm count (10 /ejaculate)
P-value
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S-TL (AB)
S-TL (R)
Protamine deficiency
DNA Fragmentation
Lipid peroxidation
r
0.61
0.47
-0.56
-0.62
-0.52
p
0.007
0.004
0.01
0.01
0.03
Parameter
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Fertilization rate (%)
ACCEPTED MANUSCRIPT Mohammad Hossein Nasr-Esfahani received his PhD from the University of Cambridge, UK, in 1991 and is currently an academic member of the Royan Institute in Tehran, Iran. He has been working as laboratory director of the Isfahan Fertility and Infertility Centre since 1992 and has especial interests on male infertility. He is also the head of Royan Institute for biotechnology in Isfahan, Iran. The main research areas of the groups with which he works are stem cells with interest on neuro-
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regeneration, animal cloning, recombinant protein and male infertility. He has over 350 publications in international and national journals. The project of the first Iranian cloned sheep and transgenic
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animal was carried out under his supervision.
Evaluation of sperm telomere length in infertile men with failed fertilization after intra-cytoplasmic sperm injection Zahra Darmishonnejad , Marziyeh Tavalaee , Tayebeh Izadi , Somayeh Tanhaei , Mohammad Hossein Nasr-Esfahania PII: DOI: Reference:
S1472-6483(18)30651-5 https://doi.org/10.1016/j.rbmo.2018.12.022 RBMO 2084
To appear in:
Reproductive BioMedicine Online
Received date: Revised date: Accepted date:
7 June 2018 6 October 2018 10 December 2018
Please cite this article as: Zahra Darmishonnejad , Marziyeh Tavalaee , Tayebeh Izadi , Somayeh Tanhaei , Mohammad Hossein Nasr-Esfahania , Evaluation of sperm telomere length in infertile men with failed fertilization after intra-cytoplasmic sperm injection, Reproductive BioMedicine Online (2018), doi: https://doi.org/10.1016/j.rbmo.2018.12.022
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ACCEPTED MANUSCRIPT Evaluation of sperm telomere length in infertile men with failed fertilization after intracytoplasmic sperm injection
Zahra Darmishonnejada,b, Marziyeh Tavalaeea, Tayebeh Izadic, Somayeh Tanhaeic, Mohammad
a
ACECR Institute of Higher Education (Isfahan Branch)
b
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Hossein Nasr-Esfahaniad
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran c
d
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Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran Isfahan Fertility and Infertility Center, Isfahan, Iran
*
Corresponding author.
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CONTACT Mohammad Hossein Nasr-Esfahani [email protected] Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan
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Institute for Biotechnology, ACECR, Isfahan, Iran.
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ABSTRACT Research Question: Telomeres are non-coding, repetitive DNA sequences (TTAGGG repeats) that play an important role in maintaining genome integrity. Unlike in somatic cells, telomere length in sperm increases with men age and is considered as a molecular marker of sperm quality. Etiology of
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failed fertilization post- intracytoplasmic sperm injection (ICSI) technique is multifactorial; perhaps one of the reasons for this failure in these individuals is shortened sperm telomere length. Therefore, we aimed to assess sperm telomere length in addition to DNA damage, lipid peroxidation and
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protamine deficiency in infertile men with previously failed fertilization post-ICSI.
Design: Semen samples were obtained from infertile men with previous failed or reduced fertilization (n=10). Chromatin integrity (CMA3 staining and TUNEL assay), lipid peroxidation (Bodipy probe), and telomere length (Real time PCR) were compared with semen samples obtained
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from fertile individuals (n=10).
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Results: The results showed significantly higher mean of sperm DNA damage, lipid peroxidation and reduced telomere length in sperm of infertile men with previous failed or reduced fertilization
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compared to fertile individuals (p<0.05).
Conclusions: Low or failed fertilization rate could be related to oxidative stress resulting in short
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telomere length, and also increased sperm chromatin damage and lipid peroxidation. Based on literature, shortened telomere length may lead to detachment of chromosomes from nuclear
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membrane, the consequences of which are: defect in process of spermatogenesis, pronuclei formation, and delayed or arrested cell cycle post-ICSI. KEY MESSAGE Low or failed fertilization rate in infertile men with previously failed fertilization post-ICSI could be related to short telomere length in sperm due to a defect in spermatogenesis that results in failed pronuclei formation and delayed or arrested cell cycle post-ICSI.
ACCEPTED MANUSCRIPT Key words: sperm telomere length, protamine, oxidative stress, DNA fragmentation, ICSI, failed fertilization
Introduction Handling of human gametes outside the body is termed “assisted reproductive techniques” (ART).
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The application of these techniques has spread worldwide and is expanding day by day mainly due to increasing age of marriage and reduced quality of gametes. Intra-cytoplasmic sperm injection (ICSI) results in a high rate of fertilization and is one of the most commonly applied ART techniques, which has resulted in over 5million births globally (Palermo et al., 2012; Wiener-Megnazi et al., 2012). Notwithstanding the success of fertilization achieved by this technique, failed fertilization is
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reported in 1 to 5% of ICSI cycles. A study of literature suggests that severe male factor infertility is the main accountable factor that leads to failed oocyte activation and thereby failed fertilization (Flaherty et al., 1998; Swain et al., 2008; Kahyaoglu et al., 2014). In addition to the absence or low expression of phospholipase c zeta (PLCζ), which results in failed oocyte activation, loss of genomic integrity may also account for this dearth (Simon et al., 2010; Aghajanpour et al., 2011; Sanusi et al.,
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2015; Yelumalai et al., 2015; Javadian-Elyaderani et al., 2016; Tavalaee et al., 2016, 2018). Reduced sperm genomic integrity is related to several functional factors such as oxidative stress, apoptosis, and
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protamine deficiency, which can lead to DNA fragmentation (Nasr-Esfahani et al., 2008 and 2007; Bahreinian et al., 2015; Henkel et al., 2010; Utsuno et al., 2014). Recently, two authors have demonstrated significant negative correlations between reduced sperm telomere length and these three
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sperm functional factors (Rocca et al., 2016; Cariati et al., 2016). Among the aforementioned factors, imbalance in the oxidative state due to overproduction of reactive oxygen species (ROS) or reduced
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antioxidant capacity account for increased DNA fragmentation. In addition, aberrant histone/protamine and epigenetic anomalies render DNA sensitive to ROS and make DNA prone to fragmentation (Nasr-Esfahani et al., 2006; Tavalaee et al., 2008; Gharagozloo et al., 2011; Wright et
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al., 2014; Castillo et al., 2015). Another factor that affects chromosome and chromatin integrity is the length of DNA telomeres” (Cariati et al., 2016). Telomeres are DNA-protein complexes and single-stranded DNA, located at end of the
eukaryotic chromosome. They are heterochromatic structures of the non-coding DNA with the hexanucleutide sequence repeats 5'TTAGGG '3. In human somatic cells, telomeres have an average length of 5 to 10 kb while in germ cells telomeres are approximately 10 to 20 kb. Unlike somatic cells in which the length of telomeres decreases with cell divisions over time, in male germ cells the length of telomeres increases with age, emphasizing the importance of telomere in the maintenance of
ACCEPTED MANUSCRIPT genomic integrity and passage of intact genetic codes from one generation to the next (Thilagavathi et al., 2013a). The number of studies assessing the role of telomeres in human reproduction or ART outcomes are limited. The overall conclusion derived from these studies is that in semen samples with reduced sperm quality, and even in infertile men with normal semen parameters, the telomere length of sperm is reduced. Some of the studies show that this defect subsequently affects developmental competency of embryos (Rocca et al., 2016; Yang et al., 2015; Thilagavathi et al., 2013 b&c).
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However, to our knowledge there is no study that shows that shortened telomere length in sperm is another factor involved in human failed fertilization. Therefore in this study, in addition to assessing sperm protamine deficiency, DNA integrity and lipid peroxidation, we also assessed the telomere length in individuals with previous failed fertilization post-ICSI. Our results for the first time reveal that sperm telomere length is significantly reduced in men with previous failed fertilization post ICSI
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that prevalence of these infertile men in infertility in society is reported to be around 3%.
Materials and methods
This study was approved by the Ethical Board of Royan Institute
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(IR.ACECR.ROYAN.REC.1397.40). For this study, couples with previous low (< 20%) fertilization rate within the last 12 months (November 2010 to September 2014) were contacted and asked to
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participate in this study. Out of 28 infertile men that were contacted, only10 men accepted and/or were included to participate and provide a semen sample for analysis. Concomitant with the time that these individuals provided semen samples, 10 fertile men who requested pre-implantation genetic
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screening (PGS) for family balancing, were also accepted to participate in this study as the control group. All the individuals that participated in the study signed an informed consent form. Considering
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the low prevalence of failed fertilization (Kahyaoglu et al., 2014; Flaherty et al., 1998) post-ICSI (3%), and according to sample size formula, the minimum number of cases required was estimated to
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be 10.
Males aged over 50 years, and men with leukocytospermia (more than 1 million leukocytes in
1 ml of semen), azoospermia, Klinefelter syndrome and cancer patients were not included for this study. To reduce female factors, couples with fewer than 6 mature normal looking oocytes, polycystic ovarian diseases or endometriosis were also not included.
Semen samples collection
ACCEPTED MANUSCRIPT Semen samples were collected by masturbation into a sterile specimen container following 3– 5 days of sexual abstinence. These samples were maintained to liquefy for a period of 15–30 min at room temperature. Then, semen analysis was performed according to World Health Organization protocol (WHO, 2010). An aliquot of semen sample was used for assessment of sperm parameters. Sperm concentration and percentage of sperm motility was assessed by computer-aided sperm analysis (CASA) system (Video Test, Version Sperm 2.1©, Russia). The control of the system is carried out by a calibration test by a Neubauer chamber and microscopic ruler.
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For each sperm sample, 10 μl was placed on a sperm counter (Sperm Processor, Aurangabad, India) and sperm concentration and motility for each sample were analyzed and reported. In addition, sperm morphology was assessed by Diff-Quik staining (WHO, 2010) and analyzed by CASA system. A normal sperm has an oval head with an acrosome covering 40%–70% of the head area. Normal sperm have head length of 5-6 µm and diameter of 2.5-3.5 µm, with a single long tail. For each
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sample, 200 sperm were assessed and percentage of abnormal sperm morphology was reported.
The remaining sample was used for DNA extraction (Cawthon et al., 2002; O'Callaghan et al., 2011) and assessment of protamine deficiency (Chromomycin A3 staining) as a marker for sperm chromatin immaturity, DNA fragmentation (TUNEL assay), and sperm lipid peroxidation (bodipy
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probe) (Tavalaee et al., 2017; Aitken et al., 2007; Nasr-Esfahani et al., 2001).
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DNA extraction and telomere length assay by Quantitative Real-time PCR Semen samples were washed with phosphate-buffered saline (PBS) and, extraction of genomic DNA from washed sperm and peripheral blood leukocytes was performed using QIAamp
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DNA Mini Kit (QIAGEN, Milan, Italy) according to manufacturer’s recommendations. Sperm telomere length (STL) and leucocyte telomere length (LTL) were assessed using quantitative real-
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time polymerase chain reaction (qPCR) as previously described by Cawthon (Cawthon et al., 2002). Each sample was run in triplicate and telomere length was reported as relative and/or absolute value. The relative telomere length was calculated by the telomere to single-copy gene
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(T/S) ratio and expressed as [2^ (-ΔΔct)]. In regard to the assessment of absolute telomere length, two standard curves were created by serial dilutions of known amounts of oligomer standards (36B4 and telomere) in each reaction, and the data were expressed as kbp (O'Callaghan et al., 2011).
Assessment of sperm DNA fragmentation A droplet of washed sperm was smeared onto slides and fixed by freshly prepared 4% methanol-free paraformaldehyde for 25 minutes at room temperature. Next, slides were washed twice with PBS and then permeabilization of sperm was performed 0.2% Triton X-100 in PBS for 5
ACCEPTED MANUSCRIPT minutes. After equilibration of sperm with equilibration buffer for 7 minutes, a detection kit (Apoptosis Detection System Fluorescein, Promega, Germany) that contained nucleotide mix, rTdT and equilibration buffer was used and the process was continued according to the manufacturer's manual. Lastly, sperm were assessed using a fluorescence microscope (BX51, Tokyo, Japan) and for each sample, at least 500 sperm were counted. Sperm with green nuclei were considered as TUNEL positive (containing fragmented DNA) while sperm with red nuclei had intact
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DNA (Tavalaee et al., 2017).
Assessment of sperm protamine deficiency
Briefly, a droplet of washed sperm was fixed with Carnoy’s solution (methanol: glacial acetic acid, 3:1) and after 5 minutes, smears were prepared onto the slides. Then, slides were incubated with
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100 μl of Chromomycin A3 (CMA3) solution [0.25 mg/ml CMA3 in McIlvaine buffer (7 ml citric acid (0.1 M) 32.9 ml Na2HPO4.7 H2O (0.2 M), pH 7.0, containing 10 mM MgCl2)] for 20 minutes and washed twice with PBS. Samples were observed by using an Olympus fluorescence microscope (BX51, Tokyo, Japan) with the appropriate filters (460–470 nm) and at least 200 sperm cells were counted for each sample. Sperm with light yellow stain were considered as CMA3-positive
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content (Nasr-Esfahani et al., 2001).
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spermatozoa (protamine deficiency) while sperm with dark yellow stain had normal protamine
Assessment of sperm lipid peroxidation using BODIPY
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For sperm lipid peroxidation, BODIPY C11 loading BODIPYw 581/591 C11 (D3861, Molecular Probes) was used and added to 2 ×106 spermatozoa at a final concentration of 5 mM (30
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min at 37° C). Then, sperm were washed twice with PBS buffer and the percentage of lipid peroxidation or BODIPY positive spermatozoa was evaluated using a FACSCalibur flow
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cytometer (Becton Dickinson, San Jose, CA, USA) (Aitken et al., 2007).
Statistical analysis All statistical analyses were performed using SPSS software, version 22 (Chicago, IL, USA). The data was checked for normality with Shapiro-Wilk test. Then, an independent samples T test was used for comparison of mean values between groups. The data were expressed as mean ± standard error of mean (SEM). In addition, Pearson analysis was used to assess the correlations between
ACCEPTED MANUSCRIPT different parameters. P-values of less than 0.05 were considered as statistically significant. For this study, sample size was determined according to sample size formula:
n=
. In this formula, P was considered as percentage prevalence of failed fertilization
post-ICSI (0.03), ɑ and d were equal 0.05, and 0.1, respectively. Accordingly, the minimum number of cases in each group was considered to be 10.
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Results
In this study, semen samples were collected from 10 infertile men with previous failed or low fertilization and 10 fertile individuals with high fertilization. Sperm parameters and male age were compared between two groups (Table 1). Among these parameters, the mean of male age and semen volume were not significantly different between two groups. Mean of sperm concentration (38.14±8.9
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vs. 84.58±11.34), sperm count (86.27±10.07 vs. 314.27±65.7), and percentage of sperm motility
(44.00±6.01 vs. 58.71±2.8) were significantly lower in infertile men with failed or low fertilization compared to fertile individuals with high fertilization (p<0.05). In addition, percentage of abnormal sperm morphology was insignificantly higher in infertile men with failed or low fertilization than
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fertile individuals with high fertilization (p>0.05).
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Comparison of relative and absolute telomere length in sperm and leukocytes Telomere lengths in sperm and leukocytes were assessed by real-time PCR, and the data were
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analyzed via two approaches; relative and absolute method. Unlike leukocytes, the mean of relative telomere length in sperm was also significantly lower in infertile men with failed or low fertilization
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(0.74 ± 0.15) than fertile individuals (1.24 ± 0.18). Relative telomere length in leukocytes was not different between the two groups (Figure 1A). Similar to relative analysis, the mean of absolute telomere length in sperm was significantly lower in infertile men with failed or low fertilization (4.75
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± 0.84) compared to fertile individuals with high fertilization (8.80 ± 1.2) (Figure 1B).
Comparison of sperm lipid peroxidation and chromatin status Sperm lipid peroxidation, protamine deficiency and DNA fragmentation were assessed by bodipy probe, CMA3, and TUNEL staining, respectively (Figure 2). Mean percentage of lipid peroxidation (27.40 ± 4.30 vs.14.14 ±1.22; p=0.014), protamine deficiency (48.06 ±6.45 vs. 20.46
ACCEPTED MANUSCRIPT ±4.89; p=0.005), and DNA fragmentation (34.00 ± 5.43 vs. 12.65 ± 1.18; p=0.01) were significantly higher in infertile men with failed or low fertilization compared to fertile individuals (Figure 3).
Relationship between sperm parameters, telomere length, lipid peroxidation and chromatin status In this study, relationship between sperm parameters with absolute and relative telomere
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length in sperm and blood were assessed. Unlike sperm count, motility, and morphology, only sperm concentration showed a positive significant correlation with telomere length [absolute (r=0.586;
p=0.01) and relative (r=0.487; p=0.04)]. When relationship between lipid peroxidation and chromatin status were analyzed with telomere length in sperm and blood, there were negative significant
correlations between percentages of sperm lipid peroxidation with absolute and relative telomere
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length in sperm (Figure 4).
Significant negative correlations were also observed between sperm count with percentage of DNA damage (r= -0.547; p=0.028) and protamine deficiency (r= -0.477; p=0.04). Additionally, a negative significant correlation was observed between percent sperm motility and DNA damage (r=-0.815;
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p<0.001). We did not observe any significant correlations among other parameters (data not shown).
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Relationship between age (male age and maternal age at conception) with sperm parameters, lipid peroxidation, chromatin status and telomere length
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Negative significant correlations were observed between male age and percentage of lipid peroxidation (r=-0.530; p=0.029). With regard to maternal age at conception, there were negative
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significant correlations between absolute and relative telomere length in sperm with this parameter (r=
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-0.537; p=0.02, r= -0.515; p=0.02) respectively.
Relationship between fertilization rate and sperm telomere length, lipid peroxidation, and chromatin status Positive significant correlations were observed between fertilization rate with sperm telomere length, and negative significant correlations were also observed between fertilization rate with percentage of sperm lipid peroxidation, protamine deficiency, and DNA damage (Table 2).
ACCEPTED MANUSCRIPT Discussion In addition to numerous functions envisaged for telomeres in somatic cells, telomeres play important roles in both meiosis and early reproduction. In meiosis, telomeres ensure tethering of homologous chromosomes together and their attachment to the nuclear envelope. They also play important roles in synapsis and recombination events (Scherthan et al., 2007). Telomeres are among the first structures to contribute in the early event of male pronuclear formation after oocyte activation. This activity is mainly attributed to their attachment to the nuclear envelope (Scherthan et
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al., 2007; Zalenskaya et al., 2000). The telomeric part of the chromosome that attaches to the nuclear envelope has a nucleosomal organization rather than compaction via protamine (Zalenskaya et al., 2000).
The chromosome attaches to the nuclear envelope via two proteins, SUN1 and KASH5,
present on the inner and outer nuclear membrane, respectively. Attachment of chromosomes to the
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nuclear envelope is mediated via the histone rich region of telomere (Boateng et al., 2013; Link et al 2013, Ding etal., 2007; Morimoto et al., 2012). Therefore, a shortened telomere jeopardizes the proper attachment of chromosome to these proteins, the consequence of which would likely to be the higher rate of aneuploidy and failure of pronuclear formation (Garcia-Cruz et al., 2009). In this regard, there was significant association between sperm telomere length and fertilization rate and good quality
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embryos in IVF candidates (Yang et al., 2015). Despite these conclusions, other researchers believe that paternal telomere length is likely to be modified by recombination events in the oocyte through the blastocyst stage. Therefore, these authors concluded that there was no association between
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telomere length in human spermatozoa with sperm parameters and DNA integrity (Liu et al., 2002; Rodríguez et al., 2005; Liu and Li., 2010; Cariati et al., 2016; Turner and Hartshorne, 2013).
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Considering the importance of sperm telomere length in the initiation of pronuclear formation, we aimed to compare the length of sperm telomere in infertile men with previous low fertilization rate in
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comparison with fertile individuals with high fertilization rate. Several studies assessed only correlation of sperm telomere length with fertilization rate in infertile couple candidate for ICSI/ IVF/and or IUI. The advantage of this study is special selection of infertile couples with previous
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failed or low fertilization.
The results of this study revealed that in addition to lower sperm parameters in these individuals, both absolute and relative sperm telomere length were significantly shorter in infertile men with previous low fertilization rate than in fertile individuals. These results indicate that there might be an association between sperm telomere length and its ability to participate in pronuclear formation. These results are consistent with the proposed theory that sperm with shortened telomere length are unable to bind SUN1 and KASH5 on the nuclear membrane to induce nuclear decondensation and thereby pronuclear formation (Boateng et al., 2013; Link et al., 2013., Ding et al., 2007; Morimoto et
ACCEPTED MANUSCRIPT al., 2012). Some authors have suggested that shortened sperm telomere length is a compensatory mechanism by which oocytes ensure that development is initiated with a sperm with intact chromatin integrity as chromatin integrity is superimposed on telomere integrity, with telomere integrity more prone to DNA damage due to nucleosomal organization compared to protamine packed region of chromosomes (Derijck et al., 2008; Liu et al., 2002; Rodríguez et al., 2005; Liu and Li., 2010; Cariati et al., 2016; Turner and Hartshorne, 2013). The difference between the two groups is consistent with previous studies showing the association between fertilization rate and sperm telomere length while
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other studies did not observe such association (Yang et al., 2015; Turner et al., 2013). In this regard, a recently published paper by Kahyaoglu (2018) demonstrated that shortened sperm telomere length cannot effect fertilization and pregnancy outcome in ICSI cycles (Torra-Massana et al., 2018). These differences could be likely related to the cohort of individuals or couples recruited in their studies (IVF or ICSI candidates or idiopathic infertility) (Torra-Massana et al., 2018; Yang Q et al., 2015,). In this regard, Kurjanowicz et al., (2017) stated that type of analysis (comparative vs. correlative) in
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addition to patient characteristics like male and female age, BMI or oocyte quality, which affect
oocyte repair capacity (Yang et al 2016; Ferlin et al., 2013) account for the controversial differences observed between studies. In addition, they also stated significant differences for most measured parameters are observed, if the studies are comparative or if the number of samples are above 81 in the correlative studies and include individuals with normal semen parameters in their systemic review.
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This conclusion, therefore, appear to apply to samples that did not observed significant correlations. Our study was comparative and we observed differences for sperm telomere length between low
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versus high fertilization and hold the criteria stated by Kurjanowicz et al. (2017). It was interesting to note that we did not observe such a difference in leucocyte telomere length between the two groups, indicating that the observation is specific to sperm. Unlike shortened telomere length affecting the
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fertilization potential of sperm, recently Denomme et al (2017) demonstrated that alterations in the sperm histone-retained epigenome does not affect fertilization capacity but it may affect the
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developmental competency of resultant embryos (Denomme et al 2017). Therefore, normal length of sperm telomere can be considered as potential marker for competent fertilization. It is also important
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to note that Kurjanowicz et al. (2017) stated that method of DNA isolation which particularly reflects its effect on the telomere population assessed (Chromosomes vs. Chromosomes+ extrachromosomal telomere-specific sequences), could also affect the results. Isolation of DNA in current study was chromosomes+ extrachromosomal telomere-specific sequences which is less specific than chromosome DNA isolation. However, it is noticeable that we also presented two methods of sperm telomere analysis; absolute and relative. Since same procedure was applied for both group, we hope this may not undermine our results. To further investigate the relation between DNA integrity and sperm telomere length, we also compared the mean of sperm DNA fragmentation, chromatin compaction and lipid peroxidation as main inducer of DNA fragmentation between the two groups and our results revealed that these
ACCEPTED MANUSCRIPT parameters significantly were higher in infertile men compared to fertile individuals. These results may further correlate to the fact that sperm with short telomere may not be able to bind the nuclear envelope through SUN1 and KASH5 and such a sperm are unable to induce nuclear decondensation and, result in proper formation of pronuclei (Zalenskaya et al., 2000; Gineitis et al., 2000; Churikov et al., 2004; Reig-Viader et al., 2016).
Among the three aforementioned parameters only sperm lipid peroxidation and sperm
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concentration showed significant correlations with sperm telomere length. This is likely related to fact that the telomeric region has nucleosomes organization compared to protamine packed region of chromosomes which is less prone to DNA damage (Zalenskaya et al., 2000; Bahreinian et al., 2015). In addition, ROS damages to shelterin can induce cell cycle arrest via ATM/Chk1 (De Lange, 2009). In mouse, cell cycle arrest or delay was induced by hydrogen peroxide-treated epididymal
mouse spermatozoa which could be related to activation ATM/Chk1 through reduction of telomere
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length and inability of shelterin proteins to protect the telomere region and thereby the integrity of DNA (Wang et al., 2013). In addition, the significant negative associations observed between fertilization and the percentage of sperm DNA damage, lipid peroxidation and protamine deficiency also emphasize that these phenomena are highly related and may account for low or delayed fertilization observed in ICSI cycles.
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In conclusion, this result, for the first time, shows that one of the causes of failed fertilization after ICSI could be due to the reduction of sperm telomere length in the original sperm. Reduced sperm
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telomere length could be indirectly associated with an increase in the level of DNA fragmentation, deficiency of protamine and lipid peroxidation in these individuals. Therefore, reduction of sperm telomere length in infertile men with previous low or failed fertilization could be accompanied with a
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defect in pronuclei formation, and delayed or arrest in cell cycle post ICSI. One of the shortcomings of this study is the low number of cases due to the low prevalence of failed fertilization in infertile
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men, especially in centers practicing artificial oocyte activation. We also have not yet established a sequence of causality, though it seems likely that telomere length is an important starting point. More
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studies are needed for further verification of the proposed hypothesis.
Acknowledgement This study was supported by Royan Institute and we would like to express our gratitude to staff of Isfahan Fertility and Infertility for their full support. In addition, we also thank Dr. R.A. Lockshin for critical review of the manuscript.
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Figure 1 - Comparison of absolute (A) and relative (B) sperm telomere length (STL) and leucocyte telomere length (LTL) between infertile men with failed fertilization and fertile
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individuals with high fertilization. * indicates P< 0.05.
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Figure 2 - Flow cytometric dot plot of sperm lipid peroxidation assessed by Bodipy in a fertile man with a high fertilization rate (A) and an infertile man
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with low fertilization rate (B). Figure C show fluorescence images of CMA3 staining for protamine deficiency (dark yellow stain is considered normal for protamine content while spermatozoa with light yellow stain are considered as protamine deficient). Figure 4D show fluorescence images of TUNEL positive sperm (red stain spermatozoa is considered as intact while spermatozoa with green stain are considered as containing fragmented DNA).
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P=0.005 50
P=0.01 P=0.01
30
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Percentage
40
20
0
Lipid peroxidation
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10
Protamine deficiency Fertile
DNA damage
Failed fertilization
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Figure 3 - Comparison of mean percentage of sperm lipid peroxidation, protamine
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deficiency, and DNA damage between infertile men with failed or low fertilization rate and
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fertile individuals with high fertilization.
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Figure 4 - Correlation between absolute (AB) and relative (R) sperm telomere
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length (S-TL) with sperm concentration (A) and sperm lipid peroxidation (B).
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Parameters
Failed fertilization
Fertile
Male age (Year)
38.1±4.17
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Table 1- Comparison of male age and sperm parameters between infertile men with failed fertilization (n=10) and fertile individuals with high fertilization (n=10).
Sperm concentration (106/ml)
38.14±8.9
6
86.27±10.07
Sperm motility (%)
44.00±6.01
Semen volume (ml)
2.95±0.35
Abnormal sperm morphology (%)
97.10±0.65
40.11±3.14
0.249
84.58±11.34
0.005
314.27±65.7
0.002
58.71±2.8
0.049
3.83±0.7
0.260
96.7±0.5
0.551
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Sperm count (10 /ejaculate)
P-value
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S-TL (AB)
S-TL (R)
Protamine deficiency
DNA Fragmentation
Lipid peroxidation
r
0.61
0.47
-0.56
-0.62
-0.52
p
0.007
0.004
0.01
0.01
0.03
Parameter
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Fertilization rate (%)
ACCEPTED MANUSCRIPT Mohammad Hossein Nasr-Esfahani received his PhD from the University of Cambridge, UK, in 1991 and is currently an academic member of the Royan Institute in Tehran, Iran. He has been working as laboratory director of the Isfahan Fertility and Infertility Centre since 1992 and has especial interests on male infertility. He is also the head of Royan Institute for biotechnology in Isfahan, Iran. The main research areas of the groups with which he works are stem cells with interest on neuro-
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regeneration, animal cloning, recombinant protein and male infertility. He has over 350 publications in international and national journals. The project of the first Iranian cloned sheep and transgenic
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animal was carried out under his supervision.