- Email: [email protected]
Evaluation of Sperm Deoxyribonucleic Acid (DNA) Damage and Effects on Embryo Development Using a Mouse Cryptorchidism Model
Basic and Translational Science Evaluation of Sperm Deoxyribonucleic Acid (DNA) Damage and Effects on Embryo Development Using a Mouse Cryptorchidism Model Seung-Hun Song, Jung Jin Lim, Jeong Kyoon Bang, Soo Kyung Cha, Dong Ryul Lee, You Shin Kim, Tai Young Ahn, and Tae Ki Yoon OBJECTIVE MATERIALS AND METHODS
RESULTS
CONCLUSION
To investigate the effects of sperm deoxyribonucleic acid (DNA) damage on fertilization and embryo development using a mouse cryptorchidism model of sperm DNA damage induction. Male ICR mice (aged 5-6 weeks) underwent cryptorchidism on their left testicles and sham operations on their right testicles. Spermatogenesis and sperm DNA fragmentation were assessed after 1, 2, and 4 weeks using hematoxylin-eosin staining, terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate-biotin nick end labeling assays. Intracytoplasmic sperm injection into the oocytes of BDF1 females (aged 4-6 weeks) was performed using DNA-damaged sperm and normal sperm, and the fertilization rates and embryonic development were compared. The testicular weight and size gradually decreased after induction of cryptorchidism, with progressive reduction of spermatogenesis and increased DNA damage after 1, 2, and 4 weeks. After intracytoplasmic sperm injection, the fertilization and blastocyst development rates were significantly lower in the cryptorchidism group; however, about one quarter of the embryos arising from DNA-damaged sperm continued to develop. This was an in vivo animal study to evaluate the effects of sperm DNA damage using a cryptorchidism model. Sperm DNA damage increased significantly over time after cryptorchidism. This model could be useful in investigating male factor infertility and evaluating the biologic effects of paternal DNA damage on fertilization and future embryonic development. UROLOGY 82: 743.e17e743.e23, 2013. 2013 Elsevier Inc.
M
ale factor infertility is present in up to 50% of infertile couples, making it increasingly important in their treatment.1,2 Many aspects of male infertility, however, remain poorly understood, and medical therapy for these patients has largely been unsuccessful. In contrast, recent advances in in vitro fertilization (IVF) techniques, especially intracytoplasmic sperm injection (ICSI), require only a small number of
Seung-Hun Song and Jung Jin Lim contributed equally as first authors to this work. Financial Disclosure: The authors declare that they have no relevant financial interests. Funding Support: This study was supported by the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family affairs, Seoul Korea (grant A084923). The sponsor had no role in the design and conduct of the study, data collection, analysis, or interpretation, or preparation, review, or approval of our report. From the Department of Urology, Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea; Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea; the Department of Obstetrics and Gynecology, Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea; and the Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Reprint requests: Tae Ki Yoon, M.D., Department of Obstetrics and Gynecology, Fertility Center, CHA Gangnam Medical Center, CHA University, 650-9 Yeoksam-1 dong, Gangnam-gu, Seoul 135-913 Korea. E-mail: [email protected] Submitted: December 10, 2012, accepted (with revisions): May 8, 2013
ª 2013 Elsevier Inc. All Rights Reserved
viable sperm and have revolutionized the treatment of men with severe male factor infertility by allowing infertile men with severely compromised semen parameters to achieve fatherhood.3-6 Most IVF pregnancies will proceed uneventfully and will result in the birth of healthy infants. However, studies have consistently identified an increased absolute risk of problems with IVF and IVF plus ICSI pregnancies and deliveries.7-9 It has been assumed that many cases of male factor infertility might be the result of genetic factors, including cytogenetic abnormalities and microdeletions of the Y chromosome.10 Now, much evidence has shown that infertile men possess substantially greater sperm deoxyribonucleic acid (DNA) damage than do fertile men.11,12 This is clinically important, given that most patients with severe male factor infertility would be treated with assisted reproductive techniques such as ICSI, and sperm DNA damage was recently reported to be associated with increased risks of pregnancy loss after IVF and ICSI.13 In addition, it has been reported that spermatozoa with defective DNA can fertilize an oocyte and produce a high-quality, early-stage embryo; however, as the extent 0090-4295/13/$36.00 743.e17 http://dx.doi.org/10.1016/j.urology.2013.05.015
of the DNA damage increases, the likelihood of a successful pregnancy to term decreases.14 Therefore, studies that induce sperm DNA damage and evaluate the biologic effects of sperm DNA damage on the next generation are very important. To evaluate the effects of sperm DNA damage on fertilization and embryo development, we used a mouse cryptorchidism model to induce damage to sperm DNA in vivo.
MATERIAL AND METHODS
the oocytes were maintained in KSOM medium at 37 C in 5% carbon dioxide in air until ICSI. Testicular sperm from male mice with induced cryptorchidism were collected in M2 medium by excising with a pair of fine scissors and forceps, followed by squeezing in a 50-mL drop of M2 under mineral oil. The sperm were allowed to disperse at 37 C for 30 minutes. Each suspension was transferred to a 50-mL Eppendorf tube for centrifugation at 1500 rpm for 5 minutes. Motile spermatozoa were allowed to swim up in the supernatant for 30 minutes. Both cryptorchidism and control sperm were mixed with 40 mL of 10% polyvinyl-pyrrolidone (PVP-360) in M2, followed by placement in a culture dish for microinjection.
Mice Male ICR mice (aged 5-6 weeks) and BDF1 female mice (aged 4-6 weeks) were obtained from IcrTacSam (Samtaco Korea, Osan, Korea). The mice were housed in a temperature- and humidity-controlled room with a 12/12-hour light/dark cycle and were provided ad libitum food and water. The Institutional Animal Care and Use Committee of CHA University approved the protocols for the use of the mice in these experiments.
Preparation of Cryptorchidism Mouse Model The surgical procedure for cryptorchidism has been described previously.15 In brief, male ICR mice (weight 30-32 g) were deeply anesthetized by intraperitoneal injection of xylazine (10 mg/kg) and ketamine (90 mg/kg), and the left testicle of each mouse was exposed through an incision above the inguinal canal. The epididymal fat pad was grasped, and the testicle was transferred into the abdominal cavity. The inguinal canal was closed by sutures to prevent descent of the testicle into the scrotum. The right testicle of each mouse underwent sham operation as the control group. The mice were killed 1, 2, and 4 weeks later by intraperitoneal injection of 200 mg/kg pentobarbital. The testicles and epididymis were removed, weighed, fixed in paraformaldehyde (4% vol/vol in Dulbecco’s phosphatebuffered saline [DPBS]) for 24 hours and embedded in paraffin. The sections were stained with hematoxylin-eosin.
Hormonal (Testosterone, Luteinizing Hormone, Follicle-stimulating Hormone) Assays Blood samples were taken from the anesthetized cryptorchidism mice by cardiac puncture and collected in serum separate tubes. Serum was prepared using centrifugation for 20 minutes at 3000 rpm. Serum hormonal levels were measured using a commercially available double antibody radioimmunoassay kits using the Access Immunoassay System (UniCel Dxl 800 system, Beckman Coulter, Chaska, MN).
Collection of Oocytes and Sperm The BDF1 female mice were superovulated by injection of 5 IU pregnant mare serum gonadotropin (Folligon, Intervet International, Boxmeer, The Netherlands), followed 48 hours later by injection of human chorionic gonadotropin (Chorulon, Intervet International). At 14 hours after human chorionic gonadotropin, the oviducts were removed from the female mice and placed in a Petri dish containing modified human tubal fluid medium (Irvine Scientific, Santa Ana, CA) at room temperature. After washing, the oviducts were placed in fresh medium, and cumulus-oocyte complexes were released from the ampulla using Dumont forceps. In the ICSI experiments, the cumulus cells were dispersed by incubation for 3-5 minutes in tubal fluid medium containing 350 IU/mL of hyaluronidase. After washing, 743.e18
ICSI and Embryo Culture One volume of each sperm preparation was mixed with 5 volumes of M2 medium containing 10% PVP at room temperature to decrease stickiness. Each ICSI dish contained 1 drop of M2 medium, 1 drop of sperm solution in M2 containing 10% PVP, and 1 drop of M2 containing 10% PVP for needle cleaning. Injections were performed with a PMM-150 FU piezoimpact unit (Prime Tech) and micromanipulators using a bluntended, mercury-containing pipette (inner volume, 6-7 mL). After 15 minutes of recovery at room temperature in M2 medium, the surviving oocytes were returned to mineral oilcovered KSOM and cultured at 37 C in an atmosphere of 5% carbon dioxide in air. For embryo culture, 50 mL of KSOM covered with mineral oil were equilibrated overnight at 37 C in a humidified atmosphere of 5% carbon dioxide. The oocytes were scored for pronucleus formation (fertilization) 6 hours after the initiation of culture, and the numbers of blastocysts were counted after 92-96 hours in culture.
Detection of Damage to Sperm DNA Terminal Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate-biotin Nick End Labeling Assay. DNA fragmentation was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assays (TUNEL) assays (In Situ Cell Death Detection Kit, TMR red, Roche Diagnostics, Indianapolis, IN). After washing 3 times with DPBS, aliquots of sperm were fixed in 1% paraformaldehyde in DPBS for 1 hour, and permeated with 0.2% Tween-20 in DPBS for 1 hour. The sperm were incubated in TUNEL reaction medium (50 mL) for 1 hour at 37 C in the dark and stained with 10 mL of 1 mg/mL 40 ,60 - diamidino 2-phenyindiol (Sigma-Aldrich) in phosphate-buffered saline for 10 minutes at 37 C. The supernatants were removed by centrifugation at 1500 rpm for 5 minutes, and excess deoxyuridine triphosphate nick-end labeling mixture was removed by washing 3 times in DPBS. The slides were examined under a fluorescence microscope, with the presence of red fluorescence at the head of the sperm considered indicative of damaged DNA. Negative (omitting the enzyme terminal transferase) and positive (using deoxyribonuclease I, 1 IU/mL for 20 minutes at room temperature) controls were performed in each experiment. A total of 200 sperm cells of each aliquot were examined. In the negative controls, none of the cells showed fluorescent staining, but in the positive controls, 100% of the cells showed DNA fragmentation. Caspase-3 Assay. Sperm apoptosis was assessed using caspase-3 activities (Caspase 3 Assay Kit, colorimetric; Abcam, Cambridge, UK). After washing 3 times with DPBS, aliquots of UROLOGY 82 (3), 2013
Figure 1. Gross morphologic changes and serum testosterone levels after cryptorchidism. (A) Gross morphologic changes showing decreases over time in testicular size and weight after cryptorchidism. (B) Serum testosterone levels 1, 2, and 4 weeks after cryptorchidism. sperm were lysed in a lysis buffer. The lysed sperms were centrifuged at 14,000 rpm for 10 minutes and then transferred supernatant (cytosolic extract) to a fresh tube. Then, 50 mg of protein was incubated with 50 mL of 2 reaction buffer and 5 mL of caspase-3 (4 mM DEVD-p-NA) colorimetric substrate at 37 C for 1-2 hours. The optical density of the reaction mixture was quantitated spectrophotometrically at a wavelength of 405 nm using a 96-well plate reader (Perkin Elmer spectrofluorometer, Victor 3). The x-fold increase in caspase 3 activity was determined by comparing these results with the level of the normal mouse sperm or substrate-omitted wells.
scanning microscope (LSM 510, Carl Zeiss, Oberkochen, Germany) with fluorescence at 400 magnification. The micrographs were stored in LSM (Zeiss LSM Image Browser, version 2.30.011; Carl Zeiss Jena GmbH, Jena, Germany).
Statistical Analysis Unless otherwise specified, each experiment was performed using 3 replicates. The statistical significance of differences among treated groups was evaluated by 1-way analysis of variance using a log-linear model in the Statistical Analysis System (SAS Institute, Cary, NC). P <.05 was considered statistically significant.
Detection of Leydig Cell and Apoptosis To investigate the colocalization of Leydig cells and apoptotic cells in the cryptorchidism mouse testicle, we performed doubleimmunohistochemistry using 3b-HSD (3-b-hydroxy-steroiddehydrogenase, Leydig cell-specific marker; Santa Cruz Biotechnology, Santa Cruz, CA) and TUNEL substrate. Each cryptorchidism testicular sections were rehydration and heatmediated antigen retrieval. For permeabilization, the sections were incubated in 0.1% Triton X-100 in DPBS for 10 minutes. After washing 3 times with DPBS, nonspecific binding of antibodies was suppressed by incubation in blocking solution (4% normal goat serum in DPBS) for 30 minutes at room temperature. After additional washing 3 times with PBS, immunohistochemistry staining was performed by incubating the fixed samples with 3b-HSD antibody diluted to 1:200 with DPBS containing 1% bovine serum albumin for 2 hours at room temperature. After incubation, the sections were incubated in TUNEL reaction medium (50 mL) for 1 hour at 37 C. The 3b-HSD was detected using 5 mg/mL of fluorescein isothiocyanate-conjugated secondary antibodies. Finally, the sections were counterstained using 1 mg/mL 40 ,60 - diamidino 2-phenyindiol (Sigma-Aldrich). After the washes, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The resulting staining was viewed on an inverted confocal laser UROLOGY 82 (3), 2013
RESULTS Fifteen male ICR mice were used for the cryptorchidism model (cryptorchidism duration 1, 2, and 4 weeks, n ¼ 5 each). An additional 15 mice, with a cryptorchidism duration of 2 weeks, were used for the ICSI procedure. Oocytes were collected from 45 BDF1 female mice. In the cryptorchidism model, the left testicle of each mouse was fixed to the lower abdominal wall, and the right testicle was allowed to move freely. We observed gradual reductions in the left testicular weight and size at 1, 2, and 4 weeks after the induction of cryptorchidism (Fig. 1A). Although the serum testosterone levels decreased after cryptorchidism, the levels were not significantly different (Fig. 1B). Neither were the serum follicle-stimulating hormone and luteinizing hormone levels (data not shown). Histologic analysis showed sequential reduction of spermatogenesis at 1, 2, and 4 weeks postoperatively (Fig. 2). At 1 week after surgery, normal spermatogenesis, as indicated by the presence of the heads of elongated sperm, was maintained. At 2 weeks after surgery, 743.e19
Figure 2. Hematoxylin-eosin staining of testicle and epididymis. Immunohistochemistry of seminiferous tubules. (A) Histologic changes in testicle and epididymis at 1, 2, and 4 weeks after cryptorchidism showing decrease over time in spermatogenesis. The red box indicates the magnified area. (B) 3b-HSD and TMR-red (apoptotic cells) localization patterns in cross-section of cryptorchidism mouse testicle. 3b-HSD immunoreactivity detected in interstitial cells and Leydig cells. However, no TMR-red signaling was detected in these cells. Some TMR-redepositive cells were detected in spermatogonia or spermatocytes. N-Cont., negative control; Mock first ab, not treated with primary antibodies; Mock enzymes, not treated with deoxyuridine triphosphate-biotin nick-end labeling mixture. Scale bars ¼ 100 mm. 743.e20
UROLOGY 82 (3), 2013
Figure 3. Detection of damaged sperm. (A) Photomicrographs of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-stained sperm after cryptorchidism. TUNEL staining showed that the number of apoptotic cells was significantly increased in the cryptorchidism testicle in each group compared with the normal testicle. Scale bars ¼ 30 mm. (B) Graphic representation of stained TUNEL-positive sperm (P <.05). (C) Sperm lysates were assayed for caspase-3 activity at 1, 2, and 4 weeks after cryptorchidism. Graphic representation of relative caspase-3 activity.
spermatogenesis had markedly decreased. After 4 weeks, the Sertoli cells were predominant, with scarce spermatocytes. However, the Leydig cells still maintained their characters after 4 weeks of cryptorchidism (Fig. 2B). The TUNEL and caspase-3 assay showed that the percentage and activity of DNA-damaged sperm gradually increased after the induction of cryptorchidism (Fig. 3). To evaluate the effects of damage to sperm DNA on fertilization and embryo development, we compared the results of the ICSI procedures using morphologically normal sperm from control testicles and cryptorchid testicles 2 weeks after surgery. We found that the fertilization rate and embryo development were within the normal ranges in the control group but were markedly decreased in the cryptorchidism group. However, despite the poor fertilization rate, about one quarter of embryos fertilized by sperm from the cryptorchidism group continued to develop further (Fig. 4).
COMMENT During the previous 30 years, the treatment of infertility has been revolutionized by the development of new UROLOGY 82 (3), 2013
assisted reproductive technologies, including IVF and ICSI. The introduction of ICSI in 1992 offered the opportunity for parenthood to men with severe oligozoospermia or azoospermia and revolutionized the treatment of male infertility.3 Unlike most therapeutic procedures used in medicine, however, fertilization by ICSI never underwent rigorous safety testing before clinical use. The level of sperm DNA fragmentation was recently reported to be higher in men with severe male factor infertility, including those with severe oligozoospermia and nonobstructive azoospermia.11,12 It has also been suggested that idiopathic severe male factor infertility might be a progressive disease, showing aggravation from severe oligozoospermia to complete azoospermia.16 Therefore, testing the biologic effects of sperm DNA damage on fertilization and embryonic development are very important for studying male factor infertility. We have used a mouse cryptorchidism model to induce sperm DNA damage in vivo. In mice with cryptorchidism, an increase in scrotal temperature has been regarded as the primary cause of sperm DNA damage in the testicles. To our knowledge, this is the first in vivo animal study to evaluate the effects of cryptorchidism-induced 743.e21
Figure 4. Effects of sperm deoxyribonucleic acid (DNA) damage on embryonic development after intracytoplasmic sperm injection. Embryonic development was significantly lower in the cryptorchidism group than in the normal group.
damage to sperm DNA on fertilization and embryonic development. During ICSI, using sperm from patients with severe male factor infertility, a significant proportion of the spermatozoa injected into oocytes can contain fragmented DNA. Therefore, the animal model used in the present study might be useful in investigating male factor infertility and in evaluating the biologic effects of paternal DNA damage on fertilization and embryonic development. We selected morphologically normal sperm, determined by microscopy under 200 magnification, for the ICSI procedures. Sperm DNA integrity during ICSI can be critically important, because this procedure bypasses multiple, apparently redundant mechanisms that have evolved to ensure the selection of high-quality sperm cells for fertilization. Most infertility clinics around the world do not perform sperm DNA fragmentation analysis before ICSI. Our findings suggested that morphologically normal sperm selected by microscopic examination can carry fragmented DNA. After ICSI, the fertilization rate and embryonic development were within the normal ranges in the control group, but both were markedly reduced in the cryptorchidism group. Despite the reduced fertilization rate, about one quarter of embryos generated from the latter group continued to develop further. Therefore, the biologic effect of abnormal sperm chromatin structure depends on the combined effects of the extent of DNA or 743.e22
chromatin damage in the spermatozoa and the capacity of the oocyte to repair that damage. Additional studies of embryos generated from DNA-damaged sperm, including embryo transfer to the female mouse uterus and assays of birth rates and development are needed to evaluate the long-term effects of sperm DNA damage. Many studies have analyzed the effects of sperm DNA damage on assisted reproductive technique pregnancies.17 Recently, sperm DNA damage was reported to be associated with an increased risk of pregnancy loss after IVF and ICSI,13 suggesting the importance of developing strategies to reduce sperm DNA damage in humans, such as reducing exposure to environmental toxins and testicular hyperthermia, supplementation with antioxidant vitamins, and varicocele repair.18-20 To date, few animal studies have assessed the effects of induced sperm DNA damage on fertilization and embryo development. Using a mouse model system, sperm with defective DNA were shown to fertilize oocytes and produce high-quality, early-stage embryos; however, the extent of DNA damage was inversely related to the likelihood of bringing a successful pregnancy to term.14 In addition, a recent animal study evaluating the long-term effects in adult offspring of DNA fragmented sperm, induced by freeze thawing of spermatozoa in the absence of a cryoprotector, found that some of these effects only emerge during later life, including aberrant growth, premature aging, abnormal behavior, and the development UROLOGY 82 (3), 2013
of mesenchymal tumors.21 In other studies, sperm DNA damage was induced by direct short-term scrotal exposure to heated water.22,23 In the in vivo cryptorchidism model used in the present work, we believe that sperm DNA damage was induced under more physiologic stress conditions. Our findings suggest that the extent of sperm DNA damage was related to the duration of cryptorchidism. Therefore, this animal model could be used to evaluate the threshold of sperm DNA damage for safe fertilization and development. The present study had several limitations, included providing little information on the development of the offspring. This was because of several reasons, including technical difficulties. Moreover, the ICSI procedure itself might have deleterious effects on fertilization and early embryo development. Additional studies evaluating the development of offspring generated by fertilization with DNA-damaged sperm and strategies reducing damage to sperm DNA before fertilization are warranted.
CONCLUSION This was an in vivo animal study to evaluate the effects of sperm DNA damage using a cryptorchidism model. Sperm DNA damage increased significantly over time after cryptorchidism operation. This model might be useful in investigating male factor infertility and evaluating the biologic effects of paternal DNA damage on fertilization and future embryonic development. References 1. Bhasin S, de Krester DM, Baker HW. Clinical review 64: pathophysiology and natural history of male infertility. J Clin Endocrinol Metab. 1994;79:1525-1529. 2. Andersen AN, Goossens V, Gianaroli L, et al. Assisted reproductive technology in Europe, 2003: results generated from European registers by ESHRE. Hum Reprod. 2007;22:1513-1525. 3. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992;340:17-18. 4. Nagy ZP, Liu J, Joris H, et al. The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod. 1995;10:1123-1129. 5. Devroey P, Liu J, Nagy Z, et al. Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril. 1994;62:639-641.
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6. Silber SJ. Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia. Hum Reprod. 2000;15:2278-2284. 7. Klemetti R, Gissler M, Sevon T, et al. Children born after assisted fertilization have an increased rate of major congenital anomalies. Fertil Steril. 2005;84:1300-1307. 8. Olson CK, Keppler-Noreuil KM, Romitti PA, et al. In vitro fertilization is associated with an increase in major birth defects. Fertil Steril. 2005;84:1308-1315. 9. Reddy UM, Wapner RJ, Rebar RW, et al. Infertility, assisted reproductive technology, and adverse pregnancy outcomes: executive summary of a National Institute of Child Health and Human Development workshop. Obstet Gynecol. 2007;109:967-977. 10. Lilford R, Jones AM, Bishop DT, et al. Case-control study of whether subfertility in men is familial. BMJ. 1994;309:570-573. 11. Irvine DS, Twigg JP, Gordon EL, et al. DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 2000;21:33-44. 12. Zini A, Fischer MA, Sharir S, et al. Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology. 2002;60: 1069-1072. 13. Zini A, Boman JM, Belzile E, et al. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2008;23:26632668. 14. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool. 1999;284:696-704. 15. Rossi LM, Pereira LA, de Santis L, et al. Sperm retrieval techniques in rats with suppressed spermatogenesis by experimental cryptorchidism. Hum Reprod. 2005;20:443-447. 16. Song SH, Bak CW, Lim JJ, et al. Natural course of severe oligozoospermia in infertile male: influence on future fertility potential. J Androl. 2010;31:536-539. 17. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril. 2008; 89:823-831. 18. Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci. 2000;22:169-189. 19. Silver EW, Eskenazi B, Evenson DP, et al. Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men. J Androl. 2005;26:550-556. 20. Werthman P, Wixon R, Kasperson K, et al. Significant decrease in sperm deoxyribonucleic acid fragmentation after varicocelectomy. Fertil Steril. 2008;90:1800-1804. 21. Fernandez-Gonzalez R, Moreira PN, Perez-Crespo ML, et al. Longterm effects of mouse intracytoplasmic sperm injection with DNAfragmented sperm on health and behavior of adult offspring. Biol Reprod. 2008;78:761-772. 22. Banks S, King SA, Irvine DS, et al. Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa. Reproduction. 2005; 129:505-514. 23. Perez-Crespo M, Pintado B, Gutierrez-Adan A. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol Reprod Dev. 2008;75:40-47.
743.e23
RESULTS
CONCLUSION
To investigate the effects of sperm deoxyribonucleic acid (DNA) damage on fertilization and embryo development using a mouse cryptorchidism model of sperm DNA damage induction. Male ICR mice (aged 5-6 weeks) underwent cryptorchidism on their left testicles and sham operations on their right testicles. Spermatogenesis and sperm DNA fragmentation were assessed after 1, 2, and 4 weeks using hematoxylin-eosin staining, terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate-biotin nick end labeling assays. Intracytoplasmic sperm injection into the oocytes of BDF1 females (aged 4-6 weeks) was performed using DNA-damaged sperm and normal sperm, and the fertilization rates and embryonic development were compared. The testicular weight and size gradually decreased after induction of cryptorchidism, with progressive reduction of spermatogenesis and increased DNA damage after 1, 2, and 4 weeks. After intracytoplasmic sperm injection, the fertilization and blastocyst development rates were significantly lower in the cryptorchidism group; however, about one quarter of the embryos arising from DNA-damaged sperm continued to develop. This was an in vivo animal study to evaluate the effects of sperm DNA damage using a cryptorchidism model. Sperm DNA damage increased significantly over time after cryptorchidism. This model could be useful in investigating male factor infertility and evaluating the biologic effects of paternal DNA damage on fertilization and future embryonic development. UROLOGY 82: 743.e17e743.e23, 2013. 2013 Elsevier Inc.
M
ale factor infertility is present in up to 50% of infertile couples, making it increasingly important in their treatment.1,2 Many aspects of male infertility, however, remain poorly understood, and medical therapy for these patients has largely been unsuccessful. In contrast, recent advances in in vitro fertilization (IVF) techniques, especially intracytoplasmic sperm injection (ICSI), require only a small number of
Seung-Hun Song and Jung Jin Lim contributed equally as first authors to this work. Financial Disclosure: The authors declare that they have no relevant financial interests. Funding Support: This study was supported by the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family affairs, Seoul Korea (grant A084923). The sponsor had no role in the design and conduct of the study, data collection, analysis, or interpretation, or preparation, review, or approval of our report. From the Department of Urology, Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea; Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea; the Department of Obstetrics and Gynecology, Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea; and the Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Reprint requests: Tae Ki Yoon, M.D., Department of Obstetrics and Gynecology, Fertility Center, CHA Gangnam Medical Center, CHA University, 650-9 Yeoksam-1 dong, Gangnam-gu, Seoul 135-913 Korea. E-mail: [email protected] Submitted: December 10, 2012, accepted (with revisions): May 8, 2013
ª 2013 Elsevier Inc. All Rights Reserved
viable sperm and have revolutionized the treatment of men with severe male factor infertility by allowing infertile men with severely compromised semen parameters to achieve fatherhood.3-6 Most IVF pregnancies will proceed uneventfully and will result in the birth of healthy infants. However, studies have consistently identified an increased absolute risk of problems with IVF and IVF plus ICSI pregnancies and deliveries.7-9 It has been assumed that many cases of male factor infertility might be the result of genetic factors, including cytogenetic abnormalities and microdeletions of the Y chromosome.10 Now, much evidence has shown that infertile men possess substantially greater sperm deoxyribonucleic acid (DNA) damage than do fertile men.11,12 This is clinically important, given that most patients with severe male factor infertility would be treated with assisted reproductive techniques such as ICSI, and sperm DNA damage was recently reported to be associated with increased risks of pregnancy loss after IVF and ICSI.13 In addition, it has been reported that spermatozoa with defective DNA can fertilize an oocyte and produce a high-quality, early-stage embryo; however, as the extent 0090-4295/13/$36.00 743.e17 http://dx.doi.org/10.1016/j.urology.2013.05.015
of the DNA damage increases, the likelihood of a successful pregnancy to term decreases.14 Therefore, studies that induce sperm DNA damage and evaluate the biologic effects of sperm DNA damage on the next generation are very important. To evaluate the effects of sperm DNA damage on fertilization and embryo development, we used a mouse cryptorchidism model to induce damage to sperm DNA in vivo.
MATERIAL AND METHODS
the oocytes were maintained in KSOM medium at 37 C in 5% carbon dioxide in air until ICSI. Testicular sperm from male mice with induced cryptorchidism were collected in M2 medium by excising with a pair of fine scissors and forceps, followed by squeezing in a 50-mL drop of M2 under mineral oil. The sperm were allowed to disperse at 37 C for 30 minutes. Each suspension was transferred to a 50-mL Eppendorf tube for centrifugation at 1500 rpm for 5 minutes. Motile spermatozoa were allowed to swim up in the supernatant for 30 minutes. Both cryptorchidism and control sperm were mixed with 40 mL of 10% polyvinyl-pyrrolidone (PVP-360) in M2, followed by placement in a culture dish for microinjection.
Mice Male ICR mice (aged 5-6 weeks) and BDF1 female mice (aged 4-6 weeks) were obtained from IcrTacSam (Samtaco Korea, Osan, Korea). The mice were housed in a temperature- and humidity-controlled room with a 12/12-hour light/dark cycle and were provided ad libitum food and water. The Institutional Animal Care and Use Committee of CHA University approved the protocols for the use of the mice in these experiments.
Preparation of Cryptorchidism Mouse Model The surgical procedure for cryptorchidism has been described previously.15 In brief, male ICR mice (weight 30-32 g) were deeply anesthetized by intraperitoneal injection of xylazine (10 mg/kg) and ketamine (90 mg/kg), and the left testicle of each mouse was exposed through an incision above the inguinal canal. The epididymal fat pad was grasped, and the testicle was transferred into the abdominal cavity. The inguinal canal was closed by sutures to prevent descent of the testicle into the scrotum. The right testicle of each mouse underwent sham operation as the control group. The mice were killed 1, 2, and 4 weeks later by intraperitoneal injection of 200 mg/kg pentobarbital. The testicles and epididymis were removed, weighed, fixed in paraformaldehyde (4% vol/vol in Dulbecco’s phosphatebuffered saline [DPBS]) for 24 hours and embedded in paraffin. The sections were stained with hematoxylin-eosin.
Hormonal (Testosterone, Luteinizing Hormone, Follicle-stimulating Hormone) Assays Blood samples were taken from the anesthetized cryptorchidism mice by cardiac puncture and collected in serum separate tubes. Serum was prepared using centrifugation for 20 minutes at 3000 rpm. Serum hormonal levels were measured using a commercially available double antibody radioimmunoassay kits using the Access Immunoassay System (UniCel Dxl 800 system, Beckman Coulter, Chaska, MN).
Collection of Oocytes and Sperm The BDF1 female mice were superovulated by injection of 5 IU pregnant mare serum gonadotropin (Folligon, Intervet International, Boxmeer, The Netherlands), followed 48 hours later by injection of human chorionic gonadotropin (Chorulon, Intervet International). At 14 hours after human chorionic gonadotropin, the oviducts were removed from the female mice and placed in a Petri dish containing modified human tubal fluid medium (Irvine Scientific, Santa Ana, CA) at room temperature. After washing, the oviducts were placed in fresh medium, and cumulus-oocyte complexes were released from the ampulla using Dumont forceps. In the ICSI experiments, the cumulus cells were dispersed by incubation for 3-5 minutes in tubal fluid medium containing 350 IU/mL of hyaluronidase. After washing, 743.e18
ICSI and Embryo Culture One volume of each sperm preparation was mixed with 5 volumes of M2 medium containing 10% PVP at room temperature to decrease stickiness. Each ICSI dish contained 1 drop of M2 medium, 1 drop of sperm solution in M2 containing 10% PVP, and 1 drop of M2 containing 10% PVP for needle cleaning. Injections were performed with a PMM-150 FU piezoimpact unit (Prime Tech) and micromanipulators using a bluntended, mercury-containing pipette (inner volume, 6-7 mL). After 15 minutes of recovery at room temperature in M2 medium, the surviving oocytes were returned to mineral oilcovered KSOM and cultured at 37 C in an atmosphere of 5% carbon dioxide in air. For embryo culture, 50 mL of KSOM covered with mineral oil were equilibrated overnight at 37 C in a humidified atmosphere of 5% carbon dioxide. The oocytes were scored for pronucleus formation (fertilization) 6 hours after the initiation of culture, and the numbers of blastocysts were counted after 92-96 hours in culture.
Detection of Damage to Sperm DNA Terminal Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate-biotin Nick End Labeling Assay. DNA fragmentation was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assays (TUNEL) assays (In Situ Cell Death Detection Kit, TMR red, Roche Diagnostics, Indianapolis, IN). After washing 3 times with DPBS, aliquots of sperm were fixed in 1% paraformaldehyde in DPBS for 1 hour, and permeated with 0.2% Tween-20 in DPBS for 1 hour. The sperm were incubated in TUNEL reaction medium (50 mL) for 1 hour at 37 C in the dark and stained with 10 mL of 1 mg/mL 40 ,60 - diamidino 2-phenyindiol (Sigma-Aldrich) in phosphate-buffered saline for 10 minutes at 37 C. The supernatants were removed by centrifugation at 1500 rpm for 5 minutes, and excess deoxyuridine triphosphate nick-end labeling mixture was removed by washing 3 times in DPBS. The slides were examined under a fluorescence microscope, with the presence of red fluorescence at the head of the sperm considered indicative of damaged DNA. Negative (omitting the enzyme terminal transferase) and positive (using deoxyribonuclease I, 1 IU/mL for 20 minutes at room temperature) controls were performed in each experiment. A total of 200 sperm cells of each aliquot were examined. In the negative controls, none of the cells showed fluorescent staining, but in the positive controls, 100% of the cells showed DNA fragmentation. Caspase-3 Assay. Sperm apoptosis was assessed using caspase-3 activities (Caspase 3 Assay Kit, colorimetric; Abcam, Cambridge, UK). After washing 3 times with DPBS, aliquots of UROLOGY 82 (3), 2013
Figure 1. Gross morphologic changes and serum testosterone levels after cryptorchidism. (A) Gross morphologic changes showing decreases over time in testicular size and weight after cryptorchidism. (B) Serum testosterone levels 1, 2, and 4 weeks after cryptorchidism. sperm were lysed in a lysis buffer. The lysed sperms were centrifuged at 14,000 rpm for 10 minutes and then transferred supernatant (cytosolic extract) to a fresh tube. Then, 50 mg of protein was incubated with 50 mL of 2 reaction buffer and 5 mL of caspase-3 (4 mM DEVD-p-NA) colorimetric substrate at 37 C for 1-2 hours. The optical density of the reaction mixture was quantitated spectrophotometrically at a wavelength of 405 nm using a 96-well plate reader (Perkin Elmer spectrofluorometer, Victor 3). The x-fold increase in caspase 3 activity was determined by comparing these results with the level of the normal mouse sperm or substrate-omitted wells.
scanning microscope (LSM 510, Carl Zeiss, Oberkochen, Germany) with fluorescence at 400 magnification. The micrographs were stored in LSM (Zeiss LSM Image Browser, version 2.30.011; Carl Zeiss Jena GmbH, Jena, Germany).
Statistical Analysis Unless otherwise specified, each experiment was performed using 3 replicates. The statistical significance of differences among treated groups was evaluated by 1-way analysis of variance using a log-linear model in the Statistical Analysis System (SAS Institute, Cary, NC). P <.05 was considered statistically significant.
Detection of Leydig Cell and Apoptosis To investigate the colocalization of Leydig cells and apoptotic cells in the cryptorchidism mouse testicle, we performed doubleimmunohistochemistry using 3b-HSD (3-b-hydroxy-steroiddehydrogenase, Leydig cell-specific marker; Santa Cruz Biotechnology, Santa Cruz, CA) and TUNEL substrate. Each cryptorchidism testicular sections were rehydration and heatmediated antigen retrieval. For permeabilization, the sections were incubated in 0.1% Triton X-100 in DPBS for 10 minutes. After washing 3 times with DPBS, nonspecific binding of antibodies was suppressed by incubation in blocking solution (4% normal goat serum in DPBS) for 30 minutes at room temperature. After additional washing 3 times with PBS, immunohistochemistry staining was performed by incubating the fixed samples with 3b-HSD antibody diluted to 1:200 with DPBS containing 1% bovine serum albumin for 2 hours at room temperature. After incubation, the sections were incubated in TUNEL reaction medium (50 mL) for 1 hour at 37 C. The 3b-HSD was detected using 5 mg/mL of fluorescein isothiocyanate-conjugated secondary antibodies. Finally, the sections were counterstained using 1 mg/mL 40 ,60 - diamidino 2-phenyindiol (Sigma-Aldrich). After the washes, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The resulting staining was viewed on an inverted confocal laser UROLOGY 82 (3), 2013
RESULTS Fifteen male ICR mice were used for the cryptorchidism model (cryptorchidism duration 1, 2, and 4 weeks, n ¼ 5 each). An additional 15 mice, with a cryptorchidism duration of 2 weeks, were used for the ICSI procedure. Oocytes were collected from 45 BDF1 female mice. In the cryptorchidism model, the left testicle of each mouse was fixed to the lower abdominal wall, and the right testicle was allowed to move freely. We observed gradual reductions in the left testicular weight and size at 1, 2, and 4 weeks after the induction of cryptorchidism (Fig. 1A). Although the serum testosterone levels decreased after cryptorchidism, the levels were not significantly different (Fig. 1B). Neither were the serum follicle-stimulating hormone and luteinizing hormone levels (data not shown). Histologic analysis showed sequential reduction of spermatogenesis at 1, 2, and 4 weeks postoperatively (Fig. 2). At 1 week after surgery, normal spermatogenesis, as indicated by the presence of the heads of elongated sperm, was maintained. At 2 weeks after surgery, 743.e19
Figure 2. Hematoxylin-eosin staining of testicle and epididymis. Immunohistochemistry of seminiferous tubules. (A) Histologic changes in testicle and epididymis at 1, 2, and 4 weeks after cryptorchidism showing decrease over time in spermatogenesis. The red box indicates the magnified area. (B) 3b-HSD and TMR-red (apoptotic cells) localization patterns in cross-section of cryptorchidism mouse testicle. 3b-HSD immunoreactivity detected in interstitial cells and Leydig cells. However, no TMR-red signaling was detected in these cells. Some TMR-redepositive cells were detected in spermatogonia or spermatocytes. N-Cont., negative control; Mock first ab, not treated with primary antibodies; Mock enzymes, not treated with deoxyuridine triphosphate-biotin nick-end labeling mixture. Scale bars ¼ 100 mm. 743.e20
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Figure 3. Detection of damaged sperm. (A) Photomicrographs of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-stained sperm after cryptorchidism. TUNEL staining showed that the number of apoptotic cells was significantly increased in the cryptorchidism testicle in each group compared with the normal testicle. Scale bars ¼ 30 mm. (B) Graphic representation of stained TUNEL-positive sperm (P <.05). (C) Sperm lysates were assayed for caspase-3 activity at 1, 2, and 4 weeks after cryptorchidism. Graphic representation of relative caspase-3 activity.
spermatogenesis had markedly decreased. After 4 weeks, the Sertoli cells were predominant, with scarce spermatocytes. However, the Leydig cells still maintained their characters after 4 weeks of cryptorchidism (Fig. 2B). The TUNEL and caspase-3 assay showed that the percentage and activity of DNA-damaged sperm gradually increased after the induction of cryptorchidism (Fig. 3). To evaluate the effects of damage to sperm DNA on fertilization and embryo development, we compared the results of the ICSI procedures using morphologically normal sperm from control testicles and cryptorchid testicles 2 weeks after surgery. We found that the fertilization rate and embryo development were within the normal ranges in the control group but were markedly decreased in the cryptorchidism group. However, despite the poor fertilization rate, about one quarter of embryos fertilized by sperm from the cryptorchidism group continued to develop further (Fig. 4).
COMMENT During the previous 30 years, the treatment of infertility has been revolutionized by the development of new UROLOGY 82 (3), 2013
assisted reproductive technologies, including IVF and ICSI. The introduction of ICSI in 1992 offered the opportunity for parenthood to men with severe oligozoospermia or azoospermia and revolutionized the treatment of male infertility.3 Unlike most therapeutic procedures used in medicine, however, fertilization by ICSI never underwent rigorous safety testing before clinical use. The level of sperm DNA fragmentation was recently reported to be higher in men with severe male factor infertility, including those with severe oligozoospermia and nonobstructive azoospermia.11,12 It has also been suggested that idiopathic severe male factor infertility might be a progressive disease, showing aggravation from severe oligozoospermia to complete azoospermia.16 Therefore, testing the biologic effects of sperm DNA damage on fertilization and embryonic development are very important for studying male factor infertility. We have used a mouse cryptorchidism model to induce sperm DNA damage in vivo. In mice with cryptorchidism, an increase in scrotal temperature has been regarded as the primary cause of sperm DNA damage in the testicles. To our knowledge, this is the first in vivo animal study to evaluate the effects of cryptorchidism-induced 743.e21
Figure 4. Effects of sperm deoxyribonucleic acid (DNA) damage on embryonic development after intracytoplasmic sperm injection. Embryonic development was significantly lower in the cryptorchidism group than in the normal group.
damage to sperm DNA on fertilization and embryonic development. During ICSI, using sperm from patients with severe male factor infertility, a significant proportion of the spermatozoa injected into oocytes can contain fragmented DNA. Therefore, the animal model used in the present study might be useful in investigating male factor infertility and in evaluating the biologic effects of paternal DNA damage on fertilization and embryonic development. We selected morphologically normal sperm, determined by microscopy under 200 magnification, for the ICSI procedures. Sperm DNA integrity during ICSI can be critically important, because this procedure bypasses multiple, apparently redundant mechanisms that have evolved to ensure the selection of high-quality sperm cells for fertilization. Most infertility clinics around the world do not perform sperm DNA fragmentation analysis before ICSI. Our findings suggested that morphologically normal sperm selected by microscopic examination can carry fragmented DNA. After ICSI, the fertilization rate and embryonic development were within the normal ranges in the control group, but both were markedly reduced in the cryptorchidism group. Despite the reduced fertilization rate, about one quarter of embryos generated from the latter group continued to develop further. Therefore, the biologic effect of abnormal sperm chromatin structure depends on the combined effects of the extent of DNA or 743.e22
chromatin damage in the spermatozoa and the capacity of the oocyte to repair that damage. Additional studies of embryos generated from DNA-damaged sperm, including embryo transfer to the female mouse uterus and assays of birth rates and development are needed to evaluate the long-term effects of sperm DNA damage. Many studies have analyzed the effects of sperm DNA damage on assisted reproductive technique pregnancies.17 Recently, sperm DNA damage was reported to be associated with an increased risk of pregnancy loss after IVF and ICSI,13 suggesting the importance of developing strategies to reduce sperm DNA damage in humans, such as reducing exposure to environmental toxins and testicular hyperthermia, supplementation with antioxidant vitamins, and varicocele repair.18-20 To date, few animal studies have assessed the effects of induced sperm DNA damage on fertilization and embryo development. Using a mouse model system, sperm with defective DNA were shown to fertilize oocytes and produce high-quality, early-stage embryos; however, the extent of DNA damage was inversely related to the likelihood of bringing a successful pregnancy to term.14 In addition, a recent animal study evaluating the long-term effects in adult offspring of DNA fragmented sperm, induced by freeze thawing of spermatozoa in the absence of a cryoprotector, found that some of these effects only emerge during later life, including aberrant growth, premature aging, abnormal behavior, and the development UROLOGY 82 (3), 2013
of mesenchymal tumors.21 In other studies, sperm DNA damage was induced by direct short-term scrotal exposure to heated water.22,23 In the in vivo cryptorchidism model used in the present work, we believe that sperm DNA damage was induced under more physiologic stress conditions. Our findings suggest that the extent of sperm DNA damage was related to the duration of cryptorchidism. Therefore, this animal model could be used to evaluate the threshold of sperm DNA damage for safe fertilization and development. The present study had several limitations, included providing little information on the development of the offspring. This was because of several reasons, including technical difficulties. Moreover, the ICSI procedure itself might have deleterious effects on fertilization and early embryo development. Additional studies evaluating the development of offspring generated by fertilization with DNA-damaged sperm and strategies reducing damage to sperm DNA before fertilization are warranted.
CONCLUSION This was an in vivo animal study to evaluate the effects of sperm DNA damage using a cryptorchidism model. Sperm DNA damage increased significantly over time after cryptorchidism operation. This model might be useful in investigating male factor infertility and evaluating the biologic effects of paternal DNA damage on fertilization and future embryonic development. References 1. Bhasin S, de Krester DM, Baker HW. Clinical review 64: pathophysiology and natural history of male infertility. J Clin Endocrinol Metab. 1994;79:1525-1529. 2. Andersen AN, Goossens V, Gianaroli L, et al. Assisted reproductive technology in Europe, 2003: results generated from European registers by ESHRE. Hum Reprod. 2007;22:1513-1525. 3. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992;340:17-18. 4. Nagy ZP, Liu J, Joris H, et al. The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod. 1995;10:1123-1129. 5. Devroey P, Liu J, Nagy Z, et al. Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril. 1994;62:639-641.
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6. Silber SJ. Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia. Hum Reprod. 2000;15:2278-2284. 7. Klemetti R, Gissler M, Sevon T, et al. Children born after assisted fertilization have an increased rate of major congenital anomalies. Fertil Steril. 2005;84:1300-1307. 8. Olson CK, Keppler-Noreuil KM, Romitti PA, et al. In vitro fertilization is associated with an increase in major birth defects. Fertil Steril. 2005;84:1308-1315. 9. Reddy UM, Wapner RJ, Rebar RW, et al. Infertility, assisted reproductive technology, and adverse pregnancy outcomes: executive summary of a National Institute of Child Health and Human Development workshop. Obstet Gynecol. 2007;109:967-977. 10. Lilford R, Jones AM, Bishop DT, et al. Case-control study of whether subfertility in men is familial. BMJ. 1994;309:570-573. 11. Irvine DS, Twigg JP, Gordon EL, et al. DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 2000;21:33-44. 12. Zini A, Fischer MA, Sharir S, et al. Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology. 2002;60: 1069-1072. 13. Zini A, Boman JM, Belzile E, et al. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum Reprod. 2008;23:26632668. 14. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool. 1999;284:696-704. 15. Rossi LM, Pereira LA, de Santis L, et al. Sperm retrieval techniques in rats with suppressed spermatogenesis by experimental cryptorchidism. Hum Reprod. 2005;20:443-447. 16. Song SH, Bak CW, Lim JJ, et al. Natural course of severe oligozoospermia in infertile male: influence on future fertility potential. J Androl. 2010;31:536-539. 17. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril. 2008; 89:823-831. 18. Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci. 2000;22:169-189. 19. Silver EW, Eskenazi B, Evenson DP, et al. Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men. J Androl. 2005;26:550-556. 20. Werthman P, Wixon R, Kasperson K, et al. Significant decrease in sperm deoxyribonucleic acid fragmentation after varicocelectomy. Fertil Steril. 2008;90:1800-1804. 21. Fernandez-Gonzalez R, Moreira PN, Perez-Crespo ML, et al. Longterm effects of mouse intracytoplasmic sperm injection with DNAfragmented sperm on health and behavior of adult offspring. Biol Reprod. 2008;78:761-772. 22. Banks S, King SA, Irvine DS, et al. Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa. Reproduction. 2005; 129:505-514. 23. Perez-Crespo M, Pintado B, Gutierrez-Adan A. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice. Mol Reprod Dev. 2008;75:40-47.
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