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A successful pregnancy and live birth after intracytoplasmic sperm injection with globozoospermic sperm and electrical oocyte activation
CASE REPORT A successful pregnancy and live birth after intracytoplasmic sperm injection with globozoospermic sperm and electrical oocyte activation Akiyoshi Egashira, M.S.,a Masao Murakami, Ph.D.,a Kosuke Haigo, M.S.,a Toshitaka Horiuchi, Ph.D.,b and Takeshi Kuramoto, M.D.a a
Kuramoto Women’s Clinic, Fukuoka, and b Department of Life Sciences, Prefectural University of Hiroshima, Hiroshima, Japan
Objective: To present the effectiveness of intracytoplasmic sperm injection (ICSI) using globozoospermic sperm and assisted oocyte activation by electrical stimulation. Design: A case report. Setting: A private IVF center in Japan. Patient(s): A man with globozoospermia. Intervention(s): Acridine orange (AO) test, mouse oocyte activation test, and ICSI with electrical oocyte activation. Main Outcome Measure(s): Fertilization, pregnancy, and live birth. Result(s): In the first ICSI attempt, neither of the two injected oocytes fertilized. Staining of the patient’s sperm with AO showed that only 2.9% of the sperm emitted a green fluorescence at the characteristic round head (sperm with native DNA content). The mouse oocyte activation test using the roundheaded sperm showed that the normal fertilization rate was 78.9% when SrCl2 was used for assisted oocyte activation; however, it was 6.0% without assisted oocyte activation. We confirmed that the sperm had defective ability to activate oocytes. In the second ICSI attempt, human oocytes were activated electrically with use of a single square direct current pulse after microinjection. All the seven injected oocytes fertilized normally, and two eight-cell embryos were transferred on day 3. Clinical pregnancy was confirmed, and a healthy girl weighing 2362 g was delivered at 37 weeks of gestation by cesarean section. Conclusion(s): This is the first successful outcome of ICSI using globozoospermic sperm and electrical oocyte activation. The electroactivation obviates the need for the use of potentially harmful drugs for activation. (Fertil Steril 2009;92:2037.e5–e9. 2009 by American Society for Reproductive Medicine.) Key Words: Globozoospermia, round-headed sperm, ICSI, assisted oocyte activation, electrical stimulation, fertilization
Globozoospermia is an uncommon condition observed in infertile men; the major morphologic anomaly in this condition is the dominance of roundheaded sperm that lack an acrosome. The condition most probably arises because of disturbed spermiogenesis, and the existence of a family history suggests a genetic basis (1). These acrosomeless sperm are unable to bind to the zona pellucida or fuse with the oocyte oolemma. Therefore, the condition was considered intractable until the introduction of intracytoplasmic sperm injection (ICSI). A normal live birth has been achieved by ICSI with globozoospermic sperm (2–5); however, there Received June 30, 2009; revised August 1, 2009; accepted August 4, 2009; published online October 1, 2009. A.E. has nothing to disclose. M.M. has nothing to disclose. K.H. has nothing to disclose. T.H. has nothing to disclose. Reprint requests: Takeshi Kuramoto, M.D., Kuramoto Women’s Clinic, 1-1-19 Hakataeki-higashi, Hakata-ku, Fukuoka 812-0013, Japan (FAX: 81-92-482-1415; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.08.013
are a significant number of patients in whom ICSI fails completely because of limited or failed fertilization. Fertilization failures have been attributed to the defective ability of the roundheaded sperm to activate oocytes (6). Rybouchkin et al. (7) demonstrated that the rates of fertilization and embryo development in ICSI using roundheaded sperm improved when a calcium ionophore was used for oocyte activation after microinjection. This activation method would be effective to achieve live birth by ICSI using sperm from men with globozoospermia with previously failed fertilization attempts (8–11). However, the potential toxic effects of ionophores on human oocytes or embryos have not yet been estimated adequately in clinical ICSI. The use of a nonchemical activation method such as electrical stimulation would obviate the need to use insufficiently tested drugs for activation (12, 13). The effectiveness of electroactivation on globozoospermic sperm has not been reported to date. In this report, we present a successful case of pregnancy
Fertility and Sterility Vol. 92, No. 6, December 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
2037.e5
and live birth after ICSI and electrical oocyte activation using sperm from a man with globozoospermia. CASE REPORT A 35-year-old woman and her 38-year-old husband who had a 10-year duration of primary infertility visited our center. Informed consent was obtained from the couple before the study was conducted, and the study protocol was approved by the Institutional Review Board. The couple had undergone a total of five IUI procedures and three IVF attempts in other IVF centers; however, pregnancy did not occur because of fertilization failures. The couple was healthy and had no physical problems, other than the husband’s sperm aspects. The wife had no fertility problems. Analysis of a semen sample revealed the following: volume 2.6 mL; sperm concentration 43106/mL; 32.6% motility. All the sperm were roundheaded (Fig. 1A). The karyotype was 46,XY on the peripheral lymphocyte. The couple decided to undergo ICSI. The ovaries were stimulated with use of a long protocol of GnRH analogue (Suprecur; Mochida, Tokyo, Japan) and FSH (Fertinorm P; Merck Serono, Tokyo, Japan). Six oocytes were retrieved under transvaginal ultrasound guidance, of which two oocytes in metaphase II were microinjected with the motile roundheaded sperm; however, two pronuclei (2PN) were not observed in any of these oocytes 18 hours after injection. In the next step, the roundheaded sperm were stained with acridine orange (AO; Sigma, St. Louis, MO) according to previous methods to assess the integrity of the chromatin structure (14). After staining, sperm with native DNA content emitted a distinct green fluorescence under blue light of wavelengths 450 to 490 nm (green fluorescent sperm), whereas sperm heads that emitted a fluorescence spectrum ranging from yellowish green to red were considered as denatured (abnormal DNA) (Fig. 1B) (15). The duration of illumination was limited to 40 seconds per field. Approximately 500 sperm were assessed per group. The percentage of sperm with normal DNA content was calculated by dividing the number of green fluorescent sperm by the total number of sperm and multiplying that value by 100. Moreover, the ability of the roundheaded sperm to activate oocytes was assessed by heterologous ICSI of the sperm into mouse oocytes (mouse oocyte activation test), as described previously (11, 12). In a separate group, mouse oocytes injected with the roundheaded sperm were activated with 10 mmol/L SrCl2 (Nacalai, Kyoto, Japan) for 30 minutes (assisted oocyte activation) (11). Fertilization of these mouse oocytes was assessed 5 hours after the injection, and only zygotes that exhibited two polar bodies and 2PN were considered normal. In the abovementioned studies (AO test and mouse oocyte activation test), sperm samples from a fertile donor were used as controls and were treated in the same manner. Data were analyzed by using the c2 test. The AO test revealed that the percentage of green fluorescent sperm (sperm with native DNA content) was significantly 2037.e6
Egashira et al.
FIGURE 1 (A) A light micrograph showing the characteristic roundheaded sperm. Original magnification, 600. (B) A fluorescence micrograph of the roundheaded sperm after AO staining. The sperm heads that emitted red light were considered to have denatured DNA. Original magnification, 1,000.
Egashira. Live birth in a patient with globozoospermia. Fertil Steril 2009.
lower (P<.05) in the patient than in the fertile donor (2.9% [16/543] vs. 58.7% [291/496]). The patient’s sperm exhibited the typical round head (Fig. 1B). In the mouse oocyte activation test, the normal fertilization rate of the roundheaded sperm from the patient was 6.0% (Table 1); however, this rate increased significantly (P<.05) when assisted oocyte activation was applied (78.9%). The fertilization rate after assisted oocyte activation was similar to that of the fertile donor’s sperm that was not subjected to assisted oocyte activation (74.5%). Therefore, this suggested that artificial oocyte activation methods should be used to increase the fertilization rate. The couple was informed that a larger number of fertilized oocytes could be obtained if an oocyte activation treatment was combined with ICSI. They provided their written informed consent and permission for oocyte activation.
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Vol. 92, No. 6, December 2009
TABLE 1 Mouse oocyte activation after ICSI with the roundheaded sperm. No. (%) of oocytes with 2PB and Sperm source Activation n Patient Fertile donor
þb
50 57 47
2PN
1PN
No. (%) of total activated oocytesa
3 (6.0)c 45 (78.9)d 35 (74.5)d
3 (6.0) 10 (17.5) 3 (6.4)
6 (12.0)c 55 (96.5)d 38 (80.9)d
Note: 2PB ¼ first and second polar body. a Mouse ocytes with two polar bodies and one or two pronuclei. b Mouse oocytes that were activated with 10 mmol/L SrCl2 for 30 minutes after microinjection. c,d Values within columns with different superscripts are significantly different (P< .05). Egashira. Live birth in a patient with globozoospermia. Fertil Steril 2009.
The electrical oocyte activation method used in this case was a modified version of that described elsewhere (12). On the basis of the experience of the researchers, we first tested the electroactivation method in a pilot study and achieved several normal pregnancies in patients who had previous failed ICSI fertilization attempts (unpublished observations). In the second ICSI attempt 4 months later, all the seven oocytes retrieved were in metaphase II and were microinjected with the motile roundheaded sperm. After 30 minutes, the injected oocytes were activated electrically in a buffer containing 0.3 mol/L mannitol, 0.05 mmol/L calcium, and 0.1 mmol/L magnesium with a single square direct current (DC) pulse of 750 V/cm for 50 microseconds delivered by an electrofusion unit (LF101; Nepa Gene, Ichikawa, Japan). After activation, all the oocytes remained intact and were fertilized normally. Three embryos reached the eight-cell stage, and two of them were transferred 3 days after microinjection. This attempt was successful. Pregnancy was confirmed by the detection of hCG in the plasma 14 days after ET and was confirmed later by ultrasonography. Amniocentesis was performed at 16 weeks of gestation, and the results were normal. A healthy female infant weighing 2,362 g without any congenital abnormality was delivered at 37 weeks of gestation by cesarean section. DISCUSSION On the basis of the observation that fertilization failures after ICSI can be overcome by artificially increasing the free intracellular Ca2þ concentration (16), ionophore-boosted oocyte activation has been applied widely in cases of ICSI with globozoospermic sperm (7–11). However, the clinical use of ionophores in assisted reproduction is limited by insufficient knowledge about their potential cytotoxic, teratogenic, and mutagenic effects on oocytes and embryos (17). In this report, ICSI with roundheaded sperm followed by electrical oocyte activation resulted in normal fertilization, embryo development, and pregnancy. This is the first case of a successful pregnancy after the use of electroactivation for Fertility and Sterility
assisted oocyte activation in ICSI with globozoospermic sperm. It has not yet been proved that electrical stimulation is the most efficient and safest method for oocyte activation in human beings. However, in rats, chemical (SrCl2) activation was compared with the use of DC pulses after ICSI with round spermatid (18). These results indicated that DC was better than SrCl2 for oocyte activation with regard to two-cell embryos that developed into blastocysts and resulted in live births. Moreover, a study of parthenogenetic oocyte activation in cows revealed that the quality of the generated blastocysts, measured by the number of viable cells, was higher after electrical activation than chemical activation with ionophores (19). The use of electrical stimulation instead of ionophores alleviates the concerns regarding the possible harmful effects that these insufficiently tested drugs have on human embryos. On the other hand, it was shown that, in pigs, reactive oxygen species (ROS) were produced in the embryos after electrical oocyte activation, and the ROS generation was higher in the activated embryos than in IVF embryos (20). The authors indicated that ROS generation was increased by application of higher voltage or longer duration of electrical pulses for activation and by higher Ca2þ concentrations in the activation media. Moreover, addition of the antioxidant glutathione to the media in which the activated embryos were cultured resulted in a reduction in ROS production. In this case, the intensity and length of the DC pulse applied for activation (750 V/cm for 50 microseconds) were half of those described previously (12); however, the effect of such modifications on ROS generation was not examined. The optimum settings for electrical oocyte activation need to be determined; moreover, further studies are required to evaluate the clinical significance and safety of electroactivation. The AO test has been used to assess sperm susceptibility to nuclear chromatin instability, although the results of this testing alone do not predict sperm fertility (15). Sperm heads with stable deoxyribonucleoprotein or compact chromatin structure emit a green fluorescence when AO intercalates 2037.e7
with double-stranded (native) DNA. Because AO has a highbinding affinity for single-stranded (denatured) DNA, sperm heads with unstable deoxyribonucleoprotein or decompacted chromatin emit a spectrum of colors ranging from yellowish green to red (21). In this case, the number of roundheaded sperm that emitted a green fluorescence at the head after AO staining was very low, suggesting that a high proportion of the globozoospermic sperm contained denatured DNA. Sperm DNA damage is more common in infertile men and may affect IVF and ICSI outcomes; however, numerous studies have shown that fertilization is possible despite a high rate of DNA fragmentation in the sperm population (22, 23). Mouse oocytes provide a useful model for assessing the capacity of human sperm to activate oocytes (24). In this case, we compared the fertilization rate of mouse oocytes injected with roundheaded sperm with and without SrCl2-induced oocyte activation. SrCl2 is used widely for oocyte activation, especially in mice (25). However, SrCl2 is not yet approved for human oocyte activation in the case of globozoospermia (11). On the basis of our results of limited fertilization after heterologous ICSI, we confirmed that roundheaded sperm might have defective ability to activate oocytes. In addition, as the fertilization rate increased after SrCl2-induced oocyte activation, assisted oocyte activation was considered to be effective in improving the fertilization rate after ICSI with roundheaded sperm. The mouse oocyte activation test with or without assisted oocyte activation would allow for discrimination between sperm- and oocyte-related fertilization failures and also may reveal the effectiveness of assisted oocyte activation. It is still unclear whether patients whose ejaculate contains both normal and globozoospermic sperm have a variation of globozoospermia (6). In fact, variable fertilization rates after ICSI have been demonstrated in a case of globozoospermia, indicating variation in the fertilizing capabilities of the roundheaded sperm within the same individual (4). Although the affected men have reduced fertility or infertility, no other physical characteristics can be associated with this syndrome. Besides defective oocyte activation capabilities, the roundheaded sperm were shown to have an irregular chromatin structure (6) and chromosomal aneuploidies (26). Nonetheless, the number of spontaneous abortions or congenital defects associated with pregnancies after ICSI has not increased. These sperm may lack normal centrosomes, which reduces embryo cleavage after ICSI (27, 28). In addition, they may have other anomalies, including anomalies in the nuclear membrane, midpiece, and mitochondria (27). Recently, it was shown that male mice deficient in protein interacting with C kinase 1 (PICK1) are infertile and have globozoospermic sperm (29). PICK1 is localized at the Golgi-derived proacrosomal granules in spermatids, and the phenotype is a likely consequence of abnormal trafficking of the granules that leads to malformed acrosomes. More research is needed to elucidate the etiology of human globozoospermia and to further understand the general mechanism underlying spermiogenesis and spermatogenesis. 2037.e8
Egashira et al.
In summary, we report a successful pregnancy and live birth from a transfer of embryos after performing electrical oocyte activation in ICSI with globozoospermic sperm. The electrical stimulation would obviate the need to use insufficiently tested drugs, including ionophores. Long-term follow-up studies are required to ensure the safety of this method. Acknowledgments: The authors thank Dr. Koji Shiraishi (Yamaguchi University School of Medicine) for his critical review of the manuscript.
REFERENCES 1. Carrell DT, Emery BR, Liu L. Characterization of aneuploidy rates, protamine levels, ultrastructure, and functional ability of round-headed sperm from two siblings and implications for intracytoplasmic sperm injection. Fertil Steril 1999;71:511–6. 2. Trokoudes KM, Danos N, Kalogirou L, Vlachou R, Lysiotis T, Georghiades N, et al. Pregnancy with spermatozoa from a globozoospermic man after intracytoplasmic sperm injection treatment. Hum Reprod 1995;10:880–2. 3. Kilani ZM, Shaban MA, Ghunaim SD, Keilani SS, Dakkak AI. Triplet pregnancy and delivery after intracytoplasmic injection of round-headed spermatozoa. Hum Reprod 1998;13:2177–9. 4. Stone S, O’Mahony F, Khalaf Y, Taylor A, Braude P. A normal livebirth after intracytoplasmic sperm injection for globozoospermia without assisted oocyte activation: case report. Hum Reprod 2000;15:139–41. 5. Bechoua S, Chiron A, Delcleve-Paulhac S, Sagot P, Jimenez C. Fertilisation and pregnancy outcome after ICSI in globozoospermic patients without assisted oocyte activation. Andrologia 2009;41:55–8. 6. Dam AH, Feenstra I, Westphal JR, Ramos L, van Golde RJ, Kremer JA. Globozoospermia revisited. Hum Reprod Update 2007;13:63–75. 7. Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P, Dhont M. Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity. Fertil Steril 1997;68:1144–7. 8. Kim ST, Cha YB, Park JM, Gye MC. Successful pregnancy and delivery from frozen-thawed embryos after intracytoplasmic sperm injection using round-headed spermatozoa and assisted oocyte activation in a globozoospermic patient with mosaic Down syndrome. Fertil Steril 2001;75:445–7. 9. Check JH, Levito MC, Summers-Chase D, Marmar J, Barci H. A comparison of the efficacy of intracytoplasmic sperm injection (ICSI) using ejaculated sperm selected by high magnification versus ICSI with testicular sperm both followed by oocyte activation with calcium ionophore. Clin Exp Obstet Gynecol 2007;34:111–2. 10. Tejera A, Molla M, Muriel L, Remohi J, Pellicer A, De Pablo JL. Successful pregnancy and childbirth after intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient. Fertil Steril 2008;90:1202.e1–5. 11. Kyono K, Nakajo Y, Nishinaka C, Hattori H, Kyoya T, Ishikawa T, et al. A birth from the transfer of a single vitrified-warmed blastocyst using intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient. Fertil Steril 2009;91:931. e7–11. 12. Yanagida K, Katayose H, Yazawa H, Kimura Y, Sato A, Yanagimachi H, et al. Successful fertilization and pregnancy following ICSI and electrical oocyte activation. Hum Reprod 1999;14:1307–11. 13. Mansour R, Fahmy I, Tawab NA, Kamal A, El-Demery Y, Aboulghar M, et al. Electrical activation of oocytes after intracytoplasmic sperm injection: a controlled randomized study. Fertil Steril 2009;91:133–9. 14. Katayose H, Yanagida K, Hashimoto S, Yamada H, Sato A. Use of diamide-acridine orange fluorescence staining to detect aberrant protamination of human-ejaculated sperm nuclei. Fertil Steril 2003;79(Suppl 1):670–6.
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15. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 1984;42:87–91. 16. Tesarik J, Sousa M. More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil Steril 1995;63:343–9. 17. Tesarik J, Rienzi L, Ubaldi F, Mendoza C, Greco E. Use of a modified intracytoplasmic sperm injection technique to overcome sperm-borne and oocyte-borne oocyte activation failures. Fertil Steril 2002;78:619–24. 18. Kato M, Ishikawa A, Hochi S, Hirabayashi M. Effect of activation regimens for rat oocytes on full-term development after round spermatid injection. Contemp Top Lab Anim Sci 2004;43:13–5. 19. Milazzotto MP, Feitosa WB, Coutinho AR, Goissis MD, Oliveira VP, Assumpcao ME, et al. Effect of chemical or electrical activation of bovine oocytes on blastocyst development and quality. Reprod Domest Anim 2008;43:319–22. 20. Koo OJ, Jang G, Kwon DK, Kang JT, Kwon OS, Park HJ, et al. Electrical activation induces reactive oxygen species in porcine embryos. Theriogenology 2008;70:1111–8. 21. Khalifa TA, Rekkas CA, Lymberopoulos AG, Sioga A, Dimitriadis I, Papanikolaou T. Factors affecting chromatin stability of bovine spermatozoa. Anim Reprod Sci 2008;104:143–63. 22. The Practice Committee of the American Society for Reproductive Medicine. The clinical utility of sperm DNA integrity testing. Fertil Steril 2006;86:S35–7.
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23. Lin MH, Kuo-Kuang Lee R, Li SH, Lu CH, Sun FJ, Hwu YM. Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates. Fertil Steril 2008;90:352–9. 24. Rybouchkin A, Dozortsev D, Pelinck MJ, De Sutter P, Dhont M. Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes. Hum Reprod 1996;11:2170–5. 25. Kishigami S, Wakayama T. Efficient strontium-induced activation of mouse oocytes in standard culture media by chelating calcium. J Reprod Dev 2007;53:1207–15. 26. Ditzel N, El-Danasouri I, Just W, Sterzik K. Higher aneuploidy rates of chromosomes 13, 16, and 21 in a patient with globozoospermia. Fertil Steril 2005;84:217–8. 27. Battaglia DE, Koehler JK, Klein NA, Tucker MJ. Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm. Fertil Steril 1997;68:118–22. 28. Nakamura S, Terada Y, Horiuchi T, Emuta C, Murakami T, Yaegashi N, et al. Analysis of the human sperm centrosomal function and the oocyte activation ability in a case of globozoospermia, by ICSI into bovine oocytes. Hum Reprod 2002;17:2930–4. 29. Xiao N, Kam C, Shen C, Jin W, Wang J, Lee KM, et al. PICK1 deficiency causes male infertility in mice by disrupting acrosome formation. J Clin Invest 2009;119:802–12.
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Kuramoto Women’s Clinic, Fukuoka, and b Department of Life Sciences, Prefectural University of Hiroshima, Hiroshima, Japan
Objective: To present the effectiveness of intracytoplasmic sperm injection (ICSI) using globozoospermic sperm and assisted oocyte activation by electrical stimulation. Design: A case report. Setting: A private IVF center in Japan. Patient(s): A man with globozoospermia. Intervention(s): Acridine orange (AO) test, mouse oocyte activation test, and ICSI with electrical oocyte activation. Main Outcome Measure(s): Fertilization, pregnancy, and live birth. Result(s): In the first ICSI attempt, neither of the two injected oocytes fertilized. Staining of the patient’s sperm with AO showed that only 2.9% of the sperm emitted a green fluorescence at the characteristic round head (sperm with native DNA content). The mouse oocyte activation test using the roundheaded sperm showed that the normal fertilization rate was 78.9% when SrCl2 was used for assisted oocyte activation; however, it was 6.0% without assisted oocyte activation. We confirmed that the sperm had defective ability to activate oocytes. In the second ICSI attempt, human oocytes were activated electrically with use of a single square direct current pulse after microinjection. All the seven injected oocytes fertilized normally, and two eight-cell embryos were transferred on day 3. Clinical pregnancy was confirmed, and a healthy girl weighing 2362 g was delivered at 37 weeks of gestation by cesarean section. Conclusion(s): This is the first successful outcome of ICSI using globozoospermic sperm and electrical oocyte activation. The electroactivation obviates the need for the use of potentially harmful drugs for activation. (Fertil Steril 2009;92:2037.e5–e9. 2009 by American Society for Reproductive Medicine.) Key Words: Globozoospermia, round-headed sperm, ICSI, assisted oocyte activation, electrical stimulation, fertilization
Globozoospermia is an uncommon condition observed in infertile men; the major morphologic anomaly in this condition is the dominance of roundheaded sperm that lack an acrosome. The condition most probably arises because of disturbed spermiogenesis, and the existence of a family history suggests a genetic basis (1). These acrosomeless sperm are unable to bind to the zona pellucida or fuse with the oocyte oolemma. Therefore, the condition was considered intractable until the introduction of intracytoplasmic sperm injection (ICSI). A normal live birth has been achieved by ICSI with globozoospermic sperm (2–5); however, there Received June 30, 2009; revised August 1, 2009; accepted August 4, 2009; published online October 1, 2009. A.E. has nothing to disclose. M.M. has nothing to disclose. K.H. has nothing to disclose. T.H. has nothing to disclose. Reprint requests: Takeshi Kuramoto, M.D., Kuramoto Women’s Clinic, 1-1-19 Hakataeki-higashi, Hakata-ku, Fukuoka 812-0013, Japan (FAX: 81-92-482-1415; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.08.013
are a significant number of patients in whom ICSI fails completely because of limited or failed fertilization. Fertilization failures have been attributed to the defective ability of the roundheaded sperm to activate oocytes (6). Rybouchkin et al. (7) demonstrated that the rates of fertilization and embryo development in ICSI using roundheaded sperm improved when a calcium ionophore was used for oocyte activation after microinjection. This activation method would be effective to achieve live birth by ICSI using sperm from men with globozoospermia with previously failed fertilization attempts (8–11). However, the potential toxic effects of ionophores on human oocytes or embryos have not yet been estimated adequately in clinical ICSI. The use of a nonchemical activation method such as electrical stimulation would obviate the need to use insufficiently tested drugs for activation (12, 13). The effectiveness of electroactivation on globozoospermic sperm has not been reported to date. In this report, we present a successful case of pregnancy
Fertility and Sterility Vol. 92, No. 6, December 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
2037.e5
and live birth after ICSI and electrical oocyte activation using sperm from a man with globozoospermia. CASE REPORT A 35-year-old woman and her 38-year-old husband who had a 10-year duration of primary infertility visited our center. Informed consent was obtained from the couple before the study was conducted, and the study protocol was approved by the Institutional Review Board. The couple had undergone a total of five IUI procedures and three IVF attempts in other IVF centers; however, pregnancy did not occur because of fertilization failures. The couple was healthy and had no physical problems, other than the husband’s sperm aspects. The wife had no fertility problems. Analysis of a semen sample revealed the following: volume 2.6 mL; sperm concentration 43106/mL; 32.6% motility. All the sperm were roundheaded (Fig. 1A). The karyotype was 46,XY on the peripheral lymphocyte. The couple decided to undergo ICSI. The ovaries were stimulated with use of a long protocol of GnRH analogue (Suprecur; Mochida, Tokyo, Japan) and FSH (Fertinorm P; Merck Serono, Tokyo, Japan). Six oocytes were retrieved under transvaginal ultrasound guidance, of which two oocytes in metaphase II were microinjected with the motile roundheaded sperm; however, two pronuclei (2PN) were not observed in any of these oocytes 18 hours after injection. In the next step, the roundheaded sperm were stained with acridine orange (AO; Sigma, St. Louis, MO) according to previous methods to assess the integrity of the chromatin structure (14). After staining, sperm with native DNA content emitted a distinct green fluorescence under blue light of wavelengths 450 to 490 nm (green fluorescent sperm), whereas sperm heads that emitted a fluorescence spectrum ranging from yellowish green to red were considered as denatured (abnormal DNA) (Fig. 1B) (15). The duration of illumination was limited to 40 seconds per field. Approximately 500 sperm were assessed per group. The percentage of sperm with normal DNA content was calculated by dividing the number of green fluorescent sperm by the total number of sperm and multiplying that value by 100. Moreover, the ability of the roundheaded sperm to activate oocytes was assessed by heterologous ICSI of the sperm into mouse oocytes (mouse oocyte activation test), as described previously (11, 12). In a separate group, mouse oocytes injected with the roundheaded sperm were activated with 10 mmol/L SrCl2 (Nacalai, Kyoto, Japan) for 30 minutes (assisted oocyte activation) (11). Fertilization of these mouse oocytes was assessed 5 hours after the injection, and only zygotes that exhibited two polar bodies and 2PN were considered normal. In the abovementioned studies (AO test and mouse oocyte activation test), sperm samples from a fertile donor were used as controls and were treated in the same manner. Data were analyzed by using the c2 test. The AO test revealed that the percentage of green fluorescent sperm (sperm with native DNA content) was significantly 2037.e6
Egashira et al.
FIGURE 1 (A) A light micrograph showing the characteristic roundheaded sperm. Original magnification, 600. (B) A fluorescence micrograph of the roundheaded sperm after AO staining. The sperm heads that emitted red light were considered to have denatured DNA. Original magnification, 1,000.
Egashira. Live birth in a patient with globozoospermia. Fertil Steril 2009.
lower (P<.05) in the patient than in the fertile donor (2.9% [16/543] vs. 58.7% [291/496]). The patient’s sperm exhibited the typical round head (Fig. 1B). In the mouse oocyte activation test, the normal fertilization rate of the roundheaded sperm from the patient was 6.0% (Table 1); however, this rate increased significantly (P<.05) when assisted oocyte activation was applied (78.9%). The fertilization rate after assisted oocyte activation was similar to that of the fertile donor’s sperm that was not subjected to assisted oocyte activation (74.5%). Therefore, this suggested that artificial oocyte activation methods should be used to increase the fertilization rate. The couple was informed that a larger number of fertilized oocytes could be obtained if an oocyte activation treatment was combined with ICSI. They provided their written informed consent and permission for oocyte activation.
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Vol. 92, No. 6, December 2009
TABLE 1 Mouse oocyte activation after ICSI with the roundheaded sperm. No. (%) of oocytes with 2PB and Sperm source Activation n Patient Fertile donor
þb
50 57 47
2PN
1PN
No. (%) of total activated oocytesa
3 (6.0)c 45 (78.9)d 35 (74.5)d
3 (6.0) 10 (17.5) 3 (6.4)
6 (12.0)c 55 (96.5)d 38 (80.9)d
Note: 2PB ¼ first and second polar body. a Mouse ocytes with two polar bodies and one or two pronuclei. b Mouse oocytes that were activated with 10 mmol/L SrCl2 for 30 minutes after microinjection. c,d Values within columns with different superscripts are significantly different (P< .05). Egashira. Live birth in a patient with globozoospermia. Fertil Steril 2009.
The electrical oocyte activation method used in this case was a modified version of that described elsewhere (12). On the basis of the experience of the researchers, we first tested the electroactivation method in a pilot study and achieved several normal pregnancies in patients who had previous failed ICSI fertilization attempts (unpublished observations). In the second ICSI attempt 4 months later, all the seven oocytes retrieved were in metaphase II and were microinjected with the motile roundheaded sperm. After 30 minutes, the injected oocytes were activated electrically in a buffer containing 0.3 mol/L mannitol, 0.05 mmol/L calcium, and 0.1 mmol/L magnesium with a single square direct current (DC) pulse of 750 V/cm for 50 microseconds delivered by an electrofusion unit (LF101; Nepa Gene, Ichikawa, Japan). After activation, all the oocytes remained intact and were fertilized normally. Three embryos reached the eight-cell stage, and two of them were transferred 3 days after microinjection. This attempt was successful. Pregnancy was confirmed by the detection of hCG in the plasma 14 days after ET and was confirmed later by ultrasonography. Amniocentesis was performed at 16 weeks of gestation, and the results were normal. A healthy female infant weighing 2,362 g without any congenital abnormality was delivered at 37 weeks of gestation by cesarean section. DISCUSSION On the basis of the observation that fertilization failures after ICSI can be overcome by artificially increasing the free intracellular Ca2þ concentration (16), ionophore-boosted oocyte activation has been applied widely in cases of ICSI with globozoospermic sperm (7–11). However, the clinical use of ionophores in assisted reproduction is limited by insufficient knowledge about their potential cytotoxic, teratogenic, and mutagenic effects on oocytes and embryos (17). In this report, ICSI with roundheaded sperm followed by electrical oocyte activation resulted in normal fertilization, embryo development, and pregnancy. This is the first case of a successful pregnancy after the use of electroactivation for Fertility and Sterility
assisted oocyte activation in ICSI with globozoospermic sperm. It has not yet been proved that electrical stimulation is the most efficient and safest method for oocyte activation in human beings. However, in rats, chemical (SrCl2) activation was compared with the use of DC pulses after ICSI with round spermatid (18). These results indicated that DC was better than SrCl2 for oocyte activation with regard to two-cell embryos that developed into blastocysts and resulted in live births. Moreover, a study of parthenogenetic oocyte activation in cows revealed that the quality of the generated blastocysts, measured by the number of viable cells, was higher after electrical activation than chemical activation with ionophores (19). The use of electrical stimulation instead of ionophores alleviates the concerns regarding the possible harmful effects that these insufficiently tested drugs have on human embryos. On the other hand, it was shown that, in pigs, reactive oxygen species (ROS) were produced in the embryos after electrical oocyte activation, and the ROS generation was higher in the activated embryos than in IVF embryos (20). The authors indicated that ROS generation was increased by application of higher voltage or longer duration of electrical pulses for activation and by higher Ca2þ concentrations in the activation media. Moreover, addition of the antioxidant glutathione to the media in which the activated embryos were cultured resulted in a reduction in ROS production. In this case, the intensity and length of the DC pulse applied for activation (750 V/cm for 50 microseconds) were half of those described previously (12); however, the effect of such modifications on ROS generation was not examined. The optimum settings for electrical oocyte activation need to be determined; moreover, further studies are required to evaluate the clinical significance and safety of electroactivation. The AO test has been used to assess sperm susceptibility to nuclear chromatin instability, although the results of this testing alone do not predict sperm fertility (15). Sperm heads with stable deoxyribonucleoprotein or compact chromatin structure emit a green fluorescence when AO intercalates 2037.e7
with double-stranded (native) DNA. Because AO has a highbinding affinity for single-stranded (denatured) DNA, sperm heads with unstable deoxyribonucleoprotein or decompacted chromatin emit a spectrum of colors ranging from yellowish green to red (21). In this case, the number of roundheaded sperm that emitted a green fluorescence at the head after AO staining was very low, suggesting that a high proportion of the globozoospermic sperm contained denatured DNA. Sperm DNA damage is more common in infertile men and may affect IVF and ICSI outcomes; however, numerous studies have shown that fertilization is possible despite a high rate of DNA fragmentation in the sperm population (22, 23). Mouse oocytes provide a useful model for assessing the capacity of human sperm to activate oocytes (24). In this case, we compared the fertilization rate of mouse oocytes injected with roundheaded sperm with and without SrCl2-induced oocyte activation. SrCl2 is used widely for oocyte activation, especially in mice (25). However, SrCl2 is not yet approved for human oocyte activation in the case of globozoospermia (11). On the basis of our results of limited fertilization after heterologous ICSI, we confirmed that roundheaded sperm might have defective ability to activate oocytes. In addition, as the fertilization rate increased after SrCl2-induced oocyte activation, assisted oocyte activation was considered to be effective in improving the fertilization rate after ICSI with roundheaded sperm. The mouse oocyte activation test with or without assisted oocyte activation would allow for discrimination between sperm- and oocyte-related fertilization failures and also may reveal the effectiveness of assisted oocyte activation. It is still unclear whether patients whose ejaculate contains both normal and globozoospermic sperm have a variation of globozoospermia (6). In fact, variable fertilization rates after ICSI have been demonstrated in a case of globozoospermia, indicating variation in the fertilizing capabilities of the roundheaded sperm within the same individual (4). Although the affected men have reduced fertility or infertility, no other physical characteristics can be associated with this syndrome. Besides defective oocyte activation capabilities, the roundheaded sperm were shown to have an irregular chromatin structure (6) and chromosomal aneuploidies (26). Nonetheless, the number of spontaneous abortions or congenital defects associated with pregnancies after ICSI has not increased. These sperm may lack normal centrosomes, which reduces embryo cleavage after ICSI (27, 28). In addition, they may have other anomalies, including anomalies in the nuclear membrane, midpiece, and mitochondria (27). Recently, it was shown that male mice deficient in protein interacting with C kinase 1 (PICK1) are infertile and have globozoospermic sperm (29). PICK1 is localized at the Golgi-derived proacrosomal granules in spermatids, and the phenotype is a likely consequence of abnormal trafficking of the granules that leads to malformed acrosomes. More research is needed to elucidate the etiology of human globozoospermia and to further understand the general mechanism underlying spermiogenesis and spermatogenesis. 2037.e8
Egashira et al.
In summary, we report a successful pregnancy and live birth from a transfer of embryos after performing electrical oocyte activation in ICSI with globozoospermic sperm. The electrical stimulation would obviate the need to use insufficiently tested drugs, including ionophores. Long-term follow-up studies are required to ensure the safety of this method. Acknowledgments: The authors thank Dr. Koji Shiraishi (Yamaguchi University School of Medicine) for his critical review of the manuscript.
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