CASE REPORT Successful pregnancy and childbirth after intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient Alberto Tejera, Ph.D., a Marta Molla, Ph.D., b Lourdes Muriel, Ph.D., b Jose Remohı, M.D., c Antonio Pellicer, M.D., c,d and Jose Luis De Pablo, Ph.D. a a Instituto Valenciano de Infertilidad (IVI) Bilbao, In Vitro Fertilization Laboratory, Vizcaya; b IVI Vigo, IVF Laboratory, Vigo; c IVI Valencia, University of Valencia, Valencia; and d Department of Obstetrics and Gynaecology, Hospital Dr. Pesset, Valencia, Spain Objective: To check the effectiveness of intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA) in a globozoospermic patient. Design: Case report. Setting: Instituto Valenciano de Infertilidad, Valencia, Spain. Patient(s): A patient with globozoospermia. Intervention(s): ICSI was administered in 14 oocytes. ICSI combined with AOA, in which a small amount of calcium was injected followed by calcium ionophore exposure, was done in 9 oocytes. Main Outcome Measure(s): Fertilization rate and embryo quality was assessed in both groups. Result(s): Chemical activation increased fertilization rate (55.6% vs. 35.7%) and the number of embryos with less multinucleation on day 2 (0 vs. 60%). Two embryos generated from AOA were transferred into the uterus (on day 3), resulting in a pregnancy and a healthy newborn. Conclusion(s): The AOAwith calcium ionophore treatment improved fertilization rate and quality of the embryos, and was found to be an effective method for AOA in this patient with a low fertilization rate after previous ICSI treatment. (Fertil Steril Ò 2008;90:1202.e1 e5. Ó2008 by American Society for Reproductive Medicine.) Key Words: Globozoospermia, ICSI, AOA, calcium ionophore, fertilization failure Globozoospermia is a very rare condition observed in <1% of infertile patients, where the major morphological anomaly is the absence of an acrosomal cap in sperm. The sperm head appears small and round due to the failure of the acrosome to develop (1). It is still unclear whether patients whose ejaculate contains both normal and globozoospermic cells (partial globozoospermia) suffer from a variation of the same syndrome. Although affected men suffer from reduced fertility or even infertility, no other physical characteristics can be associated with the syndrome. Chromatin compaction appears to be disturbed but is not consistently overcondensed or undercondensed. In some cases, an increased number of cells with Received June 22, 2007; revised November 19, 2007; accepted November 20, 2007. A.T. has nothing to disclose. M.M. has nothing to disclose. L.M. has nothing to disclose. J.M. has nothing to disclose. A.P. has nothing to disclose. J.L.D.P. has nothing to disclose. Reprint requests: Alberto Tejera, Ph.D., In Vitro Fertilization Laboratory, IVI-BILBAO, Paseo Landabarri n 1, LEIOA 48940, Bilbao, Spain (FAX: 944806029; E-mail: atejera@ivi.es). DNA fragmentation have been observed. The analysis of the cytogenetic composition can reveal an increased aneuploidy rate in some cases. Nonetheless, no increased number of spontaneous abortions or congenital defects has been reported in pregnancies conceived after intracytoplasmic sperm injection (ICSI). The pathogenesis of globozoospermia most probably originates in spermatogenesis, more specifically in acrosome formation and sperm head elongation (2). The absence of the acrosome renders globozoospermic spermatozoa unable to bind to the zona pellucida (ZP) (3) or fuse with the oocyte oolemma (4, 5). Therefore, men affected by globozoospermia are infertile and untreatable by artificial insemination or conventional IVF. But when globozoospermic spermatozoa bypass the ZP and oocyte oolemma with ICSI, normal fertilization, embryo development (6), and pregnancy (7 10) may occur. However, globozoospermia reduced ICSI fertilization rates compared with other forms of teratozoospermia (6, 11). Fertilization failures have been attributed to a deficiency in oocyte activation capacity (12). In this study we have evaluated the effect of oocyte activation after ICSI using calcium ionophore on fertilization rate 1202.e1 Fertility and Sterility â Vol. 90, No. 4, October 2008 0015-0282/08/$34.00 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2007.11.056
and embryo quality in a globozoospermic patient with a previous low fertilization treatment. This is the first case in Spain that has finished in an offspring with this technique. CASE REPORT The couple was informed that a larger number of fertilized oocytes might be obtained if assisted oocyte activation (AOA) treatment was combined with ICSI, and then they provided by written informed consent and agreed to oocyte activation after ICSI. Infertility History (anamnesis) A couple with an infertility history of 3 years due to globozoospermia is presented. This couple had undertaken one attempt with low fertilization rate (15%) and pregnancy did not ensue in another IVF center. The patient was 32 years old and polycystic ovarian syndrome (PCOS) was diagnosed when she came to our center. An endometrial polypectomy had been done in 2001. The husband was 34 years old and his sperm revealed a count of 45 10 6 /ml, 17% motility, and 0 normal morphology according to the criteria of the World Health Organization (13), with all round-headed and acrosomeless sperm. This abnormal morphology (Fig. 1) was not diagnosed before. The patient presented a normal 46, XY karyotype, as examined on peripheral lymphocytes. In physical examination mild varicocele was observed on the left side of the scrotum. Sperm nuclei were analyzed by fluorescence in situ hybridization analysis on the sperm. Dual fluorescence in situ hybridization for chromosomes 13 and 21 and triple FIGURE 1 Spermatozoa showing the characteristic round or globe head. Original magnification, 100. fluorescence in situ hybridization for chromosomes 18, X, and Y were performed. We studied DNA fragmentation by sperm chromatin disperse test (Halosperm kit; INDAS laboratories, Madrid, Spain). Controlled Ovarian Stimulation A long protocol of GnRH analogue (Procrin; Abbott, Madrid, Spain) was used. After using a long luteal down-regulation with leuprolide acetate (LA) (Procrin) of 1 mg/day, the wife was induced for controlled ovarian hyperstimulation (COH) using recombinant FSH/LH (Puregon; Serono, Madrid, Spain) at 75/75 IU/day starting on the fourth day of the menses and after 6 days she continued with 125 IU/day of FSH for 9 more days. Finally, after 15 days of ovarian stimulation, when seven follicles were >17 mm, eight follicles were >14 mm, and six follicles were between 12 and 14 mm, 6,500 IU of hcg (Ovitrelle; Serono) was provided. Twenty-seven oocytes were retrieved by transvaginal follicle aspiration under ultrasonographic monitoring, 36 hours after hcg injection. The luteal phase was supported by vaginal suppositories of micronized P (Utrogestan; Seid, Madrid, Spain). Assisted Oocyte Activation in Clinical Intracytoplasmic Sperm Injection Because all sperm were globozoospermic, those appearing to have the best motility were selected for injection. The ionophore (ionomycin; cat.no. I9657; Sigma Aldrich, Madrid, Spain) was dissolved in cell culture-tested dimethyl sulfoxide (DMSO; Sigma Aldrich) at a concentration of 1 mmol/l stock solution. The final solution containing 10 mmol/l ionophore was prepared just before ICSI by diluting the stock solution with IVF medium (Vitrolife, Kungsbacka, Sweden). An immobilized sperm was first aspirated in the injection needle and brought to the tip of the needle. In a separate drop, 5 picoliters of 0.1 mmol/l ionophore was aspirated and injected along with the spermatozoon into the ooplasm. After sperm injection, the oocytes in the activation treatment group were activated in a culture medium containing calcium ionophore for 10 minutes. These oocytes were individually transferred into a 50-mL drop of IVF medium under mineral oil in a plastic disk, and incubated at 37 C under 5% CO 2 air atmosphere. On day 1 (16 20 hours after injection), fertilization was assessed and only zygotes presenting two pronuclei (2PN) and two polar bodies were considered normal. On days 2 and 3, embryos were checked for the number and size of blastomeres, as well as for the percentage of fragmentation. Embryo transfer was carried out on day 3. Tejera. Pregnancy in a globozoospermic patient. Fertil Steril 2008. RESULTS The sperm chromatin disperse test resulted in 45.7% fragmentation index, and although it is a high value (normal value is around 30%) (14, 15), we suggested that the couple try it with his semen because the results from assisted reproductive Fertility and Sterility â 1202.e2
techniques (ART) (IVF and ICSI) in connection to sperm DNA damage are controversial and it has been demonstrated that successful pregnancies in IVF/ICSI cycles can be obtained using semen samples with a high proportion of DNA damage. In addition some reports demonstrated that significantly higher clinical pregnancy (PR) and delivery rates were seen after ICSI compared with IVF when semen samples with high levels of sperm DNA damage were used (16). These data are in agreement with other reports showing that sperm DNA damage is more predictive in IVF and much less so in ICSI (15, 17, 18). The results of the fluorescence in situ hybridization study revealed 0.66% numerical abnormalities of the sexual chromosomes (disomy), and although there were significant differences compared with a control population (0.37%) (19), the couple refused to undergo preimplantation genetic diagnosis because the percentage of sperm abnormalities was too low. Twenty-three of 27 oocytes, obtained after oocyte retrieval, were in metaphase II. Fourteen of them were injected by ICSI (group I) and nine were treated with a combination of ICSI and calcium ionophore (group II). Chemical activation increased the fertilization rate, 35.7% with ICSI versus 55.6% with ICSI AOA, and improved the development and quality of the embryos. The embryos without calcium ionophore presented slower development, poor quality, and three of them had multinucleated blastomeres, whereas embryos with AOA did not have multinucleation and reached a better cleavage rate on days 2 and 3 (Table 1). Two embryos with eight cells on day 3 were transferred into the uterus from group II. The b-hcg value was 189 miu/ml 13 days after embryo transfer. The transvaginal ultrasonography (TVS) revealed one sac with cardiac activity at 7 weeks of pregnancy, resulting in a healthy female baby at 40 weeks gestation. DISCUSSION Intracytoplasmic sperm injection is the most powerful tool in ART. Although the fertilization rate of ICSI is typically the highest of all assisted reproduction treatments, fertilization failure (complete fertilization failure or a low fertilization rate) after ICSI has been recognized in rare cases. The frequency of complete fertilization failure after ICSI has been reported at 1.29% (20) and 3% (11). Additional reports purport that oocyte activation does not occur in approximately 70% of unfertilized oocytes after ICSI (21, 22) despite accurate injection of the spermatozoon into the oocyte. Globozoospermia is a rare type of teratozoospermia, first described in 1971 (23). Biochemically, the spermatozoa are characterized by the absence, or reduced activity, of acrosin (acrosome protease) and of calicin (a cytoskeletal protein) (24). The mode of inheritance for this condition has not yet been established (25). Dominant inheritance, homozygous autosomal gene defect, and a possible environmental effect have been suggested (9, 26). Battaglia et al. (27) postulated that spermatozoa from patients with this condition may be deficient in oscillin, a factor that is normally expressed by fertilizing spermatozoa. It causes the release of calcium ions, which are needed for oocyte activation during fertilization, from intracellular stores in the oocyte (28). During physiological fertilization, a transient increase in intracellular calcium occurs after TABLE 1 Results of ICSI and ICSI D AOA in terms of fertilization and embryo quality. Group I (ICSI) Group II (AOA) Fertilization rate (%) 5/14 (35.7) 5/9 (55.6) No. of embryos with MNB on day 2 (%) 3/5 (60) 0 (0) No. of embryos with 2 cells on day 2 (%) 3/5 (60) 1/5 (20) No. of embryos with 3 cells on day 2 (%) 1/5 (20) 2/5 (40) No. of embryos with 4 cells on day 2 (%) 1/5 (20) 2/5 (40) No. of arrested embryos on day 2 (%) 0 (0) 0 (0) Fragmentation average rate on day 2 5% 6.4% No. of embryos with MNB on day 3 (%) 0 (0) 0 (0) No. of embryos with 4 cells on day 3 (%) 1/5 (20) 0 (0) No. of embryos with 5 cells on day 3 (%) 1/5 (20) 0 (0) No. of embryos with 6 cells on day 3 (%) 0 (0) 1/5 (20) No. of embryos with 7 cells on day 3 (%) 3/5 (60) 1/5 (20) No. of embryos with 8 cells on day 3 (%) 0 (0) 3/5 (60) No. of arrested embryos on day 3 (%) 0 (0) 0 (0) Fragmentation average rate on day 3 7% 2% Note: AOA ¼ assisted oocyte activation; ICSI ¼ intracytoplasmic sperm injection; MNB ¼ multinucleated blastomere. Tejera. Pregnancy in a globozoospermic patient. Fertil Steril 2008. 1202.e3 Tejera et al. Pregnancy in a globozoospermic patient Vol. 90, No. 4, October 2008
spermatozoon egg fusion. This is followed by calcium oscillations, which continue for 3 4 hours. One to several transient calcium increases are required for oocyte activation. Calcium fluxes promote the exocytosis of cortical granules that are superficially situated in the oocyte (29). Although the function of these calcium oscillations is not clear, they also affect embryonic development. Cheung et al. (30), Alberio et al. (31), and Bos-Mikich et al. (32) demonstrated that calcium oscillations, during mitosis and exit from meiosis, increased the number of inner cell mass cells of the blastocyst. Others studies have shown that repeated ICSI failures can be linked to sperm DNA damage (33) and meiotic disorders are more common in patients with severe sperm abnormalities (34). There are some reports that show a possible correlation between globozoospermia and chromosomal aneuploidies. Ditzel et al. (35) observed that aneuploidy rates in spermatozoa from a patient with globozoospermia were significantly higher than in a normospermic man. In that study they demonstrated a positive correlation between globozoospermia and higher chromosomal aneuploidies of chromosomes 13, 16, and 21 (35). Viville et al. (36), Carrell et al. (25), and Zeyneloglu et al. (37) also reported higher aneuploidy rates in this type of patient compared with controls. But Martin and Rademaker (38) and Celik-Ozenci et al. (39) evaluated the potential relationship between the frequency of chromosomally and morphologically abnormal sperm, and the results evidenced that a morphological assay is not a good indication of chromosomal abnormalities in human sperm. Calcium ionophore has been used for oocyte activation in humans since the 1990s. Tesarik and Testart (40) reported the activation of human oocytes after ICSI by calcium ionophore. In 1995, Hoshi et al. (41) reported the first pregnancy using ICSI with calcium ionophore treatment as AOA to increase the fertilization rate. Using calcium ionophore treatment, Rybouchkin et al. (42) reported the first pregnancy for a couple with complete fertilization failure due to a round-headed sperm defect associated with deficient oocyte activation capacity. Kim et al. (43), Eldar-Geva et al. (44), Chi et al. (45), and Murase et al. (46) reported pregnancies and deliveries with this technique. Assisted oocyte activation has been recommended as an efficient treatment option in cases of complete fertilization failure and low fertilization rates after ICSI (21, 42 48). Enhanced fertilization rates and normal embryo cleavage (27) resulted after oocyte activation using calcium ionophore injection and subsequent ionophore treatment exposure. Therefore, if oocyte activation is achieved, globozoospermic spermatozoa may support fertilization and subsequent development comparable to that of other forms of teratozoospermia (11). Chi et al. (45) recently reported a study where they split ICSI to demonstrate a significant difference in the rate of fertilization between treated and untreated oocytes. In that study, a calcium ionophore treatment was also applied as AOA to improve the efficacy of ICSI. In that couple with a history of low fertilization rate, an attempt was made to evaluate the influence of chemical oocyte activation on the outcome of ICSI in terms of fertilization and embryo development on days 2 and 3. Our results are in agreement with Battaglia et al. (27), who reported that cleavage rates were also compromised and they suggested that these sperm may lack normal centrosomes. After application of AOA during clinical ICSI they were able to restore fertilization and embryo cleavage, and developmental rates were brought to a normal level in these globozoospermic patients. In conclusion, pregnancy and live birth can be achieved with chemical activation in a globozoospermic patient with a low fertilization previous cycle. We detected an improvement in the fertilization rate, development, and quality of the embryos generated with AOA. Two embryos of this cohort were transferred into the uterus, resulting in a pregnancy and healthy newborn. Acknowledgments: The authors thank Dr. B. Heindryckx and his team for their cooperation. REFERENCES 1. Sun F, Ko E, Martin RH. Is there a relationship between sperm chromosome abnormalities and sperm morphology? Reprod Biol Endocrinol 2006;4:1 5. 2. Dam AHDM, Feenstra I, Westphal JR, Ramos L, Van Golde RJ, Kremer JAM. Globozoospermia revisited. Hum Reprod Update 2007;13:63 75. 3. Aitken RJ, Kerr L, Bolton V, Hargreave T. Analysis of sperm function in globozoospermia: implications for the mechanism of sperm zona interaction. Fertil Steril 1990;54:701 7. 4. Syms AJ, Johnson AR, Lipshultz LI, Smith RG. Studies on human spermatozoa with round-headed syndrome. Fertil Steril 1984;42:431 5. 5. Dale B, Iaccarino M, Fortunato A, Gragnaniello G, Kyozuka K, Tosti E. A morphological and functional study of fusibility in round-headed spermatozoa in the human. Fertil Steril 1994;61:336 40. 6. Bourne H, Liu DY, Clarke GN, Baker HW. Normal fertilization and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm. Fertil Steril 1995;63:1329 32. 7. Lundin K, Sjogren A, Nilsson L, Hamberger L. Fertilization and pregnancy after intracytoplasmic microinjection of acrosomeless spermatozoa. Fertil Steril 1994;62:1266 7. 8. Liu J, Nagy Z, Joris H, Tournaye H, Smith J, Camus M, et al. Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm injection cycles. Hum Reprod 2005;10:2630 6. 9. 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. 10. 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. 11. Liu J, Nagy Z, Joris H, Tournaye H, Devroey P, Van Steirteghem A. Successful fertilization and establishment of pregnancies after intracytoplasmic sperm injection in patients with globozoospermia. Hum Reprod 1995;10:626 9. 12. Rybouchkin A, Dozortsev D, Pelinck MJ, De Sutter P, Dhont M. Analysis of the oocyte activating capacity and chromosomal complement of Fertility and Sterility â 1202.e4
round-headed human spermatozoa by their injection into mouse oocytes. Hum Reprod 1996;11:2170 5. 13. World Health Organization. WHO laboratory manual for the examination of human semen and sperm cervical mucus interaction. Cambridge, UK: Cambridge University Press, 1999. 14. Larson KL, De Jonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 2000;15: 1717 22. 15. Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril 2003;80:895 902. 16. Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl 2006;8:11 29. 17. Hammadeh ME, Stieber M, Haidl G, Schmidt W. Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program. Andrologia 1998;30:29 35. 18. Host E, Lindenberg S, Smidt-Jensen S. The role of DNA strand breaks in human spermatozoa used for IVF and ICSI. Acta Obstet Gynecol Scand 2000;79:559 63. 19. Blanco J, Rubio C, Simon C, Egozcue J, Vidal F. Increased incidence of disomic sperm nuclei in a 47, XYY male assessed by fluorescent in situ hybridisation (FISH). Hum Genet 1997;99:413 6. 20. Esfendiari N, Javed MH, Gotlieb L, Casper RF. Completed failed fertilization after intracytoplasmic sperm injection: analysis of 10 years data. Int J Fertil Womens Med 2005;50:187 92. 21. Yanahida K. Complete fertilization failure in ICSI. Hum Cell 2004;17: 187 93. 22. Heindryckx B, Van der Elst J, De Sutter P, Dhont M. Treatment option for sperm- or oocyte-related fertilization failure: assisted oocyte activation following diagnostic heterologous ICSI. Hum Reprod 2005;20:2237 41. 23. Schirren CG, Hostein AF, Chirren C. Uber Die morphogenese rund-kopfiger spermatozoen des menschen. Andrologia 1971;3:117 25. 24. Florke-Gerloff SF, Topfer-Petersen E, Muller-Esterl W, Mansouri A, Schatz R, Schirren C, et al. Biochemical and genetic investigation of round-headed spermatozoa in infertile men including two brothers and their father. Andrologia 1984;16:187 202. 25. Carrel 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. 26. Kilani Z, Ismail R, Ghunaim S, Mohamed H, Hughes D, Brewis I, et al. Evaluation and treatment of familial globozoospermia in five brothers. Fertil Steril 2004;82:1436 9. 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. Swann K, Ozil JP. Dynamics of the calcium signal that triggers mammalian egg activation. Int Rev Cytol 1994;152:183 222. 29. Tesarik J, Sousa M, Mendoza C. Sperm-induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery. Mol Reprod Dev 1995;41:257 63. 30. Cheung A, Swann K, Carroll J. The ability to generate normal Ca2þ transients in response to spermatozoa develops during the final stages of oocyte growth and maturation. Hum Reprod 2000;15:1389 95. 31. Alberio R, Zakhartchenko V. Mammalian oocyte activation: lessons from sperm and implications for nuclear transfer. Int J Dev Biol 2001;45:797 809. 32. Bos-Mikich A, Swann K, Whittingham DG. Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes. Mol Reprod Dev 1995;41:84 90. 33. Tesarik J, Mendoza C, Greco E. The activity (calcium oscillator?) responsible for human oocyte activation after injection with round spermatids is associated with spermatid nuclei. Fertil Steril 2000;74: 1245 7. 34. Barri PN, Vendrell JM, Martınez F, Coroleu B, Aran B, Veiga A. Influence of spermatogenic profile and meiotic abnormalities on reproductive outcome of infertile patients. Reprod Bio Med 2005;10:735 9. 35. 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. 36. Viville S, Mollard R, Bach ML, Falquet C, Gerlinger P, Warter S. Do morphological anomalies reflect chromosomal aneuploidies? Case report. Hum Reprod 2000;15:2563 6. 37. Zeyneloglu HB, Baltaci V, Duran HE, Erdemli E, Batioglu S. Achievement of pregnancy in globozoospermia with Y chromosome microdeletion after ICSI. Hum Reprod 2002;17:1833 6. 38. Martin RH, Rademaker A. The relationship between sperm chromosomal abnormalities and sperm morphology in humans. Mutat Res 1988;207:159 64. 39. Celik-Ozenci C, Jakab A, Kovacs T, Catalanotti J, Demir R, Bray- Ward P, et al. Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations. Hum Reprod 2004;19:2052 9. 40. Tesarik J, Telstart J. Treatment of sperm-injected human oocytes with Ca2þ ionophore supports the development of Ca2þ oscillations. Biol Reprod 1994;51:385 91. 41. Hoshi K, Yanagida K, Yazawa H, Katayose H, Sato A. Intracytoplasmic sperm injection using immobilized or motile spermatozoon. Fertil Steril 1995;63:1241 5. 42. Rybouchkin AV, Van der Straeten F, Quatacker J. 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. 43. Kim ST, Cha YB, Park JM, Gye MC. Successful pregnancy and delivery from frozen-thawed embryos after intracytoplasmic sperm injection using patient with mosaic Down syndrome. Fertil Steril 2001;75: 445 7. 44. Eldar-Geva T, Brooks B, Margalioth EJ, Zylber-Haran E, Gal M, Silver SJ. Successful pregnancy and delivery after calcium ionophore oocyte activation in a normozoospermic patient with previous repeated failed fertilization after intracytoplasmic sperm injection. Fertil Steril 2003;79:1656 8. 45. Chi HJ, Koo JJ, Song SJ, Lee JY, Chang SS. Successful fertilization and pregnancy after intracytoplasmic sperm injection and oocyte activation with calcium ionophore in a normozoospermic patient with extremely low fertilization rates in intracytoplasmic sperm injection cycles. Fertil Steril 2004;82:475 7. 46. Murase Y, Araki Y, Mizuno S, Kawaguchi C, Naito M, Yoshizawa M, et al. Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report. Hum Reprod 2004;19:1604 7. 47. Vanderzwalmen P, Zech H, Birkenfeld A, Yemini M, Bertin G, Lejeune B, et al. Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum Reprod 1997;12:1203 13. 48. 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. 1202.e5 Tejera et al. Pregnancy in a globozoospermic patient Vol. 90, No. 4, October 2008