Successful pregnancy and childbirth after intracytoplasmic sperm injection with calcium ionophore oocyte activation in a globozoospermic patient

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1 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, 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: ; 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 /08/$34.00 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 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 The husband was 34 years old and his sperm revealed a count of /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 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

3 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 e3 Tejera et al. Pregnancy in a globozoospermic patient Vol. 90, No. 4, October 2008

4 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. 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Pregnancy in a globozoospermic patient Vol. 90, No. 4, October 2008