Case report Birth and follow-up of babies born following ICSI using SrCl 2. oocyte activation

RBMOnline - Vol 17 No 1. 2008 53-58 Reproductive BioMedicine Online; www.rbmonline.com/article/3284 on web 19 May 2008 Case report Birth and follow-up of babies born following ICSI using SrCl 2 oocyte activation Dr Kyono graduated from Fukushima Medical College, receiving his MD in 1978 and his PhD in 1984 from in obstetrics and gynaecology (reproductive biology) in Tohoku University. He was a member of the Tohoku University team that succeeded in producing the first IVF pregnancy and delivery in Japan in 1983, and also succeeded the first delivery following oocyte cryopreservation in Japan in 2001 (Ladies Clinic Kyono). He is currently President of Kyono ART Clinic in Sendai. Dr Kyono s long-term research interests include studies on cryopreservation (oocyte, embryo ovarian tissue, and testicular spermatozoa), invitro maturation, in-vitro culture, andrology (MD-TESE, IMSI), and endocrinology (ovarian stimulation). Dr Koichi Kyono Koichi Kyono 1,3, Shima Kumagai 1, Chikako Nishinaka 1, Yukiko Nakajo 1, Hirofumi Uto 1, Mayumi Toya 1, Junichi Sugawara 1, Yasuhisa Araki 2 1 Kyono ART Clinic, 3F Mitsuiseimei Sendai Honcho Building, 1 1-1 Honcho, Aobaku, Sendai 980 0014; 2 The Institute for Advanced Reproductive Medical Technology, 909 21, Ishii, Fujimi, Gunma 371 0105, Japan 3 Correspondence: Tel: +81 22 7228841; Fax: +81 22 7228840; e-mail: info@ivf-kyono.or.jp Abstract This report describes six successful pregnancies (five healthy children from four deliveries and two miscarriages) with SrCl 2 oocyte activation using spermatozoa from nine patients with repeated fertilization failure. Oocytes were artificially activated by SrCl 2 30 min after intracytoplasmic sperm injection (ICSI). Oocytes were placed in 10 mmol/l of SrCl 2 medium for 1 h, rinsed several times, and then cultured in Universal IVF medium. Developmental characteristics of five resulting children until 1 year old were assessed according to the maternal and children health hand book issued by Mothers and Children s Health Organization in Japan. Mean fertilization rate, mean frequency of good cleaved embryos, pregnancy rate, and implantation rate after artificial activation in nine couples were increased from 21.7 to 64.5% (P < 0.001), from 0 to 15.4%, from 0 to 40.0% and from 0 to 25.0% respectively. Five healthy children were born following ICSI and artificial activation between February 2005 and March 2006. Physical and mental development of the children from birth to 12 months was normal. These suggest the utility and safety of SrCl 2 for patients with repeated failed fertilizations following ICSI and artificial activation. Keywords: failed fertilization, follow-up study, healthy babies, ICSI, oocyte activation, strontium chloride (SrCl 2 ) Introduction Intracytoplasmic sperm injection (ICSI) facilitates the fertilization of oocytes from patients whose partners have extremely low numbers of viable spermatozoa and a very low probability of achieving fertilization in vitro. However, one occasionally encounters unusual cases in which two pronuclei do not develop despite ICSI. The reason for this phenomenon may be a partial or complete inability of the spermatozoa to activate oocytes, sperm protamine deficiency, and an inability of the oocytes to decondense spermatozoa (Sakkas et al., 1996; Schmiady et al., 1996; Nasr-Esfahani et al., 2007). Oocytes that were not fertilized with ICSI have been subsequently activated using chemical substances or electroporation, and these oocytes have been able to form pronuclei (Tesarik and Sousa, 1995a; Battaglia et al., 1997; Rybouchkin et al., 1997; Yanagida et al., 1999, 2006; Zhang et al., 1999; Kim et al., 2001; Nakagawa et al., 2001; Eldar-Geva et al., 2003; Murase et al., 2004; Moaz et al., 2006). However, optimal methods and substances have yet to be confirmed, despite the extreme importance of this technique for rescue of unfertilized oocytes. This report therefore evaluated the efficacy of artificial oocyte activation using SrCl 2 in patients with low fertilization and achieved successful pregnancies. In addition, the safety of SrCl 2 oocyte activation was assessed by follow-up of the children for 1 year after birth. Materials and methods Patients A total of nine couples were treated with ICSI and artificial oocyte activation after failure of conventional ICSI. The couples who succeeded in achieving live births are described in detail. 53 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

Case report - Chemical oocyte activation for ICSI - K Kyono et al. 54 Case 1 A 28-year-old woman and her 32-year-old husband with severe oligo-asthenospermia were referred to the clinic (Kyono ART Clinic, Miyagi, Japan) after two attempts at achieving pregnancy via ICSI. Case 2 A 36-year-old woman presented with normal findings. The 39-yearold husband s semen analysis was severe oligo-asthenospermia. At another clinic, three previous cycles had been performed. Case 3 A 31-year-old woman displayed bilateral tubal occlusion and endometriosis. Her husband was 34 years old and semen analysis yielded normal results. At another clinic, IVF had been performed twice, but both attempts failed. This study was performed with the approval of the Ethical Review Committee at Kyono ART Clinic and with the informed consent of the patients. Ovarian stimulation and luteal support Ovarian stimulation was achieved using a combination of gonadotrophin-releasing hormone (GnRH) agonist (Nasanyl; Yamanouchi, Japan), GnRH antagonist (Cetrorelix; Serono, Switzerland) and human menopausal gonadotrophin (HMG) (Pergogreen; Serono, Switzerland). The brand of clomiphene citrate was Clomid (Shionogi, Japan). An injection of 5000 IU of human chorionic gonadotrophin (HCG) (Profasi; Serono, Switzerland) was administered when the dominant follicle reached a mean diameter of 20 mm. Vaginal ultrasound-guided follicle puncture was performed 35 h after HCG injection. Retrieved oocytes were cultured for several hours in Queen Advantage Cleavage medium (Sage, USA) at 37 C in an atmosphere of 6% CO 2, 5% O 2, and 89% N 2 under humidified conditions. All oocyte handling procedures were conducted on warm stages using conventional methods. For luteal support regarding the fresh embryo transfer cycle, the patient was administered oral oestradiol at 2 mg (oestradiol tablets, USP; Mylan Pharmaceuticals, Morgantown, USA) and a 600 mg progesterone suppository (Prometrium; Solvay Pharmaceuticals, Marietta, USA) per day until confirmation of pregnancy following embryo transfer. Clinical pregnancy was determined by the presence of a gestational sac and fetal heartbeat via transvaginal ultrasonography. Strontium treatment of oocytes ICSI was performed on metaphase II (MII) oocytes. These oocytes were cultured in Universal IVF Medium (Medicult, Denmark) for 30 min and then activated in SrCl 2 (Sigma-Aldrich; 10 mmol/l), 10% synthetic serum supplement (Irvine Scientific, USA), and Dulbecco s modified Eagle s medium (Gibco, USA) 20 µl/drop under 6% CO 2, 5% O 2, and 89% N 2 under humidified conditions for 60 min. Oocytes were subsequently rinsed several times in culture medium. Oocytes were then transferred back to Universal IVF Medium, and cultured for 18 h. Oocytes with a second polar body and two pronuclei were considered to be normally fertilized. For blastocyst cultivation, embryos were transferred to sequential medium Quinn Advantage (Sage, USA) or Multi-Blasto (Irvine, Scientific) medium on day 3. Assessment of baby development Prior to the child development assessment, informed consent was also obtained from the three couples and they were then sent written questionnaires to assess child development according to the maternal and children health hand book issued by the Mothers and Children s Health Organization in Japan from birth to 12 months (Kaneda, 2000). The questionnaire, which assessed baby development in terms of physical and mental health, was completed by their parents and paediatricians. The babies were checked at birth and characteristics by questionnaire at 3, 6, 9 and 12 months. Statistical analysis Statistical analysis was performed using Fisher s exact test. Results Case 1 Physical and gynaecological examinations yielded results within normal limits, including hysterosalpingography and routine blood tests. Menstrual cycles were regular. Mean results of semen analyses for the husband showed: volume, 5.5 ml; count, 3.8 10 6 /ml; motility, 26%; abnormal morphology, 43%. The first and second ICSI cycles were performed at another clinic; therefore, no data were available, except for the fact that no pregnancy was achieved. The third treatment involved stimulation of the ovaries by GnRH agonist-long protocol; 22 oocytes were collected, including eight mature oocytes, and ICSI was used on all eight oocytes, but none was fertilized. Four months later, at the fourth treatment, the ovaries were stimulated with GnRH agonistlong protocol, and 34 oocytes were collected of which 19 were mature. Only five of the 19 oocytes were fertilized (26.3%). All five were poorly cleaved embryos with >50% fragmentation. Three embryos were transferred, but no pregnancy occurred. Six months later, at the fifth treatment, the ovaries were stimulated with GnRH antagonist protocol. Thirty oocytes were collected, including 19 mature oocytes. Informed consent was obtained regarding the fact that oocyte activation by SrCl 2 represented an experimental step for which safety had not yet been confirmed. After ICSI, oocytes were activated and nine of the19 oocytes were fertilized (47.4%). However, all were poorly cleaved embryos with fragmentation. One poor-quality embryo (6-cell stage with >50% fragmentation) was transferred on day 3, and the remaining eight embryos were cultured for 3 more days. On day 6, one of the eight embryos developed to blastocyst grade 5 CC according to Gardner s criteria (Gardner et al., 2000). At this time, one poor-quality blastocyst was also transferred. Despite the fact that both transferred embryos were of poor quality, a successful pregnancy resulted, and the patient spontaneously

Case report -Chemical oocyte activation for ICSI - K Kyono et al. delivered a single 3152 g healthy boy (46,XY) at 41 weeks and 4 days of gestation. Subsequently, this couple attempted the same procedure again, with ICSI treatment using SrCl 2 oocyte activation. Ovarian stimulation was performed using the GnRH antagonist protocol. A total of 22 oocytes (nine mature oocytes) were retrieved, and ICSI was subsequently performed before oocyte activation using SrCl 2. Three of the nine mature oocytes were fertilized. A 4-cell embryo (grade 2) was transferred on day 2 and a blastocyst (grade 3BB) was transferred on day 5. Pregnancy again resulted and a 3298 g healthy boy (46,XY) was spontaneously delivered at 39 weeks and 5 days of gestation. Case 2 Physical and gynaecological examinations were within normal limits, including hysterosalpingography and routine blood tests. Menstrual cycles were regular. Mean results of semen analyses for the husband were: volume, 3.0 ml; count, 0.8 10 6 /ml; motility, 25%; abnormal morphology, 25%. Three cycles had been preformed at another clinic. Fertilization rates were 30.0 (3/10), 22.2 (2/9) and 33.3% (1/3), but no pregnancy resulted. The ovaries were initially with a GnRH antagonist and collected two oocytes, one of which was mature. ICSI was performed, but fertilization did not occur. One month later, at second treatment, the ovaries were stimulated by the long protocol using a GnRH agonist, and seven oocytes (all mature) were collected. Oocyte activation was attempted following ICSI, and six of the seven mature oocytes were fertilized (85.7%). Despite findings of poorly cleaved embryos in early-stage culture, four of the five embryos developed to the blastocyst stage. One blastocyst, classified as grade 3BB by Gardner s criteria (Gardner et al., 2000), was transferred. Pregnancy resulted, but ended in spontaneous abortion. Five months later, at the third treatment, the ovaries were again stimulated with the long protocol using a GnRH agonist. Eight oocytes were collected, five of which were mature. Oocytes were activated after ICSI, three of the five oocytes were fertilized (60%), and all became well-cleaved embryos. These two embryos developed to one morula, one blastocyst grade 5BB, but another seven cell embryo on day 3 remained in arrest on day 5. These two developed embryos were transferred and subsequently two gestational sacs were identified on ultrasound. The patient delivered dizygotic male twins (2266 and 1950 g; both 46,XY) at 38 weeks and 2 days of gestation by Caesarean section. Case 3 This couple visited another clinic, but although IVF was performed twice, both attempts failed. The two cycles were performed using conventional IVF and resulted in 0% fertilization rate. Next, the third cycle was performed at Kyono ART Clinic using ICSI and resulted in retrieval of nine oocytes, including six MII oocytes. Two of these six oocytes were fertilized (33.3%), but both embryos displayed arrested development, so embryo transfer was cancelled. The fourth cycle obtained nine MII oocytes, which were then activated by SrCl 2, and fertilization rate increased to 67% (6/9) and blastocysts were obtained. One early blastocyst embryo was transferred on day 5, but pregnancy was not achieved. The fifth cycle retrieved eight oocytes, and six of eight oocytes were MII. Fertilization rate of 83.3% (5/6) was obtained after activation and embryos developed to the blastocyst stage. One early blastocyst and one good-quality blastocyst (3BA) were transferred, resulting in pregnancy. A healthy girl weighing 2854 g was born at 39 weeks of gestation (Table 1). Table 1. Results of oocyte activation in three couples who achieved live births. Case 1 Case 2 Case 3 Conventional IVF Fertilization rate (%) 1) 0 (0/7) 2) 0 (0/6) Conventional ICSI Fertilization rate (%) 1) No data a 1) 30.0 (3/10) 1) 33.3 (2/6) 2) No data a 2) 22.2 (2/9) 3) 0 (0/8) 3 )33.3 (1/3) 4) 26.3 (5/19) 4) 0 (0/1) ICSI with activation Fertilization rate (%) 1) 47.4 (9/19) 1) 85.7 (6/7) 1) 66.7 (6/9) Transferred embryos 6 cell (day 3) 3BB blastocyst EB (day 5) 5CC blastocyst (day 5) (day 6) Outcome 3152 g, male Spontaneous abortion No pregnancy (6 weeks gestation) Fertilization rate (%) 2) 33.3 (3/9) 2) 60.0 (3/5) 2) 83.3 (5/6) Transferred embryos 4-cell (day 2) Morula (day 5) EB (day 5) 3BB blastocyst (day 5) 5BB blastocyst (day 5) 3BA blastocyst (day 5) Outcome 3298 g, male 2266 g, male, 1950 g, male 2854 g, female a The first and the second intracytoplasmic sperm injections (ICSI) were performed at another clinic, and the data were not available. EB = early blastocyst. 55

Case report - Chemical oocyte activation for ICSI - K Kyono et al. Overall, for the nine patients treated with artificial oocyte activation following ICSI, the mean fertilization rate after activation was significantly increased from 21.7 (15/69) to 64.5% (71/110) (P < 0.001; Table 2). Frequency of goodquality embryos on day 3 was improved from 0 (0/15) to 15.4% (10/65). Pregnancy rate and implantation rate in ICSI with artificial activation group were 40.0 (6/15) and 25.0% (7/28), compared with 0 (0/6) and 0% (0/10) in the conventional ICSI group respectively (Table 2). Assessment of baby development Physical and mental development was assessed for the five resulting children at 0 (newborn), 3, 6, 9 and 12 months. Characteristics observed at specific time points comprised: visible anomalous characteristics at 0 months; holding the head up, laughing when playing with voice reaction and ability to eat soup at 3 months; turning over in bed, sitting up, extending hand to toy, vocal expression, and reaction to television at 6 months; crawling, hanging on and standing up, grip development, playing alone, and turning in response to whispering at 9 months; and walking with support, mimicking gestures, moving to music, understanding some adult words, showing pleasure when meeting others at 12 months. According to characteristic emotions at time points, all children displayed satisfactory emotional development, so physical and mental development was considered normal. Discussion IVF has been beneficial for a variety of infertile patients, and ICSI has been particularly helpful in cases of severe male infertility. However, a 100% success rate is unattainable, as a small percentage of oocytes remain unfertilized despite insertion of high-quality spermatozoa. Most fertilization failures following ICSI reportedly manifest as non-activated oocytes (Sousa and Tesarik, 1994; Tesarik and Sousa, 1995a). However, it is not known whether failed fertilization is due to sperm factors, such as sperm protamine deficiency and inability of spermatozoa to activate the oocyte, or oocyte factor (inability of oocyte to become activated by spermatozoa) (Nasr-Esfahani et al.2007). The spermatozoa of infertile male patients possesses minimal ability to form pronuclei, even if these spermatozoa are normal in number. Furthermore, if both motility and morphology are abnormal, fertilization rate is low. This low rate of pronuclear formation might be attributable to total or partial deficiency in sperm capacity to activate oocytes, or to an inability of the oocytes to decondense spermatozoa. If infertile male spermatozoa are unable to form a pronucleus after insemination, one cause might be due to a very weak oocyteactivating sperm factor in patient spermatozoa. Several investigators have reported success after prior fertilization failures by artificially activating oocytes (Tesarik and Sousa, 1995b; Battaglia et al., 1997; Rybouchkin et al., 1997; Yanagida et al., 1999, 2006; Zhang et al., 1999; Kim et al., 2001; Nakagawa et al., 2001; Eldar-Geva et al., 2003, Murase et al., 2003). Such artificial oocyte activation in humans has included methods using calcium ionophores, electrical stimulation and puromycin. Calcium ionophore treatment is the most commonly applied method of oocyte activation in clinical trials (Tesarik and Testart, 1994; Hoshi et al. 1995; Rybouchkin et al., 1997; Moaz et al., 2006), and causes a single transient increase in intracellular calcium (Ca 2+ ). In the oocyte, this transduction pathway induces oocyte activation through signal transduction mechanisms (Swann and Ozil, 1994). Conversely, during physiological fertilization, a transient increase in intracellular Ca 2+ occurs after sperm egg fusion, followed by calcium oscillation that continues for 3 4 h. Bos-Mikich et al. (1995) demonstrated that calcium oscillations during both mitosis and exit from meiosis increase the number of inner cell mass cells in the blastocyst. Table 2. Results of oocyte activation in couples who underwent intracytoplasmic sperm injection (ICSI) with artificial oocyte activation after previous failure with conventional ICSI. Conventional ICSI ICSI with artificial activation Patients (n) 9 9 Maternal age (median years) 33.0 34.0 ICSI cycles (n) 10 18 Total no. of oocytes 111 167 Fertilization rate a (%) 21.7 (15/69) 64.5 (71/110) Day-3 good quality embryo (%) 0 (0/15) 15.4 (10/65) Embryo transfer rate (%) 60.0 (6/10) 83.3 (15/18) Implantation rates (%) 0 (0/10) 25.0 (7/28) Pregnancy rates/embryo transfer (%) 0 (0/6) 40.0 (6/15) Miscarriage rate (%) 0 33.3 (2/6) 56 Values in parentheses are numbers. a P < 0.001 for ICSI with artificial oocyte activation versus conventional ICSI.

Case report -Chemical oocyte activation for ICSI - K Kyono et al. In mice, SrCl 2 treatment is used in place of calcium ionophore treatment and electrical stimulation as the method of oocyte activation. Calcium oscillation has been confirmed in the mouse model following SrCl 2 treatment (Kline and Kline, 1992). Calcium oscillation patterns with SrCl 2 treatment thus appear closer to the pattern seen with spontaneous fertilization than artificial activation by calcium ionophore. In mice, oocytes injected with spermatozoa and treated with SrCl 2 resulted in normal fertilization (92.9%) and good offspring rate (49.2%) of transferred embryos (Suganuma et al., 2005). It is not known whether SrCl 2 could be as useful for activation as calcium ionophore treatment for human oocytes. However, Yanagida et al. (2006) recently reported SrCl 2 treatment of an infertile patient resulting in successful pregnancy and delivery using fresh embryo and cryopreserved thawed embryo transfer. This suggests that SrCl 2 treatment is useful for activating human oocytes, which frequently fail to fertilize following IVF. In the present data, nine infertile couples failed to achieve fertilization and pregnancy with previous treatments, but artificial oocyte activation with SrCl 2 resulted in three women successfully delivering five babies (three singleton pregnancies and one twin pregnancy). For these nine patients, mean fertilization rate after activation was significantly increased from 21.7 to 64.5% (P < 0.001). Frequency of good-quality embryos on D3 was improved from 0 to 15.4%. According to the maternal and children health hand book issued by Mother s and Children s Health Organization in Japan, physical and mental development of the five children was normal. Artificial oocyte activation using SrCl 2 is therefore recommended as a safe and effective method for unfertilized oocytes in infertile couples. So far as is known, the only previous use of SrCl 2 treatment in humans was reported by Yanagida et al. (2006). Although the safety of SrCl 2 treatment was clarified in children up to 1 year old, further studies are needed to confirm the safety of this oocyte activation. The long-term effects of ICSI on the resulting children remain largely unknown. It is important that the established protocol is revised often, and that the role of ICSI in infertility therapy is continually re-evaluated (Varghese et al., 2007). The same applies to the use of artificial oocyte activation with ICSI. The incidence of chromosomal abnormalities increases with the degree of abnormal sperm morphology and the presence of these abnormalities impairs oocytogenesis (Yoshida et al., 1996). Chromosome analysis is recommended for infants delivered after ICSI obtained by oocyte activation with severe infertility. 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