Case report Vitrified human day-7 blastocyst transfer: 11 cases

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1 RBMOnline - Vol 17 No Reproductive BioMedicine Online; on web 3 October 2008 Case report Vitrified human day-7 blastocyst transfer: 11 cases Kenichiro Hiraoka graduated from Hiroshima Prefectural University, Japan, in In 2000, he moved to Kinutani Women s Clinic, Hiroshima, Japan, where he has been working as an embryologist. He was awarded his PhD in 2007 from Hiroshima Prefectural University for research on cryopreservation of human expanded and hatched blastocysts by vitrification. His current interest focuses on cryopreservation of human oocytes and ovarian tissues. Dr Kenichiro Hiraoka Kenichiro Hiraoka 1,3, Megumi Fuchiwaki 1, Kaori Hiraoka 1, Toshitaka Horiuchi 2, Shinichiro Okano 1, Masayuki Kinutani 1, Kazuo Kinutani 1 1 Kinutani Women s Clinic, F, Hondori, Naka-ku, Hiroshima , Japan; 2 Graduate School of Applied Biosciences, Hiroshima Prefectural University, Hiroshima, , Japan 3 Correspondence: Tel: ; Fax: ; hiraoka@chive.ocn.ne.jp Abstract This case report describes successful pregnancies after vitrification of human day-7 blastocysts. A total of 16 day-7 blastocysts were vitrified and warmed. All 16 blastocysts survived after warming and were transferred to 11 patients. Six of the women (55%) became clinically pregnant and the implantation rate was 44% (7/16). Among these women, one woman delivered a healthy baby, two pregnancies ended in miscarriage, and three pregnancies are ongoing at 10, 29 and 34 weeks of gestation. This is the first report of successful pregnancies after vitrification of human day-7 blastocysts. Keywords: blastocyst, cryopreservation, day 7, human, transfer, vitrification Introduction With the introduction of sequential culture system, blastocyst culture is being adopted by many IVF clinics as a means to increase pregnancy rates, while minimizing multiple gestations (Gardner et al., 1998). Therefore, a reliable procedure for cryopreservation of supernumerary blastocysts is needed. Since the first pregnancy after vitrification of a human blastocyst was reported using cryostraws (Yokota et al., 2000), most attention has focused on using very small volumes of cryoprotectant. This greatly increases the cooling and warming rate, while reducing chilling injuries and ice crystal formation (reviewed by Kasai and Mukaida, 2004). The efficacy of vitrification in small volumes is demonstrated by good survival rates of human blastocysts with the cryotop (Hiraoka et al., 2004a c, 2007a c; Kuwayama et al., 2005; Stehlik et al., 2005; Liebermann and Tucker, 2006), the cryoloop (Reed et al., 2002; Mukaida et al., 2003a,b), electron microscope grids (Choi et al., 2000; Son et al., 2003, 2005) or the hemi-straw (Vanderzwalmen et al., 2003; Zech et al., 2005). However, Park et al. (2006) reported that the vitrification procedure may cause damage to blastocyst cells resulting in an increase in DNA fragmentation and apoptosisrelated gene transcription, reducing developmental capacity of vitrified bovine blastocysts. Conversely, Huang et al. (2007) reported that the number of dead cells was not significantly different between fresh and vitrified bovine blastocysts. Moreover, based on the results of pregnancies and live births from vitrified human blastocysts (Mukaida et al., 2003a; Vanderzwalmen et al., 2003; Liebermann and Tucker, 2006), the vitrification procedure itself may not cause fetal DNA fragmentation and apoptosis-related gene transcription and reduction in the developmental capacity of the blastocysts. Despite the advances in human blastocyst vitrification, much remains to be learned regarding the limits of current extended human embryo culture techniques and the clinical usefulness of later-stage cryopreservation. In fresh blastocyst transfer, previous investigators have found superior implantation rates with fresh transfer occurring at day 5 as compared with day 6 (Shapiro et al., 2001; Barrenetxea et al., 2005) and at days 5 and 6 as compared with day 7 (Gorrill et al., 2001; Utsunomiya et al., 2004). On the other hand, blastocysts vitrified on day 5 have a pregnancy potential superior (Stehlik Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 690 et al., 2005; Liebermann and Tucker, 2006) or similar (Mukaida et al., 2003a; Hiraoka et al., 2004b) to those of day- 6 blastocysts. However, as far as is known, there are no reports on pregnancies following the transfer of day-7 vitrified and warmed blastocysts. Two reports were found in which day- 7 blastocysts were frozen by a slow-freezing method (Sills et al., 2003; Richter et al., 2006). This case report presents successful pregnancies after vitrification of human day-7 blastocysts. Materials and methods Patient treatment All couples who entered this vitrified day-7 blastocyst transfer programme had at least two previous failures of embryo transfers and had unexplained infertility. The treatment was conducted with patients following informed consent and according to the guidelines of the Japan Society of Obstetrics and Gynecology (JSOG). There is no Institutional Review Board (IRB) in the (private) clinic. S Okano, K Kinutani and M Kinutani are members of JSOG and Kinutani Women s Clinic has been registered as a certified fertility centre by JSOG. Women were treated with gonadotrophin-releasing hormone (GnRH) analogue buserelin acetate (Mochida, Tokyo, Japan) from either the preceding mid-luteal phase in a long treatment protocol or second day of the cycle in a short treatment protocol. Ovarian stimulation was carried out with human menopausal gonadotrophin (Nikken, Tokyo, Japan) or urinary FSH (Fertinorm; Serono, Japan). Follicular development was monitored with serial vaginal ultrasound examinations and serum oestradiol measurements. Women were administered human chorionic gonadotrophin (HCG; Teizo, Tokyo, Japan) when dominant follicles reached a diameter of 18 mm. Oocytes were collected 35 h after HCG administration using a vaginal ultrasound-guided procedure and were incubated in human tubal fluid (HTF) medium (Irvine Scientific, CA, USA) containing 10% (v/v) serum substitute supplement (SSS; Irvine). Sperm preparation was carried out using discontinuous ISolate (Irvine) gradient. Mature oocytes were either inseminated with spermatozoa 5 7 h after oocyte retrieval at a concentration of 100, ,000 motile spermatozoa/ml for 5 10 oocytes or microinjected with a single spermatozoon. The day of insemination/injection was considered as day 0. Fertilization was confirmed at h after insemination (day 1) by the presence of two pronuclei. Embryo culture and grading of blastocysts Fertilized oocytes were washed well and cultured in Blast Assist Medium 1 (Medicult, Jyllinge, Denmark) until day 3, and then placed in Blast Assist Medium 2 (Medicult) until day 5. On day 5, the embryos were examined for development into blastocysts and then placed in fresh Blast Assist Medium 2 (Medicult) until day 7. All oocytes and embryos were incubated at 37 C in an atmosphere of 6% CO 2, 5% O 2 and 89% N 2. In two cases, one or two expanded blastocysts were transferred to the patient on day 5. Of these, one woman became pregnant and delivered a healthy baby. In another nine cases, one or two morulae were transferred to the patient on day 5. However, no women became pregnant. After embryo transfer on day 5, surplus embryos that developed to the expanded blastocyst stage (diameter 160 µm) were cryopreserved. For expanded blastocysts, the development of the inner cell mass (ICM) and trophectoderm was assessed. The ICM grading was as follows: A: tightly packed, many cells; B: loosely grouped, several cells; C: very few cells. The trophectoderm grading was as follows: A: many cells forming a tightly knit epithelium; B: few cells; C: very few cells forming a loose epithelium (Gardner and Schoolcraft, 1999). Only expanded blastocysts scoring B or higher for both ICM and trophectoderm grades (i.e. BB) were vitrified (Figure 1a). In all cases, most surplus embryos were at morula or early blastocyst stage on day 6 and expanded blastocysts were available for cryopreservation only on day 7. Vitrification of blastocysts The expanded blastocysts were vitrified by the method developed by Kuwayama et al. (2005) using a cryotop (Kitazato Supply Co., Fujinomiya, Japan), albeit with slight modifications, and has been described previously (Hiraoka et al., 2004b). The cryotop consists of a 0.4 mm wide 20 mm long 0.1 mm thick polyethylene strip attached to a plastic handle and equipped with a cover straw. As the base medium, modified HTF medium HEPES (Irvine) plus 20% (v/v) SSS (Irvine) was used. The equilibration solution contained 7.5% (v/v) ethylene glycol (Sigma Chemical Co., MO, USA) and 7.5% (v/v) dimethyl sulphoxide (Kanto Chemical Co., Tokyo, Japan). The vitrification solution was composed of 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulphoxide and 0.5 mol/l sucrose (Nacalai Tesque, Inc., Kyoto, Japan). Before starting the vitrification procedure, artificial shrinkage of expanded blastocysts was performed in the equilibration solution. First, pipetting of the expanded blastocyst was started immediately after placing the embryo in 1 ml of 30 C equilibration solution with a glass pipette slightly smaller in diameter (~140 µm) than the expanded blastocyst. After confirmation of slight shrinkage of the blastocoele, pipetting was performed with a pipette slightly smaller in diameter than the first one (~ µm). This procedure was repeated two to three times until the blastocoele collapsed completely. After blastocoele contraction, the blastocysts were equilibrated in the same equilibration solution for another 2 min before exposure to the vitrification solution. The blastocysts were then incubated in 1 ml of 30 C vitrification solution and loaded, within 45 s, onto the tip of the cryotop with ~1 µl of cryoprotectant solution. Then the cryotop was immediately submerged into liquid nitrogen which had been filter sterilized through a 0.22-µm filter (Millipore, Cork, Ireland) (Vajta et al., 1998) and, under the liquid nitrogen, the plastic cover was placed over the strip to provide protection during storage. Warming of blastocysts The warming procedure was done as follows. The protective cover was removed in liquid nitrogen and the end of the polypropylene strip was immersed directly into 1 ml of 37 C 1.0 mol/l sucrose solution for 1 min. The blastocysts were then transferred into 1 ml of 37 C 0.5 mol/l sucrose solution for 3 min and washed twice in the base medium for 5 min.

3 Assisted hatching method As soon as warming of blastocysts had completed, assisted hatching was performed while the warmed blastocysts remained collapsed (within 5 min after warming procedure completed) by the method as described previously (Hiraoka et al., 2007c). Briefly, embryos were placed under mineral oil within a 50 µl microdroplet of 37 C sperm washing medium (Irvine) in a Petri dish and positioned on the phase-contrast inverted microscope stage. Total removal of the zona pellucida was performed using a laser (Zilos-tk Laser, Hamilton Thorne Research, Beverly, MA, USA) and mechanical pipetting. Embryos were stabilized with a holding pipette held at 9 o clock position, and positioned with the laser target located on the outer edge of the zona. The power of laser was 100% and the pulse duration was 500 µs. By using this setting, a 10 µm hole was formed in the zona by one laser shot. Multiple irradiations along the convex periphery of the zona from outside to inside were used to form a 10 µm opening. Similarly, another opening was formed next to the first one. This procedure was repeated until a large opening was formed in the zona. The size of the opening was 60 90% of the circumference of the zona, estimated from an area of empty perivitelline space. It took about 2 min per embryo to complete this procedure. After forming a large opening, the embryo was released from the holding pipette and aspirated out from the opened zona by mechanical pipetting and the zona was removed. Assessment of survival After the assisted hatching procedure was completed, warmed blastocysts were rinsed several times and were cultured in Blast Assist Medium 2 (Medicult) for further culture until transfer. The post-warming survival of blastocysts was observed 1 2 h after warming under a microscope, and re-expanded blastocysts were judged to have survived (Figure 1b). Endometrial preparation and assessment of pregnancy Warmed blastocyst transfer was performed in hormonal replacement treatment cycles. All women received transdermal oestradiol (Estraderm mg, Kissei, Tokyo, Japan) for 2 days with gonadotrophin-releasing hormone analogue for the preparation of the endometrium. The administration of progesterone (vaginal 400 mg daily) was initiated when endometrial thickness exceeded 10 mm. Embryo transfer was scheduled on day 5 after the initiation of progesterone treatment. The time from warming to transfer ranged from 2 3 h. One to two surviving blastocysts were transferred into the patient s uterus. A serum pregnancy test was performed 10 days after embryo transfer. A serum HCG concentration >2 miu/ ml was considered positive. A clinical pregnancy was defined as a pregnancy when a gestational sac was recognized in the uterus by ultrasound. The implantation rate was determined by dividing the number of gestational sacs by the number of embryos transferred. Results In all, 28 day-7 blastocysts from 18 patients and 761 day-5 and day-6 blastocysts from 290 patients were vitrified between July 2002 and February Of these, data are documented from 16 vitrified day-7 blastocysts from 11 patients. The results of human vitrified day-7 blastocyst transfer are summarized in Table 1. A total of 16 day-7 blastocysts were vitrified and warmed. All 16 blastocysts (100%) survived after warming and were transferred to 11 patients. The HCG positive pregnancy rate was 72% (8/11), the clinical pregnancy rate was 55% (6/11) and the implantation rate was 44% (7/16). Of six clinical pregnancies, one pregnancy revealed two sacs with one cardiac activity and resulted in delivery of a healthy male (3050g) at 41 weeks of gestation. Another five pregnancies were diagnosed as singletons. Of these, two pregnancies spontaneously aborted a b Figure 1. Two blastocysts observed on day 7 after insemination: (a) before vitrification; (b) 2 h after warming. Following zonaremoval assisted hatching at the time of warming, blastocysts were re-expanding without zona (b). Both of the warmed blastocysts implanted after embryo transfer. The patient delivered a healthy male. 691

4 692 Table 1. Results of human vitrified day-7 blastocyst transfer. Parameter at 7 and 8 weeks of gestation, respectively, and the other three pregnancies are ongoing at 10, 29 and 34 weeks of gestation at the time of writing. Discussion Value No. of women 11 Mean age of women at warming (years) ± SD 35.5 ± 4.4 Mean no. of prior failed embryo transfers ± SD 3.6 ± 2.2 Cycles of warming 11 Blastocysts warmed 16 Blastocysts survived 16 (100) Mean no. of blastocysts transferred ± SD 1.5 ± 0.5 HCG-positive pregnancies 8 (72) Clinical pregnancies 6 (55) Embryos implanted 7 (44) Clinical pregnancy outcomes Delivery 1 Abortion 2 Ongoing 3 Values are number (percentage) unless otherwise stated. HCG = human chorionic gonadotrophin. This report documents six cases of successful pregnancy following the transfer of vitrified human day-7 blastocysts from 11 transfers in hormone replacement treatment cycles combined with total zona removal assisted hatching. The survival rate of vitrified day-7 blastocysts in the present report was high as 100% (16/16). In the author s clinical experience, artificial shrinkage of blastocoele has proved to be effective for increasing the survival rate of vitrified day-5 and day-6 zona-intact expanded blastocysts (98%; 162/166) (Hiraoka et al., 2007c) and that of vitrified day-5 and day-6 zona-free hatched blastocysts (100%; 4/4) (Hiraoka et al., 2007a). Therefore, the same vitrification protocol was applied to the day-7 blastocysts, which could be why a high survival rate of vitrified day-7 blastocysts in the present study was observed. The survival, pregnancy and implantation rates of vitrified day-7 blastocysts in the present report (100%, 16/16; 55%, 6/11; 44%, 7/16) were similar to those of blastocysts vitrified on day 5 (97%, 33/34; 63%, 12/19; 45%, 15/33) and day 6 (96%, 23/24; 53%, 9/17; 48%, 11/23) in hormone replacement treatment cycles combined with total zona removal assisted hatching groups in a previous report (Hiraoka et al., 2007c). However, the abortion rate of vitrified day-7 blastocysts in the present report (33%; 2/6) was higher than that of blastocysts vitrified on day 5 (25%; 3/12) and day 6 (22%; 2/9) of the above same groups in the previous report. These results suggest that, although more slowly developing blastocysts may be innately compromised to some extent, day-7 blastocysts have similar implantation potential to day-5 and day-6 blastocysts and can be vitrified without impairing their ability to implant. There are two reports of live births following fresh (Sagoskin et al., 2002) and cryopreserved (Sills et al., 2003) day-7 blastocyst transfers in both of which assisted hatching was performed. Similarly, in this report, assisted hatching was performed for all warmed day-7 blastocysts. The rate of blastocyst development appears to be related to the ultimate ability for blastocysts to hatch free from their zona pellucida (Porter et al., 2000). It has been proposed that more slowly forming blastocysts may benefit from assisted hatching to compensate for their poorer potential to escape the zona pellucida (Tucker, 1999) and thereby improve implantation, presumably by both guaranteeing hatching and hastening contact of the trophectoderm with the endometrium after uterine transfer. On the other hand, the vitrification procedure may cause the hardening of the zona pellucida of human blastocysts. Resistance to enzymatic removal of zona pellucida by pronase has been reported to be significantly increased after vitrification compared with fresh human blastocysts (Vanderzwalmen et al., 2003; Hiraoka et al., 2007b). Moreover, Vanderzwalmen et al. (2003) reported that assisted hatching after warming of vitrified blastocysts significantly improved the pregnancy and implantation rates. Similarly, it has been previously reported that assisted hatching after warming of vitrified human day-5 and day-6 blastocysts significantly improved the pregnancy and implantation rates (54%, 13/24; 37%, 15/41) as compared with the control group (13%, 1/8; 7%, 1/15) (Hiraoka et al., 2004c). Consequently, it is plausible that assisted hatching for vitrified day-7 blastocysts contributed to the positive outcome observed here. Murata et al. (2005) reported that significantly higher clinical pregnancy results were obtained in cryopreserved day-6 blastocyst transfers at hormonal replacement treatment cycles (55%, 22/40) compared with fresh day-6 blastocyst replacements (11%, 4/37). In addition, the pregnancy rate of vitrified day-7 blastocyst transfers at hormonal replacement treatment cycles of the present report (55%, 6/11) was significantly higher than that of fresh day-7 blastocyst transfers of the reports of Gorrill et al. (2001) (4%, 1/25) and Utsunomiya et al. (2004) (10%, 3/31). While the importance of embryo quality has been clearly demonstrated, another cause of implantation failure may be impairment of endometrial receptivity. This suggestion is supported by higher implantation rates in hormonal replacement treatment cycles (Edwards et al., 1991). Pinopodes have been proposed as an accurate marker for endometrial receptivity (Psychoyos, 1986). The pinopodes appear about 1 week after ovulation and they develop and regress within just 2 days (Develioglu et al., 1999; Nikas and Aghajanova, 2002; Nikas and Makrigiannakis, 2003). On the other hand, hatching of human embryos occurs between day 5 and day 9 after fertilization (Porter et al., 2000). These observations suggest that slower growing fresh embryos cannot implant due to asynchrony with the implantation window and that embryo transfer onto a hormone-stimulated endometrium assists the implantation of cryopreserved day-7 blastocysts. A limitation of this report is that the number of treatment cycles was small. Only 18 cohorts of day-7 blastocysts have been vitrified and 11 cohorts warmed between July 2002 and February The other seven remain in storage and have not yet been warmed. Therefore, it will be difficult to increase the number of treatment cycles from this group in a short period. However, the information of vitrified day-7 blastocyst transfer is lacking and embryo culture termination and failure to cryopreserve

5 until day 7 may have occurred in many IVF clinics. That would have resulted in the loss of viable supernumerary embryos. Accordingly, although the number of treatment cycles was small, the present report has profound clinical value in knowing that more slowly developing blastocysts can be vitrified as late as day 7, warmed and result in a delivery. In conclusion, this is the first report of successful pregnancies after vitrification of human day-7 blastocysts. These results suggest that vitrified human day-7 blastocyst transfer can contribute to increasing cumulative pregnancy rates in assisted reproduction. However, it will require more data on vitrified day-7 blastocyst transfer to determine the survival, implantation, and live birth rates from these more slower developing embryos. In addition, the risk of biochemical pregnancies, spontaneous abortions and birth defects must be determined. 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6 the open pulled straw (OPS) vitrification method. Cryo Letters 19, Vanderzwalmen P, Bertin G, Debauche Ch et al Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Human Reproduction 18, Yokota Y, Sato S, Yokota M et al Successful pregnancy following blastocyst vitrification. Human Reproduction 15, Zech NH, Lejeune B, Zech H et al Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification. Reproductive BioMedicine Online 11, A report of one of the transfers was presented orally in Japanese at the 48th Annual Meeting of the Japanese Society of Mammalian Ova Research, Yamanashi, Japan, May 26, Declaration: The authors report no financial or commercial conflicts of interest. Received 2 January 2008; refereed 28 January 2008; accepted 16 June