Article Zona pellucida removal and vitrified blastocyst transfer outcome: a preliminary study

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1 RBMOnline - Vol 15. No Reproductive BioMedicine Online; on web 16 May 2007 Article Zona pellucida removal and vitrified blastocyst transfer outcome: a preliminary study 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 on the subject of 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, Tomoyo Murakami 1, Masayuki Kinutani 1, Kazuo Kinutani 1 1 Kinutani Women s Clinic, F, Ohtemachi, 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 The objective of this study was to investigate whether a change in assisted hatching technique from partial opening to total removal of the zona pellucida improved the outcome of vitrified blastocyst transfer. This was a preliminary observational study conducted from November 2003 to April Partial opening using acid Tyrode s solution was performed in 45 cycles, while total removal using a laser and mechanical pipetting was performed in 57 cycles. The clinical pregnancy, implantation, and delivery rates were higher in the total removal group than in the partial opening group (67% versus 42%, P < 0.02; 55% versus 30%, P < 0.01; 56% versus 36%, P < 0.04, respectively). These results suggest that total removal of the zona pellucida is associated with higher pregnancy, implantation and delivery rates compared with partial opening for vitrified blastocyst transfer. Keywords: assisted hatching, blastocyst, cryopreservation, human, vitrification, zona pellucida Introduction 68 With the introduction of the 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; Marek et al., 1999; Milki et al., 2000). 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; Kuwayama, 2005). The efficacy of vitrification in small volumes is demonstrated by good survival rates of human blastocysts with the cryotop, the cryoloop, electron microscope grids or the hemi-straw (Choi et al., 2000; Kuwayama, 2000, 2001; Cho et al., 2002; Mukaida et al., 2002, 2003, 2006; Reed et al., 2002; Son et al., 2003, 2005; Vanderzwalmen et al., 2003; Hiraoka et al., 2004a c, 2006, 2007a,b; Huang et al., 2005; Stehlik et al., 2005; Kuwayama et al., 2005; Zech et al., 2005; Liebermann and Tucker, 2006). On the other hand, the vitrification procedure may cause the hardening of the zona pellucida (ZP) of human blastocysts. Resistance to enzymatic removal of ZP by pronase has been reported to be significantly increased after vitrification compared with fresh human blastocysts (Vanderzwalmen et al., 2003; Hiraoka et al., 2007b). Using scanning electron microscopy, Moreira da Silva and Metelo (2005) observed that the diameter of the pores in the outer surface of the ZP of vitrified bovine morula/blastocyst embryos were significantly smaller compared with fresh embryos. They also observed that the number of pores of vitrified embryos were significantly fewer compared with fresh embryos. These results indicated that the vitrification procedure causes alterations in the ZP that may cause its 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK

2 hardening. However, few reports are available on the effect of assisted hatching for human vitrified blastocyst transfers. As far as is known, only two reports have analysed the effect of assisted hatching for human vitrified blastocyst transfers. It has been reported that partial opening of the ZP of vitrified human blastocysts after warming considerably improved the implantation and pregnancy rates (Vanderzwalmen et al., 2003; Hiraoka et al., 2004c). However, it was noted in vitro that partial opening of the ZP, in some cases, can also impede the hatching of vitrified human blastocysts, making some blastocysts unable to hatch and retain their integrity. Considering that partial opening may potentially be associated with entrapment of the blastocyst, this study wanted to know whether assisted hatching with total removal of the ZP is preferable to partial opening of the ZP for vitrified blastocyst transfer. However, there are no studies comparing the relative effectiveness of each method for human vitrified blastocyst transfer. The aim of this study was to evaluate retrospectively the results of two different methods of assisted hatching (partial ZP opening using acid Tyrode s solution and total ZP removal using a laser and mechanical pipetting) for human vitrified blastocyst transfer. Materials and methods Patient treatment All patients who entered this vitrified blastocyst transfer programme had previous multiple failures of conventional day 2 or day 3 embryo transfers. The day of oocyte retrieval was considered as day 0. The experiment 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 in the (private) study clinic; Drs 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) at 37 C in an atmosphere of 6% CO 2, 5% O 2 and 89% N 2. Sperm preparation was carried out using discontinuous Isolate (Irvine) gradient. Mature oocytes were either inseminated with spermatozoa 5 7 h after oocyte retrieval at the concentration of 100,000 to 200,000 motile spermatozoa/ml for 5 to 10 oocytes or microinjected with a single spermatozoon. Fertilization was confirmed at h after insemination 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 6. In conventional cases, one or two blastocysts were transferred to the patient on day 5 or day 6. After blastocyst transfer, surplus embryos that developed to the expanded blastocyst stage (diameter 160 μm), which were not hatching or had not hatched from the ZP, were cryopreserved on day 5 or day 6. In some cases, all fresh blastocysts were cryopreserved in order to reduce the risks of ovarian hyperstimulation syndrome. For expanded blastocysts, the development of the inner cell mass (ICM) and trophectoderm can be 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. Vitrification of blastocysts The expanded blastocysts were vitrified by the method developed by Kuwayama (2001) 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 69

3 70 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. Assisted hatching methods As soon as warming of blastocysts was completed, assisted hatching was performed while the warmed blastocysts remained collapsed (within 5 min of completing the warming procedure). 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. Partial opening of the ZP was performed as described by Cohen et al. (1992) with modifications. Briefly, embryos were stabilized with a holding pipette held at the 9 o clock position and a 10 μm pipette containing acid Tyrode s solution and connected with a 3 ml syringe was oriented at the 3 o clock position next to an area of empty perivitelline space. A μm diameter defect in the ZP was then created by blowing acid Tyrode s solution over the external surface of the ZP. This was a modification and it may increase the amount of solution expelled above that of the original protocol. Total removal of the ZP was performed using a laser (Zilostk Laser, Hamilton Thorne Research, Beverly, MA, USA) and mechanical pipetting. Embryos were stabilized with a holding pipette held at the 9 o clock position (Figure 1a), and positioned with the laser target located on the outer edge of the ZP. The power of the laser was 100% and the pulse duration was 500 μs. By using this setting, a 10 μm hole was formed in the ZP by one laser shot. Multiple irradiations along the convex periphery of the ZP from outward to inward were used to form a 10 μm opening (Figure 1b). Similarly, another opening was formed next to the first one. This procedure was repeated until a large opening was formed in the ZP (Figure 1c). The size of the opening was 60 90% of the circumference of the ZP, 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 (Figure 1d) and aspirated out from the opened ZP by mechanical pipetting (Figure 1e) and the ZP was removed (Figure 1f). 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 3 h after warming under a microscope, and re-expanded blastocysts were judged to have survived (Figure 2a,b). Endometrial preparation and assessment of pregnancy Warmed blastocyst transfer was performed in natural or hormone replacement treatment cycles. In natural cycles, embryo transfer was scheduled on day 5 after ovulation in the spontaneous cycles, irrespective of whether they had been day 5 or day 6 at the time of vitrification. In hormone replacement treatment cycles, all women received transdermal oestradiol (Estraderm mg; Kissei, Tokyo, Japan) for two 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 irrespective of whether they had been day 5 or day 6 at the time of vitrification. The time from warming to transfer ranged from 2 5 h. One to three surviving blastocysts were transferred into the patient s uterus. Pregnancy was first assessed by urinary HCG 9 days after blastocyst transfer, and then clinical pregnancy was confirmed by the presence of fetal heart activity 30 days after blastocyst transfer. Statistics The Mann-Whitney test, unpaired Student s t test, chi-squared test and Fisher s exact test were used as appropriate to determine statistical differences between groups. A P-value of <0.05 was considered significant. Results Between November 2003 and April 2006, 105 consecutive patients entered this vitrified blastocyst transfer programme. During this period, no blastocysts survived for three patients and embryo transfer was cancelled for these patients. Partial opening of the ZP using acid Tyrode s solution was performed for 45 patients between November 2003 and January Total removal of the ZP using a laser and mechanical pipetting was conducted for 57 patients between February 2005 and April Table 1 summarizes the patients demographic characteristics. There were no differences between the partial opening and total removal groups in the age of women at vitrification or warming, number of prior stimulated cycles, primary infertility, secondary infertility or cause of infertility. The number of oocytes retrieved, maturation rate, the percentage of conventional IVF or intracytoplasmic sperm injection, fertilization rate, cleavage rate, the number of blastocysts and the number of blastocysts vitrified were also comparable between the two groups (Table 2). Table 3 summarizes the outcomes of vitrified blastocyst transfer. The mean number of prior failures of embryo transfers, type of endometrial preparation, percentage of day 5 and day 6 blastocysts, morphology scores of blastocysts and survival rate were similar between the two groups, while a significantly (P < 0.05) lower mean number of blastocysts were transferred in the total removal group. On the other hand, clinical pregnancy, implantation, and delivery rates were significantly higher in the total removal group compared with the partial opening assisted hatching group (67% versus 42%, P < 0.02; 55% versus 30%, P < 0.01; 56% versus 36%, P < 0.04 respectively). No difference in the multiple pregnancy rate was found between the two groups. All multiple pregnancies in both groups were twin and dizygotic. In all, 57 infants have been born, 19 from the partial opening group and 38 from the total removal group. All women have delivered infants that have had normal physical profiles up to the present.

4 a b c d e f Figure 1. A warmed human blastocyst: (a) stabilized with a holding pipette; (b) at the beginning of assisted hatching (a 10 μm opening is formed); (c) after assisted hatching with multiple laser shots (the size of opening is about 60% of circumference of zona pellucida); (d) released from holding pipette; (e) during zona pellucida removal by mechanical pipetting; (f) removed and empty zona pellucida. Bar represents 100 μm. a b Figure 2. Warmed human blastocysts 2 h after warming: (a) embryos treated by partial opening of the zona pellucida using acid Tyrode s solution; (b) embryos treated by total removal of the zona pellucida using a laser and mechanical pipetting. Both assisted hatching procedures were conducted at the time of warming. Bar represents 100 μm. 71

5 Table 1. Demographic characteristics of women in the partial opening and total removal assisted hatching groups. Characteristic Partial Total opening removal No. of women Mean age of women at vitrification (years) ± SD 33.9 ± ± 4.2 Mean age of women at warming (years) ± SD 34.4 ± ± 4.1 Mean no. of prior stimulated cycles ± SD 2.5 ± ± 1.8 No. with primary infertility No. with secondary infertility 9 13 Cause of infertility Tuboperitoneal (%) 6 (13) 11 (19) Endometriosis (%) 4 (9) 5 (9) Male (%) 13 (29) 13 (23) Unexplained (%) 22 (49) 28 (49) There were no statistically significant differences between the two groups. Table 2. Comparison of embryo development in stimulated cycles and number of vitrified blastocysts between the partial opening and the total removal assisted hatching groups. Parameter Partial Total opening removal Mean no. of oocytes 14.0 ± ± 7.0 MII oocytes (%) Insemination method Conventional IVF (%) 6 (13) 8 (14) ICSI (%) 39 (87) 49 (86) Fertilization rate (% of total oocytes) Cleavage rate (%) Mean no. of day 5 blastocysts ± SD (%) 2.5 ± 1.8 (32) 2.7 ± 3.2 (36) Mean no. of day 5 and 6 blastocysts ± SD (%) 4.0 ± 2.1 (50) 3.7 ± 3.1 (49) Mean no. of blastocysts vitrified ± SD 3.0 ± ± 2.9 There were no statistically significant differences between the two groups. MII = metaphase II; ICSI = intracytoplasmic sperm injection. 72

6 Table 3. Comparison of outcomes of vitrified blastocyst transfer cycles between the partial opening and the total removal assisted hatching groups. Parameter Partial Total P-value opening removal Mean no. of prior failed embryo transfers ± SD 4.1 ± ± 2.7 NS No. of cycles for warming Type of endometrial preparation Natural cycle (%) 17 (38) 21 (37) NS HRT cycle (%) 28 (62) 36 (63) NS No. of blastocysts warmed No. of day 5 blastocysts (%) 41 (53) 50 (57) NS No. of day 6 blastocysts (%) 37 (47) 38 (43) NS Blastocyst morphology a AA (%) 24 (31) 28 (32) NS AB (%) 18 (23) 17 (19) NS BA (%) 5 (6) 6 (7) NS BB (%) 31 (40) 37 (42) NS No. of blastocysts survived (%) 77 (99) 85 (97) NS Mean no. of blastocysts transferred ± SD 1.7 ± ± No. of clinical pregnancies (%) 19 (42) 38 (67) No. of multiple pregnancies (%) 3 (16) 9 (24) NS No. of embryos implanted (%) 23 (30) 47 (55) No. of deliveries (%) 16 (36) 32 (56) HRT = hormone replacement treatment; NS = not significant. a For details of grading, see Materials and methods. Discussion This study suggests that assisted hatching with total removal of the ZP using a laser and mechanical pipetting significantly improves the implantation (P < 0.01), pregnancy (P < 0.02) and delivery (P < 0.04) rates of vitrified blastocysts as compared with partial opening of the ZP using acid Tyrode s solution. Lower pregnancy and implantation rates (42% and 30%) were observed after the transfer of partial ZP opening vitrified blastocysts in the present study. These results are congruent with those reported by Vanderzwalmen et al. (2003) (38% and 22%) and Liebermann and Tucker (2006) (46% and 31%). Both research groups performed assisted hatching with partial ZP opening for vitrified human blastocysts after warming. In the present study and the report of Liebermann and Tucker (2006) a μm hole was drilled in the ZP (Sills et al., 2003). In the report of Vanderzwalmen et al. (2003), two μm length slits were created in the ZP. On the other hand, Lyu et al. (2005) reported that when the slit size for assisted hatching of fresh human blastocysts was μm, significantly more of the larger ICM became trapped by the ZP opening during hatching than the smaller ICM. Therefore, it is suggested that opening the ZP with a hole of μm for assisted hatching of vitrified human blastocysts is inadequate for the completion of hatching in some cases: however, it can be considerably improved by total removal of the ZP. Partial ZP opening using acid Tyrode s solution (Cohen et al., 1992; Tucker et al., 1993; Check et al., 1996) and a laser (Ng et al., 2005; Balaban et al., 2006; Jones et al., 2006) is normally performed at the cleavage stage. However, assisted hatching was performed with either acid Tyrode s solution (partial opening) or a laser (total removal) at the blastocyst stage in the present study. In a previous study at the clinic, when partial opening assisted hatching at the blastocyst stage with either acid Tyrode s solution or a laser were compared for human blastocysts developed from frozen cleaved embryos, no difference was found with regard to pregnancy rate (45%; 9/20 and 40%; 8/20 for acid Tyrode s solution and laser, respectively), implantation rate (28%; 9/32 versus 27%; 8/30, respectively) and delivery rate (40%; 8/20 versus 35%; 7/20, respectively) (unpublished data). Therefore, it can be assumed that if a laser had been used in both groups in the present study, it would have made no difference to the outcome of the partial opening group. To remove the ZP, multiple shots of the laser beam were used. This procedure may pose potential harm to embryos. Hartshorn et al. (2005a) demonstrated that embryos at the 8-cell stage are able to respond to thermal shock by activating heat shock protein (hsp) production, as shown by the sharp increase in hsp70i transcription that follows embryo exposure to elevated temperature. They also reported that in 8-cell mouse embryos, laser drilling of the ZP did not stimulate hsp70i expression, even in the blastomeres closest to the laser beam (Hartshorn et al., 2005b). In addition, the results for total ZP removal by a laser demonstrated high survival, implantation, pregnancy and delivery rates. However, the fact that a pregnancy is established does not preclude the presence of underlying anomalies. 73

7 74 Therefore, further long-term studies that monitor birth defects and other anomalies occurring later in life, are needed to confirm and assess the safety of total ablation of the ZP using a laser. In handling mouse embryos, hatched blastocysts are more likely to stick to the inner surface of the cryostraw (Zhu et al., 1996) and the culture dishes (authors observation). However, human blastocysts with the ZP removed did not stick to the surface of the culture dishes or the inner surface of embryo replacement catheter. Therefore, these blastocysts could be handled like intact blastocysts. The preferred outcome of assisted reproductive technology is a singleton delivery. To achieve this, it is evident that a single embryo should be transferred. Gardner et al. (2004) proposed that before considering single blastocyst transfers, it would appear prudent to transfer two blastocysts to ensure that the clinic can establish acceptable implantation and pregnancy rates. Rates could be set arbitrarily at >45% and >60% for implantation and pregnancy, respectively. In this study, it has been confirmed that high implantation and pregnancy rates (55% and 67% respectively) can be attained with the transfer of an average of 1.5 vitrified blastocysts after total removal of the ZP. These results suggest that if the safety of total ZP removal using either laser or another method is confirmed, the move to single vitrified blastocyst transfer will be possible. In conclusion, this is the first report comparing the results of two different methods of assisted hatching for human vitrified blastocyst transfer. In this study, the number of treatment cycles was small because this study is preliminary and was conducted for a short period. Further prospective randomized trials comparing the effectiveness of partial opening versus total removal of the ZP using a laser in vitrified blastocyst transfer may be needed. 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