Mechanically expanding the zona pellucida of human frozen thawed embryos: a new method of assisted hatching

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1 Mechanically expanding the zona pellucida of human frozen thawed embryos: a new method of assisted hatching Cong Fang, Ph.D., Tao Li, Ph.D., Ben-Yu Miao, M.D., Guang-Lun Zhuang, M.D., and Canquan Zhou, M.D. Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-Sen University, Guangdong, People s Republic of China Objective: To determine whether a new assisted hatching (AH) method increases the implantation and clinical pregnancy rates of frozen thawed day-3 (D3) embryos. Design: Prospective study. Setting: A university hospital in vitro fertilization (IVF) program. Patient(s): Patients who had their first IVF/intracytoplasmic sperm injection (ICSI) cycles between June 1, 2006, and December 31, 2008, with fresh IVF embryo transfer failures or without fresh embryo transfer. Intervention(s): The couples were randomized into thawed embryo transfer after AH versus no AH. In the AH group, the zona pellucida (ZP) of D3 frozen thawed embryos was expanded by injected hydrostatic pressure after thawing. In the control group, embryos were pierced by ICSI needles without expanding the ZP. Main Outcome Measure(s): Clinical pregnancy and implantation rates. Result(s): The morphologic features of the blastomeres were carefully monitored and recorded. In the AH group, 244 embryos were thawed, and 178 (73.0%) survived; in the control group, 259 embryos were thawed, and 190 (73.4%) survived. Despite the transfer of a similar number of embryos, the AH group resulted in statistically significantly higher implantation and clinical pregnancy rates compared with the no AH group. Conclusion(s): Mechanically expanding the ZP of frozen thawed D3 embryos with injected hydrostatic pressure after thawing increases the implantation rate compared with control embryos. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Assisted hatching, frozen-thawed embryo, implantation, zona pellucida Hatching of the blastocyst from the zona pellucida (ZP) is critical for embryo implantation. If the blastocyst does not hatch within the time period in which the uterus is receptive to implantation, pregnancy will not occur (1). For both fresh and cryopreserved thawed embryos, impaired hatching may be due to the extended time in culture in an artificial environment causing a hardening of the ZP, as evaluated on the basis of ZP dissolution times (2). For cryopreserved thawed embryos, the freeze thaw process might further exacerbate hardening of the ZP (3, 4), although this has not been specifically demonstrated. The artificial rupture or thinning of the ZP before embryo transfer assisted hatching (AH) has been proposed to foster spontaneous hatching and improve embryo implantation Received June 26, 2009; revised and accepted August 7, 2009; published online September 26, C.F. has nothing to disclose. T.L. has nothing to disclose. B-Y.M. has nothing to disclose. G-L.Z. has nothing to disclose. C.Z. has nothing to disclose. Supported by Guangdong Natural Science Foundation (No /No ); the National Basic Research Program of China (973 Program, No. 2007CB948103). Cong Fang and Tao Li contributed equally to this work. Reprint requests: Tao Li, Ph.D., Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-Sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, People s Republic of China (FAX: ; lilitao@gmail.com). rates (5). Techniques for AH in IVF programs introduced over the years include mechanical (1), chemical (5), enzymatic (6), and laser manipulation (7). Although different devices and procedures have been used for AH, methods can be classified into two types: breaching the ZP or thinning the ZP (8). Despite initial promising results, it has been suggested that complete perforation of the ZP may have adverse effects, such as abnormal blastocyst expansion, loss of blastomeres through the breached ZP, and disappearance of the protective effect of the ZP against infectious or immunologic aggression (9, 10). Similarly, the benefit of ZP thinning has also been debated. Indeed, some studies have reported that failure of ZP thinning greatly enhances human embryo implantation (11). In fact, some investigators have suggested that the bilayered human ZP needs to be fully breached to enhance the implantation ability of such embryos (12). To obviate such potential drawbacks, a new kind of mechanical AH method has been developed, which was inspired by the natural expanding effects of blastocysts on the ZP. This mechanical AH neither thins nor breaches the ZP; rather, it expands/stretches the ZP via the injected hydrostatic pressure to assist with the embryo hatching process. In our study, the new method was applied to frozen thawed day-3 (D3) embryos to determine whether it could enhance their implantation ability. It was assumed that ZP hardening in frozen thawed embryos is more pronounced than in fresh embryos Fertility and Sterility â Vol. 94, No. 4, September /$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 TABLE 1 Experimental data from zona expanding on human embryos. AH group Control group P value Total no. of survived day-3 embryos a Embryos with 6 8 blastomeres survived Embryos with 3 5 blastomeres survived Mean no. of intact blastomeres/embryo No. of day-5 blastocysts (%) 18 (31.0) 15 (25.9).681 Hatching/hatched blastocysts (%) 13 (22.4) 4 (6.9).033 b No. of day-6 blastocysts (%) 25 (43.1) 23 (39.7).851 Hatching/hatched blastocysts (%) 19 (32.8) 7 (12.1).013 b a Embryos with R50% intact blastomeres after thawing were indicated as survived embryos. b Statistically significantly different. MATERIALS AND METHODS Preliminary Experimental Work We performed a randomized study on donated frozen thawed human embryos to evaluate the hatching efficiency and safety of this new mechanical AH method before introducing it into our AH program. The frozen embryos were voluntarily donated for research from consenting couples who had achieved pregnancies upon completion of an IVF or ICSI embryo transfer between March 2000 and September In the AH group, the ZP was expanded by injected hydrostatic pressure using an ICSI injection needle. The morphology of the blastomeres was carefully monitored and recorded. Table 1 showed that this method was effective, and no detrimental effects were manifested for embryo development. Therefore, we began our clinical work, mechanically expanding the ZP of frozen-thawed human embryos for AH. Patient Selection Criteria Patients who had their first IVF/ICSI cycles between 2006 and 2008 with fresh IVF-ET failures or without fresh embryo transfer were considered for participation in the study. The patients were recruited once, with only the first frozen thawed embryo transfer (FET) attempts included in the study, and were randomly allocated to the treatment and control groups. Patients with only one frozen embryo available for transfer after thawing were excluded from the study. The study was approved by the ethics committee of the Faculty of Medicine of Sun Yat-Sen University, and informed consent was obtained from all couples. Embryo Freezing Embryos were cryopreserved at the cleavage stage, 3 days after oocyte retrieval, by the use of a programmable freezer (Planer Products Ltd., Sunbury-on-Thames, Middlesex, United Kingdom). Only good quality embryos with <50% fragments and R5 blastomeres on D3 were cryopreserved. The freezing program for embryos in our unit was as follows: starting temperature, 20 C; rate of cooling, 2 C/minute from 20 Cto 7 C; manual seeding. After seeding, the embryos were further cooled to 30 C at 0.3 C/minute, and then to 150 Cat10 C/minute. Embryo Thawing Procedure Embryos were thawed in the morning of the day of transfer following the manufacturer s instructions (Embryo Thawing Pack; Sage, Trumbull, CT). All embryologic parameters of the frozen thawed embryos were evaluated immediately after thawing, and reevaluated after 3 hours of in vitro culture by an experienced embryologist who was blinded to the group assignment. The total number of clearly visible blastomeres, the presence of lysed blastomeres, and the embryo scores were recorded. AH Procedure The conventional ICSI holding pipette, injection pipette, and the ICSI manipulator were used for the new AH method. For the procedure, a Falcon 1006 dish was prepared by adding 50- ml microdroplets of HTF-HEPES medium (Sage), one drop for each embryo to be expanded. The HTF-HEPES medium was withdrawn into the injection needle until a length of 200 mm of the injection needle was filled with medium (Fig. 1A). The embryo was firmly attached to the holding pipette by suction, showing a blastomere-free area, which was toward the 3 o clock position. Once the injection needle pierced through the entire ZP, HTF-HEPES medium was injected into the perivitelline space until the preloaded medium was dispelled from injection pipette (see Fig. 1B). After that, the injection needle was gently withdrawn from the embryo. The treated embryos were cultured for an additional 3 hours until reevaluation of embryo morphology before transfer. In the control group, the embryos were pierced by ICSI needles without expanding the ZP. Fertility and Sterility â 1303

3 FET Cycles Three hours after AH, the embryo transfer was performed. The number of embryos transferred varied from two to three. All transfers were performed gently using a Cook embryo replacement catheter (Cook Ob/Gyn, Spencer, IN). After thawing, the frozen embryos were transferred in hormonal replacement cycles. Statistical Analysis Patient age between the hatched and nonhatched groups was compared using a two-sample t-test. Clinical pregnancy and implantation rates were analyzed with the use of a chi-square test. P<.05 was considered statistically significant. FIGURE 1 (A) HTF-HEPES medium was withdrawn into the injection needle until a length of 200 mm ofthe injection needle was filled with medium (magnification: 40). (B) The zona pellucida expanded abruptly when HTF-HEPES medium was injected into the perivitelline space (magnification: 200). RESULTS The prestudy results are summarized in Table 1. There were 159 D3 cryopreserved embryos donated for the preliminary study. After thawing, 116 embryos survived. The surviving embryos were randomized into the AH and no AH groups. Prethaw and postthaw embryo quality and cryosurvival were similar between the two groups. No blastomeres were damaged during the AH procedure. Though the blastocyst formation rate was not statistically significantly different between the two groups, statistically significantly more embryos hatched in vitro in the AH group (P<.05). This randomized prospective study included 131 FET cycles performed between January 1, 2007, and December 31, The couples were randomized into two groups (thawed embryo transfer after AH versus no AH). Due to the thawing procedure, four cycles were excluded from the AH group, and two cycles were excluded from the control group. Table 2 summarizes the demographic data and number of frozen embryos. There were no differences in the age of women at embryo freezing/thawing, duration of infertility, primary/secondary infertility, cause of infertility, basal serum FSH concentration, or the number of embryos frozen between the AH group and the control group. In the AH group, 244 embryos were thawed, and 178 survived (73.0%) survived, and in the control group, 259 embryos were thawed, and 190 survived (73.4%). The two groups had a comparable number of blastomeres, fresh embryo grades, presence of cell lysis, and degree of fragmentation of frozen embryos (Table 2). Therefore, we compared AH, which was performed on 178 embryos from 61 transfer cycles with a control group of 190 embryos from 64 transfer cycles. In the AH group, the ZP was expanded and thinned for a very short time (%30 seconds) during the AH procedure (see Fig. 1B). The ZP quickly returned to its original diameter. No blastomere damage was detected during the AH procedure. Moreover, after 3 hours of culture before ET, we did not note a reduction in the number of blastomeres when we reevaluated the quality of the transferred embryos. The clinical results of thaw transfer cycles are summarized in Table 3. Despite the transfer of an equal number of embryos, AH resulted in statistically significantly higher implantation and clinical pregnancy rates as compared with no hatching. DISCUSSION Thinning of the ZP with pronase/tyroid acid treatment or laser manipulation has the advantage of dissolving or drilling off the outer layer. The whole ZP or part of the ZP is thinned, but the integrity is maintained (13, 14). In theory, it would be easier for embryos to hatch out of a thinned ZP. However, some studies have failed to show any beneficial effect of ZP thinning methods in terms of implantation and live birth rates (15). The inner layer of the ZP is somewhat unaffected or left intact with this type of AH method, which may explain the lack of efficacy of the methods observed in these studies. Although the inner layer of the ZP is completely breached 1304 Fang et al. New mechanical assisted hatching method Vol. 94, No. 4, September 2010

4 TABLE 2 Patients characteristics in the zona-expanding and no-expanding groups. Assisted hatching group No assisted hatching group P value No. of embryo transfer cycles Age of women at embryo freezing (y) NS Duration of infertility (y) NS Primary/secondary infertility 37/24 34/30 NS Basal FSH level (miu/ml) NS Cause of infertility Male NS Tubal NS Endometriosis 3 5 NS Unexplained 4 2 NS Mixed 9 7 NS Note: NS ¼ not statistically significant. with other methods, it has been shown that complete perforation of the ZP may have adverse effects on subsequent embryonic development (1, 16). Considering that hydrostatic pressure exerted on the ZP through the expansion of the blastocyst has been shown to play an important role for hatching (17, 18), we advanced a new mechanical AH method, which mimics via the injected hydrostatic pressure the expanding effect blastocysts exert on the ZP. Traditional AH methods have focused on surgery involving the outer layer of the ZP or even the entire ZP, but the main focus of this new mechanical AH method is hatching dynamics and ZP ultrastructure. In the method we have described, the ZP expanded abruptly once medium was injected into the perivitelline TABLE 3 Comparison of embryo characteristics and outcomes of frozen thawed embryo transfer cycles between the zona-expanding group and the control group. Assisted hatching group No assisted hatching group P value No. of thawed embryos Cryosurvival (%) a 178 (73.0) 190 (73.4).918 No. of embryos with all blastomeres survived No. of embryos with 1 necrotic blastomere No. of embryos with 2 necrotic blastomeres No. of transfer cycles Total no. of transferred embryos Mean no. of transferred embryos Mean no. of intact blastomeres/transferred embryo No. of transferred embryos with 6 8 blastomeres survived No. of transferred embryos with 3 5 blastomere survived No. of clinical pregnancies b 23 (37.7) 13 (20.3).032 c Implantation rate per transferred embryo (%) d 25/178 (14.0) 14/190 (7.4).038 c a Embryos with R50% intact blastomeres after thawing were indicated as survived embryos and were transferred back to patients. b Number of sacs divided by embryo transfer cycles. c Statistically significantly different. d Number of sacs divided by total number of embryos transferred. Fertility and Sterility â 1305

5 space (see Fig. 1B). However, the expansion of the ZP could be maintained for no longer than 30 seconds. The ZP quickly returned to its original diameter (within 1 minute) after a period of short expansion, even with continued injection of media, which easily flushed out of the enlarged ZP mesh. Moreover, in a preliminary study, we demonstrated that the ZP could not be reexpanded by hydrostatic pressure after 8 and 24 hours in vitro (data not shown). Thus, transient expansion of the ZP resulted in an irreversible change in the ZP ultrastructure. We assumed that the initial expansion of the ZP might be critical for the embryo hatching process owing to the change in the ZP ultrastructure, thus making the subsequent blastocyst expansion and ZP thinning occur more easily. Furthermore, if there were some errors with the first expansion, embryo hatching might not occur. In the preliminary study, our results clearly demonstrated that the blastocyst hatching rate was statistically significantly higher in the AH group than in the control group. The ultrastructural change of the ZP might also occur naturally in the hatching blastocyst. After being expanded and stretched by the blastocyst several times, the ZP becomes increasingly thinned and easily broken. However, artificial expansion of the ZP with injected hydrostatic pressure occurred only once, which did not completely mimic the expanding effects of the blastocysts on the ZP. It would perhaps be clinically beneficial if the new mechanical AH technique could expand the ZP more than once. Even though natural expanding/stretching of the ZP by the contracting/expanding blastocyst begins on days 4 to 6, preexpanding the ZP of frozen thawed D3 embryos might result in irreversible changes in the ZP ultrastructure, and assist the subsequent physiologic ZP thinning and embryo hatching on days 5 to 6. Our prestudy showed that the hatching rate of blastocysts was increased by this AH method. The clinical results also indicated that the implantation and pregnancy rates were higher in the AH group than the control group. Indeed, this is the first AH method to be described that uses natural hatching dynamics without thinning or breaching of the ZP. With this AH method, the injected hydropressure not only expands the ZP of frozen thawed embryos but also exerts pressure on the embryo blastomeres. It is possible that some blastomeres could be damaged or crushed by the injected hydrostatic pressure. However, no blastomere lysis was observed during the AH process or 3 hours of in vitro culture. Furthermore, the extraordinarily good results of FET cycles in the AH group make it clear that this theoretical damage was of minor consequence for the survival and developmental potential of the frozen thawed embryos. It was difficult to characterize the exact changes in ZP ultrastructure after it was expanded/stretched by the injected hydrostatic pressure. A comparison of the ZP ultrastructure between artificially and naturally expanded ZP through electron microscopic analysis might help to determine the effects of injected hydropressure on the ZP and the hatching efficiency of this AH method. Although our prestudy showed the increased hatching rate of frozen thawed embryos by this AH method, injected medium might further flush out residual cryoprotectants from thawed embryos, possibly accounting, in part, for the increased implantation and pregnancy rates. The beneficial effects of this new AH method need to be further confirmed on fresh embryos and compared with other AH methods. Our study demonstrated that mechanical expansion of the ZP of frozen thawed D3 embryos with injected hydrostatic pressure after thawing could increase the implantation rate compared with control embryos. 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