Synchronization between embryo development and endometrium is a contributing factor for rescue ICSI outcome

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1 Reproductive BioMedicine Online (2012) 24, ARTICLE Synchronization between embryo development and endometrium is a contributing factor for rescue ICSI outcome Li Ming, Liu Ping *, Qiao Jie, Lian Ying, Zheng Xiaoying, Ren Xiulian, Huang Jin, Wu Yuqi a Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing , People s Republic of China; b Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing , People s Republic of China * Corresponding author. address: pingliu7703@sina.com (P Liu). Professor Liu Ping, MD is Associate Chief Physician and Associate Director of the Center of Reproductive Medicine, Peking University Third Hospital, Beijing. She specializes in diagnosis and treatment of male and female infertility with assisted reproductive treatment, including IVF and single sperm microinjection, and in reproductive endocrine diseases in gynaecology. She participated as a major staff member for the culture of the first live-born IVF and gamete intra-fallopian transfer babies as well as the first baby from a freeze thawed embryo in mainland China. She is a young-group member of the board of the Chinese Medical Association s branch of obstetrics and gynaecology. Abstract Recent evidence shows that the outcome of rescue intracytoplasmic sperm injection (ICSI) is unsatisfactory on account of a poor clinical pregnancy rate. These outcomes may be due to either the in-vitro ageing of cultured oocytes before ICSI or the asynchrony between the embryo developmental stage and the endometrial secretory pattern. To address the latter issue, this study performed a retrospective analysis of 534 fresh cycles after rescue ICSI and 64 frozen thawed cycles in subsequent treatment. Rescue ICSI cycles were divided into three groups: group I included 469 fresh embryo-transfer (FET) cycles; group II included 74 FET cycles in which supernumerary good-quality embryos were also cryopreserved; and group III included 64 frozen thawed transfer cycles. Group III was considered to have achieved better synchronization than group II. As a result, significantly higher clinical pregnancy (29.69%, 19/64 versus 10.81%, 8/74) and implantation (13.33%, 22/165 versus 5.13%, 8/156) rates were achieved in group III compared with group II (both P < 0.05). Therefore, synchronization of embryo development with the endometrium is considered a contributing factor for rescue ICSI outcome. It is recommended that embryos derived from rescue ICSI cycles should be cryopreserved and subsequently used in frozen thawed cycles. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cryopreservation, endometrium, implantation rate, pregnancy rate, rescue ICSI, synchronization /$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 528 L Ming et al. Introduction Unexpected fertilization failure sometimes occurs in conventional IVF cycles. To salvage fertilization failure, intracytoplasmic sperm injection (ICSI) of unfertilized 1-day-old oocytes, called rescue ICSI has been frequently suggested (Kuczynski et al., 2002; Lombardi et al., 2003; Lundin et al., 1996; Morton et al., 1997; Nagy et al., 1993; Yuzpe et al., 2000). Recent studies showed that the outcome of rescue ICSI was unsatisfactory on account of a poor clinical pregnancy rates (Chen and Kattera, 2003; Kuczynski et al., 2002), although some pregnancies and live births have been reported in fresh cycles (Lundin et al., 1996; Morton et al., 1997; Yuzpe et al., 2000;) or frozen thawed cycles (Lombardi et al., 2003; Sermondade et al., 2010; Yuzpe et al., 2000). These outcomes may be due to either the in-vitro ageing of cultured oocytes before ICSI or the asynchrony between the embryo developmental stage and the endometrial secretory pattern. The majority of studies have paid attention to the ageing of cultured oocytes, which could cause increased cytogenetic abnormalities (Chen and Kattera, 2003; DeUgarte et al., 2006; Pehlivan et al., 2004), lower fertilization rate, fewer normal viable embryos and poorer pregnancy rates. Meanwhile, few studies (Sermondade et al., 2010) have focused on the synchronization of the embryo development with the endometrium. To address the latter issue, the current study performed a retrospective analysis of 534 fresh cycles after rescue ICSI (from January 2006 to January 2011) and 64 frozen thawed cycles in subsequent treatment (from January 2006 to May 2011) in this infertility centre. Materials and methods A total of 24,855 cycles was performed in the infertility centre from January 2006 to January 2011 and 15,162 (61.00%) of these used conventional IVF. Of these, a total of 534 (3.52%, 534/15,162) rescue ICSI cycles were performed after total fertilization failure in conventional cycles from January 2006 to January Embryo transfer was not performed in 65 cycles due to fertilization failure after rescue ICSI (n = 28), no embryos suitable for transfer (n = 34), risk of ovarian hyperstimulation syndrome (n = 2) and fever on the day of embryo transfer (n = 1). There were 469 fresh embryo transfers (FET) on day 3 after oocyte retrieval. Among the 534 rescue ICSI cycles, 77 cycles included cryopreservation of cleavage-stage embryos. FET was not performed in three of the cycles in which embryos were cryopreserved due to risk of ovarian hyperstimulation syndrome (n = 2) and fever on the day of embryo transfer (n = 1). Of the 74 FET and cryopreservation cycles, 64 frozen thawed transfers of cleavage-stage embryos were performed in subsequent cycles from January 2006 to May Ovarian stimulation for oocyte retrieval was performed by standard procedures as previously described (Zheng et al., 2008). Oocytes were inseminated by conventional IVF 3 4 h after oocyte retrieval. Spermatozoa were collected by the swim-up technique, using 100,000 motile spermatozoa per insemination dish, each containing 3 5 oocytes. Oocytes were examined for evidence of fertilization between h after insemination. When fertilization failure was detected, rescue ICSI was performed as previously described (Palermo et al., 1992). Spermatozoa from the original insemination were used in all rescue ICSI procedures. Oocytes were examined for evidence of fertilization h after insemination and good-quality embryos (4 cells and 10% fragmentation) were transferred 48 h after rescue ICSI. Serum human chorionic gonadotrophin (HCG) concentration was measured 2 weeks after embryo transfer and clinical pregnancy was defined as the presence of a gestational sac on ultrasound examination on day 35 after transfer. Supernumerary good-quality embryos were cryopreserved by slow freezing 48 or 72 h after rescue ICSI and were transferred in a subsequent thawing cycle. The slow-freezing and thawing protocols were performed by standard procedures as previously described (Zheng et al., 2008). Frozen thawed embryo transfers were performed in either natural monitored cycles or programmed artificial cycles. Natural cycles were used for women with regular ovulatory menstrual cycles. Thawed embryo transfer was scheduled for 2 or 3 days after ovulation. Luteal support was provided with intramuscular injections of progesterone (progesterone; Shanghai General Pharmaceutical Company, China) in oil (20 40 mg) from the night of transfer to day 14 when the serum HCG test was performed. In cycles treated with hormone replacement therapy, endometrial development was achieved with oral oestradiol. When oestradiol concentrations and endometrial thickness were suitable, this phase was complemented by administration of progesterone. Embryo transfer was performed on day 3 or 4 of progesterone administration. Hormone replacement therapy was continued until the pregnancy test. Serum HCG concentration was determined 14 days after embryo replacement and 1 week later. If pregnancy was initiated, steroid supplementation was maintained until week 12 of gestation. Clinical pregnancy was defined as the presence of a gestational sac on ultrasound examination on day 35 after transfer (Zheng et al., 2008). This study included cleavage-stage embryo transfers in fresh and frozen thawed cycles and excluded blastocyst-stage embryo transfers. The cycles were divided into three groups: group I included 469 FET on day 3 after oocyte retrieval (on day 2 after rescue ICSI), group II included 74 cycles from the 469 FET cycles in which supernumerary good-quality embryos were cryopreserved on day 3 or 4 after oocyte retrieval (on day 2 or 3 after rescue ICSI); and group III included 64 frozen thawed transfer cycles. Depending on the number of embryos, developmental stage and degree of cytoplasmic fragmentation, embryos were either frozen on day 3 after oocyte retrieval (on day 2 after rescue ICSI) or on day 4 after oocyte retrieval (on day 3 after rescue ICSI). Group III was therefore divided into two subgroups: group A, in which embryos were frozen on day 3 after oocyte retrieval (on day 2 after rescue ICSI) and thawed embryos were transferred on day 2 after ovulation (33 cycles); and group B, in which embryos were frozen on day 4 after oocyte retrieval (on day 3 after rescue ICSI) and thawed embryos were transferred on day 3 after ovulation (31 cycles). The group A protocol was used when there were at least two embryos (four cells, 10% fragmentation) on day 3 after oocyte retrieval. It is sometimes difficult to

3 Endometrial synchronization is a contributing factor for rescue ICSI outcome 529 decide whether embryos should be cryopreserved, for example when there are less than two embryos (four cells, 10% fragmentation) or when there were more than six embryos on day 3 after oocyte retrieval. Under these circumstances, the group B protocol was used and embryo culture was continued for one more day. If embryos went on developing for 1 day, resulting in at least two embryos (five cells, 20% fragmentation), the developing embryos were frozen on that day (day 4 after oocyte retrieval). The study was approved by the Ethics Committee of Peking University Third Hospital (reference no ) and all patients signed written informed consent. Data were analysed using the chi-squared test. All analyses were performed with Statistical Package for Social Sciences version 17.0 (SPSS, USA). Statistical significance was defined as P < Results A total of 534 rescue ICSI cycles were performed after total fertilization failure in conventional IVF cycles from January 2006 to January There were 5797 unfertilized oocytes in these cycles, of which 4824 (83.22%, 4824/5797) were metaphase-ii oocytes chosen for rescue ICSI. Normal fertilization was observed in 2170 (44.98%, 2170/4824) oocytes after rescue ICSI. Finally, there were 1038 embryos available for FET on day 3 after oocyte retrieval and 378 for cryopreservation. In group I, there were 469 FET of 2.13 ± 0.58 embryos and 40 clinical pregnancies resulted (pregnancy rate (PR) 8.53%, 40/469; implantation rate (IR) 4.50%, 45/1000). In group II, there were 74 FET of 2.11 ± 0.31 embryos and eight clinical pregnancies resulted (PR 10.81%, 8/74; IR 5.13%, 8/156) (Table 1). The 74 cryopreserved embryo cycles performed after rescue ICSI subsequently resulted in 64 frozen thawed transfer cycles (group III). A total of 266 embryos were thawed, of which 184 survived (survival rate 69.17%). This resulted in 64 frozen thawed transfer cycles of 2.58 ± 0.87 embryos and 19 clinical pregnancies resulted (PR 29.69%, 19/64; IR 13.33%, 22/165) (Table 1). In group I (women s age ± 4.34 years), the 40 clinical pregnancies resulted in 26 singleton and five twin gestations, seven missed abortions, one ectopic pregnancy and one late termination of pregnancy because of fetal congenital eye abnormality. To date (June 2011), 16 healthy infants have been delivered (nine girls and seven boys) and 15 pregnancies are ongoing. In group II (women s age ± 3.38 years), all eight clinical pregnancies were singleton. To date (June 2011), five healthy infants have been delivered (two girls and three boys) and three pregnancies are ongoing. In group III (women s age ± 3.27 years), the 19 clinical pregnancies included 13 singletons, one twin, one triplet and four missed abortions. To date (June 2011), 12 healthy infants have been delivered (nine girls and three boys) and three pregnancies are ongoing. A significant difference (P < 0.05) was found in the women s age between groups I and III (33.21 ± 4.34 years versus ± 3.27 years); women s age was similar in groups II and III (Table 1). Since women s age is a primary factor affecting the IVF-transfer outcome (Maheshwari et al., 2008; Malizia et al., 2009), the differences in outcomes between groups II and III are noteworthy. Group III was divided into groups A and B according to when embryos resulting from rescue ICSI were frozen and transferred in a subsequent cycle. No significant differences were found between the outcomes in these two groups (Table 2). Discussion In this retrospective study, of the 266 embryos thawed, 184 (69.17%) survived the thawing process, which was similar to that of the 3467 normal frozen thawed cycles in this infertility centre from January 2006 to May 2011 (70.11%, 9534/13,599). These outcomes demonstrated that good-quality embryos obtained after rescue ICSI of 1-day-old oocytes were able to tolerate the process of cryopreservation. Thus, it is suggested that the good-quality embryos derived from rescue ICSI should be cryopreserved for future treatment. In group I, the clinical PR was 8.53% in fresh rescue ICSI cycles, which was basically in agreement with the findings in previous studies (Lundin et al., 1996; Morton et al., 1997; Yuzpe et al., 2000). The low PR in fresh rescue ICSI cycles could be related to the in-vitro ageing of cultured oocytes (Kuczynski et al., 2002). Besides, some studies have shown that there was a dramatic increase in embryonic chromosomal abnormalities when the time interval between oocyte retrieval and fertilization was extended (DeUgarte et al., 2006; Pehlivan et al., 2004). Another factor likely to contribute to low PR in fresh rescue ICSI cycles could be the Table 1 Outcome of rescue intracytoplasmic sperm injection (ICSI) cycles in groups I, II and III. Group I (n = 469) Group II (n = 74) Group III (n = 64) P-value Women s age (years) ± ± ± 3.27 <0.05 a ;NS b Clinical pregnancy rate/transfer cycle 8.53 (40/469) (8/74) (19/64) <0.05 b Implantation rate/embryo transferred 4.50 (45/1000) 5.13 (8/156) (22/165) <0.05 b Values are mean ± SD or % (n/total). Group I = fresh cleavage-stage embryo transfers after rescue ICSI; group II = fresh cycles in group I that included cryopreservation of cleavage-stage embryos following rescue ICSI; group III = frozen thawed transfers of cleavage-stage embryos in subsequent cycles for group II. a Between groups I and III. b Between groups II and III.

4 530 L Ming et al. Table 2 Outcome of rescue intracytoplasmic sperm injection (ICSI) cycles in subgroups A and B of group III. Group A (n = 33) Group B (n = 31) Women s age (years) ± ± 2.23 Clinical pregnancy rate/transfer cycle (9/33) (10/31) Implantation rate/embryo transferred 12.5 (11/88) (11/77) Values are mean ± SD or % (n/total). The 64 frozen thawed transfers of cleavage-stage embryos in group III were divided into two groups: group A = embryos frozen on day 3 after oocyte retrieval (on day 2 after rescue ICSI) and thawed embryos transferred on day 2 after ovulation; group B = embryos frozen on day 4 after oocyte retrieval (on day 3 after rescue ICSI) and thawed embryos transferred on day 3 after ovulation. There were no statistically significant differences between the two groups. asynchrony between the embryo development stage and the endometrial secretory pattern (Sermondade et al., 2010). It has been recognized that the endometrium is receptive during a limited period of time, called the implantation window (Wilcox et al., 1999). Based on Noyes criteria, some studies have shown that endometrial maturation on the day of oocyte retrieval in IVF cycles is advanced by 2 4 days compared with natural cycles. In those series, no ongoing clinical pregnancy was achieved when the endometrial advancement surpassed 3 days (Kolibianakis et al., 2002; Ubaldi et al., 1997). FET in day-1 rescue ICSI cycles is more likely to miss the implantation window compared with normal IVF cycles; therefore, better results can be achieved owing to a better endometrial synchronization in frozen thawed cycles. The current study found that the PR obtained in group III was significantly higher than that in group II (29.69% versus 10.81%), which is in accordance with the above assumption. These results suggest that synchronization of embryo development with the endometrium could be a major factor contributing to the outcome of rescue ICSI; therefore, it is important to get a better endometrial synchronization in subsequent frozen thawed cycles. In order to improve synchronization of embryo development with the endometrium, two methods were applied in group III, giving the two groups, A and B, as described in the Materials and Methods. The clinical pregnancy rates were 27.27% and 32.25% in groups A and B, respectively, and did not differ significantly. These results were acceptable compared with the clinical PR of 10.81% in group II. In conclusion, a better PR in frozen thawed rescue ICSI cycles can be obtained by using either method, which may achieve a better synchronization of embryo development with the endometrium. It is remarkable to find that, of the 534 rescue ICSI cycles from January 2006 to January 2011, there were only 77 cycles (14.42%, 77/534) with cleavage-stage embryos frozen. In contrast, around 55% of normal IVF cycles in this study centre had cleavage-stage embryos frozen from January 2006 to January The number of viable embryos in rescue ICSI cycles was much less than in normal IVF cycles, as, presumably, in-vitro ageing of the oocyte led to poor developmental potential of embryos. These results suggest that both the in-vitro ageing of cultured oocytes and synchronization of the embryo development with the endometrium are factors of concern in relation to rescue ICSI outcome. Several studies have reported infants born after rescue ICSI, including fresh (Lundin et al., 1996; Morton et al., 1997; Yuzpe et al., 2000;) and frozen thawed cycles (Lombardi et al., 2003; Sermondade et al., 2010; Yuzpe et al., 2000). No genetically abnormal infants have been reported in these studies. In the current study, 28 infants were born after rescue ICSI and no birth defects have been found. It is worth noting that one pregnancy was terminated because of congenital eye malformations in a FET cycle; however, no conclusions can be drawn due to the small number of such infants. Some investigators suggested that rescue ICSI should be applied in combination with preimplantation genetic diagnosis to achieve a successful pregnancy and that prenatal genetic diagnosis should be performed for patients conceiving after rescue ICSI (Morton et al., 1997; Pehlivan et al., 2004; DeUgarte et al., 2006). In conclusion, good-quality embryos obtained after rescue ICSI of 1-day-old oocytes are able to tolerate the procedure of cryopreservation and should be cryopreserved for future treatment. This study demonstrates that synchronization of the embryo development with the endometrium is a contributing factor for rescue ICSI outcome. It might improve the rescue ICSI outcome by achieving a better synchronization of embryo development with the endometrium in frozen thawed cycles, which can consequently result in viable pregnancies and healthy infants. It is recommended that embryos derived from rescue ICSI cycles be cryopreserved and subsequently used in frozen thawed cycles. Acknowledgements This work was supported in part by Natural Sciences Foundation of China (Grant Nos and /H0426). References Chen, C., Kattera, S., Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF. Hum. Reprod. 18, DeUgarte, C.M., Li, M., Jordan, B., Hill, D., Rescue intracytoplasmic sperm injection and preimplantation genetic diagnosis in combination can result in pregnancy. Fertil. Steril. 86, Kolibianakis, E., Bourgain, C., Albano, C., Osmanagaoglu, K., Smitz, J., Van Steirteghem, A., Devroey, P., Effect of ovarian stimulation with recombinant follicle-stimulating hormone,

5 Endometrial synchronization is a contributing factor for rescue ICSI outcome 531 gonadotropin releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil. Steril. 78, Kuczynski, W., Dhont, M., Grygoruk, C., Pietrewicz, P., Redzko, S., Szamatowicz, M., Rescue ICSI of unfertilized oocytes after IVF. Hum. Reprod. 17, Lombardi, E., Tiveron, M., Inza, R., Valcarcel, A., Young, E., Bisioli, C., Live birth and normal 1-year follow-up of a baby born after transfer of cryopreserved embryos from rescue intracytoplasmic sperm injection of 1-day-old oocytes. Fertil. Steril. 80, Lundin, K., Sjogren, A., Hamberger, L., Reinsemination of 1-day-old oocytes by use of intracytoplasmic sperm injection. Fertil. Steril. 66, Maheshwari, A., Hamilton, M., Bhattacharya, S., Effect of female age on the diagnostic categories of infertility. Hum. Reprod. 23 (3), Malizia, B.A., Hacker, M.R., Penzias, A.S., Cumulative live-birth rates after in vitro fertilization. N. Engl. J. Med. 360, Morton, P.C., Yoder, C.S., Tucker, M.J., Wright, G., Brochkman, W.D.W., Kort, H.I., Reinsemination by intracytoplasmic sperm injection of 1-day-old oocytes after complete conventional fertilization failure. Fertil. Steril. 68, Nagy, Z.P., Joris, H., Liu, J., Staessen, C., Devroey, P., Van Steirteghem, A.C., Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes. Hum. Reprod. 8, Palermo, G., Joris, H., Devroey, P., Van Steirteghem, A.C., Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, Pehlivan, T., Rubio, C., Ruiz, A., Navarro, J., Remohi, J., Pellicer, A., Simón, C., Embryonic chromosomal abnormalities obtained after rescue intracytoplasmic sperm injection of 1-day-old unfertilized oocytes. J. Assist. Reprod. Genet. 21, Sermondade, N., Hugues, J.N., Cedrin-Durnerin, I., Poncelet, C., Benzacken, B., Lévy, R., Sifer, C., Should all embryos from day 1 rescue intracytoplasmic sperm injection be transferred during frozen thawed cycles? Fertil. Steril. 94, Ubaldi, F., Bourgain, C., Tournaye, H., Smitz, J., Van Steirteghem, A., Devroey, P., Endometrial evaluation by aspiration biopsy on the day of oocyte retrieval in the embryo transfer cycles in patients with serum progesterone rise during the follicular phase. Fertil. Steril. 67, Wilcox, A.J., Baird, D.D., Weinberg, C.R., Time of implantation of the conceptus and loss of pregnancy. N. Engl. J. Med. 340, Yuzpe, A.A., Liu, Z., Fluker, M.R., Rescue intracytoplasmic sperm injection (ICSI)-salvaging in vitro fertilization (IVF) cycles after total or near-total fertilization failure. Fertil. Steril. 73, Zheng, X., Liu, P., Chen, G., Qiao, J., Wu, Y., Fan, M., Viability of frozen thawed human embryos with one-two blastomeres lysis. J. Assist. Reprod. Genet. 25, Declaration: The authors report no financial or commercial conflicts of interest. Received 6 January 2012; refereed 31 January 2012; accepted 1 February 2012.