Extended culture of vitrified warmed embryos in day-3 embryo transfer cycles: a randomized controlled pilot study

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1 Reproductive BioMedicine Online (2013) 26, ARTICLE Extended culture of vitrified warmed embryos in day-3 embryo transfer cycles: a randomized controlled pilot study Rentao Jin a, *,1, Xianhong Tong a,1, Limin Wu a, Lihua Luo a, Hongbing Luan a, Guixiang Zhou a, Lars Johansson b, Yusheng Liu a, * a Reproductive Medical Center, Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui, China; b Centre for Assisted Reproduction, Codra Hospital, Podgorica, Montenegro * Corresponding authors. addresses: jackyandcherry@gmail.com (R Jin), shengzhizhongxin@126.com (Y Liu). 1 These authors contributed equally to this work. After a 10-year career in the Reproductive Medical Center in Anhui Provincial Hospital in China, Mr. Jin is now a MD candidate in immunology at Anhui Medical University. He has worked as an embryologist and has performed a number of IVF treatments. He also takes part in several research projects on fields of interest such as vitrification, repeat pregnancy loss and in-vitro maturation. His major interests include reproductive immunology and cryopreservation. Abstract Synchronization between embryonic stage and endometrium is vital to achieve a successful pregnancy. The objective of this study was to assess the implantation, clinical pregnancy and live birth rates of cryopreserved embryo transfer cycles using embryos after extended culture for 16 h. A prospective randomized controlled pilot study was performed on women who underwent vitrified warmed embryo transfer. Of the 540 women assessed for eligibility, 479 were randomly allocated to either extended culture for h (EC group, n = 242) or conventional culture for 2 h (control group, n = 237). Endometrial preparation was the same in both groups. No significant differences were found between the extended culture and control groups respectively in clinical pregnancy rate per embryo transfer (42.48% versus 40.95%), implantation rate (21.79% versus 20.82%) or live birth rate per embryo transfer (37.61% versus 34.05%); however, the spontaneous reduction rate was lower in the extended culture group (10.04% versus 20.80%; P = 0.032) In conclusion, extended culture of day-3 cleavage embryos for 16 h would not influence the pregnancy outcome of day-3 cryopreserved embryo transfer cycles. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cryopreserved embryo transfer, endometrium, extended culture, implantation, pregnancy outcome, synchronization /$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

2 Extended culture of vitrified warmed embryos has no effect on pregnancy outcome 385 Introduction Selective embryo transfer, in combination with cryopreservation of surplus high-quality embryos (Grady et al., 2012), significantly reduces multiple pregnancy risks and cryopreserved embryos can subsequently be used for later transfer, thus improving cumulative pregnancy rate of a single IVF cycle. Also, in cases of ovarian hyperstimulation syndrome (Griesinger et al., 2007; Griesinger et al., 2011) and premature luteinization (Bosch et al., 2003; Van Vaerenbergh et al., 2011), the retrieved oocytes and generated embryos can be vitrified and transferred into a more favourable uterine environment (Krikun et al., 2005; Liu et al., 2008). For a successful implantation to be achieved in a cryopreserved embryo transfer cycle, it is crucial to transfer embryos in the implantation window, which is limited to 4 8 days after endogenous or exogenous progesterone stimulation. Using ultrasound, endometrial thickness (Kehila et al., 2010), endometrial morphological aspects (Bourgain and Devroey, 2007) and endometrial and sub-endometrial blood flow (Wang et al., 2010) are useful indicators to predict endometrial receptivity to embryonic implantation. However, although most precautions are undertaken, approximately two-thirds of embryo transfers fail to result in implantation, possibly due to inadequate endometrial receptivity or defects of the embryo endometrium crosstalk (Altmae et al., 2012; Simon et al., 1998). An alternative hypothesis is that the endometrium only plays a generally permissive role; the decisive step is the actual synchronization between the viable embryo and the receptive endometrium. Previous studies on endometrial receptivity show that the out-of-phase endometrium is detrimental to embryo viability and implantation, but to maintain the synchronization is difficult in practice. It has been suggested that the impairment of synchronization is due to the high concentration of steroids resulting from ovarian stimulation affecting the endometrium (Krikun et al., 2005; Liu et al., 2008; Wilcox et al., 1999), suboptimal culture conditions (Waldenstrom et al., 2009) and media (Gruber and Klein, 2011) which affect embryo quality. Asynchronization, influenced by a delay in development of the embryo or endometrial advancement, induces implantation failure. A degree of asynchronization between the endometrium and embryo is acceptable within a certain range (Imbar and Hurwitz, 2004). On the other hand, many kinds of drugs such as human chorionic gonadotrophin (HCG) (Papanikolaou et al., 2010), progesterone (Kumar et al., 1998) and oestradiol (Ma et al., 2003) disturb endometrial development. If endometrial advancement is less than 3 days, a satisfactory outcome can be achieved (Papanikolaou et al., 2010). Knowledge of the degree of synchronization may be useful in improving implantation rates of cryopreserved embryo transfers. In addition, because early implantation helps to improve pregnancy outcome (Wilcox et al., 1999), patients may benefit from prewarming of their cryopreserved embryos. This study is a prospective randomized controlled trial, where the effects of 2 h versus 16 h culture of vitrified embryos and transfer into the same phase of endometrium, were compared. Materials and methods The study was performed from January 2010 to February According to their previous indication, the regimen was selected. In brief, in patients with ovulation disorders, cryopreserved embryo transfer was carried out either in a stimulated or hormone-replacement cycle. In patients with spontaneous ovulation and a normal menstrual cycle, transfers were performed in a natural cycle, a mild stimulation cycle or an artificial cycle. All patients undergoing cryopreserved embryo transfer were allocated with random assignment provided by the Research Randomizer ( randomizer.org/) into the extended culture (EC) or the control group. Embryos were warmed and cultured overnight for 16 h in the EC group and for 2 h in the control group. All cryopreserved embryos were transferred to a well-prepared endometrium on day 3 of progesterone supplementation (Figure 1). The inclusion criteria were: (i) age <38 years; (ii) vitrification performed on day 3; and (iii) insemination complete within 2 6 h on day of oocyte retrieval. The exclusion criteria were: (i) repeated IVF failure; and (ii) disorder of uterine Figure 1 Schematic representation of the trial protocol. All embryos were cryopreserved on day 3 of the fresh cycles. In the extended culture group, embryos were warmed on day 2 of progesterone supplementation, cultured overnight and then transferred (at day 4 of preimplantation development). In the control group, embryos were warmed on day 3 of progesterone supplementation and transferred within 2 h of warming (at day 3 of preimplantation development). ET = embryo transfer.

3 386 R Jin et al. cavity. The study was approved by the Ethics Committee of the hospital on 5 January 2009 (Reference No ). All patients signed informed consent before the treatment procedure. Ovarian stimulation and embryo culture As the drug regimen has no influence on survival or success rates of cryopreserved embryo transfers (Bahceci et al., 2009; Heijnen et al., 2007; Medved et al., 2006), different stimulation protocols in fresh cycles including gonadotrophin-releasing hormone agonist and antagonist were enrolled. In fresh cycles, HCG was administrated when the dominant follicles reached mm, and average serum oestradiol reached around pg/ml. Oocytes were retrieved 36 h after HCG injection using a single-lumen needle (Cook Medical, Sydney, Australia). The collected oocytes were inseminated either via conventional IVF or via intracytoplasmic sperm injection (ICSI) on the day of the retrieval. Sydney IVF Fertilization Medium was used during the fertilization process, and Sydney IVF Cleavage Medium was used to culture all embryos (Cook Medical). Criteria for selection of embryos for vitrification The embryos were evaluated according to the number of blastomeres, their symmetry and cytoplasmic fragmentation. Embryos with >50% fragmentation and embryos with no cell division between days 2 and 3 were not eligible for vitrification or transfer and were discarded. All other surplus embryos were cryopreserved on day 3. Vitrification of embryos Embryos were vitrified on day-3 post insemination or injection, using the Cryotop method (Kuwayama et al., 2005) and with commercially available medium (Kitazato, Shizuoka, Japan). The vitrification solutions were warmed at room temperature for at least 1 h and the embryos incubated for 5 min in the equilibration solution followed by a maximum of 60 s in the vitrification solution before plunging into liquid nitrogen. Warming of embryos Before warming of the embryos, 1.0 mol/l sucrose solution was warmed to 37 C in an incubator and 0.5, 0.25 and mol/l sucrose solutions, as well as the base medium, were warmed to room temperature for about 1 h before use. The warming procedure was carried out as follows: the Cryotop with the embryos was plunged directly into the warm 1.0 mol/l sucrose solution and the embryos incubated for 1 min, and then the embryos were then transferred to 0.5, 0.25 and mol/l sucrose solutions and incubated for 2.5 min in each solution and finally incubated in the base medium for 5 min. None of the warmed embryos underwent artificial hatching post warming and all were at day 3 of preimplantation development. In the EC group, all the embryos were cultured overnight in Sydney IVF blastocyst medium (Cook Medical) and embryos at day 4 of preimplantation development were transferred at h culture post warming. In the control group, embryos at day 3 of preimplantation development were transferred within 2 h of warming. Preparation of endometrium In a natural cycle, the recipients were monitored to detect the dominant follicle and endometrial thickness. When the leading follicle had reached 17 mm and endometrial thickness was >7 mm, 10,000 IU HCG (Livzon, Zhuhai, China) was administered. Progesterone supplementation (20 mg/ day i.m.; progesterone in oil; Xianju Pharma, Hangzhou, China) was started on the following day after HCG day and embryos were transferred 4 days after HCG day. During artificial cycles, patients took oral oestradiol (2 mg, Progynova; Bayer HealthCare, Germany) twice per day, starting on day 2 of their menstrual cycle. A transvaginal sonogram was performed on day 9 or 10 of the menstrual cycle and if the endometrial thickness was >7 mm and a triple line endometrium was present, progesterone supplementation as for natural cycles was started. If, on the contrary, the endometrium was <7 mm in thickness, patients continued to take oral oestradiol 3 mg twice daily until the endometrium was >7 mm, at which point they began to receive progesterone. If the endometrial thickness was <7 mm by day 20 the cycle was cancelled (n = 6). In all cases, embryos were transferred 3 days after the initiation of progesterone. Patients who were allocated to a stimulated protocol received human menopausal gonadotrophin injections (HMG; 75 IU; Livzon) every second day from day 3 of their menstrual cycle. Ultrasound and hormone assays were performed on day 9 or 10 and HMG was continued in patients with a thin endometrium if follicle size was <15 mm. Ultrasound and hormonal surveillance was continued until the endometrial thickness was >7 mm and a dominant follicle reached mm, at which time 10,000 IU HCG was administered. Cycles with endometrial thickness significantly <7 mm by day 20 were cancelled (n = 2). Injections of progesterone in oil (20 mg) were commenced on the day following HCG administration. Assessment of pregnancy outcome Serum bhcg was measured 14 days post embryo transfer, and if the test was positive, daily progesterone supplement was continued until the 10th week of pregnancy. Pregnant patients were followed with serial ultrasound evaluations, and the numbers of gestational sacs with fetal cardiac activity were determined 4 5 weeks after embryo transfer. Cell stage of embryo and pregnancy In order to determine unequivocally the relationship between cell stage of transferred embryo and endometrial receptivity, only those cycles in which all transferred embryos with equivalent culture growth characteristics were included in the EC group. The equivalent culture growth meant that all transferred embryos were at the same cell stage of development through overnight culture. The cell stage was divided into four subcategories: <8 cell, 8 16 cell, morula and blastocyst.

4 Extended culture of vitrified warmed embryos has no effect on pregnancy outcome 387 Statistical analysis Variables were analysed using the Statistical Package for Social Sciences version 16.0 for Windows (SPSS, Chicago, USA). All continuous variables, such as age and number of embryos transferred, were presented as mean ± SD and were compared between treatment groups by means of a two-tailed, unpaired t-test. All categorical variables, such as pregnancy, implantation and live birth rates, were presented as n and percentages and were compared between treatment groups by chi-squared test. All analyses were considered significant at P < Where required, a modified chi-squared test was applied to test for within-subject effects. Results As shown in the CONSORT flow diagram (Figure 2), a total of 540 couples were assessed for their eligibility for inclusion in the study. Of those, 237 and 242 cycles were allocated to the EC group or the conventional culture (control group) group, respectively. Among them, 11 in the EC group were excluded due to failure of endometrial preparation and other reasons and 10 were excluded in the conventional culture group. Finally, 232 and 226 cycles, respectively, were included to analyse the pregnancy outcome. As shown in Table 1, couples who underwent cryopreserved embryo transfer in the two groups showed similar baseline characteristics, including age and duration of infertility. In addition, there were also no significant differences between the two groups regarding the proportion of stimulation cycles using ICSI. Both groups of patients were comparable with respect to indication of infertility. Table 2 compares the distribution of endometrial preparation in the two groups. There was no difference across the groups. Endometrial thickness was 9.63 ± 0.56 mm in the EC group, and 9.44 ± 0.27 mm in the control group. No statistical difference was observed in endometrial thickness between two groups. Table 3 depicts the pregnancy outcomes in each of the experimental protocols. In the EC group, the clinical pregnancy rate was 42.48% versus 40.95% in the control group. The implantation rates were 21.79% versus 20.82% and the live birth rates per embryo transfer were 37.61% versus 34.05%, respectively. There were also no statistically Figure 2 CONSORT statement flow diagram.

5 388 R Jin et al. Table 1 Variable Comparison of patients characteristics in the extended culture and control groups. Extended culture (n = 226) Control (n = 232) Age (years) ± ± 4.31 Duration of infertility (years) 4.81 ± ± 3.12 ICSI 66 (29.20) 64 (27.59) Primary infertility 124 (54.87) 112 (48.28) Secondary infertility 102 (45.13) 120 (51.72) Parity 43 (19.03) 42 (18.10) Unexplained infertility 23 (10.18) 25 (10.78) Male factor 46 (20.35) 45 (19.40) Tubal factor 50 (22.12) 59 (25.43) Polycystic ovarian syndrome 19 (8.41) 23 (9.91) Endometriosis 45 (19.91) 38 (16.38) Values are mean ± SD or n (%). There were no statistically significant differences between the two groups. Table 2 Comparison of endometrial preparation regimes for cryopreserved embryo transfer in the extended culture and control groups. Variable Extended culture (n = 226) Control (n = 232) Natural cycles Hormone-replacement cycles Stimulated cycles Endometrial thickness (mm) 9.63 ± ± 0.27 Values are n or mean ± SD. There were no statistically significant differences between the two groups. Table 3 Comparison of cryopreserved embryo transfer outcomes in the extended culture and control groups. Variable Extended culture Control Cycles Transfer cycles Embryos transferred 2.62 ± ± 0.55 Top-quality embryos post warming 2.05 ± ± 0.41 Clinical pregnancies/transfer cycle 96 (42.48) 95 (40.95) Miscarriages/pregnancy 10 (10.42) 14 (14.74) Ectopic pregnancies/pregnancy 1 (1.04) 2 (2.11) Implantations/embryo transferred 129 (21.79) 127 (20.82) Spontaneous reduction rate a 14 (10.94) 26 (20.80) Live birth deliveries/transfer 85 (37.61) 79 (34.05) Values are n, mean ± SD or n (%). There were no statistically significant differences between the two groups. a Spontaneous reduction rate was calculated as the number of gestational sacs spontaneously lost after the first trimester divided by the number of gestational sacs observed in the first trimester; here ectopic gestational sacs were excluded (one and two ectopic gestational sacs in the extended culture and control groups, respectively). Spontaneous reductions include spontaneous reduction in multiple pregnancy and spontaneous loss in single pregnancy. The spontaneous reduction rate was significantly higher in the control group (P = 0.032). significant differences between the two groups for miscarriage and ectopic pregnancy rates. The spontaneous reduction rate was significantly lower in the EC group compared with the control group (10.94% versus 20.80%, P = 0.032). Implantation rates were significantly different for the different categories of embryo developmental stage (<8-cell stage 8.64%, 8 16-cell stage 27.37%, morula stage 26.92%, blastocyst stage 13.04%; P = 0.006; Figure 3). When these results were compared within subgroups, the implantation rate was significantly (P < 0.05) lower in those cycles in which all of the transferred embryos had <8 cells. Although there was a slight decrease in the blastocyst group, no significant difference in implantation rate was found with respect to those embryos that had >8 cells.

6 Extended culture of vitrified warmed embryos has no effect on pregnancy outcome 389 Figure 3 Implantation rates in the EC group according to the cell stage of transferred embryos. Only cycles in which all the transferred embryos were at the same developmental stage were included. n = number of embryos transferred. * P < 0.05; ** P < R C contingency table analysis is adopted by chi-squared tests. Because P < 0.05 (P = 0.006), a modified chi-squared statistic is applied for further testing within each subject. Discussion Cryopreserved embryo transfer, as a useful tool in assisted reproductive technology, has greatly increased the chance of cumulative pregnancy. Evidence from recent studies indicates that implantation potential is comparable between cryopreserved embryos and fresh embryos (Aflatoonian et al., 2010; Shapiro et al., 2011; Zhu et al., 2011). It provides clinical doctors with alternative approaches in certain clinical situations, such as prevention of ovarian hyperstimulation syndrome, dysfunction of uterine cavity and other disorders during treatment. Since the advent of vitrification has dramatically decreased the damage to cryopreserved embryos (Rezazadeh Valojerdi et al., 2009; Stehlik et al., 2005), the remaining pivotal problem is how to set up an optimal procedure to improve implantation. Among them, synchronization between the embryo and the endometrium plays a vital role. The results revealed that, with the prewarming of cryopreserved embryos combined with overnight culture, the pregnancy outcome was not affected by the transfer of a day-4 embryo into a day-3 endometrium (Table 3). In the natural cycle, the blastocyst implantation occurs 5 6 days after ovulation. During implantation, the receptive endometrium helps with the proliferation and differentiation of the trophoblast cells (Barnea, 2001), then the trophoblast cells gain the invasive capabilities via a series of morphological and biochemical changes before the completion of implantation. Meanwhile, preimplantation factors derived from embryo help the endometrium to facilitate implantation (Barnea, 2001). The presumptive optimal synchronization has been considered as equivalent on the day of embryo development and the day of progesterone stimulation, while evidence from endometrial biopsies performed on women undergoing ovarian stimulation supports the need for histological endometrial advancement compared with natural cycles (Kolibianakis et al., 2005; Papanikolaou et al., 2010; Sharma et al., 1990; Van Vaerenbergh et al., 2009). A satisfying pregnancy outcome can be achieved in transfer cycles within 3 days of endometrial advancement (Kolibianakis et al., 2002; Papanikolaou et al., 2005), and extreme endometrial advancement is associated with pregnancy failure. For those cycles in which endometrial advancement was initiated by HCG, progesterone and oestradiol, prewarming may create a workable strategy. Embryonic age plays an important role in synchronization. While poor outcomes are seen in fresh cycles using rescue ICSI of 1-day-old oocytes (Sermondade et al., 2010), the use of cryopreservation for synchronization prior to transfer reverses this trend (Ming et al., 2012; Sermondade et al., 2010). Similarly, implantation rates with day-6 or -7 blastocysts is comparable with day-5 blastocysts in vitrified warmed embryo transfer cycles but lower in fresh cycles (Hiraoka et al., 2009). The seemingly contradictory synchronization between embryonic age and endometrium reveals the importance of the embryonic stage. The optimal

7 390 R Jin et al. endometrial receptivity needs the embryo to be at a certain cleavage stage instead of at a particular day of development. Embryos at different days of development in cryopreserved embryo transfer cycles showed comparable synchronization with the endometrium (Shapiro et al., 2008). Another important factor is that the highest-quality embryos are usually selected for transfer in fresh cycles, therefore the excess embryos may have lower development potential. In a study by Dayal et al. (2006), only 20% of excess embryos remaining in culture following day-3 embryo transfers developed to the blastocyst stage, including those with slower development. So, the slower embryo needs more culture time to reach preimplantation status before implantation (Hiraoka et al., 2009). Although the study centre carefully checks the embryo before transfer, it cannot know the exact information about subsequent divisions and elapsed time to reach the hatching blastocyst stage, which can be really useful for implantation. In addition, some studies have found synchronization between endometrium and embryo development to be not absolutely crucial for implantation. A warmed embryo can implant into an endometrium with a short exposure to progesterone (Imbar and Hurwitz, 2004). The current study showed that transfers of embryos with >8 cells were strongly associated with implantation potential compared with <8-cell embryos (Figure 3) and that implantation rates in <8-cell embryos were lower (8.64%). It is universally accepted that a 6 8-cell embryo on day 3 has implantation potential. In this study, embryos with <8 cells following overnight culture implied poor viability; the reason for the lower implantation rate in this group may be the embryos per se (Edgar et al., 2001). Those embryos with >8 cells implied further development and higher implantation potential. Among embryonic factors associated with successful implantation, single cryopreserved embryo transfers have indicated that embryos with subsequent cleavage are associated with higher success rates (Edgar et al., 2007; Van der Elst et al., 1997; Ziebe et al., 1998), findings that are consistent with the current study. In the EC group, prewarmed embryos probably reached the hatched stage early during the implantation window, allowing the possibility of early implantation. This may contribute to the reduced rate of spontaneous reduction (Wilcox et al., 1999). Surprisingly, blastocysts in the EC group showed a reduction of implantation potential (Figure 3). One probable reason is a lack of synchronization with the endometrium. It has been verified that out-of-phase conditions may result in failure of the embryo to implant (Barnes, 2000). Besides, the results of endometrial research have indirectly demonstrated a detrimental effect if the time interval between endometrial and embryonic development is too large (Papanikolaou et al., 2010). It should be noted that, to stress the absence of a difference between two groups of patients, the power of a study is clearly important. As far as continuous variables are concerned, sample sizes of around 220 are likely to be quite adequate. With regard to discrete variables used in the comparison of proportions, the following results suggest that the current sample sizes are adequate to detect important differences with a satisfactory probability. Thus if the unknown parent proportions are 0.1/0.2, a one-sided 5% test would detect that difference with a probability (power) of The corresponding power value for the comparison of proportions of 0.2/0.3 is 0.75 and for the proportions 0.3/0.4 is Therefore, it is believed that these negative findings represent a valuable addition to current knowledge on this topic. In summary, this study has set forth the effect of extended culture on cryopreserved embryo transfer. The findings suggest that overnight culture is not harmful to clinical outcome. Accordingly, the extended culture protocol provides not only the flexible warming/thawing procedure, but also a possible method of decreasing the spontaneous reduction rate. However, this was only a pilot study with no formal statistical assumptions and/or sample size; further confirmatory and well-powered studies need to be carried out to confirm the findings of this study. 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