Introduction

Blastocyst transfers in IVF protocols led to an increase in implantation rates, higher pregnancy rates, and a decrease of high-order multiple gestations resulting from a reduction in the number of embryos transferred compared with embryos transferred at the cleavage stage [1]. Thus, the idea of cryopreservation of the superfluous embryos evolved. There are two basic techniques currently applied in embryo cryopreservation: the slow-freezing technique and the vitrification technique. Vitrification is thought to be safer and more cost effective than slow freezing. During the rapid freezing process with high concentration of cryoprotectants, less intracellular ice crystal formation reduces cellular damage and results in better reproductive potential after thawing [2, 3].

Though, the procedures of freezing and thawing have some adverse effects on the embryos, there are significantly higher ongoing pregnancy, clinical pregnancy and implantation rates in cycles with vitrified-thawed day 2 embryos than in cycles with fresh day 2 embryos [4]. The cycles of thawed bipronuclear (2PN) oocytes subjected to post-thaw extended culture to the blastocyst stage also resulted in a higher clinical pregnancy rate than did fresh embryo transfer cycles [5]. The vitrified blastocysts retained good developmental competency after thawing. High pregnancy and implantation rates can be expected once the blastocysts go through vitrification and thawing because they have the potential to develop as fresh blastocysts [6].

The developmental potential of cryo-thawed blastocysts is a concern due to synchronization of endometrial receptivity. Natural endometrial preparation is superior to hCG and hormonally manipulated endometrial preparation [3, 7]. Better synchronization between the embryo and endometrium is believed in frozen-thawed natural cycles than in stimulated cycles [4, 5].

The objective of this study was to compare the clinical outcomes between fresh and vitrified-thawed day 5 blastocyst transfers with the same morphologic quality of embryos.

Methods

An institutional review board approved this study before initiation. The registration number is 11MMHIS081. The study is a retrospective case control study to review the IVF/ICSI cycles from June 1, 2009 to November 30, 2011 at Mackay Memorial Hospital. It included 118 cycles of fresh blastocyst transfer and 59 cycles of vitrified-thawed blastocyst transfer. The inclusion criteria were age, 38 years of younger and good-quality blastocyts (morphology better than or equal to 3BB, according to Gardner’s criteria). The exclusion criteria were fresh or vitrified day 6 blastocysts and post-thaw extended culture from day 3 embryo to day 5 blastocyst.

Our vitrification and thawing protocols were applied from the Cryotop safety kit and modified from the Cryotop method. ( Kitazato BioPharma Co., Ltd., Fuji city, Shizuoka, Japan).

Pregnancy definition

Clinical pregnancy which was defined as ICMART and WHO revised glossary of ART terminology was proven by the appearance of a gestational sac in the ultrasound examination [8]. The ongoing pregnancy was defined as pregnancy beyond 12 gestational weeks. Abortion was defined as any clinical pregnancies that did not continue to the point of an ongoing pregnancy.

Statistical analysis

The main outcome measures were compared between the groups using the Chi-square test or Fisher exact test for comparison of percentages and Student’s t test for comparison of mean values. Statistical analyses were performed with PASW statistical software, version 18. P < 0.05 was considered significant for all measures.

Results

Of the 145 cycles in the fresh blastocyst transfer group, 27 cycles were abandoned due to poor quality blastocysts and of the 156 cycles in the vitrified-thawed blastocyst group, 97 cycles were excluded due to poor quality blastocysts, vitrified on Day 3 or Day 6. The characteristics of patients in the two groups are listed in Table 1. There was no significant difference in the proportion of ICSI between the two groups. Of the 118 cycles in the fresh group, 234 blastocysts were transferred (mean number of embryos transferred per cycle: 1.98 ± 0.130). The clinical pregnancy rate was 66.1 % and the implantation rate was 50.9 %. The ongoing pregnancy rate was 56.8 % and the rates of singleton and twin pregnancies were 53.7 % and 44.8 %, respectively. Of the 59 cycles in the vitrified-thawed blastocyst group, 111 blastocysts were transferred (mean number of embryos transferred per cycle: 1.88 ± 0.326). The clinical pregnancy rate was 59.3 % and the implantation rate was 43.2 %. The ongoing pregnancy rate was 47.5 % and the rates of singleton and twin pregnancies were 60.7 % vs. 39.3 % (Table 2).

Table 1 Patient characteristics by type of blastocyst transfers
Full size table
Table 2 Clinical outcomes of fresh blastocyst and vitrified-thawed blastocyst transfers
Full size table

Discussion

There was no significant difference in clinical pregnancy rates, implantation rates, or ongoing pregnancy rate between the fresh blastocyst transfer group and the vitrified-thawed blastocyst transfer group. This data is meaningful to us because all vitrified blastocysts were surplus after fresh embryo transfer cycles. To achieve a better live birth rate, the selection methods call for choosing the best fresh embryos for transfer. Thus, the remaining embryos that survive the freezing and thawing procedures supposedly have a reduced chance of implantation. Nevertheless, the implantation and pregnancy rates were not reduced in the vitrified-thawed group compared with the fresh blastocyst transfer group in this investigation.

To explain the similar outcomes between the fresh and thawed embryo transfers, multiple factors may be considered. First, the improved techniques of vitrification results in better survival and developmental potential after thawing. Kuc et al. reported that the clinical pregnancy rates of the vitrification group and slow-freezing group of day 5 or day 6 blastocysts were significantly different. The clinical pregnancy rates of the vitrification and slow-freezing groups were 50.4 % and 25.9 % (P < 0.05), respectively [9]. Due to the high concentration of cryoprotectant and fast cooling rate, the vitrification process avoids intracellular ice crystal formation during cooling and prevents injury to the cryopreserved cells. Additionally, it protects the cells from damage due to ice crystals during thawing [2].

Initially, we were not sure whether the high concentration of cryoprotectant was chemically toxic to the cryopreserved cells. Since the vitrification procedure of murine embryos was first presented in 1985, the protocol and solutions have been modified to be less toxic and more efficient [10]. Hong et al. reported improved clinical results between two cohorts in the same laboratory. They modified the protocol due to the finding that the permeation of the cryoprotectant into the embryo was faster at a higher temperature [6]. Minimum vitrification solution is used in Cryotop method. This approach increases the cooling and warming rates, which improve post-thaw survival and development of the embryo. Larman et al. analyzed gene expression of cryopreserved mouse blastocysts using microarrays. The gene expressions, including involvement of protein metabolism, transcription, cellular biosynthesis, and development were only significantly influenced by slow-freezing. There was no significantly influenced gene expression in non-cryopreserved or vitrification groups [11].

Endometrial receptivity and synchronization between the embryo and endometrium are very important in cryopreserved-thawed embryo transfer cycles and in fresh embryo transfer cycles, regardless of the cleavage stage, post-thaw extended culture, or blastocyst stage. Cryopreserved-thawed embryos transferred in a natural ovulatory cycle resulted in better clinical outcomes than fresh embryos transferred in stimulated cycles [3–5, 7, 12]. Gene expression, various cytokine signaling pathways, the complement and coagulation factor cascade, and leukocyte transendothelial migration are different in natural and stimulated cycles [4]. Haouzi et al. reported that the TGFβ signaling, leukocyte transendothelial migration, and the cell cycle of the endometrium in stimulated cycles were defective [13]. The same laboratory also analyzed the gene expression of endometrium between the natural cycle and stimulated cycles, including the GnRH agonist long protocol and GnRH antagonist protocols by DNA microarray analysis. There were significantly different expression patterns of endometrial chemokines and growth factors between stimulated cycles in comparison with natural cycles. Especially, the differences were more obvious in GnRH agonist long protocol than in the GnRH antagonist protocols. Among the down-regulated genes specific to the GnRH agonist protocol, numerous genes play important roles in cell-cycle function and more especially in checkpoint regulation [14].

Zhu et al. reported higher pregnancy and implantation rates in vitrified-thawed blastocysts than in fresh blastocyst transfer cycles. The clinical pregnancy rate of fresh and vitrified blastocyst transfer groups were 36.4 % and 55.1 %, respectively (P < 0.05) and the implantation rate of the fresh and vitrified group was 25.2 % and 37.0 % (P < 0.05) [12]. We also had similar clinical pregnancy and implantation rates (59.3 % and 43.2 %, respectively) in the vitrified-thawed blastocyst transfer group.

In our current study, the implantation rate, which reflects the endometrial synchronization and receptivity between the blastocyst and endometrium, was 50.9 % in the fresh blastocyst transfer group and 43.2 % in the vitrified-thawed group; they did not differ significantly. Thus, single embryo transfer may be considered in fresh cycles to decrease multiple pregnancy rates, if blastocysts of good quality are obtained. The surplus embryos would be vitrified for frozen embryo transfer to improve the cumulative pregnancy rate. In conclusion, as long as results are reliable for vitrified-thawed cycles, all available blastocysts can be vitrified in patients for whom fresh blastocyst transfer is unsuitable, such as patients at risk of OHSS, those with a history of repeat failed fresh embryo transfers, and those in need for preimplantation genomic diagnosis.