Introduction

Since achievement of the first successful pregnancy after the transfer of a frozen human embryo [1], embryo cryopreservation has greatly contributed to the progress of human-assisted reproductive technology (ART), including the prevention of ovarian hyperstimulation syndrome and efficient use of surplus embryos. Further developments in embryo cryopreservation have been achieved through the use of ultra-rapid vitrification, which was originally applied to murine embryos by Rall and Fahy [2].

The most important consideration of the vitrification procedure is to minimize the possibility of extra- and intracellular formation of ice crystals, which would impair organelles and cell membranes during the cooling and warming phases [3–5]. Toward this end, cooling and warming rates are generally maximized by using the smallest volume possible of cryoprotectant medium to surround the cells, and the specimens are then exposed directly to liquid nitrogen without any thermo-insulation. This procedure is known as an open vitrification system. This idea was initially proposed for freezing Drosophila embryos [6], and drastic improvement in viability has since been shown in both animal studies [7–10] and clinical reports [11, 12]. However, there are some potential drawbacks of the open vitrification system, such as the sterility of liquid nitrogen and the risk of cross-contamination during long-term storage [13, 14]. Such cross-contamination could arise from direct contact of the solution containing the oocytes and embryos with the liquid nitrogen.

Therefore, to avoid the possible risk of contamination, closed vitrification systems have been developed [15–25]. However, new concerns such as a potential rise in temperature caused by a heat sealer and a decrease in the cooling rate have emerged with these methods. In addition, the recovery rate of embryos after warming has been shown to be lower with the use of one type of closed vitrification system compared to an open vitrification system [20].

We recently reported no significant difference between the use of closed and open vitrification systems in the survival rate, blastulation rate, proportion of good blastocysts, mean number of cells, or implantation rate [24]. In this closed system, an embryo is inserted into a straw with super-cooled air for vitrification, and its open end is sealed using ultrasound adhesion to avoid the risks of increased temperature and contamination. Subsequently, the vitrified embryos are warmed by direct exposure to a warming solution. Therefore, these embryos are vitrified, cryopreserved, and warmed without direct exposure to liquid nitrogen. Another research group also revealed no significant difference between the use of closed and open vitrification systems in the survival rate or implantation rate [25]. Recently, a total of 114 infants were obtained from blastocysts vitrified using the same closed vitrification device [25] and another closed device [26]. However, despite this apparent success, the perinatal outcomes of embryos vitrified using the closed system following implantation remains unknown. To the best of our knowledge, there are only two reports comparing neonatal data between closed and open vitrification systems [25, 26]. However, these analyses combined data of single- and multiple-embryo transfers. Since multiple pregnancies increase the risk of complications at birth, i.e.,: the risk of extreme preterm birth (28 weeks) is increased 3-fold for twins and 13-fold for triplets, and the risk of very preterm birth (28–32 weeks) is increased by almost 5-fold for twins and 20-fold for triplets [27], it is difficult to accurately and independently assess the effect of vitrification protocols (closed vs. open) on the neonatal outcome from these data.

In the present work, we compared the neonatal outcome and clinical safety using the closed vitrification system in comparison with an open vitrification system after single blastocyst transfer.

Materials and methods

This was a retrospective cohort study that was approved by the ethics committee of the IVF Namba Clinic. The data pertaining to a total of 875 vitrified-warmed blastocysts that were single-transferred under hormone-replacement cycles between November 2011 and December 2013 were randomly divided into two groups according to the day of blastocyst vitrification (closed vitrification, n 313; open vitrification, n 562) after receiving informed consent. All embryos were obtained from stimulation cycles. Some data on the viability and implantation potential of the vitrified embryos in the present work are provided in our previous study [24].

Vitrification

The Rapid-i Kit (Vitrolife Japan; Tokyo, Japan) is a closed vitrification system containing a polymethyl methacrylate stick (Rapid-i) and a thermoplastic elastomer storage straw (RapidStraw). Rapid-i has a 50-nL loading hole designed for receiving an embryo from a pipette under microscopy [19, 24]. The Rapid-i Kit also contains a stainless steel rod inserted into RapidStraw for cooling prior to insertion of the device (a rod is removed 20–30 s before insertion of Rapid-i). Cryotop®(Kitazato Corporation; Tokyo, Japan) [15] was used as the open vitrification system.

Embryos were equilibrated in 7.5 % (v/v) ethylene glycol (EG, Wako Chemical; Osaka, Japan), 7.5 % (v/v) dimethyl sulfoxide (Sigma-Aldrich; St. Louis, MO, USA), 20 % (v/v) serum substitute supplement (SSS, Irvine Scientific; St. Ana, CA, USA), and TCM 199 medium (Invitrogen; Tokyo, Japan) for a maximum of 10 min; shrinkage and re-expansion were confirmed, and then the embryos were transferred to vitrification solution consisting of 15 % (v/v) EG, 15 % (v/v) dimethyl sulfoxide, 0.5 M sucrose (Wako Chemical), 20 % (v/v) SSS, and TCM 199 medium. Each embryo was picked up with 50 nL of vitrification solution and pipetted into a hole of Rapid-i. Then, the specimens were immediately placed in super-cooled air inside a RapidStraw dipped in liquid nitrogen. The straw was then sealed using an ultrasonic sealer as described previously [24]. The sealed straw was stored in liquid nitrogen for several weeks. For the open vitrification system, after equilibration in vitrification solution, each embryo was picked up as described for the closed vitrification system and placed on a fine polypropylene strip of the Cryotop. The strip was then immediately submerged in liquid nitrogen.

Warming

After clipping the end of the straw, the Rapid-i stick was removed and the vitrified embryos were warmed in 1 mL TCM 199 containing 20 % SSS and 1 M sucrose at 37 °C for 1 min. The specimens were diluted in TCM 199 containing 20 % SSS and 0.5 M sucrose, and then diluted twice in TCM 199 containing 20 % SSS for 5 min at room temperature. The embryos vitrified using the open system were also warmed and diluted in a similar manner.

Blastocyst quality score (BQS)

To establish a numerical blastocyst morphology grading system based on Gardner’s grading system [28], the blastocyst grade was converted to the multiplicative BQS proposed by Rehman et al. [29]. The BQS is a metric of blastocyst quality that is based on established morphological criteria, and is defined as the product of the degree of expansion and hatching status and ICM and TE grades, where grade A is given the value 3, grade B is given a value of 2, and grade C is given a value of 1. For example, for a 3AB blastocyst, the BQS is 3 × 3 × 2 = 18.

Preparation of the endometrium

The endometrium was prepared by administration of GnRH agonist (600 μg/day, Suprecur® nasal solution 0.15 %; Mochida Pharmaceutical; Tokyo, Japan) for 3 weeks followed by increasing doses of oral estradiol valerate (Progynova®; Bayer Schering Pharma; Zürich, Switzerland) from 1 to 4 mg for 2 weeks [30]. After ultrasonographical confirmation that the endometrium was thicker than 8 mm, chlormadinone acetate (Lutoral®; Shionogi & Co.; Osaka, Japan) was administered (6 mg/day). Progesterone (Progeston depot® 125 mg; Fuji Pharma Co.; Toyama, Japan) was administered intramuscularly on the day of embryo transfer, with two additional doses after conception. Blastocyst transfer was carried out on the 5th day of chlormadinone acetate administration. Daily doses of 3 mg estradiol valerate and 6 mg chlormadinone acetate were maintained until the time of pregnancy test. When pregnancy was confirmed, estradiol (2.88 mg every 2 days, Estradna®; Hisamitsu; Saga, Japan) and progesterone (400 mg/day, Utrogestan® 200 mg; Ferring Pharmaceuticals; West Drayton, UK) were administered transcutaneously and transvaginally, respectively, until 9 weeks of gestation.

Outcome variables

Implantation was determined at around 3 weeks after embryo transfer by the detection of a single intrauterine gestational sac by transvaginal ultrasound. Fetal heart beat was confirmed beyond 6 weeks of gestation by ultrasound. Fetal loss before 22 weeks was defined as miscarriage and that after 22 weeks was defined as stillbirth. In the case of abortion for any cause, the karyotype of the abortus was analyzed as described previously [31]. Slide preparations and G-banding of chromosomes were conducted according to standard protocols [32].

The neonatal outcomes were assessed by the mean gestational age, birth weight, sex, Apgar score (evaluated within 5 min of birth), and congenital anomalies.

Statistical analysis

Differences between pairs of groups were determined using an unpaired Student’s t-test or a χ 2 test. P-values < 0.05 were considered to be significant. Data are presented as mean ± SE for the t-tests. Statistical analysis was performed using StatView version 5 (SAS Institute Inc.; Cary, NC, USA).

Results

Embryo implantation potential of thawed embryos

The patients’ characteristics are provided in Table 1. The implantation rate in the closed vitrification system group was 49.7 % (150/302), which was similar to that in the open vitrification system group (49.4 %, 266/539).

Table 1 Baseline patient clinical characteristics and embryo implantation potential of thawed embryos vitrified with a closed or open vitrification system in hormone-replacement cycles
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Fetal or embryo development after implantation

Table 2 shows the developmental characteristics after the implantation in the two groups. Data were calculated based on the number of implantations (closed: 150, open: 266). There were no significant differences between the two systems in the frequencies of detection of a heartbeat (closed: 89.3 % vs. open: 89.5 %), miscarriage (closed: 22.0 % vs. open: 22.2 %), stillbirth (closed: 0.7 % vs. open: 0.0 %), monozygotic twins (closed: 0.0 % vs. open: 0.8 %), and live births (closed: 75.3 % vs. open: 77.4 %). There was no difference in the chromosomal aberration rate of the abortus between embryos vitrified using the closed (40 %, n 15) and open (67.6 %, n 34) systems.

Table 2 Fetal development after implantation and neonatal birth characteristics of thawed embryos vitrified with a closed or open vitrification system in hormone-replacement cycles
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Neonatal birth characteristics

Neonatal birth characteristics are presented in Table 2. Data were calculated based on the number of live births (closed: 113, open: 206). There were no significant differences between the two systems in the proportions of live births before 32 weeks (closed: 2.7 % vs. open: 1.0 %), from 32 to 34 weeks (closed: 0.9 % vs. open: 1.9 %), from 34 to 37 weeks (closed: 3.5 % vs. open: 4.9 %), and over 42 weeks (closed: 0.9 % vs. open: 1.9 %). The proportion of normal gestational age per live birth in the closed system group (92.0 %) was also similar to that in the open system group (89.8 %). There were no statistical differences in the mean birth weight (closed: 3127.9 g vs. open: 3056.8 g), or in the proportions of neonates with a birth weight less than 1500 g (closed: 1.8 % vs. open: 1.5 %) and 1500–2500 g (closed: 6.3 % vs. open: 7.3 %) between the two systems.

Table 3 shows the neonatal data of babies born at normal gestational age. Data were calculated based on the number of babies born at normal gestational age in each group (closed: 104, open: 185). There were no significant differences in the maternal age (closed: 34.7 years vs. open: 35.0 years) and maternal body mass index (closed: 20.7 vs. open: 20.3) between women in the two groups. There were also similarities between the groups in mean gestational age (closed: 278.4 days, open: 277.1 days), birth weight (closed: 3207.5 g, open: 3125.4 g), Apgar score (closed: 9.3, open: 9.3), proportion of Caesarian sections (closed: 36.5 %, open: 40.5 %), and proportion of male babies (closed: 43.3 %, open: 48.4 %). The congenital anomalies included 1 case of aproctia, 1 cleft lip and lymphangioma, and 1 syndactylus in the closed system group (2.8 %), and 1 case of hernia inguinalis in the open system group (0.5 %). These values were not statistically different.

Table 3 Neonatal birth characteristics at normal gestational age (259–293 days)
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Discussion

The perinatal outcomes of embryos vitrified using a closed system following implantation remain obscure because conventional research comparing neonatal data between closed and open vitrification systems [25, 26] has thus far relied on combined data of single- and multiple-embryo transfers. In fact, multiple pregnancies increase the risk of complications at birth [27]. Thus, we investigated the neonatal outcome and clinical safety using the closed vitrification system in comparison with an open vitrification system after single blastocyst transfer. The results of the present work suggested that the use of a closed vitrification system supports the full-term development of vitrified human blastocysts. The data obtained from single embryo transfer is of great value for assessing the effects of closed vitrification.

There are two steps that could cause contamination during the vitrification procedure [13, 14]. The first occurs during the rapid cooling procedure via direct contact with liquid nitrogen. The second arises during long-term preservation in liquid nitrogen. The contaminated liquid nitrogen would then cause cross-contamination. Since an embryo is vitrified in super-cooled air in a closed device and then packaged in a closed straw [19], the risk of contamination from liquid nitrogen could be decreased as compared with that in an open device [13, 14, 33], in a manner similar to that of conventional freezing [33] and packaged straw vitrification [34].

However, use of a closed vitrification device could potentially decrease the viability of embryos due to a decrease in the cooling rate. Slower cooling would increase the risk of ice crystal formation in the cells [4, 6]. The average rate of cooling is about −1220 °C/min in the Rapid-i device [35], whereas that of the Cryotop (open vitrification) device is −23000 °C/min [15]. Thus, the cooling rate in the closed system is markedly slower than that in the open system. However, the viability after vitrification and neonatal data obtained using the closed system were similar to those obtained using the open system. Recently, it has been shown that thawing rate is a more critical factor for embryo viability after vitrification and warming than cooling rate [36]. Since the vitrified embryos are warmed directly in the same warming solution in a similar manner, it is not surprising that no significant difference was found between two systems in the present study.

The fetal malformation rate in the closed system was 2.9 % (3/104), which was slightly higher than that in the open system (0.5 %), despite the lack of statistical significance. However, this value is similar to those obtained in a European large prospective study (3.38 %: 96/2840 in ICSI and 3.79 %: 112/2955 in in vitro fertilization), a US retrospective cohort study (5.4 %: 21/392), and in our previous study (2.3 %: 19/829) [37–39].

According to the latest available data, over 104,000 cryopreserved embryo replacements were performed in Europe in 2010 and more than 15,600 deliveries were reported as a result of cryopreserved embryo transfer [27]. Thus, the closed system, which enables aseptic vitrification without impairing the developmental competence of human embryos, could potentially have a large impact in ART.

Our study offers some insights into the safety of closed vitrification. Although there were no significant differences in the developmental characteristics after implantation or in the neonatal status between the closed and open vitrification groups, further study will be required to assess the subsequent growth and development of the children.