Equivalent clinical outcome after vitrified‐thawed blastocyst transfer using semi‐automated embryo vitrification system compared with manual vitrification method

Abstract Purpose This study compared Gavi®, an automated system for the equilibration and dehydration steps of vitrification, and a manual vitrification procedure in terms of effects on clinical outcomes. Methods The authors retrospectively compared survival rate, and clinical and perinatal outcomes after vitrified‐thawed single blastocyst transfer between Gavi® (G method) in 398 cases and Cryotop® (C method) in 208 cases. Results With C and G methods, survival rates were 98.6% (208/211) and 99.3% (398/401), total pregnancy rates were 34.3% (72/208) and 33.4% (133/398), and total miscarriage rates were 22.2% (16/72) and 24.8% (33/133), respectively. Among women <35 years old, pregnancy rates were 41.1% (30/73) and 40.5% (62/153) and miscarriage rates were 13.3% (4/30) and 16.1% (10/62) with C and G methods, respectively. Among women ≥35 years old, pregnancy rates were 31.1% (42/135) and 29.0% (71/245) and miscarriage rates were 28.6% (12/42) and 32.4% (23/71) with C and G methods, respectively. C and G methods showed no significant differences in any trials, including gestational age, cesarean section rate, or birthweight (P > .05 each). Conclusions Gavi® showed comparable clinical outcomes to the manual vitrification method and can be considered an alternative vitrification procedure in assisted reproductive technology.


| INTRODUC TI ON
The history of embryo freezing started with a report in 1972 by Whittingham et al, 1 showing that mouse blastocysts survived a freeze-thaw cycle. Since then, the need for embryo freezing in assisted reproductive technology (ART) has gained recognition. Currently, vitrification has emerged as the more reliable method for cryopreservation. As a result, fewer blastocysts are transferred, allowing reductions in the frequency of multiple pregnancies and an increased chance of healthy transplant.
To preserve embryos in good condition, it is necessary to maintain (a) reversible metabolic arrest in the embryo; (b) the structure of the embryo itself (including DNA); (c) an acceptable survival rate; and (d) normal embryo growth after thawing. Furthermore, the cryopreservation technique must be stable and allow high reproducibility. A key problem in embryo cryopreservation is to avoid the formation of ice crystals in cells, as these can cause physical and chemical damage to the embryo.
Vitrification involves preventing the formation of ice crystals inside cells and freezing water in a glassy state to arrest molecular conversions without inducing any structural reorganization.
Vitrification is defined as a method for solidification of a liquid by raising its consistency in the process of freezing at an extremely low temperature. 2,3 Generally, a vitrification solvent comprises a cryoprotectant that remains unfrozen even with quite rapid cooling to an ultra-low temperature. In addition, freezing and thawing speeds show inverse correlations with cryoprotectant concentration in the vitrification process.
Seki et al 4,5 reported that thawing speed plays a more important role than freezing speed in the survival rate of mouse oocytes. In 1985, Rall et al 6 first reported the blastocyst freezing method without ice crystals. In 2000, Yoon et al reported a healthy pregnancy and live birth of a human embryo using vitrification. 7,8 By preventing ice crystal formation, blastocysts have shown an extended survival rate after thawing compared to programmable rate freezing used in ART. 9 In fact, clinics familiar with vitrification reportedly show around a 90% blastocyst recovery rate and pregnancy and live birth rates equal to or higher than those with fresh embryo transfer. [10][11][12] The vitrification method is very simple and does not require particularly expensive equipment, and the blastocysts are placed in a very small amount of vitrification solution that can be frozen quickly, compared with traditional closed straw or vial devices. However, the toxicity of high concentration of cryoprotectant can present an obstacle to embryologists unfamiliar with vitrification technology.
Basically, the vitrification media is more toxic than any cryopreservation media used in slow freezing methods. This places practical limitations on the speed of freezing used to attain vitrification and also biological limitations on the concentration of cryoprotectant in cells.
While vitrification is a widely accepted cryopreservation method in ART, the actual procedure requires the manual performance of several steps by the embryologist and requires a long learning curve to obtain the necessary level of skill. Results are thus extremely dependent on the embryologist and clinic. For those reasons, ART clinics try to maintain trained and skillful embryologists for consistent results. [13][14][15] 2 | MATERIAL S AND ME THOD

| Study design
After obtaining sufficient informed consent, blastocysts were vitrified by C method in 181 patients of 208 cases from February to April 2017 and by G method in 302 patients of 398 cases from June to December 2017. Right after thawing, blastocyst survival rate was compared between C method and G method.
In C and G methods, patients were diagnosed as ART indication because of oviductal factor, male factor, ovulation factor, implantation failure, and unexplained infertility.
After single vitrified-thawed blastocyst transfer, we retrospectively compared pregnancy rate, miscarriage rate, gestational age, birthweight, and cesarean section rate.

| Ovarian stimulation
Patients were stimulated by controlled ovarian stimulation protocol (GnRH antagonist, short GnRH agonist, or long GnRH agonist) or modified mild ovarian stimulation protocol (clomiphene citrate, letrozole). For final maturation stimulation, patients were administered human chorionic gonadotropin (hCG) or GnRH agonist when dominant follicles reached a diameter of 18 mm. In our clinic, the blastocyst vitrification is conducted following Gardner's criteria with early stage blastocysts classified 1 or 2, or that with more than CC grade in Day 5 and good morphology blastocysts are considered more than equal 3BB grade.

| In vitro fertilization and embryo culture
Clinical pregnancy was defined as recognition of a gestational sac on transvaginal ultrasonography at around gestational week 5. Miscarriage was defined as spontaneous abortion by gestational week 10.

| Blastocyst vitrification and thawing
Gavi ® can automate the procedure for equilibration and dehydration steps in vitrification ( Figure 1A). In Gavi, the equilibration and dehydration process is conducted in a closed-type vitrification device called a "Gavi pod" ( Figure 1B). After dehydration, Gavi pod is put on "cassette" as an attachment holding up Gavi pods. This cassette containing Gavi pod is then dumped into liquid nitrogen storage for completion of vitrification ( Figure 1C,D).
The vitrification step using Gavi ® was conducted following the protocol specified by each manufacturer. Briefly, blastocysts were held in a slit on the Gavi pod with a small amount of HEPES buffered medium, and the pod was then attached to the cassette. The embryologist then pressed the button to start blastocyst vitrification mode.
All steps of discarding or adding solvent were automatically conducted with disposable sterilized tips. HEPES buffer medium was discarded, and the equilibration solvent was continuously added. After equilibration steps, the solvent was discarded and the dehydration step was conducted. After discarding the dehydration medium, each Gavi pod was heat-sealed to a lid and the cassette was stored in a liquid nitrogen F I G U R E 1 Images of the Gavi system and its consumable components. A, Appearance of the whole Gavi system with the automated pipette inside. B, Gavi pod, which can preserve oocyte, embryo, and blastocyst on a slit in the pod. C, Vitrification media, cassette, and disposable tip and seal. D, After the dehydration step, the Gavi pod is dumped into liquid nitrogen to complete the vitrification step storage unit before being placed in a liquid nitrogen tank. For embryologists, this protocol is very simple: set the embryo in the Gavi pod, press the start button, and after the dehydration step, dump the cassette containing the Gavi pods into liquid nitrogen storage.
For C and G methods, vitrified blastocysts were thawed using Cryotop thawing kit VT506 (Kitazato Corporation) and Gems Warming Set (Genea), respectively, following the manufacturer's protocol. Briefly, for C method, the Cryotop ® was removed from the protective cover in liquid nitrogen, and the end of the strip was placed directly into 1.0 mL of 37°C thawing solution for 1 minute.
The blastocysts were subsequently transferred into 500 μL of diluent solution for 3 minutes and washed twice with 500 μL of washing solution for 5 minutes at room temperature. After thawing, all blastocysts were cultured in blastocyst medium for up to 4 hours and if greater than 50% of the cells were intact and re-expansion had been observed, blastocysts were considered as survived. All vitrified-thawed operations with C method and G method in this study were conducted by the same embryologist, who had experience conducting more than 1000 cases using the C method before the start of this study.

| Embryo transfer
For embryo transfer, endometrium preparation was conducted by the hormone-replacement therapy. Briefly, transdermal E2 (Estrana ® ; Hisamitsu Pharmaceutical) was administered from days 1-3 of menstruation cycle, and after the endometrial thickness was reached at least 6.5 mm, progesterone (UTOROGESTAN vaginal capsules ® ; Fuji Pharmaceutical) was administered until 9 weeks of pregnancy.
After thawing, single blastocyst was transferred into uterus after 6 days of progesterone treatment using Kitazato ET catheter ® (Kitazato Corporation) under ultrasound guidance.
All the blastocysts performed laser-assisted hatching before embryo transfer. The presence of a gestational sac was confirmed via transvaginal ultrasonography at around 5 weeks. Pregnancy and miscarriage rates were analyzed for the overall cohort, and also for the <35-year-old and ≥35-year-old patient subgroups.

| Statistical analysis
Statistical analysis was conducted using chi-square test for patient's proportion, survival rate, pregnancy rate, miscarriage rate, and cesarean section rate. For other parameters, Student's t test was applied for statistical analysis. P-value <.05 was considered significant.
No significant differences in pregnancy or miscarriage rates were evident between C and G methods for any age-groups. There was no significant difference between C and G methods in each age-group.

| Perinatal outcomes
We also examined perinatal outcomes following vitrified-thawed single blastocyst transfer (Table 4). With C method, total follow-up data for perinatal outcomes were collected from 30 cases, with a mean gestational age of 38 weeks 6 days, a cesarean section rate of 46.7%, and a mean birthweight of 3103 g. With G method, total follow-up data for perinatal outcomes were collected from 36 cases, with a mean gestational age of 39 weeks 4 days, a cesarean section rate of 41.6%, and a mean birthweight of 3226 g. In this study, no multiple pregnancy was observed in G and C methods. No significant differences in gestational age, cesarean section rate, or birthweight were evident between C and G methods.

| D ISCUSS I ON
In this study, survival rate, pregnancy rate, and miscarriage rate after vitrified-thawed single blastocyst transfer were compared between C and G methods. No significant differences were observed between the two groups. Similarly, no significant differences in perinatal outcomes were observed between groups.