Effect of equilibration time on clinical and neonatal outcomes in human blastocysts vitrification

Abstract Purpose Prolonged exposure to equilibration solutions may be detrimental to an embryo's developmental potential, whereas a shorter exposure may affect the penetration of cryoprotectants into blastomeres. The purpose of this study was to evaluate the effects of different equilibration times on the clinical and neonatal outcomes of human blastocyst vitrification. Methods This is a retrospective study based on data collected between November 2008 and November 2015. A total of 192 blastocysts (80 non‐expanded and 112 expanded) obtained from 167 patients were analyzed. The blastocysts were divided into two groups according to their equilibration time: 8‐11 minutes or 12‐15 minutes. The clinical and neonatal outcomes of warmed blastocysts were evaluated. Results The survival, implantation, and live birth rates of non‐expanded blastocysts were not different between the two groups, but they significantly improved for the expanded blastocysts in the 12‐15 minutes group compared to the 8‐11 minutes group. The results were similar for the neonatal outcomes after vitrified embryo transfer, when partitioned by equilibration time and blastocyst stage at vitrification. Conclusions For the non‐expanded blastocysts, a shortened equilibration time (8‐11 minutes) is sufficient for effective vitrification.

solutions is unclear. Commercial vitrification media often contains the CPAs ethylene glycol (EG) and dimethyl sulfoxide (DMSO).
A critical consideration is the duration of blastocyst suspension in the vitrification solution, which should be strictly controlled within 1 minute to reduce the cytotoxic effects of the highly concentrated CPAs. On the other hand, the duration of blastocyst suspension in the equilibration solution is flexible. To simplify the vitrification process, some embryologists adopt a fixed equilibration time (Table 1).
These differences suggest that there is no universally agreed upon equilibration duration for human blastocysts, thus leading us to pose the following question: To what extent does the equilibration time influence the clinical outcomes of human blastocyst vitrification?
Moreover, knowledge is still limited regarding the safety of vitrification in terms of neonatal outcomes of babies delivered from vitrified embryos.
Prolonged exposure to equilibration solutions may be harmful to an embryo's developmental potential, whereas a shorter exposure may affect the penetration of CPAs into blastomeres. The purpose of this study was to evaluate the effects of different equilibration times on the clinical and neonatal outcomes of human blastocyst vitrification.

| Experimental data
In this retrospective observational study, data were collected between November 2008 and November 2015 from 167 infertility patients. Causes of infertility included male factors, female factors (oviduct and endometrial), and/or unexplained infertility. This study was approved by the Ethics Committee at Kurashiki Medical Center, Japan, and was conducted electronically via a web site, which included additional explanatory information and an opt-out option.

| Blastocyst preparations
In the oocyte retrieval cycle, ovarian stimulation was achieved using standard gonadotropin-releasing hormone and agonist/folliclestimulating hormone (FSH) protocols or an antagonist/FSH protocol.
Vaginal ultrasound-guided follicle puncture was conducted 36 hours after an injection of human chorionic gonadotropin (Mochida, Tokyo, Japan). The retrieved oocytes were inseminated by conventional in vitro fertilization or intracytoplasmic sperm injection in accordance with a previously reported method. 7 and C: very few cells forming a loose epithelium. These scores differentiated between "good" (AA), "fair" (AB, BA, BB, AC, CA), and "poor" (BC, CB, CC) graded blastocysts.

| Blastocyst vitrification and warming
The Cryotop ® method (Kitazato Cryotop ® method; Kitazato Corporation, Shizuoka, Japan) was used for blastocyst vitrification and has been described elsewhere. 10  Collapsing procedures of the blastocoel such as artificial shrinkage or trophectoderm biopsy and assisted hatching procedures were not performed on any embryos.

| Transfer of post-warmed blastocysts
Blastocyst survival was defined as a blastocyst that was partially intact after warming and re-expansion following culture in vitro before transfer. Each surviving blastocyst was transferred to a patient's uterus. Common modalities for blastocyst transfer were natural cycles or hormonal replacement cycles for endometrial preparation.
Blastocyst transfer was performed under ultrasound guidance using an embryo transfer (ET) catheter.

| Follow-up and evaluation index
To confirm the establishment of a clinical pregnancy, an ultrasound examination was performed in order to visualize a gestational sac and a fetal heartbeat. The loss of a fetus with a gestational age of <20 weeks was considered a spontaneous abortion. The live birth rate was calculated by dividing the number of live birth delivery cycles by the number of transfer cycles. A preterm birth was defined as <37 gestational weeks. A birth weight <2500 g was defined as low birth weight. The duration of pregnancy, mode of delivery, and weight and sex of the child were recorded as neonatal outcomes.

| Statistical analysis
The primary outcome was the live birth rate per warming cycle.
Secondary outcomes were embryo survival and implantation rates. Analyses were performed using the Statcel 2 program (OMS Publishing was determined using an online calculator. P < .05 was considered statistically significant.

| RE SULTS
In all cycles, single ET was completed. The average interval time from warming to transfer was comparable for the two groups (8-11 and 12-15 minutes groups) of non-expanded and expanded blastocysts (14.5 ± 6.7 vs 14.5 ± 6.7 hours and 15.1 ± 7.0 vs 13.1 ± 7.5 hours, respectively). The average equilibration duration for the 8-11 minutes group in non-expanded and expanded blastocysts was 9.65 ± 0.86 and 9.65 ± 0.86 minutes, and for the 12-15 minutes group in non-expanded and expanded blastocysts was 13.7 ± 1.25 and 13.4 ± 1.34 minutes. There were no significant differences among the groups.
Patient characteristics are summarized in Table 2

| Clinical outcomes of non-expanded and expanded blastocysts
In all cycles, 192 total blastocysts were warmed, from which 43 babies were born (live birth rate: 22.4%).
Results for the two groups (

| Neonatal outcomes of non-expanded and expanded blastocysts
The neonatal outcomes of 43 children born after blastocyst transfer are summarized in Table 4.
For the non-expanded blastocysts, 5 babies were born in the were also not different.  Note: Values presented as number.

| D ISCUSS I ON
Abbreviations: CI, confidence interval; HRT, hormone replacement therapy. a Blastocyst morphology when the blastocyst was vitrified.
b Defined as a gestational sac identified with ultrasound.
reduced. This allows for sufficient permeation of the CPAs and a more rapid equilibration before vitrification. Non-expanded blastocysts in this study were identified when the blastocoels occupied less than half of the embryo volume; they were < 150 µm in diameter. Consequently, the non-expanded blastocysts survived after the vitrification procedure. Unnecessary prolonged CPA exposure could have harmful effects to embryonic development, so a shorter exposure to equilibration solution is preferable for non-expanded blastocyst vitrification.
In the expanded blastocysts, a shorter equilibration time  We acknowledge that the manipulation skills of each embryologist may affect the study's outcomes. However, in this investigation, only embryologists with more than 5 years' clinical experience undertook embryo vitrification, and their results were statistically comparable. Therefore, the differences in technique between embryologists probably did not influence the study's outcomes.
In conclusion, a short equilibration time (8-11 minutes) is sufficient for blastocyst vitrification when the blastocoel cavity is small.
However, these results should be interpreted with caution due to the small study size and the high risk of bias. Further randomized controlled trials that examine clinical and neonatal outcomes are necessary to adequately judge the efficacy and safety of vitrification.

Shingo Mitsuhata, Yoshitaka Fujii, Yuji Endo, Momoko Hayashi and
Hiroaki Motoyama declare that they have no conflict of interest.

H U M A N R I G HT S S TATE M E NT A N D I N FO R M E D CO N S E NT
All the procedures were followed in accordance with the ethical standards of the institutional ethical committee and with the Helsinki Declaration of 1964 and its later amendments. All the study's participants provided informed consent, and the study design was approved by the appropriate ethics committee of Kurashiki Medical Clinic, Okayama, Japan.

A N I M A L S TU D I E S
This article does not contain any studies with animal participants performed by any of the authors.