Effects of vitrified cryopreservation duration on IVF and neonatal outcomes

Study design and participants

The data were collected from the Center for Reproductive Medicine at The Third Affiliated Hospital of Guangzhou Medical University in Guangzhou, China. We retrospectively reviewed patient and neonatal outcomes following the first frozen embryo transfer (FET) cycles during in vitro fertilization (IVF) with vitrified cryopreserved embryos. The study period was from January 2004 to August 2019. The inclusion criteria were as follows: embryos cryopreserved by VF, procedures corresponding with the first frozen embryo transfer (FET) cycle, and availability of complete follow-up information (including situation of HCG, clinical pregnancy, multiple pregnancy, live birth, ectopic pregnancy, the fetus gender, birth height, birth weight, congenital anomalies). There were no restrictions on age or BMI. There was no donor oocytes cycle in this study. Participants were grouped according to the embryo storage time as follows: Group 1 (n = 20,926),1–90 days; Group 2 (n = 6,472), 91–180 days; Group 3 (n = 2,237), 181–365 days; Group 4 (n = 746), 366–730 days; and Group 5 (n = 762), > 731 days. Patients with slow thawing, non-first thaw and incomplete information were excluded. Women were excluded if they underwent PGT. The study design was approved by the ethics committee of the Third Affiliated Hospital of Guangzhou Medical University, and written informed consent was obtained from each participant.

ART procedures

Women were monitored and managed according to the hospital’s clinical protocols. Various controlled ovarian stimulation (COS) protocols were used, with 150–450 IU/day of recombinant FSH or human menopausal gonadotropin in a gonadotropin-releasing hormone antagonist protocol, a long agonist protocol, or a short agonist protocol. The protocols were determined according to each patient's characteristics (age, body mass index (BMI), AFC and AMH). Transvaginal oocyte retrieval was scheduled 35–36 h after HCG injection. ART was performed per standard operating procedure of the hospital.

Conventional IVF or intracytoplasmic sperm injection was performed depending on the semen characteristics and history of previous fertilization attempts. For conventional IVF, cumulus-oocyte complexes were inseminated with progressively motile spermatozoa in fertilization culture medium (G-IVF PLUS, Vitrolife, Gothenburg, Sweden). For intracytoplasmic sperm injection, oocytes were denuded 2–3 h after ovum pickup, and sperm microinjection was performed 5–6 h after oocyte retrieval. Fertilization was checked approximately 16 h after insemination/injection and was determined by the presence of two pronuclei. Embryos were placed in the incubator (K-MINC-1000, Cook Medical, Bloomington, IN, USA) and cultured at 6% CO2, 5% O2, and 37 °C. G-1 PLUS and G-2 PLUS (Vitrolife, Gothenburg, Sweden) was used for culturing cleavage-stage and blastocyst-stage embryos, respectively. The quality of cleavage-stage embryos was assessed approximately 68 h after insemination/injection, and the quality of blastocyst-stage embryos was evaluated approximately 116 h or 140 h after insemination/injection.

Good-quality cleavage-stage embryos were defined as those with 7–9 symmetrical blastomeres without obvious fragmentation [14]. Good-quality blastocysts were defined as those having reached at least grade 3 expansion and grade A or B for the inner-cell mass and trophectoderm parameters. Poor-quality cleavage-stage embryos were defined as those with blastomere numbers greater than four, fragmentation < 30%, and positive size differences. Poor-quality blastocysts were defined as those having reached at least grade 3 expansion but having grade C inner-cell mass or trophectoderm parameters [15].

Fertilized oocytes were cultured and observed for three days after oocyte retrieval. The embryos destined for freezing were selected according to the embryonic stage and embryo count. For all embryo freezing procedures, 1–2 cleavage-stage embryo VF was performed if embryos were available. If not, blastocyst culture was performed and blastocysts are frozen if available. In cases of fresh ET, when the number of day3 available embryos ≥ 5, blastocysts were cultured and perform fresh ET at the blastocyst stage. When fewer than five embryos are available on day3, the fresh ET is performed at the cleavage stage.

The luteal phase was supported by vaginal administration of micronized progesterone (400 mg/d) initiated on the day of ovarian puncture.

Embryo cryopreservation techniques and thawing protocols

The embryos were frozen and thawed in accordance with the protocols for the Vitrification Kit (Kato Corp., Shizuoka, Japan). Initially, the embryos were exposed to an equilibration solution (ES)for 5 min at room temperature (cleavage-stage embryo) or 2 min at 37 °C (blastocyst). The embryos were then transferred into a vitrification solution (VS) and incubated for 45 s. Finally, the embryos were set on a Cryotop strip (Kitazato Corp., Japan) in a small volume and immediately plunged into liquid nitrogen. Each storage container contained between one and two embryos. The embryologists have been well trained to perform vitrification technically. The embryos were stored at a constant temperature of -196 °C in the liquid phase of liquid nitrogen tank (Taylor Wharton HC35, Theodore, AL, USA). The level of liquid nitrogen was kept under constant manual surveillance by experienced embryologists to prevent suboptimal storage conditions, and the liquid nitrogen tank was manually refilled twice a week.

Vitrified embryos were thawed by a rapid thawing method on the morning of embryo transfer. For the thawing process of vitrified embryos, the embryos unloaded from the carriers were immediately submerged into the thawing solution for 1 min at 37 °C. Then, the embryos were transferred into the diluent thawing solution for 3 min at room temperature. At the final step, the embryos were moved to the wash solution twice for 3 min at room temperature. After that, the embryos were cultured in culture medium at 37 °C under the gas phase of 5% CO2 and 5% O2 in an incubator until transfer. If the embryo demonstrated at least 70% of intact cell (up to 30% degraded cell), it was considered viable and was transferred. Embryos showing less than 70% of essential cells, were considered non-viable and were discarded [16].

The laboratory procedures and cryopreservation protocols remained unchanged throughout the study period. The same storage tanks and pieces of technical equipment were used over the years included in the study period. In addition, the clinical freeze-all indication has not changed too.

Endometrial preparation for FET cycle, and embryo transfer

Endometrial preparation for FET cycle in this study was achieved by natural cycle (NC) or hormone replace treatment (HRT) programs. The ovulation in NC program was determined by monitoring follicular development with transvaginal ultrasonography and hormone levels. The patients in HRT-FET cycles were treated with daily oral estradiol valerate tablets (Progynova, Bayer, Germany) since the second to fourth day of menstruation. When the endometrial thickness reached 7 mm or thicker, 40 mg/day progesterone was intramuscularly administered daily. All embryos transfer treatments were performed on the 4th (cleavage-stage embryo)/6th (blastocyst) day of progesterone exposure or 3(cleavage-stage embryo)/5(blastocyst) days after ovulation using a soft-tipped Wallace (PortexLed., Hythe, United Kingdom) catheter under ultrasound guidance. All patients received luteal support with progesterone after embryo transfer. If transvaginal ultrasound showed gestational sac and embryonic heartbeat 4–6 weeks after embryo transfer, luteal support was continued until 10 weeks of gestational age.

Outcome measure

The primary outcomes of this study included embryo survival rate, live birth rate and occurrence of congenital conditions. Secondary outcome variable included implantation rate, positive HCG rate, clinical pregnancy rate, multiple pregnancy rate, ectopic pregnancy rate, sex ratio (male/female), birth weight and birth height. Clinical pregnancy was defined as the detection of gestational sacs by transvaginal ultrasound 28 days after ET. The implantation rate was defined as the number of observed gestational sacs divided by the number of transferred embryos. The clinical pregnancy rate was calculated as the number of clinical pregnancies divided by the number of patients. Live birth was defined as the delivery of any viable neonate with a gestational age of 28 weeks or older. Low and high birth weights were defined as birth weights < 2500 g or > 4000 g, respectively.

Statistical analysis

The statistical analysis was performed using the Statistical Package for Social Science (SPSS) version 22.0. Descriptive data are presented as mean with 1 SD. The differences between groups were tested using the ANOVA test for continuous variables and the Pearson’s chisquare test for categorical variables. In addition, storage group was included as a categorical variable, and Group 1 was used as the reference. We select indicators with statistical differences from ANOVA results and include them in multivariate regression analysis. Multivariable logistic regression was performed to explore the effect of storage time on pregnancy outcome or neonatal outcome after controlling for potential confounders. P < 0.05 was considered statistically significant.

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