Devices
The EC6-230LE dry incubator (Astec, Japan) and APM-50 D humid incubator (Astec, Japan) were used in this study. The temperature, CO2 and O2 concentration were determined per day before the experiments.
Subjects
The IVF-ET related data were collected from 501 subjects presenting to the Reproductive Medicine Center, Zhejiang Provincial People’s Hospital between November 2016 and October 2017. The inclusion criteria were as follows: (i) female aged ≤38 yrs; (ii) those received frozen-thawed embryo transfer (FET) as the first embryo transfer; (iii) those received transfer of D3 high-quality embryos. Those with the following conditions were excluded from this study: (i) any individual of the couples presenting chromosomal anomaly; (ii) those with rescue intracytoplasmic sperm injection (ICSI) cycles after the IVF.
Experimental design
Twenty-one cycles with oocytes retrieval of ≥20 was collected in this study. The oocytes in each cycle were randomly divided into DC group (N=262 oocytes) and HC group (N=263 oocytes). Then we determined the in vitro culture efficiency including the fertilization rate, cleavage rate and high-quality embryo rate. In order to eliminate the effects of other factors on the clinical outcome, we analyzed the clinical outcome of the embryo and offspring conditions based on different culture model. Eventually, 352 cycles met the inclusion criteria, and 320 cycles were finally included in this study, consisting of DC group (N=184) and HC group (N=136), after excluding 5 with female chromosome abnormality, 5 with male chromosome abnormality, and 22 with rescue ICSI.
IVF therapy
The oocytes retrieval, IVF and embryo culture were conducted in our lab using the GnRH antagonist protocol, long protocol, and minimal stimulation protocol according to the previous description [9, 10]. The follicle-stimulating hormone (FSH), human menopause gonadotropin (HMG) and clomiphene were utilized to stimulate the follicular development according to the ovary reserve and response. In cases of a diameter of >17 mm in two follicles, or a diameter of >18 mm in one follicle, human chorionic gonadotrophin (HCG) and/or GnRH-a were administrated, followed by oocytes retrieval within 36-38 hrs.
In order to avoid the influences of repetitive door open during the culture procedure, the chamber was performed independently for the in vitro culture cycle. The COOK sequential media (Cook Medical, Brisbane, Australia) was utilized for the IVF and embryo culture. The droplets prepared based on fertilization medium (FM, 80µl) and cleavage medium (CM, 30µl) were transferred to petri dishes (60×15, 353652, Falcon), followed by covering with 8.5 ml embryo culture oil (ART-4008-5P, Origio). Upon oocytes retrieval, conventional IVF or ICSI was performed. On day 3, the high-quality embryo was selected according to the ASBIR criteria [11] for the subsequent embryo cryopreservation.
The endometrial preparation protocol of the FET cycle consists of natural cycle, hormone replacement therapy protocol and ovarian stimulation protocol. Based on the endometrium development conditions, one or two D3 high-quality frozen embryo was thawed, followed by transfer. Then the serum HCG was measured on day 12 to determine whether the female was pregnant. The clinical pregnancy was defined as presence of gestational sac by B-ultrasonography on day 35 after transfer. Those presenting no fetal heart or loss of fetal heart at week 12 were defined as early abortion. Ongoing pregnancy was defined as development of gestational sac for at least 12 weeks.
Statistical analysis
SPSS 19.0 software was used for the data analysis. Measurement data that were normally distributed were presented as mean ± standard deviation. Inter-group comparison was conducted using Student’s t-test. The comparison of numeration data was conducted using the Chi square test. Multi-variate logistic regression analysis was used to analyze the effects of female age, fertilization protocol, number of transferred embryos, and the culture mode on the pregnancy. p<0.05 was statistically significant.