**2.5 Blastocyst biopsy for PGT-A**

Blastocysts were biopsied at Days 5, 6 and 7 for PGT-A in some patients based on patients' requests and FET were performed in these patients if there were euploid blastocysts. After biopsy, blastocysts were vitrified individually, and biopsied samples were analyzed with DNA microarray or next generation sequencing by commercial genetic testing companies.

#### **2.6 Blastocyst vitrification, warming and transfer**

Blastocysts were vitrified and warmed using commercial vitrification and warming kits (Fujifilm-Irvine Scientific). For vitrification, both ES and VS were warmed in original vials at 37°C for at least 30 min before use. Briefly, collapsed blastocysts by a laser pulse were equilibrated in 100 μl drop (without oil cover) of ES for 2 min, and then 45 seconds in 100 μl drop (without oil cover) of VS (both steps were performed on a 37°C warming stage) before loading to vitrification devices. The devices were then immersed to liquid nitrogen for vitrification and all samples were stored in liquid nitrogen until warming for FET.

For warming, blastocysts were exposed to a TS at 37°C for 1 min, transferred to a DS for 3 min and finally to a WS for 10 min with a solution change after 5 min at room temperature. After completion of the warming process, blastocysts were cultured in Global medium supplemented with 10% SPS for 2–4 h before transfer. For blastocyst transfer, we selected the best quality of blastocyst for transfer regardless of Day 5, 6 or 7. However, if the blastocysts had the same quality, Day 5 blastocyst is preferred than Days 6 and 7 blastocysts. If embryos after PGT-A were transferred, we used same embryo selection criteria but only euploid blastocysts were transferred.

#### **2.7 Patient preparation for fresh and frozen/warmed blastocyst transfer**

All patients for embryo transfer received estradiol orally and transvaginally. Intramuscular administration of progesterone oil was initiated at about Day 14 of estradiol treatment. Endometrium thickness was measured on the day of progesterone administration. Embryo transfer occurred on the sixth or seventh day of progesterone administration and progesterone was continued until the first serum β-hCG test two weeks after transfer. Pregnancy was assessed 14 days after embryo transfer by a serum β-hCG assay. When the β-hCG was >5 mIU/ml, the patients were regarded as having a biochemical pregnancy and pregnancies were supported by continued estradiol and progesterone. Four weeks after embryo transfer, when a gestational sac and a heartbeat appeared ultrasonographically, the patients were diagnosed as having a clinical pregnancy. Live birth rates were calculated based on healthy baby delivery per transfer.

*Efficiency of Autologous Egg Cryopreservation: Eight Years' Experiences and Clinical Outcomes DOI: http://dx.doi.org/10.5772/intechopen.98675*

#### **2.8 Statistical analysis**

Interval data was analyzed by one-way analysis of ANOVA. The differences between groups were compared with chi square test. If the P value was less 0.05, the difference was considered to be statistically significant.
