**2. Methods**

#### **2.1 Ethics approval and consent to participate**

All patients undergoing oocyte cryopreservation, warming for IVF, embryo culture, and embryo transfer signed informed consents for all laboratory and clinical procedures. All procedures were approved by Houston Fertility Institute's research and clinical committee. The data were retrospectively collected from the medical records and patients' information were not included in the data presentation, so the IRB was waived for this study.

## **2.2 Patients and data collection**

Autologous oocyte cryopreservation was assessed in women whose oocyte cryopreservation and warming were performed between 2009 and 2017. Women's age at the time of oocyte cryopreservation was divided into 3 groups, <35, 35–37 and > 37 years old. Based on these age groups, data were compared between fresh blastocyst transfer and frozen/warmed blastocyst transfer.

There were mainly three reasons for patients to cryopreserve their oocytes: 1) all oocytes were cryopreserved because there was no motile sperm or no semen sample being collected at the time of oocyte retrieval; 2) partial oocytes were cryopreserved because no enough motile sperm was found to inseminate all of the oocytes, or patients required to inseminate a portion of oocytes and purposely required to cryopreserve remaining oocytes; and 3) all oocytes were cryopreserved for single women for fertility preservation. Therefore, the data were further analyzed based on these three categories.

#### **2.3 Oocyte cryopreservation, warming and insemination**

Oocyte cryopreservation and warming were based on the procedures previously reported [11] by using commercial vitrification and warming kits (Fujifilm-Irvine Scientific, CA, USA). Briefly, for cryopreservation, matured oocytes were vitrified 4–5 hours after retrieval with initial equilibration of the oocytes in equilibration solution (ES) for 9 mins, and then in vitrification solution (VS) for 90 seconds until vitrification in Cryotop.

For warming, Cryotops were removed from liquid nitrogen and the tips with oocytes were quickly placed in 1 ml thawing solution (TS) at 37°C for 1 min. Oocytes were then transferred to 0.5 ml dilution solution (DS) for 3 min and then to a 0.5 ml washing solution (WS) for 10 min with a solution change after 5 min. After warming, oocytes were washed in Global medium (IVFonline, CT, USA) supplemented with 10% serum protein substitute (SPS, IVFonline) and then cultured in the same medium until insemination. Oocyte survival was evaluated based on morphology after completion of the warming.

## **2.4 Insemination, fertilization assessment, embryo culture and fresh blastocyst transfer**

All oocytes were inseminated by intracytoplasmic sperm injection (ICSI) 2–3 hours after warming. We chose this time for ICSI as it has been reported that most functions, such as meiotic spindle recovery, mitochondria activity and ATP level recovery in frozen/warmed oocytes, take about 2–3 hours after warming [29–31] and it has been found that ICSI time (9 ± 2 h) after oocyte retrieval in the vitrified human oocytes does not affect clinical outcomes [32].

Fertilization was examined 16–18 h after ICSI and normally fertilized zygotes were cultured in Global medium supplemented with 10% SPS at 37°C in a humidified atmosphere of 5.5% CO2, 6% O2 and balanced nitrogen until Day 7 (some patients' embryo culture was extended to Day 7 if morula or early blastocysts were observed at Day 6). On Day 3, embryo cleavage status was examined, and all embryos that divided to two cells and above were considered as cleaved embryos and were transferred to freshly prepared culture medium. On Day 5, embryo development was evaluated and the best 1 or 2 embryos were transferred.
