Single Step Protocol and Multi-step Warming Protocol for Blastocyst FET

The Live Birth Rate Between Single and Multi-step Warming Protocol Applied in Blastocyst Vitrification: a Randomized Controlled Trial

The multi-step thawing protocol with a reduction of non-permeable cryoprotectant concentrations to reduce osmotic shock caused by the rapid influx of water. Recent studies have shown that a simplified warming protocol by only a thawing solution gave a comparable survival rate but increased pregnancy rate, reduced patients' waiting time, and decreased the workload of embryologists.

Nowadays, vitrification is the gold standard method in freezing human embryos, using different commercial brands of ready-to-use kits. Removing cytotoxic cryoprotectants and rehydration to prevent osmotic shock has been a fundamental principle in cryobiology. This minimized damage during the vitrification/thawing (V/T) process. However, the entire process is time-consuming and labor-intensive in the IVF laboratory. Especially, some laboratories have difficulty ordering the same brand of medium for V/T kits. Because of the long period of cryopreserved embryos, it may be that embryos were vitrified and warmed with different kits with a potentially different kind and concentrations of cryoprotective agents. Recently, the combinations of the two different V/T commercial kits have shown comparable survival, blastulation, and implantation rates in both own and donor oocyte cycles.

Additionally, there remains an opportunity and a necessity to continue improving the warming protocol. The key factors for thawing require a fast warming rate, a gradually decreasing concentration of intracellular cryoprotectant, and embryologist skills to secure the survival rate.

Based on previous work, one option would be shortening the time necessary to rehydrate. A study by Seki and Mazur has shown that embryo survival is almost entirely dependent on the warming rate rather than the extracellular cryoprotectant concentration used. A recent study by Liebermann showed that simplifying warming procedures in one step by using 1M sucrose only is possible with an encouragingly higher ongoing pregnancy rate and comparable clinical outcomes when compared to the same conventional multi-step warming protocol, showing a significantly lower miscarriage rate (4.0% vs. 7.6%). These results lead to a faster, safer, and more cost-effective procedure.

This study aims to investigate the effectiveness and safety of a new combination of V/W solutions-single and multi-step thawing protocol- on live birth rate (LBR), as well as embryo transfer, obstetric, and neonatal outcomes.

Inclusion Criteria:

• Women aged from 18
• Undergoing no more than 3 previous IVF/ICSI cycles
• Had at least a single good-quality blastocyst frozen.
• Endometrium preparation using artificial cycle
• Agree to single blastocyst transfer
• Not participating in any interventional studies at the same time

Exclusion Criteria:

• Embryos from cycles after in-vitro maturation, pre-implantation genetic testing (PGT)
• Having contraindications for exogenous hormone administration (e.g., breast cancer, thromboembolic disease)
• Having uterine abnormalities (e.g., adenomyosis, intrauterine adhesions, unicornuate/ bicornuate/ arcuate uterus; unremoved hydrosalpinx or endometrial polyp)