Single Step Protocol and Multi-step Warming Protocol for Blastocyst FET (ONESTEP)

Mỹ Đức Hospital

Status: Enrolling

Conditions: IVF; Frozen Embryo Transfer (FET); Embryo Thawing Protocol

Treatments: Procedure: Single-step warming protocol by thawing solution only; Procedure: Standard warming protocol

Study type: Interventional

Funder types: Other

Identifiers

NCT07088640

07/25/DD-BVMD

Details and patient eligibility

About

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.

Full description

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.

Enrollment: 816 estimated patients

Sex: Female

Ages: 18+ years old

Volunteers: No Healthy Volunteers

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)

Trial design

Primary purpose: Treatment

Allocation: Randomized

Interventional model: Parallel Assignment

Masking: None (Open label)

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816 participants in 2 patient groups

Single-step thawing protocol

Experimental group

Description:

For the warming phase, vitrified blastocysts are exposed to the thawing solution of a commercial embryo thawing kit (Irvine Scientific Inc., USA) at 37°C for one minute. Immediately following this, embryos will be rinsed through a 35mm diameter dish of 2ml of pre-equilibrated thawing solution before being placed in culture media in the incubator for at least 2 hours before transfer.

Treatment:

Procedure: Single-step warming protocol by thawing solution only

Multi-step thawing protocol

Experimental group

Description:

For the Multi-step (MS) protocol, thawing kits were equilibrated overnight in a 37°C incubator. Warming procedures utilized the kits (Cryotech RtU, Japan). To remove the cryoprotectants, blastocysts were warmed, and cryoprotectants were diluted in a three-step process. The warming process starts with the exposure of blastocysts to thaw solution (TS) with 1M trehalose for one minute, set at a temperature to ensure 37°C. Subsequently, blastocysts will be transferred to a second well containing dilution solution (DS) 0.5M trehalose for a two-minute rinse at room temperature. This is followed by two additional three-minute and 30 seconds rinses in the wash solution (WS) at room temperature. The timeline for standard warming of blastocysts requires a total of 6.5 min. After thawing, embryo will be placed in the incubator at least 2 hours before transfer.

Treatment:

Procedure: Standard warming protocol