Embryo Thawing Protocol, Frozen Embryo Transfer (FET), IVF
Conditions
Brief summary
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.
Detailed 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.
Interventions
PROCEDURESingle-step warming protocol by thawing solution only
Potentially eligible patients' vitrified blastocysts will be thawed by a single-step thawing protocol. 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 in 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.
PROCEDUREStandard warming protocol
For the 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 at 37°C. Subsequently, the blastocyst will be transferred to a second well containing a dilution solution (DS) of 0.5M trehalose for a two-minute rinse at room temperature. This is followed by two additional three-minute and 30-second 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.
Sponsors
Mỹ Đức Hospital
Study design
Allocation
RANDOMIZED
Intervention model
PARALLEL
Primary purpose
TREATMENT
Masking
NONE
Eligibility
Sex/Gender
FEMALE
Age
18 Years to No maximum
Healthy volunteers
No
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)
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Live birth rate | At 22 weeks of gestation | Live birth is defined as the complete expulsion or extraction from a woman of a product of fertilization, after 22 completed weeks of gestational age; which, after such separation, breathes or shows any other evidence of life, such as heart beat, umbilical cord pulsation or definite movement of voluntary muscles, irrespective of whether the umbilical cord has been cut or the placenta is attached. A birth weight of 500 grams or more can be used if gestational age is unknown |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Cancellation rate | Any day during endometrium preparation days before embryo transfer. | Cancellation due to: The blastocyst cells are lysed after thawing. |
| Positive pregnancy test | At 2 weeks after embryo placement | Serum ß-hCG ≥25mIU/mL |
| Implantation rate | At 3 weeks after embryo placement | The implantation rate is explained as the number of gestational sacs per number of embryos transferred |
| Clinical pregnancy | At 5 weeks after embryo placement | diagnosed by ultrasonographic visualization of one or more gestational sacs or definitive clinical signs of pregnancy at 6 weeks or more after the onset of the last menstrual period. In addition to intra-uterine pregnancy, it includes a clinically documented ectopic pregnancy. |
| Ectopic pregnancy | At 7 weeks of gestation | A pregnancy outside the uterine cavity, diagnosed by ultrasound, surgical visualisation, or histopathology |
| Ongoing pregnancy | At 10 weeks after embryo placement | Having at least one gestational sac on ultrasound at 12 weeks' gestation with heart beat activity |
| Miscarriage | before 22 completed weeks of gestational age | The spontaneous loss of an intra-uterine pregnancy before 22 completed weeks of gestational age |
| Preterm delivery | At 22, 28, 32 weeks and 37 weeks of gestation | Multiple definitions, defined as delivery at \<24, \<28, \<32, \<37 completed weeks |
| Major congenital abnormalities | At birth | Structural or functional disorders that occur during intra-uterine life and can be identified prenatally, at birth or later in life. Congenital anomalies can be caused by single gene defects, chromosomal disorders, multifactorial inheritance, environmental teratogens and micronutrient deficiencies. The time of identification should be reported. Any congenital anomaly will be included as followed definition of congenital abnormalities in Surveillance of Congenital Anomalies by Division of Birth Defects and Developmental Disabilities, NCBDDD, Centers for Disease Control and Prevention (2020). |
| Survival rate | At least 2 hours after thawing. | The survival rate by the presence of blastocoel re-expansion before embryo transfer. |
| Low birth weight | At the time of delivery | Weight \< 2500 gm at birth |
| Very low birth weight | At the time of delivery | Weight \< 1500 gm at birth |
| High birth weight | At the time of delivery | Weight over than 4.500 g for women with diabetes, and a threshold of 5.000 g for women without diabetes |
| Admission to NICU | At birth | The admittance of the newborn to NICU |
| Multiple pregnancy | At 6 to 8 weeks' gestation | ≥2 gestational sac at early pregnancy ultrasound |
| Multiple delivery | At 22 weeks' gestation | Birth of more than one baby beyond 22 weeks |
| Still birth | At 20 weeks' gestation | The death of a fetus prior to the complete expulsion or extraction from its mother after 20 completed weeks of gestational age. The death is determined by the fact that, after such separation, the fetus does not breathe or show any other evidence of life, such as heartbeat, umbilical cord pulsation, or definite movement of voluntary muscles. |
| Neonatal mortality | within 28 days of birth | Death of a live-born baby within 28 days of birth. This can be divided into early neonatal mortality, if death occurs in the first seven days after birth, and late neonatal if death occurs between eight and 28 days after delivery |
| Direct costs to live birth | At the time of delivery | Total direct cost to have a live birth after embryo transfer. Direct cost include medical consultations, ovulation stimulation drugs, laboratory and embryology services, ultrasound scanning, medical procedures such as oocyte retrieval and embryo transfer, hospital charges, nursing and counselling services and administrative and overhead charges. Cost data will be collected for a supplementary analysis and will be reported in a separated paper. |
| Birth weight | At the time of delivery | Weight of singletons and twins |
Countries
Vietnam
Contacts
Primary ContactTam TM Luu, MD
Backup ContactVu NA Ho, PhD
Outcome results
None listed