P-249: Successful clinical pregnancy following the transfer of embryos frozen and thawed twice: A case report

Abstract

To report the ongoing clinical pregnancy of an IVF patient following the transfer of embryos frozen/thawed at the two pronucleus stage (2PN) then cultured to day 5 and frozen/thawed again at the blastocyst stage. Case Report. An IVF cycle was performed using a normal ovarian stimulation protocol with GnRH agonist down regulation, recombinant FSH stimulation and recombinant βhCG ovulation-induction. Half of oocytes retrieved underwent standard insemination and the remaining half was fertilized by intracytoplasmic sperm injection (ICSI). Embryo culture was performed using Quinn’s Advantage Cleavage Media (QAC) supplemented with 10% v/v Serum Substitute Supplement (SSS) in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 37° C. All resulting 2PN’s were cryopreserved using a standard propanediol and sucrose slow freezing protocol in a controlled-rate liquid nitrogen (LN2) freezer. 2PN’s were thawed with a multi-step thaw protocol in sucrose solutions at room temperature. The zygotes were then cultured again in QAC Medium for 48 hrs under similar culture conditions. Blastocyst culture was performed using Quinn’s Blastocyst Medium also in a humidified low oxygen environment at 37°C to Day 5 and Day 6. Blastocyst cryopreservation was performed on Day 6 on all remaining blastocysts of grade 3BB or better using a two-step slow-freezing protocol (Menezo) with glycerol and sucrose in a controlled rate LN2 freezer. Blastocysts were thawed with a two-step thaw protocol in sucrose solutions (Menezo). Recovery and re-expansion was conducted in Quinn’s Blastocyst Medium supplemented with 20% SSS in a humidified low oxygen environment at 37°C for two hours before embryo transfer. Laser assisted hatching was performed on all frozen embryos transferred. A 34-year-old female (gravida 0) with a diagnosis of polycystic ovarian disease and BMI of 37.6 underwent controlled ovarian hyperstimulation for IVF. The cycle resulted in 29 oocytes which were split between standard insemination and ICSI. 17- 2PN’s, 8 from standard insemination and 9 from ICSI, were observed on Day 1 all of which were cryopreserved due to risks associated with ovarian hyperstimulation syndrome. Since then, three FET cycles followed. First 5 zygotes from the IVF group were thawed yielding the transfer of 2 cleavage stage embryos and 3 embryos cultured to the blastocyst stage. One blastocyst (5AB) was re-frozen. After a negative pregnancy test the patient underwent a second FET cycle. 5 zygotes from the ICSI group were thawed yielding the transfer of 3 cleavage stage embryos and 2 embryos cultured to the blastocyst stage. 2 blastocysts were frozen (4AA, 3BA). Again a negative pregnancy test ensued. The third FET cycle was composed of a blastocyst thaw using the one blastocyst (5AB) from the first FET cycle and both of the blastocysts (4AA,3BA) from the second FET cycle. All 3 blastocysts survived (90%, 70%, 70%) the thaw to be transferred following laser assisted hatching. A positive pregnancy test resulted and subsequent 6 and 8 week ultrasound scans revealed a viable singleton intrauterine pregnancy. Human embryos are capable of withstanding more than one freeze/thaw cycle without impairing their ability to develop and implant. Furthermore, additional data relating to the favorable outcome of fetuses born from twice frozen/thawed embryos is needed before such practices are recommended routinely.