Case report: The live birth of a normal healthy baby from blastocysts frozen and thawed twice

Abstract

To report the birth of a normal healthy baby following a frozen embryo transfer (FET) utilizing blastocysts that were frozen and thawed twice. Case report. IVF-ET was performed using a normal ovarian stimulation protocol with GnRH agonist down-regulation, recombinant FSH stimulation and recombinant βhCG ovulation-induction. Oocyte insemination and embryo culture were performed with G1.2/G2.2 media (Vitrolife) in a 37°C incubator with 6% CO2. The embryo transfer was performed with 2 blastocysts on Day 6. Blastocyst freezing was performed on Day 6 on all remaining expanded/ hatching/hatched blastocysts with a visible inner cell mass (ICM) using a two-step slow-freezing protocol with glycerol and sucrose as cryoprotectants (Menezo-modified) in a controlled-rate LN2 freezer. Thawing was performed with a two-step rapid-thaw protocol in sucrose solutions (Menezo-modified). The embryos were then incubated in G2.2 media for 4 hrs at 37°C with 6% CO2. Only blastocysts that demonstrated re-expansion after the 4 hr incubation were used for the FETs. A 26-year-old female (gravida 0) with a diagnosis of male factor infertility and polycystic ovarian disease underwent IVF-ET. The IVF cycle resulted in 12 blastocysts after 6 days of culture in vitro. Two hatched zona-free (HZF) blastocysts were transferred to the uterus on Day 6. Eight remaining blastocysts were frozen on Day 6. The fresh transfer resulted in a twin pregnancy that spontaneously reduced to a singleton pregnancy after 17 weeks gestation. The patient gave birth to a normal healthy baby (male) weighing 2069g after 32.5 weeks gestation. Thirty-six months later, FET #1 was performed. All 8 frozen blastocysts were thawed. At 4 hrs post-thaw, only 1 blastocyst had re-expanded. This HZF-blastocyst was transferred to the uterus and the remaining 7 were cultured in G2.2 media overnight (18 hrs). The next morning, 4 of the 7 blastocysts had re-expanded and were refrozen. FET #1 resulted in a singleton pregnancy that ended in a spontaneous abortion after 7 weeks gestation. Six months later, FET #2 was performed. All 4 refrozen blastocysts were thawed. At 4 hrs post-thaw, all 4 had re-expanded (3 HZF-blastocysts and 1 expanded blastocyst) and were transferred to the uterus. FET #2 resulted in a singleton pregnancy and the birth of a normal healthy baby (male) weighing 3005g after 38 weeks gestation. Human blastocysts can be refrozen/thawed and produce a normal healthy baby following FET. The finding suggests some novel approaches to FETs in the future.