Case Report: The Birth of a Normal Healthy Baby from Blastocysts Frozen and Thawed Twice

L. Keith Smith, Ellen H. Roots and M. Janelle Odom DorsettThe Centre for Reproductive Medicine, Lubbock TX

IntroductionThe culture of human embryos to the blastocyst stage in vitrohas been an important advancement in ART, resulting inincreased implantation rates and a reduction in the numberof embryos transferred in IVF-ET cases (1,2). Supernumeraryblastocysts are normally cryopreserved, giving the patient thepotential of a future pregnancy with a frozen embryo transfer(FET). Many live births occur each year using blastocysts thathave been frozen/thawed once. However, little is known aboutthe implantation potential of human blastocysts frozen/thawedmore than once. We report here the case of a successfulpregnancy and the live birth of a normal healthy baby fromhuman blastocysts frozen/thawed twice at the blastocyst stage.

ObjectiveReport the birth of a normal healthy baby following an FETwith blastocysts that were frozen/thawed twice.

DesignA case report.

Materials/MethodsIVF-ET was performed using a normal ovarian stimulationprotocol. The patient was placed on oral contraceptives anda GnRH agonist (20-10 IU/day) added after 14 days.Recombinant FSH was begun on cycle day 1 and ovulationinduced with a single dose of recombinant hCG (5,000 IU).Thirty-six hours post-hCG, an ultrasound-guided transvaginaloocyte aspiration/retrieval was performed with a 17g x 30 cmoocyte retrieval needle and 20 mL syringes.

Oocyte insemination was performed with 200,000 motile sperm/mL in 100 µL drops of G1.2 media (Vitrolife) overlaid withmineral oil for 18 hrs. All 2PN embryos were cultured in 50µLdrops of G1.2 media overlaid with mineral oil for 48 hrs. OnDay 3, all viable embryos were transfered to 50 µL drops of G2.2media (Vitrolife) overlaid with mineral oil and cultured to Day 6.All embryos were cultured at a density of 1 embryo/drop in a37°C incubator with 6% CO2.

Blastocyst freezing was performed on Day 6 on all expanded/hatching/hatched blastocysts with a visible inner cell mass( 4BB) using a two-step, slow freeze protocol (Menezo Modified2-Step). Blastocysts were incubated 10 min in a HEPES- buffered5% glycerol solution followed by 10 min in a HEPES-buffered 9%glycerol solution with 0.2 M sucrose. During the incubations, thetemperature was lowered from 37°C to 20°C by opening the IVFchamber door and turning the heater off. The blastocysts werefrozen in cryopreservation vials with 200 µL of the HEPES-buffered 9% glycerol solution with 0.2 M sucrose using acontrolled-rate LN2 vapor freezer.

The freezing program lowered the temperature from 20.0°Cto -7.0°C at -2.0°C/min. The temperature was held at -7.0°C for10 min, the vials manually seeded with 1,1,1,2-tetrafluoroethanespray and maintained at -7.0°C another 10 min. The temperaturewas then lowered to -37.0°C at -0.3°C/min and the vials plungedin LN2 at -196°C.

Blastocyst thawing was performed using a two-step rapid-thawprotocol (Menezo Modified 2-Step). The vials were incubated atroom temperature for 1 min, followed by 2 min in a 30°C waterbath. The embryos were then incubated 3 min in HEPES-buffered 0.5 M sucrose solution followed by 2 min in HEPES-buffered 0.2 M sucrose solution. During the incubations, thetemperature was increased from 30°C to 37°C by closing theIVF chamber door and turning the heater on. The embryoswere then incubated in 1 mL of G2.2 media for 4 hrs at 37°Cwith 6% CO2. Only blastocysts that demonstrated re-expansionafter 4 hrs were used for the FETs. Embryo transfers wereperformed with an 18 cm Wallace embryo transfer cathetercontaining 15 µL of G2.2 media.

The patient was prepared for the FETs using an E2 patchprotocol. She was placed on OCPs and a GnRH agonist(20 IU/day) was added after 14 days. Transdermal E2 patches(0.1-0.4 mg/3 days) were started at cycle day 1. After 14 days,daily IM injections of progesterone in oil (50 mg) were startedalong with E2 patches (0.2 mg/3 days). The FETs were performedon the 6th day of progesterone injections. Both progesterone andE2 patches were continued until the 10th week of pregnancy.

ResultsA 26-year old female (gravida 0) with a diagnosis of male factorinfertility and polycystic ovarian disease underwent IVF-ET.The oocyte aspiration/retrieval resulted in 19 metaphase II oocytes;fifteen of these fertilized normally (2PN) following insemination.Twelve embryos reached the blastocyst stage, with 8 exhibitingspontaneous hatching in vitro after 6 days in culture. Two hatchedzona-free (HZF) blastocysts were transferred to the uterus on Day 6.Eight other blastocysts were frozen on Day 6. The embryo transferresulted in a twin pregnancy that spontaneously reduced to asingleton pregnancy after 17 weeks gestation. The patient gavebirth to a normal healthy baby (male) weighing 2069g after 32.5weeks gestation.

Thirty-six months later, FET #1 was performed with all 8 frozenblastocysts being thawed. At 4 hrs post-thaw, only 1 blastocyst hadre-expanded and was transferred to the uterus. The remaining 7were cultured in 1 mL of G2.2 media overnight (18 hrs). The nextmorning, 4 of the 7 blastocysts had re-expanded (3 HZF blastocysts+ 1 expanded blastocyst) and were refrozen.

Examples of Day 6 Blastocysts:

Expanded Hatching Hatched Zona- Blastocyst Blastocyst Free Blastocyst

DiscussionWe report here one of the first pregnancies and live birthsfrom human embryos frozen/thawed twice at the blastocyststage. Two other groups have previously reported pregnanciesand live births from embryos frozen/thawed twice at earlierstages of development (3,4). Further studies will be requiredto determine the survival, implantation and live birth rateswith refrozen/thawed human blastocysts.

ConclusionsHuman blastocysts can be refrozen/thawed and producea normal healthy baby following an FET.

References1. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Stevens J, Schoolcraft WB.Culture and transfer of human blastocysts increases implantation rates and reducesthe need for multiple embryo transfers. Fertil Steril 1998;69:84-88.2. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J.A prospective randomized trial of blastocyst culture and transfer in in-vitrofertilization. Hum Reprod 1998;13:3434-3440.3. Farhat M, Zentner BS, Lossos F, Bdolah Y, Holtzer H, Hurwitz A.Successful pregnancy following replacement of embryos previouslyrefrozen at the blastocyst stage. Hum Reprod 2001;16:337-339.4. Yokota Y, Yokota H, Yokota M, Sato S, Araki Y.Birth of healthy twins from in vitro development ofhuman refrozen embryos. Fertil Steril 2001;76:1063-1065.

FET #1 resulted in a singleton pregnancy that ended in aspontaneous abortion after 7 weeks gestation.

Six months later, FET #2 was performed with all 4 refrozenblastocysts being thawed. At 4 hrs post-thaw, all 4 had re-expanded and were transferred to the uterus. Ten days later,the patient’s serum hCG was 197 mIU/mL. Three weeksafter the positive hCG, a transvaginal ultrasound revealed2 sacs, one with cardiac activity (153 bpm). The singletonpregnancy progressed uneventfully with the patient givingbirth to a normal healthy baby (male) weighing 3005g after38 weeks gestation.