Delivery of a healthy baby following more than 17 years of pronuclear stage embryos cryopreservation

Abstract

We would like to report a case of pregnancy achieved after a long period of embryos cryostorage. A couple (the female 24 and the male 34 years old) attended our clinic in 1993 due to primary infertility. Investigation revealed that the husband had oligo-terato-asthenozoospermia. An IVF cycle yielded 22 oocytes which were inseminated using standard IVF, resulting in the development of 16 embryos. A transfer of four embryos was done 48 h after the ovum pickup. The 12 surplus embryos were cryopreserved (8 at two-pronuclear stage and 4 at cleavage stage) using a slow freeze technique in freezing media prepared in our laboratory. 1,2-Propanediol (PROH) and sucrose were used as cryoprotectants. After equilibration in the cryopreservation media, the embryos were loaded and frozen in a programmable freezer (Planer; Middlesex, UK). The freezing program was as follows: starting temperature 25 °C; rate of cooling −2.8 °C/min from 25 to −7 °C; soak at −7 °C for 5 min; manual seeding; hold the temperature at −7 °C for 10 min; rate of cooling: −0.3 °C/min from −7 to −30 °C; rate of cooling: −50 °C/min from −30 to −150 °C. The embryos were stored in liquid nitrogen. No pregnancy resulted from the fresh cycle. The couple abandoned fertility treatments but during the next years conceived naturally twice and delivered two healthy daughters. Twelve years after the treatment, two cycles of frozen-thawed embryo transfer were done but pregnancy was not achieved. In 2011, after more then 10 years of secondary infertility, the couple requested the transfer of the last cryopreserved embryos. The endometrium was primed using beta estradiol (Estrofem, Novo Nordisk, 6 mg/d) and progestin (Endometrin, Ferring Pharmaceuticals Israel, 200–300 mg/d). One day prior to the transfer, 4 zygotes were removed from liquid nitrogen and held in the air at room temperature for 30 s, followed by immersion into a water-bath at 30 °C for 40–50 s. Cryoprotectants were removed by sequential dilution containing decreasing concentrations of cryoprotectants. Four embryos (graded 3–3.5 out of 4) were transferred on cycle day 15 using a soft pass catheter (K-SOFT-5000 Cook Medical). Two weeks later beta-HCG levels were 124 IU/L. Ultrasound done after amenorrhea of 5 weeks revealed 2 gestational sacs. At 8 weeks one sac containing a viable embryo was observed. The hormonal treatment was continued until the 12th week. A single umbilical artery was observed on a fetal anomaly scan, which was otherwise normal. At 37 weeks’ gestation polyhydramnios was observed (amniotic fluid index of 27). Labor was induced at 38 weeks. A healthy girl weighing 3382 g was delivered vaginally 18 years after ovum pickup.