Center for Human Reproduction, Los Angeles, California, 18370 Burbank Blvd, Suite 301, Tarzana, CA 91356, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: delivery/embryo/extended cryopreservation/human/pregnancy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report the birth of a healthy, full-term boy following the transfer of embryos cryopreserved for 7.5 years. We performed a Medline search dating back to 1985 as well as a MEVYL® magazine, journal, newspaper current contents search dating back to 1987. Keywords utilized were: human, cryopreservation, embryo, pregnancy and English language. To our knowledge, at the time of writing this is the longest reported cryopreservation period of embryos which resulted in a successful pregnancy. Recently, a similar success was reported (Go et al., 1998).
Beyond the novelty of this case report, it illustrates the importance for patients and assisted reproduction programmes to re-assess long-term cryopreserved embryos. Women who were among the first to have their embryos cryopreserved are now most likely in their forties and may well decide to attempt conception if they can be assured of safety and normality for their resulting offspring.
![]() |
Case report |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fifteen oocytes were retrieved transvaginally under ultrasound guidance and general anaesthesia. Five of the oocytes along with husband's spermatozoa were laparoscopically transferred into the Fallopian tubes. The patient conceived and delivered a full-term male infant in August 1990.
The ten remaining oocytes were inseminated with husband's spermatozoa following the retrieval. Three pre-embryos were obtained and cryopreserved at the two-pronuclear stage the day after retrieval, utilizing increasing concentrations of 1,2-propanediol (Sigma, St Louis, MO, USA) and sucrose solutions (Sigma), slow cooling to 30°C followed by immersion into liquid nitrogen (Air Liquide America Corporation, Simi Valley, CA, USA).
The cryopreservation technique utilized was modified from previously published protocols (LaSalle et al., 1985; Testart et al., 1986
). A Cryomed 1010® (Cryomed, Mount Clements, MI, USA) cell freezer was used. Seeding temperature was 7°C and was performed manually. The embryos were stored in 0.5 ml of 1.5 mol/l propanediol in freezing straws. The zygotes were stored at 196°C in liquid nitrogen for 7.5 years in manual filling storage tanks.
The cryopreserved/thawed embryos were transferred in an unstimulated cycle in June 1997. Ovulation was detected with a urinary lutenizing hormone kit (OvuQUICK®; Quidel, San Diego, CA, USA) and confirmed by a 6.1 ng/ml serum progesterone concentration on the day of transfer.
The pre-embryos were thawed utilizing modifications of prior protocols (LaSalle et al., 1985; Testart et al., 1986
). On the first post-ovulatory day in a 30°C waterbath, they were placed into progressively diluted 1,2 propanediol and sucrose solutions, and then rinsed in sucrose.
All three zygotes survived the thawing and were incubated in a 5% CO2 chamber overnight. After a 24 h incubation, on post-ovulatory day 2, the resulting 4-cell, 4-cell and 2-cell embryos (Figure 1) were transferred transcervically using a TDT® catheter (Laboratoire CCD, Paris, France). Hormonal support with daily 50 mg progesterone in oil i.m. injection and oral 2 mg 17 ß-oestradiol twice daily was administered for 7 weeks.
|
The course of pregnancy was uncomplicated, including a normal second trimester maternal serum -fetoprotein and amniocentesis. The patient delivered a healthy male infant, 4054 g, by repeat Caesarean section on 16 February 1998, at the age of 44 years.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report the successful outcome of a pregnancy following 7.5 years of embryo cryopreservation, the longest such published period to our knowledge. The efficacy and safety of extended cryopreservation is often presumed, but has not been definitively demonstrated in the literature until recently (Go et al., 1998). It was reported that embryo survival rates decreased from 70.6% at 1 month to 52.6% by 615 months of cryopreservation (Testart et al.,1987).
Similarly, there was a trend toward pregnancy rate reduction from 23.3% at 2 months to 15.4% at 615 months with extended cryopreservation. However, it was reported that cryopreservation under 6.5 years does not diminish embryo quality (Lin et al., 1995). They reported a pregnancy rate of 2430% for frozen embryo transfers, but did not clearly specify the number of long-term cryopreserved embryos transferred, nor the pregnancy and delivery rates or gestational/perinatal complications in this group. Nonetheless, this was a large and meaningful study. In our centre, the pregnancy rate overall after frozenthawed embryo transfer is 1520%.
In our programme, most frozen embryos are utilized within 3 months of a failed or spontaneously aborted cycle, and within 30 months of a successful delivery. Our patient forgot she had frozen embryos. In 1989, there was a relatively small number of frozen embryos in the laboratory we used, and no annual cryostorage fees.
Consequently, patients and physicians were informed by the laboratory that they had frozen embryos, but no further communication ensued. In 1997, this laboratory was closing. Therefore, letters were sent to all the patients asking what they desired to do with their embryos. This patient, of course, had the embryos transferred to our current facility and decided to attempt a frozen embryo cycle.
Major and minor malformation rates appear to be similar in children conceived from cryopreserved embryos, fresh in-vitro fertilization cycles and naturally (Sutcliffe et al., 1995). Thus, couples conceiving from cryopreserved embryos can probably follow accepted guidelines for amniocentesis. Our patient underwent amniocentesis as she was 36 years old at the time of egg retrieval and she was very concerned about the prolonged cryopreservation period.
Recently we began freezing cleaving embryos on day 3 post-retrieval or at the blastocyst stage. Too few frozen embryo transfers have been performed thus far to draw firm conclusions, though initial results are encouraging. Prior to cryopreservation of embryos, patients must sign a consent form which allows them to choose between donating versus discarding embryos which are abandoned for 5 or more years.
Additionally, nominal annual cryopreservation fees have been instituted by the laboratory to enhance intermittent communication with patients as well as assist with the cost of the cryopreservation programme.
In summary, experience regarding the safety and efficacy of extended cryopreservation of human embryos may benefit couples who need to defer childbearing for prolonged periods. The positive outcome in our report and that of Go et al.(Go et al., 1998), and the results of other studies are encouraging (Lin et al., 1995
; Sutcliffe et al.,1995). However, further studies and reports are needed before definitive conclusions can be drawn.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Jones, H., Jones, D. and Kolm, P. (1997) Cryopreservation, a simplified method of evaluation. Hum. Reprod., 12, 548553.[ISI][Medline]
Lasalle, B., Testart, J. and Renard, J. (1985) Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil. Steril., 44, 645651.[ISI][Medline]
Lin, Y., Cassidenti, D., Chacon, R. et al. (1995) Successful implantation of frozen sibling embryos is influenced by the outcome of the cycle from which they were derived. Fertil. Steril., 63, 262267.[ISI][Medline]
Sutcliffe, A., D'Souza, S., Cadman, J. et al. (1995) Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Hum. Reprod., 10, 33323337.[Abstract]
Testart, J., Lasalle, B., Belaisch-Allart, J. et al. (1986) High pregnancy rate after early human embryo freezing. Fertil. Steril., 46, 268272.[ISI][Medline]
Testart, J., Lassalle, B., Forman, R. et al. (1987) Factors influencing the success rate of human embryo freezing in an in vitro fertilization program. Fertil. Steril., 48, 107112.[ISI][Medline]
Trounson, A. and Mohr, L. (1983) Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature, 305, 707709.[ISI][Medline]
Veeck, L., Adashi, E.Y., Rock, J.A. and Rosenwaks, Z. (1996) Reproductive Endocrinology, Surgery and Technology. Lippincott-Raven, Philadelphia, pp. 23532365.
Zeilmaker, G.H., Alberta, A.T., Van Gent, I. et al. (1984) Two pregnancies following transfer of intact frozenthawed embryos. Fertil. Steril., 42, 293296.pa[ISI][Medline]
Submitted on May 21, 1998; accepted on February 11, 1999.