Full term delivery following cryopreservation of human embryos for 7.5 years: Case report

Snunit Ben-Ozer1 and Michael Vermesh

Center for Human Reproduction, Los Angeles, California, 18370 Burbank Blvd, Suite 301, Tarzana, CA 91356, USA


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Successful pregnancy in a 44 year old woman is described following the transfer of embryos which were cryopreserved for 7.5 years. The embryos were obtained during a gamete intra-Fallopian transfer (GIFT) procedure in 1989. To our knowledge this is one of the longest published periods of cryopreservation of embryos which has resulted in a healthy baby. This report illustrates the previously presumed viability and normality of human embryos undergoing long-term cryopreservation. Additionally, it emphasizes the importance for advanced reproductive technique programmes and patients to review and update their embryo status.

Key words: delivery/embryo/extended cryopreservation/human/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
The first pregnancy achieved from human embryo cryopreservation was reported after modification of animal embryo cryopreservation techniques (Trounson and Mohr, 1983Go). The first birth, in 1984 was reported (Zeilmaker et al., 1984Go). In the past 15 years, alternative protocols of embryo cryopreservation, thawing and transfer have been evaluated to optimize embryo survival and pregnancy rates. A good overview of cryopreservation was recently published (Veeck et al., 1996Go).

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., 1998Go).

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
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
A 44 year old gravida 1, para 1 Caucasian woman with a history of mild endometriosis and otherwise unexplained infertility desired to conceive using her frozen embryos. In November 1989, she underwent a gamete intra-Fallopian transfer (GIFT) cycle by one of the authors (M.V.). Pituitary down-regulation was achieved with luteal phase leuprolide acetate and was followed by ovarian stimulation with human menopausal gonadotrophins (Pergonal®; Serono, Randolph, MA, USA). Ovulation was induced with human chorionic gonadotrophin (10 000 IU Profasi®; Steris for Serono).

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., 1985Go; Testart et al., 1986Go). 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., 1985Go; Testart et al., 1986Go). 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 1Go) 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.



View larger version (104K):
[in this window]
[in a new window]
 
Figure 1. Post-ovulatory day 2 frozen–thawed embryos following 7.5 years of cryopreservation, which resulted in a healthy singleton pregnancy. Original magnification x500.

 
Pregnancy was confirmed 11 days after the frozen/thawed embryo transfer. Fetal pole and cardiac activity were detected at 4 weeks after the transfer.

The course of pregnancy was uncomplicated, including a normal second trimester maternal serum {alpha}-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
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Jones et al. (1997) eloquently described the importance and pitfalls of true expression of assisted reproduction pregnancy rates (Jones et al., 1997Go). Calculation of these rates must include the fresh and frozen embryo cycles. The latter group, however, represents a heterogeneous mixture which on occasion is difficult to assess. For example, cryopreservation duration varies from a few months to several years. Embryo transfer may be performed in a natural or an artificially supplemented cycle. The frozen embryos may be donated, and transferred into a different woman. Embryos may be transferred to outside programmes at the patient's request or due to programme restructuring, thereby increasing variability in storage, thawing and transferring techniques. Some embryos are donated for research or discarded after cryopreservation, and their pregnancy potential is never realized. Changes in freezing protocols and embryo development stage further complicate evaluation of such cycles. Nonetheless, it is generally agreed that embryo cryopreservation can significantly enhance cycle fecundity.

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., 1998Go). It was reported that embryo survival rates decreased from 70.6% at 1 month to 52.6% by 6–15 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 6–15 months with extended cryopreservation. However, it was reported that cryopreservation under 6.5 years does not diminish embryo quality (Lin et al., 1995Go). They reported a pregnancy rate of 24–30% 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 frozen–thawed embryo transfer is 15–20%.

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., 1995Go). 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., 1998Go), and the results of other studies are encouraging (Lin et al., 1995Go; Sutcliffe et al.,1995). However, further studies and reports are needed before definitive conclusions can be drawn.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Go, K., Corson, S., Batzer, F. et al. (1998) Live birth from a zygote cryopreserved for 8 years. Hum. Reprod., 13, 2970–2971.[Abstract/Free Full Text]

Jones, H., Jones, D. and Kolm, P. (1997) Cryopreservation, a simplified method of evaluation. Hum. Reprod., 12, 548–553.[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, 645–651.[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, 262–267.[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, 3332–3337.[Abstract]

Testart, J., Lasalle, B., Belaisch-Allart, J. et al. (1986) High pregnancy rate after early human embryo freezing. Fertil. Steril., 46, 268–272.[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, 107–112.[ISI][Medline]

Trounson, A. and Mohr, L. (1983) Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature, 305, 707–709.[ISI][Medline]

Veeck, L., Adashi, E.Y., Rock, J.A. and Rosenwaks, Z. (1996) Reproductive Endocrinology, Surgery and Technology. Lippincott-Raven, Philadelphia, pp. 2353–2365.

Zeilmaker, G.H., Alberta, A.T., Van Gent, I. et al. (1984) Two pregnancies following transfer of intact frozen–thawed embryos. Fertil. Steril., 42, 293–296.pa[ISI][Medline]

Submitted on May 21, 1998; accepted on February 11, 1999.