Successful pregnancy following replacement of embryos previously refrozen at blastocyst stage: Case report

Murshid Farhat, Bat-Sheva Zentner, Francine Lossos, Yuval Bdolah, Hananel Holtzer and Arye Hurwitz,1

IVF Unit, Department of Obstetrics and Gynecology, Hadassah University Hospital, Mt Scopus, Jerusalem, Israel


    Abstract
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
We present the first reported clinical pregnancy following transfer of embryos that had been subjected to two freeze–thaw cycles: the first at day 3 after insemination, and the second after culturing to the blastocyst stage. A 25-year-old woman undergoing IVF treatment for male factor infertility opted for intracytoplasmic sperm injection (ICSI). ICSI treatment resulted in the successful production of 19 early cleavage embryos, all of which were frozen. After thawing, the embryos were cultured to the blastocyst stage. Thereafter, the blastocysts were refrozen and again thawed prior to embryo transfer. Embryos surviving a day 3 freeze–thaw cycle developed to the blastocyst stage and survived the second freeze–thaw cycle. Successful clinical pregnancy resulted following two sequential freeze–thaw cycles. This finding shows that it is possible to refreeze supernumerary blastocysts for subsequent transfer.

Key words: blastocyst/human/IVF/sequential freeze-thaw


    Introduction
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
The primary objective of embryo cryopreservation is to obtain the highest possible post-thaw survival rates, as well as in-vitro or in-vivo development rates that are comparable with those achieved with unfrozen, control embryos.

Extensive experience has been gained in cryopreservation at the early stages of embryo development, including the two pronuclear (2PN) stage and also at 48 and 72 h after insemination (Mandelbaum et al., 1998Go). By comparison, growing embryos to the blastocyst stage is a relatively new development, and consequently considerably less experience has been accumulated with this technique. Nevertheless, reports have been made of satisfactory results after freezing and thawing at the blastocyst stage (Kaufman et al., 1995Go). The potential to refreeze previously frozen samples of embryos would be useful should an ampoule be thawed that contained more embryos than were needed for transfer.

We describe what we believe to be the first human clinical pregnancy following two freeze–thaw cycles, the second being at the blastocyst stage.


    Case report
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 Abstract
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 Case report
 Discussion
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A 25-year-old healthy woman was admitted to our IVF unit because of male factor infertility. Her past medical history was unremarkable. She had regular menstrual cycles of 28 days length and had been trying to conceive for 3 years before her admission.

Infertility work-up revealed normal ovulation and a uterus didelphis for which she had undergone a vaginal septectomy. The spermogram was consistent with severe oligoteratoasthenozoospermia: 3 ml volume, 0.3–2x106/ml, 10% progressive motility and 15% normal morphology (World Health Organization criteria). The couple was scheduled for IVF/intracytoplasmic sperm injection (ICSI) treatment.

A single intramuscular dose of 3.75 mg D-Trp-6-LHRH gonadotrophin-releasing hormone agonist (GnRHa) (Decapeptyl® CR; Lapidot Pharmaceuticals, Herzlia, Israel) was injected on day 21 of the preceding cycle. After pituitary down-regulation was confirmed by measurement of serum 17-ß-oestradiol concentrations <200 pmol/ml, ovarian stimulation was initiated with 2 ampoules per day of human menopausal gonadotrophin (HMG) (Pergonal®; Teva Pharmaceutical, Petah Tikva, Israel) for 5 days. After the fifth day, the dose of HMG was tailored to the patient's response, based on transvaginal ultrasound and serum concentrations of oestradiol and progesterone. When six follicles were >=18 mm in diameter and the serum oestradiol concentration was 10 923 pmol/ml, 5000 IU of human chorionic gonadotrophin (HCG) (Chorigon®, Teva Pharmaceutical) were administered.

Transvaginal oocyte retrieval was performed under sonographic guidance 36 h after HCG administration. Semen and oocyte preparation, as well as oocyte fertilization and embryo assessment, were performed according to previously reported protocols for ICSI.

Twenty-seven oocytes were aspirated, but only 22 were mature (metaphase II; MII) and suitable for insemination by the ICSI procedure. During the first 72 h the embryos were cultured in 30 µl droplets of P-1® medium (Irvine Scientific, Santa Ana, CA, USA) enriched with 10% synthetic serum substitute (SSS; Irvine Scientific) under mineral oil (Sigma Chemical Co., St Louis, MO, USA). The culture dishes were incubated at 37°C under 5% CO2 in air, each 30 µl droplet containing three embryos. Embryos were grown to day 3, as embryo transfer is usually performed at 72 h. It was decided not to perform embryo transfer as the patient was complaining of abdominal discomfort and nausea, and concern was expressed about the possible development of ovarian hyperstimulation syndrome.

The 19 day-3 embryos that developed were cryopreserved, as planned, in six aliquots containing different numbers of embryos, depending on their grade. In the first two cycles the patient elected to undergo regular embryo transfer immediately after thawing. The endometrium was prepared by using micronized oestradiol (Estrofem®; Novo Industry A/S, Copenhagen, Denmark) 6 mg/day for 1 week from day 1 of the cycle, and adjusting the dose thereafter as previously described previously (Simon et al., 1999Go). Approximately 14 days after the first menstrual cycle, when the endometrium was >=9 mm in thickness, micronized progesterone (Uterogestan; Besins Iscovesco, Paris, France) was added vaginally at a dose of 900 mg/day. Day-3 embryos were transferred after 3 days of progesterone treatment. Since the patient did not conceive in those two attempts, she elected to have the next batch thawed (third thawing cycle) and for them to be grown to blastocyst stage for better embryo selection before embryo transfer.

Three early cleavage embryos were thawed and cultured in blastocyst medium (Irvine Scientific) to the blastocyst stage (further 96 h), using a previously published protocol (Milki et al., 1994). This resulted in two day-7 blastocysts, which were transferred. Two embryos developed (biamniotic monochorionic) but unfortunately the woman aborted spontaneously. A dilatation and curettage was performed.

For the fourth attempt the patient elected to undergo a regular embryo transfer. Three day-3 early cleavage embryos were transferred, but she did not conceive.

For the fifth attempt, the patient again elected for the embryos to be grown to the blastocyst stage. Two ampoules containing six early cleavage embryos were thawed, according to her request, and cultured (as previously described) to the blastocyst stage (further 48 h); this resulted in the development of four day-5 blastocysts. Due to the patient's age and preferences, only two blastocysts were transferred. This resulted in a clinical pregnancy that aborted spontaneously in the first trimester and necessitated dilatation and curettage. The remaining two blastocysts were cryopreserved for a second time, using a modification of a published protocol (Ménézo et al., 1992Go). At the sixth attempt, the two refrozen blastocysts were thawed and transferred 4h after thawing; this resulted in a singleton pregnancy. The patient had a normal ultrasonography at 20 weeks gestation, but declined our recommendation to perform amniocentesis due to the unique circumstances of refrozen embryos. The patient delivered a healthy female at 37 weeks gestation.

Details of the six thawing cycles are summarized in Table IGo.


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Table I. Six thawing cycles: cryodamage and embryo transfer (ET) outcome
 

    Discussion
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
Only limited data are available regarding the effects of multiple freeze–thaw cycles on mammalian embryos. It has been reported (Leibo et al., 1991Go) that live young were produced from 2-cell mouse embryos subjected to two successive cycles of freezing, while others (Vitale et al., 1997Go) have detailed results showing that mouse embryos can survive even three successive freeze–thaw cycles yet still be capable of in-vitro development.

In humans, three cases of successful pregnancies after transfer of human embryos that were frozen twice before transfer have been reported. In one case (Macnamee et al., 1990Go), four 2PN embryos were frozen, thawed and allowed to cleave. The embryos were then refrozen, with two of the four surviving after the second thaw. Those two embryos were transferred and resulted in the birth of a male baby. Another case has been reported (Mowat et al., 1994Go) in which three cleaved embryos underwent the cryopreservation–thaw process twice, were transferred, and resulted in a baby girl. A report has also been made of a successful pregnancy following a second freeze at the pronuclear stage, with only 4 h of growth in culture after the first thaw (Baker et al., 1996Go).

The present report describes for the first time a clinical pregnancy after two freeze–thaw cycles involving early (day-3) stage embryos and blastocysts. The ability to refreeze embryos without endangering their developmental potential is a significant finding, with many possible ramifications.

In this case, all 19 embryos were frozen at 72 h (day 3), with 17 out of 19 reaching the 6- to 8-cell cleavage stage. Most of the embryos had <20% fragmentation. After thawing, 13 of the 19 embryos remained intact without any cryodamage. Overall, embryo survival was 95%, while blastomere survival reached 90%, indicating that this cohort of 19 embryos withstood cryopreservation well.

As a routine, we recommend freezing two to three average quality human embryos in each ampoule. In the case described, the couple—after four unsuccessful thawing cycles—requested that two ampoules be thawed simultaneously and the embryos further grown to the blastocyst stage to enable better embryo selection. With medium quality embryos the expected average blastocyst formation rate is ~30%; hence we agreed to thaw six embryos at the same time. The four blastocysts that developed were all of good quality, with distinct blastocoelic cavities, and differentiating outer trophectoderms and inner-cell-mass cells (grades 4–5) (Racowsky et al., 2000Go).

As we almost never transfer more than two embryos to young patients (age <35 years), we were left with two surplus blastocysts, which were refrozen (some laboratories would discard these). Ideally, one should not have surplus thawed early embryos or blastocysts, though this occasionally occurs.

Provided that the thawed early embryos are kept in culture long enough to repair any cryodamage, they are capable of withstanding another freeze–thaw cycle. This report shows that surplus blastocysts need not necessarily be discarded, as refreezing is possible without impairing their developmental capacity. Nevertheless, it should be borne in mind that additional data relating to the favourable outcome of fetuses born after such procedures is needed before such a procedure is recommended routinely.


    Notes
 
1 To whom correspondence should be addressed at: Hadassah University Hospital Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel. E-mail: Hurwitz{at}cc.huji.ac.il Back


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 Abstract
 Introduction
 Case report
 Discussion
 References
 
Baker, A., Check, J.H., Hourani, C. and Hoover, L.M. (1996) Pregnancy achieved with pronuclear-stage embryos that were cryopreserved and thawed twice: a case report. J. Assist. Reprod. Genet., 13, 713–715.[ISI][Medline]

Kaufman, R.A., Ménézo, Y., Hazout, A. et al. (1995) Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil. Steril., 64, 1125–1129.[ISI][Medline]

Leibo, S.P., Christian, C.R. and Malinak, L.R. (1991) In vitro and in vivo survival of twice-frozen mouse embryos. Cryobiology, 28, 534 (abstract).

Macnamee, M., Wick, K., Rainsbury, P. and Sathnadan, M. (1990) How robust are early embryos? Lancet, 336, 636.

Mandelbaum, J., Belaisch Allart, J., Junca, A.M. et al. (1998) Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum. Reprod., 13 (Suppl. 3), 161–174.[Abstract]

Ménézo, Y., Nicollet, B., Herbaut, N. and Andre, D. (1992) Freezing cocultured human blastocysts. Fertil. Steril., 58, 977–980.[ISI][Medline]

Milki, A.A., Fisch, J.D. and Behr, B. (1999) Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer. Fertil. Steril., 72, 225–228.[ISI][Medline]

Mowat, L.G., Jamieson, M.E., Ross, K.S. et al. (1994) Successful pregnancy after replacement of embryos which had been frozen and thawed at both pronuclear and cleavage stage. Abstracts of the 2nd International Meeting of the FBS, Glasgow. Hum. Reprod., 9, 22.[ISI]

Racowsky, C., Jackson, K.V. and Ceklleniak, N.A. (2000) The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. Fertil. Steril., 73, 558–564.[ISI][Medline]

Simon, A., Hurwitz, A., Pharhat, M. et al. (1999) A flexible protocol for artificial preparation of the endometrium without prior gonadotropin-releasing hormone agonist suppression in women with functioning ovaries undergoing frozen-thawed embryo transfer cycles. Fertil. Steril., 71, 609–613.[ISI][Medline]

Vitale, N.J., Myers, M.W., Denniston, R.S. et al. (1997) In-vitro development of refrozen mouse embryos. Hum. Reprod., 12, 310–316.[Abstract]

Submitted on June 14, 2000; accepted on October 20, 2000.