1 Center for Assisted Reproduction, Bedford, Texas, 76022 and 2 Colorado Center for Reproductive Medicine, Englewood, Colorado, 80110, USA
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Abstract |
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Key words: embryo culture/embryo development/implantation/pregnancy/viability
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Introduction |
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It is widely held that embryonic cleavage rate during the first 72 h following insemination is an important indicator of embryo quality and suggests that embryos with a higher cleavage rate have an increased implantation rate (Shoukir et al., 1998). Therefore, many programmes attempting blastocyst transfer elect to culture embryos with the highest cell number beyond day 3 (Milki et al., 1999
). Conversely, when faced with either a low number of fertilized oocytes or few rapidly-dividing embryos the decision is made to transfer at the cleavage stage on day 3.
In our programme prior to April 1998, cleavage stage embryo transfers were routinely performed on day 3. After March 1998, routine blastocyst transfers were completed on day 5 or day 6. The change from day 3 to day 5 transfers included all patients irrespective of age, aetiology, number of follicles, and the number of fertilized oocytes or number of cleaving embryos. Therefore, it was possible to determine the relationship between blastocyst formation and embryo cell stage on day 3, irrespective of age. In addition, the viability of blastocysts derived from embryos of different cell number on day 3 was determined. Further, the application of extended culture to poor prognosis patients with slow cleaving embryos and/or low zygote number was evaluated. The results of extended culture and blastocyst transfer in oocyte donor cases were considered and finally the ability to cryopreserve blastocysts analysed.
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Materials and methods |
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Following retrieval, oocytes were placed into human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) + 12% synthetic serum substitute (SSS; Irvine Scientific) and cultured for ~4 h. Embryo culture was performed in a humidified incubator with 5% CO2 and 95% air at 37°C. For traditional IVF, a short 2 h co-incubation of spermatozoa (0.2x106/ml) with oocytes was initiated 38 to 42 h post HCG (Gianaroli et al., 1996). After shortened co-incubation with spermatozoa, oocytes were rinsed through fresh media and moved to 100 µl micro-drops of S1/G1.2 media under oil (Squibb, Princeton, NJ, USA). Sperm injection was performed on metaphase II oocytes at 4042 h post HCG administration following hyaluronidase treatment using previously described techniques (Van Steirteghem et al., 1992
). Fertilization was evaluated 1820 h after IVF or intracytoplasmic sperm injection (ICSI).
Beginning in April 1998, extended embryo culture to day 5 in serum-free sequential media was performed in all patients undergoing IVF. From April 1998 to February 1999 embryos were cultured for 48 h in S1 (IVF Science) medium followed by 48 to 72 h culture in S2 (IVF Science, Vero Beach, FL, USA) to day 5 or day 6. In February 1999 a media change was made and G1.2 (IVF Science) and G2.2 (IVF Science) replaced S1 and S2 respectively. Fertilized oocytes were cultured in Petri dishes containing 100 µl micro-drops of S1/G1.2 medium under oil, two embryos per drop, for 48 h. On day 3, cleavage stage embryos were transferred to fresh 100 µl micro-drops of S2/G2.2 medium under oil and cultured, two embryos per drop, for an additional 48 to 72 h to day 5 or day 6. Blastocysts remaining after transfer were frozen using modified protocols (Ménézo et al., 1992). These modifications included cryopreservation of blastocyst embryos in glycerol using a two-step freeze and thaw technique.
Only good or excellent quality blastocyst embryos were frozen. Blastocyst embryos exhibiting an expanded or expanding blastocele cavity with a clearly defined inner cell mass and a trophectoderm consisting of many cells forming a cohesive epithelium were frozen. According to a previously published scoring system (Gardner and Schoocraft, 1999) all cryopreserved blastocysts were graded as 3BB or better. Blastocyst embryos for freezing were exposed to 5% glycerol (Sigma) solution of phosphate buffered saline (PBS; Irvine Scientific) and human serum albumin (HSA; Irvine Scientific) for 10 min then transferred to 10% glycerol containing 0.2 mol/l sucrose (Sigma, St Louis, MO, USA) for 2 min. Blastocysts were loaded, one embryo to each 1.2 ml Nunc vial (Nalge Nunc International) containing 300 µl 10% glycerol/0.2 mol/l sucrose solution and placed in a Bio-cool III methanol bath freezer (FTS Systems, Stone Ridge, NY, USA). Temperature was reduced at a controlled rate dropping 2.0°C/min from 20 °C to 7.0°C. Temperature was held at 7.0°C for 20 min for seeding. Following seeding, methanol temperature was further reduced at a rate of 0.3°C/min to 37°C. Blastocysts were then plunged into liquid nitrogen for storage.
Cryopreserved blastocysts were thawed by removing each vial from the liquid nitrogen. Thawing immediately ensued by placing the vial on a countertop for 1020 s, only long enough to break the seal of the vial. After the seal was broken, each vial was thawed for 3 min in a 30°C water bath. Blastocysts were recovered from each vial and placed in 10% glycerol/0.2 mol/l sucrose solution for 1020 s and then moved to 5% glycerol/0.2 mol/l sucrose solution for 3 min. Blastocysts were then transferred to 0.2 mol/l sucrose for 2 min and then moved to PBS/HSA solution for 1 min. Thawed blastocysts were then transferred to S2/G2.2 culture media and analysed for survival, embryo stage and quality. Prior to embryo transfer, ~24 h after thawing, frozen embryos were again evaluated for survival, embryo stage and quality.
An abdominal ultrasound (5 MHz) was utilized for all transfers to assist intrauterine placement of the embryo transfer catheter (Edwards-Wallace catheter; Marlow Technologies, Willoughby, OH, USA) (Gardner et al., 1998b). Patients were advised to transfer two or three blastocysts depending only on patient age and/or embryo quality. Donor oocyte recipients were advised to transfer no more than two blastocyst stage embryos. Criteria for the number of frozen blastocysts transferred were the same for fresh embryo transfer. Intramuscular luteal support with 50 mg progesterone (PCCA, Houston, TX, USA) in oil was initiated following retrieval. Ongoing pregnancies were confirmed with the use of an ultrasound to detect the presence of an intrauterine sac with fetal cardiac activity 3034 days after oocyte retrieval.
Statistical analysis
Statistical analysis was performed with Fisher's exact test or 2 to determine significant differences between day 3 versus day 5/6 ongoing pregnancy rates, implantation rates for frozen cycles. Statistical analysis was performed with Fisher's exact test or
2 to determine significant differences between embryo transfer results for day 5/6 age specific and donor group pregnancy rates, ongoing pregnancy rates, and implantation rates for fresh cycles. Analysis of variance followed by the Bonferroni procedure for multiple comparisons was performed on fresh cycles including non-donor and donor cycles to compare numbers of oocytes, pronucleate embryos and embryos transferred and cryopreserved. Independent t-tests were used to compare the mean number of frozen embryos transferred on day 3 versus day 5/6. Linear regression test was performed to determine the relationship between maternal age and blastocyst development. Values are mean ± SEM.
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Results |
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Discussion |
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It is evident from this study that a proportional relationship exists between number of blastomeres present in day 3 embryos and blastocyst formation. Blastocyst production from day 3 cleavage stage embryos was linear with a correlation coefficient approaching 1.00 in both IVF patients and oocyte donors (r = 0.96). It appears that blastocyst production from embryos containing at least eight cells begins to plateau around 80% for non-donor cases and 90% for donor cases. These data are in agreement with one previously published study (Shapiro et al., 2000) which found that the more developed embryos at 72 h were more likely to become blastocysts, but in disagreement with another (Racowsky et al., 2000
). In the report by Racowsky et al. (2000) it was proposed that patients who did not have at least one 8-cell embryo on day 3 should not have blastocyst culture. The data presented here are in contrast with this proposal and suggest that, although higher blastulation rates were observed in embryos that had progressed to at least eight cells by 72 h post insemination, a significant number of embryos with initially retarded development were able to progress to the blastocyst stage by 120 to 144 h post insemination. Further, when evaluating the implantation and pregnancy rates over the same time period when blastocyst culture is applied to poor prognosis patients with slow cleaving embryos (less than eight cells) and/or low zygote number (less than eight 2PN) acceptable rates are obtained. Although Racowksy et al. (2000) used a sequential culture system similar to that of the current study, it was not identical. Racowsky et al. (2000) used a simple medium for the first 48 h of culture (IVF50), as opposed to one containing amino acids (G1.2), which may have contributed to their less than optimum results in their poor prognosis patient group (Gardner and Lane, 1997
). In addition, it is plausible that the differences reported in blastocyst development between the programmes are due simply to other laboratory or clinic related factors.
A decrease in the average number of embryos available for extended culture was observed with increasing maternal age. The average number of embryos available for extended culture was 7.9, 6.7, and 6.1 for patients in the age categories <35, 3539, and >39 respectively (Table V). A significant linear trend of decreasing blastocyst development with increasing maternal age was observed. Such a decrease in a general population of IVF patients has previously been reported (Janny and Ménézo, 1996
) using co-culture to support embryo development. Increasing maternal age was also associated with a decrease in implantation rate, a phenomenon previously reported (Schoolcraft et al., 1999
). This is consistent with an increasing incidence of aneuploidies with advancing maternal age (Munne et al., 1995
).
Blastocyst development was not only significantly higher in the oocyte donor patients, but resulting implantation rates were also higher. As such these data indicate that oocytes from donors have a higher developmental potential both in culture and post transfer. This has been reported previously (Patton et al., 1999; Schoolcraft and Gardner, 2000
) and confirms that blastocyst culture is of great benefit when using oocyte donation. This is of significance given the potential problems that older patients would face conceiving a high order multiple gestation.
Importantly, it was shown that blastocysts cultured in commercially available sequential media could be cryopreserved and thawed successfully, resulting in implantation rates similar to those previously reported for co-cultured embryos (Kaufman et al., 1995). The ability to adequately cryopreserve human blastocysts has been proposed to be a weak component of a blastocyst transfer cycle. However, it is evident that this is not the case. With the advent of new vitrification procedures (Lane et al., 1999
) it is envisaged that the success of blastocyst cryopreservation may be further increased. Furthermore, these data show that the transfer of frozen-thawed blastocysts gave rise to significant increases in embryo survival, implantation and pregnancy rates compared to frozen-thawed embryos transferred on day 3.
In conclusion, this report demonstrates the ability of cleavage stage embryos of different cell number to develop to the blastocyst stage by day 5 or day 6, and highlights the difficulty in selecting embryos for transfer at the cleavage stage. Most importantly, these data suggest that blastocyst transfer can generate very acceptable pregnancy and implantation rates for poor prognosis patients and especially in good prognosis patients and donor oocyte IVF cycles. With the ability to identify the most viable blastocysts within a cohort (Gardner et al., 2000) it should be possible to perform single blastocyst transfers for many patients. Although the current analysis suggests many advantages of blastocyst transfer over multi-cell embryo transfer, prospective randomized clinical trials may further define the benefit and role of extended embryo culture in human assisted reproduction techniques.
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Acknowledgements |
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Notes |
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References |
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Gardner, D.K. and Lane, M. (1997) Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum. Reprod. Update, 3, 367382.
Gardner, D.K. and Schoolcraft, W.B. (1999) In-vitro culture of human blastocysts. In Jansen, R. and Mortimer, D. (eds), Towards Reproductive Certainty: Fertility and Genetics Beyond. Parthenon Press, Carnforth, pp. 378388
Gardner, D.K., Vella, P., Lane, M. et al. (1998a) Culture and transfer of human blastocyst increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril., 69, 8488.[ISI][Medline]
Gardner, D.K., Schoolcraft, W.B., Wagley, L. et al. (1998b) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod., 13, 34343440.[Abstract]
Gardner, D.K., Lane, M., Stevens, J. et al. (2000) Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil. Steril., 73, 11551158.[ISI][Medline]
Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al. (1996) Reducing the time of sperm-oocyte interaction in the human in-vitro fertilization improves the implantation rate. Hum. Reprod., 11, 166171.[Abstract]
Janny, L. and Ménézo, Y.J.R. (1996) Maternal age effect on early human embryonic development and blastocyst formation. Molec. Reprod. Dev., 45, 3137.[ISI][Medline]
Jones, G.M., Trounson, A.O., Lolatgis, N. and Wood, C. (1998) Factors affecting the success of human blastocyst development and pregnancy following in vitro fertilization and embryo transfer. Fertil. Steril., 70, 10221029.[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, 11251129.[ISI][Medline]
Lane, M., Schoolcraft, W.B. and Gardner, D.K. (1999) Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil. Steril., 72, 10731078.[ISI][Medline]
Marek, D., Langley, M., Gardner, D.K. et al. (1999) Introduction of blastocyst culture and transfer for all patients in an IVF program. Fertil. Steril., 72, 10351040.[ISI][Medline]
Ménézo, Y., Nicollet, B., Herbaut, N. and Andre, D. (1992) Freezing cocultured human blastocysts. Fertil. Steril., 58, 977980.[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, 225228.[ISI][Medline]
Munné, S., Alikani, M., Tomkin, G. et al. (1995) Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil. Steril., 64, 382391.[ISI][Medline]
Patton, P.E., Sadler-Fredd, K., Burry, K.A. et al. (1999) Development and integration of an extended embryo culture program. Fertil. Steril., 72, 418422.[ISI][Medline]
Racowsky, C., Jackson, K.J., Cekleniak, N.A. et al. (2000) The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. Fertil. Steril., 73, 558564.[ISI][Medline]
Schoolcraft, W.B. and Gardner, D.K. (2000) Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil. Steril., 74, 482486.[ISI][Medline]
Schoolcraft, W.B., Gardner, D.K., Lane, M. et al. (1999) Blastocyst culture and transfer: Analysis of results and parameters affecting outcome in two IVF programs. Fertil. Steril., 72, 604609.[ISI][Medline]
Shapiro, B.S., Harris, D.C. and Richter, K.S. (2000) Predictive value of 72-hour blastomere cell number on blastocyst development and success of subsequent transfer based on the degree of blastocysts development. Fertil. Steril., 73, 582586.[ISI][Medline]
Shoukir, Y., Chardonnens, D., Campana, A. et al. (1998) The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Hum. Reprod., 13, 676681.[Abstract]
Van Steirteghem, A.C., Liu, J., Joris, H. et al. (1992) Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. A report of a second series of 300 consecutive treatment cycles. Hum. Reprod., 8, 10551060.[Abstract]
Ziegler, D., Lu, J., Cedars, M. et al. (1987) Suppression of the ovary using a gonadotropin releasing-hormone agonist prior to stimulation for oocyte retrieval. Fertil. Steril., 48, 807810.[ISI][Medline]
Submitted on September 29, 2000; accepted on January 18, 2001.