At What Stage Can Embryos be Frozen?
Embryos may be cryopreserved at the pronuclear, cleavage, and blastocyst stage of development. The pronuclear stage (PN) is where the egg has just fertilized and the two pronuclei are seen in the cell, representing chromosomes from the mother and the father. The cleavage stage is when the embryo is actively dividing from day 2-3, and the blastocyst stage is when the embryo is ready to implant with the future placenta and embryonic pole clearly seen. In vitro growth will demonstrate embryo developmental competency. ART programs may culture embryos from 2-6 days to determine which embryos to cryopreserve. The embryonic genome is activated after the 6-8 cell stage, so extended culture will identify embryonic arrest and aid identification of healthy embryos for cryopreservation, though not all embryos that grow will be chromosomally normal.
The advantages of 2PN (i.e. pronuclear) stage cryopreservation include: 1. lack of a delicate meiotic spindle; 2, less organized nuclear material; and 3, nuclear membrane intact (1). Pronuclear freezing may be indicated in OHSS, fluid found in the uterine cavity on the day of HCG administration, or patients that develop hydrosalpinges during the stimulation where implantation may be compromised. The cumulative pregnancy rate will be increased by the addition of cryopreservation. Marrs et al (2) reported on 1408 pronuclear embryos frozen in propanediol/Sucrose. 78% survived the thaw leading to a 26% pregnancy rate per transfer in 181 cycles. ICSI cycles had 72% survival compared to 81 % survival in cycles where the oocytes were inseminated. They postulated that the zona damage caused by ICSI may be responsible for the decrease. However, Hoover et al (3) found no difference in outcomes with inseminated oocytes versus ICSI oocytes with respect to pregnancy rates. This was a smaller study with only 205 embryos reported, which may have affected the outcome. Al-Hasani et al (4) found similar results as Hoover et al (3) in a retrospective review of nearly 2500 frozen pronuclear zygotes.
Kattera et al (5) compared PN to cleavage stage embryos with respect to pregnancy rates after cryopreservation (22.8 % with cleavage and 14. % with PN embryos). Embryo morphology was the same. It was thought that the ‘best’ embryos were determined by culturing them out. In cleavage stage cryopreservation, blastomere loss is important. Edgar et al, (6) showed a 30% decrease in implantation potential when limited blastomere loss was detected. They also showed (7) that the pre-freeze growth rate correlated with implantation. 4 cell embryos on day 2 do better than 4 cell embryos on day 3. Additionally, the degree of fragmentation and distribution had a negative effect (8). Many programs will cryopreserve excess embryos at the cleavage stage in order to enhance overall outcome from ART. Because of inconsistent results with blastocyst cryopreservation, many prefer cleavage stage cryopreservation of high quality embryos.
It is logical to determine the fullest potential of embryonic growth by waiting to freeze only blastocysts. The nuclear:cytoplasmic ratio is high and more cells are present which would seem to favor freezing at this stage (1). Some of the variability in outcomes appears to be due to effects of the different culture media (i.e. sequential, co-culture), gas ratios (%CO2), osmolality and type of cryoprotectant (1). Vitrification, using very high concentrations of cryoprotectants, simplified protocols and cryoloops in open systems, may ultimately prove to be widely accepted as a means to freeze blastocysts as the data appear promising (9). Vitrification is not widely used, however.
In summary, there is no doubt that embryo cryopreservation has benefits and that the exact time to freeze embryos depends upon the specific needs of the patient and the clinic treating them.
1. Menezo Y. Cryopreservation of IVF embryo: which stage? Eur. J. Obststr. Gynecol. Rep. Med.2003.113S:S28-S32.
2. Marrs RP, Greene J, Stone BA. Potential factors affecting embryo survival and clinical outcome with cryopreserved pronuclear human embryos. 2004 AJOG. 190:1766-72.
3. Hoover L, Baker A, Check JH, Lurie D, Summers D. Clinical outcome of cryopreserved human pronuclear stage embryos resulting from intracytoplasmic sperm injection. 1997 Fertil. Steril. 67(4):621-624.
4. Al-Hasani S, Ludwig M, Gagsteiger F, Kupker W, Sturm R, Yilmaz A, Bauer O, Dedrich K. Comparison of cryopreservation of supernumary pronuclear human oocytes obtained after intracytoplasmic sperm injection (ICSI) and after conventional in-vitro fertilization. 1996 Hum. Rep. 11:604-607.
6. Edgar DH. Archer J, Gook DA, Jericho H, Wilton L, Bourne H. Survival and developmental potential of stored human early cleavage stage embryos. 2004. Eur. J. Obstetr. Gynecol. Rep. Med. 115S:S8-S11.
7. Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. 2000. Hum. Rep. 15(1):179-1798.
8. Antzak M, Van Blerkom J. Temporal and spatial aspects of fragmentation in the early embryo: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. 1999 Hum. Rep. 14:429-47.
9. Liebermann J. Recent developments in human oocyte, embryo and
blastocyst vitrification: where are we now? 2003 RBM On-line.