Evidence-based ET: Does the inclusion of sucrose in EG freezing medium improve embryo survival and pregnancy rates?

Categories: Evidence-Based ET
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Published on: June 17, 2014

Evidence-based ET

John F. Hasler

Does the inclusion of sucrose in EG freezing medium improve embryo survival and pregnancy rates?

As pointed out previously in this column, efficacious cryopreservation of bovine embryos is critical to the commercial ET industry because, as has been the case for some years now, more than 70% of embryos collected in the US are frozen, while fewer than 30% are transferred fresh. Following the published report of Voelkel and Hu in 1992 on cryopreservation with EG, the commercial bovine ET industry rather quickly switched from glycerol to EG as the major cryoprotectant in freezing media. The overall percentage of embryos frozen in EG rose rapidly starting in 1992 and reached 97% in 2008, the last year that the AETA collected data on this specific statistic.

Several companies provide 1.5 M EG freezing media with or without the inclusion of 0.1 M sucrose. The question posed here is whether including sucrose in the freezing medium makes any difference. In the normal range of ambient temperatures under which most embryos are placed in freezing media and loaded into straws, sucrose does not enter the cells.  Although sucrose readily diffuses through the zona, it remains outside the cells and, at the relatively low concentration of 0.1 M, exerts a mild osmotic imbalance, thereby pulling some water out of the cells. Whether this improves embryo survival is in question and may, in fact, be somewhat related to embryo stage of development.

Dochi et al. (1995) compared the pregnancy rates following transfer of small numbers of bovine in vivo-derived embryos frozen in 1.8 M EG with or without 0.25 M sucrose. There was not a significant difference in the pregnancy rates, although the EG (n=29) pregnancy rate was numerically higher than the EG + sucrose (n=25) pregnancy rate.

In a study involving the freezing of IVF-derived embryos in EG and their subsequent thawing and in vitro culture, I failed to find any difference in survival of embryos frozen with or without 0.25 M sucrose, as shown in Table 1 (Hasler, et al., 1997).

Table 1 – Post-thaw survival of IVF-derived bovine blastocysts frozen in 1.5 M EG with or without 0.25 M sucrose

Cryoprotectant No. thawed Blastocysts after 24 h culture
%
EG (1.5 M) 385 301 78.2
EG (1.5 M) + sucrose (0.25 M) 378 280 74.1

 

In their survey of ET practitioners, presented at the AETA convention in 1998, Leibo and Mapletoft reported virtually equal pregnancy rates for EG with or without sucrose included (Table 2).

 

Table 2 – Effect of EG with or without sucrose on pregnancy rates

No. embryos transferred No. clinics % pregnant
AETA
    EG 5,056 24 55.6
    EG + sucrose                  10,377 14 55.2
CETA
    EG 7,273 24 59.0
    EG + sucrose 4,653 13 58.8

 

Martinez, et al. (2002) reported a higher pregnancy rate following transfer of in vivo-derived bovine embryos frozen in 1.5 M EG containing 0.1 M sucrose compared to EG with 0 or 0.3 M sucrose (Table 3).

 

Table 3 – Pregnancy rates following transfer of in vivo-derived bovine embryos frozen in 1.5 M EG with 0, 0.1 or 0.3 M sucrose

EG 1.5 M No. thawed and transferred No. pregnant % pregnant
+ 0 M sucrose 108 32 29.6a
+ 0.1 M sucrose 108 52 48.1b
+ 0.3 M sucrose 107 30 28.0a

a,bValues with different letter in same column are significantly different (P<0.05)

 

The most recently published study that I found on this subject involved the freezing of IVF-derived bovine embryos in 1.5 M sucrose with or without either 0.1 M sucrose or 0.1 M xylose (Lim, et al., 2008). Sucrose is commonly known as table or cane sugar and is a disaccharide composed of the monosaccharides glucose and fructose, with the molecular formula C12H22O11 and   MW 342. Xylose is a sugar first isolated from wood and is derived from hemicellulose. It is classified as a monosaccharide of the aldopentose type, which means that it contains five carbon atoms, with the molecular formula C5H10O5 and MW 150.  At a concentration of 0.1M, each of these sugars exerts an equal, mild dehydration influence on embryo cells. However, as shown in Table 4, embryos frozen with sucrose had a higher survival rate than those frozen with xylose or without any sugar.

 

Table 4 – Influence of adding 0.1 M sucrose or xylose to 1.5 M EG on the survival of frozen-thawed bovine IVF-derived embryos

Treatment No. embryos thawed No. (%) embryos surviving after
    24 h 48 h
0.1 M sucrose 94 67 (71.3)a 48 (51.1)
0.1 M xylose 86 46 (53.5)b 39 (45.3)
None 87 45 (51.7)b 35 (40.2)

a,b Values with different letter in the same column are significantly different (P<0.05)

 

Obviously, the published accounts described above do not clearly answer the question first poised. Although I did not detect a difference in the survival of embryos frozen with or without 0.25 M sucrose, the re-expansion of the blastocysts in this study is not conclusive proof that they survived with viability adequate to establish pregnancies. The culture system in this study was a co-culture system composed of Menezo’s B-2 medium with Buffalo Rat liver cells. This system is known to robustly support trophoblastic cell division and it is not known if the re-expanded blastocysts retained equally viable inner cell masses.

The Martinez, et al. (2002) study provided evidence that 0.1 M sucrose was better than either 0 or 0.3 sucrose. Unfortunately, the census reported by Leibo and Mapletoft (1998) did not record whether there were any differences in the concentration of sucrose used by practitioners. However, there is not any hint of a difference in the pregnancy rates reported by the two groups.  Lastly, the Lim, et al. (2008) study supports the use of 0.1 M sucrose.

I think that the take home message is that there is no convincing evidence for a benefit from including a low level of sucrose in EG freezing medium, but there also is no evidence that it does any harm. About 70% of the EG sold by Bioniche-Vetoquinol contains 0.1 M sucrose. This is apparently just a reflection of personal preference and is not based on the available published evidence.  I would feel comfortable continuing to freeze bovine embryos in EG either with or without sucrose.

 

References

Dochi, O., Imai, K. and Takakura, H. 1995. Birth of calves after direct transfer of thawed bovine embryos stored frozen in ethylene glycol. Anim. Reprod. Sci. 38:179-185.

Hasler, J.F., Hurtgen, P.J., Jin, Z.Q. and Stokes, J.E. 1997. Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol. Theriogenology 48:563-579.

Leibo, S.P. and Mapletoft, R.J. 1998. Effect of transfer of cryopreserved cattle embryos in North America. Proceedings of the 17 Annual AETA Convention, pp. 91-98

Lim, K.T., Jang, G., Ko, K.H., Lee, W.W., et al. 2008. Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium. Anim. Reprod. Sci. 103:239-248.

Martínez, A.G., Brogliatti, G.M., Valcarcel, M.A. de las Heras. 2002. Pregnancy rates after transfer of frozen bovine embryos: a field trial. Theriogenology 58:963-972.

Voelkel, S.A. and Hu, Y.X. 1992. Direct transfer of frozen-thawed bovine embryos. Theriogenology 37:23-37.

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