去除囊胚blastocoele以進行玻璃化冷凍

囊胚期後期期形成之blastocoele可能對於玻璃化冷凍有不良影響
以29#針頭從3點極9點鐘推擠blastocoele使其排出後在置於抗凍液以進行下一步玻璃化冷凍

解凍後植入懷孕率48%，著床率29%

http://humrep.oxfordjournals.org/content/18/1/137.long

Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele

1. Weon‐Young Son1, 
2. San‐Hyun Yoon, 
3. Hye‐Jin Yoon, 
4. Sang‐Min Lee and
5. Jin‐Ho Lim

+Author Affiliations

1. Maria Infertility Hospital, Seoul, Korea ¹To whom correspondence should be addressed at: In Vitro Fertilization Laboratory, Maria Infertility Hospital, 103‐11, Sinseol‐dong, Dongdaemun‐gu, Seoul, Korea. e‐mail: sonyoung@mariababy.com

 

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Abstract

BACKGROUND: The purpose of this study was to examine the effects on blastocyst survival and subsequent pregnancy rate of ‘artificial shrinkage’ (i.e. induced collapse of the blastocoele) before vitrification of human blastocysts. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were cryopreserved. Before vitrification on electron microscopy (EM) grids, artificial shrinkage was induced in expanded blastocysts using a 29‐gauge needle. After thawing, transfers were performed on 25 couples. Post‐thaw survival rates and clinical outcome after the transfer of vitrified blastocysts were examined. RESULTS: Of 90 expanded blastocysts vitrified from 25 patients, 81 survived (90.0%) and 40 of them were hatched (49.4%) at the time of transfer. The implantation rate was 29.0% (20/69), and the pregnancy rate was 48.0% (12/25). Nine patients delivered 15 infants, two pregnancies are ongoing and one ended in miscarriage. CONCLUSIONS: The results suggest that artificial shrinkage is a useful technique for vitrification of expanded blastocysts on EM grids.

Artificial shrinkage of expanded blastocysts and vitrification

Artificial shrinkage of expanded blastocysts was performed with two 29‐gauge needles. After holding the expanded blastocyst with the flat side of a needle and placing the inner cell mass (ICM) at the 12 or 6 o’clock position, a needle was pushed trough the trophectoderm cell into the blastocoele cavity until it shrank (Figure 1). Contraction of the blastocysts was then observed after 30 s to 1 min. After complete shrinkage of the blastocoele, the blastocysts were equilibrated in EG20 for 1.5 min before exposure to the vitrification solution. The blastocysts were then incubated in EFS40, loaded onto the EM grid (IGC 400; Pelco International, CA, USA) and directly plunged in liquid nitrogen within ∼30 s. The EM grids containing the blastocysts were sealed in a cryovial that had previously been submerged under liquid nitrogen. The cryovials were attached in canes and stored in liquid nitrogen.

Figure 1. Freezing and warming of human expanded blastocysts on electron microscopy (EM) grids after artificial shrinkage. Human expanded blastocysts (a) before artificial shrinkage, (b) during artificial shrinkage, (c) after artificial shrinkage, (d) loaded onto EM grids, and (e) hatched blastocysts 18 h after thawing. Original magnification ×100.

Table I.

Results of human blastocyst vitrification after artificial shrinkage

Variables	Value
No. of patients (no. of treatment cycles)	25 (25)
No. of expanded blastocysts vitrified	90
No. of blastocysts survived (%)	81 (90.0)
No. of blastocysts hatched at ET (%)	40 (49.4)
Average no. of blastocysts transferred	2.8
Implantation rate (%)	20/69 (29.0)
Pregnancy rate (%)	12/25 (48.0)