使用140um pipette反覆抽吸囊胚導致囊胚之blastocoele破裂排出後再玻璃化冷凍
解凍後培養3h以達re-expansion
3-5h後植入
解凍後存活率96%
植入懷孕率50%, 著床率33%
缺點: pipetteing 時間過久，需4-10min
http://humrep.oxfordjournals.org/content/19/12/2884.long
Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts
+Author Affiliations
- 1To whom correspondence should be addressed. Email: hiraoka@chive.ocn.ne.jp
- Received April 26, 2004.
- Accepted August 12, 2004.
Abstract
BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.
Figure 2.
Cryopreserving and warming of human expanded blastocysts after artificial shrinkage. Human expanded blastocysts (a) before artificial shrinkage, (b) during artificial shrinkage by pipetting with a glass pipette ∼140 μm in diameter, (c) during artificial shrinkage by pipetting with a glass pipette ∼120 μm in diameter, (d) after artificial shrinkage, (e) loaded onto cryotop with cryoprotectant solution, and (f) 3 h after warming. Scale bars (a, b, c, d and f)= 100 μm. Scale bar (e)=700 μm.
Table I.
Results of human expanded blastocyst vitrification on day 5 or day 6 after artificial shrinkage
| Parameter | Age of blastocyst | ||
|---|---|---|---|
| Day 5 | Day 6 | Total | |
| Mean age of patients (years) | 34.8±4.1 | 33.9±4.2 | 34.3±4.1 |
| Range of age of patients (years) | 29–41 | 27–45 | 27–45 |
| No. of cycles for vitrification | 13 | 15 | 28 |
| No. of cycles for warming | 13 | 16 | 29 |
| No. of expanded blastocysts vitrified | 24 | 25 | 49 |
| Mean time (min) of artificial shrinkage | 7.4±4.5 | 8.9±4.2 | 8.2±4.4 |
| Range of time (min) of artificial shrinkage | 3–18 | 3–18 | 3–18 |
| No. of blastocysts survived (%) | 24 (100) | 24 (96) | 48 (98) |
| No. of embryos transferred | 24 | 24 | 48 |
| Mean no. of blastocysts transferred | 1.6±0.7 | 1.8±0.4 | 1.7±0.6 |
| Range of no. of blastocysts transferred | 1–2 | 1–3 | 1–3 |
| No. of embryos implanted (%) | 7 (29) | 9 (38) | 16 (33) |
| No. of cycles transferred | 13 | 15 | 28 |
| No. of clinical pregnancies (%) | 7 (54) | 7 (47) | 14 (50) |
| No. of miscarriages | 0 | 0 | 0 |
| No. of deliveries | 3 | 3 | 6 |
| No. of ongoing pregnancies | 4 | 4 | 8 |
| No. of babies (male:female ratio) | 3 (2:1) | 5 (1:4) | 8 (3:5) |
Figure 1.
The cryotop: (a) laminate film and cover straw: (b) the cryotop covered with cover straw.
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