用micro-needle穿刺blastocoele造成shrinkage後再冷凍囊胚

Blastocoele越大，內含大量水份，容易產生ice crystal，冷凍效果越差

本研究使用micro-needle穿刺blastocoele造成shrinkage後再冷凍

http://humrep.oxfordjournals.org/content/17/3/744.full

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification

1. P. Vanderzwalmen1,4, 
2. G. Bertin1, 
3. Ch. Debauche1, 
4. V. Standaert1,
5. E. van Roosendaal1, 
6. M. Vandervorst1, 
7. N. Bollen1, 
8. H. Zech2, 
9. T. Mukaida3,
10. K. Takahashi3 and 
11. R. Schoysman1

+Author Affiliations

1. ¹Schoysman Infertility Management Foundation, Vaartstraat 42, 1800 Vilvoorde, Belgium,
2. ²Institut fur In vitro Fertilisierung und Embryo Transfer, Bregenz, Austria and
3. ³Hiroshima HART Clinic, Hiroshima, Japan

• Accepted November 5, 2001.

 

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Abstract

BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol–18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Artificial shrinkage procedure

Artificial shrinkage of blastocysts and expanded blastocysts was performed before vitrification and also before a fresh transfer in order to examine if the artificial shrinkage technique had no harmful effect on the further developmental capacity of the blastocysts.

Ten to 15 min before the vitrification assay or transfer, the blastocysts and expanded blastocysts were dropped into a Petri dish containing 50 μl. drops of pre-equilibrated S2 or G2-2 medium covered with mineral oil. After holding the blastocyst with a holding pipette and placing the ICM at the 12 or 6 o'clock position, a glass micro-needle was pushed through the cellular junction of the trophectoderm into the blastocoele cavity until it shrank (Figure 1). After removing the pipette, contraction of the blastocyst was observed after 30 s to 2 min. If shrinkage was not observed, the needle was re-introduced at the opposite pole of the ICM and a slit was made in the trophectoderm cells and the zona pellucida by rubbing the needle on the holding pipette. Directly after complete shrinkage of the blastocoele, the blastocysts were vitrified or loaded in a catheter for a fresh transfer in order to assess the capacity of further development of artificially shrunk blastocysts.

Figure 1.

Artificial reduction of the blastocoelic volume: (A) immobilization of the blastocyst with the inner cell mass at 12 o'clock;(B) insertion of a needle inside the blastocoele; (C) shrinkage after 15 s; (D) shrinkage after 1 min.