胚胎施行輔助孵化後再玻璃化冷凍，可能提高冷凍胚胎懷孕率

囊胚期胚胎玻璃化冷凍存活率90%，懷孕率40%

囊胚期胚胎施行輔助孵化後再玻璃化冷凍，可能可提高冷凍胚胎懷孕率

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757025/

Assisted hatching

Pribenszky et al [39] studied the survival of zona-free mouse blastocysts. There was no difference in survival after thawing between these blastocysts and fresh control blastocysts. This experiment suggested that the intact zona pellucida can potentially negatively impact blastocyst vitrification

In lieu of using zona-free blastocysts, which may not be practical with human blastocysts, assisted hatching can be performed prior to vitrification. With assisted hatching, a small hole is created in the zona pellucida so that the blastocyst can more easily escape or "hatch." It was primarily thought to overcome the post-freezing zonal hardening preventing spontaneous hatching and it proved effective [30].

Assisted hatching has been shown to improve the outcome of vitrification of blastocysts through another mechanism. Applying assisted hatching prior to blastocyst vitrification allows better permeation of the cryoprotectants and better blastocoele dehydration [36,40]. Zech et al [40] found that vitrified warmed blastocysts that had undergone assisted hatching had significantly better survival, implantation and pregnancy rates than blastocysts with an intact zona. In concordance with Zech's findings, we have demonstrated that assisted or spontaneous hatching both have a significantly positive impact on the post-warming DNA integrity index of mice blastocysts post-warming as compared with zona-intact blastocysts [36]. These two studies show that assisted hatching is a useful and effective pre-vitrification intervention that can reduce DNA damage incurred during the vitrification process and improve clinical outcome parameters.

Table 3

Studies showing different methods of blastocyst pre-vitrification interventions and their outcome parameters

Blastocoele Evacuation
Authors, year	Species	Method	Intervention Sample size	Outcome parameter	Intervention	Control
Vanderzwalmen et al.2002[37]	Human	Micro-needle puncture	N = 75	Survival rate	70.6%	20.3%
				Pregnancy rate	20.5%	4.5%
				Implantation rate	18.4%	7.1%
Son et al. 2003[43]	Human	Micro-needle puncture	N = 90	Survival rate	90.0%	---
				Pregnancy rate	48.0%	---
				Implantation rate	29.0%	---
Hiraoka et al. 2004[44]	Human	Micropipetting	N = 48	Survival rate	98.0%	---
				Pregnancy rate	50.0%	---
				Implantation rate	33.0%	---
Chen et al. 2005[42]	Mice	Microsuction	N = 108	Survival rate	92.0%	80.0%
Mukaida et al. 2006[41]	Human	Microneedle puncture	N = 462	Survival rate	97.2%	85.0%
				Pregnancy rate	60.2%	34.1%
				Implantation rate	46.5%	---
Mukaida et al. 2006[41]	Human	Laser pulse	N = 40	Survival rate	97.5%	85.0%
				Pregnancy rate	61.5%	34.1%
				Implantation rate	48.6%	---
Kader et al. 2009[45]	Mice	Microsuction	N = 22	DNA integrity index	90.1%	77.6%
Zonal Hatching
Author, year	Species	Method	Intervention Sample size	Outcome parameter	Intervention	Control
Zech et al. 2005[40]	Human	Spontaneous and Assisted (Mechanically)	N = 38	Survival rate	82%	64%
				Pregnancy rate	35%	21%
				Implantation rate	26%	12%
Kader et al. 2009[45]	Mice	Assisted (Acidified Tyrod's)	N = 16	DNA integrity index	94.6%	84.4%
		Spontaneous	N = 12	DNA integrity index	88.5%	77.6%

Table 1

Comparison of survival, implantation and pregnancy rates according to loading device

	Loading Device	Sample Size	Survival Rate	Implantation Rate	Pregnancy Rate
Mukaida et al, 2001[8]	Cryoloop	N = 60	63%	--	31%
Cho, 2002 et al [21]	EM grid	N = 21	83%	--	34%
Reed et al, 2002[10]	Cryoloop	N = 54	100%	15%	--
Mukaida et al, 2003[9]	Cryoloop	N = 725	80%	20%	37%
Osada et al, 2003[11]	Cryotop	N = 580	99%	--	56%
Stehlik et al, 2005[12]	Cryotop	N = 41	100%	--	50%
Takahashi et al, 2005[19]	Cryoloop	N = 1129	86%	29%	44%
Kuwayama et al, 2005[18]	Cryotip	N = 5695	90%	--	53%
Liebermann et al, 2006[13]	Cryotop	N = 547	97%	31%	46%
Mukaida et al, 2008[29]	Cryoloop	N = 5412	92%	36%	49%

Different pre-vitrification interventions for blastocysts. A. Assisted hatching: An opening is created in the zona using laser pulse B. Needle blastocoele puncture: A needle is passed through the zona and blastocoele and retracted allowing the blastocelic fluid to freely leak. C. Laser blastocoele puncture: laser pulse creates an opening in the zona and a small defect in the trophectoderm causing the blastocoele to leak. D. Blastocoele aspiration: An injection needle is introduced into the blastocoele and blastocoelic volume is sucked out. E. Micropipetting: Passing the blastocysts through a narrow pipette would crack the zona and compress the blastocoele to leak through the cracked zona.

Table 2

Different studies comparing the slow preservation and/or vitrification of day 5 and day 6 blastocysts in terms of survival after warming, implantation and pregnancy rates

	Slowly frozen Day 5 Blastocysts	Slowly frozen Day 6 Blastocysts	Vitrified Day 5 Blastocysts	Vitrified Day 6 Blastocysts
Mukaida et al. 2003[9]			Survival 87%	Survival 55%
Stehlik et al. 2005[12]	Survival 83.1% Pregnancy rate 16.7%	Survival 89.5% Pregnancy rate 18.5%	Survival 100% Pregnancy rate 50%	Survival 100% Pregnancy rate 33%
Liebermann & Tucker 2006[13]	Survival 91.4% Implantation 29.6% Pregnancy rate 42.8%	Survival 94.8% Implantation 28.2% Pregnancy rate 43.1%	Survival 95.9% Implantation 33.4% Pregnancy 48.7%	Survival 97.5% Implantation 25.9% Pregnancy 42.8%
Kader et al. 2008[36]	DNA integrity index: 94.76% ± 4.70	DNA integrity index: 90.87% ± 6.16	DNA integrity index: 84.36% ± 8.76	DNA integrity index: 77.61% ± 16.65

Table 3

Studies showing different methods of blastocyst pre-vitrification interventions and their outcome parameters

Blastocoele Evacuation
Authors, year	Species	Method	Intervention Sample size	Outcome parameter	Intervention	Control
Vanderzwalmen et al.2002[37]	Human	Micro-needle puncture	N = 75	Survival rate	70.6%	20.3%
				Pregnancy rate	20.5%	4.5%
				Implantation rate	18.4%	7.1%
Son et al. 2003[43]	Human	Micro-needle puncture	N = 90	Survival rate	90.0%	---
				Pregnancy rate	48.0%	---
				Implantation rate	29.0%	---
Hiraoka et al. 2004[44]	Human	Micropipetting	N = 48	Survival rate	98.0%	---
				Pregnancy rate	50.0%	---
				Implantation rate	33.0%	---
Chen et al. 2005[42]	Mice	Microsuction	N = 108	Survival rate	92.0%	80.0%
Mukaida et al. 2006[41]	Human	Microneedle puncture	N = 462	Survival rate	97.2%	85.0%
				Pregnancy rate	60.2%	34.1%
				Implantation rate	46.5%	---
Mukaida et al. 2006[41]	Human	Laser pulse	N = 40	Survival rate	97.5%	85.0%
				Pregnancy rate	61.5%	34.1%
				Implantation rate	48.6%	---
Kader et al. 2009[45]	Mice	Microsuction	N = 22	DNA integrity index	90.1%	77.6%
Zonal Hatching
Author, year	Species	Method	Intervention Sample size	Outcome parameter	Intervention	Control
Zech et al. 2005[40]	Human	Spontaneous and Assisted (Mechanically)	N = 38	Survival rate	82%	64%
				Pregnancy rate	35%	21%
				Implantation rate	26%	12%
Kader et al. 2009[45]	Mice	Assisted (Acidified Tyrod's)	N = 16	DNA integrity index	94.6%	84.4%
		Spontaneous	N = 12	DNA integrity index	88.5%	77.6%