A. Kader

Factors affecting the outcome of human blastocyst vitrification

With single blastocyst transfer practice becoming more common in ART, there is a greater demand for a convenient and reliable cryostorage of surplus blastocysts. Vitrification has emerged in the last decade as an alternative promising substitute for slow freezing. Blastocysts represent a unique challenge in cryostorage due to their size, multicellular structure and presence of blastocoele. The continuous acquisition of experience and introduction of many different technological developments has led to the improvement of vitrification as a technology and improved the results of its application in blastocyst cryostorage. The current information concerning safety and efficacy of the vitrification of blastocysts will be reviewed along with the variables that can impact the outcome of the procedure.

Evaluation of post-thaw DNA integrity of mouse blastocysts after ultrarapid and slow freezing

OBJECTIVE To evaluate the effect of vitrification and two other methods of slow cryopreservation on DNA integrity in expanded and nonexpanded blastocysts. DESIGN Prospective in vitro study. SETTING Tertiary care academic hospital. INTERVENTION(S) 1) Twenty-two expanded blastocysts (EB) and 17 nonexpanded blastocysts (NEB) vitrified in cryotips; 2) 15 EB and 16 NEB by slow freezing using propanediol; 3) 11 EB and 16 NEB by slow cryopreservation using glycerol; and 4) 14 EB and 13 NEB as fresh control samples. MAIN OUTCOME MEASURE(S) DNA fragmentation by TUNEL and confocal imaging. RESULT(S) Blastocysts slowly cryopreserved with glycerol showed DNA integrity of 94.76 +/- 4.70% and 90.87 +/- 6.16% for NEB and EB, respectively. Propanediol cryopreservation showed values of 72.63 +/- 13.44% and 56.19 +/- 25.49% and vitrification 84.36 +/- 8.7%6 and 77.61 +/- 16.65%, respectively, for the same groups. The NEB showed less DNA fragmentation than EB in all cryopreservation techniques, but this was significant only with slow freezing using propanediol. CONCLUSION(S) All cryopreservation techniques induce DNA damage to blastocysts. Damage is maximal with propanediol and minimal with slow freezing using glycerol. The more expanded the blastocyst, the greater is the susceptibility to DNA damage during cryopreservation.

Mouse blastocyst previtrification interventions and DNA integrity.

OBJECTIVE To assess the effect of implementing different previtrification interventions on the postwarming DNA integrity of vitrified blastocysts. DESIGN Prospective in vitro study. SETTING Center for Reproductive Medicine laboratory in a tertiary hospital setting. ANIMALS A total of 70 expanded and 46 nonexpanded mouse blastocysts were used for the study. INTERVENTION(S) [1] Twenty-two expanded blastocysts were blastocele aspirated and immediately vitrified. [2] Twelve expanded blastocysts were spontaneously hatched before vitrification. [3] Twenty-two expanded blastocysts were vitrified without intervention. [4] Sixteen nonexpanded blastocysts underwent assisted hatching using acidified Tyrodes solution. [5] Seventeen nonexpanded blastocysts were vitrified without intervention. [6] Thirteen nonexpanded blastocysts and 14 expanded were used as fresh controls. Vitrification was done using a cryotip loading device. MAIN OUTCOME MEASURE(S) DNA integrity index using terminal deoxynucleotide transferase [TdT]-mediated dUTP-digoxigenin nick-end labeling staining and confocal imaging. RESULT(S) [1] Intervention by blastocele aspiration for expanded blastocysts or assisted hatching of nonexpanded blastocysts significantly improved the postwarming results in each blastocyst stage. [2] Allowing spontaneous hatching before vitrification is a noninvasive technique that can improve the postwarming integrity of expanded blastocysts similar to blastocele aspiration. CONCLUSION(S) Blastocele aspiration or spontaneous hatching of expanded blastocysts and assisted hatching of nonexpanded blastocysts before vitrification helps minimize blastomere DNA damage.

Effect of varying equilibration time in a two-step vitrification method on the post-warming DNA integrity of mouse blastocysts

OBJECTIVE To assess the effect of equilibration time on the DNA integrity of vitrified-warmed mouse blastocysts. DESIGN Prospective in vitro study. SETTING Embryology research laboratory. INTERVENTION(S) Mouse nonexpanded blastocysts (NEB) (n = 54) and expanded blastocysts (EB) (n = 56) were vitrified using cryotips. Blastocysts were vitrified using a two-step media protocol, with a first (equilibration) step of 4 minutes [NEB (n = 20), EB (n = 24)], 8 minutes [NEB (n = 17), EB (n = 16)], or 15 minutes: [NEB (n = 17), EB (n = 16)]. Control samples were fresh NEB (n = 17) and EB (n = 18) blastocysts. MAIN OUTCOME MEASURE(S) DNA integrity index (DII) using terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, confocal imaging, and viability. RESULT(S) 1) The DII of the vitrified warmed NEB and EB improved significantly with 8-minute equilibration protocol compared with 4 minutes. 2) The DII of the EB significantly decreased with 15-minute equilibration protocol compared with 4 or 8 minutes. 3) The DII in the NEB significantly improved with 8- and 15-minute equilibration protocols compared with 4 minutes, with no significant difference between 8 or 15 minutes. CONCLUSION(S) Vitrification with an 8-minute equilibration step improved DII of the NEB and EB after warming.

Vitrification of isolated mice blastomeres using a closed loading device.

Isolated blastomeres obtained by embryo biopsy serve mainly for preimplantation genetic screening. Blastomeres are undifferentiated embryonic cells that include all the embryo genetic information. A lot of developing technologies may benefit by the efficient cryopreservation of blastomeres for future potential use, especially for stem cell culture and differentiation control. We are hereby reporting for the first time the feasibility of preserving individual isolated blastomeres in microvolumes in a closed vitrification system. Using a cryotip and propagation in microvolumes, isolated mice blastomeres were vitrified and warmed with 100% post-warming survival.

Slow and ultrarapid cryopreservation of biopsied mouse blastocysts and its effect on DNA integrity index

PurposeTo evaluate the effect of slow and ultra-rapid freezing on biopsied blastocysts’ DNA integrity.MethodsForty eight mouse blastocysts were biopsied of which 16 were cryopreserved by slowly freezing and 17 by vitrification. Fourteen intact blastocysts were slowly cryopreserved and 24 were vitrified. Eighteen fresh intact blastocysts and fifteen biopsied blastocysts served as controls. The DNA integrity index of all blastocysts was evaluated using (TUNEL) staining and confocal imagingResultsBoth slow freezing and vitrification of biopsied blastocysts induced apoptosis to a similar extent. Biopsying blastocysts before vitrification resulted in less apoptosis than vitrification of intact blastocysts.ConclusionSlow freezing and vitrification are equal options for preservation of biopsied blastocysts as regards the DNA integrity index (DII). Biopsied blastocysts better tolerate vitrification than intact expanded blastocysts.

Creating A Standard of Care for Fertility Preservation

Fertility preservation options for women are currently only routinely offered to patients who face iatrogenic fertility loss, and most options are considered experimental. The most common modalities for female fertility preservation are embryo cryopreservation, oocyte cryopreservation, and ovarian tissue cryopreservation, the later two of which remain in the experimental arena. Natural fertility loss is affecting women as significantly as premature fertility loss. Increasing cancer survival and the modern reproductive trend of delaying childbearing are indications for the need and demand for fertility preservation. Advances in the field are necessary to respond to this demand and include superior cryopreservation techniques and fertility preservation technologies, customized guidelines, comprehensive care plans, and availability of more cost-efficient procedures. The obstacles to creating a standard of care for fertility preservation are as broad as the field itself. Lack of patient awareness, limited physician experience and knowledge, inadequate counseling, costs, and ethical issues are some examples of the many challenges to establishing a standard of care. With continued research and multidisciplinary collaboration, a higher quality of care may be provided to a larger patient population who wishes to maximize their fertility potential in the future.