J. Liebermann

Potential Importance of Vitrification in Reproductive Medicine

Abstract As early as 1985, ice-free cryopreservation of mouse embryos at −196°C by vitrification was reported in an attempted alternative approach to cryostorage. Since then, vitrification techniques have entered more and more the mainstream of animal reproduction as an alternative cryopreservation method to traditional slow-cooling/rapid-thaw protocols. In addition, the last few years have seen a significant resurgence of interest in the potential benefits of vitrification protocols and techniques in human-assisted reproductive technologies. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human-assisted reproduction, and human preimplantation embryo vitrification is still considered to be largely experimental. Besides the inconsistent survival rates that have been reported, another problem is the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. Clearly, however, attention needs to be paid to the inconsistent survival rates following vitrification.

Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette

The purpose of this study was to demonstrate the safety and efficacy of vitrification of human pronuclear stage (PN) embryos in the human assisted reproduction laboratory. Using single pronucleate (1PN) and three pronucleate (3PN) zygotes, the impact of vitrification in the Flexipet denuding pipette (FDP) as a carrier was assessed in terms of survival, embryonic development and blastocyst formation when compared according to the PN number, and unvitrified controls. A total of 65 1PN and 152 3PN zygotes were vitrified; after warming 82% (53/65) of 1PN and 90% (137/152) of 3PN survived. The overall percentage of warmed zygotes (1PN and 3PN) that cleaved and reached 2-cell stage did not differ (chi(2); P = 0.32) from the control groups (77%; 147/190 versus 85%; 115/136). In addition, when the cleavage behaviour was examined on day 3 for >or=4-cell stage, no significant differences (chi(2); P = 0.95) were observed between the vitrified group and the unvitrified control groups (74%; 109/147 versus 77%; 89/115). Comparing the developmental potential up to cavitation and blastocyst formation on day 5, the overall outcome of the vitrified PN was 31% compared with 33% for the controls (chi(2); P = 0.76). The simple vitrification protocol used in this study, and these data highlight the usefulness of vitrification using FDP as a consistent and effective cryopreservation method for pronuclear zygotes, and a suitable alternative to slow cryopreservation protocols.

Vitrification of human blastocysts: an update

Transfer of blastocyst-stage embryos has been shown to increase pregnancy rates while allowing for improved selection of potentially viable embryos. At this late stage of development, lower numbers of embryos can be transferred, resulting in less high-order multiple pregnancies and increased implantation rates. Between January 2004 and February 2009, 8449 blastocysts from 2453 patients were vitrified. After 1398 vitrified embryo transfers (VET) of both day-5 and day-6 blastocysts with a mean patient age of 34.6 +/- 5.0 years, the study centre has seen a survival rate of 96.3% (2730/2835), an implantation rate of 29.4% and a clinical pregnancy rate per VET of 42.8% (599 pregnancies/1398 warmed embryo transfers). After more than 5 years of vitrifying blastocysts, the perinatal outcome was, from 348 deliveries with vitrified blastocysts, the births of 431 babies (202 boys and 229 girls). One of the benefits of blastocyst vitrification is that it can be undertaken on a more flexible basis by laboratory staff. Also, vitrification may allow individual blastocysts to be cryopreserved at their optimal stage of development and expansion.

Vitrification in Assisted Reproduction: A User’s Manual and Trouble-shooting Guide

Vitrification in Assisted Human Reproduction: A Users Manual and Trouble-Shooting Guide - Libros de Medicina - Fertilidad y embarazo - 120,71

Estimation of Second Polar Body Retention Rate After Conventional Insemination and Intracytoplasmic Sperm Injection: In Vitro Observations from More Than 5000 Human Oocytes

AbstractPurpose: Tripronucleate (3pn) development after conventional insemination (CONV) or ICSI was analyzed to estimate the rate of second polar body retention giving rise to 3pn formation. Methods: Data from 453 consecutive IVF cycles were reviewed during a 6-month period. Mature oocytes were monitored in ICSI (n = 3195) and CONV (n = 2274) groups by fertilization assessment 16–18 h post-insemination. Ovulation induction protocols and in vitro culture conditions remained constant during the study interval. Results: Normal (2pn) fertilization occurred in 74.2% and 70.5% for CONV and ICSI groups, respectively (p < 0.003). 1pn formation was observed in 4.5% of CONV oocytes, and 2.5% of ICSI oocytes (p < 0.001); 3pn formation was 8.1% in the CONV group, and 2.5% in the ICSI group (p < 0.0001). We observed 4pn formation in 0.4% of oocytes in the CONV group, but in only 0.04% of oocytes fertilized with ICSI (p < 0.007). Cellular degeneration occurred in 2.4% of oocytes inseminated conventionally, and in 3.5% of oocytes fertilized by ICSI (p = 0.02). Maternal age did not impact pronuclear status. Conclusions: We found the 3pn formation rate after ICSI to be approximately one-third that observed in the CONV group. Extrapolating the ICSI data to the CONV data, it may be inferred that 2.5% of 3pn development after CONV was due to second polar body retention. This suggests that 5.6% of CONV oocytes showed dispermic fertilization. Decreasing oocyte quality with increasing maternal age had no apparent influence on any of the fertilization outcomes.

Laser-assisted human embryo biopsy on the third day of development for preimplantation genetic diagnosis: two successful case reports

OBJECTIVE To perform preimplantation genetic diagnosis (PGD) with 1.48-microm infrared diode laser assistance during embryo biopsy for two patients undergoing IVF. DESIGN Case reports. SETTING Private ART laboratory. Two couples undergoing IVF for infertility therapy, both of whom had previously delivered offspring afflicted with spinal muscular atrophy (type 1) after IVF therapy, and who underwent subsequent cycles of IVF coupled with PGD to screen for this disorder. INTERVENTION(S) Two individual IVF cases involving intracytoplasmic sperm injection (ICSI), embryo biopsy with laser assistance, and PGD. The ease and apparent safety of human embryo biopsy using a 1.48-microm infrared laser for partial zona pellucida (ZP) dissection to assist with embryo blastomere biopsy was evaluated. RESULT(S) Both couples were deemed to have some unafflicted embryos for transfer on the fifth day of development after blastomere biopsy in conjunction with PGD. Patient A had a singleton pregnancy and delivered a healthy normal singleton male. Patient B had a twin pregnancy; however, one twin was spontaneously lost at 10 weeks but she ultimately delivered a healthy normal singleton male. CONCLUSION(S) These successful outcomes help to demonstrate the efficacy and safety of laser-assisted embryo biopsy to facilitate PGD screening.

Vitrification of Embryos

Vitrification is a very promising cryopreservation method with many advantages and an ever increasing clinical track record. There exist many variables that can profoundly influence the effectiveness and the survival rates of vitrified cells. A standardized vitrification protocol applicable to all stages of the preimplantation embryo may not be realistic because of (a) different surface-to-volume ratios; (b) differing cooling rate requirements between oocytes, zygotes, cleavage stage embryos, and blastocysts; and (c) variable chill sensitivity between these different developmental stages. Currently, however, the most widely used protocol applied to any embryo stage is the two-step equilibration in an equimolar combination of the cryoprotectants ethylene glycol and DMSO, at a concentration of 15% each (v/v), supplemented with 0.5 mol/L sucrose.