Appendix G: Vitrification of Blastocysts Using VitriBlastTM and ThermoBlastTM: Nidacon.

This appendix describes the vitrification of blastocysts using VitriBlast™ (VBK) and ThermoBlast™ (TBK) from Nidacon, Mölndal, Sweden. The technique used and the reason for not including DMSO in the medium at the production stage, but including it separately in the kit, and the importance of collapsing the blastocyst prior to vitrification will be explained and described.

Vitrification of oocytes, embryos and blastocysts.

In assisted reproductive technology, cryopreservation of human oocytes and embryos has been significantly improved by refined slow-cooling and the new vitrification method. The slow-cooling method requires a programmed cryo-machine, and usually takes several hours. It is, however, difficult to eliminate injuries resulting from ice formation completely. Vitrification has become a reliable strategy because it is simple, can lead to high survival rates and viability, and has better clinical outcome. Vitrification transforms cells into an amorphous glassy state inside and outside the vitrified cell with ultra-rapid cooling and warming steps by plunging the oocytes and embryos into liquid nitrogen, instead of ice-crystal formation. Over the past decade, several advances in vitrification technologies have improved clinical efficiency and outcome. In this chapter, we focus on vitrification technologies for cryopreservation in human assisted reproductive technology.

Improved development of mouse and human embryos using a tilting embryo culture system.

Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression and friction force in the Fallopian tube before nidation. In order to apply mechanical stimuli to embryos in a conventional IVF culture system, the tilting embryo culture system (TECS) was developed. The observed embryo images from the TECS suggest that the velocities and shear stresses of TECS embryos are similar to those experienced in the oviduct. Use of TECS enhanced the development rate to the blastocyst stage and significantly increased the cell number of mouse blastocysts (P<0.05). Although not statistically significant, human thawed embryos showed slight improvement in development to the blastocyst stage following culture in TECS compared with static controls. Rates of blastocyst formation following culture in TECS were significantly improved in low-quality embryos and those embryos cultured under suboptimal conditions (P<0.05). The TECS is proposed as a promising approach to improve embryo development and blastocyst formation by exposing embryos to mechanical stimuli similar to those in the Fallopian tube.

Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study.

OBJECTIVE: To evaluate perinatal outcome of ultrarapid vitrified blastocyst transfer. DESIGN: Retrospective study. SETTING: Private IVF clinics. PATIENT(S): One hundred eight women, who delivered 147 babies. INTERVENTION(S): Supernumerary blastocysts were vitrified using cryoloop method and transferred after warming. MAIN OUTCOME MEASURE(S): Survival rate of blastocyst after vitrification, implantation and pregnancy rates, neonatal outcome and congenital birth defects. RESULT(S): A total of 1,129 vitrified blastocysts from 435 cycles were warmed and 967 survived (85.7%). In 413 cycles of transfer, the pregnancy, implantation, and abortion rates were 44.1%, 29.0%, and 22.0%, respectively. Of 108 deliveries, 34 (32.9%) were multiple pregnancies and 20 were preterm (18.5%). Out of 147 children born, 50.3% were male and congenital birth defects were observed in 1.4%. These results were similar with those of fresh blastocyst transfer program. CONCLUSION(S): The vitrification of blastocyst using cryoloop is a simple, easy, and quick method. This technique yields the same high pregnancy and implantation rates as fresh blastocyst transfer. Congenital defect rate in this study was similar to fresh blastocyst transfer, proving the method to be safe.

GnRH antagonist improved blastocyst quality and pregnancy outcome after multiple failures of IVF/ICSI-ET with a GnRH agonist protocol.

BACKGROUND: To determine the efficacy of a gonadotrophin-releasing hormone (GnRH) antagonist, cetrorelix, in improving the quality of embryos and pregnancy outcome, we performed a study in patients with a history of multiple failures of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles with a GnRH agonist (GnRHa) long protocol. METHODS: Forty women with no live births after conventional IVF or ICSI embryo transfer (ET) and subsequent blastocyst transfer (BT) with a GnRHa long protocol entered this study. The treatment protocol consisted of a daily dose of clomiphene citrate 100 mg for 5 days and gonadotrophin injections daily from cycle day 4 onward. Cetrorelix, 0.25 mg/day, was started when the leading follicle reached 14 mm. Induction of ovulation was triggered with human chorionic gonadotrophin (HCG) (N = 36) or GnRHa (N = 4). It was possible to perform BT in 38 patients. RESULTS: Comparison of the results with the results for BT with the previous GnRHa protocol showed no significant differences in number of oocytes retrieved or the zygote- and blastocyst-development rate. With the cetrorelix protocol, however, number of patients whose embryos had developed to at least one expanded blastocyst on day 5 was significantly higher than with the GnRHa protocol (25 vs. 9) (p < 0.001), and 16 of the women became pregnant (42.1%), with 7 delivering 9 infants, 4 ending in abortion (25%), and 5 in progressing. CONCLUSIONS: The use of a GnRH antagonist in controlled ovarian hyperstimulation improves the outcome of pregnancy of patients with a history of multiple failure of IVF/ICSI-ET in a GnRHa protocol, most likely due to improvement of the quality of the blastocysts generated.

Cryopreservation of animal and human embryos by vitrification.

Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. For embryo cryopreservation, the vitrification method has advantages over the slow freezing method. For example, injuries related to ice is less likely to occur, embryo survival is more likely if the embryo treatment is optimized, and embryos can be cryopreserved by a simple method in a short period without a programmed freezer. However, solutions for vitrification must include a high concentration of permeating cryoprotectants, which may cause injury through the toxicity of the agents. Since the development of the first vitrification solution, which contained dimethylsulphoxide, acetamide, and propylene glycol, numerous solutions have been composed and reported to be effective. However, ethylene glycol is now most widely used as the permeating component. As supplements, a macromolecule and/or a small saccharide are frequently added. Embryos of various species, including humans, can be cryopreserved by conventional vitrification using insemination straws or by ultrarapid vitrification using minute tools such as electron microscopic grids, thin capillaries, minute loops, or minute sticks, or as microdrops. In the ultrarapid method, solutions with a lower concentration of permeating cryoprotectants, thus having a lower toxicity, can be used, because ultrarapid cooling/warming helps to prevent ice formation.

Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique.

Human embryos have been cryopreserved mainly by slow freezing, but vitrification has also proven effective for embryos at early cleavage stages. However, clinical results on blastocyst cryopreservation have not been consistent. A feasible option appears to be ultrarapid vitrification, in which embryos are vitrified with a reduced amount of solution to achieve extremely high rates of cooling and warming. The cryoloop is a tiny nylon loop connected to the lid by a small metal tube; a metal insert on the lid enables the use of a stainless steel handling rod with a small magnet, and the loop can be stored in the cryovial. In the HART Clinic group, of 444 supernumerary human blastocysts that were vitrified by cryoloops 79% survived after warming, and of 126 recipients 36% became pregnant. The outline of ultrarapid vitrification using cryoloops is described.