Current results with slow freezing and vitrification of the human oocyte

The past decade has witnessed renewed interest in human oocyte cryopreservation (OCP). This article reviews the two general methods used for OCP, slow freezing and vitrification, compares the outcomes associated with each technique and discusses the factors that might influence success with OCP (such as oocyte selection or day of transfer). Based on available data, OCP offers a reliable, reproducible method for preservation of the female gamete and will find increasing application in assisted reproductive technology.Oocyte cryopreservation can provide a number of advantages to couples undergoing assisted reproduction or to women interested in fertility preservation. Two methods, slow freezing and vitrification, have been used successfully for oocyte cryopreservation. This article reviews and compares these methods, and discusses various factors that can impact upon success of oocyte cryopreservation.     

Closed-system solid surface vitrification versus slow programmable freezing of mouse 2-cell embryos

Purpose  To compare closed-system solid surface vitrification with slow freezing. Methods  Mouse 2-cell embryos (n = 348) were divided into vitrification, slow freezing and non-frozen groups. For vitrification, embryos were exposed to 10% ethylene glycol (EG), 10% dimethylsulfoxide (DMSO) and 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for 10 min, then transferred into 17.5% EG, 17.5% DMSO, 0.25 M trehalose and 10% FBS in PBS. They were placed on hemi-straws and inserted into 0.5 ml straws inside a previously cooled aluminum cylinder. Slow freezing was done in straws by the conventional method. Results  Vitrified embryos had significantly higher survival, further cleavage and blastocyst formation rates than those in the slow freezing group (p < 0.001) and were comparable to controls. Blastocysts in the vitrification and control groups had significantly more cells than those in the slow freezing group (p < 0.05). Conclusions  Closed-system vitrification was more effective than conventional slow freezing. Capsule   Closed system solid surface vitrification was more effective than conventional slow freezing in the cryopreservation of mouse 2-cell embryos.     

Developmental potential and ultrastructural injuries of metaphase II (MII) mouse oocytes after slow freezing or vitrification

Purpose: The purpose of this study was to determine the developmental ability and ultrastructure of MII mouse oocytes after cryopreservation by slow freezing or vitrification.Methods: Ovulated MII mouse oocytes were allocated to slow frozen, vitrified and control groups. Oocytes in the slow frozen and vitrified groups were cryopreserved using 1,2 propandiol (PROH) and ethylene glycol (EG) respectively as cryoprotectants. After thawing, the surviving MII oocytes in both cryopreserved groups and the control group were inseminated and their developmental ability was compared. The ultrastructure of MII oocytes in both cryopreserved groups was assessed immediately after thawing and 10 h post insemination at the pronuclear stage, and compared with that of the control group.Results: The survival rates were nearly identical in both cryopreserved groups. The fertilization rates were also identical and comparable to that of the control group. The further development of vitrified oocytes was similar to that of the control oocytes, whereas it was severely limited in the slow-frozen oocytes. In the slow-frozen MII oocytes, the intermediate filaments were destroyed and the oolemma and microvilli were also modified. At the pronuclear stage deterioration of mitochondria and the presence of numerous vacuoles were also observed within the ooplasm. In the vitrified MII oocytes, the intermediate filaments were the only structures affected and these cytoskeletal elements were reorganized at the pronuclear stage.Conclusions: Vitrification results in less ultrastructural damage and better post fertilization development of MII mouse oocytes than slow freezing.     

Comparison of the cryopreservation of human embryos obtained after intracytoplasmic sperm injection with a slow cooling or an ultrarapid cooling procedure

Purpose: Our purpose was to compare an ultrarapid method (URM) modified with dimethyl sulfoxide (Me₂SO) to a slow method (SM) with propanediol (PROH) for the cryopreservation of extra human embryos in a program of intracytoplasmic sperm injection (ICSI).Methods: The extra embryos of 160 patients were cryopreserved in a prospective and randomized manner (drawing lots) by a modified URM (3 M Me₂SO/0.25 M sucrose/thawing in three sucrose gradients) (Group I) or by a SM )1.5 M Propanediol/program 0-Cryologic CL863) (Group II). A total of 103 cycles has been thawed thus far. The number of thawed cycles was 58 for group I and 45 for group II.Results: The mean age (group I, 31.3 ± 4.5; group II, 31.9 ± 4.3) did not differ between the groups (P = 0.38). The number of frozen embryos (group I, 6.6 ± 3.2; group II, 6.5 ± 3.2) was similar (P = 0.49) for the two groups, as was the number of thawed embryos (P = 0.52) (group I, 6.5 ± 2.9; group II, 6.2 ± 3). The survival rate was higher (P < 0.01) for group II (83.3 ± 23%) than for group I (69.2 ± 28.7%). The cleavage rate was also higher (P < 0.01) for group II (56.8 ± 31%) compared with group I (24.2 ± 22.4%). The number of embryos transferred did not differ (P = 0.14) between the groups (group I, 3.16 ± 1.2; group II, 3.5 ± 1.0). The implantation rate (group I, 6.3%; group II, 13.8%) was significantly different between groups (P = 0.034). Pregnancy rates per thawed and transferred cycle were higher for group II (33.3 and 36.6%, respectively) compared with group I (13.8 and 16%, respectively), and these differences were significant (P = 0.03 and P = 0.03, respectively).Conclusion: The data obtained suggest that the SM is superior to the URM for the cryopreservation of extra embryos after ICSI.     

Cryopreservation of human embryos and oocytes for fertility preservation in cancer and non cancer patients: a mini review

Abstract

The term ‘cryopreservation’ illustrates the process of freezing cells and storing at very low temperature in liquid nitrogen (–196?°C). Cooling is not a physiological condition for human cells especially due to the high concentration of water in the living matter, whose conversion to ice crystals may be associated with cell death. Human oocytes are particularly sensitive to the freezing process, primarily because of their large size and the presence of the meiotic spindle, which at low temperature can degenerate. In the last decade, the cryopreservation technology has become highly important as an option for fertility preservation (FP) in women with cancer. Anticancer therapy might promote premature ovarian failure and negatively affects the reproductive outcome. Over the years, scientists have proposed different cryopreservation strategies in the effort to maintain the physiological functions of oocytes and embryo. However, despite the first success obtained in the 1980s with frozen oocytes, it was not until recently that a new approach has been proposed: the ‘Vitrification’ which allowed a breakthrough in this procedure. FP is a major determinant for cancer survivor women in the reproductive age. This article describes the FP options currently available, focusing mainly on oocyte and embryo cryopreservation.     

Vitrification versus slow freezing gives excellent survival,post warming embryo morphology and pregnancy outcomes for human cleaved embryos

Purpose  The objective of this retrospective study was to evaluate the efficacy of vitrification and slow freezing for the cryopreservation of human cleavage stage embryos in terms of post-warming survival rate, post-warming embryo morphology and clinical outcomes. Methods  The embryos of 305 patients at cleavage stages were cryopreserved either with vitrification (153 patients) or slow-freezing (152 patients) methods. After warming; the survival rate, post-warmed embryo morphology, clinical pregnancy and implantation rates were evaluated and compared between the two groups. Result(s)  In the vitrification group versus slow freezing group, the survival rate (96.9% vs. 82.8%) and the post-warmed excellent morphology with all blastomeres intact (91.8% vs. 56.2%) were higher with an odds ratio of 6.607 (95% confidence interval; 4.184–10.434) and 8.769 (95% confidence interval; 6.460–11.904), respectively. In this group, the clinical pregnancy rate (40.5% vs. 21.4%) and the implantation rate (16.6% vs. 6.8%) were also higher with an odds ratio of 2.427 (95%confidence interval; 1.461–4.033) and 2.726 (95% confidence interval; 1.837–4.046), respectively. Conclusion(s)  Vitrification in contrast to slow freezing is an efficient method for cryopreservation of human cleavage stage embryos. Vitrification provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes. Capsule Cryopreservation of human cleavage stage embryos with vitrification versus slow freezing provides better laboratory and clinical outcomes.     

A randomized controlled trial comparing two vitrification methods versus slow-freezing for cryopreservation of human cleavage stage embryos

Purpose

To compare two different vitrification methods to slow freezing method for cryopreservation of human cleavage stage embryos. Design: Prospective randomised trial. Setting: University assisted reproduction centre. Patient(s): 568 patients (mean age 33.4 ± 5.2) from April 2009 to April 2011.

Methods

1798 supernumerary good-quality cleavage stage embryos in 645 IVF cycles intended to be cryopreserved were randomly allocated to three groups: slow freezing, vitrification with the Irvine® method, vitrification with the Vitrolife® method. Main Outcome Measure(s): Embryo survival and cleavage rates, implantation rate.

Results

A total of 1055 embryos were warmed, 836 (79.2 %) survived and 676 were finally transferred (64.1 %). Post-warming embryos survival rate was significantly higher after vitrification (Irvine: 89.4 %; Vitrolife: 87.6 %) than after slow freezing (63.8 %) (p < 0.001). No differences in survival rates were observed between the two vitrification methods, but a significant higher cleavage rate was observed using Irvine compared to Vitrolife method (p < 0.05). Implantation rate (IR) per embryo replaced and per embryo warmed were respectively 15.8 % (41/259) and 12.4 % (41/330) for Irvine, 17.0 % (40/235) and 12.1 % (40/330) for Vitrolife, 21.4 % (39/182) and 9.9 % (39/395) for slow-freezing (NS).

Conclusions

Both vitrification methods (Irvine and Vitrolife) are more efficient than slow freezing for cryopreservation of human cleavage stage embryos in terms of post-warming survival rate. No significant difference in the implantation rate was observed between the three cryopreservation methods.     

Advances in the cryopreservation of mammalian oocytes and embryos: Development of ultrarapid vitrification

The cryopreservation of embryos has become a powerful tool in assisted reproduction in several mammalian species. Embryos are cryopreserved by slow freezing or by vitrification. However, consistently high survival has not been obtained in most oocytes and in some embryos. The main reasons for the low survival would be sensitivity to low temperatures, which leads to chilling injury, and low permeability of the cell membrane, which leads to the formation of intracellular ice. As a strategy aiming to overcome these injuries, modified vitrification methods have been devised in which the cooling and warming rate is markedly increased by minimizing the volume of the solution and the container. The modified methods use electron microscope grids, open-pulled straws, cryoloops, or container-less microdrops. In this article, recent developments in the ultrarapid vitrification of mammalian oocytes and embryos are reviewed based on the understanding of the mechanisms of cell injury in cryopreservation. (Reprod Med Biol 2002; 1 : 1–9)     

Reprint of: Theoretical and experimental basis of slow freezing

In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow-freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins for improvement.In human IVF, cryopreservation of oocytes has become an alternative to embryo storage. It has also shown enormous potential for oocyte donation, fertility preservation and animal biotechnology. Mouse oocytes have represented the elective model to develop oocyte cryopreservation in the human and over several decades their use has made possible the development of theoretical and empirical approaches. Progress in vitrification has overshadowed slow freezing to such an extent that it has been suggested that vitrification could soon become the exclusive cryopreservation choice in human IVF. However, recent studies have clearly indicated that human embryo slow freezing, a practice considered well established for decades, can be significantly improved by a simple empirical approach. Alternatively, recent and more advanced theoretical models can predict oocyte responses to the diverse factors characterizing an entire slow freezing procedure, offering a global method for the improvement of current protocols. This gives credit to the notion that oocyte slow freezing still has considerable margins of improvement.     

Prognostic factors for patients undergoing vitrified-warmed human embryo transfer cycles: a retrospective cohort study

We examined the prognostic factors for pregnancy in 210 vitrified-warmed embryo transfer (ET) cycles in 121 patients. The univariate analysis showed that age, gravida, the number of cycles associated with infertility caused by endometriosis, the number of previous assisted reproductive technology (ART) treatment cycles, and the number of ICSI procedures were significantly lower in pregnant cycles compared with non-pregnant cycles. The percentages of ET using at least one intact embryo and of ET using at least one embryo that had developed further after warming were significantly higher in pregnant cycles compared with non-pregnant cycles. Multivariate logistic regression analysis showed that previous ART treatment cycles, ET with at least one intact embryo, and ET using at least one embryo that had developed further were independent prognostic factors for pregnancy in vitrified-warmed ET cycles. We conclude that fewer previous ART treatment cycles, ET using at least one intact embryo, and ET with embryos that have developed further after warming might be favourable prognostic factors for pregnancy in vitrified-warmed ET cycles.     

A closed system supports the developmental competence of human embryos after vitrification

Purpose

Closed-system vitrification may enable the risk of contamination to be minimised. We performed three studies to compare the developmental competence of human embryos vitrified using either a closed vitrification system (CVS; Rapid-i®) or an open vitrification system (OVS; Cryo-top®).

Methods

The first study was performed in vitro using 66 zygotes previously vitrified at pronuclear stage. These were warmed and randomised 1:1 to revitrification using either the OVS or the CVS. After re-warming, embryo development and blastocyst cell number were assessed. For the second study, also performed in vitro, 60 vitrified–warmed blastocysts were randomised 1:1:1 into three groups (OVS or CVS revitrification, or no revitrification). The proportion of dead cells was assessed by staining. The third study was performed in vivo, using 263 high-grade blastocysts randomly assigned to vitrification using either the CVS (n = 100) or the OVS (n = 163). After warming, single blastocyst transfer was performed.

Results

There were no differences between the CVS and the OVS in survival rate (100 % vs. 97 %), blastulation rate (96 h: 50 % vs. 50 %; 120 h: 68 % vs. 56 %), proportion of good blastocysts (96 h: 32 % vs. 22 %, 120 h: 47 % vs. 41 %), or mean number of cells (137 vs. 138). The proportion of dead cells in blastocysts re-vitrified by CVS (31 %) was similar to that for OVS (38 %) and non-revitrification (32 %). In vivo, the implantation rate for blastocysts vitrified using the CVS (54 %) was similar to that with the OVS (53 %).

Conclusion

Our studies consistently indicate that human embryos may be vitrified using a CVS without impairment of developmental competence.