Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryos

Vitrification is an effective method for the cryopreservation of mammalian embryos. Nevertheless, it is unclear which embryonic developmental stage is the most suited for vitrification and would ensure maximal developmental competence upon subsequent warming. This study, therefore, compared the effects of cryotop vitrification on the developmental competence of murine morula and blastocyst stage embryos. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in two hatched blastocyst groups derived from vitrified morulae and blastocysts, respectively. The post-vitrification survival rates for mouse embryos at the morula and blastocyst stage were 95.4% (186/195) and 96.5% (195/202), respectively. The blastocyst formation rate was significantly lower for vitrified morulae (90.3%) compared with the non-vitrified control group (98.4%) (P < 0.05). The hatching rates were similar between the vitrified morula (79.6%) and the vitrified blastocyst (81.0%) groups. When further development to the fully hatched blastocyst stage was compared, fully hatched blastocysts derived from vitrified morulae had significantly higher cell counts for both the ICM and TE lineage, as compared with hatched blastocysts derived from vitrified blastocysts (P < 0.001). Cryotop vitrification of mouse embryos at the morula stage rather than blastocyst stage would thus ensure a higher degree of post-warming developmental competence.     

Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method

Purpose

The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos.

Methods

Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups.

Results

Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P?>?0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P?<?0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P?<?0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P?<?0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P?>?0.05).

Conclusions

Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.     

Highly efficient vitrification method for cryopreservation of human oocytes

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.     

Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique

OBJECTIVE: Clinical application of vitrification for the cryopreservation of human blastocysts. DESIGN: Clinical trial of vitrification of human blastocysts. SETTING: Private assisted reproductive technology clinic. PATIENT(S): Supernumerary blastocysts after fresh blastocyst transfer were vitrified for subsequent transfer. INTERVENTION(S): Culture of pronuclear embryos to the blastocyst stage in sequential media and subsequent vitrification of supernumerary blastocysts using a cryoloop technique. MAIN OUTCOME MEASURE(S): Clinical outcome after transfer of vitrified blastocysts. RESULT(S): A total of 60 vitrified blastocysts from 21 patients were warmed, and the survival rate at 2 hours after warming was 63%. Six clinical pregnancies were achieved after 19 transfers. One healthy baby was born, four pregnancies are ongoing, and one ended in miscarriage. CONCLUSION(S): Human blastocysts can be successfully vitrified by suspension on a small nylon loop and a direct plunge into liquid nitrogen. A delivery and ongoing pregnancies prove the safety of this method. This report documents the first successful pregnancy and delivery achieved by blastocyst vitrification using the cryoloop containerless technique.     

Birth of a healthy baby following vitrification of human blastocysts

OBJECTIVE: To assess vitrification of human blastocysts. DESIGN: Retrospective study of blastocyst vitrification. SETTING: A private clinic. PATIENT(S): Twenty couples with different types of infertility. INTERVENTION(S): Blastocysts were frozen with rapid vitrification and then transferred after thawing. We vitrified blastocysts using a modification of Ishimori's vitrification solution of ethylene glycol and dimethyl sulfoxide (VSED). MAIN OUTCOME MEASURE(S): After thawing, survival was defined by the embryo's development morphology after 6 hours or overnight culture. RESULT(S): Eighteen of 20 patients underwent treatment. Of 45 vitrified blastocysts, 36 survived, for a survival rate of 80% (36 of 45). The implantation rate was 21.9% (7 of 32), and the pregnancy rate (per embryo transfer cycle) was 33.3% (6 of 18). One of the pregnancies resulted in the delivery of a healthy baby. CONCLUSION(S): Supernumerary embryos were grown in culture to blastocysts, and the survival rate of vitrified-thawed blastocysts was the same as that for slow freezing of early stage embryos. Blastocyst vitrification should prove effective for clinical treatment. The present results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the future.     

A new,simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos

Purpose

This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos.

Methods

Mice oocytes (n?=?40) and embryos (8 cells, n?=?35 and blastocysts, n?=?165), bovine embryos (2PN, n?=?35), and MII oocytes (n?=?84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates.

Results

Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos’ survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts’ rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts’ rates.

Conclusion

This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.

     

Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination

Survival and development of human embryos was compared following slow cooling versus vitrification involving more than 13,000 vitrified embryos. In addition, the efficacy of an open system, the Cryotop, and a closed vitrification system, the CryoTip(trade mark), were compared using human blastocysts. One hundred percent of vitrified human pronuclear stage embryos survived and 52% developed to blastocysts as compared with 89% survival and 41% blastocyst development after slow cooling. Similar survival rates were seen with vitrification of 4-cell embryos (98%) as compared with slow cooling (91%). Furthermore, 90% of vitrified blastocysts survived and resulted in a 53% pregnancy rate following transfer, as compared with 84% survival and 51% pregnancy rates following slow cooling. All corresponding values were significantly different. When the closed and open vitrification systems were compared, no difference was found with regard to supporting blastocyst survival (93 and 97% for CryoTip and Cryotop respectively), pregnancies (51 versus 59% respectively) and deliveries (48 versus 51% respectively). Vitrification is a simple, efficient and cost-effective way to improve cumulative pregnancy rates per cycle. The use of the closed CryoTip system eliminates the potential for embryo contamination during cryopreservation and storage without compromising survival and developmental rates in vitro and in vivo.     

Closed vitrification of human oocytes and blastocysts: outcomes from a series of clinical cases

Purpose

High survival rates and clinical outcomes similar to those from fresh oocytes and blastocysts have been observed with open oocyte vitrification systems. It has been suggested that the extremely fast cooling rates that are only achieved with open systems are necessary for human oocyte and blastocyst vitrification. However, there is a potential risk of introducing contamination with open systems. The aim of this study was to assess whether similar survival and subsequent implantation rates could be achieved using a closed vitrification system for human oocytes and blastocysts.

Methods

Initially, donated immature oocytes that were matured in vitro were vitrified using the cryoprotectants ethylene glycol (EG) + dimethyl sulphoxide (DMSO) + sucrose and either a closed system (Rapid-i®) or an open system (Cryolock). The closed system was subsequently introduced clinically for mature oocyte cryopreservation cases and blastocyst vitrification.

Results

Using in vitro matured oocytes, a similar survival was achieved with the open system of 92.4 % (73/79) and with the closed system of 89.7 % (35/39). For clinical oocyte closed vitrification, high survival rate of 90.5 % (374/413) and an implantation rate of 32.7 % (18/55) from the transfer of day 2 embryos was achieved, which is similar to fresh day 2 embryo transfers. Blastocysts have also been successfully cryopreserved using the Rapid-i closed vitrification system with 94 % of blastocysts having an estimated ≥75 % of cells intact and a similar implantation rate (31.5 %) to fresh single blastocyst transfers.

Conclusion

Closed vitrification can achieve high survival and similar implantation rates to fresh for both oocytes and blastocysts.

     

Human oocyte vitrification: in-vivo and in-vitro maturation outcomes

This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.     

H19 and MEST gene expression and histone modification in blastocysts cultured from vitrified and fresh two-cell mouse embryos

Vitrification of embryos is a routine procedure in many IVF laboratories. The effect of vitrification on gene expression and some modifications of H3 histone in H19 and MEST imprinted genes in blastocysts produced in vitro from non-vitrified and vitrified two-cell embryos was investigated. The expression level of the chosen imprinted genes increased significantly (P < 0.05) in experimental groups compared with in-vivo blastocysts (control group). H3K9me2 decreased, whereas H3K9ac increased in the experimental group compared with the control group. The increases in the expression levels of the imprinted genes, and the attendant changes in histone and chromatin status associated with in-vitro culture of embryos from the two-cell stage, are unaffected by prior vitrification and warming. In the present study, it was shown that such changes are solely caused by the effect of in-vitro culture, irrespective of vitrification. Although these genes are sensitive to environmental changes, vitrification seems to have no additional effect on these genes and on the histone marks, and can threfore be considered to be a process with minimum damage for embryo cryopreservation in assisted reproductive technology applications.     

Selective vitrification of euploid oocytes markedly improves survival, fertilization and pregnancy-generating potential

Enthusiasm for oocyte cryopreservation has been limited by poor pregnancy rates per thawed metaphase II (MII) oocytes (<4%) and low implantation rates per embryos. The reasons relate to technical limitations in the freezing process, and the fact that <40% of oocytes are euploid and unable to produce 'competent' embryos. Comparative genomic hybridization was performed on the first polar body (PB-1) of 323 MII oocytes retrieved from 16 donors. Of these, 111 were euploid, and were vitrified. Seventy-five of 78 vitrified oocytes (96%) survived warming and were fertilized using intracytoplasmic sperm injection. Thirty-one (41%) subsequently developed into expanded blastocysts, of which no more than two were subsequently transferred per uterus to 16 out of 19 prospective embryo recipients. Twelve of 19 (63%) recipients produced 17 healthy babies (eight singletons, three twins, and one set of triplets) One twin pregnancy miscarried in the late first trimester The birth rate per transfer of a maximum of two blastocysts to 16 recipients was 75%. The implantation rate per vitrified euploid oocyte was 27%. This study showed a six-fold improvement in pregnancy rate per cryopreserved oocyte over previous reports and a marked improvement in implantation rate. If independently validated, this approach could open the door to commercial egg cryobanking, significantly expanding women's reproductive choices.     

Blastocyst transfer after aseptic vitrification of zygotes: an approach to overcome an impaired uterine environment

In some IVF cycles, no fresh embryo transfer in the stimulated cycle is advisable. The cryopreservation of zygotes and the transfer of blastocysts in a cryo-embryo transfer is an option to circumvent an inadequate uterine environment due to risk of ovarian hyperstimulation syndrome, inappropriate endometrium build up, endometrial polyps or uterine myomas. For this strategy, highly secure and safe cryopreservation protocols are advisable. This study describes a protocol for aseptic vitrification of zygotes that results in high survival rates and minimizes the potential risk of contamination in liquid nitrogen during cooling and long-term storage. In mouse zygotes, there was no difference in efficiency as compared with a conventional open vitrification system. In IVF patients, aseptically vitrified zygotes showed no difference in blastocyst formation rate as compared with sibling zygotes kept in fresh culture. A clinical study comprising 173 cryo-cycles with a transfer of blastocysts originating from vitrified zygotes showed an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification encourages the use of cryo-embryo transfer for patients with a suboptimal uterine environment in a fresh cycle.In stimulated IVF cycles, high doses of hormones are given to stimulate multifollicular growth. One drawback of the hormonal substitution is that the uterine environment is not at the same time optimally prepared for embryo implantation. A solution, which is increasingly under discussion, is to cryopreserve the embryos obtained in the stimulated cycle and to transfer them back into the optimal uterine environment in a subsequent cryo-cycle. This procedure requires highly secure and safe cryopreservation protocols in order to ensure benefits for both pregnancy and birth rates. We have established a protocol for the vitrification of zygote-stage embryos in aseptic devices, which minimize the potential risk of contamination during cooling and storage. The vitrified zygotes showed the same blastocyst development as compared with sibling zygotes in fresh culture. A clinical study comprising 173 cryo-cycles with transfer of blastocysts originating from vitrified zygotes shows an ongoing pregnancy rate of 40.9%. The live birth rate per patient was 36.8%. A combination of good clinical results and increased safety conditions due to aseptic vitrification conditions contributes to a change in transfer strategy and encourages us to increase the cryo-embryo transfer rate for an optimal uterine environment.     

Vitrification demonstrates significant improvement versus slow freezing of human blastocysts

Blastocyst culture has reduced the number of embryos transferred per cycle, whilst simultaneously creating new quandaries regarding supernumerary blastocyst cryopreservation. This retrospective study was undertaken to compare a slow freezing protocol to a vitrification protocol for cryopreservation of day 5 and day 6 human blastocysts. To demonstrate this, the survival, implantation rate and pregnancy rates were compared after thawing, assessment and embryo transfer of 86 consecutive day 5 and day 6 thawed blastocyst transfer cycles from January 1, 2002 to December 31, 2003. Seventy-one day 5 slow-frozen (SF) blastocysts were thawed and 59 embryos survived the thawing (83.1%). An average of 2.5 SF blastocysts was replaced per embryo transfer, resulting in a pregnancy rate of 16.7% (4/24). Concurrently, 41 vitrified (VIT) blastocysts were thawed and all 41 survived the thawing process (100%). An average of 2.0 VIT blastocysts was replaced per embryo transfer, resulting in a pregnancy rate of 50% (10/20). Survival, pregnancy and implantation rates of day 5 VIT blastocysts have significantly increased (P < 0.01, P < 0.02 and P < 0.01 respectively) over day 5 SF blastocysts. A similar trend was observed with day 6 blastocysts.     

The presence of 1 mM glycine in vitrification solutions protects oocyte mitochondrial homeostasis and improves blastocyst development

Purpose

Embryos generated from oocytes which have been vitrified have lower blastocyst development rates than embryos generated from fresh oocytes. This is indicative of a level of irreversible damage to the oocyte possibly due to exposure to high cryoprotectant levels and osmotic stress. This study aimed to assess the effects of vitrification on the mitochondria of mature mouse oocytes while also examining the ability of the osmolyte glycine, to maintain cell function after vitrification.

Methods

Oocytes were cryopreserved via vitrification with or without 1 mM Glycine and compared to fresh oocyte controls. Oocytes were assessed for mitochondrial distribution and membrane potential as well as their ability to fertilise. Blastocyst development and gene expression was also examined.

Results

Vitrification altered mitochondrial distribution and membrane potential, which did not recover after 2 h of culture. Addition of 1 mM glycine to the vitrification media prevented these perturbations. Furthermore, blastocyst development from oocytes that were vitrified with glycine was significantly higher compared to those vitrified without glycine (83.9 % vs. 76.5 % respectively; p?<?0.05) and blastocysts derived from oocytes that were vitrified without glycine had significantly decreased levels of IGF2 and Glut3 compared to control blastocysts however those derived from oocytes vitrified with glycine had comparable levels of these genes compared to fresh controls.

Conclusion

Addition of 1 mM glycine to the vitrification solutions improved the ability of the oocyte to maintain its mitochondrial physiology and subsequent development and therefore could be considered for routine inclusion in cryopreservation solutions.     

Vitrification of blastocysts derived from fair to poor quality cleavage stage embryos can produce high pregnancy rates after warming

Purpose

This study investigates whether certain embryos considered unsuitable for cryopreservation on day 3 might nevertheless have the potential to develop into worthwhile blastocysts that could be vitrified in the same cycle.

Methods

Retrospective study: between 2010 and 2011, embryo transfers and cryopreservation took place mainly on day 3 in our centre. Supernumerary embryos of intermediate to poor quality were reassessed on days 5/6 and any good quality blastocysts were vitrified.

Results

Out of 914 cleavage stage (day 3) embryos left in culture, 16 % were vitrified on days 5/6. Fifty blastocyst warming cycles resulted in a 76 % survival rate, 44 % clinical pregnancy rate and 39 % implantation rate. During the same time period, 213 warming cycles of good quality cleavage stage embryos rendered survival rates, clinical pregnancy and implantation rates of 97 %, 23 % and 16 % respectively.

Conclusions

Supernumerary average quality day 3 embryos should be given a second chance to be selected for cryopreservation. If blastocysts are obtained and survive vitrification, there is a good chance of implantation thus reducing embryo waste.     

Pregnancy resulting from transfer of repeat vitrified blastocysts produced by in-vitro matured oocytes in patient with polycystic ovary syndrome

This report describes a live birth produced from repeat vitrification and thawing of blastocysts derived from in-vitro matured (IVM) oocytes in a woman with polycystic ovarian syndrome. Immature oocyte retrieval was performed on day 12 of her induced menstrual cycle. The patient was administered 10,000 IU of human chorionic gonadotrophin s. c. 36 h before immature oocyte retrieval. A total of 47 immature oocytes were collected. Following IVM of these immature oocytes, 76.6% (36/47) become mature (at metaphase II stage). Thirty oocytes (30/36, 86.1%) were normally fertilized following insemination by intracytoplasmic sperm injection. The fertilized zygotes (two-pronuclear stage) were co-cultured with cumulus cells in YS medium supplemented with 10% human follicular fluid. On day 5 after insemination, three blastocysts were transferred. Unfortunately, fresh embryo transfer did not result in pregnancy. The remaining 10 embryos developed to the expanded blastocyst stage. These remaining blastocysts were vitrified with electron microscope grids following artificial shrinkage. Three months later, three blastocysts were thawed due to a clinical error. Consequently, the embryos were revitrified. After a week, the three blastocysts were warmed again. Two of them developed to hatched blastocysts. Following transfer, a full-term pregnancy resulted in the delivery of healthy twins.     

Vitrification of human blastocysts previously cryopreserved by slow controlled-rate freezing at the cleavage stage

Purpose

The objective of this study was to evaluate the efficiency of vitrified blastocysts derived from frozen-thawed cleavage stage embryos in terms of morphological survival and re-expansion status of the blastocoelic cavity.

Results

After warming 162 blastocysts derived from fresh embryos (= control group) and 90 blastocysts from frozen-thawed cleavage stage embryos (= study group) and after 2–3 h of in vitro culture the percentage of blastocysts with morphological survival was not different between the two groups. After 24 h of in vitro culture, the percentage of fully expanded, hatching or hatched blastocysts was not different between both groups.

Conclusion(s)

The results show that blastocysts derived from frozen-thawed cleavage stage embryos can be cryopreserved successfully a second time by vitrification method. Re-cryopreservation by vitrification still needs to be approached with some caution because little data on long term safety of multiple freezing is available.     

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.     

Cryopreservation of human oocytes,zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification) methods

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.     

Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing?

Cryopreservation of human oocytes and embryos is a necessary tool in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing costs. Vitrification is a cryopreservation technique that leads to a glass-like solidification, with rapid cooling of cells or tissues. Nowadays vitrification is claimed to be the future of cryopreservation of human embryos due to improved survival rates and clinical outcomes. This study was conducted at a university clinic to assess the safety and efficiency of vitrification of human zygotes as a routine procedure. A total of 849 pronuclear-stage (PN) zygotes were vitrified between March 2004 and July 2006. During this period, 103 cycles of cryopreserved embryo transfer were completed. In total, 339 PN zygotes were thawed resulting in an 89% survival rate (302 PN zygotes). The mean number of embryos per transfer was 2.2. The pregnancy rate obtained was three times higher (36.9%) than that obtained with the slow-rate freezing method (10.2%) used previously in the same centre. In conclusion, vitrification of human zygotes at the pronuclear stage seems to be a successful and reliable method with favourable outcomes and can be recommended as a routine technique for cryopreservation of human embryos.