From a Backup Technology to a Strategy-Outlining Approach

The Success Story of Cryopreservation

Gábor Vajta; Anikó Reichart; Filippo Ubaldi; Laura Rienzi

Disclosures

Expert Rev of Obstet Gynecol. 2013;8(2):181-190. 

In This Article

Cryopreservation-based New ART Strategies

MII Oocytes

Cryopreservation of in vivo-matured oocytes has been a demanding task and a frustratingly inefficient procedure for decades, and accordingly this option was not considered as viable in assisted reproduction. Even the theoretical opportunity of a possible application was disregarded; accordingly, the occurrence of an efficient method was followed by more neglect than enthusiasm. During informal discussion, many embryologists asked: "nice achievements, but do we really need it? What for?"

Fortunately, the situation has changed markedly, although the geographical distribution of babies born after oocyte vitrification still shows a strange variation, depending mostly – but not entirely – on the legal situation. In some countries including Italy, where embryo cryopreservation was banned, embryologists turned energetically to oocyte cryopreservation, while others have almost entirely neglected this possibility. Other countries (especially those in Latin America), where financial compensation for oocyte donors was legally permitted or tolerated, have adopted vitrification methods with great success, and Latin American embryologists have also propagated the success to other regions. On the other hand, progress in regions including the USA, Australia and especially north western Europe was extremely slow, especially compared with the most advanced stage of ART in these countries.

Although lately promising signs show increasing application all over the world – for example, according to recent reports, more than half of North American ART clinics offer oocyte cryopreservation[50,51] – it is probably worth summarizing the possible areas and situations where oocyte cryopreservation could be considered as an option or even the only solution. These include fertility preservation for medical reasons (treatment of malignant diseases, surgical ovary removal, premature menopause, and so on), or for social reasons to expand the fertile age.[52,53] This latter indication may be the subject of debate, however, considering the handicapped situation of women in reproduction with more suffering but fewer possibilities (fewer gametes, shorter fertile period), as well as the changing role of females in modern society, so any nonmedical restriction in this area may be regarded as a dictate of the majority and the neglect of a reasonable need of a (rather considerable) minority.

Additional indications may include logistic reasons, for example, lack of sperm after successful oocyte aspiration. Oocyte cryopreservation can also be part of routine infertility treatments, with efficiency comparable to embryo cryopreservation,[41,54] and may help to overcome legal restrictions, religious concerns or problems with the fate of embryos of divorced couples. Thus far, the most exploited application of oocyte cryopreservation is oocyte donation,[55] where oocyte banks may offer higher safety to prevent disease transmission, less waiting, more choice and more efficiency to use appropriately the valuable biological material.[56]

Blastocysts

As already mentioned, a decade ago one important argument against blastocyst culture was the low efficiency of cryopreservation in that stage. Introduction of vitrification has not only resolved the problem, but according to concordant observations of many clinics, considerable improvement in the overall efficiency (i.e., pregnancy or birth rates) can be achieved. Apart from the very unlikely possibility that vitrification improves the in vivo developmental competence of blastocysts, the most feasible explanation is the more appropriate uterine environment. According to the unpublished observation of the authors, the improvement can be as high as 15% in pregnancy rates. According to other groups, higher implantation rates can be achieved with transfer of cryopreserved embryos both in normal and high responders. Impaired endometrial receptivity can account for most implantation failures in the fresh group.[57,58] Natural cycles provide better results than even mild hormonal preparation.[59–61]

Additional benefits of transfer of cryopreserved embryos are the decreased risk of ovarian hyperstimulation syndrome[62,63] and perinatal morbidity,[45] therefore increased safety for mother and child,[31] although this statement is obviously valid for oocyte cryopreservation as well.[41,54] Transfer of cryopreserved single blastocysts was also found to decrease the risk of ectopic pregnancy.[64] Moreover, blastocyst cryopreservation reduces the number of embryos to be cryopreserved.[65] Considering all these benefits, a new embryo transfer strategy has been proposed of cryopreserving all embryos in the stimulated cycle and performing transfer exclusively in the subsequent cycles.[59]

Induced Blastocyst Collapse

Artificial shrinkage of blastocysts, after or especially before cryopreservation[66,67] may further improve the overall efficiency of cryopreservation. Although this approach was introduced as early as 10 years ago, it was mostly disregarded for a long time, predominantly because the improved outcome could not be detected in vitro, where the best methods resulted in up to 100% survival rates. Recent data, however, confirm the observation of the first report; that artificial shrinkage increases implantation rates, especially when applied at the expanded blastocyst stage.[68,69] Two mechanisms are supposed to explain this improvement. First, the large volume of the blastocoel does not allow proper equilibration during the short exposition to the concentrated cryoprotectant solution, and ice may be formed inside the blastocoel causing mechanical damage. Second, the prolonged toxic effect of cryoprotectants slowly leaving the blastocoel at the highly fragile stage after vitrification. Different methods were suggested to induce artificial shrinkage: incubation in sucrose solution is probably the simplest but least efficient approach; microsuction and application of a laser shot on the trophectodermal layer may be equally efficient, but the latter one is much easier.[67,69,70]

The laser shot may also be accompanied to assisted hatching. Although results are still controversial,[71] some authors found increased survival, implantation and pregnancy rates after opening the zona pellucida.[72]

Blastocyst Biopsy

The two procedures above are intrinsic elements or can be easily associated with blastocyst biopsy performed for either preimplantation genetic diagnosis or screening. While the former one has clear indication and is widely used, the initial enthusiasm towards preimplantation genetic screening was not supported by clinical data: embryo biopsy, genetic screening and transfer of embryos without detected chromosomal abnormalities did not increase implantation, pregnancy and live-birth rates.[73,74] However, most of these studies were performed on day 3 precompaction-stage embryos by using FISH for sample evaluation, and mostly fresh embryos were transferred.

On the other hand, blastocyst biopsy may create less harm; the mosaicism, although still existing, is less common than in earlier stages,[75] and with multiple cells, the molecular analysis is safer, especially when performed with more sophisticated methods including array comparative genomic hybridization, single-nucleotide polymorphism or quantitative PCR. Additionally, blastocyst biopsy significantly reduces the number of embryos and samples to be biopsied and analyzed, respectively. Although combination of these procedures resulted in increased pregnancy rates even after fresh transfer,[76,77] it may be a better strategy to combine the procedures with blastocyst cryopreservation – preferably by vitrification[78] – to obtain the possible combined benefits of all related procedures: blastocyst biopsy for selection of the best embryos, improved embryo evaluation, cryopreservation in an unstimulated cycle and increasing the chance of implantation and birth with the blastocoel collapse and probably also assisted hatching. The study of Schoolcraft et al. clearly demonstrates the viability of such approaches.[79]

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