EMBRYO CRYOPRESERVATION

TECHNIQUES

Frozen

The primary goal of a freezing program is to cause the least damage possible at the moment when gametes and embryos are exposed to a very low, non-physiological temperature. Protocols used nowadays involve, essentially, dry-freezing methods, which allow cell dehydration to prevent intracellular ice formation. Intracellular ice can cause mechanical damage to oocytes and embryos by fracturing and dispersing organelles, or by tearing the cytoplasmic membrane. This is why cryopreservation techniques are based on cryoprotectant agents and controlling ice formation in critical temperatures. That only comes to show that, when human cells are placed in a medium containing na intracellular cryoprotection agent, intracellular water immediately leaves the cell, due to high concentration of extracellular cryoprotectants. This causes the cell to shrink until it reaches the osmotic equilibrium by the slow cryoprotectant diffusion into the cell. Once this balance is attained, the cell resumaes a normal appearance. Cryoprotectant and water penetration rates depend on the temperature, balance is quickly reached in high temperatures. That’s why oocytes and embryos are usually placed in cryoprotectant medium at room temperature. Howewer, as some cryoprotectants like dimethylsulfoxide (DMSO) are toxic in high concentrations, they are used in low temperatures to reduce their bad efects.

Cryoprotectants are beneficial because of their ability to lower the solution freezing point. Solutions may be kept unfrozen until temperatures as low as –5o to –15oC because of super freezing (cooling to the maximum temperature below freezing point, wqithout formation of extra cellular ice). When solutions are super-frozen, cells aren’t properly dehydrated, which doesn’t cause increase in osmotic pressure with formation of extra-cellular ice crystals.

To prevent a super-freezing, na ice crystal is introduced, in a control process called seeding. This contributes to intra-cellular dehydration, through which water leaves the cell to acivate the extra-cellular ambient balance. If the freezing rate is too quick, the water can’t leave the cell fast enough, and, as temperature continues falling, a point is reached where the intra cellular solution concentration is not high enough to prevent the formation of ice crystals. Mammal oocytes and embryos, which possess relatively low proportion between area of superficies and volume contained therein, and contain high quantity cellular water, are usualy cooled down slowly (0.1o – 1o C/min), to permit adequate dehydration. With the use of cryoprotectants, membrane permeability is different for oocyte, embryo and blastocyst. It has been found, on the other hand, that certain the different cryoprotection agents are more convenient in certain different stages of gamete and embryo cryoprotection. DMSO and 1,2 propanodiol (PROH) are are frequently used for the freezing of embryos in a stage of little cell division, whereas propylene glycol (glycerol) is always used for blastocysts. All three cell agents have reasonable amount of small molecules able to easily penetrate the cell membrane. In addition to those agents, there are other extra cellular substances that help dehydrate and protect the cell. The most frequently used is sucrose, which possesses big non permeable molecules and exerts na osmotic effects that can help acelerate cell dehydration. Sucrose cannot be used alone, it is frequently used together with other usual intra-cellular cryoprotectants.

Freezing

If freezing is terminal at a relatively high temperature (> -30oC), the cell will carry more intra cellular ice than it would if frozen over a long period of time reaching lower temperatures (< -80oC). To protect the cells in this situation, it must be thawed quickly to induce rapid ice dispersion. Inversely, samples frozen at <-80oC must be thawed more slowly to permit gradual rehydration. If water penetrates the cell rapidly, the cell may swell or burst out. In this way, thawed samples are usually exposed pogressively in lower cryprotectant dilutions to remove the latter lightly and gently from the cell.

Vitrification

The idea of vitrification is to protect the cell totally avoiding ice crystal formation. In order to do this, cryoprotectant solutions must be risen to 40% (weight/volume) or more. DMSO is frequently used, although PROH, glycerol and other agents have been tested. Because high cryoprotectant concentrations are toxic at room temperature, embryos are exposed to the agents at 0oC. Samples are placed directly into liquid nitrogen (LN2) without first introducing the seed; viscosity is so high that the solutions solidify as in a state of vitrification. Vitrificated samples must be thawed in salt water, which can be inconvenient. Other attempts using this technique have been done in animals.

Ultra-rapid freezing

Vitrification and ultra rapid reezing are, in fact, very techniques. If no ice crystal forms during this last process, the result is vitrification. However, the difference is that samples are handled at room temperature before they go to the ice baths.

With ultra-rapid freezing, samples are exposed for short periods of time (2-3 min) in relatively high DMSO (3.5M) and sucrose (0.25M) concentrations, followed by immediate immersion into LN2. Samples are quickly unfrozen in Mary’s bath at 37oC for the removal of the cryoprotectant in one only step. A certain number of children have been born using simple and and quick techniques like that. Gordts et al. Have reported before 1990 4 pregnancies after ultra rapid freezing of oocytes in pronuclear stage. In this stage, high oocyte survival was observed in oocyted having pronuclei, as opposed to cleaved embryos. This finding was also reported by other investiogators. In contrast, Lai et al. Reported imn 1996 a survival rate of 83% (with at least one intact blastomere) and a 16% birth rate for cleaved embryos frozen by the ultra rapid freezing technique, and then thawed. The distribution of mitochondria and global subcellular structures are described as normal after this type of freezing.

Oocytes

Oocytes are easily frozen if they possess a nucleus. Studies using human oocytes in the vesicula germinativa stage, collected from stimulated and non stimulated cycles, have shown that this stage has acceptable survival and maturation rates after thawing. Furthermore, they have shown that the oocyte percentage in the vesicula germinativa stage, frozen and thawed, if they have a normal meiotic spindle, is similar to the control group which hasn’t been frozen. This finding is not the same due to a low percentage of of normal meiotic bundle observed in oocytes frozen and thawed in metaphase II. Other abnormalities have been described in thawed mature oocytes including: plasmatic membrane rupture, extense ooplasm unorganization, lack of nucleus, frequent triploidy. However, concern about aneuploidy potential has been the major issue to discourage most clinical programs to utilize technique with mature oocytes.

In spite of this preoccupation, many pregnancies have been reported after mature oocyte freezing and thawing, but few pregnancies have been described in the last 10 years. It is expected that ultra-rapid freezing enhances future results.

The ability to freeze unfertilized human oocytes may be priceless in some cases. A young woman under radition treatment, or one that has to undergo na ovarian loss, may benefit a lot from it. Similarly, na older woman willing to stock multiple oocytes before losing ovarian function can be helped by this technology. Donated oocyte banks may be created in the same way as sperm banks, to serve the groing population of women in need for donated eggs.

Pre-zygotes

Oocytes penetrated by spermatozoa represent a completely different treatment option. The success of freezing this cell stage lingers for over a decade and culminated in thousands of births. This idea of losing the spindle in the pre-zygote is for the most part responsible for the excellent survival and implantation potential. It is easy to determine whether the pre-zygote survived the thawing or not. When the membrane is not intact, the cell seems to be flat and presents a dark color. Left in culture for 15-24 hours, a healthy oocyte with pronuclei enters syngamy, completes the fertilization process and goes on to the first cleavage. Cell division is the true indicator of survival after thawing; < 5% of pre-zygotes that appear to be healthy after thawing fail to proceed in this pattern. In spite of the good results found after freezing the cell at this stage, there are some disadvantages. Because pre-zygotes are frozen before cleavage, there is no pattern for the morphological parameters to help in this selection. Consequently, pre-zygotes having poor development potential are sometimes frozen. It is disappointing to freeze a large number of pre-zygotes knowing that those ones that remained in culture showed morphological and developmental alteration after 2 to 3 days. In those cases, it would be more indicated to wait one or two days for freezing. However, it is important to freeze the pre-zygote before the pronuclei disappear, since waiting too long has a negative effect on the results. The urgency to begin freezing may be inconvenient for some programs without the adequate scientific team.

The thawed pre-zygote morphology is generally similar to its appearance before freezing. But occasionally the cytoplasm is clearer and there is a reduced number of organelles around the pronuclear structures. After thawing nucleoles are seen scattered inside the the pronuclei, in spite of their previous alignment in the pronuclear junctions before freezing. Na interesting observation (often made by the author) is where the two pronuclei condense into one big pronucleus during this procedure.

Pre-embryos

The first report of birth after freezing and thawing originated from a frozen pre-embryo. As is true for oocytes in pronuclear stage, pre-embryos in cleavage stage develop after thawing and contribute to acceptable pregnancy rates. Aslmost any cleavage stage can be successfully frozen, from the two cell stage to the blastocyst. Pre-embryo freezing is convenient because, differently from pre-zygotes, there is no time limit.

Besides, morphology and growth rate are known, permitting the selection of totally viable embryos. It’s becoming more end more common to select best embryos for fresh transference and freeze all other ones having good morphology, but only after fresh ones are selected.

Survival is sometimes difficult to evaluate, because not all blastomeres survive the severities of freezing and thawing. Dead blastomeres may be scattered among the living ones, but are easily removed during the hatching procedure. In general, na embryo that has over 50% living blastomeres is considered to be a survivor. There is no convincing evidence that the loss of one or two blastomeres is clearly harmful for the embryos in their initial developmental stages. Despite this lack of evidence, it has been reported that completely intact human pre-embryos demonstrate higher implantation rates than those ones partially intact.

Blastocysts

Blastocysts have been of great interest in the last years. There is a growing trend in the direction of routine culture until this stage and, as a consequence, a routine of freezing expanded blastocysts. Many groups have reported successful freezing and living births, and many among them have used co-culture systems to sustain pre-embryo growth. Most reports on ongoing pregnancy between 10 to 20 % per transference surprisingly did not show significantly higher rates if compered to american statistics on initial stage freezing, but this could be related to the transference of a smaller number of blastocysts or a the indication of a blastocyst – uterus assinchrony. One study described the admirable rate of 100% survival with frozen blastocyst, but the pregnancy rate was just 16% per tranference. Although the pregnancy rate for blastocyst was higher than that with pre-embryos thawed in the initial stages (6%), it is difficult to determine the reasons for the low incidence of pregnancy in this last group.

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