A. Sperm Cryopreservation
Freezing of sperm is a simple and useful technique to keep a seminal samplethat could be used in the future. The Cryopreservation of semen is done in the following way:
- Sample Collection:The patientmust have sexual abstinence for between 3 or 4 days. It is not suitable for many days more (the limits are not less than 2 or more than 5 days).
The sample should be obtained by masturbation in the clinic where the sperm will be treated. If the sample had to be takenat the patient’s home, it should be delivered at the clinic within one hour from the collection and kept at body temperature all the time.
The sample is collected in a sterile container, prior tohand washing and ensuring you do not miss part of the sample. If this occurs you must inform the clinic.
- Sample Analysis:The Andrology laboratory will perform a semen analysis to determine if the ejaculated sample has sufficient quality to be frozen.
- Freezing of the sample:once we have determined that the sample is valid to be frozen, it is added to a cryoprotectant medium and will be frozen with liquid nitrogen (- 196 ° C) vapour, in straws which will be stored in our sperm bank where they will remain perfectly identified until required its use.
Freezing of sperm is indicated in the following cases:
- Patients who are subjected to treatments of chemotherapy or radiation therapy which will therefore decrease and even undothe reproductive capacity of the male, or before undergoing a vasectomy.
- For assisted reproduction treatments (though a fresh sample is used), it is important to have a frozen sample for any unexpected events providing adequately peace of mind for the couple, sincesometimes it is not possible to obtain a satisfactory sample the day of fertilization.
- When there is a progressive deterioration due to known or unknown characteristics of the sperm. Over time it can drasticallydecrease the spermquality, or even reach azoospermia.
- When the number of spermatozoa are extremely lowso it is advisable to obtain sperm accumulation from different ejaculates, which will be freeze for later use. For example, in an artificial insemination.
B. Cryopreservation of oocytes
According to the research carried outinternationally, women reproductive capacity start declining from 35 years old, with a clear decrease in the quantity and quality of their oocytes. It is somewhat unfair since it is the age where the woman is more stable from the psychological, physical, intellectual, and professional point of view.
But that is the feminine nature;consequentlythe reproduction is very improbable at the age of 40 and almost impossible from 43 years old. On the other hand, there is not any case of maternity for woman older than 45 years old, after using their own oocytes in assisted reproduction.
In this regard, fertility treatment has achieved notably progress allowing women to have children at an older age that nature would allow.
One of the ways to preserve fertility is storing ovarian tissue or their oocytes (immature or mature) to fertilize them in the future. A utopia that is possible.It consists in freezing ovarian tissue or vitrification of oocytes.
Cryopreservation of ovarian tissuedirectly preserves part of the crust of the ovary to be later ended in the patient.There have already been many babies born thanks to this method, the first in the United States.
Vitrification of oocytes is the easiest and a more effective method and consists of super cooling of the ovule at high speed, preventing the formation of ice crystals inside the oocytes, since they are cells with a high content in water and ice crystals could damage the internal structures of cells causing them to die. In this way, the survival of these oocytes will be highly effective after thawing.
We will perform the fertilization of the oocytes at the moment a woman decides she is ready to get pregnant, resulting in a high rate of pregnancy, according to our results.
Thus, women can preserve the oocytes from their youth and use them at another time of their life. This will provide women with cancer who have to receive radio- or chemo-therapy, with the possibility of having their own baby in the future, and it is also an alternative for those who want to postpone motherhood, whether for professional or other reasons.
C. Cryopreservation of pre-embryos:
The pre-embryos with 2-6 or 8 cells that are not transferred to the uterus, in assisted reproduction treatment, are frozen in special tanks with liquid nitrogen at – 196 ° C. After that, the appropriated moment to defrost them will be carefully decided in order to transfer them into the patient uterus, to donate them to other couples or to donate them for research, according to the couple’s desire.
It is recommended that only good quality embryos are frozen. Once embryos have been defrosted, they will be left in a culture media for 24 hours to see if it is evolutionary, since it would continue to divide.
During the defrosting process the embryo blastomere could die. It has been previously noted that, at least, half plus one of the blastomere must remain alive, so that the embryo is evolutionary. That is to say, if the embryo had 8 cells prior the freezing process, after thawing at least 5 cells should survive. And above all, leave it for 24 hours in culture to see if it continues to divide.
It has been found that the freezing of embryos does not have any adverse effects on the rate of abnormalities at birth (1.8%) and that if there is a good program of embryonic freezing the pregnancy rate is very acceptable and with a lower risk of multiple pregnancy.
The transfers of frozen embryos can be done in both natural and artificial or replaced cycles, in which hormones are used for the preparation of the endometrium (estrogens and progesterones with or without analogues).
The freezing process
Spanish legislation establishes the obligation to cryopreserve all remaining viable embryos from assisted reproduction cycle. In addition, assisted reproduction centres should be equipped with the human and material resources as well as the appropriate technical resources and a mandatory insurancefor banks of pre-embryos.
There is aninformation leaflet and a consent formabout the treatments available for the couple or patients. In any case of existing frozen embryos, these will belong to the couple for a period of 5 years and at the end of the period, and if the couple do not express their interest in them, they will become property of the assisted reproduction clinic, with the aim of including these embryos in embryo donation protocol.
Both frozen pre-embryos and their possible use are regulated in the Spanish law 14/2006 of May 26th, about assisted human reproduction techniques.
In terms of technical and biophysical aspects of the cell freezing process, this requires an initial cell dehydrationas much as possible, in order to avoid, during the process, the formation of ice crystals in its interior that could damage it. In order to achieve this aim, cryoprotective substances will be employed, replacing the water that comes out of the cell. These permeable cryoprotectantsagents are the 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) and glycerol. This is usually used in conjunction with other non-permeable cryoprotectants, such as sucrose, which acts a hyperosmolar effect. To avoid intracellular ice crystals formation it will be necessary to inducethe formation of ice in the media away from the cells or embryos. This process is known as seedingprocess. Finally, albumin is frequently added to the freezing media to avoid the hardening of the pellucid zone during the freezing process.
The outcomes in terms of survival in the thawing of the pre-embryo may vary between 60-80% and pregnancy rates between 30-60% according to the different treatments.
D. Cryopreservation of pre-embryos to 2 pronuclei:
The protocol to freeze embryos in early stages of development, i.e. in the stadium of pronuclei, is similar to that it is usedfor embryos but with other freezing ramp times. In addition, all the considerations that have been indicated to freeze embryos must be applied rigorously including theaccomplishment of times and temperatures, the placement of the embryos in the straw, the realization of seeding and the rapid location of the embryos in theirchamber for storage. All these factors will influence the survival of embryos when thawing them.If the embryos to be frozen are of low quality, the survival and implantation will be also low.
Outcomes in terms of survival during the thawing of the pre-embryos to two pronuclei can vary between 80-100% and pregnancy rates between 50-80% according to the different treatments.
E. Cryopreservation of blastocysts
The blastocyst is an embryonic structure formed in the early gestation (embryogenesis), occurring about 5 or 6 days after fertilization and before implantation in the endometrium. It is formed by a prominent cavity, and between 70-100 cells. The ] blastocyst cells are pluripotent, i.e. the cells from the inner mass cell can become any type of tissue.
Only between 40 and 60% of the embryos manage to reach blastocyst, so the number of embryos to be transferred and freeze is also reduced.
Actually, the cultivation and freezing of blastocysts in vitro fertilization programs began in the early 1990s when the culture media were optimized and made possible to obtain good blastocysts with the cocultivos. Then there was a small transition and adaptation to use sequential culture media.
From the beginning, the freezing of blastocysts has been done in glycerol with multi-step slow protocols so that there was a good spread of the cryoprotectant, since the number of cells increased and thus changes in the surface/volume ratio. However, protocols with short or two stepsare already been employed with glycerol and sucrose providing the same or better results.
In our case, we use vitrification for the freezing of the blastocysts which consists in an ultra fast drop in temperature that “solidifies” the blastocyst with a score of 59% clinical pregnancy rate.
As it happens for embryos with 4-8 cells, the quality of the blastocyst that will be frozen will mark the result obtained when these are defrosted, i.e. the better the quality of blastocysts (blastocyst at day 5 or 6, expanded and with good quality inner mass cell) the greater probability of success.
In the same way, it is necessary to leave an incubation time of at least 4 hours after the defrosting process, to give time to the blastocyst to recover and then to observe their survival rate, before being transferred to the uterus.