Cryopreservation

….the frozen life in reproductive medicine!
Why is cryopreservation vital in reproductive medicine?
The science of reproductive medicine cannot advance if this technique does not develop; in fact good cryopreservation should be an integral part of any assisted conception program.

Cryopreservation of a cell involves cooling of a cell & its storage at a temperature, where all the metabolic cell functions are arrested. There exists a biological clock for all the events related to reproduction from the formation of gametes to their maturation, fertilization, cleavage till implantation and then to further growth of the fetus. Cryopreservation allows us to intervene in this timed procedure to curtail the further development of the cell by stopping this biological clock and recovering the cell in its same form and vitality when ever thawed.

History of cryopreservation
First live human birth using cryo-preserved sperm by slow freezing was in 1953 (Bunge RG et al). Ultra rapid freezing (Vitrification) was developed and applied to human sperms in 1942 by Pincus
The first pregnancy from frozen-thawed human embryos by slow freezing was reported in 1983 by Trounson et al, and then in 1985, Rall & Fahy, reported the efficacy of the vitrification (the rapid freezing) method, a simpler and better alternative for slow cryopreservation
In 1986, Chen reported the first pregnancy from a frozen thawed mature oocyte and in 1998, Tucker reported human pregnancy following freezing of immature oocytes.

What does cryopreservation of gametes & embryos involve?
Exposure to cryoprotectants and cooling to sub-zero temerature.
Storage in liqiud nitrogen at -196 degree centigrade for as long as desired.
Thawing successfully with no or minimal damage to the cell.

What all can be cryo-preserved and what is easier to freeze presently, is in the following sequence
1. Spermatozoa – ejaculated or surgically retrieved from testes
2. Preimplantation Embryos – pronuclear stage, cleavage stage and blastocyst
3. Oocytes – mature as well immature
4. Testicular tissue
5. Ovarian tissue

What are the benefits of gamete or embryo cryo-preservation?
Semen:

• Fertility preservation for one self
• Donor semen for sperm banking

Oocyte :

• Fertility preservation for one self, especially in women who don.t have a partner
• Oocyte banking/ donation
• Non-availability of semen after an oocyte retrieval in IVF
• Religious, legal or ethical objections to embryo cryopreservation as in some European countries like Germany and Italy

Embryo:

• Surplus embryos in an IVF cycle to be used subsequently in another cycle to create pregnancy without having to stimulate the women with injectable gonadotropins
• Fertility preservation in females having a partner
• Cycle cancellation due to risk of OHSS or sub-optimal endometrium
• Inappropriate synchronization between oocyte donor and recipient

Ovarian/Testicular tissue:
Fertility preservation by banking ovarian tissue

Drawbacks of cryopreservation is due to cryo-injury to the cell which leads to the following results

• Decreased motility & viability of the sperm by 10 – 20 %
• Approximately 90% embryos survive post thaw by the new rapid method of vitrification
• than 50% oocytes survive and even less fertilize successfully after ICSI with much lower pregnancy rates as compared to frozen embryos.

Drafted ICMR guidelines 2010

• ‘Gamete donation whether of sperms or oocytes must be anonymous’ (this is mandatory for the psychological health of the couple and the baby- not to have gametes from a known person. In life table analysis this has been routed as a reason for marital disharmony and breakdown, family breakups, disowning the child or parent and social discordance).
• Quarantine period of 6 months for all donated gametes is mandatory to prevent transmission of sexually transmitted diseases due to the donor being in the window period of diseases such as HIV 1&2 or hepatitis B
• Storage of Gametes & Embryos is allowed for a maximum of 5 years
• Sperm of a single donor not to be used more than 75 times in India to prevent higher chance of consanguinity
• Oocyte donation less than 6 times by a woman in her life with at least 3 months interval between OPU to prevent health hazards due to ovarian hyper-stimulation

Where does cryopreservation stand today?

• Most commonly & successfully used for semen and is the basis of semen banking
• Increasingly successful use for embryos because of decreases in rate of multiple pregnancies, decrease in cycle cancellation and decrease in overall cost & increases the cumulative pregnancy rates.

Why is cryopreservation less successful and difficult for oocytes ?

Cryopreservation difficult: This is because the oocyte is a large sized cell with an exposed meiotic spindle which contains the complete genetic material from the mother hence damage to this cell and meiotic spindle is very likely.

Collection difficult: For sperm banking anonymity is easy to maintain as semen can be collected at any time by a person himself whereas, oocyte collection involves stimulating ovaries by a series of daily injections, monitoring ovarian response and then collecting the oocytes under sedation or anesthesia surgically from the ovaries by ultrasound guided needles.

Complications more common: Ovarian stimulation also involves certain degree of discomfort due to ovarian hyper-stimulation which if becomes severe can lead to considerable morbidity due to ovarian hyper stimulation syndrome.