Oocyte and Semen freezing-Dr.Akhila

                                             OOCYTE CRYOPRESERVATION

Human oocyte cryopreservation (egg freezing) is a procedure to preserve a woman’s eggs (oocytes). This technique was mainly developed to enable women who, due to studies or any other complication can´t deal with pregnancy during their most fertile years, to postpone their maternity until their personal situation is the right to form a family. Surprisingly, the uterus remains completely functional in most elderly women. This implies that the factor which needs to be preserved are the woman’s eggs. The eggs are extracted, frozen and stored. The intention of the procedure is that, in the future, the woman may choose to have the eggs thawed, fertilized, and transferred to the uterus as embryos to facilitate a pregnancy. The procedure’s success rate (being the chances of a live birth using frozen eggs) varies depending on the age of the woman, and range from 14.8 percent (if the eggs were extracted when the woman was 40) to 31.5 percent (if the eggs were extracted when the woman was 25).

Indications

Oocyte cryopreservation can increase the chance of a future pregnancy for three key groups of women:

1.Those diagnosed with cancer who have not yet begun chemotherapy or radiotherapy

2.Those undergoing treatment with assisted reproductive technologies who do consider embryo freezing an option

2.Those who would like to preserve their future ability to have children, either because they do not yet have a partner, or for other personal or medical reasons.

1] Chemotherapy and radiotherapy are toxic for oocytes, leaving few, if any, viable eggs. Egg freezing offers women with cancer the chance to preserve their eggs so that they can attempt to have children in the future.

2.Oocyte cryopreservation is an option for individuals undergoing IVF who object, either for religious or ethical reasons, to the practice of freezing embryos. Having the option to fertilize only as many eggs as will be utilized in the IVF process, and then freeze any remaining unfertilized eggs can be a solution.

3.Additionally, women with a family history of early menopause have an interest in fertility preservation. With egg freezing, they will have a frozen store of eggs, in the likelihood that their eggs are depleted at an early age.

Method

The egg retrieval process for oocyte cryopreservation is the same as that for in vitro fertilization. This includes one to several weeks of hormone injections that stimulate ovaries to ripen multiple eggs. When the eggs are mature, final maturation induction is performed, preferably by using a GnRH agonist rather than human chorionic gonadotropin (hCG), since it decreases the risk of ovarian hyperstimulation syndrome with no evidence of a difference in live birth rate (in contrast to fresh cycles where usage of GnRH agonist has a lower live birth rate).

[2] The eggs are subsequently removed from the body by transvaginal oocyte retrieval. The procedure is usually conducted under sedation. The eggs are immediately frozen.

The egg is the largest cell in the human body and contains a high amount of water. When the egg is frozen, the ice crystals that form can destroy the integrity of the cell. To prevent this, the egg must be dehydrated prior to freezing. This is done using cryoprotectants which replace most of the water within the cell and inhibit the formation of ice crystals.

Eggs (oocytes) are frozen using either a controlled-rate, slow-cooling method or a newer flash-freezing process known as vitrification. Vitrification is much faster but requires higher concentrations of cryoprotectants to be added. The result of vitrification is a solid glass-like cell, free of ice crystals. Indeed, freezing is a phase transition. Vitrification, as opposed to freezing, is a physical transition. Treatment with the first life birth following vitrification of oocytes achieved in 1999.

[3] Vitrification eliminates ice formation inside and outside of oocytes on cooling, during cryostorage and on warming. Vitrification is associated with higher survival rates and better development compared to slow-cooling when applied to oocytes in metaphase II (MII).

[4] Vitrification has also become the method of choice for pronuclear oocytes, although prospective randomized controlled trials are still lacking.

During the freezing process, the zona pellucida, or shell of the egg can be modified preventing fertilization. Thus, currently, when eggs are thawed, a special fertilization procedure is performed by an embryologist whereby sperm is injected directly into the egg with a needle rather than allowing sperm to penetrate naturally by placing it around the egg in a dish. This injection technique is called ICSI (Intracytoplasmic Sperm Injection) and is also used in IVF.

Immature oocytes have been grown until maturation in vitro, but it is not yet clinically available.

[5]Success rates

The percentage of transferred cycles is lower in frozen cycles compared with fresh cycles (approx. 30% and 50%).

Risks

The risks for women undergoing egg freezing can include: vaginal/uterine bleeding from the oocyte recovery procedure; developing ovarian hyperstimulation syndrome (OHSS) as a reaction to the hormones used to induce hyperovulation (producing more than one egg); and liver failure. There are indications that ovarian stimulation may increase risk of breast, uterine and other cancers, however this remains inconclusive.[12] The long-term effects of egg extraction on women’s bodies have not been well studied.

There may also be some risks to any resulting child. These include IVF-associated risks such as multiple pregnancy, pregnancy-induced high blood pressure, premature delivery, operative delivery and infants displaying low birth weight.[12] There also remain potential unknown risks caused by long-term freezing. Expanded use of Intracytoplasmic sperm injection (ICSI) to inject a single sperm into a thawed egg could additionally be a cause for concern as this method has been associated with a higher rate of birth defects.

                                                 SEMEN CRYOPRESERVATION

 Semen cryopreservation (commonly called sperm banking) is a procedure to preserve sperm cells. Semen can be used successfully indefinitely after cryopreservation. For human sperm, the longest reported successful storage is 24 years. It can be used for sperm donation where the recipient wants the treatment in a different time or place, or as a means of preserving fertility for men undergoing vasectomy or treatments that may compromise their fertility, such as chemotherapy, radiation therapy or surgery.

Freezing

The most common cryoprotectant used for semen is glycerol (10% in culture medium). Often sucrose or other di-, trisaccharides are added to glycerol solution. Cryoprotectant media may be supplemented with either egg yolk or soy lecithin, with the two having no statistically significant differences compared to each other regarding motility, morphology, ability to bind to hyaluronate in vitro, or DNA integrity after thawing.[2]

Additional cryoprotectants can be used to increase sperm viability and fertility rates post-freezing. Treatment of sperm with heparin binding proteins prior to cryopreservation showed decreased cryoinjury and generation of ROS.[3] The addition of nerve growth factor as a cryoprotectant decreases sperm cell death rates and increased motility after thawing.[4] Incorporation of cholesterol into sperm cell membranes with the use of cyclodextrins prior to freezing also increases sperm viability.[5]

Semen is frozen using either a controlled-rate, slow-cooling method (slow programmable freezing or SPF) or a newer flash-freezing process known as vitrification. Vitrification gives superior post-thaw motility and cryosurvival than slow programmable freezing.[6]

Thawing

Thawing at 40 °C seems to result in optimal sperm motility. On the other hand, the exact thawing temperature seems to have only minor effect on sperm viability, acrosomal status, ATP content, and DNA.[7] As with freezing, various techniques have been developed for the thawing process, both discussed by Di Santo et al. (2012)[8]

Refreezing

In terms of the level of sperm DNA fragmentation, up to three cycles of freezing and thawing can be performed without causing a level of risk significantly higher than following a single cycle of freezing and thawing. This is provided that samples are refrozen in their original cryoprotectant and are not going through sperm washing or other alteration in between, and provided that they are separated by density gradient centrifugation or swim-up before use in assisted reproduction technology.[9]

Effect on quality

Some evidence suggests an increase in single-strand breaks, condensation and fragmentation of DNA in sperm after cryopreservation. This can potentially increase the risk of mutations in offspring DNA. Antioxidants and the use of well-controlled cooling regimes could potentially improve outcomes.

In long-term follow-up studies, no evidence has been found either of an increase in birth defects or chromosomal abnormalities in people conceived from cryopreserved sperm compared with the general population.

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