030 Fresh and frozen ovary Transplants: Over 7 years of ovarian function with 14 babies from 12 transplants, a robust technique

Abstract

The aim of this lecture is to summarize the state-of-the-art of ovarian tissue cryopreservation and transplantation. This clinical experience will then be applied to understanding (1) the mechanism for initiation of primordial follicle development, (2) controversially why cancer cells metastases do not severely limit the transplantation of frozen ovary tissue back to cancer patients, and (3) what we can learn from comparing ovarian embryology to testis embryology. First, all nine fresh ovary transplants were between identical twins all ovulating and menstruating, resulting in eleven healthy babies in seven of the nine recipients. Recipients always reinitiated ovulatory menstrual cycles and normal Day 3 serum FSH levels by 4-1/2 months. Most conceived naturally (three of them twice or three times from the same graft). Duration of function of fresh ovarian grafts, contrary to initial expectation, indicated a very acceptable or minimal oocyte loss from ischemia time. Grafts of just modest portions of ovarian tissue have lasted more than 7 years. The same surgical techniques were then applied to 4 frozen ovary tissue transplants, up to 14 years after the ovary had been frozen, all resulting in normal ovulation and in 3 more healthy babies. Around the world, the number of healthy babies from ovary grafts has now risen to over 30, with more than 20 from frozen grafts. Although ovary freezing and transplantation has been referred to as “experimental” for preserving fertility in cancer patients, it should be noted that virtually all of the babies born from fertility preservation for cancer patients, have resulted thus far from ovary tissue freezing, rather than from egg freezing. As to the efficiency of ovarian tissue freezing compared to the use of fresh tissue, slow freeze has resulted in only a 60% loss of oocytes, and vitrification has resulted in no observable loss. For practical purposes therefore both techniques are reliable. However, in vitro studies in humans, and in vivo studies in bovine, show that vitrification of ovarian tissue, may nonetheless be an improvement over slow freeze. The basic science concept of vitrification is to completely avoid any ice crystal formation by using a very high concentration of cryoprotectant and a very rapid rate (virtually “instant”) of cooling. This is quite different from classic slow freeze cooling which relies on a partial and very gradual removal of water from the cell by encouraging ice crystal formation preferentially on the outside of the cell, drawing the water out. A comparison has to be made between vitrification for ovarian tissue versus vitrification of mature eggs and embryos. For mature eggs and embryos, there is first an equilibration in 7.5% EG and 7.5% Me 2 SO followed by a final solution of 15% EG and 15% Me 2 SO with 0.5 molar sucrose. It is very important to allow enough time for full absorption of this more concentrated cryoprotectant solution usually more than one minute, as these solutions, contrary to myth, are not toxic. However, for ovarian tissue, there must be a longer incubation in 7.5% EG and 7.5% Me 2 SO, and then a later incubation in a denser 20% EG and 20% Me 2 SO with 0.5% sucrose, to make certain there is full absorption of cryoprotectant. In all cases, the eggs or embryos or ovarian tissue must not be frozen in a droplet (even a tiny microdroplet) as this would slow the rate of freeze and thaw. The ovarian tissue must have fully absorbed the cryoprotectant, but be “dry” on the outside. Just as thawed ovarian tissue works as well as fresh, thawed embryos have just as good a success rate as fresh embryos. With vitrification, embryos can be frozen with impunity, whereas with slow freeze (like with ovarian tissue) there is some viability loss. A comparison of the embryology and anatomy of the ovary and the testis is very instructive for two issues, (1) the initiation of follicle development, and (2) the low risk of neoplastic metastasis to the ovary cortex and, therefore, the safety of ovarian cortical cryopreservation and transplantation in cancer patients. The ovarian cortex is actually identical to the tunica albuginea of the testis, except that in the male the germ cell cords to not invade the testes of boys and men with leukemia, but uncommonly the tunica albuginea of the testis. This phenomenon explains both of these issues. It is clearly possible to preserve and restore fertility, using ovary and egg or embryo freezing in young women with cancer who are undergoing otherwise sterilizing chemotherapy and radiation. But this approach can also be used for any woman who wishes to prolong her reproductive lifespan. It may thus eventually obviate the growing worldwide epidemic of female age-related decline in fertility, and even could eliminate menopause. Source of funding: None declared. Conflict of interest: None declared. silber@infertile.com