Ovarian Cryopreservation by Michael Opsahl, MD

Body: 

Ovarian Cryopreservation 
by Michael S. Opsahl, MD 

 
Cryopreservation of ovarian tissue is a technique to bank oocytes (eggs) in situations where the woman may lose all her eggs from a medical treatment, disease process or even the natural loss from natural aging. Potential uses for this technique include restoring fertility and normal ovarian hormone production without the use of medications. The technique of ovarian tissue cryopreservation and transplantation of the thawed tissue is experimental. Since the first significant publication on ovarian tissue cryopreservation and transplantation in 1994(1), over 100 publications attest to the scientific interest in this technique or treatment strategy.(2-36)

Background

Several diseases and their treatments threaten to destroy all the eggs in a woman's ovaries. Diseases rarely have a direct effect on the eggs in the ovary. Exceptions include genetic disorders such as women with a single X chromosome (Turner Syndrome) or women missing a specific piece(s) of an X chromosome, in which case the eggs die quickly and the women have premature menopause. Chemotherapy or radiation used to treat cancer or some non-cancerous disorders have the unfortunate side effect of destroying the eggs in the ovary as well as the diseased cells.

Unlike sperm production in men which is continuous, women are born with all their eggs and they do not produce any more. The natural process of each menstrual cycle consumes approximately 500-1000 eggs until the supply is exhausted (about age 51, menopause). Any treatment that accelerates the loss of eggs threatens to decrease fertility and will cause menopause at an earlier age than expected. Surgery to remove all or a portion of one or both ovaries, some chemotherapy (cyclophosphamide, doxorubicin, vinblastine, etc.), and radiation therapy all have known toxic effects on eggs. The number of eggs that die from these treatments depends primarily on the agent(s), the dose, and the age of the woman when treated. The higher the dose and the older the woman, the more likely that most eggs will be lost and that menopause will occur.

Men have been able to cryopreserve (freeze) their sperm for decades. However, women have not been able to freeze their eggs reliably because the eggs are hard to retrieve and unfertilized eggs have generally not survived freezing. With the advent of IVF, egg or embryo freezing became possible. IVF has several significant limitations. IVF takes time to complete since the hormones used to stimulate the ovary are administered at specific times of the menstrual cycle and then are administered for 2-4 weeks. Many cancer patients must begin their cancer treatment before IVF can be completed. Many cancer patients are young and unmarried; therefore, they do not have a partner to provide sperm to fertilize the eggs. Cryopreservation of unfertilized eggs is an alternative that has gained interest with newer freezing techniques and there are successful pregnancies. Further, IVF is medically inappropriate for many women with hormonally responsive tumors such as breast cancer. The ovaries can be surgically moved from the field of radiation in selected cases but this technique does not help women with chemotherapy.

Ovarian hormone suppression (gonadotropin releasing hormone agonist) during chemotherapy significantly protected human eggs in one research study. Follow-up studies by other centers and with larger numbers of women will clarify the value of this approach. Gonadotropin releasing hormone agonist administration is inexpensive, relatively safe and unlikely to compromise other treatments; therefore, it deserves serious consideration despite limited data on its effectiveness. Donor eggs, for those women who have irreversible ovarian failure, provide very high rates of successful pregnancies, if other options fail.

Perhaps the largest group of women who may benefit from egg banking, are those women who delay child-bearing, for whatever reason, until the late thirties or forties. It is widely recognized that female reproduction becomes progressively more inefficient with advancing age and pregnancies are quite rare by the mid-forties. Whether, egg banking at an early age will be practical or effective remains speculative.

For all these reasons, a technique to bank eggs would allow women to have the same reproductive options as men when faced with a serious disease that threatens to destroy their eggs.

 

Dr. Roger Gosden's published paper in Human Reproduction in 1994 demonstrated the ability to cryopreserve ovarian tissue, transplant it after thawing and obtain functioning ovarian tissue that led to successful births of healthy animals.(1) His team also demonstrated long-term functioning of transplanted cryopreserved ovarian tissue for about two years in sheep.(21) The fertility rate after ovarian cryopreservation in mice was approximately 50 - 75% after ovarian tissue autotransplantation.(27;33) Human tissue was viable after transplantation into a mouse model.(9;15;29) Whether human results will be as successful as animal results will require time and experience. Consequently, until clinical trials demonstrate the viability of this technique, ovarian tissue cryopreservation and transplantation must be considered highly experimental.

Based on preliminary animal data, human trials of ovarian tissue cryopreservation began in 1995.(30) These ongoing trials have produced limited human data since humans often require many years to be free of cancer, or they may not have partners and may not be ready for pregnancy.

 

Techniques

Ovarian tissue cryopreservation begins with laparoscopy or mini-laparotomy. Ordinarily, the surgeon removes only one ovary to allow normal ovarian hormone production from the other ovary during treatment and because the woman may have ovarian function after treatment of her disease. The laboratory staff portion sections the ovarian cortex (which contains the eggs) into thin tissue slices. The tissue slices are cryopreserved at -196°C using specialized cryoprotectants and controlled-rate freezing equipment.

When the woman and her physicians feel fertility is appropriate, transplantation of the ovarian tissue strips can be attempted. 

In animals and humans, frozen ovarian slices have a high survival rate after thawing. The location for tissue transplantation could be in the abdomen near the fallopian tube to allow natural ovulation and conception. Natural conception occurred in all animal studies. The ovarian tissue began to function within several months after transplantation. The disadvantage of transplantation into the abdominal cavity is limited access, potentially lower viable tissue since ingrowth of blood vessels occurs primarily from only one side of the tissue, and adhesion formation (scar tissue) during the recovery period after surgery.

Recently, transplantation of human ovarian tissue into the forearm resulted in follicle formation and egg retrieval with a needle. Parathyroid tissue transplanted into the forearm routinely functions normally in patients with other medical disorders. The forearm is well-vascularized, easy to access, and is a site with little surgical risk. Consequently, because the ovarian tissue is surrounded with vascularized tissues, it may have a greater probability of short and long-term function. Transplantation of tissue into the forearm precludes natural conception. Assisted reproduction with IVF using eggs retrieved from the arm would necessarily be applied. Risks Surgery is necessary to remove the ovary. Despite routine surgical and anesthetic techniques, complications invariably occur but they are uncommon. The risks are no greater for this procedure than are the risks for any other patient who undergoes ovary removal. If the patient has cancer, the oncologist should address any pre-operative medical needs.

Most cancers do not spread (metastasize) to the ovary. However, if cancer cells are in the ovarian tissue at the time of cryopreservation, they may survive freezing and thawing and they will be transplanted with the normal ovarian tissue. Transplantation of tumor containing ovaries resulted in cancer in the recipient animals. (36) You should ask your doctor for his/her opinion about the likelihood of metastatic cancer cells in the ovary.

Usually, pathology evaluates a sample of the excised ovary. However, the sample is insufficient to confirm a cancer-free ovary. In selected cases, researchers may be able to stain a small portion of the ovarian tissue for cancer markers that to detect tumor cells before transplanting the tissue strips. Other researchers explored the possibility of growing a sample of the ovarian tissue in an immune-deficient mouse to detect occult cancer in the tissue.

Most oncologists believe transmission of cancer cells should be very remote; however, the possibility is real and collaboration between the oncologist and the reproductive surgeon is essential to minimize this risk.

Currently, the process requires removal of all or a part of an ovary and less often both ovaries. The oophorectomy, by definition, will remove 50% of the woman's eggs. It follows that this should increase the probability of menopause from the chemotherapy or radiation to follow if one believes that menopause occurs earlier in women with fewer eggs. Available research suggests that complete removal of an ovary before age 30 accelerates the age of onset for menopause to 44 years, on average. Removing an ovary after age 30 has less effect, incrementally, on menopause.(37-39)

 

Future Directions

Any number of questions remain unanswered, but of critical importance, to bring this technique into mainstream use. For example: How long can the tissue remain frozen and still function after thawing?, Where is the best location to transplant the tissue strips?, How much ovarian tissue is required to provide enough eggs for successful pregnancy?, How long will the tissue function after transplantation?, and many more.

A very interesting avenue of research involves the growth of ovarian tissue from one species in another species (xenograft).(5;6;9;11;15;26) If this technique works and proves safe, several problems become less of an issue.

First, tissue grown in another animal prevents cancer in the human recipient. Even patients with ovarian cancers may be able to use this technique to recover normal eggs without any associated cancer cells. Second, the ovarian tissue contains a limited number of eggs. A xenograft may allow more efficient maturation and retrieval of eggs for IVF. Third, the limited tissue strips might be used more gradually over time for additional children.

Fourth, if only small amounts of tissue prove adequate for IVF, then a small biopsy of tissue in a young woman may be a means of banking eggs if she later finds herself infertile.

Another line of research is egg freezing. Ovarian stimulation similar to IVF allows egg retrieval. The eggs are frozen before they are inseminated and fertilized. Subsequently, when the time is right, the eggs are thawed and inseminated. Embryos derived from cryopreserved unfertilized eggs have yield embryos that developed into normal children. The number of children is limited but growing and the number of successful births clearly exceeds that with ovarian tissue cryopreservation. Only a few centers offer this technique.

 

Questions to ask your doctor and yourself before consulting a reproductive specialist.

Q: What is the likelihood that sterility will occur after treatment? Most patients view their participation in experimental therapies differently when the risk of sterility is 10% versus 90%.

Q: What is the risk of cancer cells in the ovary at the time of ovarian cryopreservation? Clearly, if the risk of metastatic cancer in the ovary is high, this technique is probably not a good idea until methods of extracting the eggs from the tissue without transplantation are available.

Q: How much time do you have before cancer therapy begins? With limited time (less than one month), IVF is impractical but surgery can be performed very quickly - within a few days if necessary. With more time, IVF for production of eggs or embryos is a realistic option.

Q: Are you single or married? Single women have less reason to pursue IVF and embryo cryopreservation unless they have a partner. Embryos formed with donor sperm may be less desirable for producing a family when later a woman finds a partner. Egg freezing may be a better option for single women. Women with a partner should consider IVF and freezing embryos. The woman may want to retain sole control of the embryos once they are frozen. Occasionally, marriages or relationships fail and the former partner could prevent the embryos from being used if the couple agrees to joint control of the embryos.

Q: Do you have a tumor sensitive to reproductive hormones? Women with breast cancer, for example, probably do not want to risk stimulation of their cancer from the high estrogen levels generated during IVF. Nevertheless, many breast cancer survivors can become pregnant safely, so some method of conserving their eggs seems reasonable.

Q: Are you willing to be part of a highly experimental research protocol? Until more experience is available, your participation in ovarian tissue cryopreservation places you on the cutting edge of science. Participation involves risks, known and unknown, and likely expenses that are not covered by insurance. If you are a healthy woman who wants to delay conception and pregnancy for any reason, the experimental and uncertain success of ovarian tissue cryopreservation makes this a very controversial technique for you. In addition to the experimental aspects of participation, the act of removing all or a portion of an ovary may actually increase the probability of an earlier menopause, which is counter-productive to the reason for participating.

Michael Opsahl, M.D. retired from a prestigious career in the United States Navy in 1994 and joined the Genetics & IVF Institute. Dr. Opsahl is board certified in Obstetrics and Gynecology and Reproductive Endocrinology.

 

Selected scientific articles:

1. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum Reprod 9[4], 597-603. 1994. 
2. Wang H, Mooney S, Wen Y, Behr B, Polan ML. Follicle development in grafted mouse ovaries after cryopreservation and subcutaneous transplantation. Am J Obstet Gynecol 187[2], 370-374. 2002. 
3. Salle B, Demirci B, Franck M, Rudigoz RC, Guerin JF, Lornage J. Normal pregnancies and live births after autograft of frozen-thawed hemi-ovaries into ewes. Fertil Steril 77[2], 403-408. 2002.
4. Schnorr J, Oehninger S, Toner J, Hsiu J, Lanzendorf S, Williams R, Hodgen G. Functional studies of subcutaneous ovarian transplants in non-human primates: steroidogenesis, endometrial development, ovulation, menstrual patterns and gamete morphology. Hum Reprod 17[3], 612-619. 2002.
5. Snow M, Cox SL, Jenkin G, Trounson A, Shaw J. Generation of live young from xenografted mouse ovaries [In Process Citation]. Science 2002 Sep 27;297[5590], 2227. 2002. 
6. Wolvekamp MC, Cleary ML, Cox SL, Shaw JM, Jenkin G, Trounson AO. Follicular development in cryopreserved Common Wombat ovarian tissue xenografted to Nude rats. Anim Reprod Sci 65[1-2], 135-147. 2001. 
7. Liu J, Van der EJ, Van den BR, Dhont M. Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation. Biol Reprod 64[1], 171-178. 2001. 
8. Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 75[6], 1049-1056. 2001. 
9. Gook DA, McCully BA, Edgar DH, McBain JC. Development of antral follicles in human cryopreserved ovarian tissue following xenografting. Hum Reprod 2001 Mar;16[3], 417-422. 2001.
10. Callejo J, Salvador C, Miralles A, Vilaseca S, Lailla JM, Balasch J. Long-term ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab 86[9], 4489-4494. 2001. 
11. Metcalfe SS, Shaw JM, Gunn IM. Xenografting of canine ovarian tissue to ovariectomized severe combined immunodeficient (SCID) mice. J Reprod Fertil Suppl 2001;57, 323-329. 2001. 
12. Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, Watson AJ, Clayton JA, Harris M, Gosden RG, Shalet SM. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma. Lancet 357[9263], 1172-1175. 2001. 
13. Kagabu S, Umezu M. Transplantation of cryopreserved mouse, Chinese hamster, rabbit, Japanese monkey and rat ovaries into rat recipients. Exp Anim 49[1], 17-21. 2000. 
14. Cox S, Shaw J, Jenkin G. Follicular development in transplanted fetal and neonatal mouse ovaries is influenced by the gonadal status of the adult recipient. Fertil Steril 74[2], 366-371. 2000. 
15. Nisolle M, Casanas-Roux F, Qu J, Motta P, Donnez J. Histologic and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice. Fertil Steril 2000 Jul;74[1], 122-129. 2000. 
16. Oktay K, Newton H, Gosden RG. Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice. Fertil Steril 73[3], 599-603. 2000.
17. Candy CJ, Wood MJ, Whittingham DG. Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Hum Reprod 15[6], 1300-1304. 2000.
18. Imthurn B, Cox SL, Jenkin G, Trounson AO, Shaw JM. Gonadotrophin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting. Mol Cell Endocrinol 163[1-2], 141-146. 2000. 
19. Shaw JM, Cox SL, Trounson AO, Jenkin G. Evaluation of the long-term function of cryopreserved ovarian grafts in the mouse, implications for human applications. Mol Cell Endocrinol 161[1-2], 103-110. 2000. 
20. Weissman A, Gotlieb L, Colgan T, Jurisicova A, Greenblatt EM, Casper RF. Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol Reprod 60[6], 1462-1467. 1999. 
21. Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C. Endocrinology 140[1], 462-471. 1999. 
22. Callejo J, Jauregui MT, Valls C, Fernandez ME, Cabre S, Lailla JM. Heterotopic ovarian transplantation without vascular pedicle in syngeneic Lewis rats: six-month control of estradiol and follicle-stimulating hormone concentrations after intraperitoneal and subcutaneous implants. Fertil Steril 72[3], 513-517. 1999. 
23. Meirow D, Fasouliotis SJ, Nugent D, Schenker JG, Gosden RG, Rutherford AJ. A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer. Fertil Steril 71[5], 948-951. 1999. 
24. Salle B, Lornage J, Demirci B, Vaudoyer F, Poirel MT, Franck M, Rudigoz RC, Guerin JF. Restoration of ovarian steroid secretion and histologic assessment after freezing, thawing, and autograft of a hemi-ovary in sheep. Fertil Steril 72[2], 366-370. 1999. 
25. Aubard Y, Piver P, Cogni Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999 Aug;14[8], 2149-2154. 1999. 
26. Gunasena KT, Lakey JR, Villines PM, Bush M, Raath C, Critser ES, McGann LE, Critser JK. Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana) ovarian tissue. Anim Reprod Sci 53[1-4], 265-275. 1998. 
27. Sztein J, Sweet H, Farley J, Mobraaten L. Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking. Biol Reprod 58[4], 1071-1074. 1998.
28. Oktay K, Newton H, Mullan J, Gosden RG. Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone. Hum Reprod 13[5], 1133-1138. 1998. 
29. Gunasena KT, Lakey JR, Villines PM, Critser ES, Critser JK. Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice. Biol Reprod 57[2], 226-231. 1997. 
30. Opsahl MS, Fugger EF, Sherins RJ, Schulman JD. Preservation of reproductive function before therapy for cancer: new options involving sperm and ovary cryopreservation. Cancer J Sci Am 3[4], 189-191. 1997. 
31. Marconi G, Quintana R, Rueda-Leverone NG, Vighi S. Accidental ovarian autograft after a laparoscopic surgery: case report. Fertil Steril 68[2], 364-366. 1997. 
32. Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden RG. Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 67[3], 481-486. 1997. 
33. Gunasena KT, Villines PM, Critser ES, Critser JK. Live births after autologous transplant of cryopreserved mouse ovaries. Hum Reprod 1997 Jan;12[1], 101-106. 1997. 
34. Nugent D, Meirow D, Brook PF, Aubard Y, Gosden RG. Transplantation in reproductive medicine: previous experience, present knowledge and future prospects. Hum Reprod Update 3[3], 267-280. 1997. 
35. Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 11[7], 1487-1491. 1996. 
36. Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 11[8], 1668-1673. 1996. 
37. Melica F, Chiodi S, Cristoforoni PM, Ravera GB. Reductive surgery and ovarian function in the human--can reductive ovarian surgery in reproductive age negatively influence fertility and age at onset of menopause? Int J Fertil Menopausal Stud 40[2], 79-85. 1995. 
38. Gosden RG, Faddy MJ. Ovarian aging, follicular depletion, and steroidogenesis. Exp Gerontol 29[3-4], 265-274. 1994. 
39. Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 7[10], 1342-1346. 1992. 

 

 

 

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