NEWS

Cell Death Genes May Hold Clues to Preserving Fertility After Chemotherapy

Tom Reynolds

In hope of finding treatments to preserve fertility and fend off premature menopause, scientists are dissecting the genetic pathway driving cell death in the oocytes of women undergoing cancer therapy.

Other researchers, meanwhile, are working toward the same goal by refining ovary cryopreservation, a technique that involves removing and freezing portions of the ovary and reimplanting them later.

And combining the two approaches might further improve the chances of success, according to leading investigators in the field.

Oocytes are among the cells most sensitive to collateral damage from both chemotherapy and radiotherapy, raising concerns and potential health risks for women surviving diseases such as breast cancer, Hodgkin's disease, and leukemia. Women relatively close to menopausal age are most vulnerable: One study of chemotherapy-treated breast cancer patients found that 92% of women over 40 had irreversible amenorrhea, compared with 31% of those under 40. The recent trend toward delayed childbearing compounds the problem.

Using oocyte and embryo cultures and knockout mice, Jonathan L. Tilly, Ph.D., of Massachusetts General Hospital in Boston, and colleagues are mapping the apoptosis signaling pathway underlying germ cell destruction in chemotherapy, and identifying key genes and proteins as potential targets for inhibitors to block the path. In a set of experiments published in the November 1997 Nature Medicine, they treated mouse oocytes and live mice with doxorubicin, a drug known to kill oocytes and reduce fertility in cancer patients. They then observed how chemical or genetic manipulations affected the drug's capacity to inflict damage.



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Dr. Jonathan L. Tilly

 
First they looked at inhibitors of ceramide, a sphingolipid molecule implicated in a variety of cells as an early messenger signaling apoptosis in response to stress. One ceramide inhibitor, sphingosine-1-phosphate, protected oocytes from death, while another inhibitor had no effect. These observations showed which of two alternate ceramide pathways is activated in drug-exposed germ cells, suggesting one point where the signal might potentially be interrupted.

Tilly and colleagues are following up with studies of mice lacking an enzyme responsible for ceramide generation. They also examined the roles of the death-susceptibility gene bax and the tumor suppressor p53. "We found that bax is key, because both in vitro and in vivo, if the Bax protein is not there, the oocytes no longer respond with death," Tilly said.

Complete Protection

"And the in vivo protection was complete: bax knockout mice had completely preserved ovaries after being given chemotherapy, while wild-type mice had a dramatic loss in the stockpile of primordial follicles." (Primordial follicles — a woman's endowment of dormant oocytes surrounded by the somatic cells that nurture them — are her insurance for future fertility and ovarian function. Because it is these that bear the brunt of damage from drugs, not the follicles growing in preparation for ovulation, the devastation may initially be silent: A woman may menstruate for months after chemotherapy, unaware that her long-term reproductive capacity has been destroyed.)

The finding that p53 is not required for drug-induced oocyte destruction "was quite striking," Tilly said, "because p53 is so critical for tumor cell death following chemotherapy. It pointed to the fact that tumor cells and germ cells aren't identical in what signals they use to get to the bottom line — death."

Less surprising was the observation that members of the caspase gene family are required for oocyte death, because caspases are thought to be the executioners of most cell death. But a study published in the May 1998 Genes and Development by Tilly, Junying Yuan, Ph.D., of Harvard Medical School, Boston, and colleagues yielded a more important finding with potential clinical value. This was the discovery that it is specifically caspase-2 that executes chemotherapy-mediated oocyte death.

"To us that's exciting because caspase-2 is relatively dispensable for most other paradigms of death in the body," Tilly explained. "It tells us that if we can design a specific caspase-2 inhibitor, it may not have any other effects on the body except for preserving germ cells."

Tilly pointed to several caveats. Before apoptosis inhibitors can safely be used to preserve fertility, scientists must ensure that they are not preserving eggs that have suffered severe genetic damage from drugs or radiation.

One cause for optimism in this regard is that women who have naturally remained fertile after cancer therapy have been able to produce healthy children without an elevated incidence of birth defects.

Fertility is not the sole reason for seeking to save a woman's ovaries. It is possible that in some women, inhibiting oocyte death may not preserve fertility but may preserve enough ovarian function to prevent early menopause and its associated problems — osteoporosis, cardiovascular disease, and sexual dysfunction. But for breast cancer patients, active, estrogen-producing ovaries may be a mixed blessing or worse: Still unresolved is the question of whether women with intact ovarian function run a higher risk of disease recurrence.

Ovarian Grafts

Approaching fertility preservation from another direction, Roger Gosden, M.D., at the University of Leeds, England, has been working for more than a decade to develop methods of freezing and transplanting ovarian tissue in women and girls who must undergo cancer therapy (see News, Sept. 4, 1996).



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Dr. Roger Gosden

 
Gosden works with animal models, primarily sheep, and with human volunteers. In the January 1999 Endocrinology, Gosden and colleagues at the University of Edinburgh reported a study in which they demonstrated extended post-transplant life for frozen and thawed ovarian grafts. They removed sheep ovaries, froze them, then transplanted strips of ovarian tissue into the same animals. Nearly 2 years after autotransplantation, all eight ewes had normal estrous cycles despite a drastic reduction in the number of primary follicles.

Gosden noted, however, that the animals' levels of follicle stimulating hormone were elevated, a signal that the grafts had only a short life span remaining. An earlier study showed ewes with grafts left in 3 to 4 months had successful pregnancies and live births.

Gosden said many possibilities remain to be explored. These include removing one of a woman's ovaries while leaving the other intact through cancer therapy. The ovary left in situ might be sterile, but because it will have an intact vasculature, it could provide a good site for grafting the frozen ovary. Another approach is grafting ovarian tissue at other sites in the body, such as under the skin of the groin or forearm. The eggs could be harvested for in vitro fertilization when a woman wants to become pregnant.

"We've done this on animals and I'm sure it will work in humans," he said. "Wherever there's a good blood supply, the ovary can work and produce hormones and grow follicles."

A potential concern with transplanting tissue back into a cancer patient is that the graft might re-introduce neoplastic cells and cause a new cancer to arise. Gosden is studying this risk, which he said is probably negligible in some diseases, such as Hodgkin's, but may be a concern with breast cancer or leukemia. This potential risk is one reason why the ideal technology would be to separate frozen and thawed ovarian tissue into individual primary follicles in the test tube, then grow them into mature eggs ready to be used in in vitro fertilization.

Premature Use

A pioneer and enthusiastic advocate of ovary cryopreservation, Gosden nevertheless does not believe it is ready for routine use in the clinic. Particularly in Europe, he said, "there are already hundreds if not a few thousand patients who have had ovarian tissue cryopreserved for clinical purposes, which I think is a bit premature outside the context of a serious clinical trial."

Movement toward widespread clinical use has been slower in the United States, he said, after the American Society for Reproductive Medicine in 1996 criticized a clinic offering the service for a fee, cautioning that the technique remains experimental. In the United Kingdom, the Royal College of Obstetrics and Gynecology is developing a voluntary code of good practice to govern tissue freezing.

Gosden plans to visit Tilly's lab to learn more about the molecular side of oocyte preservation, which could potentially improve both current preservation techniques and the prospects for in vitro oocyte growth.

"Our limitations at the moment are probably because we don't understand what cell survival molecules are needed to keep the follicles going," he said. "With grafts, we put them back in the body and they have everything they need. But in vitro we just haven't hit on the right cocktail yet — that's why we can only grow follicles so far before they start to pack up. So by using the Bax-deficient mice, for example, we might find that we'd be able to more successfully culture follicles in vitro."

Gosden's research "is a great complement to our work," Tilly added, "and we hope both approaches will prove to be fruitful."


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