Rejuvenation Biotechnology: Toward the Indefinite Postponement of Menopause
Menopause is brought on by the loss of the ovaries' ability to produce new egg cells, which in turn leads to a disruption of the normal hormonal feedback system between the ovaries and the centers in the brain that regulate their production of estrogen and other hormones. The menstrual cycle is governed by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are released by the pituitary gland in the brain; the pituitary, in turn, is governed by another brain gland, the hypothalamus, which receives hormonal feedback from the ovaries. In youth, the system of hormonal feedback between the hypothalamus, the pituitary, and the ovarian follicles regulates the maturation and release of egg cells from the egg-cell-producing follicles of the ovaries, and through them, the orchestrated release of the female sex hormones estrogen and progesterone over the course of the menstrual cycle.
For reasons we'll discuss below, number of viable egg cells and follicles in a woman's ovaries begins to decline below a threshold level in early midlife. As a result, the pituitary gland needs to pump out higher levels of FSH and LH In order to ensure the release of an egg each month, and to maintain the associated feedback signal of estrogen from the ripening follicle.
For two or three decades, rising levels of FSH is enough to stimulate one the dwindling supply of follicles to release an egg cell each month and to produce key sex hormones. But even as it does, the degenerative aging of a woman's egg cells and follicles continues silently. Not only does the follicles' ability to mature and release egg cells decline, but the integrity of those eggs becomes progressively lower as a result of aging damage. Notably, aging egg cells acquire chromosomal abnormalities, which is the main reason why older mothers are at higher risk of their children having Down's syndrome and other congenital disorders. Menopause occurs when so few viable follicles remain that the ovaries are no longer able to keep maturing and releasing eggs in response even to very high levels of LH and FSH. The monthly cycle comes to an irregular halt, and the ovaries' production of estrogen plummets, leading to the classic menopausal symptoms: hot flashes, insomnia, mood changes, and loss of vaginal lubrication, tissue elasticity and pH, as well as to the loss of bone mass and quality that culminates in osteoporosis.
SENS Research Foundation works to catalyze the development of rejuvenation biotechnology: a new class of medicines that will keep us young and healthy and forestall the disease and debility that currently accompany a long life, by targeting the root causes of age-related ill health. Menopause shares much in common with major age-related health problems, inasmuch as they all result from the accumulation of cellular and molecular damage in our tissues over time. Because this damage takes our tissues' microscopic functional units offline, aging damage gradually degrades each tissue's capacity to carry out its normal function with time. When enough of this damage accumulates in a particular tissue, specific diseases and disorders of aging characteristic of that tissue emerges, whether it's in the brain (Alzheimer's and Parkinson's disease), or the heart and circulatory system (atherosclerosis and heart failure), or the machinery controlling cellular growth (cancer) — or the ovaries (menopause). The corollary of this is that by removing and repairing this damage, rejuvenation biotechnology will restore the proper structure of the cellular machinery that keeps our tissues functioning, restoring their ability to keep us alive and with the good health that most of us enjoy at earlier ages.
So maintaining a woman's fertility and postponing or eliminating menopausal symptoms comes down to a mixture of repairing and replacing damaged cells (notably egg cells) and tissues (follicles) whose age-related degradation leads to menopause in the first place, bringing the whole system back to its youthful, functional norm. Today, researchers are pursuing several “damage-repair” approaches to realize this goal, and that's what we'll discuss in this article.
You're probably familiar with the promise of stem cells and other cell therapies to treat a variety of diseases and disorders involving cell loss, particularly diseases of aging. Cell therapy is an straightforward way to counteract the loss of viable egg cells with age, particularly in restoring a woman's fertility.
To give a woman a new supply of eggs that matches her original genetics will require that those new egg cells begin with her own cells. Scientists are now mastering a couple of ways whereby a person's ordinary, mature cells can have their developmental clocks reset, giving them the open developmental potential of embryonic stem cells. To turn those primal cells into the specific kind of cell that is needed therapeutically, scientists then use chemical cues and structured environments to coax them along a whole new pathway and mature into a different but needed cell type. This is a promising approach for diseases that centrally involve the loss of specific cell populations, such as type I (“juvenile”) diabetes and Parkinson's disease.
In 2012, Japanese scientists reported that they had taken cells from the deep layers of adult mouse skin and turned them into embryonic stem-like cells using one of these methods (induced pluripotent stem cell (iPS) technology). The researchers first used genetic nudges to first turn the iPS cells into the very early cells that develop during embryonic development and later become the precursors of egg cells, and then brought these cells together with cells from embryonic ovarian tissue that did not contain sex cells of their own, creating “reconstituted ovaries” in a lab dish. And when scientists transplanted these reconstituted ovaries into mice, they began producing early-stage egg cells of the sort that women are born with — just the kind that are lost or damaged in aging ovarian follicles. And the scientists showed that these early egg cells are fully functional, being capable of producing viable offspring when treated with in vitro fertilization (IVF) and implanted into surrogate mouse mothers. Scientists have not yet attempted to transplant these “reconstituted ovaries” directly into the ovaries and produce offspring by mating these mice, but this research is the critical advance necessary to realize those goals.
This approach is similar to cell therapy, but focuses on the larger-scale goal of replacing an entire organ or tissue instead of replacing specific, critical cell types. In an exciting study, Stanford scientists have reported the ability to generate new follicles from ovarian tissue from women with primary ovarian insufficiency, in which a woman's ovaries stop producing new eggs before the age of 40 and she enters early menopause.
After preliminary studies in mice, the researchers surgically removed a small amount of ovarian tissue from 27 such women, and simply divided the tissue into smaller units. As was previously observed in mice, this separation of the tissue reduced the expression of a regulatory gene called Hippo, whose normal function is to prevent excessive growth of tissues. With Hippo signaling turned down, a cascade of growth factors in the ovarian tissue was unleashed, and follicles began to grow. They then bathed the tissue with a drug that activates Akt, a signaling molecule that promotes survival and inhibits defensive “cellular suicide” (apoptosis). Again consistent with what they had seen in mouse studies, this treatment cause dormant “proto-follicles” in the tissue to become active, further promoting follicle growth. The researchers then transplanted this reactivated tissue with its newly-generated follicles underneath the thin outer membrane of the fallopian tubes.
Over the course of the next six months — and in several cases, within weeks — eight of the women began experiencing the growth of new follicles from the re-implanted tissue. When that happened, the scientists treated these women with FSH and human chorionic gonadotropin (HCG), another hormone that is involved in making the uterus lining ready for impregnation and that is used clinically to induce ovulation. Thirty-six hours later, they were able to retrieve egg cells from five of these women. These eggs then fertilized in vitro using each woman's husband's sperm, and the fertilized eggs were implanted back into the women, leading to two new pregnancies. The scientific report does not indicate what happened with one of these pregnancies, but astonishingly, the other woman — who had been menopausal for four years — later gave birth to a healthy seven-pound baby boy.
As of late last year, two women who were not successfully impregnated in their first round of IVF were trying again, either implanting additional embryos left over from their first round of IVF or having additional eggs harvested from their reactivated ovary transplants. The researchers say they expect to be able to use the same methods to preserve fertility in cancer patients being treated with follicle-destroying chemotherapy, and for women in their forties undergoing more “normal” age-related infertility. And a Stanford press story confirms that this isn't just idle speculation: “The researchers are planning to study the experimental treatment in women who are infertile for other reasons.”
Obviously, there's a lot of work to do before this research can lead to the ultimate vision of self-sustaining tissue-engineered ovarian tissue that would allow a woman become pregnant and give birth through intercourse as a healthy young woman would — but this powerful study goes a long way toward showing the outlines of how that vision might be realized.
Awakening “Oogonial Stem Cells”
But maybe we don't need to actually give women new ovarian tissue to revive ovarian function. Since the 1950s, it's been the dogma that women are born with a fixed supply of early-stage egg cells that are produced during embryonic development. It was from this limited “bank account” of egg cells, the understanding went, that a woman would “draw down” with each ovulation, and from which she would also lose viable eggs due to genetic and other aging damage. When her egg cell “bank balance” fell below some limit, the loss of crosstalk amongst egg cells, follicles, and the regulatory centers in the brain would cause her follicles to atrophy and stop producing estrogen, leading to the symptoms of menopause.
But this widely-accepted view has been strongly challenged in the last decade, mostly by studies from pioneering reproductive biologist Dr. Jonathan Tilly, now Chair of the Department of Biology at Northeastern University. Tilly began to question the conventional wisdom on this subject when he conducted studies in mice at different ages, and found that the math on the follicle counts didn't add up: when he compared the rate at which follicles were degenerating with age with the actual number of remaining follicles at a given age, the aging mice had more remaining follicles than could be accounted for by simply taking the number of follicles present early in life and subtracting the number that his studies showed were being lost every year to degenerative aging processes. One possible reading of this finding was that mice were continuing to produce new functional follicles, complete with egg cells, well into their adult lives, albeit at a very low rate.
Moreover, cases were being reported of women who had regained their fertility after having had it destroyed by cancer therapy: months or years after being rendered infertile and suffering with menopausal symptoms, women who had received bone marrow transplants would spontaneously begin cycling again, with several cases of spontaneous pregnancies reported.
Then in 2006, Tilly reported the results of a surprising study that offered an explanation for these phenomena. An earlier study by Tilly had suggested that stem cells in the bone marrow transplants were homing to the ovaries and somehow developing into new, donor-derived follicles. But when other investigators repeated his study using more precise ways of tracking those cells, they found that while the transplanted cells were indeed reaching the ovaries, they were behaving more or less like blood cells, with no evidence of the donated cells developing into follicle or egg cells.
In his 2006 study, Tilly found a possible way to resolve that contradiction. He simulated chemotherapy in mice, making them infertile, and then gave them bone marrow transplants, using bone marrow from mice whose cells had been modified to produce a fluorescent protein that allows scientists to track them and their genetic progeny. Then he housed these treated mice with males and let them breed.
Just as with the case studies of cancer chemotherapy patients who had suddenly become fertile again after bone marrow transplants, the mice that just months before had been infertile suddenly began getting pregnant again. But consistent with his critics' findings, the newborn mice did not bear the telltale fluorescent protein in their cells, indicating that the pups could not have been generated from egg cells that were derived from the bone marrow transplant cells.
But if cells in the bone marrow transplants weren't developing into a fresh supply of precursor egg cells, where were the eggs coming from? There were two possibilities. One was that factors in the bone marrow transplants were contributing growth factors and other molecules that were resuscitating follicles that had been inactivated by the chemotherapy, allowing them to release a supply of egg cells that were secretly lying dormant within them. The other, even more exciting possibility: the transplanted cells were homing in to the follicles and regenerating their capacity to produce completely new egg cells.
The latter explanation would explain all the unaccounted-for follicles in his early studies, but for it to make sense, there had to be some kind of very early precursor to egg cells still present in the ovaries that the bone marrow transplants was stimulating to mature into early egg cells. In 2012, Tilly performed painstaking studies of adult mouse and human ovarian tissue, and reported the presence of a rare population of cells in the ovaries that expressed genes in a pattern similar to the primitive egg cell precursors that are found in embryos, and that had the ability to replicate. And after isolating and culturing these cells, Tilly was able to get them to develop into cells that bore multiple hallmarks of being actual egg cells.
To test the human cells' ability to actually develop into egg cells, Tilly tweaked them to express a fluorescent tracking protein, and nested them in with ovarian tissue left over from women of childbearing age who had recently undergone sex-reassignment operations. Then, he took the co-cultured mystery cell/ovarian tissue cultures and injected them together into mice. Remarkably, the two tissue types together developed into follicles containing egg cells that expressed the fluorescent labeling protein, consistent with the idea that these “oogonial stem cells” (OSC) really do have the potential to develop into egg cells under the right conditions.
Other scientists have also reported the presence of rare egg-stem-like cells in adult women and mice, long after the fixed-supply model would rule them out — but others have not , and the debate goes back and forth and remains unresolved.
If Tilly's results (and his interpretation of those results) do pan out, it offers the potential that OSC may lie dormant in aging women, waiting to be revitalized with the right cocktail of cells or signaling factors. This would be expected to extend reproductive lifespan and (potentially) postpone or reverse menopausal symptoms, so long as the body could continue to generate new OSC. It is unlikely that this would be an indefinite solution, because OSC themselves (and other tissues in the follicle and the rest of the reproductive system) would continue to suffer aging damage, and so would eventually pass the point of no return. At that point, cell therapy and tissue engineering solutions would eventually be needed to keep women fertile and free of hot flashes. But Tilly's research suggests that there may be an easier route to a more modest extension of childbearing, youthful-feeling years for women while we wait for more durable damage-repair strategies to mature.
For decades, many women made the transition into menopause more bearable using hormone replacement therapy (HRT) to top up their ovaries' dwindling production of estrogen (sometimes with other hormones). This worked reasonably well as a symptomatic treatment many women, and reduced the severity of menopause-associated bone loss, but the early hope that it would protect women's hearts turned out to be false: as commonly used, it slightly increased the risk of stroke and some cancers, and even the best protocols need to be timed and dosed well just to avoid increasing a woman's risk. One of the reasons for this is that HRT is necessarily a somewhat crude instrument: no regimen of hormones that are swallowed in pills or smeared as gels can match the exquisite system of regulated feedback amongst the ovaries, the sex hormones, and the centers in the brain that govern the physiological cycles of a young woman's body.
The rejuvenation biotechnologies we've explored so far involve replacing egg cells, or whole follicles, or even whole ovaries with new tissue, which would restore both fertility and normal, youthful hormone production. But the disruption of the hormonal system that drives the symptoms of menopause is only indirectly related to the actual release of egg cells. The two cell populations involved in the production and release of release sex hormones under the orchestration of FSH and LH are part of the follicle itself, and their release is not directly tied to ovulation. If these cells could be replaced and maintained in the ovaries, they could potentially carry on producing sex hormones and maintain the normal system of feedback between the ovaries, those hormones, and the regulatory centers in the brain, even with no egg cell replacement.
Dr. Emmanuel C. Opara and colleagues at the Wake Forest Institute of Regenerative Medicine has conducted some initial work in developing a system to implant healthy replacement follicle cells in a small encapsulated device that would keep the transplanted cells separate from the recipient's own cells, but allow the passage of hormones back and forth across a membrane. A system like this would allow the implanted cells to respond to LH and FSH by producing sex hormones in the normal, regulated way, and for those sex hormones to be released in the blood, while keeping the transplanted cells isolated from the body's immune system.
The advantage of such a system is that the implanted cells could be taken from any donor (including, probably, even pigs!), without the worry of immunological rejection, since the patient's immune system would never “see” the donated cells. Similar systems are now being tested as a potential fast route to regenerative therapies for multiple conditions, particularly those in which donated cells are needed to secrete soluble factors. Most notably, there are now several such systems being tested in human clinical trials as a way to transplant fresh insulin-producing beta-cells into patients with diabetes, replacing cells destroyed by autoimmune disorders or by decades of overtaxing their capacity. If they work in humans as they do in mice, these cells will sense the rise and fall of blood sugar levels just as a person's own beta-cells do and secrete just the right level of insulin to keep blood sugar under control, eliminating the need for crude and painful insulin injections or the use of drugs that boost the flagging capacity of the few remaining beta-cells to produce insulin.
Dr. Opara has now tested several possible encapsulated follicle cell device designs, in which the two key cell populations are either intermixed in the device or kept separate in different arrangements by membranes, to see which one best facilitates the cells' normal, physiological interactions with each other. Using cells isolated from ovaries of 21-day old rats, he found that a multilayered system in which the cells are arranged in the same relative position within the device as they are in the living follicle, but separated by a permeable membrane, releases higher and more sustained levels of estrogen than the other systems, which produced it weakly, inconsistently, or out of proportion with progesterone. Chinese researchers are also working on the development of such a system. It is still early days, but this research could well lead to day when women can have their physiologically-regulated, youthful hormone levels and balance restored and maintained throughout their lives.
To fully maintain ovarian function entails women holding on to their fertility. But many women, after living five decades or so, would be quite happy to leave their child-bearing years behind them — they just don't want to feel cranky, suffer hot flashes, or have their intimate relations made awkward and physically uncomfortable by changes in their sexual organs. Establishing and maintaining replacement hormone-producing cells in a way that keeps the hormonal feedback system working youthfully could allow women who chose it to escape from the many unpleasant symptoms of menopause, without restoring their fertility and with fewer risks than HRT (although other rejuvenation biotechnologies would be required to eliminate those risks entirely).
Women Age as Whole People
The subject of this article has been ways that the rejuvenation biotechnology can be brought to bear against the cellular and molecular damage most intimately involved with menopause. As discussed, there are several damage-repair strategies that could be applied to postpone or reverse both the loss of fertility that is the core of menopause, and the more or less intense discomfort, embarrassment, and inconvenience of the symptoms that arise as the body's reproductive feedback systems work with increasing desperation to coax old ovaries into performing their young tricks. Even as this article was being written, progress was reported in a closely-related area: three of nine women in Sweden who had received transplanted wombs are at least midway through new pregnancies, and one of them has just given birth to a healthy if slightly premature boy.
But of course, a woman is more than a womb, and her aging is more than the aging of her reproductive system. Aging affects every organ, every tissue, every cell. And while specific diseases and disorders arise most recognizably when the burden of cellular and molecular damage to some particular tissue crosses a “threshold of pathology,” no organ ages in isolation. We age as whole people, with stiffening arteries damaging our kidneys and brains, failing eyesight impairing our intellectual work, and a rising burden of tissue damage across the entire body forcing all of our cells operate in a haze of oxidative stress and inflammation. In the end, women will be truly free of menopause when and only when we are all free of the entire degenerative aging process: when a comprehensive panel of rejuvenation biotechnologies is developed to remove, repair, replace, or render harmless the full range of the damage of aging, and all of our tissues are made new. SENS Research Foundation is dedicated to that mission, and a future free of age-related disease and debility.
Readers interested in rejuvenation biotechnology and Dr. Tilly's research should know that Tilly is working to initiate a geroscience initiative within his department at Northeastern University. As part of the momentum toward that initiative, the Department of Biology will honor SENS Research Foundation Chief Science Officer Dr. Aubrey de Grey to present as Northeastern University Distinguished Lecturer in Biology this November. Details are still being finalized, and will be announced closer to the event if it is open to the public.