Study Suggests Women May Be Able to Make More Eggs as They Age

Feb. 26, 2012 -- Scientists say they have found a way to use ovarian stem cells to perhaps one day help infertile women get pregnant -- or add years to a woman’s reproductive cycle.

In a study published in Nature Medicine, researchers report finding egg-producing stem cells in human ovaries. They also report being able to make some of those ovarian stem cells grow into immature eggs that may someday be useful for reproduction.

At this point, such “seed” eggs can’t be fertilized by sperm. But if scientists are able to entice them to mature and can prove they can be fertilized and grow into embryos -- a feat that has been reported in mice -- it would overturn a long-held scientific belief that women can’t make new eggs as they get older.

“What it does is really open a door into human reproduction that 10 years ago didn’t even exist,” says researcher Jonathan L. Tilly, PhD, director of the Vincent Center for Reproductive Biology at Massachusetts General Hospital, in Boston.

Outside experts agree. They say the findings could have profound importance for reproductive medicine and aging, allowing doctors not only to restore a woman’s fertility but also to potentially delay menopause.

“I think the significance of this work is like reporting that we found microorganisms on Mars,” says Kutluk Oktay, MD, who directs the Division of Reproductive Medicine and the Institute for Fertility Preservation at New York Medical College in Valhalla, N.Y.

Still, It’s a Long Way to Mars

“It’s a proof of principle that they could do it,” says David F. Albertini, PhD, director of the Center for Reproductive Sciences at the University of Kansas Medical Center in Kansas City, Kan.

“The world wants to know today if we’re ready to restore fertility in women, whether they’ve aged or been treated for cancer or whatever,” Albertini says, adding that he doesn’t think that’s on the horizon. “This is an extremely rare event, at best.”

The egg-generating stem cells the researchers were able to extract from ovaries were very rare. The researchers only came across one for every 10,000 or so ovarian cells that they counted.

But when they took those cells and implanted them back into human ovarian tissue, they divided and essentially made young eggs.

Tilly says his team stopped short of trying to make one of the eggs functional because “for a lot of reasons, as it should be,” it is illegal in the U.S. to experimentally fertilize human eggs.

“We think the evidence provided clearly indicates that this very unique, newly discovered pool of cells does exist in women,” he says.

A Lot of Potential

“It’s a really exciting result,” says Evelyn Telfer, PhD, a cell biology expert at the University of Edinburgh in Scotland.

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Future Fertility Fix? Egg-Producing Stem Cells Found in Human Ovaries

POSTED: 1:07 pm EST February 26, 2012

WASHINGTON -- For 60 years, doctors have believed women were born with all the eggs they'll ever have. Now Harvard scientists are challenging that dogma, saying they've discovered the ovaries of young women harbor very rare stem cells capable of producing new eggs.If Sunday's report is confirmed, harnessing those stem cells might one day lead to better treatments for women left infertile because of disease - or simply because they're getting older."Our current views of ovarian aging are incomplete. There's much more to the story than simply the trickling away of a fixed pool of eggs," said lead researcher Jonathan Tilly of Harvard's Massachusetts General Hospital, who has long hunted these cells in a series of controversial studies.Tilly's previous work drew fierce skepticism, and independent experts urged caution about the latest findings.A key next step is to see whether other laboratories can verify the work. If so, then it would take years of additional research to learn how to use the cells, said Teresa Woodruff, fertility preservation chief at Northwestern University's Feinberg School of Medicine.Still, even a leading critic said such research may help dispel some of the enduring mystery surrounding how human eggs are born and mature."This is going to spark renewed interest, and more than anything else it's giving us some new directions to work in," said David Albertini, director of the University of Kansas' Center for Reproductive Sciences. While he has plenty of questions about the latest work, "I'm less skeptical," he said.Scientists have long taught that all female mammals are born with a finite supply of egg cells, called ooctyes, that runs out in middle age. Tilly, Mass General's reproductive biology director, first challenged that notion in 2004, reporting that the ovaries of adult mice harbor some egg-producing stem cells. Recently, Tilly noted, a lab in China and another in the U.S. also have reported finding those rare cells in mice.But do they exist in women? Enter the new work, reported Sunday in the journal Nature Medicine.First Tilly had to find healthy human ovaries to study. He collaborated with scientists at Japan's Saitama Medical University, who were freezing ovaries donated for research by healthy 20-somethings who underwent a sex-change operation.Tilly also had to address a criticism: How to tell if he was finding true stem cells or just very immature eggs. His team latched onto a protein believed to sit on the surface of only those purported stem cells and fished them out. To track what happened next, the researchers inserted a gene that makes some jellyfish glow green into those cells. If the cells made eggs, those would glow, too."Bang, it worked - cells popped right out" of the human tissue, Tilly said.Researchers watched through a microscope as new eggs grew in a lab dish. Then came the pivotal experiment: They injected the stem cells into pieces of human ovary. They transplanted the human tissue under the skin of mice, to provide it a nourishing blood supply. Within two weeks, they reported telltale green-tinged egg cells forming.That's still a long way from showing they'll mature into usable, quality eggs, Albertini said.And more work is needed to tell exactly what these cells are, cautioned reproductive biologist Kyle Orwig of the University of Pittsburgh Medical Center, who has watched Tilly's work with great interest.But if they're really competent stem cells, Orwig asked, then why would women undergo menopause? Indeed, something so rare wouldn't contribute much to a woman's natural reproductive capacity, added Northwestern's Woodruff.Tilly argues that using stem cells to grow eggs in lab dishes might one day help preserve cancer patients' fertility. Today, Woodruff's lab and others freeze pieces of girls' ovaries before they undergo fertility-destroying chemotherapy or radiation. They're studying how to coax the immature eggs inside to mature so they could be used for in vitro fertilization years later when the girls are grown. If that eventually works, Tilly says stem cells might offer a better egg supply.Further down the road, he wonders if it also might be possible to recharge an aging woman's ovaries.The new research was funded largely by the National Institutes of Health. Tilly co-founded a company, OvaScience Inc., to try to develop the findings into fertility treatments.

Copyright 2012 by The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.

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Report: Women Have Rare Egg-Producing Stem Cells

Public release date: 26-Feb-2012
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Contact: Sue McGreevey
smcgreevey@partners.org
617-724-2764
Massachusetts General Hospital

For the first time, Massachusetts General Hospital (MGH) researchers have isolated egg-producing stem cells from the ovaries of reproductive age women and shown these cells can produce what appear to be normal egg cells or oocytes. In the March issue of Nature Medicine, the team from the Vincent Center for Reproductive Biology at MGH reports the latest follow-up study to their now-landmark 2004 Nature paper that first suggested female mammals continue producing egg cells into adulthood.

"The primary objective of the current study was to prove that oocyte-producing stem cells do in fact exist in the ovaries of women during reproductive life, which we feel this study demonstrates very clearly," says Jonathan Tilly, PhD, director of the Vincent Center for Reproductive Biology in the MGH Vincent Department of Obstetrics and Gynecology, who led the study. "The discovery of oocyte precursor cells in adult human ovaries, coupled with the fact that these cells share the same characteristic features of their mouse counterparts that produce fully functional eggs, opens the door for development of unprecedented technologies to overcome infertility in women and perhaps even delay the timing of ovarian failure."

The 2004 report from Tilly's team challenged the fundamental belief, held since the 1950s, that female mammals are born with a finite supply of eggs that is depleted throughout life and exhausted at menopause. That paper and a 2005 follow-up published in Cell showing that bone marrow or blood cell transplants could restore oocyte production in adult female mice after fertility-destroying chemotherapy were controversial; but in the intervening years, several studies from the MGH-Vincent group and other researchers around the world have supported Tilly's work and conclusions.

These supporting studies include a 2007 Journal of Clinical Oncology report from the MGH-Vincent team that showed female mice receiving bone marrow transplants after oocyte-destroying chemotherapy were able to have successful pregnancies, delivering pups that were their genetic offspring and not of the marrow donors. A 2009 study from a team at Shanghai Jiao Tong University in China, published in Nature Cell Biology, not only isolated and cultured oocyte-producing stem cells (OSCs) from adult mice but also showed that those OSCs, after transplantation into the ovaries of chemotherapy-treated female mice, gave rise to mature oocytes that were ovulated, fertilized and developed into healthy offspring.

"That study singlehandedly deflated many of the arguments from critics of our earlier Nature paper by showing that oocyte-producing stem cells exist in mice and could develop into fully functional eggs," says Tilly. Another paper from a west-coast biotechnology company, published in Differentiation in 2010, provided further independent confirmation of Tilly's earlier conclusions regarding the presence of oocyte-producing stem cells in ovaries of adult mice.

Tilly is quick to point out, however, "These follow-up studies, while providing definitive evidence that oocyte-producing stem cells exist in ovaries of adult female mammals, were not without their limitations, leaving the question open in some scientific circles of whether the adult oocyte pool can be renewed. For example, the protocol used to isolate OSCs in the 2009 Nature Cell Biology study is a relatively crude approach that often results in the contamination of desired cells by other cell types." To address this, the MGH-Vincent team developed and validated a much more precise cell-sorting technique to isolate OSCs without contamination from other cells.

The 2009 study from China also had isolated OSCs based on cell-surface expression of a marker protein called Ddx4 or Mvh, which previously had been found only in the cytoplasm of oocytes. This apparent contradiction with earlier studies raised concerns over the validity of the protocol. Using their state-of-the-art fluorescence-activated cell sorting techniques, the MGH-Vincent team verified that, while the marker protein Ddx4 was indeed located inside oocytes, it was expressed on the surface of a rare and distinct population of ovarian cells identified by numerous genetic markers and functional tests as OSCs.

To examine the functional capabilities of the cells isolated with their new protocol, the investigators injected green fluorescent protein (GFP)-labeled mouse OSCs into the ovaries of normal adult mice. Several months later, examination of the recipient mouse ovaries revealed follicles containing oocytes with and without the marker protein. GFP-labeled and unlabeled oocytes also were found in cell clusters flushed from the animals' oviducts after induced ovulation. The GFP-labeled mouse eggs retrieved from the oviducts were successfully fertilized in vitro and produced embryos that progressed to the hatching blastocyst stage, a sign of normal developmental potential. Additionally, although the Chinese team had transplanted OSCs into ovaries of mice previously treated with chemotherapy, the MGH-Vincent team showed that it was not necessary to damage the recipient mouse ovaries with toxic drugs before introducing OSCs.

In their last two experiments, which Tilly considers to be the most groundbreaking, the MGH-Vincent team used their new cell-sorting techniques to isolate potential OSCs from adult human ovaries. The cells obtained shared all of the genetic and growth properties of the equivalent cells isolated from adult mouse ovaries, and like mouse OSCs, were able to spontaneously form cells with characteristic features of oocytes. Not only did these oocytes formed in culture dishes have the physical appearance and gene expression patterns of oocytes seen in human ovaries ? as was the case in parallel mouse experiments ? but some of these in-vitro-formed cells had only half of the genetic material normally found in all other cells of the body. That observation indicates that these oocytes had progressed through meiosis, a cell-division process unique to the formation of mature eggs and sperm.

The researchers next injected GFP-labeled human OSCs into biopsied human ovarian tissue that was then grafted beneath the skin of immune-system-deficient mice. Examination of the human tissue grafts 7 to 14 days later revealed immature human follicles with GFP-negative oocytes, probably present in the human tissue before OSC injection and grafting, as well as numerous immature human follicles with GFP-positive oocytes that would have originated from the injected human OSCs.

"These experiments provide pivotal proof-of-concept that human OSCs reintroduced into adult human ovarian tissue performed their expected function of generating new oocytes that become enclosed by host cells to form new follicles," says Tilly, a professor of Obstetrics, Gynecology and Reproductive Biology at Harvard Medical School and chief of Research at the MGH Vincent Department of Obstetrics and Gynecology. "These outcomes are exactly what we see if we perform the same experiments using GFP-expressing mouse OSCs, and GFP-expressing mouse oocytes formed that way go on to develop into fully functional eggs.

"In this paper we provide the three key pieces of evidence requested by those who have been skeptical of our previous work," he adds. "We developed and extensively validated a cell-sorting protocol to reliably purify OSCs from adult mammalian ovaries, proving once again that these very special cells exist. We tested the function of mouse oocytes produced by these OSCs and showed that they can be fertilized to produce healthy embryos. And we identified and characterized an equivalent population of oocyte-producing stem cells isolated from adult human ovaries."

Among the many potential clinical applications for these findings that Tilly's team is currently exploring are the establishment of human OSC banks ? since these cells, unlike human oocytes, can be frozen and thawed without damage ? the identification of hormones and factors that accelerate the formation of oocytes from human OSCs, the development of mature human oocytes from OSCs for in vitro fertilization, and other approaches to improve the outcomes of IVF and other infertility treatments.

###

Tilly notes that an essential part of his group's accomplishment was collaboration with study co-author Yasushi Takai, MD, PhD, a former MGH research fellow on Tilly's team and now a faculty member at Saitama Medical University in Japan. Working with his clinical colleagues at Saitama, Takai was able to provide healthy ovarian tissue from consenting patients undergoing sex reassignment surgery, many in their 20s and early 30s. Co-lead authors of the Nature Medicine report are Yvonne White, PhD, and Dori Woods, PhD, of the Vincent Center for Reproductive Biology at MGH. Additional co-authors are Osamu Ishihara, MD, PhD, and Hiroyuki Seki, MD, PhD, of Saitama Medical University.

The study was supported by a 10-year MERIT Award to Tilly from the National Institute on Aging, a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health, the Henry and Vivian Rosenberg Philanthropic Fund, the Sea Breeze Foundation, and Vincent Memorial Hospital Research Funds. Tilly is a co-founder of OvaScience, Inc. (www.ovascience.com), which has licensed the commercial potential of these and other patent-protected findings of the MGH-Vincent team for development of new fertility-enhancing procedures.

Massachusetts General Hospital (www.massgeneral.org), founded in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $750 million and major research centers in AIDS, cardiovascular research, cancer, computational and integrative biology, cutaneous biology, human genetics, medical imaging, neurodegenerative disorders, regenerative medicine, reproductive biology, systems biology, transplantation biology and photomedicine.

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Mass. General researchers isolate egg-producing stem cells from adult human ovaries

Discovery of stem cells in human ovaries overturns dogma

Web edition : 2:07 pm

A newly discovered type of stem cell in the ovary could mean big things for women’s health, possibly leading to new fertility treatments and maybe even a way to delay menopause.

Since the 1950s it has been thought that women are born with all of the egg cells they will ever have. But with the discovery of egg-producing stem cells in mice and humans, it now appears that the ovary can replenish its egg supply. Researchers led by Jonathan Tilly, a reproductive biologist at Massachusetts General Hospital in Boston, report the finding online February 26 in Nature Medicine.

Other researchers hail the discovery as a genuine breakthrough with huge implications. “This is like discovering a new planet in our solar system that has a bacterium on it,” says Kutluk Oktay, a reproductive biologist at the New York Medical College in Valhalla. At the very least, he says, the cells offer hope for extending a woman’s reproductive life span.

Tilly didn’t set out to overturn the accepted dogma that women don’t make new eggs. As part of their research into the onset of menopause, he and his colleagues developed ways to track the death of egg cells over time. When the researchers counted the number of healthy egg cells in mouse ovaries, they saw a steady decline with age as expected. But the team also found that dying cells greatly outnumber the starting population of eggs. “What we had was a math problem,” Tilly says. “We refocused all of our efforts on this glaring mathematical dilemma.”

In 2004, Tilly’s group reported the answer to their math problem: There are more dying eggs than healthy ones because stem cells in mouse ovaries are constantly making more eggs, which then die off. The discovery didn’t go over well. “The vast majority of our colleagues were not very receptive,” Tilly says. Many of those who did accept the existence of egg-forming stem cells in mice didn’t think humans would have similar cells.

Tilly and his colleagues isolated stem cells from ovaries that had been removed from six women during sex reassignment surgeries at Saitama Medical Center in Japan. Only about 1.5 percent of cells in the ovaries fit the stem cell profile. The researchers compiled molecular profiles of the cells and demonstrated that the stem cells are able to make precursors to eggs when transplanted into other ovaries. 

Tilly’s group convincingly demonstrates that stem cells in human ovaries can make egg cell precursors. But it remains to be seen if the cells can make mature gametes, says Evelyn Telfer, a reproductive biologist at the University of Edinburgh.

Stopping the depletion of eggs or keeping ovaries functioning could help stave off many of the health problems women experience after menopause, Tilly says. “If we can somehow control this biological clock, to me, the possibilities are endless.”

Found in: Genes & Cells

Go here to see the original:
Eggs may be made throughout adulthood

WASHINGTON — For 60 years, doctors have believed women were born with all the eggs they'll ever have. Now Harvard scientists are challenging that dogma, saying they've discovered the ovaries of young women harbor very rare stem cells capable of producing new eggs.

If Sunday's report is confirmed, harnessing those stem cells might one day lead to better treatments for women left infertile because of disease — or simply because they're getting older.

"Our current views of ovarian aging are incomplete. There's much more to the story than simply the trickling away of a fixed pool of eggs," said lead researcher Jonathan Tilly of Harvard's Massachusetts General Hospital, who has long hunted these cells in a series of controversial studies.

Tilly's previous work drew fierce skepticism, and independent experts urged caution about the latest findings.

A key next step is to see whether other laboratories can verify the work. If so, then it would take years of additional research to learn how to use the cells, said Teresa Woodruff, fertility preservation chief at Northwestern University's Feinberg School of Medicine.

Still, even a leading critic said such research may help dispel some of the enduring mystery surrounding how human eggs are born and mature.

"This is going to spark renewed interest, and more than anything else it's giving us some new directions to work in," said David Albertini, director of the University of Kansas' Center for Reproductive Sciences. While he has plenty of questions about the latest work, "I'm less skeptical," he said.

Scientists have long taught that all female mammals are born with a finite supply of egg cells, called ooctyes, that runs out in middle age. Tilly, Mass General's reproductive biology director, first challenged that notion in 2004, reporting that the ovaries of adult mice harbor some egg-producing stem cells. Recently, Tilly noted, a lab in China and another in the U.S. also have reported finding those rare cells in mice.

But do they exist in women? Enter the new work, reported in the journal Nature Medicine.

First Tilly had to find healthy human ovaries to study. He collaborated with scientists at Japan's Saitama Medical University, who were freezing ovaries donated for research by healthy 20-somethings who underwent a sex-change operation.

Tilly also had to address a criticism: How to tell if he was finding true stem cells or just very immature eggs. His team latched onto a protein believed to sit on the surface of only those purported stem cells and fished them out. To track what happened next, the researchers inserted a gene that makes some jellyfish glow green into those cells. If the cells made eggs, those would glow, too.

"Bang, it worked — cells popped right out" of the human tissue, Tilly said.

Researchers watched through a microscope as new eggs grew in a lab dish. Then came the pivotal experiment: They injected the stem cells into pieces of human ovary. They transplanted the human tissue under the skin of mice, to provide it a nourishing blood supply. Within two weeks, they reported telltale green-tinged egg cells forming.

That's still a long way from showing they'll mature into usable, quality eggs, Albertini said.

And more work is needed to tell exactly what these cells are, cautioned reproductive biologist Kyle Orwig of the University of Pittsburgh Medical Center, who has watched Tilly's work with great interest.

But if they're really competent stem cells, Orwig asked, then why would women undergo menopause? Indeed, something so rare wouldn't contribute much to a woman's natural reproductive capacity, added Northwestern's Woodruff.

Tilly argues that using stem cells to grow eggs in lab dishes might one day help preserve cancer patients' fertility. Today, Woodruff's lab and others freeze pieces of girls' ovaries before they undergo fertility-destroying chemotherapy or radiation. They're studying how to coax the immature eggs inside to mature so they could be used for in vitro fertilization years later when the girls are grown. If that eventually works, Tilly says stem cells might offer a better egg supply.

Further down the road, he wonders if it also might be possible to recharge an aging woman's ovaries.

The new research was funded largely by the National Institutes of Health. Tilly co-founded a company, OvaScience Inc., to try to develop the findings into fertility treatments.

Copyright 2012 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.

Here is the original post:
Women have rare egg-producing stem cells

By Ryan Flinn - Sun Feb 26 18:00:00 GMT 2012

Stem cells taken from human ovaries can produce normal, healthy eggs, scientists demonstrated for the first time in an experiment that may lead to new methods to help infertile women.

The finding challenges a belief that women have a fixed number of eggs, or oocytes, from birth that are depleted by the time of menopause, and that their ovaries can't make make more. The research, led by Jonathan Tilly, director of Harvard University-affiliated Massachusetts General Hospital’s Vincent Center for Reproductive Biology, is published today in the journal Nature Medicine.

In 2004, Tilly discovered that ovarian stem cells in mice can create new eggs, similar to how stem cells in male testes produce sperm throughout a man’s life. The latest study proves the same is true in human ovaries, and may point to new ways to overcome infertility or preserve fertility by delaying the time when a woman’s ovaries stop functioning, he said.

“The 50-year-old belief in our field wasn’t actually based on data proving it was impossible, or not ongoing, it was simply an assumption made because there was no evidence indicating otherwise,” Tilly said in a telephone interview. “We have human cells that can produce new oocytes.”

A female is most endowed with oocytes as a fetus, when she has about 7 million. That number that drops to 1 million by birth, and around 300,000 by puberty. By menopause, the number is zero. Since the 1950’s, scientists thought that ovarian stem cells capable of producing new eggs are only active during fetal development.

Ovarian Stem Cells

In the study, healthy ovaries were obtained from consenting patients undergoing sex reassignment surgery. The researchers were able to identify ovarian stem cells because they express a rare protein that’s only seen in reproductive cells.

The stem cells from the ovaries were injected into human ovarian tissue that was then grafted under the skin of mice, which provided the blood supply that enabled the cells to grow. Within two weeks, early stage human follicles with oocytes had begun to form.

“This paper essentially opens the door to the ability to control oocyte development in human ovaries,” Tilly said.

About 10 percent of women of child-bearing age in the U.S., or 6.1 million, have difficulty getting pregnant or staying pregnant, according to the Centers for Disease Control and Prevention. Most cases of female infertility are caused by problems with ovulation, hormone imbalance or age.

Infertility Treatments

Infertility in women is now treated through drugs, surgery, artificial insemination or assisted reproductive technology, in which the woman’s eggs are mixed with sperm outside the body, then reinserted.

The study offers “a new model system for understanding the human egg cell,” according to David F. Albertini, director of the Center for Reproductive Services and professor in the department of molecular and integrative physiology at Kansas University. Still, “there’s a long way to go before this has real practical applications,” he said.

“I’ve spent 35 years of my life studying egg cells and this is a cell that is at least as complicated as a neuron in the brain, if not more,” Albertini said in an interview. “You will need to establish reproducibility from one lab to the next, and hopefully others will be able to confirm his work and extend it, make it into something that will make us confident that the cells are safe to use and we could actually use them to repopulate an egg-depleted ovary.”

New Therapies

The research is opening other therapeutic avenues in fertility treatment, Tilly said.

His team is exploring the development of a bank for ovarian stem cells, which can be cryogenically frozen and thawed without damage, unlike human oocytes. The researchers are also working to identify hormones and other growth factors for accelerating the production of eggs from human ovarian stem cells and ways to improve in-vitro fertilization.

“The problem we face with IVF is we don’t have many eggs to work with,” he said. “These cells are renewable. If we are successful -- and it’s a big if -- in generating functioning eggs from these cells, we can generate as many eggs as we need to on a per patient basis.”

Tilly is also collaborating with researchers at the University of Edinburgh in the U.K. to determine whether the oocytes can be developed into fully mature human eggs for fertilizing. The U.S bans creating or fertilizing embryos for experimental purposes, he said.

A company Tilly co-founded, Boston-based OvaScience Inc., has licensed the technology for potential commercial applications.

To contact the reporter on this story: Ryan Flinn in San Francisco at rflinn@bloomberg.net

To contact the editor responsible for this story: Reg Gale at rgale5@bloomberg.net

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Ovarian Stem Cells Produce Eggs in Method That May Aid Fertility Therapy