In a little-noticed study published earlier this year, scientists from Oregon Health & Science University reported the birth of three mouse pups that had been created with a never-before-used recipe for reproduction. Using a common cloning technique, researchers removed the genetic material from one female’s eggs and replaced them with nuclear DNA from the skin cells of another. Then with a novel chemical cocktail, they nudged the eggs to lose half their new sets of chromosomes and fertilized them with mouse sperm.
In a big step toward achieving in vitro gametogenesis — one of reproductive medicine’s more ambitious moonshots — the group led by pioneering fertility researcher Shoukrat Mitalipov now intends to use the same method to make artificial human embryos in a test tube.
If successful, the research holds enormous potential for treating infertility, preventing heritable diseases, and opening up the possibility for same-sex couples to have genetically related children.
“It’s one of those high-risk, high reward type of projects,” said Paula Amato, an OB-GYN and infertility specialist at OHSU who collects the human eggs used in Mitalipov’s experiments. “We have no idea yet if it will work, but age-related fertility decline remains an intractable problem in our field, so we’re eternally grateful to these private funders who are filling a real need here.”
Mitalipov directs the Center for Embryonic Cell and Gene Therapy at OHSU. Established in 2013, the center focuses on combining assisted reproductive technologies with genetic correction techniques, with the goal of one day preventing inherited disease.
The group’s work on in vitro gametogenesis (IVG) in human cells is being made possible by an award from the Open Philanthropy Project — a grant-making foundation primarily funded by Facebook co-founder Dustin Moskovitz and his wife Cari Tuna — which will supply the researchers with $4 million over the next three years. The infusion of funds and the involvement of a scientist as storied as Mitalipov makes the ethical and legal questions surrounding mass egg and sperm production more urgent, experts told STAT.
In the U.S., there are no federal laws that prohibit this type of IVG work. However, Congress has barred any research that creates, destroys, or knowingly harms human embryos from receiving federal funding. At the state level, laws governing human embryo research vary widely with 11 states banning it entirely, five states expressly permitting it, and a lot of gray areas in between.
For IVG to move from the research lab to a fertility clinic would require permission from the Food and Drug Administration. It’s still unclear if that’s something the agency would be able to consider — a spending bill rider currently prevents the FDA from receiving any requests to pursue clinical trials involving starting pregnancies with embryos that have been genetically manipulated. In 2019, Congress considered modifying the ban, following a push from scientists and advocates of mitochondrial replacement therapy, also known as three-person IVF, but ultimately renewed it. Mitochondrial replacement therapy is a procedure that combines genetic material from an egg and sperm with mitochondria from a female donor.
Somatic cell nuclear transfer for IVG could fall under the same provision, if the somatic DNA and the egg came from different people. But if they came from the same person, that might represent a loophole.
Some bioethicists worry that the easy availability of IVG could usher in a new era of eugenics, scenarios where prospective parents could create large numbers of embryos and use genetic tools to select the “best” one. IVG also raises the specter of nonconsensual parenthood — something most state laws are currently ill-equipped to handle.
“Should this become clinically available, there will be legitimate questions — about whose cells can be used and under what conditions — that will need regulatory answers,” said Hank Greely, director of the Stanford Center for Law and Bioscience, whose book, “The End of Sex,” examines the future of in vitro gametogenesis. “Will that happen? We don’t know. But Mitalipov has certainly proven himself a bold and creative scientist, and from my perspective, having his group join the effort to help people who want to have genetic babies but can’t is a good thing, provided they can do it safely and effectively.”
Mitalipov’s lab has long been an incubator for envelope-pushing science. In 2009, he and his colleagues figured out a way to swap out glitchy mitochondrial DNA for healthy versions in the egg cells of monkeys — a groundbreaking advance that paved the way for mitochondrial replacement therapy in humans. In 2013, they created lines of embryonic stem cells from cloned human embryos for the first time. A few years later, they became the first team in the U.S. to attempt to correct a genetic mutation in viable human embryos using CRISPR.
But until recently, in vitro gametogenesis, or IVG, wasn’t on his to-do list.
Gametes are the cells capable of giving rise to future generations: sperm and eggs. The idea behind IVG is to produce those kinds of cells in test tubes, rather than inside a developing animal’s body.
In recent years, scientists have made headlines producing artificial gametes from induced pluripotent stem cells. But Mitalipov’s group plans to revive a much older technology, which saw some early success in IVG before being abandoned: somatic cell nuclear transfer.
Somatic cell nuclear transfer was pioneered by researchers at the Roslin Institute in Scotland. After they succeeded in using the technique to clone the first mammal — a sheep named Dolly — scientists realized it might be used to generate artificial gametes, if they could overcome a few additional hurdles.
In cloning, the emptied egg receives a full set of chromosomes from the somatic cell donor and is stimulated in the lab to make it start dividing. Any offspring that result will be genetically identical to that somatic cell.
The procedure for making an artificial oocyte is technically similar to cloning, but would generate unique individuals after fertilization with sperm. However, in order for any resulting embryos to have the right number of chromosomes, the donor DNA has to be cut in half, a process known as haploidization. Oocytes are equipped with the machinery to make that adjustment, if the somatic DNA is introduced at the right phase of their cell cycle.
In 2000, four years after Dolly was born, researchers in Spain generated the first human artificial oocytes using this method. They fertilized three of them, and froze the resulting embryos at the two-cell stage. The plan was to transfer the frozen embryos to the uterus of a woman who had been unable to conceive, and consented to having her somatic DNA slipped into donor eggs as a last-ditch attempt to have genetically related children with her husband.
But when the same protocol was tested in mice — where its effects could be examined more closely — the chromosomes didn’t separate as intended. Shortly thereafter, somatic cell nuclear transfer for human reproduction was banned in many countries, including Spain.
The IVG field moved on, buoyed by the discovery a few years later of a method for taking any kind of cell and rewinding its developmental clock to a more primitive state. With the right chemical cues, a team of Japanese scientists nudged these pluripotent stem cells to produce functional gametes in mice; first sperm in 2011, then eggs, five years later. But they struggled to generate similar results in humans.
In 2018, the group succeeded for the first time in making immature human eggs from scratch. But the process wasn’t very efficient and it involved incubating the human stem cells in mini-ovaries they’d created in the lab from mouse embryonic cells — a resource-intensive process not exactly suited to mass manufacturing.
So when a post-doc at OHSU named Eunju Kang proposed revisiting the idea of somatic cell nuclear transfer for IVG, Mitalipov was initially skeptical. But data from her initial mouse experiments proved persuasive. Mitalipov threw his support behind the project, and teamed up with a group at Weill Cornell Medicine in New York, including reproductive endocrinologist Gianpiero Palermo, who had successfully generated artificial human oocytes using cloning technology back in 2002. They published the results of their mice experiments in Nature Communications Biology in January.
The OHSU team is now adapting those methods to see if they can generate artificial human eggs with properly separated chromosomes. If successful, they plan to then fertilize those eggs with sperm and grow the resulting embryos in the lab for five or six days to see if they develop normally.
They are betting that this method, while older, will prove better than the induced pluripotent stem cell technologies currently being advanced by artificial egg-making start-up outfits like Conception, Ivy Natal and Gameto.
That approach requires the cells to be cultured for months rather than days, which can lead to epigenetic programming errors and chromosomal instability. Mitalipov also believes that starting with natural eggs will make it easier to strip the donor DNA of its cellular memory and return it to the primitive state known as totipotency — a critical step in enabling the embryo to eventually develop all the specialized tissues that make up a human body.