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Solving reproduction mysteries has helped make paternity possible by the millions

At first, no one really understood how babies were made. Thinkers have baffled for millennia about how life arose from one generation to the next. But until the seventeenth century scientists began to seriously study the issue. At the time, preformation theory held that tiny, fully formed humans already existed, either in the mother's menstrual blood or in the father's semen, depending on whether you were "ovist" or "spermist."

Little changed until two late 19th-century scientists, Oskar Hertwig of Germany and Hermann Fol of France, independently conducted experiments on sea urchins, conclusively proving that creating new offspring takes an egg and a sperm.

Despite the initial confusion, the ancients were sure of one thing: reproduction is far from a safe bet. Today, it is estimated that 15 percent of couples around the world are unable to conceive a child naturally, which causes feelings of grief, loss, and a deep sense of inadequacy for many. A century ago, science didn’t have much to offer these couples.

People used to think that preformed humans grew inside sperm, as in this 1695 drawing.Nicolas Hartsoecker / Wikimedia Commons

The only widely available fertility intervention in 1921 was artificial insemination by donor sperm, which was morally and legally full. In the first half of the twentieth century, the practice was often considered a form of adultery; as early as 1963, an Illinois court ruled that a baby conceived in this way, even with the consent of the husband, was illegitimate.

In 1978 everything changed. The birth of Louise Brown, the world’s first “test tube baby,” showed that infertile couples had another option: in vitro fertilization. The technique consisted of removing a mature egg from the mother, mixing it in a laboratory dish with the father’s sperm, and letting the fertilized egg, called the zygote, grow for a couple of days. The zygote was returned to the mother's uterus, where it could implant and grow in an otherwise normal pregnancy.

Since Brown’s historic birth, scientists have devised a number of ways to give Mother Nature a boost in raising babies. The different methods are collectively known as assisted reproduction technology or ART. About 9 million babies worldwide were born using versions of ART.

The impact was as profound sociologically as it was medically. Now that ART has become almost routine, many of the first complaints about scientists playing God and manipulating life are gone. Fatherhood is now possible for people who never imagined it in their future, including same-sex couples and single parents, thanks to refinements such as egg donors, surrogacy and the successful freezing of eggs, sperm and embryos. And it all starts, as human life itself does, with the egg.

Good eggs

Even scientists can’t make babies without eggs. Typically, a woman produces only one mature egg each month and the quality of her eggs tends to decrease as she reaches the age of 30. Thus, the ability of researchers to recover and prepare this scarce resource for fertilization, and, if necessary, to preserve eggs by freezing, has been crucial in aiding reproduction.

Women have two ovaries, each containing thousands of immature egg follicles. During the fertile years, the ovaries usually release rotating mature eggs: a single mature egg that leaves the right ovary one menstrual cycle, the left ovary the next.

But women who use ART often rely on injections of various fertility hormones to begin the process. These vaccines will allow slow ovaries to produce eggs that can be fertilized either with intercourse or in the laboratory through IVF. In IVF the most robust candidates are chosen to implant or freeze them for later use. It turns out that this first step in ART is also one of the most complicated: choosing the right hormones to get the eggs you need.

Knowledge about hormones and how they affect ovulation dates back to 1923, when scientists Edgar Allen and Edward Doisy of the University of Washington School of Medicine in St. Louis. Louis first isolated estrogen in experimental mice and rats and found that it occurred in the ovaries. In the 1940s, scientists elucidated the flow and reflux of other hormones in laboratory and human animals (follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotropin) throughout a typical menstrual cycle.

ART usually begins with a woman giving daily injections containing a cocktail of these hormones, usually for 10 to 14 days. But for some women who want to have children but are being diagnosed with cancer, hormone injections are not an option, and the clock is ticking. They need to start cancer treatments as soon as possible, but many of the treatments are likely to damage the reproductive system.

To preserve their fertility, these women may choose to freeze their eggs before cancer treatment. But they may not have to wait 10 or more days until hormonal injections cause the ovaries to produce additional eggs; nor will they be able to take the drugs in the first place if they have a hormone-sensitive cancer, such as some breast cancers. , that ovulation-stimulating drugs can make it worse. Thus, for these women, researchers had to find ways to get a lot of eggs to mature at once and out of the body, a technique known as in vitro maturation.

In vitro maturation was first used in 1934, when Harvard researchers Gregory Pincus and E.V. Enzmann used it in rabbits (SN: 3/10/34, p. 149). The two scientists cultured immature rabbit eggs for about a day, supplementing the nutrient broth with pituitary gland extracts from the cows or with an unspecified “maturity hormone”. Both supplements helped immature eggs grow, at which point they were successfully fertilized.

In 1940, a New York Times reporter asked Pincus what the next major development in reproductive science might be. “There are no big steps, there are all small steps,” he said, refusing to make any predictions. As he said, the only thing he knew for sure was that the “big questions” of the day were: why does an egg start to develop and why does it continue to develop?

Gregory Pincus holding a rabbit in the labIn the 1930s, Harvard researchers Gregory Pincus (shown) and E.V. Enzmann farmed rabbit eggs to maturity and fertilized them in the laboratory. Pincus later developed the contraceptive pill in coding.Bettmann / Getty Images

When reproductive endocrinologists retrieve eggs from their ART patients, after stimulating the ovaries by hormone injections or maturing the eggs in the lab, they have two options: fertilize the eggs and implant the embryo immediately or save them. For women who are not yet ready to have a baby, storing eggs is the best option. This is done through freezing, which in the early days of ART was a tricky business. Eggs have a high liquid content that leads them to form crystals when they freeze; during thawing, these crystals can damage the egg, especially the delicate apparatus needed to cut the number of chromosomes in the cell in half. By dividing chromosomes, an egg plus a sperm can fuse without doubling the number of chromosomes.

In the 1980s, egg freezing worked from time to time; the first successful pregnancy with a woman's frozen eggs, which led to the birth of healthy twins, was reported in 1986 by Christopher Chen of Flinders University in South Australia in Adelaide. But freezing eggs was still a long shot. Estimates were that no more than 1 or 2 percent of thawed eggs would result in a live birth.

Then, in 1999, reports appeared of a more reliable method of freezing: vitrification, which freezes the egg so fast that ice crystals cannot form. A research team based in Australia and Italy described animal experiments in which 1 in 4 vitrified cow eggs fertilized and later grew, around day 5, to the blastocyst stage. It was only half the rate reached for fresh cow eggs, but it was still several times better than the rate of slowly frozen eggs. When it came to clinical use, some researchers put the live birth rate of vitrified eggs at between 2 and 12 percent for women under 38 years of age.

At first, vitrification was limited to people who froze eggs for medical reasons such as cancer. But in 2013, cryopreservation of eggs became an option for anyone who wants to delay motherhood for any reason, medical or not.

By 2020, estimates estimated that a growing subset of women who choose egg vitrification each year in the United States do so because they are not yet ready to have children, but ultimately expect lifestyle-related reasons known as “social freezing. ".

Although social freezing is often promoted as a way to delay procreation almost indefinitely, it turns out that most women never return to the clinic to use their frozen eggs. At McGill University in Montreal, for example, William Buckett and colleagues found that over the course of 13 years, the school’s cancer fertility preservation program treated 353 women, of whom 9 percent died, 6 percent she became pregnant spontaneously and most were either still treating her cancer or had lost contact with the clinic for unknown reasons. Only 23 women, 6.5 percent of the group, returned to McGill to use their frozen eggs or embryos. That low rate of return is also true for women who opt for social freezing.

illustration of a sperm entering an egg as more sperm swim towards itOnce a single sperm has entered an egg, a special protein layer called the pellucid zone shuts down all other sperm.Nicolle Rager Fuller

The sperm meets the egg

Compared to the human egg, sperm are quite simple. They were first observed in 1677 when Antonie van Leeuwenhoek, the Dutch inventor of one of the world's first microscopes, glanced at his own low-magnified ejaculate and noticed what he called "animalcules" swimming in the sample. But its structure and function did not come into focus until 1876, when Hertwig saw a sperm fertilize a sea urchin's egg.

Pincus and Enzmann, who were the first to bring mammal eggs to maturity in the laboratory, used rabbit sperm to achieve the first laboratory fertilization in a mammal in 1934.

It took years of struggle to make the jump from rabbits to humans. In 1951, a sperm rarity made it seem that Pincus and Enzmann were lucky. Apparently, sperm cells needed to be prepared in some way through a process called training before they could pass through the egg.

Robert Edwards of Cambridge University, one of the world's leading researchers in IVF during the 1960s and 1970s, thought training would be "a terrible obstacle to IVF," recalls Roger Gosden, an embryologist who worked in Edwards' lab. and was author of the biography of Edwards, That there is life. It recalls some frantic attempts to mimic sperm training, such as when scientists created porous chambers, about the size of an implanted IUD or intrauterine device, used for contraception. Researchers would fill a sperm chamber and insert it into a volunteer’s uterus, hoping to expose the sperm to any unknown enabling substance that exists in nature. After waiting a while, the scientists pulled on a rope attached to the camera to retrieve the sperm now “ready” to see if they managed to improve the fertilization of the eggs in the lab.

In the end, scientists found an easier way. “You just have to wash the sperm to get rid of some surface components,” Gosden says, and the sperm are ready to fertilize an egg in a lab dish. Other difficulties in clinical research turned out to be more formidable, such as measuring women’s best recovery time from eggs and modifying how the days after a man’s fertilization were best for transferring the zygote to the uterus. Edwards and his collaborators, including gynecologist Patrick Steptoe, had more than 300 failed attempts at in vitro fertilization before their first success with Louise Brown in 1978 in England (SN: 25/07/18). Edwards won the Nobel Prize in 2010 for his discoveries of IVF.

Louise Brown as a childThe 1978 birth of Louise Brown, a healthy baby girl, launched the field of in vitro fertilization. Although controversial at the time, IVF was responsible for millions of births.Stock Foto ZUMA Press Inc./Alamy

Among the beneficiaries of Edwards' outrageous work is Claudy, who was diagnosed with breast cancer at age 29 and who in a different century may never have been able to have a baby of her own. Claudy sought out Michaël Grynberg and colleagues at the fertility clinic at Antoine Béclère University Hospital on the outskirts of Paris to discuss ways to preserve it. It was 2014 and freezing eggs by vitrification was becoming more common. But bringing an immature egg to maturity in the lab was still relatively rare. From the time of the first baby born from fresh eggs matured in the laboratory in 1991 to the time of Claudy’s arrival at the Grynberg clinic, there have only been about 5,000 such births.

But Grynberg had no choice. He had to recover Claudy’s immature eggs, both for speed and to avoid aggravating his hormone-sensitive breast cancer with fertility drugs. Also, you would have to do something unprecedented in the context of cancer: freeze those mature lab eggs for later use. No one had ever been born from eggs taken from an oncology patient that had matured in the laboratory and then frozen. (There had been a baby born at McGill in 2009 from a woman who did not have cancer, whose eggs ripened in the lab and froze and then thawed).

Grynberg extracted seven immature eggs and was able to cultivate six of them until maturity over the next 48 hours. Those six were frozen, while Claudy underwent surgery and chemotherapy.

A few years later, Claudy’s oncologist told her it was safe to get pregnant and she spent a year trying to conceive. But she doesn't. So, in 2018, he returned to the Grynberg clinic, where doctors prepared to defrost him six frozen eggs.

To fertilize them, in Claudy’s case, a little more ART was required. Because their eggs had been frozen, their partner’s sperm would need help fertilizing the eggs. Vitrification causes changes in the outer membrane of the egg which makes the thawed egg especially hard for sperm cells to penetrate. This membrane, called the pellucid zone, is a formidable barrier even in nature (SN: 1/3/09, p. 15). One of the first to describe it was Sardul Singh Guraya, a biologist at the Punjab Agricultural University of India, who did his first work on field rats.

illustration of a blastocyst heading towards the uterusAfter fertilization, the blastocyst makes its way to the uterus. When an egg fertilizes in the lab, scientists can cut a cell from this developing mass to check for genetic problems without causing injury.Nicolle Rager Fuller

The pellucid area, as reported by Guraya in 1978, is a barrier around the egg made of proteins and carbohydrates, and when a sperm fails to do so, the cortical granules rearrange to close all other sperm. This ensures that the zygote will have a normal genetic complement of only two pairs of 23 chromosomes, one from the mother and one from the father, rather than a very inflated number that would occur if multiple sperm fertilized the egg.

Scientists have spent much of the next decade trying to put sperm into eggs that had been frozen using micro-manipulations described in terms as invasive as “zone perforation”. But sperm has not yet reached the nucleus for fertilization.

Then, in 1992, Gianpiero Palermo, an Italian sabbatical scientist at the University of Bari, reported an accidental discovery he made while working in a fertility laboratory at the Free University of Brussels. When he tried to inject sperm gently under the outer layer of the egg, taking care not to pierce the jealous center known as the cytoplasm, he noticed that an occasional “hole” in the membrane would allow sperm to penetrate directly into the center anyway. When this happened, the egg almost always fertilized. So, despite the general recommendation to avoid doing so, Palermo tried to inject sperm, tail and everything, directly into the cytoplasm.

Of the first 47 attempts made by Palermo and colleagues in Brussels with this approach, 38 eggs remained intact after injection, 31 were fertilized and 15 grew to embryos that could be transferred to a uterus.

microscopic image of in vitro fertilizationWhile a pipette (left) holds an egg in place, a single sperm is injected to fertilize the egg in a technique known as ICSI.Temple / Source of science

All in all, four babies were born: two healthy children from two individual pregnancies and a healthy pair of twins from boys and girls. Belgian scientists have called the procedure ICSI (pronounced ICK-see), short for intracytoplasmic sperm injection.

Nowadays, injecting a single sperm directly into an egg is even more common than the traditional form of IVF that adds sperm to an egg in a laboratory dish so that fertilization occurs on its own. The injection method is used in approximately two-thirds of ART cycles worldwide. And it is used in virtually every cycle that, like Claudy’s, begins with a frozen hard-shelled egg.

Growing up a healthy baby

In 2018, Claudy returns to the fertility clinic of the Antoine Béclère University Hospital, in the suburbs of Paris. He is 34 years old and has no cancer. Due to the unusual nature of their case, the eggs were immature when they recovered and reached maturity in the laboratory, doctors are not confident that the eggs will survive thawing and subsequent handling.

Claudy's six eggs are thawed with no apparent damage. Scientists perform ICSI on the eggs, using fresh sperm from Claudy’s partner. Five of the eggs fertilize.

These five zygotes enter an incubator so that they can develop into a ready-to-implant stage. They go through the first stages of cleavage, in which a cell becomes two, two to four, four to eight, and so on.

At many fertility clinics in other parts of the world, doctors can interrupt things at this time to remove one or two cells from the initial embryo to see if things are progressing normally. It was revolutionary to discover that this could even be done, a feat first performed in 1968 by embryologist Richard Gardner. At the time, Gardner was a graduate student working in Edwards' laboratory in Cambridge. Their work demonstrated for the first time that it was possible in rabbits to extract cells from a blastocyst without causing damage (SN: 8/3/68, p. 119).

illustration of an egg implanted in the uterine wallOnce implanted in the uterine wall, the cells of the embryo begin to differentiate, eventually forming all the tissues and organs that make up a human being.Nicolle Rager Fuller

Scientists can examine the chromosomes of those cells extracted from a human embryo, a process called preimplantation genetic diagnosis or PGD. They may be looking for a particular disease-related gene that works in the family to prevent the implantation of an affected embryo in the womb. Or maybe they are checking that a developing embryo has the right number of chromosomes and that the embryo has a good chance of implanting in the womb and leaving a baby with 10 fingers, 10 fingers and the possibility of a healthy life.

In the future, they could also use PGD to see if a desired gene fit, introduced through a gene editing technique such as CRISPR, has become effective. Without GDP, none of these approaches, from disease prevention to design babies, could take place.

Two days after putting Claudy’s five embryos in the incubator, only one remains in cleavage. That’s the embryo that doctors are moving to Claudy’s womb in the fall of 2018.

The embryo implants and continues to develop the way any embryo would, regardless of the history of its origin: a ball of a few hundred genetically identical embryonic cells that eventually differentiate into the approximately 200 cell types that make up a human. The mechanism by which this occurred was first established in 1924 by German researcher Hans Spemann, who discovered the "organizing effect" that leads specific regions of the embryo to become particular cell types.

In 1965, Beatrice Mintz created mice that bore her strange genetic lineage in her unique black and white striped coats. In his lab at the Institute for Cancer Research in Philadelphia, he created a mouse with four fathers (two mothers and two fathers) to demonstrate the genetic contribution of which fathers ended up in which region of the body (SN: 4/12/69, p. 361) .

black and white image of Beatrice Mintz looking under the microscopeBiologist Beatrice Mintz has created thousands of mice from four parents, mixing different traits to be able to trace the genetic origins of the distinctive organs that develop from the undifferentiated cells of a developing fetus.Smithsonian Institution / Flickr

Mintz fused eight-cell embryos from two different mice, one pure black embryo and one pure white, by placing them in a laboratory dish, dissolving the protective layer around each embryo, and carrying them using a glass bar. The result was a mosaic mouse: some of its cells contained genes that could be traced directly to both white-mouse parents, and some had genes from both black-mouse parents.

Other mysteries of how embryos develop were revealed using “knockout” technology, in which scientists deactivated genes in a particular region of an embryo to see what those genes controlled. In 1995, developmental biologists William Shawlot and Richard Behringer of the MD Anderson Cancer Center at the University of Texas reported using this method in mouse embryos, confirming Spemann's theory that a small region of the embryo touches changes in neighboring cells to make them particular. cell types (SN: 01/04/95, p. 197).

The embryo in Claudy's uterus develops normally; everything about her pregnancy seems normal, except how miraculous Claudy herself could feel, who must have had some doubts, like a new breast cancer patient, about whether she would ever have a baby of her own. In early July 2019, Claudy returns to Antoine Béclère University Hospital, this time to give birth. Her son is born on July 6; she and her partner call her Jules.

When Grynberg asks Claudy for permission to write her historical case in the Annals of Oncology, she is overwhelmed. “I thought about everything that had happened,” he told a reporter for the British newspaper The Telegraph as he posed for a photo with baby Jules. "And I cried when I realized how lucky I was."

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