Shortly before 10:30 on a recent evening, with a nearly full moon luminous through mile-high air, Jonathan Van Blerkom climbed into his car, eased out of his driveway, and threaded his way through a quiet Denver neighborhood to check on the fate of some precious human eggs. They had been inseminated that morning, and some of them should be one-celled embryos by now. Van Blerkom’s day had begun more than 16 hours earlier, but human development works the night shift, so Van Blerkom does too. Every evening, weekends included, he sets out on this five-minute drive to do one of the things he does best: look at very early embryos, only hours after fertilization, to decide if they are likely to become babies.
The embryos had been incubating all day in a small laboratory at Colorado Reproductive Endocrinology, a private fertility clinic where Van Blerkom, a professor at the University of Colorado, collaborates with in vitro fertilization doctors to help increase the chances that infertile couples can have children. He himself is not an “IVF doc.” He is a scientist with a passionate, if not obsessive, curiosity about the biological factors that allow an egg to create a human. Ironically, that interest has also made him an expert in all the things that can go wrong with an egg and doom a pregnancy—even before it begins.
On this particular night, Van Blerkom dropped in to check on the status of eight eggs that were harvested that morning from a persistently infertile woman and soon afterward mixed with her husband’s sperm. The woman had undergone several previous cycles of IVF at another clinic without a pregnancy, and Van Blerkom wasn’t particularly hopeful about this round, either. “She’s maybe a problem,” he said in his low, urgent voice as he moved quickly about the lab.
Van Blerkom—dressed in blue jeans and a blue button-down shirt, a fringe of long graying hair sticking out like a worn-down but beloved brush—took great pains to keep the eggs warm during this nocturnal assessment. He turned on special heaters and waited about 15 minutes until the filtered air under a protective hood—where he would inspect the nascent embryos under a microscope—had reached 95 degrees Fahrenheit. Then he removed several small plastic dishes from the incubator and began to peruse the eggs.
For the better part of the past two decades, human embryologists have been staring at eggs and early embryos trying to decide which are “good” and which are not, which embryos seem most likely to yield a viable infant after implanting and which are destined to fail. These judgments have traditionally involved more art than science, as befits a procedure with an overall success rate of less than 34 percent. Van Blerkom has spent the last 25 years trying to inject scientific logic into these snap visual judgments, which last no more than 30 seconds.
Under the microscope, these eggs appeared like dark dots in a field of cellular clutter. “She has a couple fertilized,” he remarked, removing the debris with a sharply pointed pipette. Then he moved to a second, more powerful Leica microscope attached to a video monitor. One by one, eight human egg cells, as big as the moon that Colorado night, loomed on the screen.
“This is at 10 hours after insemination,” Van Blerkom said. “There, you can see the pronuclei.” There on the screen was the huge, rotund universe of the female egg cell, its internal jelly, or cytoplasm, smooth and evenly grained, and there, just below the equator, two ghostly yolklike circles around the male and female DNA, mere mirages of genetic material, in close proximity, nearly nuzzling. Each gamete—egg and sperm—prepares its half packet of genetic material, known as the pronucleus, and one of the first organizational tasks of human development is to bring these two packets together. The glancing proximity of the male and female pronuclei on the screen represented the final stage of a daylong dance—a long latitudinal migration by the sperm’s DNA to the site of the female pronucleus, so that the male and female chromosomes can “approach each other and melt into one,” as a 19th-century embryologist poetically put it. That produces a complete set of human chromosomes and leads to the first division of the cell.
Even though the first several embryos looked smooth and even, Van Blerkom wasn’t optimistic. “She doesn’t have great stuff,” he said. When asked how he could tell, he replied, “Just by looking at the quality of the cytoplasm in the unfertilized eggs. This is in pretty bad shape. These are not normal eggs.
“Look at this one,” he continued. “This one has a lot of disorganization in the cytoplasm.” And indeed, as more of the eggs filled the screen of the monitor—some fertilized, most not—the cells frequently had large vacuoles, or fluid-filled bubbles, in their interior. From experience, Van Blerkom knew that, although such eggs may become fertilized, they rarely produce a successful pregnancy. There is even a hint of evidence that normal-looking eggs from a woman who also has these abnormal eggs may fail to yield offspring.
“You look at these eggs, and you know they’re telling a story,” Van Blerkom said later. “But you only know bits of the story. If it were an abstract notion, who’d care? But around the world, thousands of people are looking down microscopes at thousands of eggs and asking, ‘Should I keep this?’ So life-or-death decisions for the one-celled embryo are made every day. My argument is, let’s make those decisions based on biology.”
For more than 20 years, Van Blerkom has been trying to understand the story that egg cells are telling, and although the tale is far from complete, some compelling new clues to early development have emerged. As both an academic studying the basic biology of mammalian development and as an IVF consultant with access to human egg cells and human embryos for research purposes, he is one of just a few scientists in a position to push a revolution in thinking about how—and whether—life begins. It involves the way an egg cell is built and how information positioned during that construction affects the fate of the embryo.
Scientific study of this phenomenon, known as polarity, could reveal how the fate of a human embryo may be shaped—and predicted—by extremely early biological events that predate conception by days, weeks, or even months. Surprising new research findings by Van Blerkom and others raise the paradoxical possibility that the viability of life may be determined long before fertilization.
The notion of polarity is quite simple. If you imagine the female egg cell (and later, the fertilized egg) as a spherical planet with its own intrinsic biological geography, then certain characteristics of that cell—the location of protein molecules or RNA messages or biochemical traits like pH or even the internal connective structures called microtubules—will be more prominent in certain regions, like one hemisphere as opposed to the other, or near the surface rather than near the core. Polarity of this sort has been known for a long time in the embryological development of simple animals like frogs and fruit flies. For just as long, it was not thought to be relevant to development in mammals.
But in the past few years, prominent British embryologists have shown that polarity exerts tremendous influence on the early development of mouse embryos. And several biologists in this country are pushing the idea of polarity in human development to more extreme conclusions. They argue that the fate of an embryo depends on the way the egg organizes itself, and that polarity in the egg can ordain either a successful or failed pregnancy before conception. This has profound implications for our understanding of life’s origins, for our understanding of why so many embryos spontaneously abort in the first few days after fertilization, and for our understanding of why some IVF procedures may subtly affect early development, with potential long-term health consequences.
Most of all, it means that the scientists who study human development are increasingly looking at deep time, at events that shape the human embryo well before fertilization. The momentum of research, said Van Blerkom, is pushing embryology back into the realm of cell biology, because the fate of the organism is so inextricably tied to the quality of one cell above all: the egg. “In mammals,” he said, “these are things that are too important to be left to chance.” And so they are built into the eggs.
Back in the 17th century, when British physician William Harvey made his famous observation “ex ovo omnia” (“from the egg, everything”), natural philosophers believed that human development derived entirely from the egg. The sperm, in size as well as in deed, was puny by comparison. The most recent research confers molecular respectability upon Harvey’s old maxim. Contrary to the message of 20th-century genetics, the success of the embryo may have less to do with embryonic genes than with maternal proteins passed on by the mother, and less to do with the embryo’s DNA than with the maternal dowry the egg brings to conception.
The basic time course of fertilization and early development has been known for decades. When a sperm cell meets an egg cell (the oocyte), it burrows through the thick outer rind surrounding the egg (the zona pellucida), enters the internal cytoplasm of the egg (the ooplasm), and locomotes its male DNA—half of the typical number of chromosomes—to the female half within about three to four hours. During this microscopic odyssey, the sperm undergoes tumultuous transformations, using some as-yet-unknown materials in the cytoplasm to build a “beacon” to find the female pronucleus, its head of DNA swelling some five times its original size and then later condensing into chromosomes at the end of the journey. “The cytoplasm,” Van Blerkom said, “dictates what the sperm does.”
Once the two packets of DNA meld into one complete set of 46 chromosomes, the one-celled embryo begins to cleave, or divide, becoming a two-celled embryo at around 22 to 28 hours after fertilization, four cells another day later, and eight cells around day three. Only then do the embryo’s own genes fully kick into gear and begin to function. Because these cells are grouped in a loose, pebbly collection resembling a berry, this stage of the embryo is referred to as the morula (from the Latin for “little mulberry”). Around the fourth day, however, the 15-to-25-celled mulberry dramatically tightens and seals its connections with neighboring cells (a process called compaction) and begins pumping fluid into its internal cavity. Now known as a blastocyst, the embryo undergoes a dramatic division of cell fate, forming a distinct outer layer of cells and an equally distinct bulge of about 20 or 30 cells on the inside. The outer cells (the trophectoderm) become the placenta; the inner bulge of cells includes embryonic stem cells, destined to form the entire fetus. Usually by the sixth day after fertilization, the blastocyst will hatch out of the egg cell’s still-resilient rind and attach to the uterus.
The intricacy with which an early embryo divides, compacts, hatches out of the zona pellucida, ingeniously secretes molecules that penetrate the cells lining the uterine wall in order to implant in the womb, and then recruits blood vessels to nourish the placenta and the developing fetus marks one of the most awe-inspiring metamorphoses in all of nature.
But here’s the rub: It’s horribly inefficient in humans.
Much more often than not, the process fails. Although the statistics on the failure rate of human fertilization are not entirely robust, given the biological and ethical delicacy of conducting research in this area, the numbers consistently suggest that, at minimum, two-thirds of all human eggs fertilized during normal conception either fail to implant at the end of the first week or later spontaneously abort. Some experts suggest that the numbers are even more dramatic. John Opitz, a professor of pediatrics, human genetics, and obstetrics and gynecology at the University of Utah, told the President’s Council on Bioethics last September that preimplantation embryo loss is “enormous. Estimates range all the way from 60 percent to 80 percent of the very earliest stages, cleavage stages, for example, that are lost.” Moreover, an estimated 31 percent of implanted embryos later miscarry, according to a 1988 New England Journal of Medicine study headed by Allen Wilcox of the National Institute of Environmental Health Sciences.
In some respects, less scientifically sophisticated cultures may have come to terms with this conundrum in the way they grappled with the knotty question of when life begins. The medieval etymology of the word conception, said Harvard biologist John Biggers, traces it to the Latin root capio, which means to grasp, take hold, or receive into the body. In 1615 an obscure writer named Cooke noted, “Conception is nothing els but the wombs receiuing and imbracing of the seede,” suggesting that centuries-old notions of conception referred, perhaps wisely, to when an embryo survived its perilous first week and was “imbraced” by the womb.
Nonetheless, the high failure rate begs challenging ethical questions. If life begins at conception, as many believe, why are so many lives immediately taken? If, as some ethicists argue, nascent life must be protected, how do we assess the degree of moral entitlement due a nascent entity that fails to pass nature’s own muster perhaps 80 percent of the time? And if the fate of an organism is indeed inscribed in the earliest biological inklings of an egg, does life begin with the gametes?
From a purely scientific, not to mention pragmatic, point of view, the main question is more straightforward: Why do so many embryos fail to grasp the womb? That question has bedeviled developmental biologists for decades, and more recently, it has vexed clinicians who practice assisted reproductive medicine. Studying early human development in the academic setting is extremely difficult, in part because of political constraints on embryo research in the United States, so a certain amount of our knowledge is limited to inferences from animal studies.
Nonetheless, it has become increasingly clear that the fate of an embryo may be cast in the ovarian follicles, where egg cells are built. “Much of the developmental biology and ability of the human embryo is determined even before it’s fertilized,” Van Blerkom said. “This all happens by the one-cell stage, which is when the fate of the embryo is determined.”
Such thinking upends long-held assumptions in the world of biology. Mammalian development was once thought to be essentially different from embryological development in fruit flies, frogs, worms, and other laboratory organisms, where well-defined polarities in the egg—higher concentrations of a protein in one part of the egg than in another, for example—ordained such fundamental aspects of body plan as head and tail, or back and belly. Mammals seemed exempt from these rules for building a body. In the mouse, it had been shown in the 1970s and 1980s that if you split an embryo at the two-cell stage, each resulting cell had the ability to develop into a full organism. If the egg were indelibly etched with asymmetric information that unequivocably determines development, the argument went, how could two embryonic cells be separated and still produce whole, intact, normal individuals? “Animal experiments led to the conclusion that mammalian eggs do not have polarity, but I think that’s a huge fallacy,” said David Albertini, a developmental biologist at Tufts University in Boston. One possible answer, he added, is that mammalian embryos are similarly shaped by polarity but retain a certain developmental flexibility as well.
These days, as biologists like Van Blerkom, Albertini, and a superb school of British embryologists based in Oxford and Cambridge have started to look at the early embryo, they have begun to catalog a number of very early polarities that affect both the competence of the egg and the form of later embryonic development. The implications of polarity reverberate far beyond the confines of academia. For example, Van Blerkom and Albertini have a gentlemanly disagreement about recent research that may spill out into the public discourse soon because it raises the possibility that some popular IVF techniques might have subtle but long-term health implications for children conceived in a dish. Indeed, on the night that Van Blerkom inspected the fertilized eggs at the Denver clinic, he made this disagreement clear at one point by holding up a sharp micropipette for my benefit. He remarked over his shoulder, “This is what I use to take off the cells that David Albertini says I shouldn’t take off.”
And with that, he began prying away the granulosa cells clinging to the eggs, in order to get a better microscopic view of the nascent embryos to see if they were developing properly. Within three days or so, those denuded embryos would be implanted in a woman’s womb.
The Sperm Cell
Polarity begins in the sex cells. The female egg cell is a huge biochemical universe unto itself, with a complex and sophisticated cytoplasm. The sperm cell, by contrast, is little more than DNA strapped to an outboard motor. Nonetheless, of the 15 percent of couples experiencing infertility problems, about half the trouble can be traced to the male, mostly in the genetic qualities of the sperm.
Immature sperm cells form during the fourth week of embryological development but remain unfinished until puberty. At that point, the male begins to churn out haploid sperm cells—that is, sex cells with half the normal complement of 46 chromosomes. Thus, when a sperm cell delivers its genetic cargo at fertilization, the one-celled egg again possesses the full 46 chromosomes. Sperm dysfunction can arise from the way these cells are built. The sperm has an acrosome (the head and sheath), a nucleus, and a tail. Sometimes a club-shaped profile on the head disturbs the proper construction of the tail. These tail abnormalities can include looping, folding, and fusion, all of which can result in reduced motility (ability to swim).
While assisted reproductive techniques such as intracytoplasmic sperm injection (ICSI)—which involves the direct injection of sperm into the egg cell—can overcome head or tail abnormalities in sperm, recent animal research suggests that fertility doctors must use these techniques with care. Abraham Kierszenbaum of the City University of New York Medical School has conducted experiments in mice showing that even normal-looking sperm from a mutant mouse “is likely to create infertile offspring.” Hence, selection of donor sperm, he said, cannot be based on appearance alone.
Biologist Jonathan Van Blerkom of the University of Colorado published a paper in 1996 suggesting that some cases of male infertility derive from defects in a tiny structure in the sperm cell called the centrosome. When a sperm penetrates the egg, it unwraps the centrosome, an organelle that acts like a construction foreman overseeing the creation of microtubules in the cell. Sperm DNA uses these microscopic highways to find the female DNA and merge into a zygote. If a sperm has centrosome defects, Van Blerkom speculates, it can get inside the egg but then is destined to wander in the desert of the egg’s cytoplasm, unable to find its way to the female’s DNA.
—S. S. H.
While the debate over polarity is much more sophisticated these days, it is not entirely new. In the late 1930s and 1940s, Arthur Hertig, John Rock, and several colleagues did an experiment in human embryology that to this day remains without peer in terms of elegance, revelation, and chutzpah. Working at the time as a researcher at the Free Hospital for Women in Brookline, Massachusetts, Hertig persuaded eight women scheduled to have hysterectomies to record intimate details of their lives prior to the surgery to remove their wombs, including when they menstruated and had sex. Armed with such precise information, Hertig’s research team found developing embryos in either the fallopian tubes or uteruses of the women and, adapting the headlight from an automobile to illuminate their work, took photographs of early, preimplantation human embryos. Not only were they able to estimate when fertilization had occurred and also plot the time course of early human development, they also made an astonishing discovery: Half the embryos were clearly abnormal. This was the first concrete hint that most human embryos fail during the first week of development. Among other things, the paper that Hertig and Rock published in 1954 contained some of the first micrograph images of a human embryo at the two-celled stage. Hertig expressed the hunch that one of those cells was destined to be placenta, the other the developing organism.
Throughout his distinguished career (he headed the department of pathology at Harvard Medical School for two decades), Hertig suspected that there was a very early commitment by embryonic cells to become either a fetus or the placenta. He continued to explore this idea after his retirement, when Harvard set him up in an animal laboratory in the central Massachusetts town of Southborough to continue embryological research in monkeys. In the mid-1960s, the lab hired a teenager from nearby Hudson for a summer job cleaning out animal cages, and Hertig filled the kid’s ears with his theories. “I had no idea who this guy was,” the teenager would later say. “But he took me under his wing, and by the end of the summer, the guy is teaching me about ovaries and eggs.”
A print of that first micrograph of a two-celled human embryo is now framed and hangs on the wall above the desk in David Albertini’s small, crowded office at Tufts University where, 30 years after he cleaned the monkey cages in Southborough, he conducts research trying to figure out how the fate of those two cells is determined. The search keeps leading back to the mother’s eggs. “You can’t produce a healthy human unless you produce a healthy egg,” said Albertini. “What endows a healthy egg, and thus a healthy embryo?”
In some respects, a human egg takes a lifetime to mature. Each female possesses up to 2 million oocytes at the time of birth, but that number is winnowed down to about 250,000 by puberty. Roughly 400 of these unfinished oocytes will mature and be ovulated during a woman’s reproductive years, although the quality of the finished eggs declines as she ages. The vast repository of egg cells remains shelved in the follicles until the brain sends a signal in the form of monthly bursts of hormones, which trigger the final maturation cycle. From that signal, it takes approximately 110 days for an egg to grow, mature, and finally be released from the follicle.
In the late 1980s, Albertini’s group began to focus on a group of satellite cells that surround the oocyte as it begins to grow and mature in the follicle. As eggs develop, each one is surrounded by a herd of much smaller hangers-on. These are called granulosa cells, and under the microscope they look like grapes glued to a beach ball. Albertini and his colleagues noticed that the interaction between the oocyte and the cells surrounding it was not symmetrical; there were more cells—and, it would turn out, more molecular back-and-forth traffic between the egg and the granulosa cells—at certain regions on the egg.
“We proposed that these cells on the outside were imposing an asymmetry on the egg,” Albertini said. The pattern, originally identified in rodents, has now been shown to be true of cows, rhesus monkeys, and as of three years ago, humans. “Almost all animals build an egg in the ovary and position molecules in the top and bottom. This is a highly conserved evolutionary mechanism to make sure that when the cell gets subdivided, the cells at the top will become the head, for example, and the cells in the back may become a gonad. So you basically have to lay that down in the egg. And then you’re just carving up the pie. We’ve been the first to have evidence to support that in the mammal, though not in the human yet. And there is evidence in human eggs, from Van Blerkom and others, that molecules are partitioned.”
Unlike Van Blerkom, who has regular access to human eggs and embryos through his IVF-related work, Albertini works primarily with mouse and primate cells. But his lab’s animal studies have revealed that asymmetry in an immature egg is important to the development of an embryo.
Through a series of elaborate experiments with mice, Albertini and his colleagues at Tufts have shown that the small cells bunched around an egg cell in the follicles are not mere microscopic groupies. They form connections, known as gap junctions, that send tendrils much like plumbing lines into the egg. The plumbing analogy is apt because molecules flow into and out of the egg through these channels. The molecules are critical to normal development: When the genes for certain of these molecules are experimentally erased, the eggs made by female mice are invariably defective, and the errors fatally disrupt the normal choreography of egg maturation.
Moreover, Albertini’s group is exploring whether these plumbing lines, which corkscrew into the outer rind of the egg, play a role in establishing one of the most important geographic landmarks in the life of an egg cell—an event, Albertini likes to say when lecturing medical students, that marks “one of the most important days in your life.”
“When you build a big round cell,” Albertini said, “where do you put its nucleus? In most animals, you anchor it to one side, and that sets up all sorts of polarity.” This happens early in the maturation of an egg cell, he argued, and is shaped by the position of the cells surrounding the egg.
When an egg cell matures, it must reduce its complement of DNA by half. This parceling process, called meiosis, occurs twice in the egg cell—once during a woman’s fetal development and a second time as the egg is released from the ovary. During the initial phase of meiosis, as a woman’s egg cell reduces its number of chromosomes from the normal 46 to the 23 found in sex cells, it parks one expendable sack of halved DNA in a spot near the cell surface. This is called the first polar body, and it defines one of the earliest discernible landmarks in the developing egg. This so-called animal pole is where the primordial nucleus of the one-celled embryo is destined to form. Just prior to ovulation, as the egg begins its second round of meiosis, it creates a spiderweb trace of proteins called the spindle, which allows the chromosomes to separate properly and is critical to a successful pregnancy. Spindle defects are believed to be the leading cause of the chromosomal abnormalities that doom so many early embryos.
Albertini’s group now suggests not only that these outside cells tell the egg where to locate the polar body—and, therefore, the nucleus and spindle—but also that their plumbing lines soften up the egg cell’s rind in the opposite, or vegetal pole, to increase the odds that sperm will penetrate the hemisphere opposite the nucleus. “We were able to study, in human oocytes, where the chromosomes were in relation to the polar body,” Albertini said. “If the egg is born with an animal and a vegetal pole, the polarity must have come from the ovary because that’s where the egg is built. The somatic cells [those outside the egg] may impose that axis. There are more cells, more connections on one side of the egg than on the other. Basically, what we’re finding is that the side the nucleus is on has little contact with outside cells, and the further you move from the nucleus, the more connections you see.” He believes that this sets up the internal organization of the egg’s cytoplasm.
In fact, Albertini has preliminary evidence suggesting that the communication between the egg cell and its surrounding granulosa cells rises and falls in a precise monthly cycle. Since the monthly spike of a follicle-stimulating hormone seems to dampen the information exchange, he is now exploring the possibility that each ovulatory cycle not only releases a mature oocyte but also uses the monthly burst of female hormone to adjust the compass of polarity in the eggs that are still growing and will be ovulated one, two, or three months later. “We can only extrapolate to humans, but in the mouse, our data show that the whole process [of egg maturation] takes 18 to 20 days, and we can detect this asymmetry by the second or third day of the process. In humans, as an extrapolation, I’d predict that it would emerge between day 10 and day 20 in a 100-day process prior to ovulation—three full reproductive cycles before that egg would be used.” If this preliminary hint holds up, the implications for maternal health become significant. Well before a woman attempts to become pregnant, she may be exposed to environmental effects—diet, prescription drugs, alcohol, and various toxins—that could affect the construction of her eggs. “Do you remember what you were doing three months ago?” Albertini asked.
The Albertini research, while pushing the starting time earlier, joins an emerging body of research establishing the impact of polarity on embryological development. In 2001 Magdalena Zernicka-Goetz and her colleagues at the Wellcome/Cancer Research UK Institute at the University of Cambridge did a clever experiment in which they dissolved colored dyes in olive oil and then stained each of the cells of a two-celled mouse embryo a different color—one blue and the other pink. As the embryo developed, the cells of the inner cell mass and the developing organism were predominantly pink while the cells of the developing placenta were blue, suggesting that developmental fate may have been etched into these cells from the moment of their very first division. This was, in a sense, a possible molecular answer to the hunch about early mammalian fates voiced by Arthur Hertig of the two-celled embryo half a century earlier.
Embryology has come a long way since those black-and-white images by Hertig. Van Blerkom has, among many other things, elevated the biology of human conception to high art. His lab in Boulder is filled with spectacular pseudocolor images that are every bit as dramatic as the peaks of the Front Range, which practically begin at the door to his office. The images depict what might be called embryology in flagrante: micrographs of sperm cells, trailing accordion-like pleats of white zags as they streak across a vast blue ocean of ooplasm; a multihued blastocyst in the process of hatching out of the egg’s zona pellucida; and egg cells with a fringe of glowing, fate-determining proteins, looking a bit like a solar eclipse inside a cell.
These are more than just pretty pictures. Ever since the 1970s, when he worked in England with the developmental biologist Martin Johnson, Van Blerkom has sought ways to analyze, and visualize, secret compartments and regions of the human egg that may offer clues to whether it is endowed with good fortune or bad. “So many embryos don’t work in the human,” he remarked one day, in the midst of a six-hour conversation. “Why do so many go wrong? How does it go wrong? And how can you use that information? All of this stuff is going to come back to polarity. Human eggs that don’t develop normally may be an issue of polarity.” Asymmetries and polarities in both the cytoplasm and nuclear organization, Van Blerkom discovered, begin to appear even before fertilization. “It’s a huge cell!” Van Blerkom said. “It’s a 100-micron cell. And we know there are different things going on in different parts of the cell. There’s incredible shuttling within the cells. How does that happen?”
Like Albertini, Van Blerkom sensed that the most important information in the embryo was not confined to the nucleus but embedded in the cytoplasm. “If I’ve done anything in this field,” he said, “it’s to deemphasize the embryo and emphasize the egg cell. Our work has shown that it all begins with the oocyte, which can have subtle cytoplasmic defects that are actually very profound. But,” he added hastily, “you have to be careful. It’s like looking at canals on Mars. Unless you can show a consistent pattern [of polarity] and then an effect that is different as cells divide, it doesn’t have meaning.”
Van Blerkom had been seeing hints of polarity since the 1970s, but one of the major turning points occurred in 1996 when, by accident, his lab discovered that cells surrounding the developing egg—the same granulosa cells that had piqued Albertini’s interest—possessed a receptor very similar to the leptin receptor. Leptin made front-page news when it was discovered in 1994 because the molecule appeared to regulate fat metabolism and obesity. What was it doing in egg cells?
The Colorado lab discovered that granulosa cells—the cells that surround maturing eggs in the ovarian follicles—were pumping out leptin and shipping it into the egg. What’s more, the researchers showed that leptin is polarized in the egg in such a way that, after fertilization, the protein is allocated primarily to the cells that become the placenta, while it is virtually undetectable in the cells destined to become the fetus.
At first, many embryologists resisted the notion that leptin was segregated in certain parts of the egg and that this asymmetry had any significance for the fate of the embryo. “For a long time, no one believed it,” Van Blerkom said. But mice in which the leptin gene has been erased are incapable of producing embryos—the fertilized eggs die almost immediately. And various experiments tracking leptin inside the mammalian egg clearly showed a more prominent distribution in one hemisphere than in the other. It is now believed that this protein acts as a delayed silencer; it hangs around in the egg and keeps certain genes from turning on in certain parts of the embryo until days after fertilization. Again, the appearance of a protein in a certain part of the egg cell may affect embryonic development or the formation of organs days and weeks later.
Lately Van Blerkom has been intrigued by another form of polarity: the way mitochondria, the cell’s little power plants, migrate in the maturing egg cell. “It’s kind of like a lava lamp,” he says, “with these blobs of cytoplasmic elements moving up and down in the cell.” Typically, mitochondria arrange themselves along the outer edge of the egg cell. But at certain points in the reproductive cycle, they migrate en masse toward the nucleus. Wherever they gather, mitochondria change the local chemical microenvironment: They cause a lower pH, and that small change, Van Blerkom believes, can affect the local activity of certain enzymes. “It’s not a bag of cytoplasm,” he said. “It’s highly structured, and that structure is changing.”
Finally, Van Blerkom has conducted extensive work on the internal structural organization of the human oocyte. First the oocyte constructs the scaffolding of connections known as microtubules, which allow molecules to move around inside the cell. Then, toward the end of fertilization, the egg provides a kind of highway that allows the sperm to make its final approach to the female pronucleus. “There’s something in that cytoplasm that allows the sperm to know where it’s going,” he said. One of the compelling messages—and central paradoxes—to emerge from these studies of polarity is that even bad eggs can be fertilized to create an embryo, but only good eggs seem to create a successful pregnancy. The politics of embryo research, however, is one reason we don’t know more about what distinguishes good eggs from bad. Federally funded research on human embryos, although sanctioned by a congressionally mandated national bioethics commission in 1975, has faced unrelenting opposition from right-to-life groups. In 1996 Congress banned NIH funding outright for any research in which an embryo is destroyed. Van Blerkom calls the issue of when life begins the “third rail” of developmental biology. “You can find whatever you want in the embryo to support any position you have on when life begins,” he said. “A lot of people believe that life begins at conception. But life also ends at conception or shortly thereafter—hours after, a day after, four or five days after. We don’t know why that happens, and what’s gone wrong. We’d like to know the answers to those questions,” Van Blerkom said, “but we can’t do those experiments.”
If polarity and the forces that shape it play a determining role in the fate of a human egg, it’s not difficult to see the implications for making babies, whether through assisted reproductive technologies or the old-fashioned way. It becomes a particularly nettlesome question because basic research of the sort done by Van Blerkom and Albertini has historically been adapted—snatched, really—for use in IVF clinics, often before all the biological ramifications are clear.
Indeed, this is where the polite disagreement between Albertini and Van Blerkom becomes a matter of intense public and medical interest. If you believe, for example, that granulosa cells and other very early features of ovarian ecology set up the polarities that ultimately determine the quality of a human egg, as Albertini does, then certain techniques widely used in IVF may be subtly perturbing the very mechanisms that eggs use to establish a plan to build an embryo and maximize the chances that it will develop properly. “We recognized in the 1980s that many culture techniques used by assisted reproduction were reducing the quality of those eggs,” Albertini said. “My own skepticism has been growing that we therefore may be damaging things with what we’re doing to these eggs prior to embryogenesis.” Other researchers—notably Alan Handyside in England—have begun to express similar concerns.
Albertini cites a popular IVF technique known as intracytoplasmic sperm injection, or ICSI, in which sperm is injected by needle right into the middle of an egg cell. If his polarity research in mice is true for humans, with its suggestion that sperm are biased toward entering the egg at the opposite pole from the cell’s nucleus for important reasons, then ICSI injections might subtly disrupt patterns of polarity in the egg. Moreover, ICSI requires the removal of the cells surrounding the egg; Albertini thinks that might deprive the egg and early embryo of important signals or alter the time course of fertilization. Several rare, so-called imprinting disorders, including Beckwith-Wiedemann syndrome, a form of gigantism, have been found in children produced by ICSI, although the extent and significance of these links is unclear. “Ten years ago, we wouldn’t have thought about the polarity thing,” said Albertini. “It wasn’t even on the radar. But now we’re looking at how we’re making these babies.” Albertini hastened to add, “I’m certainly a proponent of human-assisted reproductive medicine, but I’m concerned that we’re rushing technologies before we’re certain they’re safe and effective.”
Van Blerkom respects Albertini’s research but expresses reservations about his clinical ruminations. “If there were really problems with manipulating eggs, you’d see it, and in fact you’d have seen it 10 or 15 years ago,” said Van Blerkom. “In the literature, there are only 26 reported cases of imprinting-associated disorders with IVF, and that is out of 1.2 million IVF births.” In some hands, he added, ICSI is now achieving fertilization rates of between 60 percent and 70 percent, even though the technique requires the removal of surrounding cells. “If these cells were so important,” he said, “you shouldn’t get such high pregnancy rates.”
Albertini replied that there might be subtle health effects, such as early onset of adult diseases like diabetes and cancer, that won’t appear until 15 or 20 years after IVF, and he pointed out that there is very little follow-up data on the health of children created through assisted reproductive medicine. Even Van Blerkom conceded that point. “There’s no systematic, organized mechanism for follow-up,” he said. “And the reason for that is that people don’t want it.”
It may seem like an arcane debate, but it has life-and-death ramifications every day, when IVF practitioners peer at egg cells through microscopes and try to predict the fate of the embryos they might become. IVF remains, at best, a hopeful art driven by the best of intentions and less than complete knowledge. About two weeks after he sorted through those eight human eggs late one moonlit night, Van Blerkom called to report, happily, that his initial hunch had been wrong.
“I’ve got good news,” he announced. “She’s pregnant.” It was a particularly felicitous way of acknowledging that, until biology provides a better crystal ball, pregnancy remains the best—and perhaps only—way to find out if an egg is good.