According to the Nobel Prize Committee, the great advances in the science of biology in the years from the middle fifties to the middle sixties involved advances in knowledge of the genetic code. The most famous hypothesis put forward and confirmed was Crick and Watson's model of DNA, but also of importance was later work, identifying, under tremendous magnification, individual genes. Biologists say that with the cracking of the genetic code and the visualization of the smallest unit in genetics, a period of intense exploration and significant discovery has come to an end; the work remaining is not speculative, but by way of filling in and reconfirming. There is a sense, among younger Len, that the "excitement" has gone out. Many have shifted their attention to the closely related field of embryology, which is rich in tantalizing problems. For example, the knowledge of how the basic genetic material reproduces itself has been helpful to the embryologist, but he still does not know exactly how the genes themselves dictate the orderly development of a fertilized egg into a complicated and highly differentiated / multi-celled organism: what makes an eye an eye, and an arm not an eye, and what puts together limbs and organs so as to make an individual creature? Not only are such questions of "pure" interest, but since they go to the center of the reproductive process, the answers to them promise to have practical application to problems of genetic defect and birth control, two worldly matters much on the minds of scientists and nonscientists alike.
The research of the last few years in embryology, or molecular and reproductive biology, has already yielded a number of interesting hypotheses, as well as laboratory experiments, such as the one called "cloning." Theoretically, since every cell of an animal carries a load of genetic information unique to that individual, it ought to be possible, by destroying (with radiation) the female complement of chromosomes in the nucleus of an unfertilized frog cell, say, and implanting in its place an entire nucleus lifted out from another cell of a second frog, to fertilize the first cell and fabricate an embryo which would grow, be born, and mature into an adult frog that would not merely resemble the donor frog but would be its absolute replica. In cloning, this theory has living proof. A whole crowd (or clone; Greek, "a throng") of identical creatures has been bred in the laboratory, literally and predictably identical in every way to the donor frog except that some of the copies are older than others. Among other things, this shows that an individual creature, previously unique and mortal, may be rid of its uniqueness and have immortality conferred on it. The experiment is dazzling, and some of the knowledge gained ("spinoff") may eventually have practical human application, but so far the major interest has been "pure": no one suggests that humans can be immortal, or that there is any good reason why they should be. Much the same thing can be said of experiments in parthenogenesis, or the asexual fertilization, by mechanical means, of mammalian eggs. The first reports of such "immaculate conception" in lower animals in the laboratory came many years ago, when the French biologist Jacques Loeb caused "traumatic parthenogenesis" by touching a sea urchin egg with dry ice, but recently the accuracy of the method has been improved, and it has been made to work on mammals such as rabbits. Again, however, the direct human application is not apparent for many reasons, among them that human eggs fertilized by parthenogenesis always deteriorate and die; that because of the chromosome business, only females are conceived; and that, taking a long view, parthenogenesis is undesirable because the machinery of adaptive evolution requires sexual reproduction.
There are many embryologists at work in many laboratories, and much going on besides cloning and parthenogenesis. Some of it focuses right on the human egg, sperm, and embryo. This work has closer applicability to human affairs; indeed, the money that pays for it comes mostly from funds allotted not for "pure" research, but for applied-specifically, research to be applied to real and actual problems of contraception, fertility, sterility, and birth defects. To gain knowledge which would lead to new and better technology and social programs in these areas of daily concern, embryologists have needed to observe, measure, and experiment with the first phases of human reproduction, which they have long suspected are crucial in many respects (such as genetic defect), but which they have had mostly to speculate about, since the events were hidden in the female body. Now, these first phases, including culturing of the human egg, its fertilization by sperm, and development as an embryo, have been carried out under the microscope, or in the scientific terminology, in vitro, "under glass."
There were an unusual number of obstacles that had to be overcome before this was accomplished. The first and by no means least was that human eggs suitable for experimentation were hard to get. While there has never been a problem getting sperm (the current rate of remuneration for a sample by masturbation is $25), eggs could only be obtained, until very recently, either from fresh cadavers or from ovarian tissue cut out during gynecological surgery and given by the surgeon to the embryologist as a favor. Both methods were unsatisfactory, yielding few and often damaged, stale, or immature eggs. It was much easier to get eggs from mice, rabbits, and hamsters, which could simply be killed at the right time in the egg-maturing cycle, and their eggs picked out of the follicles of the ovary or flushed from the oviduct. The first experiment of this kind was conducted by the German Schenk, who in 1878 put some rabbit eggs in a culture dish and added sperm; nothing happened, however. For about fifty years a small number of biologists in scattered laboratories fitfully persisted in trying to fertilize mammalian eggs outside the body. In 1934 an American, Dr. Gregory Pincus, later one of the developers of the Pill, published findings that suggested he might have succeeded in fertilizing rabbit eggs. In 1940, Dr. John Rock, another American, who was to play an important role in legalizing and propagandizing birth control, said that he had put some human eggs, which he had managed to get, into the presence of sperm, and that a small number of the eggs had been fertilized and had actually divided. Ten years later, Landrum Shettles, an obstetrician at Columbia-Presbyterian Hospital in New York, also claimed to have gotten fertilization and growth of the human egg in vitro, this time to a stage where the embryo was a solid mass of cells.
There was considerable skepticism, however, about the validity of Pincus', Rock's, and Shettles' results. One of their fellow scientists who was most skeptical, and offered the most persuasive criticism, was M. C. Chang of the Worcester Foundation for Experimental Biology in Shrewsbury, Massachusetts. Chang said that if you take an egg out of the body and just leave it alone in a culture dish, as likely as not it will show signs of "dividing"-signs that will inevitably prove misleading, for in fact, the egg will be deteriorating and dying. The events described by Pincus, Rock, and Shettles, Chang more than implied, could just as well have taken place, and therefore the case for fertilization was unconvincing. Furthermore, Chang reminded biologists that the very definition of fertilization had still not been agreed on (this was in the mid-fifties). He advanced the view, which has since been accepted by everyone, that fertilization, rather than being a single event which happens instantaneously (the mythical "moment of conception"), is a process which takes place over several hours. Fertilization, in other words, is not the simple penetration of the sperm into the outermost layer of the egg; rather, it includes a whole series of events and reactions which probably cannot be said to be complete until the division (or cleãvage) of the egg into two cells, each carrying a bad of maternal and paternal chromosomes in its nucleus.
Chang now set out to make a thorough study of the process of fertilization in mammals, with the goal of eventually devising and carrying out an experiment whose meaning no one could dispute. His diligent work during the fifties rescued this sector of embryology from the realm of the exotic. One of Chang's first and most important discoveries while working with rabbits was that the sperm. had to have something happen to it while it was in the female reproductive tract before it would penetrate an egg cell: the sperm had to be "capacitated." The precise mechanism of this physiological change in sperm is even now not completely understood, but exploiting what he had learned, and after extensive investigation in timing and mapping egg development, Chang was finally able, in 1959, to fertilize a mammalian egg outside the body, using "capacitated" sperm recovered from the uterus of a female rabbit killed soon after coitus. Chang's evidence was be-. yond dispute, because he took the egg he had fertilized and implanted it-in the uterus of another rabbit, certified not pregnant, which was segregated from males and in due course gave birth.
In the next few years Chang's experiment was successfully repeated by at least two other research teams. The reports quickened activity in in vitro fertilization in several countries. In 1961 a physiologist at the University of Bologna, Dr. Daniele Petrucci, said that he had fertilized a human egg in vitro, cultured the embryo for twenty-nine days ("a heartbeat was discernible"), and then destroyed it because "it became deformed and enlarged-a monstrosity:" He said-that as a womb substitute he had used a silicone container filled with amniotic fluid (liquid material that separates the 'growing embryo from the innermost membrane of the natural' placenta), which 'he extracted from pregnant- women. Petrucci, who professed himself a good Catholic, told the Italian newspapermen that his aim was just to find a way to culture organs that would resist the 'rejection phenomenon when transplanted. A couple of days after the story was printed, the Vatican's L'Qsservatore Romano ran an editorial which read in part, "God surrounded the act of creation of a human being with the most supreme assistances of love, nature, and conscience. It would' be monstrous to violate these conditions," which suggested to some that certain pressures had been put on Petrucci to stop. Another kind of reaction came in an editorial in Jenmin un Pao, the paper of the Chinese Communist Party:
These are achievements of extreme importance, which have opened up bright perspectives for similar research . . . . Nine months of pregnancy is no light or easy burden and such diseases as poisoning due to pregnancy are detrimental to health. If children can be had without being borne, working mothers need not be affected by childbirth. This is happy news for women.
The Russians were also impressed. Petrucci was invited to Moscow and spent two months at the Institute of Experimental Biology, returning to Bo.lognawith a Soviet medal. Presumably, he gave the Russians the benefit of his expertise, and since then, while he has desisted from further work, there have been rumors from time to time that professors Anoichin and Maiscki in Moscow, have; followed up on Petrucci, "and have got even further than he did."
But rumors only. In another context, writing about "The Ethical Basis of Science," Bentley Glass, professor of biology at the State University of New York and former president of the American Association for the Advancement of Science, said:
A full and true report is the hallmark of the scientist, a report as accurate and faithful as he can make it in every detail. The process of verification depends upon the ability of another scientist, of any other scientist who wishes to, to repeat the .procedure and to confirm the observation.
Dr. Petrucci offered no photographs; he never even published a report in a .scientific journal to describe how he had far surpassed anything that had ever been done before. Because of this, among biologists in the West who understand science roughly as Glass does, Petrucci's "experiment" is said to be "incompletely documented," which seems to be a polite formula for saying, not that there is an honest difference of opinion as there was between Shettles and Chang, but that Petrucci is a fraud.
Meanwhile, more serious men, many quite young, were initiating projects whose findings appeared in the Journal of Embryology and Experimental Morphology, Science, the International Journal of Fertility, and other sober and reputable publications. Their work would converge, toward the end of the sixties, in successful in vitro fertilization and growth of the human egg. Three of the most important projects were run by Americans:
Joseph C. Daniel, Jr., professor of biology at the University of Colorado, used rabbits, ferrets, and mink to investigate the development of the embryo just before it implants in the uterus. He found that certain proteins and other compounds are crucial at this phase. Professor Daniel was supported by grants from the National Institutes of Health and the Atomic Energy Commission.
Ralph Brinster, professor of veterinary medicine at the University of Pennsylvania, perfected a combination of nutritive substances (the "medium") in which eggs can be cultured best. He also described the changing biochemistry of the embryo during the time from fertilization to implantation, and devised an efficient incubator and culturing chamber for in vitro work, hooked up to CO. gas and maintained at 37° centigrade. Professor Brinster's work was also sponsored by the NIH.
Dr. Wesley Whitten of the Jackson Laboratory at Bar Harbor and Dr. John Biggers of Johns Hopkins cultured mice eggs from fertilization to just before implantation. For the first time, they observed the whole development of the embryo in this crucial stage when it is "free," not yet attached to the mother, and can be manipulated in vitro and reimplanted without apparent harm to the offspring. The work of Whitten and Biggers was supported by the National Institute for Child Health (in addition, Biggers' work was supported by the Population Council).
With the knowledge and experience gained from these and other experiments available to him, Dr. Robert G. Edwards of Cambridge University was ready in 1969 to try to achieve convincing in vitro fertilization, of the human egg. He had been making various preparations for this for ten years. Among other things, he and his group had plotted out the stages a human egg must go through on its own between the time it leaves the ovarian follicle and the time it is ready to be fertilized. Edwards would not have been able to do this preliminary research, let alone achieve fertilization, had he not exploited a new method of obtaining large numbers of usable human eggs.
Women volunteers were injected with a hormone, gonadotrophin, which caused them to "superovulate" many eggs (usually only one egg matures per menstrual cycle). Thirty hours after the injection, the volunteer would undergo an operation called "laparoscopy" (Greek, lapara, "flank" or "abdomen"), performed by a surgeon in Edwards' group, Patrick Steptoe. Two small punctures were made in the woman's side, into the ovary. Through one of these openings Dr. Steptoe introduced a slender hollow suction tube, and through the other an ingenious miniature optical device with a tiny flashlight which allowed him to look into the ovary. While he held the optical image steady in his left hand, with his right he maneuvered the suction probe from follicle to follicle, sucking out the eggs. In this way the Edwards group has been able to collect the basic, unsubstitutable, natural material they needed, which no other investigators had ever had in such quantity and quality.
In 1969, the eggs were incubated and washed, and three hours after collection, each one in its separate dish was put in the presence of sperm, which the volunteer woman's husband had contributed. The ratio of nutrients in the dish, the regulation of temperature, gas, and acidity followed closely the work of the Americans on mice, rabbits, and hamsters; Edwards counted, as it turned out only partly correctly, on the uniformity in timing and chemistry among eggs, sperm, and embryos of all mammals. As for "capacitation" of the sperm, Edwards was relieved to find that it would not be necessary to ask the volunteers to have intercourse, and then to operate to recover sperm from the uterus; the masturbated sperm could be capacitated simply by adding to the medium of the culturing dish some serum from the blood of a lamb fetus.
Having put eggs and sperm together, Edwards and his colleagues watched through microscopes to see what would happen. "Suddenly," Edwards was later to recall, "to our unbounded delight, the sperm started penetrating the eggs." In a minority of the dishes, there was to be definite proof of the first stage in fertilization, defined by. the formation of "pronuclei" and the expulsion from the center of the egg of something called the "second polar body." Edwards was entitled to his emotion, for this was a considerable success, but there was still no evidence that he had been able to push the process of fertilization to its acknowledged completion: the cleavage of the egg into two cells each having its own nucleus and full number of chromosomes. Possible reasons. for this failure suggested themselves, and the Edwards group prepared another series of experiments under somewhat different conditions. It was decided to take the eggs from the volunteers before they were ovulated, and to mature them in vitro, so that the timing in placing them with sperm could be exact.
In 1970, the laparoscopies were repeated, the eggs and sperm placed together under new conditions, and by thirty-eight hours after this in vitro insemination, many eggs had undergone a cleavage into two cells; by forty-six hours some had divided again into four cells; by sixty-two hours, some had divided again into eight cells; by eighty-five hours a few had divided again into sixteen cells. According to the report of the Edwards group published in the English journal Nature, none of the embryos matured past sixteen cells. When no cleavages had been seen to occur for two days, the embryos were removed from their dishes.
The photographs published in Nature show clear, jellylike mass, not much like a soccer more like a bunch of grapes.
These photographs and other data are substantial evidence that true fertilization and development took place in vitro. The evidence is not, however, indisputable. A determinedly skeptical embryologist might still insist, even in the face of the symmetry and regularity of the cleavages, that there is a chance the eggs were simply deteriorating all the time; he would not be convinced unless, as in Chang's experiment with rabbits, an egg was put back into the uterus of one of the volunteer women, she was then segregated from her husband, and eight and three quarters months later she gave birth. It should be noted that no embryologist has expressed such doubts formally; Edwards' colleagues in the field seem to be convinced by the evidence available. Yet this is not the end of it.
One or more embryos [Edwards wrote in Nature} have been produced from twenty-nine of the forty-nine patients under treatment in this work. The normality of embryonic development and the efficiency of embryo transfer cannot yet be assessed, although conditions for implantation in the treated patients should be favorable.
Edwards thus indicates that when it is taken, the next step for his group will in effect duplicate Chang's absolute test with rabbits. The question is, When will that step be attempted?
Indeed, the question may be asked, Why have Edwards and his group been at their work with human eggs, sperm, and embryos at all? Has it been just for the satisfaction of research and discovery? Edwards' answer is a definite "no." His answer, in interviews and statements, is that he wishes to relieve the suffering of the women who come to his laboratory as volunteers. These women are sterile; they have tried, and failed, to have babies. Their husbands have adequate sperm, but there has been no conception. They and their husbands are very unhappy to be childless, and much preferring to follow any hope of conceiving their own child rather than adopt one, they have been referred by obstetricians and gynecologists to the lab in Cambridge. Edwards says, "We tell these women, 'Your only hope is to help us.'" Because of their motives, his success in fertilizing and culturing eggs cannot be the end of it; rather, it is a first step, which makes them eager for the obvious next one.
It is known that roughly a fourth of sterile women fail to conceive because their oviducts are either blocked or nonexistent, thus preventing sperm from reaching the egg. In the technique used by Chang, and by now routine in lower mammals, the egg is fertilized in vitro; it completes in vitro the divisions it would normally undergo in the oviduct; then, the embryo is implanted in the uterus, where its presence provokes the growth of a placenta, within which it matures into a fetus that is eventually born and is normal. The infertility that most of Edwards' Volunteers are suffering from is attributable to blocked or absent oviducts; They come to Edwards for one reason only, to be cured of their barrenness. And the money which pays for Edwards' work (American money, Ford Foundation) is specifically awarded for research in fertility. This is why the next step is obvious.
And yet the decision to take it is not easy. What makes him hesitate, Edwards says; is that much testing remains to be done on the in vitro embryos to make sure that the manipulation of eggs and sperm does not damage the chromosomes, which would show up in more or less serious birth defects. "The last thing we want is abnormal babies." In order to do this minute checking up on the genetic material in the nuclei of the cells, a procedure called "karyotyping" (Greek, karyo, "nucleus"), the embryo has to be removed from its dish, which endangers its survival.
According to the published reports, 'all the genetic work has been done on embryos which spontaneously stopped growing. In fact, officially, the problem of deciding to take the next step is postponed and somewhat eased by the report that none of the embryos has survived in vitro past the sixteen-cell stage, while only an embryo developed well past that stage can successfully implant itself or be implanted into the uterus. However, when the Edwards group does succeed in culturing embryos to the implantation stage, the decision to implant a given embryo will have to be based on statistical evidence, and on hope-it will not be possible to karyotype the embryo itself. As Edwards' colleague, Dr. Steptoe, says, it will call for a "brave decision."