IVF and embryo transfer: historical origin and development

Reproductive BioMedicine Online (2012) 25, 118– 127

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REVIEW

IVF and embryo transfer: historical origin and development John D Biggers Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, MA 02115, USA E-mail address: [email protected] John Biggers, DSc, PhD is professor of cell biology at Harvard Medical School. His current research interests are evaporative drying of spermatozoa, vitrification, embryo culture, embryo assessment and the biography of Walter Heape. He is a former Commonwealth Fellow of St John’s College, Cambridge, past President of the Society of Reproduction, former Editor in Chief Biology of Reproduction, Chief Scientific Advisor to the Ethics Committee, US Department of Health, Education and Welfare that made recommendations on IVF and embryo transfer, Fellow of the American Association for the Advancement of Science, Hartman Award of the Society of Reproduction, Pioneer Award of the International Embryo Transfer Society, Marshall Medal of the Society for the Study of Fertility and a Life Member of the New England Fertility Society and the Society for the Study of Reproduction.

Abstract IVF and embryo transfer for the treatment of human infertility has now resulted in the birth of over 4 million babies. The

technique did not arise as a quantum event but was built on the efforts of many earlier workers in the fields of reproductive endocrinology and development. One should remember the famous saying of Isaac Newton: ‘If I have seen further than most, it is because I have stood on the shoulder’s of giants’. Ethical and moral issues have always arisen when investigators study early mammalian development, particularly human development. This paper documents these earlier studies and also draws attention to the ethical and moral arguments that inevitably arose. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: ethical issues, history, in-vitro fertilization, IVF, Gregory Pincus, John Rock

Introduction The 2010 Nobel Prize for Physiology or Medicine was awarded to Robert Edwards for developing IVF and embryo transfer (IVF/ET) to treat infertility in women with non-patent oviducts. His work resulted in the birth of the first ‘test tube’ baby in July 1978. Now, after a period of about 33 years, more than 4 million babies have been born using IVF/ET, and a new specialty of assisted reproduction has been established with its own professional societies. The history of IVF/ET is extensive and it has been recently documented in

part by Johnson (2011) and on the web (www.IVF-Worldwide.com). The technique did not arise as a quantum event but was built on the efforts of many earlier workers in the fields of reproductive endocrinology and development. One should remember the famous quotation from Isaac Newton: ‘If I have seen further than most, it is because I have stood on the shoulders of giants’. Ethical and moral issues have always arisen when investigators study early mammalian development, particularly human development. This paper documents these early studies and also draws attention to the ethical and moral arguments that inevitably arose.

1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.04.011

History of IVF and embryo transfer

IVF/ET in mythology The idea of transferring a human fetus from one mother to another can be found in a story from the Jain religion about the religious leader Mahavira. The following account is a brief summary of the full story in the Sacred Books of the East (Jaina Sutra, 1964). At one of his reincarnations the ruler of all the gods of heaven and earth, Sakra, realized that the Mahavira had been conceived by Devananda, a woman of an unacceptable caste. Sakra then commanded that the embryo carried by Devananda be transferred into the womb of Trisala, a woman of an acceptable caste, and that Trisala’s embryo be placed in Devananda’s womb. The story has been memorialized at least until the 15th century in sculpture and painting.

Treatment of non-patent oviducts prior to IVF/ET Clinicians knew, by the middle of the 19th century, that blocked oviducts resulted in sterility (Churchill, 1846). Demands for treatments were driven by the effects of infertility on the dispersal of family wealth, and since that time several surgical procedures have been tried (reviewed by Biggers, 1984). In 1849, Tyler Smith attempted to unblock the oviduct in a patient by passing a whalebone bougie through the tube (Smith, 1849). Colleagues greeted the procedure with skepticism and it was never adopted. The demonstration at the end of the 19th century that the ovaries could be transplanted to ectopic sites paved the way for the discovery of the ovarian hormones. The fact that the ovary maintains its function when placed in an ectopic site led Morris (1895), an American gynaecologist, to treat patients with blocked oviducts by grafting ovarian tissue into the oviduct or uterus below the obstruction. The method was never successful. In 1909, Estes, another American gynaecologist, introduced an operation in which the ovary was inserted through the uterine wall, keeping its pedicle containing blood vessels and nerves intact, where it was hoped ovulation would occur (Estes, 1909). The so-called Estes operation was seldom successful, although it was used until the middle of the 20th century. While IVF/ET was being developed in the 1970s, microsurgery was attracting the attention of gynaecologists for the anastomosis of the cut ends of the oviduct after removal of an obstruction. By the time the first baby was produced by IVF/ET in 1978, microsurgery procedures showed promise of being an alternative procedure (Eddy, 1981; Winston, 1981). Since then other improvements in technique have occurred so that the suggestion has been made that IVF/ET and surgery should be regarded as complimentary techniques for the treatment of tubal infertility (Gomel and McComb, 2006; Schippert et al., 2010a,b).

Basic research that paved the way to current reproductive technologies IVF/ET involves four main aspects: (i) acquisition in sufficient numbers of meiotically and cytoplasmically mature ova; (ii) fertilization of these mature ova in vitro; (iii) culture of preimplantation embryos; and (iv) embryo

119 replacement within a mother. Initially, these areas were largely studied independently and it was only later that they were linked to create the technical procedure called IVF/ET. Unfortunately, there is a tendency in historical accounts of IVF/ET, particularly among clinicians, to focus almost exclusively on the actual process of fertilization in vitro, ignoring the history of the other three components. Walter Heape reported in 1891 that he had successfully transferred mammalian (rabbit) embryos from one mother to another (Heape, 1891). He was a gentleman scientist who studied under Francis Balfour in Cambridge. Although he never earned a degree, he was later appointed a demonstrator in the embryology course where students recovered and observed living rabbit embryos (Foster and Balfour, 1883). Heape transferred two ova from an Angora doe rabbit into the Fallopian tube of a Belgian Hare recipient and obtained six young; two had Angora phenotypes and four had Belgian hare phenotypes. IVF was first attempted using rabbits and guinea pigs by Schenk (1887) at a time when the essential biological features of fertilization were being worked out independently by Van Beneden, Hertwig and Folin in rabbits, sea urchins and starfish (reviewed by Austin, 1961). Another early attempt was made by Onanoff (1893) using rabbits. Schenk’s experiments failed and Onanoff’s results were unconvincing. The first successful attempts to culture preimplantation mammalian embryos were made in 1913 by Albert Brachet, Director of the Brussels School of Embryology at the Warocque ´ Institute of Anatomy (Brachet, 1913). He studied the expansion of the rabbit blastocyst in vitro. His experiments were done only 4 years after the first report of tissue culture in which nerve cells were grown by Harrison at Yale University in 1907 (Harrison, 1907). Little attention, if any, was paid to the manipulation of mammalian development in vitro during the next 18 years other than science fiction accounts of ectogenesis – the complete production of an individual outside an organism’s body. Ectogenesis was first invoked in a lecture given in 1923 by the geneticist and biochemist JBS Haldane to the Heretics Society at the University of Cambridge. The main objective of this society was to needle religious dogma. Haldane made the tongue in cheek prediction that ectogenesis would be perfected by 1960. He imagined what a student might write in the 1960s, as follows: It was 1951 that Dupont and Schwartz produced the first ectogenetic child . . . France was the first country to adopt ectogenesis officially, and by 1968 was producing 60,000 children annually by this method. In most countries the opposition was far stronger, and was intensified by the Papal Bull ‘Nunquam prius audito’, and by the similar ‘fetwa’ of the Khalif, both of which appeared in 1960. (Haldane, 1923) A few years later, the notion of ectogenesis became widely disseminated by the book by Aldous Huxley called Brave New World, published in 1932 (Huxley, 1932). The techniques imagined by Huxley were remarkably realistic. Elsewhere I have speculated that his ideas may have resulted from conversations with Gregory Pincus, who was doing research on fertilization and development using rabbits in Cambridge at the time (Biggers, 1991).

120 The contributions of Gregory Pincus in this field were highly significant, but they have been frequently overlooked because of his major contributions to the development of the oral contraceptive pill (Ingle, 1971; Speroff, 2009). After obtaining his Doctor of Science degree in the Department of Biology, Harvard University in 1927, Pincus was awarded a National Research Council Fellowship for 3 years, the first two at Harvard and the third at the University of Cambridge in England under the reproductive biologist John Hammond and at the Kaiser Wilhelm Institute in Berlin under the geneticist R Goldschmidt. He was appointed Assistant Professor of Biology in 1931, soon after his return to Harvard, where he remained for 6 years. His first paper in reproductive biology was entitled ‘Observations on the living eggs of the rabbit’ (Pincus, 1930). In it he described the work he had done in Cambridge, England, which included a brief description of his first unsuccessful attempts to fertilize rabbit ova in vitro. Three important papers in the field were written during the 6 years he was at Harvard. In 1934, he and Enzmann reported in Proceedings of the National Academy of Sciences, USA that they had successfully produced newborn rabbits following IVF. These results were accepted by the scientific community for many years as the first demonstration of IVF. They were not challenged, as shall be discussed, until the 1950s when further information on the nature of fertilization became available. The results also attracted comments in the popular press, as happens so often with topics in reproductive biology. The results were well received by some commentators, who suggested they may eventually help in the solution of human problems. Others were critical, saying that the scientists were playing God. A year later, in 1935, Pincus and Enzmann showed that when rabbit oocytes were isolated from the Graafian follicle and placed in culture they would resume meiosis spontaneously, passing from the arrested dictyate stage to the metaphase-II stage. Four years later, in 1939, Pincus and Saunders demonstrated that isolated human oocytes would also complete meiosis in vitro, although they underestimated the duration possibly by using partially matured oocytes. This phenomenon is exploited in current IVF protocols. In 1936, Pincus published a monograph entitled ‘The eggs of mammals’ with a chapter on the culture of the initial stages of mammalian development. He used methods practised at the time in the field of tissue culture that used media based on biological fluids, such as blood serum. Also, in 1936, Pincus and Enzmann reported that they had successfully activated rabbit ova, causing them to begin the cleavage divisions (Pincus and Enzmann, 1936). This paper attracted the popular press and attendant adverse publicity. WL Laurence, writing in New York Times on 27 March 1936, speculated on some of the possible consequences of the research Pincus had done over the previous few years as follows: As rabbits and men belong to the mammalian group, the work is viewed as pointing toward the possibility of human children being brought into the world by a ‘host-mother’ not related by blood to the child. It is reasoned that eventually women capable of having children whose health does not permit them to do so may ‘hire’ other women to bear their children for them, children actually their own flesh and blood.

JD Biggers To one who desires to speculate at this point the Harvard experiment offers another possibility. Theoretically, at least, it may become possible for a woman so inclined, particularly in a country influenced by eugenic considerations, to bring into the world twelve children a year by ‘hiring’ twelve ‘host-mothers’ to bear their test-tube-conceived children for them. Advocates of ‘race betterment’ might urge such procedures for men and women of special aptitudes, physical, mental or spiritual (Laurence, 1936). The following day, an emotionally negative editorial was published in the New York Times entitled ‘Brave new world’. A few months later, JD Radcliff, wrote a sensational article for Collier’s Magazine with the title ‘No father to guide them’. Radcliff’s article contained an unflattering photograph of Pincus smoking a cigarette holding two adult rabbits that had not been produced parthenogenetically, and commented: In the resulting world man’s value would shrink. It is conceivable that the process would not even produce males. The mythical land of the Amazons would then come to life. A world where woman would be self-sufficient; man’s value precisely zero. Radcliff recorded that Pincus found such comments about his work annoying saying ‘. . . I am not interested in the implications of the work’ (Radcliff, 1937). In 1937, Pincus was granted sabbatical leave by Harvard to spend another year in Cambridge, UK, but was informed that his assistant professorship would not be renewed on his return to Harvard. Thus, when he returned to the USA he was unemployed. The sensational publicity may have been a cause of Pincus’s losing his faculty appointment. Many conservative academics probably believed that it was a threat to Harvard’s image. However, there were other causes. The President of Harvard, James B Conant, replaced Pincus’s mentor, the Chairman of the Department of Biology, with someone more oriented to a molecular approach to the subject (Speroff, 2009). 1937 was also a year when new ideas changed clinical approaches to infertility. In 1937 the following anonymous editorial was published in New England Journal of Medicine: Conception in a watch glass Contemplating this new discovery, one’s mind travels much farther. Lewis and Hartman have isolated a fertilized monkey ovum and photographed its early cleavage in vitro. Pincus and Enzmann have started one step earlier with the rabbit, isolating an ovum, fertilizing it in a watch glass, and re-implanting it in a doe other than the one that furnished the egg, and have thus successfully inaugurated pregnancy in an unmated animal. If such an accomplishment with rabbits were to be duplicated in human beings, we should, in the words of ‘flaming youth’, be ‘going places’. The difficulty with human ova has been that those recovered from tubes have regressed beyond the possibility of fertilization in vitro. But by utilizing the electrical sign we may be able to obtain them from the follicle at the peak of their maturity. If the new peritoneoscope can be developed

History of IVF and embryo transfer along the lines of the operating cystoscope, laparotomy may even be dispensed with. What a boon for the barren woman with closed tubes! Who was this anonymous writer and what is the ‘electrical sign’? John Rock, a gynaecologist at the Free Hospital for Women, Boston and Harvard Medical School, subsequently admitted he was the author (Marsh and Ronner, 2008). In the same issue of New England Journal of Medicine, Rock described the use of a potentiometer to detect the time of ovulation (Rock et al., 1937). At this time it was not known with certainty whether ovulation was associated with menstruation or whether it occurred mid-cycle between two menstrual periods. The technique would facilitate the collection of living human oocytes shortly after their release from the ovary. The following year, Rock embarked on two parallel lines of research. One was the collection and study of the earliest stages of human development, and the other was to fertilize human eggs in vitro. He hired Miriam Menkin, who had been an assistant several years earlier in Pincus’s laboratory at Harvard, working on the isolation of the two pituitary hormones FSH and LH (McLaughlin, 1982). While there she learnt to manipulate rabbit ova and embryos. Rock’s work on early human development continued for about 15 years in collaboration with Arthur Hertig, a pathologist at Harvard Medical School, and led to the famous collection of early human stages frequently referred to in nearly all textbooks on human embryology (Hertig et al., 1956). Their prodigious work on the fertilization of human ova led to the claim that human ova had been fertilized in vitro (Rock and Menkin, 1944; Menkin and Rock, 1948). Nearly 800 human follicular eggs were obtained from women undergoing surgery and 138 of these eggs were exposed to spermatozoa. During this time Pincus was consulted. Eventually two ova that had divided into 2-cell stages were obtained and one 3-cell stage. The claim was made that fertilization had occurred in vitro. The claims of Menkin and Rock were widely accepted for a few years and are frequently cited in more recent times. Although published during the Second World War, Rock and Menkin’s first paper attracted sufficient attention to be covered on the first page of Boston Globe. The newspaper viewed the work favourably, stating that it would help towards treating serious problems of infertility. Some, however, objected to the work. Rock, however, had antagonists at Harvard and their influence is said to have subverted any further studies on human embryos (McLaughlin, 1982). Several years later, in 1954, Landrum Shettles, at Columbia, claimed to have fertilized a human ovum in vitro by repeating Menkin and Rock’s protocol (Shettles, 1954). In the archives at Harvard Medical School there is a letter dated 6 June 1954 from Carl Hartman, then Director of the Ortho Research Foundation, to John Rock. He wrote: I don’t believe you ever got in vitro fertilization . . . Have a dozen reasons to question your conclusions, chief of which is the simultaneous and independent discovery by Chang, Austin and Blandau [Braden?] that ‘raw’ sperms won’t fertilize any egg even in vivo! Sperms must be ‘capacitated’ (Austin) in the female tract, either in the uterus or the tube.

121 Furthermore, Hartman encouraged Rock to continue his work, for he continued: Now, I want you to go back to the problem and clean it up and really immortalize yourself. Inject 50,000,000 sperm into a woman’s uterus. In 2 h take out the sperms and add to the ovarian egg (but only from a 16–18 mm. follicle, eggs in lesser ones are N.G.). I’m betting heavy odds on the outcome of this experiment. Rock never took up Hartman’s suggestion. By 1954, Rock had ceased working on IVF/ET. Perhaps he was discouraged by the very low success rate of their attempts to achieve IVF. He was ahead of his time because, in the next two decades, important advances in reproductive biology made success much more likely. Nevertheless, Rock’s enthusiasm for developing IVF/ET for the treatment of infertility remained because, 4 years later after a paper read by Landrum Shettles before the New York Obstetrical Society, he commented: The time may be rapidly approaching when the poor woman whose tubes had been excised, yet who still wants a baby, will rejoice that Dr Shettles will be able to extract an ovum from her ovary, probably not by laparotomy, but through an operating telescope (which can be done – we have done it); then fertilize the egg in vitro by the husband’s spermatozoa; and finally put it back in the uterus. Thus will he impregnate the woman in spite of the fact that she has no tubes. (Shettles, 1958) There is ample evidence that Rock came under pressure to discontinue work on early human embryos both locally by his colleagues at Harvard and by the Catholic Church. Meanwhile he had become associated with Pincus in another ‘hot’ subject – birth control and the development of the oral contraceptive pill. It seems likely that he could not defend two contentious issues simultaneously. The sixth and seventh decades of the 20th century produced advances in understanding the physiology of fertilization and preimplantation development. Before this period, many investigators were convinced that fertilization in vitro had been achieved by Pincus in the rabbit and Menkin and Rock in the human. The claims were questioned after it was discovered in 1951 by two independent groups, Austin (1951) in Sydney, Australia and Chang (1951) in Worcester, Massachusetts, that freshly ejaculated spermatozoa could not immediately fertilize an egg since they require a period of so-called maturation in the female genital tract. The word ‘capacitation’ was coined to denote the phenomenon by Austin (1952). How then did Pincus and Enzmann (1934) get young when they added spermatozoa to the ova immediately after the spermatozoa were collected? One possibility is that spermatozoa were carried into the uterus at transfer where they capacitated and then fertilized the ovum in vivo. The discovery of capacitation led to controversy over the nature of convincing evidence sufficient to claim successful fertilization of mammalian eggs. Rothschild (1969) defined fertilization as the incitement of an egg to development by a spermatozoon, together with the transmission of male hereditary material into the egg. The difficulty of unequivocally demonstrating that fertilization has occurred was emphasized by Austin (1961) who proposed the following requirements: (i) use of capacitated

122 spermatozoa; (ii) avoidance of aged ova; (iii) confirmation that a spermatozoon had entered the ovum; and (iv) conditions that exclude parthenogenesis. Parthenogenesis cannot be excluded by observing preimplantation stages of pregnancy since parthenogenotes can develop through these stages and beyond, though parental imprinting deficiencies precludes their development to term (reviewed by Markert, 1988). Thus, the ultimate requirement is the birth of offspring with the use of genetic markers that demonstrate characteristics transmitted by the spermatozoon. Many claims that fertilization in vitro had been successful were made by this time, but all can be criticized on the grounds that the reported evidence was insufficient. Austin (1961) cites about 30 papers in which it is claimed that fertilization in vitro was successful in the rabbit, guinea-pig, human and sheep. He concluded that only the work of Thibault and his colleagues, Moricard (1954) and Chang (1959), merited serious consideration. For example, Thibault et al. (1954) used capacitated spermatozoa to fertilize rabbit ova. They reported only cytological observations on the cleaving embryos and did not transfer the embryos into uterine foster mothers to see if they would develop into newborn young. Moricard also did similar experiments without confirming that fertilization occurred by using embryo transfer. It is now generally accepted that the first unequivocal achievement of IVF was done in the rabbit by Chang (1959), working at the Worcester Foundation where Pincus was Director. This does not mean that others had not previously achieved IVF; it means that the necessary rigorous scientific proof was not provided. In all this early work, IVF was done using biological fluids of unknown composition as the culture medium. IVF, using a chemically defined culture medium, was achieved in the mouse by David Whittingham in 1968 using a medium for embryo culture described by Whitten and Biggers (1968). In his book The Eggs of Mammals, Pincus (1936a,b) described in detail methods for the culture of preimplantation embryos. He used media based on biological fluids, such as blood serum, employed at the time in the field of tissue culture. Some successes were reported, including some pioneering attempts at determining the nutritional requirements of preimplantation embryos in vitro. A major development occurred in the field of tissue culture when chemically defined media were developed by White (1946) at the Jackson Laboratory in Maine and for animal cells by Fischer (1947) in Germany. These media allowed culture experiments to be repeated in different laboratories under relatively comparable conditions, which is not possible with biological fluids. A little-known letter was published in 1947 by John Hammond, Jr, who showed that 8-cell mouse embryos would develop into blastocysts when cultured in Krebs-Ringer bicarbonate supplemented with egg white (Hammond, 1947). Whitten in 1956 replaced the egg white with bovine serum albumin to produce the first chemically defined medium that supported development of 8-cell mouse embryos into blastocysts (Whitten, 1956). Earlier stages would not develop under these conditions. In a totally unrelated study of the classic nature versus nurture problem, McLaren and Michie (1956) had optimized the technique for embryo transfer in mice. McLaren and Biggers (1958) used this optimized protocol to transfer blastocysts,

JD Biggers produced from the 8-cell stage in Whitten’s medium, into uterine foster mothers, and showed using coat colour as a genetic marker that phenotypically normal mice developed from the cultured embryos. The scientific report published in Nature was greeted in the London Daily Telegraph under the heading ‘Brave new mice’. We received no adverse criticism of the work, although a well-known scientist from Cambridge, Cecilia Lutwak-Mann chastised us by mail for allowing our results to be published in the popular press. We were not interested, like Pincus 20 years earlier, in sensational implications of our work, but only in the scientific applications. In the popular magazine Discovery we wrote: It is inevitable that the thoughts of anyone who has worked on the subjects outlined in this article should turn to Aldous Huxley’s fantasy ‘Brave New World’, where he describes completely artificial fertilization and development of human embryos. Fortunately we are far removed from this frightening prospect. The study of the cultivation and transfer of embryos is none the less of the greatest interest, both from the point of view of pure science, and because the techniques associated with it are potentially of immense value in the investigation of many biological problems in medicine and agriculture. (Biggers and McLaren, 1958) This work first demonstrated that live-born mice could develop after being exposed to chemically defined media during the initial stages of development. While this work was ongoing, Whitten (1957) found that 2-cell mouse embryos would also develop into blastocysts if lactate was added to the medium. A few years later, Biggers et al. (1967) showed that the first cleavage division also required pyruvate in the medium. These observations established the basis for the design of several media, including medium KSOM/AA that many use to culture mouse embryos today and which has been slightly modified in one commercially available medium for the culture of human embryos (Global: IVFOnline, Guelph, Ontario, Canada). Successful IVF requires the availability of ova ready for fertilization. Pincus and Enzmann (1935) in the rabbit and Pincus and Saunders (1939) in the human had shown that oocytes isolated at the germinal vesicle stage would proceed to the metaphase-II stage spontaneously when placed in culture. In the next 30 years, the phenomenon was demonstrated in several more species: mouse, rat, hamster, rabbit, sheep, cow, pig and monkey (reviewed by Biggers, 1972). Particularly important contributions on larger animals with longer maturation times were made by Edwards (1962, 1965a), who observed the phenomenon using serum-supplemented cell-culture media. Another advance was made by Kennedy and Donahue (1969), who showed that human oocytes could complete meiosis in several chemically defined media, including those commercially available for cell culture. These media varied in complexity, from F10, which contains many components, to simple media with relatively few components based on modified, pyruvate-supplemented Krebs-Ringer bicarbonate. Pincus (1940a,b), in another pioneer paper using the rabbit, described research results with the objectives ‘to ripen as large a number of follicles as possible and to determine whether the ova obtained at ovulation were fertilizable’. The technique was needed to increase numbers of ova and

History of IVF and embryo transfer early embryos for experimental study. He succeeded in devising a technique, using crude extracts of the pituitary gland that satisfied these objectives and thus was the first to describe the technique of superovulation widely used today. By this time, it was known that the maturation of Graafian follicles and the ovum depended on two hormones from the anterior pituitary gland originally called ‘Prolan A’ and ‘Prolan B’ by Zondek (1929), now known as FSH and LH (reviewed by Lunenfeld, 2004). An important study by Fowler and Edwards (1957) worked out the protocol used to this day for the production of synchronous mouse oocytes capable of fertilization and development to term, using the sequential injection of pregnant mare serum gonadotrophin and human chorionic gonadotrophin. By the time Edwards began his work on the maturation of human oocytes in vitro, human menopausal gonadotrophin (a mixture of FSH and LH) and chorionic gonadotrophin (mainly LH) were available to stimulate follicular growth (Edwards et al., 1970). Another technique that has played an important role in human IVF is laparoscopy for the recovery of oocytes from the mature Graafian follicle. The idea of an ‘operating telescope’ originated early in the 20th century (Steptoe, 1967). Its potential usefulness to recover human oocytes was recognized by Rock in his editorial in New England Journal of Medicine in 1937. A cystoscope employed in urology, similar to a laparoscope, was first used to recover an oocyte from a single human follicle in France by Klein and Palmer (1961).

Putting it all together: Robert Edwards’ contributions Interest in human in IVF/ET recurred in the 1960s largely due to the work of Robert Edwards. In the course of a few years, Edwards and his colleagues published the following key papers that paved the way for the birth of the first ‘test-tube’ baby: (1) ‘Maturation in vitro of human ovarian oocytes’ in The Lancet (Edwards, 1965b). (2) ‘Early stages of fertilization in vitro of human oocytes matured in vitro’ in Nature (Edwards et al., 1969). (3) ‘Fertilization and cleavage in vitro of preovular human oocytes’ in Nature (Edwards et al., 1970). (4) ‘Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins’ in The Lancet (Steptoe and Edwards, 1970). Edwards’ interest in the mechanisms involved in the establishment of pregnancy arose from cytogenetic studies on oocyte maturation (reviewed by Edwards, 2001; Johnson, 2011). He extended work done in the 1930s by Pincus and his colleagues by showing that spontaneous maturation occurred in several species using the chemically defined medium 199 supplemented with 15% serum. Edwards began his initial studies on the maturation of human oocytes by incubating them for only 12 h, following Pincus and Saunders (1939). This period of incubation was too short, and it took some time before Edwards found that the required time was between 36 and 43 h after laparoscopy. Obtaining human oocytes for experimentation is difficult.

123 To get access to further material, Edwards spent 6 weeks at the Women’s Clinic in Johns Hopkins Hospital, Baltimore with Howard Jones and Georgeana Seeger Jones. This rewarding visit resulted in key paper 1, which was published from Johns Hopkins Hospital. The availability of meiotically mature human ova that resulted from this work opened the door to needed experimental work on the fertilization of human eggs in vitro. Unfortunately, attempts to fertilize these in-vitro matured oocytes with capacitated spermatozoa consistently failed (Edwards et al., 1966). Fertilization in vitro of matured human oocytes was finally reported in key paper 2. It occurred almost serendipitously. Bavister was working on the capacitation of hamster spermatozoa in a laboratory adjacent to Edwards’ laboratory. Capacitation in vitro had been observed in the hamster by Yanagamachi and Chang (1963) using a simple modified Tyrode’s balanced salt solution. Bavister’s attempts to repeat this work resulted in erratic outcomes that he showed were due to poor pH control. A modified medium supplemented with pyruvate and bovine serum albumin was finally produced in which the pH was strictly controlled at 7.6. When in-vitro matured human oocytes were incubated for 6 hours with human spermatozoa in this medium, spermatozoa were observed inside the oocytes, and one oocyte contained two pronuclei. In a letter to Nature, Rothschild (1969) pointed out that these experiments demonstrated only the initial stages of fertilization and did not satisfy the stringent requirements of Austin (1961); the results provided no proof that normal young would finally develop. Edwards and colleagues then showed that human ova penetrated by a spermatozoon could develop in modified Bavister’s medium and other media through the subsequent cleavage divisions to the blastocyst stage (key paper 3). Bavister’s original formulation was modified by increasing slightly the sodium and potassium concentrations. A second, simple, chemically defined medium was also used; the medium had been described by Whitten and Biggers (1968) for the complete culture of mouse embryos from the zygote to the blastocyst thus overcoming the 2-cell block. This medium was made available before publication to Whittingham (1968) for his successful studies on IVF in the mouse, and it was subsequently passed on to Edwards and his colleagues. Three other media were widely used in cell culture – Ham’s F10, Eagle’s 199 and Weymouth’s (modified by the addition of pyruvate) – all supplemented with inactivated fetal calf serum. Pyruvate was included in all of these media after the demonstration in the mouse that it is required for oocyte maturation and the first cleavage division (Biggers et al., 1967). Some penetration of spermatozoa into the ova, pronuclear formation and cleavage were observed with all media, although at low rates. These experiments did not rule out the possibility of parthenogenesis. Thus, it was not until 1978 and the birth of the first baby produced by IVF/ET that normal IVF in humans was finally proved. A cohort of oocytes recovered from an ovary are invariably at different stages of maturation. Key paper 4 describes the use of two gonadotrophins given sequentially to synchronize as closely as possible a cohort of oocytes at a stage normally reached just before the expected time of ovulation. The two hormones were human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin

124 (HCG). The preovulatory oocytes were then harvested for IVF/ET by puncturing the follicles with a laparoscope and aspirating them with a specially designed piece of equipment. A period of about 8 years elapsed before the first baby conceived by IVF was born in July 1978 (Steptoe and Edwards, 1978). In all but one case, the patients failed to become pregnant after embryos produced by IVF were transferred to the mother. The one exception resulted in an ectopic pregnancy. Various reasons for the failure of transferred cleavage-stage embryos to develop were considered. For example, such transfers in mice to the uterus rather than to the oviduct were rarely successful due to the inappropriate hormonal priming. However, work on the transfer of cleaving human embryos to the uterus was encouraged by the report of Marston et al. (1977) who showed that a 5-cell rhesus monkey embryo could develop into a newborn baby after transfer into the uterus of its mother. Another potential contributory cause of the failures in human patients was the trauma involved in transferring the embryos into the uterus. A further possible cause was the endocrine disturbances elicited by the HMG and HCG used for ovarian stimulation, leading to what Edwards called luteal weakness (Edwards, 2001). After trying various hormonal supplements, Edwards and Steptoe turned to the recovery of a single oocyte from a natural cycle by monitoring the patient’s LH surge near the time of ovulation. The first IVF baby, Louise Brown, was conceived in this way. The first babies produced following ovarian stimulation using clomiphene and HCG were not reported until 1981 by Carl Wood’s group (Trounson et al., 1981). Edwards’ papers generated renewed interest in IVF in the USA, particularly that of Howard Jones and Georgeanna Seeger Jones at the Woman’s Clinic at Johns Hopkins Hospital. Others who became interested in the field were Pierre Soupart at Vanderbilt School of Medicine, Nashville (Soupart and Morgenstern, 1973; Soupart and Strong, 1974) and Melvin Taymor at the Brigham Hospital in Boston (Berger et al., 1975). Soupart, in fact, had a grant application approved by a study section at the National Institutes of Health to support human IVF, an application which, in an unusual step, had been referred to an Ethics Panel. Unfortunately Soupart died before approval was given. By the end of the 1960s, Edwards decided to seek large-scale support for his work, and in 1971 he and Steptoe applied to the British Medical Research Council for a grant to set up a clinical research programme, but was rejected, a great disappointment to Edwards. In retrospect it may seem that those who decided the fate of the application were short sighted. Recently, Martin Johnson of the University of Cambridge and colleagues reviewed the deliberations within the Medical Research Council (Johnson et al., 2010). Their research revealed a complex network of conflicting interests, mainly administrative but not scientific, that resulted in the rejection. On the whole, the scientific advisors to the MRC approved of the science, although concern was expressed over the potential of producing birth defects. One reviewer suggested the technique should first be evaluated in monkeys. Rejection was due to an excessive budget, concern over what was perceived as the use of patients in clinical research, doubts about the high media profile of Edwards and Steptoe, with some hints of politicking by aspirant Directors of other

JD Biggers MRC-supported organizations who vied for the clinical research programme in their own establishment. Edwards may well have unwittingly killed his application by insisting that if the clinic was located at a site other than Cambridge he was not interested. In 1979, after the birth of Louise Brown, Edwards applied to the National Health System for state support to establish an IVF clinic, but again the application was rejected. He finally secured venture capital to set up Bourn Hall, the first IVF clinic, which was opened in September 1980 located on an estate near Cambridge. Clinical research proceeded rapidly in this new environment and it included the development of successful treatment regimens for overcoming the luteal weakness that plagued the early work. Securing government support in this field remained difficult. A second application for research funding on IVF to the Medical Research Council also failed. Nevertheless, over 1000 babies were born using IVF/ET in the first 10 years of Bourn Hall’s existence (Edwards, 2001). A controversial claim was made in India, soon after the birth of Louise Brown, that a baby girl had been born following IVF/ET. The team that made this claim were two physicians (Subhas Mukerji, leader of the team, and SK Bhattacharya) and a cryobiologist (Sunit Mukherjee). Mukerji had done research on hormone assaying at the University of Edinburgh, and Mukherjee was well trained in cryogenics at Cornell University. The case was not described in professional journals but was widely reported with considerable hoopla in the Indian press (Kumar, 1997). A six-page detailed report appeared in the popular Indian magazine Sunday under the title ‘Our very own test-tube baby’ (Mitra and McMahon, 1978). The baby was born on 3 October 1978 and thus, if the claim is true, her mother became pregnant about 6 months before Louise Brown was born. The case also attracted considerable attention since, for the first time, ovarian stimulation and cryopreservation of the embryo were reported. An official at the NIH asked me to look into the claim when I visited India to attend the International Congress of Hormonal Steroids held in New Delhi in the late fall of 1978. Dr Kenneth Ryan, Harvard Medical School, Dr Liselotte Mettler, University of Kiel, both also attending the Conference, and I interviewed Subhas Mukerji in the Ashoka Hotel on 2 November 1978. He gave us full details of the protocol that was followed. We were informed that we could not see the baby since her parents wished to remain anonymous. The protocol used seemed to follow the practices used at the time so we were not inclined to dismiss the claim out of hand. Nevertheless, the Indian doctors we talked to unanimously regarded the claim as fraudulent. During the next 20 years, the case was largely forgotten and considered questionable. Then, in 1997, Anand Kumar, a prominent Indian gynaecologist (Metha, 2010), re-examined the case in minute detail. He had gained access to the group’s laboratory and clinical notes and official government documents. He concluded that Mukerji and his team had indeed succeeded in producing a baby by IVF/ET and that bureaucratic interference had prevented Mukerji from justifying his claim in front of his scientific peers. Mukerji had summarized the details of the case in a letter dated 1 December 1978 to the Director of Health Services of the West Bengal Government, and on 28 December 1978 the Director specifically ordered Mukerji not to attend any conference without prior permission. Permission to accept

History of IVF and embryo transfer an invitation to present his case in Japan was denied (Anand Kumar, 1997). Mukerji’s death soon afterwards deprived him of any further opportunity to justify his claim.

Ethical and moral issues raised in the USA and the formation of the first clinic in Norfolk, Virginia The birth of Louise Brown was widely reported in the US press, often in a sensational manner. The negative reception received by John Rock 34 years earlier was repeated with greater intensity. Based on my own experiences, I can provide some understanding of the environment that scientists and physicians encountered in the USA. In 1978, I was director of a programme project at Harvard, supported by the Institute of Child Health and Human Development, on the biology of early pregnancy. One section of the project, headed by Dr Melvin Taymor of the Brigham and Women’s Hospital, was concerned with trying to mature human oocytes in vitro. The day the birth of Louise Brown was announced, I received a call from an NIH official freezing these funds. It was clear that some members of Congress were upset by the demonstration that a new individual could be created in a test tube. Soon after, Joseph Califano, Secretary of the then Department of Health, Education and Welfare, activated a dormant Ethics Board to advise him and President Carter on whether it was ethically acceptable for the Federal Government to support work on IVF/ET. I was appointed scientific advisor to the Board, together with a philosopher and two lawyers. The members of the Board were a very talented group of people representative of many walks of life. Several hearings open to the public were held by the Board, which eventually recommended that the Federal Government could support work on IVF/ET subjected to certain restrictions. In retrospect, it is interesting that one provision made by the Board was that in any experiment the spermatozoon and egg must be donated by a married couple. The recommendations of the Board were never accepted by the Government, however, and as far as I know the report still sits in some government pigeon hole (Biggers, 1978, 1981). In 1980, the National Institute of Child Health and Human Development invited me and Luigi Mastroianni, Chairman of Obstetrics and Gynecology at the University of Pennsylvania, to organize a small conference on the Bethesda Campus about IVF/ET as practised in animals, with the explicit instructions that discussion of human IVF/ET be disallowed (Mastroianni and Biggers, 1981). I was chairing a session when Barry Bavister jumped up at a point in the discussion and said ‘Let’s talk about the human’. After receiving frantic signals from the NIH staff in the audience, I adjourned the meeting for lunch, with the result that my friends accused me of stifling free speech. When the proceedings were published by Plenum Press, we were required not to acknowledge that the NICHD sponsored the meeting. Clearly the NICHD staff was scared by the possible reactions of some politicians in Congress. Soon after the birth of Louise Brown, the President of the Eastern Virginia School of Medicine invited Howard Jones and Georgeanna Seeger Jones to come out of retirement and rekindle their interest in IVF to form an IVF/ET clinic. A legal requirement for setting up a new clinic was getting

125 a Certificate of Need from the State of Virginia. This involved having a public hearing. Howard Jones invited me to testify at this hearing. It turned out to be a horrendous experience. The hearing took place in a hall that held 200–300 people. The audience had seated themselves in two groups on opposite sides of the hall, one group supporting IVF/ET, including couples who had not been able to conceive, and the other group, largely right-to-lifers, strongly opposing IVF/ET. The entire meeting turned out to be a shouting match, the two groups hurling insults at each other. I particularly remember a right-to-life activist from Chicago whose purpose was to attack in a particularly insulting way the motives of Georgeanna Seeger Jones, one of the most dedicated clinicians you could ever meet. Fortunately, the Certificate of Need was granted. Attacks on the proposed clinic continued to be made in the local media and this resulted in a lawsuit, which the Medical School won. The monetary settlement helped create the first IVF clinic in the USA. Soon after the clinic was formed, the Virginia Bar Association sponsored a 1-day symposium on IVF/ET. I was invited to present the first paper outlining the principles of IVF to the audience of lawyers. The meeting was held at Virginia Beach, which is also the home of the Christian Broadcasting Network run by Pat Robertson. His network organized a protest outside the hotel where the Conference was to take place. As a result, all speakers were taken to the back of the hotel and admitted through the kitchen. Public policy makers like to make the distinction between a procedure done for the benefit to the patient and a procedure done for research. In some situations including IVF/ET, the former is legal while the latter is not. I remember participating in a long, tedious conference call with Dr Taymor, who succeeded in producing the first test tube baby in Boston, and the District Attorney of Boston over how a particular procedure should be classified. No clear rules developed other than all issues should be decided on a case-by-case basis.

Conclusion IVF and embryo transfer in the human is built upon extensive basic research done by many investigators in reproductive biology for over a century. Robert Edwards built on this earlier work, making major scientific contributions. Equally important was his dogged resistance to those who opposed IVF/ET on ethical and moral grounds. His unwavering enthusiasm and perseverance led to a revolution in the treatment of human infertility and the establishment of a new branch of medicine. John Rock was a visionary who was ahead of his time because the necessary basic science in the field had not been done. An organization called IVF-Worldwide attempts to accumulate statistics on IVF around the world. At the latest count, there are at least 3221 clinics located in almost all countries in the world and almost 4 million IVF babies have been born.

Presentation This article is based on a plenary lecture given to the Society for the Study of Reproduction on 1 August 2011, in Portland, Oregon.

126

Acknowledgements The author is indebted to Drs Carol Kountz and Betsey Williams for help in researching and preparing this paper.

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