1 In Vitro Fertilization: The First Three Decades Jean Cohen International Federation of Fertility Societies, European Society of Human Reproduction and Embryology, Paris, France Howard W. Jones, Jr. Eastern Virginia Medical School, Norfolk, Virginia and Johns Hopkins University Hospital, Baltimore, Maryland, U.S.A. The birth of the world’s first baby born as a result of in vitro fertilization (IVF) in July 1978 was by no means a chance event. Indeed, in the long evolu- tion of reproduction, conception by IVF represents the end of a continuum which origi nated with childbirth wholly dependent on chance but which today is almost exclusively under human control. Today, nearly all forms of infertility can be treated by the various techniques of assisted repro- duction, which are now responsible for the birth of around two million children worldwide. THE HISTORY OF THE PAST Although the origins of our medical knowledge of human reproduction are usually attributed to Hippocrates, so often described as the ‘‘father of medi- cine,’’ we do know that in the fifth century B.C. it was believed that both males and females each produced two seminal liquors, one stronger than the other; a blend predominantly with the former would produce a male offspring, with the latter a female. In the following century, Aristotle 1 proposed that the first stage of a human be ing was indeed the egg found in females. Sperm had the power to give that egg its shape; the male would bring immaterial strength, the female material substance. For centuries, people lived with this concept of pre-formation, even after De Graaf described the follicle in 1672 and, at the same time, Leuwenhoek the sperma- tozoa. Only in 1875 would Hertwig demonstrate in the sea urchin that only one sperm cell would penetrate the egg to achieve fertilization. In 1786, Hunter performed the first artificial insemination in humans, and in 1866 Sims the first donor insemination. In 1833, the cytologist Van Beneden demonstrated that gametes had only two chromosomes in the ascaria. The two chromosomes of the male nucleus would join with the two chromosomes of the female to form the nucleus of a new zygote, thereby laying the foundations for the discovery of the hereditary principle. In 1903, a Danish pharmacist, Johannsen, coined the term ‘‘gene,’’ from which Bateson 3 years later defined the new science of ‘‘genetics.’’ Almost 50 years later, in 1953, Watson, Crick, and Wilkins discovered the double helic structure of DNA and in 1956 Tijo and Levan identified 46 chromo- somes in the human. Equally important were the advances made by gynecologists in their understanding of the physiology of reproduction. By observing the effects of ovariectomy, they were able to explain the function of the ovary and in particular the menstrual cycle; the first treatments were developed as a result of injecting extracts of ovarian tissue. The concept of ‘‘hormone’’ activity was proposed by Baylin in 1904, and the subsequent discovery of the differ- ent hormones persisted throughout the rest of the 20th century. 1950–1978 Studies of animal and then human fertilization began in the second half of the 20th century. In 1954, Thibault achieved the first fertilization in vitro in the mammal (in the rabbit); the following year, Chang (1,2) succeeded in growing rabbit embryos derived from oocytes fertilized in vitro, and in 1959 achieved a live birth by transfer of an in-vitro-fertilized oocyte. In 1965, Edwards (3) determined that human oocytes removed from ovarian biopsies required 37 hr to complete their maturation in vitro. This time was also the beginning of the gynecologist’s interest in infer- tility. It was in 1959 that the first Congress on Infertility was held in New York. Five years earlier, in 1954, the first human pregnancy derived from frozen sperm was achieved, and the following year Pincus (4), who at the time was best known for his (unsuccessful) attempts to fertilize human oocytes in vitro, published the first results on hormonal contraception (for Enovid 1 , Searle Pharmaceuticals). In 1958 and 1960, Gemz ell and Lunen- feld obtained the first pregnancies following treatment with human pituitary gonadotrophin (hPG) and human menopausal gonadotrophin (hMG), 2 Cohen and Jones respectively (5,6). In 1961, Klein and Palmer (7) described the first aspir- ation of a human oocyte during laparoscopy. However, throughout this time there was also a man working to achieve in humans what had seemed possible from work in animal models, much of it his own work in mice: IVF and embryo transfer. Eventually, in his scientific rigor and disciplined success, this man would change the face of human reproduction, demonstrating throughout persistence, self-denia l, and exceptional confidence. This man was Edwards. Edwards had completed his Ph.D. in 1958 on developmental genetics in mice. His studies, using diakinesis and metaphase-2 as markers, had shown that mice needed around 12 hr to achieve oocyte maturation, but now, as his work progressed from mouse models to the human, it was clear that human eggs required much longer. However, at the same time he and colleagues in Glasgow had produced the world’s first embryonic stem cells from rabbit embryos. Intrigued by the therapeutic potential of these stem cells, Edw ards turned to the maturation and fertilization of human oocytes in vitro—as a source of stem cells and for other research purposes. And it was from this work with human embryos—during an intense 6-wk period at Johns Hopkins Hospital in Baltimore—that Edwards found that human oocytes required 37 hr to reach full maturity, and thus 35–40 hr after ovu- lation before insemination could be carried out. By 1969, workin g with Ph.D. student Barry Bavister, Edwards was able to fertilize human eggs without any obvious need for sperm capacitation. It was at this time—in 1967—that one of us (Cohen) first met Edwards. Both (Cohen and Edwards) were attending a co nference on immunology in reproduction in Bulgaria. We met again in 1972 at an IFFS Congress in Tokyo, and here we talked of the possibilities of IVF in humans. At least, I listened, as he explained his vision of the future of human repro- duction—IVF, cryopreservation, preimplantation, and genetic diagnosis. I asked myself if he was serious, but I quickly understood that his was the vision of a true prophet. Edwards had tried unsuccessfully to collaborate with clinicians in Cambridge and London to supply him wi th human oocytes. Frustrated in these efforts, he thus turned to the United States and in 1965 had joined Georgeanna and Howard Jones at Johns Hopkins where ovarian tissue (from wedge biopsies) was more readily available. And it was here, during this 6-wk working visit, that he obtained human oocytes, confirmed the pre- cise timing of human oocyte maturation. Back in the United Kingdom, his clinical collaborations continued to prove fruitless, until his chance meeting at a London conference with the gynecologist Steptoe. At the time Steptoe was working in the small northern town of Oldham and already had much experience in the surgical use of laparoscopy. Steptoe immediately agreed to collaborate with Edwards, and so began—in 1968—the partner ship that would leave such a lasting legacy in reproductive medicine. In Vitro Fertilization 3 The story of Edwards and Steptoe is well known, but for them it was also a difficult one—the long drives of Edwards from Cambridge to Oldham (180 miles each way), the laparoscopic recovery of oocytes from the ovary, the start of embryo transfers in 1971, ovarian stimulation with hMG, clomi- phene, luteal support, and constant failure—until the first ectopic pregnancy in 1975. Finally, despite accusations of malpractice by some U.K. colleagues and after 32 embryo transfers, their first healthy pregnancy was achieved with the birth of Louise Brown on July 25, 1978 (8). I was surprised that the announcement of the world’s first IVF birth was received in such a variety of ways. Certainly, there were very few people in the world who immediately understood the huge importance of its scien- tific achievement. Many doubted it, or did not even pay it much attention. I remember that I made the trip to London in early 1979 to hear Edwards and Steptoe report their medical and scientific success to the Royal College of Obstetricians and Gynaecologists, and I remember, too, the discussions and doubts when I arrived. However, after their precise and somewhat unsettling lecture (both the biologist and the clinician presenting data), any doubts in the audience evaporated and the meeting ended to the tune of ‘‘For he’s a jolly good fellow ’’ Immediately after the birth of Brown, the attention of Edwards and Steptoe turned to extending their clinical work, but their progress was halted by the retirement of Steptoe from Britain’s nationalized health service. It took two more years before an alternative private service could be set up at Bourn Hall near Cambridge, which in time would become one of the most progressive and best known in the world. However, while Edwards and Steptoe planned their move to Bourn Hall, other groups throughout the world were inspi red by the U.K. success and set about their own efforts to repeat it. Like Edwards and Steptoe, they were contemplating a treatment for tubal infertility, with the idea that IVF would circumvent the tubal blockage if tubal surgery had failed. 1978–1982 The embryo that became Brown was derived from a natural—and not stimulated—cycle. Thus, with the success of Edwards and Steptoe showing the way, the predominant scenario of these first IVF attempts was the natural cycle, determination of the luteinizing hormone (LH) peak and follicle punc- ture during laparoscopy. There was also a new demand for the development of culture media. In Australia at the time there was already a long history in repro- ductive medicine. By 1970, Prof. Wood had established a combined research team in Melbourne involving the Royal Women’s Hospital and the University of Monash. Johnston was the Medical Director at the former, while Leeton and Talbot comprised the medical staff at the latter, with 4 Cohen and Jones Lopata and Trounson handling the biology. This joint group was worki ng with hormonally stimulated IVF cycles throughout the mid-1970s, using hPG or clomiphene and hMG. However, following the birth of Brown, the Melbourne group also turned its attention to the natural cycle. Improvements in culture media were initiated by Trounson, while the devel- opment of Teflon-lined catheters by Buttery and Kerin improved the technique of embryo transfer. Australia achieved its first IVF birth— the third in the world—in June 1980 when Candice Reed was born at the Royal Women’s Hospital. In June 1978, Howard and Georgeanna Jones had retired from Johns Hopkins—where Edwards had joined them for his 6-wk working visit in 1965—and had been asked by Andrews to set up a division of reproductive medicine at the Eastern Virginia Medical School in Norfolk. They began their IVF program in 1980, but, following 41 laparoscopies to collect oocytes, they had achieved embryo cleavage in only 13 patients, and no pregnancies following transfer. In 1981, Georgeanna Jones proposed a change to hMG and the stimu- lated cycle to obtain more oocytes, a move which yet again prompted intense debate on the relative merits of the natural or stimulated cycle in IVF. The Norfolk group had its first success in the 13th attempt in a stimulated cycle, the first American IVF baby born in December 1981. In France, two groups were making progress in friendly compe- tition. At the university hospital in Clamart, Frydman as clinician and Testart as biologist were focusing their research on the LH peak, and in 1981 developed an assay for the initial rise of LH in plasma (LHSIR) (9). This assay would allow the accurate prediction of the start of the LH surge, and thus more time for the organization of follicle puncture. In Sevres, a non-university hospital, the biologists Mandelbaum and Plachot and I found ourselves frustrated by the absence of a laboratory on site, and adopted a policy of transporting oocytes by thermos flask to the INSERM laboratory of the Hospital Necker, 30 minutes away by taxi. It was also at Sevres that Pez and I began tracking follicular growth by ultrasound. Both French groups benefited from the help of veterinary researchers at INRA (Institut National de la Recherche Agronomique), one of whom, Menezo, had developed the B2 culture medium known as the ‘‘French medium.’’ France’s first IVF babies were born at Clamart in February 1982 and at Sevres the following June. And there were now several other live births being reported from groups elsewhere—in Sweden, Finland, the Netherlands, and Germany, as well as in the United Kingdom, United States, and Australia. In Vienna, Feichtinger and Kemeter began with clo- miphene cycles in the summer of 1981 and, doing their own biology, had their first live births (twins) in August 1982. In Vitro Fertilization 5 One catalyst for the surge of activity in IVF at this time was a meeting at Bourn Hall in September 1981 organized by Edwards for those groups world- wide now actively involved and reporting results—from Bourn Hall itself, Basel, Gothenburg, Kiel, Manchester, Melbourne, Norfolk, Paris, and Vienna. It was here that many of us met for the first time, and the atmosphere was warm and friendly. Comparing experiences was reassuring for everyone, and one important conclusion did emerge—a preference for stimulated cycles, which would generate more oocytes and allow a better prediction of the tim- ing of ovulation. Now, looking back through the proceedings of that 1981 meeting and the reported discussions, I can see the following: 1. ov arian stimulation was mainly with clomiphene, 2. ultrasound was already in use (with Feichtinger) for monitoring follicular growth, 3. a concern for the effect of gas on oocyte quality during laparoscopy, 4. a concern about quality control in culture media and during lab- oratory processes, 5. an d the conviction of Edwards that his former use of Primolut 1 (norethisterone) during the luteal phase of his earlier stimulated cycles would explain the failure of his first atte mpts at IVF; most participants at the meeting seemed to agree that, if post-aspiration progesterone values were low, a progesterone supplement would be needed during the luteal phase. Primolut, Edwards concluded, was probably an abortifacient. If my descriptions of this first clinical phase of IVF seems to focus on just a few groups, it is because there was so little reporting of scientific data from elsewhere and because only the announcement of a pregnancy allowed some form of recognition from the scientific and lay communities. A fuller review of these pioneering days of IVF can be found in a series of articles in Human Reproduction Update by, Tr ounson, Dawson, Jones, Hagekamp, Nygren, Hamberger, and myself (10). This was also, let us not forget, a period of general doubt in the scien- tific integrity of IVF and in its wider clinical application. In 1982, there were only 11 reported IVF births in the world, but this does not mean that the ‘‘celebrity’’ groups were the only ones doing IVF successfully. In many cit- ies, there were young groups making their first attempts, and many of them traveled to the United Kingdom, United States, and Australia for their training. In the years which followed, they too would achieve their first preg- nancies and live births. 1982–1992 The next decade was a time of huge progress in IVF. There was an explosion in the number of centers performing IVF in many countries, and it was also 6 Cohen and Jones at this time that the first discussions on the ethics of assisted reproduction began in earnest, many of which would pave the way for subsequent legislation and guidelines. Each year saw important new clinical and scientific developments. Among the milestones were 1982: The recognition of poor and high responders to hMG, the first ultrasound-guided aspiration of follicles, and the first reports of GnRH agonist use for the downre gulation of pituitary hor- mones in IVF (11–13) 1983: Human embryo freezing (14) 1984: The first pregnancy following gamete intrafallopian transfer (GIFT) (15) 1986: The first pregnancy following zygote intr afallopian transfer (ZIFT) (16) 1986: The first human pregnancy following oocyte freezing (17) 1988: The first report of a human pregnancy following sub-zonal insemination (18) 1989: Vitrification of human oocytes (19) 1990: The first live birth following preimplantation genetic diagnosis, the detection of aneuploidy following polar body testing, and the first description of assisted hatching (20–22) 1991: The first clinical use of GnRH antagonists for the suppression of pituitary hormones (23) 1992: Intracytoplasmic sperm injection (ICSI) (24) ICSI would become the most successful technique introduced in the decade, thereafter applied throughout the world to overcome fertilization failure as a result of male factor or unexplained infertility. The success of ICSI would also be shown to be independent of the three basic sperm parameters, motility, morphology, and concentration. Throughout the decade, there was a huge increase in the use of assisted reproduction and in its success. In 1986, approximately 20 00 babies were born following IVF, with half of them conceived at Bourn Hall. However, by 1989, the first year of data presented in the initial world collaborative report at the Seventh World Congress of IVF in Paris in 1991, that total had increased to more than 18,000 (Table 1). In January 1984, Seppala had sent a questionnaire to 65 individuals or groups then working in IVF which had produced data on 10,028 cycles. Success rates according to the type of ovarian stimulation is shown in Table 2, and accordi ng to the number of embryos transferred in Table 3. In 1988, I reported a similar collaborative study at the Sixth World Congress of IVF in Melbourne which showed that, of 2342 pregnancies in the database, 24.8% were spontaneously lost and 5.2% were ectopic (Tables 4 and 5). In Vitro Fertilization 7 Table 2 Type of Ovarian Stimulation and Number of Pregnancies Achieved Stimulation No. of reporting teams No. of pregnancies/ No. of cycles Success (%) per cycle None; natural cycle 7 41/352 11.6 Clomiphene/hCG 44 256/3083 8.3 Clomiphene/hMG/hCG 50 377/3847 9.8 Clomiphene a 14 167/980 17.0 Clomiphene/hMG a 7 53/340 15.6 hMG/hCG 41 235/1591 14.8 a Spontaneous LH surge. Abbreviations: hMG, human menopausal gonadotropin; hCG, human chorionic gonadotropin. Source: From Ref. 36. Table 1 In Vitro Fertilization: 1989 General Data FR USA UK Aus/ NZ DE Scand BE JP CA ES Clinics reported 115 180 24 40 33 14 124 14 Clinics participating 50 161 35 23 37 25 14 67 10 10 Studied cycles 15,880 18,211 10,489 9345 8385 6245 4578 3726 3180 OPU cycles 15,725 15,392 8514 7356 5759 5379 3750 3438 2724 1247 Transfer cycles 10,531 (þ999) 13,523 6553 6261 4365 4581 (þ100) 3040 2571 (þ93) 2233 1158 Clinical pregnancies 2526 (þ142) 2811 1354 1040 900 997 (þ21) 741 421 (þ5) 391 244 Deliveries 1893 a 2146 982 755 646 705 a 306 a 264 194 Babies including stillborn 2531 a 2929 964 926 a 391 286 232 Total babies reported since start 11,127 11,015 4595 3275 1864 2428 552 1337 Abnormal babies 181 27 18 22 a Including frozen-thawed transfers (numbers in parentheses). FR; France, USA; United States of America, UK; United Kingdom, AU; Australia, KR; Korea, CZ; Czechoslovakia, GR; Greece, Yug; Yugoslavia, NL; Netherlands, Abbreviation: IVF, in vitro fertilization. Source: From Ref. 35. 8 Cohen and Jones KR CZ GR Yug NL SG CN BR IN PT EG TR IE Total %OPU 5587167393411!649 3 5 6 2 1 6 4 3 4 2 1 1 1 469 1456 1456 1232 769 696 617 504 383 271 243 66 >87,732 1240 1234 1000 857 656 618 509 455 322 222 168 66 34 76,030 100 1019 (þ41) 524 835 (þ21) 655 531 (þ49) 483 (þ60) 358 383 267 (þ18) 178 139 65 29 60,282 79.3 191 (þ2) 51 156 (þ1) 89 140 (þ7) 110 (þ11) 94 85 51 51 27 8 2 12,480 16.4 137 a 32 100 61 112 a 95 a 68 33 a 40 20 6 0 >8595 12.0 122 a 34 98 72 162 a 137 a 90 50 a 62 31 8 >9125 151 55 206 153 399 139 202 171 109 117 105 13 >38,013 30411502 230 0 >278 1.5 New Zealand, DE; Germany, Scand; Scandinavia, BE; Belgium, JP; Japan, CA; Canada, ES; Spain, SG; Singapore, CN; China, BR; Brazil, IN; India, PT; Portugal, EG; Egypt, TR; Turkey, IE; Ireland. It seems worthwhile to pause here and reflect on our main concerns during this decade of such great progress in reproductive medicine. First, our main scientific efforts were concentrated on fertility itself, whether to prevent pregnancy with contraception or to facilitate it with assisted repro- duction. In IVF, we were looking for ways to increase the number of oocytes available for fertilization but to decrease the number of sperm cells neces- sary to achieve it (as it was by now quite clear that the failure of fertilization was often the result of a low concentration of motile sperms). At the same time, we were also searching for ways—as reflected in the techniques of zona drilling or partial zona dissection, or indeed in GIFT or ZIFT—to bring gametes closer together in time and space, and break through the physio- logical barriers of the oocyte. However, the indications for IVF were not yet changing in any major way—and would not until the introduction of ICSI opened a door to the treatment of male infertility. From the beginning, IVF had remained indi- cated mainly for the treatment of tubal infertility as a result of blocked or damaged Fallopi an tubes. Thus, there was a lively debate in the early In Vitro Fertilization 9 NZ; 1980s following developments in microsurgery on how tubal blockage might best be treated; the microsurgeons were insistent that their new surgical techniques were potentially more effective than IVF. How ever, IVF quickly won that debate by gradually extending its indications far beyond the range of surgery, first into tubal infertility wi th patent but diseased tubes, and then into polycystic ovary disease and other idiopathic conditions. By the time the indications had been stretched to male infertility following the introduc- tion of ICSI in the early 1990s, the debate between assisted reproduction and surgery was long over. Today, ICSI accounts for around 40% of all the indications for assisted reproductive technology (ART). Table 3 Clinical Pregnancies Relative to Number of Embryos Replaced No. of embryos replaced No. of pregnancies/ No. of replacement cycles Success rate (%) One embryo 317/3321 9.5 Two embryos 366/2514 14.6 Three embryos 259/1340 19.3 Four or more embryos 197/818 24.1 Source: From Ref. 36. Table 4 Features of the Population Under Study: 2342 Pregnancies in Women of Mean Age of 33 Yr (%) Indications for IVF Type of ovarian stimulation Oocyte collection Tubal 67.9 Idiopathic 11.0 Male infertility 3.5 Other 16.7 Clomiphene 3.9 hMG 20.8 FSH 3.4 CC/hMG 62.7 hMG/FSH 7.4 Other 1.8 By ultrasound 22.6 By laparoscopy 77.4 Abbreviations: IVF, in vitro fertilization; hMG, human menopausal gonadotropin; FSH, follicle stimulating hormone. Source: From Ref. 37. 10 Cohen and Jones [...]... discrepancies remain between one registry and the next Nevertheless, most reports show that, although the mean delivery per embryo transfer increased from 22% in 1995 to 31% in 2000, some individual groups achieved published rates of 40% or more In Vitro Fertilization 13 Any progress in these results appears to have been modest in recent years, but the problems emerging in the 1990s have remained: a high... kind of eugenics under pressure of parents Since the birth of Dolly, the sheep in Scotland in 1996, the issue of reproductive or therapeutic cloning has been exposed In therapeutics, the transplantation of human embryonic stem cells now holds great promise for the treatment of diseases such as Parkinson’s or diabetes, whereas developments in stem cell biology will lead to a better understanding of infertility,... of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation Hum Reprod 1990; 5:7 23 Fleming R Gonadotrophin-releasing hormone agonist and estrogen–progestogen replacement therapy Am J Obstet Gynecol 1991; 165:1156 In Vitro Fertilization 15 24 Palermo G, Joris H, Devroey P, et al Pregnancies after intracytoplasmic injection of single... freezing and donation These paradoxes were essentially created by politicians and healthcare regulators and resulted mainly in sizable traffic of infertile couples seeking treatment beyond their own legislative borders, thereby initiating the ‘‘import and export’’ of ART Even if the legislation has changed over the years, the problem of ‘‘reproductive tourism’’ has not Thus, even from the pioneering... abnormality The possible source of these risks has been associated with ovarian stimulation, laboratory procedures, or infertility itself Prospective studies designed to identify the etiology of these problems are needed, even though most studies so far suggest that the causes of the infertility itself are the main associations with risk Since the introduction of ICSI in the early 1990s, ART has continued... respect to the new reproductive technologies In some countries, politicians and representatives of society were the final arbiters, whereas in others clinicians and scientists were left to define their own codes of practice In 1999, Jones and I, on behalf of the IFFS, published a review of the guidelines and legislation in place in 38 countries, and could not find even two of those countries sharing the same... replaced by the far less invasive, ultrasound-guided transvaginal route; and there were also at this time great improvements made in the composition of culture media and in the processes of laboratory quality control And for the patient, what was the benefit of these developments? There were still those who argued that IVF was an inefficient procedure, with success rates improving only marginally with... However, with no limit on the number of embryos transferred and clinics anxious to increase their success rates, the number (and proportion) of multiple pregnancies was seen to increase in parallel to the wider use of ovarian stimulation Multiple pregnancies would become one of the real issues of ART, both in terms of health and cost There were also major changes introduced in the technicalities of IVF:... imprinting, and meiosis The gynecologist could take part in that research, but for now the initiative lies with the scientists and the active work of the geneticist REFERENCES 1 Chang MC Fertilization and normal development of follicular oocytes in the rabbit Science 1955; 121:867 14 Cohen and Jones 2 Chang MC Fertilization of rabbit ova in vitro Nature 1959; 184(suppl 7):466 3 Edwards RG Maturation in. .. days of IVF, clinicians and biologists have shared a concern for the moral responsibility of providing IVF that was not always the same as the legislators’ Both of the leading scientific societies in reproductive medicine have continued to encourage discussions on its ethics, with debate, taskforce review, and publications However, 25 years after the birth of Brown, there remain different opinions adopted . chromosomes in the ascaria. The two chromosomes of the male nucleus would join with the two chromosomes of the female to form the nucleus of a new zygote, thereby laying the foundations for the discovery. changing in any major way—and would not until the introduction of ICSI opened a door to the treatment of male infertility. From the beginning, IVF had remained indi- cated mainly for the treatment. some kind of eugenics under pressure of parents. Since the birth of Dolly, the sheep in Scotland in 1996, the issue of reproductive or therapeutic cloning has been exposed. In therapeutics, the transplantation