Infertility: Diagnosis, Management & IVF Anil K Dubey
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1Infertility
SECTION CONTENTS
  1. A History of Clinical Embryology and Therapeutic IVF: From Pythagoras and Aristotle to Boveri and Edwards
  2. Fertility Testing and Treatment in 2020
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A History of Clinical Embryology and Therapeutic IVF: From Pythagoras and Aristotle to Boveri and Edwards1

Jacques Cohen
CHAPTER CONTENTS
  • ♦ From Preformation and Epigenesis to the Discovery of Chromosomes and Meiosis
  • ♦ Preformation and the Foundation of Modern Embryology
  • ♦ Early Cell and Germ Theories
  • ♦ The Boveri-Sutton Model and the Chromosome Theory of Inheritance
  • ♦ From Meiosis to the Concept of Ectogenesis
  • ♦ Haldane, Huxley and Rock
  • ♦ Diagnosis and Treatment of Infertility
  • ♦ Experimental Fertilization and Preimplantation Embryology
  • ♦ Fertilization In Vitro and Culturing Preimplantation Embryos
  • ♦ Development of a Culture System
  • ♦ Two Decades that Changed Human IVF
  • ♦ Establishing and Expanding the Clinical Alternative: The 1980s
  • ♦ IVF for Men
  • ♦ Gamete and Embryo Preservation
  • ♦ Preimplantation Genetic Diagnosis
  • ♦ The Evolution of Reproductive Clinical Science
“All truths are easy to understand once they are discovered; the point is to discover them.”
— Galileo Galilei (1564–1642)
 
INTRODUCTION
In vitro fertilization (IVF) has become a routine medical intervention over the past three decades, resulting in the birth of millions of children and culminating in the awarding of the 2010 Nobel Prize in physiology or medicine to Robert G Edwards. Yet, just 50 years ago IVF was considered science fiction and not at all an obvious choice for treatment of infertility and subfertility.
As astounding as this relatively quick rise may be, the future of IVF promises to be even more so. Inventor and futurist Ray Kurzweil predicts that our knowledge-base will multiply thousands of times faster during the next few decades compared to the entire history of science, technology and philosophy. In vitro fertilization is certain to see major new changes with further integration of genetics, molecular biology and physics. But to anticipate and help shape future possibilities, the past must be understood. Where did we begin and how did we arrive here?
The history of science and technology is defined as a field of history that examines how humanity understands the science and technology that has changed over time.
This now-accepted academic discipline also includes the study of cultural, economic, and political impacts of scientific innovation. In vitro fertilization is a wonderfully broad discipline that demands both historical reflection and frank discussion of complex and profound issues touching on matters of law, politics, culture and ethics.
The reader is reminded that this text is not written by a science historian. Though intended to be unbiased, the narrative draws not only on written history gleaned from historical documents, but on personal experience, as well as numerous conversations with scientists and physicians in the field.
The history of infertility treatment and IVF in particular, can be told in many different ways. Here the story is told from the perspective of basic science, with emphasis on the final steps that led to the birth of the first IVF baby in the 1970s and tribute made to those responsible for paradigm shifts in philosophy that allowed the new reproductive technologies to take form. Moreover, because no medical intervention is possible without the tools that have been made available in surgery and laboratory practice, this aspect is also covered in some detail, in the hope that future historical reflections on IVF and related technologies will include appropriate reference to this neglected area of science history.
 
FROM PREFORMATION AND EPIGENESIS TO THE DISCOVERY OF CHROMOSOMES AND MEIOSIS
 
Antiquity and Procreation
It is evident that humans have long been intrigued by questions surrounding fertilization and procreation. Symbols depicting fertility are at least 35,000 years old, dating from the early Aurignacian period shortly after the earliest representatives of Homo sapiens (Cro-Magnon) migrated to Europe (Figures 1A to F). However, it was not until well after the introduction of script that such consideration was recorded in Western thought.
The first written record of deliberations on reproduction starts with those by Greek physicians and philosophers who evidently were quite familiar with the concept of generations and embryology. They held the belief that a new organism could not only arise through sexual and asexual reproduction, but also through the process of spontaneous generation, a now obsolete principle described in detail by Aristotle (384–322 BC) (Figure 2). Earlier, Pythagoras (c570-495 BC) introduced the concept of ‘spermism’, an erroneous theory asserting that only fathers provide the essential characteristics of offspring while mothers supply only a solid substrate.
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Figures 1A and B:
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Figures 1A to F: Fine examples of so-called small Venus figurines made by European representatives of Homo sapiens of the Cro-Magnon culture during the Upper Paleolithic era of prehistory (from 40,000 BCE onwards). These figures either represent an early form of pornography or some form of worship of the female secondary sex characteristics, such as hips, breasts and vulva, possibly reflecting on the need of survival through reproduction. Facial and extremity details are under-represented or absent. Artistic and cultural interpretation may be a reflection of our modern opinion and experience
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Figures 2A and B:
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Figures 2A to F: (A) A depiction of Aristotle, the great Greek philosopher on a Drachme coin, who first introduced the concept of epigenesis. Reprinted with permission from 123RF. Reprint purchased by author; (B) Rendering of Pythagoras, the Greek mathematician and philosopher, who first formulated spermism; (C) A possible representation of Anton van Leeuwenhoek who first described spermatozoa, in Johannes Vermeer's ‘The Geographer’. Vermeer and van Leeuwenhoek knew each other in 17th century Delft, The Netherlands; (D) The figure closest to the pathologist performing the autopsy in Rembrandt's “The Anatomy Lesson” was mistakenly believed to be the great Dutch biologist Jan Swammerdam. No known portraits of Swammerdam exist; (E) Jan Swammerdam's handheld microscope; (F) Anton van Leeuwenhoek's microscope
Two millennia later, the doctrines of spermism and spontaneous generation were finally proven to be wrong through experiments and observations of Louis Pasteur (1859) who won a contest called by the French Academy of Sciences (Figure 3). However, it must be mentioned that 200 years earlier, the physician and poet Francesco Redi had already raised serious doubts about spontaneous generation by conducting an elegant set of controlled experiments that showed maggots could not arise in a jar of rotting meat covered with gauze. Aristotle's concepts were entirely replaced by germ and cell theories in the 19th century, but it took a great deal of convincing before scientists and philosophers accepted that spontaneous generation was a simply wrong theory.
Aristotle described two historically important models of development based on Pythagoras' doctrine known as the theories of ‘preformation’ and ‘epigenesis’. Preformationism held that an embryo or miniature individual already existed in either the mother's egg or the father's semen and began to grow when stimulated; spermism was the first of these models. Aristotle preferred the theory of epigenesis, which assumed that the embryo began as an undifferentiated mass and that new parts were added during development. Aristotle thought that the female parent contributed only unorganized material to the embryo.
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Figures 3A to F: (A) Francesco Redi (1629–1697) was a physician, poet and naturalist who in 1668 elegantly showed that maggots did not form from rotting meat through “spontaneous generation”; (B) Lazzaro Spallanzani (1729–1799) was an Italian catholic priest and biologist who discovered that reproduction required semen and an ovum. In frogs and dogs he performed artificial insemination, before John Hunter's experiment in humans; (C) Caspar Friedrich Wolff (1733–1794) was one of the first to reject preformationism. His work opened the doors to germ layer theory and fertilization. He discovered the mesonephros; (D) Hermann Fol (1845–1892) was a Swiss zoologist and one of at least three scientists who observed fertilization microscopically for the first time; (E) Louis Pasteur (1822–1895) was a French chemist and microbiologist, best known for developing the first vaccines against rabies and anthrax and the process of pasteurization. He provided clear evidence that spontaneous generation was not an existent reproductive process; (F) Karl Ernst von Baer (1792–1876) was a multidisciplinary German zoologist born in Estonia. He discovered the ovum in 1826 and the blastocyst later. He also accurately described the germ layer theory of development in the characteristic separation of ectoderm, endoderm and mesoderm
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The male-centric views of the day helped lead him to the conclusion that semen from the male parent provided both the form and the soul. Both Pythagoras and Aristotle were ‘spermists’.
 
PREFORMATION AND THE FOUNDATION OF MODERN EMBRYOLOGY
Aristotle's theory of epigenetic development dominated the science of embryology until the work of English physician William Harvey (1578–1657), although it took another 200 years to be considered archaic by most scientists. Harvey was inspired by the work of his teacher, Girolamo Fabrici (ca1533–1619). Some science historians consider Fabrici the founder of modern embryology, because of the significance of his embryological thesis: On the Formed Fetus and On the Development of the Egg and the Chick. Harvey's On the Generation of Animals was not published until 1651 after he completed his groundbreaking work: An Anatomical Study of the Motion of the Heart and of the Blood in Animals which explained how blood was pumped by the heart throughout the body. Although Harvey had hoped to provide experimental confirmation for Aristotle's theory of epigenesis, his observations proved that many aspects of Aristotle's theory were erroneous, yet Harvey held on to certain core beliefs of epigenesis.
Aristotle held the view that the embryo was formed by coagulation in the uterus soon after mating. Harvey's experiments in chick and deer's eggs persuaded him that the generation proceeded by epigenesis, that is, the accumulation of parts over time. Epigenesis or epigenetics is still used in biology, the contemporary sense being aspects of morphogenesis that are not encoded by genes themselves but occur by factors that control the gene activity. Many of Harvey's contemporaries and students rejected Aristotle's epigenesis and turned to the more fundamental theories of preformation.
Naturalists who favored preformationist theories (preformationism) of generation were inspired by the microscope, probably first introduced in primitive form by two Dutch spectacle makers (Hans and Zacharia Janssen around 1590) who used their knowledge of lens manufacturing. Based on this primitive compound microscope, Galileo Galilei (1564–1642) added a focusing control. Later, Anton van Leeuwenhoek (1632–1723) refined the curvature of the lenses and his upgraded device could be used to enlarge objects by as much as 260x (Figure 2). Leeuwenhoek was the first to observe bacteria, yeast and blood cells.
Marcello Malpighi (1628–1694) and Jan Swammerdam (1637–1680), two pioneers of observational microscopy, provided information that seemed to support preformation (Figure 2). Based on Swammerdam's studies of insects and amphibians, naturalists suggested that embryos pre-existed within each other and called the forms homunculi or animalcules. This phenomenon was likened to sets of Russian nesting dolls by the developmental biologist and author Pinto-Correia (1997) in her outstanding book on preformationism.1 However, the limitation of this theory was that only one parent could be the biological source of the preformed organism. At the time, philosophers were familiar with the eggs of many species, but when the microscope revealed the apparent existence of ‘little animals’ in male semen, some naturalists argued that the preformed individuals must be present in the sperm (Figure 4).
Respected scientists of the time, such as Charles Bonnet (1720–1793) and Lazzaro Spallanzani (1729–1799) supported preformationism (Figure 3). Bonnet's study of parthenogenesis in Aphids was regarded as an argument in favor of ‘ovist’ preformationism. Thus, some naturalists argued that the human race was already present in the ovaries of Eve, while others reported seeing homunculi (tiny humans) inside spermatozoa apparently derived in paternal lineage from the theological figure Adam. Clara Pinto-Correia (1997) has argued that the terminology and emphasis on this theory is the result of a more recent historical misrepresentation.1 The vivid discussions between groups of naturalists and theologians holding these two opposed views would shape the debate on the origins of life for some time to come.
 
EARLY CELL AND GERM THEORIES
Some 18th century scientists rejected both the ovist and spermist doctrines. One of the most convincing arguments was raised by Casper Friedrich Wolff (1733– 1794), who published a groundbreaking article, “Theory of Generation,” in 1759. Wolff argued that the organs of the body did not exist at the beginning of gestation, but formed from some originally undifferentiated material through a series of steps. Other naturalists became interested in this attractive model known as natural philosophy. During the 19th century, the basis of cell theory was expanded by the discovery (1827) of the mammalian (dog) ovum in Germany by Karl Ernst von Baer (1792–1876) many years after the finding that semen contained millions of individual moving cells called spermatozoa (Leeuwenhoek, approximately 1677) (described in Anton von Leeuwenhoek and his perception of spermatozoa by Ruestow).2
Historians are not always in agreement about who actually witnessed the mammalian fertilization process and sperm-egg interaction first.
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Figure 4: Left panel depicts Nicholaas Hartsoeker's homunculi (1695), the presence of a tiny already complete human in the sperm seen using Hartsoeker's primitive microscope. The right panel shows van Leeuwenhoek's sketches of spermatozoa (1677). The latter showed morphologic disparity as well as detailed head features. The differences between the observations of both microscopists may have been due to subjectivity, visualization and artistic interpretation. Hartsoeker never claimed to have actually seen the homunculi, but suggested the representations to support spermist theory. He apparently was present when Leeuwenhoek noticed spermatozoa in semen for the first time
Was it Schenk in 19783 in Vienna or the Swiss physician and zoologist Hermann Fol4 a year later (Figure 2)? What is evident is that Schenk was the first to describe the dissolution of cumulus cells in rabbit eggs held in follicular and uterine fluids after exposure to epididymal spermatozoa, thereby clearly establishing the field of experimental embryology. Interestingly, this was reported exactly 100 years before the birth of the first IVF baby in the human (Steptoe and Edwards, 1978) (Figure 5).5
Oskar Hertwig, a student of the renowned German biologist and artist Ernst Haeckel, described fertilization in the sea urchin6 two years before Schenk (in 1876) and it seems that these observations led him to emphasize the important role of sperm and egg nuclei during inheritance and the reduction of chromosomes (meiosis) during the generations. Another German biologist and artist, Theodor Boveri, published some of the most significant principles of preimplantation embryology in the late 1880s and early 1890s (Figure 5)7. Oscar Hertwig before this had already proposed that sperm and egg nuclei fuse during fertilization (fusion is typical in invertebrates studied by Hertwig, but does not occur in mammals).
 
THE BOVERI-SUTTON MODEL AND THE CHROMOSOME THEORY OF INHERITANCE
Boveri studied the maturation of egg cells of Ascaris megalocephala, the horse nematode. He observed that as eggs matured, there came a point where chromosome numbers were reduced to half. Boveri was one of the first to see evidence of the process of meiosis. Boveri7 and Sutton8 independently advanced the chromosome model of inheritance in 1902 (Figure 5). Boveri performed his studies with sea urchins, in which he found that all the chromosomes had to be present for appropriate embryonic development to occur. Sutton's work with grasshoppers demonstrated that chromosomes are organized in matched pairs of maternal and paternal chromosomes, which detach during meiosis.8 The Boveri-Sutton chromosome model (the chromosome theory of inheritance) is a fundamental conclusion in genetics. This model identifies chromosomes as the carriers of genetic material. It explains the mechanism essential to the laws of Mendelian inheritance by identifying chromosomes with paired factors as would be required by Mendel's laws.
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Figures 5A to F: (A) Patrick Steptoe and Robert (Bob) Edwards in 1969 tensely answering questions from reporters during their press conference after the announcement of obtaining proof of fertilization using human gametes in the laboratory. Reprinted with permission of Getty Images. Reprint purchased by author; (B) The first wave of IVF pioneers. From left to right: Pincus, Hamilton and Chang; (C) Walter Sutton, one of the co-pioneers of the chromosome theory of inheritance; (D) Theodor Boveri, the other co-pioneer and the most famous of experimental embryologists of his day; (E) EB Wilson who discovered the sex determining chromosomes X and Y, simultaneous with (F) Nettie Stevens
Boveri-Sutton model also argues that chromosomes must essentially be linear structures with genes located at specific sites along them. The chromosome as an organelle was discovered at least 60 years earlier by Wilhelm Hofmeister in Germany (Campbell, 1925).9 Just a few years after Boveri-Sutton, E B Wilson10 and Nettie Stevens11 (1905) independently discovered the chromosomal XY sex-determination system—that males have XY and females XX sex chromosomes (Figure 5).
Boveri and his partner Marcella Boveri were among the first true experimental embryologists. He was nominated, but never received the Nobel Prize before his sudden death in 1912. He chronicled the development of normal sea urchin eggs, but also when the egg was fertilized by two rather than one sperm cell. Boveri deducted that male sperm and female egg nuclei were similar in the amount of transmissible information. They each had a half set (haploid number) of chromosomes. As long as a set of each was present, defined as the diploid number of chromosomes, there was usually normal sea urchin development. Any more or any less and development would proceed abnormally. Mendel's laws were rediscovered in 1900. Boveri recognized the 11correlation between Mendel's findings and his own cytological evidence of how chromosomes behaved.
The centriole, which is integral to cell division and flanks the spindle, was also discovered by Boveri earlier in 1888 (Sathananthan et al. 2006).12 A pair of centrioles, one aligned perpendicular to the other, are found in the centrosome—the microtubule organizing center of animal cells (although some centrosomes, like that of the mouse are acentriolar). Boveri subsequently hypothesized that cancer was caused by errors during cell division. Although scorned at the time, Boveri was later proved to be right. In addition to playing a critical role in mitosis, the centriole apparently also provides structural support. A centriole may have its own unique genetic code, which is distinct from the code of the cell; some scientists now believe that this code allows the centrosome to double and divide with each cell cycle precisely and carry out its various functions in the cell. Boveri correctly argued that only one of the centrioles from the two gametes could survive the fertilization process, the other one being inactivated.
 
FROM MEIOSIS TO THE CONCEPT OF ECTOGENESIS
 
Embryo Transfer
Walter Heape (1890) in the UK was the first to successfully transfer a ‘segmented ova’ (cleaved) embryo from one animal to another.13 Heape used the characteristics of the Angora rabbit from which the embryos were obtained to describe the offspring after transfer into a Belgian hare. The cohort of siblings was of a mixed nature since the recipient rabbit was mated normally. The embryos were not exposed to laboratory conditions and transfer was done very quickly after washing the embryos from the oviducts. Interestingly, Heape's rabbit experiments were either performed in his laboratory in Cambridge or in Prestwick near Manchester, his family home. Bob Edwards would use a similar venue combination in the 1970s during the first series of human IVF, commuting back and forth between Cambridge and Oldham, a town near Manchester where Steptoe practiced as an NHS consultant. Heape's groundbreaking experiments in rabbits and deer and his suggestion to use the transfer procedures in farm animals in a later book are described in a concise review by Biggers (1991)14. Heape's thoughts, to use embryo transfer between two animals, apparently did not translate into the concept of artificial fertilization, at least not explicitly stated as such; however, if his experiments did not lead him to the idea directly, it may have inspired others.
 
HALDANE, HUXLEY AND ROCK
The idea of achieving extracorporeal fertilization was probably first introduced by the great British Population Geneticist, JBS Haldane,15 who in a book written for a lay audience and published in 1924 (Daedalus; or, Science and the Future) described how a process he called ‘ectogenesis’ would soon create individuals outside of the human body (Figure 6). He predicted that the first birth would occur in 1951, only slightly optimistic since the concept would become validated not too long after. Haldane's friend Aldous Huxley, an English writer, popularized reproductive technology mixed with provocative descriptions of sexuality some 600 years into the future in his famous novel, Brave New World (1932)16 (Figure 6). As has unfortunately become commonplace when the future of science is portrayed, Brave New World is a dark prophecy. Huxley only admitted to having copied the concept from Haldane's in vitro conception theory many years after the publication of his book. Now the Heape-Haldane-Huxley concept of alternative forms of procreation was out of the box and the tantalizing possibility that these could soon be available to anyone was on the horizon.
The second paradigm shift occurred with the idea of applying the ectogenesis model to women with tubal disease. This concept was introduced rather plainly in a short editorial in The New England Journal of Medicine in 1937 by Dr John Rock, who was a highly regarded ObGyn at Harvard University (Figure 6). At the time, the idea was perceived to be so outrageous that even the author avoided claiming it, and the editorial was unsigned.17 The concept had now matured from being proposed as a futuristic way of general procreation to a specific treatment for women with tubal disease.
 
DIAGNOSIS AND TREATMENT OF INFERTILITY
Infertility diagnosis and treatment before Louise Brown was more sophisticated than is sometimes believed. Infertility was already an established subspecialty well before World War II. By contrast, andrology is a very new discipline. The success of treatment was sometimes expressed as a function of the duration of infertility. Treatment rarely produced better results than no treatment. There were notable exceptions, for instance tubal disease treatment using surgical intervention was well established and quite successful. Similarly, certain endocrinological and immunological disorders could be treated occasionally. The advent of sperm transfer, artificial insemination using the semen of a donor may have occurred as early as 1790 in Scotland (Dr John Hunter).
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Figure 6: A cover of one of the later editions of ‘Brave New World’, the novel (1932) by Aldous Huxley (lower panel insert) describing a repressed society where anonymous in vitro fertilization and gestation were considered a normal reproductive routine uncoupled from sexual activity. The book was based on JBS Haldane's prophecy of ‘ectogenesis’ described in ‘Daedalus, or, science and the future’ (1924). John Rock (upper panel insert), a famous Harvard ObGyn suggested to use ectogenesis for cases of tubal infertility
In the early part of the 20th century, donor insemination was practiced sporadically until the 1950s when the procedure was first described in medical journals. Chris Polge was the first to deep-freeze spermatozoa from any mammalian species in 1949.18 Human spermatozoa were first successfully frozen in Iowa (USA) a few years later, by Jerome Sherman, who also established the world's first sperm bank in 1960.19
 
EXPERIMENTAL FERTILIZATION AND PREIMPLANTATION EMBRYOLOGY
Meanwhile scientists would complete the first steps of ectogenesis in the laboratory, planning fertilization experiments in vitro in animal models. Although MC Chang's work in 195920 is widely regarded as the first proof of IVF in a mammalian model, there were dozens of scientific publications spanning 80 years of research, which paved the way for embryologists (described by Austin in 1961).21 Of note are the remarkable early experiments by Onanoff in 1893 using eggs flushed from the uterus.22 Most experimental embryologists later used tubal eggs. Gregory Pincus, the father of the contraception pill, claimed to have fertilized rabbit eggs before World War II, however, the does were inseminated first by a buck and the eggs were flushed quickly from the fallopian tubes after which they were washed vigorously to remove spermatozoa.23 In the 1950s, when in vitro inseminations were more commonplace, it became obvious that spermatozoa could interact with the zona pellucida shortly after insemination and that excess spermatozoa could not be easily removed by washing. Other observations published by Pincus, such as the presence of two polar bodies after activation 13(disputed by Chang) and a very short interval of only 12 hours between observing the germinal vesicle and the first polar body appearing in the human (disputed by Edwards) were also reasons to perhaps consider the pre-war work in a different light.5,24
John Rock and Miriam Menkin at Harvard would collect hundreds of immature ovarian eggs from patients and attempt to fertilize them with modest results during the years after the second world war (Menkin and Rock, 1948).25 In the 1950s, Thibault in France and MC Chang in the United States carried the field forward by confirming fertilization in vitro and obtaining offspring in the rabbit following transfer of the embryos (Thibault et al. 1954; Chang, 1959).20,26 However, many of the intricate details of the IVF process were still basically unknown. For instance, it was believed that spermatozoa had to mature in the uterus first. By the time Bob Edwards became interested in treating tubal infertility by IVF in 1963, a few others had also attempted to fertilize human eggs in vitro, although fertilization was not positively proven in any of those cases.27 A number of important questions needed to be resolved first: (1) what was a suitable culture fluid or medium? (2) What was the best way of culturing the specimens? (3) How could immature eggs be matured in vitro? (4) How could mature rather than immature eggs be obtained routinely? (5) How should spermatozoa be prepared? (6) How could more than one egg be recruited? (7) How could ovulation be timed accurately? (8) At what stage should embryos be returned to the uterus? (9) How and where should embryos be transferred? Although some of these questions were being addressed by experimental embryologists working with animal models, each species had its own specific requirements. The human was very different not only because the women were older and suffered from infertility, but because oocytes were obtained from and embryos were returned to the same individual rather than the egg donor and embryo recipient being two different individuals as is routine in animal work. The concepts of clinical IVF and PGD were accurately described in 11 key points published in Edwards' remarkable 1965 paper in the Lancet.27 This paper was recently reviewed by one of his first PhD students Martin Johnson.28
 
FERTILIZATION IN VITRO AND CULTURING PREIMPLANTATION EMBRYOS
 
The First Successes in Animal Models
For several years after the success of IVF in the rabbit, as Chang24 describes, “it was felt that unless living young could be obtained after transplanting such fertilized eggs into recipient rabbits, successful fertilization in vitro could not be held to be proven, since such eggs could be abnormally fertilized or may not be fertilized at all”. This final confirmation was obtained by Chang in 1959 (Figure 5).25 He incubated newly ovulated eggs with capacitated sperm for four hours then cultured the eggs in 50% rabbit serum for another 18 hours before transferring the eggs into recipient females. These experiments resulted in the first live births following IVF and embryo transfer in mammals.
It was over a decade after the discovery of in vivo capacitation in the rabbit that sperm capacitation in vitro was first achieved in the hamster by Yanagimachi and Chang (1963).29 After another few years, IVF was achieved in the mouse by David Whittingham (1968),30 opening a tremendous avenue for research into early mammalian development since the period from egg to blastocyst could now be artificially controlled in at least one species. For experimental embryology in mammals to move forward, a reliable embryo culture method was imperative so that the fertilized eggs could be maintained in vitro through the cleavage stages. Earlier Hammond (1949) had discovered that mouse embryos collected at the 8-cell stage, but not the 2-cell stage, could be cultured to the blastocyst stage in a physiological saline solution that was supplemented with hen's egg yolk and white.31 Although the inability to culture 2-cell embryos was and remained for some time a formidable challenge, Hammond's discovery was a significant one and set the stage for abandonment of biological fluids as culture media for embryos. In 1956, Whitten (1956) replaced Hammond's medium with a modification of Krebs-Ringer bicarbonate solution.32 He supplemented the latter with glucose, antibiotics and bovine serum albumin (BSA) and showed that it could support development of 2-cell and 8-cell mouse embryos to the blastocyst stage. Two years later, McLaren and Biggers (1958) obtained normal young following transfer of blastocysts grown in Whitten's medium, proving that viable blastocysts could be produced in vitro.33
At this juncture, it was the inability to obtain large numbers of eggs at a time (and in a controlled manner) that hampered research efforts. According to Edwards (1980), the dogma of the time dictated that ovaries of adult females would not respond to gonadotrophic hormones.34 However, Fowler and Edwards (1957) successfully challenged this theory.35 They followed the work of Gates in 195436 who had artificially induced ovulation in prepubertal mice using a regimen that included injection of pregnant mare serum followed two days later by serum from a pregnant woman. Fowler and Edwards used the same method and induced 14superovulation and pregnancy in mature mice.35 Later, it was shown by Gemzell that superovulation could be achieved in the human using pituitary gonadotrophins (1962).37
 
DEVELOPMENT OF A CULTURE SYSTEM
But another hurdle had to be overcome before production, fertilization, culture, and transfer of mammalian eggs would become routine practice. A reliable and efficient embryo culture system had to be devised. In 1963, Brinster introduced culture of eggs and embryos in small drops of culture medium under a layer of paraffin oil.38 With only minor modifications, this ‘micro-drop’ method using a 19th century invention called the Petri dish, has become the most widely used and successful system for culture of mammalian embryos in vitro today. It would be difficult to think about human embryos growing in the laboratory without contemplating their artificial world and the Petri dish that is temporarily their home. The Petri dish is used in more than 99.9% of ART procedures. By inference, embryologists may have used over 100 million of them to date. In spite of that, the dish has changed little since its inception in the latter part of the 19th century. There have been few secondary changes to adapt the original plain design of the dish to areas of specialized use, such as cell tissue culture (e.g. the square four-well dish) and microbiology. The same can be said about the adaptations made to the Petri dish after its introduction to preclinical embryo research in the 40s and 50s. There have been few such alterations and usually these have been unremarkable, such as place markings for droplets or identification numbers on the bottom. The dish was developed in the latter part of the 19th century, because there was a need in vaccine research to grow micro-organisms on a solid substrate rather than in a broth. This used to be the common way of growing bacteria in culture until the famous German scientist and physician Robert Koch (1843–1910) known as the master of germ theory, suggested replacing the liquid phase. This made a huge difference to the field of germ culture and development of vaccines but the problem was that Dr Koch's assistant had difficulty using glass flasks for this purpose. Koch's assistant was Julius Richard Petri (1852–1921). He decided one day in 1887 to cut off the flask and only use the bottom for pouring the solid media into. The dishes were manufactured in glass and mammalian embryos were cultured experimentally using glass dishes well into the 1970s. In the mid-80s, a sudden increase in the cost of raw material and a better understanding of the injection molding process, allowed most manufacturers to reduce the weight of plastic Petri dishes to the 15–17 grams range. This new thin plastic Petri dish has remained largely unchanged and is now an industry standard.
As mentioned above, one of the most important steps toward contemporary embryo culture was developed by the scientist Ralph Brinster (of sperm stem-cell fame) in 1963, when he successfully cultured mouse eggs to blastocysts. He decided to do away with ‘open’ culture and protect small amounts of culture medium using a transparent viscous fluid overlaying the media. He used paraffin oil for this purpose. The advantages of this system were huge, although it essentially moved away from Koch's solid substrate approach for which the Petri dish was designed. Oil prevented most microbial infections, allowing fertilization and embryo growth events to take place in less stringent conditions. For example, gametes and embryos could be observed for longer periods since medium evaporation became a problem of the past. The method also allowed the study of minute quantities of metabolites released or absorbed by the cells and later, it facilitated the introduction of micromanipulation methods. Intracytoplasmic sperm injection (ICSI) would have been nearly impossible without the use of oil. The high heat capacity of oil also helped to maintain incubator temperature when the dishes were moved around for observation or manipulation. The problems were oil toxicity and batch-to-batch variation. Paraffin oil has now been largely replaced by other oils, such as mineral oil. This is a variation on light hydrocarbon oils, a distillate of petroleum. Toxicity has been diminished because certain mineral oils are used for human consumption as a lubricant laxative. However, batch-to-batch variation is still a problem. Brinster's technical marvel was for a long time unappreciated by human IVF specialists, as nearly all early practitioners (particularly in the USA) used either organ culture dishes or small test tubes for culture of human gametes and embryos.
 
TWO DECADES THAT CHANGED HUMAN IVF
 
The First Test Tube Baby
The basic principles of experimental animal embryology and experience gained in that area, including human oocyte maturation in vitro by Edwards in 1965.27 Bavister, Edwards and Steptoe39 using a modification of Tyrode's solution devised by Yanagimachi and Chang for hamster IVF,29 added sperm to nine human eggs and, 11 hours later, recorded the presence of a sperm tail within one egg and the presence of pronuclei in another. This was indisputable evidence of fertilization in vitro in the human (Edwards et al. 1969),40 but it was only the first step since this medium was not able to support 15further development. It was already known that seminal plasma was not supportive of fertilization and also spermatozoa had to undergo a process called capacitation first, before they could penetrate the oocyte.
The collaboration between Bob Edwards and Patrick Steptoe, one of the most fruitful collaborations ever undertaken between a scientist and clinician, started in 1968 because Steptoe had been able to introduce laparoscopy successfully after others like Palmer (1947)41 and Fikentscher and Semm (1964) provided the instruments to visualize and manipulate the ovaries.42
The first infertile patients were invited to participate in IVF treatment in 1970. Unfortunately for those volunteers, it took over 100 attempts to finally obtain a sustained pregnancy in November 1977. The first pregnancy had been achieved a year earlier in 1976, but it was ectopic and had to be terminated. Wood and Leeton in Australia also reported a biochemical pregnancy in 1975. Other teams in Sweden, Holland, the USA, India and Australia had joined in, but the two pioneers remained the most focused and determined about the work in progress often supported by a third collaborator, nurse Jean Purdy. Purdy played a crucial role in the convergence of experimental embryology and reproductive medicine. She facilitated the transformation of basic research in in vitro fertilization to a meticulous clinical discipline with a foundation in quality control. Jean Purdy is without a doubt, the founding mother of QC in clinical embryology.
Louis Joy Brown was born on July 25, 1978 and quickly became the most famous baby. Her name is still well recognized throughout the world. She represents Edwards and Steptoe's quest for knowledge and making human IVF a reality for infertile couples. After the birth of Louise, a short (and remarkably understated) letter was published in the Lancet (Steptoe and Edwards, 1978).5 Three things stood out in this publication. The first was that the transferred embryo was an 8-cell and not a later stage embryo as was the case during previous transfer attempts. The transfer of blastocysts was based on the assumption that embryos at earlier stages would be received with physiological hostility, since it was believed that the uterus normally accommodates only morulae and blastocysts. We now know that this is only true in animal models and that the human uterus can tolerate any stage of development around the time of ovulation, even prefertilization, if sperm and eggs are injected together as Ian Craft and colleagues demonstrated in 1982.43
The second surprising revelation in the Lancet letter was that Lesley Brown's (Louise's mother) diseased fallopian tubes were removed and her ovaries had been relocated into a position of easy accessibility. It escaped no one that this maneuver guaranteed that there would be no doubt about the pregnancy having occurred with the IVF embryos and not per chance from the spontaneous fertilization of a wandering egg.
A third extraordinary aspect of the announcement was that the mature egg had been retrieved from a naturally growing follicle rather than from follicles that had been developing under exogenous hormonal stimulation, as it had been the case in previous patients. The question that was then posed was whether the natural cycle was requisite to success of IVF. It was the team of Alan Trounson that provided the answer a few years later using gonadotropins and clomiphene citrate successfully.44 Earlier in 1980, another team in Melbourne achieved the first Australian pregnancies.45
It should be noted that the initial and subsequent successes of IVF occurred against an extraordinarily unfriendly background, without the support of government agencies, and under a continuous barrage of criticism. Many ethicists, religious leaders, politicians, lawyers, fellow scientists and physicians were appalled by the idea. Edwards confronted them head on and even described scenarios new to them in order to focus the debate. His defense of IVF never wavered and he has written dozens of scholarly articles about the legal, political and ethical issues surrounding reproductive technologies.
 
ESTABLISHING AND EXPANDING THE CLINICAL ALTERNATIVE: THE 1980s
At the end of 1980, Edwards and Steptoe opened the world's first IVF clinic near Cambridge in an old land-house called Bourn Hall, which became Bourn Hall Clinic; it had taken the founders some time to establish the facility due to the general lack of interest among financiers. Government funding, both locally and nationally was quite out of the question after the UK Medical Research Council (MRC) and National Health boards again refused to support IVF; an earlier refusal goes back to 1971 when the MRC declined to fund the emerging field of assisted reproduction (for an excellent review on this topic see Johnson et al. 2010).46 Later in 1983, the MRC would again refuse to grant a broad research application from Edwards and his embryologists. Nevertheless, Bourn Hall Clinic became a legendary place complete with in-patient wards, ethics and visitors committees, endocrinology, embryology, research laboratories, parlors and a dining hall. Other clinics were opened soon: at the Royal Women's Hospital (Alex Lopata) and Monash University (Carl Wood, John Leeton and Alan Trounson) in Melbourne, 16Australia with some government support, and in London, UK (Ian Craft) from private funding. At the Eastern Virginia Medical School in Norfolk, Virginia (USA), two famous reproductive gynecologists, Drs Georgianna and Howard Jones, opened the first US-based facility using funds released by the university. Other countries, such as India, Austria, France, Holland, Sweden and Spain followed swiftly and established their own clinics. By 1982, a new discipline was in the making, a field some people were referring to for the first time as ART.
The enthusiasm generated by the success of IVF in Norfolk in 1981, however, did not persuade the US government to lift the moratorium it had placed on all human embryo research a year earlier. In fact, later (in 1995) a law was enacted that prohibits the funding of ‘research in which human embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero.’ (Dickey-Wicker Amendment, 1995). The US federal government thus does not support clinics or any clinical studies and this sad situation has not changed for over a quarter century.
Although the basis of the technology was now established, many of its aspects were poorly understood. A number of important observations had been made by the first IVF pioneers. They recognized that timing of ovulation and follicular recruitment were complicated processes often limiting a team's ability to plan ahead while many patients became frustrated because of cancellations shortly before egg retrieval. Drugs were needed to recruit follicles at will and control and time ovarian stimulation. The first such family of drugs was the GnRH agonists. These drugs downregulate the secretion of gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) resulting in a dramatic decline in estradiol levels. This allowed suppression of endogenous gonadotropin production and the LH surge, and planning for egg retrieval following an injection of human chorionic gonadotropin (hCG) (Fleming et al. 1982).47
Another clinical bottleneck during the early days of IVF was the requirement for laparoscopy. Although a magnitude more efficient than laparotomy, laparoscopy had to be performed under general anesthesia in a full operating theater, and required considerable recovery time. Moreover when visualization was hindered, ovaries remained inaccessible and dominant follicles unreachable. The search for a faster and more efficient means of oocyte recovery was on. Ultrasonography, though in its infancy, had already been applied to track growing follicles in the 1970s.48 The question was whether it could be used during egg retrieval to visualize the follicle and its content. After all, the ovaries were positioned near the vaginal wall. Nevertheless, the first aspiration of a follicle using ultrasound was achieved transabdominally in 1982, a considerably longer route requiring access through the bladder.49 That same, year abdominal ultrasound was combined with vaginal follicle aspiration.50 The final and determining step was performed by the Swedish team of Hamberger and Wikland in 1985 using a new narrow vaginal ultrasound probe guiding a needle adjacent to it; this method is still in use now.51
In the laboratory meanwhile, experimental embryologists, veterinary researchers and pathology technicians were retrained as clinical embryologists. Their first task was to safely handle and observe gametes and embryos. Laboratory and equipment maintenance, and standardization of methods were other important tasks, as was meticulous record-keeping. These first clinical embryologists were surprised to notice that human embryos varied considerably (Edwards and Purdy, 1982) not just between patients, but also within cohorts.52 This variability made evaluation of embryos difficult. Even more frustrating was the fact that morphology and rate of development seemed only loosely correlated with outcome. The search for important characteristics that predict implantation has brought under examination many aspects of gamete and embryo development in culture and complicated algorithms have been developed. However, after 30 years, not a single common morphological marker has been identified that can predict the future success of an embryo with certainty. Even algorithms of multiple morphologic criteria do not reveal implanting ability with perfect accuracy. During the past 15 years, researchers have attempted to correlate clinical outcomes with embryo metrics, but only with mixed success. Certainly one of the major challenges remains the identification of accurate (and affordable) embryo selection methods, a crucial step in further reducing multiple pregnancy and facilitating single embryo transfer.53
 
IVF FOR MEN
In vitro fertilization is the first and only general treatment for infertility and subfertility; couples with male infertility can now be treated just as successfully as those with female-related infertility. However, this aspect was not generally accepted in the early 80s. It was feared that spermatozoa from men with male infertility would not be able to penetrate the zona pellucida or that if they did, fetal development could be abnormal. However, when couples with male factor infertility were selected for IVF, many had fertilized eggs although the fertilization rate 17was only a fraction of that in other groups of infertile couples (Cohen et al. 1984).54 Moreover, many men with severely reduced sperm counts could not be treated as not enough spermatozoa could be prepared for micro-droplet insemination. The notion that micromanipulation could enhance fertilization in male factor cases even further than standard IVF had already been suggested some years back. The first such experiments in some human spare eggs were conducted in Rotterdam in 1979 (Zeilmaker and Cohen, unpublished). The first birth in mice following micromanipulation was achieved by opening the zona pellucida artificially, an approach called zona drilling or dissection.55 In 1988, human babies were born from a similar mechanical zona dissection, as well as injection of spermatozoa into the perivitelline space.56,57 Though this improved the prospects for treatment of male factor infertility, fertilization rate were low due to the absence of a quick block to polyspermy on the membrane level. This meant that embryologists could only use very low concentrations of suboptimal spermatozoa after zona dissection or they could only inject one spermatozoon for subzonal insertion. Fertilization rates were improved dramatically with the introduction of ICSI by a team of researchers in Brussels, Belgium.58 ICSI is now the preferred method of treating those at risk of reduced or failed fertilization.
 
GAMETE AND EMBRYO PRESERVATION
The most exciting events in science are often marked by the merging of seemingly unrelated disciplines. The field of reproductive science had already experienced this in the 19th century, when the beliefs of both spermists and ovists were shattered by the observation that the spermatozoa penetrate the egg and that this is followed by the formation of two pronuclei in the zygote. Those lucky clinicians and scientists practicing IVF in the 1980s witnessed not one, but two revolutions. The first groundbreaking shift was the enablement of preserving extra embryos for later use. Cryopreservation of the embryo (and later the egg) allowed clinicians to reduce the number of embryos for transfer. In the human, all stages between the zygote and blastocyst were frozen, however, different cryoprotectants and freezing protocols were required.5962 Thawing of embryos later allowed transfer in the natural cycle. Some couples did not have to undergo multiple IVF treatments, since the embryos from one cohort could be enough to establish a multisibling family. The effort was well founded in science, since pioneers working with rodents and farm animals had already mastered the technology years earlier.6365 The past ten years have seen further refining of egg and embryo cryopreservation, the aims being simplification of methodology and increasing egg and embryo survival rates.
 
PREIMPLANTATION GENETIC DIAGNOSIS
The other revolution in the 1980s was genetic diagnosis of embryos through blastomere biopsy before transfer, a feat accomplished by a multidisciplinary team in London (UK).66 Interestingly, the general concept was already introduced 20 years earlier by Bob Edwards and one of his brilliant PhD students at the time, Richard Gardner.67 They performed trophectoderm biopsy in the rabbit embryo, applied a sexing technique and transferred sexed embryos to the uterus. More than 20 years later and a few years after development of the PCR, this elegant experiment would form the basis for a new field called PGD.
 
THE EVOLUTION OF REPRODUCTIVE CLINICAL SCIENCE
In vitro fertilization is now considered an industry, a field of its own. More than two thousand clinics specializing in IVF exist worldwide. The largest, in Tokyo, Japan treats more than 15,000 couples a year! A few forward-looking governments support the IVF effort financially. Other governments, such as the ones in Sweden and Belgium, support and guide the practice with smart laws based on clinical data. Many professional organizations have been formed to support the effort and special university-based training programs exist for physicians and embryologists subspecializing in IVF. It is estimated that four million babies have been born through ART; however, the road to this success has not always been easy. In 1934, Dr Gregory Pincus was a young man in his early 30s when he claimed to have achieved in-vitro fertilization in rabbits, just a few years after Haldane's prophecies and Huxley's book. While the discovery made international headlines, he was vilified in the press for his research. The New York Times depicted him as Dr Frankenstein, just like others would later describe the work on IVF by Patrick Steptoe and Robert G Edwards as a travesty. It must have been disconcerting for scientific mavericks like Pincus and Edwards to be called names for their sound scientific enquiry. Yet, maybe they found solace in the history of science, since many true innovators, Copernicus, Galileo, Darwin and Boveri among them, were frequently disparaged and often unfairly treated during their lifetime.
18
 
ACKNOWLEDGMENTS
I like to thank Dr Mina Alikani for critical review of this chapter and general encouragement and support during the preparations. This chapter is a reprint from a chapter with the same title to be published in Textbook of Clinical Embryology by Cowan and Wells (editors) in Cambridge University Press in 2013. The current chapter was reprinted with permission from Cambridge University Press.
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