【佳學(xué)基因檢測(cè)】生殖技術(shù)年鑒：基因檢測(cè)與試管嬰兒
根據(jù)《生殖技術(shù)年鑒》，1978 年是人類生殖技術(shù)發(fā)展史上值得記憶的一年。在這一年，諾貝爾獎(jiǎng)授給了宇宙微波背景輻射發(fā)現(xiàn)者，它提供了大爆炸的先進(jìn)個(gè)直接證據(jù)，而時(shí)間本身在我們的宇宙中有一個(gè)起點(diǎn)。 先進(jìn)個(gè)在線交流論壇，博客和社交媒體等所有公共傳播的前身，在芝加哥新穎亮相。 索尼發(fā)明了隨身聽(tīng)，這是先進(jìn)臺(tái)便攜式立體聲音響，也是 iPod 之父。 但毫無(wú)疑問(wèn)，與9年前人類在月球上行走相媲美的技術(shù)進(jìn)步是先進(jìn)個(gè)體外受精嬰兒的誕生。
That human gametes could be fertilized outside of the mother's uterus in an instance where reproduction would be possible no other way, leading to an embryo that could then develop as a fetus in the mother and born live changed the world. Millions upon millions of children have been born since that time who simply could not have been without the development of IVF.
This monograph chronicles the arc of development of IVF that began 40 years ago and follows the new questions asked by the beginning of this new technology and the possibilities that it created. It is designed not to be an exhaustive data-driven compendium, but a readable narrative of what has happened in the past, what is happening at present, and what may happen in the future. For those who are students of reproductive medicine and who did not experience living through the entirety of these past 40 years, it tells the story of how and why you do what you do. For those who predate 1978, it serves as a thrilling ride through all of the varied roads that IVF paved.
We begin with the laboratory, as that is where the heart of IVF was born and where the foundations of our field developed. At present our IVF laboratories appear totally different from those in 1978. Initial implantation rates that were <5% per embryo replaced were continuously increased, at present, to rates >50%. The low implantation rates of the early days was problematically addressed by increasing the number of embryos replaced into the uterus and aggressive ovarian stimulation protocols, unfortunately often uncontrolled, introducing the two main complications of IVF in these 40 years—multiple pregnancies and ovarian hyperstimulation syndrome (OHS). These challenges were intensively reported, studied, and almost completely resolved.
Access to these early embryos in the laboratory also introduced a series of medical techniques that have been tremendously useful and provide new pathways to fight against disease by applying preimplantation genetic testing. In the future perhaps we will be able to treat genetic diseases by genomic editing.
Other techniques were developed to preserve fertility in men and women with life-threatening illnesses for whom the use of life-saving but gonadotoxic treatments may compromise their future fertility. Cryopreservation of the male gamete predated IVF, but oocyte cryopreservation was far more challenging, which was finally solved a quarter century into the story of IVF. Freezing the oocyte has not only been effective in preserving fertility, but has also given practitioners the ability to split the reproductive cycle into different steps to improve outcomes and reduce complications, such as OHS. In many ways, at present, oocyte cryopreservation has changed the way we perform IVF.
The protocols of controlled ovarian hyperstimulation (COH) have also undergone substantial evolution, mainly due to the research and development of new therapeutic agents. But we also have learned that overly aggressive management of the ovaries was detrimental to a woman’s reproductive system. As a result, at present, the way we manage COH is completely different from 40 years ago. The way we monitor patients and perform oocyte retrievals has also changed—the development of vaginal ultrasound in the 1980s has been a fundamental pillar in the advancement of IVF.
Arguably the most important advance since 1978 in the trajectory of IVF was the ability to insert a single sperm into an ovum and achieve a live birth. Intracytoplasmic sperm injection (ICSI) made possible biological parenthood when only a few sperm were available in the ejaculate, and ultimately the previously unthinkable source of sperm, the testis. But this technique raised its own questions: which single sperm should one choose, how, and why? We continue to search for answers to these fundamental challenges that this remarkable reproductive tool engendered.
Of course IVF did not just fundamentally change reproductive possibilities for a mother, it did so for the father as well. From evaluating male reproductive potential to therapy, IVF opened avenues of investigation, created dilemmas, and opened doors previously unimagined. With the ability to use gametes obtained directly from the testis, we now can truly probe medical therapies as we enter an age of controlled spermatogenic stimulation.
We conclude with the big picture, how IVF created legal and ethical challenges, and how mental health and psychology play central roles in this new world of reproductive technology. Finally, luminaries in our reproductive medical societies, our places of vigorous interaction in moving our field forward, describe how they came to be and how they are propelling us into a better future.
We hope that you enjoy reading this monograph as much as we enjoyed making it, and that wherever you are in the arc of reproductive medical history, it will give you insights into the remarkable field that gives birth to the unborn.
Advances in the IVF Laboratory
How the Embryology Laboratory has Changed!
Jacques Cohen, Ph.D., H.C.L.D.
The human assisted reproduction laboratory has undergone near-complete metamorphosis since the early days of IVF (
). The increasing success rates with this technology are owed in large part to these changes, including improvements in quality control and embryo culture systems. Equally important have been the advances in personnel training and exchange of ideas and communication, which is not surprising as IVF matured during the information age. The first IVF laboratories were essentially improvised, sometimes fitted into existing surgery suites. Before 1985, there were no “add-on” procedures, no egg donation or surgical sperm retrievals, and no cryopreservation or micromanipulation. At that time, there was little governmental oversight with few licensing requirements. There was no specialized clinical or laboratory training, therefore, skills were acquired mostly through apprenticeships. The procedures in the laboratory were performed by researchers with experience in experimental embryology and veterinary science. These pioneers were establishing rules and principles, which evolved into international guidelines and standard operating procedures.
As astounding as the relatively quick rise of IVF may have been, 40 years ago IVF was rarely viewed as an obvious treatment for subfertility. Despite numerous obstacles—most important, a lack of public funding—assisted reproduction is now well established. Its path seems to follow Moore’s Law with linear increases in implantation rates, being just shy of 1% annually, corrected for maternal age (
). This linear progression has been relatively constant since the early days and may predict a time in the near future when there is no need for multiple attempts at pregnancy. It seems that IVF will become an obvious form of safe reproduction, the means to avoid deleterious mutations, inheritable or de novo, and to allow prospective parents to build their families with forethought and deliberation. This future is possible because of initiatives that started in the laboratory in 1978.
Before IVF was accepted as standard treatment for infertility, there was little to no concern about the laboratory environment, from cleanliness of work surface areas to air quality in the confines of the laboratory. Although not typical, one did come across improvised laboratories where staff was allowed to smoke and even enjoy a meal in between cases! The first dedicated clinical suite and laboratory to exclusively perform IVF was built in Cambridgeshire, United Kingdom, in the village of Bourn in 1980 by IVF pioneers Robert G. Edwards (scientific director) and Patrick Steptoe (medical director) and their senior team, which included Jean Purdy (laboratory quality control manager), John Webster (senior consultant), and Alan Dexter (financial director). The move to this private setting represented extraordinary courage, as it had taken hundreds of attempts to achieve two births before the planning of this dedicated facility (
). Other clinics were opened as well, at the Royal Women’s Hospital in Australia (Alex Lopata and Ian Johnston) and at Monash University (Carl Wood and Alan Trounson), both in Melbourne, with some government financial support, and in London, United Kingdom (Ian Craft) using private funding. At the Eastern Virginia Medical School in Norfolk, Virginia (USA), pioneers Howard and Georgiana Jones opened the first US-based facility using funds provided by the university. Other countries, such as India, Austria, France, Holland, Sweden, and Spain, followed swiftly and established their own clinics. By 1985, a new discipline was emerging, a field that was for the first time referred to as assisted reproductive technology or ART.
Laboratory equipment and instrumentation: from bell jars to time-lapse incubators
Just as formulation of the cell theory was intricately linked to the development of the microscope, IVF and its associated technologies have relied on engineering efforts by many key individuals who have played important roles but unfortunately are rarely remembered. For instance, in 1850, John Lawrence Smith, a faculty member at what is now Tulane University (New Orleans, LA, USA), engineered the inverted microscope. Robert Chambers from New York University (USA) invented the first micromanipulator for cell microsurgery in 1912. The first incubators were used for hatching chicken eggs and date back to ancient Egypt. In the 19th century, this changed to heated bell jars. Carbon dioxide incubators date from the 1960s, and warm-jacketed incubators were developed in the 1970s.
In vitro fertilization-specific instrumentation began to be introduced in the late 1980s, and this process is ongoing with many companies now specializing in the area. The pioneering laboratories relied completely on equipment and materials that were designed for somatic cell tissue culture and not human (or mammalian) gametes and embryos. This is illustrated by the presentations and discussions of the first international group of IVF clinicians and biologists, to convene at Bourn Hall in 1981, to discuss the emerging IVF technology. The 26 attendants came from Basil (Switzerland), Cambridge (United Kingdom), Gothenburg (Germany), Kiel (Germany), Manchester (United Kingdom), Melbourne (Australia), Norfolk, Virginia (USA), Paris (France), and Vienna (Austria). In her chapter on methods of fertilization and embryo culture in vitro in the proceedings of this first conference on clinical IVF, Jean Purdy wrote that, “The equipment needed in a tissue-culture laboratory has been described extensively by Paul (1970)” (
). This was reflective of a conspicuous lack of specialized equipment and disposables for IVF. Egg collection kits and ET catheters, as well as a pump that allowed gentle aspiration of follicular fluid (FF) from ovarian follicles were among the first IVF-specific instruments/devices to be developed. For the laboratory, laminar flow workstations equipped with heated surfaces were engineered. A benchtop incubator was later invented by David Mortimer and colleagues in Australia as an alternative to “big box” incubators to provide a more stable and controlled culture environment. Incubators have continued to evolve and improve and the present-day embryologists not only culture embryos for longer periods of time, and with more confidence, they can also watch development frame by frame thanks to incorporation of time-lapse microscopy into incubation systems. Close observation of embryos through superior microscopy has contributed to an understanding of the morphology and timing of developmental events and the ability, albeit with limitations, to select/deselect embryos for transfer and cryopreservation.
Culture media and culture systems: from simple salt solutions to complex optimized culture media
Tissue culture media were first developed nearly 150 years ago by Ludwig and Ringer. These were simple salt solutions, which were based on the properties of serum/blood plasma. Knowledge was gained during those years about the biochemistry of metabolism in mammals and humans, particularly osmolarity, pH, and temperature. This knowledge provided the basis for modifications to the simple salt solutions and successive generations of culture media, which were considerably more complex. The second generation of culture media was developed in the 1970s, mimicking the female reproductive tract environment. This was followed by a third generation of media, which was designed to optimize growth in vitro, to some extent ignoring existing formulations and the “back to nature” principle behind those formulations. To formulate these new media, the performance of each ingredient was evaluated separately using a “simplex optimization” process. This was first developed for mouse embryo culture by Professor John Biggers at Harvard University. His work was supported by the US National Institutes of Health, in part aiming to develop culture media for use in the human while circumventing the moratorium still in place on human embryo research. Variations on the second-generation media, called sequential and third generation simplex optimized-derived media are still in use at present and seem equally effective in supporting development of human embryos through 6 or 7 days of culture in vitro. The departure from home-brew culture media and the acceptance of commercially manufactured media, which is strictly regulated by governmental agencies, very likely has contributed to improvements in laboratory and clinical outcomes by virtually eliminating inconsistencies, manufacturing errors, and batch-to-batch variability.
Culture systems have evolved too. In the 1930s, Carrell flasks (Gregory Pincus) were used to culture embryos or perform in vitro insemination. In the 1950s, experimental embryologists like John Hammond and Wesley Whitten switched to test tubes. In 1963, Ralph Brinster introduced the culture of eggs and embryos in small droplets of culture medium under a layer of paraffin oil (
). With some modifications, this ingenious “micro-drop” method using the Petri dish has become the most widely used and successful system for culture of mammalian embryos in vitro. Brinster’s contributions were for a long time unappreciated by human IVF specialists; early on, nearly all practitioners used either organ culture dishes or small test tubes for culture of human gametes and embryos. But eventually, by the mid-1990s, most IVF laboratories adopted the “closed” under-oil culture system. There is little doubt that this system has provided a better, more stable environment and significant advantages for embryonic growth, which were lacking in the open culture systems of the past. This in turn has led to increased efficiency and efficacy of embryo culture.
Staffing the IVF laboratory: from experimental to clinical embryologists
Clinical embryology did not evolve in a vacuum but against a backdrop of a 150-year history in experimental embryology, cryobiology, and other related fields. When Bourn Hall Clinic opened in 1980, Jean Purdy hired two laboratory assistants who worked as quality control technicians. They handled the Petri dishes, completed the paperwork, and witnessed procedures. The clinical embryologists, on the other hand, were charged with the handling of gametes (including sperm preparation) and embryos and communication with patients. The pioneering embryologists were often involved in optimizing follicular recruitment protocols and timing of egg retrievals. By 1983, when Louise Brown turned 5 years, worldwide fewer than 100 embryologists were involved in clinical work. They experienced a very different work environment than exists at present—so much was unknown and there was little guidance. At present, aspiring embryologists can participate in Master of Embryology programs, and they can get training in specific technologies and techniques through courses, which provide hands-on experience. Embryologists and trainees can reinforce and expand their theoretical knowledge through a rich literature of thousands of articles, dozens of textbooks and “how-to” books, and attend one of the many specialized workshops organized annually around the world. They can watch videos on the Internet or participate in web-based journal clubs and discussion groups. In some countries, embryology directors and managers must attain postgraduate credits to keep qualification standards and their laboratories must be audited and certified. These new opportunities have provided for an expansion of the workforce and the possibility to more appropriately staff ART laboratories. Proper staffing itself is a significant contributor to improved safety and better outcomes (
).
The expanding horizons of IVF: from partial zona dissection and subzonal insertion of sperm to intracytoplasmic sperm injection, slow freezing to vitrification, morphology to morphokinetics and genetics
During the years, the IVF laboratory has incorporated many transformative technologies that have continued to be refined and perfected. Most important have been oocyte and embryo cryopreservation; assisted fertilization for treatment of male factor infertility; genetic diagnosis of embryos before transfer; and development of new embryo selection methodologies and platforms, including embryo morphokinetics using time-lapse microscopy.
Cryopreservation
Chris Polge and co-workers were the first to deep-freeze mammalian spermatozoa in 1949. Human spermatozoa were frozen in Iowa (USA) a few years later, by Raymond Bunge and Jerome Sherman. In 1971, David Whittingham, Stanley Leibo, and Peter Mazur changed the field of embryology by freezing cleavage-stage mouse embryos (
). In the early 1980s, in relatively quick succession, the human embryo was cryopreserved at all embryonic stages from the zygote to the hatched blastocyst, with minor adaptations, but survival rates remained <80% for many years. The effort was well founded in science, as basic scientists working with rodents and farm animals had already mastered the technology years earlier. The past 10 years have seen a dramatic improvement in oocyte and embryo cryopreservation with the introduction/application of vitrification. With survival rates of nearly 100%, treatment approaches that involve cryopreservation of all embryos and oocytes, have become more viable, revealing clinical advantages of delayed transfer in natural rather than stimulated cycles.
Assisted fertilization
Micromanipulation to assist fertilization was first applied successfully in 1986 in the mouse through zona drilling before insemination. During this procedure, an acidified Tyrode's solution was expelled onto the zona by a micromanipulator to cause focal zona dissolution and allowing spermatozoa easier access to the oolemma (
). In 1988, babies were born to couples with male factor infertility after application of a mechanical form of zona drilling (
). These were the first pregnancies in the human established with the micromanipulator as a surgical tool. That same year, teams in Singapore and Rome (Italy) reported on injection of spermatozoa into the perivitelline space. Although both methods improved the prospects for treatment of more extreme forms of male factor infertility, fertilization rates were low due to the absence of a quick block to polyspermy on the membrane level. However, once monospermic fertilization was achieved, implantation rates were as high or higher than after standard insemination. Assisted fertilization improved dramatically with the introduction of intracytoplasmic sperm injection (ICSI) by a team in Brussels (Belgium) (
). One important factor that led to this success was the design of the injection tool (P. Devroey, personal communication). This needle was very thin, sharp, and straight unlike the tools that were developed earlier. The design allowed nontraumatic piercing of the membrane and placement of spermatozoa in the cytoplasm with precision. It was quickly demonstrated that fertilization rates were as high as conventional IVF even in the most severe of male factor cases. At present, ICSI is being used with increasing frequency, in some cases replacing standard insemination altogether. Although successful in terms of pregnancy, live birth rates, the wisdom of nondiscriminant application of ICSI when it is not indicated continues to be debated.
Preimplantation genetic testing
Successful genetic diagnosis of single gene defects through blastomere biopsy, or preimplantation genetic testing (PGT), was first reported by Alan Handyside (
) and the team at Hammersmith Hospital in London in 1989. Basic science had again led the way in this case, after Richard Gardner, while in the Cambridge laboratories of Bob Edwards, showed in 1968 that trophectoderm biopsy and sexing of the rabbit embryo was compatible with obtaining live offspring (
). The development of PCR by Gary Mullis and his team in the 1980s were crucial to the success of rapid single cell sequencing, which was a prerequisite for PGT (
). The discovery of the high incidence of chromosomal anomalies in biopsied embryonic cells led to the approach of testing of embryos and selective transfer of only euploid embryos, a variation on PGT, now dubbed PGT-A or PGT for aneuploidy (
). The efficacy of PGT-A (in all its variations) has been the subject of debate for >20 years. Embryo biopsy has shown that manipulation of embryos at a cellular level is not always without harm and optimization of the techniques is much needed. Perhaps this can be achieved using a noninvasive biopsy-free approach? There are also concerns about the high frequency of mosaicism, but PGT has been of tremendous benefit to couples at risk for transmitting genetic disease.
Embryo selection technologies
One of the remaining challenges in the ART laboratory is the development of efficacious (and affordable) embryo selection methods, which would help facilitate routine single ET in all patient groups. Early observers of embryo development in vitro were surprised by the apparent variation, not just between patients, but among embryos of the same patient. In the mouse, small aberrations from the morphological norm were known to significantly reduce implantation, but in the human, embryo morphology and rate of development were only loosely correlated with outcome. The search for important characteristics that could 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. The recent development of time-lapse microscopy has made permanent record-keeping a reality, but reliable embryo selection using a single parameter or algorithm applicable to all patients remains elusive.
Acknowledgments
Mina Alikani is gratefully acknowledged for reading and commenting on the manuscript.
Development of In Vitro Fertilization Culture Media and the Importance of Blastocyst Transfer
David K. Gardner, D.Phil.
Deliberations on culturing the human embryo
Consideration of a cell’s physiology is a logical starting point when trying to culture it. This premise is challenging when considering the preimplantation human embryo, whose physiology and metabolism changes dramatically from fertilization to implantation. The fertilized oocyte and cleavage-stage embryo are relatively quiescent, exhibit limited oxidative capacity, and use carboxylic acids predominantly. In contrast, the blastocyst is one of the most active tissues in the body, characterized by exponential growth, expansion of a blastocoel, increased oxygen use and oxidative metabolism, a large demand for glucose (largely to support biosynthetic requirements), and a somewhat paradoxical production of lactate through aerobic glycolysis (thought to be key in embryonic signaling with the endometrium) (Fig. 1). The dynamics and complexities of preimplantation embryo physiology, therefore, help to explain the long and complex road to blastocyst transfer.
