The dawn of the future: 30 years from the first biopsy of a human embryo. The detailed history of an ongoing revolution.

Following early studies showing no adverse effects, cleavage stage biopsy by zona drilling using acid Tyrode's solution, and removal of single blastomeres for preimplantation genetic testing (PGT) and identification of sex in couples at risk of X-linked disease, was performed by Handyside and colleagues in late 1989, and pregnancies reported in 1990. This method was later used for specific diagnosis of monogenic conditions, and a few years later also for chromosomal structural and/or numerical impairments, thereby establishing a valuable alternative option to prenatal diagnosis. This revolutionary approach in clinical embryology spread worldwide, and several other embryo biopsy strategies developed over three decades in a process that is still ongoing. The rationale of this narrative review is to outline the different biopsy approaches implemented across the years in the workflow of the IVF clinics that provided PGT: their establishment, the first clinical experiences, their downsides, evolution, improvement and standardization. The history ends with a glimpse of the future: minimally/non-invasive PGT and experimental embryo micromanipulation protocols. This grand theme review outlines a timeline of the evolution of embryo biopsy protocols, whose implementation is increasing worldwide together with the increasing application of PGT techniques in IVF. It represents a vade mecum especially for the past, present and upcoming operators and experts in this field to (re)live this history from its dawn to its most likely future.

40 years of bovine IVF in the new genomic selection context.

The development of a complex technology such as in vitro fertilization (IVF) requires years of experimentation, sometimes comparing several species to learn how to create the right in vitro environment for oocytes, spermatozoa and early embryos. At the same time, individual species characteristics such as gamete physiology and gamete interaction are recently evolved traits and must be analysed within the context of each species. In the last 40 years since the birth of Louise Brown, IVF techniques progressed and are now used in multiple domestic and non-domestic animal species around the world. This does not mean that the technology is completely matured or satisfactory; a number of problems remain to be solved and several procedures still need to be optimized. The development of IVF in cattle is particularly interesting since agriculture practices permitted the commercial development of the procedure and it is now used at a scale comparable to human IVF (millions of newborns). The genomic selection of young animals or even embryos combined with sexing and freezing technologies is driving a new era of IVF in the dairy sector. The time has come for a retrospective analysis of the success and pitfalls of the last 40 years of bovine IVF and for the description of the challenges to overcome in the years to come.

Preimplantation genetic screening.

Preimplantation genetic diagnosis was first successfully performed in 1989 as an alternative to prenatal diagnosis for couples at risk of transmitting a genetic or chromosomal abnormality, such as cystic fibrosis, to their child. From embryos generated in vitro, biopsied cells are genetically tested. From the mid-1990s, this technology has been employed as an embryo selection tool for patients undergoing in vitro fertilisation, screening as many chromosomes as possible, in the hope that selecting chromosomally normal embryos will lead to higher implantation and decreased miscarriage rates. This procedure, preimplantation genetic screening, was initially performed using fluorescent in situ hybridisation, but 11 randomised controlled trials of screening using this technique showed no improvement in in vitro fertilisation delivery rates. Progress in genetic testing has led to the introduction of array comparative genomic hybridisation, quantitative polymerase chain reaction, and next generation sequencing for preimplantation genetic screening, and three small randomised controlled trials of preimplantation genetic screening using these new techniques indicate a modest benefit. Other trials are still in progress but, regardless of their results, preimplantation genetic screening is now being offered globally. In the near future, it is likely that sequencing will be used to screen the full genetic code of the embryo.

The Past, Present, and Future of Preimplantation Genetic Testing.

Preimplantation genetic testing (PGT) of oocytes and embryos is the earliest form of prenatal testing. PGT requires in vitro fertilization for embryo creation. In the past 25 years, the use of PGT has increased dramatically. The indications of PGT include identification of embryos harboring single-gene disorders, chromosomal structural abnormalities, chromosomal numeric abnormalities, and mitochondrial disorders; gender selection; and identifying unaffected, HLA-matched embryos to permit the creation of a savior sibling. PGT is not without risks, limitations, or ethical controversies. This review discusses the techniques and clinical applications of different forms of PGT and the debate surrounding its associated uncertainty and expanded use.

Oocyte developmental competence and embryo development: impact of lifestyle and environmental risk factors.

Oocyte development is the end result of a sophisticated biological process that is hormonally regulated and produced by highly specialized cellular lines that differentiate in early embryo/fetal development. Embryo development is initially regulated by maternal transcripts until replaced by embryonic genomic expression. Then, an assortment of hormones and local environmental factors in various concentrations along the reproductive tract (e.g. fallopian tube, endometrial lining) provide the protection, nutrients and means of communication for the embryo to implant and develop. Both oocytes and embryos are susceptible to environmental, occupational and lifestyle exposures that can exert direct toxic effects and disrupt hormones. While some exposures may produce reversible changes, others, especially those damaging germinal cells in utero or during prepuberty, may result in permanent sequelae that continue in future generations. This article reviews the main factors that affect female fertility and their possible influence on human reproduction. Some lifestyles, xeno-oestrogens and heavy metals are already known to compromise female reproductive function. Nonetheless, many questions remain and little is known about the effect of many other factors on female fertility.

The politics of human embryo research and the motivation to achieve PGD.

This article reports a historical study of factors influencing the achievement of clinical preimplantation genetic diagnosis (PGD) in 1990, 22 years after its first demonstration in animals. During the 1970s, research on PGD continued in large farm animals, but serious interest in human PGD was not evident until 1986. First, interest in PGD during the 1970s waned with the advent of prenatal testing, which for gynaecologists was clinically more familiar, technically simpler and ethically less challenging than IVF. Indeed, IVF was viewed with widespread suspicion until the first IVF births in 1978. Second, interest in clinical PGD was stimulated by the UK Parliamentary reaction against human embryo research that greeted the Warnock Report in 1984. This hostility led scientists to initiate a pro-research campaign, further galvanized in 1985 by MP Enoch Powell's bid to ban such research. However, while Powell abhorred embryo research, he approved of PGD, a stance that divided the anti-research lobby. Accordingly, the campaigners for research emphasized that it was needed to achieve PGD. Powell demanded evidence of such projects and PGD research increased from 1986. It is concluded that UK political debates on embryo research played a critical role in stimulating the achievement of clinical PGD. Human pregnancies following preimplantation genetic diagnosis (PGD) for embryo sex were announced in 1990, 22 years after the technique was pioneered in animals. PGD in humans required not only technological advances, such as IVF and sensitive diagnostic tests, but also the motivation to develop and apply them. Our historical analysis shows that, although research on PGD continued in large farm animals during the 1970s, and techniques of the required sensitivity were developed on mouse embryo models, interest in clinical PGD was not evident until 1986. Two factors stimulated this sudden change in motivation. First, interest in PGD was depressed during the 1970s by the advent of prenatal diagnostic techniques, which for gynaecologists were clinically, technically and ethically less challenging than IVF. IVF was then regarded with a suspicion that only started to wane in the early 1980s following the first IVF births. Second, the UK Parliamentary reaction against human embryo research that greeted the Warnock Report in 1984 provided a positive stimulus to clinical PGD by prompting scientists to form a pro-research lobby, which was further galvanized in early 1985 by MP Enoch Powell's almost-successful bid to ban human embryo research. We show that while Powell abhorred embryo research, he approved of PGD, a stance that fractured the unity of the anti-research lobby. Accordingly, the pro-research lobby emphasized that embryo research was needed to achieve PGD. Powell demanded evidence of such projects, thereby, we argue, stimulating PGD research from 1986. Our evidence shows that UK political debates about PGD played a critical role in stimulating the achievement of PGD clinically.

Invited commentary: the politics of human embryo research and the motivation to achieve PGD.

The idea that biomedical research can be influenced by political events implies a teleological basis indicating that scientific achievements occur because there is a political need. Such a concept appears to have been the reason PGD was fast-tracked to emerge as a biomedical achievement well before its due date, occurring at a time when human embryology was still struggling to reach a reasonable level of efficiency and become adopted as a clinically relevant advance around the world. One story underlying the historical achievement of the HFE Act 1990, enabling regulated embryo research, steps outside the firm ground of biomedical science and encourages the idea that Reproductive BioMedicine Online should embrace a further section enabling articles dealing with 'History, politics and personalities' where these influence biomedical research.

Preimplantation genetic diagnosis after 20 years.

Preimplantation genetic diagnosis (PGD) should not be an option only for the few couples at risk of serious genetic conditions who can afford it. We appear to have lost sight of the original driving force behind the development of PGD, which is that most couples who carry a serious genetic disorder find it more acceptable to choose to conceive with healthy embryos tested in-vitro at preimplantation stages of development within the first week following fertilization, even if that means discarding those diagnosed as affected. It has been shown using cystic fibrosis as an example, that the cost savings to the US healthcare system of providing free IVF-PGD to all carrier couples compared to the lifetime costs of medical treatment for patients affected by this disease, run to dozens of billions of dollars. With the increasing emphasis in medicine on early diagnosis and prevention of disease together with the availability of new molecular genetic diagnostic tools, a national IVF-PGD programme seems to be the next step in modern health care.

Preimplantation genetic screening: "established" and ready for prime time?

Unless attempts to improve pregnancy rates and/or diminish miscarriage rates through preimplantation genetic screening (PGS) are applied to only carefully selected patients, they will fail. Because specific PGS indications have remained undefined, PGS should be considered an experimental procedure.

Ethics and moral philosophy in the initiation of IVF, preimplantation diagnosis and stem cells.

Details of the work leading to the introduction of human IVF, animal and human stem cells, and the preimplantation diagnosis of inherited characteristics in blastocysts are outlined briefly in this paper. The progress of these studies is related to ethical issues emerging during these years. The current status of these studies is outlined, together with a brief moral philosophy as practised by the original investigators.

Changing genetic world of IVF, stem cells and PGD. A. Early methods in research.

Genetics proved essential to introduce IVF, preimplantation diagnosis (PGD) and embryo stem cells in the 1960s. Its small input in early years was confined to aspects such as timing follicle growth and ovulation. Modest understanding in the mid- to late 1980s, mostly on studies in mice, involved the actions of single genes and the balance between maternal and zygotic transcripts in preimplantation stages. Human IVF began after human oocytes were matured in vitro, and their meiotic chromosomes analysed. Their fertilization in vitro led to PGD and embryo stem cells. Unlike mouse embryos, most human embryos failed to implant, so the best had to be selected to improve IVF pregnancy rates. Initially, faster-growing embryos proved superior. Later, patterns of polarized nucleoli in pronuclei, the degree of blastomere fragmentation and growth of embryos in vitro to blastocysts provided excellent markers. Single cells could be isolated from embryos using micromanipulation. Stem cells from inner cell mass, a branch of IVF, differentiated into immortal stem cell lines in vitro if disaggregated. They formed virtually all body tissues in blastocysts cultured intact or when injected singly into recipient blastocysts. Later, the genetic controls of ES cell differentiation were assessed, together with factors switching them along specific differentiation pathways. Marker genes identified ES cells differentiating into various tissues.

Changing genetic world of IVF, stem cells and PGD. B. Polarities and gene expression in differentiating embryo cells and stem cells.

Novel genetic techniques in the later twentieth century led to new analytical methods for assessing the growth of embryos and stem cells and improve preimplantation diagnosis. Increasing attention to the nature of polarities in mouse and human embryos revealed the existence of an animal-vegetal axis in human oocytes and embryos. Combinations of meridional and transverse cleavage divisions, the latter due to spindle rotation, determined the unequal division of ooplasm to embryonic blastomeres. Blastomeres with differing functions were accordingly formed in 4-cell embryos, including founders of inner cell mass and trophectoderm. New forms of gene analysis led to the polymerase chain reaction, while fluorescence in-situ hybridization revealed astonishingly high degrees of heteroploidy in human embryos. Developmental genetics gained immense analytical power as cDNA libraries, microarrays, transcriptomes RNAi and other methods clarified the roles of hundreds of genes in pre- and early post-implantation embryos and stem cells.