A Brief History of Preimplantation Genetic Diagnosis and Preimplantation Genetic Screening

Jason Franasiak, MD, FACOG
Richard T. Scott, Jr, MD, FACOG, HCLD
Reproductive Medicine Associates of New Jersey
Rutgers, Robert Wood Johnson Medical School

Introduction

The development and enhancement of in vitro fertilization (IVF) over the last 35 years has resulted in dramatic improvements in the treatment of infertility. At present, IVF provides both the most successful and often the most cost effective approach to the care of most infertile couples [1][2].

In the short time since the first IVF birth in 1978 [3], our greater understanding of embryo development had allowed for the development of new technologies which can be implemented to enhance embryo selection. The primary goal is to distinguishing those embryos that are reproductively competent and are capable of producing a healthy child from those that cannot. The drive to select healthy embryos and avoid failed pregnancy attempts, miscarriages, and the need for pregnancy termination led to the first applications of preimplantation genetic diagnosis (PGD) in 1990 [4].

The concept of PGD is not a new one. Indeed, in a 1937 manuscript in the New England Journal of Medicine, Dr. John Rock predicted that human IVF, gender selection, and gestational carriers would be utilized in reproductive science [5]. He stated that one day science will allow for parents to obtain sons or daughters "according to specification", foreshadowing the ability to screen out detrimental disease states with PGD.

The first description of PGD came years later published by the great Dr. Robert Edwards and Dr. Richard Gardner in a 1967 Nature manuscript. In it he described the use of PGD for sexing of rabbit blastocysts [6]. Further work in animal models continued, including Marilyn Monk's work in 1987 demonstrating PGD in a murine model for Lesch-Nyan syndrome [7]. The development and implementation of several techniques in human embryology were instrumental in the application of PGD in human reproduction. First, Leeanda Wilton pioneered the cleavage stage biopsy in 1986. Then, in 1988, two additional approaches to obtaining genetic material from embryos were described with Yuri Verlinsky describing polar body biopsy and Audrey Muggleton-Harris describing trophectoderm biopsy.


The first applications for PGD came in testing monogenic disorders and sex-linked disorders. This was made possible by Elana Kontogianni's work in 1989 which showed PCR for the Y chromosome was possible from a blastomere. Focusing on X-chromosome linked diseases, amplification and detection of Y-chromosome specific repeat sequences allowed for selection of embryos that were female and thus not at risk of carrying the disease. These early approaches gave way to technologies that allowed for the detection of gene mutations on autosomes and sex chromosomes enabling clinicians to select embryos that do not harbor the mutation for embryo transfer.


The success of PGD to predict embryos which did not have genetic disease led to attempts to apply the technology more widely as a selective tool to all embryos in a particular cohort and identify those embryos with normal chromosome complements and thus a higher chance of success on a per cycle basis [8–16]. This practice became known as preimplantation genetic screening (PGS). A prominent goal in this case was to decrease the reliance on high embryo transfer order to achieve high success rates. This is an important given that the practice vastly increases the prevalence of multiple gestations, which are associated with high maternal and neonatal morbidity as compared to singletons [12].


The contribution of embryonic aneuploidy to the inefficiency of human reproduction is well established [17–19] and it seemed intuitive that assessment of the ploidy status of each embryo within the developing cohort would allow selection of only euploid embryos and would ultimately improve IVF outcomes [20]. While this premise was always valid, early attempts at embryonic aneuploidy screening were suboptimal [21,22]. The early techniques entailed molecular analysis that lacked sufficient precision to be clinically meaningful. More recently, application of newer and more powerful molecular technologies have overcome some of the early limits and produced meaningful improvements in clinical outcomes.

Applications for Genetic Testing of Embryos

The impetus to develop PGD in the clinical realm was to identify only unaffected children prior to implantation and thus eliminate the need for pregnancy termination after a diagnosis was made at a later time in the pregnancy. The first clinical application of preimplantation genetic testing was published in 1990 by Handyside et. al. detailing two couples at risk for transmission of X-linked mental retardation and anderoleukodystrophy [4]. Analysis of a blastomere at the 6-8 cell stage with polymerase chain reaction (PCR) analysis which amplified a Y chromosome specific repeat sequence allowed for transfer of female embryos.


While PGD was initially applied to a small subset of disorders with a high likelihood (25-50%) of being present, over the subsequent 15 years its use expanded. Testing for genetic disorders with low penetrance and late-onset became more common and the list of disorders tested expanded to include over 100 conditions, although the most frequent were cystic fibrosis and hemoglobinopathies [23].


The circumstances under which PGD is now utilized include an extensive list of sex-linked and autosomal single gene disorders, HLA typing, and translocations. This expansion is due in part to the way in which patients eligible for PGD are identified. Originally, couples were identified due to a history of a poor pregnancy outcome or a strong family history of disease. Now, expanded carrier screening has become more widely utilized and in many circumstances allows for detection of a transmissible genetic anomaly before it has been phenotypically apparent in patients.


Aneuploidy Screening

Utilization of PGS for aneuploidy screening was initially borne out of a desire to improve pregnancy rates in patients with advanced reproductive age. While antenatal diagnosis for aneuploidy was utilized to decrease the live birth rates of fetuses with an extra chromosome 21, 18, or 13, PGS was meant to look more widely at chromosomes to better select competent embryos and improve reproductive success for patients undergoing IVF on a per cycle basis.


The first papers evaluating human embryo chromosomes were published by Angell et. al. in 1983. They evaluated 3 8-cell embryos with 11 metaphase spreads and discovered that 2/3 of them were aneuploidy [24]. This led them the conclusion that these anomalies were the type "in early embryonic loss and probably contribute to the high failure rate after embryo transfer." This led to further work in 1986 by Angell et. all, where evidence was found of nondisjunction, resulting in trisomy, monosomy, and nullosomy; structural abnormalities; haploidy; and triploidy [25]. Additionally, they noted, importantly, that these lethal chromosome complements could not be distinguished morphologically from those with normal chromosome complements. Thus began attempts to accurately count chromosomes for the purpose of PGS that would span the subsequent decades.


FISH was initially employed with various combinations of chromosomes. However, this method was always limited by the inability to simultaneously screen for all 24 chromosomes [22]. FISH typically screened the seven chromosomes most frequently seen in miscarriage specimens (chromosomes 13, 16, 18, 21, 22, X and Y) analyzing only one or two blastomeres [26,27]. Five trials examining the impact of chromosomal screening in patients with advanced maternal age [26–28] and four trials in relatively good prognosis patients failed to show benefit when screening with FISH [29–32]. This is due in part to the limited interrogation of chromosomes and in part to the technical challenges associated with FISH for PGS.


The development of technologies for single cell whole genome amplification (WGA) allowed for analysis of all 24 chromosomes [33–35]. The first platform characterized was metaphase comparative genomic hybridization (mCGH) by Wells et. al. [36]. The mCGH proved to be quite time consuming and many other platforms utilizing WGA have evolved at a rapid pace in the past several years including, array CGH (aCGH) [37,38,16], single nucleotide polymorphism (SNP) arrays [8,39,40], oligonucleotide CGH [41] and more recently next generation sequencing (NGS) [42]. An additional method, which enables 24 chromosome evaluation without requiring whole genome amplification, is quantitative real time (qPCR) [43].
These methods which interrogate all 24 chromosomes have resulted in improvement in implantation rates and live birth rates, decreased miscarriage rate, and has changed practice patterns to allow for elective single embryo transfer without sacrifice of high success rates for patients.

Summary

Since PGD's inception in 1990, the utilization worldwide has increased and indications for its use have expanded. Originally, the goal of PGD was to detect and eliminate embryos that contained monogenic sex-liked disorders. The development of PGS followed with the original goal of increasing pregnancy success rates on a per cycle basis. Both PGD and PGS now have expanded roles in reproductive medicine. PGD allows for the identification of numerous autosomal and sex-linked disorders, many of which are now identified through expanded carrier screening. PGS allows for not only improved success on a per cycle basis, but also empowers reduced transfer order while maintaining high success rates. This reduced transfer order without sacrificing outcomes allows for a significant reduction of multiple gestations, which improves obstetric and neonatal outcomes and reduce the cost of care.

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