Preimplantation Genetic Diagnosis in Assisted Reproductive Technology



Preimplantation Genetic Diagnosis in Assisted Reproductive Technology


Nanette Santoro

Thanh Ha Luu

Liesl Nel-Themaat






History of PGT

Although the first clinical utilization of PGT was published in 1990,1 the idea was first presented in 1937.2 It is widely believed that it was Dr John Rock who became known for the first experiments in human IVF during the mid-1940s,3 who proposed in an unauthored editorial in the New England Journal of Medicine that one day parents may have children “according to specification.”2 To grasp how prescient this thinking was, one has to consider that, at the time of this prediction, the structure of DNA was not to be published for another 16 years,4 DNA sequencing5 and the first human IVF birth6 would be reported more than 40 years later, and the polymerase chain reaction (PCR)7 and fluorescent in situ hybridization (FISH)8 were only to be discovered about 50 years afterward. Both these technologies were instrumental in the evolution of PGT as we know it today.

With significant success in mammalian embryo culture procedures during the second half of the 20th century, ancillary embryo technologies started emerging, including embryo biopsy and fluorescent staining techniques, which were used in the first sexing of preimplantation embryos by Robert Edwards and Richard Gardner in 1967.9 Rabbit blastocysts produced in vivo were biopsied and the trophectoderm assessed for sex chromatin using fluorescent microscopy. The sex of resultant fetuses correlated with the determined sex of the biopsied embryos, confirming the effectiveness of the procedure. A few years later, Whittingham et al reported the first live birth from frozen-thawed embryos in mice,10 followed by the first human
pregnancy from frozen-thawed embryo transfer in 1983.11 This discovery became especially significant in the 21st century when off-site reference laboratories started doing the majority of genetic testing for clinical laboratories, necessitating cryopreservation of embryos until results became available, after which the appropriate embryos could be thawed and transferred.

Around the same time, Angell et al started analyzing the chromosomes of cleavage-stage human embryos using classic cytology fixation and staining techniques. The authors found evidence of nondisjunction, resulting in an array of chromosomal aberrations in individual blastomeres.12,13 One of the observations from these experiments was a lack of morphological correlations between euploid and aneuploid embryos, which further highlighted the need for developing an effective selection method based on ploidy. In 1987, an expert meeting held at the Ciba Foundation concluded that collaborative research would be essential to develop PGT as a clinical management alternative to abortion of genetically abnormal fetuses following prenatal testing.14

The remainder of the century saw numerous advancements, including the discovery that embryonic genome activation in humans occurs between the 4- and 8-cell stage,15 the development of cleavage-stage biopsy techniques,16,17 the first true PGT for a specific mutation in the mouse model,18 and development of the polar body biopsy technique.19 The first reported human pregnancies following embryo sex selection were in 1990, when cleavage-stage single blastomeres were sexed using PCR to amplify the DNA of the Y chromosome in patients with X-linked defects.1 Transfer of embryos deselected for the Y chromosome resulted in female-only pregnancies. Shortly after, normal births following screening of embryos for cystic fibrosis were published20 using similar techniques. Identification of specific diseases by genetic testing was subsequently coined PGD, for preimplantation diagnosis. Today it is known as preimplantation genetic testing for monogenic gene disorders (PGT-M).

Toward the turn of the millennium, improved embryo culture systems allowed blastocyst culture21 and trophectoderm biopsy. Human blastocyst biopsy was shown to be a safe alternative to cleavage-stage biopsy, causing IVF programs to switch over.22 Trophectoderm biopsy allows for a larger sample of cells and is, thus, more representative of the entire embryo, thereby circumventing many concerns raised regarding cleavage-stage biopsy (for review, see references 23,24,25).

On the molecular genetics side, new technologies continued to provide faster and more accurate diagnoses. In 1994, Munne et al described successful FISH analysis for selection of embryos based on the chromosomes X, Y, 13, 18, and 21.26,27 Many clinical IVF laboratories thus started routinely performing FISH for what was subsequently called preimplantation genetic screening and today is known as preimplantation genetic testing for aneuploidy (PGT-A). FISH PGT, however, has several key drawbacks: it requires cleavage-stage blastomere biopsy, it is a highly technical procedure, and it can only provide information on a limited number of chromosomes. Whole genome amplification of single blastomeres28 paired with DNA array-based comparative genomic hybridization29 and single-nucleotide polymorphism (SNP) microarray30 soon became the standard testing platforms, each with its unique benefits and drawbacks (see Testing Platforms section and Table 16.1). Scientists continued their pursuit of a more accurate, higher resolution platform, and in 2012, Treff et al published development of a quantitative real-time PCR method for identifying aneuploidy in blastocysts. The most recent technology to enter the PGT market (and quickly becoming the most widely used) is next-generation sequencing (NGS), which provides the highest resolution and most diagnostic power currently available.31

The use of PGT in IVF cycles has been steadily increasing to include almost 40% of all IVF cycles reported to the Society for Reproductive Technology (SART) in 2017 (www.sart.org). Therefore, it is imperative that the clinician has a sound understanding of the available options to best serve their patients’ genetic testing needs. PGT-A is more broadly utilized to optimize pregnancy outcomes with single-embryo transfers, in cases of advanced maternal age or recurrent implantation failure. PGT-A will also provide sex chromosome information and, thus, can be used for gender selection for disease risk stratification or family balancing goals. For the couple in Case 1, PGT-A will allow for the selection of only euploid embryos for transfer. This selection then permits the transfer of a single embryo, a strategy that has

contributed to the declining rate of triplet pregnancies due to assisted reproductive technology (ART) in the United States.32







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Jun 19, 2022 | Posted by in OBSTETRICS | Comments Off on Preimplantation Genetic Diagnosis in Assisted Reproductive Technology
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