Preimplantation genetic diagnosis: Its time is now

July 8, 2016

Indications for PGD have extended far beyond single-gene disorders.




Dr Simpson, a member of the Contemporary OB/GYN editorial board, is Executive Associate Dean for Academic Affairs and Professor of Obstetrics and Gynecology and Human and Molecular Genetics at Florida International University College of Medicine, Miami.

Preimplantation genetic diagnosis (PGD) is 25 years old. Its technology and novel indications have long dazzled, but some continued to feel that the field was just boutique medicine. In 2016 we can now confidently state that PGD is an integral part of not only medical genetics but also, increasingly, reproductive medicine and infertility care. Evolving technology has welded pragmatism with vision. Here I review developments that have made this transition feasible.

Obtaining DNA through trophectoderm biopsy

There have always been 3 sources for obtaining embryonic DNA for PGD: (1) polar body biopsy, prior to or at the time of fertilization, (2) blastomere biopsy from the 3-day 6- to 8-cell cleaving embryo, and (3) trophectoderm biopsy from the 5- to 6-day blastocyst. Initial work in PGD involved removal of a single blastomere, the zona pellucida traversed by mechanical or laser means.

Today, the preferred approach for PGD involves biopsy of the trophectoderm in the 5- to 6-day blastocyst, since more than a single cell can be safely removed, and thus more DNA is available. A further advantage is that the trophectoderm forms the placenta; thus, the 5–10 cells removed were never destined to be part of the embryo itself (inner cell mass).

PGD for single-gene disorders

When first performed, the intention of PGD was to diagnose single-gene disorders. Approximately 20% of PGD cases are now performed on couples at risk for one or more single-gene disorders. Despite the miniscule amount of DNA, PGD can be performed whenever the chromosomal location of the gene causing the disorder is known. The causative mutation need not even be known so long as affected and unaffected family members are available to determine markers linked to the mutation site. This allows one to deduce whether a given embryo has or has not inherited the mutation.


More than 300 different conditions have been tested worldwide. The most frequent are hemoglobinopathies, cystic fibrosis, and fragile X syndrome. In 2015, Rechitsky and colleagues tabulated their experience with single-gene disorders at Reproductive Genetic Innovations (RGI): 2982 cycles involving 1685 patients.1 This yielded 1095 pregnancies and 1118 live births; 47 pregnancies were ongoing at the time of publication.

PGD has certain advantages over traditional PGD. PGD cases can obviously detect abnormalities much earlier than can chorionic villus sampling and amniocentesis, thus avoiding termination of a clinical pregnancy. PGD is also the only practical approach for a couple in which one partner is at risk for an adult-onset disorder, wishes to remain unaware of his or her genotype, and yet does not want to transmit a potentially serious mutation to his or her offspring. Multiple embryos can be screened with only unaffected embryos transferred; the patient can, if desired, remain oblivious to diagnostic results and thus whether he/she is or is not destined to develop the disorder (nondisclosure PGD). The most common indications for nondisclosure PGD are adult-onset cancers, Huntington’s disease, and autosomal-dominant early-onset Alzheimer’s disease.

A third indication pragmatically necessitating single-gene PGD occurs when a couple is at risk for offspring with a genetic disorder involving bone marrow derivatives (eg, β-thalassemia). The couple may wish not only to avoid another abnormal child with these typically autosomal-recessive disorders, but also to ensure that the transferred embryo has a human leukocyte antigen (HLA) profile compatible with their older, perhaps moribund child. If so, the affected child’s disease could be treated by umbilical cord blood obtained following the birth of its sibling.

Stem-cell transplantation has a high success rate (95%) if the cord blood is HLA-compatible, but less so (65%) if it is not HLA-compatible. Given that only 3 of 16 embryos would be predicted to be both unaffected (3/4) and HLA-compatible (1/4), only PGD is practical.

PGD aneuploidy to increase ART success

The “game changer” in PGD is its capacity to determine presence or absence of all 24 chromosomes. It has long been reasoned that transferring only euploid embryos should increase pregnancy rates, even in women who otherwise have no genetic indications for PGD. This rationale is based on pregnancy rates in assisted reproductive technology (ART) markedly declining late in the mother’s fourth decade, primarily as a result of embryonic losses due to aneuploid embryos. Decreasing ART pregnancy rates with advancing maternal age mirror the increasing rate of miscarriages.

The first available technology to determine aneuploidy on interphase embryonic cells was fluorescent in situ hybridization (FISH). Pregnancy rates after PGD appeared to be increased in larger centers, and miscarriage rates proved no higher in older women than in younger women. However, only 5–9 chromosomes could be interrogated on a single cell, and technical expertise was required. Although larger centers performing PGD at that time (late 1990s and early 2000s) never completed a randomized clinical trial (RCT), other centers did and found no significant improvement in pregnancy rates.2 Although valid criticisms have been directed at RCTs of that era,3,4 enthusiasm predictably diminished. Still PGD aneuploidy continued in many centers, but controversy persisted.5


More recently, diagnostic prowess has improved and clinical efficacy has been demonstrated. One reason is that the currently preferred biopsy approach (trophectoderm from the 5- to 6-day blastocyst) has proven more generalizable. The even greater advance has been the ability to interrogate all 24 chromosomes, using either array comparative genome hybridization (array CGH) or 1 of several next-generation sequencing (NGS) methods utilizing either single nucleotide polymorphisms (SNP), quantitative polymerase chain reaction (PCR), or copy number variants (CNV).

Initially the protocol involved blastocyst biopsy and 24-chromosome analysis followed by transfer in the same cycle. The preferred protocol is now to biopsy all blastocysts, then analyze and freeze all normal embryos. Transfer can then occur in subsequent cycles synchronized for embryo implantation, preferably one embryo into a uterus no longer hyperstimulated.

RCTs using 24-chromosome interrogation have shown statistically significant increased pregnancy rates. As an example, Scott et al reported sustained implantation rates (leading to delivery) of 66% after 24-chromosome aneuploidy testing on blastocyst using a quantitative PCR method, compared with 48% without PGD (<0.001).6 Impressive RCT results were also shown by Yang et al.7 With PGD, aneuploidy miscarriage rates unequivocally fall dramatically,8 and pregnancy rates do not show a maternal age effect until age 42.9

Another benefit of contemporary PGD aneuploidy testing is its potential to reduce multiple gestations in ART. Multiple embryos have traditionally been transferred in ART because not all embryos generate viable pregnancies. If all actually did, multiple gestations would result. Forman and colleagues showed the benefit of PGD aneuploidy testing in a RCT involving women of mean age approximately 35 years and requiring ART.10 In one group 2 morphologically normal blastocysts were transferred without PGD; in the other group, 1 blastocyst known to be euploid by PGD and was transferred. Pregnancy rates were not different (65% vs 61%), but the rate of twins was markedly different (55% vs 0%).



Unresolved questions

Two general areas of controversy remain in PGD: 1) What should be the precise indications for PGD-aneuploidy testing when the sole goal is to increase ART pregnancy rates; and 2) Under what circumstances could mosaic aneuploid embryos be offered for transfer should no euploid embryo exist?

A. Maternal age to improve pregnancy rates

1) Given that aneuploid embryos are correlated positively with maternal age, PGD-aneuploidy testing and transfer of euploid embryos logically becomes relatively more beneficial with advancing maternal age. In 2014 the proportion of cycles not using FISH for diagnostics but array CGH or next generation sequencing (NGS) was not stated. It would be expected that 24-chromosome array CGH or NGS would increase success in any group. The age below which limited presumptive embryo damage would outweigh predicted benefit of euploid embryo transfer is still unclear. Also unclear are related indications of repeated pregnancy loss or repeated implantation failure. Both these clinical conditions may frequently be the result of aneuploidy. Indeed, in RPL miscarriage rates have long been known to decrease with PGD-aneuploidy, using FISH for only 5–9 chromosomes.

Uncertainties in setting an age threshold do not apply to known benefit of PGD when a balanced translocation is present in either parent. Without PGD the length of time to conception is greatly increased, with a mean of 4-5 years.12,13 This reflects the existence of few transferable normal embryos. Thus, with PGD both abnormal offspring and lengthy time to conception can be avoided.

B. Mosaic embryos

2) A second area requiring clarification involves status of mosaic embryos: mosaic monosomy (eg, monosomy 46, XX, -3/46, XX) or mosaic trisomy (eg, 46, XX, +16/46, XX). At first in array 24 chromosome CGH relatively few mosaic embryos were reported because sensitivity was designed mostly to detect whole aneuploidies. Next-generation sequencing (NGS) methods now used are more sensitive than array CGH, capable of detecting a single aneuploid cell among the 5–10 in a trophectoderm biopsy.

Of note, trophectoderm cells are destined to develop into the placenta. Thus mosaicism is not a surprise. Recall that confined placental mosaicism (CPM) has been recognized for decades in prenatal diagnosis. Approximately 1%–2% of CVS or amniocentesis samples are mosaic and subjected to diagnostic algorithms. At the May 2016 meeting of the Preimplantation Genetic Diagnosis International Society (PGDIS) it was reported that 20% of mosaic trophectoderm biopsies were mosaic. In turn, earlier this year, a brief letter by Florentino et al pointed out that, if transferred, some mosaic monosomies can lead to a livebirth.15 At the PGDIS meeting this rate was estimated to be 5%.



Given this small potential for success, when transferring mosaic aneuploidy blastocysts, how should the not-infrequent occurrence of mosaic aneuploid embryos be handled clinically? In most cases, if in a cohort of embryos some are clearly non-mosaic euploid, no controversy exists. It would be illogical to transfer a mosaic aneuploid embryo. If no euploid embryos exist, one should first recommend 1 or more additional cycles, perhaps “batching” embryos for PGD-aneuploidy testing at a single time in hopes of finding a euploid embryo to transfer.

Suppose, however, that there exist only mosaic aneuploid embryos. Suppose further that the patient is relatively older and unlikely to ever produce few to no non-mosaic euploid embryos. In this circumstance one can consider transfer of mosaic aneuploid embryos. Of course, discussion with the couple is obligatory, and amniocentesis recommended in the event of an ongoing pregnancy. Priority preferential sequence should be devised for transfer of mosaic embryos. For example, embryos trisomic for a chromosome capable of a viable livebirth (Nos. 13, 18, and 21) are clearly less desirable than those mosaic for a chromosome never reported to result in a livebirth.

At present, a blastocyst embryo mosaic for monosomy seems preferable to one with trisomy, because monosomic cells do not survive, leaving only euploid cells.


In 2016 PGD has a well-defined role. Indications have extended far beyond the single-gene disorders that initiated the field 25 years ago to encompass HLA-typing for umbilical and cord-blood stem cell transplantation. Twenty-four-chromosome PGD aneuploidy testing identifies euploid embryos to transfer, thus improving the pregnancy rates in ART while reducing the occurrence of multiple gestations.

Miscarriage rates in ART are greatly reduced using PGD, and transfer of euploid embryos allows pregnancy rates to remain relatively undiminished until age 42. All this enables single-embryo transfer to be practical.



1. Rechitsky S, Pakhalchuk T, San Ramos G. First systematic experience of preimplantation genetic diagnosis for single-gene disorders, and/or with advanced maternal age preimplantation human leukocyte antigen typing, combined with 24-chromosome aneuploidy testing. Fertil Steril. 2015; 103:2. 503–512.

2. Mastenbroek S, Twisk M, Goddijn M, et al. PGD – a model to evaluate efficacy? Fertil Steril. 2006; 85:2. 535-536

3. Munné S., Cohen J., Simpson, JL. In vitro fertilization with Preimplantation Genetic Screening (Letter). New Engl J Med. 2007; 357:17. 1769-1770.

4. Munné S., Gianaroli L, Tur-Kaspa I, et al. Substandard application of preimplantation genetic screening may interfere with its clinical success. Fertil Steril. 2007; 88:4. 781-784.

5. Simpson JL. What next for preimplantation genetic screening? Randomized clinical trial in assessing PGS: necessary but not sufficient. Hum Reprod. 2008; 23:10. 2179-2181.

6. Schoolcraft WB, Katz-Jaffe MG. Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age. Fertil Steril. 2013; 100:3.615–619.

7. Yang Z, Liu J, Collins, GS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet. 2012; 5:24. 1755-8166.

8. Hordes-Wertz MD, Grifo J, Ghadir S, et al. Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos. Fertil Steril. 2012; 98:3. 675-680.

9. Harton GL, Munne S, Surrey M, et al. Diminished effect of maternal age on implantation after preimplantation genetic diagnosis and array comparative genomic hybridization. Fertil Steril. 2013; 100:6. 1695-1703.

10. Forman EJ, Hong KH, Franasiak JM, Scott Jr RT. Obstetrical and neonatal outcomes from the BEST Trial: single embryo transfer with aneuploidy screening improves outcomes after in vitro fertilization without compromising delivery rates. Am J Obstet Gynecol. 2013; 210:157.e1–e6.

11. Stephenson MD, Sierra S. Reproductive outcomes in recurrent pregnancy loss associated with a parental carrier of a structural chromosome rearrangement. Hum Repro 2006;21 1076-1082.

12. Sugiura-Ogasawara M, Ozaki Y, Sato T, Suzumori N, Suzumori K. Poor prognosis of recurrent aborters with either maternal or paternal reciprocal translocations. Fertil Steril 2004; 81: 367-373.

13. PGDIS 2016 Position Statement on chromosomal mosaicism detected in PGD by blastocyst biopsy: Transfer or not transfer?

14. Greco E, Minasi MG, Fiorentino F. Healthy babies after intrauterine transfer of mosaic aneuploidy blastocysts.. New Engl J Med 2015; 373:21. 2089-2090.