Preimplantation genetic screening
According to Centers for Disease Control and Prevention (CDC) estimates, one in eight couples have trouble achieving or sustaining a pregnancy,1 and approximately 7.4 million women in the United States have received help for infertility.2 The use of ART has doubled over the past decade.3
While these statistics provide a general overview of the frequency of fertility struggles in the U.S., they don’t address the complexity that is added by the societal trend of women waiting until later in life to start a family. As a woman’s age increases, the success rates of IVF decrease. This decline is due, in part, to the increase in chromosome abnormalities, or aneuploidy, in the eggs and resulting embryos as a woman ages. For instance, women under 35 will produce 40 percent of embryos with chromosome abnormalities; women from 35 to 39 about 70 percent; women 40 to 42 close to 80 percent; and older women 90 percent or more.4 Aneuploidy is responsible for the majority of miscarriages and serious genetic disorders. As a result, the ability to screen for the chromosomal health of embryos is an exciting advancement in the field of ART and is the objective of preimplantation genetic screening (PGS) technologies.
PGS may be recommended for women who have experienced recurrent miscarriage or are over age 35, or women for whom multiple fertility treatments have failed.5 PGS technologies have a single aim: identifying chromosomally normal embryos for transfer to the womb. Euploid embryos have a higher chance of implantation, and the resulting pregnancies have a lower chance of miscarriage. The viability of the embryo selected for transfer is one of the most important factors influencing IVF success.
Over the years, genetic screening of embryos has become instrumental in helping to identify aneuploid embryos, and the field continues to advance rapidly. Fluorescence in situ hybridization (FISH) was replaced by DNA microarray analysis or array comparative genomic hybridization (aCGH) in the late 2000s, and these techniques, broadly speaking, are now being replaced by NGS. Each advance provides a more complete picture of the genetic structure of an embryo and its overall health prior to implantation.
Today, NGS is gaining in popularity as costs start to decline and the technology improves. In particular, high-resolution NGS (hr-NGS) enables even more detailed examination of chromosomes and thus better detection of abnormalities through high-resolution sequencing.
To perform NGS for PGS, the first step is whole genome amplification of the sample obtained from the embryo, which is usually about five cells. Since every biopsy is different, the DNA is then quantified so the starting material from each sample is at the correct concentration.
Next, the DNA is fragmented into smaller pieces with specific adapter sequences added to each piece. A low-cycle polymerase chain reaction (PCR) proceeds so that each sample can be individually barcoded with indexes. This is an important step because it distinguishes each sample when sequenced and is what allows attachment to the flow cell during sequencing. Size selection steps are then performed to remove unwanted library fragments and primers, and then the library is normalized to ensure equal library representation. Afterward, all samples are pooled together to create a single library that is then sequenced.
High-resolution NGS has the potential to provide the whole genome sequence of an embryo, enabling detection of chromosome count as well as inherited and de novo gene defects. Also called “high throughput sequencing,” hr-NGS is more scalable and cost-effective than previous technologies as laboratories can sequence an increased number of samples simultaneously during a single experiment.6 Combined with laboratory automation technologies that increase consistency and reduce human error, hr-NGS is a significant advancement in the field of ART.
A key differentiator for hr-NGS is its ability to reveal a wider range of mosaic embryos7—those that contain a mixture of normal and abnormal cells. Mosaicism is extremely common in early human development, affecting 30 percent of blastocyst-stage embryos.8 Recent studies suggest mosaicism plays an important role in pregnancy loss, though some mosaics do go on to become successful pregnancies. Knowledge of the presence of mosaicism may be useful to select the most viable embryos during IVF, thus increasing the likelihood of pregnancy, reducing the chances of a pregnancy loss, and improving the odds of delivering a healthy child.
hr-NGS in the future
High-resolution NGS may be the future, and in fact it is already available. The first baby using hr-NGS was born in 2013. Some studies suggest that there may be up to 60 percent fewer miscarriages using NGS than other PGS techniques.9 As hr-NGS is further validated through clinical studies, future applications may involve coupling this technology with other types of tests.
For instance, today it is possible to examine the mitochondrial DNA (mtDNA)—the tiny organelles that generate energy for the cell. Elevated mtDNA is associated with failure to implant.10 Women who use hr-NGS can add mtDNA testing to boost their chances even further of achieving a successful pregnancy. PGS and mtDNA analysis can be performed in parallel on a single blastocyst biopsy, providing a more thorough picture of embryo health without a significant change in workflow for the clinic or patient.10
Due to the rapid growth and advancement of PGS techniques, PGS is not yet recommended by major professional societies. That said, it is clear that the field of ART is changing, and changing in a way that favors use of these novel technologies. The American Society for Reproductive Medicine recommends no more than two embryos be implanted in women under 35—and doctors should consider using just one. The U.S. may be moving toward a single embryo transfer during IVF, similar to Europe, making the use of PGS technologies such as NGS even more important to increase the odds of a successful pregnancy.
- Chandra A, Copen CE, Stephen EH. Infertility and Impaired Fecundity in the United States, 1982–2010: Data From the National Survey of Family Growth. National Health Statistics Reports. Number 67, August 14, 2013. http://www.cdc.gov/nchs/data/nhsr/nhsr067.pdf.
- Centers for Disease Control and Prevention. Infertility. Key Statistics from the National Survey of Family Growth, 2006-2010. http://www.cdc.gov/nchs/fastats/infertility.htm.
- Centers for Disease Control and Prevention. National ART Surveillance System, Preliminary Data, 2014. Available at: http://www.cdc.gov/art/reports/index.html.
- Munne S, Ribustello L, Kolb B, et al. Blastocysts needed to transfer at least one euploid embryo: data from 10,852 pre-implantation genetic screening (PGS) cycles, Fertility and Sterility. 2015;104(3):e13-e14.
- Fact Sheet: Preimplantation genetic testing. American Society of Reproductive Medicine. http://www.asrm.org/uploadedFiles/ASRM_Content/Resources/Patient_Resources/Fact_Sheets_and_Info_Booklets/PGT_2014.pdf.
- Fiorentino F, Bono S, Biricik A, et al. Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles. Hum. Reprod. 2014 http://humrep.oxfordjournals.org/content/early/2014/10/21/humrep.deu277.long.
- Behjati S, Tarpey PS. What is next-generation sequencing? Arch Dis Child Educ Pract Ed. 2013 Dec; 98(6): 236–238. Published online 2013 Aug 28. doi: 10.1136/archdischild-2013-304340.
- Fragouli E, Alfarawati S, Spath K, Tarozzi N, Borini A, Wells D. Fertility and Sterility. 2015; 104(3):Supplement, Page e96. Available at: http://www.fertstert.org/article/S0015-0282(15)00799-2/abstract.
- Munne, S. New methods for embryo selection: NGS and MitoGradeTM Available at: http://www.eivf.net/documents/sum_2015/Santiago_Munne.pdf.
- Fragouli E, Cohen J, Munne S, Grifo J, McCaffrey C, Wells D. The biological and clinical impact of mitochondrial genome variation in human embryos. ASRM Scientific Program Prize Paper (unpublished).
Lia Ribustello BS, MS, serves as Laboratory Supervisor in New Jersey for Reprogenetics, Inc. She is a certified scientist in Molecular Biology through the American Society for Clinical Pathology.