New Markers Aid Determination of IVF Success, Prenatal Maturity

Sequencing based testing is already making significant inroads into obstetric care with non-invasive prenatal testing. Now, two new studies show the potential for sequencing technology to further improve success rates in reproductive medicine and neonatal care of pre-term infants.

Cell-Free RNA to Assess Fetal Maturity
Gene expression signatures in amniotic fluid (AF) can indicate organ-specific fetal maturity, according to a small study published Oct. 22 in BMC Medical Genomics. The current study identified both plausible genes associated with maturation of multiple organ systems as well as the different time points in pregnancy when the expression patterns could be utilized. The AF transcriptome may improve upon current methods to help clinicians assess potential neonatal morbidities and inform delivery planning for preterm births.

There is ongoing debate about whether fetal lung maturity testing alone is sufficient to predict postnatal readiness prior to 39 week’s gestation. Although amniocentesis is falling out of favor in light of the rapid adoption of non-invasive prenatal testing early in pregnancy, AF contains higher amounts of cell-free fetal RNA/DNA than maternal serum and researchers believe it holds clues about organ system development beyond that of lung maturity testing.

Researchers from Cincinnati Children’s Hospital Medical Center (Ohio) isolated AF RNA from 16 women at different time points in pregnancy: 18 to 24 weeks (prenatal; n=4), 34 to 36 weeks (late preterm; n=6), and 39 to 40 weeks (term; n=6). Samples were collected from patients undergoing amniocentesis for prenatal diagnosis purposes or during caesarian section delivery. RNA sequencing was performed on cell-free RNA.

Overall, the researchers found a strong correlation between cellular molecular markers in the intrauterine environment and fetal respiratory, digestive and external barrier tissues of the fetus. There were differences in RNA transcripts of 257 genes seen at different time points in pregnancy and these expression patterns were associated with distinct neonatal co-morbidities (underdeveloped lungs and immature feeding patterns), indicating fetal immaturity.

“Given the advantages of amniotic fluid being less complex than serum and containing higher amounts of cell-free RNA and DNA that more directly reflect fetal status, analysis of the amniotic fluid transcriptome is a practical first step towards the biomarker discovery that can later be translated to less invasive methods,” write the authors led by Beena Kamath-Rayne, M.D. “Taking a broader overview of fetal maturity than just focusing on the lung will better enable obstetricians to make delivery planning decisions for preterm births, and prepare pediatricians and neonatologists for the various neonatal morbidities these preterm infants may face.”

Elevated Mitochondrial DNA Lessens Success of IVF
Elevated levels of mitochondrial DNA (mtDNA) in embryos leads to lower success rates for in vitro fertilization (IVF) procedures, according to a study published June 3 in PLOS Genetics. The researchers identified an mtDNA threshold above which no viable pregnancies occur and they say that incorporating quantification of embryo mtDNA into pre-implantation testing can boost IVF success rates.

“There is an urgent need for new methods to improve the efficiency and success rates of IVF,” said Elpida Fragouli, Ph.D., laboratory director of Reprogenetics UK and lead author of the study. “The discovery of a new biomarker of embryo viability, independent of standard assessments such as morphology, is a rare event and of great clinical potential.”

This study assessed the clinical relevance of mtDNA quantification in 379 embryos (39 cleavage-stage and 340 blastocysts, which are five days post-fertilization). The embryos were examined using a combination of microarray comparative genomic hybridisation, quantitative polymerase chain reaction, and next-generation sequencing (which has the benefit of enabling evaluation of both chromosomal status and mtDNA quantification).

The researchers found that the quantity of mtDNA was significantly higher in embryos from older women and in aneuploid embryos, independent of age. By linking clinical outcomes to embryo assessment data, the researchers determined that blastocysts that successfully implanted contained lower mtDNA quantities than those failing to implant. This led to the identification of an mtDNA quantity threshold, above which implantation was never observed.

The predictive value of this threshold was confirmed in an independent blinded prospective evaluation of 42 chromosomally normal blastocysts. Fifteen embryos had mtDNA levels above the .003 threshold (relative quantity of mtDNA) and none of these resulted in a viable pregnancy. Of the 27 embryos with mtDNA levels below .003, 16 ultimately established viable pregnancies. Overall, only 38 percent of the 42 embryos resulted in a viable pregnancy, but by including mtDNA data in the selection process, the percentage was raised to 59 percent. Additionally, the authors note that the mtDNA threshold does not appear to be altered by variation in the processes used by different fertility clinics.

Reprogenetics (Livingston, N.J.) already offers preimplantation genetic testing that screens embryos for chromosomal aneuploidy, a leading cause of IVF failure, and says it plans to add mtDNA testing to its aneuploidy test. It has been reported that the company’s mtDNA Mitograde test will cost $500 as an add-on to the $2,000 preimplantation genetic screening test.

“Even the transfer of a morphologically ‘perfect’ embryo, which is additionally considered chromosomally normal following analysis of biopsied cells, cannot guarantee the initiation of a successful pregnancy (only about two thirds of such embryos actually produce a child),” the authors write in the study. “We propose that mtDNA content represents a novel biomarker with potential value for (IVF) treatment, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy. “

Takeaway: New applications of sequencing-based testing hold the potential for improving IVF success and refining measurements of fetal maturity.