Reprogenetics

Experts in Preimplantation Genetic Diagnosis

NEW YORK (GenomeWeb) – Researchers from the preimplantation genetics laboratory at Reprogenetics have shown that elevated levels of mitochondrial DNA lead to lower success rates for in vitro fertilization procedures.

In a study published recently in PLOS Genetics, the firm found that embryos with mtDNA levels above a certain threshold never successfully implant in women undergoing IVF and that quantifying mtDNA in embryos before implantation can boost IVF success rates.

Reprogenetics already offers a preimplantation genetic test that screens embryos for chromosomal aneuploidy, which is a leading cause of IVF failure. Only about 25 percent of IVF procedures result in pregnancy and some preliminary studies have suggested that screening for chromosomal aneuploidy could increase IVF success rates by about 50 percent.

Nonetheless, there is still plenty of room for improvement, and Reprogenetics now plans to add mtDNA testing to its aneuploidy test to further boost IVF success rates.

Santiago Munné, founder and director of Reprogenetics, told GenomeWeb that the firm plans to launch the test, called Mitograde, at the European Society of Human Reproduction and Embryology meeting next week. Reprogenetics will offer it as an add-on to its PGS test for an additional $500, Munné said, analyzing just the embryos that have already been found to be chromosomally normal. PGS testing typically costs around $2,000, Munné said.

The firm first became aware of the possibility that mtDNA may play a role in IVF success rates when researchers were developing a next-generation sequencing-based aneuploidy test to screen embryos with abnormal chromosome numbers. In 2013, Dagan Wells, a director at Reprogenetics and associate professor at the University of Oxford, told GenomeWeb that the researchers began to notice that embryos with chromosomal aneuploidy often had more mitochondria. At the time, he said that eventually quantifying mitochondria might serve as another marker to choose viable embryos and increase the success rates of IVF.

The PLOS Genetics study shows that mtDNA can indeed play a role in determining which embryos to implant.

The Reprogenetics team examined a total of 39 cleavage-stage embryos and 340 blastocysts using array CGH and/or next-gen sequencing. The 39 cleavage-stage embryos were all tested with array CGH, determined to be chromosomally normal, and subsequently implanted in the women undergoing IVF. Of the blastocysts, 123 were determined to be aneuploid, while 217 were normal. Of the normal ones, 131 were transferred to the uterus.

First, the researchers looked at the relationship between mtDNA and age. A comparison of 148 blastocysts from women with an average age of 34.8 years to 154 blastocysts from women with an average age of 39.8 years showed that the older women had statistically significant more mtDNA. The phenomenon was also seen when the researchers restricted their analysis to only blastocysts that were chromosomally normal as well as those that were chromosomally abnormal. In addition, in both the older and younger cohort, mtDNA levels were higher in chromosomally aberrant blastocysts than euploid blastocysts.

Next, the researchers analyzed trophectoderm samples from 203 normal and 99 aneuploid blastocyst-stage embryos, first using real-time PCR to assess mtDNA quantity and array CGH to assess chromosomal abnormalities. Again, the team found that abnormal blastocysts contained significantly higher amounts of mtDNA. They then verified their findings using whole-genome amplification and NGS, since the method would enable them to detect both chromosome status and quantify mtDNA.

In order to see whether mtDNA quantity impacted the viability of the embryo, they retrospectively analyzed data from single embryo transfers with and without implantation as well as double embryo transfers. From 89 blastocysts, 81 of which were single embryo transfers, 42 established a pregnancy and 47 failed.

Real-time PCR analysis showed that the blastocysts that implanted contained significant lower mtDNA compared to those that failed. In addition, the researchers were able to establish a threshold, where embryos with mtDNA quantities above .003 never implanted. All 42 pregnancies had mtDNA levels below .003. All 14 embryos with mtDNA levels higher than .003 failed. An additional 33 embryos had mtDNA levels below .003 but failed to implant for other reasons.

Munné said that these embryos could have failed for a variety of reasons, including genetic effects not being screened for and detected like microdeletions or single point mutations, how the embryos were transferred into the patient, or another laboratory issue.

Finally, in a prospective blinded study, the Reprogenetics team showed that measuring mtDNA levels could better predict which embryos would have a higher chance of success. The team quantified mtDNA from trophectoderm biopsies from 42 chromosomally normal blastocysts from women with an average age of 36.7 years. Fifteen embryos had mtDNA levels above the .003 threshold and none of those 15 embryos resulted in a viable pregnancy. Of the 27 embryos with mtDNA levels below .003, 16 ultimately established viable pregnancies.

Looking at the entire cohort, only 16 embryos, or 38 percent, resulted in a viable pregnancy, but including mtDNA analysis bumped that percentage up to 59 percent.

Munné said that Reprogenetics plans to introduce Mitograde as an add-on to the firm’s PGS test that analyzes chromosomal abnormalities. In the PLOS Genetics study, the team demonstrated that both PCR and NGS could successfully analyze mtDNA. Going forward, however, Munné said that the firm would likely focus on NGS, since that one technique could detect both chromosomal aneuploidy and quantify mtDNA.

Currently, he said, Reprogenetics does PGS testing by either NGS or array CGH, depending on the specific lab. The Livingston, New Jersey-based firm has laboratories in Miami, Chicago, Los Angeles, and Portland, Oregon, in addition to offices in Japan, Peru, Spain, and the UK. Increasingly, the labs are moving toward running all 10,000 of its annual PGS tests by NGS, Munné said.

The firm struck a deal last year to use Illumina’s VeriSeq PGS kit and to conduct randomized clinical trials of NGS protocols, and Munné noted that Reprogenetics and Illumina are now in the midst of a large randomized clinical trial for NGS-based PGS, the results of which are expected to be available by the end of the year.

The VeriSeq PGS kit uses a whole-genome shotgun sequencing strategy of a single or a few cells, similar to the low-coverage shotgun sequencing technique used in noninvasive prenatal testing for chromosomal aneuploidies. Munné said that adding in the Mitograde portion would require additional sequence coverage of the mitochondrial genome, which would be done in a targeted manner to keep the test cost-effective.