Optimized protocol to prevent disease transmission through maternal mitochondrial DNA carryover

By Dr. Liji Thomas, MDOct 5 2023Reviewed by Benedette Cuffari, M.Sc.

Several human diseases arise due to defects in mitochondrial DNA (mtDNA), which is distinct from nuclear DNA and is inherited only from maternal ancestors. The prevention of these diseases by mitochondrial replacement (MR) therapy involves the risk of increasing mtDNA mutations, which can subsequently lead to mitochondrial genetic drift.

A recent study published in PLOS Biology discusses a new method to potentially minimize this risk of transferring mtDNA mutations while maximizing embryo survival and development.

Study: Significant decrease of maternal mitochondria carryover using optimized spindle-chromosomal complex transfer. Image Credit: nobeastsofierce / Shutterstock.com

Introduction

Within the mitochondria, substrates undergo oxidative phosphorylation to generate adenosine triphosphate (ATP). Mitochondria contain their own DNA in the form of a double-stranded circular chromosome.

While mtDNA is very short with only 37 genes, mutations may arise from being inherited or genetic drift during embryonic development. The health of the embryo then depends on the level of proliferation of heteroplasmic mtDNA.

At high levels, mutant mtDNA may predominate and contribute to the development of clinical symptoms such as blindness, deafness, diabetes, liver failure, or muscular weakness. These symptoms may manifest at any age.

This has motivated researchers to identify approaches that can prevent these diseases by avoiding their transmission through approaches such as MR. MR involves replacing the mitochondria of gametes involved in the conception, or the embryo formed by conception, using spindle transfer or other methods.

What is SCCT?

One of the benchmark MR approaches is spindle-chromosomal complex transfer (SCCT). Herein, researchers manipulate the formation of the spindle during the metaphase II (MII) stage of cell division, during which chromosomes separate to the two poles of the cell and are attached to the spindle fibers. Paternal and maternal chromosomes segregate to the opposite poles of the cell.

In SCCT, the spindle to which the maternal-origin chromosomes are attached is removed from an unfertilized oocyte, which represents maternal-origin nuclear DNA. These chromosomes are subsequently transferred by micromanipulation into a recipient oocyte from a donor woman, from which the nucleus has been extracted from an empty oocyte.

Fertilization of the new or reconstructed oocyte then occurs. Successful fertilization and development in rhesus monkey oocytes has been reported, followed by similar experiments in human oocytes. Analysis of the blastocysts and embryonic cell lines (ESCs) in these animal models showed that mtDNA levels were undetectable.

The first human birth following conception with the use of SCCT occurred in Mexico in 2017 and involved three parents.

Even with SCCT, mitochondrial genetic drift was reported in several experiments. This might be because the spindle-chromosomal complex (SCC) came with a small amount of cytoplasm containing some mitochondria, thereby leading to the transfer of heteroplasmic or mutant mtDNA to the recipient oocyte.  This exposes oocytes constructed in this manner to the risk of genetic drift. Thus, the individual conceived from these oocytes is more vulnerable to the risk of subsequent mitochondrial disease.

The limitations of these techniques led the researchers of the current study to investigate a new method of micromanipulation during SCC. The aim of this study was to minimize the risk of mtDNA carryover, thereby preventing genetic risk and clinical mitochondrial disease. The novel method they propose in the current paper is called maximal residue removal (MRR).

What is MRR?

SCC from mouse and human oocytes were removed using a very fine micropipette. The cytoplasm surrounding the SCC was subsequently removed by a “swing-away” technique in a special mixture. Individual SCCs were evaluated to ensure that MRR had been achieved and that the mtDNA copy numbers and genetic copy number variation (CNV) were intact.

Thereafter, mouse oocytes were reconstructed with mtDNA from different cells. The development of oocytes was examined following fertilization by intracytoplasmic sperm injection (ICSI) at the blastocyst stage. The level of mtDNA heteroplasmy and chromosomal copy number were also determined.

SCCT triggered the activation of the reconstructed oocyte too early and, as a result, caused it to enter meiosis and lead to abnormal fertilization prematurely. To overcome this problem, ICSI was performed first followed by MRR.

This novel approach increased successful fertilization and reduced the time taken and number of oocytes that might have to be sacrificed. SCC also remained morphologically intact after these procedures, and CNV analysis showed normal chromosomal copy number.

This novel mitochondrial removal approach of MRR would not compromise the integrity of spindle and chromosome and could be readily performed in nuclear transfer.”

The SCC-MRR procedure did not prevent normal fertilization and subsequent embryonic development.

Fertilized oocytes were transplanted into oviducts, which resulted in healthy offspring in about 30% of cases. This is highly comparable to the efficacy of conventional SCC embryo transfer. The mtDNA heteroplasmy was about 1.5% in SCCT-MRR offspring as compared to about 4.1% in conventional SCC embryos.

These offspring grew to adulthood and mated normally with wild-type males. Second-generation offspring also exhibited low mtDNA residue at about 0.5%.

SCCT-MRR ESC lines appeared and behaved like control and stable ESC lines, whereas conventional SCCT-ESCs exhibited increased mtDNA heteroplasmy. Thus, this method did not result in mtDNA genetic drift.

The MRR-SCCT could strongly reduce the transmission of spindle-donor mtDNA to the lowest level at a stable state in mESCs.”

These manipulations were also performed during the MII stage of the oocyte, which resulted in properly developing SCCT-MRR embryos with minimal mtDNA carryover. Using this technique, mtDNA carryover declined to 0.04%, which is the lowest level ever reported in human SCCT experiments.

The high fertilization rate, proportion of embryos that progressed to the blastocyst stage, and euploidy levels were comparable to non-manipulated ICSI-derived human oocytes.

What are the implications?

The mtDNA levels consistently declined to very low and stable levels in the karyoplasts, reconstructed oocytes, and blastocyst derivatives following SCCT-MRR. Moreover, mtDNA levels were about one-sixth of those resulting from conventional SCCT in mice and remained stable in the first and second generations of offspring.

These findings support the hypothesis that greater residual mtDNA is associated with an increased risk for genetic drift. This approach also does not appear to increase the risk of spindle-chromosomal defects caused by the manipulation.

Earlier human infertility research has shown that low mtDNA carryover at the blastocyst stage was still followed by a rise from 0.8% to as high as 60% in various tissues by the time of birth. This drift in heteroplasmy has also been observed in other experiments on human ESCs.

Therefore, we believe that more investigations involving human ESCs will be necessary before the clinical translation of current MRR-SCCT strategy.”