Mitochondrial DNA content is not predictive of reproductive competence in euploid blastocysts

Abstract

Research question

Does mitochondrial DNA (mtDNA) copy number predict the reproductive potential of euploid human blastocysts?

Design

To investigate whether the amount of mtDNA in trophectoderm biopsies correlates with IVF outcome, euploid human blastocysts (n = 615) used in single embryo transfer were analysed. Furthermore, to determine whether mtDNA content is predictive of reproductive outcome within a given cohort, paired sibling embryos (n = 78) transferred in two consecutive cycles carried out in the same patient (in which one cycle failed to result in implantation and the other cycle resulted in sustained implantation) were studied. Targeted amplification followed by quantitative real-time polymerase chain reaction for two mitochondrial loci (16S and MajArc) relative to a multicopy nuclear genome locus (AluYb8) were carried out to determine relative mtDNA copy number.

Results

Sustained implantation was not associated with relative mtDNA copy number (P = 0.78), and there was no threshold value above or below which ongoing implantation was more or less likely. No correlation was observed between maternal age and relative mtDNA copy number (P = 0.39). In addition, no association was found between relative mtDNA levels of sibling embryos and ensuing implantation and delivery rates in women who underwent a successful single embryo transfer before or after a failed transfer using embryos derived from the same cohort of oocytes (P = 0.70).

Conclusions

In trophectoderm samples, mitochondrial DNA copy number analysis was not found to be predictive of euploid human embryo reproductive competence. These data do not support the use of mitochondrial DNA copy number in clinical decision making when selecting which embryo to transfer.

Introduction

The reasons why a given embryo may or may not implant and develop are numerous. One of the great challenges of contemporary reproductive medicine is the accurate assessment of these factors and how they pertain to embryonic competence within the context of assisted reproduction. Despite advances in culture systems and diagnostic methods, current techniques are not able to accurately predict which embryos will implant and progress to delivery and which will arrest in development either before or after implantation. Even among embryos assessed and selected for competence and transferred to a synchronous endometrium, overall sustained implantation rates are about two out of three (Forman et al., 2013; Scott et al., 2013). This means that one-third of embryos characterized as capable of developing will ultimately fail to deliver. This strongly implies that sources of failure not detected by the currently used embryo assessment strategies exist.

One factor that may potentially play a role in embryo viability is mitochondrial function. Mitochondria are double membrane-bound organelles that are present in the cytoplasm of almost all eukaryotic cells (Babayev et al., 2016). They vary significantly in size and number per cell, and are unique as they contain their own DNA, which encodes for 13 proteins with key roles in the electron transport chain (Babayev et al., 2016). Mitochondrial morphology and number change in a predictable manner during oocyte and early embryo development, suggesting a tightly regulated and biologically significant process. Importantly, metabolic abnormalities in oocytes caused by targeted deletion of mitochondrial stress response or dynamics (fusion and fission) genes results in infertility and follicular depletion, confirming the requirement of mitochondrial function in female reproduction (Wang et al., 2018; Zhang et al., 2019a; 2019b).

The association between embryonic mitochondrial parameters and reproductive outcomes has been evaluated by several investigators. Initial reports by Fragouli et al. (2015) and Diez-Juan et al. (2015) were promising. Fragouli et al. (2015) found that elevated relative mitochondrial DNA (mtDNA) content above a threshold level was associated with dramatically diminished clinical outcomes among euploid blastocysts. Diez-Juan et al. (2015) produced similar results and extended the association between high mtDNA content and decreased pregnancy to cleavage-stage embryos. They proposed a scale, in which increasing mtDNA copy number would predict a gradually decreasing implantation potential.

These findings are not universal and other studies have yielded different results. Victor et al. (2017) conducted a detailed analysis, which failed to find a relationship between mtDNA content and clinical outcomes among euploid embryos. Similarly, a study by our group (Treff et al., 2017) found no implantation advantage in embryos with lower relative mtDNA content. Importantly, although these studies were ongoing, some commercial laboratories have started to offer assessment of mtDNA as an adjunct to embryonic aneuploidy screening for embryo selection in assisted reproduction. It is not currently known, however, if this information is clinically relevant.

The present study seeks to clarify this critical question by approximating the methodology used in the initial study by Fragouli et al. (2015) in embryos from a large group of unselected euploid blastocysts undergoing transfer in a single centre.