Digital PCR Detection of mtDNA/gDNA Ratio in Embryo Culture Medium for Prediction of Embryo Development Potential

Abstract

Purpose: The ratio of mitochondrial DNA to genomic DNA (mtDNA/gDNA) in embryo culture medium as a predictor of embryonic development is a new method of noninvasive embryo screening. However, current tests based on this concept have proven inconsistent. The aim of this study was to define the predictive value of the ratio of mtDNA/gDNA for embryonic developmental potential.

Materials and methods: We used digital PCR to measure mtDNA/gDNA ratios in day 3 culture media of 223 embryos from 56 patients. We compared the relationship between the predictive value of mtDNA/gDNA ratio and each of embryo fragmentation, embryo morphological grade, and blastocyst formation.

Results: mtDNA/gDNA ratio decreased significantly with a decrease in embryo rating: 22.54 (44.66); 31.25 (36.97) and 46.33 (57.11); Grades A vs C, P = 0.006; B vs C, P = 0.015. mtDNA/gDNA ratio increased overall with an increase in embryo fragment content but did not differ significantly between high-, -medium, and poor-quality embryos. Interestingly, this trend differed from that of the unformed blastocysts. mtDNA/gDNA ratio of cleavage stage embryos forming blastocysts was lower (P=0.005). Trends of mtDNA/gDNA ratio differed according to inner cell mass (ICM) and trophectoderm (TE) levels, but not significantly. mtDNA/gDNA ratio in day 3 culture medium was not significantly improved over morphological scores.

Conclusion: We hereby show the correlation of mtDNA/gDNA ratio in the culture medium of developing embryos. The correlation between the mtDNA/gDNA ratio and early embryonic development was controversial. Furthermore, an increase in mtDNA/gDNA ratio might indicate reduced development potential, but the difference remains insufficient for application as a clinical predictor.

Keywords: blastocyst; digital PCR; embryo; mtDNA/gDNA.

Figure 1

mtDNA/gDNA ratio in culture medium (n=223) and negative control medium (n=5). D3 was experimental group, NC was the control groups that identically processed but did not contain embryo. One black dot or square represents one sample. Data are presented as the median with interquartile ranges (Q1-Q3). (**P <0.01).

Figure 2

mtDNA/gDNA ratio in embryo culture medium for different cleavage stages on day 3. The high-quality embryo group is categorized as Grade A (n = 62), the medium-quality group is Grade B (n = 85), and the poor-quality embryo group is Grade C (n = 76). One black dot, triangle, or square represents one sample. Data are presented as the median with interquartile ranges (Q1–Q3). (**P <0.01, *P<0.05).

Figure 3

Embryos with different fragments content. (A) Day 3 embryos developing showed fragmentation less than 5%. (B) Day 3 embryos developing showed 5–10% fragmentation. (C) Day 3 embryos developing showed 10–15% fragmentation. (D) Day 3 embryos developing showed 15–20% fragmentation. (E) Day 3 embryos developing showed 20–25% fragmentation. (F) Day 3 embryos developing showed more than 25% fragmentation. Scale bar, 60 μm.

Figure 4

Difference in mtDNA/gDNA ratio in the culture medium of embryos with different fragment content at day 3. The mtDNA/gDNA ratio corresponding to different proportions of embryo fragments in the embryo culture medium were divided into three groups: Fragmentation of embryos in the first group is less than 10% (n = 127), the fragment content of embryos in the second group is between 10% and 15% (n = 59), and in the third group, embryo fragmentation>15% (n = 37). One black dot, black triangle, or black square represents one sample. Data are presented as the median with interquartile ranges (Q1–Q3).

Figure 5

Group comparison of cleavage stage embryo fragments without blastocyst formation. Fragmentation of embryos in the first group was ≤10% (n = 37), the fragment content of embryos in the second group between 10% and 15% (n = 18), fragmentation of embryos in the third group was >15% (n = 27). One black dot, black triangle, or black square represents one sample. Data are presented as the median with interquartile ranges (Q1–Q3).

Figure 6

Comparison of mtDNA/gDNA in embryo culture medium of groups with different blastulation outcomes. Embryos were grouped according to whether they grew to the blastocyst stage or not. One black dot or black triangle represents one sample. Data are presented as the median with interquartile ranges (Q1–Q3). (**P <0.01).

Figure 7

Blastocysts divided into high-quality, non-high-quality, and unusable blastocyst groups based on classification of blastocyst ICM and TE. There were 41 high-quality blastocysts are divided into group A(AA/AB/BA/BB), 76 medium-quality blastocysts are divided into group B(BC), 24 poor-quality blastocysts are divided into group C (stage 1/stage 2/stage 3/CC). One black dot, black triangle, or black square represents one sample. Data are presented as the median with interquartile ranges (Q1–Q3).

Figure 8

ROC curve of morphological grade and the mtDNA/gDNA ratio: (A) day 3 morphological grade group undergoing IVF: area under the curve (AUC)= 0.793 [0.734–0.853], cutoff value= 3.50 with sensitivity=63.4% and specificity= 83.0%. (B) mtDNA/gDNA ratio group undergoing IVF: area under the curve (AUC)= 0.614 [0.539–0.689], cutoff value= 24.83 with sensitivity= 73.2%and specificity= 47.5%.