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Updated: Jul 18, 2025
Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
Published on: July 12, 2022
Increases in computationally predicted deleterious variants of unknown significance and sperm mtDNA copy numbers may
Yuze Pi1, Zhuo Huang1,2, Xilin Xu3
1Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China.
Background:
Mitochondria are essential for sperm motility because they provide the energy required for the movement. Changes in sperm mtDNA, such as point mutations, large-scale deletions, or copy number variations, may interfere with ATP production and reduce sperm motility. However, it is not clear if changes in mtDNA are linked to semen quality.
Objectives:
To explore the association between sperm mitochondrial DNA (mtDNA) changes and semen quality.
Materials And Methods:
Sixty-five oligo and/or astheno and/or terato patients (O/A/T) patients and 41 controls were recruited from couples undergoing assisted reproduction. Semen and blood samples were collected from the same individual on the day of oocyte retrieval to extract, isolate and purify mtDNA for next-generation sequencing. mtDNA copy numbers were assessed in 64 patient and 39 control sperm DNA samples using quantitative real-time PCR. The 4977 bp deletion was assessed in 20 patient and 20 control sperm DNA samples using polymerase chain reaction.
Results:
The mtDNA of patients was more likely to carry pathogenic variants or variants of unknown significance (VUSs) (P = 0.091) with higher heteroplasmy levels (P < 0.05) than that of controls. Interestingly, 33.85% of O/A/T patients (22 out of 65) lacked unique variants in their spermatozoa. but presented an exceptionally high mtDNA copy number (P < 0.0001). Moreover, we observed a decrease in the heteroplasmy level of common mtDNA variants shared by somatic and gamete cells (P < 0.0001) and the emergence of a very large number of de novo mtDNA variants with low-level heteroplasmy in spermatozoa.
Discussion And Conclusion:
The increases in the number of computationally predicted deleterious VUS and mtDNA copies in spermatozoa may be associated with semen quality. Exposure to environmental mutation pressure that causes novel mtDNA variants with low-level heteroplasmy may occur during spermatogenesis. Furthermore, when a certain harmful threshold is reached, male germ cells may degrade mtDNA with mutations and replicate the correct mtDNA sequence to maintain the mitochondrial function in spermatozoa.
Insights
Sperm mitochondrial DNA (mtDNA) changes, including increased copy numbers and variants of unknown significance, are linked to reduced semen quality. These alterations may arise from environmental factors during sperm development.
Area of Science:
- Reproductive Biology
- Genetics
- Mitochondrial Biology
Background:
- Mitochondria are crucial for sperm motility, providing energy for movement.
- Alterations in sperm mitochondrial DNA (mtDNA) can impair ATP production and sperm motility.
- The relationship between sperm mtDNA changes and overall semen quality remains unclear.
Purpose of the Study:
- To investigate the association between alterations in sperm mitochondrial DNA (mtDNA) and semen quality.
- To explore specific mtDNA changes such as variants and copy number variations in relation to semen parameters.
Main Methods:
- Semen and blood samples were collected from patients with oligo-, astheno-, and/or teratozoospermia (O/A/T) and controls.
- Next-generation sequencing was used to analyze mtDNA.
- Quantitative real-time PCR and polymerase chain reaction assessed mtDNA copy numbers and the 4977 bp deletion.
Main Results:
- Patients exhibited a higher likelihood of carrying pathogenic variants or variants of unknown significance (VUSs) and increased heteroplasmy levels in their mtDNA compared to controls.
- A significant portion of O/A/T patients displayed exceptionally high mtDNA copy numbers without unique variants.
- A decrease in heteroplasmy of common mtDNA variants and the emergence of numerous de novo mtDNA variants with low heteroplasmy were observed in spermatozoa.
Conclusions:
- Elevated levels of deleterious VUSs and increased mtDNA copy numbers in spermatozoa are potentially associated with impaired semen quality.
- Environmental mutation pressure may induce novel mtDNA variants during spermatogenesis.
- Spermatozoa might regulate mtDNA by degrading mutated sequences and replicating correct ones to maintain mitochondrial function.
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