It is estimated that over 186 million people are affected by infertility, and assisted reproductive technology (ART) treatments are continuously increasing worldwide (1). However, there is a challenge in that a large proportion of embryos during ART end with adverse outcomes, such as implantation failures and miscarriages (2). Abnormal gametes from parents impact embryo quality, and the effect of paternal factors has been increasingly explored in recent years (3, 4).

Previous studies have investigated the associations between seminal quality, including parameters of sperm concentration, motility, and morphology, and ART outcomes. It has been reported that both sperm concentration and motility are strongly correlated with the fertilization rate, and poor seminal quality may lead to developmental failures of embryos, mainly manifested as blastomere fragmentations (5). However, sperms with normal morphology were selected for the intracytoplasmic sperm injection (ICSI) process, and sperm concentration rarely affects this treatment. Thus, sperm motility plays a more significant role in embryo development in ICSI (6).

Researchers have investigated the relationship between sperm structural abnormalities and adverse pregnancy outcomes (7–9). As an indicator of sperm chromatin integrity, the DNA fragmentation index (DFI) has been suggested to have a negative correlation with embryo development and implantation rates in ICSI cycles (9), while some studies have indicated contrary results (10). Sperm mitochondria are essential to the normal reproductive process, as they are involved in multiple functions, including the production of adenine triphosphate and reactive oxygen species (ROS) as well as the regulation of apoptosis (11). Oxidative stress induced by excess ROS in sperm has been found to impair DNA demethylation in the paternal pronucleus and affects embryo development (8). A negative association has been observed between seminal ROS and pregnancy rates after in vitro fertilization (IVF) (12). Additionally, sperm mitochondrial DNA copy number (mtDNA-CN), a relative measure of mtDNA content, has also been reported to be negatively correlated with the fertilization rate (13). Rosati et al. (14) found that higher sperm mtDNA-CN is associated with lower pregnancy probability in couples without contraception, suggesting that mtDNA might be a potential clinical biomarker to predict male fecundity.

It remains unclear whether the measurements mentioned above, namely, DFI, mtDNA-CN, and ROS, are predictive of ART outcomes. Therefore, the present study aimed to investigate the correlations of sperm mtDNA-CN, ROS, and DFI with male fertility, embryo viability, and pregnancy outcomes.

In the present study, we found that three sperm measurements, namely, mtDNA-CN, DFI, and ROS, were negatively associated with seminal quality. In 92 cases conducted with the first ART cycle, high levels of mtDNA-CN and DFI were correlated with a poor fertilization rate. However, this correlation was not significant after adjusting for male age, female age, seminal quality, ART strategy, number of retrieved oocytes, COS protocols, and cycle rank. Moreover, pregnancy outcomes were summarized in 79 cases of transferred embryos, and no associations were found between the three measurements and the clinical pregnancy rate or the live birth rate.

During spermatogenesis, most of the cytoplasm is discarded, while a proportion of mitochondria are retained in the midpiece of mature sperm to provide energy for flagellar beating (18). The role of mitochondria in sperm function, especially sperm motility, has been widely noted. It has been reported that abnormal mitochondrial structures and functions, such as short midpieces, abnormal assemblies, and membranous defects, are associated with poor seminal quality (19, 20). As an important component of mitochondria, mtDNA encodes 22 tRNAs, two rRNAs, and 13 proteins that are crucial for oxidative phosphorylation (21). Mutations and deletions of mtDNA have been reported in asthenozoospermia and shown to be correlated with male infertility when they present beyond a certain threshold level (22). In addition, amplification of sperm mtDNA-CN has been observed in sperm samples from infertile males (23).

Consistent with previous studies in various species (23–26), higher mtDNA-CN in abnormal sperm was observed in the present study. Although the underlying mechanism remains unclear, the elevated mtDNA-CN in abnormal sperm might be explained by abnormal gene expression, mtDNA mutations/deletions, and mitochondria per se. First, abnormal expression of genes that regulate mtDNA transcription and replication, such as mitochondrial transcription factor A (TFAM), may be responsible for the elevated mtDNA-CN. Because TFAM expression is positively associated with mtDNA-CN and negatively correlated with sperm motility (27), increased TFAM expression may lead to the aberrant replication of mtDNA in abnormal sperm. Second, increased mtDNA-CN may compensate for mitochondrial dysfunction caused by mtDNA mutations or deletions (28–30). Moreover, mtDNA-CN may accumulate due to the imbalance between mitochondrial fusion and fission. In mitochondrial fission factor (Mff) mutant mice, sperm mitochondria fail to divide and mitochondrial sheaths are disjointed, resulting in abnormal sperm morphology and motility, which ultimately cause reduced fertility in mice (31).

ROS, a variety of oxygen-derived free radicals, is essential for sperm maturation, capacitation, hyperactivation, and acrosome reactions at low levels (32). However, excess ROS leads to oxidative stress and DNA damage in sperm, including DNA fragmentation, mtDNA damage, telomere attrition, epigenetic abnormalities, and Y chromosome microdeletions (33). In the present study, ROS/MS was correlated with mtDNA-CN in both normal and abnormal sperm groups, while the association of ROS/MS and DFI was not observed ( Supplementary Figure S1 ), indicating that the negative effects of ROS on mtDNA may be more serious than those on nuclear DNA. Compared to nuclear DNA, mtDNA is adjacent to the ROS source and more susceptible to oxidative stress due to a lack of protective histones and repair system. In the multivariate logistic regression analysis, the association with ROS/MS was not significant, yet mtDNA and DFI were still associated with seminal quality, suggesting that mtDNA and DFI are more predictive of seminal quality.

For embryo and pregnancy outcomes, it has been reported that abnormal seminal quality has negative paternal effects in IVF or ICSI procedures (5, 6, 34, 35). However, the effects of sperm mtDNA-CN, DFI, and ROS on embryo quality are ambiguous. In 2019, Wu et al. (13) found that sperm mtDNA-CN, as well as mtDNA deletions, is inversely associated with the odds of fertilization and high-quality embryos after adjusting for male age and measurement batches. In addition, a prospective study of couples discontinuing contraception has revealed that a higher mtDNA-CN is associated with a lower pregnancy probability (14). In contrast, it has been recently reported that sperm mtDNA-CN is not a prognostic factor for fertilization, usable blastocyst development, or live birth rates in couples who undergo ICSI (36). In this study, mtDNA-CN is negatively associated with the fertilization rate, but the association is not significant after adjusting for male age, female age, seminal quality, number of retrieved oocytes, ART strategy, and cycle rank. Thus, further studies on the role of mtDNA-CN in embryo development are needed.

The role of DFI has been investigated over a longer time span, and many meta-analyses and systematic reviews have summarized the effect of sperm DNA damage on clinical outcomes after IVF or ICSI (37–41). Overall, these studies have suggested that there is a difference between the outcomes of IVF and ICSI. Most studies have reported no significant association of DFI and clinical outcomes in ICSI. Nevertheless, increased DFI leads to a negative impact on IVF outcomes, including fertilization rate, embryo quality, implantation rate, pregnancy rate, and live birth rate (41, 42). However, the correlation analysis of DFI and IVF outcomes in these studies was not adjusted for factors, such as seminal quality, COS protocols, and number of retrieved oocytes. In the present study, the correlation of DFI with fertilization rate was also observed in the first ART cycle of couples, but this correlation was not statistically significant in the adjusted model. Notably, in the study by Pregl Breznik et al. (42), washed sperm samples during IVF procedures were analyzed for hyaluronan-binding assays, DFI, and hyperactivity, which was a good strategy to attenuate the impact of fluctuations in sperm measurements on clinical outcomes.

Sperm ROS was reported to have a greater effect on embryo development than the fertilization process (43). However, negative associations of ROS with fertilization rate and pregnancy rate have also been reported (12, 44). In the present study, however, ROS/MS was not correlated with the fertilization rate, cleavage rate, top-quality embryo rate, or clinical pregnancy rate.

The present study prospectively investigated the relationships between three sperm measurements and clinical outcomes throughout the ART procedure, including seminal quality, fertilization rate, cleavage rate, clinical pregnancy rate, and live birth rate. A major limitation of this study was the limited sample size for the analysis of embryonic/pregnancy outcomes. Moreover, the sperm samples analyzed for the three measurements and sperm quality were collected before the ART procedure. Even though the analysis of IVF/ICSI cycles was restricted to within 3 months after the semen analysis, the fluctuations in sperm measurements and parameters could not be ignored. Multicenter studies with a larger sample size are warranted to validate these findings in the future.

In conclusion, sperm mtDNA-CN, ROS/MS, and DFI were separately associated with sperm parameters, while elevated sperm mtDNA-CN and DFI jointly contributed to poor seminal quality. Moreover, mtDNA-CN was negatively correlated with fertilization rate in ART cases, which was not significant after adjusting for male age, female age, seminal quality, ART strategy, number of retrieved oocytes, COS protocols, and cycle rank. For pregnancy outcomes, sperm mtDNA-CN, ROS/MS, and DFI were not associated with clinical pregnancy rate or live birth rate. Further studies are necessary to determine the role of sperm mtDNA-CN, ROS/MS, and DFI in embryonic and fetal development.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the International Peace Maternity and Child Health Hospital Ethics Review Committee. The patients/participants provided their written informed consent to participate in this study.

WHS and MJY collected seminal samples and wrote the article. ZYZ and NXQ performed the experiments and analyzed the data. XYZ, NXX, and SCC collected the clinical outcomes of the participants. SYL and CMX designed the study and revised the article. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (Nos. 81771638, 81971344, 81871136, and 81501231) and the Shanghai Municipal Health Commission (No. GW-10.1-XK07).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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