P-046 Sperm mitochondrial DNA copy number and mitochondrial function in normozoospermic men may predict fertilization rates after intracytoplasmic sperm injection
Authors: Barragan M , Messaritaki R , Bellido R , Lafuente R , Vassena R , Zorzano A , Popovic M 
Published: June 2023

Abstract

Study question
Do sperm mitochondrial DNA copy number (mtDNAcn) and mitochondrial function correlate with fertilization rates after intracytoplasmic sperm injection (ICSI)?

Summary answer
We reveal a negative correlation between sperm mtDNAcn and fertilization rates after ICSI, while higher mitochondrial function corresponded to an increase in sperm fertilization capacity.

What is known already
Over 40% of male-factor infertility is of unknown origin (unexplained and idiopathic in nature). As current diagnoses remain largely descriptive, the identification of markers predicting the fertilization capacity of sperm will hold the key towards improved treatment. Mitochondria are fundamental for supporting spermatogenesis and fertilization. A recent study showed that altered levels of sperm mitochondrial proteins led to oocyte-activation deficiency, resulting in fertilization failure after ICSI. Poor semen parameters have also been linked to high mtDNAcn, however the relationship between mtDNAcn in sperm and oocyte-activation capacity remains unexplored. Further characterization may uncover prognostic markers associated with sperm-related fertilization failure.

Study design, size, duration
Twenty normozoospermic semen samples were included in the study. These were obtained from oocyte donation ICSI cycles, performed between March 2021 and November 2022. Both fresh and cryopreserved sperm samples were analysed. Five million sperm were collected from the fresh ejaculate for genomic DNA (gDNA) isolation and mtDNAcn quantification, while the remainder of the sample was used for mitochondrial functional analysis. These analyses were also performed on washed samples, cryopreserved after swim-up.

Participants/materials, setting, methods
Mean paternal age and BMI were 40 ± 5.8 years and 25 ± 2.3 kg/m2, respectively. We evaluated mtDNAcn using qPCR, by assessing the ratio of mitochondrial-to-nuclear genes (NT-ND4/B2M). For our functional studies, we quantified mitochondrial membrane potential (MMP) in individual spermatozoa by JC-1 staining and flow cytometry. Flow cytometry analysis was performed on ≥ 10 million sperm treated with a mitochondrial uncoupler for 15 minutes (1 µM trifluoromethoxy-carbonylcyanide-phenylhydrazone, FCCP) and controls. Spearman correlation was applied.

Main results and the role of chance
We observed variable fertilization rates in our cohort (69.0 ± 24.9%). One sperm sample resulted in total fertilization failure (TFF) after ICSI and was analyzed independently. In fresh samples, mtDNAcn (5.8 AU ± 2.61) correlated negatively with progressive motility (rs = -0.65) and fertilization rate (rs = -0.32). Notably, the sample resulting in TFF presented with the highest mtDNAcn value (13.9 AU ± 2.77). Flow cytometry analysis revealed two distinct sperm populations, corresponding to motile and immotile sperm. While immotile sperm presented with low or undetectable JC-1 aggregates (red signal), motile sperm showed different levels of MMP (ratio INTred/INTgreen) (2.3 AU ± 1.83). In motile sperm, our results revealed a positive correlation between MMP and fertilization rate (rs = 0.59). As with raw semen, we observed a negative association between MMP and mtDNAcn in motile sperm (rs = -0.53).

Limitations, reasons for caution
Cryopreservation is known to affect sperm motility and active mitochondria in sperm, however its effect on MMP intensity remains to be established. Our limited sample size warrants careful interpretation.

Wider implications of the findings
Our findings suggest a negative correlation between sperm mtDNAcn and fertilization rates after ICSI, while fertilization capacity improved with increasing MMP. Sperm mtDNAcn and MMP may have prognostic value for male fertility and ICSI outcomes. Further validation may deliver clinically useful markers, aiding infertility diagnosis, treatment and counselling.

Trial registration number
NA