P-354 Advanced Paternal Age (APA) amplifies cryopreservation-induced stress in human spermatozoa
Authors: Pérez Casasús S , Luongo F  P , Haxhiu A , Governini L , Scupoli G , Orini M , Del Canto M , Buratini J , Morgante G , Piomboni P , Luddi A 
Published: July 2024

Abstract

Study question
Does paternal age intensify the negative effects of cryopreservation on sperm quality?

Summary answer
Paternal age exacerbates the adverse impact of cryopreservation on sperm quality.

What is known already
Despite mounting evidence indicating age-related declines in sperm quality, the impact of APA on male fertility remains unclear. APA raises questions about DNA fragmentation and mitochondrial activity, a robust indicator of sperm functionality. While sperm cryopreservation is extensively employed in fertility preservation and assisted reproductive technology (ART), its potentially detrimental effects on cells compartments, including mitochondria, persist. The mechanisms of APA on sperm susceptibility to cryopreservation-induced cellular damage still need to be addressed.

Study design, size, duration
This prospective multicentric study, conducted between January and October 2023, comprised 200 men. Only normozoospermic samples were included in the research (n = 81). Then, based on their age, the participants were categorised into two groups: ages ≤35 (non-APA, n = 40) and ages ≥42 (APA, n = 41). The cohorts were stratified into distinct subgroups, encompassing both fresh and frozen/thawed specimens of APA and non-APA sperm, and subjected to evaluations encompassing mitochondrial functionality, acrosomal reaction, DNA fragmentation and Tyrosine phosphorylation.

Participants/materials, setting, methods
Semen samples were collected after 2-7 days abstinence and analysed according to WHO 2021 guidelines. Acrosomal status was determined using FITC-conjugate PSA. DNA fragmentation was examined with the Halosperm kit. Mitochondrial functionality was gauged using MitoTracker Red CMXRos through the percentage of stained cells and fluorescence intensity and PCR for mitochondrial DNA copy number (mtDNAcn). Tyrosine phosphorylation was assessed through immunofluorescence and Western Blotting.

Main results and the role of chance
Non-APA group exhibited significantly superior fresh semen characteristics. DNA fragmentation was notably elevated in fresh semen from APA group, reaching 21.7%, compared to 15.4% non-APA individuals. After cryopreservation, the DNA fragmentation index further increased in men over 42 years to 26.7%. Mitochondrial functionality analysis indicated a significant decrease in staining intensity in cryopreserved sperm, notably in the APA group, with mean fluorescence intensity decreasing from 3.35±1.5 to 1.8±0.5. In contrast, the non-APA group experienced a drop from 3.33±1.6 to 2.53±1.6. Additionally, in the APA group, a significant increase in mtDNAcn was observed in both fresh and frozen/thawed sperm compared to the younger counterparts, indicative of poor mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, with a significant decrease observed in unreacted acrosomes from 71.5% to 57.7% in the non-APA group and from 75% to 63% in the APA group. Furthermore, exploration of capacitation-like changes, like tyrosine phosphorylation, demonstrated a significant decrease with age and a negative impact of cryopreservation on tyrosine phosphorylation in sperm from young males.

Limitations, reasons for caution
Main limitations are the small number of examined samples and the inclusion of only normozoospermic patients. Therefore, future investigations are needed to provide a more comprehensive understanding of the impact of paternal age on cryopreservation-induced stress in human spermatozoa.

Wider implications of the findings
Our research sheds light on the influence of paternal age on sperm cryopreservation, revealing higher vulnerability of mitochondria and elevated DNA fragmentation in APA. These findings should be considered when advising older men on choosing cryopreservation techniques in assisted reproductive treatments.

Trial registration number
Not applicable