Implementation of an in-house flow cytometric analysis of DNA fragmentation in spermatozoa

An increased amount of DNA fragme ntation in the spermatozoa(SDF)is linked to male in fertility.The Sperm Chromati n Structure Assay(SCSA)is widely used for analysis of SDF.However,the current software(SCSASoftR)linked to this assay is licensed and often located within larger diagnostic centers.In this study,we present a protocol for using other types of software than SCSASoftR to determine the SDF index(DFI)with clinical relevance.This protocol is engineered after collect!ng and analyzing 254 samples from fertility patients and sperm donors over a 15-month period.DFI is analyzed using a strict protocol where the spermatozoa are treated with a strong acid(pH 1.2)followed by acridine orange.DFI is determined by a standard flow cytometric software,FACSDiva 6.1.3.Analysis of the outcome of the fertility treatment is included for 137 patients receiving either intrauterine inseminations(IUI)or timed coitus(TC).The results show that the chance of pregnancy decli nes as DFI in creases.We also found that the male DFI affects the chanee of pregnancy independent of the female age.We have shown that a standard flow cytometric software can be used when determi ning a clinical releva nt DFI.These findings are a sign ificant step toward impleme nting the an alysis as a part of the routi ne,in・house diag no sing of the male fertility patient and subseque ntly optimizing the treatme nt course of the couple with reduced human and financial costs.

Testis tissue cryopreservation may be considered in boys with cryptorchidism

This study assessed the feasibility of testis tissue cryopreservation (TTC) for fertility preservation in prepubescent boys with cryptorchidism. From January 2014 to December 2022, the University Hospital of Copenhagen (Rigshospitalet, Copenhagen, Denmark) implemented TTC for 56 boys with cryptorchidism to preserve their reproductive potential. Testis tissue samples were collected during orchiopexy (32 cases) or at subsequent follow-up procedures (24 cases), necessitated by an increased risk of infertility as indicated by hormonal assessments and/or findings from initial surgical biopsies. Testis samples were procured for TTC and pathological analysis. The cohort had an average age of 1.3 (range: 0.3–3.8) years at the time of orchiopexy, with 91.1% presenting bilateral cryptorchidism. The study revealed a median germ cell count of 0.39 (range: 0–2.88) per seminiferous tubule, with germ cells detected in 98.0% of the bilateral biopsies and 100% of the unilateral, indicating a substantial potential for fertility in these immature tissues. A dark spermatogonia (Ad) was detected in 37 out of 56 patients evaluated, with a median Ad spermatogonia count of 0.027 (range: 0.002–0.158) per seminiferous tubule. A total of 30.2% of the samples lacked Ad spermatogonia, indicative of potential gonadotrophin insufficiency. The median hormone levels measured were as follows: follicle-stimulating hormone (FSH) at 0.69 (range: 0.16–2.5) U l−1, luteinizing hormone (LH) at 0.21 (range: 0.05–3.86) U l−1, and inhibin B at 126 (range: 17–300) pg ml−1. Despite early orchiopexy, 20%–25% of boys with cryptorchidism remain at risk for future infertility, substantiating the necessity of TTC as a precaution. The study highlights the need for refined predictive techniques to identify boys at higher risk of future infertility.