ABSTRACT
BACKGROUND: It is now possible for infertile males to father their own genetic children through the technique of ICSI. This prospect has consequently prompted several investigations into the quality of sperm being retrieved from infertile males. One potential risk is the use of aneuploid sperm or spermatids, which might then be transferred to the fertilized oocyte. METHODS: In this investigation, aneuploidy of spermatids was assessed through immunocytochemistry using antibodies directed against chromosome centromeric regions and complexes. Three different types of infertile male mice with phenotypes closely resembling those described in human non-obstructive azoospermia [PP1cgamma-deficient mice, CREM-deficient mice and C57BL/6J.MAC-17(0--23) mice] were examined for chromosome numbers by counting the number of kinetochores in round spermatids using a CREST antiserum. RESULTS: PP1cgamma(-/-) and CREM(-/-) spermatids from infertile mice showed highly significant elevated levels in the rate of aneuploidy compared with wild-type animals (P < 0.0001). Thus infertile males with independent genetic mutations resulting in different histopathologies showed a high risk in the level of aneuploidy in their spermatids. CONCLUSIONS: These results suggest that impaired spermatogenesis may lead to production of aneuploid gametes. Analysis of aneuploidy in gametes from infertile men, coupled with appropriate genetic counselling, is recommended prior to ICSI.
Subject(s)
Aneuploidy , Infertility, Male/genetics , Repressor Proteins , Spermatids/physiology , Animals , Cell Size , Chromosome Aberrations , Cyclic AMP Response Element Modulator , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Isoenzymes/deficiency , Isoenzymes/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , Mice, Mutant Strains , Mutation/genetics , Phospholipase C gamma , Spermatids/pathology , Type C Phospholipases/deficiency , Type C Phospholipases/geneticsABSTRACT
Non-obstructive azoospermia accounts for a considerable proportion of male factor infertility. Current therapies for treatment of this kind of infertility include procedures such as intracytoplasmic sperm injection (ICSI), round spermatid injection (ROSI), round spermatid nucleus injection (ROSNI) and elongated spermatid injection (ELSI). All involve injection of haploid germ cells retrieved from testicular biopsies into recipient oocytes. We have investigated a mouse model of azoospermia for quality of haploid germ cell genomes, based on 4,6-diamidino-2-phenylindole (DAPI)/TdT-mediated dUTP nick-end labelling (TUNEL) labelling. The mouse model, a targeted mutation in the protein phosphatase 1cg gene, results in severe depletion of haploid germ cells from the round spermatid stage on. Mice homozygous for the mutation are completely infertile, and produce only the occasional spermatozoon. Spermatozoa and round spermatids retrieved from either the epididymides or the testes of mutant mice displayed very high rates of DNA fragmentation. In contrast, similar cells retrieved from heterozygous or wild-type littermates displayed low levels of DNA fragmentation. In some cases, the high rates of DNA fragmentation in mutant cells could be lowered by inclusion of antioxidants in the retrieval media. High rates of DNA fragmentation were also observed in round spermatids retrieved from testicular biospies of human patients with non-obstructive azoospermia. These results suggest that one of the features of the pathology associated with azoospermia is fragmented DNA in haploid germ cells. This raises questions about the suitability of using these cells for fertility treatment.
