Spermatid-specific linker histone H1-like protein (HILS1), transition proteins 1 and 2 (TNP1 and TNP2), and protamines 1 and 2 (PRM1 and PRM2) contribute to considerable dense packing of spermatid chromatin during spermiogenesis. We evaluated the HILS1, TNP1, and TNP2 transcript levels in spermatozoa isolated from normozoospermic and asthenozoospermic men. Human ejaculates from normozoospermic (n ¼ 70) and asthenozoospermic (n ¼ 100) donors were purified by centrifugation through a discontinuous Percoll density gradient. RNA was isolated from spermatozoa according to the Chomczyński and Sacchi method, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of HILS1, TNP1, and TNP2 transcripts was performed by real-time quantitative (RQ-PCR) SYBR green I analysis. We found significantly lower levels of HILS1, TNP1, and TNP2 transcripts in spermatozoa from asthenozoospermic men compared to normozoospermic men. Our observations suggest that a reduction in HILS1, TNP1, and TNP2 transcripts may be associated with asthenozoospermia.
The aim of this study was to prospectively investigate the spermatozoa ultrastructure in relation to the results of in vitro fertilization -embryo transer (IVF-ET).Forty-nine consecutive couples admitted for IVF-ET were prospectively evaluated for electron microscopic spermatozoa morphology and the outcome of IVF-ET. Thirty-four couples revealed successful fertilization, defined as presence of two pronuclei 14-16 hours after spermatozoa administration, while the remaining 15 formed the failure group. Spermatozoa fixed with 2.5% glutaraldehyd and embedded in Spurr's resin were analyzed with JAM 100 S transmission electron microscope (TEM) for the following ultrastructure abnormalities: head deformity, cytoplasmic residues, chromatin condensation failures, acrosomal alterations, neck defects, midpiece defects, principal piece and end-piece defects and immature forms.Successful IVF-ET couples revealed a significantly higher percentage of normal spermatozoa utrastructure (32.0 AE 13.1% versus 17.1 AE 13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8 AE 5.1% versus 5.7 AE 5.3%, p < 0.05) and tail defects (16.7 AE 11.5% versus 7.2 AE 7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r ¼ 0.35, p < 0.05).Our data suggest that spermatozoa TEM findings correlate with IVF-ET results. Ultrastructural estimation of spermatozoa can improve the diagnosis of male fertility and may explain some reasons of failure in assisted reproduction methods. We consider systematic TEM spermatozoa examination in cases with failed IVF-ET prior to intracytoplasmic sperm injection (ICSI).
KEYWORDS in vitro fertilization, spermatozoa, ultrastructureAbbreviations: IVF-ET: in vitro fertilization -embryo transfer; TEM: transmission electron microscope; ICSI intracytoplasmic sperm injection.
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