Expression analysis of MND1/GAJ, SPATA22, GAPDHS and ACR genes in testicular biopsies from non-obstructive azoospermia (NOA) patients.

BACKGROUND: High-throughput studies provide a wide spectrum of genes for use as predictive markers during testicular sperm extraction (TESE) in combination with ICSI. In this work, we used the specimens from testicular biopsies of men with non-obstructive azoospermia who underwent TESE to investigate the expression of spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR. METHODS: Testicular biopsy specimens were subdivided into three groups: hypospermatogenesis (HS); maturation arrest (MA); and Sertoli cell-only syndrome (SCO). The levels of expression of the spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR in the testes were compared among these three groups using the reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: Analysis of the expression of spermatogenic genes in human testes with abnormal spermatogenesis showed different expression patterns in patients from different groups. Fertilization rate for studied set of patients was 66% and pregnancy rate 29%. For HS group fertilization rate was 72% and pregnancy rate 32%, while for MA group fertilization and pregnancy rates were 54% and 26%, respectively. Fertilization rates in relation to the studied genes were uniformly around 70%, pregnancy rates for ACR and GAPDHS genes were surprisingly low at 6% and 8% correspondingly. CONCLUSIONS: Analysis of the expression of genes involved in spermatogenesis can be a fast additional test for the level of spermatogenesis in testicular samples.

Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies.

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

Expression of beta-tubulin epitope in human sperm with pathological spermiogram.

OBJECTIVE: To determine the location of the corresponding epitope on the tubulin molecule and to find any differences in its exposition in human sperm with normal and pathological spermiograms. The mature spermatozoon exhibits extraordinary structural compartmentalization that is related to the presence of cytoskeletal proteins and has a functional role in connection with fertilization and motility. Previously, we have shown that anti-beta-tubulin antibody TU-12 provided an unexpectedly strong reaction in human and boar sperm head. DESIGN: A retrospective study. SETTING: Academic research laboratories and private IVF center. PATIENT(S): One hundred thirteen men participating in the IVF program. INTERVENTION(S): Sperm were divided into five categories: normozoospermia, oligozoospermia, asthenozoospermia, teratozoospermia, and asthenoteratozoospermia. Well-characterized monoclonal antibodies were applied for monitoring tubulin epitope distributions in pathological spermatozoa. MAIN OUTCOME MEASURE(S): Qualitative and quantitative detection of tubulin. RESULT(S): TU-12 epitope was located in the beta-tubulin region beta 426-435. Immunoblotting revealed differences in the amount of tubulin among men with normozoospermia and pathological spermiogram. Striking differences were observed in the exposition of TU-12 epitope in heads of normal and pathological spermatozoa. CONCLUSION(S): The results suggest that tubulin epitopes could be useful biomarkers of the pathological sperm state.

Evaluation of reproductive potential after intracytoplasmic sperm injection of varied human semen tested by antiacrosomal antibodies.

OBJECTIVE: To determine whether varied human spermatozoa, as detected with monoclonal antibodies against acrosomal proteins, have an influence on fertilization, transfer, pregnancy, and implantation rates when intracytoplasmic sperm injection is used. DESIGN: A retrospective study. SETTING: A private IVF center and academic research laboratory. PATIENT(S): One thousand two hundred forty men participating in the intracytoplasmic sperm injection program. INTERVENTION(S): Sperm were divided into seven groups: oligozoospermia, oligoasthenozoospermia, and oligoasthenoteratozoospermia and fresh and frozen-thawed epididymal and fresh and frozen-thawed testicular sperm. Fertilization, transfer, pregnancy, and implantation rates were recorded in each category. Sperm were tested with antibodies for detection of the of the sperm acrosome. MAIN OUTCOME MEASURE(S): Fertilization, transfer, pregnancy and implantation rates, and percentage of acrosome-reacted cells. RESULT(S): The fertilization rate and statistical evaluation showed differences between morphologically normal and pathological sperm and other groups. The freezing-thawing procedure had no influence on the fertilization of testicular sperm, but epididymal frozen-thawed sperm had a higher fertilization rate. Immunofluorescence proved decreasing sperm quality in all groups compared with the control group. This difference is not manifested in other parameters (transfer, pregnancy, implantation rates). CONCLUSION(S): The spermatozoa with varied semen characteristics and good quality, also detected with specific antibodies, gave the best fertilization rates. The paternal effect is not proved in other parameters.