Piotr Jedrzejczak

Mosaicism for 45,X cell line may accentuate the severity of spermatogenic defects in men with AZFc deletion

Editor—Over the past 10 years, several authors have reported microdeletions in the long arm of the Y chromosome (Yq) in men with idiopathic, non-obstructive azoospermia or severe oligospermia. These microdeletions were clustered on the Yq fragment previously described as the azoospermia factor region (AZF).1 More recently, a number of genes expressed specifically in the testes and mapping to AZFa, AZFb, or AZFc subregions have been cloned.2-4 One of the approaches to understanding the role of these genes in human spermatogenesis is to look for a correlation between the lack of given AZF genes and the particular spermatogenic defect in the phenotypes of the patients. However, attempts to find such a correlation have failed so far. Instead, a broad spectrum of phenotypes ranging clinically from azoospermia to severe oligospermia and histologically from Sertoli cell only syndrome (SCOS) to hypospermatogenesis has been described in association with AZFc deletions.5 6 A recent study found chromosomal aberrations in 15% of azoospermic patients.7 However, in papers focusing on the analysis of AZF microdeletions in patients with idiopathic infertility,2 3 5 8-30 systematic, bilateral, histological, molecular, and cytogenetic analyses in the same large group of patients was rarely carried out, thus limiting information on the coexistence of AZF deletions and chromosomal aberrations. In this study, we propose and test the hypothesis that chromosomal defects may often accompany AZF deletions and cause the lack of a genotype-phenotype correlation in human male idiopathic infertility. We also attempt to evaluate the nature of the spermatogenetic failure associated with isolated AZFc deletions. For this purpose, we performed a dual genetic analysis of karyotypes and molecular status of the AZF region along with bilateral testicular histological evaluation in 94 patients with non-obstructive, idiopathic infertility and azoospermia, severe oligospermia, or oligospermia. Sixty five men with …

Caspase-3 activation and phosphatidylserine membrane translocation in human spermatozoa: is there a relationship?

The presence of biochemical signs of apoptosis in ejaculated spermatozoa suggests that apoptosis may be one of the pathways for sperm death. The relationship between the phosphatidylserine (PS) externalization and the presence of the active form of caspase-3 (CP-3) was studied in human spermatozoa after exposure to hydrogen peroxide (H(2)O(2)). Semen from 27 normozoospermic men was examined, as the neat semen, after swim-up isolation and after H(2)O(2) incubation, for the translocation of PS, activation of caspase-3 and mitochondrial membrane potential. The percentage of vital spermatozoa expressing PS translocation was lower than the percentage of vital spermatozoa with the active form of the caspase-3, either in neat (4.9 +/- 2.3% versus 19.7 +/- 6.2%, P < 0.001) or in swim-up semen (2.2 +/- 2.3% versus 4.8 +/- 2.9%, P < 0.01). After swim-up isolation, the percentage of vital spermatozoa with active caspase-3 decreased (P < 0.01). After H(2)O(2) stimulation of the swim-up semen fraction, a decrease in the mitochondrial membrane potential was observed (P < 0.001). Only the midpiece revealed PS translocation after H(2)O(2) stimulation, and it was also the only part to reveal the presence of the active form of caspase-3. All spermatozoa expressing the PS translocation revealed the presence of the active form of caspase-3.

Quantitative assessment of transition proteins 1, 2 spermatid-specific linker histone H1-like protein transcripts in spermatozoa from normozoospermic and asthenozoospermic men.

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 = 70) and asthenozoospermic (n = 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.

Potential biomarkers of nonobstructive azoospermia identified in microarray gene expression analysis

OBJECTIVE To identify potential biomarkers of azoospermia to determine a particular stage of spermatogenetic differentiation. DESIGN GeneChip Human Gene 1.0 ST microarray with validation at mRNA and protein levels. SETTING Basic research laboratory. PATIENT(S) Men with various types of nonobstructive azoospermia (n = 18) and with normal spermatogenesis (n = 4). INTERVENTION(S) Obtaining 31 testicular biopsy samples. MAIN OUTCOME MEASURE(S) Gene expression analysis using the Affymetrix Human Gene 1.0 ST microarrays on 14 selected genes according to the highest fold change, verified with quantitative polymerase chain reaction and on independent set of microarray samples. Western blot and immunohistochemistry were additionally performed. RESULT(S) The comparative analysis of gene expression profiles in the infertile and control groups resulted in the selection of 4,946 differentially expressed genes. AKAP4, UBQLN3, CAPN11, GGN, SPACA4, SPATA3, and FAM71F1 were the most significantly down-regulated genes in infertile patients. Global analysis also led to identification of up-regulated genes-WBSCR28, ADCY10, TMEM225, SPATS1, FSCN3, GTSF1L, and GSG1-in men with late maturation arrest. Moreover, the results from quantitative polymerase chain reaction and Western blot largely confirmed the microarray data. CONCLUSION(S) The set of selected genes can be used to create a molecular diagnostic tool to determine the degree of spermatogenic impairment for men with idiopathic nonobstructive azoospermia.

QUANTITATIVE ANALYSIS OF CCR5 CHEMOKINE RECEPTOR AND CYTOCHROME P450 AROMATASE TRANSCRIPTS IN SWIM-UP SPERMATOZOA ISOLATED FROM FERTILE AND INFERTILE MEN

We determined the CCR5 chemokine receptor and cytochrome P450 aromatase (P450arom) transcript copies number in swim-up sperm isolated from fertile and infertile men. The ejaculates were purified by centrifugation through discontinuous Percoll density gradient and swim-up techniques. RNA was isolated from sperm, treated with DNase I and reverse-transcribed into cDNA. Quantitative analysis of CCR5 and P450arom cDNA were performed by real-time quantitative (RQ-PCR) SYBR Green I analysis. There was a higher content of CCR5 and P450arom transcripts copy number in swim-up sperm of fertile than from infertile donors. The decrease in CCR5 and P450arom transcripts in swim-up sperm may be associated with male infertility.

Mutations of NANOS1, a human homologue of the Drosophila morphogen, are associated with a lack of germ cells in testes or severe oligo-astheno-teratozoospermia

Background The Nanos gene is a key translational regulator of specific mRNAs involved in Drosophila germ cell development. Disruption of mammalian homologues, Nanos2 or Nanos3, causes male infertility in mice. In humans, however, no evidence of NANOS2 or NANOS3 mutations causing male infertility has been reported. Although Nanos1 seems dispensable for mouse reproduction, we sought to analyse for the first time its homologue in infertile men. Methods A group of 195 patients manifesting non-obstructive azoospermia or oligozoospermia were tested for mutations of the NANOS1 gene, using single-strand conformation polymorphism and DNA sequencing. Results Three types of NANOS1 gene mutations were identified in five patients and were absent in 800 chromosomes of fertile men. Pedigree analysis indicated a dominant inheritance pattern with penetration limited to males. Two mutations caused deletions of single amino acids, p.Pro77_Ser78delinsPro and p.Ala173del, each of them identified in two unrelated patients. Both types of deletions were located in the NANOS1 N-terminus (responsible for protein interactions) and were associated with a lack of germ cells in testes. Interestingly, the Pro77_Ser78delinsPro mutation altered interaction of NANOS1 with a microRNA biogenesis factor, GEMIN3. The third identified mutation, p.[(Arg246His; Arg276Tyr)], found in the C-terminal RNA-binding domain, was present in a single oligo-astheno-teratozoospermic man. We bioinformatically demonstrated that the p.Arg246His substitution causes a decrease in the positive charge of this domain, potentially altering RNA-binding. Conclusions This is the first report describing the association of NANOS1 gene mutations with human infertility. Two different infertility phenotypes may reflect distinct functions of N-terminal versus C-terminal regions of NANOS1.

ORIGINAL ARTICLE: The Role of IL-6, IL-10, TNF-α and its Receptors TNFR1 and TNFR2 in the Local Regulatory System of Normal and Impaired Human Spermatogenesis

Problem  To investigate the expression of genes coding for selected cytokines with antagonistic functions (IL‐6, IL‐10, TNF‐α) as well as TNF‐α receptors (TNFR1 and TNFR2) in correct spermatogenesis (normal proliferation), maturation arrest (proliferation inhibited) and testicular tumors (overgrowth).

The role of IL-6, IL-10, TNF-alpha and its receptors TNFR1 and TNFR2 in the local regulatory system of normal and impaired human spermatogenesis.

Problem  To investigate the expression of genes coding for selected cytokines with antagonistic functions (IL‐6, IL‐10, TNF‐α) as well as TNF‐α receptors (TNFR1 and TNFR2) in correct spermatogenesis (normal proliferation), maturation arrest (proliferation inhibited) and testicular tumors (overgrowth).

Human Spermatozoa Ultrastructure Assessment in the Infertility Treatment by Assisted Reproduction Technique

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 Spurrs 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±13.1% versus 17.1±13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8±5.1% versus 5.7±5.3%, p < 0.05) and tail defects (16.7±11.5% versus 7.2±7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r = 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).

Effect of sperm subpopulation's kinetics on human fertilization in vitro.

The present study was carried out to investigate the relationship between sperm subpopulation kinetics on in vitro fertilization rate. The ability of human sperm to achieve fertilization oocytes was investigated in relation to particular motility parameters obtained on a computer aided sperm analysis system base. Analysis covers velocity straight linear (VSL), cross beat frequency (CBF), lateral head displacement (LHD) and homogeneity of progressive motility velocity (HPMV) of fresh semen and semen after density gradient selection. Investigation was based on sperm samples from 82 infertile couples undergoing IVF. Two subpopulations were extracted from each sample using the clustering method with respect to VSL parameter: a slow and rapid one. Comparison of obtained results before and after selection shows no significant change of subpopulations percentage. However, this method of selection strongly influences motility parameters of both subpopulations. There was found a positive correlation for VSL, LHD and HPMV and a negative correlation for CBF parameters found in slow fraction of fresh semen and percentage of fertilized oocytes. On the other hand, rapid subpopulation parameters for fresh semen and parameters found for both subpopulations in semen after selection did not correlate with one. This means that information of slow sperm subpopulation kinetics carries important prognostic value of IVF success. Since the current prognosis factors ignore motility parameters of slow sperms, our results show the importance of such an analysis.