The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART programme.

STUDY QUESTION: Could the protamine-1 to protamine-2 mRNA ratio serve as a biomarker to estimate the fertilizing capacity of sperm from men taking part in an IVF/ICSI programme? SUMMARY ANSWER: The protamine mRNA ratio clearly discriminates between fertile and subfertile men and sperm with a normal protamine mRNA ratio exhibit a higher fertilizing capacity in IVF/ICSI. WHAT IS KNOWN ALREADY: Aberrant sperm protamine ratios are associated with male factor infertility and mRNA ratio is comparable with protein ratio (due to transcriptional stop in elongating spermatids). STUDY DESIGN, SIZE, DURATION: The study population was drawn from subfertile men, whose female partners participated in IVF or ICSI programmes between September 2010 and February 2012. Normozoospermic healthy volunteers served as controls. Sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time quantitative PCR using specific primer pairs for protamine-1 and protamine-2. Relative protamine-1 and protamine-2 mRNA levels were analysed with the Mann-Whitney U-test (two-tailed). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative RT-PCR for protamines 1 and 2 has been performed in ejaculates from 32 normozoospermic volunteers (control, University Clinic Giessen, Germany) and 306 patients, whose female partners took part in an IVF (n = 76; University Clinic Hamburg, Germany and Shanghai Jiaotong University, China) or an ICSI (n = 230; University Clinic Munich, Germany and Kinderwunschzentrum Wiesbaden, Germany) programme. MAIN RESULTS AND THE ROLE OF CHANCE: The sperm protamine mRNA ratio in normozoospermic men (0.98 ± 0.3) differed significantly from that of ICSI patients (Munich 0.81 ± 0.1; Wiesbaden 0.78 ± 0.2; P < 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0 ± 0.07; Shanghai 1.0 ± 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc > 80%; P = 0.017) and ICSI (65.1% of patients with fc > 70%; P = 0.028). LIMITATIONS, REASONS FOR CAUTION: The protamine mRNA ratio in an individual sperm cell used for ICSI may be different from the overall value obtained from a semen aliquot. WIDER IMPLICATIONS OF THE FINDINGS: Data are in line with current literature and suggest the protamine mRNA ratio as a diagnostic marker to estimate the fertilizing capacity of sperm. STUDY FUNDING: The German Research Foundation (DFG) to K.S., W.W. and A.P. (STE 892/9-2), as well as to A.S. and H.C.O. (SP721/1-3). COMPETING INTEREST(S): None.

Evaluating the localization and DNA binding complexity of histones in mature sperm.

The paternal genome in many animal taxa is efficiently packaged into the sperm nucleus by switching from a histone (nucleosome)-based chromatin configuration to one using predominantly protamines. Nonetheless, various studies have shown that some nucleosomes, often containing modified histones are retained in mature sperm and bind DNA with distinct sequence compositions. Considering the significance of histone modifications in epigenetic phenomena and the fact that sperm histones and their bound DNA must be carried into the oocyte, this chapter describes methods aimed at examining and analysing the histone composition of sperm chromatin. The focus is on both microscopic visualisation and evaluation of sequence composition of histones and histone-bound DNA in human and mouse spermatozoa. However, similar methods may be applicable to the sperm of other mammalian and even non-mammalian classes.

Analysing the sperm epigenome: roles in early embryogenesis and assisted reproduction.

An understanding of the epigenetic mechanisms involved in sperm production and their impact on the differentiating embryo is essential if we are to optimize fertilization and assisted reproduction techniques in the future. Male germ cells are unique in terms of size, robustness, and chromatin structure, which is highly condensed owing to the replacement of most histones by protamines. Analysis of sperm epigenetics requires specific techniques that enable the isolation of high quality chromatin and associated nucleic acids. Histone modification, DNA methylation and noncoding RNAs have important, but so far underestimated, roles in the production of fertile sperm. Aberrations in these epigenetic processes have detrimental consequences for both early embryo development and assisted reproductive technology. Emerging computational techniques are likely to improve our understanding of chromatin dynamics in the future.

Genome wide identification of promoter binding sites for H4K12ac in human sperm and its relevance for early embryonic development.

Sperm chromatin reveals two characteristic features in that protamines are the predominant nuclear proteins and remaining histones are highly acetylated. Histone H4 acetylated at lysine 12 (H4K12ac) is localized in the post-acrosomal region, while protamine-1 is present within the whole nucleus. Chromatin immunoprecipitation in combination with promoter array analysis allowed genome-wide identification of H4K12ac binding sites. Previously, we reported enrichment of H4K12ac at CTCF binding sites and promoters of genes involved in developmental processes. Here, we demonstrate that H4K12ac is enriched predominantly between ± 2 kb from the transcription start site. In addition, we identified developmentally relevant H4K12ac-associated promoters with high expression levels of their transcripts stored in mature sperm. The highest expressed mRNA codes for testis-specific PHD finger protein-7 (PHF7), suggesting an activating role of H4K12ac in the regulatory elements of this gene. H4K12ac-associated genes revealed a weak correlation with genes expressed at 4-cell stage human embryos, while 23 H4K12ac-associated genes were activated in 8-cell embryo and 39 in the blastocyst. Genes activated in 4-cell embryos are involved in gene expression, histone fold and DNA-dependent transcription, while genes expressed in the blastocyst were classified as involved in developmental processes. Immunofluorescence staining detected H4K12ac from the murine male pronucleus to early stages of embryogenesis. Aberrant histone acetylation within developmentally important gene promoters in infertile men may reflect insufficient sperm chromatin compaction, which may result in inappropriate transfer of epigenetic information to the oocyte.

The interaction of modified histones with the bromodomain testis-specific (BRDT) gene and its mRNA level in sperm of fertile donors and subfertile men.

As histone modifications have been suggested to be involved in the regulation of gene expression after fertilisation, the present study aimed to analyze the interaction between the bromodomain testis-specific (BRDT) gene and differentially modified histones in human spermatozoa. The BRDT transcript level was studied to identify possible correlations between epigenetic changes, mRNA level and subfertility associated with impaired sperm chromatin condensation. Chromatin immunoprecipitation (ChIP) was performed with ejaculates from fertile and subfertile men using antibodies against specifically acetylated and methylated histone H3. Immunoprecipitated DNA was analysed by real-time quantitative PCR with primer pairs for BRDT. The BRDT mRNA level was screened by real-time RT-PCR. ChIP assay revealed co-localisation of acetylated and methylated histones within promoter and exon regions of the BRDT gene in fertile men. Interestingly, reduced binding of investigated modified histone modifications was observed in the BRDT promoter of subfertile patients. Different mRNA levels of BRDT have been detected in a group of infertile patients, as well as in fertile men. Enrichment of methylated histones within the BRDT promoter of fertile sperm suggests that this epigenetic mark may cause repression of BRDT after fertilisation, and may be changed in infertile patients. Our data suggest that reduced histone methylation in the promoter of BRDT may be associated with increased transcript levels in subfertile patients.

PTPIP51 mRNA and protein expression in tissue microarrays and promoter methylation of benign prostate hyperplasia and prostate carcinoma.

BACKGROUND: Protein tyrosine phosphatase interacting protein 51 (PTPIP51) shows a tissue-specific expression pattern and is associated with cellular differentiation and apoptosis in several mammalian tissues. Overexpression of the full-length protein enhances apoptosis. It is also expressed in various carcinomas. In this study the expression of PTPIP51 and its in vitro interaction partners was investigated in human benign prostate hyperplasia (BPH) and in prostate carcinoma (PCa). METHODS: Tissue microarrays of human BPH and PCa were analyzed by immunohistochemistry. For polymerase chain reaction (PCR), cryo samples of BPH and PCa were used. Bisulfite DNA treatment, followed by sequencing of PCR products was performed in order to analyze CpGs methylation within the promoter region of the PTPIP51 gene. RESULTS: PTPIP51 mRNA and protein expression was detected in prostatic epithelia of BPH and in tumor cells of PCa, respectively, and within smooth muscle cells of the stromal compartment. A stronger expression was present in nerve fibers, particularly in PCa, in immune cells and in smooth muscle and endothelial cells of vessels of BPH and PCa. On mRNA levels, a slightly elevated expression of PTPIP51 was observed in the PCa group as tested by real-time quantitative PCR analyses. Methylation experiments revealed that at least 70% of methylated CpGs in the CpG island of the PTPIP51 gene promoter region were identified in BPH samples. In contrast, a loss of methylation has been found in the PCa group. CONCLUSION: The promoter methylation status of PTPIP51 seems to influence the expression of PTPIP51, which was seen as elevated in the PCa.

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences.

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

Aberrant epigenetic modifications in the CTCF binding domain of the IGF2/H19 gene in prostate cancer compared with benign prostate hyperplasia.

Expression of the imprinted genes insulin-like growth factor 2 (IGF2) and H19 depends on the methylation pattern of their common imprinting control region (ICR) located on chromosome 11p15. As the somatic imprinting pattern may be lost during tumorigenesis due to epigenetic alterations, in the present study, we analyzed the DNA methylation and histone modifications in the differentially methylated region (DMR) of IGF2/H19 in benign prostate hyperplasia (BPH) and prostate carcinoma (PCa). Sodium bisulfite sequencing was performed on frozen tissue collected after radical prostatectomy. Thirty tumors and 17 non-cancerous tissue samples were analyzed. Histological diagnosis was, in addition, confirmed by amplification of the epithelial tumor marker alpha-methylacyl coenzyme-A racemase. Chromatin immunoprecipitation assay (ChIP) was carried out on sonificated chromatin from fresh tissue samples from 10 PCa, 10 BPH using antibodies against trimethyl histone H3K9, dimethyl histone H3K9, trimethyl H3K27 and acetyl H3K9. The methylation pattern of 17 CpGs within 227 bp of the H19 fragment was characterized from each DNA sample. All (BPH) samples demonstrated >80% methylation of CpGs. In contrast, we found 41% of CpGs methylated in 9 out of 30 PCa specimens. We observed statistically significant differences in the methylation state between PCa and BPH groups, especially in the DMR of H19 (p<0.0001) and in the ICR (p=0.0034), which corresponds to CTCF binding domain. ChIP assay revealed that dimethyl H3K9 is associated with the ICR of IGF2/H19 in BPH, but not in PCa (p<0.0001). Our data demonstrate that DNA methylation and histone methylation analysis of the ICR within the DMR of IGF2/H19 provides important insights into early steps of carcinogenesis and, therefore, may contribute to improving diagnosis of PCa.

Interleukin-1beta gene (IL-1beta) polymorphisms (SNP -511 and SNP +3953) in thyroid-associated ophthalmopathy (TAO) among the Polish population.

OBJECTIVE: The aim of this study was to evaluate whether the IL-1beta gene could be a genetic marker of the thyroid-associated ophthalmopathy (TAO) development. MATERIALS AND METHODS: The IL-1beta gene polymorphisms at -511 and +3953 regions in 117 TAO patients of Polish origin (ATA/NOSPECS class III or greater) and in 106 controls were studied. RESULTS: We found no significant differences in the frequencies of genotypes and allelic variants for SNP -511 and SNP +3953 between the controls and the studied groups. CONCLUSIONS: No association between the IL-1beta polymorphisms and the TAO existed, so those polymorphisms are not suitable genetic markers for TAO.

[Bacterial nosocomial infections in the material of the Chair of Forensic Medicine, Collegium Medicum, Medical University in Bydgoszcz in the years 2000-2006]. / Bakteryjne zakazenia szpitalne w latach 2000-2006 w materialach wlasnych Katedry Medycyny Sadowej CM UMK w Bydgoszczy.

The ever increasing number of legal claims and lawsuits associated with nosocomial infections has become a serious problem. The present authors analyzed the medicolegal opinions regarding nosocomial bacterial infections issued by the faculty of the Bydgoszcz Department of Forensic Medicine in the years 2000-2006. Within the evaluated period, the investigators observed an increase in the number of court cases concerning hospital-acquired infections. The most frequent pathogen was Staphylococcus aureus.

[The medico-legal analysis of drowning in the materials collected by Department of Forensic Medicine in Bydgoszcz in the years 1992 to 2002]. / Sadowo-lekarska analiza utoniec w materialach Zakladu Medycyny Sadowej w Bydgoszczy w latach 1992-2002.

The report presents a medico-legal and toxicological analysis of cases of drowning. Within eleven years, 184 cases of deaths by drowning were observed in our Department. The majority of victims died because of a tragic accident, while in three cases death was a result of suicide. Toxicological observations showed that most victims were under the influence of alcohol. In isolated cases, blood carboxyhemoglobin and delta9tetrahydrocanabinnol were detected.

[Negligence in a care and sudden death in children]. / Niezachowanie nalezytej starannosci w opiece a gwaltowne zgony dzieci.

Deaths in children, and particularly sudden deaths, are extremely emotionally difficult experiences for parents, caregivers and the general public. The primary objective of this study was to present negligence in care as a risk factor of sudden deaths in children. The second goal was to estimate a chance of avoiding such tragic deaths. The analyzed material was provided by protocols of body inspection and postmortem examination of children below 15 years of age examined in the years 1992-2007, where the circumstances surrounding death suggested an accident. The majority of victims were male and the greatest number of death was noted in preschool and school children. The most frequent cause of death was accidental drowning and cerebrocranial trauma due to a road traffic accident. Several homicide cases were also observed. After the analysis of the material, the authors concluded that negligence in care is a significant risk factor in sudden deaths in children.

[Deaths of children in infancy, with particular emphasis on neonaticide in materials collected by Department of Forensic Medicine in Bydgoszcz in the years 1992 to 2006]. / Zgony dzieci w wieku noworodkowym ze szczególnym uwzglednieniem dzieciobójstwa w materialach Zakladu Medycyny Sadowej w Bydgoszczy w latach 1992-2006.

The issue of sudden deaths of children in the infant period is presented in the report. The analysis included a total of 54 cases of sudden deaths of infants. Death caused by a disease, neonaticide and intrauterine necrosis were observed. In the group of disease-associated deaths, the most common cause of death was pneumonia. The most common mechanism of neonaticide was strangulation.

[The effect of the duration of corpse immersion on the occurrence of selected morphological findings]. / Wplyw czasu przebywania zwlok w wodzie na pojawienie sie wybranych zmian morfologicznych.

The report presents a method of estimation the duration of corpse immersion based on fully developed morphological findings. The authors demonstrated that the duration of body immersion affects the occurrence of marked putrid color, separation of hair, the "washerwoman's skin" effect on the macerated fingertips, separation of fingernails, fimbriated separation of epidermis involving the feet, lack of blood in the heart ventricles and putrid encephalomalacia. The report further stated that the duration of immersion does not necessarily affect the occurrence of marbling, corpse distension, separation of epidermis from the body surface, detachment of fingernails from fingernail bed, fimbriated separation of epidermis involving the hand, the "washerwoman's skin" effect on the toes, detachment of toenails from toenail bed and the presence of over 500 ml of pleural putrefied "effusion".

[The structure of homicides in Bydgoszcz area in the years 1992-2002]. / Struktura zabójstw w regionie bydgoskim w latach 1992-2002.

The authors presents an analysis of homicides based on autopsy material collected at the Institute of Forensic Medicine in Bydgoszcz in the years 1992-2002 and the comparison of the thus obtained results with the results for the years 1986-1991. In approximately 3500 analyzed postmortem examinations, this type of death accounted for 5.6% of cases. The majority of victims were men, mostly 31-50 years of age, accounting for 69% of cases. In the compared periods, the authors noted an approximately 20% increase in this type of crimes, especially apparent in rural areas. The number of homicides with firearms and serial killings was also increased. The most common injuries were stab wounds. The number of drunken perpetrators was also increased.

[Gunshot wounds in the material of Forensic Medicine Institute, Collegium Medicum, Nicolaus Copernicus University in Bydgoszcz]. / Obrazenia postrzalowe w materialach Katedry i Zakladu Medycyny Sadowej CM UMK w Bydgoszczy z lat 1995-2005.

The Medical Forensic Institute, Collegium Medicum, Nicolaus Copernicus University in Bydgoszcz annually conducts approximately 600 autopsies. Gunshot wounds constitute only a small percentage of that number. The authors of this work have conducted an analysis of autopsy protocols prepared at the Institute in the years 1995-2005. During this period, 48 people were found dead as a result of gunshot wounds. This number constitutes 0.66% of all autopsies conducted within that time-frame. The objective of this study was an attempt at assessing the character of this phenomenon with due consideration given to the following parameters: age, sex, sobriety, circumstances and locality of the event, as well as season of the year, at comparing the results with data found in the literature on the subject.

The function of interleukin 17 in the pathogenesis of rheumatoid arthritis.

Interleukin (IL)-17 is a 30- to 35-kDa homodimeric polypeptide cytokine cloned in 1993 and originally named cytotoxic T lymphocyte-associated antigen-8 (CTLA-8). Sequencing the human genome resulted in the discovery of an additional five members of the IL-17 family that were consecutively named IL-17B to IL-17F. IL-17A is exclusively produced by a newly identified CD4+ T-helper subset that was recently named Th17. Differentiation of these cells from naive CD4+ T cells requires both TGF-beta and IL-6. IL-15 and, especially, IL-23 are required for these cells' survival and efficient IL-17 production. IL-17 binding to an IL-17 receptor expressed on epithelial, endothelial, and fibroblastic stromal cells triggers the activation of transcription factor NF-kappaB and mitogen-activated protein kinase (p-38), which in turn results in the secretion of IL-1, TNF-alpha, IL-6, IL-8, or prostaglandin E2. The IL-17 family plays a key role in the regulation of immune and inflammatory response, in the homeostasis of several tissues, and the progression of autoimmune diseases. In addition, IL-17 exerts synergistic effects with TNF-alpha and IL-1 in the induction of joint inflammation and cartilage and joint destruction. Given these properties, it is not surprising that in certain pathological conditions, for example rheumatoid arthritis, Th17 cells emerge as a new pathological cell type that, by IL-17 production and release, contributes to their pathogeneses.

[Analysis of deaths caused by rail-vehicles in the materials collected by the Department of Forensic Medicine in Bydgoszcz in the years 1992-2002]. / Analiza zgonów w nastepstwie wypadków z udzialem pojazdów szynowych w materialach Katedry Medycyny Sadowej CM w Bydgoszczy w latach 1992-2002.

The analysis focused on cases of death following accidents involving rail-vehicles, in which autopsies were performed at the Department of Forensic Medicine in Bydgoszcz in the years 1992-2002. Sixty-five individuals died in train accidents (80%), 16 people (20%) were victims of tram accidents (20%). Males constituted 86% of the material, while females accounted for 14%. The highest number of rail-vehicle accidents was observed in 1992. The most common reason of death was multiorgan injury. In the subgroup of tram accidents, the authors noted no injuries of the highest severity, such as limb amputations, amputations or crushing of the head, or fragmentation or rupturing of the trunk. On the other hand, in railroad accidents, the percentage of the above injuries was 26%, 44% and 24.5%, respectively. The most severe injuries were noted in the group of people hit by a train while in prone position.

Identification of evolutionary conserved mouse sperm surface antigens by human antisperm antibodies (ASA) from infertile patients.

PROBLEM: The presence of antisperm antibodies (ASA) in semen may impair sperm function leading to immunological infertility. The aim of the study was to identify the evolutionary conserved antigens on mouse sperm surface that react with human ASA in order to study the mechanism of autoimmune infertility. METHODS OF STUDY: The binding of human ASA to mouse sperm was investigated by means of indirect immunofluorescence. 2D-electrophoresis was applied to separate the biotin-labelled mouse membrane proteins using isoelectric focusing followed by polyacrylamide gel electrophoresis. Cognate antigens of ASA from seminal plasma of infertile patients were analysed by Western blotting. Performing avidin-blots it was detected which of the proteins recognized were sperm surface proteins. The spots of interest were analysed by means of mass spectrometry. RESULTS: ASA bound most frequently (36%) to the post-acrosomal region and to the midpiece of mouse spermatozoa. About 30% of ASA recognized apo lactate dehydrogenase (LDHC4) as a cognate antigen, 30% voltage-dependent anion channel (VDAC2). ASA of 20% bound to outer dense fibre protein and 20% of samples recognized glutathione S-transferase mu5. CONCLUSIONS: Human ASA bound to specific cognate antigens of mouse spermatozoa, offering the possibility to study their functional relevance in the mouse model.