Comparing different sperm separation techniques for ART, through quantitative evaluation of p53 protein.

CONTEXT: In the last 10 years, assisted reproductive technologies (ARTs) have offered infertile couples an opportunity to complete their reproductive project. However, the high failure rate could be explained with the complex human reproduction system. In ART, the decrease of the success is due to the conditions far from the natural ones. AIMS: The aim of this study is to evaluate deoxyribonucleic acid (DNA) damage of spermatozoa before and after selection procedures, using a new technique able to quantize sperm DNA damage. SETTINGS AND DESIGN: They were involved 43 males domiciled permanently in two areas with different Environmental Impact, HEI (high environmental impact) and LEI (Low environmental impact), they are aged between 24 and 31 years with various degrees of dyspermia. SUBJECTS AND METHODS: The 43 males were divided into two groups: 21 in Group A (EIL) and 22 in Group B (EIH). The samples must be aliquoted into parts of 0.5 mL: Group (a) Control, no processing; Group (b) Swim-up (SUP) from semen; Group (c) classic SUP; Group (d) density gradient centrifugation (DGC). All samples were subjected to a quantitative dosage of p53 protein, before and after processing. STATISTICAL ANALYSIS USED: For the development of the probability and significance of the data, the Student's t-test was used. RESULTS: From our data, it emerges that Groups D and B provide a superior quality about motility, vitality, and apoptosis indexes compared to other conventional techniques. In Group B, apoptosis is comparable to Group D, but they have slightly lower about motility and vitality. Group C is the one that has lower parameters than the other techniques. Regarding the evaluation of p53 protein, the results are conflicting with the evaluation of apoptosis; in fact, in Group D, the values are significantly higher than the other techniques. CONCLUSIONS: Sperm separation is an important moment in ART techniques. From our data, it emerges a greater fragility of DNA in the male spermatozoa who reside permanently in areas with high environmental impact.

p53 Protein Evaluation on Spermatozoa DNA in Fertile and Infertile Males.

INTRODUCTION AND OBJECTIVES: Protein p53 role in the spermatogenesis is demonstrated, it guarantees both the appropriate quality and quantity of mature spermatozoa. In this observational study we evaluate the eventual correlation between "corrected" p53 concentration on human spermatozoa DNA and male fertility potential. MATERIALS AND METHODS: Our work is based on an observational study made of 169 male in a period between March 2012 and February 2017. The entire study group is composed by 208 male partners aged between 26-38 years with ejaculate volume from 0.6 to 5.8 mL and heterogeneous seminal valuation: 86/208 (41,3%) normospermic; 19/208 (9,1%) mild oligospermic; 51/208 (24,5%) moderate oligospermic to; 52/208 (25,1%) with severe oligospermic. The "control" group A includes 39 male partners considered "fertile", because we did the p53 "corrected" test on their spermatozoa after 28 ± 3,5 days from the positives of their partners pregnancy test (betaHCG> 400 m U/m L). The group B, subdivided in B1, B2 and B3, includes 169 male partners for a observational period of 60 months. This partners don't report previous conceptions, they aren't smokers, don't make use neither of alcohol nor drugs and don't present pathologic varicocele studied with ecoColorDoppler. They are all married or stable cohabitants from a period of 27-39 months and report to have frequent sex without protection with their partners. Determination of p53 procedure: To separate the spermatozoa from seminal fluid we utilized the Differex™ kit System and the DNA IQ™ kit (Promega). For the p53 test we used the direct DuoSet IC kit and quantitative (R&D System). The p53 values were corrected in respect to the spermatozoa concentration expressed in ng/millions of spermatozoa. RESULTS: Group A (39 male) presents "correct" p53 values that vary from 0.35 to 3.20 ng/millions of spermatozoa and group B presents values that vary from 0.68 to 14.53. From group B (48 male) in the observational period we have recorded 21 pregnancies with initial "correct" p53 values that vary from a minimum of 0.84 to a maximum of 3.29. In the subgroup B1 we obtained 8 pregnancies from male partners with a "correct" p53 concentration included between 0.84 to 1.34. In the subgroup B2 we obtained 13 pregnancies from male partners with a "correct" p53 concentration included between 1.66 and 3.29. In the subgroup B3 (121 male) there weren't neither pregnancies nor miscarriages and "correct" p53 values were included between 3.58 and 14.53. CONCLUSION: The results show that the member of the group A with values of 'corrected' p53 between 0.35 and 3.20 were considered "Fertile", although in the observational period 3 miscarriages happened for 3 partners. 36 partners on 39 (92,3%) had a p53 concentration inferior to 1.65, this value were considered as the extreme to identify this group. The member of the group B1 had "corrected" p53 concentration that were included in the group. In the group B2 were observe 13 pregnancies, so its member were considered "potentially fertile" In the group B3 (121 male) weren't observe neither pregnancies nor miscarriages, so its member were considered "potentially infertile". If further studies confirm these data, we will consider the p53 test ELISA inspected in "correct" p53 as a new and accurate marker of the potential of male fertility.

Biogeography of Sinorhizobium meliloti nodulating alfalfa in different Croatian regions.

Sinorhizobium meliloti is a nitrogen-fixing rhizobium symbiont of legumes, widespread in many temperate environments the high genetic diversity of which enables it to thrive as a symbiont of host legumes and free-living in soil. Soil type, together with geographic differences and host plant genotype, seem to be prominent factors in shaping rhizobial genetic diversity. While a large body of research supports the idea that the genetic structure of free-living microbial taxa exhibits a clear biogeographic pattern, few investigations have been performed on the biogeographic pattern of S. meliloti genotypes in a restricted geographic range. In the present study, a collection of 128 S. meliloti isolates from three different regions in Croatia was investigated to analyze the relationship between genetic diversity, geographic distribution, soil features and isolate phenotypes by using amplified fragment length polymorphism (AFLP) as a genome-wide scanning method. Results obtained led to the conclusion that the genotypes of isolates cluster according to the region of origin and that the differentiation of S. meliloti populations can be mainly ascribed to geographic isolation following an isolation-by-distance model, with a strong distance-decay relationship of genetic similarity with distance, in which local soil conditions are not the major component influencing the isolate phenotypes or their genomic differentiation.

Molecular characterization of acinetobacter isolates collected in intensive care units of six hospitals in Florence, Italy, during a 3-year surveillance program: a population structure analysis.

The strain diversity and the population structure of nosocomial Acinetobacter isolated from patients admitted to different hospitals in Florence, Italy, during a 3-year surveillance program, were investigated by amplified fragment length polymorphism (AFLP). The majority of isolates (84.5%) were identified as A. baumannii, confirming this species as the most common hospital Acinetobacter. Three very distinct A. baumannii clonal groups (A1, A2, and A3) were defined. The A1 isolates appeared to be genetically related to the well-characterized European EU II clone. A2 was responsible for three outbreaks which occurred in two intensive care units. Space/time population dynamic analysis showed that A1 and A2 were successful nosocomial clones. Most of the A. baumannnii isolates were imipenem resistant. The genetic determinants of carbapenem resistance were investigated by multiplex PCR, showing that resistance, independently of hospital origin, period of isolation, or clonal group, was associated with the presence of a bla (OXA-58-like) gene and with ISAba2 and ISAba3 elements flanking this gene. bla (OXA-58) appeared to be horizontally transferred. This study showed that the high discriminatory power of AFLP is useful for identification and typing of nosocomial Acinetobacter isolates. Moreover the use of AFLP in a real-time surveillance program allowed us the recognition of clinically relevant and widespread clones and their monitoring in hospital settings. The correlation between clone diffusion, imipenem resistance, and the presence of the bla(OXA-58-like) gene is discussed.

Molecular surveillance and population structure analysis of methicillin-susceptible and methicillin-resistant Staphylococcus aureus in high-risk wards.

In this study we report the results of analysis of 253 isolates of Staphylococcus aureus (132 methicillin [meticillin]-resistant S. aureus [MRSA] isolates and 121 methicillin-susceptible S. aureus [MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureus infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureus species, maximizing benefits and reducing the cost of infection control strategies.