Cell-free DNA in Embryo Culture Media as Non-invasive Biomarker of the Quality of Embryo Cleavage

Background: The success of in vitro-fertilization (IVF) cycles is determined in large part by the quality of embryo cleavage, which in turn, is dependent on the quality of the embryo culture media (CM). Many factors can influence the quality of embryo CM, one of which is the levels of Cell Free Deoxyribonucleic acid (DNA). Understanding the association between Cell-free DNA levels in embryo CM and the quality of embryo cleavage could help improve the quality of IVF techniques. Methods: This prospective study was conducted with 96 spent CM from patients undergoing IVF cycle, in order to determine relationships of Cell-free DNA levels in embryo CM with embryo cleavage quality on day 3. After intracytoplasmic sperm injection (ICSI), 48 embryos were evaluated on day 3 of their development, according to their cell number. Day 2 and day 3 CM corresponding to each one of the embryos was analyzed, by quantitative PCR, for estimation of Cell-free DNA levels. Results: The results revealed a significant increase in Cell-free DNA levels on day 2 CM corresponding to 4 to 6 cell embryos compared to those corresponding to 7 to 8 cell embryos (p=0.04). As for day 3 CM, the results showed no significant difference between the Cell-Free DNA levels in CM of 7-8 and those of 4-6 cell embryos (p=0.4). Also, cell free DNA levels in embryo CM, were significantly higher on day 2 compared to day 3 for both 7-8 and 4-6 cell embryos (p=0.03; p=0.04). Conclusion: We conclude that cell-free DNA levels in CM might be associated with delayed embryo cleavage.

Lipid Peroxidation Status in Embryo Culture Media: The Impact on Fertilization and Embryo Quality during IVF Cycles

Background: The process of preimplantation embryo development in vitro represents a key phase during in vitro fertilization (IVF) cycles. It involves several regulatory signaling pathways as well as an optimized in vitro culture system. The resulting embryo quality helps to determine embryo competence before implantation and pregnancy outcomes. Reactive oxygen species (ROS) are known to play a major role in influencing the process of embryo development. Their role can be reflected in the regulation of signaling pathways as second messengers or in the irreversible cell alterations due to oxidative stress following an excess of ROS levels. Methods: In this study, we investigated the association between morphological embryo quality (fertilization, cleavage quality, and fragmentation levels) and lipid peroxidation levels (Malondialdehyde) in embryo culture media. After intracytoplasmic sperm injection (ICSI), a total of 103 oocytes were evaluated on day 1 and day 3 of their development, and their corresponding culture media were analyzed by estimating MDA levels using thiobarbituric acid. Results: The results showed no significant association between MDA levels in culture media and fertilization rate (p=0.3), sperm quality (p=0.99; p= 0.17; p=0.46; p=0.30; p=0.65; p=0.44; p=0.09; p=0.15; p=0.56), embryo fragmentation levels (p=0.79; p=0.40), AMH levels (p=0.31; p=0.36) and female age (p=0.60; p=0.34). However, we revealed a significant association between cleavage quality and MDA levels in the embryo environment (p=0.03). Conclusion: We conclude that oxidative stress in IVF culture media might be mainly associated with delayed embryonic development.

Severe Oligospermia Treatment with Testicular Sperm Using 揑CSI�

Introduction: Assisted reproductive technology has been developed significantly throughout the past few years, particularly diagnosing and treating male infertility. Many studies have been performed showing that Intracytoplasmic Sperm Injection (ICSI) is a successful method to attain clinical pregnancy and live birth through impaired spermatozoa characteristics or low sperm count, such as severe oligospermia. Severe oligospermia indicates low sperm count, which in some cases leads to azoospermia. Severe oligospermia can be caused by several factors such as genetics or medication. In search of efficient treatment for couples with Severe oligospermia, numerous retrospective and prospective researches have reported high pregnancy and live birth rates through testicular sperm for men with severe oligospermia and cryptozoospermia with or without high sperm DNA damage. The research showed that the use of testicular sperm in combination with ICSI yielded a high pregnancy rate and live births over another source of sperm, such as ejaculated sperms. Moreover, the use of ICSI in severe oligospermia has shown successful fertilization and pregnancy. Methods: In search for effective treatment for couples with severe male factor, a number of small retrospective and prospective studies have reported high pregnancy and live birth rates using testicular sperm for men with necrozoospermia, cryptozoospermia and oligozoospermia with or without elevated sperm DNA damage. Although the data suggest that there may be some benefit in performing testicular sperm retrieval (TSR)-ICSI in select groups of non-azoospermic infertile men, there are potential risks involved with TSR. Clinicians should balance these risks prior to the recommendation of TSR-ICSI on the result of a semen analysis or sperm DNA test alone. Careful evaluation and management of male factor infertility is important. The use of TSR-ICSI in the absence of specific sperm DNA defects is still experimental. Discussion: In 1992 and subsequently, several reports indicated that ICSI was a successful technique to achieve clinical pregnancy and live birth using spermatozoa with severely impaired characteristics. The initial optimism over the ability of ICSI to overcome significant sperm abnormalities was later tempered by the findings of more recent publications suggesting that some sperm deficits may not be as effectively treated with ICSI. Conclusion: Severe oligospermia indicates low sperm count, which can lead to male infertility; severe oligospermia which can be overcome through ICSI. Genetic factors like microdeletions of the Y chromosome (Yq) can cause severe oligospermia or chemotherapy molecules, affecting the sperm count directly.

olecular Analysis of Genetic Diversity of Pectobacterium carotovorum Subsp Carotovorum Isolated in Morocco by PCR Amplification of the 16S-23S Intergenic Spacer Region.

Aims: Pectobacterium carotovorum is a ubiquitous bacterium that causes soft rot in different crop plants throughout the world. In Morocco, approximately 95% of the Strains isolates from potato plants with tuber soft rot are P. carotovorum. In this study, we test whether PCR ribotyping can be used to distinguish strains of Pectobacterium carotovorum isolated from soft rot potato and to differentiate among strains from different geographic regions. Place and Duration of Study: Laboratory of Virology, Microbiology and Quality / Ecotoxicology and Biodiversity, department Biology, Faculty of Sciences and Techniques, University Hassan-II Mohammedia Casablanca. Methodology: Eighty-three pectolytic enterobacteria were collected from potatoes rotten in Morocco, the strains were isolated in the Cristal Violet Pectate (CVP) medium and were purified in LPGA agar (yeast extract, peptone, glucose and agar). After purification, strains were identified by physiological and biochemical tests. The confirmation of species was performed by PCR using primers Y1 and Y2. The genetic diversity of Pectobacterium carotovoum was investigated by PCR ribotyping using primers G1/L1, which are complementary to conserved regions of the rRNA operon. Furthermore, the profiles obtained were compared by the Unweighted Pair Group Method. Results: The biochemical and physiological analysis demonstrated that the predominant pectolytic enterobacterium present in Morocco is Pectobacterium carotovorum subsp carotovorum. The specific confirmation of species P. carotovorum by PCR has yielded a 434 bp DNA fragment of the pelY gene with all isolates. Further, PCR amplification of the 16S-23S Intergenic spacer Region (ITS-PCR) has presented a specific pattern made of 2-6 fragments ranging from 300 bp to 800 bp. The UPGMA tree has shown that there is considerable genetic diversity in P. carotovorum strains, which can be divided into four distinct groups.

Prevalence and Antibiotic-Resistance of Salmonella Isolated from Food in Morocco

Background: Salmonellosis remains one of the most frequent food-borne diseases worldwide; especially in developing countries. The emergence of antimicrobial resistance in Salmonella isolates from food can potentially compromise the treatment of these infections. This investigation was conducted for the first time in Morocco both to detect the occurrence of Salmonella in foods as well as to determine the antibiotic resistance profile of the Salmonella isolates. Methodology: In total; 11;516 food samples collected from 2002 to 2005 were investigated. Isolated Salmonella were characterized by serotyping and susceptibilities were determined for 15 antimicrobial drugs using the disc diffusion assay. Results: The overall percentage of Salmonella prevalence (n=105) was 0.91with rates of 71for slaughterhouses and 9for seafood. Sixteen different serotypes were identified among 104 Salmonella enterica isolates including serotypes Infantis (n=25); Bredeney (n=13); Blokley (n=11); Typhimurium (n=9); Mbandaka (n=8); Branderup II (n=7); and Kiambu (n=6); 1 isolate of Salmonella enterica belonged to subspecies II salamae. Twenty-nine percent of isolates (n=30/105) were resistant to at least one antimicrobial. Resistance to tetracycline was the most common finding (21); followed by resistance to ampicillin (13); amoxicillin+clavulanic acid (9); streptomycin (7); chloramphenicol (4) and nalidixic acid (3;8). None of the isolates was resistant to 3rd-cephalosporin and fluoroquinolones (i.e. ciprofloxacin). Multidrug resistance (MDR) was seen in 9.5of the isolates; mainly in S. Typhimurium DT104 with R-type ACSSuT and S. Hadar. Conclusions: Despite a low frequency of Salmonella isolation; S. Typhimurium DT104 was identified in the first step of the food chain. The study points out the need control antibiotic resistance in Salmonella isolated from food in Morocco to avoid the spread of MDR

Prevalence and Antibiotic-Resistance of Salmonella Isolated from Food in Morocco

Background: Salmonellosis remains one of the most frequent food-borne diseases worldwide; especially in developing countries. The emergence of antimicrobial resistance in Salmonella isolates from food can potentially compromise the treatment of these infections. This investigation was conducted for the first time in Morocco both to detect the occurrence of Salmonella in foods as well as to determine the antibiotic resistance profile of the Salmonella isolates. Methodology: In total; 11;516 food samples collected from 2002 to 2005 were investigated. Isolated Salmonella were characterized by serotyping and susceptibilities were determined for 15 antimicrobial drugs using the disc diffusion assay. Results: The overall percentage of Salmonella prevalence (n=105) was 0.91with rates of 71for slaughterhouses and 9for seafood. Sixteen different serotypes were identified among 104 Salmonella enterica isolates including serotypes Infantis (n=25); Bredeney (n=13); Blokley (n=11); Typhimurium (n=9); Mbandaka (n=8); Branderup II (n=7); and Kiambu (n=6); 1 isolate of Salmonella enterica belonged to subspecies II salamae. Twenty-nine percent of isolates (n=30/105) were resistant to at least one antimicrobial. Resistance to tetracycline was the most common finding (21); followed by resistance to ampicillin (13); amoxicillin+clavulanic acid (9); streptomycin (7); chloramphenicol (4) and nalidixic acid (3;8). None of the isolates was resistant to 3rd-cephalosporin and fluoroquinolones (i.e. ciprofloxacin). Multidrug resistance (MDR) was seen in 9.5of the isolates; mainly in S.. Typhimurium DT104 with R-type ACSSuT and S. Hadar. Conclusions: Despite a low frequency of Salmonella isolation; S. Typhimurium DT104 was identified in the first step of the food chain. The study points out the need control antibiotic resistance in Salmonella isolated from food in Morocco to avoid the spread of MDR