Extracellular embryo genomic DNA and its potential for genotyping applications

none8noBackground: Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping. Results/methodology: We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively. Conclusion: A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout.openGalluzzi, Luca; Palini, Simone; Destefani, Silvia; Andreoni, Francesca; Primiterra, Mariangela; Diotallevi, Aurora; Bulletti, Carlo; Magnani, MauroGalluzzi, Luca; Palini, Simone; Destefani, Silvia; Andreoni, Francesca; Primiterra, Mariangela; Diotallevi, Aurora; Bulletti, Carlo; Magnani, Maur

Determination of Plasma Antioxidant Power in Capillary Blood through the Innovative system PAT (Plasma Antioxidant Test)

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Role of Kamut® brand khorasan wheat in the counteraction of non-celiac wheat sensitivity and oxidative damage

It has been suggested that ancient grains show lower immunogenic properties and therefore can be introduced in the diet of non-celiac wheat-sensitive people. In the present study we investigated the possible difference in inflammation caused by feeding ancient Kamut® wheat pasta (KP) compared to modern durum wheat pasta (WP) to rats. The effect of the two experimental diets on the oxidative status was also compared in basal condition and after an exogenous oxidative stress. In rats fed WP the histological evaluation of the duodenum morphology evidenced a flattened mucosa, an unusual shape and shortening of the villi, and a high lymphocyte infiltration, while no modifications were detected in KP fed animals. The fecal metabolite profiling was differently modified by the two diets, suggesting significant changes in the gut microflora. Furthermore, the results confirmed previous data on the antioxidant protection in rats by Kamut® wheat foods. It is conceivable that Kamut® components can act through a hormetic effect, eliciting an adaptive response that protects the organism against both oxidative stress and inflammatio

Validation of a patented method to determine the antioxidant capacity of human saliva based on the reduction of iron: the SAT test

The defense against damaging attack at mouth level caused by reactive species, in particular reactive oxygen species (ROS), is guaranteed by saliva, the main constituent of the antioxidant barrier. The aim of the performed tests was to establish the precision, linearity, and accuracy of the new patented test, SAT, on saliva samples taken from healthy volunteers. The analysis also provided useful information on storage conditions of the sample at low temperatures and on the normality range and defined the influences of interferences (in particular phosphates) in the determination

Performance evaluation of the innovative PAT test, comparison with the common BAP test and influence of interferences on the evaluation of the plasma antioxidant capacity

Antioxidants (AOs) represent the main barrier of defense against damaging aggression due to reactive species, in particular by reactive oxygen species (ROS). The plasma AO capacity is a measure of physiological, environmental, and nutritional factors (exposure to ROS and antioxidant supplementation) determining the redox status in humans and can underline the oxidative stress (OS) conditions in the progression/development of many diseases. Moreover, changes in AO plasma content after supplementation may provide information on the absorption and bioavailability of nutritional compounds and efficacy of AO therapy

A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen

The well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2',7'-dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments

Differences in blood and semen oxidative status in fertile and infertile men, and their relationship with sperm quality

Oxidative stress plays a fundamental role in the aetiology of male infertility by negatively affecting sperm quality and function. Assessment of blood and seminal plasma oxidative profiles might be a valuable tool to improve evaluation of sperm reproductive capacity and functional competence. This study examined the lipid-soluble antioxidant profile and levels of lipid peroxidation both in blood and seminal plasma samples of infertile and fertile males, in relation to semen parameters. Total antioxidant capacity (TAC) and vitamin E concentrations were significantly (P<0.05) lower in seminal plasma of infertile men compared with fertile subjects; concurrently, a significant accumulation of malondialdehyde was found in infertile patients (P=0.032 compared with controls), which was negatively correlated with sperm motility and morphology. In blood samples, infertile men presented lower concentrations of TAC, carotenoids and vitamin E than fertile subjects; TAC and carotenoids were positively correlated with sperm motility, morphology and concentration. Finally, blood TAC and vitamin E concentrations were positively correlated with the corresponding seminal values, confirming the close relationship between blood and semen antioxidants. All these results indicated the possibility of using not only seminal antioxidants but also blood antioxidants as biochemical markers to support sperm quality evaluation. Oxidative stress induced by reactive oxygen species (ROS) has been widely recognized as one of the major causes of male infertility; indeed, excessive ROS production can negatively impact sperm quality and function. The assessment of blood and seminal plasma oxidative profiles has been suggested as a valuable tool to improve the evaluation of sperm reproductive capacity and functional competence in infertile men. With this in mind, in the present study we examined the lipid soluble antioxidant profile (carotenoids and vitamins A and E) and the levels of lipid peroxidation (malondialdehyde; MDA) both in blood and seminal plasma samples of infertile and fertile males, in correlation with semen parameters namely motility, morphology and concentration. As a result, we obtained evidence that the total antioxidant capacity (TAC) and the concentrations of vitamin E of seminal plasma samples were significantly lower in infertile men than in fertile subjects; at the same time, a significant accumulation of MDA was found in infertile patients. MDA, in turn, negatively correlated with sperm motility and morphology, thus confirming that oxidative damage to lipids impairs sperm quality. In blood samples, infertile men presented lower TAC and lower concentrations of carotenoids and vitamin E than fertile subjects; interestingly, TAC and carotenoid concentrations were positively correlated with sperm motility, morphology, and concentration, confirming the close relationship between blood antioxidants and sperm quality. In conclusion, all these results suggested that the examination of blood and semen oxidative profiles might furnish useful information on sperm quality and function in infertile men

A new micro swim-up procedure for sperm preparation in ICSI treatments: Preliminary microbiological testing

Objective: This study aimed to assess the levels of microbial contamination in semen samples before and after the micro swim-up (MSU) procedure in intra-cytoplasmic sperm injection (ICSI). The new method is an upgrade to the classic wash swim-up procedure. Methods: Semen analysis and microbiological tests were carried out before and after the MSU procedure. A total of twenty semen samples were analyzed. Results: Pathogens were observed in semen samples only before MSU and never after ICSI. Microbiological tests revealed a large prevalence of gram-positive cocci [Staphylococcus spp. (n=16, 80%) and viridans streptococci (n=10, 50%)]. The results of this study indicate that direct MSU in ICSI improved the ICSI workflow. Conclusion: The new workflow is faster and more affordable, and is likely to prevent infection problems that could arise from the normal microbial flora of the semen

Influence of ovarian stimulation for IVF/ICSI on the antioxidant defence system and relationship to outcome

Ovarian stimulation is used with IVF/intracytoplasmic sperm injection (ICSI) cycles to obtain multiple oocytes and improve pregnancy rates; however, it also induces perturbation in the oxidant-antioxidant balance leading to oxidation stress. The present study monitored the plasma antioxidant status in women undergoing a long agonist protocol of ovarian stimulation at three different time points: at baseline (T0), after pituitary suppression (T1) and on the day of oocyte retrieval (T2). The antioxidant composition of follicular fluid samples collected on T2 was also evaluated. Significant decreases (P < 0.05) of plasma vitamin C, vitamin E and carotenoids were found between T1 and T2 but not between T0 and T1. At T2, high plasma vitamin E was associated with high numbers of total and mature oocytes retrieved per patient, which, in turn, were favourable for achieving pregnancy. Accordingly, women who became pregnant presented higher vitamin E concentrations both in plasma and FF than those who did not. In conclusion, this study confirmed the occurrence of significant modifications of the plasma antioxidant profile during ovarian stimulation with gonadotrophins; at the same time, it was found that both systemic and follicular antioxidant status may be related to IVF/ICSI outcome

Identifying Predictive Bacterial Markers from Cervical Swab Microbiota on Pregnancy Outcome in Woman Undergoing Assisted Reproductive Technologies

Background and aims: Failure of the embryo to implant causes about three-fourths of lost pregnancies. Female genital tract microbiota has been associated to Assisted Reproductive Technologies (ART) outcomes. The objective of this study was to analyze the microbiota of human cervical swab and to correlate these findings with the ART outcomes. Materials and Methods: In this study, 88 cervical swabs were collected from women undergoing ART cycles, with various causes of infertility, at the beginning of the ART protocols. After microbial DNA extraction, V3–V4 variable regions of the 16S rRNA gene were amplified and sequenced on the Illumina MiSeq platform. PEnalized LOgistic Regression Analysis (PELORA) was performed to identify clusters of bacterial populations with differential abundances between patients with unfavorable and favorable pregnancy outcome groups, respectively. Results: We identified a core of microorganisms at lower taxonomic levels that were predictive of women’s pregnancy outcomes. Statistically significant differences were identified at species levels with Lactobacillus salivarius, Lactobacillus rhamnosus among others. Moreover the abundance of Lactobacillus crispatus and iners, respectively increased and decreased in favorable group as compared to unfavorable group, resulted within the core of microorganisms associated to positive ART outcome. Although the predominance of lactobacilli is generally considered to be advantageous for ART outcome, we found that also the presence of Bifidobacterium (together with the other lactobacilli) was more abundant in the favorable group. Discussion: Cervix is colonized by microorganisms which can play a role in ART outcomes as seen by an overall decrease in embryo attachment rates and pregnancy rates in both fertile and infertile women. If confirmed in a larger cohort, the abundance of these bacteria can be useful not only as a marker of unfavorable pregnancy outcome but also they may open the way to new interventional strategies based on genital tract microbiota manipulation in order to increase the pregnancy rates in woman undergoing assisted reproductive technologies