Quantitative Analysis of Serum Procollagen Type I C-Terminal Propeptide by Immunoassay on Microchip

BACKGROUND: Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. METHODS AND FINDINGS: The microchip was made of cyclic olefin copolymer with straight microchannels that were 300 µm wide and 100 µm deep. For the construction of a sandwich ELISA for procollagen type I C-peptide (PICP), a biomarker for bone formation, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-PICP antibody on the surface of the microchannel. After the infusion of the mixture of 2.0 µl of peroxidase-labeled 2nd anti-PICP antibody and 0.4 µl of sample to the microchannel and a 30-min incubation, the substrate for peroxidase was infused into the microchannel; and the luminescence intensity of each spot of 1st antibody was measured by CCD camera. A linear relationship was observed between PICP concentration and luminescence intensity over the range of 0 to 600 ng/ml (r(2) = 0.991), and the detection limit was 4.7 ng/ml. Blood PICP concentrations of 6 subjects estimated from microchip were compared with results obtained by the conventional method. Good correlation was observed between methods according to simple linear regression analysis (R(2) = 0.9914). The within-day and between-days reproducibilities were 3.2-7.4 and 4.4-6.8%, respectively. This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. CONCLUSION: This assay enabled us to determine serum PICP with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis

Impact of the one‐carbon metabolism on oocyte maturation, fertilization, embryo quality, and subsequent pregnancy

Purpose: To investigate impact of the one‐carbon metabolism (OCM) on oocyte maturity and embryo development. Methods: This prospective study analyzed 18 women who agreed to participate. We measured the OCM biomarkers’ concentrations including Vitamin B12 (VB12), folic acid (FA), and homocysteine (Hcy) in serum and follicular fluid (FF), and assessed their correlation. We also evaluated the influence of such OCM biomarker concentrations in mono‐FF on oocyte maturation, fertilization, embryo quality, and consequent pregnancy after embryo transfers. Results: All biomarkers showed a high concentration variability in different follicles of each woman, but their mean levels correlated with the serum levels. Among the 106 collected oocytes, 92 were mature, 59 were fertilized, and 16 yielded good‐quality embryos. We performed 26 single embryo transfers, and 7 patients achieved clinical pregnancies. VB12 concentration (FF) was significantly lower in fertilized than unfertilized oocytes by univariate analysis. In multivariate logistic analysis, a significant correlation was found between FA concentration (FF) <14.25 ng/mL and good‐quality embryos and between Hcy concentration (FF) <4.9 nmol/mL and clinical pregnancy. Conclusion: OCM in FF may affect fertilization, embryo quality, and clinical pregnancy

Microbiome analysis in women with endometriosis: Does a microbiome exist in peritoneal fluid and ovarian cystic fluid?

Abstract Purpose To investigate the relationship between the microbiome of the female genital tract and endometriosis. Methods This prospective cohort study included 36 women who underwent laparoscopic surgery for ovarian tumor from July 2019 to April 2020. Of them, 18 had endometriosis, and 18 did not have endometriosis. Vaginal secretions, endometrial fluid, peritoneal fluid, and ovarian cystic fluid were collected during surgery. Next‐generation sequencing of bacterial 16S rRNA was performed to characterize the microbiome. Results Specific microbiomes were not detected in either peritoneal fluid or ovarian cystic fluid regardless of the presence or absence of endometriosis and the type of cyst. When the cutoff value of infectious bacterial abundance in the vagina was set as 64.3%, there were many cases more than a cutoff value in the endometriosis group significantly (p = 0.01). When the cutoff value of infectious bacterial abundance in the endometrium was set as 18.6%, there were many cases more than a cutoff level in the endometriosis cases significantly (p = 0.02). Conclusion Peritoneal fluid and ovarian cystic fluid are almost sterile, although dysbiosis may occur in the vaginal and endometrial microbiome in women with endometriosis

Comparative analysis of the values obtained with the microchip method and the conventional microtitration plate method.

<p>Linear regression analysis was used.</p

Reproducibility of microchip method for the determination of PICP in serum samples.

<p>Reproducibility of microchip method for the determination of PICP in serum samples.</p

Reproducibility of luminescence intensity of PICP in 3 different channels.

<p>Reproducibility of luminescence intensity of PICP in 3 different channels.</p