ABSTRACT
Genome size has played an important role in the evolution of plants and animals because changes in genome size seem to accompany if not facilitate evolutionary adaptation to environmental conditions. Flow cytometry (FCM) is a widespread method for determining genome size thanks to its high accuracy and speed of measurements. Nevertheless, only a few comparative studies of FCM methods exist in the field of mycology, and reviews are absent. In this study, we compared the suitability of several concentrations and RNAse A incubation times, fixatives and buffers for estimating genome size in fungi. We chose the genus Geosmithia as a model filamentous fungus. We also introduced a new standard, Aspergillus fumigatus CEA10, to determine absolute genome size. We found FCM to be an appropriate method for measuring genome size in fungi, but optimization steps showed that incorrect propidium iodide staining of nuclei can overestimate genome size due to cytoplasmic staining. We identified fixation with methanol:glacial acetic acid (3:1 v/v), 10% DMSO, 0.1% Triton-X 100, and 5 mM EDTA in combination with Tris-MgCl2 buffer as the best treatment.
Subject(s)
Eurotiales/genetics , Flow Cytometry/methods , Genome Size/genetics , Cell Nucleus/geneticsABSTRACT
OBJECTIVE: Gonadotropin-releasing hormone (GnRH) antagonist combined with the human chorionic gonadotropin hormone (hCG) is commonly used in assisted reproduction techniques (ARTs) to induce controlled ovarian hyperstimulation (COH) and to synchronize oocyte maturation. While hCG is known to have immunomodulatory properties, we aimed to assess its effect on immunological changes, with respect to HLA-G binding receptors and embryo implantation success. DESIGN: The study involved 103 subjects, including patients undergoing COH protocols (n=66), divided on the basis of the pair's fertility disorder (FD) causes (female FD, n=29; male FD, n=37), and age matched healthy women (n=37). The relative distribution of T cell (CD3+/CD4+, CD3+/CD8+) and NK cell (CD56bright/CD16-, CD56dim/CD16+) populations was evaluated together with HLA-G ligands KIR2DL4 and LILRB1 expression by flow cytometry in the peripheral blood of all subjects, as well as in patient follicular fluids. RESULTS: Both groups of patients exhibited a significant decrease of their CD4/CD8 index, a down-modulation of LILRB1-positive CD8 T cells, and increased KIR2DL4-positive NK cell distribution, when compared to the healthy donors. We attribute these changes to the COH protocol, since the only significant change between the patient groups was in the number of cytotoxic CD56dim NK cells (elevated in the female FD group). Patients with male FD causes, having an above-average CD4/CD8 index (≥3.17) and below-average KIR2DL4+/CD56bright NK cell levels(≤13.3%), exhibited higher embryo implantation rates. CONCLUSION: The GnRH antagonist/hCG protocol promotes CD3+/CD8+ and KIR2DL4+ NK cell levels, more abundant in subjects with lower implantation rates, and thus decreases the embryotransfer success in otherwise fertile women.
