A. Younis

Permeability of the rhesus monkey oocyte membrane to water and common cryoprotectants

Successful cryopreservation of oocytes of the rhesus monkey (Macaca mulatta) would facilitate the use of this valuable animal model in research on reproduction and development, while providing a stepping stone towards human oocyte cryopreservation and the conservation of endangered primate species. To enable rational design of cryopreservation techniques for rhesus monkey oocytes, we have determined their osmotic and permeability characteristics in the presence of dimethylsulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PROH), three widely used cryoprotectants. Using nonlinear regression to fit a membrane transport model to measurements of dynamic cell volume changes, we estimated the hydraulic conductivity (Lp) and cryoprotectant permeability (Ps) of mature and immature oocytes at 23.5°C. Mature oocyte membranes were most permeable to PROH (Ps = 0.56 ± 0.05 µm/sec) and least permeable to DMSO (Ps = 0.24 ± 0.02 µm/sec); the permeability to EG was 0.34 ± 0.07 µm/sec. In the absence of penetrating cryoprotectants, mature oocytes had Lp = 0.55 ± 0.05 µm/min/atm, whereas the hydraulic conductivity increased to 1.01 ± 0.10, 0.61 ± 0.07, or 0.86 ± 0.06 µm/min/atm when mature oocytes were exposed to DMSO, EG, or PROH, respectively. The osmotically inactive volume (Vb) in mature oocytes was 19.7 ± 2.4% of the isotonic cell volume. The only statistically significant difference between mature and immature oocytes was a larger hydraulic conductivity in immature oocytes that were exposed to DMSO. The biophysical parameters measured in this study were used to demonstrate the design of cryoprotectant loading and dilution protocols by computer‐aided optimization. Mol. Reprod. Dev. 76: 321–333, 2009.

Serum tumor necrosis factor-α, interleukin-6, monocyte chemotactic protein-1 and paraoxonase-1 profiles in women with endometriosis, pcos, or unexplained infertility

ObjectiveTo investigate the serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and Paraoxonase-1 (PON-1) during fertility treatment of women with endometriosis (Endo), PCOS or unexplained infertility (Unexpl).MethodsThirty-six patients with Endo, PCOS or Unexpl undergoing controlled ovarian stimulation for IVF or IUI were consented and their serum, on day-3 (baseline) and at the end of FSH treatment (peak), was collected and investigated for levels of TNF-α, IL-6, MCP-1, and PON-1. Correlations, ANOVA and Students t-test were used for statistical analysis.ResultsPeak serum levels of IL-6, MCP-1 and PON-1 were positively correlated to E2 peak levels. TNF-α levels were inversely correlated to estradiol levels and they were lower in patients who ultimately became pregnant when compared to non-pregnant (P < 0.05). Mean TNF-α levels were significantly higher in Unexpl group (P < 0.05). The mean levels of IL-6, and MCP-1 were significantly (p < 0.05) higher in women with PCOS compared with Endo and Unexpl. No differences were found between the three clinical groups in patient’s age, BMI, Day-3 FSH, PON-1 and pregnancy outcome.ConclusionCirculating cytokine levels were influenced by ovarian stimulation, as demonstrated by increased levels of IL-6, MCP-1 and PON-1, and decreased level of TNF-α at the end of controlled ovarian stimulation. While evidence of relationship between circulating cytokines with mild endometriosis was not found, PCOS was associated with elevated serum IL-6 and MCP-1 but lower TNF-α concentration. Unexplained infertility was associated with elevated TNF-α level. No relationship between serum PON-1 concentration and PCOS, mild endometriosis or unexplained infertility was noted.

Paraoxonase-1 enzyme activity assay for clinical samples: validation and correlation studies.

Background Paraoxonase-1 (PON1) enzyme is reported in various types of tissues and linked to numerous pathophysiological disorders. It is a potential biomarker in many pathological conditions such as cardiovascular diseases. Material/Methods We conducted several small-scale studies to evaluate PON1 performance as affected by sample types, storage, and interferences. We also carried out short-term studies to compare the performance of the widely used PON1 assay to the similar commercially available PON1 kit assay method; sample size for the method comparison was N=40, and the number varied for other validation experiments. Results Our studies using various types of anticoagulants show that samples collected in tubes with NaF, citrate, EDTA, clot activator, and sodium heparin have increased PON1 levels that are 49%, 24.5%, 19.8%, 11.4%, and 8%, respectively, higher compared to serum samples collected in plain tubes. However, samples collected in lithium heparin tubes demonstrated 10.4% lower PON1 levels compared to serum collected in plain tubes. Biological interference such as hemolysis has little effect on PON1 levels; however, samples spiked with lipids have shown 13% lower PON 1 levels. Our studies comparing the PON1 method commonly available for PON1 assay and a similar non-ELISA commercially available PON1 kit method showed a weak Spearman correlation coefficient of R2=0.40 for the range of 104.9–245.6 U/L. Conclusions The current study provides new validation data on enzyme PON1 performance. While no appreciable change was seen with storage, samples type affects the enzyme performance. Our results should encourage additional clinical studies to investigate other aspects of factors known to affect PON1 enzyme function and performance.