Detection Of Versican In Follicular Fluids And Its Clinical Implications

PartⅠ The Expression of Versican in Human Ovarian FolliclesObjective: There is considerable evidence that versican, member of the lectican family, plays important roles in maintaining follicular structure, follicular development, expansion of cumulus-oocyte complexes(COCs) and ovulation. In this research, we aimed to demonstrate the isoforms of versican in human ovarian follicles and understand the expression level of each isoforms in the follicular cells.Method: We collected follicular fluids from both larger follicles(≥ 18mm) and smaller follicles(≤ 10mm) from IVF or ICSI patients. The mural granulosa cells(all shorted for granulosa cells below) were separated, partly of which were used to extract the total mRNA for real-time quantitative polymerase chain reaction(q RT-PCR); and partly of which were used to extract the protein for western blot analysis. We collected cumulus cells from ICSI patients for q RT-PCR and western blot respectively.Results: The q RT-PCR showed that V0, V1, V2 and V3 mRNA were expressed in cumulus cells and granulosa cells isolated from larger follicles and smaller follicles. In the cumulus cells and granulosa cells from larger follicles, the expression level of V1 was the highest, and it was higher than that of V0, V2 and V3 mRNA respectively(P < 0.05) from the corresponding cells. In the cumulus cells, the expression level of V0 and V2 was second only to the level of V1, and the level of V3 isoforms was the lowest. In the granulosa cells from larger follicles, the level of V2 mRNA was higher than that of V0 and V3(P < 0.05),while no difference was observed of the abundance of V0 and V3 mRNA. In the granulosa cells isolated from smaller follicles, the expression level of V2 mRNA was much higher than the level of V0 and V3(P < 0.05), and the abundance of V1 was higher than V3(P < 0.05), and there was no difference between the level of V2 and V1, of V1 and V0, and of V0 and V3 isoforms.The abundance of V0 and V1 mRNA was higher in cumulus cells than that in granulosa cells from larger follicles and smaller ones(P < 0.05), and no difference was observed of the level of V0 or V1 in granulosa cells from larger follicles and granulosa cells from smaller ones. The expression level of the isoforms of V2 in cumulus cells was higher than that in granulosa cells isolated from larger follicles(P < 0.05), while there was no difference of the level of V2 between granulosa cells from larger follicles and granulosa cells from smaller follicles, between cumulus cells and granulosa cells from smaller follicles. The level of V3 in granulosa cells from larger follicles was higher than that in cumulus cells and granulosa cells isolated from smaller follicles(P < 0.05), and no difference of the level of V3 was observed between cumulus cells and granulosa cells isolated from smaller follicles.The western blot results demonstrated that there exsiting isoforms of V1, V2 and V3 protein in granulosa cells from both larger follicles and smaller ones; for V0, all of the bands were weak. Four isoforms of versican proteins were all expressed in cumulus cells. The expression level of V1, V2 and V3 was much higher in cumulus cells than that in the granulosa cells from larger follicles and smaller ones. The level of V0 is higher in cumulus cells than that in granulosa cells from smaller follicles. No difference was observed of the level of V0 between granulosa cells from larger follicles and smaller ones, between cumulus cells and granulosa cells from larger follicles. V2 and V3 isoforms of protein were enriched in follicular fluids of both larger follicles and smaller follicles.Conclusion: Four isoforms of versican mRNA and protein were all expressed in cumulus cells and granulosa cells isolated from larger follicles and smaller follicles, of which, V0, V1 and V2 isoforms of mRNA and protein were mainly expressed in cumulus cells. Therefore, cumulus cells was the mainly source of the four isoforms of versican protein. And V2 and V3 isoforms of protein were enriched in human follicular fluids from smaller follicles and larger follicles. The alteration of expression of isoforms of versican in granulosa cells isolated from smaller follicles and larger follicles may correlate with the changes of its functions during the follicular development.PartⅡ Detection of Versican in Follicular Fluids and Its Clinical ImplicationsObjective: This investigation was performed to clarify the possible relationship between the value of versican in follicular fluids from larger follicles and the clinical outcomes; and to determine the potential influencing factors of versican in follicular fluids isolated from larger follicles, namely, whether various protocols of controlled ovarian stimulation and the states of polycystic ovarian syndrome(PCOS), of endometriosis patients and of patients of poor ovarian response(POR) affected the concentration of versican in follicular fluids. And to demonstrate whether ages, basic follicle stimulating hormone(FSH) values in serum, progesterone(P) in follicular fluids, estradiol(E2) and P in serum on the day of human Chorionic Gonadotropin(h CG) administration had correlation with the value of versican in follicular fluids from larger follicles.Method: We collected samples of large follicular fluids from 327 patients. And we had detected the content of versican of these samples by ELISA(Enzyme Linked Immunosorbent Assay). And we had dectected the level of E2 from 180 specimens of follicular fluids and P from 162 follicular fluids via Radioimmunoassay. The clinical information was collected and the clinical outcomes were followed. We demonstrated the relationship between the value of versican in follicular fluids of larger follicles and the pregnant outcomes. In addition, to understand the potential influencing factors of the content of versican in follicular fluids isolated from larger follicles, including ages, basic FSH values in serum, protocols of controlled ovarian stimulation, endocrine diseases, P and E2 in follicular fluids etc.Outcomes: There were 223 patients who were pregnant, and 98 who were nonpregnant, 159 who given birth to live babies, 35 that were aborted and 28 who were ongoing pregnant subdivided from the pregnant group patients. No difference was observed of the level of versican in follicular fluids between the pregnant group(1799.00±956.01 pg/ml) and the nonpregnant group(1900.55±1040.10 pg/ml). The content of versican in follicular fluids in live birth group was 1861.09±1004.19pg/ml, and 1769.89±897.56pg/ml in the abortion group, showing little difference between the two groups.Totally, there were 194 patients undergoing long protocol administration, 49 patients undergoing rolong protocol, and 75 antagonist scheme. The value(1989.46±1006.26 pg/ml) of versican in follicular fluids in long protocol was much higher than that(1398.89 ± 930.46 pg/ml) in antagonist group(P < 0.001). And the content(1793.78 ± 920.77 pg/ml) of versican in follicular fluids was higher in rolong protocol group than that in antagonist group(P = 0.028). No difference of the level of versican in follicular fluids between the long protocol group and rolong scheme group was observed. And there were 34 PCOS, 40 endometriosis patients, 28 patients of poorly ovarian response and 208 controls. And there was no difference of the level of versican in follicular fluids between the patients of PCOS and the control group, of the endometriosis group and the control, of the POR and the control group repectively.The level of versican in follicular fluids was positively correlated with the value of E2 in follicular fluids and E2 in serum on the day of h CG administration, but it had no correlation with the ages of patients and the basic FSH value in serum, the value of P in follicular fluids and P in serum on the day of hCG administration.Conclusion: This research showed that there was no relationship between the concentration of versican in follicular fluids of larger follicles and clinical pregnancy outcome. The value of versican in follicular fluids in three different protocols(long protocol, rolong protocol and antagonist scheme) showed significant difference, suggesting that different administrations had distinct impact on the functions of secreting activities of follicular cells including granulosa cells and cumulus cells. And the value of versican positively correlated with the E2 in follicular fluids and E2 in serum, therefore the value of versican in follicular fluids could be a kind of indicator for reflecting the functional state of follicular cells.