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P-290 Inhibin-A and inhibin α-subunit are elevated in preeclamptic pregnancy
sone (DEX) suppression test with the 10 day prednisone (PDN) suppression test in the same subject. Materials and Methods: Women with moderate to severe hirsutism were referred by their dermatologist. A total of 17 women have initiated this study and 7 have completed both DEX and PDN suppression tests. All subjects received the overnight DEX suppression test (1 mg at bedtime), blood samples were obtained the morning prior to DEX and the morning after. After 1 - 2 weeks women returned for the 10 day PDN suppression test (10 mg at night for 10 consecutive days), blood was collected the morning of PDN initiation and at 1, 5 and 10 days thereafter. All blood samples were obtained between 0730 and 0930 hours. Serum was analyzed for total testosterone, free testosterone, cortisol, androstenedione, and DHEA-S using commercially available methodology. Results: The overnight DEX test results in a dramatic drop in serum cortisol to basal levels (16.11 +_ 2.93 to 1.23 _+ 0.11 #g/DL). PDN never results in this dramatic decline with some subjects, while showing a decline, remain in the normal range. Similarly, the decline in serum androgens [TTesto (0.67 +_ 0.07 to 0.22 _+ 0.04 ng/ml), fresto (1.67 _+ 0.33 to 0.74 _+ 0.11 pg/ml), Androstenedione (1.94 _+ 0.68 to 0.68 _+ 0.06 ng/ml)] is also most dramatic with the overnight DEX test and only moderately suppressed with PDN. DHEAS shows a similar degree of suppression with both tests. Conclusions: The DEX test, because of its more rapid and dramatic results, appears to be more clinically useful than the 10 day PDN test.
P-289 Estradiol I n c r e a s e s P r o s t a g l a n d i n F2a P r o d u c t i o n in H u m a n E n d o m e t r i a l Glandular Cells T h r o u g h C y c l o o x y g e n a s e - 2 Induction. 1j. C. Huang, 18. Yadollahi, 1M. Y. Dawood, 2K. K. Wu. 1Dept of OB/GYN, 2Dept of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX. Objective: We have previously demonstrated that estradiol induced the gene expression and enzyme activity of cyclooxygenase-2 (Cox-2) in cultured human endometrial stromal cells. The objective of this study was to determine the effect of estradiol on Cox-2 enzyme activity in human endometrial glandular cells. Design: Quiescent cells were first treated with estradiol, then given exogenous arachidonic acid with or without specific Cox-2 inhibitor. Prostaglandin F2a (PGF2a) in the media was then determined by specific enzyme immunoassay. Materials and Methods: Ishikawa cells, a human endometrial cancer cell line with estrogen receptors, were maintained in 6-well plate in RPMI medium supplemented with fetal calf serum. When 75% confluence was reached, cells were maintained in serum-free medium (RPMI with 0.1% bovine serum albumin, 5 #g/mL transferrin and 0.1 #g/mL insulin) for 24 hours before the experiment. The experiment consisted of a 6-hour incubation with estradiol (10 nM) followed by a 1-hour incubation with arachidonic acid (10 mM). Specific Cox-2 inhibitor
NS398 (1 #M) was added 1 hour before the addition of arachidonic acid (10 mM) in some estradiol-stimulated cells. Control cells received ethanol (0.1%) instead of estradiol. Medium was collected and assayed in duplicate using specific PGF2a enzyme immunoassay. We used analysis of variance to determine the difference among the 3 groups; the difference between any 2 groups was then determined by Bonferroni test. p < 0.05 was considered statistically significant. Results:
PGF2a (pg/mL) mean _+ SD
Control (n = 6)
Estradiol (n = 6)
Estradiol + NS398 (n = 6)
49 +_ 27 ~'c
114 _+ 41 a'b
47 _+ 19b'c
Analysis of variance: p = 0.002. Bonferroni test: a,bp < 0.05; c not significant. Conclusion: Estradiol (10 nM) stimulated a 2.3-fold increase in PGF2a biosynthesis from arachidonic acid in Ishikawa cells. The increase was completely blocked by specific Cox-2 inhibitor. We concluded that estradiol induced Cox-2 activity in Ishikawa cells.
P-290 Inhibin-A and Inhibin a - S u b u n i t Are E l e v a t e d in P r e e c l a m p t i c P r e g n a n c y . R. F. Fraser, M. E. McAsey, P.J. Coney. Department of Obstetrics & Gynecology, Southern Illinois University School of Medicine, Springfield, IL. Objective: Serum concentrations of the heterodimeric glycoprotein inhibin-A (a~A) and its a-subunit increase during pregnancy. The placenta is the predominant source of inhibin during pregnancy and an autocrine/paracrine role in the trophoblast has been suggested. The cytotrophoblast and syncytiotrophoblast undergo structural and ultrastructural changes in preeclampsia. Previous investigators have found elevated serum levels of inhibin asubunit in preeclampsia (Kalil et al., Am J Obstet Gynecol. 172:1019). The effect of preeclampsia on the production of inhibin-A is unknown. The objective of this investigation was to compare serum levels of inhibin-A and inhibin asubunit in pregnancies complicated by preeclampsia with those of normotensive pregnancies. Design: A case-control design employing cases of preeclampsia and normotensive controls was used for this investigation. Materials and Methods: Study subjects were 32 cases of preeclampsia with a single fetus at 32 to 41 weeks gestation and 37 gestational age matched normotensive controls. A single blood specimen was collected from each subject. Solid phase sandwich Enzyme Linked Immunosorbent Assays (ELISA) were used to measure dimeric inhibin-A and inhibin a-subunit in sera (Groome et al., Clin Endocrinol. 40:717; Groome et al., J Clin Endocrinol Metab. 80:2926). A two sample t-test was applied to the Abstracts
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group means and to gestational age grouped means of cases and controls. Results: Inhibin-A and inhibin a-subunit were significantly elevated in the sera of women with preeclampsia when compared to circulating inhibin levels in control patients (p < 0.001). The elevation was apparent at 32 to 37 weeks of gestation but was not demonstrated beyond 37 weeks (Fig. 1). A correlation of serum levels of inhibin-A and inhibin ~-subunit with severity of preeclampsia was not observed.
2000 -
@
Preeclampsia
•
Control
~ ' 1500
< 1000
._a
500
32-'33 34-'35 36-'67 38:39 40:41
Gestational Age (Weeks) Figure 1 Serum inhibin-A concentrations in women with either preeclamptic or normal pregnancies. Conclusion: Circulating levels of inhibin-A and inhibin a-subunit are increased in pregnancies complicated by preeclampsia. The elevated levels ofinhibin-A and inhibin a-subunit in preeclamptic pregnancy may represent a regulatory mechanism in syncytial proliferation.
P-291 Expression of Vimentin and Cytokeratin in Eutopic and Ectopic Endometrium of Women with Adenomyosis and Ovarian Endometrioma. 1I. O. Song, 2S. R. Hong, 1H. J. Yeon, 1K. H. Kim, 1j. p. Lee, 1M. K. Koong, 3y. B. Huh, 1I. S. Kang. 1Dept. of OB/GYN, 2Dept. of Pathology, Samsung Cheil Hospital, 3Dept. of Anatomy, Medical College, Kyunghee Univ., Seoul, Korea. Objective: Vimentin (VM) is present in the cells of mesenchymal origin and serves a structural function. Cytokeratin (CK) expressed in epithelium is used as a marker of cell differentiation. It has been reported that VM and CK are expressed in peritoneal endometriosis with similar pattern to those of eutopic endometrium, but to a lesser intensity, suggesting different degrees of differentiation. But, there are scanty data on the expression of VM and CK in adenomyosis and endometrioma. The aim of this study is to determine VM and CK expression and their cyclic changes in adenomyosis and endometrioma. Design: Immunohistochemical analysis of VM and CK in women with both adenomyosis and endometrioma
S232
Abstracts
Materials and Methods: Twenty patients having both adenomyosis and ovarian endometrioma requiring hysterectomy with salpingo-oophorectomy enrolled in this study. The expression of VM and CK in eutopic endometrium, adenomyosis and inner lining of endometrioma were studied using paraffin-embedded sections stained with avidinbiotin peroxidase method. Staining intensity was assessed by quantitative histologic score (H-score= Sum[P/× (i+ 1)]; i: intensity 0 - 3 , Pi: %cells for each given intensity) of 100 cells. Patients were grouped into proliferative (n=9) a n d secretory ( n = l l ) phase by Noyes criteria. Results: The mean (_+SEM) H-score of epithelial VM was significantly higher (p<0.01) in the proliferative phase than in the secretory phase in eutopic endometrium (342_+13 vs 252_+22) and adenomyosis (272_+19 vs 176-+11). In contrast, H-score of stromal VM was significantly higher (p<0.05) in the secretory phase than in the proliferative phase in eutopic endometrium (132-+9 vs 193-+21) and adenomyosis (123_+7 vs 149-+8). Neither the intensity of VM in endometrioma nor CK in epithelium of all these tissues showed a significant cyclic change. The mean intensities of epithelial VM were significantly different among the tissues, being lowest in endometrioma (185_+30, 130_+12: proliferative, secretory phase), highest in eutopic endometrium and intermediate in adenomyosis (ANOVA, Scheffe's test, p<0.05). Likewise, H-score of stromal VM in adenomyosis was lower than that of eutopic endometrium. There was no significant difference in Hscores of epithelial CK between adenomyosis (237_+6.7, 228_+ 14.4) and endometrioma (220_+ 11, 227_+ 15) but these intensities were significantly lower (p<0.01) than that of eutopic endometrium (302-+17, 305+17). Conclusions: These data demonstrate that the expression of VM may be dependent upon the hormonal influence in the eutopic endometrium and adenomyosis, but not in the endometrioma. Lower intensities of CK in adenomyosis and endometrioma than eutopic endometrium suggest that these ectopic endometrium may have lower degree of differentiation regardless of the site. The lower intensity of epithelial VM in endometrioma than in adenomyosis during proliferative phase may reflect decreased functional activity probably due to the pressure effect upon the lining epithelium within endometrioma.
P-292 D e x a m e t h a s o n e Inhibits Interleukin-l-Dependent Prostaglandin Endoperoxide Synthase-2 Gene Expression by Cultured Rat Whole Ovarian Dispersates. 1'2M. Irahara, 1'3M. Ando, 1'3E. Y. Adashi. Dept of OB/GYN, 1University of Maryland School of Medicine, Baltimore, MD, Current address: 2University of Tokushima School of Medicine, Tokushima City, Tokushima, Japan, ~University of Utah School of Medicine, Salt Lake City, UT. Objective: Ovulation may constitute a cyclic, inflammatory-like process wherein the increased biosynthesis of prostanoids may feature prominently. We demonstrated that interleukin-1 /3(IL-1/3) stimulates the production of
P-289 Estradiol I n c r e a s e s P r o s t a g l a n d i n F2a P r o d u c t i o n in H u m a n E n d o m e t r i a l Glandular Cells T h r o u g h C y c l o o x y g e n a s e - 2 Induction. 1j. C. Huang, 18. Yadollahi, 1M. Y. Dawood, 2K. K. Wu. 1Dept of OB/GYN, 2Dept of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX. Objective: We have previously demonstrated that estradiol induced the gene expression and enzyme activity of cyclooxygenase-2 (Cox-2) in cultured human endometrial stromal cells. The objective of this study was to determine the effect of estradiol on Cox-2 enzyme activity in human endometrial glandular cells. Design: Quiescent cells were first treated with estradiol, then given exogenous arachidonic acid with or without specific Cox-2 inhibitor. Prostaglandin F2a (PGF2a) in the media was then determined by specific enzyme immunoassay. Materials and Methods: Ishikawa cells, a human endometrial cancer cell line with estrogen receptors, were maintained in 6-well plate in RPMI medium supplemented with fetal calf serum. When 75% confluence was reached, cells were maintained in serum-free medium (RPMI with 0.1% bovine serum albumin, 5 #g/mL transferrin and 0.1 #g/mL insulin) for 24 hours before the experiment. The experiment consisted of a 6-hour incubation with estradiol (10 nM) followed by a 1-hour incubation with arachidonic acid (10 mM). Specific Cox-2 inhibitor
NS398 (1 #M) was added 1 hour before the addition of arachidonic acid (10 mM) in some estradiol-stimulated cells. Control cells received ethanol (0.1%) instead of estradiol. Medium was collected and assayed in duplicate using specific PGF2a enzyme immunoassay. We used analysis of variance to determine the difference among the 3 groups; the difference between any 2 groups was then determined by Bonferroni test. p < 0.05 was considered statistically significant. Results:
PGF2a (pg/mL) mean _+ SD
Control (n = 6)
Estradiol (n = 6)
Estradiol + NS398 (n = 6)
49 +_ 27 ~'c
114 _+ 41 a'b
47 _+ 19b'c
Analysis of variance: p = 0.002. Bonferroni test: a,bp < 0.05; c not significant. Conclusion: Estradiol (10 nM) stimulated a 2.3-fold increase in PGF2a biosynthesis from arachidonic acid in Ishikawa cells. The increase was completely blocked by specific Cox-2 inhibitor. We concluded that estradiol induced Cox-2 activity in Ishikawa cells.
P-290 Inhibin-A and Inhibin a - S u b u n i t Are E l e v a t e d in P r e e c l a m p t i c P r e g n a n c y . R. F. Fraser, M. E. McAsey, P.J. Coney. Department of Obstetrics & Gynecology, Southern Illinois University School of Medicine, Springfield, IL. Objective: Serum concentrations of the heterodimeric glycoprotein inhibin-A (a~A) and its a-subunit increase during pregnancy. The placenta is the predominant source of inhibin during pregnancy and an autocrine/paracrine role in the trophoblast has been suggested. The cytotrophoblast and syncytiotrophoblast undergo structural and ultrastructural changes in preeclampsia. Previous investigators have found elevated serum levels of inhibin asubunit in preeclampsia (Kalil et al., Am J Obstet Gynecol. 172:1019). The effect of preeclampsia on the production of inhibin-A is unknown. The objective of this investigation was to compare serum levels of inhibin-A and inhibin asubunit in pregnancies complicated by preeclampsia with those of normotensive pregnancies. Design: A case-control design employing cases of preeclampsia and normotensive controls was used for this investigation. Materials and Methods: Study subjects were 32 cases of preeclampsia with a single fetus at 32 to 41 weeks gestation and 37 gestational age matched normotensive controls. A single blood specimen was collected from each subject. Solid phase sandwich Enzyme Linked Immunosorbent Assays (ELISA) were used to measure dimeric inhibin-A and inhibin a-subunit in sera (Groome et al., Clin Endocrinol. 40:717; Groome et al., J Clin Endocrinol Metab. 80:2926). A two sample t-test was applied to the Abstracts
S231
group means and to gestational age grouped means of cases and controls. Results: Inhibin-A and inhibin a-subunit were significantly elevated in the sera of women with preeclampsia when compared to circulating inhibin levels in control patients (p < 0.001). The elevation was apparent at 32 to 37 weeks of gestation but was not demonstrated beyond 37 weeks (Fig. 1). A correlation of serum levels of inhibin-A and inhibin ~-subunit with severity of preeclampsia was not observed.
2000 -
@
Preeclampsia
•
Control
~ ' 1500
< 1000
._a
500
32-'33 34-'35 36-'67 38:39 40:41
Gestational Age (Weeks) Figure 1 Serum inhibin-A concentrations in women with either preeclamptic or normal pregnancies. Conclusion: Circulating levels of inhibin-A and inhibin a-subunit are increased in pregnancies complicated by preeclampsia. The elevated levels ofinhibin-A and inhibin a-subunit in preeclamptic pregnancy may represent a regulatory mechanism in syncytial proliferation.
P-291 Expression of Vimentin and Cytokeratin in Eutopic and Ectopic Endometrium of Women with Adenomyosis and Ovarian Endometrioma. 1I. O. Song, 2S. R. Hong, 1H. J. Yeon, 1K. H. Kim, 1j. p. Lee, 1M. K. Koong, 3y. B. Huh, 1I. S. Kang. 1Dept. of OB/GYN, 2Dept. of Pathology, Samsung Cheil Hospital, 3Dept. of Anatomy, Medical College, Kyunghee Univ., Seoul, Korea. Objective: Vimentin (VM) is present in the cells of mesenchymal origin and serves a structural function. Cytokeratin (CK) expressed in epithelium is used as a marker of cell differentiation. It has been reported that VM and CK are expressed in peritoneal endometriosis with similar pattern to those of eutopic endometrium, but to a lesser intensity, suggesting different degrees of differentiation. But, there are scanty data on the expression of VM and CK in adenomyosis and endometrioma. The aim of this study is to determine VM and CK expression and their cyclic changes in adenomyosis and endometrioma. Design: Immunohistochemical analysis of VM and CK in women with both adenomyosis and endometrioma
S232
Abstracts
Materials and Methods: Twenty patients having both adenomyosis and ovarian endometrioma requiring hysterectomy with salpingo-oophorectomy enrolled in this study. The expression of VM and CK in eutopic endometrium, adenomyosis and inner lining of endometrioma were studied using paraffin-embedded sections stained with avidinbiotin peroxidase method. Staining intensity was assessed by quantitative histologic score (H-score= Sum[P/× (i+ 1)]; i: intensity 0 - 3 , Pi: %cells for each given intensity) of 100 cells. Patients were grouped into proliferative (n=9) a n d secretory ( n = l l ) phase by Noyes criteria. Results: The mean (_+SEM) H-score of epithelial VM was significantly higher (p<0.01) in the proliferative phase than in the secretory phase in eutopic endometrium (342_+13 vs 252_+22) and adenomyosis (272_+19 vs 176-+11). In contrast, H-score of stromal VM was significantly higher (p<0.05) in the secretory phase than in the proliferative phase in eutopic endometrium (132-+9 vs 193-+21) and adenomyosis (123_+7 vs 149-+8). Neither the intensity of VM in endometrioma nor CK in epithelium of all these tissues showed a significant cyclic change. The mean intensities of epithelial VM were significantly different among the tissues, being lowest in endometrioma (185_+30, 130_+12: proliferative, secretory phase), highest in eutopic endometrium and intermediate in adenomyosis (ANOVA, Scheffe's test, p<0.05). Likewise, H-score of stromal VM in adenomyosis was lower than that of eutopic endometrium. There was no significant difference in Hscores of epithelial CK between adenomyosis (237_+6.7, 228_+ 14.4) and endometrioma (220_+ 11, 227_+ 15) but these intensities were significantly lower (p<0.01) than that of eutopic endometrium (302-+17, 305+17). Conclusions: These data demonstrate that the expression of VM may be dependent upon the hormonal influence in the eutopic endometrium and adenomyosis, but not in the endometrioma. Lower intensities of CK in adenomyosis and endometrioma than eutopic endometrium suggest that these ectopic endometrium may have lower degree of differentiation regardless of the site. The lower intensity of epithelial VM in endometrioma than in adenomyosis during proliferative phase may reflect decreased functional activity probably due to the pressure effect upon the lining epithelium within endometrioma.
P-292 D e x a m e t h a s o n e Inhibits Interleukin-l-Dependent Prostaglandin Endoperoxide Synthase-2 Gene Expression by Cultured Rat Whole Ovarian Dispersates. 1'2M. Irahara, 1'3M. Ando, 1'3E. Y. Adashi. Dept of OB/GYN, 1University of Maryland School of Medicine, Baltimore, MD, Current address: 2University of Tokushima School of Medicine, Tokushima City, Tokushima, Japan, ~University of Utah School of Medicine, Salt Lake City, UT. Objective: Ovulation may constitute a cyclic, inflammatory-like process wherein the increased biosynthesis of prostanoids may feature prominently. We demonstrated that interleukin-1 /3(IL-1/3) stimulates the production of