- Email: [email protected]
P-415
Financial support: Correspondence: Liang Xiaoyan e-mail: [email protected] Supported by: This research was funded by the NSFC (No. 30271367, 30300372 30571956); Guandong Natural Science Fund (No. 2002c31301,31690; 21874) and GZST (No.2004J-C0111).
P-415 DIFFERENTIAL MODULATION HSP60 AND HSP90〈 BY HRT COMPONENTS IN BREAST CANCER CELLS M. Abdullah, M. Abdallah, D. D. Taylor, S. Nakajima, C. Gercel-Taylor. Pacific Reproductive Center, Torrance, CA; University of Louisville, Louisville, KY. OBJECTIVE: Heat shock proteins (Hsps) are overexpressed in a variety of human cancers including breast cancer. They are involved in tumor cell proliferation, differentiation, invasion, metastasis, death and immune recognition. Hsp60 and Hsp90 have been associated with breast cancer carcinogenesis and outcome. Also, Hsp protein levels have been shown to be increased by estrogen metabolites. While recent studies indicate that Hormone Replacement Therapies may result in increased incidence of breast cancer particularly in those patients with existing occult tumors. The role of estrogen/progestogens components on Hsps involved in breast cancer has not been previously studied. Our objective was to study the effect of the components of HRT on Hsp60 and 90␣ expression in breast cancer cells. DESIGN: MCF-7 cells were treated in vitro with HRT components for 48 hours. The effect of these treatments on transcription of Hsp60 and Hsp 90␣ was determined. MATERIALS AND METHODS: Real time polymerase chain reaction (RT-PCR) was used to determine the effect of HRT components estradiol (E2), equilin (Eq), norethiderone acetate (NEA), medroxyprogesterone acetate (MPA) and progesterone (P) at physiologic levels (10 nM) on the transcripts of Hsp60 and Hsp 90␣. Untreated cells were used as controls. RNA was isolated with the Trizol reagent. Following first-strand synthesis primers for Hsp60 and Hsp 90␣ were utilized in RT-PCR reactions. LightCycler2 was used to perform the analysis. Experiments were performed in duplicate with multiple DNA concentrations during each run. Data were analyzed by one-way ANOVA with Tukey-Kramer multiple comparisons post test. RESULTS: HSP60 and 90␣ transcripts were induced 1.52 and 2.10 fold respectively by Eq treatment. E2 resulted in 1.11 and 1.54 increase in Hsp90␣ and Hsp60 in MCF-7 cells. When estrogens were combined with progestogens both NEA and MPA resulted in 1.61 increase in Hsp90␣, while P resulted in 1.13. Hsp 60 transcripts were also induced by 2.50, 1.75 and 2.25 with NEA, MPA and P combined with Eq. Highest level of induction of both Hsp90␣ and Hsp60 were seen with E2NEA at 1.92 and 2.45 respectively. P alone resulted in slight decrease of Hsp90␣, and except NEA, remaining progestogens resulted in decreased Hsp60 transcription. CONCLUSION: Both Hsp60 and Hsp90␣ have been associated with carcinogenesis or poor prognosis in breast cancer. Our data indicate that differential induction of these proteins may play a role in HRT associated increase in breast cancer as suggested by the in vitro model. Supported by: No financial support was provided.
P-416 DECREASED SERUM ANTI-MULLERIAN HORMONE LEVELS AND HOMOGENIZATION OF FOLLICULAR COHORT AFTER INTER-CYCLE FSH INHIBITION. J. Cunha-Filho, E. Arbo, F. Freitas, A. Facin, C. Salazar, E. Passos. Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil. OBJECTIVE: to evaluate the effect of FSH suppression in the late luteal phase of menstrual cycle with oral contraceptives pills (OCP) over the anti-mullerian hormone (AMH) levels and follicular cohort homogeneity. DESIGN: a prospective controlled trial. MATERIALS AND METHODS: We included in our trial 20 normovulatory women, with tubal obstruction or male infertility. All patients signed a pos informed term consenting in participate. In the third day of a spontaneous menstrual cycle, they were submitted to a transvaginal ultrasound examination and hormonal measurement. All antral follicles great the 2 mm
S290
Abstracts
were noticed. From the 20th day of this menstrual cycle, patients took daily OCP, containing 30 mcg of ethinil-estradiol plus 0.15 mg of desogestrel. In the third day of the following cycle, the measurements were repeated. Normally distributed data were analyzed by the Student’s t test for repeated measures and the non-parametric data by the Wilcoxon’s signed-rank test. The level of significance considered was 0.05. RESULTS: The mean age was 29 years, ranging from 23 to 35. After OCP use the levels of FSH and estradiol were significantly reduced. The means ⫹ SD for FSH were 7.21 ⫹ 1.17 mU/mL before and 1.98 ⫹ 0.4 mU/mL after OCP administration (P⬍0.001) and for estradiol were 43.09 ⫹ 7.94 and 13.46 ⫹ 4.34 pg/mL (P⬍0.001), respectively. The number of antral follicles measured at both moments did not differ (15.8 ⫹ 4.7 before and 16.6 ⫹ 3.6 after), although after OCP the follicles presented significant lower diameters (4.4 ⫹ 1.7 mm before versus 3.5 ⫹ 1.2 mm after, P⬍0.05), and a lower variance (Levene’s test for equality of variances F⫽32.212, P⬍0.001). The levels of AMH were significantly reduced after pituitary down-regulation, with medians (interquartille range) of 3.02 ng/mL (1.21 6.39) before OCP use and 2.22 ng/mL (0.9 - 3.11) after it. CONCLUSION: The inhibition of the inter-cycle FSH pituitary secretion leads to a more homogeneous follicular cohort and a reduction on follicular diameter. Moreover, this is the first report showing a decrease in AMH levels verified after FSH suppression in a prospective design. This result suggests an important physiological role of FSH on AMH secretion. Furthermore, assuming that the AMH effects during ovulation is associated with an inhibition of primordial follicle growth; this novel concept could help us to explain the increased number of retrieved oocytes after the inter-cycle FSH inhibition in controlled ovarian stimulation cycles using GnRH antagonists. Supported by: FIPE-HCPA, CNPq
P-417 NIGERIAN WOMEN RESIDING IN USA ARE MORE HIRSUTE THAN THOSE RESIDING IN NIGERIA; POSSIBLE ROLE OF ENVIRONMENTAL AND LIFESTYLE FACTORS. K. Olorunrinu, A. J. Duleba. Yale Univ. School of Medicine, New Haven, CT. OBJECTIVE: Hirsutism may be related to endocrine causes (e.g. PCOS), be familial, or be due to use of androgens. However, in a large proportion of women, no cause of hirsutism is found. In this report we compare two populations of Nigerian women to determine whether other factors, such as those possibly related to environment and lifestyle may contribute to hirsutism. DESIGN: Cross-sectional evaluation of two populations of Nigerian women living in disparate environments. MATERIALS AND METHODS: The study included one hundred and twelve Nigerian women living in the USA (for 1 to 33 years, median 7.5 years) and seventy women living in Nigeria. All women completed pictorial survey evaluating peripheral hair growth in the following body areas: upper lip, chin, chest, upper abdomen, lower abdomen and thighs; each area was scored from 0 (no significant hair growth) to 4 (severe hair growth). Total hirsutism score was calculated as a sum of individual scores. Survey also included demographic data, menstrual history, and data regarding use of hormonal treatments. Statistical comparisons between groups included ttest, nonparametric tests and chi-square test. Multiple regression analysis was carried out to identify independent predictors of peripheral hair growth. RESULTS: Comparisons between women living in USA and Nigeria are presented in the table; individual variables represent means ⫾ SEM. Women residing in USA had a 31% higher total hirsutism score than those residing in Nigeria. This difference was not related to the proportion of patients with irregular menstrual cycles. To account for possible effects of age, BMI and differences in tribal origin, multiple regression analysis was performed. Accounting for other variables, location (living in USA vs. Nigeria) remained the strongest predictor of total hirsutism score (P⫽0.01); tribal origin was also significant (P⫽0.02), while age and BMI had no independent predictive value (P⬎0.1). Living in USA vs. Nigeria remained a significant predictor in a regression model excluding women using hormonal treatments (P⫽0.02).
Vol. 86, Suppl 2, September 2006
P-415 DIFFERENTIAL MODULATION HSP60 AND HSP90〈 BY HRT COMPONENTS IN BREAST CANCER CELLS M. Abdullah, M. Abdallah, D. D. Taylor, S. Nakajima, C. Gercel-Taylor. Pacific Reproductive Center, Torrance, CA; University of Louisville, Louisville, KY. OBJECTIVE: Heat shock proteins (Hsps) are overexpressed in a variety of human cancers including breast cancer. They are involved in tumor cell proliferation, differentiation, invasion, metastasis, death and immune recognition. Hsp60 and Hsp90 have been associated with breast cancer carcinogenesis and outcome. Also, Hsp protein levels have been shown to be increased by estrogen metabolites. While recent studies indicate that Hormone Replacement Therapies may result in increased incidence of breast cancer particularly in those patients with existing occult tumors. The role of estrogen/progestogens components on Hsps involved in breast cancer has not been previously studied. Our objective was to study the effect of the components of HRT on Hsp60 and 90␣ expression in breast cancer cells. DESIGN: MCF-7 cells were treated in vitro with HRT components for 48 hours. The effect of these treatments on transcription of Hsp60 and Hsp 90␣ was determined. MATERIALS AND METHODS: Real time polymerase chain reaction (RT-PCR) was used to determine the effect of HRT components estradiol (E2), equilin (Eq), norethiderone acetate (NEA), medroxyprogesterone acetate (MPA) and progesterone (P) at physiologic levels (10 nM) on the transcripts of Hsp60 and Hsp 90␣. Untreated cells were used as controls. RNA was isolated with the Trizol reagent. Following first-strand synthesis primers for Hsp60 and Hsp 90␣ were utilized in RT-PCR reactions. LightCycler2 was used to perform the analysis. Experiments were performed in duplicate with multiple DNA concentrations during each run. Data were analyzed by one-way ANOVA with Tukey-Kramer multiple comparisons post test. RESULTS: HSP60 and 90␣ transcripts were induced 1.52 and 2.10 fold respectively by Eq treatment. E2 resulted in 1.11 and 1.54 increase in Hsp90␣ and Hsp60 in MCF-7 cells. When estrogens were combined with progestogens both NEA and MPA resulted in 1.61 increase in Hsp90␣, while P resulted in 1.13. Hsp 60 transcripts were also induced by 2.50, 1.75 and 2.25 with NEA, MPA and P combined with Eq. Highest level of induction of both Hsp90␣ and Hsp60 were seen with E2NEA at 1.92 and 2.45 respectively. P alone resulted in slight decrease of Hsp90␣, and except NEA, remaining progestogens resulted in decreased Hsp60 transcription. CONCLUSION: Both Hsp60 and Hsp90␣ have been associated with carcinogenesis or poor prognosis in breast cancer. Our data indicate that differential induction of these proteins may play a role in HRT associated increase in breast cancer as suggested by the in vitro model. Supported by: No financial support was provided.
P-416 DECREASED SERUM ANTI-MULLERIAN HORMONE LEVELS AND HOMOGENIZATION OF FOLLICULAR COHORT AFTER INTER-CYCLE FSH INHIBITION. J. Cunha-Filho, E. Arbo, F. Freitas, A. Facin, C. Salazar, E. Passos. Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil. OBJECTIVE: to evaluate the effect of FSH suppression in the late luteal phase of menstrual cycle with oral contraceptives pills (OCP) over the anti-mullerian hormone (AMH) levels and follicular cohort homogeneity. DESIGN: a prospective controlled trial. MATERIALS AND METHODS: We included in our trial 20 normovulatory women, with tubal obstruction or male infertility. All patients signed a pos informed term consenting in participate. In the third day of a spontaneous menstrual cycle, they were submitted to a transvaginal ultrasound examination and hormonal measurement. All antral follicles great the 2 mm
S290
Abstracts
were noticed. From the 20th day of this menstrual cycle, patients took daily OCP, containing 30 mcg of ethinil-estradiol plus 0.15 mg of desogestrel. In the third day of the following cycle, the measurements were repeated. Normally distributed data were analyzed by the Student’s t test for repeated measures and the non-parametric data by the Wilcoxon’s signed-rank test. The level of significance considered was 0.05. RESULTS: The mean age was 29 years, ranging from 23 to 35. After OCP use the levels of FSH and estradiol were significantly reduced. The means ⫹ SD for FSH were 7.21 ⫹ 1.17 mU/mL before and 1.98 ⫹ 0.4 mU/mL after OCP administration (P⬍0.001) and for estradiol were 43.09 ⫹ 7.94 and 13.46 ⫹ 4.34 pg/mL (P⬍0.001), respectively. The number of antral follicles measured at both moments did not differ (15.8 ⫹ 4.7 before and 16.6 ⫹ 3.6 after), although after OCP the follicles presented significant lower diameters (4.4 ⫹ 1.7 mm before versus 3.5 ⫹ 1.2 mm after, P⬍0.05), and a lower variance (Levene’s test for equality of variances F⫽32.212, P⬍0.001). The levels of AMH were significantly reduced after pituitary down-regulation, with medians (interquartille range) of 3.02 ng/mL (1.21 6.39) before OCP use and 2.22 ng/mL (0.9 - 3.11) after it. CONCLUSION: The inhibition of the inter-cycle FSH pituitary secretion leads to a more homogeneous follicular cohort and a reduction on follicular diameter. Moreover, this is the first report showing a decrease in AMH levels verified after FSH suppression in a prospective design. This result suggests an important physiological role of FSH on AMH secretion. Furthermore, assuming that the AMH effects during ovulation is associated with an inhibition of primordial follicle growth; this novel concept could help us to explain the increased number of retrieved oocytes after the inter-cycle FSH inhibition in controlled ovarian stimulation cycles using GnRH antagonists. Supported by: FIPE-HCPA, CNPq
P-417 NIGERIAN WOMEN RESIDING IN USA ARE MORE HIRSUTE THAN THOSE RESIDING IN NIGERIA; POSSIBLE ROLE OF ENVIRONMENTAL AND LIFESTYLE FACTORS. K. Olorunrinu, A. J. Duleba. Yale Univ. School of Medicine, New Haven, CT. OBJECTIVE: Hirsutism may be related to endocrine causes (e.g. PCOS), be familial, or be due to use of androgens. However, in a large proportion of women, no cause of hirsutism is found. In this report we compare two populations of Nigerian women to determine whether other factors, such as those possibly related to environment and lifestyle may contribute to hirsutism. DESIGN: Cross-sectional evaluation of two populations of Nigerian women living in disparate environments. MATERIALS AND METHODS: The study included one hundred and twelve Nigerian women living in the USA (for 1 to 33 years, median 7.5 years) and seventy women living in Nigeria. All women completed pictorial survey evaluating peripheral hair growth in the following body areas: upper lip, chin, chest, upper abdomen, lower abdomen and thighs; each area was scored from 0 (no significant hair growth) to 4 (severe hair growth). Total hirsutism score was calculated as a sum of individual scores. Survey also included demographic data, menstrual history, and data regarding use of hormonal treatments. Statistical comparisons between groups included ttest, nonparametric tests and chi-square test. Multiple regression analysis was carried out to identify independent predictors of peripheral hair growth. RESULTS: Comparisons between women living in USA and Nigeria are presented in the table; individual variables represent means ⫾ SEM. Women residing in USA had a 31% higher total hirsutism score than those residing in Nigeria. This difference was not related to the proportion of patients with irregular menstrual cycles. To account for possible effects of age, BMI and differences in tribal origin, multiple regression analysis was performed. Accounting for other variables, location (living in USA vs. Nigeria) remained the strongest predictor of total hirsutism score (P⫽0.01); tribal origin was also significant (P⫽0.02), while age and BMI had no independent predictive value (P⬎0.1). Living in USA vs. Nigeria remained a significant predictor in a regression model excluding women using hormonal treatments (P⫽0.02).
Vol. 86, Suppl 2, September 2006