- Email: [email protected]
SMAD3 pathway
TABLE 1. P4 ranges and percent outcomes
LBR, OPR (%) Missed Abortion, Biochemical pregnancies (%)
P4 (10-15)
P4 (15-20)
P4 (20-30)
P4 (30-40)
P4 (>40)
70 7
62 15
52 27
50 32
33 20
*P4 is in ng/ml CONCLUSION: P4 levels greater than 20 ng/ml on day of transfer (during FET cycles) are associated with increased incidence of missed abortion and biochemical pregnancies and decreased incidence of live birth/ongoing pregnancy.
EARLY PREGNANCY P-296 Tuesday, October 21, 2014 MICRORNA-224 STIMULATES THE EXPRESSION OF MATRIX METALLOPROTEINASE (MMP)-2 AND MMP-9 IN HUMAN TROPHOBLAST CELL LINE JEG-3 VIA TGF-b/SMAD3 PATHWAY. Z. Huang, S. Li, Q. Ma, W. Fan, Y. Wang, Z. Xiao. Obstetrics and Gynecology, West China 2nd Hospital, Sichuan University, Chengdu, Sichuan, China. OBJECTIVE: Our previous study showed that TGF-b1 treatment upregulated the expression of miR-224 and the invasive-associated genes MMP-2/9 in human trophoblast cell line JEG-3 cells. The objectives of this study is to investigate whether miR-224 is involved in TGF-b induced up-regulation of MMP-2/9 in JEG-3 cells and the possible mechanisms. DESIGN: A controlled experiment. MATERIALS AND METHODS: JEG-3 cells were transfected with miR-224 mimics, miR-224 mimics control, pretreated with SB431542 (a TGF-bIR inhibitor) for 30 minutes and then transfected with miR-224, or co-transfected with miR-224 mimics and siRNA targeting Smad3 using Lipofectamine following the manufacturer’s protocol. After transfection for 6h, the media were changed into medium with TGF-b1 at the concentration of 5ng/ml for 24-48h. Quantitative real-time PCR and Western blot analysis were used to detect the mRNA and protein levels of MMP-2 /9 in JEG-3 cells. RESULTS: Over-expression of miR-224 by transfection with miR-224 mimics led to the up-regulation of MMP-2 and MMP-9. Both the TGFbIR inhibitor SB431542 and the Smad3 siRNA induced mRNA silencing significantly attenuated the stimulatory effect of miR-224 on MMP-9 and MMP-2 mRNA expression in JEG-3 cells. The protein expression levels of MMP-2 and MMP-9 induced by miR-224 were significantly inhibited by SB431542 pretreatment or co-transfection of Smad3 siRNA. CONCLUSION: MiR-224 could enhance TGF-b induced the upregulation of invasive-associated genes MMP-2/9 of JEG-3 cell and the effect may partly dependent on the TGF-b/Smad3 signaling pathway. Supported by: This research is Supported by the National Natural Science Foundation of China (No.81200453). P-297 Tuesday, October 21, 2014 THROMBIN ALTERS DECIDUALIZATION AND MATRIX HOMEOSTASIS IN HUMAN ENDOMETRIAL STROMAL CELLS. S. N. Babayev, O. Bukulmez, B. R. Carr, R. A. Word. OB/ Gyn, UT Southwestern Medical Center, Dallas, TX. OBJECTIVE: Vaginal bleeding and subchorionic hematomas are associated with increased risk of both early and late pregnancy loss. Previous work suggests that thrombin generation may play a pivotal role in development of these complications. Specifically, in human endometrial stromal cells (HESC), thrombin acts via protease-activated receptors (e.g. PAR-1) to induce matrix metalloproteinases (MMPs) and inflammatory factors. Our hypothesis is that thrombin activates MMPs and disrupts expression of genes involved in matrix homeostasis. Further, we sought to determine if thrombin alters the decidualization process. DESIGN: HESCs (precursor of decidua) were used as an in vitro model system.
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ASRM Abstracts
MATERIALS AND METHODS: HESC were isolated from proliferative phase endometrium of premenopausal women undergoing benign hysterectomy. Cells were treated with vehicle or thrombin at baseline or during decidualization using cAMP and medroxyprogesterone acetate (MPA). Thereafter, mRNA levels were analyzed by qPCR. RESULTS: As expected, two marker of decidualization (IGF-1 and prolactin (PRL)) were induced 15 and 9-fold 72 h after initiation of cAMP/MPA. Treatment with human plasma thrombin (6 U/ml) x 24 h decreased mRNA of both IGF-1 and PRL at baseline (71-73%, P<0.04). Interestingly, thrombin completely blocked expression of IGF-1 in decidualized cells and decreased PRL mRNA from 9.71.48 to 5.470.63 R.U./GAPDH (p¼0.057). Thrombin induced MMP1 at baseline (7-fold, p<0.01) and during decidualization (11.4-fold, P<0.01). COX-2 gene expression was also induced 8-fold under baseline conditions and amplified in cells undergoing decidualization (from 4- to 7.7-fold). Thrombin also decreased expression of collagen 1a1 50-65%, but not fibronectin. Further, thrombin decreased mRNA of lysyl oxidase (LOX, the major collagen cross-linking enzyme). Decidualization increased LOX mRNA 6.8-fold which was inhibited significantly by thrombin. Recombinant thrombin mimicked the effects of purified plasma thrombin suggesting that these effects were not due to contamination of plasma thrombin. Like thrombin, PAR-1 agonist inhibited IGF-1, PRL, LOX, and Col1a1. CONCLUSION: Taken together, our data indicate that thrombin adversely affects decidualization in vitro primarily through PAR-1. Furthermore, thrombin not only activates genes involved in matrix degradation (MMPs) and inflammation (COX-2), but also suppresses genes encoding factors important for collagen formation (Col1a1, LOX). The results suggest that intrauterine bleeding and generation of thrombin impairs decidualization and endometrial support of early pregnancy. Supported by: Study was Supported by Division of Reproductive Endocrinology and Infertility, UT Southwestern Medical Center.
P-298 Tuesday, October 21, 2014 ‘‘NEGATIVE’’ SERUM HCG CAN FAIL TO DIAGNOSE ECTOPIC AND ABNORMAL INTRAUTERINE PREGNANCY AT LEVELS BETWEEN 1.0-5.0MIU/ML. B.-S. L. Maslow, A. Bartolucci, C. M. Sueldo, L. Engmann, C. A. Benadiva, J. C. Nulsen. Center for Advanced Reproductive Services/University of Connecticut Health Center, Farmington, CT. OBJECTIVE: To assess the negative predictive value (NPV) of a ‘‘negative’’ first post-embryo transfer (ET) quantative serum hCG with respect to ectopic pregnancy (EP) or abnormal intrauterine pregnancy (IUP). DESIGN: Retrospective cohort study. MATERIALS AND METHODS: We collected all first post-ET ‘‘negative’’ serum hCG results processed at a large fertility center from 6/2009-2/ 2014 and recorded clinical outcomes. ‘‘Negative’’ serum hCG was defined as <5mIU/mL. All first post-ET serum hCG were measured 14 days after oocyte retrieval and processed at our in-house laboratory with Siemens Immulite 2000. Associations were analyzed using c2 test or Fisher’s Exact test for categorical variables and student’s t-test for continuous variables. RESULTS: 1495 first post-ET serum hCG <5mIU/mL were collected. 1343 (90%) were <1.0mIU/mL and 152 results (10%) were 1.0-5.0mIU/ mL. 5.3% (8/152) of those with hCG 1.0-5.0mIU/mL were subsequently diagnosed with a pregnancy. There were 4 EP, of which 3 required salpingectomy, and 4 spontaneous abortions, one who presented with hemorrhage and hemodynamic instability, requiring surgery and transfusion. No normal pregnancies were identified with hCG 1.0-5.0mIU/mL. The mean first post-ET serum hCG in abnormal pregnancies was 2.641.27mIU/mL. There was no correlation between the concentration of hCG 1.0-5.0mIU/mL and risk of abnormal pregnancy (p¼0.6). No pregnancies were identified in the hCG<1.0mIU/mL group. Women with hCG 1.0-5.0mIU/mL were significantly more likely to be diagnosed with an abnormal pregnancy than those with hCG<1.0 (5/152 vs. 0/1343, p<0.001). The NPV of first post-ET hCG<5mIU/mL was 99.4% overall. NPV decreased to 94.7% for hCG 1.0-5.0mIU/mL. 19 women with hCG 1.0-5.0mIU/mL would need to have repeat testing 48 hours later to diagnose one abnormal pregnancy (NNT¼19). CONCLUSION: ‘‘Negative’’ serum hCG is widely accepted as <5mIU/ mL. 90% of all negative results fall below 1.0mIU/mL. However, this study demonstrates that serum hCG levels 1.0-5.0mIU/mL can fail to exclude abnormal pregnancies, with potentially severe clinical implications. This is especially true for women at high risk for abnormal pregnancy, such as those undergoing assisted reproductive technologies. We propose that a first postET serum hCG 1.0-5.0mIU/mL be considered ‘‘indeterminate,’’ and recommend repeat testing 48 hours later. Serial serum hCG levels in those whose
Vol. 102, No. 3, Supplement, September 2014
LBR, OPR (%) Missed Abortion, Biochemical pregnancies (%)
P4 (10-15)
P4 (15-20)
P4 (20-30)
P4 (30-40)
P4 (>40)
70 7
62 15
52 27
50 32
33 20
*P4 is in ng/ml CONCLUSION: P4 levels greater than 20 ng/ml on day of transfer (during FET cycles) are associated with increased incidence of missed abortion and biochemical pregnancies and decreased incidence of live birth/ongoing pregnancy.
EARLY PREGNANCY P-296 Tuesday, October 21, 2014 MICRORNA-224 STIMULATES THE EXPRESSION OF MATRIX METALLOPROTEINASE (MMP)-2 AND MMP-9 IN HUMAN TROPHOBLAST CELL LINE JEG-3 VIA TGF-b/SMAD3 PATHWAY. Z. Huang, S. Li, Q. Ma, W. Fan, Y. Wang, Z. Xiao. Obstetrics and Gynecology, West China 2nd Hospital, Sichuan University, Chengdu, Sichuan, China. OBJECTIVE: Our previous study showed that TGF-b1 treatment upregulated the expression of miR-224 and the invasive-associated genes MMP-2/9 in human trophoblast cell line JEG-3 cells. The objectives of this study is to investigate whether miR-224 is involved in TGF-b induced up-regulation of MMP-2/9 in JEG-3 cells and the possible mechanisms. DESIGN: A controlled experiment. MATERIALS AND METHODS: JEG-3 cells were transfected with miR-224 mimics, miR-224 mimics control, pretreated with SB431542 (a TGF-bIR inhibitor) for 30 minutes and then transfected with miR-224, or co-transfected with miR-224 mimics and siRNA targeting Smad3 using Lipofectamine following the manufacturer’s protocol. After transfection for 6h, the media were changed into medium with TGF-b1 at the concentration of 5ng/ml for 24-48h. Quantitative real-time PCR and Western blot analysis were used to detect the mRNA and protein levels of MMP-2 /9 in JEG-3 cells. RESULTS: Over-expression of miR-224 by transfection with miR-224 mimics led to the up-regulation of MMP-2 and MMP-9. Both the TGFbIR inhibitor SB431542 and the Smad3 siRNA induced mRNA silencing significantly attenuated the stimulatory effect of miR-224 on MMP-9 and MMP-2 mRNA expression in JEG-3 cells. The protein expression levels of MMP-2 and MMP-9 induced by miR-224 were significantly inhibited by SB431542 pretreatment or co-transfection of Smad3 siRNA. CONCLUSION: MiR-224 could enhance TGF-b induced the upregulation of invasive-associated genes MMP-2/9 of JEG-3 cell and the effect may partly dependent on the TGF-b/Smad3 signaling pathway. Supported by: This research is Supported by the National Natural Science Foundation of China (No.81200453). P-297 Tuesday, October 21, 2014 THROMBIN ALTERS DECIDUALIZATION AND MATRIX HOMEOSTASIS IN HUMAN ENDOMETRIAL STROMAL CELLS. S. N. Babayev, O. Bukulmez, B. R. Carr, R. A. Word. OB/ Gyn, UT Southwestern Medical Center, Dallas, TX. OBJECTIVE: Vaginal bleeding and subchorionic hematomas are associated with increased risk of both early and late pregnancy loss. Previous work suggests that thrombin generation may play a pivotal role in development of these complications. Specifically, in human endometrial stromal cells (HESC), thrombin acts via protease-activated receptors (e.g. PAR-1) to induce matrix metalloproteinases (MMPs) and inflammatory factors. Our hypothesis is that thrombin activates MMPs and disrupts expression of genes involved in matrix homeostasis. Further, we sought to determine if thrombin alters the decidualization process. DESIGN: HESCs (precursor of decidua) were used as an in vitro model system.
e238
ASRM Abstracts
MATERIALS AND METHODS: HESC were isolated from proliferative phase endometrium of premenopausal women undergoing benign hysterectomy. Cells were treated with vehicle or thrombin at baseline or during decidualization using cAMP and medroxyprogesterone acetate (MPA). Thereafter, mRNA levels were analyzed by qPCR. RESULTS: As expected, two marker of decidualization (IGF-1 and prolactin (PRL)) were induced 15 and 9-fold 72 h after initiation of cAMP/MPA. Treatment with human plasma thrombin (6 U/ml) x 24 h decreased mRNA of both IGF-1 and PRL at baseline (71-73%, P<0.04). Interestingly, thrombin completely blocked expression of IGF-1 in decidualized cells and decreased PRL mRNA from 9.71.48 to 5.470.63 R.U./GAPDH (p¼0.057). Thrombin induced MMP1 at baseline (7-fold, p<0.01) and during decidualization (11.4-fold, P<0.01). COX-2 gene expression was also induced 8-fold under baseline conditions and amplified in cells undergoing decidualization (from 4- to 7.7-fold). Thrombin also decreased expression of collagen 1a1 50-65%, but not fibronectin. Further, thrombin decreased mRNA of lysyl oxidase (LOX, the major collagen cross-linking enzyme). Decidualization increased LOX mRNA 6.8-fold which was inhibited significantly by thrombin. Recombinant thrombin mimicked the effects of purified plasma thrombin suggesting that these effects were not due to contamination of plasma thrombin. Like thrombin, PAR-1 agonist inhibited IGF-1, PRL, LOX, and Col1a1. CONCLUSION: Taken together, our data indicate that thrombin adversely affects decidualization in vitro primarily through PAR-1. Furthermore, thrombin not only activates genes involved in matrix degradation (MMPs) and inflammation (COX-2), but also suppresses genes encoding factors important for collagen formation (Col1a1, LOX). The results suggest that intrauterine bleeding and generation of thrombin impairs decidualization and endometrial support of early pregnancy. Supported by: Study was Supported by Division of Reproductive Endocrinology and Infertility, UT Southwestern Medical Center.
P-298 Tuesday, October 21, 2014 ‘‘NEGATIVE’’ SERUM HCG CAN FAIL TO DIAGNOSE ECTOPIC AND ABNORMAL INTRAUTERINE PREGNANCY AT LEVELS BETWEEN 1.0-5.0MIU/ML. B.-S. L. Maslow, A. Bartolucci, C. M. Sueldo, L. Engmann, C. A. Benadiva, J. C. Nulsen. Center for Advanced Reproductive Services/University of Connecticut Health Center, Farmington, CT. OBJECTIVE: To assess the negative predictive value (NPV) of a ‘‘negative’’ first post-embryo transfer (ET) quantative serum hCG with respect to ectopic pregnancy (EP) or abnormal intrauterine pregnancy (IUP). DESIGN: Retrospective cohort study. MATERIALS AND METHODS: We collected all first post-ET ‘‘negative’’ serum hCG results processed at a large fertility center from 6/2009-2/ 2014 and recorded clinical outcomes. ‘‘Negative’’ serum hCG was defined as <5mIU/mL. All first post-ET serum hCG were measured 14 days after oocyte retrieval and processed at our in-house laboratory with Siemens Immulite 2000. Associations were analyzed using c2 test or Fisher’s Exact test for categorical variables and student’s t-test for continuous variables. RESULTS: 1495 first post-ET serum hCG <5mIU/mL were collected. 1343 (90%) were <1.0mIU/mL and 152 results (10%) were 1.0-5.0mIU/ mL. 5.3% (8/152) of those with hCG 1.0-5.0mIU/mL were subsequently diagnosed with a pregnancy. There were 4 EP, of which 3 required salpingectomy, and 4 spontaneous abortions, one who presented with hemorrhage and hemodynamic instability, requiring surgery and transfusion. No normal pregnancies were identified with hCG 1.0-5.0mIU/mL. The mean first post-ET serum hCG in abnormal pregnancies was 2.641.27mIU/mL. There was no correlation between the concentration of hCG 1.0-5.0mIU/mL and risk of abnormal pregnancy (p¼0.6). No pregnancies were identified in the hCG<1.0mIU/mL group. Women with hCG 1.0-5.0mIU/mL were significantly more likely to be diagnosed with an abnormal pregnancy than those with hCG<1.0 (5/152 vs. 0/1343, p<0.001). The NPV of first post-ET hCG<5mIU/mL was 99.4% overall. NPV decreased to 94.7% for hCG 1.0-5.0mIU/mL. 19 women with hCG 1.0-5.0mIU/mL would need to have repeat testing 48 hours later to diagnose one abnormal pregnancy (NNT¼19). CONCLUSION: ‘‘Negative’’ serum hCG is widely accepted as <5mIU/ mL. 90% of all negative results fall below 1.0mIU/mL. However, this study demonstrates that serum hCG levels 1.0-5.0mIU/mL can fail to exclude abnormal pregnancies, with potentially severe clinical implications. This is especially true for women at high risk for abnormal pregnancy, such as those undergoing assisted reproductive technologies. We propose that a first postET serum hCG 1.0-5.0mIU/mL be considered ‘‘indeterminate,’’ and recommend repeat testing 48 hours later. Serial serum hCG levels in those whose
Vol. 102, No. 3, Supplement, September 2014