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399: Metabolic acidosis decreases fetal myocardial isovolumic velocities in a chronic sheep model of increased placental vascular resistance
SMFM Abstracts 397
A NOVEL KISS-1 RELATED PEPTIDE AS INDICATOR OF MURINE PREGNANCY AND POTENTIAL PRENATAL DIAGNOSTIC MARKER NIMA GOHARKHAY1, HUAIZHI YIN1, ESTHER TAMAYO1, PHYLLIS GAMBLE1, FANGXIAN LU1, ANCIZAR BETANCOURT1, KARINA VILLARREAL1, GARY D.V. HANKINS1, GEORGE R. SAADE1, MONICA LONGO1, 1The University of Texas Medical Branch, Obstetrics and Gynecology, Galveston, Texas OBJECTIVE: The KISS-1 gene produces several peptides known to play important roles in reproduction, including the regulation of gonadotropin secretion. KISS-1 has also been described in the placenta. A recent study in humans has shown increasing levels of the secretory form of KISS-1 in blood with advancing gestation. No marker for early detection of mouse pregnancy is currently available, and the presence of gestation in mice can only be detected empirically. The determination of pregnancy in mice and other laboratory animals is important for the planning of studies relating to various aspects of reproduction. The purpose of this study was to determine whether a secreted form of KISS-1 was present in the blood of mice and if it could be utilized as a marker for pregnancy. STUDY DESIGN: Mice from the CD1 strain were bred under standardized conditions. The presence of sperm plugs was recorded as day 1 of gestation. Nonpregnant mice were used as controls. Blood samples were collected on days 7 and 14 of gestation and Western blot was performed using a polyclonal antibody to KISS-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Peptide levels were determined by densitometric analysis. RESULTS: We were able to demonstrate the presence of a peptide with specific reactivity to a polyclonal KISS-1 antibody in mouse serum. The levels increased significantly from day 7 to day 14 of gestation and were almost undetectable in nonpregnant animals (p⬍0.0001) (figure). The immunoreactive peptide identified was larger than any previously described KISS-1 related protein.
www.AJOG.org 399
METABOLIC ACIDOSIS DECREASES FETAL MYOCARDIAL ISOVOLUMIC VELOCITIES IN A CHRONIC SHEEP MODEL OF INCREASED PLACENTAL VASCULAR RESISTANCE GANESH ACHARYA1, JUHA RASANEN2, KAARIN MAKIKALLIO3, TIINA ERKINARO4, TOMI KAVASMAA4, MERVI HAAPSAMO3, LUC MERTENS5, JAMES HUHTA1, 1University of South Florida, Pediatrics, St. Petersburg, Florida, 2Oregon Health Sciences University, Obstetrics and Gynecology, Portland, Oregon, 3University of Oulu, Obstetrics and Gynecology, Oulu, Finland, 4University of Oulu, Anesthesiology, Oulu, Finland, 5 University Hospital Leuven, Pediatric Cardiology, Leuven, Belgium OBJECTIVE: We hypothesized that acute fetal metabolic acidosis decreases fetal myocardial motion in a chronic sheep model of increased placental vascular resistance (Rua). STUDY DESIGN: Eleven ewes and fetuses were instrumented at 118-122 days of gestation (term 145 days). After 5 days of recovery and 24 hours of placental embolization to increase Rua, the longitudinal myocardial velocities of the right (RV) and left (LV) ventricles and interventricular septum (IVS) were assessed at the level of the atrioventricular valve annuli using tissue Doppler imaging (TDI). Ventricular outflow and inflow velocities (E and A waves) and cardiac outputs were measured. All the measurements were performed at baseline and during fetal metabolic acidosis caused by epidural anesthesia-induced maternal hypotension. RESULTS: Maternal hypotension decreased (p⬍ 0.0001) fetal arterial pH (7.27⫹/⫺0.007 vs. 7.04⫹/⫺0.13). Compared to baseline, the peak isovolumic myocardial contraction and relaxation velocities of RV (p⬍ 0.02), LV (p⬍ 0.006) and IVS (p⬍ 0.003), early relaxation velocity (E=) of both ventricles (p⬍ 0.03), and the systolic velocity of the IVS (p⬍ 0.03) decreased during metabolic acidosis. The proportion of isovolumic contraction time of the cardiac cycle increased (p⬍ 0.05) but the isovolumic relaxation and ejection time proportions, and the TDI Tei index did not change. The E/E= ratio for both ventricles was higher (p⬍ 0.03) during metabolic acidosis compared to baseline. During metabolic acidosis RV, LV and combined cardiac outputs remained unchanged compared to baseline. CONCLUSION: In sheep fetuses with increased Rua and acute metabolic acidosis, the global cardiac function is preserved. However, acute metabolic acidosis impaired myocardial contractility during the isovolumic phase and relaxation during isovolumic and early filling phases of the cardiac cycle. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.417
CONCLUSION: A KISS-1 related peptide may be useful as a reliable and specific marker for pregnancy in mice and other species. The expression profile of this peptide makes it a strong candidate as a potential marker for use in prenatal diagnosis or for assessment of placental function. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.415
398
MATERNAL FOOD RESTRICTION IMPAIRS FETAL NEPHROGENESIS BY ALTERED TRANSCRIPTION FACTOR EXPRESSION TASMIA HENRY1, AHMED ABDEL-HAKEEM1, THOMAS MAGEE1, MINA DESAI1, CYNTHIA NAST2, ROY Z. MANSANO1, JOHN TORDAY1, MICHAEL ROSS1, 1Harbor-UCLA Med. Ctr. (LABioMed), Dept. of Ob/Gyn, Torrance, California, 2Cedars-Sinai Medical Center, Dept. of Pathology & Lab. Med., Los Angeles, California OBJECTIVE: Maternal food restriction (MFR) during pregnancy results in growth restricted newborns with reduced glomerular number. However the mechanism of altered nephrogenesis is poorly understood and there are neither prophylactic or rescue therapies. Reduced nephrogenesis potentially may be programmed during embryogenesis by altered gene expression of transcription factors regulating ureteric bud branching or mesenchyme condensation. We thus investigated several transcription factors controlling these two developmental phases of fetal nephrogenesis. STUDY DESIGN: From 10 to 20 days gestation, Sprague Dawley pregnant rats (n⫽12) were randomly allocated to ad libitum food, or 50% food restricted (MFR). At embryonic day 20 (e20), mRNA expression of transcription factors regulating mesenchyme condensation (WT-1, PAX-2) and ureteric bud branching (WNT-4 and WNT-11) were determined in fetal kidneys by real time RT-PCR. RESULTS: MFR resulted in changes in mRNA expression related to both mesenchyme and ureteric bud processes. Among mesenchymal factors, WT1 was upregulated (2-fold; p⬍0.05) in association with a downregulation of PAX2 (0.5fold). Among ureteric bud factors, WNT4 and WNT11 were similarly downregulated (0.2-fold and 0.3-fold, respectively). CONCLUSION: MFR results in altered gene expression of transcription factors regulating mesenchyme condensation and ureteric bud branching during embryonic development. As WT1 is a transcriptional repressor of PAX2, and PAX2 is an activator of WNT genes, these results suggest that MFR induces an initial upregulation of WT1 initiating a transcriptional cascade resulting in impaired nephrogenesis.
400
HUMAN CHORIONIC GONADOTROPHIN (HCG) IS PRODUCED BY BOTH SYNCYTIOTROPHOBLAST AND CYTOTROPHOBLAST CELLS: A PARADIGM SHIFT VICTORIA SNEGOVSKIKH1, ERIC HODGSON1, MARK WEHRUM1, GUOYANG LUO1, EDMUND FUNAI1, YUEHONG MA1, MIZANUR RAHMAN1, ERROL NORWITZ1, 1Yale University, New Haven, Connecticut OBJECTIVE: Cytotrophoblast cells are the stem cells for syncytio-trophoblast formation in early human pregnancy. Traditional teaching is that hCG production by syncytiotrophoblast (but not cytotrophoblast) is critical to the maintenance of early pregnancy by sustaining progesterone production by the corpus luteum. Indeed, hCG is regarded as a marker of differentiation from cytotrophoblast to syncytiotrophoblast. To test this hypothesis, we measured the production of hCG by freshly isolated cytotrophoblast, syncytiotrophoblast, and immortalized human extravillous cytotrophoblast-derived HTR-8/SV neo cells. STUDY DESIGN: Term placentae were collected from elective cesarean deliveries and cytotrophoblast cells isolated by enzymatic digestion and purified using Percoll gradient. As previously described, cells were cultured in Dulbecco’s medium containing 5% fetal calf serum for 3-4 days at which time syncytiotrophoblast formation was evident. HTR-8/SV neo cells were cultured to confluence in RPMI 1640 medium containing 5% fetal calf serum. After 24h incubation in serum-free medium, cells were incubated in fresh medium for 6-24h. Levels of hCG in conditioned supernatant were measured by ELISA and/or Immulite-I and corrected for protein content. Data are expressed as mean⫾SEM and differences analyzed by ANOVA and Chi Square tests. RESULTS: As expected, high levels of hCG were measured in supernatant from syncytiotrophoblast (40.8⫾7.5 mIU/mg of protein per 24h; n⫽6). Interestingly, hCG was also produced in large amounts by freshly isolated cytotrophoblast cells (36.6⫾4.3 mIU/mg of protein per 6h; n⫽6) and in small amounts by cytotrophoblast-derived HTR-8/SV neo cells (3.2⫾1.0 mIU/mg of protein per 24h; n⫽4; p⬍0.001 compared with syncytiotrophoblast). CONCLUSION: In contrast to traditional teaching, these studies demonstrate that hCG is produced not only by syncytiotrophoblast but also by freshly isolated and immortalized cytotrophoblast cells. Further studies are needed to investigate the source and regulation of hCG production in early pregnancy. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.418
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.416
S120
American Journal of Obstetrics & Gynecology Supplement to DECEMBER 2007
A NOVEL KISS-1 RELATED PEPTIDE AS INDICATOR OF MURINE PREGNANCY AND POTENTIAL PRENATAL DIAGNOSTIC MARKER NIMA GOHARKHAY1, HUAIZHI YIN1, ESTHER TAMAYO1, PHYLLIS GAMBLE1, FANGXIAN LU1, ANCIZAR BETANCOURT1, KARINA VILLARREAL1, GARY D.V. HANKINS1, GEORGE R. SAADE1, MONICA LONGO1, 1The University of Texas Medical Branch, Obstetrics and Gynecology, Galveston, Texas OBJECTIVE: The KISS-1 gene produces several peptides known to play important roles in reproduction, including the regulation of gonadotropin secretion. KISS-1 has also been described in the placenta. A recent study in humans has shown increasing levels of the secretory form of KISS-1 in blood with advancing gestation. No marker for early detection of mouse pregnancy is currently available, and the presence of gestation in mice can only be detected empirically. The determination of pregnancy in mice and other laboratory animals is important for the planning of studies relating to various aspects of reproduction. The purpose of this study was to determine whether a secreted form of KISS-1 was present in the blood of mice and if it could be utilized as a marker for pregnancy. STUDY DESIGN: Mice from the CD1 strain were bred under standardized conditions. The presence of sperm plugs was recorded as day 1 of gestation. Nonpregnant mice were used as controls. Blood samples were collected on days 7 and 14 of gestation and Western blot was performed using a polyclonal antibody to KISS-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Peptide levels were determined by densitometric analysis. RESULTS: We were able to demonstrate the presence of a peptide with specific reactivity to a polyclonal KISS-1 antibody in mouse serum. The levels increased significantly from day 7 to day 14 of gestation and were almost undetectable in nonpregnant animals (p⬍0.0001) (figure). The immunoreactive peptide identified was larger than any previously described KISS-1 related protein.
www.AJOG.org 399
METABOLIC ACIDOSIS DECREASES FETAL MYOCARDIAL ISOVOLUMIC VELOCITIES IN A CHRONIC SHEEP MODEL OF INCREASED PLACENTAL VASCULAR RESISTANCE GANESH ACHARYA1, JUHA RASANEN2, KAARIN MAKIKALLIO3, TIINA ERKINARO4, TOMI KAVASMAA4, MERVI HAAPSAMO3, LUC MERTENS5, JAMES HUHTA1, 1University of South Florida, Pediatrics, St. Petersburg, Florida, 2Oregon Health Sciences University, Obstetrics and Gynecology, Portland, Oregon, 3University of Oulu, Obstetrics and Gynecology, Oulu, Finland, 4University of Oulu, Anesthesiology, Oulu, Finland, 5 University Hospital Leuven, Pediatric Cardiology, Leuven, Belgium OBJECTIVE: We hypothesized that acute fetal metabolic acidosis decreases fetal myocardial motion in a chronic sheep model of increased placental vascular resistance (Rua). STUDY DESIGN: Eleven ewes and fetuses were instrumented at 118-122 days of gestation (term 145 days). After 5 days of recovery and 24 hours of placental embolization to increase Rua, the longitudinal myocardial velocities of the right (RV) and left (LV) ventricles and interventricular septum (IVS) were assessed at the level of the atrioventricular valve annuli using tissue Doppler imaging (TDI). Ventricular outflow and inflow velocities (E and A waves) and cardiac outputs were measured. All the measurements were performed at baseline and during fetal metabolic acidosis caused by epidural anesthesia-induced maternal hypotension. RESULTS: Maternal hypotension decreased (p⬍ 0.0001) fetal arterial pH (7.27⫹/⫺0.007 vs. 7.04⫹/⫺0.13). Compared to baseline, the peak isovolumic myocardial contraction and relaxation velocities of RV (p⬍ 0.02), LV (p⬍ 0.006) and IVS (p⬍ 0.003), early relaxation velocity (E=) of both ventricles (p⬍ 0.03), and the systolic velocity of the IVS (p⬍ 0.03) decreased during metabolic acidosis. The proportion of isovolumic contraction time of the cardiac cycle increased (p⬍ 0.05) but the isovolumic relaxation and ejection time proportions, and the TDI Tei index did not change. The E/E= ratio for both ventricles was higher (p⬍ 0.03) during metabolic acidosis compared to baseline. During metabolic acidosis RV, LV and combined cardiac outputs remained unchanged compared to baseline. CONCLUSION: In sheep fetuses with increased Rua and acute metabolic acidosis, the global cardiac function is preserved. However, acute metabolic acidosis impaired myocardial contractility during the isovolumic phase and relaxation during isovolumic and early filling phases of the cardiac cycle. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.417
CONCLUSION: A KISS-1 related peptide may be useful as a reliable and specific marker for pregnancy in mice and other species. The expression profile of this peptide makes it a strong candidate as a potential marker for use in prenatal diagnosis or for assessment of placental function. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.415
398
MATERNAL FOOD RESTRICTION IMPAIRS FETAL NEPHROGENESIS BY ALTERED TRANSCRIPTION FACTOR EXPRESSION TASMIA HENRY1, AHMED ABDEL-HAKEEM1, THOMAS MAGEE1, MINA DESAI1, CYNTHIA NAST2, ROY Z. MANSANO1, JOHN TORDAY1, MICHAEL ROSS1, 1Harbor-UCLA Med. Ctr. (LABioMed), Dept. of Ob/Gyn, Torrance, California, 2Cedars-Sinai Medical Center, Dept. of Pathology & Lab. Med., Los Angeles, California OBJECTIVE: Maternal food restriction (MFR) during pregnancy results in growth restricted newborns with reduced glomerular number. However the mechanism of altered nephrogenesis is poorly understood and there are neither prophylactic or rescue therapies. Reduced nephrogenesis potentially may be programmed during embryogenesis by altered gene expression of transcription factors regulating ureteric bud branching or mesenchyme condensation. We thus investigated several transcription factors controlling these two developmental phases of fetal nephrogenesis. STUDY DESIGN: From 10 to 20 days gestation, Sprague Dawley pregnant rats (n⫽12) were randomly allocated to ad libitum food, or 50% food restricted (MFR). At embryonic day 20 (e20), mRNA expression of transcription factors regulating mesenchyme condensation (WT-1, PAX-2) and ureteric bud branching (WNT-4 and WNT-11) were determined in fetal kidneys by real time RT-PCR. RESULTS: MFR resulted in changes in mRNA expression related to both mesenchyme and ureteric bud processes. Among mesenchymal factors, WT1 was upregulated (2-fold; p⬍0.05) in association with a downregulation of PAX2 (0.5fold). Among ureteric bud factors, WNT4 and WNT11 were similarly downregulated (0.2-fold and 0.3-fold, respectively). CONCLUSION: MFR results in altered gene expression of transcription factors regulating mesenchyme condensation and ureteric bud branching during embryonic development. As WT1 is a transcriptional repressor of PAX2, and PAX2 is an activator of WNT genes, these results suggest that MFR induces an initial upregulation of WT1 initiating a transcriptional cascade resulting in impaired nephrogenesis.
400
HUMAN CHORIONIC GONADOTROPHIN (HCG) IS PRODUCED BY BOTH SYNCYTIOTROPHOBLAST AND CYTOTROPHOBLAST CELLS: A PARADIGM SHIFT VICTORIA SNEGOVSKIKH1, ERIC HODGSON1, MARK WEHRUM1, GUOYANG LUO1, EDMUND FUNAI1, YUEHONG MA1, MIZANUR RAHMAN1, ERROL NORWITZ1, 1Yale University, New Haven, Connecticut OBJECTIVE: Cytotrophoblast cells are the stem cells for syncytio-trophoblast formation in early human pregnancy. Traditional teaching is that hCG production by syncytiotrophoblast (but not cytotrophoblast) is critical to the maintenance of early pregnancy by sustaining progesterone production by the corpus luteum. Indeed, hCG is regarded as a marker of differentiation from cytotrophoblast to syncytiotrophoblast. To test this hypothesis, we measured the production of hCG by freshly isolated cytotrophoblast, syncytiotrophoblast, and immortalized human extravillous cytotrophoblast-derived HTR-8/SV neo cells. STUDY DESIGN: Term placentae were collected from elective cesarean deliveries and cytotrophoblast cells isolated by enzymatic digestion and purified using Percoll gradient. As previously described, cells were cultured in Dulbecco’s medium containing 5% fetal calf serum for 3-4 days at which time syncytiotrophoblast formation was evident. HTR-8/SV neo cells were cultured to confluence in RPMI 1640 medium containing 5% fetal calf serum. After 24h incubation in serum-free medium, cells were incubated in fresh medium for 6-24h. Levels of hCG in conditioned supernatant were measured by ELISA and/or Immulite-I and corrected for protein content. Data are expressed as mean⫾SEM and differences analyzed by ANOVA and Chi Square tests. RESULTS: As expected, high levels of hCG were measured in supernatant from syncytiotrophoblast (40.8⫾7.5 mIU/mg of protein per 24h; n⫽6). Interestingly, hCG was also produced in large amounts by freshly isolated cytotrophoblast cells (36.6⫾4.3 mIU/mg of protein per 6h; n⫽6) and in small amounts by cytotrophoblast-derived HTR-8/SV neo cells (3.2⫾1.0 mIU/mg of protein per 24h; n⫽4; p⬍0.001 compared with syncytiotrophoblast). CONCLUSION: In contrast to traditional teaching, these studies demonstrate that hCG is produced not only by syncytiotrophoblast but also by freshly isolated and immortalized cytotrophoblast cells. Further studies are needed to investigate the source and regulation of hCG production in early pregnancy. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.418
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.416
S120
American Journal of Obstetrics & Gynecology Supplement to DECEMBER 2007