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Effect of GnRH-I and -II on human first trimester decidual stromal cells in vitro
SMFM Abstracts S169 590 THE ROLE OF PRENATAL VERSUS POSTNATAL ENVIRONMENT ON POSTNATAL GROWTH SHANNON CLARK (F)1, MONICA LONGO1, JOSJE LANGENVELD2, G. D. V. HANKINS1, GARLAND ANDERSON1, GEORGE SAADE1, 1University of Texas Medical Branch at Galveston, Obstetrics and Gynecology, Galveston, Texas, 2 University Hospital, Maastricht, Obstetrics and Gynecology, Maastricht, Netherlands OBJECTIVE: It has been shown that an altered uterine environment affects postnatal growth. Our aim was to determine the role of cross-fostering on the altered postnatal growth in an established transgenic animal model of fetal programming induced by an abnormal uterine environment secondary to lack of endothelial nitric oxide synthase (NOS3). STUDY DESIGN: Homozygous NOS3 knockout (C57BL/6J-NOS3ÿ/ÿKO) and wild-type mice (NOS3C/CWT) were cross-bred to obtain 2 heterozygous litters that are genomically-similar but with normal (paternally-derived; NOS3C/ÿpat) or abnormal (maternally-derived; NOS3C/ÿmat) uterine environment. Within 24 hours of delivery, the NOS3C/ÿmat were switched to a wild-type foster mother (NOS3FWT/ÿmat) and NOS3C/ÿpat litters to a knockout foster mother (NOS3FKO/ÿpat). Pups from each litter were jointly counted and weighed for 21 days (weaning time), then the litters were separated by sex and weighed weekly until week 8 (adult time). The cross-fostered litters were compared with similar naturally-fostered heterozygous litters. One-way ANOVA and Newman-Keuls post hoc tests were used for statistical analysis. RESULTS: The growth curve of NOS3C/ÿpat litters was significantly higher than NOS3C/ÿmat, NOS3FWT/ÿmat, and NOS3FKO/ÿpat litters from day 13 to 21 (p ! 0.001). The growth of male NOS3C/ÿpat pups from week 4 to 8 was also significantly better compared with the other groups (p!0.05). No differences were observed between NOS3FWT/ÿmat and NOS3FKO/ÿpat litters, however these litters had significantly decreased growth at 7-8 weeks when compared to naturally-fostered NOS3C/ÿmat and NOS3C/ÿpat litters (p ! 0.05). CONCLUSION: An adverse uterine environment has a deleterious effect on postnatal growth, and its effect predominates over the postnatal environment. While an adverse postnatal environment can worsen growth, an improvement in the postnatal environment has only a slight beneficial effect. Our findings confirm the primary importance of the uterine environment and fetal programming in health and disease in later life.
592 HUMAN -DEFENSIN 2: A NATURAL ANTI-MICROBIAL AGENT PRESENT IN NORMAL AMNIOTIC FLUID PARTICIPATES IN THE HOST RESPONSE TO INTRA-AMNIOTIC INFECTION ELEAZAR SOTO1, JIMMY ESPINOZA1, JYH KAE NIEN2, JUAN PEDRO KUSANOVIC1, OFFER EREZ1, KARINA RICHANI1, JOAQUIN SANTOLAYA1, ROBERTO ROMERO2, 1Wayne State University School of Medicine, Department of Obstetrics and Gynecology, Detroit, Michigan, 2Perinatology Research Branch, NICHD, NIH, DHHS, Bethesda, Maryland OBJECTIVE: Human b-defensin 2 (HBD-2) is a potent antimicrobial peptide which is part of the innate immune response. The purpose of this study was to determine if HBD-2 is present in amniotic fluid (AF) and if its concentration changes with intra-amniotic infection (IAI) and preterm labor. STUDY DESIGN: AF was retrieved by amniocentesis from 267 patients in the following groups: 1) mid-trimester (n = 75); 2) term not in labor (n = 28); 3) preterm labor and intact membranes without IAI who delivered at term (n = 36), preterm (n = 52) and preterm labor with IAI (n = 25); and 4) preterm premature rupture of membranes (PPROM) with (n = 25) and without (n = 26) IAI. IAI was defined as a positive AF culture for microorganisms. AF HBD-2 concentrations were determined by ELISA. Non-parametric statistics were used for analysis. RESULTS: 1) HBD-2 was detected in all AF samples; 2) the concentrations of HBD-2 did not change with gestational age (mid-trimester: median: 3 ng/ml; range: 0.04-11.8 vs. term: median: 2.9 ng/ml; range: 0.3-15.1; p = 0.8); 3) patients with IAI had a higher AF HBD-2 than those with sterile AF (for preterm labor: median: 17.6 ng/ml; range: 0.96-105.2 vs. median: 5 ng/ml; range: 0.1-140; p!0.01 and for PPROM: median: 5.8 ng/ml; range: 0.57-66.7 vs. median: 3.4 ng/ml; range: 1.04-19.6; p=0.02); 4) patients with preterm labor and sterile AF who delivered preterm had a higher median AF HBD-2 than those with preterm labor who delivered at term (median: 5.5 ng/ml; range: 0.11-140.2 vs. median: 3.4 ng/ml; range: 0.57-17.05; p!0.01). CONCLUSION: 1) AF contains HBD-2, a natural anti-microbial peptide, and this may account for some of the normal anti-microbial activity of AF reported more than 20 years ago; 2) AF HBD-2 concentrations are increased in women with IAI, regardless of membrane status (intact membranes or PROM); and 3) we propose that AF HBD-2 is part of the innate immune system within the amniotic cavity.
591 PROGRAMMING OF FAT AND MUSCLE: IN UTERO UNDERNUTRITION AND POSTWEANING CAFETERIA DIET MINA DESAI1, DAVE A. GAYLE1, LINDA DAY1, STACY BEHARE1, MICHAEL G. ROSS1, 1Harbor-UCLA Med. Ctr. (LA BioMed), Dept. of Ob/Gyn, Torrance, California OBJECTIVE: Maternal nutrition plays a crucial role in the development of offspring obesity. We have shown that maternal food restriction during pregnancy results in growth retarded pups that become obese when nursed ad libitum and provided laboratory chow (LC) post-weaning. To simulate the effects of growth retarded infants fed high fat western diets, we studied the combined effects of maternal food restriction in pregnancy/lactation and a post-weaning cafeteria diet, on offspring body composition. STUDY DESIGN: From day 10 to term gestation and throughout lactation, control pregnant rats received ad libitum (AdLib) food, whereas study rats were 50% food restricted (FR). Cross-fostering allowed evaluation of pregnancy vs lactation. The groups studied were AdLib/AdLib, FR/AdLib, and AdLib/FR. At 3 weeks, offspring were weaned to LC (9% energy as fat) or cafeteria diet (16% energy as fat). Body composition was analyzed in 9 month old male offspring using DEXA. RESULTS: FR in pregnancy resulted in obese offspring, as FR/AdLib fed LC were significantly (p!0.01) heavier (742 G 15 vs 647 G 18g) with greater body fat (20 G 2 vs 12 G 2%) and reduced lean body mass (78 G 2 vs 85G3%) than controls. FR in lactation did not alter habitus, as AdLib/FR offspring had comparable body weight, fat and lean mass as controls. Controls fed cafeteria diet showed similar body weight (664 G 18g), but increased fat (20G2%) and lower lean mass (77 G 2%) as controls fed LC. The obesity predisposition of growth restricted FR/AdLib offspring was accentuated by cafeteria diet, with offspring exhibiting markedly increased body weight (816G20g) and fat (29 G 2%) but reduced lean mass (68 G 2%) as compared to both controls on cafeteria diet or FR/AdLib offspring on LC. CONCLUSION: Akin to human populations, growth retarded rat newborns are predisposed to adult obesity. The results suggest that rapid newborn catch up growth, combined with western diets, further potentiates manifestations of offspring obesity. We speculate that titrated feeding of low birth weight infants may reverse the predisposition to offspring obesity.
593 EFFECT OF GNRH-I AND -II ON HUMAN FIRST TRIMESTER DECIDUAL STROMAL CELLS IN VITRO NASTARAN FOYOUZI1, VICTORIA SNEGOVSKIKH1, FREDERICK SCHATZ1, SETH GULLER1, ELIZA MEADE1, CATALIN BUHIMSCHI1, IRINA BUHIMSCHI1, CHARLES LOCKWOOD1, ERROL NORWITZ1, 1Yale University, Obstetrics & Gynecology, New Haven, Connecticut OBJECTIVE: Hypothalamic GnRH (GnRH-I) plays a critical role in regulating mammalian reproduction. GnRH-I and a second isoform (GnRH-II) are produced also by extra-hypothalamic tissues (including placenta and decidua) and have autocrine/paracrine functions. The function of GnRH in decidua is unknown, but it has been implicated in decidualization and regulating trophoblast invasion. This study investigates the effects of GnRH-I and -II on the production of cytokines and angiogenic factors by first trimester decidual stromal cells. STUDY DESIGN: First trimester decidua was identified from pregnancy termination tissues. Stromal cells were isolated by enzymic digestion, purified, and depleted of leukocytes. Cells were pretreated with estradiol (10ÿ8 M [E2]), medroxyprogesterone acetate (10ÿ7 M [MPA]), both, or vehicle for 7 days. After 24h incubation in fresh medium, cells were stimulated with GnRH-I (1-100 nM), GnRH-II (1-100 nM), IL-1B (1 ng/mL), or thrombin (25 IU/mL) for 48h. Levels of IL-8, IL-6, MMP-3, MCP-1, uPA, PAI-1, VEGF, and sFlt1 in conditioned supernatant were measured by ELISA. Measurements were corrected for protein content. RESULTS: Neither GnRH-I nor -II significantly altered the production of cytokines or angiogenic factors regardless of the hormonal milieu. Positive controls included IL-1B (which upregulated IL-8 by 2581.5-fold; IL-6 by 1334.8-fold; MMP-3 by 10.5-fold; MCP-1 by 128.3-fold; uPA by 6.2-fold; VEGF by 3.2-fold; and sFlt-1 by 4.0-fold [P!0.05 for all]) and thrombin (which upregulated IL-8 by 74.5-fold; IL-6 by 2.5-fold; MMP-3 by 3.8-fold; MCP-1 by 3.4-fold; uPA by 14.1-fold; and sFlt-1 by 18.3-fold [P!0.05 for all]). To confirm their biologic activity, both GnRH-I (100 nM) and –II (100 nM) stimulated hCG production by freshly isolated syncytiotrophoblast cells by 2.3- and 2.7-fold, respectively (ANOVA; P!0.05). CONCLUSION: GnRH-I and -II did not significantly effect production of cytokines and angiogenic factors by first trimester decidual stromal cells. Further studies are required to better define the biologic function of GnRH in the decidua.
592 HUMAN -DEFENSIN 2: A NATURAL ANTI-MICROBIAL AGENT PRESENT IN NORMAL AMNIOTIC FLUID PARTICIPATES IN THE HOST RESPONSE TO INTRA-AMNIOTIC INFECTION ELEAZAR SOTO1, JIMMY ESPINOZA1, JYH KAE NIEN2, JUAN PEDRO KUSANOVIC1, OFFER EREZ1, KARINA RICHANI1, JOAQUIN SANTOLAYA1, ROBERTO ROMERO2, 1Wayne State University School of Medicine, Department of Obstetrics and Gynecology, Detroit, Michigan, 2Perinatology Research Branch, NICHD, NIH, DHHS, Bethesda, Maryland OBJECTIVE: Human b-defensin 2 (HBD-2) is a potent antimicrobial peptide which is part of the innate immune response. The purpose of this study was to determine if HBD-2 is present in amniotic fluid (AF) and if its concentration changes with intra-amniotic infection (IAI) and preterm labor. STUDY DESIGN: AF was retrieved by amniocentesis from 267 patients in the following groups: 1) mid-trimester (n = 75); 2) term not in labor (n = 28); 3) preterm labor and intact membranes without IAI who delivered at term (n = 36), preterm (n = 52) and preterm labor with IAI (n = 25); and 4) preterm premature rupture of membranes (PPROM) with (n = 25) and without (n = 26) IAI. IAI was defined as a positive AF culture for microorganisms. AF HBD-2 concentrations were determined by ELISA. Non-parametric statistics were used for analysis. RESULTS: 1) HBD-2 was detected in all AF samples; 2) the concentrations of HBD-2 did not change with gestational age (mid-trimester: median: 3 ng/ml; range: 0.04-11.8 vs. term: median: 2.9 ng/ml; range: 0.3-15.1; p = 0.8); 3) patients with IAI had a higher AF HBD-2 than those with sterile AF (for preterm labor: median: 17.6 ng/ml; range: 0.96-105.2 vs. median: 5 ng/ml; range: 0.1-140; p!0.01 and for PPROM: median: 5.8 ng/ml; range: 0.57-66.7 vs. median: 3.4 ng/ml; range: 1.04-19.6; p=0.02); 4) patients with preterm labor and sterile AF who delivered preterm had a higher median AF HBD-2 than those with preterm labor who delivered at term (median: 5.5 ng/ml; range: 0.11-140.2 vs. median: 3.4 ng/ml; range: 0.57-17.05; p!0.01). CONCLUSION: 1) AF contains HBD-2, a natural anti-microbial peptide, and this may account for some of the normal anti-microbial activity of AF reported more than 20 years ago; 2) AF HBD-2 concentrations are increased in women with IAI, regardless of membrane status (intact membranes or PROM); and 3) we propose that AF HBD-2 is part of the innate immune system within the amniotic cavity.
591 PROGRAMMING OF FAT AND MUSCLE: IN UTERO UNDERNUTRITION AND POSTWEANING CAFETERIA DIET MINA DESAI1, DAVE A. GAYLE1, LINDA DAY1, STACY BEHARE1, MICHAEL G. ROSS1, 1Harbor-UCLA Med. Ctr. (LA BioMed), Dept. of Ob/Gyn, Torrance, California OBJECTIVE: Maternal nutrition plays a crucial role in the development of offspring obesity. We have shown that maternal food restriction during pregnancy results in growth retarded pups that become obese when nursed ad libitum and provided laboratory chow (LC) post-weaning. To simulate the effects of growth retarded infants fed high fat western diets, we studied the combined effects of maternal food restriction in pregnancy/lactation and a post-weaning cafeteria diet, on offspring body composition. STUDY DESIGN: From day 10 to term gestation and throughout lactation, control pregnant rats received ad libitum (AdLib) food, whereas study rats were 50% food restricted (FR). Cross-fostering allowed evaluation of pregnancy vs lactation. The groups studied were AdLib/AdLib, FR/AdLib, and AdLib/FR. At 3 weeks, offspring were weaned to LC (9% energy as fat) or cafeteria diet (16% energy as fat). Body composition was analyzed in 9 month old male offspring using DEXA. RESULTS: FR in pregnancy resulted in obese offspring, as FR/AdLib fed LC were significantly (p!0.01) heavier (742 G 15 vs 647 G 18g) with greater body fat (20 G 2 vs 12 G 2%) and reduced lean body mass (78 G 2 vs 85G3%) than controls. FR in lactation did not alter habitus, as AdLib/FR offspring had comparable body weight, fat and lean mass as controls. Controls fed cafeteria diet showed similar body weight (664 G 18g), but increased fat (20G2%) and lower lean mass (77 G 2%) as controls fed LC. The obesity predisposition of growth restricted FR/AdLib offspring was accentuated by cafeteria diet, with offspring exhibiting markedly increased body weight (816G20g) and fat (29 G 2%) but reduced lean mass (68 G 2%) as compared to both controls on cafeteria diet or FR/AdLib offspring on LC. CONCLUSION: Akin to human populations, growth retarded rat newborns are predisposed to adult obesity. The results suggest that rapid newborn catch up growth, combined with western diets, further potentiates manifestations of offspring obesity. We speculate that titrated feeding of low birth weight infants may reverse the predisposition to offspring obesity.
593 EFFECT OF GNRH-I AND -II ON HUMAN FIRST TRIMESTER DECIDUAL STROMAL CELLS IN VITRO NASTARAN FOYOUZI1, VICTORIA SNEGOVSKIKH1, FREDERICK SCHATZ1, SETH GULLER1, ELIZA MEADE1, CATALIN BUHIMSCHI1, IRINA BUHIMSCHI1, CHARLES LOCKWOOD1, ERROL NORWITZ1, 1Yale University, Obstetrics & Gynecology, New Haven, Connecticut OBJECTIVE: Hypothalamic GnRH (GnRH-I) plays a critical role in regulating mammalian reproduction. GnRH-I and a second isoform (GnRH-II) are produced also by extra-hypothalamic tissues (including placenta and decidua) and have autocrine/paracrine functions. The function of GnRH in decidua is unknown, but it has been implicated in decidualization and regulating trophoblast invasion. This study investigates the effects of GnRH-I and -II on the production of cytokines and angiogenic factors by first trimester decidual stromal cells. STUDY DESIGN: First trimester decidua was identified from pregnancy termination tissues. Stromal cells were isolated by enzymic digestion, purified, and depleted of leukocytes. Cells were pretreated with estradiol (10ÿ8 M [E2]), medroxyprogesterone acetate (10ÿ7 M [MPA]), both, or vehicle for 7 days. After 24h incubation in fresh medium, cells were stimulated with GnRH-I (1-100 nM), GnRH-II (1-100 nM), IL-1B (1 ng/mL), or thrombin (25 IU/mL) for 48h. Levels of IL-8, IL-6, MMP-3, MCP-1, uPA, PAI-1, VEGF, and sFlt1 in conditioned supernatant were measured by ELISA. Measurements were corrected for protein content. RESULTS: Neither GnRH-I nor -II significantly altered the production of cytokines or angiogenic factors regardless of the hormonal milieu. Positive controls included IL-1B (which upregulated IL-8 by 2581.5-fold; IL-6 by 1334.8-fold; MMP-3 by 10.5-fold; MCP-1 by 128.3-fold; uPA by 6.2-fold; VEGF by 3.2-fold; and sFlt-1 by 4.0-fold [P!0.05 for all]) and thrombin (which upregulated IL-8 by 74.5-fold; IL-6 by 2.5-fold; MMP-3 by 3.8-fold; MCP-1 by 3.4-fold; uPA by 14.1-fold; and sFlt-1 by 18.3-fold [P!0.05 for all]). To confirm their biologic activity, both GnRH-I (100 nM) and –II (100 nM) stimulated hCG production by freshly isolated syncytiotrophoblast cells by 2.3- and 2.7-fold, respectively (ANOVA; P!0.05). CONCLUSION: GnRH-I and -II did not significantly effect production of cytokines and angiogenic factors by first trimester decidual stromal cells. Further studies are required to better define the biologic function of GnRH in the decidua.