Levels of maternal serum corticotropin-releasing hormone (CRH) at midpregnancy in relation to maternal characteristics.

BACKGROUND: Corticotropin-releasing hormone (CRH) in maternal blood originates primarily from gestational tissues and elevated levels in midpregnancy have been linked to adverse pregnancy outcomes. Investigators have hypothesized that high levels of maternal stress might lead to elevated CRH levels in pregnancy. Yet a few studies have measured maternal CRH levels among subgroups of women who experience disproportionate socioeconomic disadvantage, such as African-American and Hispanic women, and found that these groups have lower CRH levels in pregnancy. Our goal was to identify maternal characteristics related to CRH levels in midpregnancy and examine which if any of these factors help to explain race differences in CRH levels. METHODS: The Pregnancy Outcomes and Community Health (POUCH) Study prospectively enrolled women at 15-27 weeks' gestation from 52 clinics in five Michigan communities (1998-2004). Data from the POUCH Study were used to examine maternal demographics, anthropometrics, health behaviors, and psychosocial factors (independent variables) in relation to midpregnancy blood CRH levels modeled as logCRHpg/ml (dependent variable). Analyses were conducted within a sub-cohort from the POUCH Study (671 non-Hispanic Whites, 545 African-Americans) and repeated in the sub-cohort subset with uncomplicated pregnancies (n=746). Blood levels of CRH and independent variables were ascertained at the time of enrollment. All regression models included week of enrollment as a covariate. In addition, final multivariate regression models alternately incorporated different psychosocial measures along with maternal demographics and weight. Psychosocial variables included measures of current depressive symptoms, perceived stress, coping style, hostility, mastery, anomie, and a chronic stressor (history of abuse as a child and adult). RESULTS: In sub-cohort models, the adjusted mean log CRH level was significantly lower in African-Americans vs. non-Hispanic Whites; the difference was -0.48pg/ml (P<0.01). This difference was reduced by 21% (-0.38pg/ml, P<0.01) after inclusion of other relevant covariates. Adjusted mean log CRH levels were also lower among women with <12 years vs. >or=12 years of education (minimal difference=-0.19pg/ml, P<0.05), and among women with high levels of depressive symptoms who did not use antidepressants vs. women with lower levels of depressive symptoms and no antidepressant use (minimal difference=-0.13pg/ml, P<0.01). Log CRH levels were inversely associated with maternal weight (-0.03pg/ml per 10 pound increase, P<.05) but unrelated to smoking and all other psychosocial measures. Results were similar in the subset of women with uncomplicated pregnancies, except that lower CRH levels were also linked to higher perceived stress. CONCLUSION: African-American women have lower blood CRH levels at midpregnancy and the race difference in CRH levels is reduced modestly after adjustment for other maternal characteristics. CRH levels were not elevated among women with high levels of perceived stress or more chronic stressors. The inverse association between CRH levels and maternal weight is likely due to a hemodilution effect. Relations among maternal CRH levels and maternal race, educational level, and depressive symptoms are difficult to explain and invite further investigation. Our results highlight a group of covariates that merit consideration in studies that address CRH in the context of pregnancy and/or post-partum complications.

Dose-related actions of GnRH II analog in the cycling rhesus monkey.

INTRODUCTION: Gonadotropin-releasing hormone (GnRH) II expression, specific high-affinity receptors for GnRH II and its potent bioactivity in human and baboon tissues led us to hypothesize that GnRH II is a bioactive peptide in primates. We recently demonstrated the contraceptive activity of GnRH II analog in rhesus monkeys. In the present experiment, we extended those studies to the dose-related action of this analog on parameters of luteal function and conception. METHODS: GnRH II analog (0-32 microg/day) or saline was administered via osmotic minipumps for 6 days (Days 1-6 postovulation) to regularly cycling rhesus monkeys mated with fertile males around the time of ovulation. Cycle dynamics was monitored through circulating luteinizing hormone, progesterone and estradiol. Pregnancy was determined by circulating chorionic gonadotropin concentrations. RESULTS: Progesterone production (Days 3-11) was significantly less (p<.05) for animals treated with 2, 4 or 8 microg/mL GnRH II analog than for controls, yet with higher doses of GnRH II analog (i.e., 16 or 32 microg/day), luteal progesterone was not different from that of saline-treated controls. The length of the luteal phase in all treated groups was similar to that of controls. In 18 animals mated at the time of ovulation and then treated with GnRH II analog (2-32 microg/day), no pregnancies resulted. In saline-treated controls, five of eight animals (62.5%) became pregnant. Thus, the contraceptive activity of this GnRH II analog did not correlate with luteal progesterone inhibition. CONCLUSIONS: These data demonstrate a dose-related action of GnRH II analog on luteal progesterone and establish the contraceptive activity of 2-32 microg/day GnRH II analog administered postovulation.

Primary amenorrhea associated with ovarian leiomyoma in a baboon (Papio hamadryas).

A 6-y-old female baboon was examined due to absence of menstrual cycling and secondary sex characteristics and failure to reproduce. The mammary glands and vaginal introitus were hypoplastic, the clitoris was prominent, and the perineal skin was immature with lack of cyclic color alterations and sexual swelling. Evaluation of the reproductive tract revealed a hypoplastic uterus and rudimentary ovaries with the presence of an ovarian leiomyoma within the right ovary. Hormonal values (estradiol, progesterone) were low and comparable to those of adult males or ovariectomized females. Adrenal and pituitary hormones (dehydroepiandrosterone, luteinizing and follicular stimulating hormones) and pituitary structure were within normal limits for this species. The baboon had a normal 42,XX karyotype. These findings suggest primary amenorrhea due to ovarian dysgenesis. This is the first report of a case of primary amenorrhea due to ovarian dysgenesis with a normal karyotype associated with a unilateral ovarian leiomyoma in the baboon. Continued studies of noncycling female nonhuman primates in captive breeding colonies may lead to a greater understanding of the underlying causes of this condition.

Effect of ductus ligation on cardiopulmonary function in premature baboons.

RATIONALE: The role of the patent ductus arteriosus in the development of chronic lung disease in surfactant-treated premature newborns remains unclear. OBJECTIVE: To examine the effects of ductus ligation on cardiopulmonary function and lung histopathology in premature primates. METHODS: Baboons were delivered at 125 d, (term = 185 d) treated with surfactant, and ventilated for 14 d. Serial echocardiograms and pulmonary function tests were performed. Animals were randomized to ligation (n = 12) or no ligation (controls, n = 13) on Day 6 of life. Necropsy was performed on Day 14. RESULTS: Compared with nonligated control animals, ligated animals had lower pulmonary-to-systemic flow ratios, higher systemic blood pressures, and improved indices of right and left ventricular performance. The ligated animals tended to have better compliance and ventilation indices for the last 3 d of the study. There were no differences between the groups in proinflammatory tracheal cytokines (interleukin [IL] 6 and IL-8), static lung compliance, or lung histology. CONCLUSION: Although a persistent patent ductus arteriosus results in diminished cardiac function and increased ventilatory requirements at the end of the second week of life, ligation on Day 6 had no measurable effect on the histologic evolution of chronic lung injury in this 14-d baboon model.

Contraceptive action of a gonadotropin-releasing hormone II analog in the rhesus monkey.

GnRH I (mammalian GnRH) was previously thought to be the only isoform of GnRH expressed in mammals, but GnRH II (chicken II GnRH) has now been identified in tissues of numerous mammalian species. Specific high-affinity receptors, which bind GnRH II and its analogs, have been identified throughout the reproductive tract, and GnRH II regulation of progesterone and human chorionic gonadotropin have been demonstrated. Thus, we hypothesized that GnRH II acts as a paracrine factor to regulate extrahypothalamic tissue functions and could be used as an effective contraceptive agent. In these studies, we examined the effect of a stable analog of GnRH II (GnRH II analog) on ovarian steroidogenesis, implantation, and maintenance of pregnancy in the rhesus monkey. GnRH II analog or saline was administered by osmotic minipumps on d 1-6, d 6-11, or d 11-17 to cycling monkeys mated with fertile males. Circulating progesterone and estradiol were determined during the luteal phase, and the cycle length before, during, and after treatment was observed. Circulating monkey chorionic gonadotropin was used to determine implantation. In animals treated with GnRH II analog on d 1-6, no pregnancies resulted; but in saline-treated controls, five of eight animals (62.5%) became pregnant. In animals treated with analog on d 6-11, two of five (40.0%); and on d 11-17, one of three (33.3%); implanted and normal pregnancies ensued. Cycle length or progesterone production was not affected by analog treatment. These data demonstrate that this GnRH II analog can act as a contraceptive agent when administered chronically around the time of ovulation or early luteal development. These findings suggest that GnRH II may play a role in reproductive physiology and that GnRH II analogs may serve as an effective, nonsteroidal, postcoital contraceptive.

Dependence of 3',5'-cyclic adenosine monophosphate--stimulated gonadotropin-releasing hormone release on intracellular calcium levels and L-type calcium channels in superfused GT1-7 neurons.

OBJECTIVE: Immortalized GT1-7 neurons were used to characterize the interactive roles of adenylate cyclase-3',5'-cyclic adenosine monophosphate (cAMP) and L-type calcium channels on gonadotropin-releasing hormone (GnRH) release. METHODS: Dibutyryl (db)-cAMP was used as an active analog of endogenous cAMP, and forskolin was used to activate adenylate cyclase. Extracellular calcium was chelated using EGTA and L-type calcium channels were blocked using nimodipine. The selective Ca2+ ionophore A23187 was employed to increase intracellular calcium levels. GT1-7 neurons were grown on Cytodex-3 beads (Pharmacia Biotech, Uppsala, Sweden) and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 mL/h with media 199 (M-199; Gibco, Grand Island, NY; pH 7.35, 37C); effluent fractions were collected at 5-minute intervals for analysis of GnRH concentrations by radioimmunoassay. RESULTS: Basal GnRH release from superfused GT1-7 neurons ranged from 10 to 62 pg. min(-1). mL(-1). Coexposure of the cells to forskolin and A23187 produced an additive effect on stimulated release of GnRH. Cells exposed to 1 microM of forskolin (an activator of adenylate cyclase) for 5 minutes showed a 2.6-fold increase in GnRH release. Likewise, the addition of 100 microM of db-cAMP to the superfusion for 5 minutes demonstrated a 2.3-fold increase in the amplitude of GnRH secretion. Maintaining the superfused cells in medium containing 5 mM EGTA had no obvious effect on basal GnRH release but blocked the effect of db-cAMP to increase GnRH release. Similarly, the addition of 10 microM nimodipine to the superfusion medium blocked db-cAMP-stimulated GnRH release. CONCLUSIONS: These findings provide additional evidence that cAMP-mediated GnRH release from GT1-7 neurons is dependent on influx of extracellular calcium via L-type Ca2+ channels.

Treatment of immature baboons for 28 days with early nasal continuous positive airway pressure.

Using the 125-day baboon model of long-term bronchopulmonary dysplasia, we hypothesized that early use of nasal continuous positive airway pressure (nCPAP), a noninvasive ventilatory method, combined with prophylactic surfactant therapy would permit continuation of alveolar and vascular development in the lung. Retrospective human studies have shown that infants treated with nCPAP spend less time on mechanical ventilation and thereby sustain less volutrauma. After delivery by cesarean section at 125 days (term, 185 days), the infants received two doses of surfactant (Curosurf) and daily caffeine citrate. Weaning from low-volume positive pressure ventilation to nCPAP was attempted at 24 hours of age. Serial physiological parameters were recorded. Lung histopathology and morphometric measurements of nCPAP animals were done after necropsy at 28 days and data were compared with 125- and 156-day gestational controls. Documented episodes of clinical sepsis and pneumonia at postmortem examination were absent. nCPAP lungs showed enlarged thin-walled air spaces with minimal fibroproliferation and scattered secondary crests. Internal surface area and surface-to-volume ratio dimensions were similar to those of 156-day gestational control lungs, the intrauterine developmental control. nCPAP is an effective noninvasive ventilatory technique that minimizes lung injury in baboons at risk of developing bronchopulmonary dysplasia.

Distribution of corticotropin releasing hormone in the fetus, newborn, juvenile, and adult baboon.

Corticotropin releasing hormone (CRH) has previously been identified in extrahypothalamic tissues and may act in a paracrine fashion within these tissues. To date, CRH production and its role in the fetus and newborn have not been investigated. The aim of this study was to explore the distribution and ontogeny of CRH in extrahypothalamic tissues of the fetus, newborn, juvenile, and adult baboon. Pituitary, adrenal, kidney, liver, and lung tissues from baboons at 125 d gestation, 140 d gestation, 185 d gestation (term), juveniles, and adults were obtained at necropsy. The tissues were quantified for protein and immunoreactive CRH was determined by a RIA. CRH levels were normalized to the protein content of each tissue. CRH was present in all tissues and varied over a 100-fold range according to tissue type. The highest concentration of CRH was found in the pituitary, which did not differ with the gestation and/or age of the animal. In the lung tissues of 125- and 140-d gestation animals, CRH was greater than the term, juvenile, and adult lung (p < 0.02). CRH in the adrenal gland of the 125-d samples was greater than the other four ages tested (p < 0.02). Liver CRH levels were higher in the term animals compared with the juvenile baboons. Our study documents the existence of CRH in extrahypothalamic tissues of the baboon from 125 d of gestation to adulthood. Given its presence and distribution, we speculate that CRH may exert ongoing paracrine and/or autocrine actions in these tissues from the time of intrauterine life throughout adulthood.

Action of gonadotropin-releasing hormone II on the baboon ovary.

The content, binding affinity, and bioactivity of chicken II GnRH (GnRH II) and a stable analogue of GnRH II (GnRH II analogue) in the baboon ovary were studied. Although mammalian GnRH is rapidly degraded by baboon ovarian extracts, we designed a GnRH II analogue that is stable to ovarian enzymatic degradation. This analogue binds to the ovarian membranes with high affinity (41 +/- 3 nM), having 20-fold the affinity of a potent mammalian GnRH analogue. The bioactivity of GnRH II and this GnRH II analogue on the regulation of ovarian progesterone release was compared with that for a potent mammalian GnRH analogue using a baboon granulosa cell culture system. Both GnRH II and GnRH II analogue produced significant inhibition of progesterone release from the granulosa cells (P < 0.03 and P < 0.005, respectively), with a greater reduction observed using the GnRH II analogue. After 24 h in culture, this GnRH II analogue produced a 59% +/- 5% inhibition of progesterone with a concentration as low as 1 nM. Maximal inhibition of 75% +/- 1% was attained with 10 nM GnRH II analogue. The endogenous GnRH II content in the baboon ovary was 5-14 pmoles/g protein. The release of endogenous GnRH II from granulosa cells was observed throughout the 48 h in culture. These studies demonstrated the presence of high enzymatic activity for the degradation of mammalian GnRH in the ovary, whereas this GnRH II analogue was stable. High-affinity binding sites for this GnRH II analogue were also found. GnRH II and this GnRH II analogue can regulate progesterone production from baboon granulosa cells, suggesting that GnRH II is a potent regulator of ovarian function.

Pregnancy and early reproductive failure in the baboon.

We documented normal pregnancy and the rate of pregnancy failure in female baboons by measuring chorionic gonadotropin (bCG) and progesterone (P) levels in 162 mated cycles of 70 baboon females on days 10, 12, and 14 postovulation. Females were mated with males during turgescene. The presence of pregnancy was defined by bCG levels >20 µg/ml by day 14 postovulation and/or documentation of a gestational sac using ultrasonography. Of the 162 cycles, 75 were fertile. Of these animals, 33 were used in other studies and thus were not included in these analyses. The analyses are based on 43 pregnancies from 91 cycles that were untreated throughout their gestations. Twenty-six of these pregnancies had abnormal bCG and/or progesterone levels in early pregnancy. All of those pregnancies with abnormal endocrine parameters terminated with spontaneous abortion (60%). Certain abnormal bCG patterns were repeatedly observed in some animals and were correlated with repeated spontaneous abortions. Of 17 pregnancies with normal bCG and P patterns, 15 (88%) continued to term with a normal fetal outcome. In this study, a pregnancy rate per mated cycle of 47% was observed, yet 60% of untreated pregnancies abortyed spontaneously. Overall 16% of the mated cycles had continuing pregnancies with normal outcome. These studies demonstrate that a high rate of early abortions occurs in the baboon and that a single bCG determination is insufficient to define the presence of a "normal" pregnancy which might be expected to carry to term with a normal outcome.