Immunoreactive corticotropin-releasing hormone in amniotic fluid Atsushi Sasaki, MD:Osamu Shinkawa, MD,b and Kaoru Yoshinaga, MDC Wakayanagi and Sendai, Japan Immunoreactive corticotropin-releasing hormone in the amniotic fluid of both human beings and rats was measured by a specific radioimmunoassay. In human subjects the hormone was detectable in all amniotic fluid samples (obtained during the sixteenth and eighteenth weeks of gestation) (2.5 ± 1.7 fmol/ml, mean ± SO, n = 17) and the thirty-eighth to fortieth weeks (9.3 ± 5.4 fmol/ml, n = 24). The levels of concentration of this hormone in this amniotic fluid correlated significantly with the levels in both maternal plasma and placenta for each patient. Gel filtration of amniotic fluid extracts revealed two major peaks of immunoreactive corticotropin-releasing hormone, one at the elution position of the rat hormone and the other at a small-molecular-weight region. Immunoreactive corticotropin-releasing hormone was not detectable in rat amniotic fluid or placenta. We concluded that immunoreactive corticotropin-releasing hormone, which may be derived from the placenta, is present in human amniotic fluid and that its detection in the human placenta but not in rat placentas suggests that the mechanism of corticotropin-releasing hormone gene expression in the placenta is species specific. (AM J OSSTET GVNECOL 1990;162:194-8.)
Key words: Corticotropin-releasing hormone, placenta, amniotic fluid The primary structures of both ovine' and rae corticotropin-releasing hormone have been determined. The amino acid sequence of a human prepro-corticotropin-releasing hormone molecule has been predicted from the nucleotide sequence of a cloned human genomic fragment; the deduced sequence of the hormone in mature human beings, a 41 amino acid amide, is identical to that of rat corticotropin-releasing hormone. 3 It has been reported that there are several proteohormones in human amniotic fluid. For instance, pituitary hormones, such as adrenocorticotropic hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, growth hormone, and prolactin, and hypothalamic hormones, such as luteinizing hormone-releasing hormone and thyrotropin-releasing hormone, have also been detected.· Recently we reported the presence of immunoreactive corticotropin-releasing hormone in human placental tissue and suggested that it is preferentially secreted into the maternal circulation, where it provides
From the Department of Internal MediCine, Wakayanagt Hospital,' Wakayanagi and the Department of Obstetncs and Gynecology' and the Second Department of Internal MedICine,' Tohoku Univemty School of Medicine, Sendai. This work was supported by a Research Grant for Intractable Diseases from the Ministry of Health and Welfare, Japan. Receivedfor publication February 1,1989; revzsed May 18,1989; accepted July 13, 1989. Repnnt requests: Atsushl Sasakz, MD, Department of Internal MedIcine, Wakayanagi Hospital, 130-5, Kawaklta-Furukawa, Wakayanagi, Miyagi, 989-55 Japan.
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extrahypothalamic stimulation to the maternal anterior pituitary gland.5-7 In this study we extended these observations to define the presence of immunoreactive corticotropin-releasing hormone in human amniotic fluid. In addition, we also tried to find it in rat amniotic fluid and in rat placentas.
Material and methods Subjects. All subjects participating in this study gave their informed consent. All studies were performed at the Tohoku University Hospital. The protocol was approved by the Department Clinical Research Committee. Fifty-four pregnant women, 18 to 32 years old, were studied. Amniotic fluid was obtained from 17 women by suprapubic amniocentesis, 16 to 18 weeks after cessation of menstruation. Maternal blood was obtained frum another 13 women in the sixteenth to eighteenth weeks of gestation. Amniotic fluid was also obtained from 24 women during rupture of the membranes in the process of vaginal delivery (at 38 to 40 weeks of gestation). In the latter group samples of maternal blood and umbilical cord blood were simultaneously obtained at the time of delivery; term placentas were also collected simultaneously with maternal blood samples. Any specimen of amniotic fluid contaminated with blood and / or meconium was excluded from the study. Animal studies. Time-mated Sprague-Dawley rats (sperm positive = day 0) were purchased from Charles River Japan (Funabashi, Japan). Rats were housed, three to a cage, in an air-conditioned room with a controlled light cycle (lights on at 8 AM; lights off at 7 PM) .
Corticotropin-releasing hormone in amniotic fluid
Volume 162 Number I
Rat chow and water were available as desired. All animals in this study were killed between noon and 1 PM by decapitation within 30 seconds after removal from their cages. Pregnant rats were killed on day 13 (n = 7), day 16 (n = 7), or day 20 (n = 7) of gestation. Rat amniotic fluid was obtained from the latter two groups (16 and 20 days of gestation) through a puncture in the amniotic sac. Amniotic fluid was not collected from rats of 13 days' gestation because of the difficulty of sampling. Placentas were also collected from all groups, frozen on dry ice immediately after removal, and kept at - 80° C until homogenization. Eight to thirteen placentas were obtained from each rat, and the mean weights of the placentas were 0.24 gm (13 days of gestation), 0.52 gm (16 days), and 1.1 gm (20 days). Extraction. The amniotic fluid samples (human and rat) were immediately acidified with IN hydrochloric acid (final hydrochloric acid concentration, 0.1 N), frozen, and lyophilized for later measurements of immunoreactive corticotropin-releasing hormone, which was extracted from the amniotic fluid by anticorticotropin-releasing hormone immune affinity chromatography, as described previously.' The rate of recovery of synthetic rat corticotropin-releasing hormone from this column was 78.2% ± 3.5% (mean ± SD, n = 6). The amniotic values were corrected according to this recovery. To demonstrate further that the corticotropin-releasing hormone in the amniotic fluid was not destroyed in the process of amniotic sampling and extraction, we added synthetic rat corticotropinreleasing hormone to human or rat amniotic fluid just after sampling and extracted the amniotic fluid in the same manner. The gel filtration profiles of the extracts in both human amniotic fluid and rat amniotic fluid revealed only one peak in the position of the synthetic rat hormone, and recoveries were about 75%. Therefore corticotropin-releasing hormone in amniotic fluid is not destroyed by these processes of collection and extraction. Blood was collected in ice-chilled glass tubes containing ethylenediamine tetraacetic acid (disodium, Img/ml) and immediately centrifuged at 4° C. Plasma was stored at - 20° C until extraction. Plasma was extracted with Sep-Pak C-18 cartridges (Waters Associates, Milford, Mass.) for measurement of immunoreactive corticotropin-releasing hormone, as previously described. 8 The rate of recovery of the synthetic form of the hormone in rats, added to hormone-free plasma, was 72.4% ± 5% (mean ± SD, n = 4). The values were corrected according to this recovery rate. Human and rat placentas were trimmed of cord and membranes, and extensively rinsed with 0.15 moUL of sodium chloride at lO° C. Each placenta was homogenized in 5 volumes of ice-cold 50% acidified methanol (O.IN HCI-CH,OH). The homogenates were centri-
~ "'0
E
195
r =0.695 p
Key words: Corticotropin-releasing hormone, placenta, amniotic fluid The primary structures of both ovine' and rae corticotropin-releasing hormone have been determined. The amino acid sequence of a human prepro-corticotropin-releasing hormone molecule has been predicted from the nucleotide sequence of a cloned human genomic fragment; the deduced sequence of the hormone in mature human beings, a 41 amino acid amide, is identical to that of rat corticotropin-releasing hormone. 3 It has been reported that there are several proteohormones in human amniotic fluid. For instance, pituitary hormones, such as adrenocorticotropic hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, growth hormone, and prolactin, and hypothalamic hormones, such as luteinizing hormone-releasing hormone and thyrotropin-releasing hormone, have also been detected.· Recently we reported the presence of immunoreactive corticotropin-releasing hormone in human placental tissue and suggested that it is preferentially secreted into the maternal circulation, where it provides
From the Department of Internal MediCine, Wakayanagt Hospital,' Wakayanagi and the Department of Obstetncs and Gynecology' and the Second Department of Internal MedICine,' Tohoku Univemty School of Medicine, Sendai. This work was supported by a Research Grant for Intractable Diseases from the Ministry of Health and Welfare, Japan. Receivedfor publication February 1,1989; revzsed May 18,1989; accepted July 13, 1989. Repnnt requests: Atsushl Sasakz, MD, Department of Internal MedIcine, Wakayanagi Hospital, 130-5, Kawaklta-Furukawa, Wakayanagi, Miyagi, 989-55 Japan.
611115271
194
extrahypothalamic stimulation to the maternal anterior pituitary gland.5-7 In this study we extended these observations to define the presence of immunoreactive corticotropin-releasing hormone in human amniotic fluid. In addition, we also tried to find it in rat amniotic fluid and in rat placentas.
Material and methods Subjects. All subjects participating in this study gave their informed consent. All studies were performed at the Tohoku University Hospital. The protocol was approved by the Department Clinical Research Committee. Fifty-four pregnant women, 18 to 32 years old, were studied. Amniotic fluid was obtained from 17 women by suprapubic amniocentesis, 16 to 18 weeks after cessation of menstruation. Maternal blood was obtained frum another 13 women in the sixteenth to eighteenth weeks of gestation. Amniotic fluid was also obtained from 24 women during rupture of the membranes in the process of vaginal delivery (at 38 to 40 weeks of gestation). In the latter group samples of maternal blood and umbilical cord blood were simultaneously obtained at the time of delivery; term placentas were also collected simultaneously with maternal blood samples. Any specimen of amniotic fluid contaminated with blood and / or meconium was excluded from the study. Animal studies. Time-mated Sprague-Dawley rats (sperm positive = day 0) were purchased from Charles River Japan (Funabashi, Japan). Rats were housed, three to a cage, in an air-conditioned room with a controlled light cycle (lights on at 8 AM; lights off at 7 PM) .
Corticotropin-releasing hormone in amniotic fluid
Volume 162 Number I
Rat chow and water were available as desired. All animals in this study were killed between noon and 1 PM by decapitation within 30 seconds after removal from their cages. Pregnant rats were killed on day 13 (n = 7), day 16 (n = 7), or day 20 (n = 7) of gestation. Rat amniotic fluid was obtained from the latter two groups (16 and 20 days of gestation) through a puncture in the amniotic sac. Amniotic fluid was not collected from rats of 13 days' gestation because of the difficulty of sampling. Placentas were also collected from all groups, frozen on dry ice immediately after removal, and kept at - 80° C until homogenization. Eight to thirteen placentas were obtained from each rat, and the mean weights of the placentas were 0.24 gm (13 days of gestation), 0.52 gm (16 days), and 1.1 gm (20 days). Extraction. The amniotic fluid samples (human and rat) were immediately acidified with IN hydrochloric acid (final hydrochloric acid concentration, 0.1 N), frozen, and lyophilized for later measurements of immunoreactive corticotropin-releasing hormone, which was extracted from the amniotic fluid by anticorticotropin-releasing hormone immune affinity chromatography, as described previously.' The rate of recovery of synthetic rat corticotropin-releasing hormone from this column was 78.2% ± 3.5% (mean ± SD, n = 6). The amniotic values were corrected according to this recovery. To demonstrate further that the corticotropin-releasing hormone in the amniotic fluid was not destroyed in the process of amniotic sampling and extraction, we added synthetic rat corticotropinreleasing hormone to human or rat amniotic fluid just after sampling and extracted the amniotic fluid in the same manner. The gel filtration profiles of the extracts in both human amniotic fluid and rat amniotic fluid revealed only one peak in the position of the synthetic rat hormone, and recoveries were about 75%. Therefore corticotropin-releasing hormone in amniotic fluid is not destroyed by these processes of collection and extraction. Blood was collected in ice-chilled glass tubes containing ethylenediamine tetraacetic acid (disodium, Img/ml) and immediately centrifuged at 4° C. Plasma was stored at - 20° C until extraction. Plasma was extracted with Sep-Pak C-18 cartridges (Waters Associates, Milford, Mass.) for measurement of immunoreactive corticotropin-releasing hormone, as previously described. 8 The rate of recovery of the synthetic form of the hormone in rats, added to hormone-free plasma, was 72.4% ± 5% (mean ± SD, n = 4). The values were corrected according to this recovery rate. Human and rat placentas were trimmed of cord and membranes, and extensively rinsed with 0.15 moUL of sodium chloride at lO° C. Each placenta was homogenized in 5 volumes of ice-cold 50% acidified methanol (O.IN HCI-CH,OH). The homogenates were centri-
~ "'0
E
195
r =0.695 p
