The neuropeptide histidyl proline diketopiperazine throughout human pregnancy: An inverse correlation with amniotic fluid prolactin Gordon C. Wolf, PhD, MD,o,d Charles W. Hilton, MD: Chandan Prasad, MD,c Joseph M. Miller, Jr., MD,b and Ian H. Thorneycroft, PhD, MDd New Orleans, Louisiana Histidyl proline diketopiperazine values have been established in human amniotic fluid (n = 81) and maternal serum (n = 36) throughout gestation (10 to 42 weeks). Newborn cord serum (n = 10) and first-voided fetal urine (n = 10) levels were also documented. These measurements reveal increasing amniotic fluid levels with term gestation values (15,551 pg/ml) nearly thirteen-fold higher than maternal serum concentrations (1150 pg/ml). Corresponding fetal urine and cord serum concentrations were 16,781 and 2160 pg/ml, respectively. The amniotic fluid values are not influenced by fetal sex or maternal labor, nor do they correlate with amniotic fluid a-fetoprotein levels. However, there is a significant inverse correlation (r = - 0.628; P < 0.0001) between amniotic fluid prolactin and histidyl proline diketopiperazine after midgestation. The hypothesis that histidyl proline diketopiperazine may be a regulatory peptide for decidual prolactin production was tested by culturing term decidua in the presence of varying concentrations of histidyl proline diketopiperazine, but no inhibitory effect was observed. Decidual cultures did not produce measurable amounts of histidyl proline diketopiperazine. It is suggested that amniotic fluid histidyl proline diketopiperazine is derived from fetal urine. (AM J QeSTET GVNECOL 1990;162:740-5.)
Key words: Cyclo (histidyl-proline) in pregnancy, amniotic fluid cyclo (histidyl-proline), prolactin correlation with cyclo (histidyl-proline)
Histidyl proline diketopiperazine [cyclo (His-Pro)] is a neuropeptide known to elicit numerous biologic actions in mammals. I First recognized and established as the cyclic dipeptide metabolite of thyrotropin-releasing hormone,2 it has been speculated that cyclo (His-Pro) may also arise from de novo synthesis from amino acid precursors, endopeptidase cleavage of the thyrotropinreleasing hormone precursor peptide, and exogenous food sources. I The biologic activity of cyclo (His-Pro) was first established in the central nervous system and has been most extensively studied there. Proven central nervous system effects are multiple and include (1) induction of hypothermia, (2) inhibition of water intake, (3) antinociception, (4) augmentation of amphetamineinduced stereotypic behavior, (5) augmentation of the sedative effects of pentobarbital, and (6) attenuation of From the Sections of Reproductive Endocnnology" and MaternalFetal Medicine, b Department of Obstetrics and Gynecology, and the Section of Endocrinology,' Department of Medicine, Louisiana State University School of Medicme, and the Section of Reproductive Endocnnology, Department of Obstetrics and Gynecology, d Tulane University School of Medicine. Received for publication April 19, 1989; reVISed September 1, 1989; accepted October 11, 1989. Reprint requests: G. C. Wolf, MD, Section of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Tulane University School of Mediczne, 1430 Tulane Ave., New Orleans, LA 70112. 611117393
740
sedative effects of ethanol and ketamine. A review of these central nervous system effects supports the concept that cyclo (His-Pro) may function as a dopamine agonist.' Studies of the endocrine effects of cyclo (His-Pro) have primarily involved prolactin secretion, although it has been reported that cyclo (His-Pro) induced marked inhibition of insulin and glucagon secretion by rat pancreatic islets.' Inhibition of prolactin secretion has been reported in rats 5 and monkeys,6 and in human prolactinoma cell cultures. 7 Such effects were not noted, however, in human subjects. 8 Specific receptors for cyclo (His-Pro) have been identified in both the liver and adrenal cortex. The binding sites are stereospecific and are down-regulated by cyclo (His-Pro)."' 10 To date, such receptors have not been identified in the central nervous system l; whether such receptors may be found in the reproductive tract remains to be investigated. Prolactin in pregnancy appears to involve the three separate and independent compartments of mother, fetus, and amniotic fluid. l l Thus maternal and fetal serum prolactin levels increase throughout pregnancy and peak at term l2 ; alternatively, amniotic fluid prolactin levels tend to rise rapidly after 10 weeks to peak near midgestation and subsequently decline to term.13 Amniotic fluid prolactin is produced by the maternal decidua and transported across the fetal membranes
Cyclo (histidyl-proline) throughout human pregnancy
Volume 162 Number 3
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and, as such, is virtually independent of maternal prolactin. In light of the foregoing discussion and our recent identification of cyclo (His-Pro) in term amniotic fluid, 14 we hypothesized that this dipeptide may be a control mechanism for decidual prolactin. We report the results of our investigations of cyclo (His-Pro) and prolactin concentrations in amniotic fluid and maternal serum from 10 weeks' gestation to term. We also report and compare the measurements in fetal cord serum and newborn fetal urine samples. Finally, prolactin production by in vitro explant cultures of term decidua in the presence of varying concentrations of cydo (HisPro) is reported, in addition to the apparent lack of cyclo (His-Pro) production by decidual tissue.
Material and methods Patient population. Samples were collected over a 6month period from April through September, 1988 from pregnant subjects. All samples were obtained in accordance with a previously approved university institutional review board protocol. Women who were taking any medications other than vitamin or iron supplementation were excluded; thus those with diabetes or hypertension or other medical complications were not studied. Thyroid laboratory screening was performed only when clinically indicated. The cohort was nearly equally distributed between white and black women.
Sample collection. Amniotic fluid was obtained by one of three procedures. For all samples up to 22 weeks' gestation (n = 28), the fluid was isolated at the time of medically indicated amniocentesis, generally for increased maternal age, or elevated or depressed maternal serum a-fetoprotein levels. All such specimens were obtained transabdominally under ultrasonographic guidance. Any samples that were grossly bloody were discarded. Specimens in later gestation were the product of transabdominal amniocentesis for evaluation of fetal lung maturity (n = 23) or to rule out possible fetal immune sensitization (n = 6); alternatively, samples were aspirated as the uterus was opened at the time of cesarean section (n = 13), or trans vaginally at the time of spontaneous or artificial rupture of fetal membranes (n = 11). In all cases, the fluid was immediately centrifuged at 2500 rpm for 10 minutes to remove any debris, including vernix caseosa and red blood cells. No samples containing meconium were included. The supernatant was then removed and stored at - 80° C until assays were performed. Maternal serum samples were obtained by standard venipuncture techniques with an 18-gauge needle in an antecubital vein. All samples were drawn between 7 and 11 AM; the most recent food intake was not ascertained. In addition to maternal serum and amniotic fluid obtained simultaneously as described, newborn mixed cord blood was collected immediately after the infant
742 Wolf et al.
March 1990 Am J Obstet Gynecol
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lestational ale(weekl) Fig. 2. Log [eydo (His-Pro)] versus gestational age.
was delivered and the cord was cut and damped. The sample was centrifuged within 30 minutes and the serum was separated and frozen. First-voided newborn urine was obtained in the delivery room or within 6 hours of birth. As the infant spontaneously voided within minutes of birth, the sample was collected in an open container; alternatively, the specimen was collected by attaching a plastic urine bag to the neonate (Hollister, Toronto). Samples were subsequently frozen at - 80° C. Assays. Cydo (His-Pro) was measured by a radioimmunoassay as described previously for human amniotic fluid, 14 human plasma, 15 and human urine. Briefly, incubations were performed in 10 x 75 mm glass tubes for 48 hours at 4° C. The assay mixture consisted of 100 IJ.I of iodine 125-labeled cydo (His-Pro) (12,000 to 15,000 dpm), 100 IJ.I of rabbit anti-serum to cydo (His-Pro) (diluted 1: 6000 in 0.05 mol/L ethylenediaminetetraacetic acid in 0.25% phosphate-buffered saline solution), 100 IJ.I of 0.25% bovine serum albumin in phosphate-buffered saline solution, and 100 IJ.I of either standard cydo (His-Pro) (Sigma Chemical Co., St. Louis) or sample (unextracted). After incubation, free and antibody-bound cydo (His-Pro) were separated by centrifugation for 20 minutes at 2000 rpm at 4° C after 1 ml of 17.5% polyethylene glycol with 4 gm/ml bovine u-globulin was added. The supernatant was aspirated and discarded and the pellet was counted. Intraassay and interassay variation was 10% and 12%, respectively.
Prolactin was measured by a solid-phase 125 1 radioimmunoassay (Diagnostic Products Corporation, Los Angeles). The assay was standardized with World Health Organization second international reference preparation of human prolactin for radioimmunoassay (75/504). Samples of amniotic fluid were diluted 1 : 10 with phosphate buffered saline solution before assay, whereas serum and urine were measured undiluted. Intraassay coefficient of variation was 8.3% and interassay variation was 11 %. u-Fetoprotein was assayed similarly with a 1251 radioimmunoassay (Diagnostic Products Corporation). Decidual culture. Decidual tissue was obtained from fresh human chorionic membranes immediately after uncomplicated term vaginal deliveries. Explants of approximately 100 mg wet weight were minced, rinsed twice with cold phosphate-buffered saline solution, and then incubated in 1 ml of Dulbecco's modified Eagle medium supplemented with sodium pyruvate (0.11 gm/L), glucose (l gm/L), penicillin (1 x 106 U), streptomycin (1000 mg), amphotericin B (2.5 x 10- 4 U), and 10% fetal calf serum at 37° C in an incubator containing an atmosphere of 95% air and 5% carbon dioxide. The cultures were done in 24-well plates and individual experiments were carried out in quadruplicate. In addition to controls, the explant cultures were done in medium containing cydo (His-Pro) in concentrations of 1, 102 , 10., and 106 pg/ml. The media were changed at 24 and 96 hours. In separate crossover experiments, cydo (His-Pro)-containing media re-
Cyclo (histidyl-proline) throughout human pregnancy
Volume 162 Number 3
743
Table I. eydo (His-Pro) and prolactin levels in amniotic fluid and maternal serum by gestational age Amniotzc fluul Gestatzonal age (wk)
n
6-11 12-17 18-23 24-29 30-35 36-42
1 19 8 6 8 39
I
(vela (Hzs-Pro) (pglml)
865 1,985 ± 146 2,202 ± 304 5,830 ± 580 7,550 ± 977 15,551 ± 1,245
Serum Prolactin (nglml)
n
"> 8'~2 ± 9~6 ±
91S.9 1,169 920 1,150
±
±
± ±
I
Prolactzn (nglml)
Key words: Cyclo (histidyl-proline) in pregnancy, amniotic fluid cyclo (histidyl-proline), prolactin correlation with cyclo (histidyl-proline)
Histidyl proline diketopiperazine [cyclo (His-Pro)] is a neuropeptide known to elicit numerous biologic actions in mammals. I First recognized and established as the cyclic dipeptide metabolite of thyrotropin-releasing hormone,2 it has been speculated that cyclo (His-Pro) may also arise from de novo synthesis from amino acid precursors, endopeptidase cleavage of the thyrotropinreleasing hormone precursor peptide, and exogenous food sources. I The biologic activity of cyclo (His-Pro) was first established in the central nervous system and has been most extensively studied there. Proven central nervous system effects are multiple and include (1) induction of hypothermia, (2) inhibition of water intake, (3) antinociception, (4) augmentation of amphetamineinduced stereotypic behavior, (5) augmentation of the sedative effects of pentobarbital, and (6) attenuation of From the Sections of Reproductive Endocnnology" and MaternalFetal Medicine, b Department of Obstetrics and Gynecology, and the Section of Endocrinology,' Department of Medicine, Louisiana State University School of Medicme, and the Section of Reproductive Endocnnology, Department of Obstetrics and Gynecology, d Tulane University School of Medicine. Received for publication April 19, 1989; reVISed September 1, 1989; accepted October 11, 1989. Reprint requests: G. C. Wolf, MD, Section of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Tulane University School of Mediczne, 1430 Tulane Ave., New Orleans, LA 70112. 611117393
740
sedative effects of ethanol and ketamine. A review of these central nervous system effects supports the concept that cyclo (His-Pro) may function as a dopamine agonist.' Studies of the endocrine effects of cyclo (His-Pro) have primarily involved prolactin secretion, although it has been reported that cyclo (His-Pro) induced marked inhibition of insulin and glucagon secretion by rat pancreatic islets.' Inhibition of prolactin secretion has been reported in rats 5 and monkeys,6 and in human prolactinoma cell cultures. 7 Such effects were not noted, however, in human subjects. 8 Specific receptors for cyclo (His-Pro) have been identified in both the liver and adrenal cortex. The binding sites are stereospecific and are down-regulated by cyclo (His-Pro)."' 10 To date, such receptors have not been identified in the central nervous system l; whether such receptors may be found in the reproductive tract remains to be investigated. Prolactin in pregnancy appears to involve the three separate and independent compartments of mother, fetus, and amniotic fluid. l l Thus maternal and fetal serum prolactin levels increase throughout pregnancy and peak at term l2 ; alternatively, amniotic fluid prolactin levels tend to rise rapidly after 10 weeks to peak near midgestation and subsequently decline to term.13 Amniotic fluid prolactin is produced by the maternal decidua and transported across the fetal membranes
Cyclo (histidyl-proline) throughout human pregnancy
Volume 162 Number 3
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and, as such, is virtually independent of maternal prolactin. In light of the foregoing discussion and our recent identification of cyclo (His-Pro) in term amniotic fluid, 14 we hypothesized that this dipeptide may be a control mechanism for decidual prolactin. We report the results of our investigations of cyclo (His-Pro) and prolactin concentrations in amniotic fluid and maternal serum from 10 weeks' gestation to term. We also report and compare the measurements in fetal cord serum and newborn fetal urine samples. Finally, prolactin production by in vitro explant cultures of term decidua in the presence of varying concentrations of cydo (HisPro) is reported, in addition to the apparent lack of cyclo (His-Pro) production by decidual tissue.
Material and methods Patient population. Samples were collected over a 6month period from April through September, 1988 from pregnant subjects. All samples were obtained in accordance with a previously approved university institutional review board protocol. Women who were taking any medications other than vitamin or iron supplementation were excluded; thus those with diabetes or hypertension or other medical complications were not studied. Thyroid laboratory screening was performed only when clinically indicated. The cohort was nearly equally distributed between white and black women.
Sample collection. Amniotic fluid was obtained by one of three procedures. For all samples up to 22 weeks' gestation (n = 28), the fluid was isolated at the time of medically indicated amniocentesis, generally for increased maternal age, or elevated or depressed maternal serum a-fetoprotein levels. All such specimens were obtained transabdominally under ultrasonographic guidance. Any samples that were grossly bloody were discarded. Specimens in later gestation were the product of transabdominal amniocentesis for evaluation of fetal lung maturity (n = 23) or to rule out possible fetal immune sensitization (n = 6); alternatively, samples were aspirated as the uterus was opened at the time of cesarean section (n = 13), or trans vaginally at the time of spontaneous or artificial rupture of fetal membranes (n = 11). In all cases, the fluid was immediately centrifuged at 2500 rpm for 10 minutes to remove any debris, including vernix caseosa and red blood cells. No samples containing meconium were included. The supernatant was then removed and stored at - 80° C until assays were performed. Maternal serum samples were obtained by standard venipuncture techniques with an 18-gauge needle in an antecubital vein. All samples were drawn between 7 and 11 AM; the most recent food intake was not ascertained. In addition to maternal serum and amniotic fluid obtained simultaneously as described, newborn mixed cord blood was collected immediately after the infant
742 Wolf et al.
March 1990 Am J Obstet Gynecol
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was delivered and the cord was cut and damped. The sample was centrifuged within 30 minutes and the serum was separated and frozen. First-voided newborn urine was obtained in the delivery room or within 6 hours of birth. As the infant spontaneously voided within minutes of birth, the sample was collected in an open container; alternatively, the specimen was collected by attaching a plastic urine bag to the neonate (Hollister, Toronto). Samples were subsequently frozen at - 80° C. Assays. Cydo (His-Pro) was measured by a radioimmunoassay as described previously for human amniotic fluid, 14 human plasma, 15 and human urine. Briefly, incubations were performed in 10 x 75 mm glass tubes for 48 hours at 4° C. The assay mixture consisted of 100 IJ.I of iodine 125-labeled cydo (His-Pro) (12,000 to 15,000 dpm), 100 IJ.I of rabbit anti-serum to cydo (His-Pro) (diluted 1: 6000 in 0.05 mol/L ethylenediaminetetraacetic acid in 0.25% phosphate-buffered saline solution), 100 IJ.I of 0.25% bovine serum albumin in phosphate-buffered saline solution, and 100 IJ.I of either standard cydo (His-Pro) (Sigma Chemical Co., St. Louis) or sample (unextracted). After incubation, free and antibody-bound cydo (His-Pro) were separated by centrifugation for 20 minutes at 2000 rpm at 4° C after 1 ml of 17.5% polyethylene glycol with 4 gm/ml bovine u-globulin was added. The supernatant was aspirated and discarded and the pellet was counted. Intraassay and interassay variation was 10% and 12%, respectively.
Prolactin was measured by a solid-phase 125 1 radioimmunoassay (Diagnostic Products Corporation, Los Angeles). The assay was standardized with World Health Organization second international reference preparation of human prolactin for radioimmunoassay (75/504). Samples of amniotic fluid were diluted 1 : 10 with phosphate buffered saline solution before assay, whereas serum and urine were measured undiluted. Intraassay coefficient of variation was 8.3% and interassay variation was 11 %. u-Fetoprotein was assayed similarly with a 1251 radioimmunoassay (Diagnostic Products Corporation). Decidual culture. Decidual tissue was obtained from fresh human chorionic membranes immediately after uncomplicated term vaginal deliveries. Explants of approximately 100 mg wet weight were minced, rinsed twice with cold phosphate-buffered saline solution, and then incubated in 1 ml of Dulbecco's modified Eagle medium supplemented with sodium pyruvate (0.11 gm/L), glucose (l gm/L), penicillin (1 x 106 U), streptomycin (1000 mg), amphotericin B (2.5 x 10- 4 U), and 10% fetal calf serum at 37° C in an incubator containing an atmosphere of 95% air and 5% carbon dioxide. The cultures were done in 24-well plates and individual experiments were carried out in quadruplicate. In addition to controls, the explant cultures were done in medium containing cydo (His-Pro) in concentrations of 1, 102 , 10., and 106 pg/ml. The media were changed at 24 and 96 hours. In separate crossover experiments, cydo (His-Pro)-containing media re-
Cyclo (histidyl-proline) throughout human pregnancy
Volume 162 Number 3
743
Table I. eydo (His-Pro) and prolactin levels in amniotic fluid and maternal serum by gestational age Amniotzc fluul Gestatzonal age (wk)
n
6-11 12-17 18-23 24-29 30-35 36-42
1 19 8 6 8 39
I
(vela (Hzs-Pro) (pglml)
865 1,985 ± 146 2,202 ± 304 5,830 ± 580 7,550 ± 977 15,551 ± 1,245
Serum Prolactin (nglml)
n
"> 8'~2 ± 9~6 ±
91S.9 1,169 920 1,150
±
±
± ±
I
Prolactzn (nglml)
