Fetus-Placenta-Newborn

Prostaglandin endoperoxide H synthase type 1 and type 2 messenger ribonucleic acid in human fetal tissues throughout gestation and in the newborn infant David M. Olson, PhD, Jane E. Mijovic, PhD, Dean B. Zaragoza, MSc, and Jocelynn L. Cook, PhD Edmonton, Alberta, Canada OBJECTIVE: We determined the relative abundance of prostaglandin endoperoxide H synthase type 1 and type 2 messenger ribonucleic acid levels in the human fetus and newborn infant. STUDY DESIGN: We used ribonuclease protection assays and normalized values to messenger ribonucleic acid of cyclophilin. Tissues were obtained from all 3 trimesters and in the first 9 days of the newborn period. RESULTS: Prostaglandin endoperoxide H synthase type 1 and type 2 messenger ribonucleic acid is present in every fetal tissue examined (lung, kidney, intestine, heart, brain, and stomach). Kidney and lung demonstrated no changes in the expression of prostaglandin endoperoxide H synthase type 1 messenger ribonucleic acid with gestational age, whereas postnatal levels in lung were one third those in the first trimester (P < .05). A large increase (5-fold to 30-fold; P < .05) occurred throughout gestation for the expression of prostaglandin endoperoxide H synthase type 2 messenger ribonucleic acid in intestine, lung, and kidney, which extended into the newborn period for lung and kidney. CONCLUSIONS: These data imply that the expression of prostaglandin endoperoxide H synthase type 2 messenger ribonucleic acid may be responsible for prostaglandin-related effects and is coordinated in several human fetal tissues in late gestation. (Am J Obstet Gynecol 2001;184:169-74.)

Key words: Fetus, human, organs, prostaglandin endoperoxide H synthase type 2, prostaglandins

Prostaglandins regulate several fetal and newborn physiologic events,1 and they are involved in fetal organ development and maturation.2 They may be synthesized in individual organs, where their action is local,1, 2 or in the placenta,3 where they can circulate systemically to affect specific organ physiologic characteristics or development.4 In situations where fetal prostaglandin synthesis is inhibited during pregnancy because of maternal administration of nonsteroidal anti-inflammatory drugs such as aspirin or indomethacin, the fetus can have reversible oligohydramnios5 and the newborn infant is at

From The Perinatal Research Centre and the Departments of Obstetrics and Gynaecology, Pediatrics, and Physiology, The University of Alberta. Supported by the Medical Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and the Natural Sciences and Engineering Research Council of Canada. Received for publication November 12, 1999; revised February 21, 2000; accepted April 19, 2000. Reprint requests: David M. Olson, PhD, The Perinatal Research Centre, 220 HMRC, The University of Alberta, Edmonton, Alberta, Canada T6G 2S2. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/1/108078 doi:10.1067/mob.2001.108078

greater risk of having renal dysfunction, necrotizing enterocolitis, intracranial hemorrhage, and patent ductus arteriosus.6 Prostaglandin action in the fetus and newborn infant, which is dependent on prostaglandin synthesis, has not been extensively studied in human beings, especially in terms of the enzymes responsible for their regulation. The final rate-limiting step in prostaglandin synthesis is the bis-dioxygenation of arachidonic acid into 2 endoperoxide intermediates, prostaglandin G2 and prostaglandin H2, by the action of an enzyme, prostaglandin endoperoxide H synthase (PGHS, cyclooxygenase). It is now well established that 2 forms of PGHS exist, one (PGHS-1) that is considered to be constitutive and is found in most cells and one (PGHS-2) that is inducible and is found in inflammatory cells, smooth muscle cells, and endothelial cells.7 Data on the presence of these enzymes or their messenger ribonucleic acid (mRNA) in human fetal tissues (other than the fetal membrane amnion and chorion) do not exist, and such information would aid in an appreciation of the role of prostaglandins in development and physiologic characteristics and in the clinical management of patients during pregnancy. 169

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In this study we assessed the relative abundance levels of PGHS-1 and PGHS-2 mRNA in human fetal lung, kidney, and small intestine during each trimester and in the first 9 days of life in babies born at term. We observed a decrease or no change in mRNA levels for PGHS-1 from the first trimester to the third trimester, whereas PGHS-2 mRNA levels were low in the first trimester, increased significantly in the second and third trimesters, and in the case of lung and kidney increased further in the newborn infant. Material and methods Ethical approval for the study was obtained, and appropriate collection procedures were followed at each institution. Human embryonic and fetal tissues were obtained at elective abortion by the Central Laboratory for Human Embryology, University of Washington, Seattle. This laboratory supplied tissues from normal fetuses from 54 days to 20 weeks of gestation. Specimens were obtained within minutes of passage (by vacuum aspiration), and tissues were aseptically identified and immediately snap frozen in liquid nitrogen. Preparation time for the tissue was always .2) (Fig 3, A, and Fig 4, A).

PGHS-2 mRNA levels. Results indicate that the relative abundance of PGHS-2 mRNA increases with either gestational age or fetal development, or both, in the fetal lung (Fig 2, B), kidney (Fig 3, B), and intestine (Fig 4, B). For example, in the fetal lung (Fig 2, B) there is an increase in PGHS-2 mRNA expression across trimesters and into the postnatal period (F3, 15 = 27.6; P < .001). Examination by post hoc analysis revealed that fetal lung PGHS-2 mRNA levels were significantly elevated by the third trimester (P < .05) and that values tripled in the postnatal period (P < .05). A similar trimester-dependent pattern of increasing PGHS-2 mRNA levels was evident in the fetal kidney (Fig 3, B) (F3, 18 = 25.0; P < .001). Again, by the third trimester, PGHS-2 mRNA levels in the fetal kidney were significantly elevated over those levels indicative of the first trimester (P < .05), and the PGHS-2 mRNA continued to increase further during postnatal development (P < .05). The profile of fetal intestine PGHS-2 mRNA levels (Fig 4, B) across trimesters was largely similar to the patterns measured from the fetal lung and the fetal kidney. There was a main effect of trimester on PGHS-2 mRNA levels (F3, 23 = 9.48; P < .001), and by the third trimester the PGHS-2 mRNA levels had doubled from the levels of the second trimester and were significantly greater than the levels from first-trimester fetuses (P < .05). There was no further increase in the relative mRNA abundance in intestine after term birth, although levels remained elevated.

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A A

B B

Fig 2. Normalized values of relative abundance of PGHS-1 mRNA (A) and PGHS-2 mRNA (B) from human fetal lung. Respective numbers of samples for A and B were 6 and 6 (first trimester), 5 and 5 (second trimester), 3 and 3 (third trimester), and 3 and 2 (postnatal period). Superscripts a, ab, b, and c indicate statistical difference (P < .05) by 1-way analysis of variance and Tukey test.

We also examined other human fetal organs (heart, brain [cerebrum], and stomach) for PGHS-1 and PGHS2 mRNA (data not shown). Each organ contained mRNA from both isoforms of enzyme in each trimester of pregnancy, but we did not obtain enough samples to make any judgments about relative abundance levels during pregnancy. Comment The clear conclusion from this study is that a significant and many-fold increase in PGHS-2 mRNA occurs throughout pregnancy in several human fetal tissues. This is unique for PGHS-2 mRNA, because PGHS-1

Fig 3. Normalized values of relative abundance of PGHS-1 mRNA (A) and PGHS-2 mRNA (B) from human fetal kidney. Respective numbers of samples for A and B were 7 and 7 (first trimester), 6 and 6 (second trimester), 3 and 3 (third trimester), and 3 and 3 (postnatal period). Superscripts a, ab, b, and c indicate statistical difference (P < .05) by 1-way analysis of variance and Tukey test.

mRNA levels either stay constant throughout pregnancy or decrease, as in the lung. In the lung and kidney this increase in PGHS-2 mRNA expression continues through birth into the neonatal period. Although we did not plot the data on individual subjects at specific times in gestation because of the limited number of subjects and tissues available, examination of the individual data suggests a late-gestation increase in PGHS-2 mRNA expression. We recently published data demonstrating a strikingly similar pattern of a large increase in PGHS-2 mRNA expression in the human fetal membranes, amnion and chorion, beginning in late gestation, at approximately week 35.8, 11 This is in contrast to PGHS-1 mRNA expression in human fetal membranes, which is fairly constant throughout pregnancy, increasing slightly, about 4-fold, at term. It is

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also in contrast to the maternal decidua, which demonstrates no changes in either PGHS-1 or PGHS-2 mRNA expression throughout gestation.8 These data were recently confirmed in the baboon decidua, which also does not demonstrate an increase in either PGHS-1 or PGHS-2 mRNA during pregnancy.12 Furthermore, there is no increase in PGHS-1 or PGHS-2 mRNA expression in the lower segment of human myometrium in late gestation or with the onset of labor,13 although a more recent article14 presents conflicting data on human myometrium, suggesting that PGHS-2 mRNA levels do increase before the onset of labor. Nevertheless, there is a uniform pattern of expression in the fetus suggesting either that many human fetal tissues, nearly simultaneously and independently, increase their expression of PGHS-2 mRNA in late gestation or that a common control stimulus (eg, hormonal) coordinates the increased expression. There is precedent for a central mechanism. Hypophysectomy in fetal lambs diminishes the late-gestation increase in placental PGHS activity and in maternal prostaglandin E2 concentrations.15 These authors suggested that luteinizing hormone may be the factor that stimulates prostaglandin synthesis. In other studies both cortisol and dexamethasone stimulated an increase in cultured amnion cell PGHS activity and in prostaglandin E2 output.16, 17 Alternatively, ligation of the bladder in adult mice stimulates an increase in PGHS-2 expression, suggesting the possibility of stretch as a stimulus.18 Every effort was made in this study to exclude factors not associated with normal fetal development that may have influenced fetal PGHS mRNA expression; hence we excluded all samples associated with inflammation or preterm birth. It is not known which of the enzyme isoforms is necessary to produce the prostaglandins for normal fetal development and physiologic characteristics, but evidence exists to suggest that PGHS-2 is vital for certain aspects of fetal organ development and physiologic features. Knockout mice homozygous for recessive PGHS-219, 20 are anephric, display cardiomyopathies, and generally die shortly after birth or do not survive to reproduc-tive age. An important aspect of this knowledge of PGHS expression and activity in the developing human fetus has to do with tocolytic therapy to delay threatened or actual preterm labor and to prolong pregnancy in women. As evidence mounts to suggest that PGHS-2 mRNA, protein, and specific activity increase at term and before term in the fetal membranes and placental tissues of the human and other species,8, 21, 22 efforts to study the effects of specific inhibitors of PGHS-2 enzymatic activity on altering the timing of human and animal preterm labor have been made. Sawdy et al23 reported that the administration of nimesulide, a preferential PGHS-2 inhibitor, to a woman to prevent recurrent second-trimester loss was associated with an increase in amniotic fluid volume and

A

B Fig 4. Normalized values of relative abundance of PGHS-1 mRNA (A) and PGHS-2 mRNA (B) from human fetal intestine. Respective numbers of samples for A and B were 9 and 9 (first trimester), 5 and 5 (second trimester), 5 and 7 (third trimester), and 3 and 3 (postnatal period). Superscripts a, ab, c, and bc indicate statistical difference (P < .05) by 1-way analysis of variance and Tukey test.

preterm birth on withdrawal of treatment. In pregnant mice NS-398 (N-[2-(cyclohexgloxy)-4-nitro-phenyl]methane sulfonamide), another PGHS-2–selective inhibitor, prolonged normal gestation and blocked lipopolysaccharide-induced preterm labor.24 Similarly, NS-398 blocked preterm labor induced by adrenocorticotropic hormone in sheep,22 and meloxicam, a third PGHS-2 inhibitor, prolonged gestation in ewes administered the progesterone receptor blocker, mifepristone (RU 486), which normally shortens gestational length.25 The mice were born healthy, and the lambs were apparently unaffected by the inhibitors, although detailed examination of newborn organ function and prostaglandin synthesis was not carried out in any study.

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It is important in studies such as these to determine the effects of specific PGHS-2 inhibitors on fetal and neonatal organ development and function. As cited earlier, it is clear that nonspecific prostaglandin synthesis inhibitors, such as aspirin and indomethacin, have deleterious effects on human renal development and function and on ductus arteriosus function. Further, their use during pregnancy is associated with a greater risk of intracranial hemorrhage and necrotizing enterocolitis. Whether PGHS-2 mRNA increases in fetal tissues before term, at the time of preterm birth, is not known; further, its relative importance in fetal development during the second and early third trimesters is also unknown. It is probable, however, that specific inhibitors of PGHS-2, although effective in delaying preterm labor, may not be safe for the fetus. Careful examination of this aspect of tocolytic therapy is warranted. In summary, our results demonstrate that PGHS-2 mRNA increases in human fetal kidney, lung, and intestine in the third trimester and into the newborn period in some cases. The expression of PGHS-1 mRNA, on the other hand, remains constant or may decline in these tissues during gestation. The roles of prostaglandins from each of these enzyme isoforms in fetal development and physiologic features remain to be determined, as does the coordinated regulation of these increases in PGHS-2 expression. REFERENCES

1. Heymann MA. Prostaglandins in the perinatal period. New York: Grune & Stratton; 1980. 2. Torday JS, Sun H, Qin J. Prostaglandin E2 integrates the effects of fluid distension and glucocorticoid on lung maturation. Am J Physiol 1998;274:L106-11. 3. Rice GE, Wong MH, Hollingworth SA, Thorburn GD. Prostaglandin G/H synthase activity in ovine cotyledons: a gestational profile. Eicosanoids 1990;3:231-6. 4. Adamson SL, Kuipers IM, Olson DM. Umbilical cord occlusion stimulates breathing independent of blood gases and pH. J Appl Physiol 1991;70:1796-809. 5. Hickok DE, Hollenbach KA, Reilley SF, Nyberg DA. The association between decreased amniotic fluid volume and treatment with nonsteroidal anti-inflammatory agents for preterm labor. Am J Obstet Gynecol 1989;160:1525-30. 6. Norton M, Merrill J, Cooper B, Kuller J, Clyman R. Neonatal complications after the administration of indomethacin for preterm labor. N Engl J Med 1993;329:1062-7. 7. Xie W, Merrill JR, Bradshaw WS, Simmons DL. Structural determination and promoter analysis of the chicken mitogen-inducible prostaglandin G/H synthase gene and genetic mapping of the murine homolog. Arch Biochem Biophys 1993;300: 247-52. 8. Mijovic JE, Zakar T, Angelova J, Olson DM. Prostaglandin endoperoxide H synthase mRNA expression in the human amnion and decidua during pregnancy and in the amnion at preterm labour. Mol Hum Reprod 1999;5:182-7.

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9. Hirst JJ, Teixeira FJ, Zakar T, Olson DM. Prostaglandin endoperoxide-H synthase-1 and -2 messenger ribonucleic acid levels in human amnion with spontaneous labor onset. J Clin Endocrinol Metab 1995;80:517-23. 10. Gunning P, Ponte P, Okayama H, Engel J, Blau M, Kedes L. Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol 1983;3:787-95. 11. Mijovic JE, Zakar T, Nairn TK, Olson DM. Prostaglandin endoperoxide H synthase (PGHS) activity and PGHS-1 and -2 messenger ribonucleic acid abundance in human chorion throughout gestation and with preterm labor. J Clin Endocrinol Metab 1998;83:1358-67. 12. Kim JJ, Wang J, Bambra C, Das SK, Dey SK, Fazleabas AT. Expression of cyclooxygenase-1 and -2 in the baboon endometrium during the menstrual cycle and pregnancy. Endocrinology 1999;140:2672-8. 13. Moore SD, Brodt-Eppley J, Cornelison LM, Burk SE, Slater DM, Myatt L. Expression of prostaglandin H synthase isoforms in human myometrium at parturition. Am J Obstet Gynecol 1999;180:103-9. 14. Slater D, Dennes W, Campa J, Poston L, Bennett P. Expression of cyclo-oxygenase types -1 and -2 in human myometrium throughout pregnancy. Mol Hum Reprod 1999;9:880-4. 15. Deayton JM, Young IR, Thorburn GD. Early hypophysectomy of sheep fetuses: effects on growth, placental steroidogenesis and prostaglandin production. J Reprod Fertil 1993;97:513-20. 16. Mitchell MD, Lytton FD, Varticovski L. Paradoxical stimulation of both lipocortin and prostaglandin production in human amnion cells by dexamethasone. Biochem Biophys Res Commun 1988;151:137-41. 17. Potestio FA, Zakar T, Olson DM. Glucocorticoids stimulate prostaglandin synthesis in human amnion cells by a receptormediated mechanism. J Clin Endocrinol Metab 1988;67: 1205-10. 18. Park JM, Yang T, Arend LJ, Smart AM, Schnermann JB, Briggs JP. Cyclooxygenase-2 is expressed in bladder during fetal development and stimulated by outlet obstruction. Am J Physiol 1997;273:F538-44. 19. Dinchuk J, Car B, Focht R, et al. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 1995;378:406-9. 20. Morham SG, Langenbach R, Loftin CD, et al. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 1995;83:473-82. 21. Slater D, Dennes W, Sawdy R, Allport V, Bennett P. Expression of cyclo-oxygenase types-1 and -2 in human fetal membranes throughout pregnancy. J Mol Endocrinol 1999;22:125-30. 22. Wu WX, Unno N, Ma XH, Nathanielsz PW. Inhibition of prostaglandin production by nimesulide is accompanied by changes in expression of the cassette of uterine labor-related genes in pregnant sheep. Endocrinology 1998;139:3096-103. 23. Sawdy R, Slater D, Fisk N, Edmonds DK, Bennett P. Use of a cyclo-oxygenase type-2–selective non-steroidal anti-inflammatory agent to prevent preterm delivery. Lancet 1997;350:265-6. 24. Mijovic J, Olson D, Zakar T. The influence of tyrosine kinase inhibitors on term and preterm parturition in mice [abstract]. J Soc Gynecol Investig 1998;5:60A. 25. Lye J, Adamson S, Bocking A, Challis J, Riggs D, Rurak D. The COX-2 inhibitor, meloxicam, effectively blocks preterm labour in sheep without adverse effects on fetal/maternal cardiovascular or GI function [abstract]. J Soc Gynecol Investig 1999;6:50A.