A novel change in cytologic localization of human chorionic gonadotropin and human placental lactogen in first-trimester placenta in the course of gestation Takeshi Maruo, MD: Cecilia A. Ladines-Llave, MD," Hiroya Matsuo, MD: Augusto S. Manalo, MD," and Matsuto Mochizuki, MD" Kobe, japan, and Manila, Philippines Cytologic localization of human chorionic gonadotropin and human placental lactogen in developing human early placenta was analyzed by avidin-biotin immunoperoxidase techniques with an affinity-purified polyclonal antibody to l3-human chorionic gonadotropin carboxyl terminal peptide and a polyclonal antibody to human placental lactogen. In 4- to 5-week placentas human chorionic gonadotropin and human placental lactogen were found to be primarily localized to cytotrophoblasts, whereas in 6- to 12-week placentas these substances were exclusively localized to syncytiotrophoblast. We previously reported that a similar change in cytologic localization of epidermal growth factor and its receptor from cytotrophoblasts to syncytiotrophoblast in first-trimester placenta appeared between 5 and 6 weeks of gestation. Because epidermal growth factor was demonstrated to stimUlate human chorionic gonadotropin and human placental lactogen production by early placental tissues, their simultaneous expression, as well as epidermal growth factor and its receptor in the cytotrophoblast of 4- to 5-week placenta and in the syncytiotrophoblast of 6- to 12-week placenta, implies that human chorionic gonadotropin and human placental lactogen production by first-trimester placenta may be regulated in an autocrine manner, wherein epidermal growth factor may serve as the Signal. These findings suggest that in very early placenta, before 6 weeks of gestation, no sequential expression of human chorionic gonadotropin and human placental lactogen closely linked to syncytia formation may exist and that both can be expressed in the cytotrophoblast or undifferentiated stem cell of villous trophoblast in very early placenta. (AM J OSSTET GYNECOl1992;167:217-22.)
Key words: Placenta, cytotrophoblast, human chorionic gonadotropin, human placental lactogen, immunohistochemistry The villous trophoblast of human placenta is composed of two distinct layers: an inner cell layer of mononuclear cytotrophoblast and an outer cell layer of multinuclear syncytiotrophoblast. It is generally accepted that the cytotrophoblast is the undifferentiated stem cell of the villous trophoblast and that the syncytiotrophoblast is the fully differentiated, end-stage cell derived from cytotrophoblast. 1·6 The human placental trophoblast elaborates at least two peptide hormones, human chorionic gonadotropin (hCG) and human placental lactogen (hPL). A number From the Department of Obstetri{s and Gynecology, Kobe University School ofMedicine,' and the Department of Obstetrics and Gynecology, University of the Philippines, Philippine General Hospital.' Supported in part by Grants in Aid for Scientific Resear{h 63480368 and 0267074 from the Japanese Ministry of Education, Scien{e and Culture and by the Ogyaa-Donation Foundation ofJapan Association of Maternal Welfare. C.A. Ladines-Llave was supported by the Ronpaku Program of the Japan Society for the Promotion of Science. Received for publication July 8,1991; revised October 16,1991; accepted November 7,1991. Reprint requests: Takeshi Maruo, MD, Department of Obstetrics and Gynecology, Kobe University School of Medicine, Chuo-Ku, Kobe 650,japan. 6/1/34903
of immunohistochemical studies,7.lo including ours," have indicated that immunoreactive l3-hCG, hCG, and hPL are exclusively localized to the syncytiotrophoblast, whereas other immunohistochemical studies l2 , '" have suggested that the cytotrophoblast may contain immunoreactive a-hCG. Consistent with the immunohistochemical findings, Hoshina et ai.,14. 15 using in situ hybridization techniques to localize hCG (a and [3) and hPL messenger ribonucleic acids in placental tissue sections, showed that the expression of a-hCG messenger ribonucleic acid is initiated in some differentiating cytotrophoblasts, whereas the expression of [3-hCG messenger ribonucleic acid starts during the process of differentiation of syncytial trophoblast and hPL messenger ribonucleic acid is expressed only in the fully differentiated syncytiotrophoblast. However, it must be pointed out that the placental tissue materials used in the studies reported have been obtained at a gestational age later than 6 weeks, probably because of the greater availability of placental materials between 6 and 12 weeks of gestation and at term. Thus the claim that the ability of human trophoblast to synthesize hCG and hPL is tied to syncytia formation may be limited to the 217
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Fig. 1. Immunohistochemical localization of ~-hCG carboxyl terminal peptide (hCGf3-CTP) and hPL in very early first-trimester placenta. Formalin-fixed, paraffin-embedded sections of 4-week placenta were stained with anti-{3-hCG-CTP antibody (A) and with anti-hPL antibody (B). Cytotrophoblastic cells (CT) showed pronounced staining with anti-~-hCG-CTP antibody and with anti-hPL a ntibody, whereas syncytiotrophoblast (ST) had scarce staining. (Original magnification x 200.)
placenta with a gestational age > 6 weeks . Nevertheless, it is known that hPL exerts a lipolytic and glucosesparing effect on the mother to ensure that the nutritional demands of the fetus are met,IO. 17 whereas hCG is responsible for the rescue of the corpus luteum, which is indispensable for the maintenance ofvery early pregnancy during the first 4 to 6 weeks of gestation. IS Thus it is of great importance to provide insight into the expression of hCG and hPL in very early placenta before 6 weeks of gestation. On the other hand, epidermal growth factor (EGF) has been demonstrated to stimulate the production of hCG and hPL by early placental tissues through the binding to EGF receptor. 19 Furthermore, we have recently found that EGF and EGF receptor are initially expressed in the cytotrophoblasts in very early placenta before 6 weeks of gestation and thereafter expressed in the syncytiotrophoblast of 6- to 12-week placenta}O A remarkable change in cytologic localization of EGF. and EGF receptor in very early placenta appeared between 5 and 6 weeks of gestation. Thus it seems interesting to investigate whether cytologic localization of hCG and hPL in very early placenta may also change during the course of early gestation in association with the shift of cytologic localization of EGF and EGF receptor in first-trimester placenta. Hence in the current study attention was focused on the expression of hCG and hPL in very early placenta, before 6 weeks of gestation, and the possible change in cytologic localization of hCG and hPL in first-trimester placenta over the course of early gestation was examined. Material and methods
Material. First-trimester placentas were obtained from 12 patients who underwent elective abortion at 4 to 5 weeks (three cases), 6 to 7 weeks (three cases), and 8 to 12 weeks (six cases) of gestation. Third-trimester placentas were obtained from four patients who had
cesarean section between 38 and 40 weeks of gestation. The gestational age of the placenta was determined by the known date of conception or estimation from the date of the patient's last menstrual period. In all cases gestational age was further evaluated by ultrasonographic examination. Informed consent for the use of placental tissues for immunohistochemical studies was obtained from the patients before operation. Immunohistochemical staining. Placental tissues obtained were fixed in 4 % buffered neutral formalin , dehydrated, and embedded in paraffin. Sections, 5 to 6 ~m in thickness, were deparaffinized and followed by standard histologic techniques. Immunohistochemical staining was performed by avidin/biotin immunoperoxide techniques with a polyvalent immunoperoxidase kit (Omnitags, Lipshaw, Mich.) as previously described by Maruo and Mochizuki. II An affinity-purified sheep polyclonal antibody against ~-hCG carboxyl terminal peptide (Takeda Chemical Industries, Osaka) and a rabbit polyclonal antibody against hPL (Dako Corp., Santa Barbara, Calif.) were used, respectively, as the primary antibodies in this study. The anti-~-hCG~ carboxyl terminal pe ptide antibody and anti-hPL antibody were diluted 1 : 100 and 1 : 1000 before use, respectively. To assure specificity of the immunologic reactions, adjacent control sections were subjected to the same immunoperoxidase method, except that the primary antibodies to ~-hCG carboxyl terminal peptide or hPL were respectively replaced by nonimmune sheep immunoglobulin G (Miles, Elkhart, Ind.) or nonimmllne rabbit immunoglobulin G (Miles). In the controls no positive staining was observed. Results
Fig. I, A and B, shows the immunohistochemical staining for l3-hCG carboxyl terminal peptide and hPL, respectively, in very early placenta obtained at 4 weeks
Cytologic localization of hCG and hPL changes in placenta 219
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Fig. 2. Immunohistochemical localization of ~-hCG-CTP and hPL in early first trimester placenta. Formalin-fixed, paraffin-embedded sections of 6-week placenta were stained with anti-~-CTP antibody (A) and anti·hPL antibody (B). Pronounced staining for ~-hCG-CTP and hPL was observed in syncytiotrophoblast (ST), whereas cytotrophoblastic cells (CT) were negative for appreciable staining. (Original magnification x 200.)
Fig. 3. Immunohistochemical localization of ~-hCG-CTP and hPL in first trimester placenta. Formalin-fixed, paraffin-embedded sections of 9-week placenta were stained with anti-~-hCG-CTP antibody (A) and anti-hPL antibody (B). Prominent staining for f3-hCG-CTP and hPL was observed with maximal intensity in syncytiotrophoblast (ST), whereas cytotrophoblastic cells (CT) were negative for appreciable staining. (Original magnification X 200.)
of gestation. Both immunostaining with anti-~-hCG carboxyl terminal peptide and anti-hPL antibody were prominent in cytotrophoblasts , whereas syncytiotrophoblast showed scarce staining for J3-hCG carboxyl terminal peptide and hPL. A similar pattern of immunostaining for J3-hCG carboxyl terminal peptide and hPL was observed in tissue sections of 5-week placenta. Fig. 2, A and B, represents the immunohistochemical staining for J3-hCG carboxyl terminal peptide and hPL, respectively, in early placenta obtained at 6 weeks of gestation. Unlike the very early placenta obtained at 4 to 5 weeks of gestation, both immunostaining with antiJ3-hCG carboxyl terminal peptide antibody and antihPL antibody were exclusively localized to syncytiotrophoblast. No appreciable staining for J3-hCG carboxyl terminal peptide and hPL was found in cytotrophoblasts. In the case of 8- to 12-week placenta (Fig. 3, A and B), the pattern of cytologic localization of J3-hCG
carboxyl terminal peptide and hPL was similar to that in 6-week placenta, but the staining intensity for f3-hCG carboxyl terminal peptide and hPL of the syncytiotrophoblast was more prominent compared with that of 6-week placenta. Fig. 4, A and B, shows the immunohistochemical staining for J3-hCG carboxyl terminal peptide and hPL, respectively, in term placenta obtained at 40 weeks of gestation. Both immunostaining with anti-J3-hCG carboxyl terminal peptide antibody and anti-hPL antibody were focall y observed with the exclusive localization in syncytiotrophoblast. The results of cytologic localization of J3-hCG carboxyl terminal peptide and hPL in first-trimester placenta according to the type of villous trophoblast and the gestational age are summarized in Table I. A remarkable shift in cytologic localization of f3-hCG carboxyl terminal peptide and hPL from cytotrophoblast
220 Maruo et al.
July 1992 Am 1 Obstet Gynecol
Fig. 4. Immunohistochemical localization of f3-hCG-CTP and hPL in term placenta. Formalin-fixed, paraffin-embedded sections of 40-week placenta were stained with anti-f3-hCG-CTP antibody (A) and anti-hPL antibody (8). Pronounced staining for f3-hCG-CTP and hPL was focally observed with exclusive localization in syncytiotrophoblast (ST). (Original magnification x 200.)
Table I. Cytologic localization of ~-hCG-CTP and hPL in first-trimester and term placenta according to the gestational age First-trimester 4 to 5 wk (3-hCG carboxyl terminal peptide
Cytotrophoblast Syncytiotrophoblast
+
I
J
6 to 7 wk
hPL
+
(3-hCG carboxyl terminal peptide
++
8 to 12 wk
hPL
(3-hCG carboxyl terminal peptide
++
+++
J
Term (40 wk) hPL
(3-hCG carboxyl terminal peptide
+++
+
I
hPL
+
Semiquantitative scoring denotes intensity of immunostaining.
to syncytiotrophoblast was found to appear between 5 and 6 weeks of gestation.
Comment The current study demonstrated that cytologic localization of hCG and hPL in human first trimester placenta changes in the advancing course of gestation. In the very early placenta obtained at 4 to 5 weeks of gestation, hCG and hPL were primarily localized to cytotrophoblasts, whereas in early placenta obtained later than 6 weeks of gestation they were exclusively localized to syncytiotrophoblast. These findings suggest that both are initially expressed in cytotrophoblasts in very early placenta before 6 weeks of gestation and thereafter expressed in syncytiotrophoblast in the placenta during the remainder of gestation. To our knowledge, the current study is the first to demonstrate the change in cytologic localization of hCG and hPL in developing human placenta in the course of early gestation. The change in cytologic localization of hCG and hPL from cytotrophoblasts to syncytiotrophoblast in very early placenta is of great interest considering the es-
tablished finding that syncytiotrophoblast is formed from cytotrophoblasts by a process of proliferation followed by cell fusion!' 5 In situ hybridization studies 14• 15 with a-hCG, l3-hCG, and hPL probes on tissue sections of the placenta obtained later than 6 weeks of gestation have demonstrated that the villous trophoblast acquires the ability to elaborate these· substances in association with the morphologic transformation from cytotrophoblasts to syncytiotrophoblast and that the biochemical differentiation of the villous trophoblast occurs only after syncytia formation. Thus it seems evident that in the placenta with gestational age later than 6 weeks there is a sequential expression of a-heG, 13heG, and hPL closely correlated with the differentiation of the villous trophoblast. However, when Kao et al. 21 grew purified human placental cytotrophoblasts under serum-free conditions to elucidate whether syncytia formation is a prerequisite for the biochemical differentiation of the villous trophoblast, they found that the formation of syncytiotrophoblast is not a prerequisite for the acquisition of the ability to elaborate heG but is only one of the consequences of the differentiation program of the trophoblast. They demon-
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strated that purified human placental cytotrophoblasts in culture could secrete heG at the same rate under serum-free conditions whether they were plated on plastic only, which prevented syncytia formation, or fibronectin laminin or type IV collagen, which allowed syncytia formation to occur. Our results obtained with the tissue sections of very early human placenta support the notion that syncytia formation is not a prerequisite for the acquisition of a specific endocrine function of the placenta. In the case of very early placenta before 6 weeks of gestation, both heG and hPL can be expressed in cytotrophoblasts. In this regard, our recent findings in an earlier study are of considerable interest. We have demonstrated that cytologic localization of EGF and EGF receptor in developing human placenta varies over the course of gestation and that even in first-trimester placenta there is a remarkable change in the cytologic localization from cytotrophoblasts to syncytiotrophoblast at the sixth week of gestation}O Thus the change in cytologic localization of heG and hPL in very early placenta observed in the course of early gestation is closely coincident with the change in cytolocalization of EGF and EGF receptor. Because EGF has been demonstrated to exert a stimulatory effect on heG and hPL production by early placental tissues through the binding to EGF receptor,!9 the simultaneous expression of EGF and EGF receptor and heG and hPL in the cytotrophoblast of 4- to 5-week placentas and in the syncytiotrophoblast of 6- to 12-week placentas implies that the production of heG and hPL by first-trimester placenta may be regulated by an autocrine system, wherein EGF may serve as the signal. It is evident that increasing amounts of heG produced by the chorion around the time of implantation play an important role in rescuing the corpus luteum. The placenta is known to take over the corpus luteum function at the sixth week of gestation in producing progesterone and estrogen necessary for the maintenance of early gestation. It is of interest that the dynamic change in cytologic localization of heG and hPL and EGF and EGF receptor from cytotrophoblast to syncytiotrophoblast in very early placenta also occurs at the sixth week of gestation. Taking these findings into account, the sixth week of gestation seems to be critical in the functional development of the early placenta. These findings suggest that in very early placenta, before the sixth week of gestation, heG and hPL can be expressed in the cytotrophoblast (the undifferentiated stem cell of the villous trophoblast) and that there is a shift in cytologic localization of heG and hPL from cytotrophoblast to syncytiotrophoblast around the sixth week of gestation. It is therefore likely that the program of the sequential expression of the genes coding for
Cytologic localization of hCG and hPL changes in placenta 221
heG (ex and 13) and hPL correlated with the differentiation of the villous trophoblast may be established in the placenta at the sixth week of gestation. Before that time no sequential expression of heG and hPL correlated with trophoblast differentiation may exist in the placenta. Further studies with in situ hybridization with heG (ex and 13) and hPL probes on very early placental tissues will be needed to reinforce this notion. REFERENCES 1. Pierce GB Jr, Midgley ARJr. The origin and function of human syncytiotrophoblastic giant cells. Am J Pathol 1963;43:153-73. 2. Enders AC. Formation of syncytium from cytotrophoblast in the human placenta. Obstet Gynecol 1965;25:378-86. 3. Gerbie AB, Hathaway HH, Brewer JI. Autoradiographic analysis of normal trophoblastic proliferation. AM J OBSTET GVNECOL 1968; 100:640-8. 4. Nelson DM, Meister RK, Onman-Nabi J, Sparks S, Stevens VC. Differentiation and secretory activities of cultured human placental cytotrophoblast. Placenta 1986;7:1-16. 5. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF III. Purification, characterization and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 1986; 118: 1567-82. 6. Kliman HJ, Feimann MA, Strauss JF III. Differentiation of human cytotrophoblast into syncytiotrophoblast in culture. Trophoblast Res 1987;2:407-21. 7. Midgley AR, Pierce GB. Immunohistochemical localization of human chorionic gonadotropin. J Exp Med 1962; 115:289-94. 8. Thiede HA, Choate JW. Chorionic gonadotropin localization in the human placenta by immunofluorescent staining. Obstet Gynecol 1963;22:433-43. 9. Fox H, Kharkongor FN. Immunofluorescent localization of chorionic gonadotropin in the placenta and in tissue culture of human trophoblast. J Pathol 1970; 101 :277-82. 10. Watkins WB. Use of immunocytochemical techniques for the localization of human placental lactogen. J Histochem Cytochem 1978;26:288-92. 11. Maruo T, Mochizuki M. Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in human placenta: implication for trophoblast proliferation and differentiation. AM J OBSTET GvNECOL 1987;156:721-7. 12. Hoshina M, Ashitaka y, Tojo S. Immunohistochemical interaction on antisera to hCG and its subunits with chorionic tissue of early gestation. Endocrinol Jpn 1979;26:175-84. 13. Gaspard JU, Hustin J, Reuter AM, Lambotte R, Franchi mont P. Immunofluorescent localization of placental lactogen, chorionic gonadotropin and its alpha and beta subunits in organ cultures of human placenta. Placenta 1980; 1: 135-44. 14. Hoshina M, Boothby M, Boime I. Cytological localization of chorionic gonadotropin a and placental lactogen mRNAs during development of the human placenta. J Cell Bioi 1982;93:190-8. 15. Hoshina M, Boothby M, Hussa R, Pattillo R, Camel HM, Boime I. Linkage of human chorionic gonadotropin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis. Placenta 1985;6:163-72. 16. JosimovichJB. Placental lactogen and pituitary prolactin. In: Fuchs F, Klopper A, eds. Endocrinology of pregnancy. Philadelphia: Harper & Row, 1983:144-60. 17. Mochizuki M. Studies on human placental lactogen. Acta Obstet GynecolJpn 1973;25:1043-7. 18. Wentz AC. Endocrinology of early pregnancy. In: Givens
Maruo et al.
JR, ed. Endocrinology of pregnancy. Chicago: Year Book, 1981:1-34. 19. Maruo T, Matsuo H, Hayashi M, Nishino R, Mochizuki M. Induction of differentiated trophoblast function by epidermal growth factor: relation of immunohistochemically detected cellular epidermal growth factor receptor levels. J Clin Endocrinol Metab 1987;64:744-9. 20. Ladines-Llave CA, Maruo T, Manalo AS, Mochizuki M. Cytologic localization of epidermal growth factor and its
July 1992 Am J Obstet Gynecol
receptor in developing human placenta varies over the course of pregnancy. AM J OBSTET GVNECOL 1991; 165: 1377-82. 21. Kao LC, Caltabiano S, Wu S, Strauss JF III, Kliman Hl The human villous cytotrophoblast: interactions with extracellular matrix proteins, endocrine function and cytoplasmic differentiation in the absence of syncytium formation. Dev Bioi 1988; 130:693-702.
Transforming growth factor-J3 opposes the stimulatory effects of interleukin-l and tumor necrosis factor on amnion cell prostaglandin E2 production: Implication for preterm labor Kristina Bry, MD, and Mikko Hallman, MD Irvine, California, and Helsinki, Finland OBJECTIVE: In preterm labor increased concentrations of interleukin-' and tumor necrosis factor are present in amniotic fluid. These cytokines may promote labor by stimulating the production of prostaglandins by intrauterine tissues. In many biologic processes, transforming growth factor-13 modifies the actions of cytokines. We studied the effect of transforming growth factor-J3 on the cytokine-induced prostaglandin E2 production by amnion cells. STUDY DESIGN: Human amnion cells in monolayer culture were treated with interleukin-1, tumor necrosis factor, or vehicle in the presence or absence of transforming growth factor-I3. The prostaglandin E2 production was measured. RESULTS: Transforming growth factor-13 decreased the interleukin-1- or tumor necrosis factor-induced prostaglandin E2 production by 70% to 80% and the basal prostaglandin E2 synthesis by 27%. The synergistic stimulation of prostaglandin E2 production by the combination of interleukin-1 with tumor necrosis factor was inhibited by 80% in cells treated with transforming growth faclor-l3. Transforming growth factor-j31, -132, and -131,2 were equipotent. CONCLUSION: Transforming growth factor-j3 suppresses the cytokine-induced prostaglandin E2 production by amnion cells and may be an important factor in maintaining pregnancy in the face of labor-promoting cytokines. (AM J OBSTET GVNECOL 1992;167:222-6.)
Key words: Preterm labor, prostaglandins, interleukin-l, tumor necrosis factor, transforming growth factor-/3, fetal membranes Preterm labor often occurs in the setting of intrauterine infection. I The concentrations of cytokines such as interleukin-l (IL-l) and tumor necrosis factor (TNF) increase in amniotic fluid in pre term labor.2~4 ProstaFrom the Department of Pediatrics, University of California, [rome, and the Department of Pediatrics, University of Helsinki. Supported by the Foundation for Maternal and Infant Care and the Sigrid Juselius Foundation. Received for publication October 14, 1991; revised January 21, 1992; accepted January 23,1992. Reprint requests: Kristina Bry, MD, DepaTtment of Pediatrics, University of California, Imine, Med. SUTge /.. Rm 109 F, [mine, CA 92717. 611/36682
222
gland ins, major inducers of uterine contractions, are produced by intrauterine tissues in response to these cytokines. 3 5·7 The amnion, which produces almost exclusively prostaglandin £2 (PG£2),8 is an important intrauterine source of prostaglandin. The combination of [L-l with TNF has a synergistic stimulator effect on the PGE 2 production by human amnion cells in culture.~ IL-I and TNF have been implicated in the induction of preterm labor associated with infections."' 3, 10 Transforming growth fac10r-/3 (TGF-/3) represents a family of multifunctional factors that influence cell growth, differentiation, immunity, and extracellular matrix formation. II Several forms of TGF -/3 and of
Key words: Placenta, cytotrophoblast, human chorionic gonadotropin, human placental lactogen, immunohistochemistry The villous trophoblast of human placenta is composed of two distinct layers: an inner cell layer of mononuclear cytotrophoblast and an outer cell layer of multinuclear syncytiotrophoblast. It is generally accepted that the cytotrophoblast is the undifferentiated stem cell of the villous trophoblast and that the syncytiotrophoblast is the fully differentiated, end-stage cell derived from cytotrophoblast. 1·6 The human placental trophoblast elaborates at least two peptide hormones, human chorionic gonadotropin (hCG) and human placental lactogen (hPL). A number From the Department of Obstetri{s and Gynecology, Kobe University School ofMedicine,' and the Department of Obstetrics and Gynecology, University of the Philippines, Philippine General Hospital.' Supported in part by Grants in Aid for Scientific Resear{h 63480368 and 0267074 from the Japanese Ministry of Education, Scien{e and Culture and by the Ogyaa-Donation Foundation ofJapan Association of Maternal Welfare. C.A. Ladines-Llave was supported by the Ronpaku Program of the Japan Society for the Promotion of Science. Received for publication July 8,1991; revised October 16,1991; accepted November 7,1991. Reprint requests: Takeshi Maruo, MD, Department of Obstetrics and Gynecology, Kobe University School of Medicine, Chuo-Ku, Kobe 650,japan. 6/1/34903
of immunohistochemical studies,7.lo including ours," have indicated that immunoreactive l3-hCG, hCG, and hPL are exclusively localized to the syncytiotrophoblast, whereas other immunohistochemical studies l2 , '" have suggested that the cytotrophoblast may contain immunoreactive a-hCG. Consistent with the immunohistochemical findings, Hoshina et ai.,14. 15 using in situ hybridization techniques to localize hCG (a and [3) and hPL messenger ribonucleic acids in placental tissue sections, showed that the expression of a-hCG messenger ribonucleic acid is initiated in some differentiating cytotrophoblasts, whereas the expression of [3-hCG messenger ribonucleic acid starts during the process of differentiation of syncytial trophoblast and hPL messenger ribonucleic acid is expressed only in the fully differentiated syncytiotrophoblast. However, it must be pointed out that the placental tissue materials used in the studies reported have been obtained at a gestational age later than 6 weeks, probably because of the greater availability of placental materials between 6 and 12 weeks of gestation and at term. Thus the claim that the ability of human trophoblast to synthesize hCG and hPL is tied to syncytia formation may be limited to the 217
218
Maruo et al.
July 1992 Am J Obstet Gynecol
Fig. 1. Immunohistochemical localization of ~-hCG carboxyl terminal peptide (hCGf3-CTP) and hPL in very early first-trimester placenta. Formalin-fixed, paraffin-embedded sections of 4-week placenta were stained with anti-{3-hCG-CTP antibody (A) and with anti-hPL antibody (B). Cytotrophoblastic cells (CT) showed pronounced staining with anti-~-hCG-CTP antibody and with anti-hPL a ntibody, whereas syncytiotrophoblast (ST) had scarce staining. (Original magnification x 200.)
placenta with a gestational age > 6 weeks . Nevertheless, it is known that hPL exerts a lipolytic and glucosesparing effect on the mother to ensure that the nutritional demands of the fetus are met,IO. 17 whereas hCG is responsible for the rescue of the corpus luteum, which is indispensable for the maintenance ofvery early pregnancy during the first 4 to 6 weeks of gestation. IS Thus it is of great importance to provide insight into the expression of hCG and hPL in very early placenta before 6 weeks of gestation. On the other hand, epidermal growth factor (EGF) has been demonstrated to stimulate the production of hCG and hPL by early placental tissues through the binding to EGF receptor. 19 Furthermore, we have recently found that EGF and EGF receptor are initially expressed in the cytotrophoblasts in very early placenta before 6 weeks of gestation and thereafter expressed in the syncytiotrophoblast of 6- to 12-week placenta}O A remarkable change in cytologic localization of EGF. and EGF receptor in very early placenta appeared between 5 and 6 weeks of gestation. Thus it seems interesting to investigate whether cytologic localization of hCG and hPL in very early placenta may also change during the course of early gestation in association with the shift of cytologic localization of EGF and EGF receptor in first-trimester placenta. Hence in the current study attention was focused on the expression of hCG and hPL in very early placenta, before 6 weeks of gestation, and the possible change in cytologic localization of hCG and hPL in first-trimester placenta over the course of early gestation was examined. Material and methods
Material. First-trimester placentas were obtained from 12 patients who underwent elective abortion at 4 to 5 weeks (three cases), 6 to 7 weeks (three cases), and 8 to 12 weeks (six cases) of gestation. Third-trimester placentas were obtained from four patients who had
cesarean section between 38 and 40 weeks of gestation. The gestational age of the placenta was determined by the known date of conception or estimation from the date of the patient's last menstrual period. In all cases gestational age was further evaluated by ultrasonographic examination. Informed consent for the use of placental tissues for immunohistochemical studies was obtained from the patients before operation. Immunohistochemical staining. Placental tissues obtained were fixed in 4 % buffered neutral formalin , dehydrated, and embedded in paraffin. Sections, 5 to 6 ~m in thickness, were deparaffinized and followed by standard histologic techniques. Immunohistochemical staining was performed by avidin/biotin immunoperoxide techniques with a polyvalent immunoperoxidase kit (Omnitags, Lipshaw, Mich.) as previously described by Maruo and Mochizuki. II An affinity-purified sheep polyclonal antibody against ~-hCG carboxyl terminal peptide (Takeda Chemical Industries, Osaka) and a rabbit polyclonal antibody against hPL (Dako Corp., Santa Barbara, Calif.) were used, respectively, as the primary antibodies in this study. The anti-~-hCG~ carboxyl terminal pe ptide antibody and anti-hPL antibody were diluted 1 : 100 and 1 : 1000 before use, respectively. To assure specificity of the immunologic reactions, adjacent control sections were subjected to the same immunoperoxidase method, except that the primary antibodies to ~-hCG carboxyl terminal peptide or hPL were respectively replaced by nonimmune sheep immunoglobulin G (Miles, Elkhart, Ind.) or nonimmllne rabbit immunoglobulin G (Miles). In the controls no positive staining was observed. Results
Fig. I, A and B, shows the immunohistochemical staining for l3-hCG carboxyl terminal peptide and hPL, respectively, in very early placenta obtained at 4 weeks
Cytologic localization of hCG and hPL changes in placenta 219
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Fig. 2. Immunohistochemical localization of ~-hCG-CTP and hPL in early first trimester placenta. Formalin-fixed, paraffin-embedded sections of 6-week placenta were stained with anti-~-CTP antibody (A) and anti·hPL antibody (B). Pronounced staining for ~-hCG-CTP and hPL was observed in syncytiotrophoblast (ST), whereas cytotrophoblastic cells (CT) were negative for appreciable staining. (Original magnification x 200.)
Fig. 3. Immunohistochemical localization of ~-hCG-CTP and hPL in first trimester placenta. Formalin-fixed, paraffin-embedded sections of 9-week placenta were stained with anti-~-hCG-CTP antibody (A) and anti-hPL antibody (B). Prominent staining for f3-hCG-CTP and hPL was observed with maximal intensity in syncytiotrophoblast (ST), whereas cytotrophoblastic cells (CT) were negative for appreciable staining. (Original magnification X 200.)
of gestation. Both immunostaining with anti-~-hCG carboxyl terminal peptide and anti-hPL antibody were prominent in cytotrophoblasts , whereas syncytiotrophoblast showed scarce staining for J3-hCG carboxyl terminal peptide and hPL. A similar pattern of immunostaining for J3-hCG carboxyl terminal peptide and hPL was observed in tissue sections of 5-week placenta. Fig. 2, A and B, represents the immunohistochemical staining for J3-hCG carboxyl terminal peptide and hPL, respectively, in early placenta obtained at 6 weeks of gestation. Unlike the very early placenta obtained at 4 to 5 weeks of gestation, both immunostaining with antiJ3-hCG carboxyl terminal peptide antibody and antihPL antibody were exclusively localized to syncytiotrophoblast. No appreciable staining for J3-hCG carboxyl terminal peptide and hPL was found in cytotrophoblasts. In the case of 8- to 12-week placenta (Fig. 3, A and B), the pattern of cytologic localization of J3-hCG
carboxyl terminal peptide and hPL was similar to that in 6-week placenta, but the staining intensity for f3-hCG carboxyl terminal peptide and hPL of the syncytiotrophoblast was more prominent compared with that of 6-week placenta. Fig. 4, A and B, shows the immunohistochemical staining for J3-hCG carboxyl terminal peptide and hPL, respectively, in term placenta obtained at 40 weeks of gestation. Both immunostaining with anti-J3-hCG carboxyl terminal peptide antibody and anti-hPL antibody were focall y observed with the exclusive localization in syncytiotrophoblast. The results of cytologic localization of J3-hCG carboxyl terminal peptide and hPL in first-trimester placenta according to the type of villous trophoblast and the gestational age are summarized in Table I. A remarkable shift in cytologic localization of f3-hCG carboxyl terminal peptide and hPL from cytotrophoblast
220 Maruo et al.
July 1992 Am 1 Obstet Gynecol
Fig. 4. Immunohistochemical localization of f3-hCG-CTP and hPL in term placenta. Formalin-fixed, paraffin-embedded sections of 40-week placenta were stained with anti-f3-hCG-CTP antibody (A) and anti-hPL antibody (8). Pronounced staining for f3-hCG-CTP and hPL was focally observed with exclusive localization in syncytiotrophoblast (ST). (Original magnification x 200.)
Table I. Cytologic localization of ~-hCG-CTP and hPL in first-trimester and term placenta according to the gestational age First-trimester 4 to 5 wk (3-hCG carboxyl terminal peptide
Cytotrophoblast Syncytiotrophoblast
+
I
J
6 to 7 wk
hPL
+
(3-hCG carboxyl terminal peptide
++
8 to 12 wk
hPL
(3-hCG carboxyl terminal peptide
++
+++
J
Term (40 wk) hPL
(3-hCG carboxyl terminal peptide
+++
+
I
hPL
+
Semiquantitative scoring denotes intensity of immunostaining.
to syncytiotrophoblast was found to appear between 5 and 6 weeks of gestation.
Comment The current study demonstrated that cytologic localization of hCG and hPL in human first trimester placenta changes in the advancing course of gestation. In the very early placenta obtained at 4 to 5 weeks of gestation, hCG and hPL were primarily localized to cytotrophoblasts, whereas in early placenta obtained later than 6 weeks of gestation they were exclusively localized to syncytiotrophoblast. These findings suggest that both are initially expressed in cytotrophoblasts in very early placenta before 6 weeks of gestation and thereafter expressed in syncytiotrophoblast in the placenta during the remainder of gestation. To our knowledge, the current study is the first to demonstrate the change in cytologic localization of hCG and hPL in developing human placenta in the course of early gestation. The change in cytologic localization of hCG and hPL from cytotrophoblasts to syncytiotrophoblast in very early placenta is of great interest considering the es-
tablished finding that syncytiotrophoblast is formed from cytotrophoblasts by a process of proliferation followed by cell fusion!' 5 In situ hybridization studies 14• 15 with a-hCG, l3-hCG, and hPL probes on tissue sections of the placenta obtained later than 6 weeks of gestation have demonstrated that the villous trophoblast acquires the ability to elaborate these· substances in association with the morphologic transformation from cytotrophoblasts to syncytiotrophoblast and that the biochemical differentiation of the villous trophoblast occurs only after syncytia formation. Thus it seems evident that in the placenta with gestational age later than 6 weeks there is a sequential expression of a-heG, 13heG, and hPL closely correlated with the differentiation of the villous trophoblast. However, when Kao et al. 21 grew purified human placental cytotrophoblasts under serum-free conditions to elucidate whether syncytia formation is a prerequisite for the biochemical differentiation of the villous trophoblast, they found that the formation of syncytiotrophoblast is not a prerequisite for the acquisition of the ability to elaborate heG but is only one of the consequences of the differentiation program of the trophoblast. They demon-
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strated that purified human placental cytotrophoblasts in culture could secrete heG at the same rate under serum-free conditions whether they were plated on plastic only, which prevented syncytia formation, or fibronectin laminin or type IV collagen, which allowed syncytia formation to occur. Our results obtained with the tissue sections of very early human placenta support the notion that syncytia formation is not a prerequisite for the acquisition of a specific endocrine function of the placenta. In the case of very early placenta before 6 weeks of gestation, both heG and hPL can be expressed in cytotrophoblasts. In this regard, our recent findings in an earlier study are of considerable interest. We have demonstrated that cytologic localization of EGF and EGF receptor in developing human placenta varies over the course of gestation and that even in first-trimester placenta there is a remarkable change in the cytologic localization from cytotrophoblasts to syncytiotrophoblast at the sixth week of gestation}O Thus the change in cytologic localization of heG and hPL in very early placenta observed in the course of early gestation is closely coincident with the change in cytolocalization of EGF and EGF receptor. Because EGF has been demonstrated to exert a stimulatory effect on heG and hPL production by early placental tissues through the binding to EGF receptor,!9 the simultaneous expression of EGF and EGF receptor and heG and hPL in the cytotrophoblast of 4- to 5-week placentas and in the syncytiotrophoblast of 6- to 12-week placentas implies that the production of heG and hPL by first-trimester placenta may be regulated by an autocrine system, wherein EGF may serve as the signal. It is evident that increasing amounts of heG produced by the chorion around the time of implantation play an important role in rescuing the corpus luteum. The placenta is known to take over the corpus luteum function at the sixth week of gestation in producing progesterone and estrogen necessary for the maintenance of early gestation. It is of interest that the dynamic change in cytologic localization of heG and hPL and EGF and EGF receptor from cytotrophoblast to syncytiotrophoblast in very early placenta also occurs at the sixth week of gestation. Taking these findings into account, the sixth week of gestation seems to be critical in the functional development of the early placenta. These findings suggest that in very early placenta, before the sixth week of gestation, heG and hPL can be expressed in the cytotrophoblast (the undifferentiated stem cell of the villous trophoblast) and that there is a shift in cytologic localization of heG and hPL from cytotrophoblast to syncytiotrophoblast around the sixth week of gestation. It is therefore likely that the program of the sequential expression of the genes coding for
Cytologic localization of hCG and hPL changes in placenta 221
heG (ex and 13) and hPL correlated with the differentiation of the villous trophoblast may be established in the placenta at the sixth week of gestation. Before that time no sequential expression of heG and hPL correlated with trophoblast differentiation may exist in the placenta. Further studies with in situ hybridization with heG (ex and 13) and hPL probes on very early placental tissues will be needed to reinforce this notion. REFERENCES 1. Pierce GB Jr, Midgley ARJr. The origin and function of human syncytiotrophoblastic giant cells. Am J Pathol 1963;43:153-73. 2. Enders AC. Formation of syncytium from cytotrophoblast in the human placenta. Obstet Gynecol 1965;25:378-86. 3. Gerbie AB, Hathaway HH, Brewer JI. Autoradiographic analysis of normal trophoblastic proliferation. AM J OBSTET GVNECOL 1968; 100:640-8. 4. Nelson DM, Meister RK, Onman-Nabi J, Sparks S, Stevens VC. Differentiation and secretory activities of cultured human placental cytotrophoblast. Placenta 1986;7:1-16. 5. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF III. Purification, characterization and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 1986; 118: 1567-82. 6. Kliman HJ, Feimann MA, Strauss JF III. Differentiation of human cytotrophoblast into syncytiotrophoblast in culture. Trophoblast Res 1987;2:407-21. 7. Midgley AR, Pierce GB. Immunohistochemical localization of human chorionic gonadotropin. J Exp Med 1962; 115:289-94. 8. Thiede HA, Choate JW. Chorionic gonadotropin localization in the human placenta by immunofluorescent staining. Obstet Gynecol 1963;22:433-43. 9. Fox H, Kharkongor FN. Immunofluorescent localization of chorionic gonadotropin in the placenta and in tissue culture of human trophoblast. J Pathol 1970; 101 :277-82. 10. Watkins WB. Use of immunocytochemical techniques for the localization of human placental lactogen. J Histochem Cytochem 1978;26:288-92. 11. Maruo T, Mochizuki M. Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in human placenta: implication for trophoblast proliferation and differentiation. AM J OBSTET GvNECOL 1987;156:721-7. 12. Hoshina M, Ashitaka y, Tojo S. Immunohistochemical interaction on antisera to hCG and its subunits with chorionic tissue of early gestation. Endocrinol Jpn 1979;26:175-84. 13. Gaspard JU, Hustin J, Reuter AM, Lambotte R, Franchi mont P. Immunofluorescent localization of placental lactogen, chorionic gonadotropin and its alpha and beta subunits in organ cultures of human placenta. Placenta 1980; 1: 135-44. 14. Hoshina M, Boothby M, Boime I. Cytological localization of chorionic gonadotropin a and placental lactogen mRNAs during development of the human placenta. J Cell Bioi 1982;93:190-8. 15. Hoshina M, Boothby M, Hussa R, Pattillo R, Camel HM, Boime I. Linkage of human chorionic gonadotropin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis. Placenta 1985;6:163-72. 16. JosimovichJB. Placental lactogen and pituitary prolactin. In: Fuchs F, Klopper A, eds. Endocrinology of pregnancy. Philadelphia: Harper & Row, 1983:144-60. 17. Mochizuki M. Studies on human placental lactogen. Acta Obstet GynecolJpn 1973;25:1043-7. 18. Wentz AC. Endocrinology of early pregnancy. In: Givens
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JR, ed. Endocrinology of pregnancy. Chicago: Year Book, 1981:1-34. 19. Maruo T, Matsuo H, Hayashi M, Nishino R, Mochizuki M. Induction of differentiated trophoblast function by epidermal growth factor: relation of immunohistochemically detected cellular epidermal growth factor receptor levels. J Clin Endocrinol Metab 1987;64:744-9. 20. Ladines-Llave CA, Maruo T, Manalo AS, Mochizuki M. Cytologic localization of epidermal growth factor and its
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receptor in developing human placenta varies over the course of pregnancy. AM J OBSTET GVNECOL 1991; 165: 1377-82. 21. Kao LC, Caltabiano S, Wu S, Strauss JF III, Kliman Hl The human villous cytotrophoblast: interactions with extracellular matrix proteins, endocrine function and cytoplasmic differentiation in the absence of syncytium formation. Dev Bioi 1988; 130:693-702.
Transforming growth factor-J3 opposes the stimulatory effects of interleukin-l and tumor necrosis factor on amnion cell prostaglandin E2 production: Implication for preterm labor Kristina Bry, MD, and Mikko Hallman, MD Irvine, California, and Helsinki, Finland OBJECTIVE: In preterm labor increased concentrations of interleukin-' and tumor necrosis factor are present in amniotic fluid. These cytokines may promote labor by stimulating the production of prostaglandins by intrauterine tissues. In many biologic processes, transforming growth factor-13 modifies the actions of cytokines. We studied the effect of transforming growth factor-J3 on the cytokine-induced prostaglandin E2 production by amnion cells. STUDY DESIGN: Human amnion cells in monolayer culture were treated with interleukin-1, tumor necrosis factor, or vehicle in the presence or absence of transforming growth factor-I3. The prostaglandin E2 production was measured. RESULTS: Transforming growth factor-13 decreased the interleukin-1- or tumor necrosis factor-induced prostaglandin E2 production by 70% to 80% and the basal prostaglandin E2 synthesis by 27%. The synergistic stimulation of prostaglandin E2 production by the combination of interleukin-1 with tumor necrosis factor was inhibited by 80% in cells treated with transforming growth faclor-l3. Transforming growth factor-j31, -132, and -131,2 were equipotent. CONCLUSION: Transforming growth factor-j3 suppresses the cytokine-induced prostaglandin E2 production by amnion cells and may be an important factor in maintaining pregnancy in the face of labor-promoting cytokines. (AM J OBSTET GVNECOL 1992;167:222-6.)
Key words: Preterm labor, prostaglandins, interleukin-l, tumor necrosis factor, transforming growth factor-/3, fetal membranes Preterm labor often occurs in the setting of intrauterine infection. I The concentrations of cytokines such as interleukin-l (IL-l) and tumor necrosis factor (TNF) increase in amniotic fluid in pre term labor.2~4 ProstaFrom the Department of Pediatrics, University of California, [rome, and the Department of Pediatrics, University of Helsinki. Supported by the Foundation for Maternal and Infant Care and the Sigrid Juselius Foundation. Received for publication October 14, 1991; revised January 21, 1992; accepted January 23,1992. Reprint requests: Kristina Bry, MD, DepaTtment of Pediatrics, University of California, Imine, Med. SUTge /.. Rm 109 F, [mine, CA 92717. 611/36682
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gland ins, major inducers of uterine contractions, are produced by intrauterine tissues in response to these cytokines. 3 5·7 The amnion, which produces almost exclusively prostaglandin £2 (PG£2),8 is an important intrauterine source of prostaglandin. The combination of [L-l with TNF has a synergistic stimulator effect on the PGE 2 production by human amnion cells in culture.~ IL-I and TNF have been implicated in the induction of preterm labor associated with infections."' 3, 10 Transforming growth fac10r-/3 (TGF-/3) represents a family of multifunctional factors that influence cell growth, differentiation, immunity, and extracellular matrix formation. II Several forms of TGF -/3 and of
