European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 172–179
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Transient CD15-positive endothelial phenotype in the human placenta correlates with physiological and pathological fetoplacental immaturity L. Seidmann a,*, T. Suhan a, R. Unger a, V. Gerein b, C.J. Kirkpatrick a a b
Institute of Pathology, University Medical Centre of the Johannes Gutenberg University, Mainz, Germany Pediatric Clinic, Johann Wolfgang Goethe University, Frankfurt/Main, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 November 2013 Received in revised form 21 June 2014 Accepted 24 June 2014
Objective: Placental growth and villous maturation are critical parameters of placental function at the end of pregnancy. A failure in these processes leads to the development of placental dysfunction, as well as fetal and neonatal mortality and morbidity. The aim of the study was to determine the relevant diagnostic markers associated with pathological placental development. Study design: Forty tissue samples from normal placentas of different gestational age and 68 pathological term placentas with defective villous maturation (GDM, idiopathic IUFD, preeclamsia, HELLP syndrome) comprised the comparative immunohistochemical study (CD15, CD45 and CD34). Positive immunohistochemical reactions were quantitatively assessed in the chorionic plate and vessels of the villi of different histological type. Results: Physiologically immature placentas of the first and second trimester and pathologically immature term placentas were characterized by marked endothelial CD15-immunostaining. A significant loss of CD15-positive endothelium of the placentas was associated with a physiological and accelerated villous maturity. A spatio-temporal correlation was shown for CD15+ endothelial cells (ECs) and the number of CD45+ stromal cells (SCs). A negative temporal correlation was shown for CD15+ ECs and CD15+ myelomonocytes in the fetal blood. CD34 expression in the ECs was stable during the pregnancy. Conclusion: A correlation between a transient CD15-positive endothelial phenotype and a physiological and pathological fetoplacental immaturity was demonstrated. Physiological and accelerated placental maturation was accompanied by a significant disappearance of CD15-positive endothelium. We propose that ‘‘immature’’ CD15+ endothelium is an important diagnostic marker of the physiological and pathological fetoplacental immaturity. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: CD15 Placental pathologies Villous immaturity Endothelium Hematopoiesis Diagnostic marker
Introduction Placental growth and villous maturation are critical parameters of placental function at the end of pregnancy [1]. A failure in these processes leads to the development of placental pathologies, placental dysfunction, fetal and neonatal mortality and morbidity [1–4]. Reliable diagnostic tests of placental pathologies remain a major clinical challenge [4].
* Corresponding author. Tel.: +49 6131 172691.. E-mail addresses: [email protected], [email protected] (L. Seidmann). http://dx.doi.org/10.1016/j.ejogrb.2014.06.022 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Placental growth and villous development undergo significant changes during a physiological pregnancy [1]. Physiological immaturity in the first and second trimester is characterized by the development of the immature villi which provide placental growth [1,2]. Physiological villous maturation in late pregnancy (third trimester) is accompanied by restriction of the growth potential and primary development of terminal villi providing the respiratory capacity of the placenta [1,5]. Two main contrasting types of developmental disturbances of placental growth and maturation in the third trimester—accelerated and delayed maturation—have been described [1,2,6]. Pathologically accelerated villous maturation is characterized by a deficiency of immature villi with growth restriction and a predominant differentiation of terminal villi [1,2]. Maternal
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morphological examination. The main clinical and morphological data of the placentas are given in the Table 1. Excess placental material was used in these studies in accordance with the regulations of the Local Ethical Committee. In all cases, advanced written informed consent was obtained from the pregnant women for tissue used in these studies. Placental tissues were fixed in 4% neutral buffered formalin. Macroscopic and histological observations of the placenta were performed according to the Vogel principles (1996) [5]. Histological criteria of villous development and maturation were the degree of branching, stromal differentiation, vascularisation and the formation of syncytiocapillary membranes [1,5]. All normal placentas were without any macroscopic or histological abnormalities. Placentas with pathological villous maturation were divided into 2 groups: placentas with persisting villous immaturity and a deficiency of terminal villi; placentas with accelerated villous maturation, a deficiency of immature intermediate villi and an increased differentiation of the terminal villi.
preeclampsia, HELLP syndrome and about 50% of preterm deliveries are associated with this defect of maturity [1,2]. Pathologically delayed maturation or persisting villous immaturity is characterized by a predominance of immature villi, intensive growth potential and a deficiency of terminal villi [1,2]. The etiology and pathophysiology of villous immaturity are unknown. Important risk factors are gestational diabetes mellitus (GDM), viral diseases and postterm pregnancy [1–3]. About 50% of cases are idiopathic villous immaturity with a latent course of the placental insufficiency and risk of an unexpected antenatal decompensation involving fetal hypoxia (38%) and intrauterine fetal death (IUFD, 9%,) [1–3]. The fetoplacental endothelium is an essential component of placental development. Furthermore, phenotypic changes in the endothelium at different gestational ages may affect fetoplacental development [7]. In the past years increasing evidence indicates that hematopoietic function of the placenta in the presence of the phenotypic peculiarities of placental endothelium is related to the presence of transient placental hematopoiesis [8–10]. The role of endothelium in the fetoplacental interactions and maturation is still unclear. Endothelial CD15-expression is a unique phenomenon described in the fetoplacental endothelium and coincides in time with the expression of placental hematopoiesis [10–12]. CD15 (called Lewis X and stage-specific embryonic antigen 1) is a carbohydrate adhesion molecule that can be expressed on glycoproteins, glycolipids and proteoglycans [13,14]. CD15 acts as a key ligand of selectin and plays an important role in adhesion, migration and differentiation of the cells in the embryo–fetal tissues. CD15 was identified in stem cells, myeloid cells, mature blood cells and tumor cells [14–20]. CD15 is expressed in some tissues, such as epithelial cells of intestinal tissues, certain neurons and glial cells in the central nervous system. In human leukocytes, CD15 is expressed preferentially in monocytes, mature neutrophils and all myeloid cells from the promyelocyte stage onwards [21]. The aim of the study was to determine the relevant diagnostic marker associated with pathological placental development.
Immunohistochemistry For immunohistochemistry, 3 mm sections were deparaffinized, rehydrated and washed with PBS. An automated immunostaining was performed (Autostainer1, DAKO) using a labeled streptavidin-biotin immunoenzymatic antigen detection system (DAKO EnVision, System- HRP mouse/rabbit) according to the manufacturer’s instructions. 3,3-Diaminobenzidine tetrahydrochloride was used as chromogen. The following antibodies were used. Mouse monoclonal IgM against human CD15 (Ready-to-Use, DAKO, clone Carb-3). Mouse monoclonal IgG against human CD34 (Ready-to-Use, DAKO, clone QBEnd 10; antibodies reacts with human hematopoietic progenitor cells, including myeloid and lymphoid progenitors, labels capillaries of most tissues [22]). Mouse monoclonal IgG against human CD45 (Ready-to-Use, DAKO, clones 2B11 and PD7/26; interacts with all hematopoietic cells from HSCs to mature blood cells [23]). For negative controls, the primary antibodies were substituted with the PBS. Immunohistochemical reactions of endothelial and stromal cells were differentially assessed in the chorionic plate and in the vessels of the villi of different histological type. Stained vessels were counted per 100 vessels of the chorionic plate, stem villi, immature and mature villi. Their ratio was calculated as percentage of positively stained vessels. Positive reaction of ECs in the vessel was graded as: ‘‘++’’ all ECs of the vessel were stained; ‘‘+’’ individual ECs were stained. Staining of the SCs was following: ‘‘ ’’ 0 cells high-power fields (HPF), ‘‘+’’ 1–10 cells per HPF, ‘‘++’’ more than 10 cell per HPF, ‘‘+++’’ more than 20 cell per HPF.
Materials and methods Tissue specimens and histology Hundred and eight tissue samples were obtained from normal and pathological placentas of different gestation age, which underwent examination in the Department of Pathology, University Mainz from June 2010 to September 2013. Gestational ages were checked by ultrasound, as well as by clinical and
Table 1 Main clinical and morphological data of the placentas. Physiological villous immaturity
Physiological villous maturity
Pathological persisting villous immaturity
Pathological accelerated villous maturation
Trimester (N) Gestation age (weeks) Weight (g) Clinical diagnosis
I (14) 8–11 Not define Abruptio (14)
III (14) 40–41 420 46 Spontan Partus (14)
Pathological cardio-tocography in antepartum Caesarean section Fetal development Infections Embryonic/Fetal malformations Features of an umbilical cord
Not define
II (12) 16–21 170 47 Late abortion (6), Cervical incompetence (6) Not define
0
III (54) 38–40 590 31 Antenatal fetal hypoxia (54) GDM (20), idiopathic IUFD (5) 54
III (14) 38–40 371 100 Preeklampsia (8), HELLP (6) 11
0 Norm No No No
0 Norm No No No
0 Norm No No No
12 Macrosomia (4) No No No
6 IUGR (5) No No No
GDM—gestational diabetes mellitus, IUFD—Intrauterine fetal death, IUGR—Intrauterine growth restriction
[(Fig._1)TD$IG]
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Statistical analysis Statistical analysis was performed by the Chi square test using StatSoft STATISTICA 6. The data are presented as the mean SD. A p value of 50%) [2]. There was also a significant decrease in the number of the terminal villi (Fig. 5a). In this group we found the most pronounced increase of CD15+ vessels in all types of placental villi (Figs. 5b and 7b). CD15+ and CD45+ cells were observed in the fetal and maternal blood (Fig. 6d, Table 3). In addition, there was an increased number of CD45+ villous SCs compared with normal placentas (Table 3). All villous ECs expressed CD34 (Table 2).
villi. In the mature villi perivascular pericyte-like CD45+ cells were found in the mature villi (Table 3). Expression of CD15, CD34 and CD45 markers in the placentas of the third trimester with pathological accelerated villous maturity Placentas from pathological pregnancies (HELLP syndrome, preeclampsia) demonstrated villous maturity with a relatively increased number of terminal villi (>60%) (Fig. 4a). The number of immature villi was decreased. Only single ECs of the mature peripheral villi showed weak CD15-staining (Figs. 4b and 7b). The macrovascular endothelium of the arteries of the chorionic plate and stem villi was mainly CD15-negative. CD15+ and CD45+ cells were observed in the fetal and maternal blood (Figs. 4b and 6c, Table 3). All vessel structures expressed CD34 (Table 2). Low numbers of CD45+ SCs were observed in the stroma of mature and immature villi (Table 3).
Comments
Expression of CD15, CD34 and CD45 markers in the placentas of the third trimester with pathological persisting villous immaturity
We have used immunohistochemical staining of the cell surface marker CD15 as a method to evaluate placental tissue at varying stages of physiological and pathological development. In the present study we showed the association of CD15+ placental endothelium with physiological and pathological villous immaturity and the gradual stage-specific loss of CD15+ endothelium in the process of villous maturation. We have also demonstrated that the expression of CD15 by ECs reflects the physiological villous immaturity in the first and second trimesters. In early pregnancy (first trimester) almost 100% of placental ECs express CD15. Endothelium of thin-walled immature
[(Fig._4)TD$IG]
Placentas from the next group of pathological pregnancies (GDM, unexplained antenatal placental insufficiency and
[(Fig._5)TD$IG]
Fig. 4. ‘‘Mature’’ CD15-negative endothelial phenotype of the pathological placenta with accelerated villous maturity (39 weeks, HELLP syndrome). (a) Accelerated villous maturity of the placenta with a predominance of terminal villi (arrows) and deficiency of immature villi. Hematoxylin and eosin staining. (b) CD15-negative endothelium of the accelerated mature placenta (arrows). CD15- positive myelomonocytic cells of the fetal blood .Immunohistochemical staining for CD15.
Fig. 5. ‘‘Immature’’ CD15-positive endothelial phenotype of the pathological placenta with persisting villous immaturity (40 weeks, idiopathic IUFD). (a) Villous immaturity of the placenta with a predominance of immature villi (arrows) and deficiency of terminal villi. Hematoxylin and eosin staining. (b) CD15-positive endothelium of macro- and microvascular vessels in the immature placenta (arrows). Immunohistochemical staining against CD15.
[(Fig._6)TD$IG]
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Fig. 6. Temporal expression of CD15 in the fetal blood of normal and pathological placentas of different gestation age (immunohistochemical staining against CD15). (a) CD15negative cells in the fetal blood of the normal placentas from the first trimester (arrows); CD15-positive endothelium of the fetoplacental vessel (double arrows). (b) CD15positive myelomonocytic cells in the fetal blood in the term placenta with physiological maturation (arrows); CD15-negative endothelium (double arrows). (c) CD15-positive myelomonocytic cells in the fetal blood in the term placenta with accelerated maturation (arrows); CD15-negative endothelium of the fetoplacental vessel (double arrows). (d) CD15-positive myelomonocytic cells in the fetal blood of the term placentas with persisting villous immaturity (GDM) (arrows). CD15-positive endothelium of the fetoplacental vessel.
[(Fig._7)TD$IG]
Fig. 7. Spatial-temporal expression of CD15 in the endothelium of normal placentas of different gestation age and pathological term placentas with defective villous maturation. (a) Spatial–temporal CD15-expression in t normal placentas (I, II, and III trimesters). (b) Spatial–temporal CD15 expression in the term placenta (III trimester) from normal and pathological pregnancies with pathological villous maturation (Group of pathological immaturity: gestational diabetes mellitus, idiopathic intrauterine fetal death, and antenatal fetal hypoxia; group of acceleration: preeclampsia, HELLP-syndrome). Immunohistochemical reaction of endothelial and stromal cells was differentially assessed in the chorionic plate and in the vessels of the villi of different histological type. Stained vessels were counted per 100 vessels of the chorionic plate, stem villi, immature and mature villi. The data are presented as the mean SD. Statistical analysis was done by the Statistical analysis was done by Chi square test using StatSoft STATISTICA (Version 6). ** statistically significant difference versus normal placentas from the first trimester (p < 0.05); ^^ statistically significant difference versus normal term placentas (p < 0.05).
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vessels revealed CD15-immunostaining in the chorionic plate and in all types of immature villi. The endothelial phenotype of immature placenta in the second trimester notably differs from those in the first trimester. A considerable loss of CD15+ endothelium in the arteries of the chorionic plate and stem villi with a well-defined muscle layer of the vessel was observed. Microvascular ECs maintain distinctive CD15+ expression in the thin-walled, mostly subepithelial vessels of the immature villi. The data also demonstrate that the physiological villous maturation at the end of the third trimester is accompanied by a significant loss of CD15-expression by ECs in all vessels of the chorionic plate and placental villi. Only a very small number of CD15+ ECs are stained in all villous vessels including the mature microvascular segment. Thus, physiological villous and vascular immaturity is characterized by a strong expression of endothelial CD15. Villous and vascular maturity is associated with significant loss of endothelial CD15 expression. An interesting finding was that macrovascular endothelial cells of mature arteries are already lacking CD15 expression in the second trimester, whilst by contrast ECs of the peripheral segments of the vessels show strong CD15 expression. We assume that loss of CD15 on the placental endothelium reflects a biphasic fetoplacental angiogenesis with distinctive changes in vascular content and arrangement occurring around mid-gestation [6,24]. In the present study we observed that placental dysmaturity during the pathological pregnancy is accompanied by the aberration of endothelial CD15-immunophenotype as compared with normal pregnancies. Clinically relevant forms of persisting villous immaturity complicated with fetal hypoxia were associated with pronounced persistent CD15-expression in the placental endothelium. Significantly increased CD15-expression was shown in the macro- and microvascular endothelium of all villi compared with normal placentas. We also examined placentas with a pathologically accelerated villous maturation associated with maternal preeclampsia and HELLP-syndrome. These placentas were characterized by a notable decrease of CD15-expression in the ECs in contrast to normal placentas. Furthermore, a spatial–temporal correlation of CD15-positive endothelium with accumulation of hematopoietic CD45-positive SCs was shown along with a lack of mature CD15+ myelomonocytic cells in the fetal blood. We observed a distinct accumulation of CD45+ SCs in the chorionic plate and in all types of immature villi during the first and second trimesters and in the placentas with pathological immaturity. Fetal blood in the first and second trimester reflects a phase of transient extramedullary hematopoiesis and is still free of CD15+ myelomonocytic cells [25]. We have also shown that villous maturation and loss of CD15+ ECs are accompanied by low accumulation of hematopoietic CD45+ SCs and distinct appearance of the CD15+ myelomonocytic cells in the fetal blood. In the physiological and accelerated mature term placentas CD45+ SCs were detected mainly in the stem and immature villi and only a small number was observed in the mature villi. In the chorionic plate of the mature placental tissue there were no CD45+ SCs or their amount was significantly decreased compared to the placentas of the first and second trimesters. Conspicuous CD15+ cell population (myelomonocytic cells) appears in the fetal blood in the third trimester. In recent years, there has been increasing evidence of a hematopoietic potential of the placenta and its participation in the development of embryonic and fetal hematopoiesis, associated with the accumulation in the stromal CD45+ hematopoietic stem and progenitor cells [9–11,26–29]. We suggest that CD15+ endothelium marks a transitory vascular niche of ‘‘extraembryonic’’
placental hematopoiesis and mirrors functional immaturity of the fetal myeloid hematopoiesis. Similar to the CD15+ endothelium of the infantile hemangioma, CD15+ placental endothelium may also have its own hematogenic potential [30–32]. We have analyzed the expression of CD34 in the endothelium and this revealed intensive staining of the ECs in all pregnancy trimesters. Thus, the expression of CD34 by ECs does not reflect a regulation occurring during placental development. In conclusion, the present study demonstrates the association of a transient endothelial CD15+ phenotype with physiological and pathological placental villous immaturity. Fetoplacental maturation at the end of the pregnancy correlates with a disappearance of CD15+ endothelium in the placental vessels. We propose ‘‘immature’’ CD15+ endothelium as an important diagnostic marker of the physiological and pathological clinically relevant fetoplacental immaturity. The presence of the endothelial CD15+ phenotype in pathological pregnancies warrants further investigation. Condensation We demonstrate the correlation of the placental CD15+ endothelial phenotype with a physiological and pathological fetoplacental immaturity. Acknowledgments The authors thank Anne Sartoris for her excellent technical assistance. This work was kindly supported by the foundation ‘‘Dr. Valentin Gerein Stiftung–Hilfe fu¨r krebskranke Kinder, Jugendliche, junge Erwachsene’’ (Germany). References [1] Benirschke K, Kaufmann P, Baergen R. Pathology of the human placenta. 5th ed, New York: Springer; 2006. [2] Vogel M. Pathologie der Plazenta: Spa¨tschwangerschaft und fetoplazentare Einheit. In: Klo¨ppel G, Kreipe H, Remmele W, editors. Pathologie,. Heidelberg: Springer-Verlag; 2013. p. 519–39. [3] Stallmach T, Hebisch G. Rescue by birth: defective placental maturation and late fetal mortality obstetrics. Gynecology 2001;4:505–9. [4] Roescher AM, Timmer A, Hitzert MM, et al. Placental pathology and neurological morbidity in preterm infants during first two weeks after birth. Early Hum Dev 2014;1:21–5. [5] Vogel M. Atlas der morphologischen Plazentadiagnostik. 2nd ed, Heidelberg: Springer-Verlag; 1996. [6] Seidmann L, Suhan T, Unger R, Gerein V, Kirkpatrick CJ. Imbalance of expression of bFGF and PK1 is associated with defective maturation and antenatal placental insufficiency. Eur J Obstet Gynecol Reprod Biol 2013;170:352–7. [7] Wadsack C, Desoye G, Hiden U. The feto-placental endothelium in pregnancy pathologies. Wien Med Wochenshr 2012;162:220–4. [8] Gekas C, Dieterlen-Lievre F, Orkin SH, Mikkola HK. The placenta is a niche for hematopoietic stem cells. Dev Cell 2005;8:365–75. 2005. [9] Rhodes KE, Gekas C, Wang Y, et al. The emergence of hematopoietic stem cells is initiated in the placenta vasculature in the absence of circulation. Cell Stem Cell 2008;2:252–63. [10] Robin C, Bollerot K, Mendes S, et al. Human placenta is potent hematopoietic niche containing hematopoietic stem and proginetor cells throughout development. Cell Stem Cell 2009;5:386–95. [11] Challier JC, Galtier M, Cortez A, Bintein T, Rabreau M, Uzan S. Immunocytological evidence for hematopoiesis in the early human placenta. Placenta 2005;26:282–8. [12] Samokhvalov IM. Deconvoluting the ontogeny of the hematopoietic stem cells. Cell Mol Life Sci 2014;71:957–78. [13] Stocks SC, Kerr MA. Stimulation of neutrophil adhesion by antibodies recognizing CD15 (Le(X)) and CD15-expressing carcinoembryonic antigen-related glycoprotein NCA-160. Biochem J 1992;288:23–7. [14] Fox N, Damjanov I, Knowles BB, Solter D. Immunohistochemical localization of the mouse stage-specific embryonic antigen 1 in human tissues and tumors. Cancer Res 1983;43:669–78. [15] Hounsell EF, Gooi HC, Feizi T. The monoclonal antibody anti-SSEA-1 discriminates between fucosylated type 1 and type 2 blood group chains. FEBS Lett 1981;131:279–82. [16] Fox N, Damjanov I, Martinez-Hernandez A, Knowles BB, Solter D. Immunohistochemical localization of the early embryonic antigen (SSEA-1) in postimplantation mouse embryos and fetal and adult tissues. Dev Biol 1981;83:391–8.
L. Seidmann et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 172–179 [17] Solter D, Knowles BB. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci USA 1978;75:5565–9. [18] Capela A, Temple S. LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal. Neuron 2002;35:865–75. [19] Imura T, Nakano I, Kornblum HI, Sofroniew MV. Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/ CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes. Glia 2006;53:277–93. [20] Masat T, Feliu E, Villamor N, et al. Immunophenotypic and ultrastructural study in peripheral blood neutrophil granulocytes following bone marrow transplantation. Br J Haematol 1997;98:299–307. [21] Nakayama F, Nishihara S, Iwasaki H, et al. CD15 expression in mature granulocytes is determined by alpha 1,3-fucosyltransferase IX, but in promyelocytes and monocytes by alpha 1,3-fucosyltransferase IV. J Biol Chem 2001;276:16100–06. [22] Thomas ML. The leukocyte common antigen family. Annu Rev Immunol 1989;7:339–69. [23] Choi KD, Vodyanik M, Slukvin II. Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat Protoc 2011;6:296–313. [24] Mathew TM. Fetoplacentla angiogenesis during gestation is biphasic, longitudinal and occurs by proliferation and remodeling of vascular endothelial cells. Placenta 2002;10:742–50.
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[25] Marshall CJ, Thrasher AJ. The embryonic origins of human haematopoiesis. Br J Haematol 2001;112:838–50. [26] Ottersbach K, Dzierzack E. The placenta as a haematopoietic organ. Int J Dev Biol 2010;54:1099–106. [27] Mei-Dan E, Fainaru O, Hantisteanu S, Shavit T, Ellenbogen A, Hallak M. Proangiogenic immature myeloid cells populate the human placenta and their presence correlates with placental and birth weight. Am J Obstet Gynecol 2012;207:141–5. [28] Ba´rcena A, Kapidzic M, Muench MO, et al. The human placenta is a hematopoietic organ during the embryonic and fetal periods of development. Dev Biol 2009;327(1):24–33. [29] Martinez-Agosto JA, Mikkola HK, Hartenstein V, Banerjee U. The hematopoietic stem cell and its niche: a comparative view. Genes Dev 2007;21: 3044–60. [30] North PE, Waner M, Mizeracki A, et al. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch Dermatol 2001;137:559–70. [31] Itinteang T, Tan ST, Brasch H, Day DJ. Haemogenic endothelium in infantile haemangioma. J Clin Pathol 2010;63:982–6. [32] Barnes CM, Huang S, Kaipainen A, et al. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc Natl Acad Sci USA 2005;102: 19097–102.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Transient CD15-positive endothelial phenotype in the human placenta correlates with physiological and pathological fetoplacental immaturity L. Seidmann a,*, T. Suhan a, R. Unger a, V. Gerein b, C.J. Kirkpatrick a a b
Institute of Pathology, University Medical Centre of the Johannes Gutenberg University, Mainz, Germany Pediatric Clinic, Johann Wolfgang Goethe University, Frankfurt/Main, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 November 2013 Received in revised form 21 June 2014 Accepted 24 June 2014
Objective: Placental growth and villous maturation are critical parameters of placental function at the end of pregnancy. A failure in these processes leads to the development of placental dysfunction, as well as fetal and neonatal mortality and morbidity. The aim of the study was to determine the relevant diagnostic markers associated with pathological placental development. Study design: Forty tissue samples from normal placentas of different gestational age and 68 pathological term placentas with defective villous maturation (GDM, idiopathic IUFD, preeclamsia, HELLP syndrome) comprised the comparative immunohistochemical study (CD15, CD45 and CD34). Positive immunohistochemical reactions were quantitatively assessed in the chorionic plate and vessels of the villi of different histological type. Results: Physiologically immature placentas of the first and second trimester and pathologically immature term placentas were characterized by marked endothelial CD15-immunostaining. A significant loss of CD15-positive endothelium of the placentas was associated with a physiological and accelerated villous maturity. A spatio-temporal correlation was shown for CD15+ endothelial cells (ECs) and the number of CD45+ stromal cells (SCs). A negative temporal correlation was shown for CD15+ ECs and CD15+ myelomonocytes in the fetal blood. CD34 expression in the ECs was stable during the pregnancy. Conclusion: A correlation between a transient CD15-positive endothelial phenotype and a physiological and pathological fetoplacental immaturity was demonstrated. Physiological and accelerated placental maturation was accompanied by a significant disappearance of CD15-positive endothelium. We propose that ‘‘immature’’ CD15+ endothelium is an important diagnostic marker of the physiological and pathological fetoplacental immaturity. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: CD15 Placental pathologies Villous immaturity Endothelium Hematopoiesis Diagnostic marker
Introduction Placental growth and villous maturation are critical parameters of placental function at the end of pregnancy [1]. A failure in these processes leads to the development of placental pathologies, placental dysfunction, fetal and neonatal mortality and morbidity [1–4]. Reliable diagnostic tests of placental pathologies remain a major clinical challenge [4].
* Corresponding author. Tel.: +49 6131 172691.. E-mail addresses: [email protected], [email protected] (L. Seidmann). http://dx.doi.org/10.1016/j.ejogrb.2014.06.022 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Placental growth and villous development undergo significant changes during a physiological pregnancy [1]. Physiological immaturity in the first and second trimester is characterized by the development of the immature villi which provide placental growth [1,2]. Physiological villous maturation in late pregnancy (third trimester) is accompanied by restriction of the growth potential and primary development of terminal villi providing the respiratory capacity of the placenta [1,5]. Two main contrasting types of developmental disturbances of placental growth and maturation in the third trimester—accelerated and delayed maturation—have been described [1,2,6]. Pathologically accelerated villous maturation is characterized by a deficiency of immature villi with growth restriction and a predominant differentiation of terminal villi [1,2]. Maternal
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morphological examination. The main clinical and morphological data of the placentas are given in the Table 1. Excess placental material was used in these studies in accordance with the regulations of the Local Ethical Committee. In all cases, advanced written informed consent was obtained from the pregnant women for tissue used in these studies. Placental tissues were fixed in 4% neutral buffered formalin. Macroscopic and histological observations of the placenta were performed according to the Vogel principles (1996) [5]. Histological criteria of villous development and maturation were the degree of branching, stromal differentiation, vascularisation and the formation of syncytiocapillary membranes [1,5]. All normal placentas were without any macroscopic or histological abnormalities. Placentas with pathological villous maturation were divided into 2 groups: placentas with persisting villous immaturity and a deficiency of terminal villi; placentas with accelerated villous maturation, a deficiency of immature intermediate villi and an increased differentiation of the terminal villi.
preeclampsia, HELLP syndrome and about 50% of preterm deliveries are associated with this defect of maturity [1,2]. Pathologically delayed maturation or persisting villous immaturity is characterized by a predominance of immature villi, intensive growth potential and a deficiency of terminal villi [1,2]. The etiology and pathophysiology of villous immaturity are unknown. Important risk factors are gestational diabetes mellitus (GDM), viral diseases and postterm pregnancy [1–3]. About 50% of cases are idiopathic villous immaturity with a latent course of the placental insufficiency and risk of an unexpected antenatal decompensation involving fetal hypoxia (38%) and intrauterine fetal death (IUFD, 9%,) [1–3]. The fetoplacental endothelium is an essential component of placental development. Furthermore, phenotypic changes in the endothelium at different gestational ages may affect fetoplacental development [7]. In the past years increasing evidence indicates that hematopoietic function of the placenta in the presence of the phenotypic peculiarities of placental endothelium is related to the presence of transient placental hematopoiesis [8–10]. The role of endothelium in the fetoplacental interactions and maturation is still unclear. Endothelial CD15-expression is a unique phenomenon described in the fetoplacental endothelium and coincides in time with the expression of placental hematopoiesis [10–12]. CD15 (called Lewis X and stage-specific embryonic antigen 1) is a carbohydrate adhesion molecule that can be expressed on glycoproteins, glycolipids and proteoglycans [13,14]. CD15 acts as a key ligand of selectin and plays an important role in adhesion, migration and differentiation of the cells in the embryo–fetal tissues. CD15 was identified in stem cells, myeloid cells, mature blood cells and tumor cells [14–20]. CD15 is expressed in some tissues, such as epithelial cells of intestinal tissues, certain neurons and glial cells in the central nervous system. In human leukocytes, CD15 is expressed preferentially in monocytes, mature neutrophils and all myeloid cells from the promyelocyte stage onwards [21]. The aim of the study was to determine the relevant diagnostic marker associated with pathological placental development.
Immunohistochemistry For immunohistochemistry, 3 mm sections were deparaffinized, rehydrated and washed with PBS. An automated immunostaining was performed (Autostainer1, DAKO) using a labeled streptavidin-biotin immunoenzymatic antigen detection system (DAKO EnVision, System- HRP mouse/rabbit) according to the manufacturer’s instructions. 3,3-Diaminobenzidine tetrahydrochloride was used as chromogen. The following antibodies were used. Mouse monoclonal IgM against human CD15 (Ready-to-Use, DAKO, clone Carb-3). Mouse monoclonal IgG against human CD34 (Ready-to-Use, DAKO, clone QBEnd 10; antibodies reacts with human hematopoietic progenitor cells, including myeloid and lymphoid progenitors, labels capillaries of most tissues [22]). Mouse monoclonal IgG against human CD45 (Ready-to-Use, DAKO, clones 2B11 and PD7/26; interacts with all hematopoietic cells from HSCs to mature blood cells [23]). For negative controls, the primary antibodies were substituted with the PBS. Immunohistochemical reactions of endothelial and stromal cells were differentially assessed in the chorionic plate and in the vessels of the villi of different histological type. Stained vessels were counted per 100 vessels of the chorionic plate, stem villi, immature and mature villi. Their ratio was calculated as percentage of positively stained vessels. Positive reaction of ECs in the vessel was graded as: ‘‘++’’ all ECs of the vessel were stained; ‘‘+’’ individual ECs were stained. Staining of the SCs was following: ‘‘ ’’ 0 cells high-power fields (HPF), ‘‘+’’ 1–10 cells per HPF, ‘‘++’’ more than 10 cell per HPF, ‘‘+++’’ more than 20 cell per HPF.
Materials and methods Tissue specimens and histology Hundred and eight tissue samples were obtained from normal and pathological placentas of different gestation age, which underwent examination in the Department of Pathology, University Mainz from June 2010 to September 2013. Gestational ages were checked by ultrasound, as well as by clinical and
Table 1 Main clinical and morphological data of the placentas. Physiological villous immaturity
Physiological villous maturity
Pathological persisting villous immaturity
Pathological accelerated villous maturation
Trimester (N) Gestation age (weeks) Weight (g) Clinical diagnosis
I (14) 8–11 Not define Abruptio (14)
III (14) 40–41 420 46 Spontan Partus (14)
Pathological cardio-tocography in antepartum Caesarean section Fetal development Infections Embryonic/Fetal malformations Features of an umbilical cord
Not define
II (12) 16–21 170 47 Late abortion (6), Cervical incompetence (6) Not define
0
III (54) 38–40 590 31 Antenatal fetal hypoxia (54) GDM (20), idiopathic IUFD (5) 54
III (14) 38–40 371 100 Preeklampsia (8), HELLP (6) 11
0 Norm No No No
0 Norm No No No
0 Norm No No No
12 Macrosomia (4) No No No
6 IUGR (5) No No No
GDM—gestational diabetes mellitus, IUFD—Intrauterine fetal death, IUGR—Intrauterine growth restriction
[(Fig._1)TD$IG]
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Statistical analysis Statistical analysis was performed by the Chi square test using StatSoft STATISTICA 6. The data are presented as the mean SD. A p value of 50%) [2]. There was also a significant decrease in the number of the terminal villi (Fig. 5a). In this group we found the most pronounced increase of CD15+ vessels in all types of placental villi (Figs. 5b and 7b). CD15+ and CD45+ cells were observed in the fetal and maternal blood (Fig. 6d, Table 3). In addition, there was an increased number of CD45+ villous SCs compared with normal placentas (Table 3). All villous ECs expressed CD34 (Table 2).
villi. In the mature villi perivascular pericyte-like CD45+ cells were found in the mature villi (Table 3). Expression of CD15, CD34 and CD45 markers in the placentas of the third trimester with pathological accelerated villous maturity Placentas from pathological pregnancies (HELLP syndrome, preeclampsia) demonstrated villous maturity with a relatively increased number of terminal villi (>60%) (Fig. 4a). The number of immature villi was decreased. Only single ECs of the mature peripheral villi showed weak CD15-staining (Figs. 4b and 7b). The macrovascular endothelium of the arteries of the chorionic plate and stem villi was mainly CD15-negative. CD15+ and CD45+ cells were observed in the fetal and maternal blood (Figs. 4b and 6c, Table 3). All vessel structures expressed CD34 (Table 2). Low numbers of CD45+ SCs were observed in the stroma of mature and immature villi (Table 3).
Comments
Expression of CD15, CD34 and CD45 markers in the placentas of the third trimester with pathological persisting villous immaturity
We have used immunohistochemical staining of the cell surface marker CD15 as a method to evaluate placental tissue at varying stages of physiological and pathological development. In the present study we showed the association of CD15+ placental endothelium with physiological and pathological villous immaturity and the gradual stage-specific loss of CD15+ endothelium in the process of villous maturation. We have also demonstrated that the expression of CD15 by ECs reflects the physiological villous immaturity in the first and second trimesters. In early pregnancy (first trimester) almost 100% of placental ECs express CD15. Endothelium of thin-walled immature
[(Fig._4)TD$IG]
Placentas from the next group of pathological pregnancies (GDM, unexplained antenatal placental insufficiency and
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Fig. 4. ‘‘Mature’’ CD15-negative endothelial phenotype of the pathological placenta with accelerated villous maturity (39 weeks, HELLP syndrome). (a) Accelerated villous maturity of the placenta with a predominance of terminal villi (arrows) and deficiency of immature villi. Hematoxylin and eosin staining. (b) CD15-negative endothelium of the accelerated mature placenta (arrows). CD15- positive myelomonocytic cells of the fetal blood .Immunohistochemical staining for CD15.
Fig. 5. ‘‘Immature’’ CD15-positive endothelial phenotype of the pathological placenta with persisting villous immaturity (40 weeks, idiopathic IUFD). (a) Villous immaturity of the placenta with a predominance of immature villi (arrows) and deficiency of terminal villi. Hematoxylin and eosin staining. (b) CD15-positive endothelium of macro- and microvascular vessels in the immature placenta (arrows). Immunohistochemical staining against CD15.
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Fig. 6. Temporal expression of CD15 in the fetal blood of normal and pathological placentas of different gestation age (immunohistochemical staining against CD15). (a) CD15negative cells in the fetal blood of the normal placentas from the first trimester (arrows); CD15-positive endothelium of the fetoplacental vessel (double arrows). (b) CD15positive myelomonocytic cells in the fetal blood in the term placenta with physiological maturation (arrows); CD15-negative endothelium (double arrows). (c) CD15-positive myelomonocytic cells in the fetal blood in the term placenta with accelerated maturation (arrows); CD15-negative endothelium of the fetoplacental vessel (double arrows). (d) CD15-positive myelomonocytic cells in the fetal blood of the term placentas with persisting villous immaturity (GDM) (arrows). CD15-positive endothelium of the fetoplacental vessel.
[(Fig._7)TD$IG]
Fig. 7. Spatial-temporal expression of CD15 in the endothelium of normal placentas of different gestation age and pathological term placentas with defective villous maturation. (a) Spatial–temporal CD15-expression in t normal placentas (I, II, and III trimesters). (b) Spatial–temporal CD15 expression in the term placenta (III trimester) from normal and pathological pregnancies with pathological villous maturation (Group of pathological immaturity: gestational diabetes mellitus, idiopathic intrauterine fetal death, and antenatal fetal hypoxia; group of acceleration: preeclampsia, HELLP-syndrome). Immunohistochemical reaction of endothelial and stromal cells was differentially assessed in the chorionic plate and in the vessels of the villi of different histological type. Stained vessels were counted per 100 vessels of the chorionic plate, stem villi, immature and mature villi. The data are presented as the mean SD. Statistical analysis was done by the Statistical analysis was done by Chi square test using StatSoft STATISTICA (Version 6). ** statistically significant difference versus normal placentas from the first trimester (p < 0.05); ^^ statistically significant difference versus normal term placentas (p < 0.05).
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vessels revealed CD15-immunostaining in the chorionic plate and in all types of immature villi. The endothelial phenotype of immature placenta in the second trimester notably differs from those in the first trimester. A considerable loss of CD15+ endothelium in the arteries of the chorionic plate and stem villi with a well-defined muscle layer of the vessel was observed. Microvascular ECs maintain distinctive CD15+ expression in the thin-walled, mostly subepithelial vessels of the immature villi. The data also demonstrate that the physiological villous maturation at the end of the third trimester is accompanied by a significant loss of CD15-expression by ECs in all vessels of the chorionic plate and placental villi. Only a very small number of CD15+ ECs are stained in all villous vessels including the mature microvascular segment. Thus, physiological villous and vascular immaturity is characterized by a strong expression of endothelial CD15. Villous and vascular maturity is associated with significant loss of endothelial CD15 expression. An interesting finding was that macrovascular endothelial cells of mature arteries are already lacking CD15 expression in the second trimester, whilst by contrast ECs of the peripheral segments of the vessels show strong CD15 expression. We assume that loss of CD15 on the placental endothelium reflects a biphasic fetoplacental angiogenesis with distinctive changes in vascular content and arrangement occurring around mid-gestation [6,24]. In the present study we observed that placental dysmaturity during the pathological pregnancy is accompanied by the aberration of endothelial CD15-immunophenotype as compared with normal pregnancies. Clinically relevant forms of persisting villous immaturity complicated with fetal hypoxia were associated with pronounced persistent CD15-expression in the placental endothelium. Significantly increased CD15-expression was shown in the macro- and microvascular endothelium of all villi compared with normal placentas. We also examined placentas with a pathologically accelerated villous maturation associated with maternal preeclampsia and HELLP-syndrome. These placentas were characterized by a notable decrease of CD15-expression in the ECs in contrast to normal placentas. Furthermore, a spatial–temporal correlation of CD15-positive endothelium with accumulation of hematopoietic CD45-positive SCs was shown along with a lack of mature CD15+ myelomonocytic cells in the fetal blood. We observed a distinct accumulation of CD45+ SCs in the chorionic plate and in all types of immature villi during the first and second trimesters and in the placentas with pathological immaturity. Fetal blood in the first and second trimester reflects a phase of transient extramedullary hematopoiesis and is still free of CD15+ myelomonocytic cells [25]. We have also shown that villous maturation and loss of CD15+ ECs are accompanied by low accumulation of hematopoietic CD45+ SCs and distinct appearance of the CD15+ myelomonocytic cells in the fetal blood. In the physiological and accelerated mature term placentas CD45+ SCs were detected mainly in the stem and immature villi and only a small number was observed in the mature villi. In the chorionic plate of the mature placental tissue there were no CD45+ SCs or their amount was significantly decreased compared to the placentas of the first and second trimesters. Conspicuous CD15+ cell population (myelomonocytic cells) appears in the fetal blood in the third trimester. In recent years, there has been increasing evidence of a hematopoietic potential of the placenta and its participation in the development of embryonic and fetal hematopoiesis, associated with the accumulation in the stromal CD45+ hematopoietic stem and progenitor cells [9–11,26–29]. We suggest that CD15+ endothelium marks a transitory vascular niche of ‘‘extraembryonic’’
placental hematopoiesis and mirrors functional immaturity of the fetal myeloid hematopoiesis. Similar to the CD15+ endothelium of the infantile hemangioma, CD15+ placental endothelium may also have its own hematogenic potential [30–32]. We have analyzed the expression of CD34 in the endothelium and this revealed intensive staining of the ECs in all pregnancy trimesters. Thus, the expression of CD34 by ECs does not reflect a regulation occurring during placental development. In conclusion, the present study demonstrates the association of a transient endothelial CD15+ phenotype with physiological and pathological placental villous immaturity. Fetoplacental maturation at the end of the pregnancy correlates with a disappearance of CD15+ endothelium in the placental vessels. We propose ‘‘immature’’ CD15+ endothelium as an important diagnostic marker of the physiological and pathological clinically relevant fetoplacental immaturity. The presence of the endothelial CD15+ phenotype in pathological pregnancies warrants further investigation. Condensation We demonstrate the correlation of the placental CD15+ endothelial phenotype with a physiological and pathological fetoplacental immaturity. Acknowledgments The authors thank Anne Sartoris for her excellent technical assistance. This work was kindly supported by the foundation ‘‘Dr. Valentin Gerein Stiftung–Hilfe fu¨r krebskranke Kinder, Jugendliche, junge Erwachsene’’ (Germany). References [1] Benirschke K, Kaufmann P, Baergen R. Pathology of the human placenta. 5th ed, New York: Springer; 2006. [2] Vogel M. Pathologie der Plazenta: Spa¨tschwangerschaft und fetoplazentare Einheit. In: Klo¨ppel G, Kreipe H, Remmele W, editors. Pathologie,. Heidelberg: Springer-Verlag; 2013. p. 519–39. [3] Stallmach T, Hebisch G. Rescue by birth: defective placental maturation and late fetal mortality obstetrics. Gynecology 2001;4:505–9. [4] Roescher AM, Timmer A, Hitzert MM, et al. Placental pathology and neurological morbidity in preterm infants during first two weeks after birth. Early Hum Dev 2014;1:21–5. [5] Vogel M. Atlas der morphologischen Plazentadiagnostik. 2nd ed, Heidelberg: Springer-Verlag; 1996. [6] Seidmann L, Suhan T, Unger R, Gerein V, Kirkpatrick CJ. Imbalance of expression of bFGF and PK1 is associated with defective maturation and antenatal placental insufficiency. Eur J Obstet Gynecol Reprod Biol 2013;170:352–7. [7] Wadsack C, Desoye G, Hiden U. The feto-placental endothelium in pregnancy pathologies. Wien Med Wochenshr 2012;162:220–4. [8] Gekas C, Dieterlen-Lievre F, Orkin SH, Mikkola HK. The placenta is a niche for hematopoietic stem cells. Dev Cell 2005;8:365–75. 2005. [9] Rhodes KE, Gekas C, Wang Y, et al. The emergence of hematopoietic stem cells is initiated in the placenta vasculature in the absence of circulation. Cell Stem Cell 2008;2:252–63. [10] Robin C, Bollerot K, Mendes S, et al. Human placenta is potent hematopoietic niche containing hematopoietic stem and proginetor cells throughout development. Cell Stem Cell 2009;5:386–95. [11] Challier JC, Galtier M, Cortez A, Bintein T, Rabreau M, Uzan S. Immunocytological evidence for hematopoiesis in the early human placenta. Placenta 2005;26:282–8. [12] Samokhvalov IM. Deconvoluting the ontogeny of the hematopoietic stem cells. Cell Mol Life Sci 2014;71:957–78. [13] Stocks SC, Kerr MA. Stimulation of neutrophil adhesion by antibodies recognizing CD15 (Le(X)) and CD15-expressing carcinoembryonic antigen-related glycoprotein NCA-160. Biochem J 1992;288:23–7. [14] Fox N, Damjanov I, Knowles BB, Solter D. Immunohistochemical localization of the mouse stage-specific embryonic antigen 1 in human tissues and tumors. Cancer Res 1983;43:669–78. [15] Hounsell EF, Gooi HC, Feizi T. The monoclonal antibody anti-SSEA-1 discriminates between fucosylated type 1 and type 2 blood group chains. FEBS Lett 1981;131:279–82. [16] Fox N, Damjanov I, Martinez-Hernandez A, Knowles BB, Solter D. Immunohistochemical localization of the early embryonic antigen (SSEA-1) in postimplantation mouse embryos and fetal and adult tissues. Dev Biol 1981;83:391–8.
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