Autophagy in Normal and Abnormal Early Human Pregnancies
Reproductive Sciences 1-7 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719114565036 rs.sagepub.com
Laura Avagliano, MD, PhD1, Laura Terraneo, PhD1, Eleonora Virgili, BD1, Carla Martinelli PhD2, Patrizia Doi, MS1, Michele Samaja, PhD1, Gaetano Pietro Bulfamante, MD1, and Anna Maria Marconi, MD1
Abstract Autophagy is an inducible catabolic process by which cells degrade and recycle materials to survive stress, starvation, and hypoxia. The aim of this study was to evaluate autophagy at the fetal–maternal interface, to assess autophagy involvement during the early phase of human gestation, and to explore autophagic modification in case of early abnormal pregnancy outcome. Specimens were collected from first-trimester normal gestations undergoing legal termination of pregnancy and first-trimester sporadic spontaneous miscarriages. Autophagy was studied in villous and decidual samples by transmission electron microscopy, immunohistochemistry, immunofluorescence, and Western blotting. Autophagy markers were found in cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, and decidual stromal cells. Autophagy is physiologically involved in early normal gestation. Compared with normal pregnancy, spontaneous miscarriage presents an increase in autophagy expression in villous specimens due to an increment in concentration of autophagic vacuole in syncytiotrophoblast, suggesting a cytoprotective mechanism of the cells to respond to microenvironmental challenge. Keywords autophagy, apoptosis, pregnancy, trophoblast, miscarriage
Introduction Autophagy is an evolutionary conserved, intracellular inducible catabolic process by which injured organelles, damaged proteins, and pathogens are sequestered into autophagosomes, double membrane-bound vesicles that fuse with lysosomes for proteolytic degradation and material recycling.1 Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of yeast autophagy-related gene 8, intervenes in the late stages of autophagosome formation. Among LC3 isoforms, LC3-II, the phosphatidylethanolamine conjugated product of LC3-I obtained after LC3 activation,1,2 is currently used as a specific marker of autophagy for its role during autophagosome genesis.3 Previous studies have investigated the occurrence of autophagy during the early stages of mammalian pregnancy in the preimplantation period for oocyte-to-embryo transition4-6 but no extensive in vivo assessment of autophagy at the fetomaternal interface after implantation has been performed in humans. At the site of implantation, apoptosis permits placenta development,7 and a relatively hypoxic environment warrants an adequate embryonic and placental growth.8 Although many cellular and molecular events are expected to regulate the various steps of fetomaternal interactions, the mechanisms that regulate the early stage of pregnancy are not yet fully
understood. The aim of the present study was to evaluate the occurrence of autophagy during the first trimester of pregnancy to verify whether it is involved in the early phase of gestation and whether autophagy expression differs between normal pregnancy (NP) and spontaneous miscarriage (SM).
Materials and Methods Sample Collection Normal pregnancies. Tissues were obtained from 10 healthy, nonsmokers women (age 28.5 + 8.1 years) undergoing legal termination of pregnancy within 12 weeks of amenorrhea (mean gestational age 9.4 + 1.1 weeks; range 8-11 weeks); 1 Department of Health Sciences, San Paolo Hospital Medical School, Universita` degli Studi di Milano, Milan, Italy 2 Department of Biomedical Sciences for Health, Universita` degli Studi di Milano, Milan, Italy
Corresponding Author: Laura Avagliano, Unit of Obstetrics and Gynecology, Department of Health Sciences, San Paolo Hospital Medical School. University of Milano, Via A. di Rudinı` 8. 20142 Milano, Italy. Email: [email protected]
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in all cases gestational age and fetal viability were confirmed by ultrasound before suction curettage. All women gave their consent to the study. All procedures were performed in general anesthesia. At the time of suction curettage, the gestational tissues were removed in sterile conditions and macroscopically assessed, through several washes in sterile phosphatebuffered saline solution. For each woman, samples of decidua and villi were collected and stored separately: immediately after the washes, tissues collected for immunohistochemical and immunofluorescence analysis were fixed in 10% formalin solution; samples for electron microscopy were fixed in 2.5% glutaraldehyde; samples for Western blotting were frozen in liquid nitrogen and stored at 80 C for protein extraction. Spontaneous miscarriages. Tissues were obtained from archive specimens from our previous work as described.9 All cases were sporadic miscarriage. Only embryonic pregnancies were included and all cases had normal embryonic karyotype. Smokers were excluded, therefore 10 cases were eligible for this study. Spontaneous miscarriage and NPs were comparable for gestational age (gestational age 9.2 + 0.6 weeks; P ¼ .76) and maternal age (age 30.9 + 4.8 years; P ¼ .43).
Immunohistochemistry Immunohistochemical studies were carried out in all samples (n ¼ 20: 10 NPs and 10 SMs) on 4-mm thick tissue sections as described previously.10-12 Primary antibodies were as follows: rabbit polyclonal LC3 (NB100-2220, Novus Biologicals, Littleton, Colorado; dilution 1:1000), mouse monoclonal antiCK-7 (OV-TL 12/30, Dako, Glostrup, Denmark; dilution 1:100), mouse monoclonal anti-HLA-G (SC-21799, Santa Cruz Biotechnology, Dallas, Texas; dilution 1:2000), and rabbit monoclonal cleaved caspase-3 (#9664, Cell Signaling Technology, Danvers, Massachusetts; dilution 1:800).
Immunofluorescence Immunofluorescence studies were conducted in the same samples used for immunohistochemistry (n ¼ 20: 10 NPs and 10 SMs), as described previously11 using rabbit antiLC3 (NB100-2220, Novus Biologicals, Littleton, Colorado; dilution 1:500). Fluorophore-conjugated secondary antibodies were used and nuclei were subsequently counterstained with 40 ,6diamidino-2-phenylindole (Invitrogen, Carlsbad, California). Fluorescence images were viewed and captured using Imager.Z1 microscope (Zeiss, Feldbach, Switzerland).
Transmission Electron Microscopy Tissues from 8 cases (4 NPs and 4 SMs) were fixed in 2.5% glutaraldehyde in 0.13 mol/L phosphate buffer at pH 7.2-7.4; specimens were postfixed in 1% OsO4, dehydrated in ethanol plus propylene oxide, and embedded in epoxy resin. Ultrathin sections of 50 to 60 nm were counterstained with uranyl acetate and lead citrate. A Jeol JEM 1010 (Tokyo, Japan) electron microscope was used for examination. Cases used for 2
transmission electron microscopy evaluation were a subgroup of the total population due to tissue availability restrictions. Autophagic vacuoles were identified, in villi and decidua, as double membrane compartments containing material in various stages of degradation. For each case, the area of autophagic vacuoles was calculated by 2 observers (LA and CM) on 60000 printed micrographs (previously converted in digital bitmap images by Image Processing and Analysis in Java-ImageJ) in 3 randomly selected areas of villi, both in cytotrophoblast and in syncytiotrophoblast. Quantification of autophagy was measured as a ratio of autophagic vacuole area to the total cytoplasmic area. The mean ratio from the 3 areas was considered as the final value for each case.
Western Blotting Villous (n ¼ 20: 10 NPs and 10 SMs) and decidual (n ¼ 20: 10s NP and 10 SMs) samples were homogenized in a glass potter in a 1:5 ratio (weight:volume) with 50 mmol/L Tris-HCl pH 7.4, 5 mmol/L EDTA, 1 mmol/L DTT, 2% SDS, and Protease Inhibitor Cocktail (Sigma Aldrich, St. Louis, Missouri) and centrifuged at 14 000g for 15 minutes. Protein concentration within the supernatant was measured by the Coomassie Plus Protein Assay reagent Kit (Pierce, Rockford, Illinois). An equal amount of protein from each sample (30 mg) was subjected to electrophoresis in a 10% polyacrylamide gel for LC3 and Bcl2 or in 8% polyacrylamide gel for hypoxia-inducible factor 1a (HIF-1a), caspase-3, Bax, and transferred onto nitrocellulose membrane. Membranes were incubated in a blocking solution (Tris-buffered saline [TBS] containing 5% powdered nonfat milk and 0.01% Tween-20) at room temperature for 1 hour. Membranes were incubated overnight at 4 C with the primary antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibody. The used primary antibodies and dilutions were as follows: rabbit polyclonal anti-HIF-1a (H-206X: sc-10790, Santa Cruz Biotechnology, Dallas, Texas; 1:300 in TBS-Tween 1), rabbit polyclonal LC3 (NB100-2220, Novus Biologicals, Littleton, Colorado; 1:1000 in 5% bovine serum albumin [BSA]), mouse monoclonal Bcl2 (C-2: sc-7382, Santa Cruz Biotechnology, Dallas, Texas; 1:500 in TBS-Tween 1X), mouse monoclonal b-actin (A5316, Clone AC-74, Sigma Aldrich, St Louis, Missouri, 1:5000 in 5% BSA), mouse monoclonal Bax (B-9: sc-7480, Santa Cruz Biotechnology, Dallas, Texas; 1:500 in TBSTween 1), and rabbit monoclonal caspase-3 (#9665, Cell Signaling, Danvers, Massachusetts; 1:1000 in 5% BSA). After washes in TBS-Tween 1, membranes were incubated with the secondary antibodies at room temperature for 1 hour. Secondary antibodies used were as follows: peroxidaseconjugated goat antirabbit immunoglobulin (IgG; AffiniPure, Jackson Immunoresearch, West Grove, Pennsylvania), diluted 1:10 000 in 5% milk for LC3, Beclin-1, and HIF-1a, or 1:10 000 in TBS-Tween 1 for caspase-3, and goat anti-mouse IgG (AffiniPure, Jackson Immunoresearch, West Grove, Pennsylvania), diluted 1:10 000 in 5% milk for b-actin or 1:10000 in TBS-Tween 1X for Bax and Bcl2.
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Chemiluminescence was detected by incubating the membrane with LiteAblot Chemiluminescent substrate (Lite Ablot, EuroClone, Milano, Italy) followed by X-ray film exposure (Kodak X-Omat Blue XB-1 Film, Eastman Kodak Company, Rochester, New York). Protein expression was quantified densitometrically (OD/ mm2) by Quantity One 4.2.1 image analysis software (BioRad, Segrate, Italy). To correlate the blot intensity with the actual protein amount, calibration curves were run in blots within the dynamic range of the instrument. After the background signal was subtracted, densitometry data were normalized with respect to a single reference extract that was inserted in every run, thereby representing the positive control to allow normalization and encompass intra- and interrun variability. Protein levels are expressed as densitometry ratio with b-actin. Western blotting analysis permits to distinguish cytosolic and membrane-bound forms of LC3, LC3-I (18 kDa), and LC3-II (16 kDa), respectively.
Statistical Analysis Analysis of immunoblots is reported as mean and standard error of the mean. The Wilcoxon matched-pairs test and Mann-Whitney U test were used, as appropriated, to compare protein levels in villi and decidua and to compare autophagosome content in cytotrophoblast and syncytiotrophoblast. The P values .05 were considered significant. Statistical tests were performed using Instat 3, GraphPad software.
Results Normal Pregnancies Signs of autophagy were detected both in villous and decidual specimens of first-trimester pregnancies (Figure 1C, E, and G): LC3 staining was observed both in villous syncytiotrophoblast and in cytotrophoblast by immunohistochemical method; LC3 immunostaining was also observed in trophoblast-anchoring columns and extravillous trophoblast (EVT). Moreover, decidual stromal cells displayed a positive immunoreactivity both in basalis and in parietalis decidua. We confirmed the localization of autophagy by electron microscopy in cytotrophoblast, syncytiotrophoblast, and decidua by observing autophagosomes, a double-membrane structures containing cytoplasmic material at various stage of degradation (Figure 1I-L). To detect differences in autophagy between villous and decidual samples, LC3-II protein expression was measured by Western blotting analysis. The expression of LC3-II was comparable in decidual and villous specimens.
Spontaneous Miscarriages Localization of LC3 staining in SM was comparable to NP, but in syncytiotrophoblast an increased intensity of staining was observed by immunofluorescence, finding an increase in punctate dots (Figure 2A). We confirmed the increase of autophagy in syncytiotrophoblast by semiquantitative electron microscopy analysis, detecting a higher concentration of
autophagosome in syncytiotrophoblast of SMs compared to NPs (Figure 2D and E). We detected an increased LC3-II expression in villi from SMs than villi from NPs by Western blotting analysis (Figure 2B and C).
Autophagy Expression and Expression of Markers of Hypoxia or Apoptosis A proper oxygen level and cells turnover are necessary at the implantation site to warrant an adequate evolution of pregnancy. Therefore, to explore the possible correlation of autophagy with hypoxia, we measured the expression of HIF-1a, a transcription factor stabilized in hypoxic conditions, by Western blotting. The expression of HIF-1a was similarly low in villous and decidual samples of NP, whereas in cases of SM, the expression of HIF-1a increased both in villi and in decidua with the highest level in decidual samples (Figure 3A). Moreover, to compare the expression of autophagy to apoptosis, we measured the expression of Bax (that promotes programmed cells death) and Bcl-2 (that inhibits programmed cells death) in villous and decidual specimens by Western blotting analysis, using for each case the same sample of tissue previously used to investigate autophagy. The ratio of Bax–Bcl-2 protein was used to indicate the susceptibility to apoptosis. Comparable low level of apoptosis was observed both in villi and in decidua of NP, whereas an increase in apoptosis was observed in decidua of SM although without statistical significance (Figure 3B). Cleaved caspase-3 expression was also evaluated to detect apoptosis and it was observed in all samples of decidua of miscarriage, according to the trend of Bax/Bcl-2 expression (Figure 3C) and in agreement with immunohistochemical staining (Figure 3D).
Discussion The present study assess autophagy expression during early human pregnancy. Few previous studies have evaluated autophagy in placental villi13,14 or decidua15 of the first trimester of gestation. A novelty of our study is the analysis, at the same time, of both the sides of blastocyst implantation, considering the embryonic side by evaluating villi and the maternal side by evaluating decidua. Interestingly, the results of the present study show that autophagy at the fetomaternal interface is constitutively expressed in normal early human pregnancy. During normal conditions, in villous specimens, we observed autophagy expression in both the trophoblastic layers, with higher expression in cytotrophoblast than in syncytiotrophoblast. This observation is in agreement with a previous report,13 confirming the physiological occurrence of autophagy in placental villi during the first weeks of gestation. The strength of our study is the novelty of the evaluation of autophagy in SM. We observed an increased autophagy expression in villous samples due to an increment of autophagic vacuoles concentration in syncytiotrophoblast. Syncytiotrophoblast is the outer trophoblastic layer, directly in contact with
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Figure 1. Localization of autophagy in normal pregnancy. A and B, negative controls. C-H, Localization of autophagy marker (LC3) by immunohistochemical staining. (C, E, and G), LC3 staining (autophagy marker); (D and F) cytokeratin 7 staining (CK7—marker of trophoblastic cells); (H) HLA-G staining (marker of endovascular trophoblast). Signs of autophagy are detectable in villous trophoblast (C and E) in decidual samples (E and G) and in endovascular trophoblast (G). Positivity are detectable not only in trophoblastic cells but also in some stromal cells both in villous and in decidual tissues. Original magnification 40 in (A), (B), (C), (D), (G), and (H); 20 in (E) and (F). I-L, Evaluation of autophagosome by transmission electron microscopy. I, Villous sample: autophagosomes are clearly identifiable at higher magnification (inset) both in syncytiotrophoblast and cytotrophoblast. L, Decidual sample: double membrane autophagic compartment containing material in degradation is detectable at higher magnification (inset). No Ab indicates no primary antibody control; C-, slice incubated with a matching concentration of nonspecific rabbit immunoglobulin; a, anchoring villous; ac, anchoring column; e.va.t, endovascular trophoblast; f, floating villous; gl, decidual gland; MMV, microvillous membrane; S, syncytiotrophoblast; C, cytotrophoblast; Fc, fetal capillary. LC3, microtubule-associated protein light chain 3.
maternal blood. Since autophagy can intervene in placental trophoblasts during stressor conditions as a cytoprotective process,16 we suggest that the increase of autophagy in syncytiotrophoblast could reflect a prosurvival tentative of the cells to respond to the environmental changes. Moreover, we detected autophagy markers in decidua specimens, trophoblastic-anchoring columns, and EVT. It is known that EVT migrates from the columns through the decidual stroma, reaches the spiral arteries, penetrates the wall of the vessels, and colonizes the lumen forming the trophoblastic plugs and conducting to vascular remodeling.17 Deficiency in 4
EVT invasion and inadequate vascular remodeling can lead to adverse pregnancy outcome such as miscarriage, preeclampsia, and intrauterine growth restriction.18 Previous studies performed on trophoblastic cell lines suggested that autophagy is involved in trophoblast invasion,14 even if the results are conflicting: Some authors observed that autophagy induced by hypoxia supports trophoblast invasion,15,19 whereas other authors observed an increase in autophagy but a decrease in trophoblast invasion in relation to the inhibition of HIF1-a expression.20 Differences in autophagy expression between decidua from NP and SM were not detected in the present study, while
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Figure 2. Autophagy expression in normal pregnancy (NP) and spontaneous miscarriage (SM). A, immunofluorescence staining (LC3 in green) in villi (v) and decidua (d). Arrows indicate syncytiotrophoblast. Note the increase number of fluorescent punta dots in syncytiotrophoblastic layer of spontaneous miscarriage, well identifiable in the insets. White scale bar: 25 mm, Yellow scale bar: 50 mm. B, Representative Western blotting bands of autophagy markers LC3-I and II (18 and 16 kDa, respectively) and b-actin (42 kDa, housekeeping gene for normalization). Despite the high intersample variability in villous samples, the amount of LC3-II normalized onto b-actin in NP was always lower than LC3-II in SP, as shown in (C). C, Western blotting analysis showed that the level of LC3-II expression was higher in villi obtained from spontaneous miscarriage. *P ¼ .02 villi SM versus villi NP (Mann-Whitney test); **P ¼ .004 villi SM versus decidua SM (Wilcoxon test). D, Autophagosome content in placental villi, expressed as the ratio between the sum of the areas of autophagosomes and the total area, evaluated in cytotrophoblast and syncytiotrophoblast by transmission electron microscopy. * P ¼ .05 syncytiotrophoblast SM versus syncytiotrophoblast NP (Mann-Whitney test). E, Autophagosome detection by transmission electron microscopy in a case of spontaneous miscarriage. Arrow heads indicate autophagosome vacuoles in syncytiotrophoblast and cytotrophoblast. c indicates cytotrophoblast; s, syncytiotrophoblast; MMV, microvillous membrane; Fc, fetal capillary. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
apoptosis and hypoxia were increased in SM. Our finding of increased apoptosis in first-trimester decidua of SM is in agreement with a previous report where apoptosis was responsible for the pregnancy loss.21 In our samples of SM, we found an increased expression of autophagy in villous specimens but a low expression of apoptosis; on the contrary, in decidua specimens we found low levels of autophagy but an increased susceptibility to apoptosis. It is possible that the opposite trend of expression of autophagy compared to hypoxia and apoptosis markers in villi and decidua of SM might reflect the role of autophagy in antagonizing hypoxia-induced apoptosis, but further investigation is needed to clarify this issue. This study has some limitations. First, samples obtained from legal interruption of pregnancies include viable tissues, whereas samples from SMs include tissues from nonviable
pregnancies; it is therefore not possible to determine whether autophagy has been induced in vivo before or after fetal demise. Second, samples were stored in liquid nitrogen before the analysis, whereas a previous study suggested a role of cold stress in the induction of autophagy,22 therefore our results might be influenced by the freezing of samples before the analysis. Third, we could not exclude that the intersample variability in villi affected our results: Autophagy is a dynamic and continuous process and LC3-II is just a marker of one stage of it. Different stages of the evolution during the maturation from the phagophore through the autolysosome in our samples could affect the presence or the level of LC3-II. In conclusion, our study demonstrates the occurrence of autophagy in vivo at the fetomaternal interface during the first trimester of normal human gestation. A low level of autophagy
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Figure 3. Evaluation of apoptosis and hypoxia by Western blotting analysis. A, Western blotting analysis showed that HIF-1a expression (hypoxia marker) was high in decidual samples obtained from spontaneous miscarriage (SM). *P ¼ .0002 decidua SM versus decidua normal pregnancy (NP; Mann-Whitney test); on the right, representative bands of HIF-1a (120 kDa) and b-actin (42 kDa, housekeeping gene for normalization) are shown. B, Western blotting analysis showed an increased trend in the Bax/Bcl2 ratio in decidua obtained from spontaneous miscarriage. C, Western blotting analysis of cleaved caspase-3. The extent of the cleavage is comparable to the Bax/Bcl2 ratio, supporting greater apoptosis in decidua SM compared to villi SM and all samples from NP. D, Immunohistochemical expression of cleaved caspase 3. Positive cells are in brown. Note the increased number of positive cells in decidua from spontaneous miscarriage respect to decidua from normal pregnancy. HIF-1a indicates hypoxia-inducible factor. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
at this site may represent a physiological event necessary to the normal evolution of pregnancy. For the first time to our knowledge, our study demonstrates that SM specimens show higher autophagy expression in villi. Further studies are needed to clarify the role of autophagy in SM and to distinguish whether the autophagy expression changes represent the cause or the consequence of the early adverse pregnancy outcome.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
References Authors’ Note Laura Avagliano had the idea, collected and analyzed the data, and wrote the manuscript. Laurea Terraneo and Eleonora Virgili performed the immunoblotting analysis and approved the last version of the manuscript. Carla Martinelli performed the electron microscopic study and approved the last version of the manuscript. Patrizia Doi performed the immunohistochemical staining and approved the last version of the manuscript. Michele Samaja discussed the data, revised the manuscript, and approved its last version. Gaetano Bulfamante analyzed and discussed the data, revised the manuscript, and approved its last version. Anna Maria Marconi revised and approved the last version of the manuscript. 6
1. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290(5497):1717-1721. 2. Noda T, Fujita N, Yoshimori T. The late stages of autophagy: how does the end begin? Cell Death Differ. 2009;16(7):984-990. 3. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67-93. 4. Mizushima N, Levine B. Autophagy in mammalian development and differentiation. Nat Cell Biol. 2010;12:823-830. 5. Tsukamoto S, Kuma A, Murakami M, Kishi C, Yamamoto A, Mizushima N. Autophagy is essential for preimplantation development of mouse embryos. Science. 2008;321:117-120.
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6. Tsukamoto S, Kuma A, Mizushima N. The role of autophagy during the oocyte-to-embryo transition. Autophagy. 2008;4:1076-1078. 7. Jerzak M, Bischof P. Apoptosis in the first trimester human placenta: The role in maintaining immune privilege at the maternal-foetal interface and in the trophoblast remodelling. Eur J Obstet Gynecol Reprod Biol. 2002;100(2):138-142. 8. Caniggia I, Winter J, Lye SJ, Post M. Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia. Placenta. 2000;21(suppl A):S25-S30. 9. Garzia E, Clauser R, Persani L, et al. Prolactin and proinflammatory cytokine expression at the fetomaternal interface in first trimester miscarriage. Fertil Steril. 2013;100(1):108-15.e1-e2. 10. Signorelli P, Avagliano L, Virgili E, et al. Autophagy in term normal human placentas. Placenta. 2011;32(6):482-485. 11. Avagliano L, Virgili E, Garo` C, et al. Autophagy and human parturition: evaluation of LC3 expression in placenta from spontaneous or medically induced onset of labor. BioMed Res Int. 2013. Web site. www.hindawi.com/journals/bmri/2013/689768/. Published 2013. Accessed December 16, 2014. 12. Avagliano L, Danti L, Doi P, et al. Autophagy in placentas from acidotic newborns: an immunohistochemical study of LC3 expression. Placenta. 2013;34(11):1091-1094. 13. Chifenti B, Locci MT, Lazzeri G, et al. Autophagy-related protein LC3 and Beclin-1 in the first trimester of pregnancy. Clin Exp Reprod Med. 2013;40(1):33-37. 14. Hung TH, Hsieh TT, Chen SF, Li MJ, Yeh YL. Autophagy in the human placenta throughout gestation. PLoS One. 2013;8(12): e83475.
15. Nakashima A, Yamanaka-Tatematsu M, Fujita N, et al. Impaired autophagy by soluble endoglin, under physiological hypoxia in early pregnant period, is involved in poor placentation in preeclampsia. Autophagy. 2013;9(3):303-316. 16. Chen B, Longtine MS, Nelson DM. Hypoxia Induces Autophagy in Primary Human Trophoblasts. Endocrinology. 2012;153(10): 4946-4954. 17. Huppertz B, Berghold VM, Kawaguchi R, Gauster M. A variety of opportunities for immune interactions during trophoblast development and invasion. Am J Reprod Immunol. 2012;67(5): 349-357. 18. Brosens I, Pijnenborg R, Vercruysse L, Romero R. The ‘‘Great Obstetrical Syndromes’’ are associated with disorders of deep placentation. Am J Obstet Gynecol. 2011;204(3):193-201. 19. Yamanaka-Tatematsu M, Nakashima A, Fujita N, Shima T, Yoshimori T, Saito S. Autophagy induced by HIF1a overexpression supports trophoblast invasion by supplying cellular energy. PLoS One. 2013;8(10):e76605. 20. Choi JH, Lee HJ, Yang TH, Kim GJ. Effects of hypoxia inducible factors-1a on autophagy and invasion of trophoblasts. Clin Exp Reprod Med. 2012;39(2):73-80. 21. Kokawa T, Shikone T, Nakano R. Apoptosis in human chorionic villi and decidua during normal embryonic development and spontaneous abortion in the first trimester. Placenta. 1998; 19(1):21-26. 22. Bang S, Shin H, Song H, Suh CS, Lim HJ. Autophagic activation in vitrified-warmed mouse oocytes. Reproduction. 2014;148(1): 11-19.
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Reproductive Sciences 1-7 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719114565036 rs.sagepub.com
Laura Avagliano, MD, PhD1, Laura Terraneo, PhD1, Eleonora Virgili, BD1, Carla Martinelli PhD2, Patrizia Doi, MS1, Michele Samaja, PhD1, Gaetano Pietro Bulfamante, MD1, and Anna Maria Marconi, MD1
Abstract Autophagy is an inducible catabolic process by which cells degrade and recycle materials to survive stress, starvation, and hypoxia. The aim of this study was to evaluate autophagy at the fetal–maternal interface, to assess autophagy involvement during the early phase of human gestation, and to explore autophagic modification in case of early abnormal pregnancy outcome. Specimens were collected from first-trimester normal gestations undergoing legal termination of pregnancy and first-trimester sporadic spontaneous miscarriages. Autophagy was studied in villous and decidual samples by transmission electron microscopy, immunohistochemistry, immunofluorescence, and Western blotting. Autophagy markers were found in cytotrophoblast, syncytiotrophoblast, extravillous trophoblast, and decidual stromal cells. Autophagy is physiologically involved in early normal gestation. Compared with normal pregnancy, spontaneous miscarriage presents an increase in autophagy expression in villous specimens due to an increment in concentration of autophagic vacuole in syncytiotrophoblast, suggesting a cytoprotective mechanism of the cells to respond to microenvironmental challenge. Keywords autophagy, apoptosis, pregnancy, trophoblast, miscarriage
Introduction Autophagy is an evolutionary conserved, intracellular inducible catabolic process by which injured organelles, damaged proteins, and pathogens are sequestered into autophagosomes, double membrane-bound vesicles that fuse with lysosomes for proteolytic degradation and material recycling.1 Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of yeast autophagy-related gene 8, intervenes in the late stages of autophagosome formation. Among LC3 isoforms, LC3-II, the phosphatidylethanolamine conjugated product of LC3-I obtained after LC3 activation,1,2 is currently used as a specific marker of autophagy for its role during autophagosome genesis.3 Previous studies have investigated the occurrence of autophagy during the early stages of mammalian pregnancy in the preimplantation period for oocyte-to-embryo transition4-6 but no extensive in vivo assessment of autophagy at the fetomaternal interface after implantation has been performed in humans. At the site of implantation, apoptosis permits placenta development,7 and a relatively hypoxic environment warrants an adequate embryonic and placental growth.8 Although many cellular and molecular events are expected to regulate the various steps of fetomaternal interactions, the mechanisms that regulate the early stage of pregnancy are not yet fully
understood. The aim of the present study was to evaluate the occurrence of autophagy during the first trimester of pregnancy to verify whether it is involved in the early phase of gestation and whether autophagy expression differs between normal pregnancy (NP) and spontaneous miscarriage (SM).
Materials and Methods Sample Collection Normal pregnancies. Tissues were obtained from 10 healthy, nonsmokers women (age 28.5 + 8.1 years) undergoing legal termination of pregnancy within 12 weeks of amenorrhea (mean gestational age 9.4 + 1.1 weeks; range 8-11 weeks); 1 Department of Health Sciences, San Paolo Hospital Medical School, Universita` degli Studi di Milano, Milan, Italy 2 Department of Biomedical Sciences for Health, Universita` degli Studi di Milano, Milan, Italy
Corresponding Author: Laura Avagliano, Unit of Obstetrics and Gynecology, Department of Health Sciences, San Paolo Hospital Medical School. University of Milano, Via A. di Rudinı` 8. 20142 Milano, Italy. Email: [email protected]
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in all cases gestational age and fetal viability were confirmed by ultrasound before suction curettage. All women gave their consent to the study. All procedures were performed in general anesthesia. At the time of suction curettage, the gestational tissues were removed in sterile conditions and macroscopically assessed, through several washes in sterile phosphatebuffered saline solution. For each woman, samples of decidua and villi were collected and stored separately: immediately after the washes, tissues collected for immunohistochemical and immunofluorescence analysis were fixed in 10% formalin solution; samples for electron microscopy were fixed in 2.5% glutaraldehyde; samples for Western blotting were frozen in liquid nitrogen and stored at 80 C for protein extraction. Spontaneous miscarriages. Tissues were obtained from archive specimens from our previous work as described.9 All cases were sporadic miscarriage. Only embryonic pregnancies were included and all cases had normal embryonic karyotype. Smokers were excluded, therefore 10 cases were eligible for this study. Spontaneous miscarriage and NPs were comparable for gestational age (gestational age 9.2 + 0.6 weeks; P ¼ .76) and maternal age (age 30.9 + 4.8 years; P ¼ .43).
Immunohistochemistry Immunohistochemical studies were carried out in all samples (n ¼ 20: 10 NPs and 10 SMs) on 4-mm thick tissue sections as described previously.10-12 Primary antibodies were as follows: rabbit polyclonal LC3 (NB100-2220, Novus Biologicals, Littleton, Colorado; dilution 1:1000), mouse monoclonal antiCK-7 (OV-TL 12/30, Dako, Glostrup, Denmark; dilution 1:100), mouse monoclonal anti-HLA-G (SC-21799, Santa Cruz Biotechnology, Dallas, Texas; dilution 1:2000), and rabbit monoclonal cleaved caspase-3 (#9664, Cell Signaling Technology, Danvers, Massachusetts; dilution 1:800).
Immunofluorescence Immunofluorescence studies were conducted in the same samples used for immunohistochemistry (n ¼ 20: 10 NPs and 10 SMs), as described previously11 using rabbit antiLC3 (NB100-2220, Novus Biologicals, Littleton, Colorado; dilution 1:500). Fluorophore-conjugated secondary antibodies were used and nuclei were subsequently counterstained with 40 ,6diamidino-2-phenylindole (Invitrogen, Carlsbad, California). Fluorescence images were viewed and captured using Imager.Z1 microscope (Zeiss, Feldbach, Switzerland).
Transmission Electron Microscopy Tissues from 8 cases (4 NPs and 4 SMs) were fixed in 2.5% glutaraldehyde in 0.13 mol/L phosphate buffer at pH 7.2-7.4; specimens were postfixed in 1% OsO4, dehydrated in ethanol plus propylene oxide, and embedded in epoxy resin. Ultrathin sections of 50 to 60 nm were counterstained with uranyl acetate and lead citrate. A Jeol JEM 1010 (Tokyo, Japan) electron microscope was used for examination. Cases used for 2
transmission electron microscopy evaluation were a subgroup of the total population due to tissue availability restrictions. Autophagic vacuoles were identified, in villi and decidua, as double membrane compartments containing material in various stages of degradation. For each case, the area of autophagic vacuoles was calculated by 2 observers (LA and CM) on 60000 printed micrographs (previously converted in digital bitmap images by Image Processing and Analysis in Java-ImageJ) in 3 randomly selected areas of villi, both in cytotrophoblast and in syncytiotrophoblast. Quantification of autophagy was measured as a ratio of autophagic vacuole area to the total cytoplasmic area. The mean ratio from the 3 areas was considered as the final value for each case.
Western Blotting Villous (n ¼ 20: 10 NPs and 10 SMs) and decidual (n ¼ 20: 10s NP and 10 SMs) samples were homogenized in a glass potter in a 1:5 ratio (weight:volume) with 50 mmol/L Tris-HCl pH 7.4, 5 mmol/L EDTA, 1 mmol/L DTT, 2% SDS, and Protease Inhibitor Cocktail (Sigma Aldrich, St. Louis, Missouri) and centrifuged at 14 000g for 15 minutes. Protein concentration within the supernatant was measured by the Coomassie Plus Protein Assay reagent Kit (Pierce, Rockford, Illinois). An equal amount of protein from each sample (30 mg) was subjected to electrophoresis in a 10% polyacrylamide gel for LC3 and Bcl2 or in 8% polyacrylamide gel for hypoxia-inducible factor 1a (HIF-1a), caspase-3, Bax, and transferred onto nitrocellulose membrane. Membranes were incubated in a blocking solution (Tris-buffered saline [TBS] containing 5% powdered nonfat milk and 0.01% Tween-20) at room temperature for 1 hour. Membranes were incubated overnight at 4 C with the primary antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibody. The used primary antibodies and dilutions were as follows: rabbit polyclonal anti-HIF-1a (H-206X: sc-10790, Santa Cruz Biotechnology, Dallas, Texas; 1:300 in TBS-Tween 1), rabbit polyclonal LC3 (NB100-2220, Novus Biologicals, Littleton, Colorado; 1:1000 in 5% bovine serum albumin [BSA]), mouse monoclonal Bcl2 (C-2: sc-7382, Santa Cruz Biotechnology, Dallas, Texas; 1:500 in TBS-Tween 1X), mouse monoclonal b-actin (A5316, Clone AC-74, Sigma Aldrich, St Louis, Missouri, 1:5000 in 5% BSA), mouse monoclonal Bax (B-9: sc-7480, Santa Cruz Biotechnology, Dallas, Texas; 1:500 in TBSTween 1), and rabbit monoclonal caspase-3 (#9665, Cell Signaling, Danvers, Massachusetts; 1:1000 in 5% BSA). After washes in TBS-Tween 1, membranes were incubated with the secondary antibodies at room temperature for 1 hour. Secondary antibodies used were as follows: peroxidaseconjugated goat antirabbit immunoglobulin (IgG; AffiniPure, Jackson Immunoresearch, West Grove, Pennsylvania), diluted 1:10 000 in 5% milk for LC3, Beclin-1, and HIF-1a, or 1:10 000 in TBS-Tween 1 for caspase-3, and goat anti-mouse IgG (AffiniPure, Jackson Immunoresearch, West Grove, Pennsylvania), diluted 1:10 000 in 5% milk for b-actin or 1:10000 in TBS-Tween 1X for Bax and Bcl2.
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Chemiluminescence was detected by incubating the membrane with LiteAblot Chemiluminescent substrate (Lite Ablot, EuroClone, Milano, Italy) followed by X-ray film exposure (Kodak X-Omat Blue XB-1 Film, Eastman Kodak Company, Rochester, New York). Protein expression was quantified densitometrically (OD/ mm2) by Quantity One 4.2.1 image analysis software (BioRad, Segrate, Italy). To correlate the blot intensity with the actual protein amount, calibration curves were run in blots within the dynamic range of the instrument. After the background signal was subtracted, densitometry data were normalized with respect to a single reference extract that was inserted in every run, thereby representing the positive control to allow normalization and encompass intra- and interrun variability. Protein levels are expressed as densitometry ratio with b-actin. Western blotting analysis permits to distinguish cytosolic and membrane-bound forms of LC3, LC3-I (18 kDa), and LC3-II (16 kDa), respectively.
Statistical Analysis Analysis of immunoblots is reported as mean and standard error of the mean. The Wilcoxon matched-pairs test and Mann-Whitney U test were used, as appropriated, to compare protein levels in villi and decidua and to compare autophagosome content in cytotrophoblast and syncytiotrophoblast. The P values .05 were considered significant. Statistical tests were performed using Instat 3, GraphPad software.
Results Normal Pregnancies Signs of autophagy were detected both in villous and decidual specimens of first-trimester pregnancies (Figure 1C, E, and G): LC3 staining was observed both in villous syncytiotrophoblast and in cytotrophoblast by immunohistochemical method; LC3 immunostaining was also observed in trophoblast-anchoring columns and extravillous trophoblast (EVT). Moreover, decidual stromal cells displayed a positive immunoreactivity both in basalis and in parietalis decidua. We confirmed the localization of autophagy by electron microscopy in cytotrophoblast, syncytiotrophoblast, and decidua by observing autophagosomes, a double-membrane structures containing cytoplasmic material at various stage of degradation (Figure 1I-L). To detect differences in autophagy between villous and decidual samples, LC3-II protein expression was measured by Western blotting analysis. The expression of LC3-II was comparable in decidual and villous specimens.
Spontaneous Miscarriages Localization of LC3 staining in SM was comparable to NP, but in syncytiotrophoblast an increased intensity of staining was observed by immunofluorescence, finding an increase in punctate dots (Figure 2A). We confirmed the increase of autophagy in syncytiotrophoblast by semiquantitative electron microscopy analysis, detecting a higher concentration of
autophagosome in syncytiotrophoblast of SMs compared to NPs (Figure 2D and E). We detected an increased LC3-II expression in villi from SMs than villi from NPs by Western blotting analysis (Figure 2B and C).
Autophagy Expression and Expression of Markers of Hypoxia or Apoptosis A proper oxygen level and cells turnover are necessary at the implantation site to warrant an adequate evolution of pregnancy. Therefore, to explore the possible correlation of autophagy with hypoxia, we measured the expression of HIF-1a, a transcription factor stabilized in hypoxic conditions, by Western blotting. The expression of HIF-1a was similarly low in villous and decidual samples of NP, whereas in cases of SM, the expression of HIF-1a increased both in villi and in decidua with the highest level in decidual samples (Figure 3A). Moreover, to compare the expression of autophagy to apoptosis, we measured the expression of Bax (that promotes programmed cells death) and Bcl-2 (that inhibits programmed cells death) in villous and decidual specimens by Western blotting analysis, using for each case the same sample of tissue previously used to investigate autophagy. The ratio of Bax–Bcl-2 protein was used to indicate the susceptibility to apoptosis. Comparable low level of apoptosis was observed both in villi and in decidua of NP, whereas an increase in apoptosis was observed in decidua of SM although without statistical significance (Figure 3B). Cleaved caspase-3 expression was also evaluated to detect apoptosis and it was observed in all samples of decidua of miscarriage, according to the trend of Bax/Bcl-2 expression (Figure 3C) and in agreement with immunohistochemical staining (Figure 3D).
Discussion The present study assess autophagy expression during early human pregnancy. Few previous studies have evaluated autophagy in placental villi13,14 or decidua15 of the first trimester of gestation. A novelty of our study is the analysis, at the same time, of both the sides of blastocyst implantation, considering the embryonic side by evaluating villi and the maternal side by evaluating decidua. Interestingly, the results of the present study show that autophagy at the fetomaternal interface is constitutively expressed in normal early human pregnancy. During normal conditions, in villous specimens, we observed autophagy expression in both the trophoblastic layers, with higher expression in cytotrophoblast than in syncytiotrophoblast. This observation is in agreement with a previous report,13 confirming the physiological occurrence of autophagy in placental villi during the first weeks of gestation. The strength of our study is the novelty of the evaluation of autophagy in SM. We observed an increased autophagy expression in villous samples due to an increment of autophagic vacuoles concentration in syncytiotrophoblast. Syncytiotrophoblast is the outer trophoblastic layer, directly in contact with
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Figure 1. Localization of autophagy in normal pregnancy. A and B, negative controls. C-H, Localization of autophagy marker (LC3) by immunohistochemical staining. (C, E, and G), LC3 staining (autophagy marker); (D and F) cytokeratin 7 staining (CK7—marker of trophoblastic cells); (H) HLA-G staining (marker of endovascular trophoblast). Signs of autophagy are detectable in villous trophoblast (C and E) in decidual samples (E and G) and in endovascular trophoblast (G). Positivity are detectable not only in trophoblastic cells but also in some stromal cells both in villous and in decidual tissues. Original magnification 40 in (A), (B), (C), (D), (G), and (H); 20 in (E) and (F). I-L, Evaluation of autophagosome by transmission electron microscopy. I, Villous sample: autophagosomes are clearly identifiable at higher magnification (inset) both in syncytiotrophoblast and cytotrophoblast. L, Decidual sample: double membrane autophagic compartment containing material in degradation is detectable at higher magnification (inset). No Ab indicates no primary antibody control; C-, slice incubated with a matching concentration of nonspecific rabbit immunoglobulin; a, anchoring villous; ac, anchoring column; e.va.t, endovascular trophoblast; f, floating villous; gl, decidual gland; MMV, microvillous membrane; S, syncytiotrophoblast; C, cytotrophoblast; Fc, fetal capillary. LC3, microtubule-associated protein light chain 3.
maternal blood. Since autophagy can intervene in placental trophoblasts during stressor conditions as a cytoprotective process,16 we suggest that the increase of autophagy in syncytiotrophoblast could reflect a prosurvival tentative of the cells to respond to the environmental changes. Moreover, we detected autophagy markers in decidua specimens, trophoblastic-anchoring columns, and EVT. It is known that EVT migrates from the columns through the decidual stroma, reaches the spiral arteries, penetrates the wall of the vessels, and colonizes the lumen forming the trophoblastic plugs and conducting to vascular remodeling.17 Deficiency in 4
EVT invasion and inadequate vascular remodeling can lead to adverse pregnancy outcome such as miscarriage, preeclampsia, and intrauterine growth restriction.18 Previous studies performed on trophoblastic cell lines suggested that autophagy is involved in trophoblast invasion,14 even if the results are conflicting: Some authors observed that autophagy induced by hypoxia supports trophoblast invasion,15,19 whereas other authors observed an increase in autophagy but a decrease in trophoblast invasion in relation to the inhibition of HIF1-a expression.20 Differences in autophagy expression between decidua from NP and SM were not detected in the present study, while
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Figure 2. Autophagy expression in normal pregnancy (NP) and spontaneous miscarriage (SM). A, immunofluorescence staining (LC3 in green) in villi (v) and decidua (d). Arrows indicate syncytiotrophoblast. Note the increase number of fluorescent punta dots in syncytiotrophoblastic layer of spontaneous miscarriage, well identifiable in the insets. White scale bar: 25 mm, Yellow scale bar: 50 mm. B, Representative Western blotting bands of autophagy markers LC3-I and II (18 and 16 kDa, respectively) and b-actin (42 kDa, housekeeping gene for normalization). Despite the high intersample variability in villous samples, the amount of LC3-II normalized onto b-actin in NP was always lower than LC3-II in SP, as shown in (C). C, Western blotting analysis showed that the level of LC3-II expression was higher in villi obtained from spontaneous miscarriage. *P ¼ .02 villi SM versus villi NP (Mann-Whitney test); **P ¼ .004 villi SM versus decidua SM (Wilcoxon test). D, Autophagosome content in placental villi, expressed as the ratio between the sum of the areas of autophagosomes and the total area, evaluated in cytotrophoblast and syncytiotrophoblast by transmission electron microscopy. * P ¼ .05 syncytiotrophoblast SM versus syncytiotrophoblast NP (Mann-Whitney test). E, Autophagosome detection by transmission electron microscopy in a case of spontaneous miscarriage. Arrow heads indicate autophagosome vacuoles in syncytiotrophoblast and cytotrophoblast. c indicates cytotrophoblast; s, syncytiotrophoblast; MMV, microvillous membrane; Fc, fetal capillary. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
apoptosis and hypoxia were increased in SM. Our finding of increased apoptosis in first-trimester decidua of SM is in agreement with a previous report where apoptosis was responsible for the pregnancy loss.21 In our samples of SM, we found an increased expression of autophagy in villous specimens but a low expression of apoptosis; on the contrary, in decidua specimens we found low levels of autophagy but an increased susceptibility to apoptosis. It is possible that the opposite trend of expression of autophagy compared to hypoxia and apoptosis markers in villi and decidua of SM might reflect the role of autophagy in antagonizing hypoxia-induced apoptosis, but further investigation is needed to clarify this issue. This study has some limitations. First, samples obtained from legal interruption of pregnancies include viable tissues, whereas samples from SMs include tissues from nonviable
pregnancies; it is therefore not possible to determine whether autophagy has been induced in vivo before or after fetal demise. Second, samples were stored in liquid nitrogen before the analysis, whereas a previous study suggested a role of cold stress in the induction of autophagy,22 therefore our results might be influenced by the freezing of samples before the analysis. Third, we could not exclude that the intersample variability in villi affected our results: Autophagy is a dynamic and continuous process and LC3-II is just a marker of one stage of it. Different stages of the evolution during the maturation from the phagophore through the autolysosome in our samples could affect the presence or the level of LC3-II. In conclusion, our study demonstrates the occurrence of autophagy in vivo at the fetomaternal interface during the first trimester of normal human gestation. A low level of autophagy
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Figure 3. Evaluation of apoptosis and hypoxia by Western blotting analysis. A, Western blotting analysis showed that HIF-1a expression (hypoxia marker) was high in decidual samples obtained from spontaneous miscarriage (SM). *P ¼ .0002 decidua SM versus decidua normal pregnancy (NP; Mann-Whitney test); on the right, representative bands of HIF-1a (120 kDa) and b-actin (42 kDa, housekeeping gene for normalization) are shown. B, Western blotting analysis showed an increased trend in the Bax/Bcl2 ratio in decidua obtained from spontaneous miscarriage. C, Western blotting analysis of cleaved caspase-3. The extent of the cleavage is comparable to the Bax/Bcl2 ratio, supporting greater apoptosis in decidua SM compared to villi SM and all samples from NP. D, Immunohistochemical expression of cleaved caspase 3. Positive cells are in brown. Note the increased number of positive cells in decidua from spontaneous miscarriage respect to decidua from normal pregnancy. HIF-1a indicates hypoxia-inducible factor. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
at this site may represent a physiological event necessary to the normal evolution of pregnancy. For the first time to our knowledge, our study demonstrates that SM specimens show higher autophagy expression in villi. Further studies are needed to clarify the role of autophagy in SM and to distinguish whether the autophagy expression changes represent the cause or the consequence of the early adverse pregnancy outcome.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
References Authors’ Note Laura Avagliano had the idea, collected and analyzed the data, and wrote the manuscript. Laurea Terraneo and Eleonora Virgili performed the immunoblotting analysis and approved the last version of the manuscript. Carla Martinelli performed the electron microscopic study and approved the last version of the manuscript. Patrizia Doi performed the immunohistochemical staining and approved the last version of the manuscript. Michele Samaja discussed the data, revised the manuscript, and approved its last version. Gaetano Bulfamante analyzed and discussed the data, revised the manuscript, and approved its last version. Anna Maria Marconi revised and approved the last version of the manuscript. 6
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