Isolation of serum-derived placental/amnio­chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G

in Reproduction
Authors:
Uma Shinde Neuroendocrinology, ICMR-National Institute for Research in Reproductive & Child Health, Parel, Mumbai, Maharashtra, India

Search for other papers by Uma Shinde in
Current site
Google Scholar
PubMed Close
,
Aishwarya Rao Innate Immunity, ICMR-National Institute for Research in Reproductive & Child Health, Parel, Mumbai, Maharashtra, India

Search for other papers by Aishwarya Rao in
Current site
Google Scholar
PubMed Close
,
Vandana Bansal Department of Obstetrics and Gynaecology, Nowrosjee Wadia Maternity Hospital, Parel, Mumbai, Maharashtra, India

Search for other papers by Vandana Bansal in
Current site
Google Scholar
PubMed Close
,
Dhanjit Kumar Das Stem Cell Biology, ICMR-National Institute for Research in Reproductive & Child Health, Parel, Mumbai, Maharashtra, India

Search for other papers by Dhanjit Kumar Das in
Current site
Google Scholar
PubMed Close
,
Nafisa Huseni Balasinor Neuroendocrinology, ICMR-National Institute for Research in Reproductive & Child Health, Parel, Mumbai, Maharashtra, India

Search for other papers by Nafisa Huseni Balasinor in
Current site
Google Scholar
PubMed Close
, and
Taruna Madan Innate Immunity, ICMR-National Institute for Research in Reproductive & Child Health, Parel, Mumbai, Maharashtra, India

Search for other papers by Taruna Madan in
Current site
Google Scholar
PubMed Close
https://orcid.org/0000-0003-2826-2443

Correspondence should be addressed to N H Balasinor or T Madan; Email: balasinorn@nirrch.res.in or guptat@nirrch.res.in

*(U Shinde and A Rao contributed equally to this work)

DOI:
https://doi.org/10.1530/REP-23-0215
Volume/Issue:
Volume 167: Issue 4
Article ID:
e230215
Article Type:
Research Article
Online Publication Date:
22 Mar 2024
Copyright:
© the author(s) 2024
Restricted access
Rent on DeepDyve

Sign up for journal news

In brief

Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G.

Abstract

Extracellular vesicles (EVs) are membrane-bound nanovesicles secreted from the cells into extracellular space and body fluids. They are considered ‘fingerprints of parent cells’, which can reflect their physiological and functional states. During pregnancy, EVs are produced by the syncytiotrophoblasts and extravillous trophoblasts and are released into the maternal bloodstream. In the present study, placental alkaline phosphatase (PLAP)-specific extracellular vesicles were isolated from maternal serum-derived EVs (SDE) across pregnancy. Transmission electron microscopy and dynamic light scattering analysis showed that the isolated EVs exhibited a spherical morphology with ~30–150 nm size range. Nanoparticle tracking analysis indicated that the concentration of PLAP+ serum-derived EVs (PLAP+-SDE) increased across the gestation. PLAP+-SDE contained DNA with LINE1 promoter methylation pattern. C19 miRNA cluster miRNAs (miR 515-5p, 519e and 520f) were present in PLAP+-SDE along with other miRNAs (miR-133-3p, miR210-3p and miR-223-3p). PLAP+-SDE confirmed the presence of EV markers (CD63 and CD9), along with placental proteins (PLAP and cullin 7). A modified novel strategy to extract an enriched population of circulating placental/amniochorionic EVs was devised employing an additional marker of extravillous trophoblasts, human leukocyte antigen G (HLA-G), along with PLAP. The isolated pooled placental/amniochorionic (PLAP+&HLA-G+) serum-derived EVs (PP-SDE) showed ~two-fold increased protein levels of HLA-G in the third-trimester pregnant women compared to the non-pregnant controls. Future studies will be focused on validation of this novel strategy to isolate an enriched population of placental/amniochorionic EVs to facilitate a better understanding of placental physiology and pathophysiology.

Reproduction is committed to supporting researchers in demonstrating the impact of their articles published in the journal.

The two types of article metrics we measure are (i) more traditional full-text views and pdf downloads, and (ii) Altmetric data, which shows the wider impact of articles in a range of non-traditional sources, such as social media.

More information is on the Reasons to publish page.

Sept 2018 onwards Past Year Past 30 Days
Full Text Views 93 93 4
PDF Downloads 123 123 7

 

  • Collapse
  • Expand
Print ISSN:
1470-1626
Online ISSN:
1741-7899

  • Alegre E, Díaz-Lagares A, Lemaoult J, López-Moratalla N, Carosella ED & & González A 2007 Maternal antigen presenting cells are a source of plasmatic HLA-G during pregnancy: longitudinal study during pregnancy. Human Immunology 68 661–667. (https://doi.org/10.1016/j.humimm.2007.04.007)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Arai T, Kasper JS, Skaar JR, Ali SH, Takahashi C & & DeCaprio JA 2003 Targeted disruption of p185/Cul7 gene results in abnormal vascular morphogenesis. PNAS 100 9855–9860. (https://doi.org/10.1073/pnas.1733908100)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Attia JVD, Dessens CE, van de Water R, Houvast RD, Kuppen PJK & & Krijgsman D 2020 The molecular and functional characteristics of HLA-G and the interaction with its receptors: where to intervene for cancer immunotherapy? International Journal of Molecular Sciences 21 8678. (https://doi.org/10.3390/ijms21228678)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Bullerdiek J & & Flor I 2012 Exosome-delivered microRNAs of "chromosome 19 microRNA cluster" as immunomodulators in pregnancy and tumorigenesis. Molecular Cytogenetics 5 27. (https://doi.org/10.1186/1755-8166-5-27)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Dancis J 1965 The role of the placenta in fetal survival. Pediatric Clinics of North America 12 477–492. (https://doi.org/10.1016/S0031-3955(1631728-X)

  • Dong X, Sui C, Huang K, Wang L, Hu D, Xiong T, Wang R, & Zhang H & & Zhang H 2016 MicroRNA-223-3p suppresses leukemia inhibitory factor expression and pinopodes formation during embryo implantation in mice. American Journal of Translational Research 8 1155–1163.

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Donker RB, Mouillet JF, Chu T, Hubel CA, Stolz DB, Morelli AE & & Sadovsky Y 2012 The expression profile of C19MC microRNAs in primary human trophoblast cells and exosomes. Molecular Human Reproduction 18 417–424. (https://doi.org/10.1093/molehr/gas013)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Dudek NL & & Purcell AW 2016 Repertoire of nonclassical MHC I (HLA-E, HLA-F, HLA-G, and Orthologues). In Encyclopedia of Immunobiology: Reference Module in Biomedical Sciences. Academic Press, pp. 2 15–21 9. (https://doi.org/10.1016/B978-0-12-374279-7.06006-9)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Fahlbusch FB, Dawood Y, Hartner A, Menendez-Castro C, Nögel SC, Tzschoppe A, Schneider H, Strissel P, Beckmann MW, Schleussner E, et al.2012 Cullin 7 and Fbxw 8 expression in trophoblastic cells is regulated via oxygen tension: implications for intrauterine growth restriction? Journal of Maternal-Fetal and Neonatal Medicine 25 2209–2215. (https://doi.org/10.3109/14767058.2012.684166)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Fu J, Lv X, Lin H, Wu L, Wang R, Zhou Z, Zhang B, Wang YL, Tsang BK, Zhu C, et al.2010 Ubiquitin ligase cullin 7 induces epithelial-mesenchymal transition in human choriocarcinoma cells. Journal of Biological Chemistry 285 10870–10879. (https://doi.org/10.1074/jbc.M109.004200)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Gascoin-Lachambre G, Buffat C, Rebourcet R, Chelbi ST, Rigourd V, Mondon F, Mignot TM, Legras E, Simeoni U, Vaiman D, et al.2010 Cullins in human intra-uterine growth restriction: expressional and epigenetic alterations. Placenta 31 151–157. (https://doi.org/10.1016/j.placenta.2009.11.008)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Goldman-Wohl DS, Ariel I, Greenfield C, Hanoch J & & Yagel S 2000 HLA-G expression in extravillous trophoblasts is an intrinsic property of cell differentiation: a lesson learned from ectopic pregnancies. Molecular Human Reproduction 6 535–540. (https://doi.org/10.1093/molehr/6.6.535)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Gurunathan S, Kang MH, Jeyaraj M, Qasim M & & Kim JH 2019 Review of the isolation, characterization, biological function, and multifarious therapeutic approaches of exosomes. Cells 8 307. (https://doi.org/10.3390/cells8040307)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Hadley EE, Sheller-Miller S, Saade G, Salomon C, Mesiano S, Taylor RN, Taylor BD & & Menon R 2018 Amnion epithelial cell-derived exosomes induce inflammatory changes in uterine cells. American Journal of Obstetrics and Gynecology 219 478.e1–478.e21. (https://doi.org/10.1016/j.ajog.2018.08.021)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • He ZM, Li J, Hwa YL, Brost B, Fang Q & & Jiang SW 2014 The transition of LINE-1 DNA methylation status and altered expression in first and third-trimester placentas. PLoS One 9 e96994. (https://doi.org/10.1371/journal.pone.0096994)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Hong CS, Funk S, Muller L, Boyiadzis M & & Whiteside TL 2016 Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. Journal of Extracellular Vesicles 5 29289. (https://doi.org/10.3402/jev.v5.29289)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Jeppesen DK, Fenix AM, Franklin JL, Higginbotham JN, Zhang Q, Zimmerman LJ, Liebler DC, Ping J, Liu Q, Evans R, et al.2019 Reassessment of exosome composition. Cell 177 428–445.e18. (https://doi.org/10.1016/j.cell.2019.02.029)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Jin J & & Menon R 2018 Placental exosomes: a proxy to understand pregnancy complications. American Journal of Reproductive Immunology 5 e12788. (https://doi.org/10.1111/aji.12788)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Jin Y, Chen K, Wang Z, Wang Y, Liu J, Lin L, Shao Y, Gao L, Yin H, Cui C, et al.2016 DNA in serum extracellular vesicles are stable under different storage conditions. BMC Cancer 16 753. (https://doi.org/10.1186/s12885-016-2783-2)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Kalluri R & & LeBleu VS 2020 The biology, function, and biomedical applications of exosomes. Science 367 eaau6977. (https://doi.org/10.1126/science.aau6977)

  • Khambata K, Raut S, Deshpande S, Mohan S, Sonawane S, Gaonkar R, Ansari Z, Datar M, Bansal V, Patil A, et al.2021 DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss. Human Reproduction 36 48–60. (https://doi.org/10.1093/humrep/deaa278)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Knöfler M, Haider S, Saleh L, Pollheimer J, Gamage TKJB & & James J 2019 Human placenta and trophoblast development: key molecular mechanisms and model systems. Cellular and Molecular Life Sciences 76 3479–3496. (https://doi.org/10.1007/s00018-019-03104-6)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Koukoura O, Sifakis S & & Spandidos DA 2012 DNA methylation in the human placenta and fetal growth (review). Molecular Medicine Reports 5 883–889. (https://doi.org/10.3892/mmr.2012.763)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Kshirsagar SK, Alam SM, Jasti S, Hodes H, Nauser T, Gilliam M, Billstrand C, Hunt JS & & Petroff MG 2012 Immunomodulatory molecules are released from the first trimester and term placenta via exosomes. Placenta 33 982–990. (https://doi.org/10.1016/j.placenta.2012.10.005)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Lai A, Elfeky O, Rice GE & & Salomon C 2018 Optimized specific isolation of placenta-derived exosomes from the maternal circulation. In Preeclampsia. New York, NY: Humana Press, pp. 131–138.

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Le Bouteiller P, Solier C, Pröll J, Aguerre-Girr M, Fournel S & & Lenfant F 1999 Placental HLA-G protein expression in vivo: where and what for? Human Reproduction Update 5 223–233. (https://doi.org/10.1093/humupd/5.3.223)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Li P, Wang N, Zhang Y, Wang C & & Du L 2021 HLA-G/sHLA-G and HLA-G-bearing extracellular vesicles in cancers: potential role as biomarkers. Frontiers in Immunology 12 791535. (https://doi.org/10.3389/fimmu.2021.791535)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Lin J, Li J, Huang B, Liu J, Chen X, Chen XM, Xu YM, Huang LF, & Wang XZ, Huang LF & & Wang XZ 2015 Exosomes: novel biomarkers for clinical diagnosis. The Scientific World Journal 2015 657086. (https://doi.org/10.1155/2015/657086)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Luo SS, Ishibashi O, Ishikawa G, Ishikawa T, Katayama A, Mishima T, Takizawa T, Shigihara T, Goto T, Izumi A, et al.2009 Human villous trophoblast express and secrete placenta-specific microRNAs into maternal circulation via exosomes. Biology of Reproduction 81 717–729. (https://doi.org/10.1095/biolreprod.108.075481)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Lyu TS, Ahn Y, Im YJ, Kim SS, Lee KH, Kim J, Choi Y, Lee D, Kang E, Jin G, et al.2021 The characterization of exosomes from fibrosarcoma cells and the useful usage of Dynamic Light Scattering (DLS) for their evaluation. PLoS One 16 e0231994. (https://doi.org/10.1371/journal.pone.0231994)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Marić I, Tsur A, Aghaeepour N, Montanari A, Stevenson DK, Shaw GM & & Winn VD 2020 Early prediction of preeclampsia via machine learning. American Journal of Obstetrics and Gynecology MFM 2 100100. (https://doi.org/10.1016/j.ajogmf.2020.100100)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Michels KB, Harris HR & & Barault L 2011 Birthweight, maternal weight trajectories and global DNA methylation of LINE-1 repetitive elements. PLoS One 6 e25254. (https://doi.org/10.1371/journal.pone.0025254)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Mincheva-Nilsson L 2010 Placental exosome-mediated immune protection of the fetus: feeling groovy in a cloud of exosomes. Expert Review of Obstetrics and Gynecology 5 619–634. (https://doi.org/10.1586/eog.10.43)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Mitchell MD, Peiris HN, Kobayashi M, Koh YQ, Duncombe G, Illanes SE, Rice GE, & Salomon C & & Salomon C 2015 Placental exosomes in normal and complicated pregnancy. American Journal of Obstetrics and Gynecology 213(4) S173–S181. (https://doi.org/10.1016/j.ajog.2015.07.001)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Muralimanoharan S, Maloyan A, Mele J, Guo C, Myatt LG & & Myatt L 2012 MIR-210 modulates mitochondrial respiration in the placenta with preeclampsia. Placenta 33 816–823. (https://doi.org/10.1016/j.placenta.2012.07.002)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Non AL, Binder AM, Barault L, Rancourt RC, Kubzansky LD & & Michels KB 2012 DNA methylation of stress-related genes and LINE-1 repetitive elements across the healthy human placenta. Placenta 33 183–187. (https://doi.org/10.1016/j.placenta.2011.12.013)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Papuchova H, Kshirsagar S, Xu L, Bougleux Gomes HA, Li Q, Iyer V, Norwitz ER, Strominger JL & & Tilburgs T 2020 Three types of HLA-G+ extravillous trophoblasts that have distinct immune regulatory properties. PNAS 117 15772–15777. (https://doi.org/10.1073/pnas.2000484117)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Poon LC & & Nicolaides KH 2014 Early prediction of preeclampsia. Obstetrics and Gynecology International 2014 297397. (https://doi.org/10.1155/2014/297397)

  • PrabhuDas M, Bonney E, Caron K, Dey S, Erlebacher A, Fazleabas A, Fisher S, Golos T, Matzuk M, McCune JM, et al.2015 Immune mechanisms at the maternal-fetal interface: perspectives and challenges. Nature Immunology 16 328–334. (https://doi.org/10.1038/ni.3131)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Qian Z, Shen Q, Yang X, Qiu Y & & Zhang W 2015 The role of extracellular vesicles: an epigenetic view of the cancer microenvironment. BioMed Research International 2015 649161. (https://doi.org/10.1155/2015/649161)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Rao A, Shinde U, Das DK, Balasinor N & & Madan T 2023 Early prediction of pre-eclampsia using circulating placental exosomes: newer insights. Indian Journal of Medical Research 158 385–396. (https://doi.org/10.4103/ijmr.ijmr_2143_22)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Rebmann V, Busemann A, Lindemann M & & Grosse-Wilde H 2003 Detection of HLA-G5 secreting cells. Human Immunology 64 1017–1024. (https://doi.org/10.1016/j.humimm.2003.08.354)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Rodosthenous RS, Burris HH, Sanders AP, Just AC, Dereix AE, Svensson K, Solano M, Téllez-Rojo MM, Wright RO & & Baccarelli AA 2017 Second trimester extracellular microRNAs in maternal blood and fetal growth: an exploratory study. Epigenetics 12 804–810. (https://doi.org/10.1080/15592294.2017.1358345)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Romero R, Kusanovic JP, Chaiworapongsa T & & Hassan SS 2011 Placental bed disorders in preterm labor, preterm PROM, spontaneous abortion and abruptio placentae. Best Practice and Research. Clinical Obstetrics and Gynaecology 25 313–327. (https://doi.org/10.1016/j.bpobgyn.2011.02.006)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Sabapatha A, Gercel-Taylor C & & Taylor DD 2006 Specific isolation of placenta-derived exosomes from the circulation of pregnant women and their immunoregulatory consequences. American Journal of Reproductive Immunology 56 345–355. (https://doi.org/10.1111/j.1600-0897.2006.00435.x)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Salomon C, Kobayashi M, Ashman K, Sobrevia L, Mitchell MD & & Rice GE 2013 Hypoxia-induced changes in the bioactivity of cytotrophoblast-derived exosomes. PLoS One 8 e79636. (https://doi.org/10.1371/journal.pone.0079636)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Salomon C, Torres MJ, Kobayashi M, Scholz-Romero K, Sobrevia L, Dobierzewska A, Illanes SE, Mitchell MD & & Rice GE 2014 A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration. PLoS One 9 e98667. (https://doi.org/10.1371/journal.pone.0098667)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Sharma U, Pal D & & Prasad R 2014 Alkaline phosphatase: an overview. Indian Journal of Clinical Biochemistry 29 269–278. (https://doi.org/10.1007/s12291-013-0408-y)

  • Théry C, Amigorena S, Raposo G & & Clayton A 2006 Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protocols in Cell Biology, Chapter 3. (https://doi.org/10.1002/0471143030.cb0322s30)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Tian Y, Gong M, Hu Y, Liu H, Zhang W, Zhang M, Hu X, Aubert D, Zhu S, Wu L, et al.2019 Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry. Journal of Extracellular Vesicles 9 1697028. (https://doi.org/10.1080/20013078.2019.1697028)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Xu P, Ma Y, Wu H & & Wang YL 2021 Placenta-derived microRNAs in the pathophysiology of human pregnancy. Frontiers in Cell and Developmental Biology 9 646326. (https://doi.org/10.3389/fcell.2021.646326)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Zhang R, Wang YQ & & Su B 2008 Molecular evolution of a primate-specific microRNA family. Molecular Biology Evolution 25 1493–1502. (https://doi.org/10.1093/molbev/msn094)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Zhuang B, Shang J & & Yao Y 2021 HLA-G: an important mediator of maternal-fetal immune-tolerance. Frontiers in Immunology 12 744324. (https://doi.org/10.3389/fimmu.2021.744324)

    • PubMed
    • Search Google Scholar
    • Export Citation