Hostname: page-component-586b7cd67f-tf8b9 Total loading time: 0 Render date: 2024-11-26T00:07:08.889Z Has data issue: false hasContentIssue false
  • English
  • Français

Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos

Published online by Cambridge University Press:  29 October 2020

Daniel Veraguas Open the ORCID record for Daniel Veraguas [Opens in a new window] ,
Constanza Aguilera ,
Carlos Henriquez ,
Alejandra E. Velasquez Open the ORCID record for Alejandra E. Velasquez [Opens in a new window] ,
Barbara Melo-Baez ,
Pedro Silva-Ibañez ,
Fidel O. Castro  and
Lleretny Rodriguez-Alvarez
Daniel Veraguas
Affiliation:
Universidad de Concepción, Chillán, Chile
Constanza Aguilera
Affiliation:
Universidad de Concepción, Chillán, Chile
Carlos Henriquez
Affiliation:
Centro de Reproducción Asistida y Especialidades de la Mujer (CRAM), Concepción, Chile
Alejandra E. Velasquez
Affiliation:
Universidad de Concepción, Chillán, Chile Centro de Reproducción Asistida y Especialidades de la Mujer (CRAM), Concepción, Chile
Barbara Melo-Baez
Affiliation:
Universidad de Concepción, Chillán, Chile
Pedro Silva-Ibañez
Affiliation:
Universidad de Concepción, Chillán, Chile
Fidel O. Castro
Affiliation:
Universidad de Concepción, Chillán, Chile
Lleretny Rodriguez-Alvarez*
Affiliation:
Universidad de Concepción, Chillán, Chile
*
Author for correspondence: Lleretny Rodriguez-Alvarez. Universidad de Concepción, Chillán, Chile. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.

Keywords

Chromosome abnormalitiesEmbryo qualityExosomesMicrovesiclesPreimplantation diagnosis
Type
Research Article
Information
Zygote , Volume 29 , Issue 2 , April 2021 , pp. 138 - 149
Copyright
© The Author(s), 2020. Published by Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abu-Halima, M, Häusler, S, Backes, C, Fehlmann, T, Staib, C, Nestel, S, Nazarenko, I, Meese, E and Keller, A (2017). Micro-ribonucleic acids and extracellular vesicles repertoire in the spent culture media is altered in women undergoing in vitro fertilization. Sci Rep 7, 1–11.CrossRefGoogle ScholarPubMed
Al-Dossary, AA, Strehler, EE and Martin-DeLeon, PA (2013). Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm. PLoS One 8, e80181.10.1371/journal.pone.0080181CrossRefGoogle ScholarPubMed
Al-Yatama, MK, Mustafa, AS, Ali, S, Abraham, S, Khan, Z and Khaja, N (2001). Detection of Y chromosome-specific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction. Prenat Diagn 21, 399–402.CrossRefGoogle Scholar
Barbash-Hazan, S, Frumkin, T, Malcov, M, Yaron, Y, Cohen, T, Azem, F, Amit, A and Ben-Yosef, D (2009). Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential. Fertil Steril 92, 890–6.CrossRefGoogle ScholarPubMed
Bonduelle, M, Van Assche, E, Joris, H, Keymolen, K, Devroey, P, Van Steirteghem, A and Liebaers, I (2002). Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod 17, 2600–14.CrossRefGoogle ScholarPubMed
Bridi, A, Perecin, F and Silveira, JCD (2020). Extracellular vesicles mediated early embryo–maternal interactions. Int J Mol Sci 21, 1163.CrossRefGoogle ScholarPubMed
Capalbo, A, Romanelli, V, Patassini, C, Poli, M, Girardi, L, Giancani, A, Stoppa, M, Cimadomo, D, Ubaldi, FM and Rienzi, L (2018). Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions. Fertil Steril 110, 870–9.10.1016/j.fertnstert.2018.05.031CrossRefGoogle Scholar
Dahdouh, EM, Balayla, J, Audibert, F, Genetics Committee, Wilson, RD, Audibert, F, Brock, JA, Campagnolo, C, Carroll, J, Chong, K, Gagnon, A, Johnson, JA, MacDonald, W, Okun, N, Pastuck, M and Vallée-Pouliot, K (2015). Technical update: preimplantation genetic diagnosis and screening. J Obstet Gynaecol Can 37, 451–63.10.1016/S1701-2163(15)30261-9CrossRefGoogle ScholarPubMed
Daughtry, BL, Rosenkrantz, JL, Lazar, NH, Fei, SS, Redmayne, N, Torkenczy, KA, Adey, A, Yan, M, Gao, L, Park, B, Nevonen, KA, Carbone, L and Chavez, SL (2019). Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion. Genome Res 29, 367–82.CrossRefGoogle ScholarPubMed
Dragovic, RA, Gardiner, C, Brooks, AS, Tannetta, DS, Ferguson, DJ, Hole, P, Carr, B, Redman, CWG, Harris, AL, Dobson, PJ, Harrison, P and Sargent, IL (2011). Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine 7, 780–8.10.1016/j.nano.2011.04.003CrossRefGoogle ScholarPubMed
Fang, R, Yang, W, Zhao, X, Xiong, F, Guo, C, Xiao, J, Chen, L, Song, X, Wang, H, Chen, J, Xiao, X, Yao, B and Cai, L (2019). Chromosome screening using culture medium of embryos fertilised in vitro: a pilot clinical study. J Transl Med 17, 73.CrossRefGoogle ScholarPubMed
Giacomini, E, Vago, R, Sanchez, AM, Podini, P, Zarovni, N, Murdica, V, Rizzo, R, Bortolotti, D, Candiani, M and Viganò, P (2017). Secretome of in vitro cultured human embryos contains extracellular vesicles that are uptaken by the maternal side. Sci Rep 7, 5210.10.1038/s41598-017-05549-wCrossRefGoogle ScholarPubMed
Grifo, JA, Hodes-Wertz, B, Lee, HL, Amperloquio, E, Clarke-Williams, M and Adler, A (2013). Single thawed euploid embryo transfer improves IVF pregnancy, miscarriage, and multiple gestation outcomes and has similar implantation rates as egg donation. J Assist Reprod Genet 30, 259–64.CrossRefGoogle Scholar
Hammond, ER, Shelling, AN and Cree, LM (2016). Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use. Hum Reprod 31, 1653–1661.10.1093/humrep/dew132CrossRefGoogle ScholarPubMed
Harding, CV, Heuser, JE and Stahl, PD (2013). Exosomes: looking back three decades and into the future. J Cell Biol 200, 367–71.CrossRefGoogle ScholarPubMed
Hung, WT, Hong, X, Christenson, LK and McGinnis, LK (2015). Extracellular vesicles from bovine follicular fluid support cumulus expansion. Biol Reprod 93, 117.CrossRefGoogle ScholarPubMed
Ishikawa, H, Ma, Z and Barber, GN (2009). STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–92.CrossRefGoogle ScholarPubMed
Kalluri, R and LeBleu, VS (2016). Discovery of double-stranded genomic DNA in circulating exosomes. Cold Spring Harb Symp Quant Biol 81, 275–80.CrossRefGoogle ScholarPubMed
Kowal, J, Arras, G, Colombo, M, Jouve, M, Morath, JP, Primdal-Bengtson, B, Dingli, F, Loew, D, Tkach, M and Théry, C (2016). Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci 113, E968–77.CrossRefGoogle ScholarPubMed
Lee, SH, Oh, HJ, Kim, MJ and Lee, BC (2020). Canine oviductal exosomes improve oocyte development via EGFR/MAPK signaling pathway. Reproduction 160, 613–25.CrossRefGoogle ScholarPubMed
Lehmann, BD, Paine, MS, Brooks, AM, McCubrey, JA, Renegar, RH, Wang, R and Terrian, DM (2008). Senescence-associated exosome release from human prostate cancer cells. Cancer Res 68, 7864–71.CrossRefGoogle ScholarPubMed
Lopera-Vasquez, R, Hamdi, M, Maillo, V, Gutierrez-Adan, A, Bermejo-Alvarez, P, Ramírez, MÁ, Yáñez-Mó, M, Rizos, D (2017). Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro . Reproduction 153, 461–70.10.1530/REP-16-0384CrossRefGoogle ScholarPubMed
Machtinger, R, Laurent, LC and Baccarelli, AA (2015). Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum Reprod Update 22, 182–93.Google ScholarPubMed
Mariani, BD, Stern, HJ, Novik, V, Tran, KD and Stanley, WS (2014). Comparison of array CGH and next generation sequencing for detection of a cryptic translocation identified by PGD. Fertil Steril 102, e178–9.CrossRefGoogle Scholar
Marin, D and Scott, RT (2018). Extracellular vesicles: a promising tool for assessment of embryonic competence. Curr Opin Obstet Gynecol 30, 171–8.CrossRefGoogle ScholarPubMed
Mastenbroek, S, Twisk, M, van Echten-Arends, J, Sikkema-Raddatz, B, Korevaar, JC, Verhoeve, HR, Vogel, NE, Arts, EG, de Vries, JW, Bossuyt, PM, Buys, CH, Heineman, MJ, Repping, S and van der Veen, F (2007). In vitro fertilization with preimplantation genetic screening. N Engl J Med 357, 9–17.CrossRefGoogle ScholarPubMed
Mellisho, EA, Velásquez, AE, Nuñez, MJ, Cabezas, JG, Cueto, JA, Fader, C, Castro, FO and Rodríguez-Álvarez, L (2017). Identification and characteristics of extracellular vesicles from bovine blastocysts produced in vitro . PLoS One 12, e0178306.10.1371/journal.pone.0178306CrossRefGoogle ScholarPubMed
Munné, S (2012). Preimplantation genetic diagnosis for aneuploidy and translocations using array comparative genomic hybridization. Curr Genomics 13, 463–70.CrossRefGoogle ScholarPubMed
Munné, S, Magli, C, Adler, A, Wright, G, De Boer, K, Mortimer, D, Tucker, M, Cohen, J and Gianaroli, L (1997). Treatment-related chromosome abnormalities in human embryos. Hum Reprod 12, 780–4.CrossRefGoogle ScholarPubMed
Munné, S, Kaplan, B, Frattarelli, JL, Child, T, Nakhuda, G, Shamma, FN, Silverberg, K, Kalista, T, Handyside, AH, Katz-Jaffe, M, Wells, D, Gordon, T, Stock-Myer, S and Willman, S (2019). Preimplantation genetic testing for aneuploidy versus morphology as selection criteria for single frozen–thawed embryo transfer in good-prognosis patients: a multicenter randomized clinical trial. Fertil Steril 112, 1071–9.10.1016/j.fertnstert.2019.07.1346CrossRefGoogle ScholarPubMed
Ng, YH, Rome, S, Jalabert, A, Forterre, A, Singh, H, Hincks, CL and Salamonsen, LA (2013). Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation. PLoS One 8, e58502.10.1371/journal.pone.0058502CrossRefGoogle ScholarPubMed
Okun, N and Sierra, S (2014). Pregnancy outcomes after assisted human reproduction. J Obstet Gynaecol Can 36, 64–83.CrossRefGoogle ScholarPubMed
Pallinger, E, Bognar, Z, Bodis, J, Csabai, T, Farkas, N, Godony, K, Varnagy, A, Buzas, E and Szekeres-Bartho, J (2017). A simple and rapid flow cytometry-based assay to identify a competent embryo prior to embryo transfer. Sci Rep 7, 39927.CrossRefGoogle ScholarPubMed
Pavani, KC, Hendrix, A, Van Den Broeck, W, Couck, L, Szymanska, K, Lin, X, De Koster, J, Van Soom, A and Leemans, B (2019). Isolation and characterization of functionally active extracellular vesicles from culture medium conditioned by bovine embryos in vitro. Int J Mol Sci 20, 38.CrossRefGoogle Scholar
Raposo, G and Stoorvogel, W (2013). Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200, 373–83.CrossRefGoogle ScholarPubMed
Simon, B, Bolumar, D, Amadoz, A, Jimenez-Almazán, J, Valbuena, D, Vilella, F and Moreno, I. (2020). Identification and characterization of extracellular vesicles and its DNA cargo secreted during murine embryo development. Genes 11, 203.CrossRefGoogle ScholarPubMed
Stigliani, S, Anserini, P, Venturini, PL and Scaruffi, P (2013). Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation. Hum Reprod 28, 2652–60.10.1093/humrep/det314CrossRefGoogle ScholarPubMed
Stroun, M, Lyautey, J, Lederrey, C, Olson-Sand, A and Anker, P (2001). About the possible origin and mechanism of circulating DNA: apoptosis and active DNA release. Clin Chim Acta 313, 139–42.CrossRefGoogle ScholarPubMed
Takahashi, A, Okada, R, Nagao, K, Kawamata, Y, Hanyu, A, Yoshimoto, S, Takasugi, M, Watanabe, S, Kanemaki, MT, Obuse, C and Hara, E (2017). Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nat Commun 8, 15287.10.1038/ncomms15287CrossRefGoogle ScholarPubMed
Vanneste, E, Voet, T, Le Caignec, C, Ampe, M, Konings, P, Melotte, C, Debrock, S, Amyere, M, Vikkula, M, Schuit, F, Fryns, JP, Verbeke, G, D’Hooghe, T and Moreau, Y (2009). Chromosome instability is common in human cleavage-stage embryos. Nat Med 15, 577–83.CrossRefGoogle ScholarPubMed
Vyas, P, Balakier, H and Librach, CL (2019). Ultrastructural identification of CD9 positive extracellular vesicles released from human embryos and transported through the zona pellucida. Syst Biol Reprod Med 4, 273–80.CrossRefGoogle Scholar
Wade, JJ, MacLachlan, V and Kovacs, G (2015). The success rate of IVF has significantly improved over the last decade. Aust N Z J Obstet Gynaecol 55, 473–6.CrossRefGoogle ScholarPubMed
Wang, J and Sauer, MV (2006). In vitro fertilization (IVF): a review of 3 decades of clinical innovation and technological advancement. Ther Clin Risk Manag 2, 355.CrossRefGoogle ScholarPubMed
Weghofer, A, Munné, S, Brannath, W, Chen, S, Tomkin, G, Cekleniak, N, Garrisi, M, Barad, D, Cohen, J and Gleicher, N (2008). The impact of LH-containing gonadotropins on diploidy rates in preimplantation embryos: long protocol stimulation. Hum Reprod 23, 499–503.CrossRefGoogle ScholarPubMed
Wilton, L (2002). Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review. Prenat Diagn 22, 512–8.CrossRefGoogle ScholarPubMed
Xu, J, Fang, R, Chen, L, Chen, D, Xiao, JP, Yang, W, Wang, H, Song, X, Ma, T, Bo, S, Shi, C, Ren, J, Huang, L, Cai, L, Yao, B, Xie, S and Shi, C (2016). Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proc Natl Acad Sci USA 113, 11907–12.CrossRefGoogle ScholarPubMed
Yang, L, Lv, Q, Chen, W, Sun, J, Wu, Y, Wang, Y, Chen, X, Chen, X and Zhang, Z (2017). Presence of embryonic DNA in culture medium. Oncotarget 8, 67805–9.CrossRefGoogle ScholarPubMed