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Ovarian Stimulation Altered Uterine Fluid Extracellular Vesicles miRNA Affecting Implantation in Rats

  • Endometrium: Original Article
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Abstract

Uterine fluid (UF) extracellular vesicle (EV) miRNA may affect implantation and could be the potential biomarker of endometrial receptivity (ER). Ovarian stimulation (OS) could damage the ER but its mechanism is still unclear. Here, we evaluate the affections of OS on UF EV miRNA expression and implantation. Female rats were divided into three groups: natural cycle or injection with GnRH-a following HP-HMG or u-FSH. UF was collected on the 5th day of gestation. Affinity membrane columns were utilized to isolate EVs from UF, obtained during implantation flushing. The EV miRNAs were sequenced, and five of them were validated by qRT-PCR. HTR-8/Svneo cells were transfected with miR-223-3p mimic and inhibitor, followed by conducting colony formation, invasion, migration, and adhesion assays to assess the cellular functions. In OS groups, the implantation rate decreased (p < 0.05), and the pinopode was damaged in the OS groups. The EVs were isolated from UF, and the differential expression key miRNAs were involved in several regulation pathways, such as cancer, endocrine, and cell cycles, which were correlated with ER and implantation. Among the miRNAs, miR-223-5p greatly differed and was most consistent with the sequencing results, followed by miR-223-3p and miR-98-5P. miR-223-3p promoted HTR-8/SVneo cells grow and ability of invasion, migration, and adhesion. OS altered UF EVs miRNAs affecting implantation in rats, and miR-223-3p might be the key molecule.

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Data Availability

The original data presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

Abbreviations

ART:

Artificial reproduction technology

ER:

Endometrial receptivity

WOI:

Window of implantation

OS:

Ovarian stimulation

UF:

Uterine fluid

EV:

Extracellular vesicles

MV:

Microvesicles

SD rat:

Sprague–Dawley rat

TEM:

Transmission electron microscopy

DLS:

Dynamic light scattering

qRT-PCR:

Quantitative reverse transcription–polymerase chain reaction

GnRH-a:

Gonadotropin-releasing hormone agonist

HP-hMG:

High-purity human menopausal gonadotropin

U-FSH:

Urine follicle stimulation hormone

References

  1. Psychoyos A. Uterine receptivity for nidation. Ann NY Acad Sci. 1986;476:36–42.

    Article  CAS  PubMed  Google Scholar 

  2. Troncoso C, Bosch E, Rubio C, Remohí J, Simón C, Pellicer A. The origin of biochemical pregnancies: lessons learned from preimplantation genetic diagnosis. Fertil Steril. 2003;79:449–50.

    Article  PubMed  Google Scholar 

  3. Chen C, Yan Q, Liu K, Zhou X, Xian Y, Liang D, et al. Endometrial receptivity markers in mice stimulated with raloxifene versus clomiphene citrate and natural cycles. Reprod Sci. 2016;23:748–55.

    Article  CAS  PubMed  Google Scholar 

  4. Zapantis G, Szmyga MJ, Rybak EA, Meier UT. Premature formation of nucleolar channel systems indicates advanced endometrial maturation following controlled ovarian hyperstimulation. Hum Reprod. 2013;28:3292–300.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wu JL, Keller P, Kanchwala M, Xing C, Babayev SN, Carr BR, et al. Controlled ovarian stimulation protocols alter endometrial histomorphology and gene expression profiles. Reprod Sci. 2020;27:895–904.

    Article  CAS  PubMed  Google Scholar 

  6. Zhang Y, Wang Q, Wang H, Duan E. Uterine fluid in pregnancy: a biological and clinical outlook. Trends Mol Med. 2017;23:604–14.

    Article  PubMed  Google Scholar 

  7. Griffiths GS, Galileo DS, Reese K, Martin-DeLeon PA. Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model. Mol Reprod Dev. 2008;75:1627–36.

    Article  CAS  PubMed  Google Scholar 

  8. Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol. 1985;101:942–8.

    Article  CAS  PubMed  Google Scholar 

  9. Capalbo A, Ubaldi FM, Cimadomo D, Noli L, Khalaf Y, Farcomeni A, et al. MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment. Fertil Steril. 2016;105:225-235.e3.

    Article  CAS  PubMed  Google Scholar 

  10. Fereshteh Z, Bathala P, Galileo DS, Martin-DeLeon PA. Detection of extracellular vesicles in the mouse vaginal fluid: their delivery of sperm proteins that stimulate capacitation and modulate fertility. J Cell Physiol. 2019;234:12745–56.

    Article  CAS  PubMed  Google Scholar 

  11. Homer H, Rice GE, Salomon C. Review: Embryo- and endometrium-derived exosomes and their potential role in assisted reproductive treatments–liquid biopsies for endometrial receptivity. Placenta. 2017;54:89–94.

    Article  CAS  PubMed  Google Scholar 

  12. Vojtech L, Woo S, Hughes S, Levy C, Ballweber L, Sauteraud RP, et al. Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res. 2014;42:7290–304.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hong L, Zang X, Hu Q, He Y, Xu Z, Xie Y, et al. Uterine luminal-derived extracellular vesicles: potential nanomaterials to improve embryo implantation. J Nanobiotechnol. 2023;21:79.

    Article  CAS  Google Scholar 

  14. Abou-Kheir W, Barrak J, Hadadeh O, Daoud G. HTR-8/SVneo cell line contains a mixed population of cells. Placenta. 2017;50:1–7.

    Article  CAS  PubMed  Google Scholar 

  15. Nishida M. The Ishikawa cells from birth to the present. Hum Cell. 2002;15:104–17.

    Article  PubMed  Google Scholar 

  16. Thie M, Denker H-W. In vitro studies on endometrial adhesiveness for trophoblast: cellular dynamics in uterine epithelial cells. CTO. 2002;172:237–52.

    PubMed  Google Scholar 

  17. Occhipinti G, Giulietti M, Principato G, Piva F. The choice of endogenous controls in exosomal microRNA assessments from biofluids. Tumor Biol. 2016;37(9):11657–65.

    Article  CAS  Google Scholar 

  18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods. 2001;25:402–8.

    Article  CAS  PubMed  Google Scholar 

  19. Nikas G, Aghajanova L. Endometrial pinopodes: some more understanding on human implantation? Reprod Biomed Online. 2002;4:18–23.

    Article  PubMed  Google Scholar 

  20. Hsu C-Y, Hsieh T-H, Tsai C-F, Tsai H-P, Chen H-S, Chang Y, et al. miRNA-199a-5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis: miRNA99a-5p in endometriosis. J Pathol. 2014;232:330–43.

    Article  CAS  PubMed  Google Scholar 

  21. Huang K, Dong X, Sui C, Hu D, Xiong T, Liao S, et al. MiR-223 suppresses endometrial carcinoma cells proliferation by targeting IGF-1R. Am J Transl Res. 2014;6:841–9.

    PubMed  PubMed Central  Google Scholar 

  22. Wang M, Ji S, Shao G, Zhang J, Zhao K, Wang Z, et al. Effect of exosome biomarkers for diagnosis and prognosis of breast cancer patients. Clin Transl Oncol. 2018;20:906–11.

    Article  CAS  PubMed  Google Scholar 

  23. Xia H-F, Jin X-H, Cao Z-F, Shi T, Ma X. MiR-98 is involved in rat embryo implantation by targeting Bcl-xl. FEBS Lett. 2014;588:574–83.

    Article  CAS  PubMed  Google Scholar 

  24. Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352/. Cited 2020 Nov 15.

  25. Burns G, Brooks K, Wildung M, Navakanitworakul R, Christenson LK, Spencer TE. Extracellular vesicles in luminal fluid of the ovine uterus. Ye X, editor. PLoS One. 2014;9:e90913.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Chen Q, Zhang Y, Elad D, Jaffa AJ, Cao Y, Ye X, et al. Navigating the site for embryo implantation: biomechanical and molecular regulation of intrauterine embryo distribution. Mol Aspects Med. 2013;34:1024–42.

    Article  PubMed  Google Scholar 

  27. Machtinger R, Laurent LC, Baccarelli AA. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum Reprod Update. 2016;22:182–93.

    CAS  PubMed  Google Scholar 

  28. Syn NL, Wang L, Chow EK-H, Lim CT, Goh B-C. Exosomes in cancer nanomedicine and immunotherapy: prospects and challenges. Trends Biotechnol. 2017;35:665–76.

    Article  CAS  PubMed  Google Scholar 

  29. Cretoiu D, Xu J, Xiao J, Suciu N, Cretoiu SM. Circulating microRNAs as potential molecular biomarkers in pathophysiological evolution of pregnancy. Dis Markers. 2016;2016:3851054.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Shekibi M, Heng S, Nie G. MicroRNAs in the regulation of endometrial receptivity for embryo implantation. Int J Mol Sci. 2022;23:6210.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Liang J, Wang S, Wang Z. Role of microRNAs in embryo implantation. Reprod Biol Endocrinol. 2017;15:90.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Altmäe S, Martinez-Conejero JA, Esteban FJ, Ruiz-Alonso M, Stavreus-Evers A, Horcajadas JA, et al. MicroRNAs miR-30b, miR-30d, and miR-494 regulate human endometrial receptivity. Reprod Sci. 2013;20:308–17.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Cuman C, Van Sinderen M, Gantier MP, Rainczuk K, Sorby K, Rombauts L, et al. Human blastocyst secreted microRNA regulate endometrial epithelial cell adhesion. EBioMedicine. 2015;2:1528–35.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Kropp J, Salih SM, Khatib H. Expression of microRNAs in bovine and human pre-implantation embryo culture media. Front Genet. 2014;5:91.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Mirkin S, Nikas G, Hsiu J-G, Díaz J, Oehninger S. Gene expression profiles and structural/functional features of the peri-implantation endometrium in natural and gonadotropin-stimulated cycles. J Clin Endocrinol Metab. 2004;89:5742–52.

    Article  CAS  PubMed  Google Scholar 

  36. Ng YH, Rome S, Jalabert A, Forterre A, Singh H, Hincks CL, et al. Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation. Ward WS, editor. PLoS One. 2013;8:e58502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kusama K, Nakamura K, Bai R, Nagaoka K, Sakurai T, Imakawa K. Intrauterine exosomes are required for bovine conceptus implantation. Biochem Biophys Res Commun. 2018;495:1370–5.

    Article  CAS  PubMed  Google Scholar 

  38. Ararooti T, Niasari-Naslaji A, Asadi-Moghaddam B, Razavi K, Panahi F. Superovulatory response following FSH, eCG-FSH and hMG and pregnancy rates following transfer of hatched blastocyst embryos with different diameter and shape in dromedary camel. Theriogenology. 2018;106:149–56.

    Article  CAS  PubMed  Google Scholar 

  39. Moro F, Scarinci E, Palla C, Romani F, Familiari A, Tropea A, et al. Highly purified hMG versus recombinant FSH plus recombinant LH in intrauterine insemination cycles in women ≥35 years: a RCT. Hum Reprod. 2015;30:179–85.

    Article  CAS  PubMed  Google Scholar 

  40. Tabata C, Fujiwara T, Sugawa M, Noma M, Onoue H, Kusumi M, et al. Comparison of FSH and hMG on ovarian stimulation outcome with a GnRH antagonist protocol in younger and advanced reproductive age women. Reprod Med Biol. 2015;14:5–9.

    Article  CAS  PubMed  Google Scholar 

  41. Haneklaus M, Gerlic M, O’Neill LAJ, Masters SL. miR-223: infection, inflammation and cancer. J Intern Med. 2013;274:215–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dominguez F, Moreno-Moya JM, Lozoya T, Romero A, Martínez S, Monterde M, et al. Embryonic miRNA profiles of normal and ectopic pregnancies. PLoS One. 2014;9:e102185.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Li C, Chen L, Zhao Y, Chen S, Fu L, Jiang Y, et al. Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model. Gene. 2017;598:20–6.

    Article  CAS  PubMed  Google Scholar 

  44. Dong X, Sui C, Huang K, Wang L, Hu D, Xiong T, et al. MicroRNA-223–3p suppresses leukemia inhibitory factor expression and pinopodes formation during embryo implantation in mice. Am J Transl Res. 2016;8(2):1155–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Shariati MBH, Niknafs B, Seghinsara AM, Shokrzadeh N, Alivand MR. Administration of dexamethasone disrupts endometrial receptivity by alteration of expression of miRNA 223, 200a, LIF, Muc1, SGK1, and ENaC via the ERK1/2-mTOR pathway. J Cell Physiol. 2019;234:19629–39.

    Article  CAS  PubMed  Google Scholar 

  46. Piersanti RL, Horlock AD, Block J, Santos JEP, Sheldon IM, Bromfield JJ. Persistent effects on bovine granulosa cell transcriptome after resolution of uterine disease. Reproduction. 2019;158:35–46.

    Article  CAS  PubMed  Google Scholar 

  47. Zhu H-Y, Ge T-X, Pan Y-B, Zhang S-Y. Advanced role of Hippo signaling in endometrial fibrosis: implications for intrauterine adhesion. Chin Med J (Engl). 2017;130:2732–7.

    Article  CAS  PubMed  Google Scholar 

  48. Sansores-Garcia L, Atkins M, Moya IM, Shahmoradgoli M, Tao C, Mills GB, et al. Mask is required for the activity of the Hippo pathway effector Yki/YAP. Curr Biol. 2013;23:229–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors appreciate the technical expertise of the team led by Professor Wu of the Lab of Biomedical Electronic Microscopy Higher Research Center, Central South University, Central South University. We would also like to thank everyone in the Reproductive Medicine Center, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China, for their kind help and encouragement.

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Authors and Affiliations

  1. Department of Reproductive Medicine, Xiangya Hospital, Central South University, NO.87, Xiangya Road, Kaifu District, Changsha, Hunan, China

    Xi Huang, Jing Zhao, Qiong Zhang, Yonggang Wang & Yanping Li

  2. Clinical Research Center for Women’s Reproductive Health in Hunan Province, NO.87, Xiangya Road, Kaifu District, Changsha, Hunan, China

    Xi Huang, Jing Zhao, Qiong Zhang, Yonggang Wang & Yanping Li

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  1. Xi Huang
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Contributions

Y.L. was involved in study concept and supervised the project. X. H. was involved in study design, performing all experiments, data analysis, and writing manuscript. L. X. was involved in experiments. J. Z., Q. Z., and Y. W. provided experiments guide and revised manuscript and figures. All the authors listed have approved the manuscript that is enclosed and agreed to the order of authors.

Corresponding author

Correspondence to Yanping Li.

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Ethical Approval

All experiments were approved by the Institutional Animal Care and Use Committee of Central South University (animal ethics number: 2020sydw0136) and were conducted in accordance with the National Institute for Health Guide for Care and Use of Laboratory Animal.

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Huang, X., Zhao, J., Zhang, Q. et al. Ovarian Stimulation Altered Uterine Fluid Extracellular Vesicles miRNA Affecting Implantation in Rats. Reprod. Sci. (2024). https://doi.org/10.1007/s43032-023-01448-w

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