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Cai L, Lv M, Wei J, Liu C, Li Y, Liao Z, Li T, Zhang H, Xi L, Sui C. Mir-218-5p from Extracellular Vesicles of Endometrium in Patients with Recurrent Implantation Failure Impairs Pre-Implantation Embryo Development. Int J Nanomedicine 2025; 20:5661-5679. [PMID: 40331233 PMCID: PMC12052006 DOI: 10.2147/ijn.s508491] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2024] [Accepted: 04/23/2025] [Indexed: 05/08/2025] Open
Abstract
Background Recurrent implantation failure (RIF) presents a crucial obstacle to in vitro fertilization success. Previous research has shown that small extracellular vesicles (EVs) from endometrial RIF patients hinder embryo development, yet the underlying mechanism and potential solutions remain largely unexplored. In this study, we aimed to investigate the effectiveness of miR-218-5p as a molecular factor in RIF-EVs. Our findings revealed that miR-218-5p disrupted mouse embryo development, and this effect could be reversed by engineered extracellular vesicles (E-EVs) containing anti-miR-218-5p. Methods The percentage of blastocyst development and hatching rates, embryo morphology, and the total cell number were measured. RNA-sequencing was used to analyze transcriptional changes in embryos post miR-218-5p agomir treatment. The abnormal segregation genes of trophectoderm (TE) and inner cell mass (ICM) were visualized via qRT-PCR and immunofluorescence staining. The E-EVs were using the EVs derived from Human Umbilical Cord Mesenchymal Stem Cells (HUMSCs). Characteristics of the EVs were measured using Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs internalization was visualized using BODIPY TR ceramide staining. Results Mouse embryos were arrested at the morula stage and demonstrated reduced blastocyst and hatching rates following miR-218-5p agomir treatment (P < 0.001). Essential transcription factors for TE and ICM, such as Cdx2, Yap1, Sox2, Nanog, Tead4, were reduced at the mRNA level in the miR-218-5p treated morula. This was accompanied by decreased Cdx2 protein levels at the 8-16-cell stage (P < 0.001) and disruption of co-localization of Yap1 and Cdx2. The blastocyte rate was increased by anti-miR-218-5p-encapsulated E-EVs compared with miR-218-5p group (P < 0.001). Conclusion This study offers valuable insights into the potential role of miR-218-5p in RIF and presents. The utilization of engineered vesicles containing anti-miR-218-5p may present a promising avenue for patients facing challenges with RIF.
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Affiliation(s)
- Lei Cai
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China
| | - Mingwei Lv
- Department of Gynecological Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China
- National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center, Key Laboratory of the Ministry of Education, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China
| | - Jianbo Wei
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China
| | - Chang Liu
- Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medicine School, Nanjing, 210000, People’s Republic of China
| | - Yuehan Li
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China
| | - Zhiqi Liao
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China
| | - Tianhui Li
- State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong
| | - Hanwang Zhang
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China
| | - Ling Xi
- Department of Gynecological Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China
- National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center, Key Laboratory of the Ministry of Education, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China
| | - Cong Sui
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China
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Otsuki N, Kato T, Yokomura M, Urano M, Matsuo M, Kobayashi E, Haginoya K, Awano H, Takeshima Y, Saito T, Saito K. Analysis of SMN protein in umbilical cord blood and postnatal peripheral blood of neonates with SMA: a rationale for prompt treatment initiation to prevent SMA development. Orphanet J Rare Dis 2025; 20:91. [PMID: 40022154 PMCID: PMC11869478 DOI: 10.1186/s13023-025-03597-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Accepted: 02/08/2025] [Indexed: 03/03/2025] Open
Abstract
BACKGROUND Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disease caused by insufficient functional survival motor neuron protein (SMN). The SMN expression level in the spinal cord is highest during the 2nd trimester of the foetal period. We previously reported the SMN spot analysis in peripheral blood using imaging flow cytometry (IFC) as a biomarker of functional SMN protein expression. In this study, we analysed neonatal cord blood, postnatal peripheral blood, and maternal peripheral blood in presymptomatic five infants whose sibling has type 1 SMA to estimate prenatal and postnatal SMN dynamics before the onset of severe SMA. RESULTS Data from 37 untreated patients with SMA showed that SMN-spot+ cells were significantly correlated with SMA clinical classification and the copy numbers of the SMN2 gene. The range of values for cord blood, converted from each SMN2 copy number statistics, was - 0.7 to + 2.0 standard deviation (SD) (0.1-24.0%) for SMN-spot+ cells in patients with SMA. Subsequent analyses of the peripheral blood of neonates ranged from - 0.8 to + 0.8 SD (0.4-15.2%). The analysis of each maternal blood, converted from carrier statistics, ranged from - 0.2 to + 2.4 SD (1.4-25.2%). A correlation was observed between the cord blood and maternal peripheral blood. CONCLUSIONS This study suggests that the status of the motor neuron pool in the spinal cord can be presumed by cord blood SMN-spot+ cells and that SMN protein depletion determines the timing of disease onset. As the SMN spot analysis values tended to decrease with time after birth, they may eventually lead to the development of SMA. Furthermore, a correlation was found between the SMN spot analysis values of neonatal cord blood and maternal blood, which predicts disease severity after birth. In other words, the SMN protein supplied from the mother to the foetus may suppress the development of SMA in the infant at birth, and depletion of the SMN protein may occur after birth, causing the infant to develop SMA. Our findings demonstrated the effectiveness of newborn screening and the potential of maternally mediated treatment strategies by providing a rationale for prompt treatment initiation in SMA.
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Affiliation(s)
- Noriko Otsuki
- Institute of Medical Genetics, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Tamaki Kato
- Institute of Medical Genetics, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Mamoru Yokomura
- Institute of Medical Genetics, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Mari Urano
- Institute of Medical Genetics, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Mari Matsuo
- Institute of Medical Genetics, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Emiko Kobayashi
- Department of Pediatrics, Gifu Prefectural General Medical Center, 4-6-1 Noisshiki, Gifu City, Gifu, 500-8717, Japan
| | - Kazuhiro Haginoya
- Department of Pediatric Neurology, Miyagi Children's Hospital, 4-3-17 Ochiai, Aoba-ku, Sendai City, Miyagi, 989-3126, Japan
| | - Hiroyuki Awano
- Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe City, Hyogo, 650-0017, Japan
- Organization for Research Initiative and Promotion, Tottori University, 36-1 Nishi-cho, Yonago City, Tottori, 683-8503, Japan
| | - Yasuhiro Takeshima
- Department of Pediatrics, Hyogo Medical University, 1-1 Mukogawacho, Nishinomiya City, Hyogo, 663-8501, Japan
| | - Toshio Saito
- Division of Child Neurology, Department of Neurology, National Hospital Organization Osaka Toneyama Medical Center, 5-1-1 Toneyama, Toyonaka City, Osaka, 560-8552, Japan
| | - Kayoko Saito
- Institute of Medical Genetics, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan.
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Bridi A, Sangalli JR, Nociti RP, Dos Santos AC, Alves L, Bastos NM, Ferronato GDÁ, Rosa PMDS, Fiorenza MF, Pugliesi G, Meirelles FV, Chiaratti MR, da Silveira JC, Perecin F. Small extracellular vesicles derived from the crosstalk between early embryos and the endometrium potentially mediate corpus luteum function†. Biol Reprod 2025; 112:54-69. [PMID: 39388257 DOI: 10.1093/biolre/ioae143] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2023] [Revised: 10/10/2023] [Accepted: 10/08/2024] [Indexed: 10/15/2024] Open
Abstract
The first interactions among the embryo, endometrium, and corpus luteum are essential for pregnancy success. Small extracellular vesicles (sEVs) are part of these interactions. We previously demonstrated that small extracellular vesicles from in vivo- or in vitro-produced bovine embryos contain different miRNA cargos. Herein we show: (1) the presence and origin (in vivo or in vitro) of the blastocyst differentially reprograms endometrial transcriptional profiles; (2) the endometrial explant (EE) cultured with in vivo or in vitro embryos release small extracellular vesicles with different miRNA contents, and (3) the luteal explant (CLE) exposed to these small extracellular vesicles have distinct mRNA and miRNA profiles. To elucidate this, the endometrial explant were cultured in the presence or absence of a single Day-7 in vivo (EE-artificial insemination; EE-AI) or in vitro (EE-in vitro fertilization; EE-IVF) embryo. After of culture we found, in the endometrial explant, 45 and 211 differentially expressed genes associated with embryo presence and origin, respectively. Small extracellular vesicles were recovered from the conditioned media (CM) in which endometrial explant and embryos were co-cultured. Four miRNAs were differentially expressed between small extracellular vesicles from CC-EE-AI and CC-EE-IVF. Luteal explants exposed in culture to these small extracellular vesicles showed 1360 transcripts and 15 miRNAs differentially expressed. The differentially expressed genes associated with embryo presence and origin, modulating cells' proliferation, and survival. These results demonstrate that in vivo- or in vitro-produced bovine embryos induce molecular alterations in the endometrium; and that the embryo and endometrium release small extracellular vesicles capable of modifying the messenger RNA (mRNA) and miRNA profile in the corpus luteum. Therefore, the small extracellular vesicles-mediated embryo-endometrium-corpus luteum interactions possibly regulate the corpus luteum viability to ensure pregnancy success.
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Affiliation(s)
- Alessandra Bridi
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Juliano Rodrigues Sangalli
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Ricardo Perecin Nociti
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Angélica Camargo Dos Santos
- Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washington Luís, km 235, 13565-905, São Carlos, Brazil
| | - Luana Alves
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Natália Marins Bastos
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Giuliana de Ávila Ferronato
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Paola Maria da Silva Rosa
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Mariani Farias Fiorenza
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Guilherme Pugliesi
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, 05508-270, São Paulo, Brazil
| | - Flávio Vieira Meirelles
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Marcos Roberto Chiaratti
- Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washington Luís, km 235, 13565-905, São Carlos, Brazil
| | - Juliano Coelho da Silveira
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
| | - Felipe Perecin
- Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, 13635-900, Pirassununga, Brazil
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de Albornoz EC, Arroyo JAD, Iriarte YF, Vendrell X, Vidal VM, Roig MC. Non Invasive Preimplantation Testing for Aneuploidies in Assisted Reproduction: A SWOT Analysis. Reprod Sci 2025; 32:1-14. [PMID: 39433699 DOI: 10.1007/s43032-024-01698-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2024] [Accepted: 09/16/2024] [Indexed: 10/23/2024]
Abstract
The implementation of non-invasive PGT-A offers a new strategy to genetically assess the preimplantation embryo and to enhance IVF results. The extraction of DNA from the embryo culture medium has been sufficiently demonstrated, and the ability to obtain chromosomal information as a result is particularly interesting. As morphological criteria have proven to have a weak correlation with embryo ploidy status, this technique emerges as a promising alternative for embryo selection. It also appears reasonable that avoiding biopsy may enhance further embryo development. However, there are growing concerns regarding several aspects of this technique, such as the origin of this cell free DNA, the degree of representativeness of the whole embryo, the need for extended culture or the absence of standardized protocols. Despite the published data on good prognosis couples are promising, niPGT-A is yet to be considered a substitute for trophectoderm biopsy. The current SWOT analysis aims to summarize both resolved and unresolved issues, as well as limiting aspects of niPGT-A.
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Affiliation(s)
- Elena Carrillo de Albornoz
- Hospital Ruber Internacional, Madrid, Spain
- Doctoral Program in Medicine and Surgery, Universidad Autonoma of Madrid, C. Arzobispo Morcillo, Madrid, 28029, Spain
| | | | | | | | | | - María Carrera Roig
- Universidad Europea, Madrid, España.
- Universidad Complutense, Madrid, España.
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Chaves MA, Ferst JG, Fiorenza MF, Vit FF, da Silveira JC. The Influence of Ovarian-Derived Extracellular Vesicles in Reproduction. ADVANCES IN ANATOMY, EMBRYOLOGY, AND CELL BIOLOGY 2025. [PMID: 39741214 DOI: 10.1007/102_2024_9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
Abstract
In this chapter, we explore the multifaceted roles of extracellular vesicles (EVs) in ovarian biology, focusing on their contributions to folliculogenesis, oocyte competence, corpus luteum function, and immune response regulation. EVs, particularly those derived from follicular fluid (ffEVs), are crucial mediators of cell-to-cell communication within the ovarian follicle, influencing processes such as meiotic progression, stress response, and hormonal regulation. We review preexisting literature, highlighting key findings on the molecular cargo of EVs, such as miRNAs and proteins, and their involvement in regulating the function of the follicle cells. Additionally, the influence of EVs on the immune responses within the ovary was also addressed. Some attention is given to the potential of EVs as non-invasive biomarkers and therapeutic tools, particularly in addressing conditions like premature ovarian insufficiency and polycystic ovary syndrome. By discussing the existing challenges and emerging research, we hope that this chapter will provide a deeper understanding of EVs' therapeutic potential and offer insights or suggestions for advancing assisted reproductive technologies.
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Affiliation(s)
- Matheus A Chaves
- Laboratory of Molecular Morphophysiology and Development, Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil
| | - Juliana G Ferst
- Laboratory of Molecular Morphophysiology and Development, Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil
| | - Mariani F Fiorenza
- Laboratory of Molecular Morphophysiology and Development, Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil
| | - Franciele F Vit
- Laboratory of Molecular Morphophysiology and Development, Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil
| | - Juliano C da Silveira
- Laboratory of Molecular Morphophysiology and Development, Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil.
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Fonteles CSR, Enterria-Rosales J, Lin Y, Steele JW, Villarreal-Leal RA, Xiao J, Idowu DI, Burgelin B, Wlodarczyk BJ, Finnell RH, Corradetti B. Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations. Stem Cell Res Ther 2024; 15:466. [PMID: 39639397 PMCID: PMC11622670 DOI: 10.1186/s13287-024-04082-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2024] [Accepted: 11/27/2024] [Indexed: 12/07/2024] Open
Abstract
BACKGROUND Mesenchymal stem cells (MSCs) derived from gestational tissues offer a promising avenue for prenatal intervention in congenital malformations although their application is hampered by concerns related to cellular plasticity and the need for invasive, high-risk surgical procedures. Here, we present naturally occurring exosomes (EXOs) isolated from amniotic fluid-derived MSCs (AF-MSCs) and their mimetic analogs (MIMs) as viable, reproducible, and stable alternatives. These nanovesicles present a minimally invasive therapeutic option, addressing the limitations of MSC-based treatments while retaining therapeutic efficacy. METHODS MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. A physicochemical, structural, and molecular comparison was conducted with exosomes (EXOs) released from the same batch of cells. Additionally, their distribution patterns in female mice were evaluated following in vivo administration, along with an assessment of their safety profile throughout gestation in a mouse strain predisposed to neural tube defects (NTDs). The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake in two different cell types(fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams. RESULTS Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a threefold greater yield. Biodistribution patterns following intraperitoneal administration were comparable between the two particle types, with the uterus being among targeted organs. No toxic effects were observed in the dams during gestation, nor were there any malformations or significant differences in the number of viable versus dead fetuses detected. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo. CONCLUSIONS The present data confirms the potential application of EXOs and MIMs as suitable tools for prevention and treatment of NTDs and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.
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Affiliation(s)
- Cristiane S R Fonteles
- Departamento de Clínica Odontológica. Faculdade de Farmácia, Odontologia E Enfermagem, Universidade Federal Do Ceara. Rua Monsenhor Furtado, S/N-Rodolfo Teófilo, Fortaleza, Brazil
| | - Julia Enterria-Rosales
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - Ying Lin
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - John W Steele
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - Ramiro A Villarreal-Leal
- Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX, USA
- Escuela de Medicina y Ciencias de La Salud, Tecnologico de Monterrey, Monterrey, Mexico
| | - Jing Xiao
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - Daniel I Idowu
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - Beck Burgelin
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - Bogdan J Wlodarczyk
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - Richard H Finnell
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
- Departments of Molecular and Human Genetics Molecular & Cellular Biology and Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA
| | - Bruna Corradetti
- Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA.
- Department of Medicine, Section Oncology/Hematology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA.
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7
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Fonteles CSR, Steele JW, Idowu DI, Burgelin B, Finnell RH, Corradetti B. Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations. RESEARCH SQUARE 2024:rs.3.rs-4325422. [PMID: 38883749 PMCID: PMC11177991 DOI: 10.21203/rs.3.rs-4325422/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/18/2024]
Abstract
Background Mesenchymal stem cells (MSCs) from gestational tissues represent promising strategies for in utero treatment of congenital malformations, but plasticity and required high-risk surgical procedures limit their use. Here we propose natural exosomes (EXOs) isolated from amniotic fluid-MSCs (AF-MSCs), and their mimetic counterparts (MIMs), as valid, stable, and minimally invasive therapeutic alternatives. Methods MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. Physiochemical and molecular characterization was performed to compare them to EXOs released from the same number of cells. The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake (using two different cell types, fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams. Results Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a 3-fold greater yield. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein (GFP) in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo. Conclusions The present data confirms the potential application of EXOs for the prenatal repair of neural tube defects and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.
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Lacconi V, Massimiani M, Carriero I, Bianco C, Ticconi C, Pavone V, Alteri A, Muzii L, Rago R, Pisaturo V, Campagnolo L. When the Embryo Meets the Endometrium: Identifying the Features Required for Successful Embryo Implantation. Int J Mol Sci 2024; 25:2834. [PMID: 38474081 DOI: 10.3390/ijms25052834] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Revised: 02/22/2024] [Accepted: 02/27/2024] [Indexed: 03/14/2024] Open
Abstract
Evaluation of the optimal number of embryos, their quality, and the precise timing for transfer are critical determinants in reproductive success, although still remaining one of the main challenges in assisted reproduction technologies (ART). Indeed, the success of in vitro fertilization (IVF) treatments relies on a multitude of events and factors involving both the endometrium and the embryo. Despite concerted efforts on both fronts, the overall success rates of IVF techniques continue to range between 25% and 30%. The role of the endometrium in implantation has been recently recognized, leading to the hypothesis that both the "soil" and the "seed" play a central role in a successful pregnancy. In this respect, identification of the molecular signature of endometrial receptivity together with the selection of the best embryo for transfer become crucial in ART. Currently, efforts have been made to develop accurate, predictive, and personalized tests to identify the window of implantation and the best quality embryo. However, the value of these tests is still debated, as conflicting results are reported in the literature. The purpose of this review is to summarize and critically report the available criteria to optimize the success of embryo transfer and to better understand current limitations and potential areas for improvement.
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Affiliation(s)
- Valentina Lacconi
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy
- Saint Camillus International University of Health Sciences, Via di Sant'Alessandro 8, 00131 Rome, Italy
| | - Micol Massimiani
- Saint Camillus International University of Health Sciences, Via di Sant'Alessandro 8, 00131 Rome, Italy
| | - Ilenia Carriero
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy
| | - Claudia Bianco
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy
| | - Carlo Ticconi
- Department of Surgical Sciences, Section of Gynaecology and Obstetrics, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy
| | - Valentina Pavone
- Reproductive Sciences Laboratory, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Alessandra Alteri
- Obstetrics and Gynaecology Unit, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy
| | - Ludovico Muzii
- Department of Maternal and Child Health and Urological Sciences, "Sapienza" University of Rome, Policlinico Umberto I, 00161 Rome, Italy
| | - Rocco Rago
- Physiopathology of Reproduction and Andrology Unit, Sandro Pertini Hospital, Via dei Monti Tiburtini 385/389, 00157 Rome, Italy
| | - Valerio Pisaturo
- Department of Maternal and Child Health and Urological Sciences, "Sapienza" University of Rome, Policlinico Umberto I, 00161 Rome, Italy
| | - Luisa Campagnolo
- Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy
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Paul N, Maiti K, Sultana Z, Fisher JJ, Zhang H, Cole N, Morgan T, Smith R. Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood. Placenta 2024; 146:71-78. [PMID: 38190772 DOI: 10.1016/j.placenta.2024.01.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Revised: 12/14/2023] [Accepted: 01/03/2024] [Indexed: 01/10/2024]
Abstract
The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100-1000 nm) and exosomes (30-150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25-2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.
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Affiliation(s)
- Nilanjana Paul
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, The University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia; Department of Genetic Engineering and Biotechnology, The University of Dhaka, Bangladesh
| | - Kaushik Maiti
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, The University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia
| | - Zakia Sultana
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, The University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia
| | - Joshua J Fisher
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, The University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia
| | - Huiming Zhang
- Research and Innovation Division, The University of Newcastle, University Drive, Callaghan, NSW, 2308, Australia
| | - Nicole Cole
- Research and Innovation Division, The University of Newcastle, University Drive, Callaghan, NSW, 2308, Australia
| | - Terry Morgan
- Department of Pathology, Oregon Health & Science University, Portland, OR, USA
| | - Roger Smith
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, The University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia.
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10
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Toledo-Guardiola SM, Luongo C, Abril-Parreño L, Soriano-Úbeda C, Matás C. Different seminal ejaculated fractions in artificial insemination condition the protein cargo of oviductal and uterine extracellular vesicles in pig. Front Cell Dev Biol 2023; 11:1231755. [PMID: 37868907 PMCID: PMC10587466 DOI: 10.3389/fcell.2023.1231755] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Accepted: 09/21/2023] [Indexed: 10/24/2023] Open
Abstract
The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction, SRF; F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows (p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we found specific protein cargo in oEVs and uEVs according to the type of semen fraction the sow was inseminated with and whose functions these specific EVs proteins are closely associated with reproductive processes.
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Affiliation(s)
- S. M. Toledo-Guardiola
- Departamento de Fisiología, Facultad de Veterinaria, Campus de Excelencia Mare Nostrum Universidad de Murcia, Murcia, Spain
| | - C. Luongo
- Departamento de Fisiología, Facultad de Veterinaria, Campus de Excelencia Mare Nostrum Universidad de Murcia, Murcia, Spain
| | - L. Abril-Parreño
- Departamento de Fisiología, Facultad de Veterinaria, Campus de Excelencia Mare Nostrum Universidad de Murcia, Murcia, Spain
| | - C. Soriano-Úbeda
- Departamento de Medicina, Cirugía y Anatomía Veterinaria, Universidad de Léon, León, Spain
| | - C. Matás
- Departamento de Fisiología, Facultad de Veterinaria, Campus de Excelencia Mare Nostrum Universidad de Murcia, Murcia, Spain
- Instituto Murciano de Investigación Biosanitaria Pascual Parrilla (IMIB-Arrixaca), Murcia, Spain
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11
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Sakr OG, Gad A, Cañón-Beltrán K, Cajas YN, Prochazka R, Rizos D, Rebollar PG. Characterization and identification of extracellular vesicles-coupled miRNA profiles in seminal plasma of fertile and subfertile rabbit bucks. Theriogenology 2023; 209:76-88. [PMID: 37364341 DOI: 10.1016/j.theriogenology.2023.06.020] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 06/06/2023] [Accepted: 06/12/2023] [Indexed: 06/28/2023]
Abstract
Seminal plasma (SP) provides essential nutrients, transport, and protection to the spermatozoa during their journey through the male and female reproductive tracts. Extracellular vesicles (EVs) are one of the main components of the SP with several biomolecular cargoes, including miRNAs, that can influence spermatozoa functions and interact with the cells of the female reproductive tract. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SP-EVs isolated from fertile (F) and subfertile (S) rabbit bucks that could serve as fertility biomarkers. In this study, the methods to isolate and identify EVs including exosomes, from SP of 3 F and S bucks have been developed. Ultracentrifugation and size exclusion chromatography analysis were using to isolate EVs from SP of F and S males that were qualitative and quantitively characterised using transmission electron microscopy, nanoparticle tracking analysis and western blotting. In addition, total RNA, including miRNA, was isolated, sequenced and identified from SP-EVs samples. Different SP-EVs concentrations (8.53 × 1011 ± 1.04 × 1011 and 1.84 × 1012 ± 1.75 × 1011 particles/mL of SP; P = 0.008), with a similar average size (143.9 ± 11.9 and 115.5 ± 2.4 nm; P = 0.7422) in F and S males, respectively was observed. Particle size was not significantly correlated with any kinetic parameter. The concentration of SP-EVs was positively correlated with the percentage of abnormal forms (r = 0.94; P < 0.05) and with the percentage of immotile spermatozoa (r = 0.88; P < 0.05). Small-RNA-seq analysis identified a total of 267 and 244 expressed miRNAs in the F and S groups, respectively. Two miRNAs (let-7b-5p and let-7a-5p) were the top most abundant miRNAs in both groups. Differential expression analysis revealed that 9 miRNAs including miR-190b-5p, miR-193b-5p, let-7b-3p, and miR-378-3p, and another 9 miRNAs including miR-7a-5p, miR-33a-5p, miR-449a-5p, and miR-146a-5p were significantly up- and downregulated in the F compared to the S group, respectively. The SP from F and S rabbit males contains EVs with different miRNA cargo correlated with spermatogenesis, homeostasis, and infertility, which could be used as biomarkers for male fertility and potential therapies for assisted reproductive technologies.
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Affiliation(s)
- Osama G Sakr
- Dept. Animal Production, Faculty of Agriculture, Cairo University, 12613, Giza, Egypt; Dept. Agrarian Production, Technical University of Madrid, 28040, Madrid, Spain
| | - Ahmed Gad
- Dept. Animal Production, Faculty of Agriculture, Cairo University, 12613, Giza, Egypt; Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721, Liběchov, Czech Republic
| | - Karina Cañón-Beltrán
- Dept. Animal Reproduction, National Institute for Agriculture and Food, Research and Technology (INIA-CSIC), 28040, Madrid, Spain; Department of Biochemistry and Molecular Biology, Veterinary Faculty, Complutense University of Madrid (UCM), Madrid, Spain
| | - Yulia N Cajas
- Dept. Animal Reproduction, National Institute for Agriculture and Food, Research and Technology (INIA-CSIC), 28040, Madrid, Spain; Dept. de Ciencias de la Vida y la Agricultura, Universidad de las Fuerzas Armadas (ESPE), Sede, Santo Domingo, 171-5-231, Ecuador
| | - Radek Prochazka
- Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721, Liběchov, Czech Republic
| | - Dimitrios Rizos
- Dept. Animal Reproduction, National Institute for Agriculture and Food, Research and Technology (INIA-CSIC), 28040, Madrid, Spain.
| | - Pilar G Rebollar
- Dept. Agrarian Production, Technical University of Madrid, 28040, Madrid, Spain
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12
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Paul N, Sultana Z, Fisher JJ, Maiti K, Smith R. Extracellular vesicles- crucial players in human pregnancy. Placenta 2023; 140:30-38. [PMID: 37531747 DOI: 10.1016/j.placenta.2023.07.006] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2023] [Revised: 07/06/2023] [Accepted: 07/08/2023] [Indexed: 08/04/2023]
Abstract
Extracellular vesicles (EVs) are lipid-bilayer enclosed membrane vesicles released by cells in physiological and pathological states. EVs are generated and released through a variety of pathways and mediate cellular communication by carrying and transferring signals to recipient cells. EVs are specifically loaded with proteins, nucleic acids (RNAs and DNA), enzymes and lipids, and carry a range of surface proteins and adhesion molecules. EVs contribute to intercellular signalling, development, metabolism, tissue homeostasis, antigen presentation, gene expression and immune regulation. EVs have been categorised into three different subgroups based on their size: exosomes (30-150 nm), microvesicles (100-1000 nm) and apoptotic bodies (1-5 μm). The status of the cells of origin of EVs influences their biology, heterogeneity and functions. EVs, especially exosomes, have been studied for their potential roles in feto-maternal communication and impacts on normal pregnancy and pregnancy disorders. This review presents an overview of EVs, emphasising exosomes and microvesicles in a general context, and then focusing on the roles of EVs in human pregnancy and their potential as diagnostics for adverse pregnancy outcomes.
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Affiliation(s)
- Nilanjana Paul
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia.
| | - Zakia Sultana
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia.
| | - Joshua J Fisher
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia.
| | | | - Roger Smith
- Mothers and Babies Research Centre, Hunter Medical Research Institute, School of Medicine and Public Health, University of Newcastle, New Lambton Heights, New South Wales, 2305, Australia.
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13
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Guzewska MM, Witek KJ, Karnas E, Rawski M, Zuba-Surma E, Kaczmarek MM. miR-125b-5p impacts extracellular vesicle biogenesis, trafficking, and EV subpopulation release in the porcine trophoblast by regulating ESCRT-dependent pathway. FASEB J 2023; 37:e23054. [PMID: 37402070 DOI: 10.1096/fj.202300710r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2023] [Revised: 05/23/2023] [Accepted: 06/12/2023] [Indexed: 07/05/2023]
Abstract
Intercellular communication is a critical process that ensures cooperation between distinct cell types at the embryo-maternal interface. Extracellular vesicles (EVs) are considered to be potent mediators of this communication by transferring biological information in their cargo (e.g., miRNAs) to the recipient cells. miRNAs are small non-coding RNAs that affect the function and fate of neighboring and distant cells by regulating gene expression. Focusing on the maternal side of the dialog, we recently revealed the impact of embryonic signals, including miRNAs, on EV-mediated cell-to-cell communication. In this study, we show the regulatory mechanism of the miR-125b-5p ESCRT-mediated EV biogenesis pathway and the further secretion of EVs by trophoblasts at the time when the crucial steps of implantation are taking place. To test the ability of miR-125b-5p to influence the expression of genes involved in the generation and release of EV subpopulations in porcine conceptuses, we used an ex vivo approach. Next, in silico and in vitro analyses were performed to confirm miRNA-mRNA interactions. Finally, EV trafficking and release were assessed using several imaging and particle analysis tools. Our results indicated that conceptus development and implantation are accompanied by changes in the abundance of EV biogenesis and trafficking machinery. ESCRT-dependent EV biogenesis and the further secretion of EVs were modulated by miR-125b-5p, specifically impacting the ESCRT-II complex (via VPS36) and EV trafficking in primary porcine trophoblast cells. The identified miRNA-ESCRT interplay led to the generation and secretion of specific subpopulations of EVs. miRNA present at the embryo-maternal interface governs EV-mediated communication between the mother and the developing conceptus, leading to the generation, trafficking, and release of characteristic subpopulations of EVs.
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Affiliation(s)
- Maria M Guzewska
- Department of Hormonal Action Mechanisms, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
| | - Krzysztof J Witek
- Cell and Tissue Analysis and Imaging Laboratory, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
| | - Elżbieta Karnas
- Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
| | - Michał Rawski
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Ewa Zuba-Surma
- Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
| | - Monika M Kaczmarek
- Department of Hormonal Action Mechanisms, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
- Molecular Biology Laboratory, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
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14
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Segura-Benítez M, Bas-Rivas A, Juárez-Barber E, Carbajo-García MC, Faus A, De Los Santos MJ, Pellicer A, Ferrero H. Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation. Hum Reprod 2023:dead138. [PMID: 37407281 DOI: 10.1093/humrep/dead138] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2022] [Revised: 06/06/2023] [Indexed: 07/07/2023] Open
Abstract
STUDY QUESTION Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? SUMMARY ANSWER Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. WHAT IS KNOWN ALREADY Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. STUDY DESIGN, SIZE, DURATION A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n = 8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n = 3 pools) was analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. MAIN RESULTS AND THE ROLE OF CHANCE EVs measured 100-300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1-2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. LIMITATIONS, REASONS FOR CAUTION This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. WIDER IMPLICATIONS OF THE FINDINGS This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A.
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Affiliation(s)
- Marina Segura-Benítez
- Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
- Departamento de Pediatría, Obstetricia y Ginecología, Universidad de Valencia, Valencia, Spain
| | - Alba Bas-Rivas
- Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
| | | | - María Cristina Carbajo-García
- Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
- Departamento de Pediatría, Obstetricia y Ginecología, Universidad de Valencia, Valencia, Spain
| | - Amparo Faus
- Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
| | - María José De Los Santos
- Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
- IVIRMA Valencia, Valencia, Spain
| | - Antonio Pellicer
- Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
- IVIRMA Rome, Rome, Italy
| | - Hortensia Ferrero
- Fundación IVI, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
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15
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Hong L, Zang X, Hu Q, He Y, Xu Z, Xie Y, Gu T, Yang H, Yang J, Shi J, Zheng E, Huang S, Xu Z, Liu D, Cai G, Li Z, Wu Z. Uterine luminal-derived extracellular vesicles: potential nanomaterials to improve embryo implantation. J Nanobiotechnology 2023; 21:79. [PMID: 36882792 PMCID: PMC9990359 DOI: 10.1186/s12951-023-01834-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Accepted: 03/02/2023] [Indexed: 03/09/2023] Open
Abstract
Most pregnancy losses worldwide are caused by implantation failure for which there is a lack of effective therapeutics. Extracellular vesicles are considered potential endogenous nanomedicines because of their unique biological functions. However, the limited supply of ULF-EVs prevents their development and application in infertility diseases such as implantation failure. In this study, pigs were used as a human biomedical model, and ULF-EVs were isolated from the uterine luminal. We comprehensively characterized the proteins enriched in ULF-EVs and revealed their biological functions in promoting embryo implantation. By exogenously supplying ULF-EVs, we demonstrated that ULF-EVs improve embryo implantation, suggesting that ULF-EVs are a potential nanomaterial to treat implantation failure. Furthermore, we identified that MEP1B is important in improving embryo implantation by promoting trophoblast cell proliferation and migration. These results indicated that ULF-EVs can be a potential nanomaterial to improve embryo implantation.
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Affiliation(s)
- Linjun Hong
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China. .,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China. .,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China.
| | - Xupeng Zang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Qun Hu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Yanjuan He
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Zhiqian Xu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Yanshe Xie
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Ting Gu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Huaqiang Yang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Jie Yang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Junsong Shi
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Yunfu Subcenter of Guangdong Laboratory for Lingnan Modern Agriculture, Yunfu, 527300, People's Republic of China
| | - Enqin Zheng
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Sixiu Huang
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Zheng Xu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Dewu Liu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Gengyuan Cai
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China
| | - Zicong Li
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China. .,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China. .,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China. .,State Key Laboratory of Livestock and Poultry Breeding, Guangzhou, 510642, People's Republic of China.
| | - Zhenfang Wu
- National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China. .,Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China. .,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People's Republic of China. .,State Key Laboratory of Livestock and Poultry Breeding, Guangzhou, 510642, People's Republic of China.
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16
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Poh QH, Rai A, Salamonsen LA, Greening DW. Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility. Proteomics 2023; 23:e2200107. [PMID: 36591946 DOI: 10.1002/pmic.202200107] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Revised: 12/14/2022] [Accepted: 12/15/2022] [Indexed: 01/03/2023]
Abstract
Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid (UF) and embryo spent media (ESM) of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV-based diagnostic and therapeutic platforms in reproductive medicine.
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Affiliation(s)
- Qi Hui Poh
- Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia.,Department of Biochemistry and Chemistry, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia
| | - Alin Rai
- Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia.,Baker Department of Cardiovascular Research, Translation and Implementation, La Trobe University, Melbourne, Victoria, Australia.,Baker Department of Cardiometabolic Health, University of Melbourne, Melbourne, Victoria, Australia
| | - Lois A Salamonsen
- Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, Victoria, Australia.,Department of Molecular and Translational Medicine, Monash University, Clayton, Victoria, Australia
| | - David W Greening
- Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia.,Department of Biochemistry and Chemistry, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Victoria, Australia.,Baker Department of Cardiovascular Research, Translation and Implementation, La Trobe University, Melbourne, Victoria, Australia.,Baker Department of Cardiometabolic Health, University of Melbourne, Melbourne, Victoria, Australia.,Central Clinical School, Monash University, Melbourne, Victoria, Australia
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17
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Bovine embryos release extracellular vesicles with differential miRNA signature during the compaction and blastulation stages. Reprod Biol 2023; 23:100725. [PMID: 36565511 DOI: 10.1016/j.repbio.2022.100725] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 11/15/2022] [Accepted: 12/12/2022] [Indexed: 12/24/2022]
Abstract
Pre-implantation embryos release extracellular vesicles (EVs) to extracellular environment. In this work it is hypothesized that the EVs miRNA cargo will vary during pre-implantation development due to the constant changes in gene expression that take place through this period. The concentration, size and miRNA cargo of EVs secreted by competent bovine embryos during the period from compaction to blastulation (Day 3-7) were analyzed. For this analysis tow developmental windows were defined: W2 from 8-cells (D3) to morula (D5) and W3 from morula (D5) to blastocyst (D7). For W2, in vitro produced embryos were individually cultured in EVs-depleted medium from D3 to D5; culture media were collected and assigned to Group W2. Morulae were kept in culture up to blastocyst stage to determine the developmental competence. For W3, D5 morulae were collected and cultured individually in EVs-depleted medium up to blastocyst stage; culture media were assigned to Group W3, and blastocysts were kept in culture up to day 11 to define their competence. The mean size of EVs was similar between groups, however, EVs concentration was lower in W2. A total of 140 miRNAs were identified. From them, 79 were differentially expressed between the groups, 28 upregulated and 51 downregulated. miRNAs differentially detected between both developmental windows participate in the regulation of signaling pathways which crucial for embryonic development. It was concluded that the secretion of EVs is regulated by the developmental progress of the embryo during the pre-implantation period.
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18
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Adamova P, Lotto RR, Powell AK, Dykes IM. Are there foetal extracellular vesicles in maternal blood? Prospects for diagnostic biomarker discovery. J Mol Med (Berl) 2023; 101:65-81. [PMID: 36538060 PMCID: PMC9977902 DOI: 10.1007/s00109-022-02278-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Revised: 11/14/2022] [Accepted: 12/05/2022] [Indexed: 03/02/2023]
Abstract
Prenatal diagnosis of congenital disease improves clinical outcomes; however, as many as 50% of congenital heart disease cases are missed by current ultrasound screening methods. This indicates a need for improved screening technology. Extracellular vesicles (EVs) have attracted enormous interest in recent years for their potential in diagnostics. EVs mediate endocrine signalling in health and disease and are known to regulate aspects of embryonic development. Here, we critically evaluate recent evidence suggesting that EVs released from the foetus are able to cross the placenta and enter the maternal circulation. Furthermore, EVs from the mother appear to be transported in the reverse direction, whilst the placenta itself acts as a source of EVs. Experimental work utilising rodent models employing either transgenically encoded reporters or application of fluorescent tracking dyes provide convincing evidence of foetal-maternal crosstalk. This is supported by clinical data demonstrating expression of placental-origin EVs in maternal blood, as well as limited evidence for the presence of foetal-origin EVs. Together, this work raises the possibility that foetal EVs present in maternal blood could be used for the diagnosis of congenital disease. We discuss the challenges faced by researchers in translating these basic science findings into a clinical non-invasive prenatal test.
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Affiliation(s)
- Petra Adamova
- School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom St, Liverpool, L3 3AF, UK.,Liverpool Centre for Cardiovascular Science, Liverpool John Moores University, Liverpool, UK
| | - Robyn R Lotto
- Liverpool Centre for Cardiovascular Science, Liverpool John Moores University, Liverpool, UK.,School of Nursing and Allied Health, Liverpool John Moores University, Tithebarn St, Liverpool, L2 2ER, UK
| | - Andrew K Powell
- School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom St, Liverpool, L3 3AF, UK.,Liverpool Centre for Cardiovascular Science, Liverpool John Moores University, Liverpool, UK
| | - Iain M Dykes
- School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom St, Liverpool, L3 3AF, UK. .,Liverpool Centre for Cardiovascular Science, Liverpool John Moores University, Liverpool, UK.
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19
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Yaghoobi A, Nazerian Y, Meymand AZ, Ansari A, Nazerian A, Niknejad H. Hypoxia-sensitive miRNA regulation via CRISPR/dCas9 loaded in hybrid exosomes: A novel strategy to improve embryo implantation and prevent placental insufficiency during pregnancy. Front Cell Dev Biol 2023; 10:1082657. [PMID: 36704201 PMCID: PMC9871368 DOI: 10.3389/fcell.2022.1082657] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Accepted: 12/21/2022] [Indexed: 01/12/2023] Open
Abstract
Assisted reproductive techniques as a new regenerative medicine approach have significantly contributed to solving infertility problems that affect approximately 15% of couples worldwide. However, the success rate of an in vitro fertilization (IVF) cycle remains only about 20%-30%, and 75% of these losses are due to implantation failure (the crucial rate-limiting step of gestation). Implantation failure and abnormal placenta formation are mainly caused by defective adhesion, invasion, and angiogenesis. Placental insufficiency endangers both the mother's and the fetus's health. Therefore, we suggested a novel treatment strategy to improve endometrial receptivity and implantation success rate. In this strategy, regulating mir-30d expression as an upstream transcriptomic modifier of the embryo implantation results in modified expression of the involved genes in embryonic adhesion, invasion, and angiogenesis and consequently impedes implantation failure. For this purpose, "scaffold/matrix attachment regions (S/MARs)" are employed as non-viral episomal vectors, transfecting into trophoblasts by exosome-liposome hybrid carriers. These vectors comprise CRISPR/dCas9 with a guide RNA to exclusively induce miR-30d gene expression in hypoxic stress conditions. In order to avoid concerns about the fetus's genetic manipulation, our vector would be transfected specifically into the trophoblast layer of the blastocyst via binding to trophoblast Erb-B4 receptors without entering the inner cell mass. Additionally, S/MAR episomal vectors do not integrate with the original cell DNA. As an on/off regulatory switch, a hypoxia-sensitive promoter (HRE) is localized upstream of dCas9. The miR-30d expression increases before and during the implantation and placental insufficiency conditions and is extinguished after hypoxia elimination. This hypothesis emphasizes that improving the adhesion, invasion, and angiogenesis in the uterine microenvironment during pregnancy will result in increased implantation success and reduced placental insufficiency, as a new insight in translational medicine.
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Affiliation(s)
- Alireza Yaghoobi
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Yasaman Nazerian
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Arman Zeinaddini Meymand
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Ali Ansari
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | | | - Hassan Niknejad
- Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran,*Correspondence: Hassan Niknejad,
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20
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Li Y, Liu C, Guo N, Cai L, Wang M, Zhu L, Li F, Jin L, Sui C. Extracellular vesicles from human Fallopian tubal fluid benefit embryo development in vitro. Hum Reprod Open 2023; 2023:hoad006. [PMID: 36895886 PMCID: PMC9991590 DOI: 10.1093/hropen/hoad006] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2022] [Revised: 02/06/2023] [Indexed: 02/23/2023] Open
Abstract
STUDY QUESTION Do extracellular vesicles (EVs) from human Fallopian tubes exert an influence on early embryo development in vitro? SUMMARY ANSWER Human Fallopian tube EVs carrying miRNAs increase murine embryo viability in vitro. WHAT IS KNOWN ALREADY Oviductal EVs (oEVs) are recently identified key players in embryo-oviduct interactions that contribute to successful pregnancy in vivo. Their absence in current in vitro systems may partly explain the suboptimal embryo development observed; therefore, further knowledge is needed about their impact on early embryos. STUDY DESIGN SIZE DURATION The oEVs were isolated from the luminal fluid of human Fallopian tubes using ultracentrifugation. We cocultured oEVs with murine two-cell embryos until the blastocyst stage. The study was conducted between August 2021 and July 2022. PARTICIPANTS/MATERIALS SETTING METHODS A total of 23 premenopausal women were recruited for Fallopian-tubes collection, and the oEVs were isolated. The micro RNA (miRNA) contents were detected using high-throughput sequencing and their target genes and effects were analyzed. After in vitro culture with or without oEVs, the blastocyst and hatching rates were recorded. Furthermore, for the blastocysts formed, we assessed the total cell number, inner cell mass proportion, reactive oxygen species (ROS) level, number of apoptotic cells, and mRNA expression levels of genes involved in development. MAIN RESULTS AND THE ROLE OF CHANCE EVs were successfully isolated from the human Fallopian tubal fluid and the concentrations were evaluated. A total of 79 known miRNAs were identified from eight samples that had been sequenced, all involved in various biological processes. The blastocyst rate, hatching rate, as well as total cell number of blastocysts were significantly increased in the oEVs-treated groups (P < 0.05 versus untreated), while the proportion of inner cell mass showed no significant difference between groups. ROS levels and apoptotic cell proportions were decreased in the oEVs-treated groups (P < 0.05 versus untreated). The genes, Actr3 (actin-related protein 3), Eomes (eomesodermin), and Wnt3a (Wnt family member 3A) were upregulated in blastocysts in the oEVs-treated group. LARGE SCALE DATA Data are available from Gene Expression Omnibus: Accession number: GSE225122. LIMITATIONS REASONS FOR CAUTION The Fallopian tubes in the current study were collected from patients with uterine fibroids (the reason they underwent hysterectomy), and this pathological condition may affect the characteristics of EVs in luminal fluid. Also, owing to restrictions for ethical reasons, an in vitro co-culture system using murine embryos was used instead of human embryos, and the findings may not be transferable. WIDER IMPLICATIONS OF THE FINDINGS Deciphering miRNA contents in human oEVs and providing new evidence that oEVs benefit embryo development in vitro will not only increase our knowledge on embryo-oviduct communication but also potentially improve ART outcomes. STUDY FUNDING/COMPETING INTERESTS This study was supported by the National Key Research and Development Project of China (2021YFC2700603). No competing interests are declared.
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Affiliation(s)
- Yuehan Li
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Chang Liu
- Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, People's Republic of China
| | - Na Guo
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Lei Cai
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Meng Wang
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Lixia Zhu
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Fei Li
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Lei Jin
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Cong Sui
- Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
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21
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Piibor J, Dissanayake K, Midekessa G, Andronowska A, Kavak A, Waldmann A, Fazeli A. Characterization of bovine uterine fluid extracellular vesicles proteomic profiles at follicular and luteal phases of the oestrous cycle. Vet Res Commun 2022; 47:885-900. [DOI: 10.1007/s11259-022-10052-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Accepted: 12/09/2022] [Indexed: 12/24/2022]
Abstract
AbstractExtracellular vesicles (EV) have been identified in uterine fluid (UF), however the bovine UF-EV profile during different phases of the oestrous cycle has not yet been established. Therefore, we compared the UF-EV, and their protein profile at follicular and luteal phases of the oestrous cycle. UF samples were collected from healthy uteri of six live and six slaughtered cows at follicular or luteal phases. Isolation of EV was performed using tangential flow filtration followed by size exclusion chromatography. EV were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, zeta potential, and transmission electron microscopy. Mass-spectrometry was used to evaluate EV protein profile from live cows. Particle concentrations (mean ± SD) were higher (P < 0.05) at follicular than at luteal phase in both live (1.01 × 108 ± 1.66 × 107 vs 7.56 × 107 ± 1.80 × 107, respectively) and slaughtered cows (1.17 × 108 ± 2.34 × 107 vs 9.12 × 107 ± 9.77 × 106, respectively). The proportion of fluorescently labelled EV varied significantly between follicular and luteal phases across live (28.9 ± 1.9% vs 19.3 ± 2.8%, respectively) and slaughtered cows (26.5 ± 6.3% vs 27.3 ± 2 .7%, respectively). In total, 41 EV proteins were differentially expressed between the phases. Some of the proteins were involved in reproductive processes, cell adhesion and proliferation, and cellular metabolic processes. The results indicated differences in bovine UF-EV concentration and protein profile at follicular and luteal phases, which would suggest that EV modulate uterine microenvironment across the oestrous cycle. Further research is needed to understand the effect of EV changes throughout the oestrous cycle.
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22
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Integrative Proteomics and Transcriptomics Profiles of the Oviduct Reveal the Prolificacy-Related Candidate Biomarkers of Goats ( Capra hircus) in Estrous Periods. Int J Mol Sci 2022; 23:ijms232314888. [PMID: 36499219 PMCID: PMC9737051 DOI: 10.3390/ijms232314888] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 11/16/2022] [Accepted: 11/24/2022] [Indexed: 11/29/2022] Open
Abstract
The oviduct is a dynamic reproductive organ for mammalian reproduction and is required for gamete storage, maturation, fertilization, and early embryonic development, and it directly affects fecundity. However, the molecular regulation of prolificacy occurring in estrous periods remain poorly understood. This study aims to gain a better understanding of the genes involved in regulating goat fecundity in the proteome and transcriptome levels of the oviducts. Twenty female Yunshang black goats (between 2 and 3 years old, weight 52.22 ± 0.43 kg) were divided into high- and low-fecundity groups in the follicular (FH and FL, five individuals per group) and luteal (LH and LL, five individuals per group) phases, respectively. The DIA-based high-resolution mass spectrometry (MS) method was used to quantify proteins in twenty oviducts. A total of 5409 proteins were quantified, and Weighted gene co-expression network analysis (WGCNA) determined that the tan module was highly associated with the high-fecundity trait in the luteal phase, and identified NUP107, ANXA11, COX2, AKP13, and ITF140 as hub proteins. Subsequently, 98 and 167 differentially abundant proteins (DAPs) were identified in the FH vs. FL and LH vs. LL comparison groups, respectively. Parallel reaction monitoring (PRM) was used to validate the results of the proteomics data, and the hub proteins were analyzed with Western blot (WB). In addition, biological adhesion and transporter activity processes were associated with oviductal function, and several proteins that play roles in oviductal communication with gametes or embryos were identified, including CAMSAP3, ITGAM, SYVN1, EMG1, ND5, RING1, CBS, PES1, ELP3, SEC24C, SPP1, and HSPA8. Correlation analysis of proteomics and transcriptomic revealed that the DAPs and differentially expressed genes (DEGs) are commonly involved in the metabolic processes at the follicular phase; they may prepare the oviductal microenvironment for gamete reception; and the MAP kinase activity, estrogen receptor binding, and angiotensin receptor binding terms were enriched in the luteal phase, which may be actively involved in reproductive processes. By generating the proteome data of the oviduct at two critical phases and integrating transcriptome analysis, we uncovered novel aspects of oviductal gene regulation of fecundity and provided a reference for other mammals.
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23
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Leal CLV, Cañón-Beltrán K, Cajas YN, Hamdi M, Yaryes A, Millán de la Blanca MG, Beltrán-Breña P, Mazzarella R, da Silveira JC, Gutiérrez-Adán A, González EM, Rizos D. Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality. J Anim Sci Biotechnol 2022; 13:116. [PMID: 36280872 PMCID: PMC9594899 DOI: 10.1186/s40104-022-00763-7] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2022] [Accepted: 07/31/2022] [Indexed: 11/28/2022] Open
Abstract
Background In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by size exclusion chromatography. Blastocyst rate was recorded on days 7–8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by qRT-PCR. Results Blastocyst yield was lower (P < 0.05) in BSA treatments compared with dFCS treatments. Survival rates after vitrification/warming were improved in dFCS-EVs (P < 0.05). EVs increased (P < 0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls (dFCS and BSA), while lipid content was decreased in dFCS-EV (P < 0.05) and mitochondrial activity did not change (P > 0.05). Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium (PPARGC1B, LDLR, CD36, FASN and PNPLA2, P < 0.05). Levels of pHSL were lower in dFCS (P < 0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (P < 0.05). Conclusions Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, and relative changes in expression of lipid metabolism transcripts and lipase activation. Finally, EVs miRNA contents may contribute to the observed effects. Supplementary Information The online version contains supplementary material available at 10.1186/s40104-022-00763-7.
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Affiliation(s)
- Cláudia Lima Verde Leal
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain ,grid.11899.380000 0004 1937 0722Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo (FZEA-USP), Pirassununga, Brazil
| | - Karina Cañón-Beltrán
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain ,grid.442066.20000 0004 0466 9211Facultad de Ciencias Agrarias y Ambientales, Programa de Medicina Veterinaria, Fundación Universitaria Juan de Castellanos, Tunja, Colombia
| | - Yulia N. Cajas
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain ,grid.442123.20000 0001 1940 3465Laboratorio de Biotecnología de la Reproducción Animal, Facultad de Ciencias Agropecuarias, Universidad de Cuenca (UC), EC010205 Cuenca, Ecuador
| | - Meriem Hamdi
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain
| | - Aracelli Yaryes
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain
| | - María Gemma Millán de la Blanca
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain
| | - Paula Beltrán-Breña
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain
| | - Rosane Mazzarella
- grid.11899.380000 0004 1937 0722Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo (FZEA-USP), Pirassununga, Brazil
| | - Juliano Coelho da Silveira
- grid.11899.380000 0004 1937 0722Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo (FZEA-USP), Pirassununga, Brazil
| | - Alfonso Gutiérrez-Adán
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain
| | - Encina M González
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain ,grid.4795.f0000 0001 2157 7667Department of Anatomy and Embryology, Veterinary Faculty-Universidad Complutense de Madrid (UCM), Madrid, Spain
| | - Dimitrios Rizos
- grid.4711.30000 0001 2183 4846Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), 28040 Madrid, Spain
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24
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Taraschi A, Cimini C, Colosimo A, Ramal-Sanchez M, Valbonetti L, Bernabò N, Barboni B. An interactive analysis of the mouse oviductal miRNA profiles. Front Cell Dev Biol 2022; 10:1015360. [PMID: 36340025 PMCID: PMC9627480 DOI: 10.3389/fcell.2022.1015360] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Accepted: 10/06/2022] [Indexed: 11/15/2022] Open
Abstract
MicroRNAs are small non-coding molecules that control several cellular functions and act as negative post-transcriptional regulators of the mRNA. While their implication in several biological functions is already known, an important role as regulators of different physiological and pathological processes in fertilization and embryo development is currently emerging. Indeed, miRNAs have been found in the oviductal fluid packaged within the extracellular vesicles, which might act as natural nanoshuttles by transporting lipids, proteins, RNA molecules and miRNAs from the oviduct to the gametes or embryos. Here, an exhaustive bibliography search was carried out, followed by the construction of a computational model based on the networks theory in an attempt to recreate and elucidate the pathways potentially activated by the oviductal miRNA. The omics data published to date were gathered to create the Oviductal MiRNome, in which the miRNA target genes and their interactions are represented by using stringApp and the Network analyzer from Cytoscape 3.7.2. Then, the hyperlinked nodes were identified to investigate the pathways in which they are involved using the gene ontology enrichment analysis. To study the phenotypical effects after the removal of key genes on the reproductive system and embryo, knockout mouse lines for every protein-coding gene were investigated by using the International Mouse Phenotyping Consortium database. The creation of the Oviductal MiRNome revealed the presence of important genes and their interactions within the network. The functional enrichment analysis revealed that the hyperlinked nodes are involved in fundamental cellular functions, both structural and regulatory/signaling, suggesting their implication in fertilization and early embryo development. This fact was as well evidenced by the effects of the gene deletion in KO mice on the reproductive system and embryo development. The present study highlights the importance of studying the miRNA profiles and their enormous potential as tools to improve the assisted reproductive techniques currently used in human and animal reproduction.
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Affiliation(s)
- Angela Taraschi
- Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
- Istituto Zooprofilattico Sperimentale Dell’Abruzzo e Del Molise “G. Caporale”, Teramo, Italy
| | - Costanza Cimini
- Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - Alessia Colosimo
- Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - Marina Ramal-Sanchez
- Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
| | - Luca Valbonetti
- Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
- Institute of Biochemistry and Cell Biology (CNR-IBBC/EMMA/Infrafrontier/IMPC), National Research Council, Rome, Italy
| | - Nicola Bernabò
- Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
- Institute of Biochemistry and Cell Biology (CNR-IBBC/EMMA/Infrafrontier/IMPC), National Research Council, Rome, Italy
- *Correspondence: Nicola Bernabò,
| | - Barbara Barboni
- Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy
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Urzì O, Olofsson Bagge R, Crescitelli R. The dark side of foetal bovine serum in extracellular vesicle studies. J Extracell Vesicles 2022; 11:e12271. [PMID: 36214482 PMCID: PMC9549727 DOI: 10.1002/jev2.12271] [Citation(s) in RCA: 34] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/1912] [Revised: 12/12/1912] [Accepted: 12/12/1912] [Indexed: 11/06/2022] Open
Abstract
Extracellular vesicles (EVs) have been shown to be involved in cell-cell communication and to take part in both physiological and pathological processes. Thanks to their exclusive cargo, which includes proteins, lipids, and nucleic acids from the originating cells, they are gaining interest as potential biomarkers of disease. In recent years, their appealing features have been fascinating researchers from all over the world, thus increasing the number of in vitro studies focused on EV release, content, and biological activities. Cultured cell lines are the most-used source of EVs; however, the EVs released in cell cultures are influenced by the cell culture conditions, such as the use of foetal bovine serum (FBS). FBS is the most common supplement for cell culture media, but it is also a source of contaminants, such as exogenous bovine EVs, RNA, and protein aggregates, that can contaminate the cell-derived EVs and influence their cargo composition. The presence of FBS contaminants in cell-derived EV samples is a well-known issue that limits the clinical applications of EVs, thus increasing the need for standardization. In this review, we will discuss the pros and cons of using FBS in cell cultures as a source of EVs, as well as the protocols used to remove contaminants from FBS.
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Affiliation(s)
- Ornella Urzì
- Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational MedicineDepartment of SurgeryInstitute of Clinical SciencesSahlgrenska AcademyUniversity of GothenburgGothenburgSweden
- Department of BiomedicineNeurosciences and Advanced Diagnostics (Bi.N.D)University of PalermoPalermoItaly
| | - Roger Olofsson Bagge
- Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational MedicineDepartment of SurgeryInstitute of Clinical SciencesSahlgrenska AcademyUniversity of GothenburgGothenburgSweden
- Department of SurgerySahlgrenska University HospitalRegion Västra GötalandGothenburgSweden
| | - Rossella Crescitelli
- Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational MedicineDepartment of SurgeryInstitute of Clinical SciencesSahlgrenska AcademyUniversity of GothenburgGothenburgSweden
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Montjean D, Neyroud AS, Yefimova MG, Benkhalifa M, Cabry R, Ravel C. Impact of Endocrine Disruptors upon Non-Genetic Inheritance. Int J Mol Sci 2022; 23:3350. [PMID: 35328771 PMCID: PMC8950994 DOI: 10.3390/ijms23063350] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Revised: 03/15/2022] [Accepted: 03/17/2022] [Indexed: 02/06/2023] Open
Abstract
Similar to environmental factors, EDCs (endocrine-disrupting chemicals) can influence gene expression without modifying the DNA sequence. It is commonly accepted that the transgenerational inheritance of parentally acquired traits is conveyed by epigenetic alterations also known as "epimutations". DNA methylation, acetylation, histone modification, RNA-mediated effects and extracellular vesicle effects are the mechanisms that have been described so far to be responsible for these epimutations. They may lead to the transgenerational inheritance of diverse phenotypes in the progeny when they occur in the germ cells of an affected individual. While EDC-induced health effects have dramatically increased over the past decade, limited effects on sperm epigenetics have been described. However, there has been a gain of interest in this issue in recent years. The gametes (sperm and oocyte) represent targets for EDCs and thus a route for environmentally induced changes over several generations. This review aims at providing an overview of the epigenetic mechanisms that might be implicated in this transgenerational inheritance.
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Affiliation(s)
- Debbie Montjean
- Fertilys Fertility Center, 1950 Rue Maurice-Gauvin #103, Laval, QC H7S 1Z5, Canada;
| | - Anne-Sophie Neyroud
- CHU de Rennes, Département de Gynécologie Obstétrique et Reproduction Humaine-CECOS, Hôpital Sud, 16 Boulevard de Bulgarie, 35000 Rennes, France;
| | - Marina G. Yefimova
- Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223 St-Petersburg, Russia;
| | - Moncef Benkhalifa
- Fertilys Fertility Center, 1950 Rue Maurice-Gauvin #103, Laval, QC H7S 1Z5, Canada;
- Médecine et Biologie de la Reproduction, CECOS de Picardie, CHU Amiens, 80054 Amiens, France;
- UFR de Médecine, Université de Picardie Jules Verne, 80054 Amiens, France
- Peritox, Centre Universitaire de Recherche en Santé, Université de Picardie Jules Verne, 80054 Amiens, France
| | - Rosalie Cabry
- Médecine et Biologie de la Reproduction, CECOS de Picardie, CHU Amiens, 80054 Amiens, France;
- UFR de Médecine, Université de Picardie Jules Verne, 80054 Amiens, France
- Peritox, Centre Universitaire de Recherche en Santé, Université de Picardie Jules Verne, 80054 Amiens, France
| | - Célia Ravel
- CHU de Rennes, Département de Gynécologie Obstétrique et Reproduction Humaine-CECOS, Hôpital Sud, 16 Boulevard de Bulgarie, 35000 Rennes, France;
- CHU Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail)—UMR_S 1085, University Rennes, 35000 Rennes, France
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Zuccarello D, Sorrentino U, Brasson V, Marin L, Piccolo C, Capalbo A, Andrisani A, Cassina M. Epigenetics of pregnancy: looking beyond the DNA code. J Assist Reprod Genet 2022; 39:801-816. [PMID: 35301622 PMCID: PMC9050975 DOI: 10.1007/s10815-022-02451-x] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Accepted: 03/01/2022] [Indexed: 12/19/2022] Open
Abstract
Epigenetics is the branch of genetics that studies the different mechanisms that influence gene expression without direct modification of the DNA sequence. An ever-increasing amount of evidence suggests that such regulatory processes may play a pivotal role both in the initiation of pregnancy and in the later processes of embryonic and fetal development, thus determining long-term effects even in adult life. In this narrative review, we summarize the current knowledge on the role of epigenetics in pregnancy, from its most studied and well-known mechanisms to the new frontiers of epigenetic regulation, such as the role of ncRNAs and the effects of the gestational environment on fetal brain development. Epigenetic mechanisms in pregnancy are a dynamic phenomenon that responds both to maternal-fetal and environmental factors, which can influence and modify the embryo-fetal development during the various gestational phases. Therefore, we also recapitulate the effects of the most notable environmental factors that can affect pregnancy and prenatal development, such as maternal nutrition, stress hormones, microbiome, and teratogens, focusing on their ability to cause epigenetic modifications in the gestational environment and ultimately in the fetus. Despite the promising advancements in the knowledge of epigenetics in pregnancy, more experience and data on this topic are still needed. A better understanding of epigenetic regulation in pregnancy could in fact prove valuable towards a better management of both physiological pregnancies and assisted reproduction treatments, other than allowing to better comprehend the origin of multifactorial pathological conditions such as neurodevelopmental disorders.
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Affiliation(s)
- Daniela Zuccarello
- Clinical Genetics Unit, Department of Women's and Children's Health, University Hospital of Padova, Padua, Italy.
| | - Ugo Sorrentino
- Clinical Genetics Unit, Department of Women's and Children's Health, University Hospital of Padova, Padua, Italy
| | - Valeria Brasson
- Clinical Genetics Unit, Department of Women's and Children's Health, University Hospital of Padova, Padua, Italy
| | - Loris Marin
- Gynaecological Clinic, Department of Women's and Children's Health, University of Padua, Padua, Italy
| | - Chiara Piccolo
- Clinical Genetics Unit, Department of Women's and Children's Health, University Hospital of Padova, Padua, Italy
| | | | - Alessandra Andrisani
- Gynaecological Clinic, Department of Women's and Children's Health, University of Padua, Padua, Italy
| | - Matteo Cassina
- Clinical Genetics Unit, Department of Women's and Children's Health, University Hospital of Padova, Padua, Italy
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28
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Emerging in vitro platforms and omics technologies for studying the endometrium and early embryo-maternal interface in humans. Placenta 2022; 125:36-46. [DOI: 10.1016/j.placenta.2022.01.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/24/2021] [Revised: 12/09/2021] [Accepted: 01/09/2022] [Indexed: 12/11/2022]
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Cajas YN, Cañón-Beltrán K, de la Blanca MGM, Sánchez JM, Fernandez-Fuertes B, González EM, Rizos D. Role of reproductive fluids and extracellular vesicles in embryo–maternal interaction during early pregnancy in cattle. Reprod Fertil Dev 2021; 34:117-138. [PMID: 35231231 DOI: 10.1071/rd21275] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
The coordinated interaction between the developing embryo and the maternal reproductive tract is essential for the establishment and maintenance of pregnancy in mammals. An early cross-talk is established between the oviduct/uterus and the gametes and embryo. This dialogue will shape the microenvironment in which gamete transport, fertilisation, and early embryonic development occur. Due to the small size of the gametes and the early embryo relative to the volume of the oviductal and uterine lumina, collection of tissue and fluid adjacent to these cells is challenging in cattle. Thus, the combination of in vivo and in vitro models seems to be the most appropriate approach to better understand this fine dialogue. In this respect, the aim of this review is to summarise the recent findings in relation to gamete/embryo-maternal interaction during the pre-elongation period.
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Affiliation(s)
- Yulia N Cajas
- Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), Ctra de la Coruña KM 5.9, 28040 Madrid, Spain; and Laboratorio de Biotecnología de la Reproducción Animal, Facultad de Ciencias Agropecuarias, Universidad de Cuenca (UC), EC010205 Cuenca, Ecuador
| | - Karina Cañón-Beltrán
- Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), Ctra de la Coruña KM 5.9, 28040 Madrid, Spain; and Facultad de Ciencias Agrarias y Ambientales, Programa de Medicina Veterinaria, Fundación Universitaria Juan de Castellanos (JdC), 150001 Tunja, Colombia
| | - María Gemma Millán de la Blanca
- Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), Ctra de la Coruña KM 5.9, 28040 Madrid, Spain
| | - José M Sánchez
- Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), Ctra de la Coruña KM 5.9, 28040 Madrid, Spain
| | - Beatriz Fernandez-Fuertes
- Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), Ctra de la Coruña KM 5.9, 28040 Madrid, Spain
| | - Encina M González
- Department of Anatomy and Embryology, Veterinary Faculty, Complutense University of Madrid (UCM), 28040 Madrid, Spain
| | - Dimitrios Rizos
- Department of Animal Reproduction, National Center Institute for Agriculture and Food Research and Technology (CSIC-INIA), Ctra de la Coruña KM 5.9, 28040 Madrid, Spain
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30
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Fang X, Tanga BM, Bang S, Seong G, Saadeldin IM, Lee S, Cho J. Oviduct epithelial cells-derived extracellular vesicles improve preimplantation developmental competence of in vitro produced porcine parthenogenetic and cloned embryos. Mol Reprod Dev 2021; 89:54-65. [PMID: 34843136 DOI: 10.1002/mrd.23550] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 11/22/2021] [Accepted: 11/23/2021] [Indexed: 12/15/2022]
Abstract
Extracellular vesicles (EVs) carry bioactive cargoes involved in the early preimplantation development. This study investigated the effects of EVs obtained from an oviductal epithelial cell (OEC) conditioned medium on the developmental competence of in parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) porcine embryos. The OEC-EV-treated group showed significant increases in blastocyst formation and hatching rates compared to the control group (40.8% ± 2.2% and 20.1% ± 2.1% vs. 24.9% ± 2.0% and 5.3% ± 1.1%; p < 0.05), respectively. The 7 day OEC-EVs treatment group significantly increased blastocyst formation rate than the 3 day and 0 day-groups (45.0 ± 0.8 vs. 33.0 ± 0.7 and 26.7 ± 0.5; p < 0.05), respectively. SCNT revealed that the OEC-EV increased blastocyst formation rate compared to that of oviductal fluid EVs (OF-EVs) (35.4% ± 1.4% vs. 29.3% ± 1.3%; p < 0.05). Reactive oxygen species levels, apoptosis, and blastocyst lipid content were significantly decreased in the OEC-EVs group compared with the control group. OEC-EV group showed a significantly decreased BAX and increased BCL2, SOD1, POU5F1, SOX2, NANOG, GATA6, PNPLA2, LIPE, and MGLL gene expression than the control group (p < 0.05). In conclusion, OEC-EVs supplementation in embryo culture media improved the quality of porcine embryos, potentially helping porcine-cloned embryonic development possibly through transfer of messenger RNA and proteins to the early embryos.
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Affiliation(s)
- Xun Fang
- Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
| | - Bereket Molla Tanga
- Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
| | - Seonggyu Bang
- Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
| | - Gyeonghwan Seong
- Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
| | - Islam M Saadeldin
- Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea.,Research Institute of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea.,Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
| | - Sanghoon Lee
- Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
| | - Jongki Cho
- Laboratory of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
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31
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Fiorenza MF, Amaral CDS, da Anunciação ARDA, Portela VVM, Marey MA, Miyamoto A, Antoniazzi AQ. Possible impact of neutrophils on immune responses during early pregnancy in ruminants. Anim Reprod 2021; 18:e20210048. [PMID: 34745357 PMCID: PMC8562715 DOI: 10.1590/1984-3143-ar2021-0048] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Accepted: 09/24/2021] [Indexed: 12/17/2022] Open
Abstract
The interaction between early embryo and maternal immune system for the establishment of pregnancy is the focus of several studies; however, it remains unclear. The maternal immune response needs to keep a balance between avoiding any damage to the conceptus and maintaining its function in combating microbes as well. When conceptus-maternal crosstalk cannot achieve this balance, pregnancy losses might occur. Intercommunication between mother and conceptus is fundamental during early pregnancy to dictate the outcome of pregnancy. In ruminants, the embryo reacts with the maternal system mainly via interferon tau (IFNT) release. IFNT can act locally on the embryo and endometrial cells and systemically in several tissues and cells to regulate their response via the expression of interferon-stimulated genes (ISGs). Also, IFNT can induce the expression of inflammatory-related genes in immune cells. Day 7 embryo induces a shift in the maternal immune response towards anti-inflammatory (Th2) immune responses. During maternal recognition of pregnancy, peripheral mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) express markers that configure an anti-inflammatory response. However, PMNs response is more sensitive to the effects of IFNT. PMNs are more likely to express interferon-stimulated genes (ISGs), transforming growth factor-beta (TGFB), interleukin 10 (IL10), and arginase-1 (ARG1), configuring one of the most rapid immune responses to early pregnancy. This review focus on the local and peripheral immune responses during early pregnancy in ruminants, mainly the PMNs function in the immune system.
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Affiliation(s)
- Mariani Farias Fiorenza
- Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil
| | - Carolina Dos Santos Amaral
- Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil
| | | | | | - Mohammed Ali Marey
- Global Agromedicine Research Center, Obihiro University of Agricultural and Veterinary Medicine, Obihiro, Japan.,Department of Theriogenology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt
| | - Akio Miyamoto
- Global Agromedicine Research Center, Obihiro University of Agricultural and Veterinary Medicine, Obihiro, Japan
| | - Alfredo Quites Antoniazzi
- Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil
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32
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Sang L, Xiao Y, Jiang Z, Forde N, Tian XC, Lonergan P, Hansen PJ. Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium. Mol Reprod Dev 2021; 88:694-704. [PMID: 34596291 PMCID: PMC8558826 DOI: 10.1002/mrd.23534] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Revised: 09/04/2021] [Accepted: 09/06/2021] [Indexed: 01/29/2023]
Abstract
Regulation of the mammalian embryo involves cell-signaling molecules produced by the maternal oviduct and endometrium. Here, datasets on the transcriptome of the gestational Days 5 and 6 bovine morula and Day 5 maternal endometrium were examined to identify receptor genes expressed by the morula and expression of the corresponding ligand-related genes in the endometrium. A total of 175 receptor genes were identified in the morula, including 48 encoding for growth factors or WNT signaling molecules, 25 for cytokines and chemokines, 35 involved in juxtacrine and matricellular signaling and 25 encoding for receptors for small molecules. Some of the highly-expressed pairs of endometrial ligand and embryo receptor genes included MDK and its receptors ITGB1, SDC4 and LRP2, WNT5A (RYK), VEGFA (ITGB1), GPI (AMFR), and the hedgehog proteins IHH and DHH (HHIP). The most highly expressed receptors for small molecules were GPRC5C (retinoic acid receptor), PGRMC1 (progesterone), and CHRNB2 (acetylcholine). There were also 84 genes encoding for cell signaling ligands expressed by the morula, with the most highly expressed being GPI, AIMP1, TIMP1, IK, and CCN2. The atlas of receptor and ligand genes should prove useful for understanding details of the communication between the embryo and mother that underlies optimal embryonic development.
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Affiliation(s)
- Lei Sang
- Institute of Animal Husbandry and Veterinary MedicineFujian Academy of Agricultural SciencesFuzhouFujianChina
- Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics InstituteUniversity of FloridaGainesvilleFloridaUSA
| | - Yao Xiao
- Institute of Animal Science and Veterinary MedicineShandong Academy of Agricultural SciencesJinanShandongChina
| | - Zongliang Jiang
- School of Animal Sciences, AgCenterLouisiana State UniversityBaton RougeLouisianaUSA
| | - Niamh Forde
- Department of Discovery and Translational SciencesUniversity of LeedsLeedsUK
| | - Xiuchun Cindy Tian
- Department of Animal ScienceUniversity of ConnecticutStorrsConnecticutUSA
| | - Patrick Lonergan
- School of Agriculture and Food ScienceUniversity CollegeDublinIreland
| | - Peter J. Hansen
- Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics InstituteUniversity of FloridaGainesvilleFloridaUSA
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33
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Xie Y, Liu G, Zang X, Hu Q, Zhou C, Li Y, Liu D, Hong L. Differential Expression Pattern of Goat Uterine Fluids Extracellular Vesicles miRNAs during Peri-Implantation. Cells 2021; 10:cells10092308. [PMID: 34571957 PMCID: PMC8470123 DOI: 10.3390/cells10092308] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Revised: 08/29/2021] [Accepted: 08/31/2021] [Indexed: 12/22/2022] Open
Abstract
Early pregnancy failure occurs when a mature embryo attaches to an unreceptive endometrium. During the formation of a receptive endometrium, extracellular vesicles (EVs) of the uterine fluids (UFs) deliver regulatory molecules such as small RNAs to mediate intrauterine communication between the embryo and the endometrium. However, profiling of small RNAs in goat UFs’ EVs during pregnancy recognition (day 16) has not been carried out. In this study, EVs were isolated from UFs on day 16 of the estrous cycle or gestation. They were isolated by Optiprep™ Density G radient (ODG) and verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Immunostaining demonstrated that CD63 was present both in the endometrial epithelium and glandular epithelium, and stain intensity was greater in the pregnant endometrium compared to the non-pregnant endometrium. Small RNA sequencing revealed that UFs’ EVs contained numerous sRNA families and a total of 106 differentially expressed miRNAs (DEMs). Additionally, 1867 target genes of the DEMs were obtained, and miRNA–mRNA interaction networks were constructed. GO and KEGG analysis showed that miRNAs were significantly associated with the formation of a receptive endometrium and embryo implantation. In addition, the fluorescence in situ hybridization assay (FISH) showed that chi-miR-451-5p was mainly expressed in stromal cells of the endometrium and a higher level was detected in the endometrial luminal epithelium in pregnant states. Moreover, the dual-luciferase reporter assay showed that chi-miR-451-5p directly binds to PSMB8 and may play an important role in the formation of a receptive endometrium and embryo implantation. In conclusion, these results reveal that UFs’ EVs contain various small RNAs that may be vital in the formation of a receptive endometrium and embryo implantation.
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Affiliation(s)
- Yanshe Xie
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
| | - Guangbin Liu
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
| | - Xupeng Zang
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
| | - Qun Hu
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
| | - Chen Zhou
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
| | - Yaokun Li
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
| | - Dewu Liu
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
- Correspondence: (D.L.); (L.H.)
| | - Linjun Hong
- College of Animal Science, South China Agricultural University, Guangzhou 510642, China; (Y.X.); (G.L.); (X.Z.); (Q.H.); (C.Z.); (Y.L.)
- National Local Joint Engineering Research Center of Livestock and Poutry, South China Agricultural University, Guangzhou 510642, China
- Correspondence: (D.L.); (L.H.)
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Rabaglino MB, O’Doherty A, Bojsen-Møller Secher J, Lonergan P, Hyttel P, Fair T, Kadarmideen HN. Application of multi-omics data integration and machine learning approaches to identify epigenetic and transcriptomic differences between in vitro and in vivo produced bovine embryos. PLoS One 2021; 16:e0252096. [PMID: 34029343 PMCID: PMC8143403 DOI: 10.1371/journal.pone.0252096] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2021] [Accepted: 05/09/2021] [Indexed: 01/16/2023] Open
Abstract
Pregnancy rates for in vitro produced (IVP) embryos are usually lower than for embryos produced in vivo after ovarian superovulation (MOET). This is potentially due to alterations in their trophectoderm (TE), the outermost layer in physical contact with the maternal endometrium. The main objective was to apply a multi-omics data integration approach to identify both temporally differentially expressed and differentially methylated genes (DEG and DMG), between IVP and MOET embryos, that could impact TE function. To start, four and five published transcriptomic and epigenomic datasets, respectively, were processed for data integration. Second, DEG from day 7 to days 13 and 16 and DMG from day 7 to day 17 were determined in the TE from IVP vs. MOET embryos. Third, genes that were both DE and DM were subjected to hierarchical clustering and functional enrichment analysis. Finally, findings were validated through a machine learning approach with two additional datasets from day 15 embryos. There were 1535 DEG and 6360 DMG, with 490 overlapped genes, whose expression profiles at days 13 and 16 resulted in three main clusters. Cluster 1 (188) and Cluster 2 (191) genes were down-regulated at day 13 or day 16, respectively, while Cluster 3 genes (111) were up-regulated at both days, in IVP embryos compared to MOET embryos. The top enriched terms were the KEGG pathway "focal adhesion" in Cluster 1 (FDR = 0.003), and the cellular component: "extracellular exosome" in Cluster 2 (FDR<0.0001), also enriched in Cluster 1 (FDR = 0.04). According to the machine learning approach, genes in Cluster 1 showed a similar expression pattern between IVP and less developed (short) MOET conceptuses; and between MOET and DKK1-treated (advanced) IVP conceptuses. In conclusion, these results suggest that early conceptuses derived from IVP embryos exhibit epigenomic and transcriptomic changes that later affect its elongation and focal adhesion, impairing post-transfer survival.
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Affiliation(s)
- Maria B. Rabaglino
- Quantitative Genetics, Bioinformatics and Computational Biology Group, Department of Applied Mathematics and Computer Science, Technical University of Denmark, Lyngby, Denmark
| | - Alan O’Doherty
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| | - Jan Bojsen-Møller Secher
- Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Patrick Lonergan
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| | - Poul Hyttel
- Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark
| | - Trudee Fair
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| | - Haja N. Kadarmideen
- Quantitative Genetics, Bioinformatics and Computational Biology Group, Department of Applied Mathematics and Computer Science, Technical University of Denmark, Lyngby, Denmark
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Rai A, Poh QH, Fatmous M, Fang H, Gurung S, Vollenhoven B, Salamonsen LA, Greening DW. Proteomic profiling of human uterine extracellular vesicles reveal dynamic regulation of key players of embryo implantation and fertility during menstrual cycle. Proteomics 2021; 21:e2000211. [PMID: 33634576 DOI: 10.1002/pmic.202000211] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 02/19/2021] [Accepted: 02/22/2021] [Indexed: 12/22/2022]
Abstract
Endometrial extracellular vesicles (EVs) are emerging as important players in reproductive biology. However, how their proteome is regulated throughout the menstrual cycle is not known. Such information can provide novel insights into biological processes critical for embryo development, implantation, and successful pregnancy. Using mass spectrometry-based quantitative proteomics, we show that small EVs (sEVs) isolated from uterine lavage of fertile women (UL-sEV), compared to infertile women, are laden with proteins implicated in antioxidant activity (SOD1, GSTO1, MPO, CAT). Functionally, sEVs derived from endometrial cells enhance antioxidant function in trophectoderm cells. Moreover, there was striking enrichment of invasion-related proteins (LGALS1/3, S100A4/11) in fertile UL-sEVs in the secretory (estrogen plus progesterone-driven, EP) versus proliferative (estrogen-driven, E) phase, with several players downregulated in infertile UL-sEVs. Consistent with this, sEVs from EP- versus E-primed endometrial epithelial cells promote invasion of trophectoderm cells. Interestingly, UL-sEVs from fertile versus infertile women carry known players/predictors of embryo implantation (PRDX2, IDHC), endometrial receptivity (S100A4, FGB, SERPING1, CLU, ANXA2), and implantation success (CAT, YWHAE, PPIA), highlighting their potential to inform regarding endometrial status/pregnancy outcomes. Thus, this study provides novel insights into proteome reprograming of sEVs and soluble secretome in uterine fluid, with potential to enhance embryo implantation and hence fertility.
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Affiliation(s)
- Alin Rai
- Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, Australia.,Central Clinical School, Monash University, Melbourne, Victoria, Australia
| | - Qi Hui Poh
- Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, Australia.,Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia
| | - Monique Fatmous
- Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, Australia.,Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia
| | - Haoyun Fang
- Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, Australia
| | - Shanti Gurung
- Hudson Institute of Medical Research, Clayton, Victoria, Australia
| | - Beverley Vollenhoven
- Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia.,Monash IVF, Clayton, Victoria, Australia.,Women's and Newborn Program, Monash Health, Clayton, Victoria, Australia
| | - Lois A Salamonsen
- Hudson Institute of Medical Research, Clayton, Victoria, Australia.,Department of Molecular and Translational Science, Monash University, Clayton, Victoria, Australia
| | - David W Greening
- Baker Heart and Diabetes Institute, Molecular Proteomics, Melbourne, Victoria, Australia.,Central Clinical School, Monash University, Melbourne, Victoria, Australia.,Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia
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36
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Campanile G, Baruselli PS, Limone A, D'Occhio MJ. Local action of cytokines and immune cells in communication between the conceptus and uterus during the critical period of early embryo development, attachment and implantation - Implications for embryo survival in cattle: A review. Theriogenology 2021; 167:1-12. [PMID: 33743503 DOI: 10.1016/j.theriogenology.2021.02.020] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Revised: 02/15/2021] [Accepted: 02/24/2021] [Indexed: 12/16/2022]
Abstract
Early embryo development, implantation and pregnancy involve a complex dialogue between the embryo and mother. In cattle this dialogue starts as early as days 3-4 when the embryo is still in the oviduct, and it continues to implantation. Immunological processes involving cytokines, mast cells and macrophages form an important part of this dialogue. Amongst the cytokines, interleukin-6 (Il-6) and leukemia inhibitory factor (LIF) are secreted by both the embryo and uterine endometrium and form part of an ongoing and reciprocating dialogue. Mast cells and macrophages populate the uterine endometrium during embryo development and are involved in achieving the correct balance between inflammatory and anti-inflammatory reactions at the uterus that are associated with embryo attachment and implantation. Embryo loss is the major cause of reproductive wastage in cattle, and livestock generally. A deeper understanding of immunological processes during early embryo development will help to achieve the next step change in the efficiency of natural and assisted breeding.
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Affiliation(s)
- Giuseppe Campanile
- Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy.
| | - Pietro S Baruselli
- Department of Animal Reproduction, Faculty of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
| | - Antonio Limone
- Instituto Zooprofilattico Sperimentale Del Mezzogiorno, Portici, Naples, Italy
| | - Michael J D'Occhio
- School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, New South Wales, 2006, Australia
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37
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Machtinger R, Baccarelli AA, Wu H. Extracellular vesicles and female reproduction. J Assist Reprod Genet 2021; 38:549-557. [PMID: 33471231 PMCID: PMC7910356 DOI: 10.1007/s10815-020-02048-2] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2020] [Accepted: 12/21/2020] [Indexed: 01/28/2023] Open
Abstract
Extracellular vesicles (EVs) are nano-sized membrane bound complexes that have been identified as a mean for intercellular communication between cells and tissues both in physiological and pathological conditions. These vesicles contain numerous molecules involved in signal transduction including microRNAs, mRNAs, DNA, proteins, lipids, and cytokines and can affect the behavior of recipient cells. Female reproduction is dependent on extremely fine-tuned endocrine regulation, and EVs may represent an added layer that contributes to this regulation. This narrative review article provides an update on the research of the role of EVs in female reproduction including folliculogenesis, fertilization, embryo quality, and implantation. We also highlight potential pitfalls in typical EV studies and discuss gaps in the current literature.
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Affiliation(s)
- Ronit Machtinger
- Sheba Medical Center, Ramat Gan and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
- Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, 52621, Tel Hashomer, Israel.
| | - Andrea A Baccarelli
- Environmental Precision Biosciences Laboratory, Columbia University, Mailman School of Public Health, New York, NY, USA
| | - Haotian Wu
- Environmental Precision Biosciences Laboratory, Columbia University, Mailman School of Public Health, New York, NY, USA
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38
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Lee SH, Lira-Albarrán S, Saadeldin IM. Comprehensive Proteomics Analysis of In Vitro Canine Oviductal Cell-Derived Extracellular Vesicles. Animals (Basel) 2021; 11:ani11020573. [PMID: 33672125 PMCID: PMC7926305 DOI: 10.3390/ani11020573] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 01/25/2021] [Accepted: 01/26/2021] [Indexed: 11/29/2022] Open
Abstract
Simple Summary As the dog shows unique and peculiar reproductive characteristics, assisted reproductive techniques such as in vitro maturation and in vitro fertilization have not been well-established compared with those of other mammals. Our recent work demonstrated the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs. Here, we provided for the first time a comprehensive proteomic analysis of OC-EVs. A total of 398 proteins were identified in all OC-EVs samples. A functional enrichment analysis indicated that these core proteins were involved in the key cellular metabolic process related to oocyte maturation and embryonic development. The current comprehensive description of the canine OC-EVs proteome would provide a fundamental resource for further understanding canine reproductive physiology, the interaction of sperms with female counterparts during fertilization, early pregnancy, and establishing an efficient system of in vitro embryo production. Abstract Dogs (Canis lupus familiaris) have unique and peculiar reproductive characteristics. While the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs has begun to be elucidated, no study has yet provided extensive information on the biological content and physiological function of OC-EVs and their role in canine oocyte development. Here, we aimed to provide the first comprehensive proteomic analysis of OC-EVs. We identified 398 proteins as present in all OC-EVs samples. The functional enrichment analysis using Gene Ontology terms and an Ingenuity Pathway Analysis revealed that the identified proteins were involved in several cellular metabolic processes, including translation, synthesis, expression, and protein metabolism. Notably, the proteins were also involved in critical canonical pathways with essential functions in oocyte and embryo development, such as ERK/MAPK, EIF2, PI3K/AKT, and mTOR signaling. These data would be an important resource for studying canine reproductive physiology and establishing a successful in vitro embryo production system in dogs.
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Affiliation(s)
- Seok Hee Lee
- Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA;
- Correspondence: (S.H.L.); (I.M.S.); Tel.: +1-4154760932 (S.H.L.); +966-530910740 (I.M.S.)
| | - Saúl Lira-Albarrán
- Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143, USA;
| | - Islam M Saadeldin
- Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt
- Department of Animal Production, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia
- King Faisal Specialist Hospital & Research Centre, Department of Comparative Medicine, Riyadh 11211, Saudi Arabia
- Correspondence: (S.H.L.); (I.M.S.); Tel.: +1-4154760932 (S.H.L.); +966-530910740 (I.M.S.)
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Wang F, Zhao S, Deng D, Wang W, Xu X, Liu X, Zhao S, Yu M. Integrating LCM-Based Spatio-Temporal Transcriptomics Uncovers Conceptus and Endometrial Luminal Epithelium Communication that Coordinates the Conceptus Attachment in Pigs. Int J Mol Sci 2021; 22:ijms22031248. [PMID: 33513863 PMCID: PMC7866100 DOI: 10.3390/ijms22031248] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2020] [Revised: 01/15/2021] [Accepted: 01/24/2021] [Indexed: 02/06/2023] Open
Abstract
Attachment of conceptus to the endometrial luminal epithelium (LE) is a critical event for early placentation in Eutheria. Since the attachment occurs at a particular site within the uterus, a coordinated communication between three spatially distinct compartments (conceptus and endometrial LE from two anatomical regions of the uterus to which conceptus attaches and does not attach) is essential but remains to be fully characterized. Using the laser capture microdissection (LCM) technique, we firstly developed an approach that can allow us to pair the pig conceptus sample with its nearby endometrial epithelium sample without losing the native spatial information. Then, a comprehensive spatio-temporal transcriptomic profile without losing the original conceptus-endometrium coordinates was constructed. The analysis shows that an apparent difference in transcriptional responses to the conceptus exists between the endometrial LE from the two anatomically distinct regions in the uterus. In addition, we identified the communication pathways that link the conceptus and endometrial LE and found that these pathways have important roles in conceptus attachment. Furthermore, a number of genes whose expression is spatially restricted in the two different anatomical regions within the uterus were characterized for the first time and two of them (SULT2A1 and MEP1B) may cooperatively contribute to establish conceptus attachment in pigs. The results from our study have implications in understanding of conceptus/embryo attachment in pigs and other large polytocous species.
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40
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Sanchez DJD, Vasconcelos FR, Teles-Filho ACA, Viana AGA, Martins AMA, Sousa MV, Castro MS, Ricart CA, Fontes W, Bertolini M, Bustamante-Filho IC, Moura AA. Proteomic profile of pre-implantational ovine embryos produced in vivo. Reprod Domest Anim 2021; 56:586-603. [PMID: 33460477 DOI: 10.1111/rda.13897] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2020] [Revised: 01/12/2021] [Accepted: 01/14/2021] [Indexed: 12/11/2022]
Abstract
The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.
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Affiliation(s)
- Deisy J D Sanchez
- Department of Animal Science, Federal University of Ceará, Fortaleza, Brazil
| | - Fabio R Vasconcelos
- Department of Animal Science, Federal University of Ceará, Fortaleza, Brazil
| | | | - Arabela G A Viana
- Department of Animal Science, Federal University of Ceará, Fortaleza, Brazil
| | - Aline M A Martins
- Laboratory of Protein Chemistry and Biochemistry, University of Brasília, Brasília, Brazil
| | - Marcelo V Sousa
- Laboratory of Protein Chemistry and Biochemistry, University of Brasília, Brasília, Brazil
| | - Mariana S Castro
- Laboratory of Protein Chemistry and Biochemistry, University of Brasília, Brasília, Brazil
| | - Carlos A Ricart
- Laboratory of Protein Chemistry and Biochemistry, University of Brasília, Brasília, Brazil
| | - Wagner Fontes
- Laboratory of Protein Chemistry and Biochemistry, University of Brasília, Brasília, Brazil
| | - Marcelo Bertolini
- The School of Veterinay Medicine, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
| | | | - Arlindo A Moura
- Department of Animal Science, Federal University of Ceará, Fortaleza, Brazil
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41
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Capra E, Lange-Consiglio A. The Biological Function of Extracellular Vesicles during Fertilization, Early Embryo-Maternal Crosstalk and Their Involvement in Reproduction: Review and Overview. Biomolecules 2020; 10:E1510. [PMID: 33158009 PMCID: PMC7693816 DOI: 10.3390/biom10111510] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Revised: 10/29/2020] [Accepted: 10/31/2020] [Indexed: 12/18/2022] Open
Abstract
Secretory extracellular vesicles (EVs) are membrane-enclosed microparticles that mediate cell to cell communication in proximity to, or distant from, the cell of origin. Cells release a heterogeneous spectrum of EVs depending on their physiologic and metabolic state. Extracellular vesicles are generally classified as either exosomes or microvesicles depending on their size and biogenesis. Extracellular vesicles mediate temporal and spatial interaction during many events in sexual reproduction and supporting embryo-maternal dialogue. Although many omic technologies provide detailed understanding of the molecular cargo of EVs, the difficulty in obtaining populations of homogeneous EVs makes difficult to interpret the molecular profile of the molecules derived from a miscellaneous EV population. Notwithstanding, molecular characterization of EVs isolated in physiological and pathological conditions may increase our understanding of reproductive and obstetric diseases and assist the search for potential non-invasive biomarkers. Moreover, a more precise vision of the cocktail of biomolecules inside the EVs mediating communication between the embryo and mother could provide new insights to optimize the therapeutic action and safety of EV use.
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Affiliation(s)
- Emanuele Capra
- Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche IBBA CNR, 26900 Lodi, Italy;
| | - Anna Lange-Consiglio
- Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, 26900 Lodi, Italy
- Centro Clinico-Veterinario e Zootecnico-Sperimentale di Ateneo, Università degli Studi di Milano, 26900 Lodi, Italy
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Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos. ZYGOTE 2020; 29:138-149. [PMID: 33118919 DOI: 10.1017/s0967199420000593] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
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.
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Lee SH, Saadeldin IM. Exosomes as a Potential Tool for Supporting Canine Oocyte Development. Animals (Basel) 2020; 10:E1971. [PMID: 33121043 PMCID: PMC7693116 DOI: 10.3390/ani10111971] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2020] [Revised: 10/24/2020] [Accepted: 10/25/2020] [Indexed: 12/27/2022] Open
Abstract
The canine oviduct is a unique reproductive organ where the ovulated immature oocytes complete their maturation, while the other mammals ovulate matured gametes. Due to their peculiar reproductive characteristics, the in vitro maturation of dog oocytes is still not wellestablished compared with other mammals. Investigations of the microenvironment conditions in the oviductal canal are required to establish a reliable in vitro maturation system in the dog. Previous studies have suggested that the oviduct and its derivatives play a key role in improving fertilization as well as embryo development. In particular, the biological function of oviduct-derived exosomes on sperm and early embryo development has been investigated in porcine, bovine, and murine species. However, the information about their functions on canine cumulus-oocyte complexes is still elusive. Recent canine reproductive studies demonstrated how oviduct-derived extracellular vesicles such as microvesicles and exosomes interact with oocyte-cumulus complexes and how they can play roles in regulating canine cumulus/oocyte communications. In this review, we summarize the physiological characteristics of canine oviduct-derived exosomes and their potential effects on cumulus cells development as well as oocyte in vitro maturation via molecular signaling pathways.
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Affiliation(s)
- Seok Hee Lee
- Center for Reproductive Sciences, Department of Obstetrics and Gynecology, University of California San Francisco, San Francisco, CA 94143, USA
| | - Islam M. Saadeldin
- Department of Animal Production, College of Food and Agriculture Sciences, King Saud University, Riyadh 44511, Saudi Arabia;
- Department of Comparative Medicine, King Faisal Specialist Hospital & Research Centre, Riyadh 11211, Saudi Arabia
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44
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Sidrat T, Khan AA, Joo MD, Wei Y, Lee KL, Xu L, Kong IK. Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development. Int J Mol Sci 2020; 21:ijms21207589. [PMID: 33066562 PMCID: PMC7593913 DOI: 10.3390/ijms21207589] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Revised: 10/09/2020] [Accepted: 10/12/2020] [Indexed: 12/31/2022] Open
Abstract
Oviduct flushing is enriched by a wide variety of nutrients that guide the 3-4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.
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Affiliation(s)
- Tabinda Sidrat
- Department of Animal Science, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea; (T.S.); (M.-D.J.); (Y.W.); (L.X.)
| | - Abdul Aziz Khan
- Center for Discovery and Innovation, Hackensack University Medical Center, Nutley, NJ 07110, USA;
| | - Myeon-Don Joo
- Department of Animal Science, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea; (T.S.); (M.-D.J.); (Y.W.); (L.X.)
| | - Yiran Wei
- Department of Animal Science, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea; (T.S.); (M.-D.J.); (Y.W.); (L.X.)
| | - Kyeong-Lim Lee
- The King Kong Corp. Ltd., Gyeongsang National University, Jinju 52828, Korea;
| | - Lianguang Xu
- Department of Animal Science, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea; (T.S.); (M.-D.J.); (Y.W.); (L.X.)
| | - Il-Keun Kong
- Department of Animal Science, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea; (T.S.); (M.-D.J.); (Y.W.); (L.X.)
- The King Kong Corp. Ltd., Gyeongsang National University, Jinju 52828, Korea;
- Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Korea
- Correspondence: ; Tel.: +82-55-772-1942
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Banliat C, Le Bourhis D, Bernardi O, Tomas D, Labas V, Salvetti P, Guyonnet B, Mermillod P, Saint-Dizier M. Oviduct Fluid Extracellular Vesicles Change the Phospholipid Composition of Bovine Embryos Developed In Vitro. Int J Mol Sci 2020; 21:ijms21155326. [PMID: 32727074 PMCID: PMC7432015 DOI: 10.3390/ijms21155326] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2020] [Revised: 07/20/2020] [Accepted: 07/25/2020] [Indexed: 12/11/2022] Open
Abstract
Oviduct fluid extracellular vesicles (oEVs) have been proposed as bringing key molecules to the early developing embryo. In order to evaluate the changes induced by oEVs on embryo phospholipids, fresh bovine blastocysts developed in vitro in the presence or absence of oEVs were analyzed by intact cell MALDI-TOF (Matrix assisted laser desorption ionization—Time of flight) mass spectrometry (ICM-MS). The development rates, cryotolerance, and total cell number of blastocysts were also evaluated. The exposure to oEVs did not affect blastocyst yield or cryotolerance but modified the phospholipid content of blastocysts with specific changes before and after blastocoel expansion. The annotation of differential peaks due to oEV exposure evidenced a shift of embryo phospholipids toward more abundant phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingomyelins (SM) with long-chain fatty acids. The lipidomic profiling of oEVs showed that 100% and 33% of the overabundant masses in blastocysts and expanded blastocysts, respectively, were also present in oEVs. In conclusion, this study provides the first analysis of the embryo lipidome regulated by oEVs. Exposure to oEVs induced significant changes in the phospholipid composition of resulting embryos, probably mediated by the incorporation of oEV-phospholipids into embryo membranes and by the modulation of the embryonic lipid metabolism by oEV molecular cargos.
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Affiliation(s)
- Charles Banliat
- INRAE, CNRS, University of Tours, IFCE, UMR 85 PRC, F-37380 Nouzilly, France; (C.B.); (O.B.); (D.T.); (V.L.); (P.M.)
- Union Evolution, F-35530 Noyal-Sur-Vilaine, France;
| | | | - Ophélie Bernardi
- INRAE, CNRS, University of Tours, IFCE, UMR 85 PRC, F-37380 Nouzilly, France; (C.B.); (O.B.); (D.T.); (V.L.); (P.M.)
| | - Daniel Tomas
- INRAE, CNRS, University of Tours, IFCE, UMR 85 PRC, F-37380 Nouzilly, France; (C.B.); (O.B.); (D.T.); (V.L.); (P.M.)
- INRAE, Université de Tours, CHU de Tours, Plate-forme CIRE, F-37380 Nouzilly, France
| | - Valérie Labas
- INRAE, CNRS, University of Tours, IFCE, UMR 85 PRC, F-37380 Nouzilly, France; (C.B.); (O.B.); (D.T.); (V.L.); (P.M.)
- INRAE, Université de Tours, CHU de Tours, Plate-forme CIRE, F-37380 Nouzilly, France
| | | | | | - Pascal Mermillod
- INRAE, CNRS, University of Tours, IFCE, UMR 85 PRC, F-37380 Nouzilly, France; (C.B.); (O.B.); (D.T.); (V.L.); (P.M.)
| | - Marie Saint-Dizier
- INRAE, CNRS, University of Tours, IFCE, UMR 85 PRC, F-37380 Nouzilly, France; (C.B.); (O.B.); (D.T.); (V.L.); (P.M.)
- Department Agrosciences, Faculty of Sciences and Techniques, University of Tours, F-37200 Tours, France
- Correspondence: ; Tel.: +33-2-47-42-75-08
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Bauersachs S, Almiñana C. Embryo-Maternal Interactions Underlying Reproduction in Mammals. Int J Mol Sci 2020; 21:ijms21144872. [PMID: 32664189 PMCID: PMC7402305 DOI: 10.3390/ijms21144872] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2020] [Accepted: 07/06/2020] [Indexed: 02/07/2023] Open
Abstract
This Special Issue, “Embryo-Maternal Interactions Underlying Reproduction in Mammals”, gathers a collection of 23 articles, 16 original research articles and 7 up-to-date reviews, providing new findings or summarizing current knowledge on embryo–maternal interactions in seven different mammalian species including humans. Considering the different players involved in these embryo-maternal interactions, articles are mainly focused on one of these different players: the oviduct, the uterus, the embryo or the emergent extracellular vesicles. Additionally, a few articles bring up the impact of reproductive, but also non-reproductive, diseases, as well as stress factors, on the establishment of pregnancy. We hope the readers enjoy this collection of articles and that the knowledge assembled here will support and inspire current and future research investigations. We would like to thank all authors for their contributions to this Special Issue.
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Affiliation(s)
- Stefan Bauersachs
- Functional Genomics, Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, 8315 Lindau (ZH), Switzerland
- Correspondence: (S.B.); (C.A.)
| | - Carmen Almiñana
- Functional Genomics, Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, 8315 Lindau (ZH), Switzerland
- UMR85 PRC, INRAE, CNRS 7247, Université de Tours, IFCE, 37380 Nouzilly, France
- Correspondence: (S.B.); (C.A.)
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Kölle S, Hughes B, Steele H. Early embryo-maternal communication in the oviduct: A review. Mol Reprod Dev 2020; 87:650-662. [PMID: 32506761 DOI: 10.1002/mrd.23352] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 05/05/2020] [Accepted: 05/10/2020] [Indexed: 12/11/2022]
Abstract
An intact embryo-maternal communication is critical for the establishment of a successful pregnancy. To date, a huge number of studies have been performed describing the complex process of embryo-maternal signaling within the uterus. However, recent studies indicate that the early embryo communicates with the oviductal cells shortly after fertilizationand that this is important for the successful establishment of pregnancy. Only if the early embryo is capable to signal the mother within a precise timeframe and to garner a response, will the embryo be able to survive and reach the uterus. This review will give an overview of all the experimental designs which have investigated embryo-maternal interaction in the oviduct. In addition to that, it will provide a comprehensive analysis of the findings to date elucidating the morphological and molecular changes in the oviduct which are induced by the presence of the early embryo highlighting how the tubal responses affect embryo development and survival.
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Affiliation(s)
- Sabine Kölle
- Health Sciences Centre, School of Medicine, University College Dublin, Dublin, Ireland
| | - Barbara Hughes
- Health Sciences Centre, School of Medicine, University College Dublin, Dublin, Ireland
| | - Heather Steele
- Health Sciences Centre, School of Medicine, University College Dublin, Dublin, Ireland
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