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1.
Rev. bras. reprod. anim ; 47(3): 598-606, jul.-set. 2023. graf
Artigo em Português | VETINDEX | ID: biblio-1436773

Resumo

Por ser uma célula altamente especializada, o espermatozoide apresenta diferentes mecanismos epigenéticos, sendo os principais as metilações do DNA, o código de histonas, os ncRNAs (RNAs não codificadores), e a alta condensação da cromatina pela presença das protaminas. Estes mecanismos interagem entre si, contribuindo para a formação do epigenoma espermático, que modela a carga molecular espermática, que, por sua vez, pode impactar sobre as características do desenvolvimento embrionário e da progênie. Dessa forma, atualmente é consenso que o papel do espermatozoide ultrapassa a entrega de DNA de qualidade para o oócito no momento da fecundação. Pesquisas recentes de diversos grupos, incluindo o nosso, mostram que além da contribuição com DNA de qualidade, o espermatozoide entrega moléculas ao oócito no momento da fecundação que influenciam o desenvolvimento do embrião. Recentemente, essas moléculas de origem espermática (Em inglês: sperm-borne) também são associadas com alterações metabólicas e cognitivas da progênie. Embora ainda pouco se entenda como esses mecanismos podem persistir mesmo com o ciclo de reprogramação celular que ocorre logo após a fecundação, é evidente que estes podem impactar as características da progênie. Nesta revisão abordaremos sobre a modulação do epigenoma espermático e seus efeitos no desenvolvimento embrionário.(AU)


Since it is a highly specialized cell, the spermatozoa display different epigenetic mechanisms; the main ones are DNA methylation, histone code, ncRNAs (non-coding RNAs), and high chromatin condensation by the presence of protamines. These mechanisms act in synergy contributing to forming the sperm epigenome, which modulates the spermatic molecular cargo, and, may impact embryo and offspring development features. Thus, it is currently a consensus that the role of spermatozoa goes beyond delivering quality DNA to the oocyte at fertilization. Relevant findings from several research groups, including ours, have shown that sperm delivers several molecules to the oocyte at fertilization, beyond the contribution to DNA, which influences the development of the embryo. Recently, these sperm-borne molecules have also been associated with metabolic and cognitive changes in the offspring. Although the mechanism by which these changes can persist even after embryo reprogramming is not completely understood, evidence shows that sperm cell molecular content impacts embryo and offspring development. This review will mainly focus on the modulation of the sperm epigenome and its effects on embryo development.(AU)


Assuntos
Animais , Masculino , Fertilidade/genética , Epigenoma/genética , Espermatozoides , Desenvolvimento Embrionário/fisiologia
2.
Anim. Reprod. (Online) ; 19(4): e20220063, 2022. graf, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1403214

Resumo

Extracellular vesicles (EVs) derived from stem cells (SCs) have regenerative potential and the possibility of being used in treating chronic diseases. EVs present lower risk of tumorigenicity and easily to isolation and storage. Therefore, this research aims to compare the morphological characteristics of the EVs (up to 150nm) derived from stem cells obtained from canine amniotic membranes in different passages during the in vitro culture. For this, cells from the amniotic membranes were isolated, cultured, and characterized. In order to answer our aim, the number of cells was normalized at each passage to generate conditioned media for EVs separation. The cells were differentiated into adipogenic, chondrogenic, and osteogenic tissue, to characterize these cells as mesenchymal stem cells (MSC). Moreover, flow cytometry analysis was performed and showed that the MSC were positive for CD90, CD105 and negative for CD34, CD45, mesenchymal and hematopoietic markers, respectively. For EVs analysis, MSC in different passages (P0-P2) were culture until 80% of confluence, then the medium was replaced by EVs depleted medium. After 48h, culture medium was collected and centrifuged to separate EVs, followed by nanoparticle tracking analysis. The EVs were also characterized by western blot and transmission electron microscopy (TEM). EVs were positive for Alix and negative for Cytochrome C as well as presented the traditional cup-shape by transmission electronic microscopy. Our results demonstrated that the concentration in the different passages was increased in P0 compared to P1 and P2 (p<0.05). No differences were found in EVs size (P0=132nm, P1=130nm and P2=120nm). Together, these results demonstrate that P0 of MSC is enriched of EVs when compared to later passages, suggesting that this passage would be the best to be applied in pre-clinical tests. Despite that, more studies are necessary to identify the EVs content and how the cells will respond to treatment with them.(AU)


Assuntos
Animais , Células-Tronco Fetais/fisiologia , Vesículas Extracelulares , Taxa Secretória
3.
J. venom. anim. toxins incl. trop. dis ; 27: e20200187, 2021. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1351016

Resumo

Background: The efficacy of bone marrow mesenchymal stromal cells (BM-MSC) and its extracellular vesicles has been demonstrated for a broad spectrum of indications, including kidney diseases. However, BM-MSC donor characteristics and their potential are not usually considered. Therefore, the present work aims to evaluate the nephroprotective capacity of sEV secreted by BM-MSC from trained rats inunilateral ureteral obstruction (UUO) model. Methods: BM-MSC was characterized by their differentiation potential and immunophenotypic markers. The sEV were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and western blot. Its miRNA cargo was examined by quantitative PCR analysis for miR-26a, 126a, and 296. Wistar rats were submitted to UUO procedure and concomitantly treated with sEV secreted by BM-MSC from the untrained andtrained rats. The kidney tissue from all groups was evaluated for fibrosis mediators (transforming growth factor beta1 and collagen), CD34-angiogenesis marker, and hypoxia-inducible factor 1 alpha (HIF-1α). Results: Treadmill training stimulated in BM-MSC the production of sEV loaded with pro-angiogenic miR-296. The treatment with this sEVin UUO-rats was able to attenuate collagen accumulation and increase CD34 and HIF-1α in the kidney tissue when compared to untrained ones. Tubular proximal cells under hypoxia and exposed to BM-MSC sEV demonstrate accumulation in HIF-1α and NFR-2 (nuclear factor erythroid 2-related factor 2), possibly to mediate the response to hypoxia and oxidative stress, under these conditions. Conclusion: The BM-MSC sEV from trained animals presented an increased nephroprotective potential compared to untrained vesicles by carrying 296-angiomiR and contributing to angiogenesis in UUO model.(AU)


Assuntos
Obstrução Ureteral , Vesículas Extracelulares , Nefropatias , Hipóxia , Estresse Oxidativo
4.
Artigo em Inglês | LILACS-Express | LILACS, VETINDEX | ID: biblio-1484784

Resumo

Abstract Background: The efficacy of bone marrow mesenchymal stromal cells (BM-MSC) and its extracellular vesicles has been demonstrated for a broad spectrum of indications, including kidney diseases. However, BM-MSC donor characteristics and their potential are not usually considered. Therefore, the present work aims to evaluate the nephroprotective capacity of sEV secreted by BM-MSC from trained rats inunilateral ureteral obstruction (UUO) model. Methods: BM-MSC was characterized by their differentiation potential and immunophenotypic markers. The sEV were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and western blot. Its miRNA cargo was examined by quantitative PCR analysis for miR-26a, 126a, and 296. Wistar rats were submitted to UUO procedure and concomitantly treated with sEV secreted by BM-MSC from the untrained andtrained rats. The kidney tissue from all groups was evaluated for fibrosis mediators (transforming growth factor beta1 and collagen), CD34-angiogenesis marker, and hypoxia-inducible factor 1 alpha (HIF-1). Results: Treadmill training stimulated in BM-MSC the production of sEV loaded with pro-angiogenic miR-296. The treatment with this sEVin UUO-rats was able to attenuate collagen accumulation and increase CD34 and HIF-1 in the kidney tissue when compared to untrained ones. Tubular proximal cells under hypoxia and exposed to BM-MSC sEV demonstrate accumulation in HIF-1 and NFR-2 (nuclear factor erythroid 2-related factor 2), possibly to mediate the response to hypoxia and oxidative stress, under these conditions. Conclusion: The BM-MSC sEV from trained animals presented an increased nephroprotective potential compared to untrained vesicles by carrying 296-angiomiR and contributing to angiogenesis in UUO model.

5.
J. venom. anim. toxins incl. trop. dis ; 26: e20190067, 2020. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135161

Resumo

Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)


Assuntos
Animais , Búfalos/microbiologia , Doenças Transmissíveis , Theileria , Nanopartículas , Vesículas Extracelulares , Fenômenos Biológicos , Proteômica
6.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 26: e20190067, May 29, 2020. ilus, tab, graf
Artigo em Inglês | VETINDEX | ID: vti-29964

Resumo

Background: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)


Assuntos
Animais , Masculino , Búfalos/parasitologia , Vesículas Extracelulares/química , Theileria , Búfalos/sangue , Proteoma/análise , Nanopartículas/análise , Infecções por Protozoários
7.
Anim. Reprod. (Online) ; 16(1): 31-38, jan.-mar. 2019. ilus
Artigo em Inglês | VETINDEX | ID: biblio-1461422

Resumo

Intercellular communication is an essential mechanism for development and maintenance of multicellular organisms. Extracellular vesicles (EVs) were recently described as new players in the intercellular communication. EVs are double-membrane vesicles secreted by cells and are classified according to their biosynthesis, protein markers and morphology. These extracellular vesicles contain bioactive materials such as miRNA, mRNA, protein and lipids. These characteristics permit their involvement in different biological processes. Reproductive physiology is complex and involves constant communication between cells. Different laboratories have described the presence of EVs secreted by ovarian follicular cells, oviductal cells, in vitro produced embryos and by the endometrium, suggesting that EVs are involved in the development of gametes and embryos, in animals and humans. Therefore, is important to understand physiological mechanisms and contributions of EVs in female reproduction in order to develop new tools to improve in vivo reproductive events and assisted reproductive techniques (ARTs). This review will provide the current knowledge related to EVs in female reproductive tissues and their role in ARTs.


Assuntos
Feminino , Animais , Técnicas Reprodutivas/tendências , Técnicas Reprodutivas/veterinária , Vesículas Extracelulares/enzimologia
8.
Anim. Reprod. ; 16(1): 31-38, jan.-mar. 2019. ilus
Artigo em Inglês | VETINDEX | ID: vti-20907

Resumo

Intercellular communication is an essential mechanism for development and maintenance of multicellular organisms. Extracellular vesicles (EVs) were recently described as new players in the intercellular communication. EVs are double-membrane vesicles secreted by cells and are classified according to their biosynthesis, protein markers and morphology. These extracellular vesicles contain bioactive materials such as miRNA, mRNA, protein and lipids. These characteristics permit their involvement in different biological processes. Reproductive physiology is complex and involves constant communication between cells. Different laboratories have described the presence of EVs secreted by ovarian follicular cells, oviductal cells, in vitro produced embryos and by the endometrium, suggesting that EVs are involved in the development of gametes and embryos, in animals and humans. Therefore, is important to understand physiological mechanisms and contributions of EVs in female reproduction in order to develop new tools to improve in vivo reproductive events and assisted reproductive techniques (ARTs). This review will provide the current knowledge related to EVs in female reproductive tissues and their role in ARTs.(AU)


Assuntos
Animais , Feminino , Técnicas Reprodutivas/tendências , Técnicas Reprodutivas/veterinária , Vesículas Extracelulares/enzimologia
9.
Anim. Reprod. (Online) ; 16(3): 485-496, 2019. ilus
Artigo em Inglês | VETINDEX | ID: biblio-1461458

Resumo

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies.


Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Vesículas Extracelulares/genética , Oócitos
10.
Anim. Reprod. ; 16(3): 485-496, 2019. ilus
Artigo em Inglês | VETINDEX | ID: vti-22350

Resumo

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies.(AU)


Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Vesículas Extracelulares/genética , Oócitos
11.
Anim. Reprod. (Online) ; 15(3): 261-270, July-Sept. 2018. ilus
Artigo em Inglês | VETINDEX | ID: biblio-1461366

Resumo

The magnitude of oocyte’s role for embryo development is categorical. This unique cell contains the machineries and cellular components necessary to remodel male and female chromatin, to sustain early development and to, ultimately, generate a complete and complex individual. However, to gain these competences before fertilization, the oocyte undergoes several morphological, cellular and molecular changes during its lifetime enclosed in the ovarian follicle. This review will briefly revisit how the oocyte orchestrate the follicular cells, and how molecules transit to the oocyte from the innermost (cumulus) and outermost (antrum and granulosa cells) layers surrounding the follicle-enclosed oocyte. Finally, we will discuss the interferences of in vitro culture conditions in the communication of the oocyte with its surrounding cells and the potential strategies to modulate these communication systems to increase oocyte competence.


Assuntos
Folículo Ovariano/anatomia & histologia , Folículo Ovariano/crescimento & desenvolvimento , Vesículas Extracelulares , Oócitos/classificação
12.
Anim. Reprod. ; 15(3): 261-270, July-Sept. 2018. ilus
Artigo em Inglês | VETINDEX | ID: vti-734673

Resumo

The magnitude of oocytes role for embryo development is categorical. This unique cell contains the machineries and cellular components necessary to remodel male and female chromatin, to sustain early development and to, ultimately, generate a complete and complex individual. However, to gain these competences before fertilization, the oocyte undergoes several morphological, cellular and molecular changes during its lifetime enclosed in the ovarian follicle. This review will briefly revisit how the oocyte orchestrate the follicular cells, and how molecules transit to the oocyte from the innermost (cumulus) and outermost (antrum and granulosa cells) layers surrounding the follicle-enclosed oocyte. Finally, we will discuss the interferences of in vitro culture conditions in the communication of the oocyte with its surrounding cells and the potential strategies to modulate these communication systems to increase oocyte competence.(AU)


Assuntos
Folículo Ovariano/anatomia & histologia , Folículo Ovariano/crescimento & desenvolvimento , Vesículas Extracelulares , Oócitos/classificação
13.
Anim. Reprod. (Online) ; 13(3): 257-263, jul.-set. 2016.
Artigo em Inglês | VETINDEX | ID: biblio-1461225

Resumo

Ovarian functions, which involve dynamically regulated processes of selection, recruitment and dominance, are known to be regulated by an array of genes, which are expressed in spatiotemporal manner in follicular somatic cells and gametes. This differential expression of genes in mammalian follicular cells is partly regulated by posttranscriptional gene regulators named microRNAs (miRNAs). In addition to the cellular miRNAs, growing number of evidences are available on the potential role of extracellular vesicles mediated transfer of miRNAs in follicular fluid. These extracellular vesicles are shown to be involved in cellto-cell communication within the follicular environment. The molecular messages carried by the extracellular vesicles released into extracellular space are thought to be reflections of the physiological status of the cells from where they are released. Therefore due to their structural nature and potential to cargo several physiologically relevant molecules, exosomes have a great potential to be used as markers of oocyte developmental competence in follicular environment. Here, we review large sets of literatures to show the dynamic nature of miRNAs during various stages of mammalian follicular development, oocyte growth and the role of some of the miRNAs in ovarian cell functions. Moreover, the presence of microvesicle and exosome-coupled extracellular miRNAs in mammalian follicular fluid and their potential involvement in cellto-cell communication are briefly discussed.


Assuntos
Animais , Fase Folicular , Mamíferos/crescimento & desenvolvimento , MicroRNAs , Vesículas Extracelulares
14.
Anim. Reprod. ; 13(3): 257-263, jul.-set. 2016.
Artigo em Inglês | VETINDEX | ID: vti-17514

Resumo

Ovarian functions, which involve dynamically regulated processes of selection, recruitment and dominance, are known to be regulated by an array of genes, which are expressed in spatiotemporal manner in follicular somatic cells and gametes. This differential expression of genes in mammalian follicular cells is partly regulated by posttranscriptional gene regulators named microRNAs (miRNAs). In addition to the cellular miRNAs, growing number of evidences are available on the potential role of extracellular vesicles mediated transfer of miRNAs in follicular fluid. These extracellular vesicles are shown to be involved in cellto-cell communication within the follicular environment. The molecular messages carried by the extracellular vesicles released into extracellular space are thought to be reflections of the physiological status of the cells from where they are released. Therefore due to their structural nature and potential to cargo several physiologically relevant molecules, exosomes have a great potential to be used as markers of oocyte developmental competence in follicular environment. Here, we review large sets of literatures to show the dynamic nature of miRNAs during various stages of mammalian follicular development, oocyte growth and the role of some of the miRNAs in ovarian cell functions. Moreover, the presence of microvesicle and exosome-coupled extracellular miRNAs in mammalian follicular fluid and their potential involvement in cellto-cell communication are briefly discussed.(AU)


Assuntos
Animais , Fase Folicular , Mamíferos/crescimento & desenvolvimento , MicroRNAs , Vesículas Extracelulares
15.
Tese em Português | VETTESES | ID: vtt-220383

Resumo

Resumo da tese em português: A criopreservação espermática é o método mais eficiente para preservar sêmen a longo prazo em mamíferos. No entanto, o congelamento do sêmen de cachaços ainda é o maior desafio para a indústria suína devido à alta sensibilidade ao choque frio das células espermáticas desta espécie e à variação dos resultados pós-descongelamento entre indivíduos e ejaculados do mesmo reprodutor. Para resolver esse problema, investigamos se os microRNAs (miRNAs) presentes em células espermáticas e pequenas vesículas extracelulares (EVs) do plasma seminal de ejaculados de suínos podem predizer se ejaculados permanecerão com alta qualidade após passarem pelo processo de congelação-descongelação. Para tanto, foram coletados 27 ejaculados de alta qualidade de 27 machos (um de cada), obtidas amostras de miRNAs de espermatozoides e das EVs de plasma seminal in natura através de protocolos de centrifugação e, após o processo de criopreservação, determinamos dois grupos com diferentes congelabilidades considerando as análises de estrutura e funcionalidade dos espermatozoides após a descongelação (P <0,05): Alta congelabilidade (AC; n = 04) e baixa congelabilidade (BC; n = 04). Feito isso, finalmente investigamos o perfil de miRNAs de espermatozoides e EVs de plasma seminal em ambos os grupos com diferentes congelabilidades. Nossos principais resultados mostraram que três miRNAs foram diferentemente abundantes em ejaculados BC, sendo o ssc-miR-503 encontrado em níveis mais elevados em células espermáticas. O ssc-miR-130a e o ssc-miR-9 foram mais abundantes em EVs do plasma seminal (P <0,10). Por meio da análise de enriquecimento, foi possível verificar que esses miRNAs estão relacionados as modificações no desenvolvimento das células germinativas masculinas e na produção de energia utilizada pelos espermatozóides para manter sua viabilidade e funcionalidade. Portanto, por meio deste estudo, podemos demonstrar que o ssc-miR-503, ssc-miR-130a e ssc-miR-9 estão relacionados à baixa criotolerância espermática no sêmen de suínos. Deste modo, esses miRNAs podem ser usados como biomarcadores para predizer sua baixa capacidade de tolerar o processo de criopreservação.


Resumo da tese em inglês: Sperm cryopreservation is the most efficient method to preserve sêmen for the long term in mammals. However, freeze boar sêmen is still the biggest challenge for the swine industry due to the high cold shock sensitivity of boar sperm cells and the variance of post-thaw results among individuals and ejaculates from the same boar. To solve this problem, we investigate if microRNAs (miRNAs) present in sperm cells and small extracellular vesicles (E.V.s) from seminal plasma of raw boar ejaculates can predict high-quality ejaculates after underwent the freeze-thaw process. Therefore, we collected 27 high-quality ejaculates of 27 boars (one of each) obtained miRNAs samples of sperm cells and E.V.s from raw seminal plasma through centrifugation protocols. After the cryopreservation process, we determined two groups with different freezability considering the analysis post-thaw of structure and sperm functionality (P <0.05): High freezability (H.F.; n=04) and low freezability (L.F.; n=04). That done, we finally investigate the miRNAs profile of sperm cells and E.V.s from seminal plasma in both groups with different freezability. Our main results showed that three miRNAs were differently abundant in L.F. ejaculates, being the ssc-miR-503 found in higher levels in sperm cells. The ssc-miR-130a and ssc-miR-9 most abundant in E.V.s from seminal plasma (P <0.10). Through enrichment analysis, it was possible to verify that these miRNAs could be performing modifications in the development of male germ cells and in the production of energy to spermatozoa to maintain their viability and functionality. Therefore, through this study, we can demonstrate that ssc-miR-503, ssc-miR-130a, and ssc-miR-9 are related to low sperm cryotolerance in boars semen. So those miRNAs can be used as a biomarker to predict their low ability to tolerate the cryopreservation process.

16.
Tese em Inglês | VETTESES | ID: vtt-222088

Resumo

BRIDI, A. Vesículas extracelulares secretadas por embriões bovinos produzidos in vivo e in vitro: conteúdo de miRNAs e os efeitos moleculares sobre o endométrio e o corpo lúteo. 254 f. Tese (Doutorado) Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga-SP, 2021. Perfis metabólicos, padrões de expressão gênica, desenvolvimento embrionário, capacidade de estabelecer e manter gestações precoces são diferenças encontradas quando embriões bovinos produzidos in vivo e in vitro são comparados. Para que a gestação tenha sucesso é necessária uma perfeita comunicação entre o embrião e o organismo materno. Vesículas extracelulares pequenas (sEVs) fazem parte dessa comunicação e carregam moléculas bioativas, como miRNAs, que podem atuar em células-alvo dentro do sistema reprodutivo bovino. Com isso, nossa hipótese é que embriões bovinos produzidos in vivo e in vitro secretam sEVs contendo diferentes miRNAs que podem modular diferencialmente o padrão de expressão gênica endometrial e o perfil de miRNAs do corpo lúteo. Para isso, primeiramente, investigamos o conteúdo de miRNA em blastocistos eclodidos produzidos in vivo ou in vitro e, em sEVs secretadas por eles. Blastocistos eclodidos produzidos in vivo ou in vitro, no dia 9 de desenvolvimento, possuem diferentes perfis de miRNA. EVs pequenas secretadas por eles, do dia 7 até o dia 9 de desenvolvimento, contém miRNAs diferentes. Esses miRNAs diferentemente expressos são preditos por regularem vias envolvidas no desenvolvimento embrionário inicial e na receptividade endometrial. Por meio disso, as primeiras interações entre o embrião e o organismo materno podem ser modificadas, e isso pode afetar o sucesso da gestação. Em segundo lugar, investigamos as alterações no transcriptoma global do endométrio após o co-cultivo de explantes endometriais com um blastocisto produzido in vivo ou in vitro no dia 7 do desenvolvimento. Genes diferentemente expressos foram identificados no endométrio e estão associados com a presença e origem do embrião. Além disso, sEVs presentes nos meios condicionados (C.M.) por essas células embrionárias e endometriais contém diferentes miRNAs, que são preditos por modularem a via de sinalização da oxitocina. Finalmente, para compreender melhor se a comunicação entre o embrião, o endométrio e o corpo lúteo pode ser intermediada por sEVs, explantes luteais foram tratados com sEVs presentes nos C.M. por explantes endometriais cultivados sozinhos ou com um blastocisto bovino produzido in vivo ou in vitro no dia 7 do desenvolvimento. O perfil de miRNAs foi modificado nos explantes luteais tratados com essas sEVs e, nós podemos observar um efeito da presença e origem dos embriões nessas alterações. Juntos, esses resultados sugerem que sEVs intermedeiam a comunicação inicial entre o embrião, endométrio e o corpo lúteo, contribuindo para manter a viabilidade e funcionalidade luteal. Entretanto, futuros experimentos precisam ser realizados para avaliar efeitos biológicos específicos desses miRNAs carreados por essas sEVs no endométrio e no corpo lúteo. Além disso, a origem do embrião (in vivo ou in vitro) modifica as interações entre ele, o endométrio e o corpo lúteo, sugerindo fortemente que esses embriões tenham necessidades distintas para se desenvolverem.


BRIDI, A., Extracellular vesicles secreted by bovine embryos produced in vivo and in vitro: miRNAs content and molecular effects in endometrial and luteal tissues. 2021. 254 f. Tese (Doutorado) Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga-SP, 2021. Metabolic profiles, gene expression patterns, embryonic development, and the ability to establish and sustain early pregnancies are differences founded when in vivo- and in vitro- produced bovine embryos are compared. To the establishment of a successful pregnancy the perfect embryo- maternal communication is needed. Small extracellular vesicles (sEVs) are part of this crosstalk and carry bioactive molecules such as miRNAs that can act on target bovine cells within thereproductive system. Thereby, our hypothesis is that in vivo and in vitro bovine embryos secrete sEVs containing different miRNAs that can differentially modulate endometrial gene expression pattern and miRNAs profile in the corpus luteum (CL). To address this hypothesis, firstly, we investigated the miRNA content of in vivo and in vitro hatched blastocysts and in sEVs secreted by them. Day 9 in vivo or in vitro hatched blastocysts have distinct miRNA profiles. Small EVs secreted by them from day 7 up to day 9 of development contain different miRNAs. These miRNAs differently expressed are predicted to regulate pathways involved with early embryonic development and endometrial receptivity. Thereby, early embryo-maternal interactions can be modified and consequently can affect pregnancy success. Secondly, we investigated changes in the endometrial global transcriptome after the co-culture of endometrial explants with a day 7 in vivo or in vitro blastocysts. Differently expressed genes were identified in the endometrium and are associated with the embryo's presence or origin. Moreover, sEVs present in the conditioned media (C.M.) by these embryonic and endometrial cells contain different miRNAs, which are predicted to modulate the oxytocin signaling pathway. Finally, to understand if the communication among embryo, endometrium, and corpus luteum can be mediated by sEVs, luteal explants were treated with sEVs from C.M. by endometrial explants alone or cultured with a day 7 in vivo or in vitro blastocysts. MicroRNAs profile was modified in luteal explants treated with these sEVs, and we can observe an effect of the embryo presence and origin in these alterations. Together these results suggest that sEVs mediate early embryo-endometrium-corpus luteum communication, contributing to maintaining luteal viability and functionality. However, further experiments are needed to evaluate specific biological effects of these miRNAs carried by sEVs in the endometrium and in the corpus luteum. Moreover, the embryo origin (in vivo and in vitro) modify the interactions among embryo-endometrium-corpus luteum, suggesting strongly that these embryos have distinct needed to develop.

17.
Tese em Português | VETTESES | ID: vtt-220880

Resumo

Recentemente, descobriu-se que as vesículas extracelulares (VEs), presentes no fluido dos folículos ovarianos são uma importante forma de comunicação celular, com funções biológicas diversificadas. O presente estudo teve como objetivo analisar as VEs recuperadas do fluido folicular de ovários ipsilaterais e contralaterais ao corpo lúteo (CL), em fêmeas bovinas da raça holandesa (Bos taurus taurus) em dois momentos distintos: antes e após a IATF, de acordo com o status gestacional. Foram utilizadas 10 vacas holandesas de uma propriedade produtora de leite, da cidade de Arapoti-Paraná. O fluido folicular foi colhido através da técnica de ovum pick-up (OPU) de vacas lactantes, com 30 dias pós-parto, aptas a reprodução. Em seguida foram submetidas a IATF (inseminação artificial em tempo fixo). O tratamento hormonal utilizado foi a base de P4 e estradiol. A IATF foi realizada utilizando sêmen de um mesmo touro para o grupo de fêmeas deste estudo. Após cerca de 60 dias da IATF, a OPU foi realizada em fêmeas prenhes (N=5) e não prenhes (N=4). As amostras de fluido folicular foram direcionadas para isolamento e análise por sequenciamento de nanoparticulas pelo Nanosigth (NS300; NTA 3.1 Build 3.1.45; Malvern). As variáveis concentração (VEs/mL) e diâmetro (nm) das VEs foram analisadas pelo teste T de Student ou teste Mann-Whitney com significancia de p 0,05. O fluido folicular proveniente de fêmeas prenhes e não prenhes contralateral ao CL apresentou maior concentração de VEs (200nm) quando comparado as amostras obtidas ipsilateral ao CL (2,7x1010 VEs/mL vs. 1,07x1010 VEs/mL; p=0,013) dos mesmos animais. Quando analisamos o grupo de fêmeas não prenhes (N=3) separadamente, a concentração de VEs é maior para contralateral (3,12x1010 VEs/mL) se comparado com o ipsilateral (6,38x109 VEs/mL) ao CL (p=0,024). Porém, quando avaliamos o mesmo grupo (ipsilateral vs. contralateral) para as fêmeas prenhes (N=3), não foi possível observar diferença para a concentração de VEs (p=0,663). Para a avaliação entre CL ipsilateral (prenhe) vs. ipsilateral (não prenhe), as fêmeas prenhes apresentaram maior concentração de VEs (1,50x1010 VEs/mL) em relação as fêmeas não prenhes (6,38x109 VEs/mL; p=0,016). Não houve diferença entre as demais comparações realizadas e para a média do diâmetro das VEs. Em nosso estudo, foi possível concluir que a presença ou ausência de CL, bem como o status gestacional da fêmea, influencia no número de VEs presentes no fluido dos folículos adjacentes.


Recently, it was discovered that extracellular vesicles (EVs), present in the fluid of ovarian follicles are an important form of cellular communication, with diverse biological functions. The present study aimed to analyze the EVs recovered from the follicular fluid of ipsilateral (same side) and contralateral (opposite side) of the corpus luteum (CL) in Holstein cows (Bos taurus taurus) at two different times: before and after timed artificial insemination (TAI), according to the gestational status. Ten cows from a dairy farm in Arapoti-Paraná were used. The follicular fluid was collected using the ovum pick-up technique (OPU), of the lactating Holstein cows, 30 days postpartum, ready for reproduction, and then were submitted to TAI. The hormonal treatment used on the property was based on P4 and estradiol. The TAI was performed using semen from the same bull for all females in this study. After about 60 days from the TAI, the OPU was perfomed in pregnant (N=5) and non-pregnant (N=4) females. The follicular fluid samples, collected were directed to isolation and Nanoparticle tracking analysis by Nanosigth (NS300; NTA 3.1 Build 3.1.45; Malvern). The concentration (EVs/mL) and diameter (nm) of the VEs were analyzed by Student's T test or Mann-Whitney test with significance of p0.05. The follicular fluid of pregnant and non-pregnant females from the ovarian opposite the CL presented higher concentration of EVs (200nm) when compared to ovarian samples with CL (2.7x1010 EVs/mL vs. 1.07x10 10 EVs/ml; p=0.013) from the animals. When we analyzed the group of nonpregnant females (N=3) separately, the nanoparticle concentration is higher for contralateral follicular fluid (3.12x1010 EVs/mL) compared to the ipsilateral (6.38x109 EVs/mL) follicular fluid (p=0.024). However, when evaluating the same group (contralateral vs. ipsilateral) for pregnant females (N=3) was not observed difference in the concentration of EVs (p=0.663). The evaluation between ipsilateral (pregnant) vs. ipsilateral (non-pregnant) on the pregnant females had greater concentration EVs (1,50x1010 EVs/mL) compared to non-pregnant females (6,38x109 EVs/mL; p=0.016). There was no difference between the others comparisons made and for the mean EVs diameter. In our study, it was possible to conclude that the presence or absence of CL, as well as its gestational status influences the number of EVs present in the fluid of adjacent follicles.

18.
Tese em Português | VETTESES | ID: vtt-220836

Resumo

Garnica, T. K. Vesículas Extracelulares Pequenas como Potencial Marcador Preditivo Em Cães Com Linfoma Multicêntrico. 2020. 84 p. Dissertação (Mestrado em Ciências) - Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2020. O linfoma é o tipo mais comum de malignidade hematológica canina, onde a forma multicêntrica é responsável por 75% de todos os casos. O tratamento padrão é o protocolo CHOP de 19 semanas, no qual 85% dos cães alcançam resposta completa / parcial; porém uma grande parcela recidiva a doença em um período de até um ano após o diagnóstico. A avaliação da resposta terapêutica e a compreensão dos mecanismos envolvidos no processo quimioresistência tem sido os maiores desafios do linfoma canino. Diante disso, esse trabalho foi dividido em dois estudos principais: No primeiro estudo investigamos in vivo o potencial de exossomos séricos e seus miRNAs como marcadores preditivos para o linfoma canino. No segundo estudo investigamos in vitro o papel dos exossomos derivados de células quimioresistentes em linhagens humana e canina de neoplasias hematopoiéticas. Para o primeiro estudo foram utilizados vinte e dois cães (8 em Remissão Completa e 14 em Progressão da Doença). Os exossomos isolados dos cães foram avaliados quanto ao tamanho e concentração e foi realizada uma triagem de 95 oncomir em amostras selecionadas de pacientes com RC e DP. Os pacientes com DP apresentaram maior concentração de exossomos séricos no momento do diagnóstico do que os pacientes com RC (D0, P = 0,0277). A análise da curva ROC foi significativa para a concentração de exossomos para predizer a resposta ao CHOP (AUC = 0,8076, P = 0,0203) e sobrevida global (AUC = 0,8333, P = 0,0136). O miR-205 (P = 0,0384) e o miR-222 (P = 0,0578) apresentaram maior frequência no grupo RC e o mir-20a foi mais expresso em pacientes com RC (P = 0,085), enquanto o miR-93 foi mais expresso em pacientes com DP (P = 0,09). No segundo estudo induzimos a quimioresistência utilizando o protocolo CHOP nas células 3132 (linfoma canino de células B) e células Jurkat (leucemia de células T humana). Mostramos que as células resistentes 3132-CR e Jurkat-CR têm um tempo de duplicação mais lento em comparação com suas células ingênuas respectivas. As células 3132 e 3132-CR secretaram uma maior quantidade de exossomos após o tratamento quimioterápico (P = 0,0187), porém não houve diferença na quantidade de exossomos entre as linhagens (P = 0,7661). Houve um aumento na proliferação de células após o tratamento do exossomos em comparação ao controle para 3132 (P <0,001) e Jurkat (P <0,0035). No entanto, não houve diferença na proliferação comparando o tratamento usando exossomos derivados de células nativas e aqueles derivados de células CR para 3132 (P = 0,11) e Jurkat (P = 0,91). A proliferação celular foi maior após 78 horas de tratamento com exossomos para 3132 (P <0,001) e Jurkat (P <0,001). Em conclusão, os resultados gerados por esses estudos podem desencadear avanços na oncologia veterinária pela introdução da abordagem de biópsia líquida e pela compreensão de mecanismos de desenvolvimento de quimioresistência mediados por exossomos.


Garnica, T. K. The potential use of small extracellular vesicles as predictive marker in canine multicentric lymphoma. 2020. 84 p. Thesis (Master in Science) Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, 2020. Lymphoma is the most common type of canine hematological malignancy, where the multicentric form is responsible for 75% of all cases. The standard treatment is the 19-week CHOP protocol, in which 85% of dogs achieve complete / partial response; however, a large portion of the disease relapses in a period of up to one year after diagnosis. The evaluation of the therapeutic response and the understanding of the mechanisms involved in the chemoresistance process have been the biggest challenges of canine lymphoma. Therefore, this work was divided into two main studies: In the first study, we investigated in vivo the potential of serum exosomes and their miRNAs as predictive markers for canine lymphoma. In the second study, we investigated in vitro the role of exosomes derived from chemoresistant cells in human and canine lineages of hematopoietic neoplasms. Twenty-two dogs were used for the first study (8 in Complete Remission and 14 in Disease Progression). The exosomes isolated from the dogs were evaluated for size and concentration and a 95 oncomir screening was performed on selected samples from patients with CR and PD. PD patients had a higher concentration of serum exosomes at the time of diagnosis than patients with CR (D0, P = 0.0277). The analysis of the ROC curve was significant for the concentration of exosomes to predict the response to CHOP (AUC = 0.8076, P = 0.0203) and overall survival (AUC = 0.8333, P = 0.0136). MiR-205 (P = 0.0384) and miR-222 (P = 0.0578) had a higher frequency in the CR group and mir-20a was more expressed in patients with CR (P = 0.085), while miR-93 was more expressed in patients with PD (P = 0.09). In the second study, we induced chemoresistance using the CHOP protocol in cells 3132 (canine B-cell lymphoma) and Jurkat cells (human T-cell leukemia). We have shown that resistant cells 3132-CR and Jurkat-CR have a slower doubling time compared to their respective naive cells. Cells 3132 and 3132-CR secreted a greater number of exosomes after chemotherapy (P = 0.0187), but there was no difference in the number of exosomes between strains (P = 0.7661). There was an increase in cell proliferation after treatment of the exosomes compared to the control for 3132 (P <0.001) and Jurkat (P <0.0035). However, there was no difference in proliferation comparing treatment using exosomes derived from native cells and those derived from CR cells for 3132 (P = 0.11) and Jurkat (P = 0.91). Cell proliferation was greater after 78 hours of treatment with exosomes for 3132 (P <0.001) and Jurkat (P <0.001). In conclusion, the results generated by these studies can trigger advances in veterinary oncology through the introduction of the liquid biopsy approach and the understanding of mechanisms of development of chemoresistance mediated by exosomes.

19.
Tese em Português | VETTESES | ID: vtt-213840

Resumo

DE ÁVILA, A.C.F.C.M. Efeitos da comunicação entre células do sistema reprodutivo feminino e oócitos bovinos mediada por vesículas extracelulares de diferentes estágios do ciclo estral. 2019. 194 f. Dissertação (Mestrado) Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2019. As técnicas de reprodução assistida (TRA) são alternativas para infertilidade e aumento da produção de animais de interesse. Apesar de amplamente utilizadas, as TRA podem causar consequências negativas aos indivíduos gerados desde o estabelecimento da gestação até a vida adulta. A tentativa de mimetizar o ambiente in vivo é uma opção para aprimorar as TRA. Vesículas extracelulares (VEs) secretadas por células são alvos de estudos recentes devido suas funções de comunicação intercelular no ambiente in vivo. As VEs são encontradas em fluidos corporais e possuem material bioativo, como miRNAs, que regulam o funcionamento de células a partir da modulação pós-transcricional. A hipótese deste estudo é que VEs pequenas secretadas por células foliculares e células do oviduto favoreçam a maturação e a fertilização in vitro de complexos cumulus-oócitos (CCOs). O objetivo do estudo foi caracterizar e avaliar os efeitos de VEs do fluido folicular (FF) e do oviduto obtidos em diferentes momentos do ciclo estral de bovinos durante a produção in vitro. Para isso, VEs foram isoladas de FF e do lavado de oviduto bovino, caracterizadas e utilizadas como suplementação durante a maturação e a fertilização in vitro, respectivamente. Os resultados demonstraram que as VEs do fluido de folículos em diferentes momentos do ciclo estral possuem diferentes perfis de miRNAs. A suplementação com VEs pequenas de FF durante a maturação in vitro (MIV) causou a internalização dessas VEs e do seu RNA pelos CCOs, mas não afetou as taxas de blastocistos. Porém, foi demonstrado que as VEs de diferentes origens foliculares modulam vias como PI3K-Akt e de transporte de RNA e processos biológicos relacionados à maturação oocitária, à ovulação e à resposta imune em células do cumulus. As VEs obtidas do oviduto em diferentes momentos do ciclo estral não afetaram as taxas de produção e a qualidade de blastocistos no modelo utilizado. Em conclusão, este estudo demonstrou que as VEs pequenas são modificadas de acordo com sua origem folicular e que impactam processos biológicos em células do cumulus de bovinos.


DE ÁVILA, A.C.F.C.M. The effects of communication between female reproductive system cells and bovine oocytes mediated by extracellular vesicles of different estrous cycle stages. 2019. 194f. M.Sc. Dissertation Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2019. Assisted reproductive techniques (ART) are alternatives to infertility and to increase animals production. Although these techniques are widely used, they can cause negative consequences since early pregnancy until adult life. Attempts to mimic in vivo environment are applied to improve ARTs. Extracellular vesicles (EVs) secreted by cells are targets of recent studies due to their function in intercellular communication. These vesicles are in body fluids and have bioactive material, as miRNAs, that regulate cell functions by post-transcriptional modulation. The hypothesis of this study is that small EVs secreted by follicular and oviductal cells improve the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs). Our goal was to characterize and to evaluate the effects of small EVs from follicular and oviductal fluids obtained from different bovine estrous cycle stages during in vitro embryo production. For this, EVs were isolated from bovine follicular fluid (FF) and oviductal flushing, characterized and used as supplementation during in vitro maturation and fertilization, respectively. The results demonstrated that small EVs from follicles at different estrous cycle stage have different miRNA profile. The small EVs from FF and their RNA were taken up by COCs during in vitro maturation but did not change blastocyst production rates. However, we demonstrated that small EVs from different follicle origin modulate pathways such as PI3K-Akt and RNA transport and biological processes related to oocyte maturation, ovulation and immune responses in cumulus cells. Small EVs from oviduct at different estrous cycle stage did not improve embryo production rates and embryo quality in our model. Therefore, this study demonstrated that small EVs are modified according to their follicular origin and are able to impact biological processes in bovine cumulus cells.

20.
Tese em Português | VETTESES | ID: vtt-217116

Resumo

As bases moleculares envolvidas no sucesso gestacional não estão totalmente definidas em todas as espécies. O fluido uterino contém uma variedade de nutrientes, proteínas, lipídeos e vesículas extracelulares, incluindo exossomos, provenientes de diversos tipos celulares de origem materna ou fetal. Os exossomos presentes no fluido uterino atuam na comunicação materno-fetal sendo capazes de transportar proteínas, moléculas de RNAm e microRNAs. O objetivo desse estudo foi determinar o tamanho e quantificar os exossomos presentes no fluido uterino de vacas não prenhes e prenhes por inseminação artificial (IA) e por fertilização in vitro em diferentes períodos gestacionais. As microvesículas extracelulares uterinas foram isoladas por centrifugação, o tamanho e a concentração dos exossomos foram analisados utilizando o NanoSight NS300. Observou-se aumento significativo na concentração dos exossomos do grupo IA 18 para o IA 32 e uma tendência de aumento na concentração do grupo FIV 18 para o grupo FIV 32. Os dados indicam que a concentração dos exossomos uterinos aumenta com a evolução da gestação, sendo necessário estudos em outras idades gestacionais e ainda estudos que determinem o conteúdo e origem desses exossomos, tornando possível elucidar questões relacionadas a comunicação maternofetal, bem como as diferenças observadas na viabilidade de gestações oriundas de inseminação artificial e fertilização in vitro.


Molecular basis involved in successful pregnancy is not fully defined in all species. Uterine fluid contains a variety of nutrients, proteins, lipids and extracellular vesicles, including the exosomes, excreted by different cell types of maternal or fetal origin. The exosomes present in uterine fluid modulate maternal-fetal communication, transporting proteins, mARN molecules and microARNs. The objective of this study was to determine the size and quantify the exosomes present in the uterine fluid of nonpregnant and pregnant cows from artificial insemination (AI) and in vitro fertilization (IVF), in different gestational periods. The uterine fluid was collected by uterine lavage on day 18 of the estrous cycle and at 18 and 32 days of gestation. Extracellular uterine microvesicles were isolated by centrifugation; the size and concentration of the exosomes were analyzed using the NanoSight NS300. There was a significant increase in the concentration of exosomes from group AI18 to AI32 and a tendency of concentration increase from group IVF 18 to group IVF 32. Data indicate that the concentration of uterine exosomes increases with the evolution of gestation. Studies in other gestational ages and studies that determine the content and origin of these exosomes are necessary, contributing to elucidate questions related to maternal-fetal communication, as well as the differences observed in the viability of pregnancies obtained by artificial insemination and in vitro fertilization.

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