Abstract Advancements in assisted reproduction (AR) methodologies have allowed significant improvements in live birth rates of women who otherwise would not be able to conceive. One of the tools that allowed this improvement is the possibility of embryo selection based on genetic status, performed via preimplantation genetic testing (PGT). Even though the widespread use of PGT from TE biopsy helped to decrease the interval from the beginning of the AR intervention to pregnancy, especially in older patients, in AR, there are still many concerns about the application of this invasive methodology in all cycles. Therefore, recently, researchers started to study the use of cell free DNA (cfDNA) released by the blastocyst in its culture medium to perform PGT, in a method called non-invasive PGT (niPGT). The development of a niPGT would bring the diagnostics power of conventional PGT, but with the advantage of being potentially less harmful to the embryo. Its implementation in clinical practice, however, is under heavy discussion since there are many unknowns about the technique, such as the origin of the cfDNA or if this genetic material is a true representative of the actual ploidy status of the embryo. Available data indicates that there is high correspondence between results observed in TE biopsies and the ones observed from cfDNA, but these results are still contradictory and highly debatable. In the present review, the advantages and disadvantages of niPGT are presented and discussed in relation to tradition TE biopsy-based PGT. Furthermore, there are also presented some other possible non-invasive tools that could be applied in the selection of the best embryo, such as quantification of other molecules as quality biomarkers, or the use artificial intelligence (AI) to identify the best embryos based on morphological and/or morphokitetic parameters.

Abstract Given the major role of the mitochondrion in cellular homeostasis, dysfunctions of this organelle may lead to several common diseases in humans. Among these, maternal diseases linked to mitochondrial DNA (mtDNA) mutations are of special interest due to the unclear pattern of mitochondrial inheritance. Multiple copies of mtDNA are present in a cell, each encoding for 37 genes essential for mitochondrial function. In cases of mtDNA mutations, mitochondrial malfunctioning relies on mutation load, as mutant and wild-type molecules may co-exist within the cell. Since the mutation load associated with disease manifestation varies for different mutations and tissues, it is hard to predict the progeny phenotype based on mutation load in the progenitor. In addition, poorly understood mechanisms act in the female germline to prevent the accumulation of deleterious mtDNA in the following generations. In this review, we outline basic aspects of mitochondrial inheritance in mammals and how they may lead to maternally-inherited diseases. Furthermore, we discuss potential therapeutic strategies for these diseases, which may be used in the future to prevent their transmission.

Abstract 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.

Nesta revisão, apresentamos alguns problemas clínicos encontrados nos animais derivados de PIV, principalmente derivados de transferência de núcleo, e discutimos o possível papel da epigenética em tais processos. Além disso, uma vez que vesículas secretadas por células têm sido descritas como possíveis reguladores de processos reprodutivos fisiológicos importantes, tais como a foliculogênese e a fertilização, estas são aqui apresentadas como uma possível nova ferramenta para a manipulação do período embrionário pré-implantacional através de moléculas efetoras, contidas em tais vesículas.

This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.

A utilização do soro fetal bovino (SFB), embora bastante disseminada na produção in vitro (PIV) de embriões bovinos, apresenta limitações por ser um meio indefinido e por causar efeitos que prejudicam a qualidade desses embriões. Por esse motivo, nos últimos anos, grande parte das pesquisas relacionadas à PIV está voltada para a substituição do SFB por outros compostos nos meios de cultura. No presente estudo, foram utilizados como compostos protéicos a albumina sérica bovina livre de ácidos graxos (BSA-FAF) e um produto comercial denominado fluido embriônico (FE) de maneira isolada ou em diferentes combinações e concentrações, com objetivo de substituir ou diminuir a concentração do SFB durante a maturação in vitro (MIV). Para isso, oócitos bovinos foram maturados in vitro nos seguintes grupos (G) que foram delineados de acordo com a suplementação protéica recebida: G1 (controle) = 10% de SFB, G2 = 8mg/mL de BSA-FAF, G3 = 10% de FE, G4 = 6mg/mL de BSA-FAF + 5% de SFB, G5 = 6mg/mL de BSA-FAF + 3,5% de SFB + 1,5% de FE, G6 = 6mg/mL de BSA-FAF + 1,5% de SFB + 3,5% de FE, G7 = 6mg/mL de BSA-FAF + 5% de FE e G8 = 5% de SFB + 5% de FE. Após 24 horas de MIV, os oócitos foram classificados de acordo com a progressão meiótica e migração dos grânulos corticais (GC). As taxas de maturação foram avaliadas pelo teste do Qui-Quadrado (χ²) ou, quando apropriado, pelo teste exato de Fisher, e para o estudo dos efeitos dos suplementos foram realizados contrastes ortogonais. O grupo suplementado com BSA-FAF (G2) mostrou diminuição na taxa de oócitos que atingiram MII (75%) em comparação aos grupos G1, G4, G5 e G8 (88,9%, 89,6%, 87% e 86,8%, respectivamente), sem diferir do do G3 (79,8%), G6 (82,9%) e G7 (82,9%). Ademais, o G3 também apresentou diminuição na taxa de maturação nuclear quando comparado ao G4. Quanto à maturação citoplasmática, nos grupos G2, G7, G6 e G3, houve redução (p<0,05) das taxas para 43,9%, 43,2%, 43,1% e 36,5%, respectivamente, quando comparadas ao meio controle (G1), que permitiu a obtenção de valores médios de 62,4%. Por outro lado, nos grupos G8, G4 e G5, a taxa de maturação citoplasmática não foi afetada com a redução do SFB, onde 59,3%, 51,3% e 50,8% dos oócitos apresentaram os GC dispostos na periferia, respectivamente. Os resultados obtidos pelo teste de contrastes ortogonais complementam os obtidos na avaliação da maturação nuclear e migração de grânulos corticais, mostrando a necessidade do SFB durante a MIV, mesmo que em baixas concentrações, e a possibilidade de diminuir a sua concentração associando-o a BSA-FAF e/ou FE. Dessa forma, conclui-se que é possível reduzir a concentração de SFB no meio de MIV para até 3,5% sem prejuízo significativo aos índices de maturação nuclear e citoplasmática.

The use of fetal calf serum (FCS), although widely employed during in vitro production (IVP) of bovine embryos, has limitations. FCS is an undefined media and may have harmful effects on the quality of embryos. For this reason, in recent years, research efforts aimed at improving IVP of bovine embryos, have focused at the replacement of FCS by alternative compounds in culture media. In this study, fatty acid free bovine serum albumin (BSA-FAF) and embryonic fluid (EF) were used separately or in combination, in different concentrations, to replace or reduce the concentration of FCS during in vitro maturation (IVM). For this purpose, bovine oocytes were in vitro matured under different treatments and were assigned to one of the following groups (G) according to the protein supplementation: G1 (control) = 10% FCS, G2 = 8mg/mL BSA-FAF, G3 = 10% EF, G4 = 6mg/mL BSA-FAF + 5% FCS, G5 = 6mg/mL BSA-FAF + 3.5% FCS + 1.5% EF, G6 = 6mg/mL BSA-FAF + 1.5% FCS + 3.5% EF, G7 = 6mg/mL BSA-FAF + 5% EF, and G8 = 5% FCS + 5% EF). After 24 hours of IVM, oocytes were classified according to the meiotic progression and migration of cortical granules (CG) to the periphery. Maturation rates were evaluated by chi-square test (χ2) or, when appropriate, by Fisher's exact test, and orthogonal contrasts were performed to isolate effects of the compounds. The group supplemented with BSA-FAF (G2) had a lower number of oocytes that reached MII (75%) compared to the groups G1, G4, G5 and G8 (88.9%, 89.6%, 87% and 86.8%, respectively), with no difference in relation to G3 (79.8%), G6 (82.9%) and G7 (82.9%). Moreover, G3 also showed inferior nuclear maturation rate when compared to G4. Regarding cytoplasmic maturation, the rates were reduced to 43.9%, 43.2%, 43.1% and 36.5% in G2, G7, G6 and G3 groups, respectively, compared to the control group (G1; 62.4%). On the other hand, in the groups G8, G4 and G5, maturation rates were not affected by reduction of FCS, where 59.3%, 51.3% and 50.8% of the oocytes displayed CG arranged peripherally, respectively. The results obtained by the orthogonal contrast test are in accordance with the ones from the evaluation of the nuclear maturation and cortical granules migration. These data show the need of FCS on the MIV, even in low concentrations, and the possibility of decrease its concentration by associating it with BSA-FAF and/or EF. Therefore, we concluded that it is possible to reduce the concentration of FCS in IVM medium to a concentration of 3.5% without affecting nuclear and cytoplasmic maturation rates.

Embryo quality is influenced by the culture conditions that affect in vitro maturation (IVM), fertilization (IVF) and culture (IVC) rates. The present study investigated the feasibility of producing bovine embryos after the replacement of fetal calf serum (FCS) and bovine serum albumin (BSA) by ovalbumin (OVA). The IVM and IVC medium were supplemented with 10% FCS, 4 mg/mL BSA, or 4 mg/mL OVA. The IVF medium was supplemented with 6 mg/mL BSA or OVA. For IVM, supplementation with FCS, BSA, and OVA did not affect nuclear maturation or cortical granule migration. Higher rates of formation of two pronuclei were obtained when FCS was employed for IVM (79.97%), regardless of the supplement used for IVF, and when BSA was used for IVF (59.4%), regardless of the supplement used for IVM. Supplementation with OVA for IVM+IVC (20.40%) and for IVF (22.15%) was inferior to supplementation with FCS for IVM+IVC (30.47%) and with BSA for IVF (28.91%) for blastocyst development. Hatching rates were lower using OVA for IVM+IVC (23.02%) and for IVF (28.93%) compared with FCS and BSA under the same conditions (40.78 and 34.82%, respectively) and BSA for IVF (36.82%). Supplementation with OVA for IVM+IVC and IVF resulted in reduced inner cell mass, trophectoderm cells and total blastocyst cell numbers (17.29, 37.88, and 55.17, respectively). In conclusion, OVA is a protein source for bovine in vitro embryo production, although the quantity and quality of bovine blastocysts using only ovalbumin in the entire in vitro production process are lower than those obtained in the presence of FCS and BSA, when used as supplements in any step of bovine in vitro embryo production.

A proposta deste estudo foi avaliar se existem alterações nos padrões hematológicos e bioquímicos de cadelas da raça Golden Retriever portadoras do gene da distrofia muscular progressiva em comparação aos valores obtidos em cadelas não portadoras de mesma raça e idade. Foram analisados 33 animais, distribuídos em dois grupos, um composto por 19 cadelas Golden Retrievers não portadoras (GRNP) e outro composto por 14 cadelas Golden Retrievers portadoras do gene da distrofia muscular (GRP). Os dois grupos foram submetidos aos mesmos testes hematológicos e bioquímicos, com a mesma frequência e durante o mesmo intervalo de tempo. Apesar de existir diferença estatisticamente significativa entre os grupos para alguns parâmetros hematológicos avaliados, todos os resultados obtidos estavam de acordo com os valores de referência utilizados. Na avaliação dos parâmetros bioquímicos séricos a dosagem de ALT no grupo GRNP ficou levemente acima da média, porém sem grandes significados clínicos A CK também apresentou níveis elevados no grupo GRP, devido à degeneração e necrose muscular característicos da doença, as alterações encontradas nessa análise já eram esperadas. Os demais parâmetros não se alteraram.

The purpose of this study was to evaluate whether there are alterations in hematological and biochemical patterns of female Golden Retriever dogs carrying the gene for progressive muscular dystrophy compared to not carriers dogs of the same breed, age and gender. We analyzed 33 animals, divided into two groups; one consisting of 13 not carriers dogs Golden Retrievers (GRNP) and the other composed of 14 dogs Golden Retrievers carrying the gene for muscular dystrophy (GRP). Animals of both groups underwent biochemical and hematological tests with the same frequency and at the same time interval. Although there is a statistically significant difference between groups for some hematological parameters evaluated, all results were in line with the benchmarks used. In the assessment of serum biochemical parameters in the determination of ALT GRS group was slightly above average, but without major clinical significance CK also showed high levels in GRP group, due to muscle degeneration and necrosis characteristic of the disease, the changes found in this analysis were already expected. The other parameters did not change.