RESUMO
OBJECTIVE: To analyze the results of the preimplantation genetic testing for aneuploidy at the Instituto de Fertilidad Humana - Inser Bogotá, Colombia, from 2016 to 2020. METHODS: This study is an observational, retrospective, and correlative analysis of biopsies from 319 embryos (from 54 patients) submitted to preimplantation genetic testing for aneuploidy by different molecular techniques. RESULTS: Of the 54 patients included in the study, 42 provided their own oocytes, and 12 used donated oocytes. The main indication to perform the preimplantation genetic testing was advanced maternal age. We obtained 319 embryos: Ninety-one (28.5%) euploid, 197 (61.8%) aneuploid and 31 (9.7%) with no detectable DNA. The highest rate of aneuploid embryos was found in patients over 40 years (72.7%), and the euploidy rate in patients under 35 years was 37.1%. After the transfer of euploid embryos, the rates for implantation, ongoing pregnancy, live birth, and miscarriage were 40%, 50%, 40.6%, and 0%, respectively. Older maternal age correlated with higher numbers of aneuploid embryos and lower numbers of both euploid and 5-day embryos. CONCLUSIONS: There was a positive correlation between maternal age and aneuploidy rate. Complex chromosomal abnormalities were the most frequent aneuploidies, followed by mosaicism and double aneuploidies. The miscarriage rate after the transfer of euploid embryos was 0 %.
Assuntos
Aborto Espontâneo , Diagnóstico Pré-Implantação , Aneuploidia , Blastocisto/patologia , Colômbia , Feminino , Clínicas de Fertilização , Testes Genéticos/métodos , Humanos , Gravidez , Diagnóstico Pré-Implantação/métodos , Estudos RetrospectivosRESUMO
Triclocarban (TCC) is an antimicrobial compound widely used in personal care products such as soaps, toothpaste, and shampoo. This agent is incompletely removed by wastewater treatment and represents an environmental contaminant. Recent studies have shown that TCC is associated with some endocrine disruptions. The aim of the present study was to evaluate if TCC exposure during critical periods of development (gestation and lactation) could lead to adverse effects on reproductive and behavior parameters of female offspring. Pregnant female Wistar rats were divided into four groups (n = 8-11/group): Control; TCC 0.3 mg/kg (TCC 0.3); TCC 1.5 mg/kg; TCC 3.0 mg/kg (TCC 3.0); and treated daily by oral gavage from gestational day 0 to lactational day 21. The female pups (F1 generation) were weaned on post-natal day 21 and included in the study. No litter-mates were used for the same group. There was a decrease in estradiol levels in the TCC 0.3 and TCC 3.0 groups. Moreover, there was a decrease in progesterone levels and an increase in pre-implantation loss in the TCC 3.0 group in adulthood. It is suggested, in this study, that the decrease in progesterone biosynthesis could interfere with implantation process. The exposure window to TCC is an important factor, as we found alterations only in the offspring.
Assuntos
Anti-Infecciosos/toxicidade , Carbanilidas/toxicidade , Disruptores Endócrinos/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Reprodução/efeitos dos fármacos , Animais , Biomarcadores/sangue , Blastocisto/efeitos dos fármacos , Blastocisto/patologia , Implantação do Embrião/efeitos dos fármacos , Perda do Embrião , Estradiol/sangue , Feminino , Idade Gestacional , Lactação , Gravidez , Progesterona/sangue , Ratos WistarRESUMO
Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70-85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.
Assuntos
Blastocisto/patologia , Embrião de Mamíferos/patologia , Cavalos/embriologia , Reação em Cadeia da Polimerase , Diagnóstico Pré-Implantação , Análise para Determinação do Sexo , Animais , Argentina , Biópsia , Cruzamento/economia , Cruzamento/métodos , Comércio , Transferência Embrionária/economia , Transferência Embrionária/métodos , Transferência Embrionária/veterinária , Feminino , Masculino , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Gravidez , Diagnóstico Pré-Implantação/métodos , Diagnóstico Pré-Implantação/veterinária , Análise para Determinação do Sexo/métodos , Análise para Determinação do Sexo/veterinária , Medicina Veterinária Esportiva/economia , Medicina Veterinária Esportiva/métodos , Medicina Veterinária Esportiva/organização & administraçãoRESUMO
Heat stress compromises bovine oocyte developmental competence, but the effects of high temperature during oocyte maturation on embryo chromatin organization is unknown. In this study bovine oocytes were exposed to heat shock (41°C) for 12 h during in vitro maturation and then submitted to in vitro fertilization. The heat shock did not affect (P > 0.05) the cleavage but reduced (P < 0.01) the blastocyst rate on Day 7 and Day 8. No effect (P > 0.05) on total cell number was found, but the heat shock increased (P < 0.05) the proportion of apoptotic cells in blastocysts at Day 8. Immunofluorescence analysis of H3K9me3 and HP1 was performed in embryos at 52 h post in vitro fertilization. An accumulation of H3K9me3 in the nuclei of embryos derived from heat-shocked oocytes at four-cell and eight-cell stages was found. Also, a non-expected higher proportion (P < 0.05) of four-cell stage embryos displaying nuclei with increased HP1 fluorescence was observed, suggesting an abnormal chromatin compaction in embryos from heat-shocked oocytes. Embryos at eight-cell stage derived from heat-shocked oocytes displayed lower (P < 0.05) relative amount of HSP40 transcripts than control ones. In conclusion, heat shock before fertilization has an effect on embryo chromatin, influencing the accumulation of H3K9me3 and HP1 in early embryos as well as further development.
Assuntos
Blastocisto/patologia , Cromatina/química , Embrião de Mamíferos/patologia , Resposta ao Choque Térmico , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/patologia , Oogênese , Animais , Apoptose , Blastocisto/metabolismo , Bovinos , Cromatina/genética , Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismoRESUMO
PURPOSE: Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. METHODS: We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2 h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. RESULTS: In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. CONCLUSIONS: Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/administração & dosagem , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/patologia , Bovinos , Colforsina/administração & dosagem , Células do Cúmulo/efeitos dos fármacos , Feminino , Meiose/genética , Oócitos/crescimento & desenvolvimento , Oócitos/patologia , Oogênese/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Gravidez , Ribossomos/efeitos dos fármacosRESUMO
Sickle cell anemia is an inherited systemic hemoglobinopathy that affects hemoglobin production in red blood cells, leading to early morbidity and mortality. It is caused by a homozygous nucleotide substitution (c.20A>T) in the ß-globin gene (HBB) that changes a glutamic acid to a valine in the protein. We present a case report of a fertile couple, both carriers of the sickle cell anemia mutation, with one affected daughter. Six cycles of assisted reproductive techniques were performed, resulting in 53 embryos in cleavage stage. Each embryo was biopsied and analyzed for pre-implantation genetic diagnosis (PGD) by fluorescent polymerase chain reaction, using polymorphic markers of the region of interest followed by capillary electrophoresis in an automated genetic analyzer. HLA Compatible and normal embryos for the mutation represented 3 (5.66%); while the carriers and compatible 6 (11.32%); therefore, embryos matching those of the affected daughter represented 9 (16.98%). A selected embryo in blastocyst stage was transferred, resulting in a healthy male newborn, who had the umbilical cord blood cells collected and stored. The affected daughter was immunosuppressed and received transplanted cells from the umbilical cord blood of her brother; the treatment was successful. Embryo selection using PGD technologies represent the most effective treatment plan for parents who want to have a healthy child, and it could cure another child already affected by inherited hemoglobinopathy.
Assuntos
Anemia Falciforme/terapia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Teste de Histocompatibilidade/métodos , Diagnóstico Pré-Implantação/métodos , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/genética , Blastocisto/metabolismo , Blastocisto/patologia , Criança , Transferência Embrionária , Feminino , Ligação Genética , Testes Genéticos/métodos , Humanos , Recém-Nascido , Masculino , Linhagem , Gravidez , Técnicas de Reprodução Assistida , Relações entre IrmãosRESUMO
Preimplantation genetic diagnosis was carried out for embryonic analysis in a patient with multiple endocrine neoplasia type 1 (MEN1). This is a rare autosomal-dominant cancer syndrome and the patients with MEN1 are characterized by the occurrence of tumors in multiple endocrine tissues, associated with germline and somatic inactivating mutations in the MEN1 gene. This case report documents a successful preimplantation genetic diagnosis (PGD) involving a couple at-risk for MEN1 syndrome, with a birth of a healthy infant. The couple underwent a cycle of controlled ovarian stimulation and intracytoplasmic sperm injection (ICSI). Embryos were biopsied at the blastocyst stage and cryopreserved; we used PCR-based DNA analysis for PGD testing. Only one of the five embryos analyzed for MEN1 syndrome was unaffected. This embryo was thawed and transferred following endometrial preparation. After positive ßHCG test; clinical pregnancy was confirmed by ultrasound, and a healthy infant was born. PGD for single gene disorders has been an emerging therapeutic tool for couples who are at risk of passing a genetic disease on to their offspring.
Assuntos
Blastocisto/patologia , Neoplasia Endócrina Múltipla Tipo 1 , Diagnóstico Pré-Implantação/métodos , Adulto , Biópsia , Feminino , Fertilização in vitro , Testes Genéticos/métodos , Humanos , Masculino , Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/patologia , Linhagem , Gravidez , Injeções de Esperma IntracitoplásmicasRESUMO
OBJECTIVE: This study aims to increase the knowledge about monopronucleated ICSI-derived blastocysts, analyzing trophectoderm biopsies by aCGH and FISH to evaluate their chromosome constitution. METHODS: Fifteen monopronucleated ICSI-derived blastocysts were studied. Double trophectoderm biopsy was performed and analyzed by FISH and aCGH. The blastocysts were classified according to chromosome constitution. Disagreements between the two techniques were assessed. RESULTS: Results obtained after FISH and aCGH analyses showed the following: 20% (3/15) and 60% (9/15) diploid females, respectively; 26.7% (4/15) and 26.7% (4/15) diploid males, respectively; and 53.3% (8/15) and 13.3% (2/15) mosaics, respectively. No mosaic male embryos were found using FISH or aCGH. There were disagreements in 40% (6/15) of the cases due to the higher detection of mosaicism by FISH compared to aCGH. CONCLUSIONS: The combination of FISH and aCGH has been shown to be a suitable approach to increase the knowledge about monopronucleated ICSI-derived embryos. FISH analysis of blastocysts derived from monopronucleated ICSI zygotes enabled us to conclude that aCGH underestimates haploidy. Some diploid embryos diagnosed by aCGH are in fact mosaic. In cases where these embryos would be used for reproductive purposes, extra analysis of parental genome origin is recommended.
Assuntos
Blastocisto/citologia , Blastocisto/patologia , Análise Citogenética/métodos , Injeções de Esperma Intracitoplásmicas , Zigoto/citologia , Biópsia , Hibridização Genômica Comparativa , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , MosaicismoRESUMO
STUDY QUESTION: Do angiotensin (Ang)-(1-7) levels in human ovarian follicular fluid (FF) correlate with the number and proportion of mature oocytes obtained for IVF? SUMMARY ANSWER: The present study shows for the first time that Ang-(1-7) levels in human FF correlate with the proportion of mature oocytes collected upon ovarian stimulation for IVF. WHAT IS KNOWN ALREADY: Ang-(1-7) is an active peptide of the renin-angiotensin system that stimulates oocyte maturation in isolated rabbit and rat ovaries. However, its role in human ovulation remains unexplored. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study including 64 participants from a single IVF center. Sample size was calculated to achieve a statistical power of 80% in detecting 20% differences in the proportion of mature oocytes between groups. The participants were enrolled in the study during six consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Plasma samples were obtained from all subjects at Day 21 of the last menstrual cycle before starting pituitary blockade and controlled ovarian stimulation (COS). Plasma and FF samples were quickly mixed with a protease inhibitor cocktail and stored at -80°C. Ang-(1-7) was quantified in plasma and FF samples by a highly sensitive and specific radioimmunoassay, which was preceded by solid phase extraction, speed vacuum concentration and sample reconstitution in assay buffer. FF Ang-(1-7) levels were stratified into tertiles and the patients of each tertile were compared for COS/IVF outcomes using Kruskal-Wallis ANOVA. Multiple regression analysis was used to adjust correlations for potential confounders. The mRNA encoding for Mas, a receptor for Ang-(1-7), was investigated by real-time PCR in luteinized granulosa cells purified from the FF. MAIN RESULTS AND THE ROLE OF CHANCE: There was a four-fold increase in plasma Ang-(1-7) after ovulation induction (median 160.9 vs 41.4 pg/ml, P < 0.0001). FF Ang-(1-7) levels were similar to (169.9 pg/ml) but did not correlate with plasma Ang-(1-7) levels (r = -0.05, P = 0.665). Patients at the highest FF Ang-(1-7) tertile had a higher proportion of mature oocytes compared to patients at the lower FF Ang-(1-7) tertile (median 100% vs 70%, P < 0.01). There was a linear correlation between FF Ang-(1-7) and the proportion of mature oocytes (r = 0.380, P < 0.01), which remained significant after adjustment for age and duration of infertility (r = 0.447, P < 0.001). The luteinized granulosa cells expressed Mas receptor mRNA, which was positively correlated to the number of mature oocytes in women with more than three mature oocytes retrieved (r = 0.42, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an observational study, therefore, no causal relationship can be established between Ang-(1-7) and human oocyte maturation. Mas protein expression was not quantified due to limited availability of granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Since this peptide promotes oocyte maturation in other species, it deserves further investigation as a potential maturation factor to human oocytes. STUDY FUNDING AND COMPETING INTEREST(S): Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors have nothing to disclose.
Assuntos
Angiotensina I/agonistas , Fármacos para a Fertilidade Feminina/uso terapêutico , Líquido Folicular/efeitos dos fármacos , Infertilidade Feminina/terapia , Oogênese/efeitos dos fármacos , Indução da Ovulação , Fragmentos de Peptídeos/agonistas , Regulação para Cima/efeitos dos fármacos , Adulto , Angiotensina I/sangue , Angiotensina I/metabolismo , Blastocisto/citologia , Blastocisto/patologia , Estudos de Coortes , Características da Família , Feminino , Fertilização in vitro , Líquido Folicular/metabolismo , Humanos , Infertilidade Feminina/sangue , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Infertilidade Masculina , Masculino , Recuperação de Oócitos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismo , Estudos Prospectivos , Radioimunoensaio , Extração em Fase SólidaRESUMO
OBJECTIVE: To study whether embryos derived from oocytes presenting a smooth endoplasmic reticulum cluster (SERC) are less likely to develop into blastocysts and implant. DESIGN: Transversal study. SETTING: Private university-affiliated in vitro fertilization (IVF) center. PATIENT(S): Total of 7,609 oocytes obtained from 743 intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S): Oocytes split between the SERC-positive cycles (with at least one SERC-positive oocyte) and the SERC-negative cycles (only oocytes free of SERC). MAIN OUTCOME MEASURE(S): Embryo implantation. RESULT(S): A statistically significantly higher mean number of follicles (24.0 ± 10.5 vs. 19.6 ± 10.5), retrieved oocytes (17.8 ± 8.3 vs. 14.3 ± 8.0), and mature oocytes (13.5 ± 6.2 vs. 10.6 ± 5.9) were observed in the SERC-positive cycles as compared with SERC-negative cycles. The implantation rate was statistically significantly lower in SERC-positive cycles as compared with SERC-negative cycles (14.8% vs. 25.6%; odds ratio 0.61; 95% confidence interval, 0.44-0.86). When only cycles with in which none (0) or all the blastocysts transferred had implanted (100%) were analyzed, the mean implantation rate per transferred blastocyst in the SERC-negative group was 20.5%; no blastocysts derived from SERC-positive oocytes implanted. CONCLUSION(S): The occurrence of SERC impairs embryo implantation. Careful oocyte observation that takes into account the presence of SERC should be part of embryo selection strategy before transfer.
Assuntos
Blastocisto/patologia , Implantação do Embrião , Transferência Embrionária , Retículo Endoplasmático Liso/patologia , Infertilidade/terapia , Oócitos/patologia , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Adulto , Feminino , Fertilidade , Humanos , Infertilidade/patologia , Infertilidade/fisiopatologia , Masculino , Pessoa de Meia-Idade , Recuperação de Oócitos , Indução da Ovulação , Gravidez , Taxa de Gravidez , Resultado do Tratamento , Adulto JovemRESUMO
ET is a critical step in an assisted reproduction cycle. Over the past decade there has been an increasing trend to extending culture from cleavage-stage to blastocyst transfer. There has also been a trend to single ET and reporting the success of an assisted reproductive cycle as a cumulative live-birth rate after using both fresh and frozen embryos. There is low evidence that fresh blastocyst transfer is associated with improved live-birth rates compared with fresh cleavage-stage embryos. However, in the few studies that report cumulative pregnancy rates after fresh and frozen transfers, no significant difference was found. Cleavage-stage transfer is associated with greater numbers of embryos available for freezing, and blastocyst transfer is associated with increased number of cycles with no embryos to transfer. Further well-designed studies are warranted to evaluate the outcomes for blastocyst transfer including cumulative live-birth rate after fresh and frozen transfers, time to live birth, costs of the different transfer strategies, and perinatal mortality and severe perinatal morbidity.
Assuntos
Blastocisto , Fase de Clivagem do Zigoto , Implantação do Embrião , Transferência Embrionária/métodos , Infertilidade/terapia , Blastocisto/patologia , Distribuição de Qui-Quadrado , Fase de Clivagem do Zigoto/patologia , Análise Custo-Benefício , Criopreservação , Técnicas de Cultura Embrionária , Transferência Embrionária/efeitos adversos , Transferência Embrionária/economia , Feminino , Fertilidade , Fertilização in vitro/efeitos adversos , Fertilização in vitro/economia , Custos de Cuidados de Saúde , Humanos , Infertilidade/diagnóstico , Infertilidade/economia , Infertilidade/fisiopatologia , Nascido Vivo , Razão de Chances , Gravidez , Taxa de Gravidez , Medição de Risco , Fatores de Risco , Resultado do TratamentoRESUMO
OBJECTIVE: To compare the outcomes of ETs using cryopreserved embryos, cryopreserved oocytes, or fresh embryos. DESIGN: Observational, cohort study. SETTING: Private university-affiliated fertility center. PATIENT(S): This study included 8,210 mature oocytes obtained from 425 oocyte donors. Of those, 5,440 were used for the donors' own cycles (Fresh Oocyte Cycles Group), and 2,770 were cryobanked for 425 recipients (Banked Donor Egg Group). All of the oocytes were sperm injected, resulting in 4,585 embryos from the donors' own cycles and 2,128 embryos from the recipients' cycles. For the donor cycles, embryos were either cryopreserved and transferred during a subsequent cycle (Thaw Cycles Group, 3,209 embryos), or they were transferred during a fresh cycle (Fresh Cycles Group, 1,307 embryos). For the recipient cycles, embryos derived from vitrified oocytes were transferred (Vitrified Oocytes Group, n = 425 cycles, 2,128 embryos). INTERVENTION(S): Oocyte/embryo vitrification and intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Embryo quality, pregnancy, and implantation rates. RESULT(S): Decreased embryo quality and lower rates of blastocyst formation were observed among embryos derived from vitrified oocytes. The highest pregnancy and implantation rates were noted for the Thaw Cycles Group, followed by the Banked Donor Egg Group; the Fresh Cycles Group had the lowest rates. CONCLUSION(S): Oocyte vitrification followed by intracytoplasmic sperm injection leads to lower embryo developmental competence compared with when fresh insemination methods are used. However, pregnancy and implantation rates are higher when embryos are transferred into a "more receptive" endometrium, free of the adverse effects of gonadotropin. Moreover, the freeze-all method leads to exceptional clinical outcomes.
Assuntos
Blastocisto/patologia , Criopreservação , Transferência Embrionária , Infertilidade/terapia , Doação de Oócitos , Injeções de Esperma Intracitoplásmicas , Preservação de Tecido/métodos , Adulto , Implantação do Embrião , Transferência Embrionária/efeitos adversos , Feminino , Fertilidade , Humanos , Infertilidade/diagnóstico , Infertilidade/fisiopatologia , Pessoa de Meia-Idade , Doação de Oócitos/efeitos adversos , Gravidez , Taxa de Gravidez , Avaliação de Programas e Projetos de Saúde , Fatores de Risco , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Resultado do Tratamento , VitrificaçãoRESUMO
In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25µM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.
Assuntos
Antioxidantes/farmacologia , Blastocisto/efeitos dos fármacos , Portadores de Fármacos , Técnicas de Cultura Embrionária/veterinária , Fármacos para a Fertilidade Feminina/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Nanocápsulas , Espécies Reativas de Oxigênio/metabolismo , Tretinoína/farmacologia , Animais , Antioxidantes/química , Apoptose/efeitos dos fármacos , Blastocisto/metabolismo , Blastocisto/patologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Bovinos , Química Farmacêutica , Relação Dose-Resposta a Droga , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fármacos para a Fertilidade Feminina/química , Regulação da Expressão Gênica no Desenvolvimento , Nanomedicina , Fosforilação , Gravidez , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/química , Proteína X Associada a bcl-2/metabolismoRESUMO
Pregnancy rates from cryopreserved embryos remain lower than non-cryopreserved counterparts, even though these embryos appear morphologically normal. How epigenetic events, such as histone modifications, are affected by cryopreservation of embryos remains unknown. The current study evaluated the effect of conventional freezing/thawing of in vitro produced bovine blastocyst embryos on histone modifications, H3K4me3 and H3K27me3. At day 7 of in vitro culture, blastocyst stage embryos were either frozen by conventional freezing method (-0.5 °C/min in 1.5 M ethylene glycol; F/T group) or remained in culture for an additional 18 h (Ctrl). Frozen embryos were stored in liquid N2 for 14 days, thawed and placed in culture for 36 h for recovery. Control and re-expanded frozen-thawed blastocysts from both groups were fixed in 4% paraformaldehyde and stored in PBS +0.1% triton-X at 4 °C. Immunofluorescence, utilizing antibodies against H3K4me3 and H3K27me3, was conducted and staining intensity was analyzed as percentage of total DNA. Day 7 blastocyst development rate was 35.55% (352/990) with blastocyst recovery at 54.23% (77/142) 36 h post-thawing. Total cell numbers per blastocyst were not different amongst groups (117.8 ± 12.49 and 116.1 ± 14.69, F/T and Ctrl groups respectively). Global staining for the active mark, H3K4me3, was lower in F/T blastocysts compared to Ctrl (17.24 ± 2.80% vs. 34.95 ± 3.77%; P < 0.01). However, staining for the inhibitory mark, H3K27me3, was nearly 2-fold higher in F/T blastocysts (40.41 ± 3.83% vs. 21.29 ± 3.92%; P < 0.01). These results suggest that bovine blastocysts, subjected to conventional freezing methods, have altered histone modifications that may play a role in poor pregnancy rates.
Assuntos
Blastocisto/patologia , Criopreservação/métodos , Embrião de Mamíferos , Histonas/metabolismo , Animais , Bovinos , Transferência Embrionária/métodos , Etilenoglicol/farmacologia , Feminino , Fertilização in vitro , Congelamento , Lisina/metabolismo , Metilação , GravidezRESUMO
Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby.
Assuntos
Fibrose Cística/diagnóstico , Fertilização in vitro/métodos , Mutação , Diagnóstico Pré-Implantação/métodos , Adulto , Biópsia , Blastocisto/patologia , Fibrose Cística/embriologia , Fibrose Cística/genética , Feminino , Humanos , Masculino , Ilustração Médica , Gravidez , Resultado da Gravidez , Resultado do TratamentoRESUMO
Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby.
A fibrose cística é uma doença autossômica recessiva causada por mutações no gene regulador de condutância transmembrana na fibrose cística. Produz fenótipo variável, incluindo doença pulmonar, insuficiência pancreática, íleo meconial, além de agenesia bilateral dos ductos deferentes, causando azoospermia obstrutiva e infertilidade masculina. O diagnóstico genético pré-implantacional é uma alternativa diagnóstica, que permite identificar embriões portadores de fibrose cística e outras doenças genéticas. Relatamos o caso de um casal portador de fibrose cística, sendo a mulher portadora da mutação I148 T e o homem da mutação gênica Delta F508. O casal foi submetido a técnicas de fertilização in vitro associadas ao diagnóstico genético pré-implantacional, com consequente seleção de embriões saudáveis, que foram transferidos para o útero, resultando em gravidez sem intercorrências e com feto saudável, do sexo masculino.
Assuntos
Adulto , Feminino , Humanos , Masculino , Gravidez , Fibrose Cística/diagnóstico , Fertilização in vitro/métodos , Mutação , Diagnóstico Pré-Implantação/métodos , Biópsia , Blastocisto/patologia , Fibrose Cística/embriologia , Fibrose Cística/genética , Ilustração Médica , Resultado da Gravidez , Resultado do TratamentoRESUMO
BACKGROUND: Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. METHODS: For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. RESULTS: The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87.7%) were cleaved and 195 (+/- 3.2; 54.6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84.5%), and these embryos produced 193 (+/- 3.2, 49.7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity. CONCLUSION: This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.
Assuntos
Apoptose , Blastocisto/virologia , Ectogênese , Herpesvirus Bovino 5/metabolismo , Mitocôndrias/metabolismo , Peroxirredoxina III/metabolismo , Superóxido Dismutase/metabolismo , Animais , Blastocisto/metabolismo , Blastocisto/patologia , Bovinos , Doenças dos Bovinos/embriologia , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Fase de Clivagem do Zigoto/metabolismo , Fase de Clivagem do Zigoto/patologia , Fase de Clivagem do Zigoto/virologia , Feminino , Fertilização in vitro/efeitos adversos , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Infecções por Herpesviridae/embriologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 5/isolamento & purificação , Técnicas de Maturação in Vitro de Oócitos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/virologia , Oócitos/fisiologia , Oócitos/virologia , Peroxirredoxina III/genética , RNA Mensageiro/metabolismo , Superóxido Dismutase/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Esta es una revision breve de las caracteristicas morfologicas, microbiologicas y patologicas de Blastocystis homisis, considerando como un protozoario del orden Amoebida, potencialmente patogeno. Aunque se presenta como organismo normal en la mayoria de las personas, se incrimina como causante de diarrea en aquellos casos clinicos que presentan diarrea, aguda o cronica, con grandes cantidades de B. hominis en las heces y en ausencia de otros agentes virales, bacterianos o parasitarios. Por esta razon, debe informarse al medico particularmente cuando no hay otro posible explicacion para el cuadro clinico y la cantidad de formas observadas corresponde a la categoria de abundante o muy abundante segun la escala de Phillips y Zierdt. .