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1.
Cochrane Database Syst Rev ; 9: CD012192, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31529804

RESUMO

BACKGROUND: 'Infertility' is defined as the failure to achieve pregnancy after 12 months or more of regular unprotected sexual intercourse. One in six couples experience a delay in becoming pregnant. In vitro fertilisation (IVF) is one of the assisted reproductive techniques used to enable couples to achieve a live birth. One of the processes involved in IVF is embryo culture in an incubator, where a stable environment is created and maintained. The incubators are set at approximately 37°C, which is based on the human core body temperature, although several studies have shown that this temperature may in fact be lower in the female reproductive tract and that this could be beneficial. In this review we have included randomised controlled trials which compared different temperatures of embryo culture. OBJECTIVES: To assess different temperatures of embryo culture for human assisted reproduction, which may lead to higher live birth rates. SEARCH METHODS: We searched the following databases and trial registers: the Cochrane Gynaecology and Fertility (CGF) Group Specialised Register of Controlled Trials, the Cochrane Central Register of Studies Online, MEDLINE, Embase, PsycINFO, CINAHL, clinicaltrials.gov, The World Health Organization International Trials Registry Platform search portal, DARE, Web of Knowledge, OpenGrey, LILACS database, PubMed and Google Scholar. Furthermore, we manually searched the references of relevant articles and contacted experts in the field to obtain additional data. We did not restrict the search by language or publication status. We performed the last search on 6 March 2019. SELECTION CRITERIA: Two review authors independently screened the titles and abstracts of articles retrieved by the search. Full texts of potentially eligible randomised controlled trials (RCTs) were obtained and screened. We included all RCTs which compared different temperatures of embryo culture in IVF or intracytoplasmic sperm injection (ICSI), with a minimum difference in temperature between the two incubators of ≥ 0.5°C. The search process is shown in the PRISMA flow chart. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial eligibility and risk of bias and extracted data from the included studies; the third review author resolved any disagreements. We contacted trial authors to provide additional data. The primary review outcomes were live birth and miscarriage. Clinical pregnancy, ongoing pregnancy, multiple pregnancy and adverse events were secondary outcomes. All extracted data were dichotomous outcomes, and odds ratios (OR) were calculated with 95% confidence intervals (CIs) on an intention-to-treat basis. We assessed the overall quality of the evidence for the main comparisons using GRADE methods. MAIN RESULTS: We included three RCTs, with a total of 563 women, that compared incubation of embryos at 37.0°C or 37.1°C with a lower incubator temperature (37.0°C versus 36.6°C, 37.1°C versus 36.0°C, 37.0° versus 36.5°C). Live birth, miscarriage, clinical pregnancy, ongoing pregnancy and multiple pregnancy were reported. After additional information from the authors, we confirmed one study as having no adverse events; the other two studies did not report adverse events. We did not perform a meta-analysis as there were not enough studies included per outcome. Live birth was not graded since there were no data of interest available. The evidence for the primary outcome, miscarriage, was of very low quality. The evidence for the secondary outcomes, clinical pregnancy, ongoing pregnancy and multiple pregnancy was also of very low quality. We downgraded the evidence because of high risk of bias (for performance bias) and imprecision due to limited included studies and wide CIs.Only one study reported the primary outcome, live birth (n = 52). They performed randomisation at the level of oocytes and not per woman, and used a paired design whereby two embryos, one from 36.0°C and one from 37.0°C, were transferred. The data from this study were not interpretable in a meaningful way and therefore not presented. Only one study reported miscarriage. We are uncertain whether incubation at a lower temperature decreases the miscarriage (odds ratio (OR) 0.90, 95% CI 0.52 to 1.55; 1 study, N = 412; very low-quality evidence).Of the two studies that reported clinical pregnancy, only one of them performed randomisation per woman. We are uncertain whether a lower temperature improves clinical pregnancy compared to 37°C for embryo incubation (OR 1.08, 95% CI 0.73 to 1.60; 1 study, N = 412; very low-quality evidence). For the outcome, ongoing pregnancy, we are uncertain if a lower temperature is better than 37°C (OR 1.10, 95% CI 0.75 to 1.62; 1 study, N = 412; very low quality-evidence). Multiple pregnancy was reported by two studies, one of which used a paired design, which made it impossible to report the data per temperature. We are uncertain if a temperature lower than 37°C reduces multiple pregnancy (OR 0.80, 95% CI 0.31 to 2.07; 1 study, N = 412; very low-quality evidence). There was insufficient evidence to make a conclusion regarding adverse events, as no studies reported data suitable for analysis. AUTHORS' CONCLUSIONS: This review evaluated different temperatures for embryo culture during IVF. There is a lack of evidence for the majority of outcomes in this review. Based on very low-quality evidence, we are uncertain if incubating at a lower temperature than 37°C improves pregnancy outcomes. More RCTs are needed for comparing different temperatures of embryo culture which require reporting of clinical outcomes as live birth, miscarriage, clinical pregnancy and adverse events.


Assuntos
Técnicas de Cultura Embrionária/métodos , Técnicas de Reprodução Assistida , Temperatura Ambiente , Feminino , Fertilização In Vitro , Humanos , Infertilidade , Nascimento Vivo , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Gravidez Múltipla , Ensaios Clínicos Controlados Aleatórios como Assunto , Injeções de Esperma Intracitoplásmicas
2.
Zygote ; 27(5): 337-346, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31405390

RESUMO

The aim of this study was to evaluate the effects of different timing for frozen-thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen-thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.


Assuntos
Blastocisto/citologia , Blastocisto/fisiologia , Criopreservação , Técnicas de Cultura Embrionária/métodos , Oviductos/citologia , Animais , Aquaporina 3/genética , Bovinos , Técnicas de Cocultura , Células Epiteliais , Feminino , Fertilização In Vitro , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/métodos , Masculino , ATPase Trocadora de Sódio-Potássio/genética , Proteína X Associada a bcl-2/genética
3.
Methods Mol Biol ; 1965: 155-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069674

RESUMO

The chicken embryo is a versatile and effective model for studying the effects of teratogenic compounds during early development. Easy access to the embryo allows for exposure and analysis of toxicant effects during embryogenesis. This chapter will provide detailed protocols for embryonic collection and toxicant exposure techniques, including EC culture and Cornish Pasty methods, LysoTracker staining, glutathione redox potential analysis, and 2',7'-dichlorodihydrofluorescein diacetate.


Assuntos
Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Teratogênios/toxicidade , Animais , Embrião de Galinha , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Espécies Reativas de Oxigênio/metabolismo
4.
Methods Mol Biol ; 1965: 187-194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069676

RESUMO

The embryotoxicity associated with exposure to exogenous compounds such as drugs and environmental chemicals can be assessed using the mouse whole embryo culture technique. This method has several advantages over traditional in vivo studies including the exclusion of any confounding maternal and placental effects, the selection of embryos that are at similar stages of development, and the control of exposure concentrations of exogenous agents and modifiers of interest. This chapter will detail the steps involved in using this technique to assess embryotoxicity following exposure to a toxicant. Briefly, embryos are explanted from murine dams on gestational day 9.0 (vaginal plug, day 1) and cultured in CO2 saturated male rat serum for up to 24 h at 37 °C in the presence or absence of a specific toxicant. Embryonic morphological and developmental parameters (e.g., anterior neuropore closure) are then evaluated using a dissecting microscope 24 h later. Potential biochemical analyses are also listed and limitations discussed.


Assuntos
Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Teratogênios/toxicidade , Animais , Meios de Cultura/química , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Masculino , Camundongos , Gravidez , Ratos
5.
Methods Mol Biol ; 1965: 195-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069677

RESUMO

The direct effects of chemical exposures, environmental extremes, and nutrient quality/quantity have been very difficult to study in mammalian embryos due to their anatomical inaccessibility, paucity of tissue, and other factors that make human studies unethical. Many acute and chronic developmental anomalies can trace their origins to postimplantation phases of gestation, where the organs are first being established and growth and differentiation are in highly active states of flux. Most chemical insults and conditions that produce birth defects are believed to act during this period of organogenesis. The evolution of rodent whole embryo culture (WEC) techniques has provided a valuable experimental model where physiological conditions and exposures can be carefully controlled and manipulated to test hypotheses and explore biochemical and molecular mechanisms of action that would otherwise be extremely difficult. Exposure to chemicals can be controlled through their direct addition to the culture medium. Optimal in vitro culture conditions support the growth of intact, viable conceptuses (embryo and associated extraembryonic membranes) from early egg cylinder stages through the establishment of the neural plate, gastrulation, neural tube closure, onset of active heartbeat and circulation, and the initial formation of all major organ systems that occur prior to the establishment of a functional placenta. Detailed comparisons of in vivo and in vitro growth show that conceptuses grown in WEC are nearly identical, structurally and functionally, to conceptuses of the same developmental stage that are allowed to develop normally in utero during a comparable developmental period. Culture conditions and mechanical apparatuses can be modified to suit a large number of different experimental approaches and paradigms.


Assuntos
Técnicas de Cultura Embrionária/métodos , Organogênese/efeitos dos fármacos , Teratogênios/toxicidade , Animais , Meios de Cultura/química , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Masculino , Gravidez , Ratos , Testes de Toxicidade
6.
Methods Mol Biol ; 1965: 219-233, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069678

RESUMO

The rabbit is a mainstay of regulatory developmental toxicity testing; however, due to the historic absence of experimental tools for this species, there is a dearth of information about its fundamental embryology and the mechanisms underlying developmental toxicity. Relatively recently, there have been advances in the methods of rabbit whole embryo culture (WEC), and this has prompted an increase in understanding of rabbit embryogenesis. Described herein are the methods used to remove early somite-stage embryos (gestation day 9) and sustain their growth for 48 h. Although there are similarities to the well-described rodent WEC, there are also important differences. Akin to rodent WEC, the major phases of organogenesis can be investigated, including neural tube development, cardiac looping, segmentation, and the development of the anlagen of the optic and otic regions, craniofacial development, somites, and early limb bud development. Unlike the rodent, rabbit WEC requires the use of an apparatus that allows for the continuous gassing of embryos, and one may observe the expansion and closure of the visceral yolk sac around the embryo. After completion of the culture period, embryos are examined across several growth and developmental parameters including a quantitative morphological scoring system. Embryonic growth and development in the absence of maternal influences allows for the study of the direct action of agents or their metabolites on the embryo. The use of both rodent and rabbit WEC together is a powerful strategy with which to investigate species-specific vulnerabilities to specific agents.


Assuntos
Técnicas de Cultura Embrionária/métodos , Organogênese , Animais , Técnicas de Cultura Embrionária/instrumentação , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Masculino , Modelos Animais , Organogênese/efeitos dos fármacos , Coelhos , Somitos/citologia
7.
Theriogenology ; 133: 135-143, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31091484

RESUMO

The objective of these experiments was to determine the effect of l-carnitine during oocyte maturation or embryo culture on embryo development and cryosurvival. For Experiments 1-3, embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). At d 7 after insemination, embryo development was assessed, and blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Post-thaw cryosurvival was determined by re-expansion and hatching rates at 24, 48 and 72 h post-thaw. In Experiment 1, COCs were matured with or without 3.03 mM l-carnitine. There was no effect of l-carnitine supplementation during maturation on embryo development or post-thaw cryosurvival. In experiment 2, presumptive zygotes were cultured in medium supplemented with or without 5% (v/v) fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5 and 3.03 mM. There was no effect of l-carnitine treatment on embryo development, but embryos treated with l-carnitine had increased (P ≤ 0.05) post-thaw re-expansion rates, irrespective of concentration. In experiment 3, presumptive zygotes were cultured with or without 0.75 mM l-carnitine from d 1 to d 4, from d 4 to d 7 or for the entire culture period. There was no effect of l-carnitine during culture on embryo development or post-thaw cryosurvival, regardless of the timing of addition. In Experiment 4, COCs were harvested by ovum pick-up from virgin dairy heifers (n = 24) and subjected to in-vitro embryo production with presumptive zygotes cultured with or without 0.75 mM l-carnitine. At d 7 after insemination, morula and blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Lactating Holstein cows (n = 102) were used as recipients and synchronized for timed embryo transfer. At d 7 after anticipated ovulation, a single embryo was thawed and transferred to the ipsilateral uterine horn of each recipient with a corpus luteum. Pregnancy was diagnosed at d 33, 44 and 72 of gestation. l-carnitine had no effect on the percentage of cows pregnant per embryo transfer (P/ET) after transfer of a frozen-thawed embryo. In conclusion, media supplementation with l-carnitine during in vitro embryo production can improve post-thaw cryotolerance as assessed in vitro but had no effect on P/ET after transfer of frozen-thawed embryos.


Assuntos
Carnitina/farmacologia , Meios de Cultura/química , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Animais , Bovinos , Criopreservação/veterinária , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Gravidez
8.
Exp Anim ; 68(3): 361-370, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-30996149

RESUMO

In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.


Assuntos
Blastocisto/citologia , Diferenciação Celular/fisiologia , Técnicas de Cultura Embrionária/métodos , Transferência Embrionária/métodos , Células-Tronco Embrionárias Murinas/citologia , Animais , Encéfalo/fisiologia , Feminino , Células Germinativas/fisiologia , Japão , Masculino , Camundongos , Camundongos Endogâmicos ICR
9.
Fertil Steril ; 111(5): 918-927.e3, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30922642

RESUMO

OBJECTIVE: To develop a noninvasive embryo selection algorithm consisting of time-lapse morphokinetics and the oxidative status of the spent embryo culture medium determined using the Thermochemiluminescence (TCL) Analyzer. DESIGN: Retrospective cohort. SETTING: Not applicable. PATIENT(S): From women participating in the oocyte donation program, data from 505 samples of spent embryo culture media samples from 292 intracytoplasmic sperm injection cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Morphokinetic parameters assessed during incubation in the time-lapse system Embryoscope. Oxidative parameters (H1sm, H2sm, and H3sm) from the spent culture medium on day 5 of incubation measured using the TCL assay; and a combined assessment algorithm, including morphology, morphokinetics, and the embryo's culture medium oxidative status, developed as a tool for embryo selection, based on implantation success and confirmed ongoing pregnancy. RESULT(S): The levels of the oxidative parameters H1sm, H2sm, and H3sm on day 5 of incubation were statistically significantly higher in transferred and vitrified embryos compared with discarded embryos and in successfully implanted embryos compared with those that did not result in pregnancy. The assessment algorithm resulted in a hierarchical classification with six embryo quality categories (A to F), associated with implantation rates of between 76.5% and 29.2%. CONCLUSION(S): An assessment algorithm combining morphology, morphokinetics and the embryo's culture medium oxidative status may help to improve current embryo selection methods criteria and in vitro fertilization success.


Assuntos
Algoritmos , Meios de Cultura/análise , Técnicas de Cultura Embrionária/métodos , Transferência Embrionária/métodos , Estresse Oxidativo/fisiologia , Imagem com Lapso de Tempo/métodos , Adulto , Estudos de Coortes , Feminino , Humanos , Cinética , Gravidez , Estudos Retrospectivos
10.
Fertil Steril ; 111(5): 991-1001.e2, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30922649

RESUMO

OBJECTIVE: To analyze how chromosome 21 (HSA21) ploidy affects global gene expression of early human blastocysts. DESIGN: Prospective study. SETTING: University-affiliated in vitro fertilization clinic. PATIENT(S): A total of 26 high-quality donated embryos from in vitro fertilization (IVF) patients: trisomy 21 (n = 8), monosomy 21 (n = 10), and euploid (n = 8) blastocysts. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Blastocyst transcriptome changes and its associated functions. RESULT(S): Trisomy 21, monosomy 21, and euploid blastocysts were classified by comparative genomic hybridization. The global transcriptome of whole blastocysts was analyzed with small cell number RNA sequencing, and they were compared to understand the gene expression behavior at early development and its implications for embryo implantation. We identified 1,232 differentially expressed genes (false discovery rate <0.05) in monosomy 21 compared with euploid blastocysts associated with dysregulated functions in embryo development as the Rap1 signaling pathway. Curiously, Down syndrome in early development revealed fewer transcriptomic changes than expected. In addition, Down syndrome gene expression in neonates, children, and adults revealed that the number of deregulated genes increases across life stages from blastocysts to adults, suggesting a potential dosage-compensation mechanism for human chromosome 21. CONCLUSION(S): At the transcriptomic level, early development in Down syndrome is mainly dosage compensated. However, monosomy 21 is strongly transcriptionally affected because early development involving main functions is associated with embryo implantation.


Assuntos
Aneuploidia , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/genética , Estudos de Associação Genética/métodos , Monossomia/genética , Transcriptoma/genética , Adulto , Cromossomos Humanos Par 21/genética , Feminino , Humanos , Gravidez , Estudos Prospectivos , Técnicas de Reprodução Assistida
11.
Theriogenology ; 129: 146-153, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851478

RESUMO

The success of in vitro embryo production demonstrates that the oviduct can be bypassed during early embryonic development. Using an ex vivo model of porcine uterus is one of the strategies used to investigate fertilization within the oviductal environment. In this study, in vitro-matured porcine oocytes (MII) were fertilized with 7.5 × 107, 15 × 107, or 30 × 107 sperm cells for 20 min in the oviduct of a porcine uterine ex vivo model. MII oocytes used for in vitro fertilization (IVF) served as control 1; those cultured in the oviduct of the ex vivo model for 20 min before IVF served as control 2. In present study, the penetration rate, polyspermy, and fertilization efficiency, and accumulated reactive oxygen species (ROS) levels in the treatment groups were significantly decreased compared to those in the control 1 group. During embryonic development, the cleavage rates in the treatment groups were significantly lower than those in the control groups. The cleavage rate in the 30 × 107 sperm cell-treated group was higher than that in the 7.5 × 107 sperm cell-treated group. The blastocyst formation rate in control 1 and 2, and 30 × 107 sperm cell-treated groups increased compared to that in the 7.5 and 15 × 107 sperm cell-treated groups. PCNA, HSP70.2, and GLUT1 were upregulated in the treatment groups and POU5F1, BAX, GPX1 were upregulated in the treatment and control 2 groups, compared to the control 1 group. These results suggest that an ex vivo model may decrease the penetration rate and fertilization efficiency by increasing the accumulated ROS levels and inducing the expression of apoptosis- and stress-related genes. However, the model improved the monospermy rate and expression of embryo developmental competence genes. This is the first study that evaluates the effect of an ex vivo model of porcine uterus on fertilization parameters, and the development of porcine embryos.


Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Fertilização , Suínos , Animais , Técnicas de Cultura Embrionária/métodos , Feminino , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma , Útero
12.
J Assist Reprod Genet ; 36(5): 819-826, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30895497

RESUMO

In recent years, a growing body of literature has emerged investigating the clinical utility of spent embryo media (SEM) for preimplantation genetic testing for aneuploidy (PGT-A) (Hammond et al. in Fertil Steril. 107(1):220-8, 2017; Xu et al. in Proc Natl Acad Sci USA. 113(42):11907-12, 2016; Shamonki et al. in Fertil Steril. 106(6):1312-8, 2016; Feichtinger et al. in Reprod BioMed Online. 34(6):583-9, 2017; Vera-Rodriguez et al. in Hum Reprod. 33(4):745-56, 2018; Kuznyetsov et al. in PLoS One. 13(5):e0197262, 2018; Ho et al. in Fertil Steril. 110(3):467-75, 2018; Capalbo et al. in Fertil Steril. 110(5):870-9, 2018). Most of these studies have reported moderate success rates, suggesting the need for improvements in sensitivity and specificity. The concordance between spent media and embryo biopsy or whole embryo was reported to be between 30.4 and 90%, with 50-70% correlation being the most representative (Xu et al. in Proc Natl Acad Sci USA. 113(42):11907-12, 2016; Shamonki et al. in Fertil Steril. 106(6):1312-8, 2016; Feichtinger et al. in Reprod BioMed Online. 34(6):583-9, 2017; Vera-Rodriguez et al. in Hum Reprod. 33(4):745-56, 2018; Kuznyetsov et al. in PLoS One. 13(5):e0197262, 2018; Ho et al. in Fertil Steril. 110(3):467-75, 2018). Here, we will analyze all spent media testing strategies including SEM collection methods, whole genome amplification (WGA) strategies, chromosome copy number detection, and bioinformatics analysis tools. We will propose improvements to further increase the accuracy and sensitivity of the assay before bringing PGT-A with SEM into the clinical sphere.


Assuntos
Aneuploidia , Meios de Cultura/análise , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/metabolismo , Testes Genéticos/métodos , Diagnóstico Pré-Implantação/métodos , Embrião de Mamíferos/citologia , Feminino , Humanos , Gravidez
13.
Theriogenology ; 129: 70-76, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30825707

RESUMO

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-ß family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Miostatina/farmacologia , Suínos/embriologia , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Técnicas de Cultura Embrionária/métodos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos/metabolismo
14.
Reprod Domest Anim ; 54(5): 750-755, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30788874

RESUMO

The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25°C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro-produced zygotes were stored at 25°C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA-containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25°C for 24 hr was evaluated, more zygotes stored with 50 µM CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 µM CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non-stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non-stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 µM CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.


Assuntos
Blastocisto/efeitos dos fármacos , Ácido Clorogênico/farmacologia , Técnicas de Cultura Embrionária/veterinária , Zigoto/crescimento & desenvolvimento , Animais , Temperatura Baixa , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro/veterinária , Líquido Folicular/fisiologia , Soroalbumina Bovina/farmacologia , Suínos
15.
Fertil Steril ; 111(4): 753-762.e1, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30683589

RESUMO

OBJECTIVE: To develop and validate Raman metabolic footprint analysis to determine chromosome euploidy and aneuploidy in embryos fertilized in vitro. DESIGN: Retrospective study. SETTING: Academic hospital. PATIENT(S): Unselected assisted reproductive technology population. INTERVENTION(S): To establish the analysis protocol, spent embryo culture medium samples with known genetic outcomes from 87 human embryos were collected and measured with the use of Raman spectroscopy. Individual Raman spectra were analyzed to find biologic components contributing to either euploidy or aneuploidy. To validate the protocol via machine-learning algorithms, additional 1,107 Raman spectra from 123 embryo culture media (61 euploidy and 62 aneuploidy) were analyzed. MAIN OUTCOME MEASURE(S): Raman-based footprint profiling of spent culture media and preimplantation genetic testing for aneuploidy (PGT-A). RESULT(S): Mean-centered Raman spectra and principal component analysis showed differences in the footprints of euploid and aneuploid embryos growing in culture medium. Significant differences in Raman bands associated with small RNAs and lipids were also observed. Stacking classification based on k-nearest-neighbor, random forests, and extreme-gradient-boosting algorithms achieved an overall accuracy of 95.9% in correctly assigning either euploidy or aneuploidy based on Raman spectra, which was validated by PGT-A sequencing results. CONCLUSION(S): This study suggests that chromosomal abnormalities in embryos should lead to changes of metabolic footprints in embryo growth medium that can be detected by Raman spectroscopy. The ploidy status of embryos was analyzed by means of Raman-based footprint profiling of spent culture media and was consistent with PGT-A testing performed by next-generation sequencing.


Assuntos
Aneuploidia , Meios de Cultura/química , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Diagnóstico Pré-Implantação/métodos , Análise Espectral Raman/métodos , Células Cultivadas , Aberrações Cromossômicas , Meios de Cultura/metabolismo , Análise Citogenética/métodos , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/metabolismo , Fertilização In Vitro , Ensaios de Triagem em Larga Escala , Humanos , Aprendizado de Máquina , Metaboloma , Modelos Biológicos , Ploidias , Valor Preditivo dos Testes , Estudos Retrospectivos
16.
J Reprod Dev ; 65(2): 147-153, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30662011

RESUMO

Previously, we reported that neurotensin (NT), which is expressed in the uterus and oviduct, enhanced bovine sperm capacitation and acrosome reactions. As NT mRNA expression in bovine oviducts increases dramatically in the follicular phase, we hypothesized that NT modulates fertilization and subsequent conception in cattle. The objective of this study was to evaluate the effect of NT on embryo development and blastocyst quality. The rate of embryo cleavage was significantly increased by the addition of NT to the fertilization medium. Furthermore, the total number of cells and numbers of cells in the inner cell mass of blastocysts were significantly increased by NT during in vitro fertilization (IVF). These results suggested that NT enhanced the efficiency of early bovine embryo development and blastocyst quality. The expression of NT receptors (NTRs) in sperm, testes, oocytes, and cumulus cells was evaluated to determine whether NT acted via NTRs in sperm alone or in both male and female reproductive cells during IVF. Immunocytochemistry and reverse transcription polymerase chain reaction revealed that NTR1 and NTR2 were expressed in sperm and testes, but not in oocytes and cumulus cells. We propose that NT selectively acts upon sperm via NTR1 and NTR2 during IVF to improve the cleavage rate and quality of blastocysts, which are important determinants of sperm quality for successful conception. This research supports our hypothesis that NT acts as a key modulator of fertilization and conception in cattle. Further studies are necessary to apply our findings to the industrial framework of bovine reproduction.


Assuntos
Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Fertilização In Vitro , Neurotensina/farmacologia , Receptores de Neurotensina/fisiologia , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/genética , Animais , Blastocisto/fisiologia , Bovinos/embriologia , Células Cultivadas , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Fertilização/efeitos dos fármacos , Fertilização/genética , Fertilização In Vitro/veterinária , Masculino , Neurotensina/metabolismo , Neurotensina/fisiologia , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/genética , Espermatozoides/fisiologia
17.
Eur J Obstet Gynecol Reprod Biol ; 233: 58-63, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30580224

RESUMO

OBJECTIVE: Continuous monitoring of embryos via time-lapse (TL) provides more information on embryo kinetics and morphology compared to standard daily evaluation. Embryo selection by TL could support single embryo transfer (SET). With SET multiple gestations are avoided and perinatal outcome is improved. Our primary goal was to determine whether selection of a single blastocyst based on an algorithm comprising kinetic and morphologic scores assessed through continuous TL monitoring results in superior clinical outcome compared to embryo selection based on morphology alone. A secondary goal was to assess whether a time-lapse score based on kinetic and morphologic parameters was predictive of implantation. STUDY DESIGN: Randomized controlled trial performed in two private IVF centers in Hungary. Infertile couples scheduled to undergo 1st or 2nd IVF cycles were enrolled. Female age had to be under 36 years. The intervention was embryo evaluation/selection based on TL algorithm. Patients were randomized to SET with TL monitoring (TL-eSET) vs. SET with standard evaluation (control-eSET). Assuming an increase in pregnancy from 44% to 58%, a sample size of 202 per group was calculated based on the interim analysis at 10% information fraction. The primary outcome of the study was pregnancy rate. Secondary outcomes were miscarriage rates, live birth, perinatal outcome and the ability of a time-lapse score constructed based on kinetic and morphologic parameters to predict implantation. Chi-square tests, likelihood-ratio tests and exact tests were used for the analysis of categorical variables. Continuous variables were compared using independent group t-test and analysis of variance. RESULTS: The study was closed after three years. Eventually 161 patients were randomized and analyzed (N = 80 TL-eSET and N = 81 control-eSET). Pregnancy rate did not significantly differ between the groups though there was a trend favoring TL selection (TL-eSET: 46.3% vs control-eSET: 34.6%, p = 0.150; OR: 1.628 (95% CI: 0.857-3.092)). The time-lapse score based on morphologic and kinetic parameters was significantly higher for blastocysts that implanted vs. those that did not (14.5 ± 1.8 vs. 12.1 ± 2.9, p = 0.0001). There were no adverse effects of the intervention. CONCLUSIONS: Selection of a single blastocyst based on information derived from time-lapse monitoring can aid embryo selection for SET.


Assuntos
Coeficiente de Natalidade , Técnicas de Cultura Embrionária/métodos , Implantação do Embrião , Transferência de Embrião Único/métodos , Adulto , Algoritmos , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro , Humanos , Nascimento Vivo , Gravidez , Imagem com Lapso de Tempo
18.
Hum Reprod ; 34(1): 44-51, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30517719

RESUMO

STUDY QUESTION: In IVF cycles in which the entire embryo cohort is slow growing, is it optimal to perform fresh transfer in Day 5 or to extend the culture and transfer in subsequent vitrified-warmed cycles? SUMMARY ANSWER: The outcomes depend on the degree of embryo development on Day 5. WHAT IS KNOWN ALREADY: Slow-growing blastocysts have lower implantation potential when transferred in fresh cycles. It has been suggested that embryo-endometrial asynchrony could explain this finding. However, studies that compared Days 5 and 6 embryos in frozen embryo transfer (FET) cycles showed contradictory results. There is still a lack of evidence regarding the best approach, performing fresh transfer or deferring transfer and continuing culture until fully developed blastocysts are achieved, when the entire cohort of embryos is slow growing. STUDY DESIGN SIZE, DURATION: This was a retrospective study that included 477 women aged <40 years who underwent fresh Day 5 single embryo transfer of slow-growing embryos and subsequent FET cycles of fully expanded blastocysts (FEB) originating from the same IVF cycle between 2012 and 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included cycles in which the embryos either began blastulation by Day 5 of culture but did not reach the fully expanded stage (Gardner Stage III) or had delayed blastulation with only morula embryos present by Day 5 of culture. All of the subjects in the study underwent elective, single embryo transfer (slow or delayed blastocysts) on Day 5 and had at least one embryo that developed into a FEB on extended culture Day 6 that was suitable for vitrification. All subjects, regardless of the outcome of the fresh transfer, returned for at least one subsequent FET cycle of Day 6 embryos. MAIN RESULTS AND ROLE OF CHANCE: A total of 1070 embryo transfer cycles (fresh + FET) were included. Of them, 365 women had elective, fresh, single transfer of slow-growing blastocysts (Group I) and 112 had elective, fresh, single morula transfer (Group II). Groups I and II underwent a subsequent 457 and 136 FET cycles, respectively. The mean age of Group I was 33.8 ± 2.9 years, the proportion of Day 5 embryos that developed to FEB by Day 6 was 92%, and the number of blastocysts vitrified was 627 (average of 1.71 blastocysts per cycle). The outcomes of fresh and FET cycles were comparable regarding clinical pregnancy rate (CPR) (31.0 vs. 30.4%, P = 0.86) and live birth rate (LBR) (23.3 vs. 20.3%, P = 0.15). In Group II, the mean age was 35.8 ± 3.4 years and the proportion of morula embryos that developed to FEB by Day 6 was 72%. The number of blastocysts vitrified on Day 6 was 155 (1.38 per cycle). The transfer of fresh embryos in Group II resulted in significantly lower clinical pregnancy (5.3 vs. 30.1%, P < 0.001) and LBRs (1.8 vs. 20.5%, P < 0.001). The results did not change after controlling for possible confounding factors. LIMITATIONS AND REASONS FOR CAUTION: The retrospective design of the study is a major limitation. Although we compared the outcomes of embryos that originated from the same cohort, the FET cycles could have been overrepresented by older patients and those with poorer prognoses. Furthermore, the study included only cycles in which there were blastocysts available for cryopreservation on Day 6; therefore, the results were not be applicable for those who had mandatory Day 5 transfer with no embryos available for vitrification. WIDER IMPLICATIONS OF THE FINDINGS: Fresh transfer of embryos that begin blastulation on Day 5 results in similar outcomes to the transfer of FEB originating from the same cohort. However, in cases where only morula/compacting embryos are available by Day 5, extending culture until FEB are achieved and then performing subsequent FET will result in significantly higher LBRs. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.


Assuntos
Blastocisto , Criopreservação , Transferência Embrionária/métodos , /estatística & dados numéricos , Adulto , Coeficiente de Natalidade , Técnicas de Cultura Embrionária/métodos , Feminino , Humanos , Indução da Ovulação/métodos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Vitrificação
19.
Theriogenology ; 126: 8-16, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30508790

RESUMO

Application of cloning technology on a wide scale is severely limited by the very low live birth rate obtained with cloned embryos. Embryo quality is an important factor which affects the conception and live birth rate of cloned embryos. microRNA-21 (miR-21) has been implicated in the regulation of apoptosis and the expression level of several important genes which control apoptosis. We examined the effects of treatment of reconstructed buffalo embryos, produced by Hand-made cloning, with miR-21 mimic on developmental competence, quality and gene expression of cloned embryos. Expression level of miR-21, which increased from 2-cell to 8-cell stage and then decreased at the blastocyst stage, showed a similar pattern in cloned and IVF embryos. It was lower in cloned than in IVF embryos at 2-, 4- and 8-cell (P < 0.001) and blastocyst (P < 0.05) stages but not at morula stage. Treatment of reconstructed embryos with miR-21 mimic for 1 h after 1 h of electrofusion, increased (P < 0.05) the total cell number (251.3 ±â€¯10.7 vs 181.5 ±â€¯2.13). Blastocysts produced from miR-21-treated reconstructed embryos had lower (P < 0.05) apoptotic index than controls and IVF blastocysts (2.01 ±â€¯0.17, 5.46 ±â€¯0.26 and 4.19 ±â€¯0.15, respectively). The treatment also improved the inner cell mass:trophectoderm cell number ratio of blastocysts than in controls (0.21 ±â€¯0.01 vs 0.11 ±â€¯0.003) to values observed in IVF blastocysts (0.20 ±â€¯0.008). However, miR-21 mimic treatment did not affect the blastocyst rate, which was similar for treatment, control and negative control groups (36.58 ±â€¯3.64, 36.58 ±â€¯3.64 and 32.2 ±â€¯2.86%, respectively). miR-21 mimic treatment increased (P < 0.01) the expression level of apoptosis- (BCL2 and PTEN), pluripotency- (OCT4 and SOX2) and development-related genes (GLUT1, FGF4 and P53), but not that of CASPASE3 than in untreated controls in blastocysts. These results suggest that treatment of reconstructed embryos with miR-21 mimic improves blastocyst quality, reduces apoptosis and alters gene expression without improving the blastocyst rate.


Assuntos
Búfalos/embriologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , MicroRNAs/farmacologia , Animais , Apoptose/efeitos dos fármacos , Técnicas de Cultura Embrionária/métodos , Regulação da Expressão Gênica no Desenvolvimento , Marcação In Situ das Extremidades Cortadas , Técnicas de Transferência Nuclear/veterinária
20.
Fertil Steril ; 111(1): 97-104, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30458993

RESUMO

OBJECTIVE: To investigate the effect of embryo culture duration on birth weight in vitrified-warmed cycles. DESIGN: Retrospective cohort study. SETTING: Tertiary-care academic medical center. PATIENT(S): A total of 4,201 women who gave birth to 3,520, 215, and 466 live-born singletons after frozen-thawed cleavage-stage (day 3) and day 5 and day 6 blastocyst transfer, respectively. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Neonatal birth weight. RESULT(S): The mean birth weight did not differ between the three study groups. However, the gestational age- and sex-adjusted birth weight (Z-scores) of singletons and the proportion of large-for-gestational-age (LGA) babies were significantly higher after day 5 and day 6 transfer than after transfer of day 3 embryos. Furthermore, multiple linear regression analysis indicated that gestational age, parental body mass index, neonatal sex, and length of the culture period all had significant and strong impacts on birth weight of singleton newborns. CONCLUSION(S): In the vitrified-warmed transfer cycles, birth weight Z-scores and the proportion of LGA infants were both higher in singletons born after blastocyst transfer than after transfer of cleavage-stage embryos. This finding suggests that the effect of culture duration was not overcome by transfer of embryos into a more physiologic uterine environment.


Assuntos
Peso ao Nascer/fisiologia , Técnicas de Cultura Embrionária/métodos , Transferência Embrionária/métodos , Vitrificação , Adulto , Estudos de Coortes , Feminino , Humanos , Recém-Nascido , Masculino , Gravidez , Estudos Retrospectivos , Fatores de Tempo
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