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1.
Reprod Biomed Online ; 42(6): 1067-1074, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33814309

RESUMO

RESEARCH QUESTION: Is there a risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral exposure and potential cross-contamination from follicular fluid, culture media and vitrification solution within the IVF laboratory using strict patient screening and safety measures? DESIGN: This was a prospective clinical study. All women undergoing transvaginal oocyte retrieval were required to have a negative SARS-CoV-2 RNA test 3-5 days prior to the procedure. Male partners were not tested. All cases used intracytoplasmic sperm injection (ICSI). The first tube of follicular fluid aspirated during oocyte retrieval, drops of media following removal of the embryos on day 5, and vitrification solution after blastocyst cryopreservation were analysed for SARS-CoV-2 RNA. RESULTS: In total, medium from 61 patients, vitrification solution from 200 patients and follicular fluid from 300 patients was analysed. All samples were negative for SARS-CoV-2 viral RNA. CONCLUSIONS: With stringent safety protocols in place, including testing of women and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in follicular fluid, medium or vitrification solution. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.


Assuntos
Meios de Cultura/análise , Fertilização in vitro , Líquido Folicular/virologia , Laboratórios , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Feminino , Humanos , Recuperação de Oócitos , Segurança do Paciente , Estudos Prospectivos , Injeções de Esperma Intracitoplásmicas , Vitrificação
2.
Mol Hum Reprod ; 24(10): 478-494, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30085220

RESUMO

STUDY QUESTION: Which different pathways and functions are altered in rhesus monkey oocytes that fail to mature after an ovulatory stimulus? SUMMARY ANSWER: Failed to mature (FTM) oocytes complete a large portion of the transition in transcriptome composition associated with normal maturation, but also manifest numerous differences that indicate incomplete transcriptional repression and cytoplasmic maturation affecting multiple processes. WHAT IS KNOWN ALREADY: Oocyte maturation defects contribute to unexplained female infertility. Failure of some oocytes to undergo germinal vesicle breakdown or progress to second meiotic metaphase in response to an ovulatory stimulus can limit the number of high quality oocytes available for ART. STUDY DESIGN, SIZE, DURATION: The transcriptome of rhesus monkey oocytes that failed to mature (FTM; n = 11, 5 donors) in response to an ovulatory stimulus in vivo was compared to those of normal germinal vesicle stage (GV, n = 7, 2 donors) and metaphase II stage (MII, n = 7, 5 donors) oocytes by RNA-sequencing (RNAseq). PARTICIPANTS/MATERIALS, SETTING, METHODS: Female rhesus monkeys of normal breeding age (6-12 years old) and with regular menstrual cycles were used. Animals underwent a controlled ovarian stimulation protocol for the collection of oocytes by ultrasound-guided needle aspiration of follicles. MAIN RESULTS AND THE ROLE OF CHANCE: We obtained a high quality RNAseq dataset consisting of n = 7, n = 7, and n = 11 libraries for normal GV, normal MII and FTM oocytes, respectively. Total reads acquired were an average of 34 million for each GV sample, 41 million for each FTM sample and 59 million for each MII oocyte sample. Approximately 44% of the total reads were exonic reads that successfully aligned to the rhesus monkey genome as unique non-rRNA gene transcript sequences, providing high depth of coverage. Approximately 44% of the mRNAs that undergo changes in abundance during normal maturation display partial modulations to intermediate abundances, and 9.2% fail to diverge significantly from GV stage oocytes. Additionally, a small group of mRNAs are grossly mis-regulated in the FTM oocyte. Differential expression was seen for mRNAs associated with mitochondrial functions, fatty acid beta oxidation, lipid accumulation, meiosis, zona pellucida formation, Hippo pathway signaling, and maternal mRNA regulation. A deficiency DNA methyltransferase one mRNA expression indicates a potential defect in transcriptional silencing. LARGE SCALE DATA: All RNAseq data are published in the Gene Expression Omnibus Database (GSE112536). LIMITATIONS, REASONS FOR CAUTION: These results do not establish cause of maturation failure but reveal novel correlates of incompetence to mature. Transcriptome studies likely do not capture all post-transcriptional or post-translational events that inhibit maturation, but do reveal mRNA expression changes that lie downstream of such events or that are related to effects on upstream regulators. The use of an animal model allows the study of oocyte maturation failure independent of covariates and confounders, such as pre-existing conditions of the female, which is a significant concern in human studies. Depending on the legislation, it may not be possible to collect and study oocytes from healthy women; and using surplus oocytes from patients undergoing ART may introduce confounders that vary from case to case. FTM oocytes were at various stages of meiotic progression, so correlates of specific times of arrest are not revealed. All the FTM oocytes failed to respond appropriately to an ovulatory stimulus in vivo. Therefore, this analysis informs us about common transcriptome features associated with meiotic incompetence. WIDER IMPLICATIONS OF THE FINDINGS: These results reveal that some diagnostic markers of oocyte quality may not reflect developmental competence because even meiotically incompetent oocytes display many normal gene expression features. The results also reveal potential mechanisms by which maternal and environmental factors may impact transcriptional repression and cytoplasmic maturation, and prevent oocyte maturation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the National Institutes of Health Office of Research Infrastructure Programs Division of Comparative Medicine Grants R24 [OD012221 to K.E.L., OD011107/RR00169 (California National Primate Research Center), and OD010967/RR025880 to C.A.V.]; the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under the award number T32HD087166; MSU AgBioResearch, Michigan State University. Authors have nothing to disclose.


Assuntos
Oócitos/metabolismo , RNA Mensageiro/metabolismo , Animais , Feminino , Técnicas de Maturação in Vitro de Oócitos , Macaca mulatta , Oogênese/genética , Oogênese/fisiologia
3.
Reprod Toxicol ; 77: 154-165, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29505797

RESUMO

Trophoblast stem cells (TSCs) are crucial for embryo implantation and placentation. Environmental toxicants that compromise TSC function could impact fetal viability, pregnancy, and progeny health. Understanding the effects of low, chronic EDC exposures on TSCs and pregnancy is a priority in developmental toxicology. Differences in early implantation between primates and other mammals make a nonhuman primate model ideal. We examined effects of chronic low-level exposure to atrazine, tributyltin, bisphenol A, bis(2-ethylhexyl) phthalate, and perfluorooctanoic acid on rhesus monkey TSCs in vitro by RNA sequencing. Pathway analysis of affected genes revealed negative effects on cytokine signaling related to anti-viral response, most strongly for atrazine and tributyltin, but shared with the other three EDCs. Other affected processes included metabolism, DNA repair, and cell migration. Low-level chronic exposure of primate TSCs to EDCs may thus compromise trophoblast development in vivo, inhibit responses to infection, and negatively affect embryo implantation and pregnancy.


Assuntos
Disruptores Endócrinos/toxicidade , Células-Tronco/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Trofoblastos/citologia , Animais , Atrazina/toxicidade , Compostos Benzidrílicos/toxicidade , Caprilatos/toxicidade , Linhagem Celular , Movimento Celular , Citocinas/metabolismo , Reparo do DNA , Dietilexilftalato/toxicidade , Fluorocarbonos/toxicidade , Macaca mulatta , Fenóis/toxicidade , Análise de Sequência de RNA , Compostos de Trialquitina/toxicidade
4.
Hum Mol Genet ; 26(14): 2678-2689, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28444193

RESUMO

Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the ß-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.


Assuntos
Hemoglobina Fetal/genética , Globinas beta/genética , Alelos , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Caspase 9/administração & dosagem , Caspase 9/genética , Feminino , Edição de Genes/métodos , Macaca mulatta , Microinjeções , Mosaicismo/embriologia , Gravidez , RNA Mensageiro/administração & dosagem
5.
J Biol Rhythms ; 31(3): 289-98, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26888974

RESUMO

Synchronization of male and female locomotor activity plays a critical role in ensuring reproductive success, especially in semelparous species. The goal of this study was to elucidate the effects of individual chemical signals, or pheromones, on the locomotor activity in the sea lamprey (Petromyzon marinus). In their native habitat, adult preovulated females (POF) and ovulated females (OF) are exposed to sex pheromone compounds that are released from spermiated males and attract females to nests during their migration and spawning periods. In this study, locomotor activity of individual POF and OF was measured hourly in controlled laboratory conditions using an automated video-tracking system. Differences in the activity between a baseline day (no treatment exposure) and a treatment day (sex pheromone compound or control exposure) were examined for daytime and nighttime periods. Results showed that different pheromone compound treatments affected both POF and OF sea lamprey (p < 0.05) but in different ways. Spermiated male washings (SMW) and one of its main components, 7α,12α,24-trihydroxy-5α-cholan-3-one 24 sulfate (3kPZS), decreased activity of POF during the nighttime. SMW also reduced activity in POF during the daytime. In contrast, SMW increased activity of OF during the daytime, and an additional compound found in SMW, petromyzonol sulfate (PZS), decreased the activity during the nighttime. In addition, we examined factors that allowed us to infer the overall locomotor patterns. SMW increased the maximum hourly activity during the daytime, decreased the maximum hourly activity during the nighttime, and reduced the percentage of nocturnal activity in OF. Our findings suggest that adult females have evolved to respond to different male compounds in regards to their locomotor activity before and after final maturation. This is a rare example of how species-wide chemosensory stimuli can affect not only the amounts of activity but also the overall locomotor pattern in a vertebrate species.


Assuntos
Ácidos Cólicos/farmacologia , Locomoção/efeitos dos fármacos , Petromyzon/fisiologia , Atrativos Sexuais/farmacologia , Comunicação Animal , Migração Animal/efeitos dos fármacos , Animais , Ritmo Circadiano , Feminino , Masculino , Ovulação , Olfato , Gravação de Videoteipe
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