Despite the challenges in studying recurrent implantation failure, progress is currently being made in therapeutic options to help those who suffer from recurrent implantation failure. Three of the ...most promising therapeutic options for recurrent implantation failure include immune therapies such as peripheral blood mononuclear cells, platelet rich plasma and subcutaneous granulocyte-colony stimulating factor.
We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic ...oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs. Furthermore, sequence variation in the placental miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, allow us to define the repertoire of miRNA abundance in the circulation and potential uses as biomarkers.
To assess attitudes and perceptions of U.S. survey respondents regarding prevalence, causes, and emotional effects of miscarriage.
We used a questionnaire consisting of 33 questions administered in ...January of 2013 to men and women aged 18-69 years across the United States.
Participants from 49 states completed the questionnaire: 45% male and 55% female (N=1,084). Fifteen percent reported they or their partner experienced at least one miscarriage. Fifty-five percent of respondents believed that miscarriage occurred in 5% or less of all pregnancies. Commonly believed causes of miscarriage included a stressful event (76%), lifting a heavy object (64%), previous use of an intrauterine device (28%), or oral contraceptives (22%). Of those who had a miscarriage, 37% felt they had lost a child, 47% felt guilty, 41% reported feeling that they had done something wrong, 41% felt alone, and 28% felt ashamed. Nineteen percent fewer people felt they had done something wrong when a cause for the miscarriage was found. Seventy-eight percent of all participants reported wanting to know the cause of their miscarriage, even if no intervention could have prevented it from occurring. Disclosures of miscarriages by public figures assuaged feelings of isolation for 28% of respondents. Level of education and gender had a significant effect on perceptions and understanding of miscarriage.
Respondents to our survey erroneously believed that miscarriage is a rare complication of pregnancy, with the majority believing that it occurred in 5% or less of all pregnancies. There were also widespread misconceptions about causes of miscarriage. Those who had experienced a miscarriage frequently felt guilty, isolated, and alone. Identifying a potential cause of the miscarriage may have an effect on patients' psychological and emotional responses.
II.
MinION is a memory stick-sized nanopore-based sequencer designed primarily for single-molecule sequencing of long DNA fragments (>6 kb). We developed a library preparation and data-analysis method to ...enable rapid real-time sequencing of short DNA fragments (<1 kb) that resulted in the sequencing of 500 reads in 3 min and 40,000-80,000 reads in 2-4 hr at a rate of 30 nt/sec. We then demonstrated the clinical applicability of this approach by performing successful aneuploidy detection in prenatal and miscarriage samples with sequencing in <4 hr. This method broadens the application of nanopore-based single-molecule sequencing and makes it a promising and versatile tool for rapid clinical and research applications.
Piwi-interacting RNAs (piRNAs), a class of 26- to 32-nt non-coding RNAs (ncRNAs), function in germline development, transposon silencing, and epigenetic regulation. We performed deep sequencing and ...annotation of untreated and periodate-treated small RNA cDNA libraries from human fetal and adult germline and reference somatic tissues. This revealed abundant piRNAs originating from 150 piRNA-encoding genes, including some exhibiting gender-specific expression, in fetal ovary and adult testis—developmental periods coinciding with mitotic cell divisions expanding fetal germ cells prior to meiotic divisions. The absence of reads mapping uniquely to annotated piRNA genes demonstrated their paucity in fetal testis and adult ovary and absence in somatic tissues. We curated human piRNA-expressing regions and defined their precise borders and observed piRNA-guided cleavage of transcripts antisense to some piRNA-producing genes. This study provides insights into sex-specific mammalian piRNA expression and function and serves as a reference for human piRNA analysis and annotation.
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•150 piRNA genes were identified in human adult testis and fetal ovary•Adult testis and fetal ovary differentially express piRNA genes•piRNAs were not identified in non-germline tissue•Non-repetitive piRNA genes account for over 90% of piRNAs
Williams et al. identify and characterize 150 piRNA genes in the adult human testis and fetal ovary. Expression of piRNA genes differs between the sexes, and repetitive elements, including those for retrotransposons, are underrepresented among piRNA genes. No piRNA expression was detected in somatic tissue.
Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic ...applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing. miRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNA–target-mRNA interactions, target mRNA levels were negatively correlated with changes in abundance for highly expressed miRNAs in HF and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced HF, which coincided with a similar increase in cardiac troponin I (cTnI) protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 mo following initiation of LVAD support. In stable HF, circulating miRNAs showed less than fivefold differences compared with normal, and myomir and cTnI levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers.
The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and ...robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.
To determine if a handheld, nanopore-based DNA sequencer can be used for rapid preimplantation genetic screening (PGS).
Laboratory study.
Academic medical center.
Amplified genomic DNA from euploid ...and aneuploid trophectoderm biopsy samples (n=9) that was also tested using traditional next generation sequencing (NGS).
Short-read DNA library preparation and nanopore-based sequencing using a hand-held MinION sequencer.
Comparison of cytogenetic testing result from NGS and nanopore-based sequencing and the time required for library preparation and sequencing.
Multiplexed short-read DNA library preparation was completed in 45 minutes. Sequencing on a single sample was completed within 20 minutes and 5 samples were simultaneously sequenced in under 2 hours. Whole-chromosome aneuploidy screening results obtained from nanopore-based sequencing were identical to those obtained using NGS.
Here we report the first application of nanopore-based sequencing for PGS on trophectoderm biopsy samples using a novel rapid multiplxed short-read nanopore sequencing library preparation protocol. Sequencing for aneuploidy screening could be performed on a single sample in 20 minutes and on 5 samples, simultaneously, within 2 hours. Overall, nanopore sequencing is a promising tool to perform rapid PGS onsite, enabling same day testing and embryo transfer, thus obviating the need for complex, large and expensive DNA sequencers or embryo freezing.
The rapid rise of novel coronavirus disease 2019 (COVID-19) cases led the American Society for Reproductive Medicine (ASRM) to recommend immediate cessation of all new fertility treatment cycles on ...March 17, 2020. Controversial from the start, providers and patients expressed their opposition through online petitions, surveys, and other forums. While the impact of a delay in access to reproductive care is unknown, previous studies are reassuring that a delay in the timespan of months may not affect clinical outcomes. However, dropout from care during this pandemic remains a serious concern. Effective therapies against the virus and a vaccine are not on the immediate horizon. Accepting COVID-19 will likely be a part of our lives for the near future necessitates the modification of fertility protocols to keep patients, providers, and staff as safe as possible. We believe fertility treatment is an urgent, essential service that can be performed safely and responsibly during this pandemic.
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