To safely re-open economies and prevent future outbreaks, rapid, frequent, point-of-need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods ...require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing. Here, we report the development and clinical validation of High-Performance Loop-mediated isothermal Amplification (HP-LAMP), a rapid, saliva-based, SARS-CoV-2 test with a limit of detection of 1.4 copies of virus per µl of saliva and a sensitivity and specificity with clinical samples of > 96%, on par with traditional RT-PCR based methods using swabs, but can deliver results using only a single fluid transfer step and simple heat block. Testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample was 100% accurate. Thus, HP-LAMP may enable rapid and accurate results in the field using saliva, without need of a high-complexity laboratory.
Miscarriage is a frequent outcome seen in obstetrics with 1 in 5 pregnancies ending in an early pregnancy loss. Aneuploidy is the most significant single factor affecting early pregnancy failure and ...miscarriage. The risk of aneuploidy increases significantly with increasing maternal age. There has been tremendous advancement in technology that has made preimplantation genetic testing for aneuploidy reliable and accessible. For women in their mid-to-late 30s there is great utility in the use of PGT-A to facilitate single embryo transfer, reduce the risk of clinical miscarriage and ongoing aneuploidy gestations. The current data supports use of preimplantation genetic testing for aneuploidy and single embryo transfer for this population of women. At this time, more prospective data is needed to determine the effect of preimplantation genetic testing for aneuploidy on rates of miscarriage in the recurrent pregnancy loss population.
Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that ...MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples' genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.
Determination of fetal aneuploidy is central to evaluation of recurrent pregnancy loss (RPL). However, obtaining this information at the time of a miscarriage is not always possible or may not have ...been ordered. Here we report on "rescue karyotyping", wherein DNA extracted from archived paraffin-embedded pregnancy loss tissue from a prior dilation and curettage (D&C) is evaluated by array-based comparative genomic hybridization (aCGH).
A retrospective case series was conducted at an academic medical center. Patients included had unexplained RPL and a prior pregnancy loss for which karyotype information would be clinically informative but was unavailable. After extracting DNA from slides of archived tissue, aCGH with a reduced stringency approach was performed, allowing for analysis of partially degraded DNA. Statistics were computed using STATA v12.1 (College Station, TX).
Rescue karyotyping was attempted on 20 specimens from 17 women. DNA was successfully extracted in 16 samples (80.0%), enabling analysis at either high or low resolution. The longest interval from tissue collection to DNA extraction was 4.2 years. There was no significant difference in specimen sufficiency for analysis in the collection-to-extraction interval (p=0.14) or gestational age at pregnancy loss (p=0.32). Eight specimens showed copy number variants: 3 trisomies, 2 partial chromosomal deletions, 1 mosaic abnormality and 2 unclassified variants.
Rescue karyotyping using aCGH on DNA extracted from paraffin-embedded tissue provides the opportunity to obtain critical fetal cytogenetic information from a prior loss, even if it occurred years earlier. Given the ubiquitous archiving of paraffin embedded tissue obtained during a D&C and the ease of obtaining results despite long loss-to-testing intervals or early gestational age at time of fetal demise, this may provide a useful technique in the evaluation of couples with recurrent pregnancy loss.
Many eukaryotic extracellular proteins share a sequence of unknown function, called the zona pellucida (ZP) domain. Among these proteins are the mammalian sperm receptors ZP2 and ZP3, non-mammalian ...egg coat proteins, Tamm-Horsfall protein (THP), glycoprotein-2 (GP-2), α- and β-tectorins, transforming growth factor (TGF)-β receptor III and endoglin, DMBT-1 (deletd in malignant brain tumour-1), NompA (no-mechanoreceptor-potential-A), Dumpy and cuticlin-1 (refs 1,2). Here, we report that the ZP domain of ZP2, ZP3 and THP is responsible for polymerization of these proteins into filaments of similar supramolecular structure. Most ZP domain proteins are synthesized as precursors with carboxy-terminal transmembrane domains or glycosyl phosphatidylinositol (GPI) anchors. Our results demonstrate that the C-terminal transmembrane domain and short cytoplasmic tail of ZP2 and ZP3 are not required for secretion, but are essential for assembly. Finally, we suggest a molecular basis for dominant human hearing disorders caused by point mutations within the ZP domain of α-tectorin.
•Fertility preservation services are utilized in 9% of reproductive age males with cancer.•While use of fertility preservation services is increasing, rates of use remain modest.•Younger age men and ...those receiving chemotherapy and radiation are more likely to utilize fertility preservation services.
While fertility preservation is a major concern among reproductive age cancer patients, little is known about access and use of fertility preserving services. We examined use of fertility preserving services among men with common solid tumors.
A total of 3648 men age 18–40 including 2610 (71.6%) with testicular cancer, 939 (25.7%) with colorectal and 99 (2.7%) with prostate cancer were identified. Fertility preservation services were utilized in 9.3% of men overall including 4.1% who underwent fertility evaluation only and 7.8% who had a fertility preservation procedure. The rate of fertility preservation services rose from 6.6% (95%CI, 3.2–10.0) in 2008 to 12.4% (95%CI, 7.3–17.5) in 2017 (P = 0.04).
Use of fertility preservation service was more common in patients with testicular (11.6%, aRR = 3.31; 95% CI 2.22–4.92) and prostate cancer (6.1%, aRR = 3.14; 95% CI 1.28–7.70) compared to those with colon cancer (3.4%). Younger men were more likely to utilize fertility preservation services. 11.5% of men age ≤ 35 years vs. 5.2% of men 36–40 used these services (P < 0.0001). Fertility preservation services were used in 10.8% of those who received chemotherapy (aRR = 1.81; 95% CI, 1.45–2.27) and in 8.1% of those who received radiation (aRR = 1.30 95% CI, 0.98–1.73). Medicaid patients were less likely to receive fertility preservation services than those with commercial insurance (0.7% vs. 10.1%; aRR = 11.58, 95%CI 2.10–63.69). These data indicate that while use of fertility preserving services is increasing, overall use of services is low among reproductive age males with cancer.
OBJECTIVE: To develop a mathematical formula that accurately predicts the probability of a singleton, twin, and higher-order multiple pregnancy according to implantation rate and number of embryos ...transferred. DESIGN: A total of 12,003 IVF cycles from a single center resulting in ET were analyzed. Using mathematical modeling we developed a formula, the Combined Formula, and tested for the ability of this formula to accurately predict outcomes. SETTING: Academic hospital. PATIENT(S): Patients undergoing IVF. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Goodness of fit of data from our center and previously published data to the Combined Formula and three previous mathematical models. RESULT(S): The Combined Formula predicted the probability of singleton, twin, and higher-order pregnancies more accurately than three previous formulas (1.4% vs. 2.88%, 4.02%, and 5%, respectively) and accurately predicted outcomes from five previously published studies from other centers. An online applet is provided (https://secure.ivf.org/ivf-calculator.html). CONCLUSION(S): The probability of pregnancy with singletons, twins, and higher-order multiples according to number of embryos transferred is predictable and not random and can be accurately modeled using the Combined Formula. The embryo itself is the major predictor of pregnancy outcomes, but there is an influence from “barriers,” such as the endometrium and collaboration between embryos (embryo–embryo interaction). This model can be used to guide the decision regarding number of embryos to transfer after IVF.
To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from ...mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs' gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs' epigenetic memory.
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