High-Yield, Case-Based, Interactive Workshop on Telehealth and Teleneurology With Pediatric Resident Physicians.

Introduction: Increasing prevalence of neurologic disorders with an aging global population and limited availability of neurologists may lead to worse patient outcomes. As a result of the COVID-19 pandemic, telehealth services surged, and despite easing public health measures, the demand has remained. Telehealth technology has the potential to close the physical gaps in expanding the reach of care. This academic half-day workshop sought to provide a learning opportunity in response to these concerns. Methods: The workshop consisted of small- and large-group case discussions among pediatric resident physicians (PGY 1-PGY 3) moderated by two child neurology faculty physicians over Zoom. Participants received a learner document with prereading articles and questions for each case. PowerPoint presentations with video demonstrations were used to introduce the cases and guide discussions. Results: Of the 25 attendees, 14 (56% response rate) answered a nonmandatory postsession survey. Eighty-six percent of the respondents were very or extremely satisfied with the content covered and were similarly satisfied with the effectiveness of content delivery. Seventy-nine percent of the respondents found the content helpful or very helpful in preparation for the board, and 93% anticipated applying the content covered occasionally or frequently in their clinical practice. Discussion: Small-group discussions with video demonstrations are helpful in increasing proficiency with telehealth technology and in examining board-relevant cases on pediatric patients. There is strong interest in subsequent telehealth half-day workshops that incorporate teaching through group discussions on relevant patient case scenarios.

What human sperm RNA-Seq tells us about the microbiome.

PURPOSE: The study was designed to assess the capacity of human sperm RNA-seq data to gauge the diversity of the associated microbiome within the ejaculate. METHODS: Semen samples were collected, and semen parameters evaluated at time of collection. Sperm RNA was isolated and subjected to RNA-seq. Microbial composition was determined by aligning sequencing reads not mapped to the human genome to the NCBI RefSeq bacterial, viral and archaeal genomes following RNA-Seq. Analysis of microbial assignments utilized phyloseq and vegan. RESULTS: Microbial composition within each sample was characterized as a function of microbial associated RNAs. Bacteria known to be associated with the male reproductive tract were present at similar levels in all samples representing 11 genera from four phyla with one exception, an outlier. Shannon diversity index (p < 0.001) and beta diversity (unweighted UniFrac distances, p = 9.99e-4; beta dispersion, p = 0.006) indicated the outlier was significantly different from all other samples. The outlier sample exhibited a dramatic increase in Streptococcus. Multiple testing indicated two operational taxonomic units, S. agalactiae and S. dysgalactiae (p = 0.009), were present. CONCLUSION: These results provide a first look at the microbiome as a component of human sperm RNA sequencing that has sufficient sensitivity to identify contamination or potential pathogenic bacterial colonization at least among the known contributors.

Human chromatin remodeler cofactor, RNA interactor, eraser and writer sperm RNAs responding to obesity.

In the United States almost 33% of adults are considered obese (BMI > 30 kg/m2). Both animal models and to a lesser extent human studies, have associated BMI, a measure of obesity, with alterations in sperm DNA methylation and RNAs. Sperm RNAs from the Assessment of Multiple Gestations from Ovarian Stimulation trial, were isolated and sequenced. A Generalized Linear Model identified 487 BMI associated human sperm RNA elements (short exon-sized sequences). They partitioned into four patterns; a continual increase with BMI, increase once obese (BMI>30 kg/m2); a steady decrease with BMI; and decrease once overweight (BMI 25 - 30 kg/m2). Gene Ontology revealed a unique relationship between BMI and transcripts associated with chromosome organization, adipogenesis, cellular stress and obesity-related inflammation. Coregulatory networks linked by Chromatin remodeler cofactors, RNA interactors, Erasers and Writers (CREWs) were uncovered to reveal a hierarchical epigenetic response pathway.

Response to Comment on "Absence of sperm RNA elements correlates with idiopathic male infertility".

RNAs from other cell types have minimal impact on male fecundity-associated sperm RNA elements.

Absence of sperm RNA elements correlates with idiopathic male infertility.

Semen parameters are typically used to diagnose male infertility and specify clinical interventions. In idiopathic infertile couples, an unknown male factor could be the cause of infertility even when the semen parameters are normal. Next-generation sequencing of spermatozoal RNAs can provide an objective measure of the paternal contribution and may help guide the care of these couples. We assessed spermatozoal RNAs from 96 couples presenting with idiopathic infertility and identified the final reproductive outcome and sperm RNA elements (SREs) reflective of fecundity status. The absence of required SREs reduced the probability of achieving live birth by timed intercourse or intrauterine insemination from 73 to 27%. However, the absence of these same SREs does not appear to be critical when using assisted reproductive technologies such as in vitro fertilization with or without intracytoplasmic sperm injection. About 30% of the idiopathic infertile couples presented an incomplete set of required SREs, suggesting a male component as the cause of their infertility. Conversely, analysis of couples that failed to achieve a live birth despite presenting with a complete set of SREs suggested that a female factor may have been involved, and this was confirmed by their diagnosis. The data in this study suggest that SRE analysis has the potential to predict the individual success rate of different fertility treatments and reduce the time to achieve live birth.

A comparison of sperm RNA-seq methods.

A significant challenge to the effective application of RNA-seq to the complete transcript analysis of low quantity and/or degraded samples is the amplification of minimal input RNA to enable sequencing library construction. Several strategies have been commercialized in order to facilitate this goal. However, each strategy has its own specific protocols and methodology, and each may introduce unique bias and in some cases show specific preference for a collection of sequences. Our wider investigation of human spermatozoal RNAs was able to reveal their complexity despite being generally characterized by low quantity and high fragmentation. In this study, the following four commercially available RNA-seq amplification and library protocols for the preparation of low quantity/highly fragmented samples, SMARTer™ Ultra Low RNA (SU) for Illumina® Sequencing, SeqPlex RNA Amplification (SP), Ovation® RNA-Seq System V2 (OR), and Ovation® RNA-Seq Formalin Fixed Paraffin Embedded System (FFPES) were assessed using human sperm RNAs. Further investigation analyzed the effects on the end results of two different library preparation methods, Encore NGS Multiplex System I (Enc) and Ovation Ultralow Library Systems (UL), that appeared best suited to this type of RNA, along with other potential confounding factors such as FFPE preservation. Our results indicate that for each library preparation protocol, the differences in the initial amount of input RNA and choice of RNA purification step do not generate marked differences in terms of RNA profiling. However, substantial disparity is introduced by individual amplification methods prior to library construction. These significant differences may be caused by the different priming methods or amplification strategies used in each of the four different protocols examined. The observation of intra-sample variation introduced by the choice of protocol highlights the role that external factors play in planning and subsequent reliable interpretation of results of any RNA-seq experiment.

Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling.

Different semen storage and sperm purification methods may affect the integrity of isolated spermatozoal RNA. RNA-Seq was applied to determine whether semen storage methods (pelleted vs. liquefied) and somatic cell lysis buffer (SCLB) vs. PureSperm (PS) purification methods affect the quantity and quality of sperm RNA. The results indicate that the method of semen storage does not markedly impact RNA profiling whereas the choice of purification can yield significant differences. RNA-Seq showed that the majority of mitochondrial and mid-piece associated transcripts were lost after SCLB purification, which indicated that the mid-piece of spermatozoa may have been compromised. In addition, the number of stable transcript pairs from SCLB-samples was less than that from the PS samples. This study supports the view that PS purification better maintains the integrity of spermatozoal RNAs.

Stability, delivery and functions of human sperm RNAs at fertilization.

Increasing attention has focused on the significance of RNA in sperm, in light of its contribution to the birth and long-term health of a child, role in sperm function and diagnostic potential. As the composition of sperm RNA is in flux, assigning specific roles to individual RNAs presents a significant challenge. For the first time RNA-seq was used to characterize the population of coding and non-coding transcripts in human sperm. Examining RNA representation as a function of multiple methods of library preparation revealed unique features indicative of very specific and stage-dependent maturation and regulation of sperm RNA, illuminating their various transitional roles. Correlation of sperm transcript abundance with epigenetic marks suggested roles for these elements in the pre- and post-fertilization genome. Several classes of non-coding RNAs including lncRNAs, CARs, pri-miRNAs, novel elements and mRNAs have been identified which, based on factors including relative abundance, integrity in sperm, available knockout data of embryonic effect and presence or absence in the unfertilized human oocyte, are likely to be essential male factors critical to early post-fertilization development. The diverse and unique attributes of sperm transcripts that were revealed provides the first detailed analysis of the biology and anticipated clinical significance of spermatozoal RNAs.

Isolating mRNA and small noncoding RNAs from human sperm.

The isolation of spermatozoal RNA is a challenging procedure due to the intrinsic heterogeneous population of cells present in the ejaculate and the small quantity of RNA present in sperm. The transcriptome of these gametes includes a wide variety of messenger RNAs (mRNAs), small noncoding RNAs (sncRNAs), and highly fragmented ribosomal RNAs (rRNAs).The protocol described in this chapter to isolate both the mRNA and sncRNA fractions represents years of development towards automation. It combines a guanidinium thiocyanate-phenol-chloroform-based methodology to reduce the content of DNA and a column-based system. Both manual and semi-automated options are described, with preference given to automation for consistent results. A novel quality control procedure has been developed to assess the integrity and purity of the entire population of isolated mRNAs due to the absence of intact rRNAs.

Identification of artifactual microarray probe signals constantly present in multiple sample types.

The detection, identification, and quantitation of transcripts have evolved from simple Northern analysis, cDNA cloning, and sequencing to RT-PCR, microarrays, and now digital gene expression using ultra-high-throughput RNA sequencing (RNA-Seq). During the course of our studies we observed that some microarray probes show very high signal intensity values yet are discordant when compared with RNA-Seq. A total of 99 probes from approximately 30,000 were identified as consistently discordant in four human tissues or cell lines. Interestingly, this set of discordant probes appears array-dependent. Among the 99 probes identified, 70 constantly exhibited a high signal in all 713 available samples surveyed using the Illumina HumanHT-12v4 platform. Some were discordant with additional probes that annotated the same genes. Absence of a number of these transcripts was confirmed by quantitative RT-PCR (qRT-PCR). Our findings suggest that one must be cautious, as some array probes do not capture the level of the target.

Interrogating the transgenic genome: development of an interspecies tiling array.

A single expressing copy of the human protamine domain was randomly inserted into an intron of Cyp2c38. The transgenic locus was shown to recapitulate the level of expression observed in normal human testis while not perturbing endogenous protamine expression. The development of an interspecies tiling array was pursued to enable direct comparison of the orthologous protamine domains in a single experiment. Probe design was adapted to generate species-specific high resolution probe sets that would tolerate repetitive elements. Results from competitive hybridizations demonstrate that interspecies tiling arrays are a valuable tool for parallel analysis of highly similar DNA sequences. This approach provides a rapid and reliable means of interrogating samples prior to deep sequencing analysis. These arrays should readily compliment most DNA isolation and analysis techniques such as ChIP, nuclease sensitivity and nuclear matrix association assays.

The preparation of human spermatozoal RNA for clinical analysis.

The recent identification of RNA as a component of mature spermatozoa necessitated the development of a reliable isolation protocol capable of yielding a high-quality substrate. In addition to the inherent difficulties associated with isolating RNA, the procedure as applied to sperm must overcome the resilient nature and reduced RNA content found within this cell type. Further, the protocol must be suited to the clinical setting. A reliable RNA isolation procedure optimized for this unique cell type is described. Ejaculate is collected, contaminating somatic cells lysed then spermatozoal RNA released by homogenization in a chaotrope. RNA is then purified from the homogenate by chromatography using a commercially available resin. The quality of isolated samples is assessed by PCR and RT-PCR. Once purity is established samples are suitable for numerous applications including amplification and probe synthesis. The reliable and consistent isolation of high-quality RNA from mature spermatozoa will aid in the development of new tools for the clinical assessment of male-factor fertility.

Success and failure in human spermatogenesis as revealed by teratozoospermic RNAs.

We are coming to appreciate that at fertilization human spermatozoa deliver the paternal genome alongside a suite of structures, proteins and RNAs. Although the role of some of the structures and proteins as requisite elements for early human development has been established, the function of the sperm-delivered RNAs remains a point for discussion. The presence of RNAs in transcriptionally quiescent spermatozoa can only be derived from transcription that precedes late spermiogenesis. A cross-platform microarray strategy was used to assess the profile of human spermatozoal transcripts from fertile males who had fathered at least one child compared to teratozoospermic individuals. Unsupervised clustering of the data followed by pathway and ontological analysis revealed the transcriptional perturbation common to the affected individuals. Transcripts encoding components of various cellular remodeling pathways, such as the ubiquitin-proteosome pathway, were severely disrupted. The origin of the perturbation could be traced as far back as the pachytene stage of spermatogenesis. It is anticipated that this diagnostic strategy will prove valuable for understanding male factor infertility.

In silico and wet-bench identification of nuclear matrix attachment regions.

Chromatin loops are tethered at discrete regions that are approx 100-1000 bp in length. These regions of attachment serve as specific sequence landmarks, anchoring the DNA to the fibers of the chromosomal scaffold. It has been estimated that our genome contains 70,000 nuclear matrix attachment sites that serve as a dynamic nuclear organizer in both the interphase and metaphase cell. Approximately 30,000-40,000 matrix attachment regions (MARs) serve as origins of replication. MARs can also be associated with chromosomal segments densely populated with transcription factor-binding sites. This may facilitate transcription that is initiated within the region of the chromosome coincident with the surface of the nuclear matrix. Assuming an average somatic loop size of 100 kb, it is reasonable to propose that each cell utilizes 30,000 MARs to anchor each of the approx 20,000 active genic domains. This is sufficient to encompass the 30,000 functional genes in our genome that exist as members of single or multigenic families, each constituting a single chromatin domain. With the sequencing phase of various genome projects complete, in silico tools are being developed to identify the long-range control elements that modulate gene expression. This information is necessary to specifically target the time-intensive wet-bench verification and expression experiments that will provide a unified understanding of gene regulation. In this chapter we review some of the in silico strategies that are currently available and a new in vivo method based on the real-time polymerase chain reaction, to assess regions of matrix association.

Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility.

OBJECTIVE: To establish the stability of spermatozoal RNAs as a means to validate their use as a male fertility marker. DESIGN: Semen samples were randomly selected for 1 of 3 cryopreservation treatments. SETTING: An academic research environment. PATIENT(S): Men aged 19 to 55 years who had fathered a child by natural conception within the past 6 months. INTERVENTION(S): Ejaculates were collected by masturbation and total spermatozoan RNA was isolated from two semen samples of ideal quality; one sample of medium quality, having been subjected to an additional freeze-thaw cycle, and two samples of poor quality, having been subjected to a third freeze-thaw cycle. MAIN OUTCOME MEASURE(S): Labeled cDNAs were generated and then used to interrogate Atlas Nylon Human Toxicology 1.2 microarrays. The spermatozoan transcriptomes were compared using a binomial approach. RESULT(S): The analysis identified a total of 228 unique spermatozoal transcripts among all samples. The medium quality sample shared 98% and 39% of its RNAs with the ideal and poor quality samples, respectively. A set of 36 RNAs resistant to insult were observed, some of which have been implicated in regulating male fertility, when all individuals were compared. CONCLUSION(S): These results support the view that a population of spermatozoal RNAs is rapidly degraded in response to insult, whereas another population appears protected from such damage. Because spermatozoal RNAs echo the gene expression of spermatogenesis, the latter is likely to prove useful as a clinical maker of fertility status.

A suite of novel human spermatozoal RNAs.

We recently described a complex population of spermatozoal coding RNAs that are delivered to the oocyte on fertilization. These are derived throughout spermatogenesis, representing a record of past events. Recently, evidence has been provided that micro-RNAs are present in testes, suggesting that they might also be carried in ejaculate spermatozoa. To directly test this hypothesis, a unique microarray system capable of directly identifying antisense RNAs and predicted transcripts was utilized. RNA isolated from the ejaculate spermatozoa of 6 normal fertile men was directly hybridized to sense oligonucleotide arrays containing 10,000 elements. This revealed 68 shared RNAs, some of which are similar to those previously defined as micro-RNAs, whereas others were the antisense of previously in silico-predicted transcripts. The results and implications of this study are described in this communication.

Trauma-induced cutaneous pigmentation from tetracycline: a case report.

Tetracycline is well known to cause pigmentation of various body tissues, such as teeth, bone, lunulae, conjunctivae, and lymph nodes. It has not been reported to cause discoloration of the skin, unlike its semisynthetic derivative, minocycline. We report an 18-year-old girl who developed lower extremity pigmentation in areas of trauma during treatment with tetracycline.