Zosterops lateralis) to French Polynesia, which represents a potentially extreme example of population founding, we reassess the potential for morphological shifts to arise via drift alone. Despite only 80 years of separation from their New Zealand ancestors, French Polynesian silvereyes displayed significant changes in body and bill size and shape, most of which could be accounted for by drift, without the need to invoke selection. However, signatures of selection at genes previously identified as candidates for bill size and body shape differences in a range of bird species, also suggests a role for selective processes in driving morphological shifts within this population. Twenty-four SNPs in our RAD-Seq dataset were also found to be strongly associated with phenotypic variation. Hence, even under population founding extremes, when it is difficult to reject drift as the sole mechanism based on rate tests of phenotypic shifts, the additional role of divergent natural selection in novel environments can be revealed at the level of the genome.Theory predicts that when populations are established by few individuals, random founder effects can facilitate rapid phenotypic divergence even in the absence of selective processes. However, empirical evidence from historically documented colonisations suggest that, in most cases, drift alone is not sufficient to explain the rate of morphological divergence. Here, using the human-mediated introduction of the silvereye ( The speed at which divergent evolution proceeds is highly variable. However, the underlying processes driving differences in evolutionary rates are not well understood Provine . InstancZosterops lateralis) the absence of founder-induced morphological divergence following the natural colonisation of islands across the South Pacific may be explained by: establishment by founding flocks in excess of hundreds of individuals , provide the potential for rapid phenotypic divergence, such as changes in body/bill size and shape.The islands of French Polynesia contain a rich assemblage of introduced bird species following documented releases of at least fifty-nine species during the late 19th and early 20th centuries . We address the following specific questions: (1) to what extent did the French Polynesian population experience a strong and sustained population bottleneck following introduction? (2) Can the rate of phenotypic divergence between French Polynesian and New Zealand silvereyes be explained by drift alone? and (3) What is the genomic evidence for selective processes? Commenserate with the introduction of a handful of individuals, we show that compared with natural colonisations by the silvereye, the French Polynesian population has undergone a more substantial population bottleneck followed by slower population recovery. In most instances we could not rule out drift as the sole cause of observed morphological shifts. However, the identification of outlier loci at genes previously associated with morphological divergence in birds, also suggests a strong role for directional selection.t-test P values > 0.05) and therefore suggesting no strong effect attributable to different observers.We collected blood samples and morphological data from 190 silvereyes across the islands of Tahiti, Mo’orea, Huahine, Raiatea and Maupiti (Society Archipelago) in April and May 2017. Birds were caught using mist nets and 20–40 μl of blood collected from the brachial wing vein, stored in 1 ml of lysis buffer  and frozen at −20 °C  . Single Nucleotide Polymorphisms (SNPs) were identified using the HaplotypeCaller tool from the Genome Analysis Toolkit (GATK) nightly build version 2016-12-05-ga159770  and restriction site-associated DNA paired-end (RAD-PE) sequencing libraries constructed at the University of California, Los Angeles following the protocol outlined in genoscapeRtools . The VCF file was then further filtered using VCFtools to include the optimum number of SNPs and only those individuals where less than 30% of data were missing.To determine the optimum number of SNPs and individuals to retain for downstream analyses, data missingness was visualised Fig.  using geZ. l. melanops genome is only assembled to the scaffold level  using Satsuma Synteny  using the full SNP dataset. We also examined patterns of population structure by performing maximum likelihood estimation of individual admixture proportions using the program fastsimcoal v. 2.6 (Excoffier and Foll tintro was fixed to 32 generations. Population size following introduction (Nintro) was fixed between 2 and 100 individuals . Population recovery following introduction (texp) was fixed between 19 and 26 generations ago (in line with timing of documented population expansion within French Polynesia). For the three-population model the establishment of the Maupiti population (tcol) was fixed between 11 and 14 generations ago (in line with the documented colonisation timeframe) and founding population size of the Maupiti population (Ncol) fixed between 2 and 500 individuals. The three-population demographic model also incorporated a population size change within the Maupiti population (tchange) which was fixed between 2 and 11 generations ago. Contemporary population sizes were estimated for each population with initial search ranges for SNZ (NSNZ), all French Polynesia (NAll FP) and FP cluster 1 (NFP1) set to between 10,000 and 10,000,000 individuals. Given the very small geographic size of the island of Maupiti, search ranges for FP cluster 2 (Maupiti only) were bounded between 5 and 1,000 individuals. For each demographic model, we performed 100 independent runs , and chose the run with the highest likelihood as the best parameter estimates. As mutation rates for silvereyes are unknown, parameter estimates were based on estimated mutation rates for collared flycatchers : 4.6 × 10−9 mutations per nucleotide site per generation  to include only SNPs present in all individuals. VCFs were then converted to SFS format using the python script EasySFS.py (https://github.com/isaacovercast/easySFS) using flag-a to include all sites.In order to infer effective population size changes in French Polynesia, we estimated demographic parameters from the joint Site Frequency Spectrum (SFS) using the composite-likelihood and coalescent simulation approach implemented in To summarise morphological divergence between the French Polynesian silvereye population(s) and representatives of its New Zealand ancestor, we conducted a PCA of morphological data collected from 189 silvereyes sampled across the Society Archipelago (French Polynesia) and 91 from Dunedin . PCA was conducted on log-transformed data for wing length, tail length, bill length, depth and width. Differences in PC scores between South Island and French Polynesian silvereyes were tested for significance using analysis of variance (ANOVA).h2 = narrow-sense heritability of trait in question, t = number of generations, z = mean morphological shift and σ = phenotypic standard deviation of the colonised population. Calculations of heritability (h2) were based on parent offspring, full-sibling comparisons and cross-fostering experiments in the Heron Island silvereye  population. Heritability (h2) estimates for morphological traits, ranged from 0.2 and 0.6  and upper estimate (3-year generation time and h2 = 0.6) of Ne for French Polynesia calculated as harmonic means referred to as Ne(h)). The harmonic mean is always lower than the arithmetic mean and is especially sensitive to low values of Ne. Use of the harmonic mean is therefore relevant when two related demographic scenarios are suspected, an in situ population bottleneck or a founder event (Wright Ne(h) > Ne(h) were calculated when treating French Polynesia as a single population (All FP), and treating French Polynesia as two separate populations (FP1 and FP2).Morphological shifts between New Zealand and French Polynesia were calculated for each morphological trait measured. Morphological data were log-transformed, and shifts expressed as standard deviation units, using pooled standard deviation estimates. To determine if random processes alone could account for observed shifts in morphological traits, we calculated Lande’s  statistit Wright . Where Ned Lande . Both \dPCAdapt, a principal components-based method of outlier detection with a low rate of false-positive detection . Outlier SNPs were identified using the following settings comparing each French Polynesian island individually to the New Zealand population: K = 2, MinMAF = 0.1 and FDR = 0.01. As PCAdapt does not included the option to assign individuals to populations, outlier detection was conducted using individual French Polynesian islands to ensure that identified outliers reflected differences between New Zealand and French Polynesia rather than within French Polynesia differences.To detect signatures of selection at the genomic level, we scanned for outlier loci using T. guttata genome occurring within 10,000 bp of outlier SNPs were identified using the Ensembl BioMart database (https://www.ensembl.org/biomart). Genes that had been previously associated with morphological variation in birds were determined to be candidate genes underlying the observed divergence in body size and beak shape in silvereyes. Genes known to be involved in craniofacial variation/disease in non-avian species were also considered candidates.Known, novel or predicted genes of the GEMMA  as implemented in the software package Zosterops scaffolds onto T. guttata chromosomes, based on output from Satsuma Synteny and removal of unmapped/unoriented scaffolds, resulted in 63,849 SNPs (full SNP dataset). The LD-filtered dataset contained 5,414 unlinked SNPs for admixture analysis, and subsequent removal of SNPs with missing data resulted in 1587 SNPs for demographic modelling.Overall, RAD-PE sequencing resulted in an average of 227,092 paired-end reads per sample ±16,784) covering 2,128,552 variable sites. Subsequent quality filtering  reduced the total number of SNPs to 215,543. Of the 119 samples sequenced, 72 were retained after removing individuals where ≥30% of data were missing across a subsample of 64,663 SNPs. The number of individuals retained per location ranged from 8 to 21  with New Zealand and French Polynesia forming distinct groups. At K = 3  admixture results were consistent with those of the PCA , contemporary effective population sizes for New Zealand and French Polynesia were estimated as 3.73 million and 1.22 million individuals, respectively . These differences were also maintained when treating French Polynesia as two populations, with the exception wing length for FP2 (Maupiti only) which was not significantly different from New Zealand  = 28.59, P < 0.001; PC2 scores: F = 10.66, P < 0.001). PC1 and PC2 scores differed significantly between New Zealand and French Polynesian populations, with French Polynesian silvereyes showing a trend towards larger PC1 scores (increased body/bill size) and lower PC2 scores    accounted for 29.38% and 21.75% of variance, respectively. PC1 broadly summarised body/bill size , whereas PC2 summarised body/bill shape (primarily contrasting bill size and body size traits) (see Table es) Fig. .Ne(h) = 10.39). However, when using the least conservative  estimates of Ne(h) = 10.39). When treating French Polynesia as two populations, in no case was Ne(h) > Rate tests indicated that, in most instances, drift alone could not be dismissed as the sole cause of observed morphological shifts. For example, the effective population size required to explain the observed shift in wing length when treating French Polynesia as a single population  have been previously associated with bill/body size differences in birds or craniofacial morphogenesis in non-avian species  were previously associated with morphological variation or craniofacial defects in vertebrates, including birds  was identified as both a PCAdapt outlier and strongly associated with PC1 scores in the BSLMM. This SNP was located within the ZMYND11 (Zinc Finger MYND-Type Containing 11).Using ion Fig. . One hunion Fig. , of whices Table . The BSLes Table . These hds Table . Genes nThe introduction of the silvereye to French Polynesia provides a contemporary case in which to assess the potential for founder-induced drift to facilitate morphological divergence under more extreme founding conditions than those observed in natural colonisations by this species. Consistent with the silvereye’s capacity for rapid differentiation . Unlike previous studies of natural island colonisations by birds, in most instances we were unable to dismiss drift as the sole mechanism through which morphological shifts may have arisen using rate tests. Whereas natural island colonisations by birds may have reduced potential for founder-induced morphological divergence due to the establishment by large flocks  on Heron Island suggest that, in this species, increased intraspecific competition is the prevailing mechanism driving increased body size of insular forms  compared with their New Zealand ancestor, their wing lengths were shorter and tarsus lengths unchanged. Smaller wing size may suggest reduced reliance on flight and/or increase use of densely vegetated habitat Savile . HoweverAs the observed morphological shifts have taken place within a maximum of 80 years (~32 generations), our study supports the hypothesis that the bulk of differences can develop rapidly, potentially within the first couple of hundred generations after colonisation as proposed for the Capricorn silvereye   , baboons (genus: Papio), European glass eels (Anguilla anguilla) and humans . The IGF1 pathway has a well-known role in body size scaling in a range of taxa, including chickens  signalling during craniofacial development  scores. Although not all strongly associated SNPs occurred within gene coding regions, a finding which is perhaps not surprising given the density of our marker set, several did occur within or close to gene coding regions including genes that have previously been associated with morphological variation in birds, non-avian vertebrates or implicated in craniofacial disease in humans. This included: HMGA2: ‘high mobility group AT-hook 2’ , red-browed firetail  and chestnut-breasted mannikin (Lonchura castaneothorax), allowing a contrast between a range of species.Given that knowledge of contemporary natural colonisation events is limited, evolutionary insights into the early stages of divergence have been predominantly retrospective or laboratory based. However, contemporary introductions of birds to islands provide ideal systems in which to study the genomic and phenotypic changes that take place during the early stages of divergence. Taking advantage of the well-documented introduction of the silvereye to French Polynesia, we set out to assess the role of founder-induced drift in driving phenotypic divergence under more extreme founding conditions than provided by natural colonisation events in this species. While we were unable to dismiss drift as the sole cause of significant shifts in morphological traits, given that island-living is known to exert strong selective pressure on avian morphology and French Polynesian silvereyes show body size/shape changes consistent with the ‘island syndrome’, observed phenotypic shifts within the French Polynesian population likely result from a combination of drift and selective processes. Studies of colonisation in action would provide the opportunity to tease apart the roles of drift and selection in driving morphological shifts at different stages of divergence. Although, such events are extremely rare, the potential establishment of a silvereye population on the French Polynesian island Rimatara  may provide such an opportunity. Genetic and phenotypic changes associated with population founding could also be further investigated in other species introduced to French Polynesia, such as common waxbill (Supplementary Materials


Preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm (TE) biopsy samples is labour intensive, invasive, and subject to sampling bias. In this study, we report on the efficacy and factors affecting accuracy of a technique we pioneered for minimally invasive preimplantation genetic testing for aneuploidy (miPGT-A). Our technique uses cell-free embryonic DNA (cfeDNA) in spent embryo culture medium (SEM) combined with blastocoel fluid (BF) to increase the amount of assayable cfeDNA. We compared miPGT-A results (n = 145 embryos) with standard PGT-A analysis of the corresponding trophectoderm biopsy. We found that accuracy of miPGT was not related to blastocyst morphological grade. The overall concordance rate per sample for euploidy/aneuploidy status between miPGT-A and TE biopsy samples was 88/90 (97.8%), and was not different between good 47/48 (97.9%) and moderate/low quality blastocysts 41/42 (97.9%) (p > 0.05). Importantly, we also discovered that for cfeDNA analysis, the SurePlex whole genome amplification (WGA) kit can be utilized without an additional cell lysis/extraction DNA step; this efficiency likely reduces the risk of maternal contamination. Regarding origin of embryonic cfeDNA, the average amount of miPGT-A WGA-DNA we obtained from blastocysts with different morphological grades, as well as the size miPGT-A WGA-DNA fragments, suggest that it is unlikely that apoptosis and necrosis are only mechanisms of DNA release from the inner cell mass (ICM) and TE into BF and SEM. However, there are three main challenges of the preimplantation genetic testing associated with trophectoderm biopsy samples: (1) TE biopsy is labour intensive7; (2) TE biopsy is invasive9; and (3) it is subject to sampling bias - TE biopsy may not accurately represent the inner cell mass (ICM) and remainder of the TE12. Furthermore, although there has been no reported increase in the risk of adverse perinatal outcomes, such as pre-term birth and low birth weight, following invasive PGT compared with IVF without embryo biopsy, conclusive evidence regarding the long-term health of the offspring after embryo biopsy will take some time to obtain14.Preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm (TE) biopsy and next generation sequencing (NGS) as a testing platform for embryo selection has significantly improved ongoing pregnancy rates per transfer shortening the time to pregnancy in addition to the reduction of multiple pregnancies by transferring single euploid embryos, reduction in miscarriage rates and reduced risk of aneuploid pregnancies17 and spent embryo culture medium20. Non-invasive preimplantation genetic testing (NIPGT) or minimally invasive preimplantation genetic testing (miPGT) using the cfeDNA of spent embryo culture medium (SEM) and/or blastocoel fluid (BF) has the potential to eliminate the need for embryo biopsy, thereby avoiding potential risks related to that invasive procedure23. Moreover, NIPGT/miPGT is less labour intensive and potentially more cost-effective method. In addition, NIPGT-A, which is based on sequencing of cfeDNA likely released from both TE and ICMcells25, may better represent the entire embryo compared to TE biopsy alone28.Recently, cell-free embryonic nuclear DNA (cfeDNA) has been found in both blastocoel fluid29:30.Blastocoel fluid aspiration using an ICSI pipette34.Spent embryo culture medium collection35.Combined spent embryo culture medium and blastocoel fluid (SEM + BF) collection without blastocoel fluid aspirationCurrently, there are three major ongoing research approaches regarding how to collect cell-free embryonic DNA for non-invasive or minimally invasive aneuploidy testing33, cfeDNA degradation, low amounts of cfeDNA, variable DNA amplification efficacy and yield37, and short DNA fragments17.Attempts to use cfeDNA for non-invasive preimplantation aneuploidy testing bring to light several factors that could potentially affect the accuracy of this approach reducing concordance rates with TE biopsy results. These include maternal contamination by cumulus and corona cells38 and monogenic disorders39. In addition, NIPGT-A, based on sequencing of cfeDNA likely released from both TE and ICM cells, may better represent the entire embryo compared to TE biopsy alone39. The mechanism(s) underlying the release of embryonic DNA in spent embryo culture medium and blastocoel fluid remain unclear7. Several authors have hypothesized that cfeDNA is correlated with apoptotic events40. If this is true, it should follow that lower quality blastocysts, which generally have higher degrees of apoptosis, would result in a higher quantity of cfeDNA release and thus more accurate results from aneuploidy testing.Previous studies from our research group as well as others, have shown that cfeDNA testing using spent embryo culture medium on days 5 or 6 has the potential to detect chromosomal aneuploidy39, and between NIPGT-A and whole blastocysts29 were reported by several groups. However, there are several very important issues still need to be addressed before routine clinical application of NIPGT. These include: minimization of maternal DNA contamination risk, determining thefactors affectingaccuracy, and optimization of the WGA protocol for cfeDNA.High concordance rates between NIPGT-A and TE biopsy25. We have also evaluated several important factors that could influence this method, including: (1) quantity of amplified cfeDNA obtained; (2) the effect of blastocyst morphological grades on cfeDNA; (3) average size of WGA-DNA fragments from good and moderate/low quality blastocysts, and (4) whole genome amplification of cfeDNA from SEM + BF samples with or without a cell lysis/extraction enzymatic step.In this report, we have assessed the accuracy and reliability of utilizing cfeDNA in SEM + BF samples for blastocyst chromosomal status detection in comparison to corresponding TE biopsy samples in a larger cohort of fresh cultured embryos than our previous publicationTable Informative NGS results Table  were obtA detailed summary of miPGT-A and PGT-A results for paired samples of embryos with good and moderate/poor morphology are presented in Table Mosaicism results were complex when comparing PGT-A with miPGT-A Table . There wThe second objective of this study was to determine the accuracy, efficacy and reliability of whole genome amplification (WGA) to determine ploidy status of the blastocyst using combined SEM + BF samples with or without using the cell lysis/extraction enzyme step before WGA on separate aliquots from the same pool of SEM + BF collected for miPGT-A analysis.We collected 2 aliquots from SEM + BF (miPGT samples) (n = 86) from 43 additional blastocysts. We analysed the amount of amplified DNA and NGS data of the 86 miPGT samples and corresponding 43 trophectoderm biopsy samples obtained from fresh blastocysts that underwent PGT-A cycles from. The first aliquot (miPGT-1) SEM + BF sample followed the standard SurePlex WGA protocol which starts with 5ul of sample and a cell lysis step. The second aliquot from the same SEM + BF pool (miPGT-2) was amplified following a modified WGA SurePlex protocol that starts with 10ul of sample and the direct pre-amplification. WGA products of miPGT-1 and miPGT-2 samples were compared with each other, and results were compared with the corresponding trophectoderm biopsy sample used as a control.The amount of concentrated amplified cfeDNA from miPGT-1 samples was higher than in miPGT-2 samples, however this was not statistically significant. Respective blank medium (negative control) associated with each sample showed no amplification in all cases Table .Table 4AInformative NGS results Table  were obt40.This could potentially impact the analysis of embryonic DNA from spent culture media. To minimize maternal contamination in our studies, we modified the procedure steps during Day 0 to Day 4 of embryo culture to include careful removal of residual corona cells by pipetting and washing25. Using a fluorescently labelled short tandem repeat (STR) marker for the analysis of both embryonic DNA (TE cells) and miPGT samples, we were able to confirm that this step minimizes maternal contamination. This is critical to avoid misdiagnosis with NIPGT-A.It has been recently reported that only ∼8% of DNA in spent embryo culture medium is embryonic in origin25, promotes a yield of less degraded cfeDNA. This method considers the embryonic genome activation stage in human embryos42 and the number of blastomeres on Day 4 versus Day 3. Our approach was also adopted in two recent studies34 that showed the superiority of using spent embryo fresh culture media from embryos cultured from day 4 today 5/6 for non-invasive PGT-A, compared to samples collected from a more extended culture period, which likely results in a more degraded cfeDNA sample.In addition, our approach to transfer embryos into individual fresh droplets of medium on Day 4 and using SEM + BF samples obtained after Day 5/6 culture33. In other studies assisted hatching was not performed prior to conducting the TE biopsy34. Ho et al. found that assisted hatching on Day 3 did not influence cfeDNA concentration or accuracy of cfeDNA sequencing for aneuploidy screening31. CfeDNA isolated from spent embryo culture medium on Day 2/3 has a low molecular weight, where cellular fragmentation may also play a role20. Vera-Rodriguez et al. pointed out that samples isolated from spent embryo culture medium could contain high molecular weight DNA or sheared DNA33. We have not measured the amount of cfeDNA in D4-D5/6 embryo culture media, with or without using laser zona opening on Day 4 to test this hypothesis.The issue of whether nucleic acids can penetrate though the zona pellucida is still an unsolved question. We hypothesized that assisted hatching (AH) may facilitate the release of a high molecular weight cfeDNA from BF into the culture medium. In our studies AH was performed on Day 4. For better passage of embryonic DNA, some researchers have tried to use zona opening on Day 343. The most popular hypothesis is that nucleosome sized DNA fragments (~180–200 bp) are being released from cells as a result of apoptosis44. Another mechanism that could contribute cell free DNA with longer sized DNA fragments is necrosis40.The mechanism(s) underlying the release of embryonic DNA into the culture medium remain unclear and the origin of these DNA fragments is unknown44. We hypothesized that lower grade blastocysts, which tend to have a higher rate of apoptosis, will release a higher quantity of cfeDNA into the medium. Recently Ho et al. found that blastocyst morphology did not influence cfeDNA concentration in spent embryo culture medium or accuracy for aneuploidy screening31. Rule K et al., indicated that cfeDNA in blastocoel fluid positively correlates with a high embryonic morphology score, which suggests that the better the embryo morphology, the higher the cfeDNA concentration44.Previous studies have reported that the concentration of cfeDNA correlates with apoptotic eventset al.32, the single laser pulse was used at the junction of TE cells located far away from the inner cell mass. Extrachromosomal microDNAs7could also be a source of cfeDNA in spent culture medium. Production of these microDNAs is a part of normal cellular physiology and has been linked to transcriptional activity and mismatch repair. Extrachromosomal microDNAs vary in size from 60 to 2000 base pairs. They are abundant in all tissue types of mammalian cells, including sperm41. In contrast to accumulation of embryonic DNA in culture medium by apoptosis or necrosis, this mechanism would not necessarily depend on cell death7. Functional aspect of the DNA or RNA released by the developing preimplantation embryo is unknown and whether it is involved in cellular communication is still a subject of research. Extracellular vesicles (EVs) in blastocoel fluid and embryo culture medium as a transport vehicle may contain packed DNA to transmit information between the cells of trophectoderm and inner cell mass40. Similar cross-talk by means of EVs transferring miRNAs and other molecules  has been described among cells45. We have also recently reported that EVs appear to be able to traverse the zona pellucida when human embryos are cultured in vitro, which supports this hypothesis46.The results of our study, which represents the largest number of blastocysts tested using spent embryo culture medium combined with blastocoel fluid, demonstrated that the morphological grade of blastocysts does not affect the rate of informative NGS results from cfeDNA. The amount of amplified DNA from good quality blastocysts was slightly lower than that from moderate/low quality blastocysts, however the difference was not statistically significant. The concordance rate per sample for whole chromosome copy number between miPGT and TE biopsy samples Tables  and 3, fSince cell-free embryonic DNA consists of relatively short DNA fragments, the analysis of spent culture medium requires modifications of the standard WGA protocol. We hypothesized that for cfeDNA analysis, the SurePlex whole genome amplification kit can be used without the need for a cell lysis/extraction DNA step. Here we report that miPGT-1 and miPGT-2 samples Table  show a s47. A single laser pulse creates an opening in the zona pellucida at the cellular junction of trophectoderm cells located far away from the inner cell mass49. Therefore, our approach to use laser zona opening on Day 4 together with the laser (or microneedle) collapsing of blastocysts prior to TE biopsy for cfeDNA collection should have no negative impact on blastocyst development and does not require an additional laser (or microneedle) collapsing step before blastocyst vitrification as in current clinical practice49.Assisted hatching is generally performed using a laser pulse prior to blastocyst vitrification, resulting in artificial shrinkage of the blastocoel. This helps to prevent injury from intracellular ice formation and has been shown to improve clinical outcomes25. In our study, some WGA-miPGT DNA samples had chaotic or inconclusive results due to low or poor quality cfeDNA that led to noisy NGS profiles. The same issue was noted by Rubio et al.34. Noisy DNA profiles could also be attributed to maternal contamination by residual cumulus or corona cells. Therefore, if used in parallel with TE biopsy, NIPGT-A/miPGT may improve testing efficacy and accuracy by acting as a backup source of embryonic DNA in cases of inconclusive TE biopsy results; this would also obviate the need for re-biopsy.We have previously shown that collection of both spent embryo culture media and blastocoel fluid as one non-invasive sample increases the quantity and quality of cfeDNA for aneuploidy testing, compared with either spent embryo culture media only, or blastocoel fluid only25, as well as Li et al.35 and Huang et al.24, revealed that concordance rates for both embryo ploidy and chromosome copy number between NIPGT-A samples and whole blastocyst were higher than between TE biopsy and whole blastocyst. Conversely, the study of Ho et al.31 showed that the concordance rate for embryo ploidy between NIPGT-A samples and whole blastocyst was lower than between TE biopsy and whole blastocyst. In contrast, Jiao et al.32 reported similar concordance between NIPGT-A and TE biopsy samples and between NIGPT-A and the whole blastocyst. Considering results obtained for the non-invasive samples, Huang et al.24 also suggested that NIPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE biopsy PGT-A.To our knowledge, only few studies have been conducted comparing NIPGT-A/miPGT samples to corresponding TE biopsy samples and the whole blastocyst as a gold standard control. Results from our previous study26 or two sources of DNA  were analysed, reported in 201934.An embryo transfer performed on November 2017 of a euploid blastocyst tested by both TE biopsy and cfeDNA from a combined SEM + BF miPGT-A sample at the CReATe Fertility Centre, Toronto, resulted in a healthy boy, born at full term. To our knowledge, this is the first report where two sources of embryonic DNA (SEM + BF and corresponding TE biopsy) were analysed in parallel with clinical PGT-A. In this case, results of cfeDNA analysis from SEM + BF were concordant with the TE biopsy findings. Since that first birth, there are more recent reports on pregnancy outcomes where, either all three sources of DNA  were analyzed in a clinical setting on January 29th, 2018In summary, our results suggest that miPGT-A, utilizing combined blastocoel fluid and embryo culture medium has the potential to be superior to TE biopsy for PGT-A for routine clinical use in the future.This research received approval from the University of Toronto Research Ethics Board (IRB #30251). Informed consent was obtained for all patients included in this study. All experiments were performed in accordance with the relevant guidelines and regulations.50 with small modifications. In this system, grade 1 are fully expanded and grade 2 are expanding blastocysts. Good quality embryos are considered ≥BB  (n = 55), and moderate/low quality are <BB  (n = 47)  from a total of 145 fresh blastocysts and their corresponding trophectoderm (TE) biopsy samples were analyzed for this report. These samples were from 28 patients, aged 33 to 42 years (mean 36.8 +/−3.0 years) undergoing PGT-A cycles from October 2018 to January 2019 at the CReATe Fertility Centre, Toronto, Canada. Of these, 102 miPGT samples and their corresponding TE biopsy samples were used to assess the impact of static embryo morphology on efficacy and accuracy of miPGT-A. Morphology of these blastocysts was evaluated based on the SART scoring systemTo evaluate the reliability, efficacy and accuracy of the SurePlex  whole genome amplification (WGA) system and WGA with vs. without a cell-lysis step, two aliquots of combined spent embryo culture medium and blastocoel fluid and corresponding TE biopsies from the remaining 43 blastocysts were included.–oocyte complexes, the cumulus and corona radiata cells were removed by a combination of enzymatic and mechanical (pipetting) procedures. Mature metaphase II oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Following ICSI, each oocyte was placed in a culture dish containing 25 µl Sage1-Step medium with serum protein supplement  under oil and then placed into the incubator . Laser zona opening was performed on Day 4 to facilitate passage of embryonic cfeDNA into the culture media. Each laser zona-opened embryo was transferred on day 4to fresh 20 µlSage1-Step medium with serum protein supplement  and cultured until blastocyst formation.After collection of all cumulus51. In brief, when blastocyst full expansion was observed, the blastocysts were collapsed by a single laser pulse at the junction of TE cells  allowing release of blastocoel fluid (BF) into the media. After transferring the embryo to a biopsy dish, collection of the mixture of leaked BF together with embryo culture media (~5 µL) as one miPGT sample was done using sterile single use pipettes in sterile RNase–DNase-free PCR tubes and stored at −80 °C until analyzed. Control blank media samples were cultured under the same conditions and served as negative controls.Our minimally invasive and invasive preimplantation genetic testing (miPGT-A and PGT-A) workflow has been described previously51. All blastocysts were transferred to a biopsy dish containing 20 µL media under oil for biopsy. Four to six trophectoderm cells were biopsied from each blastocyst. The biopsied cells were placed immediately in RNase–DNase-free PCR tubes and stored at-80 °C until analyzed. Control blank media samples were collected as negative controls.A corresponding TE biopsy sample from each embryo was obtained using our standard protocolWhole genome amplification (WGA) was performed, according to manufacturer’s instructions, using the SurePlex WGA . The WGA starts with enzymatic lysis of biopsied cells or miPGT samples (5 ul SEM + BF) to release gDNA followed by a pre-amplification and amplification steps using degenerative primers for uniform random whole genome amplification.When a cell lysis step was not performed, WGA SurePlex protocol starts with direct pre-amplification of 10ul miPGT (SEM + BF) sample. WGA products  were quantified with the Qubit3.0-Fluorometerand their size distribution was assessed using 2100 BioAnalyzer .All samples were diluted to 0.2 ng/ul and a total of 1 ng from each sample and amplified using random primers. The kit contains 24 unique indexes added by amplification. Indexed DNA libraries were cleaned-up (AMPure XP beads 1:1 ratio) and normalized using magnetic beads. The normalized libraries were pooled, denatured, and sequenced using a MiSeq . Alignment and demultiplexing are done as part of the VeriSeq PGS protocol on MiSeq and CNV analysis and visualization were done using BlueFuse Multi (Illumina) software. Reporting was done using Hg39 reference with threshold for mosaicism of >30% and CNV changes >10 Mb.In our study, we analyzed the concordance rate for whole chromosome copy number abnormalities between miPGT samples and corresponding TE biopsy PGT-A samples. Results were statistically evaluated using Chi-squared and Fisher’s exact testing, with significance at p < 0.05.Supplementary Data.Supplementary Information.


P = 0.001 ~ 0.007) and displayed modest increase in burdens of rare deleterious SNVs (P = 0.047) and rare CNVs (P = 0.002 ~ 0.033) in genes related to BP1. We did not observe rare variants segregating in the pedigrees. These results suggest that small-to-moderate effect rare and common variants are more likely to contribute to BP1 risk in these extended pedigrees than a few large-effect rare variants.Current evidence from case/control studies indicates that genetic risk for psychiatric disorders derives primarily from numerous common variants, each with a small phenotypic impact. The literature describing apparent segregation of bipolar disorder (BP) in numerous multigenerational pedigrees suggests that, in such families, large-effect inherited variants might play a greater role. To identify roles of rare and common variants on BP, we conducted genetic analyses in 26 Colombia and Costa Rica pedigrees ascertained for bipolar disorder 1 (BP1), the most severe and heritable form of BP. In these pedigrees, we performed microarray SNP genotyping of 838 individuals and high-coverage whole-genome sequencing of 449 individuals. We compared polygenic risk scores (PRS), estimated using the latest BP1 genome-wide association study (GWAS) summary statistics, between BP1 individuals and related controls. We also evaluated whether BP1 individuals had a higher burden of rare deleterious single-nucleotide variants (SNVs) and rare copy number variants (CNVs) in a set of genes related to BP1. We found that compared with unaffected relatives, BP1 individuals had higher PRS estimated from BP1 GWAS statistics ( For BP, as for most other common disorders, SNP-based genome-wide association studies (GWAS) of large case/control samples have discovered many loci that contribute unequivocally to disease risk but that collectively explain only a small fraction of disease heritability. The most recent published BP GWAS, incorporating >20,000 cases and 30,000 controls, has reported 30 genome-wide significant SNP-associations and SNP-based heritability (h2snp) of 25% for BP12. The hypothesis that rare single-nucleotide variants (SNVs) and rare copy number variants (CNVs) could explain a substantial proportion of the “missing heritability” of complex traits3 has motivated the rapid growth of whole-exome sequencing and whole-genome sequencing (WGS) throughout biomedicine, including psychiatry. More than for other psychiatric disorders, however, sequencing efforts to identify variants with a high impact on BP risk have continued to focus on pedigrees7. This focus reflects published descriptions, over several decades, of numerous extended families in which BP is observed across multiple generations; as would be expected if these pedigrees were segregating a relatively high-penetrance susceptibility variant.Bipolar disorder (BP), consisting of episodes of mania and depression, has a heritability from twin studies estimated to be ~80%9, we aimed to systematically characterize the genetic contribution to BP disease risk in a series of such families through evaluation of variants across the allele frequency spectrum. If rare variants contribute to this risk it is expected that they would be enriched in this sample, which is, to our knowledge, the largest BP pedigree sample sequenced to date. In addition, because a wide range of evidence indicates considerable etiological heterogeneity between BP1 and milder forms of BP10, this study focused exclusively on families ascertained for multiple individuals with BP1, a strategy that we reasoned would reduce the impact of such heterogeneity. In a further effort to reduce heterogeneity, we limited the data set to pedigrees derived from two Latin American populations that are considered closely related genetic isolates; the province of Antioquia in CO and the Central Valley of CR11.Because the evidence in the literature regarding the apparent segregation of BP in extended pedigrees is mostly anecdotal12, and performed high-coverage WGS on 449 individuals, selected because identity by descent information provided by them would enable imputation of rare variants in the family members who were not sequenced. We analyzed these data to obtain high-quality genotypes for SNVs and CNVs. With this information, we sought to evaluate the impact of both common and rare variants on BP1, focusing on two major questions about its genetic etiology. First, we attempted to evaluate the overall genetic architecture of BP1 in these families by characterizing the genome-wide burden of both common and rare genetic variation. For common variants, we calculated the genome-wide burden with polygenic risk scores (PRS), using the latest BP1 GWAS summary statistics2 and compared the polygenic burden of risk alleles in affected cases and related controls. For rare variants, the genome-wide burden contrasted the burden of rare variants predicted to be deleterious in a set of genes related to BP1 between affected cases and related controls. Second, we attempted to identify rare deleterious variants segregating in the families, using a new method that we developed for this purpose.We collected microarray SNP data for 838 family members (as reported previously)17. The ascertainment and phenotyping strategy was previously reported18, and is briefly reviewed in the 19 questions related to mood or psychotic symptoms and were > 60 years of age. Individuals who were not diagnosed as BP1 or who were not considered as controls had unknown disease status. Written informed consent was obtained from all participants. Institutional Review Boards at participating institutions approved all study procedures. Using DNA extracted from whole blood we performed microarray genotyping using Illumina Omni 2.5 chips; as reported previously12 this procedure yielded data after QC for 838 individuals (206 BP1) with 2,026,257 SNPs  were sequenced including 143 BP1 , each ascertained for multiple individuals diagnosed with BP1  were either sequenced or imputed with high quality  Colombians (CLM) and ExAC33 Latinos (AMR). For CNVs, we extracted frequency information from the Database of Genomic Variants Gold Standard Variants34 for microarray CNVs and from Phase 3 of 1KG35 (AMR) for WGS CNVs. If a variant in our data set is present in an external source, we considered it rare if its MAF is < 1% in that source. For variants not present in any external source, we considered them rare if their MAF is < 10% in our data set where MAF is estimated from all sequenced individuals. Deleterious SNVs are stop-gain/loss, splice-site, and missense variants predicted damaging by PolyPhen-236.SNVs were mapped to UCSC knownGene37 with 57,180 LD-pruned SNPs. The reference populations were CEU (n = 112) and YRI (n = 113) from HapMap39, and 52 Native American samples from Central or South America who have virtually no European or African admixture40. We compared the proportion of European ancestry between BP1 individuals and controls using both a linear mixed model (LMM) based on lmekin function in coxme R package41 and a generalized linear mixed model (GLMM) based on GMMAT software42; both took into account relatedness of individuals using a kinship matrix calculated from theoretical kinship. In LMM, the dependent variable was the proportion of European ancestry, whereas the independent variable was BP1 status, and it was vice versa in GLMM.We generated estimates of admixture proportions for the 838 individuals with microarray data using ADMIXTURE43 and summary statistics from the Psychiatric Genomics Consortium (PGC) GWAS of BP12 and schizophrenia (SCZ)44 after excluding A/T and G/C SNVs and SNVs in the MHC region. Our WGS data were LD clumped, and we retained from the GWAS summary statistics the most significant SNV for each clump. We used LMM and GLMM to test association between BP1 status and PRS at each of five GWAS p value thresholds while considering relationships among individuals and global admixture proportions of European ancestry. We used logistic regression without considering relationships to estimate Nagelkerke R2 as it was not straightforward to estimate R2 using GLMM. We also did not include the admixture proportions when calculating R2 because we were interested in variance of BP1 explained only by PRS.We calculated PRS of our samples with WGS data using PRSice45 using the latest PGC BP1 GWAS summary statistics2 to identify cell-type specific promotor or enhancer regions in which BP1 heritability is enriched. Among the 10 cell-types groups tested, we observed enrichment of heritability for BP1 only in the central nervous system (CNS) group  transformed, and we compared the QN transformed residuals between BP1 individuals and controls using both LMM and GLMM while taking into account relationships among individuals and admixture proportions of European ancestry.p values to estimate the probability of having the observed segregation pattern or more extreme segregation patterns under the null hypothesis of random segregation. Our segregation statistic for a rare variant (Srare) is the sum of the number of BP1 individuals with a rare allele and the number of controls without the allele. To calculate the p value of Srare, we assume that the rare allele was introduced by certain founders in a family (denoted as Frv), enumerate many random inheritance vectors (IVs), and find the proportion of IVs that generate the same or larger Srare values , the number of tests, and type of multiple testing correction applied to each analysis in Supplementary Table 32  with every increase of 0.1 units of European ancestry, whereas we observed the opposite trend for Native American ancestry with OR of 0.67 (GLMM p = 0.0096), and African ancestry with OR of 0.61 (GLMM p = 0.026)  and at a GWAS p value threshold of 0.01 using GLMM 48, which share symptoms in common. Notably, in GWAS data from large BP case/control samples, the PRS estimated from the PGC’s SCZ GWAS results have explained up to 2.5% of BP variance47. We contrasted the mean PRS from SCZ GWAS in BP1 individuals and related controls in our pedigrees; to calculate PRS we used the GWAS summary statistics for SCZ44 . These SCZ PRS are not statistically associated with an increased risk of BP1, in the CO/CR pedigrees, at any of the GWAS p value thresholds that we examined . We also tested the burden of rare deleterious SNVs in genes defined by each of the three sources for the gene-set and observed a higher burden only in genes from the CNS cell type region (p = 0.040), but not in genes near PGC BP1 GWAS associations (p = 0.648) or in genes near linkage peaks (p = 0.399). This is expected as a majority of genes in our gene-set came from the CNS cell type region.We first identified 8,757 genes related to BP1 using the three sources of information based on the CNS cell type region where BP1 heritability is enriched, regions near PGC BP1 GWAS signals, and regions near linkage peaks from these pedigrees (see Methods). We then identified rare SNVs in those genes using both an external source of allele frequency and allele frequency observed in the CO/CR families (see Methods). We identified 25,072 rare predicted-deleterious SNVs in 8,237 of the 8,757 genes in our gene-set. For each individual, we computed the mean genome-wide burden of these SNVs, then compared these means between BP1 individuals and related controls, whereas taking into account the proportion of European ancestry in each individual, and the mean genome-wide burden of all rare SNVs in the gene-set. We observed that the mean burden of the rare deleterious SNVs was higher in BP1 individuals than in controls . For CNVs from WGS , we also observed an increased burden of genes in the BP1 gene-set affected by rare CNVs for BP1 individuals  when restricting our analysis to the subset of CNVs covered by a minimum of 10 SNPs on microarray , thus demonstrating a consistent increase in gene count burden using different methods of detection. We did not, however, observe a difference in the average number of all rare CNVs between BP1 individuals and controls (p = 0.67 for microarray CNVs and p = 0.45 for WGS CNVs using GLMM).We also performed an analysis of genome-wide burden using rare CNVs detected from microarray and WGS. Our CNV burden score was calculated as the number of genes in our BP1 gene-set intersected by rare CNVs, and similar to the burden analysis of rare deleterious SNVs, we measured the enrichment of CNV burden score using LMM and GLMM, accounting for factors known to affect global measures of CNV burden (see Methods). For CNVs from microarray , BP1 individuals had a higher CNV burden score than controls , which contained one rare missense variant . This variant did not appear in the Colombian samples within 1000 Genomes and was very rare in the Latino samples of ExAC (MAF of 0.04%). It was enriched in the CO/CR pedigrees as MAF in all 449 sequenced individuals was 0.44% accounting for relatedness49 (11× over ExAC) and 1.89%, not accounting for relatedness (47× over ExAC). GOLPH3 was the top gene in the CNV segregation results (p = 7.89e-4), with a single rare CNV  appearing only in family CO27 (MAF of 0.22% accounting for relatedness and 0.78% not accounting for relatedness). Neither of the above two rare variants segregated perfectly with BP1 status in the pedigrees in which they were present  by comparing the burden of those variants carried by affected individuals and that by related controls. We chose this set of genes because they corresponded to regions where BP1 heritability was enriched and where BP1 GWAS hits and our linkage peaks resided. This approach assumes that genes affected by rare variants overlap with those affected by common variants, which was observed in other studies such as human heightp values for rare variant segregation, because existing methods60 are not scalable to our large pedigrees and also make the simplifying assumption that only one founder has introduced a given rare variant into the pedigree. Our method relies on accurate imputation of rare variants, achieved using a family imputation approach that achieves a higher call rate and accuracy for rare alleles than population-based imputation approaches23. Our method worked well, from a technical standpoint but did not detect rare variants with strong evidence of segregation in our pedigrees after correction for multiple testing. One reason may be that there are 320 individuals who could be designated as either BP1 or controls, in the total sample of individuals (782) who are either sequenced or imputed well. If phenotypic status was more definite in a higher proportion of these individuals it could have added substantially to the power to detect such associations.We attempted to discover the specific loci and variants responsible for increased rare variant burden for BP1 in our data set. We used a new statistical approach that we developed to calculate In conclusion, results of our study point to the polygenic genetic architecture of BP1 in a well-characterized and large series of extended pedigrees, reflecting the action of a combination of many common and possibly rare variants (including both SNVs and CNVs) with small or moderate effect sizes. Rare variants with relatively large effect may contribute substantially to genetic risk of BP1 in the pedigrees although identifying associations to those rare variants is likely to require larger samples than were available in the current study. Identifying these variants may also require advances in our ability to recognize functionally important variation in non-coding parts of the genome. In addition, unlike in BP case/control samples, common SCZ risk alleles appear to contribute less than the weak effect of common variants we observed for BP1 in these families. Finally, although our new method makes it feasible to rigorously evaluate rare variant segregation in large pedigrees, our inability to identify BP1-associated coding variants suggests that non-coding variants may play an important role in BP1 risk in these pedigrees.Supplemental InformationSupplementary Table 6


To define the aerosol and droplet risks associated with endonasal drillingand to identify mitigation strategies.Simulation series with fluorescent 3-dimensional (3D) printed sinonasalmodels and deidentified cadaveric heads.Dedicated surgical laboratory.Cadaveric specimens irrigated with fluorescent tracer and fluorescent3D-printed models were drilled. A cascade impactor was used to collectaerosols and small droplets of various aerodynamic diameters under 15 µm.Large droplet generation was measured by evaluating the field forfluorescent debris. Aerosol plumes through the nares were generated vianebulizer, and mitigation measures, including suction and SPIWay devices,nasal sheaths, were evaluated regarding reduction of aerosol escape from thenose.The drilling of cadaveric specimens without flexible suction generatedaerosols ≤3.30 µm, and drilling of 3D sinonasal models consistently producedaerosols ≤14.1 µm. Mitigation with SPIWay or diameter-restricted SPIWayproduced same results. There was minimal field contamination in thecadaveric models, 0% to 2.77% field tarp area, regardless of drill burr typeor drilling location; cutting burr drilling without suction in the 3D modelyielded the worst contamination field (36.1%), followed by coarse diamonddrilling without suction (19.4%). The simple placement of a flexible suctioninstrument in the nasal cavity or nasopharynx led to complete elimination ofall aerosols ≤14.1 µm, as evaluated by a cascade impactor positionedimmediately at the nares.Given the findings regarding aerosol risk reduction, we strongly recommendthat physicians use a suction instrument in the nasal cavity or nasopharynxduring endonasal surgery in the COVID-19 era. In addition, particles between 10 and 20 µm settle morereadily, and particles >20 µm have a ballistic trajectory.3 In this study, aerosols are defined as particles with an aerodynamic diameter of≤10 µm, whereas droplets are defined with diameters >10 µm. This is in accord withdifferences in fluid dynamics between the 2 groups regarding suspension time anddeposition in different airway regions.3 Given that endoscopic drilling is a fundamental tool in rhinology and skull basesurgery, it is important to understand any associated aerosolization risks. Recently,Workman et al4 identified endonasal drilling as the greatest risk of aerosol generation using anoptical particle sizer. However, there are limited data in the literature regardingaerosolization risk with otolaryngology procedures and interventions that mitigateaerosol risk. Our project aims to define the aerosol and droplet risks associated withendonasal drilling using a cascade impactor. This study evaluates the aerosol dispersionfrom endonasal drilling and explores potential mitigation measures for aerosol anddroplet spread.The risks associated with endonasal surgery and transmission of severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2) remain unclear. Several modes of SARS-CoV-2transmission have been proposed, including direct contact, droplets, andaerosols.Suppl. Video S1 in the online version of the article). The fieldcontamination survey addresses large droplet risk while the cascade impactor trialsassess aerosol and small droplet risk (<15 µm) associated with endonasaldrilling. The simulations of aerosol dynamics highlight how various mitigationmeasures, including suction use and nasal aperture reduction, can affect aerosolescape through the nares.Overall, there are 3 main components of the study: (1) field contamination surveyassessing the distribution of fluorescent contamination on the surgical field and onthe provider’s personal protective equipment (study of large visible droplets), (2)simulation of sinonasal aerosol dynamics with nebulized vitamin B2 solution andgross visualization of mitigation measures, and (3) cascade impactor studies tospecifically record the presence of small droplet and aerosol particles generatedunder various simulation scenarios and after application of mitigation measures  of 2.8 µm.7 A combination of fluorescent3-dimensional (3D) printed sinonasal models created from Formlabs white resin(Formlabs) and deidentified cadaveric heads was used for the endonasal surgerysimulations. We chose to use models and specimens in which there was a partialseptectomy defect in order to best simulate skull base surgery, in whichendonasal drilling is most often performed in the setting of a wide surgicalcavity. SPIWay nasal sheaths 8 were tested as potential mitigation methods. The SPIWay is an endonasalsheath used to reduce mucosal trauma during endoscopic procedures and to avoidcontamination of scope lenses by surrounding debris.This study was conducted with approval from the University of PittsburghCommittee for Oversight of Research and Clinical Training Involving Decedents(CORID #888). Vitamin B2 (riboflavin) was used as the fluorescent tracer for allsimulation trials. Both 0.05-g/L and 1-g/L  vitamin B2solutions were prepared for use as drilling irrigation as well as for aerosolgeneration via an AeroEclipse II Breath Actuated Nebulizer (BAN) .10 This was used to determine the aerodynamic sizedistribution present with each simulation scenario.A Next Generation Impactor  was used to collect aerosoland small droplet particles based on specific aerodynamic sizes as they exitedfrom the nares.The specimens, 3D models with an overlying mask or cadaver heads, were placedat the edge of a black tarp, which was labeled with 6-inch increment markersusing orange tape  with the distal tip projecting into the nasopharynxas confirmed with an endoscope. Aerosols were generated using a BANnebulizer with a 1-g/L vitamin B2 solution. Under room light conditions, theaerosol plumes could be visualized exiting the nares; this was accentuatedunder UV blacklight. Different variables were studied to evaluate whetherthe aerosol plumes could be reduced or eliminated altogether: presence ofsuction, location of suction tip , type ofsuction (rigid vs flexible), finger occlusion on rigid suction channel, useof SPIWay nasal sheath,10 The NGI impactor has 8 sequential stages for collecting aerosols ofdifferent aerodynamic diameter sizes based on inertial impaction.Aerodynamic diameter relates to the settling properties of the aerosol andindicates that the aerosol has equivalent properties to a water densityaerosol of that size. It is clinically relevant in that it reflects where aparticle is most likely to collect in the airway. Smaller particles willhave a lower tendency to be collected by impaction while larger particleswill be impacted against a solid surface (filter stage).11 Aerosol deposition is influenced by multiple aerosol characteristics,including density and shape.12 The D50 refers to the cutoff diameter where collectionefficiency of the cascade impactor is 50%.12 Unlike optical particle sizers, impactors provide a directmeasurement of aerodynamic particle size11 based on the momentum of individual particles (product of density andvelocity).The Next Generation Impactor (NGI) was used to determine the presence of finedroplets and particles for each simulation scenario.50, µm) based on the flow rate: stage 1,14.1; stage 2, 8.61; stage 3, 5.39; stage 4, 3.30; stage 5, 2.08; stage 6,1.36; and stage 7, 0.98. Stage 8 is the micro-orifice collector (MOC)filter—smallest particles without a defined D50 value.13 Each filter stage has a corresponding capture chamber. Here, thesewere lined with aluminum foil to prevent accumulation of fluorescentmaterial between experiments. UV light exposed the presence of any filteredparticulate matter on the foil pieces. Between each trial, the impactorinlet nozzle, filter tray, and individual chambers were cleaned withdistilled water and new aluminum foils were placed into collection chambers.Nebulized vitamin B2 solution (1 g/L) was used as a positive control to testthe NGI . Aerosolplumes disappeared when a flexible suction (14 Fr) was placed inside the nasalcavity at either shallow or deep depths or parked in the nasopharynx. Placementof the flexible suction at the columella was effective as most of the aerosolplumes were still drawn to the suction opening; however, plume escape was notedif the suction tip was moved just off midline favoring 1 naris. Similarly, withuse of a rigid suction (9 Fr) and thumb on the relief hole, the aerosol plumesdisappeared whenever the tip was inside the nasal cavity or nasopharynx or evendirectly at the columella. Without occluding the relief hole, the rigid suctionstill eliminated all visible aerosol plumes when placed inside the nasal cavityor nasopharynx but failed when suction tip was placed just outside thenares.Nebulized vitamin B2 produced significant aerosol plumes emanating from the nares(see Suppl. Video S1 in the online version of the article). However,with any manipulation of the instruments in the lateral or vertical axis, theplumes would escape around the SPIWay itself. No combination of rubber bandapplication was found to completely prevent aerosol plume escape, nor waspushing the SPIWay outer flanges immediately inside the nares. When a flexiblesuction was parked underneath a nonconstricted SPIWay in the nasopharynx, theaerosol plumes disappeared. The SPIWay device effectively secured the flexiblesuction tip in the posterior nasal cavity without compromising full expansion ofthe SPIWay device.Directing nebulized vitamin B2 plumes into a SPIWay did not show any leak throughthe material of the SPIWay itself. When SPIWays were positioned in the nasalcavity, there were visible aerosol plumes still coming out of the nares but in afocused, nondispersed manner due to the SPIWay outer flange. When orthodonticelastics were applied to narrow the SPIWay lumen, plume volume decreased but wasnot eliminated. This remained the case when instruments were introduced afterconstricting the SPIWay opening. Placement of a rigid endoscope and anonfunctioning rigid suction in the left naris and a drill in the right narisalmost completely blocked the banded SPIWays and produced a significant decreasein aerosol plume escape (see Nebulized vitamin B2 was applied to the fluorescent blue 3D models (viaretrograde intubation into nasopharynx), and the models were drilled with coarsediamond and cutting burrs during nebulization. With both drill bits, filters 1to 8 were positive , whereas the cutting burr produced a greater distribution of largeraerosols . Reduction of the cadaveric anterior nasalaperture using banded SPIWay sheaths still yielded detectable aerosols under 3.30µm. However, use of a flexible suction parked inside the naris, outside the SPIWaydevice, resulted in elimination of all detectable aerosols under 14.1 µm (filters1-8).This study aimed to accurately define aerosol risk with endonasal drilling based onaerodynamic diameter and explore mitigation strategies. In the cadaver specimens,aerosols with DThis study proves that placing a suction inside the nasal cavity or nasopharynxsignificantly reduces aerosols. This was demonstrated both visually with sinonasalfluid dynamics simulations and objectively with the cascade impactor trials. Alltrials in which a suction was parked inside of the nose demonstrated only negativeresults. This was consistent with every condition that was aerosol generating,including the cadaver endonasal drilling trials, the 3D model drilling trials, andthe nebulized vitamin B2 fluid dynamics simulations. The nebulized vitamin B2 trialsprovided a “worst-case scenario” in which aerosols were constantly produced, andeven in these conditions, the suction resulted in a completely negative result withall 8 NGI filters empty.Regardless of suction type, rigid or flexible, or depth of suction tip placement inthe nasal cavity or nasopharynx, aerosol particles across a range of aerodynamicdiameters were successfully removed from the surgical field and were prevented fromexiting the nares. If the suction tip was placed outside of the nasal cavitycentered at the columella, this still resulted in aerosol mitigation; however, evenslight mispositioning of the suction tip off to one side caused aerosol escape fromthe contralateral nostril. Based on the findings of this study, we stronglyrecommend that physicians performing endonasal surgery place a suction instrument inthe nasal cavity or nasopharynx to mitigate aerosol risk. Provided that the suctiontip is maintained open during the case and not occluded by tissue, the aerosolplumes will be directed toward the suction tip rather than exiting the nares.Reducing aerosol production may lead to safer conditions for surgeons and operatingroom staff when treating a patient with active SARS-CoV-2 infection. Our currentpractice has been adapted to include a flexible tracheal suction catheter trappedbetween the SPIWay device and nasal cavity, therefore held outside the path ofinstrument passage, in addition to a standard 2-surgeon, 4-hands technique withrigid suction at all times during drilling.In terms of droplet risk, the amount of debris noted in the field after drillingvaried depending on whether a coarse diamond or cutting burr was used. The 3D modeltrials provided a fluorescent tracer to easily visualize particles on the field andin the air  direct evaluation of a particle’s aerodynamicdiameter using physical measurements of size and momentum; (2) consideration of eachparticle’s shape, size, and density, which all influence aerodynamic diameter; (3)minimization of detection error (aerosol loss to surroundings in a closed system orlack of proper sampling when using indirect measures in an open setting); (4)reliable measurement of particle concentration; and (5) ability to link aerosols tothe specimen via a specific tracer.Several groups have previously studied the aerosol and droplet risks associated withendonasal surgeryOur experimental method presents several advantages. The cascade impactor allowed fordirect determination of aerosol aerodynamic size, which has not been addressed byany study in the otolaryngology literature. Even though individual particle size,shape, and density were unknown, the impactor’s inertial filtering system enabled usto determine approximate aerodynamic diameters based on known device calibrations at15.0 L/min. We attempted to mimic a closed system by ensuring that the impactornozzle was almost flush against the nares; this positioning allowed for maximizingdetection of all generated aerosols and limiting aerosol escape into thesurroundings. A high flow rate (15.0 L/min) ensured that even aerosols suspendedinside of the nasal cavity or nasopharynx were likely to be detected by theimpactor. Unlike other studies, we were able to collect the aerosol particles basedon aerodynamic size so that it could be used for biochemical analysis if needed. Theaddition of vitamin B2 as a fluorescent tracer allowed us to link the aerosols tothe original specimen rather than to the provider or the surrounding environment.Our study presents the novel use of vitamin B2 as a fluorescent tracer in aerosolcontamination studies and the utility of an impactor in aerosol contamination modelswith an emphasis on filtering aerosol particles produced in otolaryngologyprocedures.There are a few limitations to our study. Although it was easy to distinguish thepresence or absence of aerosols with vitamin B2 fluorescence, we did not use aspectrophotometer to precisely quantify the relative fluorescence of each impactorstage, which showed visible collection of particles. This study does not addresswhether the captured aerosols were biologically active with the capacity to causeinfection. We are working on further trials to determine whether aerosol particlesisolated from procedures in SARS-CoV-2–positive patients have infectious potential.Despite the unclear baseline risk, we urge physicians performing endonasal surgeryto use endonasal suction placement as it is a ubiquitous and effective tool tomitigate aerosol risk.Our current practice for skull base cases has been adapted to place a flexibletracheal suction catheter in the nasopharynx; the suction tubing is situated betweenthe SPIWay sleeve and nasal cavity surface, and therefore held outside the path ofinstrument passage. In addition, a handheld suction is used during each case sobetween the 2 suctions, there is always an active suction in the nasopharynx. Forendoscopic sinus surgery cases when the SPIWay sleeve is not routinely used, theflexible suction tip is parked just inside of the nostril and held in place withtape. Some surgeons at our institution choose to park the suction in the nasopharynxor deeper in the nasal cavity for endoscopic sinus surgery cases. Using a parkedsuction does require having an extra suction set up but has not presented anydifficulties when operating, including in patients with a bleeding tendency ordiseased sinuses. The flexible suction can be maintained for the duration of eachcase without problems. The addition of a parked flexible suction to the operativesetup is quick, inexpensive, and reliable. We hope this becomes the acceptedstandard for cases involving endonasal instrumentation, especially withdrilling.This study evaluates aerosol production during endonasal drilling based onaerodynamic diameter and explores mitigation strategies. Across a range of drillingscenarios, aerosols under 15 µm were consistently generated. The simple placement ofa suction instrument in the nasal cavity or nasopharynx led to complete eliminationof all detectable aerosols, as evaluated by a cascade impactor positionedimmediately at the nares. Given the findings in our study, we strongly recommendthat physicians use a suction instrument in the nasal cavity or nasopharynx duringendonasal surgery to mitigate aerosol risk.


Approximately 90% of human tumors use telomerase to synthesize de novo telomere repeats and obtain telomere-dependent cellular immortality. Paradigm shifting data indicates that hTERT alternative splicing, in addition to transcription, plays an important role in the regulation of active telomerase in cells. Our group and others are pursuing the basic science studies to progress this emerging area of telomerase biology. Recent evidence demonstrates that switching splicing of hTERT from the telomerase activity producing full-length hTERT isoform to alternatively spliced, non-coding isoforms may be a novel telomerase inhibition strategy to prevent cancer growth and survival. Thus, the goals of this review are to detail the general roles of telomerase in cancer development, explore the emerging regulatory mechanisms of alternative RNA splicing of the hTERT gene in various somatic and cancer cell types, define the known and potential roles of hTERT splice isoforms in cancer cell biology, and provide insight into new treatment strategies targeting hTERT in telomerase-positive cancers.Alternative RNA splicing impacts the majority (>90%) of eukaryotic multi-exon genes, expanding the coding capacity and regulating the abundance of gene isoforms. Telomerase ( Nearly 40% of adults in the United States are expected to be diagnosed with cancer in their lifespan . In 2020n DNA sequences located at the ends of linear chromosomes ) that contains four specific domains necessary for producing the catalytically active telomerase enzyme: the telomerase N-terminal (TEN)-domain in exon 1, the telomerase RNA-binding domain (TRBD) spanning exons 2 and 3, a reverse transcriptase (RT)-domain spanning exons 4 through 13, and a c-terminal extension (CTE)-domain spanning exons 14 through 16. The RT-domain specifically provides the active sites necessary to catalyze telomerase enzyme activity, is involved in the proper assembly and stabilization of the hTERT/hTERC complex, and helps to facilitate telomerase enzyme processivity [hTERT gene is observed in telomerase-positive cells, including germline cells, embryonic and induced pluripotent stem cells, and in transit amplifying adult progenitor cells, but is thought to be silenced in fully differentiated somatic cells and other mature cell populations  that are telomerase negative or express very low levels of the active telomerase enzyme [hTERT gene is expressed in telomerase-positive immortal cell lines, yet remains absent or alternatively spliced to non-catalytic isoforms in telomerase-negative cancer cells that rely on ALT mechanisms [ 5p15.33 . Splicinessivity ,59,60. Ee enzyme . Similarchanisms . hTERT was transcriptionally silenced during the cellular differentiation process in somatic cells. Shortly after the discovery of hTERT in 1997, Meyerson et al. hypothesized that hTERT might also be constitutively expressed and subject to post-transcriptional modifications, such as alternative splicing [hTERT variants (bands). The longest variant detected contains exons 5–9 and is assumed to be the potentially FL, catalytically active hTERT transcript containing all 16 exons required for telomerase enzyme activity. In addition, three alternatively spliced hTERT variants were detected that are incapable of producing active telomerase [hTERT gene include: (1) minus alpha (-α)—a deletion of the first 36 nucleotides due to an in-frame cryptic splice site at the 5′ end of exon 6 that serves as a dominant-negative inhibitor of telomerase, resulting in the progressive shortening of telomeres when overexpressed in cells, and subsequently, cellular senescence or apoptotic death [The historical paradigm in the telomere/telomerase field was that splicing . Within lomerase . The thric death ; (2) minic death ; and (3)ic death . hTERT splice variants to demonstrate whether or not the hTERT gene is transcriptionally repressed during development or undergoes alternative splicing as a mechanism to tightly regulate telomerase activity [hTERC, and hTERT in the fetal liver, heart, and kidney at various stages of human fetal development, Ulaner and colleagues observed three distinct tissue-specific expression patterns. Although hTERC gene expression was observed at near constant levels in each tissue during development, expression patterns of the FL and alternatively spliced hTERT variants changed throughout development in each tissue. Likewise, telomerase activity tightly correlated with potential FL (exons 5–6–7–8–9 containing) hTERT variant expression [hTERT splicing away from the potential FL (+α+β), catalytically active variant towards alternatively spliced (-α and/or -β), catalytically inactive variants that lack portions of the RT-domain occurs differently across tissues and at various stages of cellular development. These findings further corroborate the emergent hypothesis that the hTERT gene is regulated post-transcriptionally, and that alternative RNA splicing of the hTERT gene serves as a mechanism to tightly control telomerase enzyme activity and regulate telomere length [Ulaner et al., using the same exon 5–9 RT-PCR assay as Kilian et al. , qualitaactivity . Measuripression . More spe length ,66.hTERT in somatic cells and fibroblast cell lines and did not observe evidence of hTERT expression, further supporting the idea that hTERT was transcriptionally silenced. However, Hrdlicková et al. used primers in exons located at the 5′ and 3′ end of the hTERT gene and identified several additional alternatively spliced variants in telomerase-negative fibroblasts and adult tissues [hTERT expression levels similarly to those observed in cancer cells, but the resulting transcripts never contained critical exons required for the reverse transcriptase activity of telomerase [hTERT gene as this would be an energetically wasteful process since stem cells and dividing cells may need small amounts of telomerase to slow telomere shortening during proliferation needed for tissue repair. Instead, somatic cells transcribe a low level of hTERT (one to 10 RNA transcripts per cell) and alternatively splice it to non-catalytically active forms. Additionally, since cells only need to maintain the shortest telomeres in a cell to continue to proliferate, only a few molecules of active telomerase are needed to perform this task, and FL hTERT can be kept at extremely low levels. Building upon this idea, a paradigm shifting idea has emerged to suggest that when cells transform and progress to a cancerous state they also may not need to increase transcription as long as hTERT is spliced to the FL isoform and can generate enough active telomerase to maintain the shortest telomeres in the dividing portion of tumor cells. Indeed, our laboratory has shown that cancer cells use a post-transcriptionally based mechanism to generate telomerase activity from very low levels of hTERT transcripts. These landmark studies paved the way for a new line of thinking that hTERT alternative RNA splicing can be targeted as a potential cancer therapeutic. Understanding the regulation of hTERT alternative RNA splicing is necessary to develop a new and more potent telomerase inhibitor to stop the long-term proliferation of malignant cells.Subsequent studies used the primers spanning exons 5–9 of  tissues . Additiolomerase . As suchcis regulatory sequences or trans-acting splicing proteins, modifications to Pol II transcriptional rates, and changes in the expression of and/or post-transcriptional modifications of trans-acting splicing proteins [hTERT is likely subjected to either dysregulation or the re-emergence of a developmentally regulated splicing program to maintain telomere lengths during cancer development. Cancer is characterized by aberrant regulation of alternative splicing . Changesproteins . As a reproteins . Due to hTERT variants [hTERT gene have thus far been identified [hTERT variants combine for >90% of the steady state transcripts observed in cancer cells, whereas the -α and -α-β variants typically account for <5% of the remaining steady state transcripts [Kilian et al. first demonstrated that cancer cells display an incredible diversity of alternatively spliced variants . Includientified . While tnscripts ,72,73. hTERT are in fact the most abundant in human cancers, we used publicly available RNA-sequencing data from The Cancer Genome Atlas (TCGA) splice variant database (TSVdb), a web tool which enables the acquisition of hTERT splice variant abundance across 33 tumors of which 31 cancer types had data available for hTERT [hTERT transcripts, results which are consistent with previous reports [hTERT transcripts quantified by the TSVdb as FL averages ~22% and ranges from ~1% to ~54% across all tumor samples , is the most common event. Thus, understanding the regulation of this region is critically important to elucidating telomerase regulation as the splicing mechanisms involved might also be amenable to manipulation.To determine whether or not alternative splicing events within the exons 5–9 region of or hTERT . The TSV reports . Further samples . Five ader cells  , which precludes detection using standard RT and PCR conditions, and thus limits the absolute quantification of each variant from RNA-sequencing analysis [hTERT splicing variant isoforms for the accurate interpretation of patient data. For instance, Sayed et al. used Pacific Biosciences long-read sequencing with a targeted library preparation approach to point out differences in TERT splicing between mice and men [hTERT, such as employing tiled probes along the span of hTERT exons and introns to enrich cDNA libraries for hTERT products that would allow for cataloging of hTERT isoforms with long-read sequencing technologies  [hTERT through the use of tiling probes followed by direct long-read RNA-sequencing [hTERT alternative splicing, our laboratory and others have begun to dissect the regulatory mechanisms of hTERT, thus leading the way in the development of potential therapeutic approaches for targeting telomerase. It is important to note that several limitations exist in measuring nscripts . Furtheranalysis . Therefo and men . Moving ciences) . An altequencing . Beyond hTERT in cancer suggests a need for malignant cells to tightly regulate and produce the “right amount” of telomerase to maintain telomere lengths, but not to elongate telomere lengths so as to confer a negative impact on cellular survival  [hTERT alternative splicing of the -β variant offers the possibility of developing novel anticancer agents that reduce telomerase activity by shunting transcripts from the FL hTERT telomerase activity coding isoform to catalytically dead variants and attenuate cancer growth and survival by shortening telomeres. Although hTERT gene expression has been known to be regulated by alternative RNA splicing for nearly 25 years [hTERT as a potential therapeutic target during cellular development and in cancer has only recently been investigated [hTERT [hTERT with ASO technologies, a deeper biochemical understanding of the molecular mechanisms, specifically the cis- and trans-acting factors, involved in the regulation of alternative RNA splicing is required. Alternative RNA splicing of diation) ,82. As s25 years , the rolstigated ,83. The d [hTERT . To effehTERT alternative splicing used a minigene construct spanning exons 5–10 [hTERT [Initial attempts to understand the mechanistic machinery involved in the regulation of ons 5–10 . Wong anons 5–10 . The B6,ons 5–10 ,86. Furt0 [hTERT . hTERT [cis-elements, and their potential for intramolecular interactions in the tight regulation of FL hTERT and the production of the alternatively spliced -β variant. Results from these investigations further indicate that hTERT may not follow the canonical rules involved in the regulation of alternative splicing. Likewise, they were the first to demonstrate that the intronic cis-elements B6, DR6, and DR8 may have recently evolved as an additional regulatory region to control the abundance of FL hTERT via post-transcriptional alternative RNA splicing to “fine-tune” telomerase enzyme activity, tightly regulate telomere lengths, and limit cellular replicative capacity as an advanced tumor suppressor mechanism in large, long-lived mammals [Follow-up minigene experiments containing the B6, DR6, and DR8 intronic sequences demonstrate that removing B6 from the minigene construct fails to produce significant amounts of the -β variant, whereas B6 alone (DR6 and DR8 removed) results in the enhanced skipping of exons 7 and 8 and the increased expression of the -β variant compared to the “full” minigene . While DhTERT . In a suhTERT . These f mammals ,87. cis-elements and their capacity to regulate the production of the FL hTERT and -β variants were initiated by the use of an antisense 2-O-methyl oligonucleotide against DR8 (hTERT splicing from ~40% FL and ~50% -β to predominantly -β (<15% FL and >70% -β) [cis-elements and potential trans-acting RNA binding factors across hTERT pre-mRNA. Ludlow et al. targeted the endogenous hTERT gene within two non-small cell lung cancer (NSCLC) lines – H1299 and Calu6 – using a 2’-deoxy-2’-fluoro-arabinonucleic acid ASO that recognizes the same sequence within DR8 as Wong et al. [hTERT splicing from the FL to -β variant, whereas no changes were observed in the Calu6 line [cis-element DR8 may be involved in the recruitment of or act as a binding site for trans-acting factors that regulate hTERT splicing, the capacity to manipulate splicing choice through DR8 occurs is cell type specific. Thus, the use of splice switching ASOs on various cell lines of a particular tumor type supports the notion that the splicing code is cell specific and may necessitate the development of cell specific therapeutic strategies and/or the identification of a more universal cis-element to target with ASOs. Attempts to further understand the functional role of these inst DR8 . As a re>70% -β) . Numeroug et al. ,85. Intelu6 line . These fhTERT have yet to be identified and thus more research is needed to determine if a universal element exists controlling hTERT splicing. Indeed, other groups have attempted to use splice switching ASOs beyond DR8 to switch the splicing of hTERT towards either catalytically dead, dominant-negative isoforms or isoforms that undergo nonsense-mediated decay  screen to determine the impact of 516 g choice . Transie cancers .hTERT FL splicing in the H1299 NSCLC line [hTERT pre-mRNA, thereby shifting hTERT splicing towards production of the -β variant, decreasing telomerase enzyme activity, and progressively shortening telomere lengths in cells [trans-acting RNA binding protein that directly binds to the DR8 region of hTERT pre-mRNA to promote cancer cell immortality. NOVA1 is an RNA-binding protein important for neuronal development . AlthougCLC line . Therefoin cells . These fhTERT pre-mRNA [hTERT splicing from FL primarily to -β, decreases telomerase, and progressively shortens telomere lengths in the NOVA1 positive NSCLC lines H1299 and H920 in vitro [hTERT splicing towards the -β variant and reduce telomerase enzyme activity. Using RNA pulldown assays, Sayed et al. further demonstrated that NOVA1 is both necessary and sufficient to recruit PTBP1 to DR8 and directs its binding directly downstream of the NOVA1 YCAY binding motifs. These observations indicate a NOVA1-PTBP1 splicing network that acts to promote FL hTERT, increase telomerase enzyme activity, and maintain telomere lengths in NSCLC [trans-acting RNA-binding factors , by limiting the number of cells that develop immortality. In the event of cancer diagnosis, ASOs aimed at switching the splicing of hTERT could be given concurrently as a telomerase inhibitor while patients receive first line therapies . In addition, delivery of similar anti-telomerase molecules as an adjuvant along with anti-metastatic therapeutics aimed at preventing tumor survival may act additively and/or synergistically. Such an approach would enable the application of primary anticancer therapies targeting cancer growth and survival while simultaneously providing an opportunity to shorten telomere lengths of cancerous cells. Similarly, telomerase inhibitors, hTERT splice inhibitors included, can be continued as a second line therapy to help prevent regrowth of remaining tumor cells during remission, thereby increasing rates of patient survival compared with first-line therapies alone [hTERT isoform ratios may also provide additional insights into the types of anti-telomerase therapies that could potentially be applied. Therefore, continued efforts are necessary to gain a more complete understanding of regulatory mechanisms involved in hTERT alternative RNA splicing regulation as a potential approach to developing novel anticancer therapies. Cancer is a major public health concern and understanding the regulation of es alone  , poor in vivo inhibition of the enzyme, and potential off target toxicities, including reduced platelet counts . Therefoin genes ,96.