In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells.

Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.

A Novel VACTERL Assessment Tool to Facilitate Counseling for Expectant Families.

INTRODUCTION: VACTERL is defined as 3 or more of the following congenital defects: vertebral, anorectal, cardiac, tracheoesophageal (TE), renal, and limb. The purpose of this study was to create an easy-to-use assessment tool to help providers counsel expecting families regarding the likelihood of additional anomalies and postnatal outcomes. METHODS: Employing the Kids' Inpatient Database from 2003-2016, neonates (<29 days old) with VACTERL were identified using ICD-9-CM and ICD-10-CM codes. For each unique combination of VACTERL, multivariable logistic regression was used to estimate inpatient mortality, and Poisson regression was used to estimate length-of-stay during the initial hospitalization. RESULTS: The assessment tool used in this study is available at https://choc-trauma.shinyapps.io/VACTERL. 1,886 of 11,813,782 (0.016%) neonates presented with VACTERL. 32% weighed <1,750 g, and 239 (12.7%) died prior to discharge. Associated with mortality were limb anomaly (1.8 [1.01-3.22], p < 0.05), prematurity (1.99 [1.14-3.47], p < 0.02), and weight <1,750 g (2.19 [1.25-3.82], p < 0.01). Median length-of-stay was 14 days (IQR: 7-32). Associated with increased length-of-stay were cardiac defect (1.47 [1.37-1.56], p < 0.001), vertebral anomaly (1.1 [1.05-1.14], p < 0.001), TE fistula (1.73 [1.66-1.81], p < 0.001), anorectal malformation (1.12 [1.07-1.16], p < 0.001), and weight <1,750 g (1.65 [1.57-1.73], p < 0.001). CONCLUSION: This novel assessment tool may help providers counsel families confronting a VACTERL diagnosis.

Pre-Existing Maternal Antibodies Cause Rapid Prenatal Rejection of Allotransplants in the Mouse Model of In Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative nonimmunosuppressive alternative to postnatal hematopoietic stem cell transplantation for the treatment of congenital hemoglobinopathies. Anti-HLA donor-specific Abs (DSA) are associated with a high incidence of graft rejection following postnatal hematopoietic stem cell transplantation. We determine if DSA present in the mother can similarly cause graft rejection in the fetus following IUHCT. Ten million C57BL/6 (B6, H2kb) bone marrow cells were transplanted in utero into gestational day 14 BALB/c (H2kd) fetuses. The pregnant BALB/c dams carrying these fetuses either had been previously sensitized to B6 Ag or were injected on gestational days 13-15 with serum from B6-sensitized BALB/c females. Maternal-fetal Ab transmission, Ab opsonization of donor cells, chimerism, and frequency of macrochimeric engraftment (chimerism >1%) were assessed by flow cytometry. Maternal IgG was transmitted to the fetus and rapidly opsonized donor cells following IUHCT. Donor cell rejection was observed as early as 4 h after IUHCT in B6-sensitized dams and 24 h after IUHCT in dams injected with B6-sensitized serum. Efficient opsonization was strongly correlated with decreased chimerism. No IUHCT recipients born to B6-sensitized dams or dams injected with B6-sensitized serum demonstrated macrochimeric engraftment at birth compared with 100% of IUHCT recipients born to naive dams or dams injected with naive serum (p < 0.001). In summary, maternal donor-specific IgG causes rapid, complete graft rejection in the fetus following IUHCT. When a third-party donor must be used for clinical IUHCT, the maternal serum should be screened for DSA to optimize the chance for successful engraftment.

Pediatric neurosurgeons' views regarding prenatal surgery for myelomeningocele and the management of hydrocephalus: a national survey.

OBJECTIVE: The Management of Myelomeningocele Study (MOMS) compared prenatal with postnatal surgery for myelomeningocele (MMC). The present study sought to determine how MOMS influenced the clinical recommendations of pediatric neurosurgeons, how surgeons' risk tolerance affected their views, how their views compare to those of their colleagues in other specialties, and how their management of hydrocephalus compares to the guidelines used in the MOMS trial. METHODS: A cross-sectional survey was sent to all 154 pediatric neurosurgeons in the American Society of Pediatric Neurosurgeons. The effect of surgeons' risk tolerance on opinions and counseling of prenatal closure was determined by using ordered logistic regression. RESULTS: Compared to postnatal closure, 71% of responding pediatric neurosurgeons viewed prenatal closure as either "very favorable" or "somewhat favorable," and 51% reported being more likely to recommend prenatal surgery in light of MOMS. Compared to pediatric surgeons, neonatologists, and maternal-fetal medicine specialists, pediatric neurosurgeons viewed prenatal MMC repair less favorably (p < 0.001). Responders who believed the surgical risks were high were less likely to view prenatal surgery favorably and were also less likely to recommend prenatal surgery (p < 0.001). The management of hydrocephalus was variable, with 60% of responders using endoscopic third ventriculostomy in addition to ventriculoperitoneal shunts. CONCLUSIONS: The majority of pediatric neurosurgeons have a favorable view of prenatal surgery for MMC following MOMS, although less so than in other specialties. The reported acceptability of surgical risks was strongly predictive of prenatal counseling. Variation in the management of hydrocephalus may impact outcomes following prenatal closure.

Enhanced in utero allogeneic engraftment in mice after mobilizing fetal HSCs by α4ß1/7 inhibition.

In utero hematopoietic cell transplantation (IUHCT) is a novel nonmyeloablative approach that results in donor-specific tolerance and mixed allogeneic chimerism. Clinical application is limited by low levels of donor cell engraftment. Competition from endogenous hematopoietic stem cells (HSCs) for limited "space" in fetal hematopoietic organs remains a significant barrier to successful IUHCT. AMD3100, a CXCR4 inhibitor, and firategrast, an α4ß1 and α4ß7 integrin inhibitor (α4ß1/7), have been shown to disrupt HSC retention in the postnatal hematopoietic niche. We hypothesized that maternal administration of AMD3100 and/or firategrast prior to IUHCT would mobilize endogenous HSCs from the fetal liver (FL) and result in preferential FL homing of donor HSCs and enhanced long-term engraftment following IUHCT in an allogeneic mouse model. We demonstrate that (1) both agents cross the placenta with rapidly detectable fetal serum concentrations following maternal administration; (2) firategrast treatment alone or with AMD3100 mobilizes endogenous HSCs from the FL and results in increased FL homing of donor HSCs following IUHCT; and (3) enhanced donor HSC homing following firategrast treatment translates into increased long-term multilineage donor cell engraftment. This approach highlights the potential of mobilization strategies to overcome barriers to successful engraftment and increase the clinical promise of IUHCT.

Racial Disparity among Youth Decedents of Legal Intervention Trauma.

BACKGROUND: Legal intervention trauma (LIT) is defined as injury due to any encounter with law enforcement. This study investigates associations between demographics, violent status, and law enforcement tactics among youth decedents of LIT. STUDY DESIGN: Decedents of LIT age 26 years or younger were identified using the CDC's National Violent Death Reporting System from 2003 to 2018. Decedents were classified as "violent" if they possessed a weapon, were committing a violent crime, or if law enforcement reported justified use of force. All others were classified as "nonviolent." Law enforcement tactics were stratified into "lethal" (firearm with standard ammunition) or "less lethal" (any other) force. Differences in the racial distribution across these classifications were assessed using chi-square tests of proportions. RESULTS: We identified 1,281 youth decedents of LIT; of which, 92.5% met violent criteria. Black youths were less likely than White youths to possess a weapon (71.6% vs 77.4%, p = 0.02) and were not more likely to be committing a violent crime (63.6% vs 60.4%, p = 0.27). They were, however, more likely than White youths to experience force reported as justified by law enforcement (89.9% vs 82.4%, p = 0.002) and to experience exclusively lethal force not preceded by less-lethal tactics (94.0% vs 88.7%, p = 0.001). Among the subset of 85 cases where law enforcement reported justified use of force despite the decedent not possessing a weapon or committing a violent crime, the precipitating event was more often a traffic stop for Black youths than for White youths (28.5% vs 6.66%, p = 0.02). CONCLUSIONS: These findings indicate a racial disparity among youth decedents of LIT.

Preexisting maternal immunity to AAV but not Cas9 impairs in utero gene editing in mice.

In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.

Fetal allotransplant recipients are resistant to graft-versus-host disease.

In utero hematopoietic cell transplantation (IUHCT) is an experimental treatment for congenital hemoglobinopathies, including Sickle cell disease and thalassemias. One of the principal advantages of IUHCT is the predisposition of the developing fetus toward immunologic tolerance. This allows for engraftment across immune barriers without immunosuppression and, potentially, decreased susceptibility to graft-versus-host disease (GVHD). We demonstrate fetal resistance to GVHD following T cell-replete allogeneic hematopoietic cell transplantation compared with the neonate. We show that this resistance is associated with elevated fetal serum interleukin-10 conducive to the induction of regulatory T cells (Tregs). Finally, we demonstrate that the adoptive transfer of Tregs from IUHCT recipients to neonates uniformly prevents GVHD, recapitulating the predisposition to tolerance observed after fetal allotransplantation. These findings demonstrate fetal resistance to GVHD following hematopoietic cell transplantation and elucidate Tregs as important contributors.

COVID-19 Pandemic Significantly Decreases Acute Surgical Complaints.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic significantly reduced elective surgery in the United States, but the impact of COVID-19 on acute surgical complaints and acute care surgery is unknown. STUDY DESIGN: A retrospective review was performed of all surgical consults at the Hospital of the University of Pennsylvania in the 30 days prior to and 30 days following confirmation of the first COVID-19 patient at the institution. Consults to all divisions within general surgery were included. RESULTS: Total surgical consult volume decreased by 43% in the post-COVID-19 period, with a significant reduction in the median daily consult volume from 14 to 8 (P < .0001). Changes in consult volume by patient location, chief complaint, and surgical division were variable, in aggregate reflecting a disproportionate decrease among less acute surgical complaints. The percentage of consults resulting in surgical intervention remained equal in the 2 periods (31% vs 28%, odds ratio 0.85, 95% CI 0.61-1.21, P = .38) with most but not all operation types decreasing in frequency. The rise in the COVID-19 inpatient census led to increased consultation for vascular access, accommodated at our center by the creation of a new surgical procedures team. CONCLUSION: The COVID-19 pandemic significantly altered the landscape of acute surgical complaints at our large academic hospital. An appreciation of these trends may be helpful to other Departments of Surgery around the country as they deploy staff and allocate resources in the COVID-19 era.

Regulatory T cells promote alloengraftment in a model of late-gestation in utero hematopoietic cell transplantation.

In utero hematopoietic cell transplantation (IUHCT) has the potential to cure congenital hematologic disorders including sickle cell disease. However, the window of opportunity for IUHCT closes with the acquisition of T-cell immunity, beginning at approximately 14 weeks gestation, posing significant technical challenges and excluding from treatment fetuses evaluated after the first trimester. Here we report that regulatory T cells can promote alloengraftment and preserve allograft tolerance after the acquisition of T-cell immunity in a mouse model of late-gestation IUHCT. We show that allografts enriched with regulatory T cells harvested from either IUHCT-tolerant or naive mice engraft at 20 days post coitum (DPC) with equal frequency to unenriched allografts transplanted at 14 DPC. Long-term, multilineage donor cell chimerism was achieved in the absence of graft-versus-host disease or mortality. Decreased alloreactivity among recipient T cells was observed consistent with donor-specific tolerance. These findings suggest that donor graft enrichment with regulatory T cells could be used to successfully perform IUHCT later in gestation.

Lung function and pulmonary artery blood flow following prenatal maternal retinoic acid and imatinib in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: Lung and pulmonary vascular maldevelopment in congenital diaphragmatic hernia (CDH) results in significant morbidity and mortality. Retinoic acid (RA) and imatinib have been shown to improve pulmonary morphology following prenatal administration in the rat nitrofen-induced CDH model. It remains unclear if these changes translate into improved function. We evaluated the effect of prenatal RA and imatinib on postnatal lung function, structure, and pulmonary artery (PA) blood flow in the rat CDH model. METHODS: Olive oil or nitrofen was administered alone or in combination with RA or imatinib to pregnant rats. Pups were assessed for PA blood flow by ultrasound and pulmonary function/morphology following delivery, intubation, and short-term ventilation. RESULTS: Neither RA nor imatinib had a negative effect on lung and body growth. RA accelerated lung maturation indicated by increased alveoli number and thinner interalveolar septa and was associated with decreased PA resistance and improved oxygenation. With the exception of a decreased PA pulsatility index, no significant changes in morphology and pulmonary function were noted following imatinib. CONCLUSION: Prenatal treatment with RA but not imatinib was associated with improved pulmonary morphology and function, and decreased pulmonary vascular resistance. This study highlights the potential of prenatal pharmacologic therapies, such as RA, for management of CDH.

Prenatal growth characteristics and pre/postnatal management of bronchopulmonary sequestrations.

PURPOSE: The prenatal natural history of intralobar and extralobar bronchopulmonary sequestrations (BPSs), including lesion growth patterns and need for prenatal intervention, have not been fully characterized. We review our series of BPSs to determine their natural history and outcomes in the context of the need for prenatal intervention. METHODS: A retrospective review of the pre/postnatal course of 103 fetuses with an intralobar (n=44) or extralobar BPS (n=59) managed at a single institution between 2008 and 2015 was performed. Outcomes included prenatal lesion growth trajectory, presence of hydrops, need for prenatal intervention, survival, and postnatal surgical management. RESULTS: Most extralobar (71%) and intralobar BPSs (94%) decreased in size or became isoechoic from initial to final evaluation. Peak lesion size occurred at 26-28weeks gestation. Eight fetuses developed hydrothorax, four of which (all extralobar BPSs) also developed hydrops. All four hydropic fetuses received maternal betamethasone, and three hydropic fetuses underwent thoracentesis and/or thoracoamniotic shunt placement with subsequent hydrops resolution. All fetuses survived. Forty-one intralobar (93%) and 35 extralobar BPSs (59%) were resected after birth. CONCLUSIONS: BPSs tend to decrease in size after 26-28weeks gestation and rarely require fetal intervention. Lesions resulting in hydrothorax ± hydrops can be effectively managed with maternal steroids and/or drainage of the hydrothorax. LEVEL OF EVIDENCE: IV.

Reduced oxygen concentration for the resuscitation of infants with congenital diaphragmatic hernia.

OBJECTIVE: To evaluate whether infants with congenital diaphragmatic hernia (CDH) can be safely resuscitated with a reduced starting fraction of inspired oxygen (FiO2) of 0.5. STUDY DESIGN: A retrospective cohort study comparing 68 patients resuscitated with starting FiO2 0.5 to 45 historical controls resuscitated with starting FiO2 1.0. RESULTS: Reduced starting FiO2 had no adverse effect upon survival, duration of intubation, need for ECMO, duration of ECMO, or time to surgery. Furthermore, it produced no increase in complications, adverse neurological events, or neurodevelopmental delay. The need to subsequently increase FiO2 to 1.0 was associated with female sex, lower gestational age, liver up, lower lung volume-head circumference ratio, decreased survival, a higher incidence of ECMO, longer time to surgery, periventricular leukomalacia, and lower neurodevelopmental motor scores. CONCLUSION: Starting FiO2 0.5 may be safe for the resuscitation of CDH infants. The need to increase FiO2 to 1.0 during resuscitation is associated with worse outcomes.

Use of blood biomarkers to screen for obstructive sleep apnea.

PURPOSE: Obstructive sleep apnea (OSA) is a highly prevalent disorder associated with increased risk for cardiovascular disease, diabetes, and other chronic conditions. Unfortunately, up to 90% of individuals with OSA remain without a diagnosis or therapy. We assess the relationship between OSA and blood biomarkers, and test the hypothesis that combinations of markers provide a characteristic OSA signature with diagnostic screening value. This validation study was conducted in an independent cohort in order to replicate findings from a prior feasibility study. PATIENTS AND METHODS: This multicenter prospective study consecutively enrolled adult male subjects with clinically suspected OSA. All subjects underwent overnight sleep studies. An asymptomatic control group was also obtained. Five biomarkers were tested: glycated hemoglobin (HbA1c), C-reactive protein (CRP), uric acid, erythropoietin (EPO), and interleukin-6 (IL-6). RESULTS: The study enrolled 264 subjects. The combination of HbA1c+CRP+EPO (area under the curve 0.78) was superior to the Epworth Sleepiness Scale (ESS; 0.53) and STOP-Bang (0.70) questionnaires. In non-obese subjects, the combination of biomarkers (0.75) was superior to body mass index (BMI; 0.61). Sensitivity and specificity results, respectively, were: HbA1c+CRP+EPO (81% and 60%), ESS (78% and 19%), STOP-Bang (75% and 52%), BMI (81% and 56%), and BMI in non-obese patients (81% and 38%). CONCLUSION: We verify our hypothesis and replicate our prior feasibility findings that OSA is associated with a characteristic signature cluster of biomarker changes in men. Concurrent elevations of HbA1c, CRP, and EPO levels should generate a high suspicion of OSA and may have utility as an OSA screening tool. Biomarker combinations correlate with OSA severity and, therefore, may assist sleep centers in identifying and triaging higher risk patients for sleep study diagnosis and treatment.

Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep.

A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens.

Management and outcomes of scoliosis in children with congenital diaphragmatic hernia.

PURPOSE: The purpose of this study was to evaluate the management and outcomes of CDH patients with scoliosis. METHODS: From January 1996 to August 2015, 26 of 380 (7%) CDH patients were diagnosed with scoliosis. Six (23%) were prenatally diagnosed by ultrasound, and 9 (35%) were diagnosed postnatally. The remaining 11 (42%) developed scoliosis after discharge. Mean follow-up was 6.6years. RESULTS: Among the 15 patients with congenital scoliosis, there were 2 (13%) perinatal deaths. Five of the 13 (38%) survivors required orthopedic surgery, and 2 have required bracing. The mean age at initial surgery was 7years. These five children underwent an average of 2.8 (range 1-7) expansions or revisions. All surgical patients required supplemental oxygen at 28days of life, and 1 required a tracheostomy. None of the 11 patients who developed scoliosis later in life required surgery, but 3 have required bracing. Six of the 11 (55%) required a patch repair for CDH compared to 158 of 264 (60%) CDH patients without scoliosis (p=0.73). CONCLUSIONS: Early diagnosis of scoliosis in CDH patients is associated with a high rate of surgery. There was not a higher incidence of patch repair among patients who developed scoliosis. LEVEL OF EVIDENCE: Prognosis. Retrospective study, level II.

Blood biomarkers of endocrine, immune, inflammatory, and metabolic systems in obstructive sleep apnea.

OBJECTIVE/BACKGROUND: Obstructive sleep apnea (OSA) is a common disorder, affecting over 100 million adults. Untreated OSA leads to serious health consequences and perturbations in endocrine, immune, inflammatory, and metabolic systems. Study objectives are to evaluate the association between OSA and biomarkers, and to test the hypothesis that a combination of markers may be useful in screening for OSA. PATIENTS/METHODS: A multicenter trial was conducted enrolling symptomatic male patients with suspected OSA. All subjects underwent in-laboratory overnight polysomnography. A non-symptomatic control group was also obtained. Eleven biomarkers were tested: HbA1c, CRP, EPO, IL-6, uric acid, cortisol, hGH, prolactin, testosterone, DHEA (Beckman Coulter UniCel DxC 600i Synchron® Access® Clinical Systems), IGF-1. RESULTS: 73 male subjects were enrolled; 26 had moderate/severe OSA. ROC curve analysis showed HbA1c, CRP, EPO, IL-6, and Uric Acid (AUCs: 0.76, 0.73, 0.65, 0.65, 0.61) were superior to the Epworth Sleepiness Scale (AUC: 0.52). Concurrent elevation of HbA1c and CRP provide even greater predictive power. A combination of elevated HbA1c, CRP, and EPO provided 0.08 increase in AUC (0.84 [0.75 - 0.94]) over individual markers (p<0.05), with high sensitivity (85%), and specificity (79%) for moderate/severe OSA. CONCLUSIONS: OSA induces characteristic endocrine, immune, inflammatory, and metabolic disturbances that can be detected with blood biomarkers. These biomarkers are superior to standard screening questionnaires. Various clusters of these biomarkers have an even greater association with OSA and thus may represent physiologic signatures of the disorder that may have value in initial screening for OSA as well as for follow-up of therapy response.