Prenatally Diagnosed Large Lung Lesions: Timing of Resection and Perinatal Outcomes.

INTRODUCTION: Fetuses with large lung lesions including congenital cystic adenomatoid malformations (CCAMs) are at risk for cardiopulmonary compromise. Prenatal maternal betamethasone and cyst drainage for micro- and macrocystic lesions respectively have improved outcomes yet some lesions remain large and require resection before birth (open fetal surgery, OFS), at delivery via an Ex Utero Intrapartum Treatment (EXIT), or immediately post cesarean section (section-to-resection, STR). We sought to compare prenatal characteristics and outcomes in fetuses undergoing OFS, EXIT, or STR to inform decision-making and prenatal counseling. METHODS: A single institution retrospective review was conducted evaluating patients undergoing OFS, EXIT, or STR for prenatally diagnosed lung lesions from 2000 to 2021. Specimens were reviewed by an anatomic pathologist. Lesions were divided into "CCAMs" (the largest pathology group) and "all lung lesions" since pathologic diagnosis is not possible during prenatal evaluation when care decisions are made. Prenatal variables included initial, greatest, and final CCAM volume-ratio (CVR), betamethasone use/frequency, cyst drainage, and the presence of hydrops. Outcomes included survival, ECMO utilization, NICU length of stay (LOS), postnatal nitric oxide use, and ventilator days. RESULTS: Sixty-nine percent (59 of 85 patients) of lung lesions undergoing resection were CCAMs. Among patients with pathologic diagnosis of CCAM, the initial, largest, and final CVRs were greatest in OFS followed by EXIT and STR patients. Similarly, the incidence of hydrops was significantly greater and the rate of hydrops resolution was lower in the OFS group. Although the rate of cyst drainage did not differ between groups, maternal betamethasone use varied significantly (OFS 60.0%, EXIT 100.0%, STR 74.3%; p = 0.0378). Notably, all OFS took place prior to 2014. There was no difference in survival, ventilator days, nitric oxide, NICU LOS, or ECMO between groups. In multiple variable logistic modeling, determinants of survival to NICU discharge among patients undergoing resection with a pathologic diagnosis of CCAM included initial CVR <3.5 and need for <3 maternal betamethasone doses. CONCLUSION: For CCAMs that remain large despite maternal betamethasone or cyst drainage, surgical resection via OFS, EXIT, or STR are viable options with favorable and comparable survival between groups. In the modern era there has been a shift from OFS and EXIT procedures to STR for fetuses with persistently large lung lesions. This shift has been fueled by the increased use of maternal betamethasone and introduction of a Special Delivery Unit during the study period and the appreciation of similar fetal and neonatal outcomes for STR vs. EXIT and OFS with reduced maternal morbidity associated with a STR. Accordingly, efforts to optimize multidisciplinary perinatal care for fetuses with large lung lesions are important to inform patient selection criteria and promote STR as the preferred surgical approach in the modern era. LEVEL OF EVIDENCE: Level IV.

An Original Study: Is There an Optimal Time to Complete Dedicated Research During Surgical Residency? Twelve Years of Research Experience After PGY2 or PGY3.

OBJECTIVE: We aimed to determine if there is an optimal time to complete dedicated research during surgical residency. BACKGROUND: Research is an integral part of academic general surgical residency, and dedicated research usually occurs after the 2nd or 3rd post-graduate year (PGY). The timing of dedicated research and its association with resident productivity, self-assessed competency (including technical skills), and fellowship match is not known. METHODS: PubMed was queried for publications resulting after dedicated research time for graduating surgical residents at a single institution from 2010 to 2021. Graduates were surveyed about their research experience and placed into 2 groups: research after PGY2 or PGY3. RESULTS: Sixty-six of 91 (73%) graduating residents completed dedicated research (after PGY2, n=28; after PGY3, n=38). Median number of total and first author publications was similar between groups; however, research after PGY2 was associated with an increased number of basic science publications by fellowship application deadlines (PGY2: 1.0[0-13] vs PGY3: 0.0[0-6], p=0.02). With a 79% survey response rate, there were no differences in self-assessed competencies upon return from research between cohorts. Most surveyed residents matched at their top fellowship choice (PGY2:70% vs PGY3:62%, p=0.77). CONCLUSIONS: Research after PGY2 or PGY3 had no association with residents' total number of publications, self-assessed competency, or rates of matching at first choice fellowship. As research after PGY2 had an increased number of basic science publications by time of fellowship application, surgical residents applying to fellowships that highly value basic science research may benefit from completing dedicated research after PGY2.

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.

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.

Donor cell engineering with GSK3 inhibitor-loaded nanoparticles enhances engraftment after in utero transplantation.

Host cell competition is a major barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Here we describe a cell-engineering strategy using glycogen synthase kinase-3 (GSK3) inhibitor-loaded nanoparticles conjugated to the surface of donor hematopoietic cells to enhance their proliferation kinetics and ability to compete against their fetal host equivalents. With this approach, we achieved remarkable levels of stable, long-term hematopoietic engraftment for up to 24 weeks post-IUHCT. We also show that the salutary effects of the nanoparticle-released GSK3 inhibitor are specific to donor progenitor/stem cells and achieved by a pseudoautocrine mechanism. These results establish that IUHCT of hematopoietic cells decorated with GSK3 inhibitor-loaded nanoparticles can produce therapeutic levels of long-term engraftment and could therefore allow single-step prenatal treatment of congenital hematological disorders.

In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long-Term Hematopoietic Engraftment.

In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are subtherapeutic, likely due to host fetal HSCs outcompeting their bone marrow (BM)-derived donor equivalents for space in the hematopoietic compartment. In the present study, we demonstrate that amniotic fluid stem cells (AFSCs; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favorable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly isolated or cultured AFSCs resulted in stable multilineage hematopoietic engraftment, far higher to that achieved with BM-HSCs. Intravascular IUT of allogenic AFSCs was not successful as recently reported after intraperitoneal IUT. Herein, we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSCs in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene-engineered, and in vitro expanded AFSCs could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. Stem Cells 2019;37:1176-1188.

In utero gene editing for monogenic lung disease.

Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPCI73T mutation. Embryonic expression of SftpcI73T alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant SftpcI73T gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

In utero CRISPR-mediated therapeutic editing of metabolic genes.

In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.

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.

Management of traumatic urethral injuries in children using different techniques: A case series and review of literature.

INTRODUCTION: Most pediatric urethral injuries are a result of pelvic fracture after high-impact blunt trauma, mainly due to motor vehicle accidents. The management of urethral injuries depends on if the rupture is complete or partial as well as the timing of surgical intervention. PRESENTATION OF CASES: Three male children with urethral trauma caused by motor vehicles accidents are presented in this article. Preoperative suprapubic catheterization was initially carried out in all patients. Each patient then received one of three different techniques during the deferred time to surgical intervention: anterior sagittal transanorectal approach (ASTRA) for end-to-end urethral anastomosis, perineal approach for urethroplasty using buccal mucosa, and urethroplasty with preputial skin flap. The three techniques were successfully performed. DISCUSSION: In the initial management suprapubic cystostomy has been a good solution in urgent situations. Deferred urethroplasty is the procedure of choice for the definite treatment of posterior urethral distraction defects. The anterior sagittal transanorectal approach provides excellent exposure of the posterior urethra and retrovesicular region, and allows the surgeon to perform dissection under direct vision. CONCLUSION: It's very important for the pediatric urologist to be familiar with the different techniques available in order to choose the best approach for each particular patient.

Amniotic fluid transcriptomics reflects novel disease mechanisms in fetuses with myelomeningocele.

BACKGROUND: Cell-free RNA in amniotic fluid supernatant reflects developmental changes in gene expression in the living fetus, which includes genes that are specific to the central nervous system. Although it has been previously shown that central nervous system-specific transcripts are present in amniotic fluid supernatant, it is not known whether changes in the amniotic fluid supernatant transcriptome reflect the specific pathophysiologic condition of fetal central nervous system disorders. In myelomeningocele, there is open communication between the central nervous system and amniotic fluid. OBJECTIVES: The purpose of this study was to identify molecular pathophysiologic changes and novel disease mechanisms that are specific to myelomeningocele by the analysis of amniotic fluid supernatant cell-free RNA in fetuses with open myelomeningocele. STUDY DESIGN: Amniotic fluid supernatant was collected from 10 pregnant women at the time of the open myelomeningocele repair in the second trimester (24.5±1.0 weeks); 10 archived amniotic fluid supernatant from sex and gestational age-matched euploid fetuses without myelomeningocele were used as controls (20.9±0.9 weeks). Differentially regulated gene expression patterns were analyzed with the use of human genome expression arrays. RESULTS: Fetuses with myelomeningocele had 284 differentially regulated genes (176 up- and 108 down-regulated) in amniotic fluid supernatant. Known genes that were associated with myelomeningocele (PRICKLE2, GLI3, RAB23, HES1, FOLR1) and novel dysregulated genes were identified in association with neurodevelopment and neuronal regeneration (up-regulated, GAP43 and ZEB1) or axonal growth and guidance (down-regulated, ACAP1). Pathway analysis demonstrated a significant contribution of inflammation to disease and a broad influence of Wnt signaling pathways (Wnt1, Wnt5A, ITPR1). CONCLUSION: Transcriptomic analyses of living fetuses with myelomeningocele with the use of amniotic fluid supernatant cell-free RNA demonstrated differential regulation of specific genes and molecular pathways relevant to this central nervous system disorder, which resulted in a new understanding of pathophysiologic changes. The data also suggested the importance of pathways that involve secondary disease, such as inflammation, in myelomeningocele. These newly identified pathways may lead to hypotheses that can test novel therapeutic targets as adjuncts to fetal surgical repair.

The Intravenous Route of Injection Optimizes Engraftment and Survival in the Murine Model of In Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) has the potential to treat a number of congenital hematologic disorders. Clinical application is limited by low levels of donor engraftment. Techniques that optimize donor cell delivery to the fetal liver (FL), the hematopoietic organ at the time of IUHCT, have the potential to enhance engraftment and the clinical success of IUHCT. We compared the 3 clinically applicable routes of injection (intravenous [i.v.], intraperitoneal [i.p.], and intrahepatic [i.h.]) and assessed short- and long-term donor cell engraftment and fetal survival in the murine model of IUHCT. We hypothesized that the i.v. route would promote direct donor cell homing to the FL, resulting in increased engraftment and allowing for larger injectate volumes without increased fetal mortality. We demonstrate that the i.v. route results in (1) rapid diffuse donor cell population of the FL compared with delayed diffuse engraftment after the i.p. and i.h. routes; (2) higher FL and spleen engraftment at early prenatal time points; (3) enhanced stable long-term peripheral blood donor cell engraftment; and (4) improved survival at higher injectate volumes, allowing for higher donor cell doses and increased long-term engraftment. These findings support the use of an i.v. route for clinical protocols of IUHCT.

Monitoring universal protocol compliance through real-time clandestine observation by medical students results in performance improvement.

OBJECTIVE: To measure universal protocol compliance through real-time, clandestine observation by medical students compared with chart audit reviews, and to enable medical students the opportunity to become conscious of the importance of medical errors and safety initiatives. DESIGN: With endorsement from Tufts Medical Center's (TMC's) Chief Medical Officer and Surgeon-in-Chief, 8 medical students performed clandestine observation audits of 98 cases from April to August 2009. A compliance checklist was based on TMC's presurgical checklist. Our initial results led to interventions to improve our universal protocol procedures, including modifications to the operating room white board and presurgical checklist, and specific feedback to surgical departments. One year later, 6 medical students performed observations of 100 cases from June to August 2010. SETTING: Tufts Medical Center, Boston, Massachusetts, which is an academic medical center and the principal teaching hospital for Tufts University School of Medicine. PARTICIPANTS: An operating room coordinator placed the medical students into 1 of our 25 operating rooms with students entering under the premise of observing the anesthesiologist for clinical education. The observations were performed Monday to Friday between 7 am and 4 pm. Although observations were not randomized, no single service or type of surgery was targeted for observation. RESULTS: A broad range of departments was observed. In 8.2% of cases, the surgical site was unmarked. A Time Out occurred in 89.7% of cases. The entire surgical team was attentive during the time out in 82% of cases. The presurgical checklist was incomplete before incision in 13 cases. Images were displayed in 82% of cases. The operating room "white board" was filled out completely in 49% of cases. Team introductions occurred in 13 cases. One year later, compliance increased in all Universal Protocol dimensions. CONCLUSIONS: Direct, real-time observation by medical students provides an accurate and granular assessment of compliance with specific components of the universal protocol and engages medical students in the quality improvement process, raises their awareness of the gravity of medical errors, and ensures appreciation of the importance of quality and safety initiatives.