Decreased neonatal morbidity in 'stomach-down' left congenital diaphragmatic hernia: implications of prenatal ultrasound diagnosis for counseling and postnatal management.

OBJECTIVE: To evaluate the influence of stomach position on postnatal outcome in cases of left congenital diaphragmatic hernia (CDH) without liver herniation, diagnosed and characterized on prenatal ultrasound (US), by comparing those with ('stomach-up' CDH) to those without ('stomach-down' CDH) intrathoracic stomach herniation. METHODS: Infants with left CDH who underwent prenatal US and postnatal repair at our institution between January 2008 and March 2017 were eligible for inclusion in this retrospective study. Detailed prenatal US examinations, fetal magnetic resonance imaging (MRI) studies, operative reports and medical records of infants enrolled in the pulmonary hypoplasia program at our institution were reviewed. Cases with liver herniation and those with an additional anomaly were excluded. Cases in which bowel loops were identified within the fetal chest on US while the stomach was intra-abdominal were categorized as having stomach-down CDH. Cases in which bowel loops and the stomach were visualized within the fetal chest on US were categorized as having stomach-up CDH. Prenatal imaging findings and postnatal outcomes were compared between the two groups. RESULTS: In total, 152 patients with left CDH were initially eligible for inclusion. Seventy-eight patients had surgically confirmed liver herniation and were excluded. Of the 74 included CDH cases without liver herniation, 28 (37.8%) had stomach-down CDH and 46 (62.2%) had stomach-up CDH. Of the 28 stomach-down CDH cases, 10 (35.7%) were referred for a suspected lung lesion. Sixty-eight (91.9%) cases had postnatal outcome data available for analysis. There was no significant difference in median observed-to-expected (o/e) lung-area-to-head-circumference ratio (LHR) between cases with stomach-down CDH and those with stomach-up CDH (41.5% vs 38.4%; P = 0.41). Furthermore, there was no difference in median MRI o/e total lung volume (TLV) between the two groups (49.5% vs 44.0%; P = 0.22). Compared with stomach-up CDH patients, stomach-down CDH patients demonstrated lower median duration of intubation (18 days vs 9.5 days; P < 0.01), median duration of extracorporeal membrane oxygenation (495 h vs 223.5 h; P < 0.05), rate of supplemental oxygen requirement at 30 days of age (20/42 (47.6%) vs 3/26 (11.5%); P < 0.01) and rate of pulmonary hypertension at initial postnatal echocardiography (28/42 (66.7%) vs 9/26 (34.6%); P = 0.01). No neonatal death occurred in stomach-down CDH patients and one neonatal death was seen in a patient with intrathoracic stomach herniation. CONCLUSIONS: In infants with left CDH without liver herniation, despite similar o/e-LHR and o/e-TLV, those with stomach-down CDH have decreased neonatal morbidity compared to those with stomach herniation. Progressive or variable physiological distension of the stomach over the course of gestation may explain these findings. Stomach-down left CDH is mistaken for a lung mass in a substantial proportion of cases. Accurate prenatal US characterization of CDH is crucial for appropriate prenatal counseling and patient management. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Myelomeningocele sac associated with worse lower-extremity neurological sequelae: evidence for prenatal neural stretch injury?

OBJECTIVE: To determine whether the presence of a myelomeningocele (MMC) sac and sac size correlate with compromised lower-extremity function in fetuses with open spinal dysraphism. METHODS: A radiology database search was performed to identify cases of MMC and myeloschisis (MS) diagnosed prenatally in a single center from 2013 to 2017. All cases were evaluated between 18 and 25 weeks. Ultrasound reports were reviewed for talipes and impaired lower-extremity motion. In MMC cases, sac volume was calculated from ultrasound measurements. Magnetic resonance imaging reports were reviewed for hindbrain herniation. The association of presence of a MMC sac and sac size with talipes and impaired lower-extremity motion was assessed. Post-hoc analysis of data from the multicenter Management of Myelomeningocele Study (MOMS) randomized controlled trial was performed to confirm the study findings. RESULTS: In total, 283 MMC and 121 MS cases were identified. MMC was associated with a lower incidence of hindbrain herniation than was MS (80.9% vs 100%; P < 0.001). Compared with MS cases, MMC cases with hindbrain herniation had a higher rate of talipes (28.4% vs 16.5%, P = 0.02) and of talipes or lower-extremity impairment (34.9% vs 19.0%, P = 0.002). Although there was a higher rate of impaired lower-extremity motion alone in MMC cases with hindbrain herniation than in MS cases, the difference was not statistically significant (6.6% vs 2.5%; P = 0.13). Among MMC cases with hindbrain herniation, mean sac volume was higher in those associated with talipes compared with those without talipes (4.7 ± 4.2 vs 3.0 ± 2.6 mL; P = 0.002). Review of the MOMS data demonstrated similar findings; cases with a sac on baseline imaging had a higher incidence of talipes than did those without a sac (28.2% vs 7.5%; P = 0.007). CONCLUSIONS: In fetuses with open spinal dysraphism, the presence of a MMC sac was associated with fetal talipes, and this effect was correlated with sac size. The presence of a larger sac in fetuses with open spinal dysraphism may result in additional injury through mechanical stretching of the nerves, suggesting another acquired mechanism of injury to the exposed spinal tissue. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Learning curves of open and endoscopic fetal spina bifida closure: systematic review and meta-analysis.

OBJECTIVE: The Management of Myelomeningocele Study (MOMS) trial demonstrated the safety and efficacy of open fetal surgery for spina bifida aperta (SBA). Recently developed alternative techniques may reduce maternal risks without compromising the fetal neuroprotective effects. The aim of this systematic review was to assess the learning curve (LC) of different fetal SBA closure techniques. METHODS: MEDLINE, Web of Science, EMBASE, Scopus and Cochrane databases and the gray literature were searched to identify relevant articles on fetal surgery for SBA, without language restriction, published between January 1980 and October 2018. Identified studies were reviewed systematically and those reporting all consecutive procedures and with postnatal follow-up ≥ 12 months were selected. Studies were included only if they reported outcome variables necessary to measure the LC, as defined by fetal safety and efficacy. Two authors independently retrieved data, assessed the quality of the studies and categorized observations into blocks of 30 patients. For meta-analysis, data were pooled using a random-effects model when heterogeneous. To measure the LC, we used two complementary methods. In the group-splitting method, competency was defined when the procedure provided results comparable to those in the MOMS trial for 12 outcome variables representing the immediate surgical outcome, short-term neonatal neuroprotection and long-term neuroprotection at ≥ 12 months of age. Then, when raw patient data were available, we performed cumulative sum analysis based on a composite binary outcome defining successful surgery. The composite outcome combined four clinically relevant variables for safety (absence of extreme preterm delivery < 30 weeks, absence of fetal death ≤ 7 days after surgery) and efficacy (reversal of hindbrain herniation and absence of any neonatal treatment of dehiscence or cerebrospinal fluid leakage at the closure site). RESULTS: Of 6024 search results, 17 (0.3%) studies were included, all of which had low, moderate or unclear risk of bias. Fetal SBA closure was performed using standard hysterotomy (11 studies), mini-hysterotomy (one study) or fetoscopy by either exteriorized-uterus single-layer closure (one study), percutaneous single-layer closure (three studies) or percutaneous two-layer closure (one study). Only outcomes for standard hysterotomy could be meta-analyzed. Overall, outcomes improved significantly with experience. Competency was reached after 35 consecutive cases for standard hysterotomy and was predicted to be achieved after ≥ 57 cases for mini-hysterotomy and ≥ 56 for percutaneous two-layer fetoscopy. For percutaneous and exteriorized-uterus single-layer fetoscopy, competency was not reached in the 81 and 28 cases available for analysis, respectively, and LC prediction analysis could not be performed. CONCLUSIONS: The number of cases operated is correlated with the outcome of fetal SBA closure, and the number of operated cases required to reach competency ranges from 35 for standard hysterotomy to ≥ 56-57 for minimally invasive modifications. Our observations provide important information for institutions looking to establish a new fetal center, develop a new fetal surgery technique or train their team, and inform referring clinicians, potential patients and third parties. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Curvas de aprendizaje del cierre de la espina bífida fetal mediante cirugía abierta y endoscópica: revisión sistemática y metaanálisis OBJETIVO: El ensayo del Estudio sobre la Gestión del Mielomeningocele (MOMS, por sus siglas en inglés) demostró la seguridad y eficacia de la cirugía fetal abierta para la espina bífida aperta (EBA). Las técnicas alternativas recientemente desarrolladas pueden reducir los riesgos de la madre sin comprometer los efectos neuroprotectores del feto. El objetivo de esta revisión sistemática fue evaluar la curva de aprendizaje (CA) de diferentes técnicas de cierre de la EBA fetal. MÉTODOS: Se realizaron búsquedas en las bases de datos de MEDLINE, Web of Science, EMBASE, Scopus y Cochrane, así como en la literatura gris, para identificar artículos relevantes sobre cirugía fetal para la EBA, sin restricción de idioma, publicados entre enero de 1980 y octubre de 2018. Se examinaron sistemáticamente los estudios identificados y se seleccionaron los que informaban de todos los procedimientos consecutivos y con seguimiento postnatal ≥12 meses. Los estudios se incluyeron sólo si informaban sobre las variables de resultado necesarias para medir la CA, definidas por la seguridad y la eficacia para el feto. Dos autores recuperaron los datos de forma independiente, evaluaron la calidad de los estudios y clasificaron las observaciones en bloques de 30 pacientes. Para el metaanálisis, los datos se agruparon mediante un modelo de efectos aleatorios cuando fueron heterogéneos. Para medir la CA, se usaron dos métodos complementarios. En el método de división de grupos, la competencia se definió cuando el procedimiento proporcionó resultados comparables a los del ensayo MOMS para 12 variables de resultados que representaban el resultado quirúrgico inmediato, la neuroprotección neonatal a corto plazo y la neuroprotección a largo plazo a ≥12 meses de edad. Luego, cuando se dispuso de los datos brutos de los pacientes, se realizó un análisis de suma acumulada basado en un resultado binario compuesto que definió el éxito de la cirugía. El resultado compuesto combinó cuatro variables clínicamente relevantes en cuanto a la seguridad (ausencia de parto pretérmino extremo <30 semanas; ausencia de muerte fetal a ≤7 días después de la cirugía) y eficacia (reducción de la hernia del rombencéfalo y ausencia de cualquier tratamiento neonatal de dehiscencia o derrame de líquido cefalorraquídeo en el lugar del cierre). RESULTADOS: De los 6024 resultados de la búsqueda, se incluyeron 17 (0,3%) estudios, todos ellos con un riesgo de sesgo bajo, moderado o incierto. El cierre de la EBA fetal se realizó mediante histerotomía estándar (11 estudios), mini histerotomía (un estudio) o fetoscopia, ya fuera mediante el cierre exteriorizado del útero de una sola capa (un estudio), el cierre percutáneo de una sola capa (tres estudios) o el cierre percutáneo de dos capas (un estudio). Sólo se pudieron metaanalizar los resultados de la histerotomía estándar. En general, los resultados mejoraron significativamente con la experiencia. Se alcanzó la competencia después de 35 casos consecutivos para la histerotomía estándar y se predijo que se alcanzaría después de ≥57 casos para la mini histerotomía y ≥56 para la fetoscopia percutánea de dos capas. En el caso de las fetoscopias percutánea y exteriorizada del útero de una sola capa, no se alcanzó la competencia en los 81 y 28 casos disponibles para el análisis, respectivamente, y no se pudo realizar el análisis de predicción de la CA. CONCLUSIONES: El número de casos operados está correlacionado con el resultado del cierre de la EBA fetal, y el número de casos operados necesarios para alcanzar la competencia estuvo entre 35 para la histerotomía estándar y ≥56-57 para las operaciones con mínima agresividad. Las observaciones realizadas proporcionan información importante para las instituciones que buscan establecer un nuevo centro fetal, desarrollar una nueva técnica de cirugía fetal o entrenar a su equipo, e informar a los médicos que remiten a especialistas a los posibles pacientes y a terceros. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Comparison of ultrasound and magnetic resonance imaging parameters in predicting survival in isolated left-sided congenital diaphragmatic hernia.

OBJECTIVE: To compare test characteristics of ultrasound- and magnetic resonance imaging (MRI)-derived parameters in predicting newborn survival in cases of isolated left-sided congenital diaphragmatic hernia (CDH). METHODS: This was a retrospective study involving 85 fetuses with an isolated left CDH. All had detailed prenatal evaluation, prenatal care, delivery and postnatal care at a single institution. Ultrasound images were reviewed to allow calculation of the lung-to-head ratio (LHR) and the observed/expected LHR (O/E-LHR), and MRI images were reviewed to determine the observed/expected total lung volume (O/E-TLV) and the percent herniated liver (%HL). Univariable logistic regression was used to evaluate each parameter for its ability to predict survival. Receiver-operating characteristics (ROC) curves were constructed and test characteristics were determined for each parameter as a predictor of survival. RESULTS: The overall survival for all fetuses included was 65%. Pseudo-R(2) values for all parameters were similar and were statistically significant as predictors of survival, with %HL having the highest pseudo-R(2) , of 0.28. ROC curve analysis showed ultrasound-determined parameters (LHR and O/E-LHR) to have a similar area under the curve (AUC), of 0.70, whilst MRI parameters (O/E-TLV and %HL) had AUC values of 0.82 and 0.84, respectively. At ROC-curve-determined cut-off values, MRI parameters had better test characteristics than did ultrasound parameters. At a standardized 5% false-positive rate, %HL performed best, with a sensitivity of 0.54 and a specificity of 0.95. At clinically employed cut-off values, sensitivity was similar for all parameters but MRI parameters provided the best combination of sensitivity and specificity, as evidenced by better likelihood ratios. CONCLUSIONS: A variety of measures have been proposed as antenatal predictors of survival in CDH. Ultrasound parameters function at a similar level, whereas MRI-determined parameters appear to offer better predictive value.

In utero surgery rescues neurological function at birth in sheep with spina bifida.

We hypothesize that the neurologic deficit associated with open spina bifida is not directly caused by the primary defect but rather is due to chronic mechanical and chemical trauma since the unprotected neural tissue is exposed to the intrauterine environment. We report here that exposure of the normal spinal cord to the amniotic cavity in midgestational sheep fetuses leads to a human-like open spina bifida with paraplegia at birth, indicating that the exposed neural tissue is progressively destroyed during pregnancy. When open spina bifida was repaired in utero at an intermediate stage, the animals had near-normal neurologic function. The spinal cord was deformed but largely preserved. These findings suggest that secondary neural tissue destruction during pregnancy is primarily responsible for the functional loss and that timely in utero repair of open spina bifida might rescue neurologic function.

The relationship of seizure activity and chronic epilepsy in early infancy and short-term neurodevelopmental outcome following fetal myelomeningocele closure.

We explored the relationship between seizure activity (SA) and/or chronic epilepsy (CE) and short-term neurodevelopmental outcomes following fetal myelomeningocele (fMMC) surgery. Retrospective databases and a parental questionnaire focusing on common complications of hindbrain herniation associated with MMC were used to determine the incidence of seizures following fMMC surgery. The Bayley Scales of Infant Development II was used to evaluate the neurocognitive outcomes. The available 3-year outcome data were used for analysis. 54 children underwent fMMC closure at our institution between 1998 and 2003. 48 (89%) families participated. The shunt rate was 50% (n=24). Seizures developed in 8/48 (17%) children, 2 (8%) non-shunted and 6 (25%) shunted (P=0.07). Of those six, 3 developed CE. Neurodevelopmental scores in the average range were found in both non-shunted and 3 shunted fMMC children. The remaining 3 shunted toddlers had CE and significant neurodevelopmental delays. Of those, 2 had severe intracranial hemorrhage and one developed frequent apneic spells in combination with epilepsy. The incidence of seizures in fMMC children was similar to previously reported data of postnatally repaired MMC patients. SA alone without CE was not associated with a worse neurocognitive outcome. The occurrence of severe acquired intracranial injury and CE, however, appeared to be correlated with adverse neurocognitive outcome following fMMC surgery.

Fetal Intraventricular Hemorrhage in Open Neural Tube Defects: Prenatal Imaging Evaluation and Perinatal Outcomes.

BACKGROUND AND PURPOSE: Fetal imaging is crucial in the evaluation of open neural tube defects. The identification of intraventricular hemorrhage prenatally has unclear clinical implications. We aimed to explore fetal imaging findings in open neural tube defects and evaluate associations between intraventricular hemorrhage with prenatal and postnatal hindbrain herniation, postnatal intraventricular hemorrhage, and ventricular shunt placement. MATERIALS AND METHODS: After institutional review board approval, open neural tube defect cases evaluated by prenatal sonography between January 1, 2013 and April 24, 2018 were enrolled (n = 504). The presence of intraventricular hemorrhage and gray matter heterotopia by both prenatal sonography and MR imaging studies was used for classification. Cases of intraventricular hemorrhage had intraventricular hemorrhage without gray matter heterotopia (n = 33) and controls had neither intraventricular hemorrhage nor gray matter heterotopia (n = 229). A total of 135 subjects with findings of gray matter heterotopia were excluded. Outcomes were compared with regression analyses. RESULTS: Prenatal and postnatal hindbrain herniation and postnatal intraventricular hemorrhage were more frequent in cases of prenatal intraventricular hemorrhage compared with controls (97% versus 79%, 50% versus 25%, and 63% versus 12%, respectively). Increased third ventricular diameter, specifically >1 mm, predicted hindbrain herniation (OR = 3.7 [95% CI, 1.5-11]) independent of lateral ventricular size and prenatal intraventricular hemorrhage. Fetal closure (n = 86) was independently protective against postnatal hindbrain herniation (OR = 0.04 [95% CI, 0.01-0.15]) and postnatal intraventricular hemorrhage (OR = 0.2 [95% CI, 0.02-0.98]). Prenatal intraventricular hemorrhage was not associated with ventricular shunt placement. CONCLUSIONS: Intraventricular hemorrhage is relatively common in the prenatal evaluation of open neural tube defects. Hindbrain herniation is more common in cases of intraventricular hemorrhage, but in association with increased third ventricular size. Fetal closure reverses hindbrain herniation and decreases the rate of intraventricular hemorrhage postnatally, regardless of the presence of prenatal intraventricular hemorrhage.

Transplantation of fetal hematopoietic stem cells in utero: the creation of hematopoietic chimeras.

Transplantation of normal, immature, fetal hematopoietic cells into a preimmune fetal recipient with a congenital hemoglobinopathy may allow partial reconstitution of normal hemoglobin production without the complications associated with postnatal bone marrow transplantation (immunosuppression and the occurrence of graft versus host disease). In order to test this hypothesis the naturally occurring polymorphism at the beta-hemoglobin locus of the sheep was used as a marker for engraftment and hematopoietic chimerism. Intraperitoneal injection of allogeneic fetal stem cells into normal fetal lambs resulted in hematopoietic chimerism in three of four surviving recipients. This chimerism has been sustained for 6 months after birth and 9 months after engraftment, without evidence of graft versus host disease, and without the use of immunosuppressive therapy.

Reversal of hindbrain herniation after maternal-fetal surgery for myelomeningocele subsequently impacts on brain stem function.

The aim of our study was to delineate whether the reversal of hindbrain herniation (HH) following fetal myelomeningocele (fMMC) closure subsequently reduces the incidence and severity of HH-associated brainstem dysfunction (BSD). Prior to the NIH-sponsored Management of Myelomeningocele Study (MOMS) trial, 54 children underwent fMMC closure at our institution. Forty-eight (89%) families participated in a structured survey focusing on HH-associated BSD (e.g., apnea, neurogenic dysphagia [ND], gastro-esophageal reflux disease [GERD], neuro-ophthalmologic disturbances [NOD]). Median age at follow-up was 72 months (range: 46-98). Fifty-percent required shunting. HH-related symptoms were completely absent in 15 (63%) non-shunted and 10 (42%) shunted children (P=0.15). No HH-related death occurred and none developed severe persistent cyanotic apnea. ND was reported in 2 (8%) non-shunted and 9 (38%) shunted infants (P=0.03). Mild GERD (medically managed) developed in 2 (8%) without and 6 (25%) with shunt placement (P=0.24). NOD was found in 6 (25%) and 13 (54%) of non-shunted and shunted children, respectively (P=0.07). The majority of fMMC children developed no or only mild BSD at follow-up. Our data support the hypothesis that neurodevelopmental deficits associated with MMC are at least partially acquired and that reversal of HH following fMMC surgery may help to reduce the incidence and severity of BSD.

Fetal spina bifida repair--current trends and prospects of intrauterine neurosurgery.

Myelomeningocele is a common dysraphic defect leading to severe impairment throughout the patient's lifetime. Although surgical closure of this anomaly is usually performed in the early postnatal period, an estimated 330 cases of intrauterine repair have been performed in a few specialized centers worldwide. It was hoped prenatal intervention would improve the prognosis of affected patients, and preliminary findings suggest a reduced incidence of shunt-dependent hydrocephalus, as well as an improvement in hindbrain herniation. However, the expectations for improved neurological outcome have not been fulfilled and not all patients benefit from fetal surgery in the same way. Therefore, a multicenter randomized controlled trial was initiated in the USA to compare intrauterine with conventional postnatal care, in order to establish the procedure-related benefits and risks. The primary study endpoints include the need for shunt at 1 year of age, and fetal and infant mortality. No data from the trial will be published before the final analysis has been completed in 2008, and until then, the number of centers offering intrauterine MMC repair in the USA is limited to 3 in order to prevent the uncontrolled proliferation of new centers offering this procedure. In future, refined, risk-reduced surgical techniques and new treatment options for preterm labor and preterm rupture of the membranes are likely to reduce associated maternal and fetal risks and improve outcome, but further research will be needed.

Hyaluronidase levels in urine from Wilms' tumor patients.

The pathophysiology of Wilms' tumor is associated with major alterations in hyaluronic acid metabolism. Elevated levels of both hyaluronic acid and a hyaluronic acid-stimulating activity occur in the urine and serum of patients with this tumor. In the current study, we describe elevated levels of urinary hyaluronidase in five patients with Wilms' tumor. Following surgical removal of the tumor, enzyme levels decreased toward normal. Characterization of enzyme activity indicates that hyaluronidase may be produced by the tumor itself. Alternatively, normal renal tissue may also be producing enzyme in a compensatory response to the elevated hyaluronic acid levels in these patients. We suggest that urinary hyaluronidase can be used as an additional marker for Wilms' tumor.

Hyaluronic acid-stimulating activity in the pathophysiology of Wilms' tumors.

Markedly elevated levels of hyaluronic acid occur in the serum and urine of some patients with Wilms' tumor. We have recently described a glycoprotein factor in fetal serum that stimulates deposition of hyaluronic acid. In a survey of bovine fetal tissue extracts, we have identified the fetal kidney as the source of this circulating activity. Wilms' tumors arise from transformed "rests" of fetal kidney. We demonstrate here that such tumors continue to produce this fetal factor and that the hyaluronic acid-stimulating activity is found in the urine of children with Wilms' tumors. In the three patients with Wilms' tumor who were followed, elevated levels of hyaluronic acid-stimulating activity were found in their urine before treatment. By 2 months after surgical removal of their tumors, these levels had returned to baseline. We propose that hyaluronic acid-stimulating activity is the mechanism for the elevated levels of hyaluronic acid in the sera and urine of these patients. The activity is an oncofetal protein and the first for which a function has been identified. It also is a marker for this common childhood solid tumor and has the potential for identifying children at increased risk.

Effect of hydrocortisone and dexamethasone on xylose uptake by isolated rat soleus muscle.

To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10-4 M hydrocortisone, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10-2 M, insulinstimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inhibit basal or insulin-stimulated sugar uptake.

Diagnostic and prognostic role of basic fibroblast growth factor in Wilms' tumor patients.

Basic fibroblast growth factor (bFGF) is a potent angiogenic peptide implicated in the growth and metastasis of solid tumors. Elevated concentrations of bFGF have been found in the urine of patients with bladder, prostate, and renal tumors. Furthermore, urinary bFGF levels have been shown to correlate with extent of disease. In order to test the utility of urinary bFGF as a Wilms' tumor marker, we measured bFGF levels in preoperative and postoperative urine samples from 97 patients with Wilms' tumor. Preoperative urine samples (n = 97), early postoperative samples obtained from 1 to 3 weeks after surgery (n = 43), and late postoperative samples obtained from 1 to 6 months after surgery (n = 66) were collected from Wilms' tumor patients at 30 institutions between 1989 and 1993. Urine samples from age-matched controls (n = 17) were also obtained. The bFGF levels were determined in duplicate by a competitive sandwich ELISA capable of measuring bFGF at the pg/ml level. Samples were normalized for creatinine content. Urinary bFGF was elevated in 42% of preoperative samples when compared to controls (>90th percentile of normal). Patients with stage III, IV, and V disease had significantly higher preoperative levels of urinary bFGF when compared to patients with stage I and II disease (P < 0.01). Patients with relapse or persistent disease had significantly elevated late postoperative bFGF levels when compared to disease-free patients and controls (P < 0.05). Thus, in patients with Wilms' tumor, elevated preoperative urinary bFGF levels raise the suspicion of aggressive disease while elevated postoperative levels may indicate recurrence or persistence of disease. These data suggest that bFGF is a biological marker for Wilms' tumor and may have a role in the evaluation of patients with this disease.

Syndecans-1 and -4 are induced during wound repair of neonatal but not fetal skin.

Syndecans are a family of four cell surface proteoglycans that bind to various components of the extracellular environment and can regulate many cellular behaviors including growth, adhesion, and movement. To determine whether syndecans can function during wound repair, we have examined expression of the syndecans during wound repair of adult mouse and neonatal or fetal human skin. Syndecan-1 and -4 were induced in the dermis within 12 h after incisional injury of murine or neonatal human skin. Syndecan-1 was induced primarily on endothelium, and syndecan-4 was present throughout the dermis at the site of injury. Following re-epithelialization, expression of the syndecans return to their baseline level. In marked contrast to these observations, wounded human fetal skin showed no increase in expression of syndecans. This lack of increase in the expression of syndecans by cells of the dermis correlates with prior observations that fetal skin heals without a polymorphonuclear cell infiltrate, appreciable fibrosis, or clinically apparent scar. Thus, induced expression of syndecans is not an absolute requirement for wound repair but does correlate with the occurrence of fibrosis in mature skin. These findings support the role of syndecans as regulators of cell behavior and suggest that syndecan-1 and -4 induction in the dermis may contribute to events that lead to inflammation and fibrosis.

Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing.

Chronic wounds represent a major clinical problem with significant morbidity and healthcare expenditures, but no effective therapies. Topical platelet-derived growth factor-BB trials have required large and repeated doses to achieve only a modest effect. We examined the ability of an adenovirus containing the platelet-derived growth factor-B transgene to improve the rate of wound healing through induction of platelet-derived growth factor-B overexpression in cells participating in the wound healing response. We treated excisional wounds in the ischemic rabbit ear, which have a 60% delay in healing, with vehicle, 106, or 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B per wound (n = 19). At 7 d this resulted in a decrease in the epithelial gap from 3.4 +/- 1 mm (mean +/- SD) in vehicle-treated wounds to 1.9 +/- 1.8 mm (mean +/- SD, p < 0.05) when treated with 106 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B, and 0.7 +/- 1.1 mm (mean +/- SD, p < 0.001) when treated with 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B. Ischemic excisional wounds treated with 108 plaque-forming units of an adenovirus containing the platelet-derived growth factor-B even healed more rapidly than non-ischemic excisional wounds treated with vehicle (p < 0.05). In contrast, 5 microg of platelet-derived growth factor-BB protein (n = 2) resulted in only modest granulation tissue at the margin, but no significant differences in epithelial gap (3 +/- 0.6 mm, mean +/- SD). Plaque-forming units (106 or 108) of an adenovirus containing the beta-galactosidase transgene (n = 4) impaired wound re-epithelialization with an epithelial gap of 5.11 +/- 0.69 mm, mean +/- SD, p < 0.004, and 3.8 +/- 0.57 mm, mean +/- SD, p < 0.07, respectively. Adenoviral-mediated gene transfer of platelet-derived growth factor-B overcame the ischemic defect in wound healing and offers promise in the treatment of chronic nonhealing wounds. The vulnerary effects of platelet-derived growth factor-B overexpression were sufficient to overcome the adverse effects of the adenovirus or transgene on wound healing.

Efficient gene expression in skin wound sites following local plasmid injection.

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development.

The spatial and temporal deployment of HOX homeobox genes along the spinal axis and in limb buds during fetal development is a key program in embryonic pattern formation. Although we have previously reported that several of the HOX homeobox genes are expressed during murine skin development, there is no information about developmental expression of HOX genes in human skin. We have now used reverse transcriptase polymerase chain reaction, in conjunction with a set of degenerate oligonucleotide primers, to identify a subset of HOX genes that are expressed during human fetal skin development. In situ hybridization analyses demonstrated that there were temporal and spatial shifts in expression of these genes. Strong HOXA4 expression was detected in the basal cell layers of 10 wk fetal epidermis and throughout the epidermis and dermis of 17 wk skin, whereas weak signal was present in the granular layer of newborn and adult skin. The expression patterns of HOXA5 and HOXA7 were similar, but their expression was weaker. In situ hybridization analysis also revealed strong HOXC4 and weaker HOXB7 expression throughout fetal development, whereas HOXB4 was expressed at barely detectable levels. Differential HOX gene expression was also observed in developing hair follicles, and sebaceous and sweat glands. None of the HOX genes examined were detected in the adult dermis.

Fibrotic healing of adult and late gestation fetal wounds correlates with increased hyaluronidase activity and removal of hyaluronan.

The lack of scarring and fibrosis in healing fetal skin wounds may relate to a prolonged presence of hyaluronan (HA). It has been suggested that fetal wounds may lack hyaluronidase, but the hyaluronidase levels in fetal wounds remain unknown. The size of HA influences its biological action, especially in relation to angiogenesis, which is also reduced in fetal wound healing. The present study determined the levels and size of HA, as well as hyaluronidase levels, in fetal and adult lamb wounds. Wire mesh cylinders, or polyvinyl acetate sponges, were placed subcutaneously in fetal lambs at 75, 100 or 120 days gestation. Wound fluid and wound tissue were harvested 3, 7 or 14 days later. Samples were digested with papain and both HA and hyaluronidase activity were determined in a competitive ELISA assay. Size distribution of HA was estimated using a Sephacryl S1000 column and fractions were collected for HA determination. Adult wound fluid HA remained low (4-5 micrograms/ml) over the 14 days. Fetal fluids were similar on day 3, but increased to 15-25 micrograms/ml by day 7. In 75/100-day wounds, HA remained elevated at 14 days, but in 120-day fluids decreased to levels similar to adult fluid. The HA in all fluids was polydisperse with a main peak at 200 kDa. Hyaluronidase levels were detected in all samples, reaching a peak 7 days post-wounding. In adult wound fluids hyaluronidase was much higher than the fetal wound fluids. These data suggest that lower hyaluronidase levels in fetal wounds may underlie the different pattern of HA deposition seen in fetal wounds.