Epispadias and the associated embryopathies: genetic and developmental basis.

The abnormalities in the urogenital organs are frequently observed as human developmental diseases. Among such diseases, the defects in the upper part of external genitalia are rather rare named epispadias. The cleft in the dorsal part of external genitalia often reaches to the urethra. In general, the urogenital abnormalities accompany defects in the adjacent tissues and organs. The ventral body wall and bladder can also be affected in the patients with dorsal defects of the external genitalia. Therefore, such multiple malformations are often classified as bladder exstrophy and epispadias complex (BEEC). Because of the lower frequency of such birth defects and their early embryonic development, animal models are required to analyze the pathogenic mechanisms and the functions of responsible genes. Mutant mouse analyses on various signal cascades for external genitalia and body wall development are increasingly performed. The genetic interactions between growth factors such as bone morphogenetic proteins (Bmp) and transcription factors such as Msx1/2 and Isl1 have been suggested to play roles for such organogenesis. The significance of epithelial-mesenchymal interaction (EMI) is suggested during development. In this review, we describe on such local interactions and developmental regulators. We also introduce some mutant mouse models displaying external genitalia-body wall abnormalities.

Comprehensive review of the duplication 3q syndrome and report of a patient with Currarino syndrome and de novo duplication 3q26.32-q27.2.

Partial duplications of the long arm of chromosome 3, dup(3q), are a rare but well-described condition, sharing features of Cornelia de Lange syndrome. Around two thirds of cases are derived from unbalanced translocations, whereas pure dup(3q) have rarely been reported. Here, we provide an extensive review of the literature on dup(3q). This search revealed several patients with caudal malformations and anomalies, suggesting that caudal malformations or anomalies represent an inherent phenotypic feature of dup(3q). In this context, we report a patient with a pure de novo duplication 3q26.32-q27.2. The patient had the clinical diagnosis of Currarino syndrome (CS) (characterized by the triad of sacral anomalies, anorectal malformations and a presacral mass) and additional features, frequently detected in patients with a dup(3q). Mutations within the MNX1 gene were found to be causative in CS but no MNX1 mutation could be detected in our patient. Our comprehensive search for candidate genes located in the critical region of the duplication 3q syndrome, 3q26.3-q27, revealed a so far neglected phenotypic overlap of dup(3q) and the Pierpont syndrome, associated with a mutation of the TBL1XR1 gene on 3q26.32.

Mutations in CRLF1 cause familial achalasia.

We here report a family from Libya with three siblings suffering from early onset achalasia born to healthy parents. We analyzed roughly 5000 disease-associated genes by a next-generation sequencing (NGS) approach. In the analyzed sibling we identified two heterozygous variants in CRLF1 (cytokine receptor-like factor 1). Mutations in CRLF1 have been associated with autosomal recessive Crisponi or cold-induced sweating syndrome type 1 (CS/CISS1), which among other symptoms also manifests with early onset feeding difficulties. Segregation analysis revealed compound heterozygosity for all affected siblings, while the unaffected mother carried the c.713dupC (p.Pro239Alafs*91) and the unaffected father carried the c.178T>A (p.Cys60Ser) variant. The c.713dupC variant has already been reported in affected CS/CISS1 patients, the pathogenicity of the c.178T>A variant was unclear. As reported previously for pathogenic CRLF1 variants, cytokine receptor-like factor 1 protein secretion from cells transfected with the c.178T>A variant was severely impaired. From these results we conclude that one should consider a CRLF1-related disorder in early onset achalasia even if other CS/CISS1 related symptoms are missing.

Comparison of environmental risk factors for esophageal atresia, anorectal malformations, and the combined phenotype in 263 German families.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) and anorectal malformations (ARM) represent the severe ends of the fore- and hindgut malformation spectra. Previous research suggests that environmental factors are implicated in their etiology. These risk factors might indicate the influence of specific etiological mechanisms on distinct developmental processes (e.g. fore- vs. hindgut malformation). The present study compared environmental factors in patients with isolated EA/TEF, isolated ARM, and the combined phenotype during the periconceptional period and the first trimester of pregnancy in order to investigate the hypothesis that fore- and hindgut malformations involve differing environmental factors. Patients with isolated EA/TEF (n = 98), isolated ARM (n = 123), and the combined phenotype (n = 42) were included. Families were recruited within the context of two German multicenter studies of the genetic and environmental causes of EA/TEF (great consortium) and ARM (CURE-Net). Exposures of interest were ascertained using an epidemiological questionnaire. Chi-square, Fisher's exact, and Mann-Whitney U-tests were used to assess differences between the three phenotypes. Newborns with isolated EA/TEF and the combined phenotype had significantly lower birth weights than newborns with isolated ARM (P = 0.001 and P < 0.0001, respectively). Mothers of isolated EA/TEF consumed more alcohol periconceptional (80%) than mothers of isolated ARM or the combined phenotype (each 67%). Parental smoking (P = 0.003) and artificial reproductive techniques (P = 0.03) were associated with isolated ARM. Unexpectedly, maternal periconceptional multivitamin supplementation was most frequent among patients with the most severe form of disorder, i.e. the combined phenotype (19%). Significant differences in birth weight were apparent between the three phenotype groups. This might be attributable to the limited ability of EA/TEF fetuses to swallow amniotic fluid, thus depriving them of its nutritive properties. Furthermore, the present data suggest that fore- and hindgut malformations involve differing environmental factors. Maternal periconceptional multivitamin supplementation was highest among patients with the combined phenotype. This latter finding is contrary to expectation, and warrants further analysis in large prospective epidemiological studies.

A premature termination mutation in a patient with Lowe syndrome without congenital cataracts: dropping the "O" in OCRL.

The oculocerebrorenal syndrome of Lowe is an X-linked recessive disorder characterized by the triad of congenital cataracts, mental retardation and a renal proximal tubulopathy. Although severity of phenotype might vary, congenital cataracts are part of the definition of this rare disorder.We report a 13-year-old patient with the typical cerebrorenal phenotype of Lowe syndrome, that had remained undiagnosed due to absence of any ocular involvement. OCRL gene analysis was carried.DNA analysis revealed a c.C760T (p.Gln199X) nonsense mutation in exon 8 expected to cause complete disruption of OCRL function. After sequencing the parents of the index patient and his maternal grandparents, this mutation turned out to be de novo in the mother. Furthermore, a silent variant (p.Arg35=) was identified in exon 2, that could also be identified in the mother and her 3 sisters, but not in the grandparents assuming germ cell mosaicism in either of the grandparents. RNA analysis from the patient's lymphocytes revealed presence of full-length OCRL transcripts. Western blotting from lymphocyte samples failed to detect OCRL protein even in controls.Our findings extend the phenotypic spectrum caused by OCRL mutations and illustrate that there may be selective organ involvement in Lowe syndrome.

Familial translocation t(6;20)(p21;p13) resulting in partial trisomy 6p and partial monosomy 20p: report of a new case and review of the literature.

Carriers of completely balanced chromosomal translocations have all necessary genetic information. Nevertheless, because of the possibility of maldistribution during gametogenesis, they are at increased risk for infertility, miscarriage, stillbirth or having a child with congenital anomalies including mental retardation. As postnatal clinical reports are infrequent, prediction of clinical course for specific unbalanced karyotypes diagnosed during pregnancy remains difficult. Here, we report the 6th case of partial trisomy 6p and partial monosomy 20p due to an unbalanced adjacent-1 segregation of the rare familial translocation t(6;20)(p21;p13). We give a thorough clinical description of the present case, demonstrating broad phenotypic overlap with the 5 previously published cases reviewed here, providing important data on postnatal outcome.

[Transition of adolescents with the exstrophy-epispadias complex to adult medicine: influence of long-term outcome results on management]. / Transition von Jugendlichen mit Blasenekstrophie-Epispadie Komplex in die Erwachsenenmedizin: Implikationen des Langzeitverlaufes für das Management.

Today, young individuals with rare congenital anomalies as the Exstrophy-Epispadias-Complex (EEC) are mostly monitored interdisciplinary with a high standard of care and enthusiasm during childhood. However, when growing up through adolescence to adulthood adequate care-givers are not available at the moment in adult medicine in Germany and a concrete transition process has yet not been established. Over the past years, we put much effort in systematic evaluation of long-term outcome after reconstruction of the EEC in the newborn period to further improve outcome results. Beside predictive parameters for continence and long-term bladder function, genital function and fertility, as well as postoperative pelvic floor morphology and gynecological outcome, orthopedic results and psychosexual and psychosocial development in EEC were of major interest. As a consequence we currently develop a German-wide follow-up concept in EEC patients regarding age- and gender specific outcome issues. Long-term observations of the EEC outcome however, underline the unrestricted importance of careful long-term follow-up of all EEC patients, as well as the necessity of close cooperation of pediatric urologist, pediatric surgeons, urologists, orthopedic surgeons, gynecologists, andrologists, psychologists and urotherapists from early childhood and the need of knowledge transfer and hopefully a successful transition of the EEC individuals to general medicine.

Practice of dilatation after surgical correction in anorectal malformations.

BACKGROUND: In order to prevent stricture of the neoanus after surgical correction, regular dilatation is recommended. There is a lack of knowledge about the performance of anal dilatation and the occurrence of pain. The aim of our investigation was to describe the practice of dilatation and to identify possible risk factors for painful procedures. METHODS: Congenital Uro-Rectal Malformations Network is a German interdisciplinary multicenter research network. With standard questionnaires, physicians interviewed 243 patients and/or their parents at home, additional 103 patients born since 2009 were assessed through their treating physicians. RESULTS: In total, 88 % of the patients received dilatations. Treatment lasted for 7 months in median (range 1-156 months), until the age of 13 months (range 1-171 months). In 69 % painful dilatation was reported; without a significant differences in age or gender. In 32 % bleeding was reported. In 30 % at least one dilatation was performed under general anesthesia. In 11 % some kind of analgesia was used. Type of fistula, dilatations lasting longer than 10 months and Hegar size above 15 were relevant factors for experience of pain. There were about 16 % postoperative strictures of the neoanus, without reported differences in dilatation procedures; but there was a relation to type of malformation. CONCLUSION: Considering the high number of painful treatments, predictors for painful dilatations should be further clarified through standardized documentation and prospective evaluation in order to improve follow-up.

Brain Abnormalities in Patients with Germline Variants in H3F3: Novel Imaging Findings and Neurologic Symptoms Beyond Somatic Variants and Brain Tumors.

BACKGROUND AND PURPOSE: Pathogenic somatic variants affecting the genes Histone 3 Family 3A and 3B (H3F3) are extensively linked to the process of oncogenesis, in particular related to central nervous system tumors in children. Recently, H3F3 germline missense variants were described as the cause of a novel pediatric neurodevelopmental disorder. We aimed to investigate patterns of brain MR imaging of individuals carrying H3F3 germline variants. MATERIALS AND METHODS: In this retrospective study, we included individuals with proved H3F3 causative genetic variants and available brain MR imaging scans. Clinical and demographic data were retrieved from available medical records. Molecular genetic testing results were classified using the American College of Medical Genetics criteria for variant curation. Brain MR imaging abnormalities were analyzed according to their location, signal intensity, and associated clinical symptoms. Numeric variables were described according to their distribution, with median and interquartile range. RESULTS: Eighteen individuals (10 males, 56%) with H3F3 germline variants were included. Thirteen of 18 individuals (72%) presented with a small posterior fossa. Six individuals (33%) presented with reduced size and an internal rotational appearance of the heads of the caudate nuclei along with an enlarged and squared appearance of the frontal horns of the lateral ventricles. Five individuals (28%) presented with dysgenesis of the splenium of the corpus callosum. Cortical developmental abnormalities were noted in 8 individuals (44%), with dysgyria and hypoplastic temporal poles being the most frequent presentation. CONCLUSIONS: Imaging phenotypes in germline H3F3-affected individuals are related to brain features, including a small posterior fossa as well as dysgenesis of the corpus callosum, cortical developmental abnormalities, and deformity of lateral ventricles.

De novo partial trisomy 18p and partial monosomy 18q in a patient with anorectal malformation.

Anorectal malformations (ARM) encompass a broad clinical spectrum which ranges from mild anal stenosis to severe anorectal anomalies such as complex cloacal malformations. The overall incidence of ARM is around 1 in every 2,500 live births. Although causative genes for a few syndromic forms have been identified, the molecular genetic background of most ARM remains unknown. The present report describes a patient with a de novo 13.2-Mb deletion of chromosome 18q22.3-qter and a 2.2-Mb de novo duplication of chromosomal region 18pter-p11.32 located at the telomeric end of chromosome 18q. The patient presented with ARM and the typical features of 18q- syndrome (De-Grouchy syndrome). The combination of a partial duplication of the short arm and a partial deletion of the long arm of chromosome 18 has been described in 16 previous cases. However, this is the first report of an association between this complex chromosomal rearrangement and ARM.

Epidemiological survey of 214 families with bladder exstrophy-epispadias complex.

PURPOSE: We sought to identify causative nongenetic and genetic risk factors for the bladder exstrophy-epispadias complex. MATERIALS AND METHODS: A total of 237 families with the bladder exstrophy-epispadias complex were invited to participate in the study, and information was obtained from 214 families, mainly from European countries. RESULTS: Two families showed familial occurrence. Male predominance was found among all subgroups comprising epispadias, classic bladder exstrophy and cloacal exstrophy, with male-to-female ratios of 1.4:1, 2.8:1 and 2.0:1, respectively (p = 0.001). No association with parental age, maternal reproductive history or periconceptional maternal exposure to alcohol, drugs, chemical noxae, radiation or infections was found. However, periconceptional maternal exposure to smoking was significantly more common in patients with cloacal exstrophy than in the combined group of patients with epispadias/classic bladder exstrophy (p = 0.009). Only 16.8% of mothers followed the current recommendations of periconceptional folic acid supplementation, and 17.6% had started supplementation before 10 weeks of gestation. Interestingly, in the latter group mothers of patients with cloacal exstrophy were more compliant with folic acid supplementation than were mothers of the combined group of patients with epispadias/classic bladder exstrophy (p = 0.037). Furthermore, mothers of children with cloacal exstrophy knew significantly more often prenatally that their child would have a congenital malformation than did mothers of children with epispadias/classic bladder exstrophy (p <0.0001). CONCLUSIONS: Our study corroborates the hypothesis that epispadias, classic bladder exstrophy and cloacal exstrophy are causally related, representing a spectrum of the same developmental defect, with a small risk of recurrence within families. Embryonic exposure to maternal smoking appears to enforce the severity, whereas periconceptional folic acid supplementation does not seem to alleviate it. There is a disproportional prenatal ultrasound detection rate between severe and mild phenotypes, possibly due to the neglect of imaging of full bladders with a focus on neural tube defects.

Candidate Genes for Nonsyndromic Cleft Palate Detected by Exome Sequencing.

Nonsyndromic cleft palate only (nsCPO) is a facial malformation that has a livebirth prevalence of 1 in 2,500. Research suggests that the etiology of nsCPO is multifactorial, with a clear genetic component. To date, genome-wide association studies have identified only 1 conclusive common variant for nsCPO, that is, a missense variant in the gene grainyhead-like-3 ( GRHL3). Thus, the underlying genetic causes of nsCPO remain largely unknown. The present study aimed at identifying rare variants that might contribute to nsCPO risk, via whole-exome sequencing (WES), in multiply affected Central European nsCPO pedigrees. WES was performed in 2 affected first-degree relatives from each family. Variants shared between both individuals were analyzed for their potential deleterious nature and a low frequency in the general population. Genes carrying promising variants were annotated for 1) reported associations with facial development, 2) multiple occurrence of variants, and 3) expression in mouse embryonic palatal shelves. This strategy resulted in the identification of a set of 26 candidate genes that were resequenced in 132 independent nsCPO cases and 623 independent controls of 2 different ethnicities, using molecular inversion probes. No rare loss-of-function mutation was identified in either WES or resequencing step. However, we identified 2 or more missense variants predicted to be deleterious in each of 3 genes ( ACACB, PTPRS, MIB1) in individuals from independent families. In addition, the analyses identified a novel variant in GRHL3 in 1 patient and a variant in CREBBP in 2 siblings. Both genes underlie different syndromic forms of CPO. A plausible hypothesis is that the apparently nonsyndromic clefts in these 3 patients might represent hypomorphic forms of the respective syndromes. In summary, the present study identified rare variants that might contribute to nsCPO risk and suggests candidate genes for further investigation.

[Genetic and molecular biological aspects of the bladder exstrophy-epispadias complex (BEEC)]. / Genetische und molekularbiologische Aspekte des Blasenekstrophie-Epispadie-Komplexes (BEEK).

The bladder exstrophy and epispadias complex (BEEC) is an anterior midline defect with variable expression involving the infraumbilical abdominal wall including the pelvis, urinary tract, and external genitalia. The incidence varies with regard to ethnical background, sex, and phenotypic expression, and an incidence of 1:20,000 to 1:80,000 has been observed in the middle European population. No gene defect has been attributed to BEEC thus far and chromosomal aberrations or genetic syndromes associated with BEEC have only rarely been reported. According to epidemiological data, a complex genetic as well as a multifactorial mode of inheritance could underlie BEEC. However, no single teratogenic agent or environmental factor has been identified, which could play a dominant role in the expression of the BEEC.A risk of recurrence of 0.5-3% has been described in families with one affected subject. These values correspond to an increased recurrence risk estimated to be as high as 200- to 800-fold when compared to the common population. Due to the paucity of affected sib pairs and suitable multiplex families, conventional linkage analysis to identify candidate genes causally related with BEEC appears to be unfeasible. Large association studies and consecutive linkage disequilibrium mapping should therefore lead to the identification of candidate genes. Also new methods including matrix-based comparative genomic hybridization (CGH) are promising and have successfully been used in the past (e.g., CHARGE association). Moreover, the low incidence of the BEEC requires close cooperation between clinicians in the operative and nonoperative specialties as well as geneticists for successful gene search.

[Subjective developmental outcome in bladder exstrophy and epispadias. A pilot study]. / Entwicklungsergebnis bei Blasenekstrophie und Epispadie aus Patientensicht Eine Pilotstudie.

The bladder exstrophy-epispadias complex (BEEC) belongs to one of the most devastating urological malformations affecting the complete urinary tract including the genitalia. A semi-standardized questionnaire was used to evaluate the functional and psychosocial developmental outcome in 122 affected patients recruited from German-speaking self-referral support groups. The questionnaire covered mode of reconstruction, subjective assessment of continence, milestones of child development, education, school performance, level of life satisfaction, anxieties, and in patients older than 16 years partnership experience. The results showed a mean of one surgery in the age group 0-4, of four surgeries in both age groups 5-13 and 14-20, and seven surgeries in patients aged 20 and over. The continence rates for the latter three age groups were 36, 64, and 80%, respectively. We found that the neurocognitive development was unremarkable. Most patients showed above average performance in school and in their profession. On the other hand, they exhibited psychological anxieties and worries in their experience with sexuality and partnerships. Future studies will be necessary to clarify the association of urological status, functional impairments, and psychosocial adaptation as a basis for improved approaches to comprehensive care and support.

Microdeletions including FMR1 in three female patients with intellectual disability - further delineation of the phenotype and expression studies.

Fragile X syndrome (FXS) is one of the most common causes of intellectual disability/developmental delay (ID/DD), especially in males. It is caused most often by CGG trinucleotide repeat expansions, and less frequently by point mutations and partial or full deletions of the FMR1 gene. The wide clinical spectrum of affected females partly depends on their X-inactivation status. Only few female ID/DD patients with microdeletions including FMR1 have been reported. We describe 3 female patients with 3.5-, 4.2- and 9.2-Mb de novo microdeletions in Xq27.3-q28 containing FMR1. X-inactivation was random in all patients, yet they presented with ID/DD as well as speech delay, macrocephaly and other features attributable to FXS. No signs of autism were present. Here, we further delineate the clinical spectrum of female patients with microdeletions. FMR1 expression studies gave no evidence for an absolute threshold below which signs of FXS present. Since FMR1 expression is known to be highly variable between unrelated females, and since FMR1 mRNA levels have been suggested to be more similar among family members, we further explored the possibility of an intrafamilial effect. Interestingly, FMR1 mRNA levels in all 3 patients were significantly lower than in their respective mothers, which was shown to be specific for patients with microdeletions containing FMR1.

Strong association of variants around FOXE1 and orofacial clefting.

Nonsyndromic orofacial clefting (nsOFC) is a common, complex congenital disorder. The most frequent forms are nonsyndromic cleft lip with or without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO). Although they are generally considered distinct entities, a recent study has implicated a region around the FOXE1 gene in both nsCL/P and nsCPO. To investigate this hypothesis, we analyzed the 2 most strongly associated markers (rs3758249 and rs4460498) in 2 independent samples of differing ethnicities: Central European (949 nsCL/P cases, 155 nsCPO cases, 1163 controls) and Mayan Mesoamerican (156 nsCL/P cases, 10 nsCPO cases, 338 controls). While highly significant associations for both single-nucleotide polymorphisms were obtained in nsCL/P (rs4460498: p Europe = 6.50 × 10(-06), p Mayan = .0151; rs3758249: p Europe = 2.41 × 10(-05), p Mayan = .0299), no association was found in nsCPO (p > .05). Genotyping of rs4460498 in 472 independent European trios revealed significant associations for nsCL/P (p = .016) and nsCPO (p = .043). A meta-analysis of all data revealed a genomewide significant result for nsCL/P (p = 1.31 × 10(-08)), which became more significant when nsCPO cases were added (p nsOFC = 1.56 × 10(-09)). These results strongly support the FOXE1 locus as a risk factor for nsOFC. With the data of the initial study, there is now considerable evidence that this locus is the first conclusive risk factor shared between nsCL/P and nsCPO.

VATER/VACTERL Association: Evidence for the Role of Genetic Factors.

The VATER/VACTERL association is typically defined by the presence of at least 3 of the following congenital malformations: Vertebral anomalies, Anal atresia, Cardiac malformations, Tracheo-Esophageal fistula, Renal anomalies, and Limb abnormalities. The involvement of genetic factors in the development of this rare association is suggested by reports of familial occurrence, the increased prevalence of component features among first-degree relatives of affected individuals, high concordance rates among monozygotic twins, chromosomal (micro-)aberrations or single gene mutations in individuals with the VATER/VACTERL phenotype, as well as murine knock-out models. Despite substantial efforts over the past decade, the genetic etiology of the VATER/VACTERL association in most instances remains elusive. The application of new genomic technologies such as high-resolution copy number variation studies or next-generation exome sequencing might lead to the identification of some of these causes.

[Duplication of the alimentary tract with ectopic gastric mucosa as cause of lower abdominal pain]. / Dünndarmduplikatur mit ektoper Magenschleimhaut als Ursache chronischer Unterbauchschmerzen.

Duplications of the alimentary tract are rare congenital anomalies, which may be difficult to diagnose. They cause unspecific symptoms and may mimic appendicitis. The authors report the case of a 9-year-old boy with recurrent pain in the right lower abdomen who underwent laparoscopic appendectomy. The painful episodes were, however, caused by a duplication with ectopic gastric mucosa, which had lead to an ulceration and an intussusception. Sonographically using a high resolution linear probe (7.5 MHz) the ileal intussusception could be demonstrated.

[Questionable effectiveness of autologous platelet growth factors (PDWHF) in treatment of venous ulcers of the leg]. / Fragliche Wirksamkeit autologer thrombozytärer Wachstumsfaktoren (PDWHF) in der Behandlung venöser Beinulzera.

Chronic venous insufficiency (CVI) can cause ulcers of the lower limb having the character of a full thickness wound involving the subcutaneous tissues and fat. Healing requires wound contraction, connective tissue formation and finally reepithelialization. To induce wound healing, on an underlying disturbed environment due to longterm effects of CVI, artificial stimuli may be needed. In a placebo controlled study we tried topical application of autologous PDWHF (platelet derived wound healing factors), to achieve ulcer healing and improve the microangiopathy surrounding of the ulcer area, as there are decreased number of skin capillaries and reduction in cutaneous vascular reserve. Alterations of cutaneous circulation during the course of the study were documented by capillaroscopy, transcutaneous oxygen pressure and laser Doppler flux (LDF) measurements. We were able to recruit 15 patients a I suffering from chronic nonhealing venous stasis ulcers. Eleven of the 15 patients agreed to participate in a placebo-controlled double blind study, whereas 4 patients agreed to participate only if they would be treated with PDWHF. The median age and duration of ulceration of the 6 patients (3 male/3 female) treated with placebo were 71 years and 1089 days. The median age and duration of ulceration of the 9 patients (1 male/8 females) treated with PDWHF were 66 years and 732 days. Duration of therapy for the PDWHF group was 91 days, as compared to 154 days for the placebo group. Despite 2 completely healed ulcers, the expensive treatment did not reveal any significant clinical advantage. In den PDWHF group an ulcer area of 26.9 cm2 was measured at the beginning, of 26.2 cm2 at the end; in the placebo group, 34.7 cm2 and 35.5 cm2. The nonsignificant increase of the capillary density at the ulcer border in the active group as well as the increase in the tcPO2, in contrast to little change in both parameters in the placebo group, suggests neoangiogenic abilities to PDWHF, secondarily leading to a better blood distribution with higher oxygen tension.