Nicolas Kalfa

Is hypospadias a genetic, endocrine or environmental disease, or still an unexplained malformation?

Hypospadias is one of the most frequent genital malformations in the male newborn and results from an abnormal penile and urethral development. This process requires a correct genetic programme, time- and space-adapted cellular differentiation, complex tissue interactions, and hormonal mediation through enzymatic activities and hormonal transduction signals. Any disturbance in these regulations may induce a defect in the virilization of the external genitalia and hypospadias. This malformation thus appears to be at the crossroads of various mechanisms implicating genetic and environmental factors. The genes of penile development (HOX, FGF, Shh) and testicular determination (WT1, SRY) and those regulating the synthesis [luteinizing hormone (LH) receptor] and action of androgen (5alpha reductase, androgen receptor) can cause hypospadias if altered. Several chromosomal abnormalities and malformative syndromes include hypospadias, from anterior to penoscrotal forms. More recently, CXorf6 and ATF3 have been reported to be involved. Besides these genomic and hormonal factors, multiple substances found in the environment can also potentially interfere with male genital development because of their similarity to hormones. The proportion of hypospadias cases for which an aetiology is detected varies with the authors but it nevertheless remains low, especially for less severe cases. An interaction between genetic background and environment is likely.

Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study

Prenatal diethylstilbestrol (DES)-exposed mice have raised the suspicion of a transgenerational effect in the occurrence of genital malformation in males. This nationwide cohort study in collaboration with a French association of DES-exposed women studied 529 families and showed that a significant proportion of boys born to DES daughters exhibited hypospadias with no other molecular defects identified.

Prenatal environmental risk factors for genital malformations in a population of 1442 French male newborns: a nested case–control study

BACKGROUND Over the past decades, an increasing trend in male external genital malformations such as cryptorchidism and hypospadias has led to the suspicion that environmental chemicals are detrimental to male fetal sexual development. Several environmental pollutants, including organochlorine pesticides, polychlorinated biphenyls, bisphenol A, phthalates, dioxins and furans have estrogenic and anti-androgenic activity and are thus considered as endocrine-disrupting chemicals (EDCs). Since male sex differentiation is critically dependent on the normal production and action of androgens during fetal life, EDCs may be able to alter normal male sex differentiation. OBJECTIVE The objective of this study was to determine the incidence of external genital malformations in a population of full-term newborn males in southern France. We also performed a case-control study to identify the risk factors for male external genital malformations, with a focus on parental occupational exposure to EDCs. METHODS Over a 16-month period, 1615 full-term newborn males with a birth weight above 2500 g were registered on a level-1 maternity ward, and the same pediatrician systematically examined 1442 of them (89%) for cryptorchidism, hypospadias and micropenis. For every male newborn with genital malformation, we enrolled nearly two males matched for age, parity and term. All parents of the case and control newborns were interviewed about pregnancy aspects, personal characteristics, lifestyle and their occupational exposure to EDCs using a detailed questionnaire. RESULTS We report 39 cases of genital malformation (2.70%), with 18 cases of cryptorchidism (1.25%), 14 of hypospadias (0.97%), 5 of micropenis (0.35%) and 2 of 46,XY disorders of sexual differentiation (DSD; 0.14%). We observed a significant relationship between newborn cryptorchidism, hypospadias or micropenis and parental occupational exposure to pesticides [odds ratio (OR) = 4.41; 95% confidence interval (95% CI), 1.21-16.00]. Familial clustering for male external genital malformations (OR = 7.25; 95% CI, 0.70-74.30) and medications taken by mothers during pregnancy (OR = 5.87; 95% CI, 0.93-37.00) were associated with the risk of cryptorchidism, hypospadias and micropenis, although the association was not statistically significant. CONCLUSIONS Although the causes of male genital malformation are multifactorial, our data support the hypothesis that prenatal contamination by pesticides may be a potential risk factor for newborn male external genital malformation and it should thus be routinely investigated in all undervirilized newborn males.

Hypospadias: interactions between environment and genetics

Hypospadias is one of the most common congenital malformations. It is considered to be a mild form of the 46,XY disorders of sex development (DSD), but its precise etiology remains to be elucidated. Compromised androgen synthesis or effects can cause this frequent malformation, although the mutational analyses of the genes involved in androgen actions have identified abnormalities in only a very small portion of patients. The overwhelming majority of cases remain unexplained and hypospadias may be a highly heterogeneous condition subject to multiple genetic and environmental factors. We here review the recent advances in this field and discuss the potential interactions between the environment and genetics.

Mutations of CXorf6 are associated with a range of severities of hypospadias

OBJECTIVE Mutations in chromosome X open reading frame 6 (CXorf6), a recently described candidate gene involved in the development of male genitalia, have been found in patients with complex 46,XY disorders of sexual development (46,XY DSD) including micropenis, bifid scrotum, and penoscrotal hypospadias. The objective of this work was to identify genomic variants of CXorf6 in patients with isolated hypospadias, severe or non-severe. DESIGN AND METHODS Forty-one patients with glandular to perineal hypospadias and thirty controls were studied. Direct sequencing for coding exons 3-6 of CXorf6 and their flanking splice sites was performed on DNA extracted from foreskin collected from surgery. Secondary and tertiary structures of the protein were predicted using NNpredict and Protein Homology/analogY Recognition Engine engines. RESULTS Four mutations (9.7% of cases) were identified. One missense mutation (1295T>C, V432A) and two deletions (325delG, predicted to cause a stop codon L121X) occurred in patients with penoscrotal and proximal hypospadias. One patient with subcoronal hypospadias had CAG-repeat amplification in the second polyglutamine domain of CXorf6. Secondary structure prediction indicated that this insertion occurred in a helix element of the protein. The tertiary structure prediction showed an alteration of the shape of the protein and crowding between domains. CONCLUSION CXorf6 mutations are associated with isolated hypospadias of varying severity. However, the pathophysiology of these mutations and the function of the CXorf6 gene product remain to be investigated.

Tolerance of Laparoscopy and Thoracoscopy in Neonates

Objectives. Video-surgery in neonates is recent. Data on the respiratory, hemodynamic, and thermic effects during the first month of life are still sparse. This study aimed to evaluate the tolerance of video-surgery in neonates and to determine the risk factors of per-operative complications. Methods. From 1994 to 2004, 49 neonates (mean age: 11 days; weight: 3285 g) underwent 50 video-surgical procedures. Indications for laparoscopy were duodenal atresias, volvulus with malrotation, pyloric stenosis, gastroesophageal reflux, cystic lymphangiomas, ovarian cysts, biliary atresia, and congenital diaphragmatic hernias; indications for thoracoscopy were esophageal atresias and tracheoesophageal fistula. Results. Median operative time was 79 minutes. Mean insufflation pressure was 6.7 mm Hg (range: 3–13). Oxygen saturation decreased, especially with thoracic insufflation or high-pressure pneumoperitoneum. Systolic arterial pressure, which decreased in 20% of the patients, was controlled easily with vascular expansion. Thermic loss (mean postoperative temperature: 35.6°C) was proportional to the duration of insufflation. No surgical incident was noted. Ten anesthetic incidents occurred (20%), 3 of which required temporary or definitive interruption of insufflation (O2 saturation <70%). Risk factors for an incident were low preoperative temperature, high variation of end-tidal pressure of CO2, surgical time >100 minutes, thoracic insufflation, and a high oxygen or vascular expansion requirement at the beginning of insufflation. Conclusion. The neonates high sensitivity to insufflation is an important limiting factor of video-surgery. The described profile of the neonate at risk may help to reduce the frequency of adverse effects of this technique and improve its tolerance.

Multicentric assessment of the safety of neonatal videosurgery

BackgroundComplex procedures for managing congenital abnormalities are reported to be feasible. However, neonatal videosurgery involves very specific physiologic constraints. This study evaluated the safety and complication rate of videosurgery during the first month of life and sought to determine both the risk factors of perioperative complications and the most recent trends in practice.MethodsFrom 1993 to 2005, 218 neonates (mean age, 16 days; weight, 3,386 g) from seven European university hospitals were enrolled in a retrospective study. The surgical indications for laparoscopy (n = 204) and thoracoscopy (n = 14) were congenital abnormalities or exploratory procedures.ResultsOf the 16 surgical incidents that occurred (7.5%), mainly before 2001, 11 were minor (parietal hematoma, eventration). Three neonates had repeat surgery for incomplete treatment of pyloric stenosis. In two cases, the incidents were more threatening (duodenal wound, diaphragmatic artery injury), but without further consequences. No mortality is reported. The 26 anesthetic incidents (12%) that occurred during insufflation included desaturation (<80% despite 100% oxygen ventilation) (n = 8), transient hypotension requiring vascular expansion (n = 7), hypercapnia (>45 mmHg) (n = 5), hypothermia (<34.9°C) (n = 4), and metabolic acidosis (n = 2). The insufflation had to be stopped in 7% of the cases (transiently in 9 cases, definitively in 6 cases). The significant risk factors for an incident (p < 0.05) were young age of the patient, low body temperature, thoracic insufflation, high pressure and flow of insufflation, and length of surgery.ConclusionDespite advances in miniaturizing of instruments and growth in surgeons’ experience, the morbidity of neonatal videosurgery is not negligible. A profile of the patient at risk for an insufflation-related incident emerged from this study and may help in the selection of neonates who will benefit most from these techniques in conditions of maximal safety.

Genomic Variants of ATF3 in Patients With Hypospadias

PURPOSE ATF3, an estrogen responsive gene expressed during genital development, could be implicated in the etiology of hypospadias. ATF3 is up-regulated in the foreskin of patients with hypospadias and is implicated in suppression of the cell cycle, which may interfere with urethral cell growth. We sought to investigate the sequence of ATF3 in patients with hypospadias. MATERIAL AND METHODS Direct sequencing of coding exons and splice sites of ATF3 was performed in 41 boys with hypospadias and 30 controls. In addition, ATF3 expression in 1 human fetal penis with and 1 without hypospadias was studied by immunohistochemical analysis. RESULTS A missense variant (L23M) was identified in a boy with anterior hypospadias. This amino acid is highly conserved. Three genomic variants (C53070T, C53632A, Ins53943A) were found in or close to exon 6 in patients with perineal, penoscrotal and anterior hypospadias. This important exon includes splice sites for an alternative transcript (ATF3DeltaZip) that have been implicated in regulation of the function of ATF3. None of these genomic variants was present in controls. Immunochemical analysis revealed that in normal fetuses ATF3 is not expressed in and around the urethra, while in patients with hypospadias ATF3 is over expressed in the urethral plate and subcutaneous tissue, especially around the ectopic orifice of the urethra. CONCLUSIONS Genomic variants of ATF3 are present in 10% of our patients with hypospadias. We also report an abnormal expression pattern of ATF3 in a hypospadiac fetus. The direct implication of ATF3 in the occurrence of hypospadias remains to be confirmed by functional studies of the genomic variants we describe.

Ideal timing of thoracoscopic decortication and drainage for empyema in children

Background: Thoracoscopy may be required for resistant empyema in children. This study aimed to determine the advantages of thoracoscopy performed soon after diagnosis and its ideal timing. Methods: Between 1996 and 2002, 21 children who had undergone thoracoscopy as an initial procedure or after failure of medical treatment were retrospectively reviewed. The study compared outcome of early thoracoscopy (fewer than 4 days after diagnosis) and that of later surgery. The 4-day limit was chosen for physiopathologic reasons (organization of pleuresia in 72 h). Results: In the early thoracoscopy group, the findings showed shorter operative time (p = 0.03) and postoperative hospital stay (p < 0.05), fewer technical difficulties, fewer complications, and no recourse to other surgical procedures. Conclusions: Early thoracoscopy is greatly beneficial for children with empyema by shortening disease progression. An initial short trial of medical treatment for nonorganising empyema may be attempted on the condition that thoracoscopy not be delayed more than 4 days.

Is Hypospadias Associated with Prenatal Exposure to Endocrine Disruptors? A French Collaborative Controlled Study of a Cohort of 300 Consecutive Children Without Genetic Defect

BACKGROUND Numerous studies have focused on the association between endocrine-disrupting chemicals (EDCs) and hypospadias. Phenotype variability, the absence of representative comparison groups and concomitant genetic testing prevent any definitive conclusions. OBJECTIVE To identify the role of occupational and environmental exposures to EDCs in nongenetic isolated hypospadias. DESIGN, SETTING, AND PARTICIPANTS A total of 408 consecutive children with isolated hypospadias and 302 normal boys were prospectively included (2009-2014) in a multi-institutional study in the south of France, the area of the country with the highest prevalence of hypospadias surgery. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS In patients without AR, SRD5A2, and MAMLD1 mutations, parental occupational and professional exposures to EDCs were evaluated based on European questionnaire QLK4-1999-01422 and a validated job-exposure matrix for EDCs. Environmental exposure was estimated using the zip code, the type of surrounding hazards, and distance from these hazards. Multivariate analysis was performed. RESULTS Fetal exposure to EDCs around the window of genital differentiation was more frequent in the case of hypospadias (40.00% vs 17.55%, odds ratio 3.13, 95% confidence interval 2.11-4.65). The substances were paints/solvents/adhesives (16.0%), detergents (11.0%), pesticides (9.0%), cosmetics (5.6%), and industrial chemicals (4.0%). Jobs with exposure were more frequent in mothers of hypospadiac boys (19.73% vs 10.26%, p=0.0019), especially cleaners, hairdressers, beauticians, and laboratory workers. Paternal job exposure was more frequent in the cases of hypospadias (40.13% vs 27.48%, p=0.02). Industrial areas, incinerators, and waste areas were more frequent within a 3-km radius for mothers of hypospadiac boys (13.29% vs. 6.64%, p<0.00005). Association of occupational and environmental exposures increases this risk. CONCLUSIONS This multicenter prospective controlled study with a homogeneous cohort of hypospadiac boys without genetic defects strongly suggests that EDCs are a risk factor for hypospadias through occupational and environmental exposure during fetal life. The association of various types of exposures may increase this risk. PATIENT SUMMARY Our multi-institutional study showed that parental professional, occupational, and environmental exposures to chemical products increase the risk of hypospadias in children.