ESX1 gene expression as a robust marker of residual spermatogenesis in azoospermic men.

BACKGROUND: It would be of value to identify ongoing spermatogenesis molecular markers which can predict successful sperm recovery in patients with non-obstructive azoospermia undergoing conventional or microsurgical testicular sperm extraction (TESE/microTESE). ESX1 is an X-linked homeobox gene expressed in testis, placenta, brain and lung in humans and specifically in pre- and post-meiotic germ cells of the testis in mice. METHODS: We investigated the sequence, expression (by RT-PCR) and epigenetic status (by promoter pyrosequencing) of ESX1 in testicular tissue samples, obtained from 81 azoospermic subjects in the context of surgical sperm extraction, to check a possible association between ESX1 alterations and impaired spermatogenesis, as determined by histological analysis. RESULTS: The ESX1 transcript was detected in 100% of cases diagnosed as obstructive azoospermia (33), hypospermatogenesis (18) and incomplete maturation arrest (MA) (2), and sperm recovery was also successful in 100% of these cases. ESX1 mRNA was also detected in 5 of 6 patients with incomplete Sertoli cell-only syndrome, in 4 of 6 subjects with complete MA but in only 3 of 16 cases of complete Sertoli cell-only syndrome (cSCOS), whereas sperm recovery was successful in 4 of 6, 2 of 6 and 5 of 16 of these patients, respectively. In cases of focal spermatogenesis, ESX1 expression and sperm retrieval were concordant in 14 of 19 (74%) cases subjected to TESE, but in only 3 of 11 (27%) men who underwent microTESE. With TESE, but not with microTESE, both samples originated from adjacent testicular areas. The pyrosequencing of the ESX1 CpG island revealed methylation levels that were significantly lower in ESX1 expressors when compared with non-expressors. CONCLUSIONS: ESX1 emerges as a potentially reliable spermatogenesis molecular marker, whose clinical value as a predictor of successful sperm retrieval warrants further studies.

Characterization of multi-locus imprinting disturbances and underlying genetic defects in patients with chromosome 11p15.5 related imprinting disorders.

The identification of multilocus imprinting disturbances (MLID) appears fundamental to uncover molecular pathways underlying imprinting disorders (IDs) and to complete clinical diagnosis of patients. However, MLID genetic associated mechanisms remain largely unknown. To characterize MLID in Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, we profiled by MassARRAY the methylation of 12 imprinted differentially methylated regions (iDMRs) in 21 BWS and 7 SRS patients with chromosome 11p15.5 epimutations. MLID was identified in 50% of BWS and 29% of SRS patients as a maternal hypomethylation syndrome. By next-generation sequencing, we searched for putative MLID-causative mutations in genes involved in methylation establishment/maintenance and found two novel missense mutations possibly causative of MLID: one in NLRP2, affecting ADP binding and protein activity, and one in ZFP42, likely leading to loss of DNA binding specificity. Both variants were paternally inherited. In silico protein modelling allowed to define the functional effect of these mutations. We found that MLID is very frequent in BWS/SRS. In addition, since MLID-BWS patients in our cohort show a peculiar pattern of BWS-associated clinical signs, MLID test could be important for a comprehensive clinical assessment. Finally, we highlighted the possible involvement of ZFP42 variants in MLID development and confirmed NLRP2 as causative locus in BWS-MLID.

Transferrin receptor gene and protein expression and localization in human IUGR and normal term placentas.

Iron (Fe) deficiency in pregnancy is associated to low birth weight and premature delivery while in adults it can result in increased blood pressure and cardiovascular disease. Cellular Fe uptake is mediated by the Transferrin Receptor 1 (TFRC), located in the trophoblast membranes. Here, we measured TFRC mRNA expression (Real Time PCR) and TFRC protein expression and localization (Western Blotting and immunohistochemistry) in IUGR compared to control placentas. A total of 50 IUGR and 56 control placentas were studied at the time of elective cesarean section. IUGR was defined by ultrasound in utero, and confirmed by birth weight <10th percentile. Three different severity groups were identified depending on the umbilical artery pulsatility index and fetal heart rate. TFRC mRNA expression was significantly lower in IUGR placentas compared to controls (p < 0.05), and this was confirmed for TFRC protein levels. In both experiments the most severe IUGR group presented lower expression compared to the other groups, and this was also related to umbilical venous oxygen levels. TFRC protein localization in the villous trophoblast did not differ in the groups, and was predominantly present in the syncytiotrophoblast. In conclusion, these are the first observations about TFRC expression in human IUGR placentas, demonstrating its significant decrease in IUGR vs controls. Thus, Fe transport could be limited in IUGR placentas. Further studies are needed to study components of the placental Fe transport system and to clarify the regulation mechanisms involved in TFRC expression, possibly altered in IUGR placentas.