Geospatial analysis of hypospadias and cryptorchidism prevalence rates based on postal code in a Canadian province with stable population.

INTRODUCTION: Hypospadias and cryptorchidism are hormone-mediated malformations that occur during male development. Prevalence rates of hypospadias and cryptorchidism are thought to be increasing worldwide. In-utero exposure to endocrine-disrupting chemicals (EDCs) may have a role in the occurrence of these malformations. Our group has reported significant clustering of hypospadias and cryptorchidism at the county level in areas of intense agricultural activity in the Canadian province of Nova Scotia (NS). Finer scale spatial analysis has shown clustering near urban centres. OBJECTIVES: The objectives of the study were: 1) to perform a granular geospatial analysis of hypospadias and cryptorchidism prevalence, at the postal code level, of all babies born in NS over a 26-year period; and 2) to determine whether there is spatial correlation between these conditions and industries linked to toxic output. STUDY DESIGN: Cases of hypospadias and cryptorchidism were identified based on ICD-10 codes from the Nova Scotia Atlee Perinatal Database with records of all live births in NS between 1988 and 2013. Data were geocoded and mapped based on the three first digits of the maternal postal code (Forward Sortation Area [FSA]). Regional prevalence of congenital anomalies was calculated for each of the 77 FSAs. To identify statistically significant high and low prevalence clusters for each anomaly, Local Morans I was used on the spatial data. Geospatial point data was created for industries linked to toxic output and correlation between clusters of malformations and proximity to these industries was assessed. RESULTS: During the study period, there were 1045 cases of hypospadias and 993 cases of cryptorchidism. Both hypospadias and cryptorchidism demonstrated statistically significant areas of high prevalence clusters. There was no significant spatial correlation between the local clustering of the congenital malformations and proximity to toxic industries. DISCUSSION AND CONCLUSION: Our study shows heterogeneity in the distribution of hypospadias and cryptorchidism, which is consistent with previously published works. In this follow-up, granular geospatial analysis of hypospadias and cryptorchidism prevalence in an area with stable population, we did not confirm the previous findings of high clustering in areas of intense agricultural activity. Furthermore, our analysis did not find high clustering of the congenital malformations in areas near toxic industries to support a clear environmental role in their development. Some of the limitations include underdiagnosis of hypospadias and cryptorchidism (as they both present with a clinical spectrum and are non-life threatening), and limited data currently available on the route of exposure to EDC industries in Nova Scotia.

Highly dynamic temporal changes of TSPY gene copy number in aging bulls.

The Y-chromosomal TSPY gene is one of the highest copy number mammalian protein coding gene and represents a unique biological model to study various aspects of genomic copy number variations. This study investigated the age-related copy number variability of the bovine TSPY gene, a new and unstudied aspect of the biology of TSPY that has been shown to vary among cattle breeds, individual bulls and somatic tissues. The subjects of this prospective 30-month long study were 25 Holstein bulls, sampled every six months. Real-time quantitative PCR was used to determine the relative TSPY copy number (rTSPY CN) and telomere length in the DNA samples extracted from blood. Twenty bulls showed an altered rTSPY CN after 30 months, although only 9 bulls showed a significant change (4 significant increase while 5 significant decrease, P<0.01). The sequential sampling provided the flow of rTSPY CN over six observations in 30 months and wide-spread variation of rTSPY CN was detected. Although a clear trend of the direction of change was not identifiable, the highly dynamic changes of individual rTSPY CN in aging bulls were observed here for the first time. In summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility.

Somatic Mosaicism in Bulls Estimated from Genome-Wide CNV Array and TSPY Gene Copy Numbers.

Somatic mosaicism has become a focus in human research due to the implications of individual genetic variability in disease. Here, we assessed somatic copy number variations (CNVs) in Holstein bulls in 2 respects. We estimated genome-wide CNVs and assayed CNVs of the TSPY gene, the most variable bovine gene from the Y chromosome. Somatic tissues (blood, lung, heart, muscle, testis, and brain) of 4 bulls were arrayed on the Illumina Bovine SNP50k chip and qPCR tested for TSPY copy numbers. Our results showed extensive copy number divergence in tissues within the same animal as well as significant copy number alterations of TSPY. We detected a mean of 31 CNVs per animal among which 14 were of germline origin, as they were constantly present in all investigated tissues of the animal, while 18 were specific to 1 tissue. Thus, 57% of the total number of detected CNVs was the result of de novo somatic events. Further, TSPY copy number was found to vary significantly among tissues as well as among the same tissue type from different animals in a wide range from 7 to 224% of the calibrator. Our study shows significant autosomal and Y-chromosomal de novo somatic CNV in bulls.

Systemic and local anti-Mullerian hormone reflects differences in the reproduction potential of Zebu and European type cattle.

This study was conducted to evaluate plasma anti-Mullerian hormone (Pl AMH), follicular fluid AMH (FF AMH) and granulosa cell AMH transcript (GC AMH) levels and their relationships with reproductive parameters in two cattle subspecies, Bos taurus indicus (Zebu), and Bos taurus taurus (European type cattle). Two-dimensional ultrasound examination and serum collection were performed on Zebu, European type and crossbreed cows to determine antral follicle count (AFC), ovary diameter (OD) and Pl AMH concentration. Slaughterhouse ovaries for Zebu and European type cattle were collected to determine FF AMH concentrations, GC AMH RNA levels, AFC, oocyte number, cleavage and blastocyst rate. Additionally GC AMH receptor 2 (AMHR2) RNA level was measured for European type cattle. Relationship between AMH and reproductive parameters was found to be significantly greater in Zebu compared to European cattle. Average Pl AMH mean ± SE for Zebu and European cattle was 0.77 ± 0.09 and 0.33 ± 0.24 ng/ml respectively (p = 0.01), whereas average antral FF AMH mean ± SE for Zebu and European cattle was 4934.3 ± 568.5 and 2977.9 ± 214.1 ng/ml respectively (p < 0.05). This is the first published report of FF and GC AMH in Zebu cattle. Levels of GC AMHR2 RNA in European cattle were correlated to oocyte number (p = 0.01). Crossbred animals were found more similar to their maternal Zebu counterparts with respect to their Pl AMH to AFC and OD relationships. These results demonstrate that AMH reflects differences between reproduction potential of the two cattle subspecies therefore can potentially be used as a reproductive marker. Furthermore these results reinforce the importance of separately considering the genetic backgrounds of animals when collecting or interpreting bovine AMH data for reproductive performance.

BPA exposure during in vitro oocyte maturation results in dose-dependent alterations to embryo development rates, apoptosis rate, sex ratio and gene expression.

Alterations in the oocyte's environment can negatively affect embryo development. Oocyte quality, which can determine embryonic viability, is easily perturbed, thus factors affecting normal oocyte maturation are a concern. Bisphenol A (BPA) is an endocrine disrupting chemical that elicits a variety of reproductive effects. BPA has previously been found to disrupt meiosis, however the embryonic effects in mammals are not well documented. Here, bovine oocytes were matured in vitro with and without BPA treatment. Resulting embryos exhibited decreased embryonic development rates, increased apoptosis, and a skewed sex ratio. Gene expression in blastocysts was not altered, whereas treatment with 15ng/mL BPA resulted in increased expression of several of the genes studies, however this increase was largely due to a vehicle effect. BPA exposure during oocyte maturation in vitro can therefore, in a dose-dependent way, decrease oocyte and embryo quality and developmental potential and affect gene expression of developmentally important transcripts.