Shift in Sex Ratio: Male Numbers Sink in Great Lakes Community

Sex ratio—the proportion of male to female live births—can be an important indicator of the reproductive health of a population, whether animal or human. This figure is typically fairly constant. For example, the worldwide human sex ratio ranges from 102 to 108 male births for every 100 female births; in other words, male babies make up about 50.4–51.9% of live births worldwide. Now, however, investigators have documented a significant skewing of the human sex ratio in a population located in a heavily polluted Great Lakes area [EHP 113:1295–1298]. 
 
In response to concerns about a shifting sex ratio among members of the Aamjiwnaang First Nation community near Sarnia, Ontario, a team of Canadian researchers examined birth records for the group from the years 1984–2003 as part of a broader community-based investigation. The researchers discovered that, as community members had suspected, there had been a significant and precipitous shift in the sex ratio. 
 
The expected sex ratio in Canada is 51.2% male babies to 48.8% female babies. For the period 1984–1992, that ratio held fairly constant among this community. In the period 1993–2003, however, male babies made up only 41.2% of live births. The five-year period from 1999 to 2003 showed an even sharper decline, with male babies making up 34.8% of live births. According to the researchers, although there is normal variation in sex ratio within populations, the deviation in this case appears to be outside the normal range. 
 
Although there is as yet no direct evidence linking this human sex ratio decline to environmental exposures, the circumstantial evidence suggests there may be a connection. The Chippewas of the Aamjiwnaang reserve reside within the St. Clair River Area of Concern, situated immediately adjacent to several large petrochemical, polymer, and chemical industrial plants. The area is one of Canada’s largest concentrations of industry. Prior soil and sediment assessment has shown that the reserve land is heavily contaminated with pollutants such as polychlorinated biphenyls, polyaromatic hydrocarbons, hexachlorobenzene, mirex, a variety of potentially toxic metals, volatile organic compounds, phthalates, and dioxins; many of these are known or suspected endocrine disruptors. 
 
As the investigators point out, past studies have documented reproductive outcomes in wildlife populations within the same region, including reduced hatching success, altered sexual development, and changes in sex ratios. Scientific suspicion has long been focused on environmental endocrine disruptor exposures as the root cause of these effects. 
 
The authors acknowledge that there are many other potential factors that could influence the declining sex ratio they describe. But the combination of close proximity to industrial facilities emitting known endocrine-disrupting chemicals and the documented adverse reproductive outcomes in wildlife populations in the region leads them to conclude that further investigations are warranted into the types and routes of chemical exposures—via air, water, food, soil, and sediment—for this population. A community health survey designed to explore health concerns among residents of the reserve is in progress, including information on potential covariates that may influence the sex ratio, such as parental age or smoking.

We wish to report some corrections to our study [1], none of which alters the interpretation of the data or the conclusions drawn. After publication, we noticed that one of the microarray hybridizations (on sample NB11) was performed on the same patient's material as another hybridization (sample NB4; see Table 1; a corrected version of Table 5 [1]). As this error leads to an incorrect subclassification of the patients into the 'favourable' and 'unfavourable' neuroblastoma subgroups, we would like to exclude this data point from the differential expression analysis of favorable versus unfavorable neuroblastoma given under the heading 'Differential expression analysis of favorable and unfavorable neuroblastoma' in the Results section of [1]. Careful reanalysis after exclusion of NB11 did not lead to important changes in the generated gene lists and conclusions; the changes are given in the corrected paragraph and Table 2 (a  corrected version of Table 4 [1]), and the Additional data files 1 and 2 (corrected versions of Additional data files 2 and 3 [1]) available online with this article.
We also noticed that sample NB1 is stage 1 instead of stage 4S and that sample NB2 was not localized to the adrenals (see Table 1).

Differential expression analysis of favorable and unfavorable neuroblastoma
So far, most published microarray studies on neuroblastomas mainly compared favorable with unfavorable neuroblastomas in order to identify prognostic markers or pathways that are involved in these clearly different neuroblastoma tumor types. In order to add value to such an analysis, we contrasted similar differentially expressed gene lists with the normal neuroblast expression profile (Additional data file 1). In a first step, we compared the differentially expressed genes between these two tumor types with published prognostic gene lists. We found that 23 of the 193 genes on our list were previously reported, including the well established markers MYCN, NTRK1, and CD44 (see NBGS analysis in Additional data file 2). This overlap demonstrates the validity of the selected neuroblastoma panel and their expression profile. Subsequently, we looked for the corresponding gene expression levels of the differentially expressed genes in the normal counterpart cells, aiming to select neuroblastoma candidate genes. Of the 100 genes that are more highly expressed in favorable tumors (compared to unfavorable) 41 also have a significant differential expression (either higher or lower) compared to neuroblasts, whereas 43 of the 93 genes that are more highly expressed in unfavorable tumors exhibit differential expression compared to the neuroblasts (Table 2).
From this analysis, a few putative positional tumor suppressor candidates emerge: CDC42 on 1p36, CACNA2D3 on 3p21 and DLK1 on 14q. The latter two genes are of particular interest because they are highly expressed in neuroblasts and favorable neuroblastomas and their expression is significantly lower in unfavorable neuroblastomas. Among the genes that are more highly expressed in unfavorable neuroblastomas than in favorable ones and neuroblasts, the proven oncogenic transcription factor MYCN emerges (and putative downstream genes KIFAP3, OPHN1, RGS7, ASCL1, ODC1, TWIST1 and TYMS, according to NBGS), as well as several other genes that have been identified or studied in the context of neuroblastoma such as ALK and PRAME, and positional candidates on 17q including BIRC5 and RNU2.

Additional data files
Additional data files 1 and 2 containing the corrected data available online with this article. Samples were subdivided into favorable or unfavorable type based on MYCN amplification, ploidy and age at diagnosis. *Neuroblastoma or nodular ganglioneuroblastoma. ND, not determined or unknown.

References
11p -Genes that are differentially expressed compared with neuroblasts among the differentially expressed genes in favorable neuroblastoma (NB) vs unfavorable NB, with an indication of the number of neuroblastoma microarray studies in which these genes were found through NBGS analysis. NBGS, Neuroblastoma Gene Server.