Background In studies of perfluoroalkyl acids, the validity and comparability of measured concentrations may be affected by differences in the handling of biospecimens. We aimed to investigate whether measured plasma levels of perfluoroalkyl acids differed between blood samples subjected to delay and transportation prior to processing and samples with immediate processing and freezing. Methods Pregnant women recruited at Aarhus University Hospital, Denmark, (n = 88) provided paired blood samples. For each pair of samples, one was immediately processed and plasma was frozen, and the other was delayed and transported as whole blood before processing and freezing of plasma (similar to the Danish National Birth Cohort). We measured 12 perfluoroalkyl acids and present results for compounds with more than 50% of samples above the lower limit of quantification. Results For samples taken in the winter, relative differences between the paired samples ranged between -77 and +38% for individual perfluoroalkyl acids. In most cases concentrations were lower in the delayed and transported samples, e.g. the relative difference was -29% (95% confidence interval -30; -27) for perfluorooctane sulfonate. For perfluorooctanoate there was no difference between the two setups [corresponding estimate 1% (0, 3)]. Differences were negligible in the summer for all compounds. Conclusions Transport of blood samples and processing delay, similar to conditions applied in some large, population-based studies, may affect measured perfluoroalkyl acid concentrations, mainly when outdoor temperatures are low. Attention to processing conditions is needed in studies of perfluoroalkyl acid exposure in humans.
Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE) in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes.
We previously demonstrated an association between plasma perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) and longer time to pregnancy (TTP) in a sample from the Danish National Birth Cohort (DNBC, 1996-2002). In this study we investigated this association in a new sample from the same cohort. Sample 1 consisted of 440 women, and Sample 2 consisted of 1161 women from whom we previously published the associations between PFOS or PFOA and TTP. We performed sample-specific and pooled analyses using discrete-time survival analyses to estimate fecundability ratios according to PFOS and PFOA quartiles, adjusted for potential confounders chosen guided by a directed acyclic graph. We also estimated odds ratios for infertility (TTP > 12 months or infertility treatment) according to PFOS and PFOA by multivariable logistic regression. In Sample 1 PFOS was not associated with lower fecundability ratios or infertility, and there was a tendency towards longer TTP with increasing PFOA only in parous women. In Sample 2 previously reported associations were again seen. In the pooled analyses including both parous and nulliparous women fecundability ratios were 13-22 % lower for the three higher quartiles of PFOS or PFOA compared to the reference quartile. The pooled analyses were driven by the larger old sample, but we did not corroborate our previous finding of an association between high PFOS and longer TTP in the new sample. The tendency towards an association for PFOA and TTP in parous women may be due to reverse causation. Results from the new sample are more in line with the recent literature.
Perfluoroalkyl substances (PFAS) are suggested to affect human fecundity through longer time to pregnancy (TTP). We studied the relationship between four abundant PFAS and TTP in pregnant women from Greenland, Poland and Ukraine representing varying PFAS exposures and pregnancy planning behaviors. We measured serum levels of perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) in 938 women from Greenland (448 women), Poland (203 women) and Ukraine (287 women). PFAS exposure was assessed on a continuous logarithm transformed scale and in country-specific tertiles. We used Cox discrete-time models and logistic regression to estimate fecundability ratios (FRs) and infertility (TTP >13 months) odds ratios (ORs), respectively, and 95% confidence intervals (CI) according to PFAS levels. Adjusted analyses of the association between PFAS and TTP were done for each study population and in a pooled sample. Higher PFNA levels were associated with longer TTP in the pooled sample (log-scale FR = 0.80; 95% CI 0.69-0.94) and specifically in women from Greenland (log-scale FR = 0.72; 95% CI 0.58-0.89). ORs for infertility were also increased in the pooled sample (log-scale OR = 1.53; 95% CI 1.08-2.15) and in women from Greenland (log-scale OR = 1.97; 95% CI 1.22-3.19). However, in a sensitivity analysis of primiparous women these associations could not be replicated. Associations with PFNA were weaker for women from Poland and Ukraine. PFOS, PFOA and PFHxS were not consistently associated with TTP. Findings do not provide consistent evidence that environmental exposure to PFAS is impairing female fecundity by delaying time taken to conceive.
Methylation-based non-invasive prenatal testing of fetal aneuploidies is an alternative method that could possibly improve fetal aneuploidy diagnosis, especially for trisomy 13(T13) and trisomy 18(T18). Our aim was to study the methylation landscape in placenta DNA from trisomy 13, 18 and 21 pregnancies in an attempt to find trisomy–specific methylation differences better suited for non-invasive prenatal diagnosis. We have conducted high-resolution methylation specific bead chip microarray analyses assessing more than 450,000 CpGs analyzing placentas from 12 T21 pregnancies, 12 T18 pregnancies and 6 T13 pregnancies. We have compared the methylation landscape of the trisomic placentas to the methylation landscape from normal placental DNA and to maternal blood cell DNA. Comparing trisomic placentas to normal placentas we identified 217 and 219 differentially methylated CpGs for CVS T18 and CVS T13, respectively (delta β>0.2, FDR<0.05), but only three differentially methylated CpGs for T21. However, the methylation differences was only modest (delta β<0.4), making them less suitable as diagnostic markers. Gene ontology enrichment analysis revealed that the gene set connected to theT18 differentially methylated CpGs was highly enriched for GO terms related to"DNA binding" and "transcription factor binding" coupled to the RNA polymerase II transcription. In the gene set connected to the T13 differentially methylated CpGs we found no significant enrichments.
