Exposure to Perflouroalkyl acids and foetal and maternal thyroid status: a review

Background Exposure to perfluorinated-alkyl-acids (PFAAs) is ubiquitous. PFAAs are hormone-disrupting compounds that are strongly suspected to affect mother-child-health such as fetal growth. Thyroid disruption is a plausible mechanism of action. We aim to summarize the epidemiological evidence for the relation between prenatal and postnatal exposure to PFAAs and disruption of thyroid homeostasis in mothers and/or infants. Method Fifteen original publications on PFAAs concentrations and thyroid hormones (TH) in pregnant women and/or infants were found upon a literature search in the PubMed database. Information on exposure to seven PFAAs congeners [Perfluorooctane sulfonate (PFOS), Perfluorooctanoate (PFOA), Perfluorohexane sulfonate (PFHxS), Perfluorononanoic acid (PFNA), Perfluorodecanoic acid (PFDA), Perfluoroundecanoic acid (PFUnA), and Perfluorododecanoic acid (PFDoA)] and thyroid stimulating hormone (TSH), free and total thyroxine (FT4 and TT4), free and total triiodothyronine (FT3 and TT3), T3RU (Free triiodothyronine resin uptake) and FT4-index (FT4I) levels were recorded. We evaluated sampling of maternal TH by trimester, and infant TH by sex stratification. Reported associations between mother or infant PFAAs and TH were not uniformly assessed in the selected studies. Results Ten out of the fifteen studies examined maternal PFAAs concentration and TSH level. Seven studies showed significant associations between TSH and exposure to six PFAAs congeners, most of them were positive. Maternal T4 and T3 were investigated in nine studies and five studies found inverse associations between exposure to six PFAAs congeners and TH (TT3, TT4, FT3, FT4 and FT4I) levels. Eight of the fifteen studies investigated PFAAs concentrations and infant TSH. Infant TSH level was significantly affected in four studies, positively in three studies. Nine studies investigated infant T4 and T3 and seven studies found significant associations with PFAAs exposure. However, both inverse and positive significant associations with infant TH were found eliciting no clear direction. Conclusion Results indicate a mainly positive relationship between maternal PFAAs concentrations and TSH levels, and suggestion of an inverse association with T4 and/or T3 levels. Associations of infant TH with PFAAs concentration were less consistent.

PFAAs are detected globally in human populations and wildlife [11][12][13]. Humans are mainly exposed to PFAAs through food items and drinking water, but also by inhalation and dermal exposure [14]. The mean halflife of serum elimination in humans is 5.4 years for perfluorooctane sulfonate (PFOS), 3.8 years for perfluorooctanoate (PFOA) and 8.5 years for perfluorohexane sulfonate (PFHxS) [15]. Since 2000, several western countries have made restrictions on the PFAAs production [16], but presently only PFOS and PFOA are regulated by the Stockholm Convention on Persistent Organic Pollutants [17]. Thus, human serum concentrations, especially for the regulated PFOS and to some degree PFOA, have decreased during the last decade [18,19].
PFAAs enter the foetal circulation through placenta transfer and postnatally the infant is exposed by breastfeeding [20]. Thus, the foetus is exposed to PFAAs at highly vulnerable developmental stages [21]. Disruption of hormonal pathways involving the hypothalamicpituitary-thyroid (HPT) and hypothalamic-pituitarygonadal (HPG) axes has been hypothesized as a mechanism of action (MoA) and mediating steps between the effects of PFAAs on foetal growth [22]. Maternal abnormal thyroid function during pregnancy might affect foetal growth and neural development [23][24][25][26][27][28]. The foetus' thyroid hormone (TH) production does not suffice until midgestation but rely on maternal supply mainly during the 1st trimester, implying a high demand on the maternal HPT axis [23].
Maternal thyroxine (T4) and triiodothyronine (T3) hormones crosses the placenta, but not the maternal thyroid stimulating hormone (TSH) [29,30]. In the pregnant woman, the TH production undergoes dynamic and physiological changes: TSH decreases during the 1st trimester and stabilizes in the 2nd and 3rd trimester [31]. The European Thyroid Association guidelines recommend a TSH upper reference limit of 2.5 mIU/L in the 1st trimester and 3.0 mU/ L in the second and third trimesters [32], though it is discussed whether the limits are too narrow [31]. In the absence of preexisting thyroid disease, the American Thyroid Association guideline recommends upper limit of 4mIU/L in early pregnancy week 7-12 and gradually during 2nd and 3rd trimesters a return to non-pregnancy range [33]. High human corionic gonadotropin possess a thyrotrophic effect and elicit a physiological increase in levels of T4 and T3. High oestrogen levels in pregnancy increase circulating levels of thyroxin binding globulin (TBG), and may lead to an increase in the total level of T4 and T3 [34,35]. The thyroid binding capacity is facilitated by several plasma proteins such as TBG, transthyretin (TTR) and albumin [36]. Thyroid peroxidase (TPO) is a key enzyme of TH biosynthesis in the thyroid gland [37]. Thyroid peroxidase-antibodies (TPOAb) may potentially be an important covariate. TPOAb production during pregnancy might be indicative of thyroid stress especially when levels are above a cut-off value [26].
This review will focus on exposure to PFAAs and thyroid hormone homeostasis in the mother and infant. Effect estimates were evaluated with the objective to understand whether PFAAs exposure is related to hypothyroidism (low free T4, high TSH) or hyperthyroidism (high free T4, low TSH).

Method
The PubMed database was searched, using Medical Subject Headings (MESH) terms and specific keywords, to locate relevant publications, following a specific strategy (Table 1). Last literature search was conducted on the 21th January 2020. Citations from selected publications were also investigated. This report was restricted to epidemiological studies with a sample size > 100 participants. All selected studies are summarized in Tables 2,  3 , 4, 5, 6, 7, 8, 9, and 10.

Data extraction
Following data was extracted to supply general information and study characteristics ( Table 2): Study design and population, year of enrollment, number of participants, exclusion criteria, biological matrix for exposure assessment, pregnancy trimester of exposure assessment and TH outcome measurement, PFAA compounds examined above the limits of detection/quantification (LOD/LOQ), outcome estimates and confounders. Tables 3 and 4 give the information on exposure, including trimester of PFAAs measurement, participants, median concentration (ng/mL) and % < LOD/LOQ. Tables 5 and 6 give the maternal and infant TH levels, respectively. Tables 7,8,9 and 10 show the original data on thyroid hormone outcome upon PFAAs exposure.
Thus, maternal PFAAs exposure and maternal TH levels were predominantly measured simultaneously with exception of Kato et al. who measured maternal TH in the 1st trimester, while PFAAs exposure was measured after 1st trimester [38] (Table 2). Tables 5 and 6 shows the maternal and infant TH levels, respectively. The maternal TSH median levels were in general lower than the upper reference limit with no obvious trimester differences. In general similar TT3 and FT3 levels were observed among the reviewed studies, as expected with lower FT3 (pmol/L) levels than the TT3 (nmol/L) levels. Also the FT4 and TT4 levels were in overall similar among the studies with significantly higher TT4 levels (Table 5). Four studies [39,42,43,45] also determined the maternal TPOAb finding positive levels.
As shown in Table 6, infant TSH median levels were in general within the normal reference range (i.e. being higher than the lower limit but lower than the upper limit). Moreover, as expected for the T3 (TT3 and FT3), the TT3 levels were higher than FT3. Similar values of T4 (TT4 and FT4) levels were also seen among the studies and FT4 levels were mainly within the lower limit range (Table 6).

Association between PFAAs exposure and thyroid hormone outcomes
The associations between PFAAs exposure and TH outcomes are outlined by the following order: i) maternal TSH (Table 7), ii) maternal T4 and T3 (Table 8), iii) infant TSH (Table 9) and iv) infant T4 and T3 (Table 10). Maternal TH estimates were assessed by the trimester in which they were measured. Infant TH estimates were assessed upon stratification by sex (Suppl . Tables 1 and 2).
Maternal PFAAs exposure and TSH levels ( Table 7) Two out of nine studies (Table 7) showed a significantly positive association between maternal PFHxS concentrations and TSH levels in the 3rd trimester                TH measured during multiple timepoints     [44] or at multiple time points [45]. Two out of ten studies found an inverse association between PFOS and maternal TSH levels (in the 1st [38] and 3rd trimester [46]). However, two other studies found PFOS significantly positively associated with maternal TSH levels at the 2nd trimester [41] and at multiple timepoints [43], respectively. In fact, women with the highest PFOS concentration had a significant 24% higher TSH levels than those with the lowest PFOS concentration at multiple time points (data not shown) [43]. No significant associations were found between maternal PFOA concentration and TSH levels ( Table 7).
Among nine studies, one study found a positive association between PFNA concentration and maternal TSH levels (Table 7) in the 2nd trimester [42], while another study found an inverse association within 3rd trimester TSH [46].
One out of seven studies found a significant inverse association between PFDA and maternal 3rd trimester TSH levels (Table 7) [46].
The association between maternal PFUnA and TSH was examined in six studies (Table 7), and Yang et al. 2016 [46] observed a significantly inverse association in the 3rd trimester. In the same study [46] the maternal PFDoA concentration and TSH level were as well found inversely associated in the 3rd trimester (Table 7).
Maternal PFAAs exposure and T4 and T3 by trimester (Table 8) Maternal T4 and T3 were assessed by nine studies (Table 8). Significantly inverse associations were observed between maternal FT4 effect estimates (FT4I) and PFHxS levels in the 1st trimester [39] and at multiple time points [45]. Although positive tendencies were found, none of the results were significant for PFOS exposure and maternal T4 and T3 levels ( Table 8). Out of nine studies, investigating the association maternal PFOA concentration with T4 and T3 levels (Table 8), one study found a significantly inverse association with maternal 1st trimester FT4I levels when comparing the 2nd and 4th quartiles with the lowest exposure quartile [39]. Among the eight studies evaluating maternal PFNA concentrations and T4 and T3 levels (Table 8), one study found that PFNA concentrations inversely associated with maternal FT4 and TT4 levels at the 3rd trimester [44]. Among six studies (Table 8), one study reported a positive association of PFDA and maternal TT3 in the 3rd trimester [44], while another study observed a significantly negative association of high PFDA concentration (4th quartile) and TT3 at multiple time        points [43]. Out of five studies assessing maternal T4 and T3 (Table 8), only one showed that FT3 measured at multiple time points decreased with high PFUnA concentrations (4th quartile) [43], while another study found inverse association between FT4 and TT4 and PFUnA exposure in the 3rd trimester [44]. Maternal T4 and T3 versus PFDoA exposure were assessed by three studies (Table 8); two studies observed a significantly inverse association with maternal 3rd trimester FT3, TT3, FT4 and TT4 levels [44,46].
Using cord blood or maternal 3rd trimester PFOA exposure, the association of PFOA with infant TSH was examined in eight studies (Table 9). A positively significant association was found in one study with maternal 3rd trimester PFOA concentrations [47], while negative non-significant tendencies were seen in several other studies [38,44,46,48,49].
Infant TSH was assessed upon PFNA exposure in seven studies (Table 9), and a positive association was seen with maternal PFNA concentrations at the 3rd trimester [47], while an inverse association was found with cord blood PFNA concentrations [52]. When stratifying by infant sex (Suppl . Table 1a  Infant TSH (Table 9) was found positively associated with maternal 3rd trimester PFDA concentrations [47], while an inverse association was found with cord blood PFDA concentrations [52]. Other studies also showed decreasing tendencies (in maternal and cord blood), however the associations were non-significant [44,46,49]. When stratifying by infant sex (Suppl . Table 1a and b), infant TSH was positive associated with maternal PFDA in 3rd trimester concentrations among boys [% change per doubling in PFDA male = 37.0, 95%CI (0.5, 86.8)] [47].
The relation between infant TSH level and PFUnA concentration were examined in six studies (Table 9) and one study showed a significantly inverse association with cord blood PFUnA concentrations [52].
The assessement of PFOS exposure and infant T4 and T3 found in four studies significant associations (Table 10). High PFOS concentration in the 1st trimester (4th quartile) [39] and in cord blood [51] was inversely associated with infant TT4 levels (Table 10). In contrast, maternal 3rd trimester PFOS concentration [46] was significantly and positively associated with increased infant TT3, FT3 and TT4 levels. Likewise, infant cord blood PFOS concentrations [52] was positively associated with FT3 levels (Table 10) . Table 2b).
In two out of six studies significant data was found for exposure to PFUnA and association to infant T4 and T3 levels (Table 10). Maternal 3rd trimester PFUnA concentration was associated inversely with TT3 and TT4 infant levels in one study [44], and another study found increasing infant TT4 levels [46]. Tsai et al. found no effect in the total study population but a significant positive association when examining the 60th-89th percentile of cord blood PFUnA concentration with infant T3 levels [β = 0.197, 95%CI (0.031;0.364), P < 0.05] (data not shown) [51].
For PFDoA exposure versus infant T4 and T3 levels two of five studies found significant associations (Table 10). An inverse association with maternal 3rd trimester PFDoA was found in one study [44]. Cord blood PFDoA concentration was found negatively associated with infant FT3 but positively with FT4 [52]. After stratification by infant sex,

Discussion
This literature review suggests that PFAAs exposure can influence maternal TSH levels. Moreover, several PFAAs were inversely associated with maternal T4 and T3 levels. The studies reported some associations between PFAAs exposure and infant TH levels although the data was less conclusive.
In overall, most studies showed a positive association of maternal TSH and a possible negative association of maternal T4 and T3 upon PFAAs exposure. This indicates that pregnant women may be at risk of developing a hypothyroid state with increasing PFAAs exposure. Infant TH might also be affected, mainly increasing infant TSH levels with PFOS. More studies are needed to determine the direction of the association with infant T4 and T3 levels.

Evaluation of TH across pregnancy trimesters with PFOS and PFOA exposure
During the 1st trimester, most studies on maternal TH [38,39] (Table 7) showed that TSH was inversely related with PFOS, although, only one reached statistical significance [38]. Non-significant positive relationships were found with PFOA concentrations. PFOS and PFOA concentrations was mainly non-significantly but positively associated to maternal FT4 levels [38,40] and TT4 levels [39] (Table 8). FT4I was inversely associated to PFOA but not significantly to PFOS [39].
In the 2nd trimester, studies on maternal TH [41,42] (Table 7) showed that TSH was positively associated with PFOS exposure, being statistically significant in one study [41]. No consistency was found for the relation between PFOA and TSH, with both positive and inverse non-significant associations [41,42]. Moreover, results on maternal T4 (Table 8) did not show any significant associations [42].
Two studies conducted a longitudinal assessment of TH measurements across the trimesters and postpartum (Tables 2, 7 and 8) [43,45]. Repeated measurements of maternal TSH were positively associated with PFOS exposure being significant in one study [43] (Table 7). A non-significantly inverse association was found for FT4, but positive for FT3 upon exposure to PFOS and PFOA [45] (Table 8).
The trimester data evaluation showed negative associations between PFOS and maternal TSH in the 1st and 3rd trimester, but positive during the 2nd trimester (Table 7), whereas PFOA exposure did not elicite significant association to TSH in any of the trimesters. The trimester data evaluations for maternal T4 and T3 versus PFOS and PFOA concentrations showed inverse association between PFOA and FT4I in first trimester [39] ( Table 8). Longitudinal TH studies found a positive association between TSH and PFOS [43] while data on PFOA exposure did not imply a strong association with maternal thyroid hormones. It must be noted that only few studies with information on each trimester were found, thus, the direction of the estimates might change when more studies are performed. Interestingly, it has been shown that associations of maternal TSH with exposure to PFHxS and branched PFOS isomers were strongest in early pregnancy (1st trimester) and weakened over subsequent trimesters (data not shown) [45].
PFOS concentration was positively and significantly associated with TSH levels in boys [38,47,51] and girls [38]. PFNA was found to be inversely significantly associated with TSH levels in boys [48] and girls [49], while another study found positive associations in both genders [47] (Suppl. Table 1a-b). One study observed a negative association of TSH with cord blood PFOS exposure in girls and PFDoA in boys (Suppl. Table 1a-b) but they did not perform the statistical analysis on all PFAAs congeners in each sex due to their choice of statistical model (sparse partial least squares) [52].
In boys, TT4 levels were inversely associated with three PFAAs congeners (PFHxS, PFOS and PFOA) measured in maternal blood and cord blood samples (Suppl .  Table 2a) [39,51] and PNFA maternal concentrations were positively associated with FT4I [47]. In girls, TT3 levels were positively associated with cord blood PFHxS [49], and FT4I and TT4 levels positively associated with maternal PFOS and PFOA and cord blood PFOA (highest quartile) [47,50] (Suppl . Table 2b). In boys, a study found significant positive associations for FT4 with several cord blood PFAAs (PFOA, PFNA, PFDA, PFDoA) concentration (Suppl .Table 2a), the analysis was however not performed in girls due to their choice of statistical model (sparse partial least squares) [52]. In the pooled infant analyses (Table 10) this study observed similar significant positive association between FT4 level, but inversely association with FT3 level, and cord blood PFDoA concentration [52].
The reviewed studies do not allow us to establish a distinct tendency or greater sensitivity regarding infant sex. Further studies must be conducted to state any conclusion on gender specific sensitivity for PFAAs exposure and TH disruption.

Previous review literature
The possible link between PFAA exposure and thyroid outcomes in pregnant women was previously reviewed by Ballesteros et al. in 2017 [53]. They included 10 epidemiological studies on PFOS, PFOA, PFHxS and PFNA exposure during prenatal life, childhood and adolescence (up to 19 years), with sample size ranging from 40 to > 10.000 participants. In accordance to this current review, Ballesteros et al. suggested a positive association between PFHxS and PFOS and maternal TSH levels, as well as, between PFNA and TSH levels measured in boys aged > 11 years. Compared to Ballesteros et al. [53], the current review includes recent epidemiological studies (enrolment year: 1996-2016 vs. 1987-2013), more studies (n = 15 vs. n = 10), bigger sample size (participants > 100 vs. > = 43) and covers more PFAAs congeners (7 vs. 4), thus it might add a stronger study power and support to the previous observations. We also found stronger evidence of a positive relationship between PFAAs exposure (especially PFOS) with maternal TSH, and a possible inverse relationship with maternal T4 and T3 levels.

Biological plausibility
PFAAs are endocrine disrupting compounds that impact on endogenous hormone homeostasis [54]. Different pathways have been suggested to explain thyroid disrupting effects: PFAAs may induce increased excretion of T4 by alteration of TBG due to competitive binding [55], and/or increased conversion of T4 to T3 by type 1 de-ionidase in hepatic cells, as well as increased hepatic metabolism of T4 [56]. PFAAs could furthermore alter the responsiveness of the HPT axis [57], as well as, alter the HPG homeostasis and their signaling pathways which might affect foetal growth [58,59].
Studies have suggested that thyroid and oestrogenic/ androgenic pathways may be a part conjoined mechanisms. In vitro studies have shown that the HPT axis is affected by PFAAs exposure by inhibiting thyroid receptors, antagonizing thyroid cell proliferation [57]. Furthermore, PFOS and PFOA decreased TPO activity, blocking the ionizing process of thyroglobin [37], leading to reduced T4 and T3 levels. Cell proliferation of the THdependent rat pituitary GH3 cells was shown to be dependent on the involvement of the oestrogen receptor [68]. Moreover, in vitro studies have also shown alterations in the biosynthesis of oestrogens and interference with the oestrogen receptor, after exposure of PFNA, PFDA, PFHxS, PFOS and PFOA, due to their oestrogenic and anti-androgenic potential [2,[69][70][71]. Extracted PFAA mixtures from human serum samples also activated oestrogenic receptors [72,73] and in an epidemiological study serum PFAAs mixture induced estrogenic activity in pregnant women was associated with significantly lower birth weight and birth length [58]. Most studies examined the effect of single PFAA congeners. More studies are needed for the combined cocktail effect of PFAAs mixtures and/or other POPs, which may have additively and/or synergistic actions [58,73].

PFAAs exposure and TH outcome assessment
All the reviewed studies determined PFAA concentrations, using small variations of the same methodology (liquid chromatography-tandem mass spectrometry), in mother and/or infant. Maternal biological matrix was either blood plasma or serum and for infant concentrations either cord blood plasma or serum. The LOD and LOQ values were different among studies. The percentages of samples below LOD/LOQ varied among studies, especially for PFHxS, PFNA, PFDA in maternal serum/ plasma samples (Table 3) and for most PFAA congeners in cord blood (Table 4). Nevertheless, most selected studies handled the PFAA concentrations below LOD/ LOQ with replacement by LOD/LOQ divided by the square root of 2. The sensitivity of the used methods might influence the estimated effect but the exposure levels is expected to be the main factor for the exposure -outcome associations.
Umbilical cord blood samples were also used to determine both infant exposure and thyroid function having the benefit being of representing the fetal environment. However, there are also some limitations of using cord blood for infant thyroid function assessment. Firstly, cord blood thyroid hormone levels likely reflects a combination of maternal and fetal hyroid hormones. Secondly, maternal and umbilical cord blood PFAAs concentrations are highly correlated [74,75], and the ability of PFAAs to cross the placenta differs between compounds (i.e. PFOA passes more easily than PFOS [20,75,76]. Maternal PFAA concentrations might not be constant throughout pregnancy due to physiological hemodynamic changes. PFAAs concentrations decrease during pregnancy due to increased plasma volumes [77] and higher renal PFAAs excretion [78]. Thus, measurement of PFAAs in late pregnancy might not be directly compared with PFAAs concentration in 1st trimester [77]. Moreover, women are able to transfer some of their PFAAs burden to their offspring by placental [21,79] and post-partum breast feeding [80]. This complicates comparison between studies with different pregnancy time points of exposure assessment [19]. Even more, one study measured the thyroid hormone outcome before exposure (PFAAs) [38], which could cause concern of causality, although some retrospective consistency for PFAAs exposure can be assumed due to their persistent nature (long half-lives in human serum) [15,18]. However, a study showed the PFAA concentrations measured in the 1st and 3rd trimester were highly correlated (r > 0.64) [81] and argued that a single exposure measurement might be considered as a representative of serum levels during pregnancy.
In most of the studies, the exposure was evaluated as single congeners and not as the real mixture/combined exposure scenario. PFAAs are often highly intercorrelated. It is very diffecult to control for confounding by other PFAAs when assessing the effect of single PFAA congeners and interpret the association in light of the high mutual correlation among PFAA congeners. However, given the structural analogy and similar expected modes of action, PFAAs should be regulated as a chemical family rather than isolated compounds [82,83]. In this regard, the complementation of exposure biomarkers with biomarkers of combined biological activity to PFAS mixtures in epidemiologic studies may be of great help [58]. Berg et al. (2017) [84] did not find an association between summed PFAAs exposure (sum of PFHpS, PFHxS, PFOA, PFOS, PFNA, PFDA and PFUnA) and TH outcome. Whereas, high exposure (4th quartile) to the summed POP (sumPFAAs + sumPCBs [sum of PCB 99,118, 138, 153, 163, 170, 180 and 187] + DDE + HCB + cis-and trans-nonachlor) was significantly positively associated with maternal TSH [84]. A study found no effect of the summed PFAAs exposure on maternal risk of hypothyroxinaemia (low free T4 without the compensatory rise in TSH) [85]. Furthermore, one study examined branched isomers of PFOS and found several significant findings, but not with the total PFOS [45].
The selected studies in this review investigated FT4I, FT4, FT3, TT4, TT3, T3RU and TSH in mothers and infants using different methods of chemoluminescent-, radioor enzyme linked immunosorbents assays, with variable sensitivity (e.g. LOD (TSH) = 0.50 μU/mL [38] vs. LOD (TSH) = 0.01 μU/mL [41]). The radio immune sorbent assay seems to be the most reliable method for the quantification of thyroid hormones [86], but all methods showed good sensitivity, specificity, and accuracy. However, the use of immunosassay for hormone determination has been criticized. Animal studies suggested that the effect of PFOS to decrease FT4 level might be due to negative bias in analog techniques resulting from competitive displacement of FT4 and the labeled analog from serum and assay binding proteins in the presence of PFOS [61,87]. Nevethrthless, such bias was not observed in human study and such different result between human and animal study might be related to the difference of PFAA level, protein binding to T4 (in rats: TTR, in human: TBG) and the interaction of proteins and PFAAs [88]. Based on these reported data, the use of different methods seems not to be of concern in measurements of human thyroid hormones.
Most studies examined TH at a single time point during pregnancy, impeding the capture of an effect on the thyroidal system during pregnancy since levels vary substantially during different pregnancy stages and stabilises approximately 6 weeks postpartum [34,89]. However, longitudinal studies measure TH outcomes multiple times having a greater chance of witnessing an effect on the thyroid system [42,43,45].
TSH was examined in all studies except one [50], and T4 and T3 parameters were less consistently studied.
The free TH reflects the biologically active hormones, which may give a better estimate of the exposure effect on the thyroid regulatory systems [90]. The FT4I is an estimate of circulating free T4 levels, which accounts for changes in T4 levels due to changes in thyroid binding protein levels and saturation, such as increases in TBG during pregnancy [91], being a better estimate, but only assessed in one single study [39]. Some of the studies only investigated a single TH parameter [41,48,50], however to understand the effect of PFAA exposure on the TH equilibrium at least T4 and TSH measurements are required.
The extent of adjustment for potential confounders varied largely between studies. General confounders were pre-pregnancy BMI, maternal age, socio-occupational status, parity, alcohol and smoking ( Table 2). Eight studies excluded participants with thyroid diseases or related medication [38, 39, 41-44, 46, 48] and one study adjusted for these confounders [50]. However, few studies also took into account other thyroid parameters such as iodine status, TPOAb or thyroid binding capacity. Berg et al. demonstrated that several significant effect estimates lost significance after adjusting for thyroid binding capacity in the statistical analysis [43], suggesting an area that should be further studied.
FT4I was suggested as a more reliable FT4 estimate during pregnancy [39]. Weiss et al. found evidence that PFAAs might decrease T4 due to their affinity to thyroid hormone transport proteins using transthyretin (TTR) as carrier protein [55]. However, Preston et al. did not support this theory, after showing lower saturation of plasma binding proteins with higher PFAAs exposure [39]. The incoherent results may be due to the fact that experimental studies use TTR as carrier protein, although the major carrier protein in humans is TBG, which has lower PFAA affinity than TTR [92].
Three studies stratified for TPOAb-status and maternal TSH was associated with several PFAAs in the high TPOAb group [39,42,45]. However, the high TPOAb groups differed in sample size (e.g. 14 vs. 98 participants), gestational week (GW) of exposure sampling (median GW 9.6, GW 15-18 and multiple time points), TPO-Ab threshold (Webster et al. and Reardon et al.: > 9 IU/mL vs. Preston et al.: > 2 U/mL) and PFAA concentrations (lower in Reardon et al). Even though results went in different directions, it might indicate a more susceptible group among women with thyroid stress, whom may not be able to compensate for the PFAAs-induced thyroid affections [42]. If these women are highly exposed to PFAAs, which are strongly suspected to affect the thyroid gland, it could increase their risk of immune reactivity and affect the TH production necessary for a normal foetal development.

Conclusion
In overall, most studies supported a positive association of maternal TSH and a possible negative association of maternal T4 and T3 upon PFAAs exposure. Infant TH might also be affected. The trimester data evaluation showed different direction of associations between PFOS and maternal TSH in different trimester. A positive association between TSH and PFOS was observed in longitudinal studies. The associations of PFAAs and TH in infant gender differed and were PFAA congeners dependent.
PFAAs exposure has a stronger influence on maternal than infant TH levels. Scientific evidence seems to favour an increase in maternal TSH and decrease T4 and T3 estimates upon PFAA exposure. The reviewed studies reported some associations between PFAAs and infant TH levels, although these data were less conclusive. The data did not indicate a clear trend in infant gender sensitivity. This review also showed a tendency of trimester dependency of maternal TSH levels, but more studies are needed longitually or during the 1st trimester with follow-up at birth with samples from mother and child (cord blood).
Future studies need a uniform method of examining the associations. We suggest future studies to focus on the 1st trimester with longitially followup in nulliparous women and include a broad range of PFAA congeners.
Additional file 1. Association of PFAAs with TSH and TH levels by infant sex.