Victor Pop1*, Johannes Krabbe2, Wolfgang Maret3 and Margaret Rayman4

1. 1Department of Medical and Clinical Psychology, Tilburg University, 500 LE Tilburg, The Netherlands
2. 2Department of Clinical Chemistry and Laboratory Medicine, Medisch Spectrum Twente, Medlon BV, Enschede, The Netherlands
3. 3Department of Nutritional Sciences, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King s College London, London SE1 9NH, UK
4. 4Department of Nutritional Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, UK (Submitted 14 April 2020 – Final revision received 14 June 2020 – Accepted 7 July 2020 – First published online 14 July 2020)

Abstract

The present study reports on first-trimester reference ranges of plasma mineral Se/Zn/Cu concentration in relation to free thyroxine (FT4), thyrotropin (TSH) and thyroid peroxidase antibodies (TPO-Ab), assessed at 12 weeks’ gestation in 2041 pregnant women, including 544 women not taking supplements containing Se/Zn/Cu. The reference range (2·5th–97·5th percentiles) in these 544 women was 0·72–1·25 μmol/l for Se, 17·15–35·98 μmol/l for Cu and 9·57–16·41 μmol/l for Zn. These women had significantly lower mean plasma Se concentration

1. (0·94 (SD 0·12) μmol/l) than those (n 1479) taking Se/Zn/Cu supplements (1·03 (SD 0·14) μmol/l; P < 0·001), while the mean Cu (26·25 μmol/l) and Zn (12·55 μmol/l) concentrations were almost identical in these sub-groups. Women with hypothyroxinaemia (FT4 below reference range with normal TSH) had significantly lower plasma Zn concentrations than euthyroid women. After adjusting for covariates including supplement intake, plasma Se (negatively), Zn and Cu (positively) concentrations were significantly related to logFT4; Se and Cu (but not Zn) were positively and significantly related to logTSH. Women taking additional Se/Zn/Cu supplements were 1·46 (95 % CI 1·09, 2·04) times less likely to have elevated titres of TPO-Ab at 12 weeks of gestation. We conclude that first-trimester Se reference ranges are influenced by Se-supplement intake, while Cu and Zn ranges are not. Plasma mineral Se/Zn/Cu concentrations are associated with thyroid FT4 and TSH concentrations. Se/Zn/Cu supplement intake affects TPO-Ab status. Future research should focus on the impact of trace mineral status during gestation on thyroid function.

Key words: Selenium: Copper: Zinc: Pregnancy: Thyroid function

Adequate maternal thyroid function during pregnancy is crucial for optimal fetal development, especially during the first 20 weeks when the fetal thyroid does not produce its own thyroid hormones (TH)(1). Proper thyroid function is highly relevant because the development of the fetal central nervous system is dependent on TH(1). Nutritional (trace) elements such as Se, Zn and Cu are involved in a diversity of mechanisms including: (i) the regulation of cellular function, (ii) growth and maintenance, and (iii) neuromodulation(2). A deficiency of trace elements may result in alterations in immune function, cognition and growth and development(3,4).

For adult females, normal plasma Se concentrations vary according to location. In Europe, they are around 1·1 μmol/l, whereas in the USA, they are more often 1·6 μmol/l(5). Except perhaps for regions with high plasma Se concentration, plasma

Se may fall in pregnancy owing to plasma volume expansion(5). Zn is important for many physiological functions such as growth, immune function and reproduction(6). As part of the adaptive immune system, T cells are particularly sensitive to deficiency of Zn that is needed for their maturation and the maintenance of a balance between different T-cell subsets(4). Cu has a crucial role in mitochondrial respiration, erythropoiesis, myelin formation, hormone synthesis, antioxidant protection and immune function(7). Cu is important for the functioning of other dietary elements including Fe. While Se and Cu have a role only in 2–3 dozen proteins, Zn is a constituent of about every tenth protein, that is, over 3000 human proteins are believed to be Zn proteins(8). For the trace elements Zn and Cu, the plasma reference ranges for adult females are 10–18 and 10–24 μmol/l, respectively(6,8). In a study from China, reference ranges for Cu

Abbreviations: FT4, free thyroxine; hCG, human chorionic gonadotropin; NHANES, National Health and Nutrition Examination Survey; TH, thyroid hormone;

TPO-Ab, thyroid peroxidase antibodies; TSH, thyrotropin. * Corresponding author: Victor Pop, email [email protected]

unidentified (sub)clinical thyroid dysfunction or developing thyroid dysfunction(1). The relationship between plasma mineral (Se, Zn and Cu) concentrations during pregnancy and TPO-Ab status has scarcely been addressed.

and Zn were established in different groups of women at a number of gestational ages and were compared with those of 552 non-pregnant matched controls(9). It was unclear whether the figures referred to plasma or serum concentrations and supplement intake was not recorded. The reference range for Cu was significantly lower for non-pregnant women (aged 21–38 years), at 7·9–24·9 μmol/l, than for any assessment during gestation. The non-pregnant reference range of Zn was 9·7–16·9 μmol/l which only slightly differed from that in pregnancy(9). In a recent Norwegian study of 2982 pregnant women, the median plasma Se/Zn/Cu concentrations assessed at 18 weeks’ gestation were 1·29, 7·35 and 24·2 μmol/l, respectively(10). However, as the authors reported themselves, this was a highly selected subgroup of the original cohort of 112 789 pregnant women accrued over a period of 10 years (1999–2008) making a bias likely(10). A major limitation in determining standard reference ranges of trace elements during pregnancy is the widespread use of supplement intake during that life stage. In several Western countries, up to 70 % of women take pregnancy supplements (folic acid, vitamin D, specific trace elements)(11). Thus, a large sample size is needed in order to find subgroups of women not taking supplements containing these trace elements. These sub-groups should preferentially share similar characteristics with the main group, enabling researchers to adapt the reference ranges to the general pregnant population.

The primary aim of the present study was to evaluate firsttrimester standard reference ranges of plasma mineral (Se, Zn and Cu) concentrations in a large cohort of healthy pregnant women, adjusting for supplement intake. The secondary aim was to investigate possible cross-sectional relationships between plasma mineral (Se, Zn and Cu) concentrations in subgroups of women with different forms of thyroid dysfunction and TPO-Ab status. A tertiary aim was to compare plasma mineral (Se, Zn and Cu) concentrations with TH concentrations during early gestation, adjusted for important determinants such as smoking, parity, BMI and family history of thyroid dysfunction. Fourth, we aimed to investigate the possible relationship between plasma mineral (Se, Zn and Cu) concentrations and TPO-Ab status.

Materials and methods

The present report is part of the longitudinal prospective HAPPY project (Holistic Approach to Pregnancy and the first Postpartum Year), the details of which have been described elsewhere(17,18).

Participants and procedure

Se, as selenocysteine, is at the active centre of the selenoproteins(5). The iodothyronine deiodinases DIO1 and

From January 2013 to September 2014, pregnant women who had their first antenatal visit at one of the seventeen participating community midwife offices in the South-East of the Netherlands were invited to participate in the HAPPY project. During the 18 months of the recruitment period, approximately 4150 women visited the participating midwife offices. Only Dutch speaking women (n 3475) were invited to participate. Women (n 3159) were eligible of whom 2275 provided written informed consent (response rate = 72 %)(16). Of these, all obstetric and biological data were available in 2041 women. The study was approved by the Psychology Ethics Committee of Tilburg University (protocol number EC-2012.25) and additionally evaluated by the Medical Ethical Committee of the Máxima Medical Center in Veldhoven.

1. DIO2 are selenoproteins that convert thyroxine (T4) to the active hormone, triiodothyronine (T3); the deiodinases DIO1 and
2. DIO3 are also able to convert T4 to inactive, reverse T3(5). A further important role for the glutathione peroxidase selenoprotein (GPX3) is the removal of excess hydrogen peroxide that, together with thyroid peroxidase, is needed for TH synthesis(5). Low Se status has been associated with newly diagnosed Graves’ disease and Hashimoto’s thyroiditis(12,13). Zn is also involved in thyroid hormone metabolism including regulation of deiodinase activity, thyroid releasing hormone and TSH (thyrotrophin) synthesis(14). Low plasma Zn concentration has been associated with hypothyroidism and high concentration with hyperthyroidism(13). Especially relevant in pregnancy is the effect of Zn deficiency on the offspring’s neuro-psychological functioning, activity and motor development(15). Previously, a correlation between serum Se, Zn and Cu concentrations and thyroid function was reported in the general US population(16). In females, Cu was significantly associated with log-transformed total thyroxine (logTT4), while Se was associated with logFT3 at the 90 % significance level. No data were reported for pregnant women; women with elevated thyroid peroxidase antibodies (TPO-Ab) were excluded from the analyses(16). Because pregnancy is characterised by fundamental changes in immune function and increasing demands for maternal TH to ensure proper fetal development, it is important to evaluate the possible role of these trace elements on gestational thyroid function.

Assessments

This study is reported according to the STROBE guidelines(19). At 12 weeks of gestation, as part of the standardised nationwide early-pregnancy blood assessment, non-fasting blood samples were collected in BD Vacutainer PST-tubes and women completed a set of questionnaires that asked about demographic and obstetric features and lifestyle habits.

Thyroid function assessment

At the 12th week of gestation, TSH, free thyroxine (FT4) and TPO-Ab concentrations were determined in heparinised plasma using electrochemiluminescence assays (Cobas_e 601; Roche Diagnostics). The non-pregnant reference range of TSH is 0·40–4·0 mU/l, of FT4 10·0–24·0 pmol/l and of TPO-Abs <35 IU/l. According to the American Thyroid Association guidelines, the reference ranges of TSH and FT4 during pregnancy

Up to 8–15 % of women of fertile age have elevated titres of TPO-Abþ(1). TPO-Abþ titres during early gestation definitely imply elevated titres before conception and are one of the most obvious signs of a thyroid that is compromised by an autoimmune process; these women are at high risk for having

Statistical analysis was performed using the IBM SPSS Statistics for Windows version 25.0 (IBM Corp.). The skewness and kurtosis of Se data were 0·62 and 1·59, those of Zn 0·51 and 0·71 and those of Cu 0·35 and 0·31, respectively; therefore, parametric testing was used to compare means between subgroups. Descriptive statistics (t test: T (df) = value, P), χ2 (χ2 (df) = value, P and ANOVA: F (df) = value, P) were used to analyse the prevalence of abnormal thyroid dysfunction in relation to plasma mineral (Se, Zn and Cu) concentrations. We then compared plasma mineral (Se, Zn and Cu) concentrations with log-transformed FT4 and TSH values adjusted for confounders, using linear regression. Finally, within the TPOAbþ group, we compared plasma mineral (Se, Zn and Cu) concentrationsandtherelationshipbetweenSe/Zn/Cusupplement intake and TPO-Ab status, using logistic regression analysis.

were defined in TPO-Ab-negative women at the 12th gestational week using the 2·5th and 97·5th percentiles to define the lower and upper limits of normal thyroid function(20). The following sub-groups of thyroid function were defined: (i) euthyroid (TSH and FT4 within the reference limit); (ii) overt thyroid dysfunction (TSH and FT4 outside the reference limits); (iii) sub-clinical thyroid dysfunction (TSH outside the reference limit with normal FT4); (iv) hypothyroxinaemia (FT4 <2·5th or <5th percentile with normal TSH) and finally, (v) hyperthyroxinaemia (FT4 >95th, or >97·5th percentile with normal TSH concentration)(20).

Selenium, zinc and copper

Plasma Se, Zn and Cu concentrations were measured at the 12th gestational week in heparinised plasma samples by inductively coupled plasma MS using a Nexion 300X instrument (PerkinElmer) in the kinetic energy discrimination mode. Samples were diluted 30 times in 0·5 % HNO3 and 0·01 % triton in MiliQ AquaDest. Se, Cu and Zn were measured employing helium at a flow rate of 1·0 ml/min as the kinetic energy discrimination gas to remove polyatomic interferences. Within- and betweenrun variation were assessed by CLSI guideline EP5-A2 and found to be <3 % for low and high concentrations of Zn and Cu in plasma and <5 % for Se. According to CLSI guideline EP 17-A2, the lower limit of quantitation was calculated at 0·15,

Results

1. Table 1 shows the characteristics of 2041 women with thyroid parameters at 12 weeks’ gestation.
2. Table 2, different subgroups are shown with the corresponding plasma mineral (Se, Zn and Cu) concentrations according to supplement intake.

1. 0·30 and 0·27 μmol/l for Se, Zn and Cu, respectively. Certified referencematerials Seronorm L1 or L2(Nycomed)wereusedto monitor accuracy. During measurement of the samples of this study, for L1 (lot 1309438), values for Se, Zn and Cu were 0·99 μmol/l (reported analytical value 1·10 μmol/l with a range 0·96–1·25 μmol/l), 17·4 μmol/l (reported analytical value 16·8 μmol/l and range 14·6–19·0 μmol/l) and 17·9 μmol/l (reported analytical value 17·1 μmol/l and range 15·7–18·5 μmol/l), respectively. For L2 (lot 1309416), values for Se, Zn and Cu were 1·58 μmol/l (reported certified mean 1·75 and 1·52–1·98 μmol/l), 25·7 μmol/l (reported certified mean 24·7 μmol/l range 21·5–28·0 μmol/l) and 29·4 μmol/l (reported certified mean 29·1 μmol/l and range 26·7–31·5 μmol/l), respectively. Moreover, pooled sera were measured as well in order to monitor within-run variation at low and high concentrations.

Supplement intake

At 12 weeks’ gestation, supplement intake was carefully investigated. Women were asked whether they used any supplement on a daily basis. In the Netherlands, most supplement tablets including Se/Zn/Cu belong to 2–3 widely used products in which the daily dose is carefully reported (as requested by law). These different tablets were all recorded, including the dose of the trace elements.

The women not taking supplements containing Se (groups 1, 3, 4) showed significantly lower mean plasma Se concentrations than those who did (group 2): ANOVA (2037) F = 34·7, P < 0·001, while the mean plasma Cu and Zn concentrations were almost identical in these sub-groups. Based on these figures, we defined two groups: those taking supplements with Se/Zn/Cu (group 2, n 1479, 73·3 %) and those who did not (groups 1 þ 3 þ 4: n 544, 26·7 %). Women not taking Se/Zn/Cu supplements had significantly lower mean Se concentration

Statistics

1. (0·96 (SD 0·14) μmol/l) than those with supplement intake
2. (1·03 (SD 0·14) μmol/l), T (2039): 9·9, P < 0·001), while mean concentrations of Zn (12·55 v 12·57 μmol/l) and Cu (26·25 v 26·29 μmol/l) were almost identical in both groups. There were no significant differences in demographics, parity, educational

The most recent guidelines of the Clinical Laboratory Standard Institute (vol. 28 no. 30, 2012) for defining reference ranges in the clinical laboratory advise that an adequate sample size for reliable numbers should at least contain 120 samples(21). The current sample size of 2041 amply meets this criterion.

Table 1. Characteristics of 2041 women with thyroid-hormone parameters assessed at 12 weeks’ gestation (Numbers and percentages; mean values and standard deviations; median values and ranges)

Mean SD n % Median Range

Demographic features Caucasian 2021 99 Age (years) 30·5 3·5 21–40 Educational level

Low 578 28·3 Medium 114 5·6 High 1349 66·1

Marital status

With partner 1931 94·6 Single 110 5·4

Obstetric features

Primiparous 1002 49·1 Previous miscarriage 508 24·9

Lifestyle habits during pregnancy Smoking 126 6·2 Any alcohol intake 73 3·6 Pre-pregnancy BMI (kg/m2) 23·8 3·7 BMI between 25 and 30 kg/m2 469 23 BMI between 30 and 35 kg/m2 142 7 BMI > 35 kg/m2 41 2

Thyroid parameters TSH (mIU/l) 1·76 2·93 1·47 0·01–27·6 logTSH (mIU/l) 0·14 0·34 0·16 –2·0 to 1·44 FT4 (pmol/l) 14·5 2·48 14·34 4·6–32·8 log FT4 (pmol/l) 1·16 0·05 1·16 0·66–1·52 TPO-Ab > 35 IU/l 179 8·6 History of thyroid dysfunction in parents 204 10

Low, primary education or secondary pre-vocational education; medium, secondary education or vocational education; high, Bachelor or Master’s degree; TSH, thyrotropin; FT4, free thyroxine; TPO-Ab, thyroid peroxidase antibodies.

1. Table 2. Plasma concentration of selenium, zinc and copper (μmol/l) in healthy pregnant women, categorised by supplement use during early gestation* (Numbers and percentages; mean values and standard deviations; median values and ranges)

Total sample Se Zn Cu n % Mean SD Median Mean SD Median Mean SD Median

1. 1. No supplement use 252 12·3 0·94 0·12 0·94 12·42 1·59 12·43 26·50 4·95 26·05
2. 2. Daily supplements with Se, Zn and Cu 1497 73·3 1·03 0·14 1·02 12·56 1·82 12·46 26·25 4·75 25·87
3. 3. Folic acid (alone or vitamin D) 240 11·8 0·96 0·13 0·95 12·60 1·82 12·32 26·20 4·42 25·79
4. 4. Vitamin D alone 52 2·5 0·95 0·20 0·94 12·90 2·26 12·40 25·71 4·79 26·26

* On daily base intake of Se: 27·5 55 μg; of Zn: 5 10 mg and of Cu: 1 mg.

level, smoking habits, alcohol intake, age and BMI between these two groups.

Based on the guidelines of the American Thyroid Association(20), the different sub-groups of women with thyroid dysfunction areshown withtheirTPO-Ab status andcorresponding mean and standard deviation Se/Zn/Cu values for those subgroups, regardless of TPO-Ab status (Table 3).

With regard to plasma Se concentration, using the euthyroid group as the reference group, there was no significant difference between the different sub-groups and the reference group (t test). With regard to plasma Zn concentration, the lowest mean concentration, that is, 11·97 (SD 1·58) μmol/l, was found in hypothyroxinaemic women, which was significantly different from that of the euthyroid group (T (1885) = 2·3, P = 0·019). In the present study, the highest plasma Zn concentrations were found in women with clinical (overt) thyroid dysfunction (hypothyroidism n 11, and hyperthyroidism n 17). Taking these

women together (n 28), women with overt thyroid dysfunction had significantly higher plasma Zn concentrations than the euthyroid group (T (1861) = 2·2, P = 0·026). Comparing the women with normal TSH but FT4 at the upper and lower ends of the spectrum, women with hypothyroxinaemia (n 52) had significantly lower plasma Zn concentrations than those with hyperthyroxinaemia (n 32, T (80) = 2·3, P = 0·023). With regard to plasma Cu, women with clinical hypothyroidism had the lowest mean concentrations and those with clinical hyperthyroidism the highest, and these were significantly different: T (26) = 2·6, P = 0·016. Also, women with clinical hyperthyroidism had significantly higher plasma Cu concentrations than the euthyroid group: T (1850) = 2·1, P = 0·027. There were no differences in the occurrence of thyroid dysfunction sub-groups between the groups with and without Se/Zn/Cu supplement intake.

Table 4A shows several linear regression analyses with logFT4 and logTSH as dependent variables and plasma Se/Zn/Cu

1. Table 3. Different sub-groups of first-trimester thyroid (dys)function in 2041 pregnant women and their concentrations of plasma mineral selenium, zinc and copper (Numbers and percentages; mean values and standard deviations)
2. Table 4. Linear regression with log free thyroxine (logFT4) and log thyrotropin (logTSH) as dependent variables, adjusted for intake of selenium, zinc and copper supplements, parity, BMI, smoking, age and family history of thyroid dysfunction (Standardised β values and P values; n 2041)

were 1·46 times less likely (95 % CI 1·09, 2·04) to have an elevated titre of TPO-Ab at 12 weeks’ gestation.

Discussion

LogFT4 logTSH Standardised β P Standardised β P

The present study reports reference ranges (2·5th–97·5th percentiles), means and median concentrations of plasma mineral (Se, Zn and Cu) in an appropriately large sample of healthy pregnant women not taking supplements of these trace elements. It also shows that intake of Se/Zn/Cu supplements significantly affects plasma Se concentration but not plasma Zn and Cu concentrations. Moreover, it shows that, at 12 weeks’ gestation, different sub-groups of women with thyroid dysfunction showed different concentrations of plasmaZn and Cu, thoughnot Seconcentration, than did euthyroid women. Plasma Zn concentration was significantly lower in women with hypothyroxinaemia than in euthyroid women. Women with overt hyperthyroidism showed the highest plasma Cu concentrations. After adjustment for covariates (including the intake of Se/Zn/Cu supplements), linear regression showed that plasma Cu and Zn concentrations separately and in combination were positively related to FT4. Plasma Cu concentration was inversely related to TSH, while plasma Se concentration was positively correlated with TSH. TPO-Ab status at 12 weeks’ gestation was not related to plasma mineral Se/Zn/Cu concentrations. However, women taking Se/Zn/Cu supplements were less likely to have elevated titres of TPO-Ab at 12 weeks’ gestation.

1. A. Independent separately Se –0·030 0·20 0·060 0·009 Zn 0·084 <0·001 –0·018 0·41 Cu 0·079 0·001 –0·062 0·008
2. B. Independent together: Se, Cu and Zn Se –0·058 0·014 0·075 0·001 Zn 0·096 <0·001 –0·032 0·14 Cu 0·091 <0·001 –0·072 0·002

concentrations separately as dependent variables, adjusted for parity, BMI, smoking, family history of thyroid dysfunction and age. We also adjusted for intake of Se/Zn/Cu supplements.

Plasma Se was not related to logFT4 but significantly, independently and positively related to logTSH; higher plasma Se concentration was related to higher TSH. Plasma Zn and Cu concentrations were both positively and independently related to logFT4; higher plasma Zn and Cu concentrations were associated with higher FT4. Plasma Cu (but not Zn) concentration was negatively associated with logTSH. Because of a possible interaction between the trace elements, we subsequently entered Se, Zn and Cu together into the regression (Table 4B) adjusting for the same covariates. Plasma Se (negatively), Zn and Cu (both positively) concentrations were all related to logFT4; plasma Se and Cu (but not Zn) concentrations were also related to logTSH. The Cu/Zn plasma concentration ratio was not related to logFT4 nor to logTSH (data not shown).

The reference range of plasma Se concentration in the present study of the total sample (0·75–1·31 μmol/l) is similar to that in a previous study in the same area in a sample of 1000 women, although assessed in serum: 0·77–1·32 μmol/ l(22). However, in the sub-group without trace element intake in the present study, the reference range was lower (0·72– 1·25 μmol/l) than in the previous study, which was performed in 2003–2004. At that time, supplement intake was not carefully assessed, so there are no comparable data available. The median of 0·94 μmol/l plasma Se in the sub-group of the present study not taking these trace elements is low, but the Netherlands (as is Europe in general) is known as a (mildly) Se-deficient area(23). Another possibility is that during the first trimester, there is an

We finally looked at the TPO-Ab status. Mean plasma mineral Se/Zn/Cu concentrations did not differ between TPO-Abþ and TPO-Ab− women. However, the number of women with elevated TPO-Ab titre at 12 weeks’ gestation in those not taking Se/Zn/Cu supplements was 61 (11·2 %) compared with 118 (7·9 %) in those taking these supplements (χ2 (1) = 5·5, P = 0·019). Women taking additional Se/Zn/Cu supplements

lower: 9·5–25·8 μmol/l(9). These comparisons illustrate the differences between countries in reference ranges of several trace elements, which are mostlyto be explainedby eating habits and probably by ethnic differences.

increased turnover of Se due to immune tolerance processes or increased thyroid hormone production. In the group of women with Se supplement intake, the median was much higher

1. (1·02 μmol/l) suggesting that a daily dose of 27·5–55 μg Se is able to increase plasma Se concentrations (there were no differences in baseline characteristics between the two groups). In the USA National Health and Nutrition Examination Survey (NHANES) 2011–2014, the plasma Se reference range in women was

Several studies demonstrated a decrease in the concentration of serum Zn concentration and, as found in other studies, an increase in the concentration of plasma and serum Cu concentrations with progression of pregnancy(27–29). The decrease in serum Zn concentration can be explained by maternal–fetal transfer and disproportionate increase of plasma volume, while the increase in copper binding proteins, that is, ceruloplasmin, the secretion of which is stimulated by maternal oestrogen as well as decreased biliary Cu excretion, might explain the increase in Cu concentration throughout the pregnancy(9).

1. 1·83–3·10 μmol/l (144·2–244·6 μg/l) but without correction for possible supplement intake(16). It is obvious that in the present study, the reference ranges are sex- and age-specific, that is, they relate to women of fertile age. In the NHANES study, the Se reference range increased with age and was higher in males(16). A recent study from Australia reported a mean serum Se concentration of 0·73 μmol/l at 15 weeks’ gestation in 894 primiparous women without fertility problems(24). As in the present study, over 90 % were white Caucasian women but the mean age (23·4 (SD 5·0) years) was substantially lower. There were no data on women not taking Se-containing supplements. The study of Alvarez et al. in Spanish pregnant women showed a much higher mean serum Se at first trimester, that is, 1·38 μmol/l, which decreased significantly to 1·08 μmol/l at term(25). However, no intake of supplements was mentioned in that study either.

Plasma Se concentration was positively (and independently) associated with TSH – the higher the Se, the higher the TSH – while it was not correlated with FT4. This is difficult to explain because Se is important for the synthesis of thyroid hormone(5). The most likely explanation is that human chorionic gonadotropin(hCG) at 10–12 weeks’ gestation ishighly elevated for several reasons, including the transformation of the post-ovulatory ovaryinto the gravid corpusluteum for the productionof progesterone and oestradiol as well as for the augmentation of immune tolerance by promoting regulatory T-cell recruitment(30). The glycoprotein, hCG, has an α-subunit displaying homology with TSH; therefore, during early gestation, the ‘classical’ negative feedback loop between TSH and FT4 is partly overwhelmed by hCG(1,31). This will result in lower TSH and higher FT4 concentrations ‘contaminating’ the definition of hyperthyroidism. Thus, high Se would normally result in appropriate amounts of FT4, downsizing TSH, but during early gestation, the TSH action of hCG might interfere with Se-dependent thyroid hormone synthesis.

The reference range for plasma Zn concentration (9·6–16·4 μmol/l) in women not taking Zn supplements is comparable to that in another study in adult non-pregnant females, that is, 10–18 μmol/l(26). In the USA (NHANES 2011–2014 data), the 2·5th–97·5th percentiles of serum Zn concentration in 659 non-pregnant women between 19 and 30 years of age were 8·5–16·5 μmol/l (55·7–107·9 μg/dl) which is also close to that of the present study(26). Again, intake of Zn-containing supplements in the present study did not result in higher mean or median plasma Zn concentration than in women not taking Zn. This seems to be in line with the US study where serum Zn concentrations were not related to dietary or supplemental Zn intake(26). The mean serum Zn concentration in the Australian study was 9·4 (SD 2·2) μmol/l, which is comparable with the value found in our study(24).

In the NHANES 2011–2014 study, serum Cu concentration was also positively associated with TT4 and TT3 in non-pregnant females with no association for either serum Zn or Se (with TSH) concentrations(16). To the best of our knowledge, there are no previous reports suggesting that plasma Zn concentrations in pregnant women with hypothyroxinaemia and overt thyroid dysfunction (hypo- as well as hyperthyroidism) differ from those in euthyroid women. This finding is intriguing but somewhat contrary to what would be expected. A ‘direct’ effect was suggested in a recent review that suggested that Zn had a role in thyroid-hormone metabolism by regulating deiodinase activity and the synthesis of thyroid releasing hormone and TSH(14); that could explain the association found in the present study between hypothyroxinaemia and relatively low plasma Zn concentrations. A recent meta-analysis showed that patients with autoimmune disorders (including thyroid disorders) predominantly show lower serum and plasma Zn concentrations than in healthy controls(32). In Table 3, women with (sub)clinical hypothyroidism had the highest incidence of elevated TPO-Ab titre, suggesting an autoimmune origin of poor thyroid function. In the NHANES 2011–2014 study, the low serum Zn concentration cut-off for females >10 years of age was between 9·2 and 10·7 μmol/l (60–70 μg/dl), depending on the time of day when the assessment was carried out(26). In the present study, there were twenty-eight women with plasma Zn concentrations

Interestingly, the reference range for plasma Cu concentrations of the total group in the present study, 17·6–36·4 μmol/l, was almost the same as that of the sub-group that was not taking Cu supplements that is, 17·2–36·0 μmol/l. The standard dose of Cu in almost all pregnancy supplements in the Netherlands is 1 mg/d. There are several explanations for not finding a change with supplement taking: the Netherlands is Cu sufficient, which explains why further Cu intake will not increase plasma Cu concentrations. But a recent review showed that serum Cu seems to respond to supplementation only at a higher dose(7). During the first 3 months of pregnancy, the modest dose of the Cu supplements of 1 mg/d does not seem to meet the substantial physiological drop in serum Cu concentration. It should be noted that the plasma Cu concentration reference range in the present study is much higher than in the reference population of adult females (10–24 μmol/l)(7). In the Australian study mentioned above, the mean serum Cu concentration was 30·3 (SD 5·4) μmol/l which is higher than that in the present study (26·5 μmol/l)(24). In the Chinese study, the reference range of serum Cu concentrations in the first trimester was substantially

below the cut-off of 9·2 μmol/l. Those women were 3·6 times (95 % CI 1·12, 12·4, P = 0·032) more likely to have subclinical hypothyroidism at 12 weeks’ gestation. It could be hypothesised that low Zn concentrations enhance (autoimmune) destruction of thyroid cells, as is the case in both Hashimoto’s thyroiditis and Graves’ disease(1,3). However, the highest mean plasma Zn concentrations were found in women with overt hyperthyroidism (Table 3) though none of these women showed elevated concentrations of TPO-Ab, suggesting another origin of thyroid dysfunction than auto-immunity. However, a possible relationship between plasma Zn concentrations and hCG (both of which are important immune modulators) remains to be established. This also seems to be the case for Cu: the highest mean plasma Cu concentration was also found in these hyperthyroid women without elevated TPO-Ab titres.

decrease in serum Zn concentrations, which is cumulative following repeated meals, whereas overnight and daytime fasting result in increased circulating Zn concentrations(26). In the Netherlands, there is a nationwide blood assessment at 12 weeks’ gestation. Therefore, standardised tubes are used which do not meet the guidelines of the International Zinc Nutrition Consultative Group to avoid contamination. Another limitation is that ferritin concentrations were not assessed. Low ferritin levels are common during pregnancy, and some research has suggested an opposite effect on Zn and Cu concentrations. However, in a Norwegian study of 2982 pregnant women, low plasma ferritin concentration (<12 μg/l, assessed at 18 weeks’ gestation) was significantly associated with lower plasma Se concentration but not significantly with (higher) plasma Zn and Cu concentrations(10). Also, the cross-sectional design of the present study does not enable us to draw conclusions with regard to the direction of associations that were found. Our sample consisted primarily of white Caucasian women, which prohibits the generalisability of the findings to the 20 % of Dutch pregnant women of non-Caucasian origin. Studies in the USA show significant differences in trace element concentrations by ethnic background(16). Against this background, comparisons of the present study with US and Chinese studies should be interpreted with caution. Another limitation is that iodine intake, the most important determinant of adequate thyroid function, was not assessed. However, the Netherlands, unlike the UK, for example, is known to be an iodine-sufficient country(18,36). Because both iodine and Se are crucial for adequate thyroid function(5), it is reasonable to suggest that in iodine-deficient countries, poor iodine status might affect the status of other trace elements such as Se. The subgroups of women with thyroid dysfunction were rather small which means that several (non) existing associations between these sub-groups and plasma Se/Zn/Cu mineral concentrations should be interpreted with caution. A final limitation that must be mentioned is that all studies of plasma/serum mineral Se/Zn/Cu concentrations are subject to the systemic inflammatory response where serum/plasma Se and Zn concentrations fall and Cu concentration rises so that values do not reflect the true trace element status(37); even normal pregnancy is an inflammatory state, as evidenced by high cytokine concentrations(38,39).

The mean concentration of the plasma mineral Se/Zn/Cu was not related to TPO-Ab status. However, women with Se/Zn/Cu supplement intake were 1·4 times less likely to have elevated TPO-Ab titre at 12 weeks’ gestation. A recent review highlighted the effect of serum/plasma Zn concentrations on the maturation of T cells and the balance between different T-cell subsets(33). Similarly, Se and Cu are involved in the stimulation of T-reg cell formation, crucial for pregnancy immune tolerance(4,33). It might be hypothesised that the intake of these trace elements will benefit pregnancy immune tolerance to the fetal allograft. It is also known that an epi-phenomenon of immune tolerance is the decrease in antibodies (markers of auto-immune syndromes), including TPO-Ab, which might explain the lower prevalence of an elevated titre in women taking these trace elements(1).

The present study has several strengths and limitations. A major strength is the relatively high number of pregnant women enabling detection of a group of 544 women not taking Se/Zn/ Cu supplements and that is sufficiently large to define accurately the 2·5th–97·5th reference ranges. Also the total number of women in the sample enabled us to define first-trimester-specific reference ranges of TSH and FT4, and sub-groups of hypo- and hyperthyroxinaemia with appropriate power.

A major limitation of the study is that pre-pregnancy trace element concentrations were not available; however, previous reports showed that first-trimester serum/plasma Se concentrations accurately reflect pre-pregnancy concentrations(34). In the Chinese study, serum Cu and Zn concentrations were compared with those of a matched non-pregnant control group(9). Throughout pregnancy, serum Cu concentrations were significantly higher than in non-pregnant controls but the intake of pregnancy supplements was not assessed. Serum Zn concentrations were significantly lower during mid-gestation than in non-pregnant controls(9). With increasing gestation, serum Cu concentrations increased but there was no correlation between serum Zn concentrations and gestational age(9). During pregnancy, there is a substantial expansion of blood volume but predominantly after the first trimester. During the first 10–12 weeks, the average weight gain is about 0·5–2 kg, which will not explain a possible drop of trace element concentrations due to substantial blood volume expansion(35). Also, in the present study, nonfasting assessments of Zn were analysed but, as shown in the NHANES 2011–2014 study, meal consumption results in a

In conclusion, the first-trimester reference range (2·5th 97·5th percentiles) for plasma Se is largely dependent on supplement intake which is not the case for Cu and Zn reference ranges. Plasma Zn concentrations were particularly related to several categories of thyroid dysfunction. Women taking Se/Zn/Cu supplements were less likely to have an elevated TPO-Ab titre in the first trimester.

Acknowledgements

We thank the pregnant women who participated into the study and the midwives for their important role of recruitment of the pregnant women.

No funding was received. V. P. was responsible for the design of the study. J. K. was

responsible for the analyses of trace elements. V. P. – and the

1. statement: guidelines for reporting observational studies. BMJ 335, 806–808.
2. 20. Alexander EK, Pearce EN, Brent GA, et al. (2017) 2017 Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and the postpartum. Thyroid 27, 315–389.
3. 21. ClSI (2008) Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline, 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute.
4. 22. Rayman MP, Wijnen H, Vader H, et al. (2011) Maternal selenium status during earlygestation and risk for preterm birth. CMAJ 183, 549–555.
5. 23. Stoffaneller R & Morse NL (2015) A review of dietary selenium intake and selenium status in Europe and the Middle East. Nutrients 17, 1494–1537.
6. 24. Grieger JA, Grzeskowiak LE, Wilson RL, et al. (2019) Maternal selenium, copper and zinc concentrations in early pregnancy, and the association with fertility. Nutrition 11, 1609.
7. 25. Alvarez SJ, Castanon SG, Ruata MLC, et al. (2007) Updating of normal levels of copper, zinc and selenium in serum of pregnant women. J Trace Elem Med Biol 21, 49 52.
8. 26. Hennigar S, Lieberman H, Fuolgoni V, et al. (2018) Serum zinc concentrations in the US population are related to sex, age, and time of blood draw but not dietary or supplemental Zn. J Nutr 148, 1341–1351.
9. 27. Liu J, Yang H, Shi H, et al. (2010) Blood copper, zinc, calcium, and magnesium levels during different duration of pregnancy in Chinese. Biol Trace Elem Res 135, 31–37.
10. 28. Tabrizi FM & Pakdel FG (2014) Serum level of some minerals during three trimesters of pregnancy in Iranian women and their newborns: a longitudinal study. Indian J Clin Biochem 29, 174–180.
11. 29. Khoushabi F, Shadan MR, Miri A, et al. (2016) Determination of maternal serum zinc, iron, calcium and magnesium during pregnancy in pregnant women and umbilical cord blood and their association with outcome of pregnancy. Mater Sociomed 28, 104–107.
12. 30. Schumacher A (2017) Human chorionic gonadotropin as a pivotal endocrine immune modulator initiating and preserving fetal tolerance. Int J Mol Sci 18, 2166–2175.
13. 31. Nwabuobi C, Arlier S, Schatz F, et al. (2017) hCG: biological functions and clinical applications. Int J Mol Sci 18, 2037–2052.
14. 32. Sanna A, Firinu D, Zavattari P, et al. (2018) Zinc status and autoimmunity: a systematic review and meta-analysis. Nurtients 10, E68.
15. 33. Elmadfa I & Meyer AL (2019) The role of the status of selected micronutrients in shaping the immune function. Endocr Metab Immune Disord Drug Targets 19, 1100–1115.
16. 34. Rayman MP, Bath SC, Westaway J, et al. (2015) Selenium status in U.K. pregnant women and its relationship with hypertensive conditions of pregnancy. Br J Nutr 113, 249–258.
17. 35. Kominiarek MA & Peaceman AM (2017) Gestational weight gain. Am J Obstet Gynecol 217, 642–651.
18. 36. Bath SC (2017) Iodine supplementation in pregnancy in mildly deficient regions. Lancet Diabetes Endocrinol 5, 840–841.
19. 37. Oakes EJ, Lyon TD, Duncan A, et al. (2008) Acute inflammatory response does not affect erythrocyte concentrations of copper, zinc and selenium. Clin Nutr 27, 115–120.
20. 38. Kristensen K, Wide-Swensson D, Lindstrom V, et al. (2009) Serum amyloid a protein and C-reactive protein in normal pregnancy and preeclampsia. Gynecol Obstet Invest 67, 275–280.
21. 39. Buxton MA, Meraz-Cruz N, Sánchez BN, et al (2020) Repeated measures of cervicovaginal cytokines during healthy pregnancy: understanding “normal” inflammation to inform future screening. Am J Perinatol 37, 613–620.

department of statistics of Tilburg University – and J. K. were responsible for data analyses. V. P., J. K., W. M. and M. R. were responsible for the writing of the manuscript.

The authors declare that there are no conflicts of interest.

References

1. 1. Korevaar TIM, Medici M, Visser TJ, et al. (2017) Thyroid disease in pregnancy: new insights in diagnosis and clinical management. Nat Rev Endocrinol 13, 610–622.
2. 2. Maret W (2018) Metallomics: the science of biometals and biometalloids. Adv Exp Med Biol 1055, 1–20.
3. 3. Saeed F, Nadeem M, Ahmed R, et al. (2016) Studying the impact of nutritional immunology underlying the modulation of immune responses by nutritional compounds – a review. Food Agric Immunol 27, 205–229.
4. 4. Wessels I, Maywald M & Rink L (2017) Zinc as a gatekeeper of immune function. Nutrients 9, 25.
5. 5. Rayman MP (2019) Multiple nutritional factors and thyroid disease, with particular reference to autoimmune thyroid disease. Proc Nutr Soc 78, 34–44.
6. 6. Andreini C, Banci L, Bertini I, et al. (2006) Counting the zinc-proteins encoded in the human genome. J Proteome Res 5, 196 201.
7. 7. Bost M, Houdart S, Oberli M, et al. (2016) Dietary copper and human health: current evidence and unresolved issues. J Trace Elem Med Biol 35, 107–115.
8. 8. Lowe NM (2016) Assessing zinc in humans. Curr Opin Clin Nutr Metab Care 19, 321–327.
9. 9. Zhang Z, Yuan E, Liu J, et al. (2013) Gestational age-specific reference intervals for blood copper, zinc, calcium, magnesium, iron, lead, and cadmium during normal pregnancy. Clin Biochem 46, 777–780.
10. 10. Caspersen IH, Thomsen C, Haug LS, et al. (2019) Patterns and dietary determinants of essential and toxic elements in blood measured in mid-pregnancy: The Norwegian Environmental Biobank. Sci Total Environ 671, 299 308.
11. 11. Mousa A, Naqash A & Lim S (2019) Macronutrient and micronutroient intake during pregnancy: an overview of recent evidence. Nutrition 11, 1–20.
12. 12. Wu Q, Rayman MP, Lv H, et al. (2015) Low population selenium status is associated with increased prevalence of thyroid disease. J Clin Endocrinol Metab 100, 4037–4047.
13. 13. Winther KH, Rayman MP, Bonnema SJ, et al. (2020) Selenium in thyroid disorders – essential knowledge for clinicians. Nat Rev Endocrinol 16, 165–176.
14. 14. Severo JS, Morais JBS, de Freitas TEC, et al. (2019) The role of zinc in thyroid hormones metabolism. Int J Vitam Nutr Res 89, 80–88.
15. 15. Adamo AM & Oteiza PI (2010) Zinc deficiency and neurodevelopment: the case of neurons. Biofactors 36, 117–124.
16. 16. Jain RB (2014) Thyroid function and serum copper, selenium and zinc in general US population. Biol Trac Elem Res 159, 87–98.
17. 17. Truijens SEM, Meems M, Kuppens SM, et al. (2014) The HAPPY study (Holistic Approach to Pregnancy and the first Postpartum Year): design of a large prospective cohort study. BMC Pregnancy Childbirth 14, 312.
18. 18. Pop VJ, Broeren M & Wiersinga WM (2014) The attitude toward hypothyroidism during early gestation: time for a change of mind. Thyroid 10, 1541–1546.
19. 19. Von Elm E, Altman DG, Egger M, et al. (2007) Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)