Two prospective studies were consecutively conducted in two maternal clinics located in Yokohama City, Japan between July 2010 and October 2014. The distance between the two clinics was approximately 13 miles. Healthy pregnant women who consecutively attended and were without a known history of thyroid dysfunction or taking thyroid hormone or anti-thyroid drugs, and their newborn infants were randomly recruited in the study. The first study (Study 1) was conducted from July 2010 to October 2011 in Ogawa Clinic and 149 mothers and 146 infants were included. The second study (Study 2) was conducted again in Ogawa Clinic from October to December 2013 and in Nakamachidai Ladies Clinic from March to October 2014 in the same way. After excluding the subjects with severe obstetric complications and high serum thyroid autoantibody (ThAb) values or the infants with perinatal complications, a total of 178 mother–infant pairs, 103 from Ogawa Clinic and 75 from Nakamachidai Ladies Clinic, were combined together because there was no statistically significant difference in the mother’s age, body weight, height and dietary iodine intake (DII) as well as the infant’s gestational age, birth weight, height, male to female ratio and thyroid function (Table 1). Gestational dates were confirmed by ultrasound in the first trimester of pregnancy. A total number of 327 mothers and their 324 of their infants were included in the studies. Seventy-five of 327 women were delivered by Caesarean section due to various obstetric complications. Cesarean section delivery includes both elective and emergent Caesarean delivery and these two subtypes were not differentiated in this study.
Iodine-containing disinfectants were not used routinely in the maternal and neonatal unit. The type of disinfectant employed for perineal preparations prior to deliveries was 0.025% benzalkonium chloride (BZC), while during Caesarean deliveries 10% povidone-iodine (PVP–I), i.e., ISODINE® solution 10%, Shionogi Healthcare Co., Ltd. Osaka, Japan (distributor) and Mundipharma K.K., Tokyo, Japan (manufacturer) was used for abdominal skin sterilization which is distributed as BETADINE® outside Japan. The skin in the lower abdomen was swabbed three times by using cotton balls presoaked with exactly 100 mL of 10% PVP–I solution which contains 1g of effective iodine. To avoid prolonged contact with PVP–I, after it dried, the skin was wiped off with a sodium thiosulfate-ethanol swab until all of the color from the iodine had disappeared. The time from applying PVP–I solution on the mother’s skin to cutting off the umbilical cord of the newborn infant was usually 10 minutes.
Based on the type of disinfectant used prior to delivery, all subjects including the neonatal infants were divided into two groups, i.e., Cesarean section delivery with prepared povidone-iodine application for abdominal preparation (CS–PVP–I group) and vaginal delivery prepared with benzalkonium chloride (VGD–BZC group) (Table 1), and the parameters of iodine status and thyroid function were compared between the two groups. The mothers and infants who were not exposed to iodine-containing disinfectant and serially provided four urine samples were included in a longitudinal study on perinatal change of urinary iodine excretion.
Sampling for analyses
In Study 1, a total of four spot urine samples for determining urinary iodine concentration (UIC) were collected from the pregnant women at the 28–37th week of gestation, during the active phase of the delivery, on day 2 after delivery, and the first month after delivery. Venous blood samples were obtained together with the first and third urine samples and serum thyrotropin (TSH), free thyroxine (FT4) and iodine concentrations were measured. Three serum ThAbs, i.e., TSH receptor antibody (TRAb), thyroperoxidase antibody (TPOAb) or thyroglobulin antibody (TgAb) were exclusively measured, when their TSH and/or FT4 values in the samples obtained at the initial study visit were out of the reference range. Neonatal urine was collected using a small self-adhesive sterile bag (Atom Medical, Tokyo Japan) during the first 24 h, on day 4 or 5 and at 28 days after birth. A nurse placed a bag on the perineum of the infants at birth and checked every 2 hours until the first voiding, and then 2 mL of clean urine not soiled by feces was collected using a 5 mL disposable syringe. The last urine sample of infants was collected at home using a plastic bag by each of the infants’ mothers within 24 h before her postpartum visit to the clinic and kept refrigerated.
In Study 2, urine and breast milk samples were collected from the lactating mothers on day 4 and one month after birth. Breast milk samples were obtained by manual expression and stored at –20 ˚C.
As part of the National Neonatal Screening Program a heel-prick blood sample was taken on filter paper on postnatal day 5 from all infants in both studies, and TSH, FT4 concentrations were measured. All serum and urine samples were kept frozen at –30 ˚C before analysis.
Calculation of dietary iodine intake and urinary iodine excretion for mothers and infants
Maternal dietary iodine intake (DII) was assessed before and after delivery by using a semi-quantitative food frequency questionnaire (FFQ). The daily DII from each food was calculated on the basis of daily, weekly or monthly frequency of food consumption and iodine content of the specific food and given in μg iodine per day .
Newborn infants were fed with 10 ml of 5% glucose at 6 to 8 h after birth, followed by 10 ml of breast milk or formula milk increasing the amount every three hours thereafter. Each infant’s feeding status (type and ratio of breast milk) was obtained from interviews with their mothers at the infant checkup on day 2 and one month after birth. The daily DII in infants during the early neonatal period was calculated with his or her own mother’s breast milk iodine concentration (BMIC) and the sucked amount of breast milk or formula milk by weighing the infant’s body weight before and after feeding. The formula milk used in the two hospitals is the same product, “Hohoemi”, Meiji Co., Ltd., Tokyo. We have measured the iodine content in this milk and the mean iodine concentration was 51.35μｇ/100g. The most reliable method to evaluate urinary iodine excretion (UIE) is a 24–h collection of urine, however, in this study the estimated 24–h UIE of infants was calculated based on the UIC in single specimens, assuming a daily urine volume in neonatal infants of 90 or 250 mL according to a previous report on Asian term-infants .
Serum TSH and FT4 were measured by electrochemiluminescence immunoassay (ECLIA) using ECLusys TSH and FT4 (Roche Diagnostics K.K., Tokyo, Japan). The detection limit and reference ranges for Japanese given by the manufacturers were: TSH, 0.005 mIU/L, 0.50–5.0 mIU/L; FT4, 0.01 ng/dL, 0.90–1.70 ng/dL. TRAb, TPOAb and TgAb in maternal serum were measured by ECLIA using ECLusys TRAb, ECLusys TPOAb and ECLusys TgAb (Roche Diagnostics K.K., Tokyo, Japan), respectively. Serum ThAbs values above the manufacturer’s reference limit (2.0 IU/L for TRAb, 16 IU/mL for TPOAb and 28 IU/mL for TgAb) were considered positive. Neonatal blood TSH, and FT4 were measured in the dried heel blood spot samples by enzyme-linked immunosorbent assay (ELISA) using Cretin TSH ELISA II ‘Eiken’ (Eiken Chemical Co., Ltd. Tokyo, Japan) and Enzaplate N-FT4 (Siemens Healthcare Diagnostics Inc. Tokyo, Japan), respectively. The detection ranges were: TSH, 0.5–100.0 mIU/L; FT4, 0.20–10.0 ng/mL. The intra-assay and inter-assay coefficients of variation for TSH were 5.9–7.7% and 6.2–7.9%, respectively. The intra-assay and inter-assay coefficients of variation for FT4 were 4.11–5.29% and 4.21–5.47%, respectively.
The iodine concentration of serum and urine samples was measured at Hitachi Chemical Co., Ltd., Kawasaki, Japan using the modified microplate method based on the ammonium persulfate digestion on microplate (APDM) method with spectrophotometric detection of the Sandell-Kolthoff reaction . The analytical sensitivity for iodine was 13.9 μg/L and the intra-assay and inter-assay coefficients of variation were 1.8–6.3 and 1.5–6.9%, respectively. This laboratory is controlled by its participation in the Ensuring the Quality of Urinary Iodine Procedures (EQUIP) program of the Centers for Disease Control and Prevention, Atlanta, U.S.A. . The creatinine (Cr) concentration in urine was estimated by colorimetric enzymatic assay.
Breast milk samples were sonicated for 15 min in a 37 ℃ water bath by using Ultrasonic Cleaner, ASU-20, AS ONE Corporation，Osaka, Japan (oscillation frequency: 40 kHz, high frequency output power: 360W). Iodine in breast milk was extracted and measured in accordance with “The official method for determining the iodine content of foods” published by Minister of Health, Labour and Welfare, Japan . Five hundred microliters of the sample solution were weighed using a precision balance with the minimum display of 0.1–0.01 mg, Shimadzu AUW120D, Shimadzu Corporation, Kyoto, Japan, and added to a polypropylene tube (DigiTUBE®, GL Sciences, Tokyo, Japan) up to a volume of 50 mL with 0.5% TMAH solution for extraction at 60 ℃ for 12 h. After cooling to room temperature, the suspension was centrifuged by 3,000 rpm for 10 min, then 15 mL of the supernatant was collected, and stored at –70℃ until analysis. The total iodine content was determined by using inductively coupled plasma-mass spectrometry (ICP–MS), iCAP Q (serial # ICAPQ 01668) with Cetac ASX260, Thermo Fisher Scientific K.K., Tokyo, Japan. The limit of detection (LOD) and background equivalent concentration (BEC) of this assay were 0.005 and 0.187 ppb, respectively. The intra-assay coefficient of variation was 5%. All analytical procedures were carried out at the Central Laboratory, Teikyo University, Tokyo, Japan.
Ultrapure water was prepared using a pure water production system for laboratory analysis, PURELAB Ultra Ionic, Organo Corporation, Tokyo, Japan, with a specific electrical resistance value of 18.2MΩ･cm at 25 ℃. The reagents used were: 25% tetramethyl ammonium hydroxide solution (TMAH), Wako Pure Chemical Industries, Ltd., Japan; Certified reference material for iodine, AS–I9–2Y 1,000 μg/mL Iodide (Lot No. 4–25I–2Y) SPEX CertiPrep, LLC. NJ, U.S.A.; Calibration standard for ICP-MS as internal standard solution, Tellurium (Te), 1,000 μg/mL and Indium (In), 1,000 μg/mL, SCP Science, Canada. The standard reference materials for non-fortified human milk, SRM 1953 (Iodine: 193±2 mg/Kg), the National Institute of Standards and Technology (NIST), U.S.A. was used for analytic quality control.
All samples were assayed in duplicate. BMIC was expressed as µg/100g. UIC was expressed relative to creatinine excretion (UI/Cr, μg/gCr) or as a concentration (μg/L, 1μg/L=0.00788 nmol/L for conversion to S.I. units). In newborn infants UI/Cr was not used since it is reported that in the first week of life the UI/Cr value in a spot urine sample is highly variable and not useful to standardize iodine excretion of newborn infants .
The results were presented as median, range, percentile or mean with SD. The UIC, UI/Cr, BMIC, DII, serum iodine, serum or blood TSH and FT4 concentrations were not normally distributed. Differences between paired data or groups were examined using one-way analysis of variance (ANOVA) with Friedman’s Multiple Comparison Test. Differences between two unmatched groups for normally and non-normally distributed data were tested using the unpaired t test and Mann-Whitney test, respectively. A P-value less than 0.05 was considered as “statistically significant”. All data were processed and statistically analyzed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, U.S.A.).
Epidemiological criteria of iodine status in pregnant and lactating women and children
The median UIC expressed as micrograms of iodine per liter (μg/L) is used for better comparison of the population’s iodine status with the WHO–defined deficiency grades. The iodine intake in pregnant, lactating women and infants is regarded as follows: insufficient, below 150 μg/L; adequate, 150 to 249 μg/L; more than adequate, 250 to 499 μg/L; excessive, 500 μg/L or higher for pregnant women and insufficient, below 100 μg/L; adequate, 100 μg/L or higher for both lactating women and infants less than two years old.