Relation Between Subclinical Hypothyroidism and Outcome of Pregnancy
Relation Between Subclinical Hypothyroidism and Outcome of Pregnancy
Studies have demonstrated a relationship between subclinical hypothyroidism and obstetrics
 complications like preterm delivery, pre-eclampsia, placental abruption and stillbirth.
 Subclinical hypothyroidism and positive thyroperoxidase antibodies (TPOab) may increase the
 risk of early spontaneous abortion before 12 weeks of gestation. But there is not a consensus
 if the prevalence of TPOab should be treated before and during pregnancy when the level of
 thyroid-stimulating hormone (TSH) is normal.

 Thyroid hormones are regulators of the mitochondrial activity and our research group has
 previously shown that subclinical hypothyroidism affects mitochondrial activity.

 The hypothesis:

 Subclinical hypothyroidism causes early spontaneous abortion and or complications in
 pregnancy like pre-eclampsia because of mitochondrial dysfunction
The aim is to estimate the relationship between SH and spontaneous abortion and/or obstetric
 complications because of mitochondrial dysfunction.

 1. A prospective study where data from the General Suburban Population Study in Naestved
 will be analyzed to measure the frequency of subclinical hypothyroidism (SH) and TPOab.
 SH is defined as raised serum concentrations of TSH > 3,4 mU/l and normal levels of free
 T4 and T3. A cut-off of 60 mU/l is used for TPOab.

 2. To estimate the relationship between SH or TPOab and early spontaneous abortion and
 pregnancy outcome. Inclusion of women who have experienced a minimum of one early
 spontaneous abortion and between 20 and 50 years old: 50 women who have SU and/or
 TPOab-positive and 50 women who have normal levels of TSH and TPOab-negative. The basal
 oxygen consumption will be measured and mitochondrial functions will be determined by
 flow cytometry.

 3. To estimate the prevalence of gestational hypertension and pre-eclampsia among pregnant
 women with subclinical hypothyroidism and estimate the mitochondrial function. A minimum
 of 100 pregnant women without any thyroid disease or diabetes will be included.

 Early spontaneous abortion is defined as abortion before 12 weeks of gestation. Obstetrics
 complications are defined as pre-eclampsia (proteinuria, oedema, hypertension),
 pregnancy-induced hypertension, placental insufficience (IUGR) and abruption, preterm
 delivery and stillbirth. Those complications will be analyzed from the data of the obstetric
 journals.

 Measurement of oxygen consumption:

 Respiratory gas analysis and volume measurements were performed with a canopy connected by a
 triple V (flow⁄gas sensor) to an Oxycon apparatus (Jaeger Oxycon Champion, Breda, the
 Netherlands). The equipment was calibrated before each test to compensate for ambient
 conditions such as air pressure, temperature and humidity. Basal VO2 was measured after at
 least 6 hours fasting. Before the test the individual resting pulse was established and the
 participants rested for about 30 min in a chair. The test room was quiet and the lighting was
 subdued. The subjects were given sufficient time to become familiarized with the equipment
 and the procedures and the test was initiated when the resting pulse was achieved. Oxygen
 consumption was measured when the uptake per minute remained constant. Measurements were
 performed for repeated periods of 30 seconds for 20 minutes. The method for recording oxygen
 uptake had a within-day variation of 0.8 % and a day-to-day variation of 5.8 %.

 Measurement of mitochondrial mass (MM) and mitochondrial membrane potential (MMP) by flow
 cytometry:

 Venous blood was drawn from the patient in EDTA Vacutainer tubes. From each patient 2 samples
 of 100 µl of blood were processed. One unstained control tube and one stained were analyzed.

 The samples to be stained were added 1 µl Pacific Blue Mouse Anti-Human anti-CD14 (BD
 Biosciences, Denmark) and mixed. The tubes were incubated for 30 minutes at room temperature
 protected from direct light and subsequently washed.

 The stained cells were resuspended in 890 µl PBS and added 10 µl 1 µM MTG (MitoTracker Green
 FM, Invitrogen A/S, Denmark) and 100 µl 1 µM TMRM (Tetramethylrhodamine methyl ester,
 Invitrogen A/S, Denmark). All stock solutions were dissolved in DMSO (dimethyl sulfoxide) and
 working solutions were diluted in PBS. After mixing samples were incubated for 30 minutes at
 room temperature in the dark.
Part 2:

 Inclusion Criteria:

 Women who had experienced a minimum of one early spontaneous abortion and between 20 and 50
 years old: 80 women who have subclinical hypothyroidism defined as raised serum
 concentrations of TSH > 3,4 mU/l and normal levels of free T4 and T3 or TPOab-positive and
 80 women who have normal levels of TSH and TPOab-negative.

 Exclusion Criteria:

 -  Women with metabolic disease, diabetes or psychiatric disease.

 -  Women using medicine against hypo- or hyperthyroidism.

 -  Women who never give birth or age more than 50 years.

 Part 3:

 Inclusion Criteria:

 -  Pregnant women (3rd trimester)

 Exclusion Criteria:

 -  Women with metabolic disease, diabetes or psychiatric disease.

 -  Women using medicine against hypo- or hyperthyroidism.