Early pregnancy study

2.8.1 Subjects

This study utilized plasma samples from an archival collection of women undergoing frozen embryo transfer (Meyer et al., 2016). This was a prospective, observational study conducted at Glasgow Royal Infirmary and was approved by the Local Research Ethics Committee (07/S0704/49). Plasma samples were collected from women undergoing frozen embryo transfer (FET) treatment for infertility and were recruited from the Assisted Conception Unit between October 2007 and April 2010 (Meyer et al., 2016). Samples were stored at -80ºC.

2.8.2 Study design

Women were eligible for the study if they had a regular menstrual cycle. Patients who had ovulation stimulation or induction were excluded. No progesterone supplementation or other hormonal supplements were given. All women were recommended to take 400 mg per day of folic acid in line with World Health Organization guidelines. Women were informed of the study when notifying the clinic nurse of their last menstrual period (LMP) date with a view to booking FET treatment. At day 10 after LMP, the women attended the Assisted Conception Unit to commence daily hormonal sampling to detect the luteinizing hormone (LH) surge in order to time embryo replacement. At this point women provided written informed consent and a basal blood sample. Embryo transfer was performed on day 3 post- LH surge. Information on patient demographics and fertility history was collected from patient notes. Patient height and weight data were collected at the pre-LH surge visit. Body mass index (BMI) was calculated as weight in kg divided by height in meters squared. Fasting blood samples were collected at day 10 after LMP (pre- LH surge) and on days 18, 29, and 45 post-LH surge. Plasma was collected by low- speed centrifugation and frozen at -80°C within 2 hours.

2.8.2.1 Preparation of density solution

1.006 g/ml density solution was prepared by adding 11.4g NaCl, 0.1g EDTA, 1000ml distilled water (dH2O) and 1ml N NaOH. The solution density was checked using a

PAAR DMA 35 Density meter (cat. no. 84138, Paar Scientific Ltd).

2.8.2.2 Isolation of VLDL by sequential density ultracentrifugation

EDTA plasma (500 μL) was overlaid with 500 μl density solution (density (d) <1.006 g/mL) in 11*34mm Thick wall Polycarbonate Optima Ultracentrifuge tubes (cat. no. 343778, Beckman Instruments Inc., UK). The samples were then transferred to a TLA 120.2 rotor and the lid secured. Using a Optima TLX Table-Top Ultracentrifuge the samples were centrifuged at 100,000 rpm (224000g) at 23 oC for 2.5 hours with 4 deceleration.

Using a drawn-out glass pipette, the top 500 µL fraction was removed from each tube by placing the pipette on the edge of the meniscus to ensure removing the very top fraction. The isolated VLDL (500 μL) was removed and divided into two tubes;

both were stored at -20oC. One was sent for lipoprotein composition analysis, and the other was used for FA extraction and later gas chromatography (GC) analysis.

2.8.2.3 Lipoprotein composition analysis

VLDL lipoprotein fraction triglycerides (TG), phospholipid (PL), total cholesterol (TC), free cholesterol (FC), cholesteryl ester (CE), were analysed by autoanalyzer and this was carried out by Mrs. Josephine Cooney, in department of Metabolic Medicine, University of Glasgow.

2.8.3 Fatty acid extraction

Methanol: toluene (4:1), with containing C21 fatty acid internal standard and butylated hydroxytoluene (BHT) was prepared via the following steps. First, 0.02 g of Heneicosanoic acid (H-5149, Sigma) was measured and transferred to a glass bottle. Second, in the fume hood, 100 ml of toluene (BDH, 102846G) was measured and added to the bottle. Third, 400 ml of methanol (BDH, 101586B) were added and mixed with amagnetic stirrer. Finally, 0.05g of butylated hydroxy toluene (BHT) (B1378, Sigma) were added and mixed with the previous solution under the fume hood. Potassium carbonate (K2CO3) 10% w/v solution (101964H, BDH) was

prepared by mixing 100g of K2CO3 with 1L of distilled water.

2.8.3.2 Procedure

Fatty acids were extracted as described previously (Lepage and Roy, 1986). In a fume hood, 2 mL of methanol: toluene 4:1 (w/v) + 0.01% BHT and 0.2 mL of 0.2 mg/mL 21:0 internal standard (Sigma-Aldrich, St. Louis, MO, USA) was added to 200 μL of VLDL in a Pyrex glass culture tube with a Teflon lined screw cap (Bibby Sterilin, Staffordshire). Then 200 μL of acetyl chloride (100055V, BDH) was added slowly while vortexing and sealed with Teflon tape (Z-104388, Sigma). The tubes were heated on a heating block (DB.3D, TECHNE DRI-block) for 1 hour at 100oC in the fume hood. The tubes were then cooled in a water bath in a metal rack for 15 minutes, after which 3 mL of 10% K2CO3 w/v solution was added and the tubes

vortexed. Then,100μl toluene were added and mixed. Tubes were centrifuged for 8min, at 3000 rpm at 5C using Jouan CR412 refrigerated centrifuge. The upper

toluene phase was transferred to a GC vial (Supelco) using a glass Pasteur pipette. Tubes were stored at - 20C until required for injection or transport.

After VLDL isolation and FA extraction was carried out by the researcher, samples were shipped to our collaborator at Wollongong University in Australia and Gas Chromatography analysis was carried out by Nicola Zamai.

2.8.4 Gas Chromatography (GC)

The VLDL fatty acids were analysed by flame-ionization gas chromatography (model GC-17A; Shimadzu, Rydalmere, NSW, Australia). A 50 m x 0.25 mm internal diameter capillary column was used. Oven temperature was initially set at 150oC and rose to 170oC at a rate of 10oC/min, then to 200oC at a rate of 2oC/min, and finally to 211oC at a rate of 1.3oC/min. A 1 μL of sample was injected into the column, and individual fatty acids were identified by comparison with known fatty acid standards (Nu-Chek and Sigma, Sydney, NSW, Australia) and quantified by comparison with the 21:0 internal standard using Shimadzu analysis software (Class-VP 7.2.1 SP1).