Figure 1 – Summary of the methodology
All pregnant women with a period of gestation of less than 12 weeks who
registered with public health midwives from early July-end of September
2019 were recruited from all MOH areas in Anuradhapura district for the
study. Collection of socio-demographic data was performed by medical
undergraduates in the third year of training, and clinical data
collection and the examination were performed by MBBS-qualified medical
officers. Baseline data was collected by using an
interviewer-administered questionnaire. Pre-tested interviewer
administered questionnaire was used for data collection on
sociodemographic, obstetric, gynecological and medical backgrounds.
Those with a history of diabetes mellitus, uncertain period of
amenorrhea (POA), known liver diseases (except NAFLD) and history of
using steatogenic drugs were excluded from the initial recruitment.
Clinical examination was performed to elicit any signs of pre-existing
diseases. Blood pressure was recorded using a high precision automated
blood pressure measuring instrument (Omron Corporation) as the mean of
two readings taken 5 min apart from both arms it and was categorized as
normal and high values for the first and second trimesters according to
the NICE guidelines 2019 28. Height (Ht), weight (Wt),
body mass index (BMI), waist circumference (WC), hip circumference (HC)
and waist to hip ratio (WHR) was taken as anthropometric measurements.
Weight was measured by using a digital weighing scale and height was
measured by using a portable stadiometer. According to standard formula
of BMI, categorized as overweight, obesity and morbid obesity and
according to standard cutoff levels for Asian population by WHO
guidelines. Waist circumference (WC) was measured by placing a
non-stretchable fibre-glass measuring tape around the waist midway
between the last rib and iliac crest with the subject in the standing
position. Two measurements were taken for improvement of the accuracy of
measurement. Hip circumference (HC) was measured the maximum
circumference of buttocks. Two measurements were taken and sum of the
measurement will be taken as WC and HC. Some data was obtained from
maternal pregnancy record. This entered data was standardized to make
sure the routine data are high quality 29. During
first trimester of pregnancy there are very minimal changes of weight
gain, and changes of waist circumference. Therefore we use standard
calculation methods and ranges for Asian adult population to calculate
BMI, cut off levels of obesity, waist circumference pregnancy period.
Baseline investigations were done to identify common metabolic
complications and to exclude any pathological conditions not related to
NAFLD. These investigations include routine basic investigations of
mothers should be done their antenatal period and investigations for
diagnose dyslipidemia and liver related disorders. Blood samples were
collected by using standard guidelines by well qualified public health
nursing sisters. Pre requisites for sample collection were informed to
mothers prior to the blood collection date. Venipuncture was done at
ante-cubital fossa under aseptic conditions and universal precautions.
Internal quality control was performed before each and every analytical
run. Peer group comparison was done every month during sample collection
and period of analysis of collected blood samples.
An oral glucose tolerance test (OGTT) was performed on all participants
at the recruitment and second trimester. Diagnoses of diabetes mellitus
in pregnancy (DIP) and gestational diabetes mellitus (GDM) were
performed using WHO (2016) criteria 30. GDM was
defined as fasting blood sugar (FBS) of 92-125 mg/dl and/or
2nd-hour plasma glucose of 153-199 mg/dl by a 75-g
glucose test anytime in pregnancy. Those with FBS ≥ 126 mg/dl and/or
2nd-hour plasma glucose ≥ 200 mg/dl were labelled as
having diabetes mellitus in pregnancy 30. Level of
serum aminotransferaces, gamma glutamyl transferaces and serum lipid
level was done as baseline screening tests to identify any liver related
pathological conditions and pre-existing dyslipidemia.
After obtaining written informed consent, all eligible study
participants were offered an ultrasound scan (USS) abdomen. USS was
performed by competent and qualified investigators. Liver echogenicity
was compared with the ipsilateral renal cortex and the spleen, and the
attenuation of waves, loss of demarcation of the diaphragm and poor
demarcation of the intrahepatic architecture were examined. Thus, fatty
liver was graded as 20; Grade 0: Normal liver
echogenicity, Grade I: Diffusely increased hepatic echogenicity
but periportal and diaphragmatic echogenicity is still
appreciable, Grade II: Diffusely increased hepatic echogenicity
obscuring periportal echogenicity but diaphragmatic echogenicity is
still appreciable, and Grade III: Diffusely increased hepatic
echogenicity obscuring periportal and diaphragmatic echogenicity.
Although comparison of fatty liver between pregnancy and the general
population is not a major objective of the study, a small sample of an
age-matched randomly selected females was subjected to USS to overcome
the possible bias of early pregnancy liver changes.