To compare the means and changes over time of intra-abdominal fat (IAF) and subcutaneous abdominal fat (SAF) measured by abdominal ultrasound (US) and computerized tomography (CT).
To compare the means and changes over time of intra-abdominal fat (IAF) and subcutaneous abdominal fat (SAF) measured by abdominal ultrasound (US) and computerized tomography (CT).
Prospective cohort study of 53 women with obesity and infertility undergoing a lifestyle program.
The Pearson's correlation between IAF measurement by US compared to CT was good at baseline, month 3 and 6 (all r ≥ 0.72). The correlation of SAF measurement by US compared to CT was reasonable at baseline (r = 0.54; 95%CI 0.30-0.78) and weak at month 3 and 6 (all r ≤ 0.39). The correlation between the changes in IAF over 3 and 6 months by US compared to CT was reasonable and significant respectively (all r > 0.48). US could not measure the changes of SAF over time. The Bland–Altman plot showed good agreement between US and CT for IAF measurements (−1.1 [95%CI -3.9-1.6] cm lower mean in US) at baseline. For changes of IAF over time, mean estimates were in agreement.
In women with obesity and infertility, measuring IAF by US is in good agreement with the CT scan methodology but the measurement of SAF by US is unreliable.
Increased accumulation of fat around the abdomen, called abdominal obesity, contributes strongly to the metabolic consequences of obesity on health and it plays a role in female infertility and pregnancy complications . Waist circumference (Wc) measurement is used to identify individuals with abdominal obesity [6, 7] but it cannot differentiate between intra-abdominal fat (IAF) and subcutaneous abdominal fat (SAF) accumulation . The reliable measurement of IAF and SAF is not only important as a tool to predict cardiovascular and metabolic disease risk, but it is also essential to evaluate the effect of these fat compartments on female reproductive function. IAF accumulation is related to insulin resistance in women with polycystic ovary syndrome (PCOS)  and in these women the resulting hyperinsulinemia contributes to anovulation . Obese anovulatory women with PCOS who resume ovulation during a 6-month lifestyle program lose more IAF with no difference in the change of SAF compared to the women who did not resume ovulation . On the other hand, in women with obesity and infertility, SAF and not IAF was associated with anovulation after correcting for BMI, insulin, and testosterone . Increased IAF during early pregnancy is associated with insulin resistance and increased diastolic blood pressure  and it can predict glucose intolerance in later pregnancy .
Considering the worldwide obesity epidemic and its consequence on female reproduction, more studies are needed on the influence of the changes of IAF and SAF on female reproduction. Abdominal ultrasound (US) might be a good tool for the measurement of IAF and SAF due to the absence of radiation exposure, low cost and its general availability in fertility and antenatal clinics.
Several studies in various populations have confirmed that US is a reliable tool for the measurement of IAF and SAF when compared to the gold standard of abdominal computerized tomography scan (CT) or magnetic resonance imaging (MRI) . However, these validation studies were mostly conducted in older populations and have not been performed in women of reproductive age before. It is relevant to do so, since body composition and abdominal fat distribution change with age, potentially affecting the reliability of the US measurement technique. Furthermore, only one study tried to validate the measurement of the changes of IAF and SAF between US and CT in study subjects undergoing weight loss . Before introducing US as a tool for studying the effect of the changes of IAF and SAF on the reproductive outcome in women undergoing lifestyle intervention, validation of the US technique by comparing CT and US measurement is required in obese women of reproductive age.
The first aim of this methodological study was to investigate the correlation and agreement of the measurement of IAF and SAF between US and CT in women with obesity and infertility at fixed time points (baseline, month 3 and 6) of a lifestyle program to achieve weight loss. The second aim of the study was to evaluate the correlation of the measurement of the changes of IAF and SAF between US and CT between baseline and month 3 and baseline and month 6 of the lifestyle program.
Participants were women with obesity and infertility attending the Fertility clinic of the University Medical Center Groningen (UMCG) between 2005 and 2008. All women with a BMI > 29 kg m−2 who met the inclusion criteria (infertility ≥1 year, age < 38 years, partner with total motile sperm concentration/ejaculate ≥10 million) were approached to participate in a lifestyle program. Informed written consent was obtained and the study was approved by the Medical Ethics Committee of the UMCG. The lifestyle program comprised dietary intervention, increased physical activity and behavior modification as described before [14, 23].
Anthropometric measurements and US and CT measurements of IAF and SAF were performed at baseline, at month 3 and 6 of the lifestyle program. At these three respective time points all measurements of each patient were performed on the same day. Pregnancy was excluded before each CT. For this analysis, 53 participants for whom both the US and CT data were available were selected from the total cohort of 57 women undergoing the lifestyle program as published previously in .
Body weight (up to nearest 0.1 kg) and height (up to nearest 1 cm) were measured using a calibrated scale and stadiometer (SECA model 764, Seca, Birmingham, UK) with participants wearing light indoor clothes and no shoes. BMI was calculated as weight in kg divided by height in square meters (m2). Waist circumference (Wc) was measured (up to the nearest 0.5 cm) at the narrowest part of the torso located between the lower rib and the iliac crest using a CEFES®-CONTROL tape measure (HOECHTMASS Balzer GmBH, Sulzbach, Germany).
Using a Harpenden Skinfold Caliper (Baty International, Burgess Hill, West Sussex, UK), four skinfold thickness (SFT) measurements were performed in duplicate on the right side of the body with all subjects in a standing position. The SFT measurements were performed according to the Anthropometric Standardization Reference Manual  and included the triceps, biceps, subscapular and supra-iliac measurements and the total sum of the four skinfolds was calculated.
US was performed using a ALOKA SSD-1000 (Aloka, Tokyo, zJapan) ultrasound machine with a 3.5 MHz convex-array abdominal transducer to measure IAF and SAF. A validated protocol was followed to perform the measurements . In short, all measurements were performed at the level of the Wc. To avoid distorting of the abdominal cavity, all measurements were taken at the end of a quiet expiration. IAF measurements were performed in the midline and the right and left longitudinal lines (10 cm to the left and right of the midline, respectively) in a longitudinal plane. IAF was measured as the distance in cm from the anterior boundary of the lumbar vertebra and the peritoneal boundary of the anterior abdominal wall. SAF was measured in the midline, in a transversal plane at the level of the Wc, as the distance in cm from the cutaneous boundary to the linea alba. A standardized image capture of SAF was applied at the end of a quiet expiration when the transducer just had contact with the skin to avoid compression of the subcutaneous adipose tissue layer. See Figure 1 for an illustration of the US measurements of IAF and SAF. All measurements were obtained by a single trained observer, blinded to the measurements by CT. The data are presented in cm (to the nearest 0.1 cm) as SAF midline, IAF midline, IAF midline and right and IAF mean (mean of midline, right and left). The left measurement of IAF was often difficult to obtain due to reflection by gas in the descending colon. We therefore evaluated IAF by US with and without the measurement of the left longitudinal plane separately. The intra-observer variability was previously shown to be 1.8-2.9% for IAF and 0.6-3.0 % for SAF .
A single sliced abdominal CT scan with a total slice thickness of 18mm (consisting of three to four subslices) was performed at the level of the umbilicus (corresponding with lumbar vertebrae 4-5) and the measurement of the IAF and the SAF volume (cm3) was calculated as published previously by us . For the method comparison analysis, IAF was measured on the most cranial subslice of the CT scan as a distance in cm from the anterior boundary of the lumbar vertebra and the peritoneal boundary of the anterior abdominal wall in the midline and 10 cm to the left and right of the midline, respectively. The data are presented in cm (to the nearest 0.1 cm) as IAF midline, IAF midline and right and IAF mean (mean of midline, right and left). Distance measurement of the SAF was not possible due the distortion of SAF by the umbilicus in the midline of the CT scans. See Figure 2 for an illustration of the distance measurements of IAF. Although distance measurement of IAF on the CT scan is inferior to volume measurement, the distance measurement was necessary for the Bland–Altman plot which requires similar units of measurement for both methods. All measurements were performed by a single observer blinded for the results of the US measurements. The edited data were archived in Rogan.
Data were expressed as mean ± standard deviation. Pearson's correlation coefficients were used for comparison of the methods. Cross-sectional comparisons were made at fixed time points (baseline, month 3 and 6) of the lifestyle program. The changes of IAF and SAF between baseline and month 3 and baseline and month 6 were calculated as change from baseline and tested using unpaired Student's t test. The Pearson's correlation coefficients were calculated for the changes measured by US and CT. Method comparison analysis was performed by constructing Bland–Altman plots to evaluate the extent to which the IAF measurement by US and CT agreed. All analyses were performed using SPSS, versions 17 and 18 (SPSS, Chicago, IL). A P value of <0.05 was considered statistically significant.
The baseline data of the 53 included women (mean BMI 37.0 ± 4.9 kg m2) are presented in Table 1. At month 3 and 6 of the lifestyle program, 35 and 27 participants remained for analysis due to pregnancy and drop-out (11 pregnant and seven drop-outs at month 3; one pregnant and seven additional drop-outs at month 6). All participants were Caucasian, except for one woman of Asian origin.
|Method||Baseline||Month 3||Month 6||Month 3||Month 6|
|n = 53||n = 35||N = 27||n = 24||n = 24|
|Age (years)||29.7 ± 4.3|
|BMI (kg m−2)||37.0 ± 4.9||36.6 ± 4.9||35.2 ± 5.5||−1.1%||−4.9%|
|Body weight (kg)||106.2 ± 15.5||106.7 ± 14.5||102.9 ± 16.2||−3.3%||−6.0%|
|Waist circumference (cm)||110 ± 11||109 ± 13||107 ± 14||−0.9%||−2.6%|
|US||n = 53||n = 33||N = 24|
|SAF (cm) midline||5.4 ± 1.2||5.3 ± 1.0||5.2 ± 1.0||−0.6%||−1.6%|
|IAF (cm) midline||7.4 ± 2.0||7.1 ± 1.9||6.4 ± 1.9||−10.0%||−14.7%|
|IAF (cm) midline and right||7.9 ± 2.0||7.6 ± 1.9||6.9 ± 1.8||−9.9%||−12.6%|
|IAF (cm) mean||7.8 ± 1.9||7.7 ± 2.0||6.9 ± 1.9||−10.1%||−13.0%|
|CT||n = 53||n = 35||N = 27|
|SAF CT scan (cm3)||956 ± 198||929 ± 158||896 ± 208||−2.8%||−6.3%|
|IAF CT scan (cm3)||202 ± 62||197 ± 56||182 ± 69||−2.5%||−9.8%|
|IAF midline (cm)||8.2 ± 2.2||8.3 ± 2.2||7.4 ± 2.0||−0.3%||−9.2%|
|IAF midline and right (cm)||8.9 ± 2.1||9.0 ± 2.0||8.3 ± 1.8||−0.2%||−8.6%|
|IAF mean (cm)||9.1 ± 2.0||9.2 ± 2.0||8.5 ± 1.7||−0.2%||−6.1%|
|SFT||n = 53||n = 35||N = 27|
|Biceps (mm)||29.3 ± 5.8||27.8 ± 6.5||27.0 ± 7.4||−5.7%||−6.9%|
|Triceps (mm)||32.9 ± 6.3||32.9 ± 6.2||31.5 ± 6.3||−0.6%||−1.1%|
|Subscapular (mm)||38.9 ± 5.4||37.2 ± 7.0||34.2 ± 9.1||−6.9%||−13.9%|
|Supra-iliacal (mm)||37.7 ± 6.9||36.9 ± 6.9||33.7 ± 6.0||−4.7%||−10.7%|
|Sum of total (mm)||138.7 ± 16.9||134.0 ± 20.3||128.7 ± 26.6||−6.2%||−9.8%|
The 6-month lifestyle program resulted in considerable weight loss (mean −6.0%; range 6.25 to −24.4%). The mean loss of SAF measured by US between baseline and month 6 was small (mean −1.6%; range 23.7 to −28.1%) compared to the mean loss of SAF measured by CT (mean −6.3%; range 32.9 to −26.2%). Measurement of the loss of IAF by US between baseline and month 6 (mean −13.2%; range 15.3 to −36.5%) was in agreement with the volume measurement by CT (mean −9.8; range 18.6 to −20.9%).
Table 2 shows that the US measurements of IAF and SAF correlated well with the CT measurements of IAF (range r = 0.74 to r = 0.80) and SAF (r = 0.54; 95% CI 0.30-0.78) at baseline. At month 3 and 6 the correlation between the US and CT measurement of SAF was weak (r = 0.39; 95%CI 0.06-0.72 and r = 0.33; 95%CI -0.10-0.76, respectively). The correlations between the measurement of IAF by US and CT remained strong (range r = 0.72 to r = 0.90) at 3 and 6 months. The median BMI of the total group (36.5 kg m−2) was chosen to select two BMI groups for a subanalysis. The correlation of the IAF measurements between US and CT at baseline were comparable in participants with a BMI ≥ 36.5 kg m−2 (range r = 0.66 to r = 0.76) as compared to a BMI < 36.5 kg m−2 (range r = 0.74 to r = 0.75). The correlation of the SAF measurement between US and CT was however less accurate (r = 0.24; 95%CI -0.08-0.69) in participants with a BMI ≥ 36.5 kg m−2 compared to those with a BMI < 36.5 kg m−2 (r = 0.59; 95% CI 0.29-0.79). The Bland–Altman plot (method comparison analysis) between the measurement of IAF by US and CT in distance at baseline showed a mean negative bias for IAF of −1.1 cm (95% limits of agreement: −3.9 to 1.6) (notsignificant) (Figure 3).
|Baseline (n = 53)||Month 3 (n = 33)||Month 6 (n = 24)|
|r (95%CI)||R2||P value||r (95%CI)||R2||P value||r (95%CI)||R2||P value|
|SAF by US in cm||SAF by CT cm3|
|SAF midline||0.54 (0.30-0.78)||0.29||<0.001||0.39 (0.06-0.72)||0.15||0.020||0.33 (-0.10-0.76)||0.11||0.13|
|SF by SFT in mm||SAF by CT cm3|
|Biceps||0.26 (0.00-0.52)||0.07||0.052||0.46 (0.15-0.76)||0.21||0.005||0.38 (0.00-0.76)||0.15||0.05|
|Triceps||0.41 (0.17-0.66)||0.17||0.002||0.51 (0.21-0.80)||0.26||0.001||0.73 (0.45-1.00)||0.54||<0.001|
|Subscapular||0.44 (0.20-0.69)||0.19||0.001||0.56 (0.27-0.85)||0.31||<0.001||0.58 (0.25-0.92)||0.34||0.001|
|Suprailiacal||0.29 (0.03-0.55)||0.05||0.029||0.33 (0.00-0.65)||0.11||0.048||0.71 (0.42-1.00)||0.51||<0.001|
|Sum of total||0.51 (0.27-0.74)||0.26||<0.001||0.60 (0.32-0.88)||0.36||<0.001||0.75 (0.48-1.00)||0.56||<0.001|
|IAF by US in cm||IAF by CT cm3|
|IAF midline||0.74 (0.55-0.93)||0.55||<0.001||0.72 (0.47-0.96)||0.51||<0.001||0.90 (0.70-1.00)||0.81||<0.001|
|IAF midline and right||0.75 (0.56-0.94)||0.56||<0.001||0.73 (0.48-0.98)||0.53||<0.001||0.90 (0.70-1.00)||0.81||<0.001|
|IAF mean||0.80 (0.62-0.97)||0.64||<0.001||0.73 (0.46-0.99)||0.53||<0.001||0.90 (0.69-1.00)||0.81||<0.001|
|IAF by CT in cm||SAF by CT cm3|
|IAF midline||0.78 (0.61-0.95)||0.61||<0.001||0.79 (0.58-0.99)||0.62||<0.001||0.82 (0.57-1.00)||0.66||<0.001|
|IAF midline and right||0.79 (0.62-0.96)||0.62||<0.001||0.79 (0.59-1.00)||0.63||<0.001||0.82 (0.57-1.00)||0.67||<0.001|
|IAF mean||0.79 (0.63-0.96)||0.63||<0.001||0.79 (0.59-1.00)||0.63||<0.001||0.83 (0.59-1.00)||0.68||<0.001|
Changes in IAF and SAF were recorded between baseline and month 3, and baseline and month 6 using US and CT. The change in SAF over time measured by US did not correlate with the change measured by CT (Figure 5). To exclude the possibility that loss of SAF was too little to measure the change of SAF accurately, SFT measurement was added. The sum of the SFT measurement showed comparable loss of SAF between SFT and CT (Table 1) and a significant correlation between the CT and SFT measurements of SAF at month 3 (r = 0.35; 95%CI 0.15-0.68) and month 6 (r = 0.43; 95%CI 0.13-0.71) (Figure 5).
The correlation between the changes of IAF measured by US and CT was weak between baseline and month 3 (range r = 0.24 to r = 0.30) and significant (range r = 0.49 to r = 0.58) between baseline and month 6 (Figure 5). The Bland–Altman plot between the measurement of the changes of IAF by US and CT in distance between baseline and month 6 showed a mean negative bias for IAF of −0.2 cm (95% limits of agreement: −2.1 to 1.7) (not significant) (Figure 4).
Figure 6 summarizes the most important correlations with 95%CI's presented in the Table 2 and Figure 5. Figure 6B in particular, illustrates that the measurement of IAF by US correlated well with the measurement by CT. As shown in Figures 6C and D, the correlation of the changes over time measured by US and CT between baseline and month 6, were stronger for IAF than for SAF.
The most important finding of the present study is that in women with obesity and infertility, the measurement of IAF by US is accurate and comparable to the measurement of IAF by CT. Considering the detrimental role of IAF accumulation on female reproductive function and pregnancy outcome, the measurement of IAF by US can be a valuable tool in reproductive research on IAF.
The correlation found in our study between the measurement of IAF by US and CT at the three fixed time points of the lifestyle program is comparable to the findings of previous validation studies in different patient populations and different BMI and age groups . In agreement with the previous validation studies, we have also shown that the correlation of the measurement of SAF by US and CT is weaker than the measurement of IAF by these two methods. A subanalysis in participants with different BMI levels (BMI ≥ 36.5 kg m−2 versus BMI < 36.5 kg m−2) revealed that the findings remained unchanged for the measurement of IAF, but that the correlation of the measurement of SAF by US and CT was less accurate in the higher BMI group.
The detection of changes in IAF over time by US compared to the CT measurements, were not as strong as the correlation of the measurements at the fixed time points (cross-sectional). An important observation was that the detection of changes in IAF was weak at month 3 and significant at month 6 with 2.5 and 9.8% loss of IAF measured by CT, respectively. This suggests that the use of US for the measurement of the changes in IAF is only reliable when the change in IAF over time is large enough. The Bland–Altman plot (method comparison analysis) at baseline, showed a mean negative bias of −1.1cm of the measurement of IAF by US compared to CT in distance (as a surrogate for the volume measurement) (Figure 3). The Bland–Altman plot of the measurement of the changes of IAF between baseline and month 6 showed a good agreement with a mean negative bias for IAF of −0.2 cm between US and CT (Figure 4). The Bland–Altman plots showed a lack of change in the bias as the mean IAF (Figure 3) or the mean change in IAF (Figure 4) increased, suggesting good agreement of the methods irrespective of the IAF thickness. Considering the wide range of the limits of agreement between the measurement of IAF by US and CT (Figures 3 and 4), the accuracy of US is not sufficient to be used for diagnostic purposes in individual patients, but may perform well in a cohort-based analysis or in an epidemiological setting to investigate the role of IAF in female reproduction.
Our study confirms the findings of the only previous validation study showing that US can measure changes in IAF but not in SAF compared to CT . In this study subjects lost on average 1.9 cm of IAF on US compared to 1 cm (over 6 months) of IAF on US in our study. The changes in IAF in this study was measured by CT as change in surface (cm2) compared to change in volume (cm3) as measured in our study. Therefore, the changes in IAF and SAF on CT cannot be compared between these two studies.
In contrast with the findings for IAF, the correlation of SAF measurements and the changes over time in SAF by US was weaker compared to the CT measurements. Subcutaneous fat depots are heterogeneous and changes in one depot might not reflect the changes in the other depots. As illustrated by the CT scan (Figure 2) there is a large variation in the distance measurement of SAF with most SAF accumulation appearing on the back. This raises the question, whether the supra-umbilical anatomic location used to measure SAF by US in the present study, is suitable in obese women of reproductive age. Measurement of SAF by US at one single location may therefore not reflect the true volume and change in the volume of SAF during weight loss. This weak performance of the US measurement of the changes of SAF is illustrated by the fact that US only showed −1.6% loss of SAF compared to −6.1% loss of SAF measured by CT. A subanalysis (data not presented) of 10 women who lost a mean of 12.7 kg of body weight, also showed that even with greater weight loss, US is not able to detect the changes in SAF reliably. Too little loss of SAF in general as one explanation for the poor performance of US for the measurement of the change in SAF is unlikely, because the sum of skinfold measurements also showed comparable loss of subcutaneous fat as measured by CT. With regard to methodological aspects, in the present study, a 3.5 MHz convex-array abdominal transducer was used to measure IAF and SAF. A 5-12 MHz linear transducer is however more accurate to visualize the abdominal wall structures and SAF . In addition to evaluating the location of SAF measurements by US, we recommend that future studies should investigate whether measurement of SAF by US at multiple locations using a 5-12 MHz linear transducer improves the accuracy of the measurement of SAF.
Obesity identified by BMI only does not reflect the heterogeneous nature of this disease with 10-25% of obese individuals having a metabolically healthy obese phenotype . Body fat distribution can indicate high risks groups, with accumulation of fat around the abdomen (abdominal obesity or upper body obesity) being associated with more detrimental health risks than total body fat . Waist circumference measurement can identify high risk individuals with abdominal obesity but it cannot identify individuals with excess risk due to predominant IAF accumulation. IAF accumulation especially is associated with insulin resistance and increased cardiometabolic risk whilst SAF might even have a protective effect [8, 11]. US measurement of IAF is therefore a very attractive tool to identify individuals with obesity with an excess cardiometabolic risk for targeted intervention in a research setting.
The following limitations of this study should be considered. IAF and SAF distribution were captured by a single sliced abdominal CT scan at the level of L4-L5, whilst the outcome depends on the individual variation of IAF and SAF distribution which can be measured more accurately by whole abdominal CT or MRI scan [28, 29]. Future validation studies should therefore use multiple slices of abdominal CT or MRI to calculate the volume of IAF and SAF. A study published after the present study was launched , indicates that in women the measurement of IAF by single sliced CT scan at the level of L2-L3 shows better correlation with whole abdomen CT scan (r = 0.95) than single sliced CT scan at the level of L4-L5 (r = 0.89).
It was noted that the left IAF measurement by US was often disturbed and difficult to perform due to gas in the descending colon. This study shows that the left measurement of IAF can be omitted, because the medial, and the mean of the medial and right measurements of IAF still show good correlation with the CT measurement (Table 2 and Figure 6).
In conclusion, in women with obesity and infertility, there is a strong correlation between the measurements of IAF by US and CT. Furthermore, US can only measure the changes of IAF over time if sufficient loss of IAF has occurred. Considering the safety and general availability of abdominal US machines at fertility clinics and antenatal clinics, the use of US is an affordable tool for further research on the role of IAF on female reproductive function. Measurement of SAF by US proved not to be reliable enough for implementation in its present form and further studies are required to investigate whether measurement of SAF at other anatomic locations will improve its accuracy as compared to CT.
Thanks and appreciation to Mr. Wim Tukker, radiological technician of the department of Radiology for performing the IAF and SAF measurements of the CT so meticulously.