Study design

This was a longitudinal study conducted in Athens-Clarke County and surrounding counties of the State of Georgia between November 2005 and December 2006. Participants recruited from the above mentioned areas reported to the Maternal and Child Nutrition Research Lab (MCNRL), Department of Foods and Nutrition, at The University of Georgia (UGA), USA, for interviews and body composition measurements. Participants were interviewed using a structured questionnaire containing both closed- and open-ended questions, which was followed by body composition measurements.

Immediately after the interview, the participant changed into a swimsuit for the body composition measurement. Height and weight were measured before entering the BOD POD for the body composition measurement. The study involved five testing days over a period of four months (36 weeks pregnancy, 2, 4, 8, and 12 weeks postpartum). All participants gave written consent before being recruited into the study after being thoroughly informed of the purpose, requirements and procedures of the study. They were also given the opportunity to read the informed consent form. The study protocol and consent form were approved by the Human Subjects Institutional Review Boards (IRBs) of the University of Georgia (UGA) and the Athens Regional Medical Center (ARMC).

Research participants were recruited via newspaper advertisements, flyers posted throughout the UGA campus, mailings to specific listservs, study brochures at doctors' offices and by word of mouth. Participants were screened for the study inclusion and exclusion criteria.

The primary outcomes were postpartum maternal weight and percent body fat changes, but we also assessed dietary intake and physical activity levels. Sample size was based on the detection of differences in postpartum maternal weight and percent body fat changes among two groups (exclusive breastfeeding and formula feeding) equivalent to 1% body fat. With a type 1 error of 0.05 and a 0.8 probability of detecting a true difference (1 – β), the required sample size per group was 8 [18]. However, because many participating mothers who were formula feeding also gave some breast milk, we ended up with exclusively breastfeeding and mixed feeding groups instead of a formula feeding group.

Eligibility and exclusion criteria

To be eligible for participation, a participant had to be at least 18 years of age, in their third trimester and not less than 36 weeks pregnant. They had to be non-smokers and not on medication which could affect their body composition such as steroids. Exclusion criteria included complications during pregnancy and delivery, a diagnosis of hypertension, pre-eclampsia or gestational diabetes, as well as pre-term delivery (≤ 37 weeks).

Study implementation and questionnaires

We collected baseline data at the time of recruitment and follow-up data at 2, 4, 8, and 12 weeks postpartum. All interviews and measurements took place in the MCNRL. During each evaluation, interviews were conducted using both closed and open – ended questionnaires to collect information on health history, infant feeding practice, and physical activity – the type of exercise, its intensity and frequency. Physical activity level was assessed based on the recommendations of the 2005 Dietary Guidelines for Americans [19]. A score for physical activity was derived by multiplying the number of days one exercised within a week by the duration of the activity [20]. For example, a participant who exercised 30 minutes a day, 3 times a week would have an activity score of 1.5 (3 × 0.5). Sedentary or inactive was defined as having an activity score of 0 – 1.4, moderately active was 1.5 – 2.9 and very active was > 3.0. Information was also gathered on dietary habits. On each visit we performed a 24 hour (24-h) dietary recall and calculated the energy and nutrient intake from foods consumed. We also used a food frequency questionnaire (FFQ) alongside the 24-h dietary recall to assess general food intake over a month period. With the 24-h dietary recall, participants were asked to recall every food they ate from morning until evening of the day before evaluation. The recall also included information on portion size as well as brand name and preparation method for each food. Overall energy intake, major macro- and micro-nutrient intakes were assessed using the Nutrition Data System for Research software. Intake of alcohol and dietary fiber were also assessed. The FFQ asked questions about specific foods and how often they were consumed (daily, weekly or monthly), and this information was used as a validation of the information collected by the 24-h dietary recall.

Body composition measurement

Body composition measurements were conducted using the BOD POD body composition system version 2.30 (Life Measurement Inc, Concord, CA). Participant height was measured to the nearest 1.0 mm using Seca 214 portable stadiometer (Itin Scale Inc, Brooklyn NY) and done in duplicates. Participant weight was measured wearing minimal clothing (bathing suit) and without shoes or jewellery on the body. Weight was measured to the nearest 0.01 kg using a calibrated electronic scale connected to the BOD POD system. Weight measurement was done during the 2-point calibration procedure for the BOD POD, described elsewhere [21].

Once these measurements were done, the computer calculated the corrected body volume (Vb_corr) which was used in calculating body density (Db) [22]. The BOD POD software then calculated the percent body fat using the Siri equation [23] for Caucasians and the general population or the Ortiz equation [24] for African Americans.

Outcome variables

The main outcome variables were changes in maternal total body weight and percent body fat. These were assessed at each evaluation time point. Changes in weight and percent body fat loss were calculated by subtracting weight or percent body fat at 2 weeks postpartum from that recorded at each evaluation time point.

Independent variables

Different one-time covariates were assessed at 36 weeks pregnancy and others at 2 weeks postpartum. These included educational background, delivery type, parity, marital status, age, pregnancy weight gain, and ethnicity. A number of time varying covariates were also assessed at each evaluation time point. These included dietary/energy intake, physical activity and infant feeding practice. The categories of infant feeding investigated in this study were exclusively breastfeeding and mixed feeding. Exclusive breastfeeding was defined as the "infant receiving only breast milk; no other liquid or solid is fed." Mixed feeding was defined as the "infant receiving some breast milk and formula as well as some solids whatever the case may be".

Statistical analysis

We performed all data entry and analyses using SPSS for Windows version 15.0 (SPSS Inc, Chicago, IL). Dietary information was analyzed using the Nutrition Data System for Research software (Nutrition Coordinating Center, University of Minnesota School of Public Health). Differences in variables by group were evaluated using t tests and analysis of variance (ANOVA) for continuous variables, and chi-square tests for categorical variables. Chi-square analyses were used to examine bivariate associations between the outcome variable and independent variables. Since the study has a longitudinal design with repeated measures over time, we conducted repeated measures analysis to assess trends in body composition changes. Multiple linear regression analysis was conducted to identify the relationship between maternal characteristics and the changes in maternal percent body fat over time. Data are reported as means ± SD. P value of ≤ 0.05 was used as a criterion for reporting statistical significance.

Participant characteristics

Prepregnancy and postpartum body weight changes

Mean self-reported prepregnancy weight for study participants was 63.4 ± 10.9 kg. MF mothers weighed more than EBF mothers (68.4 kg vs. 61.4 kg; p = 0.159) and also had higher BMIs (25.9 kg/m² vs. 22.1 kg/m²; p = 0.04). There was no significant difference in infant birth weight between the two groups (Table 2). Weight change among the two groups was compared with respect to self-reported prepregnancy weight and total weight at delivery. Although there was weight loss within both groups, the weight loss with respect to prepregnancy weight within the EBF group was significantly (p < 0.05) and consistently more at all time points than in the MF group (Figure 1a). There was no significant difference in weight loss both within and between groups with weight at delivery as the reference (Figure 1b). With respect to maternal weight at 2 weeks postpartum, the rate and amount of weight loss was greater for EBF than MF mothers throughout the duration of the study except at 4 weeks when weight loss was higher among MF groups (Figure 2; p = 0.072). Although, the difference in weight loss between the two groups was not significantly different, the magnitude of weight loss was greater among EBF mothers than MF mothers.

	Exclusive breastfeeding (n = 17) Mean ± SD	Mixed feeding (n = 7) Mean ± SD
Prepregnancy weight (kg)	61.4 ± 8.1	68.4 ± 15.6
Height (cm)	165.8 ± 6.6	160.0 ± 6.0
Prepregnancy BMI (kgm-2)	22.1 ± 2.8	25.9 ± 6.2
Weight at 36 wks pregnancy (kg)	75.6 ± 9.6	81.1 ± 13.2
Pregnancy weight gain (kg)	14.8 ± 4.2	13.7 ± 3.3
Delivery weight (kg)	76.8 ± 9.8	81.9 ± 13.6
Infant birthweight (kg)	3.4 ± 0.3	3.3 ± 0.4

* Differences between the groups were not statistically significant.

Changes in body composition

Even though the percent body fat loss was higher in MF than EBF mothers, our repeated measures analysis showed that the rate and trend of percent body fat loss across time was statistically significant for EBF mothers (p = 0.011) but not for MF mothers (p = 0.082) (Figure 3). A similar trend was found for fat mass.

Dietary intake and physical activity

EBF mothers consumed more calories than MF mothers (1980 ± 618 kcal vs. 1541 ± 196 kcal p = 0.08). They also tended to consume more fat, protein, carbohydrates, dietary fiber, calcium, iron and vitamin D than their MF counterparts (data not shown). Alcohol consumption, however, was higher among MF mothers than EBF mothers (4.8 ± 10.2 g vs. 2.06 ± 4.4 g; p = 0.3) although the difference was not statistically significant. Physical activity assessed based on the 2005 Dietary Guidelines for Americans showed MF mothers exercised more than their EBF counterparts (1.8 ± 0.7 vs. 1.0 ± 0.3). Exercise activities engaged in during pregnancy included aerobics, biking, running, walking, weight-lifting, walking on treadmill and yoga. Participants' engagement in physical activity increased with advancing postpartum period. The most common activity participants engaged in was walking. About 33% of participants engaged in walking at 36 weeks, 22% at 2 weeks, and 38%, 42% and 38% at 4, 8, and 12 weeks, respectively. Other common activities were running/jogging (17%) and aerobics (13%).

Regression analysis

Infant feeding and weight changes

Findings from this study show that mothers with higher prepregnancy BMI chose to mixed feed their newborns. They also lost more percent body fat during the early postpartum period. We found that infant feeding influenced postpartum maternal weight and body composition changes over time, although, the differences seen between the groups were not statistically significant due to the small sample size and short duration of the study. Our results show that weight loss among EBF mothers tended to be higher and at a faster rate compared to their MF counterparts. This finding is in agreement with the results from other studies [2, 4] which have found significant postpartum weight loss in exclusively breastfeeding mothers compared to formula feeding mothers. The greatest differences in weight loss, according to these studies, were detected around 3 months postpartum which is confirmed by this study. The amount of weight loss after delivery has been linked to pregnancy weight gain: the more weight gained during pregnancy, the more weight retention after delivery [2]. We found the opposite of the trend reported by Janney et al. [2]. In the current study, we found mothers who gained more weight during pregnancy lost more after delivery.

Weight changes and dietary intake

In agreement with the findings of Chou et al., [9] this study found that caloric intake was higher in EBF mothers than MF mothers. Their mean energy intake, however, did not exceed the 2000 kcal recommendation for women between the ages 19–30 years, [25] the age range of most of our participants. The low dietary intake recorded in the groups could be due to underreporting by participants. We feel confident about our dietary data as this involved five 24-hour dietary recalls taken over a 12 week period that included at least the intake of one weekend day. There were no statistically significant differences between reported intakes. We also observed a strong correlation between data from the different 24-hr dietary recalls (r > 0.967) for both within and between groups. Even though the energy spent in breast milk production is estimated to be about 500 kcal/day, the US Institute of Medicine recommends that breastfeeding mothers consume an extra 330 kcal/day to make up for the energy used in breast milk production with the difference of 170 kcal/day coming from stored fat [26]. This therefore brings the daily recommended energy intake for breastfeeding mothers to 2330 kcal. EBF mothers did not meet this recommendation. It will therefore be important to ascertain the dietary/energy intake of these women before pregnancy. This will help evaluate the current Recommended Dietary Allowances (RDAs) to determine whether that is contributing to the obesity epidemic facing the U. S. Although EBF mothers were found to exercise less than their mixed feeding counterparts, their lower activity did not affect their weight loss trend. The result from measuring weight loss with respect to weight prior to pregnancy supports the protective association of breastfeeding with postpartum weight loss reported in other studies [1–4, 27]. All of these studies used prepregnancy weight as a reference for weight loss or weight retention after delivery; they did not use other time points such as weight at delivery which was used in this study.

Infant feeding and body composition changes

In both groups, weight loss was derived more from fat mass than lean mass though EBF mothers lost more lean mass than MF mothers. This supports what has been reported by Chou et al. [9] who used the dual energy X-ray absorptiometry (DXA) and skin-fold measurements to measure and compare postpartum body composition of 20 women who were either exclusively breastfeeding or formula feeding. We found no difference in fat weight loss between the two groups by 12 weeks postpartum. Even though MF mothers seemed to have lost more percent body fat than EBF mothers in the current study, the trend in percent body fat loss was significant over time among EBF mothers compared to MF mothers according to the repeated measures analysis. The MF mothers may have lost more fat mass because they had a higher prepregnancy weight and BMI which may be an indicator of higher percent body fat compared to EBF mothers. The difference may also be due to the high dietary fat consumption and low physical activity rates of EBF mothers. Adequate Intake (AI) of dietary fat for women aged 19 – 30 years based on a 2000 kcal diet is between 25 – 35% [25]. EBF mothers were found to consume more than the recommended intake for fat (Table 4). Multiple regression analysis also showed that more calories consumed led to less percent body fat loss. Most of our participants (EBF and MF mothers) met and/or even exceeded the recommendation for macronutrient intake but not for micronutrient intake. EBF mothers especially consumed very high amounts of dietary fat which supports the finding by Chou et al. [9].

Effect of other covariates

Different studies have identified several covariates that may affect body composition besides infant feeding. These include maternal age, parity, prepregnancy weight, pregnancy weight gain, dietary intake and physical activity [1, 2, 4, 10, 12, 13]. In this study, we used multiple regression analysis to identify independent factors that affect maternal percent body fat besides infant feeding methods. Maternal characteristics that were identified to have independent effects included prepregnancy BMI and dietary intake. Identification of energy intake in the current study is consistent with what has been reported by Chou et al. [9]. To the best of our knowledge, none of the previous studies in this area have investigated the effect of BMI on maternal body composition. Regression analysis in this study showed that mothers with higher prepregnancy BMI lost more percent body fat after delivery.

Exercising during pregnancy has been found not to affect birth weight but, rather, to have other physiological benefits such as improved labor [28]. Even though this study did not investigate the physiological benefits of exercising during pregnancy, we found that women who were physically active during pregnancy engaged in exercises recommended by the American College of Obstetricians and Gynecologists [29]. Similar to a previous study by Zhang et al., [30] our participants engaged in activities such as walking, running or jogging and aerobics during pregnancy. About 75% of participants engaged in these exercises even at 36 weeks pregnancy. This percentage reduced to about 21% two weeks after delivery but gradually started increasing by 4 weeks postpartum. At 12 weeks postpartum, none of the new mothers had engaged in any high impact activity such as running.