Dietary intervention prior to pregnancy reverses metabolic programming in male offspring of obese rats

Abstract

Obesity involving women of reproductive years is increasing dramatically in both developing and developed nations. Maternal obesity and accompanying high energy obesogenic dietary (MO) intake prior to and throughout pregnancy and lactation program offspring physiological systems predisposing to altered carbohydrate and lipid metabolism. Whether maternal obesity-induced programming outcomes are reversible by altered dietary intake commencing before conception remains an unanswered question of physiological and clinical importance. We induced pre-pregnancy maternal obesity by feeding female rats with a high fat diet from weaning to breeding 90 days later and through pregnancy and lactation. A dietary intervention group (DINT) of MO females was transferred to normal chow 1 month before mating. Controls received normal chow throughout. Male offspring were studied. Offspring birth weights were similar. At postnatal day 21 fat mass, serum triglycerides, leptin and insulin were elevated in MO offspring and were normalized by DINT. At postnatal day 120 serum glucose, insulin and homeostasis model assessment (HOMA) were increased in MO offspring; glucose was restored, and HOMA partially reversed to normal by DINT. At postnatal day 150 fat mass was increased in MO and partially reversed in DINT. At postnatal day 150, fat cell size was increased by MO. DINT partially reversed these differences in fat cell size. We believe this is the first study showing reversibility of adverse metabolic effects of maternal obesity on offspring metabolic phenotype, and that outcomes and reversibility vary by tissue affected.

Introduction

Obesity represents an ever increasing epidemic in developing and developed countries that also involves women in their reproductive years. According to WHO the percentage of obese females (BMI greater than 30) in Mexico rose from 21 to 28% between 1999 and 2006 while the increase in USA was even greater, from 19.7 to 33% (World Health Organisation, 2006). Maternal obesity increases maternal obstetric complications (gestational diabetes and preeclampsia) and poor fetal outcomes (macrosomia and stillbirth). Human epidemiological and well-controlled animal research studies provide a clear association between developmental programming of offspring postnatal metabolic, cardiac and endocrine function and maternal obesity during pregnancy and lactation (Barker, 2002; Nathanielsz, 2006; Armitage et al. 2008a,b; Catalano et al. 2009). When adult, the offspring of obese rats become obese and hypertensive, presenting a phenotype with insulin resistance and increased plasma leptin (Samuelsson et al. 2008; Kirk et al. 2009). By 11 years of age, children exposed to maternal obesity during pregnancy are at twice the risk of developing metabolic syndrome (Boney et al. 2005).

Whether developmental programming of offspring physiology resulting from maternal obesity and high calorie diets can be reversed by dietary interventions introduced before conception remains an unanswered question of considerable physiological and clinical interest and importance. Although rodent models of offspring metabolic developmental programming by maternal obesity and excessive maternal nutrition have been extensively investigated (Armitage et al. 2005), we know of no studies designed to reverse unwanted developmental programming effects by dietary intervention prior to pregnancy. To rectify the lack of information on this important question, we induced maternal obesity in non-pregnant female rats by feeding them a high fat diet from weaning through pregnancy and lactation, and determined outcomes in relation to offspring metabolic variables at weaning and 120 days postnatal life, and adipose tissue at weaning and 150 days postnatal life. In a separate group of females, we determined the extent to which dietary intervention by transferring MO rats back to normal chow diet 1 month before mating could reverse the adverse offspring outcomes. An obese maternal phenotype results from multiple and complex interactions between the mother's genetic predisposition and her own programming by factors in her own developmental environment pre- and post-natally. This rich, mechanistic complexity is a major confounding factor in interpretation of human epidemiological data and increases the need for data from well-controlled animal studies. We have embraced this need by rigorously controlling both genetic stock and phenotype of the mothers of the pregnant rats whose male offspring were our study subjects.

We hypothesized that dietary intervention beginning before pregnancy would be able to reverse at least some of the adverse consequences in the offspring of female rats eating a high energy, obesogenic diet from the time they themselves were weaned.

Methods

Care and use of animals

Standardization of phenotype of mothers of females used to produce the pregnancies studied

Female albino Wistar rats born and maintained in the colony of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INNSZ), Mexico City, Mexico held in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited light-controlled facility (lights on from 07:00 to 19:00 h at 22–23°C) and fed normal laboratory chow (Zeigler Rodent RQ 22-5, USA) containing 22.0% protein, 5.0% fat, 31.0% polysaccharide, 31.0% simple sugars, 4.0% fibre, 6.0% minerals and 1.0% vitamins (w/w), energy 4.0 kcal g⁻¹. At age between 14 and 16 weeks, when they weighed 220 ± 20 g (mean ±s.e.m.), females were bred to randomly assigned, non-litter mate, proven male breeders. On postnatal day 2 after delivery (postnatal day 0) litters were culled to 10 pups. At weaning (postnatal day 21), offspring females were randomly assigned to either a control (CTR; n= 5) group that received the laboratory chow or to a maternal obesity group (MO; n= 10) fed a high energy, obesogenic diet containing 23.5% protein, 20.0% animal lard, 5.0% fat, 20.2% polysaccharide, 20.2% simple sugars, 5.0% fibre, 5.0% mineral mix, 1.0% vitamin mix (w/w), energy 4.9 kcal g⁻¹. Only one female from any one litter was assigned to a group.

At postnatal day 90, 1 month before breeding, five MO females were selected at random for the dietary intervention group (DINT) and placed back on the control diet for the rest of the study, including pregnancy and lactation. The remaining five MO females continued the high fat diet. At postnatal day 120 all three groups were bred and were fed their pre-pregnancy diet throughout pregnancy and lactation. All rats delivered by spontaneous vaginal delivery. Day of delivery was considered as postnatal day 0. Food and water were available ad libitum. All procedures were approved by the Animal Experimentation Ethics Committee of the INNSZ, Mexico City. Pregnant and lactating rats were weighed every day through pregnancy and until pups were removed at weaning.

Maintenance of offspring

Litter size and pup weight were recorded at birth. Ano-genital distance, anterior–posterior abdominal distance and head diameter were measured with calipers. Our published data indicate that ano-genital distance is 1.67 ± 0.13 mm (n= 291 pups from 43 litters; mean ±s.e.m.) in female pups and 3.26 ± 0.22 mm (n= 252 pups from 43 litters) in males at birth (Zambrano et al. 2005). Since a value of 2.5 mm is more than 2 s.d.s from the mean of either group, sex was judged according to whether the ano-genital distance was greater than (male) or less than (female) 2.5 mm. Litters of over 14 were excluded. To ensure homogeneity of offspring evaluated, all litters studied were adjusted to 10 pups per dam except for one MO litter that contained only 9 pups all of which were retained with the mother. The sex ratio was maintained as close to 1:1 as possible. Pups continued to be weighed every week.

Offspring blood samples and retrieval of organs

At postnatal day 21, mothers were removed and pups fasted for 4 h. Two male offspring were chosen at random from each litter and trunk blood samples obtained following rapid decapitation by experienced personnel trained in the procedure using a rodent guillotine (Thomas Scientific, USA). Morphometric measurements were made on the neonates. Subcutaneous fat, the most plentiful site at this stage of development, was scraped clean from the skin and abdominal wall tissue from the axilla to iliac crest, weighed and either frozen in liquid nitrogen or fixed for histology. At postnatal day 120, following an overnight fast, blood was removed from the tail vein of two male rats chosen at random from each litter. At postnatal day 150, following an overnight fast, rats were rapidly killed by decapitation as described above and perigonadal fat was excised.

Quantification of adipocyte size

Paraffin sections of fixed fat were stained with haematoxylin and eosin and analysed with a Leica Qwin – 500 W microscope equipped with a digital camera. For each sample, four areas and 10 cells in each area were evaluated. Cell areas were obtained using Leica software for digital imaging processing.

Biochemical analyses

Blood glucose measurement

Serum glucose was determined spectrophotometrically using the enzymatic hexokinase method (Beckman Coulter, Co., Fullerton, CA, USA). Intra- and inter-assay coefficients of variations (CV) were <2% and <3%, respectively.

Insulin radioimmunoassay (RIA)

Serum insulin concentrations were determined by RIA (Linco Research, Inc., St Charles, MO, USA; Cat. no. RI-13K). The intra- and inter-assay CVs were <4% and <6%, respectively.

Triglycerides and cholesterol measurement

Serum triglycerides were determined enzymatically (Synchron CX auto analyzer, Beckman Coulter). Intra- and inter-assay CVs were <6%.

Leptin radioimmunoassay

Serum leptin was determined by RIA (Linco Research, Inc., Cat. no. RL-83K). The intra- and inter-assay CVs were <4% and <5%, respectively.

Measurement of food intake at postnatal day 120 to 150

Offspring food intake was measured for 14 consecutive days between 120 to 150 days of age. Two male rats from the same experimental group were housed per cage. Food was provided in the form of large flat biscuits. The amount of food provided each day was weighed as was the amount remaining after 24 h. The amount consumed was averaged between the two rats.

Statistical analysis

Litter sizes were normalized to 10 pups per litter on postnatal day 2 and all measures were made in two randomly selected males per litter, and data from these offspring averaged for analysis to provide an n= 5 litters per group. All data are presented as mean ±s.e.m. To conform to common practice we performed the conventional analyses used by ourselves (Zambrano et al. 2006) and others (Nivoit et al. 2009), in this type of study of equal numbers of subjects per litter in which n refers to the number of litters. The effect of diet before and during pregnancy as well as differences between groups of offspring was assessed by one-way analysis of variance (ANOVA) with the Tukey post hoc test where appropriate. P < 0.05 was regarded as significant. Confirmation of significant differences was obtained using a mixed linear model to analyse the data with dam as a random effect using data from all the pups rather than litters in which n= 10 pups per group. With this method we obtained the same significant changes as with the conventional method. Insulin resistance index (IRI) was assessed by the homeostasis model assessment (HOMA) calculated from the formula IRI = glucose (mmol l⁻¹) × insulin (μU ml⁻¹)/22.5 (Nandhini et al. 2005).

Results

Maternal phenotype

One month prior to breeding, non-pregnant females on the high energy obesogenic diet were 22% heavier than controls (Fig. 1 and Table 1). When bred at postnatal day 120, the MO group was 16% heavier than the control females while the dietary intervention group was intermediate in weight and was now only 9% heavier than controls. At postnatal day 21, the time the pups were weaned, maternal serum leptin was higher in the MO group in comparison with the control mothers. Leptin levels in the DINT group were similar to controls (Table 1).

Figure 1. Weight curves for the three groups of mothers.

A, pre-pregnancy growth curves from weaning to mating, B, maternal weight during pregnancy and lactation. 22-0 means parturition day: 22 end of pregnancy and 0 beginning of lactation. Mothers: control (•), #P < 0.05 different from both obese mothers (MO, ○) and dietary intervention mothers (DINT, ▾), *P < 0.05 versus MO (n= 5 per group). Data are mean ±s.e.m.

Table 1.

Maternal and Offspring phenotype

	Control	MO	DINT
Maternal phenotype
Weight 1 month before breeding (g)	178.3 ± 7a	218.5 ± 8b	217.8 ± 7b
Weight at breeding (g)	213 ± 9a	248 ± 13b	232 ± 7ab
Weight at the end of pregnancy (g)	316 ± 16	325 ± 1	307 ± 7
Weight at delivery (g)	239 ± 17a	265 ± 8b	233 ± 6a
Weight at weaning (g)	280 ± 19	253 ± 10	267 ± 15
Serum leptin at weaning (ng ml−1)	0.8 ± 0.1a	3.8 ± 0.1b	1.2 ± 0.1a
Offspring phenotype
Weight at birth (g)	6.2 ± 0.1	6.0 ± 0.1	6.0 ± 0.1
Length (mm)	51.1 ± 0.1	51.3 ± 0.4	50.8 ± 0.3
Head diameter (mm)	11.2 ± 0.07	11.3 ± 0.04	11.2 ± 0.08
Abdominal diameter (mm)	12.3 ± 0.05	12.3 ± 0.05	12.4 ± 0.09
Head:abdominal ratio	0.93 ± 0.01	0.92 ± 0.01	0.90 ± 0.01
Ano-genital distance (mm)	3.71 ± 0.05	3.88 ± 0.07	3.72 ± 0.07
Ano-genital distance (mm g−1)	0.59 ± 0.02	0.65 ± 0.01	0.62 ± 0.02
Body weight at weaning (g)	40 ± 1.6	42 ± 2.2	37 ± 2.7
Body weight at 120 d (g)	300 ± 10	308 ± 2	313 ± 10
Body weight at 150 d (g)	348 ± 12	354 ± 9	369 ± 10
Food intake (g day−1) between 120 and 150 d	17.9 ± 0.6	17.1 ± 0.7	18.5 ± 0.4

Data are mean ±s.e.m. from n= 5 mothers per treatment group. P < 0.05 for data not sharing at least one letter. MO: maternal obesity, DINT: dietary intervention.

Offspring phenotype at birth

At birth there were no differences in offspring between groups in birth weight or other morphometric variables measured (Table 1).

Offspring phenotype at weaning

There were no differences in body weight in the pups between groups at weaning (Table 1). However, MO pups had more subcutaneous fat tissue, and higher serum triglycerides, leptin and insulin than control. Dietary intervention returned all four measures to control levels (Fig. 2). While serum glucose did not differ between the three groups of offspring, serum insulin was elevated in MO offspring and returned to CTR levels in the DINT group indicating the presence of insulin resistance in the MO offspring.

Figure 2. Offspring phenotype on postnatal day 21.

A, subcutaneous fat mass; B, serum triglycerides; C, serum leptin; D, glucose; E, insulin; F, IRI. Data showing different letters are significantly different, P < 0.05 between groups. See Methods for description of maternal diets for control (CTR), obese (MO) and dietary intervention (DINT) mothers (n= 5 litters per group). Data are mean ±s.e.m.

Insulin resistance index at 120 days postnatal age

At 120 days postnatal age, offspring of MO mothers had elevated fasting serum glucose and insulin and increased insulin resistance when compared with control offspring (Fig. 3). In the dietary intervention offspring, insulin remained elevated above the control group while blood glucose did not differ from either of the two other groups. As a result, recuperation of insulin resistance was intermediate between the control and MO groups and statistically different from both (P < 0.05), indicating partial recovery with a degree of persisting insulin resistance.

Figure 3. Fasting insulin, glucose and insulin resistance index (IRI) at postnatal day 120.

A, fasting glucose; B, fasting insulin; C, insulin resistance index. Data showing different letters are significantly different, P < 0.05. See Methods for description of maternal diets for control (CTR), obese (MO) and dietary intervention (DINT) mothers (n= 5 litters per group). Data are mean ±s.e.m.

Adipose tissue characteristics at postnatal day 150

There were no differences in body weight in any of the groups of offspring at postnatal day 150 (Table 1). Offspring of MO mothers had a greater amount of body fat, larger fat cell size and higher leptin concentrations than controls (Fig. 4). Serum leptin in the DINT group was no longer different from controls. However, although the dietary intervention significantly lowered both fat depot mass and fat cell size, these were still significantly higher than controls.

Figure 4. Adipose tissue characteristics at postnatal day 150.

A, serum leptin; B, perigonadal fat mass, C, fat cell size; and D, example of haematoxylin and eosin staining of fat cells. The scale bar (50 μm) in the bottom histology panel applies to all. Data are mean ±s.e.m. Data showing different letters are significantly different, P < 0.05. See Methods for description of maternal diets for control (CTR), obese (MO) and dietary intervention (DINT) mothers (n= 5 litters per group).

Discussion

A recent review on effects of maternal obesity in pregnancy concludes ‘The escalation of obesity amongst women of reproductive age and the complications both short and long term for the mother and child has provided the stimulus for rapid development of an intervention to improve outcomes. To date, none has been validated for clinical use. Undoubtedly, the most successful intervention will be that which prevents development of obesity before the reproductive years’ (Nelson et al. 2010). We sought to determine if effective intervention could be established. We and other investigators have demonstrated that male offspring are more predisposed to the adverse outcomes resulting from exposures to challenges such as poor maternal nutrition and stress on glucose and insulin homeostasis (Desai et al. 1997; Sugden & Holness, 2002; Zambrano et al. 2006) as well as offspring lipid levels (Lucas et al. 1996). This increased susceptibility has been attributed to the faster growth and consequently more critical nutritional need (Lucas et al. 1996). Since developmental programming of offspring outcomes by maternal obesity may result from changes in uterine and ovarian function (including egg quality) prior to conception, the optimum intervention should reverse unwanted changes that occur before pregnancy begins.

The increased maternal weight and offspring adipose tissue mass indicates clearly that the experimental diet was able to produce an increase in weight and other maternal characteristics seen in maternal obesity in human pregnancy. If a direct conversion is made from weight to BMI – an extrapolation that must be made with great caution, and control mothers represent a woman with a BMI of 25, the top of the normal range, the obesogenic diet induced an increase to reach a BMI equivalent of 30.8, exceeding the overweight range and entering the lower end of the obese category. Dietary intervention returned the DINT rats to the equivalent of BMI 27.3 – the middle of the overweight range. Maternal serum leptin was still elevated in the MO group at the end of the weaning period and was completely recuperated by the dietary intervention.

The failure to observe differences in birth weights in the three groups is consistent with findings in many models of effects of maternal diet on developmental programming (Armitage et al. 2005, 2008a). In one study, birth weight was lower in offspring of mothers with diet-induced obesity due primarily to larger litter sizes of 14.5 pups as against 10.6 per litter in control litters (Nivoit et al. 2009). Thus, evaluation of outcomes, especially in the early stages of neonatal life, should be based on body composition rather than weight, which is a poor measure of the quality of the intra-uterine environment.

Studies on the effects of maternal obesity have shown similar altered glucose and insulin-related, as well as fat metabolism changes, in MO offspring outcomes to those shown here (Samuelsson et al. 2008; Kirk et al. 2009) and thus this model has value in determining ability of the dietary intervention introduced to reverse these basic adverse outcomes. It was not the purpose of this study to determine the mechanisms involved either in the developmental programming observed or its recuperation. The primary purpose was to establish, for the first time, the possibility of reversal of well-established adverse offspring outcomes of maternal obesity and high energy diets.

Although offspring basal fasting glucose levels at weaning were not different in the three groups, insulin levels were raised by maternal obesity, indicating a degree of increased peripheral resistance even at this young age. DINT completely reversed the increased insulin resistance. It is of interest that insulin levels in 21-day-old offspring were 20 times values observed at 120 days of postnatal life. High neonatal serum insulin has been previously reported (Aguayo-Mazzucato et al. 2006). By 120 days postnatal life, offspring insulin levels as well as insulin resistance index were significantly elevated in both the MO and DINT groups. Delay in emergence of altered phenotype resulting from challenges during development is a major feature of developmental programming. In addition, while recuperation appeared complete at 21 days of postnatal life, there were persistent metabolic changes since DINT did not return either the serum insulin or the insulin resistance index to control levels at 120 days postnatal life. From the point of view of translation of our observations to programming of human life time health, it is important that these animals were maintained on what is a relatively low fat, low energy normal rodent laboratory chow compared with human diets. It would be important to see if the outcomes differ following high energy dietary challenges such as provided by modern junk food. The explanation of residual effects remain to be determined but it is possible that the initial period of maternal obesity has produced epigenetic changes in the oocyte or other reproductive functions that are not completely reversed in the limited period of dietary intervention imposed.

As with the carbohydrate metabolism variables, dietary intervention completely reversed the increased adipose tissue mass and elevated triglycerides observed in MO offspring at 21 days of life. In contrast, fat cell size at postnatal day 150 was not completely reversed, again showing persistence of adverse effects of maternal obesity in the presence of the dietary intervention.

Maternal obesity resulted in increased offspring leptin concentrations at postnatal days 21 and 150. An extensive literature exists that indicates that leptin is predominantly produced by adipose tissue and acts on arcuate nucleus neurons to inhibit food intake by stimulating secretion of the anorexogenic neuropeptides POMC and CART (Bouret & Simerly, 2007). Neonatal serum leptin in rodents has a characteristic profile that demonstrates a peak between postnatal day 8 and 21 (Elias et al. 1998; Elmquist et al. 1998; Proulx et al. 2001; Yura et al. 2005; Bautista et al. 2008; Delahaye et al. 2008; Kirk et al. 2009). Though the determination of the timing and duration of the leptin peak varies between studies and depends on the precise experimental conditions (e.g. sampling frequency), the existence of a peak is now well established. The postnatal peak differs within rodent species and strains, occuring a couple of days earlier in mice than rats. The timing and trajectory of the postnatal leptin surge in rodents is critical to the development of obesity in later life (Vickers et al. 2005, 2008; Bautista et al. 2008). One very recent study shows that the leptin peak is amplified and prolonged in offspring of rats made obese by eating a high fat diet. These offspring demonstrated a hyperphagic, obese phenotype in later life (Kirk et al. 2009). In the present study, serum leptin was still elevated in offspring of the MO mothers at postnatal day 21 but was recuperated by DINT. However, it will be necessary to examine the whole neonatal profile with more frequent sampling.

Multiple mechanisms are potentially responsible for immediate and delayed offspring outcomes resulting from maternal obesity and over-nutrition. These include increased nutrient delivery to the fetus and newborn, altered growth factor function in mother, placenta and fetus, and inflammatory changes during pregnancy accompanied by obesity (Challier et al. 2008). Since an increase in maternal weight depends on increased maternal calorie intake, in both human and animal studies, it is difficult (perhaps impossible) to separate mechanistic pathways resulting from obesity per se from those due to the high calorie diet. Reduction of calorie intake is the simplest and most physiological intervention for reducing maternal weight. However, both maternal weight and diet are reduced simultaneously when dietary intake is reduced. These simultaneous changes make differentiation of outcomes due to the individual components impossible, even in experimental models. This limitation may not be a major concern in obtaining data that translate to human pregnancy since multiple factors operate in pregnant women who reduce calorie intake. Thus, our data support future conduct of similar interventions in women and provide an evidence-based background and rationale for human translation studies. Other potential interventions such as increased exercise and stimulation of calorie utilization may also provide an opportunity to differentiate the effects of weight and diet but they would also introduce other confounds.

There are currently several clinical trials on-going that are attempting to determine potential maternal behavioural and dietary modifications in obese pregnant women to improve outcomes. However, the human situation is complex and involves behavioural modifications that are very individual and the end points to be evaluated are hotly contested – weight gain and/or insulin resistance, for example (Nelson et al. 2010). There is a need to determine optimal timing, nature and extent of interventions. We have taken the view that the optimal time for recuperation would be prior to pregnancy and have sought to develop a model to show the ability and extent of the simplest of interventions, reducing global intake, to produce beneficial results. It could be argued that, regardless of the success of any experimental intervention, women will not be willing to take similar, effective action prior to pregnancy. The available evidence indicates that women do not spontaneously alter their dietary patterns when pregnant (Crozier et al. 2009). Interventions in pregnancy, as all major health areas, therefore need to be based on firm, reproducible scientific evidence. Evidence to persuade obese women to decrease their BMI either before or during pregnancy must convince them of two things. First that maternal obesity is harmful to mother and offspring in many ways and, second, that appropriately lowering their BMI and food intake will provide significant benefit to themselves and their children. The experience with the efforts that led to a dramatic decrease in cigarette smoking suggests that the strongest of compulsive behaviours can be modified when firm, incontrovertible information on benefit is provided. In the best known of all human epidemiological studies lasting over 50 years, Doll demonstrated the connection between cigarette smoking and lung cancer (Doll et al. 2004). One of the most persuasive pieces of evidence in those studies was the demonstration in men born around 1920, that while smoking from early adult life tripled mortality rates, giving up smoking at age 50 halved the risk and stopping at age 30 removed virtually all the risk. The parallel with maternal obesity would be that adjustment of life style with concomitant decrease in obesity would avoid the maternal and offspring hazards. Human studies indicate that maternal pre-pregnancy BMI is a major determinant of adverse offspring metabolic outcomes resulting from maternal obesity (Catalano et al. 2009). Therefore, in the absence of any human intervention studies, we chose to begin by recuperating the diet in our rat model before pregnancy and maintaining the recuperation throughout pregnancy and lactation. While this protocol does not allow determination of critical windows, it is an essential first step in demonstrating that recuperation can be achieved by intervention.

In summary, this study is, to our knowledge, the first to attempt to develop a model of dietary recuperation in maternal obesity in an extensively studied rodent model and provides some of the first evidence that unwanted developmental programming effects on offspring that result from maternal obesity are at least partially reversible by dietary intervention prior to pregnancy. We present these data in the hope that they provide a first step in showing the benefits of pre-pregnancy modifications that improve maternal diet and BMI. One of the key findings of this study is that it was not necessary to return the maternal weight to the level of the controls for benefit to accrue. Further studies comparing different degrees of recuperation of maternal weight will be of interest and importance.