Newborn body composition after maternal bariatric surgery.

INTRODUCTION: In pregnancy after Roux-en-Y gastric bypass (RYGB), there is increased risk of low birthweight in the offspring. The present study examined how offspring body composition was affected by RYGB.
MATERIAL AND METHODS: Mother-newborn dyads, where the mothers had undergone RYGB were included. Main outcome measure was neonatal body composition. Neonatal body composition was assessed by dual-energy X-ray absorptiometry scanning (DXA) within 48 hours after birth. In a statistical model offspring born after RYGB were compared with a reference material of offspring and analyses were made to estimate the effect of maternal pre-pregnancy body mass index (BMI), gestational weight gain, parity, gestational age at birth and newborn sex on newborn body composition. Analyses were made to estimate the impact of maternal weight loss before pregnancy and of other effects of bariatric surgery respectively. The study was performed at a university hospital between October 2012 and December 2013.
RESULTS: We included 25 mother-newborn dyads where the mothers had undergone RYGB and compared them to a reference material of 311 mother-newborn dyads with comparable pre-pregnancy BMI. Offspring born by mothers after RYGB had lower birthweight (335g, p<0.001), fat-free mass (268g, p<0.001) and fat% (2.8%, p<0.001) compared with reference material. Only 2% of the average reduction in newborn fat free mass could be attributed to maternal pre-pregnancy weight loss whereas other effects of RYGB accounted for 98%. Regarding reduction in fat mass 52% was attributed to weight loss and 47% to other effects of surgery.
CONCLUSION: Offspring born after maternal bariatric surgery, had lower birthweight, fat-free mass and fat percentage when compared with a reference material. RYGB itself and not the pre-pregnancy weight loss seems to have had the greatest impact on fetal growth.

Introduction

Maternal obesity has both short- and long-term consequences for the offspring and increases the offspring’s risk of developing overweight and obesity [1-6]. Once established, severe obesity is difficult to treat. Roux-en-Y gastric bypass (RYGB) has been proven to be an effective way to reverse or lower the degree of obesity and the related morbidity and mortality [7, 8]. Women of child-bearing age also undergo bariatric surgery [9].

Birthweight and newborn body composition are affected by maternal BMI and gestational weight gain (GWG), and both maternal obesity and excessive weight gain is associated with a higher birthweight and higher fat mass in the offspring [10-12]. We have previously shown that infants of obese mothers have higher fat mass at birth and an abdominal fat accumulation compared with infants of normal weight women. Furthermore, low birthweight was associated with a lower crude abdominal fat mass, but a higher proportion of fat mass placed abdominally [13].

The adverse body composition tracks into childhood and may contribute to obese women’s offspring increased risk of developing overweight and obesity [4, 14]. Interventional programs which aimed to limit the GWG in obese women did not seem to affect offspring weight and body composition at birth [15].

Women who undergo bariatric surgery before pregnancy have a lower risk of developing gestational diabetes mellitus, lower offspring birthweight and lower risk of large for gestational age (LGA) infants, but a higher risk of small for gestational age (SGA) infants than weight matched non-operated women. In contrast the risk of preterm delivery and having SGA infants is higher. Some studies have also found that perinatal mortality may be increased in pregnancy after RYGB [16-19]. There are no studies on how newborn body composition is affected by maternal bariatric surgery.

The aim of the present study was to examine how term newborn infant total and abdominal body composition is affected by maternal bariatric surgery, with the hypothesis that offspring born after maternal bariatric surgery have a lower total fat mass, and that they accumulate a relative higher proportion of their fat tissue abdominally. We also wanted to examine the impact of bariatric surgery on offspring birthweight and if body composition of the newborn was associated with maternal weight loss before pregnancy or surgery induced metabolic changes.

Material and methods

Pregnant women with previous RYGB and their offspring were included consecutively between the 2nd of October 2012 and the 1st of December 2013 at Copenhagen University Hospital Hvidovre, Denmark, with more than 7000 deliveries annually. All term singleton, RYGB pregnancies were offered inclusion, there were no exclusion criteria other than maternal chronic disease such as insulin dependent diabetes or pre-eclampsia. A mixed group of 80 normal weight and 231 obese mother-newborn dyads was used as reference material. None of the women in the reference material had undergone RYGB or other bariatric surgery. We consecutively recruited 80 singletons, healthy infants born at term (>258 days of gestation) and their mothers within 48 hours after delivery. The obese mothers had participated in the Treatment of Obese Pregnant (TOP) study at Copenhagen University Hospital Hvidovre [15]. In this subset of the TOP-study cohort the intervention did neither affect birthweight nor newborn body composition [13]. Exclusion criteria: Mothers with a chronic disease or pre-eclampsia were excluded. Infants requiring admission to neonatal intensive care unit or suffering from congenital diseases were also excluded. Furthermore, children born prematurely were excluded.

Maternal and infant data

Information on parity, maternal social and smoking status, exercise habits was collected from a questionnaire filled in during the first trimester survey. For women with previous RYGB the date for the bariatric surgery, preoperative weight and the weight loss before pregnancy onset was registered.

Pre-pregnancy weight was self-reported. All women were weighed at gestational week 36–37 at the hospital wearing light clothing and no shoes (Seca digital scales, Seca, Germany). GWG was calculated as the difference between pre-pregnancy weight and weight at 36–37 weeks of gestation. Height was collected through hospitals files at inclusion. Pre-pregnancy body mass index (BMI) was calculated using the self-reported weight. Gestational age was determined by ultrasound at the nuchal transluciency scan in week 11 to 14.

The infants were weighed recumbent (Seca 727, digital baby scales, Seca, Germany). Length and head circumference were measured with non-stretchable measuring tape according to WHO guidelines [20]. Abdominal circumference was measured by non-stretchable measuring tape during mid-expiration at umbilical level in the supine position. Size for gestational age was calculated based on the reference from Marsal et al. [21].

Body composition assessment of the newborn offspring

Within 48 hours after birth, newborn body composition was assessed using DXA scanning (DXA, Hologic 4500, Bedford, MA, USA). This method calculates fat and fat-free body mass as well as bone mass. Total X-ray dose for a whole-body scan was 10.5 μSv, equivalent to 1 to 2 days of background radiation. We used the same criteria as Cooke et al. when evaluating the scans, and only scans that met predefined quality criteria were included in the analysis [22]. Fat (%) was calculated as fat mass/total mass.

To estimate abdominal fat mass and fat-free mass, two abdominal regions were identified using the paediatric DXA software. The thoracic diaphragm and both upper iliac crests limited the first region, while the second region was limited laterally by the upper iliac crests and caudally by the femoral heads. The sum of the two regions constitutes the abdominal region. Abdominal/total fat ratio was calculated as abdominal fat mass/total fat mass. From duplicate scans in 58 infants, the test-retest variability concerning DXA-derived fat and fat-free mass was calculated to 11.8% and 7.1%, respectively [23].

Ethics

The Ethics Committee of the Capital Region of Denmark (H-D-2008-119) approved the study, and written informed consent was obtained from both parents before the mother and infant were included in the study.

Statistical analysis

Newborn offspring of women with previous RYGB where compared with a reference material consisting of a mixed group of obese and normal weight women’s infants. Means and standard deviations (SD) were calculated for all normally distributed outcome variables. Data were compared by Student’s t-test. Differences in proportions were tested using the Chi-square test. Simple linear regression analyses were also performed, where the effect of bariatric surgery (as a dichotomous outcome) were evaluated. In the analyses infant birth weight, fat-free mass and fat percentage were used as dependent variables. Analyses were adjusted for maternal age, smoking, primiparity, pre-pregnancy BMI, GWG, and infant sex and gestational age at birth.

In order to explore the effect of RYGB on foetal growth, body composition compartments in newborns of mothers with RYGB were compared with expected values. The expected values were calculated from the previous derived regression coefficients based on data in the reference material [13]. We used the reference material to calculate estimates of expected values of birthweight and each body composition compartment in infants of women with RYGB. For each infant, we made two sets of estimated values, one based on the maternal pre-surgery BMI and one based on the post-surgery, pre-pregnancy BMI. Furthermore GWG, parity, infant sex and gestational age at birth were incorporated in the calculations. These estimates were compared with actual measured values by means of paired t-test.

To illustrate our method, we present, as example, the regression used to calculate expected birthweight:

The impact of weight loss and RYGB on body composition in compartments in percent, were derived from the regressions models. The difference in the compartments between pre-surgery and pre-pregnacy BMI were compared to actual measured values. The difference that could not be explained by the change in pre-pregnancy BMI, was analysed as it derived from changes induced by bariatric surgery.

P values < 0.05 were set as significant (SPSS Statistics, version 19.0, Chicago, IL, USA).

Results

In the study period, there was 53 women pregnant with previous bariatric surgery. We included 25 (47%) bariatric surgery mother-newborn dyads (Fig 1). Women who delivered at another hospital (n = 13), who gave birth prematurely (n = 6) and births where the infant was admitted to the neonatal intensive care unit (NICU) (n = 1) were excluded. Eight women declined participation. There were no differences in baseline maternal characteristics in women who gave birth premature and the case that was admitted to NICU (n = 7) when compared to included women (data not shown). No data were collected on the women that delivered at another hospital.

Fig 1

Inclusion of women with previous bariatric surgery.

The reference material consisted of 311 mother-newborn dyads (n = 231 obese and n = 80 normal weight mothers). There were no differences in maternal characteristics including pre-pregnancy BMI and GWG when comparing women with previous RYGB with the reference material. However, there were large differences in newborn characteristics (Table 1).

Table 1

Baseline characteristics for mothers with or without RYGB and for their newborns.

Maternal characteristics	Bariatric surgery mothers n = 25	Reference material n = 311	p-value
Maternal age (years)1	30.3 (4.1)	31.2 (4.6)	0.35
Pre-pregnancy BMI (kg/m2) 1	28.8 (4.8)	30.9 (6.0)	0.07
Gestational weight gain (kg) 1	13.2 (8.3)	11.4 (6.1)	0.15
Primipara n (%)2	13 (52)	188 (60)	0.41
Maternal smoking (n) (%)	4 (5.2)	30 (9.6)
Placental weight (g)1	645 (91)	665 (151)	0.33
Pre-surgery BMI (kg/m2) 1	44.0 (5.4)
Weight loss after surgery until conception(kg) 1	40.8 (12.9)
Time from surgery to birth (months) 1	29.7 (11.6)
Newborn characteristics
Birthweight (g) 1	3284 (327)	3619 (523)	<0.001
Birth length (cm) 1	51.0 (1.5)	52.2 (2.3)	0.01
Head circumference (cm) 1	34.2 (1.6)	35.1 (1.6)	0.007
Abdominal circumference (cm) 1	32.0 (2.2)	33.4(2.2)	0.002
Sex 2
Male (n) (%)	13 (52)	161 (52)
Female (n) (%)	12 (48)	150 (48)	0.98
Gestational age at birth (days) 1	277 (9)	280 (9)	0.14
Birthweight Z-score *1	-0.45 (1.0)	0.12 (1.10)	0.01
Size for gestational age(n)(%) *
SGA	1 (4)	13 (4)
AGA	24 (96)	276 (89)
LGA	0 (0)	22 (7)
Birthweight (n) 2
<2500 g	0	8
2500–4000 g	24	229
>4000 g	1	74	0.04
Fat-free mass (g) 1	3062 (308)	3330 (398)	<0.001
Fat mass (g) 1	260 (126)	407 (204)	< 0.001
Fat (%) 1	7.7 (3.6)	10.5 (4.3)	< 0.001
Abdominal fat mass (g)1	32 (17)	53 (26)	< 0.001
Abdominal/total fat mass (%)1	11.2 (3.8)	12.9 (3.2)	0.02
Body mass index (kg/m2)	12.6 (1.3)	13.2 (1.3)	0.02

1 Mean (±SD), Student’s t-test.

2 Proportion, chi-square test.

* Normalised birthweight adjusted for gestational age at birth and sex, according to Marsal et al. SGA (small for gestational age), AGA (appropriate for gestational age) and LGA (large for gestational age) [21].

Offspring born after bariatric surgery were in average 9.3% lighter, 2.3% shorter, had 2.6% smaller head circumference, and had 36.1% lower fat and 8% lower fat-free mass compared with the reference material. There were no differences in newborn sex and gestational age at birth (Table1). The effect of RYGB on birth weight (-298 g, p = 0.002), fat-free mass (-253 g, p = 0.001) and fat percentage (-2.5%, p = 0.002) persisted when performing linear regression, adjusted for factors known to influence infant growth; maternal age, smoking, pre-pregnancy BMI, GWG, primiparity, smoking, infant sex and gestational age at birth (Table 2).

Table 2

Difference in infant birth weight, fat-free mass and fat (%) after bariatric surgery (RYGB) (n = 25), compared with reference material (n = 311).

Dependent variable: Newborn characteristics	RYGB infant, n = 25, Estimate (95% CI)	p-value
Birth weight (g)	-298 (-498–-106)	0.002
Fat-free mass (g)	-253 (-398–-109)	0.001
Fat (%)	-2.5 (-0.9–-4.1)	0.002

Results are derived from linear regression. Estimate based on linear regression. Analyses adjusted for maternal age, smoking, pre-pregnancy BMI, GWG, primiparity, infant sex and gestational age at birth.

The risk of preterm birth was higher in offspring born after RYGB (6/53) versus the reference material (8/404) (p<0.001).

Estimated impact of maternal weight loss and of other effects of RYGB

Maternal RYGB before pregnancy resulted in an average reduction in fat free mass of 283 g (value estimated based on pre-surgery BMI 3345g- actual measured value 3062g) in the newborn offspring. Based on the previously reported regression coefficients of associations between maternal pre-pregnancy BMI and GWG and offspring weight, and fat distribution, we calculated that 2% (5g/283g) of the average reduction in newborn fat free mass could be attributed to maternal weight loss before pregnancy (difference between estimated pre-surgery BMI fat-free mass and pre-pregnancy BMI fat-free mass was 5 g). Thus, other effects of the bariatric surgery accounted for 98% (278g/283g) (Table 3).

Table 3

Body composition in offspring of mothers after RYGB.

Newborn characteristics	RYGB mothers n = 25	Expected values based on pre-pregnancy BMI	Difference between expected value based on pre-pregnancy BMI and measured value	Expected values based on pre-surgery BMI	Difference between expected value based on pre-surgery BMI and measured value
Birthweight	3284 (374)	3582 (262)	299 (141–456)*	3788 (270)	504 (334–665)*
Fat-free mass (g)	3062 (308)	3340 (89)	283 (161–405)**	3345 (87)	278 (171–385)*
Fat mass (g)	260 (127)	405 (73)	145 (86–204)*	536 (86)	276 (212–340)*
Fat (%)	7.7 (3.4)	10.5 (1.4)	2.7 (1.2–4.3)**	13.4 (1.7)	5.7 (4.1–7.3)*
Abdominal fat mass (g)	32 (21)	52 (9)	20 (11–29)*	70 (13)	38 (8–68)*

Actual measured values in first column, expected values based on maternal pre-pregnancy BMI in second column and expected values based on pre-surgery BMI in fourth column.

Data presented as mean (standard deviation) except for last column presented as mean (95% confidence interval).

Expected values were derived by linear regression using a reference material [13]. Calculated based on pre-pregnancy pre-surgery BMI. Parity, GWG, gestational age at birth and offspring sex were included in the analysis. The difference between the actual measured value and pre-pregnancy estimated value, was set as the effect induced by bariatric surgery.

Differences between the actual and estimated values compared using paired t-test.

*p-value <0.001,

**p-value = 0.001.

Regarding reduction in fat mass of 276 g (value estimated based on pre-surgery BMI 536g- actual measured value 260g), 47% (131g/276g) was attributed to weight loss (difference between estimated pre-surgery BMI fat mass and pre-pregnancy BMI fat mass was 131 g). Other effects of RYGB lowered fat mass with 52% (145g/276g) (Table 3) (Fig 2a and 2b).

Fig 2

(a) Total fat mass in the newborn offspring: Expected values in gram calculated in the reference material using respectively presurgery BMI and prepregnancy BMI and measured values in the group where mothers had previous RYGB. (b) Total fat-free mass in the newborn offspring: Expected values in gram calculated in the reference material using respectively presurgery BMI and prepregnancy BMI and measured values in the group where mothers had previous RYGB.

Discussion

In the present study, we found that newborn offspring of mothers with RYGB before pregnancy had a lower birthweight, a lower fat-free mass, lower fat mass, lower fat percentage, and a lower part of the fat distributed abdominally. The infants were shorter in length and had a smaller head circumference. The infants in the reference material were born to mothers with comparable pre-pregnancy BMI, parity, GWG and infant sex and gestational age, all factors known to determine infant birthweight and body composition [10, 11, 13].

We found that the effect of bariatric surgery was larger than that anticipated by weight loss alone. The pathophysiology behind the effect of bariatric surgery on birthweight and newborn body composition is not fully understood but possibly mediated through glucose metabolism, comprehensive hormonal alterations and altered absorption of nutrients from the intestines [6, 24, 25]. This reduces fetal growth. The consequences hereby are not known. It could, potentially, be harmful. Two small follow up studies have found that bariatric surgery does not induce long-term growth restriction [26, 27]. To our knowledge, there is only one study examining cognitive function in infants born after maternal bariatric surgery. This study observed that offspring born after bariatric surgery had impaired speech development. The authors conclude that this was not induced by bariatric surgery since the affected children were appropriate of size at birth [28]. There is no knowledge on long-term effects of the altered body-composition and fat-distribution. And future follow-up studies on the offspring are needed.

A higher proportion of offspring of obese women are LGA. There are benefits related to a normalized birthweight; a reduction of numbers of birth traumas and neonates with hypoglycaemia [1, 6].

A central question that remains unanswered is whether lowered birthweight and altered body composition, with lower total and abdominal fat-mass as well as lower fat percentage affects future risks for obesity. A large meta-analysis has shown that birthweight above 4000 g doubles the subsequent risk of becoming overweight and obese [29]. There are (as far as we are aware of) only two small studies examining (follow up 2–18 years) consequences of bariatric surgery on the offspring risk of developing overweight and obesity, in both studies siblings born before maternal bariatric surgery were used as reference. The results are contradictive. One shows lowered risk for overweight and obesity (n = 45 sibling pairs), whereas the other finds no effect of bariatric surgery on offspring risk of becoming overweight and obese (n = 39 sibling pairs) [26, 27]. More and larger studies on this subject are warranted to be able to draw proper conclusions.

We have previously shown that infants with a low birthweight have a lower total fat mass, and tend to accumulate their fat mass abdominally [13]. The long-term consequences of this is not known, but it might be linked to the low birthweight infants’ abdominal fat accumulation as children and adults, as well as their long-term increased risk of developing type II diabetes [30-32]. In this study, there were no sign of that the infants born after maternal RYGB accumulated fat mass abdominally. There was, however, only one SGA infant in the bariatric surgery group. We only included infants born at term. So potential pregnancies with severe fetal growth restriction and delivery before gestational age 37 might not be represented in our study. In a Danish study from 2013 there were 7.1% SGA infants among mothers with previous bariatric surgery, compared with 2.9% in the matched reference material. In our small study only one (4%) of the offspring of mothers with RYGB before pregnancy were SGA [16]. One should bear in mind that a possible beneficial effect of bariatric surgery on offspring risk of developing overweight and obesity can thus be replaced by an increased risk of morbidity related to being SGA at birth.

Strengths and limitations

We only examined 25 term born infants born after bariatric surgery, but despite this, we found significant differences in both birthweight and body composition. Our findings should, however, be interpreted with caution due to the low number included. Our study was restricted to term born infants, and six were excluded due to premature birth. The risk of preterm birth was higher in pregnancies after bariatric surgery than in the reference material, and this relates well with previous findings. In this cohort there is a low proportion of SGA infants and this may influence the analyses on the effect of RYGB on newborn fat distribution [33, 34].

In our further analyses, we have chosen to use both maternal pre-pregnancy BMI and pre-surgery BMI. By using pre-pregnancy BMI we believe to estimate the gross metabolic effect of bariatric surgery on newborn body composition. We also included an estimate of the effect of post-surgery weight loss in our analyses, by making an estimate of newborn infants body composition compartments based on maternal pre-surgery BMI. One should bear in mind that we used the same GWG in both pre-surgery and pre-pregnancy estimates of infants body composition compartments and we have not accounted for the fact that GWG is in numerous previous studies shown to be inversely related to pre-pregnancy BMI [12].

The reference material was partly recruited through an interventional program which aimed to lower GWG. The program had no effect on mean GWG, birthweight and newborn body composition in the subgroup included in our study. But the fact that the women were followed through pregnancy might have elevated their awareness regarding life-style issues, leading to a healthier life-style, which might lead to and underestimation of the effect of bariatric surgery on birthweight and body composition. The cohort as a whole was a rather homogenous group, which reduce the risk of bias, but lower the external validity of our findings.

Conclusion

Offspring of mothers with RYGB before pregnancy born at term have lower birthweight, fat-free mass and fat percentage when compared with reference material. There is nothing that indicates aberrant fat deposition in this group of term born, infants. It seems like RYGB itself and not the pre-pregnancy weight loss had the greatest impact on offspring body composition. The long-term consequences of these findings need to be further examined.

25 Nov 2019

PONE-D-19-29375

Newborn body composition is severely affected by maternal bariatric surgery

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Methods:

- Were there any exclusion criteria for the RYGB group regarding e.g. maximum time between RYGB and birth or intermittent pregnancies?

- Was it checked whether any of the reference mothers had had RYGB previously?

- How were SGA and LGA calculated?

- l.140-152: This approach is quite unusual. Why did the authors not simply calculate regression models on all mothers and report the regression coefficient for RYGB as a binary variable?

Results:

- Figure 1 should be described in more detail in the main text. Did the RYGB mothers who were excluded differ from those included in certain characteristics?

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- Table 1: Suggest to provide all p-values, also the NS ones. The authors should report n(%) for all categorical variables. The chi-square test is not appropriate for maternal smoking, SGA/AGA/LGA and birth weight due to too low expected cell numbers.

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- l.183/184: The preterm rate in the control children seemed unusually low. What could be a potential explanation for this low rate?

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Reviewer #1: ¬¬¬Review of manuscript - PONE-D-19-29375

Thank you for the opportunity to review this manuscript. This is a Danish study on offspring body composition after bariatric surgery compared with control births. In general, this is a very interesting and well conducted study of importance for the research field.

General comments

1. How was the model for expected values of birth weight developed? Does it use the Marsal curves as reference? Is it appropriate to use gestational weight gain in the model given that this could be a mediator between RYBG and the outcome under study? Could the model be overfitted?

2. The study included both obese and normal weight comparators. Is there a risk that the comparison would not be optimal? Would a matched analysis be superior where women of the same BMI would be compared?

3. How was the attribution of the average reduction in new-born free fat mass calculated?

4. Almost half of the study participants were excluded and did not perform the DXA analysis. Did they differ in maternal and neonatal characteristics including birth weight for gestational age? Could this have influenced the findings?

5. Were there any missing data for the variables included in the study? If yes, how was this handled in the analysis?

Specific comments

1. Please include proportions (%) in Table 1 for size for gestational age and birthweight.

2. In Table 1, was it weight loss from surgery to conception or birth? Please include this for the variable.

3. In Table 2, include as a footnote how the expected measures were obtained.

4. Please exclude paired t-test from the title of Table 2.

5. Could not early pregnancy weight be retrieved from the antenatal medical record instead of having it self-reported?

6. The discussion on the biological reason for the results could be further developed. What metabolic, nutritional and hormonal differences could induce such an effect?

Reviewer #2: In this manuscript, Malchau Carlsen et al. compared newborn's body composition measured by DXA within 48 h of birth in neonates born after maternal bariatric surgery (RYGB) (N=25) with measures obtained from a reference sample of newborns from obese and normal-weight mothers (similar pre-pregnancy BMI). They found that neonates born after maternal RYGB had a lower birthweight, a lower fat-free mass, lower fat mass, lower fat percentage, and a lower part of the fat distributed abdominally than neonates from the reference sample.

This study reports novel data, and answers a clinically relevant question, that has not been assessed previously in litterature. Whereas the sample size is rather small (N=25) for the post- bariatric surgery sample, the study uses standard measurements of newborn's body composition.

Major:

1. The selection of the "reference material" is not clearly explained. Some details are not described in the method and are important for comprehension and interpretation of the data: How the mothers in reference sample were recruited and how mothers included in this specific analysis were selected ? Were the procedures exactly the same in the two studies? Was the reference cohort matched for any characteristics of the RYGB cohort?

2. I suggest that the statistical analysis should be reviewed by a statistician, as I am not in measure to assess the quality of the analysis proposed. The interpretation of the results of the expected body fat mass based on previously described associations as a % contribution from each variable needs to be commented by a statistician as it may not be appropriate to interpret directly as the contribution of a variable to the outcome. As this is an important message from the manuscript, interpretation of these models should be done with caution.

3. The authors should explain the sample size determination for both groups.

Minor:

Line 153: RYGB instead of RYGBP

Line 199-200: these interpretation of data should be consider in the discussion rather than results section.

Line 220-223: a reference for glucose metabolism and hormonal alteration's impact on body fat mass would be appreciated

**********

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Reviewer #1: Yes: Olof Stephansson

Reviewer #2: No

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8 Feb 2020

Editors comments:

General:
- I understand that the data cannot be made publicly available. However, at least the analysis code could be put online, e.g. at www.osf.io, with the exact site being mentioned in the manuscript.

First of all: Thank you for a detailed review of our manuscript that certainly improved the quality of our work.

We are, unfortunately not, familiar with the website mentioned. The analysis will be available by contacting the corresponding author. Please, refer to comment under acknowledgement.

- Please provide a STROBE statement and adjust the manuscript accordingly where necessary.
Strobe statement is added, and the checklist is attached (Appendix 1).

Methods:
- Were there any exclusion criteria for the RYGB group regarding e.g. maximum time between RYGB and birth or intermittent pregnancies?

No exclusions, all pregnancies were included after informed oral and written consent was retrieved.

- Was it checked whether any of the reference mothers had had RYGB previously?

Yes, there was no woman in the reference material that had undergone bariatric surgery of any kind, it has been added to the methods section.

- How were SGA and LGA calculated?

Sorry for not making this clear. We used Marsal’s reference, which is the reference used by Danish neonatologists (and used in most Nordic countries). This is more clearly stated in the manuscript now, please see line 118-119.

- l.140-152: This approach is quite unusual. Why did the authors not simply calculate regression models on all mothers and report the regression coefficient for RYGB as a binary variable?
We chose this approach since it was the only way we could estimate the effect of weight loss induced by surgery and the effect of bariatric surgery on foetal growth during pregnancy.

A linear regression, with RYGB as a binary variable, has been calculated and added as Appendix 2 to this review and is of course also informative.

We, however, believe that the analyses we made are better to illustrate the enormous effect bariatric surgery has on fetal growth since it is this and not the weight loss that induce the primary changes. We find it interesting to see that the women that had undergone bariatric surgery has the same mean gestational weight gain and there is also no difference I placental weight. Despite this, their offspring is significantly smaller.

Results:
- Figure 1 should be described in more detail in the main text. Did the RYGB mothers who were excluded differ from those included in certain characteristics?

We did not collect data on the women who gave birth at another hospital (n=13). There was no difference in baseline maternal characteristic in the women that gave birth premature or the got admitted to NICU (n=7). This also added to the text.

- Figure legends should be presented below the figures, not in the main text.

It has been changed.

- Table 1: Suggest to provide all p-values, also the NS ones. The authors should report n(%) for all categorical variables. The chi-square test is not appropriate for maternal smoking, SGA/AGA/LGA and birth weight due to too low expected cell numbers.

All p-values are now added as well as n(%) for all categorical values. The Chi-square test for maternal smoking, SGA/AGA/LGA and birthweight has been removed.

- Table 2: Please provide 95% confidence intervals and state more clearly how the p-values were calculated.

The 95 CI% and SD are added. We have tried to clarify how the p-values were calculated.

Discussion:
- l.183/184: The preterm rate in the control children seemed unusually low. What could be a potential explanation for this low rate?

We cannot explain the low rate of prematurity in the control children and we were also surprised by this finding.

- l.265-268: Could the exclusion of the RYGB preterm children have biased the main findings? It may be worth to include them in a sensitivity analysis which could also be adjusted for preterm birth.

We did not dexa-scan the premature born children. The incidence of growth restriction is significantly higher in the premature born babies and our aim was to examine how term newborn body composition was affected by bariatric surgery. Therefore, women with pre-eclampsia were also excluded.

- Conclusion (and title): Wording implying causation should be avoided in cross-sectional studies.

Sorry, the title has been changed and the wording in the conclusion has been changed.

Reviewer #1: 
Thank you for the opportunity to review this manuscript. This is a Danish study on offspring body composition after bariatric surgery compared with control births. In general, this is a very interesting and well conducted study of importance for the research field.
General comments

Thank you for reviewing our article, which improved our work.
1. How was the model for expected values of birth weight developed? Does it use the Marsal curves as reference? Is it appropriate to use gestational weight gain in the model given that this could be a mediator between RYBG and the outcome under study? Could the model be overfitted?

We calculated expected values using our reference material. We used linear regression. For each woman, expected values for birth weight and body composition compartments were calculated based on the pre-surgery and pre-pregnancy BMI. The expected value was compared to the actual measured value. We used the actual measured gestational weight gain (GWG).

As an example, we show you the regression used to calculate birth weight (this is also presented in the statistics section):

Expected birthweight = (- 3086.1)+ (pre-preganancy BMI or pre-surgery BMI*13.8)+(GWG*22.9)+(days of gestation at birth*22.3)+(if primiparity -192)+(if female sex -63.8).

It is important to notice that there was no difference in mean GWG comparing the RYGB women with the reference material and we do not believe this mediate the change in birth weight and newborn body composition since the RYGB women delivered smaller babies despite an appropriate mean GWG. One should also notice that there is no difference in mean placental weight.

We have tried to clarify our method under the statistics section.

2. The study included both obese and normal weight comparators. Is there a risk that the comparison would not be optimal? Would a matched analysis be superior where women of the same BMI would be compared?

We considered a matched analysis when we started the work. We believe the method used is appropriate, using linear regression we calculate an expected outcome in the reference material based on BMI, GWG, gestational age at birth, parity and newborn sex. The problems with a matched analysis would be to obtain enough controls with high pre-pregnancy BMI, few women have pre-pregnancy BMI of 44, uncomplicated pregnancy and uneventful delivery. The mean pre-surgery BMI was 44.0.

3. How was the attribution of the average reduction in new-born free fat mass calculated?

We have tried to explain this in the statistics section and clarify it in the results section. Sorry for not doing it properly. The difference in the compartments between pre-surgery and pre-pregnacy BMI were compared to actual measured values. We concluded that the difference between the actual measured value and the estimated value based on the pre-pregnancy BMI was induced by the effect of RYGB during pregnancy. The babies are smaller and leaner than expected by the maternal pre-pregnancy BMI and GWG.

4. Almost half of the study participants were excluded and did not perform the DXA analysis. Did they differ in maternal and neonatal characteristics including birth weight for gestational age? Could this have influenced the findings?

Please see the answer to the editors question above. We did not obtain neonatal characteristics on excluded infants. We would guess that a proportion of babies delivered premature have lower birth weight SDS. Including them, might have done the differences more pronounced.

5. Were there any missing data for the variables included in the study? If yes, how was this handled in the analysis?

We only included women for whom the offspring had a successful dexa-scan. The data are therefore complete.

Specific comments
1. Please include proportions (%) in Table 1 for size for gestational age and birthweight.

Proportions is now included.

2. In Table 1, was it weight loss from surgery to conception or birth? Please include this for the variable.

It was until conception, and this is now included.

3. In Table 2, include as a footnote how the expected measures were obtained.

The footnote now include how expected values were obtained.

4. Please exclude paired t-test from the title of Table 2.

Paired t-test is removed from title and added to footnote.

5. Could not early pregnancy weight be retrieved from the antenatal medical record instead of having it self-reported?

In Denmark women do not visit their midwife/doctor until gestational week 8-10, therefore we have self-reported weight. This pre-pregnancy weight is also self-reported in the reference material.

6. The discussion on the biological reason for the results could be further developed. What metabolic, nutritional and hormonal differences could induce such an effect?
Based on current literature I am not sure we can further develop this, but if there are any reference we have not included that are relevant, we would like to do so.

Reviewer #2:

In this manuscript, Malchau Carlsen et al. compared newborn's body composition measured by DXA within 48 h of birth in neonates born after maternal bariatric surgery (RYGB) (N=25) with measures obtained from a reference sample of newborns from obese and normal-weight mothers (similar pre-pregnancy BMI). They found that neonates born after maternal RYGB had a lower birthweight, a lower fat-free mass, lower fat mass, lower fat percentage, and a lower part of the fat distributed abdominally than neonates from the reference sample.
This study reports novel data, and answers a clinically relevant question, that has not been assessed previously in litterature. Whereas the sample size is rather small (N=25) for the post- bariatric surgery sample, the study uses standard measurements of newborn's body composition.

Thank you for considerate input, which have improved our paper.

Major:
1. The selection of the "reference material" is not clearly explained. Some details are not described in the method and are important for comprehension and interpretation of the data: How the mothers in reference sample were recruited and how mothers included in this specific analysis were selected ? Were the procedures exactly the same in the two studies? Was the reference cohort matched for any characteristics of the RYGB cohort?
The reference material was recruited in the same manner as the RYGB women. The reference cohort was not matched in any way. The reference material was recruited before the RYGB cohort. For further detail please read reference 13 and 15.

2. I suggest that the statistical analysis should be reviewed by a statistician, as I am not in measure to assess the quality of the analysis proposed. The interpretation of the results of the expected body fat mass based on previously described associations as a % contribution from each variable needs to be commented by a statistician as it may not be appropriate to interpret directly as the contribution of a variable to the outcome. As this is an important message from the manuscript, interpretation of these models should be done with caution.
We have consulted the statistician at our hospital, but any external review would also be welcome. Please see answer to reviewer 1 first comment.

3. The authors should explain the sample size determination for both groups.

We wanted to describe differences in birth weight and body composition after bariatric surgery, and there is no previous data on this, therefore sample size calculation was difficult.

Minor:
Line 153: RYGB instead of RYGBP
Thank you, it has been changed.

Line 199-200: these interpretation of data should be consider in the discussion rather than results section.

It has been deleted.

Line 220-223: a reference for glucose metabolism and hormonal alteration's impact on body fat mass would be appreciated

We have added two references.

Submitted filename: PLOS_one answer to reviewers_final.docx

Click here for additional data file.

6 Mar 2020

PONE-D-19-29375R1

Newborn body composition after maternal bariatric surgery

PLOS ONE

Dear Dr Carlsen,

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Andreas Beyerlein

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The authors did well to revise their manuscript in response to most of my questions. However, a few issues remain:

- l.197-205: These results should be described in much more detail. In particular, the birthweight findings should be mentioned, and the calculations of how much impact the surgery vs. weight loss had should be explained more clearly. Besides, I do not understand the authors' conclusion that 2% of the reduction in fat free mass could be attributed to weight loss, because the estimated difference based on pre-surgery compared to pre-pregnancy BMI was 5 g lower and not higher.

- l.209: Also the third column shows 95% CIs.

- I am still in doubt the analyses presented in table 2 are sound. In particular, I am wondering whether the authors took the expected values as fixed, which would mean that the 95% CIs for the differences between expected and measured values did not take the uncertainty in the estimation of the expected values into account. The authors should clarify this and/or revise their analyses accordingly.

- The results from Appendix 2 should be presented in suitable fashion, i.e. as scientific tables with explanations, and the results should also be mentioned in the main text and set into context to the results from Table 2.

- The authors should provide a statement in the main text why the analysis code cannot be made publicly available.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: No additional comments, my comments have been addressed properly. The manuscript is in my opinion ready for publication.

Reviewer #2: (No Response)

**********

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Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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24 Mar 2020

The authors did well to revise their manuscript in response to most of my questions. However, a few issues remain:

- l.197-205: These results should be described in much more detail. In particular, the birthweight findings should be mentioned, and the calculations of how much impact the surgery vs. weight loss had should be explained more clearly. Besides, I do not understand the authors' conclusion that 2% of the reduction in fat free mass could be attributed to weight loss, because the estimated difference based on pre-surgery compared to pre-pregnancy BMI was 5 g lower and not higher.

Thank you for your very useful comments, we are sorry we haven’t made this clear during the first review. We have tried to make it clearer in the text and hope we now have improved the description.

We looked at the difference in our estimates. Pre-surgery BMI estimates for mean fat-free mass was 3345 g, pre-pregnancy BMI estimates for fat-free mass 3340 g, the difference is hence 5 g, the effect of weight loss before pregnancy. The difference between pre-surgery fat-free mass and actual measured fat-free mass (3345 g - 3062 g) is 283 g. Therefore, weight loss only accounts for a small reduction in fat-free 5 g/283 g = 2%. The effect of bariatric surgery during pregnancy (276g/283g) induced the greatest change.

- l.209: Also the third column shows 95% CIs.

That is correct.

- I am still in doubt the analyses presented in table 2 are sound. In particular, I am wondering whether the authors took the expected values as fixed, which would mean that the 95% CIs for the differences between expected and measured values did not take the uncertainty in the estimation of the expected values into account. The authors should clarify this and/or revise their analyses accordingly.

Good point. We did not use the values as fixed, so the uncertainty is included in the 95%CI, which also are rather wide.

- The results from Appendix 2 should be presented in suitable fashion, i.e. as scientific tables with explanations, and the results should also be mentioned in the main text and set into context to the results from Table 2.

The results from appendix 2 are incorporated in our text now, and presented in a new table 2. The old table 2 is renamed table 3. The findings in table 2 are in line with table 3 (difference between pre-pregnancy BMI values and actual measured values).

- The authors should provide a statement in the main text why the analysis code cannot be made publicly available.
A short statement has been added. Please see line 320.

Submitted filename: Answer to second reviewRYGB_final.docx

Click here for additional data file.

27 Mar 2020

Newborn body composition after maternal bariatric surgery

PONE-D-19-29375R2

Dear Dr. Carlsen,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Well done. Please take care that the minus signs in the 95% CIs in table 2 will not get lost in the during production.

Reviewers' comments:

1 Apr 2020

PONE-D-19-29375R2

Newborn body composition after maternal bariatric surgery

Dear Dr. Carlsen:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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