Comparison between participants versus non-participants. \* *<sup>p</sup>* < 0.05 between intervention and control. <sup>+</sup> Exclusive breastfeeding for three or more months. BMI = Body mass index, kg = kilogram, IUI = Intra-uterine insemination, IVF = In Vitro fertilization, ICSI = Intracytoplasmic sperm injection, CRYO = Cryotherapy.

As our primary analysis, we compared outcome measures (BMI Z score, BF%, BP Z score, PWV, serum lipids, glucose and insulin concentrations and HOMA-IR) of children from mothers in the intervention and control groups by means of Student's *t*-test or Fisher's exact test, as appropriate (Table 2). We adhered to the randomized trial design in our analyses. We tested in multivariable linear regression analyses whether an adjustment for the maternal- or pregnancy-related characteristics that were found to be different between group characteristics (i.e., birth weight), would alter the outcomes.

**Table 2.** Cardiometabolic outcome values of children of mothers from the intervention and control group.


BMI = Body mass index, SBP = Systolic blood pressure, DBP = Diastolic blood pressure, LDL = Low-density lipoprotein, HDL = High-density lipoprotein, HOMA-IR = Homeostatic model of insulin resistance.

As exploratory analyses for potential offspring sex differences in effects of maternal obesity, we compared outcome measures for boys and girls separately (Supplementary Table S1). Furthermore, we assessed whether children of women who successfully lost weight (i.e., 5–10% weight reduction or achieving a BMI < 29 kg/m2) differed in outcomes from children of mothers who were unsuccessful, independent of randomization (Supplementary Table S2).

Values are presented as means and standard deviations (±SD) for continuous data and as frequency distributions for categorical data. We considered *p*-values of less than 0.05 statistically significant.

#### **3. Results**

Figure 1 shows the flowchart of the included participants. Out of the 163 women who conceived within 24 months in the invention group, 15 children were from twin/triplet pregnancies and three children were deceased, leaving 145 eligible children. From the 178 conceiving women in the control group, 14 children were from twin pairs and four children were deceased, leaving 160 children eligible. Despite intensive efforts, many parents did not respond or declined participation. A total of 51 parents provided informed consent; however, not all were able/willing to undergo measurements. Thus, we report

on 17 children whose mother was randomized to the intervention group and 29 from the control group (15%).

**Figure 1.** Flowchart of included participants. Mo = Months.

As shown in Table 1, there were no differences between the maternal baseline characteristics and the pregnancy-related characteristics between the mothers of participating children and eligible children that did not participate. Additionally, the baseline and pregnancy-related characteristics of the mothers of participating children did not differ between the intervention and control groups. In the original trial, analyzing women who conceived and who did not conceive, the women in the intervention group had improved their lifestyle, lost weight and improved metabolic indices over 6 months [21]. Up to 6 years after the intervention, the differences abated between the intervention and control groups; however, beneficial effects were still present in the women who achieved the lifestyle intervention goals (5–10% weight loss or a BMI < 29 kg/m2) [22]. In our selected sample, all the women reduced their BMI slightly, but between the intervention and control groups there were no significant differences in the change in BMI between baseline and time of conception. Furthermore, maternal weight gain during pregnancy was equally high in both groups.

In the children, the mean birth weight of those who participated in the follow-up was lower in the intervention group compared to the control group (3234 g vs. 3652 g, *p* < 0.05; Table 1), while there was no statistically significant difference in the original trial (3312 g vs. 3341 g), and there were no differences in gestational age [19].

The mean (±SD) age of the participating children was 4.6 (±1.0) years (range 3.2–6.5 years). There were 22 boys (48%). Overall, the BMI Z score was 0.65 (±1.26), and the SBP and DBP Z scores were 0.51 (±0.59) and 0.94 (±0.60), respectively. Table 2 shows the cardiometabolic health indices of children in the intervention and control groups. We found no differences between the children of mothers from the intervention group compared to the children of mothers from the control group in childhood outcome measures (BMI Z score, BF%, BP Z score, PWV, serum lipids, glucose and insulin concentrations and

HOMA-IR). Adjusting for confounders such as the child's birth weight did not alter the effect estimates in multivariate analyses.

In exploratory analyses, cardiometabolic outcome values of boys and girls did not differ according to the maternal allocation to lifestyle intervention (Supplementary Table S1). Furthermore, there were no differences in childhood cardiometabolic outcomes between children of mothers who successfully lost weight (*n* = 8) compared to those whose mothers did not (Supplementary Table S2). The latter analysis was independent of the assigned groups by randomization. In both exploratory analyses, the groups had very small numbers prohibiting conclusions to be drawn, and too few lab values were available for the children of mothers who successfully lost weight.

#### **4. Discussion**

We could not detect differences in offspring cardiometabolic health at age 3–6 years in the follow-up of a preconception maternal lifestyle intervention trial. Despite numerous efforts to enhance participation (e.g., measurements near the participant's home, multiple phone calls, layman information leaflets), our study was hampered by high attrition rates, which reduced the statistical power substantially. In our sample, the maternal BMI at conception and gestational weight gain (GWG) throughout pregnancy were not different between the intervention and control groups. We were unable to examine maternal cardiovascular and metabolic factors at the time of conception in our selected sample. However, we do know from previous publications by our group, that in all the women in the original trial, regardless of their conception status, the intervention increased physical activity, reduced snacking and sugary drinks intake, led to weight loss (approximately 4 kg) and halved the odds of metabolic syndrome after 6 months [20,21]. Furthermore, up to six years later, the intervention led to decreased caloric intake, and those women that were deemed successful in the primary trial showed improved BMI and cardiometabolic indices [20,22].

A range of experimental animal studies and observational human studies have shown that during embryonic developmental, even small environmental changes will have lasting effects (Table 3) [13]. In animals, changing the maternal lifestyle before and during pregnancy, and, in turn, comparing between in utero exposure to maternal obesity or reduced weight, was associated with improved adiposity and lipid levels in offspring [14,15]. In humans, environmental factors and the lifestyle of mothers impacted the fetal and placental metabolism, oxidative stress and interactions of these, inflicting epigenetic changes that are suggested to have lasting effects [30,31]. Furthermore, in assisted reproduction variations in embryo culture conditions have led to altered metabolic and epigenetic regulation, resulting in altered growth and cardiometabolic profiles of offspring [32,33]. In a large cohort study, children born after assisted reproduction had different growth patterns in their first few years, but ended up at a grossly similar height and weight in adolescence compared to their naturally conceived peers [34]. Although these findings seem reassuring, such alterations in growth during early life are linked to a predisposition of poor cardiometabolic health later in life, suggested by early life echocardiograms alterations in cardiac shape and function in assisted reproduction offspring [35].

This follow-up was based on the first randomized controlled trial in obese women examining the effects of a preconception lifestyle intervention. Due to the randomized design, confounding factors related to maternal infertility and/or obesity were equally divided between groups. Hence, we consider this population of infertile women valid to explore the effects of maternal preconception lifestyle change and weight loss on offspring. Despite the fact that most maternal- and pregnancy-related factors were similar between the groups, there was selective participation in our follow-up sample. This was indicated by a lower birth weight in children in the intervention group, a difference that was not present in the original trial [19]. Since our study had a null result and adjusting our analyses for birth weight did not change our results, we consider it unlikely that this selection has led to bias.


**Table 3.** Summary of selected current (pre-)pregnancy lifestyle intervention studies in animal and human settings and effects on offspring's health.

More importantly, in contrast to the original trial, participating women from both groups had similar weight loss between randomization and periconception. Additionally, the GWG was above the recommended levels for obese women (5–9 kg) in both groups [46]. This resulted in a very limited contrast in maternal body weight at time of conception between the groups, which may have contributed to our null finding. While the intervention induced effects on maternal parameters other than body weight, i.e., glucose metabolism or nutritional quality [20,22], these changes may not have been able to mitigate the detrimental effects of maternal obesity and/or excessive GWG on the offspring's cardiometabolic health [47]. Since studies assessing children born after their mothers had bariatric surgery showed improved cardiometabolic health [48,49], more substantial maternal weight loss may be needed to elicit changes in childhood health outcomes. On the other hand, observational evidence suggests a graded 'dose response' association between maternal BMI and offspring's cardiometabolic health [47,50], indicating even modest weight changes could carry positive effects, but we were not able to detect any effect.

To provide more reliable conclusions about the potential effects of maternal lifestyle change before conception on children, future studies should aim to maximize follow-up rates and power calculations based on childhood outcomes, which should account for high attrition. Compared to our study, a higher participation rate of 52% was present 3 years after a different lifestyle intervention during pregnancy [51]. Still, attrition in follow-up studies is generally high [52], and reasons why often remain unknown. Although parents were not required to state their reasons for declining participation, those that did indicated that they refused due to time restraints, did not want to burden their child or that they mainly wanted to become pregnant and were not interested in a further follow-up. Some stated that they were aware of the negative association of maternal obesity with childhood health, while many others were not previously aware; many women indicated that they did not want to contribute to further evidence of that association. While we attempted to involve participants in the planning of the study, only a few provided limited information on topics of interest to them that corresponded to the outcomes we were considering. The future investigation of offspring health-related themes considered relevant by obese women and their partners may provide guidance into a strategy that achieves lower attrition rates in follow-up studies such as our own.

Individual interventions in obese adults have been marginally successful [52]; community-based interventions and policies could thus be better suited to optimize the health of women prior to pregnancy. While the WHO has made an important step in global obesity prevention by formulating nine voluntary targets to prevent non-communicable diseases [53], and policy change has shown some local improvements [54], these have not yet been able to counteract the overall worldwide burden of obesity. Since pregnant women with obesity indicated they were mostly unaware of the effects of obesity on their (future) child [55], there is an urgent need to improve awareness in the general public of the consequences of obesity before and during pregnancy. As health behaviors are not solely individually determined and depend on environmental factors [56], and knowledge of healthy habits do not directly translate to changes in behavior [57], policies focusing on improving nutrition, physical activity and sports involving the home, school/work environment and the community are needed to curb the intergenerational cycle of obesity.

#### **5. Conclusions**

We could not detect any effect of a preconception lifestyle intervention—that did improve lifestyle, induced weight loss and improved cardiometabolic health in women 6 months after randomization—on the offspring's cardiometabolic health at age 3–6 years. Our study was hampered by limited statistical power, perhaps due to the children's age or the subject matter, as well as the minimal difference in the maternal periconception weight between the groups of participants in contrast to the original trial. Future studies should account for these factors to maximize follow-up rates to be able to draw conclusions about the potential of preconception lifestyle interventions to affect offspring cardiometabolic health. Whether there is no effect of lifestyle interventions in women with obesity prior to conception on their offspring's cardiometabolic health needs to be confirmed in larger studies.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/cells11010041/s1, Supplementary Table S1: Cardiometabolic outcome values of boys and girls of mothers from the intervention and control group, Supplementary Table S2: Cardiometabolic outcome values of children of mothers who successfully\* lost weight compared to children of mothers who did not successfully lose weight, pooled data independent of the randomization group.

**Author Contributions:** A.H. is the principal investigator (PI) of the Lifestyle trial, and T.J.R. is the PI of the follow-up studies. S.M., M.N.M.v.P., H.G., A.H., B.W.M., R.C.P., R.J.B.J.G. and T.J.R. planned the study procedures. S.M. collected and analyzed the data. S.M., M.N.M.v.P., R.J.B.J.G. and T.J.R. interpreted the data. S.M. wrote the manuscript. M.N.M.v.P., H.G., A.H., B.W.M., R.C.P., R.J.B.J.G. and T.J.R. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by a grant of the Dutch Heart Foundation (2013T085) and by a DynaHealth H2020 grant (agreement No 633595). The funders had no role in the design, conduct and interpretation of the study.

**Institutional Review Board Statement:** The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guidelines on human experimentation (CCMO), and with the Helsinki declaration of 1975, as revised in 2008, and has been approved by the Medical Ethical Committee of University Medical Centre Groningen, the Netherlands (NL24478.042.08). Parental consent was attained for all participants.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. This manuscript has been presented as a preprint in the University of Amsterdam repository for Ph.D. thesis.

**Acknowledgments:** This manuscript has been presented as a preprint in the University of Amsterdam repository for Ph.D. thesis. We thank all the women and children who participated in this study. We are grateful to all members of the WOMB-project who contributed to the follow-up study, with special thanks to our colleague Ph.D. students, post-docs, research assistants and students. This follow-up study would not have been possible without the original LIFEstyle study; therefore, we thank all participating hospitals for their contribution to this study, and the lifestyle coaches, research nurses, research midwives and office members of the Dutch Consortium (www.studies-obsgyn.nl, accessed on 18 October 2021) for their hard work and dedication.

**Conflicts of Interest:** S.M., M.N.M.v.P., H.G., A.H., B.W.M., R.C.P., R.J.B.J.G. and T.J.R. declare no conflict of interest relevant to this study. Outside the work submitted, A.H. declares consultancy fees and grants by Ferring Pharmaceutical BV. B.W.M is supported by a NHMRC practitioner Fellowship and reports consultancy for ObsEva, Merck, Merck KGaA and Guerbet. R.P. reports board membership for the Dutch Gezondheidsraad NVVH guideline committee, and travel expenses reimbursement for ESHRE meeting Barcelona 2018 (June).

#### **References**


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