University of Bergen

Nov 20, 2023

Abstract This longitudinal study investigated the impact of actigraphy-measured maternal physical activity on yolk sac size during early development. The yolk sac, a transient extraembryonic organ, plays a crucial role in embryonic development and is involved in metabolism, nutrition, growth, and hematopoiesis. Prospectively collected data from 190 healthy women indicated that their total daily physical activity, including both light and moderate-vigorous activity, was associated with yolk sac growth dynamics depending on embryonic sex and gestational age. Higher preconception maternal physical activity was linked to a larger yolk sac at 7 weeks (95% CI [0.02–0.13 mm]) and a smaller yolk sac at 10 weeks’ gestation (95% CI [− 0.18 to − 0.00]) in male embryos; in female embryos, the yolk sac size was increased at 10 weeks’ gestation (95% CI [0.06–0.26]) and was, on average, 24% larger than that in male embryos (95% CI [0.12–0.38]). Considering the pattern of other maternal effects on yolk sac size—e.g., body composition and sleep duration—we suggest that physiological yolk sac adaptations occur in short, sex-specific time windows and can be influenced by various maternal factors. Introduction The ability of an embryo and fetus to adapt to the intrauterine environment, including maternal factors, is considered to diminish over time 1 , 2 , 3 . Even before conception, maternal health factors are relevant for optimal endometrial preparation 4 and implantation 5 . In addition, the response to environmental and maternal factors can vary between the sexes as early as the moment of conception based on their specific genetic and epigenetic potential 2 , 6 , 7 , 8 . The rapid stages during early gestational development might be accompanied by corresponding rapid shifts in sensitivity to maternal and environmental cues. More detailed insight into the effect of specific factors and the sequence of events may reveal mechanisms of interest beyond fundamental knowledge, such as public health measures and clinical management 1 . The secondary yolk sac is a prominent structure during early human embryonic development that is easily visualized using ultrasound imaging during the first trimester. It is located in the exocoelomic cavity and remains connected to the developing embryo by the vitelline duct and its vessels (Fig. 1 a,b). The yolk sac is involved in gastrointestinal tract formation, protein synthesis, stem cell production, and hematopoiesis 9 , 10 , 11 , 12 ; for example, it is the origin of macrophage subtypes with high plasticity for epigenetic programming 13 . Through surface diffusion and transport proteins, the yolk sac membrane also facilitates gas exchange and provides nutrients until the placenta is sufficiently developed 9 , 10 , 11 , 12 , 14 . Figure 1 (a) 3D ultrasound of a 10-weeks’ embryo with a secondary yolk sac (YS). (b) Graphical illustration of the embryo-yolk sac connection with the yolk sac localized outside the amniotic cavity in the extraembryonic coelom. * Uterine glands secrete amino acids, ions, carbohydrates (glucose), lipids, proteins (e.g., cytokines, enzymes, hormones, growth factors, proteases and their inhibitors, and transporters) 61 . ** Yolk sac membrane with a vascular plexus envelope is involved in transport or resynthesis and exocytosis of nutrients, either directly into the surrounding blood vessels or the yolk sac cavity 11 . (c) Yolk sac size was assessed by two perpendicular outer-to-outer diameters measured thrice, and the mean was entered into the statistics. Recently, our group found that in a healthy pregnancy, the yolk sac at approximately 8 weeks’ gestation is larger when the maternal height and weight are low, suggesting a compensatory adaptation to maintain embryonic growth within an optimal trajectory, and the effect was essentially observed in female embryos 15 . In a second study, a shorter maternal sleep duration was linked to a larger yolk sac at 7 weeks of gestation, but this was essentially limited to male embryos 16 . This brings attention to another maternal factor, physical activity, which is related to healthy weight gain, improved maternal glucose control 17 , 18 , and favorable obstetric outcomes 18 , 19 , 20 , 21 , 22 . More specifically, maternal physical activity is associated with a larger placental volume, villous surface area and vascular volume 23 and modulates factors related to placental angiogenesis 24 , 25 . Physical activity also downregulates genes involved in placental fatty acid and insulin transport, upregulates genes involved in amino acid transport across the placenta, and reduces oxidative stress 18 , 26 . Based on this background, we speculate that physical activity in healthy women before and during early pregnancy affects the intrauterine environment. Thus, we hypothesize that these effects are reflected in the size of the yolk sac, which is involved in embryonic growth regulation, and that the effects are sex specific. Results The cohort consisted of 436 eligible participants (Fig. 3 and Table 1 ), of whom 190 (43.6%) became pregnant and provided sufficient data for inclusion in the present study (all study data with keys are supplied in Supplementary Tables S1 and S2 ). These 190 women had regular menstrual cycles with a median of 28 days (range 24–35 days, interquartile range (IQR) 1 day) and successful pregnancies resulting in live-born neonates with a median pregnancy length of 281 days (IQR 12 days) according to the date of the last menstrual period (LMP) or 278.5 days (IQR 11.8 days) based on the embryonic crown-rump length (CRL) in the first trimester 27 . Generally, the rate of pregnancy complications was low, e.g., gestational hypertension (3.2%), gestational diabetes (3.7%), preterm birth (3.2%), and a 5-min Apgar score less than seven (1.1%). The demographic characteristics of the study cohort are provided in Table  1 . Table 1 Descriptive statistics of the participants 16 are presented as the mean, standard deviation (SD), range (Min, Max), and interquartile range (IQR). Discussion This study of low-risk human pregnancies demonstrated that maternal physical activity before and during early pregnancy affects embryonic development, in this case, yolk sac size. A graded yolk sac response based on maternal physical activity duration was observed across all activity levels, encompassing both light and moderate-vigorous activities. Notably, we found that the effect was sex dependent, with different time windows and directions of impact (Fig. 3 b, 4 ). Additionally, the sex-specific effect on yolk sac growth rate between gestational weeks 7 and 10 revealed the dimension of an inverse effect on yolk sac growth dynamics for male and female embryos. Therefore, we hypothesize that high physical activity levels may strain the intrauterine environment and cause compensatory enlargement of the yolk sac surface at different GAs to ensure adequate nutritional support for embryonic growth, determined by embryonic sex. Based on the two previous studies 15 , 16 and the present study, a distinct pattern emerges: sensitive windows in embryonic development seem short, and the timing and effects are sex-specific (confer overview in Fig. 6 ). For example, at 8 weeks of gestation, a larger yolk sac size in female embryos develops when the maternal height and weight are low 15 . On the other hand, at 7 weeks’ gestation, a larger yolk sac is seen in male embryos when the maternal sleep duration is short 16 . The present study showed that an extended maternal physical activity leads to a larger yolk sac in male embryos at 7 weeks’ gestation, while in female embryos, an extended physical activity is associated with a smaller yolk sac at 10 weeks’ gestation. This figure illustrates not only the sex-specific modifications of the observed effects in terms of timing and direction but also underscores the importance of precise and frequent observations—during a phase of rapid progression through consecutive developmental stages—to capture such effects. Animal studies have also provided some evidence supporting the effect of environmental factors, such as temperature, nutrition, and noise, on the yolk sac 28 , 29 , 30 , 31 , but yolk sac development and implantation mechanisms vary among species 5 , 10 , 11 . Figure 6 Effect of preconception maternal factors on yolk sac size: i.e., the effect of maternal sleep duration 16 , total maternal physical activity duration, and maternal body size (weight and height) 15 . The figure illustrates the sex and time-dependent effects during the 1st trimester. The time windows, where these effects can be observed, are short. Nevertheless, the findings of the present study support the concept of the compensatory enlargement of the yolk sac surface to ensure adequate nutritional support for embryonic growth 15 . In nonpregnant individuals, exercise is known to reduce visceral blood flow to meet the metabolic demand of the working muscles 32 . This reduction in visceral blood flow is accompanied by increased vasodilatation through the release of nitric oxide (NO) 33 and endothelium-dependent hyperpolarization 34 . It has also been suggested that physical activity induces shear stress and intermittent fluctuations in substrate and oxygen delivery, resulting in hypoxic strain, which generates a repetitive stimulus triggering a feto-maternal response with increased placental vascularization 26 , 35 . However, compared with the increase in yolk sac size, the association of physical activity and fetal body composition 36 , 37 , 38 , 39 , fetal growth, placental size 36 , 40 , 41 , 42 , 43 , and placental circulation 23 are relatively late pregnancy responses to multiple factors and events. A significant feto-maternal connection via placental circulation is not established before twelve weeks of gestation 44 and therefore is unlikely to explain variations in yolk sac size. Nevertheless, the underlying mechanisms may be similar because they both occur within the same organ, the uterus, with the same supplying vasculature, myometrium, and endometrium that includes glands surrounded by vessels. Therefore, it is plausible that fluctuations capable of influencing placental development may also influence histotrophic nutrition at earlier stages of pregnancy via the uterine glands and vasculature 10 , 11 . Furthermore, sex steroid levels, which are associated with physical activity in women 45 , are widely recognized to influence both the menstrual cycle and the timing of ovulation, as well as the composition of the endometrium and its glands. The sex-specific response in size and growth dynamics shown in the present study is in line with other reports on sexual dimorphism in response to environmental factors during pregnancy in animal and in vitro studies 2 . We envisage that physical activity might act as a natural stress factor leading to the physiological adaptive response of the yolk sac (i.e., increasing size and thereby surface area that facilitates gas exchange and nutrient uptake during the period before the placenta is sufficiently developed). Study strengths The strengths of this study lie in its prospective longitudinal design, which includes maternal data from the preconception period, and the inclusion of many healthy women who conceived naturally, without the confounding influence of hormonal treatments commonly used in assisted reproduction. Furthermore, the observed opposite effects on male and female embryos at 10 gestational weeks provide robust evidence for a sex-dependent effect of maternal physical activity on yolk sac size. Additionally, the effect on yolk sac size at gestational weeks 7 and 10 is corroborated by the sex-specific effect on the yolk sac growth rate and strengthens the internal validity of the study. Another strength is the utilization of alternative statistical models and quantile regression models, which consistently yielded the same results. This demonstrates that the results were not dependent on skewed data, systematic distribution differences, or extreme values. To ensure the accuracy of yolk sac measurements, intra- and interobserver variability were calculated, confirming sufficient measurement precision, which was unbiased by any observer. Confounding observer effects are unlikely since maternal physical activity is not inherently related to the ultrasound procedure itself. The ultrasound operators were blinded to embryonic sex, as embryonic sex was unknown at the time of observation. Multiple regression analyses examining the association between maternal physical activity and yolk sac size did not reveal any significant observer effects (ultrasound operator) or effects of maternal age, parity, weight, height, BMI, lean body mass, body fat percentage, GA, time of inclusion, or the inclusion of the few participants with pregnancy complications. Study limitations Due to the study design, we cannot be certain that the activity patterns recorded before pregnancy, although close to conception (Table  2 ), continued into early pregnancy. In addition, the study included only two yolk sac measurements (gestational weeks 7 and 10). Both factors imply that we cannot infer whether the variation in yolk sac observations stems from pre- or periconception variations in the intrauterine environment (i.e., the vasculature, myometrium, and endometrium with endometrial glands) or whether we observed an ongoing sex-specific impact of maternal physical activity during gestation within the specific time windows of gestational weeks 7 and 10. An additional use of activity diaries or continuous measurement as well as more frequent yolk sac measurements would have strengthened the conclusions and provided deeper insight. However, the effect of different intensities of physical activity on the yolk sac could also be traced later in pregnancy (at 13 weeks’ gestation), suggesting that the physical activity pattern was similarly distributed in the population throughout the entire period. Other challenges to control for are sex steroid levels, mental stress, and maternal nutrition. Stress causes a hormonal response similar to that of exercise, and nutrition is closely related to energy metabolism; thus, both might be confounders. The study population, however, consisted of healthy women with no history of chronic diseases or risk factors, and the chance of chronic psychological stress in this population should therefore be low. Confounding by differences in maternal nutrition also seems unlikely, as maternal body composition, which is closely related to energy metabolism and nutrition, did not significantly affect our results (Supplementary Tables S4 – S7 ). Conclusion Normal human embryonic development is sensitive to maternal cues. Here, we showed that maternal physical activity influences human yolk sac development in a graded fashion. Second, embryonic sex determines the timing, degree, growth dynamics, and direction of the effect. Third, the time frames for these effects seem to be rapidly changing, short phases at this stage of pregnancy. Methods We studied the effects of maternal physical activity on the yolk sac in a prospective, longitudinal study of healthy nonsmoking women who planned to conceive naturally. The study is embedded in the ongoing CONIMPREG research program 16 , 46 . Data collection During the period 2014–2020, women aged 20–35 years with a BMI of 18–30 kg/m2 were recruited through social media (targeted Facebook® advertisements) and posters, provided that they had an uncomplicated obstetric history, a regular menstrual cycle, did not use contraceptives during the month before study entry and had no chronic diseases or fertility problems. If the women did not conceive within six sampling cycles, they were excluded from the study. The participants were assessed at four consecutive study visits (Fig. 3 a). At the first visit—before conception—maternal height and body composition were measured, immediately followed by the first actigraphy recording. The second visit was scheduled—based on the first day of the LMP—at 7 ± 1 weeks’ gestation. At this time, we confirmed the viability of the embryo and the length of gestation 47 and assessed the yolk sac. At the third visit (10 ± 1 weeks’ gestation), the yolk sac measurements were repeated, and finally, at the fourth visit (13 ± 1 weeks’ gestation), maternal body composition and activity duration were reassessed. Height, weight, and maternal body composition Before conception, height was measured with a wall-mounted stadiometer 48 , and weight was measured digitally using bioelectrical impedance analysis (model BC-418, Tanita, Tokyo, Japan). The percentage of body fat was estimated using the instrument’s computer software, and lean body mass was calculated by subtracting body fat mass from total body weight. Measurements were carried out as recommended by the manufacturer 49 . Physical activity Maternal physical activity was recorded before conception and at gestational week 13 using the SenseWear Mini Armband Actigraph (model MF-SW, BodyMedia, Pittsburgh, PA, USA). This wireless, noninvasive activity monitor incorporates triaxial accelerometry, heat flux, galvanic skin response, skin temperature, and near-body temperature measurements with a sampling frequency of 32 Hz. All information, plus information on sex, age, height, and weight, is considered in proprietary algorithms to predict physical activity at the level of 1.4 metabolic equivalents (METs) 50 . In accordance with the Sedentary Behavior Research Network (SBRN) consensus and American College of Sports Medicine (ASM) guidelines, the recordings were classified as light activity at ≥ 1.5 METs < 3.0, moderate at ≥ 3.0 METs < 6.0, and vigorous at ≥ 6.0 METs 51 , 52 . The monitor was worn on the upper posterior part of the nondominant arm for 4 days 53 , and the recording started at midnight. Raw data were processed and summarized using SenseWear Pro analysis software (SenseWear Professional, version 8.0.0.2903, Body Media) and exported into Excel workbooks (Microsoft Office, Excel version 2016, Redmond, WA, USA). Sampling days were excluded from the statistical analyses when data loss in a single day exceeded 6%. This or earlier versions of this actigraph have been validated for physical activity measurements 50 , 54 , 55 , including measurements during pregnancy 56 , 57 . The results of the included pregnant women were in good agreement with results from earlier versions of this monitor and other actigraphs or methods 46 . Embryonic measurements At gestational weeks 7 and 10, ultrasound measurements were carried out by a group that consisted of seven obstetricians using a 6–12 MHz transvaginal transducer (Voluson Expert E8; GE Medical Systems, Kretz Ultrasound, Zipf, Austria). The transducer output power was set to be low, with a thermal index (TI) always below 1.0 58 . Viability of the embryo was ensured by employing clinical guideline safety criteria 59 , and the length of gestation was confirmed by the CRL 47 determined as the mean of three measurements. The yolk sac size was determined as the average of two perpendicular outer diameters measured thrice 15 (Fig. 1 c). Inter- and intraobserver variability of the yolk sac measurements To calculate the inter- and intraobserver variability of yolk sac size measurements, we expanded our study in 2023 by utilizing prospectively collected data (Supplementary Tables with keys in S10 and S11) from the same study cohort (CONIMPREG). Embryonic yolk sacs (n = 19) were assessed either at gestational week 7 or week 10, and video sequences (ultrasound loops) were generated and stored in the machine’s local archive. All seven ultrasound operators were instructed to select the best yolk sac image from the sequence and measure the yolk sac using the previously described method. This involved measuring the perpendicular diameters three times and calculating the mean size. After a minimum of one day, the procedure was repeated to assess intraobserver variability (repeatability). Statistics Statistical analysis was performed using R (Foundation for Statistical Computing, version 4.1, Vienna, Austria) and R-studio (Integrated development for R, Boston, MA, USA) software. The mean and standard deviation (SD) with minimum and maximum values were calculated for each continuous variable, and frequencies and proportions were calculated for categorical variables. When the distribution was asymmetric, the median and IQR are reported. In addition, the 95% CIs of the mean were calculated for the recorded physical activity intensities, the number of days with recorded data, the frequency of physical activity on weekend days, and the CRL and yolk sac size with GA at the time of the measurements. Ordinary least square linear (OLS) regression models were used to analyze the association of yolk sac size with maternal physical activity duration before pregnancy and at the end of the first trimester (week 13). Linearity assumptions and normal distribution of the residuals were ascertained. The regression models were fitted with and without embryonic sex stratification. In addition, we tested the effect of embryonic sex on the maternal physical activity-yolk sac relation by adding the interaction term (embryonic sex*maternal physical activity) to the OLS model. OLS regression results were compared with results from quantile regression, including iterated reweighted least squares regressions (Huber weights and bisquare weighting), and heteroskedastic methods (sandwich variance estimators). In the subanalysis of our main findings, we replaced the yolk sac diameter with the yolk sac Z score that was calculated employing multilevel growth models, accounting for repeated measurements and GA (Supplementary Equation EQ1 and code C1). In addition, we controlled for physical activity effects in the original OLS model for GA. Likewise, maternal age, parity, and body composition parameters (i.e., height, weight, body mass index, lean body mass, and body fat percent) were added one by one to the primary model and were included if they notably altered the effect size of the association. We also stratified by time of inclusion and assessed the effect of three equally sized time categories between 2014 and 2020 by adjusting the regression model for these strata. Finally, we performed regression analyses with and without participants who experienced complications or unfavorable obstetric outcomes (i.e., hypertensive complications, gestational diabetes, preterm birth, and a 5-min Apgar score less than seven). As measures of fit, the adjusted R-squared and Akaike information criterion were calculated. Differences between the regression models were tested using analysis of variance (ANOVA) methods. Differences in variables from the summary statistics were tested with unpaired and paired parametric or nonparametric tests. The assessment of intra- and interobserver variability, along with the associated SEM (SEM-intraobserver and SEM-interobserver), as well as the minimum detectable difference, was conducted using a two-way ANOVA method, as outlined by Popović and Thomas 60 . The necessary variances were derived either directly or indirectly by utilizing the variances expressed as multiple squares for the various factors of the model (i.e., observer, subject, the interaction between the observer and subject) and the residual variation (Code is provided in Supplementary Code C2). Ethics declaration and consent The study was approved by the Regional Committee for Medical Research Ethics Southeast Norway (REK Southeast, ref. 2013/856a). E-mail: rek-sorost@medisin.uio.no. Written informed consent was obtained from all participants, and all research was performed in accordance with relevant guidelines and regulations. Data availability All data generated or analyzed during this study are included in this published article (and its supplementary information files). References Hanson, M. A. & Gluckman, P. D. Early developmental conditioning of later health and disease: physiology or pathophysiology. Physiol. Rev. 94, 1027–1076 (2014). Fiksen, Ø. & Folkvord, A. Maternal effects and the benefit of yolk supply in cod larvae in different environments—A simulation model. ICES Council Meeting, 1–6 (1999). Lara, R. A. & Vasconcelos, R. O. Impact of noise on development, physiological stress and behavioural patterns in larval zebrafish. Sci. Rep. 11, 6615 (2021). Acknowledgements The authors express their gratitude to the women who participated in this study and their families. We are thankful for valuable advice from Professor Anders Goksøyr, Department of Biological Sciences, University of Bergen. We acknowledge the contributions in managing included participants, logistics, measurements, and data collection provided by Rita Sollien and Norunn Solvang (Reg. midwives), Carol Cook (MBE), Henriette Odland-Karlsen (MD PhD), and Synnøve Lian Johnsen (MD PhD). Funding Open access funding provided by University of Bergen. The study received financial support from the Western Norway Health Trust, Norway; University of Bergen, Norway; Wayne University, Detroit, Michigan, USA; and the National Institutes of Health, USA. This work was also supported, in part, by the Pregnancy Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States Department of Health and Human Services (NICHD/NIH/DHHS); and, in part, by federal funds from NICHD/NIH/DHHS (Contract No. HHSN275201300006C). Dr. Roberto Romero contributed to this work as part of his official duties as an employee of the United States Federal Government. Author information Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .