Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=iaan20 Systems Biology in Reproductive Medicine ISSN: 1939-6368 (Print) 1939-6376 (Online) Journal homepage: https://www.tandfonline.com/loi/iaan20 Effect of the induction of transgenerational obesity on maternal-fetal parameters Thaigra Sousa Soares, Ana Paula Andreolla, Carolina Abreu Miranda, Eduardo Klöppel, Luhara Silva Rodrigues, Rafaianne Queiroz Moraes-Souza, Débora Cristina Damasceno, Gustavo Tadeu Volpato & Kleber Eduardo Campos To cite this article: Thaigra Sousa Soares, Ana Paula Andreolla, Carolina Abreu Miranda, Eduardo Klöppel, Luhara Silva Rodrigues, Rafaianne Queiroz Moraes-Souza, Débora Cristina Damasceno, Gustavo Tadeu Volpato & Kleber Eduardo Campos (2018) Effect of the induction of transgenerational obesity on maternal-fetal parameters, Systems Biology in Reproductive Medicine, 64:1, 51-59, DOI: 10.1080/19396368.2017.1410866 To link to this article: https://doi.org/10.1080/19396368.2017.1410866 Published online: 11 Dec 2017. Submit your article to this journal Article views: 302 View Crossmark data https://www.tandfonline.com/action/journalInformation?journalCode=iaan20 https://www.tandfonline.com/loi/iaan20 https://www.tandfonline.com/action/showCitFormats?doi=10.1080/19396368.2017.1410866 https://doi.org/10.1080/19396368.2017.1410866 https://www.tandfonline.com/action/authorSubmission?journalCode=iaan20&show=instructions https://www.tandfonline.com/action/authorSubmission?journalCode=iaan20&show=instructions http://crossmark.crossref.org/dialog/?doi=10.1080/19396368.2017.1410866&domain=pdf&date_stamp=2017-12-11 http://crossmark.crossref.org/dialog/?doi=10.1080/19396368.2017.1410866&domain=pdf&date_stamp=2017-12-11 RESEARCH ARTICLE Effect of the induction of transgenerational obesity on maternal-fetal parameters Thaigra Sousa Soares a,b, Ana Paula Andreollaa, Carolina Abreu Miranda a,b, Eduardo Klöppela,b, Luhara Silva Rodriguesa, Rafaianne Queiroz Moraes-Souzaa,b, Débora Cristina Damasceno b, Gustavo Tadeu Volpato a,b, and Kleber Eduardo Campos a,b aLaboratory of System Physiology and Reproductive Toxicology, Institute of Biological and Health Sciences, Federal University of Mato Grosso (UFMT), Barra do Garças, Mato Grosso State, Brazil; bGynecology, Obstetrics and Mastology Graduate Course, Laboratory of Experimental Research on Gynecology and Obstetrics, Botucatu Medical School, Univ Estadual Paulista_Unesp, Botucatu, São Paulo State, Brazil ABSTRACT Maternal obesity can cause complications for both women and their offspring for generations. Therefore, we intended to verify the repercussions of induction of transgenerational obesity on biochemical parameters, reproductive performance, and congenital anomaly frequency in Wistar rats. Female rats were used from successive generations. The female rats of parental generation (F0, n=10) were mated to obtain their offspring (F1 generation). F1 female rats received a mono- sodium glutamate (MSG) solution to induce obesity (n=07) or vehicle (control, n=06) during the neonatal period. These adult female rats were classified as normal or obese using the Lee Index, mated, and delivered offspring (F2 generation), which were also evaluated for obesity using the Lee Index in adult life (F2MSG, n=13, born from obese dams) or non-obesity status (F2Control, n=12, born from control dams), and were mated in adulthood. During pregnancy, glycemia and an oral glucose tolerance test (OGTT) were analyzed. At term pregnancy, the females were sacrificed for serum biochemical profile, maternal reproductive outcomes, and fetal development. In F2MSG rats, body weight gain at early pregnancy, glycemia by OGTT, total cholesterol, high- density-lipoprotein, and alanine transaminase activity were higher compared with those of F2Control rats. F2MSG rats also presented a lower implantation number and gravid uterus weight, increased pre-implantation loss and anomaly frequency in their fetuses (F3 generation) compared with those of F2Control rats. Therefore, even without significant changes in body weight gain, obesity was established at the end of pregnancy of Wistar rats using other biomarkers. Additionally, these rats showed multiple adverse reproductive outcomes, confirming the deleter- ious effects that lead to obesity. ARTICLE HISTORY Received 4 July 2017 Revised 12 November 2017 Accepted 13 November 2017 KEYWORDS Fetus; obesity; rat; transgenerational Introduction Obesity and its related comorbidities have become one of the main conditions negatively impacting public health, representing an alarming global problem. The epidemic proportions of obesity are mainly resulting from lifestyle changes, including increased high calorie intake and decreased physical activities [Flegal et al. 2015]. Obesity and being overweight have also increased in women of reproductive age [McDonald et al. 2010], and have increased strongly in populations with low and average incomes, especially in urban areas of developed countries [Nelson et al. 2010]. During pregnancy, obesity can cause complications for both women and their offspring, which may result in still- birth and congenital anomalies [Begum et al. 2011]. The impaired maternal intrauterine environment may induce critical changes in fetal growth and develop- ment, contributing to the risk of developing disease in later life [Barker 2007; Gluckman et al. 2008]. Clinical and experimental studies show that maternal obesity induced intrauterine changes may influence the fetal organism leading to metabolic adaptations and/or complications [Gluckman et al. 2008], such as glucose intolerance, obesity, and meta- bolic syndrome in adult life [Barker 2007; Campos et al. 2007; Desai et al. 2013]. Obese women present an increased risk of birth defects in their offspring [Stothard et al. 2009]. In experimental models, obe- sity is related to an abnormal biochemical profile, with obese rats presenting with increased serum tri- glyceride levels and glucose intolerance in their off- spring [Chen et al. 2014]. CONTACT Prof. Dr. Gustavo Tadeu Volpato gtvolpato@gmail.com Laboratório FisioTox, Instituto de Ciências Biológicas e da Saúde - UFMT, Rodovia Valdon Varjão, 6390, Barra do Garças – MT. SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 2018, VOL. 64, NO. 1, 51–59 https://doi.org/10.1080/19396368.2017.1410866 © 2017 Taylor & Francis http://orcid.org/0000-0003-4402-0241 http://orcid.org/0000-0002-4402-1466 http://orcid.org/0000-0002-7003-9643 http://orcid.org/0000-0002-4753-3264 http://orcid.org/0000-0001-6847-4577 https://crossmark.crossref.org/dialog/?doi=10.1080/19396368.2017.1410866&domain=pdf&date_stamp=2018-01-03 Studies regarding the transgenerational effects of obesity are being conducted to evaluate the offspring outcomes after maternal and/or paternal exposures of their respective parents or ancestors [Vickers 2014; Hanafi et al. 2016]. However, no study of transgenera- tional obesity, pregnancy, and teratogenesis in rats has yet been conducted. Therefore, we began to test our hypothesis that obesity might negatively affect the maternal and fetal organisms in future generations. The objective of the present study was to verify the repercussions during the induction of transgenerational obesity on biochemical parameters, reproductive per- formance, and congenital anomaly frequency in Wistar rats. Results and discussion Four generations of rats were used to study the reper- cussions of inducing transgenerational obesity in female rats as shown in Figure 1: F0 (parental genera- tion); F1 (obesity induction); F2 (for maternal study); and F3 (for fetal study). The F1 rats showed 0% obesity compared to the control group and 100% obesity com- pared to the monosodium glutamate (MSG) group, and all F1 rats became pregnant. The F2 dams in Table 1 showed maternal obesity biomarkers, and the F2MSG group presented an increased body weight gain in the first week of pregnancy, total percentage of obesity by Lee index classification at days 0 and 21 of pregnancy, and increased periovarian adipose tissue weight at term pregnancy, but also showed decreased weight gain in the third week of pregnancy and gravid uterus weight compared to the F2Control group. Similarly, women that become pregnant with higher body weight can present a lowering of this parameter until the end of the gestational period. Moreover, pregnant women with Figure 1. Experimental design. F0: Healthy female rats mated to obtain F1; F1 (obese rats): Female newborn induced to obesity by monosodium glutamate (days 2, 4, 6, 8, and 10 of life) and mated (90 days old); F2: Female offspring from obese rats and mated (90 days old); F3: Fetuses of F2 generation. Table 1. Obesity biomarkers during pregnancy of adult off- spring from obese and non-obese dams. Groups F2Control (n=12) F2MSG (n=13) Weight gain in pregnancy (g) 1st week 16.9 ± 1.8 24.1 ± 6.5* 2nd week 19.8 ± 3.4 20.8 ± 4.4 3rd week 81.9 ± 4.9 55.9 ± 10.8* Total body weight gain 118.6 ± 13.2 95.9 ± 15.1 Gravid uterus weight (g) 76.2 ± 2.5 51.9 ± 10.2* Positive obesity (%)a Day 0 0.0 100.0* Day 21 8.3 100.0* Periovarian adipose tissue weight (g) 3.3 ± 0.6 5.2 ± 0.6* Data shown as mean± standard error of mean (SEM); *p<0.05 - compared to F2 Control group (Student´s t test; aFisher´s Exact test). 52 T. S. SOARES ET AL. morbid obesity may present lower or no body weight gain [Rasmussen et al. 2010; Thangaratinam et al. 2012]. The Lee index is an anthropological biomarker for obesity classification used in experimental studies [Bernardis and Patterson 1968; Fernandes et al. 2012]. The higher periovarian fat weight and 100% obesity of F2MSG rats are consistent with obesity in our study. Several different biological mechanisms for obesity development, such as epigenetic factors influencing phenotype parameters, even in the absence of genetic or environmental alterations [Desai et al. 2015], affect metabolic pathways and promote obesity throughout generations [Armitage et al. 2008]. The early pregnancy period in the F2MSG group showed greater body weight gain, and this was expected to be maintained throughout the pregnancy. However, even with obesity confirmation, a lesser body weight gain in the last week of pregnancy was verified, which is directly related to the embryo loss at the beginning of pregnancy. This finding is associated with the reduced gravid uterus weight shown in this study. Moreover, the anthropometric parameters are considered obesity bio- markers [Novelli et al. 2007]. The intrabdominal adi- pose tissue accumulation (periovarian and periepididymal tissue) is often referred to as visceral obesity in rats and is traditionally associated with this syndrome [Hishikawa et al. 2005; Tchernof and Després 2013]. The increased visceral fat supports stu- dies using MSG-induced obese rats [Von Diemen et al. 2006; Suárez-Román et al. 2016], as MSG neonatal treatment leads to a series of endocrine disturbances, such as an inhibitory regulatory effect of the adrenal glands, which increases glucocorticoid levels and fat mass [Perello et al. 2003; Moreno et al. 2006; Von Diemen et al. 2006; Zubiría et al. 2014]. Blood glucose was increased in early pregnancy in F2MSG rats compared with those of control dams. Moreover, blood glucose levels at fasting and 120 min- utes (min) timepoints in the oral glucose test tolerance (OGTT) were higher compared with those of F2Control rats. At 30 and 60 min of OGTT, glycemia was increased in both groups compared to fasting glycemia in the same group, but the glycemia was increased at 120 min compared to fasting glycemia only in the F2MSG group. The area under the curve (AUC) of OGTT was also increased in F2MSG rats in relation to that of F2Control rats (Table 2). Obesity is strongly associated with an imbalance in glucose homeostasis [Hotamisligil et al. 1993], which is related to abnormal insulin secretion and/or action that leads to glucose intolerance [Campos et al. 2007]. Although our study showed no differences in serum glucose on specific days during the gestational period, the transgenerational obesity model showed higher blood glucose levels in OGTT and AUC, suggesting a classical glucose intolerance [Foti et al. 2005; Campos et al. 2007]. The impairment of glucose tolerance is dynamic by a considerable number of inducing factors, and shown in other studies using transgerational obe- sity model in rats, by obesogenic diet in the parental generation [Hanafi et al. 2016], and in mice using a combination of high chaloric feeding and low dose streptozotocin treatment in the previous generation [Wei et al. 2014] or a low protein diet inducing altered glucose homeostasis from F1 to F3 mice [Frantz et al. 2011]. In all these studies, including this one, glucose tolerance alteration provoke changes in uterus home- ostasis in the parental generation. Pregravid obesity is associated with a systemic inflammatory state that con- tributes to worsening of pregnancy and imbalance of glucose homeostasis. Moreover, glucose is the main energy source for development and fetal growth in later pregnancy can be impaired responding to glucose uptake from the maternal organism [Herrera and Ortega-Senovilla 2010]. Glucose intolerance and insulin resistance are related to a decrease of insulin-stimulated IR, IRS1, and IRS2 tyrosine phosphorylation, and asso- ciated to the substrates PI-3K in liver and muscles. In adipose tissue, a decreased association of insulin-stimu- lated IRS1 tyrosine phosphorylation and IRS1/PI-3K is evident. Both effects impair GLUT-4 translocation in cell membranes [Foti et al. 2005; Prada et al. 2007]. Table 3 presents maternal biochemical parameters. F2MSG rats presented a decreased total cholesterol and high-density lipoprotein (HDL-c). The reduced total Table 2. Glycemic levels and oral glucose tolerance test (OGTT) during pregnancy of adult offspring from obese and non-obese dams. Groups F2Control (n=12) F2MSG (n=13) Glycemic level on pregnancy (mg/dL) Day 0 99.1 ± 7.1 119.2 ± 4.5* Day 7 107.2 ± 7.8 115.8 ± 3.9 Day 14 97.7 ± 10.7 98.0 ± 3.7 Day 21 94.6 ± 3.4 88.0 ± 3.4 Glycemic level on OGTT (mg/dL) Fasting timepoint 69.3 ± 2.6 82.0 ± 2.1* 30 min timepoint 113.4 ± 8.4# 119.6 ± 4.7# 60 min timepoint 97.0 ± 3.1# 100.3 ± 4.2# 120 min timepoint 65.3 ± 2.7 91.3 ± 5.1*# Area under the curve (mg/dL/120min) 10765.7 ± 349.7 12067.5 ± 368.8* OGTT: oral glucose test tolerance. Data shown as mean± standard error of mean (SEM); * p<0.05 - compared to F2 Control group (Student´s t test); # p<0.05 – compared to fasting timepoint (ANOVA followed by Dunnett’s test). SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 53 cholesterol and HDL-c levels are related to the fluc- tuating anabolism during the first two thirds of preg- nancy by fat deposit accumulation. During the last third, a catabolic status is observed when fat deposit breakdown is enhanced [Herrera and Ortega- Senovilla 2010]. Serum lipid changes during the pregnancy can affect the outcomes at the end of the gestational period [Scifres et al. 2014]. Although there is a classic increase of triglyceride levels in the obesity state [Knopp et al. 1970], our study showed pregnancy preferentially induced catabolism of car- bohydrates and lipids for fetal development, con- firmed by unchanged blood glucose levels and decreased serum triglycerides. The tendency for the hypoglycemic state found in late pregnancy is com- pensated by the uptake of circulating triglycerides, leading to an impaired lipoprotein lipase activity in maternal tissues [López-Luna et al. 1991; Desai et al. 2013]. Moreover, the increase of hepatic enzymes in blood are important indicators of liver injury, like alanine aminotransferase activity (ALT) [Milinković et al. 2005]. ALT was reduced in F2MSG rats, show- ing a value within a normal range [Messias et al. 2010], suggesting no hepatic change. The adult female offspring born to obese rats (F2MSG) presented reduced implantation and increased embryonic loss before implantation in rela- tion to those of F2Control rats (Table 4). Suárez- Román et al. [2016] also verified a lesser implanted embryo number and Campos et al. [2008] observed a reduced fertility rate in MSG-induced obese rats. Similarly, obese women are three times more probable to undergo low fertility compared with women pre- senting with an adequate body mass index [Rich- Edwards et al. 1994], which is also related to insulin resistance and hyperandrogenemia [Dağ and Dilbaz 2015]. Similar to humans, obesity also induces reduced implantation and pregnancy rates, impairs reproductive functions by affecting both ovaries and endometrium, mainly by changes in luteinizing hor- mone and androstenedione levels [Dağ and Dilbaz 2015]. Then, excess body fat impairs female reproduc- tive functions in humans and in laboratory models [Hall et al. 2005]. It is suggested that a mechanism is the transmission by altering the epigenome through somatic or germ cells [Bazzano et al. 2015]. An abnor- mal maternal intrauterine environment directly affects the fetus and their germ cells, which are the origin of the next generation [Aiken and Ozanne 2014]. However, the exact mechanisms by which excess body fat interferes with reproductive function are still not fully understood. In the F2MSG group, it was verified that nine rats finished at term pregnancy, nine of them finished at term pregnancy. No differences in fetal and placental weights, or placental efficiency were observed (Table 5). Moreover, the total number of fetuses with anomalies showed no difference between the experimental groups. The rate of fetuses with incomplete ossification of the sternebra, abnormally shaped sternebra, and a dis- tended trachea were increased in the F3MSG group compared with those of control rats (Table 6; Figure 2). In a study of pre-gestational obesity in rats, mater- nal obesity led to an embryotoxic effect and reduced somite numbers [Suárez-Román et al. 2016]. The changes found in F3MSG newborns, such as incom- plete ossification of sternebrea and abnormal ster- nebrea, are considered variations, which were Table 3. Maternal serum biochemical parameters, triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-c), very low-density lipoprotein (VLDL-c), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), at 21st day of pregnancy of adult offspring from obese and non-obese dams. Groups F2 Control (n=12) F2 MSG (n=13) TG (mg/dL) 120.6 ± 7.9 146.2 ± 13.3 TC (mg/dL) 133.5 ± 11.2 100.0 ± 4.7* HDL-c (mg/dL) 50.7 ± 4.7 35.7 ± 3.9* VLDL-c (mg/dL) 24.1 ± 1.6 27.9 ± 1.8 ALT (U/L) 62.5 ± 7.2 44.9 ± 3.4* AST (U/L) 106.0 ± 10.5 109.4 ± 11.1 Data shown as mean± standard error of mean (SEM). * p<0.05 - compared to F2 Control group (Student´s t test). Table 4. Maternal reproductive outcome of adult offspring from obese and non-obese dams. Groups F2 Control (n=12) F2 MSG (n=13) Pregnant rats 12 13 At term pregnancy 12 9 Corpora lutea Total (N) 152 197 Mean ± SD 12.67 ± 0.40 15.15 ± 1.01* Implantation Total (N) 145 106 Mean ± SD 12.08 ± 0.43 8.15 ± 1.69* Live fetuses Total (N) 134 101 Mean ± SD 11.17 ± 0.41 7.77 ± 1.65 Dead fetuses Total (N) 2 0 Mean ± SD 0.17 ± 0.17 0.00 ± 0.00 Resorptions Total (N) 9 5 Mean ± SD 0.45 ± 0.46 0.38 ± 0.18 Litter sizeb 11.33 11.22 Live birth index (%)c 98.53 100.00 Pre-implantation loss (%)a 4.61 46.19* Post-implantation loss (%)a 7.58 4.72 Data shown as mean±standard deviation (SD) and proportions (%)a. * p<0.05 - compared to F2 Control group (Student´s t test;aFisher´s Exact test); b Litter size: Total number of pups delivered (live and still- born)/Number of dams that delivered; c Live birth index: (Number of pups born alive/Total number of pups born) x 100. 54 T. S. SOARES ET AL. merely observed and represent no teratological effect. However, tracheal abnormality is classified as a mal- formation, leading to functional consequences [Solecki et al. 2003]. Human and animal studies show similar outcomes in an inadequate intrauterine environment, which leads to an increased suscept- ibility to obesity in later life [Vickers 2014]. The results of this study improve the understanding of the transgenerational biological mechanisms to the metabolic disorder transmission. As the main limiting factor of our study, we high- light the difficulty of mating in F1 obese dams. Due to the reduced number of at term pregnant obese dams, the F2 female pups collected to F1 pregnant obese dams were 1 to 3 even though the final data collected were satisfactory. The experimental rat systems constantly evolve and the appropriate methodologies are the best way to collect better data [Iannaccone and Jacob 2009]. The use of a transgenerational obesity model to study reproductive parameters can be a starting point to further research, using treatment to decrease the obe- sity index and improve the reproductive biomarkers, or to comprehend the action mechanism of pre-implanta- tion loss with evaluation of blastocyste or uterine morphometry. Obesity induces biological changes in the maternal pregnancy period as the fetus develops. It is important to evaluate health conditions before considering preg- nancy, aiming at reducing the abnormal maternal intrauterine environment-induced negative effect on their offspring, which may interfere with fetal program- ming. When heritable, obesity affects the embryo, its perinatal development, and the adult life of these off- spring. So, even without relevant changes in body weight gain at term pregnancy, the female rats born to obese dams presented with abnormal glucose toler- ance and triglyceride levels at the end of pregnancy, and their obesity was established by other biomarkers. Additionally, multiple adverse reproductive outcomes and impaired fetal development were demonstrated, showing the deleterious effects that may lead to trans- generational obesity. Materials and methods Parental generation rats (F0) Wistar rats weighing about 200 grams (g) (90 d of life) were adapted for seven days in the vivarum of the Laboratory of System Physiology and Reproductive Toxicology (FisioTox). The rats were kept in collective cages in controlled conditions of temperature (22 ± 2°C), light (12h light/dark cycle), and relative humidity (60 ± 5%). The animals were fed laboratory chow 3.0 Kcal/g (Presence Rat Chow®, Paulínia, Brazil) and tap water ad libitum. The procedures and animal handling were per- formed in accordance with the guidelines provided by the Brazilian College of Animal Experimentation in agreement with the International Guiding Principles for Biomedical Research Involving Animals promulgated by the Society for the Study of Reproduction. The Ethical Table 5. Fetal and placental weights, and placental efficiency of adult offspring from obese and non-obese dams. Groups F2 Control (n=12) F2 MSG (n=09) Fetal body weight (g) Mean ± SE 5.16 ± 0.04 5.08 ± 0.03 Placental weight (g) Mean ± SE 0.42 ± 0.01 0.41 ± 0.01 Placental efficiency Mean ± SE 12.65 ± 0.18 13.07 ± 0.22 Data shown as mean± standard error (SE). p > 0.05: non-significant statis- tical difference. Table 6. Fetal anomaly frequency of adult offspring from obese and non-obese dams. Groups F3 Control (n=134) F3 MSG (n=101) External anomalies Number fetuses examined (litter) 134 (12) 101 (9) Total number of fetuses (%) with alteration 1 (0.7%) 2 (2.0%) Mean % fetuses with alteration per litter (mean ± SE) 0.8 ± 0.8 2.0 ± 1.3 Hidropisia 0 (0.0 %) 1 (1.0 %) Gastrosquises 1 (0.7 %) 1 (1.0 %) Skeletal anomalies Number fetuses examined (litter) 65 (12) 53 (9) Total number of fetuses (%) with alteration 30 (46.1%) 28 (53.8%) Mean % fetuses with alteration per litter (mean ± SE) 45.6 ± 7.4 54.0 ± 5.5 Incomplete ossification of cranius 1 (1.5%) 1 (1.9%) Supranumerary rib 4 (6.1%) 5 (9.4%) Wavy rib 2 (3.1%) 0 (0.0 %) Costela reduzida 1 (1.5%) 0 (0.0 %) Sternebra agenesis 2 (3.1%) 0 (0.0 %) Unossified sternebrae 13 (20.0%) 8 (15.1%) Incomplete ossification of sternebrae 8 (12.3%) 18 (34.0%)* Abnormally shaped sternebrae 14 (21.5%) 25 (47.2%)* Visceral anomalies Number fetuses examined (litter) 62 (12) 48 (9) Total number of fetuses (%) with alteration 21 (33.9%) 23 (47.9%) Mean % fetuses with alteration per litter (mean ± SE) 32.4 ± 7.9 47.3 ± 8.7 Distended Trachea 0 (0.0%) 13 (27.1)* Extended artery Anonymous 2 (3.2%) 0 (0.0%) Kidney agenesis 1 (1.6%) 0 (0.0%) Calice extended 1 (1.6%) 0 (0.0%) Hydroureter 18 (29.0%) 16 (33.3%) Hydronephrosis 0 (0.0%) 2 (4.2%) Sinuosis Ureter 1 (1.6%) 0 (0.0%) Ectopic testicle 1 (1.6%) 0 (0.0%) Data shown as mean± standard error (SE) and proportions (%).* p<0.05: compared to F3 Control group (Fisher´s Exact Test). SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 55 Committee for Animal Research of the UFMT Brazil approved all protocols under process number 23108.045215/2014-39. For offspring obtained for obesity induction, females (F0 generation, n = 10) were caged overnight with males. Mating was confirmed by the presence of sperm in the vaginal smear the next morning. Pregnant rats were kept in individual cages during the pregnancy period (21 d), including vaginal delivery, and lactation periods (21 d). The experimental protocol followed four generations (F0 to F3) as shown in Figure 1. F1 generation rats - obese and non-obese rats From total number of offspring, only female newborns (NB) were kept until the number of eight with their own dams (one newborn/nipple) from d 0 to the end of the lactation period (21 d). When the number of females would not reach eight, male NB were placed in the litter. This procedure was used to obtain better and equal feeding for all NB. Female offspring were randomly distributed into two groups: rats that received saline solution (0.9% NaCl) subcutaneously (sc) at postnatal d 2, 4, 6, 8, and 10 of life (Control group, n=6) and rats given a solution of monosodium glutamate (MSG - C5H8NNaO4.xH2O G1626 Sigma- Aldrich®, St Louis, USA) with a dose of 4.0 mg/g body weight, sc. at the same days as those of the Control group (MSG group, n=7) [Campos et al. 2007, Suárez- Román et al. 2016]. After weaning, all female rats were transferred to collective cages (four animals per cage) and maintained until adult life (90 d of life) in con- trolled conditions. At 90 d of life, the parameter of obesity was obtained by the Lee Index, defined as the cube root of body weight (g) x 10/nasoanal length (cm), for which a value equal to or less than 0.300 was classified as normal. Rats presenting values higher than 0.300 were classified as obese [Bernardis and Patterson 1968; Fernandes et al. 2012] and included in the MSG group. The exclusion criteria were applied when the Lee Index values were higher than 0.300 for rats neonatally treated with saline; or values equal or lower than 0.300 for rats neonatally treated with MSG. After the Lee Index, all female rats were mated overnight with normal males and the sperm pre- sence in vaginal smears the next morning was defined as d zero (0) of pregnancy and included in their respective group. The pregnant rats were kept in individual cages during pregnancy (21 d) and weaning (21 d) periods. F2 generation rats – offspring from obese and control dams The offspring obtained from F1 rats is known as F2 genera- tion. These rats were kept in controlled conditions until adult age. At d 90 of life, all F2 female rats were mated with normal male rats, and after the confirmation of pregnancy (d 0), the rats were classified as normal or obese by the Lee Index. During pregnancy, the rats were maintained in individual cages and in two experimental groups, following their respective F1 generation: F2Control - offspring from non-obese dams (n=12), and F2MSG - offspring fromobese dams (n=13). Course of pregnancy of F2 rats At d 0 (early pregnancy), 7 (embryonic period), 14 (fetal period), and 20 of pregnancy (end of pregnancy – at term pregnancy), maternal body weight and glycaemia were evaluated. Blood samples were obtained by venous punc- ture of the tail for blood glucose concentrations by conven- tional glucometer (One TouchUltra - Johnson& Johnson®, São José dos Campos, Brazil) and the values were presented inmg/dL. Total bodyweight gainwas determined using the difference of the last and the first day of body weight measured in the pregnancy period. After 6 h of fasting, all the rats were submitted to OGTT at d 17 of pregnancy for evaluation of glucose intolerance status. Glucose responses during the OGTT were evaluated by the estimation of the total area under Figure 2. Main anomalies showed in F3 fetuses. (A) Normal sternebra of rat fetuses; (B) incomplete ossification of sternebra (arrow); (C) abnormally shaped sternebra (arrow); (D) section of normal rat fetus’s thorax with normal trachea (arrow); and (E) distended trachea (arrow) in fetus’s thorax section. 56 T. S. SOARES ET AL. the curve (AUC), using the trapezoidal method [Sinzato et al. 2012]. At d 21 of pregnancy, the Lee Index was performed one more time and the rats were lethally anesthetized by sodium thiopental (Thiopentax®). The periovarian adipose tissue was removed and weighted. The gravid uterus was dissected to count dead and live fetuses, resorption (embryonic death), implantation sites, and corpora lutea numbers. The rate of pre-and postim- plantation loss, the litter size, and live birth index were calculated [Volpato et al. 2015; Parker 2012]. Maternal whole blood samples obtained at d 21 of pregnancy were centrifuged at 1,200 x g to obtain serum. The serum samples were then stored at -20°C for measurement of triglyceride, total-cholesterol and high- density lipoprotein levels, and aspartate aminotransferase and alanine aminotransferase activities were determined using commercial kits (Wiener® Rosario, Argentina). The very low-density lipoprotein levels were determined by mathematical estimation [Knopfholz et al. 2014]. The weight of the F3 generation of fetuses, following the groups as their dams (F3Control rats from F2Control dams, F3MSG rats from F2MSG dams) and their placen- tas from these dams were weighed to calculate the pla- cental efficiency (fetal weight/placental weight). The fetuses were evaluated in a microscope with respect to incidence of external anomaly. After external analysis, half of the fetuses were fixed in Bodian´s solution and serial sections were prepared as described by Wilson [1965] for visceral examination. The remaining fetuses were prepared for examination of the bones by the stain- ing procedure of Staples and Schnell [1964]. Statistical analysis For comparison of the mean values between the experi- mental groups, Student’s t test was used. The propor- tions were calculated by Fisher’s exact test. ANOVA followed by Dunnett test was used to compare different timepoints during OGTT. For fetal data, litter was used as the statistical unit. Differences were considered sta- tistically significant when p < 0.05. Acknowledgments The authors wish to thank Iuri Júnor Francisco, Biomedical Sciences graduate student, for technical assistance. Declaration of interest The authors report no conflicts of interest with respect to the research, authorship, and/or publication of this article. Notes on contributors Conception and design of the study (Figure 1) were carried out by: GTV, KEC; Analysis and interpretation of reproduc- tive parameters (Tables 1, 4, 5, and 6; and Figure 2) were supported by: TSS, LSR, RQM-S, DCD, GTV; Analysis and interpretation of Metabolic parameters (Tables 2 and 3) by: APA, CAM, EK, DCD, KEC; The drafting of the article, critical revision, and final approval of the manuscript were supported by all authors. ORCID Thaigra Sousa Soares http://orcid.org/0000-0003-4402- 0241 Carolina Abreu Miranda http://orcid.org/0000-0002-4402- 1466 Débora Cristina Damasceno http://orcid.org/0000-0002- 7003-9643 Gustavo Tadeu Volpato http://orcid.org/0000-0002-4753- 3264 Kleber Eduardo Campos http://orcid.org/0000-0001-6847- 4577 References Aiken, C.E., Ozanne, S.E. (2014) Transgerational develop- mental programming. Hum Reprod Update 20(1):63–75. Armitage, J.A., Poston, L., Taylor, P.D. 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SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE 59 Abstract Introduction Results and discussion Materials and methods Parental generation rats (F0) F1 generation rats - obese and non-obese rats F2 generation rats – offspring from obese and control dams Course of pregnancy of F2 rats Statistical analysis Acknowledgments Declaration of interest Notes on contributor References