Intrauterine androgen exposure impairs gonadal adipose tissue functions of adult female rats.

Prenatal androgen exposure induces fetal programming leading to alterations in offspring health and phenotypes that resemble those seen in women with Polycystic Ovary Syndrome. It has been described that prenatal androgenization affects the reproductive axis and leads to metabolic and endocrine disorders. Adipose tissue plays a crucial role in all these functions and is susceptible to programming effects. Particularly, gonadal adipose tissue is involved in reproductive functions, so dysfunctions in this tissue could be related to fertility alterations. We aimed to investigate the extent to which prenatal hyperandrogenization is able to alter the functionality of gonadal adipose tissue in female adult rats, including lipid metabolism, adipokines expression, and de novo synthesis of steroids. Pregnant rats were treated with 1 mg of testosterone from day 16 to day 19 of pregnancy, and female offspring were followed until 90 days of age, when they were euthanized. The prenatally hyperandrogenized (PH) female offspring displayed two phenotypes: irregular ovulatory (PHiov) and anovulatory (PHanov). Regarding lipid metabolism, both PH groups displayed disruptions in the main lipid pathways with altered levels of triglyceride and increased lipid peroxidation levels. In addition, we found that Peroxisome Proliferator-Activated Receptors (PPARs) alpha protein expression was decreased in both PH phenotypes (p < 0.05), but no changes were found in PPARγ protein levels. Furthermore, regarding adipokines, no changes were found in Leptin and Adiponectin protein levels, but Chemerin protein levels were decreased in the PHiov group (p < 0.05). Regarding de novo synthesis of steroids, the PHanov group showed increased protein levels of Cyp17a1 and Cyp19, while the PHiov group only showed decreased protein levels of Cyp19 (p < 0.05). These results suggest that prenatal androgen exposure affects females' gonadal adipose tissue in adulthood, disturbing different lipid pathways, Chemerin expression, and de novo synthesis of steroids.

Parental ageing and Assisted Reproduction Technologies: analysis of risk of chronic diseases in the progeny / Pózne rodzicielstwo i rozród wspomagany: analiza ryzyka wystapienia chorób przewleklych u potomstwa.

Conception of a child at advanced parental age (> 35 years) has been steadily increasing in recent decades, especially in developed countries. Socio-economic factors, effective contraceptives, and the availability of Assisted Reproduction Technologies (ART) have a direct impact on postponing the decision to have a baby. ART enables reproductive success for people diagnosed as infertile or with reduced possibilities of becoming pregnant due to concomitant pathologies. Epidemiological studies indicate that both advanced parental age and ART are associated with pathologies of pregnancy, such as gestational diabetes, risk of pre-eclampsia, miscarriage, placental abruption, preterm labor, stillbirth, neurodevelopmental disorders and chronic disease of the offspring. In our work, we will focus on the available information on metabolic changes that increase the risk of developing cardiovascular diseases in the offspring of parents at an advanced age and conceived through ART. Finally, we will address the sources of the observed disturbances at the gamete and embryo level, related to oxygen stress, epigenetic modifications and DNA damage, considering possible rescue actions.

Fetal programming by androgen excess impairs liver lipid content and PPARg expression in adult rats.

It is known that prenatal hyperandrogenization induces alterations since early stages of life, contributing to the development of polycystic ovary syndrome affecting the reproductive axis and the metabolic status, thus promoting others associated disorders, such as dyslipidemia, insulin resistance, liver dysfunction, and even steatosis. In this study, we aimed to evaluate the effect of fetal programming by androgen excess on the hepatic lipid content and metabolic mediators at adult life. Pregnant rats were hyperandrogenized with daily subcutaneous injections of 1 mg of free testosterone from days 16 to 19 of pregnancy. The prenatally hyperandrogenized (PH) female offspring displayed two phenotypes: irregular ovulatory phenotype (PHiov) and anovulatory phenotype (PHanov), with different metabolic and endocrine features. We evaluated the liver lipid content and the main aspect of the balance between fatty acid (FA) synthesis and oxidation. We investigated the status of the peroxisomal proliferator-activated receptors (PPARs) alpha and gamma, which act as lipid mediators, and the adipokine chemerin, one marker of liver alterations. We found that prenatal hyperandrogenization altered the liver lipid profile with increased FAs levels in the PHanov phenotype and decreased cholesterol content in the PHiov phenotype. FA metabolism was also disturbed, including decreased mRNA and protein PPARgamma levels and impaired gene expression of the main enzymes involved in lipid metabolism. Moreover, we found low chemerin protein levels in both PH phenotypes. In conclusion, these data suggest that prenatal hyperandrogenization exerts a negative effect on the liver and alters lipid content and metabolic mediators' expression at adult age.

Perturbations of the hepatic proteome behind the onset of metabolic disorders in mouse offspring developed following embryo manipulation.

Assisted Reproductive Technologies (ART) allowed the births of >8 million babies worldwide. Even if ART children are healthy at birth, several studies reported that ART may cause changes in foetal programming, leading to an increased predisposition to metabolic disorders in adulthood. Previous studies on mouse model showed obesity, glucose intolerance, and hepatic lipid accumulation in ART offspring. A cumulative effect of the different components of ART protocol has been previously described, for example, in the occurrence of epigenetic defects. Here, we investigated whether there is a cumulative effect of embryo transfer (ET), in vitro culture (IVC) and blastomere biopsy (BB) in the onset of metabolic disorders in mouse offspring vs those naturally conceived (Control - CTR). To this aim, proteomic analysis was performed on the livers from adult mouse offspring developed following ET, IVC and BB vs CTR. We observed deregulated expression of proteins involved in lipid, carbohydrate, energy metabolisms and cellular processes in ART offspring. Moreover, we found increased body weight in all ART offspring while i) insulin resistance in BB male, ii) females glucose intolerance and high level of triglycerides and cholesterol in BB females and iii) low levels of interleukin-6 in BB, IVC and ET males. In conclusion, our study suggests that the use of various embryo manipulations influences the metabolic health of adult offspring, resulting in an increased predisposition to hepatic diseases and metabolic syndrome in a sex-specific manner.

The effects of assisted reproduction technologies on metabolic health and disease†.

The increasing prevalence of metabolic diseases places a substantial burden on human health throughout the world. It is believed that predisposition to metabolic disease starts early in life, a period of great susceptibility to epigenetic reprogramming due to environmental insults. Assisted reproductive technologies (ART), i.e., treatments for infertility, may affect embryo development, resulting in multiple adverse health outcomes in postnatal life. The most frequently observed alteration in ART pregnancies is impaired placental nutrient transfer. Moreover, consequent intrauterine growth restriction and low birth weight followed by catch-up growth can all predict future obesity, insulin resistance, and chronic metabolic diseases. In this review, we have focused on evidence of adverse metabolic alterations associated with ART, which can contribute to the development of chronic adult-onset diseases, such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Due to high phenotypic plasticity, ART pregnancies can produce both offspring with adverse health outcomes, as well as healthy individuals. We further discuss the sex-specific and age-dependent metabolic alterations reflected in ART offspring, and how the degree of interference of a given ART procedure (from mild to more severe manipulation of the egg) affects the occurrence and degree of offspring alterations. Over the last few years, studies have reported signs of cardiometabolic alterations in ART offspring that are detectable at a young age but that do not appear to constitute a high risk of disease and morbidity per se. These abnormal phenotypes could be early indicators of the development of chronic diseases, including metabolic syndrome, in adulthood. The early detection of metabolic alterations could contribute to preventing the onset of disease in adulthood. Such early interventions may counteract the risk factors and improve the long-term health of the individual.

Prenatal testosterone exposure induces insulin resistance, uterine oxidative stress and pro-inflammatory status in rats.

Prenatal androgen excess is considered one of the main causes of the development of polycystic ovary syndrome. In this study, we investigated the effect of prenatal hyperandrogenization (PH) on the physiology of the adult uterine tissue using a murine model of fetal programming caused by androgen excess in adult female rats. Pregnant rats were hyperandrogenized with testosterone and female offspring were studied when adult. Our results showed that PH leads to hyperglycemia and hyperinsulinemia. Consequently, PH developed insulin resistance and a systemic inflammatory state reflected by increased C-reactive protein. In the uterine tissue, levels of PPAR gamma-an important metabolic sensor in the endometrium-were found to be impaired. Moreover, PH induced a pro-inflammatory and an unbalanced oxidative state in the uterus reflected by increased COX-2, lipid peroxidation, and NF-κB. In summary, our results revealed that PH leads to a compromised metabolic state likely consequence of fetal reprogramming.

Prenatal androgen exposure affects ovarian lipid metabolism and steroid biosynthesis in rats.

Prenatal androgen exposure affects reproductive functions and has been proposed as an underlying cause of polycystic ovary syndrome (PCOS). In this study, we aimed to investigate the impact of prenatal androgen exposure on ovarian lipid metabolism and to deepen our understanding of steroidogenesis regulation during adulthood. Pregnant rats were hyperandrogenized with testosterone and female offspring were studied when adult. This treatment leads to two different phenotypes: irregular ovulatory and anovulatory animals. Our results showed that prenatally hyperandrogenized (PH) animals displayed altered lipid and hormonal profile together with alterations in steroidogenesis and ovarian lipid metabolism. Moreover, PH animals showed alterations in the PPARg system, impaired mRNA levels of cholesterol receptors (Ldlr and Srb1) and decreased expression of the rate-limiting enzyme of de novo cholesterol production (Hmgcr). Anovulatory PH animals presented an increase of ovarian cholesteryl esters levels and lipid peroxidation index. Together with alterations in cholesterol metabolism, we found an impairment of the steroidogenic pathway in PH animals in a phenotype-specific manner. Regarding fatty acid metabolism, our results showed, in PH animals, an altered expression of Srebp1 and Atgl, which are involved in fatty acid metabolism and triglycerides hydrolysis, respectively. In conclusion, fatty acid and cholesterol metabolism, which are key players in steroidogenesis acting as a source of energy and substrate for steroid production, were affected in animals exposed to androgens during gestation. These results suggest that prenatal androgen exposure leads to long-term effects that affect ovary lipid metabolism and ovarian steroid formation from the very first steps.

Prenatally androgenized female rats develop uterine hyperplasia when adult.

Prenatal hyperandrogenization (PH) is hypothesized as one of the main factors contributing to the development of polycystic ovary syndrome (PCOS). In this study, we aimed to investigate the impact of prenatal exposure to androgen excess on the uterus when animals reach their adulthood. We found that PH altered the morphology of the uteri that show a hyperplastic morphology with increased total uterine thickness as well as luminal epithelium thickness, with both enhanced and altered distribution of glands as compared with controls. Morphological alterations were associated with an unbalanced homeostasis as assessed by the expression of regulators of cell cycle progression and cell death dynamics. PH also causes disturbances in the cell cycle of the uterine tissue and dysregulates cell death and survival pathways leading to the development of uterine hyperplasia. These findings suggest that PH may have a deleterious effect on the uterus.

Fetal programming by androgen excess in rats affects ovarian fuel sensors and steroidogenesis.

Fetal programming by androgen excess is hypothesized as one of the main factors contributing to the development of polycystic ovary syndrome (PCOS). PCOS is more than a reproductive disorder, as women with PCOS also show metabolic and other endocrine alterations. Since both ovarian and reproductive functions depend on energy balance, the alterations in metabolism may be related to reproductive alterations. The present study aimed to evaluate the effect of androgen excess during prenatal life on ovarian fuel sensors and its consequences on steroidogenesis. To this end, pregnant rats were hyperandrogenized with testosterone and the following parameters were evaluated in their female offspring: follicular development, PPARG levels, adipokines (including leptin, adiponectin, and chemerin as ovarian fuel sensors), serum gonadotropins (LH and FSH), the mRNA of their ovarian receptors, and the expression of steroidogenic mediators. At 60 days of age, the prenatally hyperandrogenized (PH) female offspring displayed both an irregular ovulatory phenotype and an anovulatory phenotype with altered follicular development and the presence of cysts. Both PH groups showed altered levels of both proteins and mRNA of PPARG and a different expression pattern of the adipokines studied. Although serum gonadotropins were not impaired, there were alterations in the mRNA levels of their ovarian receptors. The steroidogenic mediators Star, Cyp11a1, Cyp17a1, and Cyp19a1 were altered differently in each of the PH groups. We concluded that androgen excess during prenatal life leads to developmental programming effects that affect ovarian fuel sensors and steroidogenesis in a phenotype-specific way.

Effects of in utero androgen excess and metformin treatment on hepatic functions.

This study aimed to evaluate the role of prenatal hyperandrogenization in liver functions and the extent of metformin as treatment. Pregnant rats were hyperandrogenized with subcutaneous testosterone (1mg/rat) between 16 and 19 of pregnancy. Prenatally hyperandrogenized (PH) female offspring displayed, at the adult life, two phenotypes; a PH irregular ovulatory phenotype (PHiov) and a PH anovulatory (PHanov) phenotype. From day 70 to the moment of sacrifice (90 days of age), 50% of the animals of each group received a daily oral dose of 50 mg/kg of metformin. We found that both PH phenotypes displayed a hepatic disruptions of insulin and glucose pathway and that metformin treatment reversed some of these alterations in a specific-phenotype manner. Our findings show, for the first time, that androgen excess in utero promotes hepatic dysfunctions and that metformin treatment is able to specifically reverse those hepatic alterations and sheds light on the possible mechanisms of metformin action.

Prenatal hyperandrogenism induces alterations that affect liver lipid metabolism.

Prenatal hyperandrogenism is hypothesized as one of the main factors contributing to the development of polycystic ovary syndrome (PCOS). PCOS patients have high risk of developing fatty liver and steatosis. This study aimed to evaluate the role of prenatal hyperandrogenism in liver lipid metabolism and fatty liver development. Pregnant rats were hyperandrogenized with testosterone. At pubertal age, the prenatally hyperandrogenized (PH) female offspring displayed both ovulatory (PHov) and anovulatory (PHanov) phenotypes that mimic human PCOS features. We evaluated hepatic transferases, liver lipid content, the balance between lipogenesis and fatty acid oxidation pathway, oxidant/antioxidant balance and proinflammatory status. We also evaluated the general metabolic status through growth rate curve, basal glucose and insulin levels, glucose tolerance test, HOMA-IR index and serum lipid profile. Although neither PH group showed signs of liver lipid content, the lipogenesis and fatty oxidation pathways were altered. The PH groups also showed impaired oxidant/antioxidant balance, a decrease in the proinflammatory pathway (measured by prostaglandin E2 and cyclooxygenase-2 levels), decreased glucose tolerance, imbalance of circulating lipids and increased risk of metabolic syndrome. We conclude that prenatal hyperandrogenism generates both PHov and PHanov phenotypes with signs of liver alterations, imbalance in lipid metabolism and increased risk of developing metabolic syndrome. The anovulatory phenotype showed more alterations in liver lipogenesis and a more impaired balance of insulin and glucose metabolism, being more susceptible to the development of steatosis.

Prenatal hyperandrogenization induces metabolic and endocrine alterations which depend on the levels of testosterone exposure.

Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. The aim of the present study was to establish if the levels of prenatal testosterone may determine the extent of metabolic and endocrine alterations during the adult life. Pregnant Sprague Dawley rats were prenatally injected with either 2 or 5 mg free testosterone (groups T2 and T5 respectively) from day 16 to day 19 day of gestation. Female offspring from T2 and T5 displayed different phenotype of PCOS during adult life. Offspring from T2 showed hyperandrogenism, ovarian cysts and ovulatory cycles whereas those from T5 displayed hyperandrogenism, ovarian cysts and anovulatory cycles. Both group showed increased circulating glucose levels after the intraperitoneal glucose tolerance test (IPGTT; an evaluation of insulin resistance). IPGTT was higher in T5 rats and directly correlated with body weight at prepubertal age. However, the decrease in the body weight at prepubertal age was compensated during adult life. Although both groups showed enhanced ovarian steroidogenesis, it appears that the molecular mechanisms involved were different. The higher dose of testosterone enhanced the expression of both the protein that regulates cholesterol availability (the steroidogenic acute regulatory protein (StAR)) and the protein expression of the transcriptional factor: peroxisome proliferator-activated receptor gamma (PPAR gamma). Prenatal hyperandrogenization induced an anti-oxidant response that prevented a possible pro-oxidant status. The higher dose of testosterone induced a pro-inflammatory state in ovarian tissue mediated by increased levels of prostaglandin E (PG) and the protein expression of cyclooxygenase 2 (COX2, the limiting enzyme of PGs synthesis). In summary, our data show that the levels of testosterone prenatally injected modulate the uterine environment and that this, in turn, would be responsible for the endocrine and metabolic abnormalities and the phenotype of PCOS during the adult life.