Placental adaptations supporting fetal growth during normal and adverse gestational environments.

NEW FINDINGS: What is the topic of this review? How the placenta, which transports nutrients and oxygen to the fetus, may alter its support of fetal growth developmentally and with adverse gestational conditions. What advances does it highlight? Placental formation and function alter with the needs of the fetus for substrates for growth during normal gestation and when there is enhanced competition for substrates in species with multiple gestations or adverse gestational environments, and this is mediated by imprinted genes, signalling pathways, mitochondria and fetal sexomes. ABSTRACT: The placenta is vital for mammalian development and a key determinant of life-long health. It is the interface between the mother and fetus and is responsible for transporting the nutrients and oxygen a fetus needs to develop and grow. Alterations in placental formation and function, therefore, have consequences for fetal growth and birthweight, which in turn determine perinatal survival and risk of non-communicable diseases for the offspring in later postnatal life. However, the placenta is not a static organ. As this review summarizes, research from multiple species has demonstrated that placental formation and function alter developmentally to the needs of the fetus for substrates for growth during normal gestation, as well as when there is greater competition for substrates in polytocous species and monotocous species with multiple gestations. The placenta also adapts in response to the gestational environment, integrating information about the ability of the mother to provide nutrients and oxygen with the needs of the fetus in that prevailing environment. In particular, placental structure (e.g. vascularity, surface area, blood flow, diffusion distance) and transport capacity (e.g. nutrient transporter levels and activity) respond to suboptimal gestational environments, namely malnutrition, obesity, hypoxia and maternal ageing. Mechanisms mediating developmentally and environmentally induced homeostatic responses of the placenta that help support normal fetal growth include imprinted genes, signalling pathways, subcellular constituents and fetal sexomes. Identification of these placental strategies may inform the development of therapies for complicated human pregnancies and advance understanding of the pathways underlying poor fetal outcomes and their consequences for health and disease risk.

Placental structure, function, and mitochondrial phenotype relate to fetal size in each fetal sex in mice†.

Fetal growth depends on placental function, which requires energy from mitochondria. Here we investigated whether mitochondrial function in the placenta relates to the growth of the lightest and heaviest fetuses of each sex within the litter of mice. Placentas from the lightest and heaviest fetuses were taken to evaluate placenta morphology (stereology), mitochondrial energetics (high-resolution respirometry), mitochondrial regulators, nutrient transporters, hormone handling, and signaling pathways (qPCR and Western blotting). We found that mitochondrial complex I and II oxygen consumption rate was greater for placentas supporting the lightest female fetuses, although placental complex I abundance of the lightest females and complexes III and V of the lightest males were decreased compared to their heaviest counterparts. Expression of mitochondrial biogenesis (Nrf1) and fission (Drp1 and Fis1) genes was lower in the placenta from the lightest females, whilst biogenesis-related gene Tfam was greater in the placenta of the lightest male fetuses. In addition, placental morphology and steroidogenic gene (Cyp17a1 and Cyp11a1) expression were aberrant for the lightest females, but glucose transporter (Slc2a1) expression was lower in only the lightest males versus their heaviest counterparts. Differences in intra-litter placental phenotype were related to changes in the expression of hormone-responsive (androgen receptor) and metabolic signaling (AMPK, AKT, and PPARγ) pathways. Thus, in normal mouse pregnancy, placental structure, function, and mitochondrial phenotype are differentially responsive to the growth of the female and male fetus. This study may inform the design of sex-specific therapies for placental insufficiency and fetal growth abnormalities with life-long benefits for the offspring.

Gaps in the knowledge of thyroid hormones and placental biology†.

Thyroid hormones (THs) are required for the growth and development of the fetus, stimulating anabolism, and oxygen consumption from the early stages of pregnancy to the period of fetal differentiation close to delivery. Maternal changes in the hypothalamic-pituitary-thyroid axis are also well known. In contrast, several open questions remain regarding the relationships between the placenta and the maternal and fetal TH systems. The exact mechanism by which the placenta participates in regulating the TH concentration in the fetus and mother and the role of TH in the placenta are still poorly studied. In this review, we aim to summarize the available data in the area and highlight significant gaps in our understanding of the ontogeny and cell-specific localization of TH transporters, TH receptors, and TH metabolic enzymes in the placenta in both human and rodent models. Significant deficiencies also exist in the knowledge of the contribution of genomic and nongenomic effects of TH on the placenta and finally, how the placenta reacts during pregnancy when the mother has thyroid disease. By addressing these key knowledge gaps, improved pregnancy outcomes and management of women with thyroid alterations may be possible.

Pregnancy-induced changes in ß-cell function: what are the key players?

Maternal metabolic adaptations during pregnancy ensure appropriate nutrient supply to the developing fetus. This is facilitated by reductions in maternal peripheral insulin sensitivity, which enables glucose to be available in the maternal circulation for transfer to the fetus for growth. To balance this process and avoid excessive hyperglycaemia and glucose intolerance in the mother during pregnancy, maternal pancreatic ß-cells undergo remarkable changes in their function including increasing their proliferation and glucose-stimulated insulin secretion. In this review we examine how placental and maternal hormones work cooperatively to activate several signalling pathways, transcription factors and epigenetic regulators to drive adaptations in ß-cell function during pregnancy. We also explore how adverse maternal environmental conditions, including malnutrition, obesity, circadian rhythm disruption and environmental pollutants, may impact the endocrine and molecular mechanisms controlling ß-cell adaptations during pregnancy. The available data from human and experimental animal studies highlight the need to better understand how maternal ß-cells integrate the various environmental, metabolic and endocrine cues and thereby determine appropriate ß-cell adaptation during gestation. In doing so, these studies may identify targetable pathways that could be used to prevent not only the development of pregnancy complications like gestational diabetes that impact maternal and fetal wellbeing, but also more generally the pathogenesis of other metabolic conditions like type 2 diabetes.

Suppression of uterine and placental ferroptosis by N-acetylcysteine in a rat model of polycystic ovary syndrome.

The mechanisms that link hyperandrogenism and insulin (INS) resistance (HAIR) to the increased miscarriage rate in women with polycystic ovary syndrome (PCOS) remain elusive. Previous studies demonstrate that increased uterine and placental ferroptosis is associated with oxidative stress-induced fetal loss in a pre-clinical PCOS-like rat model. Here, we investigated the efficacy and molecular mechanism of action of the antioxidant N-acetylcysteine (NAC) in reversing gravid uterine and placental ferroptosis in pregnant rats exposed to 5α-dihydrotestosterone (DHT) and INS. Molecular and histological analyses showed that NAC attenuated DHT and INS-induced uterine ferroptosis, including dose-dependent increases in anti-ferroptosis gene content. Changes in other molecular factors after NAC treatment were also observed in the placenta exposed to DHT and INS, such as increased glutathione peroxidase 4 protein level. Furthermore, increased apoptosis-inducing factor mitochondria-associated 2 mRNA expression was seen in the placenta but not in the uterus. Additionally, NAC was not sufficient to rescue DHT + INS-induced mitochondria-morphological abnormalities in the uterus, whereas the same treatment partially reversed such abnormalities in the placenta. Finally, we demonstrated that NAC selectively normalized uterine leukemia inhibitory factor, osteopontin/secreted phosphoprotein 1, progesterone receptor, homeobox A11 mRNA expression and placental estrogen-related receptor beta and trophoblast-specific protein alpha mRNA expression. Collectively, our data provide insight into how NAC exerts beneficial effects on differentially attenuating gravid uterine and placental ferroptosis in a PCOS-like rat model with fetal loss. These results indicate that exogenous administration of NAC represents a potential therapeutic strategy in the treatment of HAIR-induced uterine and placental dysfunction.

Increased uterine androgen receptor protein abundance results in implantation and mitochondrial defects in pregnant rats with hyperandrogenism and insulin resistance.

In this study, we show that during normal rat pregnancy, there is a gestational stage-dependent decrease in androgen receptor (AR) abundance in the gravid uterus and that this is correlated with the differential expression of endometrial receptivity and decidualization genes during early and mid-gestation. In contrast, exposure to 5α-dihydrotestosterone (DHT) and insulin (INS) or DHT alone significantly increased AR protein levels in the uterus in association with the aberrant expression of endometrial receptivity and decidualization genes, as well as disrupted implantation. Next, we assessed the functional relevance of the androgen-AR axis in the uterus for reproductive outcomes by treating normal pregnant rats and pregnant rats exposed to DHT and INS with the anti-androgen flutamide. We found that AR blockage using flutamide largely attenuated the DHT and INS-induced maternal endocrine, metabolic, and fertility impairments in pregnant rats in association with suppressed induction of uterine AR protein abundance and androgen-regulated response protein and normalized expression of several endometrial receptivity and decidualization genes. Further, blockade of AR normalized the expression of the mitochondrial biogenesis marker Nrf1 and the mitochondrial functional proteins Complexes I and II, VDAC, and PHB1. However, flutamide treatment did not rescue the compromised mitochondrial structure resulting from co-exposure to DHT and INS. These results demonstrate that functional AR protein is an important factor for gravid uterine function. Impairments in the uterine androgen-AR axis are accompanied by decreased endometrial receptivity, decidualization, and mitochondrial dysfunction, which might contribute to abnormal implantation in pregnant PCOS patients with compromised pregnancy outcomes and subfertility. KEY MESSAGES: The proper regulation of uterine androgen receptor (AR) contributes to a normal pregnancy process, whereas the aberrant regulation of uterine AR might be linked to polycystic ovary syndrome (PCOS)-induced pregnancy-related complications. In the current study, we found that during normal rat pregnancy there is a stage-dependent decrease in AR abundance in the gravid uterus and that this is correlated with the differential expression of the endometrial receptivity and decidualization genes Spp1, Prl, Igfbp1, and Hbegf. Pregnant rats exposed to 5α-dihydrotestosterone (DHT) and insulin (INS) or to DHT alone show elevated uterine AR protein abundance and implantation failure related to the aberrant expression of genes involved in endometrial receptivity and decidualization in early to mid-gestation. Treatment with the anti-androgen flutamide, starting from pre-implantation, effectively prevents DHT + INS-induced defects in endometrial receptivity and decidualization gene expression, restores uterine mitochondrial homeostasis, and increases the pregnancy rate and the numbers of viable fetuses. This study adds to our understanding of the mechanisms underlying poor pregnancy outcomes in PCOS patients and the possible therapeutic use of anti-androgens, including flutamide, after spontaneous conception.

Placental mitochondrial function in response to gestational exposures.

Poor environmental conditions, including malnutrition, hypoxia and obesity in the mother increase the risk of pregnancy complications, such as pre-eclampsia and gestational diabetes mellitus, which impacts the lifelong health of the mother and her offspring. The placenta plays an important role in determining pregnancy outcome by acting as an exchange interface and endocrine hub to support fetal growth. Mitochondria are energy powerhouses of cells that fuel placental physiology throughout pregnancy, including placental development, substrate exchange and hormone secretion. They are responsive to environmental cues and changes in mitochondrial function may serve to mediate or mitigate the impacts of poor gestational environments on placental physiology and hence, the risks of pregnancy complications. Thus, a more integrated understanding about the role of placental mitochondria in orchestrating changes in relation to environmental conditions and pregnancy outcome is paramount. This review summarises the functions of mitochondria in the placenta and findings from humans and experimental animals that demonstrate how mitochondrial structure and function are altered in different gestational environments (namely complicated pregnancies and adverse environmental conditions). Together the available data suggest that mitochondria in the placenta play a major role in determining placental physiology, fetal growth and pregnancy outcome.

TLR4-Associated IRF-7 and NFκB Signaling Act as a Molecular Link Between Androgen and Metformin Activities and Cytokine Synthesis in the PCOS Endometrium.

CONTEXT: Low-grade chronic inflammation is commonly seen in polycystic ovary syndrome (PCOS) patients with elevated levels of inflammatory cytokines in the endometrium. OBJECTIVE: This work aimed to increase the limited understanding of the mechanisms underlying cytokine synthesis and increased endometrial inflammation in PCOS patients. METHODS: Endometrial biopsy samples were collected from non-PCOS (n = 17) and PCOS (n = 22) patients either during the proliferative phase of the menstrual cycle or with hyperplasia. Endometrial explants were prepared from PCOS patients and underwent pharmacological manipulation in vitro. The expression and localization of toll-like receptor 2 (TLR2)/4, key elements of innate immune signal transduction and nuclear factor κB (NFκB) signaling pathways, and multiple cytokines were comprehensively evaluated by Western blotting, immunohistochemistry, and immunofluorescence in endometrial tissues. RESULTS: We demonstrated the distribution of protein expression and localization associated with the significantly increased androgen receptor, TLR2, and TLR4-mediated activation of interferon regulatory factor-7 (IRF-7) and NFκB signaling, cytokine production, and endometrial inflammation in PCOS patients compared to non-PCOS patients with and without endometrial hyperplasia. In vitro experiments showed that 5-dihydrotestosterone (DHT) enhanced androgen receptor, TLR4, IRF-7, and p-NFκB p65 protein expression along with increased interferon α (IFNα) and IFNÉ£ abundance. The effects of DHT on IRF-7, p-NFκB p65, and IFN abundance were abolished by flutamide, an antiandrogen. Although 17ß-estradiol (E2) decreased p-IRF-7 expression with little effect on TLR-mediated IRF7 and NFκB signaling or on cytokine protein levels, exposure to metformin alone or in combination with E2 suppressed interleukin-1 receptor-associated kinase 4 (IRAK4), p-IRF-7, IRF-7, IκB kinase α (IKKα), p-NFκB p65, IFNÉ£, and tumor necrosis factor α protein expression. CONCLUSION: Cytokine synthesis and increased endometrial inflammation in PCOS patients are coupled to androgen-induced TLR4/IRF-7/NFκB signaling, which is inhibited by metformin treatment.

Hyperandrogenism and insulin resistance modulate gravid uterine and placental ferroptosis in PCOS-like rats.

Women with polycystic ovary syndrome (PCOS) have hyperandrogenism and insulin resistance and a high risk of miscarriage during pregnancy. Similarly, in rats, maternal exposure to 5α-dihydrotestosterone (DHT) and insulin from gestational day 7.5 to 13.5 leads to hyperandrogenism and insulin resistance and subsequently increased fetal loss. A variety of hormonal and metabolic stimuli are able to trigger different types of regulated cell death under physiological and pathological conditions. These include ferroptosis, apoptosis and necroptosis. We hypothesized that, in rats, maternal hyperandrogenism and insulin-resistance-induced fetal loss is mediated, at least in part, by changes in the ferroptosis, apoptosis and necroptosis pathways in the gravid uterus and placenta. Compared with controls, we found that co-exposure to DHT and insulin led to decreased levels of glutathione peroxidase 4 (GPX4) and glutathione, increased glutathione + glutathione disulfide and malondialdehyde, aberrant expression of ferroptosis-associated genes (Acsl4, Tfrc, Slc7a11, and Gclc), increased iron deposition and activated ERK/p38/JNK phosphorylation in the gravid uterus. In addition, we observed shrunken mitochondria with electron-dense cristae, which are key features of ferroptosis-related mitochondrial morphology, as well as increased expression of Dpp4, a mitochondria-encoded gene responsible for ferroptosis induction in the uteri of rats co-exposed to DHT and insulin. However, in the placenta, DHT and insulin exposure only partially altered the expression of ferroptosis-related markers (e.g. region-dependent GPX4, glutathione + glutathione disulfide, malondialdehyde, Gls2 and Slc7a11 mRNAs, and phosphorylated p38 levels). Moreover, we found decreased expression of Dpp4 mRNA and increased expression of Cisd1 mRNA in placentas of rats co-exposed to DHT and insulin. Further, DHT + insulin-exposed pregnant rats exhibited decreased apoptosis in the uterus and increased necroptosis in the placenta. Our findings suggest that maternal hyperandrogenism and insulin resistance causes the activation of ferroptosis in the gravid uterus and placenta, although this is mediated via different mechanisms operating at the molecular and cellular levels. Our data also suggest that apoptosis and necroptosis may play a role in coordinating or compensating for hyperandrogenism and insulin-resistance-induced ferroptosis when the gravid uterus and placenta are dysfunctional.

Hyperandrogenism and insulin resistance-induced fetal loss: evidence for placental mitochondrial abnormalities and elevated reactive oxygen species production in pregnant rats that mimic the clinical features of polycystic ovary syndrome.

KEY POINTS: Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanisms remain unknown. Herein, pregnant rats chronically treated with 5α-dihydrotestosterone (DHT) and insulin exhibited hyperandrogenism and insulin resistance, as well as increased fetal loss, and these features are strikingly similar to those observed in pregnant PCOS patients. Fetal loss in our DHT+insulin-treated pregnant rats was associated with mitochondrial dysfunction, disturbed superoxide dismutase 1 and Keap1/Nrf2 antioxidant responses, over-production of reactive oxygen species (ROS) and impaired formation of the placenta. Chronic treatment of pregnant rats with DHT or insulin alone indicated that DHT triggered many of the molecular pathways leading to placental abnormalities and fetal loss, whereas insulin often exerted distinct effects on placental gene expression compared to co-treatment with DHT and insulin. Treatment of DHT+insulin-treated pregnant rats with the antioxidant N-acetylcysteine improved fetal survival but was deleterious in normal pregnant rats. Our results provide insight into the fetal loss associated with hyperandrogenism and insulin resistance in women and suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy. ABSTRACT: Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanism of PCOS-induced fetal loss during pregnancy remains obscure and specific therapies are lacking. We used pregnant rats treated with 5α-dihydrotestosterone (DHT) and insulin to investigate the impact of hyperandrogenism and insulin resistance on fetal survival and to determine the molecular link between PCOS conditions and placental dysfunction during pregnancy. Our study shows that pregnant rats chronically treated with a combination of DHT and insulin exhibited endocrine aberrations such as hyperandrogenism and insulin resistance that are strikingly similar to those in pregnant PCOS patients. Of pathophysiological significance, DHT+insulin-treated pregnant rats had greater fetal loss and subsequently decreased litter sizes compared to normal pregnant rats. This negative effect was accompanied by impaired trophoblast differentiation, increased glycogen accumulation, and decreased angiogenesis in the placenta. Mechanistically, we report that over-production of reactive oxygen species (ROS) in the placenta, mitochondrial dysfunction, and disturbed superoxide dismutase 1 (SOD1) and Keap1/Nrf2 antioxidant responses constitute important contributors to fetal loss in DHT+insulin-treated pregnant rats. Many of the molecular pathways leading to placental abnormalities and fetal loss in DHT+insulin treatment were also seen in pregnant rats treated with DHT alone, whereas pregnant rats treated with insulin alone often exerted distinct effects on placental gene expression compared to insulin treatment in combination with DHT. We also found that treatment with the antioxidant N-acetylcysteine (NAC) improved fetal survival in DHT+insulin-treated pregnant rats, an effect related to changes in Keap1/Nrf2 and nuclear factor-κB signalling. However, NAC administration resulted in fetal loss in normal pregnant rats, most likely due to PCOS-like endocrine abnormality induced by the treatment. Our results suggest that the deleterious effects of hyperandrogenism and insulin resistance on fetal survival are related to a constellation of mitochondria-ROS-SOD1/Nrf2 changes in the placenta. Our findings also suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy.

Hyperandrogenism and insulin resistance induce gravid uterine defects in association with mitochondrial dysfunction and aberrant reactive oxygen species production.

Women with polycystic ovary syndrome (PCOS) are at increased risk of miscarriage, which often accompanies the hyperandrogenism and insulin resistance seen in these patients. However, neither the combinatorial interaction between these two PCOS-related etiological factors nor the mechanisms of their actions in the uterus during pregnancy are well understood. We hypothesized that hyperandrogensim and insulin resistance exert a causative role in miscarriage by inducing defects in uterine function that are accompanied by mitochondrial-mediated oxidative stress, inflammation, and perturbed gene expression. Here, we tested this hypothesis by studying the metabolic, endocrine, and uterine abnormalities in pregnant rats after exposure to daily injection of 5α-dihydrotestosterone (DHT; 1.66 mg·kg body wt-1·day-1) and/or insulin (6.0 IU/day) from gestational day 7.5 to 13.5. We showed that whereas DHT-exposed and insulin-exposed pregnant rats presented impaired insulin sensitivity, DHT + insulin-exposed pregnant rats exhibited hyperandrogenism and peripheral insulin resistance, which mirrors pregnant PCOS patients. Compared with controls, hyperandrogenism and insulin resistance in the dam were associated with alterations in uterine morphology and aberrant expression of genes responsible for decidualization (Prl8a2, Fxyd2, and Mt1g), placentation (Fcgr3 and Tpbpa), angiogenesis (Flt1, Angpt1, Angpt2, Ho1, Ccl2, Ccl5, Cxcl9, and Cxcl10) and insulin signaling (Akt, Gsk3, and Gluts). Moreover, we observed changes in uterine mitochondrial function and homeostasis (i.e., mitochondrial DNA copy number and the expression of genes responsible for mitochondrial fusion, fission, biogenesis, and mitophagy) and suppression of both oxidative and antioxidative defenses (i.e., reactive oxygen species, Nrf2 signaling, and interactive networks of antioxidative stress responses) in response to the hyperandrogenism and insulin resistance. These findings demonstrate that hyperandrogenism and insulin resistance induce mitochondria-mediated damage and a resulting imbalance between oxidative and antioxidative stress responses in the gravid uterus.

Regulating needs: Exploring the role of insulin-like growth factor-2 signalling in materno-fetal resource allocation.

During pregnancy, the fetus requires nutrients supplied by the mother to grow and develop. However, the mother also requires sufficient resources to support the pregnancy, as well as, to maintain her health. Failure to regulate resource allocation between the mother and fetus can lead to pregnancy complications with immediate and life-long consequences for maternal and offspring health. This review explores the role of insulin-like growth factor (IGF)-2 in regulating materno-fetal resource allocation, particularly via its regulation of placental development and function.