Effect of prenatal hyperandrogenization on the uterine function.

El Síndrome del Ovario Poliquístico (SOP) es una patología reproductiva de alta incidencia en mujeres en edad fértil. La etiología del SOP se desconoce, pero actualmente se postula que un exceso androgénico intrauterino genera programación fetal. El SOP es una de las principales causas de infertilidad. Esto puede atribuirse no solo a disfunción ovárica ya que aun restableciendo la función ovulatoria o mediante técnicas de reproducción asistida, existe una alta tasa de aborto espontaneo lo que sugiere un problema de receptividad uterina. En este trabajo, nos propusimos estudiar los efectos de la hiperandrogenización prenatal (PH) sobre el tejido uterino en un modelo murino de programación fetal por exceso androgénico, el cual nos permite emular características de tipo SOP en ratas adultas. Nuestros resultados revelan que la PH conlleva a la expresión de dos fenotipos: ovulatorio irregular y anovulatorio, los cuales presentan hiperandrogenismo, quistes ováricos y alteraciones hormonales y metabólicas. En el útero, las vías de señalización responsables de la homeostasis celular se encontraron alteradas. Como consecuencia, se han observado cambios en la histomorfología uterina que se evidencian por un aumento del grosor uterino, displasia del epitelio luminal e hiperplasia glandular que se asocian con la presencia de un estado pro-oxidante y un estado pro-inflamatorio elevado. En conclusión, estos resultados revelan que la PH programa a largo plazo las vías de señalización responsables de la proliferación, supervivencia y muerte celular, generando una desregulación homeostática que se traduce en cambios en la histomorfología del útero lo que condicionaría su funcionalidad.Polycystic ovary syndrome (PCOS) is the most common endocrine-reproductive disorder that affects women during their reproductive years. The etiology of PCOS is unclear but current theories postulate that an excess of androgens in the intrauterine environment leads to fetal programming. PCOS is a leading cause of female infertility. PCOS women have high rates of pregnancy loss even if the ovulation has been restored, or by using assisted reproduction techniques, suggesting a problem in the uterine receptivity. In the present work, we aimed to study the effect of prenatal hyperandrogenization (PH) on the uterine tissue in a murine model of fetal programming caused by androgen excess, which let us recreate PCOS features in adult rats. Our results show that PH led to two phenotypes according to the estrous cycle: irregular ovulatory and anovulatory, accompanied by hyperandrogenism, ovarian cysts, and hormonal and metabolic alterations. In the uterine tissue, signaling pathways that maintain homeostasis were found altered. As a consequence, changes in the histomorphology have been observed and are evidenced through the increased uterine thickness, luminal epithelial dysplasia, and glandular hyperplasia. Moreover, a pro-oxidant and pro-inflammatory status was found. In conclusion, these results revealed that PH programs several signaling pathways responsible for proliferation, survival, and cell death, leading to homeostatic deregulation evidenced by altered histomorphology, which could likely condition its functioning.Fil: Ferreira, Silvana Rocío. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Effect of prenatal hyperandrogenism on fertility

Introducción: El Síndrome de Ovario Poliquístico (SOP) es una de las endocrinopatías reproductivas más frecuente en mujeres en edad fértil. Dado que la infertilidad es un problema que afecta a numerosas parejas con deseo de concepción en el presente trabajo nos centramos en el estudio del útero, órgano encargado de mantener y proteger al feto en desarrollo durante la gestación. Objetivo: Evaluar cómo la reprogramación fetal afecta la funcionalidad del tejido uterino durante la etapa puberal y durante la etapa adulta. Material y Métodos: Se utilizó un modelo murino de hiperandrogenización prenatal (HA). Para ello se utilizaron ratas Sprague-Dawley las cuales fueron inyectadas en los días 16 a 19 de gestación de manera subcutánea con aceite vegetal (grupo Control -C-) o con 2 mg de testosterona (grupo -HA-). Un grupo fue sacrificado a los 60 días -etapa puberal- realizándose una caracterización de la funcionalidad uterina y otro grupo fue estudiado a los 90 días -etapa adulta- evaluándose la fertilidad por vía natural y bajo estimulación gonadotrófica. Resultados: Durante la etapa puberal el grupo HA prenatalmente presentó un estado proinflamatorio elevado caracterizado por un aumento de la Prostaglandina E (PGE) uterina y la enzima limitante de la síntesis de PGs, Ciclooxigenasa 2 (COX2). El estrés oxidativo del tejido uterino evaluado a través de peroxi­dación lipídica no mostró diferencias entre C y HA como así tampoco el metabolito antioxidante, Glutation. El receptor activado por proliferadores peroxisomales gama (PPAR γ) se encontró aumentado con respecto al grupo C. En la etapa adulta el grupo HA presentó disminuida su capacidad reproductiva y esto se revirtió luego del tratamiento con gonadotrofinas. Conclusiones: Bajo este modelo, el HA condiciona la funcionalidad uterina disminuyendo la capacidad re­productiva de animales HA. El tratamiento con gonadotrofinas restablece dicha función, permitiendo a los animales HA alcanzar la preñez.Introduction: Polycystic ovary syndrome (PCOS) is a common endocrine-reproductive disorder that affects women in their reproductive age. Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. PCOS is the commonest cause of anovulatory infertility. PCOS is not only a reproductive pathology, since it includes metabolic disorders (insulin resistance, impaired glucose tolerance, type 2 diabe­tes mellitus, high risk factor for cardiovascular diseases) and psychological symptoms (depression, increased anxiety, low self-esteem). However; the pathophysiology of PCOS is complex and remains unclear. Objectives: 1) To evaluate how hyperandrogenism (HA) affects the function of the uterine tissue at puber Pregnant Sprague-Dawley rats were prenatally injected daily with 2 mg free testosterone (HA group) or vehicle (Control group) from day 16 to day 19 of gestation. Female offspring were study at pubertal and adult age. The uterine oxidative stress was quantified by lipid peroxidation index (LP) and antioxidant glutathione (GSH) content. No differences were found between LP and GSH. The protein expressions of peroxisome proliferator-activated receptor gamma (PPAR γ), and the limiting enzyme of the PG synthesis, cyclooxygenase 2 (COX2) and the uterine PGE content increased in the HA group with respect to the C group. We also studied fertility at adult stage with and without hormone induction. The fertility rate decreased in HA. Hormone induction reversed the fertility rate similarly to controls. Conclusions: HA affects the uterine function in PCOS. HA increases protein levels of PPAR gamma, which modulates the pro-inflammatory status. The uterine oxidative stress was balanced. Fertility was decreased in the HA group and this was reversed by gonadotropin stimulation.Fil: Ferreira, Silvana Rocío . Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires; ArgentinaFil: Velez, Leandro Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires; ArgentinaFil: Motta, Alicia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires; Argentin

Prenatal hyperandrogenism induces alterations that affect liver lipid metabolism

Prenatal hyperandrogenism is hypothesized as one of the main factors contributing to26 the development of polycystic ovary syndrome (PCOS). PCOS patients have high risk27 of developing fatty liver and steatosis. This study aimed to evaluate the role of prenatal28 hyperandrogenism in liver lipid metabolism and fatty liver development. Pregnant rats29 were hyperandrogenized with testosterone. At pubertal age, the prenatally30 hyperandrogenized (PH) female offspring displayed both ovulatory (PHov) and31 anovulatory (PHanov) phenotypes that mimic human PCOS features. We evaluated32 hepatic transferases, liver lipid content, the balance between lipogenesis and fatty acid33 oxidation pathway, oxidant/antioxidant balance and pro-inflammatory status. We also34 evaluated the general metabolic status through growth rate curve, basal glucose and35 insulin levels, glucose tolerance test, HOMA-IR index and serum lipid profile.36 Although neither PH group showed signs of liver lipid content, the lipogenesis and fatty37 oxidation pathways were altered. The PH groups also showed impaired38 oxidant/antioxidant balance, a decrease in the pro-inflammatory pathway (measured by39 prostaglandin E2 and cyclooxygenase-2 levels), decreased glucose tolerance, imbalance40 of circulating lipids and increased risk of metabolic syndrome. We conclude that41 prenatal hyperandrogenism generates both PHov and PHanov phenotypes with signs of42 liver alterations, imbalance in lipid metabolism and increased risk of developing43 metabolic syndrome. The anovulatory phenotype showed more alterations in liver44 lipogenesis and a more impaired balance of insulin and glucose metabolism, being more45 susceptible to the development of steatosis.Fil: Abruzzese, Giselle Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Heber, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Ferreira, Silvana Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Velez, Leandro Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Reynoso, Roxana María. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Endocrinología; ArgentinaFil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Motta, Alicia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

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

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Effect of prenatal hyperandrogenization on the uterine function.

El Síndrome del Ovario Poliquístico (SOP) es una patología reproductiva de alta incidencia en mujeres en edad fértil. La etiología del SOP se desconoce, pero actualmente se postula que un exceso androgénico intrauterino genera programación fetal. El SOP es una de las principales causas de infertilidad. Esto puede atribuirse no solo a disfunción ovárica ya que aun restableciendo la función ovulatoria o mediante técnicas de reproducción asistida, existe una alta tasa de aborto espontaneo lo que sugiere un problema de receptividad uterina. En este trabajo, nos propusimos estudiar los efectos de la hiperandrogenización prenatal (PH) sobre el tejido uterino en un modelo murino de programación fetal por exceso androgénico, el cual nos permite emular características de tipo SOP en ratas adultas. Nuestros resultados revelan que la PH conlleva a la expresión de dos fenotipos: ovulatorio irregular y anovulatorio, los cuales presentan hiperandrogenismo, quistes ováricos y alteraciones hormonales y metabólicas. En el útero, las vías de señalización responsables de la homeostasis celular se encontraron alteradas. Como consecuencia, se han observado cambios en la histomorfología uterina que se evidencian por un aumento del grosor uterino, displasia del epitelio luminal e hiperplasia glandular que se asocian con la presencia de un estado pro-oxidante y un estado pro-inflamatorio elevado. En conclusión, estos resultados revelan que la PH programa a largo plazo las vías de señalización responsables de la proliferación, supervivencia y muerte celular, generando una desregulación homeostática que se traduce en cambios en la histomorfología del útero lo que condicionaría su funcionalidad.Polycystic ovary syndrome (PCOS) is the most common endocrine-reproductive disorder that affects women during their reproductive years. The etiology of PCOS is unclear but current theories postulate that an excess of androgens in the intrauterine environment leads to fetal programming. PCOS is a leading cause of female infertility. PCOS women have high rates of pregnancy loss even if the ovulation has been restored, or by using assisted reproduction techniques, suggesting a problem in the uterine receptivity. In the present work, we aimed to study the effect of prenatal hyperandrogenization (PH) on the uterine tissue in a murine model of fetal programming caused by androgen excess, which let us recreate PCOS features in adult rats. Our results show that PH led to two phenotypes according to the estrous cycle: irregular ovulatory and anovulatory, accompanied by hyperandrogenism, ovarian cysts, and hormonal and metabolic alterations. In the uterine tissue, signaling pathways that maintain homeostasis were found altered. As a consequence, changes in the histomorphology have been observed and are evidenced through the increased uterine thickness, luminal epithelial dysplasia, and glandular hyperplasia. Moreover, a pro-oxidant and pro-inflammatory status was found. In conclusion, these results revealed that PH programs several signaling pathways responsible for proliferation, survival, and cell death, leading to homeostatic deregulation evidenced by altered histomorphology, which could likely condition its functioning.Fil: Ferreira, Silvana Rocío. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Prenatal hyperandrogenism and lipid profile during different age stages: an experimental study

Objective The present study investigates the effect of prenatal hyperandrogenization on lipid metabolism and oxidant/antioxidant balance. Design Experimental study. Setting Research institute. Animal(s) Pregnant Sprague Dawley rats were subcutaneously injected with 2 mg free T between days 16 and 19 of pregnancy, and controls (C) received vehicle (0.1 mL of sesame oil). Prenatally hyperandrogenized female offspring (T2) had a condition that resembles polycystic ovary (PCO). Animals were weighed and killed at 21 and 60 days of age (N = 15 rats/group). Intervention(s) Ovarian tissue and truncal blood were obtained from the C and T2 groups. Main Outcome Measure(s) Circulating lipid profile (total cholesterol, high-density lipoprotein [HDL], low-density lipoprotein [LDL] cholesterol, and triglycerides) was quantified by colorimetric-enzymatic methods. Ovarian oxidative stress was evaluated by quantifying lipid peroxidation and glutathione content by spectofotometric assays. Ovarian fat content was evaluated by Red Oil staining and ovarian messenger RNA (mRNA) expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) by real-time polymerase chain reaction (PCR). Result(s) At 60 days of age, 100% of group C rats and 20% of group T2 rats ovulated. At 21 days of age the T2 rats displayed lower body weight than C rats; however, at 60 days of age T2 and C rats showed similar body weights. The lipid profile (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides) was altered in the anovulatory and ovulatory phenotype of the T2 group, but the levels were higher in the anovulatory phenotype. Lipid peroxidation of rats at 21 and 60 days of age from T2 was similar to C but the antioxidant glutathione level was decreased in 21-day-old rats compared with C rats. The lipid content of ovarian tissue, determined by Red Oil staining, was higher in the T2 than in the C group. The mRNA expression of ovarian PPAR-γ, quantified by real time PCR, decreased in anovulatory rats at 60 days of age from T2 compared to C rats. Conclusion(s) Our findings reveal the importance of evaluating the complete lipid profile, especially at early stages of life after the prenatal hyperandrogenism condition. In addition, we demonstrated that the antioxidant-reduced glutathione would represent a good marker of oxidative stress as it is altered before lipid peroxidation. Prenatal hyperandrogenization also alters the gene expression of PPAR-γ in rats. Here we demonstrated for the first time that abnormalities in PPAR-γ and lipid profile were higher in rats showing an anovulatory phenotype than those displaying an ovulatory phenotypeFil: Heber, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Ferreira, Silvana Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Velez, Leandro Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Motta, Alicia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentin

Prenatal hyperandrogenization and glucose homeostasis.

<p>As a measurement of glucose homeostasis, dynamic studies were performed in female offspring of Sprague Dawley rats prenatally injected with 2 mg testosterone (T2 group), 5 mg testosterone (T5) or vehicle (C). Blood sample followed by itraperitoneal injection of 2 g dextrose/kg body weight was collected at 0, 30, 60, 90 and 120 post-injection. Each column represents the mean+SEM from ten different animals, N = 20 animals/group.</p

Prenatal hyperandrogenization and ovarian steroidogenesis.

<p>(A) Serum progesterone levels, (B) Serum estradiol levels and (C) Serum testosterone levels from female offspring of Sprague Dawley rats prenatally injected with 2 mg testosterone (T2 group), 5 mg testosterone (T5) or vehicle (C). CP: rats from the control group at proestrous stage of the estrous cycle, T2P: rats from the T2 group of treatment at proestrous stage of the estrous cycle CD: rats from the control group at diestrous stage of the estrous cycle, T2D: rats from the T2 group of treatment at diestrous stage of the estrous cycle, T5: rats from the T5 group of treatment. a vs b; a vs c and b vs c P<0.0001 by ANOVA test. Each column represents the mean+SEM from ten different animals, N = 20 animals/group.</p

Prenatal hyperandrogenization and ovarian morphology.

<p>(A) A representative ovarian tissue section from rats prenatally injected with vehicle (Control group), ×200. (B) Detail of granulosa (GC), theca cells (TC) and luteinized cells (LC) of ovarian tissue from rats prenatally injected with vehicle (Control group), ×400. (C) Magnification of conserved oocyte of ovarian tissue from rats in the Control group, ×1000. (D) Detail of conserved oocytes (O) and distribution of granulosa (GC) and theca cells (TC) of ovarian tissue from rats in the Control group, ×400. (E) Detail of luteinized cells (LC) of ovarian tissue from rats in the Control group, ×400 (F) Magnification of conserved luteinized cells (LC) of ovarian tissue from rats in the Control group, ×1000. (G) A representative ovarian tissue section from rats prenatally injected with 2 mg testosterone (T2 group), PF = primary follicle, SF = secondary follicle, ×400. (H) A representative atretic oocyte (AO) of ovarian tissue from rats in the T2 group, ×400. (I) A representative follicular cyst (FC) of ovarian tissue from rats in the T2 group, ×400. (J) Magnification of granulosa (GC) and theca cell (TC) of the follicular cyst, ×1000. (K) Aspect of luteinized cells (LC) of ovarian tissue from rats in the T2 group, ×1000. (L) A representative section of ovarian tissue from rats prenatally injected with 5 mg testosterone (T5 group), AF = antral follicle, ×200. L.C. = luteal cells (M) Follicular cysts (FC) in ovarian tissue from rats in the T5 group, ×400. (N) A detail of altered organization of granulosa (GC) and theca cells (TC) and atretic oocytes (AO) of ovarian tissue from rats in the T5 group, N = ten ovaries per group.</p