Sperm Flow Cytometry: Beyond Human Fertilization and Embryo Development

Male infertily is a contributing factor in up to 50% of all infertility cases, a solo cause in about 30% of them. Therefore, new and improved diagnostic methods that reduce operator variability regarding sperm defects that are not accesible by the conventional microscope scoring should be evaluated. Assisted reproductive technology (ART) has been involved in the description of alternative pathways in basic cellular functions. it is important to know that it is also related to the peri-implantatory processes that involve the sperm-oocyte interaction, cellular changes observed during fertilization, and the early and late embryo development. Several pathways have been involved at the early stages of human gametogenesis. The spermatozoon has demonstrated an intricate correlation during the fertilization process, as a transfected vector on genetic material, and as interacting with other inner components (RNAm, mitochondrial organelles, etc.). Spermatogenesis is affected by programmed death cell pathways from its packaging process through the elongated cytoplasmic structures during spermiogenesis. Flow cytometry (FC) has been an outstanding tool with the capability to select human gametes to achieve a better reproductive condition. It has been applied as a diagnostic and therapeutic tool allowing a measurable and objective selection and discrimination of spermatozoa from subfertile subjects. Using FC, we are able to know that early distribution of organelles such as mitochondria has an impact in embryo quality before genetic activation on the eight-cell stages occurs. This chapter will let the readers know the current knowledge on sperm fertilization and the relation between the embryo development and the offspring and all the tools now available for an early diagnosis and to identify therapeutic options with FC

Interacción entre la función reproductiva y metabólica: caracterización del papel de factores neuroendocrinos y el sensor energético, proteína quinasa activada por AMP (AMPK)

La reproducción es un proceso costoso en términos de consumo energético que, si bien es esencial para la supervivencia de las especies, es prescindible a nivel individual. Por lo tanto, la maduración y la función del eje reproductor están estrechamente conectadas con el estado energético del organismo y se encuentran bajo el control de una red sofisticada de factores reguladores, donde señales neuronales, hormonales y ambientales cooperan para garantizar su correcta maduración, durante distintas etapas del desarrollo, y su mantenimiento funcional en la edad adulta, siempre que las condiciones nutricionales y metabólicas sean adecuadas. El éxito de este proceso está dirigido por la acción integrada de las señales del denominado eje Hipotálamo- Hipófiso-Gonadal (HHG), en el que las neuronas GnRH ocupan una posición destacada controlando la reproducción y su modulación mediante señales metabólicas (1, 2). Las neuronas Kiss1, por su parte, son elementos clave en el control de la reproducción, ya que regulan la liberación de GnRH y, por ende, de las gonadotropinas, y además son capaces de transmitir información metabólica a las neuronas GnRH, estimulando o inhibiendo su síntesis (3). En este contexto, en las últimas décadas se han descubierto un gran número de señales de origen central y periférico, responsables de la adecuación de la función reproductora a la disponibilidad de recursos energéticos del organismo. En líneas generales, puede asumirse que las señales de suficiencia energética (anorexigénicas) estimulan la puesta en marcha y funcionamiento del eje reproductor, mientras que las señales que informan de la escasez de recursos energéticos (orexigénicas) inhiben el funcionamiento del eje reproductor. De esta forma, se han desarrollado mecanismos sofisticados durante la evolución para permitir una inhibición específica del eje reproductor en condiciones energéticas desfavorables (3). Dichas condiciones metabólicas adversas pueden concurrir en situaciones tanto de déficit como de exceso de los depósitos energéticos del organismo. Así, tanto en países desarrollados como en países en vías de desarrollo, la prevalencia de la obesidad y las patologías asociadas está aumentando a un ritmo de proporciones epidémicas. Las razones se deben a una combinación entre la predisposición genética y los factores sociales y ambientales, que conducen a un desequilibrio en el balance energético, y se asocia con la hipertensión, la diabetes tipo 2, el hígado graso y una variedad de trastornos conocidos como síndrome metabólico. De otra parte, diversas condiciones, algunas de prevalencia creciente, se asocian a un déficit energético severo, tales como la anorexia nerviosa, la caquexia tumoral y otras patologías consuntivas, o incluso programas de entrenamiento físico intensivo, tales como los propios de deportistas profesionales. En unas y otras condiciones, dependiendo del momento en el que concurran, tanto la pubertad como la fertilidad y la función gonadal pueden verse severamente afectadas (4). A su vez, la propia función gonadal puede influenciar el estado metabólico del organismo, de suerte que las secreciones gonadales, como los andrógenos y los estrógenos, son potentes moduladores de la homeostasis metabólica y del peso corporal. Por todo ello, diversas evidencias experimentales apuntan que no sólo cambios nutricionales y otras situaciones de estrés metabólico, sino también niveles inapropiados de hormonas sexuales en determinados periodos del desarrollo, pueden contribuir a desencadenar alteraciones metabólicas y a modificar de manera permanente la función reproductora (5, 6). Sin embargo, los mecanismos neurohormonales que subyacen a esta interacción bidireccional entre el metabolismo y el eje reproductor permanecen aún en gran medida desconocidos. Por otro lado, además de distintas señales centrales y periféricas, evidencias recientes sugieren la participación de sensores energéticos celulares en el control metabólico de la función reproductora. Entre ellos, destaca la proteína quinasa activada por AMP, denominada AMPK, un sensor energético clave que se activa en condiciones de déficit energético y está implicado en la homeostasis energética del organismo, principalmente a través de la regulación de la ingesta y el gasto energético, ejerciendo su acción en los núcleos hipotalámicos, arcuato (ARC) y ventromedial (VMN), respectivamente (7). Además, estudios recientes han sugerido que AMPK opera como una vía clave que informa y modula al eje HHG en situaciones de insuficiencia energética, si bien las evidencias que apoyan dicho papel son aún fragmentarias y no concluyentes, sin una caracterización clara del papel de la señalización de AMPK en poblaciones neuronales clave para el control metabólico de la función reproductora (1). En suma, aun cuando es evidente que existe un claro acoplamiento entre el estado energético y metabólico del organismo, y la maduración y función del eje reproductor, aún no se conocen en profundidad las redes neuronales y las bases hormonales y moleculares de los mecanismos que intervienen en este fenómeno fisiológico. Todo ello hace necesario un mejor conocimiento de las complejas interacciones bidireccionales entre el estado metabólico y la función reproductiva, las bases neuroendocrinas de dichas interacciones, y los mecanismos moleculares que conectan ambos sistemas corporales relevantes. Teniendo en cuenta lo expuesto previamente, el objetivo general de esta Tesis Doctoral ha sido profundizar en la caracterización de las interacciones dinámicas entre el estado metabólico del organismo y aspectos clave de la maduración y la función reproductora. En este contexto, en esta Tesis se ha prestado especial atención a la definición del impacto de cambios en la función gonadal y la exposición a factores obesogénicos sobre el perfil endocrino-metabólico en la edad adulta y a la evaluación del posible papel del sensor energético celular, AMPK, actuando en las neuronas GnRH y Kiss1, en el control metabólico de la función reproductora

Effect of Epas1 and Pcx inactivation in pancreatic β-cell formation and function.

According to the consensus model for GSIS (Glucose-Stimulated Insulin Secretion), glucose is rapidly metabolized and coupled to insulin secretion involving a substantial number of cofactors and metabolites intermediates such as ATP, NADPH and citrate, among many others (1). In particular, pyruvate carboxylase (PC) is a fundamental enzyme in redox cycling between NADH and NADPH and also participates in an intricate process known as “pyruvate cycling” which allows the anaplerotic entry of pyruvate in the krebs cycle (2).Pancreatic β-cells express abnormally high levels of pyruvate carboxylase (PC) and insignificant levels of phosphoenolpyruvate carboxylase, the enzyme necessary for gluconeogenesis. This implies that PC must play a different role in β-cells, such as insulin secretion, which is required for the "metabolic switch" from glycolytic to aerobic metabolism during β-cell maturation. It is also known that pyruvate carboxylase activity is elevated in mature β-cells but diminished under diabetic conditions. Recent studies have revealed that the Hypoxia Inducible factor (HIF) pathway plays an important role of in β-cell function (cita algun articulo nuestro). Both overexpression and inactivation of HIF-1α in β cells cause defects in insulin secretion. However, the role of HIF-2α in β-cell formation and function has been largely ignored despite been reported to be activated during diabetic conditions.In this project, we hypothesize that HIF-2α and pyruvate carboxylase activity during late pancreatic HIF-2α formation is critical for the metabolic switch that ocurrs in β-cell during early postnatal development and thus for proper β-cell function.In this study, we will analyze the expression of Epas1 (the gene encoding HIF-2α and Pcx) at different prenatal and postnatal stages by mRNA TaqMan essay. Using Cre/lox technology in mice, we will inactivate Epas1 and Pcx specifically in β-cells. Immunofluorescence and immunohistochemical assays will be carried out to determine specific markers of cell identity, vascularization, proliferation, and polarity in pancreatic tissue of Epas1- and Pcx-deficient mice. Finally, we will also evaluate the in vivo behavior of pancreatic β-cells in transgenic mice through glucose and insulin tolerance assays (GTT and ITT). This will help us understand the relationship between HIF-2 and PC activity and β-cell development and function, as well as whether HIF-2 and PC activity play a role in β-cell failure during diabetes

Development and validation of a method for precise dating of female puberty in laboratory rodents: The puberty ovarian maturation score (Pub-Score)

Puberty is a key developmental event whose primary regulatory mechanisms remain poorly understood. Precise dating of puberty is crucial for experimental (preclinical) studies on its complex neuroendocrine controlling networks. In female laboratory rodents, external signs of puberty, such as vaginal opening (VO) and epithelial cell cornification (i.e., first vaginal estrus, FE), are indirectly related to the maturational state of the ovary and first ovulation, which is the unequivocal marker of puberty. Whereas in rats, VO and FE are almost simultaneous with the first ovulation, these events are not so closely associated in mice. Moreover, external signs of puberty can be uncoupled with first ovulation in both species under certain experimental conditions. We propose herein the Pubertal Ovarian Maturation Score (Pub-score), as novel, reliable method to assess peripubertal ovarian maturation in rats and mice. This method is founded on histological evaluation of pre-pubertal ovarian maturation, based on antral follicle development, and the precise timing of first ovulation, by retrospective dating of maturational and regressive changes in corpora lutea. This approach allows exact timing of puberty within a time-window of at least two weeks after VO in both species, thus facilitating the identification and precise dating of advanced or delayed puberty under various experimental conditions

Molecular analysis of prolactinoma formation in Pten-deficient mice.

Pituitary tumors are abnormal masses developed in the pituitary gland. Although they are generally benign, between 40-50% of pituitary adenomas cannot be removed by surgery alone due to local invasion. Moreover, they are associated with hormonal dysregulation. Prolactinoma is the most common type (50-60%), followed by somatotropic cell adenoma (10-15%), corticotropic cell adenoma (5-10%) and finally thyrotropinoma (less than 1%) (Cano González et al., 2015).Previous descriptive studies have suggested a possible role for the PI3K/AKT/mTOR signaling pathway in the formation of pituitary adenomas. In this study, we used genetic mouse models to assess the oncogenic capacity of this signaling pathway in the pituitary. For this purpose, conditional knockout mice have been generated in which the Pten gene is inactivated specially in the pituitary, indirectly causing the AKT overexpression. To accomplish this, a HesX1-Cre mouse line, whose expression is controlled by a pituitary-specific promoter and which is present in very early stages of embryonic development (Rizzoti, 2015) were crossed with mouse lines in which the Pten gene is floxed by two LoxP sequences.We have analyzed the pituitary in Pten-deficient mice at three different ages: 12, 6 and 1 month of age, comparing genotype and sex. At young ages, Pten-deficient mice show pituitary hyperplasia. After 12 months of age, Pten-deficient mice develop pituitary tumors. However, this is only observed in mutant female mice, whereas male mice simply display pituitary hyperplasia. Data from immunohistochesmistry, immunofluorescence, and blood hormones show that Pten-deficient mice developed prolactinomas. These tumors show high rates of cell proliferation as well as alterations in the expression levels of several cell cycle inhibitors

Educação Permante em Saúde: uma estratégia para capacitação de profissionais da Estratégia de Saúde da Família / Continuing Education in Health: a strategy for training professionals of the Family Health Strategy

O Brasil apresenta uma grande capacidade de desenvolvimento de tecnologias e sabe, principalmente para formação técnico-científica e capacitação no setor de plantas medicinais e fitoterápicos. Desta forma, o trabalho teve como objetivo capacitar profissionais na orientação do uso de plantas medicinais e medicamentos fitoterápicos do Centro de Saúde da Família Agente Comunitária de Saúde Fracinilda Mendes, em Sobral, Ceará. Trata-se de uma pesquisa qualitativa. O estudo aconteceu com todos os profissionais da equipe de saúde através de educação permanente em saúde, realizando-se em três encontros. Com a realização das educações permanentes, obtiveram um maior conhecimento quanto ao uso e terapêutica das plantas medicinais e fitoterápicas, bem como, almeja-se que esta prática complementar possa ter uma maior visibilidade para o município e assim fortalecer as políticas públicas, principalmente as Práticas Integrativas e Complementares no âmbito da Estratégia de Saúde da Família

Educação popular em saúde: desmistificando o pré-natal odontológico em um grupo de práticas corporais / Popular health education: demystifying dental prenatal care in a group of body practices

A educação em saúde bucal possui um forte potencial de modificação do quadro de saúde populacional, através de ações que objetivam a apropriação do conhecimento sobre o processo saúde-doença. O trabalho tem como objetivo relatar uma atividade de educação popular em saúde sobre o período gestacional e o pré-natal odontológico no grupo de práticas corporais do Centro de Saúde da Família Dr. Everton Francisco Mendes Mont’Alverne, no município de Sobral, Ceará. Trata-se de um relato de experiência de abordagem qualitativa descritiva. Através de metodologias participativas e reflexivas, foi possível um diálogo sobre o período gestacional e o pré-natal odontológico com diversas pessoas da comunidade, visando à transformação local na perspectiva de promoção da saúde. Desta maneira as participantes foram ouvidas e se sentiram reconhecidas como parte fundamental no processo de transmissão de saberes relacionado ao pré-natal na comunidade.

Interplay between gonadal hormones and postnatal overfeeding in defining sex-dependent differences in gut microbiota architecture

Aging is associated with a decline in sex hormones, variable between sexes, that has an impact on many different body systems and might contribute to age-related disease progression. We aimed to characterize the sex differences in gut microbiota; and. to explore the impact of depletion of gonacial hormones, alone or combined with postnatal overfeeding, in rats. Many of the differences in the gut microbiota between sexes persisted after gonadectomy, but removal of gonadal hormones shaped several gut microbiota features towards a more deleterious profile, the effect being greater in females than in males, mainly when animals were concurrently overfed. Moreover, we identified several intestinal miRNAs as potential mediators of the impact of changes in gut microbiota on host organism physiology. Our study points out that gonadal hormones contribute to defining sex-dependent differences of gut microbiota, and discloses a potential role of gonadal hormones in shaping gut microbidta, OS consequence of the interaction between sex and nutrition. Our data suggest that the changes in gut microbion, observed in conditions of sex hormone decline, as those caused by ageing in men and menopause in women, might exert different effects on the host organism, which are putatively mediated by gut microbiota-intestinal miRNA cross-talk

Dicer ablation in Kiss1 neurons impairs puberty and fertility preferentially in female mice

Kiss1 neurons, producing kisspeptins, are essential for puberty and fertility, but their molecular regulatory mechanisms remain unfolded. Here, we report that congenital ablation of the microRNA-synthesizing enzyme, Dicer, in Kiss1 cells, causes late-onset hypogonadotropic hypogonadism in both sexes, but is compatible with pubertal initiation and preserved Kiss1 neuronal populations at the infantile/juvenile period. Yet, failure to complete puberty and attain fertility is observed only in females. Kiss1-specific ablation of Dicer evokes disparate changes of Kiss1-cell numbers and Kiss1/kisspeptin expression between hypothalamic subpopulations during the pubertal-transition, with a predominant decline in arcuate-nucleus Kiss1 levels, linked to enhanced expression of its repressors, Mkrn3, Cbx7 and Eap1. Our data unveil that miRNA-biosynthesis in Kiss1 neurons is essential for pubertal completion and fertility, especially in females, but dispensable for initial reproductive maturation and neuronal survival in both sexes. Our results disclose a predominant miRNA-mediated inhibitory program of repressive signals that is key for precise regulation of Kiss1 expression and, thereby, reproductive function.Kiss1 neurons are essential for puberty and fertility. Here, the authors show that canonical microRNA biosynthesis in Kiss1 neurons plays an essential role in the control of puberty and fertility, especially in females, likely via repression of repressors on the Kiss1 gene.</p

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection