SIN001, drug for improvement of the embryonic implantation. Study of endometrial markers in clinical regulatory phase

Motivation:The study of endometrial receptivity has opened a new approach in the world of assisted reproduction, allowing the development of genetic tests, drugs and techniques that increase the rate of “child at home” mainly in couples who are attending to assisted reproduction programmes. Genetic tests about endometrial receptivity allow to evaluate the “window of implantation” (WOI) estimating the best moment for successful embryo transfer, decreasing the maternal factor. Due to the progress of these tests it can be considered if drugs like SIN001, improve endometrial receptivity in clinical phase. Methods:Through the endometrial biopsy before and after the intake of the drug SIN001 of 32 women in a natural cycle, the transcriptome is analysed, after extracting RNA, using quantitative PCR in microfluidics technique (Fluidigm technology). It consists of a dynamic array where the samples and the primers of the genes, in this case 192 markers previously described and related to the endometrial receptivity, are combined in a real-time PCR. A pre-treatment and treatment histological determination can be obtained due to the endometrial biopsy, for the expression and localization of some of the proteins that have been defined as important in endometrial receptivity.Results: In the preclinical in vitro tests obtained with the drug SIN001, the toxicity was dismissed and the adhesion increase was observed in cell culture models and in primary cultures models of endometrial epithelium. In this clinical phase is expected to improve the WOI in women after the intake of the drug and therefore the embryo implantation. In addition, to evaluate the receptive status of the women with the endometrial receptivity test and also it is expected to establish the expression profile of the 192 genes in the transcriptomics, as well as an evaluation of the proteins obtained in immunohistochemistry.Conclusions: Currently there are no drugs improving embryo implantation, this means that the discovery of a substance like SIN001, significantly increase the probability of success in reproduction clinics, since the drug is expected to cover three phases: (i) endometrial preparation; (ii) embryo implantation; (iii) placentation and prevention of spontaneous miscarriage. Therefore, the next steps to scale up the drug to market would be to increase the cohort of the study and studies focusing on the action mechanism of the drug SIN001 in endometrium

Encapsulación de células endometriales para el co-cultivo de embriones

Motivación: En la fecundación in vitro (FIV) se transfiere el embrión el dia 5, en el estadío de mórula-blastocito, para asegurar que se desarrolle correctamente y que haya menor probabilidad de aneuploidias. En pacientes con fallo de implantación, se desarrollo un método seguro, ético y efectivo para el co-cultivo con células epiteliales de endometrio (Simón y cols., 1999). Estos se siguen realizando de rutina en muchas clínicas, pero presentan una serie de desventajas como las contaminaciones bacterianas o fúngicas y la imposibilidad de llevarse a cabo en laboratorios externos que dificultan su trabajo. Este proyecto esta encaminado a implantar un nuevo método en las clínicas por sus ventajas: mejora de la calidad embrionaria, ahorro de tiempo en los laboratorios del FIV, mejora de la calidad del co-cultivo evitando posibles contaminaciones y un abaratamiento del sistema de co-cultivo al no tener que realizar el cultivo en los laboratorios FIV. Métodos: Se toman pequeñas biopsias endometriales mediante una fina cánula procedentes de donantes en los laboratorios FIV. Tras su obtención, se sigue un protocolo para la separación de epitelio y estroma (Simón y cols., 1999), en las que la mitad de las células epiteliales son encapsuladas en alginato cálcico y la otra mitad son sembradas en monocapa. Actualmente, estamos recogiendo los medios de cultivo tanto de las células encapsuladas como de las células en monocapa, los días 2, 4, 8 y 10 tras alcanzar la confluencia una vez han sido sembradas después de la encapsulación. Se analizarán en estos medios la presencia de factores que previamente han sido descritos en medios de cultivo de células epiteliales endometriales como IL-6. Se realizarán ensayos de ELISA para las proteínas secretadas y de Western para las células recogidas. Se correlacionarán los niveles de proteínas existentes en el medio con los medios de cultivo de las células en monocapa. Conclusiones: Tras la obtención y procesamiento de biopsias como se indico anteriormente, se demostró la viabilidad de las células endometriales en capsulas de alginato cálcico. Este sistema ofrece la posibilidad de desarrollar un nuevo método de implantación en las clínicas FIV mejorando los sistemas de co-cultivo actuales

Application of functional genomics to primate endometrium: insights into biological processes

Endometrium is a dynamic tissue that responds on a cyclic basis to circulating levels of the ovarian-derived steroid hormones, estradiol and progesterone. Functional genomics has enabled a global approach to understanding gene regulation in whole endometrial tissue in the setting of a changing hormonal milieu. The proliferative phase of the cycle, under the influence of estradiol, has a preponderance of genes involved in DNA synthesis and cell cycle regulation. Interestingly, genes encoding ion channels and cell adhesion, as well as angiogenic factors, are also highly regulated in this phase of the cycle. After the LH surge, different gene expression profiles are uniquely observed in the early secretory, mid-secretory (window of implantation), and late secretory phases. The early secretory phase is notable for up-regulation of multiple genes and gene families involved in cellular metabolism, steroid hormone metabolism, as well as some secreted glycoproteins. The mid-secretory phase is characterized by multiple biological processes, including up-regulation of genes encoding secreted glycoproteins, immune response genes with a focus on innate immunity, and genes involved in detoxification mechanisms. In the late secretory phase, as the tissue prepares for desquamation, there is a marked up-regulation of an inflammatory response, along with matrix degrading enzymes, and genes involved in hemostasis, among others. This monograph reviews hormonal regulation of gene expression in this tissue and the molecular events occurring therein throughout the cycle derived from functional genomics analysis. It also highlights challenges encountered in using human endometrial tissue in translational research in this context

Early Developing Pig Embryos Mediate Their Own Environment in the Maternal Tract

The maternal tract plays a critical role in the success of early embryonic development providing an optimal environment for establishment and maintenance of pregnancy. Preparation of this environment requires an intimate dialogue between the embryo and her mother. However, many intriguing aspects remain unknown in this unique communication system. To advance our understanding of the process by which a blastocyst is accepted by the endometrium and better address the clinical challenges of infertility and pregnancy failure, it is imperative to decipher this complex molecular dialogue. The objective of the present work is to define the local response of the maternal tract towards the embryo during the earliest stages of pregnancy. We used a novel in vivo experimental model that eliminated genetic variability and individual differences, followed by Affymetrix microarray to identify the signals involved in this embryo-maternal dialogue. Using laparoscopic insemination one oviduct of a sow was inseminated with spermatozoa and the contralateral oviduct was injected with diluent. This model allowed us to obtain samples from the oviduct and the tip of the uterine horn containing either embryos or oocytes from the same sow. Microarray analysis showed that most of the transcripts differentially expressed were down-regulated in the uterine horn in response to blastocysts when compared to oocytes. Many of the transcripts altered in response to the embryo in the uterine horn were related to the immune system. We used an in silico mathematical model to demonstrate the role of the embryo as a modulator of the immune system. This model revealed that relatively modest changes induced by the presence of the embryo could modulate the maternal immune response. These findings suggested that the presence of the embryo might regulate the immune system in the maternal tract to allow the refractory uterus to tolerate the embryo and support its development

Study of the endometrial immune status: a key factor in women infertility

Motivation: The concept of unexplained infertility arises when all known causes of male and female infertility have been discarded. This reproductive disorder affects 40% of all infertile couples and represents 30% of the total cases of female infertility. Currently, several studies approach the problem from an immunological point of view, focusing on cases in which assisted reproduction techniques have failed recurrently. In this immunological aspect, the endometrial tissue takes great relevance, since it can detect as a natural sensor when the organism of a woman is going through a high inflammation or pathologic process. For this reason, studying the immune status of the endometrium becomes an indispensable strategy to determine if the absence of pregnancy in these women can result from a previous health problem detected by this tissue. Methods: Peripheral blood and endometrial biopsy samples from 15 women with unexplained infertility are taken to determine, by flow citometry, the count of NK cells, macrophages, and LB1 cells. In addition, the endometrial biopsy is used to analyze the gene expression profile of 192 genes associated with endometrial receptivity and immune response. This analysis is performed, after RNA extraction from the biopsies, using quantitative PCR in the microfluidics technique (Fluidigm technology), a dynamic matrix where the samples and the primers of the genes are combined in a real-time PCR.Results: If the patients have an immune disorder, it is expected that the levels of immune cells will be altered, detecting whether they present an inflammatory, autoimmune, or immunosenescent profile. In case there is an immunological alteration, the patient would be given a personalized immunomodulatory treatment to regulate this immunological imbalance and thus, increase the chances of pregnancy. To check the effectiveness of this treatment, another immunological study will be performed accompanied by gene expression analysis to check whether aspects such as endometrial receptivity have changed and if the infertility problem has been solved.Conclusions: When the origin of infertility problems remains unknown, 8 out of 10 reproductive failures are associated with an imbalance of the immune system.Specifically, the immune reaction that occurs in the endometrium is key to a successful pregnancy, so it becomes necessary to develop a diagnostic tool that evaluates the endometrial immune status to solve unexplained infertility problems