Sebastián Martínez

Human Endometrial Side Population Cells Exhibit Genotypic, Phenotypic and Functional Features of Somatic Stem Cells

During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for womens cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.

Next-generation manufacturing systems: key research issues in developing and integrating reconfigurable and intelligent machines

Throughout history it has been proved that tendencies in the manufacturing floor are a reflection of the changes in the customers demands. Today, markets increasingly require more customized products, with shorter life cycles. In response, manufacturing systems have evolved from mass production techniques, through flexible automation, and to mass customization to produce ‘one-of-a-kind production’ at mass production costs. This paper argues that manufacturing systems of the next generation will have to incorporate more flexibility and intelligence, evolving towards reconfigurable manufacturing systems. In particular, the concept of ‘intelligence’ becomes more relevant because of the need to maintain effective and efficient manufacturing operations with minimum downtime under conditions of uncertainty. This paper presents a comprehensive literature review of the field of reconfigurable and intelligent machines. The relevant research issues for the development and introduction of reconfigurable machines tools are presented.

Mitochondrial DNA content as a viability score in human euploid embryos: less is better.

OBJECTIVE To investigate the clinical relevance of mitochondrial DNA (mtDNA) content as a viability score in human euploid embryos. DESIGN Retrospective analysis of mtDNA content of transferred euploid embryos. SETTING Reproductive genetics laboratory. PATIENT(S) Single-embryo transfer in 270 patients who underwent preimplantation genetic screening (205 day-3 blastomere biopsies, and 65 day-5 trophectoderm biopsies), and 10 patients with double-embryo transfer (male-female). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Normalized mtDNA content versus nuclear DNA (nDNA) from transferred euploid embryos. RESULT(S) A high mtDNA copy number in euploid embryos is indicative of lower embryo viability and implantation. Using the normalized mtDNA content, we created the mitochondrial score or Mitoscore (Ms). Day-3 embryos with <34 (MsA) had an implantation rate (IR) of 59% (n = 51); those with 34-52 (MsB) had an IR of 44% (n = 52); those with 52-97 (MsC) had an IR of 42% (n = 50); and those with >97 (MsD) had an IR of 25% (n = 52). Embryos with Ms >160 (n = 22) never implanted. Day-5 embryos with <18.19 (MsA) had an IR of 81%; those with 18.19-24.15 (MsB) had an IR of 50% (n = 16); those with 24.15-50.58 (MsC) had an IR of 62% (n = 16); and those with levels >50.58 (MsD) had an IR of 18% (n = 17). Embryos with levels >60 (n = 7) never implanted. CONCLUSION(S) An increased amount of mtDNA in euploid embryos is related to poor implantation potential and may be indicative of reduced metabolic fuel during oocyte maturation. We are implementing Ms in our preimplantation genetic screening platform to prospectively analyze its clinical relevance.

CXCL10 and IL-6 induce chemotaxis in human trophoblast cell lines

The investigation of trophoblast chemoattractive molecules in humans is of high interest for the reproductive field. Current evidence in ruminants demonstrates that CXCL10, formerly the interferon-gamma-inducible protein 10 (IP-10), is a potent chemotactic molecule implicated in the migration of trophoblast cells during early gestation. The aim of this work was to explore the existence of CXCL10/CXCR3 in the human model. Furthermore, chemotaxis assays were performed to demonstrate CXCL10 chemotactic activity in the human trophoblast cell lines JEG-3 and AC-1M88. Surprisingly, the conditioned media from epithelial endometrial cells (EEC) induced the highest trophoblast migration rate. Cytokine and chemokine membrane protein arrays were used to identify the secreted protein profile of EEC-conditioned media, and IL-6 was found to be the most abundant and CXCL13 the second most abundant molecule. Using a chemotaxis assay on AC-IM88, IL-6 antibody blocked the effect of EEC, indicating IL-6 to be an effective chemoattractive factor for trophoblast cells in the human model.

miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization In Vitro

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.

Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome

During embryo implantation, the blastocyst interacts with and regulates the endometrium, and endometrial fluid secreted by the endometrial epithelium nurtures the embryo. Here, we propose that maternal microRNAs (miRNAs) might act as transcriptomic modifier of the pre-implantation embryo. Microarray profiling revealed that six of 27 specific, maternal miRNAs were differentially expressed in the human endometrial epithelium during the window of implantation – a brief phase of endometrial receptivity to the blastocyst – and were released into the endometrial fluid. Further investigation revealed that hsa-miR-30d, the expression levels of which were most significantly upregulated, was secreted as an exosome-associated molecule. Exosome-associated and free hsa-miR-30d was internalized by mouse embryos via the trophectoderm, resulting in an indirect overexpression of genes encoding for certain molecules involved in the murine embryonic adhesion phenomenon – Itgb3, Itga7 and Cdh5. Indeed, this finding was supported by evidence in vitro: treating murine embryos with miR-30d resulted in a notable increase in embryo adhesion. Our results suggest a model in which maternal endometrial miRNAs act as transcriptomic modifiers of the pre-implantation embryo. Summary: Maternal miRNAs are differentially expressed in the human endometrium and are released into the endometrial fluid, suggesting that they may act as transcriptomic modifiers of the pre-implantation embryo.

Human Endometrial CD98 Is Essential for Blastocyst Adhesion

Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window.

Inhibition of Histone Deacetylase Activity in Human Endometrial Stromal Cells Promotes Extracellular Matrix Remodelling and Limits Embryo Invasion

Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion.

Comparison of Cryotip vs. Cryotop for mouse and human blastomere vitrification.

OBJECTIVE Compare the efficiency of two vitrification methods for single-blastomere cryopreservation with mouse or human embryos. DESIGN Experimental prospective controlled study. SETTING Research center. PATIENT(S) Human Blastomeres were obtained after biopsy. INTERVENTION(S) Mouse blastomeres were obtained after diluting the zona pellucida of embryos with Tyrode acid and manual isolation. Individual human blastomeres were biopsied from embryos following established clinical protocols. The modified open Cryotop and classical closed Cryotip vitrification systems were assayed. After thawing, some mouse blastomeres were fixed and analyzed for apoptotic markers annexin V and caspase 3 with the use of immunofluorescence and confocal microscopy. Ultrastructural morphology was examined using electron microscopy. The human blastomere division rate was assessed 24 hours after thawing. MAIN OUTCOME MEASURE(S) Blastomere survival and division rates after thawing, apoptotic markers, and electron microscopy; adhesion and outgrowth rates of human blastomeres. RESULT(S) After thawing, survival rates in mouse blastomeres using Cryotop vs. Cryotip were 38.46% vs. 85.41%, respectively. As expected, thawed morphologically alive blastomeres were classified negative for annexin V and caspase 3, whereas degenerated blastomeres were positive for both. Further, nuclear chromatin was compacted. Survival rates of human blastomeres vitrified with Cryotop were 22.78% vs. 53.77% with Cryotip. Division capabilities were 16.6% and 47.16%, respectively, in Cryotop and Cryotip. CONCLUSION(S) The closed system is more efficient in preserving mouse and human blastomeres in terms of acceptable survival and division rates, and it also complies with European Union directives.

The Lin28/Let-7 System in Early Human Embryonic Tissue and Ectopic Pregnancy

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7–9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans.