Effect of ulipristal acetate on gene expression profile in endometrial cells in culture and in vivo upon post-ovulatory administration in fertile women.

PURPOSE: To analyse the effect of ulipristal acetate (UPA) as emergency contraception (EC) on the gene expression of human endometrial cell line (HEC-1A) and endometrium from fertile women treated with UPA after ovulation. MATERIALS AND METHODS: HEC-1A cells were treated with UPA, and endometrial tissue from four healthy women was collected in cycles before, during and 2 months after post-ovulation pill intake. Ovulation and luteal phase were monitored, and endometrial biopsies were obtained at day LH + 7 in each cycle. In all cases, we analysed the expression profile of 192 genes associated to endometrial receptivity. RESULTS: We observed a significant change in total transcriptomic activity of UPA-treated HEC-1A cells compared to controls. In vivo, we also observed a trend to down-regulation of genes in the UPA-treated cycle that was partially restored in the post-treatment cycle. Altogether, our results supported a partially reversible effect of UPA in gene expression associated with uterine receptivity. CONCLUSIONS: When UPA was administered after ovulation, it seems to induce a down-regulation of the main genes involved in conditioning the endometrium for implantation. This effect is partially restored two months after pill intake. The action of UPA on the endometrium for users of EC should be further investigated.

Milestones of Precision Medicine: An Innovative, Multidisciplinary Overview.

Although the concept of precision medicine, in which healthcare is tailored to the molecular and clinical characteristics of each individual, is not new, its implementation in clinical practice has been heterogenous. In some medical specialties, precision medicine has gone from being just a promise to a reality that achieves better patient outcomes. This is a fact if we consider, for example, the great advances made in the genetic diagnosis and subsequent treatment of countless hereditary diseases, such as cystic fibrosis, which have improved the life expectancy of many of the affected children. In the field of oncology, the development of targeted therapies has prolonged the survival of patients with breast, lung, colorectal, melanoma, and hematological malignancies. In other disciplines, clinical milestones are perhaps less well known, but no less important. The current challenge is to expand and generalize the use of technologies that are central to precision medicine, such as massively parallel sequencing, to improve the management (prevention and treatment) of complex conditions such as cardiovascular, kidney, or autoimmune diseases. This process requires investment in specialized expertise, multidisciplinary collaboration, and the nationwide organization of genetic laboratories for diagnosis of specific diseases.

Using outcome data from one thousand mosaic embryo transfers to formulate an embryo ranking system for clinical use.

OBJECTIVE: To study how the attributes of mosaicism identified during preimplantation genetic testing for aneuploidy relate to clinical outcomes, in order to formulate a ranking system of mosaic embryos for intrauterine transfer. DESIGN: Compiled analysis. SETTING: Multi-center. PATIENT(S): A total of 5,561 euploid blastocysts and 1,000 mosaic blastocysts used in clinical transfers in patients undergoing fertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation (gestational sac), ongoing pregnancy, birth, and spontaneous abortion (miscarriage before 20 weeks of gestation). RESULT(S): The euploid group had significantly more favorable rates of implantation and ongoing pregnancy/birth (OP/B) compared with the combined mosaic group or the mosaic group affecting only whole chromosomes (implantation: 57.2% vs. 46.5% vs. 41.8%; OP/B: 52.3% vs. 37.0% vs. 31.3%), as well as lower likelihood of spontaneous abortion (8.6% vs. 20.4% vs. 25%). Whole-chromosome mosaic embryos with level (percent aneuploid cells) <50% had significantly more favorable outcomes than the ≥50% group (implantation: 44.5% vs. 30.4%; OP/B: 36.1% vs. 19.3%). Mosaic type (nature of the aneuploidy implicated in mosaicism) affected outcomes, with a significant correlation between number of affected chromosomes and unfavorable outcomes. This ranged from mosaicism involving segmental abnormalities to complex aneuploidies affecting three or more chromosomes (implantation: 51.6% vs. 30.4%; OP/B: 43.1% vs. 20.8%). Combining mosaic level, type, and embryo morphology revealed the order of subcategories regarding likelihood of positive outcome. CONCLUSION(S): This compiled analysis revealed traits of mosaicism identified with preimplantation genetic testing for aneuploidy that affected outcomes in a statistically significant manner, enabling the formulation of an evidence-based prioritization scheme for mosaic embryos in the clinic.

Endometrial transcriptome analysis indicates superiority of natural over artificial cycles in recurrent implantation failure patients undergoing frozen embryo transfer.

Little consensus has been reached on the best protocol for endometrial preparation for frozen embryo transfer (FET). It is not known how, and to what extent, hormone supplementation in artificial cycles influences endometrial preparation for embryo implantation at a molecular level, especially in patients who have experienced recurrent implantation failure. Transcriptome analysis of 15 endometrial biopsy samples at the time of embryo implantation was used to compare two different endometrial preparation protocols, natural versus artificial cycles, for FET in women who have experienced recurrent implantation failure compared with fertile women. IPA and DAVID were used for functional analyses of differentially expressed genes. The TRANSFAC database was used to identify oestrogen and progesterone response elements upstream of differentially expressed genes. Cluster analysis demonstrated that natural cycles are associated with a better endometrial receptivity transcriptome than artificial cycles. Artificial cycles seemed to have a stronger negative effect on expression of genes and pathways crucial for endometrial receptivity, including ESR2, FSHR, LEP, and several interleukins and matrix metalloproteinases. Significant overrepresentation of oestrogen response elements among the genes with deteriorated expression in artificial cycles (P < 0.001) was found; progesterone response elements predominated in genes with amended expression with artificial cycles (P = 0.0052).

Effects of adipocyte-secreted factors on decidualized endometrial cells: modulation of endometrial receptivity in vitro

Obesity is defined as an excessive accumulation of adipose tissue that may lead to health complications. Mounting evidence indicates that obesity has a negative impact on fertility. Yet, the link between adipose tissue biology and infertility remains unclear. We aimed to investigate the communication between the adipose tissue and the reproductive system and the importance of this cross talk for the development of a receptive endometrium. To that end, we generated an in vitro model with endometrial and adipocyte cell lines. Sexual hormones, progesterone and estradiol, were used to decidualize endometrial cells and sensitize adipocytes. Decidualization produced a simultaneous increase of adipokine receptors in endometrial cells paralleling changes in their receptivity status. Furthermore, sensitization of 3T3-L1 adipocytes increased mRNA levels of leptin and resistin and decreased the expression of adiponectin and chemerin levels. This was accompanied by increased isoproterenol-induced lipolysis and reduced insulin-stimulated glucose uptake. Lastly, conditioned culture medium of those sensitized adipocytes was used to feed endometrial cells. This treatment resulted in (i) upregulation of genes previously identified as positive regulators of endometrial receptivity, such as leukemia inhibitory factor and glutathione peroxidase 3, and (ii) downregulation of interleukin-15 and mucin1, both genes negatively related with endometrial receptivity. Our results indicate that the endocrine communication between adipose tissue and the reproductive system is bidirectional and stress the importance of the adipose tissue to modulate the reproductive fitness

Effects of adipocyte-secreted factors on decidualized endometrial cells: modulation of endometrial receptivity in vitro.

Obesity is defined as an excessive accumulation of adipose tissue that may lead to health complications. Mounting evidence indicates that obesity has a negative impact on fertility. Yet, the link between adipose tissue biology and infertility remains unclear. We aimed to investigate the communication between the adipose tissue and the reproductive system and the importance of this cross talk for the development of a receptive endometrium. To that end, we generated an in vitro model with endometrial and adipocyte cell lines. Sexual hormones, progesterone and estradiol, were used to decidualize endometrial cells and sensitize adipocytes. Decidualization produced a simultaneous increase of adipokine receptors in endometrial cells paralleling changes in their receptivity status. Furthermore, sensitization of 3T3-L1 adipocytes increased mRNA levels of leptin and resistin and decreased the expression of adiponectin and chemerin levels. This was accompanied by increased isoproterenol-induced lipolysis and reduced insulin-stimulated glucose uptake. Lastly, conditioned culture medium of those sensitized adipocytes was used to feed endometrial cells. This treatment resulted in (i) upregulation of genes previously identified as positive regulators of endometrial receptivity, such as leukemia inhibitory factor and glutathione peroxidase 3, and (ii) downregulation of interleukin-15 and mucin1, both genes negatively related with endometrial receptivity. Our results indicate that the endocrine communication between adipose tissue and the reproductive system is bidirectional and stress the importance of the adipose tissue to modulate the reproductive fitness.

In the secretory endometria of women, luminal epithelia exhibit gene and protein expressions that differ from those of glandular epithelia.

OBJECTIVE: To characterize the transcriptome of luminal epithelia (LE) of fertile secretory endometria and compare the results with those from glandular epithelia (GE). DESIGN: Endometrial samples were collected at 2 and 7 days after initial blood LH surge in separate menstrual cycles. LE were obtained with the use of laser microdissection. mRNA was amplified with the use of linear polymerase chain reaction and hybridized to Agilent 4×44 microarrays. Gene analysis was used to identify differentially expressed mRNAs. Immunohistochemistry was used to assess nine proteins. SETTING: One IVF clinic. PATIENT(S): Seven Caucasian fertile cycling women. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cycle dating with the use of blood endocrinologic markers, microarrays of laser-microdissected LE, immunohistochemical analysis. RESULT(S): One hundred sixty-one (of 401) differentially expressed mRNAs in LE were identified from the metabolism pathway. Increased selective protein expression in LE at 7 days after initial LH surge was observed. LE mRNA expression was the converse of that in GE. The two cell types each had a different significant biologic pathway identified. CONCLUSION(S): Our results introduce a new concept that LE differentially expressed mRNAs are in the converse direction to that of GE, indicating different biologic processes despite the GE being continuous with the luminal monolayer. This probable distinction of biologic roles has not been noted previously. Further investigations must take cognizance of this observation.

Characterization of secreted proteins of 2-cell mouse embryos cultured in vitro to the blastocyst stage with and without protein supplementation.

PURPOSE: To identify the secreted proteins of murine embryos grown in vitro. METHODS: Two-cell mouse embryos (n=432) were randomly allocated to culture to the blastocyst stage in protein-free and in protein-supplemented (3 % BSA) media. Proteins were separated by SDS-PAGE; bands were visualized by coomassie staining, followed by in-gel trypsin digestion and liquid chromatography-tandem mass spectrometry. RT-PCR and confocal microscopy were used to confirm gene/protein expression in blastocysts. RESULTS: Of all individually identified proteins, 34 and 23 were found in embryos cultured without and with BSA, respectively, and 20 were common. Identified proteins having an N-terminal secretory sequence or transmembrane domains located on the extracellular backbone were postulated as secreted proteins. Gene and protein expression for two selected molecules were confirmed. Functional analysis revealed over-represented processes related to lipid metabolism, cyclase activity, and cell adhesion/membrane functions. CONCLUSIONS: This study provided evidence to further characterize secreted proteins by mouse embryos grown from the 2-cell to the blastocyst stage in vitro. Because of homology between murine and human, these results may provide information to be translated to the clinical setting.

Guidelines for the design, analysis and interpretation of 'omics' data: focus on human endometrium.

BACKGROUND: 'Omics' high-throughput analyses, including genomics, epigenomics, transcriptomics, proteomics and metabolomics, are widely applied in human endometrial studies. Analysis of endometrial transcriptome patterns in physiological and pathophysiological conditions has been to date the most commonly applied 'omics' technique in human endometrium. As the technologies improve, proteomics holds the next big promise for this field. The 'omics' technologies have undoubtedly advanced our knowledge of human endometrium in relation to fertility and different diseases. Nevertheless, the challenges arising from the vast amount of data generated and the broad variation of 'omics' profiling according to different environments and stimuli make it difficult to assess the validity, reproducibility and interpretation of such 'omics' data. With the expansion of 'omics' analyses in the study of the endometrium, there is a growing need to develop guidelines for the design of studies, and the analysis and interpretation of 'omics' data. METHODS: Systematic review of the literature in PubMed, and references from relevant articles were investigated up to March 2013. RESULTS: The current review aims to provide guidelines for future 'omics' studies on human endometrium, together with a summary of the status and trends, promise and shortcomings in the high-throughput technologies. In addition, the approaches presented here can be adapted to other areas of high-throughput 'omics' studies. CONCLUSION: A highly rigorous approach to future studies, based on the guidelines provided here, is a prerequisite for obtaining data on biological systems which can be shared among researchers worldwide and will ultimately be of clinical benefit.

MicroRNAs miR-30b, miR-30d, and miR-494 regulate human endometrial receptivity.

MicroRNAs (miRNAs) act as important epigenetic posttranscriptional regulators of gene expression. We aimed to gain more understanding of the complex gene expression regulation of endometrial receptivity by analyzing miRNA signatures of fertile human endometria. We set up to analyze miRNA signatures of receptive (LH + 7, n = 4) versus prereceptive (LH + 2, n = 5) endometrium from healthy fertile women. We found hsa-miR-30b and hsa-miR-30d to be significantly upregulated, and hsa-miR-494 and hsa-miR-923 to be downregulated in receptive endometrium. Three algorithms (miRanda, PicTar, and TargetScan) were used for target gene prediction. Functional analyses of the targets using Ingenuity Pathways Analysis and The Database for Annotation, Visualization and Integrated Discovery indicated roles in transcription, cell proliferation and apoptosis, and significant involvement in several relevant pathways, such as axon guidance, Wnt/ß-catenin, ERK/MAPK, transforming growth factor ß (TGF-ß), p53 and leukocyte extravasation. Comparison of predicted miRNA target genes and our previous messenger RNA microarray data resulted in a list of 12 genes, including CAST, CFTR, FGFR2, and LIF that could serve as a panel of genes important for endometrial receptivity. In conclusion, we suggest that a subset of miRNAs and their target genes may play important roles in endometrial receptivity.

Gene and protein expression signature of endometrial glandular and stromal compartments during the window of implantation.

OBJECTIVE: To map the changes in messenger RNA (mRNA) and protein abundance during the window of implantation in specifically endometrial stromal and glandular epithelial cells obtained using laser microdissection microscopy (LDM). DESIGN: Endometrial samples were collected from two menstrual cycles at 2 and 7 days after first significant rise in blood LH, and separate cell populations were obtained using LDM. A new generation linear polymerase chain reaction (PCR) amplified the mRNA, which were hybridized to both Affymetrix U133 Plus2 and Agilent 4x44K microarrays followed by gene set analysis. Immunohistochemistry assessed protein expression between the two collection times. SETTING: In vitro fertilization clinic. PATIENT(S): Nine Caucasian, fertile, cycling women. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cycle dating using blood markers; microarrays on laser microdissected glands and stroma; dual platform microarray confirmation; immunohistochemical analysis of cell cycle proteins. RESULT(S): The two microarray platforms showed concordance. During the window of implantation, a statistically significant network of 22 mRNA associated with the cell cycle was down-regulated. Immunohistochemistry identified altered localization in stroma. CONCLUSION(S): Microarrays demonstrated glands and stroma have distinct mRNA signatures, each dependent on the day of the cycle. We characterized two compartments of the receptive endometrium with a transcriptomic signature identifying regulation of only the cell cycle. Immunohistochemical analysis of cell cycle proteins identified a signature staining pattern of nuclear relocalization of a group of cyclins of stromal cells, which may be clinically applicable.

Hormonal and molecular characterization of follicular fluid, cumulus cells and oocytes from pre-ovulatory follicles in stimulated and unstimulated cycles.

BACKGROUND: The use of ovarian stimulation, to stimulate a multi-follicular response for assisted reproduction treatments, may force the production of oocytes from follicles that do not reach optimal maturation, possibly yielding oocytes that are not fully competent. The present study aimed to define the follicular environment and oocyte competence of unstimulated pre-ovulatory follicles, to compare it with that of similar-sized stimulated follicles. For this purpose, we analyzed the follicular hormonal milieu, the oocyte meiotic spindle, the embryo development and the cumulus cells gene expression (GE) profiles. METHODS AND RESULTS: The study population was divided in two groups: (i) 42 oocyte donors undergoing unstimulated cycles and (ii) 18 oocyte donors undergoing controlled ovarian stimulation cycles (COS). Follicular fluid was analyzed to quantify the concentrations of estradiol (E2), progesterone (P), FSH, LH, testosterone (T) and androstendione (Δ4). T was higher in the COS group, while Δ4, E2 and LH were significantly higher in unstimulated cycles. The cumulus oophorus cells (CC) surrounding the oocyte were removed and their GE profiles were analyzed with microarrays. There were 18 differentially expressed genes in CC: 7 were up-regulated and 11 were down-regulated in the COS cycles. The microarray was validated by qRT-PCR. The analysis of spindle structure revealed no significant differences between the groups, except for the parameter of length which presented differences. The fertilization ability and embryo morphology on Days 2, 3 and 4 did not show any significant differences between groups. CONCLUSIONS: The use of ovarian stimulation induces changes in the follicular fluid and in CC GE that may affect immune processes, meiosis and ovulation pathways. Although these differences do not seem to relate to early-stage embryo morphology, the implications of some of the molecules, especially ALDH1A2, CTSL and ZNF33B at the CC level, deserve to be addressed in future studies.

A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids.

There is an urgent need to develop optimized experimental models to examine human implantation. These studies aimed to (i) establish a human endometrium-like three-dimensional (3D) culture system, and (ii) examine the attachment of trophoblast-like Jar spheroids to the culture. In the present work, 3D endometrial cultures were constructed with fibrin-agarose as matrix scaffold, and using epithelial and stromal cells from both human primary cultures and established cell lines. An attachment assay between trophoblast cells and the 3D culture was developed. Epithelial cells (cytokeratin(+)) concentrated on top of the matrix forming a monolayer, and stromal cells (vimentin(+)) resided within the matrix, resembling the normal endometrial structure. The capability of primary epithelial cells to form glands spontaneously was observed. Human trophoblast cells (Jar cells) were hCG(+) by immunostaining, allowed to form spheroids, and confirmed to secrete hCG into the medium. Time-dependent experiments demonstrated a high rate of attachment of Jar spheroids to the epithelium, and adhesion was strongly related to the various cell types present in the 3D culture. An architecturally and functionally competent 3D endometrial culture system was established, that coupled with Jar spheroids mimicking trophoblast cells, provides a unique in vitro model for the study of certain aspects of human implantation.

Differential gene expression between inbred Roman high- (RHA-I) and low- (RLA-I) avoidance rats.

Microarray technology was used to explore differences in brain gene expression under basal conditions in two strains of psychogenetically selected rats which differ in anxiety/stress responses, the inbred Roman High-(RHA-I) and Roman Low-(RLA-I) Avoidance rats. Microarray analysis detected 14 up-regulated and 24 down-regulated genes in RLA-I vs. RHA-I rats functionally related to neurobiological processes. The differentially expressed genes CAMKK2, CRHBP, EPHX2, HOMER3, NDN, PRL and RPL6 were selected for microarray validation using qRT-PCR. EPHX2, CAMKK2 (both up-regulated in RLA-I vs. RHA-I rats) and HOMER3 (down-regulated in RLA-I vs. RHA-I rats) showed a similar tendency and fold-change both in microarray and RT-PCR analyses; PRL (up-regulated in RLA-I vs. RHA-I rats), CRHBP and RPL6 (both down-regulated in RLA-I vs. RHA-I animals) showed a similar tendency but a different order of magnitude of change among experiments; finally, NDN was validated neither in tendency nor in magnitude of change.

Adenomyosis does not affect implantation, but is associated with miscarriage in patients undergoing oocyte donation.

OBJECTIVE: To evaluate the effect of adenomyosis on endometrial gene expression and its correlation with clinical outcome. DESIGN: Transcriptomic analysis of the endometrium of women with adenomyosis during the window of implantation. Retrospective matched cohort study of the impact of adenomyosis on oocyte donation (OD) outcome. SETTING: University-affiliated infertility clinic (2005-2009). PATIENT(S): Endometrial samples were analyzed using microarrays in women with adenomyosis and healthy controls. The clinical study included three groups: adenomyosis, endometriosis, and control. INTERVENTION(S): Endometrial biopsies in natural cycles 7 days after the LH peak; controlled ovarian stimulation in donors; ET in recipients after replacement therapy. MAIN OUTCOME MEASURE(S): Differentially expressed genes; implantation, pregnancy, miscarriage, and term pregnancy rates in OD. RESULT(S): There is a similar endometrial gene expression pattern in both the adenomyosis group and controls, and nonparametric tests revealed 34 dysregulated genes in adenomyosis patients but none belonged to the group of window of implantation genes. Implantation in OD did not differ among the three groups. However, miscarriage was significantly higher in the adenomyosis group vs. the adenomyosis + endometriosis and control groups. Term pregnancy rate was also significantly lower in the adenomyosis group compared with others. CONCLUSION(S): Clinical and molecular data show that implantation is not affected by adenomyosis, but the higher miscarriage rates associated with this condition lead to lower term pregnancy rates, indicating a clear negative effect on the final outcome of OD.

Transcriptomic signature of trophoblast differentiation in a human embryonic stem cell model.

Identification of genes involved in trophoblast differentiation is of great interest in understanding cellular and molecular mechanisms involved in placental development and is relevant clinically to fetal development, fertility, and maternal health. Herein, we investigated differentiation of human embryonic stem cells (hESCs) down the trophoblast lineage by culture with bone morphogenetic protein 4 (BMP4) over a 10-day period. Within 2 days, the stemness markers POU5F1 and NANOG were markedly down-regulated, followed temporally by up-regulation of the CDX2, KRT7, HLA-G, ID2, CGA, and CGB trophoblast markers. To understand, on a global scale, changes in the transcriptome during the differentiation of hESCs down the trophoblast lineage, a large-scale microarray analysis was performed. Through whole-genome analysis, more than 3800 genes displayed statistically significant and 2-fold or greater changes in expression during the time course. Of those genes that showed the largest increases, many were involved in processes associated with trophoblast biology; however, novel genes were also identified. Some of them are hypothesized to be associated mainly with extracellular matrix remodeling (e.g., NID2) and cell migration and invasion (e.g., RAB25). Using Ingenuity pathways analysis software to identify signaling pathways involved in trophoblast differentiation or function, we discovered that many genes are involved in WNT/beta-catenin, ERK/MAPK, NFKB, and calcium signaling pathways, suggesting potential roles for these families in trophoblast development. This work provides an in vitro functional genomic model with which to identify genes involved in trophoblast development.

A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature.

OBJECTIVE: To create a genomic tool composed of a customized microarray and a bioinformatic predictor for endometrial dating and to detect pathologies of endometrial origin. To define the transcriptomic signature of human endometrial receptivity. DESIGN: Two cohorts of endometrial samples along the menstrual cycle were used: one to select the genes to be included in the customized microarray (endometrial receptivity array [ERA]), and the other to be analyzed by ERA to train the predictor for endometrial dating and to define the transcriptomic signature. A third cohort including pathological endometrial samples was used to train the predictor for pathological classification. SETTING: Healthy oocyte donors and patients. PATIENT(S): Healthy fertile women (88) and women with implantation failure (5) or hydrosalpinx (2). INTERVENTION(S): Human endometrial biopsies. MAIN OUTCOME MEASURE(S): The gene expression of endometrial biopsies. RESULT(S): The ERA included 238 selected genes. The transcriptomic signature was defined by 134 genes. The predictor showed a specificity of 0.8857 and sensitivity of 0.99758 for endometrial dating, and a specificity of 0.1571 and a sensitivity of 0.995 for the pathological classification. CONCLUSION(S): This diagnostic tool can be used clinically in reproductive medicine and gynecology. The transcriptomic signature is a potential endometrial receptivity biomarkers cluster.

The bone marrow-derived human mesenchymal stem cell: potential progenitor of the endometrial stromal fibroblast.

The cellular sources that contribute to the renewal of human endometrium are largely unknown. It has been suggested that endometrial stem cells originate from bone marrow-derived mesenchymal stem cells (MSC), with subsequent development into endometrial stromal fibroblasts (hESF). We hypothesized that if bone marrow-derived MSC contribute to endometrial regeneration and are progenitors of hESF, their treatment with agents known to regulate hESF differentiation could promote their differentiation down the stromal fibroblast lineage. To this end, we treated bone marrow-derived MSC with estradiol and progesterone, bone morphogenetic protein 2 (BMP2), and activators of the protein kinase A (PKA) pathway and investigated specific markers of hESF differentiation (decidualization). Furthermore, we investigated the transcriptome of these cells in response to cAMP and compared this to the transcriptome of hESF decidualized in response to activation of the PKA pathway. The data support the idea that MSC can be differentiated down the hESF pathway, as evidenced by changes in cell shape and common expression of decidual markers and other genes important in hESF differentiation and function, and that bone marrow-derived MSC may be a source of endometrial stem/progenitor cells. In addition, we identified MSC-specific markers that distinguish them from other fibroblasts and, in particular, from hESF, which is of biologic relevance and practical value to the field of endometrial stem cell research.

The protein kinase A pathway-regulated transcriptome of endometrial stromal fibroblasts reveals compromised differentiation and persistent proliferative potential in endometriosis.

Intrinsic abnormalities in transplanted eutopic endometrium are believed to contribute to the pathogenesis of pelvic endometriosis. Herein we investigated transcriptomic differences in human endometrial stromal fibroblasts (hESFs) from women with (hESF(endo)) vs. without (hESF(nonendo)) endometriosis, in response to activation of the protein kinase A (PKA) pathway with 8-bromoadenosine-cAMP (8-Br-cAMP). hESF(nonendo) (n = 4) and hESF(endo) (n = 4) were isolated from eutopic endometrium and treated +/- 0.5 mm 8-Br-cAMP for 96 h. Purified total RNA was subjected to microarray analysis using the whole-genome Gene 1.0 ST Affymetrix platform. A total of 691 genes were regulated in cAMP-treated hESF(nonendo) vs. 158 genes in hESF(endo), suggesting a blunted response to cAMP/PKA pathway activation in women with disease. Real-time PCR and ELISA validated the decreased expression of decidualization markers in hESF(endo) compared with hESF(nonendo). In the absence of disease, 8-Br-cAMP down-regulated progression through the cell cycle via a decrease in cyclin D1, cyclin-dependent kinase 6, and cell division cycle 2 and an increase in cyclin-dependent kinase inhibitor 1A. However, cell cycle components in hESF(endo) were not responsive to 8-Br-cAMP, resulting in persistence of a proliferative phenotype. hESF(endo) treated with 8-Br-cAMP exhibited altered expression of immune response, extracellular matrix, cytoskeleton, and apoptosis genes. Changes in phosphodiesterase expression and activity were not different among experimental groups. These data support that eutopic hESF(endo) with increased proliferative potential can seed the pelvic cavity via retrograde menstruation and promote establishment, survival, and proliferation of endometriosis lesions, independent of hydrolysis of cAMP and likely due to an inherent abnormality in the PKA pathway.

Controlled ovarian stimulation induces a functional genomic delay of the endometrium with potential clinical implications.

CONTEXT: Controlled ovarian stimulation induces morphological, biochemical, and functional genomic modifications of the human endometrium during the window of implantation. OBJECTIVE: Our objective was to compare the gene expression profile of the human endometrium in natural vs. controlled ovarian stimulation cycles throughout the early-mid secretory transition using microarray technology. METHOD: Microarray data from 49 endometrial biopsies obtained from LH+1 to LH+9 (n=25) in natural cycles and from human chorionic gonadotropin (hCG) +1 to hCG+9 in controlled ovarian stimulation cycles (n=24) were analyzed using different methods, such as clustering, profiling of biological processes, and selection of differentially expressed genes, as implemented in Gene Expression Pattern Analysis Suite and Babelomics programs. RESULTS: Endometria from natural cycles followed different genomic patterns compared with controlled ovarian stimulation cycles in the transition from the pre-receptive (days LH/hCG+1 until LH/hCG+5) to the receptive phase (day LH+7/hCG+7). Specifically, we have demonstrated the existence of a 2-d delay in the activation/repression of two clusters composed by 218 and 133 genes, respectively, on day hCG+7 vs. LH+7. Many of these delayed genes belong to the class window of implantation genes affecting basic biological processes in the receptive endometrium. CONCLUSIONS: These results demonstrate that gene expression profiling of the endometrium is different between natural and controlled ovarian stimulation cycles in the receptive phase. Identification of these differentially regulated genes can be used to understand the different developmental profiles of receptive endometrium during controlled ovarian stimulation and to search for the best controlled ovarian stimulation treatment in terms of minimal endometrial impact.