Decidualization involves the differentiation of the endometrial tissue into the decidual tissue of the pregnant uterus in several species including humans and rodents. This differentiation occurs only after the onset of implantation in mice and can be artificially-induced causing the formation of deciduomal tissue. The purpose of this study was to identify a group of differentially expressed genes between the developing decidua and deciduoma and study their expression as it may relate to conceptus influenced changes in endometrial gene expression during decidualization. In this study we artificially induced decidualization by transferring blastocyst-sized ConA-coated agarose beads into the uterus on Day 2.5 of pregnancy as we had previously found this model to be more "physiological". Total RNA was isolated from implantation sites of the uteri of pregnant mice as well as pseudopregnant mice that received beads. This RNA was then used for microarray analysis using Mouse Illumina Beadarray chips. This revealed potential differential mRNA levels of over 1,000 genes between the decidua and bead-induced deciduoma tissues of Day 7.5 pregnant and pseudopregnant mice, respectively. Of these, the mRNA levels of 102 genes were 2-fold greater in the decidual tissue while almost twice as many were 2-fold greater in the deciduoma. The broad functions of the protein encoded by the mRNAs included protein binding (e.g. Copz1, Gjb2, Dctn1, Islr, Nisch, Wwc1, Cdc20, Rxrb, Klhl7, Adam10), calcium transport (e.g. Anxa6, Itga11, Clta, Smoc2, Vdr), hydrolase/peptidase activity (e.g. Klk5, Klk26, Klk24, Tmprss4, Ptpn14, Ddx3x, Atp1a2, Usp25, Smarca1), ligase activity (e.g. Iars, Farsla, Ube2v1, Cbll1, Rnf19, Mccc1), and transcription (e.g. Irf1, Hip1, Bhlha15, Supt6h, Scand1, Myocd, Sp3, Mitf, Papolg). We confirmed the differential mRNA levels of a number of gene transcripts using quantitative RT-PCR. Finally, the level and localization of some of the mRNA's identified by our microaray analysis were examined in the mouse uterus during decidualization in more detail and included: Aldh3a1, Bcmo1, Guca2b, GCC, and Inhbb. Localization of mRNA expression in the Day 7.5 implantation site occurred in the mesometrial region near the lumen (Aldh3a1), luminal and glandular epithelia (Guca2b), and endothelial cells lining the sinusoids (Inhbb). This study provides the identity and expression analysis of steady-state mRNA levels of genes whose expression may be influenced by the conceptus using a physiological model for implantation.

One of the critical periods of time for the establishment of pregnancy is after the onset of implantation but before the establishment of the placenta. In fact, evidence strongly suggests that when abnormalities occur during this period of pregnancy in humans there may be dire consequences such as termination of pregnancy, pre-eclampsia later in pregnancy and small for gestational weight offspring that are less healthy in later life. Therefore, what occurs in the endometrium during the progression of implantation can be crucial for proper fetal development later in pregnancy as well as the health of the offspring. One of the evolving ideas coming from research on the biology of what occurs during early pregnancy is the existence of key bi-directional communications between the conceptus and uterus that may be required for successful pregnancy. Amazingly, some common aspects of this signaling might exist to some extent between mammalian species that have very different modes of implantation and where quite different placentas are formed. Although some conceptus-uterine communication during the progression of decidualization is starting to be worked out, we still have much to learn about the precise details of the entire repertoire of the molecular signals responsible. In the rodent we still have to clearly find and define the biology of important paracrine factors produced by the conceptus that target cells of the endometrium during this period of time when the endometrium is undergoing decidualization. This review focuses on some of the approaches that have been used in the past and what has been learned about the effect of the conceptus on the decidualization of the rodent uterus. Where possible, this is compared and contrasted to what is currently known in other species that exhibit quite different modes of implantation.

Blastocyst implantation is the process in which a competent blastocyst acquires the ability to tether into the mother endometrium. At the same time, the endometrial tissue undergoes the process of decidualization to support the anchoring of the blastocyst and provides the blastocyst with nutrition until the fully functional placenta is formed. Although the process of implantation and decidualization are under control of progesterone and estrogen, the precise mechanisms involved in this regulation are not fully understood. Here, we report the expression and function of a transcription factor, HAND2, in sensitizing mouse uterus for implantation and decidualization. In mouse, HAND2 expression was localized mainly in the endometrial stromal cells even before the blastocyst implantation. The expression of HAND2 increased after blastocyst implantation and correlated with the increase in decidual compartment. The expression of HAND2 depended on progesterone but not estrogen. Further investigation using conditional knockout mouse revealed that HAND2 was important for both implantation and decidualization. Hand2d/d mice were infertile and had defects in decidualization. It seemed that HAND2 was an important factor that mediates the anti-estrogenic effect of progesterone on luminal epithelial proliferation. The abnormal in expression of Mucin 1, Calcitonin and E-Cadherin in Hand KO uterus may be responsible for defects in the uterine receptivity. The expression of HAND2 was also critical in decidualization in vitro. Silencing and over-expression HAND2 disclosed the roles of HAND2 in regulating the expression of FOXO1A, IGFBP1, BMP2 as well as WNT4. It seemed that HAND2 promoter worked in tissue specific manner and although both HOXA10 and cAMP binding sites were found in proposed HAND2 promoters, its activity was stimulated by cAMP and steroid hormones rather than the expression of HOXA10.

The function of the endometrium is to mediate implantation of the embryo. During the early stages of implantation, the endometrial stroma undergoes differentiation known as decidualization, a process critical for successful embryo implantation. The precise mechanisms involved are not clearly understood but extracellular matrix (ECM) remodelling is a key feature. Fibronectins (FNs) are large glycoproteins abundant in the ECM of the human endometrium. Up to twenty isoforms of FNs are generated from alternative splicing, including the EDIIIA+ and EDIIIB+ variants. This thesis investigated changes in endometrial stromal ECM levels, in particular FN and its splice variants, during decidualization and in response to the endometrial cytokines and growth factors that drive the implantation process. Furthermore, the influence of these splice variants on the functional properties of FN was explored, including cell attachment, spreading and proliferation, integrin binding and focal adhesion kinase activation. Structural studies including crystallization trials were carried out to investigate how the insertion of EDIIIA modulates the conformation of FN and accessibility of its integrin binding sites. These combined studies allow us to test the hypothesis that the regulation of alternative splicing provides a biological mechanism for modulating function of FN in the endometrium. The main findings from this study can be summarized as follows: Immunocytochemistry and immunoblotting demonstrated reduced endometrial stromal levels of EDIIIA+FN, total FN and tenascin during in vitro decidualization. Substrate-associated FN production by endometrial stromal cells was reduced in response to the endometrial cytokine TNFalpha as detected by ELISA. Recombinant FIII7-12A±B± fragments were expressed, purified and mediated endometrial stromal cell adhesion. Inclusion of EDIIIA in the recombinant FIII7-12 fragment decreased binding affinities to integrin alpha5beta1. These findings suggest that production of FNs in the endometrial stroma is modified during in vitro decidualization and in response to endometrial TNFalpha. This modification in ECM composition is likely to result in modulation of cellular processes, perhaps to allow for cellular differentiation and migration that is required for invasion of the implanting embryo.

Maternal age is a significant risk factor for adverse pregnancy outcomes, and is strongly associated with an increased risk of aneuploidy of the conceptus, as well as a significantly higher frequency of serious pregnancy complications known as the "Great Obstetrical Syndromes", including miscarriage, pre-eclampsia and fetal growth restriction. In the last 40 years average maternal age has increased considerably in many wealthy countries, and in the UK the number of babies born to women aged 35 and over is set to surpass those born to women under 25. The high incidence of aneuploidy in older mothers can be attributed to abnormalities in the oocyte and embryo, however the "Great Obstetrical Syndromes" do not appear to be related to the oocyte and may instead be linked to abnormal development of the placenta. In this thesis, I show that advanced maternal age in the mouse is associated with a drastically increased variability of developmental progression in utero, including developmental delays and growth restriction, severe embryonic abnormalities and higher resorption rates. I find that these embryonic defects are always accompanied by gross morphological and transcriptomic abnormalities in the placenta. Notably, I show that the increased risk of these complications can be rescued by transfer of embryos from aged females to a young surrogate mother, thus implicating the aged maternal uterus as the basis for embryonic and placental defects. Transcriptomic analysis of the decidua compartment in placentas from aged pregnancies revealed abnormal expression of genes involved in the decidualization process, which occurs during early pregnancy and facilitates implantation and development of the conceptus. I show that these defects are already obvious in the peri-implantation window, with endometrial stromal cells from aged females being unable to mount an adequate decidualization response due to a decline in their ability to respond to pregnancy hormones. This blunted decidualization reaction in turn may lead to abnormal development of the placenta. These age-associated decidualization defects are cell-intrinsic and can be recapitulated in vitro. The detected insufficient activation levels and abnormal intracellular distribution of phospho-STAT3, combined with highly variable progesterone receptor expression, may be possible causes of these defects. In addition, I examined the possible effects of ageing on the epigenome as a potential contributor to the decline in endometrial function. My results indicate that ageing of the uterus displays some of the common epigenetic hallmarks of tissue ageing. However, more importantly, decidual cells of aged females exhibit abnormal distributions of the histone modification H3K4me3, and are refractory to the profound DNA methylation remodeling that I find takes place during pregnancy. These age-related changes in the epigenome may underpin, or contribute to, the observed decline in uterine function during pregnancy. Understanding the mechanism underlying these epigenomic and functional changes in the ageing reproductive tract may pave the way for new therapeutic strategies to improve maternal and fetal outcomes of pregnancy in older mothers.

La période de réceptivité endométriale chez l’humain coïncide avec la différentiation des cellules stromales de l’endomètre en cellules hautement spécifiques, les cellules déciduales, durant le processus dit de décidualisation. Or, on sait qu’une transformation anormale des cellules endométriales peut être à l’origine de pertes récurrentes de grossesses. LRH-1 est un récepteur nucléaire orphelin et un facteur de transcription régulant de nombreux évènements relatif à la reproduction et comme tout récepteur, son activation promouvoit l’activité transcriptionnelle de ses gènes cibles. Nous avons déjà montré que LRH-1 et son activité sont essentiels pour la décidualisation au niveau de l’utérus chez la souris et nous savons qu’il est présent dans l’utérus chez l’humain au moment de la phase de prolifération mais aussi de sécrétion du cycle menstruel, et que son expression augmente dans des conditions de décidualisation in vitro. Notre hypothèse est alors la suivante : LRH-1 est indispensable à la décidualisation du stroma endométrial, agissant par le biais de la régulation transcriptionnelle de gènes requis pour la transformation de cellules stromales en cellules déciduales. Afin d’explorer le mécanisme moléculaire impliqué dans la régulation transcriptionnelle effectuée par l’intermédiaire de ce récepteur, nous avons mis en place un modèle de décidualisation in vitro utilisant une lignée de cellules stromales de l’endomètre, cellules humaines et immortelles (hESC). Notre modèle de surexpression développé en transfectant les dites cellules avec un plasmide exprimant LRH-1, résulte en l’augmentation, d’un facteur 5, de l’abondance du transcriptome de gènes marqueurs de la décidualisation que sont la prolactine (PRL) et l’insulin-like growth factor binding protein-1 (IGFBP-1). En outre, la sous-régulation de ce récepteur par l’intermédiaire de petits ARN interférents (shRNA) abolit la réaction déciduale, d’un point de vue morphologique mais aussi en terme d’expression des deux gènes marqueurs cités ci-dessus. Une analyse par Chromatin ImmunoPrécipitation (ou ChIP) a démontré que LRH-1 se lie à des régions génomiques se trouvant en aval de certains gènes importants pour la décidualisation comme PRL, WNT 4, WNT 5, CDKN1A ou encore IL-24, et dans chacun de ces cas cités, cette capacité de liaison augmente dans le cadre de la décidualisation in vitro. Par ailleurs, des études structurelles ont identifié les phospholipides comme des ligands potentiels pour LRH-1. Nous avons donc choisi d’orienter notre travail de façon à explorer les effets sur les ligands liés à LRH-1 de traitements impliquant des agonistes et antagonistes à notre récepteur nucléaire. Les analyses par q-PCR et Western blot ont montré que la modulation de l’activité de LRH-1 par ses ligands influait aussi sur la réaction déciduale. Enfin, des études récentes de Salker et al (Salker, Teklenburg et al. 2010) ont mis en évidence que les cellules stromales humaines décidualisées sont de véritables biocapteurs de la qualité embryonnaire et qu’elles ont la capacité de migrer en direction de l’embryon. La série d’expériences que nous avons réalisée à l’aide de cellules hESC placées en co-culture avec des embryons de souris confirme que la migration cellulaire est bien dirigée vers les embryons. Cette propriété quant à l’orientation de la migration cellulaire est notoirement diminuée dans le cas où l’expression de LRH-1 est déplétée par shRNA dans les hESC. Nos données prouvent donc que LRH-1 régule non seulement la transcription d’un ensemble de gènes impliqués dans le processus de décidualisation mais agit aussi sur la motilité directionnelle de ces cellules hESC décidualisées in vitro. / The period of endometrial receptivity in humans coincides with the differentiation of endometrial stromal cells into highly specialized decidual cells through a process known as decidualization. This transformation of endometrial cells is abnormal in recurrent pregnancy loss patients. Liver homolog receptor 1 (LHR-1, NR5A2) is an orphan nuclear receptor and a transcription factor that regulates many reproductive events. The activation of this receptor leads to transcriptional activation of its target genes. We have previously shown that it is essential for decidualization in the mouse uterus. LRH-1 is expressed in the human uterus in both proliferative and secretory phases of the menstrual cycle and its expression increases during in vitro decidualization. We hypothesize that LRH-1 is indispensable for proper decidualization of the endometrial stroma, acting through the transcriptional regulation of genes required for transformation of stromal cells into decidual cells. To explore the molecular mechanism of transcriptional regulation mediated by this receptor, we established an in vitro model of decidualization, using an immortal human endometrial stromal cell line (hESC). An overexpression model developed by transfecting the cells with a plasmid constitutively expressing Lrh-1, resulted in 5 fold increases in abundance of transcripts for the decidualization marker genes prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Furthermore, the downregulation of the receptor using short hairpin RNA (shRNA) abrogates the decidual reaction, from both a morphological point of view and in terms of expression of the two marker genes. Chromatin immunoprecipitation (ChIP) analysis showed that LRH-1 binds to genomic regions upstream of genes important for decidualization such as PRL, wingless-type MMTV integration site family, member 4 (WNT4), wingless-type MMTV integration site family, member 5 (WNT5), cyclin-dependent kinase inhibitor 1A (p21, CDKN1A) and interleukin-24(IL-24). For each of these genes, the binding increased during in vitro decidualization. Structural studies have identified phospholipids as potential LRH-1 ligands. We therefore explored the effect of ligand treatment on LRH-1 with an agonist and an inverse agonist for the nuclear receptor. Analysis by quantitative polymerase chain reaction (qPCR) and Western blot demonstrated that the modulation of LRH-1 activity by its ligands also affects the decidual reaction. Recent studies have shown that decidualized human stromal cells are biosensors of embryo quality and that they have the capacity to migrate towards the embryo. Our time-lapse evaluation of hESC cells co-cultured with mouse embryos indicates directed migration of the cells toward the embryo. This effect is markedly diminished when LRH-1 is depleted by shRNA in hESC. Our data provide evidence that LRH-1 regulates not only the transcription of a set of genes involved in decidualization but also the directional motility of these cells in vitro.

L’implantation embryonnaire repose sur une synchronisation spatio-temporelle entre un placenta fonctionnel et un endomètre réceptif. La réceptivité endométriale requiert la différenciation des cellules stromales en cellules déciduales sous l’effet des hormones ovariennes (œstrogènes et progestérone). Le placenta est un organe transitoire constitué de deux types cellulaires. Le cytotrophoblaste villeux est responsable des échanges fœtaux maternels et de la fonction endocrine du placenta. Le cytotrophoblaste extravilleux présente des propriétés invasives et assure ainsi l’ancrage du placenta dans l’endomètre maternel. Un dialogue paracrine complexe entre les cellules placentaires et endométriales s’établit au cours des premières étapes de l’implantation.L’adiponectine est une adipokine produite majoritairement par le tissu adipeux. Elle contrôle le métabolisme glucido-lipidique et joue le rôle d’hormone insulino-sensibilisatrice. Dans de nombreux tissus, l’adiponectine exerce des effets anti-prolifératifs, pro-invasifs et pro-différenciants. L’adiponectine et ses récepteurs ADIPOR1 et ADIPOR2 sont présents à l’interface fœto-maternelle. Le placenta et l’endomètre sont donc des tissus cibles de l’adiponectine.Au cours de ce travail, nous nous sommes intéressés aux effets directs de l’adiponectine à l’interface foeto-maternelle au cours du premier trimestre de grossesse.Dans une première partie, nous avons observé que l’adiponectine exerce des effets anti-différenciants et anti-invasifs dans les cellules stromales endométriales.Dans un second temps, nous avons démontré que l’adiponectine favorise la production de glycogène dans les cellules déciduales. Inversement, l’adiponectine semble limiter l’entrée du glycogène dans les cellules placentaires. Ces résultats démontrent que l’adiponectine pourrait contrôler la nutrition histiotrophe du foetus.Dans une dernière partie, nous avons observé que l’adiponectine diminue l’expression des transporteurs de nutriments et exerce une action pro-apoptotique dans le trophoblaste villeux. Ces derniers résultats pourraient permettre de mieux comprendre le rôle de l’adiponectine dans les pathologies placentaires telles que le retard de croissance intra-utérin qui se caractérise par une diminution du poids foetal et une augmentation de l’apoptose des cellules trophoblastiques.L’ensemble de ces résultats montre que l’adiponectine est un acteur clé du dialogue foeto-maternel au cours de la grossesse précoce en contrôlant la maturation d’un endomètre fonctionnel et en régulant les échanges nutritifs transplacentaires. / Embryo implantation requires a spatiotemporal synchronization between a functional placenta and a receptive endometrium. Endometrium receptivity based on the differentiation of stromal cells into decidual cells, under the influence of ovarian hormones (estrogens and progesterone). The placenta is a transient organ composed of two cell types. Villous trophoblast ensures fetal-maternal exchanges and the endocrine functions. Extravillous trophoblast acquire an invasive phenotype resulting in the placenta anchoring in the endometrium. Then, a complexe paracrine dialog between placental cells and endometrial cells is established during the first stages of the embryo implantation.Adiponectin is an adipokine predominantly produced by the adipose tissue. This cytokine has an important role in the control of energy metabolism and displays an insulin-sensitizing action. In some cell types, adiponectin limits proliferation, but promotes invasion and differentiation. Adiponectin and its receptors ADIPOR1 and ADIPOR2, are expressed at the fetal-maternal interface. Thus, endometrium and placenta are adiponectin targets.In this work, we aimed to determine adiponectin direct effects at the human fetal maternal interface during the first trimester of pregnancy.In a first part, we observed that adiponectin limits differentiation and invasion in endometrial stromal cells.In a second part, we showed that adiponectin promotes glycogen production by decidual cells. Conversely, adiponectin seems to limit glycogen uptake by placental cells. These results demonstrate that adiponectin could regulate histiotrophic nutrition to the fetus.In a last part, we demonstrated that adiponectin down-regulates the expression of nutrient transporters and promotes apoptosis in villous trophoblast. These last results could help to better understand the adiponectin roles in some placental pathologies, as intrauterine growth restriction, characterized by a decreased fetal weight and a enhanced trophoblastic apoptosis. Altogether, these results demonstrate that adiponectin is a key regulator of the fetal-maternal dialog by controlling the differentiation of a functional endometrium and by regulating transplacental nutrient exchanges.