- Email: [email protected]
Human endometrial receptivity: a genomic approach
RBMOnline - Vol 6. No 3. 332–338 Reproductive BioMedicine Online; www.rbmonline.com/Article/784 on web 12 February 2003
Articles Human endometrial receptivity: a genomic approach
Dr Carlos Simón
Carlos Simón, MD, is Professor in the Obstetrics and Gynaecology Department at Valencia University and Scientific Director of the Instituto Valenciano de Infertilidad (IVI). He was post-doctoral research fellow at Stanford University, CA, USA from 1991 to 1994. Research in his laboratory focuses on human embryonic implantation and uterine receptivity, leading to the publication of over 100 per review papers and six books. Dr Simón has received awards from the Spanish Fertility Society in 1991, the Society for Gynecological Investigation (SGI) in 1993 and the Society of Reproductive Endocrinologist of the American Society of Reproductive Medicine (ASRM) in 1993, 1995, 1997 and 1999. He is a member of the Editorial Board of several leading journals in the field as well as being an ad hoc reviewer for several more. Scientific international appointments include membership of the HRP Scientific and Ethical Review Group of the World Health Organization, the International Committee of the Society of Gynecological Investigation, and the FIGO expert advisory panel on reproductive medicine.
F Domínguez1, J Remohí 1,2, A Pellicer1,2, C Simón1,2,3 1Instituto Valenciano de Infertilidad Foundation (FIVIER), Plaza de la Policia Local 3, 46015, Valencia. Spain 2Department of Pediatrics, Obstetrics and Gynecology, School of Medicine. Valencia University, Valencia, Spain. 3Correspondence: Tel: +34 963624555; Fax: +34 963694735; e-mail: [email protected]
Abstract The endometrium is a specialized tissue, hormonally-regulated, that is non-adhesive for embryos throughout most of the menstrual cycle in humans and other primates. Thus, endometrial receptivity is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status. The luminal EE acquires the ability to adhere (receptivity) the developing human blastocyst during this period due mainly to the presence of progesterone after appropriate 17β-oestradiol priming. This status is a key element for embryonic implantation and appears to be closely associated with morphological and biochemical changes of EE cells. This specific time window is thought to be open after 4–5 days and closes after 9–10 days of progesterone production or administration, creating a physiological window of receptivity limited to days 19–24 of the menstrual cycle in humans. The scientific knowledge of the endometrial receptivity process is fundamental for the understanding of the human reproduction, but, so far, none of the proposed biochemical markers for endometrial receptivity have been proved clinically useful. In this work new strategies are presented based on molecular biology technologies that aim to clarify the fragmented information in this field using differential display, quantitative PCR and cDNA microarray analysis of endometrial epithelial-derived cell lines and endometrial samples to investigate the hierarchy at the mRNA level of molecules implicated in the process of endometrial receptivity.
Keywords: cDNA array, endometrial receptivity, implantation, microarrays
Introduction
332
Endometrial receptivity is a self-limited period in which the endometrial epithelium acquires a functional and transient ovarian steroid-dependent status allowing blastocyst adhesion (Psychoyos, 1973). In humans, the luminal endometrial epithelium acquires this status simultaneously with the development of the decidualization process in the stromal compartment (Popovici et al., 2000) due mainly to the presence of progesterone after appropriate 17β-oestradiol priming.
to days 19–24 of the menstrual cycle in humans (Navot et al., 1991) and between 8–10 days post ovulation in other primates (Fazleabas, 1999). Indeed, the administration of progesterone antagonist (Hegele-Harting et al., 1992; Banaszak et al. 2000) or oestradiol antiserum (Ravindranath and Moudgal, 1990) during the pre-implantation period disrupted endometrial receptivity in primates. Using this concept of oestradiol and progesterone priming, a clinical endometrial receptivity window is induced routinely in ovum donation programmes to synchronize the timing of embryo transfer (Remohí et al., 1997).
This period, termed the ‘window of implantation’, occurs 4–5 days after, and closes 9–10 days after, progesterone production or administration. Therefore, the receptive window is limited
Steroids acting through their nuclear receptors in the endometrial epithelial cells (EEC) induce the acquisition of a receptive phenotype. EEC suffers structural and functional
Articles - Genomic approach to endometrial receptivity - C Simón et al.
changes. The morphological changes include modifications in the plasma membrane (PM) (Murphy, 2000) and cytoskeleton (Martin et al., 2000). The apical PM develops transitional adhesive properties by undergoing structural changes; long thin, regular microvilli are gradually converted into irregular, flattened projections and this process is named as the plasma membrane transformation. The remodelling of the epithelial organization, from a polarized to a non-polarized phenotype might prepare the apical pole for cell-to-cell adhesion (Thie et al., 1995). This PM remodelling requires the participation of the underlying cytoskeleton, and a relevant function of the Ezrin family in the regulation of cell polarity to change from the non-adhesive to the adhesive status has been suggested (Martin et al., 2000). These changes occur with the complicity of the decidualization process that occurs in the stromal compartment and at the endometrial vasculature. A number of
biochemical markers for endometrial receptivity have been proposed over the years (Irwin et al., 1989) and more recently (Giudice, 1999) although none of them have been proved clinically useful. The scientific knowledge of the endometrial receptivity process is fundamental for the understanding of the mechanism that governs embryonic implantation and human reproduction. Recent advances in molecular biology and gene technology as well as the sequence of the human genome, invite us to re-consider the endometrial receptivity process from a genomic perspective. However, a hierarchical perspective of the genes modified during this relevant process in humans is still lacking. In this review current data are presented using molecular biology technologies that aim to clarify the fragmented information in this field.
Figure 1. Schematic diagram of cDNA array protocol.
333
Articles - Genomic approach to endometrial receptivity - C Simón et al.
Data are presented that were obtained using three different scientific approaches: first, macroarray technology which allows us to analyse the expression pattern of 300–400 genes; second, microarray technology that provides us with a hierarchical global view of genes up- or down-regulated during human endometrial receptivity by analysing simultaneously the expression pattern of 12,000 genes in prereceptive (LH+2) versus receptive (LH+7); and finally, to establish a possible differential gene expression pattern for novel gene discovery, differential display-PCR analysis (DDPCR) has been used.
Macroarray analysis of cytokines, chemokines and growth factors genes in pre-receptive versus receptive human endometrium combined with specific cell lines Profiling RNA on cDNA arrays provides a method to screen the hierarchical contribution of the genes investigated in a given situation. Human endometrial receptivity is characterized by two main features: first, it occurs in a specific period of time in which this tissue acquires a differential functional and transient status, and second, this new status is characterized by the development of adhesiveness to the blastocyst. Therefore, this approach was used to profile the similarity between the differential transcriptional response in two related models: (i) receptive (LH+7) versus pre-receptive (LH+2) human endometrium and (ii) human endometrial cell lines with higher (RL95–2) and lower (HEC-1-A) adhesiveness to trophoblast-derived cells (JAR) cells and mouse blastocysts.
334
The human endometrial cell line RL95–2 is an epithelial cell line derived from a moderately-differentiated endometrial adenocarcinoma (Way et al., 1983) with specific morphological and biological characteristics (Tinel et al., 2000). This cell line exhibits more pronounced adhesiveness for JAR cells (John et al., 1993) and mouse blastocysts (Martin et al., 2000) than any other human EEC line, including HEC-1-A and primary epithelium. The HEC-1-A cell line, in contrast, has poor adhesive properties, and exhibits a polarized distribution of integrins, meanwhile RL95–2 cell line show atypical features in adherins junctions, with non-polarized actin cytoskeleton and integrin distribution (Thie et al., 1996). Embryonic adhesion experiments using mouse blastocysts demonstrate that RL95–2 and HEC-1-A cells showed pronounced receptive and nonreceptive phenotypes (81 versus 46% of blastocyst adhesion, respectively), compared with an intermediate adhesion rate present in primary EEC cultured on extracellular matrix, (67% of blastocyst adhesion) (Martin et al., 2000). Therefore, these cell lines have been used as in vitro models for higher receptivity (RL95–2) and lower receptivity (HEC-1-A). Using both models, the expression pattern of 375 human genes have been differentially analysed; these include cytokines, chemokines, adhesion molecules, and their receptors (Human Cytokine Expression Array, R and D). Briefly, human endometrial samples were obtained in the luteal phase at LH+2 and LH+7 days. A portion of each specimen was dated according to the criteria of Noyes et al. (1950). Total RNA was extracted from whole endometrial biopsies treated with 5µl of DNase I and cDNA probes were synthesized from purified total RNA using [α32-P] dATP, dCTP, dGTP and dTTP. After pre-hybridization during 30 min the probes were hybridized overnight with the cDNA array filters (Figure 1). The hybridization was performed three times with different LH+2 and LH+7 samples.
Figure 2. A. cDNA array of pre-receptive (LH+2) and receptive (LH+7) endometrium. B. cDNA array of RL95–2 and HEC-1A. Hybridization spots of the major up-regulated genes in both states are shown.
Articles - Genomic approach to endometrial receptivity - C Simón et al.
These results indicate that seven genes were up-regulated more than two-fold and four genes were down-regulated in LH+7 versus LH+2 (Dominguez et al., 2003). Among the list of regulated genes, genes were identified that were already known to be differentially expressed during the receptive phase compared with the pre-receptive phase such as PP14, osteopontin, integrin α3, and IL-1RtI (Figure 2). However, a number of genes were also identified for which the differential expression between the pre-receptive (LH+2) and the receptive (LH+7) endometria or even the presence in human endometrium has not been described before. These genes can be classified into different groups such as: extracellular matrix proteins (decorin), heparin-binding molecules (pleiotrophin), genes related to tyrosine kinases (EFNA2) and growth factors (BMP-7). Other genes well studied in human endometrium are upregulated in the receptive endometrium such as protease inhibitors (TIMP1, 2 and 3), matrix metalloproteinase 13 (MMP-13), insulin growth factor II (IGFII) and others. Only four genes were minimally down-regulated in the receptive endometrium. These genes were two interleukin receptors (IL15Rα and IL-9R), bone morphogenetic protein 7 (BMP-7) (a growth factor of the TGFβ family), and ephrin-A2 (a tyrosine kinase ligand) (Figure 2). The same experiments have been repeated using adhesive versus non-adhesive cell lines. The comparative results obtained after the cDNA array hybridization of the endometrial cell lines, HEC-1-A versus RL95–2 shows that the two highly expressed genes in RL95–2 cell line were neurite growthpromoting factor 2 (NEGF2/midkine) and IGFBP-rP1. The rest of the up-regulated genes were chemokines (GRO oncogene 1 and 2), growth factor receptors (erbB1 and TNFRSF16) and growth factors (TGFα) (Figure 2). The group of adhesion molecules appears minimally up-regulated and includes EpCAM, integrins β1, α4 and α1. Remarkably IGFBP-rP1 was the second most up-regulated gene in the two models investigated, receptive versus prereceptive endometria and in high-adhesive versus lowadhesive cell lines respectively. RT-PCR was performed using the same RNA of both cell lines and biopsies to confirm the RNA expression of the cDNA arrays. IGFBP-rP1 expression in both models exhibit a clear trend similar to the results obtained in the cDNA arrays. Further corroboration of these findings was obtained by quantitative fluorescent RT-PCR (QF-PCR) and immunohistochemistry, the expression pattern and distribution of IGFBP-rP1 in the human endometrium across the menstrual cycle conforming a profile consistent with a marker of endometrial decidualization (Dominguez et al., 2003). Although the functional proof of the concept remains to be demonstrated, this work strongly suggests that IGFBP-rP1, also known as IGFBP-7 or mac25, might be implicated in human endometrial receptivity. It is unlikely that the possible role that this molecule may play in endometrial receptivity was as IGFBP due to its low affinity for IGF. However, several independent functions in vascular biology have been attributed to this molecule that may be involved in endometrial receptivity. IGFBP-rP1 has been reported as prostacyclin-
stimulating factor (PSF) in vascular endothelial cells (Yamauchi et al., 1994) and to contribute to the organization of new capillary vessels in tumour tissues by modulating the interaction of endothelial cells with type IV collagen (Akaogi et al., 1996). IGFBP-rP1 contains an amino-terminal domain with homology to IGFBP that is responsible for low-affinity binding to IGF. This sequence is followed by a follistatin-like module that has a low but significant homology with the cysteine-rich follistatin-like module of hevin/SC1, which is known to mediate cytokine binding in other proteins. These two modules in IGFBP-rP1 are likely to be involved in growth factor binding and may facilitate the retention of growth factors or chemokines (Gunn et al., 1998). This molecule has also been defined as tumour-derived adhesion factor (TAF, recently renamed angiomodulin) (Akaogi et al., 1994). At the ultrastructural level, a noteworthy feature associates IGFBPrP1 with microvillous structures that constitute the initial point of contact between the endothelium and the adherent lymphocytes. It has been demonstrated that this molecule may harbour chemokines (Middleton et al., 1997) and adhesion molecules (Girard et al., 1999), suggesting that this molecule may contribute to the lymphocyte migration through high endothelial venules endothelial cells (HEVEC). Therefore, in addition to its vascular functions, these properties indicate that this molecule may be a good candidate for the presentation of adhesion-triggering cytokines to the lymphocytes rolling on, or migrating across, in and through the human endothelial epithelium, having this process remarkable similarity with the implantation process.
Microarray analysis of human receptivity The gene expression studies with microarray technology use a more global strategy in which more than 10,000 genes are analysed in only one experiment. During this year three studies have investigated the differential gene expression pattern between a pre-receptive and receptive endometrium using microarray technology with different study designs and results. Kao et al. (2002) have investigated global gene expression during the window of implantation in human endometrial biopsies timed to the LH surge, compared with the late proliferative phase of the menstrual cycle. Statistical analysis revealed 156 significantly up-regulated genes and 377 significantly down-regulated genes in the implantation window. Up-regulated genes included those for cholesterol trafficking and transport (apolipoprotein (ApoE) being the most induced gene, 100-fold), prostaglandin (PG) biosynthesis (PLA2) and action (PGE2 receptor), proteoglycan synthesis (glucuronyltransferase), secretory proteins (glycodelin), IGF binding protein (IGFBP), and TGFβ superfamilies, signal transduction, extracellular matrix components (osteopontin, laminin), neurotransmitter synthesis (monoamine oxidase) and receptors (γ aminobutyric acid A (GABA) receptor π subunit), numerous immune modulators, detoxification genes (metallothioneins), and genes involved in water and ion transport (CPE1-R). Down-regulated genes included intestinal trefoil factor (ITF) (the most repressed gene, 50-fold), matrilysin, members of the G protein-coupled receptor signalling pathway, frizzled-related protein (FrpHE, a Wnt
335
Articles - Genomic approach to endometrial receptivity - C Simón et al.
Table 1. Comparative results by families between genes up- and down-regulated in the receptive phase with a fold change greater than 3.0 in the studies by Kao et al. (2002) (n = 60 up-regulated and n = 87 down-regulated), Carson et al. (2002) (n = 35 upregulated and n = 39 down-regulated) and Riesewijk et al. (2003) with the same criteria. In those genes in which the accession number is different, both codes are indicated. Boldface type indicates genes identified in all three studies. Family/Accession number
Up-regulated genes comparison Secretory protein AB020315 Transporter AB000712 Extracellular matrix/Cell adhesion molecules AF052124 Other cellular functions J02611 Down-regulated genes comparison Vasoactive substance J05081 Other cellular functions U79299
Gene name
Riesewijk et al. (2003)
Kao et al. (2002)
Carson et al. (2002)
Dickkopf/DKK1 (hdkk-1) Claudin 4/CEP-R Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) Apolipoprotein D
+
+
+
+ +
+ + J04765
+ + J04765
+
+
+
Endothelin 3 (EDN3) Olfactomedin-related ER localized protein
+ +
+ +
+ + X52001
antagonist), transcription factors, TGFβ-signalling pathway members, immune modulators (major histocompatibility complex class II subunits) and other cellular functions. To validate the data, two approaches were used, first RT-PCR and Northern blot analysis using RNA isolated from late proliferative and window of implantation endometrial tissue samples and second RT-PCR using cultured human endometrial glandular and stromal cells. RT-PCR data demonstrate clearly the up-regulation of IGFBP-1, glycodelin, Dkk-1 and other genes in the implantation window. Following Northern blot analysis, the mean values of relative expression were calculated for each mRNA on each blot confirming the up-regulation of Dkk-1, IGFBP-1, glycodelin and other genes. RT-PCR experiments using RNA from cultured endometrial epithelial cells revealed that glycodelin, Dkk-1, GABAA receptor, mammaglobin and others were all high expressed in these cells. CPE-1R, Dkk-1, ApoD and IGFBP-1 were all upregulated in stromal cells.
336
Other recent work based on microarray technology compared the expression of the early luteal (pre-receptive) and mid-luteal phase (receptive) (Carson et al., 2002). In this study more than 12,000 genes have been analysed using the hybridization chip from Affimetrix. Comparing pre-receptive versus receptive endometria, 370 genes decrease from 2- to 100-fold while 323 genes were up-regulated ranging from 2- to 45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change between the proliferative and mid-luteal phases. In addition, novel genes were identified in this work: genes encoding cell surface receptors, adhesion and extracellular matrix proteins, and growth factors. Northern blot analysis was performed confirming the up-regulation of claudin 4 or osteopontin gene in receptive endometrium. Immunohistochemical studies of prostaglandin receptor EP1 and leukotriene B4 receptor were performed to localize these proteins in human endometrium. Claudin 4 strongly stained both luminal and glandular epithelia but not in the stroma.
The authors and their group, in collaboration with Organon International, have investigated the gene expression pattern of human endometrial receptivity using a hybridization chip from Affimetrix containing 12,000 genes (Riesewijk et al. 2003). Comparison of the gene expression pattern of receptive (LH+7) versus pre-receptive (LH+2) endometria in the same women reveals a consistent pattern of differentially expressed genes. Identification of 220 regulated genes and 12 expressed sequence tags (EST) has been made. In total, 172 of these were specifically up-regulated in the LH+7 samples whereas 60 of them were down-regulated. Using more stringent criteria of regulation in all five patients, only 83 genes were identified as being up-regulated at least 3fold in all five patients at LH+7 whereas 10 genes were downregulated. In the list of regulated genes, genes were identified that were already known to be differentially expressed during the receptive phase, compared with the pre-receptive phase, such as glycodelin, osteopontin, IGFBP-3, crystallin αB and integrin α3 (Riesewijk et al. 2003). However, a number of genes were also identified for which the differential expression between the pre-receptive (LH+2) and the receptive (LH+7) endometria or even the presence in human endometrium has not been described before. These genes can be classified into different groups such as: immune modulatory genes, adhesion molecules, genes related to oxidative stress, cytoskeletal proteins and others. Further corroboration was obtained by investigation of the expression pattern of three differentially up-regulated genes. Moreover, for these genes a role in human endometrial receptivity had not been previously described. The selected genes were GPx-3, (glutathione peroxidase 3), CEPR (Claudin 4) and SLC1A1 (neuronal/epithelial high affinity glutamate transporter, system Xag member 1). Comparison of the up- or down-regulated genes in these three studies is shown in Table 1. Here the up- or down-regulated genes found in this study that match the first 50 genes found in
Articles - Genomic approach to endometrial receptivity - C Simón et al.
Figure 3. A. General view of the differential display 4.5% denaturing gel; genes associated to the receptive phenotype are displayed. Currently 60 differentially-expressed bands have been purified, both qualitatively and/or quantitatively. B, C. Re-amplification PCR were performed to obtain sufficient amount of DNA for cloning. Approximately 25% of them have been cloned and sequenced. the other two studies are represented. Genes present in the three papers are shown in bold. The mouse has become an indispensable model organism for the study of endometrial receptivity and implantation. Nevertheless, the comparison of the present study with elegant microarray-based studies in the mouse (Reese et al., 2001; Yoshioka et al., 2000) indicates the existence of important differences in the genomics of endometrial receptivity and implantation between human and mouse. Firstly, there are few genes that were mutually identified in these two models and more importantly, genes functionally crucial for implantation in mice such as LIF (Stewart et al., 1992) or COX-2 (Lim et al., 1997), as demonstrated by the different knockout models, were not detected as regulated genes in this human study. Even more intriguing is the fact that these genes were not detected in the mouse model during implantation using a similar genome-wide approach (Reese et al., 2001). As the authors pointed out this may be due to highly spatially restricted expression around the implanting blastocyst. It should be mentioned that, in the human, the timing is not as restricted as it is in the mouse with a window of receptivity of approximately 3 days.
Discovery of new genes associated to endometrial receptivity using differential display-PCR. Finally, another approach to isolate and characterize receptivity-related genes was the employment of the differential display associated to PCR (DD-PCR) using cDNA preparations from RL95–2 and HEC-1-A cells, which shows distinct patterns of gene expression (Martin et al., 2000). For this purpose total RNA was extracted and treated with DNase I. To obtain the cDNA from cell lines reverse transcription was performed using [α-33P] dATP, 12 oligo(dT) anchored 3′ primers (AP) individually with aliquots of each total RNA sample in combination with four arbitrary 5′ primers (ARP).
Following DD-PCR, radiolabelled cDNA fragments were electrophoretically separated on a polyacrylamide gel under denaturing conditions. (Figure 3a) For the re-amplification and cloning of differentiallyexpressed cDNA fragments the autoradiogram was superimposed on the dried gel to locate the cDNA fragments of interest. Differential bands were excised, eluted and reamplified. (Figure 3b) The obtained products were ligated to the cloning vector (pGEM-T) after Not I/Nco I digestion. Positive colonies were used to purify the DNA. After this cDNA fragments were purified and sequenced. To date, 60 differentially-expressed bands have been purified by the authors, both qualitatively and/or quantitatively. Currently, work is progressing to sequence and identify these bands. Most of them are known genes but possibly some bands obtained may correspond to unknown genes related to receptivity. Current work involves identification of the genes by database analysis before going forward with functional analysis. This approach should lead to improved understanding of endometrial receptivity and to the optimization of treatment of infertile patients or to its modification as an interceptive procedure.
Conclusion Studies previously published, including this study, have been performed on complete endometrial tissue but nowadays there is no available information of the specific contribution to the general gene pattern from different compartments of the human endometrium such as stromal cells, glandular or luminal epithelium, etc. So it is suggested that further experiments following this direction should be performed to unravel the answers to these questions.
References Akaogi K, Okabe Y, Funahashi K et al. 1994 Cell adhesion activity of a 30-kDa major secreted protein from human bladder
337
Articles - Genomic approach to endometrial receptivity - C Simón et al.
338
carcinoma cells. Biochemical and Biophysical Research Communications 198, 1046–1053. Akaogi K, Okabe Y, Sato J et al. 1996 Specific accumulation of tumour-derived adhesion factor in tumour blood vessels and in capillary tube-like structures of cultured vascular endothelial cells. Proceedings of the National Academy of Sciences USA 93, 8384–8389. Banaszak S, Donnelly KM, Brudney A et al. 2000 Modulation of the action of chorionic gonadotrophin on the baboon endometrium by a progesterone receptor antagonist (ZK 137.316). Biology of Reproduction 63, 819–823. Carson DD, Lagow E, Thathiah A et al. 2002 Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Molecular Human Reproduction 8, 871–879. Dominguez F, Avila S, Martín J et al. 2003 A combined approach for gene discovery identifies IGFBP-rP1 as a new gene implicated in human endometrial receptivity. J Clinical Endocrinology and Metabolism (in press). Fazleabas A, Donnelly KM, Srinivasan S et al. 1999 Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity. Proceedings of the National Academy of Sciences USA 96, 2543–2548. Girard JF, Baekkevold ES, Yamanaka T et al. 1999 Heterogeneity of endothelial cells. The specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization. American Journal of Pathology 155, 2043–2055. Giudice LC 1999 Potential biochemical markers of uterine receptivity. In: Simón C, Pellicer A, Remohí J (eds) Emerging concepts on human implantation. Human Reproduction Suppl. 14, pp. 3–16. Gunn MD, Tangemann K, Tam C et al. 1998 A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proceedings of the National Academy of Sciences USA 95, 258–263. Hegele-Harting C, Mootz U and Beier M 1992 Luteal control of endometrial receptivity and its modification by progesterone antagonists. Endocrinology 131, 2446–2460. Irwin J, Kirk D, King R et al. 1989 Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertility and Sterility 52, 761–768. John NJ, Linke M, Denker HW 1993 Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers. In vitro Cellular and Developmental Biology. Animal 29, 461–468. Kao LC, Tulac S, Lobo S et al. 2002 Global gene profiling in human endometrium during the window of implantation. Endocrinology 143, 2119–2138. Lim H, Paria B, Das SK et al. 1997 Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91, 197–208. Martín J, Ávila S, Jiménez O et al. 2000 Detection of novel genes associated to high versus low endometrial receptivity status. Journal of the Society of Gynecologic Investigation (suppl.) 8, 229. Martin JC, Jasper MJ, Valbuena D et al. 2000 Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression. Biology of Reproduction 63, 1370–1376. Middleton J, Neil S, Wintle J et al. 1997 Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91, 385–395. Murphy CR 2000 The plasma membrane transformation of uterine epithelial cells during pregnancy. In: Simón C, Pellicer A, Doberska C (eds) Human Implantation: Recent Advances and Clinical Aspects. Journal of Reproduction and Fertility. Supplement 55, pp. 23–28. Navot D, Bergh PA, Williams M et al. 1991 An insight into early reproductive processes through the in vivo model of ovum donation. Journal of Clinical and Endocrinology and Metabolism 72, 408–414.
Noyes RW, Hertig AT, Rock J 1950 Dating the endometrial biopsy. Fertility and Sterility 1, 3–25. Popovici RM, Kao LC, Giudice LC 2000 Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology. Endocrinology 141, 3510–3513. Psychoyos A 1973 Endocrine control of egg implantation. In: Greep RO, Astwood EB (eds) Handbook of Physiology, Sect. 7, Endocrinology Vol. II, Pt II. Am Physiol Soc, Washington, DC, pp. 187–215. Ravindranath N, Moudgal R 1990 Effect of a specific oestrogen antibody on pregnancy establishment in the bonnet monkey (Macaca radiata). Fertility and Sterility 54, 1162–1167. Reese J, Das S, Paria B et al. 2001 Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation. Journal of Biological Chemistry 276, 44137–44145. Remohí J, Gartner B, Gallardo E et al. 1997 Pregnancy and birth rates after oocyte donation. Fertility and Sterility 67, 717–723. Riesewijk A, Martín J, Van Os R et al. 2003 Gene expression profiling of human endometrial receptivity by microarray technology. Molecular Human Reproduction, in press. Stewart C, Kaspar P, Brunet L et al. 1992 Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359, 76–79. Thie M, Fuchs P, Butz S et al. 1996 Adhesiveness of the apical surface of uterine epithelial cells: the role of junctional complex integrity. European Journal of Cell Biology 70, 221–232. Thie M, Harrach-Ruprecht B, Sauer H et al. 1995 Cell adhesion to the apical pole of epithelium: a function of cell polarity. European Journal of Cell Biology 66, 180–191. Tinel H, Denker HW, Thie M 2000 Calcium influx in human uterine epithelial RL95–2 cells triggers adhesiveness for trophoblast-like cells. Model studies on signalling events during embryo implantation. Molecular Human Reproduction 6, 1119–1130. Way DL, Grosso DS, Davis JR et al. 1983 Characterization of a new human endometrial carcinoma (RL95–2) established in tissue culture. In vitro 19, 147–158. Yamauchi T, Umeda F, Masakado M et al. 1994 Purification and molecular cloning of prostacyclin-stimulating factor from serumfree conditioned medium of human diploid fibroblast cells. Biochemical Journal 303, 591–598. Yoshioka K, Matsuda F, Takakura K et al. 2000 Determination of genes involved in the process of implantation: application of GeneChip to scan 6500 genes. Biochemical and Biophysical Research Communications 272, 531–538. Declaration: The authors have declared they have no financial interests in any products arising from the work reported in this article. Paper based on contribution presented at the Serono Symposium ‘Toward Optimizing ART: a Tribute to Howard and Georgeanna Jones’ in Williamsburg, VA, USA, April 2002. Received 4 October 2002; refereed 13 November 2002; accepted 14 January 2003.
Articles Human endometrial receptivity: a genomic approach
Dr Carlos Simón
Carlos Simón, MD, is Professor in the Obstetrics and Gynaecology Department at Valencia University and Scientific Director of the Instituto Valenciano de Infertilidad (IVI). He was post-doctoral research fellow at Stanford University, CA, USA from 1991 to 1994. Research in his laboratory focuses on human embryonic implantation and uterine receptivity, leading to the publication of over 100 per review papers and six books. Dr Simón has received awards from the Spanish Fertility Society in 1991, the Society for Gynecological Investigation (SGI) in 1993 and the Society of Reproductive Endocrinologist of the American Society of Reproductive Medicine (ASRM) in 1993, 1995, 1997 and 1999. He is a member of the Editorial Board of several leading journals in the field as well as being an ad hoc reviewer for several more. Scientific international appointments include membership of the HRP Scientific and Ethical Review Group of the World Health Organization, the International Committee of the Society of Gynecological Investigation, and the FIGO expert advisory panel on reproductive medicine.
F Domínguez1, J Remohí 1,2, A Pellicer1,2, C Simón1,2,3 1Instituto Valenciano de Infertilidad Foundation (FIVIER), Plaza de la Policia Local 3, 46015, Valencia. Spain 2Department of Pediatrics, Obstetrics and Gynecology, School of Medicine. Valencia University, Valencia, Spain. 3Correspondence: Tel: +34 963624555; Fax: +34 963694735; e-mail: [email protected]
Abstract The endometrium is a specialized tissue, hormonally-regulated, that is non-adhesive for embryos throughout most of the menstrual cycle in humans and other primates. Thus, endometrial receptivity is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status. The luminal EE acquires the ability to adhere (receptivity) the developing human blastocyst during this period due mainly to the presence of progesterone after appropriate 17β-oestradiol priming. This status is a key element for embryonic implantation and appears to be closely associated with morphological and biochemical changes of EE cells. This specific time window is thought to be open after 4–5 days and closes after 9–10 days of progesterone production or administration, creating a physiological window of receptivity limited to days 19–24 of the menstrual cycle in humans. The scientific knowledge of the endometrial receptivity process is fundamental for the understanding of the human reproduction, but, so far, none of the proposed biochemical markers for endometrial receptivity have been proved clinically useful. In this work new strategies are presented based on molecular biology technologies that aim to clarify the fragmented information in this field using differential display, quantitative PCR and cDNA microarray analysis of endometrial epithelial-derived cell lines and endometrial samples to investigate the hierarchy at the mRNA level of molecules implicated in the process of endometrial receptivity.
Keywords: cDNA array, endometrial receptivity, implantation, microarrays
Introduction
332
Endometrial receptivity is a self-limited period in which the endometrial epithelium acquires a functional and transient ovarian steroid-dependent status allowing blastocyst adhesion (Psychoyos, 1973). In humans, the luminal endometrial epithelium acquires this status simultaneously with the development of the decidualization process in the stromal compartment (Popovici et al., 2000) due mainly to the presence of progesterone after appropriate 17β-oestradiol priming.
to days 19–24 of the menstrual cycle in humans (Navot et al., 1991) and between 8–10 days post ovulation in other primates (Fazleabas, 1999). Indeed, the administration of progesterone antagonist (Hegele-Harting et al., 1992; Banaszak et al. 2000) or oestradiol antiserum (Ravindranath and Moudgal, 1990) during the pre-implantation period disrupted endometrial receptivity in primates. Using this concept of oestradiol and progesterone priming, a clinical endometrial receptivity window is induced routinely in ovum donation programmes to synchronize the timing of embryo transfer (Remohí et al., 1997).
This period, termed the ‘window of implantation’, occurs 4–5 days after, and closes 9–10 days after, progesterone production or administration. Therefore, the receptive window is limited
Steroids acting through their nuclear receptors in the endometrial epithelial cells (EEC) induce the acquisition of a receptive phenotype. EEC suffers structural and functional
Articles - Genomic approach to endometrial receptivity - C Simón et al.
changes. The morphological changes include modifications in the plasma membrane (PM) (Murphy, 2000) and cytoskeleton (Martin et al., 2000). The apical PM develops transitional adhesive properties by undergoing structural changes; long thin, regular microvilli are gradually converted into irregular, flattened projections and this process is named as the plasma membrane transformation. The remodelling of the epithelial organization, from a polarized to a non-polarized phenotype might prepare the apical pole for cell-to-cell adhesion (Thie et al., 1995). This PM remodelling requires the participation of the underlying cytoskeleton, and a relevant function of the Ezrin family in the regulation of cell polarity to change from the non-adhesive to the adhesive status has been suggested (Martin et al., 2000). These changes occur with the complicity of the decidualization process that occurs in the stromal compartment and at the endometrial vasculature. A number of
biochemical markers for endometrial receptivity have been proposed over the years (Irwin et al., 1989) and more recently (Giudice, 1999) although none of them have been proved clinically useful. The scientific knowledge of the endometrial receptivity process is fundamental for the understanding of the mechanism that governs embryonic implantation and human reproduction. Recent advances in molecular biology and gene technology as well as the sequence of the human genome, invite us to re-consider the endometrial receptivity process from a genomic perspective. However, a hierarchical perspective of the genes modified during this relevant process in humans is still lacking. In this review current data are presented using molecular biology technologies that aim to clarify the fragmented information in this field.
Figure 1. Schematic diagram of cDNA array protocol.
333
Articles - Genomic approach to endometrial receptivity - C Simón et al.
Data are presented that were obtained using three different scientific approaches: first, macroarray technology which allows us to analyse the expression pattern of 300–400 genes; second, microarray technology that provides us with a hierarchical global view of genes up- or down-regulated during human endometrial receptivity by analysing simultaneously the expression pattern of 12,000 genes in prereceptive (LH+2) versus receptive (LH+7); and finally, to establish a possible differential gene expression pattern for novel gene discovery, differential display-PCR analysis (DDPCR) has been used.
Macroarray analysis of cytokines, chemokines and growth factors genes in pre-receptive versus receptive human endometrium combined with specific cell lines Profiling RNA on cDNA arrays provides a method to screen the hierarchical contribution of the genes investigated in a given situation. Human endometrial receptivity is characterized by two main features: first, it occurs in a specific period of time in which this tissue acquires a differential functional and transient status, and second, this new status is characterized by the development of adhesiveness to the blastocyst. Therefore, this approach was used to profile the similarity between the differential transcriptional response in two related models: (i) receptive (LH+7) versus pre-receptive (LH+2) human endometrium and (ii) human endometrial cell lines with higher (RL95–2) and lower (HEC-1-A) adhesiveness to trophoblast-derived cells (JAR) cells and mouse blastocysts.
334
The human endometrial cell line RL95–2 is an epithelial cell line derived from a moderately-differentiated endometrial adenocarcinoma (Way et al., 1983) with specific morphological and biological characteristics (Tinel et al., 2000). This cell line exhibits more pronounced adhesiveness for JAR cells (John et al., 1993) and mouse blastocysts (Martin et al., 2000) than any other human EEC line, including HEC-1-A and primary epithelium. The HEC-1-A cell line, in contrast, has poor adhesive properties, and exhibits a polarized distribution of integrins, meanwhile RL95–2 cell line show atypical features in adherins junctions, with non-polarized actin cytoskeleton and integrin distribution (Thie et al., 1996). Embryonic adhesion experiments using mouse blastocysts demonstrate that RL95–2 and HEC-1-A cells showed pronounced receptive and nonreceptive phenotypes (81 versus 46% of blastocyst adhesion, respectively), compared with an intermediate adhesion rate present in primary EEC cultured on extracellular matrix, (67% of blastocyst adhesion) (Martin et al., 2000). Therefore, these cell lines have been used as in vitro models for higher receptivity (RL95–2) and lower receptivity (HEC-1-A). Using both models, the expression pattern of 375 human genes have been differentially analysed; these include cytokines, chemokines, adhesion molecules, and their receptors (Human Cytokine Expression Array, R and D). Briefly, human endometrial samples were obtained in the luteal phase at LH+2 and LH+7 days. A portion of each specimen was dated according to the criteria of Noyes et al. (1950). Total RNA was extracted from whole endometrial biopsies treated with 5µl of DNase I and cDNA probes were synthesized from purified total RNA using [α32-P] dATP, dCTP, dGTP and dTTP. After pre-hybridization during 30 min the probes were hybridized overnight with the cDNA array filters (Figure 1). The hybridization was performed three times with different LH+2 and LH+7 samples.
Figure 2. A. cDNA array of pre-receptive (LH+2) and receptive (LH+7) endometrium. B. cDNA array of RL95–2 and HEC-1A. Hybridization spots of the major up-regulated genes in both states are shown.
Articles - Genomic approach to endometrial receptivity - C Simón et al.
These results indicate that seven genes were up-regulated more than two-fold and four genes were down-regulated in LH+7 versus LH+2 (Dominguez et al., 2003). Among the list of regulated genes, genes were identified that were already known to be differentially expressed during the receptive phase compared with the pre-receptive phase such as PP14, osteopontin, integrin α3, and IL-1RtI (Figure 2). However, a number of genes were also identified for which the differential expression between the pre-receptive (LH+2) and the receptive (LH+7) endometria or even the presence in human endometrium has not been described before. These genes can be classified into different groups such as: extracellular matrix proteins (decorin), heparin-binding molecules (pleiotrophin), genes related to tyrosine kinases (EFNA2) and growth factors (BMP-7). Other genes well studied in human endometrium are upregulated in the receptive endometrium such as protease inhibitors (TIMP1, 2 and 3), matrix metalloproteinase 13 (MMP-13), insulin growth factor II (IGFII) and others. Only four genes were minimally down-regulated in the receptive endometrium. These genes were two interleukin receptors (IL15Rα and IL-9R), bone morphogenetic protein 7 (BMP-7) (a growth factor of the TGFβ family), and ephrin-A2 (a tyrosine kinase ligand) (Figure 2). The same experiments have been repeated using adhesive versus non-adhesive cell lines. The comparative results obtained after the cDNA array hybridization of the endometrial cell lines, HEC-1-A versus RL95–2 shows that the two highly expressed genes in RL95–2 cell line were neurite growthpromoting factor 2 (NEGF2/midkine) and IGFBP-rP1. The rest of the up-regulated genes were chemokines (GRO oncogene 1 and 2), growth factor receptors (erbB1 and TNFRSF16) and growth factors (TGFα) (Figure 2). The group of adhesion molecules appears minimally up-regulated and includes EpCAM, integrins β1, α4 and α1. Remarkably IGFBP-rP1 was the second most up-regulated gene in the two models investigated, receptive versus prereceptive endometria and in high-adhesive versus lowadhesive cell lines respectively. RT-PCR was performed using the same RNA of both cell lines and biopsies to confirm the RNA expression of the cDNA arrays. IGFBP-rP1 expression in both models exhibit a clear trend similar to the results obtained in the cDNA arrays. Further corroboration of these findings was obtained by quantitative fluorescent RT-PCR (QF-PCR) and immunohistochemistry, the expression pattern and distribution of IGFBP-rP1 in the human endometrium across the menstrual cycle conforming a profile consistent with a marker of endometrial decidualization (Dominguez et al., 2003). Although the functional proof of the concept remains to be demonstrated, this work strongly suggests that IGFBP-rP1, also known as IGFBP-7 or mac25, might be implicated in human endometrial receptivity. It is unlikely that the possible role that this molecule may play in endometrial receptivity was as IGFBP due to its low affinity for IGF. However, several independent functions in vascular biology have been attributed to this molecule that may be involved in endometrial receptivity. IGFBP-rP1 has been reported as prostacyclin-
stimulating factor (PSF) in vascular endothelial cells (Yamauchi et al., 1994) and to contribute to the organization of new capillary vessels in tumour tissues by modulating the interaction of endothelial cells with type IV collagen (Akaogi et al., 1996). IGFBP-rP1 contains an amino-terminal domain with homology to IGFBP that is responsible for low-affinity binding to IGF. This sequence is followed by a follistatin-like module that has a low but significant homology with the cysteine-rich follistatin-like module of hevin/SC1, which is known to mediate cytokine binding in other proteins. These two modules in IGFBP-rP1 are likely to be involved in growth factor binding and may facilitate the retention of growth factors or chemokines (Gunn et al., 1998). This molecule has also been defined as tumour-derived adhesion factor (TAF, recently renamed angiomodulin) (Akaogi et al., 1994). At the ultrastructural level, a noteworthy feature associates IGFBPrP1 with microvillous structures that constitute the initial point of contact between the endothelium and the adherent lymphocytes. It has been demonstrated that this molecule may harbour chemokines (Middleton et al., 1997) and adhesion molecules (Girard et al., 1999), suggesting that this molecule may contribute to the lymphocyte migration through high endothelial venules endothelial cells (HEVEC). Therefore, in addition to its vascular functions, these properties indicate that this molecule may be a good candidate for the presentation of adhesion-triggering cytokines to the lymphocytes rolling on, or migrating across, in and through the human endothelial epithelium, having this process remarkable similarity with the implantation process.
Microarray analysis of human receptivity The gene expression studies with microarray technology use a more global strategy in which more than 10,000 genes are analysed in only one experiment. During this year three studies have investigated the differential gene expression pattern between a pre-receptive and receptive endometrium using microarray technology with different study designs and results. Kao et al. (2002) have investigated global gene expression during the window of implantation in human endometrial biopsies timed to the LH surge, compared with the late proliferative phase of the menstrual cycle. Statistical analysis revealed 156 significantly up-regulated genes and 377 significantly down-regulated genes in the implantation window. Up-regulated genes included those for cholesterol trafficking and transport (apolipoprotein (ApoE) being the most induced gene, 100-fold), prostaglandin (PG) biosynthesis (PLA2) and action (PGE2 receptor), proteoglycan synthesis (glucuronyltransferase), secretory proteins (glycodelin), IGF binding protein (IGFBP), and TGFβ superfamilies, signal transduction, extracellular matrix components (osteopontin, laminin), neurotransmitter synthesis (monoamine oxidase) and receptors (γ aminobutyric acid A (GABA) receptor π subunit), numerous immune modulators, detoxification genes (metallothioneins), and genes involved in water and ion transport (CPE1-R). Down-regulated genes included intestinal trefoil factor (ITF) (the most repressed gene, 50-fold), matrilysin, members of the G protein-coupled receptor signalling pathway, frizzled-related protein (FrpHE, a Wnt
335
Articles - Genomic approach to endometrial receptivity - C Simón et al.
Table 1. Comparative results by families between genes up- and down-regulated in the receptive phase with a fold change greater than 3.0 in the studies by Kao et al. (2002) (n = 60 up-regulated and n = 87 down-regulated), Carson et al. (2002) (n = 35 upregulated and n = 39 down-regulated) and Riesewijk et al. (2003) with the same criteria. In those genes in which the accession number is different, both codes are indicated. Boldface type indicates genes identified in all three studies. Family/Accession number
Up-regulated genes comparison Secretory protein AB020315 Transporter AB000712 Extracellular matrix/Cell adhesion molecules AF052124 Other cellular functions J02611 Down-regulated genes comparison Vasoactive substance J05081 Other cellular functions U79299
Gene name
Riesewijk et al. (2003)
Kao et al. (2002)
Carson et al. (2002)
Dickkopf/DKK1 (hdkk-1) Claudin 4/CEP-R Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) Apolipoprotein D
+
+
+
+ +
+ + J04765
+ + J04765
+
+
+
Endothelin 3 (EDN3) Olfactomedin-related ER localized protein
+ +
+ +
+ + X52001
antagonist), transcription factors, TGFβ-signalling pathway members, immune modulators (major histocompatibility complex class II subunits) and other cellular functions. To validate the data, two approaches were used, first RT-PCR and Northern blot analysis using RNA isolated from late proliferative and window of implantation endometrial tissue samples and second RT-PCR using cultured human endometrial glandular and stromal cells. RT-PCR data demonstrate clearly the up-regulation of IGFBP-1, glycodelin, Dkk-1 and other genes in the implantation window. Following Northern blot analysis, the mean values of relative expression were calculated for each mRNA on each blot confirming the up-regulation of Dkk-1, IGFBP-1, glycodelin and other genes. RT-PCR experiments using RNA from cultured endometrial epithelial cells revealed that glycodelin, Dkk-1, GABAA receptor, mammaglobin and others were all high expressed in these cells. CPE-1R, Dkk-1, ApoD and IGFBP-1 were all upregulated in stromal cells.
336
Other recent work based on microarray technology compared the expression of the early luteal (pre-receptive) and mid-luteal phase (receptive) (Carson et al., 2002). In this study more than 12,000 genes have been analysed using the hybridization chip from Affimetrix. Comparing pre-receptive versus receptive endometria, 370 genes decrease from 2- to 100-fold while 323 genes were up-regulated ranging from 2- to 45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change between the proliferative and mid-luteal phases. In addition, novel genes were identified in this work: genes encoding cell surface receptors, adhesion and extracellular matrix proteins, and growth factors. Northern blot analysis was performed confirming the up-regulation of claudin 4 or osteopontin gene in receptive endometrium. Immunohistochemical studies of prostaglandin receptor EP1 and leukotriene B4 receptor were performed to localize these proteins in human endometrium. Claudin 4 strongly stained both luminal and glandular epithelia but not in the stroma.
The authors and their group, in collaboration with Organon International, have investigated the gene expression pattern of human endometrial receptivity using a hybridization chip from Affimetrix containing 12,000 genes (Riesewijk et al. 2003). Comparison of the gene expression pattern of receptive (LH+7) versus pre-receptive (LH+2) endometria in the same women reveals a consistent pattern of differentially expressed genes. Identification of 220 regulated genes and 12 expressed sequence tags (EST) has been made. In total, 172 of these were specifically up-regulated in the LH+7 samples whereas 60 of them were down-regulated. Using more stringent criteria of regulation in all five patients, only 83 genes were identified as being up-regulated at least 3fold in all five patients at LH+7 whereas 10 genes were downregulated. In the list of regulated genes, genes were identified that were already known to be differentially expressed during the receptive phase, compared with the pre-receptive phase, such as glycodelin, osteopontin, IGFBP-3, crystallin αB and integrin α3 (Riesewijk et al. 2003). However, a number of genes were also identified for which the differential expression between the pre-receptive (LH+2) and the receptive (LH+7) endometria or even the presence in human endometrium has not been described before. These genes can be classified into different groups such as: immune modulatory genes, adhesion molecules, genes related to oxidative stress, cytoskeletal proteins and others. Further corroboration was obtained by investigation of the expression pattern of three differentially up-regulated genes. Moreover, for these genes a role in human endometrial receptivity had not been previously described. The selected genes were GPx-3, (glutathione peroxidase 3), CEPR (Claudin 4) and SLC1A1 (neuronal/epithelial high affinity glutamate transporter, system Xag member 1). Comparison of the up- or down-regulated genes in these three studies is shown in Table 1. Here the up- or down-regulated genes found in this study that match the first 50 genes found in
Articles - Genomic approach to endometrial receptivity - C Simón et al.
Figure 3. A. General view of the differential display 4.5% denaturing gel; genes associated to the receptive phenotype are displayed. Currently 60 differentially-expressed bands have been purified, both qualitatively and/or quantitatively. B, C. Re-amplification PCR were performed to obtain sufficient amount of DNA for cloning. Approximately 25% of them have been cloned and sequenced. the other two studies are represented. Genes present in the three papers are shown in bold. The mouse has become an indispensable model organism for the study of endometrial receptivity and implantation. Nevertheless, the comparison of the present study with elegant microarray-based studies in the mouse (Reese et al., 2001; Yoshioka et al., 2000) indicates the existence of important differences in the genomics of endometrial receptivity and implantation between human and mouse. Firstly, there are few genes that were mutually identified in these two models and more importantly, genes functionally crucial for implantation in mice such as LIF (Stewart et al., 1992) or COX-2 (Lim et al., 1997), as demonstrated by the different knockout models, were not detected as regulated genes in this human study. Even more intriguing is the fact that these genes were not detected in the mouse model during implantation using a similar genome-wide approach (Reese et al., 2001). As the authors pointed out this may be due to highly spatially restricted expression around the implanting blastocyst. It should be mentioned that, in the human, the timing is not as restricted as it is in the mouse with a window of receptivity of approximately 3 days.
Discovery of new genes associated to endometrial receptivity using differential display-PCR. Finally, another approach to isolate and characterize receptivity-related genes was the employment of the differential display associated to PCR (DD-PCR) using cDNA preparations from RL95–2 and HEC-1-A cells, which shows distinct patterns of gene expression (Martin et al., 2000). For this purpose total RNA was extracted and treated with DNase I. To obtain the cDNA from cell lines reverse transcription was performed using [α-33P] dATP, 12 oligo(dT) anchored 3′ primers (AP) individually with aliquots of each total RNA sample in combination with four arbitrary 5′ primers (ARP).
Following DD-PCR, radiolabelled cDNA fragments were electrophoretically separated on a polyacrylamide gel under denaturing conditions. (Figure 3a) For the re-amplification and cloning of differentiallyexpressed cDNA fragments the autoradiogram was superimposed on the dried gel to locate the cDNA fragments of interest. Differential bands were excised, eluted and reamplified. (Figure 3b) The obtained products were ligated to the cloning vector (pGEM-T) after Not I/Nco I digestion. Positive colonies were used to purify the DNA. After this cDNA fragments were purified and sequenced. To date, 60 differentially-expressed bands have been purified by the authors, both qualitatively and/or quantitatively. Currently, work is progressing to sequence and identify these bands. Most of them are known genes but possibly some bands obtained may correspond to unknown genes related to receptivity. Current work involves identification of the genes by database analysis before going forward with functional analysis. This approach should lead to improved understanding of endometrial receptivity and to the optimization of treatment of infertile patients or to its modification as an interceptive procedure.
Conclusion Studies previously published, including this study, have been performed on complete endometrial tissue but nowadays there is no available information of the specific contribution to the general gene pattern from different compartments of the human endometrium such as stromal cells, glandular or luminal epithelium, etc. So it is suggested that further experiments following this direction should be performed to unravel the answers to these questions.
References Akaogi K, Okabe Y, Funahashi K et al. 1994 Cell adhesion activity of a 30-kDa major secreted protein from human bladder
337
Articles - Genomic approach to endometrial receptivity - C Simón et al.
338
carcinoma cells. Biochemical and Biophysical Research Communications 198, 1046–1053. Akaogi K, Okabe Y, Sato J et al. 1996 Specific accumulation of tumour-derived adhesion factor in tumour blood vessels and in capillary tube-like structures of cultured vascular endothelial cells. Proceedings of the National Academy of Sciences USA 93, 8384–8389. Banaszak S, Donnelly KM, Brudney A et al. 2000 Modulation of the action of chorionic gonadotrophin on the baboon endometrium by a progesterone receptor antagonist (ZK 137.316). Biology of Reproduction 63, 819–823. Carson DD, Lagow E, Thathiah A et al. 2002 Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Molecular Human Reproduction 8, 871–879. Dominguez F, Avila S, Martín J et al. 2003 A combined approach for gene discovery identifies IGFBP-rP1 as a new gene implicated in human endometrial receptivity. J Clinical Endocrinology and Metabolism (in press). Fazleabas A, Donnelly KM, Srinivasan S et al. 1999 Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity. Proceedings of the National Academy of Sciences USA 96, 2543–2548. Girard JF, Baekkevold ES, Yamanaka T et al. 1999 Heterogeneity of endothelial cells. The specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization. American Journal of Pathology 155, 2043–2055. Giudice LC 1999 Potential biochemical markers of uterine receptivity. In: Simón C, Pellicer A, Remohí J (eds) Emerging concepts on human implantation. Human Reproduction Suppl. 14, pp. 3–16. Gunn MD, Tangemann K, Tam C et al. 1998 A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proceedings of the National Academy of Sciences USA 95, 258–263. Hegele-Harting C, Mootz U and Beier M 1992 Luteal control of endometrial receptivity and its modification by progesterone antagonists. Endocrinology 131, 2446–2460. Irwin J, Kirk D, King R et al. 1989 Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertility and Sterility 52, 761–768. John NJ, Linke M, Denker HW 1993 Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers. In vitro Cellular and Developmental Biology. Animal 29, 461–468. Kao LC, Tulac S, Lobo S et al. 2002 Global gene profiling in human endometrium during the window of implantation. Endocrinology 143, 2119–2138. Lim H, Paria B, Das SK et al. 1997 Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91, 197–208. Martín J, Ávila S, Jiménez O et al. 2000 Detection of novel genes associated to high versus low endometrial receptivity status. Journal of the Society of Gynecologic Investigation (suppl.) 8, 229. Martin JC, Jasper MJ, Valbuena D et al. 2000 Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression. Biology of Reproduction 63, 1370–1376. Middleton J, Neil S, Wintle J et al. 1997 Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91, 385–395. Murphy CR 2000 The plasma membrane transformation of uterine epithelial cells during pregnancy. In: Simón C, Pellicer A, Doberska C (eds) Human Implantation: Recent Advances and Clinical Aspects. Journal of Reproduction and Fertility. Supplement 55, pp. 23–28. Navot D, Bergh PA, Williams M et al. 1991 An insight into early reproductive processes through the in vivo model of ovum donation. Journal of Clinical and Endocrinology and Metabolism 72, 408–414.
Noyes RW, Hertig AT, Rock J 1950 Dating the endometrial biopsy. Fertility and Sterility 1, 3–25. Popovici RM, Kao LC, Giudice LC 2000 Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology. Endocrinology 141, 3510–3513. Psychoyos A 1973 Endocrine control of egg implantation. In: Greep RO, Astwood EB (eds) Handbook of Physiology, Sect. 7, Endocrinology Vol. II, Pt II. Am Physiol Soc, Washington, DC, pp. 187–215. Ravindranath N, Moudgal R 1990 Effect of a specific oestrogen antibody on pregnancy establishment in the bonnet monkey (Macaca radiata). Fertility and Sterility 54, 1162–1167. Reese J, Das S, Paria B et al. 2001 Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation. Journal of Biological Chemistry 276, 44137–44145. Remohí J, Gartner B, Gallardo E et al. 1997 Pregnancy and birth rates after oocyte donation. Fertility and Sterility 67, 717–723. Riesewijk A, Martín J, Van Os R et al. 2003 Gene expression profiling of human endometrial receptivity by microarray technology. Molecular Human Reproduction, in press. Stewart C, Kaspar P, Brunet L et al. 1992 Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359, 76–79. Thie M, Fuchs P, Butz S et al. 1996 Adhesiveness of the apical surface of uterine epithelial cells: the role of junctional complex integrity. European Journal of Cell Biology 70, 221–232. Thie M, Harrach-Ruprecht B, Sauer H et al. 1995 Cell adhesion to the apical pole of epithelium: a function of cell polarity. European Journal of Cell Biology 66, 180–191. Tinel H, Denker HW, Thie M 2000 Calcium influx in human uterine epithelial RL95–2 cells triggers adhesiveness for trophoblast-like cells. Model studies on signalling events during embryo implantation. Molecular Human Reproduction 6, 1119–1130. Way DL, Grosso DS, Davis JR et al. 1983 Characterization of a new human endometrial carcinoma (RL95–2) established in tissue culture. In vitro 19, 147–158. Yamauchi T, Umeda F, Masakado M et al. 1994 Purification and molecular cloning of prostacyclin-stimulating factor from serumfree conditioned medium of human diploid fibroblast cells. Biochemical Journal 303, 591–598. Yoshioka K, Matsuda F, Takakura K et al. 2000 Determination of genes involved in the process of implantation: application of GeneChip to scan 6500 genes. Biochemical and Biophysical Research Communications 272, 531–538. Declaration: The authors have declared they have no financial interests in any products arising from the work reported in this article. Paper based on contribution presented at the Serono Symposium ‘Toward Optimizing ART: a Tribute to Howard and Georgeanna Jones’ in Williamsburg, VA, USA, April 2002. Received 4 October 2002; refereed 13 November 2002; accepted 14 January 2003.