Determinants of Endometrial Receptivity JOSÉ A. HORCAJADAS,a ANNE RIESEWIJK,b FRANCISCO DOMÍNGUEZ,a ANA CERVERO,a ANTONIO PELLICER,a,c AND CARLOS SIMÓNa,c aIVI
Foundation, C/Guadassuar, 1, 46015 Valencia, Spain
bNV
Organon, Departments of Target Discovery and Pharmacology, Oss, The Netherlands cDepartment
of Pediatrics, Obstetrics, and Gynecology, Faculty of Medicine, Valencia University, 46010 Valencia, Spain
ABSTRACT: Understanding the molecular changes that occur during the window of implantation is fundamental to our knowledge of human reproduction. Lately, the development of microarray technology has allowed this process to be studied from a global molecular perspective. In the last 2 years, researchers have focused their efforts on throwing light on the gene expression profile of the receptive endometrium. The genes hold the key to the development of the endometrium at any stage, and we have focused our work on the window of implantation. The four most recently published works in this field have revealed a long list of genes that are up- or downregulated at the time of implantation. Although these studies have been conducted using varying approaches, collectively these studies identify new candidate markers that can be used to accurately diagnose the receptive state of the endometrium. The next step is to perform functional analysis for confirming the importance of these genes. In this article, we gather together these recent findings to provide an overview of the current knowledge regarding the genetic functioning of human endometrial receptivity and related processes. KEYWORDS: endometrial receptivity; gene expression profile; microarray
INTRODUCTION The implantation process requires several conditions. The embryo must be healthy and should have reached the blastocyst stage. A receptive endometrium and a molecular dialogue between it and the embryo are also prerequisites. Endometrial receptivity is a self-limiting period in which the endometrial epithelium acquires a functional and transient ovarian steroid-dependent status that allows blastocyst adhesion.1 A normal human endometrium is controlled by the ovarian sex steroid hormones estrogen and progesterone, which elicit their actions by binding to specific high-affinity receptors. These act as ligand-dependent transcription factors2 that modulate the transcription of a high number of proteins. The action of these hormones results in a period of receptivity 7–9 days after ovulation during the midAddress for correspondence: Carlos Simón, C/ Guadassuar, 1, 46015 Valencia, Spain. Voice: +34-96-345-55-60; fax: +34-96-345-55-12.
[email protected] Ann. N.Y. Acad. Sci. 1034: 166–175 (2004). © 2004 New York Academy of Sciences. doi: 10.1196/annals.1335.019 166
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secretory or midluteal phase.3 Steroids, acting through their nuclear receptors in the endometrial epithelial cells (EECs), induce the formation of a receptive phenotype. EECs undergo specific structural and functional changes. Morphological changes include modifications in the plasma membrane4 and cytoskeleton.5,6 These changes occur as part of the complex decidualization process that takes place in the stromal compartment7 and endometrial vasculature. Moreover, several biochemical markers for endometrial receptivity have been proposed over the years,8 although so far none of them has proved to be clinically useful. The optimum markers of the receptive state would be those molecules directly involved in the initial stages of the implantation process; however, to localize these marker molecules would require functional testing, an impractical approach in humans. Until now, the identification of these markers has been, in most cases, the result of careful examination of the expression of specific proteins or mRNAs based on preconceived ideas or results obtained in animal models. The recent advent of high-throughput microarray screening of the expression of human genes has permitted a new approach for identifying changes in global gene expression in a specific physiological or pathological situation.9 Here, we review the most recently published work on endometrial receptivity in humans, not merely that focused on the natural process of endometrial receptivity, but also those studies whose approaches indirectly provide information about the genes involved in embryonic implantation. In this article, we analyze the recent developments in the area of genomics in our field and the significance of the results of the latest publications when contemplated together. We consider the present and the future of research on human reproduction and the importance of functional analysis in a process as crucial as that of embryonic implantation.
MICROARRAYS: NATURAL ENDOMETRIAL RECEPTIVITY DNA microarray technology has broad applications as it is directed toward the study of global gene expression.9 Using established cell lines and DNA microarrays, it is possible to identify genes that are up- and downregulated in any process in humans. However, the biological functions of many of the sequenced genes remain unknown or have been predicted based purely on their homology with genes whose functions are better known. We have some strategies based on molecular biology technologies to analyze groups of genes. Profiling RNA on cDNA macroarrays provides a method to screen the hierarchical contribution of a group of related genes in a given situation. The studies with microarray technology use a more global strategy in which more than 10,000 genes are analyzed in only one experiment. Another approach used in our group to isolate and characterize receptivity-related genes was differential display associated with PCR (DD-PCR) using cDNA preparations from two different cell types or situations. Although many protocols and types of microarrays are available, the basic technique involves extraction of RNA from biological samples in either normal or interventional states. This RNA or, in some protocols, isolated messenger RNA is copied, while incorporating either fluorescent nucleotides or a tag that is later stained with fluorescent. The labeled nucleic acid then is hybridized to a microarray for a period
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FIGURE 1. Different approaches in genome-wide analysis.
of time, after which the excess is washed off and the microarray is scanned under laser light. In the case of oligonucleotide microarrays, to which all probes have been designed to be theoretically similar for hybridization temperature and binding affinity, each microarray measures a single sample and provides an absolute measurement level for each RNA molecule, although this absolute measurement might not correlate exactly with the concentration in terms of micrograms per unit volume. cDNA microarray, where each probe has its own hybridization characteristic, measures two samples and provides a relative measurement level for each RNA molecule. This has been the technique most commonly used in endometrial receptivity studies. Whatever the technique, the end result is 4,000–50,000 measurements of gene expression per biological sample10 (FIG. 1). A recent study on endometrial receptivity by Domínguez et al.22 compared the gene expression pattern in receptive versus prereceptive human endometria and contrasted the results with gene expression in the highly adhesive cell line RL95-2 versus the considerably less adhesive cell line HEC-1A. The authors, using a macroarray containing 375 human cytokines, chemokines, and related factors, found a new gene implicated in human endometrial receptivity, the insulin-like growth factor–binding protein related 1 (IGFBP-rP1).23 In addition to microarray technology, the group also used quantitative PCR and in situ hybridization to confirm the endometrial localization and regulation of IGFBP-rP1 mRNA. At the protein level, IGFBP-rP1 was localized, using immunohistochemistry, at the apical zone of the luminal and glandular epithelium and in stromal and endothelial cells. This publica-
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TABLE 1. Characteristics of the four works published on endometrial receptivity Study
RNA Samples pooled
First sample
Second sample
Foldchange
Up
Down
Microarray
11
No
Proliferative phase (8–10)
LH+(8–10)
>2.0
156
377
Affymetrix HG-U95A
6
Yes
LH+(2–4)
LH+(7–9)
>2.0
323
370
Affymetrix HG-U95A
Borthwick13
10
Yes
Proliferative phase (9–11)
LH+(6–8)
>2.0
90
46
Affymetrix HG-U95A-E
Riesewijk14
10
No
LH+2
LH+7
>3.0
153
58
Affymetrix HG-U95A
Kao12
Carson11
Used with permission from Horcajadas, J.A. et al. 2004. Global gene expression profiling of human endometrial receptivity. J. Reprod. Immunol. 63: 41–49.
tion demonstrates that there is not a single strategy for obtainin g positive results in genomics. In the last 2 years, four studies focusing on endometrial gene expression profile during receptivity have been published: Carson et al.,11 Kao et al.,12 Borthwick et al.,13 and our own article, Riesewijk et al.14 All used the same technology and, more importantly, the same type of array, acquired from the same company (Affymetrix). However, there do exist some differences in the experimental design and in the data analysis. The four studies can be differentiated principally for the day of the cycle on which samples were collected, pooling or nonpooling of the isolated RNA, and the number of samples used. Furthermore, the cutoff used by which a gene was considered to be regulated varies among said studies. Three of the studies11–13 established a minimal fold-increase of 2.0 as evidence of gene regulation. We14 have used a 3.0-fold increase as a parameter, whereas it also must be noted that we have analyzed samples from the same woman at two different stages of the menstrual cycle. These differences in study design are reflected in the lists of differentially expressed genes identified. TABLE 1 summarizes the main differences between the four works. We compared the results as a whole with respect solely to those genes up- or downregulated in the receptive phase with a fold change of >3.0. Only one down-regulated gene is present in the four studies, the olfactomedin, a tissue-specific secreted glycoprotein involved in the maintenance, growth, and differentiation of chemosensory cilia on the apical dendrites of olfactory neurons.15 In the lists of up-regulated genes, three were identified in all four works: osteopontin,16 apoliprotein D,17 and Dickkopf. The presence of the third in all the studies is significant. Dickkopf-1 belongs to the human Dickkopf gene family18 and is an inhibitor of Wnt signaling. It is known that Wnt7A (–/–) null mice are infertile.19 Recent publications have identified, characterized, and revealed the regulation of the canonical Wnt signaling pathway in human endometrium,20 but the role of the Wnt family in this tissue and in implantation needs to be the focus of future research. It is also remarkable that glycodelin (also called placental protein 14),21 a secreted protein, was the most up-regulated gene in two of the four works and appeared in all but one. The overall comparison of these studies is represented in TABLE 2. We have also included those genes that we identi-
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TABLE 2. Comparison between the results obtained in the four works published on endometrial receptivity Accession number (function)
Gene name
Riesewijk
Kao
Carson Borthwick
Upregulated genes present in the four works AF052124 (structural protein)
Osteopontin
✓
✓
✓
✓
J02611 (transporter)
Apolipoprotein D
✓
✓
✓
✓
AB020315 (signaling)
Dickkopf/DKK1 (hdkk-1)
✓
✓
✓
✓
Upregulated genes present in Riesewijk’s work and in two out of the other three works J04129 (secretory protein)
Placental protein14/glycodelin
✓
✓
✓
M31516 (immunomodulator)
Decay accelerating factor for complement (CD55, Cromer blood group system)
✓
✓
✓
M84526 (complement protein)
Adipsin/complement factor D
✓
✓
✓
M55543 (GTPbinding protein)
Guanylate-binding protein 2, interferon-inducible
✓
AB000712 (receptor)
Claudin 4/CEP-R
✓
✓
AA420624 (signaling)
Monoamine oxidase A (MAOA)
✓
✓
✓
M60974 (regulatory protein)
Growth arrest and DNA-damageinducible protein (gadd45)
✓
✓
✓
AB002365 (cell death factor)
Nip2
✓
Total genes analyzed (>3.0)
✓
✓
✓
✓
✓
153
60
120
85
✓
✓
✓
✓
58
87
153
40
Downregulated genes present in the four works U79299 (secretory protein) Total genes analyzed (>3.0)
Olfactomedin-related ER localized protein
Used with permission from Horcajadas, J.A. et al. 2004. Global gene expression profiling of human endometrial receptivity. J. Reprod. Immunol. 63: 41–49.
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fied and thar appeared in two of the other three articles (eight genes in total). We consider that all the published data are complementary, despite variations in the design of the studies and differences in the software and statistics used for analysis of the hybridization data. Taken in their entirety, these results provide valuable insight into the complexity of endometrial receptivity and the high number of both known and newly discovered molecules involved in successful embryo implantation.
GENOME-WIDE ANALYSIS IN ENDOMETRIOSIS Kao et al.24 used another strategy to obtain information regarding genes implicated in implantation in patients with endometriosis. Endometriosis is a gynecological disorder affecting 10–15% of women of reproductive age.25 It is clinically associated with infertility. Implantation failure is strongly suggested as the underlying cause of said infertility.26 Kao et al.24 used high-density oligonucleotide arrays to compare regulated genes in endometrium from women with and without endometriosis. They found that more than 200 genes were up- or down-regulated where there was endometriosis. Some of these were genes that are regulated during the window of implantation (WOI), for example, cell adhesion molecules such as integrin α2 (1.8-fold down) and endometrial epithelial secreted proteins such as glycodelin (51.5-fold down). When the authors compared the WOI genes with those regulated in endometriosis, they found that a high number were deregulated. The data suggest that the deregulation of WOI genes leads to an inhospitable environment for the embryo and one in which implantation is difficult. These negative conditions include the affecting of genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses. The leptin system has always been involved in reproductive function acting at endocrine and paracrine levels. Recently, deregulation of this gene family has been linked to endometrial changes caused by endometriosis. Another recent work investigated the role of leptin and leptin receptors in endometriosis.27 The characterization of these genes and their functions will contribute to the uncovering of previously unknown mechanisms underlying implantation failure in women with endometriosis and with infertility in general. These are obvious potential new targets in embryonic implantation.
GENOME-WIDE ANALYSIS STUDIES IN CONTROLLED OVARIAN HYPERSTIMULATION CYCLES Clinical evidence indicates that uterine receptivity is more deteriorated when controlled ovarian hyperstimulation (COH) is used in in vitro fertilization (IVF) treatment compared with hormonal replacement therapy (HRT) and natural cycles.28,29 Therefore, we studied the gene expression profile of endometrial tissue during controlled ovarian hyperstimulation (COH), and we compared, on the one hand, the differences between LH+2 and LH+7 (prereceptive vs. receptive status) and on the other, the differences in the gene expression profile of a stimulated cycle (hCG+7) versus a normal cycle (LH+7) in IVF treatment. All these studies were con-
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ducted using microarray technology from Affymetrix (GeneChip HG_U133A, with more than 22,000 human DNA fragments). We found that COH results in considerable differences in endometrial gene expression when compared with the previous natural cycle in the same patient. In total, 558 genes on the Affymetrix chip showed differential expression, of which 281 were upregulated when compared with the natural cycle (166 genes two- to threefold, 74 genes three- to fivefold, and 41 more than fivefold). Downregulation was identified in 277 genes (161 genes two- to threefold, 72 genes three- to fivefold, and 44 more than fivefold). Comparison of LH+2 and LH+7 revealed similar gene expression profiles to those previously obtained with the HG_U95A chip. This study demonstrated that the gene expression of the COH hCG+7 samples is deregulated compared with that of the natural cycle at LH+7. An important number of genes implicated in endometrial receptivity show a very different expression in COH. In fact, genes regulated during the window of implantation in the natural cycle are more comparable to those at LH+2 than at the LH+7 patterns of the COH cycle.30 However, Mirkin and Oehninger 31 recently reported that minimal changes exist in the gene expression profile during the window of implantation in COH compared with natural cycles.
FUNCTIONAL ANALYSIS: RNA INTERFERENCE Taken as a whole, the abovementioned studies have borne fruit in a significant amount of information about the genes implicated in endometrial receptivity. Logically, it is necessary to filter all this information to obtain a manageable list of genes. The lists that are currently available contain several genes that always appear in the receptive status and disappear or are deregulated in nonreceptive conditions. However, understanding the differential expression of these genes in the endometrium is not sufficient for evaluating the real importance of these genes/proteins in endometrial receptivity. It is necessary to test, via functional analysis, their real implication in this process. One of the primary techniques for investigating gene function is RNA interference (RNAi). The phenomenon of RNAi was first discovered in the nematode worm Caenorhabditis elegans as a response to double-stranded RNA (dsRNA), which resulted in sequence-specific gene silencing.32 With this technique, it is possible to knock out or knock down a specific mRNA target in a cell or organism and to observe the importance of the encoded protein in a specific process. Apart from the technique, an adequate system, the appropriate organism or cell line, is necessary for working. The human endometrial cell line RL95-2 is an epithelial cell line derived from a moderately differentiated endometrial adenocarcinoma33 with specific morphological and biological characteristics.34 This cell line exhibits more pronounced adhesiveness for trophoblast-derived cells (JAR cells)35 and mouse blastocysts6 than any other human endometrial epithelial cell (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, whereas the RL95-2 cell line shows atypical features in adherens junctions, with nonpolarized actin cytoskeleton and integrin distribution.36 Embryonic adhesion experiments using mouse blastocysts showed pronounced receptive and nonreceptive phenotypes in RL95-2 and HEC-1-A cells (81% vs. 46% of blastocyst adhesion, respectively),
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when compared with an intermediate adhesion rate in primary EEC cultured on extracellular matrix (67% of blastocyst adhesion).6 Therefore, these cell lines are considered good in vitro models of higher (RL95-2) and lower (HEC-1-A) receptivity. Once an appropriate candidate’s genes have been selected, they can be analyzed using RNAi. By blocking the expression of the genes in question, it is possible to evaluate, one by one, the increase or decrease of the mice blastocyst adhesion rate. The results obtained with these assays should considerably expand the information available for the functional importance of these proteins in the implantation process.
CONCLUSIONS Microarray technology has changed the way researchers design their experiments. The enormous amount of information generated by this technique has led to a necessity for a more detailed analysis of the data. It is also necessary to reach a consensus in the understanding of the results. Parameters must be established for considering, objectively, when a gene is regulated or not. As an example, in the four aforementioned articles published on endometrial receptivity,11–14 only that of Riesewijk et al.14 established a cutoff of 3.0-fold for considering a gene to be regulated. The “list of genes” resulting from a microarray analysis should not be viewed as an end in itself; its real value increases only as that list moves through biological validation, ranging from the numerical verification of expression levels with alternative techniques, to ascertaining the meaning of the results, such as finding promoter regions or biological relationships between the genes. There are, in our opinion, several important points in these types of works to obtain interesting and real results: • Design the study properly (number and type of samples, duplicates, double labeling, etc.). • Define a stringent criteria to identify regulated genes. • Validate the microarray data with alternative techniques such as quantitative PCR or TaqMan probes. • Analyze the correlation between gene and function, if known. • Translate the obtained information into designs of functional analysis that show the importance of the changes observed. RNAi, together with other techniques or tools, such as knockout animals, that block the mRNA of a determined gene, can help the abovementioned functional analysis. They are not a perfect model for studying the function of one protein in a tissue or cell line. One model is the best if it can answer our questions about its function in a specific physiological or pathological situation. It is very important to acquire the technology as soon as available and learn the new techniques to improve the number and quality of our results in the laboratory. The knowledge in embryonic implantation has increased enormously in the last years. However, the “implantation molecule” has not been discovered yet, maybe because nature has provided the necessary redundancy for important functions that can be assessed only by “clusters of implantation molecules.”
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REFERENCES 1. PSYCHOYOS, A. 1973. Endocrine control of egg implantation. In Handbook of Physiology, Section 7, Endocrinology, Vol II, Pt II. R.O. Greep & E.B. Astwood, Eds. :187– 215. Washington, DC. American Physiological Society. 2. O’MALLEY, B.M. & M.J. TSAI. 1992. Molecular pathways of steroid receptor action. Biol. Reprod. 2: 163–167. 3. WILCOX, A.J., D.D. BAIRD & C.R. WEINBERG. 1999. Time of implantation of the conceptus and loss of pregnancy. N. Engl. J. Med. 340: 1796–1799. 4. MURPHY, C.R. 2000. Human implantation: recent advances and clinical aspects. J. Reprod. Fertil. Suppl. 55: 23–28. 5. THIE, M., B. HARRACH-RUPRECHT, H. SAUER, et al. 1995. Cell adhesion to the apical pole of epithelium: a function of cell polarity. Eur. J. Cell Biol. 66: 180–191. 6. MARTÍN J.C., M. JASPER, D. VALBUENA, et al. 2000. Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression. Biol. Reprod. 63: 1370–1376. 7. IRWIN, J., D. KIRK, R. KING, et al. 1989. Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil. Steril. 52: 761–768. 8. GIUDICE, L.C. 1999. Emerging concepts on human implantation. Hum. Reprod. 14 (Suppl.): 3–16. 9. SCHENA, M., D. SHALON, R.W. DAVIS & P.O. BROWN. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270: 467–470. 10. BUTTE, A. 2002. The use and analysis of microarray data. Nat. Rev. Drug Discov. 1: 951–960. 11. CARSON, D.D., E. LAGOW, A. THATHIAH, 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. Mol. Hum. Reprod. 8: 971–979. 12. KAO, L.C., S. TULAC, S. LOBO, et al. 2002. Global gene profiling in human endometrium during the window inplantation. Endocrinology 143: 2119–2138. 13. BORTHWICK, J.M., D.S. CHARNOCK-JONES, B.D. TOM, et al. 2003. Determination of the transcript profile of human endometrium. Mol. Hum. Reprod. 9: 19–33. 14. RIESEWIJK, A., J. MARTÍN, R. VAN OS, et al. 2003. Gene expression profiling of human endometrial receptivity on days LH+2 versus LH+7 by microarray technology. Mol. Hum. Reprod. 9: 253–264. 15. YOKOE, H. & R.R.H. ANHOLT. 1993. Molecular cloning of olfactomedin, an extracellular matrix protein specific to olfactory neuroepithelium. Proc. Natl. Acad. Sci. USA 90: 4655–4659. 16. APPARAO, K.B., M.J. MURRAY, M.A. FRITZ, et al. 2001. Osteopontin and its receptor alpha (v) beta (3) integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J. Clin. Endocrinol. Metab. 86: 4991–5000. 17. DRAYNA, D., C. FIELDING, J. MCLEAN, et al. 1986. Cloning and expression of human apolipoprotein D cDNA. J. Biol. Chem. 261: 16535–16539. 18. KRUPNIK, E., J.D. SHARP, C. JIANG, et al. 1999. Funtional and structural diversity of the human Dickkopf gene family. Gene 238: 301–313. 19. MILLER, A., A. PABLOBA & D.A. SASSOON. 1998. Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle. Mech. Dev. 76: 91–99. 20. TULAC, S., N.R. NAYAK, L.C. KAO, et al. 2003. Identification, characterization, and regulation of the canonical Wnt signalling pathway in human endometrium. J. Clin. Endocrinol. Metab. 88: 3860–3866. 21. JULKUNEN, M., R. KOISTINENE, J. SJÖBERG, et al. 1986. Secretory endometrium synthesizes placental protein 14. Endocrinology 118: 1782–1786. 22. DOMÍNGUEZ, F., S. ÁVILA, A. CERVERO, et al. 2003. A combined approach for gene discovery identifies insulin-like growth factor-binding protein-related protein 1 as a new gene implicated in human endometrial receptivity. J. Clin. Endocrinol. Metab. 88: 1849–1857. 23. JONES, J.I. & D.R. CLEMMONS. 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocrinol. Rev. 16: 3–34.
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24. KAO, A., S. GERMEYER, S. TULAC, et al. 2003. Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility. Endocrinology 144: 2870–2881. 25. GIUDICE, L.C., S.I. TAZUKE & L. SWIERSZ. 1998. Status of current research on endometriosis. J. Reprod. Med. 43: 252–262. 26. ARICI, A., E. ORAL, O. BUKULMEZ, et al. 1996. The effect of endometriosis on implantation: results from the Yale University in vitro fertilization and embryo transfer program. Fertil. Steril. 65: 603–607. 27. LIMA, I., A. CERVERO, F. BONILLA-MUSOLES, et al. Endometrial leptin and leptin receptor expression in women with severe/moderate endometriosis. Mol. Hum. Reprod. 10: 777–782. 28. PAULSON, R.J., M.V. SAUER & R.A. LOBO. 1990. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil. Steril. 53: 870–874. 29. MACKLON, N.S. & B.C. FAUSER. 2000. Impact of ovarian hyperstimulation on the luteal phase. J. Reprod. Fertil. Suppl. 55: 101–108. 30. HORCAJADAS, J.A., A. RIESEWIJK, J. POLMAN, et al. 2004. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Submitted to Mol. Hum. Reprod. 31. MIRKIN, S. & S. OEHNINGER. 2004. Gene expression profiling of the human endometrium during the early and mid luteal phase: analysis of natural and COH (controlled ovarian hyperstimulation) cycles. J. Soc. Gynecol. Invest. 11 (Suppl.): 307A. 32. FIRE, A. et al. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. 33. WAY, D.L., D.S. GROSSO, J.R. DAVIS, et al. 1983. Characterization of a new human endometrial carcinoma (RL95-2) established in tissue culture. In Vitro 19: 147–158. 34. TINEL, H., H.W. DENKER & M. THIE. 2000. Calcium influx in human uterine epithelial RL95–2 cells triggers adhesiveness for trophoblast-like cells. Model studies on signalling events during embryo implantation. Mol. Hum. Reprod. 6: 1119–1130. 35. JOHN, N.J., M. LINKE & H.W. DENKER. 1993. Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers. In Vitro Cell. Dev. Biol. Anim. 29: 461–468. 36. THIE, M., P. FUCHS, S. BUTZ, et al. 1996. Adhesiveness of the apical surface of uterine epithelial cells: the role of junctional complex integrity. Eur. J. Cell Biol. 70: 221–232.
