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Global Gene Expression Analysis to Identify Molecular Markers of Uterine Receptivityand Embryo Implantation

Jeff Reese1,3, Sanjoy K. Das2,3, Bibhash C. Paria1,3 , Hyunjung Lim3, Haengseok Song3,Hiromichi Matsumoto3, Kevin L. Knudtson4, Raymond N. DuBois5 and Sudhansu K. Dey3

1Departments of Pediatrics, 2Obstetrics and Gynecology, and 3Molecular and IntegrativePhysiology, University of Kansas Medical Center, Kansas City, KS 66160.4Department of Internal Medicine, Diabetes and Endocrinology Research Center, University ofIowa, DNA Facility, 323 Eckstein Medical Research Bldg., Iowa City, IA 52242.5Departments of Medicine and Gastroenterology, Room C-2104, MCN BuildingVanderbilt University Medical Center, Nashville, TN 37232

Running Title: Markers of Uterine Receptivity during Implantation

Corresponding author: S. K. Dey, Department of Molecular and Integrative Physiology,University of Kansas Medical Center, MRRC 3013, 3901 Rainbow Blvd. Kansas City, KS,66160. Phone: (913) 588-6213; Fax: (913) 588-5677

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 10, 2001 as Manuscript M107563200 by guest on June 2, 2020

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Summary

Infertility and spontaneous pregnancy losses are an enduring problem to women’s health.

The establishment of pregnancy depends on successful implantation, where a complex series of

interactions occur between the heterogeneous cell types of the uterus and blastocyst. Although a

number of genes are implicated in embryo-uterine interactions during implantation, genetic

evidence suggests that only a small number of them are critical to this process. To obtain a

global view and identify novel pathways of implantation, we used a dual screening strategy to

analyze the expression of nearly 10,000 mouse genes by microarray analysis. Comparison of

implantation and interimplantation sites by a conservative statistical approach revealed 36

upregulated genes and 27 downregulated genes at the implantation site. We also compared the

uterine gene expression profile of progesterone-treated, delayed implanting mice to that of mice

in which delayed implantation was terminated by estrogen. The results show upregulation of

128 genes and downregulation of 101 genes after termination of the delayed implantation. A

combined analysis of these experiments showed specific upregulation of 27 genes both at the

implantation site and during uterine activation, representing a broad diversity of molecular

functions. In contrast, the majority of genes that were decreased in the combined analysis were

related to host immunity or the immune response, suggesting the importance of these genes in

regulating the uterine environment for the implanting blastocyst. Collectively, we identified

genes with recognized roles in implantation, genes with potential roles in this process and genes

whose functions have yet to be defined in this event. The identification of unique genetic

markers for the onset of implantation signifies that genome-wide analysis coupled with

functional assays is a promising approach to resolve the molecular pathways required for

successful implantation.

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Introduction

Early loss of pregnancy is a significant clinical problem for women and their health care

providers. Successful embryo implantation depends upon bi-directional communication between

the blastocyst and the uterus. Recent advances in our ability to define complex biochemical and

genetic pathways have begun to unfold the molecular mechanisms underlying the regulation of

implantation (1,2). Although numerous factors involved in implantation have been identified (3-

5), targeted mutations in mice have revealed only a few genes that are essential to this process

(6-11).

Implantation is defined as a process by which the blastocyst makes the first physical and

physiological contact with the maternal uterine luminal epithelium. Under the influence of

ovarian steroid hormones, an optimal "window" for implantation is created when the activated

state of the developing blastocyst overlaps with a brief period of uterine receptivity (12,13). In

pregnant mice, removal of preimplantation estrogen secretion by ovariectomy postpones the

onset of implantation and induces blastocyst dormancy (14). A single injection of estrogen can

reactivate the signaling network resulting in implantation in the progesterone (P4)-primed uterus.

This model, termed delayed implantation , has provided insights into the cellular

communication pathways between the uterine and embryonic cell types and led to the

identification of embryo-induced uterine genes that correspond to early steps in the establishment

of pregnancy (2).

The blastocyst and uterus generate various factors during implantation, but it is likely that

the molecular "cross-talk" between them involves many more yet unknown factors. Indeed, it is

more realistic to view the process of implantation as a condition of equilibrium in the

upregulation and downregulation of a diverse set of genes. Identification of other essential

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regulatory steps is necessary to further understand the biologic basis for the establishment of

pregnancy or the underlying causes of pregnancy failures. In this respect, two recent reports

highlight gene expression profiling in the post-implantation period (15,16). However to our

knowledge, no such analysis at the onset of implantation has been reported. To address this

issue, we employed two complementary strategies using murine GeneChip Expression Arrays

(Affymetrix, Santa Clara, CA) to determine global gene expression profiles during implantation

in mice. The first approach compared RNAs from implantation and interimplantation sites to

identify genes that are specifically up- or downregulated at the implantation site. A second

analysis compared RNA from P4-primed pregnant uteri with delayed implantation with that of

P4-primed uteri after estrogen activation. Several genes with known expression status at the

implantation site were detected. In addition, cell-specific expression patterns in the implantation

and interimplantation site were observed for four candidate genes, confirming the validity of this

approach. Mice with delayed implantation expressed a large number of genes associated with

immunity or immune responses. The suppression of these genes at the implantation site also

suggests that this site is immunologically privileged during early pregnancy and that modulation

of the immune response is an active process during implantation. There were 81 genes whose

expression was affected in both analyses. These results suggest that pan-genomic gene

expression profiles are a promising approach for the identification of markers of uterine

receptivity during implantation, and that multiple screening strategies yield a distinct set of

candidate genes that appear to be critical in early pregnancy.

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Experimental Procedures

Animals

All experiments were conducted in accordance with NIH standards for the care and use of

animals. Mice were killed by cervical dislocation or were anesthetized for survival surgery with

avertin. Adult virgin CD-1 female mice were mated with fertile males of the same strain to

induce pregnancy (day 1 = vaginal plug).

Increased stromal vascular permeability at the site of initial contact of the blastocyst with

the uterine luminal epithelium is the first visible sign of the implantation process (2300-2400 h

on day 4) and can be monitored by an intravenous injection of a blue dye (12,13,17). For the

first analysis, implantation and interimplantation sites were divided by sharp dissection at 2300-

2400h on day 4 (n=12 mice). Uterine segments included uterine myometrium, stroma and

epithelium. Implantation sites also included blastocysts. Because there are normal variations in

the timing of implantation, only those uteri with uniformly distinct blue bands were included,

while regions with embryo crowding were discarded (Figure 1).

In the second analysis, P4-primed uteri of mice with delayed implantation were compared

to P4-treated uteri after estrogen activation. To induce delayed implantation, pregnant females

were ovariectomized on the morning of day 4 of pregnancy (0900 h) and given daily

subcutaneous injections of P4 (2 mg/mouse in 0.1 ml sesame oil) from days 5-7 (13,14). To

terminate delayed implantation and induce blastocyst activation, a single subcutaneous injection

of estradiol-17β (25 ng/mouse in 0.1 ml oil) was given to one group of animals at the same time

as P4 injection on the third day of delay (day 7). Whole uteri (n=6) were collected from each of

these two groups 12 h after the last injection of steroids.

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Sample Preparation

Uterine tissues were flash frozen at the time of dissection and stored at —80C. Specimens

of implantation and interimplantation regions, and of delayed and activated uteri were separately

pooled and total RNA was extracted in Trizol reagent (Life Technologies, Gaithersburg, MD)

according to the manufacturer’s recommendations. An additional RNA cleanup step was

performed using the Qiagen (Chatsworth, CA) RNeasy total RNA isolation kit. Total RNA (10

µg) from each group was used to generate cDNA using the Superscript Choice System (Life

Technologies). First-strand synthesis was performed using a T7-(dT)24 primer (Sigma-Genosys,

Woodlands, TX). The resulting cDNA was used to synthesize biotin-labeled cRNA via in vitro

transcription (IVT) using the ENZO BioArray HighYield RNA transcript labeling kit

(Affymetrix, Inc.). The cRNA was fragmented in fragmentation buffer (40 mM Tris (pH 8.1),

100 mM KOAc, and 30 mM MgOAC, final concentration) by heating to 94C for 35 min. The

quality of each cRNA preparation was assessed by analysis with a Test2 array (Affymetrix, Inc.)

and all preparations met Affymetrix s recommended criteria for use on their expression arrays.

GeneChip Hybridization and Statistical Analysis

Each cRNA (15 µg) preparation was used to inoculate Murine U74A GeneChip

Expression Arrays (Affymetrix, Inc.) and the hybridization, staining, scan and analysis were

conducted per recommended protocols. An Affymetrix software filter was applied to mask

transcripts with incorrect orientation in the public databases. Although numerous ESTs were

differentially expressed, only those transcripts with known identities are reported herein. Three

replicate hybridizations were performed using each of the four pooled RNA samples

(implantation, interimplantation, delayed, activated) to establish the reproducibility of our

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results. Alterations in RNA transcript levels were analyzed using the Affymetrix Analysis Suite

4.0 software. Differences in levels of fluorescent intensity, which represents levels of

hybridization, between the 25 base pair oligonucleotides and their mismatches, were analyzed by

multiple decision matrices to determine the Presence or Absence of gene expression and to

derive an Average Difference score representing the relative level of gene expression.

Background and noise corrections account for nonspecific binding and minor variations in

hybridization conditions. Values for the mean and standard deviation of the three replicate

Average Difference scores were calculated for each gene on the GeneChip. Comparison

between groups was performed by Student s t-test (P<0.05 considered significant). The Fold

Change in expression between groups was calculated from the mean Average Difference scores.

A second approach was used to verify the identification of differentially expressed genes.

Affymetrix algorithms produced statistical decisions for an Increase or Decrease in expression

when comparing the hybridization results of any two samples. Thus, paired comparisons of three

replicate hybridizations resulted in 9 possible outcomes for a single analysis (e.g., implantation

vs interimplantation). Transcripts with statistically significant expression (by t-test) that were

also differentially expressed in 4 or more of the nine pair-wise comparisons (Increase or

Decrease) were considered candidates for further evaluation. Others have used this counting

approach using somewhat higher cut-off points (18). However at higher threshold levels, we

noted that a number of genes, known to be expressed at the implantation site, were excluded.

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In Situ Hybridization

In situ hybridization was performed as previously described (17). 35S-labeled cRNA

probes were generated for Sik-SP, Bip, Cyp1b1, and EP4 with the appropriate polymerases (19-

21).

Results

Gene expression is altered at the onset of natural implantation (implantation vs

interimplantation sites)

Hybridization intensity to the arrays was uniform for housekeeping genes such as

GAPDH, cyclophilin, and a large number of ribosomal proteins, indicating that the expression

data in the array hybridization experiments are consistent with other standards for studying gene

expression.

The relative levels of gene expression at the implantation and interimplantation sites were

first compared by plotting the Average Difference values for one individual array hybridization

experiment against another and determining the Presence or Absence of gene expression for the

entire array (Figure 2). Genes that were considered Absent or Marginally expressed were

widely dispersed (black points), whereas genes that were declared Present in both hybridizations

were tightly grouped (red points). An increase of more than 2-fold in the Average Difference

score indicated genes with significantly higher expression at the implantation site (above the

curve) or at the interimplantation region (below the curve) (Figure 2). These results show that a

vast majority of the genes on the chip have similar expression patterns (Present, Absent or

Marginal) and their relevance to implantation is questionable. A small number of genes were

Present in both (red points) or Present in one but Marginal or Absent in the other (blue points)

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that also had greater than a two-fold difference in the level of expression. These genes were

considered as potential candidates for further evaluation. Surprisingly, a symmetrical

distribution of these candidate genes was observed, suggesting that an equal number of genes are

upregulated in the implantation and interimplantation regions.

Genes with statistically significant differences in expression at implantation versus

interimplantation sites were identified by comparison of replicate hybridizations and by a

statistical decision for an Increase or a Decrease in gene expression. By t-test alone, there were

293 upregulated and 370 downregulated genes at the implantation site. A second statistical

approach was performed to provide an additional objective analysis. We used a threshold value

of 4 of the 9 possible outcomes, which resulted in 49 upregulated and 60 downregulated genes

and included several known genes that are expressed during implantation or are considered

biologically relevant to the implantation process. A combination of t-test and counting

approaches identified 36 genes that were upregulated at the implantation site and 27 genes that

were downregulated (Tables 1, 2).

Differentially expressed genes were categorized based on the best available information

regarding their biologic functions. Genes with multiple functions were assigned to a single

category. Many genes that are known to be associated with the implantation process fall into

categories similar to the genes we detected with increased expression at the implantation site,

including growth factors/cytokines and their receptors, transcription factors, genes encoding

structural proteins, or genes associated with cell proliferation. We also observed upregulation of

a group of calcium-related genes, including Bip, Sik similar protein (Sik-SP), and calcineurin-

and calcyclin-related proteins. Genes encoding Bip and Sik-SP showed highly localized

expression at the implantation site, confirming our array results (Figure 3). These genes are of

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interest since calcium is an essential modulator of enzyme functions and signal transduction, and

there is evidence that genes involved in calcium regulation play an important role during

implantation (21-25).

Previous efforts to identify novel genes during the periimplantation period have primarily

focused on the implantation site, with less attention paid to genes that are expressed at the

interimplantation region. Genes with increased expression at the interimplantation site may act

to guide the blastocyst to specific sites for implantation or be important for embryo spacing.

Aberrant expression of these genes may be as detrimental to implantation as the loss of genes

that are expressed at the implantation site. In our gene array experiments, we observed a 5-fold

decrease in levels of Cyp1b1 expression at the implantation site. Cyp1b1 is a member of the

cytochrome P450 system that converts primary estrogens to their active metabolites,

catecholestrogens, which are important for blastocyst activation (20). In situ hybridization

showed that Cyp1b1 is restricted to the subepithelial stroma of the interimplantation region

(Figure 3), in agreement with the decreased expression levels seen in the array experiments. A

similar expression pattern was noted for the PGE2 receptor subtype, EP4, which showed a nearly

2-fold decrease by gene array. The overall diversity of genes with this pattern of expression

suggests that further investigation of interimplantation-specific genes is warranted.

Gene expression is altered in an experimental model of implantation (delayed versus

induced implantation)

To identify genes that are upregulated at the time of blastocyst activation for

implantation, RNAs from P4-primed delayed implanting uteri and P4-primed uteri after estrogen

treatment were obtained for comparative analysis. Scatterplot analysis of genes considered

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Absent or Present showed that overall gene expression patterns in delayed and activated uteri

were closely correlated (Figure 2). The tight distribution of overall expression is similar to that

seen in the implantation versus interimplantation analysis. The overall similarity of the two

different scatterplots is not surprising, since the ultimate outcome, implantation, is similar in both

models and the experiments were designed to be complementary and provide a dual approach to

identify novel implantation-specific genes.

Statistical analysis by t-test alone identified 409 upregulated and 550 downregulated

genes in comparisons of delayed versus activated uteri. However, our combined statistical

approach reduced the number of candidate genes to 128 upregulated and 101 downregulated

transcripts after termination of the delayed implantation by estrogen (Supplement, Tables 1, 2).

Mice with delayed implantation expressed a large number of genes associated with host

immunity or the immune response (n = 48) compared to the uteri of mice after estrogen

activation (n = 3) (Supplement, Table 1). Furthermore, nearly 50% of the genes with

significant expression during delayed implantation have some immune-related function (48/101).

Specific roles for these genes during implantation have not been described.

There were striking differences in the functional categories of delayed versus activated

genes. In general, more DNA processing, cell cycle-associated genes and a larger number of

enzymes were observed after initiating the process of implantation (Supplement, Table 2). This

shift in gene diversity suggests that embryonic activation and uterine preparation for the onset of

implantation is mediated by a subset of genes that requires estrogen. In this respect, the gap

junction proteins, connexin 26 and connexin 43, are implicated in implantation and their

regulation is influenced by steroid hormones (26). In our array experiments, the expression of

connexin 26 increased over 8-fold, while the expression of connexin 43 showed a greater than

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2.5-fold increase after estrogen activation. The PGE2 receptor subtype EP2, is the only other

gene that was detected in our delayed versus activated gene array whose expression is also

known to be induced at the site of the implanting blastocyst after termination of delayed

implantation (27). Overall, our results show that delayed implantation is a valuable model to

dissect the molecular aspects of implantation, and compare the distribution of genes that are

expressed during dormancy or active implantation.

Comparison of gene expression in natural and delayed implanting models of pregnancy

Although implantation is the eventual outcome of the both models, it is unclear whether

molecular mechanisms underlying natural implantation and induced implantation are similar. To

determine whether genes upregulated in the delayed uterus after estrogen activation are similar to

the group of genes with increased expression at the implantation site, the results of both

hybridization analyses were combined to highlight genes that were differentially expressed in

both comparisons. The intersection of these sets revealed 244 genes with significantly altered

expression (t-test only) in both models (Figure 4). Considering only those transcripts with a

greater than 2-fold difference in Average Difference scores, there were 54 genes that had

significant expression at the interimplantation site and during progesterone-primed implantation

delay (Table 3). By similar criteria, we also observed 27 genes that had increased expression at

the implantation site and after estrogen activation (Table 4). Among these, only connexins 26

and -43, amphiregulin and nexin-1 are associated with implantation (26,28,29). The importance

of the remaining genes in implantation awaits further investigation. Nonetheless, these results

show that a dual screening strategy identified a small number of candidate genes that are likely to

have significant roles in implantation.

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Discussion

The survival of any species depends on stable mechanisms for reproduction. Thus, it is

assumed that essential mechanisms for embryo implantation must be supported by redundant

pathways to ensure the conception of new offspring. This predicts that a large number of genes

that are important for implantation remain to be identified. Previous approaches to investigate

implantation have generally relied on the analysis of individual candidate genes or gene families.

We used DNA microarray technology to screen a large cross-section of the murine genome to

identify novel implantation-specific genes. Our present investigation has identified genes with

recognized roles in implantation, genes with potential roles in this process and genes whose

functions have yet to be defined. In addition, a small number of genes showed significantly

altered expression during both natural and induced implantation.

The process of implantation involves cell-cell interactions between the blastocyst and

uterus, cell-type specific proliferation and differentiation of the uterus, and immunological

responses of the mother to the semi-allogenic embryo. Our data show a broad diversity of

genes that are modulated during implantation. A recent report described a microarray-based

approach to identify genes in the uterus during the post-implantation period (15). They observed

192 genes with increased expression and 207 genes with decreased expression levels. Similar to

our results, genes typically showed 1.5 to 3-fold induction at the implantation site. Surprisingly,

there are very few genes that were mutually identified in both studies. However, they compared

uterine gene expression profiles on the evening of day 4 to those on day 6 of pregnancy. With

this approach, both implantation and interimplantation regions would be included in a single

sample so that no distinction could be made for gene localization around the implanting

blastocyst. Our in situ hybridization results show the importance of differentiating these two

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sites. In addition, uterine horns were flushed in this study (15), and the uteri were split and the

luminal surface was scraped to remove conceptuses. Physical disruption of the uterine

epithelium would likely result in different gene expression profiles. We designed our

experiments to include the implanting blastocyst in both analyses. The presence of blastocysts in

our first (implantation vs interimplantation) and second (delayed vs activated) analyses serves to

strengthen our approach by including the embryonic genome and profiling the expression of

embryonic factors that may be significant to implantation. Moreover, we analyzed intact uterine

horns to preserve an undisturbed relationship between the uterine myometria, stroma, and

epithelium and its intimate contact with the blastocyst. In contrast, Yoshioka et al. (15), focused

on two distinct time points in pregnancy, resulting in the identification of genes with differential

expression between implantation and decidualization, rather than implantation site-specific

genes.

We also identified genes that are differentially expressed in delayed versus estrogen-

activated uteri. There are previous reports that genes encoding the EGF-like growth factors,

cytokines and other inflammatory mediators, extracellular matrix proteins, cell cycle molecules

and immunoregulatory proteins are expressed at the site of estrogen-induced implantation in a

pattern similar to their expression in natural implantation (17,27,30,31-38). In this respect, our

microarray results are consistent with the previously described expression of several steroid

hormone-sensitive and implantation-specific genes, including Cyp1b1, connexin 26 and

connexin 43, Sik-SP, the prostaglandin receptor EP2, and histidine decarboxylase

(20,21,26,27,39). However, we failed to detect the anticipated changes in the expression of

cyclooxygenase-2, perlecan, trophinin, HB-EGF, LIF and a number of other hormone-

responsive, implantation-associated genes at the implantation site. This is perhaps due to their

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highly restricted expression around the implanting blastocyst, resulting in the dilution of

implantation-specific RNAs in a large pool of RNAs derived from other uterine cell types.

Indeed, we have previously observed that changes in the expression of cyclooxygenase-2 and

HB-EGF and several other implantation-specific genes could not be detected by Northern

hybridization, but showed discrete upregulation at the implantation site as observed by in situ

hybridization (17,30-32).

A significant shift was noted in the diversity of genes expressed in delayed implantation

uteri compared to estrogen-activated uteri. In particular, mice with delayed implantation

expressed a large number of genes associated with immunity and/or the immune response. The

suppression of these genes at normal implantation sites and after estrogen-activation (Table 3)

suggests that the implantation site is immunologically protected. Although large numbers of

maternal natural killer cells are recruited to the uterine deciduum 48 hours after embryo

attachment (40), leukocytes and other bone marrow-derived cells migrate away from the site of

blastocyst attachment at an earlier time during the onset of implantation (41,42). Reduced

expression of numerous immune-related genes at the implantation site suggests that

immunomodulatory cells, even if present, remain quiescent with the onset of implantation. Thus,

reduced expression of these genes is an important finding, since it is still unclear how the embryo

escapes maternal immunological responses during pregnancy (43). The mechanism of

downregulation of these genes at the implantation site is unknown, but elaboration of

immunosuppressive signals from active blastocysts cannot be ruled out (44,45). Our data

suggest that modulation of the immune response is an active process prior to or during blastocyst

implantation.

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There were 81 genes with differential expression at the implantation site during both

natural and induced implantation, suggesting their importance for implantation. Connexin 26

and -43 are gap junction proteins that are influenced by ovarian steroids, accumulate in the

stroma around the implantation site, and are upregulated during experimentally induced

decidualization (26). Their expression was significantly increased at the implantation site and in

the uterus after estrogen activation. These observations coincide with a growing body of

evidence for the role of structural genes in the establishment of pregnancy (46). Amphiregulin

and nexin-1 were the only other implantation-associated genes that had increased expression

during both natural and induced implantation (Table 4). Amphiregulin is a member of the EGF-

family of growth factors that becomes intensely localized to the uterine luminal epithelium

surrounding the blastocyst at the onset of implantation (28). Nexin-1 is a serine protease

inhibitor that regulates processing of plasmin, thrombin, urokinase, plasminogen activators and

other proteases, and is upregulated during implantation (29). Tight regulation of proteases and

their inhibitors is considered an important aspect of embryo-uterine interaction at the site of

implantation (47). Collectively, our results suggest that other genes identified in the composite

analysis are physiologically relevant to the implantation process.

The remaining genes identified by the combined screening approach do not yet have any

recognized roles in implantation. However, many of the genes identified with increased

expression in both analyses (Table 4) are associated with cell proliferation (spermidine synthase,

PCNA), cell cycle regulation (cyclin E2), inflammation or tumor biology (ribonucleotide

reductase M2, NM23), and DNA replication, synthesis, and/or repair (ribonucleotide reductase

M2, CDC46 and Mcm homologs, topoisomerase, PCNA, FEN-1). Since the process of

implantation is considered a proinflammatory response and involves uterine proliferation,

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differentiation and apoptosis, these genes could well be very important for various aspects of this

process. It is interesting to note that PCNA can directly bind to FEN-1 to stimulate its nuclease

activity during base excision repair of damaged DNA (48), and that ribonucleotide reductase is

the rate-limiting enzyme that provides the essential deoxynucleotides for new DNA synthesis

and DNA repair. All three of these genes showed upregulated expression during implantation

and may act in a concerted fashion for remodeling of the uterine epithelium and stroma during

blastocyst attachment and invasion. However, the diverse functions for each of these genes

preclude any speculation as to their specific significance to implantation.

In summary, we employed a global gene expression strategy to identify novel genes in

the implantation process. Our results show an equal number of genes that are upregulated or

downregulated at the implantation site and a small number of candidate genes that have

significant changes in their expression during natural and estrogen-induced implantation. A

better understanding of the molecular mechanisms of embryo-uterine interactions during

implantation will provide insight into the high rate of spontaneous pregnancy losses. Attempts to

resolve these complex signaling networks will likely benefit from a genome-wide approach

coupled with functional assays, and facilitate new methods to address infertility and

contraceptive challenges in women’s health.

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Acknowledgements

We are grateful to Jian Tan and Xuemei Zhao for their assistance with in situ hybridization andto Richard Melvin for his assistance with microarray analysis. This work was supported in partby The Mellon Foundation, National Institutes of Health grants HD37677 (J.R.), ES07814(S.K.Das), HD37394 (B.C.P.), DK47297 (R.N. DuBois), HD12304, HD29968 (S.K.Dey) and byan NICHD Mental Retardation and Developmental Disabilities center grant (HD02528).S.K.Dey is the recipient of an NICHD MERIT award.

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Table 1. Genes with significantly increased expression at the implantation site.RNAs from implantation and interimplantation sites were obtained for GeneChip hybridization.The fold change in gene expression was determined by comparison of mean Average Differencescores. Differentially expressed genes were identified by two statistical methods. *, genes withknown expression during the peri-implantation period.

GeneBank

Accession No. Gene Name Fold Change Functional CategoryX83601 Pentraxin related gene 5.76 Inflamm/primary responseY11666 Hexokinase II 3.16 EnzymeX59769 * Interleukin 1 receptor, type II 3.07 ReceptorAF091432 Cyclin E2 2.88 Cell cycleM63801 *Connexin 43 (alpha-1 gap junction) 2.58 Structural proteinM14223 Ribonucleotide reductase M2 subunit 2.55 DNA/chromatin-relatedX62154 Mcm 3 homologue 2.14 DNA/chromatin-relatedM20658 * Interleukin 1 receptor, type I 2.12 ReceptorL40406 Heat shock protein, 105 kDa 2.07 ChaperoneM35970 Tumor metastatic process-associated protein (NM23) 2.02 NeoplasiaD55720 Karyopherin ( importin) alpha 2 2.02 Protein proc./transportJ04627 NAD-dependent methylenetetrahydrofolate dehydr. 1.94 EnzymeX01756 Cytochrome c, somatic 1.91 EnzymeX72310 Transcription factor Dp 1 1.90 Transcription factorM95604 Snail homolog 1.83 Transcription factorM81445 *Connexin 26 (beta-2 gap junction) 1.82 Structural proteinAF053232 *SIK similar protein 1.79 Calcium-relatedZ29532 *Follistatin 1.77 Growth factor/cytokineM61007 CCAAT/enhancer binding protein beta (C/EBP) 1.75 Transcription factorU14648 Splicing factor, arginine/serine-rich 1 0 1.72 RNA processingM31885 Inhibitor of DNA binding 1 1.69 DNA/chromatin-relatedL26320 Flap structure specific endonuclease 1 (FEN-1) 1.66 DNA/chromatin-relatedD26089 Mcm 4 homolog (cdc21) 1.65 DNA/chromatin-relatedZ67748 Spermidine synthase 1.61 EnzymeX56045 RAN binding protein 1 1.60 Protein proc./transportX56304 *Tenascin C 1.60 Structural proteinD85904 Apg-2 1.59 ChaperoneM31418 Interferon activated gene 202 1.55 Growth factor/cytokineU97327 Calcyclin binding protein 1.54 Calcium-relatedAF041476 BAF53a 1.54 DNA/chromatin-relatedJ05479 Calcineurin catalytic subunit 1.54 Calcium-relatedX91656 Splicing factor, arginine/serine-rich 3 (SRp20) 1.53 RNA processingU27830 mSTI1 1.52 ChaperoneJ04633 *Heat shock protein 8 6 1.49 ChaperoneX57800 Proliferating cell nuclear antigen (PCNA) 1.45 Cell cycleAJ002387 *BiP 1.40 Calcium-related, Chaperone

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Table 2. Genes with significantly decreased expression at the implantation site.RNAs from implantation and interimplantation sites were obtained for GeneChip hybridization.The fold change in gene expression was determined by comparison of mean Average Differencescores. Differentially expressed genes were identified by two statistical methods. By definition,genes with decreased expression at the implantation site also have increased expression in theinterimplantation region (see Figure 3). *, genes with known expression during the peri-implantation period.

GeneBank

Accession No. Gene Name Fold Change Functional CategoryAF080469 Putative glycogen storage disease type 1b protein -5.74 EnzymeX78445 *Cyp1-b-1 cytochrome P450 -5.08 EnzymeM88694 Thioether S-methyltransferase -3.78 EnzymeAJ242912 Disintegrin metalloprotease (decysin) -3.57 EnzymeM20878 Murine T-cell receptor beta-chain mRNA, VDJ region -3.07 Immune-relatedU37465 Protein tyrosine phosphatase, receptor type -2.66 EnzymeX81581 * Insulin-like growth factor binding protein 3 -2.43 HormoneU77364 Hoxd4 -2.37 Transcription factorAJ131851 Cathepsin F -2.37 EnzymeU20366 *Hoxa11, opposite strand transcript -2.23 Transcription factorL47335 Branched chain alpha ketoacid decarboxylase E1a -2.21 EnzymeU65747 Interleukin 13 receptor, alpha 2 -2.03 ReceptorD13458 *Prostaglandin E receptor EP4 -1.97 ReceptorU88909 Apoptosis inhibitor 2 -1.93 ApoptosisAB028921 NAKAP95 -1.89 Protein proc./transportX69942 Enhancer trap locus 1 -1.85 Transcription factorAB006960 mRECK -1.85 NeoplasiaU09504 Thyroid hormone receptor alpha -1.81 ReceptorU92068 RecA-like protein (mREC2) -1.81 DNA/chromatin-relatedAF085745 Nuclear orphan receptor LXR-alpha -1.78 ReceptorM26071 Coagulation factor III -1.72 HematologicAF031127 Inositol trisphosphate receptor type 2 (Itpr2) -1.67 ReceptorAF089751 *ATP receptor P2X4 subunit -1.64 ReceptorU32329 *Endothelin receptor type B -1.62 ReceptorY00629 Histocompatibility 2, T region locus 2 3 -1.55 Immune-relatedU49507 Lisch7 -1.44 Transcription factorU95826 Cyclin G2 -1.34 Cell cycle

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Table 3. Fold change of genes that showed decreased expression at the implantation site andafter initiation of estrogen-induced implantation.Differentially expressed genes in the first (implantation vs interimplantation) and second (activatedvs delayed) analyses were compared to identify common patterns of gene regulation. Genes withsignificant alterations in expression level (t-test only) and at least a 2-fold change in one of theanalyses are shown. Negative fold change values indicate decreased expression relative to thebaseline condition (interimplantation sites or delayed uteri). By definition, these genes also haveincreased expression at the interimplantation site and during P4-induced delayed implantation. *,genes with known expression during the peri-implantation period.

GeneBank

Accession No. Gene/EST name

Implantationvs.

Interimplantation

Activatedvs.

Delayed

Gene

FunctionM81591 Membrane metallo endopeptidase -2.15 -25.65 EnzymeAJ007971 IIGP protein -1.72 -13.53 Unspecified functionL38444 T-cell specific protein mRNA -1.29 -12.68 Immune-relatedD44456 Low molecular mass polypeptide complex -4.53 -8.24 Unspecified functionL02914 Aquaporin 1 -1.56 -6.10 TransporterX98055 Glutathione S-transferase, theta 1 -1.46 -4.63 EnzymeJ05663 Androgen regulated vas deferens protein -1.88 -4.41 Gamete/sex-specificAJ007970 mGBP-2 protein -1.30 -4.35 Unspecified functionX67210 Rearranged Ig gamma 2b heavy chain -1.17 -4.18 Immune-relatedY00629 Histocompatibility 2, T region locus 2 3 -1.55 -4.14 Immune-relatedAF042798 Immunoglobulin heavy chain, CDR3 region -1.35 -4.10 Immune-relatedAJ131851 Cathepsin F -2.37 -3.94 EnzymeU73029 Interferon regulatory factor 6(mirf6) -1.30 -3.88 Transcription factorU55641 Anti-DNA immunoglobulin light chain IgG -1.33 -3.84 Immune-relatedAJ007972 GTPI protein -1.25 -3.72 Unspecified functionU09504 Thyroid hormone receptor alpha -1.81 -3.44 ReceptorZ70661 Artificial single chain antibody scFv -1.23 -3.26 Immune-relatedAF059706 Immunoglobulin heavy chain VDJ region -1.32 -3.17 Immune-relatedL33943 Germline Ig variable region heavy chain precursor -1.32 -3.05 Immune-relatedAF025445 Ig heavy chain variable region precursor -1.25 -2.94 Immune-relatedJ04696 *Glutathione S-transferase class mu (GST5-5) -1.16 -2.93 EnzymeM21932 MHC class II H2-I-A-beta (k haplotype) -1.70 -2.84 Immune-relatedU73037 Interferon regulatory factor 7 (mirf7) -1.33 -2.78 Transcription factorX02466 Germline immunoglobulin V(H)II (H17) -1.36 -2.76 Immune-related L33954 Germline Ig variable region heavy chain precursor -1.18 -2.72 Immune-related L19932 Transforming growth factor beta induced -1.25 -2.71 Growth factor/cytokineU82758 Lung-specific membrane protein -1.67 -2.69 Unspecified functionU30241 Anti-DNA antibody Ig kappa chain, V-J region -1.25 -2.65 Immune-relatedX16740 Immunoglobulin heavy chain variable -1.28 -2.64 Immune-relatedX58609 MHC (Qa) Q2-k class I antigen -1.30 -2.63 Immune-relatedAJ223208 Cathepsin S -1.36 -2.59 EnzymeL47335 Branched chain α ketoacid decarboxylase E1a -2.21 -2.49 EnzymeX94418 IgA V-D-J-heavy chain (2F7) -1.22 -2.38 Immune-relatedX00651 Ig-kappa light chain V-J kappa 5 joining region -1.20 -2.33 Immune-relatedY13090 Caspase-12 -1.91 -2.31 ApoptosisY14296 BTEB-1 transcription factor -1.78 -2.23 Transcription factor

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L09192 Pyruvate carboxylase -1.44 -2.21 EnzymeAJ132922 Methyl-CpG-binding protein -1.30 -2.20 DNA/chromatin-relatedU27462 BS4 peptide -1.27 -2.20 Unspecified functionL20315 MPS1 -1.36 -2.20 Unspecified functionM13226 *Granzyme A -1.36 -2.15 EnzymeM24417 P glycoprotein 3 -1.61 -2.14 Structural proteinU32329 *Endothelin receptor type B -1.62 -2.13 ReceptorX00496 Ia-associated invariant chain -1.32 -2.11 Immune-relatedAF031955 Krupple-related zinc finger protein (Emzf1) -2.22 -2.11 Transcription factorAF072249 Methyl-CpG binding protein MBD4 (Mbd4) -2.30 -2.09 Transcription factorD14883 CD82 antigen -1.23 -2.06 Immune-relatedM26071 Coagulation factor III -1.72 -2.05 HematologicX88903 Variable light chain -1.24 -2.04 Immune-relatedY08135 ASM-like phosphodiesterase 3a -1.43 -2.03 EnzymeU42467 *Leptin receptor -1.60 -2.03 ReceptorAB028921 NAKAP95 -1.89 -2.01 DNA/chromatin-relatedAF029261 Ig kappa light chain variable region precursor -1.20 -2.01 Immune-relatedAF047704 Tuftelin -1.23 -2.00 Structural protein

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Table 4. Fold change of genes that showed increased expression at the implantation site andafter initiation of estrogen-induced implantation.Differentially expressed genes in the first (implantation vs interimplantation) and second (activatedvs delayed) analyses were compared to identify common patterns of gene regulation. Genes withsignificant alterations in expression level (t-test only) and at least a 2-fold change in one of theanalyses are shown. *, genes with known expression during the peri-implantation period.

GeneBankAccession No. Gene/EST name

Implantation

vs.Interimplantation

Activated

vs.Delayed

GeneFunction

AF091432 Cyclin E2 2.88 39.8 Cell cycleM81445 *Connexin 26 (beta-2 gap junction) 1.82 8.93 Structural proteinU01915 Topoisomerase (DNA) II alpha 1.44 7.56 DNA/chromatin-related M64086 Spi2 proteinase inhibitor (spi2/eb4) 1.27 6.38 InhibitorD87908 Nuclear protein np95 1.59 5.93 DNA/chromatin-relatedD26090 CDC46 protein 1.84 4.59 DNA/chromatin-relatedL26320 Flap structure specific endonuclease 1 (FEN-1) 1.66 3.80 DNA/chromatin-relatedX83601 Pentraxin related gene 5.76 3.68 Growth factor/cytokineAB025409 Sid1334p 1.61 3.13 Unspecified functionX62154 Mcm 3 homolog 2.14 3.04 DNA/chromatin-related M35970 Tumor metastatic process-associated (NM23) 2.02 3.03 NeoplasiaD86725 Mcm 2 1.63 2.97 DNA/chromatin-relatedM14223 Ribonucleotide reductase M2 subunit 2.55 2.94 DNA/chromatin-relatedX57800 Proliferating cell nuclear antigen (PCNA) 1.45 2.76 Cell cycleM63801 *Connexin 43 (alpha-1 gap junction) 2.58 2.72 Structural proteinL41352 *Amphiregulin 1.92 2.52 Growth factor/cytokineX70296 *Serine protease inhibitor 4 (nexin-1) 1.46 2.34 InhibitorD26089 Mcm 4 homolog (cdc21) 1.65 2.30 DNA/chromatin-relatedL40156 Surfactant associated protein D 1.52 2.26 Immune-relatedAJ005559 Small proline-rich protein 2A 1.16 2.24 Structural proteinU14648 Splicing factor, arginine/serine-rich 1 0 1.72 2.13 RNA processingAB027012 Galactokinase 1.42 2.11 EnzymeU42385 Fibroblast growth factor inducible 1 6 1.45 2.09 Growth factor/cytokine Z83956 Spermidine synthase 1.87 2.02 EnzymeL32752 Ran GTPase 1.47 2.02 Protein proc./transportV00755 Interferon beta 1.13 2.01 Growth factor/cytokineL40406 Heat shock protein, 105 kDa 2.07 1.39 Chaperone

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Figure Legends

Figure 1. Microarray analysis to identify novel implantation-specific genes. Increased vascularpermeability at the site of blastocyst attachment demarcates implantation sites after injection of amacromolecular blue dye. Top panel (natural pregnancy), comparison of RNAs fromimplantation and interimplantation sites at 2300 — 2400h on day 4 of pregnancy. Bottom panel(induced pregnancy), comparison of RNAs from mice with delayed implantation (progesteroneonly) and estrogen-induced termination of delayed implantation (progesterone + estrogen).

Figure 2. Levels of gene expression during implantation. Fluorescent-labeled cRNAs werehybridized to the Affymetrix murine U74A GeneChip. X and Y-axes indicate values for theAverage Difference score (arbitrary units) of a single GeneChip hybridization. The AverageDifference score reflects the relative level of gene expression for a given transcript. The resultsof a single representative experiment are shown as a scatterplot. Algorithms for data analysisindicate genes whose expression is Present, Absent, or Marginal in either experiment. Linesindicate 2-fold and 10-fold differences in the level of gene expression from the mean. Toppanel, Comparison of implantation and interimplantation RNAs. Transcripts that were calledMarginal or Absent (black, n = 6,600), Present in one but Absent or Marginal in the other (blue,n = 1317), or Present in both analyses (red, n = 4668) are shown at the time of initial blastocystattachment (day 4, 2300 - 2400h). Bottom panel, Comparison of uterine RNAs from delayedimplantation and estrogen-activated mice. Transcripts that were called Marginal or Absent(black, n = 7186), Present in one but Absent or Marginal in the other (blue, n = 2358), or Presentin both analyses (red, n = 3044) are shown.

Figure 3. Expression of implantation and interimplantation-specific genes. In situhybridization with 35S-labeled Sik-SP and Bip shows concentration of autoradiographic signals inthe stroma surrounding the implanting blastocyst (arrows) (top panel, 40X). Bip signals are alsonoted in the glandular and luminal epithelium, whereas Sik-SP signals are absent in the luminalepithelium and subepithelial stroma in the implantation bed. In contrast, 35S-labeled Cyp1b1and EP4 signals accumulated in the interimplantation regions, with marked decreases in signalintensity at the implantation site (bottom panel, 20X). s, stroma; le, luminal epithelium; ge,glandular epithelium; myo, myometrium.

Figure 4. Expression levels of candidate implantation-specific genes. X and Y-axes indicatevalues for the Average Difference score (arbitrary units). Combined analysis of AverageDifference scores from both implantation models (implantation vs interimplantation, delayed vsactivated) showing only those transcripts that have statistically significant differential expressionin both models (t-test only). Lines indicate 2-fold and 10-fold differences in the level of geneexpression from the mean.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Supplement Table 1. Genes with significantly decreased expression during estrogen-induced termination of delayed implantation.RNAs from P4-primed delayed implanting uteri and delayed implanting uteri after estrogentreatment were obtained for GeneChip hybridization. The fold change in gene expression wasdetermined by comparison of mean Average Difference scores. Differentially expressed geneswere identified by two statistical methods. Negative fold change values indicate decreasedexpression relative to estrogen-activated uteri. By definition, these genes also have higherexpression during delayed implantation. *, genes with known expression during the peri-implantation period.

GeneBank

Accession No. Gene Name Fold Change Functional CategoryX67210 Rearranged immunoglobulin gamma 2b heavy chain -30.11 Immune-relatedM81591 Membrane metallo endopeptidase -25.65 EnzymeM33266 Macrophage interferon inducible protein 10 (IP-10) -14.28 Growth factor/cytokineL38444 T-cell specific protein -12.68 Immune-relatedJ03298 *Lactotransferrin -10.34 Growth factor/cytokineU43084 Interferon-induced protein with tetratricopeptide repeats -8.98 Growth factor/cytokineX56602 Interferon-induced 15-KDa protein -8.58 Growth factor/cytokineD44456 Low molecular mass polypeptide complex subunit 2 -8.24 Unspecified functionU43086 Glucocorticoid-attenuated response 49 (GARG-49/IRG2) -7.90 Inflamm/primary responseX97991 *Calcitonin -7.05 Calcium-relatedL02914 Aquaporin 1 -6.10 TransporterM36120 Keratin complex 1, acidic -5.27 Structural proteinM84487 Vascular cell adhesion molecule -5.17 Structural proteinAF036738 Immunoglobulin heavy chain variable region precursor -4.61 Immune-relatedL14553 Ig light chain V-region CC49 rearranged -4.38 Immune-relatedAJ007970 mGBP-2 protein -4.35 Unspecified functionY00629 Histocompatibility 2, T region locus 2 3 -4.14 Immune-relatedAF042798 Immunoglobulin heavy chain, CDR3 region -4.10 Immune-relatedL28060 Ig B cell antigen receptor -3.95 Immune-relatedU73029 Interferon regulatory factor 6 (mirf6) -3.88 Transcription factorU55641 Anti-DNA immunoglobulin light chain IgG -3.84 Immune-relatedJ00592 Germline Ig lambda-2-chain V-region (V-J) -3.73 Immune-relatedAJ007972 GTPI protein -3.72 Unspecified functionAF059706 Immunoglobulin heavy chain VDJ region -3.17 Immune-relatedL28059 Ig B cell antigen receptor -3.17 Immune-relatedAF065324 Immunoglobulin heavy chain variable region -3.10 Immune-relatedM15520 Ig V-kappa10-Ars-A kappa chain -3.05 Immune-relatedX52643 Histocompatibility 2, class II antigen A, alpha -3.00 Immune-relatedAF025445 Immunoglobulin heavy chain variable region precursor -2.94 Immune-relatedJ04696 *Glutathione S-transferase class mu (GST5-5) -2.93 EnzymeX02466 Germline immunoglobulin V(H)II H17 -2.76 Immune-relatedU55576 Anti-DNA immunoglobulin light chain IgM -2.75 Immune-relatedM90766 Ig active joining chain (J chain) of the b allele -2.74 Immune-relatedL33954 Germline Ig variable region heavy chain precursor -2.72 Immune-relatedL19932 Transforming growth factor, beta induced, 68 kDa -2.71 Growth factor/cytokineX66402 *Matrix metalloproteinase 3 -2.69 Enzyme

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X14759 Homeobox, msh-like -2.68 Transcription factorU10410 Antineuraminidase single chain Ig VH and VL domains -2.66 Immune-relatedU30241 Anti-DNA antibody Ig kappa chain , V-J region -2.65 Immune-relatedX58609 MHC (Qa) Q2-k for class I antigen -2.63 Immune-relatedAF036736 Immunoglobulin heavy chain variable region precursor -2.63 Immune-relatedAJ22 3208 Cathepsin S -2.59 EnzymeX61232 Carboxypeptidase H -2.50 EnzymeU50413 PI 3-kinase, regulatory subunit, polypeptide 1 -2.49 Cell signaling/effectorsD90146 Q8/9d gene -2.47 Unspecified functionM12660 Complement component factor h -2.44 Immune-relatedM27134 Histocompatibility 2, K region locus 2 -2.40 Immune-relatedX94418 IgA V-D-J-heavy chain -2.38 Immune-relatedAJ235940 IgVk aj4 gene -2.38 Immune-relatedX00651 DNA for Ig-kappa light chain V-J kappa 5 joining region -2.33 Immune-relatedAB017349 Immunoglobulin light chain V region -2.31 Immune-relatedM18837 MHC class I Q4 beta-2-microglobulin (Qb-1) -2.26 Immune-relatedM21065 Interferon regulatory factor 1 -2.23 Growth factor/cytokineX00246 Set 1 repetitive element for a class I MHC antigen -2.21 Immune-relatedU22033 Large multifunctional protease 7 -2.13 EnzymeX06368 *Clony stimulating factor 1 receptor -2.13 ReceptorM29008 Complement factor H-related protein -2.11 Immune-relatedX00496 Ia-associated invariant chain (Ii) fragment -2.11 Immune-relatedAB001489 Phosphatidylinositol glycan, class R -2.08 Structural proteinM26071 Coagulation factor III -2.05 Immune-relatedM58566 TIS11 primary response -2.03 Inflamm/primary responseJ00475 Germline IgH chain , DJC region -1.97 Immune-relatedD49473 SRY-box containing 17 -1.94 Gamete/sex-specificL43568 Antigen, B-cell receptor -1.94 Immune-relatedJ03520 *Tissue plasminogen activator ( tPA) -1.91 HematologicU28960 Phospholipid transfer protein -1.90 TransporterAF004666 Solute carrier family 8 (sodium/calcium exchanger) -1.90 TransporterX65128 Growth arrest specific 1 (Gas-1) -1.89 Cell cycleX62940 Transforming growth factor beta 1 induced transcript -1.88 Growth factor/cytokineAF077861 Inhibitor of DNA binding 2 -1.87 DNA/chromatin-relatedM23362 Fibroblast growth factor receptor 2 -1.86 ReceptorAJ250489 Receptor activity modifying protein 1 (Ramp1) -1.85 Calcium-relatedU19315 Immunoglobulin kappa light chain variable region -1.83 Immune-relatedL20450 DNA-binding protein -1.83 Transcription factorX65627 D-E-A-D box polypeptide 5 -1.82 ReceptorM38381 CDC-like kinase -1.82 EnzymeM27266 Fyn proto-oncogene -1.79 NeoplasiaM21050 Lysozyme M -1.77 EnzymeU76253 Integral membrane protein 2 B -1.77 Structural proteinU69262 Matrilin-2 precursor -1.76 Structural proteinX00652 DNA for Ig-kappa light chain V-J kappa 5 joining region -1.75 Immune-relatedAF060565 RW1 protein -1.73 Immune-relatedM55561 CD80 antigen -1.71 Immune-relatedJ03952 *Glutathione transferase (GT8.7) -1.70 EnzymeX51547 Lysozyme P -1.69 Enzyme

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AF010254 Complement component 1 inhibitor -1.68 Immune-relatedX78989 Testin -1.67 Structural proteinM60474 Myristoylated alanine rich protein kinase C substrate -1.66 NeurologicAF014010 Polycystic kidney disease 2 -1.65 TransporterX57437 *Histidine decarboxylase cluster -1.62 EnzymeX00958 I-E(beta-b) -1.61 Immune-relatedM80423 Mus castaneus IgK chain , C-region -1.61 Immune-relatedU72941 Annexin IV -1.61 Structural proteinM35247 Histocompatibility 2, T region locus 1 7 -1.60 Immune-relatedM58661 CD24a antigen -1.60 Immune-relatedAF031127 Inositol trisphosphate receptor type 2 (Itpr2) -1.59 ReceptorM22531 Complement C1q B chain -1.58 Immune-relatedM29260 Histone 1-0 -1.57 DNA/chromatin-relatedM17327 Endogenous murine leukemia virus - provirus DNA -1.51 NeoplasiaX16202 Q4 class I MHC (exon 5 ) -1.46 Immune-relatedAF083464 DNA polymerase zeta catalytic subunit -1.43 DNA/chromatin-related

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Supplement Table 2. Genes with significantly increased expression during estrogen-induced termination of delayed implantation.RNAs from P4-primed delayed implanting uteri and delayed implanting uteri after estrogentreatment were obtained for GeneChip hybridization. The fold change in gene expression wasdetermined by comparison of mean Average Difference scores. Differentially expressed geneswere identified by two statistical methods. *, genes with known expression during the peri-implantation period.

GeneBank

Accession No. Gene Name Fold Change Functional CategoryL38281 Immunoresponsive 1 19.36 Immune-relatedK02927 Ribonucleotide reductase M1 16.30 DNA/chromatin-relatedF034610 Nuclear autoantigenic sperm protein 13.81 Gamete/sex-specificAJ005564 Small proline-rich protein 2F 12.62 Structural proteinM22527 Cytotoxic T lymphocyte-specific serine protease CCPII 8.96 EnzymeM81445 *Connexin 26 (beta 2 gap junction) 8.93 Structural proteinU01915 Topoisomerase (DNA) II alpha 7.56 EnzymeX57487 Paired box 8 6.96 Transcription factorY12657 Cytochrome P450, retinoic acid 6.80 EnzymeD13545 DNA primase, p58 subunit 6.66 RNA processingU83902 Mitotic checkpoint component Mad2 6.54 Cell cycleU00937 GADD45 protein (gadd45) 6.48 Cell cycleM64086 Spi2 proteinase inhibitor (spi2/eb4) 6.38 InhibitorAJ005567 Small proline-rich protein 2I 6.37 Structural proteinD13139 Dipeptidase 1 (renal) 6.35 EnzymeM38724 Cell division cycle control protein 2a 6.20 Cell cycleD87908 Nuclear protein np95 5.93 DNA/chromatin-relatedD00812 Rad51 homolog. 5.30 DNA/chromatin-relatedM15501 Alpha actin, cardiac 5.25 Structural proteinJ02652 Malic enzyme, supernatant 4.76 EnzymeJ04620 Primase p49 subunit (priA) 4.64 RNA processingD26090 CDC46 protein 4.59 DNA/chromatin-relatedAJ005560 Small proline-rich protein 2B 4.55 Structural proteinU49513 Small inducible cytokine A9 4.31 Growth factor/cytokineAJ005563 Small proline-rich protein 2E 4.09 Structural proteinX81580 * Insulin-like growth factor binding protein 2 3.96 Growth factor/cytokineX90647 *Hsd11b2 3.83 EnzymeL26320 Flap structure specific endonuclease 1 (FEN-1) 3.80 DNA/chromatin-relatedD45889 *Chondroitin sulfate proteoglycan 2 3.76 Structural proteinX83601 Pentraxin related gene 3.68 Inflamm/primary responseX77731 Deoxycytidine kinase 3.68 EnzymeL34570 Arachidonate 15-lipoxygenase 3.60 EnzymeAJ005565 Small proline-rich protein 2G 3.51 Structural proteinAB025409 Sid1334p 3.13 Unspecified functionX62154 Mcm 3 homolog 3.04 DNA/chromatin-relatedM35970 Tumor metastatic process-associated protein (NM23) 3.03 NeoplasiaD86725 Mcm 2 2.97 DNA/chromatin-relatedM14223 Ribonucleotide reductase M2 subunit 2.94 DNA/chromatin-related

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U79550 Slug, chicken homolog 2.90 Transcription factorAJ223087 Cdc6-related protein 2.90 Cell cycleAJ010108 Cytosolic adenylate kinase 2.90 EnzymeX03505 Serum amyloid A 3 2.88 Inflamm/primary responseX57800 Proliferating cell nuclear antigen (PCNA) 2.76 Cell cycleX91864 Glutathione peroxidase 2 2.74 EnzymeM63801 *Connexin 43 (alpha-1 gap junction) 2.72 Structural proteinL48015 Activin type IB receptor (ALK-1) 2.52 ReceptorX52102 p16K 2.47 NeoplasiaX70296 *Serine protease inhibitor 4 (nexin-1) 2.34 InhibitorAB015978 Oncostatin receptor 2.33 NeoplasiaD26089 Mcm 4 homolog 2.30 DNA/chromatin-relatedM33988 Histone H2A.1 2.29 DNA/chromatin-relatedAF064749 *Type VI collagen alpha 3 subunit 2.29 Structural proteinU28656 Eukaryotic translation initiation factor 4E binding protein 2.29 DNA/chromatin-relatedX67668 High mobility group protein 2 2.27 Transcription factorAJ005559 Small proline-rich protein 2A 2.24 Structural proteinD42048 Squalene epoxidase 2.24 EnzymeJ02980 *Alkaline phosphatase 2, liver 2.18 EnzymeM36901 *Granzyme E 2.17 EnzymeZ84471 G6pd-2 2.17 EnzymeU52951 Putative transcriptional regulator mEnx-1 2.16 Transcription factorX56683 mRNA coding for modifier 2 protein 2.15 DNA/chromatin-relatedU14648 Splicing factor, arginine/serine-rich 1 0 2.13 RNA processingAB027012 Galactokinase 2.11 EnzymeU42385 Fibroblast growth factor inducible 1 6 2.09 Growth factor/cytokineD31863 Phosphatidylinositol glycan, class A 2.07 Protein proc./transportL10244 *Spermidine/spermine N1-acetyl transferase 2.05 EnzymeL32752 Ran GTPase 2.02 Protein proc./transportV00755 Interferon beta, fibroblast 2.01 Growth factor/cytokineZ67748 Spermidine synthase 1.98 EnzymeAB021491 p100 co-activator 1.97 Unspecified functionL32836 S-adenosyl homocysteine hydrolase (ahcy) 1.93 EnzymeU65747 Interleukin 13 receptor, alpha 2 1.93 ReceptorD26091 mCDC47 1.92 Cell cycleD13543 DNA polymerase alpha 1 1.92 DNA/chromatin-relatedU49350 CTP synthetase 1.91 EnzymeZ11974 Mannose receptor, C type 1.90 ReceptorM32490 Cyr61 1.88 Growth factor/cytokineX68193 Nucleoside diphosphate kinase B 1.88 EnzymeM31885 Inhibitor of DNA binding 1 1.87 DNA/chromatin-relatedU11027 Sec61 protein complex gamma subunit 1.85 Protein proc./transportAJ001633 Annexin III 1.82 Inflamm/primary responseX67644 Gly96 1.81 Inflamm/primary responseM17516 Lactate dehydrogenase A-4 1.81 EnzymeAF053232 *SIK similar protein 1.80 Calcium-relatedU64450 Nucleoplasmin 3 1.79 Protein proc./transportX56045 RAN binding protein 1 1.78 Protein proc./transportU06834 eph-related receptor protein tyrosine kinase (ephB4) 1.78 Cell signaling/effectors

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D89063 Oligosaccharyltransferase 1.77 EnzymeU81052 Defender against Apoptotic Death (Dad1) 1.76 ApoptosisAJ222586 NEFA protein 1.76 Immune-relatedX72310 Transcription factor Dp 1 1.76 Transcription factorX61431 Diazepam-binding inhibitor 1.75 Cell signaling/effectorsU11274 RNA-binding protein AUF1 1.74 RNA processingM15668 X chromosome-linked phosphoglycerate kinase (pgk-1) 1.73 EnzymeAJ249987 TAFII30 for mTAFII30 protein 1.73 Cell cycleAB025049 Sid393p 1.72 Unspecified functionD87990 UDP-galactose transporter related isozyme 1 1.71 TransporterL27453 Pre B-cell leukemia transcription factor 1 1.71 Transcription factorAF021031 DiGeorge syndrome chromosome region 6 1.71 Calcium-relatedU42443 MECA39 1.68 Cell cycleU88327 Suppressor of cytokine signalling-2 (SOCS-2) 1.68 Cell signaling/effectorsU35142 Retinoblastoma-binding protein (mRbAp46) 1.65 Cell cycleAF016583 Checkpoint kinase Chk1 (Chk1) 1.64 Cell cycleM61007 CCAAT/enhancer binding protein beta (C/EBP) 1.64 Transcription factorL20509 Chaperonin subunit 3 (gamma) 1.64 ChaperoneU20365 Gamma actin, smooth muscle 1.61 Structural proteinX99572 C-fos induced growth factor 1.58 Growth factor/cytokineU13174 Solute carrier family 1 2 1.58 TransporterAF011644 Oral tumor suppressor homolog (Doc-1) 1.56 NeoplasiaAF026481 EIF-1A 1.55 Transcription factorD14485 DbpA murine homologue 1.55 Cell cycleAB025048 Sid6061p 1.55 Unspecified functionAB007696 *Prostaglandin E receptor subtype EP2 1.54 ReceptorU09659 Chaperonin 1 0 1.51 ChaperoneAF097511 Zyxin related protein-1 (Zrp1) 1.51 Unspecified functionU92454 WW domain binding protein 5 1.50 Unspecified functionAF077527 Zyntenin 1.48 Cell signaling/effectorsM32599 Glyceraldehyde-3-phosphate dehydrogenase 1.48 EnzymeAB012276 ATFx 1.47 Transcription factorAF093853 1-Cys peroxiredoxin protein 2 1.47 EnzymeAJ005983 cAMP-regulated phosphoprotein (ARPP-19) 1.44 Cell signaling/effectorsAF100694 Pontin52 1.44 Cell signaling/effectorsD31717 MARib ribophorin 1.43 Protein proc./transportAB025015 Elongin A 1.43 RNA processingM21332 MHC class III RD (H2-d and H2-Sk haplotypes) 1.38 Immune-relatedU28932 Smooth muscle calponin 1.37 Calcium-relatedX54327 Glutamyl-tRNA synthetase 1.36 EnzymeAF034092 Nuclear localization signal containing protein (Nlvcf) 1.35 Cell signaling/effectors

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M Matsumoto, Kevin L. Knudtson, Raymond N. DuBois and Sudhansu K. DeyJeff Reese, Sanjoy K. Das, Bibhash C. Paria, Hyunjung Lim, Haengseok Song, Hiromichi

and embryo implantationGlobal gene expression analysis to identify molecular markers of uterine receptivity

published online September 10, 2001J. Biol. Chem. 

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