Molecular Human Reproduction, Vol. 5, No. 9, 866-873,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular events in the endometrium |
Regulation of HOXA-10 and its expression in normal and abnormal endometrium
Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of North Carolina, Chapel Hill, NC, USA
Abstract
HOXA-10 is a member of a family of genes that serve as transcription factors during development and have been shown to be important for uterine function. Using immunohistochemistry and RNAse protection assays (RPA), HOXA-10 was shown to be expressed in both epithelial and stromal cells with increased expression during the window of implantation. By in-vitro culture of isolated endometrial epithelium or stroma, HOXA-10, expression was increased after treatment with oestradiol (10–8 mol/l) with or without progesterone (10–6 mol/l). In stromal cells, oestradiol and progesterone both appeared to increase HOXA-10 expression and were additive. Relaxin (30 ng/ml) appeared to further increase stromal HOXA-10 expression. HOXA-10 expression during the window of implantation was compared in normal menstrual cycles to endometrium from women with endometriosis and suspected defects in uterine receptivity. Little or no difference was seen in luminal, glandular or endothelial HOXA-10 expression but a significant reduction in stroma HOXA-10 expression was noted in women with endometriosis. In conclusion, HOXA-10 is a hormone-regulated endometrial transcription factor that appears to be responsive to both ovarian steroids and relaxin. The appearance of this nuclear protein during the window of implantation in epithelium and stroma may offer new insight into the regulation of uterine receptivity and assist in the identification of other genes that are critical to the establishment of a successful pregnancy.
endometriosis/HOXA-10/implantation/integrins/relaxin
Introduction
HOXA-10 is a member of the `posterior' abdominal B-related (AbdB) subclass of homeobox genes, first described in Drosophila melanogaster (Lewis, 1978
). These genes are characterized by a conserved region of 183 bp known as the homeobox (Gehring, 1987). The products of these genes function as transcription factors that regulate a myriad of other developmental genes. The HOXA family of genes is responsible for segmental development and the genes are expressed in restricted domains from the rostral to the caudal portions of the embryo (Favier and Dollé, 1997). The mammalian AbdB HOXA family of genes consists of 16 separate genes, of which HOXA-10 is a member. This gene has attracted attention recently for its role in the development of the uterus. Targeted mutation of this gene in mice has shown a failure of implantation associated with the loss of this gene (Satokata et al., 1995). Characterization of these mice have shown morphological changes in the proximal oviduct and a defect in the uterine decidual response (Benson et al., 1996). Furthermore, developmental defects, such as those associated with in-utero exposure to diethylstilboestrol may be tied to the inappropriate expression of this homeobox gene (Ma et al., 1998). More recently, HOXA-10 was shown to be expressed in the human endometrium and to be regulated by ovarian steroids (Taylor et al., 1998). Peak expression occurred during the window of implantation in the human endometrium and suggests a potential role for this gene product in implantation and as a potential marker of uterine receptivity.
We have been interested in the role of endometrial proteins in the regulation of uterine receptivity (Lessey et al., 1992; Ilesanmi et al., 1993). During a 28 day cycle, implantation is thought to occur at or around cycle day 20, based on studies from the 1950s (Hertig et al., 1956) and more recent data from assisted reproductive technology studies (Bergh and Navot, 1992). An increasing number of proteins have been shown to be expressed during the window of implantation and/or to be critical to the implantation process, including leukaemia inhibitory factor (LIF) (Stewart et al., 1992; Cullinan et al., 1996), integrins (Lessey et al., 1992, 1994a; Tabibzadeh, 1992), Cox-2 (Chakraborty et al., 1996; Lim et al., 1997), calcitonin (Kumar et al., 1998; Zhu et al., 1998), trophinin (Fukuda et al., 1995), and others (Ilesanmi et al., 1993). As a developmental and regulatory transcription factor, it was of interest to further examine the expression of HOXA-10 in human endometrium and compare it with another developmentally-regulated product, the 
vß3 integrin which is an increasingly accepted marker of uterine receptivity in the human (Lessey, 1997). The loss of uterine receptivity has been classified into at least two distinct subtypes. In type I defects, the absence of appropriate markers relates to histological delay due to hormone insufficiency or luteal phase defect (LPD; Lessey et al., 1992, 1996b), while type II defects have been described in certain infertile patients, including those with endometriosis (Lessey et al., 1994b), hydrosalpinges (Meyer et al., 1997), and unexplained infertility (Lessey et al., 1995). In the latter, the histological progression of the endometrium is normal and `in phase' with an inappropriate delay or loss of this marker of receptivity.
Both HOXA-10 and vß3 have been reported to appear at the time of implantation in the human endometrium (Lessey et al., 1992; Taylor et al., 1998) and both have been reported to be decreased in women with endometriosis and infertility (Lessey et al., 1994b; Taylor et al., 1997). During the transition from the early to mid-secretory phases of the menstrual cycle there is a cell-specific decrease in oestrogen and progesterone receptors (ER and PR respectively) on the endometrial epithelium (Garcia et al., 1988; Lessey et al., 1988; Press et al., 1988). This putative loss of epithelial responsiveness to ovarian steroids is associated with the expression of other implantation specific markers such as vß3, and a delay in the loss of ER and PR due to hormonal inadequacy may postpone the appearance of such markers (Lessey et al., 1996b). While the expression of some endometrial proteins follow the expression of ER and PR (Hild-Petito et al., 1996), other peptides thought to be regulated by oestrogen and/or progesterone, paradoxically appear on the endometrial epithelium beyond cycle day 20 when epithelial ER and PR have all but disappeared. Such observations suggest a paracrine role for the stroma in regulation of key implantation events (Cooke et al., 1997). Based on the hypothesis that both ovarian steroids and proteins regulate implantation through endocrine and paracrine mechanisms, we examined the regulation of HOXA-10 in the human endometrium and examined its expression during the window of implantation in normal and abnormal menstrual cycles.
Materials and methods
Endometrial samples
Human endometrium was obtained by pipelle suction curettage from healthy volunteers and from women with infertility, timed in their cycle using urinary luteinizing hormone (LH) surge prediction kits (Ovuquik; Quidel, San Diego, USA). Additional endometrial samples were obtained from women undergoing hysterectomy or at the time of bilateral tubal ligation. A total of 73 endometrial samples were evaluated from various times in the menstrual cycle including 65 samples timed to the window of implantation from normal cycles (n = 17; in phase histology with normal integrin expression), type I defects (histological delay consistent with LPD; n = 7) and type II defects (in phase histology with absent vß3 integrin expression; n = 41). All 41 patients with type II defects had been diagnosed with endometriosis. Thirty-five samples from women without known endometriosis were obtained throughout the menstrual cycle including five from the proliferative phase, 27 from the secretory phase and three from early pregnancy. All tissue was obtained in accordance with the Committee for the Protection of Human Subjects at the University of North Carolina. Samples of endometrium were either snap-frozen in liquid nitrogen and stored for RNA isolation or immunohistochemistry or placed in 10% buffered formalin prior to embedding in paraffin. Paraffin sections were sectioned and stained with haematoxylin and eosin and dated according to established criteria (Noyes et al., 1950).
Immunohistochemistry
Serial sections (8 µm) of frozen endometrium were placed on glass slides. Cryosections were lightly fixed in 3.7% formaldehyde in phosphate-buffered saline. Immunohistochemistry was performed using Vectastain Elite® ABC kits (Vector Laboratories, Burlingame, CA, USA) as previously described (Lessey et al., 1992, 1994a). Polyclonal antibodies to HOXA-10 (BabCo, Richmond, CA, USA) and monoclonal antibodies to the ß3 integrin subunit (SSA6) were used at concentrations determined by limiting dilution.
The resulting staining was evaluated on a Nikon microscope, by a single blinded observer (BAL). The HSCORE was calculated using the following equation:
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). Photomicrographs were taken on Kodak 100 ASA film.
Isolation and culture of human endometrial stromal and epithelial cells
Reagents were obtained from the Sigma Chemical Company (St Louis, MO, USA) except where otherwise specified. Human endometrium obtained from normally cycling women undergoing hysterectomy for benign disease was finely minced and digested with collagenase (Type 1A, 0.1 mg/ml; Sigma) for 2 h at 37°C in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco Laboratories, Grand Island, NY, USA) supplemented with 5% heat-inactivated fetal calf serum (FCS) (Flow Laboratories, Mclean, VA, USA) and penicillin/streptomycin. The digested material was added to a stacked sterile wire sieve assembly with #100 wire cloth sieve (140 µm size; USA Standard Sieve Series, Newark Wire Cloth Company, Newark, NJ, USA) followed by passing through a #400 wire sieve (37 µm). The epithelial cells and glands were retained in the #100 and #400 sieves while the stromal cells passed through to the receptacle below. The stromal cells remaining with the glands and epithelial cells were further separated by selective adherence to plastic tissue culture dishes for 1 h.
Stromal cells were collected from the lower receptacle and centrifuged at 400 g for 8 min, and the pellet was resuspended in 1 ml of fresh medium. The volume was carefully placed on 3 ml of Ficoll–Paque (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) and centrifuged at 400 g for 10 min. The interface contained an enriched fraction of endometrial stromal cells depleted of erythrocytes. Stromal and epithelial cells were resuspended in DMEM/F12, plated in 100 mm dishes (Nunc, Roskilde, Denmark) and grown to confluence at 37°C in 5% CO2 and 95% air. The culture media were changed every 3 days.
Hormone treatments
Isolated endometrial stromal or epithelial cells were cultured in the presence or absence of exogenous hormones. Control media consisted of DMEM/F12 Phenol Red-free media with 5% heat inactivated charcoal-stripped FCS (Flow Laboratories). Other cells received oestradiol-17ß (10–8 mol/l), progesterone (10–6 mol/l), or relaxin (30 ng/ml), alone or in combination for 1–14 days. In the time-course studies, all samples including the controls were cultured for the same number of days, though the time of exposure to hormones varied. In some experiments cells were incubated in the presence of ovarian steroids and antagonists of oestradiol and progesterone including ICI 182780 (10–7 mol/l) or RU 486 (10–5 mol/l).
PCR cloning of cDNA fragment of human HOXA-10 and integrin subunit ß3
Total RNA was isolated from Ishikawa cells, a well-differentiated human endometrial cell line with functional oestrogen and progesterone receptors (Lessey et al., 1996a), using TRI REAGENT (Molecular Research Center, Cincinnati, OH, USA). First-strand complementary DNA was synthesized from total RNA with cDNA Cycle Kit (Invitrogen, San Diego, CA, USA). Reverse transcription–polymerase chain reaction (RT–PCR) was performed in a Perkin-Elmer Cetus PCR Thermocycler under the following conditions: 95°C for 1 min, 60°C for 2 min and 72°C for 3 min for 30 cycles. The primers were, for integrin ß3 subunit: GGAAAGATTGGCTGGAGGAA (sense) and GGCATACCCCACACTCAAAG (antisense); for HOXA-10: AGCCCCTTTCTCCCTCCCACAC (sense) and AAACACAGCCCAGCACTCCAGG (antisense). The expected sizes obtained for both the ß3 integrin subunit and HOXA-10 were 683 and 395 bp respectively.
The PCR products of integrin ß3 and HOXA-10 were extracted from 1% agarose gel with QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned into pCR 2.1 vector with Original TA Cloning Kit (Invitrogen). Positive bacterial colonies was selected with X-gal plates. Restriction analysis and sequencing confirm its inserting orientation. The sense plasmid containing cDNA fragment of ß3 and HOXA-10 was linearized with BamHI as the DNA template for RNA riboprobe synthesis.
Ribonuclease protection assay (RPA)
The in-vitro transcription and labelling reaction was carried out using 80 µCi [-32P]-UTP (Amersham Life Science Inc, Arlington Heights, IL, USA) and the Maxiscript T7 polymerase Kit (Ambion, Austin, TX, USA). To isolate the probe, it was run on a 5% polyacrylamide 8 mol/l urea gel. The band containing the full-length probe was excised and eluted from the gel in 350 µl of elution buffer (Ambion) by incubation overnight at 37°C. A riboprobe for ß-actin (Ambion) was used as the internal standard against which to compare integrin ß3 and HOXA-10 mRNA concentrations. The century RNA marker plus from Ambion was used as the molecular weight marker.
Total RNA from endometrial tissues, stromal and epithelial cells was isolated with RNAgent Total RNA Isolation System (Promega, Madison, WI, USA). A Ribonuclease Protection Assay Kit (Ambion) was used to quantify the fluctuation of integrin ß3 and HOXA-10 mRNA in endometrial stromal cells treated with steroids or tissues during the menstrual cycle. Equal amounts of total RNA (30–100 µg) from endometrial tissues and cells were hybridized in hybridization buffer containing ~2x105 c.p.m. of [-32P]-labelled integrin ß3 or HOXA-10 and 5x104 c.p.m. of [-32P]-labelled ß-actin probe by incubation overnight at 45°C. After hybridization, single-stranded RNA was digested with RNase A/T at 37°C for 30 min. The remaining RNA duplexes were separated on 5% polyacrylamide 8 mol/l urea gel. The hybridization signal was detected by autoradiography.
Western blot assay
For Western blotting, stromal cells were homogenized in RIPA buffer [10 mm Tris–HCl, pH 8.0, 10 mmol/l EDTA, pH 8.0, 0.15 mol/l NaCl, 1% NP–40, 0.5% sodium dodecyl sulphate (SDS), 1 µg/ml Aprotinin, 1 mmol/l phenyl methyl sulphonyl fluoride (PMSF)]. The homogenate was clarified by centrifugation at 15 000 g for 15 min and the concentration of protein in homogenates was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Aliquots (30 µg) of homogenate protein were separated by discontinuous 8% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). The separated proteins were electroblotted onto a Hybond ECL nitrocellulose membrane (Amersham) at 200 mA for 2 h.
After blocking the non-specific binding sites with non-fat dry milk in TBST buffer (5 mmol/l Tris–HCl, pH 7.4, 136 mmol/l NaCl, 0.1% Tween 20) for 1 h at room temperature, the blots were incubated overnight at 4°C with a 1:1000 dilution of polyclonal antibody against HOXA-10 (BabCo). The blots were then washed three times with TBST buffer, incubated for 2 h at room temperature with horseradish peroxidase-linked sheep anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) at a dilution of 1:5000 and, after further washing, the immunoreactive proteins were revealed using the ECL reagents (Amersham) and quantified by densitometry.
Statistical analysis
Western blots were scanned and densitometry readings used to compare the relative intensity of each treatment group, in duplicate. Results of immunohistochemical HSCORE and Western blotting were compared using analysis of variance with Scheffé's correction when making multiple comparisons with significance based on the 95th percentile confidence intervals.
Results
HOXA-10 expression during the menstrual cycle
HOXA-10 protein was examined in 35 endometrial samples throughout the menstrual cycle using immunohistochemistry. As shown in Figure 1A, epithelial HOXA-10 expression was low during the proliferative phase, similar to a negative control immunostained with preimmune serum (Figure 1B). Expression of HOXA-10 increased in the secretory phase on both luminal (arrowhead) and epithelial cells (arrow) (Figure 1C). Comparison of HSCOREs showed a significant increase in the secretory phase (P < 0.05). There was strong positive immunostaining seen on the endothelium (asterisk) in both the proliferative and secretory phase (Figure 1A and D) that did not appear to change during the menstrual cycle. Stromal cells appeared to express this transcription factor throughout the menstrual cycle but increased expression was noted in the pseudodecidual cells of the secretory phase and in the decidua of early pregnancy (not shown). Using the RNase protection assay (RPA), expression of mRNA for HOXA-10 was examined throughout the cycle comparing it with the expression of another cycle-dependent endometrial protein, the ß3 integrin subunit. HOXA-10 appears to increase in the early secretory phase with peak expression during the mid-secretory phase, concomitant with maximal expression of ß3 expression (Figure 2).
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Comparison of HOXA-10 expression during the window of implantation in normal and abnormal endometrium
Expression of HOXA-10 by glandular and luminal epithelium, stroma and endothelial cells was studied during the window of implantation in normal and abnormal endometrium from normal fertile women and women with infertility, using RPA and immunohistochemistry. By RPA, mRNA isolated from similar samples of normal versus type II defects showed a significant decrease in ß3 expression but overall the change in HOXA-10 expression was less evident (Figure 3
). The reason a greater change is not apparent by RPA could be due to the use of whole tissue homogenates since there is expression of HOXA-10 mRNA by most of the cell types present in the endometrium. Relative differences in HOXA-10 distribution were compared using the semi-quantitative HSCORE (Budwit-Navotny, 1986). Samples were defined as abnormal if they lacked another marker of uterine receptivity, the vß3 integrin, as previously described (Lessey et al., 1992, 1995). Two groups of abnormal endometrium were examined and included samples exhibiting histological delay of 
3 days relative to the known time of biopsy, consistent with LPD (type I defects) and samples without histological delay lacking vß3 expression in women with endometriosis (type II defects). There was no apparent difference in the expression of glandular or luminal epithelium (arrows, Figure 4A,B), nor in the relative expression of endothelial HOXA-10 between groups. In contrast, we noted a significant reduction in stromal HOXA-10 (P < 0.01) specifically in the group with type II defects and endometriosis (Figure 4A–C).
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In-vitro regulation of epithelial and stromal HOXA-10 expression
To investigate the factors that could account for the observed cycle-dependent HOXA-10 expression in vivo, primary epithelial cells were isolated from proliferative endometrium and cultured on plastic surfaces. Cells were treated with either no hormones (no Tx) or oestradiol, progesterone or a combination of oestradiol and progesterone. HOXA-10 mRNA expression was determined in each group using RPA (Figure 5
). HOXA-10 mRNA values increased with both oestradiol and progesterone treatment with maximal expression when cells were treated with both hormones.
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Stromal cells were treated in a similar manner with or without oestradiol and progesterone and with or without relaxin. Similar to epithelial cells, oestradiol and progesterone both increased HOXA-10 mRNA expression by RPA (Figure 6
). Treatment with both oestradiol and progesterone together, further increased HOXA-10 expression. While relaxin had little effect on HOXA-10 expression by itself, relaxin plus oestradiol and progesterone appeared to further increase HOXA-10 expression. Western blot analysis was used to examine HOXA-10 protein values after similar treatments. These data, shown in Figure 7, illustrate again the cumulative effect of oestradiol and progesterone (P < 0.05) and a reduction in HOXA-10 expression to baseline using the specific oestradiol and progesterone antagonists, ICI 182780 and RU 486. Dose–response curves demonstrated maximal stimulation at oestradiol concentrations of 10–8 mol/l and progesterone at 10–6 mol/l respectively. Time-course studies demonstrated a maximal effect of treatment by 7 days using RPA analysis (Figure 8).
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Discussion
There is increasing interest in the role of the endometrium and uterine receptivity in the early events of implantation. Understanding the biochemical basis for the establishment of a successful pregnancy requires an appreciation of the role of many endometrial peptides during the menstrual cycle. In the present study we have investigated the temporal and spatial distribution of the HOXA-10 gene in human endometrium. Using both RPA and immunohistochemistry we have shown that this transcription factor is regulated during the menstrual cycle with peak expression prior to, and during, the window of implantation. As a hormonally stimulated transcription factor, HOXA-10 is an excellent candidate protein to study as a pivotal regulator of endometrial function. Evidence points to such a role for this peptide during embryonic development (Satokata et al., 1995) and as a critical element for implantation (Benson et al., 1996).
Steroid regulation of HOXA-10 by oestrogen and progesterone has been demonstrated in the mouse uterus (Ma et al., 1998). It now appears that the HOXA-10 is also expressed in a hormone-dependent manner in human endometrium and that its appearance is correlated with the time of implantation in women (Taylor et al., 1998). These authors have shown that HOXA-10 is expressed during the mid-secretory phase, in agreement with the present study. In addition, using primary stromal cells they reported an increase in HOXA-10 expression in response to both oestrogen and progesterone. Studies were not performed in primary epithelial cells, however, using instead the well differentiated (Ishikawa) cell line. Results using either primary epithelial cells or Ishikawa cells were generally comparable.
In this study, we confirmed that HOXA-10 is also regulated in human endometrial stromal cells by both oestradiol and progesterone, with the greatest effect by progesterone in a dose dependent process. Interestingly, a greater effect on HOXA-10 expression was seen when relaxin was added to the oestrogen and progesterone treatment. Relaxin, a peptide hormone produced by the corpus luteum (Seppälä and Tiitinen, 1995) had no apparent effect when administered by itself. These data argue that oestrogen and progesterone priming may sensitize the stromal cells to the effects of relaxin, perhaps through induction of the putative relaxin receptor. Similar results have been reported for prolactin, another marker of decidualization (Huang et al., 1987). A time-course of these effects suggest that maximal induction of HOXA-10 was seen by 7 days of treatment. The effects of ovarian steroids on HOXA-10 expression in vitro could be antagonized by specific steroid antagonists, including ICI 182780 and RU 486. In each case, the effects of oestradiol and progesterone or their combination were blocked by the addition of the appropriate antagonist, demonstrating the direct effect of steroids on stromal HOXA-10 expression.
Primary endometrial epithelium also demonstrated hormonal regulatation of HOXA-10 expression in vivo and in vitro. Progesterone appeared to have the dominant effect though the combination of oestradiol and progesterone had the greatest effect on HOXA-10 expression. The expression of HOXA-10 changed in normal endometrium throughout the cycle, demonstrating temporal and spatial differences between endometrial epithelium and stroma. Since both ER and PR have been shown to decrease at the time of implantation (Garcia et al., 1988; Lessey et al., 1988), the continued expression of epithelial HOXA-10 expression was somewhat surprising. The strong expression in luminal and glandular epithelium at this time argues for other factors, e.g. epidermal growth factor (EGF) or EGF-like molecules (e.g. heparin binding-EGF), that may regulate this gene, similar to that noted for the endometrial vß3 integrin (Somkuti et al., 1997).
In the mouse, null mutants for HOXA-10 are infertile, exhibiting a phenotype of implantation failure (Satokata et al., 1995; Benson et al., 1996). We questioned whether women with otherwise unexplained infertility and suspected defects in uterine receptivity might also exhibit reduced or absent HOXA-10 expression during the window of receptivity. To test this hypothesis we examined LH-timed endometrial samples from normal women and compared these with endometrial biopsies from women with infertility, endometriosis and lack of endometrial expression of another marker of uterine receptivity, vß3. This integrin has been extensively described in cycling endometrium (Lessey et al., 1992, 1994a; Albers et al., 1995; Rai et al., 1996) and was found to be aberrantly expressed in women with endometriosis (Lessey et al., 1994b), hydrosalpinges (Meyer et al., 1997), and unexplained infertility (Lessey et al., 1995). In the present study we found that stromal HOXA-10 was indeed decreased in the endometrium from women with type II defects and endometriosis, but found little difference in the expression in samples from women with absent vß3 integrin due to histological delay (type I defects). These data support our hypothesis that occult defects in uterine receptivity exist and are associated with various diagnoses including endometriosis, hydrosalpinges and unexplained infertility. These data, coupled with the finding that HOXA-10 null mutant female mice fail to decidualize, suggest that stromal HOXA-10 may be critical to both stromal and epithelial differentiation, through autocrine and paracrine mechanisms. Specific gene products that result from HOXA-10 activity may be responsible for these down-stream events.
HOXA-10 is a transcription factor that appears to be critical for the programmed expression of proteins in the endometrium during development and during the menstrual cycle. It is currently not known which genes are regulated by this nuclear protein, but based on these studies there may be a role for stromal HOXA-10 in the expression of other cycle-specific markers. In mice, both heparin-binding EGF and LIF expression were found to be normal in HOXA-10 null mutants (Benson et al., 1996). These are two other endometrial proteins that appear to be critical for implantation and expressed proximal to the time of uterine receptivity in both mice (Stewart et al., 1992; Das et al., 1994) and humans (Cullinan et al., 1996; Yoo et al., 1997). Future studies will be needed to extend our understanding of HOXA-10 or HOXA-11 (another similar gene that appears critical for implantation) and the HOXA-dependent genes that are expressed in the human endometrium at the time of implantation.
Acknowledgments
Supported by grants HD 35041 and HD 34824 (BL).These studies were funded in part by the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation and the Fogerty International Fellowship.
Notes
1 To whom correspondence should be addressed 
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Submitted on January 11, 1999; accepted on May 28, 1999.
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