Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, PO Box 208063, New Haven, CT 065208063, USA
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Abstract |
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Key words: endometriosis/endometrium/homeobox genes/HOX/implantation
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Introduction |
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HOXA10 and HOXA11 are homeobox genes that mediate embryonic development (Krumlauf, 1992; McGinnis and Krumlauf, 1992
) including the development of the reproductive tract (Favier and Dolle, 1997
; Taylor et al., 1997
). They are translated into transcription factors that regulate a battery of downstream genes necessary for growth and differentiation. We have recently demonstrated that HOX genes play an analogous role in endometrial development during the adult menstrual cycle (Taylor et al., 1998
, 1999
). HOX gene expression possibly regulates the growth and development of the human endometrium (Taylor et al., 1997
). HOXA10 and HOXA11 gene expression varies in response to sex steroids during the menstrual cycle, with dramatic up-regulation in the mid-secretory phase, the time of implantation.
Expression of each of these Hox genes is necessary for implantation in the mouse. Mice with a targeted mutation of either of these genes have uterine factor infertility, producing normal embryos, but with a uterus which lacks the ability of wild-type embryos to implant (Hsieh-Li et al., 1995; Satokata et al., 1995
; Favier and Dolle, 1997
). We have recently shown that HOXA10 and HOXA11 likely play a similar role in human implantation (Taylor et al., 1997
, 1998
, 1999
). HOXA10 and HOXA11 are expressed in the adult human endometrial stroma and glands and are differentially expressed in the developing endometrium during the menstrual cycle. HOX genes may affect endometrial development in a way analogous to their role in embryonic development, leading to endometrial growth, differentiation and receptivity. In women HOXA10 and HOXA11 are up-regulated in the mid-secretory endometrium, at the time of implantation. The extraordinarily high conservation of HOX gene function and the spatial and temporal expression pattern of HOXA10 and HOXA11 in the endometrium suggest that these HOX genes play an essential role in human implantation. In this study we determined the levels of expression of HOXA10 and HOXA11 in the eutopic endometrium of patients with endometriosis. Alterations of the HOXA10 and HOXA11 genes, whose expression is necessary for implantation, may provide evidence of molecular alterations in the endometrium of these patients, and would suggest a defect in the development and receptivity of the endometrium in patients with endometriosis.
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Materials and methods |
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Northern blot analysis
Tissues were individually homogenized in 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarkosyl, and 0.1 M 2-mercaptoethanol. Total RNA was size-fractioned on a 1% agarose-0.66 M formaldehyde gel and sequentially hybridized with a 32P-labelled riboprobe as described below. Hybridization was performed overnight at 60°C in 50% formamide, 1x sodium chloride/sodium citrate (SSC), 5x Denhardt's reagent, 0.2% tRNA, and 32P-labelled riboprobe at 2x106 cpm/ml. The filter was washed twice at 86°C for 30 min in 0.1x SSC and 0.1% SDS. Kodak (Rochester, NY, USA) X-Omat AR film was exposed overnight at 70°C.
Probe preparation
Plasmids used for probe preparation were a generous gift from E.Boncinnelli. pGEM plasmids containing sequence from the 3' untranslated region of either human HOXA10 or HOXA11 were linearized with EcoRI or HindIII (New England Biolabs, Beverly, MA, USA), ethanol precipitated and used as a template for generation of riboprobes. Radiolabelled RNA probes were generated by in-vitro transcription using the Promega Riboprobe Kit (Promega, Madison, WI, USA). Antisense probes were generated using the appropriate RNA polymerase (T7 or SP6) and labelled with -[32P]-UTP (Amersham, Arlington Heights, IL, USA).
Statistical analysis
The autoradiographic bands were quantified using a laser densitometer (Molecular Dynamics Inc, Sunnyvale, CA, USA). Each HOXA10 or HOXA11 band was normalized to the value obtained from the same lane hybridized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed using analysis of variance. P < 0.05 was considered to be statistically significant.
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Results |
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Endometria from 40 patients with endometriosis were analysed in the same fashion. The eutopic endometria obtained from patients with endometriosis failed to show the equivalent up-regulation of either the HOXA10 or HOXA11 genes at the time of implantation. Representative samples are shown in Figure 1. Densitometry was performed on the Northern blot analysis of all samples normalized to GAPDH, and summarized in Figure 2
. Levels of HOXA10 expression were similar in the proliferative and early secretory phases and not statistically different. In both the mid- and late segments of the secretory phase, a statistically significant difference was noted in endometrial HOXA10 expression between patients with or without endometriosis (P < 0.01). A similar difference was noted with HOXA11 (Figure 3
). This failure to increase HOX gene mRNA levels did not depend on the stage of the disease and occurred despite in-phase endometrial histology.
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Discussion |
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Alterations in other molecules expressed in the endometria have been reported in endometriosis (Isaacson et al., 1990; Lessey et al., 1994
; Noble et al., 1996
; Shifren et al., 1996
; Fujimoto et al., 1997
; Sharpe Timms, 1997
; Sillem et al., 1997
). In the absence of histological alteration, molecular defects in the endometrium may be responsible for failure of implantation. In mice with a targeted disruption of either the Hoxa10 or Hoxa11 gene, implantation cannot occur despite a histologically normal endometrium (Hsieh-Li, et al., 1995
; Satokata et al., 1995
). Similarly a defect in HOX expression in patients with endometriosis may lead to a decrease in implantation without an appreciable pathology noted on histological examination. Very few molecules are known to affect implantation specifically when a targeted mutation is produced in mice; it is interesting to note that these defects are often undetectable on histological examination. Molecular markers may be a more valuable way to assess the receptivity of the endometrium.
Hox genes function as transcription factors and are early regulators of tissue identity in embryonic development (Krumlauf, 1992; McGinnis and Krumlauf, 1992
). It is likely they function in an analogous role in cyclic endometrial development (Taylor et al., 1998
, 1999
). Other molecular markers of implantation or of endometrial development are likely downstream target genes of Hox genes either direct or indirect. It will be interesting to determine if any of the structural molecules or growth factors that are involved in implantation are regulated by HOXA10 or HOXA11. Alterations in HOX genes can be expected to produce a cascade of other defects in the expression of downstream target genes. Hox genes may be important early initiators of signal transduction that lead to the proper molecular development of the endometrium and to endometrial receptivity. It is likely many of the molecular, ultrastructural and clinical alterations seen in patients with endometriosis are mediated through alterations in HOX gene expression.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 2, 1998; accepted on January 22, 1999.