Decreased endometrial HOXA10 expression associated with use of the copper intrauterine device.

Intrauterine devices (IUDs) provide long-lasting, highly effective contraception to approximately 150 million women worldwide ( 1 ). Although this form of birth control is one of the most commonly used reversible contraceptives, the exact mechanism of action remains incompletely characterized ( 2 , 3 ). Endometrial morphology and biochemical composition are important factors in reproduction; modification of such factors result in the interference of processes required for spermatozoa capacitation or endometrial receptivity. The copper IUD alters the intrauterine environment by eliciting a local foreign body reaction characterized by a significant influx in polymorphonuclear leukocytes, mast cells, and macrophages. Traditionally, influx of inflammatory cells resulting from copper concentrations in the endometrial tissue has been postulated to create a hostile environment for the embryo, thus contributing to the IUD mode of action. ( 4 – 7 ) Timonen and Kosonen investigated copper release from copper-T IUDs and the corrosive lifespan of copper in utero respectively; they observed sustained low pregnancy rates even when the device was approaching the end of the copper releasing lifespan, raising the question as to whether the mode of action can be explained by the release of copper alone( 8 , 9 ). Copper affects the fertilizing capacity of human spermatozoa by interfering with sperm migration, viability, and acrosomal reaction in vitro( 10 – 14 ). Recovery of viable spermatozoa near the site of fertilization is decreased in IUD users when compared to non-IUD users, yet hindrance of sperm migratory ability only diminishes the chance of fertilization. Chang and Tatum studied the effect of intrauterine copper in rats, demonstrating the transfer of blastocysts from a copper exposed uterus to a normal uterus resulted in normal implantation, whereas normal blastocysts transferred to a copper influenced uterus failed to implant, concluding the altered intrauterine environment was the mechanism underlying the contraceptive effect ( 15 ). Hox genes (HOX in human) encoded evolutionarily conserved transcription factors, which are essential to embryonic development, endometrial development and endometrial receptivity ( 16 – 19 ). During the embryonic period, HOX gene expression is necessary for directing developmental identity along the paramesonephric duct where HOXA9, A10, A11 and A13 are expressed in the developing fallopian tubes, uterus, cervix and upper vagina respectively. ( 20 – 25 ) In the adult human uterus, HOXA10 expression is apparent in endometrial stroma and glands, where it is regulated by the sex steroid hormones 17β-estradiol and progesterone ( 22 , 26 ). We have previously demonstrated the differential expression pattern of HOXA10 protein in human endometrial biopsies throughout the menstrual cycle( 26 , 27 ). HOXA10 expression increases throughout the menstrual cycle, most dramatically during the mid secretory phase, corresponding to the time of implantation.( 26 , 28 ]) Hoxa10 is essential for embryo implantation in mice and humans. Maternal disruption of Hoxa10 results in impaired decidualization and uterine factor infertility due to defective endometrial receptivity. ( 29 – 31 ) In the current study, we investigated the effect of copper containing Paraguard T380A on endometrial HOXA10 gene expression and characterized a novel mechanism of IUD action. As demonstrated by its role in endometrial differentiation and receptivity, decreased endometrial HOXA10 expression, in response to IUD usage would be expected to lead to impaired implantation.

In women using a copper containing IUD, endometrial stromal HOXA10 expression, independent of menstrual cycle stage, was localized to the nucleus and markedly decreased when compared to non-IUD users ( Fig 1a ). Endometrial stromal HOXA10 expression has been previously well characterized in non-IUD users and served as a control and basis for comparison. Endometrial biopsies collected from controls throughout the menstrual cycle, demonstrated relatively high levels of endometrial stromal HOXA10 expression ( Fig 1b ). The mean H score for stromal expression of HOXA10 in women using Paraguard T380A IUD was 0.21 compared to 2.2 in the control endometrial biopsies ( Fig 2 ). The glandular expression of HOXA10 was absent in the endometrium exposed to the IUD. In non-IUD users, endometrial glandular expression was observed in the late proliferative phase and peaked during the mid- late secretory phase. Endometrial HOXA10 expression was lower in all IUD exposed specimens at any point in the menstrual cycle compared to controls. Clinical variables were correlated with endometrial HOXA10 expression using multiple linear regression analysis. Patient age and last menstrual period were significant predictors of endometrial HOXA10 expression, p = 0.008 and p = 0.012 respectively. Duration of IUD usage and prolonged vaginal bleeding associated with use had no effect on endometrial HOXA10 expression in IUD users. ( Table 1 )

Copper containing IUDs are the most widely used reversible contraceptive worldwide( 1 – 3 ). Copper-T IUD’s demonstrate a persistently low failure rate, even at the end of the corrosive lifespan of copper in utero; thus suggesting that the mode of action cannot be explained by copper alone( 8 , 9 ). Alterations in endometrial morphology and biochemical composition disrupt processes required for endometrial receptivity and implantation( 34 ). Blastocyst implantation fails in copper exposed endometrium, while transfer of copper exposed blastocysts to a normal endometrium results in successful implantation; demonstrating the direct endometrial effect of the IUD ( 15 ). Previous studies from our laboratory demonstrate the essential contribution of endometrial HOXA10 expression to implantation and thus suggested a possible mechanism underlying the IUD’s contraceptive effect. As copper release is not essential for maximal contraceptive efficacy, the effect of the IUD on HOXA10 expression is likely independent of the copper itself. The use of non-hormonal IUD contraceptives results in a local foreign body effect characterized by a significant influx of inflammatory cells including polymorphonuclear leukocytes, mast cells, and macrophages( 4 – 6 ). IL-1β, a pleiotropic macrophage- derived cytokine, is involved in the inflammatory immune response( 41 ). We have previously characterized the effects of inflammatory cytokines on HOX gene expression where we reported the effect of IL-1β on HOXA10 expression in decidual cells. Exposure of decidual cells to IL-1β resulted in an approximate 90% decrease in HOXA10 expression. ( 42 ) We propose the contraceptives local foreign body effect increases inflammatory cytokines, such as IL-1β, resulting in a significant decrease in endometrial HOXA10 expression. Adult endometrial cyclic HOXA10 expression is required to achieve endometrial receptivity( 26 – 28 ). Maternal Hoxa10 expression is necessary for decidualization and embryo implantation. Mice with targeted Hoxa10 disruption demonstrate uterine factor infertility. Hoxa10 knockout mice produce a normal number of embryos, yet neither these embryos nor wild-type embryos implant in Hoxa10 (−/−) uteri. ( 29 – 31 ) Similarly, altered HOXA10 gene expression is associated with decreased implantation in humans. Endometriosis, a poorly understood gynecological disorder, is characterized by the presence of endometrial tissue outside of the uterus and often results in reproductive infertility. Endometrial expression of HOXA10 and HOXA11 are downregulated in women with endometriosis.( 35 , 36 ) In addition, decreased endometrial HOXA10 expression is apparent in women with hydrosalpinges, polycystic ovary syndrome and submucosal uterine leiomyomas; all associated with reduced implantation rates ( 37 – 40 ). Diminished endometrial HOXA10 expression is a common mechanism by which multiple gynecological disease states impact endometrial receptivity. The variables confounding uterine HOXA10 expression in IUD users include patient age and last menstrual period. The inverse relationship between age and uterine HOXA10 expression correlates with the well know association between age and fertility. The cyclic variation of endometrial HOXA10 expression is well characterized; therefore it is not surprising that menstrual cycle stage influences HOXA10 expression, even in IUD users( 26 ). The variables including duration of usage and prolonged vaginal bleeding had no influence on HOXA10 expression or contraceptive efficacy; this correlates with both immediate and prolonged efficacy of IUDs. Duration of use doesn’t improve or hinder IUD efficacy. Similarly, prolonged vaginal bleeding in the presence of a copper IUD does not impair contraceptive efficacy. Decreased endometrial HOXA10 expression in IUD users suggests a novel mechanism of copper IUD mode of action involving the suppression of genes required for endometrial receptivity, primarily endometrial HOXA10 expression.

Twenty-four women using Paraguard T380A were recruited under an approved Human Investigation Committee protocol. Informed consent was obtained from all subjects. At the time of IUD removal, adherent endometrial tissue was collected from the device at variable times during the menstrual cycle. The average patient age was 29 years (range 19–43). The average duration of IUD usage was 17 months (range 1–122 months). Control endometrial biopsies were obtained from 10 normally cycling who were not using an IUD or hormonal contraceptive method. Endometrium was obtained by collecting tissue incidentally shed at the time of IUD removal. The tissue samples were then fixed in formalin, embedded in paraffin, cut into 5μm sections, and mounted on glass slides. Slides were deparaffinized and hydrated through a series of three 10 minute xylene and ethanol washes respectively, followed by permeabilization in cold 95% ethanol. After rinsing with distilled water, antigen retrieval was performed by steaming the slides at 90°C in 0.01M sodium citrate for 20 minutes and at room temperature for an additional 20 minutes. Slides were rinsed in PBS for 3 minutes, followed by an additional 5 minute wash in PBS containing 0.1% Tween 20 (PBST). A 3 minute rinse in 3% hydrogen peroxide was performed to quench endogenous peroxidase activity. To block non-specific binding, tissue sections were incubated at room temperature with 1.5% normal horse serum diluted in PBST for 1 hour. Slides were then incubated overnight at 4°C with HOXA10 polyclonal primary antibody (sc-17159) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Following a 5 minute rinse in PBST, slides were incubated for 1 hour at room temperature with biotinylated secondary antibody, anti-goat horse IgG (BA-9500), purchased from Vector Laboratories (Burlingame, CA). Slides were rinsed in PBST for 5 minutes. To increase stain intensity and reduce background stain, slides were incubated for 15 minutes in VECTASTAIN ® Elite ABC, purchased from Vector Laboratories. After a 5 minute rinse in PBST tissue sections were incubated for 5 minutes in 3,3′-diaminobenzidine (DAB), purchased from Vector Laboratories. To counterstain nuclei, slides were exposed to hematoxylin for 12 seconds and immediately rinsed in distilled water. Finally, slides were rehydrated by a series of 3, 3 minute washes in ethanol and xylene respectively, and then mounted with Permount. Normal horse IgG and anti-goat IgG were used as negative controls. Analysis of HOXA10 expression was performed independently by three evaluators blinded to specimen source and quantified using the H-Score. The H-Score was used to calculate glandular and stromal cell staining intensity and was calculated using the following equation: H-Score = Σ Pi (i + 1), where the Pi represents the percentage of stained cells (0–100%) and stain intensity (i) is assigned a value of 1, 2, or 3 (weak, moderate, or strong respectively)( 32 , 33 ). H-Score results for endometrial stromal and glandular staining obtained from each evaluator were averaged. Statistical analysis was performed using Mann Whitney Rank Sum Test, where statistical significance was defined as P< 0.05. Linear regression analysis was performed using Statistical Analysis Software (SAS) to identify variables confounding endometrial HOXA10 expression.