ENDOMETRIAL POLYPS AFFECT UTERINE RECEPTIVITY

Narrative abstract

This case-control study evaluated the effect of hysteroscopically identified endometrial polyps on endometrium using HOXA10 and HOX11, known molecular markers of endometrial receptivity. Uteri with endometrial polyps demonstrated a marked decrease in HOXA10 and HOXA11mRNA levels that may impair implantation; these findings suggest a molecular mechanism to support clinical findings of diminished pregnancy rates in women with endometrial polyps.

Endometrial polyps are localized hyperplastic overgrowths of endometrium that contain both endometrial glands and stroma. The large majority of these lesions are benign. Polyps have a variable presentation; they can occur as individual or multiple lesions, range in size from millimeters to centimeters, and can be sessile or pedunculated. Although endometrial polyps may be identified during evaluation of abnormal bleeding, many polyps are asymptomatic and only discovered during the infertility evaluation. Up to 25% of women with unexplained infertility have endometrial polyps on hysteroscopy (1–3). However, their effect on endometrial receptivity and fertility is unclear.

Established markers of endometrial receptivity, HOXA10 and HOXA11, were utilized to investigate the effect of endometrial polyps on endometrium. HOXA10/Hoxa10 (human/mouse) is a homeobox-containing transcription factor that is essential for embryonic uterine development, essential for proper adult endometrial development during each menstrual cycle, and necessary for endometrial receptivity (4–11). Targeted mutation of Hoxa10 produces mice that experience implantation failure, likely the sequela of absent Hoxa10 during embryonic uterine development and the direct effect of absent Hoxa10 during cyclic adult endometrial development (12). Furthermore, decreased Hoxa10 expression correlates with diminished implantation, which suggests that an altered level of Hoxa10 regulates the degree of endometrial receptivity (5). Similarly, a targeted mutation of Hoxa11 results in uterine factor infertility and poor endometrial glandular development (13, 14). Hoxa11 heterozygotes have lower levels of Hoxa11 expression and diminished implantation rates (14).

In humans, HOXA10 and HOXA11 are expressed in proliferative phase endometrial glands and stroma, and expression of these genes is markedly upregulated in the midsecretory glands at the time of implantation (8, 15–18). Both estrogen and progesterone upregulate HOXA10 and HOXA11 expression (8, 17). The pattern of HOXA10 and HOXA11 expression suggests a critical role in the implantation process. Furthermore, downstream target genes involved in implantation regulated by HOXA10 include β₃ integrin, EMX2, and IGFBP-1, and KLF9 (19–22). Defective HOXA10 expression has been demonstrated in conditions associated with implantation such as endometriosis, polycystic ovarian syndrome (PCOS), hydrosalpinges and submucosal myomas (9, 15, 23–25). This study investigated the effect of endometrial polyps on endometrium by evaluating established markers of endometrial receptivity, HOXA10 and HOXA11.

This case-control study included 30 subjects, 21 with endometrial polyps and 9 with a normal uterine cavity without polyps. Subjects were identified at the time of hysteroscopy for evaluation of the endometrial cavity prior to infertility treatment. One-half of the subjects had a known endometrial abnormality identified during a sonohysterogram, and nine subjects were considered controls after finding a normal uterine cavity. At the time of surgery, the following data were obtained: age, obstetric and gynecologic history, medical conditions, medications, surgical history, last menstrual period, preoperative and postoperative diagnoses, and operative findings. Subjects had not used hormonal medications for at least 3 months prior to surgery, and had regular menstrual periods. Subjects did not have any confounding medical conditions known to affect endometrial receptivity such as endometriosis, PCOS, hydrosalpinges or submucosal myomas. This study was approved by the Yale University School of Medicine Human Investigation Committee.

All subjects underwent hysteroscopy in the proliferative phase of the menstrual cycle. When an endometrial polyp was present, it was excised under hysteroscopic guidance. Endometrial curettage was performed in all procedures, and the endometrial specimen was divided for pathology evaluation and laboratory investigation.

At surgery, each endometrial tissue sample was stored in 1 ml of RNAlater solution (Qiagen, Valencia, CA) at −80°C until RNA isolation. RNA was processed as previously described (25). Messenger RNA levels were analyzed by quantitative real-time RT-PCR using the Bio-Rad iCycler iQ system (Bio-Rad Laboratories, Hercules, CA). For each sample, 500ng of total RNA was reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Real-time RT-PCR was performed using a MyiQ Single Color Real-Time PCR Detection System and iQ SYBR Green Supermix (both, Bio-Rad). The sequences of all primers and the real time RT-PCR reaction conditions have been previously published (25). Melting curve data were collected and analyzed. Each assay was run in duplicate with each set of primers, and samples without mRNA were included as negative controls. HOXA10 and HOXA11 gene expression was normalized to the expression of β-actin for each sample. Relative mRNA expression levels for each gene were calculated using the 2^−ΔΔC_T method (26). Differences in endometrial HOXA10 and HOXA11 expression between the polyp and control groups were determined using Student’s t-test; p<0.05 was considered statistically significant.

Mean subject age was 33.6 years (range 22–41 years). There was no difference in mean age between the two groups. Twenty-one subjects had endometrial polyps, and polyp size ranged from 3–20mm. The number of polyps ranged from 1–4, and 6 of 21 subjects (28.6%) had more than one polyp.

All samples underwent histological evaluation; benign endometrial polyps were identified, and curettage samples revealed normal proliferative endometrium. Endometrium from subjects with and without polyps was evaluated for HOXA10 and HOXA11 mRNA expression.

Compared to controls, endometrial HOXA10 mRNA expression (normalized to β-Actin expression) was significantly decreased in uterine cavities with polyps (0.334× control; p=0.016; Figure 1). Similar findings were seen for endometrial HOXA11 mRNA expression in the presence of a polyp compared to controls (0.183× control; p=0.03; Figure 1). When polyp samples were compared based on polyp size [<8mm polyps (n=15) versus ≥8mm polyps (n=6], there were no differences in HOXA10 (p=0.354) or HOXA11 (p=0.11) expression. Similarly, the presence of single (n=15) versus multiple (n=6) polyps did not affect HOXA10 (p=0.092) or HOXA11 (p=0.795) expression.

Figure 1.

(A) Endometrial HOXA10 gene expression in control and polyp groups; (B) Endometrial HOXA11 gene expression in control and polyp groups.

The effect of asymptomatic endometrial polyps on infertility is unclear. However, it is plausible that polyps can cause infertility due to mechanical interference with sperm and embryo transport, impairment of embryo implantation or altered endometrial receptivity. Furthermore, the size, number or location of polyps may influence any effect on reproductive outcomes.

Several studies have investigated the effect of endometrial polyps on pregnancy rates for infertile women who underwent intrauterine insemination (IUI) or in vitro fertilization (IVF), or who conceived spontaneously, and disparate results were obtained. One prospective randomized trial (n=215) identified significant improvement in pregnancy rates after hysteroscopic polypectomy and up to four cycles of controlled ovarian hyperstimulation with gonadotropins plus IUI (63% vs. 28%, p<0.001) (27). In this study, all polyps were visualized hysteroscopically, and the study group underwent hysteroscopic polypectomy and the control group underwent hysteroscopic biopsy of the polyp. Additionally, several nonrandomized studies of women with unexplained infertility and polyps demonstrated an improvement in spontaneous pregnancy rates after undergoing hysteroscopic polypectomy (3, 28, 29). In contrast, several studies of women undergoing IVF have not demonstrated impairment of pregnancy rates in the presence of polyps <2cm in size (30, 31), although one study (31) noted an increased miscarriage rate in the polyp group and recommends embryo cryopreservation if a polyp is identified. These IVF studies included 15 and 49 subjects, respectively, with polyps who underwent IVF, and may have been underpowered to detect a difference in pregnancy rates. Hence, a better understanding of the effect of polyps on infertility and pregnancy is warranted.

The effect of polyps on the endometrium was evaluated using HOXA10 and HOXA11, established molecular markers of endometrial receptivity. Significantly lower HOXA10 and HOXA11 expression was identified in endometrium from uteri with polyps compared to controls, suggestive of impaired endometrial receptivity in uteri with polyps. The presence of a polyp may alter HOX gene expression throughout the endometrium, and changes in endometrial signaling pathways lead to impaired endometrial receptivity. These findings provide molecular data to support some clinical findings that pregnancy rates improve after hysteroscopic polypectomy. Furthermore, these findings mirror our findings of impaired endometrial receptivity in uteri with submucosal myomas (25). This study did not have the power to assess differences in these markers based on the number of polyps or polyp size, and future studies should investigate these questions. It is not known if there is a threshold size above which a polyp causes a significant clinical effect.

Research on uterine myomas and infertility may help guide recommendations for the management of polyps in infertile women. It has been established that submucosal myomas negatively impact fertility and pregnancy rates, and surgical removal of submucosal myomas leads to improved pregnancy rates (32). Furthermore, when a submucosal myoma is present, endometrial receptivity is globally impaired throughout the uterine cavity (25), and this finding may explain the reproductive dysfunction observed with submucosal myomas. Here we show that endometrial polyps have an effect on markers of endometrial receptivity that is similar to the effect seen with fibroids; these two conditions likely both affect endometrial receptivity through similar molecular mechanisms.

Our finding of impaired endometrial receptivity in uteri with one or more endometrial polyps supports the results from the one randomized trial demonstrating the negative effect of polyps on fertility (27). Detailed evaluation of the uterine cavity is essential in infertile women, and if an endometrial polyp is identified, hysteroscopic polypectomy prior to infertility treatment is reasonable and recommended as this intervention may improve fertility. Further studies are warranted to evaluate if markers of endometrial receptivity normalize after removal of the polyp.