Pelvic organ prolapse (POP) results from the failure of the support and support mechanisms of the pelvic viscera, resulting in vaginal protrusion and the fall of the pelvic organs through the vaginal canal [1]. The pathophysiology of genital prolapse is multifactorial, in which environmental factors such as lifestyle, parity, and childbirth interact with molecular, endocrine, and genetic factors (2). Several risk factors have been identified in the genesis and/or progression of POP and, depending on the combination of risk factors in a person, prolapse may or may not develop during their lifetime [3]. The uterosacral-cardinal complex, including the uterosacral ligament (USL), is responsible for the apical support of the vagina and pelvic organs, with its loss of support being associated with advanced prolapse (4, 5). USLs are composed of collagen, smooth muscle, elastin and nerve bundles (6, 7). On POP there are histomorphological changes characterized by decreased smooth muscle content, decreased cellularity, alterations in the extracellular matrix (ECM), increased apoptosis, increased inflammation and increased adipocytes (7, 8, 9, 10, 11, 12, 13). The ECM dysfunction is characterized by alterations in metabolism and distribution of the main proteins, like changes in the proportions of collagen subtypes (14,15,16,17).
Hoxa11 is responsible for development of the female reproductive system and is responsible for the development of the uterosacral ligaments, lower uterine segment, and cervix, and also acts in collagen type III synthesis and matrix metalloproteinase 2 (MMP2) synthesis (18, 19, 20). HOXA11 regulates morphology and integrity of USLs by promoting cell proliferation and attenuating apoptosis through the suppression of the p53 gene, a tumor suppressor gene that is associated with the formation of extracellular matrix (13, 21, 22, 23). Studies have shown reduced expression of HOXA11 associated with reduced expression of collagens and increased MMP2 and low cellularity in women with POP (13, 23). 
It is our hypothesis that due to the low expression of HOXA11 in women with prolapse, there are changes in the uterosacral ligament, as well as the organization of the connective tissue, proportion of smooth muscle and cellularity. Therefore, the objective of this study is to analyze the expression of the HOXA11 gene in the uterosacral ligament in women with pelvic organs.

A prospective cross-sectional study was carried out with a consecutive sampling of 50 women with genital prolapse (POP) and 50 women from the control group (without prolapse) assisted at the Women's Hospital at the State University of Campinas (UNICAMP). This study was approved by the Research Ethics Committee (protocol 64599516.4.0000.5404). Uterosacral ligaments and vaginal wall biopsies were performed during hysterectomy for POP or other benign gynecologic indications than POP. Samples were subjected to morphological analyses using hematoxylin and eosin and immunohistochemistry. Hoxa11 quantification of immunostained area and morphology analysis was performed using ImageJ. Analysis and correlation of clinical, sociodemographic, and quantitative data of Hoxa11 gene expression were performed. Descriptive analysis were performed and t-Student test or Fisher test were performed for continuous and dichotomous variables, respectively.

In the prolapse (POP) group, we observed a more frequent family history of POP (p=0.046), more previous surgeries (p=0.007), gynecological surgeries (p=0.001), pregnancies, and deliveries, with an average number of vaginal deliveries per woman with prolapse being 3.6 (95% CI: 2.8-4.3) versus 0.8 (95% CI: 0.5-1.2) in those without prolapse (p<0.05). The mean age of the control group was 44.6 years (95% CI: 42.8-46.5), meanwhile women in the prolapse group were 21 years older with mean age of 65.9 years (95% CI: 63-68.9) (p<0.05) and therefore more likely to be in menopause. We found a higher prevalence of arterial hypertension (p=0.001), diabetes mellitus (p=0.046), and medication use (p=0.005) in the POP group, with no difference in BMI between the groups (p=0.09). In the POP-Q measurements, there was a significant difference between all points measured during the Valsalva maneuver and in the measurement of the genital hiatus, which was 1.2 cm larger (p<0.05) in the POP group, with a mean of 3.9 cm (95% CI: 3.5-4.3) versus 2.7 cm in the control group (95% CI: 2.6-2.9). In the quantitative analysis of Hoxa11 expression by immunohistochemistry, the control group (n=16) presented higher Hoxa11 expression with a mean of 21.60% immunostained area, while the POP group (n=15) presented a mean of 10.20% immunostained area (p = 0.0043). The morphology analysis revealed no difference on fibroblast count between groups (p = 0.6356), also there was no difference on smooth muscle area (p = 0.5629). USLs in POP group (n=13) in comparison with the control group (n=10) have extracellular matrix disorder with larger area of loose connective tissue (p = 0.0099) and smaller area of dense connective tissue (p = 0.0065). The mean area percentage of loose connective tissue on POP was 48% while on the control group was 24%. The mean percentage area of dense, connective tissue on POP was 6% whereas on the control group was 26%.

This is to our knowledge, the study with the largest sample studying the HOXA 11 gene between women with and without POP, and we have confirmed the findings from the first pilot studies performed by Connell et al. and other researchers. It is unclear whether this altered pattern of the HOXA 11 gene will be similar with other molecular studies, such as western blot and RT-PCR analysis, but we believe these data are promising with regard to expecting another marker for POP. This is a cross-sectional study, and one of the weaknesses is that we cannot stablish causality between POP and HOXA11 expression, and future cohort studies are needed.

Women with genital prolapse have a more frequent family history of prolapse, older age, higher parity, more previous surgeries, increased genital hiatus, more comorbidities, and medication use. HOXA11 gene expression is decreased in women with POP by IHC. USLs in POP have extracellular matrix disorder with prevalence of loose connective tissue and reduction of dense connective tissue.

Alperin M, Moalli PA. Remodeling of vaginal connective tissue in patients with prolapse. Current opinion in obstetrics & gynecology. 2006;18(5):544-50.
Ma Y, Datar A, Hennessey A, Cardenas I, Johnson J, et al. Knockdown of Hoxa11 in vivo in the uterosacral ligament and uterus of mice results in altered collagen and matrix metalloproteinase activity. Biol Reprod 2012;86 (4):100
Connell KA, Guess MK, Chen HW, Lynch T, Bercik R, Taylor HS. HOXA11 promotes fibroblast proliferation and regulates p53 in uterosacral ligaments. Reproductive sciences. 2009;16(7):694-700.