Mutation Study Of EMX2 Gene In Women With M(?)llerian Duct Anomalies And HOXA10,EMX2,TENM1 Expression In The Mid-secretory Endometrium Of Infertile Women With A M(?)llerian Duct Anomaly

Objective To investigate whether the involvement of EMX2 gene in müllerian duct anomalies.At first,direct sequencing of EMX2 gene exons,exon–intron boundaries and flanking sequence.Functional studies was conducted to clarify whether the mutation affects the ability of EMX2 gene.Methods Genomic DNA were extracted from 162 women with müllerian duct anomalies and 100 controls were used for EMX2 gene mutation analysis by using standard methods from peripheral blood leukocytes. Three coding regions of EMX2 gene along with exon–intron boundaries and flanking sequence were amplified by polymerase chain reaction(PCR) using specific primers. PCR products were sequenced on an automated DNA Sequencer(ABI 3730 XL DNA sequencer).Mutations were found using Chromas(version 2.31). Two luciferase reporter constructs was to clarify the effect of the novel mutation.Results EMX2 gene coding regions were screened,exon–intron boundaries and flanking sequence for mutations in 162 women with müllerian duct anomalies and 100 unrelated unaffected controls. Sequence analysis of EMX2 gene in this patient’s DNA found a novel heterozygous T to G mutation in position 6 of flanking sequence(3’untranslated region). The variant was not found in 100 unrelated controls. By methods of bioioformatics and luciferase reporter constructs,we validated the binding sites of micro RNA4307 and micro RNA1258 in the 3’UTR of EMX2 gene.However, the region including the heterozygous mutant in the 3’UTR of EMX2 gene did not bind with micro RNA4307 and micro RNA1258.Conclusions The research hsa found that there is a novel mutation in position 6 of3’UTR of EMX2 gene in the women of müllerian duct anomalies and found a novel mutation in position 6 of 3’untranslated region. But this mutation is not involved in the pathogeny of müllerian duct anomalies.None mutation of EMX2 gene coding regions was found in the patients and controls.Micro RNA4307 and micro RNA1258 can modulate the expression of EMX2 gene by binding its 3’untranslated region.Objective Homeobox A10(HOXA10) and empty spiracles homeobox 2(EMX2) are two transcription factors necessary for female Müllerian duct differentiation and development. They are thought to play important roles in embryo implantation in mice and humans. The EMX2 gene is a known direct target of HOXA10 in the reproductive tract. Human TENM1 is directly regulated by EMX2 and is expressed during embryonic pattern formation and morphogenesis. This study aimed to investigate expression patterns of HOXA10, EMX2 and TENM1 in the mid-secretory endometrium of infertile patients with a Müllerian duct anomaly causing a partially septate uterus.Methods Mid-secretory endometrial biopsies were collected from 13 infertile women aged with a partially septate uterus. Both transvaginal ultrasound and a midcyclesurge in urinary LH were used to determine the midsecretory phsae. Association of Gynaecological Endoscopy system, a partially septate uterus is defined as when a septum divides the uterine cavity incompletely above the internal cervical orifice.Hysteroscopy was carried out during the midsecretory phsae(days 19–23) of the menstrual cycle, and the samples were taken from endometrium lining the lateral wall of the uterus with microforceps. Twelve nulliparous premenopausal women volunteered to take part in this study as a control group. They all had a normal uterus, cervix, vagina and ovaries, but their husbands were infertile with non-obstructive azoospermia. In the control group, mid-secretory endometrial tissues from the lateral wall of the uterus were also collected during the midsecretory phsae of the menstrual cycle under hysteroscopy.HOXA10,EMX2, TENM1 m RNA expression levels were analyzed by quantitative real-time PCR.HOXA10,EMX2, TENM1 protein expression levels were analyzed by w estern blo t.Results The endometrial tissues expressed HOXA10, EMX2 and TENM1. The expression levels of HOXA10, EMX2 and TENM1 by real-time quantitative PCR in 13mid-secretory endometrial tissue samples from women with a partially septate uterus and from 12 women with a normal uterus ascontrols are shown in this paper. The m RNA expression level of HOXA10 in the tissues from the cases was significantly lower than incontrols(P <0.01). EMX2 was expressed at significantly higher levels in the tissues from cases compared with controls(P <0.01), as was TENM1(P <0.01).Then, to test whether the changes in HOXA10, EMX2 and TENM1 m RNA levels were associated with changes inprotein levels; we carried out Western blot analysis on identicallytreated samples. The HOXA10 protein expression levels in the mid-secretory endometrial tissues from women with a partially septate uterus were statistically significantly lower than those in controls(P = 0.002). The cases had statistically significantly higher levels of EMX2(P <0.01)and TENM1(P <0.01)proteins compared with thecontrols.Conclusions The mid-secretory endometrial tissues expressed HOXA10, EMX2 and TENM1.Changes in HOXA10, EMX2, and TENM1 expression levels might act in infertile women with Müllerian duct anomaly to cause a partially septate uterus.