The Role Of HOXA Genes In The Pathogenesis Of Human Mullerian Duct Abnormalities (MDAs) And Pregnancy Outcome In Women With MDAs

PartⅠThe role of HOXA10mutation in the pathogenesis of human Mullerian duct abnormalitiesBackgroudMullerian duct abnomalities (MDAs) refer to congenital absence or severe malformations of female reproductive tracts, caused by defects at any stage of the mullerian duct developments. Previous studies revealed a series of genes involved in the pathogenesis of MDAs. HOXA genes are important developmental regulatory genes participating in body segmental identity, and many other organ developments. The HOXA5’genes are mainly expressed in the reproductive system.During embryogenesis, HOXA10was expressed in the primodial of uterus and in female adults in the copus of uterus. Experiments on knock-out mice revealed that the functional loss of Hoxa genes might lead to abnormalities of mullerian ducts. Hoxa10gene-/-mice has a transformation of the upper uterus to fallopian tubes. Exposure to DES during pregnancy would also reduce HOXA10expression and result in the "T" shape uterus. HOXA10is an attractive candidate gene for MDAs.ObejectiveThe present study was to determine whether HOXA10variants contribute to MDAs in192Chinese Han females.Methods A total of192women with MDAs of Han Chinese (n=192) were recruited. Ethnically matched healthy women with normal developed uterus served as controls. All were screened for variants in the HOXA10gene by Sanger sequencing. Novel Variants were sequence-aligned and functional predicted by various online tools. Subsequently, transactivation activity assay using Dual-Luciferase Reporter Assay System, and protein expression and nuclear localization with GFP vector were conducted to determine the causative role of the novel variants in vitro.ResultsA novel heterozygous missense variant (c.394G>A, p.Gly132Arg) was identified in a patient with MRKH. The Y57C mutation was verified in three MDAs patients. The p.G132R and Y57C were highly conserved among orthologs. G132R impaired ranscriptional activation on EMX2and ITGB3gene, as shown by transactivation assays. However, no dominant negative effect was observed, nor was there impact on protein expression and nuclear localization. ConclusionTwo heterozygous variants in four patients with MDAs were identified in192Chinese Han populations. Mutations in HOXA10might not be common explanation for MDAs in Chinese. G132R mutation is presumed to result in haploinsufficiency and impair transactivation activity on target genes of HOXA10so as to participate in the development of MDAs. Part IIThe role of HOXA11variants in the pathogenesis of Chinese patients with MDAsBackgroundThe etiology of MDAs is genetically heterogenous with various clinical manifestations. HOXA11is one of the key regulatory factors formed protein complexes with Meis1to bind the promoter region of downstream genes, participating in the process of mullerian duct development, kidney formation, limb development and hemotogenesis. HOXA11is expressed in lower uterus and cervix both in embryogenesis and adults. Hoxall knock-out lead to sterility, with decreased endocrine glands and leukemia inhibitory factor (LIF) secretion, as well as skeleton malformations. Deletions in chromosome7p including HOXA11was identified in patients with phenotype similar to hand-foot-genitila syndrome or severe developmental delay. HOXA11seemed good candidate gene for MDAs.ObjectiveThe aim of the present study was to determine whether HOXA11variants account for MDAs in Han Chinese.MethodsA large cohort of of Chinese women with MDAs (n=270) were recruited to screen for HOXA11variants with direct sequencing. For the identified novel missense mutation, functional prediction by Align GVGD, Polyphen-2, Mutation Taster and SIFT were performed. Protein expression and nuclear localization with GFP vector and transactivation activity assay using Dual-Luciferase Reporter Assay System were conducted to determine the role of the novel variants.ResultsA novel heterozygous missense variant c.260G>A (p.Glu87Gly, p.E87G) was identified in one of two hundred and seventy MDAs patients in Han Chinese, plus a synonymous mutation (c.774G>A) and SNP rs201081293. E87G, located in an unknown region of HOXA11, was discorved in a woman with MRKH syndrome. This point was highly conserved among othlogs, with a prediction of "probably damaging". However, in vitro analysis revealed no impairment on HOXA11protein expression, localization and transactivation activity.ConclusionsA novel heterozygous variant E87G was indentified in the large cohort of Chinese women with MDAs. Preliminary in vitro analysis revealed no effect on HOXA11expression, subcellular localization and transactivation, which was contradictory to the functional prediction. HOXA11gene variants were not common in Chinese Han patients with MDAs. Part ⅢMutation screening of HOXA7and HOXA9genes in Chinese women with MDAsBackgroundSeveral factors have been suggested to contribute to the cause of MDA, such as environmental factors, intrauterine exposure to radiation or drugs and chromosomal abnormalities. However, the mechanisms for most MDA cases were unknown. Highly conserved between species, homeodomain proteins direct the early morphogenesis and spatial identity along the anterior-posterior axis. HOXA7is expressed specifically in regions of the neuoectoderm and mesoderm in mouse embryos, and the epithelium of fallopian tubes and endometrium in humans. HOXA9is expressed in the primordia of fallopian tubes during embryogenesis and oviducts in adults. Aberrant expression and methylation of HOXA9was found in patients with MRKH. Previous studies have revealed that patients carrying deletions located at chromosome7p15or7p21.1-p14.3, covering the HOXA7gene, have either features similar to the hand-foot-genital syndrome or severe developmental delay. Few studies had been conducted on HOXA7and HOXA9genes, except for mutational screening in six patients with MRKH.ObjectiveIn this study,192Chinese patients with MDA were examined to elucidate whether HOXA7and HOXA9mutation was causative for the malformation.MethodsA total of192patients with MDA and192controls were recruited among patients attending the Center for Reproductive Medicine, Provincial Hospital affiliated to Shandong University between January2009and December2012because of infertility. Genome DNA was extracted and direct sequencing to scan for gene variants of HOXA7and HOXA9.ResultsRs2301721and rs2301720in HOXA7, rs35355140and rs7810502in HOXA9 were identified in patients with MDA and controls. One rare single nucleotide polymorphism rs189587233in3’UTR of HOXA9gene was detected in one patient with didelphic uterus and absent in the192controls. This polymorphism, however, is known to exist in the normal Chinese population.ConclusionsOur results indicated that variants in the HOXA7and HOXA9genes were not common in Chinese women with Miillerian duct abnormalities. Part ⅣReproductive performances of patients with unicornuate uterus undergoing IVF/ICSI-ETBackgroudUnicornuate uterus is defined as the normal development of only one mullerian duct. It covers a wide range of anatomic variability according to the differentiation of the other mullerian duct and account for about20%of all MDAs. Increased rates of preterm birth, ectopic pregnancy and cesarean section have been reported for unicornuate uterus. However, there remained many concerns with regard to its reproductive performance, including:whether it increases infertility; the concrete gestational week of miscarriage and preterm birth; the maternal complications and newborn characteristics.ObjectiveTo investigate the effect of unicornuate uterus on the obstetrical outcomes undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) treatment.MethodsOne hundred and fifty-five females with unicornuate uterus and two hundred eighty-six controls, who underwent IVF/ICSI-ET treatment between2009and2011in a university-affiliated hospital, were recruited for this retrospective cohort study. Trans-vaginal ultrasound, hysterosalpingography, hysteroscopy and laparoscopy were performed for diagnosis. Strict inclusion criteria and matching applied for both studies cases and normal controls to exclude possible confounding factors.ResultsHigher rates of biochemical miscarriage (14.5%vs.7.7%, adjusted OR1.963[1.015-3.798], p<0.05), preterm birth (33.3%vs.15.6%, adjusted OR2.448[1.088-5.506], p<0.05), and lower clinical pregnancy rate (42.8%vs.55.6%, adjusted OR0.603[0.396-0.918], p<0.05) were observed in patients with unicornuate uterus compared with controls.70.6%of of preterm birth occurred between34-36gestational weeks. Among patients those suffering from primary infertility were more apt to poorer pregnancy outcomes including increased rate of preterm birth, and decreased clinical pregnancy rate and take-home-baby rate. However, the rates of maternal complications (includinggestational hypertension, preterm rupture of membrane and placenta previa) and newborn characteristics (including gestational weeks at birth, mean birth weight, and percentage of low birth weight) were comparable in the two groups. Pregnancy outcomes including pregnancy rate, miscarriage rate, and preterm birth rate in patients accepting single embryo transfered were similar with that in controls with one embryo transfered.ConclusionsOur results suggest that unicornuate uterus impair both embryo implantation potential and maintenance of late-term pregnancy while primary infertile patients are more challenged. Single embryo transfer is optimal for patients with unicornuate uterus to get better obstetrical outcomes. Part ⅤPregnancy loss characteristics of Chinese patients with MDAs undergoing IVF-ETBackgroundMDAs might have adverse effects on pregnancy outcomes, such as recurrent pregnany loss and premature birth. It has been reported that for patients with septum and bicornuate uterus who had a history of recurrent pregnancy loss, the miscarriage rate occurred at second-trimester was inceased. Due to its clinical heterogeneity, different kinds of MDAs might result in abonormal pregnancy outcome at different gestational week. However, there was little knowledge about the characteristics of pregnancy outcome and miscarriage in patients with MDAs.ObjectiveThis retrospective study was performed to explore the characterics of pregnancy loss characteristics of different categories of MDAs undergoing IVF-ET.MethodsA retrospective cohort study was conducted with a large sample of MDAs in Han Chinese (n=277) and matched controls (n=368). Pregnancy loss during the whole gestational weeks was recorded. Pregnancy loss pattern were analysed for different MDA type. Possible confounding factors including prior spontaneous abortion, IVF indication, and endometrium thickness on HCG day were adjusted and then subgroup analyzed.ResultsThe prevalence of prior spontaneous abortion history was higher for MDAs patients. After COH theray, they were with thinner endometrial thickness (1.02±0.21cm vs.1.12±0.17cm, p<0.01) and elevated level of E2on HCG days (p<0.05).The total pregnancy loss rate in the MDAs group was17.9%, similar to that of the controls (15.8%). MDAs patients were with increased rate of biochemical pregnancy (13.3%vs.4.9%,p<0.01) and decreased rate of miscarriage after clinical pregnancy (4.6%vs.10.9%, p<0.01). The MDAs patients were with reduced early miscarriage rate(2.9%vs.9.0%,p<0.01, adjusted OR0.294[0.118-0.733]), increased late miscarriage rate (2.5%vs.0, p<0.01) and similar ectopic pregnancy rate (1.7%vs.1.9%)Subgroup analysis revealed that unicornuate uterus increased the risk of biochemical pregnancy and late miscarriage rate after clinical pregnancy though their early miscarriage rate was lower. Uterus septum was also shown to be with inceased biochemical pregnancy. For arcurate uterus, the late miscarriage rate after clinical pregnancy was elevated.ConclusionsPregancy failure or loss in patients with MDAs patients usually occurred at the very early stage of embryo implantation and late pregnancy. Thinner endometrium might have adverse effect on pregnancy failure or loss. Patients with unicornuate uterus are more susceptible to biochemical prengnancy loss and late miscarriage. Patients with septate uterus have higher risk of biochemical pregnancy loss, while arcurate uterus was associated with higher risk of late miscarriage.