HOXA10基因在子宫内膜异位症不孕患者内膜组织中的表达及意义

目的:探讨HOXA10基因在子宫内膜异位症(EMs)不孕中的作用及作用机制。方法:以行腹腔镜手术并经病理确诊为EMs合并不孕症的患者为研究对象(18例,A组),以同期行腹腔镜手术的输卵管性不孕患者(18例,B组)、正常生育组(15例,C组)为对照。分别应用荧光定量PCR、免疫组织化学和Western blotting等方法从mRNA及蛋白质水平检测HOXA10基因在EMs不孕症患者在位、异位子宫内膜以及对照组在位子宫内膜的表达。结果:HOXA10 mRNA及蛋白表达水平在C组的内膜较高;B组稍低于C组,但两者相比差异无统计学意义(P>0.05);A组的在位及异位内膜表达水平较C组明显降低(P<0.01);但A组的在位内膜与异位内膜基因和蛋白的表达水平均无统计学差异(P>0.05)。结论:HOXA10基因在EMs不孕患者在位子宫内膜中的表达显著降低,可能是EMs不孕患者子宫内膜容受性降低,进而引起不孕的重要因素之一。     

HOXA9、HOXA11、LIF在输卵管中的表达及与输卵管妊娠的关系

目的:探讨同源框基因HOXA9、HOXA11以及白血病抑制因子(1eukemia inhibitory factor,LIF)在输卵管异位着床中的作用。方法:用免疫组织化学染色方法分别检测36例异位妊娠输卵管和34例正常输卵管HOXA9、HOXA11和LIF蛋白的表达。结果:①HOXA9、HOXA11强阳性(+++)表达率在异位妊娠输卵管中最高,且输卵管着床部位和非着床部位表达率无明显差异;增生期输卵管HOXA9、HOXA11强阳性表达率明显较低,在分泌期输卵管中表达率为0。②LIF强阳性表达仅见于妊娠输卵管,着床部位的表达率高于非着床部位;增生期和分泌期输卵管呈中低水平表达。结论:HOXA9、HOXA11及LIF蛋白表达增加与输卵管妊娠相关。     

In vitro fertilization outcome in women with endometrial tuberculosis and tubal tuberculosis

Abstract

Purpose: To evaluate in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) outcome in women with genital tuberculosis (GTB).

Methods: One hundred and fifty-five women with genital tuberculosis constituted the study group (Group A), consisting of 25 patients with endometrial tuberculosis (Group A1) and 130 patients with tubal tuberculosis (Group A2). Women with non-tuberculous tubal infertility were matched by age and study period served as controls (Group B).

Results: Patients with GTB had significantly reduced endometrial thickness, high-quality embryos rate, implantation rate as compared with controls (p?<?.05), no differences were found in other pregnancy parameters. In addition, the endometrial thickness, fertilization rate, high-quality embryos rate and implantation rate were also significantly lower in women with endometrial tuberculosis as compared with controls. And the cumulative pregnancy rate in endometrial tuberculosis was significantly decreased compared with tubal tuberculosis and controls (p?<?.05). However, IVF/ICSI pregnancy outcomes in patients with tubal tuberculosis showed no difference as compared with controls (p?>?.05). Also, rates of miscarriage, preterm birth, obstetrical complications, and neonatal problems did not differ among three groups.

Conclusions: In conclusion, IVF/ICSI-ET remains the most optimal method for the treatment of female infertility associated with tubal tuberculosis. However, patients with endometrium tuberculosis showed significantly reduced fertilization, implantation and cumulative pregnancy rates.     

Ovarian Response to Gonadotropins in Patients With Tubal Factor Infertility: Salpingectomy Versus Nonsalpingectomy

Study ObjectiveTo evaluate the effects of salpingectomy on the ovarian response to gonadotropins and in vitro fertilization–embryo transfer (IVF-ET) cycle outcomes in women with tubal factor infertility.DesignA retrospective study (Canadian Task Force Classification II-3)SettingAn in vitro fertilization laboratory in a university hospital in Taiwan.PatientsWe analyzed the outcomes of 288 consecutive fresh IVF-ET cycles in 251 consecutive women with tubal factor infertility from January 2001 to December 2011. Two hundred eighty-eight cycles were divided into 2 groups comprising 103 cycles with laparoscopic salpingectomy and 185 cycles with prior bilateral tubal sterilization, laparoscopic tuboplasty, or proximal tubal occlusion as the control group.InterventionsControlled ovarian hyperstimulation and IVF-ET.Measurements and Main ResultsThe main outcome was measured by comparing the duration of stimulation, number of gonadotropin ampoules per cycle, number of follicles, number of oocytes retrieved, fertilization rate, implantation rate, clinical pregnancy rate, and live birth rate. We observed no significant difference in any ovarian response parameter between the salpingectomy and nonsalpingectomy groups. Implantation rates, clinical pregnancy rates, and live birth rates were similar. The mean numbers of follicles and oocytes retrieved ipsilateral to the operated side in the salpingectomy group were similar to the numbers of follicles and oocytes retrieved from the nonoperated ovary.ConclusionsLaparoscopic salpingectomy did not have a negative effect on the ovarian response in women with tubal factor infertility.     

Hydrosalpinx fluid diminishes endometrial cell HOXA10 expression

OBJECTIVE: To determine the effect of hydrosalpinx fluid on the expression of HOXA10, an essential regulator of endometrial receptivity.DESIGN: In vitro study.SETTING: Academic medical center.PATIENT(S): Patients with unilateral or bilateral hydrosalpinx.INTERVENTION(S): Hydrosalpinx fluid was aspirated from 10 patients at laparoscopy. The fluid was serially diluted in minimum essential medium. Ishikawa cells (an endometrial adenocarcinoma cell line, representative of endometrial epithelium) were incubated with this fluid at concentrations of 10% and 50% for 48 hours. Cells were also incubated in undiluted minimum essential medium (MEM) and in 10% serum as controls. After incubation, the cells were lysed in Trizol, and total RNA was extracted and analyzed by Northern blot using a 32P-labeled HOXA10 riboprobe. A 32P-labeled G3PDH probe was used as a control for loading.MAIN OUTCOME MEASURE(S): HOXA10 mRNA expression.RESULT(S): HOXA10 mRNA expression in endometrial cells decreased with increasing concentrations of hydrosalpinx fluid. Densitometric analysis of the northern blot revealed that HOXA10 mRNA expression was different from control at both concentrations (P<.007).CONCLUSION(S): HOXA10 is necessary for implantation in the murine model. HOXA10 expression is diminished by hydrosalpinx fluid. This effect on HOXA10 is a potential molecular mechanism by which implantation rates are diminished in women with hydrosalpinges.     

子宫内膜异位症内膜组织中HOXA10基因异常DNA甲基化及意义

目的:检测子宫内膜异位症(EMs)在位及异位内膜HOXA10基因的表达及DNA的甲基化,探讨HOXA10基因异常表达及DNA异常甲基化在EMs发病及不孕机制中的状态和作用。方法:选择2009年1月至2010年8月在我院行腹腔镜手术治疗的卵巢子宫内膜异位囊肿患者20例(10例合并不孕)、非EMs患者20例(卵巢良性囊肿10例,输卵管因素不孕10例)。分别采取其在位、异位内膜及正常子宫内膜组织,采用荧光定量PCR方法检测HOXA10基因mRNA的表达,甲基化特异性聚合酶链反应(MSP)检测HOXA10基因3个启动子区F1、F2、F3的甲基化状态。结果:EMs在位和异位内膜组的HOXA10基因mRNA表达水平明显低于正常内膜(P<0.01),EMs在位内膜组总甲基化率45%(9/20),合并不孕患者甲基化率50%(5/10)。EMs异位内膜组总甲基化率40%(8/20),合并不孕患者甲基化率40%(4/10)。正常内膜组仅有1例卵巢成熟性畸胎瘤患者发生了甲基化,甲基化率5%(1/20)。EMs在位、异位内膜组总甲基化率及合并不孕患者甲基化率的差异无统计学意义(P=0.58,P=0.32),但与正常内膜组的差异均有显著统计学意义(P<0.01)。结论:HOXA10基因在EMs在位及异位内膜组织中均呈低表达状态,EMs中存在HOXA10基因DNA异常甲基化,EMs合并不孕患者内膜组织中HOXA10基因的DNA甲基化明显高于非EMs不孕患者。EMs中HOXA10基因的DNA异常甲基化可能与其发病及不孕机制有关,深入研究EMs中HOXA10基因DNA异常甲基化,有望为阻断EMs发病或治疗其引起的不孕提供新的思路。     

HOXA10 protein expression in the endometrium of normally menstruating women after receiving GnRH antagonist

Objective(s)

To compare HOXA10 protein expression in the endometrium between natural control cycles and GnRH antagonist-treated cycles obtained during the window of implantation of normally menstruating women.

Study design

This study was conducted at the Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Thirty-five volunteers were recruited into this prospective, self-controlled study, which was divided into two cycles, the first a natural control cycle and the second a GnRH antagonist-treated cycle. The two cycles were separated by one resting cycle. In the GnRH antagonist-treated cycle, when the leading follicle was 15 mm, ganirelix (Orgalutran^®) 0.25 mg was administered daily. In both cycles, ovulation was induced when the largest follicle reached 18 mm in diameter. Finally, endometrial biopsy was performed on day 6 after documented ovulation, which corresponds to the window of implantation. Endometrial HOXA10 protein expression, a marker of endometrial receptivity, was analyzed by immunohistochemistry. The protein expression was compared between the two cycles regarding their percentage of immunostained cells and IHC-scores (percentage of stained cells × intensity of nuclear staining).

Results

HOXA10 protein was exclusively localized in the stromal compartment of the endometrium. The percentage of HOXA10 nuclear staining in the endometrium collected from GnRH antagonist-treated cycles was higher than that of the natural cycles, whereas the IHC-scores showed no difference between the two cycles.

Conclusion(s)

GnRH antagonists may have no effect on HOXA10 protein expression in the endometrium obtained during the implantation window of normally menstruating women.     

Influence of various tubal surgeries to serum antimullerian hormone level and outcome of the subsequent IVF-ET treatment

Abstract

Objective: To compare the influence of various tubal surgeries to ovarian reserve via serum level of antimullerian hormone (AMH) and the subsequent in vitro fertilization and embryo transplantation (IVF-ET) outcome in patients with simple tubal infertility.

Study design: A prospective cohort study was conducted on 134 IVF cycles undegone by 26 and 34 cases with bilateral and unilateral salpingectomy, respectively, 23 cases with bilateral oviducts interrupted in the proximal and 51 cases with bilateral oviducts obstruction without intervention as controls.

Results: Serum AMH displayed its great superiority to traditional markers of ovarian reserve in correspondence with antral follicles count and decisive effect for the number of oocytes retrieved after stimulation in each group. No significant differences on ovarian reserve and responsiveness or IVF-ET outcome existed among four groups comparable on essential characteristics, except for numerically higher clinical pregnancy rate and live birth rate after various tubal surgeries versus no intervention for bilateral oviducts obstruction. Especially, bilateral salpingectomy precursed the statistically highest implantation rate (51.0% versus 28.0%, 39.1%, 30.4%) and numerically best IVF outcome.

Conclusion: Tubal surgical procedures have some beneficial effect for improving IVF outcome without significant impact on ovarian reserve or responsiveness. Bilateral salpingectomy appears to be an appropriate procedure before IVF treatment for bilateral salpingitis, especially hydrosalpinx.     

Preretrieval predictors of pregnancy in IVF

Purpose: This study was designed to evaluate the predictive value of preretrieval parameters of ovarian stimulation in patients undergoing IVF-ET. Methods: Women diagnosed with infertility due to tubal factor were compared to women with other and/or multiple diagnoses. Stepwise logistic regression evaluated 389 cycles to identify the best predictors of pregnancy among the following variables: age, primary or secondary infertility, cycle number, type and dose of gonadotropin, duration of gonadotropin administration, serum estradiol level, and number and size of follicles. Results: In the tubal disease group, probability of pregnancy was greater in cycles with serum estradiol levels below 1100 pg/ml on the day of hCG (odds ratio, 4.7) and with administration of gonadotropins for less than 10 days (odds ratio, 3.7). In contrast, in the other/mixed diagnoses group, a serum estradiol below 1100 pg/ml was associated with a decreased probability of pregnancy (odds ratio, 0.6). Conclusions: Optimal parameters of ovarian stimulation may vary according to the etiology of infertility. In patients with tubal disease, the beneficial effects of greater stimulation, and thus the greater number of available oocytes, may be offset by adverse effects on the endometrium and on the quality of oocytes and embryos. In contrast, in other diagnostic groups, the advantage of an increased number of oocytes may outweigh the potential adverse effects of prolonged stimulation and higher estradiol levels.     

HOXA10基因在子宫内膜组织中的表达及与不孕的关系

目的　探讨HOXA10基因在正常育龄妇女及不明原因不孕患者子宫内膜中的表达 ,在着床过程中的作用及与不孕的关系。方法　采用原位杂交和逆转录聚合酶链反应 (RT PCR)技术 ,检测 5 2例正常育龄妇女及 38例不明原因不孕患者子宫内膜组织中HOXA10基因mRNA的表达。其中 ,正常育龄妇女子宫内膜周期为 10例增殖早期、10例增殖晚期、9例分泌早期、16例分泌中期、7例分泌晚期 ,不明原因不孕患者子宫内膜周期为增殖早期 7例、增殖晚期 8例、分泌早期 6例、分泌中期 11例、分泌晚期 6例。结果　 (1)HOXA10基因mRNA在正常育龄妇女子宫内膜腺体及间质中均有表达。原位杂交法检测 (以显色区灰度阳性单位表示 )显示 ,分泌中期腺体为 5 6 9± 0 5 7、间质为 7 48± 0 6 7,分泌晚期腺体为 5 99± 0 40、间质为 7 98± 1 0 8,显著高于增殖早期 (腺体 3 0 4± 0 30 ,间质3 2 5± 0 31)、晚期 (腺体 3 35± 0 2 0 ,间质 3 2 0± 0 37)和分泌早期 (腺体 3 0 7± 0 2 6 ,间质 3 18± 0 2 7)(P <0 0 1) ;分泌中、晚期间质表达高于腺体 (P <0 0 1)。RT PCR法检测显示 ,HOXA10基因mRNA表达 ,分泌中期为 (5 7 0± 3 4) %、晚期为 (5 6 2± 2 9) % ,显著高于增殖早期的 (31 8± 2 6 ) %、晚期的(32 2± 2 3) %和     

Cisplatin decreases HOXA13 and alphaVBeta3 integrin levels in the uterus

ObjectiveTo examine the effects of cisplatin on uterine histology and implantation molecules and the possible protective role of recombinant Klotho administration on uterine histology and uterine receptivity in mice exposed to cisplatin.Materials and methodsThis study was conducted using thirty-two adult female mice assigned to four groups with 8 mice in each group. Saline was given to the 1st group, cisplatin to the 2nd group, recombinant mouse Klotho to the 3rd group and recombinant mouse Klotho plus cisplatin to the 4th group. Uterine tissues were examined for damage histologically and immunobiologically for the uterine receptivity markers HOXA13 and alphaVBeta3 integrin.ResultsApoptosis, degeneration, decrease in uterine thickness and uterine absence of gland scores were higher in the cisplatin group (3rd group) compared to the saline group (1st group) (cisplatin vs. saline p < 0.0001 for all parameters). In the recombinant Klotho plus cisplatin group (4th group), scores of apoptosis, degeneration, reduction in uterine thickness and uterine absence of gland were lower than the group receiving only cisplatin (cisplatin plus recombinant Klotho vs cisplatin, p = 0.006 for apoptosis; p = 0.017 for degeneration; p = 0.011 for the reduction in uterine thickness; p = 0.002 for the absence of gland). However, HOXA13 and alphaVBeta3 integrin staining levels were not different between the cisplatin group (group 3) and the cisplatin plus recombinant Klotho group (group 4) (p = 0.980 and p = 0.762, respectively.)ConclusionCisplatin has adverse effects on the uterus. Administration of recombinant Klotho was found to attenuate the cisplatin-induced damage but failed to preserve levels of the implantation molecules HOXA13 and alphaVbeta3. Further studies examining the effect of cisplatin toxicity using other implantation markers along with functional studies are needed.     

苗勒管发育异常对生育结局的影响及HOXA13基因的突变研究

目的:分析苗勒管发育异常患者不同表型对生育结局的影响,探讨这些患者HOXA13基因的第二号外显子编码区是否存在突变。方法:回顾分析348例本中心苗勒管发育异常患者的不同表型对生育结局的影响;应用聚合酶链反应(PCR)扩增目的片段后,自动化测序分析HOXA13基因的第二号外显子编码区,与NCBI提供的正常序列比较,分析HOXA13基因的第二个外显子是否存在突变。结果:(1)苗勒管发育异常患者中不同表型对是否妊娠的影响有统计学差异(χ2=32.656,P<0.0001);(2)苗勒管发育异常患者的不同表型对妊娠结局的影响有统计学差异(χ2=12.817,P=0.005);(3)本研究中,所有患者第二号外显子均无突变发生。结论:(1)苗勒管发育异常患者的不同表型对生育结局可能有不同的影响;(2)苗勒管发育异常可能与HOXA13基因的第二个外显子编码区突变无明显关系。     

HOXA-11基因与不孕及妊娠失败关系的研究

目的 :研究HOXA 11基因与不孕及妊娠失败的关系。方法 :选择 4 1例不明原因不孕症患者及 30例难免流产患者 ,同时选择 2 8例正常未孕者及 18例正常早期妊娠者分别作为对照组 ,留取子宫内膜或蜕膜组织。子宫内膜标本通过组织学检查分为增生期或分泌期。难免流产及正常妊娠组均妊娠 6～ 9周。采用原位杂交方法检测所有子宫内膜或蜕膜组织中的HOXA 11基因mRNA的表达。结果 :整个月经周期子宫内膜腺上皮细胞及间质细胞中均有HOXA 11基因mRNA表达 ,并因月经周期不同而有所波动。HOXA 11基因mRNA在分泌中晚期子宫内膜间质细胞中阳性表达率为 10 0 % ,在增生期子宫内膜间质细胞中的阳性表达率为 6 3.6 % ,差异有显著性 (P <0 .0 5 ) ;在不明原因不孕症患者中HOXA 11基因失去了它在正常子宫内膜表达的周期性变化 ,而且基因表达缺失明显。HOX A 11基因在正常增生期及分泌期子宫内膜腺上皮细胞或间质细胞中的阳性表达率分别为90 .9%、82 .4 %和 6 3.6 %、10 0 % ,在不孕症增生期及分泌期子宫内膜两种细胞的阳性表达率分别为 38.9%、4 7.8%和 2 2 .2 %、39.1% ,两组分别比较 ,差异均有显著性 (P <0 .0 5 ) ;HOXA 11基因在正常早期妊娠蜕膜细胞中持续表达 ,其阳性表达率均为 10 0 % ,在难免流产蜕膜细胞中的阳性表达     

miRNA-135a对子宫内膜异位症HOXA10基因表达的调控的研究

目的:探讨miRNA-135a对子宫内膜异位症(EMs)中HOXA10基因表达的调控作用。方法:选取2013年1月到12月在天津市中心妇产科医院因EMs和单纯卵巢囊肿行腹腔镜手术的患者各80例。留取EMs患者的宫腔内膜组织(EMs在位内膜组)和异位内膜组织(EMs异位内膜组)以及单纯卵巢囊肿患者的宫腔内正常内膜组织。分别采用转染技术、实时荧光定量PCR技术检测miRNA-135a和HOXA10 mRNA的表达。结果:miRNA-135a和HOXA10 mRNA在不同月经周期表达水平不同。增殖期和分泌中期,EMs在位和异位内膜组织中miRNA-135a表达水平显著高于正常内膜,HOXA10 mRNA表达水平显著低于正常内膜,差异均有统计学意义(P0.01);EMs在位和异位内膜组织比较,差异均无统计学意义(P0.05)。经miRNA-135a、miRNA-135a抑制剂转染后,子宫内膜间质细胞中HOXA10 mRNA表达水平显著下降和升高,差异均有统计学意义(P0.01)。结论:EMs患者子宫内膜中HOXA10异常表达受miRNA-135a调控,miRNA-135a高表达抑制了HOXA10基因表达。     

The effect of adenomyosis types on clinical outcomes of IVF embryo transfer after ultra-long GnRH agonist protocol

Research questionWhat is the effect of adenomyosis types on IVF and embryo transfer (IVF-ET) after ultra-long gonadotrophin-releasing hormone (GnRH) agonist protocol?DesignPatients who underwent the first cycle of IVF-ET with ultra-long GnRH agonist protocol were included in this retrospective cohort study. They were divided into three groups: (A) 428 patients with diffuse adenomyosis; (B) 718 patients with focal adenomyosis; and (C) 519 patients with tubal infertility. Reproduction outcomes were analysed.ResultsLogistic regression analysis revealed that, compared with focal adenomyosis and tubal infertility, diffuse adenomyosis was negatively associated with clinical pregnancy and live birth (clinical pregnancy: A versus B: OR 0.708, 95% CI 0.539 to 0.931, P = 0.013; A versus C: OR 0.663, 95% CI 0.489 to 0.899, P = 0.008; live birth: A versus B: OR 0.530, 95% CI 0.385 to 0.730, P < 0.001; A versus C: OR 0.441, 95% CI 0.313 to 0.623, P < 0.001), but positively associated with miscarriage (A versus B: OR 1.727, 95% CI 1.056 to 2.825, P = 0.029; A versus C: OR 2.549, 95% CI 1.278 to 5.082, P = 0.008). Compared with patients with tubal infertility, focal adenomyosis was also a risk factor for miscarriage (B versus C: OR 1.825, 95% CI 1.112 to 2.995, P = 0.017).ConclusionsCompared with patients with focal adenomyosis or tubal infertility, the reproduction outcomes of IVF-ET in patients with diffuse adenomyosis seems to be worse.     

Zygote intrafallopian transfer in patients with tubal factor infertility after repeated failure of implantation with in vitro fertilization-embryo transfer

OBJECTIVE: To evaluate the efficacy of zygote intrafallopian transfer (ZIFT) in terms of implantation and pregnancy rates in patients with tubal factor infertility and repeated implantation failure in IVF-ET cycles. DESIGN: Retrospective analysis of ZIFT cycles. SETTING: An IVF unit in a university hospital. PATIENT(S): Criteria for patient selection for ZIFT included at least four failures of implantation in IVF-ET cycles in which at least 3 embryos were replaced per transfer and a cause of infertility diagnosed as male, unexplained, or tubal factor with proof of one patient tube. INTERVENTION(S): Four to six zygotes were transferred by laparoscopy into the fallopian tube 24-26 hours after oocyte retrieval. MAIN OUTCOME MEASURE(S): Implantation and pregnancy rates were determined in 112 ZIFT cycles performed in 81 patients with repeated failure of implantation. Results were further stratified for patients with tubal factor (n = 15) and patients without tubal factor (n = 66). RESULT(S): The pregnancy and implantation rates for all ZIFT cycles were 35.1% and 11.1%, respectively. Pregnancy and implantation rates per cycle in patients with tubal factor versus patients without tubal factor were 26.6% versus 37.1% and 9.4% versus 11.4%, respectively. CONCLUSION(S): ZIFT can be considered as a mode of treatment for patients with repeated failure of implantation in IVF-ET and with tubal factor with proved patency of one tube.     

Human Embryo Development and Pregnancies in an Homologous Granulosa Cell Coculture System

Purpose: Our purpose was to determine the effects of thecoculture of embryos on human granulosa cells (GCs) inpatients in the first cycle of IVF-ET treatment and in patientswith repeated implantation failures and to investigate thepresence of specific proteins in a 48-hr GC conditionedmedium and the GC ultrastructural characteristics. Methods: Eighteen patients with tubal or idiopathicinfertility were enrolled in this study: 7 patients (Trial 1) were inthe first cycle of IVF-ET treatment and 11 patients (Trial 2)had repeated implantation failures (one to five). Embryosfrom each patient were cocultured randomly either onhomologous granulosa cells or on a conventional culturemedium. Results: At the end of the coculture period (day 5 or 6),50% of the embryos (Trial 1) reached the blastocyst stage,with respect to 35% in Trial 2. The pregnancy rate perretrieval was 14.2 and 9%, respectively, in Trial 1 and inTrial 2. Many conditioned media showed proteins of 24–29kDa. and some of them showed additional proteins of 90kDa. The ultrastructural analysis of GCs showed healthy,metabolically active, protein-synthesizing, and mostlysteroidogenic cells. Conclusions: GC cultures improve embryo development butnot pregnancy rates both in Trial 1 and in Trial 2.