不影响宫腔形态的子宫肌瘤和子宫腺肌病对IVF/ICSI结局的影响
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摘要
第一部分
不影响宫腔形态的子宫肌瘤和子宫腺肌病对IVF/ICSI结局的影响
研究背景：
子宫肌瘤是最常见的子宫良性肿瘤,子宫肌瘤可以分为粘膜下肌瘤、浆膜下肌瘤和肌壁间肌瘤三类。子宫肌瘤在不孕症女性中发病率可达5%-13%,其中可能只有2%～3%的人子宫肌瘤可能是唯一的生育能力受损的原因。子宫肌瘤的组成、大小、位置和数量等因素可能影响女性生育力和妊娠并发症,这可能是因为子宫内膜受挤压,导致子宫内膜容受性改变和子宫内膜发育异常。但目前的证据尚未证明子宫肌瘤与不孕间有直接因果关系,有些结论甚至是相互矛盾的。不孕症中有粘膜下肌瘤者较无子宫肌瘤者临床妊娠率、胚胎着床率、活产率等显著降低,而自然流产率显著增高。浆膜下肌瘤似乎不影响ART的结果,浆膜下肌瘤切除对IVF亦无明显益处。然而,肌壁间肌瘤对生育和IVF/ICSI治疗结局的影响尚无定论。为进一步明确不影响宫腔形态子宫肌瘤对IVF/ICSI结局的影响,进行更大样本的深入研究和发现可能对IVF有不良影响的肌壁间肌瘤患者亚群是有必要的。另一方面,子宫腺肌病的特征是在子宫肌层存在异位子宫内膜腺体和间质,它可能是一种表观遗传性疾病。关于子宫腺肌病与不孕的关系研究比较热门,但结论都具有争议性,并没有达成共识,最新的包含5项研究的一个系统评价研究没能发现关于子宫腺肌病对辅助生殖技术预后的影响的任何明确结论。我们拟进行了一个样本数量相对较大的队列研究来消除混杂因素和探讨子宫腺肌病对IVF/ICSI的影响程度。
研究目的：
研究不影响宫腔形态的子宫肌瘤、子宫腺肌病对IVF/ICSI结局的影响,以期指导临床治疗。
研究方法：
本研究回顾性分析了本中心2009-2011年10,268例行IVF/ICSI鲜胚移植的不孕症患者的完整资料,根据入选标准筛选出研究组病例,设立严格的配对标准,对每例研究组患者从剩余病例中选择具有相近年龄(±1岁),相同移植周期和不孕类型(原发或继发性不孕)的无子宫肌瘤或腺肌病的不孕者,并尽量兼顾不孕原因、合并症、促排方案、既往病史等因素,进行1：1配对,对肌瘤的病例根据肌瘤位置、大小又分成了若干亚组,分析各组患者的一般状况、基本病例特点、促排卵相应数据及主要IVF/ICSI结局等。
研究结果：
共发现符合入选标准的经阴B超诊断的子宫腺肌病77例,<6cm的子宫肌瘤249例,其中浆膜下肌瘤51例和肌壁间肌瘤198例。肌瘤方面,研究发现肌瘤组与对照组在年龄、体重指数、不孕时间、卵巢储备、控制性促排卵方案、获卵数目、移植胚胎数目等方面无显著性差异,肌瘤组月经第3天促卵泡生成素(FSH)水平显著高于对照组(p=0.030),调整了FSH后肌瘤组与对照组的周期取消率、临床妊娠率、胚胎着床率、分娩率等均无显著差异。在亚组分析中,虽然浆膜下肌瘤的最大直径显著大于肌壁间肌瘤(p=0.000),但周期取消率、临床妊娠率、分娩率等均无显著差异。198例肌壁间肌瘤的研究中,受试者工作特征曲线(ROC)分析显示,单一肌瘤直径(SD)和肌瘤总直径(TD)在预测分娩率方面的截断值分别是2.85cm和2.95cm,肌瘤直径小于2.85cm或肌瘤直径之和小于2.95cm者IVF/ICSI分娩率显著高于相应的具有更大子宫肌瘤的患者；针对肌壁间肌瘤组内比较结果显示,肌瘤的SD大于2.85cm或者是TD大于2.95cm的患者,相对于SD小于2.85cm或者是TD小于2.95cm者,有较大的年龄和体重指数,经过调整体重指数和年龄后,logistic回归分析显示肌瘤较大组的分娩率显著低于肌瘤较小组(分别的p=0.033,p=.006)。根据肌壁间肌瘤的位置、数目、大小分为不同亚组,与相应的对照组比较并进行logistic回归,发现相对于相应的非肌瘤对照组,直径大于2.85cm的子宫肌瘤组分娩率显著降低(p=0.043,post-hoc power=0.91。根据肌瘤数目分组,有一个或多个肌壁间肌瘤的患者和配对的对照组相比较,临床妊娠率和分娩率也没有观察到显著的统计学差异。TVS提示有19例患者属于合并有3型肌瘤,与有4型和5型肌壁间肌瘤的其他179例患者相比,有3型肌瘤的病人有相对较低的分娩率和临床妊娠率(15.8%与33.5%,36.8%与47.5%)和相对较高的流产率(21.0%与13.9%),但经过统计并未显示出有统计学差异。腺肌病方面,研究发现腺肌病组与对照组在年龄、体重指数、不孕时间、卵巢储备、控制性促排卵方案、获卵数目、移植胚胎数目等方面无显著性差异,腺肌病组月经第3天雌激素(E2)水平显著高于对照组(p=0.039),合并子宫内膜异位症的比例显著高于对照组(p=0.022),每周期促性腺激素使用总量显著高于对照组(p=0.020),临床妊娠率略低于对照组但无统计学差异(p=0.09),流产率略高于对照组但无统计学差异(p=0.103),但分娩率显著低于对照组(p=0.022)。
研究结论：
总体来讲不影响宫腔形态的小于6cm的子宫肌瘤IVF/ICSI主要结局无不良影响,但亚组分析显示直径大于2.85cm的单个肌瘤对IVF/ICSI的分娩率有显著不良影响,当肌壁间肌瘤最大直径小于2.85cm时,可不必为了行IVF/ICSI而切除子宫肌瘤。子宫腺肌病对IVF/ICSI的主要妊娠结局分娩率有不良影响,在进一步提高子宫腺肌病诊断水平的同时,建议对腺肌病严重程度进行进一步细化分析,寻找较好的参数,以进一步研究。本研究对指导不孕症患者合并子宫肌瘤或子宫腺肌病的治疗方案选择有重要参考意义。
第二部分
H19/Iet-7轴调节肿瘤细胞的迁移和侵袭
研究背景：
长非编码RNA (IncRNA)、基因印记H19与人类多种癌症有关系,但潜在的机制仍然不甚了解。H19内含4个let-7结合位点,可以像“分子海绵”一样通过与let-7结合“吸附”let-7,抑制其功能。HMGA2, cMyc和Imp3是let-7的靶基因,已经被证明了能够调节多种下游效应基因参与细胞转移和侵袭。
研究目的：
本研究拟通过在肿瘤细胞中干扰H19的表达,研究H19对肿瘤细胞转移和侵袭的作用并证明这种作用与let-7的关系。
研究方法：
培养人卵巢癌细胞系A2780. TaraR127和子宫内膜癌细胞系ARK2细胞,转染特异性作用于H19的siRNA siH19来下调H19水平,通过体外transwell定量试验检测H19下调对肿瘤细胞迁移和侵袭的影响,同时检测let-7靶基因HMGA2, cMyc和Imp3的表达改变。将包含siHH19或Let-7抑制剂(iLet-7)的混合物转染A2870细胞,分析基因表达和细胞功能。检测A2780和TaraR127细胞H19过表达以后,HMGA2, cMyc,和Imp3基因表达改变及细胞迁移侵袭能力改变。
研究结果：
A2780、TaraR127和ARK2细胞转染siHH19后HMGA2, cMyc,和Imp3的mRNA和蛋白表达水平均下降,细胞的迁移和侵袭能力亦显著下降,加入iLet-7后,可部分或全部逆转这种现象。A2780、TaraR127细胞过表达H19后HMGA2, cMyc, Imp3表达显著上升,细胞的迁移和侵袭力亦显著上升。
研究结论：
我们的研究显示H19通过抑制let-7促进肿瘤细胞转移和侵袭。下调H19会抑制而其过表达会增强肿瘤细胞的活力和侵袭能力。这种现象发生至少部分原因是通过影响let-7介导的对肿瘤转移促进基因(包括HMGA2, cMyc和Imp3)的调控。我们的研究结果揭示了一种新的机制关于H19介导的肿瘤转移并且可以解释为什么在某些情况下let-7的表达增加与不良预后有关。针对这种新发现的途径可能会提供治疗机会。
PartⅠ
Effect of fibroids not distortingthe endometrial cavity and adenomyosis on the outcome of IVF/ICSI treatment
Background:
Uterine fibroids are the most common benign tumor of the uterus.Fibroids are classically described as submucosal fibroids (SM), intramural fibroids (IM), or subserosal fibroids (SS).Estimated to be present in5-10%of infertile women, they may be solely responsible for Impaired fertility outcomes in2-3%of cases. The composition, size, location, and number of fibroids may affect a woman's fertility and pregnancy complications. This may be due to distortion of the endometrial cavity, causing abnormal endometrial receptivity, hormonal milieu, and altered endometrial development For SM fibroids that distort the uterine cavity, there is consensus that a negative Impact exists on both clinical pregnancy rates and delivery rates after IVF/ICSI treatment. Conversely, SS fibroids do not appear to Impact IVF outcomes.The myomectomy of SS does not show any benefit either.However, the effect of IM fibroids on the outcome of IVF/ICSI treatment remains incompletely understood, with studies yielding conflicting results. To further determine the effect of fibroids (SS plus IM) on IVF/ICSI in the absence of uterine cavity distortion and identify potential subgroups of women with IM fibroids who experience deleterious outcomes when undergoing IVF/ICSI, a large sample size study is necessary. On the other hand,uterine adenomyosis is characterized by the presence of heterotypic endometrial glands and stroma in the myometrium and may be an epigenetic disease. Recently reports have focused on the relationship between adenomyosis and infertility but with controversial conclusions and no consensus. The most recent systematic review of five existing studies failed to draw any definite conclusions about the effect of adenomyosis on outcomes of assisted reproductive technology because of major differences in study design and populations.Here we intend to conduct a case-control study with a relatively large sample size to eliminate many confounding factors and to explore the extent of the effect of adenomyosis on IVF/ICSI.
Objective:
To investigate the effect of fibroids that do not distort the endometrial cavity and adenomyosis on IVF/intracytoplasmic sperm injection (ICSI) outcomes in order to guide clinical practice.
Methods:
We performed a retrospective, database-searched cohort study based on10,268patients undergoing controlled ovarian hyperstimulation and IVF/ICSI between2009and2011in our unit. Fibroids and adenomyosis were diagnosed by transvaginal ultrasound. A high-quality1:1matched cohort study with strict inclusion criteria was conducted, including age(±year),cycle and type of infertility, and also keep other factors matched as much as possible like cause of infertility, comorbidities and ovary stimulating protocols etc. We compared the basic characteristics and main IVF/ICSI outcomes between the two groups. In order to identify any subset of women with deleterious IVF/ICSI outcomes, patients were further classified into several subgroups. We also analyzed the predictive accuracy of fibroid size in identifying adverse effects on the delivery rate by calculating the appropriate receiver operating characteristic (ROC) curves.
Results:
We identified77patients with adenomyosis and249patients with fibroids less than6cm including51subserosal and198intramural fibroids who underwent IVF/ICSI. There were no significant differences between the two groups in terms of age, body mass index (BMI), duration of infertility, or ovarian reserve, ovary stimulation protocol, oocytes retrieved and embryo transferred. Higher day3FSH levels were found in women with fibroids compared with in control subjects (p=0.030). There was no significant difference in cycle cancellation rate, clinical pregnancy rate or delivery rate after adjusting for day3FSH levels between the two groups. Although SD in the subserosal group was larger than in the intramural group, there were no significant differences with regards to cycle cancellation rate, clinical pregnancy rate or delivery rate. After the ROC analysis, we identified2.85cm as the cutoff value for SD and2.95cm for TD. No significant differences were found in IVF/ICSI outcomes between the two groups. Patients with fibroids with SD>2.85cm or TD>2.95cm tended to have significantly higher age and BMI and lower DR compared with patients with lower diameter. Our results also showed that patients with larger BMI and age had more and larger fibroids. A significant negative effect on delivery rate was noted when intramural fibroids with the largest diameter greater than2.85cm were considered, compared with matched controls without fibroids (p=0.043, post-hoc power=0.91).We studied the effect of fibroid number on IVF/ICSI outcome. From our result, patients with single or multiple fibroids had similar IVF/ICSI outcomes19patients were found to have type3fibroids on TVS, compared with179patients with type4and5fibroids, patients with type3fibroids had relatively lower DR and CPR (15.8%vs33.5%,36.8%vs47.5%) and higher clinical MR (21.0%vs13.9%) but these differences did not reach a statistical significance. There were no significant differences between the adenomyosis group and the control group in terms of age, body mass index, duration of infertility, or ovarian reserve, ovary stimulation protocol, oocytes retrieved and embryo transferred. Higher day3estrogen levels (p=0.039) and a higher total of gonadotropin stimulation doses (p=0.020) were found in women with adenomyosis compared to control subjects. Patients with adenomyosis had a nonsignificant trend toward a lower clinical pregnancy rate (p=0.09) and a higher miscarriage rate (p=0.103). The delivery rate was significantly lower in the adenomyosis group in comparison to the matched controls (p=0.022)
Conclusions:
Our results suggest that although non-cavity-distorting fibroids (less than6cm) do not affect IVF/ICSI outcomes, intramural fibroids greater than2.85cm in size significantly Impair the delivery rate of patients undergoing IVF/ICSI. It is definitely unessary to do myomectomy before IVF/ICSI when SD is less than2.85cm.Our results also suggest that transvaginal ultrasound-diagnosed adenomyosis could have a negative Impact on the main IVF/ICSI outcomes. Improvement of the diagnosis and scoring of disease severity in patients with adenomyosis might add to the strength of these observations. Our study has Important strength in guiding the treatment of infertility patients combined with fibroids or adenomyosis before IVF/ICSI.
Part Ⅱ
H19/let-7axis regulates tumor cell migration and invasion Abstract
Background:
The Imprinted, developmentally regulated H19long noncoding RNA (lncRNA) has been Implicated in the pathogenesis of diverse human cancers, but the underlying mechanisms have remained poorly understood., HMGA2, cMyc, and Imp3are proved let-7target and have been demonstrated to promote metastasis via regulating various downstream effector genes directly involved in cell migration and invasion.
Objective:
The aim of this study is to investigate the role of H19on promoting tumor cell migration and invasion by changing the expression of H19in tumor cells.Methods:We transfected ovarian cancer cell lines A2780and TaraR127and endometrial cancer cell line ARK2cells with siH19or H19plasmid.We tested the motility and invasiveness of tumor cells using transwell method. We also tested the expression of let-7down regulating oncogenes such as HMGA2, cMyc, and Imp3.We co-transfected let-7inhibitor,iLet-7,to see if this phenomena could be rescued.
Resluts:
The motility and invasiveness of tumor cells were Impaired by transfecting ovarian cancer cell lines A2780and TaraR127and endometrial cancer cell line ARK2cells with siH19,together with the down regulation of let-7targeted genes HMGA2, cMyc, and Imp3.Co-transfecting iLet-7could partially or completely rescue this phenomona. H19over-expression showed opposite results.
Conclusion:
We show that H19depletion impairs, whereas its overexpression enhances the motility and invasiveness of tumor cells. These phenomena occur, at least in part through affecting let-7-mediated regulation of metastasis-promoting genes, including HMGA2, cMyc, and Imp3.Our results reveal a novel mechanism underpinning H19-mediated regulation in metastasis and may explain why in some cases increased let-7expression unexpectedly correlates with poor prognosis. Targeting this newly identified pathway might offer therapeutic opportunities.
不影响宫腔形态的子宫肌瘤和子宫腺肌病对IVF/ICSI结局的影响
研究背景：
子宫肌瘤是最常见的子宫良性肿瘤,子宫肌瘤可以分为粘膜下肌瘤、浆膜下肌瘤和肌壁间肌瘤三类。子宫肌瘤在不孕症女性中发病率可达5%-13%,其中可能只有2%～3%的人子宫肌瘤可能是唯一的生育能力受损的原因。子宫肌瘤的组成、大小、位置和数量等因素可能影响女性生育力和妊娠并发症,这可能是因为子宫内膜受挤压,导致子宫内膜容受性改变和子宫内膜发育异常。但目前的证据尚未证明子宫肌瘤与不孕间有直接因果关系,有些结论甚至是相互矛盾的。不孕症中有粘膜下肌瘤者较无子宫肌瘤者临床妊娠率、胚胎着床率、活产率等显著降低,而自然流产率显著增高。浆膜下肌瘤似乎不影响ART的结果,浆膜下肌瘤切除对IVF亦无明显益处。然而,肌壁间肌瘤对生育和IVF/ICSI治疗结局的影响尚无定论。为进一步明确不影响宫腔形态子宫肌瘤对IVF/ICSI结局的影响,进行更大样本的深入研究和发现可能对IVF有不良影响的肌壁间肌瘤患者亚群是有必要的。另一方面,子宫腺肌病的特征是在子宫肌层存在异位子宫内膜腺体和间质,它可能是一种表观遗传性疾病。关于子宫腺肌病与不孕的关系研究比较热门,但结论都具有争议性,并没有达成共识,最新的包含5项研究的一个系统评价研究没能发现关于子宫腺肌病对辅助生殖技术预后的影响的任何明确结论。我们拟进行了一个样本数量相对较大的队列研究来消除混杂因素和探讨子宫腺肌病对IVF/ICSI的影响程度。
研究目的：
研究不影响宫腔形态的子宫肌瘤、子宫腺肌病对IVF/ICSI结局的影响,以期指导临床治疗。
研究方法：
本研究回顾性分析了本中心2009-2011年10,268例行IVF/ICSI鲜胚移植的不孕症患者的完整资料,根据入选标准筛选出研究组病例,设立严格的配对标准,对每例研究组患者从剩余病例中选择具有相近年龄(±1岁),相同移植周期和不孕类型(原发或继发性不孕)的无子宫肌瘤或腺肌病的不孕者,并尽量兼顾不孕原因、合并症、促排方案、既往病史等因素,进行1：1配对,对肌瘤的病例根据肌瘤位置、大小又分成了若干亚组,分析各组患者的一般状况、基本病例特点、促排卵相应数据及主要IVF/ICSI结局等。
研究结果：
共发现符合入选标准的经阴B超诊断的子宫腺肌病77例,<6cm的子宫肌瘤249例,其中浆膜下肌瘤51例和肌壁间肌瘤198例。肌瘤方面,研究发现肌瘤组与对照组在年龄、体重指数、不孕时间、卵巢储备、控制性促排卵方案、获卵数目、移植胚胎数目等方面无显著性差异,肌瘤组月经第3天促卵泡生成素(FSH)水平显著高于对照组(p=0.030),调整了FSH后肌瘤组与对照组的周期取消率、临床妊娠率、胚胎着床率、分娩率等均无显著差异。在亚组分析中,虽然浆膜下肌瘤的最大直径显著大于肌壁间肌瘤(p=0.000),但周期取消率、临床妊娠率、分娩率等均无显著差异。198例肌壁间肌瘤的研究中,受试者工作特征曲线(ROC)分析显示,单一肌瘤直径(SD)和肌瘤总直径(TD)在预测分娩率方面的截断值分别是2.85cm和2.95cm,肌瘤直径小于2.85cm或肌瘤直径之和小于2.95cm者IVF/ICSI分娩率显著高于相应的具有更大子宫肌瘤的患者；针对肌壁间肌瘤组内比较结果显示,肌瘤的SD大于2.85cm或者是TD大于2.95cm的患者,相对于SD小于2.85cm或者是TD小于2.95cm者,有较大的年龄和体重指数,经过调整体重指数和年龄后,logistic回归分析显示肌瘤较大组的分娩率显著低于肌瘤较小组(分别的p=0.033,p=.006)。根据肌壁间肌瘤的位置、数目、大小分为不同亚组,与相应的对照组比较并进行logistic回归,发现相对于相应的非肌瘤对照组,直径大于2.85cm的子宫肌瘤组分娩率显著降低(p=0.043,post-hoc power=0.91。根据肌瘤数目分组,有一个或多个肌壁间肌瘤的患者和配对的对照组相比较,临床妊娠率和分娩率也没有观察到显著的统计学差异。TVS提示有19例患者属于合并有3型肌瘤,与有4型和5型肌壁间肌瘤的其他179例患者相比,有3型肌瘤的病人有相对较低的分娩率和临床妊娠率(15.8%与33.5%,36.8%与47.5%)和相对较高的流产率(21.0%与13.9%),但经过统计并未显示出有统计学差异。腺肌病方面,研究发现腺肌病组与对照组在年龄、体重指数、不孕时间、卵巢储备、控制性促排卵方案、获卵数目、移植胚胎数目等方面无显著性差异,腺肌病组月经第3天雌激素(E2)水平显著高于对照组(p=0.039),合并子宫内膜异位症的比例显著高于对照组(p=0.022),每周期促性腺激素使用总量显著高于对照组(p=0.020),临床妊娠率略低于对照组但无统计学差异(p=0.09),流产率略高于对照组但无统计学差异(p=0.103),但分娩率显著低于对照组(p=0.022)。
研究结论：
总体来讲不影响宫腔形态的小于6cm的子宫肌瘤IVF/ICSI主要结局无不良影响,但亚组分析显示直径大于2.85cm的单个肌瘤对IVF/ICSI的分娩率有显著不良影响,当肌壁间肌瘤最大直径小于2.85cm时,可不必为了行IVF/ICSI而切除子宫肌瘤。子宫腺肌病对IVF/ICSI的主要妊娠结局分娩率有不良影响,在进一步提高子宫腺肌病诊断水平的同时,建议对腺肌病严重程度进行进一步细化分析,寻找较好的参数,以进一步研究。本研究对指导不孕症患者合并子宫肌瘤或子宫腺肌病的治疗方案选择有重要参考意义。
第二部分
H19/Iet-7轴调节肿瘤细胞的迁移和侵袭
研究背景：
长非编码RNA (IncRNA)、基因印记H19与人类多种癌症有关系,但潜在的机制仍然不甚了解。H19内含4个let-7结合位点,可以像“分子海绵”一样通过与let-7结合“吸附”let-7,抑制其功能。HMGA2, cMyc和Imp3是let-7的靶基因,已经被证明了能够调节多种下游效应基因参与细胞转移和侵袭。
研究目的：
本研究拟通过在肿瘤细胞中干扰H19的表达,研究H19对肿瘤细胞转移和侵袭的作用并证明这种作用与let-7的关系。
研究方法：
培养人卵巢癌细胞系A2780. TaraR127和子宫内膜癌细胞系ARK2细胞,转染特异性作用于H19的siRNA siH19来下调H19水平,通过体外transwell定量试验检测H19下调对肿瘤细胞迁移和侵袭的影响,同时检测let-7靶基因HMGA2, cMyc和Imp3的表达改变。将包含siHH19或Let-7抑制剂(iLet-7)的混合物转染A2870细胞,分析基因表达和细胞功能。检测A2780和TaraR127细胞H19过表达以后,HMGA2, cMyc,和Imp3基因表达改变及细胞迁移侵袭能力改变。
研究结果：
A2780、TaraR127和ARK2细胞转染siHH19后HMGA2, cMyc,和Imp3的mRNA和蛋白表达水平均下降,细胞的迁移和侵袭能力亦显著下降,加入iLet-7后,可部分或全部逆转这种现象。A2780、TaraR127细胞过表达H19后HMGA2, cMyc, Imp3表达显著上升,细胞的迁移和侵袭力亦显著上升。
研究结论：
我们的研究显示H19通过抑制let-7促进肿瘤细胞转移和侵袭。下调H19会抑制而其过表达会增强肿瘤细胞的活力和侵袭能力。这种现象发生至少部分原因是通过影响let-7介导的对肿瘤转移促进基因(包括HMGA2, cMyc和Imp3)的调控。我们的研究结果揭示了一种新的机制关于H19介导的肿瘤转移并且可以解释为什么在某些情况下let-7的表达增加与不良预后有关。针对这种新发现的途径可能会提供治疗机会。
PartⅠ
Effect of fibroids not distortingthe endometrial cavity and adenomyosis on the outcome of IVF/ICSI treatment
Background:
Uterine fibroids are the most common benign tumor of the uterus.Fibroids are classically described as submucosal fibroids (SM), intramural fibroids (IM), or subserosal fibroids (SS).Estimated to be present in5-10%of infertile women, they may be solely responsible for Impaired fertility outcomes in2-3%of cases. The composition, size, location, and number of fibroids may affect a woman's fertility and pregnancy complications. This may be due to distortion of the endometrial cavity, causing abnormal endometrial receptivity, hormonal milieu, and altered endometrial development For SM fibroids that distort the uterine cavity, there is consensus that a negative Impact exists on both clinical pregnancy rates and delivery rates after IVF/ICSI treatment. Conversely, SS fibroids do not appear to Impact IVF outcomes.The myomectomy of SS does not show any benefit either.However, the effect of IM fibroids on the outcome of IVF/ICSI treatment remains incompletely understood, with studies yielding conflicting results. To further determine the effect of fibroids (SS plus IM) on IVF/ICSI in the absence of uterine cavity distortion and identify potential subgroups of women with IM fibroids who experience deleterious outcomes when undergoing IVF/ICSI, a large sample size study is necessary. On the other hand,uterine adenomyosis is characterized by the presence of heterotypic endometrial glands and stroma in the myometrium and may be an epigenetic disease. Recently reports have focused on the relationship between adenomyosis and infertility but with controversial conclusions and no consensus. The most recent systematic review of five existing studies failed to draw any definite conclusions about the effect of adenomyosis on outcomes of assisted reproductive technology because of major differences in study design and populations.Here we intend to conduct a case-control study with a relatively large sample size to eliminate many confounding factors and to explore the extent of the effect of adenomyosis on IVF/ICSI.
Objective:
To investigate the effect of fibroids that do not distort the endometrial cavity and adenomyosis on IVF/intracytoplasmic sperm injection (ICSI) outcomes in order to guide clinical practice.
Methods:
We performed a retrospective, database-searched cohort study based on10,268patients undergoing controlled ovarian hyperstimulation and IVF/ICSI between2009and2011in our unit. Fibroids and adenomyosis were diagnosed by transvaginal ultrasound. A high-quality1:1matched cohort study with strict inclusion criteria was conducted, including age(±year),cycle and type of infertility, and also keep other factors matched as much as possible like cause of infertility, comorbidities and ovary stimulating protocols etc. We compared the basic characteristics and main IVF/ICSI outcomes between the two groups. In order to identify any subset of women with deleterious IVF/ICSI outcomes, patients were further classified into several subgroups. We also analyzed the predictive accuracy of fibroid size in identifying adverse effects on the delivery rate by calculating the appropriate receiver operating characteristic (ROC) curves.
Results:
We identified77patients with adenomyosis and249patients with fibroids less than6cm including51subserosal and198intramural fibroids who underwent IVF/ICSI. There were no significant differences between the two groups in terms of age, body mass index (BMI), duration of infertility, or ovarian reserve, ovary stimulation protocol, oocytes retrieved and embryo transferred. Higher day3FSH levels were found in women with fibroids compared with in control subjects (p=0.030). There was no significant difference in cycle cancellation rate, clinical pregnancy rate or delivery rate after adjusting for day3FSH levels between the two groups. Although SD in the subserosal group was larger than in the intramural group, there were no significant differences with regards to cycle cancellation rate, clinical pregnancy rate or delivery rate. After the ROC analysis, we identified2.85cm as the cutoff value for SD and2.95cm for TD. No significant differences were found in IVF/ICSI outcomes between the two groups. Patients with fibroids with SD>2.85cm or TD>2.95cm tended to have significantly higher age and BMI and lower DR compared with patients with lower diameter. Our results also showed that patients with larger BMI and age had more and larger fibroids. A significant negative effect on delivery rate was noted when intramural fibroids with the largest diameter greater than2.85cm were considered, compared with matched controls without fibroids (p=0.043, post-hoc power=0.91).We studied the effect of fibroid number on IVF/ICSI outcome. From our result, patients with single or multiple fibroids had similar IVF/ICSI outcomes19patients were found to have type3fibroids on TVS, compared with179patients with type4and5fibroids, patients with type3fibroids had relatively lower DR and CPR (15.8%vs33.5%,36.8%vs47.5%) and higher clinical MR (21.0%vs13.9%) but these differences did not reach a statistical significance. There were no significant differences between the adenomyosis group and the control group in terms of age, body mass index, duration of infertility, or ovarian reserve, ovary stimulation protocol, oocytes retrieved and embryo transferred. Higher day3estrogen levels (p=0.039) and a higher total of gonadotropin stimulation doses (p=0.020) were found in women with adenomyosis compared to control subjects. Patients with adenomyosis had a nonsignificant trend toward a lower clinical pregnancy rate (p=0.09) and a higher miscarriage rate (p=0.103). The delivery rate was significantly lower in the adenomyosis group in comparison to the matched controls (p=0.022)
Conclusions:
Our results suggest that although non-cavity-distorting fibroids (less than6cm) do not affect IVF/ICSI outcomes, intramural fibroids greater than2.85cm in size significantly Impair the delivery rate of patients undergoing IVF/ICSI. It is definitely unessary to do myomectomy before IVF/ICSI when SD is less than2.85cm.Our results also suggest that transvaginal ultrasound-diagnosed adenomyosis could have a negative Impact on the main IVF/ICSI outcomes. Improvement of the diagnosis and scoring of disease severity in patients with adenomyosis might add to the strength of these observations. Our study has Important strength in guiding the treatment of infertility patients combined with fibroids or adenomyosis before IVF/ICSI.
Part Ⅱ
H19/let-7axis regulates tumor cell migration and invasion Abstract
Background:
The Imprinted, developmentally regulated H19long noncoding RNA (lncRNA) has been Implicated in the pathogenesis of diverse human cancers, but the underlying mechanisms have remained poorly understood., HMGA2, cMyc, and Imp3are proved let-7target and have been demonstrated to promote metastasis via regulating various downstream effector genes directly involved in cell migration and invasion.
Objective:
The aim of this study is to investigate the role of H19on promoting tumor cell migration and invasion by changing the expression of H19in tumor cells.Methods:We transfected ovarian cancer cell lines A2780and TaraR127and endometrial cancer cell line ARK2cells with siH19or H19plasmid.We tested the motility and invasiveness of tumor cells using transwell method. We also tested the expression of let-7down regulating oncogenes such as HMGA2, cMyc, and Imp3.We co-transfected let-7inhibitor,iLet-7,to see if this phenomena could be rescued.
Resluts:
The motility and invasiveness of tumor cells were Impaired by transfecting ovarian cancer cell lines A2780and TaraR127and endometrial cancer cell line ARK2cells with siH19,together with the down regulation of let-7targeted genes HMGA2, cMyc, and Imp3.Co-transfecting iLet-7could partially or completely rescue this phenomona. H19over-expression showed opposite results.
Conclusion:
We show that H19depletion impairs, whereas its overexpression enhances the motility and invasiveness of tumor cells. These phenomena occur, at least in part through affecting let-7-mediated regulation of metastasis-promoting genes, including HMGA2, cMyc, and Imp3.Our results reveal a novel mechanism underpinning H19-mediated regulation in metastasis and may explain why in some cases increased let-7expression unexpectedly correlates with poor prognosis. Targeting this newly identified pathway might offer therapeutic opportunities.
引文
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51 Kallen, A.N., Zhou, X.B., Xu, J., Qiao, C., Ma, J., Yan, L., Lu, L., Liu, C., Yi, J.S., Zhang, H. et al. The Imprinted H19 IncRNA antagonizes let-7 microRNAs. Mol Cell.2013;52(1):101-12.
52 Boyerinas B1, Park SM, Shomron N, Hedegaard MM, Vinther J, Andersen JS, Feig C, Xu J, Burge CB, Peter ME. Identification of let-7-regulated oncofetal genes.2008;68(8):2587-91.
53 Tan EJ, Thuault S, Caja L, Carletti T, Heldin CH, Moustakas A.Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition.J Biol Chem. 2012;287(10):7134-45.
54 Shu J, Xia Z, Li L, Liang ET, Slipek N, Shen D, Foo J, Subramanian S, Steer CJ. Dose-dependent differential mRNA target selection and regulation by let-7a-7f and miR-17-92 cluster microRNAs.RNA Biol.2012;9(10):1275-87.
55 Barsyte-Lovejoy, D., Lau, S.K., Boutros, P.C., Khosravi, F., Jurisica, I., Andrulis, I.L., Tsao, M.S. and Penn, L.Z. The cMyc oncogene directly induces the H19 noncoding RNA by allele-specific binding to potentiate tumorigenesis. Cancer Res.2006; 66,(10) 5330-37.
56 Vercellini P, Cribiu FM, Del Gobbo A, Carcangiu ML, Somigliana E, Bosari S. The oncofetal' protein IMP3:a novel biomarker and triage tool for premalignant atypical endometriotic lesions.2013,99(7):1974-9.
57 Samanta S, Sharma VM, Khan A, Mercurio AM. Epub 2012 Jan 23.Regulation of IMP3 by EGFR signaling and repression by ERβ:Implications for triple-negative breast cancer.Oncogene.2012; 31(44):4689-97.
58 Lu, L., Katsaros, D., de la Longrais, I.A., Sochirca, O. Yu, H. Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis. Cancer Res,2007,67(21):10117-22.
59 Tang, Z., Ow, G.S., Thiery, J.P., Ivshina, A.V. and Kuznetsov, V.A. Meta-analysis of transcriptome reveals let-7b as an unfavorable prognostic biomarker and predicts molecular and clinical subclasses in high-grade serous ovarian carcinoma.Int J Cancer.2014; 134(2):306-18.
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53 Tan EJ, Thuault S, Caja L, Carletti T, Heldin CH, Moustakas A.Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition.J Biol Chem. 2012;287(10):7134-45.
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56 Vercellini P, Cribiu FM, Del Gobbo A, Carcangiu ML, Somigliana E, Bosari S. The oncofetal' protein IMP3:a novel biomarker and triage tool for premalignant atypical endometriotic lesions.2013,99(7):1974-9.
57 Samanta S, Sharma VM, Khan A, Mercurio AM. Epub 2012 Jan 23.Regulation of IMP3 by EGFR signaling and repression by ERβ:Implications for triple-negative breast cancer.Oncogene.2012; 31(44):4689-97.
58 Lu, L., Katsaros, D., de la Longrais, I.A., Sochirca, O. Yu, H. Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis. Cancer Res,2007,67(21):10117-22.
59 Tang, Z., Ow, G.S., Thiery, J.P., Ivshina, A.V. and Kuznetsov, V.A. Meta-analysis of transcriptome reveals let-7b as an unfavorable prognostic biomarker and predicts molecular and clinical subclasses in high-grade serous ovarian carcinoma.Int J Cancer.2014; 134(2):306-18.