不明原因不孕妇女分泌期子宫内膜HOXA10基因的表达
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摘要
前言
随着生殖医学的飞速发展，人类对不孕的诊治水平已大大提高，但仍有一部分不明原因不孕妇女的生育问题尚未得到解决，其中胚胎着床失败是问题的关键之一，而胚胎能否着床则要看胚胎质量和子宫内膜容受力及其二者是否同步。虽然评价子宫内膜的金标准仍是黄体期内膜活检，但已有越来越多的证据表明临床分期与组织学分期一致的子宫内膜并不能完全代表其功能正常或反映其容受力。一些细胞因子如整合素(α_γβ_3)、环化合酶-2(COX-2)、降钙素(CT)、白血病抑制因子(LIF)等均在分子水平上影响着着床期的子宫内膜容受力。通过针对性的基因治疗，可以提高着床成功率。目前，有关子宫内膜容受力的分子水平的研究已成为生殖医学的研究热点。
同源框基因(HOX)是一类转录调节基因，其蛋白通过激活或抑制下游基因的表达在胚胎发育中起着促进体节发育、决定组织分化的作用，HOXA10基因是HOX基因家族中AbdB亚家族成员之一。在小鼠，HOXA10基因缺陷表现为子宫性不孕，故其在生殖内分泌领域的作用引起学者的关注。目前，国内外对于HOXA10基因在人类这方面作用的研究甚少。Taylor等发现其在人类子宫内膜的增生期和分泌早期低表达，而于分泌中期和晚期表达水平急剧上升，这种特征性的表达时相提示HOXA10基因在人类子宫内膜容受力形成方面可能具有重要作用。通过研究HOXA10基因在不明原因不孕妇女子宫内膜上的表达特点，可能为进一步阐明其在子宫内膜容受力形成方面的作用提供理论依据。
实 验 材 料
不孕门）组子宫内膜组织取自于本院妇产科生殖中心就诊
的28例不明原因不孕妇女，监测排卵确定排卵日，于排卵后刮取
近宫底处子宫内膜，分泌中期诊刮者同时行血孕酮水平测定，以排
除黄体功能不全（LPD人正常生育（NF）组子宫内膜组织取自因
单发子宫肌瘤而于本院妇产科住院手术的已育患者20例，无自然
流产史，月经规律，未置环，于分泌期手术刮取正常肌壁侧近宫底
处内膜组织。标本取自 2002年 1月－10月，各组年龄无显著性
差异，术前3个月末用过激素类药物。所取之内膜组织立即用
4％多聚甲醛固定，石蜡包埋。行HE染色后光镜下依Noyes标准
判定组织学分期，继后进行分组。
实 验 方 法
采用分子原位杂交技术，检测28例IF妇女和20例NF妇女
分泌早中期子宫内膜上HOXA10 mRNA的表达水平。石蜡切片
脱腊至水，蛋白酶消化后，加人HOXA10 mRNA寡核昔酸探针，使
其与组织内HOXA10 mRNA杂交，之后加人一系列抗体，最后经
DAB显色。同时设空白对照片，除不加探针外，其余步骤同上。
胞浆内出现棕黄色颗粒为阳性表达，再用图像分析系统测定显色
区灰度阳性单位（PU）相对值的平均值，应用统计分析软件进行分
析。
结 果
HOXA10 mRNA在两组子宫内膜的腺体和间质上均有表达，
·2·
定位于胞浆，呈棕黄色颗粒，核旁胞浆表达较集中。IF组分泌早
中期子宫内膜上 HOXA10 mRN＊表达的 PU值在腺体为 6．61。0．
17、6．60 L 0．22，间质为6．40 t 0．52、6．30 t 0．18，两期表达无显著
差异仲＞O．05h＊F组分泌早中期子宫内膜上＊0＊AIO m RNRNA表
达的 PU值在腺体为 6．66。口．豆二、10．95。0．95，间质为 6．76 L 0．
13上．49ti．33，分泌中期 HO）［A10 mRNA表达的PU值显著高
于分泌早期h＜0．05八 IF组分泌中期 HOXA10 mRNA表达的 PU
值显著低于 NF组同期水平h＜0．05人
结 论
＊不明原因不孕妇女分泌中期子宫内膜上HOXA10 mRNA
的表达水平显著下降。
2．子宫内膜HOXA10基因在分泌中期的高表达可能和子宫
内膜容受力密切相关，其表达缺陷可能是导致不明原因不孕的重
要原因之一。
Introduction
With the rapidly development of genesiologe, the level of making on diagnosis and giving treatment to infertility had improved greatly. But there are still some unexplained infertile women not to be cured. Among infertile causes, implanting failure is key problem. Embryo with high quality N endometrial receptivity and their coordination are major factors of implantation. Although the golden criterion to evaluate endometrial function is still pathologic examination on luteal phase en-dometrium, more and more evidence indicate that endometrium with consistent clinical and histological phase can't fully represent its normal function and receptivity. Some cytokin such as Integrin COX-2,CT,LIF and so on effect on endomerial receptivity during implanting phase on molecular level. By aimed genie therapy, implanting rate may be raised. Now on molecular level studies about endometrial receptivity are being focal point of genesiological studies.
HOX genes are transcriptional regulators that play promoting body segment development and determinating histodifferentation roles in embryonic development by activating or repressing downstream genes.
HOXA10 gene is a member of AbdB subgroup of HOX genes family. In the mouse, HOXA10 gene expression is essential for fertility. HOXA10 gene defect exhibit uterine factor infertility. Thus its effect on reproductive endocrinology domain has brought scholars' interest. At present, studies of HOXA10 gene effecting on human's reproduction and endocrine are few either at home or at abroad. Taylor reported that expression of HOXA10 gene was low during proliferative and early secretory endometrium, but it dramatically increased during the mid-secretory and late secretory endometrium of human being. The especially timing of expression points to a probable essential role for HOXA10 gene in leading to endometrial receptivity. By studying expression characteristics of HOXA10 gene in endometrium of unexplained infertile women, theoretical basis can be provided for further clarifying its effect on endometrial receptivity formation.
Materials
28 cases human endometria were obtained from unexplained infertile (IF) women in our center of reproductive endocrinology. Ovu-latory day was determined by monitoring ovulation and endometrium closing to uterine fundus was taken by curettage after ovulation. In order to remove luteal phase defect ( LPD) , blood progesterone level was examined at the same time of curettage in middle secretory phase. 20 cases endometria of normal fertile (NF) group were obtained from normal fertile women without habitual abortion history undergoing operation for their simple leiomyorna who had normal menstrual cycle. They underwent operation during secretory phase and their endometria on normal side closing to uterin fundus were curetted. All of samples
were obtained from 2002.1-2002.10. Ages of two group hadn't significantly difference and women hadn' t taken hormonal medicine 3 months before operation. Total tissue was immediately fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histological phase of samples was confirmed using the criteria of Noyes. Then each group was divided into early and middle secretory phase.
Methods
HOXA10 mRNA expressions in early and middle secretory endo-metria of 28 cases IF and 20 cases NF women were examined by in situ hybridization. After paraffin sections were treated with proteinase, HOXA10 mRNA antisense riboprobes were added to sections to hybridize. Then they were added to a series of antibodies. At last they were stained by DAB. Brown and yellow granulae emerging in cytoplasm defined as positive expression. We detected positive unites (PUs) of stained area using image analysis system. Data were analyzed using SPSS software.
Results
HOXA10 mRNA were expressed in glandula cells and- stromal cells of endometria of two groups. In the IF group, PUs of the expression of HOXA10 mRNA in early and mid secretory phase endometria were 6. 61+
随着生殖医学的飞速发展，人类对不孕的诊治水平已大大提高，但仍有一部分不明原因不孕妇女的生育问题尚未得到解决，其中胚胎着床失败是问题的关键之一，而胚胎能否着床则要看胚胎质量和子宫内膜容受力及其二者是否同步。虽然评价子宫内膜的金标准仍是黄体期内膜活检，但已有越来越多的证据表明临床分期与组织学分期一致的子宫内膜并不能完全代表其功能正常或反映其容受力。一些细胞因子如整合素(α_γβ_3)、环化合酶-2(COX-2)、降钙素(CT)、白血病抑制因子(LIF)等均在分子水平上影响着着床期的子宫内膜容受力。通过针对性的基因治疗，可以提高着床成功率。目前，有关子宫内膜容受力的分子水平的研究已成为生殖医学的研究热点。
同源框基因(HOX)是一类转录调节基因，其蛋白通过激活或抑制下游基因的表达在胚胎发育中起着促进体节发育、决定组织分化的作用，HOXA10基因是HOX基因家族中AbdB亚家族成员之一。在小鼠，HOXA10基因缺陷表现为子宫性不孕，故其在生殖内分泌领域的作用引起学者的关注。目前，国内外对于HOXA10基因在人类这方面作用的研究甚少。Taylor等发现其在人类子宫内膜的增生期和分泌早期低表达，而于分泌中期和晚期表达水平急剧上升，这种特征性的表达时相提示HOXA10基因在人类子宫内膜容受力形成方面可能具有重要作用。通过研究HOXA10基因在不明原因不孕妇女子宫内膜上的表达特点，可能为进一步阐明其在子宫内膜容受力形成方面的作用提供理论依据。
实 验 材 料
不孕门）组子宫内膜组织取自于本院妇产科生殖中心就诊
的28例不明原因不孕妇女，监测排卵确定排卵日，于排卵后刮取
近宫底处子宫内膜，分泌中期诊刮者同时行血孕酮水平测定，以排
除黄体功能不全（LPD人正常生育（NF）组子宫内膜组织取自因
单发子宫肌瘤而于本院妇产科住院手术的已育患者20例，无自然
流产史，月经规律，未置环，于分泌期手术刮取正常肌壁侧近宫底
处内膜组织。标本取自 2002年 1月－10月，各组年龄无显著性
差异，术前3个月末用过激素类药物。所取之内膜组织立即用
4％多聚甲醛固定，石蜡包埋。行HE染色后光镜下依Noyes标准
判定组织学分期，继后进行分组。
实 验 方 法
采用分子原位杂交技术，检测28例IF妇女和20例NF妇女
分泌早中期子宫内膜上HOXA10 mRNA的表达水平。石蜡切片
脱腊至水，蛋白酶消化后，加人HOXA10 mRNA寡核昔酸探针，使
其与组织内HOXA10 mRNA杂交，之后加人一系列抗体，最后经
DAB显色。同时设空白对照片，除不加探针外，其余步骤同上。
胞浆内出现棕黄色颗粒为阳性表达，再用图像分析系统测定显色
区灰度阳性单位（PU）相对值的平均值，应用统计分析软件进行分
析。
结 果
HOXA10 mRNA在两组子宫内膜的腺体和间质上均有表达，
·2·
定位于胞浆，呈棕黄色颗粒，核旁胞浆表达较集中。IF组分泌早
中期子宫内膜上 HOXA10 mRN＊表达的 PU值在腺体为 6．61。0．
17、6．60 L 0．22，间质为6．40 t 0．52、6．30 t 0．18，两期表达无显著
差异仲＞O．05h＊F组分泌早中期子宫内膜上＊0＊AIO m RNRNA表
达的 PU值在腺体为 6．66。口．豆二、10．95。0．95，间质为 6．76 L 0．
13上．49ti．33，分泌中期 HO）［A10 mRNA表达的PU值显著高
于分泌早期h＜0．05八 IF组分泌中期 HOXA10 mRNA表达的 PU
值显著低于 NF组同期水平h＜0．05人
结 论
＊不明原因不孕妇女分泌中期子宫内膜上HOXA10 mRNA
的表达水平显著下降。
2．子宫内膜HOXA10基因在分泌中期的高表达可能和子宫
内膜容受力密切相关，其表达缺陷可能是导致不明原因不孕的重
要原因之一。
Introduction
With the rapidly development of genesiologe, the level of making on diagnosis and giving treatment to infertility had improved greatly. But there are still some unexplained infertile women not to be cured. Among infertile causes, implanting failure is key problem. Embryo with high quality N endometrial receptivity and their coordination are major factors of implantation. Although the golden criterion to evaluate endometrial function is still pathologic examination on luteal phase en-dometrium, more and more evidence indicate that endometrium with consistent clinical and histological phase can't fully represent its normal function and receptivity. Some cytokin such as Integrin COX-2,CT,LIF and so on effect on endomerial receptivity during implanting phase on molecular level. By aimed genie therapy, implanting rate may be raised. Now on molecular level studies about endometrial receptivity are being focal point of genesiological studies.
HOX genes are transcriptional regulators that play promoting body segment development and determinating histodifferentation roles in embryonic development by activating or repressing downstream genes.
HOXA10 gene is a member of AbdB subgroup of HOX genes family. In the mouse, HOXA10 gene expression is essential for fertility. HOXA10 gene defect exhibit uterine factor infertility. Thus its effect on reproductive endocrinology domain has brought scholars' interest. At present, studies of HOXA10 gene effecting on human's reproduction and endocrine are few either at home or at abroad. Taylor reported that expression of HOXA10 gene was low during proliferative and early secretory endometrium, but it dramatically increased during the mid-secretory and late secretory endometrium of human being. The especially timing of expression points to a probable essential role for HOXA10 gene in leading to endometrial receptivity. By studying expression characteristics of HOXA10 gene in endometrium of unexplained infertile women, theoretical basis can be provided for further clarifying its effect on endometrial receptivity formation.
Materials
28 cases human endometria were obtained from unexplained infertile (IF) women in our center of reproductive endocrinology. Ovu-latory day was determined by monitoring ovulation and endometrium closing to uterine fundus was taken by curettage after ovulation. In order to remove luteal phase defect ( LPD) , blood progesterone level was examined at the same time of curettage in middle secretory phase. 20 cases endometria of normal fertile (NF) group were obtained from normal fertile women without habitual abortion history undergoing operation for their simple leiomyorna who had normal menstrual cycle. They underwent operation during secretory phase and their endometria on normal side closing to uterin fundus were curetted. All of samples
were obtained from 2002.1-2002.10. Ages of two group hadn't significantly difference and women hadn' t taken hormonal medicine 3 months before operation. Total tissue was immediately fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histological phase of samples was confirmed using the criteria of Noyes. Then each group was divided into early and middle secretory phase.
Methods
HOXA10 mRNA expressions in early and middle secretory endo-metria of 28 cases IF and 20 cases NF women were examined by in situ hybridization. After paraffin sections were treated with proteinase, HOXA10 mRNA antisense riboprobes were added to sections to hybridize. Then they were added to a series of antibodies. At last they were stained by DAB. Brown and yellow granulae emerging in cytoplasm defined as positive expression. We detected positive unites (PUs) of stained area using image analysis system. Data were analyzed using SPSS software.
Results
HOXA10 mRNA were expressed in glandula cells and- stromal cells of endometria of two groups. In the IF group, PUs of the expression of HOXA10 mRNA in early and mid secretory phase endometria were 6. 61+
引文
1、Coulifonis C, Onighodun A, Coukos G. Integrins, endometrial maturation & human embryo implantation. Semin Reprod Endocrinol, 1998, 16(12): 219-229
2、Ma L, Yao M, Mass RL. Genetic control of uterine receptivity during implantation. Reprod Endocrinol, 1999, 17(3): 205-216
3、Zhu LJ, Cullinan-Bore K, Polihronis M, et al. Calcitonin is a progesterone-regulated marker that forecasts the receptive state of endometrium during implantation. Endocrinology, 1998, 139(17): 3923-3934
4、Chen JR, Cheng JG, Shatzer T, et al. Leukemia inhibitory factor can substitute for nidatory estrogen and is essential to inducing a receptive uterus for implantation but is not essential for subsequent embryogenesis. Endocrinology, 2000, 141(12): 4365-4372
5、Krumlauf R. Hox genes in vertabrate develepment. Cell, 1994, 78(2): 191
6、Benson GV, Lim H, Paria BC, et al. Mechanisms of reduced fertility in Hoxa - 10 mutant mice: uterine homeosis and loss of maternal Hoxa - 10 expression. Development, 1996, 122(9): 2687-2696
7、Taylor HS, Arici A, Olive D, Igarashi P. Hoxa10 is response to sex steroids at the time of implantation in the human endometrium. J Clin Invest, 1998, 10(7): 1379-1384
8、Shen Hong. Study on the quantitative method of immunohisto-
chemistry. Chinese Journel of Histochemitry and Cytochemistry (in Chinese), 1995, 4(1): 89-92
9、Apiou F, Flagiello D, Cillo C, et al. Fine mapping of human Hox gene clusters. Cytogenet Cell Genet, 1996, 73 (1-2): 114-115
10、Chang CP, Brocchieri L, Shen WF, et al. Pbx modulation of Hox hemedomain N - terminal arms establishes a gradient of DNA - binding specificities across the Hox locus. Mol Cell Biol, 1996, 16: 1734-1745
11、Shen WF, Rozenfeld S, Lawrrence HJ, et al. The Abd - B -like homeodomain proteins can be Chang CP, Brocchieri L, Shen WF, et al. Pbx modulation of Hox homeodomain N - terminal arms subdivided by the ability to from complexes with Pbxla on a novel DNA target. J Biol Chem, 1997, 272(13): 8198-8206
12、Taylor HS, Vanden GB, Igarashi P. A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes. Biol Reprod, 1997, 57(6): 1338-1344
13、Lim H, Ma L, Ma WG, et al. Hoxa-10 regulation uterine stromai cell responsiveness to progesterone during implantation and decidualization in the mouse. Mol Endocrinol, 1999, 13(6): 1005-1017
14、Daftary GS, Troy PJ, Bagot CN, et al. Direct regulation of beta3-integrin subunit gene expressionby HOXA10 in endometrial cells. Mol Endocrinol, 2002, 16(3): 571-579
15、Sutherland AE, Calarco PG, Damshy CH. Developmental regulation of integrin expression at the time of implantation in the mouse embryo. Development, 1993, 119(1): 175
16、Bagot CN, Troy PJ, Taylor HS. Alteration of materal Hoxa10 ex-
pression by vivo gene transfection affects implantation. Gene ther, 2000, 7(16): 1378-1384
17、Shen Hongling, Sun Yingpu, Dong Changhong. Immunohistochemical study on estrogen and progesterone receptors in endometrium of women with luteal insufficiency. J Reprod Med (in Chinese), 1998,7(1): 33-37
2、Ma L, Yao M, Mass RL. Genetic control of uterine receptivity during implantation. Reprod Endocrinol, 1999, 17(3): 205-216
3、Zhu LJ, Cullinan-Bore K, Polihronis M, et al. Calcitonin is a progesterone-regulated marker that forecasts the receptive state of endometrium during implantation. Endocrinology, 1998, 139(17): 3923-3934
4、Chen JR, Cheng JG, Shatzer T, et al. Leukemia inhibitory factor can substitute for nidatory estrogen and is essential to inducing a receptive uterus for implantation but is not essential for subsequent embryogenesis. Endocrinology, 2000, 141(12): 4365-4372
5、Krumlauf R. Hox genes in vertabrate develepment. Cell, 1994, 78(2): 191
6、Benson GV, Lim H, Paria BC, et al. Mechanisms of reduced fertility in Hoxa - 10 mutant mice: uterine homeosis and loss of maternal Hoxa - 10 expression. Development, 1996, 122(9): 2687-2696
7、Taylor HS, Arici A, Olive D, Igarashi P. Hoxa10 is response to sex steroids at the time of implantation in the human endometrium. J Clin Invest, 1998, 10(7): 1379-1384
8、Shen Hong. Study on the quantitative method of immunohisto-
chemistry. Chinese Journel of Histochemitry and Cytochemistry (in Chinese), 1995, 4(1): 89-92
9、Apiou F, Flagiello D, Cillo C, et al. Fine mapping of human Hox gene clusters. Cytogenet Cell Genet, 1996, 73 (1-2): 114-115
10、Chang CP, Brocchieri L, Shen WF, et al. Pbx modulation of Hox hemedomain N - terminal arms establishes a gradient of DNA - binding specificities across the Hox locus. Mol Cell Biol, 1996, 16: 1734-1745
11、Shen WF, Rozenfeld S, Lawrrence HJ, et al. The Abd - B -like homeodomain proteins can be Chang CP, Brocchieri L, Shen WF, et al. Pbx modulation of Hox homeodomain N - terminal arms subdivided by the ability to from complexes with Pbxla on a novel DNA target. J Biol Chem, 1997, 272(13): 8198-8206
12、Taylor HS, Vanden GB, Igarashi P. A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes. Biol Reprod, 1997, 57(6): 1338-1344
13、Lim H, Ma L, Ma WG, et al. Hoxa-10 regulation uterine stromai cell responsiveness to progesterone during implantation and decidualization in the mouse. Mol Endocrinol, 1999, 13(6): 1005-1017
14、Daftary GS, Troy PJ, Bagot CN, et al. Direct regulation of beta3-integrin subunit gene expressionby HOXA10 in endometrial cells. Mol Endocrinol, 2002, 16(3): 571-579
15、Sutherland AE, Calarco PG, Damshy CH. Developmental regulation of integrin expression at the time of implantation in the mouse embryo. Development, 1993, 119(1): 175
16、Bagot CN, Troy PJ, Taylor HS. Alteration of materal Hoxa10 ex-
pression by vivo gene transfection affects implantation. Gene ther, 2000, 7(16): 1378-1384
17、Shen Hongling, Sun Yingpu, Dong Changhong. Immunohistochemical study on estrogen and progesterone receptors in endometrium of women with luteal insufficiency. J Reprod Med (in Chinese), 1998,7(1): 33-37