- Email: [email protected]
HOXA10 gene expression in human fallopian tube and ectopic pregnancy
American Journal of Obstetrics and Gynecology (2004) 190, 1404e6
www.elsevier.com/locate/ajog
HOXA10 gene expression in human fallopian tube and ectopic pregnancy Sana M. Salih, MD, Hugh S. Taylor, MD* Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn Received for publication August 20, 2003; revised December 30, 2003; accepted January 21, 2004
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Homeobox genes Fallopian tube Ectopic pregnancy Endometrium
Objective: The molecular mechanisms underlying ectopic implantation have not been well characterized. Here we investigate HOXA10 gene expression at the site of ectopic implantation as compared with the endometrium and with the normal fallopian tube. Study design: Northern blot analysis was used to evaluate HOXA10 gene messenger RNA level in various segments of normal pregnant and nonpregnant human fallopian tube, ectopic pregnancy, and endometrium. Results: Normal human fallopian tube expressed minimal levels of HOXA10 gene messenger RNA in the nonpregnant state. A trend toward a greater expression of HOXA10 gene was observed in the normal fallopian tube during pregnancy, but the difference was not statistically significant (P = .075). HOXA10 gene messenger RNA expression was up-regulated significantly at the site of implantation in ectopic pregnancy (P!.001), and its expression approached that of the endometrium during normal pregnancy (P = .33). Conclusion: HOXA10 gene expression is up-regulated at the ectopic implantation site in the fallopian tube, approaching that of the endometrium in normal intrauterine gestation. Inherently increased HOXA10 gene expression in the fallopian tube or dysregulation of HOXA10 gene expression by an abnormally implanting blastocyst may play a role in ectopic implantation. Ó 2004 Elsevier Inc. All rights reserved.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Ectopic pregnancy is the leading cause of maternal death in the first trimester of pregnancy. Ectopic pregnancy often results from salpingitis and tubal damage. More than 50% of the cases of ectopic pregnancy occur in fallopian tubes without macroscopically evident disease. The molecular mechanisms underlying ectopic implantation in such cases have not been well character-
* Reprint requests: Hugh S. Taylor, MD, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St, PO Box 208063, New Haven, CT 06520-8063. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.01.066
ized. We investigated HOXA10 expression at the site of ectopic implantation as compared with the endometrium and with the normal fallopian tube. The HOXA10 gene belongs to a large family of transcription factors that share a highly conserved 183-nucleotide sequence, encoding a 61-amino acid domain, the homeodomain. These homeodomains are related structurally to the helix turn-helix motif of prokaryotic DNA-binding proteins and have sequence-specific DNA binding activity.1 HOXA10 is a homeobox gene that is expressed both during embryogenesis and in the adult human reproductive tract.2 Uterine HOXA10 expression is necessary for endometrial receptivity to
Salih and Taylor
1405
blastocyst implantation, as demonstrated by targeted mutation or by HOXA10 antisense treatment.3,4 Mice with targeted disruption of HOXA10 are fully viable but have uteri that do not support implantation and pregnancy. We investigated the expression of HOXA10 in the human fallopian tube and the possible association with ectopic implantation. We compare HOXA10 messenger RNA (mRNA) expression in ectopic pregnancy with the expression in the normal fallopian tube in nonpregnant and pregnant states and in the endometrium.
Material and methods Segments of human fallopian tubes were obtained from 22 patients under an approved Human Investigations Committee protocol. These included 6 premenopausal patients who underwent hysterectomy and bilateral salpingo-oophorectomy for benign disease, 4 patients who underwent postpartum tubal ligation during cesarean delivery, 4 patients who underwent an interval laparoscopic tubal ligation, 2 patients with cesarean hysterectomy for intractable postpartum hemorrhage for placenta accreta, and 6 patients who underwent salpingectomy for ruptured ectopic pregnancy (not previously treated with methotrexate). Endometrium in addition to fimbria, ampulla, ischmus, and cornual segments of the fallopian tube were collected from the hysterectomy specimens and were stored separately. Samples from the ampullary region of the fallopian tube were obtained from patients who underwent postpartum and interval tubal ligation. In cases of ectopic pregnancy, samples were obtained from the ectopic implantation site of the fallopian tube. All tissue samples were snap frozen in liquid nitrogen and stored at ÿ80(C before isolation of RNA with trizol. Twenty micrograms of RNA was used for Northern blot analysis, as previously described.2,5 A well-characterized pGEM plasmid that contained 103 base pairs of the 3 prime untranslated region of HOXA10 was used as a template to generate the riboprobe. Northern blot hybridization signal that corresponded to HOXA10 and 18 S ribosomal RNA were quantified by laser densitometry. Each HOXA10 band was normalized to the value that was obtained from 18 S ribosomal RNA in the same lane. Data were analyzed with Kruskal-Wallis analysis of variance on ranks with Dunn’s post hoc test when the expression in segments of fallopian tube was compared. Unpaired t-test was used to compare the ectopic implantation site with the endometrium. Statistical significance was defined as a probability value of !.05. This experiment was validated twice with the use of different samples.
Results To determine the level of HOXA10 messenger RNA expression in the fallopian tube, Northern blot analysis of different areas of normal non-regnant fallopian tube were
Figure A, Representative Northern blot of HOXA10 mRNA expression in ectopic pregnancy, endometrium, and normal fallopian tubes. B, Each of HOXA10 band was normalized to 18 S ribosomal RNA and the average results from all samples were displayed as histograms. The source of tissue from which the data were obtained is listed on the X axis. The average ratio of the HOXA10 band density to 18 S ribosomal RNA is shown on the Y axis in arbitrary densitometry units. Error bars are SEM. The asterisk indicates statistically significant difference. Lanes 1-4 contain RNA from different segments of normal fallopian tube (fimbria [1], ampulla [2], ischmus [3], and cornua [4]). Lane 5 contains RNA from ectopic implantation site of the fallopian tube; lane 6 contains RNA from endometrium in late pregnancy, and lane 7 represents normal fallopian tube in term pregnancy.7 HOXA10 expression was down-regulated in normal nonpregnant fallopian tubes (lanes 1-4). HOXA10 expression was up-regulated in the fallopian tube at the ectopic implantation site (lane 5) and was twice as high compared with normal unaffected fallopian tube (P!.001). HOXA10 expression was highest in the endometrium in late pregnancy (lane 6); the expression in the endometrium was statistically higher than normal nonpregnant tube (P = .002) but was similar to the expression in ectopic pregnancy (P = .33). HOXA10 expression was also slightly up-regulated in the fallopian tube in term pregnancy (lane 7); when compared with normal nonpregnant fallopian tube, the difference was not statistically significant (P > .075).
compared with the endometrium. HOXA10 bands were normalized to 18 S ribosomal RNA. Figure 1 shows representative Northern blot results and histograms that demonstrate the average density of all HOXA10 band measurements that were obtained from each segment of the fallopian tube. The fimbria, ampulla, ischmus, and cornual segments of normal fallopian tube are shown in lanes 1-4, respectively. HOXA10 expression was similar in different areas of the normal fallopian tube (P = .838). HOXA10 expression in each area of the fallopian
1406 tube was significantly less than the expression in the endometrium (Figure, lane 6; P = .002). To compare HOXA10 mRNA expression in ectopic pregnancy and normal fallopian tube, specimens from ectopic implantation site were used. Figure shows Northern blot results and histograms of average HOXA10 expression normalized to 18 S ribosomal RNA in normal fallopian tube, ectopic pregnancy, and endometrium collected from cesarean hysterectomy specimens (lane 4-6, respectively). HOXA10 expression was 2-fold higher in the ectopic implantation site (lane 5) when compared with normal fallopian tube in nonpregnant states (P!.001). HOXA10 mRNA expression in ectopic pregnancy was not statistically different from the expression in the endometrium of term intrauterine pregnancy (P = .33). To study HOXA10 expression in the normal fallopian tube during pregnancy, specimens were collected from patients who underwent cesarean delivery with bilateral tubal ligation at term and were compared with normal nonpregnant fallopian tubes. Figure (lane 7) shows that there was an apparent increase in HOXA10 expression in the normal fallopian tube during pregnancy. The increase, however, was not statistically significant when compared with normal nonpregnant fallopian tube (P = .075) and was not as high as endometrium or fallopian tube with ectopic implantation.
Comment The major cause of ectopic pregnancy is disruption of normal tubal anatomy from pelvic inflammatory disease; however, 55% of ectopic pregnancy occurs in macroscopically normal fallopian tubes.6,7 Hormonal imbalances (such as abnormally elevated levels of estrogen, as seen in ovulation induction, or progesterone with the use of progesterone-releasing intrauterine device) are known to increase the rate of ectopic implantation. Structural and chromosomal abnormalities of embryonic development can also cause ectopic gestation. The exact molecular mechanism underlying ectopic implantation is not yet well elucidated. We investigated HOXA10 expression in the fallopian tube and its association with ectopic implantation. Previously we showed that the nonpregnant fallopian tube expresses HOXA9 and that HOXA10 normally is expressed minimally. We have shown that HOXA10
Salih and Taylor mRNA expression in the fallopian tube is up-regulated at the site of implantation in ectopic pregnancy. HOXA10 expression in the fallopian tube in ectopic pregnancy approaches that of the endometrium during normal intrauterine pregnancy. Expression was less than in the normal fallopian tube in late pregnancy; however, we were not able to compare to normal fallopian tube of comparable gestational age. Increased HOXA10 expression in the fallopian tube or aberrant regulation of HOXA10 expression by an abnormal blastocyst may play a role in ectopic implantation. Furthermore, the molecular mechanisms that control implantation may be the same, regardless of the site of implantation (uterus vs tube) because HOXA10 expression, an implantation marker, is increased both in eutopic and ectopic pregnancies. Our data do not address the cause of increased HOXA10 expression in the fallopian tube in ectopic pregnancy. Abnormal HOXA10 expression could represent an abnormal constitutive expression by the abnormal fallopian tube before ectopic implantation or could be driven by an abnormally implanting embryo. Ectopic implantation may be mediated by a HOXA10dependent mechanism, similar to that established necessary for eutopic implantation.
References 1. Gehring WJ, Muller M, Affolter M, Percival-Smith A, Billeter M, Qian YQ, et al. The structure of the homeodomain and its functional implications. Trends Genet 1990;6:323-9. 2. Taylor HS, Vandenheuvel 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;57:1338-45. 3. Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes in Hoxa10 deficient mice. Nature 1995;374:460-3. 4. Bagot CN, Troy PJ, Taylor HS. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation. Gene Ther 2000;7:1378-84. 5. Taylor HS, Arici A, Olive D, Igarashi P. HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium. J Clin Investig 1998;101:1379-84. 6. CDC for Disease Control and Prevention. Ectopic pregnancy: United States, 1990-1992. MMWR Morb Mortal Wkly Rep 1995; 44:46-8. 7. Westrom L, Bengtsson LP, Mardh PA. Incidence, trends, and risks of ectopic pregnancy in a population of Women. BMJ 1981;282: 15-8.
www.elsevier.com/locate/ajog
HOXA10 gene expression in human fallopian tube and ectopic pregnancy Sana M. Salih, MD, Hugh S. Taylor, MD* Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn Received for publication August 20, 2003; revised December 30, 2003; accepted January 21, 2004
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Homeobox genes Fallopian tube Ectopic pregnancy Endometrium
Objective: The molecular mechanisms underlying ectopic implantation have not been well characterized. Here we investigate HOXA10 gene expression at the site of ectopic implantation as compared with the endometrium and with the normal fallopian tube. Study design: Northern blot analysis was used to evaluate HOXA10 gene messenger RNA level in various segments of normal pregnant and nonpregnant human fallopian tube, ectopic pregnancy, and endometrium. Results: Normal human fallopian tube expressed minimal levels of HOXA10 gene messenger RNA in the nonpregnant state. A trend toward a greater expression of HOXA10 gene was observed in the normal fallopian tube during pregnancy, but the difference was not statistically significant (P = .075). HOXA10 gene messenger RNA expression was up-regulated significantly at the site of implantation in ectopic pregnancy (P!.001), and its expression approached that of the endometrium during normal pregnancy (P = .33). Conclusion: HOXA10 gene expression is up-regulated at the ectopic implantation site in the fallopian tube, approaching that of the endometrium in normal intrauterine gestation. Inherently increased HOXA10 gene expression in the fallopian tube or dysregulation of HOXA10 gene expression by an abnormally implanting blastocyst may play a role in ectopic implantation. Ó 2004 Elsevier Inc. All rights reserved.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Ectopic pregnancy is the leading cause of maternal death in the first trimester of pregnancy. Ectopic pregnancy often results from salpingitis and tubal damage. More than 50% of the cases of ectopic pregnancy occur in fallopian tubes without macroscopically evident disease. The molecular mechanisms underlying ectopic implantation in such cases have not been well character-
* Reprint requests: Hugh S. Taylor, MD, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St, PO Box 208063, New Haven, CT 06520-8063. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.01.066
ized. We investigated HOXA10 expression at the site of ectopic implantation as compared with the endometrium and with the normal fallopian tube. The HOXA10 gene belongs to a large family of transcription factors that share a highly conserved 183-nucleotide sequence, encoding a 61-amino acid domain, the homeodomain. These homeodomains are related structurally to the helix turn-helix motif of prokaryotic DNA-binding proteins and have sequence-specific DNA binding activity.1 HOXA10 is a homeobox gene that is expressed both during embryogenesis and in the adult human reproductive tract.2 Uterine HOXA10 expression is necessary for endometrial receptivity to
Salih and Taylor
1405
blastocyst implantation, as demonstrated by targeted mutation or by HOXA10 antisense treatment.3,4 Mice with targeted disruption of HOXA10 are fully viable but have uteri that do not support implantation and pregnancy. We investigated the expression of HOXA10 in the human fallopian tube and the possible association with ectopic implantation. We compare HOXA10 messenger RNA (mRNA) expression in ectopic pregnancy with the expression in the normal fallopian tube in nonpregnant and pregnant states and in the endometrium.
Material and methods Segments of human fallopian tubes were obtained from 22 patients under an approved Human Investigations Committee protocol. These included 6 premenopausal patients who underwent hysterectomy and bilateral salpingo-oophorectomy for benign disease, 4 patients who underwent postpartum tubal ligation during cesarean delivery, 4 patients who underwent an interval laparoscopic tubal ligation, 2 patients with cesarean hysterectomy for intractable postpartum hemorrhage for placenta accreta, and 6 patients who underwent salpingectomy for ruptured ectopic pregnancy (not previously treated with methotrexate). Endometrium in addition to fimbria, ampulla, ischmus, and cornual segments of the fallopian tube were collected from the hysterectomy specimens and were stored separately. Samples from the ampullary region of the fallopian tube were obtained from patients who underwent postpartum and interval tubal ligation. In cases of ectopic pregnancy, samples were obtained from the ectopic implantation site of the fallopian tube. All tissue samples were snap frozen in liquid nitrogen and stored at ÿ80(C before isolation of RNA with trizol. Twenty micrograms of RNA was used for Northern blot analysis, as previously described.2,5 A well-characterized pGEM plasmid that contained 103 base pairs of the 3 prime untranslated region of HOXA10 was used as a template to generate the riboprobe. Northern blot hybridization signal that corresponded to HOXA10 and 18 S ribosomal RNA were quantified by laser densitometry. Each HOXA10 band was normalized to the value that was obtained from 18 S ribosomal RNA in the same lane. Data were analyzed with Kruskal-Wallis analysis of variance on ranks with Dunn’s post hoc test when the expression in segments of fallopian tube was compared. Unpaired t-test was used to compare the ectopic implantation site with the endometrium. Statistical significance was defined as a probability value of !.05. This experiment was validated twice with the use of different samples.
Results To determine the level of HOXA10 messenger RNA expression in the fallopian tube, Northern blot analysis of different areas of normal non-regnant fallopian tube were
Figure A, Representative Northern blot of HOXA10 mRNA expression in ectopic pregnancy, endometrium, and normal fallopian tubes. B, Each of HOXA10 band was normalized to 18 S ribosomal RNA and the average results from all samples were displayed as histograms. The source of tissue from which the data were obtained is listed on the X axis. The average ratio of the HOXA10 band density to 18 S ribosomal RNA is shown on the Y axis in arbitrary densitometry units. Error bars are SEM. The asterisk indicates statistically significant difference. Lanes 1-4 contain RNA from different segments of normal fallopian tube (fimbria [1], ampulla [2], ischmus [3], and cornua [4]). Lane 5 contains RNA from ectopic implantation site of the fallopian tube; lane 6 contains RNA from endometrium in late pregnancy, and lane 7 represents normal fallopian tube in term pregnancy.7 HOXA10 expression was down-regulated in normal nonpregnant fallopian tubes (lanes 1-4). HOXA10 expression was up-regulated in the fallopian tube at the ectopic implantation site (lane 5) and was twice as high compared with normal unaffected fallopian tube (P!.001). HOXA10 expression was highest in the endometrium in late pregnancy (lane 6); the expression in the endometrium was statistically higher than normal nonpregnant tube (P = .002) but was similar to the expression in ectopic pregnancy (P = .33). HOXA10 expression was also slightly up-regulated in the fallopian tube in term pregnancy (lane 7); when compared with normal nonpregnant fallopian tube, the difference was not statistically significant (P > .075).
compared with the endometrium. HOXA10 bands were normalized to 18 S ribosomal RNA. Figure 1 shows representative Northern blot results and histograms that demonstrate the average density of all HOXA10 band measurements that were obtained from each segment of the fallopian tube. The fimbria, ampulla, ischmus, and cornual segments of normal fallopian tube are shown in lanes 1-4, respectively. HOXA10 expression was similar in different areas of the normal fallopian tube (P = .838). HOXA10 expression in each area of the fallopian
1406 tube was significantly less than the expression in the endometrium (Figure, lane 6; P = .002). To compare HOXA10 mRNA expression in ectopic pregnancy and normal fallopian tube, specimens from ectopic implantation site were used. Figure shows Northern blot results and histograms of average HOXA10 expression normalized to 18 S ribosomal RNA in normal fallopian tube, ectopic pregnancy, and endometrium collected from cesarean hysterectomy specimens (lane 4-6, respectively). HOXA10 expression was 2-fold higher in the ectopic implantation site (lane 5) when compared with normal fallopian tube in nonpregnant states (P!.001). HOXA10 mRNA expression in ectopic pregnancy was not statistically different from the expression in the endometrium of term intrauterine pregnancy (P = .33). To study HOXA10 expression in the normal fallopian tube during pregnancy, specimens were collected from patients who underwent cesarean delivery with bilateral tubal ligation at term and were compared with normal nonpregnant fallopian tubes. Figure (lane 7) shows that there was an apparent increase in HOXA10 expression in the normal fallopian tube during pregnancy. The increase, however, was not statistically significant when compared with normal nonpregnant fallopian tube (P = .075) and was not as high as endometrium or fallopian tube with ectopic implantation.
Comment The major cause of ectopic pregnancy is disruption of normal tubal anatomy from pelvic inflammatory disease; however, 55% of ectopic pregnancy occurs in macroscopically normal fallopian tubes.6,7 Hormonal imbalances (such as abnormally elevated levels of estrogen, as seen in ovulation induction, or progesterone with the use of progesterone-releasing intrauterine device) are known to increase the rate of ectopic implantation. Structural and chromosomal abnormalities of embryonic development can also cause ectopic gestation. The exact molecular mechanism underlying ectopic implantation is not yet well elucidated. We investigated HOXA10 expression in the fallopian tube and its association with ectopic implantation. Previously we showed that the nonpregnant fallopian tube expresses HOXA9 and that HOXA10 normally is expressed minimally. We have shown that HOXA10
Salih and Taylor mRNA expression in the fallopian tube is up-regulated at the site of implantation in ectopic pregnancy. HOXA10 expression in the fallopian tube in ectopic pregnancy approaches that of the endometrium during normal intrauterine pregnancy. Expression was less than in the normal fallopian tube in late pregnancy; however, we were not able to compare to normal fallopian tube of comparable gestational age. Increased HOXA10 expression in the fallopian tube or aberrant regulation of HOXA10 expression by an abnormal blastocyst may play a role in ectopic implantation. Furthermore, the molecular mechanisms that control implantation may be the same, regardless of the site of implantation (uterus vs tube) because HOXA10 expression, an implantation marker, is increased both in eutopic and ectopic pregnancies. Our data do not address the cause of increased HOXA10 expression in the fallopian tube in ectopic pregnancy. Abnormal HOXA10 expression could represent an abnormal constitutive expression by the abnormal fallopian tube before ectopic implantation or could be driven by an abnormally implanting embryo. Ectopic implantation may be mediated by a HOXA10dependent mechanism, similar to that established necessary for eutopic implantation.
References 1. Gehring WJ, Muller M, Affolter M, Percival-Smith A, Billeter M, Qian YQ, et al. The structure of the homeodomain and its functional implications. Trends Genet 1990;6:323-9. 2. Taylor HS, Vandenheuvel 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;57:1338-45. 3. Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes in Hoxa10 deficient mice. Nature 1995;374:460-3. 4. Bagot CN, Troy PJ, Taylor HS. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation. Gene Ther 2000;7:1378-84. 5. Taylor HS, Arici A, Olive D, Igarashi P. HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium. J Clin Investig 1998;101:1379-84. 6. CDC for Disease Control and Prevention. Ectopic pregnancy: United States, 1990-1992. MMWR Morb Mortal Wkly Rep 1995; 44:46-8. 7. Westrom L, Bengtsson LP, Mardh PA. Incidence, trends, and risks of ectopic pregnancy in a population of Women. BMJ 1981;282: 15-8.