Decreased occurrence of endometriosis in women with Chlamydia trachomatis infection.

PROBLEM: Despite abundant reports on the risk role of uterine outflow tract obstruction in endometriosis, information on the occurrence of endometriosis in women with Chlamydia trachomatis infection causing fallopian tube obstruction is unknown. We investigated the role of Chlamydia trachomatis infection with or without fallopian tubal patency in the occurrence of endometriosis. METHODS: This is a retrospective case-controlled cohort study with 539 women who had laparoscopic surgery for several indications during the period between January, 2003 and June, 2010. Women with ectopic pregnancy, uterine anomaly, chromosomal abnormality, primary amenorrhea, and perimenopausal women were excluded. Endometriosis was diagnosed by laparoscopic inspection and confirmed by histopathology. Tubal patency was diagnosed by HSG or laparoscopic chromopertubation test. Presence of chlamydia infection was examined by RT-PCR and serological test. RESULTS: Two-hundred and seven women were enrolled. Eighty-six (41.5%) women had chlamydia infection. Tubal patency and occurrence of endometriosis were significantly decreased among women with chlamydia infection comparing to women without it (P = .005 and P = .0008, respectively). Even among women with patent tube, laparoscopic detection of endometriosis was significantly decreased in chlamydia infected comparing to non-infected women (P = .02). Multiple logistic regression model revealed that previous history of chlamydia infection significantly decreased the occurrence of endometriosis, and was independent of age, menstrual status, parity and tubal patency (odds ratio .44; 95% confidence interval .24-.80; P = .007). CONCLUSION: A decreased occurrence of peritoneal endometriosis was observed in women with Chlamydia trachomatis infection. The possible impairment of retrograde menstrual flow by chlamydia-infected tubal damage may decrease the risk of endometriosis.

Effect of GnRH agonist therapy on the expression of human heat shock protein 70 in eutopic and ectopic endometria of women with endometriosis.

OBJECTIVES: Human heat shock protein 70 (HSP70) has been reported to enhance Toll-like receptor 4-mediated pelvic inflammation and growth of endometriotic cells. However, information on tissue expression of HSP70 before and after treatment with estrogen suppressing agent is scanty. Here, we investigated tissue expression HSP70 in the eutopic/ectopic endometria of gonadotropin-releasing hormone agonist (GnRHa)-treated and -non-treated women with endometriosis. STUDY DESIGN: Biopsy specimens were collected from peritoneal lesions, cyst walls, and corresponding endometria of 20 women with peritoneal endometriosis, 35 women with ovarian endometrioma, and 15 control women during laparoscopy. Fifteen women with ovarian endometrioma were treated with GnRHa for a variable period of 4-6 months before laparoscopy. The immunoexpressions of HSP70 and CD68-positive macrophages (Mφ) in endometria, peritoneal lesions, and cyst walls were examined by immunohistochemistry. The immunoreactivity of HSP70 in tissues was analyzed by quantitative-histogram (Q-H) score. RESULTS: Tissue expression of HSP70 was found to be the highest in the menstrual phase than in other phases of the menstrual cycle. A significantly higher immunoreactivity of HSP70 was found in the eutopic endometria of women with peritoneal and ovarian endometriosis than in controls and in opaque red lesions than in other peritoneal lesions. A significant correlation between Q-H scores of HSP70 and CD68-positive Mφ numbers was found in the endometria derived from women with peritoneal endometriosis (r=0.481) and in ovarian endometrioma (r=0.560) but not in control women. The Q-H scores of HSP70 expression were significantly lower in cyst walls, coexisting peritoneal lesions and corresponding endometria of women with ovarian endometrioma after GnRHa treatment comparing to similar tissues derived from women without GnRHa treatment. CONCLUSION: These results suggest that as a marker of tissue stress reaction, immunoexpression of HSP70 was highly increased in pathological lesions and endometria of women with endometriosis and had a significant correlation with tissue inflammatory reaction. The higher tissue expression of HSP70 can be effectively suppressed after GnRHa treatment.

Role of prostaglandin E2 in bacterial growth in women with endometriosis.

STUDY QUESTION: Can prostaglandin E(2) (PGE(2)) in menstrual and peritoneal fluid (PF) promote bacterial growth in women with endometriosis? SUMMARY ANSWER: PGE(2) promotes bacterial growth in women with endometriosis. WHAT IS KNOWN ALREADY: Menstrual blood of women with endometriosis is highly contaminated with Escherichia coli (E. coli) compared with that of non-endometriotic women: E. coli-derived lipopolysaccharide (LPS) promotes the growth of endometriosis. STUDY DESIGN, SIZE AND DURATION: Case-controlled biological research with a prospective collection of body fluids and endometrial tissues from women with and without endometriosis with retrospective evaluation. PARTICIPANTS/MATERIALS, SETTING AND METHODS: PF and sera were collected from 58 women with endometriosis and 28 women without endometriosis in an academic research laboratory. Menstrual blood was collected from a proportion of these women. Macrophages (Mφ) from PF and stromal cells from eutopic endometria were isolated in primary culture. The exogenous effect of PGE(2) on the replication of E. coli was examined in a bacterial culture system. Levels of PGE(2) in different body fluids and in the culture media of Mφ and stromal cells were measured by ELISA. The effect of PGE(2) on the growth of peripheral blood lymphocytes (PBLs) was examined. MAIN RESULTS AND THE ROLE OF CHANCE: The PGE(2) level was 2-3 times higher in the menstrual fluid (MF) than in either sera or in PF. A significantly higher level of PGE(2) was found in the MF and PF of women with endometriosis than in control women (P < 0.05 for each). Exogenous treatment with PGE(2) dose dependently increased E. coli colony formation when compared with non-treated bacteria. PGE(2)-enriched MF was able to stimulate the growth of E. coli in a dilution-dependent manner; this effect was more significantly enhanced in women with endometriosis than in control women (P < 0.05). PGE(2) levels in the culture media of LPS-treated Mφ/stromal cells were significantly higher in women with endometriosis than in non-endometriosis (P < 0.05 for each). Direct application of PGE(2) and culture media derived from endometrial Mφ or stromal cells significantly suppressed phytohemagglutinin-stimulated growth of PBLs. LIMITATIONS AND REASONS FOR CAUTION: Further studies are needed to examine the association between PGE(2)-stimulated growth of E. coli and endotoxin level and to investigate the possible occurrence of sub-clinical infection within vaginal cavity. WIDER IMPLICATIONS OF THE FINDINGS: Our findings may provide some new insights to understand the physiopathology or pathogenesis of the mysterious disease endometriosis and may hold new therapeutic potential. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan. There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: Not applicable.

Cell proliferation effect of GnRH agonist on pathological lesions of women with endometriosis, adenomyosis and uterine myoma.

BACKGROUND: We recently demonstrated the effect of gonadotrophin-releasing hormone agonist (GnRHa) on tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma. Here, we investigated expression of GnRH receptors (GnRHRs) and effect of GnRHa on the proliferation of cells derived from endometria and pathological lesions of women with these reproductive diseases. METHODS: Biopsy specimens were collected from lesions and corresponding endometria of 35 women with pelvic endometriosis, 45 women with ovarian endometrioma, 35 women with adenomyosis and 56 women with uterine myoma during laparoscopy or laparotomy. The gene and protein expressions of GnRHR in eutopic/ectopic cells and tissues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The immunoreactivity of GnRHR in tissue was analysed by quantitative-histogram (Q-H) scores. The exogenous effect of GnRHa on cell proliferation was examined by 5-bromo-2-deoxyuridine incorporation assay. The Ki-67-immunoreactive cell proliferation index was analysed in biopsy specimens derived from GnRHa-treated and -non-treated women. RESULTS: Types I and II GnRHRs mRNA and proteins were expressed in eutopic endometria and pathological lesions derived from women with endometriosis, adenomyosis and uterine myoma. GnRHR expression was the highest in the menstrual phase when compared with other phases of the menstrual cycle. Higher Q-H scores of GnRHR immunoreaction were found in blood-filled opaque red lesions than in other peritoneal lesions. Exogenous treatment with GnRHa significantly suppressed the proliferation of cells derived from respective endometria and pathological lesions when compared with GnRHa-non-treated cells. CONCLUSIONS: Local tissue expression of GnRHR was detected in endometriosis, adenomyosis and uterine myoma. In addition to a hypo-estrogenic effect, a direct anti-proliferative effect of GnRHa may be involved in the regression of these reproductive diseases with consequent remission of clinical symptoms.

Escherichia coli contamination of menstrual blood and effect of bacterial endotoxin on endometriosis.

To test the hypothesis that bacterial contamination of menstrual blood could be a local biologic event in the development of endometriosis, menstrual blood was cultured and bacterial endotoxin was measured in menstrual blood and peritoneal fluid. Our results suggest that compared with control women, higher colony formation of Escherichia coli in menstrual blood and endotoxin levels in menstrual fluid and peritoneal fluid in women with endometriosis may promote Toll-like receptor 4-mediated growth of endometriosis.

Effect of human seminal fluid on the growth of endometrial cells of women with endometriosis.

OBJECTIVES: We investigated the effect of human seminal fluid on the growth of endometrial cells derived from women with and without endometriosis. STUDY DESIGN: Seminal plasma (SP) was collected from 18 healthy fertile men. Serum, peritoneal fluid (PF) and tissue specimens of eutopic and ectopic endometrium were collected from 45 women with endometriosis and 20 women without endometriosis during laparoscopic surgery. Prostaglandin (PG) E2, hepatocyte growth factor (HGF), and estradiol (E2) levels in each sample of SP, serum and PF were measured by enzyme-linked immunosorbent assay. The growth pattern of cells derived from eutopic and ectopic endometria in response to SP was examined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. RESULTS: Seminal plasma was able to significantly stimulate the growth of epithelial cells and stromal cells derived from the eutopic and ectopic endometria of women with endometriosis (2-3-fold) when compared with control media. The SP-promoted proliferation of both gland cells and stromal cells derived from eutopic endometria was also remarkably higher in women with endometriosis than that of women without endometriosis. Although levels of PGE2, HGF and E2 in SP were variable when compared with other body fluids, the levels of PGE2 and HGF in SP were significantly higher than those in either peritoneal fluid or serum of women with or without endometriosis. Pretreatment of cells with individual anti-PGE2 antibody, anti-HGF antibody and two selective estrogen receptor modulators, tamoxifen and raloxifene was unable to suppress SP-mediated growth of endometrial cells. However, pretreatment of cells with combined anti-PGE2 antibody plus anti-HGF antibody or combined anti-PGE2 antibody plus anti-HGF antibody plus tamoxifen or raloxifene was able to significantly suppress SP-promoted growth of eutopic and ectopic endometrial cells. CONCLUSION: Human seminal fluid enriched with different macromolecules may promote the growth of endometrial cells derived from women with endometriosis. Our findings may suggest some detrimental effect of unprotected sexual intercourse in women with endometriosis.

Seroepidemiological survey of cytomegalovirus infection among pregnant women in Nagasaki, Japan.

BACKGROUND: Epidemiology of cytomegalovirus (CMV) infection varies widely, depending on ethnicity and socioeconomic status. A seroepidemiological survey was conducted to determine CMV infection among pregnant women in Nagasaki Prefecture, Japan. METHODS: We measured serum CMV-specific immunoglobulin (Ig) M and IgG at the first and third trimesters. IgG avidity was determined when both CMV-IgG and CMV-IgM were positive. RESULTS: Of 339 pregnant women, 296 (87.3%) were CMV-IgG-positive at the first trimester. Of 267 paired sera, one (0.37%) had CMV-IgG seroconversion, another one (0.37%) had CMV-IgM seroconversion, and 12 had both CMV-IgG and CMV-IgM, two (0.75%) of whom had low IgG avidity, suggesting recent infection. Thus, the incidence of primary CMV infection during pregnancy was 0.74-1.5%. Assuming the rate of in utero transmission following maternal primary infection to be approximately 40%, the incidence of congenital infection is estimated to be 0.3-0.6%. CONCLUSION: Although CMV seroprevalence among pregnant women has been decreasing in industrialized regions including other parts of Japan, CMV-seroprevalence remains high in Nagasaki. Thus, epidemiology of CMV infection seems variable within Japan, a country generally considered to be ethnically and socioeconomically homogeneous. However, 40-80 infants may be congenitally infected and 15-27% (or 6-22) of them may ultimately suffer from certain neurological sequelae annually in Nagasaki Prefecture, so where annual live births are approximately 13,300, congenital CMV infection seems to be a significant public health problem in such an apparently low-risk region as Nagasaki.

Changes in tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma after GnRH agonist therapy.

BACKGROUND: Information is limited regarding the multifunctional role of GnRH agonist (GnRHa) therapy in reproductive diseases. We investigated the pattern of changes in inflammatory reaction, micro-vessel density and apoptosis in the tissues collected from women with endometriosis, adenomyosis and uterine myoma who were treated with or without GnRHa therapy. METHODS: Biopsy specimens were collected from lesions, myometria and corresponding endometria of 45 women with ovarian endometrioma, 35 women with adenomyosis and 56 women with uterine myoma. A fraction of these women were treated with GnRHa therapy for a variable period of 3-6 months before surgery. We performed immunohistochemical analysis of CD68, a macrophage (Mvarphi) marker and von Willebrand factor (VWF), a vessel marker, using respective antibodies. Changes in apoptosis were examined using TdT-mediated dUTP-biotin nick end-labeling assay and by the immunoexpression of activated caspase-3 in tissues after GnRHa therapy. RESULTS: The infiltration of CD68-positive Mvarphi and VWF-positive micro-vessel density were significantly decreased in the endometria of women with endometriosis, adenomyosis and uterine myoma in the GnRHa-treated group when compared with that in the non-treated group. Marked decreases in inflammatory and angiogenic responses were observed in lesions and myometria of these diseases. When compared with the non-treated group, a significant increase in apoptotic index (apoptotic cells per 10 mm(2) area) and quantitative-histogram scores of activated caspase-3 after GnRHa therapy were observed in the eutopic endometria, lesions and myometria of these diseases. CONCLUSIONS: GnRHa was able to markedly reduce the inflammatory reaction and angiogenesis and to significantly induce apoptosis in tissues derived from women with endometriosis, adenomyosis and uterine myoma. These multiple biological effects at the tissue level may be involved in the regression of these reproductive diseases.

Toll-like receptors in innate immunity: role of bacterial endotoxin and toll-like receptor 4 in endometrium and endometriosis.

Macrophages, dendritic cells, and Toll-like receptors (TLRs) are integral components of the innate immune system. This rapidly reactive system responds immediately to infectious or other non-self agents, thereby inducing an inflammatory response to protect the host until the activation of the slower adaptive immune system. The fundamentals of the innate immune system, functional characteristics of TLRs, and signaling pathways of TLR4 are discussed for the easy understanding by readers. Studies showed that the growth and progression of endometriosis continue even in ovariectomized animals. This indicates that besides ovarian steroid hormones, the growth of endometriosis can be regulated by the innate immune system in the pelvic environment. As a component of the innate immune system, increased infiltration of macrophages has been described in the intact tissue and peritoneal fluid of women with endometriosis. In this review article, we discuss the role of bacterial endotoxin and TLR4 in endometrium and endometriosis and outline the involvement of endotoxin in causing adverse reproductive outcome.

Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

BACKGROUND: We investigated the role of human heat-shock protein 70 (Hsp70) in Toll-like receptor 4 (TLR4)-mediated growth of endometriosis. METHODS: TLR4 expression was examined in macrophages (M) isolated in primary culture from the peritoneal fluid of women with and without endometriosis. The production of a number of macromolecules by non-treated M, Hsp70-treated M and after treatment with anti-TLR4 antibody was examined by enzyme linked immunosorbent assay (ELISA). The single and combined effects of Hsp70 and lipopolysaccharide (LPS) on the growth of endometrial stromal cells were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation study. Hsp70 levels in eutopic and ectopic endometria were measured by ELISA. RESULTS: TLR4 was detected in isolated M at protein and gene level. Hsp70 (10 microg/ml) significantly stimulated the production of hepatocyte growth factor, vascular endothelial cell growth factor, interleukin-6 and tumor necrosis factor alpha by M derived from women with endometriosis compared with M derived from women with no endometriosis (P < 0.05 for each). This effect of Hsp70 was abrogated after pretreatment of M with anti-TLR4 antibody. BrdU incorporation indicated that Hsp70 significantly enhanced the growth of endometrial stromal cells ( approximately 50% increase) from women with endometriosis compared to non-treated cells. A synergistic effect on cell proliferation was observed between exogenous Hsp70 and LPS and this was significantly suppressed by pretreatment of cells with anti-TLR4 antibody (P < 0.05). Tissue levels of Hsp70 were significantly higher in the eutopic endometria (P < 0.05) and opaque red lesions (P < 0.01) derived from women with endometriosis than from other peritoneal lesions or from women with no endometriosis. CONCLUSIONS: A prominent stress reaction was observed in blood-filled opaque red peritoneal lesions. Human Hsp70 induces pelvic inflammation and may be involved in TLR4-mediated growth of endometrial cells derived from women with endometriosis.

Long-term effects of polychlorinated biphenyls and dioxins on pregnancy outcomes in women affected by the Yusho incident.

BACKGROUND: Maternal exposure to polychlorinated biphenyls (PCBs) is associated with increased proportions of spontaneous abortion and stillbirth in animal studies. In Japan in 1968, accidental human exposure to rice oil contaminated with PCBs and other dioxin-related compounds, such as polychlorinated dibenzofurans (PCDFs), led to the development of what was later referred to as Yusho oil disease. OBJECTIVE: The aim of this study was to investigate the association of maternal PCB and dioxin exposure with adverse pregnancy outcomes in Yusho women. METHODS: In 2004, we interviewed 214 Yusho women (512 pregnancies) about their pregnancy outcomes over the past 36 years. Pregnancy outcomes included induced abortion, spontaneous abortion, preterm delivery, and pregnancy loss. RESULTS: In pregnancy years 1968-1977 (within the first 10 years after exposure), the proportions of induced abortion [odds ratio adjusted for age at delivery (ORadj) = 5.93; 95% confidence interval (CI), 2.21-15.91; two-tailed p < 0.001) and preterm delivery (ORadj = 5.70; 95% CI, 1.17-27.79; p = 0.03) were significantly increased compared with the proportions in pregnancy years 1958-1967 (10 years before the incident). Spontaneous abortion (ORadj = 2.09; 95% CI, 0.84-5.18), and pregnancy loss (ORadj = 2.11; 95% CI, 0.92-4.87) were more frequent (OR = 2.18; 95% CI, 1.02-4.66), but these were not significant (p = 0.11 and p = 0.08, respectively) in pregnancy years 1968-1977. We found no significant increases in the proportions of these adverse pregnancy outcomes in pregnancies occurring during 1978-1987 or 1988-2003 compared with those in pregnancies before 1968. CONCLUSION: High levels of PCB/PCDF exposure had some adverse effects on pregnancy outcome in Yusho women.

Immunopathogenesis of pelvic endometriosis: role of hepatocyte growth factor, macrophages and ovarian steroids.

Endometriosis, a chronic disease characterized by endometrial tissue located outside the uterine cavity is associated with chronic pelvic pain and infertility. However, an in-depth understanding of the pathophysiology of endometriosis is still elusive. It is generally believed that besides ovarian steroid hormones, the growth of endometriosis can be regulated by innate immune system in pelvic microenvironment by their interaction with endometrial cells and immune cells. We conducted a series of studies in perspectives of pelvic inflammation that is triggered primarily by bacterial endotoxin (lipopolysaccharide) and is mediated by toll-like receptor 4 and showed their involvement in the development of pelvic endometriosis. As a cellular component of innate immune system, macrophages were found to play a central role in inducing pelvic inflammatory reaction. We further report here that peritoneal macrophages retain receptors encoding for estrogen and progesterone and ovarian steroids also participate in producing an inflammatory response in pelvic cavity and are involved in the growth of endometriosis either alone or in combination with hepatocyte growth factor (HGF). As a pleiotropic growth factor, HGF retains multifunctional role ometriosis. We describe here the individual and step-wise role of HGF, macrophages and ovarian steroid hormones and their orchestrated involvement in the immunopathogenesis of pelvic endometriosis.

Re-evaluation of the indication for and limitation of laparoscopic salpingotomy for tubal pregnancy.

OBJECTIVE: We investigated the outcome of laparoscopic salpingotomy for tubal pregnancy by follow-up hysterosalpingography (HSG) or second-look laparoscopy (SLL) and reexamined the indication for and limitation of this conservative surgery. STUDY DESIGN: From April 1991 to December 2003, we treated 181 cases of tubal pregnancy using laparoscopic salpingotomy. The tubal patency was assessed by either HSG or SLL performed at 3 months post-surgery. The patients with a successful initial operation and confirmed ipsilateral patent tubes at follow-up were classified as truly successful cases (group I). Even after successful operation, if the treated tubes were found to be occluded, they were considered as unsuccessful cases. Therefore, those cases that were unsuccessful at initial surgery as well as at follow-up were categorized as group II. RESULTS: One hundred and thirty-four cases (74%) were successfully treated by salpingotomy at initial laparoscopy and 85 of them (63.4%) were found to be truly successful at follow-up (group I). The remaining 47 cases (26.0%) were unsuccessful at initial surgery and 18 (13.4%) cases at follow-up (group II). Thirty-one other patients refused to accept a tubal patency test or were not examined for personal reasons or were lost to follow-up. No difference in surgical outcome was observed between these two groups of patients with regard to gestational age, intra-operative hemorrhage, size or anatomic location of the pregnancy mass, and pre-operative adhesions of the fallopian tube. However, pre-operative serum levels of hCG were significantly higher in group II than in group I. In addition, the unsuccessful cases were more frequently associated with positive fetal heart beat (FHB), tubal rupture, and pre-operative serum levels of hCG of more than 10,000 IU/l (p<0.05, chi2 test). The log-rank test indicated a higher pregnancy success rate in group I (p<0.05) than in group II in those who desired future pregnancy. CONCLUSION: Laparoscopic salpingotomy may be practised as conservative surgery for proximal ectopic pregnancy, and gestational mass size is not as important and is not a relative contraindication for conservative laparoscopic surgery, as previously reported. Low pre-operative HCG levels, absence of FHB, absence of tubal rupture initially or minimal rupture may be considered suitable parameters for successful surgery and for achieving future pregnancy.

Effect of placenta previa on blood loss in second-trimester abortion by labor induction using gemeprost.

OBJECTIVES: The study was conducted to determine whether placenta previa increases bleeding during gemeprost-induced termination of second-trimester pregnancy. METHODS: We carried out a retrospective study of 158 second-trimester terminations between 12 and 21 weeks' gestation. We compared the intraoperative blood loss among three groups: women without placenta previa undergoing gemeprost termination, women with placenta previa undergoing gemeprost termination and women with placenta previa undergoing dilatation and evacuation (D&E). RESULTS: Eleven of 158 women (7.0%) had placenta previa; four underwent D&E and seven had gemeprost termination. There was no statistical difference in mean intraoperative blood loss among the three groups, although one of the seven women with placenta previa who underwent gemeprost termination developed serious bleeding requiring blood transfusion. CONCLUSION: The use of gemeprost for second-trimester pregnancy termination in women with placenta previa seems to be relatively safe and does not increase intraoperative blood loss in the majority of cases.

Role of DNA methylation and histone H3 lysine 27 methylation in tissue-specific imprinting of mouse Grb10.

Mouse Grb10 is a tissue-specific imprinted gene with promoter-specific expression. In most tissues, Grb10 is expressed exclusively from the major-type promoter of the maternal allele, whereas in the brain, it is expressed predominantly from the brain type promoter of the paternal allele. Such reciprocally imprinted expression in the brain and other tissues is thought to be regulated by DNA methylation and the Polycomb group (PcG) protein Eed. To investigate how DNA methylation and chromatin remodeling by PcG proteins coordinate tissue-specific imprinting of Grb10, we analyzed epigenetic modifications associated with Grb10 expression in cultured brain cells. Reverse transcriptase PCR analysis revealed that the imprinted paternal expression of Grb10 in the brain implied neuron-specific and developmental stage-specific expression from the paternal brain type promoter, whereas in glial cells and fibroblasts, Grb10 was reciprocally expressed from the maternal major-type promoter. The cell-specific imprinted expression was not directly related to allele-specific DNA methylation in the promoters because the major-type promoter remained biallelically hypomethylated regardless of its activity, whereas gametic DNA methylation in the brain type promoter was maintained during differentiation. Histone modification analysis showed that allelic methylation of histone H3 lysine 4 and H3 lysine 9 were associated with gametic DNA methylation in the brain type promoter, whereas that of H3 lysine 27 regulated by the Eed PcG complex was detected in the paternal major-type promoter, corresponding to its allele-specific silencing. Here, we propose a molecular model that gametic DNA methylation and chromatin remodeling by PcG proteins during cell differentiation cause tissue-specific imprinting in embryonic tissues.

Clinical outcome of infants with confined placental mosaicism and intrauterine growth restriction of unknown cause.

The purpose of this study was to know a role of confined placental mosaicism (CPM) in perinatal outcome and postnatal growth and development of infants with intrauterine growth restriction (IUGR). We selected 50 infants with IUGR (<-2.0 SD) from 3,257 deliveries in a regional medical center during the past 10-year period, and carried out cytogenetic and molecular analyses in their placenta and cord blood. Of the 50 infants, 8 had CPM (CPM group) and were composed of five single (CPM2, 7, 13, 22, and 22), one double (CPM7/13), and one quadruple trisomy (CPM2/7/15/20), and one partial monosomy [del(2)(p16)]. The origin of an extra chromosome of trisomy was maternal in six cases of CPM, paternal in one, and undetermined in one. Uniparental disomy in disomic cell lines was ruled out in all these mosaics. We also compared clinical parameters for perinatal outcome between CPM group and infants without evidence of CPM (non-CPM group), such as maternal and gestational age, birth weight, Apgar score, cord blood pH, gender, and uterine artery patterns by Doppler ultrasonography, as well as weight, height, and developmental quotient (DQ) by Denver Developmental Screening Test at age 12 months. Phenotypic abnormalities were noted in two infants with CPM and three infants of non-CPM group: One with CPM22 had ASD and hypospadias, one with CPM7/13 had Russell-Silver syndrome (RSS), and one without CPM had polydactyly, and two without CPM had RSS. All but one infant with CPM are alive at age 12 months. Among the clinical parameters, the detection rate of a notch waveform pattern of the uterine artery was significantly higher in the CPM group (P < 0.05). However, no significant difference was noted in perinatal outcome of pregnancy and in DQ at age 12 months between the two groups. Interestingly, short stature (<-2 SD) at age 12 months was more frequently seen in CPM group (7/8 infants with CPM vs. 8/15 infants without CPM), although no statistically significant difference was obtained. The information obtained will be useful for perinatal care and genetic counseling for infants with IUGR and CPM.

Differential infiltration of macrophages and prostaglandin production by different uterine leiomyomas.

BACKGROUND: The association between uterine myoma and infertility is still controversial. The anatomical defect of endometrium by uterine fibroids could be a factor for reducing pregnancy rates and increasing miscarriage rates. However, pregnancy and implantation rates were found to be significantly lower in women with intramural myomas (IMMs), when there was no deformity of uterine cavity. This could be due to other biological factors such as increased accumulation of inflammatory cells within fibroid tissue and corresponding endometrium that might impair fertility. Therefore, we tried to investigate the pattern of macrophage (Mvarphi) accumulation in different uterine fibroids and the production of chemokine and prostaglandin (PG) by these tissues. METHODS: The selection criteria of uterine fibroids were based on the classification of European Society of Hysteroscopy. Biopsy specimens were collected from respective nodules and autologous endometrium of 20 women with submucosal myoma (SMM), 29 women with IMM and 18 women with subserosal myoma (SSM). CD68 immunoreactive Mvarphis were identified in these tissues by immunohistochemistry. A fraction of corresponding tissues were homogenized, and levels of monocyte chemotactic protein-1 (MCP-1) and PGF(2alpha) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Mvarphi infiltration in the myoma nodule and corresponding endometrium of women with SMM and IMM was significantly higher than that of women with SSM or control women (P<0.01 and P<0.05, respectively). This tissue accumulation of inflammatory cells was independent of the sizes of the myoma nodules and phases of menstrual cycle. The tissue concentration of MCP-1 corresponded to increased Mvarphi infiltration and was significantly higher in women with SMM and IMM than that in women with SSM (P<0.05 for each). A positive correlation was observed between MCP-1 concentration and accumulated Mvarphi numbers in the endometrium of women with SMM and IMM but not in women with SSM. The tissue levels of PGF2alpha were also significantly higher in the nodule and corresponding endometrium of women with SMM and IMM than that in SSM or control women (P<0.05 for each). CONCLUSIONS: Higher production of MCP-1 could be responsible for the increased accumulation of Mvarphi in women with SMM and IMM. The augmented inflammatory reaction in endometrium and increased PGF2alpha levels might be detrimental to reproductive outcome in women with SMM or IMM.

Identification of estrogen receptor beta-positive intraepithelial lymphocytes and their possible roles in normal and tubal pregnancy oviducts.

BACKGROUND: Although intraepithelial lymphocytes (IELs) in human oviductal epithelium have been implicated in the regulation of local immunity, the precise kinetics and mechanism of steroid regulation of IEL are largely unknown. METHODS: We examined the localization of estrogen receptors (ERs) and progesterone receptors (PRs) in 41 human oviducts by immunohistochemistry. These tissues were obtained from various menstrual cycles, also from both post-menopausal women and tubal pregnancies. The expressions of ERbeta mRNA and membrane (m)PR mRNA were examined by in situ hybridization and RT-PCR, respectively. RESULTS: Most of the IEL expressed ERbeta at both mRNA and protein levels. The number of ERbeta-positive IEL, which were identified as CD8-positive T lymphocytes and also were mPR positive, was increased in the late proliferative, the mid-secretory and late secretory phases in normally cycling women (P < 0.05). Interestingly, in tubal pregnancy, ERbeta-positive IELs were consistently abundant. In addition, we found a high Ki-67-labelling index for IEL, although ERalpha was entirely absent in the tubal pregnancy oviducts. CONCLUSIONS: These results suggest that the number of IEL fluctuated because of estrogen and progesterone levels probably through ERbeta and mPR, respectively. ERbeta-positive IEL may be involved in regulating immune tolerance in tubal pregnancy oviducts.

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid.

Cell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.