Decreased pregnancy rate is linked to abnormal uterine peristalsis caused by intramural fibroids.

BACKGROUND: The relationship between fibroids and infertility remains an unsolved question, and management of intramural fibroids is controversial. During the implantation phase, uterine peristalsis is dramatically reduced, which is thought to facilitate embryo implantation. Our aims were to evaluate (i) the occurrence and frequency of uterine peristalsis in infertile women with intramural fibroids and (ii) whether the presence of uterine peristalsis decreases the pregnancy rate. METHODS: Ninety-five infertile patients with uterine fibroids were examined using magnetic resonance imaging (MRI). Inclusion criteria were as follows: (i) presence of intramural fibroids, excluding submucosal type; (ii) no other significant infertility factors (excluding endometriosis); and (iii) regular menstrual cycles, and MRI performed at the time of implantation (luteal phase day 5-9). The frequency of junctional zone movement was evaluated using cine-mode-display MRI. After MRI, patients underwent infertility treatment for up to 4 months, and the pregnancy rate was evaluated prospectively. RESULTS: Fifty-one patients fulfilled the inclusion criteria, and 29 (57%) and 22 (43%) patients were assigned to the low (0 or 1 time/3 min) or high frequency (≥ 2 times/3 min) uterine peristalsis group, respectively. Endometriosis incidence was the same in both groups. Ten out of the 29 patients (34%) in the low-frequency group achieved pregnancy, compared with none of the 22 patients (0%) in the high-frequency group (P< 0.005). Comparing pregnant and non-pregnant cases, 4 of 10 patients (40%) and 9 of 41 patients (22%), respectively, had endometriosis (not significant). CONCLUSIONS: A higher frequency of uterine peristalsis during the mid-luteal phase might be one of the causes of infertility associated with intramural-type fibroids.

Isolation of estrogen-responsive genes with a CpG island library.

In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched. CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells. Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively. Both transcripts were increased by estrogen in MCF-7 cells. The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochrome c oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5' upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5' upstream region of the chicken beta-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

Ontogenetic changes in the expression of estrogen receptor alpha and beta in rat pituitary gland detected by immunohistochemistry.

The physiological effects of estrogen on the pituitary, including cellular proliferation and regulation of hormone synthesis, are mediated by the nuclear estrogen receptor (ER). The purpose of this study was to determine ontogenetic expression of two types of ERs (ERalpha and ERbeta) in the pituitary using specific antibodies, monoclonal antibody (1D5) for ERalpha and polyclonal antibody generated against ERbeta. First, we confirmed the detection of 66- and 55-kDa bands for ERalpha and ERbeta, respectively, in the rat pituitary extract by Western blotting. Then immunostaining with these antibodies was performed using fetal and adult Wistar rat tissues, combined with PRL or LHbeta immunohistochemistry. Intense ERbeta signal was detected throughout the pituitary from day 12 of gestation. However, staining for ERalpha only became detectable from day 17 of gestation. In contrast with the fetal period, nuclei stained for ERalpha were widely distributed in the anterior lobe in the adult rat, whereas ERbeta-positive cells were restricted in the anterior lobe. LHbeta, but not PRL, was colocalized in ERbeta-positive cells. Our results indicated that the major population of ER subtypes in the rat pituitary gland has changed around the day of birth and that the expression of ERbeta may be involved in the differentiation of pituitary cell function to synthesize a specific hormone.

Demonstration of angiogenin in human endometrium and its enhanced expression in endometrial tissues in the secretory phase and the decidua.

Angiogenesis is thought to be crucial for normal physiology of the endometrium, where dynamic vascular remodeling occurs during the menstrual cycle and pregnancy. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human endometrium. Western blot analysis demonstrated that angiogenin protein expression increased by 3- to 4-fold in the endometrium in the mid and late secretory phases and in early gestation relative to that during the proliferative phase. Quantitative mRNA analysis showed the similar tendency in the expression of angiogenin mRNA in the endometrium, with the highest levels observed in the mid and late secretory phases and early gestation. An immunohistochemical study showed that angiogenin was expressed in both stromal cells and epithelial cells, with indistinguishable intensity between these cells regardless of phases of the menstrual cycle. In support of the Western blot analysis, the intensity of staining appeared to be highest in the mid to late secretory phases relative to other phases. Consistent with these in vivo results, decidualized cultured stromal cells, after treatment with progesterone or progesterone plus E2, exhibited the capacity to secrete significantly increased amounts of angiogenin compared with untreated or E2 alone-treated control group. Both the treatment with (Bu)2cAMP and hypoxic conditions stimulated angiogenin secretion by stromal cells. For isolated epithelial cells, hypoxia stimulated angiogenin secretion, whereas (Bu)2cAMP had no appreciable effect. In summary, we demonstrated the presence of angiogenin in human endometrium and its possible local regulatory factors, such as progesterone, cAMP, and hypoxia. These findings along with its enhanced expression in the endometrium in the secretory phase and in decidual tissues raise the possibility that angiogenin may play a role in establishing pregnancy.

Expression of estrogen, progesterone and androgen receptors in the oviduct of developing, cycling and pre-implantation rats.

To determine expression and localization of receptors for estrogen (ER), progesterone (PR) and androgen (AR), detailed immunohistochemical evaluations were performed in the Sprague-Dawley rat oviduct during pre- and neonatal development, estrous cycle and pre-implantation period. In addition, real-time RT-PCR studies were conducted to evaluate changes in ERalpha, ERbeta, total PR (PR-A+B), PR-B and AR mRNA expressions. All receptors except for ERbeta were detected in epithelial, and stromal or mesenchymal cells of the fetal and neonatal oviduct, and increased with development. During the estrous cycle and early pregnancy, ERalpha and PR-A+B were expressed in epithelial, stromal and muscle cells throughout the oviduct region, and showed changes in expression predominantly in the isthmus. Only a few epithelial cells in the infundibulum (inf) and ampulla (AMP) showed ERbeta staining. AR was detected in stromal and muscle cells throughout the oviduct region, and in epithelial cells of the inf/AMP. Taken together, ERalpha, PR-A+B and AR were detected in the epithelium of the inf/AMP region, but all of these receptors were expressed in a distinct subset of epithelial cells which were negative for beta-tubulin IV, a ciliated epithelial cell marker. These results contribute to a better understanding of the respective roles of ERs, PRs and AR in the rat oviduct.

Changes in ontogenetic expression of estrogen receptor alpha and not of estrogen receptor beta in the female rat reproductive tract.

To evaluate ontogenetic expression and localization of estrogen receptor (ER) alpha and beta in fetal female rat reproductive tract, competitive RT-PCR and immunohistochemistry were performed. Expression levels for Müllerian ERalpha, ERbeta1 and ERbeta2 mRNAs were determined by competitive RT-PCR. ERalpha expression on gestational day (GD) 15 x 5 increased 4 x 4-fold by GD 21 x 5, whereas both ERbeta1 and ERbeta2 gene expression were maintained at lower constant levels compared with ERalpha during development. ER immunolocalization was evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. Nuclear ERalpha was localized predominantly in proximal Müllerian epithelium, and middle and caudal Müllerian mesenchyme on GDs 15 x 5-21 x 5. Staining intensity for ERalpha increased with development in all regions. However, ERbeta immunoreactivity was not detected in any region during prenatal life after separate staining with three different polyclonal anti-rat ERbeta antibodies. These findings provide fundamental information critical for clarifying the species-specific physiological roles of ER subtypes during fetal development and for investigating the tissue-specific mechanisms underlying the prenatal response to estrogen and estrogen receptor agonists.

Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus.

In order to investigate the localization of estrogen receptor (ER) alpha and ERbeta in the reproductive organs in the rat, polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ERalpha antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ERbeta antibody detected a 55 kDa band in rat ovary, uterus and prostate. The ligand-independent nuclear localization of the two receptors was verified by immunocytochemistry. By immunohistochemistry, the nuclei of glandular and luminal epithelial cells in the uterus were stained with anti-ERalpha antibody, whereas only the nuclei of glandular epithelium cells were stained with anti-ERbeta antibody. In rat ovary, positive signals were shown with anti-ERbeta antibody in the nuclei of granulosacells. No specific immunostaining was observed with anti-ERalpha antibody. Although ERbeta was immunostained at the proestrous, metestrous and diestrous stages, the immunoreactivity of ERbeta was hardly detected at the estrous stage in rat ovary. Thus, we show differential expression of ERalpha and ERbeta in rat uterus and ovary at the protein level, which may provide a clue for understanding the roles of the two receptors in reproductive organs.

NMDA receptor type 2D gene as target for estrogen receptor in the brain.

Although it is well known that estrogen exerts its effect in the brain, the direct target genes transcriptionally regulated by estrogen or rather estrogen receptor (ER) are almost unknown. During the search for estrogen receptor-binding sites from human CpG island library, we found one genomic DNA fragment corresponding to the putative 3'-untranslated region of human NMDA receptor subunit 2D (NR2D) gene. It contained at least four half palindromic estrogen responsive elements (hEREs) within two hundred nucleotides, which was conserved also in the rat. Interestingly, the NR2D mRNA is co-localized with ERalpha and/or ERbeta mRNA in a number of regions of rat brain. We have also demonstrated that NR2D mRNA is up-regulated in rat hypothalamus by estrogen possibly via hEREs identified here. Thus, we suggest that NR2D is one of the direct targets of estrogen receptors which are involved in reproductive as well as non-reproductive actions in the brain.

An earlier menopause as clinical manifestation of granulosa-cell tumor: a case report.

We present a case of a granulosa-cell tumor, which can cause menopause at an earlier than normal age. The hormonal profiles were characterized by undetectable FSH levels associated with an estradiol level compatible with the level seen in perimenopausal women and by a significant increase in the inhibin level.

Hyperemesis gravidarum associated with thyrotoxicosis and a past history of an eating disorder.

We present a case of severe hyperemesis gravidarum (HG) associated with thyrotoxicosis in a woman with a past history of an eating disorder. She had developed persistent HG from early pregnancy until about at the end of the second trimester with a body loss of 14 kg. Total parenteral nutrition was effective in alleviateing HG. It is suggested that even a past history of an eating disorder could be at risk of developing HG.

Mucinous adenocarcinoma arising in a neovagina using the sigmoid colon thirty years after operation: a case report.

BACKGROUND: Because of a relative rarity of the cases with an artificial vagina, the incidence of a case with malignant disease arising in the neovagina is extremely rare. A case of adenocarcinoma arising from a neovagina is presented with a review of the literature. CASE: A neovagina was constructed using the sigmoid colon at the age of 23 for congenital agenesis of the vagina, Rokitansky-Küster-Hauser syndrome. Subsequently, the patient had regular sexual intercourse for about 20 years. At the age of 53, she came to our outpatient clinic with a complaint of vaginal bleeding, and adenocarcinoma was found at the anterior wall of the neovagina adjoining the introitus. Total resection of the neovagina and adjuvant radiotherapy was performed. The pathological diagnosis was mucinous adenocarcinoma. CONCLUSIONS: In view of relatively low incidence of mucinous carcinoma arising in the sigmoid colon along with the ectopic localization, this case may have implications for the understanding of pathogenesis of sigmoid colon cancer.

Differential interactions of bisphenol A and 17beta-estradiol with estrogen receptor alpha (ERalpha) and ERbeta.

Bisphenol A (BPA), a monomer of plastic used in consumer products, is abundant in the environment and enters the body by ingestion or adsorption. We developed a cell based transcription assay system using a reporter gene under the transcriptional control of estrogen receptor alpha (ERalpha) as well as ERbeta and performed chloramphenicol acetyltransferase (CAT) assay on HeLa cells transfected with either human ERalpha cDNA or ERbeta cDNA to characterize the estrogenic effect of BPA. Estrogenic activity of BPA was detectable at a concentration of 10(-9) M and the activity increased in a dose dependent manner between concentrations of 10(-9) M and 10(-6) M of BPA for both ERalpha and ERbeta. The estrogenic activity of 17beta-estradiol at a concentration of 10(-8) M was almost compatible with that of BPA at the concentration of 10(-6) M of BPA for ERalpha as well as ERbeta. CAT activity was significantly decreased when cells expressing ERalpha were incubated with 10(-6) M of BPA and 10(-8) M of 17beta-estradiol while the activity was essentially the same for ERbeta in the same condition, indicating that BPA exhibits only agonistic action for ERbeta whereas it has dual actions as an agonist and antagonist of estrogen for ERalpha. These results indicates that BPA exerts its effects in ER subtype specific way, thus suggesting that the mode of action of endocrine disruptors are more complex than thought.

Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos.

The effects of bisphenol A, a xenoestrogen widely used in industry and dentistry, were studied in early preimplantation mouse embryos. Two-cell mouse embryos were cultured with 100 pM to 100 microM bisphenol A with or without 100 nM tamoxifen and evaluated at 24-h intervals for their development to eight-cell and blastocyst stages. At 72 h, blastocysts were cultured for another 48 h without bisphenol A, and surface areas of trophoblast spread were measured. At 24 h, more embryos exposed to 3 nM bisphenol A than to controls had reached the eight-cell stage. At 48 h, more embryos exposed to 1 nM and 3 nM bisphenol A than to controls had become blastocysts. At 100 microM, bisphenol A decreased frequency of development to blastocysts. Tamoxifen counteracted both stimulatory and inhibitory effects of bisphenol A on blastocyst formation. Although bisphenol A did not alter blastocyst morphology or cell number, early exposure to 100 microM bisphenol A increased subsequent trophoblast areas. These findings suggest that bisphenol A may not only effect early embryonic development via estrogen receptors even at low, environmentally relevant doses, but also exert some late effects on subsequent development of these embryos.

Agonistic effect of tamoxifen is dependent on cell type, ERE-promoter context, and estrogen receptor subtype: functional difference between estrogen receptors alpha and beta.

To investigate the functional differences between estrogen receptor (ER) alpha and beta subtypes, we studied the expression and the transcription stimulating activities of these receptors. RT-PCR has demonstrated that ER alpha is expressed at a high level in MCF-7 cells derived from human breast cancer. Both ER alpha and ER beta were expressed at a lower level in HOS-TE85 and Saos2 cells derived from human osteosarcoma. Chloramphenicol acetyltransferase reporter assay detected the transcriptional activation by the endogenous receptor only in MCF-7 cells. Agonistic effect of tamoxifen was observed as strong as that of 17beta-estradiol on ERE activation in MCF-7 cells at the concentration of 10(-7) M when ERE-containing reporter is constructed with beta-globin promoter. The effect of tamoxifen was not apparent when the reporter was constructed with thymidine kinase promoter, suggesting that the differential gene activation between tamoxifen and estrogen may take place depending upon ERE-promoter context. Agonistic activity of tamoxifen was also detected in COS-7 and Saos-2 cells, but not in HEC-1 cells derived from human endometrial carcinoma via exogenously expressed ER. Interestingly, this effect was ER alpha specific. Thus, we demonstrate that agonistic effect of tamoxifen depends on the cell type, ERE-promoter context, and ER subtype. These parameters would explain at least a part of the tissue specific effects of antiestrogens in vivo.

Molecular cloning and characterization of mouse EBAG9, homolog of a human cancer associated surface antigen: expression and regulation by estrogen.

We previously identified a human estrogen-responsive gene, EBAG9 (ER-binding fragment-associated antigen9) (Watanabe, T. et al., Mol. Cell. Biol. 18, 442-449, 1998). It was later reported as RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) that induced apoptosis and suppressed the growth of several cells such as activated T cells (Nakashima, M. et al., Nat. Med. 5, 938-942, 1999). Here, we have isolated both cDNA and genomic DNA of mouse EBAG9/RCAS1. Mouse EBAG9 gene spans about 30 kb in genomic DNA and consists of 7 exons. Mouse EBAG9 cDNA encodes a protein that contains the transmenbrane segment and coiled-coil domain. An alignment between the predicted mouse and human EBAG9 shows a high degree of homology at the amino acid level (98%). Northern and Western blot analyses demonstrate that EBAG9 is expressed in several tissues including the heart, brain, spleen, liver, kidney, and testis, and also in developing embryo. In the uterus, a target organ for estrogen, the EBAG9 was shown to be upregulated in vivo by 17beta-estradiol. To determine the biological action of mouse EBAG9, NIH3T3 fibroblastic cells were incubated with recombinant EBAG9 protein, resulting in suppression of cell growth. These findings suggest that EBAG9 is an in vivo estrogen-responsive gene that inhibits the cell growth.

The complete primary structure of human estrogen receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in vitro.

Human estrogen receptor beta (hER beta) cDNA that encodes the full-length amino acid sequence has been isolated from testis poly(A)+ RNA with the combination of cDNA screening and reverse transcription-PCR. It is composed of a 1590-bp open reading frame and a segment of the 5'- and 3'-untranslated region (UTR) and encodes an additional 53 amino acids in the N-terminal region compared with the previously reported one. Protein interaction between ER alpha and ER beta was demonstrated in vitro by GST pull-down assay and in vivo by immunoprecipitation. Thus, this study indicates that ER alpha and ER beta can interact in vivo, cross-signaling each other.

Evidence for the presence of hepatocyte growth factor expression in human ovarian follicles.

The presence of hepatocyte growth factor (HGF) in follicular fluid (FF) relative to concentrations of sex steroid hormones and human chorionic gonadotrophin (HCG) was investigated. A total of 69 FF samples were obtained during oocyte retrieval for in-vitro fertilization (IVF) from 11 patients with no apparent endocrine disorders. The concentrations of HGF, oestradiol, progesterone, HCG and testosterone in FF samples were measured by enzyme-linked immunosorbent assay. Transcription of HGF and its receptor, c-met, was detected by reverse transcription-polymerase chain reaction (RT-PCR). Human FF samples contained approximately 90-fold higher amounts of HGF (24.2 +/- 1.2 ng/ml), compared with those of serum (0. 28 +/- 0.04 ng/ml). Concentrations of HGF in FF were positively correlated with those of progesterone (r = 0.649, P < 0.0001) and HCG (r = 0.264, P = 0.026) concentrations in FF. However, HGF concentrations were not significantly correlated with oestradiol and testosterone. HGF in FF was detected by Western blotting, as a single 90 kDa band, corresponding to a single chain form. Additionally, mRNA for both HGF and its receptor were detected in a crude granulosa cell preparation from the pre-ovulatory follicles. These findings suggest that HGF is produced locally in human ovarian follicles and may have a physiological role as an autocrine/paracrine factor.

Stage-specific expression of estrogen receptor subtypes and estrogen responsive finger protein in preimplantational mouse embryos.

In hope of understanding possible roles of estrogen during early embryogenesis, we examined the expression of both estrogen receptor alpha (ER alpha) and ER beta, a recently cloned novel subtype, in mouse oocytes and preimplantation embryos by means of reverse transcription polymerase chain reaction (RT-PCR). To investigate whether estrogen actually exerts its action, we further determined the expression of efp (estrogen-responsive finger protein), a newly characterized estrogen responsive gene belonging to the RING finger family. ER alpha mRNA was detected in whole ovaries, cumulus-oocyte complexes, denuded oocytes, 2-cell and 4-cell embryos, whereas it was undetected in 8-cell embryos. Interestingly it reappeared in morulae and blastocysts. ER beta mRNA was detected similarly to ER alpha except for the absence of ER beta mRNA in morulae. The efp mRNA was detected in whole ovaries, cumulus-oocyte complexes, 4-cell embryos, morulae and blastocysts. The stage specific expression of ER alpha and ER beta along with detection of the product of the estrogen responsive gene in early preimplantation embryos may indicate the possible physiological roles of estrogen in early embryogenesis.

A fetus with Prader-Willi syndrome showing normal diurnal rhythm and abnormal ultradian rhythm on heart rate monitoring.

Clinical features of Prader-Willi syndrome in neonates are marked hypotonia with the absence of crying and feeding difficulty so that prenatal diagnosis of Prader-Willi syndrome is strongly hoped in order to provide appropriate medical and psychological care for neonates and their families. However, the clinical picture of Prader-Willi syndrome in utero has not been well described. We report a pregnancy associated with Prader-Willi syndrome manifesting polyhydramnios, large biparietal diameter of the fetus and characteristic fetal heart rate pattern: prolonged inactive periods and diurnal variation of the incidence of heart rate accelerations. These findings may offer a clue to the prenatal diagnosis of Prader-Willi syndrome, although molecular cytogenetics is mandatory for the definite diagnosis.

Underdeveloped uterus and reduced estrogen responsiveness in mice with disruption of the estrogen-responsive finger protein gene, which is a direct target of estrogen receptor alpha.

The biological roles of estrogen-responsive finger protein (efp) in vivo were evaluated in mice carrying a loss-of-function mutation in efp by gene-targeted mutagenesis. Although efp homozygous mice were viable and fertile in both sexes, the uterus that expressed abundant estrogen receptor alpha exhibited significant underdevelopment. When the ovariectomized homozygotes were subjected to 17beta-estradiol treatment, they showed remarkably attenuated responses to estrogen, as exemplified by decreased interstitial water imbibition and retarded endometrial cell increase, at least, attributable to the lower ratio of G1 to S-phase progression in epithelial cells. These results suggest that efp is essential for the normal estrogen-induced cell proliferation and uterine swelling as one of the direct targets of estrogen receptor alpha.