Genotyping of congenital adrenal hyperplasia due to 21-hydroxylase deficiency presenting as male infertility: case report and literature review.

We describe here two infertile male patients who were referred to our hospital with azoospermia at the ages of 33 and 30 years, respectively. Hormonal examinations led to a diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency in both patients. Genotyping revealed that the patients had a homozygous I172N and a heterozygous compound I172N/IVS2-13A/C>G mutation, respectively. Glucocorticoid replacement therapy succeeded in improving the seminal status of one patient, but not the other. For the latter patient and his wife, a pregnancy was achieved by testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) following genetic counseling. It is important to investigate genotyping and to classify patients on the basis of genotypic information in order to arrive at better treatment strategies for male infertility; especially in counseling of TESE-ICSI.

Requirement for transglutaminase in progesterone-induced decidualization of human endometrial stromal cells.

Differentiation of endometrial stromal cells (decidualization) is essential for embryo implantation and maintenance of pregnancy. By sequential complementary DNA subtractive hybridization, one of the messenger RNAs (mRNA) induced by progesterone in human endometrial stromal cells decidualized in vitro was identified as that of a tissue transglutaminase type II (TGase). TGase mRNA was induced within 6 h after the addition of progesterone to the culture, and the effect was dose dependent. Both the TGase inhibitor monodansylcadaverine and oligodeoxynucleotide complementary to the TGase mRNA inhibited the decidualization, as assessed by PRL production and morphological transformation. Expression of TGase mRNA in human decidua and endometria exposed to high levels of progesterone in vivo was demonstrated by Northern blotting and in situ hybridization. These data suggest that TGase is necessary for the decidualization of human endometrial stromal cells and that clarification of the mechanism of action of TGase will facilitate further insight into the diagnosis and treatment of infertility.

Detection of antiendometrial antibodies in patients with endometriosis by cell ELISA.

PROBLEM: To determine whether infertile patients with endometriosis have serum antiendometrial antibodies. METHODS: Sera from 40 infertile patients with or without endometriosis were tested by cell enzyme-linked immunosorbent assay (ELISA), in which endometrial cancer cells were used as endometrial antigens, and uterine cervix cancer cells as control antigens. As a negative control, eight healthy adult males were included. The level greater than the mean +/- 2 standard deviations (SD) of the male control group was judged positive. RESULTS: The mean value of antiendometrial antibody level was significantly higher in patients with endometriosis than in those without endometriosis (ANOVA, P < 0.01). The frequency of antiendometrial antibody-positive patients was also higher in the former than in the latter (chi 2 test, P < 0.05). However, when uterine cervix cancer cells were used as antigens, no difference was observed in the mean antibody levels or in the positive rates between the two groups. CONCLUSIONS: Endometriosis seems to be associated with autoantibody production against the endometrium-related antigen(s).

Induction of tissue inhibitor of metalloproteinase 3 gene expression during in vitro decidualization of human endometrial stromal cells.

Endometrial stromal differentiation (decidualization) is essential for implantation of the developing blastocyst. To investigate the process of progesterone (P)-induced decidualization of human endometrial stromal cells (ESC), a complementary DNA library enriched with P-induced genes was constructed from cultured human ESC by subtractive hybridization and the polymerase chain reaction. One of the isolated clones was the complementary DNA for the tissue inhibitor of metalloproteinase-3 (TIMP-3), a recently identified member of the human TIMP family. When human ESC were cultured in the presence of P for 6 days, the induction of TIMP-3 messenger RNA (mRNA) expression was observed by Northern blotting. In contrast, the marked induction of PRL mRNA expression and morphological changes were observed after 9 days of culture. P-induced TIMP-3 mRNA expression was dose dependent, and this induction was inhibited by the antiprogestin RU486. Estrogen did not induce TIMP-3 mRNA expression under similar conditions. In situ hybridization analysis of endometria from nonpregnant women revealed that the TIMP-3 mRNA expression was restricted to predecidualized stromal cells. At the feto-maternal interface, TIMP-3 expression was observed in fetal extravillous trophoblasts that had invaded the maternal decidual tissues as well as in the maternal decidual cells. These findings suggest that TIMP-3 is a sensitive indicator of ESC decidualization, and that the induction of TIMP-3 expression in decidual cells and trophoblasts may be important in the regulation of trophoblast invasion.

Expression of vav proto-oncogene by nonhematopoietic trophoblast cells at the human uteroplacental interface.

Vav is a signal transducing molecule containing SH2 and SH3 domains and a guanine nucleotide releasing factor-like domain. Its expression is thought to be highly specific for hematopoietic cells. Here we describe the expression of vav transcripts in human nonhematopoietic trophoblasts. By northern blotting, expression of 2.8-kb vav mRNA was detected in human decidual, placental, and chorionic villous tissues and in a choriocarcinoma cell line BeWo. By in situ hybridization, vav mRNA was found to be expressed in the cytotrophoblast shell and columns and in the extravillous trophoblasts in the maternal decidua from the first through third trimesters. Vav mRNA was also detected in villous syncytiotrophoblasts during the second and third, but not the first, trimesters. When 1 microM oligodeoxynucleotide antisense to the vav mRNA was added to the medium, growth of BeWo cells was significantly inhibited. These results suggest that vav plays an important role for successful implantation and placental development by regulating development of trophoblasts.

Isolation and structural determination of seminal vesicle-specific peptides of the terrestrial isopod, Armadillidium vulgare.

During the course of purifying the androgenic gland hormone of the terrestrial isopod, Armadillidium vulgare, that induces post-embryonic sex differentiation, four structurally related peptides were obtained and their structures determined by a combination of microsequence and mass spectral analyses. These peptides were found to exist speciffically in the seminal vesicle and vas deferens by a Western blot analysis, therefore being designated as seminal vesicle-specific peptides (SVSPs). They had essentially the same amino acid sequences but differed from one another in the truncation of several residues at the N-terminus and of one residue at the C-terminus, and in the modification of glutamine to pyroglutamate at the N-terminus. The longest peptides, SVSP-4, consisted of 60 amino acid residues with two intramolecular disulfide bridges. There is no significant homology with any other vertebrate or invertebrate peptides.

Expression of leukaemia inhibitory factor (LIF) receptor in human placenta: a possible role for LIF in the growth and differentiation of trophoblasts.

Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.

Regulation of sex steroid receptor gene expression by progesterone and testosterone in cultured human endometrial stromal cells.

Progesterone (P) is known to regulate sex steroid receptors in uterine cells. However, its precise regulation at the messenger ribonucleic acid (mRNA) level is unclear. In this study we examined the effects of P and testosterone (T) on the regulation of sex steroid receptors in cultured human endometrial stromal cells (ESC), using the quantitative reverse transcriptase polymerase chain reaction method. We isolated ESC from human endometrial tissues and cultured them with or without P (10(-6) mol/L) or T (10(-8) mol/L) for 9 days. Incubation with P decreased progesterone receptor (PR), estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.56 +/- 0.04-, 0.53 +/- 0.08-, and 0.84 +/- 0.04-fold (mean +/- SE), respectively. T also decreased PR, estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.48 +/- 0.06-, 0.52 +/- 0.05-, and 0.82 +/- 0.04-fold (mean +/- SE), respectively. These decreases by P and T occurred in a dose-dependent manner. We also examined the sex steroid receptor levels in human ESC cultured for 0, 3, 6, and 9 days. The PR mRNA level in ESC without P was increased in a time-dependent manner. This increase was also inhibited by P, and the mRNA level in the presence of P was almost constant throughout the culture period. Our results demonstrated that P or T is a regulator of sex steroid receptors in ESC and that this regulation may influence the responsiveness to P of decidual change in ESC.

Hormonal regulation in the production of macrophage colony-stimulating factor and transforming growth factor-beta by human endometrial stromal cells in culture.

The effects of gonadal steroids on the secretion and gene expression of macrophage colony-stimulating factor (M-CSF) and on the secretion of transforming growth factor (TGF)-beta 1 and TGF-beta 2 by human endometrial stromal cells (ESCs) were examined by an in vitro system of ESC differentiation (decidualization). M-CSF production by ESCs was dose-dependently enhanced by the addition of progesterone or testosterone, while estradiol treatment had no effect. TGF-beta 2 secretion by ESCs was inhibited by progesterone, estradiol and testosterone treatment, and on the contrary, slight enhancement by estradiol was observed in TGF-beta 1 secretion. These findings indicate that human ESCs produce cytokines of M-CSF and TGF-beta s, which are important for the growth and differentiation of the peri-implantation embryo as well as local immune cells under direct control of gonadal steroidal actions, and suggest a novel network between endocrine and immune systems in the human endometrium.

Progesterone enhances macrophage colony-stimulating factor production in human endometrial stromal cells in vitro.

Increasing evidence suggests that macrophage colony-stimulating factor (M-CSF) is produced in the uterine endometrium and that it plays an important role in the reproductive process. In the present study, using an in vitro decidualization model and human endometrium, we investigated M-CSF messenger RNA (mRNA) expression in human endometrial stromal cells (ESC) by Northern blotting and in situ hybridization. The secreted M-CSF in the culture medium of ESC was measured by enzyme-linked immunosorbent assay. ESC were cultured in the presence of progesterone (P) or estrogen. After a 9-day culture with P, when in vitro decidualization was confirmed by the production of PRL, M-CSF mRNA and protein levels were 3.1 +/- 0.5- and 3.2 +/- 0.8-fold (mean +/- SEM) higher, respectively, than those in cultures without P (P < 0.01). The P-induced increase was dose dependent. On the other hand, estrogen did not increase M-CSF mRNA expression. M-CSF mRNA expression in the first trimester deciduae that expressed PRL mRNA was higher than that in the endometria. By in situ hybridization, ESC as well as epithelial cells were shown to express M-CSF both in vitro and in vivo. These findings indicate that human ESC (decidua cells) express M-CSF mRNA and suggest that they secrete M-CSF in a P-dependent manner during the process of decidualization.

Progesterone-dependent secretion of macrophage colony-stimulating factor by human endometrial stromal cells of nonpregnant uterus in culture.

Recent studies have suggested that macrophages colony-stimulating factor (M-CSF), a hematopoietic glycoprotein essential to the proliferation and differentiation of mononuclear phagocytes and their progenitor cells, is also involved in the reproductive process in mice and humans. In this study, we examined, by enzyme-linked immunosorbent assay, the supernatants of stromal cell-enriched fraction (SF) of human nonpregnant endometrium for the presence of M-CSF during culture with progesterone (P) or estrogen. The bioactivity of M-CSF was assessed in a colony-forming assay of murine bone marrow cells. In addition, the M-CSF level in the culture supernatant of SF further purified by subculture, of epithelial cell-enriched fraction purified from human endometrium, and of peripheral blood lymphocytes, including about 10% monocytes, was examined with or without P, because SF is contaminated by epithelial cells and macrophages, both of which are suggested to secrete M-CSF. During 2-week culture, the level of M-CSF in the supernatants of SF cultured with P was markedly higher than that of control culture and estrogen-treated culture on any day tested, except for the first 2 days. P had a dose-dependent effect on M-CSF production by SF. Estrogen also enhanced M-CSF production by SF, but did not show dose dependency. The SF culture supernatants showed a colony-forming activity that was completely blocked by neutralizing anti-M-CSF antibody. SF subcultured three times, which was confirmed to be of more than 99% purity, secreted M-CSF in a P-dependent manner. M-CSF was also detected in the culture supernatants of epithelial cell-enriched fraction and peripheral blood lymphocytes, but P-dependent M-CSF production was not shown in these cultures. These results suggest that human endometrial stromal cells themselves can secrete bioactive M-CSF in a P-dependent manner in vitro, indicating that the M-CSF reported to be present in human endometrium is secreted in part by stromal cells and may play a role in the regulation of endometrial function under P control.

Expression of leukemia inhibitory factor in human endometrium and placenta.

Leukemia inhibitory factor (LIF), a cytokine that induces macrophage differentiation in the murine M1 myeloid leukemia cell line, is essential for blastocyst implantation in mice. However, its expression and the role it plays in the human uterus are unknown. To clarify these issues, we examined LIF gene expression in the human uterus by Northern blot hybridization and by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Analysis of LIF mRNA showed two hybridization bands, with estimated mRNA sizes of about 4.0-kb pairs and 1.8-kb pairs. LIF mRNA was detected at high levels in endometrial tissue and decidua, but at low levels in the chorionic villus in first trimester and term placenta. In the secretory phase, the endometrial tissue showed higher LIF expression than in the proliferative phase (9.5-fold; p < 0.01). The endometrial tissues were separated into a stroma-enriched fraction (SF) and an epithelium-enriched fraction (EF), and the LIF mRNA levels in each fraction were examined by quantitative RT-PCR. These levels were higher in the EF than in the SF (3.3-fold; p < 0.05). These findings suggest that, in humans, LIF plays a role in uterine function during the menstrual cycle, as well as during pregnancy.

Androgens induce prolactin production by human endometrial stromal cells in vitro.

Although there is a significant quantity of androgens in the endometrium, the function of these hormones has not been clarified, except for being estrogen precursors. Human endometrial stromal cells (ESC) were cultured in the presence of testosterone (T) and 5 alpha-dihydrotestosterone. Following culture, prolactin (PRL), a biochemical marker of stromal cell differentiation (decidualization) which is produced by ESC, was examined. T induced PRL production in a time- and dose-dependent manner, as reported previously for progesterone (P) stimulation. In addition, 5 alpha-dihydrotestosterone, which cannot be converted to estrogens, similarly induced PRL production. T in combination with P enhanced PRL production in cultured ESC significantly more than either P or T stimulation alone. A specific androgen receptor blocker, flutamide, when added to cultures containing T, inhibited PRL production in a dose-dependent manner, but did not affect the production of PRL induced by P. These results indicate that in vitro PRL production by human ESC is induced not only by P, but also by androgens through specific receptors and further suggest that androgens play an important role in human endometrial differentiation.

[A case of advanced gastric cancer with liver metastasis completely responding to a combined immunochemotherapy with UFT, mitomycin C and lentinan].

A 74-year-old man with advanced gastric cancer of Borrmann type III and liver metastasis was treated by combined administration of UFT (400 mg/day, p. o.), Mitomycin C (14 mg/body/4w., i.v.) and Lentinan (2 mg/w., i.v.). Five and half months after the therapy, endoscopic examination and ultrasonography showed the primary and liver-metastatic lesions had completely disappeared. Ten months after the therapy, total gastrectomy and intraoperative liver wedge biopsy were performed and complete disappearance of cancer cells was histologically confirmed. The total dose of UFT, MMC and LNT administered until the operation was 76.4 g, 42 mg and 74 mg, respectively. However, the patient eventually died of the recurrence of liver metastases three years after the initial immunochemotherapy.

The expression and localization of mRNA for colony-stimulating factor (CSF)-1 in human term placenta.

A 4-kb mRNA for colony-stimulating factor 1 (CSF-1) was detected in normal human placenta at term by Northern blot analysis. In-situ hybridization revealed that the mRNA for CSF-1 was localized in the mesenchymal cells of the chorionic villous stroma, but not in the trophoblasts or capillary epithelial cells. Because there are significant numbers of tissue macrophages (Hofbauer cells) in the placental stroma and because the receptor for CSF-1 (the c-fms proto-oncogene product) is known to be expressed by trophoblasts, our results suggest that CSF-1 produced by placental stromal cells may act as a growth and survival factor for human placental macrophages and trophoblasts.

The origin of extragonadal teratoma: case report of an immature teratoma occurring in a prenatal brain.

We describe a massive congenital intracranial teratoma (MCIT), which had a normal chromosome banding pattern 46,XY karyotype and a normal diploid DNA histogram, and which produced a variety of carcinoembryonic antigens. The volume density of primitive neural components (primitive neural tubes, small undifferentiated neuroepithelial cells, immature glial fibers and pigment cell components without neurofibrillar differentiation) was estimated to be 45%. We discuss the histogenesis, pathobiology and cell cycle kinetics.