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To analyze how the synthesis and release of glycosylated PRL (G-PRL) is regulated, we transfected human PRL complementary deoxyribonucleic acid (cDNA) into three different cell lines consisting of GH3 cells that originated in rat pituitary tissue, Chinese hamster ovary cells, and COS-1 cells generated from monkey renal tissue. 35S-labeled PRLs produced by the cells were immunoprecipitated with anti-human PRL antiserum, and the ratios of G-PRL to total PRL were compared. PRLs of 23 kDa and 25 kDa were detected in the cell lysate and medium. The 25-kDa PRL was confirmed to be a glycosylated form by endoglycosidase treatments. The ratios of G-PRL/total PRL were 0.17-0.33, which were similar in lysates and media and among different cell lines. Pulse-chase experiments revealed that the autonomaous secretion rates of G-PRL and non-glycosylated PRL were almost identical. These results indicate that synthesis and secretion kinetics of human PRL may not be affected by its glycosylation in the cells transfected with PRL cDNA.

To examine the effects of an intermittent injection regimen of exogenous gonadotropin for controlled ovarian hyperstimulation on follicular development and on in vitro fertilization (IVF) outcome, 120 women who were candidates for IVF received intermittent injection (II) or consecutive injection (CI) regimens with a "short" protocol (SP) or a "long" protocol (LP) of gonadotropin releasing hormone agonist (GnRHa). Pure follicle stimulating hormone (pFSH) was injected to the women in the II groups on the first, second, and fifth days of the stimulation cycle and every other day thereafter. The women in the CI groups received a daily injection of pFSH. An additional 16 patients who were treated with both II-SP and CI-LP were also analyzed.Although the cancellation rate in the II-LP group was higher than those in the other groups, follicular development and IVF outcomes in the II-SP group were similar to those in the CI groups. The number of injections in the II-SP group was about half that in the CI groups.These results indicate that an intermittent pFSH injection regimen with a "short" protocol of GnRHa may be beneficial for patients in terms not only of being a less painful treatment but also causing less physical and mental stress than daily injections.

To assess the neonatal and maternal outcomes of pregnancy complicated by previable preterm premature rupture of membranes (PPROM).Retrospective study.Tertiary referral hospital. Sample. Forty-five women having aggressive intervention with antibiotics, amnioinfusion, cerclage and tocolysis.The hospital database between July 2001 and December 2009 was reviewed for women with singleton fetuses and PPROM before 23(+0) weeks of gestation. We analysed maternal and neonatal characteristics.Neonatal survival without major morbidity.Thirty-eight infants were delivered alive and seven were stillborn. Ten infants died in the neonatal intensive care unit and one in the labor ward. Twenty-seven live-born infants survived to discharge from hospital. The survival rate of pregnancies with aggressive management was 60% (27 of 45); that of live-born infants was 71.1% (27 of 38). The median gestational age at PPROM and at delivery were significantly lower in the non-surviving group than the surviving group. Thirty-seven women (82.2%) had an amniotic neutrophil elastase level0.15 μg/mL. Only four women (8.9%) developed clinical chorioamnionitis. Overall, 90.7% of the women showed histological evidence of chorioamnionitis. Eighty-three per cent of the surviving children had bronchopulmonary dysplasia. Nine infants had serious sequelae at a corrected age of one and a half years. Maternal complications were uncommon.An aggressive treatment protocol for women with previable PPROM resulted in a high neonatal survival rate. Neonatal survival was associated with higher gestational age at delivery and with more frequent use of antenatal corticosteroids. The prognosis is still bad in PPROM before 22(+0) weeks of gestation.

We found 9 proteases in human placenta. Our studies showed that these placental proteasesmetabolize vasoactive and immunomodulating peptides, possibly derived from the fetus, and protect the exchange of peptide hormones across the placenta in order to maintain feto-placental homeostasis. We clarified the pregnancy serum oxytocinase discovered by Fekete in 1930 and angiotensinase by Page in 1947, respectively. In addition we showed the pregnancy serum bradykininase. Changes in maternal serum protease activities were useful for monitoring of pre-elcampsia and predicting the onset of labor. In addition, the ratio of peak systolic over least diastolic pressure of uterine or umbilical artery assessed by the Doppler technique was closely correlated with the levels of maternal serum proteases in preeclampsia, which suggested that placental proteases might control uteroplacental circulation via the regulation of concentrations of vasoactive peptides in uteroplacental circulation. The degradation of immunomodulating peptides by placental protease also suggests the possible involvement of placental protease in the immunological aspect of pregnancy.

The aim of the present study by a retrospective chart review was to examine the recurrence rate after placental abruption. Between 1985 and 1998, 81 patients had a placental abruption. We had 2-year follow-up information about 31 patients and 27 of them had a total of 34 subsequent pregnancies. Recurrent placental abruption was observed in 6 pregnancies in 6 patients (6/27, 22.2%). Of the 6 recurrent placental abruptions, the gestational age was 1-3 weeks earlier than that of previous abruption in 4 patients. One patient delivered a healthy baby after her first abruption and then experienced a second abruption. We conclude that careful management is needed after 30 weeks in pregnant women with a previous history of placental abruption.

To evaluate the five-tier classification of fetal heart rate (FHR) tracings recently proposed by Japan Society of Obstetricians and Gynecologists (JSOG).The database between January and June 2009 was reviewed for women in active labor at ?36 + 0 gestational weeks, with singleton fetuses in cephalic presentation and with umbilical artery blood gas analyses. Continuous FHR tracings were assessed according to the five-tier classification proposed by JSOG, where level 1 is normal, level 2 is subnormal and levels 3?5 are abnormal patterns.A total of 341 parturient women were eligible for this study protocol. The median (range) of the levels in the first and the second stage of labor were 1 (1-4) and 2 (1-4), respectively (p0.001). Both pH and base excess of umbilical artery decreased with higher levels of FHR tracings interpretation (p0.01). Interventions resulting in delivery were more necessary in the first stage of labor than in the second stage of labor in cases of levels 3 and more.Five-tier system for FHR tracing interpretation proposed by JSOG intercorrelates with the fetal acid-base balance well. Categorization of FHR tracings by uniform diagnostic criteria will be useful to standardize therapeutic strategy by sharing common perception among obstetrical staff.

To clarify the expression of PLAP during the course of pregnancy, the amount of PLAP mRNA and its activity in normal placental villi were measured. Both PLAP and its mRNA were found in placentae of as early as 7 weeks of gestation, and they continued to increase throughout pregnancy. But they showed different patterns of increase. The amount of PLAP mRNA began to increase dramatically around 13th week and probably continued to increase gradually until term. PLAP activity per gram of villi showed a gradual increase from around 13th week and a marked increase was observed after about 20th week. PLAP levels in sera from pregnant women were also measured, and they showed a pattern of increase imilar to that of PLAP activity per gram of villi. The continuous increase in the expression of PLAP throughout pregnancy suggests that PLAP may play a role in feto-maternal metabolism and placental differentiation.

Although paraaortic lymphadenectomy is one of the standard treatments for gynecological cancer in Japan, it is very invasive so one must examine its safety for patients. Paraaortic lymphadenectomy was performed in 215. Two hundred and fifteen gynecologic cancer patients at our hospital between January 1991 and August 2005. We evaluated operation time, estimated blood loss and the incidence of operative injury, wound complication, and postoperative ileus. It was revealed that the mean operation time was 364 minutes and the estimated blood loss was increased at the operation around the vena cava or renal vein. After we adopted Kocher's technique, the mean blood loss was decreased. The incidence of postoperative ileus was 13.3%, but almost all of the patients were cured within seven days without surgical treatment. The incidence of wound complication was within 10%.

To test the hypothesis that counting heart rate is dispensable and other components of the Apgar score are satisfactory to evaluate the physical condition of infants shortly after birth in routine deliveries.The database of the Japanese Red Cross Nagoya First Hospital was reviewed for newborn infants whose Apgar scores were marked by trained neonatologists attending delivery.The scores of respiratory effort, muscle tone, and reflex irritability increased parallel to Apgar scores. Heart rate gained higher marks even in lower Apgar scores, whereas color demonstrated lower marks even in higher Apgar scores. Correlation coefficients were higher among respiratory effort, muscle tone, and reflex irritability. In contrast, both heart rate and color exhibited lower correlation coefficients against other components and total Apgar scores. High correlation coefficients were shown between the sum of the four components other than heart rate and the total score.Heart rate and color play more independent roles in the Apgar score. Heart rate seems to be dispensable and the other components of the Apgar score are satisfactory to evaluate the physical condition of infants shortly after birth, and hence, counting heart rate may be omitted in routine deliveries when a newborn infant is apparently well.

We report a rare case of fetal intraventricular bleeding possibly due to maternal vitamin K deficiency. A 20-year-old woman was admitted to our hospital due to impending premature delivery and loss of dietary intake at 28 weeks of gestation. Her blood examination showed metabolic alkalosis, prolonged prothrombin time, and extremely high level of plasma des-gamma-carboxyprothrombin (protein induced by vitamin K absence, PIVKA-II). Intraventricular hemorrhage was demonstrated by ultrasonography 6 days after admission. She delivered a 2,288-gram girl infant at 40 weeks of gestation. Cranial computerized tomography and magnetic resonance images obtained postnatally demonstrated a reduced cerebral parenchyma adjacent to the interior side of the right lateral ventricle and the deficit of left cerebellum. The infant’s head control was insufficient and central impaired hearing was noted at 6 months of life.

Chorioamnionitis is considered to be one of the main causes of preterm labor and has been associated with an adverse perinatal outcome in preterm infants. The controversy about the benefits/risks of delaying labor is a critical issue concerning the management of chorioamnionitis.The database between July 2001 and March 2006 was reviewed for women with singleton pregnancies between 22 and 28 weeks of gestation and with chorioamnionitis diagnosed on admission by amniotic fluid neutrophil elastase level. Women were classified according to the severity of chorioamnionitis (group A, amniotic fluid neutrophil elastase level of 0.15-1 microg/ml; B, 1-10 microg/ml; and C,or = 10 microg/ml). During expectant management, serum C-reactive protein levels monitored the remission and aggravation of chorioamnionitis. Following deliveries, placentas were examined for histologic chorioamnionitis.One hundred women were enrolled (group A, 38; B, 34; C, 28). The latency period until delivery was significantly longer in group A than in groups B and C. C-reactive protein levels just before delivery were higher than those on admission in 61% of the overall cases. Histologic chorioamnionitis and funisitis were manifested in 90.4% and 65.5%, respectively. Intrauterine fetal demise (4 cases) and neonatal and postneonatal deaths during admission (10 cases) were observed. Bronchopulmonary dysplasia was the most common major morbidity noted in groups B and C.Chorioamnionitis could be controlled but is hard to cure. Higher levels of amniotic fluid neutrophil elastase are associated with a shorter interval from admission to delivery in women with subclinical chorioamnionitis.

A 32-year-old woman presented for evaluation of primary infertility. Because she had a history of iodine-induced hypothyroidism, conventional hysterosalpingography was contraindicated. Tubal patency was assessed by magnetic resonance imaging (MRI) after infusion of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). Visualization of the contrast medium in the peritoneal cavity revealed tubal patency. Our case indicates that MRI with gadolinium-diethylenetriamine pentaacetic acid is a safe, simple, and easy way to confirm that at least one tube is patent when a patient is at risk for hysterosalpingography. To our knowledge, this is the first report that tubal patency was diagnosed on MRI.

Neoadjuvant chemotherapy (NAC) with intra-arterial infusion was performed in the treatment for 53 patients with advanced cervical squamous cell carcinoma. After NAC with intra-arterial infusion of the anticancer agents including cisplatin via internal iliac artery or uterine artery, 42 patients received radical hysterectomy. The response to therapy was observed in 45 of all patients (84.9%) clinically, and 36 of 42 patients (85.7%) pathologically. Cancer cells disappeared in 11.9% of patients with cervical invasion, 69.2% with vaginal wall invasion and 39.4% with parametrium invasion after NAG. Five-year survival rates were 100% in stage I, 71.5% in stage II, 52.2% in stage II and 0% in stage IV. The group of patients without cancer in the parametrium after NAC showed a significantly better 5-year survival rate than the group with residual cancer in the parametrium. According to the results, the elimination of cancer invasion to the parametrium by NAC is thought to be important for improvement of the prognosis in advanced cervical cancer.

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. hCGbeta is unique among beta-subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of alpha-subunit and CTP, i.e. a variant with CTP at the N-terminal region (Calpha) and another analog with CTP at the C-terminus (alphaC) of the alpha-subunit. To address whether O-glycosylation is influenced by the heterodimer formation, Calpha and alphaC were expressed alone or with FSHbeta-subunit in Chinese hamster ovary cells. The O-linked glycosylation was assessed by continuous labeling with [(35)S]methionine/cysteine, immunoprecipitation with anti-alpha or anti-FSH serum, serial digestion with endoglycosidase-F and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-F was greater in Calpha than that in alphaC after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-F, the dimeric alphaC migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of Calpha was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s).

The authors present a case report of primary adenocarcinoma of the vulva. An ulcerated tumor on the left side of the vulva in a 59-year-old woman was presented. The tumor was excised, and histological examination revealed both papillary and tubulocystic patterns of adenocarcinoma. In some areas, the tumor was composed of cells with a clear cytoplasm. Lymph nodes showed positive in bilateral superficial inguinal nodes but negative in pelvic nodes, showing FIGO stage IVa. Following the operation, both chemotherapy and irradiation therapy were undergone to her. She has been without evidence of disease for more than 5 years following the completion of the treatment. Because primary adenocarcinoma of the vulva is very rare, therapeutic modality to date is individually selected.

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA and WGA increases with blastocyst formation and earlier expression is observed with WGA. The present study clearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.

The aim of the present study was to elucidate functions of the interleukin (IL)-8 at ovulation and luteinization in vivo. To compare the morphological differences between human chorionic gonadotropin (hCG) and IL-8 stimulation, scanning electron microscopy was employed to study rat ovarian vascular corrosion casts. Follicular growth and increased capillary vessel densities around the follicles were seen in vascular corrosion casts after IL-8 injection, similar to the result of hCG administration. This result indicated that exogenous IL-8 could play a role in the neovascularization during follicular development as an angiogenetic factor. Many fenestrations were observed in the vascular endothelium by hCG administration. In contrast, no fenestrations were observed with IL-8 injection, indicating that IL-8 may not be sufficient to increase the vascular permeability directly. Although germinal vesicle breakdown (GVBD) occurred at rates of 82% after the hCG injection, only 20% GVBD was observed after the IL-8 injection. The present study indicated that IL-8 might have important effects on rat follicles at ovulation and luteinization via vascularization in a similar manner to hCG. However, IL-8 was not effective on vascular permeability and oocyte maturation, which were different from hCG. Thus, we can conclude that IL-8 can participate in follicular development in part and may play important roles in ovulation and luteinization as one of some mediators induced by endogenous luteinizing hormone.

Assisted reproductive technology is a widely accepted treatment for infertile women with endometriosis. The presence of an ovarian endometrial cyst reduces the quality of oocytes, while surgical resection of endometrioma may reduce the ovarian reserve for ovarian stimulation by exogenous gonadotropins. To determine what pretreatment should be performed for ovarian endometrial cyst before IVF-ET, we analyzed IVF outcomes with or without pretreatment in patients with endometrioma. Infertile women with endometrioma who underwent IVF-ET were divided into 3 groups, including patients who had received laparotomy or laparoscopy, patients for whom the endometrioma content had been aspirated and treated with or without alcohol fixation, and patients who did not undergo pretreatment. The number of retrieved oocytes, rate of mature oocytes, and fertilization rate were compared among groups. The results showed that pretreatment for endometrioma reduces the number of retrieved oocytes. Although oocyte quality as a rate of mature oocytes was not affected by the presence of an ovarian endometrial cyst, the fertilization rate was improved by cyst aspiration. We propose that surgical pretreatment is not necessary for ovarian endometrial cyst before IVF-ET, but cyst aspiration may be beneficial after several failed attempts of IVF.

A retrospective study regarding the relationship between serum hormonal levels and bone mineral density (BMD) was performed in 125 women with hormone replacement therapy (HRT). Serum estradiol (E(2)), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and BMD were evaluated before and at 12 and 24 months of HRT.There was a significant increase in E(2) and decrease in FSH at both 12 (E(2), 39.3+/-76.6 pg/ml to 71.0+/-67.9 pg/ml; FSH, 67.9+/-36.3 mIU/ml to 47.9+/-29.0 mIU/ml) and 24 months (E(2), 68.3+/-54.5 pg/ml; FSH, 45.3+/-24.4 mIU/ml). LH level was high at baseline (26.5+/-16.1 mIU/ml) and decreased at 12 months (22.9+/-14.0 mIU/ml). On the contrary, it increased from 12 to 24 months (27.4+/-14.9 mIU/ml). In the lumbar spine BMD, a significant rise was seen only in the first 12 months (0.933+/-0.157 g/cm(2) to 0.938+/-0.152 g/cm(2)). When percentage change was analyzed, a significant positive correlation was found between E(2) and BMD and a negative correlation between gonadotropin levels and BMD.These data demonstrate that serum gonadotropin levels, especially FSH, are a good marker to predict BMD in women with HRT.

Objective: To describe a patient with congenital cervical atresia who became pregnant through IVF and thawed transmyometrial ET and then experienced a missed abortion. Design: Case report. Setting: University hospital. Patient(s): A patient with congenital cervical atresia who underwent reconstructive surgery at 23 years of age and underwent IVF twice at 28 and 30 years of age. Intervention(s): Abortion management. Main Outcome Measure(s): Medical follow-up of IVF-ET, resultant pregnancy, and abortion. Result(s): After the second cycle of IVF with frozen-thawed transmyometrial ET, the patient became pregnant but then experienced a missed abortion. Serum β-hCG levels decreased, the two gestational sacs disappeared, and genital bleeding without signs of infection occurred 14 weeks after diagnosis of the abortion. The abortion was managed conservatively. Conclusion(s): When assisted reproductive techniques are used in patients with congenital cervical atresia, the risks (including those relating to the management of an abortion) should be explained in detail to the couple and sufficient informed consent should be obtained before starting IVF-ET procedures.

The present study was undertaken to investigate whether hCG beta-core fragment (hCGbeta cf) was directly produced by cancer cells. Fifteen cell lines, including four choriocarcinoma and five ovarian cancer cell lines, were tested, and immunoreactivity of hCGbeta cf was present in the culture media of five of the cell lines. It was also present in the culture media of Chinese hamster ovary (CHO) cells transfected with hCGbeta gene. In addition to hCGbeta cf, gel chromatography and Western blot analysis of the culture media showed the presence of an hCGbeta cf immunoreactive material with a molecular weight of approximately 40 kDa. In an in vivo study, hCGbeta cf immunoreactivity was detected in the sera of the mice transplanted with NaUCC-3 choriocarcinoma cells, although the ratios of hCGbeta cf/hCG and hCGbeta cf/free hCGbeta were lower than those in the culture medium. Incubation experiments of purified hCGbeta cf in the serum showed no substantial decrease in its values, ruling out the possibility that formation of a macromolecule with serum components may mask hCGbeta cf immunoreactivity in the serum. Taken together, these results indicate that hCGbeta cf immunoreactive materials are directly produced by cancer cells and hCGbeta cf is not a urinary metabolite of hCG or hCGbeta alone. Also, reduced levels of hCGbeta cf in the serum compared with that of intact hCG or free hCGbeta are likely due to its short half-life.

The authors present a case report of a 62-year-old woman who experienced irregular genital bleeding. Although the serum hCG level was extremely low, transvaginal ultrasonography revealed vesicle pattern in the uterine cavity, suggesting trophoblastic disease. Hysterectomy was performed and histological diagnosis was adenomyomatous polyp. To our knowledge, this is the first report that the adnomyomatous polyp can demonstrate the vesicle pattern on ultrasonography.

CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. Despite the considerable homology between LHbeta and CGbeta, we previously demonstrated that, when expressed in GH(3) cells, the secreted form of LHbeta showed mispaired disulfide-linked aggregation in addition to monomer, whereas no aggregation was observed in CGbeta. To determine the domains which are associated with the LHbeta-aggregation and which prevent CGbeta-aggregation, mutant beta-subunits in glycosylation and carboxy-terminus were expressed in GH(3) cells, and the occurrence of aggregation was assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-hCGbeta serum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. No aggregation was seen when N-linked oligosaccharides were attached to the Asn(13) of LHbeta. Removal of the carbohydrate unit at the Asn(13) of CGbeta caused aggregation, although the amount was less than 10% of monomer. The carboxy-terminal regions of neither LHbeta nor CGbeta were associated with their aggregation. Both CGbeta wild-type (WT) and CGbeta lacking N-glycosylation at Asn(13) (CGbeta-N13) showed aggregates in lysate. However, in contrast to CGbeta-N13, CGbetaWT revealed no aggregation in medium. These results indicate that the backbone structure consisting of 114 amino acids and N-linked glycosylation at Asn(30) is involved in the aggregation of LHbeta. Moreover, N-glycosylation at Asn(13) does not prevent such aggregation, but instead plays an important role in correct folding for both LHbeta- and CGbeta-subunits to be secreted as monomer.

Asialoglycoprotein (ASGP) receptor, which exists in hepatic parenchymal cells, plays important roles in the clearance of desialylated glycoproteins from the circulation. To study the expression of ASGP receptor in human fetal liver, specimens were obtained after therapeutic/spontaneous abortion or at autopsy with informed consent. Reverse transcription polymerase chain reaction and immunohistochemical staining showed that ASGP receptor was present as early as 15 weeks of gestation in human fetal liver. In liver at 15 weeks-gestation it was stained uniformly in cytoplasm as seen in HepG2 cells. As gestation proceed the ASGP receptor was frequently observed on the cell surface and intracellular staining showed a spotted distribution. We conclude that the expression of ASGP receptor is an early event in the developmental process of fetal liver. Furthermore, the distribution of ASGP receptor changes as the liver develops.

We reviewed 1246 vaginal hysterectomies performed at Handa City Hospital between January 1984 and December 1996. We divided the patients into 2 groups: those with leiomyomas (n = 893) and those with adenomyosis (n = 353). There was no difference in operative time and estimated blood loss between the 2 groups when analyzed by uterine weight. However, adenomyosis was associated with an increased risk of bladder injury.

The common alpha subunit of glycoprotein hormones contains five disulfide bonds. Based on the published crystal structure, the assignments are 7-31, 59-87, 10-60, 28-82, and 32-84; the last three comprise the cystine knot, a structure also seen in a variety of growth factors. Previously, we demonstrated that the efficiency of secretion and the ability to form heterodimers by alpha subunits bearing single cysteine residue mutants in the cystine knot were significantly reduced. These results suggested that the cystine knot is critical for the intracellular integrity of the subunit. To assess if the presence of the free thiol affected the secretion kinetics, we constructed paired cysteine mutants of each disulfide bond of the alpha subunit. The secretion rate for these monomers was comparable with wild type except for the alpha-10-60 mutant, which was 40% lower. The recovery of the alpha7-31 and alpha59-87 mutants was greater than 95%, whereas for the cystine knot mutants, it was 20-40%. Co-expression of the wild-type chorionic gonadotropin beta subunit with double cysteine mutants did not enhance the recovery of alpha mutants in the media. Moreover, compared with wild-type, the efficiency of heterodimer formation of the alpha10-60 or alpha32-84 mutants was less than 5%. Because subunit assembly is required for biological activity, studies on the role of these disulfide bonds in signal transduction were not possible. To bypass the assembly step, we exploited the single chain model, where the alpha and beta subunits are genetically fused. The recovery of secreted tethered gonadotropins bearing mutations in the cystine knot was increased significantly. Although dimer-specific monoclonal antibodies discriminated the conformation of single chain alpha10-60 and alpha32-84 mutants from the native heterodimer, these mutants were nevertheless biologically active. Thus, individual bonds of cystine knot are important for secretion and heterodimer formation but not for in vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a prerequisite for receptor binding or signal transduction.

Apoptosis has recently been proposed to represent programmed cell death. Several investigations have revealed that uterine endometrium in mammals can be regulated by apoptosis. However, apoptosis may be less likely to occur in human eutopic endometrium. On the other hand, immunoreactivity of Bcl-2 that may serve a regulatory function in protecting from undergoing apoptosis was observed in uterine endometrial glandular cells in the proliferative phase through to the early secretory phase. In endometriotic tissues, although apoptosis was detected in all the samples from ovarian endometriosis, it was indicated in only a few tissues from adenomyosis. Bcl-2 was negative in almost all samples from ovarian endometriosis, whereas it was positive in all adenomyotic tissues from cases in the proliferative phase, but in none of tissues from cases in the secretory phase. Thus, ovarian endometriosis can be discriminated from adenomyosis based on the existence of apoptosis though both represent a type of ectopic endometrial tissue, and the pathogenesis or condition of the cells may be different between ovarian endometriosis and adenomyosis.

Purpose: To analyze the effects of glucocorticoid treatment for patients with abnormal autoimmunity on IVF-ET outcomes, low-dose predonisolone or dexamethasone was administered in 51 IVF-ET cycles of 41 patients with positive antinuclear antibody (ANA), anti-DNA antibody, and/or lupus anticoagulant (LAC). Rates of clinical pregnancy and implantation in these patients were compared with those in 48 cycles without corticosteroid therapy. Autoantibody-negative patients were also treated with IVF-ET combined with (29 cycles) or without (57 cycles) glucocorticoid administration, and the pregnancy and implantation rates were investigated. Results: Without glucocorticoid treatment, the pregnancy rate per cycle and implantation rate per embryo in antibody-positive patients were 10.4 and 3.8%, respectively. Significant increases in pregnancy (35.3%) and implantation (13.2%) rates were observed with corticosteroid treatment. In antibody-negative patients, the rates of pregnancy and implantation showed no significant differences with versus without the glucocorticoid administration. Conclusions: Our results indicate that because autoimmune abnormalities may be at least one cause of implantation failure following IVF-ET, the combined use of low-dose corticosteroid can be effective for autoantibody-positive women.

Objective To evaluate the clinical significance of the LH consisting of a mutant β -subunit (Trp 8 to Arg 8 and Ile 15 to Thr 15 ). Design Clinical and biochemical studies. Setting Fertility center at the University Hospital and its research laboratory. Patients Fifty-one patients with menstrual disorders and three homozygote cases and two heterozygote cases of the mutant LH who were reported previously. Interventions Nucleotide mutations of the LH β gene in patients with menstrual disorders were screened using techniques of the polymerase chain reaction and restriction fragment length polymorphism. Immunologic and biologic activities of the mutant LH and endocrinologic profiles in the affected women were evaluated. Main Outcome Measures Serum LH levels measured with different immunoassay kits; serum FSH and LH on the GnRH test; serum thyroid-stimulating hormone, PRL, T, and androstenedione; ultrasound examination of the ovaries; clinical hyperandrogenic symptoms; and biologic activity of LH. Results Two cases of homozygotes and four of heterozygotes affected by the LH β gene mutations were discovered in the current study through screening of patients with menstrual disorders. Serum LH levels in the homozygote cases were undetectable using a LH immunoassay kit, whereas levels in the heterozygote cases showed reduced detectability with the kit. However, the ratio of the mutant LH values in the bioassay to those in the immunoassay was higher in the homozygote group than that in the control subjects. Response patterns of serum gonadotropins to GnRH in the homozygote were similar to those in patients with polycystic ovary syndrome. Conclusion The mutations of LH β -subunit might be related to menstrual disorder in some patients.

CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunit. Processing of the N-linked oligosaccharide of the glycoprotein family is both tissue and dimer specific. LH, TSH, and free alpha synthesized in pituitary bear oligosaccharide terminating with sulfate (SO4) and N-acetylgalactosamine (GalNAc), whereas the termination of oligosaccharide in CG synthesized in placenta and FSH is sialic acid and galactose (Gal). Using site-directed mutagenesis and gene transfer, we studied the role of the Pro-Leu-Arg motif, which has been shown to be a recognition marker of glycoprotein hormone-specific GalNAc transferase, in sulfation of N-linked oligosaccharide in alpha-subunit. The wild-type or mutated alpha gene was transfected into GH3 cells. Our data revealed that substitution of the Pro-Leu-Arg motif by Ala-Leu-Ala did not affect the sulfation of N-linked oligosaccharide, but generated the attachment of O-linked oligosaccharide. alpha-Subunit containing either of the two N-linked glycosylations is also sulfated. We conclude that in GH3 cells, the Pro-Leu-Arg motif plays no role in the sulfation of oligosaccharide in alpha-subunit, and both N-glycosylations are terminated with SO4.

In the past, it has been difficult for female patients with primary hypopituitarism to achieve successful pregnancies because these women have poor physical constitutions, induced by growth hormone (GH) deficiency and/or anovulation caused by a lack of gonadotropin secretions. Although some women with hypopituitarism have recently been able to conceive through replacement therapies using human GH and gonadotropins, some negative gonadotropin-stimulation effects, including multiple pregnancies and ovarian hyperstimulation syndrome (OHSS), occur. We report the case of a patient with primary hypopituitarism who achieved pregnancy and delivery through IVF and ET.

The effects of estradiol and progesterone on the binding of rat placental angiotensin II receptors were examined. Sex steroid (progesterone estradiol plus progesterone) decreased the total number of rat placental angiotensin II receptors, while sex steroid (estradiol plus progesterone) increased angiotensin levels. Our present results suggest that sex steroids may play an important role in the control of the number of the angiotensin binding sites during pregnancy.

Pregnant Wistar rats were injected with bestatin, a specific inhibitor of aminopeptidase M. Placental aminopeptidase M activity was inhibited by injection of bestatin, and fetal body weight was statistically lower than that in the saline-injected or control group. Our present data suggest that placental aminopeptidase M plays an important role in fetal growth.

The degradation of opioid peptides by human placental aminopeptidase M was studied by measuring liberated amino acids by high performance liquid chromatography. The purified placental aminopeptidase M actively degraded Met-enkephalin (Met-Enk) and Leu-enkephalin (Leu-Enk) completely into constituent 5 amino acids. The activities as a function of tyrosine liberation were observed for Met-Enk followed by Leu-Enk, beta-neoendorphin, dynorphin, and beta-endorphin.

Mammalian cells require cholesterol as a structural component of plasma membranes. It is also required for placental steroid synthesis. De novo synthesis of cholesterol is limited in human placenta and cholesterol is obtained mainly from plasma low density lipoprotein (LDL). Cholesterol delivery from LDL is mediated by receptor-mediated uptake and the receptor amount is the most important factor for cellular delivery. Thus, the regulation of receptor synthesis is important for placental development and function. Since the regulation of LDL receptor gene expression has not been studied in human placenta, LDL receptor mRNA was measured in placentae of 5-40 weeks of gestation by hybridization of RNA with 32P-labeled cDNA for human LDL receptor. Two mRNA species for LDL receptor were demonstrated by Northern blot analysis. The longer mRNA [5.3 kilobases (kb)] was much more abundant than the shorter mRNA (3.7 kb). The amount of 5.3 kb mRNA was highest early in gestation and decreased during pregnancy. However, the amount of 3.7 kb mRNA did not change appreciably during gestation. Dot blot analysis of 26 placental mRNAs obtained from various stages of gestation revealed a negative correlation between LDL receptor mRNA and gestation (r = -0.76, P less than 0.001). Considering the rapid growth of the trophoblast during gestation, especially in the first and the second trimester, increased expression of the LDL receptor gene and subsequent translation are expected for efficient cholesterol uptake to provide a sufficient substrate for cell growth. Possible mechanisms for the appearance of two mRNA species for LDL receptor are also discussed.