Comparison of human menopausal gonadotropin stimulation with and without clomiphene for in-vitro fertilisation in poor-responders.

This study aimed to determine the effectiveness of human menopausal gonadotropin (hMG) with and without clomiphene citrate (CC) for ovarian stimulation and oocyte retrieval in poor-responders. A total of 66 cycles in 13 patients met the inclusion criteria of 20 cycles of hMG stimulation and 46 cycles of CC + hMG stimulation. Mean total hMG dose per patient was significantly lower during the stimulation cycle with CC + hMG than with hMG alone. Mean oestradiol level at oocyte retrieval and mean number of oocytes retrieved per patient showed no significant differences between the two cycles. Four of the 13 patients achieved pregnancy with the CC + hMG cycles. The absence of a significant difference in oestradiol level at oocyte retrieval or in the number of oocytes retrieved between the two cycles, despite a significantly lower total hMG dose during the CC + hMG stimulation cycles, suggests a greater benefit of CC + hMG stimulation for poor-responders.

Role of pituitary adenylate cyclase-activating polypeptide in modulating hypothalamus-pituitary neuroendocrine functions in mouse cell models.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally identified as a hypothalamic activator of cyclic adenosine monophosphate production in pituitary cells. PACAP and its receptor are expressed not only in the central nervous system, but also in peripheral organs, and function to stimulate pituitary hormone synthesis and secretion as both a hypothalamic-pituitary-releasing factor and an autocrine-paracrine factor within the pituitary. PACAP stimulates the expression of the gonadotrophin α, luteinising hormone (LH) ß and follicle-stimulating hormone (FSH) ß subunits, as well as the gonadotrophin-releasing hormone (GnRH) receptor and its own PACAP type I receptor (PAC1R) in gonadotrophin-secreting pituitary cells. In turn, GnRH, which is known to be a crucial component of gonadotrophin secretion, stimulates the expression of PACAP and PAC1R in gonadotrophs. In addition, PAC1R and PACAP modulate the functions of GnRH-producing neurones in the hypothalamus. This review summarises the current understanding of the possible roles of PACAP and PAC1R in modulating hypothalamus and pituitary neuroendocrine cells in the mouse models.

Evaluation of acrosome reaction and viability of human sperm with two fluorescent dyes.

OBJECTIVE: To evaluate the usefulness of the Hoechst 33258/FITC-Pisum sativum agglutinin (FITC-PSA) staining for simultaneous assessment of viability and acrosome reaction rate (%AR) of human sperm. MATERIAL AND METHODS: Fresh sperm was collected 13 fertile donors provided fresh semen. We used motile sperm selected by the "swim-up" procedure using modified HTF. Hoechst 33258 was added and co-incubated with sperm for 10 min. Samples were washed free of unbound stain and the sperm were mounted as smears on glass slides. After drying, sperms were incubated with FITC-PSA for 30 min. Sperm were examined by fluorescence microscopy. Also, FITC-Concanavalin A (FITC-ConA) staining and vital staining with yellowish eosin were performed simultaneously. The correlation of viability and %AR were analyzed. RESULTS: Four different staining patterns were observed and clearly distinguished as follows: a) Viable acrosome-reacted sperm, b) Viable acrosome-intact sperm, c) dead acrosome-reacted sperm, d) dead acrosome-intact sperm. There was significant correlation between the results obtained by Hoechst 33258 and vital staining methods in viability of human sperm (r = 0.927, P < 0.001). There was significant correlation between the two methods (FITC-PSA and FITC-ConA) in %AR of human sperm (r = 0.92, P < 0.001). CONCLUSION: Viability and acrosome status of a human sperm can be easily assessed simultaneously by Hoechst 33258/FITC-PSA staining method. This combination method is considered useful to evaluate sperm function.

Successful pregnancy in a woman with ovarian failure associated with mutation in the beta-subunit of luteinizing hormone.

BACKGROUND: We report a successful pregnancy in a woman with severe ovarian dysfunction and infertility associated with a variant beta-subunit of luteinizing hormone (LH). METHOD/OUTCOME: A 35-year-old woman consulted our unit for infertility. Laparoscopy and ultrasonography showed obstruction of the right tube and ovulation from the right ovary only. Human menopausal gonadotrophin (hMG) therapy was used for six subsequent cycles, but did not result in conception. Subsequently, marked elevation of follicle-stimulating hormone (FSH) and testosterone, together with polycystic ovary (PCO) were noted. The patient failed to respond to ovarian stimulation by hMG. Severe ovarian dysfunction such as premature ovarian failure (POF) was strongly suspected. Sequence analysis of the LH beta-subunit gene indicated heterozygosity for point mutations Trp(8) to Arg(8) and Ile(15) to Thr(15) in the coding sequence. LH hypersecretion resembling that seen in PCO syndrome was observed. Induction of ovulation by hMG was successful in the first cycle in which the basal LH and FSH were well controlled with gonadotrophin-releasing hormone analog following estrogen-progesterone replacement. She conceived and delivered a healthy male infant at term. CONCLUSION: Clinicians should be clinically aware of patients with immunologically anomalous LH variant who might be at risk of developing ovarian failure within a relatively short time span. Pertinent treatment should be applied without delay in such cases.

Involvement of mitogen-activated protein kinase in cyclic adenosine 3',5'-monophosphate-induced hormone gene expression in rat pituitary GH(3) cells.

We examined whether mitogen-activated protein (MAP) kinase was activated by stimulation of the cAMP pathway and whether MAP kinase activation was involved in synthesis of PRL and GH in GH(3) cells. Treatment of the cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP (CPT-cAMP), activated MAP kinase and increased PRL at both the protein and messenger RNA levels. The protein and messenger RNA of GH were decreased by the treatment. We constructed the luciferase reporter genes after the promoters of PRL and GH and found the activation of both promoters by the CPT-cAMP treatment. We confirmed that overexpression of the catalytic subunit of cAMP-dependent protein kinase had essentially the same effects on MAP kinase activation and synthesis of PRL and GH as the CPT-cAMP treatment. Furthermore, treatment of the cells with pituitary adenylate cyclase-activating polypeptide 27 activated MAP kinase. The activation of PRL promoter by CPT-cAMP and pituitary adenylate cyclase-activating polypeptide 27 was abolished by pretreatment with PD098059 and H89. Although the increase in PRL and GH secretion by CPT-cAMP was inhibited by H89, PD098059 had no effect on secretion. These results suggest that cAMP-induced MAP kinase activation is essential for PRL gene expression, but not for secretion of PRL and GH.

Benign or malignant ovarian neoplasms and ovarian endometriomas.

STUDY OBJECTIVE: To investigate clinical features and biologic behavior of ovarian cancer that might be closely related to endometrioma and/or endometriosis. DESIGN: Retrospective study (Canadian Task Force classification II-2). SETTING: University hospital. PATIENTS: All 324 women who were operated for endometriomas and/or ovarian tumors 5 cm or greater in diameter between January 1988 and December 1997. INTERVENTION: One hundred twelve women underwent laparoscopic surgery and 212 had laparotomy. MEASUREMENTS AND MAIN RESULTS: All tissues were evaluated histologically. Clinical examinations including ultrasound and serum tumor makers were performed in all patients preoperatively. No malignancies were found at laparoscopic surgery (76 endometriomas, 36 ovarian tumors). The frequency of endometriosis in benign, borderline malignant, and malignant tumors was 9.7%, 12.5%, and 11.4%, respectively. Endometriosis was present most frequently (40%) in women with endometrioid adenocarcinoma. It was present in 81 patients with endometriomas and 25 with ovarian neoplasms. Of these, nine women (8.5%) had malignant tumors, including borderline malignancy. Among patients with malignant tumors, those without endometriosis were significantly older (mean +/- SD age 54.9 +/- 16.2 yrs) than those with endometriosis elsewhere in the pelvis (45.9 +/- 8.9 yrs). CONCLUSION: Endometriosis may be closely related to ovarian tumors such as endometrioid adenocarcinoma. Surgeons should be aware of this possibility, and candidates for laparoscopic surgery should be carefully selected based on preoperative evaluations.

A case of a pregnant woman with luteal insufficiency and a mutation in the beta-subunit of luteinizing hormone.

We report a case of a pregnant woman with luteal insufficiency and infertility associated with a variant luteinizing hormone (LH) beta-subunit and normal follicle-stimulating hormone (FSH) concentration. A 29-year-old woman presented to our hospital with infertility. Given the presence of low mid-luteal serum progesterone concentration and out-of-phase endometrial biopsy, luteal insufficiency was suspected. Several forms of treatment (clomiphene citrate therapy and human menopausal gonadotrophin therapy) were administered, but did not improve the patient's luteal insufficiency. However, administration of hCG during the luteal phase could distinctly improve the luteal insufficiency. She became pregnant after luteal support with progesterone. Sequence analysis of the patient's LH beta-subunit gene indicated heterozygosity for the point mutations Trp8 to Arg8 and Ile15 to Thr15 in the coding sequence. LH hypersecretion resembling that seen in polycystic ovary syndrome was observed. Serum concentrations of variant LH showed a more rapid increase in response to gonadotrophin-releasing hormone and reached a higher apparent value than did those of normal LH. Therefore, abnormal bioactivity and mistimed secretion of variant LH at mid-cycle may have a deleterious effect on the completion of oocyte maturation, ovulation, and subsequent corpus luteum function.

Pituitary response to luteinizing hormone-releasing hormone in women with variant luteinizing hormone.

OBJECTIVE: To assess the LH response of the pituitary gland to GnRH stimulation in healthy women with a mutant beta-subunit (Trp8 to Arg8 and Ile15 to Thr15). DESIGN: Clinical study. PATIENTS: We studied 40 healthy non-pregnant Japanese women of known zygosity for the LH beta-subunit gene (3 homozygotes for the mutant gene, 17 heterozygotes, and 20 homozygotes for the wild type). All women had normal ovulatory cycles. MEASUREMENTS: Serum LH status was determined by comparing LH immunoassays results using a monoclonal antibody recognizing only wild-type LH with those from a polyclonal antibody assay recognizing both variant and wild-type LH. The ratio of monoclonal to polyclonal immunoassay results determined the serum LH status. LH secretion in response to a GnRH stimulation test was measured. RESULTS: All women with the wild-type LH showed a normal response of LH to GnRH according to both assays. Over the time course of the response, the ratios in women with wild-type LH showed no remarkable changes. The response curves in women heterozygous for the mutant peaked 15-30min after GnRH injection; their response patterns included a statistically significant decrease in the rates of response at 15min after injection. CONCLUSIONS: There are the differences in circulatory kinetics between the two LH forms and in regulation of the two types of LHbeta genes. The maximal response of the variant LH to pituitary stimulation with GnRH appears to be greater than that of wild-type LH.

Expression of cyclooxygenase 2 by prostaglandin E(2) in human endometrial adenocarcinoma cell line HEC-1B.

The regulation of expression of cyclooxygenase 2 (COX-2) was investigated by treatment with PGE(2) in human endometrial adenocarcinoma cell line HEC-1B. One microM PGE(2) could stimulate the expression of COX-2 approximately twofold in this cell line. The same concentration of PGE(2) also stimulated activation of mitogen-activated protein kinase (MAP kinase) and protein kinase B (PKB). PGE(2)-induced MAP kinase activation was sensitive to a MAP kinase kinase (MEK) inhibitor, PD098059, and a protein kinase A inhibitor, H-89. PD098059 and H-89 also partially inhibited the expression of COX-2 stimulated by PGE(2). PGE(2) could stimulate the activation of PKB, which was sensitive to phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. Whereas wortmannin alone partially inhibited the expression of COX-2, a combination of wortmannin and PD098059 totally inhibited PGE(2)-mediated COX-2 expression. These results suggest that MAP kinase and PI3K pathways are stimulated with PGE(2), and that both of these pathways are involved in the expression of COX-2. In addition, they also suggest that protein kinase A remains upstream of PGE(2)-induced activation of MAP kinase in HEC-1B cells.

Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells.

Bromocriptine, a dopamine D(2) receptor agonist, is a therapeutic agent for patients with prolactinoma and hyperprolactinemia. In this study we demonstrated that bromocriptine induced activation of p38 mitogen-activated protein (MAP) kinase, with concomitant induction of apoptosis in rat pituitary adenoma cell line GH3 cells. Treatment of GH3 cells for 48 h with bromocriptine increased the p38 MAP kinase activity up to 3- to 5-fold and simultaneously increased the number of apoptotic cells. Inclusion in the medium of SB212090 or SB203580, specific p38 MAP kinase inhibitors, completely abolished the bromocriptine-induced activation of p38 MAP kinase and significantly reduced the number of apoptotic cells. The bromocriptine-induced p38 MAP kinase activation was not prevented by S(-)-eticropride hydrochloride, a specific D(2) receptor antagonist. Treatment with either epidermal growth factor (EGF) or thyrotropin-releasing hormone (TRH), which stimulates p44/42 MAP kinase, rescued cells from the bromocriptine-induced apoptosis, with concomitant inhibition of the bromocriptine-induced p38 MAP kinase activation. These results suggest that bromocriptine induces apoptosis in association with p38 MAP kinase activation, and that the p44/42 MAP kinase signaling through EGF and TRH receptors has an opposing effect on p38 MAP kinase activation as well as on apoptosis induced with bromocriptine in GH3 cells.

Safety and efficacy of oestriol for symptoms of natural or surgically induced menopause.

To assess the safety and efficacy of oestriol in relieving post-menopausal symptoms 53 post-menopausal Japanese women with climacteric symptoms, 27 with natural menopause (group I) and 26 with surgically induced menopause (group II), received oral oestriol, 2 mg daily for 12 months. Clinical parameters including Kupperman index (KI) and the degree of satisfaction with symptomatic relief; serum concentrations of oestradiol, FSH and LH; serum lipids; blood pressure; bone mineral density, serum calcium (Ca), alkaline phosphatase (ALP), and urinary Ca were compared between the two groups. Oestriol improved KI in groups I and II by 49 and 80% respectively. Satisfaction with treatment was 85% in group I and 93% in group II. For both parameters, values were significantly different between groups I and II (P < 0.05 for both). Serum concentrations of oestradiol, FSH and LH changed in group I versus group II 6 months after initiation. A significant decrease in serum ALP and Ca/Cr was observed in group I at 6 months. Except for serum triglycerides, oestriol had no significant effect on lipids. Systolic and diastolic blood pressures were significantly decreased in group I at 3 months versus baseline. Slight vaginal bleeding occurred in 14.3% of group I. Histological evaluation of the endometrium in all women of group I and ultrasound assessment of the breasts following 12 months of oestriol treatment found normal results in all women. Therefore, oestriol appeared to be safe and effective in relieving symptoms of menopausal women. The beneficial biochemical effects of oestriol were marked in the natural menopause. Overall, oestriol may serve as a good choice for hormone replacement therapy to protect against other climacteric symptoms in post-menopausal women who do not need medication for osteoporosis or coronary artery disease.

Efficacy and safety of oral estriol for managing postmenopausal symptoms.

OBJECTIVE: to assess the therapeutic efficacy and safety of oral estriol for the treatment of climacteric symptoms in postmenopausal women. METHODS: 68 postmenopausal women with climacteric symptoms received oral estriol, 2 mg/day, daily for 12 months. We evaluated the degree of climacteric complaints with estriol therapy; serum levels of gonadotropins, estradiol (E2) and lipids; biochemical markers of bone metabolism; blood pressure; and side effects both at baseline and during treatment. Climacteric symptoms were assessed according to the menopausal index (MI), a version of the Kupperman index that had been modified for Japanese women. RESULTS: oral estriol therapy significantly reduced total MI scores. The greatest relief was noted for hot flushes, night sweats, and insomnia. Estriol treatment significantly lowered serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations but did not affect any of the other parameters (lipids, bone, liver and blood pressure) during the study period. Slightly vaginal bleeding occurred in 14.3% of those who underwent natural menopausal women. Histologic evaluation of the endometrium and ultrasound assessment of the breasts following 12 months of estriol treatment found normal results in all women. CONCLUSION: Estriol is a safe and effective alternative for relieving climacteric symptoms in postmenopausal Japanese women.

Estradiol-17beta measurement in women receiving conjugated estrogens. Dissociation between two commercial methods.

To investigate problems associated with measurement of estradiol-17beta (E2) in hormone replacement therapy (HRT), two commercial immunometric methods (Coat-A-Count E2 and Immulyze E2) were used to assay E2 concentrations and the two results were expressed as E2 ratios. Samples were obtained from 97 Japanese women receiving HRT and 168 unmedicated women. The largest differences between methods (P < 0.001) occurred in patients receiving oral conjugated estrogen (CE), while the best concordance was found in unmedicated women; like these controls, patients receiving oral estriol or transdermal E2 showed no significant difference between methods. Defining an E2 ratio > or = 2.0 as an abnormal discordance, the mean E2 ratio and the frequency of abnormal discordance in the CE group were 2.15 +/- 1.18 and 43.6%, respectively. No abnormal discordance for E2 occurred in other groups. In serial serum samples from the control group, no significant difference was seen between the mean E2 ratio at first measurement and those at a subsequent measurement. Similarly, no significant difference in the ratio was seen when two serial samples from CE patients were compared. However, E2 ratios after prescription of CE were significantly higher than before treatment in all patients. In conclusion, although measurement of E2 is important in patients receiving HRT, validity of the test methods must be carefully weighed for patients receiving CE.

Mitogen-activated protein kinase activation by stimulation with thyrotropin-releasing hormone in rat pituitary GH3 cells.

We examined whether mitogen-activated protein (MAP) kinase is activated by thyrotropin-releasing hormone (TRH) in GH3 cells, and whether MAP kinase activation is involved in secretion of prolactin from these cells. Protein kinase inhibitors--such as PD098059, calphostin C, and genistein--and removal of extracellular Ca2+ inhibited MAP kinase activation by TRH. A cAMP analogue activated MAP kinase in these cells. Effects of cAMP on MAP kinase activation were inhibited by PD098059. TRH-induced prolactin secretion was not inhibited by levels of PD098059 sufficient to i activation but was inhibited by wortmannin (1 microM) and KN93. Treatment of GH3 cells with either TRH or cAMP significantly inhibited DNA synthesis and induced morphological changes. The effects stimulated by TRH were reversed by PD098059 treatment, but the same effects stimulated by cAMP were not. Treatment of GH3 cells with TRH for 48 h significantly increased the prolactin content in GH3 cells and decreased growth hormone content. The increase in prolactin was completely abolished by PD098059, but the decrease in growth hormone was not. These results suggest that TRH-induced MAP kinase activation is involved in prolactin synthesis and differentiation of GH3 cells, but not in prolactin secretion.

Increased prevalence of luteinizing hormone beta-subunit variant in patients with premature ovarian failure.

OBJECTIVE: To evaluate the significance of an LH variant with a mutant beta-subunit (Trp8 to Arg8 and Ile15 to Thr15) in gynecologic disease, including infertility. DESIGN: Clinical study. SETTING: Department of Obstetrics and Gynecology, Shimane Medical University Hospital, Izumo, Japan. PATIENT(S): Two hundred forty-five Japanese women with endocrine disorders and/or gynecologic disease and 153 healthy, nonpregnant, fertile Japanese women. INTERVENTION(S): A blood sample was collected. MAIN OUTCOME MEASURE(S): The ratio of LH values from the SPAC-S and Immulyze assays (LH ratio: SPAC-S LH/Immulyze LH) was used to determine variant (< or =0.5) or wild-type (>0.5) LH status according to a demonstrated relation between the ratio and the sequence of the LH beta-subunit gene. RESULT(S): The LH ratio was lower (0.80+/-0.31) in the 245 patients than in the controls (1.00+/-0.38), and the variant was more frequent in the patients (18.4%) than in the controls (8.5%). We found no difference in the frequency of the variant between infertile and fertile patients. The prevalence of infertility did not differ between patients with variant LH and patients with normal LH. Ovulatory disorders, hyperprolactinemia, premature ovarian failure, menstrual disorders, and luteal insufficiency were significantly more frequent in patients with the variant. CONCLUSION(S): Variant LH may contribute to female reproductive disorders, including infertility and premature ovarian failure.

Increased prevalence of luteinizing hormone beta-subunit variant in Japanese infertility patients.

To determine the frequency of a common luteinizing hormone variant in a Japanese population and to evaluate its significance in infertility, serum samples were collected from 169 healthy non-pregnant Japanese women, 105 healthy adult Japanese men and 97 female Japanese infertility patients. The luteinizing hormone variant includes two point mutations in the beta-subunit gene (Trp8 to Arg8 and Ile15 to Thr15). DNA from blood cells was studied in 10 healthy women, 10 men and five patients using polymerase chain reaction and direct sequencing. In immunoassays, results with a monoclonal antibody recognizing only the wild-type hormone and a polyclonal antibody recognizing the variant as well were compared as a ratio; ratios in heterozygotes and in individuals with only wild-type alleles ranged from 0.19 to 0.50 and from 0.56 to 1.21, respectively, and 0.50 was considered a 'cut-off' value for identifying individuals with the variant. For the larger subject groups, the frequency of the variant was 9.5% in normals. The mean ratio (0.80 +/- 0.35) in infertility patients was significantly lower (P < 0.01) than in healthy women (1.09 +/- 0.56), and the variant occurred more frequently in infertility patients (16.5%) than in healthy women (8.3%; P < 0.05). The variant was more frequent in patients with ovulatory disorders (43.8%) than other patients (16.0%; P < 0.05).