Association of primary ovarian insufficiency with a specific human leukocyte antigen haplotype (A*24:02-C*03:03-B*35:01) in Japanese women.

Primary ovarian insufficiency (POI) is a heterogeneous condition defined by the triad of oligo/amenorrhea, elevated gonadotropins and estrogen deficiency in women under the age of 40 years. Although autoimmune abnormalities appear to be involved in the development of POI, there are only a few studies with respect to human leukocyte antigen (HLA). The objective of this study was to identify an HLA allele(s) and/or haplotype(s) constituting a susceptibility factor(s) for POI. We examined 83 Japanese women with apparently idiopathic isolated POI. For controls, Japanese HLA reference data registered in the HLA Laboratory were utilized. No significant association was found for a total of 94 alleles for HLA-A, B, C, DRB1, and DQB1 loci, after both stringent Bonferroni correction and less stringent Benjamini- Hochberg (B-H) correction for multiple comparisons. By contrast, of 86 haplotypes identified for MHC class I (HLA-A, B, and C) and 31 haplotypes detected for MHC class II (HLA-DRB1 and DQB1), a single haplotype (A*24:02-C*03:03-B*35:01) remained significant after Bonferroni and B-H corrections (frequency: 4.82% in women with POI and 1.06% in the control data; p = 0.00049). The results imply that a specific HLA haplotype (A*24:02-C*03:03-B*35:01) constitutes a susceptibility factor for apparently isolated POI in Japanese women.

Establishment and characterization of two cell lines (HEC-155, HEC-180) derived from uterine papillary serous adenocarcinoma.

Uterine papillary serous adenocarcinoma (UPSC) is an uncommon histologic subtype of endometrial cancer that characteristically behaves aggressively with a poor prognosis. We established two novel cell lines derived from UPSC designated HEC-155 and HEC-180. Both cell lines have been growing steadily in monolayer cultures for over ten years. Overexpression of p53, Ki67 and p27 was detected in both cell lines by immunohistochemistry. Using a DNA sequencing technique, a point mutation of p53 was detected in exon 8, codon 286 in HEC-155 and in exon 6, codon 195 in HEC-180. These newly established cell lines should be useful for investigating the characteristics of UPSC.

Gonadotropin-releasing hormone increases melatonin release in the pineal gland of the female rat in vitro.

To evaluate the effect of gonadotropin-releasing hormone (GnRH) on melatonin ( N-acetyl-5-methoxytryptamine) release and its synthesizing enzyme activities in pineal glands, pineals from adult female rats during diestrus were organ-cultured in a medium containing 10 -12, 10 -10, or 10 -8 M GnRH for 6 h. Melatonin release increased significantly in pineals cultured with 10 -10 and 10 -8 M GnRH compared to controls. However, in pineal glands that were organ-cultured in a medium containing 10 -12 to 10 -8 M GnRH, the activity of arylalkylamine N-acetyltransferase, which is the key regulatory enzyme in melatonin biosynthesis, showed no significant difference from controls. Likewise, GnRH at these concentrations had no significant effect on the activity of pineal hydroxyindole- O-methyltransferase, which catalyzes the final step of melatonin biosynthesis. These results show that GnRH stimulates pineal melatonin release, but suggest that GnRH does not affect its melatonin synthesis.

Stimulatory effects of LH on release of melatonin and activities of its synthesizing enzymes NAT and HIOMT in organ-cultured pineal glands of female rats.

The pineal hormone melatonin (N-acetyl-5-methoxytryptamine) exerts antigonadotropic effects in some mammalian species. To evaluate the effect of luteinizing hormone (LH) on melatonin release and its synthesizing enzyme activities in pineal glands, pineals of adult female rats undergoing diestrus were organ-cultured in a medium containing 10(-12), 10(-10) or 10(-8) M LH for 6 h. Melatonin release increased significantly in pineals cultured with 10(-12) and 10(-10) M LH, as compared to control values. Similarly, the activity of arylalkylamine N-acetyltransferase (NAT), the key regulatory enzyme in melatonin biosynthesis, was significantly higher in pineals cultured with 10(-12) and 10(-10) M LH for 6 h, while LH at 10(-8) M had no effect. Although LH at 10(-10) M increased pineal hydroxyindole-O-methyltransferase (HIOMT) activity, which catalyzes the final step of melatonin biosynthesis, LH at 10(-12) and 10(-8) M had no effect. These results demonstrate that at relatively low physiological levels, LH stimulates pineal melatonin synthesis and release, mainly by increasing NAT activity.

FISH analysis for apparently simple terminal deletions of the X chromosome: identification of hidden structural abnormalities.

We report on fluorescence in situ hybridization (FISH) analysis in 30 mosaic or nonmosaic females diagnosed as having apparently simple terminal X deletions by standard G-banding analysis. FISH studies for DXZ1, the Xp and Xq telomere regions, and the whole X chromosome painting were carried out for the 30 females, indicating rearranged X chromosomes with signal patterns discordant with terminal deletions in 6 cases: one dic(X)(DXZ1++) chromosome, two der(X)(qtel++) chromosomes, one Xq- (qtel+) chromosome, and two der(X)(ptel++) chromosomes. Additional FISH studies were performed for the 6 cases using probes defining 12 loci on the X chromosome, showing large Xp deletion and small Xp duplication in the dic(X)(DXZ1++) chromosome, partial Xp deletions and partial Xq duplications in the two der(X)(qtel++) chromosomes, an interstitial Xq deletion in the Xq- (qtel+) chromosome, and partial Xq deletions and partial Xp duplications in the two der(X)(ptel++) chromosomes. Clinical assessment of the 6 cases revealed tall and normal stature in the two mosaic cases with the der(X)(ptel++) chromosomes that were shown to be associated with SHOX duplication. The results suggest that unusual X chromosome rearrangements are often misinterpreted as simple terminal X deletions, and that FISH analysis is useful for precise structural determination and better genotype-phenotype correlation of the X chromosome aberrations.

Stress responses during laparoscopy with CO2 insufflation and with mechanical elevation of the abdominal wall.

STUDY OBJECTIVE: To compare the intensity of stress responses caused by laparoscopy combined with CO2 insufflation (CI) with those caused by mechanical elevation of the abdominal wall (MEA). DESIGN: Prospective study (Canadian Task Force classification I). SETTING: Tertiary care university hospital. PATIENTS: Thirty-one women. Intervention. Laparoscopy, 16 with CI and 15 with MEA. MEASUREMENTS AND MAIN RESULTS: Circulating levels of norepinephrine, epinephrine, cortisol, and interleukin (IL)-6 were compared in the two groups. Arterial partial pressure of CO2 (PaCO2) did not increase during laparoscopy by either method. Plasma norepinephrine and epinephrine levels increased after extubation in the MEA group but not in the CI group. Circulating cortisol levels in both groups increased during laparoscopy; mean intraoperative levels were higher in the CI group. Serum IL-6 levels increased after extubation in the MEA but not the CI group. Mean heart rate increased during laparoscopy in both groups, whereas blood pressure increased only in the CI group. More patients in the MEA group required postoperative analgesia. CONCLUSION: Laparoscopy with MEA caused more pronounced adrenosympathetic and cytokine responses than that with CI when PaCO2 was maintained within the range of normocapnia.

Differential growth inhibitory effect of melatonin on two endometrial cancer cell lines.

The effect of melatonin on endometrial cancer cell growth was investigated using two cell lines, SNG-II and Ishikawa, which are different in their estrogen receptor status. A physiological concentration of melatonin (10(-9) M) showed no growth inhibitory effect on SNG-II cells, which are estrogen receptor-negative at all cell densities and incubation times. In contrast, melatonin significantly inhibited Ishikawa cells, which are estrogen receptor-positive at all cell densities tested after 96 hr incubation. The greatest inhibition of Ishikawa cell growth was observed at 10(-9) M melatonin, compared with other supra (10(-6), 10(-8) M) or subphysiological concentrations (10(-10), 10(-12) M). This growth inhibitory effect of melatonin on Ishikawa cells was completely blocked by 10(-10) to 10(-8) M concentrations of 17-beta estradiol administration. Pretreatment with luzindole, which is a selective melatonin receptor antagonist, prior to the addition of melatonin also blocked the inhibitory effect of melatonin on Ishikawa cells. This is the first study to demonstrate an anti-proliferative effect of physiological melatonin on endometrial cancer cells in vitro. The present study revealed that melatonin also inhibits the growth of estrogen receptor positive endometrial cancer cells and that this effect of the pineal indole may be mediated by both steroid and melatonin receptors.

Melatonin secretion from organ-cultured pineal glands of rats: modulation by gonadectomy and gonadotropin-releasing hormone agonist administration.

The objective of the study was to evaluate the effect of pretreatments such as gonadectomy in male and female rats, and gonadotropin-releasing hormone agonist (GnRHa) administration in female rats, on levels of secretion of melatonin, using an organ culture of pineal glands. Gonadectomy 2 weeks before the animal was killed increased the amount of melatonin secreted into the medium by the pineal glands of female rats but not of male rats. The increase in in vitro melatonin secretion after ovariectomy in female rats was prevented by estrogen replacement. Ovariectomy 3 and 4 weeks before death also significantly increased the amount of melatonin secretion. Administration of GnRHa 2 weeks before decapitation significantly decreased serum estradiol concentrations and significantly increased melatonin secretion by the pineal glands of female rat. GnRHa administration 3 or 4 weeks before decapitation also significantly decreased serum estradiol concentrations, but did not increase pineal secretion of melatonin. The results indicate that ovariectomy increases melatonin secretion from organ-cultured pineal glands and that this increase is suppressed by estrogen in adult female rats. In contrast, orchiectomy in male rats does not influence in vitro secretion of melatonin. These results suggest that the GnRH-gonadotropin system may participate in the regulation of pineal melatonin secretion in adult female rats.

Plasma catecholamine responses during laparoscopic gynecologic surgery with CO(2) insufflation.

STUDY OBJECTIVE: To compare adrenergic-sympathetic responses during pelvic laparoscopic surgery with CO(2) insufflation with those during laparotomy. DESIGN. Prospective study (Canadian Task Force classification II-1). SETTING: Tertiary care university hospital. PATIENTS: Twenty-one infertile women with a clinical diagnosis of endometriosis. INTERVENTION: Plasma norepinephrine (NE) and epinephrine (E) levels were measured in patients undergoing laparoscopic or open pelvic surgery. MEASUREMENTS AND MAIN RESULTS: During laparoscopic surgery, increases in NE and E levels were correlated with arterial tension of CO(2) (PaCO(2)) and were greater in patients with a larger increase in PaCO(2) than in those undergoing laparotomy. Hemodynamic changes were also more evident during laparoscopic surgery, with larger PaCO(2) increases. CONCLUSION: The PaCO(2) increases due to CO(2) insufflation are associated with adrenergic-sympathetic activation and hemodynamic changes during laparoscopic pelvic surgery.

The effect of melatonin on in vitro fertilization and embryo development in mice.

To examine the effect of melatonin on in vitro fertilization and embryonic development, mouse embryos after insemination in vitro were cultured in a physiological medium with or without melatonin. Melatonin increased the fertilization rate significantly at a concentration between 10(-6) and 10(-4) M (27.6 vs. 43.9 or 40.4%, P < 0.01). Furthermore, a significant increase in the rate of embryos reaching the four-cell stage (16.0 vs. 26.7%, P < 0.01), the eight-cell stage (12.1 vs. 25.8 or 23.5%, P < 0.01), and blastulation (8.9 vs. 23.5 or 17.5%, P < 0.01) was observed when the embryos were cultured in a medium containing 10(-8) or 10(-6) M melatonin. These results demonstrate that melatonin supports fertilization and early embryo development after in vitro fertilization.

Noradrenaline concentrations in human preovulatory follicular fluid exceed those in peripheral plasma.

The concentrations of noradrenaline in preovulatory follicular fluid obtained from women undergoing in vitro fertilization were determined by high-performance liquid chromatography with fluorometric detection and compared with those in peripheral plasma obtained concurrently. All of the follicular fluid samples contained noradrenaline at concentrations substantially higher than those in the corresponding plasma samples. A positive correlation was found between noradrenaline levels in follicular fluid and plasma in each woman (n=11; r=0.952; p<0.001). However, there was no significant difference between noradrenaline concentrations in follicular fluid aspirated from follicles with or without an oocyte [mean+/-SEM, 0.207+/-0.002 ng/microl for follicular fluid samples with an oocyte (n=44), and 0.221+/-0.003 ng/microl for follicular fluid samples without an oocyte (n=13)]. The data indicate that noradrenaline accumulates in follicular fluid, supporting the physiological significance of noradrenaline in the local regulation of human ovarian functions.

Postoperative delirium and plasma melatonin.

The molecular mechanisms of postoperative delirium are not understood in detail yet. This condition is similar to cases of mental symptoms in interferon therapy or hemodialysis. We propose that postoperative delirium is caused by a deficiency of serotonin, an important neurotransmitter, and review the evidence supporting our hypothesis. The serotonin deficiency results from a decrease in tryptophan, the serotonin precursor, and from an increase in melatonin, a serotonin metabolite. Our hypothesis may be applicable to the mechanisms of mental symptoms in interferon therapy and hemodialysis. In addition, we discuss to the relationship between homeostasis and biorhythms because postoperative delirium may be a dysrhythmia.

Anti-nuclear antibodies in patients with premature ovarian failure.

We examined the prevalence of anti-nuclear antibodies (ANA) in 32 consecutive patients with premature ovarian failure with and without chromosomal abnormalities. Blood samples were taken for karyotype determination as well as detection of autoantibodies, X-terminal microdeletions and spontaneous follicular growth. The correlation between ANA positivity and the age at onset of amenorrhoea, as well as the presence of karyotype abnormalities, X-terminal microdeletions and follicular growth was determined. Ten of the 24 patients with normal karyotype and none of the 8 patients with karyotype abnormalities were ANA positive. ANA were found more frequently in patients with premature ovarian failure with normal karyotypes than in control amenorrhoeic patients (42 versus 6, P < 0.01). ANA were found in 77% (10/13) of premature ovarian failure patients with normal karyotypes who developed amenorrhoea at or under the age of 30 years, but not in the patients who developed amenorrhoea later in life. Follicular growth was evident in 50% (5/10) of karyotypically normal patients with ANA, 71% (10/14) of karyotypically normal patients without ANA and 38% (3/8) of patients with karyotype abnormalities. X-terminal microdeletions were not found in any of the patients studied. These results suggest that patients with premature ovarian failure and ANA are an aetiologically and clinically distinct group.

Melatonin, its precursors, and synthesizing enzyme activities in the human ovary.

The presence of melatonin (N-acetyl-5-methoxytryptamine) and its precursors, serotonin (5-hydroxytryptamine) and N-acetylserotonin, was demonstrated in extracts of human ovary using reverse-phase high-performance liquid chromatography coupled with fluorometric detection. In addition, activities of two melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were found in human ovary homogenates. The apparent Michaelis constants for the substrates of NAT and HIOMT in the human ovary were similar to those reported for the pineal glands of humans and other mammals. These findings strongly suggest that the human ovary, like the pineal gland, may synthesize melatonin from serotonin by the sequential action of NAT and HIOMT.

Ovulation induction in a woman with premature ovarian failure resulting from a partial deletion of the X chromosome long arm, 46,X,del(X)(q22).

OBJECTIVE: To report successful ovulation induction in a woman with premature ovarian failure (POF) resulting from a partial Xq deletion. DESIGN: An uncontrolled study. SETTING: University hospital. PATIENT(S): A 27-year-old woman with 46,X,del(X)(q22) who had hypergonadotropic secondary amenorrhea. INTERVENTION(S): Injections of hMG (225 IU/d) for 8 consecutive days after endogenous gonadotropin suppression with a long-acting GnRH agonist (900 micrograms/d) for 12 weeks, together with cyclic sex steroid replacement therapy. MAIN OUTCOME MEASURE(S): Serum concentrations of E2 and P as well as ultrasonography. RESULT(S): Folliculogenesis and ovulation. CONCLUSION(S): Ovulation induction is possible in patients with POF caused by X chromosome aberrations.

[A study on the role of ultrasonography in antenatal care in Thailand with focus on the appropriate technology].

The authors investigated the state of antenatal care, outcome of deliveries and use of ultrasonography at the Maternal and Childhood Health Hospital in Khonkane province, Thailand. The study was conducted before (stage I) and after (stage II) the introduction of the second ultrasonographic equipment in an attempt to set up a proper standard of technological level of clinical laboratory services and to investigate the usefulness of introducing ultrasonography into the prenatal care in developing countries. As a result, about 60% of first antenatal visits were in their second or third trimester, about 80% of pregnancies terminated by normal vaginal deliveries, parity, rate of cesareans sections and pre-term labors were not significantly different in both stages. Ultrasonography was performed on 16% of the first antenatal visits in stage I, while, in stage II, it was performed on 31.3% of them. Ultrasonography was used most frequently to determine the gestational age and presentation of the fetus in both years (70.3% and 79.1% respectively in stage I and stage II). In most of the cases, ultrasonography was performed only once during the gestation. Thus, it was proved that technical training must be provided with introduction of new equipment to medical facilities and utilization of medical technologists for ultrasonography was another concern in developing countries. Beside, it was important to determine the proper period during the gestation when ultrasonography should be applied and to adopt an effective public health service including health education and health insurance to promote antenatal visits in time during the proper periods.

[Prolonged neuromuscular blockade with vecuronium in patient with triple pregnancy treated with magnesium sulfate].

A 33 year-old parturient with triplet pregnancy underwent emergency cesarean section at 35 week of gestation under general anesthesia. The patient had received magnesium sulfate to prevent uterine contraction immediately before the cesarean section. Although serum magnesium value was not beyond therapeutic levels (3.3 mEq.l-1), the neuromuscular blocking effects with vecronium were strengthened. It was not likely that volatile anesthetic enhanced neuromuscular blockade produced by vecuronium because the onset time of vecuronium had already been faster than that in pregnant patients untreated with magnesium before she was exposed to isoflurane. In addition, it is possible that magnesium could interfere with postpartum uterine contractions because of its tocolytic properties. Magnesium sulfate therapy has several implications to anesthetic agents. We, anesthesiologists, should know about the biophysiological effects of magnesium and control the interaction between anesthetic agents and this electrolyte.

Detection of melatonin and serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase activities in rat ovary.

Melatonin (N-acetyl-5-methoxytryptamine) and the activities of two melatonin-synthesizing enzymes, serotonin N-acetyltransferase (acetyl coenzyme A: arylalkylamine N-acetyltransferase EC 2.3.1.87; NAT) and hydroxyindole-O-methyltransferase (S-adenosyl-L-methionine: N-acetylserotonin-O-methyltransferase EC 2.1.1.4; HIOMT), were assayed in extracts of ovaries obtained from virgin Wistar-derived rats (7-9 week-old) during the light period of a 12 h light/12 h dark cycle. Melatonin was detected in the rat ovary using reverse-phase high-performance liquid chromatography (HPLC) coupled with fluorometric detection and radioimmunoassay (RIA). In addition, NAT and HIOMT activities were found in rat ovary. The apparent Michaelis constants (Km) for the substrates of NAT and HIOMT in the rat ovary were similar to those reported for the pineal gland and retina. These data suggest that the rat ovary, like the pineal gland and the retina, may synthesize melatonin from serotonin by the sequential action of NAT and HIOMT.