[Female pseudohermaphroditism].

Patients with female pseudohermaphroditism have female internal genitalia and karyotype (XX) and various degree of external genitalia virilization. External genitalia is musculinized congenitally when female fetus is exposed to excess androgenic environment. Congenital adrenal hyperplasia (CAH), mostly 21-hydroxylase deficiency, is the most common cause. Maternal androgen excess due to maternal ovarian tumor or drug intake also causes female pseudohermaphroditism. Combination of hormonal therapy and surgical correction is required for CAH. When appropriate treatments were given, normal puberty, fertility and child bearing are possible. HLA typing in patient's family is useful for identifying heterozygote and homozygote, because of close linkage of 21-hydroxylase gene and HLA gene. Prenatal diagnosis and genetic diagnosis for female pseudohermaphroditism due to 21-hydroxylase deficiency can be performed, however prenatal treatment is not completely established.

Dissociation of collagenase-tissue inhibitor of metalloproteinases-1 (TIMP-1) complex--its application for the independent measurements of TIMP-1 and collagenase activity in crude culture media and body fluids.

Collagenase-tissue inhibitor of metalloproteinases-1 (TIMP-1) complex was prepared from activated collagenase and TIMP-1 purified from culture media of human skin fibroblasts. After having been confirmed to be a complex by zinc chelate chromatography, the complex was demonstrated to dissociate by passage through an anti-TIMP-1 monoclonal antibody-affinity column. On the basis of above evidence, a simple strategy was set up for the independent measurements of TIMP-1 concentration, and both active and total collagenase activities in crude culture media and body fluids.

Source of prolactin in human follicular fluid.

To analyze whether prolactin (PRL) in human follicular fluid (FF) is synthesized locally or derived from the circulation, PRL concentrations of plasma and FF were determined in the patients after ovarian stimulations. The amounts of PRL messenger ribonucleic acid (mRNA) in the follicular tissues during different menstrual phases were also determined. The FF PRL concentration was correlated positively with plasma PRL and highest estradiol levels during the stimulatory cycle. No PRL mRNA sequence was detected in the RNAs extracted from follicles at any stage in the menstrual cycle, although beta-actin mRNA was detected in all samples. In a comparison with pituitary RNA, the PRL mRNA concentration in ovarian follicular tissues seemed to be 10,000 times less than that in the pituitary. These results suggest that FF PRL may not be synthesized locally, but derived from the pituitary via the circulation through passive diffusion, and thus regulated by estrogen.

Menotropin stimulation after prolonged gonadotropin releasing hormone agonist pretreatment for in vitro fertilization in patients with endometriosis.

Two protocols were scheduled for in vitro fertilization and embryo transfer (IVF-ET) in patients with various stages of endometriosis who were resistant to conventional therapies. In the ultralong protocol (21 patients), gonadotropin releasing hormone agonist (Gn-RHa) was administered for at least 60 days prior to ovarian stimulation along with menotropin until human chorionic gonadotropin was injected. In the long protocol (11 patients), Gn-RHa was started at the midluteal phase and exogenous gonadotropin was commenced between the third and the seventh day of the menstrual cycle after pituitary suppression. The estradiol response and the number of retrieved oocytes, fertilized oocytes, cleaved oocytes, and transferred embryos were similar in both groups but the clinical pregnancy rate per transfer was superior in the ultralong protocol (67 vs 27%). The miscarriage rate was 14% (2/14) in the ultralong protocol. Prolonged Gn-RHa suppression of ovarian function before superovulation may overcome some causes of infertility in patients with endometriosis.

Induction by cortisol of aminopeptidases production from the human placenta in tissue culture.

Human placental aminopeptidase M and A and post-proline endopeptidase are known to act as degrading enzymes of bioactive peptides such as angiotensin II, oxytocin and endogenous opioids. We tested the effects of cortisol on the activities of human placental aminopeptidase A and M and post-proline endopeptidase using short-cultured placental tissues. From 34.5 nM to 3.45 microM of cortisol significantly increased the activities of 3 enzymes. Our present data suggest a possible important role of cortisol in the growth of human placenta via induction of placental aminopeptidases.

Identification of two subtypes of protein kinase C in human placenta.

A purified protein kinase C (PKC) has been isolated from term human placental tissue, which is phospholipid and Ca(2+)-dependent. Two subtypes of the enzyme were identified by hydroxyapatite column chromatography and using monoclonal antibodies with immunohistochemical techniques; subtype III is present in higher concentration than subtype II. Their ratio of 2.5 is very similar in second and third trimester placentas, but is higher, 6.5, in the first trimester. The subtype I was never expressed.

Effects of low molecular weight peptides and divalent cations on degradation and binding of angiotensin II.

The effects of peptide inhibitors (bestatin and amastatin) and divalent cations (Ca2+ and Co2+) on the velocity of Asp1 liberation from angiotensin II (A-II) by human placental membrane fractions and binding of 125I A-II to human placental membranes were tested at 22 degrees C and 4 degrees C. Asp1 liberation was measured by high performance liquid chromatography. As expected, the degradation and binding of A-II were temperature sensitive, with both being at 4 degrees C than at 22 degrees C. While amastatin (10(-4) M) and bestatin 10(-6) M) significantly reduced the velocity of Asp1 liberation from A-II to about 45%, amastatin (10(-4) M) and bestatin (10(-4) M) increased 125I A-II binding to 125% and 130%, respectively. Ca2+ (10 mM) and Co2+ (10 mM) activated the velocity of Asp1 liberation from A-II to 140% and 120%, respectively at 22 degrees C. Ca2+ (10(-1) M) and Co2+ (10 mM) also enhanced 125I A-II binding about 130%. Previously we showed that the A-II degrading activity found in human placental membrane fractions is mainly due to aminopeptidases A and M. Since amastatin and bestatin are the specific inhibitors for aminopeptidases A and M, and since Ca2+ and Co2+ are the activators for aminopeptidase A and aminopeptidase M, respectively, it is conceivable that the enzymes regulate the levels of A-II and, therefore, that they may play an important role in the binding of A-II to human placental membrane fractions.

Increased serum kininase I activity in pregnancy complicated by pre-eclampsia.

Serum kininase I activity was measured in normal pregnancy and pre-eclampsia. The mean value in nonpregnant controls was 180 +/- 25 (SD) nmol/min/ml. Kininase I activity during normal pregnancy significantly increased after week 14, reaching the highest value (240 +/- 20 nmol/min/ml) at weeks 38 and 40. The kininase I activity in pregnancy complicated by severe pre-eclampsia was higher than that in normal pregnancy. The possible role played by elevated kininase I levels in pre-eclampsia was discussed.

A comparison of the binding of angiotensin II to human placenta between normal and severe pre-eclamptic pregnancy.

We compared the binding of angiotensin II (A-II) to human placenta between normal and severe pre-eclamptic pregnancy. Our data did not show any statistically significant difference between normal and pre-eclamptic pregnancy in both the equilibrium dissociation constant (Kd) and the total number of placental A-II receptors. Therefore, changes in the binding of A-II to placenta might not be involved in the possible cause of activation of the renin-angiotensin system in the feto-placental unit in severe preeclampsia.

Expression of low density lipoprotein receptor gene in human placenta during pregnancy.

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.

Increased sensitivity to bradykinin in pregnant rats.

We tested the effects of late pregnancy on the depressor response to bradykinin (100, 200 and 400ng) in rats. All experiments were performed under anesthesia by intraperitoneal injection of pentobarbital (50 mg/kg). Catheters were connected to the arterial and venous lines for blood pressure recording and administration of drugs. Late pregnancy (19- to 21-day) showed a hypersensitivity of the depressor response to bradykinin. The effects of captopril, Kininase II inhibitor, on the depressor response to bradykinin were examined in rats. Since captopril (50, 100 and 200 micrograms) notably increased the depressor response to bradykinin both in nonpregnant and 19- to 21-day pregnant rats, kininase II acts as a bradykinin metabolizing enzyme in vivo. Captopril (50, 100, 200 micrograms), however, resulted in the augmented parallel increases of depressor response to bradykinin (100ng) in both nonpregnant and pregnant rats. Our data may suggest that kininase II is not involved in the hypersensitivity to bradykinin in late pregnancy in rats.

Effects of pregnancy, delivery and lactation in hyperprolactinemia with prolactin producing pituitary adenoma.

The effects of pregnancy, delivery and lactation on changes in serum prolactin (PRL) values were investigated in patients with hyperprolactinemia. Thirty-seven patients with hyperprolactinemia who wished to become pregnant were treated by transsphenoidal surgery, bromocriptine therapy, or a combination of the two. In 33 patients whose pre-pregnancy serum PRL concentration exceeded 30ng/ml, only in two did serum PRL return to the normal range below 30ng/ml after pregnancy, delivery and lactation. However, the serum PRL concentration was decreased in 28 patients. When classified according to the pre-pregnancy serum PRL concentrations, PRL less than or equal to 100 (Group A), 100 less than PRL less than or equal to 200 (Group B) and 200 less than PRL (Group C), patients with the greatest pre-pregnancy serum PRL concentration showed the greatest reduction. The ratios of post-pregnancy serum PRL to pre-pregnancy PRL in group A, B and C were 91.4 +/- 22.1%, 81.5 +/- 7.0% and 65.0 +/- 6.5% (Mean +/- SE), respectively. Group C with the highest pre-pregnancy serum PRL concentration consisted almost entirely of patients with macroadenoma. Thus, the reduction in serum PRL after pregnancy, delivery and lactation was considered to be the result of a decrease in the size of the adenoma due to adenoma enlargement over the sella turcica through the estrogen effects during pregnancy, and from impairment of pituitary circulation.

A comparison of the hypotensive effects of Met-enkephalin in normotensive and spontaneously hypertensive rats.

To elucidate the role of Met-enkephalin in the hypertension in spontaneously hypertensive rats, the depressor response to Met-enkephalin was compared between normotensive (Wistar) and spontaneously hypertensive rats by administration of bestatin (1.25 mg) as a component of a mixture of enkephalinase inhibitors. All experiments were performed under anesthesia by intraperitoneal injection of pentobarbital (50mg/kg). Catheters were connected to the arterial and venous lines for blood pressure recording and administration of drugs. Hypotensive effects of injected Met-enkephalin were noticeably enhanced by administration of bestatin compared with pre-bestatin responses at each dose of Met-enkephalin(10, 25, 50, and 100n mol). We could not find any statistically significant difference between blood pressure responses to 25n mole Met-enkephalin in Wistar and spontaneously hypertensive rats both before and after administration of bestatin. Our present study dose not suggest the active role of Met-enkephalin in the hypertension of spontaneously hypertensive rats.

The ontogeny of growth hormone in the human fetal pituitary.

To examine the ontogeny of growth hormone synthesis and secretion in human fetus, growth hormone messenger ribonucleic acid was measured in 11 pituitaries from fetuses of 16 to 27 weeks of gestation by hybridization of cytosol ribonucleic acid with complementary deoxyribonucleic acid labeled with phosphate 32. Pituitary growth hormone content and serum growth hormone, thyroxine, and cortisol concentrations were assessed by radioimmunoassay. Growth hormone messenger ribonucleic acid content in the fetal pituitary increased from the early midtrimester, reaching a level 15.3 times higher at 27 weeks of gestation than the value at 16 weeks. However, growth hormone content in the pituitary showed no evident change during 16 to 21 weeks of gestation and started to increase after 22 weeks. Serum concentration of growth hormone was variable but always greater than 50 ng/ml, and the maximal level was observed at 20 weeks of gestation (141 ng/ml). Although serum thyroxine concentration in the fetuses showed no correlation with pituitary growth hormone content or serum growth hormone concentration, serum concentration of cortisol was correlated positively with growth hormone content in the fetal pituitary. These results suggest that the maturation of the growth hormone synthesis and secretion system in the fetal pituitary occurs after 22 weeks of gestation and that cortisol may play some role in the ontogenesis of growth hormone.

Purification and properties of microsomal carboxypeptidase N (kininase I) in human placenta.

Carboxypeptidase N (kininase I, EC 3.4.17.3) was found in human placenta and purified 600-fold. The enzyme was solubilized from membrane fractions with Triton X-100 and was purified by affinity chromatography with histargin, a potent inhibitor of this enzyme. The pH optimum of the enzyme was 7.8. The Km values for L-hippuryl-L-lysine and bradykinin were 1.25 and 0.43 mmol/l, respectively. The apparent molecular mass (Mr) of the enzyme determined by gel filtration was estimated to be 280,000, which is identical to that of the human serum enzyme. We propose that the placenta is a major source of carboxypeptidase N and thus may be involved in the physiological control of fetal circulation by regulating the kallikrein-kinin and renin-angiotensin systems.

In vitro degradation of opioid peptides by human placental aminopeptidase M.

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.

Positive effect of estradiol and progesterone in severe pre-eclampsia.

We have used estradiol benzoate (E) and Progesterone (P) for the treatment of severe pre-eclampsia and obtained good results in 8 cases. To assess the placental function, we serially measured leucine aminopeptidase (placental type, P-LAP) in serum. As a new trial, we used E and P in place of the hypotensive and diuretic drugs in 8 cases of severe pre-eclampsia. In a typical case, a patient at week 31 of gestation suffered from highly elevated blood pressure (190/130 mmHg) and proteinuria; at this time we started the treatment with E (20 mg/day) and P (80 mg/day), and continued it for 23 days with increasing doses. By this treatment the blood pressure was gradually decreased and the edema improved; the serum P-LAP increased gradually. At week 35, the P-LAP activity decreased suddenly despite the steroid treatment suggesting a placental dysfunction. At this stage we made Cesarean section and a male infant weighing 1.750 g (Apgar score 8) was delivered alive. Until now we have experienced 7 other cases of the successful treatment with the E and P.

Simultaneous assay for individual sulphated 3 alpha- and beta-hydroxysteroids in serum using high-performance liquid chromatography combined with 3 alpha- and beta-hydroxysteroid dehydrogenases immobilized on one column.

A high-performance liquid chromatographic method for the simultaneous determination of individual sulphated 3 alpha- and beta-hydroxysteroids in serum using 3 alpha- and beta-hydroxysteroid dehydrogenases (3 alpha-HSD and beta-HSD, respectively) immobilized on one column and a fluorimeter to detect the resulting NAD+ to NADH transformation is described. Individual sulphated 3 alpha- and beta-hydroxysteroids in serum are extracted with ethanol, solvolysed with sulphuric acid in ethyl acetate and then separated by high-performance liquid chromatography. The hydroxysteroids thus separated are subsequently mixed with NAD+ and then passed through the column in which the following catalytic reaction occurs: (formula, see text) The detection limits are as low as 0.5-1.0 microgram/dl for sulphated 3 alpha- or beta-hydroxysteroids in serum. The present assay method is highly specific, reliable and reproducible and is thus applicable to a clinical study on the metabolism of sulphated 3 alpha- and beta-hydroxysteroids in patients with adrenal or gonadal diseases.

[Clinical assessment of body hair growth in Japanese women. The relationship between a grade of hirsutism and the menstrual status].

Three hundred and sixty-nine volunteer women and 308 outpatients were studied to clarify the criteria of hirsutism and the relationship between a grade of hirsutism and menstrual status in Japanese women. The body hair growth was assessed according to the modified score system described by Ferriman and Gallway. The menstrual status was classified into six groups based on basal body temperature. The hair growth score calculated in volunteers tended to be lower than that of western women in most sites. Therefore one criterion of hirsutism in Japanese women should be a score of more than 6 points in our system. There was a close correlation between body hair growth and menstrual status. As a result, it can be considered that the systematic assessment of body hair growth is useful in diagnosing and treating some irregular menstrual patients.

Serum CA 125 levels in patients with endometriosis: changes in CA 125 levels during menstruation.

We investigated serum CA 125 levels in 120 patients with benign gynecologic disease, and examined variations in CA 125 levels during the menstrual cycle in six normally cycling women. CA 125 levels were elevated transiently during menstruation, suggesting that blood samples taken during menstruation can give a false-positive result. Whereas the CA 125 values during the nonmenstrual part of the cycle were significantly higher in the patients with adenomyosis and advanced endometriosis (stages III and IV) than in those with a normal pelvis, there was no difference between the patients with mild endometriosis (stages I and II) and the normal-pelvis group. CA 125 levels during menstruation were significantly elevated, not only in women with adenomyosis and advanced endometriosis, but also in those with mild endometriosis, compared with those with a normal pelvis. These data suggest that the measurement of serum CA 125 during menstruation may increase the rate of detection of endometriosis over that in studies done during other phases of the cycle.