Decreased peak bone mass is associated with a 3-bp deletion/insertion of the CYP19 intron 4 polymorphism: preliminary data from the GOOS study.

Finding that estrogen plays an important role in bone homeostasis in men prompted research on relationship of polymorphism at the CYP19 gene and the bone mass. Therefore, influence of 3-bp deletion/insertion polymorphism of CYP19 (TTTA)7 allele on the peak bone mass attainment in males was studied. Fifty-eight unrelated male participants, aged 21-35, were selected depending on the presence of (TTTA)7 (no.=19) or (TTTA)7-3 (no.=39) alleles from the initial cohort of 92 young males. Heterozygotes (TTTA)7/(TTTA)7-3 (no.=13) were not included in the analysis. Serum levels of estradiol, free testosterone, 25-hydroxyvitamin D, bone alkaline phosphatase, osteocalcin, and beta-crosslaps were measured. Bone mass was measured by DXA at the hip and at the spine. (TTTA)7-3 allele was associated with significantly lower femoral neck bone mineral density (BMD) (p=0.02). Logistic regression model indicated strong association of (TTTA)7-3 allele with low BMD in the range of osteopenia/osteoporosis (p=0.014, odds ratio 12.36, confidence intervals 1.65-92.46). In the present study association of 3-bp deletion polymorphism of the (TTTA)7 allele with decreased peak bone mass in males is reported for the first time. However, further studies are necessary to elucidate the functional relevance of this polymorphism.

Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium.

Members of the Wnt family of signaling molecules are important in cell specification and epithelial-mesenchymal interactions, and targeted gene deletion of Wnt-7a in mice results in complete absence of uterine glands and infertility. To assess potential roles of the Wnt family in human endometrium, an endocrine-responsive tissue, we investigated in the proliferative and secretory phases of the menstrual cycle, endometrial expression of several Wnt ligands (Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, and Wnt-8b), receptors [Frizzled (Fz)-6 and low-density lipoprotein receptor-related protein (LRP)-6], inhibitors [FrpHE and Dickkopf (Dkk)-1], and downstream effectors (Dishevelled-1, glycogen synthase kinase-3beta, and beta-catenin) by RT-PCR, real-time PCR and in situ hybridization. No significant menstrual cycle dependence of the Wnt ligands (except Wnt-3), receptors, or downstream effectors, was observed. Wnt-3 increased 4.7-fold in proliferative compared with secretory endometrium (P < 0.05). However, both inhibitors showed dramatic changes during the cycle, with 22.2-fold down-regulation (P < 0.05) of FrpHE and 234.3-fold up-regulation (P < 0.001) of Dkk-1 in the secretory, compared with the proliferative phase. In situ hybridization revealed cell-specific expression of different Wnt family genes in human endometrium. Wnt-7a was exclusively expressed in the luminal epithelium, and Fz-6 and beta-catenin were expressed in both epithelium and stroma, without any apparent change during the cycle. Both FrpHE and Dkk-1 expression were restricted to the stroma, during the proliferative and secretory phase, respectively. These unique expression patterns of Wnt family genes in different cell types of endometrium and the differential regulation of the inhibitors during the proliferative and secretory phase of the menstrual cycle strongly suggest functions for a Wnt signaling dialog between epithelial and stromal components in human endometrium. Also, they underscore the likely importance of this family during endometrial development, differentiation and implantation.

Design and synthesis of 13,14-dihydro prostaglandin F(1alpha) analogues as potent and selective ligands for the human FP receptor.

The in vitro evaluation of a new class of potential bone anabolic agents for the treatment of osteoporosis is described. These compounds are potent and selective ligands for the human prostaglandin F receptor (hFP receptor). The compounds lack the olefin unsaturation required for potency in the natural ligand PGF(2)(alpha) yet retain binding affinity for the hFP receptor in the nanomolar to micromolar range. Removal of the alkenes also results in a better selectivity ratio for the hFP receptor over the other prostaglandin receptors tested. A rationale for the selectivity differences of various analogues, based on ligand docking experiments to a putative hFP receptor model, is also described.

The role of dopamine in the formation of gastric ulcers in rats.

A single dose of the dopamine agonists L-dopa, bromocriptine or apomorphine produced a protective effect by significantly shortening of the length of stomach ulcerations. A single dose of the dopamine antagonists haloperidol, sulpiride or domperidone potentiated the ulcerogenic effect by extending the length of stomach ulcerations. These results point to the fact that dopamine is involved in the rise and development of experimental stomach ulcers.

Dopamine antagonists induce gastric lesions in rats.

Gastric lesions were provoked in all rats that had received intraperitoneally a single dose of the dopamine antagonists haloperidol, metoclopramide or domperidone 24 h before. Dose-dependence was demonstrated for haloperidol. This drug induced gastric lesions as early as 90 min after its application. The ulcerogenic effect of haloperidol was completely prevented or markedly reduced by simultaneous applications of dopamine agonists bromocriptine or L-dopa. We conclude that the model of gastric lesions induced by dopamine antagonists could be successfully applied in further investigations of the role of dopamine in the pathogenesis of ulcer disease.

The influence of dopamine receptor agonists and an antagonist on acute pancreatitis in rats.

The influence of the dopamine receptor-stimulating agent, bromocriptine, the dopamine-releasing drug, amantadine, and the dopamine antagonist, domperidone, on acute pancreatitis was studied in rats. Acute pancreatitis was induced by ligation of the bile duct at its point of entry into the duodenum. Each drug was applied intraperitoneally 1 h before induction of acute pancreatitis and all the surviving animals were killed 24 h thereafter. The control, saline-pretreated animals exhibited the mortality rate, macroscopical and histological changes, as well as increase of serum amylase levels that were consistent with acute pancreatitis. Domperidone induced a large increase in serum amylase which was significantly reduced by the simultaneous administration of bromocriptine. However, both bromocriptine and amantadine, when given separately did not prevent the increase of serum amylase levels to the control levels. Statistical analysis showed that the difference between the mortality rate in the control and treated groups did not reach the level of significance probably due to the rather limited number of animals used. On the other hand, application of bromocriptine as well as amantadine successfully reduced the onset of acute pancreatitis whereas domperidone, a rather specific peripheral dopamine receptor blocker, had the opposite effect. Both bromocriptine and amantadine significantly reduced the mortality rate from acute pancreatitis in domperidone-pretreated rats. Since the aggravating effect of domperidone was successfully reduced by simultaneous application of bromocriptine, we think that these effects are mediated by peripheral dopamine receptors. However, the mechanisms whereby dopamine receptor-stimulating and dopamine-releasing drugs produce their beneficial effects remain to be elucidated.

The influence of dopamine agonists and antagonists on indomethacin lesions in stomach and small intestine in rats.

Dopamine agents (saline in control groups) were coadministered with indomethacin by either single or repeated application. The ulcerogenic effect (erosions and/or ulcers) of repeated given indomethacin on gastric mucosa differed clearly from that on intestinal mucosa. The effect on intestinal mucosa was markedly greater than after a single dose. The effects of dopamine agents appeared to be more consistent. Domperidone and haloperidol, given as single or repeated doses, strongly aggravated both the gastric and intestinal lesions. Bromocriptine and amantadine had a protective effect. The adverse effects of both dopamine antagonists (increased after repeated administration) were strongly inhibited by the simultaneous administration of either bromocriptine or amantadine. The involvement of the dopamine system (central or peripheral) in the mechanisms that maintain gastric (probably related to cytoprotection also) and intestinal mucosa integrity is therefore suggested.

The influence of dopamine agonists and antagonists on gastric lesions in mice.

The dopamine agonist bromocriptine and L-dopa significantly inhibited whereas dopamine antagonist haloperidol aggravated the gastric lesions induced by pylorus ligation in mice as found earlier for rats. Furthermore, the successful use of a dopamine antagonist alone for the induction of gastric lesions also in mice was demonstrated, since the gastric lesions were induced by a single dose of haloperidol without any additional noxious treatment. Bromocriptine successfully inhibited both the gastric lesion-potentiating as well as the gastric lesion-inducing effect of haloperidol.

Follicular fluid contents of hyaluronic acid, follicle-stimulating hormone and steroids relative to the success of in vitro fertilization of human oocytes.

OBJECTIVES: To determine the concentrations of hyaluronic acid, FSH, P, and E2 in the follicular fluid (FF) obtained from IVF-ET patients and to assess the value of these measurements in predicting the outcome of fertilization. DESIGN: One hundred eleven samples were retrospectively analyzed for the hyaluronic acid and hormone contents. SETTING: University-based tertiary care center. PATIENTS: Preovulatory FF samples were collected from 67 women undergoing IVF-ET treatment because of tubal absence or obstruction. MAIN OUTCOME MEASURES: The FF hyaluronic acid and hormone concentrations were compared according to the type of ovulation induction, follicular development, and IVF outcome. RESULTS: According to the type of ovulation induction, a significantly lower hyaluronic acid concentration was found in FF harvested from the patients treated with GnRH agonist-hMG. No significant correlation was found between FF hyaluronic acid and either morphological maturity of the oocyte-cumulus complex or fertilizability of oocytes. The level of FSH was significantly higher in FF, yielding a mature oocyte-cumulus complex and from which the oocyte obtained successfully fertilized and cleaved. A significant increase in the E2 concentration was found in FF in which mature cumuli oophori were present. The levels of hyaluronic acid significantly correlated with FSH in FF. CONCLUSIONS: Expansion of the human oocyte-cumulus cell complex is an FSH-dependent phenomenon. The data are in agreement with the hypothesis that intrafollicular FSH plays an important role in the secretion of hyaluronic acid by granulosa cells and may act synergistically with E2 to enhance cytoplasmic maturation, resulting in successful fertilization.

Histochemical demonstration of a delta 5,3 beta-hydroxysteroid dehydrogenase activity of cumulus cells related to the maturity and developmental potential of recovered oocytes.

A simple and rapid histochemical technique is described for demonstration of delta 5,3 beta-hydroxysteroid dehydrogenase activity in cumulus cells from preovulatory follicles aspirated for in vitro fertilization (IVF) of corresponding oocytes. Histochemical activity of delta 5,3 beta-hydroxysteroid dehydrogenase was demonstrated in samples of cumulus obtained from 62 oocytes recovered from 24 women. Patients were treated with clomiphene citrate in combination with human menopausal gonadotropins and human chorionic gonadotropin injections. The cumulus was found to contain small and large cell types. Small cells possessed more delta 5,3 beta-hydroxysteroid dehydrogenase activity predominantly in the area near the oocyte. Cytoplasmic vacuolation has been noted in large, pale cells with moderate or low enzyme activity. The most active cells were predominant in cumulus from which oocytes were fertilized. Significant differences have been found between high and low delta 5,3 beta-hydroxysteroid dehydrogenase activity of cumulus cells from mature oocyte-corona-cumulus complexes leading to a successful fertilization and cleavage of oocytes and between groups with different histochemical activity when aspirated complexes were scored immature and the IVF of oocytes has failed.

Milk versus TEST-yolk--preincubated sperm; in vitro fertilization outcome.

OBJECTIVE: Preincubation of sperm in TEST-yolk medium enhances in vitro fertilization (IVF) outcome. However preincubation of sperm in milk at 5 degrees C enhances the results of sperm penetration assay and hemizona assay. This study was therefore performed to determine the influence of milk on in vitro fertilization rate of human oocytes, as compared with TEST-yolk medium. STUDY DESIGN: Forty-one consecutive couples undergoing an IVF procedure were randomized. Of these 20 couples were admitted for the milk study (group 1) and 21 couples for the TEST-yolk study (group 2). Each ejaculate was Percoll-processed and the sperm pellet was resuspended in 0.5 ml of culture medium. An equal volume of heat-inactivated homogenized cow's milk (95 degrees C, 10 min) was added to sperm suspension from group 1 and an equal volume of TEST-yolk medium was added to sperm suspension from group 2. After 2 h of incubation at 5 degrees C and washing with culture medium at 37 degrees C, oocytes were inseminated. Oocytes from group 1 couples were inseminated with milk-treated spermatozoa and those from group 2 couples with yolk-treated spermatozoa. Oocytes were evaluated for fertilization after 18 h. RESULTS: Sperm preincubated in milk fertilized 75 out of 100 mature oocytes (75%). TEST-yolk-treated sperm fertilized 45 out of 64 mature oocytes (70%). The difference was not statistically significant. CONCLUSIONS: Preincubation of spermatozoa in milk, as compared with preincubation in TEST-yolk medium yields a similar IVF outcome, so milk may be a suitable alternative medium for preincubation of spermatozoa to enhance its fertilizing potential.

Chromosomal preparations of human triploid zygotes and embryos fertilized in vitro.

Forty-eight zygotes with more than two pronuclei were identified after in vitro fertilization, representing 6.1% of all fertilized oocytes. The chromosome preparations from pronuclear stage to the cleaved human embryos were examined. Prophase was found in eight out of ten zygotes. The spreading of chromosomes allowed an adequate counting in only two cases. Six of the eight preparations displayed a late prophase. In this stage each haploid group of chromosomes can be analysed separately. Kariogamy usually occurred 4 to 5 h after the pronuclei had disappeared, and polyploid number of chromosomes were found in well-spread metaphases. The chromosomal preparations were made for eleven human embryos arising from zygotes with three pronuclei. Out of ten preparations, where the chromosomes could be counted, seven embryos (70%) contained hypodiploidic groups of chromosomes. In two of the cases, however, triploid metaphases were found, and in the last one a triploid/diploid mosaicism.

Prostaglandin F2 alpha, progesterone and estradiol concentrations in human follicular fluid and their relation to success of in vitro fertilization.

Prostaglandin F2 alpha (PGF2 alpha), progesterone (P4) and estradiol-17 beta (E2) in follicular fluid were measured by radioimmunoassay in patients undergoing in vitro fertilization and embryo transfer. Follicular growth was induced using clomiphene citrate-hMG-hCG (15 patients) and FSH-hMG-hCG (4 patients). There was no significant difference in follicular fluid PGF2 alpha and P4 concentrations relative to oocyte maturity as assessed morphologically. The highest PGF2 alpha concentration was found in fluid from FSH-hMG-hCG cycles where fertilization occurred. The value is significantly higher (p less than 0.002) than in fluid from clomiphene-hMG-hCG cycles whether fertilization took place or not. There was no significant difference in P4 and E2 levels in relation to the type of ovarian induction or success in fertilization. Positive correlation between P4 and E2 in follicular fluid was found (r = 0.404). The positive correlation between total dose of hMG given to the patients and PGF2 alpha concentration in their preovulatory follicular fluid (r = 0.434) suggests that PGF2 alpha is secreted locally as the result of hMG and hCG stimulation. It is proposed that PGF2 alpha could be a biochemical marker for assessing the success of in vitro fertilization.

Number of follicles, oocytes and embryos in human in vitro fertilization is relative to serum estradiol and progesterone patterns during different types of ovarian hyperstimulation.

Preovulatory serum estradiol and progesterone levels as well as their ratio were compared in different types of ovulation induction in order to determine whether these findings could be used to predict the number of preovulatory follicles, number of oocytes aspirated and embryos obtained. Significantly more oocytes were retrieved by follicular aspiration and significantly more embryos developed in patients receiving gonadotropin-releasing hormone agonist and human menopausal gonadotropins than in those given other ovulation inductors. On days -2 and -1 of the cycle, serum estradiol levels were significantly lower in pure follicle-stimulating hormone induction. Serum progesterone was significantly higher in pure follicle-stimulating hormone cycles on days -4 and -3. In clomiphene citrate and human menopausal gonadotropin induction, progesterone levels were significantly lower on days -2 and -1, and on the day of follicular aspiration. Ratios of estradiol/progesterone were lower in pure follicle-stimulating hormone group from day -3 to day -1 of the cycle. A significant correlation was found between estradiol and progesterone serum levels and the numbers of preovulatory follicles, oocytes and embryos. The study revealed the usefulness of serum estradiol and progesterone determinations in assisted reproduction.

Comparison of protective media and freezing techniques for cryopreservation of human semen.

OBJECTIVE: To evaluate the influence of cryopreservation medium and freezing-thawing techniques on human sperm motility and morphology. STUDY DESIGN: 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST-yolk ¿zwitterion-citrate-egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]¿ and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST-yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40-50 and >50%) in fresh semen. RESULTS: Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST-yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean+/-standard deviation) was 49+/-15.7, 43+/-15.2 and 52+/-16.8% when TEST-yolk was used and 34+/-17.8, 32+/-18.2 and 50+/-13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST-yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57+/-26.4, 38+/-8.6 and 38+/-17.3% in groups with >50, 40-50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using SAS/STAT software. CONCLUSIONS: Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST-yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.

The triple-marker test in predicting fetal aneuploidy: a compromise between sensitivity and specificity.

OBJECTIVE: to review the contribution of unconjugated estriol in Down's syndrome detection, and influence of maternal age, cut-off choice, and population specificity on the balance between triple-marker test sensitivity and specificity. STUDY DESIGN: Prenatal karyotyping was performed in 2833 pregnant women, 73% of them over the age of 34. Duration of gestation was determined by ultrasound in 98% of women. Prior to amniocentesis, the serum levels of alpha-fetoprotein, human chorionic gonadotropin and unconjugated estriol were evaluated and corrected for weight. The risk of trisomy 21 was calculated for the first 986 subjects using default medians, and for 1847 by our population-specific medians. The cut-off was initially 1:300 at term, but the 1:100 and 1:200 risks were also tested. Down syndrome risk was calculated with alpha-fetoprotein and human chorionic gonadotropin combination as well. Linear logistic regression model was performed to test the ability of aneuploidy markers to classify patients into normal and trisomic groups. RESULTS: There were twelve cases of Down's syndrome, seven of trisomy 18, four of trisomy 13, and one trisomy 22. Four cases of aneuploidy (16.7%) referred to women younger than 35. With the cut-off risk of 1:300, detection rate was 87.5% and specificity 63.3%, and with 1:100, 66.7% and 79.5%, respectively. The sensitivity for Down's syndrome was from 75% for cut-off=1:100 to 92% for cut-off=1:300, while detection of other trisomies was less successful (58% and 83%, respectively). Exclusion of unconjugated estriol MoM from the risk calculations reduced detection rate by 33% and improved specificity by 4% independently of cut-off choice. Linear logistic regression analysis showed that only unconjugated estriol was able to correctly classify patients between normal and trisomy 21 (p=0.011, odds ratio=1.445), and normal and trisomy 18 (p=0.0023, odds ratio=1.96) groups. CONCLUSIONS: The unconjugated estriol significantly contributes in Down's syndrome detection with cost of slightly reduced specificity. The 1:300 risk caused an unfavorable compromise between sensitivity and specificity. A higher cut-off, 1:100, would indicate performance of amniocentesis in women aged 39 years and older, and in those aged 35-39 only after the screen-positive result.

Protein concentration in pre-ovulatory follicular fluid related to ovarian stimulation.

Sixty follicular fluids obtained from 26 women with either clomiphene citrate and human menopausal gonadotropins (hMG) or hMG-induced ovulation were analyzed for the contents of total proteins, fibrinogen, plasminogen, antithrombin III, ceruloplasmin, alpha-2 macroglobulin, alpha-1 antitrypsin and immunoglobulins (IgG, IgA, IgM). Concentrations of these proteins was correlated to the type of ovarian follicle growth induction. Follicular fluids from patients stimulated with clomiphene citrate-hMG contained significantly higher concentrations of ceruloplasmin than those treated with hMG alone. No significant differences in the concentrations of other proteins were noted between the two types of ovarian induction. A multivariate data analysis resulted in three Varimax factors (VRX I) suggesting that proteins with antiprotease activity in the follicular fluid may play a role in human follicle maturation. Follicular fluid Ig may reflect the degree of follicular wall permeability under hMG treatment. Accordingly, it may be assumed that a combination of different proteins described by VRX factors could be used for evaluation of ovarian stimulation.

Contact allergy in children.

With a view to determining the frequency of contact allergy in children, a group of 100 subjects 5 to 15 years of age with various dermatoses was examined. The study group included children with allergic dermatoses (atopic dermatitis, eczema, and others) as well as nonallergic skin diseases (alopecia areata, psoriasis, vitiligo). The frequency of contact allergy occurrence in the individual groups has been determined. Contact allergy occurred the most frequently in children with various forms of eczema (60 percent), the least frequently in those with nonallergic dermatoses (32 percent). Children with eczema most often displayed a polyvalent allergy. Most frequently, positive patch test reactions occurred with potassium dichromate (in 21 percent of children), with cobalt chloride (in 11 percent), and with neomycin sulfate (in 10 percent).

Intramolecular coupling of BrO stretching vibrations in solid bromates, infrared and Raman spectroscopic studies on M(BrO3)2.6H2O (M = Mg, Co, Ni, Zn) and Ni(ClO3)2.6H2O.

Infrared and Raman spectra of the isostructural cubic halate hexahydrates M(BrO3)2.6H2O (M = Mg, Co, Ni, Zn) and Ni(ClO3)2.6H2O (space group, Pa3; Z = 4) are presented. They are discussed with respect to the strength of the O-H...OXO2 hydrogen bonds (hydrogen bond acceptor capability, synergetic effect) and the order of the BrO stretching modes. In the case of undistorted bromate ions, e.g. at C3 lattice sites, the order of the symmetric (v1) and asymmetric (v3) XO stretching modes is v1 < v3 as for ClO3- but in contrast to IO3- with v1 > v3. The relative order of v1 and v3 of halate ions is mainly governed by the specific masses of the halogen atoms and the angles of the XO3- ions. The latter decrease in the sequence ClO3- (107degrees) > BrO3-> IO3- (< 100 degrees). The Raman scattering intensities of the asymmetric XO stretching modes v3 of the title compounds are unusually low (< 5% those of v1).

Effect of pulse Doppler ultrasound on placental hormone excretion in vitro.

There is no literature data on the effects of ultrasound on the metabolism of placental tissue. In the present study, the authors have investigated the possible influence of pulsed field Doppler ultrasound on hormonal excretion of human placental trophoblasts in vitro. For the detection of placental hormones, specific immunoassays for chorionic gonadotropin (hCG), human placental lactogen (hPL), estrogen and progesterone were used. Compared to control trophoblast hormone excretion, pulsed field ultrasound exposure induced no changes in the excretion of the hormones in vitro. The authors were unable to demonstrate any alterations caused by pulsed field Doppler ultrasound in this in vitro model. Additional studies are required to deepen our understanding of the effect of pulsed Doppler ultrasound on other biomolecules and their metabolism.