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Biomedical subjects

M DiMattina

Publications and source records attributed to M DiMattina.

9 recordsLinked to original sources
PubMed1995

Extracorporeal development and ultrarapid freezing of human fetal ova.

PURPOSE: The present study was performed to culture human fetal ova to determine whether they can be matured and cryopreserved using ultrarapid freezing. METHODS: Thirty-three pairs of fetal ovaries were obtained from fetuses of 16-20 weeks' gestation following elective abortion. Ovarian tissues were minced into approximately 1-mm sizes and cultured in Waymouth media either before or after ultrarapid freezing. The Waymouth medium was supplemented with 15% (v/v) fetal bovine serum, 0.03 IU/ml FSH and 35 ng/ml insulin. The tissue was cultured at 37 degrees C in 5% CO2 in air for 5-25 days in Falcon dishes and 30-40 days in Costar Transwell-COL membranes prior to induction of final maturation in the presence of LH and human follicular fluid. Minced tissues were also frozen by ultrarapid freezing in M199 with 4.2 M dimethylsulfoxide (DMSO) and 0.35 M sucrose and plunged directly into liquid nitrogen. For thawing, the straws were plunged into a 37 degrees C water bath for 5 s. The contents were then expelled and diluted 1:5 with thawing medium containing 0.42 M sucrose. After washing the thawed tissues were cultured as described for the fresh tissues. RESULTS: Patches of monolayer consisting of fibroblasts had formed within 2-3 days of culture of fresh tissues. After 1 week of culture, follicles separated out from the ovarian tissue but remained attached to the monolayer. The maximal number of follicles separating out from the tissue appeared about 1 week after initiating the culture (154 follicles per 10 fields at Day 5 and 61 and Day 25). After 40 days of culture in Costar dishes, 34% of the ova reached a diameter of more than 80 microns, which was significantly higher than at the beginning of culture (6%; P < 0.05). Among these ova, 34% were found to be surrounded by the zona pellucida, which was not observed at the beginning of culture. Following induction of final maturation, extrusion of the first polar body was noted in 25% of ova grown in Costar dishes for 40 days. Twelve percent of the oocytes showed the first polar body when they were grown in Costar dishes for less than 30 days. For frozen-thawed tissues, 14% of minced ovarian tissues displayed central necrosis immediately after thawing. Following digestion and Trypan blue staining, 75% of ova and 85% of somatic cells survived ultrapid freezing. Nineteen percent of the ova which have been cultured as described for fresh tissues displayed extrusion of the first polar body, comparing favorably with the 25% maturation rate observed with the fresh tissue (P > 0.05). CONCLUSION: This study demonstrates that morphologically normal, mature human ova can be obtained from primordial follicles in vitro development. Using a simple, quick ultrarapid freezing method, human fetal ova can be cryopreserved in the form of minced tissue without significantly compromising their ability to grow in vitro.

Animals↗
PubMed1994

Zona-opening of hamster oocytes: a comparative study using macro- and micro-manipulation methods.

The present study compared a new macro-manipulation technique for zona-opening of hamster oocytes with existing micro-manipulation techniques. In experiment I the zona pellucida of hamster oocytes was partially opened with a 32 gauge steel needle (macro-manipulation) and these oocytes were then co-incubated with human spermatozoa at 36.5 degrees C in 5% CO2 in air for 3.5 h. Zona-free and zona-intact oocytes similarly inseminated served as controls. Of 113 oocytes, 30 (26%) lysed following zona-opening with the macro technique. The sperm penetration rates of zona-opened and zona-free oocytes were 37% (31/83) and 60% (49/81) respectively (P < 0.01), with 12 and 28% oocytes respectively showing polyspermia (P < 0.05). No sperm penetration occurred in zona-intact oocytes. In experiment II the zona pellucida of hamster oocytes was partially opened by macro- or micro-manipulation techniques before the oocytes were coincubated with human spermatozoa as in experiment I. Nine of 156 (6%) macro-manipulated and 11 of 133 (8%) micro-manipulated oocytes lysed following zona-opening. Of 147 remaining macro-manipulated and 122 remaining micro-manipulated oocytes, 80 (55%) and 79 (64%) respectively were penetrated (P > 0.05), with 30 and 37% respectively showing polyspermia (P > 0.05). These results demonstrate that macro-manipulation results in similar penetration and polyspermia rates as compared to conventional micro-manipulation.

Animals↗
PubMed1992

An improved medium for long-term culture of human embryos overcomes the in vitro developmental block and increases blastocyst formation.

OBJECTIVE: To determine if a culture medium, designated CZB after the authors who first described it, which is supplemented with 0.11 mM ethylenediaminetetraacetic acid, 1.0 mM glutamine, 31.30 mM lactate, and 0.27 mM pyruvate and is lacking glucose for the initial stages of culture that overcomes the in vitro two-cell block of mouse embryos, can improve the rate of blastocyst formation of human embryos in long-term cultures and increase the pregnancy rate (PR) when used in a clinical in vitro fertilization (IVF) program. DESIGN: The study is in two parts. Initially, excess oocytes from IVF and gamete intrafallopian transfer patients were fertilized in vitro and then placed in long-term culture of either CZB plus 10% heat-inactivated human serum (32 zygotes) or Earle's balanced salt solution (EBSS) supplemented with 0.45 mM pyruvate plus 10% human serum (28 zygotes) to determine if CZB medium could enhance in vitro development and increase blastocyst formation when compared with EBSS. Subsequently, CZB or EBSS medium was used for short-term cultures of embryos in a clinical IVF program to determine if the use of CZB could increase the clinical IVF PR. SETTING: Private practice of one author (M.D.). PATIENTS: The excess oocytes were donated by couples not wishing to have cryopreservation. In the clinical trial, 49 couples presenting with tubal or male factor infertility and who had three or more fertilized zygotes were randomly assigned to one of the culture media being used. RESULTS: In long-term cultures, embryos were observed at 42, 66, 90, 114, and 138 hours after fertilization and scored for blastomere number, degree of fragmentation, and developmental arrest. When CZB- and EBSS-cultured embryos were evaluated over 138 hours, there was a significant increase in the number of CZB-cultured embryos reaching the blastocyst stage (56% versus 20%; P less than 0.009) and less fragmentation of CZB-cultured embryos (18.8% versus 50%; P less than 0.01). In short-term cultures, pronuclear stage embryos from patients undergoing IVF were cultured in CZB or EBSS for 24 hours, graded, and then used in embryo transfers. Of the 28 patients assigned to EBSS, 6 became pregnant (21.4%), and of the 21 assigned to CZB, 5 attained pregnancy (23.8%). These results were not significantly different. CONCLUSIONS: The use of CZB for the long-term culture of human embryos is highly beneficial and increases the rate of blastocyst formation, but its use in an IVF program does not increase the clinical PR.

Analysis of Variance↗
PubMed1992

Serum beta-human chorionic gonadotropin, estradiol and progesterone as early predictors of pathologic pregnancy.

We prospectively studied 110 asymptomatic female infertility patients with serial serum measures of beta-human chorionic gonadotropin (hCG), estradiol (E2) and progesterone (P) to determine their sensitivity, specificity, predictive value and test efficiency, alone or in combination, for the prediction of pathologic gestations prior to five weeks after ovulation. Circulating levels of serum beta-hCG, E2 and P were measured at 48- or 72-hour intervals. Seventy-four patients (67%) had viable pregnancies, for which the abnormal changes in steroid levels were defined as: a beta-hCG rise of less than 66% in 48 hours or less than 120% in 72 hours, an E2 decline of greater than 15% in 48 hours or greater than 20% in 72 hours, or a P decline of greater than 25% in 48 hours or greater than 33% in 72 hours. Thirty-six women (33%) had pathologic pregnancies, which included ectopic pregnancies (8), spontaneous or missed abortions (7), blighted ova (anembryonic gestation, 20) and hydatidiform mole (1). For the detection of pathologic pregnancies in this asymptomatic infertility population, the sensitivity of beta-hCG, E2 and P, singly or in combination, ranged from 34% to 78%, and the test efficiency ranged from 68% to 88%. Beta-hCG alone provided the highest sensitivity (78%) and test efficiency (88%). When compared to measuring serial beta-hCG alone, serum E2 or P did not enhance the test efficiency and lowered the sensitivity for the detection of pathologic pregnancies in an asymptomatic infertility population.

Adult↗
PubMed1989

The inhibition of human adrenal steroidogenic enzyme activities by suramin.

Suramin has recently been used to treat patients with acquired immune deficiency syndrome because of the action of this drug on reverse transcriptase. Patients so treated developed the symptoms and hormonal profiles of adrenal insufficiency. To evaluate the mechanism of action of suramin on adrenalcortical function, adrenal mitochondrial and microsomal preparations from five subjects were assayed for steroidogenic enzyme activity in the presence and absence of suramin. Specifically, 3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, 21-hydroxylase, 11 beta-hydroxylase, and 17,20-desmolase activities were measured in the presence of 0-5000 mumol/L suramin concentrations. In all assays, enzyme activities decreased in a dose-dependent fashion as suramin concentrations increased. The drug doses (calculated) that caused 50% inhibition of enzyme activity were: 21-hydroxylase activity, 50 mumol/L; 17 alpha-hydroxylase activity, 25 mumol/L; 17,20-desmolase activity, 50 mumol/L; 11 beta-hydroxylase, 2 mumol/L, and 3 beta-hydroxysteroid dehydrogenase/isomerase, 1200 mumol/L. These results suggest that suramin has a concentration-dependent inhibitory effect on the key P-450-regulated enzymatic steps in adrenal glucocorticoid steroidogenesis, which may explain the development of adrenal insufficiency in acquired immune deficiency syndrome patients treated with suramin.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Ketoconazole inhibits multiple steroidogenic enzymes involved in androgen biosynthesis in the human ovary.

Ketoconazole (KZ) has been shown to inhibit testicular and adrenal steroidogenesis and is useful in the medical management of gonadotropin-independent precocious puberty, prostatic cancer, and Cushing's syndrome. To determine whether KZ similarly affects ovarian steroidogenesis, the authors examined its effect on the activity of the human ovarian 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), 17-hydroxylase (17-OH), and aromatase (AR) in vitro. A dose-dependent decrease in the activities of 3 beta-HSD and 17-OH was observed with increasing amounts of KZ. With 10, 50, and 100-fold excess KZ, the activity of 3 beta-HSD decreased by 59% (P less than 0.001), 73% (P less than 0.005), and 85% (P less than 0.005), respectively. At equimolar concentrations with substrate (50 microM), KZ inhibited 17-OH by 70% (P less than 0.01). No significant effect on ovarian AR activity was observed, except at the highest concentration of KZ tested. The authors conclude that low concentrations of KZ profoundly inhibit the activity of human ovarian 3 beta-HSD and 17-OH in vitro. These observations suggest that KZ might be useful in the medical management of women with hyperandrogenism, but further experimentation and clinical trials will be necessary.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

The effect of ketoconazole on steroidogenesis. II. Adrenocortical enzyme activity in vitro.

The activity of five adrenocortical steroidogenic enzymes, 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), 17-hydroxylase (17-OHase) 17,20 desmolase (17,20D), 21-hydroxylase (21-OHase) and 11-hydroxylase (11-OHase), were measured in vitro in purified mitochondria or microsomes from rhesus monkey (Macaca mulata) and human adrenal tissue in the presence and absence of graded concentrations of ketoconazole. Rhesus 3 beta-HSD activity was unaffected by ketoconazole at concentrations up to 5000 microM. However, human adrenal 3 beta-HSD was inhibited by approximately 40% (p less than .01) at concentrations of 500 microM and by 80% at 100 microM. 17-OHase and 17,20D were significantly inhibited in the human at 5 microM (p less than .001) and in the rhesus monkey at 50 microM (p less than .001). A similar inhibitory effect was found on microsomal 21-OHase, with significant inhibition at 5 microM ketoconazole in the human and rhesus monkey (p less than 0.001). Mitochondrial 11-OHase was also significantly inhibited by ketoconazole in both the human (p less than .005) and rhesus (p less than .001) at 2.0 microM. These results represent documentation of the specific adrenal steroidogenic steps affected by ketoconazole and confirm the observations that this imidazole derivative is a powerful inhibitor of enzymes in the glucocorticoid pathway.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Effect of the antiprogestin RU486 on human ovarian steroidogenesis.

Previous studies in the authors' laboratory suggest that the antiprogestin RU486 may directly affect human ovarian progesterone production. The possibility that this compound could affect other steps in human ovarian steroidogenesis was examined by studying its effects on estrogen production in cultured human granulosa cells and on human ovarian aromatase (AR) and 17-hydroxylase (17-OH) activities in vitro. RU486 had no effect on media estradiol (E2) levels as measured by radioimmunoassay (RIA) over a 24-hour incubation period. Furthermore, no effect on ovarian AR activity occurred at concentrations of RU486 100 times substrate. However, a dose-dependent decrease in the activity of 17-OH was observed with increasing amounts of drug. RU486 decreased 17-OH activity by 12 and 29% below that of basal activity at concentrations equal to and ten times substrate. At 50- and 100-fold excess, RU486 further decreased 17-OH activity by 42 (P less than 0.01) and 48% (P less than 0.005). In conclusion, RU486 directly inhibits human ovarian 17-OH activity, but does not affect AR activity or E2 production in vitro. Clinically observed decreases in serum E2 levels may be due to inhibition of enzymatic steps proximal to E2 synthesis. These findings support the authors' previous observations suggesting that RU486 has a direct affect on human ovarian steroidogenesis.

Adult↗