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Biomedical subjects

A Bongso

Publications and source records attributed to A Bongso.

At least 37 records · Page 2Linked to original sources
PubMed1996

Evaluation of motility, freezing ability and embryonic development of murine epididymal sperm after coculture with epididymal epithelium.

Murine sperm from the caput, corpus and cauda epididymis were cocultured with epididymal epithelial cells of their own region or more distal regions, in the presence and absence of androgens (testosterone and dihydrotestosterone). Epithelial cell cultures were used 3 or 10 days after preparation in a complex tissue culture medium (Chang's) as plated tubules. The coculture studies involving spermatozoa and oocytes with epithelial cells were carried out in T6 medium. Motility of caput spermatozoa was maintained for 24 h in the presence of day 3 corpus and cauda epithelial cells and hormones but not under other conditions. Likewise, the motility of corpus spermatozoa was maintained for 24 h in the presence of day 3 cauda epithelial cells and hormones but not other conditions. Fertilization of zona-intact oocytes by epididymal spermatozoa was not affected by their coculture for 24 h with epithelial cells but fertilization rates for zone-free oocytes were increased for caput spermatozoa cocultured with more distal epithelial cells. Fertilization rates for both zona-intact and zone-free oocytes were increased for corpus spermatozoa cocultured with more distal cauda epithelial cells. The developmental capacity of embryos derived from caput spermatozoa was not significantly increased by coculture with epithelial cells but those derived from corpus spermatozoa cocultured with cauda epithelial cells were significantly increased. We conclude that the presence of more distal epithelial cells of the mouse epididymis maintains motility in culture, increases the ability of caput and corpus spermatozoa to fertilize zona-free oocytes and increases the developmental capacity of embryos formed from corpus spermatozoa. These observations demonstrate the function of epididymal regions in the maturation of murine spermatozoa for fertilization and embryo development.

Animals↗
PubMed1996

Update on micromanipulation techniques for assisted conception.

This article reviews the progress in the field of micromanipulation in the past year. The major event has been the wide application of intracytoplasmic sperm injection. Other advances include haploid spermatid injection and the development of successful mouse models for intracytoplasmic sperm injection. There is now increasing awareness of the genetic basis of male infertility and its possible impact on how we offer treatment.

Animals↗
PubMed1995

Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals.

The objective of this investigation was to determine whether intracytoplasmic sperm injection (ICSI) can be performed in the mouse. Metaphase II oocytes were obtained from F1 hybrid mice (C57BL x CBA) by i.p. injections of 10 IU pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) administered 48 h apart. Oocytes with cumulus oophorus were retrieved 13-14 h post HCG. Cumulus was dispersed with 0.1% hyaluronidase. Mouse spermatozoa were obtained from the cauda epididymides of males of the same strain. The spermatozoa were processed by the standard swim-up procedure. The harvested spermatozoa were then incubated for 1.5 h to allow capacitation. Healthy oocytes were injected with 3-4 pl 5 mM Ca2+, followed by one live morphologically normal spermatozoon into the cytoplasm at intervals of 0, 0.5, 1, 2 and 3 h. The proportion of 2-cell embryos that developed from oocytes injected with Ca2+ and spermatozoa ranged between 29.5 and 36.5% in all groups, with no statistical difference between treatments. Chromosomal analysis showed that two-thirds of the ICSI-derived 2-cell embryos were diploid. The proportion of parthenogenetically activated embryos in the ICSI groups was similar to that in the control group (8-10%) which was injected with Ca2+ and polyvinyl pyrrolidone only. The proportion of blastocysts that developed in culture from the ICSI-derived 2-cell embryos was of the order of 36-42%. Some blastocysts were used for cell number counts. There was a significant increase in total and inner cell mass counts of blastocysts in which the spermatozoon was injected at 2 and 3 h following Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Human embryonic behavior in a sequential human oviduct-endometrial coculture system.

Embryonic behavior to blastocyst, hatching, and hatched stages were evaluated in 77, four-cell human embryos that were first grown in oviductal cell coculture and then equally allotted at the eight-cell stage to two coculture systems in a serum-free medium (34 continued on oviductal monolayers, 32 on endometrium monolayers). Sixty-three percent and 40% of embryos expanded and hatched in the sequential oviductal-endometrial coculture system when compared with 41% and 9% in the oviductal system alone, respectively. The sequential coculture system appears to be an improved system over the single human oviductal coculture system.

Culture Techniques↗
PubMed1994

Early sperm-egg interaction after sperm microinjection.

The early events of sperm-egg interaction occurring 1-3 h after multiple sperm injection into the perivitelline space (PVS) and after direct injection into the ooplasm of human oocytes are reported. The sperm acrosome reaction occurred in the PVS but was not detected within the ooplasm. Sperm in the PVS were incorporated into the ooplasm in the usual manner described in vitro, after completion of the acrosome reaction, and a block to polyspermy was evident at the oolemma. However, sperm incorporation into the ooplasm was not clearly defined and requires further investigation. Sperm were also incorporated into oolemma-bound vacuoles within the ooplasm and breaches in the ooplasm were seen after intracytoplasmic injection. Both normal and abnormal sperm were found in the PVS and ooplasm, even though sperm from donors with normal semen parameters were used. The effect of freezing in liquid nitrogen on demembranation of sperm for microinjection is also reported.

Acrosome↗
PubMed1994

Isolation and culture of inner cell mass cells from human blastocysts.

Totipotent non-committed inner cell mass (ICM) cells from human blastocyts, if demonstrated to be capable of proliferating in vitro without differentiation, will have several beneficial uses, not only in the treatment of neurodegenerative and genetic disorders, but also as a model in studying the events involved in embryogenesis and genomic manipulation. Nine patients admitted to an in-vitro fertilization programme donated 21 spare embryos for this study. All 21 embryos were grown from the 2-pronuclear until blastocyst stages on a human tubal epithelial monolayer in commercial Earle's medium (Medicult, Denmark) supplemented with 10% human serum. The medium was changed after blastocyst formation to Chang's medium supplemented with 1000 units/ml of human leukaemia inhibitory factor (HLIF) and the embryos left undisturbed for 72 h to allow the hatched ICM and trophoblast to attach to the feeder monolayer. Nineteen of the 21 embryos from nine patients produced healthy ICM lumps which could be separated and grown in vitro. Two of the lumps differentiated into fibroblasts while the remaining 17 (eight patients) produced cells with typical stem cell-like morphology, were alkaline phosphatase positive and could be maintained for two passages. It was possible to retain the stem cell-like morphology, alkaline phosphatase positiveness and normal karyotype through the two passages in all of them using repeated doses of HLIF every 48 to 72 h. This is the first report on the successful isolation of human ICM cells and their continued culture for at least two passages in vitro.

Adult↗
PubMed1994

An interesting case of intersex: case report.

Intersex is a rare problem. Ambiguous genitalia, a frequent presentation of intersex, is seldom due to true hermaphroditism. This case report describes the findings in such a rare case.

Adolescent↗
PubMed1994

Differential regulation of cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzyme mRNA expression by gonadotrophins and cyclic AMP in human granulosa cells.

The co-ordinated biosynthesis of progesterone and oestradiol in the human ovary is critical for reproductive cyclicity and eventual pregnancy. The crucial regulatory enzymes for progesterone and oestradiol biosynthesis in granulosa cells are the cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzymes respectively. We utilized the cDNA sequences encoding P450arom and P450scc to examine the roles of FSH and LH, and their intracellular second messenger, cyclic AMP (cAMP), in regulating steroidogenic gene expression. Mature granulosa cells (aspirated before the onset of the endogenous LH surge) and granulosa lutein cells (obtained after an ovulatory dose of human chorionic gonadotrophin) were cultured for 4 days with FSH, LH or dibutyryl cAMP (dbcAMP). After the period of culture, total RNA was extracted from granulosa cells and Northern analyses were performed utilizing 32P-labelled cDNAs encoding P450arom and P450scc. Spent culture media were analysed for steroid and cAMP content. Both FSH and LH strongly stimulated P450arom mRNA expression and oestradiol production in mature granulosa cells. On the other hand, P450scc mRNA expression and progesterone biosynthesis were weakly induced by FSH; maximal synthesis occurred only in the presence of LH. With both gonadotrophins at equivalent concentrations, LH generated a 30-fold higher level of cAMP than FSH. Furthermore, the differential effects of FSH and LH on P450 mRNA expression were reproduced by the presence of low and high concentrations of dbcAMP respectively. LH (and high levels of dbcAMP) increased P450arom mRNA expression in mature granulosa cells but inhibited its accumulation in granulosa lutein cells. In contrast, it stimulated P450scc mRNA expression and progesterone synthesis in both mature granulosa and granulosa lutein cells. Therefore, FSH/low cAMP levels stimulated P450arom gene expression and oestradiol production, while LH/high cAMP levels maximally induced P450scc gene expression and function, in a development-related manner consistent with steroid production in vivo. These findings support the hypothesis that one set of genes (like P450arom) in human granulosa cells is regulated by FSH/low cAMP levels and another (like P450scc) by LH/high cAMP levels.

Aromatase↗
PubMed1994

Evaluation of inhalational anaesthetics on murine in vitro fertilization.

Chronic exposure to inhalational anaesthetics has been implicated in producing adverse reproductive effects in exposed personnel. A longer duration of general anaesthesia during laparoscopic oocyte retrieval for human in vitro fertilization programmes was associated with a decrease in fertilization and cleavage rates. In this study, a mouse model was used to evaluate the effects of clinical concentrations of nitrous oxide, isoflurane and halothane on in vitro fertilization. Mouse oocytes were exposed to 3 inhalational anaesthetic mixtures, oxygen/nitrous oxide, oxygen/nitrous/isoflurane 0.5% or oxygen/nitrous oxide/halothane 0.5% just prior to insemination and their subsequent development was assessed. The fertilization (2-cell) rate and early embryonic growth rate, i.e., 4-cell and morula rates were not statistically different from those of controls which were exposed to air. This study showed that short exposures to clinical concentrations of nitrous oxide, isoflurane and halothane had no deleterious effect on in vitro fertilization and early embryonic growth up to the morula stage in the mouse. Their effects on subsequent implantation and foetal teratogenecity are unknown.

Anesthetics, Inhalation↗
PubMed1993

Mitogenic and cytogenetic evaluation of transforming growth factor-beta on murine preimplantation embryonic development in vitro.

Slow cleavage rate has been a major contributory factor influencing embryo morphology in in vitro fertilization (IVF) programs. The role of transforming growth factor-beta (TGF beta 1) in improving this characteristic was evaluated using the murine model. Replicate batches of eight-cell compacting embryos from superovulated mice were divided into three groups. Group A were treated with 0.3 ng/ml TGF beta 1 at the initial compacting stage, followed by a second treatment of 0.1 ng/mL 22 h later at the cavitating stage; group B received 0.3 ng/ml TGF beta 1 at the cavitating stage; group C were controls. The percentages of treated embryos reaching fixed embryonic stages, total cell number (TCN), mitotic index, and incidence of chromosome anomalies were monitored. The percentage of embryos reaching the cavitating, expanded, hatching, and hatched stages in both treatment groups were not significantly different from control (96.6% +/- 4.2% to 37.7% +/- 12.7% vs. 95.3% +/- 7.3% to 47.0% +/- 3.5%; P > 0.05). Values between the two treatment groups were also not significantly different. Embryos in groups A and B produced significantly greater TCN at expanded blastocyst and hatching stages compared to controls (Group A: 107.0 +/- 18.9 vs. 89.9 +/- 17.4, P < 0.05 and 125.5 +/- 16.4 vs. 113.9 +/- 12.1, P < 0.05; Group B: 107.9 +/- 14.0 vs. 89.9 +/- 17.4, P < 0.05 and 124.9 +/- 17.4 vs. 113.9 +/- 12.1, P < 0.05). Values, however, were not significantly different between treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Fetal blood sampling and its complications related to the indications for fetal blood sampling.

A prospective study on fetal blood sampling (FBS) was conducted in the Fetomaternal Medicine Division of the Department of Obstetrics and Gynaecology at the National University Hospital, Singapore. FBS was performed on 159 occasions in 156 women between January, 1988 and December, 1991. The aim of this study was to identify the factors that were associated with an adverse outcome following the procedure. Twenty four abnormal pregnancies were terminated; of the remaining 132 desired pregnancies the overall pregnancy loss was 44 (33.3%), which included those within 2 weeks and those after 2 weeks of the procedure and neonatal deaths. Fetal loss occurring within 2 weeks of the procedure is considered a procedure-related loss which occurred in 19 (14.3%) of the 132 pregnancies. When the fetal loss occurred within 2 weeks of the procedure 89% had a major abnormality on ultrasonographic scanning. The conclusion from our study is that the risks of FBS were increased in abnormal pregnancies, most likely due to the underlying pathology.

Blood Specimen Collection↗
PubMed1993

The effect of coculture on human zygote development.

It is well over a decade since the birth of the first test-tube baby and still the in-vitro conditions for early embryonic development remain suboptimal. The ideal culture medium to increase longevity and improve viability of human embryos is not available. Since the metabolic requirements of the human embryo changes from one cleavage stage to another, the development of a single culture medium for all stages could not be expected. The use of helper cells (coculture) in vitro offers much promise as there are numerous documentations in both man and animals describing their ability to increase blastulation rates and improve embryo viability. This paper reviews the effect of coculture on human zygote development. The selection and establishment of cell-lines, biologic actions of coculture of gametes and zygotes, the outcome, and future prospects are discussed.

Cell Line↗
PubMed1992

In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation.

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and beta-mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center.

Animals↗
PubMed1992

Embryonic behavior of two-cell mouse embryos frozen by the one- and two-step ultrarapid techniques.

PURPOSE: A modified two-step ultrarapid freezing technique was compared to the one-step ultrarapid freezing technique. Two-cell mouse embryos were frozen-thawed using the two freezing protocols, and postthaw cryoprotectant removal was carried out in either a single- or a multiple-step procedure. RESULTS: Statistically similar cryosurvival (96.95-100%) and blastocyst formation rates (87.95-91.47%) were obtained with both freezing groups. In addition, the method of cryoprotectant removal did not have any significant effect on the survival and development of the frozen-thawed embryos in both groups. Blastocysts formed following single-step cryoprotectant removal had significantly lower inner cell mass counts in the one-step than in the two-step group (26.14 and 27.59, respectively; P < 0.05). Embryo transfer studies showed that the implantation and fetal formation rates of embryos frozen by the two-step technique (61.67 and 60.0%, respectively) were similar to those of embryos frozen by the one-step technique (74.12 and 71.76%, respectively). CONCLUSION: These results demonstrate that the ultrarapid two-step technique is as effective in cryopreserving two-cell mouse embryos as the ultrarapid one-step technique.

Animals↗
PubMed1992

Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm.

OBJECTIVE: To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. DESIGN: Prospective. SETTING: Assisted reproductive technology (ART) program. PATIENTS: Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. RESULTS: Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P less than 0.01). Zona-free oocytes had higher fertilization rates than zona-intact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization). CONCLUSIONS: One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients.

Cell Division↗