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Biomedical subjects

A Bongso

Publications and source records attributed to A Bongso.

At least 19 recordsLinked to original sources
PubMed1998

The first 2 case reports of frozen embryo donation twin pregnancies in Singapore: hormonal profiles and obstetrical outcome.

A 35 year old women with premature ovarian failure and another 30 year old women with gonadal dysgenesis were the recipients of donated supernumerous frozen embryos after successfully prepared with cyclic steroid replacement therapy as described previously. One patient received 4 and the second 2 frozen embryos, transferred transcervically on the 3rd day of progesterone administration. Both patients had viable twin pregnancies. The plasma beta hCG levels for both patients at 2 weeks post replacement (4 weeks gestation) were lower than the median values in our normal, uncomplicated singleton pregnancy for the same gestation. The level after 4 weeks post-replacement (6 weeks gestation) became comparable. Plasma progesterone profiles suggested a level of above 70 ng/ml would be enough to support the twin pregnancies. The first patient developed antepartum haemorrhage of unknown origin at 34 weeks of gestation preceding preterm premature rupture of membranes and subsequently had preterm labour. The second patient developed proteinuric hypertension at 33 weeks of gestation. Both ended in a lower segment cesarean section. Both sets of twins and their mothers were discharged well.

Adult↗
PubMed1997

Ongoing normal pregnancy after transfer of zona-free blastocysts: implications for embryo transfer in the human.

Recent reports suggest that transfer of day 5 blastocysts improves implantation rates in in-vitro fertilization programmes. This paper reports a successful ongoing pregnancy after the transfer of zona-free day 6 expanded and hatching blastocysts. The patient was 37 years old and had undergone six stimulated and two thaw cycles previously, all of which had failed. Stimulation was by down-regulation and oocytes recovered transvaginally by ultrasound guidance. Two pronuclear embryos were co-cultured on Vero cells to day 6. The zonae of two hatching and two fully expanded blastocysts were removed using 0.5% pronase, and the zona-free blastocysts were then transferred. Pregnancy was confirmed on day 18 with a positive human chorionic gonadotrophin (HCG) and ultrasound at 6 weeks showed a single healthy fetal heart inside a clear sac. At 14 weeks a triple test (oestriol, J-HCG and alpha-fetoprotein) was normal and at 22 weeks a detailed ultrasound scan showed no congenital anomalies. This is the first report in the human of a normal ongoing pregnancy after the transfer of zona-free day 6 embryos.

Adult↗
PubMed1997

Analysis of cystic hygroma, ascitic, and pleural fluids by conventional lymphocyte culture and fluorescent in situ hybridization.

Serous fluids from cystic hygromas, pleural effusions, and ascites are an easily accessible and plentiful source of lymphocytes. The feasibility and reliability of using these as alternative sources to conventional amniotic fluid or fetal blood cultures have been studied here. In some cases of prenatal diagnosis, especially in pregnancies complicated by the presence of cystic hygromas and fetal hydrops, obtaining amniotic fluid or fetal blood can be difficult due to obstruction by the cyst or oligohydramnios. A total of 14 cases with fetal hydrops detected ultrasonigraphically between 15 and 33 weeks of pregnancy over a period of 1 year have been subjected to conventional amniotic fluid or fetal blood karyotyping, along with samples of fluids from cystic hygromas, ascites or pleural effusions as obtained. Pleural fluids (n = 4), cystic hygroma fluids (n = 5), and ascitic fluids (n = 6) were obtained. The culture failure rate was low, 2/14. Karyotypically, two of the fluids, both from cystic hygromas, were 45,X; the rest were normal. A rapid 1-day additional test of fluorescent in situ hybridization (FISH) was carried out on uncultured cells of the alternative fluids using probes for the most commonly occurring aneuploidies, 13, 18, 21, X and Y, with good results.

Ascitic Fluid↗
PubMed1997

Fluorescent in-situ hybridization--some of its applications in clinical cytogenetics.

Fluorescent in-situ hybridization (FISH) is becoming more and more relevant as an important future tool in prenatal and pre-implantation genetic diagnosis and cancer cytogenetics. This review describes the FISH technique as applied to whole chromosome spreads and interphase cells and discusses its applications in clinical cytogenetics. Information is presented on the various types of probes and the subsequent hybridization and detection procedures. The potential use of this novel FISH technique in the diagnosis of numerical and structural chromosomal aberrations in routine karyotyping for prenatal diagnosis, tumour cytogenetics and pre-implantation genetic diagnosis is outlined.

Chromosome Aberrations↗
PubMed1996

Intracytoplasmic injection of human sperm into the hamster oocyte (hamster ICSI assay) as a test for fertilizing capacity of the severe male-factor sperm.

OBJECTIVE: Our objective was to investigate the fertilizing ability of human sperm from severe male-factor patients, by microinjection of single sperm into the hamster oocyte. DESIGN: Semen samples of severe male factor either with a 0% penetration rate in the zona-free hamster test or with a very low number of motile sperm for which performing the standard penetration test was impossible were used. For the control study, oligozoospermic semen samples with at least 10% penetration rate in zona free hamster test were used. SETTING: All materials were collected from the National University Hospital, Singapore. METHODS: There were 10 patients in both the experimental and the control groups. Intracytoplasmic sperm injection (ICSI) was carried out. The main outcome measures were sperm head decondensation and pronuclear formation. RESULTS: Twenty-one percent of the injected sperm could decondense and undergo male pronuclear formation. This rate was not significantly different from that in the control study group (28%; P = 0.13). A small proportion of the oocytes was damaged during the procedure (9.2 and 8.75% in experimental and control groups, respectively). CONCLUSIONS: Hamster-ICSI assay may be of benefit in predicting the sperm's ability for further development before allowing the patient to undergo the clinical program.

Animals↗
PubMed1996

Fertilization, embryonic development and implantation of mouse oocytes with one or two laser-drilled holes in the zona, and inseminated at different sperm concentrations.

The zonae pellucidae of mouse oocytes were photo-ablated by an ultraviolet laser (337.1 nm) to create one or two 10 microm holes. The pulse energy used was approximately 3 microJ/s, with a frequency of 10 pulse/s. These laser zona-drilled (LZD) oocytes with one hole (LZD1) or two holes (LZD2) and the zona-intact controls were inseminated with spermatozoa at standard concentrations of 5x10(4), 5x10(5), 1x10(6) and 2x10(6)/ml. Fertilization was significantly improved at all sperm concentrations in LZD1 and LZD2 oocytes as compared to the controls. However, there was no significant difference in fertilization rates between LZD1 and LZD2. LZD2 produced significantly higher numbers of blastocysts at day 5. Hatching was significantly enhanced in the presence of either one or two holes in the zona. Polyploidy was generally absent, except in LZD2 oocytes (1%) inseminated at higher sperm concentrations. Differential cell counts of expanded LZD blastocysts were similar to those of the controls. Significantly fewer LZD2 blastocysts implanted and produced viable fetuses than LZD1 and control blastocysts. Morphological abnormalities of the fetuses were absent in all three groups. Laser zona-drilling using the ultraviolet laser was shown to be fast, efficient and safe.

Animals↗
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↗