Development of quantitative-fluorescence polymerase chain reaction for the rapid prenatal diagnosis of common chromosomal aneuploidies in 1,000 samples in Singapore.

INTRODUCTION: We aimed to develop a rapid quantitative-fluorescence polymerase chain reaction (QF-PCR) to detect common foetal aneuploidies in the Singapore population within 48 hours of sample collection in order to alleviate parental anxiety. METHODS: DNA from 1,000 foetal samples (978 amniotic fluids, 14 chorion villi and eight foetal blood samples) was analysed using a QF-PCR of 19 microsatellite markers located on chromosomes 13, 18, 21, X and Y. A total of 523 samples were archived before the QF-PCR analysis (archived), while QF-PCR was performed and the results obtained within 48 hours of sample collection in the remaining 477 samples (live). The results were confirmed with their respective karyotypes. RESULTS: In total, 47 autosomal trisomies (T) were found: 30 among the archived (three T13, 12 T18, 15 T21) and 17 among the live (four T18, 13 T21) samples. The QF-PCR results were verified with their respective karyotypes. We achieved 100 percent sensitivity (lower 95 percent confidence interval [CI], 92.8 percent) and specificity (lower 95 percent CI, 99.5 percent), and the time taken from sample collection to the obtaining of results for the 477 live samples was less than 48 hours. CONCLUSION: Prenatal diagnostic results of common chromosomal abnormalities can be released within 48 hours of sample collection using QF-PCR. Parental anxiety is alleviated and clinical management is enhanced with this short waiting time.

Carbetocin versus syntometrine for the third stage of labour following vaginal delivery--a double-blind randomised controlled trial.

OBJECTIVE: Prevention of postpartum haemorrhage is essential in the pursuit of improved health care for women. However, limited literature is available for comparing the use of oxytocin agonist carbetocin with syntometrine in women undergoing vaginal deliveries. We aimed to compare intramuscular carbetocin with intramuscular syntometrine for the routine prevention of postpartum haemorrhage in women who deliver vaginally. DESIGN: Prospective double-blind randomised controlled trial. SETTING: Tertiary referral centre. POPULATION: Pregnant women with no contraindication for vaginal delivery recruited from January 2005 to April 2008. METHODS: Participants were randomised to receive either syntometrine or carbetocin during the third stage of labour. MAIN OUTCOME MEASURES: Primary outcome measure was postpartum haemorrhage requiring additional uterotonics. Secondary outcome measures were the incidence of postpartum haemorrhage (> or =500 ml), severe postpartum haemorrhage (> or =1000 ml) and adverse effects profile. RESULTS: Women in the carbetocin group (13.5%) and in the syntometrine group (16.8%) had postpartum haemorrhage requiring additional uterotonics (P = 0.384). 1.6% of women in each group had postpartum haemorrhage (P = 1.0) and the estimated blood loss during the third stage of labour was similar between the two groups (P = 0.294). Women who had syntometrine were four times more likely to experience nausea (RR = 4.2; 95% CI 2.2-7.8) and vomiting (RR = 4.3; 95% CI 1.9-9.5) compared with women who had carbetocin. Tremor, sweating, retching and uterine pain were also more likely in the syntometrine group compared with the carbetocin group (P < 0.05). CONCLUSIONS: Carbetocin has an efficacy similar to syntometrine for prevention of postpartum haemorrhage, but is associated with less adverse effects.

Probiotic supplementation in the first 6 months of life in at risk Asian infants--effects on eczema and atopic sensitization at the age of 1 year.

BACKGROUND: The role of probiotics in allergy prevention remains uncertain but has been shown in some studies to have a possible protective effect on eczema. OBJECTIVE: We aimed to assess the effect of probiotic supplementation in the first 6 months of life on eczema and allergic sensitization at 1 year of age in Asian infants at risk of allergic disease. METHODS: A double-blind, placebo-controlled randomized clinical trial involving 253 infants with a family history of allergic disease was carried out. Infants received at least 60 mL of commercially available cow's milk formula with or without probiotic supplementation [Bifidobacterium longum (BL999) 1 x 10(7) colony forming unit (CFU)/g and Lactobacillus rhamnosus (LPR) 2 x 10(7) CFU/g] daily for the first 6 months. Clinical evaluation was performed at 1, 3, 6 and 12 months of age, with serum total IgE measurement and skin prick tests conducted at the 12-month visit. The primary and secondary end-points were eczema and allergen sensitization, respectively. RESULTS: The incidence of eczema in the probiotic (22%) group was similar to that in the placebo group (25%) (P=0.53). The median Scoring Atopic Dermatitis score at 12 months was 17.10 (9.74) in the probiotic group and 11.60 (8.40) in the placebo group (P=0.17). The prevalence of allergen sensitization showed no difference (probiotic=24% vs. placebo=19%, P=0.26). The total IgE geometric mean (95% confidence interval) was 18.76 (12.54-24.98) kU/L in the probiotic group and 23.13 (16.01-30.24) kU/L in the placebo group (P=0.15). Atopic eczema (with sensitization) in the probiotic (7.3%) group was comparable to the placebo group (5.8%) (P=0.86). CONCLUSION: Early life administration of a cow's milk formula supplemented with probiotics showed no effect on prevention of eczema or allergen sensitization in the first year of life in Asian infants at risk of allergic disease. Further work is needed to determine whether timing of supplementation, dose and probiotic strain are important considerations.

In vivo model to determine fetal-cell enrichment efficiency of novel noninvasive prenatal diagnosis methods.

OBJECTIVE: To develop an in vivo model to determine fetal-cell enrichment efficiency of novel noninvasive prenatal diagnosis methods. METHODS: Efficiency of our three-step enrichment protocol was determined in vitro before fetal nucleated red blood cells (FNRBCs) were enriched from first-trimester maternal blood samples collected from the same patients pre- and postsurgical termination of pregnancy (TOP) (n = 10). FNRBCs enriched were identified using embryonic epsilon-globin immunocytochemistry and chromosomal fluorescence in situ hybridization. RESULTS: We recovered 37% of spiked FNRBCs (95% confidence interval (CI) 28.5-45.6; n = 8) in in vitro experiments. We show a consistent threefold increase in the number of epsilon + FNRBCs in maternal blood obtained immediately post-TOP (p = 0.005). A mathematical relationship was derived: observed number of pretermination primitive FNRBCs = 0.6 + 0.31 (coefficient between pretermination/post-termination primitive FNRBCs, 95% CI 0.12-0.49; p = 0.005) x observed number of post-termination primitive FNRBCs (R2 = 0.65). CONCLUSION: Our data demonstrate that maternal blood obtained immediately post-TOP would be a good in vivo model to determine the enrichment efficiency of novel protocols and methods for noninvasive prenatal diagnosis.

The 75 and 50g OGTT response in normal pregnant women.

The 75 and 50g OGTT (Oral Glucose Tolerance Test) responses were studied in 86 normal healthy pregnant women (mean age 28.7 +/- 0.4 (SEM) years) at 28 and 32 weeks respectively. Of these were 50 Chinese, 20 Malays and 16 Indians. Mean glucose responses at fasting, 1 and 2h post glucose load were 78.3 +/- 0.7, 132.2 +/- 2.8 and 116.2 +/- 2.1 mg/dl respectively for the 75g OGTT and 78.5 +/- 0.7, 130.5 +/- 2.5 and 106.7 +/- 1.8 mg/dl respectively for the 50g OGTT. Except for the 2h responses, corresponding responses between both OGTTs were not significantly different. All races showed a similar OGTT response. Using a set criteria for diagnosis of abnormals resulted in gross inconsistency in the number of abnormals diagnosed for both OGTTs. However, the use of 95th percentile shows a closer agreement in the diagnosis of abnormal cases for both OGTTs. Also, the 2h OGTT response for the 75g OGTT is higher than that of WHO's criteria for impaired glucose tolerance. This emphasizes the need to establish our own reference range.

Lipid profiles during and after normal pregnancy.

Lipid profiles were estimated in two groups of normal healthy women comprising 39 non-pregnant women and 67 pregnant women at 28 and 32 weeks gestation and 6 weeks after delivery. Triglycerides, total, HDL and LDL cholesterols were high during pregnancy. Except for LDL cholesterols which remained constant, all the above decreased at 6/52 postnatally, but levels were still above those of normal non-pregnant women. Compared to the later, the atherogenic index, LDL/HDL cholesterol ratio was unchanged during pregnancy but significantly increased at 6/52 postnatally. These results may suggest that while total lipid levels increase during pregnancy, this is balanced by an even distribution of the lipoprotein fractions. After delivery, though lipid levels had decreased, the decrease in HDL cholesterol and increase in LDL cholesterol caused unfavourable changes in lipid-lipoprotein ratios. These changes may be due to the change in metabolism of the pregnant women as well as diet during and after pregnancy.

Serum fructosamine concentrations in Singapore pregnant women.

Serum fructosamine levels in women at pregnancy (28 weeks' gestation) were determined. The women (n = 99) also participated in an oral glucose tolerance test (OGTT) with a 75g liquid glucose load, to determine their glucose tolerance. At 32 weeks' gestation, seventy-nine of them repeated the oral glucose tolerance test and fructosamine measurement. Results showed that fructosamine levels in pregnant women with normal glucose tolerance (2.20 +/- 0.19 mmol/l, n = 76), were not statistically different from those with glucose intolerance (gestational diabetes: 2.19 +/- 0.22 mmol/l, n = 23) at 28 weeks' and also 32 weeks' gestation. However, serum fructosamine levels in pregnant women were lower than those in non-pregnant subjects. Serum fructosamine measurement is not a sufficiently sensitive test for diagnosis of gestational diabetes.

Improved quality of human embryos when co-cultured with human ampullary cells.

Cultured human, ampullary, epithelial cells obtained from fertile women undergoing hysterectomy were evaluated for the support of human embryonic cleavage and growth in vitro. Twelve patients provided 23 embryos for co-culture with subcultured ampullary cells grown in T6 + 15% patient's serum and 18 embryos for growth in T6 + 15% patient's serum alone (controls). Of embryos co-cultured with ampullary cells, 78% cleaved to the compacted embryo stage and 69% cavitated as compared with 50 and 33% respectively for controls (P less than 0.01). Only 30% of co-cultured embryos reached the expanded blastocyst and 26% underwent hatching as compared with 28% for both stages in controls. At the 2 - 4- and 6 - 8-cell stages, 91 and 87% of co-cultured embryos showed an absence or slight fragmentation as compared with 72 and 61% respectively for embryos grown in medium alone (P less than 0.01). None of the co-cultured embryos showed unequal-sized blastomeres while 22% of controls showed unequal cleavage. Embryos grown with ampullary cells cleaved slightly faster than controls. Scanning electron micrographs showed that ampullary cells collected from co-cultures were all of the secretory type with several microvilli and apical protrusions. It is clear that subcultured human ampullary cells support human embryonic cleavage and yield a reasonable number of good quality embryos up to the cavitation stage. Development past the expanded blastocyst and hatching stages seems to involve another critical phase with its own specific requirements.

Establishment of human ampullary cell cultures.

Epithelial cells from the ampulla of healthy oviducts from 16 women aged 33-41 years and at different phases of their menstrual cycles were used to establish primary cultures and the continuation of a cell line. The morphology and behaviour of these cells in vitro were evaluated using Nomarski's inverted optics, scanning and transmission electron microscopy. Cells from all patients produced confluent monolayers in 6-7 days with no significant relationship of cell growth with stage of cycle. Fourteen primary cultures were of the epithelioid type while two showed mixed epithelioid and fibroblast-like growth. Two distinct cell types (ciliated and secretory) were observed in primary culture. Secretory cells showed several microvilli of different lengths and distribution. Secretory cells predominated over ciliated cells in all patients, but maximum ciliation occurred around the time of ovulation. Structural features of the cells in vitro were remarkably similar to those described in vivo. Ampullary cells could be maintained in vitro through four to six passages with 3-4 days of growth between passages. Sub-cultured cells were all secretory and were of two types (I and II) based on ultrastructure. Secretory vesicles containing electron-dense material and lipids were observed in these cells. The method described allows for the use of ampullary cells as feeder layers for IVF and support of cleaving human embryos and the evaluation of the biochemical events surrounding fertilization and ectopic pregnancies.

Rapid karyotyping of spontaneous abortions with trophoblastic villi.

24-hour short term culture of trophoblastic villi provided rapid and accurate karyotypes in spontaneous first trimester abortions. Good quality metaphases were obtained in 60.0% of villous cultures if these were set up within 8 hours of uterine evacuation, in contrast to the 10.3% success rate with intervals longer than 8 hours. 21% (4 of 19) of first trimester pregnancy losses were chromosomally abnormal, 3 of which were examples of autosomal trisomy; in 2 cases, the mothers were more than 37 years of age. The results of successful karyotyping reported here compare well with previous reports of long term culture of minced fetal material. Further, the risks of infected cultures, maternal contamination and pseudomosaicism are minimal with the short-term culture technique described here. Chorionic villi isolated from spontaneously aborted material are a simple and practicable source of fetal material for rapid and accurate cytogenic diagnosis in early spontaneous abortions.

Follicular-phase response in two clomiphene-human menopausal gonadotropin regimes for an in vitro fertilization program.

Two clomiphene-human menopausal gonadotropin regimes were assessed for our in vitro fertilization and embryo replacement (IVF and ER) program since September 1983. Clomiphene, 50 mg bd, was taken from day 2 for 5 days. Human menopausal gonadotropin (hMG) was given from day 6; for the first regime, 75 IU/day was given for the first 3 days, and for the second, 150 IU/day. The subsequent dosages were dependent on the estradiol response. There were 9 cases for the first regime and 10 cases for the second. The mean number of hMG ampoules given was 16.5 and 19.25, respectively. The number of follicles seen on ultrasound was 3.0 +/- 0.5 and 3.4 +/- 1.2 (mean +/- SD), respectively. There was no statistical difference in the estradiol response up to the day of laparoscopic ova recovery for the two regimes. However, a spontaneous luteinizing hormone (LH) surge was observed in 4 of 9 cases in the first group and 6 of 10 cases in the second group. When a comparison was made between cases that had a spontaneous LH surge and cases that were given human chorionic gonadotropin (hCG), there was a higher estradiol level on the day of the laparoscopy in the hCG group with the lower hMG regime (P less than 0.05). There were no other differences. Our small series shows a 52.6% incidence of spontaneous LH surge with clomiphene-hMG. Hence such stimulated regimes can result in a high proportion of spontaneous LH surges; this may be an index of satisfactory endocrinological control in spite of an increase in the number of follicles.