A.C. Roy

Influence of serum paraoxonase polymorphism on serum lipids and apolipoproteins.

One hundred and sixty‐three healthy Chinese subjects of both sexes were studied for serum paraoxonase (PON) polymorphism, and levels of lipids and apolipoproteins in order to examine effects of PON alleles on these parameters. The level of serum triglyceride was significantly higher in high activity allele (PON*B) compared with that in low activity allele (PON*A) in both sexes (P<0.01). The subjects with PON A had significantly higher LDL cholesterol (P<0.05) and lower Apo A‐II and ApoB levels. The influence of serum paraoxonase on serum lipids was estimated further by Spearmans rank correlation. In the males, there was a significant negative correlation of serum paraoxonase activity with total (P<0.05) and LDL (P<0.01) cholesterol levels, and positive correlation with HDL cholesterol and Apo A‐II levels (P< 0.05). Serum paraoxonase activity had a high positive correlation with serum triglyceride levels in both sexes (P< 0.001). Serum ApoB level had a positive correlation with the enzyme activity only in females (P<0.01). The allelic effect of PON on these parameters was studied by multiple regression analysis. The high activity allele (PON*B) was associated with higher serum triglyceride level (P<0.001) and ApoB (P<0.001), while it had lowering influence on total cholesterol (P<0.05) and LDL cholesterol (P<0.005) in men. The average allelic effect of PON was found to be about 22% for serum triglycerides, 11% for LDL cholesterol, 14% for Apo A‐II and 19% for Apo B in the present study. This study suggests a possible significant role of serum paraoxonase alleles in the metabolism of serum lipids and apolipoproteins.

A new molecular variant of luteinizing hormone associated with female infertility

OBJECTIVE To investigate whether the newly described G1502 to A1502 mutation in exon 3 of the LH beta-subunit gene, causing the amino acid substitution of Ser102 for Gly102, is related to female infertility. DESIGN Screening of fertile and infertile women for the G1502 to A1502 mutation in the LH beta-subunit gene. SETTING Clinics and laboratories of the National University Hospital obstetrics and gynecology department, Singapore. PATIENT(S) Two hundred twelve healthy fertile women; 40 infertile women with menstrual disorders, polycystic ovary syndrome, and endometriosis; and 12 women with idiopathic infertility. INTERVENTION(S) Exon 3 of the LH beta-subunit gene was analyzed using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and PCR-mediated direct DNA sequencing. MAIN OUTCOME MEASURE(S) The PCR products of patients were analyzed by RFLP, and the results were compared with those of fertile controls. DNA sequencing radiographs were compared between two mutation-bearing patients and four controls. RESULT(S) The mutation was identified in only two infertile women with endometriosis; other women studied were found to be negative for this mutation. CONCLUSION(S) The missense mutation in the LH beta-subunit gene may be implicated in female infertility, possibly endometriosis-associated infertility in some women.

Quality assessment of two lactate test strip methods suitable for obstetric use.

Accuracy of lactate determinations in cord blood was tested for one reflectometric (Accusport) and one amperometric (Lactate Pro) microvolume test strip lactate meter. Both meters, using a whole blood sample, measure lower levels of lactate than a reflectometric device considered as a reference method, which analyses lactate in plasma. Readings were unaffected irrespective of lactate concentrations for the Lactate Pro, whilst the Accusport overestimated low lactate concentrations and underestimated high values. Both lactate meters underestimated lactate concentrations at high hematocrits, as compared with the reference method. The Lactate Pro has a fixed sample volume of 5 microliters while the Accusport uses random blood drop as sample volume. However, in analyses with less than 20 microliters sample volume considerable underestimation was found with the Accusport. Coefficient of variation was 3.8-8.9% for the Accusport and 3.1-4.0% for the Lactate Pro within lactate concentrations between 2.1 and 5.3 mmol/l. The amperometric device, the Lactate Pro, performed best in these tests dealing with fetal blood lactate concentrations. The new technique can be a useful tool in perinatal research as well as in obstetric practice.

Mitochondrial gene mutations in gestational diabetes mellitus

Mitochondrial DNA mutations have been implicated in many diseases including diabetes mellitus. Although gestational diabetes mellitus (GDM) has been suggested to have genetic determinant and to be etiologically indistinct with non-insulin-dependent diabetes mellitus (NIDDM), its association with mitochondrial gene mutations is still unknown. In this study, 137 patients with GDM and 292 non-diabetic pregnant controls were examined for mitochondrial DNA mutations from the nucleotide 3130-4260 encompassing tRNA-Leu gene and adjacent NADH dehydrogenase 1 gene by polymerase chain reaction, single-stranded conformation polymorphism, restriction fragment length polymorphism and DNA sequencing. One heteroplasmic mutation at the position of 3398 (T-C), which changed a highly conserved methionine to threonine in NADH dehydrogenase subunit 1, was identified in 2.9% GDM patients but not in the controls, indicating its association with GDM (P = 0.01). Two novel mutations, a heteroplasmic C3254A and a homoplasmic A3399T, were also found in GDM subjects, the functional meaning of which merits further investigation. G3316A and T3394C mutations implicated in NIDDM, were seen at higher frequencies in patients with GDM than the controls. Our results suggest that mitochondrial DNA mutations may contribute to the development of GDM in some patients.

Association of molecular variants of luteinizing hormone with menstrual disorders

Luteinizing hormone (LH) promotes ovulation and luteinization of the ovarian follicle, and stimulates steroidogenesis in the ovaries. It is known to be present in different molecular forms, and secretion of abnormal LH has been implicated in menstrual disorders and infertility. The purpose of this study was to determine any association of two recently described LH variants with menstrual disorders in Singapore Chinese women. One of these variants had Trp8 to Arg8 and Ile15 to Thr15 replacements in the LH β‐subunit, while the second variant possessed Ser102 substitution for Gly102.

Identification of seven novel mutations in LH beta-subunit gene by SSCP.

Seven new point mutations have been identified from LH β-subunit gene by PCR-mediated SSCP, and sequencing. One mutation was found changing amino acid from Gln102 to Ser102. The remaining six mutations, which did not change the codings, were in complete linkage disequilibrium. SSCP can be used in the diagnosis of LH-related disorders.

Complete androgen insensitivity due to a splice-site mutation in the androgen receptor gene and genetic screening with single-stranded conformation polymorphism *

OBJECTIVE To characterize the genetic defect in a family with complete androgen insensitivity syndrome and to determine whether single-stranded conformation polymorphism (SSCP) can be used to detect subtle mutations in the androgen receptor (AR) gene. DESIGN Amplification, subcloning where appropriate, and sequencing of the AR gene in members of the affected family and to use SSCP to differentiate rapidly mutant from normal alleles. SETTING Reproductive endocrinology clinic and laboratory in a university hospital. PATIENTS A family of which two sisters (46XY) have complete androgen insensitivity syndrome. RESULTS A novel single base (G --> A) mutation in the exon G-intron 7 junction of the AR gene caused an abnormal donor splice site leading to complete androgen insensitivity in both affected siblings. Their mother was demonstrated to be the heterozygous carrier of this mutation while the other two males in the family carried the normal allele. Single-stranded conformation polymorphism proved useful for defining the normal, mutant, and heterozygous carrier status of each member of this family. CONCLUSIONS This new mutation of the human AR gene illustrates the importance of exon G in receptor function. Single-stranded conformation polymorphism is a simple and rapid screening technique that can be used to detect unknown subtle mutations in the AR gene.

Validation of a Laboratory Method of Measuring Postpartum Blood Loss

Laboratory methods give more accurate measurement of blood loss in the postpartum period than visual estimation. In order to evaluate a laboratory method used to quantify blood loss postpartum, blood lost at gynecological operations was collected in a measuring bottle. The measured amount of blood (50–1,000 ml) was then poured onto absorbent paper towels and sanitary pads, in order to mimic conditions when measuring blood loss in clinical trials in the postpartum period. The amount of blood absorbed onto the absorbent paper and sanitary pads was measured by a rapid method of automatic extraction and photometric measurement of alkaline hematin. The study shows that the method provides a reliable and accurate means of measuring blood loss. The error in each case was less than 10% with an intraclass correlation coefficient of almost 1.

Association of AccI polymorphism in the follicle-stimulating hormone β gene with polycystic ovary syndrome ☆

OBJECTIVE To search for FSH beta-subunit gene mutations in patients with polycystic ovary syndrome (PCOS) and determine the association between the mutations and the syndrome. DESIGN Clinical and molecular studies. SETTING Clinics and laboratories of the National University Hospital Obstetrics and Gynecology Department in Singapore. PATIENT(S) One hundred thirty-five patients with PCOS and 105 normal control subjects. INTERVENTION(S) Exons two and three were screened for mutations by single-stranded conformational polymorphism and DNA sequencing. MAIN OUTCOME MEASURE(S) Polymerase chain reaction followed by restriction enzyme analysis. RESULT(S) No missense mutation was found in the functional units of the FSHbeta gene in patients with PCOS, but a thymine-cytosine substitution in exon 3 (codon 76, TAT to TAC) was identified. The nucleotide change led to creation of an AccI digestion site. The distribution pattern of AccI polymorphism in the patients was significantly different from that in the control group, and the occurrence of homozygous carriers was significantly higher in patients (12.6%) than in the control group (3.8%). The frequency of polymorphism and prevalence of homozygosity were significantly higher in patients with PCOS with obesity (0.50% and 31.0%, respectively) than in those with menstrual disorders only (0.366% and 8.5%, respectively), which correlated with significantly higher androgen levels in the obese patients. CONCLUSION(S) The AccI polymorphism in FSHbeta gene may be associated with PCOS in some women, especially those with obesity.

Hypoxia up-regulated angiogenin and down-regulated vascular cell adhesion molecule-1 expression and secretion in human placental trophoblasts

Objective: Many processes that are involved in cellular invasion, including blastocyst implantation, placental development, and rapidly growing tumors, occur in reduced oxygen environments. It has been surmised that oxygen tension could regulate the cytotrophoblast ability to differentiate and, as a consequence, to express proteins that are critical for placentation. The objective of the current investigation was therefore to test the hypothesis that placental tissues and trophoblast cells in culture, under low oxygen tension, release angiogenic factors that could affect vascular behavior and invasive potential, thus providing a link between abnormal placentation and maternal vascular abnormality. Methods: Functionally active term placental explant culture and trophoblast cultures were used to demonstrate the secretion profiles of angiogenin and vascular cell adhesion molecule-1 (VCAM-1), and the real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique was employed to demonstrate the mRNA expression under both normoxic and hypoxic conditions. Results: A significant increase in the secretion (P <.01) and mRNA expression (P <.01) of angiogenin and a signficant decrease in the secretion (P <.04) and mRNA expression (P <.03) of VCAM-1 from both term placental explants and trophoblast cultures subjected to hypoxia in vitro were observed. Conclusion: Because the primary defect in uteroplacental insufficiency is placental maldevelopment probably associated with hypoxia in situ, this study provides molecular evidence to indicate that the differential expression and secretion of angiogenic factors may play an important role in these pathologic conditions.