Preimplantation genetic diagnosis for hemophilia A using indirect linkage analysis and direct genotyping approaches.

BACKGROUND: Preimplantation genetic diagnosis (PGD) is an appealing option for couples at risk of having a child with hemophilia A (HA). Although many clinics offer PGD for HA by gender selection, an approach that detects the presence of the underlying F8 mutation has several advantages. OBJECTIVES: To develop and validate analysis protocols combining indirect and direct methods for identifying F8 mutations in single cells, and to apply these protocols clinically for PGD. METHODS: A panel of microsatellite markers in linkage disequilibrium with F8 were validated for single-cell multiplex polymerase chain reaction. For point mutations, a primer extension genotyping assay was included in the multiplex. Amplification efficiency was evaluated using buccal cells and blastomeres. Four clinical PGD analyses were performed, for two families. RESULTS: Across all validation experiments and the clinical PGD cases, approximately 80% of cells were successfully genotyped. Following one of the PGD cycles, healthy twins were born to a woman who carries the F8 intron 22 inversion. The PGD analysis for the other family was complicated by possible germline mosaicism associated with a de novo F8 mutation, and no pregnancy was achieved. CONCLUSIONS: PGD for the F8 intron 22 inversion using microsatellite linkage analysis was validated by the birth of healthy twins to one of the couples. The other family's situation highlighted the complexities associated with de novo mutations, and possible germline mosaicism. As many cases of HA result from de novo mutations, these factors must be considered when assessing the reproductive options for such families.

Evidence that oxytocin is a physiological component of LH regulation in non-pregnant women.

BACKGROUND: Regulation of the LH surge is central to the functioning of the female ovulatory cycle. In animal models, oxytocin has been shown to alter LH activity. Oxytocin advanced the LH surge and, conversely, oxytocin receptor antagonists inhibited full production of the LH surge in rats. Few data exist on the possibility that oxytocin modulates LH in women. METHODS: Ten non-pregnant women participated in this study over two menstrual cycles. One cycle was a control cycle, and the other a trial cycle; the two were separated by at least one cycle. When the diameter of an ovarian follicle was >15 mm, a subject was allocated at random into either a control or treatment group. In a control cycle, volunteers received normal saline; in a treatment cycle, volunteers received an oxytocin antagonist (atosiban). RESULTS: For treatment cycles, the maximum LH concentration was significantly less than that in control cycles (42.1 +/- 6.2 versus 60.3 +/- 8.3 IU/l respectively; P < 0.05). Maximum FSH and estradiol concentrations were not significantly different between the two groups. CONCLUSIONS: The results indicated that inhibition of endogenous oxytocin affects the endocrinology of the ovulatory cycle in women, and strongly suggest that oxytocin has a role in the physiological processes of LH regulation.

The utility of plasma CRH as a predictor of preterm delivery.

It has been suggested that CRH is a placental clock that controls the duration of pregnancy and that the timing of the rise in CRH may permit prediction of the onset of labor. We have performed a prospective longitudinal study, in 297 women, to examine the utility of a single second-trimester plasma CRH measurement to predict preterm delivery. Venous blood samples were taken at 4-weekly intervals, beginning at 16-20 wk gestation, until delivery for CRH and its binding protein. A time point at which a single plasma CRH test might give optimal data to predict preterm delivery was determined. Thirty-one subjects delivered prematurely (10.4%). Sampling for plasma CRH at 26 wk gestation seemed the optimal time point to maximize sensitivity and specificity of the test. The mean (+/- SD) plasma CRH in women at this gestation who eventually delivered after spontaneous labor within 1 wk of their due date (39-41 wk, n = 127) was 34.7 +/- 27.0 pM. A plasma CRH of more than 90 pM at 26 wk gestation had a sensitivity of 45% and a specificity of 94% for prediction of preterm delivery. The positive predictive value was 46.7%. Calculation of free CRH did not improve these figures. In conclusion, a single measurement of plasma CRH, toward the end of the second trimester, may identify a group at risk for preterm delivery, but over 50% of such deliveries will be unpredicted. These data do not support the routine clinical use of plasma CRH as a predictor of preterm labor.

Association of a novel human mtDNA ATPase6 mutation with immature sperm cells.

This study reports the first clearly defined heteroplasmic mutation in immature human sperm cells. The human sperm mitochondrial genome from residue 8186-9341 was analysed with the aim of identifying point mutations which may be associated with human male infertility. The semen samples analysed were obtained from 88 fertile men, 19 with oligozoospermia, and 12 with severe oligozoospermia. Using single strand conformation polymorphism analysis a heteroplasmic T to C transition was detected in the ATPase6 gene, at nucleotide position 8821, in semen samples from one out of 12 (8%) severely oligozoospermic men, but not in oligozoospermic men or normospermic men. This mutation changed the amino acid serine to proline at residue 99 of the mitochondrial ATPase6 in a region which is highly conserved in other vertebrates including rat, bovine, chicken, salmonids and Xenopus. The mutation was detected in semen samples collected from the same man 9 months apart and in peripheral blood lymphocytes. Single sperm cell analyses did not find this mutation in the mature sperm, but the mutation was detected in 7% of immature spermatids. Our finding suggests that immature spermatids with this mutation fail to develop fully.

The effect of recombinant follicle stimulating hormone (Gonal-F) on endogenous luteinizing hormone secretion in women.

Parenteral administration of follicle stimulating hormone (FSH) has been shown to lower luteinizing hormone (LH) concentrations in women undergoing ovulation induction. This study was designed to explore the physiological mechanism of this effect. Seven healthy women were recruited into a double-blind placebo-controlled study. LH secretion, after the administration of variable i.v. boluses (37.5, 75 and 150 IU) of recombinant FSH (Gonal-F), was evaluated. LH was measured at 10 min intervals for 2 h before and 4 h after the FSH/placebo infusion. LH pulse frequency and amplitude were evaluated and there was no significant difference between control and trial cycles for each subject. A linear regression analysis revealed that in the group receiving 150 IU FSH, the mean plasma LH concentration decreased significantly due to a reduction tonic LH secretion. This could be a result of the suppression of secretion or an alteration of clearance. This decrease was not seen in the other dosage groups, revealing that above a dosage threshold, FSH reduced non-pulsatile LH secretion. Therefore the effect of FSH in this study exposed the likely presence of two components of LH concentration: an FSH-sensitive, non-pulsatile tonic secretion and a gonadotrophin-releasing hormone-stimulated, pulsatile release that is unaffected by FSH. Although an indirect effect involving ovarian regulation is not excluded, the rapidity of the effect suggests that FSH acts directly on the pituitary gland.

Pre-ovulatory oxytocin administration promotes the onset of the luteinizing hormone surge in human females.

Luteinizing hormone (LH) secretion during the ovulatory cycle is believed to be predominantly regulated by gonadotrophin-releasing hormone. Investigations in animals have strongly suggested that oxytocin also participates in LH control and the physiological events controlling LH surge initiation. In the human female, however, there has been no evidence supporting oxytocin's involvement in the processes leading to ovulation. In this study the effect of a preovulatory infusion of oxytocin on the onset of the LH surge was investigated in women aged 20-35 years who had natural ovulatory menstrual cycles of lengths between 25-35 days. Vaginal ultrasound scanning monitored follicular growth during the late follicular phase. When a follicle > 14 mm in diameter was observed each woman was randomized into one of two groups. One group (n = 8) received an oxytocin infusion of 256 mIU/min for 2 h, the other group (n = 8) received normal saline. The women who were administered oxytocin at this late follicular stage had an earlier onset of the LH surge than those who had received saline (P < 0.01). The results indicate that oxytocin promotes the onset of the LH surge in humans.

Nutrition in pregnancy in the Wellington region.

The diets of 115 pregnant women in the Wellington region were assessed for nutrient intake using 24 h dietary recall. Assessment was performed in both the second and third trimester. Women came from three ethnic groups, European (61), Maori (29) and Pacific Islanders (25). Comparisons of nutrient intake were made between these groups. The overall energy intake was similar between the groups (range 4.8-19.7 MJ/d) but Maori (p less than 0.05) and Pacific Islanders (p less than 0.02) had a significant decrease in energy intake from second to third trimester. Pacific Islanders consumed significantly more starch (121 g/d, p less than 0.05) whereas Maori women consumed significantly more sucrose (86 g/d, p = 0.0002). The mean intake in Pacific Islanders contained significantly less calcium (882 mg/d, p = 0.0002) and zinc (9.0 mg/d, p = 0.014). Forty-four percent Europeans, 28% Maori and 51% Pacific Islanders had an estimated iron intake below the minimum safe intake for pregnancy. However dietary iron intake did not relate to the presence of anaemia nor whether iron supplements were given.

Meconium aspiration--role of obstetric factors and suction.

Meconium aspiration occurred in 66 of 120 infants with meconium-stained liquor admitted to a Neonatal Unit. Four of the 66 infants died, whilst 17 other required ventilatory support. Meconium aspiration syndrome, especially when severe, was more likely if the mother was a smoker, hypertensive, anaemic, Maori or the pregnancy was greater than 42 weeks' gestation. Meconium aspiration was also associated with thick meconium, prolonged labour, late fetal heart decelerations and inadequate suctioning of the airway at birth. Although meconium-stained liquor is a common occurrence in labour, a bad neonatal outcome will only be obviated by vigilant intrapartum care and active suctioning of the infant's airway at birth. Once aspiration has occurred, intensive treatment is often required and sometimes fails.

Amniotic fluid endocrine changes during maternal hyperalimentation.

Ten women with low estriol excretion received hyperalimentation prior to induction of labor. Six received an amino acid mixture (5% Aminofusin) and 25% dextrose, two received the amino acid mixture, and two received 25% dextrose. Amniotic fluid obtained before and after hyperalimentation was assayed for fetal surfactant production, thyroid, pituitary, and carbohydrate regulating hormones. In the combined amino acid/dextrose infusion group the amniotic fluid palmitic acid levels increased significantly post infusion; rT3 also increased significantly but T3 and T4 showed no significant change. The pituitary hormones growth hormone, prolactin, and ACTH showed no significant change, but beta-endorphin-like activity was significantly elevated. No thyroid-stimulating hormone was detected in any of the samples. All the carbohydrate regulating hormones, insulin, cortisol, and cAMP, showed significant increases but cGMP showed a significant decrease. The amino acid and dextrose only groups gave similar results. Seven of the infants showed some degree of intrauterine growth retardation but no neonatal complications attributable to the hyperalimentation.

Septic abortion in Wellington 1960-1979.

A retrospective analysis was made of 283 cases of septic abortion admitted to Wellington Hospital from 1960-1979. There was a substantial reduction in the annual numbers of admissions occurring at the end of the study period compared with the beginning. Morbidity as measured by hospital stay, haemorrhage and septicaemia also fell. Possible causes for the reduction are discussed.

PIP: 283 records of all Wellington, New Zealand, Hospital patients categorized as having had a septic abortion from 1960 to 1979 were retrieved and reviewed; 7 records could not be found. Morbidity was assessed by duration of hospital stay and the incidence of complications. Septicemia was defined by a positive blood culture rigors. Hypotension was assumed to be due to hemorrhage unless endotoxic shock was positively identified. Significant hemorrhage was also diagnosed if a blood transfusion was required. The annual number of admissions for septic abortion over the 20-year period decreased substantially: 203 cases from 1960 to 1969 and 80 cases from 1970 to 1979. The total number of abortion admissions of all types also decreased over the same period. During this period the total number of births in Wellington public hospitals initially increased and then declined, suggesting some degree of independence between total abortions and birthrate. The peak of total abortions in 1978-79 represents the initial increase in hospital therapeutic abortions under the Contraception, Sterilization and Abortion Act 1977. The gestational age at the time of septic abortion ranged from 7 to 26 weeks. The mean gestation by year ranged from 8.6 to 15 weeks. No trend was obvious. The mean duration of hospital stay declined, e.g., 7.3, 7.6, 6.0, and 5.3 days in successive 5-year periods. Morbidity was greater during the 1st decade but there were 2 patients who in 1974 and 1975 stayed 23 and 28 days, respectively. Septicemia patients numbered 39 (35%) in the first 5 years, and fell successively to 31 (33%), 17 (36%), and 6 (17%) in each succeeding 5-year period. A similar trend was noted for significant hemorrhage: 38 (34.5%), 39 (42%), 13 (28%), and 3 (8.5%) between 1975 and 1979. Only maternal death due to septic abortion occurred in 1960. The patient was noted to be fairly "feebl minded" and the diagnosis was delayed because she hid her symptoms. She went into acute renal failure, fitted, then collapsed and died quite suddenly. The Wellington admissions for septic abortion represented 1 in 16 of the New Zealand total in 1965 and 1 in 28 in 1979. It would seem that the trend of decreased septic abortion admissions in Wellington is a mirror of the national trend.

The biochemical effects of a maternal hyperalimentation during pregnancy.

Six patients each with a very low 24-hour urine oestriol excretion were infused with hypertonic dextrose and amino acids for 48 hours prior to delivery. Amniotic fluid samples were studied before and after infusion. Lecithin sphingomyelin ratios and insulin levels were increased following infusions in all patients. There was usually a fall in amniotic fluid glucose and an increase in ammonia, amino acid nitrogen and osmolality following infusions. Maternal plasma oestriol, human placental lactogen, cystyl aminopeptidase and urine oestriol excretion were unchanged during the infusions.