Evaluating sex chromosome content of sorted human sperm samples with use of dual-color fluorescence in situ hybridization.

OBJECTIVE: Although most methods for selecting the sex of offspring by sorting spermatozoa are ineffective at shifting the ratio of Y- to X-containing cells, some commercial sources continue to offer such services. Our objective was to evaluate commercially "sorted" samples with use of dual-color fluorescence in situ hybridization and to identify variations in assessment by comparing motile and total sperm populations, donors, observers, and fluorescence in situ hybridization probes. STUDY DESIGN: Cryopreserved sperm from seven anonymous donors were processed as for insemination. Sperm cells from each total sample or motile subfraction were prepared for fluorescence in situ hybridization by incubation with disulfide-reducing agents to expand sperm nuclei. Two sets of X and Y chromosome-specific, fluorophore-labeled deoxyribonucleic acid probes were used. At least 400 nuclei from each preparation were classified independently by three blinded observers. Hybridization efficiency, aneuploidy, and sex chromosome content were evaluated in subsets of five unsorted, five female-oriented, and five male-oriented samples. Total and motile subfractions were compared with eight samples. Fluorescence in situ hybridization probes were compared in five paired unsorted samples. RESULTS: No differences were detected between washed samples and paired motile subfractions. No differences in hybridization and aneuploidy were detected between groups of sorted samples. The Y/X ratio was significantly different between the sorted groups. However, male-oriented samples had a lower Y/X ratio than female-oriented samples did. Observer and probe choice accounted for small but significant variations that did not alter conclusions about the X/Y ratio for sorted samples. CONCLUSION: In a series of 10 sorted samples from one commercial source, dual-color fluorescence in situ hybridization demonstrated a small but significant shift in the sex chromosome ratios among samples. However, this shift was opposite to that expected by the orientation of the sorted samples.

Fresh frozen plasma prophylaxis for hereditary angioedema during pregnancy. A case report.

BACKGROUND: Hereditary angioedema (HAE) is an uncommon disorder of the complement system due to a deficiency or dysfunction of the inhibitor of the first component of complement (C1 INH). Clinically, HAE is characterized by episodic abdominal pain or edema of the extremities, face, larynx and vulva. Laryngeal edema is potentially lethal and accounts for mortality rates as high as 30%. Therapy is divided into short-term (fresh frozen plasma) and long-term (antifibrinolytic agents, hormonal therapy) prophylaxis, or acute treatment (epinephrine, steroids, antihistamines). CASE: An 18 year-old, primiparous woman presented with a history of multiple episodes of abdominal pain, swelling of abdomen and extremities. A diagnosis of hereditary angioedema was made based on the patient's and family's history and on decreased levels of C1 INH and complement component 4. Standard prophylactic methods were contraindicated due to potential teratogenic effects. Fresh frozen plasma given twice weekly over 2.5 months resulted in reduction in the number and severity of attacks and allowed successful completion of pregnancy. CONCLUSION: This is the first report of fresh frozen plasma therapy as a means of long-term prophylaxis in a pregnant woman. Fresh frozen plasma may serve as an alternate mode of long-term prophylaxis if the standard agents (antifibrinolytic, hormone) are unsuccessful or contraindicated.

Puerperal cerebral venous thrombosis associated with decreased free protein S: a case report.

BACKGROUND: Cerebral venous thrombosis is a rare event with multiple risk factors. This condition can be associated with a hypercoagulable state, such as that seen with dysfunctional or decreased plasma protein C and protein S. Both plasma proteins are naturally occurring, vitamin K-dependent anticoagulants that are important for regulation of the clotting cascade. CASE: A postpartum patient presented with complaints of a severe headache and hypertension and later developed status epilepticus. She was found to have superior sagittal sinus and right internal jugular vein thrombosis and protein S deficiency. CONCLUSION: The combination of protein S deficiency and the further reduction due to pregnancy may enhance the risk of major thrombotic episodes in the peripartum period.

Maternal and fetal effects of laparoscopic insufflation in the gravid baboon.

STUDY OBJECTIVE: To test the hypothesis that intraabdominal pressures (IAP) associated with abdominal insufflation for laparoscopic procedures can alter pulmonary and hemodynamic values in the pregnant baboon and hemodynamic values in the fetus. DESIGN: A descriptive physiologic study. SETTING: Animal research facility at Scott and White Memorial Hospital, Temple, TX. PARTICIPANTS: Four pregnant baboons at 120 +/- 7 days' gestation. INTERVENTIONS: The baboons underwent general anesthesia, Swan-Ganz and arterial catheter placement, and abdominal insufflation at 10 and 20 mm Hg IAP for 20-minute intervals at each pressure. The following end points were measured: maternal heart rate (MHR), mean arterial pressure (MAP), cardiac output (CO), pulmonary capillary wedge pressure (PCWP), pulmonary artery pressures (PAP), central venous pressure (CVP), systemic vascular resistance (SVR), ventilator rate (VR), oxygen saturation, and end-tidal carbon dioxide (CO2). Fetal heart rate and fetal growth values were measured, and umbilical artery Doppler flow studies were performed. MEASUREMENTS AND MAIN RESULTS: The PCWP (p <0.026), CVP (p <0.0012), and PAP (p <0.046) were significantly increased at 20 mm Hg IAP; CO decreased as IP increased. The MAP, MHR, and SVR did not change significantly with increased IAP. The only significant change in pulmonary values was the increase in peak airway pressure (p <0.001). The VR was increased from an average of 18 to 41 breaths/minute in an attempt to maintain adequate oxygen saturation and to normalize end-tidal CO2 when IAP was increased to 20 mm Hg. Respiratory acidosis (pH <7. 35, partial pressure of carbon dioxide >50 torr) was demonstrated in three of four animals within 20 minutes at an IAP of 20 mm Hg. Results of Doppler flow studies on the effects of the fetuses were unaltered immediately after this procedure compared with baseline measurements. Normal interval growth was demonstrated 2 weeks after the procedure. CONCLUSION: The baboon mothers and fetuses had no adverse effects at an IAP of 10 mm Hg, but may have significant cardiovascular and respiratory alterations associated with IAP of 20 mm Hg.

Intravenous penicillin for antenatal syphilotherapy.

A 21 year old woman (G(2) P(0101)) of 24 weeks gestation presented with syphilis of unknown duration. Sonography revealed fetal hydrops and placental thickening. Weekly intramuscular injections of 2.4 million U Bicillin for 3 weeks was initiated as recommended by the Centers for Disease Control. Repeat sonogram 1 week after starting treatment revealed increased ascites and a new pericardial effusion. Due to the worsening fetal condition, therapy was altered and the patient was admitted for IV penicillin. She received a continuous infusion of 18 million U penicillin G daily for 10 days. Serial sonograms showed improvement offetal ascites and pericardial effusion with 10 days of IV therapy, and complete resolution of hydrops was noted within 3 weeks. The fetus was born at term with no stigmata of congenital syphilis on newborn exam, and laboratory tests suggested adequate treatment in utero.

Magnesium sulfate versus phenytoin for seizure prophylaxis in pregnancy-induced hypertension.

Seizure prophylaxis is standard intrapartum therapy for patients with pregnancy-induced hypertension. Magnesium sulfate is used in the United States in spite of limited literature comparing its efficacy with other anticonvulsants. Fifty patients with pregnancy-induced hypertension were prospectively randomized to receive magnesium sulfate or phenytoin for seizure prophylaxis. Patients were observed for toxicity, side effects, and labor outcomes, and the neonates were evaluated for side effects of the therapy. Three patients were excluded with adverse reactions to medications (one in magnesium sulfate group, two in phenytoin group). No differences were found in patient tolerance, adverse reactions, or neonatal outcomes between groups. Maternal free phenytoin levels were 13.0% +/- 0.4% of total phenytoin (serum albumin, 2.5 to 3.5 gm/dl), significantly higher than in nonpregnant patients. Neither free phenytoin levels nor percentage of total phenytoin that was free correlated significantly with maternal albumin levels. The pharmacokinetics of phenytoin loading in the massively obese pregnant patient may differ and require further evaluation. Phenytoin is a well-tolerated alternative to magnesium sulfate for seizure prophylaxis in the patient with mild pregnancy-induced hypertension.

Effect of propofol on the auditory evoked response and oesophageal contractility.

Six patients were anaesthetized with 70% nitrous oxide in oxygen supplemented by infusion of propofol 40, 80, 120, 160 and 200 micrograms kg-1 min-1 sequentially in successive 10-min periods. Auditory evoked response (AER) and lower oesophageal contractility (LOC) were monitored. The AER findings were consistent with those noted in previous studies of i.v. agents. Early cortical waves showed attenuation of Pa and Nb amplitude (P less than 0.01) and increase in Pa and Nb latency (P less than 0.01; P less than 0.05) with increasing blood concentrations of propofol. Brainstem waves were not affected significantly. LOC, provoked and spontaneous, showed no consistent relationship with blood concentration of propofol. The two variables AER and LOC were not related.

A retrospective study on the effects of steroid administration and prolongation of the latent phase in patients with preterm premature rupture of the membranes.

To address the issues of whether corticosteroid treatment and prolongation of the latent phase improve the outcome of pregnancy in patients with preterm premature rupture of the membranes, we studied 96 patients with premature rupture of the membranes who were delivered of infants of adequate weight for gestational age with birth weights between 751 and 2000 gm and who had a latent period longer than 48 hours. Of these 96 infants, 53 received treatment with steroids and tocolytic agents and 43 received no treatment. We found a significant decrease in perinatal mortality and in the incidence of moderate and severe hyaline membrane disease in infants whose mothers received glucocorticoids. The protective effect of glucocorticoid therapy was limited to infants with a birth weight between 751 and 1000 gm or with a gestational age of 27 to 28 weeks. We also found a significant increase in perinatal mortality, mainly due to infection, when the latent phase was prolonged for greater than 7 days, regardless of the type of management.

Production of insulin-like growth factors by ovarian granulosa cells.

Evidence for ovarian secretion of somatomedins or insulin-like growth factors (IGF's) was generated by two approaches. First, porcine granulosa cells were shown to produce IGF's and an IGF-binding protein under serum-free conditions in vitro. The ovarian IGF's were recognized in two competitive binding assays specific for IGF's, a RIA using antibodies to human IGF-I and a radioreceptor assay using rat liver plasma membranes. IGF secretion was maintained for at least 10 days in culture. Second, ovarian production of IGF's in vivo was suggested by studies which showed that IGF levels in follicular fluid from preovulatory follicles were significantly greater than those in either serum or immature follicles. In contrast, similar low levels of insulin were observed in the follicles and serum. In conjunction with previous evidence of IGF action on granulosa cells, the present studies suggest the possibility of an autocrine role of IGF's in regulating follicular growth and development.

Ultrasound guidance for human embryo transfer.

Embryo transfer is facilitated with ultrasound guidance. The 16 ultrasound-guided transfers were rated easier, and there was less catheter distortion, in comparison with 12 transfers guided by "clinical feel." The benefits we ascribe to ultrasound guidance are as follows: (1) transfers can be done with the patient supine in the lithotomy position; (2) the catheter tip is accurately positioned in the fundus of the uterine cavity; (3) the ejection of the transfer bubble into the uterus and its persistence during catheter removal and documented; and (4) the continuance of the bubble is comforting to the patient. Greater experience is needed before the impact of ultrasound-guided transfer can be correlated with the rate of pregnancy continuance.

Endogenous digoxin immunoactivity is elevated in hypertensive pregnancy.

We have detected an endogenous digoxin-immunoreactive substance in the blood of third trimester pregnant women, of newborn infants and in amniotic fluids. In this report we present data showing an increase in blood levels of this endogenous substance in pregnant women with time of gestation (45 +/- 12, 125 +/- 32, and 195 +/- 27 pg/ml digoxin-equivalent for the 1st, 2nd, and 3rd trimesters, respectively, mean +/- SE). Preliminary evidence suggests the blood level of this substance is elevated in third-trimester hypertensive pregnant women (315 +/- 29 pg/ml). The immunoactive substance isolated from pregnant subjects is water soluble and reversibly bound to serum proteins. Heating the serum increases the measured immunoactivity. Exploration of the physiologic relevance of this endogenous substance or family of substances is in progress.

Endogenous digoxin-immunoreactive substance in human pregnancies.

We report the presence of an immunoreactive digoxin-like substance in blood from third trimester pregnant women. The sera from 51 women in the third trimester of pregnancy were analyzed by 4 commercially available digoxin RIAs. None of these patients was receiving digoxin. Digoxin immunoreactivity was detected in all patients by 3 of 4 assays. The measured values, in nanograms per ml digoxin equivalent, were (mean +/- SD): method A, 0.27 +/- 0.05; method B, 0.28 +/- 0.07; method C, 0.01 +/- 0.01; and method D, 0.15 +/- 0.06. Method B measured values greater than 0.50 ng/ml in sera from 5 patients. Digoxin immunoactivity was not detectable 24 h postpartum, suggesting a half-life in serum of 6 h or less. Exogenous digoxin added to these serum samples resulted in quantitatively additive increments above the endogenous measured levels. Three of 4 digoxin RIAs did not distinguish between true digoxin and the endogenous substance present in the sera of third-trimester pregnant patients. Preliminary evidence suggests that the endogenous digoxin immunoactivity is not due to elevation of levels of major known steroids in the blood of these women. Clinical management of women requiring digoxin therapy during pregnancy, therefore, is complicated by the inability to assume the same therapeutic range of digoxin in serum during the third trimester of pregnancy as in adult nonpregnant individuals.

Receptors for insulinlike growth factor I are defective in fibroblasts cultured from a patient with leprechaunism.

We previously have demonstrated that fibroblasts from a patient with leprechaunism exhibited markedly decreased insulin binding to insulin receptors and that the ability of insulin to stimulate glucose incorporation in the patient's cells was greatly impaired. In addition, the insulinlike growth factor, multiplication-stimulating activity (MSA), also exhibited an impaired ability to stimulate glucose incorporation in the patient's fibroblasts, although in normal fibroblasts this response appears to be mediated by an insulinlike growth factor receptor. The present study examines 125I-labeled insulinlike growth factor I (IGF-I) binding to patient's and control fibroblasts. 125I-labeled IGF-I binds to a specific IGF-I receptor in normal fibroblasts. At steady state, binding was inhibited by unlabeled IGF-I, IGF-II, MSA III-2, MSA II, insulin, and proinsulin, in order of potency, but not by high concentrations of epidermal growth factor and human growth hormone, chemically unrelated polypeptides 125I-labeled IGF-I binding to patient's cells was decreased by approximately 75%, whereas binding of epidermal growth factor to its cell surface receptors was unaffected. Computer curve-fitting of untransformed equilibrium binding data suggests that the decreased binding resulted from a decreased Ka for IGF-I. The ability of the patient's IGF-I receptor to recognize insulin also appears to be altered. Impaired IGF-I binding by the leprechaun patient's fibroblasts may contribute to the abnormal biological response to insulinlike growth factors observed in vitro and to the in utero growth retardation.

Stimulation of glucose incorporation and amino acid transport by insulin and an insulin-like growth factor in fibroblasts with defective insulin receptors cultured from a patient with leprechaunism.

Fibroblasts cultured from an infant with leprechaunism and insulin resistance have been reported to exhibit profound, selective defect in insulin binding. We now examine the effect of this defect on two acute metabolic actions of insulin thought to be mediated by the insulin receptor, glucose incorporation and N-methyl-alpha-aminoisobutyric acid (Me-AiBu) uptake. In the patient's fibroblasts, maximal insulin-stimulated glucose incorporation was less than 25% of that in control fibroblasts, whereas stimulation by hydrogen peroxide, an insulinomimetic agent that acts distal to the insulin receptor, was normal. By contrast, insulin stimulated Me-AiBu uptake to the same extent in patient's and control fibroblasts. Impaired glucose incorporation and relatively normal Me-AiBu uptake also were observed in the patient's cells with multiplication-stimulating activity, an insulin-like growth factor, despite the fact that multiplication-stimulating activity appeared to stimulate both responses in normal fibroblasts via an insulin-like growth factor receptor. The divergent effects on two hormone-stimulated functions in the patient's cells suggests differences in the coupling of a receptor to different effectors. The same coupling mechanisms appear to be used by insulin receptors and receptors for insulin-like growth factors.

Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: relation to human somatomedins and insulin.

The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component of adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.

Maternal pulmonary edema resulting from betamimetic and glucocorticoid therapy.

Four cases are presented of maternal pulmonary edema occurring in patients who had no primary cardiac disease but who were receiving terbutaline and glucocorticoids or terbutaline alone for treatment of premature labor. Fluid overload was the event that triggered this decompensation. The physiologic high-output cardiac state of pregnancy is described and the manner in which betamimetic drugs and corticosteroids exacerbate this situation and cause congestive heart failure is shown. Methods of management to avoid this complication of premature labor therapy are suggested.