Collection and cryopreservation of cord blood for the treatment of hematopoietic disorders: the obstetrician's overview.

Bone marrow transplantation provides successful treatment for many diseases of the immune and hematopoietic systems. The main therapeutic ingredient in this procedure is stem cells collected from the bone marrow. Recently, it has been demonstrated that stem cells from human umbilical cord blood can serve as an alternative to bone marrow transplantation in children. Although cord blood transplantation in adults has not yet been attempted, it appears that there are enough stem cells present in cord blood for successful engraftment in adults. Obstetric health care providers should be aware that many familial conditions are treatable by cord blood stem cell transplantation in children. Obstetric health care providers caring for patients with familial disorders should consider counseling such patients regarding the collection and storage of cord blood for potential future uses in autologous or allogeneic transplantation.

Twin transfusion syndrome: reassessment of ultrasound diagnosis.

OBJECTIVE: To assess the antenatal ultrasonic diagnosis of the twin transfusion syndrome, an enigmatic disorder that results in an imbalance in the blood flow between monochorionic twins. STUDY DESIGN: Retrospective review of 16 cases of twin transfusion syndrome confirmed by placental pathology and neonatal data. RESULTS: A disparity in antenatal fetal weights, size difference between the two amniotic sacs and a single placenta were present in 81% (13/16) of confirmed cases. The finding of two separate umbilical cords with a disparity in the size or number of vessels was rare. Evidence of hydrops in either fetus or findings of congestive cardiac failure in the recipient twin were uncommon. CONCLUSION: Our data strongly suggest that the twin transfusion syndrome can be diagnosed in a significant number (81%) of cases. The syndrome is variable, and it is rare to observe all the diagnostic criteria in one case.

Indomethacin in severe twin-to-twin transfusion syndrome.

Despite advances in maternal fetal medicine, the management of severe twin-to-twin transfusion syndrome in the second trimester presents a significant challenge. Presently, there is no uniformly accepted management protocol that is available for the treatment of this syndrome. We report the use of indomethacin in three cases of severe twin-to-twin transfusion syndrome in the second trimester. In the three cases of severe twin-to-twin transfusion syndrome no reduction of amniotic fluid in either sac was demonstrated. Two cases were complicated by single intrauterine fetal death within 72 hours of initiating indomethacin therapy. Because of our experience with these three cases, we conclude that indomethacin does not prevent perinatal mortality in patients with severe twin-to-twin transfusion syndrome.

A rapid visual test for predicting fetal lung maturity.

A rapid bedside test has been devised that enables an untrained observer to predict (p less than 0.001) when amniotic fluid will be greater than or equal to 0.15 at an optical density of 650 nm, lecithin/sphingomyelin ratio will be greater than or equal to 2.0, or when phosphatidylglycerol will be present. By a visual comparison of the turbidity of unspun amniotic fluid against positive (mature) or negative (immature) controls, technicians and resident physicians who had had no special training were able to classify correctly 87.2% (82/94) of unknown amniotic fluid samples. The sensitivity of the new test is 90.8% (58/65); the specificity is 70.3% (23/29). Thus, when more sophisticated methods are not readily available, we believe that this easily performed and accurate test can provide supplemental or preliminary data for patient management. In remote geographic areas our method could serve as the primary source of information about fetal lung maturity.

Isolation of Chlamydia trachomatis from amniotic fluid.

A 26-year-old white woman had premature rupture of membranes at 35 weeks' gestation. Cervical specimens initially demonstrated group B streptococci and Chlamydia trachomatis. Amniocentesis was performed and the amniotic fluid was positive for chlamydia by direct fluorescent antibody stain. The Gram stain was negative. The patient had an elevated white blood cell count. Labor was induced because of suspected chorioamnionitis. A 2120-g female infant was delivered with an Apgar score of 9 at 1 and 5 minutes. The infant's eye and nasopharyngeal specimens were positive for chlamydia by direct fluorescent antibody stain. After delivery, both Ureaplasma urealyticum and C trachomatis were isolated from the amniotic fluid and fetal membranes. This is the first reported case of chlamydial isolation in amniotic fluid.

Antepartum management of twin gestation.

Twin gestation is now an area of vital concern to the perinatologist. Within the last decade a substantial reduction in perinatal mortality has been achieved, largely through advances in neonatal intensive care. However, preterm birth and its consequences remain the most important causes of perinatal morbidity and mortality. Twin pregnancy accounts for approximately 10% of all premature deliveries. From the data currently available, there is no justification for the routine use of hospital bedrest, prophylactic tocolytic agents, or elective cerclage in the management of twin pregnancy. Programs directed at the prevention of perterm delivery in twin pregnancy will be the focus of more scientific research. The use of ultrasound and antepartum testing in the form of the nonstress test, the biophysical profile, and Doppler ultrasound may lead to major advances in the diagnosis of intrauterine growth retardation and the twin-to-twin transfusion syndrome in twins. Therefore, it is reasonable to expect that fetal death and neonatal death from intrauterine growth retardation can be prevented.

Antimicrobial activity of amniotic fluid against Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum.

Amniotic fluids obtained by amniocentesis at 16 weeks to term were examined for the presence of Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum. Of 140 fluids tested, none harbored chlamydiae, and only one harbored mycoplasma, M. hominis. A number of amniotic fluids were subsequently tested for their ability to inhibit the growth of these microorganisms. Amniotic fluids and chlamydial suspensions in a 2:1 ratio were incubated 30 to 90 minutes before their inoculation in McCoy cells. Procedures were followed for chlamydial isolation. Genital mycoplasmas were incubated with amniotic fluid samples for 24 or 48 hours at 35 degrees C. Growth in amniotic fluid specimens was compared with growth in pseudoamniotic fluid and broth controls. Fourteen amniotic fluid specimens collected from gestations of 16 to 40 weeks, were found to be inhibitory to the formation of inclusions of C. trachomatis in McCoy cells. Ten amniotic fluid specimens (16 to 39 weeks, gestation) demonstrated various degrees of inhibition against M. hominis, and three fluids were inhibitory to the growth of Ureaplasma. The inhibitor was heat and protease resistant and activity was proportional to concentration. The molecular weight of the inhibitor was probably greater than 10,000 daltons, and pH, although perhaps a contributing factor, was not the cause of the inhibition.

Effect of bacterial growth on the bursting pressure of fetal membranes in vitro.

By mounting a layer of chorioamniotic membrane on a specially designed reaction vessel, we studied the effect of Escherichia coli and/or group B streptococcus growing on the decidual surface of the membranes in tissue culture or bacteriologic medium. The organisms grew equally well in either medium. When growing in tissue culture medium, either organism significantly weakened the membranes as compared with controls (membranes incubated in the absence of either organism). Membranes derived from pregnancies delivered vaginally or abdominally responded similarly. When organisms were grown in bacteriologic medium, bursting pressures did not decrease. Addition of bacteriologic medium (20-60%) to tissue culture medium did not affect bacterial growth, but inhibited significantly the lowering of bursting pressures. Bacteriologic medium also inhibited the peroxidase-H2O2-halide system in vitro. Heat-killed bacteria and/or supernatants of culture medium previously inoculated with bacteria were not effective in weakening membranes. The results suggest that live bacteria in conjunction with active membrane metabolism lead to a weakening and eventual rupture of the membranes.

Positive correlation between mature amniotic fluid optical density readings and the absence of neonatal hyaline membrane disease.

Fetal pulmonary maturity is generally determined by analyzing amniotic fluid for surfactants. This task is accomplished by lipid extraction of the fluid and resolution, identification and quantitation of the isolated lipids with thin-layer chromatography. These methods are lengthy, cumbersome and often not available on demand. A quick, simple, reliable and economical test therefore would be highly desirable. We have been able to correlate an optical density (OD) reading of amniotic fluid at 650 nm greater than or equal to 0.15 with the absence of hyaline membrane disease (HMD). For 428 fluids in which an OD reading of greater than or equal to 0.15 was found and delivery occurred within 48 hours, HMD was present in only two infants. The accuracy of the test was 99.53% , with a false-positive rate of 0.47%. With the use of this simple and accurate test one can satisfy the requirement of an on-demand test to determine fetal pulmonary maturity.

Infection and phagocytosis as possible mechanisms of rupture in premature rupture of the membranes.

The concept that premature rupture of the membranes is due to an infectious process is well accepted. However, no definitive data implicating a particular microorganism or a mechanism of action have been advanced. By the use of our recently developed experimental in vitro amnion-chorion reaction vessel model we have studied the effect of the peroxidase-hydrogen peroxide-halide antimicrobial system on these membranes. We have noted that amnion, chorion, decidua, and placental macrophages all possess peroxidase activity. Tissues collected from deliveries following labor (vaginal) are significantly higher in activity than those collected from deliveries with no labor (cesarean section). A mobilization of enzyme from macrophages to amnion appears to occur in the laboring patient. Increased protein hydrolysis is noted in membranes collected from patients without labor subjected to the peroxidase-hydrogen peroxide-halide cytotoxic system when compared with membranes from laboring patients. Bursting pressures of membranes collected from patients without labor are shown to be decreased when the membranes were incubated in the presence of lysolecithin or in the presence of amniotic fluid and phospholipase A2.

Correlation of amniotic fluid optical density at 650 nm and lecithin/sphingomyelin ratios with phosphatidylglycerol.

In this study we have attempted to correlate amniotic fluid optical density measurements and lecithin/sphingomyelin (L/S) ratios with the presence of phosphatidylglycerol. An 85% correlation (126 of 148) of all three parameters was noted. When the assumption was made that phosphatidylglycerol is indicative of fetal pulmonary maturity, 93 of 94 fluid samples having an optical density at 650 nm greater than or equal to 0.15 had demonstrable phosphatidylglycerol for a 1% false positive rate. In 53 fluid samples having an optical density at 650 nm less than 0.15, phosphatidylglycerol was present in 11 and absent in 42, a 21% false negative rate. The same analyses with lecithin/sphingomyelin ratios showed a 5% false positive rate and a 31% false negative rate.

Phagocytosis and onset of human labor.

An in vitro experimental model has been developed which allows the study of amnion lysosomal enzyme release under controlled conditions. Briefly, a layer of human amnion membrane mounted on a specially designed reaction vessel serves as the reaction surface. We have noted that the addition of particulate material to these membranes incubating in pseudoamniotic fluid results in an increased release of the lysosomal marker enzyme N-acetylglucosaminidase when compared to the release in the absence of particles. This release is completely inhibited by iodoacetate and slightly by azide. A similar increased release is also noted with the use of term amniotic fluid as incubation medium when compared to centrifuged (30,000 g/20 min) amniotic fluid. Lecithin and lysolecithin were effective in releasing increasing amounts of enzyme. This increased release was noted only from membranes of placentas collected from subjects who had undergone cesarean section prior to labor. Membranes collected from vaginal deliveries after labor showed a baseline increased release but no further stimulation upon the addition of any of the substances. These results suggest that the release of lysosomal enzymes from amnion membranes is brought about by substance(s) present in amniotic fluid. Very probably, these are surfactants. The interaction of these substances with amnion cells would eventually result in an exponential burst of prostaglandin synthesis, which would result in labor.

Premature rupture of the membranes at term. A comparison of induced and spontaneous labors.

Analysis of 150 patients at term (greater than 36 weeks) with premature rupture of the membranes (PROM) was made. In the group followed conservatively, 87.5% (98 of 112) began labor within 48 hours, and only 3.6% (4 of 112) had a latent period of greater than seven days. The incidence of cesarean section in patients with induced and spontaneous labors was compared in corrected groups. The induced group had a 39% (15 of 38) cesarean section rate as compared to 12% (11 of 91) in the spontaneous labor group (p = less than 0.01). None of the patients in the study showed signs of sepsis by central cultures, and only 12.7% (19 of 150) showed febrile morbidity. A conservative approach to term patients with PROM and an unfavorable cervix for induction seems to decrease the incidence of cesarean section.

Surfactants, L/S ratio, amniotic fluid optical density and fetal pulmonary maturity.

Optical density readings of amniotic fluids of 0.15 or greater at 650 nm have been noted to correlate with fetal pulmonary maturity. The amniotic fluid absorbance has been shown to be due not only to lecithin and sphingomyelin but also to other surfactants, including phosphatidyl glycerol and inositol. The addition of lecithin and sphingomyelin to previously centrifuged amniotic fluid (i.e., optical) density less than 0.001: L/S ratio, nondetectable) results in an increase in absorbance. At any simulated L/S ratio, the addition of phosphatidyl glycerol and/or phosphatidyl inositol results in a further increase in optical density. It is suggested that optical density readings represent more closely the surfactant composition of amniotic fluid than L/S ratios; therefore, it appears that optical density measurements are a better predictor of fetal pulmonary development than are L/S ratios.

A microtechnique for studying chemiluminescence response of phagocytes using whole blood and its application to the evaluation of phagocytes in pregnancy.

Previous investigators have demonstrated that polymorphonuclear neutrophils exhibit intense chemiluminescence (CL) during phagocytosis and the CL response can be used to study the cellular and humoral aspects of the phagocytic process. A microtechnique that used 10 microliters of whole blood as a source of phagocytes was developed and used to measure the phagocytic CL response during pregnancy. Increased sensitivity was achieved by the use of a high concentration of luminol (0.5 mM), prepared by sonication, as a CL amplifier. The high intensity of CL produced with luminol permitted the use of a scintillation counter in the IN coincidence mode, avoiding the necessity of dark-adapting the counting vials and reagents and working under subdued red light. The CL response was dose dependent on the number of phagocytes and/or particles (polystyrene spherules, opsonized zymosan, and E coli). The CL response was decreased by inhibitors that prevent particle uptake (iodoacetate and fluoride), inhibitors that prevent free-radical production (sodium benzoate and superoxide dismutase), and by inhibitors that inactivate myeloperoxidase (cyanide and azide). Results suggested that the phagocytic CL response in our assay system was dependent on O2 activation followed by the activated O2 species reacting with myeloperoxidase and chloride. The new technique was used to demonstrate a progressive increase with gestation in the phagocytic CL response in pregnancy and a rapid decrease to normal values at 1 week postpartum.

The effect of cervical/vaginal secretions on measurements of lecithin/sphingomyelin ratio and optical density at 650 nm.

Free-flowing amniotic fluid collected vaginally can be used in a reliable way for determination of fetal pulmonary maturity. Lavaging the vaginal/cervical area with sterile saline and examining the lavage fluid for lecithin/sphingomyelin (L/S) spots showed no detectable spots in the supernatants (one exception) and barely detectable L/S spots in the sediment. Vaginal-cervical saline-wash fluids did not affect fluid L/S ratios. Lavaging the vaginal-cervical area with abdominal amniotic fluid did not affect the L/S ratio of the original amniotic fluid.