Substance In Amniotic Fluid Research Articles

Chemical Identification of "Maternal Signature Odors" in Rat.

Newborn altricial mammals need just after birth to locate their mother's nipples for suckling. In this precocious behavior, including for the human baby, maternal odor via the olfactory process plays a major role. Maternal odor emitted by lactating females or by amniotic fluid (AF) attracts pups, but the chemical identity of this attractant has not yet been elucidated. Here, using behavioral tests and gas chromatography coupled with mass spectrometry (GC-MS) techniques, we show that AF extracts from rat pregnant female, nipples, ventral skin, milk, and nest extracts of mother contained 3-6 active substances. AF extracts contained 3 active compounds: ethylbenzene, benzaldehyde, and benzyl alcohol, and their mixture in similar proportions to those found in AF extracts, in a ratio, respectively, of 1:1:12 (700 ng), attracts pups as putative maternal attractant substances (MAS). These 3 AF substances have already been identified in milk, nipples, ventral wash, and nest extracts of mother, but not in feces. Moreover, anethole flavor incorporated in pregnant rat and mother's diet is also detected in AF, nipples, milk, and nest extracts and the pups are attracted to anethole odor, but not in the case of the no-anethole pups. MAS, combined with diet flavors present in the AF bath, represent olfactory signals as "maternal signature odors" (MSO) that are learned by fetus and pups. These findings open the way to improved understanding of the neurobiology of early olfactory learning and of the importance of evolutionarily conserved survival behavior in many mammal species.

Therapeutic amnioinfusion for chronic abruption‐oligohydramnios sequence: A possible prevention of the infant respiratory disease

Chronic abruption-oligohydramnios sequence (CAOS), characterized by chronic vaginal bleeding and oligohydramnios, is associated with adverse pregnancy outcomes, including preterm delivery and lung problems in the infant. Fetal lung damage may be induced by not only oligohydramnios but also iron-induced oxidative stress through chronic aspiration of bloody substances in amniotic fluid. We describe a pregnancy complicated with CAOS that was managed with repeated amnioinfusions. This is the first report showing that amnioinfusions succeeded in a significant reduction in high concentrations of iron, lactose dehydrogenase, and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage, in the amniotic fluid complicated by CAOS. The baby, born at 26 weeks' gestation via cesarean, was discharged home without supplemental oxygen 116 days after birth.

Strip of the Month: February 2011

Strip of the Month: February 2011

Index of suspicion.

Index of suspicion.

Frigen II improves the reliability of measurement of interleukin‐1 related substances in amniotic fluid

To investigate the role of interleukin-1 related substances in amniotic fluid in normal term labor without intrauterine infection. Amniotic fluid samples were collected from forty-one patients with various backgrounds. A novel pre-assay treatment using Frigen II was introduced to improve the recovery rates of cytokines, i.e., interleukin-1alpha, interleukin-1beta and interleukin-1 receptor antagonist, prior to ELISA assay. Urine samples from newborn infants were also tested. The concentrations of interleukin-1alpha, interleukin-1beta and interleukin-1ra were significantly higher in samples from normal vaginal delivery. The higher levels of interleukin-1alpha were also observed in samples from preterm labor without infection. Preterm infants produced more of interleukin-1 receptor antagonist in urine compared with term infants. This study provides evidence of the possible involvement of interleukin-1 related substances in labor without signs of infection. The data from newborn urine suggests that amniotic interleukin-1ra originates from the fetus. However, amniotic fluid interleukin-1alpha and interleukin-1beta may be derived from maternal tissue, such as decidua.

Frigen II improves the reliability of measurement of interleukin-1 related substances in amniotic fluid

Frigen II improves the reliability of measurement of interleukin-1 related substances in amniotic fluid

The influence of amniotic fluid on prostaglandin synthesis and metabolism in human fetal membranes

To investigate the effect of amniotic fluid on prostaglandin synthesis and metabolism in the fetal membranes. A cell culture study of amnion and chorion obtained at elective cesarean section incubated with amniotic fluid collected following either spontaneous labor and delivery, or elective cesarean section. Forty-eight pregnant women at 3742 weeks gestation: 24 in spontaneous labor and 24 delivered by elective cesarean section. Significantly more PGE2 and PGF2alpha were produced by amnion and chorion treated with amniotic fluid from spontaneous labor compared with elective cesarean section. Spontaneous labor amniotic fluid favors PGE2 and PGFM production by amnion and chorion respectively; while elective section fluid stimulates PGE2 synthesis by both tissues (reflected as PGEM in chorion). Amniotic fluid, from either spontaneous labor or elective section, had no effect on the metabolism of exogenous PGE2 or PGF2alpha by chorion cells. Spontaneous labor is associated with the presence of a substance in amniotic fluid which facilitates prostaglandin synthesis in the fetal membranes, but which is without effect on prostaglandin metabolism.

Effects of keratinocyte conditioned medium, amniotic fluid and EGF in reepithelialization of human skin wounds in vitro

Growth factors and combinations of growth promoting substances in amniotic fluid, platelet extracts or factors secreted by cultured human keratinocytes have been shown to stimulate cell proliferation and wound healing. In this report, the possibility of studying reepithelialization of wounds in human skin, using small biopsies maintained in vitro, was examined. The effects of fetal calf serum (FCS), keratinocyte conditioned medium (KCM), amniotic fluid and epidermal growth factor (EGF) on the reepithelialization process was investigated after seven days. KCM alone did not affect healing, but when added to a suboptimal concentration of FCS (2%), KCM induced reepithelialization of the wounds. Amniotic fluid (25%) alone stimulated the formation of a new epidermis, whereas EGF (10 ng/ml) alone or added to 2% FCS did not induce reepithelialization. The model used in this study includes an autologous matrix comprising living fibroblasts and endothelial cells and may thus be used to study aspects of wound healing in human skin.

Growth-factor-like substance in amniotic fluid in the rat: effect on the development of fetal colonic goblet cells.

Extra- and intrauterine fetuses were studied to explore the effect of amniotic fluid on the development of colonic goblet cells. Significant differences in the density of goblet cells in developing colon were observed during fetal days 19-22 between these two groups, suggesting that amniotic fluid affects the development of the colonic goblet cells, especially during the period between days 19 and 21 when rat fetuses began to swallow amniotic fluid, as demonstrated by injecting India ink into the amniotic cavity. By immunological dot blot analysis, the amniotic fluid and gastric juice from intrauterine fetuses were positive for epidermal growth factor (EGF), whereas maternal abdominal serous fluid and gastric juice from extrauterine fetus were negative for EGF. The present results indicate that the amniotic fluid during late gestation greatly affects the development of the colonic goblet cells in rat fetuses and that the trophic factors for the cells seems to be EGF or an EGF-like substance in the amniotic fluid.

Isolation of multiple substances in amniotic fluid that regulate amnion prostaglandin E 2 production: The effects of gestational age and labor

The present study was undertaken to evaluate the effects of whole amniotic fluid (AF) and fractions of AF on amnion cell prostaglandin E 2 (PGE 2) production. Amnion cells were grown to confluence and then incubated in the presence of AF, or fractions thereof, obtained at 17–19 weeks gestation (MID), at term prior to the onset of labor (NIL), and at term after spontaneous onset of labor (LABOR). All whole AFs were stimulatory to amnion cell PGE 2 production (p < 0.001) but the stimulation by NIL and LABOR AFs was significantly greater (p < 0.001) than stimulation by the MID AF. Fractionation of the AFs from the three groups (n=9–10 per group) revealed multiple discernable peaks of stimulatory activity in each group. The majority of peaks had retention times that were similar among the three groups, and peak stimulatory activities were greater in NIL and LABOR samples than in MID samples.

Placental opioid-enhancing factor (POEF): Generalizability of effects

A substance in amniotic fluid and placenta (POEF for Placental Opioid- Enhancing Factor) has been shown to enhance opiate- or opioid-mediated analgesia in rats. Recent studies have only touched on the generalizability of the phenomenon. The present studies further tested the generalizability of the POEF effect: they examined sex specificity of the mechanism; whether POEF activity exists in afterbirth material of species other than the rat; whether POEF activity exists in tissue other than afterbirth material; whether POEF activity could be demonstrated after injection rather than ingestion of afterbirth material; and whether POEF enhances all opioid-mediated phenomena. We found that (a) POEF is effective in male rats as well as in female rats; (b) POEF activity exists in human and dolphin afterbirth material; (c) ingestion of pregnant-rat liver does not produce enhancement of opioid-mediated analgesia; (d) POEF does not seem to be effective when amniotic fluid is injected either IP or SC; and (e) POEF does not modify morphine-induced hyperthermia.

Amniotic fluid ingestion before vaginal/cervical stimulation produces a dose-dependent enhancement of analgesia and blocks pseudopregnancy

A substance in amniotic fluid (AF) and placenta has been shown to enhance analgesia produced by morphine, late pregnancy, footshock, and vaginal/cervical stimulation (VS). When morphine-induced analgesia was assessed previously, the degree of enhancement by ingestion of AF or placenta was found to be a function of the amount of analgesia being generated. We have extended these results to include the analgesia produced by VS. Analgesia induced by 75, 125, 175, or 225 g of vaginal/cervical pressure was measured in rats pretreated with 0.25 ml (by orogastric infusion) of either AF or saline. AF infusion enhanced the analgesia produced by 125 g VS, but did not affect the analgesia produced by 75, 175, or 225 g VS. Unexpectedly, we also found that infusion of AF shortly before the application of VS prevents VS-induced pseudopregnancy (PsP). Whereas the incidence of PsP following 75, 125, or 175 g VS was less than 19% and not statistically different for AF and saline pretreatments, the incidence of PsP after 225 g VS was 44% in saline-pretreated rats, but only 10% in AF-pretreated rats. Protection from the induction of pseudopregnancy, which could be caused by mechanical stimulation of the cervical area during delivery, may be an additional benefit of parturitional ingestion of placenta and amniotic fluid (placentophagia).

Detection of Early Pregnancy Factor‐Like Activity in Human Amniotic Fluid

Early pregnancy factor (EPF), an immunosuppressive substance, appears in mammalian maternal serum at a very early stage after fertilization. To investigate the normal distribution of EPF in the human body, the rosette inhibition test has been used to detect EPF in human amniotic fluid. The mean value of rosette inhibition titer (RIT) was significantly higher (5.93 +/- 0.43 SE) in 52 amniotic fluid samples (16-30 weeks' gestation) than in 23 nonpregnant sera (3.54 +/- 0.11), indicating that something in amniotic fluid inhibits rosette formation. This inhibitory factor is recognized as EPF or EPF-like substance for its activity significantly correlated with that of EPF in serum from the same donor (r = 0.756: P less than 10(-6]. The immunological significance of the EPF-like substance in amniotic fluid may be important to the survival of semiallogeneic conceptus.

Digoxin-like substance in amniotic fluid--fact or artifact?

A digoxin-like immunoreactive substance (DLIS) has been reported in the amniotic fluid. Since radioimmunoassay kits are standardized using serum-based standards, we hypothesized that measurement of DLIS may be an artifact related to the low protein content of amniotic fluid. We analyzed 12 amniotic fluid samples before and after supplementation with lyophilized human serum. The means +/- SDs for DLIS (nmol/l) at protein concentrations of 0, 32 and 63 g/l were 1.4 +/- 0.16, 0.6 +/- 0.09, and 0.4 +/- 0.09 nmol/l, respectively. We, therefore, hypothesize that DLIS in amniotic fluid may in part be explained by a technical artifact.

Digoxin- and digitoxin-like immunoreactive substances in amniotic fluid, cord blood, and serum of neonates.

Using four different digoxin kits, it was disclosed that the majority of various samples including amniotic fluid, cord blood, and serum from neonates contained substantial levels of digoxin-like immunoreactive substance. The differences in data seemed to be due to the range of epitopes which are recognized by antidigoxin antiserum. The day-to-day studies on sera serially obtained from infants at birth to 48 days old revealed that the level of the substance (0.31 +/- 0.12 ng/ml) in sera of the 1-day-old neonates rapidly declined to the level of 0.1 ng/ml by the 2nd postnatal wk and thereafter gradually declined. The immunological specificity and accuracy of the detection of digoxin-like immunoreactive substance was confirmed by a sample dilution test, a recovery test for standard digoxin, and an absorption test with antidigoxin antiserum. The amniotic fluid and cord blood also contained four to eight times more of a digitoxin-like immunoreactive substance than they did digoxin-like immunoreactive substance. A significant correlation was observed between the levels of digoxin-like immunoreactive substance and of digitoxin-like immunoreactive substance (p less than 0.01).

The presence of alpha-interferon in human amniotic fluid.

Almost all the samples of amniotic fluid from 62 pregnant women from the 16th week to the end of the pregnancy contained detectable amounts of alpha-type interferon. The presence of this substance in amniotic fluid during pregnancy raises the question of the physiological significance of this finding. It is postulated that the amniotic type of alpha-interferon might be a product of a constitutive gene, rather than induced by latent virus infection.

Genital mycoplasmas and inhibitory substances in amniotic fluid: A negative result

BECAUSE OF THE POTENTIAL importance of genital mycoplasmas as a cause of amnionitis, we cultured 50 amniotic fluid samples obtained at the time of routine amniocentesis for :~f~c~~fdasrnu hominis and Ureaplasma rr~crl$icum. In addition. we studied 25 amniotic fluid samples for inhibitory stubstances to either of these mycoplasmas. ,4mniotic fluids were kept at -70” C until they were thawed for culturing. After thawing the fluid was spun at 2,000 rpm for 10 minutes; 0.6 ml was removed from the bottom of the tube. and 0.2 ml was distributed into each of three vials: urea broth, urea broth with lincomycin, and arginine broth with erythromycin. Cultures were incubated at 37” C and checked twice daily for a color change consistent with a rise in pH. Such vials were subcultured onto mycoplasma agar. All .N specimens were negative for either mycoplasma species. In order to ascertain whether amniotic fluid might contain an inhibitory substance to genital mycoplasmas. filter paper disks. 6 mm in diameter, were impregnated with 25 ~1 samples of amniotic fluid and the disks were placed on plates which had been seeded with a known strain of ,Lf. hominis and I,‘. urc~alyticum. The 25 amniotic fluid samples selected for these experiments were derived from pregnancies of 15 to 43 weeks’ gestation with a mean of 35.5 weeks. These plates were incubated in an atmosphere of 90%’ nitrogen and 10% carbon dioxide and, beginning 23 hours after incubation, were inspected for anv inhibition of mycoplasma growth around the disks. None of the 25 amniotic fluid samples demonstrated any inhibition of either mycoplasma species. Chorioamnionitis has been associated with infant, but not maternal. colonization with U. urenlyticum.’ It is thought that amnionitis. which occurs with intact membranes. is mainly a result of invasion of these membranes in the area of the cervical OS. After rupture of membranes ,M. ho~fi~b has been cultured from amniotic fluid,’ but, just as we observed, Klein and Milunsky were unable to culture mycoplasmas from 32 amniotic fluid specimens. It might be surmised that the antibacterial polypeptide that has been found in amniotic fluid would also have a role in preventing the

Lewis and ABH substances in amniotic fluid obtained by amniocentesis.

Extract: When eight amniotic fluid samples obtained by transabdominal amniocentesis at various ages of gestation (15–38 weeks) were tested, Lewis substances were present in considerable concentration as early as 15 weeks of gestation. The amniotic Lewis substances found corresponded to the Lewis and secretor phenotypes of the fetus ascertained by determination of the erythrocyte and salivary phenotypes of the mother-infant pairs after delivery. These findings indicate strongly that the amniotic Lewis substances are exclusively of fetal origin. Although not conclusive, the data also indicate that the amniotic ABH substances are the expression of the fetal ABH and secretor status. Speculation: This preliminary study shows that the fetal Lewis and secretor phenotype can be determined antenatally by amniocentesis. This information could be clinically significant in the prenatal detection of hereditary conditions exhibiting linkage to the Lewis and secretor loci. In further research on this subject, Lewis phenotypes of erythrocytes in infants should be determined at appropriate postnatal age with due consideration for the maturation process and differential tissue expressivity of the Lewis and secretor gene systems. Amniotic fluid samples should be obtained preferably by transabdominal amniocentesis to avoid contamination by maternal secretions and saliva from infants should be obtained several days postnatally.

PREDICTION OF NEONATAL RESPIRATORY DISTRESS BY ESTIMATION OF AMNIOTIC-FLUID LECITHIN

A simple chemical method for estimating the total lipid, phospholipid, and lecithin content of amniotic fluid is described. It has been used to test the hypothesis that neonatal respiratory function is related to the pre-delivery levels of these substances in amniotic fluid. 171 samples of amniotic fluid taken at different stages of pregnancy from 156 patients were assayed; 110 samples were obtained in the 24 hours before delivery of the infant. Amniotic-fluid lecithin levels of around 3·5 mg. per 100 ml. would seem to be critical, for pre-delivery levels above this value were in every case associated with normal respiratory function, even in 12 infants who were "small for dates " or premature. By contrast the pre-delivery amniotic-fluid lecithin concentration was below 3·5 mg. per 100 ml. (range 0·6-3·4 mg. per 100 ml.) in 12 out of 13 infants who developed respiratory-distress syndrome. The lecithin concentration in the amniotic fluid seems to be a sensitive index of fetal lung maturity and of the potential risk of respiratory distress developing in the baby.

Identification of a pressor substance in amniotic fluid: I. Role of epinephrine and norepinephrine

Unidentified pressor material has been isolated from amniotic fluid of toxemic patients by several investigators. Other investigators have not been able to confirm these substances. This paper deals with the isolation and identification of a specific pressor agent (norepinephrine) in amniotic fluid of normal pregnancies. Details are enumerated pointing out the necessity of meticulous handling of this fluid if data are to be valid. Catecholamine values, i.e., epinephrine and norepinephrine, are altered by pH change, temperature, preservatives, interfering substances, storage in deep freeze, and an enzyme inhibitor. A specific method is outlined to overcome these problems so that valid values are obtained. Amniotic fluid represents an amount equivalent to 2 days’ output of fetal urinary catecholamines, approximately 1.0 μg per liter of epinephrine and 3.0 μg per liter of norepinephrine. A pressor agent in amniotic fluid has been identified as norepinephrine; however this probably has no effect on the maternal organism. This report is the first in the English language to identify a specific pressor agent. Unidentified pressor material has been isolated from amniotic fluid of toxemic patients by several investigators. Other investigators have not been able to confirm these substances. This paper deals with the isolation and identification of a specific pressor agent (norepinephrine) in amniotic fluid of normal pregnancies. Details are enumerated pointing out the necessity of meticulous handling of this fluid if data are to be valid. Catecholamine values, i.e., epinephrine and norepinephrine, are altered by pH change, temperature, preservatives, interfering substances, storage in deep freeze, and an enzyme inhibitor. A specific method is outlined to overcome these problems so that valid values are obtained. Amniotic fluid represents an amount equivalent to 2 days’ output of fetal urinary catecholamines, approximately 1.0 μg per liter of epinephrine and 3.0 μg per liter of norepinephrine. A pressor agent in amniotic fluid has been identified as norepinephrine; however this probably has no effect on the maternal organism. This report is the first in the English language to identify a specific pressor agent.