Fetal maturity: Biochemical analyses of amniotic fluid JERRY L. MYERS, PH.D. MARY JANE P. HARRELL, B.A. FREDDIE L. HILL, B.S. Galveston, Texas

One hundred and three samples of amniotic fluid from 21 normal and 64 medically complicated pregnancies were used for evaluating fetal maturity by measuring one or more of the following: the lecithin-sphingomyelin ratio (L/S), bilirubin concentration, creatinine concentration, and percentage of fetal fat cells. The rapid foam test for surfactant was evaluated in 20 of the samples and found to be unreliable in predicting the risk of respiratory distress syndrome. The test for fat cells gave a large percentage of false negative results, and the creatinine concentration failed to show a good correlation with gestational age. Both the bilirubin concentration and LIS ratio showed good correlation with gestational age. How ever, since the major cause of death in a premature infant is the respiratory distress syndrome, the maturity of the fetal lung, which is best estimated by the LIS ratio, has to be the prime concern.

E v A L u A T I o N o F fetal maturity is important when delivery prior to term is indicated to assure survival of the infant. Biochemical and cytologic studies of amniotic fluid are being used with increasing frequency for this evaluation. Attention here is focused on four such analyses which assess the functional maturity of individual fetal organs and can be used to predict gestational age. Earlier studies on amniotic fluid components established the relationship of bilirubin, 1 creatinine, 2 and fetal fat cellsa with fetal maturity. These tests contribute valuable information about the maturity of the fetal liver, kidney, and skin, respectively. However, because the survival of a premature infant depends primarily on the maturity of the fetal lung, an estimation of the functional maturity of this organ is necessary. From the Departments of Pediatrics and Obstetrics and Gynecology, University of Texas Medical Branch. Supported in part by The National Foundation March of Dimes Grant No. C-129. Received for publication June 6, 1974. Accepted june 17, 1974.

Reprint requests: Dr. ferry L. Myers; Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas 77550.


Respiratory distress syndrome is the major cause of death in the premature infant. This disease is directly related to the maturity of the lung. Pulmonary alveolar stability is dependent on the amount of surfactant produced by the alveolar membranes of the fetus. 4 It has been shown that lung maturity can be estimated by a ratio of two phospholipids, lecithin and sphingomyelin, both having surfactant properties. 5 However, as the lung matures, the rapid synthesis of lecithin produces an abundant amount of this compound resulting in a ratio of more than 2: 1 when the fetal lung is mature." Recently, a simple foam test for estimation of surfactant in amniotic fluid was reported. 6 This test is based on the formation of a stable foam when the amniotic fluid is mixed with ethanol. The test is rapid and inexpensive; however, it has not been established as a valid means of predicting the risk of the respiratory distress syndrome. The data presented here were obtained when amniotic fluid was analyzed by these four tests in an effort to establish a correlation between the test values and gestational age. At the same time an evaluation is made of the reliability of the foam test for surfactant in predicting the risk of the respiratory distress syndrome.


April 1, 1975

Myers, Harrell, and Hill

Am. ]. Obstet. Gynecol.

Table I l.Jurn.be-r Tv/JP- nf gestation

Normal Abnormal


I Amniocenteses

21 64



Table II. Selection of patients for fetal maturity studies* Com Plication

No. of patients

Anticipated cesarean section Rh sensitization Hypertension Pre-ec 'ampsia Diabetes Asthma s;ckle cell disease Premature labor Postmaturity Congenital heart disease Flacen tal insufficiency

21 17 12 20 10 2 'J

*Three patients are in two categories.

Materiai and methods One hundred and three amniotic fluid specimens were obtained from 85 patients by transabdominal puncture performed under aseptic conditions (Table I). Amniocentesis was done on these women because of clinical conditions (Table II) which would probably require preterm delivery. Three pregnancies were complicated by two factors; five pregnancies involved twin births. Four methods of analysis were chosen for the estimation of fetal maturity, that is, lecithin-sphingomyelin ratio (L/S), bilir~bin co~centratio; ( sp~c­ trophotometric scan), creatinine concentration, and fetal fat cell count. Samples of amniotic fluid containing gross amounts of blood or meconium were excluded from this study, as were improperly prepared mailed-in specimens and fluids from pregnancies resulting in stillborn infants. Gestational age in weeks was calculated from the date of the last normal menstrual period. After delivery the maturity of the infant was estimated by a physical examination 7 performed by the attending pediatrician. L/S ratio. The amniotic fluid sample was chilled in ice immediately, then centrifuged (2,000 x g) at 4 degrees C. to remove cell debris and any red blood cells present. Equal volumes of amnioti~ fluid and methanol were mixed, then two volumes of chloroform were added to extract the phospholipids.

The lower chloroform layer was separated by centrifugation. The method of Gluck and co-workers 5 was modified at this point to include the filtration of the chloroform layer through Na 2 S0 4 to remove any traces of moisture before evaporation to dryness under nitrogen at 60 degrees C. The residue was moistened with 10 ,ul of chloroform and the phospholipids precipitated by the addition of 0.4 ml. of cold dry acetone. The precipitate was dissolved in 50 ,ul of chloroform and 10 ,ul were spotted on 250 fLM silica gel H plates. The chromatogram was developed in a chloroform-methanol-water system (60:30:5), then dried, sprayed with 50 per cent HzSO,, charred on a hot plate at 160 to 190 degrees C., and scanned with the use of a densitometer.* The L/S ratio was calculated from the areas of the lecithin and sphingomyelin peaks. Foam test. The procedure of Clements and coworkers6 was followed as closely as possible. Three minor modifications were made: ( 1) The test tubes were fitted with Teflon-lined screw tops instead of rubber stoppers. ( 2) Seventeen of the 20 sam pies were tested in only three dilutions (1:1, 1:2, and 1:4) instead of five dilutions. ( 3) Only once was a standard solution of dipalmitoyl lecithin prepared as a control. Three amniotic fluid samples that had been positive in a 1:2 dilution were frozen and subsequently used as controls, and four times no control was used. All samples were kept chilled in ice if the testing was done on the same day as the amniocentesis. Otherwise, they were frozen for later testing. Bilirubin peak at 450 m,u. The amniotic fluid sample was protected from light at all times. The fluid was centrifuged for ten minutes in an analytic centrifuge and the supernatant liquid was filtered through Whatman No. 2 filter paper. Optical densities (O.D.) in the visible range were measured with a Beckman DC spectrophotometer and plotted on semilogarithmic graph paper. The llO.D. 450 m,u is the difference in the optical densities as shown by the actual reading at 450 m,u and the point at which a straight line drawn between 375 m,u and 525 m,u intersects the 450 m.,u ordinate.' Creatinine. The amniotic fluid was centrifuged and the supernatant liquid used for determination of creatinine with the use of a modification of the procedure of Folin and Wu.!l Fat cells. A smear was made of the amniotic fluid *Helena La-boratories, Beaumont, Texas.

Volume 121 Number 7

Fetal maturity 963

Fig. 1. Lecithin-sphingomyelin ratio in the amniotic fluid during gestation. • = Individual single sample. •-• = Sequential samples on the same patient. &. = Sample from pregnancies involving twin births.

cells obtained by centrifugation. One or two drops of a 0.1 per cent aqueous solution of Nile blue sulfate were added and a coverslip was applied. The· wet preparation was examined under intermediate objective (lOx) and high-power objective ( 43x). Fetal fat cells were identified as those cells that stained orange. Two hundred celis were counted and the percentage of orange-stained cells was calculated.2 Results

Changes in the values with gestational age in weeks. The L/S ratio was determined in 102 samples of amniotic fluid. The change in this ratio during gestation is represented in Fig. 1. Before 35 weeks the L/S ratio was less than 2:1 except in three samples. The clinical data from the pregnancies involving these samples are given in Table III. Of particular interest is the sample at 33 weeks that had an L/S ratio of 13:1. This amniotic fluid was obtained from an Rh-sensitized patient whose fetus had had an intrauterine transfusion 12 days prior to this sample. After 35 weeks' gestation the L/S ratio was greater than 2:1 except in three cases. Table III also shows the clinical data for these three pregnancies. The optical density difference at 450 mfL was determined on 78 samples of amniotic fluid. Samples obtained from Rh-sensitized pregnancies which terminated with infants affected with moderate or severe hemolytic disease of the newborn were ex-

Fig. 2. The optical density difference at 450 niotic fluid during gestation.


of am-

eluded from the scattergram shown in Fig. 2 because in these cases the bilirubin is more representative of hemolysis of the fetal red cells than of fetal maturity. The ~O.D. 450 mp. decreased with gestationa! age, and after 35 weeks' gestation the value was less than 0.04 in all but six cases. In three samples of amniotic fluid obtained before 35 weeks' gestation, the aO.D. 450 m,u value was less than 0.04, while in three other cases after 35 weeks it

964 Myers, Harrell, and Hill



April!, 1975 Obstet. Gynecol.

Table III. L/S ratio and clinical data on amniotic fluid samples greater than 2:1 before 35 weeks or less than 2: 1 after 35 weeks Patient B.McC.

J. F.

]. s.

Y.G. M.W. D.L.

LIS 4.1 13.1 7.8 0.6 1.9t 1.6 5.8t 0.8

Maternal complication Diabetes Rh Chronic hypertension Diabetes Chronic hypertension Placental insufficiency

Days to delivery ')


2 3



Weeks' gestation*

Gestational aget

33 33 34 34 36 36 41 37

35-36 34 34 35 38 Unknown

*Obstetrician. t Pediatrician. tRepeat amniocentesis.

Table IV. aO.D. 450 mfl. values and the clinical data on amniotic fluid samples that were less than 0.04 before 35 weeks' gestation or greater than 0.04 after 35 weeks' gestation Patient B. McC.

E.Q. C. H. S.D.



t..O.D. 0.008 0.015 0.040 0.060 0.065 0.090

Maternal complication Diabetes Chronic hypertension Rh sensitization Sickle cell disease Congenital heart disease Anticipated cesarean section

Days to delivery

Weeks' gestaeion*

Gestational aget

2 17 13 3 4 4

33 34 34 37

35 37 36



36 38


*Obstetrician. t Pediatrician.

Table V. Accuracy of the four tests* in predicting fetal maturityt % Accuracy

Observation L/S ratio AO.D. 450 m_. Creatinine Fetal fat cells

Mature value 3.0 0.020 2.0 20%

Normal I Abnormal gestation gestation 100


56 50

82 69 88


*All tests done within one week of delivery. tFetal maturity defined as 37 weeks or more and/or 2,500 grams or greater.

was greater than 0.04. The clinical data identifying these amniotic fluids are presented in Table IV. Fig. 3 illustrates the creatinine concentration in amniotic fluid samples from 79 pregnancies during late gestation. At 32 \a:eeks' gestation the values were 1.3 and 1.5 mg. per cent; at 34 weeks the values ranged from 1.3 to 2.5 mg. per cent. After 35 weeks the average value was 2 mg. per cent. The percentage of fetal fat cells was found to be less than ten per cent until 35 weeks' gestation. After this time the value varied from one to

90 per cent as shown in the scattergram in Fig. 4. Accuracy of the four tests for predicting fetal maturity. In evaluating the accuracy of each indi· vidual test used to assess fetal maturity, only those analyses done within one week of delivery were used. An infant was considered "mature" if the pediatrician's estimation of gestational age was 37 weeks or more and/or the birth weight was 2,500 grams or greater. An L/S ratio of 3:1 or greater, a aO.D. 450 mJJ. of 0.02 or less, a creatinine concentration of 2.0 mg. per cent or greater, and a fetal fat cell count of 20 per cent or more were the values chose by the obstetricians to represent fetal maturity. As shown in Table V, in medically uncomplicated pregnancies creatinine concentration, aO.D. 450 mJJ., and percentage of fat cells were accurate in only 50 to 56 per cent of the analyses. The L/S ratio correctly predicted a mature iPJant of 37 weeks or more in all samples of normal amniotic fluid assayed. In the high-risk pregnancies, identified as being complicated by maternal diabetes, Rh sensitization, pre-eclampsia, and/or hypertension, a creatinine value greater than 2.0 mg. per cent predicted maturity in 88 per cent of

Volume 121 Number 7

Fetal maturity


Table VI. L/S ratios and foam tests in 20 samples of amniotic fluid

I 401


. ... .. .... .... .. ... .. :· .....




2.0 0~



Foam test LIS ratio ~8.0

14.0 13.0 12.5 11.5 i0.9 8.0 7.8







WEEKS 1 GESTATION Fig. 3. Creatinine concentration in a.n1niotic fluid during gestation.

5.8 5.2 4.5 4.1 3.0 3.0 2. 7 2.0 1. 9 0.8


48 Hours or more later

Positive Intermediate

Positive Intermediate







24 Hours later

Same day

Positive Positive Negative Positive Intermediate Positive* Intermediate Intermediate Intermediate Negative


Positive* Negative Negative Negative Negative Negative


*Sample frozen for later use as a control. tSample drawn at cesarean section delivery two days after determination of L/S ratio and frozen for later use as a control.

ji001 0 80 ....

1t ...1

;:: 60 UJ



~ 40

~ 20+-----------------------------------------,. [










j. j:







Fig. 4. The percentage of fetal fat cells in amniotic fluid during gestation.

the analyses while the L/S ratio greater than 3:1 was accurate in 82 per cent of the analyses. L/S ratio, foam test, and the respiratory distress syndrome. Since the L/S ratio and the foam test are both considered indicators of lung maturation, they were compared as to their ability to predict the risk of the respiratory distress syndrome. The results of the foan1 tests and the conco1nitant L/S ratios are shown in Table VI for 20 samples of ~mnlntir

,.,....,._.._,..._..._ .. ..., ...... "-'

fh.;rl Tht=). ............. .._.. • ..-. i.4'--'


Fetal maturity: biochemical analyses of amniotic fluid.

Fetal maturity: Biochemical analyses of amniotic fluid JERRY L. MYERS, PH.D. MARY JANE P. HARRELL, B.A. FREDDIE L. HILL, B.S. Galveston, Texas One hu...
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