Surface properties of the amniotic fluid in normal pregnancy ERICH JURI KARL

MULLER-TYL, LEMPERT,

M.D.

STEINBEREITHNER,

HERBERT Vienna,

M.D.

BENZER,

M.D. M.D.

Austria

The surface properties of the amniotic fluid were determined between weeks 23 and 41. The y-max, y-mitt, hysteresis area, and as parameters for evaluation of the surface tension area diagrams. against the gestation week and a curve for the normal course

in 96 normal Pregnancies stability index were used These results were plotted of surface tension of the

amniotic @id during the second half of gestation was obtained. The injluence blood, vernix, and meconium to amniotic @id as well as that of changes dilution were examined. The results were unaffected by either blood, vernix, The slight scatter of the y-min values makes this parameter particularly determining fetal lung maturity.

I N R E c E N T years it was demonstrated with chemical methods that during the progress of pregnancy there is a rise in the concentration of surfactant components (lecithin, sphingomyelin, etc.) in the amniotic fluid.“, 9, I2 In 1967 Scarpelli demonstrated for the first time that amniotic fluid at term and lung extracts from diseased newborn infants with fully developed lungs showed similar surface properties when examined with the surface balance.13 Since preliminary results have shown that there is a steady rise in the surface activity of amniotic fluid during the last 20 weeks of pregnancy,l’* I4 we also considered it worthwhile to test whether amniotic fluid surface properties are influenced by presence of blood, meconium, and vernix or by changes in the pH and by dilution. In addition we intended to systematically examine a considerable number of amniotic fluid samples from normal pregnancies for surface activity during gestation in From the I. Department of Obstetrics-Gynecology and Experimental Division of the Department Anesthesiology, University of Vienna. Received

for publication

Accepted

September

July

order to obtain data sufficiently accurate for establishing standards that might be used as a basis for future assessment of fetal lung development. Material and methods Sampling of the amniotic fluid. Amniotic fluid (12 to 15 ml.) during pregnancy was obtained by percutaneous amniocentesis7 or, at term, by transcervical amniotomy. Measurements in the surface balance. The surface properties of the amniotic liquid were measured in a surface (Wilhelmy) balance.*, 5 Since often only limited quantities of amniotic fluid are available a specially designed small Teflon trough (15 by 20 by 105 mm.) was used. The amniotic fluid was allowed to age for 30 minutes; the duration of a cycle was 4 minutes; the measurements were performed at room temperature. Surface tension area diagrams (SAD) were recorded on a x/y plotter. The following parameters were evaluated : surface tension-at 100 per cent surface (y-max) and at 20 per cent of the initial surface (y-min) and hysteresis area (cm.*). The stability index was calculated5 :

of

30, 1974.

14, 1974.

Reprint requests: Dr. Juri Lempert, Znstitut fiir Anaesthesiologie der Universitiit Wien, Experimentelle Abteilung, Spitalgasse A-1090 Wien, Austria.

of addition of in the pH and or pH changes. suitable for

S=

23,

295

2 ( y-max y-max

- y-min ) -. + y-min

256

hr,ti!‘er--y’

et a;.

Table I. The influence of whole and hemolysed l)H on the surface properties of amniotic liquid ~---__--__ ’ y-mm y-min p of Control 0.5 ml. whole blood 445 mg./lOO ml. free HB Vernix Meconium pH 6.5” pH 6.90 PH 7.33

(445 ng. free HR), value of 6 tests f

p of difference

vernix, SD.)

meconilml,

and

p Of difference

I idyn./cn.)

di flerence

48.3 48.3

2 1.3 k 2.0

NS

15.” 14.3

f 2.2 2 2.0

NS

1.05 1.09

2 0.14 -c o.09

NS

27.8 “7.8

-t L’.TI f 4.1

h-s

48.7

2 1.5

NS

14.2

+ 1.8

NS

1.10

? 0.10

NS

28.5

+ 3.7

‘is

48.5 48.6 48.7 48.4 48.3

2 2 + i 5

NS NS NS NS NS

13.8 13.5 15.5 14.3 14.3

+ ? + c -c

NS NS NS NS NS

1.12 1.13 1.06 1.10 1.09

k + + k t-

NS NS NS NS NS

28.3 41.0 “8.5 “8.8 ‘7.1

* i+ + +

1.1 1.5 1.5 1.0 1.5

Cdyn./cm.)

blood (mean

1.8 1.1 3.4 9 5 ‘17

Possible sources of error and their influence on the surface properties of amniotic fluid. Clear amniotic fluid of a normal pregnancy-“standard”was used for determination of initial (control) values. The effect of presence of blood, vernix, meconium and of changes in pH and dilution was examined in six tests each. Amniotic fluid with whole blood and hemolysed blood added. The hemoglobin content of a strongly hemorrhagic amniotic fluid was determined by spectral photometry. The hemoglobin content was 445 mg. per 100 ml. and was used as a basis for the tests. This concentration was obtained by addition of whole and hemolysed blood (0.5 ml.) to 12 ml. of “standard” amniotic fluid. Amniotic fluid with vernix added. “Standard” amniotic fluid (12 ml.) is mixed with 1 Gm. of vernix and saturated by shaking and stirring. Coarse filtration through a layer of gauze prevents the vernix lumps from getting into the trough. Amniotic fluid with meconium added. “Standard” amniotic fluid ( 12 ml.) is stirred with 0.5 Gm. of meconium. Saline solution with meconium added. To study the surface properties of meconium by itself, 0.5 Gm. of meconium was added to 12 ml. of 0.9 per cent saline and dissolved by shaking and stirring. Serial dilution of amniotic fluid. “Standard” amniotic fluid was diluted with saline in ratios 1: 2, 1:4, 1:8, 1:16, and 1:32. Influence of pH changes in the amniotic fluid. The pH of amniotic fluid is in the range of 6.90 to 7.20 Our tests were done at pH 6.52, 6.90, and 7.33. These values were obtained by addition of l.ON HCl or l.ON NaOH. Clinical material. Ninety-six amniotic fluid sam-

s

0.10 0.06 0.14 0.12 0.12

4.3 ‘1.6 2.8 5.0 2.6

NS < n.nrl1 NS NS NS

ples were obtained by transabdominal or vaginal (in labor) amniocentesis between weeks 23 and 42 of gestation. Surface tension parameters were plotted against the corresponding week of pregnancy. Amniotic fluid from normal pregnancies with a delivery at term of an infant with fully developed lungs only was used. Periods of four weeks each were grouped together. Statistical treatment. For all surface tension parameters a mean value i SD. was calculated; the difference of the means was tested by the t test. Results

Neither the addition of whole and hemolyzed blood nor vernix and changes in the pH in the range tested caused any significant change in the surface properties of amniotic fluid (Table I) . The addition of meconium to amniotic fluid resulted in a significant increase of the hysteresis area of SAD, while y-max and y-min values remained unchanged (Table I, Fig. 1, B) ; saline containing meconium has only a slight surface activity (Fig. 1, C) Surface activity of amniotic fluid decreases steadily with increasing dilution (Fig. 2) . In the second half of pregnancy there is a continuous rise of surface activity of amniotic liquid (Fig. 3). Comment

The rising concentration of surfactant cornponents during the course of pregnancy has been proved chemically and used for assessment of fetal lung maturity.lg, I’, I8 Physical methods were also used to verify the presence of surfactant in the amniotic fluid. As early as 1958 Agostoni and associate? studied the surface

Volume

12

Number

3

Surface

I

I

-60

,

I

properties

of

amniotic

1

I

I

100% 1

fluid

297

’

II

iij~~~yy-(

-10

20

100%

I

20

Fig. 1. Surface tension y(dyn./cm.) same amniotic fluid with 0.5 ml.

100%

area meconium

I

20

(4) diagrams. A, .4mniotic fluid at term; B, for the added; C, for saline solution with meconium added.

,O.A

0.c

0.6

.l.O Y (DYN/C#) 1.2

L -

30

-

5

-

1

1~32

Fig.

-CM2

-.-.-

t0

2. The

stability

index

I

1:16

surface

tension

?? of amniotic

1:s

parameters fluid

y-max,

plotted

tension of cat, goat, and guinea pig amniotic fluid. In 1964 Enhorning” was the first to test human amniotic fluid at term, using his own technique of maximum bubble pressure. Because of certain methodological shortcomings, these investigations were not followed up. In 1972 Clements and associates” introduced another technique for the assessment of surfactant in the amniotic fluid. This method is based on the property of the surfactant of generating a stable foam after shaking with ethanol. In 1967 Scarpelli” observed a considerable de-

1:4

y-min

against

(dyn./cm.),

dilution

(mean

1:2

hysteresis values

1:l

area

(cm.?),

and

of 6 tests -C 1 S.D.).

gree of similarity between the surface properties of amniotic fluid at term and of extracts from the fully developed lungs of dead infants in the surface balance. Within a test designed to provide general orientation about the surface properties of amniotic fluid during pregnancy, evidence was furnished of a continuous rise in surface activity during the second half of gestation.“, IA There is one disadvantage shared by all chemical techniques proposed to determine the surfactant in the amniotic fluid: the impact of meconium and

i

Am. J. Ot,\r:.l

Fig.

3. Surface

tension

(cm.3), and stability 42 weeks of gestation

parameters index (mean

of amniotic

(3) during 5 1 S.D.).

the

blood present in the amniotic fluid on the results. Our own investigations with whole and hemolyzed blood as well as with vernix established that the surface activity of the amniotic fluid was not influenced by these substances. This agrees with earlier studies about bubble stability squeezed out of pulmonary tissue into saline solution and into whole and hemolyzed blood.” Vernix, too, cannot be considered a source of error since it contains only traces of phospholipids which, moreover, are clearly outside of the surface activity range of amniotic fluid. Meconium had no effect on the y-max and y-min values but the hysteresis of the SAD rose significantly. It is therefore possible to evaluate meconium-containing amniotic fluid using the y-max and y-min values. Whereas chemical techniques do not yield reliable results in the presence of blood and meconium, this method makes it possible to obtain data about the surface activity of “contaminated” amniotic fluid. Surface properties of biologic material can be altered by quantitative or qualitative changes. Rising surface activity of the amniotic fluid during pregnancy may therefore be due to both these fac-

hysteresis

area

23 to 26, 27 to 30, 31 to 34, 35 to 38, and

fluid

y-max,

y-min

(dyn./cm.),

39 to

I

1u-7:

(r\~nero!.

tors. The dilution test series may explain this phenomenon, as well as the impact of pathologic quantities of amniotic fluid on its surface properties. As our results show, higher dilution is accompanied by a linear decrease in surface activity. The similarity of the amniotic fluid surface activity in the dilution series and during the progress of pregnancy allows the conclusion that rising surface activity of amniotic liquid during pregnancy is a matter of concentration of surface active substances. With polyhydramnios we expect a diminition of amniotic surface activity because of the larger amniotic fluid volume. Just the opposite happens in oligohydramnios; because of the reduced amniotic fluid volume, surface activity rises. In these cases exact measurements are not possible, but 100 to 200 per cent increases in fluid volume are not likely to affect the results since the 1 : 2 dilution of amniotic liquid does not affect its surface properties (Fig. 2) ; furthermore, if less than 12 ml. of amniotic liquid are available a dilution up to the volume required is possible. When testing the effect of pH on amniotic fluid surface activity within the normal pH range, the

Volumr Number

122 3

Surface

--

L

I

L:S

Ti

Y

to

I

I

I

I

I

I

I

I

I

I

I

I

I

properties

I

I

of

amniotic

I

,

I

- . - . - L:s . .... . . . .. L ---m-T,

fluid

I

rr

299

I

I

.

,I,

2

,

24

I

I

26

,

I

28

I

I

30

III

I

32

34

I

I

36

I

I

38

Fig. 4. Lecithin concentration in milligrams per 100 ml. according to Bhagwanani and ates,” lecithin-sphingomyelin ratio according to Gluck and associates,” dilution titer curve semiquantitative bubble stability test according to Clements and associates,5 and y-min amniotic fluid during the second half of gestation (mean 2 1 S.D.)

difference was not significant. Even with pH values beyond the normal range, no change of surface activity was noticed. These observations agree with findings of Sutnick and associates,l” who did not record any changes of the surface tension of lung extracts within the pH range from 4.6 to 8.2. We are unable to offer an accurate explanation of the absence of a pH effect on the amniotic surface properties; however, according to Sutnick a buffer mechanism might be involved. Surface tension values y-max and y-min, stability index ($1, and hysteresis of the SAD may be used for the evaluation of the amniotic surface properties; y-min seem particularly useful for the evaulation of surface activity of amniotic fluid because it undergoes a substantial change during pregnancy and has a minimal scatter. Fig. 4 represents a comparison between the am-

REFERENCES

1. 2.

3.

Agostoni, E., Taglietti, A., Agostoni, F., and Setnikar, I.: J. Appl. Physiol. 13: 344, 1958. Baum, M., Benzer, H., Lempert, J., Regele, H., Stiihlinger, W., and Tiille, W.: Respiration 28: 409, 1971. Benzer, H., and Lempert, J.: Wien. Klin. Wochenschr. 45: 828, 1967.

II

14

40

G.P

associof the in the

niotic fluid y-min and the L/S ratio,” lecithin content,” and semiquantitive Clements method.F The only difference between the y-min curve and the other techniques is the more gradual drop of the y-min values after week 32 of pregnancy. This may be so because with the surface balance the total activity of the surfactant is measured whereas the other techniques are confined to measuring the individual components. On the basis of this study, an exact assessment of the amniotic fluid surface activity is possible, but no conclusion as to fetal lung maturity. The correlation of the surface activity of amniotic fluid with the fetal lung and with the clinical condition of the newborn infant for establishing criteria for assessment of the degree of fetal lung maturity is the subject of the subsequent paper.

4. 5. 6.

Bhagwanani, S. G., Fahmy, D., and Turnbull, A. C.: Lancet 1: 159, 1972. Clements, J. A., Brown, E. S., and Johnson, R. P.: J. Appl. Physiol. 12: 262, 1958. Clements, J. A., Platzker, A. C. G., Tierney, D. F., Hobel, C. J., Creasy, R. K., Margolis, A. J., Thibeault, D. W., Tooley, W. H., and Oh, W.: N. Engl. J. Med. 286: 1077, 1972.

7.

Crvstle,

(2. D., and Rigsby, W. C.: AM. J. OBSTET. 106: 310, 1970. :3. EnhGming, G.: AM. J. OBSTET. GYNECOL. 88: 519, 1964. 9. Gluck, L., and Kulovich, M. V., Borer, R. C., Brenner, P. H., Anderson, G. G., and Spellacy, W. N.: AM. J. OBSTET. CYNECOL. 109: 444, 1971. 10. KGtter, I)., and Riifer, R.: Respiration 25: 35, 1968. I I. Lempert, J., Miiller-Tyl, E., Benzer, H., Baum, M., and Kraus, U.: Wien. Klin. Wochenschr. 85: 678, 1973. 12. Parkinson, C. E., and Harvey, D. R.: J. Obstet. Gynaecol. Br. Commonw. 80: 406, 1973. GYNECOL..

13. 14. 15. 16. 17. 18.

Scarpelli, E. M.: Pediatrics 40: 951, 1967. Shelley, S. A., Takagi, L. R., and Balis, J. c.: ~\XI J. OBSTET. GYNECOL. 116: 369, 1973. Spellacy, W. N., and Buhi, W. C.: Ah1. J O~s71r.l. GYNECOL. 3% 852, 1972. Sutnick, A. I., Cronlund, M. M., and Soloff. L A.: Proc. Sot. Exp. Biol. Med. 133: 506, 1969. Wag-staff, T. I., and Bromham, D. R.: J. Obstvt. Gynaecol Br. Commonw. 80: 412. 1973. Whitfield, C. R., Chan, W. H., Sproule. M. B.. and Stewart, A. D.: Br. Med. J. 8 April, 8j, 197:.