Pediatric Annals

Methods of Determining Fetal Maturity

Maurice L Druzin, MD

Abstract

INTRODUCTION

Fetal maturity is a functional term reflecting the ability of the newborn to adapt to extrauterine life. This term is vague in that it encompasses all the organ systems of the body. While maturity of the gastrointestinal tract, nervous system and excretory system are certainly important, functional maturity of the respiratory system is mandatory for successful adaptation to life ex utero.

A major cause of neonatal morbidity and mortality is the infant respiratory distress syndrome (IRDS). This syndrome is often, but not exclusively, the clinical expression of the pathological entity of hyaline membrane disease (HMD). The ventilatory support that is required to maintain adequate gaseous exchange in infants with IRDS may range from minimally invasive procedures with low complication rates (eg, increased inspired oxygen by hood) to invasive procedures with potentially serious complications such as endotracheal intubation and respirator use. The placement of intravenous and intra-arteria! catheters, feeding tubes and numerous monitoring devices all lead to increased infectious morbidity and potential trauma to the neonate. The increased incidence of pneumothorax and intraventricular hemorrhage in these newborns is often secondary to efforts at maintenance of adequate gaseous exchange in the respiratory system. The long term complications of retrolental fibroplasia and bronchopulmonary dysplasia are added risks to the neonate who develops IRDS.

IRDS is most often a result of inadequate amounts of surface active material which allow the alveoli to remain expanded during respiratory movement. While infants with adequate amounts of this material do sometimes develop IRDS, this is the exception and our discussion will address methods of determining fetal respiratory maturity which is most accurately done by determining quantities of surface active material.

The goal of the obstetrician is to attempt to maintain the fetus in utero until adequate amounts of surface active material have developed to maintain respiratory function. Other structural components of the lung are important for adequate gas exchange, but surface active material remains the key element in predicting development of IRDS.

A decision to deliver a fetus at high risk for developing IRDS should only be made when the chance of intrauterine compromise or death exceeds the risk of the complications of IRDS. Antepartum surveillance with biochemical means, such as estriol determinations, or biophysical techniques such as antepartum fetal heart rate testing and ultrasound, allows maintenance of the fetus in utero safely in the majority of cases. Early detection of fetal compromise is an integral part of modern obstetrics.

METHODS OF DETERMINING FETAL MATURITY

Prior to the era in which measurement of certain constituents of the amniotic fluid was possible by means of amniocentesis, indirect methods of assessing fetal maturity were utilized.

The method most commonly used, but often inaccurate, was dating the pregnancy based on the date of the last menstrual period (LMP). Yerushalmy1 reported that only 84% of patients deliver within 2 weeks of the estimated date of confinement. In three other studies,2-4 the information supplied by the mother on the LMP was considered unreliable in 22%, 40% and 80% of cases respectively. Detection of fetal quickening by the mother and of fetal heart tones by the physician are open to many variables and inconsistencies, involving maternal obesity, fetal movement, lack of maternal interest and physician skill. These clinical signs, if available and internally consistent, are useful but leave room for error. In one study, 12% of all infants with IRDS admitted to a NICU were born after elective intervention based on the obstetrical assumption of maturity.5 Obstetrical dating was three or more weeks greater than pediatric estimates in more than 50% of these infants.

Other indirect methods of assessing fetal maturity such as…

INTRODUCTION

Fetal maturity is a functional term reflecting the ability of the newborn to adapt to extrauterine life. This term is vague in that it encompasses all the organ systems of the body. While maturity of the gastrointestinal tract, nervous system and excretory system are certainly important, functional maturity of the respiratory system is mandatory for successful adaptation to life ex utero.

A major cause of neonatal morbidity and mortality is the infant respiratory distress syndrome (IRDS). This syndrome is often, but not exclusively, the clinical expression of the pathological entity of hyaline membrane disease (HMD). The ventilatory support that is required to maintain adequate gaseous exchange in infants with IRDS may range from minimally invasive procedures with low complication rates (eg, increased inspired oxygen by hood) to invasive procedures with potentially serious complications such as endotracheal intubation and respirator use. The placement of intravenous and intra-arteria! catheters, feeding tubes and numerous monitoring devices all lead to increased infectious morbidity and potential trauma to the neonate. The increased incidence of pneumothorax and intraventricular hemorrhage in these newborns is often secondary to efforts at maintenance of adequate gaseous exchange in the respiratory system. The long term complications of retrolental fibroplasia and bronchopulmonary dysplasia are added risks to the neonate who develops IRDS.

IRDS is most often a result of inadequate amounts of surface active material which allow the alveoli to remain expanded during respiratory movement. While infants with adequate amounts of this material do sometimes develop IRDS, this is the exception and our discussion will address methods of determining fetal respiratory maturity which is most accurately done by determining quantities of surface active material.

The goal of the obstetrician is to attempt to maintain the fetus in utero until adequate amounts of surface active material have developed to maintain respiratory function. Other structural components of the lung are important for adequate gas exchange, but surface active material remains the key element in predicting development of IRDS.

A decision to deliver a fetus at high risk for developing IRDS should only be made when the chance of intrauterine compromise or death exceeds the risk of the complications of IRDS. Antepartum surveillance with biochemical means, such as estriol determinations, or biophysical techniques such as antepartum fetal heart rate testing and ultrasound, allows maintenance of the fetus in utero safely in the majority of cases. Early detection of fetal compromise is an integral part of modern obstetrics.

METHODS OF DETERMINING FETAL MATURITY

Prior to the era in which measurement of certain constituents of the amniotic fluid was possible by means of amniocentesis, indirect methods of assessing fetal maturity were utilized.

The method most commonly used, but often inaccurate, was dating the pregnancy based on the date of the last menstrual period (LMP). Yerushalmy1 reported that only 84% of patients deliver within 2 weeks of the estimated date of confinement. In three other studies,2-4 the information supplied by the mother on the LMP was considered unreliable in 22%, 40% and 80% of cases respectively. Detection of fetal quickening by the mother and of fetal heart tones by the physician are open to many variables and inconsistencies, involving maternal obesity, fetal movement, lack of maternal interest and physician skill. These clinical signs, if available and internally consistent, are useful but leave room for error. In one study, 12% of all infants with IRDS admitted to a NICU were born after elective intervention based on the obstetrical assumption of maturity.5 Obstetrical dating was three or more weeks greater than pediatric estimates in more than 50% of these infants.

Other indirect methods of assessing fetal maturity such as radiological assessment of fetal epiphyseal centers and measurement of biparietal diameter by ultrasonography have been used. The high false positive rate of these methods has led to the development of direct methods of determining fetal lung maturity.

Measurement of various constituents of amniotic fluid obtained by amniocentesis is currently the most reliable method of judging whether IRDS will complicate elective delivery.

PHOSPHOLIPIDS

Phospholipid indicators have been found to reflect maturity of the fetal lung. Gluck et al described the relationship of two phospholipids, lecithin and sphingomyelin, in the first systematic investigation of amniotic fluid phospholipids.6 The lecithin sphingomyelin ratio (L: S ratio) remains the standard against which all other tests are judged in assessment of lung maturity.

Prior to differentiation of the lung alveoli at 24 weeks, supporting structures of lung, trachea and bronchi to the level of respiratory bronchioles are formed. During the development of alveoli, differentiation of glycogencontaining columnar epithelial-lining cells takes place. Type I and type II alveolar cells differentiate in the following weeks. The type I cell is primarily a structural supporting cell. Type II cells secrete the phospholipids that constitute the major part of the surfactant complex which lines the alveoli and decreases surface tension. Without surfactant to decrease the surface tension, the pressure on the alveoli would become so high that alveoli would collapse and gas exchange would be impaired.

Surfactant primarily contains phospholipids of which the most abundant is lecithin. Sphingomyelin, a sphingosine compound with a fatty acid amide, is another important constituent of the surfactant complex. Other important phospholipids include phosphatidylinositoL phosphatidylserine, phosphatidylethanolamine and phosphatidylglycerol. Phosphatidylglycerol does not appear in significant quantities in alveolar fluid until the lung is mature.

Surfactant production increases slowly throughout pregnancy until approximately 35 weeks gestation, when there is a surge of production, followed by stability of lung. Surfactant action begins with the first breath taken by the newborn infant when, on expiration, the lungs do not collapse but retain about 50% of the air in the alveoli as the functional residual capacity.

The Lecithin:Sphingomyelin Ratio (L:S ratio)

The concentration of lecithin and sphingomyelin approximate each other until 32 weeks of gestation when the lecithin concentration rapidly rises indicating the development of the mature form of surfactant. The concentration of sphingomyelin changes relatively little and decreases slightly after 32 weeks, thus serving as a useful internal control. By recording the ratio of these two substances, the effect of changes in amniotic fluid volume on lecithin concentrations is eliminated. While the aforementioned sequence of events occurs in normal pregnancies, different rates of production can occur in complicated pregnancies. Acceleration of the time of appearance of mature L:S ratio occurs in situations of chronic intrauterine stress such as hypertensive disease, conditions leading to intrauterine growth retardation, and diabetes complicated by vascular disease. Delayed lung maturation is seen in diabetes, class A, B and C (Whites classification).7

The assay technique described by Gluck et al is the standard method used.8 After centrifugation of 2 ml to 5 ml amniotic fluid at 5400 rpm for five to ten minutes, the supernatant is extracted with methanol and chloroform, the extract is dried and then precipitated with cold acetone.

Lecithin and sphingomyelin are separated by thin layer chromatography on silica gel prepared with ammonium sulfate. The spots are visualized by charring and measured by reflectance densitometry. Polar planimetry has also been used with similar results.

With the use of two dimensional thin-layer chromatography, a lung profile can be recorded. This consists of measurement of the L:S ratio, and percentages of disaturated lecithin, phosphatidylinositol and phosphatidylglycerol. This method has been suggested as being a more accurate indication of biochemical maturation.

Phosphatidylglycerol

The measurement of phosphatidylglycerol, which appears at 26 weeks gestation and increases to term, greatly enhances the information derived from amniocentesis. It is possibly the most reliable indicator of pulmonary function, even in the diabetic pregnancy where an increased incidence of false positive (mature) tests are seen. Measurement of phosphatidylglycerol also reduces the incidence of false immature results, as some infants with L:S ratios of less then 2:1 do demonstrate phosphatidylglycerol.9 IRDS had not been demonstrated when phosphatidylglycerol was present in amniotic fluid, until recently.10'11 Another advantage of phosphatidylglycerol is that it is found only in pulmonary surfactant so that contamination of the amniotic fluid with blood, meconium or vaginal secretions does not confuse the interpretation.

Shake Test (Foam Stability)

Clements et al12 have described a simple and rapid screening test for lung maturity. Serial dilutions of amniotic fluid are shaken after the addition of ethanol to exclude the action of other surface-active materials, and the persistence of bubbles at the liquid-air interface represents the concentration of surfactant. This test, when positive (persistent foam at dilutions of ^ 1:2), indicates pulmonary maturity with a degree of accuracy equivalent to the L: S ratio. The intermediate test is associated with a variable and unpredictable risk of RDS. The negative test (no foam with undiluted amniotic fluid) indicates significant risk of RDS.

The advantages of simplicity, time-saving (less than 30 minutes) and economy are offset by the invalidation of results when blood, meconium or vaginal secretions are present. The non-specificity of an intermediate test mandates further study by means of a more accurate test.

Fluorescence Depolarization (FD Technique)

The relative lipid content of amniotic fluid may be determined by FD analysis. A lipid-soluble dye is incubated with the amniotic fluid specimen for 30 minutes and the amount of dye absorbed into phospholipid membrane structures is assessed by measuring the fluorescence of polarized light. The FD value falls as the L: S ratio rises and correlates with the presence of phosphatidylglycerol. Various numerical values (p values) are assigned to the FD measurement. This technique gives few false positive results but has significant false negative results.13

CREATININE

Amniotic fluid creatinine rises gradually until 32 to 34 weeks, then more rapidly until term, when it is two to four times the mid-pregnancy level. There is a linear increase in fetal urine production during the last eight weeks of pregnancy and it is thought that creatinine is transferred to amniotic fluid mainly through fetal urine but the placenta also seems to playa role in production. Amniotic fluid levels of 2 mg/ 100 ml or greater have been correlated with gestational age of 37 weeks or greater in 94% of cases.14 Amniotic fluid creatinine reflects fetal muscle mass and renal function. Maternal serum creatinine levels also affect the amniotic fluid levels. At present, serum creatinine is only used as supportive evidence of maturation as it does not reflect lung maturity directly.

STAINING OF CELLS

Epithelial cells are shed into amniotic fluid from fetal skin and possibly the amniotic membranes. Cells appear in increasing numbers as pregnancy progresses. The proportion of lipid-containing cells from the fetal epidermis increases from 28 weeks gestation to term and a percentage of lipid-containing cells greater than 20% has been considered indicative of fetal maturity. These cells are identified by staining with Nile blue sulfate which causes lipid-containing cells to turn orange whereas cells without lipids stain blue. The margin of error with this technique is considered too great for clinical application but a recent report has documented its usefulness in combination with other methods of assessing fetal maturity.15

OTHER CONSTITUENTS

In normal pregnancies, amniotic fluid bilirubin levels are maximum at 20 to 24 weeks and decline thereafter. When the amniotic fluid bilirubin reaches undetectable levels, ? OD 450 of zero, the fetus is assumed to be mature.

Measurement of amniotic fluid density of 650 mm (OD 650) has shown to correlate with lung maturity as defined by the L:S ratio.16

Measurements of amniotic fluid palmitic acid, stearic acid, total Cortisol17 and activity of the enzyme phosphotidate phosphohydrolase (PAPase)18 have all been correlated with lung maturity with varying degrees of accuracy.

All of the methods described have false positive and false negative rates. Complications of pregnancy, particularly diabetes mellitus, have been shown to alter the predictive reliability of tests for fetal maturity. Knowledge of the limitation of each of these methods is a prerequisite for intelligent application to clinical situations.

REFERENCES

1. Yerushalmy J: Relation of birth weight, gestational age, and the rate of intrauterine growth to perinatal mortality. Clin Obstet Gynecol 1970; 13:107.

2. Dewhurst JC, Beazfey TM, Campbell S: Assessment of fetal maturity and dysmaturity. Am J Obstet Gynecol 1972; 113:141.

3. Sabbagha RE: Ulstrasound in managing the high risk pregnancy. Spellacy WN (ed): in Management of High Risk Pregnancy. Baltimore, University Park Press, 1976.

4. Hertz RH.Sokol RJ, Knoke JD. et al: Clinical estimation of gestational age: Rules for avoiding preterm delivery. Am J Obstet Gynecol 1978; 131:395.

5. Hack M, Fanaroff AA, Klaus MH, et al: Neonatal respiratory distress syndrome following elective delivery. A preventable disease. Am J Obstet Gynecol 1976; 126:43.

6. Gluck L. Kulovich MV. Bover RC, et al: Diagnosis of the respiratory distress syndrome by amniocentesis. Am J Obstet Gynecol 1971; 109:440.

7. Gluck L. Kulovich MV: L:S ratio in amniotic fluid in normal and abnormal pregnancy. Am J Obstet Gynecol 1973; 1 15:539.

8. Gluck L. Kulovich MV, Borer RC Jr Estimates of fetal lung maturity. Nesbitt REL (ed): Clinics in Perinatology. Philadelphia, WB Saunders Co, 1974. vol I. ? 125.

9. Hallman M. Feldman BH. Kirkpatrick E. et al: Absence of phosphatidylglycerol (PG) in respiratory distress syndrome in the newborn. Pediair Res I977;11:7I4.

10. Whittle MJ. Wilson Al. Whitfield CR. et al: Amniotic fluid phospholipid profile determined by two-dimensional thin-layer chromatography as index of fetal lung maturation. Br Med J 1981; 282-428.

1 1. Anderson CW. Conrad L, Cordero L: Neonatal respiratory distress in the presence of amniotic fluid phosphatidylglycerol. Am J Obstet Gynecol 1 982; 143:233.

12. Clements JA. Platzker ACG. Tiemey DH. et al: Assessment of risk of the respiratory distress syndrome by a rapid new test for surfactant in amniotic fluid. .V EngtJ Med. 1972: 268:1077.

13. Golde SH. Vogt JF. Gabbe SG. et al: Evaluation of the FELMA microviscommeter in predicting fetal lung maturity. Obstet Gynecol 1977; 98:1135.

14. Pitkin RM. Zwirek SJ: Amniotic fluid creatinine. Am J Obstet Gynecol I967;98:1135.

15. Morrison JC. Whybrew WD. Bucovaz ET. et al: Amniotic fluid tests for fetal maturity in normal and abnormal pregnancies. Obstet Gynecol 1977; 49:1.

16. Hill CM. Harkes A. Donnai P. et al: Amniotic fluid optical density determination as a rapid test for assessment of fetal lung maturity. Br J Obstet Gynaecol 1979; 86:773.

1 7. Doran J A. Ford J A. Allen IC. et al: Amniotic fluid L:S ratio, total Cortisol, creatinine, and percentage of lipid-positive cells in assessment of fetal maturity and fetal pulmonary maturity: A comparison. Am J Obstet Gynecol 1979; 133:3.

18. Herbert WNP. Johnston JM. McDonald DPC. et al: Human amniotic fluid phosphatidate phosphohydrolase activity through normal gestation and its relation to the L:S ratio. Am J Obstet Gynecol 1978: 132:4.

10.3928/0090-4481-19830101-01

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