Amniotic fluid lecithin/sphingomyelin complicated pregnancies DONALD
ratio in
DYSON
MARY
BLAKE
GEORGE
CASSADY
Birmingham,
Alabama
Amniotic jluid (AF) lecithin/sphingomyelin (L/S) ra t ios were obtained in 2.23 pregnancies. Gestations complicated by maternal diabetes (71), vascular disease (50), and fetal growth retardation (47) were included. Elevated L/S ratios at 34 to 36 weeks’ gestation were observed to accompany maternal vascular disease; higher ratios were also related to fetal sex (female) and race (black). Declining L/S ratios were observed in 14 of 35 diabetic patients studied serially (40 per cent) and mere accompanied by increased perinatal morbidity and deaths. These data suggest an urgent need for further studies concerning the clinical usefulness of a serial decline in the L/S ratio as a predictor of fetal compromise.
ESTIMATES OF AMNIOTIC fluid (AF) phospholipids are of current clinical importance in predicting fetal pulmonary maturity. The extensive, meticulous studies of Gluck and others’.” have shown a relation between the relative AF concentrations of lecithin and sphingomyelin (L/S ratio) and alveolar stability after birth. It has also been suggested that deveIopmenta1 maturation of fetal surfactant production may be altered by such factors as maternal diabetes,“-” vascular disease,“-lz4 rupture of fetal membranes,‘“-” fetal growth retardation,7s I” severe fetal hemolytic disease,“. ‘. !I, lx and maternalfetal drug therapy.‘+” The exact sequence of events by which these factors affect fetal lung development is unclear. Furthermore, because of the small numbers of paFrom the Perinatal Research Laboratory, Departments of Pediatrics and Obstetrics, University of Alabama, Birmingham.
Table I. Number Pregnancy complications
Accepted
for
publication
September
September 30,
II,
samples, and pregnancy
No. of patients
Diabetic patients Class A Class AD Class B Class C Class D IGR Vascular disease Other Achondroplastic Total
71 53 8 4 4 2 29 50 72 1 223
No. of amniotic fluid samples 148 106 19 10 11 2 47 69 109 5 378
tients reported, the extent and variability of these factors remain unclear. During the past 11 months. WC have had the opportunity to obtain a large number of amniotic fluid samples from women with a variety of complications of pregnancy. The purpose of this study is to analyze the relation of maternal disease to the timing of fetal pulmonar) maturation as reflected by amniotic fluid L/S ratio.
The
Supported in part by Grant, No. MC-R-01-0071-01-0 from the Health Services and Mental Health Administration, Department of Health, Education, and Welfare, and Grant No. 5M01-RR00032-13 from the National Institutes of Health. Received
of patients, in the study
complications
Material
1974.
and
methods
Three hundred and seventy-eight samples were obtained by transabdominal amniocentesis from 223 kvomen. Details of pregnancy complications in these women and their relative frequency in this series
1974.
Reprint requests: Dr. George Cassady, Department of Pediatrics, University of Alabama, University Station, Birmingham, Alabama 36294. 772
Volume Number
122 6
L/S
ratio
in complicated
pregnancies
773
I ~20-30
31-33
34-36 GESTATION
Fig. 1. Relation between L/S ratio and gestational 72 patients without major pregnancy complications. S.D.; number of samples in parentheses.)
are provided in Table I. One hundred and fortyeight samples were obtained from 71 diabetic women; for the most part, these women had gestational diabetes. Criteria for diagnosis of gestational diabetes were similar to those of O’Sullivan and associates,‘g the diagnosis of Class A being made when whole blood glucose exceeded 110 mg. per cent (fasting), 170 mg. per cent (one hour), 120 mg. per cent (2 hours), or 110 mg. per cent (three hours) after administration of 100 Gm. of oral glucose (GTT) . Class AD (“gestational diabetes with carbohydrate decompensation”) was diagnosed when whole blood glucose exceeded 120 mg. per cent (fasting) or 180 mg. per cent (2 hours) after GTT ,or if whole blood glucose exceeded 300 mg. per cent at any time. Severity of insulin-requiring diabetes was judged with the criteria of White and colleagues. ” Sixty-nine AF samples were obtained from 50 women with vascular disease (diastolic blood pressure 2 90 mm. Hg on two successive occasions and/or proteinuria) . This group included patients with pre-eclampsia or eclampsia as well as those with inter-pregnancy renovascular and/or cardiovascular disease. A total of 47 samples from 29
37-
39
z42wks
40-42
age in 109 amniotic (Horizontal lines =
fluid samples from means; bars = + 1
patients whose fetuses were intrauterine growthretarded (IGR, below the tenth percentile of expected weight for gestational age”) were studied. Five samples from a pregnancy in an achondroplastic woman were also studied. Finally, there were 109 AF samples from 72 women with none of these complications. Upon receipt, fluids were centrifuged at 3,200 r.p.m. for 5 to 10 minutes. The supernate was then removed and either analyzed immediately or frozen at -20’ C. The L/S ratio for each AF sample was estimated by the standard method of Gluck and cow0rkers.l. ” The only modification of this technique was the dilution of AF : methanol : chloroform at 3 to 6: 5 : 10 instead of a precise 5 : 5 : 10 volume ratio, respectively. Relative densities of lecithin and sphingomyelin were measured with a reflectance densitometer* and L/S ratios were then calculated planimetrically on the basis of density from these scans. Reproducibility of this technique was i 0.1 in our laboratory. *Scanning densitometer, Transidyne S. Wagner Rd., Ann Arbor, Michigan
General 48103.
Corp.,
462
774
Dyson,
Blake, and Cassady
I < 28-30
Fig. 2. Relation between 71 pregnancies complicated of samples in parentheses; separately: ? 1 S.D. range
31-33
34-36 GESTATION
37-39
40-42
L/S ratio and gestational age in by diabetes. (Horizontal lines = individual values from two patients from Fig. 1 overplotted.)
Results
The amniotic fluid L/S ratio is related to gestational age in Fig. 1, in which the mean + 1 S.D. range for L/S ratios at grouped gestational ages is provided for 109 fluid samples from 72 patients without major pregnancy complications. These data are subsequently employed as an overplot in Figs. 2 to 4 for comparison with results from pregnancies complicated by maternal diabetes, fetal growth retardation, and maternal vascular disease. In Fig. 2 the relation of gestational age to the L/S ratio in 148 fluid samples from 71 pregnancies complicated by diabetes is shown. Differences related to severity of diabetes (Class A, AD, B, or C) were not observed, and these data are combined as the mean L/S ratio + S.D. by grouped gestational age in Fig. 2. Included within these figures are 19 fluid samples from 11 women with diabetes as well as vascular disease. As exclusion of values from these patients did not significantly affect the results, they are included here rather than later in the “vascular disease” group. The similarity of these
148 amniotic means; bars with Class
~42
wks
fluid samples from = 5 1 S.D.; number D diabetes indicated
L/S ratios to those observed in pregnancies not complicated by diabetes is evident. The decline at > 42 weeks (mean 1.8 versus 3.3 in others) is not significant (p > 0.05). L/S ratio values for fluid samples from two women with Class D diabetes are indicated separately in Fig. 2; at 34 to 36 weeks’ gestation, the mean for these was significantly higher than in uncomplicated gestations (2.2 versus 1.0; 1’ =
42wks
age in 47 amniotic fluid samples from 29 (Horizontal lines = means; bars = -‘- 1 range from Fig. 1 overplotted. Individual woman are indicated separately.)
grouped gestational age, pregnancy complication, and race, are provided in detail in Table II. Mean L/S ratios prior to 37 weeks’ gestation were consistently higher with female than male fetuses (0.70 versus 0.56 at < 28 to 30 weeks; 0.83 versus 0.71 at 31 to 33 weeks; 1.37 versus 1.33 at 34 to 36 weeks), but none of these differences was statistically significant. Ratios in female and male fetuses were nearly identical at 37 to 39 weeks (1.73 versus 1.74, respectively) and 40 to 42 weeks (2.08 in both groups) but were higher in female fetuses (but not significantly so) at >42 weeks’ gestation (3.59 versus 2.46). Serial L/S ratios were obtained in 84 patients (35/71 with diabetes, 11/29 with fetal growth retardation, 15/50 with maternal vascular disease, and 23/73 others). Stable or declining L/S ratios were observed in 15 of these patients, all but one of whom were in the diabetic group. Therefore, failure of terminal rise or actual decline in L/S ratio was quite common in the diabetic pregnancies studied (N/35 or 40 per cent) but very uncommon in
776
Dyson,
Bake,
and
Iuly
Cassady
Am. J. Ohstrt.
5.0
4.0
3.0 L/S
2.0
1.0 .
I
HI x28-30
31-33
34-36
37-39
> 42
40-42
wks
GESTATION
Fig. 4. Relation between L/S ratio and gestational age in 69 amniotic fluid samples from 50 pregnancies complicated by maternal vascular disease. (Horizontal lines = means; bars = + 1 S.D.; number of samples in parentheses; f 1 S.D. range from Fig. 1 overplotted. Individual value from patient given diphenylhydantoin indicated separately.)
3.0 -
~28-30
31-33
34-36
~42 wks
40-42
37-39
GESTATION
Fig. 5. Mean open
circles
=
L/S ratios at grouped gestational ages for black; dashed lines and x’s = white.
ail
samples
by
race.
Solid
line
and
Ii
I 19i’,
Gyn~rol.
Volume Number
12 6
L/S
ratio
in complicated
pregnancies
777
4.0’ \
9
3.0 7
\
9
L/S
2.0
4\
9
1.0 -
*w
I
1
1
35
37
39
41
43 wks
GESTATION Fig. 6. Individual refer to patient
L/S ratios in nine patients numbers in Table III. Values
Table II. Relation
of race to amniotic in parentheses
fluids indicated
fluid
L/S
with prior
ratio
declines during serial sampling. to decline are excluded.
within
Patient Uncomplicated
Gestation (weeks)
ratio in
DYSON
MARY
BLAKE
GEORGE
CASSADY
Birmingham,
Alabama
Amniotic jluid (AF) lecithin/sphingomyelin (L/S) ra t ios were obtained in 2.23 pregnancies. Gestations complicated by maternal diabetes (71), vascular disease (50), and fetal growth retardation (47) were included. Elevated L/S ratios at 34 to 36 weeks’ gestation were observed to accompany maternal vascular disease; higher ratios were also related to fetal sex (female) and race (black). Declining L/S ratios were observed in 14 of 35 diabetic patients studied serially (40 per cent) and mere accompanied by increased perinatal morbidity and deaths. These data suggest an urgent need for further studies concerning the clinical usefulness of a serial decline in the L/S ratio as a predictor of fetal compromise.
ESTIMATES OF AMNIOTIC fluid (AF) phospholipids are of current clinical importance in predicting fetal pulmonary maturity. The extensive, meticulous studies of Gluck and others’.” have shown a relation between the relative AF concentrations of lecithin and sphingomyelin (L/S ratio) and alveolar stability after birth. It has also been suggested that deveIopmenta1 maturation of fetal surfactant production may be altered by such factors as maternal diabetes,“-” vascular disease,“-lz4 rupture of fetal membranes,‘“-” fetal growth retardation,7s I” severe fetal hemolytic disease,“. ‘. !I, lx and maternalfetal drug therapy.‘+” The exact sequence of events by which these factors affect fetal lung development is unclear. Furthermore, because of the small numbers of paFrom the Perinatal Research Laboratory, Departments of Pediatrics and Obstetrics, University of Alabama, Birmingham.
Table I. Number Pregnancy complications
Accepted
for
publication
September
September 30,
II,
samples, and pregnancy
No. of patients
Diabetic patients Class A Class AD Class B Class C Class D IGR Vascular disease Other Achondroplastic Total
71 53 8 4 4 2 29 50 72 1 223
No. of amniotic fluid samples 148 106 19 10 11 2 47 69 109 5 378
tients reported, the extent and variability of these factors remain unclear. During the past 11 months. WC have had the opportunity to obtain a large number of amniotic fluid samples from women with a variety of complications of pregnancy. The purpose of this study is to analyze the relation of maternal disease to the timing of fetal pulmonar) maturation as reflected by amniotic fluid L/S ratio.
The
Supported in part by Grant, No. MC-R-01-0071-01-0 from the Health Services and Mental Health Administration, Department of Health, Education, and Welfare, and Grant No. 5M01-RR00032-13 from the National Institutes of Health. Received
of patients, in the study
complications
Material
1974.
and
methods
Three hundred and seventy-eight samples were obtained by transabdominal amniocentesis from 223 kvomen. Details of pregnancy complications in these women and their relative frequency in this series
1974.
Reprint requests: Dr. George Cassady, Department of Pediatrics, University of Alabama, University Station, Birmingham, Alabama 36294. 772
Volume Number
122 6
L/S
ratio
in complicated
pregnancies
773
I ~20-30
31-33
34-36 GESTATION
Fig. 1. Relation between L/S ratio and gestational 72 patients without major pregnancy complications. S.D.; number of samples in parentheses.)
are provided in Table I. One hundred and fortyeight samples were obtained from 71 diabetic women; for the most part, these women had gestational diabetes. Criteria for diagnosis of gestational diabetes were similar to those of O’Sullivan and associates,‘g the diagnosis of Class A being made when whole blood glucose exceeded 110 mg. per cent (fasting), 170 mg. per cent (one hour), 120 mg. per cent (2 hours), or 110 mg. per cent (three hours) after administration of 100 Gm. of oral glucose (GTT) . Class AD (“gestational diabetes with carbohydrate decompensation”) was diagnosed when whole blood glucose exceeded 120 mg. per cent (fasting) or 180 mg. per cent (2 hours) after GTT ,or if whole blood glucose exceeded 300 mg. per cent at any time. Severity of insulin-requiring diabetes was judged with the criteria of White and colleagues. ” Sixty-nine AF samples were obtained from 50 women with vascular disease (diastolic blood pressure 2 90 mm. Hg on two successive occasions and/or proteinuria) . This group included patients with pre-eclampsia or eclampsia as well as those with inter-pregnancy renovascular and/or cardiovascular disease. A total of 47 samples from 29
37-
39
z42wks
40-42
age in 109 amniotic (Horizontal lines =
fluid samples from means; bars = + 1
patients whose fetuses were intrauterine growthretarded (IGR, below the tenth percentile of expected weight for gestational age”) were studied. Five samples from a pregnancy in an achondroplastic woman were also studied. Finally, there were 109 AF samples from 72 women with none of these complications. Upon receipt, fluids were centrifuged at 3,200 r.p.m. for 5 to 10 minutes. The supernate was then removed and either analyzed immediately or frozen at -20’ C. The L/S ratio for each AF sample was estimated by the standard method of Gluck and cow0rkers.l. ” The only modification of this technique was the dilution of AF : methanol : chloroform at 3 to 6: 5 : 10 instead of a precise 5 : 5 : 10 volume ratio, respectively. Relative densities of lecithin and sphingomyelin were measured with a reflectance densitometer* and L/S ratios were then calculated planimetrically on the basis of density from these scans. Reproducibility of this technique was i 0.1 in our laboratory. *Scanning densitometer, Transidyne S. Wagner Rd., Ann Arbor, Michigan
General 48103.
Corp.,
462
774
Dyson,
Blake, and Cassady
I < 28-30
Fig. 2. Relation between 71 pregnancies complicated of samples in parentheses; separately: ? 1 S.D. range
31-33
34-36 GESTATION
37-39
40-42
L/S ratio and gestational age in by diabetes. (Horizontal lines = individual values from two patients from Fig. 1 overplotted.)
Results
The amniotic fluid L/S ratio is related to gestational age in Fig. 1, in which the mean + 1 S.D. range for L/S ratios at grouped gestational ages is provided for 109 fluid samples from 72 patients without major pregnancy complications. These data are subsequently employed as an overplot in Figs. 2 to 4 for comparison with results from pregnancies complicated by maternal diabetes, fetal growth retardation, and maternal vascular disease. In Fig. 2 the relation of gestational age to the L/S ratio in 148 fluid samples from 71 pregnancies complicated by diabetes is shown. Differences related to severity of diabetes (Class A, AD, B, or C) were not observed, and these data are combined as the mean L/S ratio + S.D. by grouped gestational age in Fig. 2. Included within these figures are 19 fluid samples from 11 women with diabetes as well as vascular disease. As exclusion of values from these patients did not significantly affect the results, they are included here rather than later in the “vascular disease” group. The similarity of these
148 amniotic means; bars with Class
~42
wks
fluid samples from = 5 1 S.D.; number D diabetes indicated
L/S ratios to those observed in pregnancies not complicated by diabetes is evident. The decline at > 42 weeks (mean 1.8 versus 3.3 in others) is not significant (p > 0.05). L/S ratio values for fluid samples from two women with Class D diabetes are indicated separately in Fig. 2; at 34 to 36 weeks’ gestation, the mean for these was significantly higher than in uncomplicated gestations (2.2 versus 1.0; 1’ =
42wks
age in 47 amniotic fluid samples from 29 (Horizontal lines = means; bars = -‘- 1 range from Fig. 1 overplotted. Individual woman are indicated separately.)
grouped gestational age, pregnancy complication, and race, are provided in detail in Table II. Mean L/S ratios prior to 37 weeks’ gestation were consistently higher with female than male fetuses (0.70 versus 0.56 at < 28 to 30 weeks; 0.83 versus 0.71 at 31 to 33 weeks; 1.37 versus 1.33 at 34 to 36 weeks), but none of these differences was statistically significant. Ratios in female and male fetuses were nearly identical at 37 to 39 weeks (1.73 versus 1.74, respectively) and 40 to 42 weeks (2.08 in both groups) but were higher in female fetuses (but not significantly so) at >42 weeks’ gestation (3.59 versus 2.46). Serial L/S ratios were obtained in 84 patients (35/71 with diabetes, 11/29 with fetal growth retardation, 15/50 with maternal vascular disease, and 23/73 others). Stable or declining L/S ratios were observed in 15 of these patients, all but one of whom were in the diabetic group. Therefore, failure of terminal rise or actual decline in L/S ratio was quite common in the diabetic pregnancies studied (N/35 or 40 per cent) but very uncommon in
776
Dyson,
Bake,
and
Iuly
Cassady
Am. J. Ohstrt.
5.0
4.0
3.0 L/S
2.0
1.0 .
I
HI x28-30
31-33
34-36
37-39
> 42
40-42
wks
GESTATION
Fig. 4. Relation between L/S ratio and gestational age in 69 amniotic fluid samples from 50 pregnancies complicated by maternal vascular disease. (Horizontal lines = means; bars = + 1 S.D.; number of samples in parentheses; f 1 S.D. range from Fig. 1 overplotted. Individual value from patient given diphenylhydantoin indicated separately.)
3.0 -
~28-30
31-33
34-36
~42 wks
40-42
37-39
GESTATION
Fig. 5. Mean open
circles
=
L/S ratios at grouped gestational ages for black; dashed lines and x’s = white.
ail
samples
by
race.
Solid
line
and
Ii
I 19i’,
Gyn~rol.
Volume Number
12 6
L/S
ratio
in complicated
pregnancies
777
4.0’ \
9
3.0 7
\
9
L/S
2.0
4\
9
1.0 -
*w
I
1
1
35
37
39
41
43 wks
GESTATION Fig. 6. Individual refer to patient
L/S ratios in nine patients numbers in Table III. Values
Table II. Relation
of race to amniotic in parentheses
fluids indicated
fluid
L/S
with prior
ratio
declines during serial sampling. to decline are excluded.
within
Patient Uncomplicated
Gestation (weeks)
