Assessment of foetal maturity - (A review and our preliminary study)Varsha R Munshi, KR Juvale, Usha R Krishna, VN Purandare
Department of Obstetrics and Gynaecology, K. E. M. Hospital, Parel, Bombay-400 012., India
Amniotic fluid of 262 patients was studied to assess foetal maturity by estimating fat cell percentage and creatinine content. A fat cell count of 25% was noted at 36 weeks and 36% at 40 weeks while creatinine levels of 2.3 mg% were found after 36 weeks of gestation. Methodology, accuracy and usefulness of the various tests used to determine the foetal maturity have been discussed.
There are numerous occasions when it is necessary to know the functional maturity of a foetus. The problem is more significant when complications of pregnancy like placental insufficiency, toxaemia, ante-partum haemorrhage, diabetes, rhesus isoimmunisation, threaten the life of the foetus in utero. In such cases an early termination of pregnancy with a certain degree of prematurity is a desirable alternative to more dire possibility for the foetus, and a balance has to be struck between the risk of intra-uterine death and the hazards of prematurity and neonatal death. Less urgent is the problem of elective repeat caesarean section and elective induction of labour. As recently as 1969, Benson et al  reported that 8.4% of infants delivered by elective caesarean section, weighed less than 2500 gms. A reliable estimate of foetal maturity is therefore mandatory before induction of labour.
In the past, the foetus was evaluated by its size and gestational age. It is now known that measurements of foetal size do not necessarily correlate with gestational age, and gestational age in turn, does not necessarily correlate with maturity and may not necessarily predict how the foetus will function physiologically once born.
Maturity must be defined in terms of extra-uterine viability rather than strictly on the basis of age, size and weight. In India, prematurity has been defined on the basis of birth weight of less than 2000 gms. while in the West, the figure is 2500 gms. This does not take into account a 2 kg. 42 week foetus of a mother with chronic hypertension or a 3 kg. 34 weeks foetus of a diabetic or Rh immunised mother. Ideally, tests for foetal maturity should provide information relating to the capacity of the foetus to survive and develop normally outside the uterus. There are a number of tests which assess the functional, maturity of the vital organs like the lungs, liver, kidneys which will determine the ability of the foetus to survive the extra-uterine environment.
Amniocentesis was done on 262 patients in the obstetric department of the K. E. M. Hospital in a period of one year. A preliminary study was conducted to assess foetal maturity by estimating the percentage of fat cells and the creatinine content. Most of the patients were antenatal cases or those admitted with false pains, while in 43 cases it was done during labour.
All the patients were of 32 weeks or longer gestation, the gestational length being calculated on the basis of menstrual history and physical examination.
In 40 patients where maturity studies were done during labour, the results were correlated with foetal weight.
Collection of liquor
10 cc of Amniotic fluid was collected after taking the necessary aseptic precautions. The samples were collected at the time of caesarean section in 13 patients, by amniocentesis in 234 patients and amniotomy in the remaining 15.
Of these, 15 patients had blood or meconium stained liquor and hence the investigations were not done.
There were no complications of amniocentesis.
Amniotic fluid cytology was studied by staining with 0.1% Nile Blue Sulphate by the method of Brosens et al.  One drop of Nile Blue stain was added to one drop of amniotic fluid, mixed by shaking the slide, and slightly warmed on a flame.
500 cells were counted under the low power microscope. Two types of cells were seen
(1) Blue stained cells with well defined outline, dark staining pyknotic nucleus and no tendency to clumpingthe foetal epithelial cells.
(2) Orange stained anucleated vesicular cells which show a tendency to clumping, originate from the foetal sebaceous glands. According to the size of the clump, the cells were counted approximately. Brosens et al  made attempts to disintegrate such clusters but it only complicated the technique without increasing the accuracy. Clusters were found to be present only when the percentage of orange cells exceeded 10 and were numerous when it exceeded 50.
The percentage of orange cells was calculated and related to foetal maturity.
Amniotic fluid creatinine was estimated by the Jaffe Picric acid technique  and expressed in mg. per 100 ml.
Amniotic fluid of 247 patients was examined for estimating the fat cell percentage and creatinine content at varying stages of gestation after 32 weeks. Of these, 17 patients were studied at 32 weeks, 24 at 36 weeks and 206 at 40 weeks. The average values of fat cell per cent and creatinine were calculated at 32, 36 and 40 weeks.
[Table 1] shows that the average fat cell count was 1% at 32 weeks, 24.97% at 36 weeks and 36.6% at 40 weeks. The creatinine values at the corresponding periods of gestation were 1.91 mg%, 2.32 mg% and 2.36 mg% respectively.
Age, parity and complications of pregnancy like anaemia and toxaemia had no effect on these values.
[Table 2] shows the correlation of maturity tests with the birth weight in 40 cases where the tests were performed during: labour. Of infants weighing 2100 to 2500 gms, 50% had a fat cell count of more than 21%; 41% the infants had creatinine content exceeding 2.1 mg% whereas of the 24 babies born with birth weight exceeding 2600 gms. 70% had fat cell count of greater than 21% and 83% of the infants had creatinine values over 2.1 mg%.
The last 25 cases also had a shake test of Clements  and the results were found to correlate well with foetal maturity.
In the last few years, numerous methods have been devised for determining foetal maturity in utero, arisen out of a need to know the earliest point at which a foetus can do as well in the nursery as in the uterus. There are a series of tests which include clinical assessment, physical methods like radio logy and ultrasonar, amniotic fluid analysis and a few maternal indicators like vaginal cytology.
Till recently, history and physical examination were mainly used to estimate the gestational period of the foetus. The first day of the last menstrual period gives an estimate of the menstrual age of the foetus but it is inaccurate in 10% to 40% of women as it is often not well recorded. There are physiological menstrual variations. Isolated episodes of coitus may determine the date of conception especially if basal body temperature charts are maintained. Onset of pregnancy symptoms and the time of quickening are subject to individual variations.
An early bimanual examination in the absence of fibroids or adnexal masses may serve as a useful guideline. The most frequently utilised clinical parameter is the serial measurement of the height of the uterine fundus in relation to landmarks on the abdominal wall.  However, direct measurements of fundal height have been found to be more accurate. One popular approach is the McDonald's method  where the duration of gestation in weeks is calculated by the formula-fundal height in centl meters x 8/7. Johnson  devised a formula of estimating foetal weight in grammes by the formula.
[ (fundal height in cms) - n] x k
where n = 12 if the station of the foetal head is below the level of the ischial spines.
n = 11 if the station is above the level of the ischial spines
k = constant (155)
This applies only to vertex presentations and is an approximation since only 50% will be within 240 gms of the calculated weight.
Visualisation of the distal femoral epiphyses implies that the foetus has reached or passed 36 weeks while the upper tibial epiphysis is seen only at 40 weeks. Wolf and Greenberg  state that of the newborn babies born with weight greater than 2500 gms., the distal femoral epiphyes is present in 99% and the proximal tibiall in 70%. Schreiber et al  noted that the presence of both centers correctly indicate maturity in 92% cases but the interpretation of negative or inconclusive findings remains difficult. Miller and Futrakul  have found a seven fold increase of respiratory distress syndrome among foetuses without ossification centers in the knee joint. There being wide deviations from the general rules, many authors consider them to be unreliable as compared to newer methods. Other disadvantages are the technical difficulties in locating the areas and the mutagenic factor.
This has proved to be perhaps the most useful method for prenatal measurements of body dimensions.  Measurement of the biparietal diameter remains one of the most reliable laboratory test which gives exact dating of gestational age, providing a 95% accuracy if first measured in 20 to 30 weeks range. With a single determination, 84% accuracy is possible. Cohen" compared measurements of biparietal diameter radiologically to ultrasound and found the former to be incorrect in 22% as opposed to only 3% with ultrasound. The other advantages are that the hazard of radiation is eliminated and it can be used repeatedly without any harm.
Amniotic fluid reflects the condition of the foetus and its analysis yields valuable information of foetal maturity. A large number of parameters are available which indicate maturity of different organs. These include the percentage of lipid laden cells which gives skin maturity, creatinine content reflecting the skeletal muscle mass and the number of functional glomeruli.  Spectrophotometry and calculating the optical density at 450 mµ indicates liver maturity while the phospholipids, lecithin and sphinogomyelin which are important components of the surfactant system of the lungs, when related as a ratio will show pulmonary maturity. Foam test of Clements  is a simple, inexpensive bedside method to determine the maturity of the lungs.
A high percentage of fat laden cells appears to be a reliable index of foetal maturity but a low proportion does not necessarily indicate prematurity. It is directly related to foetal maturity and not to birth weight. Many authors like Jakob vits et al  and Bishop and Corson  showed that an orange cell count of 10-20% indicates a foetus weighing over 2000 gms. and a gestational age of 36 weeks and a count of 30% indicates a foetus weighing over 2500 gms. and a gestational age beyond 37 weeks.
Sharma and Trusell  stated that the orange cells first appear at 32 to 34 weeks and reach a 50% concentration by the 40th week with a marked tendency to clumping and cluster formation. They proposed that three factors must be present to confirm 38 week maturity, (1) mature number of fat cells (2) free vernix particles and (3) free orange staining droplets.
Post dated pregnancies show a steep rise of 60%, to 90% of the fat cells. Age, parity, pre-eclampsia, twins, IUGR, diabetes, Rh immunisation, APH do not influence the results. In hydramnios the number of cells are diluted.
Using Papanicolou's stain, Floyd et al  distinguished five categories of cells. (1) Anucleated (2) Cornified squamous (3) Precornified squamous (4) Intermediate squamous and (5) Parabasal cells. As gestation progressed they found a preponderance of cornified and precornified squamous cells, with a virtual absence of parabasal cells after 36 weeks.
[Table 3] compares the results of the present study with the results of two other studies , from India. All of them show a fat cell count of less than 1% at 32 weeks and over 35% at 40 weeks. Bros sens et al  give their results as 1% at 34 weeks, to 10% at 34 to 37 weeks and 11% to 50%, at 38 to 40 weeks.
Pitkin and Zwirek  say that creatinine levels show a slow increase from 0.8 mg% to 1.2 mg,, between the 20th and 34th weeks and a fast rise thereafter. Concentration of 2 mg % was a reliable borderline value as is found in 97% over 37 weeks. Chandiok et al,  found that at or before 36 weeks, concentration was less than 2 mg% in 73% cases and above 37 weeks it was more than 2 mg% in 80% cases. Begneaud et al  stated that values less than 1.5 mgt o indicate an immature foetus and those between 1.5 to 2 mg.% are not clinically useful.
Some authors state that values of 3 mg% or more indicate a postmature foetus and induce labour with a level of 3.5 to 4 mg%, while Pitkin and Zwirek  say there is no progressive increase after 37 weeks and it does not diagnose postmaturity.
Age, parity, diet, twins, IUGR, diabetes, Rh immunisation, APH, pyelonephritis have no effect on these levels. Its value in pre-eclampsia is controversial. Sinha, O'Leary and Bezjan  found significantly higher levels in severe cases of preeclampsia as compared to mild cases with a progressive increase in both groups towards term. While Pitkin and Zwirek  and Begneaud et al  have noted no difference. As compared to the present study Chaudhary et al  has shown the creatinine content to be 0.9, 1.8 and 2.6 mg% at 32, 36 and 40 weeks respectively.
Bilirubin first appears in the amniotic fluid late in the first trimester reaching a peak in the 2nd trimester and begins to decline at 30 weeks. In non Rh sensitised pregnancies it cannot be detected by the 36th week as seen by the disappearance of the 450 mµ peak on spectrophotometry. Mandelbaum et al  found that 85% of babies showing no bilirubin in amniotic fluid weighed over six pounds and none weighed less than five pounds.
The major phospholipids, Lecithin and Sphingomyelin are important components of the surfactant system of the lung and prevent respiratory distress syndrome. Gluck et al  related lecithin to sphinogomyelin as a ratio using thin layer chromatography and showed maturation of the lungs associated with a L/ S ratio of 2: 1 which is reached at about 35 weeks.
Dunn and Bhatnagar  showed that in foetuses with a mature pattern of 2: 1 ratio, in 99% cases RDS would not develop. Immature pattern of 1: 1 ratio was associated with normal respiratory function in 50% RDS in 33% and respiratory death in 15%. L/S ratio of 7 or more is associated with postmaturity. Caesarean section did not influence development of RDS with a mature L/S ratio.
Maturation of the lungs occurs independent of either gestational age or birth weight especially in abnormal pregnancies. Gluck and Kulovichi  reported a 3.3 kg. infant of 38 weeks gestation who had L/S ratio of 0.75 and severe RDS while a 920 gms. infant of 28 weeks gestation with a mature L/S ratio had no RDS. He found each infant with L/S ratio less than two had some clinical RDS and the lower the L/S ratio, more severe was the RDS.
Gluck and Kulovich  studied L/S ratio in high risk pregnancies. He found that some maternal diseases were associated with delayed maturation to about 3612 weeks of gestation. These include mild and gestational diabetes mellitus, hydrops foetalis, chronic renal disease without hypertension and the smaller of the identical but non parasitic twins.
Maturation acceleration occurring before 32 weeks and as early as 26 to 28 weeks was noted in degenerative diabetes mellitus, chronic renal disease with hypertension, hypertension associated with severe toxaemia or cardiovascular disease, sickle cell disease, primary placental disease, chronic abruptio placenta and drug addicts to heroin, morphine. In these cases, a mature L/S ratio not only indicates lung maturity but may also indicate acceleration of liver and neurological function.
A sudden sharp increase in L/S ratio reflects acute stress e.g. placental infarction or rupture of membranes. Rupture of membranes causes an abrupt rise within 72 hours to mature ratios but is not associated with maturation acceleration of other organs. Incidence of RDS is reduced, the longer the membranes have ruptured.
This test, described by Clements et al  is a simple, inexpensive, bedside method to determine the pulmonary maturity and the results correlate well with the L/S ratio. It depends on the ability of lecithin to generate a stable foam in the presence of ethanol. The amniotic fluid is centrifuged at 2,000 rpm for 10 minutes. One ml. of the clear supernatant is pipetted into one tube and 0.5 ml. into the second tube. 0.5 ml. of saline is added to the second tube.
One ml. of 95% ethanol is then added to both the tubes, stoppered and shaken for 15 seconds. They are then placed upright for 15 minutes. A ring of bubbles completely around the meniscus after 15 minutes, in both the tubes gives a positive test result and indicates a mature foetus. A similar ring in only the first tube but not the second gives an intermediate result while an incomplete ring at both dilutions gives a negative result, indicating immaturity.
Clements et al  found that the infants delivered within 24 hours of the positive test, did not develop RDS while all with negative test developed RDS and eight of the 13 infants with intermediate results developed RDS.
Numerous other tests for foetal maturity using amniotic fluid have been described with varying results. These include volume measurements of the amniotic fluid which is 1000 cc. at 38 weeks falling to 800 cc. at 40 weeks but is subject to unpredictable individual variations. There is a gradual decrease of osmolality during gestation and Miles and Pearson 2 l found values of 250 millios-moles per kg. or less giving a reliable index. Protein concentration decreases progressively from 4 to 8 gms/L at 28 weeks to 2 to 6 gm/L. at term. Seppala and Ruoslahti  found decreasing concentrations of alpha-foetoproteins towards term. Uric acid levels of over 8.5 mg% indicate a foetus of more than 38 weeks gestation. Nelson and Freedman  found that triglyceride values of 2 mg% or greater showed only a 4% false positive rate of maturity but a 42% false negative rate. Fernandes deCastro et al  found an abrupt rise of amylase after 36 weeks. Oestriol in amniotic fluid shows a steep rise from 8 mg% at 32 weeks to 65 mg% at 38 weeks and 100 mg% at term but is not useful in patients with diabetes, pre-eclampsia, rhesus immunisation.
A few maternal indicators of foetal maturity have also been studied. Among them, vaginal cytology has been found to be most useful. A predominance of intermediate cells in the maturation index is a favourable sign while parabasal cells are considered omnious. It mainly indicates whether labour will ensue within 14 days or not irrespective of gestational age.
Urinary oestriol, human chorionic sotnatomamotropin and heat stable alkaline phosphatase show rising titres towards term but are indicators of foetal well-being rather than maturity.
Many authors have described combined methods which give a better accuracy of foetal maturity. Well known among them is the modified O'Leary and Bejzian's  score which takes into account biparietal diameter on sonar, amniotic fluid studies of creatinine, fat cells and L/S ratio, with the clinical estimation of foetal weight. A score of six or more indicates maturity while that of five or less indicates prematurity. Moltz et a1  used a combination of amniotic fluid creatinine, fat cell count and optical density at 450 mµ and found that if only one test was positive for maturity it gave a 24% reliability, 2 positive tests gave 92% reliability and if all were positive it gave a 100% assurance of foetal maturity.
We thank Dr. C. K. Deshpande, Dean of the K.E.M. Hospital for allowing us to publish the hospital data.
[Table 1], [Table 2], [Table 3]