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S.H.Ramet al
Calicut Medical Journal 2009; 7(4):e2
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Original Article / Clinical Research
Amniotic fluid optical density at spontaneous onset of labor and its correlation with
gestational age, birth weight, functional maturity and vernix caseosa of new born.
Samartha Ram H, Sandhya ram S, Shankar Ram H.S, Shobhana Mohandas*.
Department of Obstetrics and Gynaecology, Sandhya Ram Hospital, Palghat, Kerala
*Elite Mission Hospital Thrissur Kerala.
____________________________________________________________________________
Abstract
Objective: To establish the correlation between the
Amniotic fluid (AF) Optical density (OD) at the
onset of spontaneous labor and the functional
maturity, gestational age and birth weight of the
newborn.
Methods: Uncentrifuged fresh amniotic fluid
samples from 360 singleton pregnancies were
collected during artificial rupture of membranes or
amniotomy at LSCS after onset of labor for AFOD
estimation at 650 nm .
Results: The mean AFOD at spontaneous labor
was found to be 0.98±0.27 (n=360). The study
population was divided into 7 different groups
according to gestational age (GA) (35 1/7 -36 wks,
36 1/7-37wks, 37 1/7-38 wks, 38 1/7-39 wks, 39
1/7-40 wks , 401/7-41wks, and above 41 wks of
GA respectively). In different GA groups, the mean
AFOD at spontaneous labor ranged from
0.50±0.13 to 1.03±0.24. Between any two groups
among 2 to 7 (i.e. after 36 wks GA) when cases of
respiratory distress syndrome (RDS) was
excluded, there is no essential change in mean
AFOD values (range 0.85 to 1.03). The results
were same after adjusting for birth weights. All the
babies delivered at AFOD value of mean ± SD
0.98 ±0.27 (0.40 to 1.55 CI 95 %) ,were fully
functionally mature and did not develop RDS.
Babies born with AFOD < 0.40 (n=8) had varying
degrees of RDS with birth weights ranging from
2300 to 3000 gms. Also they had more vernix on
their body than the non RDS babies The mean
birth weights progressively increased from group 1
to 7.
Synopsis: Spontaneous normal labor takes
place with complete fetal functional maturity at an
optimum AFOD value of 0.90 irrespective of the
GA and birth weight in our study population. The
correlation of AFOD with functional maturity
supports the concept of individual term
Key words: Fetal functional maturity,
spontaneous labor , amniotic fluid optical density
Introduction
Harmanni Boerhaave (1774) formulated a way of
calculating the expected date of delivery, which is
today known as Naegle’s rule. Although this rule
is widely practiced , only 4% of the women deliver
on the 280
th
day i.e expected date of delivery
(EDD/EDC) from last menstrual period (LMP)
(1).In practice we observe that spontaneous onset
of labor are spread widely from 36 weeks to 42
weeks with complete functional maturity of the
fetus. Even the most sophisticated ultrasound
imaging equipment fails to provide more accurate
information concerning individual birth dates, than
does Naegle’s Rule (95% confidence interval ± 3
weeks)(2). The mean GA at the onset of
spontaneous labor is variable with ethnicity
(3).The GA at spontaneous labor in the same
woman with different uncomplicated pregnancies
is also not the same
The International convention that 37 – 40 weeks
is term appears to be only a group correlation
(4).Where as individually the Six-week period of
birth occurrence in a human being is too large for
obstetrical management to be determined by
statistical methods. Functional Maturity of the fetus
S.H.Ramet al
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is an independent time spatial event that follows
auxonomic laws and quantum mechanics. Fetal
maturity quanta is ideally expressed in Vertical
time unlike the USG based measurements which
are widely biased on horizontal time.(2,5). It is not
an uncommon observation that few babies born
preterm are fully functionally mature and do not
develop RDS, where as few babies born after term
are functionally premature and develop RDS
(Term RDS). At least 3% of preterm births occur
beyond 37 gestational calendar week .This
percentage is doubled owing to induction of labor
only because the observed pregnancy has
reached its 287th or 294th day .GA by itself does
not always exclude the possibility of RDS
(2,6,7,8,9,). Even after 40 weeks “full term” an
incidence of RDS about 0.25 % have been
recorded (10). In one large study about 0.05%
who were delivered electively between 37 to 40
weeks required mechanical ventilation
(11).Though the percentage is small the number
increases to significant proportions when
extrapolated to larger population many of which
are avoidable (12).The American College of
Obstetricians and Gynecologists (ACOG)
recommends that obstetricians confirm fetal
pulmonary maturity prior to prelabor elective
delivery less than 39 weeks’ gestation to avoid
iatrogenic prematurity when there is a reason to
suspect delayed maturation and for documentation
as well (13).Using a similar set of guidelines prior
to that of ACOG , Frigoletto et al reported an
incidence of 0.13 % of iatrogenic RDS over three
year span covering more than 1500 repeat
Cesarean sections(14)
The lung skin interactions by the surfactant,
causes the induction of vernix detachment from
the fetal skin surface which is the primary factor
leading to increase in AF turbidity (15).Skin
appears to be the last organ to mature after lung
(16).Amniotic fluid absorbance at 650 nm is
considered as one of the standard investigations
for lung maturity evaluation in third trimester
(17).However unnecessary amniocentesis can be
avoided to large extent if the characteristics of
amniotic fluid particles are considered despite
limitations (18).Though exact measurements of
OD is not possible the range of AFOD values can
be assessed by amniotic fluid particle score with
best correlation within AFI of 9-16 (19). An
Amniotic Fluid index (AFI) ranging from 5-25 cms
is included in the current study.
The Ultrasound measurements evaluate the
chronological age which is different from biological
age and the later may be ahead or lag behind for
an individual fetus. (5).To measure the biological
EDC we need AFOD or AFOD equivalent which is
a qualitative parameter. In our study we confirm
this phenomenon in terms of AFOD which
measure lung maturity, skin maturity as well as
biological EDC (i.e complete functional maturity).
Materials and methods:
360 singleton pregnant women who underwent
first trimester scan and Crown rump length
estimation, and who were on spontaneous labor
were selected for this observational study. Under
aseptic precautions AF samples were collected
while doing amniotomy after 3-4 cm dilatation of
cervix by 22 G Spinal needle fitted with 2ml
disposable syringe. AF samples were also
collected while doing caesarean section after
careful hysterotomy from the bulging membranes.
The color and turbidity of fresh uncentrifuged AF
samples thus obtained were quantified by
colorimetry. The measurement of AFOD was done
at 650 nm after the reading of control test tube.
Birth weights were recorded for all babies in
electronic weighing machine. AFOD values were
correlated with functional maturity, gestational
age, and birth weight of the new born babies.
Informed consent was obtained from all women at
spontaneous labor prior to ARM and LSCS. The
reasons for LSCS were Cephalopelivic
disproportion, malpresentation, primi breech, post
intrauterine resuscitation of fetal distress within
half an hour.
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Classical clinical signs of respiratory distress
(tachypnea >60 breaths/min, grunting, retraction
of ribs ,sternum ,low spo2 exceeding more than 2
hrs requiring 0
2
to maintain sp0
2
(to exclude
transient tachypnoea of new born), chest x ray
findings appearing within 24 hrs, has been taken
as RDS .Other factors like sepsis, pneumonia,
meconium aspiration, intra uterine fetal distress,
and congenital abnormalities, macrosomia /
Gestational diabetes mellitus have been excluded
as cause of RDS. The severity has been
measured clinically by Downes or silverman
retraction score. Grade 1 silverman retraction
score or Downe’s score <4 were taken as Mild
RDS. (The above protocol was followed). Oxygen
supplementation was done for new borns who did
not require CPAP or IPPV. In our study bag and
mask ventilation was followed by nasal O
2
on
response to therapy for cases of mild RDS.
Protocol adopted from Hein et al (20) is shown
below
S.H.Ramet al
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Exclusion criteria.
1) Blood stained & Meconium stained AF samples
2) Medical disorders complicating pregnancy. 3)
Intrauterine growth restriction 4) Pregnancy
induced hypertension 5) Premature rupture of
membranes 6) Preterm premature rupture of
Membranes 7) Amniotic fluid index < 5 and > 25
8) History of corticosteroid therapy
Analysis and results.
Analysis was done with SPSS 15.0 and Microsoft
Excel 2007. GLM procedure (Matrix i/j method)
was done for comparison between groups. A p-
value < 0.01 was considered significant and a
value of < 0.05 as probably significant. The results
are summarized in table 1.
Figure 1 : Bar diagram shows the mean AFOD and Birth weight (kgs) in different Gestational Age groups (n=360)
Table 1:
Characteristics of AFOD and Birth weight in different gestational age groups. *mild RDS ^ Severe RDS
Group
No
1
2
3
4
5
6
7
Non RDS
Total
GA(wks)
35+1
-
36
36+1
-
37
37+1
-
38
38+1
-
39
39+1
-
40
40+1
-
41
>41
GA (days)
246
-
252
253
-
259
260
-
266
267
-
273
274
-
280
281
-
287
>
288
No: of Cases
(N)(% of Total
women delivered )
3
(0.8%)
20
(5.5%)
52
(14.4%)
138
(38.3%)
106
(29.4%)
34
(9.4%)
7
(1.9%)
352
(97.7%)
360
(100%)
AFOD mean±SD
0.50±
0.13
0.75±
0.31
0.93±
0.28
1.01±
0.27
1.03±
0.24
0.99±
0.25
1.02±
0.27
1.00±
0.25
0.9
8±
0.27
(n)AFOD<0.40
0
3^
2
1
0
2*
0
0
8
BW(gms)
Mean±SD
2283±
189
2590±
369
2832±
350
2965±
363
3087±
468
3140±
453
3221±
346
2995±
414
2987±
414
BW/GA
Gms/days
9.1±
0.74
10.1±
1.27
10.8±
1.2
10.9±
1.3
11.1±
1.63
11.17±
1.41
11.1±
1.2
11.0±
1.4
10.98±
1.43
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Figure 2.The graph below shows AFOD plotted against Gestational age in days. Blue dots represent cases
of RDS. The lines represent mean and individual prediction intervals within 95% CI (n =352) .
Figure 3. Histogram with bell curve showing physiological variation in duration of pregnancy at spontaneous
labor.( n=352 women who delivered normal babies)
Gestational Age days
300290280270260250240
Frequency
60
40
20
0
Mean =272.01
Std. Dev. =7.595
N =352
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In this observational study comprising 360
pregnant women, spontaneous onset of labor has
taken place at different gestational ages ranging
widely from 256 to 288 days (CI 95%).Only 14
women, ie. 3.8% delivered at 280
th
day by
Naegle’s rule. 82.1% women delivered between
37 to 40 wks, 11.3% delivered after 40 wks, and
6.3% delivered before 37 wks. The mean GA at
the onset of spontaneous labor was found to be
271.89±7.68 days. The mean birth weights
progressively increased from group 1 to 7 (Table
1).The mean AFOD value at the onset of
spontaneous labor was found to be 0.98±0.27
(n=360, 0.40 to 1.55 CI 95%). The mean AFOD at
spontaneous labor in different GA groups 1 to 7
(Table 1), ranged from 0.50± 0.13 to 1.03±0.24.
The Mean AFOD between groups(3-,4,6,7) (4-
3,5,6,7),(5-6,7),(6-7) ; p >.05 . Between groups
( 1- 3,4,5,6,7), (2-3,4,5,6) p <0.01,(2-7) p= 0.02,(3-
5);p = 0.033. Mean AFOD (Adjusted for Birth
weight) between groups (3-,4,6,7) (4-3,5,6,7),(5-
6,7),(6-7);p >.05.Between groups ( 1- 3,4,5,6,7),(2-
3,4,5,6) p <0.01,(2-7) p= 0.01,(3-5): p = 0.038.
The mean AFOD of non RDS cases (n=352) 1.006
± 0.25was found to be significantly higher when
compared to RDS cases (n=8) 0.25 ± 0.07 ; p
<0.001 (Independent t test ). The mean GA was
266.75 days for RDS cases (Frequency
256,258,259,261,266,270,281,283) with median
of 263 days .The mean GA for non RDS cases
was (272.01± 7.5 days).Those babies who
developed RDS with OD < 40 had more amount of
vernix (more than 60 % of body surface area) than
normal babies (chi square test - significant p <
0.001).The mean Birth weight /GA ratio ranged
from 9.1 to 10.98 and no significant difference was
found between the groups when cases of RDS
were excluded. The difference among RDS and
non RDS cases remained insignificant.
No statistically significant difference was found in
the mean AFOD values between primi (n=158)
and multi gravidae (n=202); p-0.79. For naked eye
appearance, the amniotic fluid was less turbid and
watery when AFOD value was below 0.40, and
gradually changed to milky, butter milk like and
finally curd like as the AFOD value advanced.
Discussion
In spite of great scientific advancement, the
secrets behind the gestational age at which
spontaneous onset of labor takes place with each
pregnancy and complete fetal functional maturity
is attained with each fetus is evading the
obstetrician. The physiology of onset and
progression of labor is undoubtedly multi factorial
involving various rate limiting complex sequential
inter related and mutually supportive cascades. A
minor natural variation at any level can affect the
duration of pregnancy. Some women have genetic
pre disposition to deliver pre term due to
differences at molecular level. Non-infected
“preterm” cervical ripening is an inflammatory
process like that of term labor. Polymorphisms in
several genes regulating cytokines (21) genetic
susceptibility to infections of low virulence,
mutations of collagen synthesis, oxytocin
receptors, BMI, parity
and age are also involved
(22).These factors vary from race to race and also
between each feto-maternal unit, resulting in
physiological variation in duration of pregnancy
(23-24).
In the current study between any two groups
among 3 to 7 there is no radical change in mean
AFOD values when cases of RDS were included
for analysis and remained same after adjusting for
birth weights. The values ranged from 0.93 to 1.03
.No significant difference in mean AFOD values
were noted after exclusion of RDS cases among
group 2 to 7.The mean AFOD at onset of labor
ranged from 0.85 ±0.21 to 1.03± 0.24 after 36
weeks GA among different groups. All the babies
born with AFOD value of mean ± SD 0.98 ±0.27 (
0.40 to 1.55 CI 95 % ;) were fully functionally
mature. Their skin was mature, pink with very little
vernix caseosa adherent to the surface. None of
the babies developed RDS when AFOD was
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more than 0.40. On the other hand, babies born
with AFOD value < 0.40 (n=8) were functionally
premature and developed varying degrees of RDS
and had birth weights ranging from 2300 to 3000
gms .Their skin was premature, thin, shiny and red
in color with plenty of vernix caesosa adherent to
the surface. The largest AFOD value below 0.40 at
which babies developed RDS was 0.35. With a
safe margin added we recommend a value around
0.40. as cut off value for uncentrifuged samples at
all Gestational ages. Initially the studies of optical
density were done with centrifuged samples. The
cut off was determined to be 0.15 (25).The values
are variable depending on centrifugation speed
and time. The uncentrifuged samples with OD
around 0.40 on centrifugation at 2000 rpm for 10
mins gives the OD reading around 0.15 (26).
We observed that Babies born with AFOD value <
0.40, at gestational age less than 39 weeks. (n=6)
developed moderate to severe and prolonged
RDS as compared to babies born with AFOD
value < 0.40 after 39 wks gestational age who
developed milder RDS for shorter duration. The
findings are in accordance with the previous
studies (27-28).AFOD represents indirectly the
amount of surfactant. Hence severity depends on
the how low the AFOD value is or in other words
how low the surfactant phospholipids are and not
always how low the chronological age is. There
could be other factors accounting for less severity
of RDS like trial of labor (29) or unknown
constitutional factors at advanced gestational age
despite low surfactant levels as revealed by low
AFOD values. How ever the severity could be
more than expected on rare occasions (30).
Figure 4 – Shows skin maturity changes with increasing AFOD values from left to right .Pictures were taken
immediately after birth.1,2 represents premature skin with lot of adherent Vernix, 3-4 shows normal skin and
5,6 shows postmature skin changes with near absence of Vernix .
Though,0.40 AFOD value is just enough to
prevent RDS, Fetal maturity is complete only when
all systems attain complete functional maturity.
Prematurity in GI system results in necrotizing
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enterocolitis, in respiratory system results in RDS,
and finally skin results in failure to maintain
temperature (31).
AF Lecithin levels increase from 43 micro
grams\ml at 34-35 wks gestation to 147 micro
grams/ml at term before labor. Further its levels
are known to increase up to 232 micro grams\ml at
term labor (32).There is a surge in sebaceous
gland hyperplasia producing sebum, which is a
primary constituent of vernix caseosa ,coupled
with the desquamation of fetal corneocytes during
the last trimester.This vernix separation has major
contribution to increase AFOD during third
trimester .The lung matures parallel over the same
period (15,33).At higher OD values there were
less amount of vernix on body surface of new
born. The inverse relation to AFOD values is
proved in this study.
The skin maturation follows lung maturity resulting
in complete functional maturity of fetus.
Considering skin maturity as reliable endpoint
(34), prematurity, optimum maturity and post
maturity are part of a spectrum which can happen
at any time within 36 to 42 weeks GA during
which birth is taking place. Sometimes a failure or
delay of mechanism of initiation of labor can result
in post maturity as revealed by post mature skin
changes seen at high AFOD values > 1.55(fig 3-
5,6) which was noted in 2 % of study population.
At a particular day of pregnancy at chronological
“term” there could be fetuses who are in the
process of obtaining complete functional maturity.
Conversely a fetus which is functionally mature at
36 weeks, becomes post mature if the delivery is
delayed. The understanding of AFOD prevents
iatrogenic prematurity as well as complications of
post maturity thereby optimizing labor.
Levels of pro labor cytokines like IL-6, IL-8, IL-1
beta, Epidermal growth factor in amniotic fluid and
maternal serum which are elevated during the
progress of labor are produced by the human
amniotic fluid cells which are shed by the fetal skin
(35-40).The onset of labor is more closely related
to fetal functional maturity than either gestational
age or birth weight (41).The gestational age at
delivery is unique to each feto-maternal unit. This
concept of individual term was introduced by
Klimek (Let man be born at his own due
time).There are gestational age and birth weight
independent scoring systems and computer aided
methods to know the functional or biological
gestational age of the fetus (42) Klimek and
Ballard scoring systems are the commonly used
for the postnatal assessment of Gestational age
(43).The Child survival and safe motherhood
programme guidelines in India advocate that
newborn without any signs of illness can be
managed at home with special care even if the
birth weight is as low as 1800 gms. .(44) In Indian
Scenario the cut off value for Low Birth Weight
(LBW) need to be readjusted as 2000 gms instead
of 2500 gms of international standard (45)
The significance of AFOD is not only to know
about lung maturity .But much of importance lies in
defining events taking place beyond lung
maturation which include skin maturity and onset
of labor. Studies have shown that induced (non
spontaneous) labors have higher oxytocin
requirements and increased incidence of PPH
(46,47). Presuming the inductions as inappropriate
in time, the iatrogenic complications and suffering
could have been probably avoided if the inductions
were AFOD guided. Inductions just based only on
chronological age of the fetus might be the reason
for requirement of heavy doses of prostaglandins,
dysfunctional labor prolonged induction delivery
intervals , more number of instrumental deliveries,
pain & larger doses of narcotic analgesia, neonatal
respiratory depression, perineal tears and
increased caesarean section rates. Such maternal
and neonatal morbidities are the result of
mechanization of natural process of labor.
Induction of labor needs to be reviewed in light of
AFOD values or its equivalent indicators which
determine the preparedness of labor and hence
the biological gestational age that is more relevant
than “EDC” which itself is a misnomer.
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We hypothesize that, each fetus has got its own
maturity potential in terms of AFOD. Therefore,
each feto-maternal unit is unique requiring an
individualistic approach .The mean AFOD at
uncomplicated labor range between 0.85 to 1.03
after 36 wks of gestation. We infer that complete
functional maturity and onset of labor takes place
at Individual term with mean AFOD around 0.98
irrespective of birth weight and Gestational age in
our study population.
Acknowledgement:
The authors would like to thank all the women who
participated in the study, and all the staff nurses in
the antenatal clinic, and the labor ward and
medical records dept whose involvement made
this study possible.
______________________________________________________________________________________
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_______________________________________________________________________________________
Corresponding author:
Samartha H Ram
Consultant, Department of Obstetrics and Gynecology.
Sandhya Ram Hospital.
Palakkad-678633India
Email:drsamartharam@gmail.com
