Serum levels of human placental lactogen and human chorionic gonadotropin in early pregnancy: A maturational index of the placenta LOL~I.5

L.

PEETERS,

M.D.*

JAMES

A.

LEMONS,

M.D.

GORDON

D.

F’RF.DERICK

NISWENDER, C.

PH.D.

BATTAGLIA,

M.D.

Betweevt wee!zs 10 and 20 of gestation, there are rapid and opposing changes in blood lwels of two hormones produred by the svncytiotrophoblast: hPL increases and KG &crea,ies. In this study, the relationship hetuleen these tnfo chav@ng hormone lez~el,~wan exumined, rather than the serum concentrations thevnsek~es, a~ a potential test jar plawntal maturation. The relationship betujeen the hCG and hPL concentrations attd GA4 determined in a selected group of patients was described by the folh~ng equation: G.4 (in day) =97.3. + 24.8 ln [hPL] - 14.5 In [hCG]. Thi endocrine ez~a/uation of piucental maturation by comparkon of [hCG] and [hPL] zn earls pregvlancv- ,shows prorni,se as a simple, s&e, and inexpensive clinical tool for GA awssrwnt. (‘4~. J. OBSTET. GYNECOL. 126: 707, 1976.)

1 N RE cE.N'r vears there has been a delineation of the pre- and postnatal problems associated with deviations in intrauterine growth rate. In addition, there has been an increased cautiousness in obstetrics to avoid inadvertant delivery of a preterm baby. Thus, the prenatal assessment of gestational age (GA) has assumed increasing importance. Approximately 15 per cent of all pregnancies in a general population are characterized by unreliability of dates.’ Therefore, a concern in obstetrics has been to develop methods for determin,

ing the chronological age of the fetus. Although chronological age shouId not be equated with either maturation of individual organ systems or the svnchronized maturation of the total organism, it is important as an indication of both. It is surprising that the dramatic changes in hormone levels which occur during early pregnancy have never been evaluated as predictors of placental matumtion and thus, indirectly, of GA. Between weeks IO and 20 of gestation, rapidly increasing hPL concentrations ([hPL]) are coupJed with decreasing hCG concentrations ([hCG]). ‘* ’ Because there is great interand intraindividual variation of these hormone concentrations, the measurement of the concentration of either hormone alone cannot he wed to predict the duration of pregnancv nor the degree of placental maturation.33 ’ However, measurement :md comparison of the levels of these two hormones has not yet been evaIuated in pregnant wornen. Since concentrations of both hormones are changing rapidly, and in opposite directions, the ratio of the two concentrations mav be a sensitive index of placental maturation. This study was designed to examine the reliability and accuracv of a method which considers levels of these two hormones in relation to each other-rather than each

Division qj Perinatal Medicine, Departments of Pediatrics, Obst~tr~.~-Gynecolo~, and Physiology, L’niwrs,ity of Colorado Medical Center, and Deflartment II/ Phvnolocgy and Biometrics, Colorado State Univer.&. This u*ork was ,supported in part b National Foundation-March of Dimes Grant CRBS-?02, Public Health Suwke Research Grant HD-00781, and Public Health Service Training Grant HD-00429. Rwpizjpd

for

.4ccepted

May

pub&ration

Februaql

23* 1976.

19, 1976.

Reprint requests: Dr. Loui.s L. Pee&s, University of Colorado Medtial Center, Division of Perinatal Mediciw, Contuiner B-l 98, 4200 E. Ninth Ave., Denver, Colorado 80220. *Dr. Peeters was supported by a Grant of the Stichting “De Drie Lichtew,” The Hague, Holland.

707

Table

I. Characteristics

of patient

Race ch?ujl

A. Accurate dates B. Unreliable dates

groups Parity

Age CY~.)

Wk.&

Black

15-19

2f3-24

25-29

9

0

2

3

4

3 ti 0

9

2

4

4

3

7 3 1

1 2

3

individually-in determining the degree of placental maturation, and thus indirectly the GA, in a population of women with very accurate last menstrual dates.

Materials and methods Selection of patients. Twenty-five pregnant women receiving care at Colorado General Hospital’s obstetric clinic were selected randomly. All women satisfied the following criteria: (1) the first prenatal visit was made no later than week 14 of gestation and (2) there were no complications with pregnancy at the time of initial evaluation. From these 25 women, a group of nine patients were selected who satisfied these additional criteria: (3) the menstrual cycle was regular within 2 days per cycle, (4) the pregnancy remained uncomplicated throughout the study, (5) the patient was followed throughout pregnancy, including parturition, and (6) the GA estimated from the examination of- the neonate using the criteria of Dubowitz, Dubowitz, and Goldberg5 was in agreement within 2 1 week with the estimate based on the date of the last menstrual period (LMP). Of the original 25 women, five patients were not included in the study for the following reasons: two developed pregnancy complications and three were lost to follow-up. Nine patients met the accurate-dates criteria (Table I), and the data from these patients (Group A) were used to evaluate the relationship between GA and combined hPL and hCG levels. To obtain an assessment of this test in an obstetric population with questionable LMP dates, 1 I patients (Group B) were evaluated who had been rejected from the accurate dates group because of failure to meet criterion (3) or (6) (Table I). Study period. The experimental period was chosen to include the time in gestation when maximal rates of change in hPL and hCG concentrations were occurring and included weeks 10 through 25 of gestation. Clinic visits were conducted every 2 weeks on all patients. Whenever possible, samples were drawn at the same hour and on the same day of the week. To avoid bias, no clinical assessment of GA was performed during study visits; the clinical course during this period of gest&on was evaluated by the physicians responsible

for the subject’s care, independent from thib study. :I[ each of the patient’s five to eight visits, 8 ml. of’ \j hol(, blood was collected from an antecubital vein. TIK blood was allowed to clot for 4 to 2-l IIOL~ a~ :I temperature of +4’ C., followed by centrifugation fat 15 minutes at 3,000 r.p.m. Then the serum lt as quickI! frozen with acetone and dry ice and stored aI - EC C. Radioimmunoassay. All samples were analyzed at the same time in a single assay. hPL leas measured in duplicate bv a double-antibody radioiriinl~lIlo~~ssav” vielding an intro-assay variability of 6.2 per cent. Inhibition curves obtained with the addition of’ Larving quantities of serum were par-dllel to the standard cur\ e. Preparations of hTSH, hFSH, hLH. and h(X did not influence the estimation of [hPL]. Finally, exogenous hPL could be recovered quantitatively tvhen carving quantities were added to serum. Reagents for the hCG radioimln~l~l(~assay were provided bv the l’ituitarp Hormone Distribution Program. Measurement of [hCG] were performed in triplicate bv a radioingmunoassay technique7 giving an intra-assay variabilitt of 6.5 per cent. This method is specific f&- hCG. \\ith negligible luteinizing hormone cross-reactivitv. Neurologic examination of the neonate. All the neonates in this population were examined t\vice. between 24 and 48 hours after delivery, in order to estimate the GA bv the technique of’ Dubolvitz, Dubowitz, and Goldberg.’ Using this technique, the actual age after conception can be determined with an error of 2 10 days. All examinations were performed by the same neonatologist, without knowledge of the endocrine results.

Results The hormone levels were determined a total of five times for each Group .4 patient, between 70 and 145 davs of gestation (weeks 10 through 2 I) except for two samples which had to bc obtained at week 23 of’ gestation. Figs. I and 2 represent the relationship between the GA and the natural logarithm (In) of’ tht [hCG] and [hPL], respectively, for the nine Group ,1 patients. Data points from the same patients were connected. Using the data for each woman, a rnuttipie linear regression line was estimated, with the GA as the dependent variable and the ln [hPL] ancl in [HCG] as the independent variables.’ Regression lines for each patient are computed based on the assumption that the nine regression lines are normally distributed as represented in Fig. 3. An equation for the mean line can be calculated: GA = 97.3 + 24.8 ln [hPL]

- 14.5 In [hCG]

Two sources of variability can be distinguished. 1. The residual variability around the individual

(1)

Serum

hPL

and hCG

in early

pregnancy

709

so 40 3c 20

?O

90 GEST

calculated

110

130

ATIONAL from

last

AGE

150

70

170

90

l3Q

GESTAlhMlAL

(days)

menstrual

110

period

calculated

Fig. I. Relationship between serum concentrations and gestational age in the nine Group A patients.

of hCG

lines (described as variance following equation:

by the

S,‘), determined

frcwn

150

AGE last

$70

(days)

menstrual

period

Fig. 2. Relationship between the serum concentrations and the gestational age in the nine Group 4 paricnts.

of hl’1,

180 -

160 -

//

140 -

where the number of degrees of freedom (d.f.) was obtained b\ multiplying the number of subjects times the difference between the number of observations per subject and the number of parameters in the regression d.f. = 9 x (5 - : 50).

from

AGE last

(days)

menstrual

period

Fig. 3. Comparison of the placental tnat.uration calculated with Eq. 1 (see text) vs. the gestational age in the nine Group A patients.

The

line

of identity

(.,....)

is included,

The two variances SIz and S2’ can be c,ombined the following equations:

resulting

in

a standard

deviation

(S,D?:

bv using

710

Peeters

et al.

Table II. Estimated gestational age in Group B patients using different techniques. All estimates are in days and are extrapolated to the day of delivery. The assumption has been made of a constant relationship between chronological age and organ maturation size,if he uterus Day.5 after LMP 283 275 293 311 288 276 308 279 284 288 204

bfeLwn uwk.s lOand of ptation 286 280 263 282 290 268 250 274 285 288 205

Endorriw Physical exam oj the mother I wk. prior to delivery 280 240 250 259 260 2a9 291 294 290 288 204

This SD. applied only to the time interval from 85 to 120 days (12 to 17 weeks of gestation), where the second source of variability was smallest. L4 single sample, drawn from each of the nine accurate-dates patients at approximately 100 days of gestation, provided an estimate of GA which proved to be accurate within 2 18.8 days (equal to ?2 SD.). In the 11 patients with unreliable dates, three different estimates of GA were compared: the clinical estimate derived from the physical examination of the mother 1 week prior to labor, the estimate derived from the endocrine assessment of placental maturation based upon a single blood sample obtained at approximatelv 100 days of gestation by dates, and an estimate from the pediatric examination of the newborn infant (‘Fable 11). Applying a paired t test, the endocrine estimate correlated significantly better with the neonate examination in this group of patients than did the estimate made by physical examination of the mother (P < 0.05). Because the [hCG] were scattered and the [hPL] were clustered tightly during the period between 70 and 145 days. the question arose whether the level of hPL alone, in this particular time interval, would correlate better with the GA than the two hormone levels combined. From a linear regression with the GA as the dependent variable and the In [hPL] as the independent variable the following equation for the Group A suejects was obtained: GL4 (in days) = 68 + 36 In [hPL]

estimate of muturatiorl &rived,from .sin& blood .sampLe

plarfvdal

(41

The first snurce of variability (the residual variabilitv around each sueject’s line; S,‘) revealed a variance of 87. The second source (the variability of the nine individual lines around the calculated mean line; Sz’) showed a variance of 65 in the period from 85 to 104

284 273 268 278 %I 284 276 276 284 291 182

Physical and neurologC maturation qf neonate 286 269 283 274 287 271 287 276 2ao 282 196

days of gestation (12 to 15 weeks-after 15 weeks, the variance increased very rapidly). By adding the two variances, the mean S.D. for the time period could be calculated for Eq. 4: S.D. =fl

=m

= 12.3 days

Thus. use of hPL levels alone did not correlate as well with the GA as did the endocrine estimate of placental maturation using the two hormone levels determined simultaneously.

Comment The chronological age of a fetus is an absolute which would be knokn with precision only if conception could be dated precisely. In an attempt to arrive at an estimate of chronological age of the fetus, physicians have relied upon measurements of variables which bear only an indirect relationship to chronological age. Such measurements include L/S ratios and creatinine concentrations in aminotic Huid, the size of the uterus, the growth of the fetal head as determined by ultrasound, etc. It shoulcl be emphasized that there is no reason why a test of maturation of organ function should correlate in anv precise way with chronological age. The point can perhaps be made most vividly by considering the onset of puberty in childhood. In some children this may begin at 11 years of age: in others, at 14 years of age. 0ne would not then conclude that their birth dates must be incorrect because puberty had not started at the “average” postnatal age. Similarly, in fetal and neonatal development, one should not expect that at the same chronological age children will show the same neurologic development, although this is precisely the assumption made in most attempts to estimate GA from the neonate examination. The measurement and comparison of hCG and hP1.

Serum

concentrations in ~w/~ pregnancv has encouraging possibilities as a clinical tool. The method is simple, inexpensiveand safe for mother and fetus. Unfortunatelv, the estimate of CA4 thus obtained still has an error of approximately ?2?G weeks, when a single blood sample is used. However, this preliminary stud) may not indicate the true reliabilitv of the combined hormone method for two reasons: (1) the number of sub-jects was small and (2) the reference point was not a precise knowledge ofthe date of conception, but rather an accurate LMP. Considering the variability in the time of ovulation from the first dav of the LMP, this reference point has its orvn inherent error, III this studs, vve have suggested that the rapidlv falling concentration of hCC in earlv pregnancv,

hPL and

hCG

in early

pregnancy

711

coupled 1, ith be rapidh increasing (WI< et11WI ion of hPL mav provide us some ~wi~s~llwlll 01 placm~~l maturation and thus. indirecth!, of- CA, ~i’ij