THE

Journal of Obstetrics & Gynaecology of India

VOLUME XXV, No. 5 OCTOBER 1915

HORMONES AS INDICES OF FETO-PLACENTAL FUNCTION

by

K. R. LAUMAS

and

s. A. RAHMAN

Western countries have succeeded in reducing the perinatal mortality rate within the last few decades to a great degree, but in our country the rate still remains considerably high. The little decline in perinatal mortalit,y that has taken place in this country may be at­tributed to better antenatal care. Diag­nostic aids represent a valuable aspect of the antenatal care. An accurate assess­ment of the feto-placental function during gestation is essential to determine the optimal chances of fetal survival if the pregnancy has to be terminated.

Considerable information is available on various aspects of the endocrinology of pregnancy. Human pregnancy is ac­companied by a spectacular rise in both the steroidal and non-steroidal hormones. Since the work of Halban (1905) and Philip (1936) evidence indicating that

Department of r cprcductive Biology All India Institute of Medical S ciences New De/hi-110016 (India) .

Received jo1· pu blication on 20-3-74 .

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the contents of the pregnant uterus are the most likely source of these hormones, has been reviewed extensively (Zondek and Sulman, 1945; Diczfalusy and Troen, 1961; Brody, · 1969; Diczfalusy and Man­cuso, 1969; Solomon and Y ounglai, 1969 and Lauritzen, 1971). Based upon this information, a series of procedures, measuring the secretions of the £eta­placental unit, have been introduced in the last few decades to evaluate the £eta­placental function during pregnancy. In addition to physical approaches, these in­clude a large number of prognostic tests on both the enzymatic and hormonal products of feto-placental origin. How­ever, hormones having a considerably faster disappearance :rate (in minutes) than the enzymes (in days) have been generally preferred to minimize the time lost between feto-placental compromise and its earliest possible detection.

S\ince estrogen production depends on both the fetus and the placenta (Dicz­falusy and Mancuso, 1969) tests for this group of hormones are useful in a variet,y

560 JOURNAL OF OBSTETRICS AND GYNAECOLOGY OF INDIA

of fetal complications. The estimation of estriol has been usually preferred over estrone (E1 ) and Estradiol (E2 ), because its production predominantly depends on the fetal function. However, the fact that about 60 per cent of the total E~ synthesized by the placenta during preg­nancy is derived from fetal precursors (Siiteri and MacDonald, 1966 and Jaffe et al, 1968), the estimation of 'E2 as feto­placental function test 1s being evaluated (Townslay et al, 1973 and Rahman et al, 1974a and d).

Unlike estrogens, the human placental lactogen (HPL), human chorionic gona" dotrophin (HCG) and progesterone are specific products of placenta. Therefore, the estimation of these hormones in the maternal circulation reflects the fetal function indirectLy. It is now recognised that the fetus and placenta are inter­dependent and some of the abnormalities of pregnancy are due to placental insuffi­ciency. It is, therefore, not surprising to note that in the prediction of fetal risk the estimation of placental hormones com­pares favourably well with estrogens (Beischer et al, 1968).

In this paper, various hormqnal para­meters used as feto-placental function indices are criticalLy reviewed.

Human ChoTionic Gonadotrophin as an index of feto-placental function

The estimation of human chorionic gonadotrophin (HCG) either by im­muno-, bio- or radioimmunoassay, during pregnancy, appears to carry little or no clinical value as a feto-placental function index (Loraine and Mathew, 1950 and Samaan et al, 1969). Although high levels of serum and urinary HCG in pregnancies associated with toxaemia and diabetes have been reported (Smith

and Smith, 1934 and 1948; Loraine, 1958 and White, 1959), a significant increase ~

in HCG level has only been confirmed in the case of severe toxaemia (Loraine and Mathew, 195(} 1and Samaan et al, 1969).

In cases of threat~ed abortion, the serum, HCG determination appears to have some diagnostic value. Hon and Morris (1956); Vermelin et al (1957) and Brody and Carlstrom (1962) have shown that reduction in HCG level is invariabl,y associated with abortion. However, in cases of intra-uterine fetal death without placental damage, the HCG levels may remain within the normal range for some time (Klapper, 1969). Moreover, a large category of abortions may not be asso­ciated with a fall in HCG early enough for such assays to be of any clinical value (Samaan et al, 1969).

Human Placental Lactogen as an index of feto-placental function

Human placental lactogen (HPL) is a protein hormone synthesized by the syncytiotrophoblast cells of the chorionic villi. In recent years, much interest has oentered around the estimation of HPL in the serum of pregnant women as a possible means of monitoring the placen­tal endocrine function · (Saxena et al, 1969; El- Tomi et al, 1970; Spona ~!ld Janisch, 1971; Varma et al, 1971; Rahman et al, 1974b and Spellacy et al, 1974). HPL is measurable in maternal serum as eady as three to four weeks after con­ception. The levels show a progressive increase until 33 to 34 weeks of preg­nancy and then plateau to term (Table I).

Serial estimation of serum HPL levels in toxaemic pregnancies as compared with the range in normal pregnancies

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HORMONES AS INDICES OF FETO-PLACENTAL FUNCTION 561

TABLE I The Levels of Urinary Estriol, Serum Free Estradiol-17 B and Human Placental Lactoge'lb is

Normal Preg'lbancy

Pregnancy Urinary Estriol in weeks

(mg/24 hour)

13-14 1.98 ± 0.35 15-16 2.10 ± 0.21 17-18 2.64 ± 0.22 19-20 4.40 ± 0.23 21-22 5.14..± 0.21 23-24 6.47 ± 0.32 25-26 7.59 ± 0.40 27-28 9.76 ± 0.68 29-30 9.44 ± 0.59 31-32 11.72 ± 1.18 33-34 14.66 ± 1.40 35-36 16.99 ± 1.46 37-38 18.75 ± 1.46 3lf-40 21.79 ± 1.01

All values are expressed as Mean ± S.E.

shows low levels with a gradual but slow rise in pregnancies terminating with a live birth (Fig. 1). In cases of severe toxaemia leading to intra-uterine fetal death, the HPL level shows a sharp fall after fetal death. However, a sharp fall in HPL level prior to the fetal death is not a ·consistent finding (Samaan et al, 1969). In our series of four cases of intra­uterine fetal death, only in one case (M.D.) the HPL level showed a decline prior to detectable fetal death (no heart sound). Although, higher values of HPL in toxaemia than in normal pregnancies have been reported (Singer et al, 1970), it is generally agreed that serum HPL level is a good indicator of placental function (Saxena et al, 1969; Spellacy et al, 1971; Letchworth and Chard, 19'72; Rahman et al, 1974b :md d and Lindberg and Nilson, 197'3). We found that in toxaemia, the fetal death occurred only in cases where maternal HPL values were less than 3.0 p.g/ml serum after the 30th week of preg-

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Estradiol-17 .B

(ng/ml serum)

3.47 ± 0.59 4.31 ± 0.88 4.44 ± 0.54 5.54 ± 1.02 7.84 ± 0.65 7.74 ± 0.56

10.96 ± 0.96 13.36 ± 0.94 15.73 ± 1.25 16.72 ± 1.68 17.97 ± 1.46 20.65 ± 1.46 21.38 ± 2.63 23.62 ± 1.72

Human placental Lactogen

(JL/ml serum)

0.99 ± 0.16 1.59 ± 0.27 1.89 ± 0.21 2.13 ± 0.25 2.55 ± 0.27 3.10 ± 0.31 2.67 ± 0.29 3.99 ± 0.23 4.45 ± 0.24 4.56 ± 0.30 5.41 ± 0.291 5.64 ± 0.37 5.49 ± 0.43 5.97 ± 0.34

nanc1y (Fig. 1). The level of HPL in the maternal serum may be regarded as a true reflection o£ the endocrine func­tional integrity of the placenta through­out pregnancy. Ho~ver, the serum HPL level may not reflect the true fetal con­ditions. The ·estimation of HPL would be most useful in pregnancies where a pri­mary defect in the placental function is endangering the fetal health.

In pregnancies complicated with ane­mia, we found low levels of .serum HPL compared with the range in normal pregnancy (Fig. 2). On the other hand, elevated levels of serum HPL in both the uncomplicated diabetic and twin pregnancies was observed (Fig. 2.). Al­though, no significant difference in mat­ernal serum HPL levels in diabetic com­pared with the normal subjects has been reported (Beck et al, 1965 and Spona and Janisch, 1971), yet higher levels o£ serum HPL in diabetic and tw~n preg­nancies is a consistent finding (Saxena

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JOURNAL OF OBSTETRICS AND GYNAECOLOGY OF INDIA

et al, 1968 and 1969 and Varma et al, 1971). It may be concluded that the serum HPL level is closely related to the growth and size of the placenta. Since fetal death is known to result from meta­bolic ·changes besides placental dyfnnc­tion (Samaan et al, 1971), a simultaneous determination of estrogens, in addition to serum HPL, in high-risk pregnancies may be useful.

The fact that a rapid"rise in serum HPL starts about 5-6 weeks earlier than a similar rise in estrogens (Table 1) sug­gests that the estimation of HPL may be useful in case of spontaneous abortion in the early stages of pregnancy. The results of Samaan et al (1969) ; Saxena et al (1969). Spona and Janisch (1971) and Ylikorkala and Jouppila (11973) have shown a good correlation between de­creased HPL levels and the outcome of pregnancies threatened with abortion. Thus, the estimation of serum HPL appears to be a good prognostic aid in the follow up of early pregnancy.

P~·ogesterone and. its metabolites as index of feto-placental function

The assay of the excreted progesterone metabolite-pregnandiol in the urine as an index of feto-placental function dur­ing pregnancy, has been used extensive­ly in the past. Reliable methods for the estimation of serum progesterone are also available. However, the estimation of neither urinary pregnanediol nor serum progesterone has gained much importance.

In a large number of toxemic women blood progesterone level failed to show any difference from normal, regardless of the severity of the condition (Eton and Short, 1960). Subnormal levels of pro­gesterone before fetal death have been

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observed in two cases of pre-eclampsi. (Coyle et al, 1962). However, the values did not show any fall after fetal death In these cases pregnanediol excretion :. urine also did not provide any guidance to the eminence of fetal death. Johans­son (1969) and Jonasson Johansson (1971) have concluded that the nteasure­ment of progesterone, either in plasma or in the amniotic fluid did not provide any help in the evaluation of fetal status in the last trimester of pregnancy.

The estimation of both serum progest­erone (Robertson et al, 1971) and uri­nary preganediol (R,awlings and Kriger, 1959; Shearman, 1959 and Patti et al, 1963) has been used in predicting the outcome of threatened abortion in early pregnancy. As a diagnostic aid in late pregnancy, the ·estimation of urinary pregnanediol, has been used extensively in the past with little success. It is doubt­ful if urinary pregnanediol can provide useful information regarding the fetal health more accurately than progest- .-. erone itself (Samaan et al, 1969 and 1971). Russel et al (1957) and Coyle et al (1962) have concluded that the mea­surement of urinary pregnandiol did not provide any accurate informatiDn on the fetal health. ,

Serum estradiol as an index of feto-placental function ...-

The estimation of serum estradiol (E2) as an index of £eta-placental function P,as not gained much importance because of the general belief that E2 synthesis depends predominantly on the placental rather than fetal function. How­ever, it needs to be emphasized that about 60 per cent of the total E2 synthe­sized by the placenta during pregnancy is derived from fetal precursors (Siiten

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HORMONES A.S INDICES OF FETO-PLACENTAL FUNCTION 563

md MacDonald, 1966 and Jaffe et al, 196~). The usefulness of serum E2 as a ~to-placental function test has been ~valuated in the last few years when radioimmunoassay methods for the esti· mation of E 2 have become available.

Fig. 3 shows the level of free E2 in the serum of toxemic pregnancies as com­pared with the normal range. The low levels of E 2 in toxaemic pregnancies with gradual though slow rise in pregnancies terminat ing with live births may be observed. In cases of severe toxaemia leading to intra-uterine fetal death, the E2 level declined sharply before detect­able fetal death (no fetal heart sound). Similar results have been -reported by Tu1chinsky and Korenmann (1971); Fischer-Resmussen (1971) and Syhulski and Maughan (1972). In a recent study, Townsley et al (1973) have examined the relationship of maternal E~ to total serum E3 , 24 hour urinary E3 and uri­nary Ea/creatinine ratio, in normal and complicated pregnancies. A good agree­ment betw1een E'2 and other estrogen indices was observed in general. Thus, the serum E2 level appears to reflect the feto-placental function quite satis­factorily.

Figure 4 shows the levels of serum free E2 in pregnancies associated with anemia, diabetes and twin pregnancies. The levels of serum E:.! closely follow the pattern of urinary E 3 in normal and in pregnancies associated with various pathological conditions. Moreover, the lowered serum E 2 levels in toxaemie pregnancies have been shown to reflect the true steroid synthesizing ability of the toxaemic placenta (Rahman et al, 1974c and 1975). Thus it may be conclud-

_., ed that serum E2 estimation is as useful -<>s urinary E 3 and a serial estimation

would indicate whether fetal jeopardy is present.

Serum estriol as an ind.ex of feto-placen­tal function

The measurement of estriol (E3 ) levels in maternal circulation is an acceptable guide to the evaluation of fetal health (Schiffer et al, 1970; Corker and Neftolin, 1971; Tulchinsky et al, 1971; Mathur et al, 1973). It is generally agreed that a change in serial plasma E3 concentrations precedes the clinical manifestations of toxaemia and that the severity of the con­dition and fetal prognosis can he predict­ed from plasma E3 concentration (Mas­son, 1973 and Mathur et al, 1973).

The additional advantage of serum over urinary E 3 estimation as the index of feto-placental function is that serum E3 levels are relatively independent of alteration in the kidney clearanee of E 3.

However renal involvement in toxaemia may also increase the plasma E 3 due to a diminished renal clearance and there­fore, may not reflect the severity of the placental insufficiency accurately (Roy et al, 1964; Nachtigall et al, 1968; Selin­ger and Levitz, 1969 and Carrington et al, 1970). This is also true with class D diabetes as a result of the diabetic nephropathy (Ratnasopa et al, 1967) . It may be concluded that the estimation of blood E3 level would be ari additional help in high-risk pregnancies, especially when the urinary E3 is undependable. However, it would still be unreasonable to replace the urinary E 3 with plasma .E3

estimation. Detailed comparative investi­gation of plasma E3 assay vis-a-vis the urinary E3 estimation as diagnostic tool is needed.

Urinary estriol as an index of feto-pla­cental function

The estimation of the urinary estriol

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564 JOURNAL OF OBSTETRICS AND GYNAECOLOGY OF INDI~

demise to be associated with a norma urinary E3 level. The . determination u. current and futur e fetal well being rna be the most important use of urinary J.. _ Thus the estimation of urinary E 3 would be useful i~ knowing when the pregnancy needs to be terminated in order to save the fetus.

Simple and rapid colorimetric method for estimation of urinary estriol

(E3 ) as a fet~-placental function index has been used extensively (Banerjea, 1962; Martin and Hahnel, 1964; Klapper, 1965 and 1968; Laumas and co-workers, 1966, 1968 and 1'917 4d; Frandsen, 1969'; Galbraith et al, 1970 and Ostergard, 1973'). It is generally believed that urinary E3

levels show a good correlation with the fetal health, especially in the last trimes­ter of pregnancy. However, a variation of about 30 per cent even in normal pregnancies has been recorded (Klop, per, 1964) and therefore, a serial estima­tion may be essential.

During toxaemia of pregnancy, the reduction in the urinary E 3 excretion is generally related to the outcome of the pregnancy. As might be expected, not only the decline of E 3 in toxaemia, but also the degree to which it may be de­clined is related to the severity of the disease (Fig. 5). The cases with abnor­mally low levels of urinary E 3 may indi­cate severe foetal distress and placental dysfunction. Such cases lead to the intra­uterine fetal death. Klapper (196,5) has reported that the reduction in mild toxae­mia was slight but in severe toxaemia the excretion of urinary E3 fell to 4 7 per cent of the normal 7-10 days before the death of the fetus in utero.

During the course of our study, urinary estriol has been estimated by a simple and rapid colorimetric method for preg- l nancy urine (Laumas et al, 1966). The method is a modification of Brown (1965) and Rouke et al, (1968).

Extraction: An aliquot of a 24 hour urine was taken in Kober tube and di­luted with equal amount of water. Con­centrated HCl was added until the pH was 2 or below (pH paper), 2.5 g of NaCl was then added. After thoroughly mixing on vortex for 2 minutes ethyl acetate --­(,6.0 ml) was added and the mixture was vorte:xed again. After centrifugation at 2000 rpm for 10 minutes at 4°C, duplicate aliquots of the solvent phase were trans-ferred to Kober tubes and dried under ~

In pregnancies complicated with anemia and diabetes the pattern of E3 excretion correlates well with the outcome of preg­nancy (Fig. 6). A normal E3 level is a reliable indicator of intra-uterine fetal well being. A variable number of fetal death have been encountered with a de­crease in urinary E 3 excretion (Heling, 1963; Corson and Bolognese, 1968; Frand­sen, 1969 and Schwarz et al, 1969).

nitrogen.

Kober colour reaction: To each tube, 0.2' ml of 2% quinol in ethanol was added and dried under nitrogen. 2.1 ml Kober • reagent (2 % purified quinol in 76% H2S04 ) was added, heated in boiling water bath for 20 minutes and cooled in ice bath. 1.1 ml of distilled water was added, heated for 6 minutes and cooled again for 10 minutes in ice bath. 3.5 ml of distilled water was added to each tube.

The critical question is the clinical The tubes were left standing in the ice usefulness of urinany E 3 assays for eva- bath for 3 minutes, shaken and left for luating the health of the fetus in utero. another 3 minutes. 4.0 ml chloroform con­It is quite unusual for intra-uterine fetal taining 2.% p-nitrophenol and 1% ethanoJ

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~ORMONES AS. INDICES OF FETO-PLACENTAL FUNCTION 565

vas added, left for 3 minutes in the ice ath, vortexed for 20 seconds and imme­

liately centrifugated at 2000 rpm for 2 1inutes at 4°C. The aqueous phase was

removed and optical density (O.D.) of the solvent phase recorded at 500, 538 and 576 nm. The corrected O.D. was calculat­ed by the following formula: Corrected O.D. = 2 x O.D. at 538---(0.D. at 500 + O.D. at 576).

Standard curve: Estriol .in concentra­tion of 0, 1, 5, 10, 15, 20 and 25 P.g was transferred in duplicate to Kober tubes, Kober colour reaction performed and the optical density was recorded as mention­€d above. The corrected optical density was plotted against the various doses of estriol on a simple graph paper. A new standard curve was plotted on every 15 days interval.

Qttantitation of est1·iol: With each set of pregnancy urine samples, a tube con­taining 10 p.g of standard estriol was taken and processed exactly as the sample. The corrected O.D. obtained for the urine sample was read from the standard curve and corrected for recoveny.

Evaluation of the urinary estriol assay method

Sensitivity: The linearity of the rela­tionship between graded doses of E3 and the corrected O.D. has been presented in Fig. 7. The calculation of least detectable dose showed that 1 p.g of E 3 was the lower limit of sensitivity.

Accuracy: 5 to 20 f.Lg of standard E 3

was added to aliquots of pregnancy urine. After Kober colour reaction, the deve­loped colour was quantitated as mention­ed earlier. The amount of E 3 already pre­sent in each aliquot of urine was sub­tracted from each observation. Figure 8 shows the amount of E 3 measured for every addition of standard E 3• It may be

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observed that the method is quite accu­rate.

Precision: Multiple determinations of urinary E 3 in 4 samples from· different weeks of pregnancy have been presented in Table II. The co-efficient of variation, ranging between 4.7 to 10.4 per cent may show a satisfactory precision.

TABLE II

The P1·ecision of Rapid Colorimetric Method for the Estimation of Estriol During Pregnancy

Pregnant Mean SD. C.V.* women

s.s. 4.7 0.49 10.4 .S.D. 7.1 0.69 9.7 G.A. 15.4 0.91 5.9 K.R. 20.0 0.94 4.7

* C.V. =Co-efficient of variation= S .D . j mean X 100.

Concluding Remarks

The different hormonal indices may provide useful guidelines for proper man­agement and control of pregnancies associated with various pathological con­ditions. The use of human placental lac­togen, serum estradiol-17 B and urinary estriol estimations have been extensively practiced by us with considerable llm­provement in antenatal care.

The fact that a rapid rise in serum HPL starts about 5-6 weeks earlier than a similar rise is noted in both urinary E3

and serum E2 ; the estimation of HPL appears to be more useful in cases of spontaneous abortion in the early stages of pregnancy. The estimation of HPL would also be valuable in pregnancies where a primary defect in the placental function is endangering the fetal health.

The level of serum E2 or Ea closely follows the pattern of urinary E3 in nor­mal pregnancy and in pregnancies asso­ciated with various pathological condi-

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566 JOURNAL OF OBSTETRICS AND GYNAECOLOGY OF INDil

tions. Therefore, it is evident that serum E2 or E~ is as useful as urinary E 3 . Serial measurement of the hormones would in­dicate whether fetal jeopardy is present, and timely intervention may be done .to save the fetus. Thus the measurement of serum HPL, E 2 or E 3 would be of addi­tional help in high-risk pregnancies as well as in instances when the urinary E 3

is suspected of providing a misleading information.

The estimation of circulating hormone levels, involves radioimmunoassay tech­niques. The technical skill and sophisti­cated instruments required for this tech­nique may limit its utility to only those laboratories where these facilities exist. On the o~h2·r hand, ithe estima:tion of urinary E 3 is simple, rapid and does not require any major instrumentation. The collection of urine sample is easy and vene-puncture is not required. Moreover, a single technical hand can process as many as 10 urine samples every day. it is concluded that the urinary E 3 estima­tion by a simple and rapid method, such as the one used by us, may be more practical and useful as a routine diag­nostic test for monitoring fete-placental function for any hospital or clinical labo­ratory.

Acknowledgement

The published and unpublished work presented in this paper has been sup­ported by grants from the Indian Council of Medical Research, New . Delhi; Depart­ment of Atomic Energy, Government of India; Ford Foundation, New York and the World Health Organization, Geneva.

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HORMONES AS: INDICES OF FETO-PLACENTAL FUNCTION 567

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64. Sybulski, S. and Maughan, G. B.: Amer. J. Obst. & Gynec. 113: 310, 1972.

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71. Zondek, B. and Sulman, F.: Vitamins and Hormones. 3: 297, 1945.

See Figs. on Art Paper I-II

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