British Journal of Obstetrics and Gynaecology November 1977. Vol84. pp 819-823

AMNIOTIC FLUID INSULIN AND GLUCAGON IN NORMAL PREGNANCY AND PREGNANCY COMPLICATED BY RHESUS ISOIMMUNIZATION nY

G. GERLINI Istituto di Puericultura

F. FALLUCCA Cattedra di Medicina Costituzionale ed Endocrinologia A.

PACHI’

M. BRESADOLA I I Clinica Ostetrica e Ginecologica C . OSSICINI Istituto di Puericultura A. Russo Cattedra di Medicina Costituzionale ed Endocrinologia AND

c. SBRACCIA Istituto di Puericultura, Universith di Roma, Roma Summary Immunoreactive insulin (IRT) and immunoreactive glucagon (IRG) were determined in the amniotic fluid from 28 rhesus isoimmunized pregnancies with moderately affected fetuses and from 15 normal pregnancies; in umbilical arterial plasma from nine newborn infants with rhesus haemolytic disease of moderate degree and from 19 normal infants; in plasma from their respective mothers at delivery; and in the urine of 13 normal infants at birth. Levels of IRI and I R G in amniotic fluid from rhesus cases were not different from those of normal pregnancies. IRG was detected in the first voided neonatal urine.

IN normal amniotic fluid at term, and in

Lowy, 1970) of newborn infants affected by rhesus haemolytic disease of the newborn (HDN).

amniotic fluid from rhesus immunized pregnancies, immunoreactive insulin (IRI) has been detected at concentrations comparable to those in the plasma of adults (Casper and Benjamin, 1970; Vidnes and Finne, 1977) and elevated insulin levels have been reported in the plasma (Barret and Oliver, 1968; Raivio and Osterlund, 1969) and urine (Schiff and

HDN seems to affect both beta and alpha pancreatic cells in the infant, with islet hyperplasia and a normal beta/alpha cell ratio (Van Assche et al, 1970), and functional evidence of hyperinsulinaemia (Barret and Oliver, 1968; Raivio and Osterlund, 1969) and 819

820

GERLINI, FALLUCCA, PACHI', BRESADOLA, OSSICINI, RUSSO AND SBRACCIA

suppressibility of glucagon by glucose (Massi Benedetti et al, 1975). Since there have been few reports of the behaviour of glucagon in HDN during the first hours of life (Fallucca et al, 1977; Massi Benedetti et al, 1975; Milner et a,, 1972) and its presence in amniotic fluid has not yet been reported in either normal or abnormal pregnancies, it was the purpose of this study to investigate its presence and behaviour in the amniotic fluid of rhesus immunized pregnancies. MATERIAL AND METHODS Amniotic fluid was sampled from 28 pregnant women with rhesus isoimmunization in a total of 51 amniocenteses between the 25th and 39th weeks of gestation; the controls were 15normal pregnant women having amniocentesis between the 37th and 40th weeks of gestation to assess fetal maturity, who subsequently delivered normal infants. Relevant data of these two groups are presented in Table I. Location of the placenta by ultrasound preceded each amniocentesis which was performed early in the morning after an overnight fast ; blood-stained samples were discarded. Each sample was submitted to direct spectrophotometry for the determination of the optical density difference (ODD) at 450 nm, according to Liley (1961). In addition, blood samples were collected at delivery from the umbilical artery and vein of 19 normal infants and their

mothers and of nine infants with HDN of moderate degree (cord haemoglobin greater than 11 g/dl) and their mothers from the rhesus amniocentesis group. The time interval between amniocentesis and delivery was between 1 and 11 days. The first urine voided after birth was collected in 13 normal infants of normal pregnancies. All samples of amniotic fluid, blood and urine were collected with plastic syringes or bags, mixed with EDTA-Trasylol (1.2 mg and 500 U/ml respectively), centrifuged at +4 "C and immediately frozen at -20 "C until examination. Glucose was determined using the glucose oxidase method of the commercial Boehringer kit; immunoreactive insulin (IRI) by the method of Hales and Randle (1963), immunoreactive glucagon (IRG) by the method of Aguilar Parada et aZ(1969) using Unger's 30 K antiserum. The reliability of glucagon and insulin assays in amniotic fluid were verified by standard curves for the two hormones. Recovery for glucagon was 83.2 per cent (for 62.5 pg added), 76 per cent (for 125 and 250 pg added) and 53 per cent (for 500 pg added). Dilution experiments adding 70 pg of glucagon to amniotic fluid in which no hormone was measurable gave the following results: 1 : 1 = 70 pg, 1 : 2 = 82 pg and 1 : 4 = 76 pg. Little interference seemed therefore to operate below 250 pg.

TABLEI Clinical features of patients ~~

Mothers

Rhesus (n Mean Range

=

Controls (n Mean Range

~

~~~~

~

Newborn infants

Age (years)

Parity

Birth weight (kg)

Cord haemoglobin (g/dl)

Cord bilirubin (mg/dl)

31.5 21-41

3.4 1-8

3.25 2.60-3 80

14.1 11-18 ' 5

3.6 1'7-5

29 25-35

2.3 1-4

3.38 2.68-3 '95

17.8 14-21.3

1.4 0.4-3

28)*

=

15)t

* There were 12 male and 16 female infants in this group. t There were 9 male and 6 female infants in this group.

AMNIOTIC FLUID INSULIN AND GLUCAGON

821

Recovery for insulin was: 75-5 per cent (for 6.25 pU added), 76.5 per cent (for 25 pU added) and 82.4 per cent (for 50 pU added). The assay for I RG in urine was used without modification since, according to the observations of Day (1972), Assan (1972), Lefebvre et al (1974) and Bilbrey et al (1974), little or no interference is seen under normal physiological conditions. The statistical evaluation was made using Student’s ‘t’ test. RESULTS Results obtained in the study are presented in Table I1 and Figures 1 and 2. In Table 11, umbilical arterial values are presented as more representative of the fetal compartment ; although hormone levels in artery and vein were not significantly different. For technical reasons all the variables could not be determined in some samples. No differences were observed in amniotic fluid at term (37 to 40 weeks of gestation) between rhesus isoimmunized and normal pregnancies for glucose, IRI or IRG (Fig. 1). Mean glucose levels in the amniotic fluid were much lower than in the umbilical artery or

1

r

IRG

G ms/c

:,$r

weeks of pregnancy

IRI

Pg l 70

T

I

LO 50

FIG.2 Changes of glucose (G), immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) in the amniotic fluid of rhesus isoimmunized pregnancies at various stages of gestation. In parentheses are the number of patients. Asterisks indicate statistically significant differences.

40

in maternal venous plasma at delivery (Table 11); mean values of IRI in the amniotic fluid of rhesus isoimmunized pregnancies were in the 10 range of those observed in the umbilical artery 10 of HDN infants while those of control pregnancies were lower than in control infants ; I RG G IRI IRG in both rhesus and normal amniotic fluid, FIG.1 IRI appeared consistently lower than in maternal Values of glucose (G), immunoreactive insulin (IN) plasma. and immunoreactive glucagon (IRG) in amniotic fluid Mean values of IRG in amniotic fluid and at term (37 to 40 weeks) of rhesus isoimmunized and maternal plasma were similar in both rhesus control pregnancies. In parentheses are the number and control groups and in HDN infants; they of patients. 30

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GERLINI, FALLUCCA, PACHI’, BRESADOLA, OSSICINI, RUSSO AND SBRACCIA

TABLE I1 Glucose, immunoreactive insulin (IRI) and immunoreactive glucagon (ZRG) in amniotic fluid at term, in plasma umbilical artery and from mothers at delivery in rhesus isoimmunized and control patients. Numbers are shown in brackets Amniotic fluid

Glucose (rngidl) Mean+SEM

IRI (pU/ml) MeanfSEM

Rhesus

Controls

Rhesus

Controls

9.7& 2 . 1 (1 3)

8.45 2.1

4814 (9)

63 5 4

1.6

7 . 6 % 1.2

lOk0.6 (7)

14% 1.8 (15)

8.8*

(14) JRG (pg/ml) Mean%SEM

Umbilical arterial plasma

63*12 (13)

(15)

(14)

59+12.7 (15)

were consistently higher in the umbilical arterial plasma of control infants. Changes in the composition of amniotic fluid of rhesus isoimmunized pregnancies from the 25th week of gestation are presented in Figure 2. Glucose showed a progressive and significant decline from the 29th week to term; mean IRI values tended to increase from the 25th week and the elevation was significant during the last month of pregnancy. IRG was detectable in all five cases examined at 25 to 28 weeks of gestation; mean values showed a subsequent increase which was significant after 32 weeks of pregnancy. No correlation was found between either IRI or 1RG values and the ODD at 450 nm of the amniotic fluid at any stage of gestation values of ODD were scattered through the first and the second Liley zones. The mean value of I R G in neonatal urine was 89+8, range: 40-145 pg/ml.

DISCUSSION The origin, turnover and maternal and fetal contributions to various amniotic fluid components are still the subject of controversy and investigation (Cassady, 1974). Of the pancreatic hormones, insulin has been reported in the amniotic fluid from normal pregnancies (Casper and Benjamin, 1970; Spellacy et al, 1973) and in rhesus isoimmunization (Vidnes and Finne, 1977) but data are completely lacking about I R G

53~t9.6 (9)

(18)

105514 (19)

Maternal plasma Rhesus

*

Controls

89.5 11 (9)

96%6.3 (19)

19.5* 4 . 2 (9)

21~t1.8 (19)

58.3513.6 (9)

63k8.6 (19)

in the amniotic fluid of either normal or pathological pregnancies. The fetal origin of amniotic fluid insulin is supported by its inability to cross the human placenta (Adam et al, 1969; Wolf et al, 1969) and its filtration in urine (Rubenstein, 1969; Schiff and Lowy, 1970), which contributes conspicuously to amniotic fluid in the last trimester of pregnancy (Vladimiroff et al, 1976). Compared to normal pregnancy our patients with moderately severe rhesus isoimmunization showed no elevation of amniotic fluid IRI in the last month of pregnancy; furthermore, the progressive increase of values during the last four months of pregnancy observed in our series is comparable with that reported for normal pregnancies by others (Casper and Benjamin, 1970; Spellacy et al, 1973). It seems, therefore, that rhesus isoimmunization of moderate degree does not appreciably influence beta cell function. With regard to fetal TRG metabolism little information is presently available and its presence in amniotic fluid has not yet been investigated. The predominantly fetal origin of the hormone in amniotic fluid can be reasonably inferred from the impermeability of the placenta to it (Adam et al, 1972) and by our results which show its presence in the first voided neonatal urine. In normal pregnant women I RG was detected in amniotic fluid during the last four weeks of gestation. In infants moderately affected by H D N we observed in a previous study (Fallucca et af,

AMNIOTIC FLUID INSULIN AND GLUCAGON

1977) a significant yet transitory alpha cell depression at birth (see also Table 11); it was therefore possible that a derangement in IRG production, if any, could be demonstrated prenatally. In the amniotic fluid from pregnancies with moderately affected fetuses, I R G could be detected throughout the stages studied, a brisk elevation being observed during the eighth month of gestation to values which at term were not different from those of controls. It seems that, as for IRI, no appreciable modifications of I R G can be detected in amniotic fluid from pregnancies with rhesus isoimmunization of moderate degree. That suggests that the modified neonatal alpha pancreatic behaviour in moderate HDN (Massi Benedetti et al, 1975; Fallucca et al, 1977) is a postnatal phenomenon.

REFERENCES Adam, P. A. J., Teramo, K., Rahia, N., Gitlin, D., and Schwartz, R. (1969): Diabetes, 18,409. Adam, P., King, K., Schwartz, R., and Teramo, K. (1972) : Journal of Clinical Endocrinology and Metabolism, 34, 772. Aguilar Parada, E., Eisentraut, A. M., and Unger, R. H. (1969): American Journal of Medical Science, 257, 415. Assan, R. (1972) : Glucagon: Molecular Physiology, Clinical and Therapeutic Implications. Edited by P. Lefebvre, and R. H. Unger. Pergamon Press, Oxford, p 45. Barret, C. T., and Oliver, T. K. (1968): New England Journal of Medicine, 278, 1260.

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Bilbrey, G. L., Faloona, G. R., White, M. G., and Knochel, J. P. (1974): Journal of Clinical Investigation, 53, 841. Casper, D. J., and Benjamin, F. (1970): Obsfetrics and Gynecology, 35, 389. Cassady, G. (1974): Clinics in Perinatology (W. B. Saunders Company, Philadelphia/London/Toronto), Vol I, No. 1, p 81. Day, D. L. (1972): Journal of Endocrinology, 53, XII. Fallucca, F., Gerlini, G., Ossicini, C., Russo, A., Sbraccia, C., Caccamo, C., and Andreani, D. (1977): Rivista italiana di pediatria, in press. Hales, C. N., and Randle, P. J. (1963): Biochemical Journal, 88, 137. Lefebvre, P. J., Luyckx, A. S . , and Nizet, A. M. (1974): Metabolism, 23, 753. Liley, A. W. (1961): American Journal of Obstetrics and Gynecology, 82, 1359. Massi Benedetti, F., Marini, A., Caccamo, M. L., and Falorni, A. (1975): Acta paediatrica scandinavica, 64, 113. Milner, R. D. G., Fekete, A. M., and Assan, R. (1972): Archives of Disease in Childhood, 47, 186. Raivio, K. O., and Osterlund, K. (1969): Pediatrics, 43, 217. Rubenstein, A. H. (1969): Journal of the American Medical Association, 209, 254. Schiff, D., and Lowy, C. (1970): Pediatric Research, 4, 280. Spellacy, W. N., Buhi, W. C., Bradley, B., and Holsinger, K. K. (1973): Obstetrics and Gynecology, 41, 323. Van Assche, F. A,, Gepts, W., de Gasparo, M., and Renaer, M. (1970): Biology of the Neonate, 15, 176. Vidnes, J., and Finne, P. H. (1977): Biology of the Neonate, 31, 1. Vladimiroff, J. W., van Otterlo, L. C., and Wallenburg, H. C. S. (1976): Abstracts of the 5th European Congress of Perinatal Medicine. Uppsala, p 16. Wolf, H., Sabata, V., Frerichs, H., and Stubbe, P. (1969): Hormone and Metabolic Research, 1, 274.

Amniotic fluid insulin and glucagon in normal pregnancy and pregnancy complicated by rhesus isoimmunization.

British Journal of Obstetrics and Gynaecology November 1977. Vol84. pp 819-823 AMNIOTIC FLUID INSULIN AND GLUCAGON IN NORMAL PREGNANCY AND PREGNANCY...
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