Serum Levels of Mullerian Inhibiting Substance in Preterm and Term Male Neonates

Vol. 158,610-612, August 1997 Printed in U S A .

SERUM LEVELS OF MULLERIAN INHIBITING SUBSTANCE IN PRETERM AND TERM MALE NEONATES BERNADETTE SCHWINDT, LEX W. DOYLE

AND

JOHN M. HUTSON

From the F. Douglas Stephens Surgical Research Labomtory, Royal Children's Hospital Research Foundation,and Department Obstetrics and Gynecology, Royal Women's Hospital, Melbourne, Australin

of

ABSTRACT

Purpose: Mullerian inhibiting substance, also called anti-miillerian hormone, is responsible in the embryo for the regression of the miillerian structures. During the second and third trimesters the physiological functions that miillerian inhibiting substance may have after the period of miillenan duct regression are poorly understood. We obtained information on miillerian inhibiting substance levels in the male newborn during this period of gestation. Materials and Methods: Mdlerian inhibiting substance was measured by an enzyme immunoassay in cord blood obtained at birth in 27 preterm (25to 36 weeks of gestation) and 92 term (37 to 42 weeks) male neonates. Results: Cord serum miillerian inhibiting substance concentrations were relatively high from 25 to 31 weeks (mean plus or minus standard deviation 86.4 -+- 36.1 ng./ml.) and then they decreased from 32 weeks to term (mean 24.2 5 14.0ng./ml.). Conclusions: The decline early in the third trimester may be consistent with mullerian inhibiting substance having a function during the second but a diminished role in the third trimester. Kcr WORDS:midlerian ducts, hormones, fetal blood, fetus

al.3 Briefly, an anti-miillerian inhibiting substance monoclonal antibody was raised against purified bovine miillerian inhibiting substance, and a polyclonal antibody was raised against recombinant human miillerian inhibiting substance. Recombinant human miillerian inhibiting substance w m used to construct the standard curve. A450 Microplate Reader* was used to measure the color intensity, and the Microplate Manager software* was used to plot the standard curve and calculate sample concentrations. Some samples (25,26, 29 and 31 weeks of gestation) were assayed using our original sandwich enzyme immunoassay method3 before recent modifications, which were minor and included changes to some incubation times, temperature, and concentrations of the monoclonal and polyclonal antibodies. As a control, cord sera from 13 female neonates were included. Gender of individuals was not known to the research laboratory before assay. All female cord sera had undetectable levels of miillerian inhibiting substance. Data analysis. Data were edited and analyzed using a statistical software package, Means were contrasted by t SUBJECTS AND METHODS test. The relationship between 2 or more continuous variSubjects. Cord sera were collected at birth from 27 preterm ables was determined by linear regression. Birth weight ratio (gestational ages 25 to 36 weeks) and 92 term (gestational was calculated by dividing individual birth weight by the ages 37 to 42 weeks)male neonates born at the Royal Wom- expected birth weight for gestational age.4.5 en's Hospital. All newborns were free of acute or chronic RESULTS endocrine or metabolic diseases, and all had normal descent of the testes for gestational age, except for 1 patient with an Of the 119 neonates assayed for mdlerian inhibiting subexomphalos and bilateral undescended testes. This newborn stance, 1 had a value of zero, which we believe was a labeling was not excluded h m the random population, since levels of error and we excluded this female subject from our analysis. miillerian inhibiting substance were similar to another sub- The mean gestational age plus or minus standard deviaject of the same gestational age born without any anomalies tion was 37.7 5 3.8 weeks (range of 25 t o 42).Mean birth and, therefore, was deliberately kept as part of our data. weight was 3,111 t 948 gm.(range of 420 to 5,560) and mean Methods. Samples were stored at -7OC until assayed. Se- birth weight ratio was 1.04 5 0.18 (range of 0.34to 1.55). rum miillerian inhibiting substance immunoreactivity was Most subjects in this study had birth weights appropriate for measured by sandwich enzyme immunoassay, using minor gestational age, except for 11 with ratios below 0.8 and 16 modifications of the assay previously described by Baker et with ratios above 1.2,and they are considered to be small

Miillerian inhibiting substance, also known as antimiillerian hormone, is a 140 kDa. glycoprotein produced by the Sertoli cells of the testis and the granulosa cells of the ovary.1 Secretion of human miillerian inhibiting substance by the Sertoli cells of the differentiating testis occurs aRer approximately 8 weeks of gestation when sexual differentiation normally commences and causes regression of the miillerian ducts.2 Testicular production of miillerian inhibiting substance is maximal during the period of miillerian duct regression in male subjects, although it can be detected throughout the remainder of gestation and even during postnatal life.3 The ontogeny of miillenan inhibiting substance in the male fetus together with key events during fetal development may provide clues to discovering its possible functions before birth. Serum levels of miillerian inhibiting substance were measured in cord blood of preterm and term neonates in an attempt to relate those key events that occur during the second and third trimesters of fetal development to the temporal expression of miillerian inhibiting substance.

* Bio-Rad, Richmond, California.

Accepted for publication February 21,1997.

610

SERUM LEVELS OF MULLERLAN INHIBITING SUBSTANCE IN PRETERM AND TERM MALE NEONATES

(less than 10th percentile) and large (greater than 90th percentile) for gestational age, respectively.4~5Birth weight ratio was not significantly related to gestational age (12 = 1.0%, t = 1.1,fig. 1). The mean mullerian inhibiting substance serum concentration plus or minus standard deviation was 86.4 4. 36.1 ng./ml. in neonates between 25 and 31 weeks of gestation, which was significantly higher than the mean serum concentration of 24.2 5 14.0 ng./ml. for those 32 or more weeks of gestation (t = 12.0, p <<0.0001). The median between 25 to 31 and 32 to 42 weeks of gestation is represented in figure 2 by the line within the box, the margins of the box show the interquartile range and the bars show the range of the data up to 1.5 times the interquartiIe range from the median. There were significant negative relationships between miillerian inhibiting substance concentration and gestational age (r2 = 40.5%, t = 8.9,p <
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GESTATIONAL AGE

FIG. 2. Box plot grouping miillenan inhibiting substance (MIS)

concentrations of neonates at 25 to 31 weeks and 32 to 42 weeks of gestation. Line within box represents median, margins of box show interquartile range and bars show range of data, up to 1.5 times interquartile range from median. 160 I

DISCUSSION

Hormonal activity of the human fetal testis is highest during the first 2 trimesters (fig. 4).6 The high levels of miillerian inhibiting substance found in male neonates towards the end of the second trimester suggest that it may have a function at this time. During male fetal development the testes descend from the abdomen to the inguinal zings between 8 and 15 weeks of gestation. After 25 to 28 weeks the testes descend rapidly through the inguinal canals, then migrate to the scrotum and enter the scrotum 35 to 40 weeks of gestation. A proportion of male neonates born with undescended testes have lower levels of miillerian inhibiting substance7.8 and many with the persistent miillerian duct syndrome have undescended testes.g.lo These studies correlate abnormal testicular descent with alterations in miillerian inhibiting substance levels, and have led to speculation that

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F b . 3. Individual serum midlerian inhibiting substance (MIS) concentrations as function of birth weight. l4I

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miillerian inhibiting substance may have a role in initiating the transabdominal phase of testicular descent.10 A putative role for miillerian inhibiting substance in the gubernacular swelling reaction remains controversial, as there is considerable evidence to the contrary.11-13 The values obtained in our study support the preliminary findings reported by Josso et d.14 In our study it appears that 8 f serum miillerian inhibiting substance concentration was high towards the end of the second trimester and declined early in the third trimester to a low level, which remained consistent to the end of gestation. With this in mind, it should be noted that some authors suggest that miillerian inhibiting substance may affect lung development in the third trimes. 2 2 24 26 28 30 32 34 36 38 40 42 44 ter, since male newborns have a higher incidence of the respiratory distress syndrome associated with decreased pulmonarv surfactant than do female newborns.*6.18 The effect GESTATIONAL AGE (completed weeks) of miilierian inhibiting substance on lung development was FIG. 1. Birth weight ratio in male neonates as function of gestational age in completed weeks. Lines at birth weight ratios 1.2 and examined when Catlin et incllb&fd female fetd rat They found that ma0.8 represent approximately 90th and loth percentiles, respectively. with miillenan inhibiting sub8tan~e.l~

612

SERUM LEVELS OF m L E R I A N INHIBITING SUBSTANCE IN PRETERM AND TERM MALE NEONATES

Recombinant human miillerian inhibiting substance wm supplied by Dr.Patricia K. Donahoe, Massachusetts General Hospital, Boston, Massachusetts. REFERENCES

0

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-1 aee (weeks) FIG.4. Timing of embryolo 'cal events that are proved or pro-

posed functions of miillenan &biting substance in relation to qualitative miillenan inhibiting substance levels throughout gestation.2.6.10.18

lerian inhibiting substance suppresses the accumulation of phosphatidylcholine, the major phospholipid of surfactant, compared to lungs cultured with ovaries or buffer. High miillenan inhibiting substance serum concentrations in the fetus during the period of late lung maturation may be undesirable for surfactant production, which is consistent with our finding of low miillerian inhibiting substance levels in the third trimester of gestation. In the normal fetus there is a surge of surfactant production, as determined by an increase in the phosphatidylcholine-to-sphingomyelinratio, at about 33 to 34 weeks of gestation.18 Miillerian inhibiting substance concentration was significantly negatively related independently to gestational age, birth weight and birth weight ratio. However, birth weight and gestational age are highly correlated, and when all were entered together into a multiple regression analysis, only the negative relationships with gestational age and birth weight ratio remained. In other words, for a given gestational age, newborns who are smaller than expected have higher miillerian inhibiting substance concentrations. However, it is possible that the concentration of miillerian inhibiting substance may be higher in growth retarded newborns merely because total body water or extracellular fluid is lower in them. The serum measured in our subjects was obtained from the umbilical circulation at birth, which assumes that the neonatal cord blood miillerian inhibiting substance levels at 25 to 42 weeks of gestation are an accurate estimate of peripheral serum levels of miillerian inhibiting substance in the fetus. However, this !inding could only be confirmed by measuring miillerian inhibiting substance in fetal cord blood samples. CONCLUSIONS

The ontogenyof miillerian inhibiting substance in the male fetus may provide clues to discovering i t s possible functions before birth. The decline early in the third trimester may be consistent with miillenan inhibiting substance having a function during the second but a reduced role in the third trimester.

1. Vigier, B., Picard, J. Y., Tran, D., Legeai, L. and Josso, N.: Production of anti-Miillenan hormone: another homology between Sertoli and granulosa cells. Endocrinology, 1 1 4 1315, 1984. 2. Josso, N., Picard, J. Y.and Tran, D.: The antimiillenan hormone. Rec. Prog. Horm. Res.. 9s: 117,1977. 3. Baker, M. L.,Metcalfe, S. A. and Hutson, J. M.: Serum levels of Mdlerian inhibiting substance in boys from birth to 18 years, as determined by enzyme immunoassay. J . Clin. Endocr. Metab., 70: 11, 1990. 4. Kitchen, W.H., Robinson, H. P. and Dickinson, A. J.: Revised intrauterine growth curves for an Australian hospital population. Aust. Paed. J., 1 9 157,1983. 5. Morley, R.,Brooke, 0. G., Cole, T. J., Powell, R. and Lucas, A.: Birthweight ratio and outcome in preterm infants.Arch. Dis. Child., Special No.65.30.1990. 6. Voutilainen, R.:Differentiation of the fetal gonad. Horm. ha., suppl., 38:66, 1992. 7. Donahoe, P. K, Ito, Y.,Morikawa, Y. and Hendren, W. H.: Miillenan inhibiting substance in human testes after birth. J. Ped. Surg., 1 2 323,1977. 8. Yamanaka, J., Baker, M., Metcalfe, S. and Hutson, J. M.: Serum levels of Miillerian inhibiting substance in boys with c r y p torchidism. J. Ped. Surg., 26: 621,1991. 9. Hutson, J. M. and Donahoe, P. K: The hormonal control of testicular descent. Endocr. Rev., 7:270, 1986. 10. Hutson, J. M., Metcalfe, S. A., MacLaughlin, D. T., Cate, R. L., Cigarroa, F., Ueno, S. and Donahoe, P. K: Miillerian inhibiting substance. In:The Testis, 2nd ed. Edited by H. Burger and D. de Kretaer. New York Raven Press Ltd., chapt. 4, pp. 143-179, 1989. 11. -an, D.,Picard, J. Y., Vigier, B., Berger, R. and Josso, N.: Persistence of miillenan duds in male rabbits passively immunized against bovine anti-midlerian hormone during fetal life. Dev. Biol., 116 160, 1986. 12. Guemer, D., Tran, D., Vanderwinden, J. M., Hideux, S., Van Outryve, L., Legeai, L., Bouchard, M., Van W e t , G., De Laet, M. H., Picard, J. Y., Kahn, A. and Josso, N.: The persistent Miillenan duct syndrome: a molecular approach. J. Clin. End m . Metab., 88: 46, 1989. 13. Josso, N.,Picard, J. Y., Imbeaud, S., Carre-Eusebe, D., Zeller, J. and Adamebaum,C.:The persisteut Mullerian duct syndrome: a rare cause of cryptorchidism. Eur. J. Ped., suppl., 152 S76, 1993. 14. Josso, N., Lamarre, I., Picard, J. Y., Berta, P., Davies, N., Morichon, N., Peschanski, M. and Jeny, R.: Anti-mdlerian hormone in early human development. Early Hum. Dev., 33: 91,1993. 15. Torday, J. S.,Nielsen, H. C., Fencl, M. de M. and Avery, M. E.: Sex differences in fetal lung maturation. Amer. Rev. Resp. Dis., 123: 205, 1985. 16. Catlin, E. A, Powell, S. M., Manganaro, T. F., Hudson, P. L., Ragm, R. C., Epstein, J. and Donahoe, P. K.: Sex-specificlung development and miillerian inhibiting substance. Amer. Rev. Resp. Dis., 141:466,1990. 17. Catlin, E. A., Manganaro, T. F. and Donahoe, P. K.: Mdlerian inhibiting substance depresses accumulationin vitro of disaturated phosphatidylcholine in fetal rat lung. h e r . J. Obst. Gynec., 15tk 1299,1988. 18. Gluck, L. and Kulovich, M. V.: Lecithin-sphingomyelinratios in amniotic fluid in normal and abnormal pregnancy. Amer. J. Obst. Gynec., 116: 539, 1973.