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Original Article |
Pediatric Endocrine Unit (M.M.) and Pediatric Surgical Research Laboratory (D.T.M., P.K.D.), Massachusetts General Hospital for Children and Harvard Medical School, Boston, Massachusetts 02114; and Pediatric Endocrine Division, Duke University Medical Center (M.M.L.), Durham, North Carolina 27710
Address all correspondence and requests for reprints to: Mary M. Lee, M.D., Pediatric Endocrine Division, Box 3080, Bell Building, Duke University Medical Center, Durham, North Carolina 27710. E-mail: . lee00140{at}mc.duke.edu
Abstract
Mullerian inhibiting substance (MIS) is a gonadal hormone expressed in a sexually dimorphic pattern. In males, serum MIS reflects Sertoli cell function and provides an estimate of seminiferous tubular integrity. We examined the role of MIS determination in the evaluation of boys with microphallus (n = 62) and/or cryptorchidism (n = 156). MIS was normal in 69.2% of boys with isolated microphallus compared with 38.1% of boys with microphallus and coexisting cryptorchidism (P < 0.05). In the cryptorchid group, MIS was normal in 46.8%, low in 24.4%, and absent in 28.8%. Normal values for age were associated with testicular tissue, whereas undetectable values were indicative of anorchia, except for two boys with MIS gene mutations (persistent Mullerian duct syndrome). These data demonstrated that a basal MIS measurement is more specific and has a higher positive predictive value than stimulated testosterone values for ascertaining the absence of testes (anorchia). In summary, a normal serum MIS concentration in the prepubertal child is a reliable determinant of testicular tissue, whereas an undetectable value is a highly sensitive initial screening test for anorchia. We conclude that preoperative measurement of MIS facilitates the management of children with cryptorchidism and intersex disorders and offers a measure of Sertoli cell function.
MULLERIAN INHIBITING SUBSTANCE (MIS) (reviewed in Refs. 1 and 2), also known as anti-Mullerian hormone (3), is a gonadal hormone that causes regression of the Mullerian structures during normal male sexual differentiation. MIS is a 140-kDa glycoprotein member of the TGFß family of peptide hormones that regulates cellular growth and differentiation (3, 4). MIS expression is limited to testicular Sertoli cells and ovarian granulosa cells and occurs in a developmentally regulated, sexually dimorphic pattern. Prenatally, MIS expression is high in males and negligible in females (5, 6). At birth, serum concentrations of MIS in males are low, but rapidly rise to peak values by late infancy, then slowly decrease to the adult range at puberty (7, 8, 9, 10, 11). In females, MIS is undetectable at birth, then gradually increases to overlap with the male range at puberty (7).
The predominant somatic cell types of the testis, the Leydig and Sertoli cells, produce hormones that are critical for both sexual differentiation and postnatal gonadal development. Recently recognized paracrine actions of MIS on Leydig cells may underlie the inverse relationship between serum concentrations of MIS and testosterone (12). MIS has been shown to inhibit the differentiation and proliferation of Leydig cell precursors (13, 14) and to down-regulate androgen steroidogenesis in mature Leydig cells (14, 15) and Leydig cell tumor lines (16, 17). Thus, MIS may function as a negative regulator of Leydig cell differentiation and testicular maturation during pubertal development and may modulate androgen production in the mature testis.
Testicular function has been evaluated traditionally by assessing Leydig cell androgen production (18, 19, 20, 21) and gonadotropins (22, 23). Measurement of gonadotropin values is particularly useful during infancy and the pubertal period for ascertaining the absence of functional testicular tissue (22, 23). More recently, there has been a growing appreciation of the value of assessing Sertoli cell function as well. Hormones such as inhibin (24) and MIS (2, 25, 26, 27) specifically reflect Sertoli cell function and may offer a noninvasive measure of seminiferous tubular integrity. Assessment of Sertoli cell function is particularly useful in the older infant and prepubertal child, in whom basal levels of gonadotropins and testosterone are low, thereby requiring hCG stimulation to evaluate androgen production. At these ages, constitutive levels of MIS are quite elevated. The present study explores the role of MIS measurement in the evaluation of boys with microphallus and/or cryptorchidism.
Subjects and Methods
Patients studied
We studied 176 boys from 1 d to 20 yr of age with microphallus, cryptorchidism, or both conditions, who were initially evaluated at Massachusetts General Hospital for Children (MGH; n = 25) or several other institutions (n = 151) between December 1995 and April 2000. None of these subjects was included in our previous study (28). Blood samples for MIS determination were drawn after verbal consent was obtained from parents, according to approved institutional guidelines. Clinical findings, hCG stimulation test results, karyotype, operative details, and pathological findings were obtained by review of charts and referring letters or clinical summaries. Long-term outcome data were available in 80 of these boys.
We compared constitutive values of MIS to stimulated testosterone values in a subgroup of 56 boys who all underwent subsequent surgical exploration and/or biopsy or had spontaneous testicular descent. Stimulated testosterone values were defined as levels obtained after hCG administration and/or levels obtained during the infantile minipuberty, when gonadotropin levels are endogenously elevated and basal testosterone values increase to the pubertal range.
MIS ELISA
Serum MIS concentrations were measured in an ELISA as described previously (7, 9, 29). This assay is specific for primate MIS and does not detect other members of the gene family. The limit of sensitivity is 0.3 ng/ml, and the intra- and interassay coefficients of variation are 9% and 15%, respectively. MIS values that fall at or below the limits of detection of the assay were assigned a zero value (undetectable MIS). MIS values were categorized as low or normal compared with established normal ranges for age for this particular assay (7). Values below the male range for age were considered low.
hCG stimulation test
Serum testosterone concentrations were measured in commercial clinical assays. The limit of detection for assays outside our institution varied from 0.10–0.35 nmol/liter (3–10 ng/dl). At MGH, testosterone was measured in a coated tube immunoassay with a detection limit of 0.17 nmol/liter (5 ng/dl; Diagnostic Products, Los Angeles, CA). The between-test coefficients of variation using control sera (Bayer Corp., Walpole, MA) are 8% at 5.00 nmol/liter (144 ng/dl), 6.6% at 12.78 nmol/liter (368 ng/dl), and 7.8% at 33.92 nmol/liter (977 ng/dl).
At MGH, hCG (1500 IU/m2) was administered twice every other day, with blood drawn at baseline, 48 h after the first injection, and 72 h after the second injection (21). A positive response was defined as a doubling of the testosterone value with each blood draw (with a peak response of 
1.74 nmol/liter, 50 ng/dl) (18). If basal testosterone was below the detection limit of the assay, a post-hCG response of 1.74 nmol/liter (50 ng/dl) or more was considered positive. In prepubertal age children, an absent rise in testosterone was confirmed with a 2-wk course of every other day hCG injections. At other institutions the test was performed according to institutional protocols (two to six injections of hCG, a total of 5,000–25,000 IU hCG over 1–3 wk). Our definition of a positive response was applied to values of basal and peak testosterone provided to us at referral.
Because the serum testosterone response to hCG was not for assessment of Leydig cell biosynthetic activity, but to confirm the presence of any testicular tissue, we selected a low cut-off value of 50 ng/dl to signify a positive response (18). For comparison, the more stringent criterion of a rise in testosterone of 3.47 nmol/liter (100 ng/dl) or more was also used to analyze the test parameters.
Statistical analysis
The differences in proportions of boys with normal, low, or undetectable MIS were calculated using Fisher’s exact test. The sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals were calculated using a two-way contingency table analysis of Javastat and the program Matlab (binofit.m), produced by The MathWorks, Inc. (Newton, MA). The calculations of Javastat were computed from methods described by Fleiss (30), and the Matlab program used methods described by Johnson and colleagues (31).
Results
Patient characteristics
All 176 subjects with microphallus and/or cryptorchidism had a 46,XY karyotype. Subjects were categorized clinically in two groups: group A, microphallus (n = 62); and group B, cryptorchidism (n = 156), with clinical overlap in 42 children (Table 1
). Of the 156 patients with cryptorchidism, only 16 had unilateral undescended testis. These 2 categories were subgrouped according to associated genitourinary findings to correlate the clinical phenotype with MIS expression (Table 1).
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Microphallus.
Serum MIS was measurable in all subjects with either isolated microphallus or coexisting hypospadias (Table 1
). MIS was in the normal range for the majority of boys in these two subgroups (69.2% with isolated microphallus and 71.4% with associated hypospadias) in contrast to only 38.1% of those with coexisting cryptorchidism (P < 0.05). In this latter subgroup (microphallus and cryptorchidism), MIS was undetectable in 21.4% and low in 40.5% of subjects.
Cryptorchidism.
Of the 156 boys with cryptorchidism, the percentage of subjects with normal, low, or absent MIS values was similar among the three subgroups (Table 1) and between boys with unilateral or bilateral cryptorchidism. Overall, 46.8% of MIS values were normal, whereas 24.4% were low (below the male range for age), and 28.8% were undetectable. Thus, the associated findings of microphallus or hypospadias did not significantly affect serum MIS concentrations in boys with cryptorchidism. Eighty boys had cryptorchid testes that were distinguishable as palpable (inguinal or retractile; n = 46) or nonpalpable (n = 34). MIS was normal for age in 65% of those with palpable testes and 25% of those with nonpalpable testes. Boys with nonpalpable testes had the highest percentage (50%) of undetectable MIS values.
Correlation of MIS values with gonadal outcome
Testicular outcome was known in half the boys with cryptorchidism (80 of 156). MIS was normal in 39 (48.8%), low in 17 (21.3%), and undetectable in 24 (30.0%), comparable to that in the total group. Of the 39 boys with normal values of MIS, 37 had normal testes, and 2 had hypoplastic testes. The 17 boys with low MIS values had normal testes (n = 9), hypoplastic testes (n = 6), testicular atrophy (n = 1), and anorchia (n = 1). The boy with anorchia (5.9 yr of age) had a low MIS value of 2.3 ng/dl, basal testosterone below 0.35 nmol/liter (<10 ng/dl), LH of 2.2 U/liter, and an elevated FSH of 84.9 U/liter. No testes were identified laparoscopically, but the detectable MIS value was concerning for fibrotic remnants with residual Sertoli cells (26, 28, 32), warranting continued monitoring and possibly reexploration. Of 24 boys with undetectable MIS, no gonadal tissue was identified in 12, fibrotic remnants were found in 10, and normal testes with Mullerian structures were found in 2 with persistent Mullerian duct syndrome (PMDS). These findings demonstrated that MIS values generally correlate with gonadal status. Testes were normal in boys with normal MIS values and absent in those with undetectable serum MIS, except in boys with MIS gene defects.
Comparison of MIS and stimulated testosterone values
To compare the value of basal MIS determination to that of stimulated testosterone testing for testicular assessment, we analyzed data from a subgroup of 56 children with cryptorchidism in whom both studies were available. All 56 subjects underwent subsequent surgical exploration and biopsy or had spontaneous testicular descent. Of these, 35 had a positive testosterone response to hCG with a mean ± SE of 10.88 ± 1.44 nmol/liter (313.2 ± 41.4 ng/dl) and a range of 2.08–29.13 nmol/liter (60–839 ng/dl). Of these, testosterone was 3.47 nmol/liter (100 ng/dl) or more in 30 and between 1.74 and 3.47 nmol/liter (50 and 100 ng/dl) in 5, 3 of whom had hypoplastic testes. Twenty-one boys had a low rise in testosterone with a mean ± SE of 0.51 ± 0.21 nmol/liter (14.6 ± 0.6 ng/dl) and a range of 0.10–1.56 nmol/liter (3–45 ng/dl; Table 2A).
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). Of 7 children with a low MIS value but a positive hCG stimulation test, 3 had hypoplastic testes, and 4 had serum samples that had been shipped at room temperature. Of 8 subjects with detectable MIS but a negative hCG stimulation test, 4 had hypoplastic testes. The other 4 with normal testes at orchiopexy were all in the prepubertal age group (2–10 yr of age).
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). This analysis showed that although both tests were highly sensitive and equally predictive for the presence of testes, MIS had a higher specificity and positive predictive value for the absence of testicular tissue. Although a low testosterone response was 57.1% predictive of anorchia, an unmeasurable MIS value was 92.3% predictive. The test parameters were also calculated using a stimulated testosterone cut-off value of 3.47 nmol/liter (100 ng/dl) or more as the lower limit for a positive response. Using this criterion, the specificity of a low stimulated testosterone value decreased from 79.5% to 68.2%, and its positive predictive value for diagnosing anorchia decreased from 57.1% to 46.2%.
MIS and basal gonadotropin values
Gonadotropin values were available in 47 boys with known gonadal outcome. Of these 47 boys, 22 were less than 2 yr of age, 21 were 2–10 yr, and four were more than 10 yr of age. Five boys in the less than 2-yr-old group and six in the 2–10 yr age range were anorchic. MIS values were undetectable in all boys with anorchia, except the child described above under gonadal outcome. In the 5 boys under 2 yr of age with anorchia, FSH values were uniformly elevated (66–254 U/liter) and were diagnostic of anorchia, whereas LH values ranged from 2.9–90 U/liter. In the 6 anorchic boys between 2–10 yr of age, FSH values ranged from 0.1–84.9 U/liter (0.1, 6.6, 7.2, 22.2, 37.8, and 84.9 U/liter), and LH values ranged from 0.2–2.4 IU/liter (0.2, 0.2, 0.2, 1.2, 2.2, and 2.4 U/liter). Thus, in the prepubertal child beyond infancy, FSH values were unequivocally elevated in only 50% of cases, and LH values were not elevated. In this age group, gonadotropins are less helpful diagnostically for anorchia, as previously reported (22, 23).
Discussion
MIS determination offers an index of Sertoli cell function in children with diverse gonadal and genital disorders (2, 26, 28, 29, 32, 33). Serum concentrations of MIS levels are much greater in prepubertal males than females and reflect the high constitutive expression of MIS in the prepubertal testis. Our data confirm the utility of MIS measurement in the evaluation of children with cryptorchidism. Almost all cases with normal MIS values had normal-appearing testicular tissue at exploration or upon spontaneous descent of testes, and an undetectable MIS value was highly predictive of anorchia. Of boys with bilateral cryptorchidism, 74% of those with nonpalpable gonads and 35% of those with inguinal gonads had low or undetectable MIS, indicative of Sertoli cell dysfunction. With the exception of subjects with PMDS, boys with low or unmeasurable serum MIS in the face of palpable inguinal gonads either had histologically abnormal testes (hypoplastic or fibrotic) or had serum specimens shipped at room temperature. In contrast, serum MIS was normal for age in the majority of children with microphallus, indicating that the seminiferous tubular compartment is probably unaffected in most cases.
A critical clinical issue in the management of children with cryptorchidism is whether to proceed with surgical exploration. If testes are present, early orchidopexy is recommended to preserve testicular function and facilitate long-term monitoring for malignant transformation. On the other hand, if testicular tissue is absent, no surgical intervention is required. Current methods to identify cryptorchid testes are suboptimal. Sonograms and magnetic resonance imaging sometimes fail to identify small prepubertal abdominal gonads (34, 35), and laparoscopic examinations can miss dysgenetic or infantile testes (35). In infants and pubertal children, elevated serum gonadotropin and low testosterone levels are suggestive of anorchia or nonfunctioning gonads. In prepubertal children, however, gonadotropin and testosterone levels in males and females are indistinguishable, even in children with anorchia, and therefore are nondiagnostic (22, 23). Thus, stimulation testing with hCG has been traditionally used to evaluate testicular endocrine function (18, 19, 20, 21). In the prepubertal child, Leydig cells are relatively refractory to hCG; consequently, a prolonged stimulation test with several weeks of hCG injections may be needed to adequately assess androgen production. MIS, on the other hand, is constitutively high in prepubertal boys, so a single basal serum sample is sufficient for MIS determination.
In our study MIS is more specific and has a higher positive predictive value than the hCG stimulation test for distinguishing between anorchia and undescended testes. A normal MIS value is consistent with the presence of structurally normal testes, and early orchidopexy should be performed in these boys to help preserve testicular function (Table 4). A low MIS value is equivocal, with variable gonadal findings on biopsy or exploration. Hypoplastic gonads comprise an important diagnostic possibility in this category; therefore, gonadal biopsy should be considered (28). When MIS is undetectable, testicular tissue is absent, except in rare cases of PMDS resulting from a MIS gene mutation (33, 36, 37, 38) or as an artifact of sample handling. MIS values may be falsely low if serum samples are shipped at room temperature rather than at 4 C or frozen. We previously reported that three freeze-thaws resulted in a 50% decrease in measured MIS (7). To avoid misdiagnosing anorchia in these two situations, a confirmatory hCG stimulation test is recommended in individuals with undetectable MIS unless elevated gonadotropins also support the diagnosis of anorchia (22, 23).
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Stimulated testosterone levels were low in four prepubertal children with normal testes. All four of these boys were between the ages of 2 and 10 yr. In this age group fetal Leydig cells have already dedifferentiated, and the adult Leydig cell population is still immature; therefore, fewer LH receptor-positive Leydig cells are present. Thus, the prepubertal testis is refractory to short-term hCG stimulation testing, and prolonged administration of hCG may be necessary to recruit steroidogenically active Leydig cells (40). Moreover, abdominally positioned testes also appear to be less responsive to hCG stimulation, compounding the difficulty of relying on Leydig cell androgen production to identify testicular tissue. The Sertoli cells appear to retain MIS expression even when the testis is dysgenetic or hypoplastic (32), and serum MIS is detectable with minimal residual testicular tissue (2, 6, 26, 27, 28, 29). In this age group the determination of MIS may be particularly advantageous as a simple, noninvasive method of identifying testicular tissue.
We conclude that measurement of MIS facilitates management of boys with gonadal disorders such as bilateral cryptorchidism and/or microphallus. Although most children with isolated microphallus have normal MIS values, a low or undetectable serum MIS level raises the possibility of an associated abnormality of the testes. In boys with cryptorchidism, if MIS values are in the normal range, and the testes remain undescended at the end of the first year, orchidopexy is recommended to preserve testicular function and facilitate future testicular examination for malignant changes (Table 4). A low MIS value is suggestive of dysgenetic or hypoplastic gonads, and surgical exploration and biopsy are usually warranted. If MIS is undetectable, and gonadotropin levels are elevated, anorchia is likely. In a prepubertal age child, gonadotropin levels may not be elevated; therefore, a confirmatory hCG stimulation test is necessary to validate the diagnosis of anorchia and exclude the rare cases of PMDS. We conclude that MIS measurement offers a simple, but highly predictive, test to help guide the initial management of the child with cryptorchidism or microphallus.
Acknowledgments
Footnotes
This work was supported in part by National Institute of Child Health and Human Development (NICHD) Grants HD-01367 and HD-36768 (to M.M.L.), National Institutes of Health Nutrition Training Grant DK-07703 (to M.M.), National Cancer Institute Grant CA-17393 (to P.K.D. and D.T.M.), and NICHD Grant HD-31223 (to P.K.D.).
Abbreviations: MGH, Massachusetts General Hospital for Children; MIS, Mullerian inhibiting substance; PMDS, persistent Mullerian duct syndrome.
Received October 19, 2001.
Accepted May 5, 2002.
References
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G. Vinci, M.-N. Anjot, C. Trivin, H. Lottmann, R. Brauner, and K. McElreavey An Analysis of the Genetic Factors Involved in Testicular Descent in a Cohort of 14 Male Patients with Anorchia J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6282 - 6285. [Abstract] [Full Text] [PDF]
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M. Misra, D. T. MacLaughlin, P. K. Donahoe, and M. M. Lee The Role of Mullerian Inhibiting Substance in the Evaluation of Phenotypic Female Patients with Mild Degrees of Virilization J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 787 - 792. [Abstract] [Full Text] [PDF]
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