Diagnosis of Abnormalities in Gonadal Development

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 12, No. 4 Copyright 1982, Institute for Clinical Science, Inc. Diagnosis of Abnormalities in Gonadal Development BERNARD GONDOS, M.D. Department of Pathology, University of Connecticut, Farmington, CT ABSTRACT The role of the clinical laboratory in the diagnosis of abnormalities in gonadal development is reviewed, beginning with a description of the normal differentiation of the ovary and testis and the major types of disorders encountered. The conditions are classified as resulting from abnormal gonadal differentiation, defective endocrine function or excessive endocrine activity. Germ cell neoplasms are also reviewed. Laboratory procedures utilized in evaluation of gonadal abnormalities include cytogenetic, hormonal, and histopathologic studies. Standard procedures are described as well as newer methods which have undergone increasing use in recent years and other specialized procedures which are under investigation for possible clinical application. Introduction The role of the laboratory in the diagnosis of abnormalities in gonadal developm ent is particularly important. Because of the many varieties of such disorders and their complex pathogenesis, the types of laboratory tests utilized are quite varied. The applications and significance of these tests should be clearly understood, since proper utilization and evaluation may be critical in determining gender role assignm ent and the need for specific forms of therapy. The complexity of this area is a result of the many etiologic and pathogenetic factors involved. Genetic, chromosomal, anatomic, histologic, and hormonal factors may all play a role in the development of structural and functional abnormalities of gonadal differentiation. As a result, classifications of intersex disorders and abnormalities of hormone production are often confusing. The present report reviews the laboratory diagnosis of disorders of gonadal development, beginning with a consideration of normal gonadal differentiation and a brief summary of the main categories of abnormalities. Clinical aspects are considered only insofar as they relate to the selection and evaluation of laboratory procedures, and detailed information in this area should be sought elsewhere. The emphasis in this review is on a description of tests currently available as well as /82/ $01.80 Institute for Clinical Science, Inc.

2 A B N O R M A L IT IE S IN G O N A D A L D E V E L O P M E N T new er methods which have begun to be used in recent years or are still under investigation for possible clinical application. Gonadal Differentiation The gonads arise as outpouchings into the coelomic cavity from the genital ridge early in embryonic development. Germ cells migrate to the developing gonads from the yolk sac region and increase in number by mitotic division. While the gonads are still sexually undifferentiated, the Wolffian and Mullerian ducts begin development, the urogenital sinus forms, and the urogenital tubercle, urethral fold, and groove appear. Genetic sex is established at the time of fertilization, but sexual differentiation of the gonads becomes evident only at the end of the second month of gestation with the development in the male of testicular cords. The establishment of testicular differentiation has generally been associated with the presence of the Y chromosome, but recent evidence suggests that H-Y antigen, a cell surface component that is present in males of all mammalian species, correlates more closely with the formation of a testis than the presence of a Y chromosome.60 T e s t i s Testicular differentiation occurs at six to seven weeks gestation with the formation of cords of primitive germ cells and Sertoli cells and the developm ent of the tunica albuginea. The cytoplasm of Sertoli cells includes organelles associated with protein production, and these cells are responsible for the production of anti- Miillerian hormone, a glycoprotein present in the fetal seminiferous cords.36 Regression of the Mullerian ducts begins shortly after the appearance of the fetal Sertoli cells. At eight weeks, Leydig cells with characteristic ultrastructural features of steroid-secreting cells appear in the interstitial regions,46 associated with the onset of testosterone production.47 This is followed by growth of the Wolffian ducts, lengthening of the urogenital distance fusion of the labioscrotal swelling, and closure of the urethral groove. Differentiation of the Wolffian ducts into the epididymis, vas deferens, and seminal vesicles occurs under the control of testosterone, while its reduced form, dihydrotestosterone (DHT), regulates the differentiation of the external genitalia.63 At mid-gestation, the Leydig cells undergo regression,58 and there is a corresponding fall in testosterone production.53 The testis continues to rest against the anterior abdominal wall and the tip of the gubemaculum projects into the scrotum. The testis descends into the scrotum during the third trimester, and there is growth of the external genitalia. In the newborn period, there is a transient redifferentiation of Leydig cells31 and rise in testosterone production.19 After a few months the levels of serum testosterone fall and remain low until the onset of puberty.3,64 Similarly, the interstitial tissue consists principally of undifferentiated mesenchymal cells throughout childhood. Germ cells are of prespermatogenic type, and Sertoli cells of adult type are not present during this period.28 Maturational changes in Sertoli cells, including formation of occlusive intercellular junctions responsible for the blood-testis barrier, occur at the time of onset of spermatogenesis at puberty. O v a r y Ovarian differentiation can be predicted early in embryonic development by the presence of Barr bodies indicating more than one X chromosome. However, distinctive structural changes are not evident until the onset of meiosis and the beginning of follicle formation. After a period of extensive mitotic activity involving oogonia, the ovarian germ cells

3 278 G O N D O S enter meiosis at 11 to 12 weeks. Numerous oocytes can be seen in various stages of meiotic prophase throughout the ovarian cortex during the remainder of fetal development and into the neonatal period.58 The first follicles begin to form during the fifth month of gestation, as individual oocytes become surrounded by granulosa cells. During the early developmental period, ultrastructural features associated with steroidogenic activity can be seen in scattered interstitial cells28 and histochemical evidence of steroidogenic activity can be found in granulosa cells and interstitial cells.6 Recent observations indicate that steroid conversions can occur in the ovary early in fetal development,22 but demonstration of a significant endocrine role for the fetal ovary remains to be established. Differentiation of theca interna and formation of medium-sized and large follicles occur during the third trimester and neonatal period. Large follicles can be seen in newborns and infants. Ovarian production of hormones is minimal in the neonatal period. Plasma TABLE I C l a s s i f ic a tio n o f Gonadal D iso rd e rs in Newborns and In fa n ts Abnormal Gonadal Differentiation Sex chromosome disorders Autosomal abnormalities True hermaphroditism Testicular regression syndrome Germ cell aplasia Cryptorchidism Defective Endocrine Function Androgen insensitivity Defects in testosterone synthesis 5a-Reductase deficiency Leydig cell hypoplasia Hypogonadotropic hypogonadism Defective Sertoli cell function Excessive Endocrine Activity Leydig cell hyperplasia Leydig cell tumor Ovarian follicle cysts Granulosa cell tumor Hilar cell hyperplasia Germ Cell Neoplasms estradiol and estrone are low throughout childhood and increase only during the initial stages of breast growth.33 Urinary excretions of estrone, estradiol, and estriol increase with sexual maturation. Androgen activity present in young girls is considered to be principally of adrenal origin, although ovarian hilar cells have the capacity for androgen production. Types of Disorders Classifications of abnormalities in gonadal development have been based on phenotypic appearance, clinical manifestations, genetic factors, anatomic characteristics, biochemical defects, and gonadal abnormalities. The most successful classifications have stressed pathogenetic considerations.18, ,49 As indicated in table I, the disorders can be grouped as follows: (1) abnormalities of gonadal differentiation, indicating structural problems appearing early in development; (2) defects in endocrine function, occurring during both fetal and postnatal development; (3) excessive endocrine activity, manifested generally by precocious developmental changes in infancy and childhood. A fourth group, germ cell neoplasms, not necessarily a result of abnormal gonadal development, is included because of similarities in clinical manifestations and because developmental aspects may play an important role in certain gonadal tumors. The list of disorders is general and not comprehensive. Abnormalities which become clinically evident only at the tim e of puberty and afterward are not included. Emphasis is placed on those disorders which can be diagnosed in newborns and infants. A b n o r m a l G o n a d a l D i f f e r e n t i a t i o n Abnormalities in this group are generally a result of chromosomal disorders. Most of these are related to the sex chromosomes, but autosomal abnormalities

4 A B N O R M A L IT IE S IN G O N A D A L D E V E L O P M E N T 279 may also affect gonadal differentiation. The sex chromosome disorders include a great variety of possible defects including additional X or Y chromosomes, a single X chromosome (Turner s syndrome), mosaicism, and structural abnormalities.23 Genetic factors may also be involved in the absence of detectable karyotypic abnormalities. True hermaphroditism, indicating a condition in which both ovarian and testicular elem ents are present, may be associated with chromosomal abnormalities, but most cases demonstrate a 46,XX karyotype indistinguishable from a normal female. The term, gonadal dysgenesis, has been used for many of the chromosomal disorders but has also been applied to disorders resulting from abnormalities of endocrine function, thus covering a range of pathogenetic categories. Developmental abnormalities not necessarily related to genetic or chromosomal defects also occur. Testes may be absent, rudimentary, or abnormally formed in individuals with normal 46,XY karyotype. A variety of terms, including gonadal agenesis, anorchia, and Swyer s syndrome, have been used for such abnormalities. The term, testicular regression syndrome, has been suggested to designate these disorders14,16 and appears to be most appropriate in indicating the probable pathogenesis. Germ cell aplasia (Sertoli cell-only syndrome, del Castillo syndrome) is a cause of male infertility generally diagnosed in the adult period, but potentially detectable at an earlier age. The condition is usually idiopathic and the cause unknown. Some cases can be attributed to radiation injury or other causes of germ cell degeneration. In most cases, there is probably a developmental defect in germ cell formation, migration, or differentiation or a failure in germ cell development because of excessive degeneration. Cryptorchidism, reflecting a developmental abnormality in testicular descent, is diagnosed clinically and in most instances does not involve the laboratory. However, because of the increased incidence of germ cell neoplasms in undescended testes, the pathologist may be called upon to evaluate such specimens and should be aware of the histologic appearance of cryptorchid testes at different ages. In addition, a recently described condition of intratubular germ cell hyperplasia, a precursor to germ cell neoplasms, has been observed in undescended and atrophic testes.55 D e f e c t i v e E n d o c r i n e F u n c t i o n Since active hormone production occurs in the fetal testis and not the fetal ovary, abnormalities in this group are generally restricted to genetic males. However, because of defective androgen production, the individuals are often phenotypic females. Major disorders in this group are caused by gene-related defects in hormone synthesis or action and may have a familial pattern. In other conditions, the cause is not known. The most well known abnormality in this group is the so-called testicular feminization syndrome. A preferred term is androgen insensitivity syndrome. The defect is not in the gonad itself but at the level of target cells in their lack of response or resistance to androgen action. The individuals have female-type external genitalia and breast developm ent in spite of having a normal male 46,XY karyotype and active Leydig cell function. The androgen insensitivity of target tissues is evidently an X-linked disorder.44 More specifically related to testicular function are abnormalities of testosterone synthesis. These involve defects in enzymatic activity at one of five different steps in the steroidogenic pathway. The resulting deficiency in testosterone formation is associated with clinical manifestations suggesting such terms as incomplete testicular feminization. As has been pointed out, these disorders should be

5 280 G O N D O S classified in terms of the specific enzymatic defect which can be determ ined by appropriate laboratory analysis.24 Another related condition involves a defect in 5a-reductase activity which is responsible for conversion of testosterone to DHT.32,62 This is associated with normal testosterone levels so that the internal genital tract structures develop normally, but the deficiency in DHT results in abnormal formation of external genitalia. Although this condition is manifested clinically at the time of puberty, because of the strong familial pattern it can be suspected and diagnosed earlier by hormonal studies, so that a decision can be made in regard to gender assignment. Deficiency in testosterone production may be a result of Leydig cell hypoplasia.8 The paucity of Leydig cells is in contrast to the abundance of Leydig cells seen in the conditions previously described. This condition, as would be expected, is associated with abnormal differentiation of internal and external genitalia. Hypogonadotropic hypogonadism occurs in both males and females. The effects on gonadal function are secondary to defective pituitary gonadotropin production. This is readily diagnosed by finding low luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels. In some cases, isolated gonadotropin deficiency has also been found. The abnormalities in gonadotropin production may be related to intrinsic pituitary defects or to secondary effects of abnormal hypothalamic function. The role of hypothalamic releasing factors in regulating pituitary activity requires special investigation in these conditions. With the recognition that Sertoli cells are responsible for producing the substance that causes regression of the Mullerian ducts, it could be postulated that in 46,XY individuals with persistent uterine and tubal structures defective Sertoli cell function might be involved. Several cases supporting such an interpretation had been reported in the past. Further support comes from a recent report describing a patient with a single uterine horn and normal Fallopian tube on one side and correlation of the appearance of Sertoli cells in the ipsilateral gonad with the morphology of the internal genitalia.5 E x c e s s i v e E n d o c r i n e A c t i v i t y Most cases in which excessive hormonal activity results in genital tract abnormalities in infants and children are not a result of intrinsic gonadal disorders. For example, the appearance of ambiguous genitalia in a newborn female is most likely related to congenital adrenal hyperplasia, maternal steroid hormone administration, or virilizing ovarian tumor. Precocious puberty developing in young children is most commonly idiopathic or neurogenic in origin. A few types of disorders are related to abnormal gonadal function and these must be distinguished from the other types in order to determ ine proper evaluation and treatment. In males, evidence of precocious sexual development may appear in the first few years of life as a result of excessive Leydig cell activity and consequent elevated testosterone levels. This may be a result of hyperplasia or tumor. Leydig cell hyperplasia, which is bilateral, must be distinguished from adrenal abnormalities. Measurement of adrenal androgens and testing for dexamethasone suppression and gonadotropin stimulation are particularly helpful in this regard. Leydig cell tumors in this age group are generally benign.7 In females, ovarian follicle cysts or granulosa cell tumors may account for evidence of precocious puberty. It should be pointed out that follicle cysts are frequently found in young children and their presence in a case of precocious puberty does not necessarily indicate an etiologic

6 A B N O R M A L IT IE S IN G O N A D A L D E V E L O P M E N T 281 relationship. This is borne out by the observation that removal of such cysts often fails to reverse the symptoms. Juvenile granulosa cell tumors are only rarely found in infants, generally appearing at an older age,52 and therefore would be an unlikely cause of precocious puberty at an early age. Hilar cell hyperplasia with progressive hirsutism resulting from elevated testosterone production has been reported in a patient with gonadal streaks.38 G e r m C e l l N e o p l a s m s The most common testicular tumor found in infants is the yolk sac tumor. This is a germ cell derivative representing an extraembryonic line of differentiation. It is also known as endodermal sinus tumor, which is the more frequent designation for the identical tumor occurring in the ovary. In the testis, tumors diagnosed at an early age, that is under two years, have a relatively good prognosis.7 This is in contrast to the ovary in which the tumors behave very aggressively,52 although in recent years use of combination chemotherapy has greatly improved survival. The tumors produce large amounts of alphafetoprotein which can be identified in blood and urine by radioimmunoassay as well as in the tumor tissue by immunohistochemistry. Another germ cell tumor occurring in young boys is the teratoma, a neoplasm composed of tissues from all three germ layers. This tumor also shows a sharp sex difference in clinical behavior. In males in the childhood period teratomas are generally benign without evidence of metastasis, while in girls teratomas are frequently highly malignant. This situation becomes reversed in adults. The reason for the discrepancy may relate to the difference in timing of oogenesis and spermatogenesis.17 The gonadoblastoma is often included in discussions of germ cell neoplasms, although it is actually not a germ cell tumor but rather an abnormality of gonadal differentiation that may evolve into a germ cell neoplasm. Gonadoblastomas generally occur in phenotypic females with sex chromosome disorders, often with mosaic patterns and often, but not always, including a Y chromosome.51 The important consideration in the evaluation of such patients is that the predilection to develop germ cell neoplasms shows a strong correlation with the presence of a Y chromosome.50 This is generally true of individuals with sex chromosomal disorders. It has therefore been suggested that in patients with a Y chromosome and abnormal gonadal development, the gonads should be removed prior to puberty when there is an appreciable rise in the developm ent of malignant tumors.42 Laboratory Diagnosis The indications for performing specific laboratory tests will depend on the clinical findings and evaluation. In some cases, a single procedure may establish the correct diagnosis. However, in most instances of abnormality of gonadal function, multiple tests will be required. Selection and interpretation will depend on a thorough knowledge of the considerations discussed above. Currently available tests (table II) generally fall into three main categories, including cytogenetic, hormonal, and histopathologic studies. C y t o g e n e t i c S t u d i e s Initial screening is done with a buccal smear to detect the presence or absence of Barr bodies, correlating with the number of X chromosomes. This is a simple procedure which may be helpful but is generally of limited value in most diagnostic problems. Procedures to demonstrate Y chromosome fluorescence are also available. These can indicate the presence or ab

7 282 G O N D O S sence of the Y chromosome as well as possible abnormalities. The definitive procedure for detection of chromosomal disorders is the karyotype analysis which will demonstrate the precise chromosomal pattern. Absent or additional chromosomes, structural abnormalities, and abnormal marker chromosomes can be identified. This procedure is most frequently performed on peripheral blood lymphocytes. Where indicated, karyotype studies should also be done on other tissues, including skin fibroblasts and both gonads. H o r m o n a l S t u d i e s Vaginal cytology can be used as a screening test for abnormal estrogen production in young girls. As with the buccal smear, this is a simple procedure of relatively limited usefulness. In difficult diagnostic problems, specific hormonal assays should be performed. Steroid hormone analysis by radioimmunoassay is available for a number of substances. Testosterone and estradiol levels are the most specific indicators of gonadal secretion, but numerous other steroids involved in biosynthetic conversions and metabolic breakdown may also be determ ined as indicated. Serum and urine determinations are most frequently employed, but other fluids and tissue fractions can also be analyzed. Among the most frequent diagnostic problems involving steroid hormone analysis is the distinction betw een gonadal and adrenal origin. This is particularly the case in abnormalities of sexual differentiation. Familiarity with the different patterns of steroid metabolism in the different endocrine organs is obviously critical. Pregnanetriol levels will be of particular value in indicating adrenal hyperfunction. In addition to direct hormone measurements, provocative tests are especially useful. Testicular capacity, specifically Leydig cell function, can be assessed by human chorionic gonadotropin (hcg) stimulation of testosterone production. Dexamethasone suppression of androgen levels will distinguish betw een adrenal and testicular origin. Steroid receptor studies are also important, for example, in the androgen insensitivity syndrome, as well as in other conditions. Gonadotropin levels determ ined by radioimmunoassay, including LH, FSH and hcg, are important in evaluating problems in gonadal development. Specific identification of the polypeptide hormones is facilitated by the analysis of beta-subunits. In addition to the gonadotropins, other pituitary hormones such as prolactin and growth hormone have been implicated in the regulation of gonadal maturation,45 although their significance in this regard remains to be determined. Some question has also arisen recently regarding the biological relevance of gonadotropin radioimmunoassays in the developmental period. It has been found, for example, that there is considerable discrepancy betw een radioimmunoassay and bioassay determinations of FSH and LH in the prepubertal period.39 The bioassay procedures are discussed later. As previously described, radioimmunoassay for alphafetoprotein is particularly useful in the diagnosis and monitoring of patients with yolk sac tumors. H i s t o p a t h o l o g i c St u d i e s In most of the abnormalities described, the diagnosis will be established by clinical findings, cytogenetic analysis, and hormonal studies. Occasionally, biopsy is required for specific confirmation, to exclude neoplasm or in certain primary gonadal defects.65 In the case of neoplastic disorders, the pathologist plays a key role and therefore should have a thorough understanding of germ cell tumors, endocrine tumors, and other types of neoplasms occurring in the ovary and testis of

8 A B N O R M A L IT IE S IN G O N A D A L D E V E L O P M E N T 283 infants and children. Several reviews on this subject are available.7,52,56 The use of electron microscopy should be considered in difficult diagnostic problems. Ultrastructural examination may be critical in evaluating developmental defects in which subtle distinctions in stages of germ cell and Sertoli cell maturation may be difficult to assess by routine light microscopy.28 Similarly, determination of Leydig cell differentiation is best evaluated by electron microscopic analysis. Although the use of electron microscopy in studying gonadal neoplasms is not required in most cases, such studies have greatly enhanced understanding of the morphologic characteristics and pathogenesis of these tumors. Application of electron microscopy in cases of abnormal gonadal maturation should receive careful consideration on a selective basis. Recent Developments Newer methods developed in recent years have added special capabilities in evaluating disorders of gonadal development (table II). Some of these methods are available for clinical application in certain laboratories and utilization of these procedures may be essential in the diagnostic work-up of specific cases. Other methods have been used principally in the research laboratory but offer promise for application in the clinical laboratory. Additional methods are described which at present are restricted to experimental protocols; however, they are included because they offer insight into possible modifications for clinical use. C y t o g e n e t i c M e t h o d s Analysis of H-Y antigen has taken on special importance with the recognition that this may be the key factor in gonadal sex differentiation.61 Serologic methods using sperm cytotoxicity26 and mixed hemadsorption-hybrid antibody tests40 are available in several reference labora- TABLE I I L a b o ra to ry D iagnosis o f A b n o rm a litie s in G onadal Development* Cytogenetic Studies Buccal smear H-Y antigen Y fluorescence Amniocentesis Karyotype analysis Hormonal Studies Vaginal cytology Steroid binding proteins Steroid determinations Gonadotropin releasing Gonadotropin radio- hormone immunoassay Gonadotropin bioassay Alphafetoprotein Anti-Müllerian hormone Receptor studies Meiosis regulating substances Histopathologic Studies Light microscopy Immunoperoxidase Electron microscopy Freeze-fracture Standard procedures are indicated on left, newer methods on right. tories. Utilization of H-Y antigen analysis is considered an essential part of the evaluation in patients with gonadal developmental disorders of uncertain origin. Amniocentesis has now become established as a reliable, clinically useful procedure, as performed by those possessing the required expertise.25 As a result, karyotype analysis can be done on amniotic fluid specimens, allowing diagnosis of chromosomal disorders during fetal development. H o r m o n a l M e t h o d s Steroid binding protein assays have been applied in experimental studies and have been found to provide important new information on the metabolism of steroid hormones.48 Only a small fraction of the total concentration of unconjugated steroid hormones exists in free form in the blood. Recent developments in understanding the properties of sex hormone binding globulin (SHBG) have introduced a new dimension to the understanding of the activity of sex hormones.2 Sex hormone binding globulin binds to both testosterone and estradiol. This apparently represents an important mecha

9 284 G O N D O S nism for regulating and maintaining sex differentiation.11 Disturbance of this mechanism may lead to abnormalities of sex differentiation. A key role for gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH) and luteinizing releasing factor (LRF), in regulating pituitary gonadotropin secretion has been recognized for a num ber of years.4,30 Radioimmunoassay techniques are available for measuring GnRH activity in the early developmental period.13 Measurement of this hypothalamic hormone is now considered an essential part of the laboratory evaluation in certain clinical disorders, using both direct measurements and provocative studies.54 Bioassays for gonadotropin activity have been in use in the research laboratory and may find clinical application. As indicated previously, studies in children have shown considerable variation in values obtained by radioimmunoassay and bioassay. An assay for LH based on stimulation of testosterone production by rat Leydig cell cultures is considered an accurate indicator of LH activity.15 Similarly, the effect of FSH in stimulating androgen binding protein production by rat Sertoli cells57 provides a reliable assay for FSH activity using rat Sertoli cell cultures.21,43 These procedures are much more detailed and time-consuming than radioimmunoassay (RIA), but the fact that the more easily performed RIA procedures may not truly reflect biological activity should be recognized. The anti-mullerian hormone (AMH) has now been studied in a num ber of species using a bioassay in which regression of fetal Mullerian ducts is assessed. Recently, an antibody against AMH has been produced, opening up the possibility for development of a radioimmunoassay procedure.59 Studies on the regulation of meiosis in the developing ovary and testis have suggested that a meiosis-inducing substance and a meiosis-preventing substance control the entry of ovarian germ cells into meiosis and the prevention of onset of meiosis in the fetal testis.9 The precise manner of action and characterization of these substances remain to be determined, but it was recently reported that the meiosis-inducing substance appears to be a steroid.1 Further work in this area may provide more specific information on the pathogenesis of certain disorders of germ cell differentiation. H i s t o p a t h o l o g i c M e t h o d s The immunoperoxidase technique has found wide application in surgical pathology in the past several years. It is now possible to identify specific cell products of various types in paraffin-embedded tissue. Among the substances that can be detected in ovarian and testicular tissue, particularly germ cell neoplasms, are hcg and alphafetoprotein.41 These determinations enhance diagnostic accuracy and in some cases are needed to establish the correct diagnosis as to tumor type or types present. In addition, immunoperoxidase methods for measuring estrogen receptors have begun to be employed34 and may find clinical application. Freeze-fracture studies have been found to be of particular value in the definition of the blood-testis barrier. Occlusive Sertoli cell junctions regulating passage of materials from the circulation into the testis are clearly identified by this technique. Possible clinical applications are suggested by the observation that in germ cell deficient individuals abnormalities in blood-testis barrier formation occur.10,12 Summary The clinical laboratory has a key role in the diagnosis of disorders of gonadal development. These conditions are associated with specific genetic, chromosomal, biochemical, endocrine, and histologic abnormalities which can be identified by

10 A B N O R M A L IT IE S IN G O N A D A L D E V E L O P M E N T 285 appropriate laboratory procedures. Because of the complex activity of the gonads in germ cell production and hormonal function, clinical manifestations may be varied and present difficult problems in differential diagnosis. Understanding of the pathogenesis of the different disorders and the proper application of laboratory tests will aid considerably in arriving at the correct diagnosis. The methods available include a wide range of cytogenetic, hormonal and histopathologic procedures. Additional new er tests which have undergone increasing clinical use in recent years and other procedures which are still under investigation for possible clinical application offer further possibilities for evaluating and understanding this complex group of disorders. References 1. An d e r s e n, C. Y., By s k o v, A. G., and G r in - STED, J.: Partial purification of the meiosis inducing substance (MIS). Development and Function of the Reproductive Organs. Byskov, A. G. and Peters, H., eds. Amsterdam, Excerpta Medica, in press. 2. An d e r s o n, D. C.: Sex-hormone-binding-glob- ulin. Clin. Endocrinol. 3:69-96, A u g u s t, G. P., G r u m b a c h, M. M., and Ka p l a n, S. L.: Hormonal changes in puberty: III. Correlation of plasma testosterone, LH, FSH, testicular size and bone age with male pubertal development. J. Clin. Endocrinol. Metab. 34: , B l a c k w e l l, R. E. and G u i l l e m i n, R.: Hypothalamic control of adenohypophysial secretions. Ann. Rev. Physiol. 35: , Bo n a v e n t u r a, L. M., R o t h, L. M., and CLEARY, R. E.: The Sertoli cell in mixed gonadal dysgenesis. Obstet. Gynec. 53: , B r a n d a u, H. and L e h m a n n, V.: Histoenzymatische Untersuchungen an menschlichen Gonaden wahrend derintrauterinen Entwicklung. Geburts. Gynak. 273: , BROSMAN, S. A. and G o n d o s, B.: Testicular tumors in children. Reviews in Pediatric Urology. Goodwin, W. 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