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ELEVATED PLACENTAL ESTRADIOL: A POSSIBLE ETIOLOGICAL FACTOR OF HUMAN CRYPTORCHIDISM
0022-5347/00/1645-1694/0 THE JOURNAL OF UROLOGY® Copyright © 2000 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 164, 1694 –1695, November 2000 Printed in U.S.A.
ELEVATED PLACENTAL ESTRADIOL: A POSSIBLE ETIOLOGICAL FACTOR OF HUMAN CRYPTORCHIDISM ´ R. GENETO F. HADZ˘ISELIMOVIC,
AND
L. R. EMMONS
From the University Clinics, Children’s Hospital, Basel and KinderTagesKlinik, Liestal, Switzerland
ABSTRACT
Purpose: It has been convincingly argued that the increasing incidence of reproductive abnormalities in human males may be associated with increased estrogen exposure during gestation. We documented the expression of estradiol in the syncytiotrophoblast and placenta of males born with cryptorchidism and normal genitalia, respectively. Materials and Methods: Tissue from newborn placentas was fixed in glutaraldehyde and embedded in ePON for immunohistological procedures. Cryptorchidism was unilateral in 6 males and bilateral in 1. Semithin histological sections of placental biopsies of these 7 males and 7 randomly selected, normal male placentas were analyzed immunohistochemically with a polyclonal anti-estradiol-17 , 6-keto antibody. Results: The weak expression of estradiol in the placentas of normal males was localized predominantly at the basal part of the syncytiotrophoblast in the terminal placental villi. In contrast, all placentas of cryptorchid males had strong expression of estradiol at the basal portion of the syncytiotrophoblast. Conclusions: The increased expression of estradiol in the syncytiotrophoblast may have an impact on testicular descent. KEY WORDS: testis, cryptorchidism, placenta, estradiol
Since 1938, the crucial involvement of estradiol in the etiology of experimental cryptorchidism has been well established.1 Maternal consumption of estrogen during the initial 4 months of pregnancy was implicated in cryptorchidism.2, 3 In our study we documented estradiol expression in the syncytiotrophoblast and placenta of males born with cryptorchidism and normal genitalia, respectively. PATIENTS AND METHODS
Males born at the University Obstetric Clinic, Basel between August 1978 and 1979 were examined immediately after birth. All neonates were born within weeks 38 to 40 of gestation and weighed 2,900 to 4150 gm. (table 1). Placental tissue was obtained from the mothers of neonates with cryptorchidism, or retractile or vanishing testes. For comparison placental tissue was obtained from the mothers of 20 newborns with normal genitalia born during the same period. Accepted for publication May 26, 2000.
The birth of all males was without complications and they were normal for gestational age (table 1). At that time the testes were located outside of the scrotum in 17 neonates, of whom 7 had retractile and 2 prescrotal testes. In all cases the testes spontaneously descended during year 1 of life. All boys with cryptorchidism were otherwise healthy and none had spina bifida. Orchiopexy was subsequently done in 6 boys, including 4 with no hormonal treatment before surgery, while the remainder received luteinizing hormone releasing hormone and/or human chorionic gonadotropin. Hormonal therapy induced testicular descent in patient Z. N. (table 2). A vanishing testis was discovered during surgery in 1 boy and none of those with cryptorchidism had panhypopituitarism. Boys with unilateral or bilateral cryptorchidism were reexamined after 1 year and followed until treatment. Newborn placental tissue was fixed in glutaraldehyde and embedded in ePON for immunohistological procedures. Semithin histological sections of placental tissue were ana-
TABLE 1. Pertinent characteristics of cryptorchid and normal males at birth Pt.
Mother Age
Gestational Age (wks.)
Cryptorchid males: ZN 29 40 BM 27 39 TA 24 39 PN 37 38 BMA 22 40 BP 25 40 SA 31 39 Normal males: WA 28 40 OS 21 40 VA 33 40 GC 26 39 CR 30 40 BR 22 40 ST 26 40 In normal boys testes were descended bilaterally.
Parity
Apgar Scores
Birth Wt. (gm.)
Birth Length (cm.)
Undescended Testis Side
1 2 3 2 2 2 1
9, 10, 10 5, 10, 10 9, 10, 10 9, 10, 10 8, 10, 10 7, 8, 10 8, 10, 10
3,620 3,150 3,500 2,960 3,570 3,180 3,110
53 49 51 47 47 49 49
Lt. Bilat. Rt. Rt. Lt. Lt. Lt.
1 1 1 2 2 2 2
9, 10, 10 9, 10, 10 8, 10, 10 9, 9, 10 8, 9, 10 7, 9, 10 9, 10, 10
3,470 3,330 3,100 3,130 4,150 3,080 3,820
50 51 48 47 52 49 52
1694
1695
ELEVATED ESTRADIOL AND CRYPTORCHIDISM TABLE 2. The number of germ cells per seminiferous tubule cross section in males with cryptorchidism at orchiopexy Pt.—Age at Surgery (yrs.)
No. Germ Cells/Tubular Cross Section
BM—1.2 BMA—2 SA—2.5 BP—2.2 TA—4 PN—2
Lt. 0.7, rt. 0.9 0.25 0.29 0.34 0.12 0.10
lyzed immunohistochemically with a commercially available, rabbit anti-estradiol-17, 6-keto polyclonal antibody kit labeled with the antibody, goat antirabbit ␥-globulin conjugated with peroxidase complex. The primary antibody provides positive and specific immunostaining of human estradiol containing cells, such as those of mammary gland carcinoma and ovarian androlslastoma, but it does not cross react with estrone, estriol or other placental hormones. RESULTS
There was a significant decrease in the mean number of germ cells in all boys who underwent orchiopexy (0.33 ⫾ 0.29 germ cells per tubular cross section) (table 2). Fetal spermatogonia were the most numerous germ cells (greater than 50%) and Ad spermatogonia occurred in less than 5%. Most Leydig cells were atrophied. These findings were indicative of typical cryptorchid testis. The terminal placental villi in all 7 cryptorchid males had a normal microscopic anatomical structure. The weak expression of estradiol in the placentas of normal males was localized predominately at the basal part of the syncytiotrophoblast (part A of figure). In contrast, all placentas of cryptorchid males strongly expressed estradiol at the basal portion of the syncytiotrophoblast and around the fetal blood vessels (part B of figure). DISCUSSION
Since 1938, the crucial involvement of estradiol in the etiology of experimental cryptorchidism has been well established.1 Green et al injected estradiol into pregnant rodents, resulting in unilateral or bilateral cryptorchidism in 75% to 100% of male offspring.1, 4 Treatment caused a significant decrease in testicular testosterone and Leydig cell atrophy due to impaired gonadotropin secretion.4, 5 Human chorionic gonadotropin administered with estradiol normalized testosterone secretion, indicating that estradiol mainly affects the hypothalamic pituitary axis.6 Leydig cells in the majority of cryptorchid cases are atrophied as a result of deficient go-
nadotropin secretion.4 –7 This deficiency also impairs the transformation of gonocytes into Ad spermatogonia,5 affecting subsequent sperm production.6 Increased expression of estradiol in the placentas of males with cryptorchidism may indicate a concomitant increase in estradiol in the fetal plasma during gestation. This condition may be similar to that in experiments with female mice treated with estradiol during pregnancy.1, 4 In rodents ␣-fetoprotein (AFP) binds to and effectively sequesters estrogen in the fetal and neonatal circulation.8 AFP in human placenta may serve as a modulator of intracellular estrogen rather than as a strict barrier to hormonal entry.9 Thus, the significant increase of AFP in the placenta of males with cryptorchidism5 may ostensibly control the estradiol level in the fetal circulation, preventing cryptorchidism and feminization of the developing male brain. CONCLUSION
Our finding of increased estradiol expression at the basal part of the syncytiotrophoblast in cryptorchidism may have an impact on testicular descent. It may be responsible for the development of hypogonadotropic hypogonadism, which is present in the majority of cryptorchid cases.5, 10 REFERENCES
1. Green, R. R., Burill, M. W. and Ivy, A. C.: Experimental intersexuality. The production of feminized male rats by antenatal treatment with estrogens. Science, 88: 30, 1938 2. Depue, R. H.: Cryptorchidism, and epidemiologic study with emphasis on the relationship to central nervous system dysfunction. Teratology, 37: 301, 1988 3. Sharpe, R. M. and Skakkebaek, N. E.: Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet, 341: 1392, 1993 4. Hadz˘iselimovic´, F.: Estrogen-induced cryptorchidism. In: Cryptorchidism: Management and Implications. New York: Springer-Verlag, p. 25, 1983 5. Hadz˘iselimovic´, F.: Kryptorchismus. In: Hodenerkrankungen im Kindesalter. Edited by F. Hadz˘iselimovic´ and B. Herzog. Stuttgart: Hippokrates Verlag, p. 24, 1990 6. Hadz˘iselimovic´, F., Herzog, B. and Seguchi, H.: Surgical correction of cryptorchism at 2 years: electron microscopic and morphometric investigations. J Pediatr Surg, 10: 19, 1975 7. Job, J. C., Garnier, P. E., Chaussain, J. L. et al: Effect of synthetic luteinizing hormone-releasing hormone on release of gonadotropins in hypophysogonadal disorders of children and adolescents IV. Undescended testes. J Pediatr, 84: 371, 1974 8. MacLusky, N. J. and Naftolin, F.: Sexual differentiation of the central nervous system. Science, 211: 1294, 1981 9. Toran-Allerand, C. D.: Coexistence of alpha-fetoprotein, albumin and transferrin immunoreactivity in neurones of the developing mouse brain. Nature, 286: 733, 1980 10. Cortes, D.: Cryptorchidism—aspects of pathogenesis, histology and treatment. Scand J Urol Nephrol, suppl., 196: 1, 1998
FIG. 1. Phase contrast photomicrograph of estradiol expression. A, placental tissue from normal male. B, staining is appreciably more intense in basal part of syncytiotrophoblast in males with cryptorchidism. Reduced from ⫻400.
Vol. 164, 1694 –1695, November 2000 Printed in U.S.A.
ELEVATED PLACENTAL ESTRADIOL: A POSSIBLE ETIOLOGICAL FACTOR OF HUMAN CRYPTORCHIDISM ´ R. GENETO F. HADZ˘ISELIMOVIC,
AND
L. R. EMMONS
From the University Clinics, Children’s Hospital, Basel and KinderTagesKlinik, Liestal, Switzerland
ABSTRACT
Purpose: It has been convincingly argued that the increasing incidence of reproductive abnormalities in human males may be associated with increased estrogen exposure during gestation. We documented the expression of estradiol in the syncytiotrophoblast and placenta of males born with cryptorchidism and normal genitalia, respectively. Materials and Methods: Tissue from newborn placentas was fixed in glutaraldehyde and embedded in ePON for immunohistological procedures. Cryptorchidism was unilateral in 6 males and bilateral in 1. Semithin histological sections of placental biopsies of these 7 males and 7 randomly selected, normal male placentas were analyzed immunohistochemically with a polyclonal anti-estradiol-17 , 6-keto antibody. Results: The weak expression of estradiol in the placentas of normal males was localized predominantly at the basal part of the syncytiotrophoblast in the terminal placental villi. In contrast, all placentas of cryptorchid males had strong expression of estradiol at the basal portion of the syncytiotrophoblast. Conclusions: The increased expression of estradiol in the syncytiotrophoblast may have an impact on testicular descent. KEY WORDS: testis, cryptorchidism, placenta, estradiol
Since 1938, the crucial involvement of estradiol in the etiology of experimental cryptorchidism has been well established.1 Maternal consumption of estrogen during the initial 4 months of pregnancy was implicated in cryptorchidism.2, 3 In our study we documented estradiol expression in the syncytiotrophoblast and placenta of males born with cryptorchidism and normal genitalia, respectively. PATIENTS AND METHODS
Males born at the University Obstetric Clinic, Basel between August 1978 and 1979 were examined immediately after birth. All neonates were born within weeks 38 to 40 of gestation and weighed 2,900 to 4150 gm. (table 1). Placental tissue was obtained from the mothers of neonates with cryptorchidism, or retractile or vanishing testes. For comparison placental tissue was obtained from the mothers of 20 newborns with normal genitalia born during the same period. Accepted for publication May 26, 2000.
The birth of all males was without complications and they were normal for gestational age (table 1). At that time the testes were located outside of the scrotum in 17 neonates, of whom 7 had retractile and 2 prescrotal testes. In all cases the testes spontaneously descended during year 1 of life. All boys with cryptorchidism were otherwise healthy and none had spina bifida. Orchiopexy was subsequently done in 6 boys, including 4 with no hormonal treatment before surgery, while the remainder received luteinizing hormone releasing hormone and/or human chorionic gonadotropin. Hormonal therapy induced testicular descent in patient Z. N. (table 2). A vanishing testis was discovered during surgery in 1 boy and none of those with cryptorchidism had panhypopituitarism. Boys with unilateral or bilateral cryptorchidism were reexamined after 1 year and followed until treatment. Newborn placental tissue was fixed in glutaraldehyde and embedded in ePON for immunohistological procedures. Semithin histological sections of placental tissue were ana-
TABLE 1. Pertinent characteristics of cryptorchid and normal males at birth Pt.
Mother Age
Gestational Age (wks.)
Cryptorchid males: ZN 29 40 BM 27 39 TA 24 39 PN 37 38 BMA 22 40 BP 25 40 SA 31 39 Normal males: WA 28 40 OS 21 40 VA 33 40 GC 26 39 CR 30 40 BR 22 40 ST 26 40 In normal boys testes were descended bilaterally.
Parity
Apgar Scores
Birth Wt. (gm.)
Birth Length (cm.)
Undescended Testis Side
1 2 3 2 2 2 1
9, 10, 10 5, 10, 10 9, 10, 10 9, 10, 10 8, 10, 10 7, 8, 10 8, 10, 10
3,620 3,150 3,500 2,960 3,570 3,180 3,110
53 49 51 47 47 49 49
Lt. Bilat. Rt. Rt. Lt. Lt. Lt.
1 1 1 2 2 2 2
9, 10, 10 9, 10, 10 8, 10, 10 9, 9, 10 8, 9, 10 7, 9, 10 9, 10, 10
3,470 3,330 3,100 3,130 4,150 3,080 3,820
50 51 48 47 52 49 52
1694
1695
ELEVATED ESTRADIOL AND CRYPTORCHIDISM TABLE 2. The number of germ cells per seminiferous tubule cross section in males with cryptorchidism at orchiopexy Pt.—Age at Surgery (yrs.)
No. Germ Cells/Tubular Cross Section
BM—1.2 BMA—2 SA—2.5 BP—2.2 TA—4 PN—2
Lt. 0.7, rt. 0.9 0.25 0.29 0.34 0.12 0.10
lyzed immunohistochemically with a commercially available, rabbit anti-estradiol-17, 6-keto polyclonal antibody kit labeled with the antibody, goat antirabbit ␥-globulin conjugated with peroxidase complex. The primary antibody provides positive and specific immunostaining of human estradiol containing cells, such as those of mammary gland carcinoma and ovarian androlslastoma, but it does not cross react with estrone, estriol or other placental hormones. RESULTS
There was a significant decrease in the mean number of germ cells in all boys who underwent orchiopexy (0.33 ⫾ 0.29 germ cells per tubular cross section) (table 2). Fetal spermatogonia were the most numerous germ cells (greater than 50%) and Ad spermatogonia occurred in less than 5%. Most Leydig cells were atrophied. These findings were indicative of typical cryptorchid testis. The terminal placental villi in all 7 cryptorchid males had a normal microscopic anatomical structure. The weak expression of estradiol in the placentas of normal males was localized predominately at the basal part of the syncytiotrophoblast (part A of figure). In contrast, all placentas of cryptorchid males strongly expressed estradiol at the basal portion of the syncytiotrophoblast and around the fetal blood vessels (part B of figure). DISCUSSION
Since 1938, the crucial involvement of estradiol in the etiology of experimental cryptorchidism has been well established.1 Green et al injected estradiol into pregnant rodents, resulting in unilateral or bilateral cryptorchidism in 75% to 100% of male offspring.1, 4 Treatment caused a significant decrease in testicular testosterone and Leydig cell atrophy due to impaired gonadotropin secretion.4, 5 Human chorionic gonadotropin administered with estradiol normalized testosterone secretion, indicating that estradiol mainly affects the hypothalamic pituitary axis.6 Leydig cells in the majority of cryptorchid cases are atrophied as a result of deficient go-
nadotropin secretion.4 –7 This deficiency also impairs the transformation of gonocytes into Ad spermatogonia,5 affecting subsequent sperm production.6 Increased expression of estradiol in the placentas of males with cryptorchidism may indicate a concomitant increase in estradiol in the fetal plasma during gestation. This condition may be similar to that in experiments with female mice treated with estradiol during pregnancy.1, 4 In rodents ␣-fetoprotein (AFP) binds to and effectively sequesters estrogen in the fetal and neonatal circulation.8 AFP in human placenta may serve as a modulator of intracellular estrogen rather than as a strict barrier to hormonal entry.9 Thus, the significant increase of AFP in the placenta of males with cryptorchidism5 may ostensibly control the estradiol level in the fetal circulation, preventing cryptorchidism and feminization of the developing male brain. CONCLUSION
Our finding of increased estradiol expression at the basal part of the syncytiotrophoblast in cryptorchidism may have an impact on testicular descent. It may be responsible for the development of hypogonadotropic hypogonadism, which is present in the majority of cryptorchid cases.5, 10 REFERENCES
1. Green, R. R., Burill, M. W. and Ivy, A. C.: Experimental intersexuality. The production of feminized male rats by antenatal treatment with estrogens. Science, 88: 30, 1938 2. Depue, R. H.: Cryptorchidism, and epidemiologic study with emphasis on the relationship to central nervous system dysfunction. Teratology, 37: 301, 1988 3. Sharpe, R. M. and Skakkebaek, N. E.: Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet, 341: 1392, 1993 4. Hadz˘iselimovic´, F.: Estrogen-induced cryptorchidism. In: Cryptorchidism: Management and Implications. New York: Springer-Verlag, p. 25, 1983 5. Hadz˘iselimovic´, F.: Kryptorchismus. In: Hodenerkrankungen im Kindesalter. Edited by F. Hadz˘iselimovic´ and B. Herzog. Stuttgart: Hippokrates Verlag, p. 24, 1990 6. Hadz˘iselimovic´, F., Herzog, B. and Seguchi, H.: Surgical correction of cryptorchism at 2 years: electron microscopic and morphometric investigations. J Pediatr Surg, 10: 19, 1975 7. Job, J. C., Garnier, P. E., Chaussain, J. L. et al: Effect of synthetic luteinizing hormone-releasing hormone on release of gonadotropins in hypophysogonadal disorders of children and adolescents IV. Undescended testes. J Pediatr, 84: 371, 1974 8. MacLusky, N. J. and Naftolin, F.: Sexual differentiation of the central nervous system. Science, 211: 1294, 1981 9. Toran-Allerand, C. D.: Coexistence of alpha-fetoprotein, albumin and transferrin immunoreactivity in neurones of the developing mouse brain. Nature, 286: 733, 1980 10. Cortes, D.: Cryptorchidism—aspects of pathogenesis, histology and treatment. Scand J Urol Nephrol, suppl., 196: 1, 1998
FIG. 1. Phase contrast photomicrograph of estradiol expression. A, placental tissue from normal male. B, staining is appreciably more intense in basal part of syncytiotrophoblast in males with cryptorchidism. Reduced from ⫻400.