Surgical and genetic aspects of persistent müllerian duct syndrome

Surgical

and Genetic Aspects

of Persistent

Miillerian

Duct Syndrome

By Deborah S. Loeff, Sandrine Imbeaud,

Hernan M. Reyes, Janet L. Meller, and Ira M. Rosenthal Chicago, Illinois and Montrouge, France

0 Persistent miillerian duct syndrome (PMDS) is characterized by the presence of a uterus, cervix, and fallopian tubes in an otherwise normally differentiated 46,XY male. During embryogenesis, regression of miillerian structures in normal males is mediated by antimiillerian hormone (AMH), also called miillerian inhibiting substance (MIS), produced by fetal Sertoli’s cells. PMDS has been attributed to deficient AMH activity or to abnormalities in the AMH receptor. The authors report on two patients with PMDS in whom the abnormalities were discovered during surgery for inguinal hernia and cryptorchidism. During the initial operations in each case, testicular biopsies were obtained, and the gonads and miillerian elements were replaced in the pelvis. A second operative procedure, performed several months later, included proximal salpingectomies with dissection of the vasa deferentia on pedicles of myometrium. This permitted excision of the vestigial uterine corpus, leaving a tiny remnant of cervix with the vasa deferentia. The testes were further mobilized so that bilateral orchidopexies could be completed. In the first case, a molecular abnormality was present at position 377 of the first exon of the AMH gene. Thymine replaced cytosine, which altered a CGG arginine codon to a TGG tryptophan codon, rendering the AMH molecule unstable. The molecular abnormality in the first case differs from the first abnormality in AMH reported by Knebelmann et al, thus indicating heterogeneity in this condition. The molecular basis for deficient AMH activity in the second patient has not yet been defined. No molecular abnormalities were found in the exons of this patient’s AMH gene. Copyright ~11994 by W. 6. Saunders Company INDEX WORDS: Miillerian duct structures; hernia uteri inguinalis; cryptorchidism; antimiillerian hormone; miillerian inhibiting substance.

deficiency of AMH activity or by an abnormality in its receptor. Purification of AMH, cloning of its gene, development of enzyme-linked immunosorbant assay (ELBA) tests for its presence in serum, and localization of the gene to chromosome 19 have further clarified the pathogenesis of PMDS.7-” It has been shown that in males, serum levels of AMH measured by ELBA technique are fairly high until 2 years of age. Measurable levels persist until puberty and then become undectectable. Moreover, in several siblings with PMDS and deficient AMH, a molecular defect in the AMH gene was reported by Knebelmann et ai,’ characterized by a stop codon in the fifth exon. We previously reported three cases of PMDS, including two cases in monovular twins and one case with transverse testicular ectopia.“‘,” Herein we report two additional cases and discuss the current approach to diagnosis and surgical management, as well as the genetic aspects of this syndrome. CASE

Case I A Z-month-old

P

JournalofPediatrrc

Surgery,

Vol29,

No 1 (January),

1994: pp 61-65

black male was found

sided cryptorchidism

associated

and scrotum

were

normal

related,

there

were

and

fallopian when

tubes,

traction

biopsies

were

were replaced

ERSISTENT miillerian duct syndrome (PMDS) is an uncommon form of male pseudohermaphroditism characterized by the presence of a uterus and fallopian tubes in an otherwise differentiated male with a 46,XY karyotype.‘-j Cases are usually discovered during surgery for an inguinal hernia or cryptorchidism, or by the presence of transverse testicular ectopia. Approximately 150 cases have been recorded, many in adults; and a familial association has been found in some cases. With the development of early surgery for inguinal hernia and undescended testes, more cases are being diagnosed in young children. This necessitates knowledge of PMDS, including pathogenesis, genetics, anatomic variations and options for surgical management. Antimiillerian hormone (AMH) or miillerian inhibiting substance (MIS), produced by fetal testicular Sertoli’s cells, is responsible for the involution of embryonic miillerian structures in normal males.‘,” It is evident that PMDS can be caused by either a

REPORTS

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Pediutric.s,

C’ook C’otcnt~

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Departments

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December 2. 1992.

reprint Department

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61

62

LOEFF ET AL

persistent miillerian duct structures when the hernia sac and left testis were mobilized. The first operation was similar to that performed in case 1. As with the first case, chromosome studies on peripheral blood lymphocytes showed a 46,XY karyotype, and testicular biopsies showed normal histology for age. AMH serum activity was markedly depressed. Postoperatively. pelvic ultrasound again demonstrated the vestigial uterus. At IS months of age, definitive surgery was performed as in the first case, and the recovery period was uncomplicated. A testicular biopsy showed marked diminution in AMH activity. Studies for the molecular basis of the defect in AMH in this patient showed no mutation in the entire length of the gene. A promoter mutation affecting the level of transorption is a possible explanation for the patient’s very low serum AMH level.

DISCUSSION

Fig 1. A 2-month-old boy (case I) with testes, a uterus, and fallopian tubes found during a right inguinal herniorraphy.

Partial excision of the uterine fundus was performed, leaving the vasa deferentia intact on thin pedicles of myometrium. The vasa deferentia were easily separated to the level of the cervix, and the gonadal vessels were mobilized, permitting bilateral orchidopexies. In snap-frozen testicular tissue, no immunoreactive AMH was apparent by immunocytochemistry.r~ The patient had an uneventful recovery. Studies designed to detect the molecular basis for PMDS were performed on snap-frozen testicular biopsy material. Southern blots of genomic DNA were performed; Sl nuclease mapping of the initiation of the transcription of AMH as well as amplification of genomic DNA were accomplished as described by Knebelmann et al.’ On Southern blots of genomic DNA, no abnormality was noted, suggesting that a point mutation, minideletion, or insertion was responsible for PMDS in this AMH-negative patient. Normal results were found with Sl nuclease mapping of the initiation of transcription of AMH, with the major initiation site found 10 bases before the ATG codon; three upstream minor sites were also noted. Amplification of genomic DNA and subsequent treatment of the amplification product for the purpose of detection of single-strand conformation polymorphism resulted in the findings of abnormal migration in some Stul and Cfol digested fragments of the first amplified fragment. This fragment contains the first exon and part of the first intron. Cloning of the abnormal fragments and sequencing gave the following similar results in one sense and six antisense strands. At position 1.56, the thymine in the ATC codon producing an isoleucine is changed to a guanine, producing an AGC serine codon. At position 377, cytosine is replaced by a thymine, changing a CGG arginine codon to a TGG tryptophan. Direct sequencing of the product of polymerase chain reaction gave identical results. Prediction of secondary structure of normal and mutated AMH indicated that the mutation that changes an arginine to a tryptophan residue changes the predicted structure from an alpha helix to a beta strand, resulting in instability of the AMH molecule. In contrast, substitution of serine for isoleucine does not modify the predicted structure and is a silent mutation.

Case 2 An 1 l-month-old Mexican-American boy underwent surgery for a left inguinal hernia and bilateral undescended testes. The parents of this child were not related. He was found to have

Mtillerian structures are present in male fetuses until the eighth week of gestation. Mullerian duct regression in males is mediated by antimullerian hormone (AMH), also called miillerian inhibiting substance (MIS) or miillerian inhibiting factor (MIF). AMH is a glycoprotein produced by fetal Sertoli’s cells. The human gene for AMH has been mapped to chromosome 19.9 It has been cloned, contains five exons, and codes for a protein consisting of 560 amino acids.7 Absent or abnormal AMH as well as defects in its receptor causes persistence of mullerian duct structures in male fetuses. In consideration of the differential diagnosis of PMDS, one must take into account the normal mechanisms of male sexual differentiation, which are driven by discrete fetal testicular hormones.‘” Testosterone causes the virilization of Wolffian ducts into epididymes, vasa deferentia, and seminal vesicles. Formation of the urogenital sinus and male external genitalia require the in situ conversion of testosterone to dihydrotestosterone.Ih Persistence of mullerian duct derivatives in other forms of male pseudohermaphrodism is uncommon. I7 In some cases, in addition to a deficiency in AMH, there is a lack of testosterone and dihydrotestosterone, with effects characterized by micropenis, hypospadias, and often the presence of the inferior portion of the vagina. I8These individuals frequently have a 45,X/46,XY mosaic karyotype. Patients with this type of testicular dysgenesis have an increased risk of gonadal neop1asia.i”~” In contrast, PMDS is characterized by a normal 46,XY karyotype and normal masculinization of external genitalia. The presence and duration of cryptorchidism will affect otherwise normal testicular histology. Testicular malignancy has been described in only one case of PMDS.‘O Routine castration is not recommended; however, careful monitoring of the testes for neoplasia is indicated (as with any child) after orchidopexy.

MANAGEMENT

63

OF MtiLLERlAN DUCT SYNDROME

There are three anatomic variants found in patients with PMDS. In the most common “male” type, also called “hernia uteri inguinalis,” one testis is usually found within the scrotum. The uterus and ipsilateral fallopian tube are either in the inguinal canal or can be brought into it by gentle traction on the presenting testis. In some cases, the contralateral testis and tube are also in the hernia sac. Transverse testicular ectopia also occurs in PMDS.“%“J’ In fact, the presence of transverse testicular ectopia should alert the surgeon to the strong possibility of PMDS, because it is the most common cause of this abnormality. The least common form, or “female” type, is characterized by bilateral cryptorchidism with testes embedded in the broad ligaments in an “ovarian” position with respect to the uterus, which is fixed in the pelvis.” The vasa deferentia are intimately adherent to the lateral walls of the uterus and course along the cervix in all three anatomic forms of this syndrome. Correct surgical management of PMDS requires recognition of the condition by the surgeon and confirmation with testicular biopsies and chromosomal studies. Because PMDS is usually discovered incidentally during surgery for undescended testis or inguinal hernia, the initial procedure includes testicuIar biopsies, herniorraphy, and replacement of the gonads and miillerian structures within the pelvis. After confirmation of the diagnosis of PMDS, definitive surgery should be performed to remove the corpus of the uterus and fallopian tubes, enabling fixation of the testes in the scrotum. Previous recommendations for surgery included complete removal of the mullerian structures, with obligatory vasectomy, because the vasa deferentia course parallel to and within the lateral uterine wallsz4 We believe that every effort should be made to preserve fertility and hormone function, especially in patients less than 2 years of age. The optimal surgical approach was originally proposed by GUeJJkJ et al.‘” Preservation of the mullerian derivatives is incompatible with successful orchidopexies because the testes and vasa deferentia are tethered to the uterus. Furthermore, with sexual maturation, the uterus may hypertrophy and cause discomfort, or may present as a mass whose origin is unknown.” The preferred operation for this syndrome includes proximal salpingectomies, leaving the fimbriae with the epididymes, thus releasing the testes (Fig 2). The vasa deferentia are then dissected from the lateral uterine walls and left intact with pedicles of myometrium. After this, a corporeal hysterectomy is performed, with or without removal of the vestigial cervix (Fig 3). Preservation of the tiny

Fig 2. Definitive surgical management of PMDS. Proximal salpingectomies are performed, leaving the fimbriae with the testes. The vasa deferentia are dissected and left intact with pedicles of myometrium.

cervix lessens the chance of injury to the vasa deferentia; however, it may be transected longitudinally to fully separate the vasa deferentia and facilitate orchidopexies. Further mobilization of the gonadal blood vessels may be necessary for completion of the orchidopexies. We believe the female type of PMDS can be corrected in a manner similar to that of the usual male type; however, correction of the female

Fig 3. Excision of the uterine fundus, with preservation vestigial cervix in preparation for bilateral orchidopexies.

of the

64

LOEFF ET AL

type requires careful division of the vestigial broad and round ligaments. In patients with transverse testicular ectopia, after proximal salpingectomies and corporeal hysterectomy, the ectopic testis may be brought through the median raphe of the scrotum and fixed in the contralateral side. PMDS is a genetic disorder. The cases of 12 sets of siblings (including a pair of monovular twins) have been reported. l”~loHowever, as previously noted, it appears that the positions of the uterus, fallopian tubes, and testes are not genetically determined because, in some families, male and female types have been found in brothers.?” There are two categories of PMDS: AMH-negative cases, in which serum and testicular levels of AMH are either nondetectable or extremely low; and AMH-positive cases, in which AMH concentrations are normal. In the latter cases, the defect presumably is in the AMH receptor or in postreceptor activity. The mode of transmission of PMDS has been somewhat obscure because AMHpositive and AMH-negative cases formerly were lumped together. Most cases are compatible with the autosomal recessive male-restricted transmission expected in a condition with a mutation in an autosomal chromosome. The apparent sex-linked transmission reported in two families is surprising.‘s.?h The serum AMH levels of these families are unknown. X-linked transmission could be explained in AMH-positive PMDS if the gene for the AMH receptor were located on the X chromosome. Contrary to this hypothesis is the fact that in dogs, AMH-positive PMDS is transmitted as an autosomal recessive trait.” One would expect X-linkage to be common of both species. Knowledge of the biochemical structure of AMH and cloning of its gene, combined with recent advances in molecular biology techniques, have permitted investigation of the molecular basis of AMHnegative PMDS. Among the possibilities to be considered are large gene deletions or insertions and point mutations affecting gene regulation, transcription, or translation. It should be noted that point mutation, microdeletion, or insertion are not detected by Southern blot analysis.‘8 A major advance in AMH-negative PMDS was the report by Knebelmann et al’ of a mutation in the AMH gene in one family. A

guanine-to-thymine transversion in the fifth exon of the AMH gene changed a triplet coding for glutamic acid to a stop codon that arrests translation. In addition, there was a silent mutation coding for glycine. The truncated AMH protein could be visualized in the culture media of transfected Chinese hamster cells. In our first case, two point mutations were detected. At position 377 in the first exon, a cytosine-tothymine mutation changed an arginine triplet to one encoding for tryptophan. We called this mutation AMH Arg 123 + Trp, numbering amino acid residues from the initating methionine. This is a nonconservative change. Arginine is a polar aminoacid with a preference for the protein surface, whereas tryptophan, an aromatic hydrophobic residue, is a major component of the hydrophobic core within the protein.ZN The mutation apparently changed the predicted secondary structure of the molecule by affecting its foiding, and thus its stability. In contrast, the change of isoleucine to serine at position 156 did not modify predicted structure. This could be anticipated because bovine AMH also carries serine at this position.’ It is possible that this change represents variation in the normal active gene at this site, which ordinarily would be silent. It is likely that the change is a silent mutation representing variation in the normal active gene at this site. In our second case, no mutation was found in the entire length of the AMH gene. A mutation or deletion in the promoter is a possibility and will be explored. Discovery of other mutations in the AMH gene in AMH-negative PMDS is anticipated. Because of the rarity of PMDS, one would expect to find homozygous mutations only in inbred families. With two different AMH gene mutations in the offspring of nonrelated parents, compound heterozygotes would be anticipated. Suprisingly, case 1 appears to be homozygous, although consanguinity between the parents was denied. Studies of AMH using the techniques of molecular biology and modern genetics are clarifying the pathogenesis of AMH-negative PMDS. Understanding relationships between structure and function afforded by the study of additional AMH mutations should help to elucidate the basic structure responsible for the biological activity of AMH.

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MANAGEMENT

nature

OF MtiLLERlAN

of the hormone

65

DUCT SYNDROME

and of its disultide-bonded

structure.

Mol

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