Familial gonadotropin-releasing hormone resistance and hypogonadotropic hypogonadism in a family with multiple affected individuals.

OBJECTIVE: To characterize the phenotype of idiopathic hypogonadotropic hypogonadism due to compound heterozygous GnRHR gene mutations (Arg262Gln/Tyr284Cys). DESIGN: Retrospective review. SETTING: Tertiary medical center. PATIENT(S): Family containing four siblings (three female and one male) with complete idiopathic hypogonadotropic hypogonadism. INTERVENTION(S): Baseline and stimulated laboratory studies. One patient received GnRH treatment and one received human menopausal gonadotropins. MAIN OUTCOME MEASURE(S): Clinical phenotype vs. genotype is assessed by endocrine studies, karyotype, pedigree, and review of pathology slides of ovarian neoplasm. RESULT(S): With GnRH stimulation, two patients with idiopathic hypogonadotropic hypogonadism had maximum LH < 10 mIU/mL, and two others had peak LH > 10 mIU/mL. With repeated GnRH stimulation 24 hours later, gonadotropin levels in all patients were increased. Stimulation of thyroid-releasing hormone and tests for insulin-induced hypoglycemia were normal. One affected patient did not ovulate after GnRH treatment, but her sister ovulated with gonadotropin treatment. Another affected sibling had bilateral oophorectomy for seromucinous cystadenomas, and her hypogonadotropic state remained after castration. The man with idiopathic hypogonadotropic hypogonadism and his unaffected brother had a ring chromosome 21. CONCLUSION(S): All patients with complete idiopathic hypogonadotropic hypogonadism had the same GnRHR mutations, but clinical presentations and endocrinologic responses were heterogeneous. Gonadotropin levels remained low in patients with idiopathic hypogonadotropic hypogonadism after castration, and ring chromosome 21 was present, suggesting that sequences from this chromosome could affect the idiopathic hypogonadotropic hypogonadism phenotype.

Pelvic adhesion formation after intraperitoneal installation of gallstones in a rabbit model.

OBJECTIVE: To investigate whether intraperitoneal gallstones increase the risk of pelvic adhesions in a rabbit model. DESIGN: Prospective, randomized, blinded, sham and human antigen controlled trial. SETTING: An academic research environment. SUBJECT(S): Twelve New Zealand white rabbits. INTERVENTION(S): Twelve rabbits were divided into three groups of four each; a sham operation group, a gallstone and bile group (study group), and a human serum albumin and bile group (antigenic control). Three weeks after the operation individual subjects were randomized, with groups concealed to observers, and a necropsy was performed on each rabbit. MAIN OUTCOME MEASUREMENT(S): Necropsy was performed on each rabbit, and the adhesions were scored for extent, type, tenacity, inflammation, and gallstone involvement. RESULT(S): There was a statistically and biologically significant increase in gallstone involvement in adhesions, especially pelvic adhesions, in the study group. CONCLUSION(S): This study, along with an increasing number of case reports, suggests that gallstones inadvertently left in the peritoneal cavity may increase the morbidity of laparoscopic cholecystectomy. In females of reproductive age these gallstones may induce pelvic adhesions that may interfere with fertility or be associated with pelvic pain.

Nonfunctioning pituitary macroadenoma presenting with mild hyperprolactinemia and amenorrhea.

OBJECTIVE: To describe a patient with a clinically nonfunctioning pituitary macroadenoma who presented with mild hyperprolactinemia and amenorrhea. DESIGN: Case report. SETTING: Tertiary care medical facility. PATIENT(S): A 44-year-old woman with a 6-month history of amenorrhea. INTERVENTION(S): Pituitary testing, magnetic resonance imaging of the sella turcica, and transsphenoidal surgery. MAIN OUTCOME MEASURE(S): Pituitary function testing, magnetic resonance imaging, and return of menstrual cycles. RESULT(S): Baseline laboratory data revealed a serum prolactin level of 34 ng/mL (normal range, 3-20 ng/mL), normal thyroid function test results, and an FSH level of 6.7 mIU/mL. A second fasting prolactin level was 48 ng/mL. Magnetic resonance imaging of the sella turcica revealed a pituitary macroadenoma measuring 1.4 x 3.2 cm. Further testing of baseline pituitary function revealed normal findings. The patient underwent an uncomplicated transsphenoidal resection of the pituitary tumor and maintained normal pituitary function. Pathologic evaluation revealed a pituitary adenoma that stained positive for FSH and focally for the alpha subunit. The adenoma stained negative for GH, prolactin, ACTH, LH, and TSH. CONCLUSION(S): This patient had a nonsecreting gonadotroph macroadenoma that resulted in hypogonadotropic hypogonadism along with mild hyperprolactinemia, presumably secondary to interruption of normal transport down the pituitary stalk.

Steroid 5alpha-reductase 2 gene melting polymorphisms in male subjects with azoospermia or oligospermia.

OBJECTIVE: This study was designed to test the hypothesis that mutations in the gene for type 2 steroid 5alpha-reductase (SRD5A2) may be the cause of a phenotype characterized primarily by oligospermia or azoospermia. STUDY DESIGN: Deoxyribonucleic acid from control subjects and subjects with oligospermia (n = 12) and azoospermia (n = 6) were evaluated for mutations in SRD5A2. Methods used for mutation analysis included polymerase chain reaction, Southern blotting, and denaturing gradient gel electrophoresis. RESULTS: Denaturing gradient gel electrophoresis of all 5 amplified exons resulted in similar migration patterns in samples from both control and study subjects. Genomic deoxyribonucleic acid subjected to denaturing gradient gel electrophoresis after restriction digest revealed melting polymorphisms. Direct sequencing of the gene in a single patient with a unique melting polymorphism yielded a normal sequence. CONCLUSIONS: Melting polymorphisms for SRD5A2 were detected in a group of patients with oligospermia or azoospermia. Sequence analysis did not demonstrate functional mutations in the coding sequence of this gene.

Androgens and bone health.

Osteoporosis is one of the most common metabolic bone diseases in the adult population and its prevalence will continue to rise as our population grows older. In both sexes, hypogonadism is associated with accelerated loss of bone and development of osteoporosis. Adrenal and gonadal androgen levels decline with advancing age in both sexes. Androgens act by either directly binding to androgen receptors, or by aromatization of androgens to estrogens and subsequently interacting with estrogen receptors. Both pathways are important for skeletal health. Direct androgen binding to an androgen receptor may play a more important role in early skeletal development and determination of sexual dimorphic traits. While bone remodeling, which is important in maintaining healthy bone through life, is primarily stimulated by estrogen, studies in the rat and human support the complex action of androgens and estrogens in bone modeling and remodeling, and hence the development and maintenance of healthy bone. In postmenopausal females, the addition of androgens to hormone replacement therapy results in significant additional improvement in bone mineral density compared to estrogen replacement alone. Accumulating evidence indicate that androgens play an important role in the health of bone and the potential benefit of adding these agents to hormone replacement regimens.

Human chorionic gonadotrophin-beta gene sequences in women with disorders of HCG production.

Women with recurrent abortion, primary unexplained infertility, and gestational trophoblastic neoplasia (GTN) manifest disordered human chorionic gonadotrophin (HCG) secretion. Mutations in the HCG beta/luteinizing hormone (LH) beta gene complex could cause aberrant HCG production in these disorders. The purpose of this study was to determine whether HCG beta gene deletions occur in women with recurrent abortion or primary unexplained infertility, and whether HCG beta gene duplications are present in women with GTN. DNA was extracted from 10 patients with unexplained recurrent abortion, 10 patients with unexplained primary infertility, 12 patients with GTN, three partners of women with GTN, and 30 controls. Southern blots were constructed and hybridized with DNA probes for HCG beta-5 and the LH beta gene. No gene deletions were identified in patients with recurrent abortion or primary unexplained infertility. Likewise, no gene duplications were identified in women with GTN. A previously described Mbol restriction fragment length polymorphism (RFLP) was identified in both patients and controls. A new Pstl RFLP was also characterized, but was present in patients and controls. Deletion/duplication mutations in the HCG beta/LH beta gene complex do not appear to be common causes of aberrant HCG production in humans with these disorders.

Paternal somatic and germ-line mosaicism for a sex-determining region on Y (SRY) missense mutation leading to recurrent 46,XY sex reversal.

OBJECTIVE: To determine the etiology for recurrent 46,XY sex reversal in a family with two Swyer siblings. DESIGN: Deoxyribonucleic acid (DNA) from peripheral lymphocytes and sperm were analyzed for duplication of the dosage sensitive sex locus (DSS) and for mutations in sex-determining region on Y (SRY). SETTING: An academic teaching hospital. PATIENTS: A family consisting of mother, father, and five phenotypic daughters, of which two were 46,XY sex-reversed females. INTERVENTION: Deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR), Southern blotting, dosage densitometry, single-strand conformation polymorphism (SSCP), and sequencing. MAIN OUTCOME MEASURE: Comparison of control and subject DNA. RESULTS: Deoxyribonucleic acid (DNA) analysis of SRY in genomic DNA from the 46,XY sex-reversed siblings revealed identical missense mutations (T-->G) in both sisters. Analysis of the SRY gene in paternal lymphocyte and sperm DNA revealed mosaicism for wild and mutant (T-->G) SRY sequences. SRY analysis of sperm DNA also demonstrated the same mosaicism for the T-->G missense mutation. CONCLUSION: A postembryonic SRY mutation gave rise to paternal mosaicism for two distinct cell populations (SRY+/SRY-). The presence of a wild type SRY in the somatic cell line may account for a normal pattern of male sexual differentiation, whereas the presence of a mutated SRY in the germ line resulted in two 46,XY sex-reversed offspring. These results confirm a proposed mechanism for the condition of recurrent 46,XY sex-reversed females.

Massive ovarian enlargement in primary hypothyroidism.

OBJECTIVE: To report a case of ovarian cyst formation and myxedematous infiltration of the ovary in a subject with primary hypothyroidism. DESIGN: Retrospective case report. SETTING: University hospital. PATIENT(S): A 16-year-old female adolescent with pelvic pain, galactorrhea, irregular menses, and ovarian cysts on pelvic examination. INTERVENTION(S): Laparotomy with bilateral ovarian wedge resection and thyroid replacement therapy. MAIN OUTCOME MEASURE(S): Ovarian histopathology, thyroid function tests, and menstrual history. RESULT(S): Resolution of patient's pain, galactorrhea, and resumption of normal menses. CONCLUSION(S): Ovarian cyst formation may accompany primary hypothyroidism in the child with accelerated or delayed sexual maturation. To date, the underlying pathophysiology of the morphological changes in the ovary remain enigmatic. This case report provides the first insight into the actual histologic changes that occur in ovaries of subjects with primary hypothyroidism without secondary ovarian pathology such as torsion. There is clear evidence of myxedematous infiltration into the ovarian stroma without luteinization of the theca interna. These microscopic findings suggest that local changes occurring independent of gonadotropin stimulation may contribute significantly to altered morphology of the ovaries in primary hypothyroidism.

Ovarian cholelithiasis after laparoscopic cholecystectomy associated with chronic pelvic pain.

OBJECTIVE: To report a case of chronic pelvic pain associated with ovarian cholelithiasis and discuss prevention and management of this condition. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 39-year-old woman who presented with right lower abdominal pain after a laparoscopic cholecystectomy. INTERVENTION(S): Diagnostic laparoscopy followed by laparotomy with lysis of adhesions and removal of three to four dozen gallstones. MAIN OUTCOME MEASURE(S): Patient's subjective report of pain. RESULT(S): Resolution of patient's pain. CONCLUSION(S): Gallstones spilled into the peritoneal cavity may migrate and adhere to the dependent portions of the pelvis, potentially resulting in pelvic pain or infertility. This suggests the importance of removing inadvertently spilled gallstones at the time of surgery or using nonsurgical methods of gallstone management in reproductive-aged females.

Follicle-stimulating hormone beta gene structure in premature ovarian failure.

OBJECTIVE: To determine whether mutations in the gene for FSH beta are present, and possibly etiologic, in some patients with 46,XX premature ovarian failure (POF). DESIGN: DNA samples obtained from 18 study patients with POF and two menopausal fertile controls were studied by Southern blot analysis. DNA sequencing was performed in one patient. SETTING: Patients were seen in a reproductive endocrinology clinic and studied in a medical school laboratory setting at the Medical College of Georgia and Tufts University. MAIN OUTCOME MEASURES: Restriction fragment sizes on autoradiographs were compared between the study group and controls. DNA sequencing radiographs were compared between one study patient and five controls. RESULTS: Fragment sizes obtained with the restriction enzymes EcoRI, DraI, HincII, PstI, KpnI, BglI, BamHI, and BglII were similar size in both study subjects and controls using the probes pFSH beta-1.4 and pFSH beta-1.2. A previously described HindIII polymorphism was present using pFSH beta-1.2, but HindIII fragment sizes were identical in patients with ovarian failure and controls using pFSH beta-1.4. DNA sequencing of the FSH beta gene in one patient was normal. CONCLUSIONS: No mutations in the gene for FSH beta were identified in women with POF. DNA sequencing of the exons and promoter region of the FSH beta gene in one woman with POF was normal. This does not entirely exclude the possibility that smaller deletions, insertions, or point mutations of the FSH beta could be etiologic in some women with POF. The HindIII polymorphism does not appear to segregate with 46,XX POF.

Absence of the testicular determining factor gene SRY in XX true hermaphrodites and presence of this locus in most subjects with gonadal dysgenesis caused by Y aneuploidy.

OBJECTIVES: The purpose of our study was to discover whether the testicular determining factor gene SRY (sex-determining region on Y) is present or absent in XX true hermaphrodites and in subjects with gonadal dysgenesis caused by Y aneuploidy. STUDY DESIGN: We screened five XX true hermaphrodites and 24 subjects with gonadal dysgenesis caused by Y aneuploidy for the presence or absence of SRY. With the polymerase chain reaction technique, the sequence coding the 80 amino acid-conserved motif was amplified. The 0.9 kb Hincll pY53.3 subclone, which covers the open reading frame of SRY, serves as a probe for Southern blot analysis. RESULTS: Test results for all five XX true hermaphrodites were negative for SRY. Conversely, 22 of the 24 individuals with 45,X/46,XY gonadal dysgenesis were positive for SRY, including the 10 subjects with only bilateral streak gonads. CONCLUSIONS: The absence of SRY in XX true hemaphrodites and the presence of SRY in 10 subjects with 45,X/46,XY constitution who harbored only bilateral streak gonads seem to indicate that multiple genes are involved in gonadal differentiation.

Gonadotropin-releasing hormone-associated peptide gene sequences in women with hyperprolactinemia.

OBJECTIVE: To determine if mutations in the structural gene for gonadotropin-releasing hormone (GnRH)-associated peptide are present in women with hyperprolactinemia. DESIGN: Patients with hyperprolactinemia and controls were studied retrospectively for GnRH-associated peptide gene mutations. SETTINGS: Patients seen in a clinical setting were studied at a medical school laboratory setting. PATIENTS: Fifteen women with hyperprolactinemia and two fertile controls with normal prolactin levels were studied. INTERVENTIONS: Genomic deoxyribonucleic acid (DNA) was extracted from each patient and subjected to Southern blot analysis and polymerase chain reaction (PCR). For Southern blot analysis, DNA was digested with EcoRI, XbaI, BglII, PstI, and BamHI and hybridized to two DNA probes for GnRH-associated peptide. Exons II to IV, which encode for the structural gene, were amplified by PCR. MAIN OUTCOME MEASURES: Fragment sizes from autoradiographs were compared among patients and controls. Amplified PCR products of exons II to IV of the GnRH-associated peptide were also compared. RESULTS: No large deletions, insertions, or polymorphisms were identified in women with hyperprolactinemia or controls by Southern blotting. Each of the exons was present and of normal size by PCR in the study patients and controls. CONCLUSIONS: No large deletions of the GnRH-associated peptide gene appear to be present in our patients with hyperprolactinemia. Small deletions, insertions, or point mutations are not excluded by this analysis.

Etiologic factors of recurrent abortion and subsequent reproductive performance of couples: have we made any progress in the past 10 years?

OBJECTIVES: We hypothesize that the diagnostic yield and pregnancy outcomes of patients with recurrent abortion have improved over the past 10 years. STUDY DESIGN: The study was performed in an academic medical center. Diagnoses and outcomes for group A, a published series of 100 patients investigated for recurrent abortion in the section between 1968 and 1977, was compared with those for group B, the 131 patients seen between 1987 and 1991. A standardized protocol was followed, enhanced by new techniques and autoimmune investigations in the latter group. Results were compiled retrospectively. Descriptive statistics and chi 2 analysis were used. RESULTS: No cause could be found in 37% of patients in group A compared with 24% of couples in group B (p less than 0.05). No clear difference could be shown in the subsequent outcomes of pregnancies. CONCLUSIONS: Our ability to establish a cause of recurrent abortion has improved slightly over the past 15 years. The gain is not yet reflected in successful pregnancy rates. Multicenter trials are indicated.

The presence of the testicular determining sequence, SRY, in 46,XY females with gonadal dysgenesis (Swyer syndrome).

Subjects with 46,XY gonadal dysgenesis (Swyer syndrome) have a distinctive phenotype. They are normal or tall in stature, lack somatic anomalies, and possess bilateral rudimentary gonads. Critical Yp deletions have been described in some cases, but in the majority no defects at the molecular level have been reported. To verify the presence or absence of SRY, the putative testicular-determining factor gene, specific primers were designed to amplify the conserved region of the SRY gene. Deoxyribonucleic acid from control males (n = 10) and sex-reversed females with the Swyer syndrome phenotype (n = 5) generated the anticipated 310 bp band. This Y-specific band was absent in the deoxyribonucleic acid from control females (n = 9). To search for possible point mutations, the amplified products of all study subjects and one control male were sequenced in both orientations. The base pair sequences were all identical and similar to the previously published report.

Evidence for a partial deletion in the androgen receptor gene in a phenotypic male with azoospermia.

Androgen resistance is thought to vary phenotypically from a normal female to an infertile male. Previous evaluation of infertile males has been limited to androgen receptor-binding affinity. The androgen receptor gene has been isolated, cloned, and studied extensively in patients with complete androgen insensitivity syndrome, but no comparative data are available on infertile males. To address this matter, the androgen receptor gene was studied in seven azoospermic males by use of the polymerase chain reaction and Southern blot hybridization. A partial gene deletion was found in one patient. This study provides the first molecular evidence of an abnormality in the androgen receptor gene in a phenotypic male with azoospermia.

Probing genomic deoxyribonucleic acid for gene rearrangement in 14 patients with androgen insensitivity syndrome.

Androgen insensitivity appears to involve mutations in the X-linked androgen receptor (AR) gene in genetic males. In this study; 14 patients with androgen insensitivity syndrome (unrelated patients [n = 6]; related patients [n = 8]) were studied. Ten patients had complete and 4 had partial insensitivity to androgens. Deoxyribonucleic acid samples from controls and study subjects were examined with probes specific for the AR gene domains (hAR1, hAR2, hAR3). In one subject with complete androgen insensitivity syndrome, a reduction in size of the 2.4 kilobase band hybridizing to hAR1 was noted. Southern blot analysis of these subjects, however, did not detect deletions or gene rearrangement. These results suggest that deletions detectable by Southern method are infrequent mutants of the AR gene in patients with androgen insensitivity syndrome.

Correlation of the testicular determinant factor sequence zinc finger Y with varying gonadal phenotypes in a series of 13 subjects with gonadal dysgenesis due to Y aneuploidy.

Deoxyribonucleic acid samples from a series of 13 subjects with 45,X/46,X,altered Y, and varying gonadal phenotypes (streak-streak, n = 9; streak-testis, n = 2; testis-testis, n = 2) were analyzed for the presence of the candidate testicular determinant factor sequence zinc finger Y. The Y-specific probes Y97 mapped to Y centromere, pDP105 A,B mapped to Yp and distal Yq11, respectively, hybridized with the deoxyribonucleic acid from all the 13 study subjects. The same deoxyribonucleic acid samples were analyzed for the presence of the zinc finger Y sequence. Eleven of the 13 subjects were positive for the zinc finger Y sequence. Four zinc finger Y-positive subjects had unilateral (n = 2) or bilateral (n = 2) testicular differentiation. Among the nine subjects with bilateral streak gonads, seven showed the presence of this sequence. The lack of testicular differentiation in the presence of quantitatively normal or almost normal zinc finger Y bands could not be explained by mosaicism alone. Mutations not detectable by analysis with the method of Southern with pDP1007, may occur in the testicular determinant factor gene vitiating testicular development.

Deoxyribonucleic acid analysis of the zinc finger Y gene in 45,X/46,XY subjects.

The deoxyribonucleic acid from nine subjects with a 45,X/46,XY karyotype with a cytogenetically intact Y chromosome and phenotypically presenting with bilateral streak gonads, streak and testis, or bilateral scrotal testes along with a control male and female were analyzed for the presence of the zinc finger Y sequence through the molecular probe pDP1007. This particular probe is thought to constitute part of the putative testicular-determining factor gene. All the study subjects demonstrated the presence of zinc finger Y. Laser densitometry studies confirmed a correlation between the intensity of the zinc finger Y band and the percentage of Y cell lines. This study supports the fact that individuals with mixed gonadal dysgenesis and cytogenetically intact Y chromosomes will tend to have intact zinc finger Y sequences.

Screening of seven putative 45,X subjects with deoxyribonucleic acid probes to detect low-level mosaicism for Y cell lines.

The frequency of monosomy X in cytogenetically abnormal abortion material (10% to 15%) suggests that viable 45,X subjects might have covert mosaicism for X or Y cell lines. The deoxyribonucleic acid samples from seven 45,X subjects with Turner syndrome were examined with three Y-specific deoxyribonucleic acid probes. Successive hybridizations with each of these three sensitive deoxyribonucleic acid probes did not reveal any Y-specific band.

Molecular scanning of Yq11 (interval 6) in men with Sertoli-cell-only syndrome.

Data suggesting that probes pDP105/B and 50f2/C,E may identify sequences on distal Yq11 (interval 6) that are critical for spermatogenesis stimulated a study of this region by means of these two probes in azoospermic 46,XY men with biopsy-proved Sertoli-cell-only syndrome. Deoxyribonucleic acid samples from controls and study subjects were digested with the restriction enzymes TaqI, EcoRI, and BamHI. These samples were blotted and hybridized with pDP105/B, 50f2/C,E, and two more proximal Yq11 probes 4B-2 and pAS1. The sequence hydridizing to 50f2/C was absent in one study subject. No deletions were detected with pDP105/B and the two more proximal probes.