Human gene mutations causing infertility.

The identification of gene mutations causing infertility in humans remains noticeably deficient at present. Although most males and females with infertility display normal pubertal development, nearly all of the gene mutations in humans have been characterised in people with deficient puberty and subsequent infertility. Gene mutations are arbitrarily categorised into four different compartments (I, hypothalamic; II, pituitary; III, gonadal; and IV, outflow tract). Diagnoses of infertility include hypogonadotrophic hypogonadism (compartments I and II), hypergonadotrophic hypogonadism (III), and obstructive disorders (compartment IV). Most gene mutations identified to date affect gonadal function, but it is also apparent that a large number of important genes in normal fertility have yet to be realised.

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.

Essential genetics for the obstetrician/gynecologist.

The science of genetics has become increasingly important in the practice of medicine. This article reviews the practical, clinical aspects of genetics relevant to obstetrics and gynecology. The basic fundamentals of molecular biology techniques currently used in DNA diagnostic tests are discussed.

Mutations of follicle stimulating hormone-beta and its receptor in human and mouse: genotype/phenotype.

The pituitary gonadotropin follicle stimulating hormone (FSH) interacts with its membrane-bound receptor, to produce biologic effects. Traditional functions of FSH include, follicular development and estradiol production in females and the regulation of Sertoli cell action and spermatogenesis in males. FSHbeta knock-out mice and transgenic mice, serve as models for FSH deficiency and excess, respectively. In addition, mutations of both FSHbeta and FSHR genes have been characterized in humans, although phenotypic effects of the ligand appear to be more profound than those of its receptor. FSH is essential for normal puberty and fertility in females, particularly ovarian follicular development beyond the antral stage. In males, FSH is necessary for normal spermatogenesis and when FSH function is completely absent, infertility occurs. With partial FSH deficiency in males, spermatogenesis is affected, but fertility may still be possible. FSH may also be necessary for normal androgen synthesis in males and females.

Mutations in the follicle-stimulating hormone-beta (FSH beta) and FSH receptor genes in mice and humans.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein synthesized in the anterior pituitary gland, is important for the production of sex steroids and gametes. FSH-beta (FSH beta) and FSH receptor (FSHR) knockout mice display impaired ovarian follicular development and infertility in females and small testes, oligospermia, and fertility in males. Humans with FSH beta gene mutations tend to have a more severe phenotype than those with FSHR gene mutations, although infertility and varying degrees of impaired sex steroid production occur in both types of mutations. Data from human and mouse mutations in the FSH beta and FSHR genes suggest that FSH is necessary for normal pubertal development and fertility in males and females.

Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay.

Although delayed puberty is relatively common and often familial, its molecular and pathophysiologic basis is poorly understood. In contrast, the molecular mechanisms underlying some forms of hypogonadotropic hypogonadism (HH) are clearer, following the description of mutations in the genes KAL, GNRHR, and PROP1. Mutations in another gene, DAX1 (AHC), cause X-linked adrenal hypoplasia congenita and HH. Affected boys usually present with primary adrenal failure in infancy or childhood and HH at the expected time of puberty. DAX1 mutations have also been reported to occur with a wider spectrum of clinical presentations. These cases include female carriers of DAX1 mutations with marked pubertal delay and a male with incomplete HH and mild adrenal insufficiency in adulthood. Given this emerging phenotypic spectrum of clinical presentation in men and women with DAX1 mutations, we hypothesized that DAX1 might be a candidate gene for mutation in patients with idiopathic sporadic or familial HH or constitutional delay of puberty. Direct sequencing of DAX1 was performed in 106 patients, including 85 (80 men and 5 women) with sporadic HH or constitutional delay of puberty and patients from 21 kindreds with familial forms of these disorders. No DAX1 mutations were found in these groups of patients, although silent single nucleotide polymorphisms were identified (T114C, G498A). This study suggests that mutations in DAX1 are unlikely to be a common cause of HH or pubertal delay in the absence of a concomitant history of adrenal insufficiency.

Mutation analysis of the EMX2 gene in Kallmann's syndrome.

OBJECTIVE: To investigate the possibility that a mutation in the human EMX2 gene may be involved in Kallmann's syndrome. DESIGN: In vitro experiment. SETTING: Academic Medical Center. PATIENTS: One hundred and twenty patients with Kallman's syndrome or idiopathic hypogonadotrophic hypogonadism (IHH). INTERVENTION: Peripheral blood leukocytes were used to obtain DNA. MAIN OUTCOMES MEASURES: Single-stranded conformational polymorphism (SSCP) analysis was used to identify possible mutations of the EMX2 gene. RESULTS: One hundred and twenty patients with Kallmann's syndrome or IHH, had no mutations noted in this gene. CONCLUSION: It is unlikely that EMX2 mutations are a clinically significant cause of IHH or Kallman's syndrome.

The molecular basis of human hypogonadotropic hypogonadism.

Patients with hypogonadotropic hypogonadism (HH) present with delayed puberty, infertility, and low serum gonadotropins. The molecular basis for most cases of HH is unknown, but single gene mutations have been described for some hypothalamic and pituitary genes. Kallmann syndrome due to KAL gene mutations and adrenal hypoplasia congenita/HH caused by AHC gene mutations are both X-linked recessive disorders. Mutations in the gonadotropin releasing hormone receptor, leptin, and the leptin receptor cause autosomal recessive HH. In addition, isolated deficiencies of follicle stimulating hormone and luteinizing hormone in the corresponding specific beta-subunit genes and PROP1 gene mutations represent pituitary deficiency states, resulting in a phenotype of HH. Despite these remarkable advances in our understanding of human HH, the cause of approximately 90% remains unknown.

Molecular analysis of the gonadotropin-releasing hormone receptor in patients with polycystic ovary syndrome.

OBJECTIVE: To determine whether a mutation in the GnRH receptor gene is responsible for polycystic ovary syndrome (PCOS). DESIGN: Molecular analysis of human genomic DNA. SETTING: Academic research environment. PATIENT(S): Eighty patients with PCOS. INTERVENTION(S): Extraction and polymerase chain reaction (PCR) analysis of genomic DNA, confirmation of PCR products by ethidium bromide staining of agarose gels after electrophoresis, denaturing gradient gel electrophoresis of PCR products, and photography. MAIN OUTCOME MEASURE(S): Mutations in the GnRH receptor of women with PCOS. RESULT(S): Denaturing gradient gel electrophoresis revealed no mutations in the exonic sequence encoding the open reading frame of the GnRH receptor. CONCLUSION(S): A mutation in the GnRH receptor gene is unlikely to be the underlying cause of PCOS in most patients. The molecular basis of this disorder remains unknown.

Administration of progesterone before oocyte retrieval negatively affects the implantation rate.

OBJECTIVE: To compare the efficacy of two clinically accepted methods of progesterone supplementation during IVF. DESIGN: Prospective randomized trial. SETTING: A university-based IVF program. PATIENT(S): Three hundred fourteen stimulated IVF cycles between January 1993 and October 1994. INTERVENTION(S): Patients were assigned to one of two luteal phase progesterone regimens by a random permuted block design. In protocol A, 12.5 mg of IM progesterone was given 12 hours before oocyte retrieval; in protocol B, 25 mg of IM progesterone was given on the day of oocyte retrieval. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Patient demographic characteristics, including age, diagnosis, number of oocytes retrieved and fertilized, and number of embryos transferred, were not different between the two groups. There was no difference in the rate of cycle cancellation between the groups. One hundred forty ETs were performed in patients assigned to protocol A and 142 in patients assigned to protocol B. The clinical pregnancy rate in group A was 12.9% compared with 24.6% in group B. CONCLUSION(S): The administration of progesterone before oocyte retrieval is associated with a lower pregnancy rate than the administration of progesterone after oocyte retrieval.

Mutations in human gonadotropin genes and their physiologic significance in puberty and reproduction.

OBJECTIVE: Human gene mutations provide an opportunity to study the pathophysiology of the disease process as well as normal physiology. The purpose of the present report was to review known human gene mutations that affect gonadotropin secretion. DESIGN: A retrospective analysis of studies of human gene mutations that affect hypothalamic, pituitary, and gonadal function was conducted. RESULT(S): Mutations have been identified for at least three genes that cause inherited hypogonadotropic hypogonadism. In addition, gene mutations for the beta-subunits of FSH and LH have been characterized. Both activating and inactivating mutations have been identified for the gonadotropin receptor genes. CONCLUSION(S): The identification of human gene mutations has furthered our understanding of the normal processes of pubertal development and fertility.

Genetics of human hypogonadotropic hypogonadism.

Humans with hypogonadotropic hypogonadism (HH) manifest irreversible pubertal delay, infertility, and low serum levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Although the genetic basis of this condition is largely unknown, mutations have been identified in approximately 5-10% of HH patients. Mutations in the KAL gene (Kallmann syndrome) and the AHC gene (adrenal hypoplasia congenita/HH) cause X-linked recessive HH. Autosomal recessive HH may be brought about by mutations in the gonadotropin-releasing hormone receptor, leptin, and the leptin receptor genes. Isolated deficiencies of the gonadotropins FSH and LH are due to corresponding beta-subunit genes. PROP1 gene mutations lead to combined pituitary deficiency, and HESX gene mutations result in septo-optic dysplasia, both of which include HH. These identified gene mutations advance our understanding of normal hypothalamic-pituitary-gonadal function.

Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene.

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication.

The Finnish follicle-stimulating hormone receptor gene mutation is rare in North American women with 46,XX ovarian failure.

OBJECTIVE: To determine whether FSH receptor gene missense mutation in Finnish women with premature ovarian failure (POF) is present in North American women with POF. DESIGN: Analysis of DNA from patients and controls. PATIENT(S): Thirty-five women with POF and ten normal controls. INTERVENTION(S): Extraction of DNA with subsequent digestion by the enzyme BsmI, polyacrylamide gel electrophoresis, ethidium bromide staining, and photography. MAIN OUTCOME MEASURE(S): After restriction enzyme digestion, the frequencies of the normal allele (two fragments of 51 and 27 base pairs) and the mutant allele (a single 78-base pair fragment) were determined. RESULT(S): BsmI digestion was noted for all 35 affected individuals and 10 controls, thus demonstrating homozygosity for the normal FSH receptor allele. No patient or control was heterozygous or homozygous for the mutant allele. CONCLUSION(S): The missense mutation in the human FSH receptor gene in Finnish women with POF is uncommon in North American women with POF. The molecular basis of ovarian failure for most patients remains unknown.

Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation.

OBJECTIVE: To determine the extent of paternal and maternal chromatin decondensation in unfertilized eggs after intracytoplasmic sperm injection (ICSI). DESIGN: Eggs that failed to show two pronuclei (2-PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). PATIENT(S): Forty-nine patients undergoing IVF cycles. MAIN OUTCOME MEASURE(S): The unfertilized eggs were studied by chromatin staining. RESULT(S): The average fertilization rate from all ICSI cycles in these two groups was 45%. The fertilization rates in groups A and B were 35% and 59%, respectively. In group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin, whereas 45% were partially decondensed. In these two groups, no sperm chromatin was detected in 24% of the unfertilized eggs. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. CONCLUSION(S): These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause of failure of fertilization rates in ICSI cycles. Rather, it is likely to be the failure to complete both the maternal and paternal chromatin transitions that occur with normal fertilization.

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.

Relevance of molecular medicine to clinical obstetrics and gynecology.

Understanding molecular biology can improve the clinical acumen of the practicing obstetrician/gynecologist. An area of basic research now becoming clinically relevant involves the G proteins and G protein-coupled receptors. Clinicians already manipulate G protein-coupled receptors in their daily practice. Examples include the administration of oxytocin (oxytocin receptors), beta-2 tocolytic agents (beta 2-adrenergic receptors), GnRH agonists (GnRH receptors), exogenous gonadotropins (FSH and LH receptors), and bromocriptine (dopamine receptor). Clinically important disorders presenting to the obstetrician/gynecologist include some forms of precocious puberty, delayed puberty, premature ovarian failure, and pituitary adenomas which are due to mutations of G proteins and G protein-coupled receptors. The importance of these proteins is demonstrated by the fact that G protein-related genes comprise about 1 percent of the human genome. Additionally, the knowledge that some G protein gene mutations are present in the germ line, and others are somatic cell in origin (and not heritable), aids in more accurate genetic counseling to patients.