Lawrence C. Layman

Delayed Puberty and Estrogen Resistance in a Woman with Estrogen Receptor α Variant

Although androgen resistance has been characterized in men with a normal chromosome complement and mutations in the androgen-receptor gene, a mutation in the gene encoding estrogen receptor α (ESR1) was previously described only in one man and not, to our knowledge, in a woman. We now describe an 18-year-old woman without breast development and with markedly elevated serum levels of estrogens and bilateral multicystic ovaries. She was found to have a homozygous loss-of-function ESR1 mutation in a completely conserved residue that interferes with estrogen signaling. Her clinical presentation was similar to that in the mouse orthologue knockout. This case shows that disruption of ESR1 causes profound estrogen resistance in women. (Funded by the National Institutes of Health.).

The follicle-stimulating hormone receptor gene is polymorphic in premature ovarian failure and normal controls *

OBJECTIVE To examine the FSH receptor gene for detectable abnormalities in women with premature ovarian failure. DESIGN Study of genomic DNA from controls and from patients with 46,XX premature ovarian failure (POF). SETTING Clinics and laboratories of university gynecology and obstetrics departments. PATIENTS Twenty-one women with 46,XX POF and 40 normal fertile controls. INTERVENTIONS Deoxyribonucleic acid was analyzed in patients and controls by Southern blot analysis, polymerase chain reaction (PCR), and denaturing gradient gel electrophoresis. Southern blots were hybridized with the FSH receptor complementary DNA and other smaller DNA probes. Exons 1, 5 to 6, and 10 were amplified by PCR and electrophoresed on agarose gels. Polymerase chain reaction products from exons 1 and 10 were electrophoresed on denaturing gradient gels. MAIN OUTCOME MEASURES Fragments obtained by Southern blot analysis and PCR were compared in patients and controls. Polymerase chain reaction fragments electrophoresed on denaturing gels also were compared in patients and controls. CONCLUSIONS No FSH receptor gene deletions or other mutations were identified in women with POF. Southern blots containing PstI- and HindIII-digested DNA revealed restriction fragment length polymorphisms in both patients and controls. Denaturing gradient gel electrophoresis analysis of PCR fragments of exon 10 also demonstrated DNA sequence polymorphisms in both patients and controls. Follicle-stimulating hormone receptor gene deletions are not common in women with POF, although the gene is polymorphic. We cannot exclude point mutations in other regions of the FSH receptor gene in some patients with POF.

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.

Genetic considerations in the patient with Turner syndrome - 45,X with or without mosaicism

Turner syndrome (TS) is a complex developmental disorder in individuals with short stature who possess a 45,X cell line, with or without mosaicism. Because the single X chromosome is maternally derived in 80% of patients, the genesis of the 45,X karyotype is due to instability of the Y chromosome leading to its loss during meiosis. Phenotypic features vary depending on the mode of ascertainment, with postnatal presentation usually generating a more severe phenotype than a prenatal one. Although patients with pure 45,X present with delayed puberty more often than those with mosaicism for 46,XX or 47,XXX cell lines, the chromosomal complement cannot reliably predict the clinical presentation. Most living TS patients are mosaics, whereas nearly all first-trimester TS fetuses have a single 45,X cell line. Exclusion of a Y cell line, the presence of which increases the risk of gonadoblastomas and subsequent gonadal germ cell tumors, is best accomplished by karyotype, fluorescence in situ hybridization, and DNA analysis if necessary. The precise genetic etiology of TS has not been elucidated, but it does appear that deletion of the short arm of the X chromosome is sufficient to result in the TS phenotype, thereby implicating haploinsufficiency of multiple genes, including SHOX.

The prevalence of intragenic deletions in patients with idiopathic hypogonadotropic hypogonadism and Kallmann syndrome

Idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) are clinically and genetically heterogeneous disorders caused by a deficiency of gonadotrophin-releasing hormone (GnRH). Mutations in three genes—KAL1, GNRHR and FGFR1—account for 15–20% of all causes of IHH/KS. Nearly all mutations are point mutations identified by traditional PCR-based DNA sequencing. The relatively new method of multiplex ligation-dependent probe amplification (MLPA) has been successful for detecting intragenic deletions in other genetic diseases. We hypothesized that MLPA would detect intragenic deletions in ∼15–20% of our cohort of IHH/KS patients. Fifty-four IHH/KS patients were studied for KAL1 deletions and 100 were studied for an autosomal panel of FGFR1, GNRH1, GNRHR, GPR54 and NELF gene deletions. Of all male and female subjects screened, 4/54 (7.4%) had KAL1 deletions. If only anosmic males were considered, 4/33 (12.1%) had KAL1 deletions. No deletions were identified in any of the autosomal genes in 100 IHH/KS patients. We believe this to be the first study to use MLPA to identify intragenic deletions in IHH/KS patients. Our results indicate ∼12% of KS males have KAL1 deletions, but intragenic deletions of the FGFR1, GNRH1, GNRHR, GPR54 and NELF genes are uncommon in IHH/KS.

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.

Ultrasound prediction of follicle volume: is the mean diameter reflective? *

OBJECTIVE To evaluate the relationship between 2 dimensional sonographic measurement of ovarian follicles and their actual volume. DESIGN Prospective clinical study. SETTING The in vitro fertilization (IVF) program of a University based, tertiary care hospital. PATIENTS AND INTERVENTIONS Sonographic categorization by shape, and measurement of 96 individual ovarian follicles immediately prior to aspiration for IVF. Each follicle was aspirated under direct ultrasound guidance and the volume recorded. The 96 follicles were visualized in a total of 14 patients from whom 2 to 27 oocytes were obtained. MAIN OUTCOME MEASURE Total volume of each follicle. RESULTS Round and polygonal follicles exhibited a highly significant relationship between sonographically measured mean diameter and total follicle volume. The volume of follicles that were categorized as ellipsoid was not predicted by measurement of the longest diameter, shortest diameter or mean diameter. CONCLUSION The mean diameter of round and polygonal follicles accurately predicts total follicular volume. However, clinical decisions in ovulation induction should be modified when the follicle shape is predominantly ellipsoid because the traditionally held belief that the sonographic measurement of the follicular diameter correlates with the follicular volume does not apply in those circumstances.

Genotype and Phenotype of Patients with Gonadotropin-Releasing Hormone Receptor Mutations

Human mutations in the gonadotropin-releasing hormone receptor (GNRHR) gene cause autosomal recessive, normosmic idiopathic hypogonadotropic hypogonadism (IHH). At least 19 different mutations have been identified in this G-protein-coupled receptor, which consist mostly of missense mutations. The Gln106Arg and Arg262Gln mutations comprise nearly half of the identified alleles. Most mutations impair ligand binding and all compromise cell signaling events. Some of the mutations also adversely affect activation of gonadotropin subunit or Gnrhr gene promoters. Interestingly, a number of the mutant GnRHRs can be rescued in vitro from misfolding and degradation within the cell by the addition of a GnRHR antagonist IN3. Most affected patients have compound heterozygous GNRHR mutations that may cause either complete IHH (no evidence of puberty) or incomplete IHH (partial evidence of puberty), although some genotypes are associated with mild disease in some families and severe disease in others. GNRHR mutations also appear to cause constitutional delay of puberty, and one genotype (homozygosity for Gln106Arg) may be reversible in patients with IHH. Mutations in the human GNRHR gene have contributed greatly to the understanding of normosmic IHH, as well as the structure and function of the GnRHR.

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.

The detection of Hind III restriction-fragment length polymorphisms using a deoxyribonucleic acid probe for the beta subunit of follicle-stimulating hormone

Molecular diagnosis of disorders of follicle-stimulating hormone (FSH) production may become possible now that the gene for FSH beta has been characterized. Restriction-fragment length polymorphism (RFLP) analysis provides a means of organized search for molecular variants of FSH. The purpose of this study was to screen controls for the presence of RFLPs using the deoxyribonucleic acid (DNA) probe pFSH beta -1.4. Genomic DNA was digested with 12 different restriction endonucleases; Southern blots were constructed and hybridized to pFSH beta -1.4. No polymorphisms were identified with 11 enzymes. Three of 24 (12.6%) Hind III digests demonstrated a polymorphic fragment of either 5.2, 4.7, or 4.3 kb. These are the first RFLPs identified for the FSH beta gene with pFSH beta -1.4. RFLPs for FSH beta constitute the first step in the molecular analysis of disorders of FSH production.