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Flame and graphite-furnace atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry were used to study the metallic ions in the digestive gland-gonad complex (DGG) of Helisoma trivolvis snails infected with the daughter rediae of Echinostoma trivolvis and in uninfected DGG. Seven metals were found to be present in infected and uninfected DGG at concentrations above the detection limits of the analytical methods. Of these, sodium was present in significantly higher amounts (Student's t-test, confidence level of 95%) in the infected versus uninfected DGG; magnesium and manganese occurred in significantly lower amounts in the infected DGG. Our results were compared with those from a Bulgarian study in which neutron activation analysis was used to determine elements in Lymnaea stagnalis snails infected with the intramolluscan stages of the 37-collar-spined echinostome E. revolutum. The Bulgarian study also reported a significant elevation of sodium but reduction of zinc in the hepatopancreas (i.e., digestive gland) of infected snails. Other differences between the two studies are discussed.

Objective: To determine the extent of biochemical changes in the zona pellucida (ZP) and cortical granule release in eggs failing to fertilize in IVF., Design: After insemination, unactivated eggs without two pronuclei (PN) were studied by high resolution microscopy and fluorescent probes to determine cortical granule density and meiotic stage. After ZP isolation, proteins from individual ZPs were biotinylated, electrophoresed, and visualized by western blots with avidin-chemiluminescence. Controls included mouse unfertilized and fertilized eggs and human germinal vesicle stage oocytes and > or = 3PN eggs., Setting: University medical center and hospital tertiary care IVF-ET program., Results: Many of the unfertilized eggs were in metaphase and had relatively low cortical granule densities indicative of cortical granule loss. Approximately one half of the ZPs showed evidence of biochemical hardening with a modification of a 90 to 100 x 10(3) molecular (weight) ratio (M(r)) ZP protein. The 3PN eggs had few cortical granules and their ZPs had a pronounced 90 to 100 x 10(3) M(r) modification that was not detected in germinal vesicle stage ZP controls., Conclusion: The observed changes in ZP biochemistry and cortical granule quantitation demonstrate that failed fertilization is frequently associated with spontaneous cytoplasmic, but not nuclear, activation. In affected eggs, these changes could prevent fertilization in routine IVF and in cases of reinsemination. The relationship of changes in the ZP and cortical granules to infertility and in vitro culture requires further investigation. The 90 to 100 x 10(3) M(r) ZP protein modification can be detected biochemically with 1/10 of a human ZP.

Through the study of naturally occurring mutations in humans, the creation of mutations by site-directed mutagenesis, and the production of transgenic knockout mice, further understanding of molecular reproductive endocrinology had been achieved. Mutations in the aromatase gene in females have confirmed that its deficiency results in a previously unrecognized form of sexual ambiguity with a 46,XX karyotype, and delayed puberty with multicystic ovaries. It has long been known that estrogen is necessary for skeletal growth and epiphyseal closure in the female, but aromatase and estrogen receptor gene mutations in men have demonstrated for the first time, that estrogen is important for epiphyseal closure in the male. Mutations in the steroidogenic acute regulatory protein have been recently described that demonstrate the cause for lipoid congenital adrenal hyperplasia, a disorder characterized by the complete lack of steroid production. New gene mutations in gonadotropin beta-subunits, pituitary hormones, G-protein coupled receptors, G-proteins, steroid enzymes and their receptors have also been characterized recently. Site-directed mutagenesis experiments and transgenic knockout mice have been increasingly used to study the effects of normal endocrine function. Normal functions of steroid receptor genes (steroidogenic factor-1, estrogen receptor, progesterone receptor) the glycoprotein alpha-subunit, luteinizing hormone beta, and proto-oncogenes such as RET have been better characterized by creating knockout models. Molecular biology techniques permit these types of studies which may be difficult, if not impossible, to perform otherwise in physiologic settings.

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.

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.

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.

We report the first case of dicavitary twin pregnancy, following clomiphene citrate therapy, in a patient with uterus bicornis bicollis and anovulation. A review of the literature is presented, and obstetric outcomes and management of these rare pregnancies are discussed.

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.

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.

Objective: To determine whether restriction fragment length polymorphisms are present using a deoxyribonucleic acid (DNA) probe for human luteinizing hormone beta subunit (hLH-beta). If the gene for hLH-beta is polymorphic, genetic diagnosis of disorders of luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) production could become possible., Design: Study of genomic DNA from controls with a variety of restriction enzymes to identify polymorphisms., Setting: Laboratories of the Department of Obstetrics and Gynecology, Department of Oral Biology, Medical College of Georgia, Augusta, Georgia., Patients: Unrelated control men and women seen in clinics at the Medical College of Georgia., Interventions: Genomic DNA was extracted from patients and digested with eight different restriction enzymes for the study of the hLH-beta gene by Southern analysis., Main Outcome Measure: Fragment (band) sizes on radiographs from Southern blots were compared with those from molecular weight standards., Conclusions: Restriction fragment length polymorphisms were identified for four of the restriction enzymes, DraI, HincII, MboI, and KpnI. These polymorphisms may be useful in the diagnosis of disorders of hLH and hCG production.

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.

Recombinant DNA techniques have increased our understanding of genetic disorders and facilitated their diagnosis and may eventually lead to therapy. Expression may be assessed and quantified by Northern blot analysis, whereas in situ hybridization may localize the transcript. Southern blotting has been the standard method to identify gene mutations and RFLPs, which are useful in the diagnosis of many different disorders. Special multiallelic RFLPs produce a DNA fingerprint particularly useful in paternity testing and forensic medicine. PCR has greatly reduced the time and expense to diagnose genetic disorders, and has an enormous number of applications both clinically and at the research level. These techniques along with DNA sequencing are currently being used in the human genome project, which may some day allow "DNA typing" of individuals for any gene of interest. There is no doubt that new techniques in molecular biology will continue to evolve so that the goal of gene therapy for many disorders may be possible in the future.

Objective: To determine if the genes for gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone beta (FSH beta), and luteinizing hormone beta (LH beta) are present, and if so, whether gene structure is normal in patients with idiopathic hypogonadotropic hypogonadism (IHH)., Design: Patients with clinical and laboratory characteristics of IHH were studied at the deoxyribonucleic acid (DNA) level to assess gene structure., Setting: This study took place in an academic setting., Patients: Human volunteers with documented IHH and fertile controls were studied., Interventions: Genomic DNAs were extracted from each patient, Southern blots were constructed and hybridized to DNA probes for GnRH, FSH beta, and LH beta. DNA samples were also subjected to polymerase chain reaction analysis., Main Outcome Measures: Gene structure was assessed by analysis of autoradiographs and gel electrophoresis of polymerase chain reaction products in both the study patients and controls., Results: Each analysis for FSH beta, LH beta, and GnRH demonstrated the same sized fragments in both the study group and control group. A 1.2-kilobase fragment containing the coding region for GnRH was present in all patients with IHH and controls by polymerase chain reaction., Conclusions: The genes for GnRH, LH beta, and FSH beta are present in patients with IHH. No large deletions or rearrangements of any of these genes were identified in any of these patients.

Ionized Ca (Ca2+) is essential for maintaining physiologic functioning in the cardiovascular system (CVS). Sepsis affects the CVS by several mechanisms and is associated with decreased serum Ca2+. The pharmacodynamic response of the CVS to serum Ca2+ alteration was compared in acutely septic and nonseptic dogs at serum Ca2+ levels of 50%, 100%, 150% and 200% of normal baseline values. Serum Ca2+ alterations caused similar changes in both groups, although finite differences existed between septic and nonseptic subjects. Ca manipulation produced limited differences in the response of mean arterial pressure, cardiac output, left ventricular dP/dtmax, systemic (SVR) and pulmonary (PVR) vascular resistance between septic and nonseptic subjects. PVR and SVR demonstrated opposite responses during hypocalcemia; PVR was lower than baseline in both groups, whereas SVR was higher at the 50% level. No difference was evident for total oxygen consumption (VO2) or heart rate. In view of the limited differences in response and the failure to improve systemic VO2, serum Ca2+ supplementation does not afford any additional benefit in this experimental model of acute sepsis.

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.

Of 3,918 obstetric patients who underwent scanning during the first 20 weeks of gestation, 94 had sonolucent adnexal masses identified. These represented predominantly the corpus luteum of pregnancy. The incidence of such a mass significantly declined after the tenth gestational week. This pattern of degeneration is physiologic and represents the normal course of events for the ovary in the first half of pregnancy. Characteristics of these masses and indications for operative intervention are discussed. The obstetric outcome for eight patients who underwent surgical treatment was uncomplicated. Guidelines for management of significant adnexal masses in pregnancy is similar to that of the nonpregnant state.

Presented is the case of a triplet pregnancy with conjoined twins diagnosed antenatally with sonography. Prenatal diagnosis has the potential to improve outcome for the normal infant.