Chromatin intact human sperm recovery is higher following glass wool column filtration as compared with density gradient centrifugation.

The sperm DNA quality as determined by chromatin integrity has been reported to be associated with in vivo and in vitro fertility. However, previous studies have evaluated preparation procedures to select motile, morphologically normal and mature spermatozoa, but not the spermatozoa with intact sperm chromatin. To determine which technique yields a population of spermatozoa with improved DNA quality, split ejaculate was processed with density gradient centrifugation (DGC) procedure and glass wool column filtration (GWF) procedure. The processed samples were analysed for sperm DNA quality using the acridine orange staining method on flow cytometry. The GWF procedure decreases the percentage of spermatozoa with DNA fragmentation resulting in more intact chromatin in the processed sample. There is a need to design a clinical study with GWF for assisted reproductive technology.

Elevated serum levels in human pregnancy of a molecule immunochemically similar to eosinophil granule major basic protein.

We have shown that serum levels of a molecule immunochemically similar to eosinophil granule major basic protein (MBP) are elevated in pregnant women throughout gestation. MBP levels increase during gestation and plateau at approximately 7,500 ng/ml by the 20th wk (greater than 10-fold above normal). Levels return to normal after delivery, with a T1/2 of 13.7 d. The MBP in pregnancy serum is remarkably similar to the eosinophil granule MBP in that: (a) pregnancy MBP fully inhibits the binding of radiolabeled MBP standard in a double antibody radioimmunoassay; (b) this inhibition reaction is specific for human MBP because pregnancy serum produces no inhibition of the binding of radiolabeled guinea pig MBP in the guinea pig MBP radioimmunoassay; (c) in a two-site immunoradiometric assay for MBP, slopes of dose-response curves for pregnancy serum, purified MBP, and serum from a patient with hypereosinophilic syndrome are identical, and maximal binding is comparable; (d) reduction and alkylation of pregnancy sera increases measured MBP 100-fold, as previously shown for eosinophil granule MBP in serum; and (e) the MBP in pregnancy serum demonstrates the same pattern of heat lability as has been previously reported for MBP. Four observations have raised the possibility that the eosinophil is not the source of the MBP in pregnancy serum: (a) no correlation between serum MBP level and peripheral blood eosinophil count exists in pregnant women, in contrast to previous studies of patients with eosinophilia; (b) levels of three other eosinophil-associated proteins are normal or low in pregnancy sera, whereas the serum levels of these proteins are elevated in patients with eosinophilia; (c) the slopes of dose-response curves for pregnancy sera and MBP standards differ in the double antibody radioimmunoassay; and (d) the molecule in pregnancy serum elutes from Sephadex G-50 columns at the void volume, while eosinophil granule MBP and the MBP in serum of patients with eosinophilia elute at a volume consistent with the previously established molecular weight of 9,300. These findings suggest that the MBP in pregnancy serum is derived from a source other than the eosinophil.

Evidence of eosinophil granule major basic protein in human placenta.

A protein immunochemically related to the eosinophil granule major basic protein (gMBP) is found in increased concentration in the plasma of pregnant women and has been localized to placental trophoblasts by immunofluorescence. Pregnancy MBP (pMBP) is indistinguishable from gMBP in its reactivity with polyclonal antisera and a panel of 14 mouse mAbs. We report the purification of pMBP from human placenta by: (a) affinity chromatography over mAb immobilized on Sepharose, (b) gel filtration in 6 M guanidine.HCl buffer, and (c) reversed-phase HPLC. Purified pMBP and gMBP are biochemically indistinguishable in that both: (a) bind to DNA, (b) polymerize and bind to carrier proteins via disulfide linkages, (c) have a molecular weight of 14,000, (d) have isoelectric points greater than 10.6, (e) comigrate in two-dimensional gels, (f) coelute during reversed-phase HPLC on C18 columns, (g) have identical peptide maps after three different digestions, and (h) have partial amino acid sequence identity. This physicochemical identity has important implications as to the role of pMBP in human placentation.

Localization of a molecule immunochemically similar to eosinophil major basic protein in human placenta.

We have recently reported that human pregnancy is characterized by a 10- to 20-fold elevation of eosinophil major basic protein (MBP) immunoreactivity in maternal blood. Here we show, by immunofluorescence, that placental tissue specifically binds antibody to MBP in and around the placental X cells and placental-site giant cells and, using thin plastic sections, that placenta has no infiltrating eosinophils. The X cells line the inner aspects of placental septal cysts, and the cyst fluid, obtained by aspiration, contains immunoreactive MBP at concentrations of 100 micrograms/ml, a sixfold greater concentration than the highest levels measured in maternal blood. The soluble MBP immunoreactivities in placental homogenates and in maternal serum chromatograph identically on Sephadex G-50, and both these gestational MBP molecules migrate as though substantially larger than the MBP found in serum from patients with hypereosinophilic syndrome or purified from the eosinophil granule. Our inability to demonstrate eosinophils in maternal blood or placental tissue, coupled with the large quantities of immunoreactive MBP highly localized in placental cysts and the chromatographic behavior of this molecule, suggest that the MBP detected in human gestation is produced by placenta.

Vascular endothelial growth factor gene polymorphism and implantation failure.

Implantation failure is the most frequent cause of lack of pregnancy after IVF and embryo transfer. Successful implantation requires the invading blastocyst to stimulate its own blood supply through angiogenesis. Vascular endothelial growth factor (VEGF) is the best-characterized regulator of angiogenesis. Since one polymorphism of the VEGF gene, -1154 G/A, has been previously suggested to be associated with recurrent spontaneous abortion, the present study was undertaken to determine whether VEGF -1154 G/A genotype is also related to recurrent implantation failure. Buccal swabs were obtained from 70 women with a history of recurrent implantation failure after IVF-embryo transfer and from 73 control women. DNA was extracted from the buccal swabs and analysed for the presence of the VEGF -1154 A/A gene. The frequency of homozygosity of the VEGF -1154 A/A gene was significantly higher among women experiencing recurrent implantation failure compared with fertile control women (19% versus 5%, P = 0.02). It is concluded that homozygosity of the VEGF -1154 A/A gene may serve as a susceptibility factor affecting the chances of recurrent implantation failure.

Acanthosis nigricans in a patient with streak gonads.

Acanthosis nigricans (ACN) is associated with ovarian disorders or abnormalities of carbohydrate metabolism. We saw a 21-year-old woman who had primary amenorrhea and ACN. Results of endocrine studies showed anovulation and low serum estradiol levels with increased gonadotropin concentrations. Laparoscopic examination disclosed bilateral streak gonads; the ovaries were completely replaced by fibrous tissue. Cultures of peripheral lymphocytes, skin fibroblasts, and the right and left ovaries demonstrated normal female karyotype 46XX in all mitoses studied. An autoimmune disorder was excluded by the absence of antithyroid and antiadrenal antibodies. Specific antibodies against ovarian proteins were also absent. A glucose tolerance test demonstrated fasting hyperinsulinemia and exaggerated serum insulin response to the glucose load. The observations in this patient exclude a primary pathogenic role for the ovary in the production of ACN; they also lend further support to a connection between the cutaneous disorder and insulin resistance.

A nuclear binding assay to assess the biological activity of steroid receptors in isolated animal and human tissues.

This paper describes a nuclear binding assay (NB assay) which measures not only the presence of a steroid receptor in a tissue, but also the quantity of that receptor which is biologically active or functional, i.e. able to bind to nuclear acceptor sites. The assay involves the isolation viable cells from tissues and their incubation with an excess of radiolabeled steroid to encourage the activation and nuclear binding of all cellular receptors. The nuclei are isolated under conditions that remove unactivated (unbound) steroid-receptor complexes. This NB assay demonstrates, in both animal and human steroid target tissues, a saturable, tissue- and steroid-specific, and temperature- and time-dependent nuclear binding of radiolabeled steroids. These properties support a receptor-dependent nuclear binding of steroids. This assay is reproducible and requires relatively small amounts of tissue. The patterns of nuclear binding of the progesterone receptor, achieved with the assay in the avian oviduct model system, are shown to correlate with the nuclear binding of progesterone in vivo, the ability of the steroid to alter transcription, and the expression of a specific gene product, the protein avidin. The assay has been used to identify the existence of nonfunctional steroid receptors in endometrial and breast carcinomas. Therefore, this NB assay combined with the standard charcoal/hydroxylapatite methods of quantitating total cellular receptors should provide a means of assessing changes in the regulation of the biological activity of steroid receptors. Further, the assay should be useful to assess the ability of steroid analogs to properly activate their respective receptors for subsequent nuclear binding.

Reproductive immunology: biomarkers of compromised pregnancies.

The objective of this paper is to consider several categories of biomarkers of human pregnancy. The design of the report is to discuss useful and promising markers and techniques. Research gaps, needs, and priorities are also defined. Useful markers are mixed lymphocyte culture reactions, measures of lymphocytotoxic antibodies, histocompatibility (HLA) typing, and immunohematological evaluations. Promising markers are measures of major basic protein and early pregnancy factor, as well as determinations of trophoblast-lymphocyte cross-reactive (TLX) antigens. Promising techniques are fluorescence-activated cell-sorter analysis of maternal blood for fetal and extraembryonic tissues and immunotherapy with TLX and other antigens to prevent spontaneous abortion. It is concluded that immunology has much to offer the development of biomarkers of human pregnancy.

Immunologic mechanisms in the maternal-fetal relationship.

Until recently, the immunologic tolerance between a mother and her allogeneic fetus has been a highly speculative and poorly understood phenomenon. New data indicate that maternal acceptance of the fetal allograft necessitates a specific, protective immune response. This specific recognition apparently involves unique placental antigens with genes that are closely linked to HLA loci. Lack of recognition of these antigens, as in matings between partners with similar HLA profiles, may result in repeated spontaneous abortions caused by unsuppressed rejection mechanisms.

Vaginal flora in asymptomatic women.

Four groups of 25 asymptomatic women--pregnant, premenopausal and taking oral contraceptives, premenopausal and not taking oral contraceptives, and postmenopausal--were studied for the presence in vaginal specimens of aerobic bacteria, anaerobic bacteria, fungi, Mycoplasma, Chlamydia, herpes simplex virus, mycobacteria, and Trichomonas. No significant differences in microbial flora were found among the groups.

A search for antibodies to luteinizing hormone receptors in premature ovarian failure.

We recently published evidence of the presence of circulating antibodies in the sera of patients who had the diagnosis of premature menopause syndrome. This led to the question: Is there a circulating antibody in the sera of patients with premature menopause syndrome which interferes with gonadotropin-luteinizing hormone (LH) receptor interaction on the luteal cell surface? Serum samples from 14 women with the diagnosis of Premature menopause syndrome were examined to see whether they would block the binding of 125I-labeled human chorionic gonadotropin (hCG) to human corpora lutea. All patients had spontaneous cessation of menses before the age of 35 years and documentation of increased gonadotropin secretion and failure of estrogen secretion. When the sera of the study group were incubated with 125I-labeled hCG and LH receptor from human corpora lutea, no difference in binding was observed when compared with controls. This suggests that there is no circulating antibody that interferes with hCG-LH receptor interaction and that would thereby lead to loss of ovarian function in premature menopause syndrome.

A rapid, sensitive assay for analyzing defects in metabolism of steroids in normal and diseased human tissues.

In this report, we describe a simple, rapid biopsy-steroid metabolism assay that is applicable to any steroid tissue system. It consists of mincing the sample, tissue culture incubation, extraction of the steroids and their metabolites from the tissue, and fractionation of the metabolites by high-pressure liquid chromatography (HPLC). Radioimmunoassay is used to verify the elution patterns of certain steroids. Studies of the metabolism of [3H]progesterone in the avian oviduct showed the generation of metabolites that eluted from the HPLC system in a pattern similar to androgens, estrogens, and glucocorticoids. Studies of the metabolism of [3H]testosterone in the human foreskin showed the production of metabolites that eluted from the HPLC system similar to 5 alpha-dihydrotestosterone and 5 alpha-androstane-3,17-dione (androstanedione) from the parent [3H]testosterone. In the production of the metabolite that eluted as androstanedione in samples of foreskin from normal subjects, a significant (P less than 0.001) correlation was found with the age of the donor. Preliminary studies of patients with hypospadias showed a significant (P less than 0.005) decrease in the production of "androstanedione" compared with that in normal subjects. Because of the wide range in rates of metabolism of testosterone in the patients with hypospadias, the effect of age does not seem to be the sole determinant of a low rate of metabolism in these patients. Some samples of hypospadias foreskin had a decreased rate of production of a metabolite that eluted as dihydrotestosterone in comparison with normal foreskin, even when the age of the donor was considered. The assay described herein should be applicable to any surgical biopsy specimen and to all steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary adenoma in Olmsted County, Minnesota, 1935--1977. A report of an increasing incidence of diagnosis in women of childbearing age.

There has been a recent, rapid increase in the number of pituitary adenomas diagnosed in women of childbearing age in Olmsted County, Minnesota. The incidence rates in men of all ages and in women aged 45 and over have remained stable and are similar to rates reported from elsewhere. Since 1970 the mean annual incidence per 100,000 women aged 15 through 44 years has been 7.1, whereas it was 0.7 in the population of the same age and sex during the period 1935 through 1969.

Testicular regression syndrome.

Recently, the testicular regression syndrome has been reported as being representative of a clinical range of XY agonadal persons. A patient with aberrant fetal sexual development is described. Of particular interest is that all of the tissues examined--the rudimentary uterine horn and the 2 fallopian tubes--displayed an XY chromosome complement. Correlation was made between the known temporal events of embryogenesis of the testes and the apparent time of regression of the testes. In this patient, the insult to testicular development, with subsequent testicular regression, occurred between 48 and 60 days after fertilization. This review supports the previously reported thesis that in certain XY persons the testes are irreparably damaged at a critical stage in fetal development, and that this critical stage is represented by a range of abnormalities of genital development. Possible causes and simplified nomenclature for such gonadal damage are presented.

Chronic anovulation syndrome and associated neoplasia.

Ovarian hormones have been implicated in a number of neoplastic conditions. Chronic anovulation syndrome, a spontaneous biologic experiment of unopposed secretion of estrogen by the ovaries, was identified in a cohort of 1270 patients, and the risk of these patients having neoplasia was studied. Of the 1270 patients studied, 30 had a subsequent malignancy develop; the expected number was 29.8. When the individual types of subsequent malignancy were analyzed, the endometrium was the only site at increased risk. The relative risk of developing carcinoma of the endometrium after the diagnosis of chronic anovulation syndrome is 3.1 (95% confidence interval 1.1 to 7.3). The long-term risk of developing adenocarcinoma of the endometrium might be considered when treating patients who have this syndrome.

Incidence of ectopic pregnancy in Rochester, Minnesota, 1950-1981.

Trends in the occurrence of ectopic pregnancy from 1950 through 1981 were evaluated in the defined population of Rochester, Minnesota. The average ratio of ectopic pregnancies to live births was 1:171. The ratio declined from 1:141 to 1:217 from 1955-1959 to 1970-1974 and increased sharply to 1:122 by 1975-1980. Similarly, the average age-adjusted incidence rate of ectopic pregnancies per 100,000 women of childbearing age was 49 over the period studied. A peak rate of 79 per 100,000 was reached in 1955 to 1959, and a trough of 30 per 100,000 occurred in 1970 to 1974; this was followed by a sharp increase to 49 per 100,000 in 1975 to 1981. Methods of measuring ectopic pregnancy are contrasted, and possible risk factors are suggested.

Specific binding of human chorionic gonadotropin to human corpora lutea in the menstrual cycle.

Luteal tissue from 26 premenopausal women undergoing surgery for nonendocrine disease was examined for specific binding to human chorionic gonadotropin. Results showed a nadir in the percentage of binding on day 10, with a gradual increase until maximal binding was obtained on days 22 through 26. Binding then markedly declined to that seen on day 1 of the menses and decreased gradually to its lowest level on day 10.

Anovulation with estrogen production: receptor disorders?

Receptor antibodies have been implicated in the pathogenesis of several autoimmune disorders such as Graves disease. The authors hypothesized that serum antibodies against the luteinizing hormone (LH) receptor existed in women with chronic anovulation syndrome and continuously stimulated the ovaries. Twenty patients with the clinical diagnosis of anovulation with estrogen production were studied. The effects of serum globulins on the binding of 125I-labeled human chorionic gonadotropin (hCG) to ovarian receptors in the patients were compared with the effects in age-matched controls. These studies did not demonstrate significant inhibition of 125I-hCG binding to ovarian receptors by immunoglobulins of patients with anovulation and estrogen production in comparison with those of control subjects. This finding suggests that the cause of anovulation with estrogen production is not an autoimmune phenomenon involving the LH receptor of the human ovary.

Incidence of premature ovarian failure.

To assess the occurrence of premature ovarian failure, the age-specific incidence rates of natural menopause were determined for a cohort of 1858 women born between 1928 and 1932. These women were identified as Rochester, Minnesota residents in 1950 and were followed for date and type of menopause. A total of nine experienced natural menopause before age 40 years, which represents a 1% risk of natural menopause to age 40. The annual incidence rates of natural menopause per 100,000 person-years were ten for ages 15 to 29 and 76 for ages 30 to 39. In the age group 40 to 44, the incidence of natural menopause increased greatly to 881 per 100,000 person-years at risk.

Comparison of estrogen receptors in human premenopausal and postmenopausal uteri using isoelectric focusing.

Previous studies have suggested that the estrogen receptor in the postmenopausal uterus, although capable of binding estrogen, may be inactive, that is, incapable of translocating and binding to nuclear acceptor sites. The present study uses isoelectric focusing to characterize differences in the estrogen receptor between premenopausal and postmenopausal uterine tissue. Human myometrium (15 premenopausal and ten postmenopausal samples) was examined by isoelectric focusing in the presence and absence of diisopropylfluorophosphate (DFP) and molybdate, which inhibit proteolysis and stabilize the receptor. As demonstrated previously, receptor forms could be identified in two regions of the pH gradient, one in the pH 4 to 5 region and another at pH 6 to 7. When prepared without DFP and molybdate, the percentage of receptors focusing in the pH 4 to 5 range was significantly greater for premenopausal than postmenopausal tissue (P less than .01). The addition of DFP and molybdate significantly altered the focusing pattern of postmenopausal tissue (P less than .01), increasing the percentage of binding in the pH 4 to 5 range. In the presence of DFP and molybdate, the focusing patterns of receptor from premenopausal and postmenopausal tissue were not significantly different. These data suggest that endogenous proteases are more prevalent in postmenopausal tissue and can degrade the receptor to forms that focus at a higher pH. With the inhibition of these proteases, the focusing patterns of receptors in premenopausal and postmenopausal tissue become very similar, representing the undegraded and perhaps native form of the receptor.