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Biomedical subjects

D R Stewart

Publications and source records attributed to D R Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1995

Laboratory methods for evaluating early pregnancy loss in an industry-based population.

Laboratory methods were adapted or developed to analyze approximately 70,000 daily urine samples collected during more than 2,500 menstrual cycles from 448 women working in the semiconductor industry. An immunoenzymometric assay (IEMA) for human chorionic gonadotropin (hCG) was employed for screening cycles in order to optimize laboratory resources and to reduce the number of samples requiring analysis by less efficient methods. The presence of hCG in urine was confirmed by the definitive immunoradiometric assay (IRMA). The screening assay eliminated 78% of cycles from further analysis because there was no evidence of conception. Thirty-eight of 448 cycles identified as having significant levels of hCG with the IEMA were confirmed as hCG positive with the IRMA. HCG-positive cycles were further evaluated by examination of daily diary data and by laboratory assays for ovarian and pituitary hormones. As a result of these evaluations, 17 of the 38 cycles identified by the IRMA as positive for hCG were found to be nonconceptive cycles. These results demonstrate the effectiveness of screening assays for hCG, as well as the importance of using multiple urinary biomarkers for the detection of early fetal loss with daily urine samples.

Abortion, Spontaneous↗
PubMed1995

Dissociation of relaxin and progesterone secretion from the primate corpus luteum by acute administration of a 3 beta-hydroxysteroid dehydrogenase inhibitor during the menstrual cycle.

The factors regulating relaxin production by the primate CL during spontaneous menstrual cycles and in early pregnancy are poorly understood. Since the CL produces steroids, notably progesterone, and expresses progesterone receptors, luteal progesterone may act locally to regulate relaxin production. For the current study, either trilostane (600 mg daily)--a 3 beta-hydroxysteroid dehydrogenase inhibitor--or vehicle was administered to rhesus monkeys during the midluteal phase (Days 6 and 7) of spontaneous menstrual cycles to examine the effects of reduced luteal progesterone synthesis on relaxin secretion. Trilostane treatment reduced serum concentrations of progesterone within 3 h of initial administration and maintained low levels typical of the follicular phase (< 1 ng/ml), causing premature menses without significant alteration in serum bioactive LH levels. Nevertheless, the patterns and levels of circulating relaxin, as measured by homologous macaque ELISA, were not different between trilostane- and vehicle-treated monkeys, with relaxin levels peaking in both groups by Day 13 of the luteal phase. To determine if chorionic gonadotropin (CG) injections simulating early pregnancy could stimulate relaxin production in a progesterone-depleted environment, trilostane or vehicle was administered as described above, followed by injections in increasing dosages of human (h) CG beginning 3 days after initial trilostane administration. Serum progesterone levels in trilostane-treated animals were significantly reduced prior to and during hCG treatment when compared with vehicle-treated animals. However, serum relaxin levels were comparable between these groups; relaxin levels peaked approximately 10-fold above pre-hCG levels in both trilostane- and vehicle-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

The relationship of corpus luteum volume to relaxin, estradiol, progesterone, 17-hydroxyprogesterone and human chorionic gonadotropin levels in early normal pregnancy.

Our purpose was to characterize the growth pattern of the corpus luteum of early normal human pregnancy and correlate this growth with the corpus luteum hormone products: relaxin, progesterone, estradiol and 17-hydroxyprogesterone. A prospective study of seven patients was initiated at a mean gestational age of 4 weeks and 2 days. Corpus luteum volume and hormone concentrations were determined for each study patient every 48 h for a period of 2 weeks. Transvaginal imaging of the corpus luteum was performed by a single observer. Corpus luteum volume was calculated using the formula for an ellipsoid (4/3 pi abc/8). Correlation between corpus luteum volume and hormone concentrations was tested using Pearson's r. There was a mean three-fold increase in corpus luteum volume between 4 and 6 weeks' gestational age. Concomitantly, relaxin and estradiol concentrations increased, 17-hydroxyprogesterone declined slightly, progesterone remained stable and human chorionic gonadotropin (hCG) increased exponentially. Mean positive correlations were shown between corpus luteum volume and relaxin (r = 0.72), corpus luteum volume and hCG (r = 0.68), and hCG and relaxin (r = 0.82). However, there was a lack of correlation between corpus luteum volume and estradiol, progesterone and 17-hydroxyprogesterone. We have shown that a rapid increase in the corpus luteum volume occurs in early normal human pregnancy without a parallel rise in the classic corpus luteum steroid products. We interpret these findings to suggest that growth of the corpus luteum in early human pregnancy is largely derived from the proliferation of non-steroid secreting cells. The precise role of these cells in controlling steroidogenesis in this gland has yet to be defined.

17-alpha-Hydroxyprogesterone↗
PubMed1994

Differences between young and aged mice in susceptibility to Friend virus.

Friend virus (FV) is a murine leukemia virus that infects progenitor red blood cells and causes an erythroleukemia in susceptible mouse strains, resulting in splenomegaly. Several genetic loci of the host have been identified that affect erythroleukemia development, differentiation status of target cells and virus replication. Since age may change expression of these loci, age may affect FV disease. To explore this possibility, FV expression in four genetically diverse strains of mice of different ages was examined. Extent of viral replication and of disease were evaluated by measuring spleen focus forming units (SFFU), spleen weight and reverse transcriptase (RT) activity in target organs. Young DBA/2 and (C57BL/6 x DBA/2)F1 mice exhibited a greater level of virus expression than their aged counterparts in all parameters investigated. Young CBA/Ca mice had slightly higher spleen weights and SFFU values than aged CBA/Ca mice, but a definitive age-related change was not observed in the RT activity of the target organs. C57BL/6 mice, which are genetically resistant to the development of FV-induced erythroleukemia, exhibited a limited degree of virus replication that was not effected by the age of the animal. Our results indicate that the age of the mouse, as well as the genetic background, can contribute to the level of susceptibility to FV.

Aging↗
PubMed1994

Immunocytochemical localization of nonluteal ovarian relaxin.

Relaxin has been demonstrated to be produced by the corpus luteum of the menstrual cycle and pregnancy and is also produced by the endometrium and decidua, although these nonluteal sources may not contribute to circulating relaxin concentrations. The reports of luteal production of relaxin have failed to consider nonluteal ovarian sources. To look for sources of nonluteal ovarian relaxin, human ovaries were collected from patients who underwent removal of the ovary for a variety of reasons. Tissues were fixed in formalin and embedded in paraffin. Two monoclonal antibodies were used for immunocytochemical staining, one directed against human relaxin and the other against the C peptide of prorelaxin. In addition to the expected staining of corpora lutea, the luteinized theca interna but not granulosa of developing follicles from ovaries with an active corpus luteum of the cycle also stained positive for both relaxin and prorelaxin. Ovaries from term pregnant women with luteinized theca also demonstrated staining for relaxin and prorelaxin. In addition to luteal and thecal cell staining, small clusters of pseudodecidual cells in the periphery of the ovary stained positive for relaxin and prorelaxin. These data indicate that the ovary contains theca interna-derived structural elements in addition to the corpus luteum that produce relaxin when a corpus luteum is active, while granulosa-derived elements do not. This suggests that luteal production of relaxin is from theca-derived elements and may explain instances of independent relaxin and progesterone secretion.

Adult↗
PubMed1993

The relationship between hCG and relaxin secretion in normal pregnancies vs peri-implantation spontaneous abortions.

OBJECTIVE: We determined the ovarian response to human chorionic gonadotrophin (hCG) in terms of relaxin and progesterone secretion during the peri-implantation period of normal and failing pregnancies. We wished to test the hypotheses that relaxin production in failing pregnancies is different from that in normal pregnancies, that relaxin is a reliable, quantitative indicator of the biological activity of endogenous hCG, and that relaxin is a useful predictor of peri-implantation spontaneous abortions. DESIGN: Daily blood samples were collected in a prospective longitudinal study from insemination patients. PATIENTS: Women undergoing artificial insemination in natural cycles with non-frozen donor semen at a University clinic. MEASUREMENTS: Serum LH, hCG, relaxin and progesterone were measured and the relationship between hCG and the ovarian hormones was evaluated in the peri-implantation period of normal pregnancies and spontaneous abortions. RESULTS: Nine of 23 conceptive cycles resulted in a spontaneous abortion between 16 and 70 days after the LH peak. In all normal and failing pregnancies there was a close qualitative relationship between hCG secretion and relaxin production. Six of nine failing pregnancies were associated with abnormally low hCG secretion. Six of the spontaneous abortions were associated with rates of relaxin secretion which were higher than the mean of 14 normal pregnancies. No such alterations in progesterone concentrations were observed. In cases where hCG was extremely low, the quantitative relationship between hCG and relaxin was different from that in cases of normal hCG concentrations. CONCLUSIONS: There is a close temporal relationship between the secretion of trophoblastic hCG and ovarian secretion of relaxin in the peri-implantation period of normal and failing pregnancies. In failing pregnancies there is substantial variability in the quantitative relationship between relaxin and hCG, indicating that relaxin is not a reliable quantitative indicator of hCG bioactivity. Contrary to previous reports, relaxin concentrations in failing pregnancies tended to be higher than or equal to concentrations in normal pregnancies until the loss was imminent. Because of this relaxin is not a useful predictor of peri-implantation spontaneous abortions.

Abortion, Spontaneous↗
PubMed1993

Measurement of periimplantational relaxin concentrations in the macaque using a homologous assay.

Circulating relaxin concentrations in the human rise in the late luteal phase and increase further in response to increasing circulating CG concentrations immediately after implantation. Similar events have not been documented in the laboratory macaque because of the lack of sensitivity of heterologous assay systems. A homologous enzyme-linked immunosorbent assay for authentic macaque relaxin was developed and validated. Using this enzyme-linked immunosorbent assay, relaxin concentrations were measured in peripheral and ovarian venous blood collected from cynomolgus and rhesus macaques. Relaxin concentrations rose in the late luteal phase of nonconceptive menstrual cycles in cynomolgus macaques, but it was not detected at other times in the cycle. In conceptive cycles, relaxin concentrations rose rapidly in close association with the appearance of mCG 13-14 days after mating. Pregnant rhesus macaques also had elevated relaxin concentrations in blood samples collected on days 15-17 postbreeding. Relaxin concentrations disappeared immediately after luteectomy or ablation of the trophoblast by either surgery or administration of methotrexate. The rise of relaxin paralleled the rise of mCG until 20-25 days postbreeding, while progesterone concentrations declined during this same time period. The lack of correlation between relaxin and progesterone secretion profiles suggests that either the cellular origins or the intracellular mechanisms promoting the secretion of these hormones are different. The periimplantational profile of serum relaxin in macaques was similar to the profile of relaxin observed during early human pregnancy.

Animals↗
PubMed1993

Enhanced ovarian steroid secretion before implantation in early human pregnancy.

Previous studies have compared ovarian steroid production in the luteal phase of nonconceptive and conceptive cycles. Some investigators reported higher preimplantational levels of progesterone in conceptive cycles vs. nonconceptive cycles, but other studies have found no differences. Many of these results were difficult to interpret because the studies included infertile women and/or women who received exogenous hormones. In this study we have characterized the profiles of gonadotropin secretion and ovarian steroid response during early pregnancy in a population of spontaneously ovulating women and compared them to those in nonconceptive cycles of recently fertile women. Blood samples were collected daily during the luteal phase from 24 women during 51 cycles of artificial insemination with donor semen. Cycles were segregated to those from women who had a successful term pregnancy (normal group) and those having an early spontaneous abortion (SAB group) and were also classified as nonconceptive or conceptive based on measurements of hCG. Serum LH and FSH did not show marked differences between nonconceptive and conceptive cycles in the periimplantation period in either the normal or SAB group. In the normal group, estradiol concentrations were significantly higher in conceptive cycles than in nonconceptive cycles beginning 6 days after the LH peak and continuing through the end of the cycle, while differences in progesterone concentrations bordered on or exceeded significance during the same time period. In the SAB group, preimplantation differences in pituitary gonadotropin and ovarian steroid secretion were not observed, whereas the postimplantation hCG concentrations in the SAB group were significantly lower than those in the normal group. It is reasoned that embryos with defective post-implantation hCG secretion may have had this defect before detection of hCG in serum, thus accounting for the lack of stimulation of steroid secretion in these pregnancies. These findings suggest that the enhanced ovarian steroid secretion in conceptive cycles may be due to a gonadotropic stimulus from the preimplantation embryo.

Abortion, Spontaneous↗
PubMed1993

Pregnancy failure in cats associated with long-term dietary taurine insufficiency.

The effect of long-term dietary taurine insufficiency on reproductive function was studied in adult female domestic cats (n = 11). Cats were time-mated during taurine-deficient (6 months) and refed (6 months) states, and the outcome of ovulatory cycles and breeding was analysed. Serum progesterone and relaxin concentrations were evaluated in order to characterize pregnancies, including those resulting in resorption of fetuses, and pseudopregnancies. Increased resorption of fetuses, reduced litter size, and increased incidence of stillborn kittens was observed in queens while on taurine-deficient diets, as well as after refeeding of a taurine-enriched diet. Overall, 30% of the ovulatory cycles resulted in the delivery of kittens, with mean live and stillborn litter sizes of 2.2 +/- 0.4 and 0.8 +/- 0.4 kittens (mean +/- SEM), respectively. The remaining ovulatory cycles resulted either in pregnancies in which fetuses were resorbed (38%), or in pseudopregnancies (32%). Ovulatory cycles resulting in resorbed fetuses were characterized by the appearance of relaxin on day 20 of gestation, but with a subsequent decrease to non-pregnant concentrations by day 25 of gestation. These results suggest that reproductive failure in domestic cats exposed to long-term nutritional taurine deficiency is associated with a postovulatory defect manifest within the first 10 days after implantation, and that this defect is not reversible upon refeeding of a taurine-enriched diet for 6 months.

Animals↗
PubMed1992

Purification and sequence determination of canine relaxin.

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C18 column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.

Amino Acid Sequence↗
PubMed1992

Analysis of the basis for persistence of herpes simplex virus type 1 in undifferentiated U937 cells.

Replication of herpes simplex type 1 (HSV-1) is inhibited in the human monocyte-like cell line, U937, when the cells are in the undifferentiated state, but when the cells are stimulated to differentiate by treatment with the phorbol ester, phorbol 12-myristate 13-acetate virus is replicated. Because HSV-1 has been shown to persist in these cells and in their in vitro counterparts freshly isolated human blood monocytes, we initiated an analysis of viral persistence in undifferentiated U937 cells. No appreciable HSV-1 DNA replication was observed in undifferentiated U937 cells compared with differentiated U937 cells and with fully permissive Vero cells. However, using in situ hybridization, we established that a significant percent of the undifferentiated U937 cells contained viral DNA sequences. Interestingly, when analyzed by Southern blot hybridization, this DNA was found to have assumed a nonlinear configuration similar to that found in latently infected neurons. Analysis of viral proteins in undifferentiated U937 cells revealed a marked absence of proteins of all three kinetic classes. However, in transient transfection assays, the major viral transactivating protein ICP4, functioned normally, whereas ICP0, a promiscuous transactivator of both viral and cellular genes, was unable to transactivate viral promoters in undifferentiated U937 cells. Thus, a subtle dysfunction in the activity of ICP0 may account, at least in part, for the inability of undifferentiated U937 cells to support replication of HSV-1.

Animals↗
PubMed1992

Breed differences in circulating equine relaxin.

Equine relaxin has been previously determined in a small number of pregnant Thoroughbred mares. To better define the normal pregnancy pattern of relaxin, the current study reports on a much larger number of mares. It also was designed to determine if all equids have the same gestational pattern of relaxin secretion. Plasma samples were collected weekly in 24 Standardbred mares, every 7-10 days in 10 pony mares, and daily in late pregnancy from 16 burros. Standardbreds had higher concentrations of relaxin than that reported for Thoroughbreds during most of gestation and did not exhibit the midpregnancy nadir in relaxin concentrations observed in Thoroughbreds. Relaxin concentrations in Standardbreds showed a small but steady decline from Day 150 until delivery. Pony mares had lower relaxin concentrations throughout pregnancy than other mares and had continuously increasing concentrations during gestation. Burros had relaxin concentrations intermediate to ponies and other mares in late gestation. Burros induced to foal with oxytocin showed a sharp increase in relaxin concentrations. No effect of the sex of the offspring was observed in relaxin profiles in Standardbred mares. Each of three Standardbreds with abnormal termination of pregnancy exhibited abnormally low relaxin concentrations at some point in the gestation prior to termination of the pregnancy. Thus, relaxin may be an indicator of placental functioning and used to assess at-risk pregnancies in mares.

Animals↗
PubMed1992

Serum relaxin concentrations in patients with out-of-phase endometrial biopsies.

When luteal phase relaxin concentrations were summed to give an integrated measure (pg/mL per cycle), relaxin was found to be significantly lower in those cycles with an out-of-phase endometrial biopsy. In addition, peak relaxin concentrations were lower in out-of-phase cycles compared with normal cycles. These data indicate that relaxin secretion may be related to normal luteal function and suggest that shortening of the luteal phase results in reduced relaxin production. Measurement of circulating relaxin may prove to be useful in making the diagnosis of out-of-phase biopsy and needs to be assessed for its usefulness in diagnosing abnormal luteal function.

Biopsy↗
PubMed1991

Affinity purification and sequence determination of equine relaxin.

Relaxin, a polypeptide hormone normally associated with pregnancy, has been purified from many species, and the sequence determined for a growing number. Equine relaxin has been previously purified by acetone extraction, gel filtration, and ion exchange chromatographies. In an attempt to develop a more rapid and efficient method for relaxin purification, the use of affinity chromatography coupled with HPLC was explored. Monoclonal antibodies were raised against highly purified equine relaxin; large quantities of antibody were obtained by ascites production and attached to a solid phase support. An extract of term equine placentas was passed through the affinity column and washed, and relaxin was eluted by a change in pH. The isoforms of equine relaxin were separated by HPLC using a C18 (ODS) reverse phase column and a linear gradient of 25-30% acetonitrile. Four major and several minor isoforms of equine relaxin were obtained. The isoforms share similar mol wt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), are composed of subunits (SDS-PAGE under reducing conditions), and have similar charges (native PAGE). Five isoforms tested positive for biological activity in the mouse interpubic ligament bioassay. Equine relaxin was sequenced by Edman degradation, and the sequence was confirmed by fast atom bombardment mass spectrometry. The isoforms of equine relaxin were found to be due to heterogeneity of the C-terminus of the B-chain. Equine relaxin appears to be the smallest relaxin sequenced. The A-chain consists of 20 residues, and the B-chain has 28 residues, with a total mol wt of 5253. Equine relaxin shares the greatest sequence homology with porcine relaxin (67% identity).

Amino Acid Sequence↗
PubMed1991

Determination of the source of relaxin immunoreactivity during pregnancy in the dog.

We investigated the source of immunoreactive relaxin (IR) in the dog during pregnancy using the following: (1) controls, (2) dogs ovariectomized during pregnancy and maintained on progesterone, (3) dogs hysterectomized during pregnancy or immediately postpartum, (4) corpora lutea, uteri, and placentas collected at various times during pregnancy for determination of IR, and (5) maternal (overian and uterine vein) and fetal (cardiac) blood and amniotic and allantoic fluid. Plasma IR patterns remained unchanged in animals subjected to ovariectomy, whereas in the controls IR was first detected at about Day 20 of pregnancy, peaked on Day 35, and was then remained at the same level until parturition. Hysterectomy, on the other hand, resulted in IR values becoming undetectable within 2 to 3 days. The highest tissue concentrations of IR were found in the placenta. These findings indicate that the source of relaxin in the pregnant dog is the placenta.

Animals↗
PubMed1990

Relaxin in the peri-implantation period.

The time of appearance of relaxin in peripheral blood was determined in conceptive and non-conceptive cycles using a sensitive and specific double-antibody enzyme-linked immunoassay for human relaxin. For study of relaxin in early pregnancy, daily plasma samples were collected from women receiving artificial insemination of donor semen. The day of ovulation was determined by daily LH monitoring and ultrasound observation. In three conceptive cycles, relaxin was significantly elevated over baseline 9-10 days following the LH peak. Relaxin concentrations quickly rose over the next 15 days of observation to over 800 pg/ml. Relaxin was observed to increase 1 to 2 days prior to the first detectable increase in plasma hCG as measured by enzyme-linked immunosorbent assay. To compare the relaxin profile in conceptive cycles with normal luteal phase concentrations, relaxin was also measured in daily plasma samples collected from women contracepting with barrier methods, bilateral tubal ligation, or abstinence. A small but consistent rise in relaxin in the late luteal phase was observed in nine of eleven women, which began 6-9 days after the LH peak, averaged approximately 50 pg/ml, and was declining by the next menses. It is concluded that a small but measurable rise in plasma relaxin is associated with the normal luteal phase and that relaxin secretion is accelerated around the time that hCG is first detected in conceptive cycles. This acceleration of relaxin secretion which is associated with the onset of hCG may provide additional evidence for identification of transient early pregnancy.

Chorionic Gonadotropin↗
PubMed1987

Determination of the source of immunoreactive relaxin in the cat.

We have recently reported the secretory profile of relaxin throughout gestation in the cat. Because the appearance of relaxin begins at about Day 20 (Day O = ovulation) and because implantation begins shortly before this at Days 13-14, we hypothesized that relaxin was of feto-placental origin. To test this hypothesis, we used 4 experimental groups: 1) Control (laparotomy-only at Day 23 or 42, n = 4); 2) Early Ovariectomy (Ovx, bilateral ovariectomy between Days 23 and 26, n = 4); 3) Late Ovx (bilateral ovariectomy between Days 40 and 44, n = 4); 4) Tissue Removal (removal of feto-placental units, uterus, and one ovary on Days 16, 21, 28 and 35, n = 1 per day). Pregnancies were maintained in both Ovx groups by progesterone administration. Relaxin secretory patterns in Ovx groups were similar to the Control data. Relaxin was detectable in plasma beginning at about Day 20, with maximum concentrations reached by Day 30. Relaxin concentrations were highest (immunoactivity per mg tissue) in homogenates of placental tissues as compared to luteal, fetal, or uterine tissues. Altogether, these data indicate that the feto-placental unit is the source of relaxin in the cat.

Animals↗