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Biomedical subjects

I L Sargent

Publications and source records attributed to I L Sargent.

At least 55 records · Page 3Linked to original sources
PubMed1998

Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies.

OBJECTIVE: To investigate whether syncytiotrophoblast microvilli (STBM) are shed into the maternal circulation in increased amounts in pre-eclamptic pregnancies as a possible cause of maternal vascular endothelial dysfunction. DESIGN: A time-resolved fluoroimmunoassay was developed to measure STBM levels in peripheral and uterine venous plasma from normal pregnant and pre-eclamptic women. Three colour flow cytometry was used to assess the microparticulate nature of the STBM in pregnancy plasma. The effects of these plasmas on endothelial cell proliferation was compared and a correlation with the levels of STBM detected was sought. SETTING: A laboratory investigation using clinical samples obtained from an obstetric practice in a teaching hospital. SAMPLES: Peripheral venous plasma from 20 women with established pre-eclampsia, 20 normal pregnant women matched for age, gestation and parity, and 10 nonpregnant women of reproductive age. Paired uterine and peripheral venous plasma taken at caesarean section from 10 women with pre-eclampsia and 10 unmatched normal pregnant women. RESULTS: STBM were detected in the plasma of pregnant women by both flow cytometry and time-resolved fluoroimmunoassay. Significantly higher levels of STBM were found in women with established pre-eclampsia (P=0.01). STBM concentrations were higher in uterine venous plasma than in concurrently sampled peripheral venous plasma, confirming their placental origin. A significant correlation was found between the amount of STBM in the plasma and endothelial cell inhibitory activity. CONCLUSIONS: STBM are shed into the maternal circulation (microvillous deportation) and are present in significantly increased amounts in pre-eclamptic women. They may contribute to the endothelial dysfunction underlying the maternal syndrome of pre-eclampsia.

Adult↗
PubMed1997

Presence of fetal DNA in maternal plasma and serum.

BACKGROUND: The potential use of plasma and serum for molecular diagnosis has generated interest. Tumour DNA has been found in 'the plasma and serum of cancer patients, and molecular analysis has been done on this material. We investigated the equivalent condition in pregnancy-that is, whether fetal DNA is present in maternal plasma and serum. METHODS: We used a rapid-boiling method to extract DNA from plasma and serum. DNA from plasma, serum, and nucleated blood cells from 43 pregnant women underwent a sensitive Y-PCR assay to detect circulating male fetal DNA from women bearing male fetuses. FINDINGS: Fetus-derived Y sequences were detected in 24 (80%) of the 30 maternal plasma samples, and in 21 (70%) of the 30 maternal serum samples, from women bearing male fetuses. These results were obtained with only 10 microL of the samples. When DNA from nucleated blood cells extracted from a similar volume of blood was used, only five (17%) of the 30 samples gave a positive Y signal. None of the 13 women bearing female fetuses, and none of the ten non-pregnant control women, had positive results for plasma, serum or nucleated blood cells. INTERPRETATION: Our finding of circulating fetal DNA in maternal plasma may have implications for non-invasive prenatal diagnosis, and for improving our understanding of the fetomaternal relationship.

Case-Control Studies↗
PubMed1997

Detection of antisperm antibodies in seminal plasma by flow cytometry: comparison with the indirect immunobead binding test.

OBJECTIVE: To compare flow cytometry with the established indirect immunobead binding test (IBT) for the detection of antisperm antibodies in seminal plasma. DESIGN: A prospective, comparative study. SETTING: University-based andrology unit. PATIENT(S): One hundred and fifty-eight men with suspected male factor subfertility. INTERVENTION(S): Seminal plasma samples were incubated with antisperm antibody-negative donor sperm. Surface-bound antibody was detected with fluorescence-labeled antihuman antibody in the flow cytometry assay or with immunobead-labeled antihuman antibody in the IBT. MAIN OUTCOME MEASURE(S): The percentage of sperm that tested positive for surface-bound antibody was determined in the two assays. Seminal plasma was antisperm antibody-positive when > or = 20% of the sperm were antibody-bound, and clinically significant levels were present when > or = 50% of the sperm were antibody-bound. RESULT(S): Of 71 samples that were negative by the IMT, 66 (93%) also were negative by flow cytometry. Of 63 samples that had > or = 50% immunobead binding, 55 had equivalent results by flow cytometry. Overall statistical analysis showed a good correlation between the two assays. CONCLUSION(S): There is a good correlation between the indirect IBT and indirect flow cytometry for the detection of antisperm antibodies in seminal plasma.

Autoantibodies↗
PubMed1997

Human placental syncytiotrophoblast microvillous membranes impair maternal vascular endothelial function.

OBJECTIVE: To investigate the hypothesis that, should there be an increase in deported syncytiotrophoblast microvillous membrane fragments in pre-eclampsia, it may cause maternal vascular endothelial dysfunction. DESIGN: Syncytiotrophoblast microvillous membrane (STBM) vesicles, prepared from normal term placentae, were perfused through small subcutaneous arteries isolated from fat biopsies obtained at caesarean section. Endothelial function of these arteries was studied by determining acetylcholine-induced relaxation after preconstriction with noradrenaline. As controls, physiological buffer or red blood cell membranes in physiological buffer were used and endothelial function similarly estimated. Transmission electron microscopy was performed on arteries after perfusion. SAMPLE: STBM vesicles, isolated from the placentae of three healthy women undergoing elective caesarean section for reasons unrelated to pre-eclampsia, were suspended in physiological buffer. Subcutaneous fat arteries were obtained from a separate group of 13 normotensive pregnant women, also undergoing elective caesarean section at term. RESULTS: Perfusion with red blood cell membranes or physiological buffer had no significant effect on the concentration dependent relaxation in arteries preconstricted with noradrenaline. However, after 2 h perfusion with STBM vesicles, arteries showed a significant reduction in relaxation to acetylcholine, indicative of altered endothelial function. Transmission electron microscopy of arteries perfused with STBM vesicles confirmed endothelial disruption. CONCLUSIONS: STBM vesicle perfusion specifically altered the relaxation response of preconstricted maternal subcutaneous fat arteries to acetylcholine, suggesting an alteration in endothelial dependent relaxation. Deported microvilli may therefore be capable of producing endothelial cell damage and endothelial dysfunction observed in the maternal syndrome of pre-eclampsia.

Arteries↗
PubMed1996

Two-way cell traffic between mother and fetus: biologic and clinical implications.

The bilateral trafficking of nucleated cells between the fetus and the mother was studied using polymerase chain reaction (PCR)-based systems sensitive enough to detect 1 target cell in 100,000 background cells. Sixty-six mother-baby pairs were recruited; maternal and cord blood samples were collected at delivery for DNA extraction. Cell trafficking was studied in informative cases using PCR-genotyping of polymorphic regions in the beta-globin cluster, the glutathione S-transferase M1 locus and the angiotensin converting enzyme gene. In addition, Y-PCR was also used in conjunction with these systems for the detection of fetal cells in maternal circulation. Fetal cells were detected in maternal peripheral blood in 26 of 51 cases whereas maternal cells were detected in 16 of 38 fetal umbilical cord blood samples. The proportion of umbilical samples with detectable maternal sequences was much higher than previously reported. In the 28 cases informative for both mother and baby, there was no obvious correlation between the cell traffic from mother to baby as compared to that from baby to mother. These findings may have implications for the use of cord blood for bone marrow transplantation, the vertical transmission of infectious agents, and the physiology of the feto-maternal relationship.

Blood Cells↗
PubMed1996

Endothelial cell proliferation is suppressed by plasma but not serum from women with preeclampsia.

OBJECTIVES: Evidence has been sought for a circulating factor derived from the placenta that suppresses endothelial cell proliferation and hence contributes to the maternal endothelial cell disturbances of preeclampsia. STUDY DESIGN: The effects of sera and plasmas from women with proteinuric preeclampsia and from matched normal pregnant control women on endothelial cell proliferation were compared. The recovery of endothelial cell inhibitory activity from syncytiotrophoblast microvesicles added to male blood and prepared as plasma or serum was determined to investigate the possible placental origin of the inhibitory factor. RESULTS: Sera from women with preeclampsia did not inhibit endothelial cell proliferation. In contrast, plasma from preeclamptic women significantly suppressed endothelial cell growth at 20% dilution compared with controls, and suppression was more pronounced in severe preeclampsia. The inhibitory activity of syncytiotrophoblast microvesicles added to blood could not be recovered from serum, only from plasma, which may explain why there was no suppression with sera from preeclamptic women. CONCLUSIONS: These results confirm that there is a blood-borne endothelial cell suppressive factor in preeclampsia that may be derived from the placenta.

Adolescent↗
PubMed1996

Does large loop excision of the transformation zone of the cervix predispose to the development of antisperm antibodies in women?

OBJECTIVE: To determine whether large loop excision of the transformation zone of the uterine cervix for cervical intraepithelial neoplasia predisposes to the development of female isoimmunity to human spermatozoa. DESIGN: A prospective, controlled study. SETTING: Colposcopy and Andrology units at the John Radclife and Churchill Hospitals, Oxford, United Kingdom. INTERVENTIONS: Serum samples were collected from 33 women before large loop excision of the transformation zone of the cervix and repeated at a minimum time interval of 4 months after the procedure. Women were questioned regarding the procedure and subsequent reproductive function. A control population of 30 women not undergoing cervical surgery also underwent serial serum screening for antisperm antibodies. MAIN OUTCOME MEASURE(S): The detection of serum antisperm antibodies by flow cytometry. RESULTS: None of the serum samples before large loop excision of the cervical transformation zone had clinically significant levels of antisperm antibodies. There was, however, a significant rise in antisperm antibody levels in women following large loop excision of the transformation zone. Apparent risk factors for the development of antisperm antibodies included a short duration of sexual abstinence and the use of nonbarrier contraception after surgery. There was no rise in antisperm antibody levels in the control population. CONCLUSION: Large loop excision of the transformation zone of the cervix is a risk factor for the development of antisperm antibodies in women. Women should be advised to use barrier contraception or avoid sexual intercourse until complete healing of the cervix has occurred.

Adult↗
PubMed1996

Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium.

The aim of this study was to investigate the effect of leukaemia inhibitory factor (LIF) on human blastocyst formation rates in vitro. To do this, it was first necessary to devise a complex serum-free medium (CSFM) in which to test its activity. Blastocyst formation rates in CSFM microdrops (18.4%) showed no difference to those obtained previously for embryos cultured in 1 ml T6 medium containing 10% serum (25%). The majority of blastocysts were of optimal blastocyst grade (BG1/BG2) as compared to the poor grade (BG3). The percentage of BG1/BG2 blastocysts was decreased in CSFM microdrops (10.2%) compared to that observed in T6 medium (24.8%). Addition of 1000 IU/ml LIF to CSFM microdrops increased the blastocyst formation rate from 18.4 to 43.6% (P = 0.025) and increased the percentage of BG1/BG2 blastocysts (33%; P = 0.025) to levels comparable with those observed in T6 medium. Thus LIF significantly increased the quality and number of human blastocysts formed in CSFM medium, increasing the potential for blastocyst transfer in human in-vitro fertilization (IVF).

Blastocyst↗
PubMed1996

The role of pregnancy-specific beta-1 glycoprotein (SP1) in assessing the human blastocyst quality in vitro.

The advent of new culture techniques resulting in more than 60% of embryos developing in vitro to the blastocyst stage suggests that blastocyst transfer in humans with its potential to increase in-vitro fertilization success rates could be a feasible option. Blastocyst quality markers, however, are required to ensure that an increase in numbers is not achieved at the expense of lowering quality. We have previously reported a morphology based method for grading blastocysts. The current study sought to determine whether the secretion of pregnancy-specific beta-1-glycoprotein (SP1) (a trophoblast product known to be associated with fetal well-being) by blastocysts of differing quality was reflected in the morphological grading. SP1 concentrations were measured in the culture medium of grade 1 (n = 19), grade 2 (n = 4) and grade 3 (n = 4) blastocysts as well as vacuolated morulae (n = 6) daily from day 1 to day 14. Cumulative SP1 concentrations secreted by blastocysts were significantly higher than those secreted by vacuolated morulae. However, SP1 levels could not distinguish between blastocysts of differing morphological grades. The inconsistent pattern of secretion suggests that at this early stage of development, SP1 secretion is probably not sufficiently defined to allow any differences in levels to be reflected by the morphological grading.

Biomarkers↗
PubMed1996

Embryotoxicity as a marker for recurrent pregnancy loss.

Some cases of unexplained recurrent pregnancy loss may be due to embryotoxic factors present in the mother's serum. In vitro assays have been devised to study the effect of these factors on pre-implantation, peri-implantation, and somite stage rodent embryos. Embryotoxic activity has been found in a high percentage of recurrent aborters but is also present in women with endometriosis and unexplained infertility, suggesting that there may be a range of factors acting at different stages of development. Embryotoxic activity may be predictive of pregnancy outcome and has been shown to be decreased after hormone and immunotherapy. However, its true value can only be assessed when the nature of the toxic factors is understood. Preliminary characterisation studies suggest that toxic activity may due to both immunological (antibodies or cytokines) and nutritional factors.

Abortion, Habitual↗
PubMed1995

Methylation status and transcriptional expression of the MHC class I loci in human trophoblast cells from term placenta.

Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated syncytiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane.

Base Sequence↗
PubMed1995

Expression of TAP1 by human trophoblast.

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Anti-transporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G+ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

An investigation of methods for enriching trophoblast from maternal blood.

Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.

Cell Count↗
PubMed1994

Micromanipulation of human embryos to assist hatching.

OBJECTIVE: To determine the effects of zona slitting and subsequent embryo transfer performed on different days of in vitro human embryonic development to allow appropriate application of assisted hatching techniques to clinical IVF-ET. DESIGN: A microsurgical technique was used to make a standardized slit in the zona pellucida of bipronucleate human embryos on day 2, 3, or 5 after oocyte retrieval. A mock ET was performed after the procedure and the rate of blastocyst formation and the process of hatching in each embryo was monitored. SETTING: All embryos used were donated for research by patients attending the IVF Unit at the John Radcliffe Hospital, Oxford, United Kingdom. MAIN OUTCOME MEASURES: The effects of the mock ET on the integrity of zona-slit embryos was documented. The embryos were cultured further and parameters such as blastocyst formation, initiation, and completion of hatching were monitored in all three groups of embryos. RESULTS: The mock ET did not have a detrimental effect on embryos as assessed by their morphology and subsequent blastocyst formation rate. The technique used in this study to make an opening in the zona increased the rate and number of blastocysts hatching in all three groups compared with nonmanipulated controls. However, the day on which the slit was made did not significantly alter the outcome. CONCLUSION: A gap in the zona pellucida of human embryos significantly increases the rate of hatching. Micromanipulative techniques such as zona slitting can be suitably used for this purpose without loss of blastomeres through the gap in the zona or trapping of the embryo during the process of hatching.

Blastocyst↗