PubMed Health⌕ Search

Biomedical subjects

I L Sargent

Publications and source records attributed to I L Sargent.

At least 73 records · Page 4Linked to original sources
PubMed1994

Meiotic progression of mouse oocytes throughout follicle growth and ovulation in vitro.

Intact ovarian follicles can be grown in culture but the normality of their oocyte development has not yet been established. This work examined meiotic progression in oocytes from mouse ovarian follicles grown in vitro and in vivo. Follicles of initial diameter 112-279 microns were isolated from ovaries of B6 CBA F1 mice and cultured individually in 25 microliters minimal essential medium-alpha containing 5% mouse serum, 100 mIU/ml follicle stimulating hormone and various supplements. Oocytes were isolated from follicles of various sizes after culture for < or = 5 days and oocyte chromatin was examined by fluorescence microscopy with Hoechst 33258. The stage of meiosis was classed as prophase I [germinal vesicle (GV) stages I-IV], metaphase I, anaphase I or metaphase II and the results were compared with oocytes from fresh follicles of unprimed or gonadotrophin-primed immature mice, and mature mice in oestrus. Small pre-antral follicles grew in vitro to intact pre-ovulatory follicles and some ovulated spontaneously despite negligible luteinizing hormone (LH). Progression through GV stages I-IV in vitro was related to initial and final follicle diameter, follicle growth rate and antrum formation. All the normal stages of GV development were observed in vitro; however, the cultured follicles were significantly larger than the freshly dissected follicles for stages II and III of GV development. GV breakdown had occurred in 7/11 analysable ovulated oocytes and 3/69 intra-follicular oocytes. We conclude that, under the conditions employed, the majority of oocytes cultured in intact immature follicles retain the GV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Growth rates and antrum formation of mouse ovarian follicles in vitro in response to follicle-stimulating hormone, relaxin, cyclic AMP and hypoxanthine.

The culture of individual intact follicles in vitro, from small pre-antral to pre-ovulatory stages, will improve our ability to perform controlled experiments studying follicle growth and female gamete development. This study was undertaken to characterize further the conditions required for physiological follicle growth in vitro. We cultured a total of 398 pre-antral follicles of immature mice (aged 26-28 days) with an initial diameter of 140-340 microns for 4 days in vitro, using individual micro-cultures under paraffin oil. Summarizing the results of all groups, 50 follicles were damaged (12.6%) and, of those remaining intact (n = 348), 60 (17.2%) became atretic, 195 (56.0%) became antral and 26 (7.5%) ovulated. The most advanced follicles grew to 400-500 microns diameter. The presence of follicle-stimulating hormone (FSH) in the medium significantly stimulated follicle growth in vitro (P < 0.03), in a manner proportional to the initial diameter over the range of 140-250 microns initial diameter, with larger follicles being refractory. FSH also significantly increased the proportion of follicles forming antra (P < 0.001) and their likelihood of ovulating in vitro (P < 0.01), and reduced the frequency of atresia (P < 0.01). Dibutyryl-cyclic AMP mimicked FSH, significantly stimulating growth of large follicles (P < 0.05) and antrum formation (P < 0.01). Hypoxanthine also stimulated antrum formation (P < 0.01) but did not significantly affect follicle growth. Porcine relaxin had no significant effect on mouse follicle growth or antrum formation. The optimal conditions for mouse follicle growth in vitro have not yet been defined, but selection of follicles of < 250 microns diameter and inclusion of FSH or dibutyryl-cyclic AMP in the culture medium are recommended.

Animals↗
PubMed1993

Sera from women with unexplained infertility inhibit both mouse and human embryo growth in vitro.

OBJECTIVES: To compare the effect of sera from women with reproductive disorders on the in vitro growth of mouse and human embryos up to the blastocyst stage and to determine the influence on human pregnancy outcome. DESIGN: The growth of mouse embryos and in vitro fertilized human embryos up to the blastocyst stage was compared in sera from women with unexplained infertility, and these results were correlated with pregnancy outcome. Also the growth of mouse embryos in sera from women with a history of recurrent abortions was correlated with their pregnancy outcome. PATIENTS, SETTING: Women with unexplained infertility were attending the IVF Unit at the John Radcliffe Hospital, Oxford, United Kingdom. Women with a history of recurrent abortions were attending the high risk pregnancy unit at the same hospital. MAIN OUTCOME MEASURES: Human embryo growth was monitored by daily morphological assessment and mouse embryo growth by both morphological assessment and tritiated thymidine uptake. RESULTS: In 15 women with unexplained infertility, poor mouse embryo development correlated with poor human embryo quality and impaired blastocyst formation when cultured in the same serum, as well as failure to achieve a pregnancy. In 11 women with a history of recurrent abortion, inhibition of mouse embryo growth correlated with unsuccessful pregnancy outcome. DISCUSSION: Assessment of both morphological development and cell proliferation in mouse embryos may be a useful test to determine the suitability of maternal sera for human IVF embryo culture, predicting pregnancy outcome and for categorizing women with reproductive disorders for future clinical management.

Abortion, Habitual↗
PubMed1993

Human blastocyst grading: an indicator of developmental potential?

We have observed marked variation in the in-vitro development of individual human embryos to the blastocyst stage which was poorly associated with the grade of the embryo on the day of transfer (day 2). Therefore we also graded embryos at the blastocyst stage to determine if their development potential can be better predicted at this stage. A total of 41 blastocysts were categorized into three grades depending on their morphology. Various parameters such as hatching, adherence, growth and amount of human chorionic gonadotrophin (HCG) secreted were documented. The number of nuclei in another 34 blastocysts belonging to the three grades were measured. Also morula stage embryos with large vacuoles but no morphological differentiation were included in a separate group called 'vacuolated morulae'. Overall, there was no significant difference between grade 1 and 2 blastocysts as assessed by their development to day 14, cumulative HCG secretion and nuclei counts. Grade 3 blastocysts reflected 'poor' morphology, secreted significantly lower levels of HCG and had significantly fewer nuclei than the other two grades. Vacuolated morulae did not resemble blastocysts in their morphological development, secreted no HCG and had significantly fewer nuclei than grade 1 and 2 blastocysts. This study illustrates the need for selecting blastocysts for transfer to the uterus or for cryopreservation to allow a better assessment of the success of these techniques. In addition, the data will be useful for evaluating the effects of techniques such as co-culture with feeder cells on the development of embryos to the blastocyst stage and beyond.

Blastocyst↗
PubMed1993

The effect of placental syncytiotrophoblast microvillous membranes from normal and pre-eclamptic women on the growth of endothelial cells in vitro.

OBJECTIVES: To determine if placental syncytiotrophoblast microvillous (STBM) membranes contain factors which could cause the maternal endothelial cell disturbance thought to be central to the pathophysiology of the maternal syndrome of pre-eclampsia. DESIGN: STMB membranes isolated from pre-eclamptic or normal placentae were added to cultures of endothelial cells and their effect on the proliferation (measured by 3H-thymidine incorporation), viability (measured by 51Cr release) and growth as a monolayer of these cells was determined. Membranes prepared from red blood cells, and non-endothelial adherent and nonadherent cell lines were used as specificity controls. SUBJECTS: STBM membranes were isolated from the placentae of primigravid women, 10 having caesarean sections for breech presentations and 10 for pre-eclampsia. RESULTS: STBM membranes from the placentae of normal and pre-eclamptic women suppressed endothelial cell proliferation to a similar extent and disrupted the cell monolayer to form a honeycomb-like pattern. This change in morphology was seen before significant endothelial cell death occurred. Red blood cell membranes had no effect on either endothelial cell proliferation, viability or monolayer integrity. Endothelial cells from human umbilical arteries and bovine adrenal capillaries were similarly suppressed, but comparable concentrations of STBM membranes had no effect on non-endothelial cell lines. CONCLUSIONS: Syncytiotrophoblast microvillous membranes specifically interfered with endothelial cell growth in vitro. Our results demonstrate that there are trophoblast products which could cause the maternal syndrome of pre-eclampsia through endothelial cell damage.

Cell Division↗
PubMed1993

Maternal and fetal immune responses during pregnancy.

Pregnancy in the human presents a paradox for the mother's immune system as the mechanisms which are essential to protect her from infection have the potential to destroy her antigenically foreign fetus. The maternal decidua is comprised principally of immune cells and it is into this tissue that the fetal trophoblast must invade to establish the placenta. The major factor which appears to prevent the rejection of the trophoblast is its expression of HLA-G, a nonpolymorphic transplantation antigen. Both local and systemic nonspecific suppressor mechanisms have been described which may down-regulate maternal immune responses without significantly impairing the ability to fight infections, but there is little evidence to suggest that specific blocking factors (antibodies and suppressor cells) play an essential role. The placental barrier restricts the traffic of cytotoxic cells to the fetus, and cytotoxic antibodies are removed by the placenta before they reach the fetal circulation. Thus a combination of immune adaptations ensures the success of the pregnancy.

Cell Movement↗
PubMed1992

Isolation and characterisation of a subpopulation of human chorionic cytotrophoblast using a monoclonal anti-trophoblast antibody (NDOG2) in flow cytometry.

Human cytotrophoblast cells, isolated from term amniochorion by enzymic digestion and Percoll gradient centrifugation, were characterised by flow cytometry. A panel of 12 anti-trophoblast monoclonal antibodies was screened for labelling of these cells in flow cytometry and the results compared with immunoperoxidase labelling of cytospin preparations and tissue sections. All 12 antibodies were positive for trophoblast on tissue sections, 11/12 were positive on cytospins but only two (NDOG2 and GB25) gave consistent results in flow cytometry. Two-colour labelling with NDOG2 and W6/32, an antibody to HLA-A, -B, -C, demonstrated that 88% of the NDOG2-positive cells also express Class I major histocompatibility complex (MHC) antigens. The NDOG2-positive cytotrophoblast subpopulation was isolated by flow cytometry in sufficient purity (greater than 95%) and yield (3.1 x 10(6)) for use in functional studies in vitro.

Antibodies, Monoclonal↗
PubMed1991

Detection of fetal cells in maternal blood.

We report the detection of fetal cells in the maternal circulation by enzymatic amplification of a single copy gene sequence that was fetal-specific. Fetal HLA-A2-positive cells were sorted from maternal HLA-A2-negative cells by flow cytometry and confirmed by demonstration of a fetal-specific HLA-DR4 sequence. However, this sequence could not be detected in unenriched maternal DNA prepared at 28 and 32 weeks' gestation. The sensitivity of detection was 1 HLA-DR4-positive cell in 10(5) HLA-DR4-negative cells. We conclude that prenatal diagnosis of paternally inherited autosomal-dominant genetic defects may be possible by selective gene amplification of maternal peripheral blood. However, preliminary enrichment for fetal cells may be necessary.

Female↗
PubMed1991

An approach to preimplantation diagnosis of beta-thalassaemia.

Using the polymerase chain reaction (PCR), it was possible to amplify a single copy fragment of the beta-globin gene from 2-32 human embryonic cells obtained from arrested preimplantation embryos. For the detection of beta-thalassaemia mutations, allele specific priming of the PCR using nested primers was employed using approximately 10 pg of DNA from individuals known to carry these mutations. This approach was successful in detecting the presence or absence of five Asian Indian beta-thalassaemia mutations that were selected for this study. In spite of meticulous precautions against contamination, false-positive amplification was observed, a problem that will have to be overcome before this approach can be used in clinical practice.

Base Sequence↗
PubMed1991

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua.

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.

Choriocarcinoma↗
PubMed1991

Human trophectoderm biopsy and secretion of chorionic gonadotrophin.

We have previously developed a technique of trophectoderm biopsy to obtain cells from human blastocysts for preimplantation genetic diagnosis. To determine whether this technique affects the subsequent development of the blastocyst, 45 manipulated blastocysts were observed from days 3 to 14 in culture, the amount of human chorionic gonadotrophin (HCG) secreted by each embryo was measured and these results were compared with those of 26 non-manipulated controls documented in a previous study. A slit was made in the zona pellucida opposite the inner cell mass in 18 of the 45 blastocysts. This increased the rate of hatching but the other morphological changes up to day 14 were similar to those seen in the non-manipulated controls. There was no difference in the mean cumulative HCG secretion by these zona-slit controls (149.8 +/- 45.7 mIU/ml) compared to the non-manipulated controls (146.2 +/- 23.7 mIU/ml). A slit was also made in the zona pellicida of the other 27 blastocysts. Approximately 12-18 h later, in 18 blastocysts a biopsy of the herniating trophectoderm cells (5-30) was performed. The rate of hatching and adherence to the culture dish was similar to the non-manipulated controls. The mean cumulative HCG secretion decreased significantly (57.5 +/- 16.2 mIU/ml, P less than 0.01) after the biopsy procedure. However, if a small biopsy was performed (less than 10 cells removed) the decrease in HCG secretion (87.6 +/- 24.8 mIU/ml) was not significant, whereas when a large biopsy was performed (greater than 10 cells), HCG levels fell to 19.9 +/- 9.1 mIU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopsy↗
PubMed1991

The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro.

Micromanipulation of human oocytes and embryos has provided new opportunities for both the treatment of infertility and the preimplantation diagnosis of genetic disease. It is important to determine whether manipulated embryos develop normally in vitro, as an indication of their suitability for transfer. However, at present there is little information on the development of non-manipulated embryos in vitro for comparison. We have therefore monitored morphological changes and human chorionic gonadotrophin (HCG) secretion in 36 non-manipulated human embryos, including 26 blastocysts and 10 cavitating morulae, daily from day 3 to day 14 of culture. Hatching was observed in 10 (38.5%) blastocysts and five of these adhered to the culture dish and appeared viable until day 14. The secretion of HCG was first detected on day 8, peaked at day 10 (51.11 +/- 8.7 mIU/ml) and then declined but was still detectable in four blastocysts on day 14. There was no overall difference in HCG secretion by hatched blastocysts and those which remained within the zona. However, those hatched blastocysts which showed adherence had significantly increased (P less than 0.05) HCG secretion. For individual blastocysts, the pattern of HCG secretion correlated well with the assessment of morphology. These data provide the basis for comparative studies of morphological changes and HCG secretion in manipulated embryos.

Blastocyst↗
PubMed1990

Flow cytometric characterisation of cell populations in human pregnancy decidua and isolation of decidual macrophages.

Methods have been developed for isolating human tissue macrophages from first trimester or term pregnancy decidua. After a two stage enzymic digestion, viable cells were separated from cellular debris by velocity sedimentation at unit gravity or by Percoll centrifugation. Cell populations were analysed by flow cytometry after labelling with monoclonal antibodies. In term decidua, 47% of the cells were of bone marrow origin, comprising 18% macrophages, 3% large granular lymphocytes and 8% T cells. The remaining cells, the proportion of which varied between individuals, were CD16-positive granulocytes. Macrophages were isolated flow cytometrically from both first trimester and term decidual cell dispersions after labelling with an antibody to MHC class II. Yields of up to 4 X 10(6) macrophages, greater than 95% pure, were routinely obtained.

Antibodies, Monoclonal↗
PubMed1990

Trophectoderm biopsy in human blastocysts.

Trophectoderm biopsy was carried out on 47 human blastocysts. A slit was made in the zona pellucida opposite the inner cell mass by micromanipulative techniques. The human blastocyst zona offered more resistance to slitting compared to that of the mouse. After 18-24 h, controlled herniation of the trophectoderm cells was observed. These cells were biopsied when the diameter of the herniation was approximately equal to that of the blastocyst. The size of the slit and the stage of embryonic development at which slitting was performed were important for successful herniation to occur. After slitting, 76% of day 5-6 blastocysts showed herniation whilst only 42% of day 7-8 blastocysts herniated. Further development of the manipulated embryos was not apparently impaired, as hatching occurred in 44% of the former and 20% of the latter, compared with 18.1% in non-manipulated controls. The biopsied cells (approximately 10-30) usually remained in a clump but 14% formed vesicles on the day after biopsy. There was, however, no evidence of adherence to the dish or formation of monolayers. These results demonstrate the feasibility of trophectoderm biopsy in human blastocysts and that sufficient extra-embryonic material can be obtained by this technique for preimplantation diagnosis of genetic disorders.

Biopsy↗
PubMed1990

Localization of anti-endometrial antibody binding in women with endometriosis using a double-labelling immunohistochemical method.

Anti-endometrial antibody binding was localized using a double-labelling immunohistochemical method on frozen sections of endometrium taken from a woman without pelvic disease. Serum from 40 women with endometriosis was tested and, as controls, serum samples from 20 adult males and 20 umbilical cords. The method allowed compensation for endogenous immunoglobulins in endometrium and accurate localization of anti-endometrial antibody binding in the cytoplasm of the glandular epithelium. Significantly more women with endometriosis (14/40) were found to have anti-endometrial antibodies than controls (1/40) (P less than 0.001; chi 2). There was no correlation between disease severity and the presence of anti-endometrial antibodies or the intensity of staining.

Adult↗
PubMed1990

Cell populations in the human early pregnancy decidua: natural killer activity and response to interleukin-2 of CD56-positive large granular lymphocytes.

Large granular lymphocytes (LGL) have been shown previously to be the most abundant cell type in the first trimester human decidua. Purified populations of decidual LGL were prepared by flow cytometry of cell dispersions labelled with NKH1 (CD56), an antibody specific for peripheral blood LGL, and the functional properties of CD56-positive cells, CD56-negative and unsorted decidual cells compared. Both CD56-positive cells and unsorted decidual cells have cytotoxic activity against the natural killer (NK) cell target K562 which was weak compared with that of peripheral blood mononuclear cells (PBMC). The CD56-negative cells had no cytotoxic activity against K562. All three decidual cell populations proliferated in response to recombinant human interleukin-2 (rIL-2), but none produced detectable levels of IL-2 in culture. When unsorted decidual cells were cultured for 7 days in rIL-2 the proportion of CD56-positive cells increased and NK activity against K562 was augmented. The NK activity of purified CD56-positive decidual cells was also augmented by culturing in rIL-2. The potential role of decidual LGL in regulating the development of the semi-allogeneic placenta is discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Antiendometrial antibodies in endometriosis measured by an enzyme-linked immunosorbent assay before and after treatment with danazol and nafarelin.

No previous study has investigated prospectively the quantitative effect of treatment on antiendometrial antibody levels in endometriosis. We measured antiendometrial antibody levels by an enzyme-linked immunosorbent assay in 35 women with laparoscopically proved endometriosis who were treated for 6 months with danazol (N = 11) or nafarelin (N = 24) in a randomized double-blind study. Levels before treatment were significantly higher than in a control group (P less than .001). Levels were lowered by treatment, but to a significant extent only in the nafarelin group (P less than .001) (danazol group: P = .091). Our data suggest that in relation to antiendometrial antibodies, nafarelin has a suppressive effect which has previously been ascribed only to danazol.

Adult↗