A critical evaluation of the magnetic cell sorter and its use in the positive and negative selection of CD45RO+ cells.

In this paper, we report on our year-long experience with the magnetic cell sorter (MACS), and present a critical evaluation of its pitfalls and benefits. Satisfactory separation of lymphocytes into subsets with preservation of function can be achieved, but there are several drawbacks: in comparison with Dynal beads, MACS results in a higher cell loss due to the increased number of separation steps and requires depletion of plastic-adherent cells as these will engulf microbeads and contaminate the enriched fraction, and is more expensive. The advantage of MACS over Dynal beads is that the microbeads are biodegradable and do not interfere with proliferation assays: both the depleted and enriched fractions of cells can therefore be used in culture immediately following separation. We used MACS for the positive and negative selection of CD45RO cells: the enriched fraction was of high purity (greater than 98%), but a depleted fraction of greater than 90% purity could not be obtained even after running the same sample over three separating columns. Dynabeads, on the other hand, achieved 98% pure CD45RO-depleted fractions after three separation runs.

Collagen phagocytosis by human extravillous trophoblast: potential role in trophoblastic invasion.

OBJECTIVES: To study collagen phagocytosis by human extravillous trophoblast. METHODS: First-trimester extravillous trophoblastic cell lines and primary trophoblast cell preparations were cultured in vitro with collagen-coated fluorescent latex beads and fluorescent-labeled collagen. Confocal microscopy was used to demonstrate internalization of collagen and beads. The effect of cytochalasin B, temperature, metabolic inhibitors, and cytokines was studied by culturing trophoblast cells with tritiated collagen. Acridine orange was used to stain for lysosomal compartments, and histochemical methods were used to demonstrate acid phosphatase in trophoblast cells. RESULTS: Both cell lines and primary culture cells internalize collagen and beads. Confocal microscopy unequivocally localized the phagocytosed material to the intracellular compartment. Inhibition by cytochalasin B and culture at 4C of uptake of [3H] collagen suggested that the process was phagocytosis. Cytokines and growth factors did not affect phagocytosis. Lysosomal compartments and acid phosphatase appear to colocalize. CONCLUSIONS: The continuous remodeling and turnover of collagen which occur in a wide variety of tissues under both physiologic and pathologic conditions are thought to be mediated by two pathways: one external (involving release of proteolytic enzymes), and the other internal (involving phagocytosis). Similar remodeling events are likely to occur during trophoblast invasion. Although current views emphasize the importance of the extracellular pathway, we postulate, on the basis of our findings, that both pathways are used, with the internal pathway probably being dominant. We hypothesize that the proteolytic enzymes (extracellular pathway) disrupt collagen matrices, thereby facilitating phagocytosis. It is teleologically sound to conceive of a dominant intracellular pathway, as it allows for more precise control of the process of invasion and is economical, as the products of collagen degradation can be used as energy sources or building blocks.

Effect of 17 beta-oestradiol on lymphocyte subpopulations, delayed cutaneous hypersensitivity responses and mixed lymphocyte reactions in post-menopausal women.

High-dose steroids are known to be potent modulators of the immune response. We accordingly investigated the effect of therapeutic doses of 17 beta-oestradiol (E2) on cellular immune responses in post-menopausal women. Fifteen (15) healthy women who had undergone a natural menopause were treated with E2 in the form of 100 mg estraderm patches applied twice weekly for 3 out of every 4 weeks over a 3-month period, followed by combined oestrogen and progestogen formulations as long-term therapy. Blood samples were taken on two occasions prior to treatment and at weeks 1, 3, 4, 7, 9, 12 and 24 after commencing therapy. Lymphocyte subsets (CD2, CD4, CD8, CD19, HLA-DR and NK) were studied in each blood sample using a monoclonal antibody kit and a two-colour fluorescence flow-cytometer. One-way mixed lymphocyte reactions (MLRs) were performed using the same stimulator throughout. Delayed hypersensitivity skin tests (DHTs) were carried out twice before treatment and at weeks 3, 4, 12 and 24 using Multitest 7-antigen kits (Institut Mérieux). Lymphocyte subsets did not change significantly with treatment, but both the MLRs and the DHTs were significantly depressed, maximally so by the third week of treatment. We conclude that therapeutic doses of E2 modulate certain immune responses. The significance of this is discussed in the light of the increasing use of long-term oestrogen replacement therapy.

Obstructive uropathy from endometriosis after hysterectomy and oophorectomy; two case reports.

Two patients with obstructive uropathy as a result of post menopausal endometriosis are reported. They both illustrate the potential problems of hormone replacement therapy after bilateral salpingo-oophorectomy and hysterectomy in patients who have had extensive endometriosis. It is suggested that in such patients the potential of reactivation of the disease with hormone-replacement therapy should be recognised and ureteric involvement be excluded by intravenous urography or renograms.

Massive obstetric hemorrhage due to placenta previa/accreta with prior cesarean section.

There is a high association between anterior placenta previa, placenta accreta and previous cesarean section. We report three cases which illustrate the particular danger of massive hemorrhage posed by placenta previa/accreta in a scarred uterus. As the incidence of cesarean section continues to rise worldwide, the problem of placenta previa/accreta is likely to become more common. We emphasize the need for each obstetric unit to have a protocol for dealing with massive hemorrhage.

Red cell distribution width (RDW) changes in pregnancy.

OBJECTIVE: As part of an exercise in establishing normograms of hematological parameters in pregnancy, we studied the red cell distribution width (RDW) in healthy pregnant women. METHODS: A longitudinal study of RDW measurements in 121 pregnant women at 16 and 34 weeks gestation and during labor and at Days 3 and 7 postpartum. All the women had uncomplicated pregnancies, minimum hemoglobin (Hb) of 11.0 g/dl at recruitment and took iron supplements from 16 weeks of gestation and until 7 days after delivery. All subjects went into spontaneous labor, 110 achieving a normal vaginal delivery while the remaining 11 were delivered by cesarean section. Two-way analysis of variance was used to study the changes in RDW between any given gestations to test the variability between and within subjects. RESULTS: RDW increased significantly (P < 0.0001) between 34 weeks of gestation and the onset of labor. No significant changes occurred between 16 and 34 weeks gestation, or during the 7 days postpartum. CONCLUSION: This is the first longitudinal study analyzing the between and within women changes in RDW with progression of pregnancy. The unexpected rise in the RDW during the last 4-6 weeks leading up to the onset of labor suggests increased bone marrow activity. The stimulus is unknown, but as RDW changes are highly significant there may well be a useful indicator of impending parturition.

Third consecutive complete posterior uterine rupture. A case report.

BACKGROUND: Management of pregnancy in a woman who has had a ruptured uterus on more than one occasion presents a great clinical and ethical challenge to the obstetrician. CASE: This appears to be the first report on complete uterine ruptures in three consecutive pregnancies. CONCLUSION: Prolonged hospital admission, intensive antenatal surveillance, antenatal steroid administration and elective premature delivery may give the best chance for a good outcome in these pregnancies. However, despite all these efforts, there is still a high risk of an unfavorable outcome.

Low participation rates amongst Asian women: implications for research in reproductive medicine.

The last two decades have witnessed tremendous advances in the field of reproductive medicine, especially assisted reproductive technology and stem cell research. As research continues in future, it is vital to ensure that individuals from all ethnic backgrounds are represented in the study populations so that the findings of the research can be generalised for the benefit of all. Many studies, however, have noted a trend of low participation rates amongst Asian women in reproductive research. Inequalities in the ethnicity of research participants can be a source of substantial bias, and have major ethical and scientific ramifications. Several factors such as educational status, fear of wrong-doing, communication barriers, and socio-cultural beliefs have been suggested to play a role. There is a need for further exploration of the factors influencing Asian women's decision to accept or decline participation in reproductive research and for development of effective targeted strategies for research recruitment with the aim of encouraging research participation as well as donation of cryopreserved embryos or other reproductive tissues.

The hysterectomy story in the United Kingdom.

Gynaecologists in India should be deeply concerned by the message broadcast on the BBC by Jill McGivering (6(th) February 2013) in which she suggested that hysterectomy is abused in India, with the overwhelming number of women being subjected to the operation unnecessarily. Their counterparts in the UK should be no more complacent because although the hysterectomy rates have fallen over the years, yet there is widely and wildly varying rates between regions, hospitals and individual gynaecologists. Until research can be undertaken to establish what the true rates of hysterectomy should be, clear simple guidelines could go a long way to ensuring that women are not subjected to an operation they do not need, while those who would benefit are not denied an intervention that can improve their quality of life.

The phagocytic activity of human first trimester extravillous trophoblast.

It has been suggested previously that phagocytic activity in the human placenta is confined to cells of the macrophage lineage. However, earlier studies were hampered by the paucity and poor viability of cells inherent in primary trophoblast cell cultures, contamination by other cell types which themselves have phagocytic activity, lack of reliable markers of trophoblasts, and by limitations of methods available to demonstrate unequivocally the internalization of particulate material. We have overcome these limitations by using: (i) DNA transfection to provide unlimited supplies of pure trophoblast cell lines; (ii) human placental lactogen as a marker unique to trophoblast; and (iii) confocal microscopy to demonstrate unequivocally the intracellular locality of phagocytosed material. We found that both untransfected primary culture extravillous trophoblast cells, as well as the cell lines, had the capacity to phagocytose sheep red blood cells, Staphylococcus aureus and baker's yeast cells, and that this activity was inhibited by cytochalasin B and by culture at 4 degrees C. Phagocytic activity in trophoblast cells was less avid than that seen in a professional phagocyte. In physiological and pathological situations where tissue remodelling occurs, such as the rapid turnover in the periodontal ligament or during inflammation, epithelial cells and other cells that are not considered professional phagocytes actively phagocytose components of the extracellular matrix. We postulate that phagocytosis by human trophoblasts may play an important role in the extensive tissue remodelling that occurs during trophoblastic invasion of the decidua.

The effect of delayed pushing in the second stage of labor with continuous lumbar epidural analgesia.

We studied primigravid women in spontaneous labor at term and given epidural analgesia. Two hundred such women giving birth in 1983 were compared with similar groups who gave birth in 1985 and 1987 after the introduction of 'delayed pushing' into our labor ward protocol. Four hundred controls were obtained in 1983 and 1985 by selecting from the labor ward register the next normal primigravid woman in spontaneous labor without epidural analgesia. The assisted delivery rate was significantly higher in all three epidural groups than in the controls (p less than 0.001). Among epidural labors, there was no significant difference between 1983 and 1987 in the incidence of rotational or non-rotational forceps, or of cesarean section. In each epidural group, women who waited more than 60 min were less likely to experience a normal spontaneous delivery than were those who did not (p less than 0.001 in 1983; p = 0.006 in 1985 p = 0.035 in 1987). We conclude that to delay pushing beyond 60 min confers no benefit regarding mode of delivery.

Efficient, rapid and reliable establishment of human trophoblast cell lines using poly-L-ornithine.

OBJECTIVE: Human trophoblast cells in primary culture are difficult to use for the rigorous study of trophoblast function because of contamination with other cell types, paucity of numbers, poor viability and inter-experiment variation engendered by the need to prepare fresh cells for each experiment. DNA-transfection to produce immortalized cells, or cells with extended life-span, has been the obvious approach to solve this problem. Although there have been a few reports in the literature describing trophoblast cell lines generated in this way, it is clear that to date the methods are difficult and few lines have been generated. The basic problem is that transfection efficiencies of different methods are cell type-specific. The objectives of this study were therefore to compare the transfection efficiencies of three commonly used techniques, to use the best technique to generate trophoblast cell lines, and to conduct preliminary characterization studies. METHODS: We have compared calcium phosphate co-precipitation, DEAE-dextran and poly-L-ornithine (PLO) DNA transfection protocols. We then used the most efficient to transfect human extravillous trophoblast with pSV3neo. RESULTS: Our modification of the PLO method has a transfection efficiency greater than 30 times that of the next best method. Several cell lines were established which had an extended life span and displayed an invasive phenotype, including the expression of MHC Class I framework antigens, human placental lactogen and human chorionic gonadotrophin, and thus have characteristics of extravillous trophoblast. In addition these cells express the integrin subunits b1, a1, and a3, all of which are known to be expressed in human trophoblast, and respond to IL-1alpha by increased secretion of GM-CSF. CONCLUSIONS: PLO is a highly efficient, rapid, reliable, simple and low-cost technique for the procurement of human trophoblast cell lines which retain most, if not all, the phenotype of the parental cell. These lines are potentially powerful tools in the rigorous study of trophoblast function.

The influence of suture material on vaginal vault granulations following abdominal hysterectomy.

A questionnaire was circulated to 1147 members (consultant status) and fellows of the Royal College of Obstetricians and Gynaecologists to survey the current management of the vaginal vault following abdominal hysterectomy. Of the 56% who responded, the majority (87%) routinely close the vault. An interrupted mattress suture is used by 60%, a continuous suture by 43% and the Lembert suture by 2%. Chromic catgut is favoured by 60%, a synthetic polymer by 33% and plain catgut by 6%. We then organized a prospective, randomized trial involving 114 women who underwent total abdominal hysterectomy for benign indications. No. 1 polygalactide (Vicryl) was used in 58 women, and No. 1 chromic catgut in 56. Overall, 37 (32%) had vault granulations 6 weeks post-operatively. Chromic catgut had been used in 25 (68%) and polygalactide in the remaining 12 (32%) (P = 0.01). Only 2 (5%) of these women denied any symptoms, while the rest complained of vaginal discharge, bleeding or both. Neither the patients' age, weight or parity, nor the status of the surgeon influenced the occurrence of granulations. We conclude that the widespread use of chromic catgut revealed by our survey is unacceptable: polygalactide, a synthetic polymer, is associated with fewer vaginal vault granulations than chromic catgut.