Mrinal K. Sanyal

Preembryo biopsy and analysis of blastomeres by in situ hybridization

We developed a method for the biopsy of preimplantation mouse embryos (preembryos) at the four- to eight-cell stage, which uses partial zona pellucida dissection. The preembryos were collected in calcium- and magnesium-free phosphate-buffered saline solution with 0.01% ethylenediaminetetraacetic acid, 0.1 mol/L sucrose, and 4 mg/ml of bovine serum albumin to facilitate removal of blastomeres. This allows entry of a fine micropipette into the perivitelline cavity with subsequent removal of a single blastomere by gentle suction. The majority of embryos (75%) from which biopsy specimens were obtained in this fashion developed to the blastocyst stage. The blastomeres obtained were mainly intact and they were fixed to glass slides. After permeabilization, in situ hybridization was performed with chromosome X- and chromosome 3-specific probes. Human unfertilized eggs and blastomeres from human polyspermic embryos also have been analyzed by in situ hybridization with chromosome specific probes. The combination of nondestructive embryo biopsy and in situ hybridization is a possible approach for preimplantation genetic diagnosis.

Augmentation of polynuclear aromatic hydrocarbon metabolism of human placental tissues of first-trimester pregnancy by cigarette smoke exposure

OBJECTIVE Polynuclear aromatic hydrocarbons are important components of cigarette smoke. The toxicity of polynuclear hydrocarbons depends on their metabolic biotransformation by tissues. This study was performed to assess the effects of cigarette smoke exposure on polynuclear aromatic hydrocarbon metabolism in the human placental tissues of first-trimester pregnancy. STUDY DESIGN The expression of essential enzymes that metabolize polynuclear aromatic hydrocarbons and regulate toxic metabolism, aryl hydrocarbon hydroxylase, epoxide hydrolase, and glutathione S-transferase, were determined by immunocytochemical staining of the specific enzymes in first-trimester placental samples from both smoker and nonsmoker donors. The overall polynuclear aromatic hydrocarbon metabolism by such tissues was quantitated by a radiometric assay with benzo[a]pyrene substrate in placental villi tissues and also in isolated trophoblast cells or in cultured trophoblast cells in the presence of polynuclear aromatic hydrocarbon agent. RESULTS Immunocytochemical staining revealed that aryl hydrocarbon hydroxylase was localized on trophoblast cells of first-trimester placentas from smoker donors. Epoxide hydrolase was present in stromal and trophoblast cells, and glutathione S-transferase (pi) was present in trophoblast cells of both nonsmoker and smoker subjects. In addition, the overall metabolism of polynuclear aromatic hydrocarbon xenobiotics in such tissues (8 to 11 weeks) of donors who smoked cigarettes was observed to be increased compared with that of nonsmokers by radiometric assay of metabolic products. The increased polynuclear aromatic hydrocarbon metabolism from such exposure was also shown in isolated and purified trophoblast cells of first-trimester placental villi and in culture of such trophoblast cells of nonsmoker donors with polynuclear aromatic hydrocarbon by the same assay procedure. CONCLUSIONS Therefore, contrary to previous assumptions, these data demonstrate that cigarette smoke exposure increases the polynuclear aromatic hydrocarbon metabolism of placentas even during the early stages of pregnancy. Augmented polynuclear aromatic hydrocarbon metabolism may produce genotoxic metabolites deleterious to conceptus development.

Metabolism of polynuclear aromatic hydrocarbon in human term placenta influenced by cigarette smoke exposure.

The relative contributions of biologic and environmental factors on embryo-fetal development were elucidated in a population of pregnant women who were exposed to varying amounts of active cigarette smoke and women who were not exposed to cigarette smoke. The neonatal weight at birth, placental weight at delivery, duration of pregnancy, and placental xenobiotic (polynuclear aromatic hydrocarbon, PAH) metabolism potential were assessed in this population. The overall metabolic capability in exposed and unexposed placental tissue was measured by in vitro assays using microsomes and a PAH substrate, benzo[a]pyrene (B[a]P). Toxicity potential was determined by B[a]P-metabolite-DNA adduct generation under the same incubation condition. Cigarette smoke exposure increased the overall PAH metabolism potential in placental tissues by approximately 200% (nonsmoker 176.2 +/- 33.6, n = 25; smoker 524.5 +/- 75.5, n = 32 pmol/mg protein) whereas PAH-DNA adduct formation potential did not increase significantly over the basal level (nonsmoker 5002 +/- 830, n = 15; smoker 6172 +/- 1443, n = 22 fmol B[a]P equivalent/mumol DNA/mg protein). Exposure to cigarette smoke during pregnancy is deleterious to fetal development as reflected by reduced neonatal weight at birth. In contrast, placental weight reduction is indistinct, but placentae expressed markedly augmented overall xenobiotic (PAH) metabolism capability in response to cigarette smoke exposure during pregnancy, indicating placental metabolism may be an important mediator of adverse effects induced by such xenobiotic exposure.

A hypothesis concerning the general basis of organogenetic congenital anomalies

Evidence supports the idea that it is the degree of metabolic imbalance present in diabetic gravid women during the period of organogenesis that accounts for organogenetic congenital defects. In light of the proved and inferred metabolic instability occurring during early pregnancy, we propose that metabolic imbalances may occur that result in organogenetic congenital defects in offspring of apparently normal gravid women.

HLA-DR antigen on human trophoblast.

ABSTRACT: To assess the presence of human leukocyte antigens (HLA) on first trimester human trophoblast cells, frozen sections of villous trophoblast and monolayer cultures of isolated cells from placental villi were prepared and exposed to a mouse monoclonal antibody directed against HLA‐DR and then incubated with fluorescein‐conjugated goat anti‐mouse antibodies. Fluorescence microscopy demonstrated that HLA‐DR antigens were present on only the small polygonal epithelioid cells of the monolayer culture. The crescentic staining pattern was consistent with widespread distribution of antigen on the cell membrane. There was no staining over giant multinucleated structures or on fibroblasts of such cell cultures. No HLA‐DR was detected when this indirect immunofluorescent technique was applied to tissue sections of villous trophoblast. Existence of high concentrations of hCG in culture supernatants and coincident localization of both hCG and HLA‐DR using antibodies conjugated with rhodamine or fluorescein on the polygonal epithelioid cells indicate the trophoblastic origin and expression of HLA‐DR antigen under in vitro monolayer culture conditions.

Immunoregulatory activity in supernatants from cultures of normal human trophoblast cells of the first trimester.

Supernatants from isolated trophoblast cell cultures (trophoblastic fluid) derived from first-trimester human placentas were assessed for immunoregulatory activity. Trophoblastic fluid at different days of culture consistently inhibited the blast transformation of allogenic lymphocytes. This suppressor effect had no apparent correlation with biosynthesis of human chorionic gonadotropin by trophoblast cells, since this hormone was secreted into the culture fluid only for the initial 3 days. However, the culture fluids of such purified trophoblast cells contained an immunosuppressive factor, pregnancy-associated plasma protein A, which was measurable throughout the culture period of 8 days. The presence of pregnancy-associated plasma protein A in significant amounts in trophoblastic fluid collected at daily intervals indicated a continuous secretion ability of pregnancy-associated plasma protein A by trophoblast cells in culture parallel to the suppressive immunoregulatory effect of the fluid. Such immunosuppressive effect was absent in the culture fluids of control BeWo malignant trophoblast cells; the BeWo cell culture fluids had markedly reduced levels of pregnancy-associated plasma protein A. The culture supernatant of normal trophoblast cells of placentas from first-trimester pregnancy activated in vitro the generation of a population of suppressor lymphocytes. This effect is generally considered responsible for immunologic tolerance. Therefore demonstration of immunosuppressive effects and the presence of relatively high levels of pregnancy-associated plasma protein A in trophoblastic fluid indicate that such proteins secreted by the trophoblast cells may be important in the local immunoregulatory processes of the fetal allograft.

In vitro production of pregnancy-associated plasma protein-A (PAPP-A) by trophoblastic cells

SummaryPregnancy-associated plasma protein-A (PAPP-A) a high molecular weight glycoprotein, is found in high concentration in the maternal circulation during pregnancy. Immunohistochemically, PAPP-A can be localized in the trophoblast and in the decidua. Short term cultures of trophoblastic and decidual explants produce PAPP-A in vitro. It was thus of interest to see if long term cultures of primary monolayers of human trophoblast cells were capable of producing PAPP-A. Under our in vitro conditions, trophoblastic monolayers were producing both PAPP-A and βhCG. During the first 3 days of culture PAPP-A levels increased in the medium whereas βhCG levels decreased. The production of both proteins could be inhibited by cycloheximide. These results strongly suggest that the trophoblast is a source of PAPP-A in vivo.

Insulin modulates neuronal plasma membrane development in human fetal spinal cord neurons in culture

The number of intramembrane protein particles (IMP) in the protoplasmic face of the perikaryal plasma membrane was evaluated in neurons from 9 week-old and 12 week-old human fetal spinal cord in culture. An increased number of IMP was observed in membranes from 12 week-old fetal neurons when compared to membranes from 9 week-old fetal neurons. The addition of insulin (100-2500 microU/ml) to the culture media resulted in a significantly increased number of IMP in neuronal membranes. Incubation with glucose (1.5-6 mg/ml) did not modify the number of IMP and glucose did not potentiate the effect of insulin when both glucose (3 mg/ml) and insulin (500 microU/ml) were added to the culture media. The results suggest that insulin may modulate the development of neuronal membranes and that this effect is not mediated by an increased glucose utilization.