Characterization of pure human first-trimester cytotrophoblast cells in long-term culture: growth pattern, markers, and hormone production

Pure long-term cytotrophoblast cultures were established from human first-trimester placentas by growing chorionic villus explants without enzymatic digestion. Cytoplasmic human chorionic gonadotropin was detectable in all (100%) cells in culture when labeled with a polyclonal anti-human chorionic gonadotropin antibody and in 71% to 83% of cells labeled with a monoclonal anti-alpha-human chorionic gonadotropin antibody. Most of the cells expressed cytokeratin and surface Trop-1 and Trop-2 antigens (89% to 95%), but none expressed cytoplasmic vimentin or surface 63D3 antigens. Study of the ultrastructure of the cells demonstrated epithelial morphologic features. The average doubling time of the trophoblast was 48 to 96 hours. Some of the lines have been continuously propagated for 8 months. They produced variable amounts of human chorionic gonadotropin (50 to 710 mIU/ml per 10(5) cells per 24 hours). The basal level of progesterone secreted by trophoblast (444.4 +/- 32.4 pg/ml per 10(5) cells per 24 hours) doubled in the presence of pure human chorionic gonadotropin (100 ng/ml). They produced small amounts of 17 beta-estradiol (less than 20 pg/ml per 10(5) cells per 24 hours); human chorionic gonadotropin had no effect on the estradiol production. Trophoblast-derived human chorionic gonadotropin acted as a growth factor because trophoblast proliferation (measured by uptake of thymidine labeled with tritium) was reduced by 60% in the presence of an anti-human chorionic gonadotropin antibody. Availability of pure, functionally competent human cytotrophoblast in long-term cultures is relevant for further studies in reproduction biology.     

Human placental cytotrophoblast cells: identification and culture

Summary Methods of disaggregation of human placental tissue were assessed with the aim of maximising the yield of cytotrophoblast cells and minimising contamination with other cell types. Brief exposure to crude trypsin was found to be the best way to balance yield of trophoblast cells against contamination by cells of the villous core. Much higher yields of all cell types could be obtained by digestion with other enzymes. Staining for NADH diaphorase activity coupled with general morphology was found to be a reasonably specific, rapid and simple method of distinguishing cytotrophoblast cells in disaggregated mixtures. Alkaline phosphatase activity was an unreliable marker of trophoblast tissue in early placentas, and of the putative cytotrophoblast cells in mixtures of disaggregated cells. Cultures of cells obtained from term placentas were fairly homogeneous, whereas placentas of 6–12 weeks gestation gave heterogeneous cell cultures which became overgrown with fibroblasts.     

Inwardly rectifying K(+) current and differentiation of human placental cytotrophoblast cells in culture

Ion transport is important for driving nutrient transport across the syncytiotrophoblast and yet is poorly understood. We have examined K(+)currents under basal conditions in cultured cytotrophoblast cells, at various stages of differentiation, using the whole cell patch clamp technique. Cytotrophoblast cells were isolated from human term placenta and maintained in culture for up to 3 days. Cells were studied at four stages of progressive morphological differentiation: (i) mononuclear cells, (ii) mononuclear cells in aggregates, (iii) small multinucleate cells and (iv) large multinucleate syncytiotrophoblast-like cells. In the conditions of whole cell recording the only K(+) selective current identified in all cell types was a strong inwardly rectifying current which was sensitive to Ba(2+) and Cs(+). This current was unaffected by intracellular ATP whereas intracellular GTPgammas caused either run down of the current or activated a linear current. The characteristics of the current described are consistent with those of the inwardly rectifying K(+) channel Kir2.1. The inwardly rectifying K(+) current was observed in three out of 19 (16 per cent ) mononuclear cells, seven out of 21 (33 per cent ) mononuclear aggregates, eight out of 21 (38 per cent ) small multinucleate cells and 16 out of 19 (84 per cent ) large multinucleate cells. This inwardly rectifying K(+) current is likely to have an important role in determining net K(+) diffusion across the syncytiotrophoblast cell membrane, perhaps increasing in importance as the cells terminally differentiate.     

Effect of in vivo GnRH agonist and GnRH antagonist on hCG and insulin-stimulated progesterone production by human granulosa-lutein cells in vitro

Purpose : To investigate hCG and insulin-stimulated progesterone (P) production by human granulosa-lutein cells (hGLC) in vitro. Methods : hGLCs were isolated from patients undergoing IVF-ET cycles in which GnRH agonist or GnRH antagonist was used to prevent a midcycle gonadotropin surge. The cells were cultured for 3 days, and then treated with hCG 0.5, 1, and 10 IU/I, and insulin 0.01, 0.1, and 1 M in serum free conditions. In vitro P production was measured by enzyme immunoassay. Results : hCG stimulated P production by hGLCs from cycles in which GnRH antagonist was used, but a blunted response was seen in GnRH-agonist treated cycles. Insulin-stimulated P production was similar in cells from cycles in which GnRH-agonist or GnRH-antagonist treatment was used. Conclusions : Because insulin and hCG may share common pathways beyond the level of receptor activation, we hypothesize that GnRH agonist, but not GnRH antagonist, may affect the expression and/or activation of LH receptors in the hGLCs.     

Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A

AIM: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. RESULTS: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 microg/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. CONCLUSIONS: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.     

Prostacyclin production by human placental cells in short-term culture

Human placentae of varying gestational ages have been cultured in vitro with little variation in cell type and pattern of growth found. Cell types found are similar morphologically and histochemically to those previously described. The biological specificity of the cells in culture was also confirmed by human placental lactogen production. Placental cells in culture appear to synthesize and release PGI2 which can be identified by gas chromatography-mass spectrometry. Production of PGI2 was routinely measured by radioimmunoassay (RIA) for 6-oxo-PGF1alpha and 13,14-dihydro-6,15-dioxo-PGF1alpha in culture supernatants. Good agreement was found between RIA and gas chromatography-mass spectrometry measurements. PGI2 production in culture was not affected by mode of delivery, and synthetic capability was found to increase with gestational age. Production of PGI2 by cells from preterm and term placentae was similar but significantly greater than that of first-trimester cells. As the proportion of PGI2 produced in culture supernatants as 13,14-dihydro-6,15-dioxo-PGF1alpha changed with time of incubation, it appears pertinent to measure this metabolite when assessing total PGI2 production. Synthesis of PGI2 wa inhibited by the cyclo-oxygenase inhibitors indomethacin and aspirin and PGI2 synthetase inhibitor 15 hydroperoxyarachidonic acid. However, in the culture system tranylcypromine, a putative specific inhibitor of PGI2 synthetase, produced weak inhibition only before becoming cytotoxic. The cell culture system appears to offer a reliable and reproducible means for measuring placental PGI2 production in vitro and in which to study factors controlling its production and metabolism.     

Effect of ketoconazole on steroidogenic human granulosa-luteal cells in culture

Ketoconazole (KCZ), a widely used antifungal drug, has been reported in humans to inhibit adrenal and testicular steroidogenesis by interfering with the cytochrome P-450-dependent enzymes. The purpose of this study was to investigate the drug effect on steroidogenic human granulosa-luteal cells, obtained by follicular aspiration from mature follicles of gonadotropin-treated women. Cells were cultured in long-term monolayers, and the steroid production was assayed by radioimmunoassay. A profound inhibition of ovarian cell secretion of progesterone (P), testosterone (T) and estradiol was found. At a low concentration (5 micrograms/ml), KCZ failed to inhibit the conversion of pregnenolone to P, mediated by the non-cytochrome 3 beta-hydroxysteroid dehydrogenase-isomerase enzyme (3 beta-HSDH). At a similar concentration, P secretion by human chorionic gonadotropin (hCG; 100 mIU/ml) -treated cells was decreased by 68% (P less than 0.001) and therefore, an inhibitory effect of KCZ on the cholesterol side-chain cleavage enzyme (P-450SCC) was assumed. A similar marked inhibitory effect (81%) (P less than 0.001) on T secretion was observed for hCG-stimulated cells given pregnenolone as substrate. The P-450 aromatase was profoundly inhibited (86%) (P less than 0.001) in a reversible manner, by a similar concentration (5 micrograms/ml) of KCZ. These findings suggest that KCZ has the capability to suppress human ovarian steroidogenesis similarly as in testis and adrenal.     

Oxygen tension directs the differentiation pathway of human cytotrophoblast cells

During placental development, human cytotrophoblast cells can differentiate to either villous syncytiotrophoblast cells or invasive extravillous trophoblast cells. We hypothesize that oxygen tension plays a critical role in determining the pathway of cytotrophoblast differentiation. A highly purified preparation of cytotrophoblast cells from human third trimester placenta was cultured for 5 days in either 20% or 1% oxygen tension. The cells incubated at 20% oxygen formed a syncytium as determined by immunohistochemistry using an anti-desmosomal protein antibody that identifies cell membranes. In addition, the mRNA was markedly induced for syncytin, a glycoprotein shown to be essential for syncytiotrophoblast formation, and for human placental lactogen (hPL), which is a specific marker for syncytiotrophoblast cells. In contrast, the cell incubated at 1% oxygen tension did not fuse by morphologic analysis and did not express syncytin or hPL mRNA. However, these cells expressed abundant amounts of HLA-G, a specific marker for extravillous trophoblast cells, which was not seen in cells incubated at 20% oxygen tension. These results suggest that low oxygen tension directs differentiation along the extravillous trophoblast cell pathway while greater oxygen tension directs differentiation along the villous trophoblast cell pathway.     

抗子宫内膜抗体对人早孕绒毛滋养细胞凋亡及FasL蛋白表达的影响

目的:探讨体外培养人早孕绒毛滋养层细胞在游离抗子宫内膜抗体(Em-Ab)环境中,细胞凋亡指数(apoptosis index,AI)及凋亡相关因子配体(factor associated suicide ligend,FasL)的表达。方法:培养经胰蛋白酶/DNA酶Ⅰ联合消化,通过Percoll细胞分离液纯化得到绒毛滋养细胞。用TUNEL法测定EmAb(+)血清处理组和EmAb(-)血清处理组绒毛滋养细胞培养0、48、72、96h时的细胞凋亡指数,同时用免疫细胞化学法测定FasL蛋白的表达。结果:EmAb(+)血清处理组于培养72、96h时间点测得的AI明显高于EmAb(-)血清处理组(P<0.01);FasL蛋白表达明显低于EmAb(-)血清处理组;而培养48h AI及FasL蛋白表达无显著差异。结论:绒毛滋养细胞在EmAb(+)环境下AI增加,FasL蛋白表达减少。     

Effect of cryopreservation on the properties of human endometrial stromal cells used in embryo co-culture systems

Purpose

Along with comparative investigation of the decidualization potential and IL-6 secretion by fresh and frozen ESCs, we also aimed to evaluate the effectiveness of co-culture systems based on fresh or frozen ESCs in terms of clinical pregnancy rates.

Methods

Outcome analysis of a total of 215 IVF cycles with co-culture with fresh or frozen ESCs was performed. Endometrial tissue was obtained from 17 healthy donors. Concentrations of secreted prolactin, IGFBP-1, and IL-6 in conditioned media from cultured fresh and frozen ESCs (decidualized or not) were measured using ELISA or ECLIA.

Results

Embryo co-culture with frozen ESCs resulted in a much lower pregnancy rate compared to the alternative system using fresh ESCs. Furthermore, cultivated frozen ESCs showed considerably decreased release of prolactin, IGFBP-1, and IL-6 compared to fresh ESCs, indicating that cryopreservation negatively affects their decidualization potential and cytokine production.

Conclusions

Altogether, this data illustrates the need for optimization and improvement of the existing autologous endometrial co-culture systems.

     

激活素A和卵泡休止素对人滋养层细胞增殖和激素分泌的调节作用

目的: 探讨激活素A和卵泡休止素对人早孕绒毛滋养层细胞的增殖和人绒毛膜促性腺激素及孕酮分泌的调节作用。方法: 早孕绒毛用胰蛋白酶与胶原酶联合消化后,再用小牛血清白蛋白密度梯度离心,分离纯化后所得的滋养层细胞进行体外原代培养。将不同浓度激活素A加入细胞培养液中作用48 h,观察其对滋养细胞生长和人绒毛膜促性腺激素及孕酮分泌的影响。向滋养层细胞培养液中加入激活素A和不同浓度的卵泡休止素,培养24 h,观察相互作用。结果: 激活素A和卵泡休止素都不影响滋养层细胞的增殖。激活素A以剂量依赖的方式促进滋养层细胞绒毛膜促性腺激素的分泌和孕酮的分泌,滋养细胞分别经30 ng/mL,100 ng/mL激活素A处理后,培液中绒毛膜促性腺激素和孕酮水平达到高峰(P均<0.01)。激活素A对滋养层细胞激素分泌的影响,可以被其特异结合蛋白卵泡休止素以剂量依赖方式阻断。结论: 激活素A和卵泡休止素以自分泌方式调节滋养层细胞绒毛膜促性腺激素和孕酮分泌,但并不影响细胞增殖,激活素A与卵泡休止素在人早期妊娠中起重要的生理作用。     

Effects of melatonin on progesterone production by human granulosa lutein cells in culture.

OBJECTIVE: To test the hypothesis that melatonin modulates steroid synthesis in the human ovary. DESIGN: Granulosa lutein cells obtained from in vitro fertilization cycles were cultured in medium containing melatonin and human chorionic gonadotropin (hCG). RESULTS: Progesterone (P) secretion by granulosa lutein cells increased progressively in both basal and hCG-stimulated conditions, up to 96 hours in culture, plateaued at 144 and decreased thereafter. Melatonin (10(-7), 10(-9), 10(-11) M) had no effect on basal P or 17 beta-estradiol production. The addition of melatonin to the hCG-treated granulosa lutein cells significantly (P less than 0.05) potentiated the stimulatory effect of hCG on P production. The effect was most prominent after 144 and 196 hours of incubation. CONCLUSION: This observation suggests a role for melatonin in the intraovarian control of P production in the human ovary.     

Identification of cytotrophoblast colonies in cultures of human placental cells using monoclonal antibodies

The morphological appearance of four distinctive types of colony in human placental cultures is described. Using a panel of monoclonal antibodies, one of these colonies is found to comprise cells with the phenotypic characteristics which allow them to be defined as cytotrophoblast with a reasonable degree of certainty. A second type of colony also contains cells of epithelial origin, but the trophoblast lineage of these cells is more difficult to ascertain. The remaining two types of colony are derived from mesenchymal elements representing either macrophages or fibroblasts. It is hoped that the precise identification of cytotrophoblast colonies will provide a useful yardstick to assess the efficiency of any future techniques which are devised for the selective growth and propagation of human trophoblast in vitro.     

Effect of cryopreservation on quality and fertilization capacity of human sperm

OBJECTIVE: To analyze retrospectively the effect of cryopreservation on donor's sperm. STUDY DESIGN: Data were collected on 178 cryopreserved-thawed sperm specimens from 44 donors and 624 oocytes from 62 women, which underwent in vitro fertilization-embryo transfer (IVF-ET) treatments with donor's sperm. Data on fresh sperm, 175 sperm specimens from 76 couples which underwent IVF-ET treatments, were used as a control group. Semen analysis was done by cell concentration, percent of motility, and quality of motility according to the World Health Organization (WHO) recommendation. Sperm quality parameters which had the strongest impact on fertilization capacity were determined using the statistical response surface model and conjoint analysis. RESULTS: Passing sperm through Percoll column decreased sperm concentration, with no improvement in sperm motility but with a slight increase in quality of motility. Quality of motility of donor's sperm had the strongest impact on fertilization capacity. CONCLUSION: Current freezing-thawing protocols of sperm cause a decrease in sperm parameters without affecting fertilization capacity. Furthermore, quality of motility of frozen-thawed sperm seems to be a significant measure of sperm fertilization capacity.