| Objective (1) A minimally invasive approach was used to obtain fetaltrophoblast cells from endocervix and to investigate the presence oftrophoblast cells.(2)To investigate feasibility of trophoblast cells culture in vitro by applicationof differential adherent culture of the cells obtained from women in earlypregnancy.(3) Sort, proliferate and purify fetal trophoblast cells by combination ofdifferential adherent culture and immunomagnetic beads method.Investigate the efficiency of this method in enriching fetal trophoblast cell.(4)To investigate the application feasibility of fetal trophoblast cells fromendocervix in noninvasive prenatal diagnostic in early pregnancy.Methods (1) Selecting170pregnant women who visit the obstetricout-patient service in the first affiliated hospital of Anhui medical Universityduring October2010to May2011as subjects in this experiment (include30cases of preliminary experiment,120cases of normal pregnancy requiring thetermination of pregnancy,20cases of Embryos stops growing). They meet thefollowing conditions：menopause，urine pregnancy test positive，gynecologicalexamination and B-mode ultrasonography confirmed in early pregnancy andare volunteer to terminate pregnancy. Combined the last menstrual andultrasound method to calculate gestational age. If the embryo length can bemeasured by ultrasound, formulas for gestational weeks=embryo length (cm)+6.5, or calculated according to the maximum diameter of the gestationalsac, formula for gestational weeks=gestational sac maximum diameter (cm)+3. Early embryo stops specimens is arrested embryos in B ultrasound (nopulse tube, cannot see the germ and so on). After getting their informedconsents, obtained cervical canal cells noninvasively before artificial abortion by using the special cell brush. At the same time acquisited thecorresponding chorionic tissues sterilily for chorionic villus cell culture.Cervix exfoliativied cells were obtained by washing the cell in sterile saline.Proper amount of collagenase was added to digest cervical mucus in thespecimen. Cervix trophoblast cells were identified after HE stain.(2) Differential adherent culture method was used to culture the cervical cellspecimens and increase trophoblast cell ratio. After2-3days culture, cellswere proliferating. Successful cultured cell suspension specimens of normalpregnancy group of67patients were treated with HE stain. Identify theexistence and ratio of trophoblast cells in specimens.(3) MACS method was used to sort out of the fetal trophoblast cells fromspecimens which have more cell positive ratio and volume. Compare the ratioof trophoblast cell before and after MACS.Results1. Results of pre-experiment(1) Trophoblast cell was found in19cases in30specimens which were getform early pregnancy group and treated by HE stain, the positive ratio was63.3%. The average number of visible trophoblast cells was7observed undermicroscope.(2) In19specimens,15cases were successfully cultured after differentialadherent culture. Trophoblast cell was found in8cases after the followed HEstain. Successful ratio of trophoblast cell culture between the two groupwere compared through Chi-square test,2=5.53， p<0.05. The averagenumber of visible trophoblast cells was32.4observed under microscope. Thenumber is4.6-fold of that before differential adherent culture.2. Results of formal experiment(1) In120cases of normal early pregnancy specimens stained by HE,trophoblast cells were positive in81cases (67.5%) and negative in39cases(32.5%); specimen positive rate (trophoblast persists) in each gestationalweek was compared by using the chi square test(χ~2),2=1.68P=0.641>0.05,they has no statistically significant difference. Analysis of variance for the trophoblast cell number of each gestational week positivespecimens, F=4.636, P=0.005<0.05, the collection of positive samples in thetrophoblast cell number difference has statistics significance. Number oftrophoblast cell in specimen increased with the gestational age in6-10weeksof gestation. Trophoblast cell was found in10cases in20specimens whichwere get form in early pregnancy embryo stop group and treated by HE stain,the positive ratio was50.0%. The average number of trophoblast cell inspecimen increased with the gestational age. The number of trophoblast cellof normal early pregnancy group and early pregnancy embryo stop groupspecimens in gestational age has no significant difference by using one-wayANOVA analysis.(2) Chi-square test (2) was used to study success rate of specimens whichtreated by differential adherent culture in each gestational week,2=0.291,P=0.962>0.05, the difference was not statistically significant. This resultindicate that culture success rate of specimen in different gestational age aresimilar. In the specimens which were successfully cultured, the chi-squaretest for positive samples rate of differential adherent of each gestationalweek from2=19.009, P=0<0.005, the difference was statisticallysignificant. With gestational weeks increasing, the trophoblast cells`successrate of differential adherent culture is decline according to the positivespecimen rate after differential adherent culture. In10positive specimens ofearly pregnancy embryo stop group,4cases were successfully cultured afterdifferential adherent culture. No trophoblast cell was found after thefollowed HE stain.(3) HE stain in cell suspension specimens of67cases by successful differentialadherent, fetal trophoblast cells present in36cases (specimens positive rate53.7%, trophoblastic cell number≥1). Analysis of variance for comparingtrophoblast cell number of each gestational week positive samples bydifferential adherent culture, F=2.139, P=0.115>0.05, the difference wasnot statistically significant; However, analysis of variance for comparingtrophoblast cell ratio of each gestational week positive samples bydifferential adherent, F=4.374, P=0.011<0.05, the difference was statistically significant.(4) Fifteen HE stain positive specimens (trophoblast ratio≥15%) aftersuccessfully differential adherent culture, were purified by immunomagneticseparation (MACS). The purified cells were treated by HE stain to identify theratio of trophoblastic cell in specimen. One-way ANOVA anaylsis was used tocompare the average trophoblastic ratio before and after MACS. F=6.061，p=0.02<0.05, the difference was statistically significant.Conclusions(1) Trophoblastic cells of embryonic origin from fetal are presentin the cervical canal of early pregnancy women. Trophoblast cells are stablein6-10weeks of gestation and not affected by the quality of embryonicdevelopment.(2) Normal early pregnancy women have a higher success culturing rate oftrophoblast cell, compared with early pregnancy embryo stop women.(3) Short-term culture could increase the number and ratio of trophoblasticcell from cervical canal, but it reduced the positive rate of specimen.(4) Trophoblast cells can be efficiently enriched by combination ofdifferential adherent and immunomagnetic beads sorting cell method. It haspossibility in applying in early pregnancy minimally invasive rapid prenataldiagnosis.(5)The method of isolation and purification of fetal trophoblast cells fromendocervix in early pregnancy by the immunomagnetic microbeads is feasible,but it still need higher specificity monoclonal antibodies to raise theseparation efficiency. |
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