Table 1.
Characteristics of the primers used for SYBR green Q-PCR assays.
Table 2.
Antibodies used in the study.
Figure 1.
Quantitative RT-PCR of normal pregnancy and miscarriage placenta.
Data are expressed as “fold changes” in relative gene expression measured in miscarriage and term placentas compared with control tissue (first trimester placenta). mRNA expression profiles of a) CD100, b) CD72 and c) CD45 show transcript down-regulation in pregnancy at term (p<0.001) and the highest values in second miscarriage placenta (p>0.05). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***).
Figure 2.
Tissue from a representative first miscarriage, 9th week of gestation.
Paraffin serial sections. A subpopulation of Hofbauer cells residing in the villous stroma, identified by CD68 (panel b,d,f), are positive for CD100 (panel a, red asterisks), CD72 (panel c, red asterisk) and CD45 (panel e, red asterisks). a,b,c,d,e,f,: Bar = 60 µm.
Figure 3.
Representative first trimester placenta (control).
Paraffin serial sections of representative first trimester placenta (9th week of gestation) from a voluntary termination of pregnancy. A subpopulation of Hofbauer cells, identified by CD68 (panel b), are positive for CD100 (panel a, red asterisks). Staining for CD72 (panel c) and CD45 (panel d) is largely negative. a,b,c,d: Bar = 60 µm.
Figure 4.
Positive and negative control tissue.
Tonsil tissue was used as a positive control for CD100 (panel a), CD45 (panel b), CD72 (panel c) and CD68 (panel d). A positive reaction for CD100 (panel a, see inset), CD45 (panel b, see inset) and CD72 (panel c, see inset) was detected in lymphocytes, and a positive reaction for CD68 was seen in macrophages (panel d, see inset). Negative control is shown in panel e. a,b,c,d,e: Bar = 30 µm; insets: Bar = 300 µm.
Figure 5.
Native CD100 protein expression in normal pregnancy and in miscarriage.
Representative immunoblot. MC = miscarriage. 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). β-actin: housekeeping protein. nCD100: native CD100.
Figure 6.
Densitometric analysis of native CD100 protein expression relative to β-actin in normal pregnancy and in miscarriage.
a) Native CD100 protein expression is significantly (p<0.01) lower in the first trimester of gestation than at term as well as in first trimester vs first and second miscarriage (both p<0.01). Its expression level is highest in first and second miscarriage and decreases subsequently. b) In the first trimester and in all miscarriage groups, native CD100 expression increases from the 8th to the 10th week of gestation; these differences are significant only for third (p<0.01) and fourth (p<0.05) miscarriage. A reduction in native CD100 expression after the 10th week is significant for first trimester and for third miscarriage (both p>0.05). Different levels of native CD100 are seen in all miscarriage groups between the 8≤ w.g. <9 and the w.g. >10 subgroups, but not in first trimester. c) In all weeks of gestation, native CD100 expression is significantly lower in first trimester vs first and second miscarriage (both p<0.001). Before the 9th week of pregnancy, it is significantly greater in second vs third and fourth miscarriage (both p<0.001). Between the 9th and the 10th week, native CD100 expression is significantly lower in first trimester placenta vs third and fourth miscarriage (both p<0.01), and significantly higher in second and third miscarriage vs fourth miscarriage (both p<0.05). After the 10th week, its expression is significantly lower in first trimester vs fourth miscarriage (p<0.001). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10).
Figure 7.
CD72 protein expression in normal pregnancy and in miscarriage.
Representative immunoblot. MC = miscarriage. 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). β-actin: housekeeping protein.
Figure 8.
Densitometric analysis of native CD72 protein expression relative to β-actin in normal pregnancy and in miscarriage.
a) CD72 is more abundant in first and second miscarriages and absent in term placenta. Significant differences in CD72 expression are detected only in first trimester vs first miscarriage (p<0.05) and in first vs third miscarriage (p<0.01). b) In first trimester and in first, second and third miscarriage, CD72 expression shows a significant increase from the 8th to the 10th week of gestation and subsequently a significant reduction after the 10th week (both p<0.001). Similar CD72 expression levels are found in first trimester and in first, second and third miscarriage between the 8≤ w.g. <9 and the w.g. >10 subgroups. c) Trend of CD72 expression in first trimester and in the four miscarriage groups at approximately the same gestational week. Before the 9th and after the 10th week, CD72 expression is significantly greater in first miscarriage vs first trimester (p<0.001) and significantly lower in second, third and fourth miscarriage vs first miscarriage (p<0.001). Between the 9th and the 10th week, CD72 expression is significantly lower in first trimester vs first and second miscarriage (both p<0.001) and significantly higher in second miscarriage vs third and fourth miscarriage (both p<0.001). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10).
Figure 9.
CD45 protein expression in normal pregnancy and in miscarriage.
Representative immunoblot. MC = miscarriage. 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). β-actin: housekeeping protein.
Figure 10.
Densitometric analysis of native CD45 protein expression relative to β-actin in normal pregnancy and in miscarriage.
a) CD45 expression is significantly (p<0.01) lower in the first trimester of gestation than at term. Its expression is highest in second miscarriage and declines subsequently. Differences are significant only in first trimester vs second miscarriage (p<0.01). b) In the first trimester and in all miscarriage groups, CD45 expression increases from the 8th to the 10th week; these differences are significant only for first (p<0.05), second (p<0.05) and third (p<0.01) miscarriage. A reduction in CD45 expression after the 10th week is significant for first trimester (p<0.01) and for first (p<0.05), second (p<0.05) and third (p<0.001) miscarriage. Similar CD45 expression levels are found in all miscarriage groups between the 8≤ w.g. <9 and the w.g. >10 subgroups, with the exception of third miscarriage and first trimester, where they are significantly lower in the samples with gestational age >10th week (both p<0.01). c) In all weeks of gestation, CD45 expression is significantly lower in first trimester vs first (p<0.001) and second (p<0.001) miscarriage and in first vs second miscarriage (p<0.05). Before the 9th and after the 10th week, it is significantly greater in second miscarriage vs third and fourth miscarriage (both p<0.001). Between the 9th and the 10th week, CD45 expression is significantly decreased not only in second vs third (p<0.05) and fourth miscarriage (p<0.001), but also in third vs fourth miscarriage (p<0.001). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10).