Uterine DCs are crucial for decidua formation during embryo implantation in mice (original) (raw)
uDCs accumulate in the IS and undergo characteristic changes during pregnancy progression. DCs have been reported to accumulate in the maternal tissue surrounding the implanting embryo (12, 16). However, the role of these cells for implantation and pregnancy maintenance remains poorly understood. Our first objective was to characterize the distribution of uDCs during pregnancy. For this, we took advantage of transgenic mice that harbor uDCs that are labeled by expression of a GFP reporter (CD11c:diphtheria toxin receptor [CD11c:DTR] and Cx3cr1gfp mice; refs. 17, 18) (Figure 1, A and D). Flow cytometry analysis of uDCs prior to (naive) and during implantation (E3.5 until E5.5) demonstrated an increase in the number of uDCs with pregnancy progression (Figure 1, A and B). Furthermore, GFP immunostaining revealed that uDCs were concentrated at the IS and were particularly abundant in the outer rim of the decidua (Figure 1C). In order to visualize uDCs in live tissue, we used the Cx3cr1gfp model (18), in which uterine CD11chiMHCIIhi DCs that express CX3CR1 are GFP labeled (Figure 1D). Analysis of uDC localization using this model confirmed the concentration of the uDCs in the decidual rim (Figure 1E). Two-photon microscopic analysis of E5.5 ISs revealed the distinct dendritic morphology of uDCs, localized at the IS, that were in close proximity to blood vessels (Figure 1F, Supplemental Video 1, and Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI36682DS1). Next, we characterized the phenotype of uDCs by flow cytometry analysis. At E5.5, 5%–6% CD11chi DCs were observed at the IS (Figure 2A). This population displayed relatively low expression of the classical macrophage marker F4/80 (19), exhibited high MHCII levels, and expressed low levels of the DC maturation molecules CD86 and CD40. Furthermore, E5.5 uDCs expressed the mucosal DC marker CD103 (20). To probe for the existence of uDC subpopulations, we analyzed the cells for CD11b and CD8α expression, i.e., molecules that characterize the 2 main subsets of conventional splenic DCs (21). At E5.5, uDCs could be subdivided into CD11bhiCD8αlo and CD11bloCD8αlo cells. The above-described uDC characteristics were unique to the early pregnancy, as analysis of uDCs at mid- (E12.5) and late (E18.5) pregnancy revealed distinct phenotypes. Thus, on E12.5, CD11chi uDCs expressed more F4/80 and were activated, as indicated by their upregulation of MHCII and expression of CD86 and CD40 (Table 1). While we still observed 2 main subpopulations, the CD11bhi DCs expressed higher levels of CD8α (Figure 2B). uDCs at E18.5 were all of the CD11bhiCD8αlo phenotype (Figure 2C), expressed high levels of F4/80, and exhibited an immature phenotype (CD86intCD40lo) (Table 1). Interestingly, the different pregnancy stages also showed a distinct anatomic distribution of uDCs in the uterus. While on E5.5 and E12.5 of pregnancy, uDCs were found mainly in the decidualized tissue at the embryo IS (Figure 1A) and decidua basalis (Figure 2D), respectively, uDCs on E18.5 were mostly localized to the nondecidualized uterine tissue (Figure 2D). Moreover, two-photon microscopy revealed that uDCs on day E12.5 (Figure 2E, Supplemental Video 2, and Supplemental Figure 1B) and E18.5 (Figure 2E, Supplemental Video 3, and Supplemental Figure 1C) were even more closely associated with blood vessels as compared with E5.5 uDCs. The distinct phenotypes and localization of uDCs at the IS suggested that these cells might have distinct roles during different pregnancy stages and specifically during embryo implantation, a decisive phase of pregnancy.
Anatomic localization of uDCs accumulating during embryo implantation. (A) Flow cytometry analysis of E5.5 IS of a CD11c:DTR transgenic mouse. Note the CD11chi uDC population, which expresses the DTR/GFP transgene. (B) Mean uDC percentages prior to and during implantation. uDCs of CD11c:DTR transgenic mice were defined as in A. Naive uteri (n = 3), E3.5 uteri (n = 2), E4.5 uteri (n = 5; *P = 0.007), and E5.5 uteri (n = 5; †P = 0.009). (C) Anti-GFP immunostaining (brown) of E5.5 uDCs of CD11c:DTR transgenic mouse (2 upper panels). Pie chart (lower panel) demonstrating quantifications of uDC distribution in the decidua. IS center, radius of 250 μm; middle rim, radius of 375 μm; outer rim, radius of 250 μm. The percentage of cells in the center is significantly smaller as compared with that in the middle (P = 0.009) and outer (P = 0.04) rims. (D) FACS analysis of Cx3cr1gfp/+ IS. uDCs are gated as CD11chiMHCIIhi cells. Histograms represent uDCs (black line) and non-uDCs (filled). (E) Fluorescence microscopy analysis of ex vivo decidual tissue demonstrating localization of GFP+ uDCs (green). (F) Two-photon microscopy demonstrating localization and morphology of uDCs (green) and blood vessels (red) in E5.5 IS. myo, myometrium; dec, decidua; e, embryo.
Phenotypic and localization differences of uDCs during pregnancy progression. (A) Flow cytometry profile of uDCs isolated from untreated mice (CD11c:DTR transgenic, C57BL/6). uDCs are gated as CD11chiGFP+ cells (R1) and stained for CD11b and CD8α. Cells gated in the R2 and R3 regions are CD11bhi and CD11blo uDCs. Histograms represent CD11chiGFP+ cells from ISs at E5.5 that are stained with the indicated antibodies (black line) and respective isotype controls (filled) (R1, 4.5%; R2, 77%; R3, 23%) (E5.5; n = 3). (B) Flow cytometric profile of cells from E12.5 decidua (R1, 5.5%; R2, 85%; R3, 15%) (E12.5; n = 3). (C) Flow cytometric profile of cells from E18.5 myometrium (R1, 4%; R2, 94%; R3, 6%) (E18.5; n = 3). (D) Flow cytometry data for decidual and nondecidual tissues from E12.5 and E18.5 ISs. (E) Two-photon microscopic analysis of E12.5 and E18.5 endometrium of Cx3cr1gfp/+ mice, demonstrating the morphology of uDCs (green; cells with dendrites) and their localization in close proximity to blood vessels (red).
Analysis of uDC phenotypes during different pregnancy stages
Conditional ablation of uDCs during the implantation window results in embryo resorption. To investigate the role of uDCs during embryo implantation, we took advantage of the CD11c:DTR mouse model, which allows the conditional ablation of CD11chi DCs by diphtheria toxin (DTx) administration (17). We originally chose to work with a semiallogeneic model, as DCs were hypothesized to have a tolerogenic role in pregnancy. Semiallogeneic pregnant mice were treated by i.p. injection of DTx on E.3.5, i.e., just before implantation, and were analyzed on E4.5 and E5.5 (Figure 3). DTx injection led to the rapid depletion of CD11chi DCs within 8 hours and lasted for at least 2 days (17), thus covering the implantation window (which starts on E4 and concludes on E5.5). For control, we injected either E3.5 CD11c:DTR mice with PBS or E3.5 WT mice with DTx (Supplemental Figure 2). It is noteworthy that uterine macrophages remained unaffected by the DTx administration (Supplemental Figure 3, A and B). uNKs included both DTR/GFP+ and DTR/GFP– subpopulations (Supplemental Figure 3D); however, under the conditions used (1 ng/g body weight DTx), uNKs were not ablated (Supplemental Figure 2C).
Conditional uDC ablation results in embryo resorption. Flow cytometry and histological analysis of ISs of controls and DTx-injected mice. (A and B) E4.5 control. (C and D) E4.5 uDC-depleted uterus. (E and F) E5.5 control. (G and H) E5.5 uDC-depleted uterus. (I and J) E5.5 partially uDC-depleted uterus. (K) Morphometric analysis of IS diameter at E5.5. Size of uDC-depleted IS (DTx) is presented as percentage of control IS (DTx: n = 3 mice, n = 6 ISs; control: n = 3 mice, n = 10 ISs; *P = 4.4 × 10–6). Of note, in uDC-depleted uteri, no normally developed embryos were detected.
Uterine sections of E4.5 control mice bearing uDCs (Figure 3A) were characterized by the presence of developing implantation chambers. We could observe embryos attached to the uterine wall initiating the process of invasion toward the uterine stroma, which showed characteristics of proliferation and differentiation into a decidua (Figure 3B). In contrast, DTx-treated CD11c:DTR transgenic mice lacking uDCs (Figure 3C) harbored nonreceptive uteri, in which the implantation chamber failed to form adequately. The uterine lumen was open, the decidualization process was severely impaired, and embryos neither attached to nor invaded the uterine epithelium (Figure 3D). On E5.5, control mice exhibited significantly expanded deciduae, and the embryo developed into the egg cylinder stage (Figure 3, E and F). In contrast, the uDC-depleted uteri exhibited reduced decidualization, and only rarely were embryos found in the uterus, mostly detached from the uterine lumen and undergoing resorption (Figure 3, G and H).
In order to study the phenotype induced by uDC depletion in more detail, we titrated the DTx dose to achieve a partial depletion of uDCs (50%–70%; Figure 3I). Thus, some decidual tissue remained, and investigation of the mechanism underlying the embryo resorption was facilitated. Deciduae of mice that were subjected to this protocol were significantly reduced in size as compared with those of controls, and the respective embryos had undergone resorption (Figure 3, J and K). This indicated a linear correlation between the number of uDCs at the IS and decidua development. Compilation of the results from independent experiments showed that while control mice displayed normal implanted embryos, two-thirds of the DTx-treated CD11c:DTR mice had resorptions or completely lacked ISs, one-third had smaller and nondeveloped ISs, while none had normal ISs (Table 2).
Analysis of the effect of uDC depletion on embryo ISs
To exclude the possibility that the systemic DC ablation had an indirect effect on the pregnancy, we performed a local uDC depletion. At E3.5, right uterine horns of mated females were injected with either DTx or PBS (control). Two days later (E5.5), the lower part of each horn was retrieved for histological examination, while the upper part was taken for flow cytometry (Figure 4J). FACS analysis confirmed a reduction in uDCs in the DTx-injected uterine horn (Figure 4G) but not in the contralateral control horn (Figure 4E). Histological sections revealed that the injected horn exhibited significantly smaller and malformed IS (Figure 4I), with retarded decidualization, and no embryos could be detected (Figure 4H). The contralateral uterine horn of the same mouse (Figure 4F), as well as the PBS-injected and noninjected horns of control mice that retained uDCs (Figure 4, A and C), exhibited normal IS with developed deciduae and embryos (Figure 4, B and D). Furthermore, we tested the effect of uDC depletion after successful implantation, i.e., on E5.5. Notably, uDCs were also readily depleted in this time point; however, there was no effect on the embryo fate or decidua development (Supplemental Figure 4). This result suggests a specific critical role for uDCs during the time window of embryo implantation and excludes nonspecific effects that the DTx administration or apoptosis of uDCs may have. Collectively, these results suggest that uDCs have a specific and direct role in decidual development and embryo implantation.
Local uDC depletion results in embryo resorption. On E5.5, all mice were sacrificed, and the lower parts of each horn were retrieved for histology, while the upper parts were taken for flow cytometry analysis. (A and B) Left noninjected uterine horn versus (C and D) right uterine horn, locally injected with PBS on E3.5 as a control. (E and F) Left noninjected uterine horn (non inj) versus (G and H) right uterine horn, locally injected with DTx on E3.5. (I) Morphometric analysis of IS diameter at E5.5. Size of uDC-depleted IS (DTx) is presented as percentage of control (PBS-noninjected) IS (DTx: n = 2, 2 injected IS, 2 noninjected IS; PBS: n = 2, 3 injected IS, 4 noninjected IS; *P = 0.03, DTx right versus PBS right). Note that size of locally DTx-treated IS is significantly reduced compared with that of all other ISs (†P < 0.04 versus DTx left and PBS right and left). uDCs are identified as CD11chiGFP+, and their percentages are indicated. (J) Experimental scheme. Uterine photograph (of a nonpregnant mouse) is only for illustration. OV, ovary.
Embryo resorption does not result from breakage of immunological tolerance or adaptive immunity. It is widely assumed that DCs in the uterus are required to establish tolerance toward the semiallograft fetus (22, 23). To test this possibility, we compared the effect of uDC depletion we had obtained in the allogeneic system to a syngeneic pregnancy model. Interestingly, analysis of uDC-depleted uteri on E5.5 (Figure 5A) revealed similar phenotypes in the allogeneic and syngeneic pregnancy models. In both cases, morphometric analysis showed a significant reduction in IS sizes in uDC-depleted as compared with control uteri (Figure 5B). These results argue against a role of uDCs in tolerance induction during the implantation period. This notion is further supported by results from additional experiments involving pregnant females that lack T and B cells (CD11c:DTR _Rag_–/–). Also in this setting, uDC depletion resulted in embryo resorption and a significant reduction in the size of the IS (Figure 5, C and D).
Decidualization failure is independent of immunity and occurs in absence of embryo. Representative histological sections of E5.5 uteri or ovaries and morphometric analysis. (A and B) Syngeneic model: CD11c:DTR transgenic females mated with C57BL/6 males (DTx: n = 3 mice, n = 5 ISs; control: n = 2 mice, n = 3 ISs; *P = 0.001). (C and D) Lymphocyte-deficient model: Rag2_–/– CD11c:DTR transgenic females mated with BALB/c males (DTx: n = 2 mice, n = 8 ISs; control: n = 3 mice, n = 12 ISs; *P = 0.001). (E and F) Artificial decidualization: Females were mated with vasectomized BALB/c males and their uteri sutured on E3.5 (DTx: n = 4 mice, n = 6 artificial ISs; control: n = 2 mice, n = 4 artificial ISs; †_P = 0.00001). (G and H) P4 administration in the allogeneic model. Pregnant females were injected s.c. with P4 on E3.5 and E4.5 (DTx: n = 3 mice, n = 9 ISs; control: n = 3 mice, n = 13 ISs; ‡P = 0.02). (I) Ovaries of control versus DTx-injected females. Note that experimental groups were injected with 1 ng/g body weight DTx, resulting in partial ablation of uDCs.
Impaired decidualization is induced by uDC depletion even in the absence of embryo. To establish whether the implantation defect caused by uDC depletion depends on the embryo or whether uDCs have a specific role in the decidua formation, we induced artificial decidualization in the absence of embryos (24). Thus, C57BL/6 CD11c:DTR females were mated with vasectomized BALB/c males, and the uteri of plug-positive females were sutured on E3.5 to induce decidualization. Animals were then administered either PBS or DTx. When analyzed on E5.5, uDC-depleted uteri exhibited malformed deciduae as compared with the well-developed artificial control deciduae (Figure 5E). Morphometric analysis showed significant impairment in the development of the deciduae in uDC-depleted uteri (Figure 5F). These results suggest that the effect of uDCs on the development of the decidua is embryo independent, further emphasizing the irrelevance of embryo allogeneity in the process. Importantly, these results directly link uDCs to the decidualization process and suggest that the embryo resorption is caused by impaired uterine receptivity.
Progesterone does not rescue the implantation disorder caused by DC-depletion. Corpus luteum–derived progesterone (P4) plays a crucial role in embryo implantation and subsequent pregnancy development by maintaining decidual viability and inhibiting myometrial contractility (25). To test whether the implantation failure observed in response to uDC depletion is a result of corpus luteum insufficiency, we tried to rescue the pregnancies by P4 administration on E3.5 and E4.5. P4 treatment did not prevent embryo resorption induced by uDC depletion (Figure 5, G and H). Furthermore, evaluation of the ovaries demonstrated that the uDC-depleted females exhibited corpora lutea with normal morphology as compared with control mice (Figure 5I). Therefore, we conclude that the implantation failure in the absence of uDCs is not due to lack of P4 production.
Impaired proliferation, decidual differentiation, and vascular expansion in uDC-depleted ISs. Our results suggest that decidualization depends on uDCs. To elucidate the underlying mechanism, we decided to analyze two hallmarks of decidua formation: cell proliferation and differentiation. The proliferation status of decidual cells was investigated on partially uDC-depleted uteri, which still retain some decidual cells. Immunostaining for phospho–histone H3 (Figure 6, A and B) as a proliferation marker (26) revealed extensive cell proliferation in the control mice. In contrast, uDC-depleted deciduae were largely devoid of proliferation. To study the impact of the uDC depletion on decidual differentiation, we investigated the induction of connexin 43 (Cx43), a gap junction protein expressed at the onset of embryo implantation, which is an established decidual differentiation marker (27). Immunohistochemical analysis for Cx43 in the control revealed prominent expression in the primary decidual zone (Figure 6, C and D). In contrast, Cx43 expression was significantly reduced in the uDC-depleted uteri, indicative of impaired decidual differentiation. Finally, we looked at the impact of uDC absence on decidual vascular expansion, a major prerequisite for adequate implantation. Immunostaining for endothelial cells using lectin (Figure 6, E and F) revealed reduced capillary density of uDC-depleted ISs versus control, implying an angiogenic disorder associated with uDC absence. Overall, uDC depletion caused severe impairment of all major characteristics of decidualization: stromal cell proliferation and differentiation, as well as vascular expansion were severely damaged, thus preventing the formation of an adequate decidual tissue.
Proliferation, decidual differentiation, and vascular expansion characterizing decidualization are impaired in uDC-depleted ISs. (A) Immunohistochemistry for phospho–histone H3 indicated upon reduced cell proliferation in the decidua of uDC-depleted ISs. (B) Quantification analysis of phospho–histone H3 staining in uDC-depleted ISs, shown as percentage of control (DTx: n = 2 mice, n = 3 ISs; control: n = 2 mice, n = 3 ISs; *P = 0.022). (C) Cx43 staining indicative of reduced cell proliferation in the decidua of uDC-depleted ISs. (D) Quantification of Cx43 staining in uDC-depleted IS, shown as percentage of control (DTx: n = 2 mice, n = 3 ISs; control: n = 2 mice, n = 3 ISs; †P = 6.5 × 10–5). (E) Immunostaining for endothelial cells using lectin (brown) of control versus uDC-depleted (DTx) E5.5 ISs. Note the reduced vessel density of uDC-depleted ISs versus control. (F) Quantification of lectin staining in uDC-depleted ISs, shown as percentage of control (DTx: n = 2 mice, n = 3 ISs; control: n = 2 mice, n = 3 ISs; ‡P = 0.044). e, direction of embryo location. In uDC-depleted uteri, embryo location was not indicated, since the embryo is resorbed in these specific histological preparations.
Functional analysis of decidual vascular function revealed impaired angiogenesis in uDC-depleted ISs. The reduced capillary density exhibited after uDC depletion implied a major perturbation in decidual angiogenesis — a dynamic, precisely timed process that critically affects uterine receptivity. Decidual angiogenesis is characterized by increased vascular permeability on E4.5 followed by a rise in blood volume on E5.5 and can be investigated noninvasively by dynamic macromolecular-contrast enhanced MRI (28). Interestingly, we were unable to detect E4.5 uDC-depleted ISs by MRI, indicating the presence of low angiogenic activity below quantifiable levels compared with controls (Supplemental Figure 5A). On E5.5, uDC-depleted ISs were significantly smaller and showed reduced contrast enhancement relative to controls, as detected by MRI (Figure 7, A, B, and E, and Supplemental Figure 5B). The blood volume fraction (fBV) calculated using the MRI data was significantly lower in uDC-depleted ISs (Figure 7C). However, the permeability surface area product (PS) of uDC-depleted ISs was similar to that of the control (Figure 7D). This suggested that the transcapillary leak of plasma proteins was higher in E5.5 uDC-depleted ISs, i.e., that blood vessels in the uDC-depleted deciduae were more permeable than those of the controls. The MRI results were corroborated by fluorescence analysis. E5.5 decidual blood vessels were undetectable in uDC-depleted ISs, 3 minutes after MR contrast material injection, and at 15 minutes, only a minor part of the contrast material had extravasated in uDC-depleted sites as compared with control (Figure 7F). Quantitative analysis (Figure 7G) revealed significantly reduced fluorescence of the decidual part in uDC-depleted ISs, indicating reduced vessel density and similar permeability. E4.5 ISs exhibited similar, although less pronounced trends than E5.5 ISs, as the decidua was less extended (Supplemental Figure 5, C and D). Overall, dynamic contrast-enhanced MRI–assisted studies revealed reduced blood volume and enhanced capillary leak, suggesting a role for uDCs not only in decidual vascular expansion but also in subsequent vascular maturation and attenuation of vascular permeability. This finding is consistent with the reduced density of vascular smooth muscle cells, immunostained using anti–α-SMA (Figure 7, H and I), of control versus uDC-depleted E5.5 ISs. The absence of mature blood vessels (with pericyte or vascular smooth muscle cell coating) from the rim of the uDC-depleted IS may account for the elevated transcapillary leak of plasma proteins in uDC-depleted deciduae.
Functional analysis of decidual vascular function reveals impaired angiogenesis in uDC-depleted ISs. (A and B) 3D gradient-echo MRI maximal intensity projections, 24 minutes after biotin-BSA-GdDTPA injection. (C) fBV and (D) PS (control: n = 3 mice, 10 ISs; DTx: n = 2 mice, 6 ISs; *P = 0.03, †P = 0.89). (E) Quantitative analysis of IS size measured by MRI. Size of uDC-depleted ISs (DTx) is presented as percentage of control ISs (control: 3 mice, n = 10 ISs; DTx: 2 mice, n = 6 ISs: ‡P = 0.0011). (F and G) ISs were retrieved 3 and 15 minutes after biotin-BSA-GdDTPA injection and stained with avidin-FITC (n = 2 mice, n = 3 ISs per time point; ‡P = 1.6 × 10–5, §P = 0.002). Gray trend lines indicate permeability (signal intensity [SI] at 15 minutes minus SI at 3 minutes: control, 4.4; DTx, 3.8). Yellow arrows, IS; white arrows, embryo location; B, bladder; K, kidney; e, embryo; ni, nonimplanted uterine site. (H) Immunostaining for smooth muscle cells using anti–α-SMA (red) of control versus uDC-depleted (DTx) E5.5 ISs. Note the absence of mature blood vessels (with α-SMA coating) from the edges of the uDC-depleted IS. Myometrium stained positive for α-SMA. m, myometrium; mbv, maternal blood vessel. (I) Quantitation of α-SMA–positive capillary number per IS showing uDC-depleted IS compared with control (DTx: n = 2 mice, n = 4 ISs; control: n = 2 mice, n = 9 ISs; ¶P = 0.026).
uDCs directly control decidual angiogenesis by regulating vascular maturation. The results of the MRI analysis and the α-SMA of uDC-depleted ISs revealed a strikingly enhanced capillary leak and the presence of immature blood vessels. In search of a molecular mechanism to explain the requirement of uDCs in the implantation process, we decided to focus on a potential link of uDCs to angiogenesis and vascular maturation. Vascular plasticity during angiogenesis is driven by VEGF, which elevates vascular permeability. The activity of VEGF is in turn under the control of a physiological “VEGF trap” in the form of a secreted VEGF receptor 1 (Flt1) (29). Interestingly, quantitative RT-PCR analysis of FACS-sorted uDCs and fractionated decidual tissue revealed that in the IS, this soluble Flt1 (sFlt1) is expressed by uDCs but not by decidual NK cells (Figure 8, A and B). This result suggests that one role of uDCs in the decidua is to fine-tune angiogenesis and vascular permeability by secretion of sFlt1. This potential scenario is further strengthened by the immunohistochemical analysis of ISs: here we found a significantly decreased immunoreactivity to sFlt1-specific antibody in uDC-depleted deciduae (Figure 8, C and D) specifically in the decidual rim, where uDCs accumulate (Figure 8E). Our expression analysis of decidual cells further revealed that uDCs express TGF-β1 (Figure 8F), a cytokine with pleiotropic functions that can directly affect vascular maturation (30) and promotes endothelial cell survival. As opposed to expression of sFlt1, TGF-β1 expression was shared with uNKs. However, interestingly, the RT-PCR analysis of uDC-depleted ISs revealed a global reduction in TGF-β1 expression (Figure 8, G and H). Taken together, our results suggest that uDCs are directly involved in the fine-tuning of angiogenesis in the decidua through provision of two critical factors, sFlt1 and TGF-β1, that act synergistically to promote coordinated vascular expansion and maturation.
Evidence for a direct role for uDCs in decidual angiogenesis by regulating vascular maturation. (A) Flow cytometry analysis of decidual cells (All) sorted for uNKs and for uDCs. (B) Semiquantitative RT-PCR for sFlt1 of the sorted cells in A. (C) Immunostaining for sFlt1 (brown) of control versus uDC-depleted (DTx) E5.5 ISs. Note that in the control ISs, sFlt1 staining is most abundant in the outer decidual rim, which is the localization of uDCs and also the localization of α-SMA–positive mature vessels (Figure 7H). In uDC-depleted deciduae, the sFlt1 staining is absent. Decidual tissue is circled. (D) Quantification of sFlt1 staining in uDC-depleted IS is shown as percentage of control (DTx: n = 2 mice, n = 3 ISs; control: n = 2 mice, n = 3 ISs; *P = 0.0035). Data quantification was performed on the whole IS (not only the decidua) to avoid bias. (E) Quantifications of sFlt1 distribution in the decidua. Radius of IS center, 240 μm; middle rim, 350 μm; outer rim, 240 μm. Note that the percentage of cells in the outer rim is significantly higher than that in the middle (P = 0.0012) and center (P = 0.0018). (F) Semiquantitative RT-PCR for TGF-β1 of the sorted cells in A. The bands were run on the same gel at the same time but were not contiguous. (G) Semiquantitative RT-PCR for TGF-β1 of uDC-depleted and control IS and (H) quantitative analysis of 3 different experiments. †P < 0.05.