Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However, whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here, we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua, where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy, we used Homeobox a11 (Hoxa11)-deficient mice, having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11 −/− mice results in stromal expansion, gland formation, and marked decidualization otherwise absent in Hoxa11 −/− mice. Moreover, in Hoxa11 +/− mice, which have increased pregnancy losses, BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively, these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma, thereby playing important roles in decidualization and pregnancy.

In this study, we utilized our previously described non-gonadotoxic BM transplant (BMT) regimen [ 24 ] to investigate the nonhematopoietic physiologic contribution of adult BMDCs to decidual stroma during pregnancy in wild-type (WT) mice. In addition, to explore the functional importance of BMDCs to implantation and pregnancy, we have used Homeobox a11 (Hoxa11) genetic knockout (KO) mice models, which are associated with endometrial stromal defects leading to decidualization failure and lack of pregnancy in homozygous ( −/− ) and pregnancy loss in heterozygous ( +/− ) mice. We chose the Hoxa11 KO model because Hoxa11 has been shown to be expressed in the BM exclusively in nonhematopoietic mesenchymal stem/stromal cell populations [ 25 ], allowing us to dissect the potential nonhematopoietic BMDCs functional contribution to pregnancy. Our data show that embryo implantation and pregnancy are associated with an increase in mobilization of mesenchymal stem cells (MSCs) to the circulation, and recruitment of BMDCs to the uterus, where BMDCs are a source of functional nonhematopoietic decidual cells. In addition, transplantation of BM from Hoxa11-expressing WT donors leads to endometrial stromal expansion, gland formation, and decidualization in Hoxa11 −/− mice. Moreover, BMT from WT donors results in normalization of uterine expression of numerous decidualization-related genes, leading to rescue of pregnancy losses in Hoxa11 +/− mice. Collectively, these data highlight the important nonhematopoietic role that adult BMDCs play in implantation and pregnancy maintenance.

It is well known that uterine implantation sites are the site of infiltration of many peripherally derived immune cells [ 6 ], including natural killer (NK) cells, macrophages, and T cells, which play important roles at the maternal–fetal interface to promote successful pregnancy [ 7 – 10 ]. While it is well established that many decidual immune cells originate in the bone marrow (BM) [ 7 , 11 ], it remains unknown whether adult BM-derived cells (BMDCs) can give rise to nonhematopoietic cells in the decidua. Kearns and Lala have previously proposed that BMDCs may also give rise to stromal decidual cells in pregnancy based on reconstitution of BM cells in the fetal period [ 12 , 13 ]. However, others could not find such evidence [ 14 ], and this concept was further challenged by investigators demonstrating that resident endometrial stromal cells differentiate into decidual cells [ 15 ]. Moreover, because BM reconstitution was performed during fetal life, it remained unknown whether BMDCs migrating to the uterus during adult life can give rise to nonhematopoietic decidual cells. Adult BMDCs have been shown to travel in the circulation and contribute to tissue repair and regeneration of various organs [ 16 ]. In the uterus, adult BMDCs have been detected in both human [ 17 – 19 ] and mouse [ 18 , 20 – 23 ] endometrium, and demonstrated to give rise to various nonhematopoietic endometrial cells, including epithelial, stromal, and endothelial cells (ECs) in the nonpregnant state, suggesting that BMDCs may serve as a source of progenitor cells for endometrial regeneration. However, whether adult BMDCs give rise to nonhematopoietic decidual cells, their temporal and spatial nonhematopoietic contribution to implantation and pregnancy, and whether implantation is a stimulus for the migration of these progenitors to the uterus remain uncharacterized. Moreover, the functional importance of such cells to embryo implantation and pregnancy development is unknown.

The decidua is a transient tissue lining the uterus of mammals with hemochorial placenta (mice, humans, and numerous other mammalian species) and is essential for pregnancy in these species. In humans, decidualization, the transformation of endometrial stromal cells into epitheloid decidual cells, occurs throughout the endometrium at the end of the implantation window (approximately 10 days after ovulation) independently of pregnancy. This differentiation process is dependent on 3′,5′-cyclic AMP (cAMP) and progesterone signaling pathways that lead to profound transcriptome and proteome changes [ 1 ]. The differentiated decidua exhibits only a short period of receptivity known as “the window of implantation,” when embryo attachment and implantation is possible [ 2 ]. In the absence of blastocyst implantation, progesterone levels decline and the decidualized endometrium undergoes timely destruction, leading to menstruation. With blastocyst implantation, the decidua grows and is maintained throughout gestation [ 1 ]. In mice, endometrial decidualization occurs later, initiated by blastocyst attachment to the uterine epithelium on embryonic day 4.5 (E4.5) and is localized to the implantation sites [ 3 ]. Although embryo quality is an important determinant of implantation, temporally coordinated differentiation of endometrial stromal cells into decidual cells to attain uterine receptivity, and a synchronized dialog between maternal and embryonic tissues are crucial for successful implantation [ 2 ]. These specialized decidual cells play key roles in nutritional sensing, endocrine regulation, immune tolerance, and evaluation of embryo quality [ 4 , 5 ].

To further investigate the underlying mechanism responsible for the decidualization effects in KO mice following WT BMT, we analyzed expression of Hoxa11 and other associated genes that are known to be essential for implantation and decidualization, such as leukemia inhibitory factor (Lif) [ 47 ], prolactin [ 59 ], and MSH homeobox 1 (Msx1) [ 54 ], in the uterus on E5.5. Hoxa11 mRNA and protein expression were observed in the uterus of KO mice receiving WT BMT, but not KO mice receiving KO BMT ( Fig 7M and 7N ). In addition, we found that the implantation-related genes Msx1, Prl8a2, and Lif were significantly up-regulated in the KO WT BMT as compared with KO KO BMT mice. Moreover, immunofluorescence analysis demonstrated the presence of Hoxa11-expressing cells in the decidua of KO WT BMT but not KO KO BMT mice ( Fig 7I ), suggesting that WT Hoxa11-expressing BMDCs were recruited to the uterus of KO mice receiving WT BMT. To exclude the possibility that the Hoxa11-expressing cells were merely circulating immune cells, we performed colocalization with the pan-leukocyte marker CD45, which demonstrated that the Hoxa11-expressing cells were nonhematopoietic (CD45-negative) stromal cells ( Fig 7I ). This is consistent with our flow cytometry data showing that Hoxa11 is not expressed on hematopoietic cells but is only expressed in CD45-negative nonhematopoietic cells ( S10 and S11 Figs). Taken together, these data suggest that Hoxa11-expressing BMDCs can affect gene expression at the uterine implantation site, leading to profound effects on decidualization, as evidenced by promotion of decidual differentiation, cell proliferation, and angiogenesis.

(A-H) Sections of E5.5 implantation sites from WT WT BMT , KO WT BMT , and KO KO BMT stained with HE (A and B), CD31 (C and D), PR (E and F), and PCNA (G and H). B, D, F, and H are higher magnification photomicrographs of A, C, E, and G, respectively. Black arrows point to embryos. (J, K and L) Quantification of pecent PR-positive cells (J), percent PCNA-positive cells (K), and mean blood vessel luminal area (μm 2 ) in the endometrium (n = 3–4 mice/group). (I) Sections of E5.5 implantation sites from WT WT BMT , KO WT BMT , and KO KO BMT co-stained with Hoxa11 (green), CD45 (red), and counterstained with DAPI (blue). Arrows point to Hoxa11-expressing cells. (M) Relative uterine mRNA expression of HoxA11 and implantation-related genes Prl8a2, Lif, and Msx1 normalized to GAPDH in WT WT BMT versus KO WT BMT versus KO KO BMT on E5.5 (n = 4–5 mice/group). (N) Uterine Hoxa11 protein expression in KO WT BMT versus KO KO BMT on E5.5. Bar graphs represent mean ± SEM. p-values are noted on the graphs. Scale bars, 100 μm (A, C, E, G), 50 μm (B, D, F, H). Underlying data are available in S1 Data . BM, bone marrow; BMT, BM transplant; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HE, hematoxylin–eosin; Hoxa11, Homeobox a11; KO, knockout; PCNA, proliferating cell nuclear antigen; PR, progesterone receptor; WT, wild-type.

Despite lack of normal implantation sites in the Hoxa11 KO mice, areas of increased swelling and vascularization in the uterus were observed on E5.5 only in the group that received WT BMT, suggesting that some uterine reaction to the embryo has taken place ( Fig 3D ). Histological analysis of uterine sections on E5.5 from KO WT BMT revealed significant decidual reaction. Embryos were noted attached to the uterine wall initiating the process of invasion toward the uterine stroma, which was expanded and showed characteristics of proliferation and differentiation into a decidua ( Fig 7A and 7B ). In contrast, KO KO BMT demonstrated non-receptive uteri without any sign of decidual reaction or embryo attachment ( Fig 7A and 7B ). To further characterize the impact of BMT from WT donors on decidualization in KO mice, we analyzed the three hallmarks of decidualization: decidual differentiation, cell proliferation, and vascular expansion. Immunohistochemical analysis for PR, an established decidualization marker, revealed prominent expression in the decidua of KO WT BMT , similar in extent to the WT WT BMT mice ( Fig 7E, 7F and 7J ). In contrast, PR expression was significantly reduced in the uteri of KO KO BMT , consistent with impaired decidualization. Immunostaining for PCNA ( Fig 7G, 7H and 7K ), a proliferation marker, demonstrated extensive cell proliferation in the KO WT BMT , in contrast to KO KO BMT deciduae, which were largely devoid of proliferation. Finally, we looked at the impact of BMT from WT donors on decidual vascular expansion, a major prerequisite for adequate implantation. Immunostaining for ECs using CD31 ( Fig 7C, 7D and 7L ) revealed increased decidual blood vessel area and mean luminal area in KO WT BMT versus KO KO BMT . Overall, uteri of Hoxa11 KO mice receiving WT BMT exhibited marked improvement in decidualization and its characteristics: stromal cell proliferation and differentiation, as well as vascular expansion. In contrast, uteri of KO mice receiving KO BMT were characterized by severe impairment of all major characteristics of decidualization, thus preventing the formation of an adequate decidual tissue.

(A and B) Uterus photographs (A) and mean uterine weight (B) of nonpregnant KO WT BMT and KO KO BMT mice. (C) HE and cytokeratin immunostaining (brown) of uterine sections from KO WT BMT and KO KO BMT mice. The endometrial area is encircled in the left panel and shown at higher magnification in the middle (HE) and right (cytokeratin) panels. Glands are seen only in KO WT BMT mice and are surrounded by black dashed circles in the middle panel, corresponding to the cytokeratin-positive (brown) areas in the right panel. The luminal epithelium (Lu) is encircled by a red dash. Scale bars, 100 μm (left) and 50 μm (middle and right panels). (D-F) Quantification of total stromal area (μm 2 ) (D), percent stromal area out of endometrial area (E), and percent mice with endometrial glands (F) in KO WT BMT and KO KO BMT groups. n = 6–7/group. (G) GFP immunostaining of uterine sections from KO WT BMT showing localization of GFP + BMDCs (brown) in the stroma. The Lu) is encircled by a red dash and the gland is encircled by black dash. Bar graphs represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. See also S15 Fig . Underlying data are available in S1 Data . BM, bone marrow; BMDC, BM-derived cell; BMT, BM transplant; GFP, green fluorescent protein; HE, hematoxylin–eosin; Hoxa11, Homeobox a11; KO, knockout; Lu, luminal epithelium; WT, wild-type.

Hoxa11 −/− mice have abnormally small uteri characterized histologically by minimal endometrial stroma and complete absence of glands [ 42 , 47 ]. To evaluate whether Hoxa11-expressing BMDCs can repair the endometrial defects of KO mice, BMT was performed into KO mice either from WT or KO donors. Examination of the nonpregnant uteri of KO animals 1 month following BMT showed a significant increase in uterine weight of KO WT BMT compared with KO KO BMT mice ( Fig 6A and 6B ). This was associated histologically with profound endometrial stromal expansion in the KO WT BMT group as compared with KO KO BMT , noted in all estrus phases (Figs 6C–6E and S15 ). Furthermore, gland formation was seen only in the KO WT BMT group ( Fig 6C and 6F ). BM transplantation from GFP (WT) donors into KO mice revealed the presence of GFP + BMDCs in the uterine stroma but not in endometrial glands or luminal epithelial cells ( Fig 6G ). These data indicate that endometrial glandular regeneration in Hoxa11 −/− mice is likely induced by BMDCs paracrine-mediated mechanisms rather than direct cellular contribution of BMDCs to glandular or luminal epithelium.

To investigate the possibility that immune cell populations are altered in Hoxa11 +/− pregnancy as an underlying reason for their reproductive phenotype, we compared the immune populations in the implantation site between pregnant Hoxa11 +/− and WT mice ( S11 Fig ). No differences were found in the total number of leukocytes or in proportions of NK1.1 + (NK) cells, F4/80 macrophages, total CD3 + T cells, CD4 + CD25 + T regulatory (Treg) cells, or Ly6G + granulocytes in the implantation site between the two groups ( S14 Fig ). Taken together, these data suggest that BMDCs recruitment to the uterus and uterine immune cell populations are unaltered in Hoxa11 +/− mice, and it is unlikely that the rescue effect of WT BMT on resorptions in Hoxa11 +/− mice is mediated by the transplanted immune cell populations.

It was shown that triple mutant mice with simultaneous KO of Hoxd9, Hoxd10, and Hoxd11 have increased number of leukocytes and altered proportions of immune cell in the uterus, with increase in myeloid lineage populations of macrophages and granulocytes [ 58 ]. To evaluate whether Hoxa11 deficiency affects uterine leukocyte populations or recruitment of BMDCs to the uterus, flow cytometric analysis was performed on nonpregnant or pregnant uteri of KO and Hoxa11 +/− mice, respectively, which underwent BMT from transgenic tdTomato-expressing BM donors. WT mice that underwent BMT from tdTomato donors served as controls. No differences were found in numbers of leukocytes or in numbers of BMDCs recruited to the nonpregnant uteri of KO mice or the implantation site of pregnant Hoxa11 +/− mice, as compared with WT controls ( S13 Fig ). In addition, no difference in the total number of BM or splenic cells was found between nonpregnant KO and WT mice, and between pregnant Hoxa11 +/− and WT mice.

Uterine Hoxa11 mRNA and protein expression on implantation day (E5.5) was greater in WT WT BMT mice as compared with Hoxa11 +/−KO BMT and Hoxa11 +/−WT BMT mice, but similar between the two latter groups ( Fig 5E and 5F ). However, close examination of the Hoxa11 immunostaining pattern in uterine sections revealed that Hoxa11-positive cells were more abundant in the primary decidual zone (PDZ) surrounding the embryo in Hoxa11 +/−WT BMT as compared with Hoxa11 +/−KO BMT ( Fig 5G and 5H ). In addition, WT WT BMT control mice showed greater abundance of Hoxa11-expressing cells around the implantation site as compared with both Hoxa11 +/− mice groups ( Fig 5G and 5H ). No histological differences were noted in implantation sites between Hoxa11 +/− mice WT BMT versus KO BMT. Taken together, these data suggest that BMDCs can affect global gene expression at the implantation site, favoring pregnancy maintenance and ultimately leading to rescue of pregnancy losses arising from endometrial stromal defects.

(A-C) RNA-seq analysis of implantation sites on E5.5 in Hoxa11 +/−WT BMT , Hoxa11 +/−KO BMT , and WT WT BMT . (A) Heat map of the expression fold change of 498 commonly DEGs in the comparisons of Hoxa11 +/−KO BMT versus Hoxa11 +/−WT BMT , and Hoxa11 +/−KO BMT versus WT WT BMT . (B) The top 10 KEGG pathways enriched in genes commonly differentially expressed in the comparisons of Hoxa11 +/−KO BMT versus Hoxa11 +/−WT BMT , and Hoxa11 +/−KO BMT versus WT WT BMT . (C) Three-way Venn diagram of the comparisons of Hoxa11 +/−KO BMT versus Hoxa11 +/−WT BMT , Hoxa11 +/−KO BMT versus WT WT BMT , and Hoxa11 +/−WT BMT versus WT WT BMT . (D) RT-PCR validation of gene expression of select genes from the 498 commonly DEGs that have important roles in decidualization. *p ≤ 0.05 and **p ≤ 0.01 versus Hoxa11 +/−KO BMT (n = 3–5/group). (E-H) Hoxa11 expression in implantation sites on ED5.5 in Hoxa11 +/−WT BMT , Hoxa11 +/−KO BMT , and WT WT BMT (n = 5–6/group). (E) Hoxa11 mRNA expression and (F) Hoxa11 protein expression in ED5.5 implantation sites. (G and H) Sections of implantation sites from ED5.5 stained with Hoxa11 antibody (brown) (H). Inset (below) shows high magnification photomicrograph of the dashed area and the embryo (e). The primary decidual zone (PDZ) and secondary decidual zone (SDZ) are noted. (G) Corresponding quantification of Hoxa11-positive cells in the PDZ. Bar graphs represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. Scale bars, 200 μm (upper panel), 100 μm (lower panel). See also S12 and S16 Fig . Underlying data are available in S1 Data . BM, bone marrow; BMT, BM transplant; DEG, differentially expressed gene; Hoxa11, Homeobox a11; KEGG, Kyoto Encyclopedia of Genes and Genomes; KO, knockout; PDZ, primary decidual zone; RNA-seq, RNA sequencing; SDZ, secondary decidual zone; WT, wild-type.

The maternal component of the implantation site consists of stromal cells of the uterine endometrium that undergo a tightly controlled process of decidualization upon contact with the early embryo. These stromal cells undergo proliferation and differentiation into large epitheloid decidual cells in a process that is critical to the establishment of fetal–maternal communication and pregnancy maintenance. To gain insight into the mechanism by which BMDCs lead to pregnancy loss prevention in Hoxa11 +/− mice, we focused on profiling gene expression by RNA-seq in the uterine tissue of the implantation site on E5.5, a time of decidualization which is critical for successful embryo implantation. To this end, we assessed expression patterns in Hoxa11 +/−WT BMT and WT WT BMT when they were compared with Hoxa11 +/−KO BMT . There were fewer differentially expressed genes (DEGs) in the comparison of WT WT BMT versus Hoxa11 +/−WT BMT (530 genes), as compared with between WT WT BMT versus Hoxa11 +/−KO BMT (795 genes) ( Fig 5C ). We found a total of 498 genes that were significantly commonly differentially expressed (false discovery rate [FDR]-adjusted p-value < 0.05) in the comparisons of Hoxa11 +/−WT BMT versus Hoxa11 +/−KO BMT and WT WT BMT versus Hoxa11 +/−KO BMT ( Fig 5A and S2 Data ). Remarkably, 98.6% of the 498 commonly DEGs were either up- or down-regulated in the same direction in Hoxa11 +/−WT BMT and WT WT BMT mice ( Fig 5A and S2 Data ), suggesting that BMT from WT donors normalized the uterine implantation transcriptome of Hoxa11 +/−WT BMT in favor of normal implantation. Interestingly, the 498 commonly DEGs were significantly enriched (N = 27; Fisher's exact test p-value = 1.29 × 10 −9 ) for decidualization genes, based on a combined list of 323 genes with known role in mouse decidualization according to Mouse Genome Informatics (MGI) database and the literature [ 48 ]. Those 27 genes included key regulators of decidualization, such as prolactin superfamily members (Prl3c1, Prl6a1, Prl8a2) [ 40 , 49 , 50 ], Wingless/Integrated (Wnt) family members (Wnt4, Wnt6, Wnt16, Fzd6) [ 51 – 53 ], Msx1 [ 54 ], Ptges [ 55 ], Igfbp2, A2m [ 56 ], and Foxa2 [ 57 ]. We independently confirmed differential expression of selected candidates by quantitative RT-PCR using additional biological replicates ( Fig 5D ). Using the list of commonly differentially regulated genes for gene ontology (GO) and pathway analysis revealed that the top 10 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included many pathways known to be important for implantation such as metabolism, chemokine signaling pathway, proteoglycans in cancer, cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, leukocyte transendothelial migration, cAMP signaling pathway, signaling pathways regulating pluripotency of stem cells, and MAPK signaling pathway ( Fig 5B ). In addition, major canonical pathways enriched in the Ingenuity Pathway Analysis (IPA) included pathways known to play important roles in implantation such as leukocyte extravasation, planar cell polarity (PCP) pathway, eicosanoid signaling, chemokine signaling, mouse embryonic stem cell pluripotency, regulation of epithelial-mesenchymal transition, and WNT/β-catenin ( S12 Fig ).

Next, we investigated whether the observed difference in litter size following treatment was related to early pregnancy (implantation) and/or later pregnancy (resorptions) events. To this end, timed pregnancies following the same treatment protocols were ended either on E5.5 or E12.5 for assessment of implantation and resorption, respectively. On E5.5, there were no differences in the number of implantations between the Hoxa11 +/− mice receiving either WT or KO BMT, and the control WT mice receiving WT BM genotype. In contrast, no distinct implantation sites were noted in the KO mice receiving either treatment ( Fig 4D and 4E ). On E12.5, heterozygous Hoxa11 +/−WT BMT mice showed a decreased resorption rate (4.4%) versus Hoxa11 +/−KO BMT mice (28.4%) and had a comparable resorption rate to that of WT controls (5.5%) ( Fig 4F and 4H ). Similarly, the mean number of viable implantations in Hoxa11 +/−WT BMT mice was greater than Hoxa11 +/−KO BMT but the same as WT control mice (8.0 versus 4.9 versus 7.9, respectively) ( Fig 4G ). Furthermore, the percentage of dams with resorptions was greater in the Hoxa11 +/−KO BMT mice as compared with both the Hoxa11 +/−WT BMT group as well as the WT control group (83% versus 30% versus 27%, respectively) ( Fig 4I ). These data indicate that BMT treatment of Hoxa11 +/− mice from WT donors results in normalization of litter size due to prevention of pregnancy loss following implantation.

BMTs were performed from WT into Hoxa11 +/− (Hoxa11 +/−WT BMT ), WT into HoxA11 −/− (Hoxa11 −/−WT BMT ), Hoxa11 −/− into Hoxa11 +/− (Hoxa11 +/−KO BMT ), and HoxA11 −/− into Hoxa11 −/− (Hoxa11 −/−KO BMT ). WT mice receiving BMT from WT donors (WT WT BMT ) served as controls. (A-C) Effect of BMT from different genotypes on (A) litter size, (B) time to delivery, and (C) mean pup weight per litter following breeding (n = 11–15/group). *p < 0.01. +p < 0.05 versus Hoxa11 +/−KO BMT . (D and E) Representative uterine images (D) and quantitation of mean number of implantations per mouse on E5.5 (E) in WT, Hoxa11 +/− , and HoxA11 −/− mice that received BMT from either WT or KO donors (n = 10–16/group). (F-I) Pregnancy resorptions on ED 12.5 in WT and Hoxa11 +/− mice that received BMT from either WT or KO donors (n = 10–12/group). (F) Representative images of uteri showing resorption sites (black arrows). (G) Mean number of viable implantations per mouse. (H) Resorption rate per mouse pregnancy. (I) Percentage of dams with at least one resorption. In all panels, bar graphs represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. Underlying data are available in S1 Data . BM, bone marrow; BMT, BM transplant; Hoxa11, Homeobox a11; KO, knockout; WT, wild-type.

KO and Hoxa11 +/− female mice underwent BMT from either WT or KO mice following the same 5-FU–based submyeloablation protocol. WT mice undergoing BMT from WT donors served as controls. First, we examined the reproductive performance of the mice following a 1-month harem breeding trial. Heterozygous Hoxa11 +/− mice receiving BMT from WT mice (Hoxa11 +/−WT BMT ) had comparable mean litter size to WT WT BMT animals and had significantly greater mean litter size, as compared with Hoxa11 +/− mice receiving KO BMT (Hoxa11 +/−KO BMT ) (8.2 versus 8.3 versus 4.0, respectively) ( Fig 4A ). The KO mice receiving either BMT treatment delivered no litters nor had any visible signs of pregnancy ( Fig 4A ). While overall pregnancy rates were the same in the WT and Hoxa11 +/− groups, differences were noted in time to conception. The Hoxa11 +/−WT BMT mice had comparable time to conception with WT WT BMT control animals but shorter time to conception as compared with Hoxa11 +/−KO BMT mice ( Fig 4B ). No differences were noted in mean pup weight per litter between the various groups ( Fig 4C ).

To investigate the functional importance of the nonhematopoietic contribution of BMDCs to implantation and maintenance of pregnancy, we took advantage of the Hoxa11 −/− (KO) and Hoxa11 +/− mouse models. Homeobox-containing (Hox) genes are developmentally regulated transcription factors belonging to a multigene family. Among these, Hoxa10 and Hoxa11 are crucial for reproductive tract development, endometrial growth and differentiation, and embryo implantation by mediating some functions of sex steroids [ 42 – 46 ]. Hoxa11 KO female mice have abnormally small uteri characterized by stromal atrophy and absence of glands and are sterile due to an endometrial receptivity defect [ 42 , 47 ]. Upon mating with WT males, they form embryos but fail to mount a decidual reaction, leading to implantation failure [ 42 , 47 ]. In Hoxa11 +/− mice, implantations occur but are characterized by increased resorptions (pregnancy loss) and, ultimately, reduced litter sizes [ 42 ]. In our experience, Hoxa11 +/− mice also had reduced litter sizes (mean 5.4, n = 78) compared with WT mice (mean 8.3, n = 96). Based on our observation of a significant contribution of BMDCs to nonhematopoietic cellular content of the decidual stroma, we hypothesized that BMT from a WT mouse may rescue the reproductive defects in these mice. We chose the Hoxa11-deficient mouse model for our investigations because it was shown that Hoxa11 expression in the BM is restricted to mesenchymal stem/stromal cell progenitors and it is not expressed in hematopoietic cells [ 25 ], thus allowing us to interrogate the role of nonhematopoietic BMDC contribution to pregnancy. Using Hoxa11 +/− mice, in which the Hoxa11 gene is replaced by EGFP knock-in (Hoxa11-GFP), we performed flow cytometry, which confirmed the absence of Hoxa11 expression in hematopoietic cells of BM, spleen, and peripheral blood and expression of Hoxa11 in a small subset of nonhematopoietic (CD45 − ) tibial BM cells ( S10 Fig ), consistent with its regional-restricted expression in the zeugopod [ 25 ]. Hoxa11 was abundantly expressed in the uterine implantation site of these mice exclusively in nonhematopoietic (CD45 − ) cells. To confirm the ability of nonhematopoietic BMDCs to migrate to the uterus and contribute to nonhematopoietic decidual Hoxa11 + cell population, we analyzed WT mice transplanted with BM from Hoxa11-GFP donors using the same 5-FU–based submyeloablation regimen. Multicolor flow cytometry analysis of the E9.5 implantation site of transplanted mice showed the presence of BM-derived GFP + Hoxa11 + cells, which were all CD45 negative in addition to being negative for NK and myeloid markers NK1.1 and CD11b, respectively ( S11A and S11B Fig ). These GFP + Hoxa11 + cells accounted for 0.5% of total cells in the implantation site. In contrast, Hoxa11 + GFP + cells were not found in nonpregnant uterus ( S11A and S11B Fig ).

Next, we wished to explore the dynamics of BM-derived MSCs and HSCs in BM and circulation during mouse pregnancy. Flow cytometry analysis was performed on BM and peripheral blood from nonpregnant as well as pregnant E5.5 and E9.5 mice, timing of peak BMDCs recruitment to the uterus. Circulating MSCs were found to be significantly increased in the circulation of pregnant mice from 0.002% in the nonpregnant state to 0.007% on E5.5, and further increased to 0.014% on E9.5 (approximately 7-fold compared with nonpregnant) ( S9A Fig ). In contrast, circulating HSCs remain unchanged from nonpregnant through E9.5 ( S9A Fig ). BM MSCs and HSCs were not significantly different between nonpregnant and pregnant E9.5 ( S9B Fig ). This evidence indicates that BM MSCs are increasingly mobilized to the circulation during pregnancy, supporting our findings of increased numbers of BM-derived CD45 − cells in the pregnant uterus.

Following our observations that adult BMDCs give rise to stromal decidual cells in our BMT model, we wished to investigate whether GFP + BM MSCs, the putative source of BM-derived DSCs, are reconstituted in BM of the transplanted mice following our non-gonadotoxic 5-FU–based submyeloablation protocol. Irradiation has been shown to be associated with severe and permanent damage of BM stromal cells, limiting engraftment of transplanted hematopoietic stem cells (HSCs) and MSCs [ 36 ], while 5-FU preconditioning results in a transient damage to the BM stromal cell niche, followed by extensive remodeling of MSCs in the BM supporting donor cell engraftment [ 37 ]. For this, we extracted BM from mice that underwent BMT from GFP donor according to our model. Samples were gated on Lineage (Lin) − /Sca-1 + /CD45 − to define MSCs using a broad definition, consistent with prior works [ 38 , 39 ]. Flow cytometry analysis of BM cells showed the presence of putative MSCs within the transplanted GFP + cell population (GFP + Sca1 + Lin − CD45 − ) ( S2A Fig ). GFP + BM MSCs accounted for 0.01% of total BM cells and were less abundant than GFP + HSCs (GFP + Sca1 + Lin − CD45 + , 0.19%). GFP + MSCs were also found in peripheral blood, albeit at much lower frequency (0.002%) ( S3A Fig ). We cultured the BM cells and further analyzed these GFP + cells for the three established criteria for definition of multipotent mesenchymal stromal cell. BM GFP + cells were adherent to plastic and were able to grow in culture conditions supporting MSC expansion ( S2C Fig ); these cultured GFP + cells were analyzed for the presence of multiple MSC markers by flow cytometry, demonstrating that a fraction of GFP + cells co-express established MSC markers Sca-1, CD29, and CD44 ( S2B Fig ). Cultured BM GFP + cells were shown to have trilineage differentiation ability in vitro to adipogenic, osteogenic, and chondrogenic lineages ( S2D–S2F Fig ). Taken together, these data indicate engraftment of GFP + MSC populations in the BM of the 5-FU BMT model. In addition, these cultured BM cells were treated with established decidualization agents medroxyprogesterone acetate (MPA) and/or 8-bromo-cAMP (cAMP), and their ability to undergo decidualization in vitro was evaluated. Cultured BM cells showed characteristic decidual morphological changes from elongated shape (control) to broad hexagonal shape in response to decidualization stimuli, which was most prominent in the combined cAMP+MPA treatment group ( S2G Fig ). Moreover, expression of DPRP mRNA (Prl8a2), a specific marker of decidualization in the mouse [ 40 , 41 ], was up-regulated on days 8 and 14 of culture in the BM cells subjected to decidualization treatment ( S2H Fig ). Taken together, decidualization of BM MSCs demonstrated in vitro is consistent with our in vivo observations of BMDC transformation to DSCs.

Cell fusion is known to occur between BMDCs and cancer cells at variable frequencies [ 33 ]. Cell fusion between BMDCs and DSCs can potentially lead to misidentification of GFP + BMDCs as stromal cells. To investigate whether cell fusion occurs between BMDCs and host decidual cells, we utilized the well-established Cre-Lox P recombination approach [ 34 , 35 ] with a dual color fluorescent mT/mG system. In this system, prior to Cre recombination, cell membrane tdTomato (mT) is expressed. Upon Cre recombinase expression in the cell, membrane enhanced green fluorescent protein (EGFP) (mG) fluorescence expression replaces the mT expression. mT/mG mice co-expressing Cre recombinase transgene ubiquitously under β-actin-Cre promoter were used as positive controls, confirming the efficiency of Cre-mediated conversion from mT to mG in the blood as well as uterus in this system ( S8 Fig ). mT/mG transgenic mice were used as BM donors. BMT was performed following the same 5-FU–based BMT regimen into β-actin-Cre mice. The BM transplants from mT/mG donor into WT mice served as negative controls. Flow cytometry demonstrated that only BMDCs expressing mT and not mG were found at the implantation site on E9.5 ( S8 Fig ), timing of peak BMDCs recruitment, in both Cre as well as WT control mice. This indicates that the nonhematopoietic phenotype of decidual BMDCs is not explained by cell fusion with resident decidual cells.

To confirm the decidual stromal identity of GFP + cell population subsets, we assessed expression of decidual-specific markers. Decidual prolactin-related protein (DPRP) is a pregnancy-specific protein produced and secreted exclusively by decidual stromal cells (DSCs) [ 30 ], which is first detected on the antimesometrial side after day 5, followed by the mesometrial side from day 8 onwards [ 30 ]. While DPRP expression was absent in the nonpregnant uterus and on E5.5 (prior to decidualization), up to 29.5% of GFP + cells in the decidua were positive for DPRP on E9.5 and thereafter, indicating that a substantial population of BMDCs recruited to the uterus had differentiated into functional prolactin-producing decidual cells ( Fig 2H and 2I ). Remarkably, the proportion of DPRP + cells in the decidua was greater in the GFP + population versuss the non-GFP resident cells throughout pregnancy on E9.5 (29.5% versus 12.8%), and nonsignificantly increased on E13.5 (22% versus 14.0%) and E17.5 (22.2% versus 17.0%) ( Fig 2I ), indicating that, compared with the resident cells, recruited BMDCs in decidua differentiated preferentially into decidual prolactin-immunoreactive cells. Moreover, we performed immunostaining for progesterone receptor (PR), a nuclear receptor that is specifically expressed on DSCs without expression in uterine immune cells [ 31 ] and is considered a hallmark of decidualized stromal cells. Interestingly, the percentage of GFP + cells acquiring PR expression was lower than in the GFP − cell population but followed the same trend of progressive increase to mid-gestation, reaching up to approximately 30% PR positivity on E9.5 and E13.5 ( Fig 2J–2M ), further corroborating their decidual stromal identity. To further establish the decidual stromal phenotype of GFP + BMDC cell subsets, we performed single-cell RNA sequencing (scRNAseq) analysis of the E9.5 mouse implantation site, timing of peak BMDCs, using the same GFP BMT model. Differential gene expression analysis of a total of 7,275 cells single cells guided by established markers identified the major clusters of immune cells, DSCs, fibroblasts (FBs), and ECs ( S6A Fig ). A sub-analysis of immune cells and DSCs by graph-based clustering method identified five clusters of DSCs visualized by Uniform Manifold Approximation and Projection (UMAP) ( S6B Fig ). Importantly, GFP + BMDCs were found within all five DSC clusters ( S6C Fig ), and GFP + cells comprised from 1.6% to 15.9% of total DSCs. The GFP + BMDCs within the DSC clusters were found to differentially express multiple DSC markers, including DPRP (Prl8a2), Hoxa10, IGFBP-4, BMP-2, Hand2, collagen isoforms, and others ( S6D Fig ), in contrast to the immune cells. Notably, there was variability in expression of some decidual stromal markers between the five different DSC clusters. This is consistent with a recent scRNAseq study of human decidua that demonstrated graded expression of many of the DSC markers in DSCs along the DSC trajectory [ 32 ]. Within each DSC cluster, GFP + BMDCs displayed a similar expression profile of DSC markers to the GFP − resident DSCs ( S6D Fig ). Overall, expression of proliferation markers Mki67 and Pcna was similar between immune and DSCs clusters ( S7A and S7B Fig ). Among GFP + BMDCs, no significant differences were found in expression of the proliferation markers Mki67 and Pcna between immune and DSC subsets ( S7C Fig ). The recruitment of nonhematopoietic BMDCs to decidua during implantation and early pregnancy and their stromal decidual phenotype suggested that these cells may play an important role during pregnancy.

(A and B) Low-magnification immunofluorescence photomicrographs of E9.5 decidua, showing (A) GFP + BMDCs (green) found predominantly in the mesometrial side but also on the antimesometrial side, while (B) DBA-lectin-positive NK cells (red) are found exclusively on the mesometrial side. (C) Quantification of DBA surface marker on BM-derived (GFP + ) cells and non–BM-derived (GFP − ) cells on E9.5 (n = 4). (D-F) Higher-magnification immunofluorescence photomicrographs of (D) E9.5 mesometrial decidua, (E) antimesometrial decidua, or (F) nonpregnant mice uteri sections demonstrating co-staining of a subset of GFP + BMDCs (green) with NK cell–specific stain DBA-lectin (red). Sections were counterstained with DAPI showing nuclei (blue). The images on the right of each panel are higher magnification of the corresponding dashed areas. (G) Uterine sections from nonpregnant, E 9.5, or ED 13.5 pregnant mice co-stained with PAS (purple) and GFP antibody (brown) showing PAS + NK cells (black arrow), BM-derived NK cells (red pointer) and other non-NK BMDCs (black pointer). Inset shows a higher magnification of the same photomicrograph. Scale bar, 20 μm. Bar graphs represent mean ± SEM. *p < 0.0001. Underlying data are available in S1 Data . BM, bone marrow; BMDC, BM-derived cell; DBA, Dolichos biflorus agglutinin; GFP, green fluorescent protein; NK, natural killer; PAS, Periodic Acid Schiff.

Because NK cells followed by macrophages are the most abundant immune cells in the decidua during pregnancy, and some of these immune populations may not express CD45, we further confirmed the nonhematopoietic nature of the majority of GFP + CD45 − cell population using flow cytometry for other immune/hematopoietic markers as well as specific immunostaining. As some populations of uterine NK cells do not express NK1.1 [ 26 ], we used CD335 as an additional NK cell marker because it is known to be uniformly expressed on all NK cell subsets [ 27 ]. Flow cytometry analysis showed that the vast majority of GFP + CD45 − cells (>95%) were negative for the NK markers NK1.1, CD335, macrophage marker F4/80, as well as other hematopoietic markers (CD34 and CD44) ( Fig 2C and 2D ), indicating their nonhematopoietic nature. Moreover, the beta1 integrin cell surface receptor CD29, which is highly expressed on decidual cells [ 28 ], was found on the majority of this putative stromal decidual cell population (GFP + CD45 − ) and expressed on a significantly higher proportion of cells than uterine GFP + CD45 + cells (BM-derived leukocytes) ( Fig 2C and 2D ). As expected, uterine BM-derived leukocytes (GFP + CD45 + ) demonstrated high expression of typical immune (NK1.1, CD335, F4/80) and hematopoietic markers (CD34, CD44) ( Fig 2C and 2D ). Immunofluorescence analysis of implantation site sections on E9.5 confirmed that many GFP + cells were Sca1 + but CD45 − and CD31 − and located in the decidual stroma, indicating that they were stromal cells and not hematopoietic or ECs ( Fig 2E–2G ). Dolichos biflorus agglutinin (DBA) lectin is the commonly used marker for mouse uterine NK cells as it stains both cytoplasmic granules as well as the cell membrane, thus identifying also immature, agranular NK cells. As expected, DBA immunofluorescence co-staining demonstrated specific localization of NK cells only on the mesometrial side of the implantation site ( Fig 3B ). GFP + BMDCs were abundant on the mesometrial side ( Fig 3A ), where 46.6% of GFP + cells were found to be DBA + NK cells ( Fig 3C–3F ). However, GFP + BMDCs were also found on the antimesometrial side, indicating that the majority of GFP + cells were not NK cells. Periodic Acid Schiff (PAS) reagent is another NK-specific marker staining cytoplasmic granules of uterine NK cells. As some populations of NK cells are DBA negative but PAS + , PAS has been shown to be more inclusive of all decidual NK cell populations [ 29 ]. Co-immunostaining with PAS corroborated our findings that the majority of GFP + cells were not NK cells ( Fig 3G ). In addition, immunostaining with F4/80 macrophage marker identified some GFP + to be macrophages ( S5 Fig ), consistent with our flow cytometry data.

(A) Flow cytometry analysis of BMDCs in uterine implantation site on E9.5. Cells gated in R2 are BM derived (GFP + ) uterine cells, while cells gated in R1 are non–BM-derived resident (GFP − ) uterine cells. Histograms represent GFP + cells (R2) from E9.5 implantation sites that are stained with the indicated antibodies (green line) and respective isotype controls (filled) (n = 4). (B) Quantification of percentage of BM-derived (GFP + ) and non–BM-derived (GFP − ) uterine cells expressing the various cell surface markers shown in (A) (n = 4), *p < 0.05. (C) Flow cytometry analysis of E9.5 BM-derived (GFP + ) uterine cells (R2) using CD45 in combination with various surface marker antibodies. (D) Quantification of surface markers shown in (C), on BM-derived nonhematopoietic decidual cells (GFP + CD45 − ) and BM-derived hematopoietic cells (GFP + CD45 + ) (n = 4–5), *p < 0.01. (E, F, G, H, K) Immunofluorescence photomicrographs of E9.5 mesometrial decidua sections or nonpregnant mice uteri sections demonstrating co-staining of GFP + BMDCs (green) with (E) CD45 (red), (F) CD31 (red), (G) Sca-1 (red), (H) DPRP (red), (K) progesterone receptor (PR) (pink), and CD45 (yellow). Sections were counterstained with DAPI showing nuclei (blue). Insets show higher magnification photomicrographs. White arrows point to BMDCs colocalizing with their respective markers. White dashes are encircling endometrial glands. Scale bars, 50 μm. (L and M) A z-stack series (L) and a 3D image (M) of the inset from (K) demonstrating a single BM-derived GFP + cell co-expressing PR but negative for CD45 (white arrow). (I) Quantification of BMDCs (GFP + ) or non-BMDCs (GFP − ) positive for DPRP throughout gestation (n = 3). *p ≤ 0.01. (J) Quantification of BMDCs (GFP + ) or non-BMDCs (GFP − ) positive for PR throughout gestation (n = 3–5). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. In all panels, bar graphs represent mean ± SEM. See also S3 – S5 Figs. Underlying data are available in S1 Data . BM, bone marrow; BMDC, BM-derived cell; DPRP, decidual prolactin-related protein; GFP, green fluorescent protein; PR, progesterone receptor; Sca-1, stem cell antigen-1; SSC, side scatter; VEGFR2, vascular endothelial growth factor receptor 2.

During early mouse pregnancy, there is a large influx of NK cells to the developing decidua, with NK cells comprising up to approximately 70% of all uterine immune cells at mid-gestation. It is well established that these NK cells concentrate in the decidua exclusively on the mesometrial side of the implantation site and no NK cells are found on the antimesometrial side [ 6 ]. Our histological analysis of immunosections stained for GFP revealed that BMDCs were localized abundantly in the decidua on the mesometrial side, but also on the antimesometrial side, indicating that these BMDCs were not just NK cells ( S4 Fig ). To characterize the phenotype of decidual BMDCs during pregnancy, we utilized flow cytometric analysis ( Fig 1D ). In the nonpregnant uterus, BM-derived GFP + cells were mostly positive for the pan-leukocyte marker CD45 (94.8%), indicating that the majority of this population consisted of immune cells. In contrast, only a small subset of non–BM-derived resident (GFP − ) uterine cells in the nonpregnant uterus were positive for CD45 marker (28.3%). During pregnancy, however, the proportion of CD45 + cells within the total GFP + uterine cell population gradually decreased, reaching a nadir at mid-gestation on E9.5 (72.4%) ( Fig 1D and 1G ). This trend was despite a marked overall increase in the total number of GFP + cells in the uterus ( Fig 1F ), suggesting that many BMDCs recruited to the uterus in pregnancy were progenitors that differentiated into nonhematopoietic (CD45 − ) cells. Flow cytometric analysis of implantation sites on E9.5, the time of peak concentration of BMDCs (24.1%), revealed that the BM-derived GFP + uterine cells (R2) expressed the cell surface markers CD29 (96.3%) and stem cell antigen-1 (Sca-1) (78.8%) most abundantly, followed by the hematopoietic markers CD45 (68.8%) and CD34 (25.7%). The cell surface markers CD44 (15.8%), CD146 (22.7%), CD90 (3.9%), and CD73 (0.6%) were expressed on a subset of these cells, while very low expression of the EC markers CD31 (2.0%), CD105 (0.2%), and vascular endothelial growth factor receptor 2 (VEGFR2) (0.09%) was observed ( Fig 2A and 2B ). Importantly, the cell surface marker profile of uterine GFP + cells was markedly different from circulating GFP + cells in the peripheral blood (which were mostly CD45 + CD29 − Sca1 − ) ( S3B Fig ) but was very similar to resident uterine decidual GFP − cells, which were mostly CD29 + Sca1 + CD45 − ( Fig 2B ), suggesting that the decidual BM-derived CD45 − progenitors became phenotypically and functionally similar to resident decidual cells.

(A) Biodistribution of BM-derived (GFP + ) cells showing preferential recruitment to the pregnant uterus as compared with other body organs. Top panel, PBS nonpregnant control; middle panel, nonpregnant BMT; bottom panel, BMT at E9.5. (B) Engraftment of BMDCs (green) in uteri in nonpregnant state and throughout pregnancy in WT mice receiving BMT from GFP donors. PBS-injected pregnant mouse (E9.5) is shown as negative control, and GFP transgenic mouse is shown as positive control. White arrows indicate the preferential localization of BMDCs to the implantation site. (C) Uterine tissue sections of pregnant mice stained with anti-GFP antibody (brown) showing the localization of BMDCs in decidua during the course of pregnancy. Yellow arrows point to the maternal–fetal interface demarcating the maternal decidua basalis (DB) from fetal placenta (P). Scale bars, 100 μm (top panel) and 50 μm (bottom panel). (D) Representative graphs of flow cytometry of uterine cells demonstrating the temporal changes in hematopoietic (CD45 + ) and nonhematopoietic (CD45 − ) GFP + BMDCs populations during the course of pregnancy. Numbers in each quadrant indicate percentage of cells. (E) Immunofluorescence of uterine tissue sections showing colocalization of PCNA-positive proliferating cells (red) and GFP-positive BMDCs (green) during the course of pregnancy; sections were counterstained with DAPI (blue). Scale bar, 50 μm. (F) Summary of flow cytometry analysis of percentage of GFP + cells in the uterus during pregnancy (n = 5–7). **p ≤ 0.01 versus E9.5, E13.5, and E17.5. *p ≤ 0.05 versus E9.5. (G) Summary of flow cytometry analysis of percentage of GFP + and GFP − cells in the uterus that are either CD45 + or CD45 − during the course of pregnancy (n = 5–7). *p < 0.01, **p < 0.001. (H) Quantification of proliferating PCNA + GFP + BMDCs in the uterus during the course of pregnancy (n = 4–7). All bar graphs are mean ± SEM. *p ≤ 0.05. See also S1 Fig . Underlying data are available in S1 Data . BM, bone marrow; BMDC, BM-derived cell; BMT, BM transplant; DB, decidua basalis; E, embryonic day; GFP, green fluorescent protein; NP, nonpregnant; P, fetal placenta; PCNA, proliferating cell nuclear antigen; WT, wild-type.

To explore the contribution of adult BMDCs to the uterus during implantation and pregnancy development, we utilized our previously described non-gonadotoxic BMT model [ 24 ] ( S1A Fig ). WT female mice underwent submyeloablation using a regimen consisting of 5-fluorouracil (5-FU) and stem cell factor followed by BMT from green fluorescent protein (GFP)-expressing syngeneic donors. Successful BM engraftment was confirmed by flow cytometry analysis of peripheral blood on day 21 post-BMT, demonstrating approximately 45% donor chimerism ( S2A and S3A Figs), consistent with our previous experience using this model [ 24 ]. Our first objective was to characterize the spatial and temporal distribution of BMDCs in the uterus during pregnancy. A pregnancy time course experiment was performed, and mice were killed at various gestational time points. Biodistribution analysis of GFP signal showed that BM-derived GFP + cells migrated preferentially to the uterus during pregnancy compared with other organs ( Fig 1A ). The lung was the only organ that showed some nonspecific autofluorescence, as seen in the phosphate-buffered saline (PBS)-injected control. Moreover, visualization of the intact uterus showed GFP signal in pregnant uteri, with signal enhancement at sites of implantation ( Fig 1B ). Dissection of the pregnant uterus demonstrated that the GFP + cells localized to the maternal side of the uterus and were not arising from the placenta or embryo ( S1B Fig ). Histological analysis of implantation site sections by GFP immunostaining confirmed these findings, revealing that BM-derived GFP + cells were concentrated specifically in the implantation site on the maternal side, reaching a peak at mid-gestation (E9.5) ( Fig 1C ). GFP + cells were localized abundantly in the stroma of the decidua on the mesometrial side, but also on the antimesometrial side ( S4 Fig ). Flow cytometry analysis of uterine implantation sites following removal of the placenta/embryo demonstrated that the percentage of BM-derived GFP + cells in the implantation site increased as pregnancy progressed to mid-gestation; it increased significantly on the day of implantation (E5.5) (15.8%) as compared with the nonpregnant state (9.2%), peaking on E9.5 (24.1%), followed by a gradual decrease on E13.5 (16.7%) and peripartum (E17.5) (13.1%) ( Fig 1D and 1F ). As the observed increase in GFP + cells in the decidua during pregnancy could be due to local proliferation of BMDCs and not only to increased recruitment, we assessed cell proliferation using proliferating cell nuclear antigen (PCNA) staining. The percentage of proliferating cells in the nonpregnant uterine stroma was minimal and not different between the GFP + (BM-derived) (2.6%) and resident GFP − (non–BM-derived) cells (2.7%) ( Fig 1E and 1H ). In contrast, cell proliferation in the pregnant uterine stroma gradually increased throughout gestation, peaking on E9.5, and was significantly greater in the GFP + subpopulation versus the GFP − resident subpopulation at all gestational time points ( Fig 1E and 1H ), suggesting that the increase in the BMDC population in the decidua during pregnancy is at least partly due to preferential local proliferation of recruited BMDCs.

Discussion

It is well established that uterine implantation sites are areas of infiltration of many BM-derived immune cells, which play important roles at the maternal–fetal interface to promote successful pregnancy (reviewed in [6]). Adult nonhematopoietic BMDCs have been detected in both human [17–19] and mouse [18,20–23] uterine endometrium, but the use of gonadotoxic irradiation for myeloablation likely precluded pregnancy investigations using those models. Thus, the nonhematopoietic contribution of adult BMDCs to pregnant decidua as well as its potential functional importance to pregnancy has remained unknown. Here, using GFP-labeled BM cells in a non-gonadotoxic BMT mouse model, we show that implantation and early gestation are strong stimuli for adult BMDCs recruitment to the uterus, where some BMDCs acquire a decidual stromal phenotype, expressing PR, and differentiate into functional prolactin-immunoreactive decidual cells, providing a physiologic contribution to nonhematopoietic stromal decidual cell compartment. Importantly, the nonhematopoietic contribution of BMDCs to the uterus in pregnancy is strikingly different than in the nonpregnant state, as demonstrated by both our GFP BMT and Hoxa11-GFP BMT models. A recent study by Ong and colleagues using an irradiated mouse BMT model reported all BMDCs in the nonpregnant uterus to be immune cells [60]. They found that BMDCs were either positive for the pan-leukocyte marker CD45 and/or positive for the macrophage marker F4/80 [60]. Similar to these findings, in our study most BMDCs in the nonpregnant uterus were CD45+ immune cells (approximately 95%). However, during pregnancy the proportion of CD45+ cells within the BMDC population in the pregnant decidua gradually decreased, reaching a nadir (approximately 70%) at mid-gestation, whereas a substantial population of BMDCs, albeit a minority population, differentiated into nonhematopoietic decidual cells (CD29+CD45−) (approximately 30%) and expressed surface markers similar to the resident decidual cells, indicating that pregnancy drives the differentiation of BMDCs into stromal decidual cells. It was important to exclude the possibility that these CD45− BMDCs were NK cells, macrophages, or other hematopoietic cells, and for this end, we utilized both flow cytometry as well as immunofluorescence with various immune and hematopoietic markers. In addition, we confirmed the identity of BM-derived stromal decidual cells by single-cell RNAseq analysis, demonstrating their clustering within DSC clusters. We also excluded the possibility that the BM-derived stromal decidual cells were a result of cell fusion.

Here, we show that a greater proportion of BMDCs are proliferating as compared with the resident decidual cells. This differential proliferation was restricted to the pregnancy period and observed at all gestational time points. The high rate of cellular division in these BMDCs suggests an important role in keeping up with the demands of rapid growth and turnover in the developing pregnant uterus. Interestingly, the proliferation rate was not found to be different between hematopoietic and nonhematopoietic BMDCs. Moreover, during pregnancy a greater proportion of BM-derived decidual cells were expressing the DPRP as compared with the resident decidual cells. DPRP is a heparin-binding cytokine that is abundantly expressed in uterine decidua and is essential for pregnancy-dependent adaptations to physiological stressors [61]. Taken together, these data suggest that the BM-derived stromal decidual cell population is functional within the developing decidua.

Over the last decade, several studies in both humans and mice have provided evidence that BMDCs give rise to various nonhematopoietic cellular compartments of the nonpregnant endometrium, including epithelial luminal, glandular, and stromal, albeit in very small numbers. These BMDCs have been suggested to play an important role in physiological endometrial tissue regeneration as well as pathologies such as endometriosis (reviewed in [62] and [63]), but their role in implantation and pregnancy maintenance has remained unknown. To study the functional nonhematopoietic contribution of BMDCs to implantation and pregnancy, we utilized two mouse models harboring endometrial stromal cell-specific defects; first, we used Hoxa11−/− null mice, which have abnormally small uteri with stromal atrophy and absence of endometrial glands and are infertile due to lack of decidualization, leading to embryo implantation failure; and second, we used heterozygous Hoxa11+/− mice, which have subfertility characterized by increased pregnancy resorptions and reduced litter size. We demonstrate that BMT from WT mice into syngeneic Hoxa11−/− mice results in endometrial stromal expansion and gland formation, as well as marked decidualization and its characteristic hallmarks, decidual differentiation, cell proliferation, and angiogenesis, compared with the complete lack thereof in Hoxa11−/−KO BMT. Hoxa11-expressing stromal cells were identified at the implantation sites of Hoxa11−/−WT BMT mice and were associated with increased uterine Hoxa11 mRNA and protein expression, as well as up-regulation of genes known to be crucial for implantation in the uterus, including Lif [47], prolactin [59], and Msx1 [64]. Both LIF and prolactin are known transcriptional targets of Hoxa11 [47,59], while MSX1 regulation by Hoxa11 has not been previously described. Our results suggest that Hoxa11 positively regulates, either directly or indirectly, MSX1 in the uterus. Uterine glands are critical for implantation and decidualization in the mouse due to secretion of LIF in the preimplantation period [65]. Absence of LIF or loss of endometrial glands in the mouse uterus results in implantation and decidual defects [65,66], which can be rescued in both cases by LIF administration [65,66]. Therefore, the induction of gland formation in Hoxa11−/− mice following WT BMT is likely responsible for the up-regulation of LIF expression and associated decidualization effects observed in our model. In addition, WT BMT into heterozygous Hoxa11+/− mice also resulted in up-regulation of LIF to levels comparable to WT (S16 Fig), suggesting that LIF up-regulation is not only due to restoration of glands but independently occurs at the time of decidualization, induced by Hoxa11-expressing BMDCs. Our findings are also consistent with the critical role of Hoxa11 in uterine stromal cell proliferation [67]. Interestingly, BMDCs were found in the uterine stroma but not in endometrial glands or luminal epithelium of Hoxa11−/− mice. This suggests that endometrial glandular regeneration was induced by BM-derived stromal paracrine-mediated mechanisms rather than direct cellular contribution to glandular or luminal epithelium. Taken together, these data suggest that gene expression of uterine BMDCs leads to major downstream effects on uterine processes governing implantation.

In the present study, we show that BMT from WT donors into Hoxa11+/− mice results in normalization of litter size through the rescue of pregnancy loss, as compared with Hoxa11+/−KO BMT. Global Hoxa11 mRNA or protein expression was not substantially different between the Hoxa11+/− mice receiving WT or KO BMT, likely due to the low absolute numbers of BMDCs expressing Hoxa11, as demonstrated in our Hoxa11-GFP BMT model. However, Hoxa11 is an important transcription factor and the presence of normal Hoxa11-expressing cells, albeit at small numbers, is likely to have downstream gene expression amplification effects. Moreover, expression of Hoxa11 in the PDZ was more prominent in the Hoxa11+/−WT BMT as compared with Hoxa11+/−KO BMT, suggesting the importance of the spatial expression pattern of this transcription factor to pregnancy maintenance. RNA-seq analysis of implantation site uterine tissues demonstrated that Hoxa11+/−WT BMT and WTWT BMT mice exhibited common differential expression of numerous genes when compared with Hoxa11+/−KO BMT, with significant enrichment for transcripts with known critical roles in decidualization. Importantly, one of the pathways highly enriched in our IPA was the WNT/β-catenin pathway, which includes several genes (Wnt4, Wnt6, Wnt16, Fzd6, Foxa2) found to be dysregulated in Hoxa11+/−KO BMT as compared with WTWT BMT and Hoxa11+/−WT BMT. The Wnt genes encode secreted glycoproteins that are homologous to the Drosophila segment polarity gene wingless (wg) and control essential developmental processes, such as embryonic patterning, cell growth, migration, and differentiation [68]. A subset of Wnt genes (Wnt4, Wnt5a, Wnt6, Wnt7a) are involved in female reproductive tract development and are critical for decidualization and implantation [51–53]. Interestingly, it was shown that Wnt7a-null mice have defective patterning of the uterus and absence of glands associated with loss of uterine Hoxa11 expression [69], suggesting that Wnt7a is upstream of Hoxa11. Moreover, mice with conditional KO of Wnt7a in the uterus using the PgrCre mouse model have defective decidualization and implantation, which is associated with aberrant uterine gene expression during implantation, including increased Wnt4 and decreased Wnt16 and Foxa2 [70]. Consistent with these studies, we observed decreased expression of Wnt16, Fzd6, and Foxa2 in the Hoxa11+/−KO BMT as compared with Hoxa11+/−WT BMT. Another signaling pathway with a critical role in decidualization that was significantly dysregulated in Hoxa11+/−KO BMT but normalized by BMT from WT donors is prolactin signaling. Several members of the prolactin superfamily with known roles in decidualization (Prl3c1, Prl6a1, Prl8a2) [40,49,50] were increased in Hoxa11+/−KO BMT as compared with Hoxa11+/−WT BMT and WTWT BMT mice. It is plausible that Hoxa11 dysregulation in Hoxa11+/−KO BMT and associated decidual dysfunction leads to compensatory increase in these decidualization factors. Taken together, our findings suggest that BMDCs transform the uterine implantation transcriptome of Hoxa11+/− mice in favor of normal decidualization and are crucial for pregnancy maintenance.

Lineage tracing of nonhematopoietic BM populations was not performed in this study, as Hoxa11 expression in the BM is restricted to mesenchymal stem/stromal cell progenitors and it is not expressed in hematopoietic cells [25]. Thus, Hoxa11 expression in transplanted BM cells should be restricted to the nonhematopoietic BM subpopulation. Our flow cytometry results in Hoxa11+/− mice with GFP knock-in are consistent with this, showing specific expression of Hoxa11 in nonhematopoietic BM and uterine stromal cells and absence of Hoxa11 expression in hematopoietic BM cells. Moreover, our Hoxa11-GFP BMT model demonstrated that all Hoxa11+GFP+ BMDCs recruited to the pregnant uterus were nonhematopoietic. In contrast, Hoxa11+GFP+ cells were not found in the nonpregnant uterus. Taken together, these data suggest that the positive effects of BMT on uterine stromal expansion and decidualization in Hoxa11 KO mice, and pregnancy loss prevention in Hoxa11+/− mice in our study, are induced by the nonhematopoietic BMDC Hoxa11-expressing subpopulation rather than the hematopoietic BMDCs. Moreover, recruitment of BMDCs to the uterus, total leukocyte numbers, as well as immune cell subpopulations in the uterus did not appear to be altered in nonpregnant Hoxa11−/− or pregnant Hoxa11+/− mice, further suggesting that hematopoietic and/or immune dysregulation is unlikely to explain the phenotypes in these mice.

While this study does not define all the BM cell subsets that give rise to DSC population in pregnancy, our study provides several lines of evidence in support of BM-MSC as a BM origin of these nonhematopoietic cells: GFP+ BM-MSC populations are reconstituted in the BM following BMT in our model; these BM-MSCs are found to be mobilized to the circulation in response to pregnancy; BM-MSCs are able differentiate into stromal decidual cells in vitro; and Hoxa11+GFP+ nonhematopoietic BMDCs are found in the pregnant uterus. Another study similarly showed that human BM-MSCs can differentiate in vitro into prolactin-producing stromal cells [71]. In this study, we used established BM culture techniques for expansion and enrichment of MSCs, which would include mesenchymal stromal cell populations. The use of specific markers such as CD140a in the isolation procedure would have refined the MSC population. Previously, it was shown that endothelial progenitor cells (EPCs) are increased during human pregnancy [72] and that EPCs directly contribute to ECs within decidual vasculature by vasculogenesis in mouse pregnancy [73]. Other studies in mice and rats showed that hypoxia, ischemia, and liver injury induced the mobilization of MSCs to peripheral blood [38,74,75], suggesting that systemic signals trigger the release of MSCs from the BM. Interestingly, hypoxia- and ischemia-induced mobilization appears to be specific for MSCs because total circulating HSCs were not significantly increased [38,74]. Similarly, pregnancy-induced mobilization in our study was specific for MSCs, as HSCs were not affected.

Previous studies have shown that factors driving the recruitment of BM-derived nonhematopoietic cells to the endometrium include uterine ischemia and injury [22,76], which increased engraftment of BMDCs to the stromal compartment by 2-fold, and estrogen, which increased the incorporation of BM-derived endothelial progenitors into uterine vasculature [77]. In contrast, exposure to cigarette smoke, which has been linked to infertility, decreases the recruitment of both stromal and epithelial BMDCs [78]. Our study shows that implantation and the developing pregnancy are very strong physiological stimuli for BMDC engraftment into the uterus (an approximately 4-fold increase on E9.5 compared with the nonpregnant state). As it was demonstrated that CXCL12 ligand and its CXCR4 receptor are important in mediating migration of BMDCs towards endometrial stromal cells in vitro [79], one may speculate that the CXCL12/CXCR4 axis plays a role in mediating homing of BMDCs to the decidua at the time of implantation and pregnancy. However, future studies are warranted to investigate the exact mechanism(s) responsible for BMDC recruitment to the uterus in response to pregnancy.

Recurrent pregnancy loss (RPL) in humans, defined as the loss of three or more consecutive pregnancies prior to 20 weeks of gestation, affects 1% to 2% of couples and in over 50% of cases its cause remains unexplained [80]. With the advent of in vitro fertilization (IVF), recurrent implantation failure (RIF), when embryos fail to implant after several IVF treatment attempts, is an increasingly recognized clinical condition [81]. The Hoxa11-deficient mice used herein model both of these clinical conditions; the Hoxa11−/− mice have a more severe and earlier defect, leading to recurrent failure of implantation, while the Hoxa11+/− mice have partial Hoxa11 expression characterized by apparently normal implantations followed by pregnancy losses. Hoxa11 expression has also been implicated in human implantation, and several studies have shown uterine Hoxa11 expression to be decreased in conditions associated with pregnancy failure, such as submucosal leiomyomas, endometriosis, and pregnancy loss [46,82–84]. Moreover, it was reported that women with RPL and obesity-associated reproductive failure have decreased endometrial clonogenic cell populations and accelerated stromal cell senescence, suggesting endometrial stem cell dysfunction [85,86]. Our findings that Hoxa11-expressing BMDCs promote changes in the uterine transcriptome leading to partial correction of decidualization defect in Hoxa11−/− mice and full rescue of pregnancy loss in Hoxa11+/− mice raise the possibility that BM stem cell dysfunction may be underlying RIF and/or RPL, and that stem cell therapy may prove to be a viable approach to treat these conditions.

In summary, our study provides evidence that BMDCs have a nonhematopoietic physiologic contribution to the decidual stroma and play an important role in implantation and pregnancy maintenance. Importantly, nonhematopoietic BMDCs have the ability to influence the decidual molecular milieu and overcome implantation defects. Although it remains to be established to what extent these findings in the mouse translate to the situation in humans, our data raise the prospect that BMDCs dysfunction may contribute to implantation failure or pregnancy loss in women.