Effects of autoimmune abnormalities on fertility and placental morphology in mice

Figures & data

Figure 1. Experimental protocol, indices for systemic immunity and fertility, and placental gene expression associated with pregnancy.

(A) Experimental protocol. (B) Maternal spleen weight (SPW). (C) Maternal serum levels of anti-double-stranded (ds) DNA antibodies. (D) The uterus, placenta, and fetus of MRL/MpJ (MpJ) and MRL/MpJ-Fas^lpr/lpr (Lpr) mice. The uterus includes the placenta (orange arrowheads) and the fetus (blue arrowheads). The green arrowhead shows an absorption scar observed in the uterus. The right panel shows a placental remnant and an absorbed fetus in Lpr. Bars = 1 cm. (E) Number of implantations and fetuses. The number of implantations was defined as the total number of fetuses, abnormal fetuses, absorption scars, and placental remnants. (F) The ratio of the number of abnormal fetuses to the number of implants. (G) Placental and fetal weights. (H) Pregnancy-associated placental mRNA expression moMpJ: MRL/MpJ (MpJ) mother mated with Lpr father. moLpr: Lpr mother mated with MpJ father. Slc7a3: solute carrier family 7 (cationic amino acid transporter, y + system) member 3. Krt15: keratin 15. Sprr2a3: small proline-rich protein 2A1. Each bar represents the mean of each group. The number of animals or organs analyzed is summarized in Table 1. Significance for other strains at the same age and gestational day (GD) (Mann-Whitney U-test, *: P < 0.05, ** P < 0.01).

Table 1. The number of samples used in this study.

Measurements	MRL/MpJ	MRL/MpJ-Fas^lpr/lpr	Application
Mother
Spleen weight	6	6	Table 2, 3, Figure 1B
Serum anti-dsDNA antibody	5	5	Table 2, 3, Figure 1C
Number of implantation*	6	6	Table 2, 3, Figure 1E, F
Number of foetus*	6	6	Table 2, 3, Figure 1E, F
Foetus, placenta
Placenta weight	15	18	Table 2, 3, Figure 1G
Foetus weight	15	18	Table 2, 3, Figure 1G
Placenta layers	12	10	Figure 2C
Natural killer cell	7	8	Table 3, Figure 3B
Neutrophil	11	8	Table 3, Figure 3B
Macrophage	12	10	Table 3, Figure 3B
B cell	7	10	Table 3, Figure 3B
T cell	14	11	Table 3, Figure 3B
Gene expression*	14	14	Table 2, 3, Figure 1E, F, Fig, 3 C, Figure 4A, Supplemental Figure 1A

* For microarray analysis, eight RNA samples from four pregnant MRL/MpJ mice and five RNA samples from three pregnant MRL/MpJ-Fas^lpr/lpr mice were collected, and one pooled sample from each group was used. dsDNA: double stranded DNA.

Table 2. The correlations between indices for maternal autoimmune disease and fertility.

Parameter		Group	Fertility parameter					Genes expressed in the placenta
			No.Im	No.Fe	AbFR	PW	FW	Slc7a3	Krt15	Sprr2a3
AID	SPW	All mice	−0.281	−0.173	−0.101	−0.085	−0.261	−0.608**	−0.430*	−0.418*
		moMpJ	−0.559	−0.429	−0.759	0.535*	−0.178	0.393	0.115	0.477
		moLpr	−0.714	−0.783	0.676	0.652**	0.370	−0.599	−0.254	−0.218
	Anti-dsDNA antibody	All mice	−0.333	−0.240	0.157	−0.311	−0.227	−0.731**	−0.477*	−0.397
		moMpJ	−0.616	−0.500	−0.671	0.641*	0.433	0.057	0.418	0.533
		moLpr	−0.900*	−0.975**	0.894*	0.042	0.151	−0.617	−0.626*	−0.329
Genes expressed in the placenta	Slc7a3	All mice	0.230	0.110	0.206	0.408	0.373	NA	NA	NA
		moMpJ	0.205	0.365	−0.258	−0.022	−0.181	NA	NA	NA
		moLpr	0.674	0.656	−0.316	0.214	0.286	NA	NA	NA
	Krt15	All mice	−0.151	−0.057	−0.225	0.492**	0.242	NA	NA	NA
		moMpJ	−0.376	−0.171	−0.616*	0.648*	0.352	NA	NA	NA
		moLpr	0.250	0.409	−0.540*	−0.160	−0.086	NA	NA	NA
	Sprr2a3	All mice	−0.071	−0.072	−0.047	0.528**	0.300	NA	NA	NA
		moMpJ	−0.378	−0.145	−0.695**	0.679**	0.090	NA	NA	NA
		moLpr	0.344	0.433	−0.194	−0.081	0.055	NA	NA	NA

Spearman’s rank correlation coefficients (ρ).

* : p < 0.05.

** : p < 0.01.

The number of analyzed mice is summarized in Table 1. AID: autoimmune disease. dsDNA: double-stranded DNA. moMpJ: MRL/MpJ (MpJ) mother mated with MRL/MpJ-Fas^lpr/lpr (Lpr) father. moLpr: Lpr mother mated with MpJ father. No.Im: number of implantations. No.Fe: number of fetuses. AbFR: ratio of number of abnormal fetuses to No.Im. PW: placental weight. FW: fetal weight. SPW: spleen weight of mother. NA: not applicable.

Figure 2. Morphological analysis of the placenta.

(A) Placental histology with the labyrinth (Lb), junctional zone (Jz), and decidua (Dc) observed on the fetal side. Hematoxylin and eosin staining (HE). Bars = 100 μm. (B) Immunohistochemistry (IHC) of placental vimentin (vim). Lb and Dc were positive (⁺) for Vim but negative for Jz. Bars = 500 μm. (C) The ratio of each layer area to the total placental area. (D) The histology of each placental layer. Abundant maternal blood was observed (asterisk) in the Lb, which was surrounded by trophoblast cells (blue arrowheads). Several nucleated fetal red blood cells were also observed (orange arrowheads). In IHC for Vim, fetal vascular endothelial (Ve) cells were Vim⁺. In Jz, glycogen cells (asterisks), spongy trophoblast cells (orange arrowheads), and giant trophoblast cells (blue arrowheads) were observed; however, no Vim⁺ cells were observed. In Dc, abundant natural killer (NK) cells (black arrowheads) and decidual cells (orange arrowheads) were observed. In IHC, decidual cells (orange arrowheads) and maternal Ve were Vim⁺. HE stain and IHC. Bars = 100 μm. moMpJ: MRL/MpJ (MpJ) mice mated with MRL/MpJ-Fas^lpr/lpr (Lpr) mice. moLpr: Lpr mother mated with MpJ father. Each bar represents the mean of each group. The number of animals or organs analyzed is summarized in Table 1.

Figure 3. Immune cells observed in the placenta.

(A) Histological analysis of placental immune cells. In the decidua (Dc), natural killer (NK) cells contained eosinophilic granules (black arrowheads). Immunohistochemistry (IHC) revealed Gr-1-positive (⁺) neutrophils in the extravascular space and blood vessels of the Dc. B220⁺ B cells in blood vessels (black arrowheads), CD3⁺ T cells in the extravascular area (orange arrowheads), and blood vessels (blue arrowheads) in the labyrinth (Lb). Squares show high-magnification images of each positive cell. Hematoxylin and eosin staining. IHC. Bars = 100 μm. (B) The number of immune cells in the placenta. (C) The mRNA expression of genes associated with T cells. moMpJ: MRL/MpJ (MpJ) mice mated with MRL/MpJ-Fas^lpr/lpr (Lpr) mice. moLpr: Lpr mother mated with MpJ father. Th: T helper cells. Tc: T killer cells. Tfh: T follicular helper cells. Tregs: Regulatory T cells. Each bar represents the mean of each group. The number of animals or organs analyzed is summarized in Table 1. Significance for another strain at the same age and gestational age (Mann-Whitney U-test, *: P < 0.05, ** P < 0.01).

Figure 4. Placental gene expression and protein location associated with T cells.

mRNA expression associated with T cells in the placenta. (B) CCL5 localization in the moLpr placenta. (C) CXCL9 localization in the placenta. moMpJ: MpJ mother mated with Lpr father. moLpr: Lpr mother mated with MpJ father. ND: not detected. CCL5: C-C motif chemokine 5. CXCL9: Chemokine (C-X-C motif) ligand 9. Each bar represents the mean of each group. The number of animals or organs analyzed is summarized in Table 1. Significance for another strain at the same age and gestational age (Mann-Whitney U-test, *: P < 0.05, ** P < 0.01).

Table 3. The correlations among indices for autoimmune disease, fertility, immune cell quantities, and T cell.

Parameter		Group	Immune cell				T cell function associated genes
			Neutrophil	NK cell	B cell	T cell	Gata3	Bcl6	Ccl5	Cxcl9
AID	SPW	All mice	−0.200	−0.154	0.259	0.684**	0.453*	0.370	0.705**	0.625**
		moMpJ	−0.600	0.436	0.156	−0.080	−0.163	−0.324	0.227	0.007
		moLpr	1.000**	0.096	0.543	0.585	0.126	0.164	0.673*	0.247
	Anti-dsDNA antibody	All mice	−0.067	−0.359	0.332	0.744**	0.594**	0.484*	0.551*	0.798**
		moMpJ	−0.300	0.147	0.798*	0.414	0.365	0.271	−0.299	0.218
		moLpr	0.800	−0.029	0.752*	0.255	0.221	0.282	0.610	0.407
Fertility	No.Im	All mice	0.175	−0.446	−0.527*	−0.045	−0.109	−0.077	−0.042	−0.075
		moMpJ	0.765	−0.561	−0.324	0.158	−0.053	0.041	−0.066	−0.106
		moLpr	−0.900*	−0.458	−0.765**	−0.502	−0.013	−0.047	−0.162	−0.116
	No.Fe	All mice	−0.055	−0.375	−0.574*	0.098	−0.029	−0.016	−0.050	0.058
		moMpJ	0.657	−0.582	−0.248	0.301	−0.043	0.011	−0.098	−0.014
		moLpr	−0.900*	−0.368	−0.804**	−0.559	−0.073	−0.140	−0.482	−0.222
	AbFR	All mice	0.748**	−0.409	0.279	−0.163	−0.161	−0.105	−0.093	−0.160
		moMpJ	0.880*	−0.267	−0.405	−0.254	−0.002	0.139	0.009	−0.235
		moLpr	0.671	−0.495	0.543	0.262	0.238	0.281	0.585*	0.452
	PW	All mice	0.018	0.100	0.405	−0.004	−0.199	−0.066	−0.241	−0.194
		moMpJ	−0.771	0.600	0.600	0.261	0.095	0.169	−0.269	0.182
		moLpr	0.900*	−0.314	0.486	0.771	0.178	0.231	0.678*	0.400
	FW	All mice	0.318	−0.055	0.469	0.140	−0.462*	−0.338	−0.387	−0.125
		moMpJ	0.086	−0.600	−0.200	0.609	−0.248	−0.297	−0.637*	0.357
		moLpr	0.900*	−0.257	0.771	0.943**	−0.099	0.015	0.692*	0.382

Spearman’s rank correlation coefficients (ρ).

* : p < 0.05.

** : p < 0.01.

The number of analyzed mice is summarized in Table 1. AID: autoimmune disease. SPW: spleen weight of mother. dsDNA: double-stranded DNA. No.Im: number of implantations. No.Fe: number of fetuses. AbFR: ratio of number of abnormal fetuses to No.Im. PW: placental weight. FW: fetal weight. NK: natural killer. Th2: T helper 2. Tfh: T follicular helper. moMpJ: MRL/MpJ (MpJ) mother mated with MRL/MpJ-Fas^lpr/lpr (Lpr) father. moLpr: Lpr mother mated with MpJ father.

Hematoxylin

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon request.