Abstract
There are around 300 million adolescent pregnancies worldwide, accounting for 11% of all births worldwide. Accumulating evidence demonstrates that many adverse perinatal outcomes are associated with adolescent pregnancies. However, how and why these abnormalities occur remain to be defined. In this study, pregnancy at different stages was compared between 25- and 30- day-old and mature female mice. We found that the litter size of adolescent pregnancy is significantly decreased from F1 to F3 generations compared to mature pregnancy. On days 8 and 12 of pregnancy, multiple abnormalities in decidual and placental development appear in F3 adolescent pregnancy. On days 5 and 8, uterine endoplasmic reticulum stress is dysregulated in F3 adolescent pregnancy. Embryo implantation and decidualization are also compromised in adolescent pregnancy. Many genes are abnormally expressed in adolescent estrous uteri. The abnormal endocrine environment and abnormal implantation from uterine immaturity may result in multiple pregnancy failures in adolescent pregnancy. The aim of this study is to shed light on human adolescent pregnancy.
Introduction
In 2015, there were about 300 million adolescent pregnancies worldwide and 16 million adolescents gave birth, accounting for 11% of all births worldwide (Holness 2015). Furthermore, nearly one-fifth of adolescents become pregnant in Africa (Kassa et al. 2018). In the industrialized world, the United States continues to have a high rate of adolescent pregnancy (Jiskrova & Vazsonyi 2019). Adolescent pregnancy will still be a target for prevention in many countries (Marvin-Dowle & Soltani 2020). Accumulating evidence demonstrates that many adverse perinatal outcomes are associated with adolescent pregnancies, such as preterm birth, low birth weight babies, preeclampsia, intrapartum death, and miscarriage (Leftwich & Alves 2017). Compared with women between 20 and 24.15 years old, the risk of stillbirth is four times higher in teens aged below 15 years and 50% higher in teens aged between 15 and 19 years (Kramer & Lancaster 2010). In adolescents aged <15 years, the median gestational week is lower, and the rates of threatened abortion and preeclampsia are higher compared to the control group (Karata et al. 2019). The possibility of premature rupture of membranes and preterm premature rupture of membranes in adolescent pregnancy was significantly higher compared to adult pregnancy (Markovic et al. 2020). Although it is shown that the incidence of meiotic errors and aneuploid embryos is higher during adolescence (age < 20) as is the miscarriage rate compared with adult women (age between 20 and 30) (Gruhn et al. 2019), most of the perinatal complications in these newborns occur without any chromosomal abnormality. However, most pregnancy complications associated with gestation usually have a relationship with incorrect implantation and placentation, such as preterm labor, low birth weight stillbirth, and preeclampsia (Woods et al. 2017). Embryo implantation, decidualization, and placenta development are key processes during pregnancy (Hemberger et al. 2020). In assisted reproductive technologies, a low implantation rate is a major problem during infertility treatments (Dey et al. 2004). Apart from embryo implantation, decidual and placental dysfunction also contribute to poor pregnancy outcomes. Impaired decidualization is closely related to preeclampsia (Zhou et al. 2019). In aged mice, there are abnormal steroid hormone responses and decline of embryo implantation (Li et al. 2017). Defective decidualization and placental development also lead to abnormal embryo development and reproductive decline (Woods et al. 2017).
Based on the available data, we hypothesize that adolescent pregnancy may be associated specifically with defective decidualization and placental development. In this study, a mouse model was used to explore the factors in adolescent pregnancy leading to abnormal pregnancy outcomes. Our study showed that pregnancy outcomes are significantly compromised in adolescent pregnancy, primarily due to delayed embryo implantation and uterine immaturity.
Materials and methods
Mice and treatments
All animal experiments were approved by the Institutional Animal Care and Use Committee of South China Agricultural University. CD1 mice were housed in a controlled environment with a 14 h light:10 h darkness cycle. Virgin females were co-caged with male CD1 mice of 8–16 weeks old. The day when vaginal plug was detected was designated as day 1 of pregnancy. The vaginal opening in CD1 virgin mice occurs about 25 days after birth (Wu et al. 2011). This is comparable to 14–16 years of age in humans (Kercmar et al. 2014). To explore the effects of adolescent pregnancy on pregnancy outcomes, this study focused on immature female mice 25–30 days after birth (PND25–PND30) and mature adult mice (12 weeks). Pregnant females were designated as F0 mice, and their offsprings were designated as F1 mice. F1 mice were mated at first estrus (25–30 days old), and their offspring were designated as F2 mice. F2 mice were mated at first estrus (25–30 days old), and their offspring were designated as F3 mice. Mature 'adult' females were 8–12 weeks old and designated as 3M. Artificial decidualization was performed as previously described (Gu et al. 2016). Pregnant mice were intraperitoneally injected with tunicamycin (0.1 or 0.001 mg/kg in saline, 5 mg/mL dissolved in DMSO and diluted in saline, 100 mL per mouse, Millipore) on days 6 and 7 at 9:00 and 21:00 h, respectively.
Mice were sacrificed by cervical dislocation after they were anesthetized. On day 12 of pregnancy, uteri were collected. Placenta used for histology, mRNA analysis, and protein analysis and conceptus used for analysis of weight were separated carefully from uteri. On day 8 of pregnancy, deciduoma was separated for the analysis of histology and weight. Decidual tissue was obtained after embryos, and myometrium was separated from uteri.
RT-qPCR
Total RNAs were isolated with TRIzol reagent kit (T9109, TaKaRa) and reverse transcribed with PrimeScript reverse transcriptase reagent kit (R233, Vazyme, Nanjing, China) according to the manufacturer’s instructions. RT-qPCR was performed using SYBR (Q311, Vazyme, Nanjing, China) on a Bio-Rad CFX96 Touch™ Real-Time System thermocycler using gene-specific primers (Supplementary Table 1, see section on supplementary materials given at the end of this article). Gene expression was analyzed, and the Ct values were normalized to Rpl7 housekeeping gene.
Analysis of spliced Xbp1
Total RNAs were isolated with TRIzol reagent kit (T9109, TaKaRa) and reverse transcribed with PrimeScript reverse transcriptase reagent kit (R233, Vazyme, Nanjing, China) according to the manufacturer’s instructions. XBP1 primers were designed to contain the 26 bp fragment for analysis of spliced and unspliced XBP1 mRNA. Products were separated on 2.5% agarose gel. The primer sequences are described in Supplementary Table 1.
Isolation and culture of mouse uterine stromal cells
Stromal cells were isolated from mouse uteri. In brief, mouse uteri on day 4 of pregnancy were digested with 1% trypsin (0458, AMRESCO) and 6 mg/mL dispase (0494207801, Roche) in Hanks’ balanced salt solution (H4891, Sigma). After luminal epithelial cells were removed by washing, the remaining uteri were incubated with 0.15 mg/mL collagenase I (17100017, Gibco). The isolated stromal cells were cultured in DMEM/F12 (D2906, Sigma) containing 10% charcoal-treated FBS (Biological Industries, Israel).
In vitro decidualization of endometrial stromal cells was induced with 1 mM progesterone and 10 nM 17β-estradiol as described (Gu et al. 2016).
Histology and immunohistochemistry
Tissues were fixed in 10% PBS-buffered formalin for 24 h, dehydrated, and embedded in paraffin. Paraffin sections (5 µm) were deparaffinized and stained with hematoxylin and eosin for histology analysis. The number of sections of day 8 of pregnancy was collected to detect the length of embryos as previously described (Li et al. 2015). Immunohistochemistry was performed as previously described (Hu et al. 2019). After blocked with 10% horse serum at 37°C for 1 h, paraffin sections were incubated with primary antibodies (Supplementary Table 2) overnight at 4°C. The same concentration of non-specific rabbit IgG was used for negative control. Following PBS washing, sections were incubated with biotinylated secondary antibody for 30 min and streptavidin-HRP complex (Zhongshan Golden Bridge) for 30 min at room temperature. The positive signals were visualized through DAB horseradish peroxidase color development kit (Zhongshan Golden Bridge). Images were obtained by microscope (Leica, DM2500) with LAS V4.3 system.
uNK cells were identified as previously described (Chen et al. 2012). Briefly, paraffin sections (5 µm) were deparaffinized and antigen-retrieved with 10 mM sodium citrate buffer (pH 6.0). After blocked with 10% horse serum at 37°C for 1 h, paraffin sections were incubated with biotinylated dolichos biflorus agglutinin (DBA) (Sigma–Aldrich, L6533) overnight at 4°C. Following PBS washing, sections were incubated with streptavidin–HRP complex (Zhongshan Golden Bridge) for 30 min at room temperature. The positive signals were visualized through DAB Horseradish Peroxidase Color Development Kit (Zhongshan Golden Bridge). Images were obtained by microscope (Leica, DM2500) with LAS V4.3 system.
Immunofluorescence
Immunofluorescence was performed as previously described (Hu et al. 2019). Briefly, paraffin sections were hydrated, permeabilized with 1% Triton X-100 in PBS for 10 min, and blocked with 10% horse serum at 37°C for 1 h. Sections were then incubated with primary antibodies (Supplementary Table 2) overnight at 4°C. The same concentration of non-specific rabbit IgG was used for negative control. After washing in PBS, sections were incubated with the corresponding secondary antibody conjugated with FITC for 30 min at room temperature. Sections were counter-stained with propidium iodide (PI, Sigma) and mounted for fluorescence analysis. Images were obtained by confocal (Leica TCS SP8) with Leica Application Suite X.
Western blot
Tissues or cultured cells were collected for protein extraction, and protein lysates were separated by SDS-PAGE. After proteins were transferred onto PVDF membranes, membranes were incubated with each primary antibody overnight at 4°C. The primary antibodies used in this study are listed in Supplementary Table 2. After membranes were incubated with the corresponding HRP-conjugated secondary antibody for 1 h, signals were detected through ECL Chemiluminescent kit (Millipore).
RNA-seq
The decidual tissues were isolated from day 8 pregnant uteri following removing embryos and myometrium before RNA extraction. Total decidual RNAs from three mice were mixed and used for RNA sequencing in each group. RNA-seq was performed by Novogene (Tianjin, China). Briefly, total RNAs were isolated and quantitated by Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies). A total of 3 μg RNAs from each sample were used for library preparation using NEBNext® UltraTM RNA Library Prep Kit for Illumina®. Library preparations were sequenced with an Illumina Hiseq platform, and 125 bp/150 bp paired-end reads were generated. After raw data were first processed by in-house Perl scripts, clean data were obtained by removing reads containing adapter. FPKM (fragments per kb per million) of genes was calculated based on the length of genes and reads count mapped to genes. Differential expression analysis of two groups was performed by the DESeq2 R package (1.16.1). After P-values were adjusted using the Benjamini–Hochberg’s approach, genes with an adjusted P-value of <0.05 were defined as differentially expressed genes. Gene Ontology (GO) enrichment analysis of differentially expressed genes was performed through the cluster Profiler R package. GO terms with a corrected P value of <0.05 were considered as significantly enriched.
Statistical analysis
Data were shown by mean ±s.e.m. Statistical analysis was performed with an unpaired Student’s t-test or two-way ANOVA followed with Bonferroni post hoc test. At least three independent repeats were performed in all groups. P < 0.05 was considered statistically significant.
Results
Pregnancy outcome and placental development
In order to investigate adolescent pregnancy in mice, adult pregnant females were designated as F0 mice, and their offsprings were designated as F1 mice. F1, F2, and F3 mice were mated at first estrus (25–30 days old). Mature 'adult' females were 8–12 weeks old and designated as 3M. After examining pregnancy outcomes for each group, we found that average litter size was significantly decreased compared to 3M with F2 (P = 0.0067) and F3 (P =0.0004) mice, while no changes were showed from 3M to F1 mice (P = 0.0636) (Fig. 1A). Interestingly, the weight of the pups showed no significant difference between the two groups (Supplementary Fig. 1A). Because the reduction of average litter size was obvious in F3, our further analysis focused on the comparison between F3 and 3M groups. It has been shown that a decrease in litter size could result from dysfunctional uteri or decreasing embryo number (Ye et al. 2005, Kim et al. 2019), and most problems during embryonic development are associated with placentation defects (Copp 1995). In examining day 12 (D12) pregnant mice, F3 mice were revealed to have abnormal uteri compared with 3M mice (Fig. 1B). Both the litter size and weight of conceptuses on D12 were decreased in F3 compared to 3M (Fig. 1C and D). There were also more abnormal conceptuses that were identified by a morphological appearance in F3 mice than in 3M (Fig. 1E and F). Therefore, we analyzed the histological structures of placentas of 3M and F3 females. Compared to 3M, the labyrinth and junctions were not significantly different from that in F3 mice (Fig. 1F and Supplementary Fig. 1B, C). We then examined the expression of Essrb and Eomes, markers of stem-like trophoblast progenitor cells differentiated from the mural trophectoderm (Woods et al. 2017, Hemberger et al. 2020). Both Essrb and Eomes were decreased in F3 abnormal conceptuses, while Essrb was decreased and Eomses was increased in F3 mice with normal conceptus compared to the 3M group (Fig. 2A and B). Stem-like trophoblast progenitor cells in the placenta can differentiate into trophoblast giant cells and spongiotrophoblasts. We then examined the expression levels of Tpbpa (a marker of trophoblast giant cells) and Prl2c2 (a marker of spongiotrophoblasts) (Woods et al. 2017, 2018). Both Prl2c2 and Tpbpa were decreased in F3 abnormal conceptuses, while Prl2c2 was increased and Tpbpa was decreased in F3 normal conceptuses (Fig. 2C and D). Ctsq and Sybn, markers of the labyrinth layer (Woods et al. 2017), were decreased in F3 normal and abnormal conceptuses compared to the 3M group (Fig. 2E and F).
Pregnancy outcomes and placental development. (A) The average number of newborn mice in 3M (adult group, n = 12), F1 (adolescent F1, n = 17), F2 (adolescent F2, n = 10), and F3 (adolescent F3, n = 12). Statistical differences are analyzed by ANOVA followed by Tukey’s multiple comparisons post hoc test. Data are present as mean ± s.e.m. **P < 0.01. (B) The uterine morphology on day 12 of pregnancy. (C) The average number of conceptuses on day 12. (D) The average weight of conceptuses on day 12. (E) The rate of abnormal conceptuses (n = 12). (F) The placental morphology on day 12 in 3M, normal F3, and abnormal F3 (F3-A) groups; scale bar: 1 mm; D, decidualization; J, junction; L, Labyrinth.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Pregnancy outcomes and placental development. (A) The average number of newborn mice in 3M (adult group, n = 12), F1 (adolescent F1, n = 17), F2 (adolescent F2, n = 10), and F3 (adolescent F3, n = 12). Statistical differences are analyzed by ANOVA followed by Tukey’s multiple comparisons post hoc test. Data are present as mean ± s.e.m. **P < 0.01. (B) The uterine morphology on day 12 of pregnancy. (C) The average number of conceptuses on day 12. (D) The average weight of conceptuses on day 12. (E) The rate of abnormal conceptuses (n = 12). (F) The placental morphology on day 12 in 3M, normal F3, and abnormal F3 (F3-A) groups; scale bar: 1 mm; D, decidualization; J, junction; L, Labyrinth.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Pregnancy outcomes and placental development. (A) The average number of newborn mice in 3M (adult group, n = 12), F1 (adolescent F1, n = 17), F2 (adolescent F2, n = 10), and F3 (adolescent F3, n = 12). Statistical differences are analyzed by ANOVA followed by Tukey’s multiple comparisons post hoc test. Data are present as mean ± s.e.m. **P < 0.01. (B) The uterine morphology on day 12 of pregnancy. (C) The average number of conceptuses on day 12. (D) The average weight of conceptuses on day 12. (E) The rate of abnormal conceptuses (n = 12). (F) The placental morphology on day 12 in 3M, normal F3, and abnormal F3 (F3-A) groups; scale bar: 1 mm; D, decidualization; J, junction; L, Labyrinth.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Placental development and uNK cells on day 12 of pregnancy in 3M (n = 6), normal F3 (n = 6), and abnormal F3 (F3-A, n = 6) groups. (A and B) The mRNA levels of Essrb and Eomes, markers of trophoblast stem cell genes. (C and D) The mRNA levels of Prl2c2, marker of trophoblast giant cells, and Tpbpa, marker spongiotrophoblast (SpTr). (E and F) The mRNA level of Ctsq and Synb, markers of placental labyrinths. (G) The distribution of uNK cells. (H) GzmB immunostaining; scale bar: 1 mm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Placental development and uNK cells on day 12 of pregnancy in 3M (n = 6), normal F3 (n = 6), and abnormal F3 (F3-A, n = 6) groups. (A and B) The mRNA levels of Essrb and Eomes, markers of trophoblast stem cell genes. (C and D) The mRNA levels of Prl2c2, marker of trophoblast giant cells, and Tpbpa, marker spongiotrophoblast (SpTr). (E and F) The mRNA level of Ctsq and Synb, markers of placental labyrinths. (G) The distribution of uNK cells. (H) GzmB immunostaining; scale bar: 1 mm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Placental development and uNK cells on day 12 of pregnancy in 3M (n = 6), normal F3 (n = 6), and abnormal F3 (F3-A, n = 6) groups. (A and B) The mRNA levels of Essrb and Eomes, markers of trophoblast stem cell genes. (C and D) The mRNA levels of Prl2c2, marker of trophoblast giant cells, and Tpbpa, marker spongiotrophoblast (SpTr). (E and F) The mRNA level of Ctsq and Synb, markers of placental labyrinths. (G) The distribution of uNK cells. (H) GzmB immunostaining; scale bar: 1 mm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
During placental development, NK cells regulate decidual angiogenesis, vascular adaptations, and trophoblast invasion (Yougbare et al. 2017). Compared to 3M, the number of uNK cells was lower in F3 normal placenta and higher in F3 abnormal placenta (Fig. 2G). GZMB and NKP46 are essential to NK cell function. The number of GZMB-positive cells was reduced in F3 mice with normal conceptuses and increased in F3 mice with abnormal conceptuses (Fig. 2H). The mRNA expression of GzmB showed a similar pattern (Fig. 3A). NKP46, an NK cell-related molecule, also showed a similar change with GzmB (Fig. 3B). Eng and Vegfa, the pro-angiogenic molecules (Yougbare et al. 2017), decreased in the F3 group, and Endoglin, an anti-angiogenic factor, increased (Fig. 3C and E). Decidualization genes, Prl8a2 increased, while Bmp2 decreased in the F3 group (Fig. 3F and G). The expression of Hoxa10 was reduced in F3 mice with normal conceptuses and increased in F3 mice with abnormal conceptuses (Fig. 3H). These data demonstrate that a dysfunctional placental may result in a decrease in litter size in the F3 group.
Decidualization changes in placenta on day 12 of pregnancy in 3M (n = 6), normal F3 (n = 6), and abnormal F3 (F3-A, n = 6) groups. (A) GzmB mRNA level. (B) NKP46 protein level. (C) End mRNA level. (D) Eng mRNA level. (E) Vegf mRNA level. (F) Prl8a2 mRNA level. (G) Bmp2 mRNA level. (H) Hoxa10 mRNA level.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidualization changes in placenta on day 12 of pregnancy in 3M (n = 6), normal F3 (n = 6), and abnormal F3 (F3-A, n = 6) groups. (A) GzmB mRNA level. (B) NKP46 protein level. (C) End mRNA level. (D) Eng mRNA level. (E) Vegf mRNA level. (F) Prl8a2 mRNA level. (G) Bmp2 mRNA level. (H) Hoxa10 mRNA level.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidualization changes in placenta on day 12 of pregnancy in 3M (n = 6), normal F3 (n = 6), and abnormal F3 (F3-A, n = 6) groups. (A) GzmB mRNA level. (B) NKP46 protein level. (C) End mRNA level. (D) Eng mRNA level. (E) Vegf mRNA level. (F) Prl8a2 mRNA level. (G) Bmp2 mRNA level. (H) Hoxa10 mRNA level.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidualization during early pregnancy
The mature placenta in rodents consists of the maternal decidua on the outside, the junctional zone, and the innermost labyrinth (Hu & Cross 2010). Decidualization is a critical process for establishing fetal–maternal communication (Gellersen & Brosens 2014). Because there were abnormalities for both embryonic and placental development on day 12 of pregnancy, we went further to examine decidualization and embryo development on day 8 of pregnancy. The morphological characteristics and the number of embryos were similar between F3 and 3M groups (Fig. 4A and B). However, the size and of the embryos and the weight of implantation sites decreased in the F3 group compared to the 3M group (Fig. 4C and D). To exclude embryonic effects, pseudopregnant mice were induced for artificial decidualization (Fig. 4E). The weight of deciduoma was reduced in the F3 group compared to the 3M group, and the expression of Prl8a2 showed an increase in the F3 group (Fig. 4F and G). However, the expression of Hoxa10 showed no changes, while the expression of the Bmp2 decreased in artificial decidualization (Supplementary Fig. 1D). In vitro decidualization was used to examine the differences. Compared to the 3M group, Prl8a2, Hoxa10, and Bmp2, decidualization-related genes, were also increased in the F3 group (Fig. 4H, I and J).
Decidual development on day 8 of pregnancy in F3 and 3M groups. (A) The uterine morphology on day 8 of pregnancy. (B) The average number of implantation sites in two groups. (C) The average length of embryo on day 8 in two groups. (D) The average weight of implantation sites on day 8 in two groups (n = 10). (E) The morphology of deciduoma on day 8 of pseudopregnancy after 5 µL of sesame oil were intraluminally injected in pseudopregnant mice. (F) The average weight of deciduoma in two groups. (G) Prl8a2 mRNA expression in two groups (n = 6). (H) Prl8a2 mRNA level under in vitro decidualization. (I) Bmp2 mRNA level under in vitro decidualization. (J) Hoxa10 mRNA level under in vitro decidualization.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidual development on day 8 of pregnancy in F3 and 3M groups. (A) The uterine morphology on day 8 of pregnancy. (B) The average number of implantation sites in two groups. (C) The average length of embryo on day 8 in two groups. (D) The average weight of implantation sites on day 8 in two groups (n = 10). (E) The morphology of deciduoma on day 8 of pseudopregnancy after 5 µL of sesame oil were intraluminally injected in pseudopregnant mice. (F) The average weight of deciduoma in two groups. (G) Prl8a2 mRNA expression in two groups (n = 6). (H) Prl8a2 mRNA level under in vitro decidualization. (I) Bmp2 mRNA level under in vitro decidualization. (J) Hoxa10 mRNA level under in vitro decidualization.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidual development on day 8 of pregnancy in F3 and 3M groups. (A) The uterine morphology on day 8 of pregnancy. (B) The average number of implantation sites in two groups. (C) The average length of embryo on day 8 in two groups. (D) The average weight of implantation sites on day 8 in two groups (n = 10). (E) The morphology of deciduoma on day 8 of pseudopregnancy after 5 µL of sesame oil were intraluminally injected in pseudopregnant mice. (F) The average weight of deciduoma in two groups. (G) Prl8a2 mRNA expression in two groups (n = 6). (H) Prl8a2 mRNA level under in vitro decidualization. (I) Bmp2 mRNA level under in vitro decidualization. (J) Hoxa10 mRNA level under in vitro decidualization.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidual tissues from both 3M and F3 groups were chosen for transcriptome analysis. Compared to 3M, there were 337 differently expressed genes in the F3 group, of which 133 were upregulated and 204 genes downregulated (Fig. 5A and Supplementary Table 3). Among the 337 differentially expressed genes, Bmp2, Cdh1, Cited2, Prl8a2 (Dtprp), and Igf1 were decidualization-related genes (Luo et al. 2016, Woods et al. 2017, Babayev et al. 2019). Prl8a2, Bmp2, and Cited2 were increased, while Cdh1 and Igf1 were decreased in the F3 group (Fig. 5B, C, D, E and F). In GO analysis, many pathways related to reproduction were found, such as reproductive structure development, reproductive system development, and endoplasmic reticulum (ER) chaperone complex (Fig. 5G). Changes in these pathways indicated that the function of decidualization is defective in F3 mice compared to 3M mice.
Decidualization-related gene expression on day 8 of pregnancy. (A) Volcano of genes differently expressed in the decidual tissues in two groups. (B) Prl8a2 mRNA level. (C) Igf 1 mRNA level. (D) Cdh1 mRNA level. (E) Bmp2 mRNA level. (F) Cited2 mRNA level. (G) GO pathway analysis in two groups. BP, biological process; CC, cellular component; MF, molecular function.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidualization-related gene expression on day 8 of pregnancy. (A) Volcano of genes differently expressed in the decidual tissues in two groups. (B) Prl8a2 mRNA level. (C) Igf 1 mRNA level. (D) Cdh1 mRNA level. (E) Bmp2 mRNA level. (F) Cited2 mRNA level. (G) GO pathway analysis in two groups. BP, biological process; CC, cellular component; MF, molecular function.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Decidualization-related gene expression on day 8 of pregnancy. (A) Volcano of genes differently expressed in the decidual tissues in two groups. (B) Prl8a2 mRNA level. (C) Igf 1 mRNA level. (D) Cdh1 mRNA level. (E) Bmp2 mRNA level. (F) Cited2 mRNA level. (G) GO pathway analysis in two groups. BP, biological process; CC, cellular component; MF, molecular function.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Dysfunction of endoplasmic reticulum stress response during decidualization
The ER plays important role in cell protein synthesis, folding, and secretion (Ron & Walter 2007). During female mammalian reproduction, proper ER stress is essential to embryo implantation and decidualization (Brosens et al. 2014, Lin et al. 2014, Gu et al. 2016). Based on our gene ontology (GO) and pathway analysis of differentially expressed genes on day 8 of pregnancy, genes involved in the ER chaperone complex were significantly enriched (Supplementary Table 4). We then examined the protein levels of ER stress-related proteins. In the F3 group, the levels of GRP78, IRE, and P-IRE were decreased compared to the 3M group, whereas CHOP levels showed no change between the two groups (Fig. 6A). In ER stress, p-IRE could splice Xbp1 to induce gene expression (Calfon et al. 2002). On day 8 of pregnancy, the level of spliced Xbp1 also showed a decrease in F3 groups (Fig. 6B). These data demonstrated that the dysfunction of GPR78-IRE-Xbp1 pathway could be one cause of the deregulation of decidualization in the F3 group compared to 3M.
Endoplasmic reticulum (ER) stress in decidua on day 8 of pregnancy. (A) The protein levels of ER stress-related genes, including GRP94, CHOP, GRP78, IRE, and P-IRE in decidua on day 8 of pregnancy. (B) The relative level of Xbp1, a downstream gene of GRP78-IRE pathway. (C) The morphology of deciduoma after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7. (D) The average weight of implantation sites after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7. (E) The protein levels of GRP78-IRE pathway-related genes at implantation sites on D8 after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7 at 9:00 and 21:00 h, respectively.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Endoplasmic reticulum (ER) stress in decidua on day 8 of pregnancy. (A) The protein levels of ER stress-related genes, including GRP94, CHOP, GRP78, IRE, and P-IRE in decidua on day 8 of pregnancy. (B) The relative level of Xbp1, a downstream gene of GRP78-IRE pathway. (C) The morphology of deciduoma after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7. (D) The average weight of implantation sites after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7. (E) The protein levels of GRP78-IRE pathway-related genes at implantation sites on D8 after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7 at 9:00 and 21:00 h, respectively.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Endoplasmic reticulum (ER) stress in decidua on day 8 of pregnancy. (A) The protein levels of ER stress-related genes, including GRP94, CHOP, GRP78, IRE, and P-IRE in decidua on day 8 of pregnancy. (B) The relative level of Xbp1, a downstream gene of GRP78-IRE pathway. (C) The morphology of deciduoma after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7. (D) The average weight of implantation sites after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7. (E) The protein levels of GRP78-IRE pathway-related genes at implantation sites on D8 after pregnant mice were treated with 0.001 and 0.1 mg/kg TM on days 6 and 7 at 9:00 and 21:00 h, respectively.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
To further examine the response of pregnant mice to ER stress, female mice were treated on days 6 and 7 at 9:00 and 21:00 h with different doses of tunicamycin (TM), an inducer of unfolded protein response by blocking N-linked glycosylation. When 3M pregnant mice were treated with 0.001 mg/kg TM, the weight of implantation sites on D8 remained unchanged compared to control. After F3 pregnant mice were treated with 0.001 mg/kg TM, the weight of embryos significantly decreased (Fig. 6C and E). When 3M mice were treated with 0.1 mg/kg TM, the weight of embryos was decreased, and both Grp78 and Ire increased compared to control. When F3 mice were treated with 0.1 mg/kg TM, embryos were obviously reabsorbed, and both GRP78 and IRE were decreased (Fig. 6C and D). These results suggest that ER stress dysfunction could be related to decidualization deregulation in F3 mice due to a differing response by uterine stromal cells.
Decreased ER stress response during embryo implantation
In rodents, embryo implantation triggers decidualization. Abnormal embryo implantation is related to the dysfunction of decidualization (Zhang et al. 2014). To explore the reason for decidualization dysfunction, we examined embryo implantation on D6. We found that embryo implantation sites were similar between F3 and 3M groups (Fig. 7A and B). However, there were fewer implantation sites on day 5 in the F3 group compared to the 3M group (Fig. 7C and D). For ER stress, the mRNA levels of Grp78 and Ire were significantly decreased in the F3 group. The levels of IRE and p-IRE proteins also showed an abnormal pattern in the F3 group (Fig. 7E and F). Furthermore, XBP1 expression was decreased in the F3 group due to the decreased p-IRE (Fig. 7G). These data suggest that embryo implantation and ER stress were deregulated in the F3 group.
Embryo implantation and ER stress on days 5 and 6 of pregnancy. (A) The representative uterine morphology of implantation sites on day 6. (B) The average number of implantation sites on day 6. (C) The representative uterine morphology of implantation sites on day 5. (D) The average number of implantation sites on day 5. (E) The mRNA levels of Grp78 and Ire at implantation sites on day 5. (F) The protein levels of GRP78, IRE, and P-IRE at implantation sites on day 5. (G) The level of spliced Xbp1 at implantation sites on day 8.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Embryo implantation and ER stress on days 5 and 6 of pregnancy. (A) The representative uterine morphology of implantation sites on day 6. (B) The average number of implantation sites on day 6. (C) The representative uterine morphology of implantation sites on day 5. (D) The average number of implantation sites on day 5. (E) The mRNA levels of Grp78 and Ire at implantation sites on day 5. (F) The protein levels of GRP78, IRE, and P-IRE at implantation sites on day 5. (G) The level of spliced Xbp1 at implantation sites on day 8.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Embryo implantation and ER stress on days 5 and 6 of pregnancy. (A) The representative uterine morphology of implantation sites on day 6. (B) The average number of implantation sites on day 6. (C) The representative uterine morphology of implantation sites on day 5. (D) The average number of implantation sites on day 5. (E) The mRNA levels of Grp78 and Ire at implantation sites on day 5. (F) The protein levels of GRP78, IRE, and P-IRE at implantation sites on day 5. (G) The level of spliced Xbp1 at implantation sites on day 8.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Delayed implantation due to receptive uterus
We further examined whether embryo implantation is normal on day 4 of pregnancy. At 22:00 h on day 4, a few implantation sites appeared in the 3M group, but no implantation sites were detected in the F3 group. At 24:00 h of day 4, implantation sites were detected clearly in the 3M group compared to the F3 group (Fig. 8A). Based on this study, we wondered whether embryonic development is normal on the morning of day 4 pregnancy. When we flushed uteri in the morning on day 4, the number of blastocysts was similar between 3M and F3 (Fig. 8B). The morphology of blastocysts was also similar between 3M and F3 groups (Fig. 8C). These results suggest that the delay of embryo implantation might be from a uterine response in the F3 group.
Embryo implantation and uterine receptivity. (A) The representative uterine morphology of implantation sites on day 4 at 22:00 and 24:00 h, respectively. (B) The average number of embryos collected from uterus at 9:00 h on D4 (3M: n = 14, F3: n = 11). (C) The representative morphology of blastocysts collected from day 4 pregnant mice at 9:00 h. (D) The serum level of estrogen on day 4. (E) The mRNA level of Ltf, an estrogen target gene. (F) The mRNA level of Muc1, an estrogen target gene. (G) The mRNA level of Pgr, an estrogen target gene. (H) Negative control with rabbit IgG in IHC, scale bar: 100 µm; (I) Estrogen receptor (ER) immunostaining on day 4 of pregnancy, scale bar: 100 µm; (J) Progesterone receptor (PR) immunostaining on day 4 of pregnancy, scale bar: 100 µm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Embryo implantation and uterine receptivity. (A) The representative uterine morphology of implantation sites on day 4 at 22:00 and 24:00 h, respectively. (B) The average number of embryos collected from uterus at 9:00 h on D4 (3M: n = 14, F3: n = 11). (C) The representative morphology of blastocysts collected from day 4 pregnant mice at 9:00 h. (D) The serum level of estrogen on day 4. (E) The mRNA level of Ltf, an estrogen target gene. (F) The mRNA level of Muc1, an estrogen target gene. (G) The mRNA level of Pgr, an estrogen target gene. (H) Negative control with rabbit IgG in IHC, scale bar: 100 µm; (I) Estrogen receptor (ER) immunostaining on day 4 of pregnancy, scale bar: 100 µm; (J) Progesterone receptor (PR) immunostaining on day 4 of pregnancy, scale bar: 100 µm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Embryo implantation and uterine receptivity. (A) The representative uterine morphology of implantation sites on day 4 at 22:00 and 24:00 h, respectively. (B) The average number of embryos collected from uterus at 9:00 h on D4 (3M: n = 14, F3: n = 11). (C) The representative morphology of blastocysts collected from day 4 pregnant mice at 9:00 h. (D) The serum level of estrogen on day 4. (E) The mRNA level of Ltf, an estrogen target gene. (F) The mRNA level of Muc1, an estrogen target gene. (G) The mRNA level of Pgr, an estrogen target gene. (H) Negative control with rabbit IgG in IHC, scale bar: 100 µm; (I) Estrogen receptor (ER) immunostaining on day 4 of pregnancy, scale bar: 100 µm; (J) Progesterone receptor (PR) immunostaining on day 4 of pregnancy, scale bar: 100 µm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Estrogen is a critical factor in determining the beginning of the implantation window. Estrogen level determines the duration of the implantation window within a very narrow range. To analyze the cause of delayed implantation, we measured estrogen levels on day 4. In the F3 group, estrogen levels were significantly higher than that of the 3M group (Fig. 8D). Interestingly, the P4 level in F3 mice was downregulated compared to 3M mice indicating that the endocrine environment should be disturbed in adolescent pregnancy (Supplementary Fig. 1E). Lactoferrin (Ltf), mucin 1 (Muc1), and (progesterone receptor (Pgr), estrogen-targeted genes, were also increased in the F3 group (Fig. 8E and G), as were immunostained levels of ER and PGR (Fig. 8H, I and J) and MUC1 and KI67 (Fig. 9A, B and C), which are closely related to embryo implantation. In order to study the uterine response to steroid hormones, ovariectomized mice were treated with E2 and P4. The target gene expression of E2 was increased, while the target genes of P4 showed no change (Supplementary Fig. 1F and G), indicating that the uterine response to steroid hormones was dysfunctional. The remodeling of the extracellular matrix is important for embryo implantation (Ye 2020). Collagen IV and Laminin a5, key components of the extracellular matrix (Lecce et al. 2011), were also increased in the F3 group. Ezrin, which is downregulated during embryo implantation (Haeger et al. 2015), was increased in F3 mice (Fig. 9D, E and F). These results illustrate that uteri receptivity was compromised in the F3 group compared to the 3M group.
Immunofluorescence of uterine receptivity-related genes on day 4 of pregnancy. (A) Negative control with rabbit IgG in IF, scale bar: 100 µm; (B) MUC1, scale bar: 50 µm; (C) KI67, scale bar: 250 µm; (D) Collagen IV, scale bar: 100 µm; (E) Laminin a5, scale bar: 100 µm; (F) Ezrin, scale bar: 50 µm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Immunofluorescence of uterine receptivity-related genes on day 4 of pregnancy. (A) Negative control with rabbit IgG in IF, scale bar: 100 µm; (B) MUC1, scale bar: 50 µm; (C) KI67, scale bar: 250 µm; (D) Collagen IV, scale bar: 100 µm; (E) Laminin a5, scale bar: 100 µm; (F) Ezrin, scale bar: 50 µm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Immunofluorescence of uterine receptivity-related genes on day 4 of pregnancy. (A) Negative control with rabbit IgG in IF, scale bar: 100 µm; (B) MUC1, scale bar: 50 µm; (C) KI67, scale bar: 250 µm; (D) Collagen IV, scale bar: 100 µm; (E) Laminin a5, scale bar: 100 µm; (F) Ezrin, scale bar: 50 µm.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Immature uterus in the F3 group
Because F3 mice were 25–30 days old, we wondered whether uteri were matured in these mice. Glands are required for embryo implantation. In F3 mice at estrus, the size of the uterus and the number of glands were significantly decreased (Fig. 10A). Except for glandular development, tight junction and the extracellular matrix are also important for uterine development. E-cadherin, an adherence junction protein (Lee et al. 2018), was strongly detected in the epithelial cells in 3M mice compared to F3 mice while the protein level decreased in F3 mice compared with 3M mice (Fig. 10B and E ). Collagen IV, a key component of the extracellular matrix, was also strongly seen in 3M mice compared to F3 mice (Fig. 10C). Compared to 3M mice, Ezrin was highly detected on the surface of epithelial cells in the F3 group (Fig. 10D and E ). These data demonstrate that uteri in F3 mice are underdeveloped compared to those in 3M mice.
The morphology and gene expression of estrous uteri in 3M and F3 mice. (A) The uterine morphology immunostained with anti-cytokeratin 8 (CK8), scale bar: 1 mm; (B) E-cadherin immunofluorescence, scale bar: 50 µm; (C) Collagen IV immunofluorescence, scale bar: 100 µm; (D) Ezrin immunofluorescence, scale bar: 100 µm; (E) Western blot analysis of E-cadherin (E-CAD) and Ezrin proteins.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
The morphology and gene expression of estrous uteri in 3M and F3 mice. (A) The uterine morphology immunostained with anti-cytokeratin 8 (CK8), scale bar: 1 mm; (B) E-cadherin immunofluorescence, scale bar: 50 µm; (C) Collagen IV immunofluorescence, scale bar: 100 µm; (D) Ezrin immunofluorescence, scale bar: 100 µm; (E) Western blot analysis of E-cadherin (E-CAD) and Ezrin proteins.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
The morphology and gene expression of estrous uteri in 3M and F3 mice. (A) The uterine morphology immunostained with anti-cytokeratin 8 (CK8), scale bar: 1 mm; (B) E-cadherin immunofluorescence, scale bar: 50 µm; (C) Collagen IV immunofluorescence, scale bar: 100 µm; (D) Ezrin immunofluorescence, scale bar: 100 µm; (E) Western blot analysis of E-cadherin (E-CAD) and Ezrin proteins.
Citation: Reproduction 162, 5; 10.1530/REP-21-0240
Discussion
Adolescent pregnancy is associated with perinatal outcomes, such as preterm birth, low birth weight babies, and preeclampsia (Brosens et al. 2015). These abnormalities could be related to both embryonic and maternal factors. In this study, we found that the litter size of F3 mice is significantly decreased. Most pregnancy complications such as low birth weight, fetal growth restriction, and preeclampsia are correlated with a failure of proper placentation (Norwitz 2006). During pregnancy, the placenta is the main regulator of nutrient supply for the growing embryo. Adequate placental function is indispensable for developmental progression during intrauterine development (Woods et al. 2018). A recent study indicated that sub-viable mouse mutants or embryonic lethal models are associated with labyrinthian defects and intrauterine growth retardation prior to death (Perez-Garcia et al. 2018). In our study, labyrinth markers are significantly decreased in abnormal embryos of F3 mice. Evidence from individual gene knockouts also demonstrated that placental labyrinth development is directly associated with fetal growth (Watson & Cross 2005). In the F3 group, the labyrinth shows defective development and fetal growth is retarded. Improper differentiation also occurs in stem-like trophoblast progenitor cells, the intermediate trophoblasts, spongiotrophoblast, and trophoblast giant cells in the F3 group. Ablation of Tpbpa-positive cells results in trophoblast invasion defects, which are related to defective remodeling of maternal spiral arteries (Hu & Cross 2011).
From days 6 to 11 of pregnancy in mice, uNK cells are recruited in large numbers to the mesometrial decidua (Kieckbusch et al. 2017). uNK cells could be the key regulator of placental vascularization, including spiral artery remodeling (Wallace et al. 2015). The combination of uNK cells and maternal β3 integrin showed profound effects on invasive trophoblasts and placental development (Yougbare et al. 2017). In our study, the decrease of uNK cell number and abnormal expression patterns of angiogenic cytokines suggest placental dysfunction, which could be the cause of lower litter sizes in F3 mice.
The correct development of placentation is closely related to uterine decidualization as the decidua functions as one part of the placenta (Ramathal et al. 2010). Although the number of implantation sites showed similar patterns in two groups on day 8 of pregnancy, the length and weight of embryos were significantly decreased in the F3 group. Dysfunction of decidualization is an important cause of preeclampsia (Garrido-Gomez et al. 2017); in women with preeclampsia, decidual cells exhibited inadequate decidualization (Sahu et al. 2019). Through microarray data analysis, the molecular pathways of decidualization were shown to be dysregulated in preeclampsia and endometrial disorders (Rabaglino & Conrad 2019). Based on our transcriptome analysis, many pathways were significantly changed in the F3 group, including the bicellular tight junction and ER chaperone complex. Artificial decidualization is also compromised in the F3 group. In aged mice, decidualization defects cause reproductive decline, key regulators of decidualization are downregulated (Woods et al. 2017), and artificially induced decidual response is also defective (Finch & Holinka 1982). The dysregulated expression of Prl8a2 suggested abnormal decidualization in F3 mice, which may lead to defective perinatal outcomes, such as preterm birth, low birth weight babies, and preeclampsia (Leftwich & Alves 2017). Previous studies indicated that defective decidualization is related to abnormal pregnancy outcome. The up-regulation of decidualization-related genes is also associated with repeated implantation failure (Berkhout et al. 2020). In preeclampsia patients, endometrial stromal cells failed to decidualize (Garrido-Gomez et al. 2017). These studies suggest that proper decidualization is essential for a successful pregnancy.
During early pregnancy in mice, GRP78-IRE-xbp1 signaling pathway of ER stress is activated in decidual cells on days 5–8 of pregnancy (Gu et al. 2016). GRP78 is expressed at implantation sites and decidual cells (Gu et al. 2016). In human decidual cells, the IRE-xbp1 signaling pathway is induced to support embryo development (Brosens et al. 2014). In patients with spontaneous labor and preterm labor, GRP78, IRE1, and spliced Xbp1 (sXbp1) are significantly increased (Liong & Lappas 2014). Compared to normal pregnancy, unspliced Xbp1 (uXbp1) is increased in decidual cells of preeclampsia patients (Lian et al. 2011). Mouse decidualization is impaired after Ire-xbp1 signaling pathway is inhibited (Gu et al. 2016). These studies suggest that ER stress is a physiological process during embryo implantation and decidualization. In our study, GRP78, IRE, P-IRE, and XBP1 which in GRP78-IRE-xbp1 signaling pathway of ER stress decreased in the F3 group, indicating that deregulation of ER stress could be one cause of dysfunctional decidualization.
TM has a concentration- and time-dependent effect on the proliferation of stromal cells (Gu et al. 2016). Treatment with 50 ng/mL or 2.5 µg/mL TM inhibits cell proliferation and growth (Shen et al. 2015). However, treatment with TM at pg/mL level has the opposite effect (Jin et al. 2016). In this study, treatment with 0.001 mg/kg TM causes a decrease of implantation sites in the F3 group but has no effects in 3M mice, and treatment with 0.1 mg/kg TM causes more severe effects in the F3 group. These results indicate that the response of ER stress is more sensitive in the F3 group than in the 3M group. In aged human skeletal muscles, many key components of the unfolded protein response are decreased, resulting in dysfunctional ER stress (Deldicque 2013). Therefore, it is possible that the young age of F3 mice may cause abnormal ER stress.
Proper embryo implantation is required for the successful establishment of pregnancy. In mice, estrogen is a key determinant for the duration of the window of uterine receptivity; specifically, a high level of estrogen will shorten the window of implantation (Ma et al. 2003). In this study, the serum estrogen level and the expression levels of estrogen-targeted genes are significantly higher in the F3 group than in the 3M group. A previous study demonstrated that estrogen levels are also upregulated in Rbpjd/d female mice, leading to abnormal decidualization and embryonic lethality (Zhang et al. 2014). In aged mice, the estrogen-responsive genes are downregulated (Li et al. 2017, Woods et al. 2017). The decrease of uterine estrogen response may contribute to the decline of pregnancy rate (Li et al. 2017). It is possible that the increase of both estrogen and uterine estrogen response may result in the decline of pregnancy in the F3 group.
Adequate uterine development is a requirement for pregnancy establishment. Uterine glands are necessary for uterine function. In the glandless uterus of Foxa2-deficient mice, embryo resorption and pregnancy failure occur (Kelleher et al. 2018). Glands are fundamental to pregnancy success as they directly connect the crypt encasing the embryo (Yuan et al. 2018). In our study, the number of glands is significantly decreased in F3 mice. Collagens, extracellular matrix components, are upregulated in human receptive endometrium (Haller-Kikkatalo et al. 2014). Collagens are important for embryo implantation (Zheng et al. 2020). In the F3 group, collagen IV was downregulated, indicating that the function of extracellular matrix may be incomplete. Cadherins are adhesion molecules expressed at the cellular membrane for controlling cell adhesion (Achache & Revel 2006). Ezrin is one ERM protein that provides a critical link between the plasma membrane and surface structures and is present in the uterine epithelium in the human endometrial cycle and downregulated during embryo implantation (Haeger et al. 2015). In F3 mice, upregulated Ezrin and downregulated E-cadherin indicated tight junction in epithelial cells that were not well developed. Based on our data on day 4 of pregnancy, the number and morphology of blastocysts flushed from day 4 uteri were similar between 3M and F3 mice, suggesting that ovulation, fertilization, and preimplantation embryonic development should be normal in F3 mice. When we examined the serum levels of E2 and P4 on day 4 of pregnancy, E2 level was increased and P4 level decreased in F3 mice. Compared to 3M mice, E-cadherin, collagen IV, and erzin were also abnormally expressed. In ovariectomized F3 mice, E2 target genes were significantly increased compared to 3M mice. Although we found reproductive abnormalities in F3 female mice, it is hard to say whether these abnormalities are caused by intrinsic uterine defects or an abnormal endocrine environment. Because de novo synthesis of estrogen in the pregnant uterus is critical for mouse decidualization (Das et al. 2009), it is possible that intrinsic uterine defects cause an abnormal endocrine environment.
In summary, this study extensively compared different stages of adolescent and normal pregnancy. We found numerous abnormalities during adolescent pregnancy, including decidual and placental defects and small litter size. These abnormalities may result from uterine immaturity, high levels of estrogen, and abnormal response of ER stress. As a result, this study sheds light on adolescent pregnancy and the underlying factors that lead to adverse perinatal outcomes, including preterm birth, low birth weight babies, preeclampsia, intrapartum death, and miscarriage, which substantially affect maternal and neonatal health.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-21-0240.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by National Key Research and Development Program of China (2018YFC1004400) and National Natural Science Foundation of China (31871511 and 31671563).
Data and materials availability
Raw data from RNA sequencing of decidua on day 8 of pregnancy have been deposited in GEO under the accession code PRJNA743694. All data of this study are available from the corresponding author on reasonable request.
Author contribution statement
C Y and Z M Y designed this study and wrote the paper. Y L, H Y Z, M Y L, and Z S Y conducted mouse work, RT-PCR, and Western blots. J M P, H Y P, and S T C performed immunofluorescence experiments. C Y and Z S Y performed RNA sequencing analyses and analyzed data.
References
Achache H & Revel A 2006 Endometrial receptivity markers, the journey to successful embryo implantation. Human Reproduction Update 12 731–746. (https://doi.org/10.1093/humupd/dml004)
Babayev SN, Kanchwala M, Xing C, Akgul Y, Carr BR & Word RA 2019 Thrombin alters human endometrial stromal cell differentiation during decidualization. Reproductive Sciences 26 278–288. (https://doi.org/10.1177/1933719118768705)
Berkhout RP, Lambalk CB, Repping S, Hamer G & Mastenbroek S 2020 Premature expression of the decidualization marker prolactin is associated with repeated implantation failure. Gynecological Endocrinology 36 360–364. (https://doi.org/10.1080/09513590.2019.1650344)
Brosens JJ, Salker MS, Teklenburg G, Nautiyal J, Salter S, Lucas ES, Steel JH, Christian M, Chan YW & Boomsma CM et al.2014 Uterine selection of human embryos at implantation. Scientific Reports 4 3894. (https://doi.org/10.1038/srep03894)
Brosens I, Benagiano G & Brosens JJ 2015 The potential perinatal origin of placentation disorders in the young primigravida. American Journal of Obstetrics and Gynecology 212 580–585. (https://doi.org/10.1016/j.ajog.2015.01.013)
Calfon M, Zeng HQ, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG & Ron D 2002 IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 420 202–202. (https://doi.org/10.1038/415092a)
Chen ZL, Zhang JH, Hatta K, Lima PDA, Yadi H, Colucci F, Yamada AT & Croy BA 2012 DBA-lectin reactivity defines mouse uterine natural killer cell subsets with biased gene expression. Biology of Reproduction 87 81. (https://doi.org/10.1095/biolreprod.112.102293)
Copp AJ 1995 Death before birth: clues from gene knockouts and mutations. Trends in Genetics 11 87–93. (https://doi.org/10.1016/S0168-9525(0089008-3)
Das A, Mantena SR, Kannan A, Evans DB, Bagchi MK & Bagchi IC 2009 De novo synthesis of estrogen in pregnant uterus is critical for stromal decidualization and angiogenesis. PNAS 106 12542–12547. (https://doi.org/10.1073/pnas.0901647106)
Deldicque L 2013 Endoplasmic reticulum stress in human skeletal muscle: any contribution to sarcopenia? Frontiers in Physiology 4 236. (https://doi.org/10.3389/fphys.2013.00236)
Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T & Wang HB 2004 Molecular cues to implantation. Endocrine Reviews 25 341–373. (https://doi.org/10.1210/er.2003-0020)
Finch CE & Holinka CF 1982 Aging and uterine growth during implantation in C57BL/6J mice. Experimental Gerontology 17 235–241 (https://doi.org/10.1016/0531-5565(8290030-4)
Garrido-Gomez T, Dominguez F, Quinonero A, Diaz-Gimeno P, Kapidzic M, Gormley M, Ona K, Padilla-Iserte P, McMaster M & Genbacev O et al.2017 Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology. PNAS 114 E8468–E8477 (https://doi.org/10.1073/pnas.1706546114)
Gellersen B & Brosens JJ 2014 Cyclic decidualization of the human endometrium in reproductive health and failure. Endocrine Reviews 35 851–905. (https://doi.org/10.1210/er.2014-1045)
Gruhn JR, Zielinska AP, Shukla V, Blanshard R, Capalbo A, Cimadomo D, Nikiforov D, Chan AC, Newnham LJ & Vogel I et al.2019 Chromosome errors in human eggs shape natural fertility over reproductive life span. Science 365 1466–1469. (https://doi.org/10.1126/science.aav7321)
Gu XW, Yan JQ, Dou HT, Liu J, Liu L, Zhao ML, Liang XH & Yang ZM 2016 Endoplasmic reticulum stress in mouse decidua during early pregnancy. Molecular and Cellular Endocrinology 434 48–56. (https://doi.org/10.1016/j.mce.2016.06.012)
Haeger JD, Hambruch N, Dantzer V, Hoelker M, Schellander K, Klisch K & Pfarrer C 2015 Changes in endometrial ezrin and cytokeratin 18 expression during bovine implantation and in caruncular endometrial spheroids in vitro. Placenta 36 821–831. (https://doi.org/10.1016/j.placenta.2015.06.001)
Haller-Kikkatalo K, Altmae S, Tagoma A, Uibo R & Salumets A 2014 Autoimmune activation toward embryo implantation is rare in immune-privileged human endometrium. Seminars in Reproductive Medicine 32 376–384. (https://doi.org/10.1055/s-0034-1376356)
Hemberger M, Hanna CW & Dean W 2020 Mechanisms of early placental development in mouse and humans. Nature Reviews: Genetics 21 27–43. (https://doi.org/10.1038/s41576-019-0169-4)
Holness N 2015 A global perspective on adolescent pregnancy. International Journal of Nursing Practice 21 677–681. (https://doi.org/10.1111/ijn.12278)
Hu D & Cross JC 2010 Development and function of trophoblast giant cells in the rodent placenta. International Journal of Developmental Biology 54 341–354. (https://doi.org/10.1387/ijdb.082768dh)
Hu D & Cross JC 2011 Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta. Developmental Biology 358 231–239. (https://doi.org/10.1016/j.ydbio.2011.07.036)
Hu W, Liang YX, Luo JM, Gu XW, Chen ZC, Fu T, Zhu YY, Lin S, Diao HL & Jia B et al.2019 Nucleolar stress regulation of endometrial receptivity in mouse models and human cell lines. Cell Death and Disease 10 831. (https://doi.org/10.1038/s41419-019-2071-6)
Jin C, Jin Z, Chen NZ, Lu M, Liu CB, Hu WL & Zheng CG 2016 Activation of IRE1 alpha-XBP1 pathway induces cell proliferation and invasion in colorectal carcinoma. Biochemical and Biophysical Research Communications 470 75–81. (https://doi.org/10.1016/j.bbrc.2015.12.119)
Jiskrova GK & Vazsonyi AT 2019 Multi-contextual influences on adolescent pregnancy and sexually transmitted infections in the United States. Social Science and Medicine 224 28–36. (https://doi.org/10.1016/j.socscimed.2019.01.024)
Karata V, Kanmaz AG, Inan AH, Budak A & Beyan E 2019 Maternal and neonatal outcomes of adolescent pregnancy. Journal of Gynecology Obstetrics and Human Reproduction 48 347–350. (https://doi.org/10.1016/j.jogoh.2019.02.011)
Kassa GM, Arowojolu AO, Odukogbe AA & Yalew AW 2018 Prevalence and determinants of adolescent pregnancy in Africa: a systematic review and meta-analysis. Reproductive Health 15 195. (https://doi.org/10.1186/s12978-018-0640-2)
Kelleher AM, Milano-Foster J, Behura SK & Spencer TE 2018 Uterine glands coordinate on-time embryo implantation and impact endometrial decidualization for pregnancy success. Nature Communications 9 2435. (https://doi.org/10.1038/s41467-018-04848-8)
Kercmar J, Tobet SA & Majdic G 2014 Social isolation during puberty affects female sexual behavior in mice. Frontiers in Behavioral Neuroscience 8 337. (https://doi.org/10.3389/fnbeh.2014.00337)
Kieckbusch J, Gaynor LM, Moffett A & Colucci F 2017 MHC-dependent inhibition of uterine NK cells impedes fetal growth and decidual vascular remodeling. Nature Communications 5 3359. (https://doi.org/10.1038/ncomms4359)
Kim BH, Ju WS, Kim JS, Kim SU, Park SJ, Ward SM, Lyu JH & Choo YK 2019 Effects of gangliosides on spermatozoa, oocytes, and preimplantation embryos. International Journal of Molecular Sciences 21 106. (https://doi.org/10.3390/ijms21010106)
Kramer KL & Lancaster JB 2010 Teen motherhood in cross-cultural perspective. Annals of Human Biology 37 613–628. (https://doi.org/10.3109/03014460903563434)
Lecce L, Lindsay LA & Murphy CR 2011 Ezrin and EBP50 redistribute apically in rat uterine epithelial cells at the time of implantation and in response to cell contact. Cell and Tissue Research 343 445–453. (https://doi.org/10.1007/s00441-010-1088-z)
Lee B, Moon KM & Kim CY 2018 Tight junction in the intestinal epithelium: its association with diseases and regulation by phytochemicals. Journal of Immunology Research 2018 2645465. (https://doi.org/10.1155/2018/2645465)
Leftwich HK & Alves MV 2017 Adolescent pregnancy. Pediatric Clinics of North America 64 381–388. (https://doi.org/10.1016/j.pcl.2016.11.007)
Li SJ, Wang TS, Qin FN, Huang Z, Liang XH, Gao F, Song Z & Yang ZM 2015 Differential regulation of receptivity in two uterine horns of a recipient mouse following asynchronous embryo transfer. Scientific Reports 5 15897. (https://doi.org/10.1038/srep15897)
Li MQ, Yao MN, Yan JQ, Li ZL, Gu XW, Lin S, Hu W & Yang ZM 2017 The decline of pregnancy rate and abnormal uterine responsiveness of steroid hormones in aging mice. Reproductive Biology 17 305–311. (https://doi.org/10.1016/j.repbio.2017.09.001)
Lian IA, Loset M, Mundal SB, Fenstad MH, Johnson MP, Eide IP, Bjorge L, Freed KA, Moses EK & Austgulen R 2011 Increased endoplasmic reticulum stress in decidual tissue from pregnancies complicated by fetal growth restriction with and with out pre-eclampsia. Placenta 32 823–829. (https://doi.org/10.1016/j.placenta.2011.08.005)
Lin PF, Jin YP, Lan XL, Yang YZ, Chen FL, Wang N, Li X, Sun YJ & Wang AH 2014 GRP78 expression and regulation in the mouse uterus during embryo implantation. Journal of Molecular Histology 45 259–268. (https://doi.org/10.1007/s10735-013-9552-1)
Liong S & Lappas M 2014 Endoplasmic reticulum stress is increased after spontaneous labor in human fetal membranes and myometrium where it regulates the expression of prolabor mediators. Biology of Reproduction 91 70. (https://doi.org/10.1095/biolreprod.114.120741)
Luo L, Wang Q, Chen M, Yuan G, Wang Z & Zhou C 2016 IGF-1 and IGFBP-1 in peripheral blood and decidua of early miscarriages with euploid embryos: comparison between women with and without PCOS. Gynecological Endocrinology 32 538–542. (https://doi.org/10.3109/09513590.2016.1138459)
Ma WG, Song H, Das SK, Paria BC & Dey SK 2003 Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. PNAS 100 2963–2968. (https://doi.org/10.1073/pnas.0530162100)
Markovic S, Bogdanovic G & Cerovac A 2020 Premature and preterm premature rupture of membranes in adolescent compared to adult pregnancy. Medicinski Glasnik 17 136–140. (https://doi.org/10.17392/1052-20)
Marvin-Dowle K & Soltani H 2020 A comparison of neonatal outcomes between adolescent and adult mothers in developed countries: a systematic review and meta-analysis. European Journal of Obstetrics and Gynecology and Reproductive Biology: X 6 100109. (https://doi.org/10.1016/j.eurox.2020.100109)
Norwitz ER 2006 Defective implantation and placentation: laying the blueprint for pregnancy complications. Reproductive Biomedicine Online 13 591–599. (https://doi.org/10.1016/s1472-6483(1060649-9)
Perez-Garcia V, Fineberg E, Wilson R, Murray A, Mazzeo CI, Tudor C, Sienerth A, White JK, Tuck E & Ryder EJ et al.2018 Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature 555 463–468. (https://doi.org/10.1038/nature26002)
Rabaglino MB & Conrad KP 2019 Evidence for shared molecular pathways of dysregulated decidualization in preeclampsia and endometrial disorders revealed by microarray data integration. FASEB Journal 33 11682–11695. (https://doi.org/10.1096/fj.201900662R)
Ramathal CY, Bagchi IC, Taylor RN & Bagchi MK 2010 Endometrial decidualization: of mice and men. Seminars in Reproductive Medicine 28 17–26. (https://doi.org/10.1055/s-0029-1242989)
Ron D & Walter P 2007 Signal integration in the endoplasmic reticulum unfolded protein response. Nature Reviews: Molecular Cell Biology 8 519–529. (https://doi.org/10.1038/nrm2199)
Sahu MB, Deepak V, Gonzales SK, Rimawi B, Watkins KK, Smith AK, Badell ML, Sidell N & Rajakumar A 2019 Decidual cells from women with preeclampsia exhibit inadequate decidualization and reduced sFlt1 suppression. Pregnancy Hypertension 15 64–71. (https://doi.org/10.1016/j.preghy.2018.11.003)
Shen MZ, Wang L, Quo X, Xue Q, Hu C, Li X, Fan L & Wang XM 2015 A novel endoplasmic reticulum stress-induced apoptosis model using tunicamycin in primary cultured neonatal rat cardiomyocytes. Molecular Medicine Reports 12 5149–5154. (https://doi.org/10.3892/mmr.2015.4040)
Wallace AE, Whitley GS, Thilaganathan B & Cartwright JE 2015 Decidual natural killer cell receptor expression is altered in pregnancies with impaired vascular remodeling and a higher risk of pre-eclampsia. Journal of Leukocyte Biology 97 79–86. (https://doi.org/10.1189/jlb.2A0614-282R)
Watson ED & Cross JC 2005 Development of structures and transport functions in the mouse placenta. Physiology 20 180–193. (https://doi.org/10.1152/physiol.00001.2005)
Woods L, Perez-Garcia V, Kieckbusch J, Wang XQ, DeMayo F, Colucci F & Hemberger M 2017 Decidualisation and placentation defects are a major cause of age-related reproductive decline. Nature Communications 8 352. (https://doi.org/10.1038/s41467-017-00308-x)
Woods L, Perez-Garcia V & Hemberger M 2018 Regulation of placental development and its impact on fetal growth-new insights from mouse models. Frontiers in Endocrinology 9 570. (https://doi.org/10.3389/fendo.2018.00570)
Wu S, Divall S, Hoffman GE, Le WW, Wagner KU & Wolfe A 2011 Jak2 is necessary for neuroendocrine control of female reproduction. Journal of Neuroscience 31 184–192. (https://doi.org/10.1523/JNEUROSCI.2974-10.2011)
Ye XQ 2020 Uterine luminal epithelium as the transient gateway for embryo implantation. Trends in Endocrinology and Metabolism 31 165–180. (https://doi.org/10.1016/j.tem.2019.11.008)
Ye X, Hama K, Contos JJ, Anliker B, Inoue A, Skinner MK, Suzuki H, Amano T, Kennedy G & Arai H et al.2005 LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing. Nature 435 104–108. (https://doi.org/10.1038/nature03505)
Yougbare I, Tai WS, Zdravic D, Oswald BE, Lang S, Zhu GH, Leong-Poi H, Qu DW, Yu LS & Dunk C et al.2017 Activated NK cells cause placental dysfunction and miscarriages in fetal alloimmune thrombocytopenia. Nature Communications 8 224 (https://doi.org/10.1038/s41467-017-00269-1)
Yuan JB, Deng W, Cha J, Sun XF, Borg JP & Dey SK 2018 Tridimensional visualization reveals direct communication between the embryo and glands critical for implantation. Nature Communications 9 603. (https://doi.org/10.1038/s41467-018-03092-4)
Zhang S, Kong SB, Wang BY, Cheng XH, Chen YJ, Wu WW, Wang Q, Shi JC, Zhang Y & Wang SM et al.2014 Uterine Rbpj is required for embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms. Cell Research 24 925–942. (https://doi.org/10.1038/cr.2014.82)
Zheng HT, Zhang HY, Chen ST, Li MY, Fu T & Yang ZM 2020 The detrimental effects of stress-induced glucocorticoid exposure on mouse uterine receptivity and decidualization. FASEB Journal 34 14200–14216. (https://doi.org/10.1096/fj.201902911RR)
Zhou Q, Yan G, Ding L, Liu J, Yu X, Kong S, Zhang M, Wang Z, Liu Y & Jiang Y et al.2019 EHD1 impairs decidualization by regulating the Wnt4/beta-catenin signaling pathway in recurrent implantation failure. EBiomedicine 50 343–354. (https://doi.org/10.1016/j.ebiom.2019.10.018)
