Endometrial decidualization refers to the process during which ESCs are gradually transformed into DSCs following stimulation by decidualization-inducing factors, and continue to proliferate and spread until the decidua is completely formed [6–9]. Unlike humans, who can spontaneously undergo decidualization without pregnancy, the decidualization process of rats can occur only after embryo implantation [13, 14]. Generally, at 22-24 days of gestation, after the blastocyst reaches the luminal epithelium and adheres to the implantation site, ESCs start to activate the decidualization process under the action of hormones [15, 16]. ESCs surrounding the blastocyst, including fibroblasts, first begin to differentiate and proliferate until the formation of the primary decidual zone (PDZ), composed of dense DSCs on GD6. Subsequently, DSCs in the PDZ region and adjacent ESCs continue to proliferate and differentiate, and finally form a secondary decidual zone (SDZ) wrapping the PDZ on GD8 . At this point, the 4-day decidualization process is completed, and the mouse endometrium is completely transformed into the pregnant decidua. Whether the decidualization process is smooth or not is closely related to the pregnancy outcome. Decidualization defects will cause a variety of adverse pregnancy outcomes, such as spontaneous abortion, premature delivery, and recurrent abortion . In this study, we selected to use an internationally recognized URSA animal model (CBA / J female * DBA / 2 male) . It was found that the level of decidualization markers in the decidua of URSA mice was significantly lower than that of normal decidua , which confirmed that decidualization defects were closely related to the occurrence of URSA disease. Furthermore, this is consistent with the research results of Blois s m and achache h [11, 12].
The decidualization process is actually the transformation and proliferation of endometrial stromal cells, which needs to be regulated by a large number of cell cycle regulators. The cell cycle is divided into the interphase (g phase) and division phase (M phase). Most of the cell life activities occur in the interphase, and the division phase accounts for only a small part of cell activities . Although the interphase includes the G1 phase (first gap), the S phase (synthesis) and G2 phase (second gap), the duration of the G1 phase is long, and the cell cycle arrest in G1 is reversible. When stimulated by certain stimuli, the cell cycle process can be restored again and the cell enters the S phase [222-224]. Cyclin D, cyclin E, CDK4, CDK6 and CDK2 are key cytokines that play a regulatory role in the G1 phase [225,226]. Therefore, this study deeply explored the dynamic changes and roles of the above cytokines in the decidualization process of URSA using animal experiments. According to previous studies, cyclin D accumulates in the G1 phase and forms cyclin D/CDK complexes after binding to CDK4 or CDK6, which can accelerate the initiation of DNA replication and play an important role in promoting cell cycle proliferation. The synthesis of cyclin E in G1 occurs slightly later than that of cyclin D, and its combination with CDK2 can also promote DNA replication and accelerate the completion of the G1-S phase transition [227-230].
Previous experimental studies and literature reports also showed that cyclin D, cyclin E and their specifically-bound CDK complexes changed dynamically during endometrial decidualization, thus regulating decidualization involving cell cycle events, such as cell proliferation, differentiation, and apoptosis. According to the research of Zhang Tan  and Tan j , cyclin E and CDK2 were expressed at low levels in pregnant mice on GD1-GD2, but the expression in the intima gradually increased with the process of decidualization from GD3, and began to decline after reaching its peak on GD5-GD8. In this study, to more accurately display the changes and trends of detection indices, we detected the mRNA and protein levels of cyclin D, CDK4, CDK6, cyclin E and CDK2 in decidual tissue during the whole process of decidualization (from GD4 to GD8). The results showed that during the decidualization of normal female rats, the expression of cyclin E mRNA increased in the decidua from GD4 to GD5, and began to decrease after its peak on GD5 to GD8. The expression trend of CDK2 is almost consistent with that of cyclin E, which is also consistent with the research results of Jian Tan et al. , showing that the expression of cyclin E and CDK2 is higher in the decidua around blastocyst embedding and early after implantation, and then decreases. The expression of cyclin D was low on GD4, but then increased rapidly until it reached its peak on GD8. From the overall trend, cyclin D levels continuously increased in the process of decidualization, with a large increase on GD4-GD6 and a small increase on GD7-GD8. The level of CDK4 increased briefly on GD4-GD5, but then gradually decreased. On the contrary, the expression of CDK6 was lower on GD4-GD5, and increased from GD6-GD8. We believe that normal mice accumulate a certain amount of cyclin E and CDK2 in the endometrium before implantation on GD4. Cyclin E/CDK2 can accelerate the entry of cells into the S phase, promote the proliferation of ESC and increase endometrial receptivity under the synergistic action of other factors, which is conducive to embryo implantation. After the decidualization reaction induced by embryo implantation, the levels of cyclin E and CDK2 increased on GD5, while those of cyclin D and CDK4 also increased on GD4-GD5. The two cyclin/CDK complexes worked together to accelerate the G1-S phase transition of DSC cells around the embryo implantation site and accelerated the proliferation of DSC cells, so the PDZ was quickly formed and blastocysts were embedded. However, after GD6, with the formation of PDZ and the formation of SDZ on GD7 and GD8, cyclin D levels in the PDZ almost disappeared, which inhibited the activity of the cyclin/CDK complex, and affected cell cycle transition from the G1 to the S phase. However, because the level of cyclin D in the SDZ is still high, the cyclin D levels in the decidua still rise slightly, and the gradual rise of CDK6 levels compensates for the decline of CDK4 levels, maintaining the activity of the cyclin D/CDK complex. Therefore, although some decidual cells continue to proliferate, some cells return to the G1-S phase. These cells break away from the cell proliferation cycle and continue to replicate in the nucleus, maintaining gene stability, limiting the life span of decidual cells, and mediating the orderly apoptosis of decidual cells, thus providing space for embryonic growth. In conclusion, we found that the cyclin/CDK levels showed an overall upward trend in the decidualization process of normal pregnant female rats, which was conducive to maintaining pregnancy.
After clarifying the change trend of cytokines in the decidua of NP mice, we detected and compared the above factors in the decidua of URSA pregnant mice using RT-PCR and Western blot. Firstly, we found that the levels of the above factors decreased in varying degrees compared to those of the normal decidua in the decidualization process of pregnant female rats with URSA. Compared to the level of NP rats, the level of cyclin E in URSA rats showed a more significant increase after GD5. However, according to the results of RT-PCR, the mRNA level of cyclin E in URSA female rats and normal female rats was significantly different on GD7 and GD8 (P < 0.05). According to the Western blot results, the protein level of cyclin E is consistent with the trend shown using PCR. The protein level of CDK2 was significantly different from that of NP rats on GD7 and GD8 (P < 0.05). This significant difference occurred later than GD6 based on the PCR results. We believe that this may be due to a low protein detection sensitivity associated with the Western blot procedure or a delay of mRNA transcription. However, in terms of the overall change trend, the expression of cyclin E and CDK2 in the decidua of URSA pregnant rats was slightly lower than that of NP rats, which had a certain impact on early cell proliferation, differentiation and PDZ formation, but this impact was small and did not affect the early implantation process of embryos. With the advancement of decidualization, the levels of cyclin E and CDK2 continued to decline, so the transformation from the G1 to the S phase could not be promoted during the proliferation of SDZ after GD6, affecting cell proliferation. In addition, the levels of cyclin D and CDK4 showed a downward trend after the formation of the PDZ on GD6-GD8, while CDK6 levels did not rise on GD6-GD8 like in normal female rats, but showed a continuous low expression throughout the decidualization process. This low level could not compensate for the maintenance of the activity of the cyclin D/CDK complex, so the cell cycle process was blocked, cells could not transition from the G1 to the S phase, and stagnated in the G1 phase for a long time, resulting in cell cycle arrest. The formation of PDZ and SDZ was affected, which eventually affected the process of decidualization and caused decidualization defects.