| Objective:To investigate the correlation between endometrial receptivity and recurrent spontaneous abortion and immune factors related to endometrial receptivity. To study the mechanism of prevention and treatment of recurrent spontaneous abortion of Pill Zhuyun, by clinical observation of the improvement of endometrial receptivity of recurrent spontaneous abortion patients after taking the pill, and experimental study on endometrial morphological, endometrial immune microenvironment and pregnancy outcome, of rats with poor endometrial receptivity and the ones that have treatment with Pill Zhuyun.Methods:1. The effect of Pill Zhuyun on endometrial receptivity and endometrial immune microenvironment of recurrent spontaneous abortion patients with spleen and kidney deficiencyCompared the endometrial thickness and blood flow index of endometrial spiral arteries (RI, PI) between recurrent spontaneous abortion patients with spleen and kidney deficiency and the normal females with normal reproductive history in the mid-luteal, and the level of E2, P, LIF, IL-2 in blood serum, as well as the percentage of CD56+CD16+ NK cells in blood. Compared the endometrial thickness and blood flow index of endometrial spiral arteries (RI, PI) of the patients before treatment,1 month after treatment, and 2 months after treatment. Compared the level of E2, P, LIF, IL-2 in serum before treatment and 2 months after treatment, as well as the percentage of CD56+CD16+ NK cells in blood. 2. The influence of Pill Zhuyun on endometrial receptivity, pregnancy outcome and immune microenvironment of kidney deficiency rat modelWe used hydroxyurea to create kidney deficiency rat model. Compared the endometrial morphological and fine structure of endometrial, the percentage of different subsets of NK cells in endometrial and blood, the level of E2, P, LIF, IL-2 in serum, and the level of LIF, IL-2 in endometrial, of rats in estrus, among normal rats group, model without herbs group, model with herbal medicine group and normal rats with herbs group. Then we created kidney deficiency and poor endometrial receptivity pregnant rat model. Compared the abortion rate, the percentage of different subsets of NK cells in decidua and blood, the level of E2, P, LIF, IL-2 in serum, and the level of LIF, IL-2 in decidua, among normal pregnant rat group, model without herbs group, model with continued herbal medicine group, model with herbal medicine post-pregnancy group, and normal pregnant rat with herbs pre/post-pregnancy group.Result:1. The endometrial thickness was thinner, the blood flow index of endometrial spiral arteries (RI) was higher, and the level of E2 in serum and the ratio of E2/P were also higher in patients pre-treatment in mid-luteal, the differences were significant compared with the results of normal females with normal reproductive history in the mid-luteal (p<0.05).2. Treated with Pill Zhuyun a month later, the blood flow index of endometrial spiral arteries (RI) was significantly lower in patients in mid-luteal, compared with the same index in patients pre-treatment in mid-luteal (p<0.05). And after 2 months treatment, the endometrial thickness was significantly thicker and the level of P was higher in patients in mid-luteal, compared with the ones pre-treatment in mid-luteal (p<0.05). But the changes of the level of IL-2 and LIF in serum, the ratio of E2/P, the percentage of CD56+CD16+ NK cells in blood and the blood flow index of endometrial spiral arteries (PI) in patients after 2 months treatment, were not significant, compared with those of pre-treatment (p>0.05).3. The rats fed with hydroxyurea showed some symptoms of kidney deficiency, such as withered fur, stool pulpy, and significant weight loss compared with the normal rats (p<0.05). The quantity of endometrial glands and endometrial glandular area both decreased in model rats, though the differences were not significant compared with the normal rats (p>0.05). Of the model rat in estrus, the distribution of microvilli at the surface of endometrial epithelial was sparse. There were more mitochondria with many vacuoles, the quantity of endoplasmic reticulum was reduced, and there was sporadic ribosomal attached to the surface of endoplasmic reticulum, compared with the normal rat in estrus. The percentage of CD56+CD16-NK cells in endometrial of model rats was significant lower (p<0.05), compared with the normal rats. The percentage of CD56-CD16+NK cells in blood was higher, and the ratios of CD56+CD16-/CD56-CD16+ both in blood and endometrial of model rats were lower, compared with the normal rats, but the differences were not significant (p>0.05). The differences of the level of E2 and P in serum between the normal rats and the model rats were not significant (p>0.05). Though the ratio of E2/P and the level of IL-2 in serum were higher in model rats compared with normal rats, the differences were not significant (p>0.05). The content of LIF in endometrial was significantly less in the model rats than that in the normal rats (p<0.05). There were no differences of the content of IL-2 in endometrial among all the groups (p>0.05). The level of LIF in serum was so little of all groups that there was no signification for statistics.4. The symptoms of kidney deficiency of model with herbal medicine group were improved. The quantity of endometrial glands and endometrial glandular area of model rats with herbal medicine both increased than the model rats, but the differences were not significant (p>0.05). Of the model with herbals medicine rat in estrus, the distribution of microvilli at the surface of endometrial epithelial was more dense. There were only a little vacuoles in mitochondria, there was more ribosomal densely attached to the surface of endoplasmic reticulum, and secretory granules could be saw in gland epithelial cells, compared with the model rat in estrus. The percentage of CD56+CD16-NK cells in endometrial and the ratio of CD56+CD16-/CD56CD16+ both in blood and endometrial of model rats with herbal medicine was higher, compared with the model rats without herbs, but the differences were not significant (p>0.05). The level of P in serum and LIF in endometrial of model with herbal medicine rats were both significantly higher than those of the model rats (p<0.05). The ratio of E2/P was a little lower and the level of IL-2 in serum was a little higher in model with herbal medicine rats than those in model rats, but the differences were not significant (p>0.05). The differences of above items between the normal rats with herbs and the normal rats without herbs were not significant (p>0.05). And the content of LIF in endometrial of the normal rats with herbs was significantly more than that of the model group (p<0.05).5. The weight of kidney deficiency pregnant model rats without herbs was significantly lighter than the normal pregnant rats (p<0.05). The quantity of embryos of the model rats was less and the abortion rate was higher of the model rats than those of normal pregnant rats, but the differences were not significant (p>0.05). There was no significantly differences of the percentage of NK cells of all the subsets in blood among the groups (p>0.05), but the percentage of CD56×CD16+NK cells was higher and the ratio of CD56+CD16-/CD56-CD16+ was lower, in decidua of the model rats, than those of the normal pregnant rats, though the differences were not significant (p>0.05). The differences of level of E2, P, IL-2 in serum was not significant between the model rats and the normal pregnant rats (p>0.05), though the level of P and IL-2 in serum were higher in the model group. The content of LIF in deciduas of the model rats was significantly less than that of the normal rats (p<0.05), but the difference of the content of IL-2 in decidua between them was not significant (p>0.05). The level of LIF in serum was also so little of all groups that there was no signification for statistics.6. The weight and the quantity of embryos of the model rats with herbal medicine pre and post pregnancy increased and the abortion rate was lower than the model rats. The difference of the quantity of embryos was significant (p<0.05). The weight and the quantity of embryos of the normal rats with herbs pre and post pregnancy increased and the ratio of abortion was lower than the model rats, the difference of the weight and abortion rate were significant (p<0.05). The percentage of CD56-CD16+NK cells in blood of the model rats with herbal medicine pre and post pregnancy, and the percentage of CD56-CD16+NK cells in decidua of the normal pregnant rats with herbs, were both lower than those of all the kidney deficiency pregnant rats, but the differences were not significant (p>0.05). The differences of the level of IL-2 in serum between the normal pregnant rats with herbs pre-pregnancy and the model rats, the differences of content of LIF in decidua between the model rats with herbal medicine pre and post pregnancy and the model rats were both significant (p<0.05).Conclusion:1. The endometrial thickness, the blood flow index of endometrial spiral arteries (RI), and the ratio of E2/P could be the important factors that influence the endometrial receptivity. The pill Zhuyun could effectively improve these factors for recurrent spontaneous abortion patient with spleen and kidney deficiency.2. We could use hydroxyurea (450mg/kg/dX8d) to create kidney deficiency rat model in mild case, that showed such as dysfunction of mitochondria and ribosomal in endometrial epithelial cells, decrease of microvilli at the surface of endometrial epithelial cells, dysfunction of secretion and signal transduction of endometrial epithelial cells, and decrease of LIF in endometrial.3. LIF in endometrial could be one of the key factors that influence the endometrial receptivity of rats. More LIF in endometrial would be good for endometrial receptivity. The increase of CD56-CD16+NK cells in blood and the decrease of CD56+CD16-NK cells in endometrial could destroy the normal phagocytosis of NK cells, and then damage endometrial receptivity.4. Pill Zhuyun could effectively adjust the disorder of endocrine-immune system of kidney deficiency model rats, improve the function of NK cells, increase the content of LIF in endometrial, and then improve the endometrial receptivity and pregnancy success rate.5. Pill Zhuyun could maintain the balance of adjustment function of endocrine-immune system of the normal rats, and then prevent abortion of normal pregnant rats, by feeding them pre or post pregnancy.
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