| Objective:To establish PCOS pregnant rats mode with Ovulation induction.On the basis of this model, the serum endocrine hormones、inflammatory factors、uterine receptivity factors and early embryo implantation rate in the peri implantation window phase of PCOS ovulation induction rats were studied. To investigate the impact of Taishan Panshi San in early luteal phase on the outcome of early pregnancy and the possible mechanism from the perspective of endometrial receptivity, expound the function way and target of Chinese medicine, so as to provide experimental basis and theoretical basis for the clinical development of the treatment for PCOS with traditional Chinese medicine and Western Medicine.Methods:Ninety-two SD female rats of 21 days age were divided into normal control group (21) and PCOS model group (71). Since the age of 23 days, the normal control group with daily subcutaneous injection of soybean oil, PCOS model group with subcutaneous injection of dehydroepiandrosterone (DHEA) and soybean oil for continuous 30 days were made separately into Normal control group and PCOS model group. After the models were finished, one ant in the normal control group and PCOS model group were selected from each group and the left ovary of selected rats were taken for HE staining.The Remaining rats were taken serum testosterone (T) determination.Since 80 days of age, the normal control group was irrigated with normal saline(NS), and PCOS model group was given with CC combined NS for 5 consecutive days. Then they were mated with male rats with 2:1 and were found the female plugs as the first day of pregnancy (dl).They were set to normal pregnancy group and PCOS pregnancy group. The 6 pregnant rats were randomly selected from two groups and were measured of Serum estradiol (E2), progesterone (P), prolactin (PRL) and tumor necrosis factor (TNF-a) by ELISA.The normal pregnancy group was set as A group, and PCOS pregnancy group was randomly divided into three groups(B, C, D). From the beginning of pregnancy (dl), group A^B^C and D were respectively administered with NS、NS、Taishan Panshi San、progesterone until death. At the 8th day of pregnancy, a part of rats from group A、B、C and D were killed to take bilateral uterine blastocyst calculation. At the 3th、4th、5th day of pregnancy, some rats of group B were selected as group d3、d4、d5, and determinated of E2、P、PRL、TNF-a by ELISA then be killed.The left uterus were obtained to be tested the expression of PR、 ICAM-1 by immunohistochemistry and right uterus and endometria to be detect the mRNA expression of HOXA10、integrinavP3、Emx2、LIF by RT-PCR. At the 5th day of pregnancy, some rats of group A、B、C、D were selected to be determinated of E2、P、PRL、TNF-a by ELISA then be killed.The left uterus were obtained to be detected the expression of PR、 ICAM-1 by immunohistochemistry and right uterus and endometria to be detect the mRNA and protein expression of HOXA10、integrinavβ3、Emx2、LIF by RT-PCR and Western blot.Results:PCOS model group serum T was significantly higher than the normal control group (P< 0.01).The ovary of PCOS model group compared with normal control group was pale, capsular thickening, subcapsular cystic dilatation of more hidden follicles and showed multiple follicular cysts.The pregnancy rate was 100% in normal control group, and that in PCOS model group was 71.43%, there was significant difference (P< 0.01).At the lth day of pregnancy, the serum levels of E2, PRL and TNF-a in normal pregnancy group were less than those in PCOS group, while P was more than that in PCOS group, there was significant difference between them (P< 0.01, P<0.05).At the 8th day of pregnancy, the rat implantation rate and implantation of group A were significantly higher than that of group A, C and D, there was significant difference (P< 0.01); there was significant difference betreen group C、D and group A (P< 0.01).In peri-implantation period:E2、P of d5 group was significantly higher than those in d3 group, the difference was significant (P< 0.05). E2/P, PRL and ICAM-1 were expressed as d5< d3< d4, but there was no difference among them (P> 0.05).PR (IOD) was expressed as d4>d5>d3, but there was no difference (P>0.05). The mRNA expression of HOXA10 and integrin avP3 in d4 group was more than that of d5 and d3 group, and there was Obvious difference (P< 0.01, P< 0.05). And d5 group was higher than d3 group in the mRNA expression of integrin avβ3, the difference was Obvious (P< 0.05). The mRNA expression of Emx2 in d4 group was significantly lower than that in d5, and the difference was obvious (P< 0.05). The mRNA expression of LIF was significantly higher than that in d4 group, and the difference was significant (P< 0.05).In implantation period:Group B、C、D about E2 much higher than Group A (P<0.01, P<0.05). Group A、C、D about P was significantly higher than group B (P<0.01).But group A、C、D about E2/P was lower than group B (P<0.01) and group A was much lower than group C、D, the difference was significant (P<0.01, P< 0.05). TNF-a in group B was higher than group A and C (P<0.01, P<0.05); TNF-a in group D was significantly higher than that in group A.The expression of PRL was group A<C<D<B, but there was no significant difference between four groups (P> 0.05). The expression of PR in group A was significantly higher than that in group B (P< 0.05). The expression of ICAM-1 in group B was significantly lower than that in group A、C、D, the difference was significant (P< 0.05). ICAM-1 and P were positively correlated (r=8.223, P<0.05); TNF-α and E2/P were positively correlated (r=5.389, P<0.05). The mRNA expression of HOXA10 in group B was significantly lower than that in group A、D, and the difference was significant (P<0.01, P <0.05). The protein expression of HOXA10 in group B was significantly lower than that in group A、C、D, and the difference was significant (P<0.01); that in group A was significantly higher than that in group C、D, and the difference was significant (P<0.05, P<0.01). Group A、C、D in the mRNA and protein expression of integrin avβ3 were higher than group B, and the difference was obvious (P<0.01); the protein expression of it in group A was significantly higher than that in group C, and the difference was so significant (P<0.01). Group A about the mRNA expression was significantly lower than group B、C, the difference was significant (P<0.01). The protein expression of Emx2 in group A、C was significantly lower than that in group B、D, and the difference was significant (P< 0.01).The mRNA expression of LIF in group B was significantly lower than that in group A、C, and the difference was significant (P <0.01). The protein expression of LIF in group B was significantly lower than that in group A、C、D, and the difference was significant (P<0.01); that in group C was significantly lower than that in group A、D, and the difference was significant (P<0.01, P<0.05).Conclusion:In PCOS model rats, it was characterized as the polycystic ovary, hyperandrogenism and Ovulation dysfunction. CC was used to correct ovulation disorders and this significantly improved the rate of pregnancy. However, the endocrine disorders (high levels of E2、PRL, low levels of P) and the inflammatory factor TNF-a in PCOS pregnancy rats may affect embryo implantation and development and lead to early abortion.PCOS pregnant rats proned to early pregnancy loss, Taishan Panshi San or dydrogesterone could effectively reduce the rate of early abortion, improve the embryo implantation rate.The peak expression of PCOS pregnant rats about uterine receptivity factor PR, ICAM-1、HOXA10、integrinavβ3、Emx2、LIF appeared sooner or later the endometrial opening time in peri-implantation, and it leaded to the time of the endometrium opening was not synchronized with the development of the embryo. This is one of the possible reasons for the loss of pregnancy.In the window of the implantation, estrogen and progesterone and the ratio of abnormal, high expression of TNF-a, low expression of endometrial PR, ICAM-1, HOXA10, integrin avβ3, LIF, the high expression of Emx2 of PCOS pregnancy rats, lowered endometrial receptivity, unsynchronized the endometrium and embryo development and resulted in low implantation rate at last.Taishan Panshi San improved endometrial receptivity by adjusting hormones, improving ovarian function; upregulation of the expression of endometrial PR, ICAM-1, HOXA10, integrinavβ3, LIF, down-regulation of Emx2 expression during the implantation window, and that may be the Possible mechanism of Taishan Panshi San to improve pregnancy outcome. |
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