Effect of Modified Cangfu Daotantang on Metabolism and Pregnancy in Patients with Polycystic Ovary Syndrome / 中国实验方剂学杂志

Objective:To observe the effect of modified Cangfu Daotantang on metabolism and pregnancy in patients with spleen deficiency and phlegm-dampness type polycystic ovary syndrome （PCOS）. Method:One hundred and twelve patients were randomly divided into control group and observation group according to the random number table. Both groups took non-pharmacological interventions， oral metformin hydrochloride， 500mg/time， 3 times/day； oral ethinyl estradiol and cyproterone tablets， 1 tablet/time， 1 time/day， starting from the third to fifth day of menstruation and lasting for twenty-one days， for a total of 3 menstrual cycles. Patients in control group additionally took Erchen pills orally， 10 g/time， 2 times/day， while patients in observation group additionally took modified Cangfu Daotantang orally， 1 dose/day. The course of treatment was six menstrual cycles in both groups （or termination after conception）. The waist-to-hip ratio （WHR）， body mass index （BMI）， insulin resistance index （HOMA-IR）， pancreatic <italic>β</italic>-cell function （HOMA-<italic>β</italic>）， triglycerides （TG）， low-density lipoprotein （LDL） and non-high-density lipoprotein （nHDL） elevation after treatment were compared. The number of ovulation cycles monitored by B-ultrasound （6 menstrual cycles）， ovulation rate， human chorionic gonadotropin （HCG） day endometrial thickness， follicle diameter， cervical mucus score>8 points and endometrial morphology type A rate were measured and recorded. The recovery of menstruation， pregnancy and early miscarriage were recorded. Luteinizing hormone （LH）， estradiol （E₂）， follicle stimulating hormone （FSH）， dehydroepiandrosterone sulfate （DHEAS）， testosterone （T）， anti-Müllerian hormone （AMH） levels， and insulin before and after treatment -Like growth factor-1 （IGF-1）， leptin （LP）， adiponectin （APN）， growth differentiation factor-9 （GDF-9） and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） levels were detected. Result:WHR， BMI and HOMA-IR levels of the observation group were lower than those of the control group （<italic>P</italic><0.05， <italic>P</italic><0.01）. HOMA-<italic>β</italic> level was higher than that in the control group （<italic>P</italic><0.01）. The increase rates of LDL， TG， and nHDL in the observation group were 19.61%（10/51），25.49%（13/51），23.53%（12/51）， respectively， lower than 41.18%（21/51），47.06%（24/51），45.10%（23/51）respectively in the control group （<italic>χ</italic>²=5.607， <italic>χ</italic>²=5.131， <italic>χ</italic>²=5.263， <italic>P</italic><0.05）. The menstrual recovery rate in the observation group was 90.20% （46/51）， higher than 72.55% （37/51） in the control group （<italic>χ</italic>²=5.239，<italic>P</italic><0.05）. The observation group had more ovulation cycles than the control group （<italic>P</italic><0.01）. The pregnancy rate in the observation group was 50.98% （26/51）， higher than 31.37% （16/51） in the control group （<italic>χ</italic>²=4.047，<italic>P</italic><0.05）. On HCG day after treatment， the endometrial thickness and follicle diameter in the observation group were better than those in the control group （<italic>P</italic><0.01）. The proportion of patients with cervical mucus score> 8 points was 78.43% （40/51） in the observation group， higher than 56.86% （29/51） in the control group （<italic>χ</italic>²=5.420，<italic>P</italic><0.05）. The intimal morphology type A rate in the observation group was 52.94% （27/51）， higher than 31.37% （16/51） in the control group （<italic>χ</italic>²=4.864，<italic>P</italic><0.05）. The levels of AMH， E₂， DHEAS， LH， T ， IGF-1， LP and TNF-<italic>α</italic> in the observation group were lower than those in the control group （<italic>P</italic><0.01）， while the APN and GDF-9 levels were superior to those in the control group （<italic>P</italic><0.01）. Conclusion:On the basis of conventional western medicine intervention， modified Cangfu Daotantang can regulate abnormal metabolism and reproductive endocrine in patients with PCOS， improve conception， and regulate the expression of IGF-1， GDF-9， adipocytokines and inflammatory factors， improve ovulation and improve pregnancy rate.

Growth differentiation factor 9 and cumulus cell supplementation in in vitro maturation culture media enhances the viability of human blastocysts / 대한생식의학회지

OBJECTIVE: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts.METHODS: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined.RESULTS: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05).CONCLUSION: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

Effect of serum containing Jinghou Zengzhi Recipe on expression of GDF9 and Bim in ovarian COCs and CCs in controlled ovarian hyperstimulation rats / 重庆医学

Objective To observe the effects of serum containing Jinghou Zengzhi Recipe (JHZZ) on the expression of growth differentiation factor 9 (GDF9) and Bim in cumulus-oocyte complexes (COCs) and cumulus cells (CCs) of controlled ovarian hyperstimulation (COH) rats,and to investigate its curative effect and mechanism.Methods The rat COH ovarian model was prepared for collecting COCs and CCs,which were respectively co-cultured in vitro with the blank serum of female COH SD rat and serum containing JHZZ,and the each group was divided into 3 subgroups:the blank serum group (blank group),serum containing JHZZ group (control group) and serum containing JHZZ plus GDF9 receptor blocker group (experimental group),total 6 subgroups.COCs and CCs were collected after 24 h.The expression levels of GDF9 and Bim mRNA were detected by real-time quantitative PCR.The GDF9 protein expression levels were detected by Western blot.Results (1) The expression level of GDF9 mRNA in control group COCs was obviously higher than that in the control group and experiment group COCs (P＜0.05);the Bim mRNA expression level in COCs of control group and experiment group was significantly lower than that in COCs of blank group (P＜ 0.05);the GDF9 protein expression level in the control group COCs was significantly higher than that in the blank group COCs (P＜0.05),and also higher than that in the experiment group COCs,but the difference was not statistically significant (P＞0.05).(2)The comparison results of GDF9 mRNA expression level in CCs among 3 groups were consistent with those in COCs;the Bim mRNA expression level in CCs of control group and experimental group was significantly lower than that in the blank group (P＜ 0.05);the Bim mRNA expression level in the control group CCs was significantly lower than that in the experimental group CCs (P＜0.05);the GDF9 protein expression level in the control group CCs was significantly higher than that in the blank group and experimental group CCs (P＜0.05).(3)The GDF9 mRNA expression level in the control group COCs was significantly higher than that in CCs (P＜0.05),and the other inter-group comparisons between COSs and CCs had no statistical difference (P＞0.05).Conclusion Serum containing JHZZ can increase the GDF9 expression level in COCs and CCs,maintain the low expression of Bim in COCs and CCs,inhibit the ovarian cells apoptosis and promote the follicular development;COCs is more conducive to the expression of GDF9 mRNA compared with CCs eliminating oocyte.

Study on ovary development in vitrification of embryos born mice and expression of growth differentiation factor 9 / 中华妇产科杂志

Objective To study ovarian development in vitrificatiou of embryos born mice and expression of growth differentiation factor 9 ( GDF-9 ) in its.MethodsThe vitrification recovery embryos (vitrified-embryo group) and fresh embryos (fresh-embryo group) were transplanted into pseudopregnant mice,respectively.The female offspring mice in two groups were sacrificcd on the 3rd,7th,14th,21st,28th and 60th day after birth,the ovarian tissues were taken,6 mice in each time point of each group.The ovarian development was observed by HE staining,the expression of GDF-9 mRNA and protein at each time point of two groups were detected by reverse transcription(RT)-PCR and western blot.ResultsHE staining showed that no abnormal ovarian development was observed in offsprings at two groups.On the 3rd,7th,14th,21st,28th and 60th day after birth,the expression of GDF-9 mRNA in vitrified-embryo group were 0.14 ± 0.07,0.42±0.16,1.00±0.24,1.59±0.28,2.05 ±0.32 and 2.23 ±0.21,respectively,which in fresh-embryo group were 0.13 ±0.06,0.45 ±0.18,1.00 ±0.21,1.56 ±0.26,2.01 0.37 and 2.26 ±0.23,respectively,there was no statistical difference between two groups ( P ＞ 0.05 ) ; the expression of GDF-9 protein in vitrified-embryo group were 0.040 ± 0.030,0.120 ± 0.060,0.170 ± 0.030,0.250 ± 0.040,0.320± 0.060 and 0.330 ± 0.010,respectively,which in fresh-embryo group were 0.030 ± 0.020,0.110 ± 0.040,0.150 ± 0.010,0.210 ± 0.020,0.360 ± 0.070 and 0.350 ± 0.030,respectively,there was no statistical difference between two groups ( P ＞ 0.05 ).Conclusion The ovarian morphology in vitrification of embryos born mice and expression of GDF-9 in ovary has no any obvious change.

Change and significance of growth differentiation factor 9 and bone morphogenetic protein expression during oocyte maturation in polycystic ovary syndrome patients with ovarian stimulation / 中华妇产科杂志

Objective To investigate the expression pattern and significance of two importantoocyte-secreted factors:growth differentiation factor 9(GDF9)and bone morphogenetic protein 15(BMP15)during oocyte maturation in women with polycystic ovary syndrome(PCOS)and infertile women due to husband factors.Methods Total of 25 oocytes[9 at germinal vesicle GV stage,9 at M Ⅰ stage and 7 at M Ⅱ stage]were obtained from 12 patients with PCOS and 82 oocytes(29 at GV stage,26 at M Ⅰ stage and 27 at M Ⅱ stage)were from 56 controls.The nested quantitative real time(RT)PCR was uscd to detect the abundance of GDF9 and BMP15 mRNA in each oocyte.Results(1)The expression level of GDF9 mRNA at the GV stage,M Ⅰ stage and M Ⅱ stage in PCOS group were 44.8(4.2-529.0),27.6(9.8-172.7)and 49.0(0.2-65.9)respectively,the expression in were 149.9(55.4-387.9),29.9(2.5-205.8)and 657.8(149.4-1376.2)in control group,respectively.The expression of GDF9 mRNA at M Ⅱ stage was significantly lower in PCOS group than in controls(P ＜ 0.01),however,the differences didn't reach statistical significant at GV or M Ⅰ stage between the two groups(P ＞ 0.05).The expression of GDF9 mRNA displayed some changes at different maturation stage in controls(P ＜ 0.05,P ＜ 0.01),however,the expression didn't demonstrate any dynamic changes in PCOS group(P ＞ 0.05).(2)The expression level BMP15 mRNA at the GV stage,M Ⅰ stage and M Ⅱ stage in PCOS group were 0.1(0.1-22.0),3.2(0.6-55.0)and 6.4(3.2-8.5),respectively,the expression were 41.6(6.5-96.1),4.0(2.0-10.4)and 49.7(2.3-139.5)in control group,respectively.The expression of BMP15 mRNA at GV stage was significantly lower in PCOS group than in controls(P ＜ 0.01),however,the differences were not significant at M Ⅰ or M Ⅱ stage between the two groups(P ＞0.05).The expression of BMP15 mRNA also displayed some changes at different maturation stage in controls(P ＜ 0.05),however,the level didn't demonstrate any dynamic changes in PCOS group(P ＞ 0.05).Conclusion It was suggested that the low expression of oocyte secreted factors in mature oocytes from PCOS patients might be associated with impaired oocyte quality and developmental competence in PCOS.