The effects of repeated controlled ovarian stimulation (COS) on the female reproductive system are still controversial. This study investigated the effects of repeated COS on the ovaries and uterus of mice and its possible mechanism. Female ICR (Institute of Cancer Research) mice were subjected to the COS using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) for 1, 3, 5, and 7 cycles. Serum hormone levels, reactive oxidative stress (ROS), 8-hydroxy-2’-deoxyguanosine (8-OHdG), total antioxidant capacity (T-AOC), and superoxide dismutase (SOD) in the mouse ovary and uterus were analyzed by ELISA. The morphology of the ovary and endometrium, ovarian apoptosis, and expressions of the vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), PI3K, AKT, Bax, and Bcl-2 in the ovarian and uterine tissues were tested by hematoxylin–eosin (HE) staining, immunohistochemistry, and western blot. The results showed that repeated COS significantly decreased the hormone level (estradiol, progesterone and anti-Müllerian hormone), high-quality of the MII oocyte ratio, oocyte and embryo number, antioxidant capacity (T-AOC, SOD activity), and the protein level of Bcl-2, LIF, and VEGF, but increased the oxidative damage (ROS, 8-OHdG content), embryo fragment ratio, and expression of pro-apoptotic protein Bax. In addition, the expressions of p-PI3K and p-AKT also decreased with the increase of COS cycle. In conclusion, repeated COS causes ovarian and uterus damage possibly through the PI3K/AKT signaling pathway, and this finding may provide some experimental basis for guiding clinical treatment.

文章总结杜惠兰教授应用分段法中药保守治疗未破损期输卵管妊娠的经验.杜教授认为输卵管妊娠发病主要为五脏气虚,强调湿热寒痰瘀滞冲任胞络贯穿于输卵管妊娠始终,提出消癥杀胚、泌浊散结和内外同治是治疗未破损型输卵管妊娠不同阶段的原则.临证胚胎存活之时应用消癥杀胚法及时杀胚祛瘀;血清人绒毛膜促性腺激素(β-HCG)下降时以泌浊散结法消癥散结,祛浊逐瘀;血清β-HCG降至正常时用化瘀通络之品以促进包块消散.

Objectives: Wenjing decoction (WJD) was widely used in the treatment for ovulatory disorder infertility (ODI) in China, while its efficacy was not clearly known. In this study, we evaluated the clinical efficacy of WJD by meta-analysis. Methods: Eight electronic databases including Cochrane Library, PubMed, Embase, Web of Science, China National Knowledge Infrastructure, WanFang Data, VIP Database, and China Biology Medicine were searched for randomized controlled trials (RCTs) published from the inception of each database to July 1, 2021, of which the interventions involve WJD and clomiphene. Outcomes included clinical efficacy rate, pregnancy rate, ovulation rate, dominant follicle diameter, endometrial thickness, estradiol, follicle-stimulating hormone, and luteinizing hormone. Meta-analysis and risk of bias were performed by RevMan 5.3 software. Results: Eleven RCTs including 915 patients, of which 476 in the intervention group and 439 in the control group. Meta-analysis showed that WJD was better than clomiphene for patients with ODI in terms of clinical effective rate (odds ratio [OR] = 1.22, 95% confidence interval [CI]: 1.08–1.34), pregnancy rate (OR = 1.54, 95% CI: 1.15–2.07), ovulation rate (OR = 1.34, 95% CI: 1.07–1.67), endometrial thickness (mean difference [MD] = 1.50, 95% CI: 0.90–2.10), and dominant follicle diameter (MD = 1.85, 95% CI: 0.68–3.02). The estradiol level (MD = 91.0, 95% CI: 80.3–101.88) in patients taking WJD was significantly higher than those taking clomiphene, while the follicle-stimulating hormone level (MD = −0.93, 95% CI: −1.13 to −0.72) and the luteinizing hormone level (MD = −4.41, 95% CI: −4.80 to −4.03) in patients taking WJD was significantly lower than those taking clomiphene. Our results also indicated that WJD combined with clomiphene was better than clomiphene alone for patients with ODI in terms of pregnancy rate (OR = 1.79, 95% CI: 1.37–2.35). Conclusions: WJD may be effective in the treatment of patients with ODI. Due to the quality and quantity of literature, RCT with large sample size and high quality need to be performed to verify our conclusion.

Cold coagulation and blood stasis (CCBS) syndrome is one of the common traditional Chinese medicine (TCM) syndromes of gynecological diseases. However, the molecular mechanism of CCBS syndrome is still unclear. Thus, there is a need to reveal the occurrence and regulation mechanism of CCBS syndrome, in order to provide a theoretical basis for the treatment of CCBS syndrome in gynecological diseases. The plasma proteins in primary dysmenorrhea (PD) patients with CCBS syndrome, endometriosis (EMS) patients with CCBS syndrome, and healthy women were screened using Label-free quantitative proteomics. Based on the TCM theory of "same TCM syndrome in different diseases," the differentially expressed proteins (DEPs) identified in each group were subjected to intersection mapping to obtain common DEPs in CCBS syndrome. The DEPs of gynecological CCBS syndrome in the intersection part were again cross-mapped with the DEPs of gynecological CCBS syndrome obtained by the research group according to the TCM theory of "different TCM syndromes in same disease" theory in the early stage, so as to obtain the DEPs of gynecological CCBS syndrome that were shared by the two parts. The common DEPs were subjected to bioinformatics analysis, and were verified by enzyme-linked immunosorbent assay (ELISA). A total of 67 common DEPs were identified in CCBS syndrome, of which 33 DEPs were upregulated and 34 DEPs were downregulated. The functional classification of DEPs involved in metabolic process, energy production and conversion, immune system process, antioxidant activity, response to stimulus, and biological adhesion. The subcellular location mainly located in the cytoplasm, nucleus, and extracellular. Gene ontology (GO) enrichment analysis showed that the upregulated DEPs mainly concentrated in lipid transport, cell migration, and inflammatory reaction, and the downregulated DEPs mostly related to cell junction, metabolism, and energy response. Protein domain enrichment analysis and clustering analysis revealed that the DEPs mainly related to cell proliferation and differentiation, cell morphology, metabolism, and immunity. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis clustering analysis showed that the upregulated DEPs were involved in inflammation and oxidative damage, while the downregulated DEPs were involved in inflammation, cell adhesion, cell apoptosis, and metabolism. The results of ELISA showed significantly increased levels of Cell surface glycoprotein MUC18 (MCAM) and Apolipoprotein C1 (APOC1), and significantly decreased levels of Vasodilator-stimulated phosphoprotein (VASP), Fatty acid-binding protein 5 (FABP5), and Vinculin (VCL) in patients with CCBS syndrome compared with healthy women. We speculated that cold evil may affect the immune process, inflammatory response, metabolic process, energy production and conversion, oxidative damage, endothelial cell dysfunction, and other differential proteins expression to cause CCBS syndrome in gynecological diseases.

目的 基于Label-free定量蛋白质组学及异病同证理论探究妇科实寒证的生物学基础.方法 纳入原发性痛经实寒证患者、子宫内膜异位症实寒证患者及健康女性各8例,分别为痛经组、内异症组、正常组.采集各组月经周期第2日的血浆样本进行蛋白提取、胰酶酶解和超高效液相色谱-质谱联用分析后得到原始数据,依据筛选标准获得差异表达蛋白,并进行交集映射,获取共有的实寒证差异表达蛋白,进行生物信息学分析.结果 痛经组与正常组共筛选出219个差异表达蛋白,内异症组与正常组共筛选出180个差异表达蛋白.经交集映射得出共有的实寒证差异表达蛋白97个,其中46个蛋白上调、51个蛋白下调.对差异表达蛋白进行功能分类统计主要涉及细胞过程、对刺激的反应、代谢过程、免疫过程、生物黏附、抗氧化活性、细胞运动等.GO富集分析显示:上调蛋白主要集中在脂质转运、炎性反应、细胞迁移、细胞运动、细胞间黏附等生物学过程;下调蛋白主要集中在细胞黏附、细胞连接等生物学过程.蛋白结构域富集分析和聚类分析显示:上调蛋白主要涉及免疫球蛋白结构域;下调蛋白主要涉及亲环蛋白型肽基-脯氨酰顺反异构酶/CLD、14-3-3蛋白等结构域.KEGG通路富集分析和聚类分析结果显示:上调蛋白主要参与Th17细胞分化、谷胱甘肽代谢等通路;下调蛋白主要参与Hippo信号通路、局灶性粘连、白细胞跨内皮迁移等通路.蛋白相互作用网络中筛选出VCL、ACTR2、ACTN1等6个核心蛋白.结论 妇科实寒证发生机制主要涉及代谢、免疫调节、炎性反应、氧化损伤、内皮细胞功能受损等生物学过程和信号通路.利用蛋白质组学获得的差异表达蛋白及其涉及的信号通路对揭示妇科实寒证的生物学基础有重要意义.

目的 观察补肾调经方对囊胚质量的影响及与有氧糖酵解相关基因表达的关系.方法 将100只动情周期正常的费城癌症研究所(ICR)雌性小鼠按照随机数字表法分为4组,分别为正常组、模型组、补肾低剂量组、补肾高剂量组,每组25只.除正常组,其余各组小鼠建立控制性超促排卵模型.正常组及模型组雌鼠每日上午9:00灌胃蒸馏水,补肾低、高剂量组雌鼠每日上午9:00灌胃补肾调经方低、高剂量(2.6、5.2 g/mL),连续10日.模型组及补肾低、高剂量组于第11日下午16:00同时腹腔注射孕马血清促性腺激素(PMSG)10 IU/只,48 h后腹腔注射入绒毛膜促性腺激素(HCG)10 IU/只;正常组于第11日下午16:00腹腔注射生理盐水0.1 mL.连续3个周期,每次间隔4天.模型组及补肾低、高剂量组均在控制性卵巢刺激(COS)第3次腹腔注射HCG后(下午16:00)立即与雄性小鼠合笼.正常组在动情期第3次腹腔注射生理盐水后(下午16:00)立即与雄性小鼠合笼.次日清晨检查阴栓,见栓的雌鼠记为妊娠.妊娠第4天(注射HCG后96 h)在体式显微镜下取出囊胚,观察囊胚质量并计数,计算优质囊胚率;免疫荧光染色、实时荧光定量PCR检测囊胚中的乳酸脱氢酶(LDH)、M2-型丙酮酸激酶(PKM2)、葡萄糖6-磷酸脱氢酶(G6PDH)、单羧酸转运蛋白4(MCT4)蛋白及mRNA的表达.结果 与正常组比较,模型组的囊胚数量增加(P＜0.05),优质囊胚率降低(P＜0.05),囊胚G6PDH、PKM2、LDH、MCT4蛋白及mRNA表达均降低(P＜0.05);与模型组比较,补肾低、高剂量组的优质囊胚率升高(P＜0.05),囊胚G6PDH、PKM2、LDH、MCT4蛋白及mRNA表达均升高(P＜0.05).结论 补肾调经方可增加超促排卵小鼠囊胚数量,提高小鼠囊胚质量,促进胚胎植入,其机制可能与上调有氧糖酵解改善早期胚胎能量代谢有关.

目的 观察补肾调经方对重复控制性卵巢刺激(COS)模型小鼠囊胚质量及凋亡的影响.方法 将160只雌性ICR小鼠按照随机数字表法分为正常组、模型组、补肾低剂量组(25.6 g/kg)和补肾高剂量组(51.2 g/kg).除正常组外,其余3组建立重复COS模型(COS 3次).每日9时正常组及模型组雌鼠灌胃蒸馏水,补肾低、高剂量组雌鼠灌胃低、高剂量补肾调经方,连续10 d,各组均于第11天16时进行腹腔注射,除正常组注射生理盐水0.1 mL外,其余3组腹腔注射孕马血清促性腺激素(PMSG)10 IU/只,并于48 h后腹腔注射人绒毛膜促性腺激素(hCG)10 IU/只.灌胃及腹腔注射连续3个周期,每次间隔4d.各组小鼠第3次腹腔注射后立即与同品系雄鼠合笼,次日清晨检查阴栓,见栓或阴道涂片见精子的雌鼠记为妊娠.妊娠小鼠于末次腹腔注射96 h后处死,获取囊胚,倒置显微镜下观察囊胚质量并计数,计算优质囊胚率;Tunel法检测囊胚细胞凋亡率;实时荧光定量PCR检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA表达;Simple Western检测凋亡相关因子Bcl-2、Bax及活化的半胱氨酸蛋白酶-3(cleaved caspase-3)的蛋白表达;免疫荧光法检测细胞色素C表达情况.结果 与正常组比较,模型组获取囊胚数增多,优质囊胚率下降(P<0.05);囊胚内细胞总数减少,凋亡率升高(P<0.05);Bcl-2、Bcl-2/Bax mRNA及蛋白比值降低,Bax mRNA及蛋白表达升高,cleaved caspase-3蛋白表达升高(P<0.05);细胞色素C从线粒体释放,共定位系数M1、M2降低(P<0.05),差异有统计学意义.与模型组比较,补肾低、高剂量组获取囊胚数差异无统计学意义(P>0.05),优质囊胚率升高(P<0.05);囊胚内细胞总数增多,凋亡率降低(P<0.05);Bcl-2、Bcl-2/Bax mRNA及蛋白比值升高,Bax mRNA及蛋白表达降低,cleaved caspase-3蛋白表达降低(P<0.05);细胞色素C无明显释放,共定位系数M1、M2升高(P<0.05).结论 补肾调经方可抑制重复COS引起的囊胚细胞凋亡,提高早期胚胎质量,其作用机制可能与线粒体凋亡途径有关.

历代医家重视对不孕症的研究,为后世留下宝贵的学术理论和丰富的临床经验.其中《傅青主女科》种子篇专论妇人不孕,介绍了10种不孕症,并立十证十方,为后世医家所推崇.笔者探析了其治疗不孕症的学术思想:重视脏腑辨证,尤其重视肾、脾、肝;辨析病位不离胞胎,运用冲任督带经络学说,揭示傅青主治疗不孕症的一大特色;灵活运用五行生克制化理论,协调脏腑气化种子.用药特点以扶正补虚为主,善用白术、人参、巴戟天,以固涩收敛为辅,活用覆盆子、山茱萸等药;用药平稳纯和,主次分明;重视对药物的炮制方法,增效引经.

目的 探讨自拟固胎煎对复发性流产小鼠蜕膜组织中叉头/翼状螺旋转录因子3(forkhead/pterygoid spiral transcription factor 3,Foxp3)、维甲酸相关核孤儿受体(retinoic acid related nuclear orphan receptor,RORγt)表达的影响.方法 将CBA/J雌鼠与BALB/c雄鼠2∶1合笼,建立正常妊娠模型(CBA/J×BALB/c组)作为对照组.CBA/J雌鼠与DBA/2雄鼠2∶1合笼,建立复发性流产模型(CBA/J× DBA/2组),并随机分为中药高、中、低剂量组、西药组以及模型组,HE染色观察各组蜕膜细胞形态,免疫组化法及Western Blot法分别检测各组小鼠蜕膜组织中Foxp3和RORγt的表达.结果 与模型组相比,自拟固胎煎中、高剂量组小鼠蜕膜组织内细胞数量及血管数量增多,胞质丰富,胞浆水肿减轻,核仁大而明显.且Foxp3表达均显著增加(P＜0.05),RORγt的表达均显著降低(P＜0.05).结论 自拟固胎煎能增加蜕膜组织Fox1p3的表达,减少RORγt的表达,改善小鼠蜕膜组织细胞局部环境,提示自拟固胎煎治疗复发性流产可能与其参与免疫调节,改善蜕膜环境作用机制相关.

目的 观察自拟固胎煎对复发性流产模型小鼠外周血白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-17(IL-17)、转化生长因子-β1(TGF-β1)表达及对妊娠结局的影响.方法 选取SPF级CBA/J雌鼠(n=48)、BALB/C雄鼠(n=4)、DBA/2雄鼠(n=20)作为研究对象.采用CBA/J雌鼠×BALB/c雄鼠建立正常妊娠模型,以CBA/J雌鼠×DBA/2雄鼠建立复发性流产模型,复发性流产模型孕鼠随机分为中药组、地屈孕酮组和复发性流产组,且将中药组分为高剂量亚组、中剂量亚组、低剂量亚组,正常妊娠组和复发性流产组给予生理盐水,中药高剂量亚组、中剂量亚组、低剂量亚组依次给予28.5 g/kg、14.25 g/kg、7.125g/kg自拟固胎煎汤剂,地屈孕酮组给予3.0mg/kg地屈孕酮溶液,连续灌胃2周,每天1次.给药结束后处死并解剖小鼠,观察子宫胚胎数量并计算胚胎丢失率,酶联免疫吸附试验(ELISA)法检测外周血IL-6、IL-10、IL-17及TGF-β1的表达水平.结果 与复发性流产组比较,中药中剂量亚组、高剂量亚组和地屈孕酮组胚胎丢失率均显著降低,差异具有统计学意义(P<0.05);与复发性流产组比较,中药中剂量亚组、高剂量亚组和地屈孕酮组的IL-6和IL-17的表达均显著降低,IL-10和TGF-β1的表达均显著升高,差异具有统计学意义(P<0.05).结论 自拟固胎煎可能通过上调IL-10、TGF-β1的表达水平,下调IL-6、IL-17的表达水平,诱导免疫耐受,减少胚胎丢失率,从而改善妊娠结局.

[目的]观察自拟固胎煎对自然流产模型小鼠脾脏Th17/Treg细胞及对妊娠结局的影响.[方法]采用CBA/J雌鼠×BALB/c雄鼠建立正常妊娠模型,以CBA/J雌鼠×DBA/2雄鼠建立自然流产模型,自然流产孕鼠分为自然流产组、地屈孕酮组、中药低、中、高剂量组,正常妊娠组和自然流产组给予生理盐水,中药低、中、高剂量组分别给予7.125、14.25、28.5 g/kg自拟固胎煎汤剂,地屈孕酮组给予3.0 mg/kg地屈孕酮溶液,连续2周灌胃,每日1次.给药结束后处死并解剖小鼠,观察子宫胚胎数量并计算胚胎丢失率,流式细胞法检测脾脏中Th17细胞、Treg细胞含量及Th17/Treg细胞比例.[结果]1)与自然流产组相比,中药低剂量组胚胎丢失率差异无统计学意义(P>0.05);中药中剂量组、高剂量组和地屈孕酮组胚胎丢失率均显著降低(P<0.05),且中药中剂量组、中药高剂量组、地屈孕酮组3组间比较,胚胎丢失率差异均无统计学意义(P>0.05).2)与自然流产组相比,中药低剂量组Th17细胞数、Treg细胞数及Th17/Treg细胞百分率比值均无统计学差异(P>0.05);中药中剂量组、高剂量组和地屈孕酮组,Th17细胞数及Th17/Treg细胞百分率比值均显著降低(P<0.05),Treg细胞数均显著升高(P<0.05);且中药中剂量组、中药高剂量组、地屈孕酮组3组间比较,Th17细胞数、Treg细胞数及Th17/Treg细胞百分率比值差异均无统计学意义(P>0.05).[结论]自拟固胎煎可降低Th17/Treg比例,诱导免疫耐受,减少胚胎丢失率,改善妊娠结局.