Female Reproduction and Metabolic Syndromes Laboratory, Division of Molecular Reproduction, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India.
Research Scholar, Research Centre, University of Kerala, Thiruvananthapuram, Kerala, India.
Hum Reprod. 2022 Jul 30;37(8):1835-1855. doi: 10.1093/humrep/deac139.
Do circadian genes exhibit an altered profile in peripheral blood mononuclear cells (PBMCs) of polycystic ovary syndrome (PCOS) patients and do they have a potential role in androgen excess?
Our findings revealed that an impaired circadian clock could hamper the regulation of peripheral steroid metabolism in PCOS women.
PCOS patients exhibit features of metabolic syndrome. Circadian rhythm disruption is involved in the development of metabolic diseases and subfertility. An association between shift work and the incidence of PCOS in females was recently reported.
STUDY DESIGN, SIZE, DURATION: This is a retrospective case-referent study in which peripheral blood samples were obtained from 101 control and 101 PCOS subjects. PCOS diagnoses were based on Rotterdam Consensus criteria.
PARTICIPANTS/MATERIALS, SETTING, METHODS: This study comprised 101 women with PCOS and 101 control volunteers, as well as Swiss albino mice treated with dehydroepiandrosterone (DHEA) to induce PCOS development. Gene expression analyses of circadian and steroidogenesis genes in human PBMC and mice ovaries and blood were executed by quantitative real-time PCR.
We observed aberrant expression of peripheral circadian clock genes in PCOS, with a significant reduction in the core clock genes, circadian locomotor output cycles kaput (CLOCK) (P ≤ 0.00001), brain and muscle ARNT-like 1 (BMAL1) (P ≤ 0.00001) and NPAS2 (P ≤ 0.001), and upregulation of their negative feedback loop genes, CRY1 (P ≤ 0.00003), CRY2 (P ≤ 0.00006), PER1 (P ≤ 0.003), PER2 (P ≤ 0.002), DEC1 (P ≤ 0.0001) and DEC2 (P ≤ 0.00005). Transcript levels of an additional feedback loop regulating BMAL1 showed varied expression, with reduced RORA (P ≤ 0.008) and increased NR1D1 (P ≤ 0.02) in PCOS patients in comparison with the control group. We also demonstrated the expression pattern of clock genes in PBMCs of PCOS women at three different time points. PCOS patients also exhibited increased mRNA levels of steroidogenic enzymes like StAR (P ≤ 0.0005), CYP17A1 (P ≤ 0.005), SRD5A1 (P ≤ 0.00006) and SRD5A2 (P ≤ 0.009). Knockdown of CLOCK/BMAL1 in PBMCs resulted in a significant reduction in estradiol production, by reducing CYP19A1 and a significant increase in dihydrotestosterone production, by upregulating SRD5A1 and SRD5A2 in PBMCs. Our data also showed that CYP17A1 as a direct CLOCK-BMAL1 target in PBMCs. Phenotypic classification of PCOS subgroups showed a higher variation in expression of clock genes and steroidogenesis genes with phenotype A of PCOS. In alignment with the above results, altered expression of ovarian core clock genes (Clock, Bmal1 and Per2) was found in DHEA-treated PCOS mice. The expression of peripheral blood core clock genes in DHEA-induced PCOS mice was less robust and showed a loss of periodicity in comparison with that of control mice.
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LIMITATIONS, REASONS FOR CAUTION: We could not evaluate the circadian oscillation of clock genes and clock-controlled genes over a 24-h period in the peripheral blood of control versus PCOS subjects. Additionally, circadian genes in the ovaries of PCOS women could not be evaluated due to limitations in sample availability, hence we employed the androgen excess mouse model of PCOS for ovarian circadian assessment. Clock genes were assessed in the whole ovary of the androgen excess mouse model of PCOS rather than in granulosa cells, which is another limitation of the present work.
Our observations suggest that the biological clock is one of the contributing factors in androgen excess in PCOS, owing to its potential role in modulating peripheral androgen metabolism. Considering the increasing prevalence of PCOS and the rising frequency of delayed circadian rhythms and insufficient sleep among women, our study emphasizes the potential in modulating circadian rhythm as an important strategy in PCOS management, and further research on this aspect is highly warranted.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the RGCB-DBT Core Funds and a grant (#BT/PR29996/MED/97/472/2020) from the Department of Biotechnology (DBT), India, to M.L. B.S.J. was supported by a DST/INSPIRE Fellowship/2015/IF150361 and M.B.K. was supported by the Research Fellowship from Council of Scientific & Industrial Research (CSIR) (10.2(5)/2007(ii).E.U.II). The authors declare no competing interests.
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多囊卵巢综合征 (PCOS) 患者外周血单个核细胞 (PBMC) 中的昼夜节律基因是否表现出改变的特征，它们在雄激素过多中是否具有潜在作用？
我们的研究结果表明，昼夜节律钟的紊乱可能会干扰 PCOS 女性外周类固醇代谢的调节。
PCOS 患者表现出代谢综合征的特征。昼夜节律紊乱与代谢性疾病和生育力低下有关。最近有报道称，轮班工作与女性 PCOS 的发病率之间存在关联。
研究设计、规模、持续时间：这是一项回顾性病例对照研究，从中获取了 101 名对照和 101 名 PCOS 受试者的外周血样本。PCOS 的诊断基于鹿特丹共识标准。
参与者/材料、设置、方法：本研究包括 101 名 PCOS 女性和 101 名对照志愿者，以及用脱氢表雄酮 (DHEA) 处理的瑞士白化小鼠，以诱导 PCOS 发展。通过定量实时 PCR 分析人类 PBMC 和小鼠卵巢和血液中的昼夜节律和类固醇生成基因的表达。
我们观察到 PCOS 外周昼夜节律基因表达异常，核心时钟基因显著减少，包括时钟输出周期 kaput (CLOCK) (P≤0.00001)、脑和肌肉 ARNT 样 1 (BMAL1) (P≤0.00001) 和 NPAS2 (P≤0.001)，以及其负反馈环基因的上调，CRY1 (P≤0.00003)、CRY2 (P≤0.00006)、PER1 (P≤0.003)、PER2 (P≤0.002)、DEC1 (P≤0.0001) 和 DEC2 (P≤0.00005)。调节 BMAL1 的另一个反馈环的转录水平表现出不同的表达模式，与对照组相比，PCOS 患者的 RORA 降低 (P≤0.008)，NR1D1 升高 (P≤0.02)。我们还在 PCOS 女性的三个不同时间点展示了 PBMC 中时钟基因的表达模式。PCOS 患者的类固醇生成酶 mRNA 水平也升高，如 StAR (P≤0.0005)、CYP17A1 (P≤0.005)、SRD5A1 (P≤0.00006) 和 SRD5A2 (P≤0.009)。在 PBMC 中敲低 CLOCK/BMAL1 会导致雌二醇产生显著减少，通过降低 CYP19A1，同时通过上调 SRD5A1 和 SRD5A2 导致二氢睾酮产生显著增加。我们的数据还表明，CYP17A1 是 PBMC 中 CLOCK-BMAL1 的直接靶标。对 PCOS 亚组的表型分类显示，时钟基因和类固醇生成基因的表达具有更高的变异性，A 型 PCOS 表现出更高的变异性。与上述结果一致，在 DHEA 处理的 PCOS 小鼠中发现卵巢核心时钟基因 (Clock、Bmal1 和 Per2) 的表达发生改变。DHEA 诱导的 PCOS 小鼠外周血核心时钟基因的表达不如对照组小鼠那样强劲，并且与对照组相比，其周期性丧失。
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局限性、谨慎的原因：我们无法评估对照与 PCOS 受试者外周血中时钟基因和时钟控制基因在 24 小时内的昼夜节律波动。此外，由于样本可用性的限制，我们无法评估 PCOS 女性卵巢中的昼夜节律基因，因此我们采用雄激素过多的 PCOS 小鼠模型来评估卵巢昼夜节律。在雄激素过多的 PCOS 小鼠模型中，我们评估了整个卵巢中的时钟基因，而不是在颗粒细胞中，这是本工作的另一个局限性。
我们的观察结果表明，生物钟是 PCOS 中雄激素过多的一个促成因素，因为它在调节外周雄激素代谢方面具有潜在作用。考虑到 PCOS 的发病率不断上升，以及女性中昼夜节律紊乱和睡眠不足的频率增加，我们的研究强调了调节昼夜节律作为 PCOS 管理的重要策略的潜力，因此非常需要进一步研究这方面。
研究资金/利益冲突：这项工作得到了 RGCB-DBT 核心基金和生物技术部 (DBT) 的资助 (#BT/PR29996/MED/97/472/2020)，用于 M.L. B.S.J. 得到了 DST/INSPIRE 奖学金/2015/IF150361 的支持，而 M.B.K. 得到了理事会科学与工业研究 (CSIR) 的研究奖学金 (10.2(5)/2007(ii))。作者没有利益冲突。
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