CReATe Fertility Centre, Toronto, ON, Canada.
CReATe BioBank, Toronto, ON, Canada.
Hum Reprod. 2021 Jun 18;36(7):1922-1931. doi: 10.1093/humrep/deab104.
Do phytocannabinoids (PCs) affect follicular endocannabinoid signalling and the epigenome in the surrounding granulosa cells (GCs)?
Exposure to PCs increases the expression of endocannabinoid receptors and reduces DNA methylation enzyme expression and global DNA methylation in naïve GCs.
Cannabis plant derivatives, known as PCs, are used for medicinal and recreational purposes. The main PC, tetrahydrocannabinol (THC), is the third most commonly used substance by women of childbearing age, hence knowledge of the effect it has on reproduction is of utmost importance. THC exerts its effects via receptors of the endocannabinoid system (ECS) and can interfere with folliculogenesis, oocyte development and ovulation. Endocannabinoids have been measured in follicular fluid (FF) obtained during oocyte retrieval and are implicated in controlling folliculogenesis. It has been established that in the placenta, PCs disrupt endocannabinoid homeostasis via impairment of the synthetic and degrading enzymes, leading to a net increase of endocannabinoid levels. Finally, previous studies have shown that THC alters methylation and histone modifications in sperm, brain and blood cells.
STUDY DESIGN, SIZE, DURATION: This study included an in vivo cohort assessment of cannabis exposure and its effects on the follicle and in vitro assays conducted to validate the in vivo findings and to explore possible mechanisms of action.
PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 318 FF samples, from 261 patients undergoing IVF treatment at a private fertility clinic who consented for biobanking biological waste material between January 2018 and July 2019, were included in this study. Concentrations of PCs and endocannabinoids were assessed in FF by liquid chromatography-mass spectrometry (LC-MS/MS). Exposure to PCs was determined based on these measured levels. Levels of both endocannabinoid receptors (CB1R, CB2R) and the de novo DNA methylating enzyme, DNMT3b, in GCs were assessed by flow cytometry both in vitro and in vivo and global DNA methylation was assessed in vitro by ELISA. In vivo effects were assessed by comparing samples positive for at least one PC, with samples negative for all measured PCs. In vitro effects were determined in naive GCs, obtained concurrently with FF samples that had tested negative for all PCs. These GCs were treated with different combinations of the main three PCs.
Overall, 17 patients (6.4%) were positive for cannabis consumption. Furthermore, the prevalence of cannabis positivity in the FF increased from 4% of the tested samples that were collected prior to national legalisation in October 2018 to 12% of those collected following legalisation. Of note, 59% of patients who tested positive for PCs (10 of 17) reported previous or ongoing exposure to cannabis upon their initial intake. Endocannabinoid levels were not affected by the presence of PCs. CB2R was more prevalent than CB1R in GCs and its expression increased following acute and chronic in vitro exposure to PCs. The expression of DNMT3b and global methylation decreased following exposure, suggesting that cannabis may affect the epigenome in the follicular niche. The acute changes were sustained throughout chronic treatment.
N/A.
LIMITATIONS, REASONS FOR CAUTION: Our study is limited by lack of details regarding mode, frequency and timing of PC consumption. Moreover, we were not able to adequately assess the effect of PCs on immediate or long-term clinical outcomes, due to the small sample size and the lack of follow up. Future, large-scale studies should focus on assess the clinical implications of cannabis exposure, validate our findings, and determine to what extent cannabis affects the epigenome ovarian follicle and the developing oocyte.
To our knowledge, this is the first study measuring PCs in FF by LC-MS/MS. We show that consuming cannabis alters the ECS in the developing follicle, and directly affects DNMT expression and global DNA methylation levels. Cannabis legalisation and use is increasing worldwide, therefore further understanding its role in female fertility and folliculogenesis is critical.
STUDY FUNDING/COMPETING INTEREST(S): All funding was provided by CReATe Fertility Centre through the reinvestment of clinical earnings. The authors declare no competing interests.
植物大麻素（PCs）是否会影响卵泡内的内源性大麻素信号转导和周围颗粒细胞（GCs）中的表观基因组？
暴露于 PCs 会增加内源性大麻素受体的表达，并降低幼稚 GC 中 DNA 甲基化酶的表达和整体 DNA 甲基化水平。
已知被称为 PCs 的大麻植物衍生物被用于药用和娱乐目的。主要的 PC，四氢大麻酚（THC），是育龄妇女中使用的第三大最常见物质，因此了解它对生殖的影响至关重要。THC 通过内源性大麻素系统（ECS）的受体发挥作用，并可能干扰卵泡发生、卵母细胞发育和排卵。已经在卵母细胞回收过程中获得的卵泡液（FF）中测量到内源性大麻素，并认为其在控制卵泡发生中起作用。已经确定，在胎盘内，PCs 通过破坏合成和降解酶来破坏内源性大麻素的内稳态，导致内源性大麻素水平的净增加。最后，先前的研究表明，THC 会改变精子、大脑和血细胞中的甲基化和组蛋白修饰。
研究设计、大小、持续时间：本研究包括对大麻暴露及其对卵泡的影响的体内队列评估，以及体外实验，以验证体内发现并探索可能的作用机制。
参与者/材料、设置、方法：共纳入 261 名在一家私人生育诊所接受体外受精治疗的患者的 318 份 FF 样本，这些患者于 2018 年 1 月至 2019 年 7 月同意对生物废物进行生物库储存。通过液相色谱-质谱联用（LC-MS/MS）在 FF 中评估 PCs 和内源性大麻素的浓度。根据这些测量水平确定 PCs 的暴露情况。通过流式细胞术在体内和体外评估 GC 中两种内源性大麻素受体（CB1R、CB2R）和从头 DNA 甲基转移酶 DNMT3b 的水平，并通过 ELISA 在体外评估整体 DNA 甲基化水平。体内效应通过比较至少有一种 PC 阳性的样本与所有测量的 PC 均为阴性的样本来评估。体外效应是在同时获得 FF 样本的幼稚 GC 中确定的，这些样本均为所有 PC 阴性。这些 GC 用三种主要 PC 的不同组合进行处理。
总体而言，17 名患者（6.4%）被检测出有大麻消费。此外，FF 中大麻阳性的患病率从 2018 年 10 月全国合法化前测试样本的 4%增加到合法化后的 12%。值得注意的是，在 17 名被检测出 PC 阳性的患者中，有 10 名（59%）在首次摄入时报告了之前或持续的大麻暴露。内源性大麻素水平不受 PCs 存在的影响。GC 中 CB2R 比 CB1R 更为普遍，其表达在体外急性和慢性暴露于 PCs 后增加。DNMT3b 的表达和整体甲基化减少，表明大麻可能会影响卵泡内的表观基因组。急性变化在慢性治疗过程中持续存在。
无。
局限性、谨慎的原因：我们的研究受到缺乏有关 PC 消费方式、频率和时间的详细信息的限制。此外，由于样本量小且缺乏随访，我们无法充分评估 PCs 对即时或长期临床结果的影响。未来，大规模研究应侧重于评估大麻暴露的临床意义，验证我们的发现，并确定大麻在多大程度上影响卵巢卵泡和发育中的卵母细胞的表观基因组。
据我们所知，这是第一项通过 LC-MS/MS 测量 FF 中 PCs 的研究。我们表明，消费大麻会改变发育中的卵泡中的 ECS，并直接影响 DNMT 表达和整体 DNA 甲基化水平。大麻合法化和使用在全球范围内正在增加，因此进一步了解其在女性生育和卵泡发生中的作用至关重要。
研究资金/竞争利益：所有资金均由 CReATe 生育中心通过临床收益的再投资提供。作者没有利益冲突。
