{"paper_id":"d4787194-5f80-4306-b5db-b756e082baf5","body_text":"Article https://doi.org/10.1038/s41467-025-63011-2\nCannabis impacts female fertility as\nevidenced by an in vitro investigation and a\ncase-control study\nCyntia Duval 1,2 , Brandon A. Wyse 1, Noga Fuchs Weizman 1,3,\nIryna Kuznyetsova 1, Svetlana Madjunkova 1 & Clifford L. Librach1,2,4,5,6\nCannabis consumption and legalization is increasing globally, raising concerns\nabout its impact on fertility. In humans, we previously demonstrated that\ntetrahydrocannabinol (THC) and its metabolites reach the ovarian follicle. An\nextensive body of literature describes THC’s impact on sperm, however no\nsuch studies have determined its effects on the oocyte. Herein, we investigate\nt h ei m p a c to fT H Co nh u m a nf e m a l ef e r t i l ity through both a clinical and in vitro\nanalysis. In a case-control study, we show that follicular fluid THC concentra-\ntion is positively correlated with oocyte maturation and THC-positive patients\nexhibit significantly lower embryo euploid rates than their matched controls.\nIn vitro, we observe a similar, but non-significant, increased oocyte maturation\nrate following THC exposure and altered expression of key genes implicated in\nextracellular matrix remodeling, inflammation, and chromosome segregation.\nFurthermore, THC induces oocyte chromosome segregation errors and\nincreases abnormal spindle morphology. Finally, this study highlights poten-\ntial risks associated with cannabis use for female fertility.\nCannabis consumption for both medicinal and recreational use and\nlegalization have been rising globally 1. Cannabis contains several\nclasses of chemicals with cannabinoids being the most prominent;\namong these, tetrahydrocannabinol (THC) is the primary psy-\nchoactive compound and the most studied\n2. Notably, the con-\ncentration of THC in cannabis products has increased signi ficantly,\nfrom an average of 3% (by weight) in the 1980s to around 15% in\n2020, with some strains reaching 30% of THC\n2. The increase in fre-\nquency, ease of availability, and escalation in potency raises con-\ncerns about broader impacts on global human health, including\nreproductive health. Indeed, the main apprehension regarding THC\nand reproductive health stems from the importance of the endo-\ncannabinoid system in human reproduction\n3. Endocannabinoids,\nincluding N-arachidonoylethanolamide and 2-arachidonoylglycerol,\nare endogenous cannabinoids that play a central role in both male\nand female reproduction\n3, whereas THC is an exogenous cannabi-\nnoid. Extensive research has documented the effects of THC on male\nreproduction, highlighting an impact on sperm deoxyribonucleic\nacid (DNA) methylation\n4–7 and sperm parameters 8 including sperm\nconcentration9–11, morphology 12–14 and motility 14. As for female\nhealth, literature reports the impact of cannabis use during preg-\nnancy on pregnancy outcomes\n15–18, placental development 18–20 and\noffspring health18,20–22. However, to our knowledge, no studies have\ninvestigated the impact of cannabis on the human female gamete,\nthe oocyte, a gap partly due to the challenge associated with\nobtaining these samples.\nDuring in vitro fertilization (IVF) treatment, exogenous gona-\ndotropins are administered in a process called “controlled ovarian\nhyperstimulation” which recruits multiple follicles and induces fol-\nlicle growth. These recruited follicles, each containing an oocyte, are\nReceived: 26 November 2024\nAccepted: 6 August 2025\nCheck for updates\n1CReATe Fertility Centre, Toronto, ON, Canada.2Department of Physiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada.3Racine\nIVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv-Yafo, Israel.4Department of Obstetrics and Gynecology, Temerty Faculty of\nMedicine, University of Toronto, Toronto, ON, Canada.5Institute of Medical Sciences, Temerty Faculty of Medicine, University of Toronto, Toronto, ON,\nCanada. 6Sunnybrook Research Institute, Toronto, ON, Canada. e-mail: cyntia@createivf.com; cyntiaduval22@gmail.com\nNature Communications|         (2025) 16:8185 1\n1234567890():,;\n1234567890():,;\n\nthen collected by a physician in a procedure called oocyte retrieval.\nOocytes are collected along with their surrounding microenviron-\nment, including follicular fluid (FF) and supportive somatic cells\n(granulosa cells). The oocytes are isolated, and mature oocytes are\nused for subsequent in vitro fertilization. Using FF, our group has\npreviously quantified Δ9-THC and its metabolites, 11-OH-THC and 11-\nCOOH-THC\n23,24, demonstrating that these compounds could reach\nthe follicular niche. This is signi ficant as it suggests that THC may\ndirectly alter the microenvironment where the oocyte matures.\nFurthermore, our group has shown that THC exposure altered\nhuman granulosa cell methylation in a concentration dependent\nmanner\n23, and in vitro exposure modulated cannabinoid receptor\ndynamics in granulosa cells24. However, no human studies and only a\nfew animal model studies have investigated the impact of cannabis\ndirectly on oocyte development with con flicting results\n25–29.\nMaturation of the oocyte is a unique and highly specialized pro-\ncess beginning in utero during fetal development. It is widely accepted\nthat female neonates are born with a finite number of oocytes, which,\nfollowing menarche, are recruited to mature in cohorts with each\nmenstrual cycle\n30. Although oocytes are protected in the ovary by the\nblood-follicle-barrier, they remain highly sensitive to environmental\nfactors\n31. Given their essential role in reproduction, any perturbations\nin their development and maturation could have profound effects on\nfertility and on future generations. Thus, understanding the impact of\nTHC on oocyte health is critical for providing informed guidance and\ncounseling to patients of the potential risks to their fertility and future\noffspring.\nIn this study, we determine the impact of physiologically relevant\nconcentrations of THC on oocyte maturation, elucidate the tran-\nscriptomic changes induced by THC exposure and its effect on chro-\nmosome segregation, and compare our findings with a retrospective\ncohort study. Our investigation will aid in bridging the knowledge gap\nin our understanding of the sex-specific reproductive consequences of\ncannabis use and contribute to more effective and evidence-based\npatient counseling.\nResults\nTHC concentrations correlate with maturation rate in IVF\nUsing a retrospective case-control design and mass spectrometry, we\nquantified the concentration of Δ9-THC and its metabolites,11-OH-\nTHC and 11-COOH-THC, in the FF of patients undergoing IVF treat-\nment to determine the reproductive consequences of THC con-\nsumption. Figure 1a illustrates the proportion of THC and its\nmetabolites measured in all samples ( n = 1059). Positivity rate was\ndefined by the presence of 11-COOH-THC in the follicular fluid (62/\n1059, 6%). 11-COOH-THC was found alone in 13% of the samples (8/\n62) while Δ9-THC was co-detected in 37% of the samples (23/62) and\n11-OH-THC co-detected in 2% (1/62). All three compounds were\nmeasured in 48% of the samples (30/62). Among the positive\npatients, 73% did not disclose their THC consumption on the patient\nintake questionnaire. The distribution of Δ9-THC and its metabolites\nshowed a predominance of 11-COOH-THC (mean = 28.8 ng/mL), fol-\nlowed by Δ9-THC (mean = 7.5 ng/mL), with 11-OH-THC being the least\nabundant (mean = 1.7 ng/mL) (Fig. 1b). Notably, concentrations of\nthese metabolites did not differ between FF and matched serum\nsamples obtained at the time of oocyte retrieval (Fig. 1c).\nA Spearman correlation analysis identi fied signi ficant correla-\ntions between THC metabolite concentrations and various clinical\nand biochemical parameters (Fig. 1d). Specifically, concentrations of\nΔ9-THC, 11-OH-THC and 11-COOH-THC were positively correlated\nwith oocyte maturation rate in the THC-positive group ( Δ9-THC:\n⍴ = 0.370, p = 0.003; 11-OH-THC: ⍴ = 0.309, p = 0.014 and 11-COOH-\nTHC: ⍴ = 0.295, p = 0.020). Interestingly, Δ9-THC levels were nega-\ntively correlated with a patient ’s Body Mass Index (BMI) ( ⍴ = −0.539,\np = 0.000053).\nIn vitro THC exposure and oocyte maturation\nPatients undergoing IVF treatment and oocyte retrieval who consented\nfor the collection of IVF waste material (immature oocytes, somatic\ncells and FF) and de-identified clinical data were included in this study.\nFor each patient, a minimum of three immature oocytes at the germ-\ninal vesicle (GV) stage were collected following the removal of somatic\ncells. GV oocytes were cultured using our standard in vitro maturation\n(IVM) protocol for 24h\n32 (control group (Ctrl), n = 96) or with the\naddition of THC (treatment groups). Oocytes were treated with either\na physiologically relevant (THC1,n = 95, 25 ng/mLΔ9-THC, 5 ng/mL 11-\nOH-THC, 50 ng/mL 11-COOH-THC) or a supraphysiologic (THC2,\nn =9 3 ,1 0 0n g / m LΔ9-THC, 50 ng/mL 11-OH-THC, 200 ng/mL 11-COOH-\nTHC) concentration where THC1 is based on the concentration ofΔ9-\nTHC and its metabolites measured in the follicularfluid of IVF patients\nand THC2 is based on previously reported concentrations in animal\nstudies\n23,25,29. Subsequently, oocytes were classi fied based on their\nprogression through key maturation checkpoints: germinal vesicle\n(GV) and Metaphase-I (MI) (after germinal vesicle breakdown (GVBD)\nand before polar body extrusion) were considered immature oocytes,\nwhile Metaphase-II (MII) oocytes (after visible polar body extrusion)\nwere considered mature (Fig.2a). Maturation rate was then calculated\nper treatment group.\nOocytes treated with THC1 showed no signi ficant change in\nmaturation rate (49/95, 52%,p = 0.6704), while THC2 exhibited a non-\nsignificant trend toward increased maturation (54/93, 58%,p = 0.1098),\ncompared to Ctrl (44/96, 46%) (Fig. 2b). Utilizing timelapse imaging,\noocyte morphology assessments were performed pre-IVM (Ctrl:n =9 2 ,\nTHC1: n = 89 and THC2:n = 85) and post-IVM (Ctrl:n =9 1 ,T H C 1 :n =8 8 ,\nand THC2: n = 83), and key maturation events were recorded: GVBD\n(Ctrl: n =7 1 , T H C 1 :n =7 2 a n d T H C 2 :n = 64) and extrusion of the first\npolar body (Ctrl: n = 28, THC1: n =3 0 a n d T H C 2 :n = 31). Examples of\ntimelapse IVM images are provided in Supplementary Fig. 1 and cor-\nresponding videos are provided as Supplementary videos (Ctrl-Sup-\nplementary video 1, THC1-Supplementary video 2 and THC2-\nSupplementary video 3). There were no signi ficant differences in\noocyte diameter between treatment groups either before (Ctrl:\n110.6 μm, THC1: 109.6μm, p =0 . 2 1 2 0a n dT H C 2 :1 0 9 . 6μm, p =0 . 2 1 2 0 )\n(Fig. 2c) or after 24 h of culture (Ctrl: 110.2 μm, THC1: 110.0 μm,\np = 0.7416 and THC2: 108.8 μm, p =0 . 1 0 6 6 ) . ( F i g .2d). Similarly, the\ntiming of GVBD (Fig. 2e) and polar body extrusion (Fig. 2f) remained\nunaffected by THC exposure. Demographic data of patients included\nin these analyses can be found in Supplementary Information - Sup-\nplementary Table 1.\nTHC exposure alters the oocyte transcriptome\nSingle MII oocytes with good morphology and normal develop-\nmental progression were sequenced using our optimized ultra-low\ninput RNA sequencing pipeline\n33 (n = 24 patients/n = 86 metaphase-II\n(MII) oocytes (28 Ctrl, 27 THC1 and 31 THC2). Differential expression\nanalysis revealed 89 genes up-regulated and 227 genes down-\nregulated greater than 2-fold (|log\n2FC| > 1) and p < 0.05 (Fig. 3a)\nwhen assessing the impact of the THC1 vs Ctrl (Supplementary\nData 1). Gene Set Enrichment Analysis (GSEA) identi fied that upre-\ngulated genes were principally associated with positive regulation of\nsynaptic transmission, axonemal dynein complex assembly, and\nglutamate receptor signaling pathway, while the downregulated\ngenes were associated with protein synthesis, expression regulation\nof SLITS and ROBOS and in flammatory processes (Fig. 3b, Supple-\nmentary Data 3). THC2 exposure induced a greater magnitude of\ntranscriptomic dysregulation, with 402 up-regulated and 62 down-\nregulated genes identi fied (Fig. 3c, Supplementary Data 2). The\nupregulated genes were associated with the immune system and\napoptotic pathways while downregulated genes were associated with\nattachment of spindle microtubules to kinetochores and in flamma-\ntory processes (Fig. 3d, Supplementary Data 3). As illustrated by the\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 2\n\nVenn diagram (Fig. 3e), THC1 exposure had 266 speci fic DEGs, while\nTHC2 exposure had 414, with 50 being common to both treatment\ngroups (gene lists are available in Supplementary Data 4). Of these 50\ncommon DEGs, 32 were protein-coding, 19 were up-regulated ( EPYC,\nRGS5, N4BP2L1, KRT19, PRR20G, KL, KCND3, ALDH3A1, SLC1A3,\nTSPAN8, PLAU, COL8A2, TFAP2E, SPTSSB, BRINP3, VANGL2, RGS18,\nRXFP1,a n d KCNMB3), 10 were down-regulated ( OR4F15, MMP9,\nPRRX2, IRS4, INFG, CCIN, IL33, NEUROD1, MT1HL1 ,a n d MT1H), and 3\ndisplayed bidirectional changes ( S100B, ACTA1 ,a n d ARHGEF19)\n(Fig. 3f) (Detailed information available in Supplementary Data 5).\nDemographic data of patients included in these analyses can be\nfound in Supplementary Information - Supplementary Table 2 and\nSequencing Quality Control metrics can be found in Supplemen-\ntary Data 6.\nTHC is harmful to chromosome segregation\nSubsets of MII oocytes from both the control and THC-treatment\ngroups were used to assess polar body ploidy status (18 Ctrl, 21 THC1,\nand 21 THC2) and for spindle morph ology (12 Ctrl, 12 THC1, and 12\nTHC2). Removal of the zona pellucida (ZP) and subsequent polar body\nbiopsy (Supplementary Fig. 2) allowed for some oocytes to be used for\nploidy determination by low-pass whole genome Next-Generation\nSequencing (NGS) aneuploidy using VeriSeq PGT-A which is specia-\nlized in detecting aneuploidy in reproductive samples\n21,34 (Supple-\nmentary Fig. 3) and meiotic spindle organization by confocal\nmicroscopy allowing for precise visualization of spindle organization\nand chromosome alignment (Fig.4a). Both THC1 and THC2 treatment\nled to a 9% increase in aneuploidy (Ctrl: 39%, THC1 and THC2: 48%,\np = 0.7479) (Fig. 4b) and a higher proportion of complex aneuploidy,\ndefined by the gain or loss of more than three chromosomes\n35 (Ctrl:\n0%, THC1 and THC2: 42%, p = 0.1029) (Fig. 4c). Figure 4dr e p o r t sa\nsubset of oocytes where both ploidy status and spindle morphology\nwere assessed ( n = 17), without stratifying by treatment group. The\nmajority of oocytes that completed meiosis I displayed normal spindle\nmorphology (euploid: n = 8/13, 62% and aneuploid: n = 3/4, 75%,\np > 0.9999) (Fig. 4d), but not all. The hallmark characteristics of\n0\n5\n10\n15\n20\n50\n100\n150\n200Concentration (ng/mL)\nΔ9-THC 11-OH-THC 11-COOH-THC\nSerum\nFollicular fluid\nab\ncd\nΔ9-THC 11-OH-THC 11-COOH-THC\n0.1\n1\n100\n1000\n10\nConcentration (ng/mL)\n-1.0\n-0.5\n0\n0.5\n1.0\nΔ9-THC\n11-OH-THC\n11-COOH-THC\nAge\nBMI\nDay 2/3 LH\nDay 2/3 FSH\nDay 2/3 FSH\nE2 on trigger\nTotal GT\nMat. Rate\nFert. Rate\nBlast. Rate\nEuploid Rate\nΔ9-THC\n11-OH-THC\n11-COOH-THC\nAge\nBMI\nDay 2/3 LH\nDay 2/3 FSH\nDay 2/3 FSH\nE2 on trigger\nTotal GT\nMat. Rate\nFert. Rate\nBlast. Rate\nEuploid Rate\nSpearman ρ\n*** *** *** **\n*** * *\n*\n*** * **\n* ** ***\n*\nPositives (n=62)\n11-COOH-THC\n8 \n(13%)\n00\n30\n(48%)\n1\n(2%)\n23\n(37%)\n0\n11-OH-THC Δ9-THC\nFig. 1 | Tetrahydrocannabinol concentrations and correlation with demo-\ngraphic data and clinical in vitro fertilization outcomes. aProportion of folli-\ncular fluid (FF) samples positive for Δ9-THC, 11-OH-THC and 11-COOH-THC\n(presence of 11-COOH-THC=positive sample).b Distribution ofΔ9-THC (n =5 3 ) ,1 1 -\nOH-THC (n = 31), and 11-COOH-THC (n = 62) concentrations in FF (data presented as\nmedian with interquartile range) andc concentrations ofΔ9-THC, 11-OH-THC, and\n11-COOH-THC in FF and matched serum samples (data presented as mean ±\nstandard deviation, fromn = 3 individual participants with matched serum samples\n(dotted fill pattern) and FF samples (plain)). d Correlation matrix of clinical and\nbiochemical parameters in THC-Positive group (n = 62). The colors represent the\ntwo-sided Spearmanρ value *p < 0.05, **p < 0.01, ***p < 0.001. AMH Anti-Müllerian\nHormone, BMI Body Mass Index, LH Luteinizing Hormone, FSH Follicle Stimulating\nHormone, E2 Estradiol, GT Gonadotropins, THC Tetrahydrocannabinol, Mat. Rate\nOocyte maturation rate, Fert. Rate Fertilization rate, Blast. Rate Blastulation rate.\nSource data are provided as a Source Data file.\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 3\n\n“normal” meiotic spindles include bipolar barrel-shaped microtubules\nwith the chromosomes aligned on the metaphase plate 36. Whereas\n“abnormal” spindles are varied in their morphology and may include\nmultipolar spindles, alterations in microtubule organization, and\nmisaligned chromosomes\n36. Spindle disorganization and chromosome\nmisalignment are shown by representative images in Fig. 4e, where\noocytes were classified as having either “normal” or “abnormal” spin-\ndles. The proportion of oocytes with abnormal spindles was higher in\nthe THC exposed groups compared to control (Ctrl (5/12), THC1 8/12),\nand THC2 (11/12), with a signi ficant increase in THC2 (Ctrl: 42% and\nTHC2: 92%, p = 0.0272) (Fig. 4f). (Spindle immunostaining negative\ncontrols ca be found in Supplementary Fig. 4)\nTHC decreases embryo euploidy rate in IVF\nFollowing pairwise case-control matching, where each THC-positive\nsample was matched to two THC-negative samples, a signi ficant\ndecrease in embryo euploidy rate was observed in the THC-positive\ngroup (n = 51, 60.0%), compared to the THC-negative group ( n =1 0 1 ,\n67.0%, p = 0.0245) (Table 1). There was no signi ficant change in\nmaturation, fertilization and blastocyst rates (Table1).\nTo further evaluate the likelihood of adverse IVF outcomes, we\nconducted multiple logistic regression analyses, focusing on clini-\ncally relevant IVF outcome thresholds\n37: maturation rate (80%),\nfertilization rate (70%), blastocyst rate (50%) and euploidy rate\n(60%). We utilized backward stepwise logistic regression, including\nthe following covariates: oocyte age, participant body mass index\n(BMI), anti-müllerian hormone (AMH), day 2/3 luteinizing hormone\n(LH), and follicle stimulating hormone (FSH), (estradiol) E2 on trig-\nger, and total gonadotropin (GT) dose. The final model for both\nblastulation and euploidy rates retained THC status as a signi ficant\nexplanatory variable, with oocyte age being a signi ficant covariate.\nIn this pairwise matched cohort, THC positivity signi ficantly\nbcd\nIn vitro maturation for 24h\n(SAGE + 75IU/μL Menopur)\nGV\nMI\nMII\n(mature)Clinical IVF \ntreatment\nTreatments \n(ng/mL)\nΔ9-\nTHC\n11-OH-\nTHC\n11-COOH-\nTHC\nCtrl 0 0 0\nTHC1 25 5 50\nTHC2 100 50 200\na\nGV\nMI\nMII\nCtrl THC1 THC2\n0\n20\n40\n60\n80\n58%\n52%\n46%\nMaturation rate 24h (%)\n96 95 93\nCtrl THC1 THC2\n90\n100\n110\n120\n130Diameter (μm)\npost IVM\nTime  (hours)\n% of oocytes at GVBD\n10 12 14 16 18 20 22 24\n0\n10\n20\n30\n40\n50\nTime  (hours)\n% of oocytes at MII\nTHC2\nTHC1\nCtrl\nCtrl THC1 THC2\n90\n100\n110\n120\n130Diameter (μm)\npre IVM\n0 4 8 1 21 62 02 4\n0\n10\n20\n30\n40\n50\nTime  (hours)\nCtrl\nTHC2\nTHC1\ne f\nFig. 2 | Impact of tetrahydrocannabinol exposure on oocyte maturation.\na Experimental design and in vitro oocyte maturation.b Maturation rate as the\npercentage of germinal vesicle (GV) oocytes that matured (progressed to\nmetaphase-II (MII)) rate (Ctrl (44/96, THC1: 49/95, THC2: 54/93). Oocyte diameter\n(c) prior to exposure to THC (Ctrl: n = 92, THC1: n = 89, THC2: n =8 5 )a n dd after\n24 h of culture (Ctrl:n =9 1 ,T H C 1 :n = 88, THC2: n = 83). Proportion of oocytes that\nunderwent key maturation events after 24 h of culture: e germinal vesicle break-\ndown (GVBD) (Ctrl: n = 71, THC1: n = 72, THC2: n = 64) and f extrusion of the first\npolar body (MII-arrested stage) (Ctrl:n = 28, THC1: n = 30, THC2: n =3 1 ) .E r r o rb a r s\nrepresent the mean ± standard deviation. Significance was assessed by two-sided\nFisher’s exact test or One-way ANOVA with two-sided Holm–Sidak multiple com-\nparison test (ns not significant). IVF In vitro fertilization, IVM In vitro maturation,\nMII metaphase II, MI metaphase I, GV germinal vesicle, GVBD germinal vesicle\nbreakdown, FF follicularfluid, THC tetrahydrocannabinol. Source data are pro-\nvided as a Source Data file.\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 4\n\ndecreased the odds of reaching a 50% blastulation rate or above\n(odds ratio: 0.45, p = 0.018) and the odds of achieving a euploidy\nrate above 60% (odds ratio: 0.47, p = 0.038) (Table 2). Age was also\nfound to signi ficantly impact blastulation and euploidy rates, with\nan odds ratio of 0.9144 ( p = 0.0010) and 0.9150 ( p = 0.0024),\nrespectively. The models for predicting blastulation rate (>50%) and\neuploidy rate (>60%) demonstrated positive predictive power, with\nareas under the curve (AUCs) of 0.68 and 0.67, respectively (Sup-\nplementary Fig. 5).\nDiscussion\nUnderstanding the impact of environmental factors and lifestyle\nchoices on female fertility is crucial for proper patient counseling.\nWith cannabis being one of the most commonly used recreational\ndrugs in the world\n1, it is critical to holistically evaluate its impact on\nmental and general health, in addition to reproductive health. This\nstudy, using donated human oocytes and an integrated multi-\ndisciplinary approach, reveals that exposure to THC affects oocyte\nmaturation, transcriptome, and induces meiotic chromosomal\nimbalances associated with altered spindle morphology. Moreover,\nour retrospective analysis revealed that exposure to THC was\nassociated with signi ficantly lower embryo euploidy rate, likely par-\ntially explained by an altered chromosomal organization as demon-\nstrated in the in vitro matured MII oocytes.\nIn our retrospective study, we measured THC concentrations in\n1059 follicular fluid samples from patients undergoing IVF treatment\nat CReATe Fertility Centre (Toronto, Canada) in a retrospective\nmatched case-control cohort. Sixty-two samples tested positive for\n11-COOH-THC, resulting in a 6% positivity rate (Fig. 1a). This rate is\nconsiderably lower than what was reported by a recent Health\nCanada survey where 23% of females reported recreational cannabis\nconsumption within 1 year of being surveyed\n38. However, these\npatients were counseled pre-treatment not to use recreational drugs\nwhile undergoing IVF. The relative concentrations of Δ9-THC, 11-OH-\nTHC, and 11-COOH-THC are consistent with metabolism of THC in\nthe liver (Fig. 1b). Δ9-THC (half-life 1.3–10 days) is metabolized to 11-\nOH-THC (half-life 20 min–2 h) and 11-OH-THC is rapidly metabolized\nto 11-COOH-THC (half-life 3–5 days), which remains in the circulation\nfor up to 30 days\n39. The consistent concentrations of THC metabo-\nlites in both the follicular fluid and serum suggests passive diffusion\nor transudation from the bloodstream into follicularfluid rather than\nactive transport into or out of the follicle (Fig. 1c).\nc\n-3 -2 -1 0 1 2\nREGULATION OF CD8-POSITIVE T CELL ACTIVATION\nPHOTODYNAMIC THERAPY INDUCED AP 1 SURVIVAL SIGNALING\nRRNA MODIFICATION IN THE NUCLEUS AND CYTOSOL\nAPOPTOSIS\nBIOCARTA_TNFR1_PATHWAY\nANTIGEN PROCESSING AND PRESENTATION OF ENDOGENOUS PEPTIDE ANTIGEN\nPROSTANOID BIOSYNTHETIC PROCESS\nAPOPTOSIS MODULATION AND SIGNALING\nPID_HDAC_CLASSIII_PATHWAY\nREGULATION OF SMOOTH MUSCLE CELL MIGRATION\nPRE IMPLANTATION EMBRYO\nCLATHRIN-DEPENDENT ENDOCYTOSIS\nINTERLEUKIN RECEPTOR SHC SIGNALING\nPHOSPHATIDYL INOSITOL PHOSPHATE PATHWAY\nREGULATION OF CHROMATIN ORGANIZATION\nEXPRESSION AND TRANSLOCATION OF OLFACTORY RECEPTORS\nREGULATION OF T-HELPER 17 TYPE IMMUNE RESPONSE\nREGULATION OF DENDRITIC SPINE MORPHOGENESIS\nRESPONSE TO ZINC ION\nATTACHMENT OF MITOTIC SPINDLE MICROTUBULES TO KINETOCHORE\nTHC2 vs Ctrl\nNES\n-3 -2 -1 0 1 2 3\nPOSITIVE REGULATION OF SYNAPTIC TRANSMISSION\nAXONEMAL DYNEIN COMPLEX ASSEMBLY\nGLUTAMATE RECEPTOR SIGNALING PATHWAY\nPOLY(A)+ MRNA EXPORT FROM NUCLEUS\nFAS SIGNALING PATHWAY ( CD95 )\nSUPERPATHWAY OF INOSITOL PHOSPHATE COMPOUNDS\nDIOL BIOSYNTHETIC PROCESS\nMETABOLISM OF COFACTORS\nCELLULAR RESPONSE TO NERVE GROWTH FACTOR STIMULUS\nNUCLEIC ACID TRANSPORT\nSYNDECAN-1-MEDIATED SIGNALING EVENTS\nPOSITIVE REGULATION OF INTERLEUKIN-6 PRODUCTION\nPOSITIVE REGULATION OF CD4-POSITIVE T CELL DIFFERENTIATION\nOXIDATIVE PHOSPHORYLATION\nATP BIOSYNTHETIC PROCESS\nCELLULAR RESPONSE TO STARVATION\nPOSITIVE REGULATION OF TUMOR NECROSIS FACTOR PRODUCTION\nNONSENSE-MEDIATED DECAY (NMD)\nREGULATION OF EXPRESSION OF SLITS AND ROBOS\nPROTEIN SYNTHESIS\nTHC1 vs Ctrl\nNES\nd\nab\ne\nEPYC\nRGS5\nN4BP2L1\nKRT19\nPRR20G\nKL\nKCND3\nALDH3A1\nSLC1A3\nTSPAN8\nPLAU\nCOL8A2\nTFAP2E\nSPTSSB\nBRINP3\nVANGL2\nRGS18\nRXFP1\nKCNMB3\nOR4F15\nMMP9\nPRRX2\nIRS4\nIFNG\nCCIN\nIL33\nNEUROD1\nMT1HL1\nMT1H\nS100B\nACTA1\nARHGEF19\nTHC1vsCtrl\nTHC2vsCtrl 0\n5\n10\nf\nTHC1 THC2\nTotal = 266\n62↑\n204↓\nTotal = 414\n369↑\n45↓\nTotal = 50\nUp-regulated (89)\nDown-regulated (227)\nNot significant (20802)\nUp-regulated (402)\nDown-regulated (62)\nNot significant (20654)\nFold \nchange\nFig. 3 | Effect of tetrahydrocannabinol exposure on oocyte transcripts after\noocyte in vitro maturation.Volcano plots of differentially expressed genes (DEGs)\ncomparing (a)T H C 1v sC t r la n db pathway analysis by gene set enrichment analysis\n(GSEA) THC1 vs Ctrl. Volcano plots of DEGs comparingc THC2 vs Ctrl andd GSEA of\nthe comparison THC2 vs Ctrl.e Venn diagram of DEGs in THC1 and THC2 compared\nto Ctrl. f Common protein-coding DEGs, colors representing fold change. DEGs\nwere defined by p < 0.05 (twp-sided Wald test resulting in unadjustedp-values) and\nlog2fold change (|log2FC)| > 1, n = 24 patients/n = 86 metaphase-II (MII) oocytes (28\nCtrl, 27 THC1 and 31 THC2). Significant pathways were defined as having a nor-\nmalized enrichment score (|NES|) > 1.5 andp < 0.05 (two-sided permutation test\nresulting in unadjusted p-values).\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 5\n\nIn a clinical IVF setting, embryologists and physicians assess\nmature oocytes based on the extrusion of thefirst polar body30,40.T h i s\nprocess, known as nuclear maturation, marks the oocyte’sp r o g r e s s i o n\nto metaphase-II and is considered the final stage of oocyte\nmaturation30,41. When we compiled the IVF outcomes of our retro-\nspective cohort, we observed a positive correlation between THC\nmetabolites and oocyte maturation (Fig. 1d). This suggests that the\nlevels of THC and its metabolites present in this cohort may support\nnuclear maturation through an unknown mechanism.\nParallel to our clinical retrospective study, we treated pairwise-\nmatched immature oocytes from patients who were negative for THC\n(naive GVs) with THC in vitro and we observed an increased proportion\nof oocytes which achieved the MII stage (Fig. 2b). Given that factors\nsuch as oocyte diameter have been previously reported to in fluence\nnuclear maturation\n42,w ec o nfirmed that there were no differences in\noocyte size distribution across the three groups (Ctrl, THC1 and THC2)\nbefore and after culture for 24 h (Fig.2c, d). Finally, we also monitored\nand recorded the timing of key maturation events using timelapse\nimaging technology and showed that the GVBD occurred slightly faster\nin THC treated oocytes (Fig. 2e), but this did not reach signi ficance,\nand there were no differences in the timing of the first polar body\nextrusion between THC-exposed and Ctrl groups (Fig. 2f). Previous\nanimal studies investigated the impact of THC on oocyte maturation\nusing bovine oocytes\n29, which is considered a better proxy to human\noocyte maturation when compared to murine model due to similar\ntemporal dynamics. López-Cardona et al. reported a 15% increase in\nmaturation rate after treating oocytes with 0.1 µM (31.45 ng/mL) Δ9-\nTHC for 12 h ( n =3 0 / g r o u p )\n29. In contrast, a more recent and larger\nstudy ( n = 164/group) concluded that a 24 h treatment with ‘recrea-\ntional cannabis doses ’ of 0.32 µM (100.63 ng/mL) and 3.2 µM\n(1,006.30 ng/mL) of Δ9-THC significantly reduced oocyte maturation\nfrom 80.1% to 65.3% and 60.1%, respectively25. Of note, the latter study\nused higher doses of Δ9-THC than what is measured in the FF of our\npatients but neither study examined the combined effects ofΔ9-THC\nand its metabolites, which might alter its effect on the growing oocyte.\nCollectively, our results suggest that THC exposure, at both\ne f\nb\nHoechst 33342/α-tubulin/Phalloidin\nNormal Abnormal\ncd\nIn vitro maturation for 24h\n(SAGE + 75IU/μL Menopur)\nGV\nMI\nMII\nTreatments \n(ng/mL)\nΔ9-\nTHC\n11-OH-\nTHC\n11-COOH-\nTHC\nCtrl 0 0 0\nTHC1 25 5 50\nTHC2 100 50 200\na\nRemove ZP\nSplit PB \nand Oo\n+\nPB\nOocyte\nWGA\nImmunofluorescence\nFrozen at -80 °C\nEuploid Aneuploid\n0\n5\n10\n15Number of oocytes\nNormal spindles\nAbnormal spindles\n38%\n62% 75%\n25%\nCtrl THC1 THC2\n0\n5\n10\n15\n20Number of oocytes\nNormal\nAbnormal\n✱\n42%\n58%\n66%\n33%\n92%\n8%\nCtrl THC1 THC20\n5\n10\n15\n20\n25Number of oocytes\nEuploid\nAneuploid\n39%\n52% 52%61%\n48% 48%\nCtrl THC1 THC2\n0\n5\n10\n15Number of aneuploid oocytes\n1\n2\n≥3\n57% 20% 40%\n43%\n40%\n20%\n40%40%\n0%\n5μm 5μm5μm\nFig. 4 | Impact of tetrahydrocannabinol exposure on oocyte spindle mor-\nphology and ploidy status. aExperimental design for polar body sequencing and\noocyte immunostaining.b Proportion of euploid oocytes deduced by polar body\nbiopsy sequencing results in Ctrl (11/18), THC1 (11/21), and THC2 (11/21).\nc Proportion of aneuploid oocytes with a gain/loss of one chromosome (white), two\nchromosomes (gray) or three and more (black) in Ctrl (1 chromosome: n =4 ,2\nchromosomes:n =3 , ≥3 chromosomes: n =0 ,t o t a ln = 7), THC1 (1 chromosome:\nn = 2, 2 chromosomes: n =4 , ≥3 chromosomes: n =4 ,t o t a ln = 10) and THC2 (1\nchromosome: n = 4, 2 chromosomes: n =2 , ≥3 chromosomes: n =4 ,t o t a ln = 10).\nd Proportion of normal morphology spindles in euploid (8/13) and aneuploid (3/4)\noocytes. e Representative images of normal (n = 12) and abnormal (n =2 4 )\nmetaphase-II-arrested oocyte spindles, chromosomes (Hoechst)are in blue, spin-\ndles (α-tubulin) are in green and cell membrane (Phalloidin) in red. Scale bar: 5µm.\nf Proportion of oocytes with abnormal spindles in Ctrl (5/12), THC1 (8/12), and THC2\n(11/12,p =0 . 0 2 7 2 ) .S i g n ificance was assessed using two-sided Fisher’s exact test. GV\ngerminal vesicle, MI metaphase I, MII metaphase II, Oo oocyte, PB polar body, THC\ntetrahydrocannabinol, WGA whole genome amplification, ZP zona pellucida.\nSource data are provided as a Source Data file.\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 6\n\nphysiological and supraphysiological concentrations, appears to\naccelerate oocyte maturation speed and completion, consistent with\nresults from the L όpez-Cardona bovine study\n29 and previous mouse\nstudies26.\nOocytes must not only successfully progress through meiosis II\nto reach metaphase-II but also need suf ficient time to reach cyto-\nplasmic maturity to support early embryo development 30,41.T h i s\nprocess involves the precise and faithful packaging of various com-\nponents, including maternal mRNA transcripts. mRNA production\nand storage in the ooplasm is critical, since, following establishment\nof the germinal vesicle, chromatin is condensed, and transcription is\nhalted\n43,44. These transcripts are critical not only for meiosis\nresumption and fertilization, but also during the first 3 days of\nembryonic development 45, since the early embryo remains tran-\nscriptionally silent, relying entirely on maternally inherited mRNA\nfrom the ooplasm to drive cellular processes prior to embryonic\ngenome activation\n46. The selection and enrichment of critical tran-\nscripts in the ooplasm have been described in the literature and are\nregulated by post-transcriptional mechanisms. These complex pro-\ncesses involve a sophisticated network of RNA binding proteins\n(RBPs), polyadenylation factors and RNA translational and degrada-\ntion machinery, which all regulate the storage, translation, and\ndegradation of oocyte mRNAs\n46.\nTo understand how THC affects stored maternal mRNA during\noocyte maturation, we identified genes associated with transcripts that\nwere differentially expressed following THC exposure (Fig. 3a, c).\nFocusing on the common protein-coding transcripts, 32 genes were\nidentified and grouped into nine principal functions: G protein-\ncoupled receptor (GPCR) signaling, extracellular matrix regulation,\nembryogenesis, cell-cell communication, inflammation, cytoskeleton,\ndetoxification, transcription factor and ion channels (Supplemen-\ntary Data 5).\nAmong these,MMP9 was significantly downregulated in both THC\ntreatment groups. MMP9 encodes for matrix metalloproteinase 9\n(MMP-9) essential for local proteolysis of the extracellular matrix and\nleukocyte migration\n47–49. In animal models, MMP-9 has well-\nestablished role in ovulation and follicle rupture 50–52, and in mice,\nprotein expression in blastocyst and early embryo is critical for\nimplantation\n53–58. In humans, several studies have investigated its role\nin implantation using various trophoblast and implantation\nmodels59–61. Dysregulation of its expression and activity is associated\nwith pregnancy complications and recurrent pregnancy loss 62–65.\nMoreover, THC has also been shown to decrease MMP-9 expression in\nhuman amniotic epithelium\n66 and endothelial cancer cells 67.T h u s ,\ndownregulation ofMMP9 in the ooplasm may negatively contribute to\nkey ovulation events needed for fertilization, embryo development,\nand implantation.\nG-protein coupled receptor (GPCR) signaling was also dysregu-\nlated following exposure to THC. GPCR signaling is crucial for oocyte\ngrowth and maturation, and many GPCRs are present at the oocyte\nsurface, including the cannabinoid receptor 1 (CB1) and 2 (CB2)\n27,68.\nGenes coding for Regulators of G protein Signaling (RGS), RGS5 and\nRGS18,w e r es i g n ificantly upregulated following THC treatment in our\nstudy. These regulators are known modulate GPCR signal\ntransduction\n69. RGS5 and RGS18 belong to the RGS R4 family and have\nbeen shown to bind with the G α proteins, thus reducing their\nTable 1 | Demographic data and in vitro fertilization outcomes of pairwise matched patients negative and positive for\ntetrahydrocannabinol\nNegative (n = 124) Median (IQR) Positive ( n = 62) Median (IQR) p-value\nTHC concentrations Δ9-THC (ng/mL) – 5.3 (9.6)\n11-OH-THC (ng/mL) – 0.1 (2.3)\n11-COOH-THC (ng/mL) – 18.1 (29.1)\nAge (years)a 30.0 (9.0) 29.5 (11) 0.4863\nBMI (kg/m2) 23.4 (4.8) 24.3 (8.3) 0.9709\nAMH (pmol/L) 24.4 (21.1) 25.8 (30.1) 0.7117\nDay 2/3 LH (IU) 1.2 (2.7) 2.1 (3.3) 0.0275\nDay 2/3 FSH (IU) 6.9 (2.5) 6.4 (3.2) 0.5714\nE2 on trigger (pmol/L) 13,445 (14,553) 12,603 (9,996) 0.5473\nTotal GT (IU) 4,088 (1,003) 3,894 (1,025) 0.2998\nMaturation rate (%)\na 72.0 (22.0) 76.0 (21.5) 0.2200\nFertilization rate (%) 82.5 (17.2) 83.0 (19.0) 0.7202\nBlastocyst rate (%)\na 59.5 (27.2) 50.0 (40.0) 0.5128\nEuploidy rate (%) 67.0 (22.0) 60.0 (26.0) 0.0245\nNormality was tested using the Shapiro–Wilk test.\nAMH Anti-Müllerian Hormone,IQR Interquartile range,THC Tetrahydrocannabinol,BMI Body Mass Index,LH Luteinizing Hormone,FSH Follicle Stimulating Hormone,E2 Estradiol,GT Gonadotropins.\naIndicates normally distributed data, all others are non-normally distributed. Significance was assessed using either a two-sided Mann–Whitney test or two-sided unpaired t-test, where appropriate.\nTable 2 | Multiple logistic regression models for blastulation and euploidy rates\nOutcomes Covariates Coef ficient (β) SE p-value OR 95% CI for OR\nLower Upper\nBlastulation rate >50% THC+ (Case) −0.803 0.338 0.018 0.448 0.229 0.864\nOocyte age −0.089 0.027 0.001 0.914 0.866 0.963\nEuploidy rate >60% THC+ (Case) −0.759 0.365 0.038 0.468 0.226 0.952\nOocyte age −0.089 0.029 0.002 0.915 0.862 0.968\nSignificance was assessed using Likelihood Ratio Test (LRT)\nSE Standard error, CI Coefficient intervals,OR Odds ratios, THC Tetrahydrocannabinol\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 7\n\ninhibitory activity70,71. They have been shown to have a crucial role in\nthe cell processing of the signal coming from the GPCRs 72. Although\nthe exact role of RGS5 and RGS18 in oocyte maturation is poorly\nunderstood, their upregulation suggests a potential impact on GPCR\nsignaling pathways in response to THC stimulation.\nImmune pathways were also overrepresented in both treatment\ngroups compared to controls. For instance, IFNG and IL33 were both\nsignificantly downregulated in THC exposed groups. It is well estab-\nlished that tight regulation of in flammatory processes is critical for\nimplantation of the embryo in the endometrium73. Interferon-γ (IFNγ)\nis a cytokine widely known for its role in in flammation and the acti-\nvation of macrophages74. Although its role during oocyte maturation is\nunclear, IFNγ secretion by the conceptus is essential for implantation\nin animal models75. On the other hand, Interleukin-33 (IL-33), a cyto-\nkine that belongs to the IL-1 family, binds to the ST2 (IL-1RL1)\nreceptor76.I nm i c e ,I L - 3 3w a sf o u n dt ob ee x p r e s s e di nt h eo o c y t ea n d\nST2 in granulosa cells and the uterus 77. In humans, altered levels or\nexpression of IL-33 or ST2 in various tissue and sample types were\nassociated with pregnancy complications like preeclampsia\n78–80, pre-\nterm birth81–83, intrauterine growth restriction84, and miscarriage85.T h e\ndownregulation of IL-33 we observed in THC-exposed oocytes sug-\ngests there may be potential disruption in the inflammatory processes\nnecessary for successful pregnancy.\nAs i g n ificant number of DEGs in THC-exposed oocytes are\ninvolved in the cytoskeletal function, includingKRT19, COL8A2, ACTA1\nand ARHGEF19 (Supplementary Data 5). In addition to these tran-\nscriptomic changes, we observed significant alterations to cytoskele-\nton machinery throughout this study. Indeed, the oocyte ’s\ncytoskeleton plays a crucial role in chromosome alignment, segrega-\ntion, and polarity establishment\n86 and without appropriate formation\nand regulation of key cytoskeletal functions, the oocyte is vulnerable\nto chromosomal abnormalities. However, the cytoskeleton-associated\nDEGs identified in this study have not been previously characterized in\noocyte development and thus require further investigation to gain a\ndeeper understanding of the effects of THC on these processes.\nTaken together, THC exposure seems to impact critical tran-\nscripts involved in key oocyte maturation processes, fertilization, early\nembryo development and implantation. While these transcriptomic\nalterations likely result from post-transcriptional processes, the spe-\ncific mechanisms by which THC affects these processes in human\noocytes remains unknown\n46.\nGiven the observed increase in nuclear maturation rate and the\naltered transcriptomic profiles related to chromosome organization,\nwe next investigated the impact of THC on chromosome segregation.\nIndeed, errors in chromosome segregation during the first meiotic\ndivision is the most frequent cause of embryo aneuploidy\n87, making\nthe faithful establishment of chromosome segregation machinery a\ncritical bottleneck in the production of a chromosomally normal\nembryo\n88,89. To investigate oocyte ploidy, we performed polar body\nbiopsy and low-pass whole genome sequencing. Notably, we observed\nthat THC exposure led to a 9% increase in aneuploidy rates (Fig. 4b).\nAdditionally, we observed an increase in the proportion of oocytes\nwith complex aneuploidies (defined as a gain or a loss of 3 or more\nchromosomes)\n35 in the THC-treated group compared to controls\n(Fig. 4c). Aneuploidies are associated with implantation failure, mis-\ncarriage, and are incompatible with life90. It has been postulated that\nmost aneuploidies arise from errors in maternal meiosis I91–96,b u to u r\ndata suggest that meiosis II may also be sensitive to perturbations as\n38% of the euploid oocytes had abnormal spindle morphology (Fig.4d)\ndetermining using confocal microscopy. We assessed spindle mor-\nphology after 24 h incubation of oocytes with and without THC. A\nnormal spindle con figuration is barrel shaped with chromosomes\naligned at the metaphase plate, while ‘abnormal’ configurations\ninclude multipolar spindles and misaligned chromosomes\n97 (Fig. 4e).\nIn this study, we demonstrated a dose-dependent decrease in the\nproportion of oocytes with normal spindle morphology following THC\nexposure (Fig. 4f). Correct chromosome segregation during oocyte\nmaturation is essential for producing euploid embryos, which have the\nhighest chance of establishing a healthy pregnancy\n98.\nTo address the primary clinical question regarding THC’s impact\non IVF outcomes, we used a pairwise case-control matching strategy,\nwhere each positive sample was matched to two negative samples\nbased on demographic data, and we compiled the resulting matched\ncohort’s IVF outcomes (Table 1). THC exposure was associated with a\nmarginal increase in maturation rate (Table 1), concordant with what\nwas obtained in our in vitro experimentations (Fig. 2b). Further, a\nsignificant decrease in embryo euploidy rate (Table 1) and reduced\nodds of obtaining a euploidy rate above 60% was observed (Table 2).\nThese results indicate that THC-positive patients may have fewer\neuploid embryos from their IVF cycle and may experience a longer\ntime to pregnancy.\nTo deepen our understanding of our findings, we must extra-\npolate what is known about THC interactions and pathways from other\ncell types. THC primarily elicits its functions through binding the\ncannabinoid 1 and 2 receptors (CB1 and CB2), which are expressed at\nall stages of oocyte maturation\n68. CB1 and CB2 are G protein-coupled\nreceptors (GPCRs) which are capable of inhibiting adenylate cyclase,\nthe enzyme responsible for catalyzing adenosine triphosphate (ATP)\nto cyclic adenosine 3’,5 ’-monophosphate (cAMP). Activation of the CB\nreceptors, through stimulation by THC, could thus lead to an inhibition\nof adenylate cyclase, resulting in lowering ooplasm cAMP levels\n99.\nAdenylate cyclase activity and the constant and high production of\ncAMP is critical to prevent premature meiotic resumption100.H e r e ,w e\npropose a hypothetical model of action of THC wherein THC binds to\nCB1/2 activating them, which in turn inhibits adenylate cyclase activity,\nreducing ooplasm cAMP concentration. Releasing the inhibition of\nmeiotic resumption, would then result in premature resumption of\nmeiosis. This untimely and premature resumption may increase the\nlikelihood of aneuploidy arising in the oocyte and resulting embryo\ndue to the premature separation of chromosomes misaligned on the\nmetaphase plate and an asymmetrical division of the chromosomes\ninto the first polar body. This hypothesis aligns with the correlation\nbetween THC concentrations and oocyte maturation rate observed in\nthe retrospective cohort (Fig.1d) and the increased oocyte maturation\nrate in vitro, as well as the associated reduction in euploid oocytes\n(Fig. 4b) and euploid embryo rates (Table 1) we observed. Further\ninvestigations are underway to dive deeper into THC signaling in the\noocyte and better refine this hypothetical model.\nTo conclude, this study comprehensively investigates and\ndemonstrate the impact of THC on the human oocyte. Herein, our\nfindings reveal signi ficant effects on oocyte maturation, tran-\nscriptomic profiles, meiotic spindle organization, and oocyte ploidy.\nCollectively, this data presents compelling evidence that cannabis\nconsumption may negatively impact female fertility. Our integrated\nand multi-faceted in vitro approach, utilizing multiple techniques and\nendpoints to assess chromosome segregation, is a major strength of\nthis study. However, it was limited by the usage of immature GV\noocytes following ovarian hyperstimulation, which are considered\nsuboptimal for reproductive purposes, since they did not mature fol-\nlowing initial stimulation. Furthermore, we acknowledge the impor-\ntance of patient age on the oocyte ability to mature in vitro, but this\nstudy was not statistically powered to analyze results based on patient\nage. This limitation arose because the majority of GV oocytes were\nretrieved from patients younger than 37 years old (81%). Also, our\nstudy focused on identifying changes in the abundance of the pre-\nstored transcripts in response to THC exposure, rather than de novo\ntranscription, limiting our ability to speculate on the impact of THC on\ngene expression before the GV stage.\nOn the other hand, our retrospective cohort objectively measured\nTHC and its metabolites to determine the impact of THC on IVF\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 8\n\noutcomes, overcoming biases inherent in self-reporting101. Indeed, 73%\nof our patients positive for THC did not report cannabis use when\ncompleting their patient intake questionnaire, potentially due to the\npersistent stigma of recreational drug consumption. A limitation of\nour retrospective study is the lack of data on cannabis consumption\nhabits (e.g. frequency, timing, dosage, route of consumption), and our\ncohort is likely not representative of the general population, as all\npatients were undergoing IVF for fertility treatment or to altruistically\ndonate their oocytes to intended parents. In addition, FF was not\nmeasured for the presence of other drugs, and even though none of\nthe patients reported concomitant use of other drugs, self-reporting\nalone cannot rule out the exposure of the follicle to these substances.\nThe limitations associated with the retrospective aspect of this study\nand the other potential contributors (e.g. lifestyle habits) to the\nobserved outcomes are compensated by our in vitro study that used at\nleast three oocytes (one in each exposure group) per patient.\nFinally, these findings underscore the need for increased aware-\nness and caution among people with ovaries, particularly those\nundergoing fertility treatments. Our study highlights the importance\nof informing patients about the potential risks associated with can-\nnabis consumption and provides a basis for regulatory bodies, medical\nprofessional societies, and public health organizations to establish\nrecommendations and guidelines regarding cannabis consumption\nduring fertility treatment.\nMethods\nEthical approval and cannabis regulatory licencing\nAll patients undergoing ART procedures were provided with the\nopportunity to participate in the collection, and future use, of biolo-\ngical waste material for research purposes. Patients were provided\nwith an Independent Review Board (IRB) approved informed consent\npackage containing information regarding the types of material that\nwould be collected following consent as well as examples of projects\nthis material may be used for. Patients did not receive compensation or\nfinancial benefit for their participation in the collection of biological\nwaste material. All patients included in this study provided informed\nconsent for the donation of their biological waste material, which\nincluded follicular fluid (FF) and immature (GV) oocytes as well as\nassociated de-identi fied demographic and clinical information,\nincluding age, Body Mass Index (BMI), ovarian reserve metrics and\ntreatment regimens (Veritas IRB Approval #16487). To be included in\nthe assessment of tetrahydrocannabinol (THC) on in vitro maturation\n(IVM), patients must have had 10 or more MII oocytes after stripping\nand a minimum of 3 GV oocytes in order to have at least one GV oocyte\nper treatment group. Last, all patients included in the assessment of\nTHC on IVM were con firmed to be negative for THC by LC-MS/MS\n(described below). Patients were excluded if: they did not meet\ninclusion criteria, had low oocyte yield (<16 oocytes), low oocyte\nmaturation rate (<62.5%), a previous cycle with poor fertilization rate\n(<75%) and/or blastulation rate (<40%), severe male factor, advanced\nmaternal age (>40 years old), or who were undergoing fertility pre-\nservation (oncofertility and/or social egg freezing). Moreover, if a\npatient was consented for the donation of their biological waste\nmaterial but the physician and embryologist deemed rescue-IVM\n(rIVM) was indicated for their clinical care, no oocytes would be col-\nlected for research purposes and patients would be informed of the\naddition of rIVM to their clinical treatment by the physician or another\nhealthcare professional. All GV oocytes included in this study were\ncollected and underwent IVM between July 2022 and January 2024. The\nrequest and use of samples and de-identified demographic and clinical\ndata for this study was approved by Veritas IRB Approval #16518. For\nthe retrospective analysis, FF was collected from all consenting\npatients undergoing IVF treatment between June 2016 and March\n2023. All samples in this study were considered “female” as they are\nhuman oocytes which are chromosomally“XX”. Gender, race, ethnicity\nor other socially relevant groupings were not considered in this study.\nThe purchase, storage and use of Δ9-THC and its metabolites for\nresearch purposes was approved by Health Canada and all procedures\nwere conducted in accordance with the ‘Cannabis Act’ and ‘Cannabis\nRegulations’ (License #LIC-A4MUR820SB-2020).\nExocannabinoid detection in FF\nMeasurements of Δ9-THC, 11-OH-THC and 11-COOH-THC in FF were\nperformed for every patient who donated GV oocytes, as previously\ndescribed, to exclude patients consuming cannabis for the IVM\ninvestigation\n23. For the retrospective study, FF and matched serum\nwere measured using the same procedure. Briefly, proteins were pre-\ncipitated using methanol (1:1 v/v), and the supernatants were assessed\nby LC-MS/MS using a QTRAP 5500 (SCIEZ, Concord, CA, USA) and\nAgilent 1290 HPLC (Agilent, Santa Clara, USA) with a calibration curve\n(0.001–200 ng) of known amount of the molecules of interest. Sam-\nples above the lower limit of quantification were considered positive.\nCannabinol (CBN) and Cannabidiol (CBD) were also measured in the\nsamples but were undetectable.\nOocyte in vitro maturation\nGV oocytes were received from consenting patients, randomly split into\nthree groups, and cultured using standard clinical IVM media (SAGE\nOne-step (CooperSurgical, Canada) + 7.5 IU/mL of Menopur (Ferring,\nCanada)): Ctrl (n = 96, only IVM media), THC1 ( n = 95, treated with a\nphysiological concentration of cannabis based on previous measure-\nments of cannabis in FF\n23:2 5 n g / m LΔ9-THC (Sigma-Aldrich, Canada),\n5 ng/mL 11-OH-THC (Sigma-Aldrich, Canada), 50 ng/mL 11-COOH-THC\n(Sigma-Aldrich, Canada)) and THC2 (n = 94, treated with THC at a con-\ncentration based on previous animal studies\n25,27,29:1 0 0 n g / m LΔ9-THC,\n50 ng/mL 11-OH-THC, 200 ng/mL 11-COOH-THC). Oocytes were\nobtained from 24 patients, and their demographic data are reported in\nthe Supplementary Table 1. Images using an inverted bright field\nmicroscope were taken prior and following incubation to assess oocyte\nmorphology. Oocytes were cultured for 24 h using the EVOS FL Auto 2\n(ThermoFisher, Canada) imaging system or cell culture incubator (5%\nCO\n2 and 37 °C). Timelapse images were taken every 15 min and used for\nthe assessment of GVBD and polar body extrusion (Ctrl: Supplementary\nvideo 1, THC1: Supplementary video 2 and THC2: Supplementary\nvideo 3). After 24 h of culture, oocytes were processed according to the\nspecific endpoint (RNAseq or immunostaining/polar body biopsies).\nSingle oocyte mRNA sequencing\nOocytes destined for single-cell RNASeq were further stripped of\nany residual cumulus cells and snap-frozen in 0.2 mL tubes in less\nthan 1 µL phosphate-buffered saline (1 X PBS). To reduce inter-\nsample variability, we selected patients with similar demographics\nand stimulation parameters (Supplementary Table 2). A total of 86\noocytes from 24 patients were selected for RNASeq, 28 in Ctrl, 27 in\nTHC1 and 31 in THC2. cDNA from single oocytes were synthesized\nusing the SMART-seq v4 Ultra Low Input RNA Kit (Takara Bio Inc.,\nJapan). The ampli fied cDNA was puri fied using Agencourt AMPure\nXP beads (Beckman Coulter, USA) and eluted. The puri fied cDNA\nwas quanti fied using the Qubit dsDNA high sensitivity assay (Ther-\nmoFisher, Canada) and length and molarity were assessed using the\nDNF-474 HS NGS Fragment Kit (1-6000 bp) with the Fragment\nAnalyzer 5200 (Agilent Technologies, USA). The ampli fied cDNA\n(0.2 ng) was used to construct sequencing libraries using a modi fied\nand miniaturized Nextera XT library preparation protocol (Illumina,\nCanada) developed for the use with the Mosquito HV liquid handling\nrobot (SPT labtech, Boston, USA). The quality of the libraries was\nassessed using the same Fragment Analyzer kit DNF-474 HS NGS\nFragment Kit (1 –6000 bp), quanti fied using Qubit dsDNA high sen-\nsitivity assay (ThermoFisher), normalized and pooled. Sequencing\nwas performed on a NovaSeq 6000 S2 flow cell (Illumina, Canada) at\nArticle https://doi.org/10.1038/s41467-025-63011-2\nNature Communications|         (2025) 16:8185 9\n\nPrincess Margaret Genomics Centre (2 × 150 bp) (Toronto, Canada).\nThe sequencing quality control metrics are compiled in the Sup-\nplementary Data 6.\nSingle oocyte RNASeq bioinformatics\nRaw sequencing reads were trimmed based on read quality (Phred >\n28) and aligned and quanti fied to hg38 using STAR (Spliced Tran-\nscripts Alignment to a Reference; v2.7.8a)102. Low abundant transcripts\nwere excluded (maximum <20) and normalized using the default\nnormalization method built into DESeq2 (v3.5)\n103. We conducted dif-\nferential expression (DE) using DESeq2 comparing THC1 vs. Ctrl and\nTHC2 vs. Ctrl. Significantly differentially expressed genes were defined\nas p-value < 0.05 and |log\n2fold change (FC)| of >1. The complete list of\nDE genes is available in the Supplementary data 1 (THC1 vs Ctrl) and the\nSupplementary data 2 (THC2 vs Ctrl)). This analysis was conducted in\nPartek Flow (version 11.0.23.1004). Gene Set Enrichment Analysis\n(GSEA)\n104,105 was conducted to determine what gene sets were impac-\nted by exposure to THC. The resulting pathway list was cross refer-\nenced with a custom gene set created and supported by the Bader Lab\n(University of Toronto) which is comprised of all GO database, KEGG,\nReactome, and Wiki pathways gene sets (v2024-01-01) ( http://\ndownload.baderlab.org/EM_Genesets/)\n106 (Supplementary Data 3) Sig-\nnificant pathways were de fined as having a |Normalized Enrichment\nScore (NES)| > 1.5 and p-value < 0.05.\nImmunostaining and imaging\nAfter 24 h in culture, the zona pellucida (ZP) was removed by incu-\nbating (30 s–2 min) with EmbryoMax® Acidic Tyrode’s Solution (Milli-\npore Sigma, CA) and polar bodies were mechanically separated from\nthe oocytes. Oocytes were immediately fixed with 3.7% paraf-\normaldehyde in PHEM (PIPES 12 mM, HEPES 5 mM, EGTA 2 mM and\nMgSO4・7H2O 0.8 mM, pH 6.9) for 30 min at room temperature (RT)\nand then permeabilized in PHEM + 0.25% Triton-X for 15 min at RT.\nAfter permeabilization, they were incubated overnight at 4 °C in\nblocking solution (3% bovine serum albumin (BSA) + 0.05% Tween-20\nin 1X PBS). On the next day, the plate was brought to RT before\ntransferring the oocytes in the primary antibody solution for 1 h at\n37 °C (mouse anti-ɑ-Tubulin (1:250), T6199, Sigma-Aldrich, USA). Then,\nthe oocytes were washed three times in the wash solution (0.5%\nBSA + 0.05% Tween-20 in PBS) at RT and moved in the secondary\nantibody solution for 2 h at 37 °C (goat anti-mouse AlexaFluor 488\n(1:200), A-11001, Invitrogen, USA and Phalloidin AlexaFluor 555 (1:500)\n(A34055, Invitrogen, USA). After secondary antibody solution, the\noocytes were washed three times in the wash solution and transferred\ninto Hoechst 33342 (1:500) for 30 min. Finally, the oocytes were\ntransferred into 1.5 µL drops in an imaging dish (Nunc Glass Bottom\nDish, ThermoScientific, CA) covered with paraf fin oil and 0.2 µm\nz-stacks were captured using Leica SP8 confocal microscope using the\n63× objective with oil and a zoom factor set at 8 at the Advanced\nOptical Microscopy Facility (Toronto, Canada). Deconvolution was\napplied on the images using Huygens software version 23.10 (https://\nsvi.nl/Huygens-Software) and images were visualized using Imar-\nisViewer 10.1.1. Negative controls consisted of GV oocytes to observe\nthe absence of spindle structure and stained MII-oocytes without the\nmouse anti-ɑ-Tubulin primary antibody (Supplementary Fig. 4).\nPB biopsy, whole genome amplification, sequencing and\nanalysis\nPolar bodies were separated from the oocytes after removal of the ZP\neliminating possible somatic cell contamination (as shown in Supple-\nmentary Fig. 3). Polar bodies were individually snap-frozen at−80 °C in\nless than 2 µL and blinded samples were sent for whole genome chro-\nmosome copy number variation assessment (CNV) to the CReATe\nReproductive Genetics sequencing platform. CNV analysis was per-\nformed by low-pass whole genome Next Generation Sequencing using\nvalidated clinical workflow on Illumina platform. Brie fly, gDNA was\namplified using SurePlex Whole Genome Amplification (WGA) (Illumina,\nCA), according to manufacturer’s instructions. Amplified gDNA was then\ntagmented and indexed using Nextera XT (Illumina, CA). The indexed\nlibraries were purified using AMPure XP beads (1:1 ratio) and normalized\nusing magnetic beads. The normalized libraries were pooled, denatured,\nand sequenced on a NextSeq 550 (paired end, 2 × 75 bp). NxClinical\nversion 6.0 (Bionano, CA) was used for chromosome CNV analysis and\ndata visualization according to our standard clinical procedure (2 mil-\nlion reads/sample, CNV resolution of >10 Mb). Optimization experi-\nmentations demonstrated the concordance between polar body\nchromosome numbers and its sister oocyte (Supplementary Fig. 3).\nStatistical analysis\nDatasets werefirst assessed for normality using the Shapiro–Wilk test.\nStatistical significance was determined using two-sided Fisher’se x a c t\ntest for contingency analysis (maturation rate, spindle morphology and\neuploid rates), One-way ANOVA with a two-sided Holm–Sidak’sm u l t i p l e\ncomparison test for continuous normally distributed datasets (oocyte\ndiameter), or Kruskal–Wallis with a two-sided Dunn’s multiple compar-\nison test for continuous non-normally distributed datasets. The specific\nstatistical test is indicated throughout table and figure legends. Sig-\nnificance was de fined as p < 0.05. For the retrospective analysis, we\nperformed pairwise case-control matching, where each THC-positive\nsample was matched to two THC-negative samples, as determined by\nt h ep r e s e n c e / a b s e n c eo f1 1 - C O O H - T H Ci nt h eF F .M a t c h i n gw a sc o n -\nducted using the FUZZY matching command in Statistical Package for\nSocial Sciences (SPSS-v29) based on the following covariates: oocyte\nage, participant body mass index (BMI), anti-müllerian hormone (AMH),\nday 2/3 luteinizing hormone (LH), and follicle stimulating hormone\n(FSH), (estradiol) E2 on trigger, and total gonadotropin (GT) dose. AMH,\nLH, FSH and E2 were quantified during the clinical assessment using the\nCobas e411 instrument (Roche, Basel, Switzerland). Matching success\nwas determined using a two-tailed Mann –Whitney test for non-\nparametric distribution. Statistical significance of the IVF outcomes\nwas determined using two-tailed Mann–Whitney test for non-parametric\ndistribution (fertilization and euploid rates) and unpaired two-tailed t-\ntest for parametric distribution (maturation and blastocyst rates) using\nGraphPad Prism 10.2.3. Numbers in parentheses in eachfigure legends\nrepresent distinct samples and not repeated measures.\nReporting summary\nFurther information on research design is available in the Nature\nPortfolio Reporting Summary linked to this article.\nData availability\nData generated in this study ar e provided in the Supplementary\nInformation/Source Data file. The normalized read count expression\nmatrix and metadata have been deposited in the Gene Expression\nOmnibus (GEO) database with accession number GSE297757.D u et o\nparticipant privacy concerns and institutional ethics restrictions, raw\nsequencingfiles (fastq) have been deposited at the European Genome-\nphenome Archive (EGA), which is hosted by the European Bioinfor-\nmatics Institute (EBI) and the Centre for Genomic Regulation (CRG),\nunder accession number EGAS50000001052. Further information\nabout EGA can be found at https://ega-archive.org. Expected time-\nframe for response to access requests is 10 business days and the data\nwill be available for the duration of the study as de fined by the Data\nAccess Agreement associated with this dataset. Source data are pro-\nvided with this paper.\nReferences\n1. Hansford, B. 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Gene set enrichment analysis: a knowledge-\nbased approach for interpreting genome-wide expression pro-\nfiles. Proc. Natl Acad. Sci. USA 102,1 5 5 4 5–15550 (2005).\n1 0 6 . M e r i c o ,D . ,I s s e r l i n ,R . ,S t u e k e r ,O . ,E m i l i ,A .&B a d e r ,G .D .\nEnrichment map: a network-based method for gene-set enrich-\nment visualization and interpretation.PLoS One 5, e13984 (2010).\nAcknowledgements\nC.D. received salary support through the University of Toronto Depart-\nment of Physiology Yuet Ngor Wong Postdoctoral Fellowship Award. All\nother funding was provided by CReATe Fertility Centre through the\nreinvestment of clinical earnings. Human biological materials and\nassociated de-identified data were collected by CReATe Biobank per-\nsonnel. The CReATe Biobank is certified by the Canadian Tissue Repo-\nsitory Network (CTRNet) Biobank Program. The authors would like to\nthank all, past and present, CReATe Biobank staff, CReATe Fertility\nCentre clinical and IVF laboratory staff, CReATe Reproductive Genetics\nstaff, and CReATe Research staff for their dedication to this study. Lastly,\nthe authors would like to thank all patients for the donation of their\nbiological material.\nAuthor contributions\nC.D., B.W. and N.F.W. participated in the design of the study. C.D. and\nB.W. established the collection ofimmature human oocytes through the\nCReATe Biobank in collaboration with IK and her team helping coordi-\nnate the oocyte collection. C.D. received, cultured and froze the oocytes\nfor all experiments. C.D. optimized and conducted oocyte staining and\nconfocal imaging, polar body biopsies, and RNA sequencing related\nmanipulations. C.D. and B.W. performed the transcriptomic analysis.\nC.D. and B.W. compiled the retrospective data and analyzed the results.\nS.M. and her team conducted the polar body sequencing to assess the\nploidy status. C.D. and B.W. wrote the manuscript. C.D. hand-drawn all\ngraphical elements. C.L. provided critical feedback on study design and\nthe manuscript. All authors provided feedback on the manuscript and\nread and approved the final manuscript.\nCompeting interests\nThe authors declare no competing interests.\nAdditional information\nSupplementary informationThe online version contains\nsupplementary material available at\nhttps://doi.org/10.1038/s41467-025-63011-2.\nCorrespondenceand requests for materials should be addressed to\nCyntia Duval.\nPeer review informationNature Communicationsthanks Norbert Glei-\ncher and the other, anonymous, reviewers for their contribution to the\npeer review of this work. A peer review file is available.\nReprints and permissions informationis available at\nhttp://www.nature.com/reprints\nPublisher’s note Springer Nature remains neutral with regard to jur-\nisdictional claims in published maps and institutional affiliations.\nOpen Access This article is licensed under a Creative Commons\nAttribution-NonCommercial-NoDerivatives 4.0 International License,\nwhich permits any non-commercial use, sharing, distribution and\nreproduction in any medium or format, as long as you give appropriate\ncredit to the original author(s) and the source, provide a link to the\nCreative Commons licence, and indicate if you modified the licensed\nmaterial. You do not have permission under this licence to share adapted\nmaterial derived from this article or parts of it. The images or other third\nparty material in this article are included in the article’s Creative\nCommons licence, unless indicatedotherwise in a credit line to the\nmaterial. 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