Cannabis impacts female fertility as evidenced by an in vitro investigation and a case-control study

THC concentrations correlate with maturation rate in IVF Using a retrospective case-control design and mass spectrometry, we quantified the concentration of Δ9-THC and its metabolites,11-OH- THC and 11-COOH-THC, in the FF of patients undergoing IVF treat- ment to determine the reproductive consequences of THC con- sumption. Figure 1a illustrates the proportion of THC and its metabolites measured in all samples ( n = 1059). Positivity rate was defined by the presence of 11-COOH-THC in the follicular fluid (62/ 1059, 6%). 11-COOH-THC was found alone in 13% of the samples (8/ 62) while Δ9-THC was co-detected in 37% of the samples (23/62) and 11-OH-THC co-detected in 2% (1/62). All three compounds were measured in 48% of the samples (30/62). Among the positive patients, 73% did not disclose their THC consumption on the patient intake questionnaire. The distribution of Δ9-THC and its metabolites showed a predominance of 11-COOH-THC (mean = 28.8 ng/mL), fol- lowed by Δ9-THC (mean = 7.5 ng/mL), with 11-OH-THC being the least abundant (mean = 1.7 ng/mL) (Fig. 1b). Notably, concentrations of these metabolites did not differ between FF and matched serum samples obtained at the time of oocyte retrieval (Fig. 1c). A Spearman correlation analysis identi fied signi ficant correla- tions between THC metabolite concentrations and various clinical and biochemical parameters (Fig. 1d). Specifically, concentrations of Δ9-THC, 11-OH-THC and 11-COOH-THC were positively correlated with oocyte maturation rate in the THC-positive group ( Δ9-THC: ⍴ = 0.370, p = 0.003; 11-OH-THC: ⍴ = 0.309, p = 0.014 and 11-COOH- THC: ⍴ = 0.295, p = 0.020). Interestingly, Δ9-THC levels were nega- tively correlated with a patient ’s Body Mass Index (BMI) ( ⍴ = −0.539, p = 0.000053). In vitro THC exposure and oocyte maturation Patients undergoing IVF treatment and oocyte retrieval who consented for the collection of IVF waste material (immature oocytes, somatic cells and FF) and de-identified clinical data were included in this study. For each patient, a minimum of three immature oocytes at the germ- inal vesicle (GV) stage were collected following the removal of somatic cells. GV oocytes were cultured using our standard in vitro maturation (IVM) protocol for 24h 32 (control group (Ctrl), n = 96) or with the addition of THC (treatment groups). Oocytes were treated with either a physiologically relevant (THC1,n = 95, 25 ng/mLΔ9-THC, 5 ng/mL 11- OH-THC, 50 ng/mL 11-COOH-THC) or a supraphysiologic (THC2, n =9 3 ,1 0 0n g / m LΔ9-THC, 50 ng/mL 11-OH-THC, 200 ng/mL 11-COOH- THC) concentration where THC1 is based on the concentration ofΔ9- THC and its metabolites measured in the follicularfluid of IVF patients and THC2 is based on previously reported concentrations in animal studies 23,25,29. Subsequently, oocytes were classi fied based on their progression through key maturation checkpoints: germinal vesicle (GV) and Metaphase-I (MI) (after germinal vesicle breakdown (GVBD) and before polar body extrusion) were considered immature oocytes, while Metaphase-II (MII) oocytes (after visible polar body extrusion) were considered mature (Fig.2a). Maturation rate was then calculated per treatment group. Oocytes treated with THC1 showed no signi ficant change in maturation rate (49/95, 52%,p = 0.6704), while THC2 exhibited a non- significant trend toward increased maturation (54/93, 58%,p = 0.1098), compared to Ctrl (44/96, 46%) (Fig. 2b). Utilizing timelapse imaging, oocyte morphology assessments were performed pre-IVM (Ctrl:n =9 2 , THC1: n = 89 and THC2:n = 85) and post-IVM (Ctrl:n =9 1 ,T H C 1 :n =8 8 , and THC2: n = 83), and key maturation events were recorded: GVBD (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 polar body (Ctrl: n = 28, THC1: n =3 0 a n d T H C 2 :n = 31). Examples of timelapse IVM images are provided in Supplementary Fig. 1 and cor- responding videos are provided as Supplementary videos (Ctrl-Sup- plementary video 1, THC1-Supplementary video 2 and THC2- Supplementary video 3). There were no signi ficant differences in oocyte diameter between treatment groups either before (Ctrl: 110.6 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 ) (Fig. 2c) or after 24 h of culture (Ctrl: 110.2 μm, THC1: 110.0 μm, p = 0.7416 and THC2: 108.8 μm, p =0 . 1 0 6 6 ) . ( F i g .2d). Similarly, the timing of GVBD (Fig. 2e) and polar body extrusion (Fig. 2f) remained unaffected by THC exposure. Demographic data of patients included in these analyses can be found in Supplementary Information - Sup- plementary Table 1. THC exposure alters the oocyte transcriptome Single MII oocytes with good morphology and normal develop- mental progression were sequenced using our optimized ultra-low input RNA sequencing pipeline 33 (n = 24 patients/n = 86 metaphase-II (MII) oocytes (28 Ctrl, 27 THC1 and 31 THC2). Differential expression analysis revealed 89 genes up-regulated and 227 genes down- regulated greater than 2-fold (|log 2FC| > 1) and p < 0.05 (Fig. 3a) when assessing the impact of the THC1 vs Ctrl (Supplementary Data 1). Gene Set Enrichment Analysis (GSEA) identi fied that upre- gulated genes were principally associated with positive regulation of synaptic transmission, axonemal dynein complex assembly, and glutamate receptor signaling pathway, while the downregulated genes were associated with protein synthesis, expression regulation of SLITS and ROBOS and in flammatory processes (Fig. 3b, Supple- mentary Data 3). THC2 exposure induced a greater magnitude of transcriptomic dysregulation, with 402 up-regulated and 62 down- regulated genes identi fied (Fig. 3c, Supplementary Data 2). The upregulated genes were associated with the immune system and apoptotic pathways while downregulated genes were associated with attachment of spindle microtubules to kinetochores and in flamma- tory processes (Fig. 3d, Supplementary Data 3). As illustrated by the Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 2 Venn diagram (Fig. 3e), THC1 exposure had 266 speci fic DEGs, while THC2 exposure had 414, with 50 being common to both treatment groups (gene lists are available in Supplementary Data 4). Of these 50 common DEGs, 32 were protein-coding, 19 were up-regulated ( EPYC, RGS5, N4BP2L1, KRT19, PRR20G, KL, KCND3, ALDH3A1, SLC1A3, TSPAN8, PLAU, COL8A2, TFAP2E, SPTSSB, BRINP3, VANGL2, RGS18, RXFP1,a n d KCNMB3), 10 were down-regulated ( OR4F15, MMP9, PRRX2, IRS4, INFG, CCIN, IL33, NEUROD1, MT1HL1 ,a n d MT1H), and 3 displayed bidirectional changes ( S100B, ACTA1 ,a n d ARHGEF19) (Fig. 3f) (Detailed information available in Supplementary Data 5). Demographic data of patients included in these analyses can be found in Supplementary Information - Supplementary Table 2 and Sequencing Quality Control metrics can be found in Supplemen- tary Data 6. THC is harmful to chromosome segregation Subsets of MII oocytes from both the control and THC-treatment groups were used to assess polar body ploidy status (18 Ctrl, 21 THC1, and 21 THC2) and for spindle morph ology (12 Ctrl, 12 THC1, and 12 THC2). Removal of the zona pellucida (ZP) and subsequent polar body biopsy (Supplementary Fig. 2) allowed for some oocytes to be used for ploidy determination by low-pass whole genome Next-Generation Sequencing (NGS) aneuploidy using VeriSeq PGT-A which is specia- lized in detecting aneuploidy in reproductive samples 21,34 (Supple- mentary Fig. 3) and meiotic spindle organization by confocal microscopy allowing for precise visualization of spindle organization and chromosome alignment (Fig.4a). Both THC1 and THC2 treatment led to a 9% increase in aneuploidy (Ctrl: 39%, THC1 and THC2: 48%, p = 0.7479) (Fig. 4b) and a higher proportion of complex aneuploidy, defined by the gain or loss of more than three chromosomes 35 (Ctrl: 0%, THC1 and THC2: 42%, p = 0.1029) (Fig. 4c). Figure 4dr e p o r t sa subset of oocytes where both ploidy status and spindle morphology were assessed ( n = 17), without stratifying by treatment group. The majority of oocytes that completed meiosis I displayed normal spindle morphology (euploid: n = 8/13, 62% and aneuploid: n = 3/4, 75%, p > 0.9999) (Fig. 4d), but not all. The hallmark characteristics of 0 5 10 15 20 50 100 150 200Concentration (ng/mL) Δ9-THC 11-OH-THC 11-COOH-THC Serum Follicular fluid ab cd Δ9-THC 11-OH-THC 11-COOH-THC 0.1 1 100 1000 10 Concentration (ng/mL) -1.0 -0.5 0 0.5 1.0 Δ9-THC 11-OH-THC 11-COOH-THC Age BMI Day 2/3 LH Day 2/3 FSH Day 2/3 FSH E2 on trigger Total GT Mat. Rate Fert. Rate Blast. Rate Euploid Rate Δ9-THC 11-OH-THC 11-COOH-THC Age BMI Day 2/3 LH Day 2/3 FSH Day 2/3 FSH E2 on trigger Total GT Mat. Rate Fert. Rate Blast. Rate Euploid Rate Spearman ρ *** *** *** ** *** * * * *** * ** * ** *** * Positives (n=62) 11-COOH-THC 8 (13%) 00 30 (48%) 1 (2%) 23 (37%) 0 11-OH-THC Δ9-THC Fig. 1 | Tetrahydrocannabinol concentrations and correlation with demo- graphic data and clinical in vitro fertilization outcomes. aProportion of folli- cular fluid (FF) samples positive for Δ9-THC, 11-OH-THC and 11-COOH-THC (presence of 11-COOH-THC=positive sample).b Distribution ofΔ9-THC (n =5 3 ) ,1 1 - OH-THC (n = 31), and 11-COOH-THC (n = 62) concentrations in FF (data presented as median with interquartile range) andc concentrations ofΔ9-THC, 11-OH-THC, and 11-COOH-THC in FF and matched serum samples (data presented as mean ± standard deviation, fromn = 3 individual participants with matched serum samples (dotted fill pattern) and FF samples (plain)). d Correlation matrix of clinical and biochemical parameters in THC-Positive group (n = 62). The colors represent the two-sided Spearmanρ value *p < 0.05, **p < 0.01, ***p < 0.001. AMH Anti-Müllerian Hormone, BMI Body Mass Index, LH Luteinizing Hormone, FSH Follicle Stimulating Hormone, E2 Estradiol, GT Gonadotropins, THC Tetrahydrocannabinol, Mat. Rate Oocyte maturation rate, Fert. Rate Fertilization rate, Blast. Rate Blastulation rate. Source data are provided as a Source Data file. Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 3 “normal” meiotic spindles include bipolar barrel-shaped microtubules with the chromosomes aligned on the metaphase plate 36. Whereas “abnormal” spindles are varied in their morphology and may include multipolar spindles, alterations in microtubule organization, and misaligned chromosomes 36. Spindle disorganization and chromosome misalignment are shown by representative images in Fig. 4e, where oocytes were classified as having either “normal” or “abnormal” spin- dles. The proportion of oocytes with abnormal spindles was higher in the THC exposed groups compared to control (Ctrl (5/12), THC1 8/12), and THC2 (11/12), with a signi ficant increase in THC2 (Ctrl: 42% and THC2: 92%, p = 0.0272) (Fig. 4f). (Spindle immunostaining negative controls ca be found in Supplementary Fig. 4) THC decreases embryo euploidy rate in IVF Following pairwise case-control matching, where each THC-positive sample was matched to two THC-negative samples, a signi ficant decrease in embryo euploidy rate was observed in the THC-positive group (n = 51, 60.0%), compared to the THC-negative group ( n =1 0 1 , 67.0%, p = 0.0245) (Table 1). There was no signi ficant change in maturation, fertilization and blastocyst rates (Table1). To further evaluate the likelihood of adverse IVF outcomes, we conducted multiple logistic regression analyses, focusing on clini- cally relevant IVF outcome thresholds 37: maturation rate (80%), fertilization rate (70%), blastocyst rate (50%) and euploidy rate (60%). We utilized backward stepwise logistic regression, including the following covariates: oocyte age, participant body mass index (BMI), anti-müllerian hormone (AMH), day 2/3 luteinizing hormone (LH), and follicle stimulating hormone (FSH), (estradiol) E2 on trig- ger, and total gonadotropin (GT) dose. The final model for both blastulation and euploidy rates retained THC status as a signi ficant explanatory variable, with oocyte age being a signi ficant covariate. In this pairwise matched cohort, THC positivity signi ficantly bcd In vitro maturation for 24h (SAGE + 75IU/μL Menopur) GV MI MII (mature)Clinical IVF treatment Treatments (ng/mL) Δ9- THC 11-OH- THC 11-COOH- THC Ctrl 0 0 0 THC1 25 5 50 THC2 100 50 200 a GV MI MII Ctrl THC1 THC2 0 20 40 60 80 58% 52% 46% Maturation rate 24h (%) 96 95 93 Ctrl THC1 THC2 90 100 110 120 130Diameter (μm) post IVM Time (hours) % of oocytes at GVBD 10 12 14 16 18 20 22 24 0 10 20 30 40 50 Time (hours) % of oocytes at MII THC2 THC1 Ctrl Ctrl THC1 THC2 90 100 110 120 130Diameter (μm) pre IVM 0 4 8 1 21 62 02 4 0 10 20 30 40 50 Time (hours) Ctrl THC2 THC1 e f Fig. 2 | Impact of tetrahydrocannabinol exposure on oocyte maturation. a Experimental design and in vitro oocyte maturation.b Maturation rate as the percentage of germinal vesicle (GV) oocytes that matured (progressed to metaphase-II (MII)) rate (Ctrl (44/96, THC1: 49/95, THC2: 54/93). Oocyte diameter (c) prior to exposure to THC (Ctrl: n = 92, THC1: n = 89, THC2: n =8 5 )a n dd after 24 h of culture (Ctrl:n =9 1 ,T H C 1 :n = 88, THC2: n = 83). Proportion of oocytes that underwent key maturation events after 24 h of culture: e germinal vesicle break- down (GVBD) (Ctrl: n = 71, THC1: n = 72, THC2: n = 64) and f extrusion of the first polar body (MII-arrested stage) (Ctrl:n = 28, THC1: n = 30, THC2: n =3 1 ) .E r r o rb a r s represent the mean ± standard deviation. Significance was assessed by two-sided Fisher’s exact test or One-way ANOVA with two-sided Holm–Sidak multiple com- parison test (ns not significant). IVF In vitro fertilization, IVM In vitro maturation, MII metaphase II, MI metaphase I, GV germinal vesicle, GVBD germinal vesicle breakdown, FF follicularfluid, THC tetrahydrocannabinol. Source data are pro- vided as a Source Data file. Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 4 decreased the odds of reaching a 50% blastulation rate or above (odds ratio: 0.45, p = 0.018) and the odds of achieving a euploidy rate above 60% (odds ratio: 0.47, p = 0.038) (Table 2). Age was also found to signi ficantly impact blastulation and euploidy rates, with an odds ratio of 0.9144 ( p = 0.0010) and 0.9150 ( p = 0.0024), respectively. The models for predicting blastulation rate (>50%) and euploidy rate (>60%) demonstrated positive predictive power, with areas under the curve (AUCs) of 0.68 and 0.67, respectively (Sup- plementary Fig. 5).

Understanding the impact of environmental factors and lifestyle choices on female fertility is crucial for proper patient counseling. With cannabis being one of the most commonly used recreational drugs in the world 1, it is critical to holistically evaluate its impact on mental and general health, in addition to reproductive health. This study, using donated human oocytes and an integrated multi- disciplinary approach, reveals that exposure to THC affects oocyte maturation, transcriptome, and induces meiotic chromosomal imbalances associated with altered spindle morphology. Moreover, our retrospective analysis revealed that exposure to THC was associated with signi ficantly lower embryo euploidy rate, likely par- tially explained by an altered chromosomal organization as demon- strated in the in vitro matured MII oocytes. In our retrospective study, we measured THC concentrations in 1059 follicular fluid samples from patients undergoing IVF treatment at CReATe Fertility Centre (Toronto, Canada) in a retrospective matched case-control cohort. Sixty-two samples tested positive for 11-COOH-THC, resulting in a 6% positivity rate (Fig. 1a). This rate is considerably lower than what was reported by a recent Health Canada survey where 23% of females reported recreational cannabis consumption within 1 year of being surveyed 38. However, these patients were counseled pre-treatment not to use recreational drugs while undergoing IVF. The relative concentrations of Δ9-THC, 11-OH- THC, and 11-COOH-THC are consistent with metabolism of THC in the liver (Fig. 1b). Δ9-THC (half-life 1.3–10 days) is metabolized to 11- OH-THC (half-life 20 min–2 h) and 11-OH-THC is rapidly metabolized to 11-COOH-THC (half-life 3–5 days), which remains in the circulation for up to 30 days 39. The consistent concentrations of THC metabo- lites in both the follicular fluid and serum suggests passive diffusion or transudation from the bloodstream into follicularfluid rather than active transport into or out of the follicle (Fig. 1c). c -3 -2 -1 0 1 2 REGULATION OF CD8-POSITIVE T CELL ACTIVATION PHOTODYNAMIC THERAPY INDUCED AP 1 SURVIVAL SIGNALING RRNA MODIFICATION IN THE NUCLEUS AND CYTOSOL APOPTOSIS BIOCARTA_TNFR1_PATHWAY ANTIGEN PROCESSING AND PRESENTATION OF ENDOGENOUS PEPTIDE ANTIGEN PROSTANOID BIOSYNTHETIC PROCESS APOPTOSIS MODULATION AND SIGNALING PID_HDAC_CLASSIII_PATHWAY REGULATION OF SMOOTH MUSCLE CELL MIGRATION PRE IMPLANTATION EMBRYO CLATHRIN-DEPENDENT ENDOCYTOSIS INTERLEUKIN RECEPTOR SHC SIGNALING PHOSPHATIDYL INOSITOL PHOSPHATE PATHWAY REGULATION OF CHROMATIN ORGANIZATION EXPRESSION AND TRANSLOCATION OF OLFACTORY RECEPTORS REGULATION OF T-HELPER 17 TYPE IMMUNE RESPONSE REGULATION OF DENDRITIC SPINE MORPHOGENESIS RESPONSE TO ZINC ION ATTACHMENT OF MITOTIC SPINDLE MICROTUBULES TO KINETOCHORE THC2 vs Ctrl NES -3 -2 -1 0 1 2 3 POSITIVE REGULATION OF SYNAPTIC TRANSMISSION AXONEMAL DYNEIN COMPLEX ASSEMBLY GLUTAMATE RECEPTOR SIGNALING PATHWAY POLY(A)+ MRNA EXPORT FROM NUCLEUS FAS SIGNALING PATHWAY ( CD95 ) SUPERPATHWAY OF INOSITOL PHOSPHATE COMPOUNDS DIOL BIOSYNTHETIC PROCESS METABOLISM OF COFACTORS CELLULAR RESPONSE TO NERVE GROWTH FACTOR STIMULUS NUCLEIC ACID TRANSPORT SYNDECAN-1-MEDIATED SIGNALING EVENTS POSITIVE REGULATION OF INTERLEUKIN-6 PRODUCTION POSITIVE REGULATION OF CD4-POSITIVE T CELL DIFFERENTIATION OXIDATIVE PHOSPHORYLATION ATP BIOSYNTHETIC PROCESS CELLULAR RESPONSE TO STARVATION POSITIVE REGULATION OF TUMOR NECROSIS FACTOR PRODUCTION NONSENSE-MEDIATED DECAY (NMD) REGULATION OF EXPRESSION OF SLITS AND ROBOS PROTEIN SYNTHESIS THC1 vs Ctrl NES d ab e EPYC RGS5 N4BP2L1 KRT19 PRR20G KL KCND3 ALDH3A1 SLC1A3 TSPAN8 PLAU COL8A2 TFAP2E SPTSSB BRINP3 VANGL2 RGS18 RXFP1 KCNMB3 OR4F15 MMP9 PRRX2 IRS4 IFNG CCIN IL33 NEUROD1 MT1HL1 MT1H S100B ACTA1 ARHGEF19 THC1vsCtrl THC2vsCtrl 0 5 10 f THC1 THC2 Total = 266 62↑ 204↓ Total = 414 369↑ 45↓ Total = 50 Up-regulated (89) Down-regulated (227) Not significant (20802) Up-regulated (402) Down-regulated (62) Not significant (20654) Fold change Fig. 3 | Effect of tetrahydrocannabinol exposure on oocyte transcripts after oocyte in vitro maturation.Volcano plots of differentially expressed genes (DEGs) comparing (a)T H C 1v sC t r la n db pathway analysis by gene set enrichment analysis (GSEA) THC1 vs Ctrl. Volcano plots of DEGs comparingc THC2 vs Ctrl andd GSEA of the comparison THC2 vs Ctrl.e Venn diagram of DEGs in THC1 and THC2 compared to Ctrl. f Common protein-coding DEGs, colors representing fold change. DEGs were defined by p 1, n = 24 patients/n = 86 metaphase-II (MII) oocytes (28 Ctrl, 27 THC1 and 31 THC2). Significant pathways were defined as having a nor- malized enrichment score (|NES|) > 1.5 andp < 0.05 (two-sided permutation test resulting in unadjusted p-values). Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 5 In a clinical IVF setting, embryologists and physicians assess mature oocytes based on the extrusion of thefirst polar body30,40.T h i s process, known as nuclear maturation, marks the oocyte’sp r o g r e s s i o n to metaphase-II and is considered the final stage of oocyte maturation30,41. When we compiled the IVF outcomes of our retro- spective cohort, we observed a positive correlation between THC metabolites and oocyte maturation (Fig. 1d). This suggests that the levels of THC and its metabolites present in this cohort may support nuclear maturation through an unknown mechanism. Parallel to our clinical retrospective study, we treated pairwise- matched immature oocytes from patients who were negative for THC (naive GVs) with THC in vitro and we observed an increased proportion of oocytes which achieved the MII stage (Fig. 2b). Given that factors such as oocyte diameter have been previously reported to in fluence nuclear maturation 42,w ec o nfirmed that there were no differences in oocyte size distribution across the three groups (Ctrl, THC1 and THC2) before and after culture for 24 h (Fig.2c, d). Finally, we also monitored and recorded the timing of key maturation events using timelapse imaging technology and showed that the GVBD occurred slightly faster in THC treated oocytes (Fig. 2e), but this did not reach signi ficance, and there were no differences in the timing of the first polar body extrusion between THC-exposed and Ctrl groups (Fig. 2f). Previous animal studies investigated the impact of THC on oocyte maturation using bovine oocytes 29, which is considered a better proxy to human oocyte maturation when compared to murine model due to similar temporal dynamics. López-Cardona et al. reported a 15% increase in maturation rate after treating oocytes with 0.1 µM (31.45 ng/mL) Δ9- THC for 12 h ( n =3 0 / g r o u p ) 29. In contrast, a more recent and larger study ( n = 164/group) concluded that a 24 h treatment with ‘recrea- tional cannabis doses ’ of 0.32 µM (100.63 ng/mL) and 3.2 µM (1,006.30 ng/mL) of Δ9-THC significantly reduced oocyte maturation from 80.1% to 65.3% and 60.1%, respectively25. Of note, the latter study used higher doses of Δ9-THC than what is measured in the FF of our patients but neither study examined the combined effects ofΔ9-THC and its metabolites, which might alter its effect on the growing oocyte. Collectively, our results suggest that THC exposure, at both e f b Hoechst 33342/α-tubulin/Phalloidin Normal Abnormal cd In vitro maturation for 24h (SAGE + 75IU/μL Menopur) GV MI MII Treatments (ng/mL) Δ9- THC 11-OH- THC 11-COOH- THC Ctrl 0 0 0 THC1 25 5 50 THC2 100 50 200 a Remove ZP Split PB and Oo + PB Oocyte WGA Immunofluorescence Frozen at -80 °C Euploid Aneuploid 0 5 10 15Number of oocytes Normal spindles Abnormal spindles 38% 62% 75% 25% Ctrl THC1 THC2 0 5 10 15 20Number of oocytes Normal Abnormal ✱ 42% 58% 66% 33% 92% 8% Ctrl THC1 THC20 5 10 15 20 25Number of oocytes Euploid Aneuploid 39% 52% 52%61% 48% 48% Ctrl THC1 THC2 0 5 10 15Number of aneuploid oocytes 1 2 ≥3 57% 20% 40% 43% 40% 20% 40%40% 0% 5μm 5μm5μm Fig. 4 | Impact of tetrahydrocannabinol exposure on oocyte spindle mor- phology and ploidy status. aExperimental design for polar body sequencing and oocyte immunostaining.b Proportion of euploid oocytes deduced by polar body biopsy sequencing results in Ctrl (11/18), THC1 (11/21), and THC2 (11/21). c Proportion of aneuploid oocytes with a gain/loss of one chromosome (white), two chromosomes (gray) or three and more (black) in Ctrl (1 chromosome: n =4 ,2 chromosomes:n =3 , ≥3 chromosomes: n =0 ,t o t a ln = 7), THC1 (1 chromosome: n = 2, 2 chromosomes: n =4 , ≥3 chromosomes: n =4 ,t o t a ln = 10) and THC2 (1 chromosome: n = 4, 2 chromosomes: n =2 , ≥3 chromosomes: n =4 ,t o t a ln = 10). d Proportion of normal morphology spindles in euploid (8/13) and aneuploid (3/4) oocytes. e Representative images of normal (n = 12) and abnormal (n =2 4 ) metaphase-II-arrested oocyte spindles, chromosomes (Hoechst)are in blue, spin- dles (α-tubulin) are in green and cell membrane (Phalloidin) in red. Scale bar: 5µm. f Proportion of oocytes with abnormal spindles in Ctrl (5/12), THC1 (8/12), and THC2 (11/12,p =0 . 0 2 7 2 ) .S i g n ificance was assessed using two-sided Fisher’s exact test. GV germinal vesicle, MI metaphase I, MII metaphase II, Oo oocyte, PB polar body, THC tetrahydrocannabinol, WGA whole genome amplification, ZP zona pellucida. Source data are provided as a Source Data file. Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 6 physiological and supraphysiological concentrations, appears to accelerate oocyte maturation speed and completion, consistent with

from the L όpez-Cardona bovine study 29 and previous mouse studies26. Oocytes must not only successfully progress through meiosis II to reach metaphase-II but also need suf ficient time to reach cyto- plasmic maturity to support early embryo development 30,41.T h i s process involves the precise and faithful packaging of various com- ponents, including maternal mRNA transcripts. mRNA production and storage in the ooplasm is critical, since, following establishment of the germinal vesicle, chromatin is condensed, and transcription is halted 43,44. These transcripts are critical not only for meiosis resumption and fertilization, but also during the first 3 days of embryonic development 45, since the early embryo remains tran- scriptionally silent, relying entirely on maternally inherited mRNA from the ooplasm to drive cellular processes prior to embryonic genome activation 46. The selection and enrichment of critical tran- scripts in the ooplasm have been described in the literature and are regulated by post-transcriptional mechanisms. These complex pro- cesses involve a sophisticated network of RNA binding proteins (RBPs), polyadenylation factors and RNA translational and degrada- tion machinery, which all regulate the storage, translation, and degradation of oocyte mRNAs 46. To understand how THC affects stored maternal mRNA during oocyte maturation, we identified genes associated with transcripts that were differentially expressed following THC exposure (Fig. 3a, c). Focusing on the common protein-coding transcripts, 32 genes were identified and grouped into nine principal functions: G protein- coupled receptor (GPCR) signaling, extracellular matrix regulation, embryogenesis, cell-cell communication, inflammation, cytoskeleton, detoxification, transcription factor and ion channels (Supplemen- tary Data 5). Among these,MMP9 was significantly downregulated in both THC treatment groups. MMP9 encodes for matrix metalloproteinase 9 (MMP-9) essential for local proteolysis of the extracellular matrix and leukocyte migration 47–49. In animal models, MMP-9 has well- established role in ovulation and follicle rupture 50–52, and in mice, protein expression in blastocyst and early embryo is critical for implantation 53–58. In humans, several studies have investigated its role in implantation using various trophoblast and implantation models59–61. Dysregulation of its expression and activity is associated with pregnancy complications and recurrent pregnancy loss 62–65. Moreover, THC has also been shown to decrease MMP-9 expression in human amniotic epithelium 66 and endothelial cancer cells 67.T h u s , downregulation ofMMP9 in the ooplasm may negatively contribute to key ovulation events needed for fertilization, embryo development, and implantation. G-protein coupled receptor (GPCR) signaling was also dysregu- lated following exposure to THC. GPCR signaling is crucial for oocyte growth and maturation, and many GPCRs are present at the oocyte surface, including the cannabinoid receptor 1 (CB1) and 2 (CB2) 27,68. Genes coding for Regulators of G protein Signaling (RGS), RGS5 and RGS18,w e r es i g n ificantly upregulated following THC treatment in our study. These regulators are known modulate GPCR signal transduction 69. RGS5 and RGS18 belong to the RGS R4 family and have been shown to bind with the G α proteins, thus reducing their Table 1 | Demographic data and in vitro fertilization outcomes of pairwise matched patients negative and positive for tetrahydrocannabinol Negative (n = 124) Median (IQR) Positive ( n = 62) Median (IQR) p-value THC concentrations Δ9-THC (ng/mL) – 5.3 (9.6) 11-OH-THC (ng/mL) – 0.1 (2.3) 11-COOH-THC (ng/mL) – 18.1 (29.1) Age (years)a 30.0 (9.0) 29.5 (11) 0.4863 BMI (kg/m2) 23.4 (4.8) 24.3 (8.3) 0.9709 AMH (pmol/L) 24.4 (21.1) 25.8 (30.1) 0.7117 Day 2/3 LH (IU) 1.2 (2.7) 2.1 (3.3) 0.0275 Day 2/3 FSH (IU) 6.9 (2.5) 6.4 (3.2) 0.5714 E2 on trigger (pmol/L) 13,445 (14,553) 12,603 (9,996) 0.5473 Total GT (IU) 4,088 (1,003) 3,894 (1,025) 0.2998 Maturation rate (%) a 72.0 (22.0) 76.0 (21.5) 0.2200 Fertilization rate (%) 82.5 (17.2) 83.0 (19.0) 0.7202 Blastocyst rate (%) a 59.5 (27.2) 50.0 (40.0) 0.5128 Euploidy rate (%) 67.0 (22.0) 60.0 (26.0) 0.0245 Normality was tested using the Shapiro–Wilk test. AMH Anti-Müllerian Hormone,IQR Interquartile range,THC Tetrahydrocannabinol,BMI Body Mass Index,LH Luteinizing Hormone,FSH Follicle Stimulating Hormone,E2 Estradiol,GT Gonadotropins. aIndicates 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. Table 2 | Multiple logistic regression models for blastulation and euploidy rates Outcomes Covariates Coef ficient (β) SE p-value OR 95% CI for OR Lower Upper Blastulation rate >50% THC+ (Case) −0.803 0.338 0.018 0.448 0.229 0.864 Oocyte age −0.089 0.027 0.001 0.914 0.866 0.963 Euploidy rate >60% THC+ (Case) −0.759 0.365 0.038 0.468 0.226 0.952 Oocyte age −0.089 0.029 0.002 0.915 0.862 0.968 Significance was assessed using Likelihood Ratio Test (LRT) SE Standard error, CI Coefficient intervals,OR Odds ratios, THC Tetrahydrocannabinol Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 7 inhibitory activity70,71. They have been shown to have a crucial role in the cell processing of the signal coming from the GPCRs 72. Although the exact role of RGS5 and RGS18 in oocyte maturation is poorly understood, their upregulation suggests a potential impact on GPCR signaling pathways in response to THC stimulation. Immune pathways were also overrepresented in both treatment groups compared to controls. For instance, IFNG and IL33 were both significantly downregulated in THC exposed groups. It is well estab- lished that tight regulation of in flammatory processes is critical for implantation of the embryo in the endometrium73. Interferon-γ (IFNγ) is a cytokine widely known for its role in in flammation and the acti- vation of macrophages74. Although its role during oocyte maturation is unclear, IFNγ secretion by the conceptus is essential for implantation in animal models75. On the other hand, Interleukin-33 (IL-33), a cyto- kine that belongs to the IL-1 family, binds to the ST2 (IL-1RL1) receptor76.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 ST2 in granulosa cells and the uterus 77. In humans, altered levels or expression of IL-33 or ST2 in various tissue and sample types were associated with pregnancy complications like preeclampsia 78–80, pre- term birth81–83, intrauterine growth restriction84, and miscarriage85.T h e downregulation of IL-33 we observed in THC-exposed oocytes sug- gests there may be potential disruption in the inflammatory processes necessary for successful pregnancy. As i g n ificant number of DEGs in THC-exposed oocytes are involved in the cytoskeletal function, includingKRT19, COL8A2, ACTA1 and ARHGEF19 (Supplementary Data 5). In addition to these tran- scriptomic changes, we observed significant alterations to cytoskele- ton machinery throughout this study. Indeed, the oocyte ’s cytoskeleton plays a crucial role in chromosome alignment, segrega- tion, and polarity establishment 86 and without appropriate formation and regulation of key cytoskeletal functions, the oocyte is vulnerable to chromosomal abnormalities. However, the cytoskeleton-associated DEGs identified in this study have not been previously characterized in oocyte development and thus require further investigation to gain a deeper understanding of the effects of THC on these processes. Taken together, THC exposure seems to impact critical tran- scripts involved in key oocyte maturation processes, fertilization, early embryo development and implantation. While these transcriptomic alterations likely result from post-transcriptional processes, the spe- cific mechanisms by which THC affects these processes in human oocytes remains unknown 46. Given the observed increase in nuclear maturation rate and the altered transcriptomic profiles related to chromosome organization, we next investigated the impact of THC on chromosome segregation. Indeed, errors in chromosome segregation during the first meiotic division is the most frequent cause of embryo aneuploidy 87, making the faithful establishment of chromosome segregation machinery a critical bottleneck in the production of a chromosomally normal embryo 88,89. To investigate oocyte ploidy, we performed polar body biopsy and low-pass whole genome sequencing. Notably, we observed that THC exposure led to a 9% increase in aneuploidy rates (Fig. 4b). Additionally, we observed an increase in the proportion of oocytes with complex aneuploidies (defined as a gain or a loss of 3 or more chromosomes) 35 in the THC-treated group compared to controls (Fig. 4c). Aneuploidies are associated with implantation failure, mis- carriage, and are incompatible with life90. It has been postulated that most aneuploidies arise from errors in maternal meiosis I91–96,b u to u r data suggest that meiosis II may also be sensitive to perturbations as 38% of the euploid oocytes had abnormal spindle morphology (Fig.4d) determining using confocal microscopy. We assessed spindle mor- phology after 24 h incubation of oocytes with and without THC. A normal spindle con figuration is barrel shaped with chromosomes aligned at the metaphase plate, while ‘abnormal’ configurations include multipolar spindles and misaligned chromosomes 97 (Fig. 4e). In this study, we demonstrated a dose-dependent decrease in the proportion of oocytes with normal spindle morphology following THC exposure (Fig. 4f). Correct chromosome segregation during oocyte maturation is essential for producing euploid embryos, which have the highest chance of establishing a healthy pregnancy 98. To address the primary clinical question regarding THC’s impact on IVF outcomes, we used a pairwise case-control matching strategy, where each positive sample was matched to two negative samples based on demographic data, and we compiled the resulting matched cohort’s IVF outcomes (Table 1). THC exposure was associated with a marginal increase in maturation rate (Table 1), concordant with what was obtained in our in vitro experimentations (Fig. 2b). Further, a significant decrease in embryo euploidy rate (Table 1) and reduced odds of obtaining a euploidy rate above 60% was observed (Table 2). These results indicate that THC-positive patients may have fewer euploid embryos from their IVF cycle and may experience a longer time to pregnancy. To deepen our understanding of our findings, we must extra- polate what is known about THC interactions and pathways from other cell types. THC primarily elicits its functions through binding the cannabinoid 1 and 2 receptors (CB1 and CB2), which are expressed at all stages of oocyte maturation 68. CB1 and CB2 are G protein-coupled receptors (GPCRs) which are capable of inhibiting adenylate cyclase, the enzyme responsible for catalyzing adenosine triphosphate (ATP) to cyclic adenosine 3’,5 ’-monophosphate (cAMP). Activation of the CB receptors, through stimulation by THC, could thus lead to an inhibition of adenylate cyclase, resulting in lowering ooplasm cAMP levels 99. Adenylate cyclase activity and the constant and high production of cAMP is critical to prevent premature meiotic resumption100.H e r e ,w e propose a hypothetical model of action of THC wherein THC binds to CB1/2 activating them, which in turn inhibits adenylate cyclase activity, reducing ooplasm cAMP concentration. Releasing the inhibition of meiotic resumption, would then result in premature resumption of meiosis. This untimely and premature resumption may increase the likelihood of aneuploidy arising in the oocyte and resulting embryo due to the premature separation of chromosomes misaligned on the metaphase plate and an asymmetrical division of the chromosomes into the first polar body. This hypothesis aligns with the correlation between THC concentrations and oocyte maturation rate observed in the retrospective cohort (Fig.1d) and the increased oocyte maturation rate in vitro, as well as the associated reduction in euploid oocytes (Fig. 4b) and euploid embryo rates (Table 1) we observed. Further investigations are underway to dive deeper into THC signaling in the oocyte and better refine this hypothetical model. To conclude, this study comprehensively investigates and demonstrate the impact of THC on the human oocyte. Herein, our findings reveal signi ficant effects on oocyte maturation, tran- scriptomic profiles, meiotic spindle organization, and oocyte ploidy. Collectively, this data presents compelling evidence that cannabis consumption may negatively impact female fertility. Our integrated and multi-faceted in vitro approach, utilizing multiple techniques and endpoints to assess chromosome segregation, is a major strength of this study. However, it was limited by the usage of immature GV oocytes following ovarian hyperstimulation, which are considered suboptimal for reproductive purposes, since they did not mature fol- lowing initial stimulation. Furthermore, we acknowledge the impor- tance of patient age on the oocyte ability to mature in vitro, but this study was not statistically powered to analyze results based on patient age. This limitation arose because the majority of GV oocytes were retrieved from patients younger than 37 years old (81%). Also, our study focused on identifying changes in the abundance of the pre- stored transcripts in response to THC exposure, rather than de novo transcription, limiting our ability to speculate on the impact of THC on gene expression before the GV stage. On the other hand, our retrospective cohort objectively measured THC and its metabolites to determine the impact of THC on IVF Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 8 outcomes, overcoming biases inherent in self-reporting101. Indeed, 73% of our patients positive for THC did not report cannabis use when completing their patient intake questionnaire, potentially due to the persistent stigma of recreational drug consumption. A limitation of our retrospective study is the lack of data on cannabis consumption habits (e.g. frequency, timing, dosage, route of consumption), and our cohort is likely not representative of the general population, as all patients were undergoing IVF for fertility treatment or to altruistically donate their oocytes to intended parents. In addition, FF was not measured for the presence of other drugs, and even though none of the patients reported concomitant use of other drugs, self-reporting alone cannot rule out the exposure of the follicle to these substances. The limitations associated with the retrospective aspect of this study and the other potential contributors (e.g. lifestyle habits) to the observed outcomes are compensated by our in vitro study that used at least three oocytes (one in each exposure group) per patient. Finally, these findings underscore the need for increased aware- ness and caution among people with ovaries, particularly those undergoing fertility treatments. Our study highlights the importance of informing patients about the potential risks associated with can- nabis consumption and provides a basis for regulatory bodies, medical professional societies, and public health organizations to establish recommendations and guidelines regarding cannabis consumption during fertility treatment.

Ethical approval and cannabis regulatory licencing All patients undergoing ART procedures were provided with the opportunity to participate in the collection, and future use, of biolo- gical waste material for research purposes. Patients were provided with an Independent Review Board (IRB) approved informed consent package containing information regarding the types of material that would be collected following consent as well as examples of projects this material may be used for. Patients did not receive compensation or financial benefit for their participation in the collection of biological waste material. All patients included in this study provided informed consent for the donation of their biological waste material, which included follicular fluid (FF) and immature (GV) oocytes as well as associated de-identi fied demographic and clinical information, including age, Body Mass Index (BMI), ovarian reserve metrics and treatment regimens (Veritas IRB Approval #16487). To be included in the assessment of tetrahydrocannabinol (THC) on in vitro maturation (IVM), patients must have had 10 or more MII oocytes after stripping and a minimum of 3 GV oocytes in order to have at least one GV oocyte per treatment group. Last, all patients included in the assessment of THC on IVM were con firmed to be negative for THC by LC-MS/MS (described below). Patients were excluded if: they did not meet inclusion criteria, had low oocyte yield (<16 oocytes), low oocyte maturation rate (<62.5%), a previous cycle with poor fertilization rate (<75%) and/or blastulation rate (40 years old), or who were undergoing fertility pre- servation (oncofertility and/or social egg freezing). Moreover, if a patient was consented for the donation of their biological waste

but the physician and embryologist deemed rescue-IVM (rIVM) was indicated for their clinical care, no oocytes would be col- lected for research purposes and patients would be informed of the addition of rIVM to their clinical treatment by the physician or another healthcare professional. All GV oocytes included in this study were collected and underwent IVM between July 2022 and January 2024. The request and use of samples and de-identified demographic and clinical data for this study was approved by Veritas IRB Approval #16518. For the retrospective analysis, FF was collected from all consenting patients undergoing IVF treatment between June 2016 and March 2023. All samples in this study were considered “female” as they are human oocytes which are chromosomally“XX”. Gender, race, ethnicity or other socially relevant groupings were not considered in this study. The purchase, storage and use of Δ9-THC and its metabolites for research purposes was approved by Health Canada and all procedures were conducted in accordance with the ‘Cannabis Act’ and ‘Cannabis Regulations’ (License #LIC-A4MUR820SB-2020). Exocannabinoid detection in FF Measurements of Δ9-THC, 11-OH-THC and 11-COOH-THC in FF were performed for every patient who donated GV oocytes, as previously described, to exclude patients consuming cannabis for the IVM investigation 23. For the retrospective study, FF and matched serum were measured using the same procedure. Briefly, proteins were pre- cipitated using methanol (1:1 v/v), and the supernatants were assessed by LC-MS/MS using a QTRAP 5500 (SCIEZ, Concord, CA, USA) and Agilent 1290 HPLC (Agilent, Santa Clara, USA) with a calibration curve (0.001–200 ng) of known amount of the molecules of interest. Sam- ples above the lower limit of quantification were considered positive. Cannabinol (CBN) and Cannabidiol (CBD) were also measured in the samples but were undetectable. Oocyte in vitro maturation GV oocytes were received from consenting patients, randomly split into three groups, and cultured using standard clinical IVM media (SAGE One-step (CooperSurgical, Canada) + 7.5 IU/mL of Menopur (Ferring, Canada)): Ctrl (n = 96, only IVM media), THC1 ( n = 95, treated with a physiological concentration of cannabis based on previous measure- ments of cannabis in FF 23:2 5 n g / m LΔ9-THC (Sigma-Aldrich, Canada), 5 ng/mL 11-OH-THC (Sigma-Aldrich, Canada), 50 ng/mL 11-COOH-THC (Sigma-Aldrich, Canada)) and THC2 (n = 94, treated with THC at a con- centration based on previous animal studies 25,27,29:1 0 0 n g / m LΔ9-THC, 50 ng/mL 11-OH-THC, 200 ng/mL 11-COOH-THC). Oocytes were obtained from 24 patients, and their demographic data are reported in the Supplementary Table 1. Images using an inverted bright field microscope were taken prior and following incubation to assess oocyte morphology. Oocytes were cultured for 24 h using the EVOS FL Auto 2 (ThermoFisher, Canada) imaging system or cell culture incubator (5% CO 2 and 37 °C). Timelapse images were taken every 15 min and used for the assessment of GVBD and polar body extrusion (Ctrl: Supplementary video 1, THC1: Supplementary video 2 and THC2: Supplementary video 3). After 24 h of culture, oocytes were processed according to the specific endpoint (RNAseq or immunostaining/polar body biopsies). Single oocyte mRNA sequencing Oocytes destined for single-cell RNASeq were further stripped of any residual cumulus cells and snap-frozen in 0.2 mL tubes in less than 1 µL phosphate-buffered saline (1 X PBS). To reduce inter- sample variability, we selected patients with similar demographics and stimulation parameters (Supplementary Table 2). A total of 86 oocytes from 24 patients were selected for RNASeq, 28 in Ctrl, 27 in THC1 and 31 in THC2. cDNA from single oocytes were synthesized using the SMART-seq v4 Ultra Low Input RNA Kit (Takara Bio Inc., Japan). The ampli fied cDNA was puri fied using Agencourt AMPure XP beads (Beckman Coulter, USA) and eluted. The puri fied cDNA was quanti fied using the Qubit dsDNA high sensitivity assay (Ther- moFisher, Canada) and length and molarity were assessed using the DNF-474 HS NGS Fragment Kit (1-6000 bp) with the Fragment Analyzer 5200 (Agilent Technologies, USA). The ampli fied cDNA (0.2 ng) was used to construct sequencing libraries using a modi fied and miniaturized Nextera XT library preparation protocol (Illumina, Canada) developed for the use with the Mosquito HV liquid handling robot (SPT labtech, Boston, USA). The quality of the libraries was assessed using the same Fragment Analyzer kit DNF-474 HS NGS Fragment Kit (1 –6000 bp), quanti fied using Qubit dsDNA high sen- sitivity assay (ThermoFisher), normalized and pooled. Sequencing was performed on a NovaSeq 6000 S2 flow cell (Illumina, Canada) at Article https://doi.org/10.1038/s41467-025-63011-2 Nature Communications| (2025) 16:8185 9 Princess Margaret Genomics Centre (2 × 150 bp) (Toronto, Canada). The sequencing quality control metrics are compiled in the Sup- plementary Data 6. Single oocyte RNASeq bioinformatics Raw sequencing reads were trimmed based on read quality (Phred > 28) and aligned and quanti fied to hg38 using STAR (Spliced Tran- scripts Alignment to a Reference; v2.7.8a)102. Low abundant transcripts were excluded (maximum <20) and normalized using the default normalization method built into DESeq2 (v3.5) 103. We conducted dif- ferential expression (DE) using DESeq2 comparing THC1 vs. Ctrl and THC2 vs. Ctrl. Significantly differentially expressed genes were defined as p-value 1. The complete list of DE genes is available in the Supplementary data 1 (THC1 vs Ctrl) and the Supplementary data 2 (THC2 vs Ctrl)). This analysis was conducted in Partek Flow (version 11.0.23.1004). Gene Set Enrichment Analysis (GSEA) 104,105 was conducted to determine what gene sets were impac- ted by exposure to THC. The resulting pathway list was cross refer- enced with a custom gene set created and supported by the Bader Lab (University of Toronto) which is comprised of all GO database, KEGG, Reactome, and Wiki pathways gene sets (v2024-01-01) ( http:// download.baderlab.org/EM_Genesets/) 106 (Supplementary Data 3) Sig- nificant pathways were de fined as having a |Normalized Enrichment Score (NES)| > 1.5 and p-value < 0.05. Immunostaining and imaging After 24 h in culture, the zona pellucida (ZP) was removed by incu- bating (30 s–2 min) with EmbryoMax® Acidic Tyrode’s Solution (Milli- pore Sigma, CA) and polar bodies were mechanically separated from the oocytes. Oocytes were immediately fixed with 3.7% paraf- ormaldehyde in PHEM (PIPES 12 mM, HEPES 5 mM, EGTA 2 mM and MgSO4・7H2O 0.8 mM, pH 6.9) for 30 min at room temperature (RT) and then permeabilized in PHEM + 0.25% Triton-X for 15 min at RT. After permeabilization, they were incubated overnight at 4 °C in blocking solution (3% bovine serum albumin (BSA) + 0.05% Tween-20 in 1X PBS). On the next day, the plate was brought to RT before transferring the oocytes in the primary antibody solution for 1 h at 37 °C (mouse anti-ɑ-Tubulin (1:250), T6199, Sigma-Aldrich, USA). Then, the oocytes were washed three times in the wash solution (0.5% BSA + 0.05% Tween-20 in PBS) at RT and moved in the secondary antibody solution for 2 h at 37 °C (goat anti-mouse AlexaFluor 488 (1:200), A-11001, Invitrogen, USA and Phalloidin AlexaFluor 555 (1:500) (A34055, Invitrogen, USA). After secondary antibody solution, the oocytes were washed three times in the wash solution and transferred into Hoechst 33342 (1:500) for 30 min. Finally, the oocytes were transferred into 1.5 µL drops in an imaging dish (Nunc Glass Bottom Dish, ThermoScientific, CA) covered with paraf fin oil and 0.2 µm z-stacks were captured using Leica SP8 confocal microscope using the 63× objective with oil and a zoom factor set at 8 at the Advanced Optical Microscopy Facility (Toronto, Canada). Deconvolution was applied on the images using Huygens software version 23.10 (https:// svi.nl/Huygens-Software) and images were visualized using Imar- isViewer 10.1.1. Negative controls consisted of GV oocytes to observe the absence of spindle structure and stained MII-oocytes without the mouse anti-ɑ-Tubulin primary antibody (Supplementary Fig. 4). PB biopsy, whole genome amplification, sequencing and analysis Polar bodies were separated from the oocytes after removal of the ZP eliminating possible somatic cell contamination (as shown in Supple- mentary Fig. 3). Polar bodies were individually snap-frozen at−80 °C in less than 2 µL and blinded samples were sent for whole genome chro- mosome copy number variation assessment (CNV) to the CReATe Reproductive Genetics sequencing platform. CNV analysis was per- formed by low-pass whole genome Next Generation Sequencing using validated clinical workflow on Illumina platform. Brie fly, gDNA was amplified using SurePlex Whole Genome Amplification (WGA) (Illumina, CA), according to manufacturer’s instructions. Amplified gDNA was then tagmented and indexed using Nextera XT (Illumina, CA). The indexed libraries were purified using AMPure XP beads (1:1 ratio) and normalized using magnetic beads. The normalized libraries were pooled, denatured, and sequenced on a NextSeq 550 (paired end, 2 × 75 bp). NxClinical version 6.0 (Bionano, CA) was used for chromosome CNV analysis and data visualization according to our standard clinical procedure (2 mil- lion reads/sample, CNV resolution of >10 Mb). Optimization experi- mentations demonstrated the concordance between polar body chromosome numbers and its sister oocyte (Supplementary Fig. 3). Statistical analysis Datasets werefirst assessed for normality using the Shapiro–Wilk test. Statistical significance was determined using two-sided Fisher’se x a c t test for contingency analysis (maturation rate, spindle morphology and euploid rates), One-way ANOVA with a two-sided Holm–Sidak’sm u l t i p l e comparison test for continuous normally distributed datasets (oocyte diameter), or Kruskal–Wallis with a two-sided Dunn’s multiple compar- ison test for continuous non-normally distributed datasets. The specific statistical test is indicated throughout table and figure legends. Sig- nificance was de fined as p < 0.05. For the retrospective analysis, we performed pairwise case-control matching, where each THC-positive sample was matched to two THC-negative samples, as determined by t 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 - ducted using the FUZZY matching command in Statistical Package for Social Sciences (SPSS-v29) based on the following covariates: oocyte age, participant body mass index (BMI), anti-müllerian hormone (AMH), day 2/3 luteinizing hormone (LH), and follicle stimulating hormone (FSH), (estradiol) E2 on trigger, and total gonadotropin (GT) dose. AMH, LH, FSH and E2 were quantified during the clinical assessment using the Cobas e411 instrument (Roche, Basel, Switzerland). Matching success was determined using a two-tailed Mann –Whitney test for non- parametric distribution. Statistical significance of the IVF outcomes was determined using two-tailed Mann–Whitney test for non-parametric distribution (fertilization and euploid rates) and unpaired two-tailed t- test for parametric distribution (maturation and blastocyst rates) using GraphPad Prism 10.2.3. Numbers in parentheses in eachfigure legends represent distinct samples and not repeated measures. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability Data generated in this study ar e provided in the Supplementary Information/Source Data file. The normalized read count expression matrix and metadata have been deposited in the Gene Expression Omnibus (GEO) database with accession number GSE297757.D u et o participant privacy concerns and institutional ethics restrictions, raw sequencingfiles (fastq) have been deposited at the European Genome- phenome Archive (EGA), which is hosted by the European Bioinfor- matics Institute (EBI) and the Centre for Genomic Regulation (CRG), under accession number EGAS50000001052. Further information about EGA can be found at https://ega-archive.org. Expected time- frame for response to access requests is 10 business days and the data will be available for the duration of the study as de fined by the Data Access Agreement associated with this dataset. Source data are pro- vided with this paper.

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C.D. received salary support through the University of Toronto Depart- ment of Physiology Yuet Ngor Wong Postdoctoral Fellowship Award. All other funding was provided by CReATe Fertility Centre through the reinvestment of clinical earnings. Human biological materials and associated de-identified data were collected by CReATe Biobank per- sonnel. The CReATe Biobank is certified by the Canadian Tissue Repo- sitory Network (CTRNet) Biobank Program. The authors would like to thank all, past and present, CReATe Biobank staff, CReATe Fertility Centre clinical and IVF laboratory staff, CReATe Reproductive Genetics staff, and CReATe Research staff for their dedication to this study. Lastly, the authors would like to thank all patients for the donation of their biological material. Author contributions C.D., B.W. and N.F.W. participated in the design of the study. C.D. and B.W. established the collection ofimmature human oocytes through the CReATe Biobank in collaboration with IK and her team helping coordi- nate the oocyte collection. C.D. received, cultured and froze the oocytes for all experiments. C.D. optimized and conducted oocyte staining and confocal imaging, polar body biopsies, and RNA sequencing related manipulations. C.D. and B.W. performed the transcriptomic analysis. C.D. and B.W. compiled the retrospective data and analyzed the results. S.M. and her team conducted the polar body sequencing to assess the ploidy status. C.D. and B.W. wrote the manuscript. C.D. hand-drawn all graphical elements. C.L. provided critical feedback on study design and the manuscript. All authors provided feedback on the manuscript and read and approved the final manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary informationThe online version contains supplementary material available at https://doi.org/10.1038/s41467-025-63011-2. Correspondenceand requests for materials should be addressed to Cyntia Duval. Peer review informationNature Communicationsthanks Norbert Glei- cher and the other, anonymous, reviewers for their contribution to the peer review of this work. A peer review file is available. Reprints and permissions informationis available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted

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