In this study, we determine the impact of physiologically relevant concentrations of THC on oocyte maturation, elucidate the transcriptomic changes induced by THC exposure and its effect on chromosome segregation, and compare our findings with a retrospective cohort study. Our investigation will aid in bridging the knowledge gap in our understanding of the sex-specific reproductive consequences of cannabis use and contribute to more effective and evidence-based patient counseling.

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 treatment to determine the reproductive consequences of THC consumption. 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), followed 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 identified significant correlations 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 negatively correlated with a patient's Body Mass Index (BMI) (⍴ = -0.539, p = 0.000053).

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 germinal 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 (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/mL Δ9-THC, 5 ng/mL 11-OH-THC, 50 ng/mL 11-COOH-THC) or a supraphysiologic (THC2, n = 93, 100 ng/mL Δ9-THC, 50 ng/mL 11-OH-THC, 200 ng/mL 11-COOH-THC) concentration where THC1 is based on the concentration of Δ9-THC and its metabolites measured in the follicular fluid of IVF patients and THC2 is based on previously reported concentrations in animal studies. Subsequently, oocytes were classified 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 significant 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 = 92, THC1: n = 89 and THC2: n = 85) and post-IVM (Ctrl: n = 91, THC1: n = 88, and THC2: n = 83), and key maturation events were recorded: GVBD (Ctrl: n = 71, THC1: n = 72 and THC2: n = 64) and extrusion of the first polar body (Ctrl: n = 28, THC1: n = 30 and THC2: n = 31). Examples of timelapse IVM images are provided in Supplementary Fig. 1 and corresponding videos are provided as Supplementary videos (Ctrl-Supplementary video 1, THC1-Supplementary video 2 and THC2- Supplementary video 3). There were no significant differences in oocyte diameter between treatment groups either before (Ctrl: 110.6 μm, THC1: 109.6 μm, p = 0.2120 and THC2: 109.6 μm, p = 0.2120) (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 = 0.1066). (Fig. 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 - Supplementary Table 1.

Single MII oocytes with good morphology and normal developmental progression were sequenced using our optimized ultra-low input RNA sequencing pipeline (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 (|logFC| > 1) and p < 0.05 (Fig. 3a) when assessing the impact of the THC1 vs Ctrl (Supplementary Data 1). Gene Set Enrichment Analysis (GSEA) identified that upregulated 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 inflammatory processes (Fig. 3b, Supplementary Data 3). THC2 exposure induced a greater magnitude of transcriptomic dysregulation, with 402 up-regulated and 62 down-regulated genes identified (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 inflammatory processes (Fig. 3d, Supplementary Data 3). As illustrated by the Venn diagram (Fig. 3e), THC1 exposure had 266 specific 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, and KCNMB3), 10 were down-regulated (OR4F15, MMP9, PRRX2, IRS4, INFG, CCIN, IL33, NEUROD1, MT1HL1, and MT1H), and 3 displayed bidirectional changes (S100B, ACTA1, and 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 Supplementary Data 6.

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 morphology (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 specialized in detecting aneuploidy in reproductive samples (Supplementary 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 (Ctrl: 0%, THC1 and THC2: 42%, p = 0.1029) (Fig. 4c). Figure 4d reports a 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 "normal" meiotic spindles include bipolar barrel-shaped microtubules with the chromosomes aligned on the metaphase plate. Whereas "abnormal" spindles are varied in their morphology and may include multipolar spindles, alterations in microtubule organization, and misaligned chromosomes. Spindle disorganization and chromosome misalignment are shown by representative images in Fig. 4e, where oocytes were classified as having either "normal" or "abnormal" spindles. 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 significant increase in THC2 (Ctrl: 42% and THC2: 92%, p = 0.0272) (Fig. 4f). (Spindle immunostaining negative controls ca be found in Supplementary Fig. 4)

Following pairwise case-control matching, where each THC-positive sample was matched to two THC-negative samples, a significant decrease in embryo euploidy rate was observed in the THC-positive group (n = 51, 60.0%), compared to the THC-negative group (n = 101, 67.0%, p = 0.0245) (Table 1). There was no significant change in maturation, fertilization and blastocyst rates (Table 1).

To further evaluate the likelihood of adverse IVF outcomes, we conducted multiple logistic regression analyses, focusing on clinically relevant IVF outcome thresholds: 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 trigger, and total gonadotropin (GT) dose. The final model for both blastulation and euploidy rates retained THC status as a significant explanatory variable, with oocyte age being a significant covariate. In this pairwise matched cohort, THC positivity significantly 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 significantly 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 (Supplementary Fig. 5).