The effects of delta-9-tetrahydrocannabinol exposure on female menstrual cyclicity and reproductive health in rhesus macaques.

Of the eight female rhesus macaques in this study, all were of reproductive age (mean, 10 years; standard deviation [SD], 2.2), all but one had proven fecundity, and none were previously exposed to THC or any other significant, known environmental exposures ( Table 1 ). All animals consumed the daily THC edible before their regular chow. The average baseline weight of all animals was 6.9 kg (SD, 0.8), and all but one animal gained weight after starting the THC treatment ( Table 1 ). At the highest THC dosing, there was an increase in average weight of all animals by 0.3 kg (SD, 0.4; P = .083) that was not significant. The animals’ behavior after THC treatment was not formally assessed but was not noted to be grossly different per veterinary and animal support staff. During the THC induction, the average plasma THC concentrations increased by 4.5 ng/mol for each mg/7 kg/day increase in THC (95% CI, 2.6–6.5 ng/mol; P <.001) ( Fig. 1 ). With increasing THC edible dosing, a positive dose-dependent effect on the average MCL was observed. The average cycle length increased 4.0 days for each mg/7 kg/day of THC (95% CI, 1.4–6.6 days; P = .002) ( Fig. 2 ). In terms of the reproductive endocrine axis, serum FSH concentration increased significantly with increasing THC dose (0.34 ng/mL for each mg/7 kg/day of THC) (95% CI, 0.14–0.57 ng/mL; P = .004), while serum E2 concentration remained stable, with an average change of −3.4 pg/mL (95% CI, −11.4–4.7 pg/mL; P = .416) and within expected values for day 3 of the macaque cycle with increasing THC dosing ( Fig. 2 ). In addition, luteinizing hormone concentrations remained flat, with an average change of 0.0 ng/mL with increasing THC dose (95% CI, −0.1–0.1 ng/mL; P = .819). Hence, there was an increase in FSH concentration without a corresponding change in basal E2 concentration as well as stable LH concentrations across increasing THC doses ( Figs. 2 and 3 ). With increasing THC dosing, AMH concentrations trended upward, although the average change was not significant (0.8 ng/mL; 95% CI, −0.7–2.3 ng/mL; P = .278). As anticipated, there was no change in progesterone concentrations because blood samples were collected in the follicular phase of the menstrual cycle ( Fig. 2 ). Additionally, with increasing THC dosing, there were no significant average changes in PRL (−13.6 ng/mL; 95% CI, −31.9–4.7 ng/mL; P = .146) ( Fig. 2 ) and TSH (−0.3 μ U/mL; 95% CI, −0.8–0.2 μ U/mL; P = .284) concentrations ( Fig. 3 ).

A cohort of sexually mature, adult female rhesus macaques ( Macaca mulatta ) (n = 8) weighing 6–8 kg with regular menstrual cycles were used in this study. Animals were pair-housed indoors under controlled conditions. Animal ages ranged from 6.7 to 12.7 years, with a mean age of approximately 10 years old. All animals were in the Oregon National Primate Research Center’s (ONPRC) time-mated breeding program and considered fertile. Animals were considered infertile if they were no longer naturally cycling, if a pelvic ultrasound revealed very few ovarian follicles, or if anti-Müllerian hormone (AMH) concentrations were very low. All animal procedures were approved by the ONPRC Institutional Animal Care and Use Committee and conformed to all applicable regulations (IP0001389). Animals were maintained on a standard chow diet (Test-Diet, St. Louis, Missouri) with a daily cookie containing THC (THC edible) made using research-grade THC obtained directly from the National Institute on Drug Abuse Drug Supply Program. Chow and tap water were available ad libitum. All cookies were administered before the animal’s daily chow to ensure they were consumed on an empty stomach and confirm complete ingestion. Animals were slowly titrated up to 2.5 mg/7 kg/day of THC over approximately a 3–4-month time period to model medical marijuana acclimation recommendations from Colorado ( 20 ). Specifically, animals were initially maintained on a dose of 0.5 mg/7 kg/day of THC for weeks 1–3, 1 mg/7 kg/day (moderate THC dose) for weeks 4–6, 2 mg/7 kg/day for weeks 7–9, and 2.5 mg/7 kg/day (heavy THC dose) for weeks 10–12. This THC dosage was calculated from the recommended THC starting dose of 5 mg for a 68-kg adult (the average rhesus macaque weighs approximately 6.5–7.5 kg), followed by titration to 10 mg for moderate users (standardized serving size for edible retail marijuana products in Colorado) and 20–30 mg for heavy users ( 20 - 22 ). Blood was sampled (2 mL) at each dose adjustment time point during THC induction, 3 hours ( 20 ) after edible consumption, to determine THC concentrations with each increase in THC dosage. Animal weight was recorded before THC induction and at the end of each dosing time point before adjustment until animals reached the maximum THC dose of 2.5 mg/7 kg/day. Vaginal swabs were performed to detect the onset and duration of menses before and during THC induction. Menstrual cycle length (MCL) was determined by counting the number of days between the onset of menstruation in one month and the onset of menstruation in the next. Day 1 of each menstrual cycle was assigned to the first day of a positive vaginal swab for blood. On cycle day 3, peripheral blood samples (2 mL collected before THC and at each THC dose) were obtained for follicular phase estradiol (E2) and progesterone concentrations as measured by radioimmunoassay (RIA) on a Roche Cobas e411 system (Roche Diagnostics, Indianapolis, IN). Additionally, concentrations of AMH, PRL, follicle-stimulating hormone (FSH), LH, and thyroid-stimulating hormone (TSH) were quantified with assays detailed in the following that were performed by the Endocrine Technologies Core (ETC) at the ONPRC and Oregon Clinical and Translational Research Institute Laboratories. Serum AMH concentrations were measured by enzyme-linked immunosorbent assay (Ansh Labs, Webster, TX). The assay range was 0.07–16.9 ng/mL. Intra-assay coefficient of variation (CV) is 0.5%–1.6% using internal controls that are provided by the manufacturer. In addition, the ETC includes an in-house nonhuman primate (NHP) serum quality control (QC) pool with each assay. Intra-assay CV for this pool in the AMH ELISA was 1.2%. Because all AMH concentrations were determined in a single assay, no inter-assay CV was determined. Follicle-stimulating hormone and LH concentrations were performed by the ETC using a double-antibody RIA procedure similar to that described by Niswender and Spies ( 23 ). The LH and FSH RIA kits were purchased from Dr. Albert Parlow (NHPP, Harbor-UCLA Medical Center, Los Angeles). These are homologous cynomolgus macaque assays with recombinant cynomolgus LH (AFP-6936A) or FSH (AFP-6940A) used for both iodination and standards. Rabbit anti-cynomolgus LH (AFP-342994) or FSH (AFP-782594) were used at final dilutions of 1:750,000 and 1:1,038,462 for LH and FSH, respectively. The standard curves ranged between 0.005 and 10 ng/tube for both assays. The detection limit of each assay was 0.005–0.02 ng/tube. Intra-assay variations were 2.6% and 5.4% for LH and FSH, respectively. Overall inter-assay variation for monkey gonadotropin RIAs in the ETC was less than 15%. Serum PRL concentrations were determined by automatic immunoassay on a Roche Cobas e411 system (Roche Diagnostics, Indianapolis, IN). Intra-assay CV using an in-house NHP serum QC pool was 2.9%. Because all PRL concentrations were determined in a single assay, no inter-assay CV was determined. Thyroid-stimulating hormone concentrations were measured using an IMMULITE 1000 (Siemens Medical Diagnostics) solid-phase two-site chemiluminescent immunometric assay (LKTS1). The Strata Impact protein precipitation plate and 2-mL collection plates were from Phenomenex (Torrance, CA). Oasis Prime elution plates and 1-mL round collection plates were from Waters (Milford, MA). Tetrahydrocannabinol and metabolites as well as their deuterated internal standards were purchased from Cerilliant (Round Rock, TX). Acetonitrile, methanol, and water were purchased from Honeywell (Mexico City, Mexico), and formic acid along with sample vials and other high performance liquid chromatography supplies were purchased from Fisher Scientific (Rockwood, TN). Human ethylenediaminetetraacetic acid plasma for standards was purchased from Innovative research (Novi, Michigan) with a voluntary drug-free affidavit, although multiple samples were tested before a drug-free matrix was obtained. Control rhesus macaque plasma was used for initial testing and QCs. For calibration, a commercial standard at 100 ng/ μ L was diluted into use stocks at 2,000, 1,000, 200,100, 20, 10, and 2 ng/mL. A volume of 50 μ L of each stock was added to 950 μ L of plasma, except for the highest standard where 100 μ L of use stock was added to 900 μ L of plasma for standard curves in the range of 0.1–200 ng/mL. Quality controls were prepared from separate dilutions at 25 and 2.5 ng/mL using 500 and 50 ng/mL of stocks. Internal standard mix was prepared using deuterated versions of all compounds at a final concentration of 1 ng/sample. Samples were thawed, and 50 μ L of standards, QCs, or samples was pipetted into an Impact Protein Precipitation plate, followed by the addition of 5 μ L of internal standard mix (1 ng) and 150 μ L of 0.1% formic acid in acetonitrile. Each sample was pipetted up and down two to three times to mix. The cleaned extract was then eluted into a 2-mL collection plate on a Bio-tage 96-well positive pressure device. The capture plate was removed from the device, and 325 μ L of water was added to each well and mixed and then transferred to an Oasis HLB Prime 2-mg u-elution plate and pushed through the plate. The plate was then washed with 2 × 250 μ L of 25:75 ratio of methanol to water. The plate was then placed onto a mass spec-compatible 1-mL collection plate, and the compounds were eluted using 2 × 25 μ L of 90:10 ratio of acetonitrile to methanol. Moreover, 50 μ L per well of water was added, and the plate was analyzed. Tetrahydrocannabinol and metabolites were analyzed using a 5500 QTRAP hybrid/triple quadrupole linear ion trap mass spectrometer (SCIEX, Framingham, MA) with electrospray ionization in positive mode. The mass spectrometer was interfaced to a Shimadzu (Columbia, MD) SIL-20AC XR autosampler, followed by two liquid chromatography (LC)-20AD XR LC pumps. The instrument was operated with the following settings: source voltage, 5,000 kV; GS1, 40; GS2, 50; CUR, 15; TEM, 700; and CAD gas, HIGH. Compounds were infused individually, and instrument parameters were optimized for each multiple reaction monitoring transition. The gradient mobile phase consisted of two solvents: A, 0.1% formic acid in water, and B, 0.1% formic acid in acetonitrile. The column used was a Phenomenex Kinetex 2.6 μ XB-C18 100 × 3 mm, with a flow rate of 0.35 mL/min. The gradient consisted of an initial concentration of organic of 55% B. Start conditions were held for 1 min, followed by an increase to 95% B over 4 min, held at 95% B for 3 min, and decreased to start condition of 55% B over 0.1 min and re-equilibration for 1.9 min. The standard curves ranged from 0.1 to 200 ng/mL; the lower limit of detection was 0.1 ng/mL, and the lower limit of quantification was 0.5 ng/mL for all compounds. We compared the mean animal weights at baseline and at the highest THC dose (2.5 mg/7 kg/day) using a paired t test. We assessed the average association between THC dose and plasma THC concentrations, MCL, and hormone concentrations using linear mixed-effects modeling with random intercepts by animal. All statistical tests were two-sided and used an alpha of 0.05. All analyses were performed using Stata version 15.1 (StataCorp, College Station, TX).

Our study design utilized a THC induction schedule that modeled published medical marijuana acclimation recommendations for humans ( 20 ). Using this model, we found a positive correlation between oral THC dosage and plasma THC concentration confirming that the dosing regimen was successful in the NHP. Additionally, the average plasma THC concentrations at the highest oral THC edible dose were within the expected contemporary dosing range reported in humans 3 hours after consuming a similar oral THC dose ( 22 , 24 ). There was no significant THC effect on weight gain observed, which is consistent with the existing literature that demonstrates an appetite stimulating effect but not always clinically meaningful weight gain ( 25 , 26 ). A notable impact of chronic THC consumption on MCL was observed in a relatively short period of exposure time. Females were exposed for a total of 3 months, with only exposure in the last month to an equivalent, heavy medical marijuana dose. This resulted in a dose response of longer MCL as well as greater variation in interanimal MCL with increased THC edible dosing. There have only been few studies examining the effects of marijuana on MCL, but the limited data suggests an association between marijuana use and disrupted menstrual cycles, including a slightly higher rate of anovulatory cycles ( 4 , 15 ). A prior rhesus macaque study utilizing daily intramuscular injections of THC of 2.5 mg/kg induced longer anovulatory cycles ( 14 ), but this dose or mode of THC delivery is not reflective of contemporary human marijuana use. With increasing THC dose, we noted a dose response of increased FSH concentrations. In our study, FSH concentrations in the follicular phase of the menstrual cycle (i.e., 3 days after the appearance of menses) increased, whereas E2 concentrations were unchanged. This suggests an effect on hypothalamic neurons (e.g., kisspeptin) or hypothalamic endocannabinoid CB 1 receptors, which would be an area for further study. Although follicular development can occur with persistent estrogen production, follicular development can be arrested before full maturation ( 27 ). This can lead to or represent an underlying disruption in ovulation and alterations in MCL and ultimately impact fecundability. Similarly, prior studies of intravenously administered acute doses of THC in rhesus macaques have additionally described significant disruption in cycle length and increasing FSH resulting in anovulation ( 14 , 28 ). Our results suggest that at increasing doses of THC, there was no significant change in AMH concentrations. Because of the animal cohort size of this pilot study and interanimal variability, there was not enough power to demonstrate a statistically significant difference. Anti-Müllerian hormone is an established predictor of ovarian reserve, with nomograms previously established for the rhesus macaques ( 29 ). It has a significant role in folliculo-genesis, with overexpression of AMH within human populations such as those with polycystic ovarian syndrome in which women are oligo-ovulatory or anovulatory ( 30 ). Although in our study AMH values remained in the previously established nomograms, the nonsignificant rise in AMH concentrations we observed could indicate follicular arrest with increasing THC doses leading to an increased number of follicles in the preantral or antral stage. Existing literature in humans suggests that there are adverse effects of chronic marijuana use implicating a shortened luteal phase ( 15 ) and reduced fertility ( 4 , 5 , 15 ) because of menstrual cycle disturbances. Serum PRL concentrations, conversely, did not significantly change with increasing THC doses. Although the existing literature on the neuroendocrine effects of chronic marijuana use is limited, this result is consistent with published findings in prior humans and rodent studies ( 11 , 31 - 33 ). Ranganathan et al. ( 31 ) reported that frequent chronic marijuana users have a lower plasma PRL concentration relative to healthy controls. Interestingly, in a small cohort of human females studying the acute effects of marijuana smoking on PRL concentrations, there were no significant changes in plasma PRL during the follicular phase of the menstrual cycle observed, but PRL concentrations were significantly lower at 60–120 and 150–180 min after marijuana smoking during the luteal phase of the menstrual cycle ( 34 ). However, these findings of acute marijuana-induced suppression of plasma PRL concentrations in females have not been observed in human males when the administration of marijuana compounds was either orally or through smoking ( 34 ). There was a slight decrease in TSH concentrations with increasing THC dosing, but this was not significant. This result is similar to reported findings in human and rat studies after recent or acute THC exposure. A prior cross-sectional study from the National Health and Nutrition Examination Survey noted that recent marijuana use was not associated with thyroid dysfunction but was significantly associated with lower TSH concentrations ( 35 ). In a rat model, reduced TSH secretion was observed after systemic injection of THC ( 36 ). To our knowledge, this is the first study to use a relevant, translational rhesus macaque model to study a dose-response effect from THC on female reproductive health. Tetrahydrocannabinol edibles were used to simulate marijuana consumption and corresponding blood concentrations in humans. Compared with other animal models, the NHP offers several advantages including a similar plasma disposition of THC ( 37 ), and rhesus macaques have a similar 28-day menstrual cycle that is regulated by mechanisms akin to women ( 38 - 40 ). In addition, our rhesus model allows precise marijuana exposure and experimental manipulation, not feasible or ethical in humans, to elucidate direct, dose-dependent biologic consequences from chronic marijuana use while methodologically controlling for potential confounders. Hence, this NHP pilot study provides otherwise unattainable insights into the relationship between increasing THC dose on MCL and reproductive endocrine function that is translatable and applicable to humans. In addition, it provides additional informative data concerning female endocrine hormone concentrations after chronic marijuana use, which has been an underinvestigated area of research. Limited existing studies have largely focused on the effects of acute marijuana exposure ( 5 , 14 , 26 , 34 , 41 ). It is possible that because some reproductive hormones are not secreted continuously and have episodic secretion patterns, some effects of chronic marijuana use were not detected because only one blood sample was obtained at each THC dose increase time point. In addition, seasonal changes in MCL have been reported in the NHP, but this has largely been observed in animals that are outdoor housed. All of the animals in this study were indoor housed and had regularly tracked cycles before undergoing THC induction. In summary, these pilot data strongly suggest a dose-response impact on female reproductive health from chronic contemporary THC use. Further studies are needed to determine whether the increases in MCL and FSH concentrations are indicative of other reproductive abnormalities such as reduced fecundity as well as the effect of a longer duration of exposure. Given the dearth of existing literature, the larger impact of chronic THC on ovarian function and fertility is significant. Our study findings may be helpful to healthcare providers when counseling patients regarding the health consequences of chronic marijuana use. In addition, these data indicate that increasing doses of chronic THC consumption results in a dose-response relationship for increased MCL and serum FSH concentrations, suggestive of ovulation dysfunction.