Stop Smoking Weed At Least 11 Weeks Before TTC

^{doi.org/10.1093/eep/dvab009}

Background

According to some studies, the use of cannabis (also known as marijuana, weed or pot) can potentially alter sperm DNA methylation in humans.

Animal studies carried out in this regard showed similar results between 9-tetrahydrocannabinol (THC), which is the main psychoactive constituent of cannabis, and altered DNA methylation.

Unsurprisingly, any alteration of sperm cells leads to potentially adverse birth outcomes, including congenital defects and neurological disorders.

Considering that the duration of human spermatogenesis is approximately 74 days, it is not unreasonable to postulate that the effects of THC exposure may be reversible. However, there is a lack of evidence to support this hypothesis.

Aim

To determine whether men who stop cannabis for 11 consecutive weeks resolves cannabis-associated epigenetic changes in their sperm.

Methodology

A total of 42 healthy males, aged between 18-40 years, were recruited from the Cannabis-Induced Potential Heritability of Epigenetics Revisions in Sperm (CIPHERS) parent study.

Among the 42 participants, 18 were frequent cannabis users (at least once daily for the past 6 months) and 24 were non-users.

Participants from both groups (users and non-users) then underwent a screening test using gas chromatography-mass spectrometry (GC-MS) to measure their cannabis use at baseline, via the primary urinary cannabis metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH) normalised to creatinine.

After the initial screening test, participants in the cannabis-user group were accepted into the study if they met the following conditions:

• THC levels >50ng/ml (non-creatinine-adjusted) 
• THCCOOH levels >15ng/ml (non-creatinine-adjusted) 
• Positive urine rapid screening test
• Willing to abstain from cannabis during the course of the study (11 weeks) 

Participants in the control group (non-users) were accepted if they met the following conditions:   

• No trace of THCCOOH (non-creatinine-adjusted)
• Did not use cannabis in the past 6 months 
• Less than 10 exposures throughout their lifetime 
• Negative urine rapid screening test 

On the other hand, participants were automatically excluded from the study if:

• there was trace of any other drug during the urine rapid screening test 
• participants were on prescribed psychoactive medications 
• participants were diagnosed with any significant psychiatric conditions (except cannabis use disorder) 
• score on the Marijuana Screening Inventory-X was >10  
• score on the Alcohol Use Disorders Identification Test was ≥8  
• expired breath carbon monoxide (CO) reading was ≥8 ppm 
• expired breath alcohol level was >0.000 
• urinary cotinine levels that are not negative or within the non-smoker level 
• estimated IQ less than 80 
• participants were unable to comply with the study requirements 
• participants were deemed unfit for participation in the study by a medical professional

Sperm and urine samples were collected from participants, at the start of the study, with another sperm sample collected at the end of 11 weeks.

Cannabis users (n=18), also had weekly visits to the clinic to provide a self-assessment report and urine samples confirming cannabis abstinence. Urine samples were examined for illicit drugs, including THC, using semi-quantitative rapid urine tests, followed by enzyme immunoassay (EIA) and liquid chromatography with tandem mass spectrometry (LC-MS MS), to measure the actual levels of THC and THCCOOH precisely.

Semen analysis included volume, appearance, viscosity, pH, white blood cell (WBC) concentration, sperm concentration, total motility, forward progression, and total motile sperm count (calculated).

At the same time, DNA from both groups sperm samples (baseline and Week 11) underwent a DNA sequencing procedure, known as Whole-Genome Bisulfite Sequencing (WGBS), to determine the DNA methylation status of participants sperm and whether or not cannabis abstinence reversed the effects.

Finally, all the differentially methylated CpG sites identified were mapped to their nearest genes and using Ingenuity Pathway Analysis software, associated diseases and gene functions were output. 

Results

Initial analysis of participant demographics revealed no significant differences between the 2 groups in terms of age, education level, intelligence quotient, race, ethnicity, employment status or marriage.  

Analysis of semen parameters also showed no significant differences between the 2 groups, although the authors noted that the mean sperm concentration trended lower in the cannabis users (87.1 × 10⁶/ml) compared to non-users (99.3 × 10⁶/ml). 

Semen parameters (mean)	Users (n=18)	Non-users (n=24)	P-Value
Volume (ml)	3.5	3.9	0.38
pH	8.1	8.2	0.18
Concentration (×106/ml)	87.1	99.3	0.49
Motility (%)	57.7	63.5	0.23
Morphology (% normal)	4.1	4.1	0.93

DNA sequencing results showed that prior to 11-weeks of abstinence, there were 163 CpG sites significantly different in the sperm from cannabis users and non-users (P < 2.94 × 10⁻⁹). The magnitude of these differences decreased in the post-study analysis, which suggests that cannabis exposure effects on sperm DNA methylation could be partly reversible.  

Interestingly, post-abstinence DNA analysis also showed that 127 CpG sites were still significantly differentially methylated between the 2 groups (P < 2.94 × 10⁻⁹), of which only 3 of those sites were common in the before and after datasets, suggesting that cannabis withdrawal also impacts DNA methylation at sites other than the 163 identified pre-abstinence.

The authors commented that spermatogenesis in men takes about 74 days, while the study lasted only 11 weeks (77 days), therefore it was quite possible at the time of DNA sequencing, a mixture of sperm cells formed after cannabis abstinence, as well as sperm cells prior to abstinence, was present in sperm samples.

Ingenuity Pathway Analysis (IPA) showed significant similarities in the disease and function annotations associated with the 163 CpG sites (prior to abstinence) and the 127 CpG sites (after abstinence) between users and non-users (controls).

Top 10 Annotations (Pre-abstinence)	Disease or function Categories	-Log(P-value)	P-value
Liver lesion	Gastrointestinal Disease	4.7	0.00002
Phagocytosis of leukemia cell lines	Cell-to-cell Signaling and Interaction	3.7	0.0002
Morphology of B-cell follicle	Hematological System	3.2	0.0006
Cell death of superior cervical ganglion neurons	Cell Death and Survival	3.0	0.0010
Cardiogenesis	Cardiovascular System	3.0	0.0010
Growth of organism	Organismal Development	3.0	0.0010
Agenesis	Nervous System	2.9	0.0013
Replications of murine herpesvirus	Infectious Disease	2.7	0.0020
Neurodevelopmental disorder	Developmental Disorder	2.6	0.0025
Cerebral disorder	Nervous System	2.6	0.0025

Top 10 Annotations (Post-abstinence)	Disease or function Categories	-Log(P-value)	P-value
Abnormal morphology of enlarged seminiferous tubule	Endocrine System Disorders	4.9	0.00001
Autosomal dominant encephalopathy	Hereditary Disorder	4.4	0.00004
Learning	Behaviour	4.1	0.00008
Cognitive impairment	Nervous System	3.5	0.0003
Organismal death	Organismal Survival	3.5	0.0003
Density of vesicles	Cellular Assembly and Organization	3.2	0.0006
Activation of alveolar macrophages	Cell to Cell Signalling & Interaction	3.1	0.0008
Quantity of pyramidal neurons	Nervous System	3.0	0.0010
Formation of hippocampus	Embryonic Development	3.0	0.0010
Area of vessel component	Cardiovascular System	3.0	0.0010

The similarity in disease and function annotations of users, before and after abstinence may be evidence of permanent changes, following exposure to cannabis, requiring longer abstinence trials to investigate further.

Comparison of both IPA analysis showed that the development of nervous and cardiovascular systems is potentially affected by cannabis use.

Limitations

1. Small study size.
2. Weekly urine tests may have missed cannabis use between testing.
3. Impurities in cannabis used could potentially affect the result.

Funding

The study was funded by The John Templeton Foundation.

Similar studies

Holloway Z R, et al. (2020). Paternal factors in neurodevelopmental toxicology: THC exposure of male rats causes long-lasting neurobehavioral effects in their offspring. https://doi.org/10.1016/j.neuro.2020.01.009

Schrott R, et al. (2020). Cannabis use is associated with potentially heritable widespread changes in autism candidate gene DLGAP2 DNA methylation in sperm. https://doi.org/10.1080/15592294.2019.1656158

Slotkin T A, et al. (2020). Paternal ∆9- tetrahydrocannabinol exposure prior to mating elicits deficits in cholinergic synaptic function in the offspring. https://doi.org/10.1093/toxsci/kfaa004

Levin E D, et al. (2019). Paternal THC exposure in rats causes long-lasting neurobehavioral effects in the offspring. https://doi.org/10.1016/j.ntt.2019.04.003

Murphy S K, et al. (2018). Cannabinoid exposure and altered DNA methylation in rat and human sperm. https://doi.org/10.1080/15592294.2018.1554521

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