P-600 cannabinoids exposure during pregnancy affects female offsprings reproduction
Authors: Zucchi A , Innocenzi E , Onorato A , Colopi A , Dolci S , Grimaldi P 
Published: June 2023

Abstract

Study question
Could in-utero exposure to cannabinoids alter the reproductive health of female offspring?

Summary answer
Prenatal exposure to cannabinoids causes a reduction of the ovarian reserve in F1 with compromised reproductive lifespan and low birth weight in F1/ F2 generations.

What is known already
Cannabis use during pregnancy is associated with adverse neonatal outcomes: preterm birth, intrauterine growth retardation, low birth weight, and neurological developmental changes. However, the impact on the germ line is still little known. Cannabis/cannabinoids exert their biological effect by activating two main cannabinoid receptors: CB1 and CB2. We previously demonstrated that fetal oocytes express CB2 receptors at a higher level than CB1. In vitro treatment of fetal oocytes with JWH-133, a selective CB2 agonist, induces an acceleration of meiosis and an increase in the percentage of γ-H2AX-positive cells and in TUNEL-positive cells (apoptotic cells).

Study design, size, duration
Five pregnant CD1 female mice were intraperitoneally injected with a single dose of JWH-133, at 1.5 mg/kg for 5 days between embryonic period (E) 12.5 to E16.5, while control pregnant females received a vehicle containing saline. At delivery, on post-natal day (PND) 2, 30 and 365, F1 pups were counted, weighted and tissues from females were collected. At adult age, F1 females were mated with control mice and F2 generation was morphologically analysed.

Participants/materials, setting, methods
Ovaries from control and in utero exposed mice were fixed in paraformaldehyde and stained with Haematoxylin and Eosin (H&E). Gonads were embedded in paraplast and sectioned at 5μm Leica-RM 2035 Microtome. Follicles in each ovary were counted serially in every third section through the entire ovary. Only healthy, non-atretic follicles with visible oocyte nuclei were scored.

Main results and the role of chance
We demonstrate that in vivo exposure to the cannabinoid JWH-133 during fetal life, through the administration of the drug to pregnant females, causes a significant reduction in the offspring body weight at birth (LBW). Histological analysis of the ovaries isolated from newborn females (PND2) shows a significant reduction of the primordial and primary follicles. This reduced ovarian reserve is observed also at PND30 and at PND365. At old adult age, these females, crossed with control males, show a drastic decrease in their litter size (F2) across the life course in comparison with control females. This suggests that the pool of primordial follicles becomes prematurely depleted in in-utero exposed F1 females, compromising their reproductive lifespan. Moreover, we find that F2 pups from prenatally exposed mothers show LBW but have an unaltered number of ovarian follicles in females. Since F2 pups have never been exposed to the drug, our results suggest that LBW is transmitted from the mother intergenerationally, while the reduced ovarian reserve is a consequence of direct exposure to the drug during fetal life of F1 females and it is not transmitted to the F2 generation.

Limitations, reasons for caution
The effects observed in mice with JWH-133 cannot be directly translated to cannabis effects since the active principle of cannabis (THC) activates both the cannabinoid receptors. Moreover, this study lacks molecular analysis of gene expression implicated in intergenerational transmission.

Wider implications of the findings
This is the first direct demonstration that prenatal exposure to cannabinoids could have long-term critical consequences for females’ reproductive health. In humans, diminished ovarian reserve is characterized by poor fertility outcomes and it represents a major challenge in reproductive medicine.

Trial registration number
not applicable