Paracetamol (N-acetyl-para-aminophenol) disrupts early embryogenesis by cell cycle inhibition.

Study question: Does paracetamol (N-acetyl-para-aminophenol (APAP) also known as acetaminophen) interfere with cell division and thereby disrupt pre-implantation embryonic development?

Summary answer: Our findings suggest that APAP exposure inhibits cell cycling during pre-implantation development (PID) through the reduction of DNA synthesis, potentially resulting in early embryonic loss.

What is known already: It is estimated that 10-40% of all human conceptions fail around the time of implantation. Genetic factors explain ∼50% of early embryonic loss, leaving a substantial portion of early losses without a known cause. Smoking and alcohol are established risk factors for spontaneous abortion, underscoring the importance of the chemical environment during embryonic development.

Study design, size, duration: To address the challenges in determining the mechanism of action and the effects of APAP during PID, we utilized a range of approaches, including in vitro, ex vivo, and in vivo methods across various models ranging from yeasts to human embryos and women of fertile age.

Participants/materials, setting, methods: A total of 90 human embryos were exposed in vitro (22 cleavage stage and 68 blastocyst-stage embryos). Endometrial tissue and uterine fluid were collected from seven women as part of an endometrial scratching procedure. Follicular fluid was collected from 26 women during transvaginal ultrasound guided aspiration of the pre-ovulatory follicles. All human material was sampled in accordance with relevant guidelines and regulations with consent from the regional scientific ethical committee of the Capital Region of Denmark and signed informed patient consent given prior to donation. All mouse experiments were approved by the Danish Animal Experiments Inspectorate and under EU directive 2010/63/EU on the protection of animals used for scientific purposes. The cultivation of the human embryonic stem cell lines H1 and HUES4 was conducted in compliance with relevant guidelines and regulations, following approval from the regional scientific ethical committee of the Capital Region of Denmark.

Main results and the role of chance: After exposure to APAP, we found an unequivocal repression of cell division across all used model systems. APAP exposure hindered cell cycle progression, likely by inhibiting ribonucleotide reductase, leading to reduced DNA synthesis and accumulation in the S-phase. At concentrations found in the reproductive system of women after standard dosing, APAP exposure decreased cell numbers in mouse and human cleavage-stage embryos or caused direct embryonic death. Similar exposure to mouse and human blastocyst-stage embryos resulted in a reduced inner cell mass and decreased DNA synthesis, respectively.

Limitations, reasons for caution: A limitation of the study is the low number of available human cleavage-stage embryos. However, the high number of human blastocysts and our translational approach, which demonstrated reproducibility across various model systems, partly addressed this limitation. Further studies are needed to confirm the potential association between APAP use and pregnancy loss in prospective cohorts.

Wider implications of the findings: Our findings indicate that the widely used mild analgesic APAP could contribute to early embryonic loss by impairing initial cell divisions. These results suggest that APAP should be used with caution by women attempting to conceive. Given that cell division is fundamental to all development, further investigation is now warranted to substantiate these findings and to elucidate possible implications for other developmental processes, such as gonadal and brain differentiation.

Study funding/competing interest(s): The research was funded by the Lundbeck Foundation (R324-2019-1881). Authors P.S. and H.K.M. were affiliated with the Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW; NNF21CC0073729). H.K.M. received a fellowship from the Novo Nordisk Foundation as part of the Copenhagen Bioscience PhD Program, supported by grant NNF19SA003544. M.H and A.D. are part of the National French Research Infrastructure France Exposome and have received funding from the European Regional Development Fund and Britanny region (Contrat Plan Etat Region, project Exposome, AIDEN 106201).

Trial registration number: N/A.