Introduction
Substances (drugs) are any psychoactive compounds that have the ability to cause harm to the health of the individual or society. These substances can be divided into different groups according to their pharmacological properties and behavioural effects, including nicotine, alcohol, cannabinoids, opioids, depressants, stimulants and hallucinogens (McLellan, Reference McLellan2017). Drug abuse refers to the habitual use of addictive drugs to alter one’s mood (Holbrook & Rayburn, Reference Holbrook and Rayburn2014). Nicotine is an addictive compound found in tobacco products. In recent years, new devices such as electronic cigarettes have entered the market, causing a shift in peoples’ exposure to nicotine compared with the past (Price & Martinez, Reference Price and Martinez2019; Yingst et al., Reference Yingst, Foulds, Veldheer, Hrabovsky, Trushin, Eissenberg, Williams, Richie, Nichols, Wilson and Hobkirk2019). Despite the notable harm of alcohol consumption on health, alcohol addiction is prevalent among people around the world (Graham et al., Reference Graham, Bernards, Knibbe, Kairouz, Kuntsche, Wilsnack, Greenfield, Dietze, Obot and Gmel2011; Labhart et al., Reference Labhart, Ferris, Winstock and Kuntsche2017). Cannabinoids are commonly used in two forms, marijuana and hashish. Although both marijuana and hashish are natural and have plant origins, a large number of synthetic cannabinoids are also available in the market. The legalization of marijuana consumption in some countries has led to an increase in the number of consumers. The main psychoactive compound in cannabis is delta-9-tetrahydrocannabinol (Δ9-THC) (Panlilio et al., Reference Panlilio, Goldberg and Justinova2015). Opioids include components such as heroin, buprenorphine, codeine, tramadol and oxycodone, which were initially introduced into the market for pain relief. Unfortunately, their unnecessary and none-medical use led to problems such as abuse, dependence, addiction and drug overdose deaths around the world (Butler & Stechlinski, Reference Butler and Stechlinski2023; McLellan, Reference McLellan2017; Roussin et al., Reference Roussin, Soeiro, Fouque, Jouanjus, Frauger, Fouilhé, Mallaret, Micallef and Lapeyre-Mestre2022; Zhao et al., Reference Zhao, Chen, Feng, Han and Zhang2019). Depressant drugs that slow down the nervous system are prescribing for the improving insomnia in people. Compounds such as benzodiazepines and barbiturates fall into this category (McLellan, Reference McLellan2017; Petrushevska & Velik Stefanovska, Reference Petrushevska and Velik Stefanovska2015). Stimulants include compounds such as cocaine, amphetamine and methamphetamine (MAMP). These compounds facilitate and increase the production of certain neurotransmitters in the nervous system while having high potential for creating dependence and addiction (Ciccarone, Reference Ciccarone2011). The use of hallucinogens such as LSD, mescaline and MDMA is increasing among young people, and over time, they cause changes in the behaviour and brain of users (Wu et al., Reference Wu, Ringwalt, Mannelli and Patkar2008).
According to the United Nations Office on Drugs and Crime (UNODC) report, drug use is more prevalent among young adults than older individuals, with some exceptions regarding traditional drug use (Drugs & Crime, 2018; Phillips et al., Reference Phillips, Ford and Bonnie2017). Both men and women engage in substance abuse. Although the prevalence of substance use is higher in men, there are increasing concerns regarding the medical, psychiatric and social consequences of substance abuse in women compared with men (McHugh et al., Reference McHugh, Wigderson and Greenfield2014). A great number of women who use drugs are in their childbearing years, resulting in a higher number of pregnant women with addiction issues (Haight et al., Reference Haight, Ko, Tong, Bohm and Callaghan2018). It is important to note that any illicit drugs that are not protein-bound can freely cross the blood-placental barrier and affect the embryos and foetus (Ganapathy, Reference Ganapathy2011; Holbrook & Rayburn, Reference Holbrook and Rayburn2014). Some women may continue drug use until they receive confirmation of their pregnancy, which means that the embryos of such women are exposed to addictive substances during critical stages of embryogenesis (Holbrook & Rayburn, Reference Holbrook and Rayburn2014).
Reproductive-aged adults appear to be more susceptible to impairments caused by drug abuse. Studies have reported that drug abuse affects the male and female reproductive systems (Barazani et al., Reference Barazani, Katz, Nagler and Stember2014; Hill et al., Reference Hill, Popov, Havlikova, Kancheva, Vrbikova, Meloun, Kancheva, Cibula, Pouzar and Cerny2005; Hugues et al., Reference Hugues, Coste, Perret, Jayle, Sebaoun and Modigliani1980; Ragni et al., Reference Ragni, de Lauretis, Bestetti, Sghedoni and Aro1988). Previous research has investigated the effects of drug use on pregnant women and their embryo (or foetus) during the post-implantation stages (Blandthorn et al., Reference Blandthorn, Leung, Loke, Lloyd-Jones, Thurman, Bowman and Bonomo2018; Corsi et al., Reference Corsi, Hsu, Fell, Wen and Walker2020; Prasad & Jones, Reference Prasad and Jones2019; Shah et al., Reference Shah, Turner, Chroust, Duvall, Wood and Bailey2022). However, there are limited studies on the effects of drug abuse during critical pre-implantation period and gamete stages. The present study aims to focus more on these subjects.
Effects of substance exposure on oocytes
Several studies have provided evidence for the presence of different opioid receptors in human oocytes. Delta, kappa and mu have been shown to be expressed in human oocytes. Kappa receptors are detected in the peripheral region of germinal vesicle (GV) oocytes. During oocyte maturation, the distribution pattern changes to a more internal location at metaphase I (MI) and becomes homogeneous at metaphase II (MII). Mu receptors also change their distribution pattern from the margin of the GV oocyte to all regions of MI and MII oocytes. Delta receptors, on the other hand, are located in the peripheral region of oocytes and do not change during oocyte maturation (Agirregoitia et al., Reference Agirregoitia, Peralta, Mendoza, Expósito, Ereño, Matorras and Agirregoitia2012). Similar distribution patterns have been observed for cannabinoid-degrading enzymes and cannabinoid receptor 1 (CB1) in human oocytes. Fatty acid amide hydrolase (FAAH), a cannabinoid-degrading enzyme, is located at the margin of GV and MI oocytes, similar to CB1 receptors. During oocyte maturation, FAAH spreads throughout all parts of MII oocytes. Monoglyceride lipase (MGLL), another cannabinoid-degrading enzyme, does not change during oocyte maturation (Agirregoitia et al., Reference Agirregoitia, Totorikaguena, Expósito, Mendoza, Matorras and Agirregoitia2016). Based on these distribution patterns, Agirregoitia et al. concluded that opioids and cannabinoid system may play a role in oocyte maturation (Agirregoitia et al., Reference Agirregoitia, Peralta, Mendoza, Expósito, Ereño, Matorras and Agirregoitia2012, Reference Agirregoitia, Totorikaguena, Expósito, Mendoza, Matorras and Agirregoitia2016). Cannabinoid receptors have been found to be expressed in various parts of the female reproductive system, including the ovaries, oviduct and uterine endometrium (Bari et al., Reference Bari, Battista, Pirazzi and Maccarrone2011; Walker et al., Reference Walker, Holloway and Raha2019 Reference Holbrook and Rayburn). Endogenous opioids (enkephalins and endorphins) are small molecules that serve as hormones and neuromodulators in the central nervous system (CNS) and exert various physiological effects in the body and also reproductive system (Böttcher et al., Reference Böttcher, Seeber, Leyendecker and Wildt2017; Faden, Reference Faden1984). Enkephalins are produced in corpus luteum and have a role in precise functions of the reproductive system. In the mouse, the presence of embryo stimulates the production of enkephalins, which in turn it helps in the transport of the embryo in the uterine tube. However, enkephalins can have other functions; for example, they may play a role in the physiology of granulosa cell or they may alter movement of fetal intestine (Buéno et al., Reference Buéno, Fargeas, Fioramonti and Menezo1986; Cupo et al., Reference Cupo, Menezo and Bueno1987).
Dell’Aquila et al. conducted a study on bovine cumulus-oocyte complexes (COCs) and mural granulosa cells and found that they have mRNA coding for the µ-opioid receptor. The supplementation of in vitro maturation (IVM) medium with hormone and β-endorphin did not result in any differences in the rates of oocytes reaching the MII stage compared with the control group. However, GV oocytes exposed to IVM hormone-free medium supplemented with β-endorphin showed a decreased rate of maturation. The inhibitory effect of β-endorphin was reversed by Naloxone. The authors reported that the mu-opioid receptor affects oocyte maturation by inducing an increase in intracellular calcium levels (Dell’Aquila et al., Reference Dell’Aquila, Casavola, Reshkin, Albrizio, Guerra, Maritato and Minoia2002). Additionally, the expression of CB1, FAAH and MGLL in human granulosa cells suggests a potential role of this system in the nuclear maturation of oocytes (Agirregoitia et al., Reference Agirregoitia, Ibarra-Lecue, Totorikaguena, Mendoza, Expósito, Matorras, Urigüen and Agirregoitia2015). In a mouse model, CB1 activation during oocyte IVM was found to modulate Akt and ERK1/2 phosphorylation status and improve embryo production. In the absence of CB1, in vivo maturation of oocytes reduced, and embryo development was delayed (López-Cardona et al., Reference López-Cardona, Pérez-Cerezales, Fernández-González, Laguna-Barraza, Pericuesta, Agirregoitia, Gutiérrez-Adán and Agirregoitia2017). Increased histone acetylation, decreased histone methylation and changes in expression of non-coding RNA(s) are some epigenetic alterations in brains of opioid users (Browne et al., Reference Browne, Godino, Salery and Nestler2020). Therefore, considering the presence of different drug receptors on oocytes and different parts of the female reproductive system, drug exposure during critical stages of oogenesis and embryogenesis can impact oocyte and embryo development.
Δ9-THC can affect the follicular phase of the menstrual cycle. Acute administration of Δ9-THC leads to a reduction in FSH levels. Consequently, follicle development, oocyte maturation and steroid production in the ovary are impaired, resulting in a lack of ovulation in the menstrual cycle and ultimately infertility (Brents, Reference Brents2016). Misner et al. investigated the effects of THC on oocyte maturation and embryo development. Immature bovine oocytes were cultured with THC for 24 h during IVM. THC significantly decreased the rate of oocyte maturation to the MII stage and subsequently reduced the cleavage rate on day 2 post-fertilization. No differences in spindle morphology were observed in the matured oocytes. Furthermore, there was no significant difference in the rate of development or the proportion of trophectoderm to inner cell mass cells at the blastocyst stage between the treatment and control groups. However, the level of apoptosis in these blastocysts increased at doses of 0.32 and 3.2 μM THC (Misner et al., Reference Misner, Taborek, Dufour, Sharifi, Khokhar and Favetta2021).
In a study conducted by Nematollahi Mahani et al, it was observed that morphine administration increased the number of atretic follicles in mouse ovaries and affected folliculogenesis. However, there were no significant differences in the volume and weight of the ovaries between the control and addicted groups (Nematollahi Mahani et al., Reference Nematollahi Mahani, Amini, Meibodi, Nabipour and Eftekhar Vaghefi2005). Similarly, the administration of cocaine in rhesus monkeys, as noted by Chen et al, disrupted the normal pulsatile secretion of gonadotropins and altered mean E2 levels during the late follicular phase (Chen et al., Reference Chen, Samuels, Luther, King, Eddy, Siler-Khodr and Schenken1998). Additionally, Potter et al. found that low-dose follicular-phase cocaine administration impaired menstrual cyclicity and folliculogenesis (Potter et al., Reference Potter, Luther, Eddy, Siler-Khodr, King and Schenken1999).
It has been shown that MAMP administration leads to an increase in the number of fragmented oocytes and a decrease in oocyte quality and embryo development (Nezhad et al., Reference Nezhad, GHAFFARI, FADAEE, Salehi, Salimi, SHAMS and Hadi2016). Another study conducted by Wang et al, investigated the long-term exposure of adolescent mice to MAMP and its effects on ovarian reserve. They found that intraperitoneal injections of MAMP at a dose of 5 mg/kg (three times per week) for 8 weeks impaired ovarian reserve. The treated mice exhibited a decrease in the number of primordial and growing follicles and an increase in atretic follicles. Furthermore, there was a decrease in the secretion of anti-Mullerian hormone (AMH), oestradiol and progesterone in granulosa cells. The ovaries treated with MAMP also showed mitochondrial swelling and degeneration in the granulosa cells, potentially leading to apoptosis in the ovarian tissue (Wang et al., Reference Wang, Qu, Dong, Huang, Kumar, Ji, Wang, Yao, Yang and Wu2016).
According to the available literature, tobacco smoke has the ability to interfere with the normal progression of folliculogenesis and development. This disruption can cause heightened levels of apoptosis or autophagy, DNA damage and abnormal connections between oocytes (immature eggs) and granulosa cells (supporting cells), ultimately resulting in the demise of ovarian follicles. In addition, epigenetic alterations including down-regulation of antioxidant genes (Gpx1 and Wnt10b) and the steroid biosynthesis gene (Fdx1) in ovarian tissue as well as down-regulation of Gja1, Lama1 and the Ferroptosis indicator (Gpx4) in granulosa cells were reported following CS exposure (Li et al., Reference Li, Wang, Xu, Hu, Miao, Zhao and Wang2022). Furthermore, there is evidence suggesting the presence of persistent oxidative stress in ovarian tissue exposed to tobacco smoke. This oxidative stress is characterized by notable increases in the levels of reactive oxygen species (ROS) within the mitochondria, lipid peroxidation and activity of the CYP2E1 detoxification enzyme. These detrimental effects contribute to a reduction in the potential for successful fertilization and can lead to overall dysfunction of the oocytes (Sobinoff et al., Reference Sobinoff, Beckett, Jarnicki, Sutherland, McCluskey, Hansbro and McLaughlin2013). The administration of high concentrations of nicotine (≥0.5 mM) to oocytes has been found to have a significant impact on their maturation. This high nicotine concentration leads to notable changes in the subsequent process of meiosis, resulting in abnormal configurations of chromosomes within the oocytes (Racowsky et al., Reference Racowsky, Hendricks and Baldwin1989).
Cigarette smoking can induce oxidative stress in granulosa cells, DNA damage in cumulus cells surrounding the oocytes, and increase thickness of the zona pellucida in women undergoing the assisted reproductive technology (ART) (Budani et al., Reference Budani, Carletti and Tiboni2017; Shiloh et al., Reference Shiloh, Baratz, Koifman, Ishai, Bidder, Weiner-Meganzi and Dirnfeld2004; Sinkó et al., Reference Sinkó, Mórocz, Zádori, Kokavszky and Raskó2005). Furthermore, exposure to 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal, a residual compound of cigarette and a secondary pollutant, has been reported to have detrimental effects on oocytes including increased DNA damage, impaired spindle morphology, epigenetic alterations and apoptosis (Liu et al., Reference Liu, Liu, Lu, Zhang, Cheng, Fu and Hou2019). Additionally, cigarette smoke exposure during pregnancy has been associated with alterations in offspring oocyte quality and histone methylation, which can affect the proper functioning of genes involved in oocyte maturation and development (Gao et al., Reference Gao, Gong, Wu, Yang, Lin and Bu2017).
There is conflicting evidence about the effect of alcohol on female infertility. Although some studies reported no association between alcohol consumption and menopausal age (Dorjgochoo et al., Reference Dorjgochoo, Kallianpur, Gao, Cai, Yang, Li, Zheng and Shu2008; Kaczmarek, Reference Kaczmarek2007), there are also other studies demonstrating that alcohol consumption tends to be correlated with postmenopausal status (Brett & Cooper, Reference Brett and Cooper2003; Cooper et al., Reference Cooper, Baird and Rebecca Darden2001; Freeman et al., Reference Freeman, Whitcomb, Purdue-Smithe, Manson, Langton, Hankinson, Rosner and Bertone-Johnson2021).
Nardo et al. demonstrated that there is no significant difference in plasma AMH concentrations and antral follicle count (AFC) between women’s alcohol consumers and non-consumers enrolled in ART (Nardo et al., Reference Nardo, Christodoulou, Gould, Roberts, Fitzgerald and Laing2007). Ozbakir & Tulay showed that the number of antral follicles was very similar in women with or without alcohol consumption. Although the number of oocytes and metaphase II (MII) oocytes tended to be higher in the control group, there was no significant difference (Ozbakir & Tulay, Reference Ozbakir and Tulay2021). Ultrastructure of ovarian tissue from rats exposed to ethanol administration revealed the harmful effects of ethanol in the primordial and secondary follicles, oocytes, granulosa cells, corona radiata cells and zona pellucida (Faut et al., Reference Faut, Rodríguez de Castro, Bietto, Castro and Castro2009).
Firns et al. demonstrated that there is no significant association between women’s alcohol consumption and fertility parameters including number of retrieved oocytes, fertilization rate, β-hCG pregnancy rate or pregnancy loss (Firns et al., Reference Firns, Cruzat, Keane, Joesbury, Lee, Newsholme and Yovich2015). The nc886 gene, a non-coding RNA transcribed via RNA polymerase III, is methylated in the oocyte and silenced on the maternal allele in nearly 75% of humans. Carpenter et al. showed that oocyte age and preconceptual alcohol consumption are associated with epigenetic imprinting of nc886 (Carpenter et al., Reference Carpenter, Remba, Thomas, Madaj, Brink, Tiedemann, Odendaal and Jones2021).
It is important to note that there is a significant gap in the literature regarding the effects of addictive drugs on oogenesis and oocyte quality. This highlights the need for further studies, particularly at the molecular level. Table 1 indicates a summary of substance effects on oocytes and oogenesis.
Summary of the substance’s effects on oocyte
Effects of substance exposure on sperm cells
The investigation of the effects of opioids on sperm cell motility and morphology highlights the importance of opioid receptors and endogenous opioid peptides in the modulation of reproduction in mammals. Albrizio et al. used western blot/indirect immunofluorescence to demonstrate the expression of delta opioid receptors on equine spermatozoa and its relationship with sperm cell physiology. The study utilized double CTC/Hoechst staining to evaluate viability, capacitation and acrosome reaction, while mitochondrial activity was assessed using MitoTracker Orange dye. The localization of the delta opioid receptor was observed in the sperm tail mid-piece as a doublet of 65 and 50 kDa molecular mass. Furthermore, the delta opioid receptor antagonist, naltrindole, was found to modulate various physiological parameters of equine spermatozoa, including motility, capacitation, acrosome reaction and viability in a dose-dependent manner (Albrizio et al., Reference Albrizio, Lacalandra, Micera, Guaricci, Nicassio and Zarrilli2010).
Another study investigated the impact of opiates on human semen quality, antioxidant capacity of seminal plasma, function and DNA integrity of spermatozoa. The results showed that opiate consumption led to a decrease in sperm concentration, catalase-like and superoxide dismutase-like activity. Additionally, sperm DNA fragmentation was found to significantly increase in opiate consumers (Safarinejad et al., Reference Safarinejad, Asgari, Farshi, Ghaedi, Kolahi, Iravani and Khoshdel2013).
Azari et al. evaluated the effects of different doses of tramadol (10 mg/kg and 20 mg/kg) on sperm parameters and testicular tissue in mice. Tramadol was injected intraperitoneally three times per week for six weeks. The results indicated that tramadol administration led to a decrease in sperm concentration, motility and vitality. Tramadol also affected the germinal layer and some seminiferous tubules, resulting in decreased spermatogenesis in the germinal epithelium of affected seminiferous tubules. More degenerative modifications were observed with a dose of 20 mg/kg tramadol at week six. However, most histopathological changes returned to the normal structure by week 12 (Azari et al., Reference Azari, Emadi, Kheirandish, Shafiei Bafti, Esmaili Nejad and Faroghi2014). One study reported reduced sperm counts, sperm vitality and free testosterone levels in patients with tramadol abuse. Higher levels of prolactin and abnormal sperm morphology were observed in these patients (Bassiony et al., Reference Bassiony, Youssef and El-Gohari2020).
López-Cardona et al. evaluated whether THC can affect the ability of spermatozoa to fertilize and produce embryos in a mice model of chronic THC treatment (10 mg/kg/day THC for 30 days). Although the expression of CB1 significantly reduced in the THC-mice cortex, CB1 mRNA was not affected in the testis. Moreover, no alterations were observed in testes histology, sperm motility or concentration. No changes were observed in the methylation of evaluated three CpG regions of CB1 in the embryos produced via in vitro fertilization (IVF) (López-Cardona et al., Reference López-Cardona, Ibarra-Lecue, Laguna-Barraza, Pérez-Cerezales, Urigüen, Agirregoitia, Gutiérrez-Adán and Agirregoitia2018). In a bovine model, THC affected motility, morphology, capacitation and mitochondrial potential of spermatozoa and also disrupted the expression of key microRNAs associated with early embryonic development (Favetta, Reference Favetta2023).
Several studies have explored the direct effects of heroin on male infertility. For instance, Nazmara et al. conducted research on men who had used heroin for at least one year to investigate its effects on semen quality. The study found that sperm motility and viability were significantly lower in the addicted group compared with the control group. Additionally, semen pH and sperm histone replacement abnormalities were significantly higher in the addicted group. However, no significant difference was found regarding serum sex hormones between the two groups. These findings suggest that the decreased quality of sperm in heroin users may be attributed to both the direct effects of heroin on opioid receptors in sperms and its indirect effects, such as increased ROS levels (Nazmara et al., Reference Nazmara, Najafi, Rezaei-Mojaz and Movahedin2019).
The activity of aminopeptidase N (APN), an essential metalloenzyme, was found to be altered and potentially contribute to male subfertility by affecting spermatogenesis, motility and viability of spermatozoa (Irazusta et al., Reference Irazusta, Valdivia, Fernández, Agirregoitia, Ochoa and Casis2004). A case-controlled study investigated the correlation between the expression of APN/CD13 and NEP/CD10 genes and semen quality in heroin-addicted men and fertile men. The study found a significant decrease in sperm progressive motility, total motility and viability in the heroin-addicted group compared with normozoospermic (normal sperm) men. Moreover, the expression levels of APN and NEP genes were lower in heroin users compared with normozoospermic men. These findings suggest a significant association between heroin addiction, asthenozoospermia (reduced sperm motility), and reduced mRNA expression levels of APN and NEP. Additionally, the duration of drug dependence was found to be associated with sperm motility and viability, as well as gene expression levels of NEP and APN. Heroin may directly decrease progressive and total sperm motility due to modification in the enkephalin-degrading enzymes (Rezaei-Mojaz et al., Reference Rezaei-Mojaz, Nazmara, Najafi, Movahedin, Zandieh, Shirinbayan, Roshanpajouh, Asgari, Abbasi and Koruji2020). In a study carried out by Gornalusse et al., the impact of heroin consumption on an important epigenetic mechanism was examined. The researchers detected an altered cargo of small RNAs (sRNAs) in human spermatozoa, which was associated with chronic heroin consumption (Gornalusse et al., Reference Gornalusse, Spengler, Sandford, Kim, Levy, Tewari, Hladik and Vojtech2023). Furthermore, heroin users were reported to experience leukocytospermia (elevated white blood cell count in semen), asthenozoospermia, increased DNA fragmentation and epigenetic alterations (Nazmara et al., Reference Nazmara, Shirinbayan, Reza Asgari, Ahadi, Asgari, Maki, Fattahi, Hosseini, Janzamin and Koruji2021).
Ebrahim et al. investigated the ultrastructural morphology of spermatozoa in diacetylmorphine-addicted patients. The findings revealed that diacetylmorphine consumption can impact the histone-to-protamine ratio, motility, viability and morphology of spermatozoa (Ebrahim et al., Reference Ebrahim, Sabry, Ali, El Fallah and Yossef2020).
One study examined the impact of codeine, the most commonly abused opioid, on sperm quality in New Zealand white rabbits. The results demonstrated that codeine treatment significantly reduced sperm membrane integrity and various sperm parameters, including normal morphology, viability, count and motility. Additionally, codeine treatment led to increased oxidative damage, caspase 3 activity and sperm DNA fragmentation. The study concluded that chronic use of codeine primarily affects sperm quality and DNA fragmentation through oxidative stress (Ajayi & Akhigbe, Reference Ajayi and Akhigbe2020). In another study, in vitro effects of different concentrations of codeine (0, 0.1, 1, 5 and 10 mM) were assessed on human spermatozoa. The findings revealed that all tested concentrations of codeine significantly decreased sperm motility and plasma membrane integrity. Additionally, the level of sperm 8-hydroxy-2-deoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, increased in a time-dependent manner (Akhigbe et al., Reference Akhigbe, Hamed, Ajayi, Anyogu and Ajayi2021).
Regarding marijuana use, significant differences in the sperm concentration and total sperm count were detected in men who had ever used marijuana compared with those who had never used it. However, no significant differences were observed in the sperm concentration between current and past marijuana smokers. Men who had ever smoked marijuana had lower sperm motility and follicle-stimulating hormone (FSH) concentrations than non-smokers, but there was no association between marijuana smoking and other reproductive hormones or sperm DNA integrity markers (Nassan et al., Reference Nassan, Arvizu, Mínguez-Alarcón, Williams, Attaman, Petrozza, Hauser and Chavarro2019). On the other hand, Gundersen et al. reported that regular marijuana smoking (more than once per week) was associated with decreased sperm concentration and total sperm count. They also found that marijuana smokers had higher levels of testosterone compared with non-smokers (Gundersen et al., Reference Gundersen, Jørgensen, Andersson, Bang, Nordkap, Skakkebæk, Priskorn, Juul and Jensen2015). Furthermore, marijuana consumption was found to negatively affect sperm motility and morphology (Carroll et al., Reference Carroll, Pottinger, Wynter and DaCosta2020).
In studies involving methamphetamine (MAMP), it was found that administration of MAMP for 7 or 14 days in rats had adverse effects on testes structure and spermatogenesis. The number of seminiferous tubule cells decreased significantly, as did the number of spermatogonia, primary and secondary spermatocytes. Moreover, various spermatogenesis indices, including the mean seminiferous tubule diameter, tubular differentiation index, repopulation index and spermiogenesis index, significantly reduced in testicular tissue (Saberi et al., Reference Saberi, Sepehri, Safi, Razavi, Jahandari, Divsalar and Salarkia2017). Another study investigated the effects of MAMP on proliferation and apoptosis in the rat seminiferous tubules. The treatment resulted in decreased cellular proliferation and the proliferation/apoptosis index ratio. The staining of rat testis with a marker of proliferation (PCNA) showed a 75% decrease in PCNA-positive spermatogonia due to MAMP administration. TUNEL results indicated an increase in TUNEL-positive spermatogonia in some seminiferous tubules. Furthermore, gaps were observed in the epithelium between the layer of spermatogonia and other layers of cells in MAMP-treated rats (Alavi et al., Reference Alavi, Taghavi and Moallem2008). Yamamoto et al. evaluated the induction of apoptosis in mouse seminiferous tubules by administering MAMP at different doses (1, 5, 10 and 15 mg/kg). The findings revealed that MAMP, in doses above 5 mg/kg, induced apoptosis in spermatogenic cells. Additionally, a dose of 15 mg/kg inhibited male copulatory behaviour by reducing serum testosterone levels (Yamamoto et al., Reference Yamamoto, Yamamoto, Hayase, Abiru, Shiota and Mori2002). The effect of MAMP on neurotransmitter secretion, such as serotonin, may be one of causes of cell apoptosis or proliferation (Alavi et al., Reference Alavi, Taghavi and Moallem2008; Kalant, Reference Kalant2001). It has also been reported that (MDMA) ecstasy can reduce the levels of Gonadotropin-releasing hormone (GnRH) and serum testosterone by affecting the hypothalamic-pituitary-testicular axis (Dickerson et al., Reference Dickerson, Walker, Reveron, Duvauchelle and Gore2008; Fronczak et al., Reference Fronczak, Kim and Barqawi2012).
Many studies conducted in recent decades have shown effects of tobacco consumption on semen and sperm parameters. Accordingly, it has been shown some alterations in sperm morphology, as well as decreases in the sperm concentration, motility and viability among individuals who smoke (Asare-Anane et al., Reference Asare-Anane, Bannison, Ofori, Ateko, Bawah, Amanquah, Oppong, Gandau and Ziem2016; Dai et al., Reference Dai, Wang and Qiao2015; Künzle et al., Reference Künzle, Mueller, Hänggi, Birkhäuser, Drescher and Bersinger2003). Additionally, tobacco smoking was found to decrease the levels of zinc and Ca+2 ATPase in seminal plasma, leading to reduced sperm motility (Kumosani et al., Reference Kumosani, Elshal, Al-Jonaid and Abduljabar2008). Individuals who smoke heavily are at a higher risk of experiencing ultrastructural abnormalities in sperm, such as alterations in axonemal microtubules and tails, which can have a detrimental impact on sperm motility (Yeung et al., Reference Yeung, Tuettelmann, Bergmann, Nordhoff, Vorona and Cooper2009; Zavos et al., Reference Zavos, Correa, Karagounis, Ahparaki, Phoroglou, Hicks and Zarmakoupis-Zavos1998). Nicotine consumption through smoking also hampers the acrosome reaction and capacitation (Shrivastava et al., Reference Shrivastava, Marmor, Chernyak, Goldstein, Feliciano and Vigodner2014; Zalata et al., Reference Zalata, Ahmed, Allamaneni, Comhaire and Agarwal2004). Consequently, smoking has been linked to compromised sperm maturation, reduced sperm function and diminished fertilization potential of sperm (Dai et al., Reference Dai, Wang and Qiao2015; Harlev et al., Reference Harlev, Agarwal, Gunes, Shetty and du Plessis2015). Besides, animal studies have shown that nicotine induces reduction in the number of germ cells, Leydig cells, and Sertoli cells, and potentially causing male infertility (Ahmadnia et al., Reference Ahmadnia, Ghanbari, Moradi and Khajeh2007; Kim et al., Reference Kim, Joo, Park, Kwon, Jang and Kim2005; La Maestra et al., Reference La Maestra, De Flora and Micale2015). Nicotine also affects the activity of testicular androgenic enzymes and plasma testosterone level. This ultimately disrupts the process of spermatogenesis and reduces fertility (Jana et al., Reference Jana, Samanta and De2010).
Tobacco smoke has been associated to cause with not only decreased semen quality, but also abnormal protein expression, genetic and epigenetic abnormalities in sperm (Linschooten et al., Reference Linschooten, Verhofstad, Gutzkow, Olsen, Yauk, Oligschläger, Brunborg, van Schooten and Godschalk2013; Marchetti et al., Reference Marchetti, Rowan-Carroll, Williams, Polyzos, Berndt-Weis and Yauk2011; Pereira et al., Reference Pereira, Juchniuk de Vozzi, Dos Santos, Vasconcelos, de Paz, Squire and Martelli2014). Both animal and human studies have shown genome instability, genetic mutations and the presence of aneuploids in the germline of individuals exposed to tobacco smoke (Beal et al., Reference Beal, Yauk and Marchetti2017; Hassold et al., Reference Hassold, Abruzzo, Adkins, Griffin, Merrill, Millie, Saker, Shen and Zaragoza1996; Omolaoye et al., Reference Omolaoye, El Shahawy, Skosana, Boillat, Loney and Du Plessis2022; Pereira et al., Reference Pereira, Juchniuk de Vozzi, Dos Santos, Vasconcelos, de Paz, Squire and Martelli2014). The process of protamination, which is crucial for fertility, is likewise affected by cigarette smoking due to the impact of various chemicals present in tobacco smoke on chromatin structure (Hamad et al., Reference Hamad, Shelko, Kartarius, Montenarh and Hammadeh2014). Additionally, smoking has been linked to decreased activity of sperm glutathione peroxidase (GPx-1, 4) and reduced mRNA expression of glutathione reductase in spermatozoa (Viloria et al., Reference Viloria, Meseguer, Martínez-Conejero, O’Connor, Remohí, Pellicer and Garrido2010). One study found that there is a distinct difference in gene expression at the mRNA and miRNA levels in the spermatozoa of men who smoke (Marchetti et al., Reference Marchetti, Rowan-Carroll, Williams, Polyzos, Berndt-Weis and Yauk2011). Another study revealed that tobacco smoke leads to specific changes in the miRNA content of spermatozoa in smoking men. These miRNA alterations are believed to play a role in the regulatory pathways crucial for maintaining healthy sperm and normal embryo development (Marczylo et al., Reference Marczylo, Amoako, Konje, Gant and Marczylo2012). Additionally, Chen et al. demonstrated that exposure to tobacco smoke for six weeks resulted in changes in the expression of sperm proteins in mice. The affected proteins were associated with energy metabolism, reproduction and the development of structural molecules (Chen et al., Reference Chen, Xu, Miao, Zhu, Dai, Chen, Fang, Wu, Nie and Wang2015). Elevated levels of ROS following smoking can disturb male fertility via damage to sperm DNA, lipid peroxidation and impaired spermatogenesis (Calogero et al., Reference Calogero, Cannarella, Agarwal, Hamoda, Rambhatla, Saleh, Boitrelle, Ziouziou, Toprak and Gul2023; Kumar et al., Reference Kumar, Chawla, Bisht, Yadav and Dada2015; Sansone et al., Reference Sansone, Di Dato, de Angelis, Menafra, Pozza, Pivonello, Isidori and Gianfrilli2018; Wright et al., Reference Wright, Milne and Leeson2014).
Effects of alcohol abuse in sperm cells have been investigated in several studies. Accordingly, it has been shown that habitual alcohol consumption caused lower quality of semen and alterations in reproductive hormones (Jensen et al., Reference Jensen, Gottschau, Madsen, Andersson, Lassen, Skakkebæk, Swan, Priskorn, Juul and Jørgensen2014). Lwow and colleagues evaluated the effect of occasional alcohol consumption on semen quality and reported no impact on semen quality. However, the percentage of macrocephalic sperm cells increased significantly in consumers (Lwow et al., Reference Lwow, Mędraś, Słowińska-Lisowska, Jóźków and Szmigiero2017). In the other study, it has been shown that alcohol consumption can decrease semen quality and also increase ROS production and DNA damage (Finelli et al., Reference Finelli, Mottola and Agarwal2021; Kotova et al., Reference Kotova, Vare, Schultz, Gradecka Meesters, Stepnik, Grawé, Helleday and Jenssen2013). Rompala et al. evaluated the effect of heavy chronic intermittent ethanol consumption on sperm cells in mice and found alterations in none-coding small RNAs, including tRNA-derived small RNA, mitochondrial small RNA and microRNA. In this way, alcohol abuse may induce epigenetic alterations in offspring (Rompala et al., Reference Rompala, Mounier, Wolfe, Lin, Lefterov and Homanics2018). In another study in mice, paternal heavy chronic alcohol consumption in periconceptional period caused foetal growth restriction via alterations in sperm inherited non-coding RNA(s) (Bedi et al., Reference Bedi, Chang, Gibbs, Clement and Golding2019).
Epigenetic alterations have been observed in sperm from male drug addicts, indicating changes in gene expression, protamine deficiency, and alterations in the miRNAs and non-coding RNAs (Chorbov et al., Reference Chorbov, Todorov, Lynskey and Cicero2011; Hamad et al., Reference Hamad, Shelko, Kartarius, Montenarh and Hammadeh2014; Marczylo et al., Reference Marczylo, Amoako, Konje, Gant and Marczylo2012; Nazmara et al., Reference Nazmara, Najafi, Movahedin, Zandiyeh, Shirinbayan, Asgari, Roshanpajouh, Maki, Bashiri and Koruji2020; Nazmara et al., Reference Nazmara, Shirinbayan, Reza Asgari, Ahadi, Asgari, Maki, Fattahi, Hosseini, Janzamin and Koruji2021; Rompala et al., Reference Rompala, Mounier, Wolfe, Lin, Lefterov and Homanics2018). These studies have demonstrated significant detrimental effects of drugs on various sperm parameters, as well as epigenetic status and sexual function. However, it is important to note that larger-scale clinical trials are recommended to provide more robust conclusions, particularly when considering other factors such as lifestyle patterns that may influence the generalizability of these findings (Srinivasan et al., Reference Srinivasan, Hamouda, Ambedkar, Arzoun, Sahib, Fondeur, Mendez, Mohammed and Arzoun2021). Table 2 indicates an overview of the effects of substances on sperm, summarizing the observed impacts on sperm parameters and reproductive health.
Summary of the substance’s effects on sperm
Effects of substance exposure on pre-implantation embryos
The expression of opioid receptors at various stages of pre-implantation embryos, from zygote to blastocyst, has been reported by Chen et al. It is interesting to note that opioid receptors are expressed both on the membrane and in the cytoplasm of pre-implantation embryos. This expression pattern suggests the involvement of opioid signalling during pre-implantation embryo development, as well as the potential detrimental effects of drug abuse on pre-implantation embryo development, implantation and pregnancy outcomes (Chen et al., Reference Chen, Kong, Tang, Fu, Wang, Zhang and Wang2014; Kalyuzhny et al., Reference Kalyuzhny, Hensleigh, Arvidsson and Elde1997). These findings suggest that the pre-implantation embryo is a direct target for the opioid system (Chen et al., Reference Chen, Kong, Tang, Fu, Wang, Zhang and Wang2014).
In mouse models, exposure to kerack during pregnancy resulted in a significant decrease in the developmental potential of the morula stage into the blastocyst stage. The addicted group exhibited a decrease in the total number of blastocyst cells and inner cell mass, as well as an increase in apoptosis rates compared with the control group (Mohammadzadeh et al., Reference Mohammadzadeh, Amjadi, Movahedin, Zandieh, Nazmara, Eslahi, Shirinbayan, Asgari, Azad and Salimi2017). However, cocaine administration in rabbits showed no effects on the number of ovarian follicles, retrieved oocytes, IVF results or cleavage rate. Nevertheless, hormonal changes were observed in the rabbits, including an increase in follicular fluid oestradiol and a decrease in progesterone levels in both serum and follicular fluid during the periovulatory stage. The authors suggested that these hormonal changes induced by cocaine administration may affect fertility through delayed granulosa cell luteinization (Kaufmann et al., Reference Kaufmann, Savoy-Moore, Sacco and Subramanian1990). When zebrafish embryos were exposed to cocaine, a very low mortality rate and no obvious abnormalities were observed. However, alterations in protein expression levels indicated detrimental effects of cocaine exposure on early embryo development (Parolini et al., Reference Parolini, Bini, Magni, Rizzo, Ghilardi, Landi, Armini, Del Giacco and Binelli2018).
In mice, intraperitoneal injection of MAMP (10 mg/kg/day) for 14 days resulted in a decrease in fertilization and cleavage rates. However, shorter-term injection of MAMP for 2 days did not impact embryo development or fertilization rates (Nezhad et al., Reference Nezhad, GHAFFARI, FADAEE, Salehi, Salimi, SHAMS and Hadi2016).
The intraperitoneal injection of morphine into mice on days 2–3 of pregnancy disrupted the expression of opioid receptors and normal development of pre-implantation embryos into blastocysts. Additionally, normal calcium oscillation was inhibited in embryos exposed to morphine (Chen et al., Reference Chen, Kong, Tang, Fu, Wang, Zhang and Wang2014). Interestingly, Chernov et al. reported that culturing embryos in a medium supplemented with β-endorphin improved the development of two-cell embryos into the blastocyst stage (Chernov et al., Reference Chernov, Kovalitskaya, Sakharova and Chailakhyan2009). The presence of mu opioid receptors has been documented in mouse oocytes and granulosa cells, with varying expression patterns depending on the stage of maturation. Furthermore, morphine has been shown to improve the development of oocytes to the blastocyst stage by modulating the PI3K/Akt and MAPK pathways (Olabarrieta et al., Reference Olabarrieta, Totorikaguena, Agirregoitia and Agirregoitia2019).
The abuse of marijuana can have an impact on IVF results. Women who have used marijuana more than 90 times exhibited a lower number of oocytes retrieved and embryos transferred compared with non-users (P < 0.05). However, mild (1–10 times) and moderate (11–90 times) marijuana use showed no significant effects on the number of oocytes retrieved. It is worth noting that even marijuana abuse by couples up to 1 year before IVF significantly decreased the number of retrieved oocytes, fertilized oocytes and embryos transferred. Furthermore, infants born to parents who abused marijuana had significantly lower birth weights (Klonoff-Cohen et al., Reference Klonoff-Cohen, Natarajan and Chen2006). Both CB receptors (cannabinoid receptors) are expressed in 2-cell embryos. However, activation of the CB1 receptor following marijuana abuse can result in arrested pre-implantation embryo development (Paria et al., Reference Paria, Ma, Andrenyak, Schmid, Schmid, Moody, Deng, Makriyannis and Dey1998; Sun & Dey, Reference Sun and Dey2008). Nonetheless, a cohort study comparing IVF outcomes between marijuana users and non-users found no significant differences in the number of retrieved oocytes and their maturity, fertilization rate, peak serum oestradiol, embryo quality, implantation rate and ongoing pregnancy between the two groups (Har-Gil et al., Reference Har-Gil, Heled, Dixon, Ahamed and Bentov2021).
A study conducted by Favetta demonstrated that THC decreased the ability of bovine oocytes to undergo nuclear maturation, leading to reduced fertilization competence and poor embryonic development (Favetta, Reference Favetta2023).
According to the literature, nicotine exposure during embryonic development can impact on embryo development. A study utilizing a time-lapse system reported impaired early embryonic development in women who smoke compared with non-smoking women (Fréour et al., Reference Fréour, Dessolle, Lammers, Lattes and Barrière2013). Nicotine-treated embryos exhibited notable variations in developmental stages compared with control embryos (Kamsani et al., Reference Kamsani, Rajikin, Chatterjee, Nor-Ashikin and Nuraliza2010). In mice, nicotine treatment caused slower transport of the embryo through follopian tube due to reduced motility which may explain the higher incidence of ectopic pregnancies in smoking women (DiCarlantonio & Talbot, Reference DiCarlantonio and Talbot1999 Reference Kamsani, Rajikin, Chatterjee, Nor-Ashikin and Nuraliza). The number of hatched blastocysts also decreased at various nicotine concentrations (Kamsani et al., Reference Kamsani, Rajikin, Khan, Satar and Chatterjee2013). Another study demonstrated that exposure to cigarette smoke led to a higher occurrence of multinucleated blastomeres in developed bovine blastocysts (Liu et al., Reference Liu, Li, Sessions, Rickords, White and Bunch2008). In a study, Banafshi and colleagues showed that smoking had a significant effect on the expression of pluripotency genes, apoptotic genes and the aryl hydrocarbon receptor (AhR) gene (Banafshi et al., Reference Banafshi, Mohammadi, Abdi, Ghaderi, Assadollahi, Erfan, Rezaei and Fathi2022). Collectively, these findings highlight the complex and dose-dependent effects of cigarette smoke on embryo development and gene expression during critical stages of embryogenesis.
Furthermore, studies have identified a correlation between active smoking in women and a delay in blastocyst expansion during ART. Women who smoke while undergoing ART procedures tend to experience a longer timeframe for blastocyst expansion in comparison to non-smokers (day 6 vs day 5) (Bourdon et al., Reference Bourdon, Ferreux, Maignien, Patrat, Marcellin, Pocate-Cheriet, Chapron and Santulli2020). In another study, nicotine exhibited adverse effects on the secondary meiotic spindle structures and overall embryonic development, with the severity of the effects being dependent on the dose (Liu et al., Reference Liu, Li, Sessions, Rickords, White and Bunch2008). A recent study conducted in 2024, Ryoma Taniguchi et al., have examined the effects of male partners’ smoking status on embryo kinetics in IVF. Despite observing abnormalities in the secondary meiotic spindle structures and impaired embryonic development in vitro due to nicotine exposure, the study detected no notable impact on blastocyst formation time, morphology of embryos or clinical outcomes (Taniguchi et al., Reference Taniguchi, Hatakeyama, Ohgi and Yanaihara2024). Similarly, another study indicated that male tobacco smoking did not have significant effects on early embryo morphology or kinetics (Frappier et al., Reference Frappier, Martinaud, Barberet, Bruno, Guilleman, Amblot, Guilloteau and Fauque2022).
Although it has been evidenced that chronic alcohol consumption during pregnancy has harmful effects on the foetus (Wilhoit et al., Reference Wilhoit, Scott and Simecka2017), the effects of alcohol consumption on pre-implantation embryo development, implantation or uterus receptivity is unclear. Studies on alcohol consumption and human embryo implantation are scarce, but in vitro studies suggested that alcohol may exert harmful effects on human placental cells and also granulosa cells (Ahluwalia et al., Reference Ahluwalia, Smith, Adeyiga, Akbasak and Rajguru1992; de Angelis et al., Reference de Angelis, Nardone, Garifalos, Pivonello, Sansone, Conforti, Di Dato, Sirico, Alviggi and Isidori2020; Wimalasena et al., Reference Wimalasena, Meehan, Dostal and de Silva1993). The presence of ethanol in the oviduct and uterine lumen of female rats following chronic alcoholization may affect pre-implantation embryo development (Sandor et al., Reference Sandor, Garban, Checiu and Daradics1981). Cebral et al. demonstrated that preconceptional chronic ethanol ingestion by prepubertal female mice can lead to retarded embryo development, morphological abnormality in embryo, impaired blastocyst hatching and embryo loss through fragmentation due to alterations induced in the oocyte (Cebral et al., Reference Cebral, Lasserre, Rettori and de Gimeno2000). Wiebold et al. showed that alcohol ingestion during the first 3 days of pregnancy had no effect on embryo development (Wiebold & Becker, Reference Wiebold and Becker1987). Lui et al. investigated the effect of ethanol and acetaldehyde on the first trimester human placental cell and showed its detrimental effects on proliferation of trophoblast cells (Lui et al., Reference Lui, Jones, Robinson, Greenwood, Aplin and Tower2014).
Early prenatal alcohol exposure can change epigenetic marks as well as impacting cell differentiation, embryo development and the adult phenotype (Wallén et al., Reference Wallén, Auvinen and Kaminen-Ahola2021). According to the mouse model, alcohol ingestion by females can influence the levels of H3K9 acetylation in pre-implantation embryos (Fang et al., Reference Fang, Li, Yong, Feng-Rui, Biao, Wen-Yong and Rong2015). Haycock et al. evaluated the effect of ethanol exposure during pre-implantation development of mouse embryos on DNA methylation at the H19 imprinting control region (ICR). Although severe growth retardation was observed in ethanol-exposed placentae and embryos, DNA methylation at maternal and paternal alleles was not affected in embryos. However, less methylation was observed in paternal alleles of ethanol-treated placentae (Haycock & Ramsay, Reference Haycock and Ramsay2009). Rao et al. evaluated the correlation between alcohol consumption and ART outcomes in a meta-analysis study. The findings showed that maternal alcohol consumption was negatively related to pregnancy after IVF/ICSI treatment (Nicolau et al., Reference Nicolau, Miralpeix, Sola, Carreras and Checa2014). Much more studies are necessary to fully understand the association between alcohol consumption and embryo implantation.
However, there is limited data available on the effects of different addictive drugs on pre-implantation embryos and IVF/ICSI outcomes, further research is essential in this field, especially at the molecular level. Table 3 illustrates the effects of substances on pre-implantation embryos based on different studies.
Summary of the substance’s effects on pre-implantation embryos
Conclusion
The existing evidence suggests that drug abuse can have negative effects on oocytes, sperm cells and pre-implantation embryos, which can negatively affect fertility. Numerous studies have indicated that drug abuse can lead to subfertility or infertility by affecting gametes and reproductive processes. Figure 1 briefly explains detrimental effects of drug abuse on pre-implantation embryos and gametes based on different studies. It has been evidenced that drug consumption in men can impair fertility by affecting semen quality, DNA integrity, antioxidant activity and hormonal disturbance. Impaired protamination, altered expression of genes and changes in non-coding RNAs are epigenetic alterations following drug abuse that can have detrimental effects on offspring health. In women, similar to men, drug abuse can lead to oxidative stress and epigenetic changes, which can contribute to reduced fertility in these individuals. Furthermore, substance exposure can disrupt ovarian follicular development and oocyte maturation, leading to lower quality and quantity of oocytes. Decreased ovarian reserve following drug abuse can be due to hormonal imbalances and impaired folliculogenesis. As mentioned, both oocytes and sperms, when exposed to drugs, can have detrimental impact on embryos and fertility. Pre-implantation embryos can also be exposed to drugs in addicted women. According to the literature, exposure to drugs before implantation can lead to poor and slow embryo development, increased apoptosis, decreased cell count, epigenetic changes in both embryo and placenta as well as poor placenta development which can subsequently lead to complications for pregnancy and foetus. However, it is important to acknowledge that there is still a significant gap in the current literature when it comes to understanding the effects of different drugs on the capability and fertilization potential of gametes especially oocytes, as well as pre-implantation embryos. More comprehensive studies, particularly at the molecular level, are needed in order to gather further insights into these complex mechanisms. Additionally, it is important to note that the available data in this field are not yet conclusive, and there are several confounding variables that need to be considered in human studies. Therefore, the conduction of large-scale clinical trials is strongly recommended in order to provide more definitive conclusions. By emphasizing the gaps in the current knowledge and the need for further research, the conclusion highlights the importance of continuing to explore the effects of drugs on fertility and reproductive processes.
Detrimental effects of drugs on pre-implantation embryos, sperm and oocyte based on different studies.
Funding
None.
Competing interests
The authors declare that they have no competing interests
