Unveiling the effects of micro and nano plastics in embryonic development

Abstract

The improper disposal and degradation of plastics causes the formation and spread of micro and nano-sized plastic particles in the ecosystem. The widespread presence of these micro and nanoplastics leads to their accumulation in the biotic and abiotic components of the environment, thereby affecting the cellular and metabolic functions of organisms. Despite being classified as xenobiotic agents, information about their sources and exposure related to reproductive health is limited. Micro and nano plastic exposure during early developmental stages can cause abnormal embryonic development. It can trigger neurotoxicity and inflammatory responses as well in the developing embryo. In embryonic development, a comprehensive study of their role in pluripotency, gastrulation, and multi-differentiation potential is scarce. Due to ethical concerns associated with the direct use of human embryos, pluripotent cells and its 3D in vitro models (with cell lines) are an alternative source for effective research. Thus, the 3D Embryoid body (EB) model provides a platform for conducting embryotoxicity and multi-differentiation potential research. Pluripotent stem cells such as embryonic and induced pluripotent stem cells derived embryoid bodies (EBs) serve as a robust 3D in vitro model that mimics characteristics similar to that of human embryos. Thus, the 3D EB model provides a platform for conducting embryotoxicity and multi-differentiation potential research. Accordingly, this review discusses the significance of 3D in vitro models in conducting effective embryotoxicity research. Further, we also evaluated the possible sources/routes of microplastic generation and analyzed their surface chemistry and cytotoxic effects reported till date.

Graphical Abstract

Highlights

• •Micro and nano plastic generation and polymer chemistry, effects of additives or plasticizers.
• •Chemical exposures during pregnancy and cellular toxicity.
• •Embryotoxicity research and micro and nano plastic induced toxic effects reported till date.
• •Micro and nano plastic induced embryotoxicity and advancements in embryotoxicity research using 3D in vitro models.

1. Introduction

Although, reduce, reuse and recycle (3Rs) are the well known strategies to control plastic pollution, the global recycling rate of plastics was reported to be low due to increasing plastic waste pollution [1]. Moreover, the dumped plastic contamination in the ecosphere and its disintegration into micro-nanoplastics that incorporates into biomass is rather difficult to address using the 3 R approach unless the 3Rs are taken care initially[2]. In light of the available reports, around 275 million metric tonnes of plastic waste was generated in 2010 from 192 coastal countries and further increased with time, recording 1.6 million tons per day generated since the outbreak of COVID-19 [3], [4]. To date, the work on microplastic pollution is contributed by the terrestrial plastic pollution of various plastic types, namely polystyrene (PS), polycarbonate (PC), poly-(methyl methacrylate) (PMMA), polypropylene (PP), polyethylene terephthalate (PET), polyethylene (PE) and polyvinyl chloride (PVC) [5]. It is believed that microplastic generation was initially reported with the occurrence of plastic debris in the marine environment [6], [7]. Later, this debris potentially disrupts the marine ecosystem, generating micro and nano-sized particles as a result of periodic accumulation and weathering [8], [9], [10]. In 2020, the presence of microplastics (MPs) was reported in the gastrointestinal tracts of South China sea fish species [11]. In light of these reports, micro and nano plastic pollution and its toxic effects have become a hot topic in the world of research and development.

Synthetic textile fibres, building material remnants, waste incineration, and landfills are some of the known contributors to microplastic generation polluting soil, water, and air. Utilization of Pyr-GC/MS for the purpose of identification of chemical composition and determination of mass of MPs, was pioneered in the year 2022 [12]. The increased deposition of atmospheric MPs made the soil microbiome susceptible to micro and nano plastic exposure. In 2018, a persistent decrease in soil enzymes including leucine-aminopeptidase and alkaline phosphatase was reported due to the accumulation of polystyrene nanoplastics [13]. In accordance with this, higher organisms particularly humans, are also prone to microplastic exposure induced toxicity from the environment. The investigation of micro and nano-plastic-induced toxicity in humans, specifically in the aspect of reproductive health, remains a less explored research area/domain. A range of adverse effects that disrupt the processes of conception, fetal development, childbirth, and the normal growth of an offspring is referred to as reproductive dysfunction. [14]. In adverse conditions, it can lead to miscarriage or spontaneous abortion [15], [16]. An increase in the prevalence of miscarriage and stillbirth was reported in Indian women, with an increased percentage of stillbirth to be 28.6 % [17]. The introductory report on the identification of MPs in human placental samples in the year 2021 intrigued concerns about studying the potential impacts of micro and nanoplastics on human embryonic development [18]. This report identified 12 microplastic fragments that majorly comprised of polypropylene. A research article communicated in the same year reported the presence of a higher amount of MPs including polyethylene terephthalate (PET) and polycarbonate (PC) in feces of infants than in adults. The median concentrations of PET and PC microplastics in infants feces are reported to be 36000 ng/g and 78 ng/g respectively while in adult feces it is around 2600 ng/g. The difference in concentration of MPs in fecal analysis of infants and adults is pointing towards possible microplastic accumulation during the gestation period [19]. Consequently, this led to a flourishing research landscape investigating the impact of micro and nano plastics on human reproductive health. This review comprehensively and sequentially addresses the genesis of micro and nano plastics, delving into their surface chemistry, potential xenobiotic effects on the cellular environment and further human reproductive health. Also, the idea of applying 3D in vitro models in embryotoxicity research to further explore the implications of micro and nano plastic induced effects in embryonic development.

2. Micro and nano plastic genesis: a scientific exploration into origin and polymer chemistry of plastics

The rise in global demand for plastics significantly increased the worldwide production and supply of the same. Using single-use plastics is one such example substantiating the high demand. In 1909, L. H. Baekeland introduced the fully synthetic plastic phenol-formaldehyde. It transpired nearly four decades subsequent to J. W. Hyatt's pioneering work on a semisynthetic plastic, cellulose nitrate [20]. Bakelite was the first made synthetic plastic, marking the inception of plastic production globally. Over the subsequent 70 years, the annual output of plastics surged by almost 230 times, reaching 460 million tonnes in 2019 [21]. The production mainly concentrates on commodity plastics, also known as thermoplastics, that can be heated and moulded repeatedly. These include polystyrene (PS), high-density polyethylene (HDPE), low-density polyethylene (LDPE), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), nylon, and acrylic each coded by a different number [21]. Plastics are made of synthetic organic compounds comprised of polymers with hydrocarbon monomers comprising long-chain carbon atoms. These monomers can be further moulded by applying different chemical or physical forces and later become solid and non-reactive at average room temperature. They offer several technical advantages including processibility, light weight, flame-retardance, lubricity, chemical resistance, and toughness depending on the type of plastic, thereby flourishing the plastic production. The polymers alone will not meet the requirements of becoming plastic. Furthermore, additives play a significant role in contributing to the substantial enhancements for the weatherability and durability of polymer products [22]. Additives are insoluble particles, classified as either organic or inorganic, which are incorporated into the base polymer to bestow specific colour and also for functional benefits upon being plastics [23]. Most of the plastic additives are not covalently bound with the polymer chain and hence capable of easy migration. Particulate additives can affect the abiotic weathering of plastics by means of creating microcracks in the plastic chain and promoting microplastic formation [24], [25]. Additives with metals can function as pro-oxidants and/or photo-oxidant and subsequently facilitate microplastic production [26], [27], [28].

Furthermore, in exploring the surface chemistry, the optimum resin adhesion is an important aspect, particularly concerning fibre-reinforced plastics like fibreglass. They are durable and offer high thermal insulation, and their applications cover the beverage industry, chemical industry, car washes, food processing, fountains and aquariums, and pulp and paper industry. The energy of adhesion per square centimetre, W_A, should be optimised to attain the desired plastic-fibre strength [29]. Generally, petroleum, salt and air are the three raw materials involved in plastic synthesis. In the later stages, they convert into small organic monomers, further joining by polymerization reactions, giving out products with the required structural strength. For example, polyethylene, widely used in packaging containers, rope, carpeting, automobile batteries, and other appliances, is made in the following two steps. The starting material is primarily from light petroleum containing alkanes (Fig. 1). Likewise, another polymer, namely, polyvinyl chloride, is made in three steps from the starting material petroleum, Fig. 2, involving an additional chlorine, and air [30]. Also, polyethylene terephthalate (polyester) is in high demand in the textile industry, food packaging, and bottles of soft drinks (Fig. 3). In light of the recent reports, processes like weathering, environmental stress, improper disposal and degradation possibly cause the generation of micro and nano-sized plastic particles in the environment [31]. Among them, the plastic recycling industry can be a major contributor to the generation of tiny plastic particles that are sub-categorized in terms of large MPs (1–5 mm) and small MPs (˂1 mm) [32]. MPs (˂1 mm) gained increased attention in research as they affect cells and other organs of organisms [33], [11]. Moreover, conventional municipal waste disposal methods such as landfills and dumping grounds generate vast amounts of MPs compared to newly adapted sustainable methods such as incineration and composting [34]. Micro and nano plastic sources are systematically classified into primary and secondary. Based on the shift towards modern lifestyle, there is an increased dependence of the basic necessities on plastic products namely, personal care products as well as cosmetics, which undergo physical, chemical and biological degradation like photooxidation, to give rise to micro and nano-plastics [35]. Most recently, a Chinese study indicated an increased presence of micro and nano-plastics in people of urban origin, as compared to rural from the same Chongqing region [36].

Fig. 1.

Two steps involved in the formation of polyethylene from petroleum.

Fig. 2.

Three steps involved in the formation of polyvinyl chloride from petroleum, chlorine and air.

Fig. 3.

Three steps involved in the formation of polyethylene terephthalate from petroleum and air.

3. Can the detection of additives alongside plastic particles aid in identifying the sources and routes of micro and nano plastic entry in humans?

Micro and nano plastic exposure can be categorised into primary and secondary depending on an individual's daily lifestyle. Primary exposure generally includes microbeads or pellets from cosmetic products [37], [38] or plastic fibres from textile industries, while the secondary generally arise via the breakdown of larger particles into smaller ones (< 5 mm) from plastic products like bottles, and cups as a result of environmental stress [39]. Their application has been extended to the manufacturing of biomedical products like prosthetics, disposable blood bags, as well [40]. In the context of child and pregnancy-related aspects, microplastic exposure can occur through placental transfer, ingestion, breastfeeding, dermal absorption, and other pathways [41]. Several toxico-kinetic studies focus on the placental transfer of plastic particles detailing their bio-distribution in the human body. The susceptibility of humans towards micro and nano plastic exposure in the environmental food chain is high as they are the higher class of organisms. Four different routes are reported for higher possibility of microplastic exposure in humans, including ingestion, inhalation, contact, and entanglement [42]. Further studies on possible sources for microplastic exposure during pregnancy are unknown and yet to be analysed and focussed more.

Certain assumptions regarding the sources of plastic exposure during pregnancy can be made using additives that are reported along with plastic particles. A recent study portrayed microplastics as vectors for the exposure of chemical additives, based on particle size [43]. The relative contribution of microplastics as vectors for plastic additive chemicals decrease with increase in the particle size. The additives commonly used in plastic polymers include plasticizers, pigments, antioxidants, heat stabilisers, and residual or unreacted monomers [44], [45]. Table 1 details some of the commonly reported additives and their respective plastic polymers. Plasticizers are generally used in polymer industries to increase the softness and plasticity of products; pigments provide colour to the respective plastic products, and antioxidants help in delaying the oxidative degradation of plastic polymers [46], [26]. Plasticizers are widely used in PP, PVC, and PE polymers. Phthalates and adipates are two of the most commonly used plasticizers and are categorized as external plasticizers, since they do not bind with the plastic polymer and can migrate due to external factors, namely pH alterations, temperature, radiations [47]. Apart from additives or plasticizers, specific monomers are also employed in manufacturing plastics. One such monomer is bisphenol-A (BPA) [48] which is employed as a monomer in the production of polycarbonate plastics used as an inner lining of aluminium cans, [49] clear plastics, water bottles, baby bottles and the lining of food packaging cans. Hence, humans can get exposed to BPA from the food and beverage packings via leaching [50] resulting in significant endocrine disruption [51]. Hence food packaging, aluminium cans and bottles used for beverages can also be significant sources of microplastic exposure during pregnancy.

Table 1.

List of some of the commonly used additives in plastics and their reported toxic effects.

Additive used	Additive type	Effect	Reference
BPA	Bisphenol	Pro-inflammatory effects, miscarriage in women, obstruction of embryonic and fetal development	[52] [53] [54]
BPS	Bisphenol	Haemolysis, Cellular depletion of leukocytes, oxidative stress, DNA damage	[55]. [56] [57]
BPF	Bisphenol	Apoptosis induction, Cellular necrosis, genotoxic, damage of lipids and proteins in PBMCs	[58] [59] [60]
BPAF	Bisphenol	Apoptosis induction, Cellular necrosis, genotoxic, damage of lipids and proteins in PBMCs, Phenotypic alterations of monocyte	[58] [59] [60] [56]
BBP	Phthalates	Haemolysis, Eryptosis, and human neural tube defects, promote fibrogenesis of lung epithelial cell	[61] [62] [63]
DBP	Phthalates	Haemolysis, Eryptosis, Reduced phagocytosis, and antigen capacity Affect human sperm functions	[64] [61] [65]
DEP	Phthalates	Cytokines and chemokines production, reproductive toxicity, Hepatic toxicity	[66] [67]
DEHP	Phthalates	Inhibitory effect on testosterone activity, Immunoglobulins inhibition	[68] [69]
DPP	Phthalates	Preterm delivery	[70]
DiP	Phthalates	Activate pro-inflammatory response, Reduce phagocytic activity	[71]
PBDE	Flame retardants	Cytotoxic and genotoxic effects on pulmonary cells, altering menstrual status, dyslipidemia and blood pressure alteration	[72] [73] [74] [75]
Benzotriazole UVSs	UV stabilizers	Antagonistic activities against human estrogen receptors (ERα/β) and androgen receptor (AR)	[76] [77]
Lead	Metal additives, UV stabililzer, inorganic pigments	Brain damage, infertility	[78]
Cadmium	Metal additive, inorganic pigments	Carcinogenesis; cellular apoptosis; DNA methylation.	[79] [78]
Chromium	Metal additives, inorganic pigments	Allergic reactions affect on cardiovascular, respiratory, and neuronal system	[80] [81]
Cobalt	Metal additives, inorganic pigments	ROS production, affect on cardiovascular and neuronal system	[81] [82].
Copper (Cu)	Metal additives, biocides	Formation of ROS, genotoxic	[81]
Arsenic	Metal additives, biocides	Carcinogen	[78] [83] [84]
Antimony	Metal additives, biocides	Metal–estrogen; cancer	[85] [86]
Per- and poly-fluoroalkyl substances (PFAS)	Water, oil and stain-resistant	Liver and kidney diseases, thyroid dysfunction, adverse effect on the reproductive system and developmental outcomes, carcinogenic	[87] [88] [89] [90]
Polycyclic aromatic hydrocarbon (PAHS)	Aromatic hydrocarbon	Carcinogenic, Genotoxic, Teratogenic, Immunotoxic	[91]. [92] [93]
Alkylphenols and alkylphenol ethoxylates	Plasticizer	Immunotoxic, neurotoxic	[94]

Phthalate, or phthalic acid ester, is a plasticizer generally used in personal-care products, PVC manufacturing, and food packaging to increase the durability and flexibility of the products. This endocrine-disrupting chemical [95] possesses non-covalent bonds with plastics and can get easily released into the environment during manufacturing, use, and disposal [96], [97]. In 2022, Zuri et al. reported the presence of dibutyl phthalate (DBP) release along with the presence of MPs from facial masks, an essential commodity during the COVID phase and afterwards globally [98]. Being a plasticizer, DBP is generally applied in vinyl fabrics, especially poly vinyl-based food wraps, for flexibility and pliability [99]. In light of these reports, it becomes evident that additives or plasticizers help navigate the main microplastic exposure routes. Adding to this, the primary sources of DBP exposure can be food wraps, aluminium cans, masks, cosmetics, etc., via ingestion and inhalation [100].

Textile industries could also be one of the sources of plastic exposure. The co-occurrence of hostopen violet, an organic compound used in textile industries, along with polypropylene microplastics is reported on the fetal side of the placental samples studied [18]. In 2016, an increase in heavy metal adsorption to plastic surfaces was reported from the marine environment as the source [101]. In this study, Brennecke et al., demonstrated the release of Cu and Zn in sea water from the antifouling paints where these metal ions are trapped/adsorbed on the polystyrene and polyvinyl chloride surface. Several studies evidenced the presence of phthalate esters (PAEs), like diethyl phthalate (DEP) and diphenyl phthalate (DPP) along with microplastics fragments obtained from river sediments. [102], [103]. The sources of DEP enroute to the cosmetic products, which can be another source for the microplastic exposure [104]. Pigments like copper phthalocyanine was identified along with polypropylene, from the maternal and fetal side of the placenta [18]. Copper phthalocyanine is a blue-coloured pigment generally used for staining plastic products, and their presence has also been reported in toothpaste as well [105], [106], [107].So the possibility of toothpaste as one of the sources for microplastic exposure cannot be ruled out quickly as plastics like polypropylene is also reported in toothpaste [108] Fig. 4.

Fig. 4.

Illustration depicting the diverse sources of plastics that can be the potential sources of micro and nano plastic exposure during pregnancy period.

4. Cellular conundrum: a comprehensive assessment of microplastic-induced cytotoxicity

In light of the 8.3 million metric tons of plastic production reported in 2017, the continuation of production and environmental accumulation can be expected to increase in the coming years [109]. The ecotoxicological effects have been well studied and reported based on the evidence to date. As the production flourishes, there comes the problem of disposal which points finally to the marine environment as plastic litter [110]. The biological effects of micro and nanoplastics on aquatic organisms and other invertebrates have been extensively documented. However, their impacts on mammalian systems needs rigorous investigation.

As discussed in the previous section, microplastic entry occurs through conventional routes, such as oral ingestion, inhalation, and skin contact [111]. Once entering the organism, it is believed that these plastic particles are excreted out through the gastrointestinal tract as well as the biliary tract [112]. This was disproved once the presence of MPs was detected in blood. Quantifiable rates of PET, PS and PE were detected in a blood sample, reaching a maximum concentration of 2.4 µg/mL, 4.8 µg/mL, 7.1 µg/mL respectively [111]. Notably, a single sample revealed the presence of up to three different polymer types. Nevertheless, cellular uptake and cytotoxicity mechanisms are contingent upon the specific cell type.

Determining the particle size and concentration is critical concerning the cytotoxicity induced by plastic particles. A recent study by Han et al. delves into the size-dependent toxicity of polystyrene nanoplastics [113] correlating the particle size and its respective penetration ability. It reported the higher penetrative ability of particles with smaller diameters compared to larger counterparts that adhere to the cell membrane. Earlier in 2003, Wilhelm et al. reported the presence of a two-step process involved in the entry of anionic nanoparticles into macrophage cells where internalisation succeeds initial cell membrane binding [114]. Later, studies found that nanoparticles with considerably smaller diameters take two different ways to traverse the cells: energy-dependent and energy-independent pathways [115], [116]. Hence, a time-dependent phagocytosis has been noted in mammalian and human gastric fibroblast cells, with diminished mitochondrial membrane potential and consequent DNA damage. More than being the powerhouse of a cell, mitochondria play a significant role in apoptosis as well [117]. The regulation of protein release by bcl2 family members from the intermembrane space of mitochondria activates caspase proteases in the cytosol, further activating apoptosis in cells. The accumulation of micro and nano plastics in mitochondria can pose a threat to bioenergetics as well as programmed cell death. The investigation done by Huang et al. reported the impact of polystyrene nano plastics (PS-NP) on mitochondrial function by exploring dopaminergic-differentiated SH-SY5Y neural cells [118]. The study identified a dose-dependent decline in cellular ATP content and a reduction in CI-NDUFB8 levels, an accessory subunit of NADH dehydrogenase (ubiquinone), through protein expression analysis resulting in the impaired electron transport chain (Illustration is provided in Fig. 5). These observations collectively suggest that heightened PS-NP dosage affects mitochondrial functioning in cells. In-depth studies on micro and nano plastics induced genotoxicity, in terms of DNA strand breaks and micronuclei formation can be the future focus [119]. Nanoparticle-induced DNA double-strand breaks in human lung epithelial cells and macrophages were studied in 2015 [120]. The report detected a significant increase in the double-strand breaks of the treated group compared with that of the control using γ-H2Ax foci detection method.

Fig. 5.

Nano plastics affects the mitochondrial functioning in cells. Pathway by which nano plastics alter the cellular ATP production as well as induce apoptosis in cells.

5. Diverse chemical exposures during pregnancy: exploring prenatal developmental toxic exposures and their impact on reproductive health

During early developmental stages, organisms exhibit heightened sensitivity compared to other phases in their life cycle, making them more susceptible to adverse environmental conditions. A successful development is determined by the cellular mechanisms that offer resilience from environmental perturbations and regulatory pathways that modify the developmental trajectory. Any disruption to these factors may result in adverse outcomes for development and, in severe instances, teratogenic effects. Each year, approximately 3.5 million births occur, with an estimated 4–6 % of congenital disabilities attributed to exposure to chemical agents in our environment [121]. The prenatal developmental period is indeed a critical and challenging period for an organism. This intricate phase of the life cycle encompasses complex biological processes and potential vulnerabilities that warrant careful consideration in research and risk assessment. Investigating the possible toxicities in the developmental process of an organism provide insights into the underlying mechanisms of embryonic and fetal development [122]. It helps to identify and understand the potential risks associated with exposure to various chemicals or environmental factors during the critical development phase. Also, it contributes for establishing regulatory guidelines and standards for chemical safety. The regulation of hazardous substances is a crucial aspect for public health and environmental protection. According to the conditions mentioned in the plastic waste management rules of 2016 in India, plastic carry bags and packaging shall be strictly adhered to the use of pigments or colorants that fall under the Indian Standard;: IS 9833:2018 [123]. Also, the effectiveness of the Indian Standards of the plastics quality have to be checked in concern with the microplastic exposure and public health [124]. Developmental toxicity tests help to analyse and evaluate the possible alterations that could be caused during the developmental path of an organism during its lifetime.

A targeted approach to exploring xenobiotic-induced disease pathogenesis can gain significance concerning fetal health. For example, K et al. conducted a study that links altered fetal blood cholesterol levels with xenobiotic exposure [125]. It details the change in maternal cholesterol metabolic function influenced by xenobiotic exposure that further affects the fetal side. Once entering the human body, a xenobiotic undergoes substantial metabolism, influenced by its structure that further involves both human and microbial processes [126]. In light of this, certain drugs can also pose risks during fetal development. We have also established the reversal of embryotoxic effects of Valproic acid using Arachidonic acid using a 3D embryoid body model of embryotoxicity [127]. Hence, pregnant individuals must consult healthcare professionals before taking any medications during pregnancy. Some drugs that are reported to have teratogenic effects [128] that interferes with normal fetal development, resulting in congenital malformations, has been listed in Table 2.

Table 2.

Drugs that are reported to cause teratogenicity based on their respective concentrations.

Name of the drug	The concentration at which the drug works as a teratogen	Scenarios when the embryos get exposed to such teratogenic drug concentrations	Reference
Valproic acid (VPA)	644 ± 310 mg/day	When a pregnant lady has epilepsy and is advised to take this anti-epileptic drug	[129]
Thalidomide analogue	100 μg/ mL, 160 μg/kg	Pregnant ladies with morning sickness earlier used this drug, which is now banned. Now, this drug is used for treating leprosy in Brazil	[130]
Alcohol	25 mM;	Pregnant ladies consuming alcohol, especially in the first trimester of pregnancy	[131]
Vitamin A	10,000 IU (International units) or 250 mcg	Often hyper Vitamin A supplementation in pregnant women	[132]
Testosterone	10 mg/mL.	Pregnant ladies consume certain drugs whose byproduct is the steroid testosterone, or else women with abnormally high levels of testosterone in their blood.	[133]
Warfarin	˃5 mg;.	Pregnant ladies who earlier had a mechanical heart-valve replacement cardiac surgery and are on lifelong warfarin medication pose a threat to the embryo.	[134]
Streptomycin	200 mg/ mL	As a prescribed medication, sometimes during pregnancy	[135]
Aspirin	˃ 60 mg/ day.	As a prescribed medication for pregnant women	[136]

The impact of micro and nano plastics in posing risk to the health of adults were reported to date, but their subsequent impact on the next generations has to focussed more[137]. Several animal models were used to study the potential transgenerational effects caused by micro and nano plastics during maternal exposure. A study reported altered cholesterol levels in the F1 generation of mice progenies, that were exposed to 0.5 and 5 μm polystyrene microplastics during pregnancy [138]. Higher effects were observed among the mice groups treated with 5um microplastic. The study discusses potential of micro and nanoplastics in causing risk to the metabolic profile new borns that could even affect the subsequent generations as well. The exposure time of micro and nano plastics can be gestational and lactation period of the female. In 2019, a mice model study detailed the hepatic fluid accumulation and potential serum metabolite changes reported in the F1 progenies [139]. The exposure of polystyrene microplastics during gestataion and lactataion period also caused intergenerational effects in F1 and F2 progenies posing risk to their metabolic profile. Polystyrene microplastics are also reported to induce ROS generation that further resulted in disrupted testicular germ cell quality leading to reduced survival of F1 larvae of freshwater prawn [140]. Other plastic polymers including polyethylene was also reported to induce transgenerational effects in other model organisms. A recent study reported the transgenerational epigenetic effects in Pimephales promelas induced by polyethylene microplastics [141]. The study detailed the changes in methylation levels across single nucleotides and differentially methylated regions (DMRs) in parent generation as well as the subsequent F1 and F2 generations of the model organism. Additives also play a crucial role in inducing transgenerational effects together with plastic polymers [142]. Polyethylene microplastic polymer together with benzophenone-3 additive was reported to significantly reduce the somatic growth and reproduction in the F3 generation of Daphnia magna [143].

6. Unravelling embryonic vulnerability: navigating the spectrum of potential embryonic hazards

The gestational period is crucial for mammals as fetal development is sensitive to various environmental chemical exposures also [144]. It is established that a singular chemical exposure during neonatal life results in altered cytochrome P450 expression profile, CYP450 isoenzymes [145]. About 99 % of the pregnant women were reported with detectable amounts of chemicals, including phthalates, perchlorate, bisphenol A, and perchlorate [146]. Reduced birth weight is a vital issue reported as a result of exposure towards various endocrine-disrupting chemicals like phthalates, bisphenol A (BPA), polybrominated diphenyl ethers (PBDEs), and organochlorine pesticides (OCPs) [147]. Growing evidence points to the fact that changes occurring during the prenatal period might result in substantial changes in maternal health as well in terms of maternal hypertensive disorders of pregnancy or HDP [146]. The long-term effects on women's health, like preeclampsia, gestational hypertension, and chronic hypertension, are well realised. Several environmentally derived chemicals detected from mothers' urine and blood samples adversely affect the health of developing fetus also [148]. This concern arose from the detection of similar chemicals found in cord blood, amniotic fluid and meconium [149]. More than 80,000 registered chemicals in the United States persist in different elements of ecosystem that can degrade and alter the homeostasis of humans. As a result of these exposures there occurs conditions like congenital disabilities, childhood morbidity, neurodevelopmental effects, and even cancer [150].

Depending on the production, process, and disposal, plastics possess positive as well as negative impacts on the environment. In other words, it acts as a carrier for various chemicals such as bisphenol A, acrolein, phthalates and other additives, followed by their release upon external stresses [151]. Several 3D in vitro models have discussed these chemicals' potential toxicity to date. One such study reported a dose-dependent attenuation of miR-134 expression, elicited by bisphenol A exposure, resulting in altered reproductive and differentiation efficiencies of both mouse embryonic stem cells and, hence, derived embryoid bodies [152]. Also, endocrine-disrupting chemicals like phthalates were frequently detected in the urine samples of pregnant women [153] that may have the potential to cross the placenta, reaching the fetal side which needs to be validated in the future.

7. Interplay of microplastics in embryonic development; exploring reproductive tract invasion of micro and nano plastics in organisms till date

Until now, all the previous sections set the stage for embryonic development and plastics. Embryotoxicity is the disruption caused during embryonic development regarding morphological and metabolic changes, including customary growth, organogenesis, and differentiation [154]. These disturbances can arise due to exposure to toxic substances or environmental xenobiotics based on their dosage regimen and the chemical or physical properties governing them [144]. The resulting accumulation could potentially be a threat to their reproductive health. This was evident from the report published in 2016 detailing a significant reduction in the oocyte number and size of oyster larvae and an alteration in fecundity caused as a result of polystyrene microplastic exposure [155]. Our group is also involved in active research on embryotoxicity using 3D embryoid body models derived from pluripotent stem cells [127].

Furthermore, research focussed on polystyrene nanoparticle exposure in Daphnia galeata estimated a low hatching rate of embryos and high lipid storage, finally marking abnormal embryonic development [156]. The reproductive toxicity in cladoceran species was extensively studied by Jaikumar et al., describing the sensitivity of different cladoceran species and the influence of MPs towards their brood sizes and finally impairing their reproductive output [157]. A synergistic effect of microplastic with other toxic chemicals and seawater acidification is deleterious for the early development of echinoderms and molluscs as well [158], [159]. Surprisingly, even biodegradable MPs such as PCL, PHB and PLA at high concentrations delayed the development and caused malformations in sea urchin embryos [160]. Most of the recent studies on MPs were done on zebrafish models. Among the various causes for embryotoxicity, oxidative stress, inflammatory responses, induction of apoptosis, endocrine disruption, and neurotoxicity were predominant in zebrafish embryo models [161], [162], [163], [164], [165]. In a pregnant mice model, where polystyrene MPs of size 5 µm were administered through drinking water at different concentrations (10² ng/L to 10⁶ ng/L) throughout the gestational period, a significant reduction in lysine and glucose levels was observed [166]. Further studies showed that polystyrene microparticles have neurotoxic effects, which is evident from the impaired social behaviours in the offspring [167]. Higher concentrations (10 mg/L) of polystyrene MPs of size 100 nm in pregnant mice resulted in abnormal cell morphologies of the placenta and fetus with a significant reduction in lipid metabolism and fetal growth [168], [169]. Endometrial thinning and uterine fibrosis can occur in such placentas with upregulation of TLR4 and NOX2 resulting in oxidative stress [170]. Using pregnant mice model, R. Zhang et al., reported the adverse pregnancy outcomes after polystyrene MPs exposure including intra uterine death and fetal growth restriction (FGR) [171]. It also caused reduction of placental junctional zones resulting in the suppressed nutritional supply to the growing fetus. These polystyrene nanoparticles can also cross the blood-brain barrier and enter the fetal thalamus, causing oxidative stress and apoptosis as well [172]. There are studies that reported the sorption of environmental contaminants to the hydrophobic surfaces of polystyrene microplastics that possibly causes enhanced toxic effects [173]. Enhanced adsorption of oxytetracycline was reported from the beached polystyrene foams than the virgin foams based on their increased surface area, porosity, and the rate of oxidation [174]. These environmental toxins may further get desorbed from the surfaces of microplastics due to external stress factors and causes toxic effects [175]. In 2023, a study reported the effect of co-exposure of polystyrene nanoplastics together with Cu that induced ROS generation in zebrafish embryos resulted in reduction in mitochondrial membrane potential and altered lipid utilization that affects the early stage development of embryos [176]. A recent study reported the behavioural aberrations, differential gene expression related to DNA damage, and downregulation of fatty acid metabolism caused due to the co-exposure of polystyrene nanoplastics with 6PPD-quinone in zebrafish model [177]. Another study has shown that exposure doses of MPs near the actual environmental exposure dose of humans, induced miscarriage in mice by activating autophagy and suppressing trophoblast cell migration [178]. In light of these reports the potential of MPs to cause reproductive toxicity that could eventually lead to miscarriage can be well understood.

The food chain significantly transfers micro and nano plastics to higher trophic levels, as evidenced by reported transfers from algae to zooplankton and ultimately to freshwater fish. [179]. A possible journey of micro and nano plastics in the ecological food chain is depicted in Fig. 6 [180], [181], [182]. On moving towards the higher trophic levels, through biomagnification, there comes a greater possibility for microplastic-induced toxicity in terms of human fetal development. Due to ethical concerns, research on the effect of MPs on human embryonic development is limited compared to the abundance of studies on non-human organisms. There have been a few observational studies in the past years in which MPs were detected in the uteroplacental unit. In a landmark study published in 2021, Ragusa et al. reported the first evidence of microplastic detection in human placental samples alerted the scientific community [18]. The placenta, being a temporary organ of the uterus, plays several vital functions, such as nutrient and oxygen supply via the maternal bloodstream to the fetus, immune protection, and waste removal. Using Raman microspectroscopy, the study identified 12 MPs in four human placentas, confirmed in both the fetal and chorioamniotic membranes. The spectroscopic data provides intriguing insights into the size, colour, and chemical characteristics of the identified MPs and their associated pigments, namely Copper phthalocyanine (Pigment Blue 15; C.I. Constitution 74160) and Hostopen violet (Pigment Violet 23; C.I. Constitution 51319). Furthermore, MPs were detected in the syncytiotrophoblast, the outermost layer of the placenta richly covered with microvilli [183]. Their presence in the endothelial cells, combined with the narrowing of the fetal capillaries, was observed along with the dilation of the endoplasmic reticulum and swollen electrodense mitochondria. These results validate the higher possibility of microplastic ingression into the embryonic structure. The health effects of exposure to micro and nano plastics during pregnancy still have received limited investigation or understanding [41]. In another study, polyamide and polyurethane MPs of 20–50 µm were detected in placentas, meconium, infant faeces, breast milk and infant formula [184]. The presumed exposure sources of MPs were drinking water and scrub cleanser or toothpaste used during the pregnancy, and for the newborn, it was breastmilk and plastic toys [184], also diapers [41], [185]. The presence of MPs is inversely related to the Chao index of the microbiota of the placenta and meconium[186]. There is a significant negative association between the levels of MPs in the amniotic fluid and the gestational age [187]. Bottled water was pointed out as one of the primary sources of MPs in most of the studies. Further investigations are needed to understand how these particles are transmitted from the mother to the fetus. Possible damages caused by MPs on human embryo and placenta has been summarised in Fig. 7. The traditional method of stem cell research involves growing cells in a two dimentional (2D) flat support [188]. But it arises with a limitation of the inability of 2D culture to accurately mimic the natural microenvironment and physiological conditions experienced by cells in vivo. Advancements in stem cell research for mitigating the issue of ethics in conducting embryotoxicity research are irreplaceable.

Fig. 6.

The potential pathway of micro and nano plastics through the ecological food chain, emphasizing the higher possibility of accumulation in organisms at higher trophic levels ultimately impacting humans, specifically pregnant women.

Fig. 7.

Potential damages induced by microplastics on the embryo and extraembryonic membranes.

8. Advancements in embryotoxicity research: exploring the potential of 3D In vitro models as innovative research

A decade ago, a research group from Switzerland demonstrated for the first time that polystyrene nanoparticles of size up to 240 nm can cross the placental barrier using an ex vivo human placental perfusion model [189], [190]. Following that, studies on in vitro models using choriocarcinoma and trophoblast cell lines assessed the physiological and toxicological parameters as well as the possible mechanisms for the transfer of polystyrene MPs across the placental barrier [191], [192], [193], [194], [195]. The mechanisms involved in the translocation of polystyrene nanoplastics was studied using choriocarcinomic human placental model [194]. Specific inhibitors of ABC transporters, including MK571, inhibitor of multidrug resistance protein (MRP-1), were applied to study the compartmental distribution of PS-NPs in the cells. The study reported with increase in PS-NPs distribution in basolateral compartment than the apical compartment. Several other in vitro human placental models are used in research, such as transwell cultures, 3D placental spheroids, placenta-on-a-chip and explant cultures [196]. A major limitation of all these models is that they focused only on the extraembryonic organs, and the embryo proper was absent. It is crucial to select the cell of interest for designing a 3D in vitro, as it reflects the cell architecture of the model. Also, factors like non-cytotoxicity, porosity, and degradability are crucial aspects to be monitored and controlled for building a 3D system [197]. A major challenge associated with culturing 3D models is ensuring adequate nutrients and oxygen supply. It is also required to ensure the periodic waste removal from the 3D models to maintain the long term viability and functionality [197]. The inconsistency in the shape and size of the 3D in vitro models represent a major limitation of the system [198]. Moreover, dynamic 3D cultures are well suited, as compared to, static cultures. Currently, various laboratories across the globe have been using various 3D dynamic culture systems namely, Synthecon rotatory cell culture system (Synthecon Inc. Texas, USA. https://www.synthecon.com/) and advanced organ-on-chip MIVO® technology for 3D humanized tissue models (React4Life, Italy https://www.react4life.com/). Most important advantages with these novel technologies is the scope in harvesting the cell culture supernatants during different time points of 3D culture for metabolomics studies and culture system modulation options such as timely supply of oxygen and nutrients.

Stem cell research holds tremendous potential for elucidating fundamental mechanisms of human development and differentiation, thereby presenting a beacon of hope for novel disease treatments. Human embryonic stem cell (hESC) research is controversial as it beholds political controversies and ethical concerns regarding using pluripotent stem cells derived from the pre-developmental stages of the embryo. Induced pluripotent stem cells (iPSCs) reprogrammed from adult tissues do not have the ethical issues that are specific for hESCs. While the use of adult stem cells as well as cord blood stem cells have lesser ethical concerns compared to hESCs and are widely used in research and other clinical studies [199]. Studying the impact of micro and nano plastics in cellular microenvironment can be effectively achieved using 3D in vitro models. 3D cell cultures are essential for understanding cellular processes in both healthy and diseased states [200]. The cells must establish close contact and adhere to the surrounding extracellular matrix within a three-dimensional framework. Thus, it can mimic natural cell characteristics and architecture [201]. Hence, 3D in vitro models can be an alternative to the in vivo models and solve the limitations of 2D models. These include differences in cell morphology, polarity, and method of division that pose significant challenges in understanding cellular behaviour [202]. Likewise, in our laboratory, we have deciphered the reactive oxygen species (ROS) generating role of the antiepileptic drug Valproic Acid (VPA) in the 3D embryoid body models derived from the human pluripotent stem cells. The embryo-cytotoxic effects of VPA were then reversed by the treatment with the bioactive lipid Arachidonic acid in a 3D model of embryotoxicity [127].

In stem cell research, various 3D in vitro models mimic the complex cellular microenvironments in living tissues. Some prominent models include spheroids, organoids, embryoid bodies, and decellularized scaffolds [198]. In 2022, Hua et al. modelled hiPSC-derived 3D human forebrain cortical spheroids to study polystyrene microplastic-induced toxicity in the human brain [203]. The results revealed adverse effects on the development of embryonic brain-like tissue followed by microplastic exposure dependent on both size and concentration. Recently, it was found that polystyrene nano plastics reduced pluripotency in hESCs and induced apoptosis by promoting mitochondrial oxidative stress [204]. It also impaired the differentiation of hESCs to cardiomyocytes and cardiac organoids. MPs can also alter the transcriptomic and epigenomic signatures of fibroblasts used to derive iPSCs and can dysregulate the metabolic and homeostatic pathways [205]. To date, several human organoids are used widely for studying microplastic-induced toxicity. They include airway organoids [206], forebrain organoids [203], intestinal organoids [207], liver organoids [208], cardiac organoids [209] and many more.

In 2022, the idea of using human iPSC-derived embryoid bodies for testing the effect of nano plastic-induced toxicity was utilised [210]. A significant amount of nanoplastic internalization was reported in the study. Furthermore, regarding pluripotency, the quantitative assessment of mRNA levels showed altered expression in the self-renewal markers, specifically Nanog. The study further concluded the absence of significant alterations affecting the differentiation potential of EBs in light of the results detailing the 3-germ layer expression and successful neural differentiation. An in vitro placental barrier model using a transwell chamber containing trophoblast cells and 3D PSC EBs in the lower chamber is the best model to study the effect of embryotoxicity of xenobiotics [211]. This model can mimic the transfer of MPs from the mother’s side to the fetus, and the cytotoxicity on both sides can be studied.

9. Conclusion and future prospects

Despite the ongoing global concern for the health implications caused by plastic pollution, a significant knowledge gap persists in understanding their impact on the ecosystem regarding their size. Their widespread accumulation followed by degradation resulted in the generation of micro and nano sized particles posing risk to environmental homeostasis. Although several reviews have been discussed concerning their effects on the ecosystems, less focus has been given to reproductive toxicity in organisms, especially humans. Research in reproductive toxicity caused by MPs strengthened when their presence was evidenced in the placenta. Further on, in depth focus is required in understanding their potential toxicity in concern with human fetal development. Embryonic development is crucial, where a single-celled zygote undergoes multiple divisions and differentiations to give rise to the final multicellular organism. The pregnancy period is so sensitive that hindrance to any sequential steps can alter the normal embryonic developmental process. Hindrance can be in the form of chemical exposures, drug toxicity, food habits or any other ways that can negatively affect fetal development. In this review, we have focussed on the possible routes of microplastic exposure that can occur during the pregnancy period using additives as the markers for the sources of microplastic generation. Further, the ecological food chain has been taken into account to study the idea of micro and nano plastic transfer through different trophic levels finally reaching the human diet. Advancements in embryotoxicity research help to understand the adverse events that can cause risk to human embryonic development. Even though many medical and scientific works reported their cytotoxic effects in different cellular environments, their involvement in the cell cycle, protein metabolism, internalisation pathway, and multidifferentiation potential are yet to be explored further. In addressing the challenges associated with studying embryotoxicity, stem cell research provides valuable tools in the form of 3D in vitro models, lighting up the hope for future research. These models provide a three-dimensional environment mimicking the characteristics of the human embryo. In the context of micro and nano plastics, 3D in vitro models offer a controlled and ethical platform to explore toxic effects on embryonic development. These models enable researchers to assess the developmental and differentiation potential in a controlled environment. Thus, with their intricate surface chemistry, the generation of micro and nano plastics builds up a complex dimension to embryotoxicity research and pregnancy. The utilization of advanced 3D in vitro models further resolves the complexity of exploring these plastic particles' role in the embryotoxicity field. Further studies in this domain will potentially benefit to explore the complex mechanism and cellular pathways by which micro and nano plastics causing risks to the embryo development. Also, public awarness on strict regulation on plastic usage and disposal, developing strategies to quantify the amount of plastic particles expected to be reported from the food and water sources are required in concern with public health. Reducing the demand of plastic packing and strengthening the reduce-reuse strategies are crucial for an effective mitigation of plastic pollution. Moreover, public educational and awareness programs are essential for the consumsers to provide information about the generation and environmental impacts of micro and nano plastics. Plastic-Waste Management (Amendment) rules 2022 should be strengthened, that warns plastic manufactures to not provide the raw materials for any unregistered producer [212]. With the support of Governmental and other non-governmental organisations effective strategies and policies can be implemented against plastic pollution. In countries like Africa and Taiwan, plastic bags prohibition bill, such as the one implemented in Nigeria, and the concerted efforts of the stakeholders have demonstrated effectiveness in reducing plastic pollution [213], [214]. Whereas, the European Union (EU) has proposed a single-use plastics directive, that aims to ensure all the plastic packaging within the European market as reusabe or recyclable by the year 2030 [215], [216]. Also, effective water treatment strategies could be possible to reduce the load of plastic fragments from different water sources. This include both ex situ, electrocoagulation and magnetic extraction, and in situ methods such as biodegradation using microorganisms and biostimulation with natural surfactants, where further research is required to improve mitigation of micro and nano plastics exposure.

Funding information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

CRediT authorship contribution statement

Bose Bipasha: Writing – review & editing, Supervision, Resources, Project administration, Conceptualization. Shenoy P Sudheer: Writing – review & editing, Project administration, Conceptualization. Gupta Sebanti: Writing – review & editing, Supervision, Conceptualization. Nair Sanjay R.: Writing – original draft. Nihad Muhammad: Writing – review & editing, Writing – original draft, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Prof. L.H. Lash

Data availability

No data was used for the research described in the article.