Is Emerging Nanomedicine a Friend or Foe to Germ Cells?

This review emphasises the dual nature of nanoparticles (NPs). In parallel with their rapid development for applications across biomedical fields, it is imperative to address their associated toxicological effects, especially those impacting reproductive health. As summarised in Table 1 , the adverse effects of various NPs, such as zinc oxide (ZnO), silver (Ag), and titanium dioxide (TiO 2 ), on both sperm and oocyte function have been shown to occur through mechanisms including oxidative stress, DNA damage, and disruption of key cellular processes; and these effects are now well-documented. Building upon this foundational knowledge, future research must pivot to address several critical areas, in order to ensure the safety and sustainable development of nanotechnologies. Table 1 Major Nanoparticles with Toxic Implications on Sperm and Oocytes Nanoparticles Species Reproductive Cells Biological Implications Ref. Zinc oxide nanoparticles Mouse (in vitro) Oocytes Induces oxidative stress and interferes with the process of meiosis [ 57 ] Mouse (in vitro and in vivo) Oocytes Induces DNA damage and affects pre- and post-natal oogenesis [ 58 ] Zebrafish (in vivo) Oocytes Activates autophagy and apoptosis, and increases oxidative stress [ 59 ] Rat (in vivo) Sperm Reduces sperm motility and increases sperm deformities [ 108 ] Human (in vitro) Sperm Increases cell death [ 100 ] Albino mice (in vivo) Sperm Decreases the weight of testes in a dose- and time-dependent manner [ 101 ] Silver nanoparticles Zebrafish (in vitro) Oocytes Induces oxidative stress, promotes apoptotic cell death [ 61 ] Mouse (in vitro and in vivo) Oocytes Induces oxidative stress, promotes apoptotic cell death [ 62 ] Albino rats (in vivo) Ovarian Reduces glutathione (GSH) and superoxide dismutase (SOD). [ 109 ] Human (in vitro) Sperm Increases ROS generation and DNA damage [ 92 ] Rat (in vivo) Sperm Causes damage to sperm plasma membrane and acrosome integrity [ 93 ] Mice (in vivo) Sperm Induces germ cell development dysfunction [ 110 ] Cerium dioxide nanoparticles Mouse (in vitro) Oocytes Increases DNA damage [ 63 ] Human (in vitro) Sperm Induces marked DNA damage [ 106 ] Mice (in vivo) Sperm Disrupts the antioxidant/oxidant balance, reducing sperm count and motility [ 105 ] Fullerenol nanoparticles Rat (in vitro) Oocytes Disrupts the tightly regulated process of meiotic resumption [ 64 ] Copper oxide nanoparticles Mice (in vivo) Oocytes Elevates ROS, increases DNA damage and causes apoptosis [ 66 ] Rat (in vivo) Oocytes Damages ovarian ultrastructural features [ 111 ] Rat (in vivo) Sperm Significantly increases the percentage of abnormal and dead sperm [ 104 ] Rat (in vivo) Sperm Distorts basement membranes of seminiferous tubules and causes seminiferous cell degeneration [ 112 ] Polystyrene nanoparticles Mouse (in vitro) Oocytes Elevates oxidative stress and reduces translation efficiency [ 68 ] Bovine (in vitro) Oocytes Impairs nuclear maturation [ 113 ] Mice (in vivo) Oocytes Induces apoptosis and autophagy, and disrupts steroidogenesis [ 107 ] Mice (in vivo) Sperm Induces oxidative stress and damages testicular microstructure and functions [ 114 ] Zero-valent iron nanoparticles Mice (in vivo) Oocytes Inhibits first polar body generation [ 70 ] Perylene nanoparticles Mice (in vivo) Oocytes Elevates ROS levels and causes apoptosis [ 71 ] Silica nanoparticles Mice (in vivo) Oocytes Disrupts meiotic recombination and increases apoptosis in oocytes [ 115 ] Mice (in vivo) Sperm Decreases RNF8 levels, suppressing histone-to-protamine exchange [ 85 ] Mice (in vivo) Sperm Inhibits the RNF8-ubH2A/ubH2B pathway resulting in incomplete histone-to-protamine exchange [ 86 ] Mice (in vivo) Sperm Elevates ROS levels, damaging mitochondrial structure, and DNA [ 87 ] Titanium dioxide nanoparticles Mice (in vivo) Sperm Capable of crossing the blood–testis barrier, and altering gene expression in testis [ 89 ] Mice (in vivo) Sperm Elevates ROS levels, inducing biochemical dysfunctions [ 90 ] Mice (in vivo) Sperm Increases ROS levels, causing DNA damage [ 91 ] Human (in vitro) Sperm Induces DNA damage [ 116 ] Gold nanoparticles Mice (in vivo) Sperm Affects sperm chromatin remodelling, increasing the rate of sperm DNA damage [ 96 ] Bovine (in vitro) Sperm Interacts with the sperm surface membrane, impairing sperm functions [ 97 ] Mouse (in vitro) Sperm Interacts with the double-helix of DNA, disturbing nuclear chromatin decondensation [ 98 ] Mice (in vitro) Sperm Spermicidal activity [ 117 ] Carbon black nanoparticles Mouse (in vivo) Sperm Increases oxidative stress and inflammation, damaging spermatogenesis [ 99 ] Iron oxide nanoparticles Boars (in vitro) Sperm Increases LPO levels, lowering sperm quality [ 118 ] Mouse (in vivo) Sperm Significantly decreases sperm motility [ 103 ] Graphene Oxide Nanosheets Human (in vitro) Oocytes Induces mitochondrial toxicity [ 119 ] Polylactic Acid Nanoplastic Mice (in vivo) Sperm Disrupts spermatogenesis and mitochondrial dysfunction [ 120 ] Major Nanoparticles with Toxic Implications on Sperm and Oocytes A primary objective should be to move beyond phenomenological observations, to facilitate establishment of clear, mechanistic structure-activity relationships that can predict the toxicity of NPs based on their physicochemical properties. This effort should be coupled with the advancement of “safe-by-design” principles, focusing on engineering NPs with enhanced biocompatibility, for instance, through surface modifications, biodegradable materials, or protective coatings, in order to mitigate unintended interactions with germ cells. Furthermore, there is an urgent need to define safe exposure thresholds and to develop more physiologically relevant in vitro models, such as reproductive organoids, for improved risk assessment. Investigating the long-term and epigenetic consequences of NP exposure on germ cells and subsequent generational health represents another vital frontier. Finally, exploration of adjuvant strategies, including the application of antioxidants to mitigate oxidative stress induced by NP exposure in germ cells, represents a critical need for mitigating the associated risks. By integrating deep mechanistic toxicology with innovative material science, the field of nanomedicine can find a way to fully harness the benefits of NPs while rigorously safeguarding reproductive health.

Nanotechnology has emerged as a transformative force in 21st century healthcare, demonstrating significant potential across various biomedical disciplines, with reproductive medicine representing a particularly promising but complex field for the application of nanomaterials. 1–4 The efficacy of reproductive function is fundamentally rooted in the highly orchestrated and sensitive processes of gametogenesis. 5 In males, spermatogenesis requires the mitotic proliferation of spermatogonia, meiosis, and the intricate transformation of haploid spermatids into mature spermatozoa, a process critically dependent on hormonal regulation and Sertoli cell support. 6 , 7 In females, oogenesis begins with primordial germ cells that develop into prophase I-arrested primary oocytes, which then resume meiosis upon gonadotropin stimulation, complete meiosis I, arrest again at metaphase II, and finish maturation during fertilization. 8 The fidelity of these processes is crucial for successful fertilization and embryonic development. Nanoparticles (NPs) are characterised by dimensions typically ranging from 1 to 100 nm, a high specific surface area, and easily modifiable surfaces, which can serve as versatile platforms for intervention in fundamental reproductive processes. 9–11 By taking advantage of these unique properties, engineered NPs can be used to precisely modulate specific physiological conditions, and enable targeted delivery of therapeutics designed to treat reproductive disorders or increase the efficacy of assisted reproduction. 12–15 Furthermore, NPs can also be applied to create advanced in vitro culture matrices, or enhance the precision of diagnostic imaging of reproductive systems. 16–18 In fact, because of these multi-faceted intervention capabilities, nanotechnology has the potential to revolutionise reproductive medicine. However, because of the physicochemical properties of nanoparticles (NPs), nanotechnology has come to be considered a “double-edged sword”, particularly in the context of reproductive health. 19 Unfortunately, the same physicochemical properties that enable therapeutic applications also facilitate unintended interactions with the delicate machinery of gametogenesis. 20 Considerable scientific evidence indicates that various NPs can traverse biological barriers, accumulate in reproductive tissues, and exert toxic effects. 21 , 22 Reports of interactions between these minute nanoparticles and the reproductive system have prompted inquiries into their impact on sperm and oocyte function, raising concerns about possible fertility disruptions. 4 , 23 Consequently, active development of nanomedical applications for reproduction, requires a deeper understanding of their interactions with the fundamental pathways of spermatogenesis and oogenesis. This review systematically evaluates the paradoxical nature of nanoparticles function in the context of reproductive biology, juxtaposing their promising applications against their potential to disrupt gametogenesis, with the goal of establishing a scientific foundation to guide the safe and sustainable advancement of nanomedicine in human reproduction.

In spite of their potential risks, emerging nanoparticles (NPs) have caused a paradigm shift in reproductive medicine, due to their superior and tuneable physicochemical properties. 24–27 The main principal advantage of NPs is that they enable direct, targeted delivery of drugs and other therapeutics. Functionalized nanocarriers can transport pharmaceuticals, genetic material, or antioxidants to specific reproductive cells or pathological sites, thereby augmenting therapeutic efficacy while circumventing systemic toxicity. 28 , 29 For example, Hao et al developed a self-assembling nanoparticle that precisely delivers siRNA to ovarian tumour sites to achieve targeted silencing of the SMARCE1 gene, affecting the activity of downstream proteases. Ingeniously, the developed NP can also monitor therapeutic response via its surface-conjugated peptide substrates, enabling non-invasive detection of SMARCE1 regulated proteases in urine in vivo. 30 Moreover, by leveraging the physicochemical properties of specific nanoparticles, Ding et al developed an intravenous, non-invasive platform for male contraception, based on iron oxide nanoparticles (IONPs) that can be magnetically guided to accumulate in the testes under external magnetic fields, in order to temporally induce localised hyperthermia and reversible suppression of fertility. 31 Currently, nanomaterials are increasingly being employed to optimise assisted reproductive technologies (ART), by enhancing gamete quality, supporting cryopreservation, and improving embryo culture systems. 14 For instance, addition of zinc oxide nanoparticles (ZnO NPs) to semen extenders has been shown to significantly improve sperm motility and membrane integrity in boar models. 15 , 16 For cryopreservation, Galmidi et al applied nanoliter-scale droplet confinement and a fundamental analysis of water diffusion, in order to develop a method that effectively reduces osmotic stress during the cryopreservation of human sperm, leading to a marked increase in post-thaw survival. 15 Furthermore, NPs can serve as potent contrast agents and biosensors for the early and accurate diagnosis of reproductive disorders. 18 , 32 Kumar et al constructed silica-coated gold nanorods conjugated with FITC, which allow for non-invasive diagnosis of deep-seated endometriosis via photoacoustic imaging, as well as the fluorescence-guided identification and thermal ablation of lesions. 33 In addition, nanotechnology has been used to develop innovative contraceptive strategies, including sustained-release hormone systems and novel approaches for reversible male contraception. 34 , 35 Yu et al designed an NIR-activated ferritin nanocage (HFn@BBT) for intravenous male contraception. This noninvasive system provides on-demand fertility control, achieving reversible or permanent infertility through a tuneable photothermal treatment. 36 These coordinated capabilities, spanning targeted therapy, ART optimisation, diagnostic imaging, and fertility regulation, collectively underscore the transformative potential of nanoscale platforms at addressing diverse reproductive health challenges. However, translation of nanotechnology and nanoparticles for clinical use in reproductive medicine necessitates ongoing rigorous safety evaluations.

Research indicates that NPs can infiltrate cellular membranes, engage with intracellular components, and induce immediate structural impairment. 37 This phenomenon has also been observed within the context of the reproductive system. For example, recent research has demonstrated that certain NPs, such as gold nanoparticles, are capable of permeating granulosa cells, and affecting their physiological functions, most notably in the context of their role in hormone secretion. 38 Furthermore, another recent investigation revealed that silica nanoparticles (SiNPs) can enter the luminal space of lysosomes within granulosa cells, and induce cytotoxic effects characterised by diminished cell viability and increased apoptosis in a dose-dependent manner. Similarly, SiNPs can induce perturbations in autophagy-associated proteins, thereby triggering autophagy; while at the same time elevating the BAX/BCL-2 ratio and increasing caspase-3 cleavage, culminating in activation of the mitochondrial-mediated caspase-dependent apoptotic signalling cascade. Also, SiNPs can elevate acidity levels within lysosomes, impairing lysosomal functionality. Through this impairment of lysosomal function, SiNPs indirectly induce dysfunctions in the cellular process of autophagy, which in turn cause follicular atresia by activating apoptotic pathways within granulosa cells. 39 Furthermore, NPs measuring 20 and 40 nm in size were rapidly internalized by epithelial cells within the upper female reproductive tract (FRT): within one hour, these NPs were detectable within the lymphatic ducts responsible for draining the FRT, as well as within the ileac lymph nodes (ILNs) and the mesenteric lymph nodes (MLNs). 40 The blood-testis barrier (BTB) is known for its exceptional tightness compared to other mammalian tissue barriers, and partitions the seminiferous epithelium into two distinct compartments, the basal and the adluminal, playing a pivotal role in facilitating spermatogenesis. Within the blood-testis barrier, tight junctions operate in conjunction with ectoplasmic specialisations, desmosomes and gap junctions, in order to establish a distinct microenvironment that is conducive for the progression of meiosis and subsequent transformation of spermatids into spermatozoa during spermiogenesis. In vitro and in vivo investigations suggest that certain nanoparticles can traverse the blood-testis barrier. Importantly, the ability of specific NPs to penetrate the BTB depends on various physicochemical attributes such as composition, morphology, size and surface coating. Recent research has addressed this issue by examining the process through which titanium dioxide nanoparticles (TiO 2 -NPs) traverse the BTB. It was revealed that exposure to TiO 2 -NPs resulted in increased oxidative stress, downregulation of TJ proteins (claudin-5, ZO-1, and occludin), disruption of TJ structure and a significant increase in the size of intercellular gaps between TM-4 cells. In fact, in TM-4 cells, the passage of TiO 2 -NPs across the BTB is facilitated by interference with actin-mediated adherens junctions. 41 Mechanistic studies revealed that nano-TiO 2 triggers structural impairment within the BTB via activation of MAPK signalling pathways, concomitant with increased levels of key BTB proteins such as F-actin, Claudin-11 and ZO-1, indicative of enhanced reactivity. Significantly, the introduction of nano-TiO 2 resulted in reduced BTB integrity, leading to decreased sperm motility and a greater incidence of sperm morphological abnormalities. 42 In addition, Au NPs undergo retrograde transport from the adluminal compartment to the interstitial compartment of the testes through Sertoli cell-mediated endocytosis and exocytosis. This process can cause damage and has been shown to trigger the release of inflammatory cytokines within mouse testis. 43

In sperm cells, the toxicity induced by nanoparticles is primarily manifested through disruptions of histone-protamine exchange, p53-mediated mitochondrial apoptosis, and increased ROS generation. Mammalian spermatogenesis is an intricate developmental procedure in which male germ cells are subjected to a sequence of precisely controlled molecular events, resulting in the formation of mature gametes capable of fertilising an oocyte. 72 During spermatogenesis, a 106-fold condensation of DNA occurs that is crucial for encapsulating the paternal genome within the compact sperm nuclei. In oocytes and somatic cell nuclei, nucleosome-based chromatin packaging involves histone octamers, resulting in a bead-on-a-string configuration. In contrast, the sperm genome is condensed by small, arginine-rich basic proteins called protamines (P1 and P2). This alternative packaging method is believed to help organise DNA into toroidal structures, achieving a chromatin condensation level 10-fold greater than that found in somatic cell nuclei. 73 Abnormalities in this transition from histones to protamines are associated with irregular chromatin packaging, which may contribute to male infertility by increasing the risk of DNA damage or causing improper epigenetic modifications. 74 Histone removal and degradation are, at least in part, regulated by the ubiquitin-proteasome system. RNF8, an E3 ubiquitin ligase, is involved in DNA damage repair mechanisms through histone ubiquitination. RNF8 is essential for monoubiquitination of histones H2A and H2B, and knockout of RNF8 results in male sterility in mice. 75 Furthermore, Lethal (3) malignant brain tumour like 2 (L3MBTL2) belongs to the family of MBT-domain proteins, which have a significant influence on chromatin remodelling that is crucial for meiosis and spermatogenesis. L3MBTL2 is significantly upregulated in pachytene spermatocytes localised within the testicular environment. Thus, not surprisingly, deletion of L3MBTL2 resulted in increased formation of aberrant sperm cells, a gradual decline in spermatozoa numbers, and premature onset of testicular dysfunction in murine models. Furthermore, the absence of L3MBTL2 also resulted in increased deposition of γH2AX within leptotene spermatocytes, which incorrectly persists on autosomes later in meiosis I. γH2AX is recognized as a highly effective biomarker for assessing DNA damage and repair processes. This phenomenon is associated with impaired crossing-over of chromosomes and synapsis processes during the pachytene phase of meiosis I, coupled with increased germ cell apoptosis. Notably, in GC2 cells, an interaction between L3MBTL2 and the histone ubiquitin ligase RNF8 was observed, and depletion of L3MBTL2 resulted in a decrease in the nuclear levels of both RNF8 and ubH2A. The absence of L3MBTL2 resulted in inhibition of RNF8 and ubH2A pathways, as well as histone acetylation, resulting in elongated spermatids. This, in turn, inhibits protamine 1 deposition and leads to impaired chromatin condensation during spermiogenesis in sperm. 76 P53 serves as a key regulator that modulates cellular reactions to diverse stress stimuli, by controlling processes such as apoptosis, cell cycle arrest, senescence, DNA repair and the preservation of genetic integrity. Over the years, extensive investigations have elucidated the multi-faceted functions of P53. 77 While traditionally recognised as a transcriptional regulator, p53 has also garnered increasing attention because of its direct involvement in non-transcriptional processes outside of the nucleus; particularly in the context of mitochondria-mediated cell death pathways. This involvement is attributed to the accumulation of p53 protein in both cytosolic and mitochondrial locations, as well as its engagement in protein-protein interactions. 78 For example, p53 interacts with the permeability transition pore complex (PTPC) on the mitochondrial membrane, inducing pore opening. In turn, pore opening in PTPC leads to osmotic swelling in the mitochondria, and subsequently mitochondrial outer membrane permeabilisation (MOMP). Cytosolic p53 can also activate multidomain proapoptotic proteins of the Bcl-2 family, such as Bax and Bak, promoting the formation of multimeric pores that facilitate release of cytotoxic proteins from the mitochondrial intermembrane space (IMS) and/or interactions between Bax/Bak and PTPC components that induce MOMP. Moreover, p53 can inhibit anti-apoptotic Bcl-2-like proteins, such as Bcl-2 and Bcl-XL, which typically regulate their pro-apoptotic counterparts such as Bax, Bak, and Bid. 79 Because of the central role of p53-mediated mitochondrial apoptosis in the elimination of sperm, this pathway has become an important target of infertility treatment. In this regard, recent studies have shown that 4-Nonylphenol (NP) induces apoptosis and hormonal deficiencies, and may impair spermatogenesis and sperm function through p53-independent Fas/FasL-Bax/Bcl-2 pathways. 80 In sperm, ROS-induced damage is a major factor that contributes to infertility in 30–80% of affected men. Recent experimental evidence suggests that ROS may induce DNA damage in sperm, thereby significantly impacting the pathogenesis of infertility. Therefore, the assessment of oxidative status, antioxidant defence mechanisms, and DNA damage could serve as valuable diagnostic and therapeutic indicators for male infertility. 81 Moreover, ROS levels also significantly influence motility in spermatozoa. In fact, it was observed that elevated ROS levels are associated with an uncoupling of electron transport and adenosine triphosphate synthesis, reducing mitochondrial respiration in sperm, and subsequently causing a decrease in spermatozoa motility. 82 Similarly, Kao et al observed a notable inverse association between sperm motility and the levels of 8-OHdG, as well as between sperm motility and lipid peroxides. In addition, they observed a positive association between sperm motility and the concentrations of ascorbate, α-tocopherol, retinol and protein thiols within seminal plasma. In support of this, elevated levels of oxidative stress and subsequent oxidative damage were observed in spermatozoa with reduced motility; while diminished antioxidant capacities were found within both the spermatozoa and seminal plasma of males with infertility or subfertility 83 ( Figure 2 ). Figure 2 Potential mechanisms of nanoparticle-induced toxic effects on sperm function, based on recent findings. 73 , 74 , 77 , 78 , 80 , 81 Created by Figdraw. Potential mechanisms of nanoparticle-induced toxic effects on sperm function, based on recent findings. 73 , 74 , 77 , 78 , 80 , 81 Created by Figdraw. Nanosilica refers to a nanostructured form (<100 nm) of silicon dioxide, or silica nanoparticles (Si-NPs), that possesses unique physico-chemical properties in contrast to its bulk counterpart: because of its reduced dimensions, Si-NPs have a greater surface-to-volume ratio and enhanced surface reactivity. 84 According to a recent investigation, Si-NPs may impact sperm maturation within the epididymis and have adverse effects on human reproductive function. Si-NPs have been associated with a reduction in sperm count and sperm motility, along with an increase in the rate of sperm abnormalities and structural damage to the testes. Furthermore, Si-NP exposure has led to a decrease in the expression of Protamine 1 (PRM1) protein and an elevation in histone levels, resulting in inhibition of chromatin condensation in sperm. In addition, Si-NP exposure markedly reduced the levels of ubiquitinated histone H2A (ubH2A)/H2B (ubH2B) and RING finger protein 8 (RNF8) within the nucleus of spermatids; while increasing RNF8 levels in the spermatid cytoplasm. Notably, by the 35th day of exposure to Si-NPs, there was a notable increase in the protein expression levels of PIWI-like protein 1 (MIWI) within late spermatids. Also, Si-NPs have the potential to reduce RNF8 levels within the spermatid nucleus, either through upregulation of MIWI expression or by suppressing its degradation. This causes sequestration of RNF8 in the cytoplasm, potentially inhibiting RNF8-mediated ubiquitination of ubH2A and ubH2B. These processes ultimately interfere with removal of H2A and H2B and chromatin condensation, and consequently inhibit the differentiation of round spermatids and chromatin remodelling, resulting in compromised sperm quality and quantity. 85 Also, exposure to Si-NPs resulted in decreased sperm motility, the appearance of histological anomalies in the seminiferous epithelium, and apoptosis of spermatogenic cells, correlating with diminished levels of Lethal (3) malignant brain tumour like 2 (L3MBTL2) and activation of DNA damage-p53-mitochondrial apoptosis pathways. Moreover, the decreased L3MBTL2 levels induced by Si-NP exposure also cause reduced expression of components of the RNF8-ubH2A/ubH2B pathway, leading to incomplete histone-to-protamine exchange. These findings indicate that suppression of Si-NP-induced L3MBTL2 not only triggers the DNA damage-p53-mitochondrial apoptosis pathway, which leads to apoptosis in spermatogenic cells, but also inhibits the RNF8-ubH2A/ubH2B pathway, resulting in incomplete histone-to-protamine exchange and negatively affecting spermatogenesis. Through the above mechanisms, Si-NPs contribute significantly to reproductive toxicity by downregulating L3MBTL2. 86 Furthermore, recent investigations have examined the impact of Si-NPs on spermatogenic processes over various time intervals. Si-NP exposure resulted in disruption of mitochondrial cristae and a concomitant decrease in ATP levels, leading to a state of oxidative stress within testicular tissues by days 15 and 35 after Si-NP exposure. However, these adverse effects appear to be transient, since complete restoration of testicular structure and function was observed by day 60. Furthermore, silica nanoparticles can induce DNA damage and reduce the quantity and quality of epididymal sperm by days 15 and 35 following exposure; however, these effects are reversed by day 60. In contrast, throughout the 60-day exposure period, no statistically significant alterations in the integrity of epididymal sperm acrosomes, the quantity of testicular spermatogonia and sperm cells, or the levels of the three primary sex hormones were observed. Based on this, Si-NPs can induce reversible damage to sperm in the epididymis without impacting overall fertility; and sperm appear to be more susceptible to silica nanoparticle toxicity than spermatogonia and spermatocytes. Through the above mechanisms, and taking into account the timeline of spermatogenesis, silica nanoparticles predominantly impact sperm maturation in the epididymis by inducing oxidative stress and mitochondrial structural damage, leading to dysfunctions in energy metabolism. 87 Titanium dioxide (TiO 2 ) NPs are produced globally on a large scale, and used for diverse applications across various industries. 88 TiO 2 NPs can traverse the blood-testis barrier, thereby gaining access to the testes and accumulating within this tissue. Accumulation of TiO 2 NPs is associated with the development of testicular lesions, sperm malformation and disruptions in the homeostasis of serum sex hormone levels. Microarray analysis revealed differential gene expression in testes following TiO 2 NP exposure. Specifically, the expression levels of 70 genes with known functions were increased, while 72 were decreased. Among the genes with altered expression levels, Prm1, Tnp2, Spata19, Tdrd6, Ly6e and Adam3 have been implicated in spermatogenesis; while Cyp2e1, Lep, Srd5a2, Sc4mol, Psmc3ip, and Mvd are linked to steroid and hormone metabolism. In fact, administration of TiO 2 NPs induces testicular toxicity in mice, which is characterised by inhibition of spermatogenesis and perturbations in gene expression. 89 In another investigation, exposure to TiO 2 NPs induced testicular and epididymal lesions, reduced sperm concentrations and sperm motility, and resulted in an increased incidence of abnormal sperm in mice. Furthermore, exposure to TiO 2 nanoparticles resulted in decreased enzymatic activities for glucose-6-phosphate dehydrogenase, succinate dehydrogenase, lactate dehydrogenase, sorbitol dehydrogenase, Ca 2+ /Mg 2+ -ATPase, Ca 2+ -ATPase, and Na + /K + -ATPase; while increasing the activities of acid phosphatase, alkaline phosphatase and total nitric oxide synthase in the testicular tissue of mice. In addition, TiO 2 NP exposure also resulted in greater ROS generation, along with elevated levels of malondialdehyde, 8-hydroxydeoxyguanosine and carbonyl, indicative of increased lipid peroxidation, DNA oxidative damage and protein oxidation within the testes, respectively. These findings suggest a potential association between TiO 2 NP-induced suppression of spermatogenesis and alterations in the activity of testicular marker enzymes, coupled with the induction of oxidative stress within the testes. 90 Notably, recent studies have shown that TiO 2 NPs can migrate from the peritoneal cavity to the scrotum, where they accumulate and exert deleterious effects on testicular histology, as well as structural and functional aspects of sperm. These effects are observable within a period of 4–8 days following injection, while the effects are less pronounced beyond a timeframe of 10–38 days post-injection. In addition, sperm motility impairments observed in this study correlated with elevated levels of ROS, implying that oxidative stress could be a causal mechanism underlying the induction of these abnormalities. 91 Human sperm samples were exposed to varying concentrations of silver nanoparticles (Ag NPs) over different durations, after which sperm viability, motility, and the proportion of abnormal to normal sperm were evaluated. These observations revealed a dose- and time-dependent reduction in both sperm viability and motility, with an increase in the ratio of abnormal to normal sperm following Ag NP exposure at a concentration of 200 μg mL −1 and 400 μg mL −1 for 30 minutes and 60 minutes, respectively. Notably, this analysis also revealed predominantly abnormal sperm morphologies, characterised by disrupted chromatin or the absence of the acrosome in the sperm head, as well as tail bending and mid-piece curvature. Furthermore, the ultrastructural features of sperm exposed to Ag NPs included disrupted chromatin with swollen, granular and vacuolar abnormalities. In addition, 60 minutes of exposure to Ag NPs at concentrations of 200 μg mL −1 and 400 μg mL −1 , was associated with increased ROS production and DNA fragmentation. Through the above mechanisms, Ag NPs can induce negative changes in human sperm parameters, emphasising the need for caution regarding the extensive application of Ag NPs. 92 Furthermore, the impact of daily exposure to silver nanoparticles during prepubertal development has also been examined. It was observed that Ag NPs decreased both acrosomal and plasma membrane integrities, decreased mitochondrial activity, and increased the prevalence of sperm abnormalities. Importantly, sperm exhibited heightened susceptibility to the cytotoxic impacts of Ag NPs, with deleterious effects associated with exposure to lower doses during the pre-pubertal stage. These findings indicate that exposure to Ag NPs during the prepubertal period can directly lead to modifications in sperm in adulthood. 93 Notably, a recent study investigated the deleterious impact of Ag NP exposure on sperm parameters and lipid peroxidation of sperm membranes in male rats. It was observed that the presence of Ag NPs led to a notable decrease in both sperm count and motility. Also, Ag NPs substantially elevated malondialdehyde levels in a dose-dependent manner within sperm membranes. Thus, it is possible that Ag NPs diminish the quality of sperm parameters in a dose-dependent fashion by augmenting lipid peroxidation. 94 A recent investigation examined the adverse impact of gold nanoparticles (Au NPs) on sperm function. In initial experiments, the presence of Au NPs was observed to have a detrimental impact on sperm motility, suggesting that they could potentially have negative fertility effects. Moreover, infiltration of Au NPs into sperm cells was observed, potentially resulting in their fragmentation. 95 In another study, M. Nazar et al conducted an investigation into the impact of Au NPs on sperm parameters and chromatin structure in mice. Based on their results, Ag NPs induced a significant reduction in both sperm motility and morphology among the tested experimental groups, particularly in those subjected to Ag nanoparticle-treatment for 35 days, in comparison to the control group. Their findings suggest that Au NPs may have a dual impact: initially affecting sperm motility and interfering with normal sperm morphology, and subsequently influencing sperm chromatin remodelling, resulting in increased chromatin instability and elevated levels of DNA damage in sperm. 96 Expanding upon these discoveries, an independent investigation also found a decrease in sperm motility, which was observed at a dose of 10 µg mL −1 of Au NPs, regardless of surface alterations. Importantly, findings from transmission electron microscopy showed the adherence of Ag nanoparticles to the sperm cell membrane, whereas molecular analysis indicated a decline in free thiol residues on the membrane post-exposure, which may explain the observed decline in sperm motility. Notably, exposure to ligand-free nanoparticles resulted in a reduction in sperm fertilization capacity, indicating that disruption of membrane properties in sperm could be detrimental to the fertilization process. These results collectively suggest that Au NPs can disrupt fundamental sperm functions by interacting with the sperm surface membrane. 97 Notably, recent research studied the effects of Ag NP exposure on gametes, which were initially treated with sodium dodecyl sulphate to remove the membrane: exposed to medium containing Au NPs caused a disruption in the chromatin decondensation process and alterations in nuclear structure. Based on these findings, the cytotoxic impact of Au NPs was postulated to be due to interactions with the double-helix of DNA molecules. 98 Carbon black nanoparticles (CBNPs) have extensive applications in the rubber industry, and are also used in a range of non-rubber products, including pigments and printing inks. During their synthesis and industrial applications, CBNPs can enter an organism via the respiratory system, spreading to critical organs including the gonads, liver, brain, kidneys and blood vessels, and inducing oxidative stress, DNA damage and cytotoxic effects. Hu et al investigated the effects of exposing mice to varying concentrations of CBNPs at distinct life stages (puberty, sexual maturity and adulthood), in order to elucidate the impact of CBNP inhalation on male reproductive physiology and spermatogenesis. After exposure to CBNPs, a notable increase in testicular oxidative stress and inflammation was observed; and these effects varied depending on the duration of the exposure. Also, significant reductions in seminiferous epithelium height (SEH), seminiferous tubule diameter (STD) and the number of Leydig and spermatogenic cells were observed, as well as reductions in sperm motility and speed. These effects varied across the different exposure doses tested. Importantly, the observed elevated levels of oxidative stress and increased inflammation within the testes adversely affected testicular morphology and impaired spermatogenesis, diminishing testosterone secretion and lowering sperm quality. Also, the observed morphological defects in the testes strongly correlated with a reduction in sperm quantity. Because of the above, CBNPs exposure decreases sperm quality and quantity in a dose-dependent fashion. 99 Recent research has investigated the cytotoxic impact of zinc oxide nanoparticles (ZnO NPs) on spermatozoa viability. Various concentrations of ZnO NPs (10, 100, 500 and 1000 µg mL −1 ) were incubated with semen samples at 37 °C for 45, 90 and 180 minutes. After intervals of 45, 90 and 180 minutes, the highest percentage of cell death observed was 20.8%, 21.2% and 33.2%, respectively. Exposure to the maximal concentration (1000 µg mL −1 ) of ZnO NPs resulted in the greatest toxicity across all incubation times. Thus, the cytotoxicity induced by ZnO NPs exhibits depended on both the dose and time of exposure. 100 Further experimentation revealed concentration-dependent and time-dependent effects associated with ZnO NPs exposure, which caused an increased incidence of sperm abnormalities within the epididymal tail. 101 Iron oxide nanoparticles (IONPs) have garnered significant attention because of their distinctive characteristics, including a high surface-to-volume ratio, superparamagnetism, increased surface area, and easy separation techniques. However, they have also been demonstrated to readily accumulate in various tissues and elicit toxicity at different exposure levels. 102 Recent investigations have revealed that administration of IONPs at a dose of 300 mg/kg/day resulted in notable reductions in various sperm-related parameters, including: motility, Leydig cells, Sertoli cells, spermatogonia, primary spermatocytes, spermatids, total length of seminiferous tubules and the volumes of testicular interstitial tissue. 103 Administration of CuO NPs resulted in a substantial decrease in serum testosterone levels, suppression of sperm concentrations, and a significant increase in the percentage of abnormal and dead sperm. Furthermore, degeneration of germ cells, Leydig cells, Sertoli cells and spermatocytes was observed in testicular tissue, accompanied by vacuolation and inflammatory cell infiltration. In conclusion, CuO NPs have a deleterious and permanent impact on testicular function and the physiological attributes of sperm, with increased detrimental effects associated with the administration of elevated doses of CuO NPs. 104 Cerium oxide nanoparticles (CeO 2 NPs) led to a reduction in both sperm motility and sperm count, while also increasing the overall incidence of sperm abnormalities in mice. 105 Furthermore, the impact of CeO 2 NP exposure on the DNA of human spermatozoa has also been recently investigated. It has been revealed that even exposure of human spermatozoa to extremely low concentrations of CeO 2 NP in vitro can lead to notable DNA damage 106 ( Figure 3 ). Figure 3 A schematic representation of nanoparticles with toxic implication on sperm and oocytes, based on findings from recent investigations. 59–62 , 64 , 66 , 68 , 69 , 83–85 , 87–99 , 101–104 , 107 Created by Figdraw. A schematic representation of nanoparticles with toxic implication on sperm and oocytes, based on findings from recent investigations. 59–62 , 64 , 66 , 68 , 69 , 83–85 , 87–99 , 101–104 , 107 Created by Figdraw.