Mitochondrial Extracellular Vesicles (mitoEVs): Emerging mediators of cell-to-cell communication in health, aging and age-related diseases.

Mitochondria are metabolic and signalling hubs that integrate a plethora of interconnected processes to maintain cell homeostasis. They are also dormant mediators of inflammation and cell death, and with aging damages affecting mitochondria gradually accumulate, resulting in the manifestation of age-associated disorders. In addition to coordinate multiple intracellular functions, mitochondria mediate intercellular and inter-organ cross talk in different physiological and stress conditions. To fulfil this task, mitochondrial signalling has evolved distinct and complex conventional and unconventional routes of horizontal/vertical mitochondrial transfer. In this regard, great interest has been focused on the ability of extracellular vesicles (EVs), such as exosomes and microvesicles, to carry selected mitochondrial cargoes to target cells, in response to internal and external cues. Over the past years, the field of mitochondrial EVs (mitoEVs) has grown exponentially, revealing unexpected heterogeneity of these structures associated with an ever-expanding mitochondrial function, though the full extent of the underlying mechanisms is far from being elucidated. Therefore, emerging subsets of EVs encompass exophers, migrasomes, mitophers, mitovesicles, and mitolysosomes that can act locally or over long-distances to restore mitochondrial homeostasis and cell functionality, or to amplify disease. This review provides a comprehensive overview of our current understanding of the biology and trafficking of MitoEVs in different physiological and pathological conditions. Additionally, a specific focus on the role of mitoEVs in aging and the onset and progression of different age-related diseases is discussed.

Building a Human Ovarian Antioxidant ceRNA Network "OvAnOx": A Bioinformatic Perspective for Research on Redox-Related Ovarian Functions and Dysfunctions.

The ovary is a major determinant of female reproductive health. Ovarian functions are mainly related to the primordial follicle pool, which is gradually lost with aging. Ovarian aging and reproductive dysfunctions share oxidative stress as a common underlying mechanism. ROS signaling is essential for normal ovarian processes, yet it can contribute to various ovarian disorders when disrupted. Therefore, balance in the redox system is crucial for proper ovarian functions. In the present study, by focusing on mRNAs and ncRNAs described in the ovary and taking into account only validated ncRNA interactions, we built an ovarian antioxidant ceRNA network, named OvAnOx ceRNA, composed of 5 mRNAs (SOD1, SOD2, CAT, PRDX3, GR), 10 miRNAs and 5 lncRNAs (XIST, FGD5-AS1, MALAT1, NEAT1, SNHG1). Our bioinformatic analysis indicated that the components of OvAnOx ceRNA not only contribute to antioxidant defense but are also involved in other ovarian functions. Indeed, antioxidant enzymes encoded by mRNAs of OvAnOx ceRNA operate within a regulatory network that impacts ovarian reserve, follicular dynamics, and oocyte maturation in normal and pathological conditions. The OvAnOx ceRNA network represents a promising tool to unravel the complex dialog between redox potential and ovarian signaling pathways involved in reproductive health, aging, and diseases.

NAD+ Metabolism and Mitochondrial Activity in the Aged Oocyte: Focus on the Effects of NAMPT Stimulation.

The ovary experiences an age-dependent decline starting during the fourth decade of life. Ovarian aging is the predominant factor driving female reproductive aging. Modern trend to postpone childbearing age contributes to reduced fertility and natality worldwide. Recently, the beneficial role of NAD+ precursors on the maintenance of oocyte competence and female fertility affected by aging has emerged. Nevertheless, age-related changes in NAD+ regulatory network have not been investigated so far. In this context, our goal was to investigate changes induced by the aging process in the expression level of genes participating in NAD+ biosynthetic and NAD+ consuming pathways and in the cellular bioenergetics in the mouse oocyte. From Ingenuity Pathway Analysis (IPA) it emerged that aging caused the downregulation of all cellular pathways for NAD+ synthesis (Kynurenine pathway, Preiss-Handler pathway and NAD+ salvage pathway) and deeply influenced the activity of NAD+-dependent enzymes, i.e. PARPs and SIRTs, with effects on many cellular functions including compromised ROS detoxification. Considering that NAMPT, the rate-limiting enzyme of NAD+ salvage pathway, was deregulated, aged oocytes were matured in the presence of P7C3, NAMPT activator. P7C3 improved spindle assembly and mitochondrial bioenergetics and reduced mitochondrial proton leak. Moreover, P7C3 influenced gene expression of NAD+ regulatory network, with Sirt1 as the central node of IPA-interfered target gene network. Finally, P7C3 effectively counteracted oocyte alterations induced by exposure to oxidative stress. Our study contributes to establish effective NAD+ boosting interventions to alleviate the effects of advanced maternal age on fertility and explore their potential in redox-related fertility disorders.

Maternal MicroRNA Profile Changes When LH Is Added to the Ovarian Stimulation Protocol: A Pilot Study.

While the use of follicle-stimulating hormone (FSH) in ovarian stimulation for in vitro fertilization (IVF) is an established practice, the use of luteinizing hormone (LH) remains debatable. MicroRNAs (miRNAs) are short, endogenous, non-coding transcripts that control a variety of cellular functions, such as gonadotrophin production and follicular development. The goal of this pilot study was to investigate whether the employment of recombinant LH (rLH) in ovarian stimulation protocols results in changes in the miRNA profiles in human oocytes. Patients were divided into two groups: seven received recombinant FSH (rFSH, 225 IU), and six received rFSH (150 IU) plus rLH (75 IU). MiRNA predesigned panels and real-time PCR technology were used to analyze the oocytes retrieved from the follicular ovarian retrieval. Among the miRNAs evaluated, a series of them evidenced upregulation or downregulation in their expression in the FSH plus LH group compared to the FSH group. Considering the results obtained from the functional and network analysis, the different maternal miRNA profiles in the two groups revealed a differential modulation of pathways involved in numerous biological functions. Overall, based on the pathways associated with most of these maternal miRNAs, the presence of LH may result in a different modulation of pathways regulating survival under the control of a Tp53-related mechanism. Interestingly, among the miRNAs differentially expressed in oocytes of the two groups, we have found miRNAs already investigated at ovarian, follicular, oocyte, and embryonic levels: hsa-miR-484, hsa-miR-222, hsa-miR-520d-5p, hsa-miRNA-17, hsa-miR-548, and hsa-miR-140. Thus, investigation into the role of these miRNAs in oocyte molecular pathways may help determine how LH affects oocyte competence and eventually leads to the clinical improvement of IVF.

Acyl-Carnitines Exert Positive Effects on Mitochondrial Activity under Oxidative Stress in Mouse Oocytes: A Potential Mechanism Underlying Carnitine Efficacy on PCOS.

Carnitines play a key physiological role in oocyte metabolism and redox homeostasis. In clinical and animal studies, carnitine administration alleviated metabolic and reproductive dysfunction associated with polycystic ovarian syndrome (PCOS). Oxidative stress (OS) at systemic, intraovarian, and intrafollicular levels is one of the main factors involved in the pathogenesis of PCOS. We investigated the ability of different acyl-carnitines to act at the oocyte level by counteracting the effects of OS on carnitine shuttle system and mitochondrial activity in mouse oocytes. Germinal vesicle (GV) oocytes were exposed to hydrogen peroxide and propionyl-l-carnitine (PLC) alone or in association with l-carnitine (LC) and acetyl-l-carnitine (ALC) under different conditions. Expression of carnitine palmitoyltransferase-1 (Cpt1) was monitored by RT-PCR. In in vitro matured oocytes, metaphase II (MII) apparatus was assessed by immunofluorescence. Oocyte mitochondrial respiration was evaluated by Seahorse Cell Mito Stress Test. We found that Cpt1a and Cpt1c isoforms increased under prooxidant conditions. PLC alone significantly improved meiosis completion and oocyte quality with a synergistic effect when combined with LC + ALC. Acyl-carnitines prevented Cpt1c increased expression, modifications of oocyte respiration, and ATP production observed upon OS. Specific effects of PLC on spare respiratory capacity were observed. Therefore, carnitine supplementation modulated the intramitochondrial transfer of fatty acids with positive effects on mitochondrial activity under OS. This knowledge contributes to defining molecular mechanism underlying carnitine efficacy on PCOS.

Modulating Morphological and Redox/Glycative Alterations in the PCOS Uterus: Effects of Carnitines in PCOS Mice.

(1) Background: Polycystic ovarian syndrome (PCOS) is a common and multifactorial disease affecting reproductive-age women. Although PCOS ovarian and metabolic features have received extensive research, uterine dysfunction has been poorly investigated. This research aims to investigate morphological and molecular alterations in the PCOS uterus and search for modulating effects of different carnitine formulations. (2) Methods: CD1 mice were administered or not with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) for 20 days, alone or with 0.40 mg L-carnitine (LC) and 0.20 mg acetyl-L-carnitine (ALC) in the presence or absence of 0.08 mg propionyl-L-carnitine (PLC). Uterine horns from the four groups were subjected to histology, immunohistochemistry and immunoblotting analyses to evaluate their morphology, collagen deposition, autophagy and steroidogenesis. Oxidative-/methylglyoxal (MG)-dependent damage was investigated along with the effects on the mitochondria, SIRT1, SOD2, RAGE and GLO1 proteins. (3) Results: The PCOS uterus suffers from tissue and oxidative alterations associated with MG-AGE accumulation. LC-ALC administration alleviated PCOS uterine tissue alterations and molecular damage. The presence of PLC prevented fibrosis and maintained mitochondria content. (4) Conclusions: The present results provide evidence for oxidative and glycative damage as the main factors contributing to PCOS uterine alterations and include the uterus in the spectrum of action of carnitines on the PCOS phenotype.

Mitochondria Biology in Reproductive Function.

Mitochondria are multitasking organelles involved in the maintenance of cell homeostasis [...].

Phytochemistry and Biological Activity of Medicinal Plants in Wound Healing: An Overview of Current Research.

Wound healing is a complicated process, and the effective management of wounds is a major challenge. Natural herbal remedies have now become fundamental for the management of skin disorders and the treatment of skin infections due to the side effects of modern medicine and lower price for herbal products. The aim of the present study is to summarize the most recent in vitro, in vivo, and clinical studies on major herbal preparations, their phytochemical constituents, and new formulations for wound management. Research reveals that several herbal medicaments have marked activity in the management of wounds and that this activity is ascribed to flavonoids, alkaloids, saponins, and phenolic compounds. These phytochemicals can act at different stages of the process by means of various mechanisms, including anti-inflammatory, antimicrobial, antioxidant, collagen synthesis stimulating, cell proliferation, and angiogenic effects. The application of natural compounds using nanotechnology systems may provide significant improvement in the efficacy of wound treatments. Increasing the clinical use of these therapies would require safety assessment in clinical trials.

Personalized Nutrition in the Management of Female Infertility: New Insights on Chronic Low-Grade Inflammation.

Increasing evidence on the significance of nutrition in reproduction is emerging from both animal and human studies, suggesting a mutual association between nutrition and female fertility. Different "fertile" dietary patterns have been studied; however, in humans, conflicting results or weak correlations are often reported, probably because of the individual variations in genome, proteome, metabolome, and microbiome and the extent of exposure to different environmental conditions. In this scenario, "precision nutrition", namely personalized dietary patterns based on deep phenotyping and on metabolomics, microbiome, and nutrigenetics of each case, might be more efficient for infertile patients than applying a generic nutritional approach. In this review, we report on new insights into the nutritional management of infertile patients, discussing the main nutrigenetic, nutrigenomic, and microbiomic aspects that should be investigated to achieve effective personalized nutritional interventions. Specifically, we will focus on the management of low-grade chronic inflammation, which is associated with several infertility-related diseases.

Carnitines as Mitochondrial Modulators of Oocyte and Embryo Bioenergetics.

Recently, the importance of bioenergetics in the reproductive process has emerged. For its energetic demand, the oocyte relies on numerous mitochondria, whose activity increases during embryo development under a fine regulation to limit ROS production. Healthy oocyte mitochondria require a balance of pyruvate and fatty acid oxidation. Transport of activated fatty acids into mitochondria requires carnitine. In this regard, the interest in the role of carnitines as mitochondrial modulators in oocyte and embryos is increasing. Carnitine pool includes the un-esterified l-carnitine (LC) and carnitine esters, such as acetyl-l-carnitine (ALC) and propionyl-l-carnitine (PLC). In this review, carnitine medium supplementation for counteracting energetic and redox unbalance during in vitro culture and cryopreservation is reported. Although most studies have focused on LC, there is new evidence that the addition of ALC and/or PLC may boost LC effects. Pathways activated by carnitines include antiapoptotic, antiglycative, antioxidant, and antiinflammatory signaling. Nevertheless, the potential of carnitine to improve energetic metabolism and oocyte and embryo competence remains poorly investigated. The importance of carnitine as a mitochondrial modulator may suggest that this molecule may exert a beneficial role in ovarian disfunctions associated with metabolic and mitochondrial alterations, including PCOS and reproductive aging.

Protective effects of a SIRT1 inhibitor on primordial follicle activation and growth induced by cyclophosphamide: insights from a bovine in vitro folliculogenesis system.

PURPOSE: Although oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs). METHODS: The effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture. RESULTS: We found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM. CONCLUSION: SIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients.

AGEs-related dysfunctions in PCOS: evidence from animal and clinical research.

Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder in women in their reproductive age. In recent years, the role of advanced glycation end products (AGEs) in PCOS has gained great attention. AGEs are highly reactive molecules that can be assumed by diet or endogenously synthesized as by-products of metabolic processes. AGE deposition increases with aging, hyperglycemia, insulin resistance, and glycotoxin-rich diet. Therefore, it has become imperative to understand the underlying mechanism of AGEs actions and its downstream effects in PCOS pathophysiology. By integrating evidence from human studies and experimental models, the present review points out that altered AGE deposition is a common feature in all PCOS phenotypes. Searching for possible mechanisms involved in the adaptive response against glycation injury in oocytes and ovaries, the role of SIRT1, the main member of the mammalian sirtuin family, has also recently emerged. Therefore, further studies based on anti-AGE interventions could be helpful in creating innovative strategies for counteracting PCOS and its effects on fertility.

Mitochondrial Sirtuins in Reproduction.

Mitochondria act as hubs of numerous metabolic pathways. Mitochondrial dysfunctions contribute to altering the redox balance and predispose to aging and metabolic alterations. The sirtuin family is composed of seven members and three of them, SIRT3-5, are housed in mitochondria. They catalyze NAD+-dependent deacylation and the ADP-ribosylation of mitochondrial proteins, thereby modulating gene expression and activities of enzymes involved in oxidative metabolism and stress responses. In this context, mitochondrial sirtuins (mtSIRTs) act in synergistic or antagonistic manners to protect from aging and aging-related metabolic abnormalities. In this review, we focus on the role of mtSIRTs in the biological competence of reproductive cells, organs, and embryos. Most studies are focused on SIRT3 in female reproduction, providing evidence that SIRT3 improves the competence of oocytes in humans and animal models. Moreover, SIRT3 protects oocytes, early embryos, and ovaries against stress conditions. The relationship between derangement of SIRT3 signaling and the imbalance of ROS and antioxidant defenses in testes has also been demonstrated. Very little is known about SIRT4 and SIRT5 functions in the reproductive system. The final goal of this work is to understand whether sirtuin-based signaling may be taken into account as potential targets for therapeutic applications in female and male infertility.

High Doses of D-Chiro-Inositol Alone Induce a PCO-Like Syndrome and Other Alterations in Mouse Ovaries.

Administration of 1000-1500 mg/day D-Chiro-Inositol (DCIns) or a combination of Myo-Inositol (MyoIns) and DCIns in their plasma molar ratio (40:1) for three or more months are among recommended treatments for metabolic syndrome and/or Polycystic Ovary Syndrome (PCOS). We previously confirmed the efficacy of this formulation (8.2 mg/day MyoIns and 0.2 mg/day DCIns for 10 days) in a mouse PCOS model, but also observed negative effects on ovarian histology and function of formulations containing 0.4-1.6 mg/day DCIns. We therefore analyzed effects of higher doses of DCIns, 5, 10 and 20 mg/day, administered to young adult female mice for 21 days, on ovarian histology, serum testosterone levels and expression of the ovarian enzyme aromatase. Five mg/day DCIns (human correspondence: 1200 mg/day) altered ovarian histology, increased serum testosterone levels and reduced the amount of aromatase of negative controls, suggesting the induction of an androgenic PCOS model. In contrast, 10-20 mg/day DCIns (human correspondence: 2400-4800 mg/day) produced ovarian lesions resembling those typical of aged mice, and reduced serum testosterone levels without affecting aromatase amounts, suggesting a failure in steroidogenic gonadal activity. Notwithstanding physiological/biochemical differences between mice and humans, the observed pictures of toxicity for ovarian histology and function recommend caution when administering DCIns to PCOS patients at high doses and/or for periods spanning several ovulatory cycles.

Pathophysiology of Mitochondrial Dysfunction in Human Spermatozoa: Focus on Energetic Metabolism, Oxidative Stress and Apoptosis.

The dogma of mitochondria as the major source of energy in supporting sperm motility should be critically reconsidered in the light of several experimental data pointing to a major role of glycolysis in mammalian spermatozoa. In this light, the reported positive correlation between the mitochondrial membrane potential (ΔΨm) and motility of ejaculated spermatozoa cannot be explained convincingly by an impaired mitochondrial ATP generation only. Evidence has been produced suggesting that, in human sperm, dysfunctional mitochondria represent the main site of generation of reactive oxygen species (ROS). Furthermore, in these organelles, a complex bidirectional relationship could exist between ROS generation and apoptosis-like events that synergize with oxidative stress in impairing sperm biological integrity and functions. Despite the activity of enzymatic and non-enzymatic antioxidant factors, human spermatozoa are particularly vulnerable to oxidative stress, which plays a major role in male factor infertility. The purpose of this article is to provide an overview of metabolic, oxidative and apoptosis-like inter-linkages of mitochondrial dysfunction and their reflections on human sperm biology.

Crocetin Mitigates Irradiation Injury in an In Vitro Model of the Pubertal Testis: Focus on Biological Effects and Molecular Mechanisms.

Infertility is a potential side effect of radiotherapy and significantly affects the quality of life for adolescent cancer survivors. Very few studies have addressed in pubertal models the mechanistic events that could be targeted to provide protection from gonadotoxicity and data on potential radioprotective treatments in this peculiar period of life are elusive. In this study, we utilized an in vitro model of the mouse pubertal testis to investigate the efficacy of crocetin to counteract ionizing radiation (IR)-induced injury and potential underlying mechanisms. Present experiments provide evidence that exposure of testis fragments from pubertal mice to 2 Gy X-rays induced extensive structural and cellular damage associated with overexpression of PARP1, PCNA, SOD2 and HuR and decreased levels of SIRT1 and catalase. A twenty-four hr exposure to 50 µM crocetin pre- and post-IR significantly reduced testis injury and modulated the response to DNA damage and oxidative stress. Nevertheless, crocetin treatment did not counteract the radiation-induced changes in the expression of SIRT1, p62 and LC3II. These results increase the knowledge of mechanisms underlying radiation damage in pubertal testis and establish the use of crocetin as a fertoprotective agent against IR deleterious effects in pubertal period.

Regulatory Functions of L-Carnitine, Acetyl, and Propionyl L-Carnitine in a PCOS Mouse Model: Focus on Antioxidant/Antiglycative Molecular Pathways in the Ovarian Microenvironment.

Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with female infertility. Based on energy and antioxidant regulatory functions of carnitines, we investigated whether acyl-L-carnitines improve PCOS phenotype in a mouse model induced by dehydroepiandrosterone (DHEA). CD1 mice received DHEA for 20 days along with two different carnitine formulations: one containing L-carnitine (LC) and acetyl-L-carnitine (ALC), and the other one containing also propionyl-L-carnitine (PLC). We evaluated estrous cyclicity, testosterone level, ovarian follicle health, ovulation rate and oocyte quality, collagen deposition, lipid droplets, and 17ß-HSD IV (17 beta-hydroxysteroid dehydrogenase type IV) expression. Moreover, we analyzed protein expression of SIRT1, SIRT3, SOD2 (superoxide dismutase 2), mitochondrial transcriptional factor A (mtTFA), RAGE (receptor for AGEs), GLO2 (glyoxalase 2) and ovarian accumulation of MG-AGEs (advanced glycation end-products formed by methylglyoxal). Both carnitine formulations ameliorated ovarian PCOS phenotype and positively modulated antioxidant molecular pathways in the ovarian microenvironment. Addition of PLC to LC-ALC formulation mitigated intraovarian MG-AGE accumulation and increased mtTFA expression. In conclusion, our study supports the hypothesis that oral administration of acyl-L-carnitines alleviates ovarian dysfunctions associated with this syndrome and that co-administration of PLC provides better activity. Molecular mechanisms underlying these effects include anti-oxidant/glycative activity and potentiation of mitochondria.

Corrigendum to "Role of Mitochondria in the Oxidative Stress Induced by Electromagnetic Fields: Focus on Reproductive Systems".

[This corrects the article DOI: 10.1155/2018/5076271.].

Methylglyoxal-Dependent Glycative Stress and Deregulation of SIRT1 Functional Network in the Ovary of PCOS Mice.

Advanced glycation end-products (AGEs) are involved in the pathogenesis and consequences of polycystic ovary syndrome (PCOS), a complex metabolic disorder associated with female infertility. The most powerful AGE precursor is methylglyoxal (MG), a byproduct of glycolysis, that is detoxified by the glyoxalase system. By using a PCOS mouse model induced by administration of dehydroepiandrosterone (DHEA), we investigated whether MG-dependent glycative stress contributes to ovarian PCOS phenotype and explored changes in the Sirtuin 1 (SIRT1) functional network regulating mitochondrial functions and cell survival. In addition to anovulation and reduced oocyte quality, DHEA ovaries revealed altered collagen deposition, increased vascularization, lipid droplets accumulation and altered steroidogenesis. Here we observed increased intraovarian MG-AGE levels in association with enhanced expression of receptor for AGEs (RAGEs) and deregulation of the glyoxalase system, hallmarks of glycative stress. Moreover, DHEA mice exhibited enhanced ovarian expression of SIRT1 along with increased protein levels of SIRT3 and superoxide dismutase 2 (SOD2), and decreased peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1α), mitochondrial transcriptional factor A (mtTFA) and translocase of outer mitochondrial membrane 20 (TOMM20). Finally, the presence of autophagy protein markers and increased AMP-activated protein kinase (AMPK) suggested the involvement of SIRT1/AMPK axis in autophagy activation. Overall, present findings demonstrate that MG-dependent glycative stress is involved in ovarian dysfunctions associated to PCOS and support the hypothesis of a SIRT1-dependent adaptive response.

Pre-conceptional maternal exposure to cyclophosphamide results in modifications of DNA methylation in F1 and F2 mouse oocytes: evidence for transgenerational effects.

Cyclophosphamide (CPM), an agent widely used in breast cancer therapy, has strong gonadotoxic effects. Female reproductive potential after therapy relies on ovulated oocytes deriving from primordial follicles surviving CPM toxic insult. In this study, we investigated in the mouse model whether pre-conceptional maternal exposure to CPM has epigenetic effects on offspring oocytes and if they are inherited. Adult female mice mated following CPM exposure, generated an offspring (F1) with delayed growth, normal fertility and altered methylation of three imprinted genes (H19, Igf2r and Peg3) in their oocytes. These alterations were present in oocytes generated by F2 mice. Pre-conceptional maternal exposure to fertoprotective agents AS101 and crocetin prior to CPM was not able to fully counteract alterations in offspring oocyte imprinting. For the first time, current study evidences that pre-conceptional CPM maternal exposure can affect the competence of offspring's oocytes and warns on possible long-term effects on the health of next generations.