Publications by authors named "Guido Macchiarelli"

70 Publications

Ultrastructure of mitochondria of human oocytes in different clinical conditions during assisted reproduction.

Arch Biochem Biophys 2021 Mar 30;703:108854. Epub 2021 Mar 30.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100, L'Aquila, Italy. Electronic address:

Infertility affects around 8% of couples with a slight change in percentage in the last years. Despite the significant efforts made in Assisted Reproductive Technologies (ARTs) in handling this disorder, oocyte quality remains a crucial factor for a positive outcome. A better understanding of the dynamics underlying oocyte maturation, fertilization, and embryo development remains one of the main areas for progress in the ARTs field. Mitochondria are believed to play an essential role in these processes. Mitochondria have a crucial part in producing energy for oocyte maturation and embryo development throughout precise cellular functions comprising Ca homeostasis regulation, glycolysis, amino acid and fatty acid metabolism, and regulation of apoptosis. Recent studies suggest that mitochondrial structure, content, and function may be related to oocyte competence, embryo viability, and implantation success during ARTs. Their defects could lead to low fertilization rates and embryonic development failure. This review aimed to provide an overview of the available literature data surrounding the correlation between changes at ultrastructural level of mitochondria or correlated-mitochondrial aggregates and oocyte quality and ARTs treatments. Our reported data demonstrated that oocyte mitochondrial ultrastructural alterations could be partial or complete recovery during the early embryo stages. However, these changes could persist as quiescent during the pre-implantation embryo development, causing abnormalities that become evident only during fetal and postnatal life. These factors led to consider the mitochondria as a crucial marker of oocyte and embryo quality, as well as a strategic target for further prospective therapeutical approaches.
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http://dx.doi.org/10.1016/j.abb.2021.108854DOI Listing
March 2021

Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor).

Reprod Domest Anim 2021 Mar 18;56(3):484-497. Epub 2021 Jan 18.

Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine, Hirosaki University, Aomori, Japan.

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 ( Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300-6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.
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http://dx.doi.org/10.1111/rda.13887DOI Listing
March 2021

Commercial oral hygiene products and implant collar surfaces: Scanning electron microscopy observations.

Can J Dent Hyg 2020 Feb 1;54(1):26-31. Epub 2020 Feb 1.

Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy.

Background: The use of medicated mouthwashes and gels in the home care maintenance of dental implants is controversial due to the possibility of residue deposition on the implant collar. The aim of this in vitro study was to analyse, by means of scanning electron microscopy (SEM), the amount of residues on dental implant collars treated with various commercial home dental care products.

Methods: Gel and mouthwash products were tested on 10 implants. The gels included sodium fluoride, amine fluoride, and sodium hyaluronate products. The mouthwashes tested contained triclosan, nimesulide, stannous fluoride, amine fluoride, and hexetidine-chlorobutanol. The SEM observations were performed at different magnifications in double modality SE (secondary electrons) and BSE (backscattered electrons) to qualitatively assess any residual products. The image quantitative analysis was performed by Image J software to assess areas occupied by residuals. All results were analysed by the same researcher with experience in electron microscopy.

Results: The fluoride-based gel products left wider areas occupied by residuals than the mouthwash products. In particular the fluoride-based and hyaluronate products left the highest amount of residues. Among mouthwashes, fluoride-based and triclosan products showed the highest amount of residuals deposition.

Discussion: Oral hygiene procedures and related professional products are fundamental to the prevention, treatment, and control of microorganisms. In the case of implants, mechanical and chemical plaque control strategies are even more important since the potentially harmful biofilm covers abiotic titanium surfaces. In situ fixture maintenance is crucial for dental implant therapy success. Correct recommendation of home care products for bacterial control is fundamental to the health of implants and their surrounding tissues.

Conclusions: Data from this experimental study showed that home care commercial products in gel formulation, especially those containing fluoride, leave more residuals on titanium smooth surfaces than mouthwash products. The longer permanence of the products may lead to a more effective plaque control than other products.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533810PMC
February 2020

Autologous Materials in Regenerative Dentistry: Harvested Bone, Platelet Concentrates and Dentin Derivates.

Molecules 2020 Nov 15;25(22). Epub 2020 Nov 15.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

The jawbone is a peculiar type of bone tissue, unique for its histological, anatomical and physiological characteristics. Therefore, a defect in the maxilla or in the mandible, because of pathological sequelae is difficult to prevent and to restore. Several biomaterials have been and are currently being developed to respond to the demands of regenerative medicine. A specific group of biomaterials used in regenerative dentistry is represented by the autologous materials. Platelet concentrates harvested bone and dentin derivates are indeed used in an attempt to minimise the alveolar resorption or in vertical ridge augmentation procedures or in sinus lift interventions. The aim of this review is to examine the properties of the above-listed materials, to compare them and to indicate eventual clinical applications.
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http://dx.doi.org/10.3390/molecules25225330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696510PMC
November 2020

Activity of Antioxidants from L. Petals: Potential Preventive Effects towards Cardiovascular System.

Antioxidants (Basel) 2020 Nov 9;9(11). Epub 2020 Nov 9.

Nutraceutical Lab, Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.

The petals of the saffron crocus ( L.) are considered a waste material in saffron production, but may be a sustainable source of natural biologically active substances of nutraceutical interest. The aim of this work was to study the cardiovascular effects of kaempferol and crocin extracted from saffron petals. The antiarrhythmic, inotropic, and chronotropic effects of saffron petal extract (SPE), kaempferol, and crocin were evaluated through in vitro biological assays. The antioxidant activity of kaempferol and crocin was investigated through the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay using rat cardiomyoblast cell line H9c2. The MTT assay was applied to assess the effects of kaempferol and crocin on cell viability. SPE showed weak negative inotropic and chronotropic intrinsic activities but a significant intrinsic activity on smooth muscle with a potency on the ileum greater than on the aorta: EC = 0.66 mg/mL versus EC = 1.45 mg/mL. Kaempferol and crocin showed a selective negative inotropic activity. In addition, kaempferol decreased the contraction induced by KCl (80 mM) in guinea pig aortic and ileal strips, while crocin had no effect. Furthermore, following oxidative stress, both crocin and kaempferol decreased intracellular ROS formation and increased cell viability in a concentration-dependent manner. The results indicate that SPE, a by-product of saffron cultivation, may represent a good source of phytochemicals with a potential application in the prevention of cardiovascular diseases.
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http://dx.doi.org/10.3390/antiox9111102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697793PMC
November 2020

Does in vitro application of pentoxifylline have beneficial effects in assisted male reproduction?

Andrologia 2021 Feb 28;53(1):e13722. Epub 2020 Oct 28.

Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Rome, Italy.

Application of nonspecific phosphodiesterases inhibitors, such as pentoxifylline (PTX), is a strategy utilised to aid sperm selection from immotile sperm samples prior to ICSI. No extensive studies have yet been performed to verify the safety of the clinical outcomes of ICSI after PTX administration. In this article, we summarise the data reported in the literature that assess the implication of in vitro usage of PTX on sperm parameters, as well as clinical outcomes during assisted male reproduction programme.
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http://dx.doi.org/10.1111/and.13722DOI Listing
February 2021

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.

Antioxidants (Basel) 2020 Sep 15;9(9). Epub 2020 Sep 15.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

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.
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http://dx.doi.org/10.3390/antiox9090867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554995PMC
September 2020

Debonding and Clean-Up in Orthodontics: Evaluation of Different Techniques and Micro-Morphological Aspects of the Enamel Surface.

Dent J (Basel) 2020 Jun 17;8(2). Epub 2020 Jun 17.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

There is currently no consensus on the best way to remove adhesive remnants from teeth following debonding. The main objective of this study is to evaluate and compare the effectiveness of four adhesive resin removal (clean-up) techniques, performed with or without the use of an operative microscope. Forty human teeth were duplicated using an epoxy resin for impregnation. Brackets were bonded to teeth and debonded from teeth. Then, the samples were randomly divided into two equal groups-the naked eye group and the magnification group-and further subdivided into four equal subgroups, in order to compare the different techniques used for the clean-up. Each subgroup was formed of five natural teeth with the respective pre- and post-bonding replicas. Macro- and micro-analysis by means of a stereomicroscope and scanning electron microscopy evaluated, qualitatively and quantitatively, the adhesive remnant index and the damage index of the enamel. Overall, the magnification improved the removal of resins compared to the naked eye ( < 0.001), and the use of magnification constantly reduced resin residual and surface damage. Enamel damage and adhesive residual from the clean-up procedures represent an ascertained risk in orthodontics. The use of a magnification system improves the quality of debonding and clean-up techniques in a significant way.
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http://dx.doi.org/10.3390/dj8020058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344892PMC
June 2020

Effective dosage of growth differentiation factor-9β in folliculogenesis and angiogenesis in the sheep ovarian tissues grafted onto chick embryo chorioallantoic membrane.

J Obstet Gynaecol Res 2020 Aug 4;46(8):1384-1392. Epub 2020 Jun 4.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

Aim: Scientists have tried to culture and transplant the ovarian tissues (OT), to preserve fertility in cancer patients. However, one of the main limitations to the applicability of this technique is the folliculogenesis disruption after transplantation. Due to the roles exerted by growth differentiation factor-9β (GDF9β), we decided to determine the most effective dose of GDF9β on promotion of folliculogenesis and angiogenesis in sheep OT grafted onto the chick chorioallantoic membrane (CAM).

Methods: Fresh sheep OT were grafted onto the CAM for 5 days, and divided into four groups based on the addition of increasing doses of GDF9β (0, 150, 200 and 250 ng/mL). Following culture, histological (hematoxylin and eosin [H&E] staining) and immunohistological studies (Ki-67) were done. Fibrotic and necrotic regions were measured using MICROVISIBLE software. For comparing the follicle development rates between the groups as well as differences in the Ki-67-positive follicles, analysis of variance was applied.

Results: In both 200 and 250 ng/mL GDF9β groups, significantly higher rates of intermediary and primary follicles were observed, also the numbers of good quality follicles increased in the aforementioned groups and the rates of fibrotic and necrotic areas decreased. Moreover, in the 200 and 250 ng/mL GDF9β groups, the number of capillaries and the proliferative activity increased. The lower dose of GDF9β (150 ng/mL) neither activated the primordial follicles nor lead to an increase in the number of growing follicles.

Conclusion: Addition of high dosages of GDF9β to the OT, grafted onto the CAM resulted in higher folliculogenesis and better transplantation features due to improvement in angiogenesis.
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http://dx.doi.org/10.1111/jog.14266DOI Listing
August 2020

Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos.

Hum Reprod 2020 06;35(6):1476

Center for Reproductive Sciences, Department of Obstetrics and Gynecology, University of California San Francisco, San Francisco, CA, USA.

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http://dx.doi.org/10.1093/humrep/deaa047DOI Listing
June 2020

Evaluation of Different Autologous Platelet Concentrate Biomaterials: Morphological and Biological Comparisons and Considerations.

Materials (Basel) 2020 May 15;13(10). Epub 2020 May 15.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

The field of regeneration interventions in oral and maxillofacial surgeries still represents a challenge for researchers and clinicians. Understanding the biological and morphological behaviour of human cells towards the materials used for the regeneration surgeries is key to successfully choosing and applying the appropriate biomaterials for specific clinical situations. The aim of the study was the biological and morphological evaluation of autologous platelet concentrate materials obtained with different protocols, in culture with human periodontal ligament fibroblasts (HPLF). The study design included the evaluation of Leukocyte-Platelet-Rich-Fibrin (L-PRF), Concentrated Growth Factors (CGF) and autologous platelet gel (APG) in contact with the HPLF cell line after 24 h, 72 h and 7 days of in vitro culture. Cell proliferation and, therefore, viability were evaluated with XTT assays. The morphological response of the cells was evaluated by light microscopy, scanning electron microscopy and confocal microscopy. The XTT assay showed an interesting response in the growth curve. In particular, the material that gave the best results was the CGF. The morphological data supported the XTT assay, showing the best results for the CGF and L-PRF. In conclusion, all the platelet-derived materials stimulated the onset of the growth of the HPLF cell line, making them promising options for periodontal regeneration interventions.
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http://dx.doi.org/10.3390/ma13102282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288147PMC
May 2020

Pre-Implantation Mouse Embryos Cultured In Vitro under Different Oxygen Concentrations Show Altered Ultrastructures.

Int J Environ Res Public Health 2020 05 13;17(10). Epub 2020 May 13.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Assisted Reproductive Technologies routinely utilize different culture media and oxygen (O) concentrations to culture human embryos. Overall, embryos cultured under physiological O tension (5%) have improved development compared to embryos cultured under atmospheric O conditions (20%). The mechanisms responsible for this remain unclear. This study aimed to evaluate the effect of physiologic (5%) or atmospheric O (20%) tension on the microscopic ultrastructure of pre-implantation mouse embryos using Transmission Electron Microscopy (TEM). Embryos flushed out of the uterus after natural mating were used as the control. For use as the control, 2-cells, 4-cells, morulae, and blastocysts were flushed out of the uterus after natural fertilization. In vitro fertilization (IVF) was performed using potassium simplex optimized medium (KSOM) under different O tensions (5% and 20%) until the blastocyst stage. After collection, embryos were subjected to the standard preparative for light microscopy (LM) and TEM. We found that culture in vitro under 5% and 20% O results in an increase of vacuolated shaped mitochondria, cytoplasmic vacuolization and presence of multi-vesicular bodies at every embryonic stage. In addition, blastocysts generated by IVF under 5% and 20% O showed a lower content of heterochromatin, an interruption of the trophectodermal and inner cell mass cell membranes, an increased density of residual bodies, and high levels of glycogen granules in the cytoplasm. In conclusion, this study suggests that in vitro culture, particularly under atmospheric O tension, causes stage-specific changes in preimplantation embryo ultrastructure. In addition, atmospheric (20%) O is associated with increased alterations in embryonic ultrastructure; these changes may explain the reduced embryonic development of embryos cultured with 20% O.
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http://dx.doi.org/10.3390/ijerph17103384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277391PMC
May 2020

The effect of low and ultra-low oxygen tensions on mammalian embryo culture and development in experimental and clinical IVF.

Syst Biol Reprod Med 2020 Aug 7;66(4):229-235. Epub 2020 May 7.

Department of Life, Health and Environmental Sciences, University of L'Aquila , L'Aquila, Italy.

Over the last forty years, many trials have been performed using mammalian embryo cultures with reduced oxygen tension (O) to encourage proper embryo development and increase the success rate for fertilization (IVF) outcome. Even if the use of atmospheric O (20%) affects embryo development and intracellular redox balance, the use of low (5% O, physiologic) and ultra-low (close or less to 5% O) O applied to embryo culture is still under debate. Numerous studies in various mammalian species have shown that embryo development improves when culturing embryos under low O, although culture conditions are not the only factors involved in the success of IVF. This article reviews the literature data of the last four decades and discusses the current evidence on the use of low and ultra-low O in embryo culture, and examines the impact of multiple factors on IVF outcomes.

Abbreviations: O: oxygen tension; IVF: fertilization; IVC: culture; ET: embryo transfer; ROS: reactive oxygen species; ARTs: assisted reproductive technologies.
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http://dx.doi.org/10.1080/19396368.2020.1754961DOI Listing
August 2020

Repeated hyperstimulation affects the ultrastructure of mouse fallopian tube epithelium.

J Reprod Dev 2020 Aug 28;66(4):387-397. Epub 2020 Apr 28.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Controlled ovarian hyperstimulation (COH) is routinary used in assisted reproductive technologies (ARTs) to increase the yields of mature oocytes. The possibility that patients with a history of failures or poor-responders may develop side-effects following these treatments is still debated. Epidemiological studies reported controversial results about pregnancy outcome and the risk of developing gynecological cancers. By using a mouse model, here we compared the ultrastructural features of fallopian tubes (FTs) obtained from mice undergoing or not (control, CTR) four (4R) and eight (8R) rounds of gonadotropin stimulation. Although the morphological characteristics of oviductal layers seemed unaffected by repeated treatments, dose-response ultrastructural alterations in the ampulla appeared in the 4R group and even more in the 8R group. The targets were oviductal ciliated (CCs) and non-ciliated (NCCs) cells, which showed damaged mitochondria and glycogen accumulations in the cytoplasm. The drastic reduction of CCs, evident after 4R, was supported by the absence of cilia. After 8R, glycogen granules were significantly reduced and massive degeneration of mitochondria, which appeared swollen and/or vacuolated, occurred in NCCs. Moreover, disintegrated mitochondria were found at the periphery of mitophagic vacuoles with evident signs of cristolysis. The morphometric analysis evidenced a significant increase in the density and frequency of damaged mitochondria after 4R and 8R. The absence of cilia, necessary to sustain oviductal transport of oocytes, spermatozoa and embryos, may originate from either mitochondrial dysfunction or glycogen consumption. These results suggest that repeated COH treatments could induce alterations impairing fertilization and embryo transport toward the uterus.
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http://dx.doi.org/10.1262/jrd.2019-147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470905PMC
August 2020

Exposure to persistent organic pollutants during tooth formation: molecular mechanisms and clinical findings.

Rev Environ Health 2020 Nov 17;35(4):303-310. Epub 2020 Apr 17.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

Persistent organic pollutants (POPs) constitute a relevant part of environmental pollution. POPs are chemical compounds that persist for a long time in the environment, bio-accumulate in the human body and determine significant adverse consequences to human health. The characteristics of these substances are lipo-affinity, semi-volatility and resistance to the degradation processes. Results deriving from several different studies attest that exposure to the main classes of POPs results in multiple toxic effects on humans and experimental animal models. Among the various alterations caused by exposition to and bio-accumulation of POPs, there are abnormalities in tooth formation and related hard dental tissue structure, especially enamel. This review aimed to describe the close association between the exposure of these compounds during the development of the tooth germ and the occurrence of tooth structural anomalies. Indeed, structural defects of the enamel have as possible consequences higher susceptibility of the tooth to caries disease and higher fragility of the crown to the occlusal trauma.
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http://dx.doi.org/10.1515/reveh-2019-0093DOI Listing
November 2020

Association between Female Reproductive Health and Mancozeb: Systematic Review of Experimental Models.

Int J Environ Res Public Health 2020 04 9;17(7). Epub 2020 Apr 9.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Mancozeb is a widely used fungicide approved for use in agriculture in many countries with long persistence in the environment and consequent bioaccumulation in tissues and biological fluids. Despite the large amount of studies published in recent years, the relationship between mancozeb exposure and female reproductive health is not fully elucidated. In order to summarize current evidence on mancozeb exposure and female reproductive disease, we performed a systematic review of literature. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to make this review. An adapted version of the National Toxicology Program's Office of Health and Assessment and Translation (OHAT) framework was used to evaluate the risk of bias. Electronic search on two databases (PubMed and Scopus) was used to find experimental studies (in vitro and in vivo) on mancozeb exposure. The database search identified 250 scientific articles, 20 of which met our inclusion criteria. Selected data were then reviewed and summarized in tables. Overall, mancozeb represents a hazard for female reproductive health, with different mechanisms of action. Undoubtedly more experimental and epidemiological studies are required to definitively validate mancozeb as reproductive toxicant.
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http://dx.doi.org/10.3390/ijerph17072580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177957PMC
April 2020

Type of protein supplement in cryopreservation solutions impacts on the degree of ultrastructural damage in frozen-thawed human oocytes.

Cryobiology 2020 08 31;95:143-150. Epub 2020 Mar 31.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media.
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http://dx.doi.org/10.1016/j.cryobiol.2020.03.010DOI Listing
August 2020

Germ cell cysts, a fetal feature in mammals, are constitutively present in the adult armadillo.

Mol Reprod Dev 2020 01 20;87(1):91-101. Epub 2019 Nov 20.

Department of Biology and Biotechnology "Lazzaro Spallanzani,", University of Pavia, Pavia, Italy.

Formation and subsequent break down of ovarian germ cell (GC) cysts is a key and an evolutionary-conserved developmental event, described in phylogenetically diverse species of invertebrates and vertebrates. In mammals, cyst break down (CBD) ends at the time of, or soon after, birth with the formation of primordial follicles enclosing single oocytes, which constitute the sole reservoir of gametes available through the whole female's reproductive life. In this study, we challenge this paradigm demonstrating the constitutive presence of a large number of cysts, enclosing two-thirty GCs, in the ovary of the adult armadillo Chaetophractus villosus, belonging to the superorder Xenarthra, one of the earliest offshoots among placentals. We also describe that (a) GCs enclosed within cysts are connected by intercellular bridges-intercellular bridges-markers of their clonal origin; (b) CBD occurs through four main phases, ending with primordial follicles containing single oocytes; (c) GCs encompass meiotic prophase I stages, from leptotene to diplotene; (d) seasonal variations in the number of GCs enclosed within cysts, suggesting the presence of a GC multiplying activity. The armadillo C. villosus''s ovary emerges as an extraordinary resource to investigate folliculogenesis and to explore the evolutionary past of the mammalian ovary.
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http://dx.doi.org/10.1002/mrd.23296DOI Listing
January 2020

Combining culture and culture-independent methods reveals new microbial composition of halitosis patients' tongue biofilm.

Microbiologyopen 2020 02 14;9(2):e958. Epub 2019 Nov 14.

Department of Operative Dentistry and Periodontology, Center for Dental Medicine, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Background: Oral malodor is a very discomforting condition deriving from the presence of volatile sulfur compounds in the expired air. In halitosis of intraoral etiology, the volatile sulfur compounds are metabolic products of the oral microorganisms within the biofilm coating the tongue dorsum as well as other tissues in the oral cavity. The aim of this study was to characterize and compare the microbial composition of tongue biofilm in volunteers suffering from halitosis and healthy volunteers by means of both the culture method and culture-independent cloning technique.

Results: A high bacterial variety (more than 80 different species) was detected using the combination of both methods. A distinct bacterial composition was revealed in the halitosis-associated biofilms compared with the health-associated biofilms. Actinomyces graevenitzii was shown to be significantly associated with the halitosis condition. The culture method identified 47 species, included Veillonella rogosae, never isolated from the tongue biofilm of halitosis patients so far. In the healthy condition, the culture-dependent method showed that the most frequent species were Streptococcus parasanguinis among the aerobes and Veillonella spp. among the anaerobes. The culture-independent cloning method detected more than 50 species. Streptococci, in particular S. mitis/oralis, S. pseudopneumoniae, and S. infantis as well as Prevotella spp., were found most frequently in halitosis patients. Streptococcus salivarius and Rothia mucilaginosa were found more frequently in the healthy condition.

Conclusions: The combination of the culture-dependent and culture-independent cloning techniques allowed for a widespread analysis of the tongue biofilm in halitosis patients. The results can support further pharmacological research for new antimicrobial agents and halitosis therapy strategies.
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http://dx.doi.org/10.1002/mbo3.958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002105PMC
February 2020

In vitro evidence supporting applications of platelet derivatives in regenerative medicine.

Blood Transfus 2020 03 8;18(2):117-129. Epub 2019 Oct 8.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

The role of platelets in haemostasis has long been known, but understanding of these cells' involvement in wound healing/tissue repair is more recent and has given rise to a multitude of translational studies. Tissue repair processes consist of complex, regulated interactions between cells modulated by biologically active molecules, most of which are growth factors released by activated platelets: this aspect represents the rationale on which the use of platelet derivatives for clinical purposes is based. In the last years, many in vitro studies have focused on the mechanisms of action by which these growth factors affect the biological activities of cells, thus supporting tissue healing. Although limited by some drawbacks (two-dimensional in vitro monocultures cannot replicate the tissue architecture and organisation of organs or the continuous interplay between different cell types), in vitro studies do have the advantages of giving rapid results and allowing precise control of platelet concentrations and other parameters.This review offers an updated overview of the data obtained from the most recent bench-top studies focused on the effects of platelet derivatives on a wide variety of human cells, highlighting their possible impact for in vivo applications. The heterogeneity of the data obtained so far is very evident. This can be explained by the different experimental settings used in each study, which may be the cause of the variability in clinical outcomes. In fact, in vitro studies suggest that the composition of platelet derivatives and the method used for their production and activation (or not) and the platelet concentration used can have profound effects on the final results.
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http://dx.doi.org/10.2450/2019.0164-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141937PMC
March 2020

Microbiological and SEM-EDS Evaluation of Titanium Surfaces Exposed to Periodontal Gel: In Vitro Study.

Materials (Basel) 2019 May 4;12(9). Epub 2019 May 4.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Inflammatory diseases affecting the soft and hard tissues surrounding an implant represent a new challenge in contemporary implant dentistry. Among several methods proposed for the decontamination of titanium surfaces, the administration of topical 14% doxycycline gel seems to be a reliable option. In the present study, we evaluated the microbial effect of 14% doxycycline gel applied on titanium surfaces and exposed to human salivary microbes in anaerobic conditions. We also examined the composition of the exposed surfaces to assess the safe use of periodontal gel on titanium surfaces. Six anatase and six type 5 alloy titanium surfaces were used and divided into two groups: The test group and the positive control group. Both were cultured with human salivary samples in anaerobic conditions. On the test groups, 240 mg of periodontal gel was applied. The microbial assessment was performed with a colony-forming unit (CFU) count and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) to identify the species. The surface integrity was assessed using scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDS). The results demonstrated the microbial efficacy of the 14% doxycycline periodontal gel and its safe use on titanium surfaces. However, the SEM observations revealed the permanence of the gel on the titanium surfaces due to the physical composition of the gel. This permanence needs to be further investigated in vivo and a final polishing protocol on the titanium surface is recommended.
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http://dx.doi.org/10.3390/ma12091448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539618PMC
May 2019

Streptococcus spp. and Fusobacterium nucleatum in tongue dorsum biofilm from halitosis patients: a fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) study.

New Microbiol 2019 Apr 29;42(2):108-113. Epub 2019 Apr 29.

Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, Coppito 2, 67100, L'Aquila, Italy.

The present study involved a qualitative and quantitative evaluation of tongue dorsum biofilms sampled from halitosis patients and healthy volunteers. The aim of the study was to quantify the distribution of Streptococcus spp. and Fusobacterium nucleatum within the oral halitosis biofilm in order to highlight the role of these bacterial members in halitosis. Tongue plaque samples from four halitosis-diagnosed patients and four healthy volunteers were analyzed and compared. The visualization and quantification of the tongue dorsum biofilm was performed combining fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Eubacteria, Streptococcus spp. and Fusobacterium nucleatum were stained using specific fluorescent probes. For a comparison of the two tested biofilm groups the Wilcoxon rank-sum test was used. Morphological analysis by CLSM illustrated the distribution of the species which were tracked. Streptococcus spp. appeared to be enclosed within the samples and always associated to F. nucleatum. Furthermore, compared to the control group the biofilm within the halitosis group contained significantly higher proportions of F. nucleatum and Streptococcus spp., as revealed by the FISH and CLSM-analysis. The total microbial load and relative proportions of F. nucleatum and Streptococcus spp. can be considered as causative factors of halitosis and thus, as potential treatment targets.
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April 2019

The impact of sperm DNA fragmentation on ICSI outcome in cases of donated oocytes.

Arch Gynecol Obstet 2019 07 2;300(1):207-215. Epub 2019 Apr 2.

Iakentro Fertility Center, IVF Laboratory, 45250, Thessaloniki, Greece.

Purpose: The aim of this study is to evaluate the sperm DNA fragmentation index (DFI) in oocyte donation cycles and correlate it with the sperm parameters, the male characteristics, the embryo quality and the outcome of intracytoplasmic sperm injection (ICSI).

Methods: A total of 150 couples participating in an oocyte donation program were included in the study. Sperm samples were assessed by conventional sperm analysis. DFI was evaluated using the Halosperm kit, a sperm chromatin dispersion test (SCD).

Results: The relations between DNA damage and epidemiological male factors (age, height, weight), standard semen parameters (concentration, total and forward motility, and morphology), and embryological and clinical parameters (fertilization rate, total blastocyst number, number of good quality blastocyst, clinical pregnancy) were analyzed. DFI was positively correlated with advanced male age (r = 0.23, p < 0.05) and negatively correlated with total sperm and forward motility (r = - 0.29, r = - 0.27, respectively; p < 0.05). DFI was not significantly correlated with pregnancy outcome in oocyte donation cycles (r = - 0.05, p > 0.05). When good quality blastocysts were chosen, a trend toward the development of good quality embryos was detected in the presence of a low DFI (r = - 0.20, p = 0.08).

Conclusions: DFI does not significantly affect the outcome of ICSI in oocyte donation cycles. Even in cases of advanced paternal age that a high DFI resulted sperm DNA fragmentation seems not to adversely affect the final outcome.
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http://dx.doi.org/10.1007/s00404-019-05133-9DOI Listing
July 2019

First successful live birth following the use of MSOME and time lapse for sperm and embryo selections in a patient with severe male factor infertility: A case report.

J Gynecol Obstet Hum Reprod 2019 Dec 18;48(10):883-885. Epub 2019 Mar 18.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

We report a case of healthy live birth from a couple diagnosed with oligoasthenoteratozoospermia (OAT). They failed three ICSI cycles in the past 11 years. In the last cycle, the ovarian stimulation was done with antagonist protocol and six oocytes were aspirated. Semen sample was prepared by one layer gradient. 10 class I spermatozoa were selected by motile-sperm organelle-morphology examination (MSOME) according to Cassuto criteria. The selected spermatozoa were then injected into the MII oocytes. 16-18 h after ICSI, three zygotes was formed and, then, cultured in the time lapse monitoring (TLM) for 3 days. Two best embryos were selected according to the morphokinetic status for transfer. A singleton pregnancy was achieved, resulting in the birth of a healthy baby. This successful outcome shows that use of high technologies of MSOME and TLM procedures are useful for selection of the best spermatozoa and embryos in case of severe male infertility.
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http://dx.doi.org/10.1016/j.jogoh.2019.03.004DOI Listing
December 2019

Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos.

Hum Reprod 2019 04;34(4):601-611

Center for Reproductive Sciences, Department of Obstetrics and Gynecology, University of California San Francisco, San Francisco, CA, USA.

Study Question: Does the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF?

Summary Answer: Embryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen.

What Is Known Already: ART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations.

Study Design, Size, Duration: Zygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested.

Participants/materials, Setting, Methods: Mitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM).

Main Results And The Role Of Chance: Embryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development.

Large Scale Data: N/A.

Limitations, Reasons For Caution: Findings in mice embryos might be different from human embryos.

Wider Implications Of The Findings: This study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming.

Study Funding/competing Interest(s): This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.
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http://dx.doi.org/10.1093/humrep/dez011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967792PMC
April 2019

VEGFR2 Expression Is Differently Modulated by Parity and Nulliparity in Mouse Ovary.

Biomed Res Int 2018 16;2018:6319414. Epub 2018 Sep 16.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Parity and nulliparity exert opposite effects on women's health, as parity is considered a protective factor for several reproductive diseases. This study is aimed at determining if ovarian VEGF and VEGFR2 expression are differently modulated in the ovaries of parous and nulliparous mice. To this end primiparous and nulliparous fertile mice were sacrificed at postovulatory stage. Whole ovaries, corpus luteum, and residual stromal tissues were analyzed to assess VEGF/VEGFR2 expression levels. Ovarian mRNA amounts of ( and ) and were comparable between primiparous and nulliparous mice; both isoforms and receptor were accumulated mainly in corpus luteum tissues. VEGF 120 and 164 protein accumulation and distribution mirrored that of mRNA. Conversely, VEGFR2 protein content was significantly higher in ovaries of nulliparous mice and was more efficiently phosphorylated in ovaries of primiparous mice. In both groups, VEGFR2 was preferentially expressed in corpus luteum, while its phosphorylated form was equally distributed in two somatic compartments. We suggest that parity influences VEGFR2/phospho-VEGFR2 expression and tissue distribution. This difference could be part of a more complex mechanism that at least in mice is activated after the first pregnancy and likely aims to preserve female health.
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http://dx.doi.org/10.1155/2018/6319414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166384PMC
January 2019

Human cumulus cell sensitivity to vitrification, an ultrastructural study.

Zygote 2018 Jun 3;26(3):224-231. Epub 2018 Jul 3.

Tel:+39 0862 433650. Fax:+39 0862 433523.

SummaryCumulus cells (CCs) play an important role in the regulation of female gamete development, meiotic maturation, oocyte-sperm interaction, capacitation and acrosome reaction. However, their role in maintaining oocyte competence after vitrification is unclear as controversial data on their protecting action against oocyte cryoinjuries are available. Here we described the effects of vitrification on the ultrastructure of human CCs collected from women undergoing assisted reproductive technologies (ARTs). In total, 50 patches of CCs, sampled from high-quality human cumulus-oocyte complexes, were randomly allocated into two groups after patient informed consent: 1, fresh CCs (controls, n = 25); 2, vitrified CCs (n = 25). Samples were then prepared and observed by transmission electron microscopy. In fresh CCs, in which small cell clusters were visible, cell membranes were joined by focal gap junctions. Microvilli were rare and short. Nuclei, mitochondria, smooth endoplasmic reticulum (SER), Golgi apparatus and lipid droplets appeared well preserved; vacuoles were scarce. After vitrification, we observed two populations of CCs: light CCs, with a smooth appearance and few short microvilli; and dark CCs, with numerous and long microvilli. In both, most of the organelles appeared similar to those of fresh CCs. Lipid droplets were denser and more numerous, with respect to fresh CCs. They were mainly located in the peri-nuclear and sub-plasmalemmal regions. Numerous packed electron-negative vacuoles were visible. The vitrification procedure did not cause alterations in the fine structure of major organelles, except for an increased amount of lipid droplets and vacuoles. This specific sensitivity of human CCs to vitrification should be considered during ARTs.
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http://dx.doi.org/10.1017/S0967199418000138DOI Listing
June 2018

Ovarian cancer-derived extracellular vesicles affect normal human fibroblast behavior.

Cancer Biol Ther 2018 08 25;19(8):722-734. Epub 2018 Apr 25.

a Department of Life, Health and Environmental Sciences , University of L'Aquila , Via Vetoio, Coppito 2, L'Aquila , Italy.

It has become clear that non-tumor cells in the microenvironment, especially fibroblasts, actively participate in tumor progression. Fibroblasts conditioned by tumor cells become "activated" and, as such, are identified as CAFs (cancer-associated fibroblasts). These CAFs remodel the tumor stroma to make it more favourable for cancer progression. The aim of this work was to verify whether EVs (extracellular vesicles - whose role as mediators of information between tumor and stromal cells is well known) released from human ovarian cancer cells were able to activate fibroblasts. EVs isolated from SKOV3 (more aggressive) and CABA I (less aggressive) cells were administered to fibroblasts. The consequent activation was supported by morphological and molecular changes in treated fibroblasts; XTT assays, zymographies, wound healing tests and invasion assays also highlighted higher proliferation, motility, invasiveness and enzyme expression. The secretome of these "activated" fibroblasts was, in turn, able to modulate the responses (proliferation, motility and invasion) of fibroblasts, and of tumor and endothelial cells. These findings support the idea that ovarian cancer cells can modulate fibroblast behaviour through the release of EVs, activating them to a CAFs-like state; the latter are able, in turn, to stimulate the surrounding cells. EVs from SKOV3 rather than from CABA I seem to be more efficient in some processes.
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http://dx.doi.org/10.1080/15384047.2018.1451286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067870PMC
August 2018

New Hydrogels Enriched with Antioxidants from Saffron Crocus Can Find Applications in Wound Treatment and/or Beautification.

Skin Pharmacol Physiol 2018 1;31(2):95-98. Epub 2018 Feb 1.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

Saffron extracts have a long history of application as skin protectant, possibly due to their ability to scavenge free radicals. In this work, the performance of a hydrogel enriched with antioxidant compounds isolated from saffron crocus (Crocus sativus L.) petals was tested. These hydrogels could be considered as new drug delivery system. Hydrogels are crosslinked polymer networks that absorb large quantities of water but retain the properties of a solid, thus making ideal dressings for sensitive skin. We tested antioxidant-enriched hydrogels on primary mouse fibroblasts. Hydrogels enriched with kaempferol and crocin extracted from saffron petals showed good biocompatibility with in vitro cultured fibroblasts. These new types of hydrogels may find applications in wound treatment and/or beautification.
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http://dx.doi.org/10.1159/000486135DOI Listing
August 2018

Increased rounds of gonadotropin stimulation have side effects on mouse fallopian tubes and oocytes.

Reproduction 2018 03 4;155(3):245-250. Epub 2018 Jan 4.

Department of LifeHealth and Environmental Sciences, University of L'Aquila, L'Aquila, Italy

In this study, it was evaluated if increased rounds of gonadotropin stimulation could affect in mice: (i) expression levels of proteins regulating cell cycle and DNA repair in fallopian tubes and (ii) meiotic spindle morphology of ovulated oocytes. To this end, adult female mice were subjected or not (Control) to 6 or 8 rounds of gonadotropin stimulation. Ovulated oocytes were incubated with anti A/B tubulin to evaluate spindle morphology. Fallopian tubes were analyzed to detect Cyclin D1, phospho-p53/p53, phospho-AKT/AKT, phospho-GSK3B/GSK3B, SOX2, OCT3/4, phospho-B-catenin/B-catenin, phospho-CHK1 and phospho-H2A.X protein levels. After 6 rounds, Cyclin D1, p53 and phospho-p53 contents were higher than Control. After 8 rounds, the contents of phosphorylated AKT, GSK3B and p53 as well as of total p53, Cyclin D1 and OCT3/4 significantly increased in comparison with Control. Conversely, SOX2 and B-catenin were similarly expressed among all experimental groups. The finding that phospho-CHK1 and phospho-H2A.X protein levels were undetectable supported the absence of extensive DNA damage. Oocytes number and percentage of normal meiotic spindles drastically decreased from 6 rounds onward. Altogether, our results demonstrated that 6 and 8 cycles of gonadotropin stimulation reduce mouse reproductive performances by inducing over-expression and over-activation of proteins controlling cell cycle progression in fallopian tubes and by impairing oocyte spindle.
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http://dx.doi.org/10.1530/REP-17-0687DOI Listing
March 2018