Guido Macchiarelli

Università degli Studi dell'Aquila, Aquila, Abruzzo, Italy

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Publications (104)173.51 Total impact

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    ABSTRACT: Background: Mancozeb (MZ) is a fungicide that belongs to the subclass of metal (Mn/Zn) ethylene-bis-dithiocarbamate pesticides. In mouse and human granulosa cells (GCs) exposed to MZ (0.01μg/ml), morphological modifications and significant alterations of p53 expression level in comparison with control GCs were recorded. Objectives: To investigate if MZ (0.01μg/ml) induces oxidative stress and alters energy metabolism in exposed mouse GCs. Results: Following fungicide exposure, GCs showed low p53 content, a depolarized mitochondrial membrane potential (ΔΨm), as well as low ATP and reduced glutathione (GSH) levels associated with increased reactive oxygen species (ROS) generation. No remarkable differences on other parameters such as ATP/ADP ratio, energy charge, as well as induction of apoptosis and DNA damage were found. The activation of AKT and PDK1 kinases in MZ-treated cells was observed. Inhibition of ROS generation by the antioxidant N-acetylcysteine (NAC) restored a normal expression level of p53. Conclusions: Our results demonstrate that the low dose of MZ here used induces a mild oxidative stress in GCs, and provides evidence for the possible involvement of AKT/PKB signaling pathway in triggering adaptive and survival response.
    Toxicology in Vitro 09/2015; DOI:10.1016/j.tiv.2015.09.018 · 2.90 Impact Factor
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    S Bianchi · G Macchiarelli · G Micara · A Linari · C Boninsegna · C Aragona · G Rossi · S Cecconi · S A Nottola ·
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    ABSTRACT: Childbearing delay contributes to the increase of subfertile couples that require assisted reproductive technology (ART). Subfertility relates with reproductive aging (RA). In vitro aging (IvA) (due to extended culture) may also impair oocyte competence. Aims of this study were to evaluate and compare the oocyte ultrastructure after RA and IvA. Cumulus-oocyte complexes (COCs) (n = 68), with metaphase II oocyte and expanded cumulus, from consenting patients (<35 years old and ≥35 years old, n = 36), were selected by phase contrast microscopy and fixed at pick up, or after 24 h culture. COCs (n = 44) were studied by light and qualitative/morphometric transmission electron microscopy. Two-way ANOVA, with age and culture as grouping factors, was applied for statistical analysis (p < 0.05). Metaphase II cumulus-free oocytes (n = 24) were selected for confocal microscopy observations. Significant decrease of mitochondria-smooth endoplasmic reticulum aggregates, increase of mitochondria-vesicle complexes size and amount, decrease of cortical granules and microvilli, and alterations of the spindle structure characterized both RA and IvA oocytes. These changes were significantly more evident in the RA oocytes submitted to IvA. RA oocytes also showed changes of the zona pellucida and occurrence of vacuoles after culture. Cumuli appeared re-compacted after culture, irrespective of the age of the patients. These data demonstrated that aging is related to decay of oocyte ultrastructural quality, and that oocytes from elder women are more sensitive to prolonged culture (IvA) than the oocytes from younger women. These morphological results should be considered when applying ART in aged patients, rescue ICSI, or artificial oocyte activation.
    Journal of Assisted Reproduction and Genetics 08/2015; 32(9). DOI:10.1007/s10815-015-0552-9 · 1.72 Impact Factor
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    ABSTRACT: Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08%respectively in prepubertals and adults; P <0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00%vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94%vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development.
    PLoS ONE 04/2015; 10(4):e0124911. DOI:10.1371/journal.pone.0124911 · 3.23 Impact Factor
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    ABSTRACT: In this chapter, we will describe the standard technical protocols used to obtain blood vascular cast for the study with SEM, as well as we will briefly review the recent advances in the observation of vascular network through the use of this tool, and its application in the study of angiogenesis mainly according to our experience in the mammalian ovary.
    Handbook of Vascular Biology Techniques, 2015 edited by Mark Slevin, Garry McDowell, 04/2015: chapter Part II: pages 153-171; Springer., ISBN: 978-94-017-9715-3 (Print) 978-94-017-9716-0 (Online)
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    ABSTRACT: Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification. We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation. Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90-100 mum in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3-4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8-9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied. Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane "recycling" that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardening.
    Reproductive Biology and Endocrinology 11/2014; 12(1):110. DOI:10.1186/1477-7827-12-110 · 2.23 Impact Factor
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    ABSTRACT: In vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries may avoid side effects connected to hyperstimulation during IVF procedures, including the risk of cancer recurrence. In humans, the scarce availability of immature oocytes limits morphological studies. The monovular ovine may represent an experimental model for IVM studies. To assess if the scarce developmental competence of prepubertal oocytes (PO) is related to morphological changes we analyzed, by light and transmission electron microscopy, cumulus-oocyte-complexes (COCs) from lambs (30-40 days old) and sheep (4-6 years old) at sampling and after 7 h, 19 h, 24 h of IVM. Meiotic progression was determined at the same time points. At sampling, the germinal vesicle (GV) of PO was round and centrally or slightly eccentrically located, whereas in adult oocytes (AO) it was irregularly shaped and flattened against the oolemma. PO, differently from AO, showed numerous trans-zonal projections. Organelles, including cortical granules (CGs), were more abundant in AO. After 7 h, the percentage of AO that underwent GVBD-MI transition increased significantly. In PO, the oolemma was juxtaposed to the ZP; in AO, it showed several spikes in correspondence of cumulus cells (CC) endings. In PO, organelles and isolated CGs were scattered in the ooplasm. In AO, groups of CGs were also present under the oolemma. After 19 h, PO underwent GVBD-MI transition; their oolemma showed several spikes, with CC projections retracted and detached from the ZP. AO underwent MI-MII transition; their oolemma regained a round shape. CGs were located beneath the plasmalemma, arranged in multiple, continuous layers, sometime discontinuous in PO. After 24 h, both groups reached the MII-stage, characterized by a regular oolemma and by expanded CCs. PO showed CGs distributed discontinuously beneath the oolemma, while AO showed a continuous monolayer of CGs. Even if PO were able of reaching morphological maturation after 24 h of IVM, our ultrastructural analysis allowed detecting the presumptive sequence of cytoplasmic alterations connected with the delay of nuclear maturation, that might explain the reduced developmental competence of such oocytes. Data from the sheep model are of interest for zootechny, and provide an experimental basis for improving human IVM technology.
    Reproductive Biology and Endocrinology 11/2014; 12(1):115. DOI:10.1186/1477-7827-12-115 · 2.23 Impact Factor
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    ABSTRACT: In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs.
    Journal of Reproduction and Development 08/2014; 60(6). DOI:10.1262/jrd.2014-027 · 1.52 Impact Factor
  • Giovanni De Gasperis · Daniele Landro · Giuseppe Stifano · Guido Macchiarelli ·
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    ABSTRACT: Academic communities need a collaborative portal to provide new distance learning opportunities and to improve personal training opportunities. An innovative portal must integrate an adaptive engine to assist learners individually, meeting them at their skill and knowledge levels. More than 300 biomedical students were enrolled to carry out the assessment of the new developed platform, which integrates a prototype adaptive engine based on Bayesian inference. Students who completed the web-based module left positive feedback and good overall satisfaction.
    FUSION 2014 - 17th International Conference on Information Fusion, IEEE, Salamanca, ES; 07/2014
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    ABSTRACT: Unlike men, who have continuous spermatogenesis throughout most of their lifetime, women are born with a fixed supply of follicles, and this number progressively declines with age until the menopause. Beside age, the speed of follicle depletion can be regulated by genetic, hormonal and environmental influences. In the course of their lives, women are exposed to multiple chemicals and radiation sources that can increase the chance of developing permanent infertility and premature ovarian failure (POF). A wealth of experimental data indicate that iatrogenic (chemotherapy, radiotherapy) and xenobiotics agents (e.g., chemicals, pharmaceuticals) are potent ovotoxicants capable of accelerating ovarian reserve depletion. In the present review we reported the negative effects exerted on mammalian ovary by some widely diffused environmental chemicals, as polycyclic aromatic hydrocarbons (PAHs) and dithiocarbamate mancozeb, and by 1-3 butadiene and 4-vinylcycloexene, two occupational chemicals known to be capable of inducing ovarian cancer and infertility. Furthermore, attention has been devoted to the consequences of chemo- and radiotherapy on the ovary, both known to affect reproductive lifespan. Our increasing understanding of metabolic alterations induced by these agents is fundamental to individuate new therapeutic strategies aimed to prevent ovarian dysfunction in fertile women.
    Current pharmaceutical design 02/2014; 20(34). DOI:10.2174/1381612820666140205145319 · 3.45 Impact Factor
  • Giovanni De Gasperis · Daniele Landro · Guido Macchiarelli ·
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    ABSTRACT: In recent years, there has been a growing interest about new tech- niques for information exchange in medical context. Academic communities need a collaborative portal with a certain degree of interactivity between speak- er and audience to provide new learning opportunities. The objective of the re- search is, therefore, addressed to provide a modular and user-friendly web por- tal that allows scientific information exchange and takes into account only the centrality of four essential elements: teachers, learners, learning-process and learning-content. This collaborative system contains also 3D learning objects to simulate highly interactive virtual laboratories. The collaborative portal is de- signed to provide an acceptable teaching tool to integrate face-to-face learning sessions with thematic web-seminars.
    3rd International Workshop on Evidence Based and User centred Technology Enhanced Learning, Trento, Italy; 09/2013
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    ABSTRACT: Abstract. The aim of this study has been to determine the effects of in vivo post-ovulatory ageing (POA) on the distribution of spindle-associated proteins, histone H3/H4 post-translational modifications and v-akt murine thymoma viral oncogene homolog 1 (Akt) expression levels. To this end, oocytes were retrieved 13, 29 and 33 h after human chorionic gonadotrophin (hCG) treatment. The presence and distribution at the meiotic spindle of acetylated tubulin, 5 g-tubulin, polo kinase-1 and Ser473/Thr308 phosphorylated Akt (pAkt) as well as histone H3 and H4 acetylation and phosphorylation levels were assayed via immunofluorescence. Akt expression levels were determined via reverse transcription–polymerase chain reaction and western blotting analyses. Spindles from oocytes recovered 13 h and 29 h after hCG treatment showed similar levels of acetylated tubulin but ageing induced: (1) translocation of g-tubulin from spindle poles to microtubules, (2) absence of Thr308- and Ser473-pAkt in 76% and 30% of oocytes, respectively and (3) a 10 significant reduction in phosphorylation levels of serine 10 on histone 3. At 29 h, a significant decrease in Akt mRNA, but not in pAkt or Akt protein expression levels, was recorded. By contrast, protein content significantly decreased 33 h after hCG. We conclude that POA impairs oocyte viability and fertilisability by altering the expression levels and spindle distribution of proteins that are implicated in cell survival and chromosome segregation. Together, these events could play a role in oocyte apoptosis.
    Reproduction Fertility and Development 04/2013; 26(4). DOI:10.1071/RD13010 · 2.40 Impact Factor
  • Giovanni De Gasperis · Guido Macchiarelli · Daniele Landro ·
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    ABSTRACT: In recent years, there has been a growing interest about new techniques for information exchange in medical context. Academic communities need a collaborative portal with a certain degree of interactivity between speaker and audience to provide new learning opportunities. The objective of the test program is to provide a modular and user-friendly web portal that allows the exchange of scientific information and takes into account only the centrality of four essential elements: teachers, learners, the learning process and the learning content. This collaborative system also contains 3D learning objects to simulate highly interactive virtual laboratories. More than 300 biomedical students were enrolled to carry out the assessment of the platform. Students who completed the web-based module left positive feedback and demonstrated good overall satisfaction with the assessment. The collaborative portal revealed an acceptable teaching alternative to integrate face-to-face instruction with thematic webinars.
    International Journal of Technology Enhanced Learning 01/2013; 5(3):198-212. DOI:10.1504/IJTEL.2013.059491
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    Mohammad Ali Khalili · Stefania A Nottola · Abbas Shahedi · Guido Macchiarelli ·
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    ABSTRACT: The use of ovarian stimulation for infertility treatment is associated with side effects of ovarian hyperstimulation syndrome (OHSS) and potential cancer risk. This is also true in high risk women such as those polycystic with ovary (PCO) and polycystic ovarian syndrome (PCOS). In vitro maturation (IVM) of oocytes was primarily developed to make IVF safe for women with PCO and at high risk of OHSS. The application of IVM of oocytes to assist clinical infertility treatment remains poor because of the reduced developmental competence of oocytes after IVM, despite several decades of research. Reduced meiotic maturation and fertilization rates, as well as low blastocyst production reveal short-term developmental insufficiency of oocytes when compared with in vivo-matured counterparts. In this review, the structural role of human oocytes, revealed by different technical approaches, to the success of IVM technology is highlighted.
    Iranian Journal of Reproductive Medicine 01/2013; 11(1):1-10. · 0.19 Impact Factor
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    ABSTRACT: Thyroxine (T4) plus gonadotropins might stimulate ovarian follicular angiogenesis in immature infertile hypothyroid rdw rats by upregulating mRNA expression of major angiogenic factors. Development of growing corpus luteum (CL) is strongly related to angiogenesis and to morphofunctional development of microcirculation. Our aim was to investigate if T4 is involved in CL angiogenesis and in the activation of capillary cells and angiogenic factors after ovulation in a spontaneous model of hypothyroidism, the rdw rat. Rdw rats were treated with T4 plus gonadotropins (equine chorionic gonadotropin plus human chorionic gonadotropin; eCG+hCG) or gonadotropins alone in order to evaluate the effects of T4 on early luteal angiogenesis, on microvascular cells and on expression of major growth factors which are involved in the regulation of angiogenesis. Wistar-Imamichi rats treated with gonadotropins were used as controls. The ovaries were collected 4 days after hCG administration and analyzed using morphologic and molecular approaches. Thyroxine plus gonadotropins stimulated the growth of CLs and follicles as in controls, differently from rdw rats treated only with gonadotropins, in which CLs were not found and only small follicles, often atretic, could be recognized. In T4 plus gonadotropin-treated rdw rats CLs showed increased microvasculature, numerous activated capillaries characterized by sprouting and other angiogenic figures, and associated pericytes. Quantitative analysis revealed that the number of pericytes in T4 plus gonadotropin-treated rdw rats was comparable with that found in control rats and was significantly higher than that found in gonadotropin-treated rdw rats. The mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor was significantly higher in control rats and in T4 plus gonadotropin-treated rdw rats than in gonadotropin-treated rdw rats. mRNA expression of tumor necrosis factor α, transforming growth factor β, and epidermal growth factor did not show significant changes. Our data originally demonstrated that T4 promoted the growth of an active microcirculation in developing CLs of gonadotropin-primed hypothyroid rdw rats, mainly by inducing sprouting angiogenesis, pericyte recruitment, and upregulation of mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor. In conclusion, we suggest that T4 plays a key role in restoring luteal angiogenesis in ovaries of immature hypothyroid rdw rats.
    Theriogenology 11/2012; 79(1). DOI:10.1016/j.theriogenology.2012.09.016 · 1.80 Impact Factor
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    M.A. Khalili · M Maione · M G Palmerini · S Bianchi · G Macchiarelli · S.A. Nottola ·
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    ABSTRACT: Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morphofunctional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microscopy has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.
    European journal of histochemistry: EJH 07/2012; 56(3):e38. DOI:10.4081/ejh.2012.e38 · 2.04 Impact Factor
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    ABSTRACT: The heart is composed by a specialized muscle, whose form and function are essentials for an adequate work and shows an amount of connective tissue which support and provide insertion for this muscle, whose collagen fibers are responsible for determination of tissue feature. Our objective was to identify the structural arrangement of the heart collagen fibers in dogs. The hearts of the dogs were submitted to the process of the controlled digestion with NaOH solution and observed by scanning electron microscope. Our results showed that the collagen fibers of the endomysial wall have structural arrangement composed by an irregular network with one layer in normal dogs but in diabetic dogs the network acquires a greater amount of the fibers and layers, looking like a "rug" of fibers modifying the relationships of the stress/strain of the tissue. Ahead of the observed results we are able to conclude that exist increase in the amount and thickness of cardiac collagen fibers, beyond the increase of layers and architectural disarrangement in the endomysial wall in the diabetic dogs.
    Microscopy Research and Technique 11/2011; 74(11):1018-23. DOI:10.1002/jemt.20988 · 1.15 Impact Factor
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    ABSTRACT: In the present work the effects of a new low frequency, high intensity ultrasound technology on human adipose tissue ex vivo were studied. In particular, we investigated the effects of both external and surgical ultrasound-irradiation (10 min) by evaluating, other than sample weight loss and fat release, also histological architecture alteration as well apoptosis induction. The influence of saline buffer tissue-infiltration on the effects of ultrasound irradiation was also examined. The results suggest that, in our experimental conditions, both transcutaneous and surgical ultrasound exposure caused a significant weight loss and fat release. This effect was more relevant when the ultrasound intensity was set at 100 % (~2.5 W/cm², for external device; ~19-21 W/cm2, for surgical device) compared to 70 % (~1.8 W/cm² for external device; ~13-14 W/cm2 for surgical device). Of note, the effectiveness of ultrasound was much higher when the tissue samples were previously infiltrated with saline buffer, in accordance with the knowledge that ultrasonic waves in aqueous solution better propagate with a consequently more efficient cavitation process. Moreover, the overall effects of ultrasound irradiation did not appear immediately after treatment but persisted over time, being significantly more relevant at 18 h from the end of ultrasound irradiation. Evaluation of histological characteristics of ultrasound-irradiated samples showed a clear alteration of adipose tissue architecture as well a prominent destruction of collagen fibers which were dependent on ultrasound intensity and most relevant in saline buffer-infiltrated samples. The structural changes of collagen bundles present between the lobules of fat cells were confirmed through scanning electron microscopy (SEM) which clearly demonstrated how ultrasound exposure induced a drastic reduction in the compactness of the adipose connective tissue and an irregular arrangement of the fibers with a consequent alteration in the spatial architecture. The analysis of the composition of lipids in the fat released from adipose tissue after ultrasound treatment with surgical device showed, in agreement with the level of adipocyte damage, a significant increase mainly of triglycerides and cholesterol. Finally, ultrasound exposure had been shown to induce apoptosis as shown by the appearance DNA fragmentation. Accordingly, ultrasound treatment led to down-modulation of procaspase-9 expression and an increased level of caspase-3 active form.
    International journal of immunopathology and pharmacology 04/2011; 24(2):411-22. · 1.62 Impact Factor
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    ABSTRACT: In vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles. The present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured in vitro for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH. The follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). It is concluded that FSH supports the in vitro growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse in vitro grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells.
    Reproductive Biology and Endocrinology 01/2011; 9(1):3. DOI:10.1186/1477-7827-9-3 · 2.23 Impact Factor

  • Reproductive biomedicine online 10/2010; 20. DOI:10.1016/S1472-6483(10)62515-1 · 3.02 Impact Factor
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    ABSTRACT: Recent studies introduced the novel concept of chemical lipolysis where phosphatidylcholine (PC), an active component of commercial preparations, plays a pivotal role. Other studies suggested that sodium deoxycholate (DOC), an excipient contained in medical preparations, could be the real active component performing an adipocytolytic action. We investigated the effects of PC and DOC on human primary adipocyte cultures and on human fresh adipose tissue. Human adipocytes isolated by Rodbell's method, were cultured onto type I collagen-coated glass coverslips, placed into 24-well tissue culture plates. Cells were incubated with or without DOC (5-7-9%), PC (5%) or DOC/PC mixture and observed under phase contrast microscope. After incubation, cells were stained with Oil Red-O and with acridine orange/ethidium bromide to observe necrotic cells with phase contrast microscope and fluorescent microscope, respectively. Histological specimens from adipose tissue biopsies were observed with phase contrast microscopy and with scanning electron microscopy. To investigate the lipid pattern variability in the different experimental conditions, culture medium obtained from the different treatments was subjected to lipid extraction and subsequently to thin layer chromatography (TLC). Microscopic observation of adipocytes showed that DOC treatment led to a detrimental morphological effect in a dose-dependent manner. PC treatment did not significantly affect adipocyte viability. On the contrary, results from experiments aimed to analyze the effects of PC/DOC combined treatment suggested a PC protective role against the DOC harmful effects on adipocytes. Results indicated that clinical effects, observed in local treatment with pharmaceutical preparation, could be due only to DOC, a detergent inducing nonspecific lysis of cell membranes following adipocyte necrosis. On the other hand, PC could likely be incorporated in the lipid bilayer, thus strongly reducing the disruptive DOC effects.
    International journal of immunopathology and pharmacology 04/2010; 23(2):481-9. · 1.62 Impact Factor

Publication Stats

1k Citations
173.51 Total Impact Points


  • 2002-2014
    • Università degli Studi dell'Aquila
      • • Department of Life, Health and Environmental Sciences
      • • Center for Microscopy
      • • Department of Experimental Medicine
      Aquila, Abruzzo, Italy
  • 2008
    • Tohoku University
      • Graduate School of Agricultural Science
      Sendai-shi, Miyagi-ken, Japan
  • 2005
    • Catholic University of Louvain
      Лувен-ла-Нев, Walloon, Belgium
  • 1989-2001
    • Sapienza University of Rome
      • Department of Internal and Specialized Medicine
      Roma, Latium, Italy
    • Toho University
      • Department of Obstetrics and Gynecology
      Edo, Tōkyō, Japan
  • 1993
    • Okayama University
      Okayama, Okayama, Japan
  • 1992
    • Università degli Studi di Messina
      • Dipartimento di Medicina Clinica e Sperimentale
      Messina, Sicily, Italy