Search

Study questionDoes 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 answerEmbryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen.What is known alreadyART 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, durationZygotes, 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, methodsMitochondrial 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 chanceEmbryos 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 dataN/A.Limitations, reasons for cautionFindings in mice embryos might be different from human embryos.Wider implications of the findingsThis 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.

Simple Summary: In 2011, the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident resulted in the release of vast quantities of radioactive substances. In this study, we performed an ultrastructural analysis of the testis of the wild large Japanese field mouse (Apodemus speciosus), inhabiting the area near the ex-evacuation zones of the FDNPP and chronically exposed to various levels of low-dose-rate (LDR) radiation, to evaluate the effects of ionizing radiation on the male reproductive system and the possible consequences for future generations. Our results showed a preserved morphology of seminiferous tubules. However, ultrastructural changes, such as wide intercellular spaces, cytoplasmic vacuolization, vacuolated mitochondria, and increased lipid droplet clusters, were found, which could be linked to the mechanisms related to spermatogenesis. Long-term chronic LDR radiation exposure associated with the FDNPP accident had no pronounced adverse effect on spermatogenesis in A. speciosus, even if testis presented some ultrastructural changes. Our findings could be used in future studies to evaluate the effects of radiation on reproductive health in wild animals. Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, great attention has been paid to the impact of chronic low-dose-rate (LDR) radiation exposure on biological systems. The reproductive system is sensitive to radiation, with implications connected to infertility. We investigated the testis ultrastructure of the wild large Japanese field mouse (Apodemus speciosus) from three areas contaminated after the FDNPP accident, with different levels of LDR radiation (0.29 µSv/h, 5.11 µSv/h, and 11.80 µSv/h). Results showed good preservation of the seminiferous tubules, comparable to the unexposed animals (controls), except for some ultrastructural modifications. Increases in the numerical density of lipid droplet clusters in spermatogenic cells were found at high levels of LDR radiation, indicating an antioxidant activity rising due to radiation recovery. In all groups, wide intercellular spaces were found between spermatogenic cells, and cytoplasmic vacuolization increased at intermediate and high levels and vacuolated mitochondria at the high-level. However, these findings were also related to the physiological dynamics of spermatogenesis. In conclusion, the testes of A. speciosus exposed to LDR radiation associated with the FDNPP accident showed a normal spermatogenesis, with some ultrastructural changes. These outcomes may add information on the reproductive potential of mammals chronically exposed to LDR radiation. [ABSTRACT FROM AUTHOR]

Highlights: What are the main findings? For the first time, the in vitro effects of simulated microgravity using a Random Positioning Machine on the ultrastructural features of human metaphase II oocytes were investigated by transmission electron microscopy. Microgravity might compromise oocyte quality affecting the ultrastructural morphology of mitochondria, endoplasmic reticulum, and cortical granules due to a possible alteration of the cytoskeleton. What is the implication of the main finding? Microgravity-induced organelle modifications potentially affect the oocyte's ability to fully mature and fertilize and develop into a viable embryo. The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria–vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocytes. [ABSTRACT FROM AUTHOR]

Simple Summary: Exposure to endocrine-disrupting pollutants, such as the fungicide mancozeb, is linked to various environmental health hazards, including female fertility. Although the dithiocarbamate mancozeb has low reported toxicity in mammals, it impairs female reproductive functions in exposed animals and humans. The specific mechanism of action of mancozeb and the damage to cell structures in the female reproductive system are still unclear. This study aims to describe the ultrastructure of mouse oocytes exposed in vitro to increasing concentrations of mancozeb (0.001–1 μg/mL) by light and transmission electron microscopy and to perform a morphometric analysis over significant organelles. While from 0.001 to 0.1 μg/mL, oocyte ultrastructure was comparable to controls, at the highest concentration (1 µg/mL), a decrease in the numerical density of mitochondria and cortical granules, an altered organelle distribution, and flattening of microvilli were observed. These results could be responsible for the adverse effect of this fungicide on mammalian reproductive performance. Mancozeb is a widely used fungicide, considered to be an endocrine disruptor. In vivo and in vitro studies evidenced its reproductive toxicity on mouse oocytes by altering spindle morphology, impairing oocyte maturation, fertilization, and embryo implantation. Mancozeb also induces dose-dependent toxicity on the ultrastructure of mouse granulosa cells, including chromatin condensation, membrane blebbing, and vacuolization. We evaluated the effects on the ultrastructure of mouse oocytes isolated from cumulus-oocyte complexes (COCs), exposed in vitro to increasing concentrations of mancozeb. COCs were matured in vitro with or without (control) low fungicide concentrations (0.001–1 μg/mL). All mature oocytes were collected and prepared for light and transmission electron microscopy. Results showed a preserved ultrastructure at the lowest doses (0.001–0.01 μg/mL), with evident clusters of round-to-ovoid mitochondria, visible electron-dense round cortical granules, and thin microvilli. Mancozeb concentration of 1 μg/mL affected organelle density concerning controls, with a reduction of mitochondria, appearing moderately vacuolated, cortical granules, and microvilli, short and less abundant. In summary, ultrastructural data revealed changes mainly at the highest concentration of mancozeb on mouse oocytes. This could be responsible for the previously described impaired capability in oocyte maturation, fertilization, and embryo implantation, demonstrating its impact on the reproductive health and fertility. [ABSTRACT FROM AUTHOR]

(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. [ABSTRACT FROM AUTHOR]

Copyright of Turkish Journal of Obstetrics & Gynecology is the property of Galenos Yayinevi Tic. LTD. STI and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

After its discovery in 1825 by the physiologist J.E. Purkinje, the human germinal vesicle (GV) attracted the interest of scientists. Discarded after laparotomy or laparoscopic ovum pick up from the pool of retrieved mature oocytes, the leftover GV was mainly used for research purposes. After the discovery of Assisted Reproductive Technologies (ARTs) such as in vitro maturation (IVM), in vitro fertilization and embryo transfer (IVF-ET) and intracytoplasmic sperm injection (ICSI), its developing potential was explored, and recognized as an important source of germ cells, especially in the case of scarce availability of mature oocytes for pathological/clinical conditions or in the case of previous recurrent implantation failure. We here review the ultrastructural data available on GV-stage human oocytes and their application to ARTs. [ABSTRACT FROM AUTHOR]

Radiofrequency electromagnetic radiation emitted from cell phone has harmful effects on some organs of the body, such as the brain, heart, and testes. This study aimed to assess the effects of cell phones on sperm parameters, DNA fragmentation, and apoptosis in normozoospermic. Normal sperm samples were divided into two groups of control and case. The samples from the case were placed for 60 min at a distance of approximately 2.5 cm from the cell phone set in the active antenna position. Control samples were exposed to cell phones without active antennas. All specimens were analysed by World Health Organization criteria. Sperm viability, sperm with chromatin abnormality and maturity, DNA fragmentation, and apoptosis were examined. Viability and motility in the case were significantly lower than the control (p <.001, p =.004 respectively). The percentage of apoptotic sperms and DNA fragmentation were significantly higher in the case when compared with the control (p =.031, p <.001 respectively). The other parameters studied such as morphology, chromatin abnormality, and maturity showed no significant difference between the case and control groups. Cell phone waves had a detrimental effect on human sperm's biological features. Therefore, it is recommended to keep the cell phone away from the pelvis as much as possible. [ABSTRACT FROM AUTHOR]

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 (137Cs) 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. [ABSTRACT FROM AUTHOR]

Background/aim: The aim of this study was to evaluate the efficiency of in vitro embryo splitting (IES) procedures. We also assessed the quality of the blastocysts developed from embryos obtained from different sources. Materials and methods: Good quality embryos at 6--8-cell stages were categorized according to their fertilization sources: 1) frozenwarmed donated embryos, 2) chromosomally abnormal embryos, 3) parthenogenetic embryos, and 4) embryos derived from fertilization of in vitro matured oocytes (rescue IVM). After IES, splitting and developmental efficiency was assessed. Furthermore, the quality of the developed blastocysts was evaluated by Hoechst and propidium iodide (PI) staining. Results: The data showed a high rate of both splitting and developmental efficiency in the frozen-warmed embryos after IES (140% and 71.7%, respectively), followed by chromosomally abnormal embryos (96.8% and 52.5%, respectively). Results of the Hoechst and PI staining showed that the mean ± SD cell numbers of the control group were higher (113.11 ± 16.01) than that of twins A (donor blastomeres embryos, 58 ± 12.2) and B (recipient blastomeres embryos, 50.4 ± 8.5), respectively. Conclusion: Chromosomally normal embryos enrolled in IES are more potent to develop into viable blastocysts. For research purposes, 1PN and 3PN embryos are the best options for splitting procedures, regardless of the poor quality of developed blastocysts. [ABSTRACT FROM AUTHOR]

Over the last forty years, many trials have been performed using mammalian embryo cultures with reduced oxygen tension (O2) to encourage proper embryo development and increase the success rate for in vitro fertilization (IVF) outcome. Even if the use of atmospheric O2 (20%) affects invitro embryo development and intracellular redox balance, the use of low (5% O2, physiologic) and ultra-low (close or less to 5% O2) O2 applied to invitro embryo culture is still under debate. Numerous studies in various mammalian species have shown that embryo development improves when culturing embryos under low O2, 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 O2 in embryo culture, and examines the impact of multiple factors on IVF outcomes. O2: oxygen tension; IVF: invitro fertilization; IVC: invitro culture; ET: embryo transfer; ROS: reactive oxygen species; ARTs: assisted reproductive technologies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, DHEAovaries 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. [ABSTRACT FROM AUTHOR]

Summary: Currently, rescue in vitro maturation (IVM) is not a routine method in assisted reproductive treatment (ART) programmes but is a promising procedure for ART to improve IVM. The aim of this study was to compare embryo morphokinetics of germinal vesicles (GV) with metaphase II (MII) oocytes from controlled ovarian hyperstimulation (COH) cycles by time-lapse photography monitoring (TLM). Morphokinetics of the same number of embryos derived from the in vivo (group I) and rescue of in vitro matured oocytes (group II) from 310 patients were analyzed and compared retrospectively. The time to form second PB extrusion (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf) and time of two to eight discrete cells (t2–t8) were assessed. Abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), trichotomous mitoses (TM), and the rates of embryo arrest were assessed. These data showed that tPB2, tPNa, tPNf, t2, t3 and t4 stages took place later in group II compared with group I (P <0.001, P =0.017, P <0.001, P <0.001, P <0.001, P <0.001, respectively). The rates of uneven blastomeres, Fu, TM, and embryo arrest were increased significantly in group II compared with group I (P =0.001, P <0.001, P =0.003, P <0.001, respectively). Based on the exact annotation of timing parameters and cleavage patterns, the present data agreed with the concept that rescue IVM of oocytes negatively influences embryo morphokinetics. Therefore, cautious use of embryos derived from rescue IVM of GV oocytes should be made. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Cumulus 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In many clinics for Assisted Reproductive Technology (ART) cleavage stage embryos are discarded prior to subsequent transfer (ET) and/or cryopreservation for a low morphological score. The aim of this study was to assess the morphology and viability of fresh and frozen-thawed human cleavage embryos, discarded at the 2nd day of in vitro culture and cultured for further 3 days. Discarded embryos (123) from 57 infertile patients were cultured for 3 days. Their morphology was daily examined. Zona pellucida (ZP) birefringence, assessed by Polscope, and cell viability, by Hoechst/propidium iodide staining, were evaluated at the 5th day. Top-quality expanded blastocysts successfully developed by poor-quality embryos from both fresh and frozen-thawed embryos. However, a normal ZP birefringence was significantly higher in fresh than in frozen-thawed embryos. Discarded embryos were able to grow up to 5 days, with aviability rate of 60.9%. In addition, the viability rate in fresh embryos was significantly higher than frozenthawed ones. In conclusion, poor-quality embryos were capable to develop to the blastocyst stage following an extended culture in vitro, thus representing an alternative choice for clinical applications. [ABSTRACT FROM AUTHOR]

The article focuses on evaluating the effects of low-dose-rate (LDR) radiation on the testis ultrastructure in the Large Japanese field mouse in areas contaminated after the Fukushima Daiichi Nuclear Power Plant accident, finding that while some modifications were observed.

The article focuses on investigating the morphological alterations in the tubal ampulla of a mouse model of polycystic ovary syndrome (PCOS) and examining the potential protective effects of L-carnitine (LC) and acyl-L-carnitine (ALC) against the PCOS phenotype.

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. [ABSTRACT FROM AUTHOR]

Objective: Polycystic ovary syndrome (PCOS) is a multisystemic endocrinopathy associated with female infertility, also characterized by polycystic ovaries. After having established an experimental model of PCOS in mice induced by DHEA and assessed the protective actions of two different L-carnitine formulations (Di Emidio et al., 2020), we here aimed to describe the microanatomy of the uterus. Methods: Adult CD1 mice were injected or not (control group) with DHEA (DHEA group) for 20 days with two different carnitine formulations: 1) L-carnitine and acetyl-L-carnitine (DHEA+C1 group), and 2) also containing propionyl-L-carnitine (DHEA+C2 group) (Di Emidio et al., 2020). Uterine horns were then collected and subjected to a) Hematoxylin-Eosin and Trichrome Mallory stainings; b) immunohistochemical analysis for MG-AGE, 4-HNE, Tomm20, Col1 and 17 β-HSD4. Results: In the DHEA group was found hyperplasia of luminal and glandular epithelium, lower stromal density, increased endometrial width, and collagen deposition, compared to controls. Both carnitine formulations reduced hyperplasia and fibrosis, with a more protective effect exerted by DHEA+C2. Compared to the DHEA group, the oral administration of Carnitines ameliorated glycative, antioxidant and energetic activities, with a reduced expression of MG-AGEs, 4-HNE and 17 β-HSD4 and increased immunostaining for the mitochondrial transporter TOMM20 in the endometrial luminal and glandular epithelium of carnitine treated groups. Conclusion: The data obtained confirmed that PCOS negatively affects uterine morphology; carnitine supplementation may reduce PCOS-induced hyperplasia and fibrosis and restore glycative, antioxidant and energetic activities. These results suggested that a diet supplemented with oral carnitines might exert protective actions on the female reproductive system. [ABSTRACT FROM AUTHOR]

Diagnostic histomorphological examination and FISH analysis in spontaneous abortion may provide the diagnosis of the cause of miscarriage, in order to planning future pregnancies and providing psychological support. To evaluate the readthrough activity of the TRID molecule in the rescue of the LRBA expression in human primary fibroblasts characterized by an homozygous stop mutation, we treated LRBAstop cells with compound 1 after 24, 48 and 72 hours of treatment. The urgent need to find novel therapeutics with both antifungal and antibiofilm activities and with minimal cytotoxicity and side effects to the host. [Extracted from the article]

The article offers information about the ultrastructural evaluation of mouse oocytes after exposure to the fungicide Mancozeb. It mentions that Mancozeb, a fungicide, a member of the Dithiocarbamates group, used in the management of fungal diseases of plants, and classified as an endocrine disruptor.

During antral follicle development mouse oocytes undergo rearrangement of granulosa cell interactions and the oocytes released from follicles at the beginning or at the end of antral development are either devoid of denuded oocytes (DO) or strictly associated with cumulus-intact (CI) cumulus cells. In this study, these two oocyte classes were analyzed before germinal vesicle (GV) and after in vitro maturation (IVM) to evaluate (a) the ultrastructural aspect of oolemma microvilli by scanning electron microscopy analysis and (b) specific morphological markers of differentiation (chromatin organization, mitochondria, cortical granules, microfilaments, and spindle of metaphase II- MII-). At GV-stage, CI oocytes exhibited remarkable differences (a) in the oolemma microvillar ultrastructure and distribution with respect to DO and (b) in the chromatin organization that was typical of meiotically competent germ cells. By contrast, homogeneous patterns of distribution of mitochondria, cortical granules, and microfilaments characterized both the oocyte classes. At the end of culture, CI oocytes, even when matured without cumulus cells, reached more efficiently the MII stage and acquired an ultrastructural microvillous configuration different from DO. In addition, MII-arrested DO had a higher percentage of meiotic spindles with abnormal morphology in comparison with preovulatory oocytes, while cortical granule and microfilament patterns revealed no appreciable differences between the groups. With regard to mitochondria, a polarized distribution of these organelles was found in 82% of DO and in 97% of CI oocytes. These observations suggested that the achievement of the full antral follicle development is a condition for the acquisition of specific qualitative properties that are essential for the production of fertilizable oocytes, both in in vivo and in vitro models as well. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

During the last years, embryo development in vitro was studied in different culture conditions and oxygen (O2) concentrations. Higher developmental blastocyst (Bl) rates were obtained with embryos cultured under a physiological O2 tension (5%), respect to those cultured under atmospheric O2 conditions (20%) [1], but the mechanisms responsible for this, during the preimplantation embryogenesis remain unclear. This study aimed to evaluate the effect of physiologic or atmospheric O2 tension on the ultrastructure of mouse Bl. In vivo, Bl were flushed out of the uterus after natural fertilization (controls). In vitro fertilization (IVF) was performed using KSOM medium and Bl were then cultured under an O2 tension of 5% and 20% for 5 days [2]. After collection, Bl were washed in PBS, fixed in 2.5% glutaraldehyde/PBS and subjected to standard preparative for transmission electron microscopy (TEM) [3]. Morphometric analysis was done on ultrathin sections. The cells of the trophoblast (TE) formed a single, continuous layer of flattened cuboidal cells. In all the group, both inner cell mass (ICM) and TE showed the presence of extensive regions of less dense, granular cytoplasm. Microvilli were distributed on the apical surface, projecting toward the zona pellucida. Nuclei were delimited by integral nuclear membranes and contained dispersed euchromatin with patches of heterochromatin. Cells in mitotic division, with well-defined chromosomes, were occasionally identified. Isolated mitochondria and vacuoles were numerous. Mitochondria, in both ICM and TE, had an elongated and tubular shape, delimited by a double electron-dense membrane. The numerical density of mitochondria was lower in vitro than in vivo, especially under 20% O2. Interestingly, this alteration in density in vitro was associated to an increased vacuolization, both at 5% and 20% O2. These results indicated that alterations in the Bl ultrastructure, especially at 20% O2, can be connected to the O2 concentration and can motivate the higher developmental rates obtained at lower O2 concentration. [ABSTRACT FROM AUTHOR]

In medical training it is fundamental to have a 3D understanding of human anatomy [1]. Body dissection is considered mandatory in most of the bio-medical schools. Medical schools around the world, constantly face the problem of availability of the cadavers. Apart from the classic methods (lectures, podcasts, atlas or photographs, models) technology advances made available new instruments to learn/teach 3D anatomy, which allow cadaverless dissection with the help of simulator software or virtual cadavers. The Anatomage® and Sectra® tables are advanced anatomy visualization systems, adopted by many of the world's leading medical schools and institutions [2,3]. Here we report our experience with Anatomage® during the Academic Year 2016-17, in the Postgraduate Courses of Medical and Surgical Specialization, Master degrees in Medicine, Dental Medicine, Biology of Health and Nutrition, as well as Basic Degrees in Nursing and Biotechnology, of the University of L'Aquila. We enrolled 30 MD postgraduate students, and 440 undergraduate students. Both postgraduate and undergraduate medical students were allowed to handle the table. The other students assisted to class table demonstrations. An evaluation test was administered to all students at the end of the Courses. Our preliminary observations suggest that the use of virtual dissection table into the anatomical curriculum improves the learning student performance. Each student have a different set of needs, and the base line skills may be different. So, the teacher should take in consideration the variable scope of practice of the specific health professions. We are currently evaluating the efficacy of this technology at the end of the examinations. In the present preliminary report, we account with our results that are indicative of a positive impact on both basic and advanced anatomical learning. [ABSTRACT FROM AUTHOR]

We investigated potential germline-specific radiosensitive biomarkers in the testes of large Japanese field mice (Apodemus speciosus) exposed to low-dose-rate (LDR) radiation after the Fukushima accident. Fukushima wild mice testes were analysed via RNA-sequencing to identify genes differentially expressed in the breeding and non-breeding seasons when compared to controls. Results revealed significant changes during the breeding season, with Lsp1 showing a considerable upregulation, while Ptprk and Tspear exhibited significant reductions. Conversely, in the non-breeding season, Fmo2 and Fmo2 (highly similar) were significantly upregulated in radiation-exposed Fukushima mice. qPCR analysis results were consistent with transcriptome sequencing, detecting Lsp1 and Ptprk regulation in the testes of Fukushima mice. While differences in gene expression were observed, these do not imply any causal association between the identified biomarkers and chronic LDR exposure, as other factors such as the environment and developmental age may contribute. This study provides valuable insights into the reproductive biology is affected by environmental radiation and highlights the value of assessing the effects of chronic LDR radiation exposure on testicular health in wild mice., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Flaxseed is a source of antioxidants utilized for female infertility treatment in traditional medicine. This study investigated the effects of flax hydroalcoholic extract and flaxseeds during prenatal and postnatal (PND) periods on folliculogenesis and serum total antioxidant capacity (TAC). Pregnant NMRI mice received 500 and 1000 mg/kg of flax extract (LE) and the same doses of flaxseed (LS). Female pups received the same regimen for 56 days. The body, ovarian morphometry, follicle development, and TAC levels were evaluated. The ovarian weight significantly increased in the LE1000 group compared to the LS500 group. The LE500 group had a considerably lower number of primary and antral follicles compared to the CTL and LS1000 groups. The number of antral follicles significantly increased in the LE1000 group compared to the LS500 and LE500 groups. The number of preovulatory follicles was higher in the LE1000 group. A significant increase in the TAC levels was detected in the LS500, LS1000, and LE1000 groups. LE showed a dose-dependent protective effect on the folliculogenesis in F1, which is more evident with the dosage of 1000 mg/kg. This could be related to the strongest antioxidant property of LE1000, as shown by the highest levels of TAC., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Fahimeh Pourjafari et al.)

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.