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Background The impact of smooth endoplasmic reticulum aggregates (SERa) on assisted reproductive technology (ART) outcomes was still controversial. Our objective is to investigate the impact of the presence of SERa on intracytoplasmic sperm injection (ICSI) outcomes. Methods This was a retrospective cohort study. A total of 1,090 fresh ICSI cycles from 944 patients between January 2016 and June 2020 were included. Outcomes from clinical, embryological and neonatal aspects were compared between SERa + and SERa- cycles as well as between SERa + and SERa- oocytes. Results The total gonadotropin (Gn) dose, number of oocytes retrieved, serum estradiol concentration and number of the available embryo were significantly higher in SERa + cycles than in SERa- cycles (P P > 0.05), but which were higher in SERa + oocytes than in SERa- oocytes (P P > 0.05). Good-quality embryo rate was statistically higher in SERa- cycles than in SERa + cycles (P P > 0.05). No statistical difference in clinical pregnancy rate, spontaneous abortion rate, live birth rate and premature delivery rate were found in SERa + and SERa- cycles as well as in SERa + and SERa- oocytes (P > 0.05). The implantation rate was comparable in SERa + and SERa- cycles (P > 0.05), but it is higher in the group of only SERa- embryo transfer when compared with the group of mixed SERa + and SERa- embryo transfer (P P > 0.05). Logistic regression analysis revealed number of oocytes and total dose of Gn were risk factors for SERa occurrence (aOR = 1.05 and 1.55, P Conclusion Oocyte's SERa is correlated with a number of oocytes retrieved and higher Gn dose, but it does not affect pregnancy outcomes and increase newborn malformation rate.

Research question What is the frequency of morphological dysmorphisms in immature human oocytes collected ex vivo from small antral follicles and matured in vitro? Design Human ovaries (n = 56) were excised for ovarian tissue cryopreservation (OTC). None of the patients had received exogenous gonadotrophins prior to the procedure. Immature oocytes released from small antral follicles were collected in connection with isolation of the cortex for OTC. The oocytes’ maturation stage and the morphological characteristics of the cytoplasm, zona pellucida, perivitelline space and first polar body were assessed after in-vitro maturation (IVM). Results A total of 1649 immature oocytes were collected: 30% of oocytes matured to the metaphase II (MII) stage after IVM, while metaphase I (MI), germinal vesicle and degenerated oocytes accounted for 20%, 24% and 26%, respectively. The percentages of oocytes without any dysmorphisms were 53%, 92%, and 97% for the MII, MI and germinal vesicle stage oocytes, respectively. The most frequently observed dysmorphisms among the MII oocytes were first polar body fragmentation (22%), homogeneously distributed cytoplasmic granularity (16%) and an enlarged perivitelline space (14%). Interestingly, none of the oocytes at any stage had clusters of smooth endoplasmic reticulum (SER). Conclusions Morphological dysmorphisms are present among in-vitro-matured oocytes at all maturation stages. The incidence of dysmorphisms increases as maturation progresses. The most frequent dysmorphism among MII oocytes after IVM was fragmentation of the first polar body. Clusters of SER were not observed in oocytes from unstimulated patients.

ObjectiveTo investigate whether the euploidy rate of blastocysts derived from smooth endoplasmic reticulum aggregates (SERa) positive cycles and oocytes are impacted.DesignRetrospective cohort study.Method(s)A total of 601 preimplantation genetic testing (PGT) cycles with at least one oocyte retrieved in our center between April 2017 and May 2021 were initially included in the study. Women>35 years and PGT cycles with chromosomal structural rearrangements (PGT-SR) were excluded. Embryological and blastocyst ploidy outcomes were compared among SERa+ oocyte, sibling SERa- oocytes and oocytes in SERa- cycles.ResultsNo significant difference was observed among the SERa+ oocyte group, sibling SERa- oocyte group, and SERa- cycle group in the normal fertilization rate (82.1% vs. 77.8% vs. 83.1%, respectively, P=0.061), blastocyst formation rate (71.0% vs. 72.5% vs. 68.4%, respectively, P=0.393), good quality blastocyst formation rate (46.4% vs. 48.3% vs. 42.6%, respectively, P=0.198). No significant difference was observed in the euploidy rate (50.0% vs. 62.5% vs. 63.3%, respectively, P=0.324), mosaic rate (12.5% vs. 9.7% vs. 13.4%, respectively, P=0.506), and aneuploidy rate (37.5% vs. 27.8% vs. 23.2%, respectively, P=0.137) among the three groups.ConclusionOur results suggest that the euploidy rate of blastocysts derived from SERa+ cycles and oocytes may not be impacted.

Background Smooth endoplasmic reticulum aggregation (SERa, SER+) has been reported to increase the risk of birth malformations and other abnormal outcomes, miscarriage, and perinatal complications. Other studies, however, suggest that SER+ embryos may develop into healthy infants. One report indicates that 25% of in vitro fertilization (IVF) centers discard SER+ oocytes. Thus, we investigated the effect of SER+ on birth outcomes in IVF and intracytoplasmic sperm injection. Methods We performed a literature search using PubMed, ScienceDirect, Cochrane, Embase, Ovid, and Scopus. We found a total of 1500 relevant studies between 1978 and 2020 and conducted a meta-analysis to study the effects of SER+ on live births, birth weight, and the number of metaphase II (MII) oocytes retrieved per cycle. Results Eleven eligible studies were included. If the SER+ zygote was evaluated again at the embryo transfer (ET) stage, SER+ did not affect birth or infant body weight. Stimulated ovaries producing too many oocytes per cycle were positively correlated with SER+ (OR = 1.28, 95% CI = 0.41–2.15; p = 0.004). SER+ was positively correlated with oocyte maturation rate, and observed heterogeneity in a previous meta-analysis was likely due to maternal age. Our data also showed that SER+ cycles produced more oocytes but achieved the same number of births from ET. Conclusions The use of SER+ MII oocytes is rare, with the collection of many oocytes in 1 cycle potentially inducing SER+. SER+ may be more common than we originally thought, as some SER+ is found in all oocytes. Although SER+ positively affected oocyte maturation rate, it did not affect births. We hypothesized that this is because the best embryos are chosen at every step of the process, and the oocytes with the poorest characteristics are removed. We therefore suggest a standard method for measuring SER+. Although embryos produced from SER+ cycles can be used, they should only be transferred when no other suitable embryos are available over several cycles.

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as “organized smooth endoplasmic reticulum” (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

SummaryWe have previously presented a stereological analysis of organelle distribution in human prophase I oocytes. In the present study, using a similar stereological approach, we quantified the distribution of organelles in human metaphase I (MI) oocytes also retrieved after ovarian stimulation. Five MI oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. Kruskal–Wallis and Mann–Whitney U-tests with Bonferroni correction were used to compare the means of relative volumes (Vv) occupied by organelles. In all oocyte regions, the most abundant organelles were mitochondria and smooth endoplasmic reticulum (SER) elements. No significant differences were observed in Vv of mitochondria, dictyosomes, lysosomes, or SER small and medium vesicles, tubular aggregates and tubules. Significant differences were observed in other organelle distributions: cortical vesicles presented a higher Vv (P = 0.004) in the cortex than in the subcortex (0.96% vs 0.1%) or inner cytoplasm (0.96% vs 0.1%), vesicles with dense granular contents had a higher Vv (P = 0.005) in the cortex than in the subcortex (0.1% vs 0%), and SER large vesicles exhibited a higher Vv (P = 0.011) in the inner cytoplasm than in the subcortex (0.2% vs 0%). Future stereological analysis of metaphase II oocytes and a combined quantitative data of mature and immature oocytes, will enable a better understanding of oocyte organelle distribution during in vivo maturation. Combined with molecular approaches, this may help improve stimulation protocols and in vitro maturation methods.

The EMBO journal 41(4), e106523 (2022). doi:10.15252/embj.2020106523, Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca$^{2+}$) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca$^{2+}$ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines., Published by Wiley, Hoboken, NJ [u.a.]

ObjectiveTo investigate whether the reproductive outcomes of oocytes with smooth endoplasmic reticulum aggregates (SERa) are impaired.MethodsA total of 2893 intracytoplasmic sperm injection (ICSI) cycles were performed between January 2010 and December 2019 in our center. In 43 transfer cycles, transferred embryos were totally derived from SERa+ oocytes. Each of the 43 cycles was matched with a separate control subject from SERa- patient of the same age ( ± 1 year), embryo condition, main causes of infertility, type of protocols used for fresh or frozen embryo transfer cycles. The clinical pregnancy, implantation, ectopic pregnancy and live birth rate were compared between the two groups.Results43 embryo transfer cycles from SERa- patient were matched to the 43 transferred cycles with pure SERa+ oocytes derived embryos. No significant difference was observed in clinical pregnancy rate (55.81% vs. 65.11%, p=0.5081), implantation rate (47.89% vs. 50.70%, p=0.8667) and live birth rate (48.84% vs. 55.81%, p=0.6659) between the SERa+ oocyte group and the matched group. No congenital birth defects were found in the two groups.ConclusionOur results suggest that the implantation, clinical pregnancy, live birth and birth defects rate of embryos derived from oocytes with SERa are not impaired.

C-type synaptic boutons (C-boutons) provide cholinergic afferent input to spinal cord motor neurons (MNs), which display an endoplasmic reticulum (ER)–related subsurface cistern (SSC) adjacent to their postsynaptic membrane. A constellation of postsynaptic proteins is clustered at C-boutons, including M2 muscarinic receptors, potassium channels, and s-1 receptors. In addition, we previously found that neuregulin (NRG)1 is associated with C-boutons at postsynaptic SSCs, whereas its ErbB receptors are located in the presynaptic compartment. Cbouton–mediated regulation of MN excitability has been implicated in MN disease, but NRG1-mediated functions and the impact of various pathologic conditions on C-bouton integrity have not been studied in detail. Here, we investigated changes inC-boutons after electrical stimulation,pharmacological treatment, and peripheral nerve axotomy. SSC-linked NRG1 clusters were severely disrupted in acutely stressedMNs and after tunicamycin-induced ER stress. In axotomized MNs, C-bouton loss occurred in concomitance with microglial recruitment and was prevented by the ER stress inhibitor salubrinal.Activatedmicroglia displayed apositive chemotaxis to C-boutons.Analysis of transgenicmice overexpressing NRG1 type I and type III isoforms in MNs indicated that NRG1 type III acts as an organizer of SSC-like structures, whereas NRG1 type I promotes synaptogenesis of presynaptic cholinergic terminals.Moreover,MN-derived NRG1 signals may regulate the activity of perineuronal microglial cells. Together, these data provide new insights into the molecular and cellular pathology of C-boutons in MN injury and suggest that distinct NRG1 isoform–mediated signaling functions regulate the complex matching between pre- and postsynaptic C-bouton elements. The authors thank Klaus A. Nave (Max-Planck-Institute of Experimental Medicine, Göttingen, Germany) for advice and for supplying neuregulin-1–mutant mice; Jesús María López (Universidad Complutense de Madrid, Madrid, Spain), Ester Desfilis, and José Antonio Moreno for providing spinal cord samples from nonrodent animals; Anaïs Panosa and Xavier Calomarde (all from Universitat de Lleida−Institut de Recerca Biomèdica de Lleida) for technical support with confocal and electron microscopy; and the Serveis Científico-Tècnics Anima Facility of the University of Lleida for mouse care and housing. This work was supported by grants to J.E.E. and J.C. from the Spanish Ministerio de Economía y Competitividad cofinanced by the Fondo Europeo de Desarrollo Regional (FEDER; SAF2015-70801-R). S.S. holds a grant from Spanish Ministerio de Educación, Cultura, y Deporte (FPU). M.H.S. holds a Heisenberg Fellowship from the Deutsche Forschungsgemeinschaft (DFG) and acknowledges funding by a DFG research grant (SCHW741/4-1). The authors declare no conflicts of interest.

Synapse clustering facilitates circuit integration, learning, and memory. Long-term potentiation (LTP) of mature neurons produces synapse enlargement balanced by fewer spines, raising the question of how clusters form despite this homeostatic regulation of total synaptic weight. Three-dimensional reconstruction from serial section electron microscopy (3DEM) revealed the shapes and distributions of smooth endoplasmic reticulum (SER) and polyribosomes, subcellular resources important for synapse enlargement and spine outgrowth. Compared to control stimulation, synapses were enlarged two hours after LTP on resource-rich spines containing polyribosomes (4% larger than control) or SER (15% larger). SER in spines shifted from a single tubule to complex spine apparatus after LTP. Negligible synapse enlargement (0.6%) occurred on resource-poor spines lacking SER and polyribosomes. Dendrites were divided into discrete synaptic clusters surrounded by asynaptic segments. Spine density was lowest in clusters having only resource-poor spines, especially following LTP. In contrast, resource-rich spines preserved neighboring resource-poor spines and formed larger clusters with elevated total synaptic weight following LTP. These clusters also had more shaft SER branches, which could sequester cargo locally to support synapse growth and spinogenesis. Thus, resources appear to be redistributed to synaptic clusters with LTP-related synapse enlargement while homeostatic regulation suppressed spine outgrowth in resource-poor synaptic clusters.

To investigate whether the rate of euploidy and pregnancy outcomes are affected by smooth endoplasmic reticulum clusters (SERc) and other metaphase II human oocyte dysmorphisms.Retrospective analysis of the morphologies of metaphase II (MII) human oocytes, which had developed into 590 biopsied blastocysts derived from 109 patients that received preimplantation genetic testing for aneuploidies (PGT-A) cycles between March 2013 and December 2017. The euploid rate of blastocysts that originated from morphologically abnormal or normal oocytes were analyzed. The chromosome status of the blastocysts was determined and analyzed by array comparative genomic hybridization (aCGH) or next generation sequencing (NGS) following trophectoderm biopsy.According to the odds ratios obtained for each oocyte morphotype, no statistically significant relationship was found between oocyte dysmorphisms and euploid rate. Specifically, although SERc-positive oocytes had a higher rate of arrest at two pronuclei, or 2 PN (26.7% vs. 19.4%, p 0.05), the blastocyst formation rate was not affected as compared with SERc-negative oocytes (40.0% vs. 38.6%, p 0.05). Among nine euploid embryos derived from oocytes with SERc, three single euploid embryo transfers were performed, of which one resulted in blighted ovum, and two resulted in the births of two healthy, singleton term babies.The results presented here suggest that oocyte dysmorphisms do not affect the euploidy rate of the blastocyst. The occurrence of SERc in the oocyte does not seem to impair the developing blastocyst nor does it interfere with good embryo formation rate and euploid rate. Thus, the embryos derived from SERc-positive oocytes could still be considered for embryo transfer if there are no other embryos available.

Jaw1/LRMP is a type II integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane. We previously reported that a function of Jaw1 is to maintain the nuclear shape as a KASH protein via its carboxyl terminal region, a component of linker of nucleoskeleton and cytoskeleton complex in the oligomeric state. Although the oligomerization of some KASH proteins via the cytosolic regions serves to stabilize protein-protein interactions, the issue of how the oligomerization of Jaw1 is regulated is not completely understood. Therefore, we focused on three distinct regions on the cytosolic face of Jaw1: the N-terminal region, the coiled-coil domain and the stem region, in terms of oligomerization. A co-immunoprecipitation assay showed that its coiled-coil domain is a candidate for the oligomerization site. Furthermore, our data indicated that the N-terminal region prevents the aberrant oligomerization of Jaw1 as an intrinsically disordered region (IDR). Importantly, the ectopic expression of an N-terminal region deleted mutant caused the formation of organized smooth ER (OSER), structures such as nuclear karmellae and whorls, in B16F10 cells. Furthermore, this OSER interfered with the localization of the oligomer and interactors such as the type III inositol 1,4,5-triphosphate receptor (IP3R3) and SUN2. In summary, the N-terminal region of Jaw1 inhibits the formation of OSER as an IDR to maintain the homeostatic localization of interactors on the ER membrane.

STUDY QUESTION Is there any association between the appearance of smooth endoplasmic reticulum aggregates (SERa) in oocytes and ovarian stimulation, embryological, clinical and neonatal outcomes of ICSI and IVF cycles? SUMMARY ANSWER A suboptimal prolonged ovarian stimulation is detrimental to oocytes by inducing the occurrence of SERa, which reduces the reproductive potential of oocytes. WHAT IS KNOWN ALREADY Controlled ovarian stimulation recruits oocytes of different qualities. Based on current evidence, it was agreed that non-homogeneous cytoplasm may represent the normal variability among oocytes rather than a dysmorphism with developmental significance. The only exception is the appearance of SERa within the ooplasm. Owing to the lack of univocal evidence in this literature about the safety of injecting oocytes with SERa and the mechanism responsible for the occurrence of SERa, this topic is still a matter of debate. STUDY DESIGN, SIZE, DURATION A retrospective, longitudinal cohort study performed at a tertiary level public infertility center. We included 1662 cycles (180 SERa+ and 1482 SERa−) from 1129 women (age: 20–44 years) who underwent IVF/ICSI treatments in 2012–2019. The SERa+ cycles had at least one SERa+ oocyte in the oocyte cohort. The SERa− cycles had morphologically unaffected oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS We collected stimulation data and embryological, clinical, neonatal outcomes of SERa− and SERa+ cycles and oocytes. MAIN RESULTS AND THE ROLE OF CHANCE Overall, 347 out of 12 436 metaphase II oocytes (2.8%) were affected by SER. We performed only 12 transfers involving at least one SERa+ embryo. Stimulation length (P = 0.002), serum progesterone (P = 0.004) and follicle size (P = 0.046) at trigger, number of retrieved (P = 0.004) and metaphase II (P = 0.0001) oocytes were significantly higher in SERa+ than SERa− cycles. Fertilization rate was significantly (P LIMITATIONS, REASONS FOR CAUTION Given that SERa+ oocytes were discarded in our center for years and transfers of embryos originating from affected oocytes were generally avoided, clinical outcomes of SERa+ cycles are largely attributable to the transfer of embryos derived from unaffected oocytes of SERa+ cycles and we did not have data about newborns from affected oocytes, since none of the transfers involving SERa+ embryos resulted in a progressive clinical pregnancy. WIDER IMPLICATIONS OF THE FINDINGS For the first time, we speculate that the late-follicular phase elevated serum progesterone caused by a suboptimal prolonged ovarian stimulation may be detrimental to the oocytes by inducing the occurrence of SERa, resulting in negative effects on their reproductive potential. This raises the question of whether some stimulation regimens could be worse than others and a change in stimulation protocol would reduce the possibility of producing oocytes with suboptimal maturation. In particular, our data highlight the importance of correct timing of the trigger in order to maximize oocyte collection, not only in terms of numerosity but also their reproductive potential. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A.

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.

The presence of smooth endoplasmic reticulum aggregates (SERa) in the ooplasm is considered as the most severe oocyte dysmorphism due to its serious and potentially lethal outcomes in offspring. In the present case report, a couple underwent their first intracytoplasmic sperm injection (ICSI) cycle using a gonadotrophin releasing hormone (GnRH) antagonist protocol, followed by fetal ultrasound scanning and amniocentesis. SERa were observed in all oocytes retrieved. A singleton pregnancy was established. The second trimester fetal ultrasound scan revealed a female fetus with overlapping fingers in both hands, and amniocentesis was performed for the detection of chromosomal abnormalities. Comprehensive genetic analysis with the combined use of array-comparative genomic hybridization (CGH), fluoresence in situ hybridization (FISH) and conventional cytogenetics revealed a complex chromosome rearrangement (CCR) involving three break points on two chromosomes, resulting in a reciprocal translocation with a cryptic 2q31 deletion. A week following amniocentesis, there was rupture of amniotic membranes and a stillborn was delivered. This is the first case in the literature to report a CCR with concomitant 2q31 deletion resulting in a well-defined and clinically recognizable contiguous gene syndrome with an abnormal phenotype in a fetus arising from a cohort of oocytes affected by SERa. It is suggested that fertilization and transfer of oocytes with SERa should be avoided, until further research establishes whether there is a causal relationship between the presence of SERa and chromosomal abnormalities in the resulting fetus.SER: smooth endoplasmic reticulum; ICSI: intracytoplasmic sperm injection; GnRH: gonadotrophin releasing hormone; CGH: comparative genomic hybridization; FISH: fluoresence in situ hybridization; FSH: follicle stimulating hormone; hCG: human chorionic gonadotrophin; OHSS: ovarian hyperstimulation syndrome; IVF: in vitro fertilization; MII: metaphase II; GV: germinal vesicle; CCR: complex chromosome rearrangement.

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4 weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11 weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies.

We tested here the ability of bee venom (BV) to interfere with spermatogenesis in rats in two experimental conditions. The histopathological changes were assessed with brightfield microscopy using a novel staining technique, based on methylene blue, orange G and ponceau xylidine. Transmission electron microscopy was also used to identify fine subcellular changes. BV injection for 30days in daily doses of 700μg BV/kg resulted in reducing testicular weight, along with significant larger diameters of seminiferous tubules and reduced number of Sertoli cells (SCs). SCs were vacuolated, detached from the basement membrane, many necrosed, leading to the basement membrane denudation. Germ cells layers were separated by empty spaces conferring a rarefied aspect to the tissue, and spermatids were detached into lumen. Thus, the seminiferous epithelium was significantly thinned. Many Leydig cells (LCs) were in a necrotic state, with disrupted plasma membrane and without smooth endoplasmic reticulum. The acute treatment with a single LD50 of 62mgBV/kg, was followed by focal disruptions of the basement membrane and localized areas of necrosis, mainly affecting the SCs. Most of the observed SCs as well as some spermatogonia were highly vacuoled, empty spaces being observed within the epithelium. The SCs count was significantly decreased. Spermatids had also the tendency of separation from the SCs, and the significant larger diameter of the tubules found was associated with a thicker epithelium. Many LCs were necrosed, with disrupted plasma membrane, swollen mitochondria, no endoplasmic reticulum and implicitly showing rarefied cytoplasm. We concluded that BV was a testicular toxicant affecting both the LCs and the seminiferous tubules. The SCs cells represented the primary target site of BV whose effects were next extended upon the germ cells. In all cells, BV triggered unspecific degenerative changes that could impaire spermatogenesis. The present study also proposes an alternative staining technique very useful in assessing the histopathological aspects of spermatogenesis.

In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII.

Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.

Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates amongst PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregates (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.

Objective To study whether embryos derived from oocytes presenting a smooth endoplasmic reticulum cluster (SERC) are less likely to develop into blastocysts and implant. Design Transversal study. Setting Private university-affiliated in vitro fertilization (IVF) center. Patient(s) Total of 7,609 oocytes obtained from 743 intracytoplasmic sperm injection (ICSI) cycles. Intervention(s) Oocytes split between the SERC-positive cycles (with at least one SERC-positive oocyte) and the SERC-negative cycles (only oocytes free of SERC). Main Outcome Measure(s) Embryo implantation. Result(s) A statistically significantly higher mean number of follicles (24.0 ± 10.5 vs. 19.6 ± 10.5), retrieved oocytes (17.8 ± 8.3 vs. 14.3 ± 8.0), and mature oocytes (13.5 ± 6.2 vs. 10.6 ± 5.9) were observed in the SERC-positive cycles as compared with SERC-negative cycles. The implantation rate was statistically significantly lower in SERC-positive cycles as compared with SERC-negative cycles (14.8% vs. 25.6%; odds ratio 0.61; 95% confidence interval, 0.44–0.86). When only cycles with in which none (0) or all the blastocysts transferred had implanted (100%) were analyzed, the mean implantation rate per transferred blastocyst in the SERC-negative group was 20.5%; no blastocysts derived from SERC-positive oocytes implanted. Conclusion(s) The occurrence of SERC impairs embryo implantation. Careful oocyte observation that takes into account the presence of SERC should be part of embryo selection strategy before transfer.

An improved unroofing method consisting of tearing off the cell membrane using an adhesive electron microscopy (EM) grid instead of vitreous ice sectioning (cryo-sectioning) has enabled us to panoramically view the membrane cytoskeleton in its native state with extremely high contrast. Grids pre-treated with Alcian blue were placed on cells, and a portion of the dorsal plasma membrane was transferred onto the grid, which was then floated in buffer solution. These membrane fragments contained sufficient cytoskeleton and were of suitable thickness for observation by cryo-EM. Many actin filaments and microtubules were clearly observed on the cytoplasmic surface of the plasma membrane with extremely high contrast because the soluble components of the cytoplasm flowed out and broke away from the cells. Actin filaments extended in all directions in a smooth contour with little branching. Microtubules spread out as far as 3 µm or more while winding gently in their native state. Upon fixation with 1% glutaraldehyde, however, the microtubules became straight and fragmented. Cryo-EM revealed for the first time a smooth endoplasmic reticulum network beneath the cell membrane in native cells. Clathrin coats and caveolae were also observed on the cytoplasmic surface of the plasma membrane, similar to those seen using freeze-etching replica EM (freeze-etching EM). Unroofing was also useful for immuno-labelling in cryo-EM. Antibody-labelled IQGAP1, one of the effector proteins facilitating the formation of actin filament networks, was localized alongside actin filaments. Freeze-etching EM confirmed the morphological findings of cryo-EM.

Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. Samples were studied by light and transmission electron microscopy. We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism.

Study question Did the revised Alpha/ESHRE consensus (Vienna, 2017) bring a real answer on managing oocytes with aggregates of smooth endoplasmic reticulum (SERa)? Summary answer According to the currently available literature, a case by case approach on the time of injecting/inseminating SERa+ oocytes may be not helpful for embryologists making a decision, so we suggest fertilizing both SERa+ and SERa- oocytes and prioritizing embryos derived from SERa- oocytes. What is known already? In 2011, the Istanbul consensus recommended not to inject/inseminate SER+ oocytes due to adverse foetal outcomes reported in literature. At the end of 2017, a panel of experts reconsidered this recommendation and advised a case by case approach. Hence, with a lack of clear recommendations, in-vitro fertilization practitioners still have heterogeneous attitudes when managing SERa+ oocytes. In this context of controversy, an updated review could be helpful in (i) forming a common language for managing cases of SERa+ oocytes and (ii) offering the most ethical practice and best care for patients seeking infertility treatment or fertility preservation. Study design, size, duration This review (with a last literature search on 1 June 2018) evaluated the effect of the SER dysmorphism on embryological and neonatal outcomes. Participants/materials, setting, methods Studies were considered for inclusion if they were prospective or retrospective cohort or case-control studies. Electronic searches of the Pubmed and Embase databases were done using the keyword combination: smooth endoplasmic reticulum, SER, oocyte and zygote. Abstracts and articles written in English and limited to humans were included. Main results and the role of chance The search returned a total of 726 studies among which 21 met the inclusion criteria. The literature does not unanimously support a negative association between SERa and embryogenesis, implantation or assisted reproductive therapy outcomes. The reviewed studies reported 112 neonatal outcomes after transfers where at least one embryo originated from oocyte affected by SERa. They included 101 healthy babies, three live births with malformations, three neonatal deaths, one stillbirth and four medical interruptions of pregnancy. After transfer of embryos exclusively derived from SERa+ oocytes, a total of 48 healthy newborns were reported along with four babies with perinatal complications (including one ventricular septal defect), one stillbirth, one neonatal death and one pregnancy termination for multiple malformations. Limitations, reasons for caution As with any review, this review was limited by the quality of the included studies especially in terms of possible methodological limitations, the limited sample size and the retrospective aspect of the studies. Among the 21 selected studies, seven were abstracts and two were case reports. Of the remaining 14 studies, only three were prospective. The tools used in identifying SERa+ oocytes may have varied from one study to another and a consequent misclassification cannot be excluded. Considering the poor resolution of light microscopy in detecting SER aggregates, we are not sure that apparently SERa- oocytes do not really exhibit such a dysmorphism if they were analysed under electronic microscopy or a time lapse system. Wider implications of the findings In the light of the existing data and the lack of a real link between fertilizing SERa+ oocytes and the occurrence of embryo aneuploidy/malformations, we think that discarding SERa+ oocytes may be not the most ethical approach even in patients with large cohorts on the day of oocyte retrieval. Avoiding the wastage of oocytes and embryos with respect to medical ethics remains a constant concern in daily IVF practice. Thus, we recommend that all mature oocytes could be fertilized and embryos originating from SERa- oocytes would be preferably transferred, even if they come from a cohort with SERa+ oocytes. The remaining embryos derived from SERa+ oocytes could be considered with a lower priority for transfer after obtaining consent from the couple if a strict follow-up of the pregnancy and the baby is performed. Study funding/competing interest(s) We have no conflict of interest to declare and no funding was received. Registration number N/A.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe β-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500 nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms.

Nevirapine (NVP) therapy is associated with a high risk of serious liver injury and skin rash. Treatment of Brown Norway rats with NVP causes an immune-mediated skin rash. Even though NVP does not cause serious liver injury in wildtype animals, incubation of hepatocytes with NVP leads to the release of presumably danger-associated molecular pattern molecules (DAMPs), which activate macrophages. In this study, we examined the liver biopsies of Brown Norway rats treated with NVP to determine the histologic correlate to the release of DAMPs by hepatocytes. In vivo, debris from necrotic hepatocytes and endothelial cells were present in the liver sinusoids, a condition that can trigger an immune response. In addition to mitochondrial, hepatocytic, and endothelial damage, the drug induced large hepatocytic inclusions composed of lipid droplets surrounded by concentric whorls of smooth endoplasmic reticulum (SER) cisternae-lipid-SER (LSER) inclusions, which were deposited in the sinusoids. NVP is lipid soluble, and these LSER inclusions may be sinks of NVP or its metabolites. LSERs are deposited in the blood stream where they may be picked up by lymph nodes and contribute to initiation of an immune response leading to serious liver injury or skin rash. LSERs migration from liver to the blood stream may signify a novel mechanism of drug exocytosis.

The present study aimed to gather information on the impact of Alpha/European Society of Human Reproduction and Embryology (ESHRE) consensus regarding oocytes with aggregates of smooth endoplasmic reticulum (SERa) on in vitro fertilization outcome. In particular, we investigated if patients undergoing intracytoplasmic sperm injection (ICSI) and whose oocytes are discarded due to SERa have a higher chance of embryo transfer cancellation compared to patients without SERa oocytes. This is a nested case–control study drawn from the cohort of women referring for in vitro fertilization with ICSI. Cases were patients showing at least one oocyte with SERa at the time of injection. Controls were subsequent patients showing no SERa oocytes and matched ratio 1:1 for age, clinical indication to in vitro fertilization (IVF), and body mass index. The main outcome was the rate of embryo transfer cancellation. The percentage of women experiencing a transfer cancellation (absence of suitable oocytes or viable embryos) in their ICSI cycle were significantly higher in cases (18 %) compared to controls (8 %) (p = 0.02); however, adjusted odds ratio for FSH and number of SERa oocytes, of follicles, of retrieved oocytes, and of inseminated oocytes were not statistically significant. We have shown that the exclusion of SERa oocytes from ICSI cycles causes an increased frequency of transfer cancellation. This effect is mostly due to the reduced number of available oocytes after exclusion of SERa oocytes.

The number of elderly patients undergoing gastrectomy for gastric cancer is increasing. Yet, although elderly patients are at high risk of postoperative pneumonia, no study has sufficiently investigated which clinicopathological factors are significant risk factors for the development of this complication after gastrectomy with lymph node dissection. We reviewed the medical records of 750 patients who underwent gastrectomy between January 2010 and May 2012, to establish the incidence of postoperative pneumonia (Clavien–Dindo grade II or higher). Univariate and multivariate analyses were performed to identify the risk factors for postoperative pneumonia. Thirty-two patients (4.3 %) suffered postoperative pneumonia, diagnosed as grades I, II, IIIa, and IVa, in 2 (0.3 %), 28 (3.7 %), 1 (0.2 %), and 1 (0.2 %) patient(s), respectively. Univariate analysis revealed that age (≥75 years), sex (male), diabetes mellitus (DM), a history of smoking, and impairment of respiratory function were significantly associated with postoperative pneumonia. Multivariate analysis revealed that age, impaired postoperative respiratory function, DM, and blood transfusion were independent risk factors for postoperative pneumonia. Age, impaired postoperative respiratory function, DM, and blood transfusion were identified as independent risk factors for postoperative pneumonia after gastrectomy.

The current study is essentially carried out to reveal the histological and ultra-structural details of the retinal pigmented epithelium (RPE) and photoreceptors cell layers of a common buzzard (Buteo buteo). The recorded results revealed that the neural retina of common buzzard consisted of seven distinct cell layers. The inner nuclear layer was markedly revealed as the thickest one among these layers. A highly melanized RPE was recorded in between the choroid and neural retina. Histologically, the RPE was represented by a single layer of cuboidal epithelial cells with centrally located nucleus. Ultrastructurally, the RPE cells showed numerous melanosomes, mitochondria, phagosomes, myeloid bodies, smooth endoplasmic reticulum (SER), but very rare rough endoplasmic reticulum (RER). The photoreceptor cell layer was represented by three categories of photoreceptor cells: few single rods, numerous single and double cones. Each double cone consisted of a short accessory cone and a long principle cone. The photoreceptor outer segment consisted of bi-membranous discs that are enclosed by outer membrane. Moreover, the inner segment of rods consisted of an ellipsoid and an inner hyperboloid. The hyperboloid was rich with RER, polysomes, Golgi apparatus and autophagic vacuoles. Furthermore, the inner segment of single cone and accessory cone consisted of an ellipsoid, paraboloid and myoid regions, while, the inner segment of principle cone lacked the paraboloid regions. At the proximal end of each inner segment for all types of cones, there was a large heterogeneous oil droplet. The paraboloid region was markedly rich with glycogen granules. The myoid region exhibited the same organelles but with little glycogen granules when compared with hyperboloid.

Does the presence of aggregates of smooth endoplasmic reticulum (SERa) impact the transcriptome of human metaphase II (MII) oocytes?.The presence of SERa alters the molecular status of human metaphase II oocytes.Oocytes presenting SERa are considered dysmorphic. Oocytes with SERa (SERa+) have been associated with reduced embryological outcome and increased risk of congenital anomalies, although some authors have reported that SERa+ oocytes can lead to healthy newborns. The question of whether or not SERa+ oocytes should be discarded is still open for debate, and no experimental information about the effect of the presence of SERa on the oocyte molecular status is available.This study included 28 women, aged38 years, without any ovarian pathology, and undergoing IVF treatment. Supernumerary MII oocytes with no sign of morphological alterations as well as SERa+ oocytes were donated after written informed consent. A total of 31 oocytes without SERa (SERa-) and 24 SERa+ oocytes were analyzed.Pools of 8-10 oocytes for both group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on oligonucleotide microarrays. Analyses were performed by R software using the limma package.The expression profiles of SERa+ oocytes significantly differed from those of SERa- oocytes in 488 probe sets corresponding to 102 down-regulated and 283 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics disclosed that genes involved in three main biological processes were significantly down-regulated in SERa+ oocytes respective to SERa- oocytes: (i) cell and mitotic/meiotic nuclear division, spindle assembly, chromosome partition and G2/M transition of mitotic cell cycle; (ii) organization of cytoskeleton and microtubules; and (iii) mitochondrial structure and activity. Among the transcripts up-regulated in SERa+ oocytes, the most significantly (P = 0.002) enriched GO term was 'GoLoco motif', including the RAP1GAP, GPSM3 and GPSM1 genes.Raw microarray data are accessible through GEO Series accession number GSE106222 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106222).Data validation in a larger cohort of samples would be beneficial, although we applied stringent criteria for gene selection (fold-change3 or1/3 and FDR0.1). Surveys on clinical outcomes, malformation rates and follow-up of babies born after transfer of embryos from SERa+ oocytes are necessary.We provide information on the molecular status of SERa+ oocytes, highlighting possible associations between presence of SERa, altered oocyte physiology and reduced developmental competence. Our study may offer further information that can assist embryologists to make decisions on whether, and with what possible implications, SERa+ oocytes should be used. We believe that the presence of SERa should be still a 'red flag' in IVF practices and that the decision to inseminate SERa+ oocytes should be discussed on a case-by-case basis.This study was partially supported by Ferring Pharmaceuticals. The authors have no conflicts of interest to declare.

STUDY QUESTION Is it time to reconsider whether oocytes affected by smooth endoplasmic reticulum aggregates (SERa) should still be destroyed? SUMMARY ANSWER At the time of writing, the literature shows that 171 apparently healthy babies have been born from SERa+ cycles amongst which 22 were from SERa+ oocytes. WHAT IS KNOWN ALREADY The SER dysmorphism has been associated with negative embryological, clinical and neonatal outcomes, which led to a recommendation in 2011 to avoid inseminating affected oocytes. The data in the literature are nevertheless conflicting and some centres have continued using SERa+ oocytes. STUDY DESIGN, SIZE, DURATION A systematic mini-review of the literature to 7 November 2013 was performed with the keywords 'Smooth Endoplasmic Reticulum' and 'oocyte', limited to humans and written in English. PARTICIPANTS/MATERIALS, SETTING, METHODS Articles (Pubmed) and major abstracts where the effect of the SER dysmorphism was studied as an individual feature on embryological, clinical or neonatal outcomes were included in this review. MAIN RESULTS AND THE ROLE OF CHANCE From a total of 297 publications identified, 13 were selected as being relevant to this review. One hundred eighty-three babies have been reported to be born from SERa+ cycles, 171 were healthy, 8 live births presented malformations, 3 were neonatal deaths, 1 was a stillborn and additionally 4 terminations of pregnancy occurred. LIMITATIONS, REASONS FOR CAUTION Data concerning SERa+ oocytes in the literature are scarce, the studies are small, heterogeneous and results are conflicting. The malformations observed could be due to over-reporting of scattered alarming results. Alternatively, an under-reporting of complications cannot be excluded. WIDER IMPLICATIONS OF THE FINDINGS Centres that have or that are including transfers of SERa+ embryos in their IVF procedures should publish their clinical and neonatal outcomes as well as the follow-up of children. The birth of healthy babies from SERa+ embryos is encouraging and might lead in the future to a revision of the current consensus on the SER dysmorphism. Further research is needed to understand the origin of this dysmorphism and help avoid its occurrence. Therefore, until we have a better understanding of the situation, transfers of affected embryos should be carried out with caution. STUDY FUNDING/COMPETING INTEREST(S) We have no conflicts of interest to declare and no funding was received. © 2014 The Author 2014.

Fast calcium transients (

Papillary renal cell carcinoma (PRCC) includes two different morphological subtypes. The differences of genetics and ultrastructure of the two subtypes have been rarely reported. Also, new biomarkers related to the diagnosis and prognosis of PRCC have still not been well elucidated. Immunohistochemistry, fluorescence in situ hybridization (FISH) and transmission electron microscopy were used systematically to determine the characteristics of 56 cases of PRCC and to reveal new diagnostic and prognostic information. Type 1 PRCC presented higher expression rates of EMA and CK7, whereas type 2 presented a higher expression rate of CD10. New immunohistochemical markers, including: p504s, PAX-2, PAX-8 and CA-IX showed extensive immunostaining in PRCC. We first revealed a distinct immunostaining pattern of CA-IX, which was located in multiple foci in PRCC. All tumors had at least one chromosomal aberration including loss of Y, gains of 7 or 17. Gain of chromosome 17 was common in type 1; losses of chromosome 18, 11 and 8 appeared in type 2. Ultrastructurally, glycogen granules and secondary lysosomes were seen in type 1, mitochondria and smooth endoplasmic reticulum were scattered in type 2. Tumor subtype, nuclear grade, TNM stage, clear cell renal cell carcinoma (CCRCC) component and sarcomatoid elements, metastasis, CAIX expression, losses of chromosome 18 and 8 were related to poor outcome of PRCC. We conclude that the two subtypes of PRCC originate from different renal cells, and arise from partially common genetic pathways. EMA, CK7, CD10, p504s, PAX-2, PAX-8 and CA-IX are helpful markers in the differential diagnosis of PRCC. CA-IX expression, losses of chromosome 18 and 8 are new prognostic factors of PRCC.

When 100 mg/kg/day of di( n-butyl) phthalate (DBP) was intragastrically administered to pregnant Sprague-Dawley rats throughout gestation days 12 to 21, the male pups had similar body weights with no apparent physical differences (e.g., litter size, sex ratio) compared to that of the vehicle group. However, prominent age-related morphological alterations in the smooth endoplasmic reticulum (sER) of testicular Leydig cells (LCs) were observed once these animals reached puberty. At weeks 5 to 7, the abundant sER with non-dilated cisternae was distributed in LCs. Subsequently, although the number of LCs significantly increased, the amount of sER was significantly decreased at 9 to 14 weeks of age and had disappeared at 17 weeks. In contrast, the number of LCs and the amount of sER in LCs of the lower dose groups (10, 30, and 50 mg/kg/day) were similar to those of the vehicle group. Further, serum testosterone levels in the 100 mg/kg dose group were significantly lower during 5 to 17 weeks of age. While their luteinizing hormone (LH) level was significantly lower at 5 to 7 weeks of age, it became significantly higher during 9 to 17 weeks. The amount of sER in LCs decreased with age with the increase in LCs proliferation and serum LH levels in rat exposed in utero to DBP in a dose-dependent manner.

Pigment aggregation in shrimp chromatophores is triggered by red pigment concentrating hormone (RPCH), a neurosecretory peptide whose plasma membrane receptor may be a G-protein coupled receptor (GPCR). While RPCH binding activates the Ca

Oocytes containing smooth endoplasmic reticulum aggregates (SERa) have been associated with reduced fertilization and clinical pregnancy rates as well as compromised neonatal outcomes. It was therefore recommended by an Alpha-ESHRE Consensus to discard oocytes presenting this dysmorphism. The data in the literature are nevertheless conflicting and healthy babies have recently been obtained from affected oocytes. The objectives of this study were to compare clinical outcomes between ICSI cycles with and without oocytes affected by smooth endoplasmic reticulum aggregates and to confirm whether affected oocytes can produce healthy babies. A prospective observational study was performed comparing 714 SERa− ICSI cycles to 112 SERa+ cycles. Among the SERa+ cycles, 518 SERa− oocytes and 213 SERa+ oocytes were analyzed. Fertilization, embryo quality, and pregnancy rates as well as neonatal outcomes were compared between SERa+ and SERa− cycles as well as between SERa+ and SERa− oocytes. The presence of SERa was not associated with an adverse effect on embryological, clinical or neonatal data for SERa+ cycles and oocytes. Seven healthy babies were born from embryos originating from SERa+ oocytes. These results are encouraging and might contribute in the future to a revision of the Alpha-ESHRE Consensus. Larger studies, including a correlation between frequency and size of SERa, clinical outcomes and malformation rates, as well as the follow-up of babies born are nevertheless necessary. In the meantime, the currently conflicting data requires caution when considering transfers of embryos affected by SERa.

Thyroid hormones (TH) are one of the key factors for normal prenatal development in mammals. Previously, we showed that subclinical maternal hypothyroidism leads to premature atresia of ovarian follicles in female rat offspring in the pre-pubertal and pubertal periods. The influence of decreased concentration of TH on primordial follicles pool formation during neonatal and early infantile period of rat pups was not investigated previously. Maternal hypothyroidism during pregnancy has irreversible negative influence on primordial follicles pool formation and population of resting oocytes in female rat offspring. The study was done on neonatal and early infantile control (n-10) and hypothyroid (n-10) female rat pups derived from control (n-6) and propylthiouracil (PTU) treated pregnant dams (n-6), respectively. Ovaries of all pups were removed and processed for light and transmission electron microscopy (TEM). Number of nests, oogonia and oocytes per nest, primordial, primary, secondary and preantral follicles were determined. Screening for overall calcium presence in ovarian tissue was done using Alizarin red staining. Morphology and volume density of nucleus, mitochondria and smooth endoplasmic reticulum (sER) in the oocytes in primordial follicles was also assessed. Caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), both markers for apoptosis, and proliferating cell nuclear antigen (PCNA) for proliferation were determined in oocytes and granulosa cells in different type of follicles. In neonatal period, ovaries of hypothyroid pups had a decreased number of oogonia, oocytes and nests, an increased number of primordial follicles and a decreased number of primary and secondary follicles, while in early infantile period, increased number of primary, secondary and preantral follicles were found. Alizarin red staining was intense in hypothyroid neonatal rats that also had the highest content of dilated sER. Number of mitochondria with altered morphology in both groups of hypothyroid pups was increased. Apoptosis markers have not shown significant difference between groups but PCNA had an increased expression in the oocytes and granulosa cells in primordial follicles of hypothyroid rats. Light and electron microscopy analysis indicate that previously detected premature ovarian follicular atresia in pre-pubertal and pubertal hypothyroid rats is preceded with premature formation of primordial follicles followed by slight changes on sER and mitochondria in examined oocytes, and increased expression of PCNA. This is the peer-reviewed version of the article: Danilović Luković, J.; Korać, A.; Milošević, I.; Lužajić, T.; Puškaš, N.; Kovačević Filipović, M.; Radovanović, A. Altered State of Primordial Follicles in Neonatal and Early Infantile Rats Due to Maternal Hypothyroidism: Light and Electron Microscopy Approach. Micron 2016, 90, 33–42. [https://doi.org/10.1016/j.micron.2016.08.007]