1,721,061 research outputs found
Apoptosis in human sperm as a quality marker for a new diagnostic approach of infertile patients
Full text linkThe quality of the sperm DNA is one of the most important molecular markers of male reproductive potential (Bosco, L et al., 2018, Environ Toxicol Pharmacol 58:243-249). The aim of this study was to evaluate sperm DNA fragmentation according to morphology, to predict the probability of selecting a sperm with normal morphology and intact DNA. An observational study was performed on 70 patients with oligoasthenoteratozoospermia. Sperm DNA Fragmentation Index (DFI) and semen parameters such as sperm density, motility and morphology were evaluated in all patients. DFI was calculated using the in situ TUNEL assay. DFI can be determined and thus used as a marker of sperm quality for a diagnostic approach of infertile patients. For each patient, out of the total of sperm DFI, we calculated the proportion between sperm with normal morphology and with abnormal morphology. Among 35 patients (A Group), the DFI was inferior than 15% (average value was 8.1%), while 35 patients (B Group) had a DFI higher than 15% (average value was 24.6%). When the analysis was restricted only to spermatozoa with normal morphology, it was observed that among patients of B Group the DFI value was 13.6%, while in A Group the average was 2.2%. DFI calculated on sperm of normal morphology can provide an important information on the risk of injecting, during ICSI procedure, a sperm with normal morphology but with DNA fragmentation. This risk is higher if the semen sample has a baseline DFI higher than 15%
Magnesium deprivation affects development and biomineralization in the sea urchin arba-cia lixula
No full textSkeletogenesis is a key morphogenetic event in the life of marine
invertebrates. Marine calcifiers secrete their calcareous skeletons
taking up ions from seawater.
Marine biominerals include aragonite and calcite, the latter of
which in some taxa (e.g. echinoderms, coralline algae) can have
a substantial magnesium (Mg) component.
Echinoderms have an extensive endoskeleton composed of high
magnesian calcite and occluded matrix proteins1. As biomineralization
in sea urchin larvae is sensitive to the
Magnesium:Calcium ratio of sea water, we investigated the
effects of magnesium deprivation on development and skeletogenesis
in the Mediterranean sea urchin Arbacia lixula.
Microscopic inspection revealed that embryos reared in Mg-free
seawater exhibited developmental delay from 6 hours post-fertilization,
complete lack of skeleton formation at 24 hours, and
severe skeleton malformations in larvae (48-72 hours). We subsequently
focused on the localization of the skeletogenic cells
(primary mesenchyme cells) and the spatial expression of associated
genes. Immunocytochemistry revealed abnormal ectopic
location of the primary mesenchyme cells (PMCs) and of the
developing skeleton of treated embryos. Expression of msp130,
an important skeleton matrix protein gene expressed only in
PMCs, detected by in situ hybridization, was normal at 24 hours,
but this gene was not down-regulated at 48 hours, as in controls2.
Strikingly, development of the pigment cells, immune cells
that, like the skeleton, are mesodermal derivatives, was also
impaired. These results suggest the essential role of Mg in skeleton
formation in sea urchin embryos with an indication that this
element is also generally important for development of mesoderm.
1. Smith AM et al. Mar Ecol Prog Ser 2016, 561:1–16
2. Martino C et al. Aq tox 2018, 194:57-66
Correlation among pAKT, pERK1/2 and DNA fragmentation index in human cumulus cells to determine oocyte competence
No full textThe aim was to correlate specific biological aspects of cumulus
cells isolated from individual cumulus-oocyte complexes (COCs) with the
clinical outcome of the related embryos
The effect of the HDACi JAHA on DNA methylation of breast cancer cells by down-regulating DNMT1 through ERK signaling
Full text linkEVALUATION OF OOCYTE QUALITY IN GRANULOSA AND CUMULUS CELLS OF PATIENTS UNDERGOING PMA
No full textBackground. We investigate the apoptosis rate of individual granulosa cell–oocyte and cumulus cell–oocyte (COC), associated with the levels of molecules playing a critical role in the regulation of cell death or survival. These molecular analyses have been done to verify the difference of competence between oocytes producing embryos able to reach the blastocyst stage compared with embryos arrested during the in vitro culture.
Methods. From each single follicle: granulosa cells were processed for Western blotting analyses, using the following antibodies: pAKT, ERK 1/2, pERK 1/2; cumulus cells were used for in situ immunofluorescence with the same antibodies. DNA fragmentation rate was measured by TUNEL assay.
Results. We have involved 58 patients and recovered 255 MII oocytes, of which 197 were fertilized and the derived embryos had the following evolution: 117 transferred, 57 vitrified and 23 arrested; 58 oocytes failed the fertilization or were in GV or MI stages. In the cumulus cells: we found a significant inverse correlation between oocytes resulting in transferred and arrested embryos in the ratio pAKT/TUNEL; nuclear localization of pERK1/2 showed a significant inverse correlation pERK1/2/TUNEL and a significant direct correlation with the intracellular accumulation of pERK1/2/pAKT. In granulosa cells: oocytes able to produce blastocysts, ERK1/2 /TUNEL ratio was higher than in cells of arrested embryos.
Conclusions. Cumulus and granulosa cells showed different levels of expression of the investigated molecules. We found that in the cumulus cells of the oocytes able to produce blastocysts, the pAKT/TUNEL ratio is higher than in cumulus cells of arrested embryos, indicating that pAKT is involved in survival pathways. Moreover, pERK1/2 has an anti-apoptotic effect, when translocated into the nucleus. In granulosa cells: ERK1/2 indicates that it is involved in survival pathways. Briefly, we demonstrated that DNA fragmentation rate related to specific molecular levels could be considered a molecular marker of oocyte competence, for the evaluation of a prognostic pattern of blastocyst formation
Sperm DNA integrity and human papillomavirus (HPV) infections: a controversy that could be resolved by a new molecular approach
No full textStudy question: The aim was to determine if HPVs affect spermatozoa DNA
integrity. To resolve the discrepancy regarding the association between HPV
infections and sperm DNA damage.
Summary answer: To elucidate if HPV impairs DNA integrity, we suggest to
investigate HPV DNA positivity in spermatozoa by a differential lysis procedure
before TUNEL assay
SELECTION OF THE BEST OOCYTES FOR INTRACYTOPLASMICSPERM INJECTION (ICSI) USING APOPTOTIC ANALYSIS OF CUMULUS CELLS
Full text linkIntroduction: We studied the apoptosis rate of the cumulus cells of individual cumulus-oocyte
complex (COC), to verify a relationship with clinical outcomes, in terms of pregnancy and
implantation rates. Usually oocytes are selected using morphological criteria. We tried to verify
if cumulus cell apoptotic rate could be used as molecular criteria in selecting oocytes with higher
implantation potentiality (1;2).
Materials and Methods: The study design consisted in two different trials: in the first, we
investigated apoptosis rate in cumulus cells of the three selected oocytes, to be fertilized by
intracytoplasmic sperm injection (ICSI); in a second trial, average apoptosis rate of the cumulus
cells coming from the three selected oocytes to be fertilized by ICSI and the pooled remaining
oocytes were compared, when more than 5 COCs were aspirated. In a first trial we included 22
consecutive couples undergoing ICSI cycles, 20 in a second one, for a total of 42 patients. We
selected the three oocytes for (ICSI) on the basis of the morphological appearance of the
cumulus, according to Veek’s criteria. The cumulus cells of each COC were submitted to
apoptotic assays (3). The patients were classified, on the basis of pregnancy success, in A Group
(pregnant patients) and B Group (patients with negative βhCG).
Results: Both trials showed that apoptosis in the cumulus cells was remarkably lower in the A
Group if compared with B Group. The apoptosis rate in the selected COCs was similar to pooled
COCs for each patient, confirming that apoptosis rate in cumulus cells is characteristic for
patient. Out of 22 patients involved in the first trial, 8 were pregnant (36.3% A Group) and 14
were not pregnant (B Group). In the second trial 4 of a total of 20 patients were pregnant (20%).
In the first trial a total of 58 metaphase II oocytes and 56 in the second trial were studied. In the
second trial 38 oocytes where pooled to compare apoptosis rate with the three selected oocytes
pools. In the first trial the incidence of DNA fragmentation, evaluated by TUNEL assay (fig. 1),
of the cumulus cells from individual treated oocytes, was lower in A Group than in B Group
(6.7% ranging between 2.2–13.3 vs 13.19% ranging between 6.2–34.9 respectively, p<0.05). To
confirm if DNA fragmentation was related to apoptosis process, we performed caspase-3
immunoassay in the same cells (fig. 2). Data showed a lower capase-3 activity in cumulus cells
of pregnant than in those of non-pregnant patients (5.2% ranging between 1.2–8.6 vs 11.8%
ranging between 5.6–14.8, p<0.05). It is noteworthy to underline that pregnant patients usually
exhibited, at least, one COC with a DNA fragmentation rate (TUNEL) less than 10% and
caspase-3 activity rate less than 7%. Four (A Group) of 20 patients involved in the second trial
were pregnant but two aborted at 8–9 weeks. The low number of pregnant patients did not allow
us to have a powerful statistical analysis of apoptotic rate in cumulus cells, but it seems evident
that a higher apoptotic rate in cumulus cells is associated to the pregnancy failure (B Group) and
in aborted patients of A Group, ranging from 10 to 60.3%.
Conclusion: The data seem to demonstrate that apoptosis may be a marker for the selection of
the best oocytes to be submitted to ICSI treatment. All pregnant patients showed a lower
apoptosis rate in cumulus cells if compared with patients with pregnancy failure.
86° CONGRESSO NAZIONALE SIBS - PALERMO 24-25 OTTOBRE 2013
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References
1. Ruvolo G, Bosco L, Pane A, Morici G, Cittadini E, Roccheri MC. Lower apoptosis rate in
human cumulus cells after administration of recombinant luteinizing hormone to women
undergoing ovarian stimulation for in vitro fertilization procedures. Fertil Steril. 2007 Mar;
87(3):542-6. Epub 2006 Nov 27.
2. Host E, Gabrielsen A, Lindenberg S, Smidt-Jensen S 2002 Apoptosis in human cumulus cells
in relation to zona pellucida thickness variation, maturation stage, and cleavage of the
corresponding oocyte after intracytoplasmic sperm injection. Fertility and Sterility 77, 511-515.
3. Bosco L, Ruvolo G, Morici G, Manno M, Cittadini E, Roccheri MC. Apoptosis in human
unfertilized oocytes after intracytoplasmic sperm injection. Fertil Steril. 2005 Nov; 84(5):1417-
23.
FIGURE 1. Apoptosis evaluation using TUNEL assay in human cumulus cells. (A1, A2, A3) A
group; (B1, B2, B3) B group; (C1, C2, C3) positive control for TUNEL assay. (A1, B1, C1)
fragmented DNA; (A2, B2, C3) propidium iodide staining; (A3, B3, C3) merge. Scale bar = 15
μm.
FIGURE 2. Apoptosis evaluation using Cleaved caspase 3 immunofluorescence in situ assay in
human cumulus cells. (A1, A2, A3) A group; (B1, B2, B3) B group; (A1, B1) Cleaved caspase 3;
(A2, B2) propidium iodide staining; (A3, B3) merge. Scale bar = 15 μm
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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