84 research outputs found
Potential role of p53 protein as a novelbiomarker of sperm quality, able to predict thesuccess of ART techniques. EcoFoodFertility Project
Introduction: Protein p53 is well known as “The guardian of genome”; it changes its concentration in human spermatozoa DNAin relation to the damage of the latter. It has been suggested thatthe role of the p53 ancestral gene was to ensure the integrity ofthe genomic germline and the fidelity of the evelopment process.The aim of this study is to evaluate if different concentrations of p53 protein in human spermatozoa could influence embryo quality and pregnancy rate and possibly representing a potential predictive marker of sperm quality for successful fertilization .Methods: From July 2013 to June 2017 we have examinatedretrospectively 79 couples with 2-5 years of infertility history.Males had an average age of 27 ± 7,5 years, sperm concentrationof 33,8 ± 6,2 mil/ml, progressive motility of 41,4 ± 8,3 and a typical morphology of 16,5 ± 3,5 according to Kruger’s method. We have divided the couples on the basis of p53 levels: Group A:0,35–1,65 ng/mil (21 males); Group B: 1,66–3,57 ng/mil (32 males);Group C: 3,58–14,53 ng/mil (26 males). We have evaluated thenumber of embryos at stage of 6–8 cells, btained at the third dayof embryo development, in these three different group. In order toevaluate the concentration of p53 protein, we first proceeded toa DNA extraction with forensic method and then to a quantification p53 protein with ELISA-immunoenzymatic assay, expressedin ng/million of spermatozoa.Results: We have observed different percentage of embryo development at stage of 6-8 cells in the third day and different pregnancy rate (PR):Group A: 101 embryos at 6-8 cells/ 147 total number of obtained embryos in this group (68,4%) and PR = 52,38%.Group B: 128/240 (53,5%); PR = 37,50%; Group C: 79/216 (36,1%);PR = 7,69%. These results support the hypothesis that an high con-centration of p53 in human sperm DNA is associated to a low percentage of embryos able to reach the stage of 6-8 cells in the third day of development and also to a lower pregnancy rate. So p53 levels can be considered as a predictive value to embryo development and pregnancy rate.Conclusions: Protein p53 is a sequence-specific transcriptionfactor that responds to a wide variety of stress signals (environ-mental insults and bad lifestyle) as we are investigating within theecofoodfertility project. Particularly quantitative research of p53could be considered as a novel biomarker of sperm quality, able topredict the success of ART echniques, and could open a new roadfor infertility diagnosis
New molecular markers for the evaluation of gamete quality
Purpose: Only 30 % of IVF cycles result in a pregnancy, so that multiple embryos need to be replaced, per treatment cycle, to increase pregnancy rates, resulting in a multiple gestation rate of 25 %. The use of new markers in the gamete selection, could reduce the number of the oocytes to be fertilized and embryos to be produced, but the tools to evidence the gamete competence remain unavailable and more studies are needed to identify bio-markers to select the best oocyte and sperm to produce embryos with higher implantation potentiality. Methods: To define oocyte competence, the apoptosis of the surrounding cumulus cells and the oxygen consumption rates for individual oocytes before fertilization seems to provide a non-invasive marker of oocyte competence and hence a quantitative assessment of the reproductive potential for the oocyte. The chromatin integrity seems to be used also as biological marker of sperm competence, together with the morphological evaluation of large vacuoles in the head. Results: The apoptosis rate of cumulus cells lower than 25 % and an higher oxygen consumption could be an evidence of an overall metabolic activity, related to a better fertilization ability and embryo cleavage quality. The apoptosis rate of the sperm chromatin, evaluated by direct Tunel in situ analysis, seems to be, also for the male gamete, a marker of competence and implantation potentiality, in particular when it is lower than 20 %. The evaluation of the presence of large vacuoles in the sperm head prior to perform ICSI seems to increase the implantation rate, but it is not associated to chromatin integrity. Conclusions: The biological concept of competence appears unrelated to any morphological parameters, so that it is necessary to investigate new molecular markers in the gamete selection. Apoptosis of cumulus cells in the oocytes and spermatozoa, revealing the presence of large vacuoles, could help to determine the competence of the gamete to be fertilize. © 2013 Springer Science+Business Media New York
SELECTION OF THE BEST OOCYTES FOR INTRACYTOPLASMICSPERM INJECTION (ICSI) USING APOPTOTIC ANALYSIS OF CUMULUS CELLS
Introduction: 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
The histone deacetylase inhibitor JAHA down-regulates pERK and global DNA methylation in MDA-MB231 breast cancer cells
The histone deacetylase inhibitor N1-(ferrocenyl)-N8-hydroxyoctanediamide (JAHA) down-regulates extracellular-signal-regulated kinase (ERK) and its activated form in triple-negative MDA-MB231 breast cancer cells after 18 h and up to 30 h of treatment, and to a lesser extent AKT and phospho-AKT after 30 h and up to 48 h of treatment. Also, DNA methyltransferase 1 (DNMT1), 3b and, to a lesser extent, 3a, downstream ERK targets, were down-regulated already at 18 h with an increase up to 48 h of exposure. Methylation-sensitive restriction arbitrarily-primed (MeSAP) polymerase chain reaction (PCR) analysis confirmed the ability of JAHA to induce genome-wide DNA hypomethylation at 48 h of exposure. Collective data suggest that JAHA, by down-regulating phospho-ERK, impairs DNMT1 and 3b expression and ultimately DNA methylation extent, which may be related to its cytotoxic effect on this cancer cytotype
Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism
Abstract
PURPOSE:
An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia.
METHODS:
In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period.
RESULTS:
After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p 15 %), while no significant variation occurred in the patients with DFI values ≤15 %.
CONCLUSIONS:
Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 %
Lower apoptosis rate in human cumulus cells after administration of recombinant luteinizing hormone to women undergoing ovarian stimulation for in vitro fertilization procedures.
Objective
To investigate the effects of recombinant (r-) LH supplementation in “low responder” patients undergoing ovarian stimulation with r-FSH for an IVF program. The apoptosis rate in cumulus cells was used as an indicator of oocyte quality.
Design
Comparison of the rate of DNA fragmentation and caspase-3 activity in cumulus cells in women stimulated with r-LH and r-FSH, versus patients treated with r-FSH alone (control).
Setting
In vitro fertilization (IVF) laboratory.
Patient(s)
Forty patients undergoing assisted fertilization programs treated with a GnRH agonist, or r-FSH treatment begun on day 3 of the cycle (control). In the r-LH group, from day 8 of gonadotropin stimulation, 150 IU per day of r-LH were administered.
Intervention(s)
Terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine-triphosphate (dUTP) nick-end labeling (TUNEL) assay, and anti-caspase-3 cleaved immunoassay, to detect apoptosis in human cumulus cells.
Main Outcome Measure(s)
Difference in DNA fragmentation rate between cumulus cells derived from r-LH treatment and cumulus cells derived from control patients.
Result(s)
No differences were observed between the two groups in the total amount of r-FSH administered and in the number of retrieved oocytes per patient. A statistically significant increase in the number of immature oocytes and in the E2 serum peak was observed in the control group. The number of transferred embryos was significantly higher in the r-LH group. Pregnancy and implantation rates were higher in the r-LH group, but without statistical significance. The apoptosis rate in cumulus cells was higher in the control group than in the r-LH group.
Conclusion(s)
This study suggests that supplementation with r-LH improves the chromatin quality of cumulus cells involved in the control of oocyte maturation
Apoptosis in human unfertilized oocytes after Intracytoplasmic Sperm Injection
Objective
To investigate the presence of programmed cell death in unfertilized oocytes after intracytoplasmic sperm injection (ICSI), assuming that previous apoptotic events could be correlated with the fertilization failure.
Design
Comparison of the rate of DNA fragmentation in human oocytes at different stages of maturation soon after pick-up (control) and in unfertilized oocytes after ICSI treatment.
Setting
In vitro fertilization (IVF) laboratory with extensive ICSI experience.
Patient(s)
Sixty-three patients undergoing assisted fertilization by ICSI.
Intervention(s)
Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay and anticaspase-3 cleaved immunoassay to detect apoptosis in control and ICSI-treated oocytes.
Main Outcome Measure(s)
Differences in the percentage of oocytes demonstrating DNA fragmentation between control oocytes and unfertilized ICSI treated oocytes at different stages of maturation.
Result(s)
The DNA fragmentation, by TUNEL assay, appeared in all the immature control oocytes, but only 37% of mature oocytes showed DNA fragmentation. This DNA fragmentation was observed in 88.8% of the oocytes unfertilized after ICSI; furthermore, DNA fragmentation appeared as well in the sperm injected into the cytoplasm.
Conclusion(s)
The study has shown DNA fragmentation in human oocytes unfertilized after ICSI. The evidence is confirmed as well in control oocytes, free from in vitro culture or manipulation stress. Caspase-3 immunoassay suggests the presence of apoptosis. The high percentage of oocytes demonstrating DNA fragmentation in the unfertilized oocytes could be correlated with fertilization failure
FSH administration reduces significantly sperm apoptosis only in the case of high DFI value: a study in idiopathic dispermic patients
Introduction: In the last decades sperm DNA quality has been recognized as one of the most
important markers of male reproductive potential (Lewis and Aitken, 2005; Ozmen, 2007; Tarozzi,
2007), in contrast to standard semen parameters as sperm density, motility and morphology, which
do not act as powerful discriminators between fertile and infertile men. DNA damage in the male
germ line is a major contributor to infertility, miscarriage and birth defects in the offspring. In
animal models, it has been unequivocally demonstrated that the genetic integrity of the male germ
line plays a major role in determining the normality of embryonic development. In humans, many
studies showed that sperm DNA damage is associated with impaired embryo cleavage (8), higher
miscarriage rates (9) and also with a significantly increased risk of pregnancy loss after in vitro
fertilization (IVF) and intracytoplasmic sperm injection (ICSI) (10). Specifically, above a threshold
of 30% of sperms with fragmented DNA, chances for pregnancy are close to zero, either by means
of natural conception or intrauterine insemination (Spano M, 2000; Bungum M, 2007). Since there
is a clear relationship between sperm DNA damage and poor assisted reproduction technology
(ART) outcomes, efforts should be directed in developing treatments to improve sperm DNA
quality to be introduced into clinical use. The aim of this observational study was to investigate the
effects of r-FSH administration on sperm DNA fragmentation of iOAT patients undergoing ICSI,
comparing the DNA fragmentation index (DFI) before and after 90 days of FSH therapy.
Matherial and Methods: Fifty-three iOAT men, with a median age of 33,6 ± 7,6 years, referred to
our clinics because of fertility problems after at least two years of natural attempts, were selected
for the study. In all patients DNA fragmentation was evaluated sperm prior to treatment with 150
IU of recombinant human FSH (GONAL-f®, Merck Serono) three times at week for at least three
months. Patients were re-evaluated after a 3-month period with semen analysis and DNA
fragmentation. Sperm DNA fragmentation index (DFI) was investigated by terminal
deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end
labelling (TUNEL) assay. Data were analysed using the paired t-test and chi-square as appropriate.
A p-value <0.05 was considered statistically significant.
Results: After 3 months of r-FSH treatment, no significant differences was observed between
baseline and post therapy semen sample parameters including sperm count, motility, and the
percentage of normal sperm forms. IThe percentage of sperm DNA fragmentation in the total of
patients dropped from 20.8 ± 9.1 to 15.1 ± 8.9 (P < 0.05) (see Tab I). Interestingly, no statistical
difference was found in sperm DFI when patients showed a baseline DFI ≤15% (10.5 ± 4.2 vs
11.4 ± 4.5). We found an evident and statistically significant DFI reduction in patients with sperm
baseline DFI value ≥15% (24.37 ± 9.6 vs 15.4 ± 4.6).
Conclusion: Our data seems to demonstrate that FSH acts as a strong anti-apoptotic agent in
reducing DNA fragmentation in iOAT patients. The therapy may be a specific pretreatment for
infertile male partners of couples undergoing ICSI, specifically in the case that basal DFI is higher
than 15%, reducing the percentage of spermatozoa with DNA integrity anomalies suggesting a
positive effect on the reproductive outcome
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