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Gosalvez J.,Autonomous University of Madrid | Ruiz-Jorro M.,Crea - Tec | Martinez-Moya M.,Centro Gutenberg | Sanchez-Martin P.,GINEMED | Caballero P.,Clinica Tambre
Revista Internacional de Andrologia | Year: 2013

Objective: The study was made to analyze the baseline levels of damage recorded in sperm DNA fragmentation (SDF) and to estimate sperm DNA longevity as observed in donors after thawing. Material and methods: Fifty donors and forty individuals attending a clinic and classified as a normo-zoospermic population were compared. The baseline SDF levels and the increasing rate of SDF (r-SDF) obtained after thawing when the sperm was incubated for a period of 24. h with different sub-sampling performed after 2, 6 and 24. h of incubation were considered as the independent variables and compared. Results: Cryopreserved donor sperm exhibited baseline SDF values approximately 2 times lower than those observed in the control group. DNA stability was 2.5 times higher than that observed in the control cohort. Baseline values of SDF of approximately 8% generates 65% sensitivity and 82% specificity to discriminate between the donors and controls. Values of increase of damage of 1.8% per hour, analyzed during the first hours of incubation, identify the donor characteristics with 77% sensibility and 65% specificity. Neither value show any correlation within the control and donor cohorts group. Conclusion: The establishment of these types of threshold values can be used to identify donors considered as "super-donors" in relation to their low levels of SDF and high chromatin stability. The donors selected from the different clinics participating in this study showed similar characteristics for these parameters. © 2013 Asociación Española de Andrología, Medicina Sexual y Reproductiva.


Nunez-Calonge R.,Clinica Tambre | Cortes S.,Clinica Tambre | Gutierrez Gonzalez L.M.,Complutense University of Madrid | Kireev R.,Complutense University of Madrid | And 6 more authors.
Reproductive BioMedicine Online | Year: 2016

The aim of this study was to determine the concentrations of oxidative stress markers, antioxidant enzymes and cytokines in the follicular fluid of young women with low response in ovarian stimulation cycles compared with high responders and fertile oocyte donors of the same age, to assess the impact of oxidative stress on ovarian reserve. The activity of follicular fluid antioxidant enzymes glutathione transferase, glutathione reductase and glutathione peroxidase was significantly lower in young women with reduced ovarian reserve compared with that in high responders and oocyte donors. Follicular fluid concentrations of oxidative stress marker malondialdehyde combined with 4-hydroxyalkenals and nitric oxide were higher in low responders than in high responders and oocyte donors. Significant differences between low responders and donors in concentrations of IL-2, IL-6, IL-8 and vascular endothelial growth factor were observed, with higher concentrations in low responders. However, IL-10 concentration was lower in low responders than in high responders and donors. No significant differences were found in follicular fluid concentrations of tumour necrosis factor alpha between the three groups. These results demonstrate that different concentrations of oxidative stress markers, oxidant enzymes and cytokines in low responders compared with high responders and oocyte donors may negatively impact ovarian response. © 2015 Reproductive Healthcare Ltd.


Nunez-Calonge R.,Clinica Tambre | Caballero P.,Clinica Tambre | Lopez-Fernandez C.,Clinica Tambre | Lopez-Fernandez C.,Autonomous University of Madrid | And 5 more authors.
Reproductive Sciences | Year: 2012

Using donor oocytes of proven fertility, the effect of sperm DNA fragmentation (SDF) and motility on reproductive success was examined in 70 couples undergoing ICSI. Both SDF and sperm motility were assessed at the time of sperm injection and using the same sperm sample that was processed for ICSI. While there was no difference in the fertilization rate, cleavage rate, embryo quality, or sperm motility between pregnant and nonpregnant couples, the SDF of nonpregnant couples (SDF = 23.9%) was higher than that of pregnant couples (SDF = 17.0%; U Mann-Whitney 347; P =.002). Using a combination of the sensitivity and specificity measures from the production of ROC (receiver-operating characteristic) curves and the Youden index, we determined a threshold SDF value for our data set of 17% for predicting pregnancy (77.8% sensitivity and 71.1% specificity). Our results suggest that proven donor oocytes in combination with SDF assessment at the time of sperm injection represent a useful experimental model for reducing the confounding influences of sperm DNA repair by the oocyte and iatrogenic sperm damage. © The Author(s) 2012.


Ramos-Medina R.,Hospital General Universitario Gregorio Maranon | Garcia-Segovia A.,Clinica Tambre | Leon J.A.,Hospital General Universitario Gregorio Maranon | Alonso B.,Hospital General Universitario Gregorio Maranon | And 14 more authors.
American Journal of Reproductive Immunology | Year: 2013

Problem: Natural killer (NK) cells play a key role in embryo implantation and pregnancy success, whereas blood and uterine NK expansions have been involved in the pathophysiology of reproductive failure (RF). Our main goal was to design in a large observational study a tree-model decision for interpretation of risk factors for RF. Methods of study: A hierarchical multivariate decision model based on a classification and regression tree was developed. NK and NKT-like cell subsets were analyzed by flow cytometry. Results: By multivariate analysis, blood NK cells expansion was an independent risk factor for RF (both recurrent miscarriages and implantation failures). We propose a new decision-tree model for the risk interpretation of women with RF based on a combination of main risk factors. Conclusions: Women with age above 35 years and >13% CD56+CD16+ NK cells showed the highest risk of further pregnancy loss (100%). © 2013 John Wiley & Sons Ltd.


Gosalvez J.,Autonomous University of Madrid | Caballero P.,Clinica Tambre | Lopez-Fernandez C.,Autonomous University of Madrid | Ortega L.,Clinica Tambre | And 4 more authors.
Asian Journal of Andrology | Year: 2013

This study compared the potential of assessing sperm DNA fragmentation (SDF) from neat semen and the subsequent swim-up (SU) procedure to predict pregnancy when conducting ICSI of fertile donor oocytes. Infertile females (n=81) were transferred embryos resulting from intracytoplasmic sperm injection (ICSI) of their partner's spermatozoa and proven donor oocytes. This model normalized the impact of female factor in putative sperm DNA repair. Semen was blindly assessed for SDF using Halosperm immediately following ejaculation (NS) and after swim-up at the time of ICSI fertilisation. There was a decrease in SDF values of the ejaculated semen sample following the swim-up protocol (P=0.000). Interestingly, pregnancy could be equally predicted from SDF values derived from either neat or swim-up semen samples. Receiver operator curves and the derived Youden's indices determined SDF cutoff values for NS and SU of 24.8% and 17.5%, respectively. Prediction of pregnancy from NS SDF had a sensitivity of 75% and a specificity of 69%, whereas for SU SDF was 78% and 73%, respectively. While increased levels of SDF negatively impact reproductive outcome, we have shown that a reduction in SDF following sperm selection using ICSI with proven donor oocytes is not mandatory for achieving pregnancy. This suggests that a certain level of DNA damage that is not detectable using current technologies could be impacting on the relative success of assisted reproductive technology (ART) procedures. Consequently, we propose a modification of the so called 'iceberg model' as a possible rationale for understanding the role of SDF in reproductive outcome. © 2013 AJA, SIMM & SJTU. All rights reserved.


Gosalvez J.,Autonomous University of Madrid | Nunez R.,Clinica Tambre | Fernandez J.L.,Complejo Hospitalario Universitario runa | Fernandez J.L.,Centro Oncologico Of Galicia | And 2 more authors.
Andrologia | Year: 2011

The rate of increase of sperm DNA fragmentation (rsDF) in fresh and frozen-thawed and processed sperm samples after a density gradient for sperm selection was analysed after 0, 0.5, 1.5, 4.5, 6, 24, 48 and 72h of incubation at 37°C, in five donors with proven fertility. The results showed that: (i) sperm DNA fragmentation (sDF) at baseline in fresh samples (14.3±3.3) was lower than that obtained after freeze-thawing and selection (19.4±4.1), significant differences; (ii) After 6h of incubation the mean sDF in fresh samples (24.2±10.2) was significantly lower than that in frozen-thawed samples (45.3±7.1); (iii) Subsequently, the rsDF in fresh semen samples was 1.6% per h after 6h of incubation, while after thawing and selection the rsDF was 4.3% per h; The tendency to increase in sDF showed high R2 values (R2=0.90) for exponential functions in case of fresh samples, whereas R2 values for linear functions were higher after sperm selection (R2=0.97). These results indicate that differences in sperm DNA fragmentation dynamics before and after storage are an important issue that must be considered for storage of sperm to be used for artificial reproduction techniques. © 2011 Blackwell Verlag GmbH.


Moraru M.,Hospital General Universitario Gregorio Maranon | Moraru M.,Hospital Univesitario Puerta Of Hierro Majadahonda | Carbone J.,Hospital General Universitario Gregorio Maranon | Alecsandru D.,Hospital General Universitario Gregorio Maranon | And 20 more authors.
American Journal of Reproductive Immunology | Year: 2012

Problem: Natural killer (NK, CD3 - CD56 +/CD16 +) and NKT-like cells (CD3 + CD56 +/CD16 +) activity is considered among the key factors for reproductive success. In the absence of immunological screening, beneficial effects of intravenous immunoglobulin (IVIG) in preventing recurrent reproductive failure (RRF) have not been reported. Here, we analyse the IVIG influence on pregnancy success in women with RRF and circulating NK or/and NKT-like cells expansion. Method of study: One hundred fifty-seven women with previous recurrent miscarriage and/or recurrent implantation failure after in vitro fertilization were consecutively studied. Sixty-four patients with CD56 + cell expansion, no apparent underlying disease and who maintained their desire to conceive were selected. Forty of them received IVIG during pregnancy. Results: Overall, the clinical pregnancy rate for the women under IVIG therapy was 92.5% and the live birth rate was 82.5%. Significantly lower pregnancy and live birth rates (25% and 12.5%, respectively) were observed for the patients with recurrent pregnancy loss and NK/NKT-like cells expansion without IVIG. After three cycles of IVIG, NK cell percentages decreased significantly and these values persisted throughout gestation. Conclusion: Intravenous immunoglobulin therapy for women with RRF and NK or NKT-like cell expansion was a safe and beneficial therapeutic strategy that associated with high clinical pregnancy and live birth rates. © 2012 John Wiley & Sons A/S.


PubMed | Complutense University of Madrid and Clinica Tambre
Type: Journal Article | Journal: Reproductive biomedicine online | Year: 2016

The aim of this study was to determine the concentrations of oxidative stress markers, antioxidant enzymes and cytokines in the follicular fluid of young women with low response in ovarian stimulation cycles compared with high responders and fertile oocyte donors of the same age, to assess the impact of oxidative stress on ovarian reserve. The activity of follicular fluid antioxidant enzymes glutathione transferase, glutathione reductase and glutathione peroxidase was significantly lower in young women with reduced ovarian reserve compared with that in high responders and oocyte donors. Follicular fluid concentrations of oxidative stress marker malondialdehyde combined with 4-hydroxyalkenals and nitric oxide were higher in low responders than in high responders and oocyte donors. Significant differences between low responders and donors in concentrations of IL-2, IL-6, IL-8 and vascular endothelial growth factor were observed, with higher concentrations in low responders. However, IL-10 concentration was lower in low responders than in high responders and donors. No significant differences were found in follicular fluid concentrations of tumour necrosis factor alpha between the three groups. These results demonstrate that different concentrations of oxidative stress markers, oxidant enzymes and cytokines in low responders compared with high responders and oocyte donors may negatively impact ovarian response.


Ramos-Medina R.,Hospital General Universitario Gregorio Maranon | Garcia-Segovia A.,Clinica Tambre | Gil J.,Hospital General Universitario Gregorio Maranon | Carbone J.,Hospital General Universitario Gregorio Maranon | And 21 more authors.
American Journal of Reproductive Immunology | Year: 2014

Problem: Recurrent reproductive failure (RRF) has been associated with expansion of circulating NK cells, key cells for maternal tolerance, decidual vasculogenesis and embryo growth. This study reports our experience in intravenous immunoglobulin (IVIg) therapy of a large cohort of women with RRF with expanded circulating NK and/or NKT-like cells (blood NKT cells are a heterogeneous subset of T cells that share properties of both T cells and NK cells). Method of study: Observational study of RRF women with NK or NKT-like expansion (>12% or 10% cutofflevels of total lymphocytes, respectively), treated with IVIg for the next gestation. Results: By multivariant logistic regression analysis after adjusting for age, NK cells subsets and other therapies, IVIg significantly improved the live birth rate to 96.3% in women with recurrent miscarriage (RM) compared with 30.6% in case not receiving IVIg (P < 0.0001). In women with recurrent implantation failure (RIF), in comparison with women not receiving IVIg, treatment increased the pregnancy rate from 26.2 to 93.8% (P ≤ 0.0001) and the live birth rate from 17.9 to 80.0% in RIF (P ≤ 0.0001). Conclusions: Immunomodulation with IVIg in our selected group of RRF patients with immunologic alterations enhanced clinical pregnancy and live birth rates. Our results may facilitate the design of future clinical trials of IVIg in this pathology. © 2014 John Wiley & Sons Ltd715 May 2014 10.1111/aji.12217 Original Article Clinical Aspects of Reproductive Immunology. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.


PubMed | Clinica Tambre
Type: Journal Article | Journal: Reproductive sciences (Thousand Oaks, Calif.) | Year: 2012

Using donor oocytes of proven fertility, the effect of sperm DNA fragmentation (SDF) and motility on reproductive success was examined in 70 couples undergoing ICSI. Both SDF and sperm motility were assessed at the time of sperm injection and using the same sperm sample that was processed for ICSI. While there was no difference in the fertilization rate, cleavage rate, embryo quality, or sperm motility between pregnant and nonpregnant couples, the SDF of nonpregnant couples (SDF = 23.9%) was higher than that of pregnant couples (SDF = 17.0%; U Mann-Whitney 347; P = .002). Using a combination of the sensitivity and specificity measures from the production of ROC (receiver-operating characteristic) curves and the Youden index, we determined a threshold SDF value for our data set of 17% for predicting pregnancy (77.8% sensitivity and 71.1% specificity). Our results suggest that proven donor oocytes in combination with SDF assessment at the time of sperm injection represent a useful experimental model for reducing the confounding influences of sperm DNA repair by the oocyte and iatrogenic sperm damage.

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