Papers by Alberto Pacheco
Fertility and Sterility, 2021
Journal of Andrology, 2006
ABSTRACT: The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile... more ABSTRACT: The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile patients with varicocele and compared with those obtained in a group of 51 normozoospermic patients, 103 patients with abnormal standard semen parameters, and 22 fertile men. The spermatozoa were processed to discriminate different levels of DNA fragmentation using the Halosperm® kit, an improved Sperm Chromatin Dispersion (SCD) test. In this technique, after an acid incubation and subsequent lysis, those sperm cells without DNA fragmentation show big or medium‐sized halos of dispersion of DNA loops from the central nuclear core. Otherwise, those spermatozoa containing fragmented DNA either show a small halo, exhibit no halo with solid staining of the core, or show no halo and irregular or faint stain of the remaining core. The latter, that is, degraded type, corresponds to a much higher level of DNA‐nuclear damage. The varicocele patients showed 32.4% ± 22.3% of spermatozoa with fragme...
Fertility and Sterility, 2012
Objective: To study the levels of four markers of oxidative stress in follicular fluid (FF) and p... more Objective: To study the levels of four markers of oxidative stress in follicular fluid (FF) and plasma of patients with infertility related to endometriosis and controls. Design: Experimental study. Setting: University-affiliated hospital and infertility center. Patient(s): Ninety-one infertile women were included in the study (23 infertile women with endometriosis and 68 controls including infertile women due to tubal factor, male factor, or healthy egg donors). Intervention(s): Blood was obtained at the time of egg retrieval, and FF from the mature follicles of each ovary was centrifuged and frozen until analysis. Main Outcome Measure(s): Vitamin C and E, malondialdehyde, and superoxide dismutase concentrations in plasma and follicular fluid. Result(s): Women with endometriosis showed a lower vitamin C concentration in FF (12.7 AE 5.9 vs. 9.7 AE 6.9 mg/mL) and lower superoxide dismutase concentration in plasma (0.9 AE 1.4 vs. 0.5 AE 0.7 U/mL) compared with controls. Vitamin E plasma levels were significantly higher in women with endometriosis (8.1 AE 3.8 vs. 5.2 AE 3.2 mg/mL). A nonsignificant trend toward a lower plasma concentration of malondialdehyde was found in women with endometriosis. Conclusion(s): These findings suggest a lower antioxidant capacity in infertile women with endometriosis. Although a certain level of reactive oxygen species is required under physiological conditions, an altered balance between pro-oxidant and antioxidant activities may have an impact on folliculogenesis and adequate embryo development. (Fertil Steril Ò 2012;98:126-30. Ó2012 by American Society for Reproductive Medicine.
Medicina Reproductiva y Embriología Clínica, 2020
Kartagener syndrome is an autosomal recessive genetic disorder associated with male infertility. ... more Kartagener syndrome is an autosomal recessive genetic disorder associated with male infertility. Mutations in genes that encode a protein called dynein affect sperm motility. A good option for these patients is to enhance the sperm selection for ICSI that could lead to improve the reproductive outcomes. For this purpose, different strategies have been used successfully in isolated cases. In the present case report, we successfully applied MACS technique to a semen sample. This immunomagnetic method of sperm selection was aimed at reducing apoptosis manifestations, including DNA fragmentation, to optimize the outcomes of the ICSI procedure. By doing so, we increased the chances of selecting spermatozoa with superior quality and higher fertilization potential in the presence of total asthenozoospermia, even after incubation with pentoxifylline. Six of ten oocytes (60%) were appropriately fertilized. Two good-quality embryos were transferred on day 3 resulting in a pregnancy and a heal...
Frontiers in Endocrinology
ContextThe widespread distribution of the Vitamin D (VitD) receptor in reproductive tissues sugge... more ContextThe widespread distribution of the Vitamin D (VitD) receptor in reproductive tissues suggests an important role for VitD in human reproduction. The assessment of patient´s VitD is based on the 25-hydroxyvitamin D (25(OH)D) metabolite measurement. However, most of the circulating 25(OH)D is bound to either VitD-binding protein (VDBP) (88%) or albumin (12%) and less than 1% circulates free.ObjectiveTo determine a possible correlation between VitD levels in serum (S) and follicular fluid (FF) and blastocyst ploidy status in patients undergoing infertility treatment.MethodsA prospective observational study was performed including couples planned for preimplantation genetic testing for aneuploidies (PGT-A) from ART Fertility Clinics. Patients were classified according to their 25(OH)D-Serum levels: VitD deficient group <20 ng/ml and insufficient/replete ≥20 ng/ml defined as VitD non-deficient group.ResultsSerum samples and 226 FF from individual follicles were collected for 25(...
Fertility and Sterility, 2019
to assess the relationship between predicted age and true age. RESULTS: Methylation data was anal... more to assess the relationship between predicted age and true age. RESULTS: Methylation data was analyzed for a total of 175 women undergoing IVF (mean age 35.26 AE 4.14 years). The Horvath-predicted age calculation for WBC samples was consistent with the true chronologic age of patients (p<0.0001). However, the predicted age from CC was significantly younger than patients' chronologic age. The mean predicted age of patients based on methylation-based calculations from CC was 8.56 AE 2.07 years. Poor response to ovarian stimulation during IVF, defined as five or fewer oocytes obtained during oocyte retrieval, did not affect the Horvath-predicted age based on calculations from WBC (p¼0.131) or CC (p¼0.502). CONCLUSIONS: In women undergoing IVF, the epigenetic algorithm described previously by Horvath accurately predicts age when applied to WBC but not to CC. The methylation-based predicted age obtained from analysis of CC is substantially younger than the true age of patients, suggesting that CC exhibit unique methylation patterns that are distinct from those demonstrated by WBC. A poor response to ovarian stimulation is not associated with predictable changes in CpG methylation sites consistent with aging within WBC or CC. CC may have their own distinct methylation pattern which changes with age and must be clearly delineated since this may have implications for reproductive lifespan. Further studies are also required to determine whether alternative CpG sites can accurately predict chronologic age or response to stimulation from CC samples. Reference: 1. Horvath S. DNA methylation age of human tissues and cell types. Genome Biol.
Journal of Endometriosis, 2009
Introduction Anti-müllerian hormone (AMH) has been recently introduced as a new marker of ovarian... more Introduction Anti-müllerian hormone (AMH) has been recently introduced as a new marker of ovarian reserve with a high sensitivity and with the added value of being independent of cycle day. Some authors have claimed that it may also provide information regarding oocyte quality. As endometriosis is a disease that mainly affects the ovary and thus, egg quality, we decided to evaluate AMH follicular fluid concentrations in three clinical groups: a) women with endometriosis and a visible endometrioma at ultrasound in one of the ovaries, b) same women with endometrioma-free contralateral ovary, and c) women without endometriosis. Material and Methods From August to December 2007, 28 women undergoing an IVF/ICSI cycle in whom an endometrioma > 2cm was clearly visible at ultrasound in one of the ovaries and not in the other, and 28 fertile egg donors as controls entered the study. Follicular fluid from the largest follicle of each ovary (with endometrioma and without endometrioma) was i...
Reproductive biomedicine online, Jan 19, 2017
Recently, growing interest in vitamin D has emerged from findings that demonstrate a low vitamin ... more Recently, growing interest in vitamin D has emerged from findings that demonstrate a low vitamin D status in populations. Similarly, much interest has been shown in the role that anti-Müllerian hormone (AMH) plays in reproductive physiology. Considerable confusion as to whether vitamin D status is related to ovarian function can be found in the literature. Our retrospective study was performed from June 2014 to April 2015. Oocyte donors were recruited and stimulated under the antagonist protocol with gonadotrophin-releasing hormone (GnRH) agonist to trigger ovulation. In 851 stimulation cycles, we determined the association among serum total and bioavailable vitamin D levels, ovarian reserve and response to ovarian stimulation and the reproductive outcome in their recipients. We showed that vitamin D levels were unrelated to ovarian reserve or ovarian response after ovarian stimulation; in oocyte recipients, gestational outcome did not differ according to a donor's vitamin D ser...
Molecular Immunology, 1998
Journal of Immunological Methods, 1996
The characterization of T cell immunodeficiencies could in part be supported by using stable cell... more The characterization of T cell immunodeficiencies could in part be supported by using stable cell lines in which biochemical and molecular studies of the defect could be carried out thereby omitting frequent bleeding of patients. First attempts to obtain such cell lines included HTLV-I transformation and exogenous IL-2 administration, but both models have important disadvantages. Recently, a virus isolated from the squirrel monkey, Herpes virus saimiri (HVS). has been reported to have the ability to transform T cells. A stable IL-2-dependent HVS-transformed T cell line from a CD3y deficient patient has been obtained; and this cell line displays both the phenotypic and the functional characteristics of the patient's lymphocytes. Moreover, the line down-modulates TCR/CD3 surface expression upon CD3 engagement, as do the patient's lymphocytes, showing that CD3y and its phosphorylation are not necessary for TCR/CD3 internalization. In addition, the abnormal staining pattern of different anti-TCR/CD3 monoclonal antibodies is preserved in the HVS-patient line. Since HVS is capable of transforming CD3y-T cells, the CD3y chain does not seem to be involved in the HVS receptor process. The fact that it is not possible to obtain a CD8+ HVS line from the CD3y-patient supports the existence of a functional anomaly in his scanty CD8+ peripheral lymphocytes. Thus, HVS transformation is a suitable model for T cell immunodeficiency studies and characterization. It may also be used in the future in cellular models for in vitro gene therapy trials.
Clinical and Experimental Immunology, 2008
SUMMARY A 2-year-old female with important signs of immune response failure against virus, bacter... more SUMMARY A 2-year-old female with important signs of immune response failure against virus, bacteria, fungi and protozoa and no obvious humoral or lymphocyte phenotypical defect was studied. Both peripheral blood mononuclear cells and IL-2-dependent T cell lines derived from the patient showed a severe selective T cell activation impairment via CD2, CD3 and CD43; however, this defect was reversible with the addition of either IL-2, or phorbol myristate acetate (PMA) or anti-CD28 antibodies, Concordantly, the induction of IL-2 (and, in part, IL-3 and IL-4) messenger RNA Vk-as severely reduced in stimulated T cells, but that of other cytokines was either normal (IL-5) or only slightly diminished (interferon-gamma (IFN–γ)). It is concluded that an activation T cell defect exists previous to protein kinase C (PKC) and between membrane receptors and the activation pathway of certain response genes encoding for interleukins involved in proliferation (i.e. lL-2, IL-3 and IL-4), but not of o...
Clinical & Experimental Immunology, 2008
SUMMARY Cytomegalovirus(CMV) infection is reported to cause transient immunosuppression in man. I... more SUMMARY Cytomegalovirus(CMV) infection is reported to cause transient immunosuppression in man. In this Study we have analysed ihe effect of CMV on T lymphocyte function in 29 children diagnosed for acute CMV infection. Peripheral blood mononuclear cells (PBMC) obtained from the palients showed a significant specific impairment in their proliferative response to enterotoxins A and C1. to coneanavalin A and to the anti-CD3 MoAb OKT3. The impaired responses were corrected with exogenous IL-2. In conlrast. slimulation using phytohaemagglutinin. as well as activation signals delivered through the surface molecules CD26 or CD28. elicited normal proliferalive responses in CMV PBMC. The results indicate that the T cell anergy associated with CMV infection is restricted to the T cell receptor/CD3 activation pathway.
Translational andrology and urology, 2017
Human assisted reproduction results have improved dramatically in the last 20 years, with lower c... more Human assisted reproduction results have improved dramatically in the last 20 years, with lower complications for the patient as well as for the newborns. This requires a precise clinical diagnosis that will allow us to individualize the best-personalized treatment for each patient or couple. But still today, the basic diagnostic test for the male is a conventional sperm analysis, even though it has been extensively discussed the difficulties that this simple test reveals to discriminate between fertile and infertile individuals. One of the male diagnostic tests that has been developed a few years ago was the analysis of the sperm DNA integrity. Due to the controversy generated by the published evidence by different research groups, with diverse subgroup of patients being analyzed, and often with different methodologies, still it has not been implemented as a routine test in most ART labs across the world, and its use has not been supported by scientific societies yet. Sperm DNA integrity defects are related with spermatogenesis alterations, varicocele, inflammatory processes and with an increase in post-testicular oxidative stress. Thus, it represents a functional diagnostic test that may complement conventional sperm analysis. From the clinical point of view, increased sperm DNA fragmentation has been correlated with suboptimal embryo development, implantation failure and recurrent miscarriage. A relevant step in the validation as a diagnostic test is to standardize the different methodologies existing nowadays that would allow comparing clinical results among groups and publications. It is required to select among the techniques one as "gold standard" for each different type of DNA damage to be evaluated. This process of standardization would allow a better discrimination of the different processes that may induce an increased sperm DNA fragmentation in the patient, and thus, individualize treatment protocols for the different types of DNA damage. It may help clinicians to select which patients may benefit from oral antioxidants therapy, select candidates for varicocelectomy, or those who may benefit from micro-TESE in severe oligozoospermia with post-testicular oxidative stress. One step further would imply to use this technology to identify sperms with unfragmented DNA and directly being selected for ICSI. New technologies as Raman spectroscopy are being developed, although still experimental. We are witnessing the transformation of the sperm DNA integrity study as "a promising diagnostic test" to a valid clinical indication. Still, standardization of the "gold standard" technique to investigate sperm DNA fragmentation is urgently needed, a test that could improve identification of the functional sperm defects, which may help to establish individualized therapeutic protocols, and at the end, to improve reproductive outcome.
Cells
Clinical outcome in assisted reproduction techniques (ARTs) is mainly influenced by the quality o... more Clinical outcome in assisted reproduction techniques (ARTs) is mainly influenced by the quality of gametes used. It is known that a high percentage of sperm DNA fragmentation (DNAf) decreases the success of ART clinical results. Therefore, techniques such as magnetic-activated cell sorting (MACS) help to improve results in cases of patients with a high percentage of DNAf. Cryopreservation of sperm in donor intrauterine insemination (D-IUI) treatments increases sperm DNAf, so patients using these sperm samples can benefit from using this technique. This prospective randomized national multicenter study analyzed clinical outcomes of 181 D-IUI treatments. MACS was performed after density gradient centrifugation (DGC) in 90 thawed semen donor samples (MACSG), whereas only DGC was performed in 91 thawed semen donor samples (CG). To our knowledge, this is the first study analyzing the effect of MACS on D-IUI cycles. Our results show no significant differences in gestation, live birth, or ...
Fertility and Sterility, 2020
Fertility and Sterility, 2021
Biology, 2021
The application of MACS non-apoptotic sperm selection in infertility clinics is controversial sin... more The application of MACS non-apoptotic sperm selection in infertility clinics is controversial since the published literature does not agree on its effect on reproductive outcomes. Therefore, it is not part of the routine clinical practice. Classical measures of reproductive success (pregnancy or live birth rates per ovarian stimulation) introduce a bias in the evaluation of a technique’s effect, since only the best embryo is transferred. This retrospective, multicenter, observational study evaluated the impact of MACS on reproductive outcomes, measuring results in classical parameters and cumulative live birth rates (CLBR). Data from ICSI cycles using autologous oocyte in Spanish IVIRMA fertility clinics from January 2008 to February 2020 were divided into two groups according to their semen processing: standard practice (reference: 46,807 patients) versus an added MACS sperm selection (1779 patients). Only when measured as CLBR per embryo transferred and per MII oocyte used was the...
Journal of Clinical Medicine, 2020
Magnetic-activated cell sorting (MACS) can be used to separate apoptotic sperm with high proporti... more Magnetic-activated cell sorting (MACS) can be used to separate apoptotic sperm with high proportions of fragmented DNA from the rest, thus improving the overall quality of the seminal sample. Therefore, the aim of this retrospective study was to investigate the efficiency of the MACS technique to increase reproductive outcomes in patients with high levels of sperm DNA fragmentation (SDF) undergoing intracytoplasmic sperm-injection (ICSI) cycles. In this study, we analyzed a total of 724 assisted-reproduction-technique (ART) cycles that were divided into two groups: the study group (n = 366) in which the MACS selection technique was performed after density-gradient centrifugation (DGC), and the control group (n = 358) in which only DGC was used for sperm selection. Reproductive outcomes were analyzed in both groups according to three different ART procedures: preimplantation genetic testing for aneuploidy (PGT-A), and autologous and oocyte-donation cycles. The MACS group showed signi...
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Papers by Alberto Pacheco