Combined Preimplantation Genetic Testing for Autosomal Dominant Polycystic Kidney Disease: Consequences for Embryos Available for Transfer.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and presents with genetic and clinical heterogeneity. ADPKD can also manifest extra-renally, and seminal cysts have been associated with male infertility in some cases. ADPKD-linked male infertility, along with female age, have been proposed as factors that may influence the clinical outcomes of preimplantation genetic testing (PGT) for monogenic disorders (PGT-M). Large PGT for aneuploidy assessment (PGT-A) studies link embryo aneuploidy to increasing female age; other studies suggest that embryo aneuploidy is also linked to severe male-factor infertility. We aimed to assess the number of aneuploid embryos and the number of cycles with transferable embryos in ADPKD patients after combined-PGT. The combined-PGT protocol, involving PGT-M by PCR and PGT-A by next-generation sequencing, was performed in single trophectoderm biopsies from 289 embryos in 83 PGT cycles. Transferable embryos were obtained in 69.9% of cycles. The number of aneuploid embryos and cycles with transferable embryos did not differ when the male or female had the ADPKD mutation. However, a significantly higher proportion of aneuploid embryos was found in the advanced maternal age (AMA) group, but not in the male factor (MF) group, when compared to non-AMA and non-MF groups, respectively. Additionally, no significant differences in the percentage of cycles with transferable embryos were found in any of the groups. Our results indicate that AMA couples among ADPKD patients have an increased risk of aneuploid embryos, but ADPKD-linked male infertility does not promote an increased aneuploidy rate.

Molecular analysis of products of conception obtained by hysteroembryoscopy from infertile couples.

PURPOSE: To analyze the molecular cytogenetic data obtained from products of conception (POC) obtained by selective biopsy of first trimester miscarriages and to estimate the rate of chromosomal anomalies in miscarriages from pregnancies achieved by natural conception (NC) or by assisted reproductive technology (ART) interventions. METHODS: We used KaryoLite™ BoBs™ (PerkinElmer LAS, Wallac, Turku, Finland) technology to analyze 189 samples from ART or NC pregnancies. RESULTS: All POC were successfully evaluated. A higher incidence of chromosomal abnormalities was observed in POC after ART using the patient's own oocytes than from NC pregnancies (62.7% vs. 40.6%; p < 0.05). The lowest incidence of chromosomal abnormalities was observed in POCs ART using donor eggs from women younger than 35 years (12.8%). No statistical differences in the percentage of abnormal miscarriages were observed in correlation with sperm concentration: a sperm concentration less than 5 million/mL produced 75% abnormal results and a concentration higher than 5 million/mL produced 51%. CONCLUSIONS: POC analysis is essential to determine the cause of pregnancy loss. Using culture-independent molecular biology techniques to analyze POCs avoids limitations such as growth failure and reduces the time required for analysis. Selective biopsy of fetal tissue by hysteroembryoscopy avoids the risk of misdiagnosis due to maternal cell contamination. Our results show that maternal age, sperm quality, and ART-assisted pregnancies are risk factors for abnormal gestations.

Prenatal diagnosis of 24 cases of microduplication 22q11.2: an investigation of phenotype-genotype correlations.

OBJECTIVE: Microduplication 22q11.2 is primarily characterized by a highly variable clinical phenotype, which ranges from apparently normal or slightly dysmorphic features (in the presence or absence of learning disorders) to severe malformations with profound mental retardation. Hence, genetic counseling is particularly challenging when microduplication 22q11.2 is identified in a prenatal diagnosis. Here, we report on 24 prenatal cases of microduplication 22q11.2. METHODS: Seventeen of the cases were also reanalyzed by microarray analysis, in order to determine copy number variations (CNVs, which are thought to influence expressivity). We also searched for possible correlations between fetal phenotypes, indications for invasive prenatal diagnosis, inheritance, and pregnancy outcomes. RESULTS: Of the 24 cases, 15 were inherited, six occurred de novo, and three were of unknown origin. Termination of pregnancy occurred in seven cases and was mainly decided on the basis of ultrasound findings. Moreover, additional CNVs were found in some patients and we try to make a genotype-phenotype correlation. CONCLUSION: We discuss the complexity of genetic counseling for microduplication 22q11.2 and comment on possible explanations for the clinical heterogeneity of this syndrome. In particular, we assessed the co-existence of additional CNVs and their contribution to phenotypic variations in chromosome 22q11.2 microduplication syndrome.

BACs-on-Beads technology: a reliable test for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples.

Gene expression in hippocampus as a function of differential trait anxiety levels in genetically heterogeneous NIH-HS rats.

To identify genes involved in the development/expression of anxiety/fear, we analyzed the gene expression profile in the hippocampus of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock is a unique genetic resource for the fine mapping of quantitative trait loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety, fearfulness or other complex traits. We selected high- and low-anxious NIH-HS rats according to the number of avoidance responses they performed in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety/fearfulness tests, i.e. the elevated zero-maze and a "novel-cage activity" test. Three weeks after behavioral testing, the hippocampus was dissected and prepared for the microarray study. There appeared 29 down-regulated and 37 up-regulated SNC-related genes (fold-change>|2.19|, FDR<0.05) in the "Low-anxious" vs. the "High-anxious" group. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, nine relevant genes (Avpr1b, Accn3, Cd74, Ltb, Nrg2, Oprdl1, Slc10a4, Slc5a7 and RT1-EC12), tested for validation through qRT-PCR, have either neuroendocrinological or neuroinmunological/inflammation-related functions, or have been related with the hippocampal cholinergic system, while some of them have also been involved in the modulation of anxiety or stress-related (neurobiological and behavioral) responses (i.e. Avpr1b, Oprdl1). The present work confirms the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis or mechanisms involved in anxiety and/or fear, and suggest that some MHC-(neuroinmunological/inflammation)-related pathways, as well as the cholinergic system within the hippocampus, may play a role in shaping individual differences in trait anxiety.

Gene expression in amygdala as a function of differential trait anxiety levels in genetically heterogeneous NIH-HS rats.

To identify genes involved in anxiety/fear traits, we analyzed the gene expression profile in the amygdala of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock has revealed to be a unique genetic resource for the fine mapping of Quantitative Trait Loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety-(or other)-related traits. We selected high- and low-anxious NIH-HS rats differing in their number of avoidances in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety tests (e.g., elevated zero-maze). Three weeks after behavioural testing, the amygdala was dissected and prepared for the microarray study. There appeared 6 significantly down-regulated and 28 up-regulated genes (fold-change >|2|, FDR<0.05) between the low- and high-anxious groups, with central nervous system-related functions. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, six relevant genes were examined with qRT-PCR, four of which (Ucn3, Tacr3, H2-M9 and Arr3) were validated. Remarkably, some of them are characterized by sharing known functions related with hormonal HPA-axis responses to (and/or modulation of) stress, anxiety or fear, and putative involvement in related neurobehavioural functions. The results confirm the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis of anxiety and fear, while suggesting the involvement of some neuropeptide/neuroendocrine pathways on the development of differential anxiety profiles.

The impact of next-generation sequencing technology on preimplantation genetic diagnosis and screening.

Largely because of efforts required to complete the Human Genome Project, DNA sequencing has undergone a steady transformation with still-ongoing developments of high-throughput sequencing machines for which the cost per reaction is falling drastically. Similarly, the fast-changing landscape of reproductive technologies has been improved by genetic approaches. Preimplantation genetic diagnosis and screening were established more than two decades ago for selecting genetically normal embryos to avoid inherited diseases and to give the highest potential to achieve stable pregnancies. Most recent additions to the IVF practices (blastocyst/trophectoderm biopsy, embryo vitrification) and adoption of new genetics tools such as array comparative genome hybridization have allowed setting up more precise and efficient programs for clinical embryo diagnosis. Nevertheless, there is always room for improvements. Remarkably, a recent explosion in the release of advanced sequencing benchtop platforms, together with a certain maturity of bioinformatics tools, has set the target goal of sequencing individual cells for embryo diagnosis to be a realistically feasible scenario for the near future. Next-generation sequencing technology should provide the opportunity to simultaneously analyze single-gene disorders and perform an extensive comprehensive chromosome screening/diagnosis by concurrently sequencing, counting, and accurately assembling millions of DNA reads.

Tecnología BACs-on-Beads™ aplicada al diagnóstico prenatal y al estudio citogenético de restos abortivos / BACs-on-Beads™ technology applied to prenatal diagnosis and the cytogenetic study of abortion remains

La tecnología de BACs-on-Beads utiliza sondas de ADN procedentes de Cromosomas Artificiales Bacterianos o BAC fijados en microesferas Luminex®. La muestra marcada y los ADN de referencia marcados se hibridan respectivamente con las mismas sondas BoBsTM complementarias. Después de la hibridación se leen las intensidades de la señal mediante el sistema instrumental Luminex® 100/200. Se puede aplicar al diagnóstico prenatal rápido con un kit diseñado para descartar aneuploidías para los cromosomas 13, 18, 21 y los cromosomas sexuales así como ganancias y pérdidas de ADN asociadas con 9 síndromes de microdeleción como son: Síndrome de DiGeorge, Síndrome de Williams-Beuren, Síndrome de Prader-Willi, Síndrome de Angelman, Síndrome de Smith-Magenis, Síndrome de Wolf-Hirschhorn, Síndrome de Cri du Chat, Síndrome de Langer-Giedion, y Síndrome de Miller-Dieker. Basados en esta misma tecnología se ha desarrollado un kit llamado KaryoLite-BoBsTM, que permite descartar aneuploidías para los 24 cromosomas y que se puede aplicar al estudio de alteraciones cromosómicas numéricas en restos abortivos. En este trabajo se presentan los resultados de nuestro grupo tras la aplicación de la tecnología de BAC-on-BeadsTM en 332 muestras de líquido amniótico, 48 muestras de vellosidades coriales y en el estudio de aneuploidías en 71 muestras de restos abortivos(AU)

BACs-on-Beads (BoBs) technology is based on DNA sequences from Bacterial Artificial Chromosomes fixed in Luminex® microspheres. The study sample and the reference DNA are labeled with similar but complementary BoBsTM probes. After the hybridisation, signal intensities were analysed using the Luminex® 100/200 instrumental system. This technology has been applied to rapid prenatal diagnosis with a kit designed to analyse aneuploidy for chromosomes 13, 18, 21 and sex chromosomes, and a panel of 9 microdeletion syndromes: DiGeorge, Williams-Beuren, Prader-Willi, Angelman, Smith-Magenis, Wolf-Hirschhorn, Cri du Chat, Langer-Giedion, and Miller-Dieker syndrome. Based on a similar technology, KaryoLite-BoBsTM kit allows the identification of aneuploidy for all 24 chromosomes, and can be applied to the analysis of products of conception, among other possibilities. In this study we present our current experience in the application of BAC-on-BeadsTM technology in 332 amniotic fluid samples; 48 chorionic villus samples and 71 samples from products of conception(AU)

Prenatal BACs-on-Beads™: the prospective experience of five prenatal diagnosis laboratories.

OBJECTIVE: We previously reported on the validation of Prenatal BACs-on-Beads™ on retrospectively selected and prospective prenatal samples. This bead-based multiplex assay detects chromosome 13, 18, 21 and X/Y aneuploidies and the nine most frequent microdeletion syndromes. We demonstrated that Prenatal BACs-on-Beads(TM) is a new-generation, prenatal screening tool. Here, we describe the experience of five European prenatal diagnosis laboratories concerning the ongoing use of Prenatal BACs-on-Beads™ . METHODS: Some 1653 samples were analyzed. All results were confirmed by conventional karyotyping or another appropriate technique. All indications for invasive prenatal diagnosis were included. Amniotic fluid and chorionic villus samples were analyzed in equivalent proportions. RESULTS: The failure rate was 3.3% and the overall abnormality detection rate was ~1/10. Eighty-five percent of the detected abnormalities were common aneuploidies. Eleven microdeletions and duplications were identified, thus giving an overall yield for microdeletion and microduplication detection of 1/145. Compared with QF-PCR, Prenatal BACs-on-Beads™ provides an additional detection rate of ~1/250 for low-risk pregnancies. The false positive and negative rates were both <1%. CONCLUSION: When associated with conventional karyotyping, the Prenatal BACs-on-Beads™ assay combines a short turnaround time (typical of rapid aneuploidy detection tests) with valuable detection of the most frequent microdeletion syndromes that cannot be detected in cytogenetic analyses.

Hormonal and molecular characterization of follicular fluid, cumulus cells and oocytes from pre-ovulatory follicles in stimulated and unstimulated cycles.

BACKGROUND: The use of ovarian stimulation, to stimulate a multi-follicular response for assisted reproduction treatments, may force the production of oocytes from follicles that do not reach optimal maturation, possibly yielding oocytes that are not fully competent. The present study aimed to define the follicular environment and oocyte competence of unstimulated pre-ovulatory follicles, to compare it with that of similar-sized stimulated follicles. For this purpose, we analyzed the follicular hormonal milieu, the oocyte meiotic spindle, the embryo development and the cumulus cells gene expression (GE) profiles. METHODS AND RESULTS: The study population was divided in two groups: (i) 42 oocyte donors undergoing unstimulated cycles and (ii) 18 oocyte donors undergoing controlled ovarian stimulation cycles (COS). Follicular fluid was analyzed to quantify the concentrations of estradiol (E2), progesterone (P), FSH, LH, testosterone (T) and androstendione (Δ4). T was higher in the COS group, while Δ4, E2 and LH were significantly higher in unstimulated cycles. The cumulus oophorus cells (CC) surrounding the oocyte were removed and their GE profiles were analyzed with microarrays. There were 18 differentially expressed genes in CC: 7 were up-regulated and 11 were down-regulated in the COS cycles. The microarray was validated by qRT-PCR. The analysis of spindle structure revealed no significant differences between the groups, except for the parameter of length which presented differences. The fertilization ability and embryo morphology on Days 2, 3 and 4 did not show any significant differences between groups. CONCLUSIONS: The use of ovarian stimulation induces changes in the follicular fluid and in CC GE that may affect immune processes, meiosis and ovulation pathways. Although these differences do not seem to relate to early-stage embryo morphology, the implications of some of the molecules, especially ALDH1A2, CTSL and ZNF33B at the CC level, deserve to be addressed in future studies.

Endometrial gene expression in the window of implantation is altered in obese women especially in association with polycystic ovary syndrome.

OBJECTIVE: To determine whether luteal phase endometrial transcriptome is altered in obese women during the window of implantation (WOI), considering the presence of infertility, fat distribution and association with polycystic ovary syndrome (PCOS). DESIGN: Prospective study. SETTING: University-affiliated infertility clinic, between May 2007 and March 2009. PATIENT(S): One control group of women with normal weight (n=4), and four study groups of obese women (n=6 each one) according to the association with infertility, PCOS, and ovarian stimulation. INTERVENTION(S): The endometrium was biopsied 7 days after LH surge or hCG administration in 28 women. MAIN OUTCOME MEASURE(S): Endometrial gene expression during the WOI. RESULT(S): One hundred and fifty-one genes were dysregulated in obese groups compared with controls. This dysregulation was more pronounced when infertility was associated. The biologic processes of these genes belonged mainly to development and regulation of different biological functions such as transcription and biosynthesis. The molecular functions overrepresented were transcription and peptide receptor activity. The endometrium of obese women with PCOS showed dysregulated genes related to biologic processes such as development, morphogenesis, and the immune system, as well as different molecular functions such as protein binding, binding, growth factor activity, and carboxylic acid transmembrane transporter activity. Some of these genes have been previously related to implantation and unexplained infertility. CONCLUSION(S): Obese women present a different endometrial gene expression than controls during the WOI, which is more pronounced when infertility or polycystic ovary syndrome are associated.

Modeling human endometrial decidualization from the interaction between proteome and secretome.

CONTEXT: Decidualization of the human endometrium, which involves morphological and biochemical modifications of the endometrial stromal cells (ESCs), is a prerequisite for adequate trophoblast invasion and placenta formation. OBJECTIVE: This study aims to investigate the proteome and secretome of in vitro decidualized ESCs. These data were combined with published genomic information and integrated to model the human decidualization interactome. DESIGN: Prospective experimental case-control study. SETTING: A private research foundation. PATIENTS: Sixteen healthy volunteer ovum donors. INTERVENTION: Endometrial samples were obtained, and ESCs were isolated and decidualized in vitro. MAIN OUTCOME MEASURES: Two-dimensional difference in-gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, Western blot, human protein cytokine array, ELISA, and bioinformatics analysis were performed. RESULTS: The proteomic analysis revealed 60 differentially expressed proteins (36 over- and 24 underexpressed) in decidualized versus control ESCs, including known decidualization markers (cathepsin B) and new biomarkers (transglutaminase 2, peroxiredoxin 4, and the ACTB protein). In the secretomic analysis, a total of 13 secreted proteins (11 up- and 2 down-regulated) were identified, including well-recognized markers (IGF binding protein-1 and prolactin) and novel ones (myeloid progenitor inhibitory factor-1 and platelet endothelial cell adhesion molecule-1). These proteome/secretome profiles have been integrated into a decidualization interactome model. CONCLUSIONS: Proteomic and secretomic have been used as hypothesis-free approaches together with complex bioinformatics to model the human decidual interactome for the first time. We confirm previous knowledge, describe new molecules, and we have built up a model for human in vitro decidualization as invaluable tool for the diagnosis, therapy, and interpretation of biological phenomena.

Differential transcriptomic profile in spermatozoa achieving pregnancy or not via ICSI.

Basic sperm analysis is limited as a method of estimating pregnancy. This study's objective was use of microarray technology to differentiate the gene expressions of spermatozoa that achieved pregnancy in an intracytoplasmic sperm injection (ICSI)cycle in an oocyte donation programme with those that did not achieve pregnancy. A study of nested cases and controls was designed to evaluate fresh and frozen spermatozoa from infertile males undergoing ICSI with donor oocytes. The global genome expression of pooled samples from each group (achieving pregnancy versus those that didn't, from fresh or frozen spermatozoa)was compared using microarray analysis. The level of expression of some of the transcripts from fresh spermatozoa was shown to differ for those that achieved pregnancy versus those that didn't. Additionally, exclusively expressed transcripts were identified for both outcome groups. Analysis of frozen spermatozoa didn't reveal differential expression, but exclusively expressed transcripts were detected. Lists of the transcripts were systematically analysed using different databases in order to provide information about them and their relationship with male fertility. The results revealed profound differences between the expression profiles of spermatozoa that resulted in pregnancy versus those that didn't. These differences may explain ICSI failure associated with male factor infertility.

Perfiles de expresión génica en hombres infértiles que se someten a reproducción asistida / Gene expression profiles of infertile men who undergo assisted reproduction

Introducción. El espermiograma tiene una capacidad diagnóstica limitada a la hora de predecir una gestación en las técnicas de reproducción asistida. Parámetros moleculares como los ARN mensajeros presentes en el esperma no se evalúan. Nuestro grupo ha realizado listados de genes diferencialmente expresados (GDE) de muestras de semen que lograban un embarazo (E) frente aquellas que no (NE), en pacientes que se sometían a ciclos de IAH o de ICSI. El objetivo era caracterizar los perfiles de expresión, usando la tecnología del microarray, de muestras de semen que logran o no embarazo en ciclos de IAH o ICSI. Material y métodos. 30 muestras de semen (10 lograron embarazo [E=10] y 10 no [NE=10]) se obtenían de pacientes que se sometían a un ciclo de IAH y 10 muestras de semen (E=5, NE=5) que se sometían a uno de ICSI con ovocitos de donantes. Finalmente se realizaron 4 microarrays con las muestras de semen que lograban embarazo frente aquellas que no para ambas técnicas evaluándose los GDE que había entre los grupos E y NE tanto en IAH como en ICSI. Resultados. El número total de genes (NTG) detectados en el microarray de IAH fue de 19.938 y 19.229 en ICSI. El número total de GDE fue de 950 en IAH y 49 en ICSI. Conclusiones. Los datos revelan que los factores moleculares requeridos para lograr embarazo son diferentes según la técnica utilizada. Conclusiones. Estas diferencias pueden ser potencialmente empleadas como marcadores de éxito en ambas técnicas y como futura herramienta terapéutica (AU)

Introduction. Basic sperm analysis has insufficient predictive power on pregnancy achievement in assisted reproductive techniques (ARTs). Molecular parameters such as as mRNA present within spermatozoa are not assessed. Our group listed differentially expressed genes (DEG) from sperm samples (SS) from that achieve pregnancy (group P) vs. those that do not (group NP) using microarray technology in couples undergoing homologous IUI and ICSI. The aim is to use microarray technology to characterise the different gene expression profiles (EP) between SS that achieve pregnancy or not in ICSI and IUI cycles. Material and methods. Ten SS (5 that achieved and 5 that did not achieve pregnancy) were obtained from IP undergoing ICSI and 20 (10 that achieved and 10 did not achieve pregnancy) IAH cycles with oocytes from young donors and their healthy female partners respectively. After freezing aliquots of the SS employed for both treatments and their respective mRNA expression profiles were compared. Finally 4 microarrays were performed in duplicate with sperm samples from group P vs. NP from both techniques and those DEG were evaluated at least twice, with statistically significant differences between P and NP SS for each group. Results. Total number of genes (TNG) in IUI microarray was 19938 and 19229 in ICSI. The total number of DEG (950) in the IAH group was much greater than in the ICSI group (49). Conclusions. These data reveal that molecular features required for pregnancy are different in IUI and ICSI procedures. Conclusions. These differences could be potentially employed to detect ICSI and IUI success markers or to improve pregnancy rates in these procedures (AU)

Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells.

During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.

Ontological evaluation of transcriptional differences between sperm of infertile males and fertile donors using microarray analysis.

PURPOSE: To catalogue Gene Ontology terms in the sperm of infertile human males vs. donors of proven fertility by analyzing five samples from each of the two groups (five aliquots from fresh sperm and five post-swim-up). METHODS: Microarray technology was employed to study the mRNA profile of both fresh and post-swim-up pooled samples from infertile males and donors. RESULTS: Genes that were differentially expressed in the two populations and expressed in only one of two were analyzed to determine the gene products in terms of their associated Gene Ontology terms. Each group presented a different number and pattern of Gene Ontology terms. CONCLUSIONS: We found differences in Gene Ontology terms between the two groups. These differences could potentially be employed to establish markers of fertility success and to identify cellular processes and complex systems related with male infertility.

The transcriptome of spermatozoa used in homologous intrauterine insemination varies considerably between samples that achieve pregnancy and those that do not.

OBJECTIVE: To differentiate transcripts' expression in the sperm from patients who achieved pregnancy in their first IUI cycle from those who did not. Basic sperm analysis is limited to forecasting pregnancies by means of assisted reproduction. New assays, such as microarray analysis, are potential predictive tools for this purpose. DESIGN: Nested case-control study. SETTING: University-affiliated private setting. PATIENT(S): Twenty sperm samples were obtained from infertile males undergoing their first IUI cycle with healthy partners. Sperm samples with which pregnancy was achieved (P; n=10) and those with which it was not achieved (NP; n=10) were identified and their respective messenger RNA expression profiles were compared. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Using microarrays, global genome expression was compared in pooled samples from each group. Results were evaluated to detect differentially expressed transcripts (TDEs; FC>2; P<0.05) and to identify those transcripts that were expressed in only one of the groups (exclusive transcripts [ETs]). RESULT(S): In group P, 756 TDEs presented increased expression, whereas 194 in group NP were found to be overexpressed. Furthermore, we found 741 ETs that were expressed only in group P and 976 that were expressed only in group NP. CONCLUSION(S): Results reveal profound differences between expression profiles of sperm samples that impregnate successfully and those that do not. These differences might improve the predictive power of sperm evaluation to estimate IUI success by complementing the basic sperm analysis.

Cigarette smoking affects specific sperm oxidative defenses but does not cause oxidative DNA damage in infertile men.

OBJECTIVE: To evaluate the effects of tobacco consumption on the oxidative defenses of sperm, the glutathione system (GS), and sperm DNA oxidation. DESIGN: Double-blind experimental study. SETTING: Andrology laboratory in a university-affiliated private setting. PATIENT(S): One hundred seventeen semen samples from infertile males. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): (a) sperm GS enzymatic activity with respect to glutathione peroxidase isoforms GPx-1 and GPx-4, glutathione reductase (GR), and cellular glutathione (GSH) content (n = 29); (b) GPx-1, GPx-4, and GR mRNA expression analysis (n = 33); (c) oxidative DNA damage quantification using OXIDNA assay kit (n = 55). Two groups were established: nonsmoking and smoking males. The t tests were employed to detect significant differences between groups. RESULT(S): We identified a significant decrease in sperm GPx-4 activity but not in GPx-1 and GSH activity in smokers compared with nonsmokers. A significant decrease was also observed in GPx-1, GPx-4, and GR mRNA expression in the former group. Interestingly, we did not observe any significant variation in the percentage of cells with oxidative damage of the DNA or in the average level of oxidation of affected cells with respect to the smoking condition of the male. CONCLUSION(S): We demonstrate that smoking has a negative impact on intracellular antioxidant enzymes but that effect does not increase oxidative DNA damage. Thus, the effects of reduced oxidative defenses in sperm as a result of cigarette smoking are yet to be elucidated.

Contribution of sperm molecular features to embryo quality and assisted reproduction success.

Semen analysis, as stated by the World Health Organization, is the only accepted tool to assess male fertility. However its predictive value to assess male capacity to initiate a pregnancy is limited. With the introduction of IVF (especially via intracytoplasmic sperm injection), infertility caused by diminished sperm production is frequently solved, but knowledge of sperm physiology remains very poor. Moreover, a percentage of males with apparently normal semen are unable to impregnate healthy women. Therefore, improvements in the diagnostic tools to assess male fertility potential are necessary. The aim of this review is to describe sperm molecular factors implicated in male fertility, demonstrated by their role in sperm physiology, the molecular differences found between fertile and infertile males, or by their influence on the results obtained in assisted reproduction treatments in terms of embryo quality and pregnancy achievement. From a search and objective evaluation of the currently available evidence, it is concluded that there is no unique factor able to predict male fertility, but several molecular factors are involved in sperm function and can potentially be considered as fertility markers. In this context, a complex molecular tool designed to analyse a battery of parameters seems to be necessary.

The human sperm glutathione system: a key role in male fertility and successful cryopreservation.

The equilibrium of the creation and scavenging of free radicals is mandatory in the spermatozoa to fertilize and initiate a full-term pregnancy. The glutathione (GSH) enzymatic system studies have discovered its relationship with oxidative stress in the ejaculate and new strategies to regulate its activity in the semen could be developed. Intracellular sperm GSH system components are altered in infertile men, and these alterations seem to be linked to sperm morphology. We have been able to correlate embryo morphology on 8 cell embryos with the sperm expression of GPx family members; this relationship appears quite promising for discovery of molecular causes of male infertility. Oxidative stress imbalance potentially leads to damage of the structure of plasma membrane. The freezing and subsequent thawing of sperm is a physically stressful process carried out during routine procedures in assisted reproduction, which results in a highly variable and unpredictable reduction of motile sperm. Subsequently, oxidative status can positively or negatively affect the motility, viability, and fertilizing capacity of thawed sperm. A reserve of glutathione, together with GPx expression, is necessary to eliminate free radicals using GSH or GPx-4 like structural protein and seems to be essential for a good post thaw recovery.