Characterization of Seminal Microbiome of Infertile Idiopathic Patients Using Third-Generation Sequencing Platform.

Since the first description of a commensal seminal microbiome using sequencing, less than a decade ago, interest in the composition of this microbiome and its relationship with fertility has been growing. Articles using next-generation sequencing techniques agree on the identification of the most abundant bacterial phyla. However, at the genus level, there is still no consensus on which bacteria are most abundant in human seminal plasma. This discrepancy may be due to methodological variability such as sample collection, bacterial DNA extraction methodology, which hypervariable regions of 16S rRNA gene have been amplified, or bioinformatic analysis. In the present work, seminal microbiota of 14 control samples and 42 samples of idiopathic infertile patients were characterized based on full-length sequencing of the 16S rRNA gene using MinION platform from Oxford Nanopore. These same samples had been analyzed previously using Illumina's MiSeq sequencing platform. Comparison between the results obtained with the two platforms has been used to analyze the impact of sequencing method on the study of the seminal microbiome's composition. Seminal microbiota observed with MinION were mainly composed of the phyla Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria, with the most abundant genera being Peptoniphilus, Finegoldia, Staphylococcus, Anaerococcus, Campylobacter, Prevotella, Streptococcus, Lactobacillus, Ezakiella and Enterococcus. This composition was similar to that found by the Illumina platform, since these 10 most abundant genera were also among the most abundant genera detected by the Nanopore platform. In both cases, the top 10 genera represented more than 70% of the classified reads. However, relative abundance of each bacterium did not correlate between these two platforms, with intraindividual variations of up to 50 percentage points in some cases. Results suggest that the effect of the sequencing platform on the characterization of seminal microbiota is not very large at the phylum level, with slightly variances in Firmicutes and Actinobacteria, but presents differences at the genus level. These differences could alter the composition and diversity of bacterial profiles or posterior analyses. This indicates the importance of conducting multi-platform studies to better characterize seminal microbioma.

TMT-Based Proteomic Analysis of Human Spermatozoa from Unexplained Recurrent Miscarriage Patients before and after Oral Antioxidant Treatment.

Recently, sperm quality and the presence of double-stranded breaks (DSB) has been pointed out as a possible cause of recurrent miscarriage, and the use of antioxidants has expanded as a treatment for male infertility. The aim of the present study was to analyze the proteomic effects of antioxidants on sperm from RM patients with high incidence of DSB. Proteomic analysis was performed using a tandem mass tag labeling technique, and subsequently compared with the PANTHER database for DEPs, and the STRING database for protein-protein interactions (PPI). Differentially expressed proteins (DEPs) both before and after antioxidant oral treatment were identified. PPI involving DEPs clustered into networks related to cell metabolism, cytoskeleton, and DNA damage. Results show that the sperm proteomic profiles before and after antioxidant treatment do not significantly differ from each other. However, some DEPs found after the antioxidant treatment shifted towards a DEPs profile typical of fertile donors. This indirect measurement suggests an improvement caused by antioxidants on the expression of several proteins. Among them were proteins involved in sperm DNA remodeling (LMO7, MMP28, BNC2, H2B, and PRDM2). The results presented here represent the first approach in the analysis and repair of the proteomic change caused by antioxidants in recurrent miscarriage patients, elucidating biomarkers that may be useful for the diagnosis and further sperm selection in this type of patient. Further studies should be conducted to validate the usefulness of these biomarkers in larger study groups.

Seminal Microbiota of Idiopathic Infertile Patients and Its Relationship With Sperm DNA Integrity.

The development of new biomarkers for human male infertility is crucial to improve the diagnosis and the prognosis of this disease. Recently, seminal microbiota was shown to be related to sperm quality parameters, suggesting an effect in human fertility and postulating it as a biomarker candidate. However, its relationship to sperm DNA integrity has not been studied yet. The aim of the present study is to characterize the seminal microbiota of a western Mediterranean population and to evaluate its relationship to sperm chromatin integrity parameters, and oxidative stress. For that purpose, 14 samples from sperm donors and 42 samples from infertile idiopathic patients were obtained and were analyzed to assess the composition of the microbiota through full-length 16S rRNA gene sequencing (Illumina MiSeq platform). Microbial diversity and relative abundances were compared to classic sperm quality parameters (macroscopic semen parameters, motility, morphology and concentration), chromatin integrity (global DNA damage, double-stranded DNA breaks and DNA protamination status) and oxidative stress levels (oxidation-reduction potential). The seminal microbiota observed of these samples belonged to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The most abundant genera were Finegoldia, Peptoniphilus, Anaerococcus, Campylobacter, Streptococcus, Staphylococcus, Moraxella, Prevotella, Ezakiella, Corynebacterium and Lactobacillus. To our knowledge, this is the first detection of Ezakiella genus in seminal samples. Two clusters of microbial profiles were built based on a clustering analysis, and specific genera were found with different frequencies in relation to seminal quality defects. The abundances of several bacteria negatively correlate with the sperm global DNA fragmentation, most notably Moraxella, Brevundimonas and Flavobacterium. The latter two were also associated with higher sperm motility and Brevundimonas additionally with lower oxidative-reduction potential. Actinomycetaceae, Ralstonia and Paenibacillus correlated with reduced chromatin protamination status and increased double-stranded DNA fragmentation. These effects on DNA integrity coincide in many cases with the metabolism or enzymatic activities of these genera. Significant differences between fertile and infertile men were found in the relative presence of the Propionibacteriaceae family and the Cutibacterium, Rhodopseudomonas and Oligotropha genera, which supports its possible involvement in male fertility. Our findings sustain the hypothesis that the seminal microbiome has an effect on male fertility.

Sperm chromatin condensation and single- and double-stranded DNA damage as important parameters to define male factor related recurrent miscarriage.

The aim of the present work is to characterize the relationship between sperm protamine deficiency and single- and double-stranded DNA damage and to assess the diagnostic potential of chromomycin A3 (CMA3). For that purpose, semen samples from 90 human males with different clinical features were included (fertile donors, patients with recurrent pregnancy loss [RPL], and infertile patients). DNA condensation was analyzed by CMA3 and different types of DNA fragmentation were analyzed through the comet assay. A positive correlation between DNA condensation and single-stranded DNA fragmentation was found (Rs = .456; p = .05). CMA3 presented differences between fertile donors and all other groups (p < .001). Interestingly, patients with RPL, who were able to achieve a pregnancy, and infertile patients showed similar values of CMA3 (p > .05). Receiver operating characteristic curves and the profiles obtained by the combination of Comet assays and CMA3 indicate that the CMA3 test may be an interesting approach to distinguish those subjects with higher pregnancy loss risk from fertile donors (CMA3 area under the curve 0.928, with a confidence interval of 0.849-1.000). The present work shows that DNA condensation is related to oxidative damage, which affects mainly protamine-rich regions. The profiles observed in different clinical groups showed that CMA3 might be useful for the diagnosis of RPL risk when combined with Comet assays.

Relationship of Seminal Oxidation-Reduction Potential with Sperm DNA Integrity and pH in Idiopathic Infertile Patients.

Seminal oxidative stress (OS) is one of the most promising factors to describe the causes of idiopathic male infertility. Redox balance is essential in several biological processes related to fertility, so alterations such as high reactive oxygen species (ROS) levels or low antioxidant agent levels can compromise it. MiOXSYS has been developed to evaluate the seminal static oxidation-reduction potential (sORP) and it has been proposed as an effective diagnostic biomarker. However, its relationship with parameters like sperm DNA fragmentation (SDF), chromatin compaction status or seminal pH requires further analysis, making it the object of this study. Semen and sORP analysis were performed for all samples. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) and Comet assay were used to assess SDF and chromomycin a3 (CMA3) test to assess sperm chromatin compaction. Regarding sORP measures, it was found that alkaline pH has an effect on sample reproducibility. To our knowledge, this unexpected effect has not been previously described. A statistical analysis showed that sORP correlated negatively with CMA3 positive cells and sperm motility, but not with SDF. As redox dysregulation, which occurs mainly at the testicular and epididymal level, causes chromatin compaction problems and leaves DNA exposed to damage, an excess of ROS could be counterbalanced further by a seminal supply of antioxidant molecules, explaining the negative correlation with CMA3 positive cells but no correlation with SDF. Our results show that the study of idiopathic infertility would benefit from a combined approach comprising OS analysis, SDF and chromatin compaction analysis.

Novel Double Factor PGT strategy analyzing blastocyst stage embryos in a single NGS procedure.

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.

An exploratory study of predisposing genetic factors for DiGeorge/velocardiofacial syndrome.

DiGeorge/velocardiofacial syndrome (DGS/VCFS) is a disorder caused by a 22q11.2 deletion mediated by non-allelic homologous recombination (NAHR) between low-copy repeats (LCRs). We have evaluated the role of LCR22 genomic architecture and PRDM9 variants as DGS/VCFS predisposing factors. We applied FISH using fosmid probes on chromatin fibers to analyze the number of tandem repeat blocks in LCR22 in two DGS/VCFS fathers-of-origin with proven 22q11.2 NAHR susceptibility. Results revealed copy number variations (CNVs) of L9 and K3 fosmids in these individuals compared to controls. The total number of L9 and K3 copies was also characterized using droplet digital PCR (ddPCR). Although we were unable to confirm variations, we detected an additional L9 amplicon corresponding to a pseudogene. Moreover, none of the eight DGS/VCFS parents-of-origin was heterozygote for the inv(22)(q11.2) haplotype. PRDM9 sequencing showed equivalent allelic distributions between DGS/VCFS parents-of-origin and controls, although a new PRDM9 allele (L50) was identified in one case. Our results support the hypothesis that LCR22s variations influences 22q11.2 NAHR events, however further studies are needed to confirm this association and clarify the contribution of pseudogenes and rare PDRM9 alleles to NAHR susceptibility.

Factores nutricionales y no nutricionales pueden afectar la fertilidad masculina mediante mecanismos epigenéticos.

Introducción: la infertilidad es un problema global en aumento. Se estima que aproximadamente un 15% de las parejas en edad reproductiva tiene dificultades a la hora de concebir. De estas, alrededor de la mitad presentan uno o varios factores masculinos asociados a infertilidad o subfertilidad, aislados o en combinación con problemas de origen femenino. Durante la última década se ha empezado a estudiar la infertilidad desde una perspectiva multifactorial, considerando las interacciones y conexiones entre diferentes situaciones genéticas, epigenéticas, bioquímicas y fisiológicas del paciente.Objetivo: la presente revisión pretende describir mecanismos epigenéticos que pueden ser modulados mediante aspectos nutricionales y que están relacionados con la etiología de la infertilidad masculina y con la herencia transgeneracional de este fenotipo.Material y métodos: se ha realizado una extensa búsqueda de publicaciones científicas en las principales bases de datos electrónicas especializadas: NBCI, Elsevier, Scielo, Scirus y Science Direct.Resultados y conclusión: varios trabajos que muestran la importancia del estado nutricional en la fertilidad del hombre y, más específicamente, la capacidad de los componentes de la dieta para modificar los perfiles epigenéticos que no únicamente pueden afectar a su fertilidad, sino que también pueden ser transmitidos a la descendencia mediante lo que se ha denominado herencia transgeneracional, ocasionándoles problemas de salud diversos entre los que también se hallan problemas en la fertilidad.

Factores nutricionales y no nutricionales pueden afectar la fertilidad masculina mediante mecanismos epigenéticos / Nutritional and non-nutritional factors may affect male infertility through epigenetic mechanisms

Introducción: la infertilidad es un problema global en aumento. Se estima que aproximadamente un 15% de las parejas en edad reproductiva tiene dificultades a la hora de concebir. De estas, alrededor de la mitad presentan uno o varios factores masculinos asociados a infertilidad o subfertilidad, aislados o en combinación con problemas de origen femenino. Durante la última década se ha empezado a estudiar la infertilidad desde una perspectiva multifactorial, considerando las interacciones y conexiones entre diferentes situaciones genéticas, epigenéticas, bioquímicas y fisiológicas del paciente. Objetivo: la presente revisión pretende describir mecanismos epigenéticos que pueden ser modulados mediante aspectos nutricionales y que están relacionados con la etiología de la infertilidad masculina y con la herencia transgeneracional de este fenotipo. Material y métodos: se ha realizado una extensa búsqueda de publicaciones científicas en las principales bases de datos electrónicas especializadas: NBCI, Elsevier, Scielo, Scirus y Science Direct. Resultados y conclusión: varios trabajos que muestran la importancia del estado nutricional en la fertilidad del hombre y, más específicamente, la capacidad de los componentes de la dieta para modificar los perfiles epigenéticos que no únicamente pueden afectar a su fertilidad, sino que también pueden ser transmitidos a la descendencia mediante lo que se ha denominado herencia transgeneracional, ocasionándoles problemas de salud diversos entre los que también se hallan problemas en la fertilidad (AU)

Introduction: Infertility rate is globally increasing. It is estimated that approximately 15% of couples in reproductive age have troubles conceiving. Half of these couples present with problems related to male infertility or subfertility, alone or in combination with female problems. During the last decade, infertility has been studied from a multifactorial perspective, which includes interactions between different genetics, epigenetics, biochemical and physiological situations of the patients. Objective: The present review aims to describe epigenetic mechanisms that can be modulated by nutritional aspects and which are related to the aetiology of male infertility and transgenerational inheritance. Materials and method: Extensive search of scientific publications was performed in specialized electronic databases: NBCI, Elsevier, Scielo, Scirus and Science Direct. Results and conclusion: Several published works have shown the importance of nutritional status in man’s fertility, and more specifically, the ability of diet components to modify the epigenetic profiles, affecting not only their fertility, but also increasing the possibility to be transmitted to the offspring. This mechanism has been called transgenerational inheritance (AU)

Analysis of invdupdel(8p) rearrangement: Clinical, cytogenetic and molecular characterization.

Inverted duplication 8p associated with deletion of the short arms of chromosome 8 (invdupdel[8p]) is a relatively uncommon complex chromosomal rearrangement, with an estimated incidence of 1 in 10,000-30,000 live borns. The chromosomal rearrangement consists of a deletion of the telomeric region (8p23-pter) and an inverted duplication of the 8p11.2-p22 region. Clinical manifestations of this disorder include severe to moderate intellectual disability and characteristic facial features. In most cases, there are also CNS associated malformations and congenital heart defects. In this work, we present the cytogenetic and molecular characterization of seven children with invdupdel(8p) rearrangements. Subsequently, we have carried out genotype-phenotype correlations in these seven patients. The majority of our patients carry a similar deletion but different size of duplications; the latter probably explaining the phenotypic variability among them. We recommend that complete clinical evaluation and detailed chromosomal microarray studies should be undertaken, enabling appropriate genetic counseling.

Multiple determinations of sperm DNA fragmentation show that varicocelectomy is not indicated for infertile patients with subclinical varicocele.

Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient's fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA) were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment). TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele.

Genetic recombination variation in wild Robertsonian mice: on the role of chromosomal fusions and Prdm9 allelic background.

Despite the existence of formal models to explain how chromosomal rearrangements can be fixed in a population in the presence of gene flow, few empirical data are available regarding the mechanisms by which genome shuffling contributes to speciation, especially in mammals. In order to shed light on this intriguing evolutionary process, here we present a detailed empirical study that shows how Robertsonian (Rb) fusions alter the chromosomal distribution of recombination events during the formation of the germline in a Rb system of the western house mouse (Mus musculus domesticus). Our results indicate that both the total number of meiotic crossovers and the chromosomal distribution of recombination events are reduced in mice with Rb fusions and that this can be related to alterations in epigenetic signatures for heterochromatinization. Furthermore, we detected novel house mouse Prdm9 allelic variants in the Rb system. Remarkably, mean recombination rates were positively correlated with a decrease in the number of ZnF domains in the Prdm9 gene. The suggestion that recombination can be modulated by both chromosomal reorganizations and genetic determinants that control the formation of double-stranded breaks during meiosis opens new avenues for understanding the role of recombination in chromosomal speciation.

Evolution of recombination in eutherian mammals: insights into mechanisms that affect recombination rates and crossover interference.

Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primates and Carnivora to better understand the dynamics of mammalian recombination. Our results suggest a phylogenetic component in recombination rates (RRs), which appears to be directional, strongly punctuated and subject to selection. Species that diversified earlier in the evolutionary tree have lower RRs than those from more derived phylogenetic branches. Furthermore, chromosome-specific recombination maps in distantly related taxa show that crossover interference is especially weak in the species with highest RRs detected thus far, the tiger. This is the first example of a mammalian species exhibiting such low levels of crossover interference, highlighting the uniqueness of this species and its relevance for the study of the mechanisms controlling crossover formation, distribution and resolution.

Differential clustering of sperm subpopulations in infertile males with clinical varicocele and carriers of rearranged genomes.

Some methods for determining sperm DNA fragmentation, such as the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion test (SCD), provide additional information about particular subgroups of spermatozoa with specific irregularities. Thus, SCSA recognizes a specific sperm subpopulation, the high-DNA stainability sperm subpopulation (HDS), and SCD recognizes the so-called DNA-degraded sperm (DDS) subpopulation. Although some studies associate the presence of these subpopulations with specific aspects related to infertility, the relationship between both sperm subpopulations and their preponderance in specific clinical groups of infertile males has not been extensively investigated. In this study, HDS and DDS subpopulations were determined in a total of 37 human males: 8 males with proven fertility, 9 infertile males with asthenoteratozoospermia, 10 carriers of chromosomal reorganizations, and 10 infertile males with clinical varicocele. Results showed a significant increase of the DDS subpopulation (P < .001) in both the varicocele patient (16.85 ± 7.24) and carrier of rearranged genome (11.6 ± 5.23) groups, but not in patients with asthenoteratozoospermia (3.88 ± 1.55) or fertile donors (2.62 ± 1.68). No statistical differences were detected for the HDS subpopulation (P = .542), but the highest values were found in the varicocele and rearranged-genome groups. However, no correlation between the HDS and DDS subpopulations were found (r = 0.196; P = .244), suggesting that both represent a different class of sperm subpopulation in the ejaculate. A significant increase in HDS, and especially DDS, can be associated with the presence of varicocele or the rearrangement of chromosomes. Specific diagnostic tests to confirm the diagnosis must be performed in patients with increased DDS and HDS values.

Detection of unbalanced chromosome segregations in preimplantation genetic diagnosis of translocations by short comparative genomic hibridization.

OBJECTIVE: To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. DESIGN: Clinical research study. SETTING: A PGD laboratory and two IVF clinics. PATIENT(S): Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a double-translocation carrier. INTERVENTION(S): After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. MAIN OUTCOME MEASURE(S): Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. RESULT(S): The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. CONCLUSION(S): Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.

Protamine 1 to protamine 2 ratio correlates with dynamic aspects of DNA fragmentation in human sperm.

OBJECTIVE: To investigate the relationship between the protamine 1 to protamine 2 (P1/P2) ratio and the rate of sperm DNA fragmentation in sperm samples from human males with proven fertility and three different cohorts of male patients. DESIGN: P1/P2 ratio was analyzed using acid-urea polyacrylamide acid-urea gels electrophoresis (PAGE). Sperm DNA fragmentation using sperm chromatin dispersion methodology was analyzed after 0, 4, 8, and 24 hours of incubation at 37°C. SETTING: University medical school and hospital. PATIENT(S): A total of 32 human males: six with proven fertility, seven carriers of chromosome reorganizations, nine clinical varicocele patients, and ten subclinical varicocele patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): P1/P2 ratio, sperm DNA fragmentation (SDF) and the rate of sperm DNA fragmentation (rSDF). RESULT(S): P1/P2 ratio correlated with SDF and rSDF. Statistical differences were detected between fertile controls and patients for the three pathologies studied. rSDF yielded information that differed from baseline SDF. No differences were detected for P1/P2 ratio among patient groups, in reference to the three pathologies studied. CONCLUSION(S): SDF and rSDF correlates with P1/P2 ratio in human sperm, and statistical differences were detected when fertile controls were compared with three different cohorts of patients.

Outcome of twin babies free of Von Hippel-Lindau disease after a double-factor preimplantation genetic diagnosis: monogenetic mutation analysis and comprehensive aneuploidy screening.

OBJECTIVE: To increase the embryo implantation rate, a double-factor preimplantation genetic diagnosis (DF-PGD) was performed, selecting for transfer potentially euploid evolved embryos free of the mutation responsible for Von Hippel-Lindau syndrome (VHL). DESIGN: Case report. SETTINGS: Medical university center and a private IVF center. PATIENT(S): A patient carrier of the R161Q mutation on the VHL gene. INTERVENTION(S): After first polar body (1PB) biopsy, it was analyzed using comparative genomic hybridization (1PB-CGH). On day +3, mutation detection using minisequencing and short tandem repeat analysis was performed in multiple displacement amplification products of a single blastomere per embryo. MAIN OUTCOME MEASURE(S): Transfering embryos free of the disease and originating from euploid oocytes. RESULT(S): Nine of the twelve oocytes obtained were successfully analyzed using 1PB-CGH. One of them was aneuploid (1PB #1: 29XX,+2,+10,+12,+17,+19), and the rest were euploid. All of the oocytes were fertilized and became evolved embryos. Six of the embryos were VHL unaffected and had good quality. Five (83%) of them were potentially euploid. According to cytogenetic results, two of the evolved and healthy embryos were transfered, achieving the birth of healthy twin babies. CONCLUSION(S): The DF-PGD can be a useful tool to increase implantation of transfered embryos in PGD for monogenic diseases.

DNA fragmentation and meiotic segregation in sperm of carriers of a chromosomal structural abnormality.

OBJECTIVE: To determine the meiotic segregation and DNA fragmentation in spermatozoa of carriers of a chromosomal structural abnormality. DESIGN: Case series. SETTING: University hospital. PATIENT(S): Thirty-seven male carriers of a chromosomal structural abnormality (21 with a balanced reciprocal translocation, 7 with a robertsonian translocation, 9 with a pericentric inversion). INTERVENTION(S): Meiotic segregation was analyzed by the human sperm-hamster oocyte fusion technique or by fluorescent in situ hybridization, and DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. MAIN OUTCOME MEASURE(S): Relationships between abnormal sperm parameters, DNA fragmentation, and meiotic mechanisms. RESULT(S): The average rates of chromosomally unbalanced spermatozoa were 55.22%, 14.09%, and 18.43% for reciprocal translocation, robertsonian translocation, and pericentric inversion carriers, respectively. The rates of DNA fragmentation were significantly higher in the whole group of carriers of a chromosomal structural abnormality and in each specific group than in the control group. No correlations between sperm DNA fragmentation and parameters of spermogram, age, or percentage of unbalanced chromosomal gametes were found. CONCLUSION(S): The DNA fragmentation rate depends solely on the presence of a chromosomal structural abnormality, and, therefore, a chromosomal structural abnormality predicts DNA fragmentation. Both meiotic segregation and DNA fragmentation studies should be integrated in the genetic exploration of male carriers of a chromosomal structural abnormality.

Birth of a healthy boy after a double factor PGD in a couple carrying a genetic disease and at risk for aneuploidy: case report.

Preimplantation genetic diagnosis (PGD) for monogenic diseases is widely applied, allowing the transfer to the uterus of healthy embryos. PGD is also employed for the detection of chromosome abnormalities for couples at high risk of producing aneuploid embryos, such as advanced maternal (>35 years). A significant number of patients requesting PGD for monogenic diseases are also indicated for chromosome testing. We optimized and clinically applied a PGD protocol permitting both cytogenetic and molecular genetic analysis. A couple, carriers of two cystic fibrosis (CF) mutations (c.3849 + 10 KbC > T and c.3408C > A) with a maternal age of 38 years and two previously failed IVF-PGD cycles, was enrolled in the study. After ovarian stimulation, six oocytes were obtained. To detect abnormalities for all 23 chromosomes of the oocyte, the first polar body (1PB) was biopsied from five of the oocytes and analyzed using comparative genomic hybridization (CGH). CGH analysis showed that 1PB 1 and 1PB 4 were aneuploid (22X,-9,-13,+19 and 22X,-6, respectively), while 1PB 2, 1PB 3 and 1PB 6 were euploid. Blastomere biopsy was only applicable on embryos formed from Oocyte 3 and Oocyte 6. After whole-genome amplification with multiple displacement amplification, a multiplex PCR, amplifying informative short tandem repeats (D7S1799; D7S1817) and DNA fragments encompassing the mutation sites, was performed. MiniSequencing was applied to directly detect each mutation. Genetic diagnosis showed that Embryo 6 was affected by CF and Embryo 3 carried only the c.3849 + 10 KbC > T mutation. Embryo 3 was transferred achieving pregnancy and a healthy boy was born. This strategy may lead to increased pregnancy rates by allowing preferential transfer of euploid embryos.