DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease

We report the world's first clinical pregnancy resulting fromDNA-based enrichment for X-bearing human spermatozoa, for preventionof X-linked hydrocephalus. Sperm separation was followed byembryo biopsy and nested multiplex polymerase chain reaction(PCR) for gender determination. Enriched populations of X-bearingspermatozoa ranging from 80 to 89% pure as determined by fluorescencein-srtu hybridization (FISH) resulted in in-vitro fertilization(IVF) rates indistinguishable from normal IVF procedures (65%).In two separate biopsy procedures, 7/9 and 15/16 of the resultingembryos were determined to be female by multiplex PCR. Embryotransfer resulted in a karyotypically normal female fetus. Thistechnique should be widely applicable to gender selection forthe prevention of genetic disorders. flow cytometry/fluorescence in-situ hybridization/gender selection/hereditary diseases/spermatozoa     

Genetics: Discontinuous Percoll gradients enrich X-bearing human spermatozoa: a study using double-label fluorescence int-situ hybridization

The aim of this study was to evaluate the efficacy of enrichingX-bearing human spermatozoa using 12-step (25–80%) discontinuousPercoll gradients. X- and Y-bearing spermatozoa were simultaneouslyidentified in neat semen (controls) and in 80% Percoll fractionsfrom the same samples using double-label fluorescence in-situhybridization and chromosome-specific DNA probes. Hybridizationand labelling efficiencies of 95–99% were obtained inall samples. The mean ratio of X- to Y-bearing spermatozoa inthe controls was 49.0: 48.2, whereas there was a significantenrichment (P < 0.0001) of X-bearing spermatozoa in the Percollfractions (mean X:Y ratio was 55.1:41.1 or 1.35). The ratiovaried from 1.1–1.5 in individual Percoll samples. Therewere no significant differences in the proportions of aneuploidspermatozoa (XX, YY, XY) between the control and Percoll fractions.We conclude that 12-step discontinuous Percoll gradients enrichX-bearing spermatozoa, but the degree of enrichment is insufficientfor use in preconceptional sex selection.     

Preliminary study of the incidence of disomy in sperm fractions after MicroSort flow cytometry

Using triple colour fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the disomy level for chromosome 21 and the sex chromosomes in flow cytometric sorted sperm samples. There were no statistically significant differences in the disomy rates when comparing the sorted samples (either for X- or Y-bearing spermatozoa) with non-sorted samples. There were no diploid spermatozoa observed in any sample group after MicroSort processing.     

Preimplantation diagnosis: Gender preselection in humans? Flow cytometric separation of X and Y spermatozoa for the prevention of X-linked diseases

Human X- and Y-chromosome-bearing spermatozoa were separatedbased on their DNA content, using modified flow cytometric cellsorting technology. The resulting separation purity of the X-bearingfrom Y-bearing spermatozoa was evaluated using in-situ hybridizationwith alpha satellite DNA probes for the X- and Y-chromosomes.In the putative X-enriched-sorted populations, an average of82% of the spermatozoa showed a hybridization signal with theX probe. Similarly, in the Y-sorted population 75% gave a signalwith the Y probe. Sorted X- and Y-bearing spermatozoa were foundto maintain their viability for several hours after sorting.These results demonstrate that the human sperm sex ratio canbe significantly shifted to favour the selection of female-producing(X) spermatozoa or maleproducing (Y) spermatozoa when spermatozoaare flow cytometrically sorted on the basis of DNA content.We propose that flow cytometrically sorted human spermatozoa,used in conjunction with in-vitro fertilization or intra-oviductalinsemination, could be used by families who are at risk forX-linked diseases to preferentially produce female offspring.Sorted spermatozoa could also be used to pre-select for maleoffspring if that were medically indicated.     

Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters

BACKGROUND: Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF. METHODS: To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy. RESULTS: FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF. CONCLUSIONS: Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.     

10, 15 reciprocal translocation in an infertile man: ultrastructural and fluorescence in-situ hybridization sperm study: case report

BACKGROUND: Peculiar sperm defects are described in a sterile man heterozygous for a balanced translocation t(10;15) (q26;q12). As this structural reorganization was absent in the parents, the translocation must have appeared de novo in the present patient. METHODS: Spermatozoa were analysed under light and transmission electron microscopy (TEM). Fluorescence in-situ hybridization (FISH) was performed on the lymphocyte karyotype. Aneuploidy frequencies of chromosomes 18, X and Y in sperm nuclei, not involved in the translocation, were investigated using three-colour FISH. Dual- colour FISH was used to evaluate segregation of chromosomes 10, 15 in decondensed sperm nuclei. Moreover, three-colour FISH, using telomeric probes for chromosomes 10, 15 was performed in order to distinguish balanced and unbalanced gametes. RESULTS AND CONCLUSIONS: Overall, structural characteristics indicate general immaturity of the germinal cells. FISH sperm analysis detected an increase in chromosome 18 disomy (0.81%) suggesting an interchromosomal effect. A high frequency of diploidies, particularly 18,18,X,X and 18,18,X,Y, was also found. FISH segregation analysis for chromosomes 10, 15 indicated that 32.8% were balanced gametes, whereas 68.2% were unbalanced. Taken together, these data demonstrate in a male carrier of a reciprocal translocation t(10;15) the presence of diffuse ultrastructural sperm alterations and a high frequency of sperm aneuploidies. The existence of a correlation among these factors is proposed.     

Diagnosing and preventing inherited disease: Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations

The discovery of nucleated erythrocytes in maternal circulationprovides a potential source for non-invasive prenatal diagnosis.We have evaluated the use of a three-stage procedure to determinethe number of cells that are of fetal rather than maternal origin.First, monoclonal antibodies specific for CD45 and CD14 wereused in conjunction with a magnetic (MACS) column to depleteunwanted leukocytes from maternal blood. This was followed bya positive MACS enrichment for nucleated erythrocytes, usingan anti-CD71 (transferrin receptor) monoclonal antibody. Todiscriminate between fetal nucleated erythrocytes and thoseof maternal origin, enriched fractions were simultaneously stainedwith an anti-fetal haemoglobin (HbF) antibody and hybridizedwith probes specific for X and Y chromosomes. Samples were thensubjected to blind analysis along with negative control samplesfrom non-pregnant volunteers. Using this dual analysis, we wereable to determine that less than one nucleated erythrocyte perml of maternal blood was of fetal origin. Small numbers of thesefetal cells were found in 87.5% of pregnancies, ranging from6 to 35 weeks gestational age. Comparison of HbF and X/Y probedata also suggests that the fetal cells are less suitable forfluorescence in-situ hybridization (FISH) analysis than similarpreparations from other sources.     

Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility

BACKGROUND: Sperm motility evaluation is associated with fertility in IVF programmes. The visual estimation of sperm motility is extremely subjective. Hence, alternative methods are required. Among them, determination of mitochondrial membrane potential (Deltapsi(m)) changes of spermatozoa using potentiometric dyes may be a reliable test to determine sperm quality. However, the use of the potentiometric dyes in sperm samples has not been compared. METHODS: We have studied sperm samples from 28 infertile patients enrolled in an IVF programme in flow cytometry after staining of spermatozoa with four commonly used potentiometric dyes. Sperm motility was evaluated visually. RESULTS: As expected, JC-1 seems to detect specifically Deltapsi(m) changes, CMX-Ros, DiOC(6)(3) and TMRE fluorescence is easily analysed and the latter three fluorochromes are particularly suitable for multiparametric staining. Irrespective of the Deltapsi(m)-dependent fluorochromes used to stain spermatozoa, a positive correlation was found between the percentage of Deltapsi(m)(high) cells and forward motility and also with high fertilization rates after IVF. CONCLUSION: The four fluorochromes may be useful for evaluation of sperm samples from infertile patients. The choice of the potentiometric dyes will depend on their fluorescence characteristics in order to use them in combination with other fluorescent markers.     

Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome

BACKGROUND: A large percentage of in-vitro generated cleavage stage human embryos are chromosomally mosaic, consisting of both normal (diploid) and abnormal (non-diploid) cells. The present study characterized mosaicism at each stage of cleavage division and examined its effect on preimplantation development in vitro. METHODS: A total of 216 normally fertilized (two-pronucleate) embryos which were not selected for transfer to the patients were analysed for chromosomal abnormalities using multi-colour fluorescence in-situ hybridization DNA probes specific for three to five of nine different chromosomes (X, Y, 2, 7, 13, 16, 18, 21, 22). RESULTS: Overall, 48.1% of embryos were mosaic. The frequency of mosaic embryos increased from 15.2 to 49.4 to 58.1%, from the 2-4-cell to 5-8-cell to morula stages respectively, and the types of non-diploid cells detected were mostly aneuploid or chaotic. The incidence of mosaicism at the blastocyst stage was 90.9%; however, most of the mosaicism comprised diploid and polyploid cells. Arrested mosaic embryos had a higher incidence of chaotic abnormalities, and higher proportions of abnormal cells compared with the non-arrested group. CONCLUSIONS: Post-zygotic errors leading to mosaicism may occur, and persist throughout preimplantation development in vitro. Our results suggest that mosaicism involving multiple chromosomal imbalances and/or imbalances affecting a high proportion of cells in an embryo appear to impair development to the blastocyst stage.     

Genetics: Constitution of semen samples from XYY and XXY males as analysed by in-situ hybridization

A brightfield microscopical in-situ hybridization (ISH) techniquewas applied to semen samples of two 47,XYY males, one 46,XY/47,XXYmale with fertility problems, and two normal 46,XY men, whoserved as controls. The use of a standardized nuclear DNA decondensationmethod, together with double-target ISH and morphological staining,allowed an accurate study of the sex chromosomal content andmorphology of spermatozoa. In the males carrying an extra sexchromosome, we detected X- and Y-bearing spermatozoa in a ratiowhich did not differ significantly from the .1:1 ratio foundin normal males. Aneuploidy for the sex chromosomes was foundin 15% of the spermatozoa of both XYY males and in 3% of theXXY male. The most striking finding was the relatively low percentageof spermatozoa in these patients, with an average of 65% inthe XYY males and 84% in the XXY male. The other cells representedimmature germ cells (IGC), including spermatogonia and spermatocytesarrested at various stages of spermatogenesis. Apparently, inXYY or XXY men, these IGC are shed into the semen to an increasedextent as compared to normal, fertile men. The sex chromosomeconstitution of these IGC was heterogeneous. However, the findingthat the majority of spermatozoa in semen of 47,XYY and 47,XXYmales carried a single sex chromosome strengthens the hypothesisthat a 46,XY germ cell line must be present, apparently witha proliferative advantage over the 47,XYY or 47,XXY cells.     

Genetics: The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility

The results and rationale of using testicular and epididymalspermatozoa with intracytoplasmic sperm injection (ICSI) forsevere cases of male infertility are reviewed. A total of 72consecutive microsurgical epididymal sperm aspiration (MESA)cases were performed for congenital absence of the vas (CAV)and for irreparable obstructive azoospermia. ICSI was used toobtain normal embryos for transfer and fertilization in 90%of the cases. The overall fertilization rate was 46% with anormal cleavage rate of 68%. The pregnancy and delivery ratesper transfer were 58 and 37% respectively. The delivery rateper cycle was 33%. In many cases, no epididymal spermatozoawere available and so testicular sperm extraction (TESE) wasused for sperm retrieval. The transfer rate was lower with TESE(84 versus 96%) and the spermatozoa could not be frozen andsaved for use in future cycles. However, there was little differencein pregnancy rates using epidiymal or testicular spermatozoa.The results were not affected by whether the obstruction wascaused by CAV or failed vasoepididymostomy. Both fresh and frozenspermatozoa gave similar results; the only significant factorappeared to be the age of the female. Because of the consistentlygood results obtained using epididymal sperm with ICSI whencompared with conventional IVF, and the similarly good resultswith testicular tissue spermatozoa, ICSI is mandatory for allfuture MESA patients. All CAV patients and their partners shouldbe offered genetic screening for cystic fibrosis; hence pre-implantationembryo diagnosis should be available in any full service MESAprogramme. It is now clear that even with non-obstructive azoospermia,e.g. Sertoli-cell only, or maturation arrest, there are usuallysome small foci of spermatogenesis which allow TESE with ICSIto be carried out. This means that even in men with azoospermiadue to absence of spermatogenesis or to a block in meiosis,there are usually a few spermatozoa available in the testesthat are adequate for successful ICSI. Finally, it is likelythat some forms of severe male factor infertility are geneticallytransmitted and although ICSI offspring have been shown to becompletely normal, it is possible that the sons of these infertilecouples will also require ICSI when they grow up and wish tohave a family.     

DNA and chromatin structure: Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the acurate and objectiveevaluation of sperm chromatin condensation and chromatin stabilityof sperm nuclei. It was also possible to determine the influenceof incubation on sperm chromatin. Different types of spermatozoawere studied: unprocessed spermatozoa at 1 and 45 min afterejaculation, after swim-up (migrated), spermatozoa incubatedfor 6 h in non-capacitating conditions (aged), or in B2 medium(capacitated) or B2 medium followed 1 h later with A23187 (reacted).All types of spermatozoa were analysed before and after treatmentwith various decondensation agents: sodium dodecyl sulphate(SDS), SDS plus EDTA and SDS plus disulphide-reducing agent[dithiotreitol (DTT)). Sperm nuclei were enzymatically isolatedand stained with propidium iodide. Three flow cytometric parameterswere then measured: forward light scatter (cellular size), sidelight scatter (cellular complexity) and fluorescence (uptakeof propidium iodide). Fluorescence was the most suitable parameterto study the degree of condensation and resistance to decondensationof DNA in the spermatozoa. Unprocessed spermatozoa 1 min afterejaculation underwent decondensation by all assessed treatments(anionic detergent, chelating or disulphidereducing agents).Unprocessed spermatozoa 45 min after ejaculation and migratedspermatozoa did not undergo decondensation with SDS treatment,but decondensation occurred after treatment with SDS + EDTAor SDS + DTT. Spermatozoa incubated for 6 h under both non-capacitating(aged spermatozoa) and capacitating conditions (capacitatedspermatozoa) and reacted spermatozoa were decondensed only aftertreatment with SDS + DTT. In conclusion, the post-ejaculationand incubation time have to be taken into account when clinicalinterpretation of the effect of different treatments on spermchromatin condensation is made.     

Andrology: Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry

Acrosomal status and viability were evaluated simultaneouslyon human spermatozoa using flow cytometry. Samples were dividedinto three aliquots and randomly assigned to one of three treatments:(i) cryopreservation; (ii) 10 µM calcium ionophore [A23187in dimethylsulphoxide (DMSO)] or (iii) DMSO alone (control).Acrosomal status was evaluated using monoclonal antibodies recognizingMH61 and CD46, respectively. Fluorescein-conjugated goat anti-mouseimmunoglobulin (IgG) was used as a second antibody. Sperm viabilitywas assessed using Hoechst 33258 (H258) exclusion. The followingfactors were analysed: (i) the specificity of the monoclonalantibodies for the human acrosome; (ii) the relative effectivenessof flow cytometry and direct fluorescent microscopy scoringand (iii) the acrosomal status and viability of the control,ionophore-treated, and cryopreserved spermatozoa. Across alltreatments, the MH61 and CD46 monoclonal antibodies resultedin acrosomal status values (acrosome-reacted/viable spermatozoa)which were not significantly different (P > 0.05): control,1.0 ± 0.3% and 1.5 ± 0.6% (mean ± SEM);A23187, 42.8 ± 3.5% and 38.1 ± 3.5%; cryopreserved,8.2 ± 2.0% and 9.9 ± 1.3%; respectively. However,acrosomal status among treatments differed significantly (P< 0.01). Flow cytometric and direct fluorescent microscopyassessments were significantly correlated (r² = 0.96, P <0.01). These results indicate that flow cytometry, using anacrosome-specific monoclonal antibody and a supravital dye,provides an objective and efficient method to evaluate humansperm acrosomal and viability status simultaneously.     

Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes

Dual fluorescent in-situ hybridization (FISH) using X and Ychromosome specific probes has been used to identify the sexof human embryos for preimplantation diagnosis of X-linked disease.With a modified spreading method and directly labelled fluorescentDNA probes, we have examined the possibility of reducing thetime of the FISH procedure from 7 to 2 h. A total of 17 normallyfertilized human embryos were disaggregated and 98 intact blastomeresobtained. The spreading efficiency was 96% and FISH signalswere obtained from 97% of nuclei. In all cases, sibling blastomeresfrom the same embryo were the same sex. Mosaicism was observedin some embryos. Five cells which lysed during the dis–aggregationprocess were spread to determine whether FISH was possible inthese cells, but in all cases the morphology of the nuclei waspoor and multiple signals were observed so that reliable diagnosisof sex was not possible. The data reported here confirm thatby using an improved spreading method in combination with directlylabelled DNA probes, we have increased the efficiency and reducedthe time required for sexing embryos for preimplantation diagnosisof X-linked disease.     

Diagnosis of CMT1A duplications and HNPP deletions by interphase FISH: Implications for testing in the cytogenetics laboratory

Charcot-Marie-Tooth (CMT) disease type 1A is an inherited peripheral neuropathy characterized by slowly progressive distal muscle wasting and weakness, decreased nerve conduction velocities, and genetic linkage to 17p12. Most (>98%) CMT1A cases are caused by a DNA duplication of a 1.5-Mb region in 17p12 containing the PMP22 gene. The reciprocal product of the CMT1A duplication is a 1.5-Mb deletion which causes hereditary neuropathy with liability to pressure palsies (HNPP). The most informative current diagnostic testing requires pulsed-field gel electrophoresis to detect DNA rearrangement-specific junction fragments. We investigated the use of interphase FISH for the detection of duplications and deletions for these disorders in the clinical molecular cytogenetics laboratory. Established cell lines or blood specimens from 23 individuals with known molecular diagnoses and 10 controls were obtained and scored using a two-color FISH assay. At least 70% of CMT1A cells displayed three signals consistent with duplications. Using this minimum expected percentile to make a CMT1A duplication diagnosis, all patients with CMT1A showed a range of 71–92% of cells displaying at least three signals. Of the HNPP cases, 88% of cells displayed only one hybridization signal, consistent with deletions. The PMP22 locus from normal control individuals displayed a duplication pattern in ∼9% of cells, interpreted as replication of this locus. The percentage of cells showing replication was significantly lower than in those cells displaying true duplications. We conclude that FISH can be reliably used to diagnose CMT1A and HNPP in the clinical cytogenetics laboratory and to readily distinguish the DNA rearrangements associated with these disorders from individuals without duplication or deletion of the PMP22 locus. Am. J. Med. Genet. 69:325–331, 1997. © 1997 Wiley-Liss, Inc.     

Potential use of human interleukin 2 as an adjunct for the therapy of neoplasia, immunodeficiency and infectious disease

Experimental and clinical systems have been developed to analyze both the in vitro and in vivo effects of highly purified interleukin 2 (IL2) as a potential therapeutic adjunct for neoplasia, immunodeficiency and infectious disease. Animal models, clinical in vitro studies, as well as preliminary clinical trials of IL2 in vivo show that this lymphokine may provide a necessary signal for the activation of thymus-derived T cells in immunologically-compromised hosts.     

The implementation of an enhanced clinical model to improve the diagnostic yield of exome sequencing for patients with a rare genetic disease: A Canadian experience

The introduction of clinical exome sequencing (ES) has provided a unique opportunity to decrease the diagnostic odyssey for patients living with a rare genetic disease (RGD). ES has been shown to provide a diagnosis in 29%–57% of patients with a suspected RGD, with as many as 70% remaining undiagnosed. There is a need to advance the clinical model of care by more formally integrating approaches that were previously considered research into an enhanced diagnostic workflow. We developed an Exome Clinic, which set out to evaluate a workflow for improving the diagnostic yield of ES for patients with an undiagnosed RGD. Here, we report the outcomes of 47 families who underwent clinical ES in the first year of the clinic. The diagnostic yield from clinical ES was 40% (19/47). Families who remained undiagnosed after ES had the opportunity for follow-up studies that included phenotyping and candidate variant segregation in relatives, genomic matchmaking, and ES reanalysis. This enhanced diagnostic workflow increased the diagnostic yield to 55% (26/47), predominantly through the resolution of variants and genes of uncertain significance. We advocate that this approach be integrated into mainstream clinical practice and highlight the importance of a coordinated translational approach for patients with RGD.     

The impact of genetic testing for Crohn's disease, risk magnitude and graphical format on motivation to stop smoking: an experimental analogue study

Genetic tests may motivate risk-reducing behaviour more than other types of tests because they generate higher risk magnitudes and because their results have high personal relevance. To date, trial designs have not allowed the disentangling of the effects of these two factors. This analogue study examines the independent impacts of risk magnitude and provenance, and of risk display type, on motivation to quit smoking. A total of 180 smokers were randomly allocated to one of the 18 Crohn's disease risk vignettes in a 3 (risk provenance: family history. genetic test mutation positive. genetic test mutation negative) × 3 (risk magnitude: 3%, 6%, 50%) × 2 (display: grouped or dispersed icons) design. The 50% group had significantly higher intentions to quit than the 3% group. A significant risk provenance × magnitude interaction showed that participants in 50% or 6% groups were equally motivated, regardless of risk provenance, while participants in the 3% group had higher intentions associated with a mutation negative result than with a result based on family history alone. Grouped icon displays were more motivating than the dispersed icons. Using genetic tests to estimate risks of common complex conditions may not motivate behaviour change beyond the impact of the numerical risk estimates derived from such tests.