Santiago Munné

A fast and efficient method for simultaneous X and Y in situ hybridization of human blastomeres

PurposeTo use a 6-hr fluorescence in situ hybridization (FISH) procedure involving fluorochrome-labeled probes to determine the gender of blastomeres from arrested biopsied human embryos.ResultsSimultaneous detection of X and Y chromosomes was performed on 68 blastomeres with this technique. The FISH efficiency for gender determination was 95.5% (65/68). In addition, rehybridization with chromosome 8-specific probes was performed to determine the ploidy of blastomeres with more than two sex chromosomes.ConclusionThis technique offers an alternative to polymerase chain reaction for the preimplantation diagnosis of X-linked diseases and can also be used for ploidy assessment.

Reduction in signal overlap results in increased FISH efficiency: Implications for preimplantation genetic diagnosis

AbstractBackground: In the absence of mosaicism, one of the problems of preimplantation genetic diagnosis with FISH is the occurrence of false-negative hybridization results. It has been hypothesized that missing signals are produced by spatial overlap of signals.nMethods and Results: To investigate the relation among cell density, signal overlap, and hybridization signal detection, 371 blastomeres and 4556 lymphocytes were fixed in different cellular concentrations and analyzed by FISH using probes for chromosomes X, Y, and 18, and their nuclear diameters and FISH results scored. The results showed that the lower the diameter of fixed nuclei, the higher the number of signal overlaps and missing signals. The minimum number of missing signals was obtained when lymphocyte and blastomere nuclei had 40 or more microns in diameter after fixation and FISH. Since blastomeres were fixed individually, results with blastomeres were invariably better than with lymphocytes.

Sex determination of human embryos using the polymerase chain reaction and confirmation by fluorescence in situ hybridization

Objective To use fluorescence in situ hybridization to corroborate the polymerase chain reaction (PCR) preimplantation diagnosis of human embryos in three couples carrying a chromosome X-linked disease. Setting Clinical and research IVF laboratories. Patients Individuals undergoing preimplantation diagnosis. Results Four ETs were performed in couples undergoing preimplantation diagnosis by multiplex PCR or fluorescence in situ hybridization, resulting in the birth of two normal female twins. The result of another is pending. A total of 22 embryos were analyzed by PCR. Embryos that were diagnosed as being at risk of carrying the genetic abnormality (n = 8), embryos that failed diagnosis (n = 4), and genetically normal embryos that arrested development (n = 4) were further analyzed by fluorescence in situ hybridization. The sex of all 16 embryos was determined and confirmed the previous 12 preimplantation diagnoses by multiplex PCR. In addition, fluorescence in situ hybridization analysis allowed the detection of two aneuploid embryos, one XO and one XYY, previously diagnosed by PCR as a normal female and male. Two mosaics were also detected. Conclusion Polymerase chain reaction and fluorescence in situ hybridization are possible for preimplantation sex determination in cases of genetic sex-linked disease. Fluorescence in situ hybridization, however, supplies additional information about sex chromosome aneuploidy and is not susceptible to contamination or misdiagnosis of monosomy X.

Origin of single pronucleated human zygotes

PurposeDiploidy in embryos developing from single pronucleated zygotes can occur following parthenogenetic activation or by asynchronous inflation of pronuclei following fertilization. To distinguish between these two mechanisms, sexing was performed.ResultsThe presence of a Y chromosome in the embryo was considered proof that fertilization had occurred. Twenty-one dividing embryos originating from single pronucleated zygotes were analyzed using the polymerase chain reaction or fluorescence in situ hybridization. In total, 43% (9/21) of the embryos showed Y chromosomes.ConclusionIt can be extrapolated that more than 80% of them originated from fertilized eggs.

Atypical activation and fertilization patterns in humans

Abstract Abnormalities of fertilization resulting in suppression of second polar body formation, activation without sperm fusion and multiple sperm fusion occur frequently following assisted (intracytoplasmic sperm injection; ICSI) and IVF in the human. Their genetic status was investigated by multiprobe fluorescence in-situ hybridization (FISH) or polymerase chain reaction (PCR) on sibling blastomeres. IVF embryos with single pronuclei at the zygote stage were usually diploid and fertilized. Embryos derived from IVF with single pronuclei can be safely replaced back to the patient. These embryos develop following formation of a single or aggregated pronucleus, a process found to occur in sea urchins. Single pronucleate ICSI zygotes are usually activated but not fertilized. The parental status of individual pronuclei was investigated in dispermic embryos. It was found that the distal pronucleus was usually male in origin and that the sex ratio was restored in enucleated zygotes, however, dispermic embryos become mosaic. Genetic heterogeneity was not restored in enucleated dispermic embryos; none of them became truly diploid. Mosaicism, on the other hand, was not common among digynic ICSI embryos and any such mosaicism originated at the third cleavage division, a pattern which is similar in mosaic monospermic embryos. Most of the digynic embryos were triploid, indicating that the first division was bipolar in origin. Most digynic embryos from which a female pronucleus was removed became diploid and their genetic condition was considered normal. From this work it is concluded that the sperm centriole is active in the human oocyte, rendering most monospermic embryos, including those that are digynic, non-mosaic. Removal of a single male pronucleus will not revert dispermic embryos to a normal status because of the activity of extra sperm centrioles. Transfer of enucleated dispermic embryos or their use as models for embryonic development should be reconsidered.

The parental origin of the distal pronucleus in dispermic human zygotes.

The purpose of this investigation was to determine the parental origin of the pronucleus furthest from the second polar body (the distal pronucleus) in dispermic human zygotes. Intact dispermic embryos (n = 53) and those from which the distal pronucleus (n = 50) was removed at the zygote stage were biopsied after cleavage. Blastomeres were sexed using either coamplification of X and Y probes using a duplex polymerase chain reaction (PCR), or simultaneous fluorescence in situ hybridisation (FISH) with directly fluorochrome-labelled probes for chromosomes X, Y and 18. The ratio X/Y was determined in both groups of embryos by assessing a minimum of two blastomeres. If the pronuclei in dispermic zygotes are topographically in a fixed position, the X/Y ratio should change from 1:3 in dispermic embryos to 1:1 in enucleated ones. The ratio of embryos containing only an X chromosome and those with X as well as Y chromosomes in the intact dispermic zygotes was 1.0:2.3 which is similar to the theoretical ratio of 1:3. This ratio was 1.0:1.3 in dispermic zygotes from which the distal pronuclei were removed. This ratio is not significantly different from the 1:1 ratio based on a statistical analysis with a sample size of 50. These sex ratios would have been considered different if more than 200 enucleations had been performed. Although the ratio X/Y was altered following removal of distal pronuclei, suggesting frequent targeting of male pronuclei, accidental removal of the female pronucleus could not be excluded. This indicates that enucleation of dispermic zygotes could produce high yields of gynogenetic and androgenetic embryos for research purposes. Clinical application aimed at producing biparental zygotes may be hazardous, since mosaicism was common among enucleated embryos.

Aneuploidy in spermatozoa using fluorescence in situ hybridization

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