Preimplantation diagnosis: Sephadex filtration and human serum albumin gradients do not select spermatozoa by sex chromosome: a fluorescent in-situ hybridization study

Vidal, Francesca; Moragas, Marta; Català, Vicenç; Torelló, M.J.; Santaló, Josep; Calderón, Gloria; Giménez, Carles; Barri, P.N.; Egozcue, J.; Veiga, Anna

doi:10.1093/oxfordjournals.humrep.a137926

Cited by 85 publications

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“…To obtain sperm nuclei, an aliquot of frozen spermatozoa was thawed and washed in NaCl 0.9% to eliminate cryoprotectant. The sample was fixed and decondensed following Vidal et al 15 Slides were maintained at 7208C until hybridisation.…”

Section: Methodsmentioning

confidence: 99%

Meiotic segregation analysis in a t(4;8) carrier: comparison of FISH methods on sperm chromosome metaphases and interphase sperm nuclei

et al. 2001

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Meiotic segregation of a t(4;8)(q28;p23) translocation carrier was determined by two different methods to compare the final results. A total of 352 sperm chromosome complements, obtained after human-hamster in vitro fertilisation, were analysed by whole chromosome painting, and 6590 sperm heads were studied by fluorescence in situ hybridisation (FISH). Frequencies of alternate, adjacent I, adjacent II and 3 : 1 segregations were, for sperm chromosomes, 35.5, 33.2, 19.9 and 11.3% respectively. For sperm head analysis, results were 30.5, 28.5, 20.5 and 19.5% respectively. There were no statistically significant differences between the two methods with respect to the observed frequencies of sperm with balanced and unbalanced chromosome constitutions. Among unbalanced gametes, the methods differed only in the frequency of 3 : 1 segregation (w 2 , P50.0001). Different factors that could explain this result are discussed. To determine possible interchromosomal effects, multicolour FISH was used on sperm heads. Disomy rates of sex and 18 chromosomes were higher in the translocation carrier than in the control. The differences observed were statistically significant (P50.0001 for chromosomes X and 18, and P=0.0091 for chromosome Y). European Journal of Human Genetics (2001) 9, 395 ± 403.

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Section: Methodsmentioning

confidence: 99%

Meiotic segregation analysis in a t(4;8) carrier: comparison of FISH methods on sperm chromosome metaphases and interphase sperm nuclei

et al. 2001

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“…By using molecular techniques, mainly fluorescence in situ hybridization (FISH), it was demonstrated that albumin gradients [10,11], Sephadex columns [10,12,13] and swim-up [13,14] techniques do not significantly alter the Y-to X-bearing spermatozoa ratio. But the discontinuous Percoll gradients technique was reported to be the only method to alter the X-and Y-bearing sperm ratio [15,16].…”

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confidence: 99%

Assessment of Bovine X- and Y-bearing Spermatozoa in Fractions by Discontinuous Percoll Gradients with Rapid Fluorescence In Situ Hybridization

Kobayashi

Oguro

Uchida

et al. 2004

Journal of Reproduction and Development

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Abstract. This study was designed to apply the method of discontinuous Percoll gradients for sex preselection in bovine semen by using a current developed molecular technique, fluorescence in situ hybridization (FISH). In addition, we attempted to amplify the level of enrichment of X-or Y-bearing spermatozoa by treating for activating sperm motility performance with 10 mM caffeine. Bovine spermatozoa were fractionated on Percoll gradients into two major subpopulations of motile spermatozoa (bottom fraction) and weak motile spermatozoa (top fraction). The percentage of Ybearing spermatozoa in the top fraction (52.9%) slightly exceeded and that in the bottom fraction (44.3%) decreased significantly (P<0.001) compared with the theoretical ratio (50:50). Washing sperm with BO medium affected a deviation between the two sex populations, whereas semen activated with caffeine showed no difference in the percentage of X-and Y-bearing spermatozoa in both fractions compared with the theoretical ratio (50:50). These results show that the proportion of X-and Ybearing bovine spermatozoa can deviate after discontinuous Percoll gradients, although the proportion of X-and Y-bearing bovine spermatozoa was affected by sperm motility of the sample applied. Key words: Percoll separation, Semen sexing, FISH, Spermatozoa, Bovine (J. Reprod. Dev. 50: [463][464][465][466][467][468][469] 2004) ince the 1970s, several investigators have made a tt e m p t s t o s e p a r at e X -a n d Y -b e ar in g spermatozoa by means of various gradient techniques such as discontinuous albumin gradients [1, 2], Shepadex column s [2,3], discontinuous Percoll gradients [4,5] and the swim-up procedure [6]. None of these methods, however, have met with success sustainable and reproducible enough to achieve separation of Xand Y-bearing spermatozoa, and only flow cytometry has been proven to be effective [7]. One o f t h e p r o b l e m s a s s o c i a t e d w i t h r e s e a r c h attempting the separation of X-and Y-bearing spermatozoa has been the difficulty of validating the proportion of X-and Y-bearing spermatozoa within putatively separated sperm populations [8]. Early studies evaluated the efficiency of sperm separation by using quinacrine staining. Recent evidence has shown that the quinacrine method is unreliable and may not necessarily detect only the Y body at the Y chromosome in spermatozoa [9].Current development of DNA probes raised against the sex chromosome specific sequence gives

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“…Sperm nuclear decondensation was performed according to the method described by Vidal et al 20 In brief, the slides were exposed to 5 mmol l 21 dithiothreitol plus 0.1 mol l 21 Tris/1% (v/v) Triton-X for at least 12 min at 37 uC, and then immediately transferred to 23strength saline-sodium citrate buffer for 3 min at room temperature, and dehydrated in an ethanol series of 70%, 90% and 100% for 2 min each. The slides were then air-dried at room temperature.…”

Section: Methods Of Sperm Selectionmentioning

confidence: 99%

No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA

Mongkolchaipak¹,

Vutyavanich²

2013

Asian J Androl

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In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria, and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method (control), high magnification at 36650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6% vs. 1.7%; P50.032), with no significant difference in aneuploidy rate (0.8% vs 0.7%; P50.583), than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7% aneuploidy and 26.8% DNA fragmentation rates, respectively). In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference (0.9%) might not be clinically meaningful. Both methods were better than the conventional method of sperm selection. Keywords: aneuploidy; DNA fragmentation; hyaluronic acid binding; intracytoplasmic sperm injection (ICSI); intracytoplasmic morphologically selected sperm injection (IMSI); motile sperm organellar morphology examination; physiologic intracytoplasmic sperm injection INTRODUCTION Intracytoplasmic sperm injection (ICSI) is a very effective method for the treatment of severe male factor infertility. It allows the use of a single motile spermatozoon to fertilise an oocyte. 1 The technique is so effective that fertilization of an oocyte can be achieved even with the use of a spermatozoon with severe DNA fragmentation. 2 Men with severe male factor infertility are known to have a higher frequency of sperm aneuploidies and DNA fragmentation than fertile men. [3][4][5][6][7][8] Although the consequences of inseminating oocytes with abnormal spermatozoa are not known for certain, there is increasing evidence that such circumstances may cause poor fertilization, defective pre-implantation embryonic development, and high rates of miscarriage and morbidity in the offspring, including childhood cancer. 8 One major challenge ICSI facing is, therefore, the selection of the best spermatozoon for micro-insemination.Many studies have shown that intracytoplasmic morphologicallyselected sperm injection (IMSI) can significantly increase the fertilization and pregnancy rate of ICSI, while decreasing its abortion rate. [9][10][11] The disadvantage...

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Preimplantation diagnosis: Sephadex filtration and human serum albumin gradients do not select spermatozoa by sex chromosome: a fluorescent in-situ hybridization study

Cited by 85 publications

References 0 publications

Meiotic segregation analysis in a t(4;8) carrier: comparison of FISH methods on sperm chromosome metaphases and interphase sperm nuclei

Meiotic segregation analysis in a t(4;8) carrier: comparison of FISH methods on sperm chromosome metaphases and interphase sperm nuclei

Assessment of Bovine X- and Y-bearing Spermatozoa in Fractions by Discontinuous Percoll Gradients with Rapid Fluorescence In Situ Hybridization

No difference in high-magnification morphology and hyaluronic acid binding in the selection of euploid spermatozoa with intact DNA

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