B. Sèle

Achievement of meiosis in XXY germ cells : study of 543 sperm karyotypes from an XY/XXY mosaic patient

Human sperm chromosomes from a 46,XY/ 47,XXY male were obtained using the technique of in vitro penetration of zona-free hamster eggs. The analysis of 543 sperm complements shows a significantly increased incidence (0.9%) of hyperhaploid gonosomal 24,XY sets, with a lack of the expected corresponding gonosomal hypohaploidies, and a normal rate of autosomal non-disjunctions. These results support the suggestion that 47,XXY cells are able to go through meiosis and to form spermatozoa. Only 24,XY sperm chromosomal constitutions were observed suggesting a preferential pairing of homologous sex chromosomes in 47,XXY spermatocytes.

Increased incidence of hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a 46,XY/47,XXY male

Meiotic segregation of gonosomes from a 46,XY/47,XXY male was analysed by a three-colour fluorescence in situ hybridisation (FISH) procedure. This method allows the identification of hyperhaploid spermatozoa (with 24 chromosomes), diploid spermatozoa (with 46 chromosomes) and their meiotic origin (meiosis I or 11). Alpha satellite DNA probes specific for chromosomes X, Y and 1 were observed on 27,097 sperm nuclei. The proportions of X-and Y -bearing sperm were estimated to 52.78% and 43.88%, respectively. Disomy (24,XX, 24,YY, 24,X or Y,+1) and diploidy (46,XX, 46,YY, 46,XY) frequencies were close to those obtained from control sperm, whereas the frequency of hyperhaploid 24,XY spermatozoa (2.09%) was significantly increased compared with controls (0.36%). These results support the hypothesis that a few 47,XXY germ cells would be able to complete meiosis and to produce mature spermatozoa.

Sperm nuclei analysis of a Robertsonian t(14q21q) carrier, by FISH, using three plasmids and two YAC probes

The meiotic segregation of chromosomes 14 and 21 was analysed in 1116 spermatozoa from an oligoasthenospermic carrier of a Robsertsonian translocation t(14q21q), and in 16 392 spermatozoa from a control donor, using two-colour fluorescence in situ hybridisation (FISH). Two YAC probes (cloned in yeast artificial chromosomes) specific for regions on the long arms of these chromosomes were co-hybridised. Of the spermatozoa, 12% were unbalanced, resulting from adjacent segregations. Chromosomes X, Y and 1 were also simultaneously detected in 1335 spermatozoa from the same carrier. Whereas gonosomal disomy rates were not significantly different from those of the control donors, disomy 1 were slightly but significantly increased to 0.7%. The diploidy rate was also slightly increased to approximately 1% in the translocation carrier.

Meiotic behaviour of sex chromosomes investigated by three-colour FISH on 35 142 sperm nuclei from two 47,XYY males

Abstract Meiotic segregation of sex chromosomes from two fertile 47,XYY men was analysed by a three-colour fluorescence in situ hybridisation procedure. This method allows the identification of hyperhaploidies (spermatozoa with 24 chromosomes) and diploidies (spermatozoa with 46 chromosomes), and their meiotic origin (meiosis I or II). Alpha-satellite probes specific for chromosomes X, Y and 1 were observed simultaneously in 35 142 sperm nuclei. For both 47,XYY men (24 315 sperm nuclei analysed from one male and 10 827 from the other one) the sex ratio differs from the expected 1:1 ratio (P < 0.001). The rates of disomic Y, diploid YY and diploid XY spermatozoa were increased for both 47,XYY men compared with control sperm (142 050 sperm nuclei analysed from five control men), whereas the rates of hyperhaploidy XY, disomy X and disomy 1 were not significantly different from those of control sperm. These results support the hypothesis that the extra Y chromosome is lost before meiosis with a proliferative advantage of the resulting 46,XY germ cells. Our observations also suggest that a few primary spermatocytes with two Y chromosomes are able to progress through meiosis and to produce Y-bearing sperm cells. A theoretical pairing of the three gonosomes in primary spermatocytes with an extra sex chromosome, compatible with active spermatogenesis, is proposed.

Meiotic segregation in males heterozygote for reciprocal translocations : analysis of sperm nuclei by two and three colour fluorescence in situ hybridization

The meiotic segregation of chromosomes was analysed in three reciprocal translocation carriers, using FISH on interphase spermatozoa. The segregation pattern was first studied in 27,844 spermatozoa from two siblings carrying the reciprocal translocation t(6;11)(q14;p14). Three centromeric probes, specific for chromosomes 6, 11 and 1, were simultaneously hybridized so that all centric fragments as well as the ploidy of each cell could be determined by three colour FISH. For both subjects, the respective frequencies of alternate/adjacent 1, adjacent 2, 3:1 and 4:0 segregation modes were 88%, 9%, 3+ and < 1%. In another reciprocal translocation t(2;14)(p23.1;q31), a two colour FISH analysis was performed on 4,610 spermatozoa, using a chromosome 2 centromeric probe and a YAC probe located on the centric fragment of chromosome 14. Frequencies of alternate/adjacent 1, adjacent 2, and 3:1 segregations were 89%, 5.2%, and 5.8% respectively. The segregation of chromosomes X, Y and 1 were also analyzed with three colour FISH on the spermatozoa from all three translocation carriers, in order to detect an interchromosomal effect. Aneuploidy rates for the X and Y chromosomes were found to be in the same range in the three translocation carriers and control donors, but disomy 1 rates were slightly increased in the translocation carriers.

Meiotic segregation of the X and Y chromosomes and chromosome 1 analyzed by three-color FISH in human interphase spermatozoa

Meiotic segregation of the X and Y chromosomes and chromosome 1 was analyzed by three-color fluorescence in situ hybridization (FISH) in 94,575 human interphase spermatozoa from four control subjects. More than 99% of the sperm cells were labeled. The proportions of X- and Y-bearing sperm were estimated to be 49.83% and 48.30%, respectively. The disomy rates were 0.04%, 0.009%, and 0.20% for the X and Y chromosomes and chromosome 1, respectively. Hyperhaploidy with an extra gonosome was found in 0.34% of spermatozoa, due to nondisjunction during meiosis I. The frequency of diploidy was 0.11% at meiosis I and 0.036% at meiosis II. Cohybridization of one autosomal and two gonosomal probes, in three-color FISH in interphase spermatozoa, seems to accurately discriminate diploidies from disomies, as well as the meiotic origin of gonosomal aneuploidies in sperm cells.

Chromosome analysis of spermatozoa from a male heterozygous for a 13;14 Robertsonian translocation

SummaryCytogenetic analysis of 78 spermatozoa from a man heterozygous for a t(13;14) Robertsonian translocation was performed. R banding was applied for chromosomal identification. Incidence of normal and balanced complements were respectively 50% and 41.3%. Six unbalanced complements (7.7%) were observed, resulting from adjacent segregation. Although alternate segregation is the most common mode of distribution, the possibility of producing unbalanced zygotes exists. The frequency of abnormalities unrelated to the translocation was 16.5% including 12.8% hypohaploïdy, 2.5% hyperhaploidy, and 1.2% of structural aberrations. An excess of t(13;14) X complements was observed (24 with X versus 14 with Y). This may result from the close association between trivalent (13;14) and X chromosome observed in the pachytene spermatocyte nucleus.

Male meiotic segregation of gonosomes analysed by two colour FISH in human interphase spermatozoa

Human meiotic segregation of X and Y chromosomes was simultaneously analysed by dual fluorescence in situ hybridization (FISH) on 10638 interphase spermatozoa from the same donor. A modified method for sperm decondensation ensured access of both X and Y probes to the sperm chromatin and a 99% hybridization efficiency. Expected sex ratios were obtained (49.30% haploidy X and 49.22% haploidy Y). The frequencies of meiotic II non-disjunctions for X and Y chromosomes (0.05%) were similar to those observed in sperm karyotypes after heterospecific fertilization of hamster eggs. In contrast, the frequency of XY bearing cells was significantly higher (0.42%). However, XY cells detected by FISH could either be diploid somatic cells, diploid germinal cells or hyperhaploid XY spermatozoa, the latter resulting from meiotic I non-disjunctions.

Human sperm chromosome analysis after microinjection into hamster oocytes

PurposeSubzonal sperm insemination (SUZI) into hamster oocytes was performed to establish the karyotypes of the fertilizing spermatozoa.MethodsSpermatozoa from two males with normal semen parameters were microinjected. Of 72 (52 + 20) analyzed sperm chromosome metaphases, only 1 (1.4%) was considered abnormal, showing a structural abnormality.ResultsNo hyperhaploidy was observed. Rates of sperm chromosomal abnormalities after microinjection were not higher than those reported previously using zona-free egg insemination, suggesting that the SUZI procedure per se does not increase sperm chromosomal abnormalities.ConclusionsThe use of subzonal insemination into hamster oocytes for the study of human sperm chromosomes in males with low sperm counts is discussed.

Syndrome de Klinefelter et AMP à l’aube de l’an 2000

RésuméParmi les anomalies cytogénétiques, le syndrome de Klinefelter est la plus fréquente des anomalies chromosomiques associées à une infertilité masculine. Il concerne environ un nouveau-né mâle sur 600. Sa fréquence est de 3% dans la population des hommes infertiles et près de 12% des sujets azoospermiques présentent un syndrome de Klinefelter. Il y a quelques années, la seule possibilité de procréation pour ces couples était de recourir au don de sperme.Depuis l’avènement des techniques de fécondationin vitro avec microinjection, il est possible d’obtenirin vitro une fécondation ovocytaire dans des cas d’oligozoospermie extrême. Par ailleurs, la possibilité de recueillir des spermatozoïdes chirurgicalement au niveau épididymaire ou testiculaire élargit les indications des infertilités pouvant bénéficier d’une AMP intraconjugale. Ainsi, il est actuellement possible de proposer une AMP intraconjugale à certains couples, dont le conjoint est porteur d’un syndrome de Klinefelter, dès lors que quelques spermatozoïdes mobiles peuvent être obtenus, soit dans l’éjaculat, soit au niveau testiculaire. Cependant, le recours à ces techniques dans le cas d’une aneuploïdie des cellules germinales pose le problème de la transmission de cette aneuploïdie à la descendance et du risque de stérilité de la descendance masculine. Ainsi, dans le cas du syndrome de Klinefelter, la transmission du chromosome X surnuméraire peut, en théorie, conduire à la naissance d’un garçon atteint de syndrome de Klinefelter ou d’une fille de caryotype 47XXX. Ce risque est directement lié au pourcentage de spermatozoïdes aneuploïdes 24XY ou 24XX. Ce pourcentage peut être évalué actuellement par la technique d’hybridationin situ en fluorescence (Fluorescent In Situ Hybridization ou FISH) sur spermatozoïdes et des résultats peu nombreux et très variables sont recensés dans la littérature.Nous présentons dans ce travail, les résultats obtenus pour un patient porteur d’un syndrome de Klinefelter à caryotype homogène et les données de la littérature concernant la ségrégation des chromosomes sexuels chez les patients proteurs de syndrome de Klinefelter. Dans le cas de notre patient, le taux de cellules aneuploïdes 24XY est environ dix fois plus élevé que le taux retrouvé pour les sujets témoins. Par ailleurs, l’analyse des données bibliographiques, apporte des éléments de réponses à quelques questions pratiques qui se posent lorsqu’on envisage une FIV avec microinjection chez ces patients:- existe-t-il des critères morphologiques de choix des spermatozoïdes à injecter rendant compte du risque d’aneuploïdie cellulaire?- le risque d’aneuploïdie augmente-t-il si l’on utilise des spermatozoïdes testiculaires? Par ailleurs, plusieurs naissances sont actuellement rapportées après FIV avec microinjection pour des patients porteurs de syndrome de Klinefelter. Enfin, dans le cadre du conseil génétique, la place du diagnostic prénatal et/ou préimplantatoire est à discuter.AbstractWith the recent advance in the techniques of assisted reproduction, it is now possible to overcome male infertility associated with severe oligozoospermia or azoospermia, with other alternatives than sperm donation. In fact, despite a deeply defective spermatogenesis, intracouplein vitro fertilization can be achieved nowadays with the help of intracytoplasmic spem injection. Spermatozoa from ejaculate or testicular biopsy can be used. Klinefelter’s syndrome defined by a somatic karyotype 47,XXY or 46, XY/47,XXY, is one of the most common sex chromosomal abnormalities in human, with an incidence of about 3% of infertile men. In most cases, a deeply defective spermatogenesis is associated with this chromosomal abnormality. Intracytoplasmic sperm injection can be proposed to these patients. However, one should then consider the risk of transmitting an aneuploidy involving sex chomosomes.We reprot here the study of sex chromosome segregation for a patient carrying a nonmosaïc Klinefelter’s syndrome (47, XXY), with oligozoospermia and who asked for intracouplein vitro fertilization.Material and MethodsThe patient is a 33 year old male, with a 47, XXY karyotype (cytogenetical investigation of 16 metaphases). Semen analysis revealed a severe oligozoospermia. (spermatozoa ≪1×106/mL) and asthenozoospermia (60% of decreased motiliy), for an ejaculate volume of 1.8mL.Three-colourIn Situ Hybridization was performed on spermatozoa recovered from his cryopreserved semen, in order to simultaneously detect the chromosome X, Y and one with specific appropriate probes. Semen from two 23 year old men were also analyzed as controls.Results502 spermatozoa were analyzed from the patient and about 10,000 from the controls. There was an increase of about ten times of the percentage of hyperhaploïd (24XY) spermatozoa in the semen of the Klinefelter patient compared to the controls.Discussion and conlustionIn a general view of IVF-ICSI practice in Klinefelter patients, we also discuss here several practical points such as (i) is there any morphological criteria which may prevent from injecting an aneuploid spermatozoa, (ii) is the risk of aneuploidy higher when using testicular spermatozoa than when using ejaculated spermatozoa, (iii) what do we know about the offspring obtained by IVF-ICSI in Klinefelter patients and (iv) when should prenatal and/or preimplantatory genetical diagnosisbe proposed in this particular context.