Frequent structural chromosome aberrations in immotile human sperm exposed to culture media

BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.     

A cytogenetic study of in-vitro matured murine oocytes after ICSI by human sperm

BACKGROUND: The purpose of this study was to investigate the chromosomal complement and developmental potential of in-vitro matured murine oocytes following ICSI by human sperm. METHODS: Heterologous ICSI fertilization between mouse oocytes and human sperm was employed in order to overcome the reduced fertilization rates observed after conventional IVF due to zona hardening during in-vitro maturation, and to assess separately maternal and paternal chromosome complements. Cytogenetic analyses were performed in four types of oocytes: (i) in-vitro matured metaphase II (MII) oocytes; (ii) in-vivo matured MII oocytes; (iii) in-vitro matured oocytes after ICSI; (iv) in-vivo matured oocytes after ICSI. RESULTS: Activation rates after ICSI of in-vitro matured oocytes was lower than that of in-vivo matured oocytes (69.9 versus 97.2%, P < 0.01), and premature chromosomal condensation was only observed in in-vitro matured oocytes. However, there were no significant differences in developmental rates after successful activation between in-vivo and in-vitro matured ICSI oocytes (69.7 versus 76.6%). The incidences of aneuploidy and structural aberrations were similar between the ICSI embryos and non-ICSI (MII) oocytes. Furthermore, the frequency of chromosomal aberrations was not associated with in-vitro or in-vivo maturation. Similar analyses of paternal chromosomes indicated that there were no significant differences in the incidence of chromosomal aberrations between the embryos derived from in-vitro and in-vivo matured oocytes. CONCLUSIONS: These results suggest that in-vitro matured oocytes following ICSI do not lead to an increase in the frequency of aneuploidy and structural aberrations when human sperm are injected into mouse oocytes.     

A novel method for chromosome analysis of human sperm using enucleated mouse oocytes

BACKGROUND: Mouse oocytes can be used in conjunction with intracytoplasmic sperm injection (ICSI) as a technique to permit chromosomal analysis of human sperm. However, chromosomes derived both from the human sperm and the mouse oocyte appear simultaneously following ICSI. The present study focused on evaluating whether or not previously enucleated mouse oocytes are usable for the analysis of human sperm chromosomes. METHODS: The metaphase chromosome-spindle complex was removed from a mouse oocyte. Human sperm from a donor with proven fertility were injected into mouse enucleated oocytes or intact oocytes. The presence of pronuclei in the oocytes was confirmed approximately 7-11 h after ICSI, and the oocytes were then fixed so that the nuclei could be observed as chromosome samples at 15-16 h after ICSI. RESULTS: The formation rate of one pronucleus in enucleated oocytes after ICSI was 93.9% (186/198) while that of two pronuclei in intact oocytes after ICSI was 85.4% (88/103). The appearance rate of metaphase chromosomes of human sperm in the enucleated oocytes, 89.4% (160/179), was significantly higher than that in intact oocytes, 78.7% (74/94) (P = 0.017). CONCLUSIONS: An efficient ICSI method using enucleated mouse oocytes was established to allow the visualization of the human sperm chromosome complement without the risk of confusion with mouse oocyte chromosomes.     

Fertilization and early embryology: The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection

Implementation of intracytoplasmic sperm injection (ICSI) inhuman in-vitro fertilization (IVF) has highlighted the needfor information about the risk of nuclear spindle damage causedby this procedure. For this purpose we studied the final productsof oocyte meiosis at the first cleavage division of multipronuclearzygotes arising after ICSI, and compared the results with abnormallyfertilized oocytes after conventional in-vitro insemination.Of 37 successfully analysed tripronuclear zygotes, 18 had threeindividual metaphases. Abnormal complements of 11 zygotes inthis group indicated that non-disjunction occurred predominantlyat the second meiotic division of the oocytes. Nine of the 37tripronuclear zygotes exhibited two individual metaphases. Sevenwere abnormal and there were some indications that non-disjunctiontook place during oocyte meiosis. Of the 37 tripronuclear zygotes,10 had a single metaphase and three showed an aneuploid numberof chromosomes. The overall rate of aneuploidy among tripronuclearmicroinjected zygotes was 56.7%. In addition, seven zygoteswith more than three pronuclei arising after ICSI displayedseverely depleted chromosome complements. The incidence of non-disjunctionin oocytes fertilized by conventional in-vitro inseminationwas significantly lower (20.0%, P < 0.01), since only fourzygotes had an aneuploid number of chromosomes. Our findingssuggest that ICSI might interfere with regular chromosome segregationat the second meiotic division of the oocytes.     

Fertilization and early embryology: Cytogenetic and fluorescent in-situ hybridization chromosomal studies on in-vitro fertilized and intracytoplasmic sperm injected 'failed-fertilized' human oocytes

This study was undertaken to establish baseline data on thechromosomal status of ‘failed-fertilized’ oocytesderived from in-vitro fertilization (IVF) or intracytoplasmicsperm injection (ICSI) procedures. A cytogenetic analysis wasundertaken on 162 IVF and 51 ICSI oocytes. In all, 82.1% (133/162)of the IVF and 78.4% (40/51) of the ICSI oocytes had metaphaseII (Mil) plates, of which 50.4% of the IVF and 47.5% of theICSI oocytes were analysed further. Chromosomes of the G-group(21–22) were identified with the majority of the anomalies.No overall significant difference in the aneuploidy rate wasfound for the IVF (37.3%) or ICSI (31.6%) oocytes, or with maternalage. However, chromosome anomalies, e.g. diploidy, fragmentedand broken chromatids, single sperm and oocyte chromatids, werefound in oocytes from IVF patients aged >36 years and inthe ICSI oocytes throughout the maternal age range (31–38years). The status of the polar body chromatin indicated thatthere was no overall significant difference in the maturationof the IVF and ICSI oocytes. Evidence of successful sperm deliverywas found in 72.5% (37/51) of the ICSI failed-fertilized oocytes.In this group there was a significant increase in the incidenceof premature chromosome condensation: 19.6% (10/51) containedsperm chromosomes, 7.8% (4/51) had swollen sperm heads, andthe remaining 45.0% had condensed sperm heads. The presenceof both sperm and Mil oocyte chromosomes was found in 19.6%(10/51) of the ICSI and 8.6% (14/162) of the IVF failed-fertilizedoocytes. Specific fluorescent in-situ hybridization DNA probeswere used to re-analyse the chromosomes of karyotyped ‘failed-fertilized’IVF oocytes and, for the first time, applied to the karyotypedchromosomes of failed-fertilized ICSI oocytes. The hybridizationefficiency was 86–95% for the centromere probe and 100%for probes 21 and 18.     

Cytogenetic abnormalities of unfertilized oocytes generated from in- vitro fertilization and intracytoplasmic sperm injection: a double- blind study

In the present study we have assessed the cytogenetic abnormalities of unfertilized oocytes from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programmes during a one year period (July 1995 to July 1996) with the cytogenetic analysis being carried out in a double-blind manner. A total of 88 unfertilized ICSI and 85 unfertilized IVF oocytes were used for the study and of these 51 and 62 oocytes, in each respective group, were suitable for analysis. The haploidy, diploidy and aneuploidy rates between ICSI (62.7, 7.8 and 5.9%) and IVF (61.3, 9.7 and 14.5%) groups were similar. A significant inter-patient variation in the incidence of hypohaploidy was observed within the IVF group. Chromosomal fragmentation or breakage was observed at a similar rate in both groups of unfertilized oocytes (23.5 and 14.5% for ICSI and IVF respectively). A significantly higher proportion of ICSI oocytes contained sperm nuclei (27/51, 52.9%) than did IVF oocytes (20/62, 32.3%, P < 0.01). The distribution and state of sperm head chromatin in relation to oocyte chromosomal complement was studied in both groups. ICSI oocytes contained decondensed or swollen sperm nuclei in association with haploid oocyte chromosomes (12/27, 44.4%) or condensed sperm heads in oocytes showing no chromosomal complements (7/27, 25.9%). In IVF oocytes sperm heads were either arrested in the condensed state (5/20, 25%), metaphase stage (3/20, 15%) or had undergone premature chromosome condensation (PCC; 6/20, 30%) in association with haploid oocyte chromosomes. The incidence of PCC was similar in the two groups. A marked variation in the incidence of total chromosomal abnormality was observed between patients within both ICSI (0-75%) and IVF (0-71%) groups indicating a possible similarity in oocyte quality between the majority of male factor and tubal infertility patients. The type of sperm used in the two fertilization procedures showed an increased incidence of chromosomal breakage with ICSI-MESA (microepididymal sperm aspiration) spermatozoa (4/6, 67%) compared to the ICSI-ejaculated (6/35, 17.1%; P < 0.05), ICSI-testicular biopsy (2/10, 20%) and IVF-normospermic (9/62, 14.5%; P < 0.01) spermatozoa. Chromosomal fragmentation may be associated with the degree of difficulty experienced at sperm injection, especially with sperm retrieved from the reproductive tract. Thus chromosomal fragmentation in ICSI may need further investigation using a larger sample size in order to assess the possible causative factors.      

Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa

An infertile couple was referred for intracytoplasmic sperm injection (ICSI) because of primary infertility and oligoasthenoteratozoospermia (OAT) in the male. It was observed that although the sperm cells presented with an unusual head size and multiple tails they were able to fertilize the oocytes after ICSI. Subsequent molecular cytogenetic analysis demonstrated de-novo chromosome abnormalities in virtually all sperm cells with 40% diploidy and 24% triploidy in addition to aneuploidy for the sex chromosomes.      

Cytogenetic study of human oocytes uncleaved after in-vitro fertilization

Chromosome analysis of oocytes uncleaved after IVF allows the cause of the failure of cleavage to be determined and shows the incidence of chromosome disorders among human oocytes. A total of 198 uncleaved oocytes fixed 40 h after insemination were successfully analysed according to Tarkowski's air-drying method: 78.3% were unfertilized and arrested in metaphase II. Among them, 70% were normal (23,X) and 30% aneuploid (16% were hypohaploid, 14% were hyperhaploid). The incidence of chromosome breaks was 18%. In 12.1% of the oocytes, sperm chromosome condensation appeared premature usually in the G1 phase. This was especially observed in idiopathic infertility (7% of fertilized oocytes versus 2% in tubal infertility cases). In 8.1% of the cases, chromosome analysis showed diploidy which may be interpreted by either an absence of extrusion or a reintrusion of the polar body or by first cleavage failure during mitosis. In 1% of the cases triploidy was observed. Our results show that the main reason for failure of cleavage is related to failure of fertilization (78.3%). However, premature condensation of sperm chromosomes at the G1 phase appears to be quite frequent. This may be involved in the aetiology of some cases of idiopathic infertility. Finally, the high rate of chromosomal disorders (30%) in human oocytes may explain the high rate of chromosomal abnormalities in preimplantation embryos.     

The high incidence of meiotic errors increases with decreased sperm count in severe male factor infertilities

BACKGROUND:The high frequency of aneuploidy sperm raises concerns that there may be an increased incidence of aneuploid offspring in ICSI programmes. In order to assess the role that chromosome complement plays in normal and abnormal fertility, detailed molecular cytogenetic studies must be done on sperm samples from men with normal and abnormal fertility. METHODS: To understand more clearly the cytogenetic make-up of sperm from oligoasthenoteratozoospermic (OAT) patients, multi-colour fluorescence in situ hybridization was used to determine numerical chromosome abnormalities. RESULTS: Increased aneuploidy frequencies for chromosomes 13, 18, 21, X and Y were detected in sperm from OAT patients. The frequencies of diploidy also increased. There were no differences in non-disjunction at meiosis I compared to meiosis II. Sperm count inversely correlated with the frequencies of diploidy, aneuploidies for chromosomes 13 and 21 in OAT patients. Twenty-two cycles of ICSI and 18 embryo transfers were performed in 20 couples. Only three cases achieved successful pregnancies. CONCLUSIONS: A higher incidence of meiotic errors and lower sperm counts was found in sperm from OAT patients.     

Andrology: Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes

The chromosome constitution of human spermatozoa was determinedafter injecting individual spermatozoa into mouse oocytes. Ofa total 279 eggs arrested at first cleavage metaphase, 200 (71.7%)were suitable for the analysis of sperm chromosomes. Incidencesof spermatozoa with numerical and structural chromosome aberrationswere 1.3 and 6.9% respectively in spermatozoa with normal headmorphology, showing values comparable with those found in previousstudies using the hamster oocyte-human sperm fusion system.The ratio of X- to Y-bearing spermatozoa did not differ significantlyfrom the expected 1:1 ratio. The incidence of structural chromosomeaberrations was about four times higher in spermatozoa withamorphous, round and elongated heads (26.1%) than in those withmorphologically normal heads, whereas the incidence of aneuploidywas not significantly different between the two groups. No increasein chromosome aberrations was found in spermatozoa with largeheads. The same was true for spermatozoa with small heads. Althoughthe sample size used in this study is rather small, the resultsnevertheless indicate that some morphological abnormalitiesin the sperm heads are associated with their chromosome defects.     

Cytogenetic analysis of human zygotes displaying three pronuclei and one polar body after intracytoplasmic sperm injection.

BACKGROUND: Digynic zygotes with three pronuclei and one polar body obtained after intracytoplasmic sperm injection (ICSI) were studied cytogenetically to elucidate the frequency and origin of chromosomal abnormalities at the earliest stage of conception. METHODS: Uncleaved, single-cell zygotes were incubated with podophyllotoxin and vinblastine and fixed by a gradual fixation air drying method. The chromosomes were stained with Giemsa. RESULTS: Twenty-two (50%) out of 44 informative zygotes revealed cytogenetic alterations, including aneuploidy (six cells, 13.6%), structural aberrations (10 cells, 22.7%) and combinations of numerical and structural abnormalities (two cells, 4.5%). In one case (2.3%), double aneuploidy or an effect of chromosomal translocation could not be distinguished and one zygote (2.3%) turned out tetraploid due to injection of a diploid spermatozoon. Two zygotes (4.5%) showed an irregular chromatid segregation between the two maternal complements. In completely analysable cells, the sex chromosome ratio XXX:XXY was 17:15. CONCLUSIONS: Digynic ICSI zygotes carry a high rate of cytogenetic abnormalities that obviously have been transmitted by the participating oocytes and spermatozoa. We also confirmed the previously reported, possibly ICSI-induced irregular oocyte chromatid segregation. The results suggest that aneuploidy in the oocytes must have been caused by predivision instead of non-disjunction.     

Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection1

The aim of this study was to determine why oocytes remain unfertilizedor develop three pronuclei after intracytoplasmic sperm injection(ICSI). Unfertilized and abnormally fertilized oocytes werefixed in glutaraldehyde,stained with Hoechst 33342 and examinedby fluorescence microscopy to identify oocyte, sperm and polarbody DNA.One-pronuclear oocytes were considered to be unfertilized.Atotal of 285 unfertilized oocytes were examined (104 ICSI cycles).Overall, 83% of these oocytes were not activated (still at metaphaseII) while 17% had activated and formed a single (female) pronucleus.About 66% of the unfertilized, metaphase II oocytes containeda swollen sperm head, indicating that the oocyte was correctlyinjected but had failed to activate and complete the secondmeiotic division. Premature chromosome condensation of the spermDNA was evident in 6% of these metaphase II oocytes (4% of theunfertilized oocytes). The swollen sperm head was located amongthe oocyte chromosomes in 5%of the metaphase II oocytes. Othercauses of failed fertilization in the metaphase II oocytes werethe failure of sperm head decondensation (11%) and ejectionof the spermatozoon from the oocyte (23%). A similar patternwas observed in one-pronuclear oocytes (52%, swollen sperm head;28%, intact, undecondensed sperm head; 20%, ejection of thespermatozoon), which indicates that asynchronous pronucleardevelopment does not explain the presence of one-pronuclearoocytes. A total of 41 threepronuclear oocytes were examinedand all had a single polar body, which indicates that the retentionof the second polar body leads to the formation of the thirdpronucleus.In conclusion, this study demonstrates that: (i)the major cause of fertilization failure after ICSI is failureof oocyte activation; (ii) ejection of the spermatozoon intothe perivitelline space is not a major cause of fertilizationfailure;and (iii) sperm head decondensation and oocyte activationafter ICSI can occur independently.     

Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI

The potential risk of transmitting chromosomally abnormal spermatozoa from infertile males into oocytes through intracytoplasmic sperm injection (ICSI) has prompted us to investigate the male pronuclei of tripronuclear zygotes (3PN) obtained after ICSI. To specify the type of anomalies, we used triple colour fluorescent in-situ hybridization (FISH) with three specific probes for chromosomes X, Y and 18. From a total of 163 paternal complements of ICSI-3PN zygotes, 90 (55.2%) had Y-chromosome signals. Eighty-three of these were normal, four had the disomy XY and three were diploid. In the remaining 73 ICSI-3PN zygotes without Y-chromosome signals, the origin of paternal pronuclei was extrapolated through chromosome constitution of the first polar body. Five anomalies were found in this group of zygotes, giving a total rate of numerical chromosome aberrations for fertilizing spermatozoa of 7.4%. In contrast to ICSI, only two disomies (1.5%) were found in the control group of IVF-3PN zygotes. Compared with the incidence of chromosome anomalies between paternal-derived pronuclei of ICSI- and IVF-3PN zygotes, the difference was statistically significant (P < 0.025). This study provides the first direct evidence of a higher incidence of numerical chromosome anomalies in sperm-fertilized human oocytes after ICSI.     

Andrology: Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa

When intracytoplasmic sperm injection (ICSI) is performed, itis important to know the capacity of sperm cells to activatethe oocytes, although knowledge of their ability to fuse withthe oocytes is not vital. Hamster oocytes are not suitable forthis purpose because they are easily activated by the injectionprocedure itself. We therefore investigated whether mouse oocytescould be used to assess the activation properties of human spermatozoa.Mouse oocytes were randomized for injection with initially motilespermatozoa, medium, heat-treated or salt-extracted spermmatozoa,and the survival and activation rates were examined. About halfof the mouse oocytes survived the intracytoplasmic injectionof a human sperm cell. Unlike hamster oocytes, the rate of activationprovoked by the injection procedure itself was acceptably low(20%), resembling in this respect the behaviour of human oocytes.Following the injection of initially motile human spermatozoa,all mouse oocytes were activated. The injection of heat-treatedor salt-extracted human spermatozoa resulted in activation ratesof 14 and 15% respectively, comparable with the results followingsham ICSI. These data support the hypothesis of a sperm-associatedoocyte activation factor. In most activated oocytes, the humansperm nucleus decondensed to form a male pronucleus. Cytogeneticanalysis at the first metaphase revealed that human sperm chromosomeswere able to undergo replication in a heterologous environment.From our data we concluded that human spermatozoa can be injectedsuccessfully into mouse oocytes, resulting in a reasonable survivalrate, and that mouse oocytes provide a useful model for boththe assessment of the sperm-associated oocyte activation factorand the cytogenetic analysis of human spermatozoa.     

The outcome of ICSI of immature MI oocytes and rescued in vitro matured MII oocytes

BACKGROUND: The use of immature oocytes is limited to cases where these are the only available oocytes, and they are usually only microinjected with sperm after having undergone maturation in vitro. This study compares the outcome of injection of sperm into metaphase I oocytes immediately after their denudation (MI) performed 2 h after their retrieval, with the outcome of injection of sperm into rescued in vitro matured metaphase II (IVM MII) oocytes after their short incubation in routine laboratory conditions. METHODS: ICSI was performed on MI oocytes, rescued IVM MII oocytes and on MI oocytes that were incubated but failed to extrude their first polar body (arrested IVM MI). Fertilization and cleavage rates were compared with those achieved in mature metaphase II oocytes (MII). RESULTS: ICSI of MI oocytes showed impaired performance compared with ICSI of rescued IVM MII oocytes and MII oocytes, in terms of oocyte degeneration rate (11 versus 6 versus 4%; P < 0.0001), fertilization rate (28 versus 44 versus 68%; P < 0.0001) and multipronucleated fertilization (10 versus 4 versus 4%; P < 0.01). The cleavage rate was lower in rescued IVM MII oocytes compared with MII oocytes (86 versus 95%; P < 0.01). Arrested IVM MI oocytes showed similar results to those of MI oocytes but had a lower cleavage rate (72 versus 96%; P < 0.01). CONCLUSIONS: The injection of rescued IVM MII oocytes is preferred to the injection of MI oocytes.     

Cytogenetic analysis of human oocytes and embryos in an in-vitro fertilization programme.

Oocytes (unfertilized and preovulatory) and embryos (normal and polypronuclear), which were donated to research by patients undergoing procedures of assisted reproductive treatment, were analysed for cytogenetic abnormalities. A total of 362 oocytes and embryos were analysed. The unfertilized oocytes with readable metaphases (53.4%) gave 25.2% chromosomal abnormality with diploidy being the main aberration observed. A high incidence of premature chromosome condensation (PCC) was observed and the incidence of PCC in oocytes exposed to colcemid was significantly higher (14/62, 22.6%) than in those not exposed to this treatment (3/41, 7.3%, P less than 0.05). When chromosomal anomalies and PCC in the unfertilized oocytes were correlated to various patient criteria such as stimulation regimen, number of human menopausal gonadotrophin ampoules, peak oestradiol levels, age of patient and number of previous attempts, none of the criteria tested had any significant relationship to the incidence of chromosomal abnormality. However a significant increase in the incidence of PCC was noted in the gonadotrophin-releasing hormone (GnRH) 'flare' group (6/15, 40.0%) compared to the GnRH 'down-regulation' group (11/88, 12.5%). The incidence of chromosomal abnormalities among preovulatory oocytes was 16.7% and diploidy was the only abnormality noted. For embryos arising from two-pronuclear oocytes, the chromosomal constitution related mainly to embryo quality. The rate of chromosomal abnormality for apparently good quality embryos was 23.5% and for poor or fragmented embryos 83.3%. The majority (77.3%) of the readable metaphase plates for polypronuclear 1-cell and cleaved embryos showed grossly abnormal chromosome complements but 19% of the cleaved embryos contained sets of normal diploid chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of aneuploidy in human gametes: comparison between human sperm and oocytes

The frequency and distribution of aneuploidy was compared in 11,615 karyotyped human sperm and 772 karyotyped human oocytes to determine if all chromosomes are equally likely to be involved in aneuploid events or if some chromosomes are particularly susceptible to nondisjunction. The frequency of hypohaploidy and hyperhaploidy was compared among different chromosome groups and individual chromosomes for human sperm and oocytes. In general, hypohaploid chromosome complements were more frequent than hyperhaploid complements, in sperm and oocytes. The distribution of chromosome loss in the hypohaploid complements indicated that significantly fewer of the large chromosomes and significantly more of the small chromosomes were lost, suggesting that technical loss predominantly affects small chromosomes. A conservative estimate of aneuploidy (2 X hyperhaploidy) was approximately 3-4% in the human sperm and 18-19% in human oocytes. All chromosome groups were represented among hyperhaploid human sperm and oocytes. For human sperm, the observed frequency of hyperhaploidy equaled the expected frequency based on the assumption that the frequency of nondisjunction is equal for all chromosome groups, with two exceptions: group G and the sex chromosomes. Among individual chromosomes in human sperm, chromosomes 1 and 21 and the sex chromosomes had a significant excess of hyperhaploidy. For human oocytes, there were fewer hyperhaploid oocytes than expected for chromosome groups C and F and more than expected for chromosome groups D and G. Among individual chromosomes there was a significant excess for chromosome 21. These results indicate that all chromosomes are susceptible to nondisjunction but that chromosome 21 is particularly prone to aneuploidy in both human sperm and oocytes. They also demonstrate that sex chromosome aneuploidy is common in human sperm but not in human oocytes.     

Successful metaphase chromosome analysis of human elongated spermatids using mouse oocytes.

Human elongated spermatids from azoospermic patients were inserted into mouse oocytes by intracytoplasmic sperm injection (ICSI). The injection resulted in survival rates of 46.5% (180 out of 387) and activation rates of 36.1% (65 out of 180). The rate of two pronuclear (2PN) formation was 35.4% (23 out of 65). Only 34.8% (eight out of 23) metaphase chromosome spreads from 2PN zygotes could be analysed; however, all were of normal karyotype. Cytogenetic analysis at the first metaphase revealed that human elongated spermatid chromosomes were able to undergo replication in a heterogeneous environment.     

Fertilization and early embryology: Premature chromosome condensation of the sperm nucleus after intracytoplasmic sperm injection

A cytogenetic-cytological study was performed on unfertilizedhuman oocytes (first polar body visible) after intracytoplasmicsperm injection (ICSI) with respect to the rate of prematurelycondensed sperm chromosomes (G₁-PCC). Out of 163 prepared oocytesderived from 41 ICSI cycles, 133 (-82%) could be analysed successfully.A total of 60 oocytes (45.1%) showed metaphase II chromosomesin the haploid range along with an intact sperm head and 27oocytes (20.3%) were missing the sperm head, but two of themshowed an approximately diploid set of chromosomes; 38 oocytes(28.6%) exhibited the maternal metaphase II chromosomes as wellas G₁-PCC of the sperm nucleus showing a remarkable variationin the degree of condensation. Ten ICSI cycles (each followedby an embryo transfer) were characterized each by 2–3oocytes demonstrating G₁-PCC. It is concluded that the maincause of failed fertilization after ICSI is the failure of oocyteactivation. When the sperm nucleus is able to act with the chromosomecondensing factors and the oocyte does not become activated,this will lead to the induction of PCC. Absence of the spermhead might be due to injection or ejection of the spermatozoonin the perivitelline space except for two cases in which fertilizationmight have occurred. Finally, the observation of both a singlechromatin region (n = 6) or two chromatin regions (n = 2) indicatedoocyte activation which, however, was followed by developmentalarrest.     

Detection of aneuploidy for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y by fluorescence in-situ hybridization in spermatozoa from nine patients with oligoasthenoteratozoospermia undergoing intracytoplasmic sperm injection.

Recent evidence suggests that infertile males donating semen for intracytoplasmic sperm injection (ICSI) may be at an increased risk of transmitting numerical (predominantly sex chromosome) abnormalities to their offspring. The present study was designed to determine aneuploidy in spermatozoa from oligoasthenoteratozoospermic (OAT) patients undergoing ICSI. Aneuploidy frequencies of 12 autosomes and the sex chromosomes were determined by fluorescence in-situ hybridization (FISH) on spermatozoa from fresh ejaculate of nine severe OAT patients and four proven fertile donors. FISH, using directly labelled (fluorochrome-dUTP) satellite or contig DNA probes specific for chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 21, X, and Y, was performed on decondensed spermatozoa. Per chromosome disomy frequencies for autosomes and sex chomosomes in OAT males were 0-5. 4%. In contrast, the disomy frequencies in controls were 0.05-0.2%. The frequency of diploid spermatozoa in OAT patients was 0.4-9.6%; controls showed a mean of 0.04%. Using recently developed formulae, the total aneuploidy in our OAT patient population was estimated to be 33-74%. In contrast, estimates of mean total aneuploidy in the spermatozoa of controls ranged from 4.1 to 7.7%, depending upon method of calculation. Six series of ICSI were performed on five of the OAT patients. Four resulted in no establishment of pregnancy; the others failed to establish ongoing pregnancies. Our cytogenetic data show significantly elevated frequencies of diploidy, autosomal disomy and nullisomy, sex chromosome aneuploidy, and total aneuploidy in OAT patients, which may contribute to the patients' infertility.