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

B. Rosenbusch1,3, M. Schneider2, R. Kreienberg1 and C. Brucker1

1 Department of Gynecology and Obstetrics, University of Ulm, Prittwitzstrasse 43, D-89075 Ulm and 2 Gregor Mendel Laboratories, 89231 Neu-Ulm, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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.

Key words: chromosomal abnormalities/digynic triploidy/ICSI/irregular chromatid segregation/tripronuclear zygotes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An inspection of pronucleus formation is routinely performed after IVF and intracytoplasmic sperm injection (ICSI) in order to detect and isolate abnormally fertilized oocytes. A common deviation from the regular bipronuclear state is the occurrence of three pronuclei (PN). Cytogenetically, this means the gain of a whole haploid chromosome complement. Subsequent cleavage of the affected zygotes can lead to the development of a triploid embryo. Triploidy plays an important role in human reproductive loss and accounts for ~15% of chromosomally abnormal spontaneous abortions (Kaufman, 1991Go).

Tripronuclear zygotes observed after conventional IVF mostly arise from dispermic fertilization including regular extrusion of the second polar body (PB). Due to the additional paternal chromosome set, this condition is termed diandric triploidy. During ICSI, only one spermatozoon is injected into a mature metaphase II oocyte, but nevertheless, occasionally three PN are formed while the presence of only one (the first) PB is maintained. It has been assumed that corresponding zygotes represent examples for digynic triploidy, the supernumerary pronucleus being a female one that results from non-extrusion of the second PB (Palermo et al., 1995Go). Analyses of uncleaved one-cell zygotes (Grossmann et al., 1997Go) and of embryos originating from tripronuclear zygotes (Staessen and Van Steirteghem, 1997Go) supported this assumption since no XYY chromosome complement was found.

Further information on the cytogenetic constitution of digynic ICSI zygotes has only been provided by Macas et al. who demonstrated a possibly ICSI-induced irregular chromatid segregation between the two maternal complements (Macas et al., 1996aGo,1996bGo). These earlier investigations largely relied on chromosome counts and the occurrence of structural chromosome abnormalities was not considered. Using three-colour fluorescent in-situ hybridization (FISH), the same research group reported an increase in numerical chromosome abnormalities in the paternal pronuclei of tripronuclear zygotes (Macas et al., 2001Go). However, the authors admitted that an exact evaluation of the overall rate of aneuploidy was not possible by this approach.

We have already shown that karyotyping of multipronuclear zygotes can serve as a model for drawing conclusions on the incidence of both numerical and structural chromosome abnormalities, at the earliest stage of conception (Rosenbusch et al., 1997Go;1998aGo). The present report summarizes our data on tripronuclear one-cell ICSI zygotes. For this purpose, two previously published single observations (Rosenbusch and Sterzik, 1996Go; Rosenbusch et al., 1998bGo) have been included.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study includes 40 couples allocated to ICSI because of impaired sperm quality (n = 31) or unsuccessful previous IVF attempts despite normal semen parameters (n = 5). Four men were azoospermic and underwent microsurgical epididymal sperm aspiration (n = 3) or testicular sperm extraction (n = 1). The mean age of the female patients was 32.5 years (range 23–43), while that of the partners was 36.2 years (range 23–61). Ovarian stimulation was performed with human menopausal gonadotrophin or FSH after pituitary suppression with a gonadotrophin-releasing hormone agonist. During one and the same treatment cycle, 29 patients provided one tripronuclear zygote, eight patients provided two and two patients provided three cells. Another three cells were obtained from one patient during two consecutive cycles. All of these zygotes were derived from oocytes injected with initially motile spermatozoa.

Our ICSI technique has been described elsewhere (Rosenbusch et al., 1998bGo; Rosenbusch, 2000Go). IVF medium (Medi-Cult, Copenhagen, Denmark or IVF Science Scandinavia, Vitrolife Productions AB, Gothenburg, Sweden) was used for incubating untreated and injected oocytes as well as pronuclear and cleavage stages. Pronucleus formation was assessed 18–20 h after ICSI. Presumed multipronuclear zygotes were examined once more to avoid confusion of PN and cytoplasmic vacuoles. Oocytes with three PN and one PB were incubated for 24–28 h in culture medium supplemented with podophyllotoxin and vinblastine (Sigma, St Louis, MO, USA) at a final concentration of 0.15 µg/ml each. Prior to fixation, the zona pellucida was digested in a 0.1% protease solution (type XIV, pronase E; Sigma). The zygotes were left in hypotonic solution (1% sodium citrate in distilled water containing 2% HSA) for 10 min and fixed according to a gradual fixation air drying method described previously (Rosenbusch et al., 1998aGo). The preparations were stained with Giemsa and karyotypes were established from photographs taken at x1000 magnification.

Our cytogenetic investigations of unfertilized or abnormally fertilized human oocytes have been approved by the ethical committee of the University of Ulm.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of 54 tripronuclear zygotes which were processed for cytogenetic analysis, 10 preparations could not be evaluated because of insufficient chromosome spreading and/or poor chromosome quality (seven cells). Two zygotes revealed incompletely condensed metaphase chromosomes whereas another cell was excluded due to presumed cell rupture during fixation and excessive chromosome scattering.

The varying arrangement of the chromosome sets was taken into consideration during slide analysis (Table IGo). Seventeen zygotes showed three separate haploid metaphases (distribution pattern n/n/n). Here, 11 cells could be completely karyotyped whereas six presented with one or two uninterpretable metaphases. Five zygotes had one haploid and one diploid chromosome set (n/2n). They included one cell in which chromosome quality of the diploid metaphase only allowed counting and two zygotes in which either the haploid or the diploid component was not analysable. In 21 zygotes the individual sets were not distinguishable (3n). In this group, one cell had chromosomes of poor quality and therefore they were only counted. Finally, one zygote with three pronuclei was in fact tetraploid. It could be karyotyped but the individual chromosome sets were not clearly separated (4n).


View this table:
[in this window]
[in a new window]
 
Table I. Cytogenetic analysis of 44 tripronuclear zygotes obtained after intracytoplasmic sperm injection (ICSI)
 
Within 44 fully or partially analysable zygotes we detected six cases (13.6%) with missing or extra chromosomes whereas two cells (4.5%) had a combination of numerical and structural abnormalities. Thus, there was a total of eight (18.2%) aneuploid cells, four (9.1%) of them being hypertriploid and four (9.1%) being hypotriploid. One zygote (2.3%) with an obviously abnormal (23,X,+C,-D) and two uninterpretable metaphases should be considered separately, since beside the given interpretation we cannot exclude that the supposed C group chromosome is in fact a product of translocation on the missing D. In total, 12 cells (27.3%) carried structural chromosome abnormalities. These included chromosome breaks (chrb) on the chromosome arms (Figure 1aGo) or in the centromeric region (cen) (Figure 1bGo), a chromatid break (chtb), acentric fragments (ace) and two deletions (del). As stated above, in two of these cells an additional numerical abnormality was present (Figure 1bGo). A particular case is the tetraploid zygote (2.3%) that has been discussed in detail earlier (Rosenbusch et al., 1998bGo). Here, we assumed injection of a diploid spermatozoon due to the presence of two Y chromosomes. Moreover, this cell revealed missing and endoreduplicated chromosomes. Finally, we discovered two unambiguous examples (4.5%) of irregular chromatid segregation between the two female PN (Figure 2Go). As suggested earlier (Rosenbusch, 1997Go), this possibly ICSI-induced phenomenon was distinguished in Table IGo from chromosomal abnormalities transmitted by male and/or female gametes. In total, 22 zygotes (50.0%) revealed some kind of cytogenetic aberration in the present material. Excluding the tetraploid and the hypotriploid (68,XX,-C or -Y) zygotes, as well as all cases with uninterpretable or counted metaphases, the sex chromosome ratio XXX:XXY was 17:15.



View larger version (45K):
[in this window]
[in a new window]
 
Figure 1. Two examples of chromosomally aberrant zygotes.(a) 3n = 69,XXY,chrb(2)(p): break in the short arm of chromosome 2 (arrow). (b) 3n = 70,XXY,chrb(C)(cen),+16: a centromeric break in a chromosome belonging to group C (arrow) and a supernumerary chromosome 16 (asterisk). Only partial karyotypes are given.

 


View larger version (36K):
[in this window]
[in a new window]
 
Figure 2. Metaphase spread (above) and karyotypes of a zygote with irregular chromatid segregation. The supernumerary chromosomes (arrowheads) of the first maternal (I.mat) set are missing (asterisks) in the second maternal (II.mat) metaphase. PBC = first polar body chromosomes; pat = paternal complement.

 
The karyotypes 23,Y,chrb(C)(cen)/47,XX,+16 and 23,X/23,X/23,Y,chrb(C)(cen) indicate that the structural chromosome abnormality in both zygotes originated from the spermatozoon whereas the supernumerary chromosome 16 must have been transmitted by the oocyte. Moreover, a female origin of the cytogenetic abnormality is evident in two completely karyotyped zygotes (23,X/22,X,-G/23,Y and 23,X/ 23,X,ace/23,Y) and may be assumed in another two cells with one uninterpretable metaphase (?/47,XX,+C and 23,X/23,X,chrb(3)/? respectively).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Both the oocyte and its first PB contain a haploid chromosome set (n = 23) with chromosomes consisting of two chromatids held together at the centromere. Sperm penetration triggers longitudinal division of the oocyte chromosomes followed by inclusion of 23 chromatids in the maternal pronucleus while the corresponding halves are extruded into the second PB. The spermatozoon also contributes 23 chromatids in the male pronucleus (Figure 3aGo). Subsequent DNA replication and disintegration of the pronuclear membranes restore diploidy (2n = 46).



View larger version (30K):
[in this window]
[in a new window]
 
Figure 3. Schematic representation of regular fertilization, digyny and development of a hypotriploid digynic zygote. (a) Normal fertilization is characterized by extrusion of the second PB and the formation of one male (m) and one female (f) pronucleus. (b) Digynic triploidy results from non-extrusion of the second PB. The corresponding chromatids remain in the ooplasm and will form an additional female pronucleus. (c) The abnormal (23,X/22,X,-G/23,Y) digynic zygote most probably results from an oocyte with a pre-divided G group chromosome. The oocyte has received only one chromatid of this chromosome because the other chromatid has been distributed into the first PB. Consequently, one of the female PN will lack this particular chromatid. For further explanations see text.

 
Tripronuclear zygotes with one PB observed after ICSI result from non-extrusion of the second PB, i.e. its chromatids remain in the ooplasm giving rise to an additional female pronucleus (Figure 3bGo). After DNA replication, three haploid chromosome sets will be present leading finally to triploidy (3n = 69). The reasons for a retention of the second PB are poorly understood and may involve the orientation or position of the second meiotic spindle in the oocyte cortex, damage to the cytoskeleton during ICSI, or reorientation of the spindle during injection (Flaherty et al., 1995Go). Possibly, clinical parameters may affect maturation of some oocytes rendering them susceptible to abnormal fertilization. Palermo et al. found a significantly higher incidence of digyny in oocytes with a specific membrane behaviour, i.e. sudden breakage without funnel formation, upon penetration of the injection needle (Palermo et al., 1996Go). These authors suggested a link between ovarian stimulation and oolemma characteristics because a significantly shorter length of stimulation and lower serum oestradiol concentrations were found in the sudden breakage group when compared with oocytes with different breakage patterns. It is conceivable that such membrane irregularities impair the oocyte metabolism and thus the function of microtubular structures needed for chromatid segregation and formation of the second PB.

Recently, Sachs et al. concluded that patients who produce at least one digynic zygote are high responders to ovarian stimulation since this group was characterized by more follicles and oocytes and higher oestradiol concentrations compared with cycles without tripronuclear oocytes (Sachs et al., 2000Go). In their study, the occurrence of three PN did not correlate with sudden breakage of the oolemma, but the number of corresponding oocytes was very small. It is evident that the factors and mechanisms responsible for a non-extrusion of the second PB need further investigation.

The postulated digynic origin of tripronuclear ICSI zygotes excludes the occurrence of an XYY sex chromosome constitution. In fact, FISH analyses failed to detect the XYY karyotype in uncleaved zygotes (Grossmann et al., 1997Go) and in uniformly triploid or mosaic embryos developing from tripronuclear zygotes (Staessen and Van Steirteghem, 1997Go). The XXX:XXY ratios were 7:8 and 34:38 respectively, compared with 17:15 in the present study. The tetraploid XXYY chromosome set observed by us can be ascribed to injection of a diploid spermatozoon and does not contradict a digynic origin of this particular zygote. It has been shown that infertile men with oligoasthenoteratozoospermia have significantly higher frequencies of diploid spermatozoa than the control groups (Aran et al., 1999Go; Ushijima et al., 2000Go). In our case, ICSI had been performed with spermatozoa from a patient with oligoteratozoospermia and borderline sperm motility (Rosenbusch et al., 1998bGo) and therefore the occurrence of a diploid male gamete is not completely unexpected. Of note, the corresponding zygote would have appeared regularly fertilized and the resulting (possibly triploid) embryo would have been transferred if the oocyte had managed to extrude its second PB. Just recently, further examples for tripronuclear zygotes carrying a diploid sperm pronucleus have been reported by Macas et al. who applied triple colour FISH with probes for chromosomes X, Y and 18 (Macas et al., 2001Go).

The parental origin of the cytogenetic abnormality can be determined in some of the aberrant zygotes studied by us. A conspicuous example is the karyotype 23,X/22,X,-G/23,Y which implies that chromosome loss has affected the oocyte. Artefactual loss during fixation appears highly improbable since we use a technique that keeps the cytoplasm intact. Also, the chromosomes within the three sets were lying close together and repeated screening of the slide yielded no indication for scattered chromosomal elements. We therefore surmise participation of an aneuploid oocyte. However, this particular oocyte could not have been hypohaploid for a whole chromosome (karyotype 22,X,-G) since this constitution would have caused two hypohaploid (22,X,-G) female PN. Instead, the abnormal zygote may have developed from an oocyte with a single G group chromatid resulting from predivision (Angell, 1997Go). According to our recently suggested nomenclature (Rosenbusch and Schneider, 2000bGo), the oocyte's karyotype would have been 23,X,-G,+Gcht indicating that one whole G group chromosome has been replaced by a single chromatid. The chromatid was distributed into one female pronucleus while the other pronucleus was lacking the corresponding half (Figure 3cGo). Thus, after DNA replication one maternal set will be normal (23,X) whereas the other will be hypohaploid (22,X,-G).

The karyotype 23,Y,chrb(C)(cen)/47,XX,+16 points to an aneuploid oocyte characterized by the presence of an additional chromatid (24,X,+16cht). Moreover, the zygote described as ?/47,XX,+C could have arisen from an oocyte with `pre-divided' chromosomes (24,X,+Ccht) if we assume a paternal origin of the uninterpretable metaphase. Considering all numerical deviations from the triploid count (hypo- and hypertriploidy) in our material, it is interesting to note that without exception only one chromosome was involved. This observation excludes non-disjunction of whole chromosomes in all maternally transmitted cases of aneuploidy since the affected zygotes would then have revealed two missing or additional chromosomes.

Concerning structural aberrations, the karyotype 23,Y,chrb(C)(cen) has been detected twice and clearly indicates a transmission of the breakage event by the spermatozoa. This is not unexpected since structural abnormalities, in particular centromeric breaks and acentric fragments, are frequently found in sperm chromosomes (Rosenbusch, 1995Go). However, we also obtained two karyotypes [23,X/23,X,ace/23,Y and 23,X/23,X,chrb(3)/?] that suggest a female origin of the structural abnormality. In the second case, we ascribed the uninterpretable metaphase to the spermatozoon because its appearance resembled one of the patterns of prematurely condensed sperm chromatin found in unfertilized oocytes after ICSI (Rosenbusch, 2000Go).

Two of the zygotes with an unambiguous separation of all participating metaphases showed an irregular chromatid segregation between the female chromosome sets. This phenomenon was first reported by Macas et al. and has been attributed to alterations of the oocyte cytoskeleton (Macas et al., 1996aGo,1996bGo). Besides a purely mechanical impact of the injection procedure, other causal mechanisms may comprise oocyte ageing, changes in temperature and an effect of calcium ions or hydrostatic pressure during injection. However, intrinsic errors during meiotic maturation of the oocytes prior to ICSI cannot be excluded with certainty (Macas et al., 1996aGo,1996bGo).

The relevance of irregular chromatid segregation lies in the fact that it could also occur in normally fertilized oocytes, i.e. those that manage to extrude the second PB. An unequal distribution of the chromatids, e.g. 24 into the female pronucleus and 22 into the second PB or vice versa, would render the oocyte aneuploid with serious consequences for the developing embryo. To our knowledge, this topic has not yet been studied in regularly fertilized ICSI oocytes. Of note, the chromosomal situation may become rather complicated as soon as an aneuploid oocyte undergoes irregular chromatid segregation and one should be able to observe such an event in digynic zygotes. Indeed, Macas et al. described a chromosome distribution pattern of 23/22/20 that can be attributed to a normal spermatozoon and irregular chromatid segregation in an oocyte with 21 chromosomes (Macas et al., 1996bGo). A comparable case was not detected in our material.

In summary, our study provides evidence for a high rate of chromosomal abnormalities in gametes capable of undergoing IVF. Moreover, the digynic situation allowed us to trace back the transmission of some chromosomal abnormalities and to confirm the previously described phenomenon of irregular chromatid segregation. Multipronuclear zygotes therefore appear as a valuable model to investigate the kind, frequency and origin of cytogenetic abnormalities at the earliest stage of conception. Finally, apart from oocytes with three PN and one PB, there are other patterns of abnormal fertilization, e.g. a combination of three PN and two PB. Some of these cells have been studied by us and they always revealed a severely hypotriploid count of ~56–59 chromosomes. This led us to suggest that the corresponding zygotes might be products of an incomplete chromatid segregation into the second PB (Rosenbusch and Schneider, 2000aGo). It will be a challenging task to analyse further, classify and explain these phenomena observed after ICSI.


    Notes
 
* The results of this study have been presented in part at the 16th Annual Meeting of ESHRE, Bologna, Italy, 2000. Back

3 To whom correspondence should be addressed. E-mail: bernd.rosenbusch{at}medizin.uni-ulm.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angell, R.R. (1997) First-meiotic-division nondisjunction in human oocytes. Am. J. Hum. Genet., 61, 23–32.[ISI][Medline]

Aran, B., Blanco, J., Vidal, F. et al. (1999) Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization–intracytoplasmic sperm injection program. Fertil. Steril., 72, 696–701.[ISI][Medline]

Flaherty, S.P., Payne, D., Swann, N. and Matthews, C.D. (1995) Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI). Reprod. Fert. Dev., 7, 197–210.[ISI][Medline]

Grossmann, M., Calafell, J.M., Brandy, N. et al. (1997) Origin of tri- pronucleate zygotes after intracytoplasmic sperm injection. Hum. Reprod., 12, 2762–2765.[Abstract]

Kaufman, M.H. (1991) New insights into triploidy and tetraploidy, from an analysis of model systems for these conditions. Hum. Reprod., 6, 8–16.[Abstract]

Macas, E., Imthurn, B., Roselli, M. and Keller, P.J. (1996a) Chromosome analysis of single- and multipronucleated human zygotes proceeded after the intracytoplasmic sperm injection procedure. J. Assist. Reprod. Genet., 13, 345–350.[ISI][Medline]

Macas, E., Imthurn, B., Roselli, M. and Keller, P.J. (1996b) The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection. Hum. Reprod., 11, 2496–2501.[Abstract]

Macas, E., Imthurn, B. and Keller, P.J. (2001) Increased incidence of numerical chromosome abnormalities in spermatozoa injected into human oocytes by ICSI. Hum. Reprod., 16, 115–120.[Abstract/Free Full Text]

Palermo, G.D., Munné, S., Colombero, L.T. et al. (1995) Genetics of abnormal human fertilization. Hum. Reprod., 10 (Suppl. 1), 120–127.[Abstract]

Palermo, G.D., Alikani, M., Bertoli, M. et al. (1996) Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by intracytoplasmic sperm injection. Hum. Reprod., 11, 172–176.[Abstract]

Rosenbusch, B. (1995) Cytogenetics of human spermatozoa: what about the reproductive relevance of structural chromosome aberrations? J. Assist. Reprod. Genet., 12, 375–383.[ISI][Medline]

Rosenbusch, B. (1997) Chromosomes of multipronuclear zygotes resulting from ICSI (letter). Hum. Reprod., 12, 1599–1600.[Free Full Text]

Rosenbusch, B. (2000) Frequency and patterns of premature sperm chromosome condensation in oocytes failing to fertilize after intracytoplasmic sperm injection. J. Assist. Reprod. Genet., 17, 253–259.[ISI][Medline]

Rosenbusch, B. and Sterzik, K. (1996) Irregular chromosome segregation following ICSI? (letter). Hum. Reprod., 11, 2337–2338.

Rosenbusch, B. and Schneider, M. (2000a) Hypotriploid tripronuclear oocytes with two polar bodies obtained after ICSI: is irregular chromatid segregation involved? (letter).Hum. Reprod., 15, 1876–1877.[Free Full Text]

Rosenbusch, B. and Schneider, M. (2000b) Predivision of chromosomes in human oocytes: a reappraisal of cytogenetic nomenclature. Cytogenet. Cell Genet., 89, 189–191.[ISI][Medline]

Rosenbusch, B., Schneider, M. and Sterzik, K. (1997) The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization. Hum. Reprod., 12, 2257–2262.[Abstract]

Rosenbusch, B., Schneider, M. and Sterzik, K. (1998a) Chromosomal analysis of multipronuclear zygotes obtained after partial zona dissection of the oocytes. Mol. Hum. Reprod., 4, 1065–1070.[Abstract]

Rosenbusch, B., Schneider, M. and Hanf, V. (1998b) Tetraploidy and partial endoreduplication in a tripronuclear zygote obtained after intracytoplasmic sperm injection. Fertil. Steril., 69, 344–346.[ISI][Medline]

Sachs, A.R., Politch, J.A., Jackson, K.V. et al. (2000) Factors associated with the formation of triploid zygotes after intracytoplasmic sperm injection. Fertil. Steril., 73, 1109–1114.[ISI][Medline]

Staessen, C. and Van Steirteghem, A.C. (1997) The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum. Reprod., 12, 321–327.[Abstract]

Ushijima, C., Kumasako, Y., Kihaile, P.E. et al. (2000) Analysis of chromosomal abnormalities in human spermatozoa using multi-colour fluorescence in-situ hybridization. Hum. Reprod., 15, 1107–1111.[Abstract/Free Full Text]

Submitted on March 3, 2001; accepted on July 24, 2001.