1 Department of Gynecology and Obstetrics, University of Ulm, Prittwitzstrasse 43, D-89075 Ulm and 2 Gregor Mendel Laboratories, 89231 Neu-Ulm, Germany
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Abstract |
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Key words: chromosomal abnormalities/digynic triploidy/ICSI/irregular chromatid segregation/tripronuclear zygotes
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Introduction |
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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., 1995). Analyses of uncleaved one-cell zygotes (Grossmann et al., 1997
) and of embryos originating from tripronuclear zygotes (Staessen and Van Steirteghem, 1997
) 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., 1996a,1996b
). 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., 2001
). 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., 1997;1998a
). The present report summarizes our data on tripronuclear one-cell ICSI zygotes. For this purpose, two previously published single observations (Rosenbusch and Sterzik, 1996
; Rosenbusch et al., 1998b
) have been included.
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Materials and methods |
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Our ICSI technique has been described elsewhere (Rosenbusch et al., 1998b; Rosenbusch, 2000
). 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 1820 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 2428 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., 1998a
). 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.
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Results |
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The varying arrangement of the chromosome sets was taken into consideration during slide analysis (Table I). 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).
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Discussion |
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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., 2000). 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., 1997) and in uniformly triploid or mosaic embryos developing from tripronuclear zygotes (Staessen and Van Steirteghem, 1997
). 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., 1999
; Ushijima et al., 2000
). In our case, ICSI had been performed with spermatozoa from a patient with oligoteratozoospermia and borderline sperm motility (Rosenbusch et al., 1998b
) 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., 2001
).
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, 1997). According to our recently suggested nomenclature (Rosenbusch and Schneider, 2000b
), 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 3c
). 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, 1995). 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, 2000
).
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., 1996a,1996b
). 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., 1996a
,1996b
).
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., 1996b). 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 ~5659 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, 2000a). It will be a challenging task to analyse further, classify and explain these phenomena observed after ICSI.
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Notes |
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3 To whom correspondence should be addressed. E-mail: bernd.rosenbusch{at}medizin.uni-ulm.de
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References |
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Submitted on March 3, 2001; accepted on July 24, 2001.