Conjoined human oocytes observed during assisted reproduction: description of three cases and review of the literature.

Conjoined oocytes derived from binovular follicles have been suspected to play a role in producing dizygotic twins, mosaicism, tetraploidy or chimeras. This issue is discussed by presenting three examples for conjoined oocytes observed in our programme of human assisted reproduction and by including a review of corresponding cases. Our material comprises two associated immature oocytes and two cases in which an immature oocyte was attached to a mature oocyte. One of the mature oocytes was fertilized and transferred after cleavage without resulting in pregnancy. In the literature, another fifteen descriptions of conjoined oocytes were found. Here, simultaneous fertilization of both gametes occurred only once and there was no evidence for pregnancies arising from binovular follicles. These data suggest that binovular follicles do not cause dizygotic twins or genetic abnormalities.

The contradictory information on the distribution of non-disjunction and pre-division in female gametes.

Valuable information on the cytogenetic constitution of female gametes has been deduced from the direct, so-called conventional analysis of oocytes remaining unfertilized in programmes of assisted reproduction. Additional, indirect conclusions have become possible by PGD of the polar bodies. Both techniques provided evidence for the co-existence of two aneuploidy-causing mechanisms during first maternal meiosis; non-disjunction (ND) of bivalents results in the loss or gain of whole chromosomes in metaphase II complements, whereas a precocious division (pre-division, PD) of univalents leads to the loss or gain of single chromatids. As to the distribution of ND and PD, however, direct oocyte chromosome studies and PGD tell surprisingly different stories. Moreover, first and second polar body analyses contradict the data derived from DNA polymorphism studies concerning the distribution of first and second meiotic division errors. An increased awareness of these problems appears necessary because important decisions are made on the basis of PGD results.

Cytogenetic analysis of giant oocytes and zygotes to assess their relevance for the development of digynic triploidy.

BACKGROUND: In order to assess the role of binucleate giant oocytes for generating digynic triploidy, we studied their frequency, maturation patterns and chromosomal complements at metaphase II (MII) or after fertilization. METHODS: Uncleaved, giant zygotes were incubated with podophyllotoxin and vinblastine, treated with hypotonic solution and fixed by a gradual fixation method. Giant MII oocytes were directly subjected to hypotonic treatment. The chromosomes were stained with Giemsa. RESULTS: A total of 7065 oocytes were collected during the study period, of which 18 (0.26%) were classified as giant cells. When considering only those patients in whom giant cells were identified (among other normal sized cells) a giant cell frequency of 18/237 (7.6%) was found. Nine cells underwent a union of the nuclei during maturation to MII and four of them became fertilized showing two pronuclei. Seven oocytes maintained the binucleate state to MII and one of them was fertilized showing three pronuclei. Ten unfertilized cells were available for cytogenetic analysis and proved to be diploid. All five giant zygotes revealed triploidy. CONCLUSIONS: The data suggest that giant oocytes may play an important, yet underestimated role in causing digynic triploidy. We recommend the exclusion of giant oocytes from IVF trials and that giant cells should be discarded, even if they carry the regular number of two pronuclei.

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.

Frequency and patterns of premature sperm chromosome condensation in oocytes failing to fertilize after intracytoplasmic sperm injection.

PURPOSE: The purpose of this study was to evaluate reasons for fertilization failure after intracytoplasmic sperm injection as a part of internal quality control and to review corresponding previous data. METHODS: One hundred injected but unfertilized oocytes were fixed and examined after Giemsa staining. RESULTS: Three oocytes (3.0%) did not show the presence of a spermatozoon and two (2.0%) contained pronuclear structures. An intact spermatozoon was found in 25 cases (25.0%), whereas the sperm nucleus had undergone premature chromosome condensation (PCC) in 70 cells (70.0%). A modified classification system was established to characterize the different PCC patterns. CONCLUSIONS: PCC indicates a correct intracytoplasmic injection and excludes technical problems as a major reason for fertilization failure in the present study. A lack of oocyte activation due to cytoplasmic immaturity is considered responsible for the occurrence of PCC. A review of the literature shows that the role of sperm chromatin abnormalities in the process of nuclear decondensation needs further investigation.

Recurrent failure of pronucleus formation after intracytoplasmic sperm injection.

We report on a couple with complete fertilization failure in three consecutive trials of intracytoplasmic sperm injection. Some of the unfertilized oocytes were fixed and decondensed sperm heads were found in all of them. It is not clear whether specific defects of the oocyte, abnormalities of sperm chromatin, or a combination of factors are responsible for the observed failure to develop pronuclei.

Chromosomal analysis of multipronuclear zygotes obtained after partial zona dissection of the oocytes.

We have attempted to analyse the chromosome constitution of multipronuclear 1-cell zygotes obtained after partial zona dissection (PZD) of the oocytes. Six cells with three pronuclei could not be evaluated whereas another one was characterized by the presence of a normal haploid and two uninterpretable metaphases. Complete karyotypes were established for 21 tripronuclear cells, taking the varying arrangement of the chromosome sets into consideration. Of the zygotes, 10 showed three separated haploid metaphases (distribution pattern n/n/n), eight zygotes had one haploid and one diploid chromosome set (n/2n) and in three cells the individual sets were not distinguishable (3n). The sex chromosome ratio XXX:XXY:XYY was 7:9:5. Chromosome abnormalities were found in eight of the completely or partially analysable tripronuclear zygotes (36.4%) and included numerical (4 cells), structural (2 cells) as well as combinations of numerical and structural alterations (2 cells). Three out of 11 zygotes with four pronuclei could not be evaluated at all. In three cases, only two chromosome sets were analysable and another cell displayed one uninterpretable set. Three out of eight completely or partially analysable zygotes with four pronuclei (37.5%) had chromosomal abnormalities. Excluding the four cells with one or two uninterpretable metaphases, the sex chromosome distribution XXXX:XXXY:XXYY:XYYY in the zygotes with four pronuclei was 0:1:1:2. Compared with previously analysed multipronuclear zygotes obtained after conventional in-vitro fertilization (IVF), the rate of aberrant zygotes as well as the incidence of aberrant (male + female) chromosome sets were not significantly changed after PZD.

Maturation of a binuclear oocyte from the germinal vesicle stage to metaphase II: formation of two polar bodies and two haploid chromosome sets.

We report on a binuclear human oocyte that underwent maturation in vitro from germinal vesicle stage to metaphase II. Following extrusion of two polar bodies, the oocyte was processed for cytogenetic analysis which revealed two separate haploid chromosome sets accompanied by the corresponding polar body chromatin. Tentatively established karyotypes were 23,X and 23,X,ace, respectively. This condition could have resulted in a tripronuclear digynic zygote after monospermic fertilization.

Tetraploidy and partial endoreduplication in a tripronuclear zygote obtained after intracytoplasmic sperm injection.

OBJECTIVE: To describe a peculiar combination of cytogenetic abnormalities in a tripronuclear zygote obtained after intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: A university hospital. PATIENT(S): A couple with a 4-year history of primary infertility. Intracytoplasmic sperm injection was performed because of male factor infertility (oligoteratozoospermia). INTERVENTION(S): Ultrasound-guided transvaginal follicular aspiration. MAIN OUTCOME MEASURE(S): Chromosomal karyotype of a tripronuclear one-cell zygote. RESULT(S): Unexpectedly, a tetraploid [92,XXYY, end3, -18, end18] chromosome complement was found, indicating injection of a diploid spermatozoon carrying two Y chromosomes. The parental origin of the other abnormalities could not be determined. The missing chromosomes may be attributed either to a hypodiploid [44,YY,-18,-18] sperm cell or to a hypohaploid [22,X,-18] oocyte. The exact tetraploid count was restored by endoreduplication of two chromosomes. This event could have occurred in one and the same or in two different pronuclei. CONCLUSION(S): Cytogenetic analysis of multipronuclear zygotes appears useful for assessing the incidence of chromosomal abnormalities at the earliest stage of conception. In addition to other methods, it also may contribute to evaluation of the transmission of aberrations by spermatozoa from infertile men.

The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization.

We have attempted to analyse the chromosome constitution of 77 multipronuclear uncleaved zygotes obtained from our in-vitro fertilization programme. Complete karyotypes could be established for 51 tripronuclear cells and eight zygotes with four pronuclei. When compiling the results, the varying arrangement of the chromosome sets was taken into consideration. Eighteen tripronuclear zygotes showed three separate haploid metaphases (distribution pattern n/n/n), 16 cells had one haploid and one diploid chromosome set (n/2n), and in 15 zygotes the individual sets were not distinguishable (3n). Two zygotes were in fact tetraploid, the distribution of metaphases on the slide being n/3n and n/n/2n, respectively. In tripronuclear zygotes the sex chromosome ratio XXX:XXY:XYY was 14:16:18, excluding the two tetraploid cells and one zygote with a 23,X/23,X/22,-C or -Y karyotype. Chromosome abnormalities were found in 16 zygotes (31.4%) and included numerical (six cells), structural (four cells) as well as combinations of numerical and structural alterations (six cells). Four of the zygotes with four pronuclei (50%) had numerical and/or structural chromosome aberrations. Excluding two cells with one uninterpretable metaphase and a 22,-C or -Y karyotype, respectively, the sex chromosome distribution XXXX:XXXY:XXYY:XYYY was 1:1:2:1 in zygotes with four pronuclei. Another zygote was found to be pentaploid after fixation. These results suggest that analysis of multipronuclear zygotes yields valuable information about cytogenetic abnormalities occurring at the earliest stage of conception.

Triploidy caused by endoreduplication in a human zygote obtained after in-vitro fertilization.

Cytogenetic analysis of a presumably tripronuclear zygote revealed that triploidy was caused by an endoreduplicated 46,XX complement. The remaining chromosomes yielded a hyperhaploid karyotype of 28,Y, +2, +3C, +D. The origin of this chromosomal composition is obscure. Besides endoreduplication in a normal 23,X oocyte pronucleus and fertilization by a normal 23,Y spermatozoon, an additional female pronucleus might have been formed due to an irregular chromosome distribution during second meiotic division. On the other hand, penetration by a hyperhaploid spermatozoon cannot be excluded with certainty.

Lack of correlation between ultrasonography and histologic staging of the endometrium in in vitro fertilization (IVF) patients.

In 53 patients of an in vitro fertilization (IVF) program with unsuccessful fertilization of oocytes, an endometrial biopsy was carried out on the day of the intended embryo transfer. The results were compared with the thickness (assessed on the very same day by means of ultrasonography) and the echo pattern of the endometrium, which was classified into four grades (A to D). We found grade A in 16 cases (30%), grade B in 22 cases (41.5%) and grade C in 15 cases (28.5%); no endometrium was assessed as grade D. The distribution of histologic findings was not significantly skewed within the respective grading categories (only 37.5% of grade A endometria, 63.5% of grade B endometria and 66.5% of grade C endometria were in phase with the menses). The in-phase and out-of-phase endometria did not display significant differences in endometrial thickness (8.8 +/- 0.29 mm vs. 9.13 +/- 0.4mm). Neither the sonographically measured endometrial thickness nor the echo pattern correlated with the histologic findings, suggesting that ultrasonography is inadequate for drawing reliable conclusions about endometrial receptivity in an IVF program.