Articles Impact of parental gonosomal mosaicism detected in peripheral blood on preimplantation embryos

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1 RBMOnline - Vol 5. No Reproductive BioMedicine Online; on web 12 September Articles Impact of parental gonosomal mosaicism detected in peripheral blood on preimplantation embryos M Cristina Magli is laboratory director at the SISMeR Reproductive Medicine Unit in Bologna, Italy. She obtained her MSc degree in Immunology at the Venezuelan Institute for Scientific Research (IVIC, Caracas) and acquired a solid experience in molecular biology techniques. She has been working with IVF for more than 10 years in collaboration with Luca Gianaroli. Together, they were the first in Europe to start clinical application of PGD for aneuploidy, with the end result of improving the success rate in patients having a poor prognosis of pregnancy. Current research interests include studies of embryo morphology and development in relation to the chromosomal condition. Dr MC Magli M Cristina Magli 1, Luca Gianaroli 1,3, Anna P Ferraretti 1, Stephan Gordts 2, Elisabetta Feliciani 1 1 SISMeR Reproductive Medicine Unit, Via Mazzini 12, Bologna, Italy 2 Leuven Institute for Fertility and Embryology, Leuven, Belgium 3 Correspondence: Tel: ; Fax: ; sismer@sismer.it Abstract This study evaluated the chromosomal condition of embryos generated by patients with an altered karyotype due to gonosomal mosaicism and the clinical outcome after preimplantation genetic diagnosis (PGD) for aneuploidy. Thirty-six patients aged 34.6 ± 3.6 years performed 54 treatment cycles and had 295 embryos diagnosed by fluorescence in-situ hybridization (FISH). Thirty-seven per cent of the embryos were chromosomally normal and generated 19 clinical pregnancies after replacement in 39 cycles. Only one pregnancy miscarried, yielding a take-home baby rate of 33.3%. Autosomal monosomy and trisomy contributed 36.1% of total abnormalities and gonosomal aneuploidy 5.9%, similar to the results detected in patients who undergo PGD for increased maternal age. Reanalysis was performed on 114 nontransferrable embryos: 41 were found to be mosaics, which were grouped in three different types, chaotic mosaics (56%), aneuploid mosaics (29%) and diploid/haploid/polyploid mosaics (15%). The incidence of aneuploid mosaics was higher than expected compared with PGD patients of the same age and resembled the condition observed in patients of advanced maternal age. These findings suggest that constitutional carriers of sex chromosome mosaicism are predisposed to autosomal mosaicism of embryos, possibly due to errors of cell division. There is an indication that this tendency is higher in female than male carriers. Keywords: aneuploidy, gonosomal mosaicism, IVF, mitotic non-disjunction, preimplantation genetic diagnosis, sex chromosomes 306 Introduction Following the first applications of preimplantation genetic diagnosis (PGD) to assess gender selection and aneuploidy (Griffin et al., 1992; Munné et al., 1993a, 1993b; Verlinsky et al., 1995; Gianaroli et al., 1997), this technical approach was then applied to the screening of structural abnormalities in carriers of balanced translocations (Munné et al., 1998a, 2002; Verlinsky and Evsikov, 1999). An altered karyotype indeed exposes the carrier to an increased reproductive risk, due to the production of a high proportion of gametes with unbalanced genetic complement. This situation has been clearly demonstrated in the case of Robertsonian and reciprocal translocations, which are associated with a high incidence of spontaneous abortions and abnormal offspring. The presence of gonosomal mosaicism (45,X; 47,XXX; 47,XXY; 47,XYY in various mosaic frequency) is another form of altered karyotype that occurs at a frequency greater than 3%, according to the data derived from prenatal diagnosis (McFadden and Kalousek, 1989). Otherwise asymptomatic, carriers of these aneuploid states are considered at reproductive risk, and genetic counselling and prenatal diagnosis in case of pregnancy are recommended. The reproductive risk related to gonosomal mosaicism cannot be established in advance and strictly depends on the possible involvement of the gonads in the production of aneuploid cell lines. Therefore, PGD has been proposed for these patients in order to decrease their reproductive risk by selecting chromosomally normal embryos for transfer.

2 The present study evaluated 54 treatment cycles for patients who underwent PGD for aneuploidy, with the aim of verifying the incidence of chromosomally abnormal embryos in carriers of gonosomal mosaicism. In addition the clinical outcome associated with the replacement of euploid embryos was assessed. Materials and methods Patients Thirty-six patients with an altered karyotype due to the presence of gonosomal mosaicism attended the SISMeR Reproductive Medicine Unit to undergo IVF treatment cycles for infertility in combination with the screening for aneuploidy on preimplantation embryos (Table 1). Eleven of these patients had a history of repeated abortions; karyotype analysis was available for five of them: two were normal, and three were trisomic (chromosomes 9, 13 and 22). In addition, one couple had a daughter with Turner s syndrome. Between September 1996 and December 2001, they underwent 54 PGD cycles. Induction of multiple follicular growth was accomplished by the administration of exogenous gonadotrophins after a long desensitization protocol with longacting gonadotrophin-releasing hormone analogues (GnRHa) (Ferraretti et al., 1996). Ovulation was induced by human chorionic gonadotrophin (HCG) administration; 34 h later, oocytes were transvaginally retrieved via ultrasound guidance and cultured in Earle s balanced salt solution (EBSS) supplemented with 10% heat-inactivated maternal serum (MS), in a 5% CO 2 humidified gas atmosphere at 37 C. Oocyte insemination was performed with intracytoplasmic sperm injection (ICSI) or conventional IVF, depending on semen sample indices. Assessment of fertilization and embryo development At approximately 16 h after insemination, oocytes were scored for the presence of pronuclei and polar bodies. Conventionally fertilized oocytes were cultured individually in EBSS 15% MS and observed at 40, 62, and 88 h post-insemination for embryo development. Number and morphology of nuclei and blastomeres, and percentage of fragmentation, were documented. Following embryo biopsy, embryos were transferred to blastocyst growing medium (Vitrolife, Gothenburg, Sweden). Only embryos diagnosed as chromosomally normal were selected for transfer and replaced into the uterine cavity. Clinical pregnancies were assessed by the presence of a gestational sac with fetal heartbeat detected at ultrasound analysis. The implantation rate represents the ratio between the number of gestational sacs with fetal heartbeat and the total number of embryos transferred. All patients were recommended to undergo conventional prenatal diagnosis in order to confirm and complete PGD results. Embryo biopsy and FISH Day 3 embryos presenting with at least four normal blastomeres and a percentage of fragmentation not greater than 50% were selected for embryo biopsy and fluorescence in-situ hybridization (FISH) analysis. Embryos were biopsied individually in HEPES-buffered medium overlaid with 36 h pre-equilibrated mineral oil. One cell was removed by using a polished glass needle, which was introduced into the perivitelline space after opening a breach of approximately 20 µm in the zona pellucida with acidic Tyrode s solution. The biopsied blastomere was transferred to hypotonic solution, fixed in methanol: acetic acid on a glass slide and dehydrated in ethanol. For the multicolour FISH analysis, probes for the chromosomes XY, 13, 14, 15, 16, 18, 21 and 22 were used in a two-round protocol for the simultaneous detection of the chromosomes most frequently involved in spontaneous abortions and trisomic pregnancies (Munné et al., 1998b). Starting from December 1998, the probe for chromosome 14 was removed from the panel and substituted with the probe for chromosome 17 in five consecutive cycles; from March 1999 the probe specific for chromosome 1 replaced the one specific for chromosome 17. These modifications were made in an effort to identify other chromosomes of which aneuploidy, although not represented in clinical pregnancies, could be so lethal as to cause an arrest of embryo development at the first divisions. In the first round of hybridization, the chromosomes 13, 16, 18, 21 and 22 were screened (Multivysion PB panel, Vysis, Downers Grove, IL, USA) followed by a second round with probes specific for chromosome X (CEP X alpha satellite, Xp11.1-q11.1), Y (CEP Y alpha satellite, Yp11.1-q11.1) and 15 (CEP 15 satellite III, 15q11.2), which were alternatively associated with those detecting chromosome 14 (binding to the 14q11.2 region), 1 (CEP 1, 1p11.1-q11.1) and 17 (CEP 17, alpha satellite, 17p11.1-q11.1). The scoring criteria used for the interpretation of FISH signals have been previously described (Munné et al., 1998c; Magli et al., 2001a). The whole procedure required 8 10 h; consequently, embryo transfer was scheduled on day 4 (Gianaroli et al., 1999b). For three patients, embryo transfer was delayed to day 5, with the aim of identifying among the FISH normal embryos the ones with the best morphology to be transferred. This approach has been adopted at SISMeR for the last 2 years when the number of transferable embryos, based both on chromosomal diagnosis and morphological evaluation, exceeds two on day 4. As an additional advantage, the cryopreservation of supernumerary embryos is reduced (Magli et al., 1999). Embryo spreading After obtaining the PGD results and performing embryo transfer, the cells from 114 non-transferable embryos were spread on day 4 following a short incubation with acidic Tyrode s solution to digest the zona pellucida. The nuclei were fixed and hybridized with the same probes used for PGD following the same protocol described above. A mean number of 8 ± 2.5 cells were analysed per embryo. The results were compared with the one-cell diagnosis in order to verify the efficiency of the technique and to estimate the overall rate of mosaicism (Gianaroli et al., 2001). Statistical analysis Data were analysed using a chi-squared test, applying the Yates correction, 2 2 contingency tables. 307

3 308 Table 1. Couples entering the study. Couple Maternal Altered Previous ID age (years) karyotype a abortions ,XY;47,XYY ,XX;45,XO ,XX;45,XO (4) ,XX;45,XO (4) ,XX;45,XO (8) ,XX;47,XXX ,XX;45,XO (8) ,XX;45,XO (8) ,XY;45,XO (6) ,XX;47,XXX ,XY;47,XXY ,XX;45,XO ,XX;45,XO ,XY;47,XXY (8) ,XX;47,XXX;45,XO ,XX;45,XO (3) ,XX;47,XXX;45,XO ,XX;47,XXX ,XX;45,XO (12) ,XX;47,XXX;45,XO ,XX;45,XO ,XX;47,XXX;45,XO ,XX;45,XO (3) ,XY;47,XXY ,XY;47,XXY (10) ,XX;47,XXX;45,XO (5) ,XX;45,XO (7) ,XX;47,XXX;45,XO ,XX;45,XO (10) ,XX;45,XO ,XX;47,XXX;45,XO ,XX;45,XO (3) ,XY;45,XO (4) ,XX;47,XXX;45,XO ,XX;45,XO (6) ,XY;47,XXY;47,XYY a Values in parentheses refer to the percentage of mosaic lines. Table 2. Overall fluorescence in-situ hybridization (FISH) and clinical results in preimplantation genetic diagnosis (PGD) cycles. No. cycles 54 Age (mean ± SD, years) 34.6 ± 3.6 No. generated embryos 361 No. FISH-analysed embryos 298 No. FISH-diagnosed embryos 295 FISH normal (%) 109 (37) FISH abnormal (%) 186 (63) No. embryos transferred (mean ± SD) 2.2 ± 0.8 No. cycles transferred 39 (72) No. of clinical pregnancies 19 (%) per transfer (49) (%) per oocyte retrieval (35) On term 16 Ongoing 2 Aborted (%) 1 (5) Implantation rate (%) 33.7 Take-home baby rate (%) 33.3 Table 3. Fluorescence in-situ hybridization- (FISH-) detected abnormalities. No. FISH abnormal embryos 186 Monosomy (%) 38 (20) Trisomy (%) 39 (21) Monosomy+trisomy (%) 2 (1) Haploidy/polyploidy (%) 16 (9) Complex abnormalities (%) 91 (49) Table 4. Frequency of specific aneuploidy. Chromosome number Frequency of aneuploidy (%) a XY a Calculated over total number of fluorescence in-situ hybridization- (FISH-) diagnosed embryos; double monosomies and trisomies were counted twice, one for each chromosome.

4 Results Table 2 represents the overall outcome of the 54 treatment cycles included in the study. In all, 361 embryos were generated and 298 were selected to undergo FISH analysis. A diagnosis was obtained for 295 embryos (99%), of which 109 were classified as chromosomally normal (37%) and the remaining 186 (63%) exhibited chromosomal abnormalities that were not compatible with either implantation or an healthy pregnancy. Following the FISH results, embryo transfer was cancelled in 15 cycles (28%) in which no euploid embryos were detected. An average of 2.2 ± 0.8 embryos were transferred to the remaining 39 cycles, resulting in 19 clinical pregnancies (49% per transferred cycle and 35% per oocyte retrieval) and an overall implantation rate of 33.7%. One pregnancy miscarried, yielding an abortion rate of 5% (fetal karyotype not available). Sixteen pregnancies have already delivered 22 healthy infants; of the two still ongoing pregnancies, prenatal diagnosis has already predicted the birth of chromosomally normal babies. Seventeen pregnant patients underwent amniocentesis or chorionic villus sampling; the PGD was confirmed in all cases. The remaining two pregnant women decided not to undergo prenatal diagnosis: one was afraid of the risk of abortion consequent to the procedure, the other had strong ethical concerns on the possibility of having therapeutic abortion. The analysis of the detected chromosomal abnormalities are shown in Table 3. Aneuploidy due to monosomy and trisomy contributed 42% of the abnormalities (79/186), and complex abnormalities were detected in 49% of the abnormal embryos (91/186). The remaining 16 embryos were either haploid (n = 4) or polyploid (n = 12). Table 4 shows the incidence of monosomies and trisomies for each chromosome calculated over the total number of FISH-diagnosed embryos. The most common aneuploidies were those for chromosomes 15, 16 and 21. Gonosomal aneuploidy was observed in 11 embryos (3.7%, 11/295): in four cases, it was the only abnormality, whereas in the other seven cases, another chromosome was also in an aneuploid condition. Table 5. Results analysed according to the carrier of gonosomal mosaicism. Women Men No. cycles No. FISH-diagnosed embryos Of diagnosed, % abnormal 141 (67) a 45 (53) a No. cycles transferred (%) No. clinical pregnancies (%) 12 (40) 7 (54) No. abortions 0 1 Implantation rate (%) Take-home baby rate (%) FISH = fluorescence in-situ hybridization; a Significantly different, P < Figure 1. Results of chromosomal analysis, reported as percentages, according to the cellular stage at the time of embryo biopsy: (a) embryos with a normal chromosomal complement; (b) chromosomally abnormal embryos. Values with same letter were significantly different ( a P< 0.005, b P< 0.05, c P< 0.01, d,f P< 0.001, e P< 0.025). 309

5 Figure 2. A 6-cell embryo was reanalysed by fluorescence in-situ hybridization (FISH) after spreading of its blastomeres. The probes used were specific for the chromosomes 13 (red), 16 (light blue), 18 (pink), 21 (green) and 22 (yellow). The three nuclei in the upper line are diploid, whereas the three in the bottom line are trisomic for chromosome The results of chromosomal analysis in relation to the cellular stage at 62 h post-insemination are reported in Figure 1. The percentage of euploidy was significantly higher in embryos at the 7 8 cell stage (51%; Figure 1A). The incidence of autosomal monosomy and trisomy over total abnormalities increased proportionally with cellular stage, whereas the rate of complex abnormalities demonstrated the opposite trend (Figure 1B). Of the nine embryos with accelerated cleavage (nine cells or more at the time of biopsy), only one was chromosomally normal; four were aneuploid, one was polyploid, and the remaining four carried complex abnormalities. Table 5 shows the results analysed according to the sex of the carrier of gonosomal mosaicism (female in 40 cycles and male in 14 cycles). The proportion of chromosomally abnormal embryos was significantly higher when the gonosomal mosaicism was female in origin (67% versus 53%, P < 0.05). The clinical pregnancy rates and implantation rates were similar between the two groups. The take-home baby rate was 30% for female carriers and 42.9% for male carriers. The results obtained from the FISH analysis of 114 nontransferable embryos after spreading of their blastomeres revealed 94.7% overall confirmation of PGD results (Table 6). Misdiagnosis occurred for six embryos: three were classified as monosomy X, trisomy 22 and polyploid respectively, by PGD but were found to be normal after re-analysis; the remaining three embryos demonstrated after re-analysis a chromosomal abnormality that was different from that originally assessed by PGD. Forty-one mosaic embryos had all their cells spread and FISH analysed: 23 were chaotic mosaics (each cell had a chromosomal complement different from the others); 12 were aneuploid mosaics generated by nondisjunction events affecting one or more chromosomes associated with a diploid cell line (Figure 2); and six were mosaics in which a diploid cell line co-existed with haploid or polyploid cell lines. In this last group, five of the six analysed embryos also presented mitotic non-disjunction of some chromosomes. The total incidence of gonosomal mosaicism in spread embryos was 1.6% (15/912 analysed cells). Discussion The purpose of this study was to evaluate whether patients with an altered karyotype due to gonosomal mosaicism could derive a benefit by undergoing an IVF cycle in combination with PGD for aneuploidy. The clinical outcome of a take-home baby rate of 33.3% was considered to be extremely positive, especially considering that 11 of the 36 couples entering the study had previously experienced repeated abortions. The relevance of selecting embryos on the basis of their chromosomal condition was anticipated by the high proportion of abnormalities detected in 63% of the diagnosed embryos (60% in couples with an history of recurrent abortions and 64% in the others). This figure is very similar to the one obtained in patients with

6 Table 6. Fluorescence in-situ hybridization (FISH) results obtained by preimplantation genetic diagnosis (PGD) and after blastomeres disaggregation and reanalysis. PGD No. embryos Re-analysis Confirmed Not confirmed Normal Other abnormalities Normal Monosomy Trisomy Haploidy/polyploidy Complex abnormalities TOTAL indications for PGD due to advanced maternal age (71%) or repeated abortions (72%) and is higher than the rate of chromosomal abnormalities detected in couples with no indications for PGD (49%; P < 0.005), making gonosomal mosaicism a condition for which PGD can be proposed (Gianaroli et al., 1997, 1999a). In agreement with previous observations, a tight relationship has been demonstrated between rate of embryo cleavage and chromosomal status (Munné et al., 1995; Magli et al., 1998, 2001b). Chromosomal abnormalities occurred more frequently in slow-cleaving embryos (69%, Figure 1) and decreased proportionally with the increase in the number of cells, having the lowest incidence in embryos with 7 8 cells at 62 h postinsemination (49%); however, an increase occurred again in embryos with an accelerated cleavage rate (11%). These findings confirm that embryos at the 7 8 cellular stage on day 3 have the greatest chances of normal development. In addition, the type of abnormality varied accordingly, with monosomy and trisomy representing the most frequent defect in embryos with 7 8 cells (65% over total abnormalities) and only 18% in slow-cleaving embryos; at the same time, complex abnormalities showed exactly the opposite trend. It seems that an impaired cleavage rate frequently occurs in the case of complex abnormalities, which mainly, as suggested by the analysis of spread embryos, originate post-meiotically, during fertilization or the initial mitotic divisions. The resulting effect is so devastating that delay or blockage in development are evident at very early stages. By contrast, aneuploidy due to malsegregation during meiosis was the main defect for embryos at the 7 8 cell stage, with similar figures for monosomies and trisomies (13% and 19% over total aneuploidy respectively); in this case, the consequences to embryo development are not so severe, and blastocyst formation can easily occur (Magli et al., 2000, 2001b; Sandalinas et al., 2001). These results altogether closely resemble those observed in patients who undergo PGD for maternal age factor (Magli et al., 2001b), suggesting that meiotic errors could occur in patients with gonosomal mosaicism at the same frequency as in women of advanced reproductive age. The incidence of aneuploidy for X and Y chromosomes (5.9%) was not increased compared with that observed in aged patients (5.5%) and in couples with previous IVF failures (7.2%) and was even lower than for testicular sperm extraction (TESE) patients (13.5%). In view of these data, it could be assumed that carriers of gonosomal mosaicism are not at risk of generating specifically X and Y cell lines, which were detected in their peripheral blood, but they have the predisposition to generate aneuploid cell lines that could probably involve any chromosome. For instance, mosaics of autosomes would have severe phenotypic effects that could range from failed implantation to spontaneous abortion or birth of an abnormal child. According to the data reported in Table 5, the effects associated with gonosomal mosaicism could be heavier when the altered karyotype is female in origin. Although not many data were generated, the incidence of chromosomally abnormal embryos was slightly higher when the woman was the carrier (67% versus 53% in male carriers; P < 0.05). Accordingly, a trend indicating a higher take-home baby rate in the case of male carriers, although no statistical significance was reached, could be considered meaningful. The reanalysis of 114 non-transferred embryos after spreading of their blastomeres provided an estimation of the error rate inherent in the FISH technique on a single cell. In agreement with previous reports (Munné et al., 1998b; Gianaroli et al., 2001), the overall rate of misdiagnosis was 5.3% (6/114), but only 2.6% (3/114) had a clinical relevance, as three euploid embryos were not considered for embryo transfer due to the diagnosis of chromosomally abnormal by PGD. Some additional information derived from the analysis of the cells belonging to 41 mosaic embryos. As already reported (Munné et al., 2002), chaotic embryos were the most common form of mosaics (56%); however, they were followed in frequency not by the diploid/haploid/polyploid mosaics that in large-scale studies have been considered to be the second most common form of mosaicism, but by aneuploid mosaics (29%) in which diploid cell lines were combined with monosomic and trisomic cell lines of one or more chromosomes. Finally, the remaining 15% of mosaics belonged to the category in which a diploid cell line co-exists with haploid or polyploid cell lines, and even in this group, aneuploid events could be observed in five of the six mosaics studied. 311

7 312 The mechanisms giving rise to aneuploid mosaics are mostly related to mitotic, anaphase lag or endo-reduplication (Munné et al., 2002), which all depend on alterations in the process of chromosome duplication and disjunction. Recently, the classical assumption about the lack of any maternal-age effect on mosaicism has been revolutionized by a report demonstrating that the frequency of mosaics with mitotic nondisjunction at the first cleavage divisions (which are still regulated by stored maternal mrna) increases with maternal age (Munné et al., 2002). This does not occur for other types of mosaics, such as chaotic mosaics or diploid/polyploid mosaics (Munné et al., 2002). If this finding is confirmed, it can be postulated that aged oocytes and oocytes that act as they were aged, as in carriers of gonosomal mosaicism, could originate aneuploid events more frequently. In conclusion, the findings reported in the present study strongly support a predisposition in patients with gonosomal mosaicism to give rise to different cell lines, possibly due to disturbances and malfunctioning of the mechanisms entering cell division. The favourable clinical outcome derived from the application of PGD suggests that this tendency is especially evident at the first cleavage divisions of embryo development, probably before or in concomitance to the time when the embryo starts to rely upon its own transcription machinery. References Ferraretti AP, Magli MC, Feliciani E et al Relationship of timing agonist administration in the cycle phase to the ovarian response to gonadotropins in the long-down regulation protocols for assisted reproductive technologies. Fertility and Sterility 65, Gianaroli L, Magli MC, Munné S et al Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? 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