Embryo development characteristics in Robertsonian and reciprocal

RBMOnline - Vol 7. No 5. 563–571 Reproductive BioMedicine Online; www.rbmonline.com/Article/951 on web 14 October 2003

Article Embryo development characteristics in Robertsonian and reciprocal translocations: a comparison of results with non-translocation cases Born in 1973, Necati Findikli obtained his BSc degree from the Bosphorus University Molecular Biology and Genetics Department in Turkey. He obtained his MSc degree from Bilkent University, Turkey in 1998, specializing in p53-regulated genes and cell cycle control in cancer. He worked as a research fellow in the Institute of Cancer Research (London, UK) between 1998 and 1999 and joined the Istanbul Memorial Hospital ART and Genetics Centre in October 2000. He is now working in the research and development section of this centre. His current research interests are somatic cell nuclear transfer and human embryonic stem cells.

Necati Findikli N Findikli1,4, S Kahraman1, Y Kumtepe2, E Donmez1, A Biricik3, S Sertyel1, H Berkil3, S Melil1 1Istanbul Memorial Hospital, Reproductive Endocrinology and ART Unit, Piyalepasa Bulvari, 80270, Okmeydani, Istanbul, Turkey; 2Ataturk University Faculty of Medicine, Obstetrics and Gynaecology Unit, Erzurum, Turkey; 3Istanbul Memorial Hospital, Genetics Unit, Istanbul, Turkey 4Correspondence: e-mail: [email protected]

Abstract The effect of translocations on embryo development was evaluated and results were compared in terms of embryo development with those of embryos obtained from standard intracytoplasmic sperm injection (ICSI) cycles. In 23 translocation carriers with 34 cycles, fertilization, pronuclear morphology scoring (PMS), developmental arrest, cleavage and blastocyst formation were evaluated and compared with embryos obtained from non-translocation cases undergoing ICSI (n = 98 cycles). In 28 cycles, preimplantation genetic diagnosis (PGD) was performed on prezygotes (first and second polar body biopsy for female carriers; n = 3) or on embryos having seven or more blastomeres (blastomere biopsy; n = 25). In six cycles for four couples, probes for translocated chromosomes were not available, so PGD could not be performed. Overall, in translocation cases, a lower fertilization rate, a higher rate of retarded embryo development, and a lower rate of blastocyst formation were observed compared with embryos of non-translocation cases. Fluorescence in-situ hybridization (FISH) analysis showed a 70.9% abnormality rate for reciprocal translocations and 55.0% for Robertsonian translocations respectively. In cases with Robertsonian and reciprocal translocation carriers, the probability of poor embryo development, which may be a result of high segregation abnormalities, may negatively affect the outcome of assisted reproductive techniques. This poor prognosis should also be considered when genetic counselling for translocation is given. Keywords: chromosomal status, embryo development, preimplantation genetic diagnosis, translocations

Introduction Of all structural chromosome abnormalities, the most clinically significant is translocations. Balanced translocations occur in 0.2% of the neonatal population; higher rates are observed among infertile couples (0.6%) and patients with recurrent abortions (9.2%) (Stern et al., 1999; Munné et al., 2000). Between 2 and 3.2% chromosomal abnormalities were also found in couples requiring intracytoplasmic sperm injection (ICSI) (Testart et al., 1996). Moreover, translocations are 10 times more frequent among infertile males (Zuffardi

and Tiepolo, 1982). Sperm counts range from normozoospermia to complete azoospermia, depending on the degree of meiotic arrest induced by the reorganization (Egozcue et al., 2000). As an alternative to prenatal diagnosis and pregnancy termination of unbalanced fetuses, preimplantation genetic diagnosis (PGD) has been offered to carriers of balanced translocations in several centres worldwide (Munné et al., 2000; Gianaroli et al., 2002; Kuliev and Verlinsky, 2002). Application of this technique, the efficiency of which depends

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Article - Embryo development in Robertsonian and reciprocal translocation - N Findikli et al.

on several key factors, is expected to reduce the rate of spontaneous abortions and minimize the risk of conceiving an unbalanced fetus. By using two proximal and one (or two) distal probes (or vice versa) for the chromosomes of interest, fluorescence in-situ hybridization (FISH) can be applied to interphase blastomeres taken from embryos with seven or more blastomeres (Scriven et al., 1998; Munné, 2002). However, several studies have reported a high percentage of genetic abnormalities in chromosomes analysed, particularly mosaicism, resulting in poor overall clinical outcomes (Bickerstaff et al., 2000; Iwarsson et al., 2000; Lim et al., 2000; Udoff et al., 2000; Xu et al., 2000; Agan et al., 2001). Spermatozoa from men with translocations can show normal, balanced or unbalanced karyotype, and none of the normal sperm tests can distinguish them in an ejaculate sample. Fertilization of an ovum can take place with these spermatozoa with or without assisted reproductive techniques; however, a high percentage result in abnormal offspring or early pregnancy loss (Egozcue et al., 2000). These high risks might justify the application of PGD when it is associated with infertility (Frydman et al., 2001). In the light of this previous work, the aim was, together with PGD, to analyse the embryo developmental pattern of couples with reciprocal or Robertsonian translocations, and to compare the outcome with that in patients undergoing standard ICSI cycles.

Materials and methods Patients Twenty-three couples undergoing 34 assisted reproduction cycles between April 2000 and November 2002 were included in this study. Routine karyotype analysis showed either reciprocal or Robertsonian translocation in one of the partners. Thirteen couples had a history of previous abortions, and 10 couples were referred for infertility treatment. The PGD Programme for Translocations was approved by an Institutional Review Board in Memorial Hospital and was offered to these patients. Written informed consent was obtained from each couple before starting the cycle. Twenty-eight PGD cycles for translocations were carried out. For four patients in six cycles, probes were not available so PGD could not be performed. Their karyotypes, origin of translocations and probes used for FISH analysis are shown in Table 1. The control group consisted of patients undergoing ICSI cycles (n = 98) in a randomly selected month within the period mentioned above. These couples were admitted and diagnosed with severe male infertility. Their karyotype analysis was found to be normal and ICSI was performed for these couples with no indications of PGD.

Ovulation induction and oocyte recovery The stimulation protocols were as outlined previously (Kahraman et al., 2002). Pituitary down-regulation was performed by using gonadotrophin-releasing hormone analogue

Table1. Translocation cases in this study with the corresponding probes used for preimplantation genetic diagnosis.

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No. patients

Translocations

Probes used

Reciprocal translocations 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

46,XY,t(1;20)(q25.1;q13.3) 46,XY,t(12;15)(p13;p11.1) 46,XY,t(2;9)(q33;q34) 46,XY,t(3;15)(p21;p11) 46,XY,t(3;5)(q29;q33.2) 46,XY,t(3;9)(q21;q34) 46,XY,t(6;21)(p11.1;q10) 46,XY,t(7;13)(q22;q12) 46,XY,t(9;21)(q11;q10) 46,XX,t(10;13)(q11.2;q12) 46,XX,t(11;14)(q11;q13) 46,XX,t(11;22)(q25;q31) 46,XX,t(7;12)(q15;q13) 46,XX,t(2;13)(q35;q14) 46,XX,t(3;12)(p2;q23) 46,XX,t(2;3)(q37;q27)

No PGD CEP 15 (SA), Tel 12p (SG), CEP 12 (SO) CEP 9 (SG), CEP 2 (SO), Tel 2q (SO) CEP 15 (SA), Tel 3p (SG), CEP 3 (SO) CEP 3 (SO), Tel 3q (SO), Tel 5p (SG) CEP 3 (SO), Tel 3q (SO), Tel 9p (SG) CEP 6 (SG), Tel 21q (SO), Tel 6q (SO) 2nd round CEP 7 (SO), Tel 7q (SO), LSI 13q (SG) No PGD CEP 7 (SO), Tel 7q (SO), LSI 13q (SG) CEP 11 (SG), Tel 14q (SO), Tel 11q (SO) 2nd round CEP 11 (SA), Tel 11q (SO), LSI 22q (SG) CEP 7 (SO), Tel 12q (SO), Tel 12p (SG) LSI 13q (SG), Tel 2p (SG), Tel 2q (SO) CEP 3 (SO), Tel 12q (SO), Tel 12p (SG) CEP 3 (SO), Tel 3q (SO), Tel 2p (SG)

Robertsonian translocations 1 2 3 4 5 6 7

45,XY,der(13;14)(q10;q10) 45,XY,der(13;14)(q10;q10) 45,XY,der(13;14)(q10;q10) 45,XY,der(13;14)(q10;q10) 45,XY,der(13;14)(q10;q10) 45,XY,der(13;22)(q10;q10) 45,XX,der(14;21)(q10;q10)

No PGD No PGD LSI 13q (SG), Tel 14q (SO) LSI 13q (SG), Tel 14q (SO) LSI 13q (SG), Tel 14q (SO) LSI 13q (SG), Tel 22q (SO) Tel 14q (SO), Tel 21q (SO)


(Buserelin Suprefact®; Hoechst AG, Frankfurt, Germany), and follicular development was then stimulated with an injection of FSH (Gonal-F®; Serono, Turkey; Puregon®; Organon, Turkey; Metrodin®; Serono, Turkey), human menopausal gonadotrophin (HMG) (Humegon®; Organon, Turkey) and human chorionic gonadotrophins (HCG; Profasi, Serono, Turkey or Pregnyl, Organon, Turkey).

ICSI and embryo culture Transvaginal ultrasound-guided oocyte retrieval was performed 36 h after the injection of 10,000 IU human chorionic gonadotrophin (HCG). After oocyte retrieval, cumulus–oocyte complexes were evaluated by inverted microscope (Olympus inverted microscope; IX70; Japan) according to the degree of expansion and the structure of surrounding cells. Approximately 2 h after oocyte retrieval, the cumulus cells and the corona radiata were removed by brief exposure (10 s) to Gamete-20 containing 40 IU/ml hyaluronidase (type VIII, specific activity 320 IU/mg, H 3757®; Sigma Chemical Co.) and ICSI was applied to MII oocytes. Immediately after ICSI, injected oocytes were taken into G1.2 medium (Vitrolife; Gothenburg; Sweden) and cultured with this medium until the morning of day 3. On day 3, embryos were taken into G2.2 medium (Vitrolife) and kept in this medium until the day of embryo transfer. Every 24 h, culture medium was changed. Fertilization was assessed at 16–18 h after injection. It was determined as normal fertilization when two clearly distinct pronuclei containing precursor nucleolar bodies (PNB) were present under inverted microscope. Each fertilized zygote was assessed according to pronuclear morphology scoring criteria (PMS), based on the alignment and positioning of prenucleolar bodies as well as the appearance and the abutting characteristics of both nuclei. After assessment, the zygotes were transferred to fresh droplets of a corresponding medium. The state of embryo cleavage and quality was assessed after a further 24, 48, 72 and 96 h of in-vitro culture. The embryos were evaluated according to the number of blastomeres present, blastomere size equality and the relative proportion of anucleate fragments by at least two experienced embryologists at ×600 magnification on an inverted microscope.

Polar body and embryo biopsies Polar body biopsy (first and second) was performed on fertilized zygotes approximately 18 h after ICSI. Zygotes to be biopsied were aligned in such a way that both polar bodies were in the same plane. Zona opening was then achieved using a 1.48 μm diyote laser (Fertilase ®; MTM Medical Technologies, Montreux SA, Switzerland) and 1 or 2 shots of 6 ms were used to ensure proper opening of the zona pellucida. Both polar bodies were then removed with the same polar body biopsy pipette and analysed by FISH. For embryo biopsy, embryos with seven or more blastomeres with good morphology (grade I or II) were selected for embryo biopsy on the morning of day 3, in the afternoon of day 3, or on the morning of day 4 of embryo development. Before blastomere removal, embryos were briefly incubated for approximately 5 min in Ca/Mg-free media (EB10; Vitrolife). Three shots of 6 ms were used for zona drilling and a 25–30 μm opening was made in order to allow the placement of the blastomere biopsy pipette (Cook IVF, Queensland, Australia). Only blastomeres with clear nuclei were biopsied.

Polar body/blastomere preparation and FISH procedure Biopsied polar bodies and blastomeres were first transferred into HCl–Tween 20. After total digestion, the remaining cytoplasm was removed with methanol/acetic acid (3:1, v/v) solution and the nucleus was fixed onto the slide. The slides were then allowed to air dry at room temperature for 15 min. For FISH analysis, different centromeric (CEP), telomeric (Tel) and locus specific (LSI) probes (Vysis Inc., Chicago, USA) were combined for each translocation type. After pepsin treatment and paraformaldehyde post-fixation, denaturation of DNA and probe mix were performed at 73°C for 5 min. Following the hybridization step in a hybridization chamber (Hybrite; Vysis) at 37°C for nearly 8 h, post-hybridization washes were performed in 0.4× SSC, 0.3% NP40 for 2 min at 73°C and 1 min in 2× SSC, 0.1% NP40 at room temperature. After drying the slides, 10 μl of DAPI was added to each slide and the slides were evaluated under a fluorescence microscope (Olympus BX 50, Japan), with recommended filters. For the second round of hybridization, slides were washed in 1× PBS for 5 min and dehydrated in different alcohol concentrations (70, 85, 100%) for 1 min in each, dried and hybridized with second probe mix for at least 12 h at 37°C after the denaturation step at 73°C for 5 min.

Statistical analysis Results were expressed as either mean ± SD or percentages with total number of embryos scored, denoted as n. Data were analysed and compared using Mann–Whitney U-test, Student t-test and Chi-squared test where available. P ≤ 0.05 was defined as statistically significant.

Results In this study, outcomes of 23 patients undergoing 34 cycles were analysed for both embryo development (n = 34 cycles) and PGD results (n = 28 cycles) and compared with a control group consisted of standard ICSI cycles (n = 98 cycles). Patient characteristics and their overall embryo development pattern for reciprocal translocations, Robertsonian translocations and the control group are shown in Table 2. There were no significant differences with respect to male age, female age and duration of infertility between these two groups of translocation carriers (38.1 ± 6.6 versus 36.8 ± 4.4; 33.6 ± 5.2 versus 33.5 ± 4.9 and 11.6 ± 8.4 versus 11.3 ± 6.5 respectively; P > 0.05). However, the mean male age as well as duration of infertility was found to be slightly lower in the control group. When overall embryo development was compared, starting from as early as fertilization, embryo development was significantly impaired in translocation carriers compared with embryos from standard ICSI cycles (Table 2). Embryos from Robertsonian translocation carriers showed a decreased fertilization rate of 54.4%, which was significantly different from the fertilization rate in the control group (71%, P ≤ 0.05). In all groups, no difference was observed according to pronuclear morphology scoring criteria. Nearly 90% of embryos in both groups were scored as having a good prognosis according to the classification published previously (Kahraman et al., 2002; data not shown). From day 2 of

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Table 2. Patient characteristics and embryo development outcome of translocation carriers compared with standard ICSI cycles.

No. patients No. cycles Male age (years) Female age (years) Duration of infertility (years) No. of patients with previous abortions Spontaneous abortions Abortion after assisted reproduction Average no. oocytes collected/patient Average MII oocytes/patient Fertilization rate (%) % of embryos with 2 cells on day 2 (n) % of embryos arrested on day 3 (n) % of embryos with >7 cells on day 3 (n) % of embryos with ≤10 cells on day 4 (n) % blastocysts on day 5 (n)

Reciprocal translocations

Robertsonian translocations

Standard ICSI cycles

16 24 38.1 ± 6.6 33.6 ± 5.2 11.6 ± 8.4

7 10 36.8 ± 4.4 33.5 ± 4.9 11.3 ± 6.5

98 98 35.5±6.2a 32.3±5.1 8.2±4.9 b

9

2

6

8 1

1 1

1 5

13.8 ± 6.5

16.4 ± 8.5

12.5 ± 8.1

10.0 ± 4.9

13.5 ± 7.2

9.7 ± 6.6

69.1 32.8 (174)

54.4c 39.5 (77)

71.0 21.1 (617)d

11.7 (162)

4.4 (69)d

13.9 (562)

31.5 (162)

37.7 (69)

44.3 (562)d

47.2 (123)

53.3 (48)

28.3 (438)d

21.1 (57)

27.2 (23)

41.9 (167)d

aP < 0.01; t-test, bP < 0.01; Mann–Whitney U-test, cP < 0.05; Chi-squared test, dP < 0.01; Chi-squared test (n): total number of embryos scored on corresponding developmental stage.

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embryo development to the day of embryo transfer, embryos of both types of translocation carriers showed slow growth. Embryos with two cells on day 2 were found to occur more frequently in the reciprocal and Robertsonian translocation groups than in the standard ICSI group (32.8 and 39.5% versus 21.1% respectively; P < 0.01). However, the numbers of embryos with more than seven cells on day 3 were higher in the standard ICSI group than the translocation groups (44.3% for standard ICSI cycles, 31.5 and 37.7% in the translocation groups; P < 0.01). In addition, the rate of blastocyst formation was dramatically lower in translocation groups (21.1 and 27.2%) compared with the standard ICSI group (41.9%; P < 0.01). Another significant difference was that the percentage of arrested embryos on day 3 was significantly lower in the Robertsonian group (P ≤ 0.01).

reciprocal translocation carriers were regrouped accordingly and the results are shown in Table 4. It was not possible to apply the same approach to Robertsonian carriers, since there was only one female carrier in this group. Mean female age, male age and duration of infertility were statistically lower in female carriers compared with male carriers (P < 0.05), but were found to be similar to the control group. In the female reciprocal translocation group, all patients had a history of abortions, whereas only two patients in the male reciprocal translocation carrier group had such histories. When embryo development was analysed, both groups showed similar developmental profiles except for fertilization (Table 4). The fertilization rate in the female reciprocal translocation carriers was found to be higher than the male translocation group (80.9 versus 64.5%; P < 0.01).

Possible effect(s) of the carrier type on embryo development were also compared in this study. First, embryo development of male reciprocal and Robertsonian translocation carriers was compared with the standard ICSI group (Table 3). Similar to the cumulative results mentioned above, embryos that belonged to male carriers of either translocation type showed similar retarded development, including lower fertilization rates, compared with the control group. Secondly, in order to assess whether there was any difference in development when the translocation carrier was male or female, embryos of

The outcomes of polar body/embryo biopsy and PGD results of translocations were analysed according to chromosomal status for given translocation type, and the results are shown in Table 5. Among blastomere biopsy procedures, the rate of embryos found to be abnormal after FISH analysis was 63.2, 78.3 and 55.0% for reciprocal male, reciprocal female and Robertsonian male translocation carriers respectively (Table 5). In the female translocation group, polar body biopsy procedure and FISH analysis were applied on 13 zygotes. Nine out of the 13 zygotes analysed (69.2%) were found to be


Table 3. Patient and embryo development characteristics of male carriers according to type of translocation.

No. patients No. cycles Male age (years) Female age (years) Duration of infertility (years) No.of patients with previous abortions Spontaneous abortions Abortion after assisted reproduction Average oocytes collected/patient Average MII oocytes/patient Fertilization rate (%) % of embryos with 2 cells on day 2 (n) % of embryos arrested on day 3 (n) % of embryos with >7 cells on day 3 (n) % of embryos with ≤10 cells on day 4 (n) % blastocysts on day 5 (n)

Reciprocal translocations

Robertsonian translocations


9 14 40.4 ± 6.7 35.1 ± 5.6 14.1 ± 9.3

6 9 37.6 ± 3.9a 34.7 ± 3.4a 12.8 ± 5.6b

98 98 35.5 ± 6.2c 32.3 ± 5.1 8.2 ± 4.9 d

2

2

6

1 1

1 1

1 5

14.3 ± 5.9

15.0 ± 5.7

12.5 ± 8.1

10.9 ± 4.6

12.7 ± 5.7

9.7 ± 6.6

64.5 29.3 (99)

58.8 43.3 (67)

71.0e 21.1 (617)f

12.9 (93)

3.2 (62)f

13.9 (562)

36.6 (93)

35.5 (62)

44.3 (562)e

47.7 (65)

57.1 (42)

28.3 (438)f

16.1 (31)

26.1 (23)

41.9 (167)f

aP < 0.05; t-test, bP < 0.05; Mann–Whitney U-test, cP < 0.01; t-test, dP < 0.01; Mann–Whitney U-test, eP < 0.05; Chi-squared test, fP < 0.01; Chi-squared test (n): total number of embryos scored on corresponding developmental stage.

chromosomally abnormal for a given translocation. Embryo developmental parameters were also assessed on day 4 and day 5 for embryos on which PGD was used for translocation; the results are shown in Table 6. Thirty-one embryos were scored for both days and classified according to FISH results as abnormal and normal or balanced. The number of embryos having more than 10 blastomeres on day 4 and blastocyst morphology on day 5 was be similar in both groups. FISH results of translocation embryos were also analysed according to the timing of blastomere biopsy. Embryos having seven or more cells and biopsied in the morning of day 3 (n = 35) showed 54.2% abnormality rate according to the translocated chromosomes, whereas 81.5% embryos having 7 or more cells in the afternoon of day 3 or in the morning of day 4, hence classified as the late biopsied group (n = 27), were found to have an abnormal chromosomal constitution (P < 0.05). Out of 24 oocyte retrievals, embryo transfer was performed in 20 cycles in male and female reciprocal translocations. Embryo transfer was cancelled in four cycles due to lack of chromosomally normal embryos (n = 3) and cleavage arrest (n = 1). Three biochemical pregnancies were obtained in male reciprocal translocations (23.1%). However, one was a

biochemical abortion and two were missed abortions. Two pregnancies were obtained in the female group. One was a biochemical abortion. The other was clinically confirmed, but unfortunately was a misdiagnosis detected by amniocentesis, and hence was aborted. For the Robertsonian translocation group, embryo transfer was successful in seven out of nine cycles for male carriers. Two pregnancies were obtained (28.6%). One delivered to term and one was aborted. Only one cycle was performed for a female Robertsonian carrier without PGD, and this resulted in early abortion. For the control group, 89 embryo transfer cycles were performed. (In nine out of 98 cycles embryo transfer was not performed, due to low oocyte retrieval and subsequent fertilization failure.) Thirty-eight biochemical pregnancies were obtained and four resulted in abortion before they were confirmed clinically. Thirty-four clinically confirmed pregnancies were obtained (38.2%), with an implantation rate of 17.6% and only one clinical abortion occurred afterwards.

Discussion Several checkpoints exist in somatic cells that control the proper alignment of chromosomes during mitosis. Cells having misaligned chromosomes usually show delayed exit from mitosis. Much less is known about the equivalent checkpoint mechanisms during meiosis, but it has been widely

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Table 4. Patient and embryo development characteristics of male and female reciprocal translocation carriers.

No. patients No. cycles Male age (years) Female age (years) Duration of infertility (years) No. of patients with previous abortions Spontaneous abortions Abortion after assisted reproduction Average oocytes collected/patient Average MII oocytes/patient Fertilization rate (%) % of embryos with 2 cells on day 2 (n) % of embryos arrested on day 3 (n) % of embryos with >7 cells on day 3 (n) % of embryos with ≤10 cells on day 4 (n) % blastocysts on day 5 (n)

Male carriers

Female carriers


9 14 40.4 ± 6.7a 35.1 ± 5.6a 14.1 ± 9.3 b

7 10 34.9 ± 5.1 31.5 ± 3.8 8.2 ± 5.6

98 98 35.5 ± 6.2 32.3 ± 5.1 8.2 ± 4.9

2

7

6

1 1

7 0

1 5

14.3 ± 5.9

14.0 ± 8.0

12.5±8.1

10.9 ± 4.6

9.4 ± 5.5

9.7 ± 6.6

64.5 29.3 (99)

80.9c 37.3 (75)

71.0 21.1 (617)d

12.9 (93)

10.1 (69)

13.9 (562)

36.6 (93)

24.6 (69)

44.3 (562)d

47.7 (65)

46.6 (58)

28.3 (438)d

16.1 (31)

28.0 (25)

41.9 (167)d

aP < 0.01; t-test, bP < 0.05; Mann–Whitney U-test, cP < 0.05; Chi-squared test, dP < 0.01; Chi-squared test (n): total number of embryos scored on corresponding developmental stage.

Table 5. PGD results of translocation cases. No. embryos biopsied

No. embryos Normal diagnosed /balanced

Abnormal

Reciprocal translocations Female carriers Female carriersa Male carriers Total

25 18 21 64

23 13 19 55

5 (21.7) 4 (30.8) 7 (36.8) 16 (29.1)

18 (78.3) 9 (69.2) 12 (63.2) 39 (70.9)

Robertsonian translocations Male carriers

23

20

9 (45.0)

11 (55.0)

a Polar body biopsy. Values in parentheses are percentages.

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Table 6. Embryo development according to chromosomal status in translocations. Abnormal

Normal

Embryos scored on day 4 (n = 31) Arrested 10 blastomeres Total

1 (4.5) 14 (63.6) 7 (31.8) 22 (70.9)

0 (0) 5 (55.6) 4 (44.4) 9 (29.1)

Embryos scored on day 5 (n = 31) Arrested/degenerated >10 blastomeres/compaction Morula/cavitation Blastocyst Total

5 (26.3) 3 (15.8) 6 (31.6) 5 (26.3) 19 (61.2)

0 (0) 6 (50.0) 3 (25.0) 3 (25.0) 12 (38.8)

Values in parentheses are percentages.

accepted that developmental arrests and cellular death are mainly related to gross chromosomal abnormalities during preimplantation development (Benkhalifa et al., 1996), and translocations are major part of these structural chromosomal abnormalities in assisted reproductive techniques. Recently, a study done on male germ cells of mice which are heterozygous for Robertsonian translocation revealed increased cell death, arrest and retarded cellular growh (Eaker et al., 2001). Likewise, a recent study shows that in human cancers, certain types of chromosome translocations display a delay in initiation and completion of chromosome replication as well as mitotic chromosome condensation. These translocated chromosomes also participate in a variety of secondary translocations and rearrangements (Smith et al., 2001). On the other hand, a relatively high frequency of gametic aneuploidy implies that even if checkpoint mechanisms may exist during meiosis they may not be effective, and resulting gamete cells show increased aneuploidy compared with somatic counterparts (Eaker et al., 2001). The current data on embryo development have shown that embryos from translocation carriers have poor developmental potential compared with standard ICSI cycles. Elimination starts at the day of fertilization and continues until blastocyst stage. The observation of blastomere compaction on the afternoon of the third day is accepted as a good prognostic factor for the progression to the blastocyst stage. In this study, differences between the control and study groups were not statistically significant but fewer embryos of translocation carriers compacted on days 3 and 4 of development (Figure 1; P > 0.05). These results are contradictory to those obtained by Evsikov et al., who found that a chromosomally unbalanced genome has no adverse effect on the viability of the preimplantation human embryo (Evsikov et al., 2000). However, in the present study, it may be concluded that most chromosomal rearrangements are selected and eliminated during the preimplantation period, and this selection may be very extensive depending on the degree and the type of the rearrangement. It is clear that not all unbalanced translocations are eliminated, since the recurrent abortion rate is high in translocation carriers. In the current study, a similar number of embryos having an abnormal and normal or balanced chromosomal constitution reached the blastocyst stage (Table

6). Although the rate of blastocyst formation has been found to be lower in translocation cases, as Evsikov et al. similarly concluded, selection of morphologically normal blastocysts cannot be used as a criterion in order to distinguish balanced embryos from unbalanced ones (Evsikov et al., 2000), although it may be helpful for selection of embryos with abnormalities (possibly secondary to translocations) other than unbalanced chromosomal constitution (Menezo et al., 1997). Malmgren et al. (2002) performed comperative genomic hybridization (CGH) in order to test structural chromosomal rearrangements in single blastomeres obtained from spare embryos that were diagnosed as unbalanced and therefore unsuitable for transfer. A total of 94 blastomeres from 28 human embryos were analysed, confirming diagnosis of the unbalanced translocation performed by FISH analysis. In addition, a high degree of numerical aberrations was found, including monosomies and trisomies for whole chromosomes or their parts. It is interesting that almost all embryos appeared to be mosaic, containing more than one chromosomally distinct cell line, or even chaotic with different chromosomal contents in each blastomere (Malmgren et al., 2002). Very recently, Gianaroli et al. investigated possible interchromosomal effects of translocations on preimplantation embryos and found that, depending on the type of translocation, a significant percentage of embryos had chromosomal abnormalities other than translocated chromosomes (Gianaroli et al., 2002). This observation supports the findings on embryo development, since chromosomal changes secondary to translocations could also be responsible for this dramatic arrest and slow growth profile. Although relatively higher pregnancy rates have been reported in the literature, pregnancy outcome may be proportional to the percentage of chromosomal abnormalities. It has been suggested that cases with ≥50% abnormal eggs or embryos achieve significantly fewer pregnancies per cycle than cases having