1 Reproductive Biology Unit, 2 Department of Cytogenetics, 3 University Department of Obstetrics and Gynaecology, Royal Women's Hospital, Carlton, 3053, Australia
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: chromosome/implantation failure/IVF/recurrent miscarriage/translocation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chromosomal translocations involve the transfer of genetic material from one chromosome to another, and can be reciprocal, involving the breakage of two non-homologous chromosomes with exchange of segments, or Robertsonian, involving breakpoints close to the centromere of two acrocentric chromosomes. The importance of translocations relates to the pattern of segregation at meiosis.
The patterns of inheritance are complex and depend on the particular chromosomes involved and the size of the rearrangements (Gardner and Sutherland, 1996). With developments in preimplantation genetic diagnosis to include testing for Robertsonian and reciprocal translocations (Cassel et al., 1997
; Munné et al., 1998a
,b
), detection of these structural chromosomal anomalies in couples having IVF treatment becomes of great importance.
We have postulated that the causes of IVF implantation failure and recurrent early pregnancy loss are the same (Stern et al., 1998) and thus some cases of persistent IVF-implantation failure may be associated with balanced parental autosomal translocations. To test this we have surveyed the karyotypes of patients with IVF-implantation failure and compared the results with those of patients with recurrent miscarriage as a positive control group and also published historical control populations of infertile patients and neonates.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patient groups
Patients were recruited from the Royal Women's Hospital IVF and Recurrent Miscarriage Clinics and from the associated private IVF facility. All were counselled and gave their informed consent.
Implantation failure (IF) group
Couples who had previously had at least 10 embryos transferred (average 17, range 1050) without achieving a clinical pregnancy were recruited. For the purpose of this study a clinical pregnancy was defined as a pregnancy diagnosed initially by biochemical means at 17 days after embryo transfer (serum ßHCG > 100 IU) with consequent evidence of gestational sac ± fetal heart observed on transvaginal ultrasound 28 days after embryo transfer. Results were available for 293 women (average age 36, range 2447) and 221 partners. For 219 couples both partners' results were available. Major infertility diagnoses for these patients were as follows: anovulation in six patients (2%), occlusive tubal disease diagnosed by laparoscopy or radiological examination in 69 patients (23%), significant endometriosis involving the ovaries, classified as revised AFS (American Fertility Society, 1985) stage 3 or 4 diagnosed by laparoscopy in 25 patients (9%), isolated male factor infertility (as per World Health Organization guidelines for subfertility) (WHO, 1992) in 86 patients (29%), combined male and tubal infertility in 14 patients (5%), and unexplained infertility in 93 patients (32%). Detailed studies of the other possible causes of IF were also undertaken, including autoantibody testing and outpatient hysteroscopy.
Recurrent miscarriage (RMC) group
We also evaluated 184 women aged 1947 years (mean age 34 years) attending the Recurrent Miscarriage Clinic. These women had all suffered at least three sequential first-trimester clinical pregnancy losses (mean 4, range 316). One hundred and thirty-five partners were also tested, and results were available for both partners in 130 couples. Again, detailed studies of the other possible causes of RMC were also undertaken, including autoantibody testing and outpatient hysteroscopy.
Control groups
In order to evaluate the significance of our findings we compared results from the patient groups with two groups of historical controls: (i) infertile control group: 500 couples referred for IVF or related fertility treatment (Hens et al., 1988), age range of women 2045 years, modal age 30 years; (ii) neonatal population control group: 94 465 infants screened in population-based screening programmes (van Assche et al., 1996
), comprising 13 751 male infants (Walzer and Gerald, 1977
), 45 804 infants (Hook and Hamerton, 1977
), and 34 910 infants (17 872 males and 17 038 females, Nielsen and Wohlert, 1991
).
Cytogenetic studies
Cytogenetic preparations were obtained from phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes. Cultures were established by routine methods (Rooney and Czepulkowski, 1992) and synchronized with 300 µg/ml bromodeoxyuridine (BrdU), a thymidine analogue, 48 h after initiation. BrdU was removed after 14 h and fresh medium was added to the cultures; cells were harvested 6 h later. Chromosome analysis was carried out on G-banded metaphases with a minimum of 10 cells routinely examined. A further 2040 cells were examined if chromosomal mosaicism was suspected. In the historical studies variable numbers of metaphases were examined.
Statistical analysis
Differences between frequencies were tested using the binomial distribution. The effects of different factors on implantation failure or recurrent miscarriage were analysed using regression analysis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
In order to investigate whether there were any particular patient or embryo characteristics that were associated with chromosomal abnormalities, Poisson regression analysis was carried out.
There was no association between infertility diagnosis and presence of a chromosomal abnormality. There was no statistically significant difference in embryo quality, i.e. proportion of good-quality embryos (grade 1 or 2) between the patients with translocations, those with IVF-implantation failure without translocations and our total IVF population (48.6, 51.7 and 47.6% respectively). None of the patients with a chromosomal abnormality was found to have other potential causes of IF on autoantibody and hysteroscopic assessment.
Translocations were found in 13/319 individuals in the recurrent miscarriage group (4.1%). Eleven of 184 female partners (6.0%) carried balanced translocations, of which seven were reciprocal and four were Robertsonian anomalies, while two reciprocal translocations were found in 135 male partners (1.5%) of women with RMC (see Table III). In one couple with RMC, both partners were found to be carrying reciprocal translocations (46,XX,t(7;22) and 46,XY,t(7;11). In twelve of 130 couples with RMC where both partners were tested, a balanced translocation was found in one or both partners (9.2%).
|
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Historical control data from screened neonatal populations have been utilized for comparison purposes in this report because it was not feasible in our study to perform our own population-based karyotype screening programme.
Of all recognized spontaneous abortions, about 50% have a chromosomal abnormality. In couples with recurrent spontaneous abortion, a balanced translocation has been found in one partner in about 57% of cases, depending on the number of previous miscarriages (Campana et al., 1986). In our series of couples with at least three previous sequential miscarriages, 9.2% were found to be carrying balanced translocations in either or both partners.
Autosomal balanced translocations have also been implicated in patients with infertility, particularly male infertility (Chandley, 1984; De Braekeleer and Dao, 1991
). There is an increased risk of males with autosomal abnormalities being found to have oligozoospermia (Yoshida et al., 1996
), and chromosome studies on spermatozoa show an unbalanced karyotype in variable proportions, e.g. 54% for reciprocal translocations (Martin and Hulten, 1993
) and 13.7% for Robertsonian translocations (Martin et al., 1992
). There is only limited information available relating to the implications of structural aberrations on oogenesis (Speed, 1988
; Tupler and Barbierato et al., 1994).
In a recent study evaluating chromosomal abnormalities in 447 couples undergoing intracytoplasmic sperm injection (ICSI), autosomal translocations were found in 2% of couples, four in the male partners and five in the female partners (Meschede et al., 1998). In another study evaluating 305 couples presenting for ICSI, translocations were found in 3.2% of couples, five reciprocal translocations and one Robertsonian translocation in the male partners and two reciprocal and two Robertsonian translocations in the female partners (van der Ven et al., 1998
). In a recent report, a male with severe oligoasthenoteratozoospermia underwent chromosomal testing after having two cycles of ICSI and was found to carry a rare de-novo t(Y;16) translocation. After genetic counselling he elected to undergo further ICSI treatment, which resulted in the birth of healthy twins, one with a 46, XX karyotype and the other with a 46,X,t,(Y;16) karyotype, the same as the father (Giltay et al., 1998
).
However, there is very little information relating to chromosomal abnormalities in couples who have repeated attempts at IVF treatment, with production of adequate numbers of good-quality embryos and apparently appropriately primed endometrium, without achieving evidence of clinical pregnancy. In these couples embryos may be lost at the cleavage stage, blastocyst stage, or even around the peri-implantation period.
It is tempting to postulate that in some cases of implantation failure, such as recurrent spontaneous miscarriage, embryos fail to progress because of unbalanced structural chromosomal defects. Most very early conceptions that fail to develop are likely to be chromosomally abnormal (Munné et al., 1994). Some abnormalities, including those that are structurally unbalanced, may allow initial development to the blastocyst stage, and even implantation, but then fail to continue development (Miller et al., 1980
; Craft et al., 1982
).
The inheritance patterns of translocations are relatively unpredictable, and are determined by various modes of segregation at meiosis I. The pattern of segregation and the implications for progeny gametes depend on the particular chromosomes involved and the size of the rearrangement (Gardner and Sutherland, 1996).
While some authors have attempted to correlate quantitative chromatin imbalance with the risk of miscarriage or having a liveborn affected child (Daniel et al., 1989; Cohen et al., 1994
), the risks of pre- and peri-implantation loss associated with balanced parental translocations are largely unknown. This makes counselling for couples with infertility and implantation failure extremely difficult.
The development of techniques allowing preimplantation diagnosis of structural anomalies (Munné et al., 1998a,b
) is of particular relevance to infertile couples with balanced autosomal translocations and may reduce the disappointment associated with chromosomally unbalanced embryos. In a recent report, co-culture on embryos from a women known to carry a de-novo balanced reciprocal translocation between chromosomes 1 and 22 was performed, in order to attempt partial selection of the embryos in vitro. Five of seven embryos arrested and on subsequent testing using three-colour in-situ hybridization were all found to have severe lethal cytogenetic anomalies related to the maternal translocation. The two embryos that developed to blastocyst stage were transferred and resulted in the birth of a singleton male child that had inherited the maternal balanced translocation (Ménézo et al., 1997
).
In conclusion, this study has demonstrated that balanced parental translocations may be implicated in the pathogenesis of IVF-implantation failure. Chromosomal evaluation should be considered as part of the investigation of these patients, and genetic counselling and consideration of preimplantation diagnosis should be an integral part of planning of further treatment strategies.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Campana, M., Serra, A. and Neri, G. (1986) Role of chromosome aberrations in recurrent abortion: a study of 269 balanced translocations. Am. J. Med. Genet., 24, 341356.[ISI][Medline]
Cassel, M.J., Munné, S., Fung, J. and Weier, H.-U.G. (1997) Carrier-specific breakpoint-spanning DNA probes: an approach to preimplantation genetic diagnosis in interphase cells. Hum. Reprod.,12, 20192027[Abstract]
Chandley, A.C. (1983) Infertility and recurrent abortion. In Emery, E.H. and Rimoin, D.L. (eds), Principles and Practice of Medical Genetics. Churchill Livingstone, Edinburgh, p. 224.
Chandley, A. (1984) Infertility and chromosomal abnormality. Oxf. Rev. Reprod. Biol., 6, 146.[Medline]
Cohen, O., Cans, C., Mermet, M-A. et al. (1994) Viability thresholds for partial trisomies and monosomies. A study of 1159 viable unbalanced reciprocal translocations. Hum. Genet., 93, 188194.[ISI][Medline]
Craft, I., Djahanbakhch, O., McLeod, F. et al. (1982) Human pregnancy following oocyte and sperm transfer to the uterus. Lancet, i, 10311033.
Crosignani, P.G. and Rubin, B.L. (eds) (1982) Genetic Control of Gamete Production and Function. Academic Press, New York.
Daniel, A., Hook, E. and Wulf, G. (1989) Risks of unbalanced progeny at amniocentesis to carriers of chromosomal rearrangements: data from United States and Canadian laboratories. Am. J. Med. Genet., 31, 1453.
De Braekeleer, M. and Dao, T. (1991) Cytogenetic studies in male infertility. Hum. Reprod., 6, 245250.[ISI][Medline]
De Kretser, D., Mallidis, C. and Bhasin, K. (1997) Y chromosome deletions and male infertility. Reprod Med. Rev., 6, 3753.
Gardner R.J.McK., and Sutherland G. R. (1996) Chromosome Abnormalities and Genetic Counselling. Oxford University Press, pp. 39, 6094.
Giltay, J., Tiemessen, C., van Inzen, W. et al. (1998) One normal child and a chromosomally balanced/normal twin after intracytoplasmic sperm injection in a male with a de-novo t(Y;16) translocation. Hum. Reprod., 13, 27452747.
Hens, L., Bonduelle, M., Liebaers, I. et al. (1988) Chromosome aberrations in 500 couples referred for in-vitro-fertilization or related fertility treatment. Hum. Reprod., 3, 451457.[Abstract]
Hook, E.B. and Hamerton, J.L. (1977) The frequency of chromosome abnormalities detected in consecutive newborn studiesdifferences between studiesresults by sex and by severity of phenotypic involvement. In Hook, E.B. and Porter, H. (eds), Population Cytogenetics: Studies in Humans. Academic Press, New York, USA, pp. 8092.
Koulisher, L and Gillerot, Y. (1985) Chromosomes and infertility: study of 7672 cases. Contraception, Fertil. Steril., 1, 195202.
Martin, R., Ko, E. and Hildebrand, K. (1992) Analysis of sperm chromosome complements from a man heterozygous for a Robertsonian translocation 45 XY, t(15q;22q). Am. J. Med. Genet., 43, 855857.[ISI][Medline]
Martin, R. and Hulten, M. (1993) Chromosome complements in 695 sperm from three men heterozygous for reciprocal translocations and a review of the literature. Hereditas, 118, 165175.[ISI][Medline]
Ménézo, Y., Bellec, V., Zaroukian, A. et al. (1997) Embryo selection by IVF, co-culture and transfer at the blastocyst stage in case of translocation. Hum. Reprod., 12, 28022803.[Abstract]
Meschede, D., Lemcke, B., Exeler, J. et al. (1998) Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injectionprevalence, types, sex distribution and reproductive relevance. Hum. Reprod., 13, 576582.[Abstract]
Miller, J., Williamson, E., Glue, J. et al. (1980) Fetal loss after implantation. A prospective study. Lancet, ii, 554556.
Munné, S., Grifo, J., Cohen, J. et al. (1994) Chromosomal abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study. Am. J. Hum. Genet., 55, 150159.[ISI][Medline]
Munné, S., Scott, R., Sable, D. et al. (1998a) First pregnancies after preconception diagnosis of translocations of maternal origin. Fertil. Steril., 69, 675681.[ISI][Medline]
Munné, S., Fung, J., Cassel, M.J. et al. (1998b) Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis. Hum. Genet., 102, 663674.[ISI][Medline]
Nielsen, J. and Wohlert, M. (1991) Chromosome abnormalities found among 34 910 newborn children: results from a 13-year incidence study in Århus, Denmark. Hum. Genet., 22, 8183.
Rooney, D.E. and Czepulkowski, (eds) (1992) Human Cytogenetics, A Practical Approach, vol. 1. IRL Press at Oxford University Press, pp. 3641.
Speed, R. (1988) The possible role of meiotic pairing anomalies in the atresia of human fetal oocytes. Hum. Genet., 78, 260266.[ISI][Medline]
Stern, C., Chamley, L., Hale, L. et al. (1998) Antibodies to ß2 glycoprotein I are associated with in vitro fertilization implantation failure as well as recurrent miscarriage: results of a prevalence study. Fertil. Steril., 70, 938945.[ISI][Medline]
Tupler, R., Barbierato, L., Larizza, D. et al. (1994) Balanced autosomal translocations and ovarian dysgenesis. Hum. Genet., 94, 171176.[ISI][Medline]
Walzer, S. and Gerald, P. (1977) A chromosome survey of 13751 male newborns. In Hook, E.B. and Porter, H. (eds), Population Cytogenetics: Studies in Humans. Academic Press, New York, USA, pp. 4561.
Van Assche, E., Bonduelle, M., Tournaye, H. et al. (1996) Cytogenetics of infertile men. In Van Steirteghem, A. Devroey, P. and Liebaers, I. (eds), Genetics and Assisted Human Conception. Hum. Reprod., 11 (Suppl. 4), 124.
Van der Ven, K., Peschka, B., Montag, M. et al. (1998). Increased frequency of congenital chromosomal aberrations in female partners of couples undergoing intracytoplasmic sperm injection. Hum. Reprod., 13, 4854.[Abstract]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge, UK.
Yoshida, A., Miura, K. and Shirai, M. (1996) Chromosome abnormalities and male infertility. Assist. Reprod. Rev., 6, 9399.
Submitted on February 8, 1999; accepted on May 7, 1999.