vitro fertilization (IVF) implantation failure, like those with recurrent miscarriage, have a higher than expected prevalence of translocations which may impact on ...
Human Reproduction vol.14 no.8 pp.2097–2101, 1999
Chromosome translocations in couples with in-vitro fertilization implantation failure
C.Stern1,4, M.Pertile2, H.Norris1, L.Hale1 and H.W.G.Baker3 1Reproductive Biology Unit, 2Department of Cytogenetics, 3University Department of Obstetrics and Gynaecology,
Royal Women’s Hospital, Carlton, 3053, Australia 4To
whom correspondence should be addressed at: 3/320 Victoria Parade, East Melbourne 3002, Australia
Recurrent miscarriage is known to be associated with parental chromosomal abnormalities, particularly balanced reciprocal and Robertsonian translocations. The aim of this study was to test the hypothesis that couples with invitro fertilization (IVF) implantation failure, like those with recurrent miscarriage, have a higher than expected prevalence of translocations which may impact on pregnancy outcome. Patients who previously had at least 10 embryos transferred without achieving clinical pregnancy were evaluated for chromosome abnormalities as part of screening investigations for implantation failure. Recurrent miscarriage patients with a history of at least three consecutive first-trimester abortions were also tested. Results were compared to reports of infertility patients presenting for treatment and population neonatal screening programmes. Chromosomal abnormalities overall were detected in 13/514 individuals with implantation failure (2.5%), and 15/319 individuals with recurrent miscarriage (4.7%). Translocations (reciprocal and Robertsonian) were found in 7/514 individuals (1.4%) and 7/219 couples (3.2%) with implantation failure (P < 0.0005 compared with infertile controls and P < 0.0001 compared with screened neonates). Translocations were found in 13/319 individuals (4.1%) and 12/130 couples (9.2%) with recurrent miscarriage. Balanced parental translocations may be implicated in the pathogenesis of IVF-implantation failure. Genetic evaluation should be considered as part of the investigation of these patients. Key words: chromosome/implantation failure/IVF/recurrent miscarriage/translocation
Introduction Although in-vitro fertilization (IVF) has revolutionized the treatment of infertility, there are still many couples who have multiple transfers of good quality embryos without achieving successful implantation. Chromosomal abnormalities, particularly translocations, are known to be implicated in various forms of reproductive failure, ranging from defective gametogenesis (Crosignani and Rubin, 1982) to recurrent spontaneous mis© European Society of Human Reproduction and Embryology
carriage (Campana et al., 1986). Although several groups have reported frequencies of chromosomal aberrations in infertile patients to be higher than those found in population screening studies (Chandley, 1983; Koulisher and Gillerot, 1985; Hens et al., 1988), with the exception of male factor infertility, chromosome analysis is not routinely performed for new patients presenting to infertility clinics. 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; Munne´ 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 Approval was obtained from the Royal Women’s Hospital Research and Ethics Committees prior to commencement of the trial. 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 10–50) 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 βHCG . 100 IU) with consequent evidence of gestational sac 6 fetal heart observed on transvaginal ultrasound 28 days after embryo transfer. Results were available for 293 women (average age 36, range 24–47) and 221
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Table I. Translocations in patient and control groups IVF-IF n 5 514 n (%)
RMC n 5 319 n (%)
Infertility patients n 5 1000 n (%)
Neonatal population n 5 94 465 n (%)
7 (1.4)*†
13 (4.1)
3 (0.3)
174 (0.2)
*P , 0.0001 compared with neonatal population; †P , 0.0005 compared with infertility patients. IVF-IF 5 in-vitro fertilization implantation failure; RMC 5 recurrent miscarriage.
Table II. Description of chromosomal translocationsa identified in patients with implantation failure referred for chromosome analysis 46,XX,t(2;18)(q31;p11.2) 46,XX,t(8;11)(p11.2;p15.1) 46,XX,t(8;16)(q11.2;q13) 46,XY,t(1;9)(p32;q22.3) 46,XY,t(5;21)(p12;q11.2) 46,XY,t(3;9)(p21.1;q22.1) 45,XY,der(13;14)(q10;q10) aTranslocations
were de novo in patients who agreed to testing; five couples, however, did not agree and therefore it is not known if translocation was de novo or familial.
Figure 1. Micrograph of karyotype in patient with IVF implantation failure showing reciprocal translocation 46,XX, t (8;11)(p11.2;p15.1).
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 19–47 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 3–16). 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 20–45 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 estab-
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Figure 2. Micrograph of karyotype in patient with IVF implantation failure showing Robertsonian translocation 45,XY, der (13;14)(q10;10). lished 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 20–40 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.
Translocations in IVF-implantation failure
Figure 3. Translocations in couples where both partners are tested.
Results Chromosomal abnormalities (excluding Turner’s syndrome and Klinefelter’s syndrome) were detected in 13/514 individuals in the IF group (2.5%) and 15/319 individuals in the RMC group (4.7%), compared with 13/1000 individuals in the historical infertile controls (1.3%) and 299/94465 historical screened neonates (0.3%). Sex chromosome abnormalities were found in 5/514 (1%) of IF patients and 1/319 (0.3%) of patients with RMC. One IF male partner was found to have a balanced paracentric inversion of chromosome 15 and one RMC patient was found to have a variant chromosome 1. Autosomal translocations were found in 7/514 individuals in the IF group (1.4%) (see Table I) and thus were significantly more common in this group than in the infertile controls (P , 0.0005) and the screened neonates (P , 0.0001). The translocations detected in the IF group included three reciprocal translocations in the female partners and three reciprocal translocations and one Robertsonian translocation in the male partners (see Table II and Figures 1 and 2). There were no couples in this group with a history of recurrent spontaneous miscarriage, but two couples had a history of one previous spontaneous miscarriage prior to development of infertility. One of the male translocations (46,XY, t (5;21)) was found in a couple who had significant male infertility and that male had also been found to be carrying a Yq microdeletion (de Kretser et al., 1997). Translocations were found in 7/219 couples (3.2%) with IF where both partners were tested (see Figure 3). When results were analysed according to sex, 3/293 females in the IF group (1.0%) were found to carry translocations, compared with 1/500 historical infertile female controls (0.2%) (P , 0.005), while 4/221 males (1.8%) in the IF group carried translocations compared with 2/500 historical infertile male controls (0.4%) (P , 0.005). 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
Table III. Description of chromosomal translocationsa identified in patients with recurrent miscarriage referred for chromosome analysis 46,XX,t(6;10)(q22.32;q11.23) 46,XX,t(6;8)(p22.2;q21.2) 46,XX,t(4;14)(p14;q31) 46,XX,t(11;18)(q23.3;q11.2) 46,XX,t(3;6)(p14.1;p21.1) 46,XX,t(7;22)(p13;q11.2)a 46,XX,t(5;11)(q15;q21) 45,XX,der(13;14)(q10;q10) 45,XX,der(13;14)(q10;q10) 45,XX,der(13;14)(q10;q10) 45,XX,der(13;14)(q10;q10) 46,XY,t(2;13)(p13;q22) 46,XY,t(7;11)(q22;q23)b aSee
footnote to Table II; bpartners.
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%). One female patient with a translocation was also found to have antiphospholipid antibodies, and no patients had hysteroscopic abnormalities. Conclusions This study has shown that couples with otherwise unexplained repeated failure of IVF-embryo implantation have a greater than expected chance of carrying a balanced chromosomal translocation. Thus it is possible that the presence of an unbalanced translocation in some gametes may predispose to preimplantation failure of embryo development, and increase the risk of repeated failure of IVF treatment. 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. 2099
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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 5–7% 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 goodquality 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 (Munne´ 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 2100
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 (Munne´ 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 (Me´ne´zo 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.
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