Introduction
Balanced reciprocal translocations are the most common chromosome rearrangements occurring 1 in 500 live births (Jacobs et al., Reference Jacobs, Melville, Ratcliffe, Keay and Syme1974). Carriers of reciprocal translocations are usually phenotypically normal; however they can have a lower chance to produce normal or balanced gametes leading to repeated spontaneous abortions and infertility (Scriven et al., Reference Scriven, Handyside and Ogilvie1998; Simopoulou et al., Reference Simopoulou, Harper, Fragouli, Mantzouratou, Speyer, Serhal, Ranieri, Doshi, Henderson, Rodeck and Delhanty2003). The chances of these carriers developing a balanced embryo depends on the positions of the breakpoints, the segregation of the rearranged chromosomes, type of translocation and gender of the carrier (Lim et al., Reference Lim, Cho, Song, Kang, Yoon and Jun2008; Lledo et al., Reference Lledo, Ortiz, Morales, Ten, De La Fuente, Garcia-Ochoa and Bernabeu2010). The chance of male carriers to produce a balanced sperm was shown to vary between 20 and 80% (Escudero et al., Reference Escudero, Abdelhadi, Sandalinas and Munne2003; Munne, Reference Munne2005). Analysis of the segregation modes in females was proven to be more difficult and it has been restricted to fetal ovarian tissue (Hartshorne et al., Reference Hartshorne, Barlow, Child, Barlow and Hulten1999) and embryos derived from female carriers undergoing preimplantation genetic diagnosis (PGD) (Munne et al., Reference Munne, Escudero, Sandalinas, Sable and Cohen2000; Ko et al., Reference Ko, Cho, Lee, Kim, Kang, Yang and Lim2013). It is important to understand the meiotic segregation in embryos derived from translocation carriers and examine the possible effect of this segregation in preimplantation embryo development to estimate the risk of implantation failure and pregnancy loss. Preliminary studies in our centre showed that the chance of an embryo transfer for a reciprocal translocation carrier is lower compared with the other patients with no chromosomal rearrangements. These carriers were also shown to have lower rates of pregnancies. It was observed that these low pregnancy rates are mainly originated from carriers with chromosome 10 rearrangements. Therefore we sought to investigate the segregation patterns in embryos derived from reciprocal translocation carriers focusing on rearrangements involving chromosome 10. We further investigated the development of embryos derived from these patients.
Materials and methods
Patient information
In total, 27 couples underwent 31 cycles of PGD for reciprocal translocations from August 2010 to May 2013 in Bahceci Assisted Reproductive Technology Centre. All the patients’ karyotypes were defined by clinical cytogeneticists using standard chromosome banding techniques. Informed consent was obtained from all the couples prior to each PGD cycle and institutional review board (IRB) approval was obtained from Bahceci Assisted Reproductive Technology Centre. Work-up was performed using the patients’ lymphocytes before the stimulation process started. Fluorescence in situ hybridisation (FISH) was optimised using one centromeric and two sub-telomeric probes targeting the translocated segments of the chromosomes. Karyotypes of each patient with probes used for each PGD case are summarised in Table 1.
Table 1 List of patient information. Patient ID with the karyotype, maternal age at the time of oocytes retrieval and the list of probes used in PGD analyses are listed
All the probes are from Cytocell (UK) and Abbott Molecular Inc. (USA).
*Represents two rounds of FISH analysis. IVF, in vitro fertilisation.
Ovarian stimulation, embryo culture and blastomere biopsy
Controlled ovarian stimulation was performed as described previously (Ulug et al., Reference Ulug, Turan, Tosun, Erden and Bahceci2007). Briefly, ovulation was induced using human chorionic gonadotrophin (hCG) injection (Ovidrelle; Merck Serono, UK). Follicles were aspirated 35–36 h post hCG injection under ultrasound guidance and oocytes were retrieved. After at least 2 h of oocyte culture, hyaluronidase treatment was performed. Discontinuous colloidal silica gel gradient (PureSperm; Nidacon, Sweden) was used to process the semen samples and the sperm pellet was washed twice with sperm washing medium. Only meiosis II (MII) stage oocytes underwent the intracytoplasmic sperm injection (ICSI) procedure. ICSI procedure was performed in mHTF solution containing HEPES. Injected oocytes were cultured in Single Step Media (SSM) supplemented with 10% synthetic serum (Irvine Scientific, Irvine, CA, USA) in a 5% CO2 and 5% O2 in air incubator (INB-203C, IKS International, The Netherlands). Fertilisation checks were performed 14–16 h post ICSI. Failed and abnormal fertilisation, which was defined as one pronucleus (1PN) or more than two pronuclei (≥3PN), was recorded. Embryo morphology was examined on day 3 of development and the number of cells, presence of even and uneven cells and expansion of cells were recorded. Only good quality embryos with at least six cells and less than 50% fragmentation were biopsied for PGD. Single blastomere was biopsied from these embryos on day 3 of development by breaching of the zona pellucida with a laser (Octax™, MTG, Germany). Culture medium was replenished on day 3 after the biopsy and embryos were kept in these conditions until the day of embryo transfer. The development of embryos obtained from age-matched patients with no chromosomal rearrangements was analysed as a control group.
Blastomere spreading and fluorescence in situ hybridisation
In total, 298 nuclei from single blastomeres were fixed on poly-l-lysine-coated slides (Thermo Scientific, Germany). A combination of alpha satellite and sub-telomeric probes to translocated segments was used to analyse the chromosomes involved in the translocation by FISH (Table 1). The nuclei were visualised under an Olympus fluorescence microscope. All the nuclei were evaluated by two experts and the type of meiotic segregation of embryos was determined. The diploid blastocysts were transferred into the uterus of the patient on day 5 of embryo development.
Statistical analysis
Statistical analyses were performed by chi-squared test using GraphPad Prism v6 software. Chi-squared test was performed to determine if there were significant differences in maturation of oocytes, normal fertilisation rates and progression of development in embryos obtained from carriers of reciprocal translocations.
Results
In total, 27 patients underwent 31 cycles of PGD for reciprocal translocations. The maturation of oocytes and development of embryos obtained from these carriers were analysed. In the same time period, the oocyte maturation and development of embryos obtained from 60 age-matched patients with no chromosomal rearrangements were analysed as a control group. Overall, 733 oocytes were collected from reciprocal translocation carriers and 84% (617) of these oocytes were microinjected by the ICSI procedure. Of these injected oocytes, 78% (482) were fertilised normally (Table 2). In the control group, 594 oocytes were collected and 93% (551) of the oocytes were microinjected. Of these fertilised oocytes 79% (438) were fertilised normally.
Table 2 Summary table for patient background. The maternal and paternal age, the number of previous IVF treatments and the number of previous miscarriages are listed
IVF, in vitro fertilisation.
Overall, there were no differences in the maturation and fertilisation of the oocytes obtained from the reciprocal translocation carriers compared with the control group. Once we analysed the progression of embryo development, it was observed that the embryos derived from carriers of translocations with chromosome 10 rearrangements was developing slower, in such only 24% (26/109) of the embryos obtained from these carriers reached the blastocyst stage (Table 3). Moreover, distinct differences were observed in the progression of embryo development between the male and female translocation carriers with chromosome 10 rearrangements. None of the embryos obtained from the male carriers with chromosome 10 rearrangements developed to the blastocyst stage, whereas 28% (26/94) of embryos derived from female carriers reached the blastocyst stage (P < 0.0001). We further investigated the meiotic segregation of the chromosomes in these embryos obtained from reciprocal translocation carriers with chromosome 10 rearrangements. It was observed that only 12% (16/135) of the embryos were balanced (Table 4) and despite the developmental incompetence in the embryos obtained from male carriers, the prevalence of 2:2 alternate segregation mode was slightly higher in these embryos compared with the ones obtained from female carriers (8 versus 5%, respectively, P = 0.5). The most common segregation pattern in the embryos obtained from these carriers with chromosome 10 rearrangements was shown to be the unbalanced 3:1 mode (44%, 59/135).
Table 3 Summary table showing the number of oocytes retrieved with the number of meiosis II (MII) stage oocytes, fertilised oocytes (2PN) and biopsied embryos are listed
Table 4 Summary of PGD cycle. Number of biopsied, diagnosed and transferable embryos are listed. Number of patients with positive β-hCG are also listed
N/A, not applicable.
Discussion
In this study, we investigated the progression of embryo development and PGD outcome in embryos obtained from reciprocal translocation carriers. A high number of patients was admitted to our clinic due to translocations involving chromosome 10 and thus our analysis was focused on the embryos obtained from these carriers with chromosome 10 rearrangements. We observed that the maturation of oocytes was similar among the control group and reciprocal translocation carriers. However, embryo development was significantly compromised in the male carriers with chromosome 10 rearrangements. The female partners of these carriers did not have an infertility problem; the detailed indication for each patient is listed in Table 1. Previous studies analysing the progression of embryo development derived from reciprocal translocation carriers showed that embryonic development may be impaired (Findikli et al., Reference Findikli, Kahraman, Kumtepe, Donmez, Biricik, Sertyel, Berkil and Melil2003). However contradictory studies focusing on only the unbalanced embryos showed that development was not disturbed and that these embryos were capable of reaching the blastocyst stage (Evsikov et al., Reference Evsikov, Cieslak and Verlinsky2000). It is well known that the embryo undergoes gradual parental demethylation at the early cleavage divisions (Mayer et al., Reference Mayer, Niveleau, Walter, Fundele and Haaf2000; Oswald et al., Reference Oswald, Engemann, Lane, Mayer, Olek, Fundele, Dean, Reik and Walter2000; Reik et al., Reference Reik, Dean and Walter2001; Beaujean et al., Reference Beaujean, Hartshorne, Cavilla, Taylor, Gardner, Wilmut, Meehan and Young2004) with the rapid demethylation of the paternal genome (Rougier et al., Reference Rougier, Bourc’his, Gomes, Niveleau, Plachot, Paldi and Viegas-Pequignot1998; Mayer et al., Reference Mayer, Niveleau, Walter, Fundele and Haaf2000; Oswald et al., Reference Oswald, Engemann, Lane, Mayer, Olek, Fundele, Dean, Reik and Walter2000; Dean et al., Reference Dean, Santos, Stojkovic, Zakhartchenko, Walter, Wolf and Reik2001; Santos et al., Reference Santos, Hendrich, Reik and Dean2002; Beaujean et al., Reference Beaujean, Hartshorne, Cavilla, Taylor, Gardner, Wilmut, Meehan and Young2004; Santos & Dean, Reference Santos and Dean2004) and gradual demethylation of the maternal genome (Monk et al., Reference Monk, Boubelik and Lehnert1987; Howlett & Reik, Reference Howlett and Reik1991; Rougier et al., Reference Rougier, Bourc’his, Gomes, Niveleau, Plachot, Paldi and Viegas-Pequignot1998; Santos et al., Reference Santos, Hendrich, Reik and Dean2002; Beaujean et al., Reference Beaujean, Hartshorne, Cavilla, Taylor, Gardner, Wilmut, Meehan and Young2004; Santos & Dean, Reference Santos and Dean2004). Therefore, it is possible that the development of an embryo with an unbalanced chromosome complement originated paternally is more compromised due to the rapid demethylation process. It is also possible that in our study the developmental incompetence of the embryos derived from male carriers with chromosome 10 rearrangements is more exaggerated due to the small number of embryos analysed.
The PGD outcome also showed that the likelihood of obtaining a balanced embryo from carriers with chromosome 10 rearrangements was low. The most common segregation pattern observed in these embryos was the unbalanced 3:1 segregation with an increased rate in embryos obtained from male carriers than female carriers. Diploid sperm could be one of the causes of this result as Van Hummelen and colleagues (Reference Van Hummelen, Manchester, Lowe and Wyrobek1997) showed a higher frequency of diploid sperm in a translocation carrier involving chromosomes 1 and 10 rearrangement (Van Hummelen et al., Reference Van Hummelen, Manchester, Lowe and Wyrobek1997). This increased rate was suggested to be due to abnormal chromosome pairing and the frequency of chiasmata leading to the absence of cytokinesis and resulting in diploid sperm (Goldman et al., Reference Goldman, Fomina, Knights, Hill, Walker and Hulten1993; Kleckner, Reference Kleckner1996). Therefore, further analysis of sperm FISH in male carriers with chromosome 10 rearrangements may shed light into the underlying reasons of obtaining such a high number of unbalanced embryos.
The main limitation of this study was the small number of embryos analysed and the technique used in PGD. Although recently comprehensive chromosomal screening using aCGH has emerged, FISH is still used to perform PGD for translocations (Scriven et al., Reference Scriven, Flinter, Khalaf, Lashwood and Mackie Ogilvie2013; Van Echten-Arends et al., Reference Van Echten-Arends, Coonen, Reuters, Suijkerbuijk, Dul, Land and Van Ravenswaaij-Arts2013). One of the reasons for this is that, depending on the breakpoints involved in the translocation, the detection sensitivity by aCGH varies. In this study, only one of the translocated segments could be detected in 33%; in 11% of the carriers none of the translocated segments could be detected even with the high resolution aCGH platforms (24 Sure+). Although high resolution aCGH could have been used in the diagnosis of the embryos where one translocated segment could be detected, there still was a risk of misdiagnosis in cases due to possible hybridisation failures of the probes on the microarray. Therefore, in our centre, we only perform PGD by aCGH when both of the translocated segments could be identified. In addition to the limitations of aCGH detection sensitivity, in most of the newly developing countries FISH is preferred for PGD for translocations due to the high costs of aCGH.
In conclusion these data show that a carrier of a translocation with chromosome 10 rearrangement has a lower chance of producing a normal or balanced embryo and that the gender of the carrier might also be an indicator of whether there is a higher chance of finding a normal or balanced embryo for transfer.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflict of interest statement
None.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.
