PGD management scheme for older females with balanced translocations: Do older females have less chance of balanced embryo transfer?

Headnote

Abstract

Objective: Carriers of reciprocal and Robertsonian translocations have a higher risk of experiencing infertility and repeated miscarriages. It is well established that with advancing maternal age, the risk of aneuploidies in embryos increases. In this study, the chance of developing balanced embryos in translocation carriers with advanced maternal age was analyzed to establish a management scheme for couples seeking fertility treatment and preimplantation genetic diagnosis (PGD).

Material and Methods: Biopsy was performed on cleavage-stage embryos. Multicolor fluorescence in situ hybridization was used for PGD. The translocation carriers underwent a total of 55 cycles of PGD. Genetics diagnosis and cycle outcomes of PGD cases were examined.

Results: This study showed that the chance of obtaining a balanced embryo from the Robertsonian translocation carriers was significantly less when the maternal age is advanced. Similar rates for balanced embryos were obtained from the reciprocal translocation carriers.

Conclusion: The results of this study show that maternal age plays an important role and that genetic counselling and planning for a PGD cycle in translocation carriers, particularly for Robertsonian carriers, must be accordingly adapted. (J Turk Ger Gynecol Assoc 2016; 17: 91-5)

Keywords: PGD, maternal age, human embryo, translocation, PGD counselling

Received: 8 March, 2016 Accepted: 6 April, 2016

Introduction

Couples with known chromosomal abnormalities and structural changes opt for preimplantation genetic diagnosis (PGD) to avoid prenatal diagnosis or miscarriages. PGD is a method of selecting unaffected embryos from patients with translocations or single gene disorders. In humans, the incidence of structural changes, including translocations, is frequent (1, 2). Translocations are grouped in two categories: reciprocal, the most common form, and Robertsonian. Reciprocal translocations occur due to an exchange of two ends of non-homologous chromosomes, whereas the rearrangement of two acrocentric chromosomes arises in Robertsonian translocation carriers occurring in 1 in 900 live births (3). The phenotype of balanced reciprocal and Robertsonian translocation carriers is normal; however, they usually have a lower chance of successful natural reproduction (1, 4). Therefore, such couples opt for PGD to select a balanced embryo and increase their implantation and pregnancy rates. PGD is a highly invasive technique that necessitates in vitro fertilization followed by polar body or embryo (cleavage or blastocyst stage) biopsy.

It is well established that there is a higher risk of aneuploidy in embryos with advancing maternal age (5). Additionally, advanced maternal age has been associated with higher levels of meiotic errors (6). Therefore, for these patients, genetic counselling holds great importance. These patients must be thoroughly informed about the risks of reproductive outcomes, and treatment strategies should be explained to these couples. In this study, we aimed to investigate if there was any effect of maternal age on the chromosomal status of embryos as well as preimplantation embryo development and to develop a management scheme for these patients.

Material and Methods

This retrospective study analyzed PGD results, the number of balanced embryos, and embryo transfer, followed by the analysis of pregnancy rates in carriers undergoing assisted reproductive technology treatments in Bahçeci ART Centre.

Patient information

Translocation carriers were subdivided according to maternal age (<35 years and advanced maternal age of ≥35 years) and translocation type, i.e., reciprocal and Robertsonian. A total of 20 couples with maternal ages of <35 years with 37 PGD cycles were analyzed within the reciprocal translocation carrier group. The advanced maternal age group of reciprocal translocation carriers included 15 couples. Twelve couples with 17 PGD cycles were involved within the Robertsonian carrier group of maternal age of <35 years and three couples with eight cycles of PGD were in the advanced maternal age group. The couples undergoing PGD were counselled by an infertility specialist and medical doctors. PGD and limitations, including the risk of misdiagnosis due to mosaicism and technical errors, were explained to the couples. Prior to PGD cycles, the patients were informed about the procedure, and the potential risks were explained to and informed consent was obtained from each patient; ethical approval was granted. Chromosome banding techniques were used to obtain the karyotypes of all patients. The translocations of carriers were confirmed using fluorescence in situ hybridization (FISH). For FISH analysis, a total of two sub-telomeric probes and one centromeric probe were used.

Controlled ovarian stimulation and embryology

Stimulation treatment was applied for each patient as previously described (7). Briefly, to induce ovulation, human chorionic gonadotrophin (hCG) (5,000 IU; Ovidrelle; Merck Serono, UK) was injected, followed by the aspiration of follicles transvaginally after 35-36 h of the hCG injection. Hyaluronidase treatment was applied to oocytes following 2 h of culturing. Sperms were washed and prepared for the Intra-cytoplasmic sperm injection (ICSI) procedure by discontinuous colloidal silica gel gradient (PureSperm; Nidacon, Sweden). Only meiosis II (MII) stage oocytes were microinjected, and the oocytes were cultured for 16-18 h following ICSI in a 5% CO2 and 5% O2 incubator (INB203C; IKS International, Netherlands). Embryo scoring was assessed in terms of the number of cells and presence of even and uneven cells.

Embryo biopsy and PGD

Embryos were biopsied on day 3 of embryonic development (OctaxTM, MTG; Munster, Germany). Embryos with less than six cells and more than 20% fragmentation were not biopsied. Three hundred eighty-nine embryos, of which 227 were from the reciprocal translocation carriers with maternal age of <35 years and 162 were from those with advanced maternal age, were biopsied. One hundred eighteen embryos, of which 131 obtained from the Robertsonian translocation carriers with maternal age of <35 years and 49 from those with advanced maternal age, were biopsied. Poly-L-lysine coated slides were used for FISH analysis, and one cell was analyzed for each embryo (Thermo Scientific; Darmstadt, Germany). Details of the translocations for each couple and the list of probes used are listed in Table 1. The poly-L-lysine coated slides were washed to remove excess probe hybridization as described by the manufacturer, and each nucleus was analyzed by counterstaining with 0.15 ng/mL of 4, 6-diamidino-2-phenylindole (DAPI, Medimiks; Istanbul, Turkey). Two expert scientists analysed the outcome of the PGD for translocations.

Statistical analysis

GraphPad prism v6 (GraphPad Software, Inc.; La Jolla, California, USA) was used for statistical analysis. The significance of obtaining balanced embryos in the translocation carriers with respect to maternal age was evaluated by the Chi-square test.

Results

Overall, 79% (1154/1457) of the oocytes collected matured to the MII stage, and 72% (832) of these were normally fertilized with two pronuclei. Seventy four percent (619) of the normally developing embryos were biopsied for PGD (Table 2). Sixty agematched patients with no chromosomal rearrangements were analyzed for the developmental rate as a control group within the same time period. Ninety three percent (551/594) of the oocytes obtained from the control group developed to the MII stage, and 79% (438) of these fertilized. The maturation and fertilization rates were similar between the two groups (Table 2).

Overall, 63 of 389 (16%) embryos from the reciprocal translocation carriers were chromosomally balanced, and eight of these were transferred, resulting in two pregnancies. The chances of obtaining balanced embryos from the reciprocal translocation carriers grouped in the maternal age<35 years (16%, 38/227) and advanced maternal age (15%, 25/162) groups were similar (Table 3). The number of balanced embr yos was further analyzed according to the sex of the carrier. From 19 female carriers, 175 embryos were obtained. Of these, 14% (24) were balanced. The chances of obtaining balanced embryos from male carriers were also at similar levels (18%, 39/214).

In the Robertsonian translocations, a total of 187 embryos were biopsied, and 42% (78) of these embryos were shown to be balanced (Table 3). When the number of balanced embryos was further investigated according to the maternal age groups, considerably fewer balanced embryos (30%, 17/56, p<0.05) were obtained from the advanced maternal age group than from the younger patients who were <35 years (45%, 59/131). Similar to the reciprocal translocation carriers, the number of balanced embryos obtained from the female (38%, 35/92) and male (45%, 43/95) carriers was similar (Table 3).

Discussion

In this study, the role of maternal age in obtaining balanced embryos from translocation carriers was investigated. Overall, among the reciprocal translocation carriers, there was no difference in the number of balanced embryos between the advanced maternal age group and younger females. However, in the Robertsonian translocation carriers, it was observed that the number of balanced embryos was significantly decreased in the advanced maternal age group. Therefore, for the Robertsonian carriers, there may be an adverse effect of maternal age on PGD outcome. Advanced maternal age is well known to increase the risk of aneuploid pregnancy (8). This may be more noticeable in Robertsonian translocation carriers because the most commonly found aneuploidies in embryos include the same chromosomes that are also involved in Robertsonian translocations, such as chromosomes 13, 14, 15, 21, and 22 (9-12). In the present study, the majority of embryos in Robertsonian translocation carriers involved chromosomes 13, 14, and 21. Therefore, it is possible that the effect of maternal age on PGD outcome is due to the fact that these chromo- somes are the most commonly observed chromosomal abnormalities in embryos. Therefore, for the Robertsonian carriers, we cannot distinguish the segregation pattern of these chromosomes from aneuploidy that may be observed in non-carriers as well. The likelihood of obtaining a balanced embryo did not seem to be affected by the sex of the carrier. Similar findings have also been previously reported for reciprocal translocation carriers (8). These findings are particularly important to counsel patients prior to and during a PGD cycle. Understanding all these factors and the possible risks and outcomes of PGD are essential for effective counselling. Furthermore, evaluating couples' motives and considerations while opting for PGD would provide valuable information for improving genetic counselling and clinical care for these couples. Therefore, the results of this study provide significant information to establish a management scheme for translocation carriers undergoing PGD.

One of the drawbacks of this study was that we only analyzed the chromosomes involved in translocation. However, mitotic recombination of these chromosomes has been suggested to interrupt segregation of the normal chromosomes, leading to aneuploidies (13-18). Therefore, some aneuploid chromosomes that were not tested by FISH may have been missed. With the use of array-based comparative genomic hybridization (aCGH), translocation as well as aneuploid embryos would have been detected. However, FISH is still widely used to detect translocations (19, 20). This is mostly due to the size of breakpoints involved in translocation because in some cases, even the high-resolution aCGH cannot detect the translocated segment (21). Additionally, neither FISH nor aCGH could distinguish a balanced embryo from a normal one.

To conclude, advancing maternal age may adversely affect the chromosome complement in embryos. The chances of attaining a balanced embryo were significantly reduced in Robertsonian translocation carriers compared to those in younger females. Therefore, it is important to counsel carriers, particularly those with advanced maternal age, undergoing PGD.

Ethics Committee Approval: Ethics committee approval was received for this study from the ethics committee (Approval number: BSG0020).

Informed Consent: Written informed consent was obtained from patients who participated in this study.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept - P.T.; Design - P.T.; Supervision - P.T., M.B.; Materials - P.T., M.G., N.F.; Data Collection and/ or Processing - P.T., M.G.; Analysis and/or Interpretation - P.T.; Literature Review - P.T., N.F.; Writer - P.T.; Critical Review - P.T., M.G., N.F., M.B.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study has received no financial support.

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