Preimplantation genetic diagnosis (PGD) is offered in many in vitro fertilization (IVF) centers to improve the reproductive outcome of specific groups of patients. PGD is used to discard affected embryos in carriers of monogenic diseases and structural chromosome anomalies. The first pregnancies following PGD were reported 20 years ago by Handyside et al. in couples with sex-linked diseases Citation[1]. Since then, indications for PGD have extended to other monogenic diseases and to the analysis of structural and numerical chromosomal abnormalities. Preimplantation genetic screening (PGS), a variant of PGD, screens for numerical chromosome anomalies in couples with normal karyotypes that experience fertility problems. PGD and PGS have been performed at different developmental stages: first and second polar body biopsy, for the detection of genetic diseases or chromosome abnormalities restricted to the female; blastomere biopsy from day 3 embryos, for the identification of both maternal and paternal contribution; and trophoectoderm biopsy at blastocyst stage, on days 5 or 6.
Current indications for the analysis of chromosomal abnormalities in preimplantation embryos can be divided into two groups:
• Couples with an abnormal karyotype (PGD): carriers of structural rearrangements (Robertsonian and reciprocal translocations, inversions) and carriers of numerical abnormalities (47,XYY and 47,XXY males, and mosaic females with varying proportions of cells with karyotypes 45,X/46,XX/47,XXX);
• Infertile couples with a normal karyotype and poor reproductive outcome (PGS).
Currently, FISH is the most widely used technique for aneuploidy screening and the analysis of structural rearrangements. However, recently, several approaches towards 24-chromosome analysis have been developed. There exists a clear need for a technique to analyze all chromosomes, while producing reliable and faithful results in a short period of time to avoid embryo cryopreservation. Comparative genomic hybridization (CGH) combined with microarrays appears to be the most suitable technique, offering better resolution than conventional CGH, along with higher rates of reproducibility and the possibility of customizing the platform. Preliminary studies of array-CGH suggest a promising future for its application to PGS aneuploidy screening, but more research is needed to improve robustness and accuracy.
PGD in carriers of structural & numerical chromosomal abnormalities
In carriers of structural chromosome rearrangements, PGD offers an opportunity to decrease miscarriages and the risk of unbalanced offspring. There is a general agreement that these patients benefit from PGD. The most common structural anomalies studied in PGD programs are translocations and inversions. We have observed that the highest incidence of unbalanced embryos results from reciprocal translocations, with almost half of the cycles having all embryos unbalanced. Robertsonian translocations and inversions were similar in their percentages of unbalanced embryos. After PGD, pregnancy and implantation rates were similar among the groups, with almost no miscarriage. For Robertsonian translocations, others reported no differences between female and male carriers after PGD Citation[2]. For reciprocal translocations, a higher risk of unbalanced segregations has been described for rearrangements involving acrocentric chromosomes Citation[3]. Interestingly, in male carriers of pericentric inversions, the percentage of unbalanced gametes has been related to the size of the inverted fragment Citation[4].
Men with altered karyotypes for sex chromosomes (47,XXY, 47,XYY and mosaic 46,XY/47,XXY) can have abnormal spermatogenesis, although frequently they do not have fertility problems. For 47,XXY men, a higher incidence of abnormal embryos was reported when compared with a control group – mostly for sex chromosome aneuploidy, but also for chromosomes 18 and 21, and for haploid embryos Citation[5]. In a group of 47,XYY patients, those with abnormal FISH on sperm showed increases of diploidy and sex chromosome disomy. The chromosomal abnormalities of their spermatozoa appear to be reflected in their embryos, with a high incidence of triploid embryos and aneuploid embryos detected for chromosomes 13, 16 and the sex chromosomes, respectively Citation[6].
Preimplantation genetic screening to improve IVF outcome
Current indications for PGS include advanced maternal age (AMA), recurrent miscarriage (RM), repetitive implantation failure (RIF), and severe male factor infertility (SMF). In PGS programs, the most widely employed technique for cytogenetic analysis of blastomeres has been FISH for a selected panel of chromosomes. The chromosomes most commonly tested are 13, 15, 16, 17, 18, 21, 22, X and Y, which detect 85% of embryo aneuploidies.
Advanced maternal age
Aneuploidies are common in early human embryos Citation[7,8]. Furthermore, aneuploidy rates are higher in oocytes and embryos from women of AMA Citation[9], probably stemming from meiotic recombination defects exacerbated by age Citation[10]. The age-related defects result in higher aneuploidy rates in the offspring, particularly for chromosomes 13, 18, 21, X and Y, and increase spontaneous abortions, thereby reducing ongoing implantation rates Citation[11]. In PGS cycles, an increase in implantation and pregnancy rates for women of AMA has been observed in several retrospective studies Citation[12–17]. However, the benefits of PGS came under debate following the publication of several prospective randomized controlled trials (RCTs).
Recurrent miscarriage
Despite the existence of known causes of RM, such as uterine, genetic, endocrine and immune factors, almost 50% of RM cases remain unexplained. An increase in numerical chromosome abnormalities has been reported in preimplantation embryos from women with RM, which may cause the poor reproductive outcome in these patients Citation[18]. PGS has been applied in these couples, with the aim of decreasing spontaneous abortions. Recently, two PGS studies have identified several subpopulations of RM patients with better outcomes: couples with two to four previous losses; couples in which an increase in sperm aneuploidy rates had been identified; couples with a previous abnormal miscarriage; and women who are 37 years old or younger Citation[19,20]. These reports are of retrospective observational studies, and no RCT has been published for this indication.
Repetitive implantation failure
Couples are diagnosed with RIF after three or more failed IVF attempts, or failed IVF treatments after cumulative transfer of more than ten good-quality embryos. Although not all RIF cases are due to embryonic defects, many have more chromosomal abnormalities in their embryos compared with control groups Citation[21]. The number of anomalies detected increases with the number of previous failed IVF cycles Citation[22]. Furthermore, among RIF patients, better results have been obtained in patients under the age of 40 years old Citation[23].
Severe male factor infertility
Numerous conditions have been considered as SMF in PGS programs: impaired sperm parameters, mainly severe oligozoospermia, azoospermia, and teratozoospermia; increased incidence of chromosome abnormalities in spermatozoa; abnormal male meiosis; and chemotherapy. PGS studies in SMF patients have reported a higher incidence of chromosome abnormalities in embryos from infertile men with altered sperm parameters. In azoospermic patients, higher rates of mosaic and chromosomally abnormal embryos have been reported, most frequently for the sex chromosomes Citation[24,25]. In teratozoospermic patients, it is harder to elucidate which morphological dysmorphism could imply a higher rate of embryo aneuploidy. Sperm aneuploidy rates and sperm head anomalies Citation[26] have been correlated with higher incidences of abnormal embryos in patients with macrocephalic spermatozoa Citation[27,28]. Kahraman et al. reported that when PGS was performed implantation rates and miscarriage rates were better than when intracytoplasmic sperm injection cycles did not include PGS Citation[27].
Prospective randomized trials
Several RCTs comparing pregnancy and live birth outcomes with and without PGS have been reported for AMA and RIF patients. Four RCTs have been published for AMA, with three of them indicating that PGS offered no benefit Citation[29–31]. The fourth study, however, described lower miscarriage rates and increased delivery rates Citation[32]. The only RCT of RIF patients concluded that no significant differences existed in clinical pregnancy rates between PGS and control groups Citation[33]. However, these studies have been criticized by several authors, who argue that some important pitfalls exist in the study methodology, such as patient inclusion criteria, embryo biopsy procedures, embryo culture conditions and type of genetic analyses performed. Some clinicians propose that more benefits would be obtained if the complete set of chromosomes could be tested. For this reason IVF programs are moving towards employing more sophisticated analyses, such as array-CGH. This approach would offer the most complete analysis of the embryo by providing information regarding all 24 chromosomes, as well as additional genomic data customized to the patient. Future developments for aneuploidy screening in preimplantation embryos may focus on polar body and trophectoderm biopsy using arrays for 24 chromosome analysis Citation[34–37].
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
References
- Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature344, 768–770 (1990).
- Keymolen K, Staessen C, Verpoest W et al. A proposal for reproductive counselling in carriers of Robertsonian translocations: 10 years of experience with preimplantation genetic diagnosis. Hum. Reprod.24, 2365–2371 (2009).
- Lim CK, Cho JW, Song IO, Kang IS, Yoon YD, Jun JH. Estimation of chromosomal imbalances in preimplantation embryos from preimplantation genetic diagnosis cycles of reciprocal translocations with or without acrocentric chromosomes. Fertil. Steril.90, 2144–2151 (2008).
- Anton E, Blanco J, Vidal F. Recombination in heterozygote inversion carriers. Hum. Reprod.22, 1192 (2007).
- Staessen C, Tournaye H, Van Assche E et al. PGD in 47,XXY Klinefelter’s syndrome patients. Hum. Reprod. Update9, 319–330 (2003).
- Rodrigo L, Peinado V, Mateu E et al. Impact of different patterns of sperm chromosomal abnormalities on the chromosomal constitution of preimplantation embryos. Fertil. Steril.94(4), 1380–1386 (2010).
- Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH, Delhanty JD. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat. Diagn.15, 41–49 (1995).
- Munne S, Cohen J. Chromosome abnormalities in human embryos. Hum. Reprod. Update4, 842–855 (1998).
- Hassold T, Chen N, Funkhouser J et al. A cytogenetic study of 1000 spontaneous abortions. Ann. Hum. Genet.44, 151–178 (1980).
- Lamb NE, Freeman SB, Savage-Austin A et al. Susceptible chiasmate configurations of chromosome 21 predispose to non-disjunction in both maternal meiosis I and meiosis II. Nat. Genet.14, 400–405 (1996).
- Boue A, Boue J, Gropp A. Cytogenetics of pregnancy wastage. Adv. Hum. Genet.14, 1–57 (1985).
- Rubio C, Simon C, Vidal F, Rodrigo L et al. Chromosomal abnormalities and embryo development in recurrent miscarriage couples. Hum. Reprod.18, 182–188 (2003).
- Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil. Steril.72, 837–844 (1999).
- Rubio C, Rodrigo L, Perez-Cano I et al. FISH screening of aneuploidies in preimplantation embryos to improve IVF outcome. Reprod. Biomed. Online11, 497–506 (2005).
- Munne S, Magli C, Cohen J, Morton P et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum. Reprod.14, 2191–2199 (1999).
- Munne S, Sandalinas M, Escudero T et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod. Biomed. Online7, 91–97 (2003).
- Munne S, Chen S, Fischer J et al. Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages. Fertil. Steril.84, 331–335 (2005).
- Pellicer A, Rubio C, Vidal F et al.In vitro fertilization plus preimplantation genetic diagnosis in patients with recurrent miscarriage: an analysis of chromosome abnormalities in human preimplantation embryos. Fertil. Steril.71, 1033–1039 (1999).
- Garrisi JG, Colls P, Ferry KM, Zheng X, Garrisi MG, Munne S. Effect of infertility, maternal age, and number of previous miscarriages on the outcome of preimplantation genetic diagnosis for idiopathic recurrent pregnancy loss. Fertil. Steril.92, 288–295 (2009).
- Rubio C, Buendía P, Rodrigo L et al. Prognostic factors for preimplantation genetic screening in repeated pregnancy loss. Reprod. Biomed. Online18, 687–693 (2009).
- Pehlivan T, Rubio C, Rodrigo L et al. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod. Biomed. Online6, 232–237 (2003).
- Gianaroli L, Magli MC, Munne S, Fiorentino A, Montanaro N, Ferraretti AP. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum. Reprod.12, 1762–1767 (1997).
- Taranissi M, El-Toukhy T, Gorgy A, Verlinsky Y. Influence of maternal age on the outcome of PGD for aneuploidy screening in patients with recurrent implantation failure. Reprod. Biomed. Online10, 628–632 (2005).
- Rodrigo L, Rubio C, Mateu E et al. Analysis of chromosomal abnormalities in testicular and epididymal spermatozoa from azoospermic ICSI patients by fluorescence in situ hybridization. Hum. Reprod.19, 118–123 (2004).
- Gianaroli L, Magli MC, Ferraretti AP, Iammarrone E. Preimplantation diagnosis after assisted reproduction techniques for genetically-determined male infertility. J. Endocrinol. Invest.23, 711–716 (2000).
- Cinar C, Yazici C, Ergunsu S et al. Genetic diagnosis in infertile men with numerical and constitutional sperm abnormalities. Genet. Test.12, 195–202 (2008).
- Kahraman S, Sertyel S, Findikli N et al. Effect of PGD on implantation and ongoing pregnancy rates in cases with predominantly macrocephalic spermatozoa. Reprod. Biomed. Online9, 79–85 (2004).
- Mateu E, Rodrigo L, Prados N et al. High incidence of chromosomal abnormalities in large-headed and multiple-tailed spermatozoa. J. Androl.27, 6–10 (2006).
- Staessen C, Platteau P, Van Assche E et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum. Reprod.19, 2849–58 (2004).
- Mastenbroek S, Twisk M, van Echten-Arends J et al.In vitro fertilization with preimplantation genetic screening. N. Engl. J. Med.357, 9–17 (2007).
- Hardarson T, Hanson C, Lundin K et al. Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial. Hum. Reprod.23, 2806–2812 (2008).
- Schoolcraft WB, Katz-Jaffe MG, Stevens J, Rawlins M, Munne S. Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial. Fertil. Steril.92, 157–162 (2009).
- Blockeel C, Schutyser V, De Vos A et al. Prospectively randomized controlled trial of PGS in IVF/ICSI patients with poor implantation. Reprod. Biomed. Online17, 848–854 (2008).
- Hellani A, Abu-Amero K, Azouri J, El-Akoum S. Successful pregnancies after application of array-comparative genomic hybridization in PGS-aneuploidy screening. Reprod. Biomed. Online17, 841– 847 (2008).
- Johnson DS, Gemelos G, Baner J et al. Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol. Hum. Reprod.25, 1066–1075 (2010).
- Geraedts J, Collins J, Gianaroli L et al. What next for preimplantation genetic screening? A polar body approach! Hum. Reprod.2, 575–577 (2010).
- Treff NR, Levy B, Su J, Northrop LE, Tao X, Scott RT Jr. SNP microarray-based 24 chromosome aneuploidy screening is significantly more consistent than FISH. Mol. Hum. Reprod.16, 583–589 (2010).