RBMOnline - Vol 11. No 4. 2005 497 506 Reproductive BioMedicine Online; www.rbmonline.com/article/1712 on web 11 August 2005 Article FISH screening of aneuploidies in preimplantation embryos to improve IVF outcome Trained in science in the University of Valencia, Spain, Dr Carmen Rubio specialized in studies in human reproduction, partly in the University of Barcelona. Becoming interested in chromosomal abnormalities in human embryos, she completed her PhD in 2004 in Valencia. She has since concentrated on human IVF and genetics, especially chromosomal genetics. She belongs to several Spanish learned societies and to ESHRE. Dr Carmen Rubio Carmen Rubio 1, Lorena Rodrigo 1, Inmaculada Pérez-Cano 2, Amparo Mercader 1, Emilia Mateu 1, Pilar Buendía 1, José Remohí 1, Carlos Simón 1, Antonio Pellicer 1,3 1 Instituto Valenciano de Infertilidad (IVI-Valencia), University of Valencia, Plaza Policía local No. 3, 46015 Valencia, Spain; 2 Instituto Valenciano de Infertilidad (IVI-Murcia), Navegante Macías del Poyo No. 5, 3007 Murcia, Spain; 3 Correspondence: Tel: +34 96 3050900; Fax: +34 96 3050999; e-mail: apellicer@ivi.es Abstract Preimplantation genetic diagnosis (PGD) has transformed the approach to the infertility patient in the IVF setting. Although the principal applications of PGD have been to prevent the transmission of sex-linked diseases, in time and with growing knowledge of the chromosomal abnormalities observed in preimplantation embryos, its applications have widened. Nowadays, apart from its implications in the prevention of transmission of chromosomal and genetic abnormalities, PGD is being used with increased frequency to improve the IVF outcome in patients with advanced maternal age ( 38 years of age), recurrent miscarriage ( 2 miscarriages), recurrent IVF failure ( 3 failed IVF attempts) and severe male infertility. A high incidence of chromosomal abnormalities has been observed in these patient groups. Keywords: advanced maternal age, aneuploidy, implantation failure, male factor, preimplantation genetic diagnosis, recurrent abortion Introduction With the introduction of preimplantation genetic diagnosis (PGD) to the field of reproductive medicine and the analysis of a single blastomere from a day 3 embryo using multicolour fluorescence in-situ hybridization (FISH), it is possible to detect chromosomal abnormalities and inherited diseases without adversely affecting the developmental (Hardy et al., 1990) or implantation potential of the embryo (Gianaroli et al., 1999; Munné et al., 2003a). As the effect of PGD in improving the outcome of IVF cycles is clarified, the indications for PGD are being extended. Approximately 30% of human embryos generated from IVF treatments have an abnormal chromosome constitution (Márquez et al., 2000; Rubio et al., 2003). This percentage may increase to 60 70% in embryos that come from poor prognosis IVF patients such as low responders, older women (Munné et al., 1995, 2002; Kahraman et al., 2000), IVF failure (Magli et al., 1998a; Gianaroli et al., 1999, Pehlivan et al., 2003; Wilding et al., 2004), or women with a history of recurrent miscarriage (Simón et al., 1998; Vidal et al., 1998; Pellicer et al., 1999; Rubio et al., 2003; Werlin et al., 2003; Wilding et al., 2004). Many efforts have been made to find a criterion good enough to select the best embryos for transfer, to improve implantation rates and also to reduce the number of embryos to be transferred without lowering success rates (Alikani et al., 2000). Morphology of human embryos has been considered an important parameter that seems to be related to chromosome anomalies. It has been shown that dysmorphic and slowly developing or arrested embryos have significantly more polyploidy and mosaicism than normally developing human embryos (Munné et al., 1995; Márquez et al., 2000). Increased incidence of aneuploidy and multinucleation has also been observed in embryos with a 497
498 high degree of fragmentation and in embryos with irregular or unevenly sized blastomeres (Hardarson et al., 2001). Regarding embryo development, only around 25% of aneuploid embryos achieved blastocyst stage compared with 62% of euploid human embryos (Rubio et al., 2003). Interestingly, a low percentage of monosomies were found at blastocyst stage, and an important percentage of trisomic embryos achieved blastocyst stage, agreeing with the results observed in spontaneous abortions (Sandalinas et al., 2001; Rubio et al., 2003). Since aneuploid human embryos may still achieve blastocyst stage, replacement of such abnormal embryos in the uterus may in part be responsible for the miscarriages and the reduction of both pregnancy and implantation rates in some IVF patients. Autosomal trisomies are the most common group of abnormalities in spontaneous abortions in couples with normal karyotypes (26.8%), followed by polyploidies (9.8%) and monosomy X (8.6%) (Hassold et al., 1996). In spontaneous abortions, chromosome 16 is the most common trisomy, followed by chromosomes 22 and 21, and more recently, a high incidence of trisomy 15 has been also reported in fetal tissue from recurrent aborters (Stephenson et al., 2002). Aneuploidy screening for chromosomes X, Y, 13, 18, 21 was first performed to eliminate the risk of trisomic offspring (Munné et al., 1993), and lately chromosomes 1, 15, 16, 17, and 22 have also been analysed to decrease spontaneous abortions and improve implantation rates (Vidal et al., 1998; Bahce et al., 1999; Munné et al., 2002). More recently, up to 13 chromosomes have been included in three hybridization rounds (Munné et al., 2003b), although only 3% of the abnormal embryos would have been missed without the third round, agreeing with the studies in fetal tissue with 83% of the total number of chromosomal abnormalities detected by FISH for chromosomes X, Y, 13, 15, 16, 18, 21 and 22 (Jobanputra et al., 2002). This manuscript presents the results of a programme for FISH screening of aneuploidies in patients with advanced maternal age (AMA), recurrent miscarriage (RM), previous IVF failures (IF) and couples with severe male factor infertility (MFI). Materials and methods Patients In this study, PGD cycles performed in the centre in Valencia from February 1996 to May 2004 were included, and the different groups are detailed below. Control group The control group included 25 PGD cycles in couples with sexlinked diseases that were performed during the same time period and were included to compare the incidence of chromosomal abnormalities with the study groups. Women were <37 years of age (mean age: 33.1 ± 2.5) and the FISH protocol was similar to the study groups. Advanced maternal age A total of 341 PGD cycles were performed in women 38 years of age (mean age: 40.5 ± 2.1). In this group, the results were also analysed separately in women aged 38 years (n = 38), 39 years (n = 71), 40 years (n = 89), 41 years (n = 41), 42 years (n = 36), 43 years (n = 23) and 44 years (n = 27). Recurrent miscarriage The inclusion criteria were couples with two or more previous miscarriages of unknown aetiology after the appropriate infertility work-up, including parental karyotypes (Thornhill et al., 2005). A total of 241 PGD cycles were included, 163 of them in women <37 years of age (mean age: 33.3 ± 2.4 and mean number of previous abortions: 3.1 ± 1.2) and 78 cycles in women 37 years (mean age: 38.7 ± 1.7 and mean number of previous abortions: 2.4 ± 1.0). Implantation failure This group included 129 cycles in women <37 years of age with 3 previous IVF failures (mean age: 33.2 ± 2.4 and mean IVF failures: 3.9 ± 1.7) and 65 cycles in women 37 years with 2 IVF failures (mean age: 39.3 ± 1.7 and mean IVF failures: 3.9 ± 1.4). In total, 194 PGD cycles were carried in couples with implantation failure. Male factor infertility A total of 105 PGD cycles were performed because of male factor infertility: obstructive azoospermia: 20 cycles (women mean age: 32.9 ± 3.3); non-obstructive azoospermia: 18 cycles (women mean age: 33.4 ± 3.0); increased incidence of sperm chromosomal abnormalities as analysed by FISH: 41 cycles (women mean age: 31.8 ± 3.0); severe oligozoospermia defined for this study as sperm concentration 5 10 6 spermatozoa/ml: 17 cycles (women mean age: 31.5 ± 3.6); teratozoospermia defined for this study as 1% of spermatozoa with normal morphology: nine cycles (women mean age: 32.5 ± 2.3). IVF and embryo biopsy Stimulation, oocyte retrieval and intracytoplasmic sperm injection (ICSI) procedures were performed as described previously (Meseguer et al., 2003). Pronuclear zygote morphology was assessed at 16 18 h post-icsi and embryo development was checked every 24 h. Embryo biopsy was performed on day 3 developing embryos with 5 nucleated blastomeres and 25% fragmentation degree. Two blastomeres were analysed when possible. For the biopsy, embryos were placed on a droplet containing Ca 2+ - and Mg 2+ -free medium (EB-10; Scandinavian IVF, Göteborg, Sweden/G-PGD; Vitrolife, Göteborg, Sweden) and Tyrode s solution (ZD-10; Scandinavian IVF) was used to perforate the zona pellucida (Rubio et al., 2003). More recently, laser technology has been incorporated in the laboratory for the drilling of the zona pellucida (OCTAX, Herbron, Germany). An average of 6 8 ms shots with a power in focus of 100 150 mw were needed to make a 30 μm hole. After the biopsy, embryos were carefully washed and co-cultured on a monolayer of endometrial epithelial cells (Mercader r et al., 2003). Euploid embryos were transferred on day 5 at morula or blastocyst stage and arrested embryos or embryos with abnormal or inconclusive results were never transferred.
FISH protocol FISH protocol for aneuploidy screening was as follows. Up to November 2002, a first round was performed using locus specific probes for chromosomes 13 and 21, in a second round and after signal elimination (Vidal et al., 1998), blastomeres were hybridized for chromosomes 16 and 22. Finally, in the third round, triple FISH was performed for chromosomes X, Y and 18. Since November 2002, FISH protocol for aneuploidy screening has changed in the laboratory and two rounds are now currently performed: the first round includes chromosomes 13, 16, 18, 21 and 22 (Multivysion PB; Vysis Inc., Downers Grove, IL, USA) and the second round included sex chromosomes. From January 2004, chromosome 15 was also included in the second hybridization round, particularly in couples with advanced maternal age and recurrent miscarriage. A third round with subtelomeric DNA probes was incorporated from the beginning of 2003 for those embryos with unclear signals in the previous rounds and also to decrease the risk of false monosomies for the tested chromosomes (Rodrigo et al., 2004). All probes were commercially available from Vysis Inc. Detection washings and signal scoring were carried out following manufacturer s instructions. Statistical analysis For statistical comparison between groups, chi-squared analysis and Fisher s exact test were used. The statistical analysis was carried out using the Graphpad Instat v. 2.05a package (Graphpad Software, San Diego, CA, USA). In this study, pregnancies were always considered as clinical pregnancies with the detection of a gestational sac on the vaginal ultrasound performed on week 5 of gestation. Results Advanced maternal age Table 1 shows a significant increase in the percentage of abnormal embryos in AMA patients compared with the control group of women <37 years of age (70.3 versus 33.1%, P < 0.05). Clinical pregnancy and implantation rates observed in advanced maternal age patients were 28.8 and 26.5% respectively and the percentage of clinical miscarriages was 15.8%. Figure 1 represents aneuploidy rates per chromosome regarding the number of embryos informative for each chromosome, and a significant (P < 0.05) increase in aneuploidy rates was observed in the tested chromosomes compared with the control group. A high incidence of abnormal embryos was also observed for chromosome 15 (21.8%) when it was included in some of the cases. Figure 2 represents the relationship between the incidence of abnormal embryos and the maternal age, ranging from 54.5% of abnormal embryos in women aged 38 years to 90% in women aged 44 years. Ongoing pregnancy rates appeared to be inversely related to maternal age, ranging from 39.4% in women of 38 years to 0% in women 44 years. Figure 3 shows the results of the PGD programme in AMA patients compared with patients of the same age in whom PGD was not performed. Recurrent miscarriage Aneuploidy screening was performed in a total of 241 cycles in couples with normal karytypes with significantly increased incidence of abnormal embryos compared with the control group (66.1 versus 33.1%, P < 0.05), high pregnancy rates (36.5%) and low miscarriage rate (12.8%) (Table 2). In the group of women <37 years of age, the incidence of chromosomal abnormalities was 63.5% and was significantly increased compared with the control group (33.1%, P < 0.05) with similar pregnancy, implantation and miscarriage rates. In this age group, there was a significant (P P < 0.05) increase in the incidence of abnormal embryos for chromosomes, 13, 16, 21 and 22, without statistical differences for sex chromosomes and chromosome 18 (Figure 4). The incidence of aneuploidies for chromosome 15 was 29%. Figure 5 shows the inverse relationship between the number of previous miscarriages and the percentage of abnormal embryos in women <37 years of age, with similar levels of abnormal embryos between the control group and RM couples with six or more miscarriages. Implantation failure Experience suggests that in a total of 194 PGD cycles of patients with previous IVF attempts, high incidence of chromosomal abnormalities (64.3%) and acceptable pregnancy and implantation rates were obtained (32.5 and 21.4% respectively), but these results were highly dependent on the age of the patients, younger women having a better prognosis (Table 3). In IF patients <37 years of age, all the tested chromosomes showed significant increases in abnormal embryos compared with the control group (P P < 0.05) and the percentage of aneuploid embryos for chromosome 15 was 23.8% (Figure 6). Maternal age effect was particularly remarkable in miscarriage rates, with a high miscarriage rate in patients 37 years (30.8%), whereas in women <37 years, this value was within the normal range (13.5%). Male factor infertility In couples with azoospermia, PGD revealed an increased incidence of abnormal embryos compared with controls, more remarkable in non-obstructive azoospermia (52.6% in obstructive azoospermia and 69.7% in non-obstructive azoospermia) (Table 4). In these cycles, it is important to mention the high incidence of mosaicism as well as sex chromomosome aneuploidy (Figure 7). Pregnancy (33.3 and 45.4%) and implantation rates (18.4 and 47.1%) in obstructive and non-obstructive azoospermia were similar to the control group; however, there was a high incidence of miscarriages in couples with non-obstructive azoospermia compared with controls (25.0 versus 8.3%). 499
Table 1. Preimplantation genetic diagnosis results in couples of advanced maternal age (AMA) compared with controls. AMA Control a No. cycles 341 25 No. embryos analysed 1375 169 % of abnormal embryos 70.3 b 33.1 Embryos transferred (mean ± SD) 1.5 ± 0.6 2.1 ± 1.1 % of transfers 58.1 96.0 % pregnancies/transfer 28.8 50.0 Implantation rate (%) 26.5 33.3 % Miscarriages (n) 15.8 (9) 8.3 (1) a Couples with sex-linked diseases: see text. b P < 0.05 compared with control group (% of abnormal embryos). Figure 1. Percentage of abnormalities for each chromosome in AMA patients compared with controls (couples with sex-linked diseases: see text). *P < 0.05 compared with control group (% abnormal embryos/embryos analysed for each chromosome). Figure 2. Percentage of abnormal embryos and ongoing pregnancy rate according to age in AMA patients. 500 Figure 3. Ongoing pregnancy rates in AMA patients with and without PGD.
Table 2. Preimplantation genetic diagnosis results in couples with recurrent miscarriage, grouped according to age, compared with controls. <37 years 37 years Total Control a No. cycles 163 78 241 25 No. embryos analysed 965 382 1347 169 % of abnormal embryos 63.5 b 72.7 b 66.1 b 33.1 Embryos transferred (mean ± SD) 1.9 ± 0.7 1.6 ± 0.7 1.8 ± 0.7 2.1 ± 1.1 % of transfers 78.5 64.1 73.1 96.0 % pregnancies/transfer 39.1 30.0 36.5 50.0 Implantation rate (%) 33.3 22.0 26.4 33.3 % Miscarriages (n) 10.0 (5) 20.0 (2) 12.3 (8) 8.3 (1) a Couples with sex-linked diseases: see text. b P < 0.05 compared with control group (% of abnormal embryos). Figure 4. Percentage of abnormalities for each chromosome in RM patients <37 years of age compared with controls (couples with sex-linked diseases: see text). *P < 0.05 compared with control group (% abnormal embryos/embryos analysed for each chromosome). Figure 5. Percentage of abnormalities in RM patients <37 years compared with control group (couples with sex-linked diseases: see text), according to number of previous miscarriages. *P < 0.05 compared with control group (% abnormal embryos/ embryos analysed for each chromosome). Table 3. Preimplantation genetic diagnosis results in couples with implantation failure compared with controls. <37 37 Total Control a No. cycles 129 65 194 25 No. embryos analysed 810 348 1158 169 % of abnormal embryos 61.2 b 71.5 b 64.3 b 33.1 Embryos transferred (mean ± SD) 1.9 ± 0.7 1.7 ± 0.9 1.9 ± 0.8 2.1 ± 1.1 % of transfers 83.6 72.3 79.4 96.0 % pregnancies/transfer 34.6 27.7 32.5 50.0 Implantation rate (%) 24.0 14.6 21.4 33.3 % Miscarriages (n) 13.5 (5) 30.8 (4) 22.5 (9) 8.3 (1) a Couples with sex-linked diseases: see text. b P < 0.05 compared with control group (% of abnormal embryos). 501
Figure 6. Percentage of abnormalities for each chromosome in IF patients compared with controls (couples with sex-linked diseases: see text). P < 0.05 compared with control group (% abnormal embryos/embryos analysed for each chromosome). Table 4. Preimplantation genetic diagnosis results in couples with severe male factor. FISH = fluorescence in-situ hybridization. Abnormal Oligozoospermia Teratozoospermia Obstructive Non-obstructive Control FISH azoospermia azoospermia No. cycles 41 17 9 20 18 25 No. embryos 195 74 59 114 76 169 analysed % of abnormal 57.4 b 43.2 55.9 b 52.6 b 69.7 b 33.1 embryos Embryos transferred 1.6 ± 0.6 2.0 ± 0.3 1.8 ± 0.6 2.2 ± 0.6 1.6 ± 0.5 2.1 ± 1.1 (mean ± SD) % of transfers 79.3 94.1 100 90.0 61.1 96.0 % pregnancies/transfer 52.2 50.0 66.7 33.3 45.4 50.0 Implantation rate (%) 42.1 31.2 56.2 18.4 47.1 33.3 % Miscarriages (n) 8.3 (1) 0 0 0 25.0 (2) 8.3 (1) a Couples with sex-linked diseases: see text. b P < 0.05 compared with control group (% of abnormal embryos). 502 Figure 7. Incidence of mosaicism and sex chromosome aneuploidy in male factor. *P < 0.05, **P < 0.05 compared with control group (% abnormal embryos/embryos analysed for each chromosome). OA = obstructive azoospermia; NOA = non-obstructive azoospermia; AbFISH = previous abnormal fluorescence in-situ hybridisation analysis; oligo = oligozoospermia; terato = teratozoospermia; control group consisted of couples with sex-linked diseases: see text.
Figure 8. Comparison of implantation rates of PGD programme in advanced maternal age (AMA), recurrent miscarriage (RM), implantation failure (IF) and male factor infertility (MFI) with implantation rates of IVF/ICSI programme in patients without these reproductive problems. PGD was performed in 41 cycles in which an increased incidence of chromosomal abnormalities was observed in previous sperm FISH studies (Table 4). The most frequent abnormality observed in spermatozoa was sex chromosome disomy, followed by increased incidence in diploid spermatozoa. In PGD cycles, the incidence of abnormal embryos was 57.4%, with a high incidence of mosaicism and sex chromosome aneuploidies compared with the control group. Pregnancy and implantation rates in these couples were 52.9 and 38.3%, with miscarriage rates similar to controls (11.1 versus 8.3%). Severe oligozoospermia (without previous FISH analysis on spermatozoa) was an indication for PGD in 17 cycles, and the incidence of chromosomal abnormalities was not significantly increased compared with controls (43.2 versus 33.1%) (Table 4). However, there was an increased incidence of abnormal embryos in patients with teratozoospermia compared with controls (55.9 versus 33.1%), with an increased percentage of embryos with sex chromosome abnormalities and mosaicism (Figure 7). Pregnancy and implantation rates were 50.0 and 31.2% respectively in oligozoospermic patients and 66.7 and 56.2% respectively in teratozoospermic patients, without any miscarriage after PGD in both groups. Additionally, Figure 8 shows the implantation rates for PGD in the indications mentioned above, compared with the results of the IVF/ICSI programme in patients without these reproductive problems. Similar implantation rates were achieved every year in the couples with poor prognosis attending the clinic, compared with the couples with better prognosis. Discussion Advanced maternal age This study showed an increased incidence of chromosomal abnormalities in AMA patients, with acceptable ongoing pregnancy rates with PGD until the age of 42 and poorer prognosis in women over 42 years of age. The results in AMA patients were quite similar in the present series to the ESHRE Consortium data (2002) in the percentage of abnormal embryos (70.3 and 66.2% respectively). Pregnancy rates were 28.8% in the present series and 35.9% in the ESHRE data collection. In evaluating this difference, it is important to underline that the patients reported at the consortium were >35 years of age, whereas the present group of patients were older ( 38), and probably displayed higher FSH concentrations and lower response to gonadotrophins, limiting the number of embryos available for analysis and transfer (2.4 in the Consortium data versus 1.5 in this series). Kuliev and Verlinsky reported their experience in more than 3000 clinical cycles indicating a positive impact of the selection and transfer of euploid embryos in women of advanced reproductive age and with an aneuploidy rate over 50% (Kuliev and Verlinsky, 2003, 2004). To properly assess the benefits of PGD in AMA patients, several prospective controlled studies have been developed. In a randomized controlled study by Gianaroli et al. (1999), patients older than 36 years of age were distributed to either PGD or assisted hatching. Ongoing implantation rates were significantly higher in the PGD group (25.0 versus 11.6) (Gianaroli et al., 1999). In the same study, when results were compared in subgroups according to age, the differences between the control group and PGD group become more obvious in patients 38 years of age. The vast drop in implantation rates normally observed in patients older than 37 years of age was overcome by the use of PGD, and resulted in implantation rates similar to young patients. In 2003, preliminary results of another prospective randomized study were published. In this study the control group was formed of day 3 or day 5 embryo transfers and pregnancy rates were 43% in the PGD group and 25% in the control group; however, no data were reported regarding miscarriage rates (Werlin et al., 2003). More recently, Staessen et al. (2004) have described higher ongoing implantation rates in PGD compared with day 5 embryo transfer, but these differences did not reached statistical significance (16.5 versus 10.4). However, they found statistical differences in the mean number of embryos transferred in each group, with higher number in the blastocyst transfer without PGD with the result 503
504 of higher risk of twin and triplet pregnancies. Therefore, PGD is considered an efficient treatment in AMA patients for two reasons: one reason is the high ongoing pregnancy and implantation rates that can be reached, decreasing the risk of multiple pregnancies, and another important reason is the diagnostic value of PGD, particularly in those couples with 100% abnormal embryos. Recurrent miscarriage These couples had a significant (P < 0.05) increase in abnormal embryos compared with controls, and with the transfer of euploid embryos miscarriage could be lowered to the values observed in the control population without this reproductive problem. The incidence of aneuploidies for chromosome 15 was 29%, showing that chromosome 15 played an important role in recurrent miscarriage as suggested by the studies in fetal tissue (Stephenson et al., 2002; Jalal et al., 2004). Another important issue in RM is the number of previous miscarriages. Cytogenetic studies in abortions have revealed that the incidence of aneuploidies is 63% in women with two miscarriages, and this figure decreased to <30% in women with six or more miscarriages (Ogasawara et al., 2000). Similar results have been observed in this RM series in patients <37 years, with aneuploidy levels that reached control values in couples with six or more miscarriages, showing that factors different to embryonic chromosomal abnormalities should be implicated in these high order aborters (Rubio et al., 2005). Compared with the last ESHRE data collection publication (2002), the results were similar, although the percentage of chromosomally abnormal embryos observed in the present patient population was higher (66.1 versus 55.7%); the pregnancy rates per transfer were similar with 36.5% in the present group and 31.6% reported in the ESHRE Consortium. The average number of embryos transferred in this series was lower than the consortium (1.8 versus 2.2). Regarding prospective studies in RM patients, there is only one publication with only 19 patients enrolled (Werlin et al., 2003). In this study, pregnancy rate per transfer in the PGD group was 63.6 and 37.5% in the control group of day 3/day 5 embryo transfer. Again in this study, although there was higher pregnancy rate in the PGD group, the number of cycles was too small to draw any clinical conclusion. Implantation failure The rate of chromosomal abnormalities observed in the present patient population was similar to the rates reported at the ESHRE Consortium (64.3 versus 60.8%); however, the pregnancy rate per transfer was strikingly higher than that reported by the consortium (32.5 versus 11%), with the average number of embryos transferred being similar. It is important to underline the possible effect of the high percentage of mosaicism observed in this patient group (Gianaroli et al., 1997, 1999; Magli et al., 1998). Since at the study centre two blastomeres are routinely biopsied when possible, it is feasible to discard more abnormalities than would be the case if only one blastomere was analysed. This fact might contribute to the successful pregnancy outcome achieved at the study centre for IF patients, since a high selection for chromosomally normal embryos was being applied, aiming at discarding mosaicism when possible. In a prospective study by Gianaroli et al. (1999), the implantation and pregnancy rates were similar in IF patients who underwent PGD and in control groups which underwent assisted hatching. In their prospective studies Werlin et al. (2003), described improved pregnancy rates with PGD in AMA and RM patients; however, they did not find this benefit in patients with more than two failed cycles compared with day 3/day 5 embryo transfer. In a recent study by Shapiro et al. (2001), pregnancy and implantation rates were shown to decline dramatically in repeated cycles of IVF with blastocyst transfer, suggesting that merely using blastocyst culture in repeated IVF attempts may not improve the clinical outcome. The culture of embryos to the blastocyst stage has been used by certain centres in an attempt to improve the outcome of implantation failure. In a previous study (Simón et al., 1999), the outcome of co-culturing embryos to the blastocyst stage in oocyte donation patients with IF showed favourable results; however, the results were not so promising when the patient s own oocytes were used. Recent studies made in preimplantation embryos show that IF patients have a higher number of chromosomally abnormal embryos; therefore, PGD in implantation failure patients is becoming widely used. Moreover, the number of failed IVF attempts stands out as a possible predictor of the chromosomal abnormality rate and is directly proportional to the number of failed IVF attempts. Whereas the chromosomal abnormality rate was around 40% in patients with two failed IVF cycles, the abnormality rate increased sharply to 50% in patients with three failed IVF cycles and to 67% in those with more than five IVF failures. These abnormalities were mainly due to mosaicism (Gianaroli et al., 1997; Magli et al., 1998). However, another study in couples with two or more previous failed cycles (Munné, 2003) did not find differences in the percentage of abnormal embryos compared with controls. Taranissi et al. (2005) found a strong effect of maternal age in the PGD results in IF patients, with higher rates of chromosomal abnormalities and lower pregnancy and implantation rates in IF patients aged 41. The present study found similar results in IF patients aged 37 with increased incidence of chromosomal abnormalities and decreased ongoing pregnancy rates. Therefore, the most effective approach in IF patients are still under debate. Male factor infertility In these couples, it is important to mention not only the percentage of abnormal embryos, but also the high incidence of mosaicism as well as sex chromosome aneuploidy. Similar results have been observed by other groups with high incidence of sex chromosome aneuploidy and mosaicism, mostly in couples in whom ICSI with testicular spermatozoa was performed (Gianaroli et al., 2000; Silber et al., 2003; Platteau et al., 2004). PGD has also been proposed in ICSI couples with severe oligozoospermia and/or teratozoospermia, showing a
correlation in the incidence of abnormal embryos, mainly for sex chromosomes, with the severity of the affected sperm parameters, (Gianaroli et al., 2000; Ludwig et al., 2001). In patients with teratozoospermia, it is difficult to find a clear relationship with particular sperm defects. There is a recent retrospective study comparing PGD versus ICSI in patients with macrocephalic spermatozoa. PGD offered improved implantation rates compared with ICSI (25.0 versus 12.3%) and overall, there was a significant decrease in miscarriage rates after PGD (14.3 versus 46.7%) (Kahraman et al., 2004). However, no prospective randomized study concerning male infertility and PGD has been published so far, and more studies would be needed to elucidate the benefits of PGD in this group of patients. Conclusions A high incidence of chromosomal abnormalities has been described in most of the groups of patients undergoing IVF with poor reproductive outcome. It is also known that blastocyst transfer does not avoid the transfer of chromosomally abnormal embryos. Therefore, PGD has been extended in clinical practice as a useful treatment in those couples to improve IVF outcome by transferring chromosomally normal embryos. Experience shows that women over 38 years of age greatly benefit from PGD, as well as women with recurrent miscarriage. However, in couples with repetitive IVF failures the benefits of PGD remains unclear, as well as in certain types of male infertility. Cumulative data, including prospective studies, show that following PGD, higher implantation rates can be achieved. Pregnancy rates without PGD can be improved to similar levels to those with PGD by increasing the number of embryos transferred, but with higher risk of multiple pregnancies (Staessen et al., 2004). Therefore, PGD would have the potential to improve the take home baby rate by decreasing abortion rates, and it has the benefit of lowering the risk of multiple pregnancies. This approach is in accordance with guidelines for good IVF practice aiming to transfer low number of embryos with high implantation potential to decrease multiple pregnancies. Additionally, there is a correlation between the first and the second cycle in terms of aneuploidy in patients undergoing PGD for aneuploidy screening, and this correlation is stronger in translocation carriers (Munné et al., 2004). It can be concluded that apart from the indications of PGD for established genetic diseases and structural abnormalities, the use of PGD for aneuploidy screening is highly recommended in some patient groups to improve their IVF outcome, although more prospective controlled studies are needed to confirm these results. 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