Article Prognostic factors for preimplantation genetic screening in repeated pregnancy loss

Transcription

1 RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 27 March 2009 Article Prognostic factors for preimplantation genetic screening in repeated pregnancy loss Trained in biochemistry at the University of Valencia, Carmen Rubio specialized in reproductive cytogenetics in the Universidad Autónoma, Barcelona. Becoming interested in chromosomal abnormalities in human embryos and spermatozoa, she completed her PhD in 2004, focussing on couples with recurrent miscarriages. She has since concentrated on human IVF and genetics and she is the director of the Preimplantation Genetic Diagnosis Unit at the Instituto Universitario-IVI in Valencia. Post-doctoral research included a period at the Hunt and Hassold laboratory at the School of Molecular Biosciences (Washington State University, USA) working on female and male meiosis. Dr Carmen Rubio Carmen Rubio 1, Pilar Buendía, Lorena Rodrigo, Amparo Mercader, Emilia Mateu, Vanessa Peinado, Arantxa Delgado, Miguel Milán, Pere Mir, Carlos Simón, José Remohí, Antonio Pellicer Instituto Universitario-IVI, Plaza Policía Local, No. 3, Valencia, Spain 1 Correspondence: c.rubio@ivi.es Abstract The objective of this study was to identify specific subgroups of recurrent pregnancy loss (RPL) patients of unknown aetiology in whom the selection of chromosomally normal embryos for transfer improves reproductive outcome in preimplantation genetic screening (PGS). A total of 428 PGS cycles were included and chromosomes 13, 15, 16, 18, 21, 22, X and Y were evaluated. In RPL patients 37 years, a lower incidence of chromosomal abnormalities (P = ) and miscarriages (P = ) was observed, and there were significantly higher pregnancy (P < ) and implantation (P < ) rates than in patients >37 years. In the former subset, results showed: (i) significantly higher implantation rates (P = ) in couples that had experienced a previous aneuploid miscarriage; (ii) similar aneuploidy, pregnancy and implantation rates in couples suffering previous miscarriages during fertility treatments and in those with previous spontaneous pregnancies; (iii) no miscarriages after PGS in couples in whom a fluorescence in-situ hybridization assay showed the male partner s sperm to be abnormal; and (iv) lower implantation rates in couples with 5 previous miscarriages, associated with a lower percentage of chromosomally abnormal embryos. It is concluded that PGS is to be strongly recommended when RPL is associated with miscarriages during infertility treatments, chromosomopathy in a previous miscarriage, up to five previous miscarriages and a high incidence of chromosomal abnormalities in spermatozoa. Keywords: aneuploidy, implantation, miscarriage, PGS, pregnancy, repeated pregnancy loss Introduction Recurrent spontaneous abortion is a distressing condition affecting up to 1% of couples of reproductive age (Stirrat, 1990). Despite the existence of well-defined causes of recurrent pregnancy loss (RPL), almost 50% of cases are classified as being of unknown aetiology following infertility work-up (Tho et al., 1979; Coulam, 1986; Clifford et al., 1994). Thus, the biological mechanisms involved in RPL remain obscure (Tulppala et al., 1993), while treatments are empirical in many circumstances and produce no real improvement in the livebirth rate (Plouffe et al., 1992). It is known that chromosomal abnormalities are implicated in first trimester spontaneous abortions. Cytogenetic evaluations of spontaneous abortions, carried out on fetal tissue, reveal an overall incidence of chromosomal abnormalities of ~50% (Boué et al., 1975; Hassold et al., 1978; Plachot, 1989; Eiben et al., 1990), most of which are trisomy, monosomy X or polyploidy. Hysteroembryoscopic biopsies for karyotyping of early missed abortions reveal an even higher abnormality rate of ~70%, and provide more accurate results than those carried out on curettage material (Ferro et al., 2003; Philipp et al., 2003). The origin of many autosomal trisomies is nondisjunction during maternal meiosis, and studies of spontaneous abortions have shown a maternal meiotic origin in all cases of trisomy 16. Trisomies 15, 21 and 22 are mostly the result of non-disjunction in maternal meiosis I, whereas maternal meiosis II contributes to an important percentage of trisomies 13 and 18. Paternal origin has been attributed to 2 15% of Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 688 autosomal trisomies, and a variable percentage of all trisomies have a post-zygotic mitotic origin (Hassold et al., 2007). For trisomies involving the sex chromosomes, in 50% of 47,XXY and in 100% of 47,XYY the origin is paternal non-disjunction (Jacobs and Hassold, 1995). Regarding monosomy for the X chromosome, the origin of the single X is usually maternal (80%), implying a paternal error during meiosis (Chandley, 1981). Maternal sex chromosome mosaicism is also thought to be responsible for some 45,X monosomies (Reinisch, 1981). Polyploidy occurs following the fertilization of a normal oocyte by more than one spermatozoon or due to meiotic errors during gametogenesis that lead to an abnormal oocyte or spermatozoon (Kajii and Niikawa, 1977). Most triploidies are of paternal origin (Egozcue et al., 2000; Zaragoza et al., 2000), and are mainly due to dispermy or the fertilization of a normal oocyte by a diploid spermatozoon. Diploidy is not so frequent in oocytes (Plachot et al., 1988), since spindle anomalies result in the production of multiple aneuploidies rather than diploidy (Eichenlaub-Ritter et al., 1999). The male contribution to recurrent miscarriage with respect to sperm chromosome abnormalities has been described in several studies. Meiotic abnormalities could originate from meiotic non-disjunction in the germ line (Egozcue et al., 1983; Vendrell et al., 1999) or as a result of the production of diploid sperm (Egozcue et al., 2000); thus, a normal blood cell karyotype does not exclude the presence of chromosomal abnormalities in the spermatozoa. Fluorescence in-situ hybridization (FISH) studies of spermatozoa have revealed an increased incidence of sperm chromosomal abnormalities in some patients with RPL (Giorlandino et al., 1998; Rubio et al., 1999, 2001; Carrell et al., 2003; Somprasit et al., 2004). With respect to repeated spontaneous abortions, the incidence of de-novo inherited chromosomal abnormalities in fetal tissue is more controversial. Whereas some authors have postulated that abnormal karyotypes are more frequent in specimens aborted by women with RPL (Ryynänem et al., 1997), others have not found such a correlation (Warburton et al., 1987; Cowchock et al., 1993; Stern et al., 1996). However, in a recent study published by Bianco et al., in which a prenatal diagnosis was performed in 46,939 women, an increased risk of karyotypic abnormality was confirmed in patients suffering a higher number of spontaneous abortions (Bianco et al., 2006). These results concur with other published studies regarding RPL, which have revealed high rates of chromosome abnormalities in cleavagestage embryos during preimplantation genetic screening (PGS) (Pellicer et al., 1999; Rubio et al., 2003, 2005; Werlin et al., 2003; Wilding et al., 2004; Munné et al., 2005; Platteau et al., 2005; Findikli et al., 2006). In some of the above mentioned reports, miscarriage rates after PGS were found to decrease to similar levels to those observed in patients without a history of RPL. However, other authors did not find evidence to support the therapeutic prescription of IVF, with or without PGS, in what was a heterogeneous group of patients, since ongoing pregnancy without further treatment in these patients was as high as ~70% (Clifford et al., 1997; Brigham et al., 1999). For these reasons, the aim in this study was to identify specific subgroups of RPL patients in whom the selection of chromosomally normal embryos for transfer improves reproductive outcome by decreasing the risk of a further miscarriage. Materials and methods Patients The PGS programme for RPL patients was initiated in the authors centre in 1996, and this report includes the results of 428 cycles performed until December PGS was performed in couples with normal parental karyotypes and at least two previous early clinical miscarriages (before 12 weeks of gestation). The aetiology of the RPL of these couples had been classified as unknown after infertility workup that included the following: vaginal ultrasound scan and hysterosalpingography/hysteroscopy; basal concentrations of FSH, LH, prolactin, thyroid stimulating hormone and glucose; screening for thrombophilia with the measurement of plasma concentrations of antithrombin, proteins C and S, and antiphospholipid antibodies; parent s karyotype and hysteroembryoscopy of previous miscarriages if possible (biochemical and ectopic pregnancies were not considered). Patients were divided into two groups based on the age of the female: 253 cycles were carried out in women 37 years (mean age: 33.5 years, SD: 2.5) with a mean number of previous miscarriages of 3.0 (SD: 1.1), and 175 cycles were performed in women >37 years (mean age: 40.2 years, SD: 1.7) with a mean number of 2.7 (SD: 0.9) previous miscarriages. In the group of patients 37 years, the incidence of chromosomal abnormalities and pregnancy, implantation and miscarriage rates were evaluated along the following lines: miscarriages from spontaneous pregnancies or from fertility treatments; presence of chromosomal abnormalities in a previous miscarriage/pregnancy; number of previous miscarriages; and presence of chromosomal abnormalities in sperm samples (defined in this paper as abnormal FISH with respect to that of controls, Rubio et al., 2001). A P-value of <0.05 was considered to be statistically significant. Analysis was carried out using the GraphPad Instat v. 2.05a (GraphPad Software, San Diego, CA, USA). FISH protocol for chromosomal studies on spermatozoa For FISH analysis on spermatozoa, the samples were centrifuged with Sperm medium (Medicult, Copenhagen, Denmark) and the pellets were fixed in methanol:acetic acid (3:1). Sperm nuclei were decondensed by slide incubation in 5 mmol/l dithiothreitol (DTT) and 1% Triton X-100. Numerical abnormalities for chromosomes 13, 18, 21, X and Y were evaluated in different slides from the same sample, using triplecolour FISH for 18, X and Y chromosomes and dual-colour FISH for chromosomes 13 and 21. Centromeric DNA probes for chromosome 18 (locus D18Z1, CEP 18 Spectrum Aqua; Vysis Inc. Downers Grove, IL, USA), chromosome X (locus DXZ1, CEP X Spectrum Green; Vysis Inc.) and Chromosome Y (locus DYZ1, CEP Y Spectrum Orange; Vysis Inc.) were used for the triple-colour FISH analysis. Locus-specific DNA probes for chromosome 13 (locus RB, LSI 13 Spectrum Green; Vysis Inc.) and chromosome 21 (loci D21S259, D21S341, D21S342, LSI 21 Spectrum Orange; Vysis Inc.) were used for the dual-colour FISH analysis. Hybridization and detection were performed according to Blanco et al. (1996).

3 Embryo biopsy and culture conditions Patients underwent ovarian stimulation following standardized protocols, and intracytoplasmic sperm injection (ICSI) was performed in all cases. Fertilization was assessed h after microinjection, and embryo cleavage was recorded every 24 h. Embryos were grown in IVF/CCM medium (1/1) (VitroLife, Göteborg, Sweden) until day 3 and subsequently cultured in CCM medium with a monolayer of endometrial epithelial cells until day 5, at which point they were transferred (Mercader et al., 2003). Embryo biopsy was performed on day 3 and can be summarized as follows: embryos were placed on a droplet containing Ca 2+ - and Mg 2+ - free medium (G-PGD; VitroLife) and the zona pellucida was perforated using acid Tyrode s solution or laser technology (OCTAX, Herbron, Germany). Only embryos with 5 nucleated blastomeres and 25% of fragmentation degree were biopsied, and one or two blastomeres were withdrawn depending on the cell number. Individual blastomeres were fixed on glass slides under an inverted microscope, using a slightly modified Tarkowski s protocol without hypotonic pretreatment. FISH protocol for PGS cycles Until the year 2003, the FISH protocol was performed as follows (Vidal et al., 1998): in a first round, locus-specific probes were employed for chromosomes 13 and 21; in a second round, following signal elimination, a centromeric probe was used for chromosome 16 and a locus-specific probe was employed for chromosome 22; finally, in a third round, triple FISH was carried out with centromeric probes for chromosomes X, Y and 18 (all probes available from Vysis Inc.). From 2003 onwards, FISH analysis was carried out for chromosomes 13, 16, 18, 21, and 22 in the first round, (Multivysion PB panel, from Vysis Inc.) and for chromosomes X, Y and 15 in the second. Detection washings and signal scoring were performed following the manufacturer s instructions. Blastomeres with two clear signals were routinely evaluated for each chromosome. Those in which there was a clear presence of one, three or more signals were considered to be abnormal. Overlapping signals separated by less than the diameter of one signal were considered to be non-informative, as were split or fibre dots. These nuclei, as well as those that showed an absence of signals for a particular chromosome, were reanalysed using subtelomeric or locus specific probes for different loci of the previously tested chromosomes (Rodrigo et al., 2004). Results The incidence of chromosomal abnormalities was higher in RPL patients >37 years (64.3 versus 72.2%, P = ). In patients 37 years of age, significantly higher pregnancy (45.0 versus 32.4%, P < ), implantation (35.1 versus 26.1%, P < ) and lower miscarriage rates (11.7 versus 24.4%, P = ) were observed than in those over 37 years of age (Table 1). PGS results were analysed in patients 37 years of age taking into account the various factors described below. Origin of pregnancy Similar aneuploidy, pregnancy and implantation rates were observed in RPL couples with previous miscarriages during fertility treatments (IVF/ICSI) and in couples with previous miscarriages from spontaneous pregnancies. A lower, although not significant, miscarriage rate was observed in the former group (Table 2). Chromosomal abnormalities in previous miscarriages/pregnancies Significantly higher implantation rates were observed in couples with previous aneuploid miscarriages/ pregnancies than in couples that did not undergo cytogenetic studies in previous miscarriages (48.1 versus 32.9%, P = ). The pregnancy rate was also higher in the group with previous chromosomopathies, which also had a lower miscarriage rate, although neither reached statistical significance (Table 3). Number of previous miscarriages Pregnancy and implantation rates decreased and miscarriage rates rose as the number of previous spontaneous abortions was higher, a tendency that was more marked in couples with five or more previous miscarriages (Figure 1). This effect was associated with a lower percentage of chromosomally abnormal embryos in couples with more than five miscarriages (44.0%) than in those with two miscarriages (65.1%, P = ), three miscarriages (65.3%, P = ) or four miscarriages (62.3%, not statistically significant). FISH studies in spermatozoa After PGS, couples in whom FISH results for spermatozoa were abnormal did not miscarry, whereas a 20.0% miscarriage rate was observed in those with normal results (Table 4). All other clinical variables were comparable between the two groups, with a higher ongoing pregnancy rate noted in couples with abnormal FISH results for spermatozoa (46.1 versus 37.2%, not statistically significant). Cytogenetic studies of fetal tissue (most of them following hysteroembryoscopy) from six patients 37 years that miscarried after a PGS cycle revealed an abnormal karyotype (47,XY+15) in one of the six abortuses. Four of the remaining abortuses possessed a normal male karyotype and one a normal female karyotype. The abnormal miscarriage occurred in a couple with previous spontaneous miscarriages, chromosomopathy in a previous miscarriage and normal FISH results for sperm. This PGS cycle was performed before 2003, during which period chromosome 15 was not included in the aneuploidy screening panel. In patients >37 years, it was possible to analyse six miscarriages, four of which presented cytogenetic abnormalities: 47,XY+7, 47,XX+10, 47,XY+15 (chromosome 15 not included in the panel), and 47, XX+17. The remaining two possessed normal male karyotypes. 689

4 Table 1. Preimplantation genetic screening results and cycle outcome in recurrent pregnancy loss patients according to women s age. Parameter Woman s age (years) Total 37 >37 No. of cycles No. of embryos analysed Abnormal embryos % 64.3 a 71.8 b 66.8 Mean embryos transferred (SD) 1.7 (0.6) 1.5 (0.6) 1.7 (0.6) Cycles with embryo transfer % 82.6 c 60.0 d 73.4 Pregnancy rate/embryo transfer % 45.0 c 32.4 d 40.8 Implantation rate % 35.1 c 26.1 d 32.3 Miscarriage rate % 11.7 c 24.4 d 18.1 a,b P < 0.05; c,d P < Table 2. Comparison of preimplantation genetic screening results in recurrent pregnancy loss patients 37 years with previous miscarriages from spontaneous pregnancies and miscarriages after IVF/ICSI cycles (ICSI). Parameter Miscarriage after Spontaneous IVF/ICSI conception conception No. of cycles Abnormal embryos % Mean embryos transferred (SD) 1.7 (0.6) 1.8 (0.6) Cycles to embryo transfer % Pregnancy rate/embryo transfer % Implantation rate % Miscarriage rate % There were no statistically significant differences between the two groups. Table 3. Comparison of preimplantation genetic screening results in recurrent pregnancy loss patients 37 years with previous abnormal miscarriages/pregnancies and patients without cytogenetic studies of the previous miscarriages. Parameter Abnormal Unknown PR/ET Implantation rate Miscarriage rate No. of cycles Abnormal embryos % Mean embryos 1.6 (0.5) 1.8 (0.6) transferred (SD) Cycles to embryo transfer % Pregnancy rate/embryo transfer % Implantation rate % 48.1 a 32.9 b Miscarriage rate % a,b P < misc (n = 105) 3 misc (n = 76) 4 misc (n = 56) 5 misc (n = 16) Figure 1. Preimplantation genetic screening results in recurrent pregnancy loss patients 37 years according to number of previous miscarriages. PR/ET = pregnancy rate/embryo transfer. Values are percentages. 690

5 Table 4. Comparison of preimplantation genetic screening results in recurrent pregnancy loss patients 37 years with a previous fluorescence in-situ hybridization (FISH) studies on spermatozoa. Parameter Abnormal FISH Normal FISH No. of cycles (%) 17 (22.7) 58 (77.3) Abnormal embryos % Mean embryos transferred (SD) 1.6 (0.6) 1.7 (0.6) Cycles to embryo transfer % Pregnancy rate/embryo transfer % Implantation rate % Miscarriage rate % There were no statistically significant differences between the two groups. Discussion The results show a high incidence of chromosomal abnormalities in embryos from couples with RPL of unknown aetiology, and are consistent with those of an earlier report in which the same tendency was observed in RPL patients undergoing repeated PGS (Pellicer et al., 1999). Overall, chromosome abnormalities were detected in all analysed embryos in ~27% of cycles demonstrating the value of PGS as a diagnostic tool in such subjects. Patient selection was a key factor of this study, since those with other known factors associated with RPL were not included in the PGS programme. Preimplantation aneuploidy screening in these couples produced significantly lower miscarriage rates than expected in natural conceptions (16.4% in the present study versus 25% in Brigham et al., 1999), and this favourable comparison with natural conceptions was previously described by Munné et al. (2005) in RPL women 35 years (16.7% in PGS versus 36.7% predicted loss rate according to the calculation of Brigham et al., 1999). Ongoing pregnancy and miscarriage rates were considered the end-points of the study and the beneficial effect of aneuploidy screening with respect to these end-points was more evident in younger RPL patients. In women 37 years, the following good prognosis factors were identified for PGS outcome: (i) miscarriages from infertility treatments; (ii) chromosomopathy in previous miscarriages/pregnancies; (iii) fewer than five previous miscarriages; and (iv) increased incidence of chromosomal abnormalities in the spermatozoa of the male partner. In recent years, several studies have described a relationship between infertility and chromosomal abnormalities in specific groups of infertile patients, such as women undergoing failed IVF cycles, severe male infertility and advanced maternal age (Gianaroli et al., 1999, 2000; Kahraman et al., 2000; Munné et al., 2003; Pehlivan et al., 2003; Silver et al., 2003; Platteau et al., 2004; Staessen et al., 2004; Rubio et al., 2005). More recently, an extensive study of 273 miscarriages following fertility treatment detected abnormal karyotypes in 64.8% of cases, and suggested that most abnormalities in the miscarried fetuses of infertile patients could be detected with PGS of at least nine chromosomes (Lathi et al., 2008). Cumulative experience in preimplantation embryos has demonstrated that some chromosomally abnormal embryos arrest before reaching the blastocyst stage; however, a considerable percentage (most of which are trisomies) do reach the blastocyst stage and subsequently implant, resulting in miscarriage or an abnormal pregnancy (Rubio et al., 2007). For this reason, a combination of morphology and PGS has until now been considered the optimal method for selecting euploid embryos in patients undergoing IVF (Munné et al., 2006, 2007), although there is now a good deal of controversy surrounding its efficacy (Moayeri et al., 2007). The findings of these previous publications describing the selection of embryos for transfer combining morphology and PGS correspond with the present results showing that infertile patients experiencing RPL after IVF/ICSI greatly benefit from PGS. An increase of aneuploidy rates in PGS cycles has been described in couples with a previous trisomic pregnancy (Munné et al., 2004). Other authors have also reported a higher risk of aneuploid pregnancy in couples with previous miscarriages (Bianco et al., 2006). The data show that, in couples in which cytogenic studies of previous miscarriages or pregnancies revealed an abnormal karyotype, the application of PGS resulted in pregnancy rates as high as 57.6% and implantation rates of 48.1%, with only 5.2% suffering miscarriages. Of all the subsets of patients analysed in the present study, this one is that which most benefited from PGS; this procedure allowed embryonic chromosomal abnormalities to be confirmed as the origin of their reproductive failure. The chromosomal abnormality rate in this study population was inversely proportional to the number of previous spontaneous abortions, in this way, the highest chromosomal abnormality rates were observed in patients with two to four previous abortions. The highest pregnancy/implantation rates and lowest miscarriage rates suggest that this group of patients would particularly benefit from PGS. The findings endorse those of a previous publication regarding spontaneous abortions, in which the frequency of abnormal embryonic karyotypes was inversely correlated with the number of previous miscarriages. A higher incidence of these abnormalities was detected in couples having suffered two to three miscarriages, while the incidence decreased with the number of previous spontaneous abortions (Ogasawara et al., 2000). 691

6 692 An increased incidence of meiotic abnormalities (Vendrell et al., 1999, Egozcue et al., 2000) and sperm aneuploidy (Giorlandino et al., 1998; Rubio et al., 1999, 2001; Carrell et al., 2003; Somprasit et al., 2004) have been reported in RPL patients; therefore, couples with recurrent miscarriages of unknown aetiology can be considered at risk for sperm chromosome abnormalities. In preliminary reports, an increased incidence of sex chromosome disomies was observed in sperm from RPL patients, and an increase in the diploidy rate was noted in a subset of patients with RPL after ovum donation (Rubio et al. 1999, 2001). The present study confirms an increased incidence in sex chromosome disomies in embryos from some of the patients with respect to controls. The identification of a sperm chromosomal factor could be a reliable indicator during PGS, since no miscarriages were observed after PGS in this subset of patients. The cytogenetic findings with hysteroembryoscopy in miscarriages following PGS demonstrate the need to screen for more chromosomes in RPL patients, since trisomies of chromosomes not included in the screening were detected, as suggested by other authors (Wilding et al., 2004; Lathi et al., 2007). Hysteroembryoscopy after PGS also revealed a high proportion of 46,XY normal karyotypes; in such couples, other causes of recurrent miscarriages should be considered, such as skewed-x inactivation (Lanasa et al., 2001; Uehara et al., 2001). The sex of the first child in secondary recurrent miscarriage has been highlighted as a prognostic factor, with less favourable reproductive outcome being related to a male first-born. The authors of the study in question suggested that maternal immunization against HY antigens may be responsible (Christiansen et al., 2004). Therefore, in couples with euploid male miscarriages after PGS, other potential causes should not be ruled out. To summarize, the results of the present study are reassuring in the sense that couples with RPL produce more abnormal embryos in vitro than couples without this problem. Patient subpopulations have been identified in which the selection of euploid embryos for transfer is of greater benefit. It is concluded that PGS is strongly recommendable when RPL is associated with miscarriages from infertility treatments, chromosomopathy in a previous miscarriage, up to five previous miscarriages, and increased incidence of chromosomal abnormalities in spermatozoa. Acknowledgements The authors would like to thank the work of the clinical team, embryologists and technicians implicated in the IVF and PGD laboratories at IVI-Valencia. The authors greatly appreciate the support and collaboration of Professor Francesca Vidal of the Universitat Autònoma of Barcelona for the development of this programme. References Bianco K, Caughey AB, Shaffer BL et al History of miscarriages and increased incidence of fetal aneuploidy. Obstetrics and Gynecology 107, Blanco J, Egozcue J, Vidal F 1996 Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization. Human Reproduction 4, Boué J, Boué A, Lazar P 1975 Retrospective and prospective epidemiological studies of 1500 karyotyped spontaneous human abortions. Teratology 12, 11. Brigham S, Conlon C, Farquharson R 1999 A longitudinal study of pregnancy outcome following idiopathic recurrent miscarriage. Human Reproduction 14, Carrell DT, Wilcox AL, Lowy L et al Elevated sperm chromosome aneuploidy and apoptosis in patients with unexplained recurrent pregnancy loss. Obstetrics and Gynecology 101, Chandley AC 1981 The origin of chromosomal aberrations in man and their potential for survival and reproduction in the adult human populations. Annual Genetics 24, Christiansen OB, Pedersen B, Nielsen HS et al Impact of the sex of first child on the prognosis in secondary recurrent miscarriage. Human Reproduction 19, Clifford K, Rai R, Regan L 1997 Future pregnancy outcome in unexplained recurrent first trimester miscarriage. Human Reproduction 12, Clifford K, Rai R, Watson H, Regan L 1994 An informative protocol for the investigation of recurrent miscarriage: preliminary experience of 500 consecutive cases. Human Reproduction 9, Coulam C 1986 Unexplained recurrent pregnancy loss. Clinical Obstetrics and Gynecology 29, Cowchock FS, Gibas Z, Jackson LG 1993 Chromosome errors as a cause of spontaneous abortion: the relative importance of maternal age and obstetric history. Fertility and Sterility 59, Egozcue S, Blanco J, Vendrell JM et al Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Human Reproduction Update 6, Egozcue J, Templado C, Vidal F et al Meiotic studies in a series of 1100 infertile and sterile men. Human Genetics 65, Eiben B, Bartels I, Bahr-Porch S et al Cytogenetic analysis of 750 spontaneous abortions with the direct preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage. American Journal of Human Genetics, 47, Eichenlaub-Ritter U, Cucurkam S, Betzendahl I et al Studies on the aneugenic properties of trichlorfon, a pesticide, vermicide and drug used in the treatment of Alzheimer patients. Human Reproduction 14 (Abstract Book 1), Ferro J, Martínez MC, Lara C et al Improved accuracy of hysteroembryoscopic biopsies for karyotyping early missed abortions. Fertility and Sterility 80, Findikli N, Kahraman S, Saglam Y et al., 2006 Embryo aneuploidy screening for repeated implantation failure and unexplained recurrent miscarriage. Reproductive BioMedicine Online 13, Gianaroli L, Magli MC, Ferraretti AP, Iammarrone E 2000 Preimplantation diagnosis after assisted reproduction techniques for genetically-determined male infertility. Journal of Endocrinological Investigation 23, Gianaroli L, Magli MC, Ferraretti AP et al Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertility and Sterility, 72, Giorlandino C, Calugi G, Laconianni L et al Spermatozoa with chromosomal abnormalities may result in a higher rate of recurrent abortion. Fertility and Sterility 70, Hassold T, Hall H, Hunt P 2007 The origin of human aneuploidy: where we have been, where we are going. Human Molecular Genetics 16, Spec No. 2: R Hassold TJ, Matsuyama A, Newlands IM et al A cytogenetic study of abortions in Hawaii. Annual Human Genetics 41, Jacobs PA, Hassold TJ 1995 The origin of numerical chromosome abnormalities. Advances in Genetics 33, Kahraman S, Bahce M, Samli H et al Healthy births and ongoing pregnancies by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure. Human Reproduction 15, Kajii T, Niikawa N 1977 Origin of triploidy and tetraploidy in man: 11 cases with chromosome markers. Cytogenetics Cell Genetics 18,

7 Lanasa MC, Hogge WA, Kubik CJ et al A novel X chromosomelinked genetic cause of recurrent spontaneous abortion. American Journal of Obstetrics and Gynecology 9, Lathi RB, Westphal LM, Milki AA 2008 Aneuploidy in the miscarriages of infertile women and the potential benefit of preimplantation genetic diagnosis. Fertility and Sterility 89, Mercader A, Garcia-Velasco JA, Escudero E et al Clinical experience and perinatal outcome of blastocyst transfer after coculture of human embryos with human endometrial epithelial cells: a 5-year follow-up study. Fertility and Sterility 80, Moayeri SE, Allen RB, Brewster WR et al Day-3 embryo morphology predict euploidy among older subjects. Fertility and Sterility 89, Munné S, Tomkin G, Cohen J 2007 Selection of embryos by morphology is less effective than the combination of aneuploidy testing and morphology observations. Fertility and Sterility 91, Munné S, Fischer J, Warner A et al Preimplantation genetic diagnosis significantly reduces pregnancy loss in infertile couples: a multicenter study. Fertility and Sterility 85, Munné 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. Fertility and Sterility 84, Munné S, Sandalinas M, Magli C et al Increased rate of aneuploid embryos in young women with previous aneuploid conceptions. Prenatal Diagnosis 24, Munné S, Sandalinas M, Escudero T et al Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reproductive BioMedicine Online 7, Ogasawara M, Aoki K, Okada S, Suzumori K 2000 Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertility and Sterility 73, Pehlivan T, Rubio C, Rodrigo L et al Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reproductive BioMedicine Online 6, 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. Fertility and Sterility 71, Philipp T, Philip K, Reiner A et al Embryoscopic and cytogenetic analysis of 233 missed abortions: factors involved in the pathogenesis of developmental defects of early failed pregnancies. Human Reproduction 18, Plachot M 1989 Chromosome analysis of spontaneous abortions after IVF. A European survey. Human Reproduction 4, Plachot M, de Grouchy J, Junca AM et al Chromosome analysis of human oocytes and embryos: does delayed fertilization increase chromosome imbalance? Human Reproduction 3, Platteau P, Staessen C, Michiels A et al Preimplantation genetic diagnosis for aneuploidy screening in patients with unexplained recurrent miscarriages. Fertility and Sterility 83, Platteau P, Staessen C, Michiels A et al Comparison of the aneuploidy frequency in embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men. Human Reproduction 19, Plouffe L, White EW, Tho SP et al Etiological factors of recurrent abortion and subsequent reproductive performance of couples: have we made any progress in the past 10 years? American Journal of Obstetrics and Gynecology 167, Reinisch LC, Silver KL, Dumars KW 1981 Sex chromosome mosaicism in couples with repeated fetal loss. American Journal of Human Genetics 33, 117 (abstract). Rodrigo L, Mateu E, Mercader A et al Rescue of false monosomies in a PGD program using subtelomeric probes. 12th International Conference on Prenatal Diagnosis and Therapy. Budapest (Hungary). Rubio C, Rodrigo L, Mercader A et al Impact of chromosomal abnormalities on preimplantation embryo development. Prenatal Diagnosis 27, Rubio C, Rodrigo L, Pérez-Cano I et al FISH screening of aneuploidies in preimplantation embryos to improve IVF outcome. Reproductive BioMedicine Online 11, Rubio C, Simón C, Vidal F et al Chromosomal abnormalities and embryo development in recurrent miscarriage couples. Human Reproduction 18, Rubio C, Gil-Salom M, Simón C et al Incidence of sperm chromosomal abnormalities in a risk population: relationship with sperm quality and ICSI outcome. Human Reproduction 16, Rubio C, Simón C, Blanco J et al Implications of sperm chromosome abnormalities in recurrent miscarriage. Journal of Assisted Reproduction and Genetics 16, Ryynänem M, Leskinen S, Heinonen S, Kirkinen P 1997 Recurrence risk of a serious, noninherited chromosomal abnormality. Fertility and Sterility 68, Silber S, Escudero T, Lenahan K et al Chromosomal abnormalities in embryos derived from testicular sperm extraction. Fertility and Sterility 79, Somprasit C, Aguinaga M, Cisneros PL et al Paternal gonadal mosaicism detected in a couple with recurrent abortions undergoing PGD: FISH analysis of sperm nuclei proves valuable. Reproductive BioMedicine Online 9, 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. Human Reproduction 19, Stern JJ, Dorfmann AD, Gutiérrez-Nájar AJ et al Frequency of abnormal karyotypes among abortuses from women with and without a history of recurrent spontaneous abortion. Fertility and Sterility 65, Stirrat GM 1990 Recurrent miscarriage I: definition and epidemiology, causes and management. Lancet 336, Tho P, Byrd JR, McDonough PG 1979 Etiologies and subsequent reproductive performance of 100 couples with recurrent abortion. Fertility and Sterility 32, Tulppala M, Palosuo T, Ramsay T et al A prospective study of 63 couples with a story of recurrent spontaneous abortion: contributing factors and outcome of subsequent pregnancies. Human Reproduction 8, Uehara S, Hashiyada M, Sato K et al Preferential X-chromosome inactivation in women with idiopathic recurrent pregnancy loss. Fertility and Sterility 76, Vendrell JM, Garcia F, Veiga A et al Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Human Reproduction 14, Vidal F, Giménez C, Rubio C et al FISH preimplantation diagnosis of chromosome aneuploidy in recurrent pregnancy wastage. Journal of Assisted Reproduction and Genetics 15, Warburton D, Kline J, Stein Z et al Does the karyotype of a spontaneous abortion predict the karyotype of a subsequent abortion? Evidence from 273 women with two karyotyped spontaneous abortions. American Journal of Human Genetics 41, Werlin L, Rodi I, De Cherney A et al Preimplantation genetic diagnosis as both a therapeutic and diagnostic tool in assisted reproductive technology. Fertility and Sterility 80, Wilding M, Forman R, Hogewind G et al Preimplantation genetic diagnosis for the treatment of failed in vitro fertilization embryo transfer and habitual abortion. Fertility and Sterility 81, Zaragoza MV, Surti U, Redline RW et al Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatiform mole. American Journal of Human Genetics 66, Declaration: The authors report no financial or commercial conflicts of interest. Received 15 August 2008; refereed 7 October 2008; accepted 7 January