Correction: Embryos of Robertsonian Translocation Carriers Exhibit a Mitotic Interchromosomal Effect That Enhances Genetic Instability during Early Development.

[This corrects the article DOI: 10.1371/journal.pgen.1003025.].

Aneuploidy and recombination in the human preimplantation embryo. Copy number variation analysis and genome-wide polymorphism genotyping.

RESEARCH QUESTION: What are the incidence and patterns of meiotic trisomies and recombination separately and in relation to each other at the blastocyst stage via single nucleotide polymorphism genotyping combined with array comparative genomic hybridization. DESIGN: Single nucleotide polymorphism microarrays were carried out on a total of 1442 blastocyst stage embryos derived from 268 fertile couples undergoing preimplantation genetic diagnosis for the purposes of avoiding transmittance of known single gene disorders to their offspring; 24-chromosome aneuploidy screening via array comparative genomic hybridization was carried out in parallel. RESULTS: One hundred per cent of meiotic trisomies identified in these embryos were of maternal origin and their incidence increased significantly with advancing maternal age (P < 0.0001). A total of 55.8% of meiotic trisomies were meiosis I-type and 44.2% were meiosis II-type. Certain chromosomes were affected more by meiosis I-type errors, whereas others experienced more meiosis II-type errors. A detailed recombination analysis was carried out for 11,476 chromosomes and 17,763 recombination events were recorded. The average number of recombination sites was 24.0 ± 0.3 for male meiosis and 41.2 ± 0.6 for female meiosis (autosomes only). Sex-specific differences were observed in the locations of recombination sites. Comparative analysis conducted between 190 euploid embryos and 69 embryos presenting maternal meiotic trisomies showed similar recombination rates (P = 0.425) and non-recombinant chromatid rates (P = 0.435) between the two categories; differences, however, were observed when analysing embryos affected with specific maternal meiotic trisomies. CONCLUSIONS: This study yielded unique data concerning recombination and the origin of aneuploidies observed during the first few days of life and provides a novel insight into these important biological processes.

The human sex ratio from conception to birth.

We describe the trajectory of the human sex ratio from conception to birth by analyzing data from (i) 3- to 6-d-old embryos, (ii) induced abortions, (iii) chorionic villus sampling, (iv) amniocentesis, and (v) fetal deaths and live births. Our dataset is the most comprehensive and largest ever assembled to estimate the sex ratio at conception and the sex ratio trajectory and is the first, to our knowledge, to include all of these types of data. Our estimate of the sex ratio at conception is 0.5 (proportion male), which contradicts the common claim that the sex ratio at conception is male-biased. The sex ratio among abnormal embryos is male-biased, and the sex ratio among normal embryos is female-biased. These biases are associated with the abnormal/normal state of the sex chromosomes and of chromosomes 15 and 17. The sex ratio may decrease in the first week or so after conception (due to excess male mortality); it then increases for at least 10-15 wk (due to excess female mortality), levels off after ∼20 wk, and declines slowly from 28 to 35 wk (due to excess male mortality). Total female mortality during pregnancy exceeds total male mortality. The unbiased sex ratio at conception, the increase in the sex ratio during the first trimester, and total mortality during pregnancy being greater for females are fundamental insights into early human development.

Causes and estimated incidences of sex-chromosome misdiagnosis in preimplantation genetic diagnosis of aneuploidy.

Preimplantation genetic diagnosis of aneuploidy (PGD-A) with comprehensive chromosome analysis has been known to improve pregnancy outcomes. Accuracy in detecting sex chromosomes becomes important when selecting against embryos at risk for sex-linked disorders. A total of 21,356 PGD-A cycles consisting of day-3 (cleavage) or day-5 (blastocyst) biopsies were received at the same laboratory for PGD-A via fluorescence in situ hybridization (FISH) or array comparative genome hybridization (aCGH) from multiple fertility centres. The misdiagnosis rates were 0.12% (Wilson 95% CI 0.05 to 0.25%) in day-3 FISH cycles, 0.48% (Wilson 95% CI 0.19 to 1.22%) in day-3 aCGH cycles and 0.0% (Wilson 95% CI 0 to 0.26) in day-5 aCGH cycles. Although rare, the likely causative biological event for true misdiagnosis is embryonic XX/XY mosaicism. Reanalysis of 1219 abnormal cleavage-stage research embryos revealed a 73% incidence of minor and major mosaicism. Only four (0.3%) embryos were found to be diploid and contained XX and XY cells that could potentially account for the misdiagnosis of sex. Our investigation identified errors leading to misdiagnosis and their attribution to specific events during PGD-A testing. The reported misdiagnosis rates suggest that PGD-A for sex determination is highly accurate, particularly when using aCGH applied to blastocyst biopsies.

Embryos of robertsonian translocation carriers exhibit a mitotic interchromosomal effect that enhances genetic instability during early development.

Balanced chromosomal rearrangements represent one of the most common forms of genetic abnormality affecting approximately 1 in every 500 (0.2%) individuals. Difficulties processing the abnormal chromosomes during meiosis lead to an elevated risk of chromosomally abnormal gametes, resulting in high rates of miscarriage and/or children with congenital abnormalities. It has also been suggested that the presence of chromosome rearrangements may also cause an increase in aneuploidy affecting structurally normal chromosomes, due to disruption of chromosome alignment on the spindle or disturbance of other factors related to meiotic chromosome segregation. The existence of such a phenomenon (an inter-chromosomal effect--ICE) remains controversial, with different studies presenting contradictory data. The current investigation aimed to demonstrate conclusively whether an ICE truly exists. For this purpose a comprehensive chromosome screening technique, optimized for analysis of minute amounts of tissue, was applied to a unique collection of samples consisting of 283 oocytes and early embryos derived from 44 patients carrying chromosome rearrangements. A further 5,078 oocytes and embryos, derived from chromosomally normal individuals of identical age, provided a robust control group for comparative analysis. A highly significant (P = 0.0002) increase in the rate of malsegregation affecting structurally normal chromosomes was observed in association with Robertsonian translocations. Surprisingly, the ICE was clearly detected in early embryos from female carriers, but not in oocytes, indicating the possibility of mitotic rather than the previously suggested meiotic origin. These findings have implications for our understanding of genetic stability during preimplantation development and are of clinical relevance for patients carrying a Robertsonian translocation. The results are also pertinent to other situations when cellular mechanisms for maintaining genetic fidelity are relaxed and chromosome rearrangements are present (e.g. in tumors displaying chromosomal instability).

Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos.

Fluorescent in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormalities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridization (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were ∼6 Mbp and ∼5 Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by array CGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for array CGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by array CGH. This study validates array CGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy.

The cytogenetics of polar bodies: insights into female meiosis and the diagnosis of aneuploidy.

Female meiosis is comprised by two cell divisions, meiosis I (MI) and II (MII) and two different stages at which the development of the oocyte is temporarily halted. In the case of MI, this pause can potentially last for four to five decades. This added layer of complexity distinguishes female gametogenesis from its male counterpart. The single most important genetic factor impacting human reproductive success is aneuploidy. Aneuploid embryos may undergo permanent arrest during preimplantation development, fail to implant or spontaneously abort. Most aneuploidies originate during female meiosis and become increasingly common with advancing maternal age. To shed further light on the nature of aneuploidy in human oocytes, we utilized comparative genomic hybridization (CGH) to provide a detailed cytogenetic analysis of 308 first and second polar bodies (PBs). These were biopsied from fertilized oocytes, generated by 70 reproductively older women (average maternal age of 40.8 years). The total oocyte abnormality rate was 70%, and MII anomalies predominated over MI (50% aneuploidy rate versus 40.3%). Both whole chromosome non-disjunction and unbalanced chromatid predivision were seen, but the latter was the dominant MI aneuploidy-causing mechanism. Chromosome losses occurred more frequently than chromosome gains, especially during MI. Chromosomes of all sizes were found to participate in aneuploidy events, although errors involving smaller chromosomes were more common. These data reveal the spectrum of aneuploidies arising after each meiotic division, indicating that oocyte-derived abnormalities present at conception differ from those observed in established pregnancies. It is also clear that advancing maternal age had a significant adverse effect on female meiosis, and that this effect is most pronounced in MII. Indeed, our data suggest that MII may be more susceptible to age-related errors than MI.

Derivation of human embryonic stem cells in xeno-free conditions.

Human embryonic stem cells (hESC) have the potential to treat a wide range of diseases. Currently, the use of existing hESC lines in human clinical applications is limited, as they are derived from blastocysts subjected to immunosurgery with animal derived antibodies, and are maintained on mouse embryonic feeder (MEF) cells, in the presence of either fetal calf serum (FCS) or on Matrigel or with conditioned media from MEFs. Successful derivation of hESCs in xeno-free conditions is crucial in advancing stem cell therapy applications. Two hESC lines, one from chromosomally abnormal embryos and another cell line from normal embryos from the inner cell mass of human blastocysts are derived using a culture media that had 20% serum replacement (SR) and human FGF2 on human foreskin fibroblasts as feeder cells. Derivation and characterization of such xenofree hESCs suitable for clinical studies is described in this chapter.

Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resulted in miscarriage or live birth using next-generation sequencing.

OBJECTIVE: To determine whether undetected aneuploidy contributes to pregnancy loss after transfer of euploid embryos that have undergone array comparative genomic hybridization (aCGH). DESIGN: Case-control study. SETTING: University-based fertility center. PATIENT(S): Cases included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in miscarriage. Controls included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in a live birth. INTERVENTION(S): Next-generation sequencing (NGS) protocols were internally validated. Saved amplified DNA samples from the blastocyst trophectoderm biopsies previously diagnosed as euploid by aCGH were reanalyzed using NGS. Cytogenetic reports of the products of conception for 20 of the pregnancies resulting in miscarriage were available for comparison. MAIN OUTCOME MEASURE(S): The incidence of aneuploidy and mosaicism using NGS within embryos resulting in miscarriage and live birth. RESULT(S): Of euploid embryos analyzed by aCGH resulting in miscarriage, 31.6% were mosaic and 5.2% were polyploid by NGS. The rate of chromosomal abnormalities was significantly higher in embryos resulting in miscarriage (36.8%) than in those resulting in live births (15.8%). The rate of mosaicism was twice as high among embryos resulting in miscarriage than those resulting in live birth, but this was not statistically significant. Next-generation sequencing detected more cases of mosaicism than cytogenetic analysis of products of conception. CONCLUSION(S): Undetected aneuploidy may increase the risk of first trimester pregnancy loss. Next-generation sequencing may detect mosaicism and triploidy more frequently than aCGH, which could help to identify embryos at high risk of miscarriage. Mosaic embryos, however, should not be discarded as some can result in live births.

Fluorescence in situ hybridization and spectral imaging analysis of human oocytes and first polar bodies.

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, <35 years, 35-39 years, and >39 years. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Therefore, for a pilot study investigating a more-comprehensive analysis of oocytes and their corresponding first polar bodies, we developed a novel eight-probe chromosome enumeration scheme using fluorescence in situ hybridization and spectral imaging analysis.

Dynamic blastomere behaviour reflects human embryo ploidy by the four-cell stage.

Previous studies have demonstrated that aneuploidy in human embryos is surprisingly frequent with 50-80% of cleavage-stage human embryos carrying an abnormal chromosome number. Here we combine non-invasive time-lapse imaging with karyotypic reconstruction of all blastomeres in four-cell human embryos to address the hypothesis that blastomere behaviour may reflect ploidy during the first two cleavage divisions. We demonstrate that precise cell cycle parameter timing is observed in all euploid embryos to the four-cell stage, whereas only 30% of aneuploid embryos exhibit parameter values within normal timing windows. Further, we observe that the generation of human embryonic aneuploidy is complex with contribution from chromosome-containing fragments/micronuclei that frequently emerge and may persist or become reabsorbed during interphase. These findings suggest that cell cycle and fragmentation parameters of individual blastomeres are diagnostic of ploidy, amenable to automated tracking algorithms, and likely of clinical relevance in reducing transfer of embryos prone to miscarriage.

Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos.

OBJECTIVE: To validate and determine the best array-comparative genomic hybridization (aCGH; array-CGH) protocols for preimplantation genetic screening (PGS). DESIGN: Embryos had one cell removed as a biopsy specimen and analyzed by one of two array-CGH protocols. Abnormal embryos were reanalyzed by fluorescence in situ hybridization (FISH). SETTING: Reference laboratory. PATIENT(S): Patients donating embryos or undergoing PGS. INTERVENTION(S): Embryo biopsy, array-CGH, FISH reanalysis. MAIN OUTCOME MEASURE(S): Diagnosis, no result rate and error rate. RESULT(S): Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after array-CGH combined with single blastomere biopsy. CONCLUSION(S): Array-CGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, array-CGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary.

The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender.

OBJECTIVE: To assess correlation between blastocyst morphology and chromosomal status. DESIGN: Observational research study. SETTING: An IVF clinic and a specialist preimplanation genetic diagnosis (PGD) laboratory. PATIENT(S): Ninety-three couples undergoing IVF treatment in combination with chromosome screening of embryos. INTERVENTION(S): Five hundred blastocysts underwent trophectoderm biopsy and comprehensive chromosome screening using comparative genomic hybridization (CGH). The morphology of the embryos was evaluated using standard methods. MAIN OUTCOME MEASURE(S): Association of aneuploidy and morphologic score. RESULT(S): A total of 56.7% of blastocysts were aneuploid. One-half of the grade 5/6 blastocysts were euploid, compared with only 37.5% of embryos graded 1/2, suggesting an effect of aneuploidy on blastocyst development. Aneuploidy also had a negative effect on inner cell mass and trophectoderm grades. Morphologically poor blastocysts had a higher incidence of monosomy and abnormalities affecting several chromosomes. The gender ratio was significantly skewed in relation to morphology. A total of 72% of blastocysts attaining the highest morphologic scores (5AA and 6AA) were found to be male, compared with only 40% of grade 3 embryos. CONCLUSION(S): Morphology and aneuploidy are linked at the blastocyst stage. However, the association is weak, and consequently, morphologic analysis cannot be relied on to ensure transfer of chromosomally normal embryos. A significant proportion of aneuploid embryos are capable of achieving the highest morphologic scores, and some euploid embryos are of poor morphology. Gender was associated with blastocyst grading, male embryos developing at a significantly faster rate than females.

Preimplantation genetic diagnosis (PGD) improves pregnancy outcome for translocation carriers with a history of recurrent losses.

OBJECTIVE: To determine if preimplantation genetic diagnosis (PGD) for translocation carriers with three or more pregnancy losses reduces loss rates. DESIGN: Retrospective review of data. SETTING: Preimplantation genetic diagnosis laboratory servicing IVF groups. PATIENT(S): Patients (n = 192) undergoing PGD for either a reciprocal translocation or Robertsonian translocation who had three or more previous pregnancy losses. INTERVENTION(S): Preimplantation genetic diagnosis for translocations. MAIN OUTCOME MEASURE(S): Pregnancy loss rate, pregnancy success rate defined as delivery of at least one child or an ongoing pregnancy in the third trimester, and length of time to success. RESULT(S): Pregnancy loss rate was significantly reduced to 13% post-PGD compared with 88.5% in previous non-PGD pregnancies and to 35% to 64% from naturally conceived pregnancies as reported in the literature. Pregnancy success rate was 87%. Conception occurred after an average of 1.4 cycles or <4 months. CONCLUSION(S): Individuals with translocations who have experienced three or more losses benefit from PGD by realizing a significant reduction in loss rate and improvement in rate of success of pregnancy. Length of time to conceive is also dramatically reduced compared with data in the literature for similar populations not undergoing PGD.

Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure.

OBJECTIVE: To identify and transfer cytogenetically normal embryos after screening all chromosomes of first and second polar bodies (PBs) or trophectoderm samples with the use of comparative genomic hybridization. DESIGN: Clinical research study. SETTING: In vitro fertilization clinic referring samples to a specialist preimplantation genetic diagnosis laboratory. PATIENT(S): Thirty-two couples with repeated implantation failure. INTERVENTION(S): Zygotes from patients with repeated implantation failure and poor response to ovarian stimulation underwent PB biopsy. Patients with repeated implantation failure who were candidates for blastocyst transfer received trophectoderm biopsy. Zygotes or blastocysts were vitrified while chromosome analysis took place. Euploid embryos were transferred during a subsequent cycle. MAIN OUTCOME MEASURE(S): Cytogenetic status and implantation and pregnancy rates. RESULT(S): The oocyte and blastocyst aneuploidy rates were 65.5% and 45.2%, respectively. Abnormalities affecting all chromosomes were detected. Implantation and pregnancy rates for the patients with PB biopsy were 11.5% and 21.4%, respectively, whereas for patients receiving blastocyst analysis they were 58.3% and 69.2%. CONCLUSION(S): Initial results for patients of advanced maternal age (39.8 years) with repeated implantation failure and poor ovarian response were encouraging. However, further study is required to confirm whether or not screening is beneficial. Blastocyst analysis was associated with high pregnancy rates, suggesting that comprehensive chromosome screening may assist patients with repeated implantation failure capable of producing blastocysts in achieving pregnancies.

Effect of infertility, maternal age, and number of previous miscarriages on the outcome of preimplantation genetic diagnosis for idiopathic recurrent pregnancy loss.

OBJECTIVE: To determine whether preimplantation genetic diagnosis (PGD) would decrease spontaneous abortion rates in patients with idiopathic recurrent pregnancy loss (RPL). DESIGN: Controlled clinical study. SETTING: IVF center and PGD reference laboratory. PATIENT(S): Patients with RPL with no known etiology. INTERVENTION(S): Preimplantation genetic diagnosis by fluorescence in situ hybridization analyzing nine chromosomes. MAIN OUTCOME MEASURE(S): The spontaneous abortion rate after PGD was compared to the patients' own expected risk of abortion. Patients were evaluated according to the number of previous losses, fertility, and maternal age. RESULT(S): Preimplantation genetic diagnosis significantly reduced spontaneous abortions in patients with RPL, especially for patients with more than two previous losses (12.8% vs. 35.9% expected). The PGD significantly reduced the rate of spontaneous abortion in both fertile (15.2% vs. 33.8% expected) and infertile patients (13.0% vs. 29.5%), which also achieved similar delivery rates (37% and 34%, respectively). The beneficial effect of PGD was less pronounced in patients <35 years than in patients > or =35 years old (13.6% vs. 34.0% expected). Overall, the PGD reduced the miscarriage rate to a similar baseline of 14%-16% across all maternal ages. CONCLUSION(S): Preimplantation genetic diagnosis improves pregnancy outcome for women with idiopathic RPL, especially those with more than two previous losses, and >35 years of age, and that improvement is not affected by fertility status.

Increased efficiency of preimplantation genetic diagnosis for infertility using "no result rescue".

OBJECTIVE: To improve preimplantation genetic diagnosis (PGD) accuracy by using "no result rescue" (NRR) consisting of the reanalysis of dubious results with additional probes binding to a locus different from the one previously analyzed. DESIGN: Prospective study of PGD cycles with and without reanalysis of inconclusive results. SETTING: PGD laboratory. PATIENT(S): Patients undergoing PGD for infertility or Robertsonian translocations. INTERVENTION(S): Nuclei from day 3 biopsied embryos were analyzed with fluorescence in situ hybridization for chromosomes X,Y, 13, 15, 16, 17, 18, 21, and 22. When inconclusive results were obtained, NRR was performed. In addition, 100 PGD cycles using NRR were matched to controls according to maternal age, previous failed cycles, number of zygotes, number of eggs, and date of retrieval. MAIN OUTCOME MEASURE(S): Determination of frequency of inconclusive results and error rate after use of additional probes. Comparison of frequency of inconclusive results with prior PGD results when NRR was not used. Assisted reproductive technology outcome was compared between PGD using NRR and controls not using PGD. RESULT(S): After analysis of 34,831 blastomeres from 34,225 embryos, 2,609 blastomeres (7.5%) showed inconclusive results. After NRR on those 2,609 blastomeres, the number of cells with inconclusive results was reduced to 3.1% (P<.001). After the introduction of NRR, fluorescence in situ hybridization errors, measured as discrepancies between the PGD diagnosis and the analysis of the nonreplaced embryo, decreased from 13.6% to 4.7% (P<.001). PGD with NRR significantly improved implantation rates, from 20% to 31%, and reduced spontaneous abortions from 27% to 6%. CONCLUSION(S): The use of NRR has been proven to be a powerful tool to reduce the error rate and the frequency of inconclusive results in PGD, both parameters of high importance to assess quality of PGD laboratories. Indeed, these parameters are two of the few measurable criteria to measure PGD laboratories. In a parallel controlled study, PGD with NRR significantly improved implantation rates and reduced spontaneous abortions, showing that PGD is more efficient in selecting embryos that will reach term.

Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies.

Preimplantation genetic diagnosis (PGD) for translocations has been shown to significantly reduce the risk of recurrent miscarriage, but because the majority of embryos produced are unbalanced, pregnancy rate is relatively low since 20% or more cycles have no normal or balanced embryos to transfer. The purpose of this study was to evaluate whether PGD could improve pregnancy outcome in translocation carriers with a history of two or more consecutive miscarriages and no live births. PGD for translocations was offered to translocation carriers with two or more previous miscarriages (average 3.5) and no live births (0/117 pregnancies) using a combination of distal and proximal probes to the breakpoints. After PGD, only 18.3% of embryos were normal or balanced. Only 5.3% of pregnancies were lost after PGD compared with 100% before PGD (P < 0.001). The cumulative pregnancy rate was 57.6% and the cumulative ongoing pregnancy rate was 54.5% in the short period of time of 1.24 IVF cycles, or 46.3% and 43.9% respectively per cycle. In conclusion, PGD significantly reduced losses and increased the number of viable pregnancies (P < 0.001). IVF plus PGD are a faster method of conceiving a live child than natural conception, at least for translocation carriers with recurrent miscarriages and no previous live births.

Self-correction of chromosomally abnormal embryos in culture and implications for stem cell production.

OBJECTIVE: To ascertain whether embryos classified by preimplantation genetic diagnosis (PGD) for infertility as abnormal and then plated to obtain stem cells would self-correct partially or totally in culture, producing disomic stem cells. DESIGN: Prospective study to determine the chromosome status of embryos on day 3 and 6, as well as cultured cells derived from inner cell masses from the same embryos when cultured up to day 12. SETTING: Research laboratory. PATIENT(S): Patients undergoing PGD of aneuploidy. INTERVENTION(S): Of 142 embryos classified by PGD for aneuploidy as abnormal, 50 were cultured to the blastocyst stage. At that stage a fraction of the embryos underwent trophectoderm biopsy to reconfirm the PGD diagnosis. After further co-culture with feeders up to day 12, 34 embryos attached to the feeder cells. Of those, 24 were analyzed by fluorescence in situ hybridization (FISH) and the rest for the expression of Oct-4, SSEA-3, SSEA-4, TRA1-60, and TRA1-80. MAIN OUTCOME MEASURE(S): Disomic cells obtained from trisomic embryos. RESULT(S): Analysis by FISH of day-12 cultures showed that 7 were totally normal, 6 were mostly abnormal, and 11 had experienced some chromosome normalization, having between 21% and 88% normal cells. Day-12 culture was positive for Oct-4 expression by reverse transcriptase polymerase chain reaction analysis and for SSEA-3, SSEA-4, TRA1-60, and TRA1-80 by immunocytochemistry. CONCLUSION(S): Chromosome self-normalization occurs in a significant proportion of chromosomally abnormal embryos, possibly because of the loss of a chromosome in trisomic cells after blastocyst stage. Thus chromosomally abnormal embryos are a potential source of disomic stem cells. Not all chromosomally abnormal embryos self-corrected. Abnormal stem cells that might be derived could be used as models to study the effect of chromosomal abnormalities on human development.

Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages.

OBJECTIVE: To determine whether preimplantation genetic diagnosis (PGD) and transfer of euploid embryos would decrease spontaneous abortion rates in recurrent miscarriage (RM) patients. DESIGN: Controlled clinical study. SETTING: In vitro fertilization centers and PGD reference laboratory. PATIENT(S): Recurrent-miscarriage patients with three or more prior lost pregnancies with no known etiology. INTERVENTION(S): Biopsy of a single blastomere from each day 3 embryo, followed by fluorescence in situ hybridization analysis. MAIN OUTCOME MEASURE(S): The rate of spontaneous abortions in RM subjects undergoing PGD were compared with [1] their own a priori expectations and [2] a comparison group of women undergoing PGD for advanced maternal age (> or =35 years). RESULT(S): Before PGD, RM patients had lost 87% (262/301) of their pregnancies, with an expected loss rate of 36.5%. After, they only lost 16.7% pregnancies. This difference was mostly due to reduction in pregnancy loss in the > or =35-years age subgroup, to 12% from an expected 44.5%. CONCLUSION(S): Preimplantation genetic diagnosis aneuploidy screening has a beneficial effect on pregnancy outcome in RM couples, especially those in which the woman is aged > or =35 years. Our data indicate that PGD reduces the risk of miscarriage in RM patients to baseline levels.