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Meiosis errors in over 20,000 oocytes studied in the practice of preimplantation aneuploidy testing

Published:September 14, 2010DOI:https://doi.org/10.1016/j.rbmo.2010.08.014

      Abstract

      This study presents the world’s largest series of over 20,000 oocytes tested for aneuploidies, involving chromosomes 13, 16, 18, 21 and 22, providing the data on the rates and types of aneuploidies and their origin. Almost every second oocyte (46.8%) is abnormal, with predominance of extra chromatid errors predicting predominance of trisomies (53%) over monosomies (26%) in the resulting embryos (2:1), which is opposite to monosomy predominance observed in embryo testing. Of the detected anomalies in oocytes, 40% are complex, so testing for a few most prevalent chromosome errors may allow detection of the majority of abnormal embryos. Chromosome 21 and 22 errors are more prevalent, while two different patterns of error origin were observed for different chromosomes: chromosome 16 and 22 errors originate predominantly from meiosis II, compared with chromosome 13, 18 and 21 errors originating from meiosis I. This provides the first evidence for the differences in the aneuploid embryo survival depending on the meiotic origin. Considering the problem of mosaicism, which is the major limitation of the cleavage-stage testing, the direct oocyte aneuploidy testing by polar body analysis may be of obvious practical value in improving accuracy and reliability of avoiding aneuploid embryos for transfer.
      As much as 95% of chromosomal disorders originate from maternal meiosis, so we present here the meiosis errors detected in aneuploidy testing of over 20,000 oocytes in practice of preimplantation diagnosis, including the rates and types of aneuploidies and their origin. Almost every second oocyte in IVF patients aged over 38 is abnormal, originating from meiosis I and meiosis II. One-third of oocytes have both meiosis I and meiosis II errors, with the potential of correction in almost half of them involving the same chromosome. Chromatid errors constitute the major source of aneuploidy, predicting predominance of trisomies over monosomies in the resulting embryos, which contrasts to monosomy predominance observed in embryo testing. Of the detected anomalies in oocytes, 40% are complex, so testing for a few most prevalent chromosome errors may detect the majority of abnormal embryos. The smaller chromosome 21 and 22 errors are more prevalent, while there are two different patterns of error origin for different chromosomes: chromosome 16 and 22 errors originate predominantly from meiosis II, compared with chromosomes 13, 18 and 21 errors originating from meiosis I. This provides the first evidence for the differences in the aneuploid embryo survival depending on the meiotic origin. The data show that direct oocyte testing by polar body analysis may be of obvious practical value in improving accuracy and reliability of avoiding the transfer of aneuploid embryos.

      Keywords

      Introduction

      Because of recent controversy in preimplantation aneuploidy testing, which is mainly due to limitations of the cleavage-stage analysis caused by high rate of mosaicism, the addition of polar body (PB)-based pre-selection of aneuploidy-free oocytes may improve the accuracy and reliability of the procedure, as it provides the information about the chromosome set of the embryos from their very onset. Also, because at least 95% of chromosomal abnormalities are of maternal origin, pre-selection of the embryos with higher developmental potential may be achieved by avoiding the transfer of the embryos deriving from the aneuploid oocytes alone. Although introduced 20 years ago (see for review,
      • Verlinsky Y.
      • Kuliev A.
      Practical Preimplantation Genetic Diagnosis.
      ), the value of this approach has only recently been fully appreciated, especially in those social settings where no embryo micromanipulation is allowed, leaving preconception testing prior to syngamy as the only preimplantation genetic diagnosis (PGD) option (
      • Gianaroli L.
      • Magli M.C.
      • Lappi M.
      • Capoti A.
      • Robles F.
      • Ferraretti A.P.
      Preconception diagnosis.
      ,
      • Kuliev A.
      • Rechitsky S.
      • Laziuk K.
      • et al.
      Pre-embryonic diagnosis for Sandhoff disease.
      ,
      • Magli C.
      • Ferraretti A.
      • Crippa A.
      • Lappi M.
      • Feliciani E.
      • Gianaroli L.
      First meiosis errors in immature oocytes generated by stimulated cycles.
      ,
      • Montag M.
      • der Ven K.
      • Dorn C.
      • Rossing B.
      • van der Ven H.
      Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening.
      ).
      First applied to PGD for single-gene disorders, the PB approach was then extended to PGD for aneuploidies, as a tool for the direct testing of the outcome of meiosis I and meiosis II errors (
      • Verlinsky Y.
      • Ginsberg N.
      • Lifchez A.
      • Valle J.
      • Moise J.
      • Strom C.M.
      Analysis of the first polar body: preconception genetic diagnosis.
      ,
      • Verlinsky Y.
      • Cieslak J.
      • Freidin M.
      • et al.
      Pregnancies following preconception diagnosis of common aneuploidies by fluorescent in-situ hybridization.
      ,
      • Verlinsky Y.
      • Cieslak J.
      • Ivakhnenko V.
      • et al.
      Birth of healthy children after preimplantation diagnosis of common aneuploidies by polar body FISH analysis.
      ). Although the most optimal approach for preconception diagnosis is testing of the outcome of both meiosis I and meiosis II, through analysis of the first and second PB (PB1 and PB2) (
      • Verlinsky Y.
      • Kuliev A.
      Atlas of Preimplantation Genetic Diagnosis. An Illustrated Texbook.
      ), PB1 testing alone was also shown to be useful for improving clinical outcome (
      • Gianaroli L.
      • Magli M.C.
      • Lappi M.
      • Capoti A.
      • Robles F.
      • Ferraretti A.P.
      Preconception diagnosis.
      ,
      • Magli C.
      • Ferraretti A.
      • Crippa A.
      • Lappi M.
      • Feliciani E.
      • Gianaroli L.
      First meiosis errors in immature oocytes generated by stimulated cycles.
      ,
      • Montag M.
      • van der Ven K.
      • Dorn C.
      • van der Ven H.
      Outcome of laser-assisted polar body biopsy and aneuploidy testing.
      ). This may be explained by the fact that the PB1 testing detects not only the meiosis I errors but also a significant proportion of the meiosis II errors originating in meiosis I (
      • Kuliev A.
      • Cieslak J.
      • Illkewitch Y.
      • Verlinsky Y.
      Chromosomal abnormalities in a series of 6733 human oocytes in preimplantation diagnosis of age-related aneuploidies.
      ,
      • Kuliev A.
      • Cieslak J.
      • Verlinsky Y.
      Frequency and distribution of chromosomal abnormalities in human oocytes.
      ).
      The experience of aneuploidy testing has been accumulated in this paper for the world’s largest series of over 20,000 oocytes. The frequency and types of chromosomal errors detected by PB1 and PB2 testing are presented, as well as the meiosis origin of the different types of aneuploidies. The paper will also explore the observed disagreement between different types of errors observed in PGD for aneuploidy by PB and blastomere analysis and discuss possible biological significance of the differences between the chromosome-specific aneuploidy origin in preimplantation embryos, spontaneously aborted fetuses and live-born children.

      Materials and methods

      Overall, 20,986 oocytes were obtained in 3953 PGD cycles, performed for indication of aneuploidy testing for 2830 IVF patients of an average age of 38.8 years. The oocytes were tested by analysis of PB1 and PB2 during 1997–2009 at Reproductive Genetics Institute in Chicago. This includes the previously reported 4584 of 6733 oocytes with five-colour probe results (
      • Kuliev A.
      • Cieslak J.
      • Illkewitch Y.
      • Verlinsky Y.
      Chromosomal abnormalities in a series of 6733 human oocytes in preimplantation diagnosis of age-related aneuploidies.
      ,
      • Kuliev A.
      • Cieslak J.
      • Verlinsky Y.
      Frequency and distribution of chromosomal abnormalities in human oocytes.
      ). The IVF treatment and micromanipulation procedures for a simultaneous mechanical removal of both PB1 and PB2 following conventional insemination or intracytoplasmic sperm injection (ICSI) have been described in detail elsewhere (
      • Verlinsky Y.
      • Kuliev A.
      Atlas of Preimplantation Genetic Diagnosis. An Illustrated Texbook.
      ). Isolated chromatin from PB1 and PB2 was tested by fluorescent in-situ hybridization (FISH) analysis, using a five-colour probe cocktail specific for chromosomes 13, 16, 18, 21 and 22 (Vysis; Downers Grove, IL): chromosome 13 (13q14) is detected in red, chromosome 16 (satellite II D16Z3) in aqua, chromosome 18 (alpha satellite D18Z1) in violet blue, chromosome 21 (LSI 21q22.13-21q22.2) in green and chromosome 22 (22q11.2) in gold.
      Visualization of signals was performed, using the appropriate single band pass filters for the probe fluorophores (red, green, blue, gold, aqua and 4,6-diamidino-2-phenylindole). Dual-band pass filters were used in distinguishing signals from nonspecific fluorescence or bleed through seen with centromeric enumeration probes that hybridize to alphoid repeat sequences resulting in large bright signals. When determining the number of signals present, the size and intensity of each signal was considered especially when in close proximity to one another.
      PB1 contains two chromatids for each chromosome, so the signals for each chromosome are paired, at least one domain apart and of equal size. However, if chromatids are in close proximity to one another, the signals may appear as one fluorescent strip, representative of two chromatids. Any deviation from this pattern was scored as error: missing one or both signals for each chromosome was scored as chromatid or chromosome loss, respectively, and one or two additional signals were scored as gain of chromatid or chromosome, accordingly. So the presence of one or three signals in PB1 for any chromosome tested depicted a chromatid error, either a missing chromatid in PB1 (one instead of two), predicting an extra chromatid in the respective MII oocyte, or an extra chromatid in PB1 (three instead of two), predicting this chromatid being missing in the respective MII oocyte. On the other hand, the total absence of relevant signals (none instead of two) or the presence of four signals depicted a chromosomal error, representing chromosomal non-disjunction.
      As PB2 contains a single chromatid for each chromosome, seen as one fluorescent signal for each chromosome tested, any deviation from a single signal for each chromosome was scored as gain or loss of chromatid, represented by extra or no relevant signal, respectively. The gain of a chromatid in PB2 infers the lack of the respective chromatid in the oocyte, predicting respective monosomy in the resulting embryo, while the absence of a chromatid in PB2 depicts an extra chromatid in the oocyte, predicting a trisomic embryo for the chromosome in question.
      Statistical analysis was performed by chi-squared test and differences at P < 0.05 were considered to be significant

      Results and discussion

      Testing for both meiosis I and meiosis II errors required for PGD of aneuploidies

      Aneuploidies were detected in 9812 (46.8%) of 20,986 oocytes studied (Table 1). As expected, the aneuploidy rates rose with increasing maternal age, from 20% in patients 35 years of age to over 40% in patients 40 years of age (Figure 1).
      Table 1Frequency of chromosomal abnormalities in human oocytes from women with average maternal age 38.8 years, detected by fluorescent in-situ hybridization (FISH) analysis using specific probes for chromosomes 13, 16, 18, 21 and 22.
      CouplesCyclesOocytes with FISH resultsNormal oocytes (n (%))Abnormal oocytes (n (%))
      2830395320,98611,174 (53.2)9812 (46.8)
      Figure thumbnail gr1
      Figure 1Aneuploid oocytes in relation to maternal age, predicted by PB1 and PB2 testing for chromosomes 13, 16, 18, 21 and 22. Number of oocytes tested for each age group is shown under the curve, which shows the increase of the overall frequency from 20% in the age group 35 years to close to 60% in the age group 43 years and over.
      As seen from Table 2, comparable proportions of detectable aneuploidies originated from meiosis I (31.1%) and meiosis II (33.7%). As seen from Table 3, only 30.4% of meiosis I and 39.8% of meiosis II errors were represented by isolated defects, while 29.8% of the chromosomally abnormal oocytes were outcomes of sequential meiosis I and meiosis II errors, suggesting that almost one-third of meiosis II errors were associated with the preceding meiosis I errors.
      Table 2Frequency and types of meiosis I and meiosis II errors.
      FISH dataMeiosis I (PB1)Meiosis II (PB2)
      n%n%
      Normal13,09769.013,63566.3
      Abnormal (total)592131.1693833.7
      Disomy151425.6272139.2
      Monosomy/nullisomy313653.0287541.4
      Complex127121.5134219.3
      Total19,01810020,573100
      Table 3Frequency and types of abnormal oocytes by polar body analysis.
      Typesn%
      PB1 + PB2292129.8
      PB1298330.4
      PB2390839.8
      Total9812100
      So, approximately half of the oocytes from IVF patients of advanced reproductive age (over 38 years) are abnormal, with a higher risk of meiotic errors with increasing reproductive age. This is in agreement with the data from the recent report of PB testing in 684 cycles from infertility patients, in which 55% of oocytes were found to be aneuploid after PB1 and PB2 testing (
      • Magli M.C.
      • Gianaroli L.
      • Crippa A.
      • Grugnetti C.
      • Ruberti A.
      • Ferraretti A.P.
      Causes of aneuploidy–polar body based PGD.
      ). The proportion of oocytes with meiosis I errors was 39% in younger than 38 years and 58% in 44-year-old patients (
      • Gianaroli L.
      • Magli M.C.
      • Lappi M.
      • Capoti A.
      • Robles F.
      • Ferraretti A.P.
      Preconception diagnosis.
      ). It is not clear to what extent the reported high prevalence of abnormalities is related to IVF treatment, involving aggressive hormonal stimulation, but preliminary data on testing of donated oocytes from young fertile women suggest that the actual prevalence may be much lower, although clearly more data is needed (
      • Fragouli E.
      • Escalona E.
      • Guttieres Mateo C.
      • et al.
      Comparative genomic hybridization of oocytes and first polar bodies from young donors.
      ).
      The data on comparable error rates in PB1 and PB2 is in contrast to the well-established concept of female meiosis I origin of chromosomal abnormalities (
      • Hassold T.
      • Merril M.
      • Adkins K.
      • Freemen S.
      • Sherman S.
      Recombination and maternal age-dependent nondisjunction: molecular studies of trisomy 16.
      ), suggesting that the observed aneuploidies originate equally from both meiosis I and II (Table 2). The fact that a significant proportion of meiosis II errors originate from meiosis I errors may be explored in light of previous considerations on the possible relationship of meiosis II errors with the increased meiotic recombination rate (
      • Lamb N.E.
      • Freeman S.
      • Savage-Austin A.
      • et al.
      Susceptible chiasmate configurations of chromosome 21 predispose to nondisjunction in both maternal meiosis I, and meiosis II.
      ). However, half of meiosis II errors are still observed as independent from meiosis I errors (Table 3), emphasizing their clinical significance, because the genotype of the resulting zygote cannot be predicted without testing the outcomes of both meiotic divisions, inferred from PB1 and PB2. The biological significance of both meiotic errors may also be obvious from the age dependence of isolated errors of meiosis I and meiosis II, as well as sequential meiosis I and meiosis II errors (
      • Kuliev A.
      • Cieslak J.
      • Verlinsky Y.
      Frequency and distribution of chromosomal abnormalities in human oocytes.
      ). The data showed the strongest age dependence of aneuploidies originating from the sequential meiosis I and meiosis II errors, which more than doubles in 40-year-old patients when compared with those of 35 years.
      Despite limitations of PB testing in which only maternally derived errors are identified, the PB testing still allows detection and avoidance of the transfer of the majority of aneuploid embryos, as approximately 95% of chromosomal errors originate from female meiosis alone. The other limitation may be that only five chromosome errors were tested, but these are the most frequent ones involved in human aneuploidies. Testing for 24 chromosomes was attempted by conventional comparative genomic hybridization (CGH) (

      Fragouli, E., Alfarawati, S., Katz-Jaffe, M., et al., 2009b. Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure. Fertil. Steril. doi:10.1016/j.fertnstert.2009.04.053.

      ,
      • Gutierrez-Mateo C.
      • Benet J.
      • Colls P.
      • et al.
      Aneuploidy study of human oocytes first polar body comparatice genomic hybridization anf metaphase II fluorescence in situ hybridization analysis.
      ), but it appeared to have significant limitations in detecting chromatid errors, which are the major source of embryo chromosomal abnormalities. This limitation has recently been overcome by the application of array-CGH, as shown in a preliminary study on the application of array-CGH for 24-chromosome testing in PB1, PB2 and resulting oocytes in cases of haploidy and triploidy (A Kuliev et al., data not shown). However, the technique is still to be validated for practical applications (Antony Gordon, personal communication).

      Inconsistency between aneuploidy types predicted by PB1 and detected by cleavage-stage testing

      Analysis of the types of errors showed a significantly higher frequency for missing (monosomy/nullisomy) chromosome/chromatid errors (53%), compared with extra chromosome/chromatid errors (disomy) in PB1 (25.6%; P < 0.001), in contrast to a comparable distribution of missing (41.4%) and extra (39.2%) chromatids in PB2 (Table 2). PB1 data showed a 72.2% chromatid error rate (46.7% missing and 25.5% extra chromatids), compared with 6.3% chromosomal errors (5.2% missing and 1.1% extra chromosomes; Figure 2). Similar to chromatid errors, missing chromosomes were more frequent compared with extra chromosomes, with an overall observation of two times higher prevalence of PB1 with chromosome/chromatid losses than chromosome/chromatid gains.
      Figure thumbnail gr2
      Figure 2Proportions of chromosome (chromatid) segregation errors in meiosis I. Upper panel: Centre: primary oocyte containing diploid set of chromosomes with the doubled amount of chromatin (4n) prior to maturation; Right: normal segregation of homologues in the first meiotic division, resulting in the extrusion of the first polar body (PB1) (smaller circle) containing one of the homologues; accordingly, the resulting secondary (metaphase II) oocyte contains the remaining homologue with two chromatids; Left: meiotic errors leading to the extrusion of PB1 containing abnormal set of chromosomes. Lower panel: (a) chromosomal non-disjunction, leading to segregation of both homologues to MII oocyte so that the extruded PB1 does not contain any material, results in a disomic oocyte; (b) chromosomal non-disjunction, leading to segregation of both homologues to PB1(smaller circle), which results in a nullisomic oocyte; (c) chromatid malsegregation, leading to an extra chromatid extrusion with PB1, which results in the lack of one chromatid in MII oocyte; (d) chromatid malsegregation, leading to a single chromatid extrusion with PB1, which results in the extra chromatid material in MII oocyte; (e) chromatid or chromosome malsegregation involving different chromosomes, involving different types of errors of different chromatids or chromosomes in MII oocyte, which results in complex errors.
      The observed phenomenon of higher frequency of chromatid over chromosome errors (10:1 ratio) is in agreement with the other relevant report mentioned (
      • Magli M.C.
      • Gianaroli L.
      • Crippa A.
      • Grugnetti C.
      • Ruberti A.
      • Ferraretti A.P.
      Causes of aneuploidy–polar body based PGD.
      ). Chromatid errors were also previously reported in traditional studies of meiotic chromosomes in MII oocytes (
      • Angel R.
      First meiotic division nondisjunction in human oocytes.
      ,
      • Pellestor F.
      • Andreo B.
      • Armal F.
      • Humeau C.
      • Demaille J.
      Mechanisms of non-disjunction in human female meiosis: the co-existence of two modes of malsegregation evidenced by the karyotyping of 1397 in-vitro unfertilized oocytes.
      ,
      • Pujol A.
      • Boiso I.
      • Benet J.
      • et al.
      Analysis of nine chromosome probes in first polar bodies and metaphase II oocytes for the detection of aneuploidies.
      ) and also observed in a mouse model (
      • Hunt P.
      • LeMaraire R.
      • Embury P.
      • Sheean L.
      • Mroz K.
      Analysis of chromosome behaviour in intact mammalian oocytes: monitoring the segregation of a univalent chromosome during female meiosis.
      ), as one of the important mechanisms of aneuploidy. This is also confirmed in a preliminary study of application of array-CGH for PB testing, as mentioned above.
      A non-random distribution of missing and extra chromatids and chromosomes (2:1 ratio, respectively; Figure 2) is also in agreement with
      • Magli M.C.
      • Gianaroli L.
      • Crippa A.
      • Grugnetti C.
      • Ruberti A.
      • Ferraretti A.P.
      Causes of aneuploidy–polar body based PGD.
      , suggesting that this might be an important biological mechanism, preventing the extrusion of extra chromosome material into the PB1 in the event of meiosis I errors. It has previously been demonstrated that there is an age dependence of both missing chromatids and missing chromosomes, increasing from 45% to 70% for missing chromatids, and from 4% to 8% for missing chromosomes, between the ages of 35 and 43 years (
      • Kuliev A.
      • Cieslak J.
      • Verlinsky Y.
      Frequency and distribution of chromosomal abnormalities in human oocytes.
      ). Although the possible relationship of missing signals due to the hybridization failure cannot be completely excluded, the age dependence of this category of abnormalities may suggest that this is a real phenomenon.
      The data are in agreement with the types of chromosomal abnormalities in spontaneous abortions, as the only autosomal monosomy observed in post-implantation development is monosomy 21. However, the predicted embryo trisomy predominance is in clear conflict with the observed monosomy predominance at the cleavage stage (
      • Munné S.
      • Bahce M.
      • Sandalinas M.
      • et al.
      Differences in chromosome susceptibility to aneuploidy and survival to first trimester.
      ). For example, analysis of aneuploidy types in the current series of 1252 embryos tested for aneuploidy by blastomere biopsy revealed 702 embryos with aneuploidy, of which 30.5% were monosomies, 27.8% trisomies, with the remaining represented by ploidy abnormalities, complex, chaotic or others types of abnormalities. It is of interest that no age dependence was revealed for these monosomies. Predominance of monosomies detected by the cleavage-stage PGD was also confirmed by PCR-based aneuploidy testing (
      • Verlinsky Y.
      • Kuliev A.
      Practical Preimplantation Genetic Diagnosis.
      ).
      The possible explanation of this discordance is that the majority of monosomies detected in embryos may derive from mitotic errors, provided that the technical causes can be excluded. In fact, a significant proportion of the cleavage-stage monosomies appeared to be euploid after their reanalysis with different probes (
      • Colls P.
      • Escudero T.
      • Cekleniak N.
      • Sadowy S.
      • Cohen J.
      • Munne S.
      Increased efficiency of preimplantation genetic diagnosis for aneuploidy by testing 12 chromosomes.
      ,
      • Uher P.
      • Baborova P.
      • Kralickova M.
      • Zech M.H.
      • Verlinsky Y.
      • Zrch N.
      Non-informative results and monosomies in PGD: the importance of a third round of re-hybridization.
      ). The fact that some of the cleavage-stage monosomies are not detected at the blastocyst stage may also suggest that some of the monosomies are either eliminated before implantation or have no biological significance, reflecting the poor viability of the monosomic embryos and their degenerative changes. However, the majority of pre-zygotically derived monosomies, as well as some of the post-zygotic ones, may still survive to the blastocyst stage and, therefore, lead to implantation failure or fetal loss (
      • Munné S.
      Chromosomal status of human embryo.
      ,
      • Verlinsky Y.
      • Kuliev A.
      Practical Preimplantation Genetic Diagnosis.
      ).
      The above inconsistency may also be due to a high prevalence of mosaicism at the cleavage stage, of which the exact prevalence and the origin has not been fully understood. The fact that the overall mosaicism prevalence does not show a relationship with maternal age (
      • Munné S.
      Chromosomal status of human embryo.
      ) may suggest that a significant proportion of mosaicism may be either artefactual and of no clinical relevance or simply transitional without affecting the embryo viability, which may be the consequence of degenerative processes in the embryos prior to embryo arrest. On the other hand, a certain fraction of mosaicism is still dependent on maternal age (
      • Munné S.
      • Sandalinas M.
      • Escudero T.
      • Marquuez C.
      • Cohen J.
      Chromosome mosaicism in cleavage stage human embryos: evidence of a maternal age effect.
      ), probably deriving from the aneuploid zygotes such as trisomics, some of which may result in disomic embryos because of selective disadvantage of abnormal cells, with also a chance of forming uniparental disomies in one-third of them. Such cases were incidentally detected in PGD for single-gene disorders as well as in the process of haplotyping for preimplantation human leukocyte antigen typing (
      • Verlinsky Y.
      • Kuliev A.
      Practical Preimplantation Genetic Diagnosis.
      ).
      The data may explain the recent controversy on the clinical impact of PGD for aneuploidies, as the majority of centres perform aneuploidy testing at the cleavage stage, which may not be ideal choice for aneuploidy detection unless it can be coupled with additional analysis, such as PB analysis or blastocyst biopsy. So, to clarify the utility of each of the approaches, further studies based on sequential PB and embryo biopsy may be useful to investigate the relative impact of each of these tests in improving the accuracy on detection of aneuploidy-free embryos for transfer.

      Complex errors and phenomenon of aneuploidy rescue in female meiosis

      Approximately one-fifth of abnormalities in PB1 and PB2 (21.5% and 19.3%, respectively) were of complex origin, represented by different types of errors, errors involving more than one chromosome or errors in both PB1 and PB2 of the same or different chromosomes (Table 2, Table 4). Of the 3881 oocytes (40%) with complex errors overall, 2438 (63%) involved simultaneous errors of different chromosomes and 1507 (37%) the same chromosome errors in both PB1 and PB2. Of 2742 (71%) with complex errors involving two or more chromosomes, 2067 (75%) involved two chromosomes and 675 (25%) involved three or more chromosomes. Of 2921 (17.0%) oocytes with both PB1 and PB2 abnormal, 1314 (45%) zygotes appeared to be balanced following these sequential errors (Table 4). This is in agreement with the other reported data (
      • Magli M.C.
      • Gianaroli L.
      • Crippa A.
      • Grugnetti C.
      • Ruberti A.
      • Ferraretti A.P.
      Causes of aneuploidy–polar body based PGD.
      ) and may represent a phenomenon of aneuploidy rescue, similar to the well-known trisomy rescue mechanism also observed in preliminary array-CGH studies.
      Table 4Oocytes with both PB1 and PB2 abnormal.
      Abnormalityn%
      Chromosome 13 only1555.3
      Chromosome 16 only2488.5
      Chromosome 18 only1274.4
      Chromosome 21 only41814.3
      Chromosome 22 only43614.9
      Different chromosomes
      The same chromosome can be involved in both meiosis I and II when two or more chromosomes are involved.
      141448.4
      Abnormal for more than one chromosome1234.2
      Total2921100
      Total balanced131445.0
      a The same chromosome can be involved in both meiosis I and II when two or more chromosomes are involved.
      A high prevalence of complex errors may also suggest that by testing for even a few most prevalent chromosome abnormalities, the errors of other chromosomes may simultaneously be detected, together with different types of errors of the same chromosome. This may indicate generalized disturbances in the meiosis process, which may be due to the age-related effect on the recombination frequency, the spindle formation errors also reported to increase with age, loss of chromosome cohesion and mitochondrial and organelle dysfunction (
      • Battaglia D.E.
      • Goodwin P.
      • Klein N.A.
      • Soules M.R.
      Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women.
      ,
      • Chatzimeletiou K.
      • Morrison E.E.
      • Prapas N.
      • Prapas Y.
      • Handyside A.H.
      Spindle abnormalities in normally developing and arrested human preimplantation embryos in vitro identified by confocal laser scanning microscopy.
      ,
      • Eichenlaub-Ritter U.
      • Vogt E.
      • Yiu H.
      • Gosden R.
      Spindles, mitochondria and redox potential in ageing oocytes.
      ,
      • Hassold T.
      • Merril M.
      • Adkins K.
      • Freemen S.
      • Sherman S.
      Recombination and maternal age-dependent nondisjunction: molecular studies of trisomy 16.
      ,
      • Lamb N.E.
      • Freeman S.
      • Savage-Austin A.
      • et al.
      Susceptible chiasmate configurations of chromosome 21 predispose to nondisjunction in both maternal meiosis I, and meiosis II.
      ,
      • Lamb N.E.
      • Feingold E.
      • Savage-Austin A.
      • et al.
      Characterization of susceptible chiasma configurations that increase the risk for maternal nondisjunction of chromosome 21.
      ,
      • Sherman S.L.
      • Peterson M.B.
      • Freeman S.B.
      • et al.
      Nondisjunction of chromosome 21 in maternal meiosis I: evidence for a maternal age-dependent mechanism involving reduced recombination.
      ). So with the testing for additional chromosomes, the prevalence of complex errors may be expected to increase, not necessarily significantly affecting the overall aneuploidy prevalence. The fact that a meiotic error of one chromosome may effect the segregation of other chromosomes was demonstrated also in a mouse model (
      • Hunt P.
      • LeMaraire R.
      • Embury P.
      • Sheean L.
      • Mroz K.
      Analysis of chromosome behaviour in intact mammalian oocytes: monitoring the segregation of a univalent chromosome during female meiosis.
      ).

      Chromosome-specific meiotic error origin and its impact on embryo viability

      The analysis of the chromosome-specific pattern showed that chromosomes 21 and 22 were much more frequently involved in female meiosis errors (25.0% and 31.8%, respectively) than chromosomes 13, 16 and 18 (12.6%, 17.8% and 12.8%, respectively; Table 5), which is in agreement with the data obtained in aneuploidy testing at the cleavage stage (
      • Munné S.
      Chromosomal status of human embryo.
      ). It was also previously demonstrated that, despite the differences in chromosome-specific aneuploidy rates, the age dependence was observed for each of these chromosome errors, almost doubling between the ages of 35 and 43 for chromosomes 16, 21 and 22 (
      • Kuliev A.
      • Cieslak J.
      • Verlinsky Y.
      Frequency and distribution of chromosomal abnormalities in human oocytes.
      ), again suggesting the overall disturbance of the meiosis process with advanced reproductive age.
      Table 5Origin of chromosome 13, 16, 18, 21 and 22 aneuploidies (based on information from both polar bodies of 8602 oocytes).
      ChromosomeTotal abnormalOriginP-value
      Chi-squared analysis between origins meiosis I and meiosis II. NS=not statistically significant with P-value >0.05.
      Meiosis IMeiosis IIMeiosis I and II
      131086 (12.6)436 (40.1)394 (36.3)256 (23.6)NS
      161531 (17.8)490 (32.0)679 (44.4)362 (23.6)<0.001
      181098 (12.8)530 (48.3)380 (34.6)188 (17.1)<0.001
      212151 (25.0)891 (41.4)790 (36.7)470 (21.9)0.002
      222736 (31.8)939 (34.3)1135(41.5)662 (24.2)<0.001
      Values are n (%).
      a Chi-squared analysis between origins meiosis I and meiosis II. NS = not statistically significant with P-value >0.05.
      Chromosome-specific origin of errors was also not similar: chromosome 16 and 22 errors originated more frequently in meiosis II (44.4% and 41.5% meiosis II errors versus 32.0% and 34.3% meiosis I, respectively), and chromosome 13, 18 and 21 errors more frequently in meiosis I (40.1, 48.3 and 41.4% in meiosis I versus 36.3, 34.6 and 36.7% in meiosis II, respectively), although the differences are not significant for chromosome 13 errors. It is of note that the proportion of oocytes with errors of both meiosis I and meiosis II origin were not significantly different for errors of different chromosomes except for chromosome 18 errors (Table 5).
      This data is opposite to that observed in spontaneous abortions and live-born children (
      • Hassold T.
      • Merril M.
      • Adkins K.
      • Freemen S.
      • Sherman S.
      Recombination and maternal age-dependent nondisjunction: molecular studies of trisomy 16.
      ,
      • Sherman S.L.
      • Peterson M.B.
      • Freeman S.B.
      • et al.
      Nondisjunction of chromosome 21 in maternal meiosis I: evidence for a maternal age-dependent mechanism involving reduced recombination.
      ) and may indicate poor viability of embryos resulting from the oocytes with the chromosome 16 and 22 errors of the second meiotic division, which may be incompatible with implantation and post-implantation development. Presently, there is no explanation of possible biological differences of aneuploidies depending on the meiotic origin, except for a loss of heterozygosity or higher homozygosity of the embryos originating from meiosis II errors for the genes located in these chromosomes, which may lead to imprinting of paternal or maternal genes of the chromosomes 16 or 22. Although there is no proof of the established imprinting genes in these chromosomes, there are case reports of a possible imprinting on chromosome 16 affecting fetal development or being associated with cancer (
      • Abu-Amero S.
      • Monk D.
      • Apostolidou S.
      • Stanier P.
      • Moore G.
      Imprinted genes and their role in human fetal growth.
      ,
      • Angel E.
      • Antonarakis S.E.
      Genomic Imprinting and Uniparental Disomy in Medicine: Clinical and Molecular Aspects.
      ,
      • Mummert S.
      • Lobanenkov V.
      • Feinberg A.P.
      Association of chromosome arm 16q loss with loss of imprinting of insulin-like growth factor-II in Wilms tumor.
      ,
      • Yong P.J.
      • Marion S.A.
      • Barrett I.J.
      • Kalousek D.K.
      • Robinson W.P.
      Evidence of imprinting on chromosome 16: the effect of uniparental disomy on the outcome of mosaic trisomy 16 pregnancies.
      ).
      The other discrepancy is related to the meiotic origin of chromosome 18 errors, which predominantly originates from meiosis I in the current data (Table 5) and is opposite to that in live-born children (
      • Fisher J.M.
      • Harvey J.F.
      • Morton N.E.
      • Jacobs P.A.
      Trisomy 18: studies of the parent and cell division of origin and effect of aberrant recombination on nondisjunction.
      ). Whatever explanation may be for the above phenomenon, this data provides the first evidence for possible viability differences dependent upon not only the chromosome involved but the meiotic origin of the error. However, this may not apply to other chromosomes, as the origin of chromosome 13 and 21 error patterns were in agreement with that observed in spontaneous abortions and live-born children (
      • Hassold T.
      • Merril M.
      • Adkins K.
      • Freemen S.
      • Sherman S.
      Recombination and maternal age-dependent nondisjunction: molecular studies of trisomy 16.
      ,
      • Sherman S.L.
      • Peterson M.B.
      • Freeman S.B.
      • et al.
      Nondisjunction of chromosome 21 in maternal meiosis I: evidence for a maternal age-dependent mechanism involving reduced recombination.
      ), although differences in the origin of chromosome 13 error patterns are not significant in the current data.

      Impact of PB testing in detection and avoidance of aneuploid embryos for transfer

      Because PB1 and PB2 have no biological significance in pre- and post-implantation development and are extruded in a normal process of oocyte maturation and fertilization, their removal and testing may become a useful tool in assisted reproduction practices to identify the aneuploidy-free oocytes. This testing in conjunction with embryo biopsy for additional chromosomes should help in the pre-selection of oocytes with the highest potential for establishing a viable ongoing pregnancy, significantly improving IVF efficiency. For example, a significant improvement in the implantation rate was reported even by PB1 pre-selection of oocytes in 553 infertile patients (
      • Gianaroli L.
      • Magli M.C.
      • Lappi M.
      • Capoti A.
      • Robles F.
      • Ferraretti A.P.
      Preconception diagnosis.
      ). So further improvement could have been achieved by detecting the remaining meiosis II errors, which represent over one-third of the overall number of abnormal oocytes. This is in agreement with previously published clinical outcome data, which demonstrated the utility of PB1 and PB2 testing in detecting and avoiding the transfer of aneuploid embryos (
      • Verlinsky Y.
      • Tur-Kaspa I.
      • Cieslak J.
      • et al.
      Preimpalntation diagnosis for chromosomal disorders improves reproductive outcome of poor-prognosis patients.
      ). The overall impact was particularly obvious in the reduction of spontaneous abortion rate, which was much lower than observed in patients of comparable reproductive age without aneuploidy testing.
      In summary, the presented results of aneuploidy testing of the world’s largest series of oocytes provide evidence for the usefulness of PB-based aneuploidy testing as part of accurate pre-selection of aneuploidy-free embryos for transfer, which should be performed by removal and analysis of both PB1 and PB2. The observed predominance of predicted trisomic embryos is in conflict with predominance of monosomies described at the cleavage stage, which may be due to post-zygotic events, some of which might not be of biological significance and not representative of the chromosomal status of the embryos tested. Finally, for the first time, as far as is known, the evidence is presented for a possible relationship between embryo viability and meiotic origin of chromosomal errors, affecting their clinical impact on preimplantation and post-implantation development. These observations may be further explored by the application of 24-chromosome testing by array-CGH analysis, which is currently underway in a number of PGD centres.

      Dedication

      This paper is dedicated to the memory of Yury Verlinsky, who introduced the polar body biopsy technique to PGD.

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