Analysis of chromosome segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes

Individuals carrying translocations suffer from reduced fertility or spontaneous abortions and seek help in form of assisted reproductive technology (ART) and preimplantation genetic diagnosis (PGD). While most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are thus unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Thus several methods have been devised to detect translocation imbalance through FISH in single cells for purpose of PGD, among them polar body chromosome painting, interphase FISH with combination of subtelomeric and centromeric probes, breakpoint spanning probes, and cell conversion. Results with PGD indicate a significant decrease in spontaneous abortions, from 81% before PGD to 13% after PGD. They also indicate very high rates of chromosome abnormalities in embryos from translocation carriers, 72% for Robertsonian translocations and 82% for reciprocal translocations. Sperm analysis was found to be a good predictor of IVF and PGD outcome, with samples with more than 60% abnormal forms indicating poor prognosis. Similarly, the predictability from first PGD cycle results for future cycles was 90%. In summary, PGD can help translocation carriers to achieve viable pregnancies, but the success of the process is conversely related to the baseline of unbalanced gametes.     

Identification of Chromosomal Errors in Human Preimplantation Embryos with Oligonucleotide DNA Microarray

A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.     

Preimplantation genetic diagnosis--an overview.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.     

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.     

Preimplantation genetic diagnosis: State of the ART 2011

For the last 20 years, preimplantation genetic diagnosis (PGD) has been mostly performed on cleavage stage embryos after the biopsy of 1–2 cells and PCR and FISH have been used for the diagnosis. The main indications have been single gene disorders and inherited chromosome abnormalities. Preimplantation genetic screening (PGS) for aneuploidy is a technique that has used PGD technology to examine chromosomes in embryos from couples undergoing IVF with the aim of helping select the chromosomally ‘best’ embryo for transfer. It has been applied to patients of advanced maternal age, repeated implantation failure, repeated miscarriages and severe male factor infertility. Recent randomised controlled trials (RCTs) have shown that PGS performed on cleavage stage embryos for a variety of indications does not improve delivery rates. At the cleavage stage, the cells biopsied from the embryo are often not representative of the rest of the embryo due to chromosomal mosaicism. There has therefore been a move towards blastocyst and polar body biopsy, depending on the indication and regulations in specific countries (in some countries, biopsy of embryos is not allowed). Blastocyst biopsy has an added advantage as vitrification of blastocysts, even post biopsy, has been shown to be a very successful method of cryopreserving embryos. However, mosaicism is also observed in blastocysts. There have been dramatic changes in the method of diagnosing small numbers of cells for PGD. Both array-comparative genomic hybridisation and single nucleotide polymorphism arrays have been introduced clinically for PGD and PGS. For PGD, the use of SNP arrays brings with it ethical concerns as a large amount of genetic information will be available from each embryo. For PGS, RCTs need to be conducted using both array-CGH and SNP arrays to determine if either will result in an increase in delivery rates.     

The Influence of Single Nucleotide Polymorphism Microarray-Based Molecular Karyotype on Preimplantation Embryonic Development Potential

In order to investigate the influence of the molecular karyotype based on single nucleotide polymorphism (SNP) microarray on embryonic development potential in preimplantation genetic diagnosis (PGD), we retrospectively analyzed the clinical data generated by PGD using embryos retrieved from parents with chromosome rearrangements in our center. In total, 929 embryos from 119 couples had exact diagnosis and development status. The blastocyst formation rate of balanced molecular karyotype embryos was 56.6% (276/488), which was significantly higher than that of genetic imbalanced embryos 24.5% (108/441) (P<0.001). No significant difference was detected in blastocyst formation rates in the groups of maternal age<30, 30–35 and >35 respectively. Blastocyst formation rates of male and female embryos were 44.5% (183/411) and 38.8% (201/518) respectively, with no significant difference between them (P>0.05). The rates of balanced molecular karyotype embryos vary from groups of embryos with different cell numbers at 68 hours after insemination. The blastocyst formation rate of embryos with 6–8 cells (48.1%) was significantly higher than that of embryos with <6 cells (23.9%) and with >8 cells (42.9%) (P<0.05). As for the unbalanced embryos, there was no significant difference of the distribution of abnormal molecular karyotypes in the subgroup of the arrest, morula and blastocyst. Thus, we conclude that embryos with balanced molecular karyotype have significant higher development potential than those with imbalanced molecular karyotype whilst maternal age, embryo gender and types of abnormal molecular karyotype have no significant influence on blastocyst formation. Compared with embryos with <6 and >8 cells, embryos with 6–8 blastomeres have higher rate of balanced molecular karyotype and blastocyst formation.     

Chromosome Segregation Analysis in Human Embryos Obtained from Couples Involving Male Carriers of Reciprocal or Robertsonian Translocation

The objective of this study was to investigate the frequency and type of chromosome segregation patterns in cleavage stage embryos obtained from male carriers of Robertsonian (ROB) and reciprocal (REC) translocations undergoing preimplantation genetic diagnosis (PGD) at our reproductive center. We used FISH to analyze chromosome segregation in 308 day 3 cleavage stage embryos obtained from 26 patients. The percentage of embryos consistent with normal or balanced segregation (55.1% vs. 27.1%) and clinical pregnancy (62.5% vs. 19.2%) rates were higher in ROB than the REC translocation carriers. Involvement of non-acrocentric chromosome(s) or terminal breakpoint(s) in reciprocal translocations was associated with an increase in the percent of embryos consistent with adjacent 1 but with a decrease in 3∶1 segregation. Similar results were obtained in the analysis of nontransferred embryos donated for research. 3∶1 segregation was the most frequent segregation type in both day 3 (31%) and spare (35%) embryos obtained from carriers of t(11;22)(q23;q11), the only non-random REC with the same breakpoint reported in a large number of unrelated families mainly identified by the birth of a child with derivative chromosome 22. These results suggest that chromosome segregation patterns in day 3 and nontransferred embryos obtained from male translocation carriers vary with the type of translocation and involvement of acrocentric chromosome(s) or terminal breakpoint(s). These results should be helpful in estimating reproductive success in translocation carriers undergoing PGD.     

The culture of human cleavage stage embryos alone or in groups: effect upon blastocyst utilization rates and implantation

The effect of cleavage-stage group culture (CGC; embryos cultured in groups of three or more for the first 3 days and then individually to blastocyst) was compared to extended single embryo culture (ESC; embryos cultured individually to the blastocyst stage). While implantation and ongoing pregnancy rates were similar between groups, the blastocyst utilization rate (number of blastocysts suitable for freezing and thawing/total number of embryos cultured to Day 5 and 6) was significantly higher when embryos were cultured in CGC for women ≤35 yrs thereby increasing the number of embryos available for clinical use for the younger women. This strategy of group culture to Day 3 would seem an ideal protocol to capitalize on an overall embryo quality in two particular settings, namely programmes wishing to (i) undertake Day 3 transfers, and (ii) keep embryos separate from Day 3 to Day5/6 for the purposes of selection. The culture system can also be applied to the embryos of older women without adverse effect, enabling the same system to be used for all embryos.     

Chromosome breakage in human preimplantation embryos from carriers of structural chromosomal abnormalities in relation to fragile sites, maternal age, and poor sperm factors

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.     

Infertile couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions

Preimplantation genetic diagnosis (PGD) may provide a feasible option for some Robertsonian translocation carriers who experience severe difficulty in achieving a normal pregnancy. We report on five PGD cycles for two such couples, 45,XY,der(13;14)(q10:q10) and 45,XX,der(13;21)(q10;q10), carried out by biopsy of two cells from day 3 post-insemination embryos generated by in vitro fertilisation. Locus-specific YAC probes for chromosomes 13, 14 and 21 were used to detect the chromosomes involved in the translocation using multicolour FISH. Three embryos transfers were carried out (two single embryo transfers and one double transfer) but no clinical pregnancies were established. In two cycles no embryos were transferred as all those biopsied were chromosomally abnormal. Combined results from both couples show 13% (6/45) of embryos analysed were normal for the translocation chromosomes and 87% (39/45) were chromosomally abnormal; these were categorised as 36% aneuploid or aneuploid mosaic and 51% chaotic where the chromosome constitution varied randomly from cell to cell. This suggests two factors may be acting to reduce fertility in these couples; the aneuploid segregation of the parental Robertsonian translocation and also a post-zygotic factor leading to uncontrolled chromosome distribution in early cleavage stages in an exceptionally high proportion of embryos. Received: 24 September 1997 / Accepted: 22 October 1997     

Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders.     

Regional assignment of seven genes on chromosome 1 of man by use of man-Chinese hamster somatic cell hybrids. I. Results obtained after hybridization of human cells carrying reciprocal translocations involving chromosome 1.

Regional localization studies of genes coding for human PGD, PPH1, PGM1, UGPP, GuK1, Pep-C, and FH, which have been assigned to chromosome 1, were performed with man-Chinese hamster somatic cell hybrids, Informative hybrids that retained fragments of the human chromosome 1 were produced by fusion of hamster cells with human cells carrying reciprocal translocations involving chromosome 1. Analysis of the hybrids that retained one of the translocation chromosomes or de novo rearrangements involving the human 1 revealed the following gene positions: PGD and PPH1 in 1pter leads to 1p32, PGM1 in 1p32 leads to 1p22, UGPP and GuK1 in 1q21 leads to 1q42, FH in 1qter leads to 1q42, and Pep-C probably in 1q42.     

The African trypanosome genome

The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.     

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300–1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7–10 hours and 63% for 1–2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained using ultrasonography in all pregnancies assessed (11/11, 100%); untreated biopsy samples were correctly diagnosed in 36 of 41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7–10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95 °C improved the overall efficiency of embryo sex determination.     

Meiotic recombinations within major histocompatibility complex of human embryos

We studied the rate and pattern of recombinations within the extended major histocompatibility complex (MHC) locus of the human embryos obtained during preimplantation genetic diagnosis (PGD) for HLA compatibility. Recombinant allele frequency was on average 5.33?%, and recombination rate was 0.44?cM/Mb in the 12.2?Mb of the extended MHC locus. Recombination rate varied up to 14-fold (0.19–2.73?cM/Mb) between cases, and maternal recombination rate was on average 3.8 times higher than paternal alleles. More than 69?% of the recombination hot spots were clustered within the extended class II region where the recombination rate was 5.4 times more than that in extended class I region. These findings indicate the potential of PGD to study the mechanisms of linkage disequilibrium within MHC locus of human embryos, demonstrate the recombination characteristics within extended MHC loci of human embryos in comparison to sperm and family studies, and point to the significance of design and interpretation of PGD for HLA compatibility to avoid misdiagnosis because of meiotic recombinations.     

Preimplantation Genetic Diagnosis and Natural Conception: A Comparison of Live Birth Rates in Patients with Recurrent Pregnancy Loss Associated with Translocation

Background

Established causes of recurrent pregnancy loss (RPL) include antiphospholipid syndrome, uterine anomalies, parental chromosomal abnormalities, particularly translocations, and abnormal embryonic karyotypes. The number of centers performing preimplantation genetic diagnosis (PGD) for patients with translocations has steadily increased worldwide. The live birth rate with PGD was reported to be 27-54%. The live birth rate with natural conception was reported to be 37-63% on the first trial and 65-83% cumulatively. To date, however, there has been no cohort study comparing age and the number of previous miscarriages in matched patients undergoing or not undergoing PGD. Thus, we compared the live birth rate of patients with RPL associated with a translocation undergoing PGD with that of patients who chose natural conception.

Methods and Findings

After genetic counseling, 52 patients who desired natural conception and 37 patients who chose PGD were matched for age and number of previous miscarriages and these comprised the subjects of our study. PGD was performed by means of fluorescence in situ hybridization analysis. The live birth rates on the first PGD trial and the first natural pregnancy after ascertainment of the carrier status were 37.8% and 53.8%, respectively (odds ratio 0.52, 95% confidence interval 0.22-1.23). Cumulative live birth rates were 67.6% and 65.4%, respectively, in the groups undergoing and not undergoing PGD. The time required to become pregnancy was similar in both groups. PGD was found to reduce the miscarriage rate significantly. The prevalence of twin pregnancies was significantly higher in the PGD group. The cost of PGD was $7,956 U.S. per patient.

Conclusions

While PGD significantly prevented further miscarriages, there was no difference in the live birth rate. Couples should be fully informed of the similarity in the live birth rate, the similarity in time to become pregnancy, the advantages of PGD, such as the reduction in the miscarriage rate, as well as its disadvantages, such as the higher cost, and the advantages of a natural pregnancy, such as the avoidance of IVF failure. The findings presented here should be incorporated into the genetic counseling of patients with RPL and carrying a translocation.     

Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis

Carriers of balanced translocations show an increased risk of infertility and spontaneous abortions, because of errors in gametogenesis, and constitute a significant fraction of patients seeking assisted reproduction. The objective of this study was to design approaches for preimplantation diagnosis of chromosome translocations and to apply such techniques to the selection of chromosomally normal or balanced embryos prior to their transfer to the mother’s womb. Three slightly different approaches were assessed by means of chromosome-specific, non-isotopically labeled DNA probes and an assay based on fluorescence in situ hybridization- to score and characterize chromosomes in single blastomeres biopsied from embryos on their third day of development. The three approaches were used for preimplantation genetic diagnosis involving four couples who had enrolled in our IVF program and in which one of the partners was a carrier of one of the following translocations: 46,XX,t(12;20)(p13.1;q13.3), 46,XY,t(3;4) (p24;p15), 45,XY,der(14;15)(10q;10q), and 46,XY,t(6;11) (p22.1;p15.3). A total of 33 embryos were analyzed, of which 25 (75.8%) were found to be either unbalanced or otherwise chromosomally abnormal. Only a single embryo could be transferred to patients A and D, whereas three embryos were transferred to patient B in a total of two IVF cycles. Transfer of two embryos to patient C resulted in an ongoing pregnancy. Re-analysis of non-transferred embryos with additional probes confirmed the initial results in 95% (20/21) of the cases. In conclusion, case-specific translocation tests can be applied to any translocation carrier for the selection of normal or chromosomally balanced embryos prior to embryo transfer. This is expected significantly to increase the success rates in IVF cycles of translocation carriers, while preventing the spontaneous abortion or birth of abnormal offspring. Received: 13 January 1998 / Accepted: 24 March 1998     

Cytogenetics in reproductive medicine: the contribution of comparative genomic hybridization (CGH)

Cytogenetic research has had a major impact on the field of reproductive medicine, providing an insight into the frequency of chromosomal abnormalities that occur during gametogenesis, embryonic development and pregnancy. In humans, aneuploidy has been found to be relatively common during fetal life, necessitating prenatal screening of high-risk pregnancies. Aneuploidy rates are higher still during the preimplantation stage of development. An increasing number of IVF laboratories have attempted to improve pregnancy rates by using preimplantation genetic diagnosis (PGD) to ensure that the embryos transferred to the mother are chromosomally normal. This paper reviews some of the techniques that are key to the detection of aneuploidy in reproductive samples including comparative genomic hybridization (CGH). CGH has provided an unparalleled insight into the nature of chromosome imbalance in human embryos and polar bodies. The clinical application of CGH for the purposes of PGD and the future extensions of the methodology, including DNA microarrays, are discussed.     

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.     

Maternal gametic transmission of translocations or inversions of human chromosome 11p15.5 results in regional DNA hypermethylation and downregulation of CDKN1C expression

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in "chromatin context".