Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH

One of the most important factors influencing embryo viability is chromosome imbalance (aneuploidy). Embryos derived from aneuploid gametes have little potential for forming a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. For more than a decade, preimplantation genetic screening (PGS) has been used to assist in the identification of aneuploid embryos. However, current strategies, based upon cell biopsy followed by fluorescent in situhybridization, allow less than half of the chromosomes to be screened. In this review, we discuss methods that overcome the limitations of earlier PGS strategies and provide screening of the entire chromosome complement in oocytes and embryos. In recent months, there has been a rapid growth in the number of PGS cycles utilizing one such method, comparative genomic hybridization (CGH). Data from IVF cycles utilizing CGH must be considered preliminary, but appear to indicate a dramatic increase in embryo implantation following comprehensive chromosomal screening. It is expected that methods based upon microarrays will yield similar clinical results and may be sufficiently rapid to permit comprehensive screening without the need for embryo cryopreservation. Some microarray platforms also offer the advantage of embryo fingerprinting and the potential for combined aneuploidy and single gene disorder diagnosis. However, more data concerning accuracy and further reductions in the price of tests will be necessary before microarrays can be widely applied

Number of embryos from each cycle and the number of chromosomes assessed along with abnormalities detected.

<p>Rec: reciprocal translocation; Rob: Robertsonian translocation; Inv: Inversion; Bla: Blastomere; PB: polar body; TE: trophectoderm cells.</p>*<p>Chromosomes involved in the rearrangement were not assessed;</p>**<p>Abnormalities affecting chromosomes involved in the rearrangement were excluded. Case 12 was the only one in which two rearrangements were present. This case was excluded from the statistical analysis.</p

Patient information.

<p>Bla: Blastomere; PB: polar body; TE: trophectoderm cells; aCGH: Microarray-CGH; Met-CGH: Metaphase CGH. Note: Some patients underwent more than one cycle of PGD and may have had analysis conducted at different stages in subsequent cycles.</p

Corrected number of errors in the patient-specific control groups.

*<p>Chromosomes involved in the matching patient's rearrangement were excluded from the assessed chromosomes.</p>**<p>Abnormalities affecting chromosomes involved in the matching patient's rearrangement were excluded.</p>***<p>Expected number in the control group if it consisted of the same number of samples as provided by the matched patient.</p

Malsegregation events detected in non-rearranged chromosomes in patient and control samples.

<p>Malsegregation events detected in non-rearranged chromosomes in patient and control samples.</p

Investigation of sperm telomere length as a potential marker of paternal genome integrity and semen quality

Recent studies have reported shorter sperm telomere length (STL) in men with idiopathic infertility. The aim of this study was to measure STL in semen samples from men to evaluate whether STL variation is associated with chromosomal abnormality, DNA fragmentation, traditional semen parameters, IVF outcome, or all four factors. A significant correlation between telomere length and diploidy was observed (P = 0.037). Additionally, STL was found to be positively associated with sperm count (P = 0.006); oligospermic samples had particularly short telomeres (0.9 ± 0.1 versus 1.4 ± 0.1; P = 0.0019). The results confirmed a link between sperm DNA fragmentation and aneuploidy, previously proposed (P = 0.009). A negative relationship was demonstrated between sperm concentration and aneuploidy and Sperm DNA framentation (P = 0.03, P < 0.0001, respectively). For a subset of 51 of the 73 sperm samples used for fertilization, IVF outcomes were known. A total of 17.6% of these samples had atypical STLs. None of these samples produced an ongoing pregnancy. In contrast, the pregnancy rate for samples that had STLs in the normal range was 35.7% (P = 0.044). In conclusion, STL has potential as a fast and inexpensive form of sperm quality assessment

Data concerning the patient-matched control groups.

<p>Bla: Blastomere; PB: polar body; TE: trophectoderm cells;</p>*<p>Chromosomes involved in the matching patient's rearrangement were excluded from the assessed chromosomes.</p>**<p>Abnormalities affecting chromosomes involved in the matching patient's rearrangement were excluded.</p

Microarray-CGH analysis of an embryo from a Robertsonian translocation carrier.

<p>Cytogenetic analysis of a cleavage stage embryo from a Robertsonian translocation carrier- 46,XY,t(13;15)(q21.3;q11.2). Microarray-CGH revealed monosomy 13, presumably resulting from a meiotic error due to problems processing the rearranged chromosomes. An additional aneuploidy unrelated to the Robertsonian translocation (monosomy for chromosome 1) was also detected. The two monosomies are indicated by the altered ratio of fluorescence related to the test (embryo) and reference (46,XY) DNA samples. All of the probes corresponding to chromosomes 1 and 13 have test/reference ratios less than 0.3.</p

The relationship between mtDNA quantity, female age and embryo chromosome constitution.

<p><b>a)</b> Data obtained during quantitative real-time PCR analysis of TE samples removed from 302 blastocysts demonstrated a statistically significant increase (P = 0.003) in the level of mtDNA in relation to advancing female age. This phenomenon was evident for both euploid and aneuploid blastocysts. <b>b)</b> Real-time PCR analysis of 39 blastomeres showed that cleavage stage embryos from reproductively younger women contained significantly (P = 0.01) higher mtDNA levels, compared to those generated by reproductively older women. <b>c)</b> Real-time PCR analysis of TE samples also demonstrated that aneuploid blastocysts (n = 99) contained significantly (P = 0.025) larger quantities of mtDNA at all ages, compared to those that were euploid (n = 203). Statistical analysis of mtDNA values took place with the use of unpaired two-tailed t-tests.</p