Genetics

Microarray / array-CGH

Homapage / Microarray / array-CGH

By using microarray technique, it is possible to diagnose all chromosomal abnormalities including aneuploidies and partial imbalances that may occur in embryos in only 12 hours. It has several advantages over FISH method where only limited chromosomes could be screened...

Preimplantation genetic diagnosis using "Microarray Technique"

By using microarray technique, it is possible to diagnose all chromosomal abnormalities including aneuploidies and partial imbalances that may occur in embryos in only 12 hours. It has several advantages over FISH method where only limited chromosomes could be screened.

Numerical abnormality in chromosomes is called as aneuploidy. Chromosomally normal human embryonic cells contain 46 chromosomes (22 pairs of autosomes and 1 pair of gonosomes) (Figure 1). Aneuploidy can originate from an excess number of chromosomes (e.g, trisomy) or from missing chromosomes (e.g. monosomy). Chromosomal abnormalities are very common in preimplantation embryos. Trisomy 21 (Down Syndrome), trisomy 16, trisomy 13 (Patau Syndrome), trisomy 18 (Edwards Syndrome); and monosomy X (Turner Syndrome) and Trisomy XXY (Klinefelter Syndrome) are the most frequent chromosomal abnormalities among humans.

Figure 1: Normal human somatic cells contain 23 pairs of chromosomes (22 autosomes and 2 gonosomes). 

Fluorescent in situ hybridization (FISH) method

FISH uses fluorescently labeled probes which are specific to each chromosome (figure 2). The steps of this technique consist of fixation of the nucleus of the biopsied cell, probe application, hybridization, washing and analysis . Every step is of key importance and requires experience and skill. With this method, embryos can be screened in 24 hours for the following chromosomes 8,13,14,15,16,17,19,18,20,21,22, X and Y. Although it is a relatively low cost, less complex method, FISH cannot detect all abnormalities, since the number of chromosomes that can be analyzed is limited. 

 Normal blastomere
Monosomy 21, Trisomy 22
 Complex Aneuploid

Figure 2: FISH images of normal (A) and abnormal (B, C) blastomeres: chromosome 13 (red), chromosome 16 (light blue), chromosome 18 (blue), chromosome 21 (green), chromosome 22 (yellow), (Images from İstanbul Memorial Hospital, Reproductive Genetics Laboratory).

Advantages of array-CGH technique over FISH 

It is known that 80% of embryos generated from advanced maternal age patients carry at least one chromosomal abnormality and 30-40% of all chromosomal abnormalities cannot be detected by FISH method. However, array comperative genomic hybridization (a-CGH) method can detect abnormalities of all chromosomes, both numerically and structurally, with high resolution. Deletions, duplications and unbalanced chromosomal regions can be easily detected using this method. With this method it is possible to diagnose all chromosomes in embryos in just 12 hours. 

Clinical indications for a-CGH 

In Istanbul Memorial Hospital a-CGH technique is mostly used for the following indications,

  • Advanced maternal age 
  • Abnormal fetus history
  • Unexplained recurrent abortions
  • Gonadal mosaicism
  • Translocation carriers 

Biopsy procedures

It is known that 80% of embryos generated from advanced maternal age patients carry at least one chromosomal abnormality. 30-40% of all chromosomal abnormalities cannot be detected by conventional flourescent in situ hybridization method (FISH). However, array comperative genomic hybridization (a-CGH) method can detect abnormalities of all chromosomes, both numerically and structurally, with high resolution. Deletions, duplications and unbalanced chromosomal regions can be easily detected using this method.  

Biopsy of embryos can be performed at 3 different stages of embryonic development: 

  • Polar body analysis prior to or after fertilization (figure 3a).
  • Blastomere analysis at cleavage stage (figure 3b)
  • Trophectoderm tissue analysis at blastocyst stage (figure 3c)

These three methods can be used individually or in combination to increase the reliability of PGD. The experience of the laboratory staff is of key importance in successful biopsy and PGD procedures.

Figure 3: Polar body biopsy (A), blastomere biopsy (B), trophectoderm biopsy (C). (Images from Istanbul Memorial Hospital, IVF Laboratory)
 

From the beginning of 2010, aCGH technique is used in combination with blastocyst stage biopsy. This  new approach decreases time needed for completion of genetic analysis by providing more samples and  decreases the analysis costs by screening only the embryos which has the high implantation capacity. This blastocyst biopsy+aCGH analysis approach helps to find the best embryo suitable for transfer and thus increases the implantation rates.  

With this method it is possible to diagnose all chromosomes in preimplantation embryos in just 12 hours.

In Istanbul Memorial Hospital Reproductive Genetics Laboratory, aCGH has been used with success since the beginning of 2011. Since the results can be obtained within 12-24 hours, freezing of the embryos is unnecessary. aCGH is the most reliable method for selecting embryos without chromosomal abnormalities.

a- CGH method

Comparative genomic hybridization (CGH) method compares the gain and loss of genetic material against a reference chromosomal system which is known to be normal. In array CGH method, the samples are hybridized against an array system (Figure 4). 

Figure 4: Summary of array-CGH method 

Arrays contain thousands of probes sopesific for each chromosome, which is very important in increasing the resolution. An image of a microarray after hybridization and scanning has been shown in figure 5. 

Figure 5: An image of a microchip

Below are the some examples of normal and abnormal embryos according to aCGH method.

Figure 6: This embryo was detected as “normal” by using aCGH method. In this method, the diagnosis has been made by comparing two reference sets in order to increase reliability of the results.  

 

 Figure 7: Abnormalities related with chromosomes 1,2,3,4,5,6,7,8,9,10,11,12,14,19,20 could be easily detected by this method, whereas FISH technique is unable to detect those abnormalities since only chromosomes 13,15,16,17,18,21,22,XY could be screened by FISH. 
 

The risk of having a child with Down’s syndrome increases dramatically by advancing maternal age.  

Figure 8: a) Trisomy 21 was detected in this embryo as shown by green color in the analysis program.

 

Figure 8: b) In this method, it is possible to enlarge the structures of chromosomes and anlyze in detail. In this image, 50 or more probes have been analyzed which have bound to chromosome 21. All of them indicates an increase compared to the reference values. The software draws those regions as “green” and indicates a “trisomy” . This result is 50 times more reliable than the FISH method in which the results are given based on only one probe. 

 

Figure 9: aCGH method is the most reliable method in diagnosing unbalanced segregation products arising from translocation carriers. In addition to that, it gives information about the whole  chromosome set and abnormalities which may rise due to other meiotic abnormalities. In this image, an unbalanced embryo in q arms of chromosomes 9 and 11 were detected in an embryo belonging to a reciprocal translocation carrier. 

 

Figure 10: It is also possible to use this method in the diagnosis of prenatal and postnatal samples. 
a) This is the genetic analysis result of an abortion material in which cytogenetic methods failed to give any result due to unsuccessfull culture. “Trisomy 6” was found as the reason of this miscarriage.  
Figure 10: b) “Trisomy 18” was detected in this amniotic fluid sample. The test gave result in 2 days with the information of all chromosomes.  

For more information,

Mol. Bio. Çağrı Beyazyürek 

(cagri.beyazyurek@memorial.com.tr)

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