DETECTION OF HUMAN EMBRYO ANEUPLOIDY IN THE PROCEDURE OF IN VITRO FERTILIZATION AFTER PREIMPLANTATION GENETIC SCREENING USING ARRAY COMPARATIVE GENOMIC HYBRIDIZATION/DETEKCIJA ANEUPLOIDIJA U HUMANIM EMBRIONIMA U POSTUPKU IN VITRO FERTILIZACIJE NAKON PREIPLANTACIONOG GENETICKOG SKRININGA ARRAY KOMPARATIVNOM GENOMSKOM HIBRIDIZACIJOM.
It is estimated that 15% of spontaneous clinical pregnancies are miscarried in the first trimester (7th to 10th gestational week), mostly as a result of chromosomal aberrations, particularly aneuploidies . According to evidence based observations, less than half of all conceptions result in childbirth, while this percentage is reduced even further with advanced maternal age [2-4]. These negative consequences of chromosomal imbalance are especially noticeable in assisted reproductive technology (ART) procedures, leading to an early arrest in cell division, embryo implantation failure or pregnancy loss, immediately after implantation, before it can be ultrasonically confirmed . Out of the total number of embryos with sustained viability after in vitro culture, only 3l% lead to a clinical pregnancy and live birth .
Abbreviations ART - assisted reproductive technology IVF - in vitro fertilization PGD - preimplantation genetic diagnosis PGS - preimplantation genetic screening FISH - fluorescent in situ hybridization DNA - deoxyribonucleic acid PCR - polymerase chain reaction CGH - comparative genomic hybridization TE - trophectoderm WGA - whole genome amplification ICSI - intracytoplasmic sperm injection ICM - inner cell mass ESHRE PGD Consortium - European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium
In in vitro fertilization (IVF) procedures, since the birth of Louise Brown in 1978, more than 5,4 million children have been born, and nearly 600.000 ART cycles are performed each year only in Europe . Embryo selection for transfer is mainly done according to morphological criteria and the developmental characteristics of the embryo. However, even after the transfer of the best quality embryos, the implantation potential is relatively low. Researches have shown that more than 50% of in vitro derived embryos contain aneuploid cells. Extended cultivation until the blastocyst stage somewhat contributes to increased implantation rates, but it has been proven that aneuploid embryos can reach high-quality blastocyst stage as well as euploid embryos [7, 8].
Understanding the mechanisms which cause changes in chromosomal constitution and their detection is of great clinical significance. In addition to traditional cytogenetic methods for the identification of chromosomal abnormalities, many IVF centers have introduced preimplantation genetic diagnosis/screening, based on molecular methods for the analysis of genetic material.
Detection of Aneuploidy in Human Embryos
Human embryo development begins with the fertilization process which simultaneously triggers the completion of the second oocyte meiosis. Fertilization is followed by the fusion of the male and female pronuclei into a zygote. Over the next several days, certain mitotic divisions take place until blastocyst formation, but these events are unsuitable for observations, as they occur in the oviducts and the uterus. Improvements in ART have enabled these events to be viewed, thus the growth and development of the embryo can be monitored and studied during all developmental phases .
It has been proven that the majority of trisomies and/or monosomies causing miscarriages are the result of errors in the first maternal meiotic division . Furthermore, it has been observed that the maternal meiotic process is more prone to errors than the paternal meiotic process. This is most likely caused by prolonged meiotic arrest at the dictyotene stage during fetal life and ends upon ovulation, i. e. several decades later . Unfertilized oocyte karyotyping after IVF has disclosed two leading mechanisms for the origin of aneuploidy. The first is homologues chromosome segregation towards the same axis during meiosis I, leading to disomic or nulisomic daughter cells. The second mechanism indicates that chromosome imbalance may occur due to untimely centromere division before anaphase I, where sister chromatids are distributed randomly towards both axis . This and other researches have also confirmed a direct correlation between advanced maternal age and increase in meiotic errors [9, 11, 12].
Although the incidence of chromosomal aberrations is ten times higher in infertile male population compared to the fertile population, 95% of infertile men have normal karyotypes . It is obvious that men with a diagnosed chromosomal imbalance have an increased risk of producing aneuploid spermatozoa; however, researches indicate that men with normal karyotype, yet with abnormal sperm morphology, are also in the risky zone. In the male population aneuploidies appear due to meiotic synapse abnormalities resulting in the creation of bivalent sister chromatids which lead to abnormal sperm morphology production, or meiotic arrest. Traditional karyotyping cannot detect meiotic synapse abnormalities .
In addition to uniformly abnormal embryos, more than 50% of human embryos contain diploid cells and aneuploid cells in addition to normal cells, and this phenomenon is called mosaicism [14, 10]. Mosaicism can greatly affect false positive or false negative results for preimplantation genetic diagnosis/screening, since the biopsy gives a small number of cells for analysis which does not represent the genetic content of the entire embryo. Clinical researches have confirmed that if an embryo contains a small portion of mosaicism (only a few abnormal cells) the implantation potential can be very good . If more than half cells in the embryo are aneuploid, the implantation potential and the survival of the implanted embryo are minimal. The most common mosaicism is of the diploid/aneuploid type--an embryo which starts its fetal life as a normal diploid and aneuploidy appears during early mitotic divisions . It is considered that the incidence of mosaicism varies depending on multiple factors, where intrafollicular and changes in laboratory conditions may increase the incidence of mosaicism .
The most common aneuploidies in the human population are trisomy 16, 21, 22 and X chromosome monosomy. Although trisomy of the 13th and 18th chromosome is not very common, these aneuploidies have a high survival rate. In general, the other imbalances are not compatible with life and result in early miscarriages or are so lethal that these embryos don't even implant . The introduction of preimplantation genetic diagnosis into clinical practice revealed that Robertsonian translocation often leads to aneuploidy. In this case, parts of acrocentric chromosomes are exchanged in a way in which the satellite region is lost and two chromosomes remain coupled, so that this embryo contains 45 chromosomes. In preimplantation genetic diagnosis, identification of this aberration is possible since specific commercial probes for Robertsonian translocation are available .
It should be noted that the greatest contribution to the studies of aneuploidy came from the treatment of embryos from infertile patients during in vitro procedures. It is in this respect that opinions are divided regarding weather high incidence of aneuploidy exists only among infertile patients or the entire population. Although embryos in ART occur after aggressive hormonal stimulation and in vitro controlled laboratory cultivation, which may affect the appearance of aneuploidy, preimplantation genetic diagnosis showed that aneuploidies are present in fertile population as well .
Preimplantation Genetic Diagnosis/Screening
Preimplantation genetic diagnosis (PGD) is a technique which allows polar body/cell genetic status assessment obtained from an embryo/oocyte biopsy in order to detect aneuploidy or mutated alleles of monogenic diseases . It was first applied in the early 1900s for identification of a genetically normal embryo in the case of an X-linked recessive mutation [16, 17]. The most important indications for PGD are high risk of transmission of hereditary diseases, several unsuccessful IVF cycles (at least 3 cycles or [greater than or equal to]10 transferred embryos without implantation), advanced maternal age (most often over 35 years of age) and several miscarriages . PGD is considered justified when it can contribute to an increase in implantation rates, decrease miscarriages, and reduce aneuploid conception . PGD can be divided into two categories: high- risk PGD in fertile patients with a diagnosed genetic disorder, and low-risk PGD for infertile patients where the aim is to increase implantation rates after the transfer of a genetically normal embryo. The second category is actually preimplantation genetic screening (PGS) . Therefore, the difference between these two categories lies in appropriate application. PGD is advised in cases where a specific aberration is isolated (single-gene disorders, inherited genetic diseases, and X-linked mutations), while PGS is carried out in infertile patients for the purpose of selecting the most viable euploid embryo . Currently, PGD is successfully used for more than 200 disorders, such as cystic fibrosis, Tay-Sach disease, Huntington disease, hemophilia, sickle cell anemia, Fragile-X syndrome, etc. .
The whole chromosome set karyotyping would be the ideal analyzing method. Due to the small amount of available genetic sample and other limiting factors (contamination, high risk of cultivation arrest, chromosome shortening due to mitotic inhibition, etc...) traditional cytogenetic methods required molecular replacement. The first available technology was fluorescent in situ hybridization (FISH) , still in use in many IVF centers. The procedure consists of the hybridization of fluorescently labeled deoxyribonucleic acid (DNA) probes (fluorochromes), with complementary target DNA, and the detection of the fluorescent signals derived after hybridization under a fluorescent microscope. This process implies cytoplasm digestion for nucleus isolation and allows the analysis of 6-9 chromosomes, whereby probes can be used during multiple rounds with a precaution due to decreasing binding efficiency of the probes with each round. While many clinics prefer this method owing to its simplicity and time-convenient procedure, FISH technology has numerous limitations. The main deficiency of this method lies in the restricted number of analyzed chromosomes, so that many aneuploidies may go undetected. In addition, this technology requires nuclei fixation which comes with at least two negative consequences: if isolated blastomere does not contain a nucleus or it is damaged during fixation, the lack of genetic material disables analysis, or if there are cytoplasmic residues after fixation, fluorescent signal evaluation will be inappropriate .
To complete analysis of all chromosomes in one blastomere, the FISH method is replaced with polymerase chain reaction (PCR) based methods. This approach enables multiplication of DNA material obtained in one cell which is sufficient for further analysis . However, PCR methods have restrictions as well, such as foreign DNA contamination, amplification failure and allele dropout. All of these limitations can be avoided by process optimization, educated personnel and strict protocols .
Comparative Genomic Hybridization
The new technology of comparative genomic hybridization (CGH) bridges the gap between molecular genetics and cytogenetics. It is based on PCR amplification (most often) of the entire genome presented from only one cell, which makes this method equally efficient for polar body, blastomere or trophectoderm (TE) cell analysis . Ploidy analysis is performed as a comparison of a test sample with a reference sample, avoiding cell cultivation. After DNA isolation of the test and reference samples, isolated DNA is labeled with two different fluorescent colors (fluorophores) followed by denaturation and hybridization with normal cell lines metaphase chromosomes (m-CGH approach) or DNA clones fixed to array (a-CGH approach). Finally, fluorescent signal is evaluated using fluorescent microscope and software .
Before the hybridization process, it is crucial to amplify a sufficient amount of genetic material for a sustained and valid analysis, especially for very small samples such as one cell obtained from biopsy. In CGH cases, the whole genome amplification (WGA) method is applied, i.e. the entire genome of one cell is nonspecifically amplified. WGA modes may be PCR-based and non PCR-based . Non PCR-based WGA is multiple displacement amplification, while for the CGH purpose, PCR-based WGAs are most common: primer extension preamplification and degenerate oligonucleotide primed-PCR .
Although microarray-CGH technology turned out to be more efficient than FISH in aneuploidy detection, it is limited by low resolution and prolonged analysis time (about 4 days). However, by introducing microarrays into clinical practice the main disadvantage of m-CGH has been overcome. The performance principle of array-CGH is basically the same as m-CGH with the difference being the use of a platform-array containing immobilized DNA fragments cloned with various vectors (bacterial artificial chromosome, yeast artificial chromosomes or P1-derived artificial chromosome), instead of metaphase chromosomes . Every microplate contains 100-200 kb clones, representing the entire genome, allowing for a more detailed analysis than m-CGH. The procedure is implemented with a laser scanner and advanced software displaying the results in the form of an algorithm. Considering the complete automatization of the process, it takes 24h for the full procedure, meaning that the biopsy can be done on the third cultivation day and blastocyst embryo transfer can be performed on the fifth cultivation day without cryopreservation required . Given that an analysis of the whole set of chromosomes can be done with an array-CGH technology many clinical trials indicate that if examinations were to be done by FISH alone, there would a high percentage of undetected aneuploidies [21-23].
Polar Body, Blastomere and Trophectoderm (TE) Cells Biopsy
The biopsy procedure starts with ablation, a process corresponding to zona pellucida, opening most widely with diode laser, enabling scroll and determination of laser path as well as the number and size of holes. Following ablation, polar body/cell biopsy is carried out via special biopsy pipette. In blastomere biopsy, prior to blastomere aspiration, embryo is exposed to the biopsy media containing no Ca[2.sup.+] i Mg2+ ions, which loosens tight cell junctions leading to relieved cell isolation. This effect is reversible, and cell connections are reestablished after displacement of an embryo into the cultivation media .
Polar body biopsy is the removal of one or both polar bodies, independently or simultaneously. Biopsy of the first polar body is performed prior intracytoplasmic sperm injection (ICSI) procedure providing insight into maternal genome only. The IVF centers performing polar body biopsy, almost always practice isolation and analysis of both polar bodies in this order: the first polar body is removed before ICSI, the second polar body after fertilization, or both polar bodies are simultaneously removed after ICSI. The biopsy of both polar bodies provides more DNA material than one polar body biopsy, but tremendous limitation still persist--information about complete genetics of an embryo is unknown regarding the developmental stadium of analyzed cell, and the fact that maternal genome controls embryonic genome until the 8-cell developmental stage . Some IVF centers still run polar body biopsy mainly due to law restrictions where embryo/blastocyst biopsy is banned (Italy, Germany, Austria) or because of potential destructive effect of cell removal on the embryo integrity .
Until recently, blastomere biopsy of an early embryo has been the most commonly used method for PGD/PGS. It is performed on the 3rd cultivation day at the 8-cell stage when one or two blastomeres with visible nucleus are removed for analysis. Biopsy at this developmental stage ensures avoidance of cryopreservation if genetic analysis doesn't take more than 48h, meaning that embryos can have extended cultivation until day 5 after biopsy and embryo transfer of euploid blastocyst in current cycle. Nevertheless, even in undisrupted culture conditions, only 60% of embryos reach blastocyst stage and this percentage decreases with disturbing embryo development in some way, by biopsy for instance .
The trophectoderm (TE) cells biopsy at blastocyst stage and the popularity of this approach constantly increases in accordance with improved culture conditions, thus the percentage of blastocyst formation is higher. At this stage, biopsy can be performed in two ways: zona pellucida is opened at 3 (rd) cultivation day, and during the next two days TE cells progress towards the hole considering their hatching tendency, so biopsy of TE cells is simplified. Negative consequence of these methods lies in great possibility of inner cell mass (ICM) extrusion. As a precaution, many IVF centers apply a different approach: zona pellucida is opened in the morning of the 5th cultivation day at the opposite side of ICM. After several hours, hatching process has started and removal of TE cells can be performed. Blastocyst biopsy provides significantly greater cell amount compared to polar body/blastomere biopsy, but this method demands cryopreservation owing to the time required for genetic analysis. However, improvement of cryopreservation protocols, especially vitrification which ensures up to 90% survival of warmed blastocysts [24-27], makes TE cells for PGD/PGS the most promising method at this moment .
Clinical Experiences in the Detection of Aneuploidy
The European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium (ESHRE PGD Consortium) was established in 1997, with the aim of collecting prospective and retrospective data about the accuracy, efficiency and safety of preimplantation genetic diagnosis/screening, defining specific guidelines for good clinical practice in PGD laboratories, and promoting continuous education. Since 1999, ESHRE PGD Consortium has published data obtained from member centers of the PGD Consortium. According to the latest report, the published data cover ten years of PGD/PGS utilization in 60 PGD centers .
Until 2009, there were 33.271 PGD/PGS cycles reported, of which 12.388 were PGD, 20.207 were PGS, and 676 were PGD with PGS. With respect to FISH technology, as the first applied method in aneuploidy detection, the number of FISH cycles was 26.052 and 6.054 were PCR. From 2009 to 2010, 6.160 PGD/PGS cycles were reported, among them 2.580 were PGD, 3.551 were PGS, and 29 were PGD with PGS. An increasing trend of PCR preimplantation genetic diagnosis has been noticed in comparison to FISH (2009-2010: 946 FISH cycles vs. 1.435 PCR cycles; 1999-2008 5.851 FISH cycles vs. 5.869 PCR cycles). For the preimplantation genetic screening purpose, most centers of the 60 members still prefer FISH than PCR technology (1999-2008, FISH 19.723 cycles vs. PCR 3 cycles; 2009-2010, FISH 3526 cycles vs. PCR 6 cycles). Until 2010, early embryo biopsy was the most frequent aspect of genetic analysis (1999-2009: 26.284 cycles; 2009-2010: 4.918 cycles) compared to polar body (1999-2009: 3.750 cycles; 2009-2010: 997 cycles) and blastocyst biopsy which was used the least often (1999-2009: 105 cycles; 2009-2010: 6 cycles) .
The newest report of the PGD Consortium is being prepared and it is expected to emphasize the recent trends in regard to the stage of embryo biopsy, particularly on the usage of advanced PCR based technologies, such as array-CGH . Defining the optimal biopsy stage depends on multiple factors. Primarily, determining the timing of biopsy, in order to precisely identify chromosomal aberrations, is essential: too early screening may leave genetic errors which affect implantation potential unidentified . It is estimated that 33% of embryo aneuploidies occur due to genetic errors in meiosis I, meaning maternal errors . These aneuploidies can be identified by polar body biopsy; however most aneuploidies appear due to improper separation of sister chromatids after meiosis II, so that polar body biopsy will not detect them . Recent papers on serial embryo biopsies (biopsy of the same embryo at all stages) indicate that little information was obtained by polar body biopsy in comparison to the blastocyst stage. Even after a biopsy of both polar bodies, 1 of 4 aberrations remains unidentified . Compared to polar body biopsy, blastomere biopsy is more invasive, but overcomes polar body biopsy limitations. In this case, the biopsy occurs after meiosis completion leading to the detection of meiosis errors regardless of their maternal or paternal origin .
Apart from accurate identification of aberrations, when choosing the timing of the biopsy, the question is whether abnormalities identified in the specimen accurately and consistently predict a corresponding abnormality in the embryo. Namely, if an embryo has the ability of self-correction, then aneuploidies detected in a biopsied sample could lead to a misdiagnosis and discarding of normal embryos. According to the latest data, mosaicism accounts for 29% of all human embryos . Since screening of one blastomere at the 3rd cultivation day is most often the case, there is a considerable risk of imprecise mosaicism detection resulting in a false positive diagnosis . Only one false positive aneuploidy is sufficient for a false positive mosaicism diagnosis. For example, assuming that genetic testing is done in one blastomere, resulting in 10% false positive diagnosis, then the analysis of 6 genetically normal blastomeres would result in 50% false positive mosaicism detections or even 70% if the analysis includes 10 blastomeres. Therefore, it is no surprise that research referring to third day biopsy (FISH on one blastomere) showed that 50-90% early embryos were mosaic, considering previous statistical analysis where 75% of false positive results were expected (8 blastomere analysis revealed 15% of false positive results). Consequently, an appropriate strategy obtaining valid mosaicism incidence in embryo is crucial . A false positive diagnosis of mosaicism could explain recent reported clinical cases of mosaic embryo implantation and the birth of healthy children . Capalbo et al. indicated that the group of authors who published these cases did not provide detailed information, i.e. original diagnosis of mosaicism turned out incorrect causing an assessment of euploid embryos as mosaic .
With respect to biopsy timing, the developmental potential of the embryo shouldn't be compromised . It has been estimated that the implantation rate of early embryos declines by 39%, in other words, 2 of 5 biopsied early embryos on which a biopsy was performed on the third day, will have negative consequences on their developmental and implantation potential due to the biopsy .
In cases of blastocyst biopsy, a negative effect on implantation and delivery rate of biopsied blastocysts compared to untouched blastocysts hasn't been found . Parallel progress in embryo cultivation to the blastocyst stage and vitrification introduction in routine practice of ARTs, has enabled efficient biopsy of TE cells with a minimal risk . In addition, significantly lower mosaicism incidence has been found after blastocyst biopsy versus early embryo biopsy, merely 4-5% . Clinical trials revealed that about 20% aneuploidies, detected by polar body biopsy and confirmed by blastomere biopsy, turned out nonexistent at blastocyst stage due to self-correction . A recently published paper noted that embryo derived from reciprocal aneuploid oocyte (untimely sister chromatid division during meiosis I, revealed after polar body biopsy) resulted in the birth of a healthy child after correct chromatid segregation during meiosis II . ESHRE PGS Task Force published that 4% embryos are euploid, despite detected aneuploidies after polar body biopsy . Another advantage of a blastocyst biopsy is reflected in the amount of obtained genetic material: 5-10 TE cells  isolated after blastocyst biopsy and successfully amplified at approximately 98%  versus 1-2 cells after blastomere biopsy and unsuccessful amplification at 20% . According to clinical data, 1 of 10 oocytes remains unexamined due to unsuccessful amplification after polar body biopsy . It is still unclear if blastocyst morphology and developmental characteristics reflect chromosome constitution. Only one study confirmed a slight correlation between poor blastocyst morphology and incidence of aneuploidy .
Although an official opinion from the PGD Consortium is still pending, thus far, biological and clinical research indicates that TE cell biopsy at the blastocyst stage followed by advanced molecular technologies in aneuploidy detection is the best choice for PGD/PGS and consequently this management has encountered increased application around the world [3, 10, 12, 38, 39].
In this paper we have shown that comparative genomic hybridization provides completely different insight into chromosomal aberrations in preimplantation embryos. The information obtained in this manner is extremely significant for patients undergoing assisted reproduction. During the in vitro fertilization process, a great number of high-quality embryos are produced, so genetic selection of healthy embryos instead of morphological selection would mean avoiding emotional and financial stress, as well as loss of time in the process of assisted reproduction, that may be completed unsuccessfully due to undetected aneuploidies.
Nevertheless, comparative genomic hybridization is still a technical challenge, especially the microarray approach which has found application in a few laboratories equipped with advanced equipment and personnel educated and experienced in molecular biology. Despite limitations of detecting structural chromosomal aberrations, that has no effect on changes in the chromosome number (balanced chromosome translocations and inversions) and mosaicism detection, the research is going towards finding a simpler and more valid comparative genomic hybridization performance, particularly when it comes to reduction of time required for analysis, and obtaining appropriate amounts of genetic material samples.
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Jelena VUKOSAVLJEVIC (1), Aleksandra TRNINIC PJEVIC (1, 2), Artur BJELICA (1, 2), Ivana JAGODIC (1), Vesna KOPITOVIC (3) and Stevan MILATOVIC (1,2)
Clinical Center of Vojvodina, Novi Sad, Department of Obstetrics and Gynecology (1)
University of Novi Sad, Faculty of Medicine (2)
Private hospital "Ferona" Novi Sad (3)
Corresponding Author: Dr Jelena Vukosavljevic, Klinicki centar Vojvodine, Klinika za ginekologiju i akuserstvo, 21000 Novi Sad, Branimira Cosica 37, E-mail: firstname.lastname@example.org
Rad je primljen 2. VI 2017.
Recenziran 3. IX 2017.
Prihvacen za stampu 4. IX 2017.
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|Title Annotation:||SEMINAR FOR PHYSICIANS/SEMINAR ZA LEKARE U PRAKSI|
|Author:||Vukosavljevic, Jelena; Pjevic, Aleksandra Trninic; Bjelica, Artur; Jagodic, Ivana; Kopitovic, Vesna;|
|Date:||Sep 1, 2017|
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