Journal of Human Reproductive Sciences

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 7  |  Issue : 1  |  Page : 25--29

Pre-implantation genetic screening using fluorescence in situ hybridization in couples of Indian ethnicity: Is there a scope?


Shailaja Gada Saxena, Kundanbala Desai, Lata Shewale, Prabhat Ranjan 
 Department of Molecular Medicine, Reliance Life Sciences Pvt. Ltd., Dhirubhai Ambani Life Sciences Centre, Navi Mumbai, Maharashtra, India

Correspondence Address:
Shailaja Gada Saxena
Department of Molecular Medicine, Reliance Life Sciences Pvt. Ltd., Dhirubhai Ambani Life Sciences Centre, Thane Belapur Road, Rabale, Navi Mumbai - 400 701, Maharashtra
India

Abstract

Context: There is a high incidence of numerical chromosomal aberration in couples with repeated in vitro fertilization (IVF) failure, advanced maternal age, repeated unexplained abortions, severe male factor infertility and unexplained infertility. Pre-implantation genetic screening (PGS), a variant of pre-implantation genetic diagnosis, screens numerical chromosomal aberrations in couples with normal karyotype, experiencing poor reproductive outcome. The present study includes the results of the initial pilot study on 9 couples who underwent 10 PGS cycles. Aim: The aim of the present study was to evaluate the beneficial effects of PGS in couples with poor reproductive outcome. Settings and Design: Data of initial 9 couples who underwent 10 PGS for various indications was evaluated. Subjects and Methods: Blastomere biopsy was performed on cleavage stage embryos and subjected to two round fluorescence in situ hybridization (FISH) testing for chromosomes 13, 18, 21, X and Y as a two-step procedure. Results: Six of the 9 couples (10 PGS cycles) conceived, including a twin pregnancy in a couple with male factor infertility, singleton pregnancies in a couple with secondary infertility, in three couples with adverse obstetric outcome in earlier pregnancies and in one couple with repeated IVF failure. Conclusion: In the absence of availability of array-comparative genomic hybridization in diagnostic clinical scenario for PGS and promising results with FISH based PGS as evident from the current pilot study, it is imperative to offer the best available services in the present scenario for better pregnancy outcome for patients.



How to cite this article:
Saxena SG, Desai K, Shewale L, Ranjan P. Pre-implantation genetic screening using fluorescence in situ hybridization in couples of Indian ethnicity: Is there a scope?.J Hum Reprod Sci 2014;7:25-29


How to cite this URL:
Saxena SG, Desai K, Shewale L, Ranjan P. Pre-implantation genetic screening using fluorescence in situ hybridization in couples of Indian ethnicity: Is there a scope?. J Hum Reprod Sci [serial online] 2014 [cited 2019 Nov 17 ];7:25-29
Available from: http://www.jhrsonline.org/text.asp?2014/7/1/25/130812


Full Text

 Introduction



An estimated total of 5 million babies have been born world-wide, since the birth of the first baby Louise Joy Brown in 1978 through in vitro fertilization (IVF). As per the European Society of Human Reproduction and Embryology press released, [1] there are approximately 1.5 million assisted reproduction technologies cycles globally annually, resulting in about 350,000 babies. India did not lag behind in the field of IVF and the first Indian IVF baby was born, just 3 months after Louise Brown. [2] As per the survey by outlook business magazine (2011), [3] there are approximately 90,000 IVF cycles performed in India alone with approximately 20,000 children born annually.

There have been tremendous advances in the field of assisted reproduction to enhance the success rate including methods to optimize embryo selection. Studies have shown a high incidence of numerical chromosomal aberration in couples with repeated IVF failure, advanced maternal age, repeated unexplained abortions. [4],[5],[6] Embryo selection based on morphological evaluation either on day 3 or day 5 of development does not ensure a normal chromosomal constitution [6],[7] and this may be the cause of low implantation potential. Pre-implantation genetic diagnosis (PGD) is a recent diagnostic modality, referring to procedures performed to identify genetic defects, in embryos generated in vitro, prior to implantation. [8] Pre-implantation genetic screening (PGS), a variant of PGD, screens numerical chromosomal aberrations in couples with normal karyotype, experiencing poor reproductive outcome including advanced maternal age, repeated IVF failures, repeated spontaneous abortions, severe male factor infertility and unexplained infertility. PGS is offered by many IVF centers to improve the reproductive outcome of the specific group of patients. [9] PGS thus brings the hope of healthy babies to couples at risk of passing heritable diseases to their offspring. PGS diagnoses the genetic aberrations at the pre-implantation stage, i.e. before implantation, [10] thus helping in negative selection of the affected embryos prior to implantation. PGS can be performed at different stages of embryo development including polar body biopsy, blastomere biopsy from cleavage stage embryos, trophectoderm biopsy from blastocyst. The diagnosis of monogenic disorders is done using the polymerase chain reaction based technique, whereas fluorescence in situ hybridization (FISH) can be used for PGS as well as diagnosis of numerical and structural chromosomal aberrations.

FISH is a molecular cytogenetic technique for numerical constitutional chromosomal picture of an embryo, to provide results within the time frame of IVF procedures. The cells, including polar body, blastomere or trophectoderm cells, are fixed on glass microscope slides and fluorescently labeled deoxyribonucleic acid (DNA) probe hybridized to an in situ chromosomal target. The probes are targeted to specific regions or genes in the chromosome and are labeled with fluorochromes. Currently, a panel of probes is commercially available for different segments of the chromosomes. However, a limited number of fluorochromes are used as labels, confining the number of chromosomes that can be analyzed simultaneously. Probe detection is accomplished by ultraviolet light excitement of a fluorochrome. Aneuploidies for chromosomes 13, 18, 21, X and Y account for 90-95% of chromosomal aberrations in live-born infants. Hence, in the present study, we perform FISH only for chromosomes 13, 18, 21 and sex chromosomes. Furthermore, increasing the number of chromosomes would increase the cost which is not always acceptable by the referring IVF specialist.

There are few centers in India offering PGD/PGS. Our laboratory is a satellite referral laboratory, where we offer PGD/PGS to various centers across India. Personnel from our team visit the IVF center on day-3 post fertilization for embryo biopsy and blastomere fixation and the slides are brought back for FISH testing on the same day. The locus specific identifier (LSI) 13 SpectrumGreen and LSI 21 SpectrumOrange probes were used to detect aneuploidies of chromosomes 13 and 21; and chromosome enumeration probe (CEP) 18 SpectrumAqua, X SpectrumGreen and Y SpectrumOrange probes were used to detect aneuploidies of chromosomes 18, X and Y. FISH is performed as a two-step procedure and the results are released by day-4 evening and hence that the embryo transfer can be performed in the same IVF cycle on day-5 at the blastocyst stage. In the present report, we present the data of the initial 10 PGS cycles performed in 9 couples for various clinical indications including male factor infertility (n = 1), recurrent spontaneous abortions (RSAs) (n = 4), repeated IVF failure (n = 3), secondary infertility (n = 1). One of the patients underwent two cycles of PGS [P6 and P7 in [Table 1]]. All these couples had normal karyotype.

 Materials and Methods



The patients underwent various stages of IVF cycle including superovulation, oocyte retrieval, in vitro embryo culture using standard protocols. Oocyte retrieval was carried out 34-36 h after administration of injectable human chorionic gonadotropin by ultrasound guided follicular aspiration. Mature oocytes were subjected to intracytoplasmic sperm injection followed by in vitro embryo culture in the incubator at 37°C and 5% CO 2 . Cleavage stage embryo biopsy on day 3 embryos at 6-10 cell of development was performed using non-contact Saturn 3™ laser system (Research Instruments, Cornwall, UK), followed by aspiration of single blastomere from each embryo. The fundamental criterion for the selection of the blastomere was the presence of a clear nucleus and maintenance of cell integrity. The embryos were manipulated singly to reduce the amount of time spent outside the incubator.

The blastomere was fixed immediately post-biopsy. The blastomere was exposed to hypotonic solution (0.075 mol/l potassium chloride supplemented with 0.6% bovine serum albumin [w/v]) for 2 min. The blastomere was then placed onto the microscope slide in a small hypotonic drop using 80 μ flexipet (Cook, Australia). A drop of chilled methanol: acetic acid (3:1) fixative was added over the blastomere, which resulted in bursting of the cytoplasm and fixation of blastomere nucleus on the slide. The nucleus was identified under the microscope and labeled with the embryo number. Approximately, 4-6 blastomeres were fixed on each slide and minimum two slides were prepared per patient. The fixed slides were then transported to the laboratory in slide box at room temperature for FISH testing. The slides were dehydrated by passing through alcohol grades (70% ethanol w/v: 1 min; 85% ethanol w/v: 1 min and 100% ethanol: 2 min). FISH was carried out as a two-step procedure. In the first round, FISH testing was carried out for chromosomes 13 and 21 and then for chromosomes 18, X and Y. 3 μl probe for chromosome 13 and 21 using LSI 13 SpectrumGreen/21 SpectrumOrange probe (Abbott, USA) was applied on the coverslip and the slide was inverted on the coverslip. The probe and nuclear DNA were denatured simultaneously at 73°C for 8-10 min and left to hybridize at 37°C overnight. To remove unbound probe, the slides were washed in wash solutions containing 0.3% Igepal in 0.4X SSC for 2 min at 73°C followed by 0.1% Igepal in 2X SSC for 1 min at room temperature. The slides were mounted in Vectarshield (Vector Laboratories, CA, USA) antifade medium containing 200 ng DAPI/ml to counterstain the nuclei and examined under Olympus AX70 microscope using appropriate filters with Cytovision software (Olympus, USA). Appropriate controls were included in the test. After analysis, the slides were washed under running tap water to wash off the probe. The slides were again dehydrated using alcohol grades and then subjected to FISH for chromosome 18, X and Y using CEP 18 SpectrumAqua/X SpectrumGreen/Y SpectrumOrange probe (Abbott, USA) following the same protocol.

Blastomere was labeled as normal diploid when two signals for each of the fluorochrosome were observed. Presence of three signals for any of the fluorochromes indicated trisomy and presence of one signal indicated monosomy for that chromosome.

 Results



In the present report, 10 PGS cycles performed in 9 couples for various clinical indications including male factor infertility (n = 1), RSAs (n = 4), repeated IVF failure (n = 3), secondary infertility (n = 1). The details of the number of embryos biopsied, the FISH results and the pregnancy outcome are listed in [Table 1].{Table 1}

In the couple with male factor infertility due to hypogonadotropic hypopituitarism (P1), 19 oocytes were retrieved of which 17 fertilized and were at 6-8 cell stage of development on day-3. Embryo biopsy was performed on all 17 embryos. Blastomere from one embryo lysed during biopsy and since the embryo was grade III embryo with a lot of fragmentation, second blastomere was not biopsied. FISH was performed for chromosomes 13, 18, 21, X and Y. Trisomy 21 was detected in 2 embryos, trisomy 18 in 2 embryos, trisomy 13 in 1 embryo and monosomy X in one embryos. Three of the 10 euploid embryos were transferred on day-5 at the blastocyst stage and in resulted in a twin pregnancy.

Another couple (P2), a 32-year-old female and 35-year-old male married for 9 years presented with secondary infertility (etiology unknown) following birth of a male child 8 years back who died at the age of 2 months due to sudden infant death syndrome. Keen to have a child, the couple underwent IVF/PGS cycle. Of the 10 oocytes retrieved, 8 fertilized and 6 embryos were at 6-8 cell stage of development on day 3 post-insemination. Embryo biopsy and FISH performed on all 6 embryos, revealed trisomy 21 in one embryo and trisomy 18 in one embryo. Two of the 4 euploid embryos were transferred and it resulted in a singleton pregnancy.

Of the 5 couples referred for PGS following adverse obstetric outcome in earlier pregnancies, two couples conceived in the same cycle and one patient conceived following frozen-thawed embryo transfer and all three patients delivered normal healthy babies at term. Another couple (P4), a 36-year-old female and 41-year-old male married for 10 years with a history of 4 spontaneous first trimester abortions. Of the 19 oocytes retrieved, 15 fertilized and 11 embryos were at 6-8 cell stage of development on day 3 post-insemination. Embryo biopsy and FISH performed on all 11 embryos, revealed triploidy in one embryo, trisomy 21 in one and trisomy 13 in one embryo. Due to some personal unavoidable reasons, the female did not have any embryo transfer in the same cycle. All the 8 euploid embryos were frozen at the blastocyst stage and two blastocysts were thawed in the subsequent cycle and resulted in a singleton pregnancy. One of the three patients with repeated IVF failure conceived and delivered one normal healthy baby at term. The details of the number of embryos biopsied and the FISH results are listed in [Table 1]. While releasing the patient report, as the Pre-natal Diagnostic Techniques (Regulation and Prevention of Misuse) Act, 1994, the results of the sex chromosome are masked.

Of the 68 embryos biopsied in the 10 PGS cycles, no results were obtained in 4 embryos. This was due to lysis of the blastomere during biopsy in two embryos, fragmentation of blastomere in during fixing in one embryo and loss of blastomere during blastomere fixation. Of the 64 blastomere subjected to FISH testing, 43 blastomere (67.18%) were found to be euploid for chromosome 13, 18, 21, X and Y. Trisomy 21 was the most common aneuploidy detected in 10 embryos (15.62%), followed by trisomy 18 in 5 embryos (7.81%), trisomy 13 in 3 embryos (4.69%), monosomy X in 2 embryos and triploidy in one embryos. Ten of the 21 aneuploid embryos were available for re-analysis on day-5. All the cells were biopsied in these embryos and subjected to FISH testing. The results of FISH testing were in concordance with the earlier results except in one embryo with trisomy 21, which was found to be mosaic.

All the six pregnant patients had prenatal diagnosis by amniocentesis and FISH/karyotyping testing performed elsewhere and the fetus were found to be normal. All the six patients have delivered normal healthy children at term.

 Discussion



Aneuploidies of chromosome 13, 18, 21, X and Y constitutes 90-95% of chromosomal aberrations in live-born infants. Hence, at present we offer PGS for these 5 chromosomes. Many centers offering PGS analyze between 8-12 chromosomes. With an increasing number of chromosomes analyzed, lower efficiency per probe has been reported. [11],[12]

There have been multiple controversial prospective randomized controls trials questioning the usefulness of FISH based PGD/PGS. Mastenbroek et al., [13] Hardarson et al., [14] and Jansen et al., [15] in their study have reported statistically detrimental results in a live birth rates in PGS group compared with the control group. Staessen et al., [6] Blockeel et al., [16] Debrock et al., [17] observed no statistical differences between the two groups; whereas Werlin et al., 2003 [18] and Schoolcraft et al., [19] in their study observed potential benefits of PGS. However, these poor outcomes may be related to high dexterity required for performing embryo biopsy and blastomere fixation and should be performed by experienced personnel. In the absence of trained embryologist performing embryo biopsy procedure, the survival and potential implantation of the embryo may get affected. [20] Blastomere fixation is the crux of the entire procedure. Improper fixation can result in poor and non-informative FISH results, making diagnosis difficult. [21],[22] In addition, FISH results need to be interpreted with caution, especially in case of overlapping signals, split signals, weak and faded signals. [23],[24]

With PGS, selection of euploid embryos for transfer helps to improves implantation and pregnancy rate and a reduced miscarriage rate. In the present report, of the 10 PGS cycles performed for various indications, pregnancy was achieved in 6 patients. Aneuploidy was observed in 32.82% embryos subjected to embryo biopsy and FISH testing. Greater than 50% of these aneuploid embryos had reached the blastocyst stage. In the absence of PGS, if these embryos had been selected for transfer based on the morphology, then the chances of implantation and subsequent pregnancy would have reduced with an increased chance of first trimester abortion. There has been an ongoing debate over the benefits of PGD/PGS world-wide. However, in our experience, PGS has shown to be beneficial in this selected group of patients.

Because the testing is only done on one or two cells from a single embryo, there is a possibility that the cells tested do not accurately represent the status of the entire embryo. Studies have shown that PGS is usually about 85% accurate, with the potential for both false-positive or false-negative results. Hence, it is recommended to confirm the results of PGS on prenatal diagnosis by amniocentesis or chorionic villus sampling testing.

Recent advances in the field of molecular biology are aiming toward developing technique allowing 24-chromosome analysis and also provide results in the time frame of an IVF cycle. These include combination of comparative genomic hybridization (CGH) combined with microarrays, offering better resolution than conventional CGH. Further research on CGH and array-CHG have improved the robustness and accuracy of the technique making it a promising technique for PGS aneuploidy screening.

The first Indian IVF baby was born just 3 months after the world's first IVF baby. However, the Indian sub-continent has failed to keep pace with the developed countries in the field of genetic testing and PGD/PGS. There is a visible lacunae that currently exists in the diagnostic services on the Indian scene and PGD/PGS tests are not available in Pathological, Academic, Research or Referral Laboratories in India. To fill this void present, our laboratory has been operating as a satellite referral laboratory for PGD/PGS both of aneuploidy screening as well as monogenic disorders with the first reported pregnancy for PGD for β-thalassemia in India. [25] However, there are no commercial laboratories including ours, which offers array-CGH as a clinical service to patient populations for PGD/PGS. The initial results on these 10 cases reported in the present study, demonstrated a beneficial effect of PGS using FISH based technology. Following this additional 21 patients have undergone PGS for aneuploidy screening (data not included), with 10 pregnancies, including 1 twin pregnancy, showing favorable outcome for PGS. It is thus imperative to integrate basic genetic services into the existing primary health care and medical services including karyotyping in couples with reproductive failure, [26] PGS using FISH in the absence of availability of microarray based technology for PGS and community control of common genetic aberrations as priority needs for biomedical intervention.

 Acknowledgement



The authors are gratefully acknowledged Reliance Life Sciences Pvt. Ltd., for their support to the project.

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