Name: exploring x chromosomal aberrations in ovarian cells by using fluorescence in situ hybridization
Uploaded: 2023-04-07T13:40:00
Description: Watch this Scientific Journal Video about Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization at JoVE.com

The authors acknowledge Marjo van Brakel, Dominique Smeets, Guillaume van de Zande, Patricia van Cleef and Milan Intezar for their expertise and technical assistance. Funding sources: Merck Serono (A16-1395), Goodlife, and Ferring.

The TurnerFertility study protocol has been approved by the Central Committee on Research Involving Human Subjects (NL57738.000.16). In this study, the ovarian cortex tissue of 93 females with TS was obtained. Materials that require safety precautions are listed in Table 1.
Table 1: Safety precautions.
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			<td>Material</td>
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<ol>
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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+abnormalities+predisposing+to+infertility,+testing,+and+management:+a+narrative+review.">Chromosomal abnormalities predisposing to infertility, testing, and management: a narrative review.</a> Bulletin of the National Research Centre. 45 (1), 65 (2021).</li><li>Foresta, C., Ferlin, A., Gianaroli, L., Dallapiccola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+appropriate+use+of+genetic+tests+in+infertile+couples.">Guidelines for the appropriate use of genetic tests in infertile couples.</a> European Journal of Human Genetics. 10 (5), 303-312 (2002).</li><li>Heard, E., Turner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sex+chromosomes+in+mammalian+fertility.">Function of the sex chromosomes in mammalian fertility.</a> Cold Spring Harbor Perspectives in Biology. 3 (10), 002675 (2011).</li><li>Reynaud, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Number+of+ovarian+follicles+in+human+fetuses+with+the+45,X+karyotype.">Number of ovarian follicles in human fetuses with the 45,X karyotype.</a> Fertility and Sterility. 81 (4), 1112-1119 (2004).</li><li>Otter, M., Schrander-Stumpel, C. T., Curfs, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple+X+syndrome:+a+review+of+the+literature.">Triple X syndrome: a review of the literature.</a> European Journal of Human Genetics. 18 (3), 265-271 (2010).</li><li>Modi, D. N., Sane, S., Bhartiya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+germ+cell+apoptosis+in+sex+chromosome+aneuploid+fetal+human+gonads.">Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads.</a> Molecular Human Reproduction. 9 (4), 219-225 (2003).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nature Reviews Genetics. 2 (4), 280-291 (2001).</li><li>Huber, D., von Voithenberg, L. V., Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH):+History,+limitations+and+what+to+expect+from+micro-scale+FISH.">Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH.</a> Micro and Nano Engineering. 1, 15-24 (2018).</li><li>Hu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH):+an+increasingly+demanded+tool+for+biomarker+research+and+personalized+medicine.">Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine.</a> Biomarker Research. 2 (1), 3 (2014).</li><li>Hwang, K., Weedin, J. W., Lamb, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+in+situ+hybridization+in+male+infertility.">The use of fluorescent in situ hybridization in male infertility.</a> Therapeutic Advances in Urology. 2 (4), 157-169 (2010).</li><li>Ramasamy, R., Besada, S., Lamb, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+of+human+sperm:+diagnostics,+indications,+and+therapeutic+implications.">Fluorescent in situ hybridization of human sperm: diagnostics, indications, and therapeutic implications.</a> Fertility and Sterility. 102 (6), 1534-1539 (2014).</li><li>Chatziparasidou, A., Christoforidis, N., Samolada, G., Nijs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+aneuploidy+in+infertile+male+patients:+a+systematic+review+of+the+literature.">Sperm aneuploidy in infertile male patients: a systematic review of the literature.</a> Andrologia. 47 (8), 847-860 (2015).</li><li>Schleedoorn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TurnerFertility+trial:+PROTOCOL+for+an+observational+cohort+study+to+describe+the+efficacy+of+ovarian+tissue+cryopreservation+for+fertility+preservation+in+females+with+Turner+syndrome.">TurnerFertility trial: PROTOCOL for an observational cohort study to describe the efficacy of ovarian tissue cryopreservation for fertility preservation in females with Turner syndrome.</a> BMJ Open. 9 (12), 030855 (2019).</li><li>Peek, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicles+of+young+patients+with+Turner's+syndrome+contain+normal+oocytes+but+monosomic+45,X+granulosa+cells.">Ovarian follicles of young patients with Turner's syndrome contain normal oocytes but monosomic 45,X granulosa cells.</a> Human Reproduction. 34 (9), 1686-1696 (2019).</li><li>Nadesapillai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+are+some+patients+with+45,X+Turner+syndrome+fertile?+A+young+girl+with+classical+45,X+Turner+syndrome+and+a+cryptic+mosaicism+in+the+ovary.">Why are some patients with 45,X Turner syndrome fertile? A young girl with classical 45,X Turner syndrome and a cryptic mosaicism in the ovary.</a> Fertility and Sterility. 115 (5), 1280-1287 (2021).</li><li>Dolmans, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reimplantation+of+cryopreserved+ovarian+tissue+from+patients+with+acute+lymphoblastic+leukemia+is+potentially+unsafe.">Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe.</a> Blood. 116 (16), 2908-2914 (2010).</li><li>Dath, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+human+ovarian+tissue+to+nude+mice:+comparison+between+four+grafting+sites.">Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites.</a> Human Reproduction. 25 (7), 1734-1743 (2010).</li><li>Cacciottola, L., Donnez, J., Dolmans, M. 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FISH analysis is a well-known technique to detect X chromosomal aberrations in lymphocytes or buccal cells of both males and females10. Several studies have described FISH on gametes of males with X chromosomal aberrations, but detailed information obtained by FISH on ovarian cells from females with X chromosomal aberrations is still lacking14. This article presents novel methods based on FISH to determine if aneuploidy is present in the ovarian cells of non-grafted and gra...

Infertility is a health issue of the male or female reproductive system, affecting approximately 186 million individuals of reproductive age worldwide1. In at least 35% of infertile couples, infertility is caused by a disorder of the female reproductive system2. There are many factors that can cause female infertility, such as genetic factors, genital tract abnormalities, endocrine dysfunction, inflammatory diseases, and iatrogenic treatment3.
Genetic abnormalities are present in approximately 10% of infertile females4,5. Of all genetic abnormalities, X chromosome aberrations are the most common cause of ovarian dysgenesis2. Several studies have reported that X chromosomal aberrations in females with Turner syndrome (TS) or Triple X syndrome are associated with premature ovarian failure due to an accelerated loss of germ cells or impaired oogenesis6,7,8.
Aberrations of the X chromosome can be divided into: 1) numerical aberrations, in which the number of X chromosomes is different but the X chromosomes are intact; and 2) structural aberrations, in which the X chromosome has gained or lost genetic material3,9. Numerical aberrations of the X chromosome are more common than structural abnormalities and are often caused by spontaneous errors during cell division3,9. When such an error occurs during meiosis, it may lead to aneuploid gametes and ultimately to offspring with chromosomal aberrations in all cells. When chromosome defects arise in somatic cells as a result of errors occurring during mitosis in the early stages of ontogenesis, it may lead to mosaicism. In these individuals, both cells with normal X chromosomal content and cells with X chromosomal aberrations are present.
In the 1980s, a cytogenetic technique called fluorescence in situ hybridization (FISH) was developed to visualize and locate specific nucleic acid sequences on metaphase and interphase chromosomes10,11. This technique uses fluorescent-labeled DNA probes to bind to a specific sequence in the chromosome, which can then be visualized by using a fluorescence microscope.
Nowadays, FISH is widely used as a clinical diagnostic tool and is considered the gold standard in detecting chromosomal aberrations10. In the field of reproductive medicine, FISH analysis on sperm has been used to gain insight into the X chromosomal content of spermatozoa in males with numerical or structural chromosomal aberrations in somatic cells12,13,14. These studies showed that males with chromosomal aberrations were more likely to have a higher frequency of aneuploid spermatozoa present in their semen compared to males with normal karyotypes12,13,14.
In contrast to spermatozoa, very little is known about the X chromosomal content of ovarian cells (including oocytes, granulosa/theca cells, and stromal cells) in individuals with a chromosomal aberration, as well as the possible consequences of aneuploidy of these cells on their reproductive potential. An important reason for the scarce information on the karyotype of ovarian cells compared to spermatozoa is the fact that women have to undergo an invasive procedure such as a follicle puncture or surgery to obtain oocytes or ovarian cortex tissue. Female gametes are, therefore, difficult to obtain for research purposes.
Currently, an observational intervention study is being performed in the Netherlands to explore the efficacy of ovarian tissue cryopreservation in young females with TS15. One fragment of the ovarian cortex tissue of the patient was available to identify the X chromosomal content of the ovarian cells16,17. As part of the study, a novel method was developed based on FISH of dissociated ovarian cortex tissue to determine if chromosomal aberrations are present in ovarian cells in females carrying a chromosomal aberration in non-ovarian somatic cells, such as lymphocytes or buccal cells. In addition, the effect of aneuploidy in ovarian cells on folliculogenesis was determined as well. To this end, an established FISH protocol was modified that enables the analysis of histological sections of ovarian cortex tissue after artificially induced folliculogenesis during long term xenotransplantation in immunocompromised mice. In this study, we present two methods based on FISH to determine the X chromosomal content in ovarian cells in non-grafted and grafted ovarian cortex tissue in females with X chromosomal aberrations, with the aim to improve basic science on this topic.

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exploring x chromosomal aberrations in ovarian cells by using fluorescence in situ hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

FISH on isolated ovarian cells prior to grafting 
Cryopreserved ovarian cortex tissue from females with 45,X/46,XX (patient A) or 45,X/46,XX/47,XXX (patient B) TS were used to illustrate the results using this protocol. In patient A, 50% of the lymphocytes had a 45,X karyotype and 50% had 46,XX. In patient B, 38% of the lymphocytes were 45,X, 28% were 46,XX, and 34% were 47,XXX. Centromere-specific probes for chromosome X (green) and chromosome 18 as the control (red) were used to determine the X ch...

Watch this Scientific Journal Video about Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization at JoVE.com

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

FISH analysis of ovarian cells from females with X chromosomal aberrations is a unique technique to gain insight into the X chromosomal content of ovarian cells in this specific group. These techniques could be helpful to learn more about reproductive potential in females with X chromosomal aberrations. It is important to mention that both methods are intended to facilitate future research, and are not designed to be used as a diagnostic tool in clinical practice.Demonstrating the procedure will be Ronald Peek, a senior researcher from our fertility laboratory, and Milad Intezar, an analyst from our pathology department. To begin, cut the cryopreserved, or thawed ovarian cortex tissue into small pieces of one by one by one millimeter using a scalpel. Enzymatically digest the tissue fragments in four liters of pre-warmed L15 for 75 minutes at 37 degrees Celsius.Pipette the digestion mixture up and down every 15 minutes. Stop the enzymatic digestion by adding four milliliters of cold L15 supplemented with 10%Fetal Bovine Serum, or FBS. Wash the dissociated tissue once with eight milliliters of cold L15 medium by centrifugation at 500 g before re-suspending in 500 microliters of L15 medium.Transfer the cell suspension containing largely individual stromal cells and small follicles to a plastic Petri dish, and examine the suspension under a stereo microscope. Pick the follicles less than 50 micrometers in size manually using a 75 micrometer plastic pipette, and transfer them to the droplet of L15 medium supplemented with 10%FBS at four degrees Celsius. Then transfer the purified follicles to a solution of 0.06%trypsin, one milligram per milliliter EDTA, and one milligram per milliliter glucose, and incubate for 20 minutes at 37 degrees Celsius.After incubation, obtain ovarian stromal cells from the cortex cell suspension using a 75 micrometer plastic pipette. For Fluorescence In Situ Hybridization, or FISH analysis of individual ovarian cells, transfer the treated ovarian follicles with trypsin/EDTA/glucose, or stromal cells to droplets of five microliters of 0.15 millimolar potassium chloride, and 15 microliters of DPBS on a slide, and incubate for 20 minutes at 37 degrees Celsius. Dry and pre-fixate the slide in 300 microliters of 0.05 millimolar potassium chloride, 7.5%acetic acid, and 22.5%methanol for two minutes.Cover the slide with three-to-one methanol acetic acid for two minutes to finalize fixation. After fixation, wash the sample twice in freshly prepared SSC at 73 degrees Celsius. Cover it with 100 microliters of 2%Proteinase K and seal it with a cover slip.Incubate the slide for 10 minutes at 37 degrees Celsius in the hybridization station. After incubation, remove the cover slip, and wash the slide for five minutes in DPBS at room temperature under shaking. Fix the sample for five minutes with 1%formaldehyde.Then wash the slide in DPBS, and serially dehydrate it in increasing ethanol concentrations for two minutes each. Air dry the sample before hybridizing it with fluorescently labeled probes. Next, add one microliter of CEPX, one microliter of CEP18, and 18 microliters of the hybridization buffer to the sample and seal with a cover slip that is glued to the slide.Transfer the slide to the hybridization station for denaturation at 73 degrees Celsius for three minutes, followed by hybridization during an overnight incubation at 37 degrees Celsius. After hybridization, remove the cover slip and any remaining glue from the slide. Wash the slide in 0.4X SSC, 0.3%Tween-20 at 72 degrees Celsius for two minutes, followed by incubation for one minute in 2X SSC and 0.1%Tween-20.Dehydrate the slide as demonstrated, and air dry it in the dark before covering it with a mounting medium containing DAPI. Place the slide at minus 20 degrees Celsius for 10 minutes before analysis by fluorescence microscopy. To hybridize paraffin sections, select new sections before, or after the section containing follicles from the paraffin ribbon.Mount one section on a glass slide, and dry it for 45 minutes on a stove at 56 degrees Celsius. Deparaffinize the section in xylene for 10 minutes, then immerse the slide in 99.5%ethanol, and rinse it for five minutes in tap water. Pre-treat the slide with the target retrieval solution for 10 minutes at 96 degrees Celsius.After cooling down, rinse the slide in distilled water. Treat the slide for five minutes with 0.01 molar hydrochloric acid, followed by pepsin digestion for 15 minutes at 37 degrees Celsius. Rinse the slide again in hydrochloric acid and subsequently in PBS.Fix the slide in 1%formaldehyde PBS for five minutes. Then briefly rinse the slide in PBS, and again in demineralized water before dehydrating it in 99.5%ethanol. Apply five microliters of probe CEP18 and CEPX on the pre-treated slide.Then apply a cover slip and seal the area with photo glue. Place the slide in a hybridizer for denaturation at 80 degrees Celsius for 10 minutes and hybridization overnight at 37 degrees Celsius. The next day, rinse the slide twice for five minutes each in SSC at 42 degrees Celsius, followed by a two minute and a one minute rinse in 0.3%Nonidet P-40 at 73 degrees Celsius.Again, rinse the slide in 2X SSC followed by rinsing in distilled water before dehydrating it with 99.5%ethanol. Air dry the slide, and mount it in DAPI-containing mounting medium. Cryopreserved ovarian cortex tissue in Patient A showed 50%of the lymphocytes, had a 45, X karyotype, and 50%had 46, XX.In Patient B, 38%of the lymphocytes were 45, X, 28%were 46, XX, and 34%were 47, XXX.Surrounding stromal cells were also analyzed for X chromosomal content. Enzymatic digestion of ovarian cortex tissue resulted in a suspension of largely dissociated cells, but leaving the primordial follicles, consisting of an intact oocyte surrounded by a single layer of granulosis cells. Small follicles provided difficulties in interpreting signals after FISH, due to the clumping of the granulosis cells.Additional digestion of the follicles with trypsin before FISH resulted in individual granulosis cells contracting into a spherical morphology on the surface of the follicles. Hematoxylin eosin staining showed morphologically normal secondary and antral follicles in ovarian cortex tissue after five months of xenografting. The X chromosomal content of granulosis cells of these follicles was determined by FISH analysis.Fixating grafted ovarian cortex tissue in Bouin solution resulted in blurred and largely obscured fluorescent signals in follicular cells due to a green haze. In contrast, ovarian cortex tissue fixed in formaldehyde provided excellent fluorescent signals. The main challenge of this protocol lies in the isolation of ovarian cells and the enzymatic digestion of the tissue.Deviating from this protocol can cause a substantial loss of ovarian cells during the process, and this should especially be avoided in females who already have a low ovarian reserve. Additional gene expression profiling could be helpful to learn more about the impact of aneuploid ovarian cells on folliculogenesis, and hence the process of premature ovarian failure in females with X chromosomal aberrations.

Research

JoVE Journal

Genetics

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