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

Summary

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.

Abstract

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.

Introduction

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 females^4,5. 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 oogenesis^6,7,8.

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 material^3,9. Numerical aberrations of the X chromosome are more common than structural abnormalities and are often caused by spontaneous errors during cell division^3,9. 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 chromosomes^10,11. 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 cells^12,13,14. 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 karyotypes^12,13,14.

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 cells^16,17. 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.

Protocol

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.

Material	Hazard
Acetic acid	Severe skin burns and irritation of the respiratory system
Collagenase	Irritating to the eyes, respiratory system and skin
DAPI	Irritating to the eyes, respiratory system and skin
DNase I	Irritating to the eyes, respiratory system and skin
Ethanol	Highly flammable
Formaldehyde	Toxic after inhalation, ingestion and skin contact
Formamide (in fluorescence probes)	May harm the unborn child
Liberase	Irritating to the eyes, respiratory system and skin
Methanol	Highly flammable, toxic by inhalation, ingestion and skin contact
Nonidet P40	Irritating to the skin or eyes
Pepsin	Irritating to the eyes, respiratory system and skin
Proteinase K	Breathing difficulties after inhalation
Xylene	Highly flammable, toxic after inhalation and skin contact. Avoid contact with the eyes.

Table 1: Materials that require safety precautions.

1. FISH on isolated individual ovarian cortex cells

1. Dissociation of ovarian cortex tissue to obtain individual cells
   1. Cut the cryopreserved/thawed ovarian cortex tissue into small pieces of approximately 1 mm x 1 mm x 1 mm using a scalpel.
   2. Enzymatically digest the tissue fragments in 4 mL of pre-warmed (37 °C) L15 medium containing 0.1 mg/mL tissue dissociation enzyme mix, 10 µg/mL DNase I, and 1 mg/mL collagenase I from C. histolyticum for a maximum of 75 min at 37 °C. Pipet the digestion mix up and down every 15 min.
   3. Stop the enzymatic digestion by adding 4 mL of cold L15 supplemented with 10% fetal bovine serum (FBS). Wash the dissociated tissue once with 8 mL of cold L15 medium by centrifugation at 500 x g and resuspend in 500 µL of L15 medium without vortexing to avoid damage to the cells.
   4. Transfer the cell suspension containing largely individual stromal cells and small follicles (oocytes surrounded by a single layer of granulosa cells) to a plastic Petri dish and examine the cell suspension under a stereomicroscope (100x magnification).
   5. Pick up the small follicles (<50 µm) manually by using a 75 µm plastic pipette and transfer the follicles to a droplet of L15 medium supplemented with 10% FBS at 4 °C to prevent the aggregation of follicles. Perform follicle pick-up for a maximum of 30 min. To improve follicular cell spreading prior to FISH analysis, transfer the purified follicles to a solution of 0.06% trypsin, 1 mg/mL ethylenediaminetetraacetic acid (EDTA), and 1 mg/mL glucose and incubate for 20 min at 37 °C.
   6. Obtain ovarian stromal cells from the cortex cell suspension using a 75 µm plastic pipette, taking special care to avoid contamination with small follicles.
2. FISH analysis of individual ovarian cell
   1. Transfer the treated ovarian follicles (recommended n = 5-20) with trypsin/EDTA/glucose or stromal cells (n > 1,000) to droplets of 5 µL of 0.15 mM KCl/15 µL of Dulbecco's phosphate buffered saline (DPBS) on a slide and incubate for 20 min at 37 °C.
   2. Dry and pre-fixate the slides in 300 µL of 0.05 mM KCl/7.5% acetic acid/22.5% methanol for 2 min at room temperature (RT). Cover the slides with methanol/acetic acid (3:1) for 2 min at RT to finalize fixation.
   3. Make 20x standard sodium citrate (SSC) by adding 876 g of sodium chloride and 441 g of tri-sodium citrate dihydrate in 5 L of distilled water. Next, add 100 mL of the 20x SSC to 900 mL of demineralized (demi) water to obtain 2x SSC. Wash the sample in 2x SSC at 73 °C, cover it with 100 µL of 2% proteinase K, and seal it with a coverslip. Incubate the slides for 10 min at 37 °C in the hybridization station.
   4. Remove the coverslip and wash the slides for 5 min in DPBS at RT. Fixate the sample for 5 min with 1% formaldehyde at RT. At this stage, the material is not yet fully attached to the glass slides and should therefore not be placed on a shaking platform.
   5. Wash the slides for 5 min in DPBS at RT, followed by dehydration in subsequent 70%, 80%, 90%, and 100% ethanol for 2 min each. Air-dry the dehydrated sample and hybridize it with fluorescently labeled probes.
   6. Select a centromere-specific probe for chromosome X and another chromosome-specific centromeric probe as a control to determine the X chromosomal content of ovarian cells. In this case, centromere-specific probes for chromosome X (CEP X [DXZ1]) directly labeled with fluorochrome SpectrumGreen and chromosome 18 (CEP 18 [D18Z1]) directly labeled with fluorochrome SpectrumOrange are used.
   7. Add 1 µL of CEP X, 1 µL of CEP 18, and 18 µL of the hybridization buffer to the sample and seal with a coverslip that is glued to the slides to prevent evaporation of the probe during hybridization. Transfer the slides to the hybridization station for denaturation at 73 °C for 3 min, followed by hybridization during an overnight incubation at 37 °C.
   8. Remove the coverslip and any remaining glue from the slide after hybridization. Wash the slides in 0.4x SSC/0.3% Tween-20 at 72 °C for 2 min, followed by incubation for 1 min in 2x SSC/0.1% Tween-20 at RT.
   9. Dehydrate the slides by subsequent 2 min incubations in 70%, 80%, 90%, and 100% ethanol and air-dry in the dark. Cover the slides with a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). Keep at -20 °C for at least 10 min before analysis by fluorescence microscopy.
3. Imaging
   1. Examine the signal(s) for the X chromosome with a fluorescence microscope linked to an image processing software.
      1. First, select fluorochrome DAPI.
         1. Acquire an image by selecting New Cell > Live > Capture at 630x magnification. A new window with the threshold will appear. Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.
         2. A new window with enhancement will appear with a suggestion for darker/brighter (12), radius (3), and depth (1). Use the suggested values or adjust them if not satisfied.
      2. Secondly, select fluorochrome SpectrumOrange and click on Capture.
         1. Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.
         2. The window with enhancement will appear with a suggestion for darker/brighter (-11), radius (2.7), and depth (0.6). Use the suggested values or adjust them if not satisfied.
      3. Finally, select fluorochrome SpectrumGreen and click on Capture.
         1. Set the blue bar of the threshold to 0 to minimize background and the red bar to maximum (255) to make the signals brighter. Click on Accept.
         2. The window with enhancement will appear with a suggestion for darker/brighter (0), radius (3), and depth (0). Use the suggested values or adjust it if not satisfied. Save the image in a newly created file.
            NOTE: Somatic cells were only evaluated when two signals of the control chromosome 18 were visible. In most oocytes, only one signal could be detected for each chromosome.
   2. Store the slides in the dark at 4 °C after analysis to prevent loss of signals.

2. FISH on paraffin sections of grafted ovarian cortex tissue

NOTE: One fragment of cryopreserved/thawed ovarian cortex tissue of 18 females with TS was xenografted into severe combined immunodeficient (SCID) mice for 5 months. The procedure of xenografting has been described previously and was conducted at the Université Catholique de Louvain (Brussels, Belgium) following the local guidelines of the Committee on Animal Research regarding animal welfare (reference 2014/UCL/MD/007)^18,19.

1. Selecting sections of xenografted ovarian cortex tissue containing follicles
   1. Fixate xenografted ovarian cortex tissue in 4% formaldehyde and embed the tissue in paraffin. Trim the blocks with a scalpel to remove extra paraffin and cut the paraffin block to 4 µm thickness on a rotation microtome.
   2. Select every seventh section of the paraffin ribbon for hematoxylin and eosin (HE) staining to determine which sections contain follicles. Put the section in a water bath at 40-45 °C and mount them on immunohistochemistry microscope slides.
2. Deparaffinization and HE staining
   1. Put the slides on a stove for 10 min at 60 °C, and thereafter immerse the slides in 100% xylene for 5 min. It is not necessary to place the slides directly on the stove. Hydrate the sections for 15 s in 100% ethanol, followed by 2 x 15 s in 96% ethanol. Rinse the slides in tap water for 2 min.
   2. Stain the slides in hematoxylin for 10 min and thereafter rinse the slides briefly in tap water. Briefly immerse the slides in a bicarbonate solution (100 g of magnesium sulfate and 10 g of sodium bicarbonate in 5 L of distilled water). Rinse the slides in tap water for 5 min.
   3. Counterstain the slides with eosin for 4 min and dehydrate the slides three times with 100% ethanol, followed by xylene. Coverslip the HE stains on the slides and evaluate the HE sections under a light microscope to select the sections with follicles (100x magnification).
3. Pre-treatment and hybridization of paraffin sections for DNA FISH
   1. Select new sections that lay before or after the section that contained follicles from the paraffin ribbon. Mount one section on a glass slide. Dry the paraffin sections for at least 45 min on a stove at 56 °C. It is not necessary to place the slides directly on the stove.
   2. Deparaffinize the sections in xylene for 10 min. Immerse the slides in 99.5% ethanol and rinse them for 5 min in tap water. Pre-treat the slides with target retrieval solution (low pH) for 10 min at 96 °C. After cooling down, rinse the slides in distilled water.
   3. Treat the slides for 5 min with 0.01 M hydrochloric acid, followed by pepsin digestion (200 U/mL) for 15 min at 37 °C. Rinse the slides again in 0.01 M hydrochloric acid and subsequently in PBS.
   4. Fixate the slides in 1% formaldehyde/PBS for 5 min. Rinse the slides briefly in PBS, and then again in demi water. Dehydrate the slides in 99.5% ethanol and let them air-dry.
   5. Select a centromere-specific probe for chromosome X and another chromosome-specific centromeric probe as a control to determine the X chromosomal content of granulosa cells. Here, chromosome 18 is used as a control.
   6. Apply 5 µL of probe CEP 18 (D18Z1) directly labeled with fluorochrome SpectrumGreen and probe CEP X (DXZ1) directly labeled with fluorochrome SpectrumRed on the pre-treated slides. Apply a coverslip and seal the area with photo glue. Place the slides in a hybridizer for denaturation at 80 °C for 10 min and hybridization overnight at 37 °C.
   7. The next day, rinse the slides for 5 min in 2x SSC at 42 °C, followed by a 2 min and a 1 min rinse in 0.3% Nonidet P40 at 73 °C. Refresh the 2x SSC and rinse the slides again for 5 min at room temperature. Cover the cuvette so that the sections are kept in the dark.
   8. Rinse the slides briefly in distilled water. Dehydrate the slides in 99.5% ethanol and let them air-dry again. Finally, mount the slides with a solution containing DAPI and mounting medium.
4. Imaging
   1. Analyze the results under a fluorescence microscope at 630x magnification. Open the image processing software on the computer. Select FISH as the profile.
   2. Check if the DAPI emission is set at 431 nm and the excitation at 359 nm, Texas red emission at 613 nm and excitation at 595 nm, and FITC emission at 519 nm and excitation at 495 nm.
   3. Acquire an image by selecting Live > Capture Single Image. Optimize the image quality by adjusting the exposure and gain by moving the Exposure Slider and Gain Slider in the Image menu on the left (e.g., exposure: 212 ms, and gain: 7.9). The required exposure and gain can vary per image; observe the changes during this process to obtain an optimized image. Save the image in a newly created file.
   4. Store the slides in the dark at 4 °C after analysis to prevent loss of signals.

Representative Results

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 chromosomal content of individual granulosa cells, stromal cells, and oocytes isolated from ovarian cortex tissue of TS patients without prior xenotransplantation (Figure 1).

Figure 1: FISH analysis of isolated ovarian cells from ovarian cortex tissue prior to grafting. Oocytes (arrow heads) and granulosa cells from single primordial follicles (A,B) and (C,D) the surrounding stromal cells were analyzed with fluorescent specific probes for chromosome X (green signals) and control chromosome 18 (red signals). White arrows indicate 45,X cell lines, yellow arrows indicate 46,XX cell lines, and red arrows indicate 47,XXX cell lines. Not all fluorescent signals are in the same plane of focus. Bars represent 10 µm. The magnification of FISH signals was set at 630x. Please click here to view a larger version of this figure.

The differences between individual primordial follicles that were treated with and without trypsin before FISH are shown in Figure 2. By using trypsin prior to the FISH analysis, the granulosa cell mass and oocytes were less clumped, allowing the analysis of the X chromosomal content of individual granulosa cells and oocytes. The DNA of oocytes can easily be distinguished from that of the surrounding granulosa cells due to the irregular shape, size, and diffuse appearance of DNA from the oocytes. In addition, only one strong FISH signal for each chromosome is observed in the oocytes of small follicles, due to the close proximity of the four sister chromatids in these cells. The X chromosomal content of oocytes can be determined by using the surface ratio of the FISH signal for chromosome X to that of chromosome 18.

Figure 2: Small follicles treated with and without trypsin prior to FISH. (A) 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 granulosa cells (arrow heads in panel A). (B) Small follicles were handpicked from the cell suspension but provided difficulties to interpret signals after FISH due to clumping of the granulosa cells (C). (D,E) Additional digestion of the isolated follicles with trypsin prior to FISH resulted in individual granulosa cells to contract into a spherical morphology on the surface of the follicles and were more likely to dissociate from the follicle to become accessible for FISH analysis. Oocyte-derived FISH signals are indicated by arrows. Black bars represent 100 µm and white bars represent 10 µm. The magnification of FISH signals was set at 630x. Panel D has been reproduced with permission from Peek et al.¹⁶. Please click here to view a larger version of this figure.

FISH analysis of granulosa cells on paraffin sections of grafted ovarian cortex tissue
Secondary and antral follicles were found to be less susceptible to enzymatic digestion, which makes the previously described method of tissue dissociation to obtain individual ovarian cells not suitable for growing follicles found in the ovarian tissue after long term xenografting. Therefore, the FISH protocol was optimized using 4 µm histological sections to determine the X chromosomal content of granulosa cells of secondary and antral follicles in ovarian cortex tissue after grafting (Figure 3). In this setting, it is unlikely that the FISH signal of either the X chromosome or the control chromosome in oocytes would be captured in a single 4 µm section, due to the large diameter of oocytes in the growing follicles.

Figure 3: Histological and FISH staining of ovarian cortex tissue sections after xenotransplantation. (A,B) Hematoxylin/eosin staining showed morphologically normal secondary and antral follicles in ovarian cortex tissue after 5 months of xenografting. (C,D) The X chromosomal content of granulosa cells of these follicles was determined by FISH analysis. In this figure, chromosome X is shown in red and chromosome 18 in green. The bar in panel A represents 50 µm, the bar in panel B represents 20 µm, and the bars in panels C and D represent 10 µm. The magnification of FISH signals was set at 630x. Please click here to view a larger version of this figure.

As a first step, experiments with different fixating techniques of the tissues were performed to determine which method results in the best FISH signals. FISH analysis was performed on grafted tissue that was fixated in both Bouin's solution and subsequently immersed in 4% formaldehyde, and on grafted tissue that was fixated in 4% formaldehyde only. Grafted tissue that was first fixated in Bouin's displayed a green haze on the image, making it difficult to accurately count the number of FISH signals (Figure 4).

Figure 4: Grafted ovarian cortex tissue fixated in Bouin's solution and formaldehyde only. (A) Fixating grafted ovarian cortex tissue in Bouin's solution resulted in blurred and largely obscured fluorescent signals in follicular cells due to a green haze. (B) Ovarian cortex tissue that was fixated in formaldehyde only provided excellent fluorescent signals that were easy to interpret. Bars represent 10 µm. The magnification of FISH signals was set at 630x. Please click here to view a larger version of this figure.

In order to determine the X chromosomal content of granulosa cells of follicles in histological sections, it is not possible to simply count the FISH signals of granulosa cells. Part of the chromosomes of an individual granulosa cell may be lost in a certain histological section, due to sectioning of the tissue. Therefore, it is necessary to first determine the percentage of FISH signals lost due to sectioning before ultimately determining the loss of X chromosomal content of the granulosa cell population due to aneuploidy in newly formed secondary and antral follicles after grafting of the tissue.

The percentage of lost FISH signals due to sectioning can be calculated by determining the number of FISH signals per granulosa cell of the non-aneuploid control chromosome 18. It is expected that the same percentage of the X chromosome is lost due to sectioning. Any additional reduction in the number of X chromosome FISH signals in the granulosa cell population is due to aneuploidy. However, using FISH signals of a control chromosome to determine X chromosomal loss due to aneuploidy requires the detection sensitivity of the FISH signals of the X chromosome and the control chromosome to be very similar. The detection sensitivity of both FISH probes in granulosa cells of growing follicles in histological sections of non-aneuploid 46,XX individuals was therefore determined. When the ratio of chromosome X/control chromosome FISH signals is close to 1, it can be assumed that the detection sensitivity of both probes are indeed very similar, and that the FISH probes can thus be used to determine the level of aneuploidy in the granulosa cell population of growing follicles in histological sections of ovarian tissue after xenotransplantation.

Example
When analyzing the number of chromosome 18 signals in 130 granulosa cells of a follicle from an ovary of a 46,XX individual, it is expected that 260 signals are present. However, in a 4 µm section of these cells, only 204 chromosome 18 signals were observed. This indicates that 21.5% of the signals are lost due to sectioning ((260-204)/260 x 100 = 21.5%). The number of chromosome 18 signals per granulosa cell is therefore reduced to 204/130 = 1.57 due to sectioning.

Next, the number of X chromosomes in granulosa cells of an antral follicle after grafting in ovarian tissue of a female with TS is analyzed. In total, 191 signals for chromosome X and 199 signals for chromosome 18 were counted. The number of granulosa cells can be determined by dividing the total number of signals for chromosome 18 by the number of chromosome 18 signals per granulosa cell (199/1.57 = 127 granulosa cells). Finally, the percentage of 45,X granulosa cells can be determined by dividing the difference in chromosome 18 and chromosome X signals with the number of granulosa cells (i.e., [199-191]/127 x 100 = 6% of the granulosa cells are 45,X, and 94% of these cells are 46, XX).

It is noteworthy that, in patients with a 47,XXX cell line, it is only possible to determine the minimum percentage of granulosa cells with a 47,XXX karyotype, since a mix of 45,X and 47,XXX granulosa cells has the same number of X chromosome signals as 46,XX granulosa cells.

Discussion

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 grafted ovarian cortex tissue from females with X chromosomal aberrations.

The main challenge of the protocol for the isolation of individual ovarian cells in non-grafted ovarian cortex tissue lies in the enzymatic digestion of the tissue, which requires some practice beforehand. In order to obtain accurate FISH signals of sufficient ovarian cells, it is important to follow the steps regarding enzymatic digestion, incubation times, and indicated temperatures strictly. Deviating from the protocol can cause a substantial loss of ovarian cells during the process, and this should especially be avoided in patients who already have a low ovarian reserve. Another critical step in this method is to treat the purified primordial follicles with trypsin to prevent oocytes and the granulosa cell mass from clumping, which precludes the analysis of individual cells. Without treatment with trypsin, the number of individual granulosa cells that can be reliably analyzed by FISH is severely reduced.

The FISH analysis on ovarian cortex tissue retrieved after long-term grafting requires a different technique, as only small follicles were found to be sufficiently susceptible to the enzymatic digestion that is necessary to obtain individual ovarian cells. In addition, similar to autotransplantation of ovarian cortex tissue, many follicles get lost after grafting due to hypoxia and a lack of nutrients before the graft is sufficiently revascularized by the host²⁰. The number of follicles after grafting is therefore expected to be considerably lower than before grafting. For this FISH technique used for histological sections, it is important to fixate the grafted tissue in 4% formaldehyde only to obtain optimal FISH signals. Fixation of ovarian cortex tissue in Bouin's fixative is routinely used in the laboratory, and while this gives excellent results when combined with standard HE staining, fixating tissue with Bouin's prior to FISH leads to weak and blurry fluorescent signals that are difficult to interpret.

Although this protocol has been proven to be successful in determining the X chromosomal content of ovarian cells, it still has some limitations. One limitation is that these methods can only be used to analyze the ovarian cells of females with numerical aberrations. Numerical aberrations can be detected by using probes directed at repetitive sequences²¹. These probes hybridize multiple repeating base pair sequences in the centromere region, resulting in strong hybridization signals. In contrast, structural aberrations can only be detected by using probes against unique single sequences. These probes hybridize to sequences that only occur once in the haploid genome, resulting in a considerably less intense FISH signal compared to numerical aberrations. Due to these relatively weak FISH signals, it is difficult to properly determine the X chromosomal content of ovarian cells in females with structural aberrations.

Secondly, FISH signals of oocytes of small follicles in non-grafted tissue are difficult to analyze, due to the close proximity of the four sister chromatids in the prophase of meiosis I²². Only one strong hybridization signal will be present in the oocytes, and therefore it is not possible to simply count the number of signals in the oocytes to determine the X chromosomal content. Instead, the surface ratio of chromosome X and 18 FISH signals should be used to determine the X chromosomal content in these cells. This can only be reliably determined if the FISH signals in the oocytes are clearly present.

Furthermore, FISH on grafted tissue can only be used to determine the X chromosomal content of granulosa cells from secondary and antral follicles, as small follicles in histological sections of grafted tissue only have a few granulosa cells that can be properly analyzed. In addition, the X chromosomal content of oocytes cannot be determined accurately using this method due to the large diameter of the oocytes.

Finally, it remains challenging to obtain female gametes compared to male gametes because invasive surgery is required to obtain ovarian cells or ovarian cortex tissue. Therefore, these methods are most likely to be applied in a research setting.

In conclusion, FISH analysis of ovarian cells of non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations is a unique and useful technique to gain insight into the X chromosomal content of ovarian cells in this specific group. These techniques show that cryopreservation of ovarian cortex tissue from females with X chromosomal aberrations is possible, and that cryopreserved primordial follicles are able to grow to secondary and antral follicles. However, it should be kept in mind that both methods are intended to facilitate future research in females with X chromosomal aberrations, and are not designed to be used as a diagnostic tool to screen reproductive outcomes of females with X chromosomal aberrations in clinical practice.

Materials

Name	Company	Catalog Number	Comments
Acetic acid	Biosolve BV	0001070602BS
Centrifuge 1200	Hettich Universal	4140
Collagenase I	Sigma	131470
Coverslip	VWR	0631-0146
DAPI	Vector	H-1200
DNase I	Roche	10104159001
Dulbecco’s Phosphate Buffered Saline	Lonza	BE17-513Q
EDTA	Merck	108421
Eosin-Y	Sigma	1159350100
Ethanol	EMSURE	1009832500
Fetal Bovine Serum (FBS)	Life technology	10100147
Fluorescence microscope for sections DM4 B	Leica Microsystems
Fluorescence microscope scope A1	Zeiss AXIO
Fluorescent labeled probes for dissociated cells	Abbott Diagnostics	CEPX (DXZ1) 05J1023 CEP18 (D18Z1) 05J0818
Fluorescent labeled probes for tissue sections	Abbott Diagnostics	CEP X (DXZ1 05J08-023 CEP 18 (D18Z1)  05J10-028
Formaldehyde	Sigma	252549
Glucose	Merck	108337
Glue (Fixogum)	Leica Microsystems	LK071A
Hematoxylin	Sigma	1159380025
Hybridization buffer	Abott Diagnostics	32-804826/06J67-001
Hybridization Station	Dako	S2451
Hydrochloric acid	Merck	1003171000
Image processing software individual ovarian cortex cells (Cytovision 7.7)	Leica Biosystems
Image processing software on paraffine sections	Leica Application Suitex (3.7.5.24914)
Immunohitochemistry microscope slides	Dako	K802021-2
L15	Lonza	12-700Q
Liberase DH	Roche	05 401 151 001
Light microscope	Zeiss West Germany
Magnesium sulphate	Merck	A335586
Methanol	Honeywell	14262-1L
Mounting medium	Vectashield, Vector	H-1000
Nonidet P40	Sigma	7385-1L
Paraffin	Poth Hile	2712.20.10
Pepsin	Sigma	P7000-25G
Phosphate-Buffered Saline (PBS)	Gibco	11530546
Plastic pipette	CooperSurgical	7-72-4075/1
Potassium chloride	Merck	1049361000
Proteinase K	Qiagen	19131
Rotation microtome HM 355S	Thermo sceintific
Scalpel	Dahlhausen	11.000.00.515
Slide for FISH on dissociated cells	Thermo scientific	J1810AM1JZ
Sodium bicarbonate	Sigma	55761-500G
Standard Sodium Citrate (SSC)	Fisher Scientific, Invitrogen	10515203
Stereomicroscope IX 70	Olympus
Target Retrieval Solution	Dako	GV80511-2
Trypsin	Sigma	T4799
Tween-20	ThermoFisher	85113
Xylene	BOOM	760518191000