Ultrarapid detection of sex chromosomeswith the use of fluorescence in situhybridization with direct label DNAprobes in single human blastomeres,spermatozoa, amniocytes, andlymphocytes

Jiaen Liu, M.D., Ph.D., Xue-Zhong Zheng, M.D., Theodore A. Baramki, M.D.,Ricardo A. Yazigi, M.D., Gail Compton, M.A.S., and Eugene Katz, M.D.

The Greater Baltimore Medical Center Fertility Center, Greater Baltimore Medical Center, Baltimore, Maryland

Objective: To assess the ultrarapid fluorescence in situ hybridization (FISH) procedure with a 1-minute hybridization timefor gender determination.

Design: Fluorescence in situ hybridization with direct label fluorescence DNA probes for chromosomes X and Y weretested with the use of different hybridization times and different cell types.

Setting: Hospital-based IVF program.

Intervention(s): The efficiency of the FISH procedure with different hybridization times was compared with the use ofmale lymphocytes. The same FISH procedure, but with only 1-minute hybridization, was carried out in human blastomeres,spermatozoa, uncultured amniocytes, male lymphocytes, and female lymphocytes.

Main Outcome Measure(s): Percentages of nuclei with positive signals.

Result(s): The percentages of nuclei with positive signals in lymphocytes with hybridization times of 1, 3, 4, 10, 30, and45 minutes were 97%, 97%, 98%, 98%, 98%, and 98%, respectively. The percentages of nuclei with positive signals afterFISH with a 1-minute hybridization time in single blastomeres, spermatozoa, amniocytes, male lymphocytes, and femalelymphocytes were 94%, 96%, 96%, 98%, and 97%, respectively.

Conclusion(s): Chromosomes X and Y of human blastomeres, spermatozoa, uncultured amniocytes, and lymphocytes canbe detected rapidly with the use of this ultrarapid FISH procedure with a 1-minute hybridization time. (Fertil Sterilt 1998;70:927–32. ©1998 by American Society for Reproductive Medicine.)

Key Words: Fluorescence in situ hybridization, gender determination, preimplantation genetic diagnosis, prenatal diagnosis

Both polymerase chain reaction (PCR) andfluorescence in situ hybridization (FISH) havebeen used for gender determination in preimplan-tation genetic diagnosis to prevent X-linkedrecessive diseases (1–6). For preimplantation ge-netic diagnosis of X-linked recessive diseasesby PCR, coamplification of X- and Y-specificsequences may prevent misdiagnosis from theanalysis of only Y-specific sequences (3, 6–9).However, the main drawback to the use of astrategy based on the coamplification of X- orY-specific sequences is the existence of aneu-ploidies that cannot be detected by PCR.

The advantages of using FISH with directfluorescence label DNA probes include [1] its

high specificity and sensitivity, [2] its morerapid performance, [3] its ability to simulta-neously detect several probes using differentfluorochromes (10), and [4] its applicability inboth interphase and metaphase nuclei. The si-multaneous detection of X and Y chromosomeswith specific probes is likely to be the mostreliable method for identifying the sex of an em-bryo and aberrations in the number of sex chro-mosomes. Therefore, the FISH technique appliedto cleavage-stage embryos has become the pre-ferred method in the preimplantation genetic di-agnosis of X-linked diseases for gender determi-nation (11).

A rapid diagnosis always is desired for the

Received February 19,1998; revised andaccepted June 10, 1998.Supported by a researchgrant from the GreaterBaltimore Medical Center,Baltimore, Maryland.Reprint requests: Jiaen Liu,M.D., Ph.D., The GreaterBaltimore Medical CenterFertility Center, 6569 NorthCharles Street, Suite 406,Baltimore, Maryland 21204(FAX: 410-512-8694;E-mail: jliu@gbmc.org).

FERTILITY AND STERILITY tVOL. 70, NO. 5, NOVEMBER 1998Copyright ©1998 American Society for Reproductive MedicinePublished by Elsevier Science Inc.Printed on acid-free paper in U.S.A.

0015-0282/98/$19.00PII S0015-0282(98)00288-X

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clinical application of preimplantation genetic diagnosis.With several modifications of the FISH technique and theuse of direct label fluorescence DNA probes, the whole FISHprocedure has been shortened to a few hours (12, 13). Themost time-consuming step in FISH is the hybridization timebetween DNA probes and their target sequences, which oftentakes from 30 minutes to overnight depending on the type ofDNA probes used.

An attempt to shorten the hybridization time in FISH to 5minutes with the use of a microwave for denaturation andhybridization was reported recently (14, 15). With the use ofa microwave, we found it difficult to determine and controlthe exact temperature in the microwave for the denaturationand hybridization of DNA and probes in the FISH proce-dures as described by Drury et al. (14) and Durm et al. (15).Instead, we report herein an ultrarapid FISH procedure usedon single human blastomeres, spermatozoa, uncultured am-niocytes, and lymphocytes in which only 1 minute is re-quired for hybridization in an automatic slide baker (HY-Brite; Vysis, Downers Grove, IL).

MATERIALS AND METHODS

This study was approved by the Institutional Ethics Com-mittee of the Greater Baltimore Medical Center.

Cell Preparations

Blastomeres

Single blastomeres were isolated from embryos obtainedfrom oocytes with three pronuclei (PN) after in vitro insem-ination in our center. The procedures used for the isolation ofa single blastomere were the same as described previously(16). An isolated blastomere was transferred into a droplet ofHEPES-buffered human tubal fluid medium (H-HTF me-dium; Irvine Scientific, Santa Ana, CA). The nucleus of eachblastomere was examined carefully under an inverted micro-scope at a magnification of3400. Only those blastomeresthat had a single nucleus each were used in this study. Onehundred sixty-five blastomeres from 38 embryos were ob-tained after conventional IVF. Of these 165 blastomeres, 146clearly had a single nucleus and were fixed successfully onglass slides. The ultrarapid FISH procedures were carried outin these 146 blastomeres.

The fixation of blastomeres was modified from themethod described by Coonen et al. (12). Briefly, a singleblastomere was placed in a premarked circle on a glass slide(Surgipath, Richmond, IL). A solution of 0.1% Tween 20 in0.01N HCl was used to remove the cytoplasm of the blas-tomere using a mouth-control glass pipette until the nucleuswas free of cytoplasm. The slide then was left at roomtemperature for at least 40 minutes before the FISH proce-dure was performed.

Spermatozoa

Fresh semen from three donors was washed in a 15-mLFalcon tube filled with 10 mL of sperm-preparation medium(HTF medium with 3% synthetic serum substrate [IrvineScientific]). After centrifugation at 3003 g for 5 minutes,the supernatant was removed. The sperm pellet was resus-pended in 5 mL of sperm-preparation medium and centri-fuged at 3003 g for 5 minutes. After the supernatant wasremoved, the sperm pellet was resuspended in approximately100 mL of sperm-preparation medium. A drop of the spermsuspension was spread on a glass slide and air-dried for 30minutes. One drop of 1N NaOH was added to the slide andretained for 8 minutes. The slide was rinsed in water andthen rinsed in 23 NaCl and Na citrate solution (SSC), andthe FISH procedure was performed immediately afterward.

Uncultured Amniocytes

Fresh amniotic fluid was obtained from three patientsundergoing midtrimester amniocentesis. Two milliliters ofthe fluid was transferred in an Eppendorf tube and centri-fuged at 3003 g for 10 minutes. The supernatant wasremoved and the pellet was resuspended in 50mL of thefluid. Ten microliters of the cell suspension was used tomake a smear on a glass slide. The slide was left at roomtemperature for 30 minutes before the FISH procedure wasperformed.

Lymphocytes

A few drops of fresh human peripheral blood (from twomen and one woman) were taken separately from a fingerneedle stick to make smears on glass slides. The slides thenwere left at room temperature for 30 minutes before theFISH procedure was performed.

Fluorescence In Situ HybridizationThe prepared slides with the nuclei of blastomeres, sper-

matozoa (after treatment with 1N NaOH), amniocytes, andlymphocytes were placed into separate Coplin jars contain-ing 0.01% pepsin (Sigma Chemical Co., St. Louis, MO) and0.01N HCl. Each jar was kept in a 37°C waterbath for 10minutes. The slides first were rinsed in water and subse-quently in 23 SSC at room temperature.

Dehydration was performed by transferring the slidessequentially into 70%, 90%, and 100% ethanol. The slidesthen were kept at room temperature for 3–5 minutes.

Direct label fluorescence Vysis CEP probes for chromo-somes X and Y were used. Five microliters of each probewas mixed with 70mL of buffer solution (Vysis) and 20mLof water. Three microliters of the mixed solution was addedto the premarked circle on each slide and a 12-mm circlecoverslip (Fisher Scientific, Pittsburgh, PA) was placed onthe drop of solution. The slides were transferred to theHYBrite. For study 1, denaturation and hybridization of theFISH procedure with lymphocytes were carried out at 80°C

928 Liu et al. Ultrarapid gender determination by FISH Vol. 70, No. 5, November 1998

for 3 minutes and at 37°C for 1, 3, 5, 10, 30, and 45 minutes,respectively, in the HYBrite. For study 2, denaturation at80°C for 3 minutes and hybridization at 37°C for 1 minutewere carried out in the FISH procedure with blastomeres,spermatozoa, amniocytes, and lymphocytes in the HYBrite.A wet paper with 60% formamide/23 SSC (pH 7) wasplaced on the slides in the HYBrite to provide humidity.

After hybridization, the slides were rinsed first in 60%formamide/23 SSC and then in 23 SSC at 42°C for 3minutes each. The slides were transferred to another jarcontaining a solution of 0.05% Tween 20 in 43 SSC andkept at room temperature for 3 minutes. After washing, theslides were dehydrated sequentially in 70%, 90%, and 100%ethanol.

Once the slides were air-dried, 10mL of a mixed solutionof 1.25 ng/mL of 49,6-diamidino-2-phenylindole (SigmaChemical Co.) and Vectashield mounting medium (VectorLaboratories Inc., Burlingame, CA) (1:1, vol/vol) was addedto each of the slides and they were covered with a 22322–mm coverslip (Richard-Allan Scientific, Richland, MI).After 5 minutes, the slides were evaluated under a fluores-cence microscope (Zeiss Axioskop, Oberkochen, Germany)at a magnification of31,000 by two investigators.

Evaluation of the ResultsFor spermatozoa, amniocytes, and lymphocytes, 100 in-

terphase nuclei were counted in each slide after FISH andexamined for the absence or presence of signal. The presenceof signal was defined as the appearance of specific coloredprobes in the nuclei after FISH. The absence of signal wasdefined as the clear observation of nuclei on the slideswithout any fluorescence signal.

StatisticsThe x2 test was used to compare the efficiency of FISH

procedures between different hybridization times in lympho-cytes and between different cell types.P,.05 was defined asstatistically significant.

RESULTS

In study 1, male lymphocytes first were used to test theinfluence of different hybridization times on the efficiency of

FISH with Vysis CEP probes for chromosomes X and Y.The percentages of nuclei with positive signals after FISHwith hybridization times of 1, 3, 4, 10, 30, and 45 minuteswere 97%, 97%, 98%, 98%, 98%, and 98%, respectively(Table 1). There was no statistically significant difference inthe rates of nuclei with positive signals between the differenthybridization times.

In study 2, the same FISH procedure was carried out, butwith only a 1-minute hybridization time. The percentages ofnuclei with positive signals in single human blastomeres,spermatozoa, amniocytes, male lymphocytes, and femalelymphocytes were 94%, 96%, 96%, 98%, and 97%, respec-tively (Table 2). There was no statistically significant differ-ence in the percentages of nuclei with positive signals be-tween these four types of cells. No Y chromosome signalwas observed in the nuclei of the female lymphocytes.

The FISH results of the uncultured amniocytes obtainedfrom the three patients indicated that one fetus was female(amniocytes with two X chromosome signals) and two fe-tuses were males (amniocytes with one X chromosome sig-nal and one Y chromosome signal). The gender of thesethree fetuses was confirmed by karyotype of culturedamniocytes from the same patients (unpublished observa-tion).

Thirty-eight embryos from oocytes with 3PN after IVFwere analyzed. As shown in Table 3, of 38 embryos, 17 hadonly X chromosome signals and 21 had X and Y chromo-some signals. The sex chromosome constitutions of the 38embryos revealed that 6 (16%) showed uniformly triploidblastomeres, 3 (8%) had the same diploid blastomeres, 8(21%) were diploid/triploid, and 21 (55%) showed mosaicand chaotic X and Y chromosome signals. In total, 146blastomeres were fixed and underwent the FISH procedure.Four (3%) nuclei from 146 blastomeres were not found afterFISH. One hundred thirty-three (94%) of 142 blastomereshad positive signals for the DNA probes.

DISCUSSION

To shorten the FISH procedure and to detect sex chro-mosomes rapidly, we have developed an ultrarapid FISH

T A B L E 1

Efficiency of ultrarapid FISH with different hybridization times using CEP probes for chromosomes X and Y in lymphocytes.

Variable

Hybridization time (min)

1 3 5 10 30 45

No. of cells examined 300 300 300 300 300 300No. of cells with signals 292 292 295 295 294 295(percentage of nuclei with signals)* (97) (97) (98) (98) (98) (98)

* There was no statistically significant difference in the percentages of nuclei with signals at different hybridization times (P.0.05 by thex2 test).

FERTILITY & STERILITY t 929

procedure that takes about 40 minutes to complete (notincluding embryo biopsy and blastomere fixation). Thepresent study demonstrates that a high efficiency of theultrarapid FISH procedure with direct label fluorescenceDNA probes for chromosomes X and Y can be obtainedusing only 1 minute for the hybridization step. Clear fluo-rescence signals always were observed under the fluores-cence microscope (Fig. 1).

The efficiency of this FISH procedure is similar to thatreported for FISH procedures with longer hybridizationtimes (with direct label or indirect label fluorescent probes)(3, 10, 17–24). With the technique described here, the tem-perature of denaturation and hybridization can be controlledmore precisely with the use of the HYBrite than with the useof a simple slide baker or a microwave. The results also

indicate that the short hybridization time of our techniquedoes not affect its efficiency. It appears that as soon as DNAand DNA probes were denatured, hybridization betweenDNA probes and target sequences occurred instantly.

In this study, direct label fluorescence probes for chro-mosomes X and Y (Vysis CEP probes) were tested in theFISH procedures. Vysis WCP probes for sex chromosomesalso can be used with our technique (unpublished observa-tion). We do not know whether this ultrarapid FISH proce-dure can be performed when other kinds of fluorescenceDNA probes are used, such as Vysis LSI probes. We cur-rently are testing this rapid FISH procedure with severalspecific probes for autosomes.

Our results indicate that human embryos from 3PN oo-cytes have a high incidence of sex-chromosome mosaicism.After IVF, a high incidence of chromosomal mosaicism hasbeen observed in preimplantation human embryos from 2PNand 3PN oocytes. The different constitutions of chromo-somes in the preimplantation embryos have been discussedpreviously (25–32). Therefore, it is recommended that twoblastomeres per embryo be analyzed in preimplantation ge-netic diagnosis with the use of either the PCR or the FISHtechnique (33, 34).

The ultrarapid FISH procedure described here is a fastand simple technique that allows the rapid detection of sexchromosomes in interphase nuclei of human blastomeres.Because this FISH procedure can be completed within anhour, it provides an obvious advantage for clinical applica-tion in preimplantation diagnosis in that ET can be carriedout at noon or in the early afternoon after embryo biopsieshave been performed in the morning.

This rapid FISH procedure also can be useful for genderdetermination in prenatal diagnosis. Our results indicate thatthis rapid FISH procedure can be carried out directly onuncultured amniocytes, which is much simpler than conven-tional karyotype analysis using cultured amniocytes.

In conclusion, a highly efficient ultrarapid FISH proce-dure with a 1-minute hybridization time can be accom-

T A B L E 2

Efficiency of ultrarapid FISH with 1-minute hybridization using CEP probes for chromosomes X and Y in singleblastomeres, spermatozoa, amniocytes, and lymphocytes.

Variable

Cell type

Blastomere Spermatozoon Amniocyte

Lymphocyte

Male Female

No. of cells examined 146 300 300 300 300No. of cells with signals 133 288 282 293 291(percentage of nuclei with signals)* (94) (96) (94) (98) (97)

* There was no statistically significant difference in the percentages of nuclei with signals between different cell types using ultrarapid FISH with1-minutehybridization and with DNA probes for chromosomes X and Y (P.0.05 by thex2 test).

T A B L E 3

Results of FISH in 38 embryos.

Chromosomal constitutionsof the embryos

No. ofembryos

All blastomeres of an embryo withonly X chromosome signalsXX 2XXX 1Mosaic

X/XX/XXX 6XX/XXX 3

Chaotic 5All blastomeres of an embryo with

X and Y chromosome signalsXY 1XXY 3XYY 2Mosaic

XX/XY 4XY/XYY 2XY/XXY 3

Chaotic 6Total 38

930 Liu et al. Ultrarapid gender determination by FISH Vol. 70, No. 5, November 1998

plished with the use of Vysis CEP probes for chromosomesX and Y. This ultrarapid FISH procedure can be useful inprenatal and preimplantation genetic diagnosis for genderdetermination, prevention of X-linked diseases, and detec-tion of sex chromosomal aneuploidy.

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F I G U R E 1

Ultrarapid FISH with 1-minute hybridization using CEP probes for chromosomes X (green signal) and Y (red signal). (A),Blastomere; (B), Amniocyte; (C), Spermatozoa; and (D), Male lymphocytes.

FERTILITY & STERILITY t 931

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