PRENATAL DIAGNOSIS, VOL. 13,349-353 (1 993)

RAPID KARYOTYPING IN ECTOPIC PREGNANCIES

DANIEL COHEN*, IRMA BRAGOS*, CESAR BERTAT, MONICA FODOR~, EDUARDO ALONSOf, CESAR NASIO*, LILIANA KREIMAN* AND RAFAEL PINEDAt

*Departamento de GenPtica Humana, Sanatorio de la Mujer, Rosario; f Servicio de Ginecologia y de SAnatornia Patolbgica, Hospital de Emergencias Dr Clemenie Alvarez, Rosario. Argentina

SUMMARY The aim of this study was to determine the feasibility of karyotyping ectopic Fallopian tube pregnancies utilizing dividing cytotrophoblast cells. Villi from 78 ectopic conceptuses were processed by the direct chromosome technique and cytogenetic diagnosis was successful in 60 cases (76.9 per cent). The amount of villi obtained, as well as villus morphology, was corre- lated with cytogenetic success rate. Histological examination of the Fallopian tube was also carried out. A total of 47 cases were chromosomally abnormal (78.3 per cent), which is the highest frequency of cytogenetic abnormalities reported to date.

KEY WORDS Ectopic Fallopian tube pregnancy Cytogenetic diagnosis Direct chromosome preparation

INTRODUCTION

Karyotyping of direct chromosome preparations of chorionic villi is a reliable diag- nostic tool in ongoing pregnancies (Simoni et al., 1985; Ford and Jahnke, 1983; Jackson et al., 1986).

In a recent report, Johnson ef al. (1990a,b) proposed this technique to karyotype missed abortions, since extraembryonic cells may remain viable after embryonic death.

We have evaluated the success rate of direct chromosome preparation on ectopic Fallopian tube pregnancies (EFTPs), assuming that an adequate mitotic index (MI) would be present a t the time of surgery.

MATERIALS AND METHODS

Seventy-eight EFTPs were studied during a 48-month period (1987-1991). Specimens were collected in patients undergoing laparotomy for an ectopic pregnancy at the Dr Clemente Alvarez Emergency Hospital (Rosario).

The material was immediately examined under a dissecting microscope and con- sidered suitable when vascularized villi and syncytial sprouts were present. Chorionic samples were weighed and direct preparations were made according to the Simoni technique (Simoni et al., 1985) with slight modifications.

After colcemid treatment in 2 ml of RPMI 1640 (Gibco), the villi were transferred to a 1 per cent sodium citrate hypotonic solution for 10 min, then passed three times

Addressee for correspondence: Daniel Cohen, MD, Departamento de GenBtica Humana, Sanatorio de la Mujer, San Luis 2493,2000 Rosario, Argentina.

0197-385 1/93/050349%05$07.50 0 1993 by John Wiley & Sons, Ltd.

Received 20 February 1992 Revised 20 August I992

Accepted 26 August I992

350 D. COHEN ETAL.

Table 1. Karyotyping success rate and the gestational age of the ectopic pregnancies

Gestational Karyotyping age* No. of success rate (days) cases (Yo)

< 30 33 19 (57.5) > 30 45 41 (91.1) Total 78 60 (76.9)

*Gestational age was determined taking into account the patient’s last menstrual period and the surgery date.

through a 1:3 acetic acid-methanol fixative, and stored at - 20°C overnight. Twenty-four hours later, chromosome spreads were obtained by dissociation through decreasing methanol concentrations and laying a drop of the cell suspen- sion on a warmed slide. GTG banding was performed by the modified Seabright (1971) method.

Histological examination of Fallopian tubes was also carried out.

RESULTS Table 1 shows the karyotyping success rate and the different gestational-age groups studied. Villi from 78 cases were processed and the karyotyping was successful in 60 cases (76.9 per cent).

A total of 33 samples were obtained within the first month of amenorrhoea and cytogenetic analysis was attained in 19 cases (57.5 per cent). After 30 days of amenorrhoea (45 cases), karyotyping was successful in all but four cases, in which metaphases were not suitable (91.1 per cent).

The amount of villi obtained and the corresponding MI are given in Table 2. With samples weighing less than 2 mg (n = 14), chromosome analysis was achieved in only one case (7.1 per cent). With 2-4 mg samples (n=47), there were no mitoses in five cases (10.7 per cent); a low MI in 15 cases (31.9 per cent), and a suitable MI in 27 cases (57.4 per cent). All samples weighing over 4 mg were successfully karyotyped (1 7 cases).

Cytogenetic findings are listed in Table 3. A total of 47 cases (78.3 per cent) were chromosomally abnormal, while 13 cases (21.7 per cent) had a normal chromosome complement.

Abnormalities included complete monosomy of the X chromosome (13 cases), autosomal trisomies (1 8 cases), triploidies (1 0 cases) and other aberrations (6 cases).

Data corresponding to histological and karyotype examination of the Fallopian tubes are presented in Table 4.

DISCUSSION Since chorionic villus sampling was developed to enhance the prenatal diagnosis of genetic diseases, direct chromosome preparation has been widely performed due to

RAPID KARYOTYPING IN ECTOPIC PREGNANCIES 351

Table 2. Sample weights and mitotic index in the 78 ectopic Fallopian tube pregnancies analysed (direct chromosome preparation)

Amount of villi (mg tissue)

MI* (No. of cases) < 2 2-4 > 4 Total

No mitoses available 13 5 0 18 Low MI (1-3 mitoses) 1 15 0 16 Adequate MI (9-22 mitoses) 0 27 17 44 Total 14 47 17 78

xz test, p <0.001. *MI=mitotic index. Defined by Romagndno ei al. (1989) as the number of

metaphases per 400 cells.

Table 3. Cytogenetic diagnosis in 60 ectopic Fallopian tube pregnancies karyotyped by the direct method

Karyot ype No. ofcases %

Diploidies 46,XX 46,XY

45,x

Trisomies of chromosome 3 9

13 16 18 20

Tnploidies 46,XY/69,XXY 69,XXY 69,XXX

45,XY, - 10

48,XXY, + 16

Others 92,XXXX

45,XY, -A

46,XX,- 16, + 13 Total

5 8 21.7

13 21.7 2 2 3 9 1 1 30.0

5 3 2 16.6 2 1 1 1 1 10.0

60 100.0

its simplicity and rapidity. O n the other hand, we have not been able to find any report regarding karyotyping of EFTPs by this method.

Since ectopic conceptuses are indeed viable, rapid karyotyping should be possible if villi are obtained a t the time of surgery.

352 D. COHEN ETAL.

Table 4. Histology of Fallopian tubes and karyotype of chorionic villus tissue

Pathological Normal No karyotype karyotype karyotype Total

Fallopian tube lesion 40 11 15 66 (84.62%)

(1 5.38 %) Normal Fallopian tube 7 2 3 12

Total 47 13 18 78

Fallopian tube lesion: unspecific chronic inflammation, specific chronic inflammation, follicu- lar salpingitis, cicatricial mucosa, granuloma due to previous surgery or ligation of the Fallopian tube, adenomyosis of the Fallopian tube, nodal isthmic salpingitis. X* test NS.

Although the success rate reported by means of ectopic embryonic tissue culture is higher (Busch and Benirschke, 1974; Elias et al., 1981), our data show the feasibility of EFTP karyotyping from rapidly dividing cytotrophoblast cells (Table 1). This obviously offers an attractive alternative since neither sophisticated nor expensive technology is needed. Besides, the mitotic index reflects the amount and mor- phology (syncytial sprouts) of the material obtained, which is in agreement with other authors’ findings (Gustavii, 1984; Verlinsky et al., 1985) (Table 2).

Moreover, the incidence of chromosomal abnormalities in our series (78.3 per cent) is the highest reported to date. In contrast, by means of the ectopic embryonic tissue culture technique, Klinger and Gasser (1977) found no chromosomal abnor- malities in 11 cases, whilst Stratford (1970) reported 63.6 per cent in 44 cases; Busch and Benirschke (1974) 36 per cent in 25 cases; Poland et al. (1976) 43.4 per cent in 76 cases; Matsunaga and Shiota (1980) 11.6 per cent in 43 cases; and Elias et al. (1981) 21.7 per cent in 23 cases.

In an attempt to explain these differences, we must take into account the cyto- genetic discrepancies between the direct technique and those employing embryonic tissue culture methods. For the most part, these discrepancies might be due to placental mosaicism of chorionic villi, which are not present in fetal cells (Kalousek and Dill, 1983; Crane and Cheung, 1988).

To support the latter statement, we have studied a series of 1250 first-trimester spontaneous abortions in which 437 were successfully karyotyped (unpublished results). The incidence of chromosomal aberrations registered in regular abortions (75-8 per cent) was similar to that observed in ectopic pregnancies (78.3 per cent) and higher than the long-term culture data reported by Bout et al. (1975) in spontaneous abortions, i.e., 50 per cent.

Furthermore, the relationship between the histology of the Fallopian tube and the direct karyotype of the ectopic chorionic tissue (Table 4), though statistically not significant, suggests the concomitant occurrence of both genetic and mechanical factors in the aetiology of the ectopic pregnancy in most cases. Thus, it is probable that an ectopic Fallopian tube pregnancy is the result of a complex process in which multiple factors, particularly mechanical and genetic ones, play important roles.

RAPID KARYOTYPING IN ECTOPIC PREGNANCIES 353

ACKNOWLEDGEMENTS

We are very grateful t o Marta Bravo Luna, MD for the revision and discussion of the manuscript.

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