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Nuclear chromatin determination in amniotic fluid cells for prenatal sex prediction in the macaque ARTHUR L. HERBST, M.D. PRISCILLA D. TAFT, M.D. STANLEY

J.

ROBBOY, M.D.*

Boston, Massachusetts

Amniotic fluid cells obtained after the ninetieth day of gestation in the macaque were analyzed for nuclear chromatin. The technique proved reliable for the prediction rif fetal sex.

IN THE couRSE or investigation of the effects of intrauterine exposure to diethylstilbestrol (DES) on the female macaque fetus, it became useful for us to establish the sex of the offspring prior to birth in order to concentrate experimental efforts on the female fetus. For many years, it has been known that a female sex chromatin body (Barr body) can be identified in the somatic cell nuclei of many mammalian species. 1 The technique for its detection in amniotic fluid cells for the

purpose of prenatal sex prediction in the human subject has also been described. 2 However, we can find no study describing the usage of this technique in the subhuman primate.

Material and methods Pregnant animals (Macaca mulatta and Macaca fascicularis) housed in the breeding colony at the New England Regional Primate Research Center were injected with DES (5 mg. in 0.5 c.c. of cottonseed oil, intramuscularly, daily) or 0.5 c.c. of the diluent alone, beginning at Day 35 to 40 of gestation and continuing for 100 days. Amniocentesis was performed on a total of 15 animals from both the experimental and control groups after the abdomen was surgically prepared, shaved, and cleaned with soap, iodine, and alcohol solutions. (This extensive skin preparation was adopted after one animal developed intrauterine sepsis early in the study.) The superior margin of the

From Vincent Memorial Hospital (Gynecology Sen•ice of the Massachusetts General Hospital), the james Homer Wright Pathology Laboratories, Massachusetts General Hospital, and the New England Regional Primate Research Center, Harvard Medical School. Supported in part by Grant 13139-02 from the National Cancer Institute and Grant ET-52 from the American Cancer Society. Reprint requests: Dr. Arthur L. Herbst, Department of Gynecology, Massachusetts General Hospital, Boston, Massachusetts 02114. *junior Faculty Fellow of the American Cancer Society.

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762

Herbst, Taft, and Robboy Am.

J.

April !, 1976 Obstet. Gvnecol.

Table I. Comparison of nuclear chromatin determination Nuclear chromatin(%)

Day of amniocentesis

Prenatal medicine

Results, gestation da_v

2 2 3

76 100 92

DES DES DES DES

Aborted. day 115 Aborted, day 86 Male, 161 Male, 156

4* 5

135 94 !JOt 148 I !2

Control DES Control Control DES

Male, !71 Male, 143 Male, 144 Female, 160 Female, 171

162 105 92

DES Control DES DES Control DES

Female, 164

88

6

24 25 2.5 28 30 32 33 35

!09:j:

!63 110§

Female, Female, Female, Female, Female,

157 165 165 170 156

Offspring status

None None Living and well Dead, intrauterine infection Living and well Living and well Living and well Living and well Dead one day after cesarean section for prolonged gestation I ~v;nrr ~nrl .... _ .. _W"Pll .. -·· Living and well Living and well Living and well Living and well Living and well ~·

,

*M. fascicularis.

tCells not interpretable on Day 82. :j:No fluid obtained on Day 54. §Cells not interpretable on Day 81.

Results

Fig. I. Sex chromatin body (arrow) in amniotic fluid cell.

(Original magnification x2400.)

pregnant uterus was palpated and held firmly while a sterile No. 22 gauge needle was introduced halfway between the top of the uterus and the symphysis pubis. Approximately 0.5 to 1 c.c. of amniotic fluid was then withdrawn. The amniotic fluid cells were collected on a membrane filter, fixed in 95 per cent ethyl alcohol, and stained by the Feulgen method. At least 100 cells were counted and evaluated for nuclear chromatin (Fig. 1).

The Barr body count of eight offspring that proved to be female varied between 24 and 35 per cent (Table I), while the count of five male offspring varied ben.veen 2 and 6 per cent. "'\mniocenteses performed on two additional animals yielded sex chromatin counts of 1 and 2 per cent, but the pregnancies were subsequently aborted, and no fetuses could be identified. During the course of amniocentesis, the bladder was occasionally entered if the needle was introduced too close to the symphysis pubis. Furthermore, it was found that amniotic fluid could not be obtained if the procedure was attempted before the tenth or eleventh week of gestation. The two specimens that were obtained before the ninetieth day of gestation (Days 8l and 82) contained cells that had degenerated and were not satisfactory for the analysis of nuclear chromatin.

Comment The data indicate that the determination of nuclear sex chromatin in amniotic fluid is a reliable technique for prenatal sex prediction in the subhuman primate. We had initially thought that specimens with 20 per cent or more nuclear chromatin bodies were consistent with a female gestation, and those with 4 per cent or fewer, with a male gestation, as in the human subject. 3 • 4 However, in this study two of the five male offspring had counts above 4 per cent (5 and 6 per cent, Table I).

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These values were well below the value for female offspring of 20 per cent, and the slight increase over the expected number of positive cells might have been due to contamination with maternal cells. Except for the introduction of intrauterine infection in one case, amniocentesis had no perceptible effect on the outcome of pregnancy in this small group of cases. In the two years prior to the present study, the M. mulatta breeding at the New England Regional Primate Research Center achieved 124 pregnancies with 15 losses (abortions or stillbirths). If the one case of preventable infection in the current study is excluded, there were two pregnancy losses among the 15 M.

Nuclear chromatin for prenatal sex prediction 763

mulattas who underwent amniocentesis, results roughly comparable to the experience of the breeding colony in general. We have no explanation for the unsatisfactory cell preparations that were obtained when amniocentesis was done on Days 81 and 82, since at this time the cells theoretically should le satisfactory for the determination of nuclear chr01r.atin. In view of these two unsatisfactory results, we have not subsequently initiated amniocentesis until after the ninetieth day of gestation. The authors wish to th mk Ms. Regina Dagata for her technical assistance in 1:arrying out this project.

REFERENCES l. Moore, K. L.: The Sex Chromatin, Philadelphia, 1966, W.

B. Saunders Company. 2. Amarose, A. P., Wallingford, A. ]., Jr., and Plotz, E. J.: Prediction of fetal sex from cytologic examination of amniotic fluid, N. Engl.]. Med. 275:715, 1966. 3. Nelson, W. 0.: Sex differences in human nuclei with

particular reference to the "Klinefelter syndrome," gonadal agenesis, and other types of hermaphroditism, Acta Endocrinol. 23: 222, 1956. 4. Greenblatt, R. B., Mateo de Acosta, 0., Vazquez, E., and Mullins, D. F., Jr.: Oral mucosal smears in detection of genetic sex, J. A.M. A. 161: 683, 1956.