Hum. Genet. 36, 265--269 (1977) t~ by Springer-Verlag 1977
Determination of Prenatal Sex Ratio in Man Masaharu Yamamoto*, Takashi Ito, and Gen-ichi Watanabe Department of Hygiene and Preventive Medicine, Niigata University School of Medicine, Niigata City, 951, Japan
Summary. The sex of a conceptus at the early embryonic stage was diagnosed in I000 induced abortions. Specimens were obtained from women who terminated their pregnancies within 12 menstrual weeks on socio-economic indications. By making use of the triple checking procedures, such as the karyotypic analysis of Giemsa-stained slides, the fluorescent Y chromosome analysis and the Y-body test in interphase nuclei, the sex ratio was determined as 106.6 (516 males/484 females). The sex distribution in the chromosomally normal embryos was 481 in males to 448 in females; that gave the ratio of 107.4. A slight excess of males was already present at this stage of pregnancy. When the ratios were calculated in relation to the maternal age, the lower sex ratio was noted for embryos born to mothers over 30 years. Taking a Ybearing embryo as male, the ratio in 71 chromosomally aberrant embryos was 97.2 (35 males/36 females). The sex ratio in the cases of chromosome abnormalities was not statistically different from that of the normal embryos.
Introduction It is of interest to know whether the sex ratio at very early stages of pregnancy is different from the ratio at birth. Several investigators reported in induced abortions a higher proportion of males by using the sex chromatin a n d / o r the karyotypic analysis (Schultze, 1961; Momoli and Volet, 1962; Serf and Ismajovich, 1963; Csordfis et al., 1963). By the same methods, however, a lower sex ratio has been noted (Makino, 1968; Sasaki et al., 1971). It is generally accepted that diagnostic errors particularly in the sex chromatin test could occur under various technical conditions, such as freshness of specimens, fixation and staining (Mikamo, 1969). By employing the karyotypic analysis of Giemsa-stained slide, the fluorescent Y chromosome analysis and the Y body test in interphase nuclei rather than the sex chromatin test alone, we tried to establish the sex ratio at an early embryonic stage from 1000 cases of induced abortions. *
To whom offprint requests should be sent
266
M. Yamamoto et al.
Materials and Methods Materials consisting of the human embryos and the chorionic villi were collected from private hospitals in Niigata City. All the cases were terminated on socio-economic indications within 12 weeks after the first day of the last menstruation. Induced abortions due to medical reasons were strictly excluded from the present investigation in an attempt to minimize possible biasing effects on the sex ratio. Specimens of an embryo proper and the chorionic villi were processed for the chromosome analysis by means of the direct method. Details of the method employed for chromosome preparations have been described before (Yamamoto et al., 1975). In most instances, the cytogenetic diagnosis was made in tissue of the chorionic villi. If no embryo was found, the embryonic chromosomal constitution was established only by the analysis of the ehorionic villi, since it was previously confirmed that the chromosomal pattern of extraembryonic specimens properly reflects the karyotype of the embryo (Yamamoto et al., 1975). Ten metaphase plates produced by the conventional Giemsa staining were selected for the study of chromosome counts and structure, and the sex determination was undertaken not only by the visual karyotyping under the microscope, but also by the examination of the Y chromosome in the photographed metaphase cells. For the analysis of Y body in interphase nuclei and the fluorescent Y chromosome, air-dried slides were stained in 0.5% aqueous solution of quinacrine hydrochloride for 5 min and washed in tap water. Then, slides were mounted in phosphate buffer solution at pH 6.8 and examined with an Olympus fluorescence microscope. In order to examine the presence of the Y body and the fluorescent Y chromosome, a total of 50 to 100 interphase nuclei and a few metaphase plates were analysed respectively by one of the authors (T.I.) without knowing the results of chromosomal sex. Then, the comparison was made between the results of Giemsa stained slides and fluorescent ones. Of I000 cases analysed, there were 43 cases with controversial results. Discrepancies in diagnosis among the three different methods were encountered mostly due to the errors of Y-body test by the presence of a brightly fluorescent part of autosomes and to the misjudgement of the Y chromosome with longer short arms as a chromosome No. 18. But we settled the true sex when the presence of the fluorescent Y chromosome was confirmed and the finding was not generally in contradiction to those of the Y-body test or of the karyotypie analysis in Giemsa-stained slides. With such technical prudence in checking, the embryo bearing the Y chromosome and the Y body was considered male. The sex of chromosomally aberrant embryos was also classified in the same manner.
Results O f 1197 cases collected d u r i n g the p e r i o d f r o m A u g u s t 1973 to J a n u a r y 1976, the c h r o m o s o m e analysis was successful in 1000 cases (83.5%). The analysis was d eri v ed f r o m 804 cases o f the c h o r i o n i c villi alone and 196 cases of e m b r y o s p r o p e r with the c h o r i o n i c villi. As to the m a t e r n a l age of induced a b o r t i o n s , the d i s t r i b u t i o n r a n g e d f r o m 17 to 46 years with a m e a n of 28 years. Th e m e a n g es t at i o n al age was 8.4 ± 1.53 ( m e a n ± S D ) weeks after the first d ay of the last m e n s t r u a t i o n . A m o n g 1000 cases e x a m i n e d , 71 (7.1%) were f o u n d to have c h r o m o s o m e a n o m a l i e s . Details o f the c h r o m o s o m e a b n o r m a l i t i e s are n o t described here, since the results of cytogenetic studies have already been reported ( Y a m a m o t o et al., 1975; 1976). I n f o r m a t i o n on the cytogenetically d e t e r m i n e d sex was available in all cases of i n d u ced a b o r t i o n s . T h e sex d i s t r i b u t i o n in the c h r o m o s o m a l l y n o r m a l e m b r y o s was 481 in males to 448 in females; that gave the ratio of 107.4. T a k i n g a Y-
Determination of Prenatal Sex Ratio in Man
267
Table 1. Sex-ratio changes of induced abortions in relation to the maternal and the gestational agesa Maternal age (years)
Gestational age (weeks) 5--8
9--12
Total
Under 30
b114.9 (154/134) c 80.0 (8/ 10) d112.5 (162/144)
120.0 (156/130) 120.0 (6/ 5) 120.0 (162/135)
117.4 (310/264) 93.3 (14/ 15) 116.1 (324/279)]
30 or over
bl01.0 (100/ 99) Cl00.0 (12/ 12) d100.9 (112/111)
94.7 (54/ 57) 112.5 (9/ 8) 96.9 (63/ 65)
98.7 (154/156)}, 105.0 (21/ 20)| 99.4 (175/176)J
Total
8109.0 (254/233) c 90.9 (20/ 22) d107.5 (274/255)
112.3 (210/187) 115.4 (15/ 13) 112.5 (225/200)
110.5 (464/420)/ 100.0 (35/ 35)]** 109.7 (499/455) ***
/
a Forty-six cases were excluded from scoring, since the maternal and/or gestational ages were not disclosed b The sex ratio of chromosomally normal embryos c~The sex ratio of chromosomally abnormal embryos; an embryo bearing Y chromosome and Y body was considered male a The sex ratio as a whole (b + c) ( ) Number of embryos examined * X2-~-1.335, P
Determination of Prenatal Sex Ratio in Man Masaharu Yamamoto*, Takashi Ito, and Gen-ichi Watanabe Department of Hygiene and Preventive Medicine, Niigata University School of Medicine, Niigata City, 951, Japan
Summary. The sex of a conceptus at the early embryonic stage was diagnosed in I000 induced abortions. Specimens were obtained from women who terminated their pregnancies within 12 menstrual weeks on socio-economic indications. By making use of the triple checking procedures, such as the karyotypic analysis of Giemsa-stained slides, the fluorescent Y chromosome analysis and the Y-body test in interphase nuclei, the sex ratio was determined as 106.6 (516 males/484 females). The sex distribution in the chromosomally normal embryos was 481 in males to 448 in females; that gave the ratio of 107.4. A slight excess of males was already present at this stage of pregnancy. When the ratios were calculated in relation to the maternal age, the lower sex ratio was noted for embryos born to mothers over 30 years. Taking a Ybearing embryo as male, the ratio in 71 chromosomally aberrant embryos was 97.2 (35 males/36 females). The sex ratio in the cases of chromosome abnormalities was not statistically different from that of the normal embryos.
Introduction It is of interest to know whether the sex ratio at very early stages of pregnancy is different from the ratio at birth. Several investigators reported in induced abortions a higher proportion of males by using the sex chromatin a n d / o r the karyotypic analysis (Schultze, 1961; Momoli and Volet, 1962; Serf and Ismajovich, 1963; Csordfis et al., 1963). By the same methods, however, a lower sex ratio has been noted (Makino, 1968; Sasaki et al., 1971). It is generally accepted that diagnostic errors particularly in the sex chromatin test could occur under various technical conditions, such as freshness of specimens, fixation and staining (Mikamo, 1969). By employing the karyotypic analysis of Giemsa-stained slide, the fluorescent Y chromosome analysis and the Y body test in interphase nuclei rather than the sex chromatin test alone, we tried to establish the sex ratio at an early embryonic stage from 1000 cases of induced abortions. *
To whom offprint requests should be sent
266
M. Yamamoto et al.
Materials and Methods Materials consisting of the human embryos and the chorionic villi were collected from private hospitals in Niigata City. All the cases were terminated on socio-economic indications within 12 weeks after the first day of the last menstruation. Induced abortions due to medical reasons were strictly excluded from the present investigation in an attempt to minimize possible biasing effects on the sex ratio. Specimens of an embryo proper and the chorionic villi were processed for the chromosome analysis by means of the direct method. Details of the method employed for chromosome preparations have been described before (Yamamoto et al., 1975). In most instances, the cytogenetic diagnosis was made in tissue of the chorionic villi. If no embryo was found, the embryonic chromosomal constitution was established only by the analysis of the ehorionic villi, since it was previously confirmed that the chromosomal pattern of extraembryonic specimens properly reflects the karyotype of the embryo (Yamamoto et al., 1975). Ten metaphase plates produced by the conventional Giemsa staining were selected for the study of chromosome counts and structure, and the sex determination was undertaken not only by the visual karyotyping under the microscope, but also by the examination of the Y chromosome in the photographed metaphase cells. For the analysis of Y body in interphase nuclei and the fluorescent Y chromosome, air-dried slides were stained in 0.5% aqueous solution of quinacrine hydrochloride for 5 min and washed in tap water. Then, slides were mounted in phosphate buffer solution at pH 6.8 and examined with an Olympus fluorescence microscope. In order to examine the presence of the Y body and the fluorescent Y chromosome, a total of 50 to 100 interphase nuclei and a few metaphase plates were analysed respectively by one of the authors (T.I.) without knowing the results of chromosomal sex. Then, the comparison was made between the results of Giemsa stained slides and fluorescent ones. Of I000 cases analysed, there were 43 cases with controversial results. Discrepancies in diagnosis among the three different methods were encountered mostly due to the errors of Y-body test by the presence of a brightly fluorescent part of autosomes and to the misjudgement of the Y chromosome with longer short arms as a chromosome No. 18. But we settled the true sex when the presence of the fluorescent Y chromosome was confirmed and the finding was not generally in contradiction to those of the Y-body test or of the karyotypie analysis in Giemsa-stained slides. With such technical prudence in checking, the embryo bearing the Y chromosome and the Y body was considered male. The sex of chromosomally aberrant embryos was also classified in the same manner.
Results O f 1197 cases collected d u r i n g the p e r i o d f r o m A u g u s t 1973 to J a n u a r y 1976, the c h r o m o s o m e analysis was successful in 1000 cases (83.5%). The analysis was d eri v ed f r o m 804 cases o f the c h o r i o n i c villi alone and 196 cases of e m b r y o s p r o p e r with the c h o r i o n i c villi. As to the m a t e r n a l age of induced a b o r t i o n s , the d i s t r i b u t i o n r a n g e d f r o m 17 to 46 years with a m e a n of 28 years. Th e m e a n g es t at i o n al age was 8.4 ± 1.53 ( m e a n ± S D ) weeks after the first d ay of the last m e n s t r u a t i o n . A m o n g 1000 cases e x a m i n e d , 71 (7.1%) were f o u n d to have c h r o m o s o m e a n o m a l i e s . Details o f the c h r o m o s o m e a b n o r m a l i t i e s are n o t described here, since the results of cytogenetic studies have already been reported ( Y a m a m o t o et al., 1975; 1976). I n f o r m a t i o n on the cytogenetically d e t e r m i n e d sex was available in all cases of i n d u ced a b o r t i o n s . T h e sex d i s t r i b u t i o n in the c h r o m o s o m a l l y n o r m a l e m b r y o s was 481 in males to 448 in females; that gave the ratio of 107.4. T a k i n g a Y-
Determination of Prenatal Sex Ratio in Man
267
Table 1. Sex-ratio changes of induced abortions in relation to the maternal and the gestational agesa Maternal age (years)
Gestational age (weeks) 5--8
9--12
Total
Under 30
b114.9 (154/134) c 80.0 (8/ 10) d112.5 (162/144)
120.0 (156/130) 120.0 (6/ 5) 120.0 (162/135)
117.4 (310/264) 93.3 (14/ 15) 116.1 (324/279)]
30 or over
bl01.0 (100/ 99) Cl00.0 (12/ 12) d100.9 (112/111)
94.7 (54/ 57) 112.5 (9/ 8) 96.9 (63/ 65)
98.7 (154/156)}, 105.0 (21/ 20)| 99.4 (175/176)J
Total
8109.0 (254/233) c 90.9 (20/ 22) d107.5 (274/255)
112.3 (210/187) 115.4 (15/ 13) 112.5 (225/200)
110.5 (464/420)/ 100.0 (35/ 35)]** 109.7 (499/455) ***
/
a Forty-six cases were excluded from scoring, since the maternal and/or gestational ages were not disclosed b The sex ratio of chromosomally normal embryos c~The sex ratio of chromosomally abnormal embryos; an embryo bearing Y chromosome and Y body was considered male a The sex ratio as a whole (b + c) ( ) Number of embryos examined * X2-~-1.335, P
