Tohoku
J. Exp.
Med.,
1992, 166, 417-427
Pericentric Prenatal
Inversion Diagnosis
of Chromosome and
9 in
Infertility
SHIGEKI UEHARA, YUKIKO AKAI, YOICHI TAKEYAMA, TOSHIFUMITAKABAYASHI, KUNIHIROOKAMURAand AKIRA YAJIMA Department of Obstetrics and Gynecology, University School of Medicine, Sendai 980
Tohoku
UEHARA,S., AKAI,Y., TAKEYAMA, Y., TAKABAYASHI, T., OKAMURA, K. and YAJIMA,A. Pericentric Inversion of Chromosome 9 in Prenatal Diagnosis and Infertility. Tohoku J. Exp. Med., 1992, 166 (4), 417-427 In order to evaluate the relation between pericentric inversion of chromosome 9 (inv(9)) and clinical problems, the characteristics of inv(9) were investigated on the basis of chromosomal analyses of fetuses and infertile couples. The incidence of such inversion in fetuses with parents having an offspring who suffered from various clinical problems was significantly higher than the basic incidence obtained in fetuses karyotyped by reason of advanced maternal age. In the chromosomal examination of the parents whose fetuses were diagnosed as inv(9), it was revealed that either parent might be the carrier. Furthermore, in the inv(9) carrying fetuses, the number of females was significantly greater than that of males. Analysis of infertile couples revealed that the incidence of such inversion in males was significantly higher than the basic incidence mentioned above. Moreover, infertile couples with an inv(9) carrier showed a significantly higher incidence of intrauterine fetal death, compared with infertile couples with a translocation carrier or those in which the etiology was unknown. These results indicate that inv(9) may often cause clinical problems in offspring of the carrier and infertility with unknown mechanisms related to sex. pericentric inversion of chromosome 9 ; prenatal diagnosis ; infertility ; repeated abortion ; sexual difference
Pericentric inversion is one type of chromosomal rearrangement, the detection of which has been greatly facilitated by the development of banding techniques. The site of such inversion of chromosome 9 (inv(9)) is now well known to be heterochromatin. In comparison with pericentric inversion of other chromosomes, pericentric inversion of chromosome 9, the incidence of which has been reported to be about 1% in the general population (Ferguson-Smith 1974; Buckton et al. 1976; Mueller and Klinger 1976), has been categorized as a minor chromosomal rearrangement which does not correlate with abnormal phenotypes Received
December
6, 1991;
revision
accepted
for publication
February
22, 1992.
Address for reprints : Shigeki Uehara, M.D., Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai 980, Japan. 417
418
S. Uehara
et al.
(Wahrman et al. 1972; DeLa Chapelle et al. 1974; Tibiletti et al. 1981). Therefore, most clinicians have considered inv(9) to be a benign chromosomal polymorphism. However, in clinical work, we have been impressed by the fact that inv(9) often shows a correlation with congenital clinical problems or reproductive failure. In addition, some previous reports have indicated that inv(9) is related to clinical problems. Therefore, in order to clarify if inv(9) is harmful or benign, we analyzed the incidence of inv(9) in fetuses who had undergone karyotyping due to various reasons and in our patients who had themselves been examined due to infertility. METHODS Subjects Chromosomal analyses were carried out at the Department of Obstetrics and Gynecology of Tohoku University Hospital from 1981 through 1991. The subjects consisted of fetuses with high risk of chromosomal aberration due to various reasons and infertile couples. They were Japanese living in northeastern Japan. Fetal karyotyping was performed in 1174 subjects examined for reasons of advanced maternal age (more than 35 years of age), a history of birth of offspring with various kinds of congenital abnormalities, abnormal findings in ultrasound examinations of the fetus and so on (Table 2). The fetal karyotypings were carried out by means of cultured fetal cells obtained by amniocentesis or fetal blood samplings. Amniocentesis was usually performed after the 15th gestational week, and fetal blood sampling was done after the 19th week. When inv(9) was detected in fetal cells, the parents were also karyotyped using peripheral blood samples and follow-up was carried out with regard to the general condition including phenotype after birth. Two hundred and twelve infertile couples were analyzed. They had experienced two or more fetal losses (spontaneous abortions, intrauterine fetal death, perinatal death). Their peripheral blood was sampled with a heparinized syringe and subjected to karyotyping. Method of karyotyping Chromosomes were analyzed after amniocyte culturing or blood cell culturing. Amniocytes were cultured in Ham F-10 media containing 10% fetal calf serum. The culture medium used for blood cells was RPM11640, also containing the same content of fetal calf serum. The cultures were always carried out in 5% CO2 in air at 3TC for an adequate number of days. Four hours after Colcemid treatment, culture cells were harvested, treated in hypotonic solution (0.075 M KC1 solution) and fixed in Carnoy's solution. All materials were analyzed by G-banding. Statistical analyses The x2 test with Yates' correction or the test of goodness of fit was applied for statistical analyses of incidences and sexual differences in this study.
RESULTS In this study analyses for karyotype determination were solely based on G-banding. The existence of pericentric inversion of chromosome 9 (inv(9)) was easily detected based on the location of heterochromatin. All inv(9)s detected in this study were between p1 and ql. However, further specification of the
Inv(9) in Prenatal Diagnosisand Infertility
419
breakpoints by band formation analysis was not possible. This limitation of the inv(9) analysis based on G-banding may be the same as that found by most investigators. Inv(9) in prenatal diagnosis Table 1 shows the profiles of all inv(9) carriers detected in the fetuses. The total number of fetal inv(9)s was 25, including 19 fetuses diagnosed by amniocyte cultures and 6 fetuses diagnosed by blood cell cultures. The follow-up showed all the 19 fetuses diagnosed by amniocyte analyses to be phenotypically normal after live birth. They grew without any severe medical problems. Abnormality in each of the 6 fetuses diagnosed as inv(9) by means of blood cell analyses was indicated by prenatal ultrasound examination before sampling. Four of those 6 fetuses were inv(9) carriers without any other chromosomal aberrations, but showing ultrasonographic abnormality, such as a fetal ovarian cyst, oligohydramnios or polyhydramnios. In the case of the fetus with an ovarian cyst, the cyst was surgically removed, and the infant grew uneventfully. In the two cases of oligohydramnios, the infants grew uneventfully after normal birth. In the case of polyhydramnios, there was a complication of tracheal atresia and the neonate died on the day of birth. The fetus who was diagnosed as 45, X, inv(9) had non-immune hydrops fetalis and cystic hygroma, and was artificially aborted at the 20th gestational week. The fetus diagnosed as 47, XY, + 13, inv(9) had a congenital heart disease and a single cord artery, and was also aborted at the 34th gestational week. The incidences of inv(9) detected by fetal karyotyping because of various reasons are presented in Table 2. The incidence of inv(9) detected in fetuses of older mothers (more than 35 years of age) was 1.3% (7/549). This value was thought to reflect the basic incidence in the general population of northeastern Japan, because it was not affected by any biases except for maternal age. The incidence of the group having a history of birth of offspring with various congenital abnormalities, such as Down's syndrome, cerebral palsy, neonatal death, 18 trisomy, and 13 trisomy or malformation, was 3.7% (10/270). This value was significaltly higher than the basic incidence (p
J. Exp.
Med.,
1992, 166, 417-427
Pericentric Prenatal
Inversion Diagnosis
of Chromosome and
9 in
Infertility
SHIGEKI UEHARA, YUKIKO AKAI, YOICHI TAKEYAMA, TOSHIFUMITAKABAYASHI, KUNIHIROOKAMURAand AKIRA YAJIMA Department of Obstetrics and Gynecology, University School of Medicine, Sendai 980
Tohoku
UEHARA,S., AKAI,Y., TAKEYAMA, Y., TAKABAYASHI, T., OKAMURA, K. and YAJIMA,A. Pericentric Inversion of Chromosome 9 in Prenatal Diagnosis and Infertility. Tohoku J. Exp. Med., 1992, 166 (4), 417-427 In order to evaluate the relation between pericentric inversion of chromosome 9 (inv(9)) and clinical problems, the characteristics of inv(9) were investigated on the basis of chromosomal analyses of fetuses and infertile couples. The incidence of such inversion in fetuses with parents having an offspring who suffered from various clinical problems was significantly higher than the basic incidence obtained in fetuses karyotyped by reason of advanced maternal age. In the chromosomal examination of the parents whose fetuses were diagnosed as inv(9), it was revealed that either parent might be the carrier. Furthermore, in the inv(9) carrying fetuses, the number of females was significantly greater than that of males. Analysis of infertile couples revealed that the incidence of such inversion in males was significantly higher than the basic incidence mentioned above. Moreover, infertile couples with an inv(9) carrier showed a significantly higher incidence of intrauterine fetal death, compared with infertile couples with a translocation carrier or those in which the etiology was unknown. These results indicate that inv(9) may often cause clinical problems in offspring of the carrier and infertility with unknown mechanisms related to sex. pericentric inversion of chromosome 9 ; prenatal diagnosis ; infertility ; repeated abortion ; sexual difference
Pericentric inversion is one type of chromosomal rearrangement, the detection of which has been greatly facilitated by the development of banding techniques. The site of such inversion of chromosome 9 (inv(9)) is now well known to be heterochromatin. In comparison with pericentric inversion of other chromosomes, pericentric inversion of chromosome 9, the incidence of which has been reported to be about 1% in the general population (Ferguson-Smith 1974; Buckton et al. 1976; Mueller and Klinger 1976), has been categorized as a minor chromosomal rearrangement which does not correlate with abnormal phenotypes Received
December
6, 1991;
revision
accepted
for publication
February
22, 1992.
Address for reprints : Shigeki Uehara, M.D., Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai 980, Japan. 417
418
S. Uehara
et al.
(Wahrman et al. 1972; DeLa Chapelle et al. 1974; Tibiletti et al. 1981). Therefore, most clinicians have considered inv(9) to be a benign chromosomal polymorphism. However, in clinical work, we have been impressed by the fact that inv(9) often shows a correlation with congenital clinical problems or reproductive failure. In addition, some previous reports have indicated that inv(9) is related to clinical problems. Therefore, in order to clarify if inv(9) is harmful or benign, we analyzed the incidence of inv(9) in fetuses who had undergone karyotyping due to various reasons and in our patients who had themselves been examined due to infertility. METHODS Subjects Chromosomal analyses were carried out at the Department of Obstetrics and Gynecology of Tohoku University Hospital from 1981 through 1991. The subjects consisted of fetuses with high risk of chromosomal aberration due to various reasons and infertile couples. They were Japanese living in northeastern Japan. Fetal karyotyping was performed in 1174 subjects examined for reasons of advanced maternal age (more than 35 years of age), a history of birth of offspring with various kinds of congenital abnormalities, abnormal findings in ultrasound examinations of the fetus and so on (Table 2). The fetal karyotypings were carried out by means of cultured fetal cells obtained by amniocentesis or fetal blood samplings. Amniocentesis was usually performed after the 15th gestational week, and fetal blood sampling was done after the 19th week. When inv(9) was detected in fetal cells, the parents were also karyotyped using peripheral blood samples and follow-up was carried out with regard to the general condition including phenotype after birth. Two hundred and twelve infertile couples were analyzed. They had experienced two or more fetal losses (spontaneous abortions, intrauterine fetal death, perinatal death). Their peripheral blood was sampled with a heparinized syringe and subjected to karyotyping. Method of karyotyping Chromosomes were analyzed after amniocyte culturing or blood cell culturing. Amniocytes were cultured in Ham F-10 media containing 10% fetal calf serum. The culture medium used for blood cells was RPM11640, also containing the same content of fetal calf serum. The cultures were always carried out in 5% CO2 in air at 3TC for an adequate number of days. Four hours after Colcemid treatment, culture cells were harvested, treated in hypotonic solution (0.075 M KC1 solution) and fixed in Carnoy's solution. All materials were analyzed by G-banding. Statistical analyses The x2 test with Yates' correction or the test of goodness of fit was applied for statistical analyses of incidences and sexual differences in this study.
RESULTS In this study analyses for karyotype determination were solely based on G-banding. The existence of pericentric inversion of chromosome 9 (inv(9)) was easily detected based on the location of heterochromatin. All inv(9)s detected in this study were between p1 and ql. However, further specification of the
Inv(9) in Prenatal Diagnosisand Infertility
419
breakpoints by band formation analysis was not possible. This limitation of the inv(9) analysis based on G-banding may be the same as that found by most investigators. Inv(9) in prenatal diagnosis Table 1 shows the profiles of all inv(9) carriers detected in the fetuses. The total number of fetal inv(9)s was 25, including 19 fetuses diagnosed by amniocyte cultures and 6 fetuses diagnosed by blood cell cultures. The follow-up showed all the 19 fetuses diagnosed by amniocyte analyses to be phenotypically normal after live birth. They grew without any severe medical problems. Abnormality in each of the 6 fetuses diagnosed as inv(9) by means of blood cell analyses was indicated by prenatal ultrasound examination before sampling. Four of those 6 fetuses were inv(9) carriers without any other chromosomal aberrations, but showing ultrasonographic abnormality, such as a fetal ovarian cyst, oligohydramnios or polyhydramnios. In the case of the fetus with an ovarian cyst, the cyst was surgically removed, and the infant grew uneventfully. In the two cases of oligohydramnios, the infants grew uneventfully after normal birth. In the case of polyhydramnios, there was a complication of tracheal atresia and the neonate died on the day of birth. The fetus who was diagnosed as 45, X, inv(9) had non-immune hydrops fetalis and cystic hygroma, and was artificially aborted at the 20th gestational week. The fetus diagnosed as 47, XY, + 13, inv(9) had a congenital heart disease and a single cord artery, and was also aborted at the 34th gestational week. The incidences of inv(9) detected by fetal karyotyping because of various reasons are presented in Table 2. The incidence of inv(9) detected in fetuses of older mothers (more than 35 years of age) was 1.3% (7/549). This value was thought to reflect the basic incidence in the general population of northeastern Japan, because it was not affected by any biases except for maternal age. The incidence of the group having a history of birth of offspring with various congenital abnormalities, such as Down's syndrome, cerebral palsy, neonatal death, 18 trisomy, and 13 trisomy or malformation, was 3.7% (10/270). This value was significaltly higher than the basic incidence (p
