Abnormal Embryonic Karyotype and Recurrent Miscarriage
Abnormal Embryonic Karyotype and Recurrent Miscarriage
This is the first study to show the prevalence of the abnormal embryonic karyotype as a cause of recurrent miscarriage. In the present study, the prevalence was 41.1%. An abnormal embryonic karyotype was detected as the commonest cause of recurrent miscarriage. The subjects in the present study can be regarded as representative Japanese patients with recurrent miscarriage because patients wishing for a second opinion after undergoing an examination at another hospital or who requested specific treatment were excluded; thus, a selection bias in the distribution of each cause was avoided.
The prevalence of abnormal embryonic karyotypes was reported to be 57% (Stern et al., 1996), 29% (Carp et al., 2001), 46% (Stephenson et al., 2002), 25.4% (Sullivan et al., 2004) and 55.1% (present study). The difference depends on women's age and the number of miscarriages. The abnormal rate increases as the women's age increases and the previous number of miscarriages decreases (Ogasawara et al., 2000).
Several reports have suggested that the abnormal embryonic karyotype predicts subsequent live birth (Ogasawara et al., 2000; Carp et al., 2001). The cumulative live birth rate (71.2%) in women with miscarriage caused by an abnormal embryonic karyotype was found to be higher than that (52.5%) in women with recurrent miscarriage of truly unexplained cause. Patients with abnormal and normal chromosomes were found to have repeat miscarriages with abnormal and normal chromosomes, respectively. This was consistent with the results of our previous study conducted with a small sample size (Mizutani et al., 2011). Karyotype analysis should be performed at least once, because the abnormal embryonic karyotype tends to repeat itself and is also a predictor of good prognosis.
Patients with recurrent miscarriage caused by the abnormal embryonic karyotype might have gene mutations associated with aneuploidy such as SYCP3. Unexplained reasons may cause the miscarriage of a euploid embryo and may continue to cause further miscarriages. SYCP3 mutations in women were found to generate an aberrant synaptonemal complex in a dominant-negative manner and to contribute to abnormal chromosomal behavior that could potentially lead to recurrent miscarriage (Bolor et al., 2009). We found no clinical significance in the routine examination of the SYCP3 mutation because only one benign mutation was ascertained in 101 patients (Mizutani et al., 2011). However, some gene mutations, such as MLH1, may influence aneuploidy because a double trisomy can be detected in recurrent but not in sporadic, miscarriage patients (Edelmann et al., 1996; Ogasawara et al., 2000). Preimplantation screening (PGS) is performed worldwide, though it is unclear whether PGS can improve the live birth rate in patients with recurrent miscarriage (Harper et al., 2010). The cumulative live birth rate in patients with an abnormal embryonic karyotype was higher because such patients could have a euploid embryo. PGS may be useful in specific patients affected by a candidate gene mutation.
According to previous reports, about half of the women at research centers are determined as having recurrent miscarriage of unexplained cause despite receiving conventional examinations to determine the cause. There have been some reports on the distribution of the causes at individual centers. The prevalences of aPL, abnormal chromosome, uterine anomaly, endocrine abnormality (DM, both PCO morphology and mid-follicular serum LH level > 10 IU/L) and unexplained causes were 14, 3.6, 1.8, 6.8 and 73.8%, respectively, in Clifford's study (mean age, 32.9 years; median miscarriages, 4; Clifford et al., 1994). The prevalences of aPL, abnormal chromosome, uterine anomaly, endocrine abnormality and unexplained causes were 20, 3.5, 16, 20 and 42.6%, respectively, in Stephenson's study (Stephenson, 1996). These previous studies did not distinguish between truly unexplained causes and an abnormal embryonic karyotype. The prevalence of unexplained causes was 69% among our patients (Fig. 1, Sugiura-Ogasawara et al., 2010). The differences in the distribution of causes at each center may depend on age, number of previous miscarriages and methods of diagnosis for each factor. We compared the distribution of the causes between the 482 couples in the present study and 1676 couples of a previous study among whom the aborted conceptuses of all the patients were not analyzed (Sugiura-Ogasawara et al., 2010, Fig. 1). The true percentage of subjects with recurrent miscarriage of unexplained cause was limited to 24.5%. Moreover, 482 patients in our present study experienced further miscarriages after undergoing a systematic examination. The patients with APS miscarried despite anticoagulant therapy. The prevalences of abnormal chromosomes in either partner and uterine anomaly were higher than the prevalences in previous study. This finding suggests that the prognosis of abnormal chromosomes and uterine anomaly is poor.
The reported live birth rate in women with APS treated with low-dose aspirin plus heparin is 70–80% (Cowchock et al., 1992; Rai et al., 1997). The cumulative live birth rate in this group in the present study was relatively low, because women with APS gave up after the first treatment and failure. The reported prevalence range, in review articles, of APS is 5–15% (Branch et al., 2010). Several reports have indicated that about 10–15% of women with recurrent miscarriage are diagnosed with APS (Clifford et al., 1994; Rai et al., 1995; Yetman and Kutteh, 1996). However, it is unclear whether the aPLs persisted according to International Criteria (Miyakis et al., 2006). The prevalence of APS according to International Criteria was found to be 2.5% in the present study, presumably because the figure represents the prevalence after one treatment failure. The single positive rate of aPLs was 10.7% and the recurrent positive rate was 4.5% in our previous study (Sugiura-Ogasawara et al., 2008). There are many methods used for the detection of aPLs. However, there are limited reports on which method might be most suitable for prediction of recurrent miscarriage or intrauterine fetal death. The positive rate might be large if methods with a large false-positive rate were used. The prevalence of 'true' APS might be <5%, although it depends on the age of the women comprising the study population and the method used for the detection of APS.
The frequency of major congenital uterine anomalies has been reported to be between 3.2 and 6.9% in women with a history of recurrent miscarriage, the variation largely depending on the method of selection and the criteria used for the diagnosis (Sugiura-Ogasawara et al., 2011). Uterine anomalies are encountered in miscarriages associated with euploidy (Sugiura-Ogasawara et al., 2010). However, the prognosis in these cases is better than that in patients presenting with recurrent miscarriage of unexplained cause.
Recently, the mean age of the population has been increasing year by year, and the proportion of women in their 40s has been increasing in Japan. APS and uterine anomalies were found to be rare in subjects with secondary recurrent miscarriage and women over 40 years old in the present study. This should be borne in mind before evaluation of the screening tests.
The prevalence of recurrent miscarriage of truly unexplained cause, in whom the embryonic karyotype was ascertained to be normal, was found to be 24.5% in this study. Kaandorp et al. (2010) demonstrated, based on the results of an RCT, that aspirin plus heparin therapy has no beneficial effect in patients with two or more miscarriages. Further studies are needed in women with recurrent miscarriage of truly unexplained cause, after excluding cases with an abnormal embryonic karyotype, to confirm the conclusion.
The prevalence of each cause differs among centers, depending on the background of the patients, such as the mean age and previous number of miscarriages of the women in the studied population and the selected method and criteria for the diagnosis.
Subjects with thrombophilia, infection, fibroid or deficiency of progesterone were excluded from the analysis, because the contribution of these factors to recurrent miscarriage has not yet been established (Branch et al., 2010).
Although the standard G-band technique is the gold standard for evaluating chromosomal abnormalities, it has several limitations, including the need for tissue culture and the possibility of maternal cell contamination. Additional analysis, such as by comparative genomic hybridization (CGH), could not be performed in the present study. Further abnormalities could be detectable and also contamination with maternal tissue could be distinguishable by CGH. A recent microarray CGH indicated that about 80% of sporadic spontaneous abortions were caused by an abnormal embryonic karyotype (Shimokawa et al., 2006). Thus, if the prevalence is similar in women with recurrent miscarring and they have no predisposing factors, the incidence of the abnormal embryonic karyotype as a cause of recurrent miscarriage can be estimated to be (0.8) in patients with n consecutive miscarriages.About 51% of patients caused by an abnormal embryonic karyotype can be expected to exist in patients with three miscarriages occasionally. The incidence might increase if the candidate gene such as SYCP3 or MLH affect.
Among subjects with abnormal chromosomes in the present study, 26.8% (19/71) carried embryos that were normal or balanced (Table I). It is possible that CGH would have revealed lack of balance, because 40% of 42 balanced translocation carriers as assessed by cytogenetic analysis were found to show a loss of balance as assessed by CGH (De Gregori et al., 2007).
Discussion
This is the first study to show the prevalence of the abnormal embryonic karyotype as a cause of recurrent miscarriage. In the present study, the prevalence was 41.1%. An abnormal embryonic karyotype was detected as the commonest cause of recurrent miscarriage. The subjects in the present study can be regarded as representative Japanese patients with recurrent miscarriage because patients wishing for a second opinion after undergoing an examination at another hospital or who requested specific treatment were excluded; thus, a selection bias in the distribution of each cause was avoided.
The prevalence of abnormal embryonic karyotypes was reported to be 57% (Stern et al., 1996), 29% (Carp et al., 2001), 46% (Stephenson et al., 2002), 25.4% (Sullivan et al., 2004) and 55.1% (present study). The difference depends on women's age and the number of miscarriages. The abnormal rate increases as the women's age increases and the previous number of miscarriages decreases (Ogasawara et al., 2000).
Several reports have suggested that the abnormal embryonic karyotype predicts subsequent live birth (Ogasawara et al., 2000; Carp et al., 2001). The cumulative live birth rate (71.2%) in women with miscarriage caused by an abnormal embryonic karyotype was found to be higher than that (52.5%) in women with recurrent miscarriage of truly unexplained cause. Patients with abnormal and normal chromosomes were found to have repeat miscarriages with abnormal and normal chromosomes, respectively. This was consistent with the results of our previous study conducted with a small sample size (Mizutani et al., 2011). Karyotype analysis should be performed at least once, because the abnormal embryonic karyotype tends to repeat itself and is also a predictor of good prognosis.
Patients with recurrent miscarriage caused by the abnormal embryonic karyotype might have gene mutations associated with aneuploidy such as SYCP3. Unexplained reasons may cause the miscarriage of a euploid embryo and may continue to cause further miscarriages. SYCP3 mutations in women were found to generate an aberrant synaptonemal complex in a dominant-negative manner and to contribute to abnormal chromosomal behavior that could potentially lead to recurrent miscarriage (Bolor et al., 2009). We found no clinical significance in the routine examination of the SYCP3 mutation because only one benign mutation was ascertained in 101 patients (Mizutani et al., 2011). However, some gene mutations, such as MLH1, may influence aneuploidy because a double trisomy can be detected in recurrent but not in sporadic, miscarriage patients (Edelmann et al., 1996; Ogasawara et al., 2000). Preimplantation screening (PGS) is performed worldwide, though it is unclear whether PGS can improve the live birth rate in patients with recurrent miscarriage (Harper et al., 2010). The cumulative live birth rate in patients with an abnormal embryonic karyotype was higher because such patients could have a euploid embryo. PGS may be useful in specific patients affected by a candidate gene mutation.
According to previous reports, about half of the women at research centers are determined as having recurrent miscarriage of unexplained cause despite receiving conventional examinations to determine the cause. There have been some reports on the distribution of the causes at individual centers. The prevalences of aPL, abnormal chromosome, uterine anomaly, endocrine abnormality (DM, both PCO morphology and mid-follicular serum LH level > 10 IU/L) and unexplained causes were 14, 3.6, 1.8, 6.8 and 73.8%, respectively, in Clifford's study (mean age, 32.9 years; median miscarriages, 4; Clifford et al., 1994). The prevalences of aPL, abnormal chromosome, uterine anomaly, endocrine abnormality and unexplained causes were 20, 3.5, 16, 20 and 42.6%, respectively, in Stephenson's study (Stephenson, 1996). These previous studies did not distinguish between truly unexplained causes and an abnormal embryonic karyotype. The prevalence of unexplained causes was 69% among our patients (Fig. 1, Sugiura-Ogasawara et al., 2010). The differences in the distribution of causes at each center may depend on age, number of previous miscarriages and methods of diagnosis for each factor. We compared the distribution of the causes between the 482 couples in the present study and 1676 couples of a previous study among whom the aborted conceptuses of all the patients were not analyzed (Sugiura-Ogasawara et al., 2010, Fig. 1). The true percentage of subjects with recurrent miscarriage of unexplained cause was limited to 24.5%. Moreover, 482 patients in our present study experienced further miscarriages after undergoing a systematic examination. The patients with APS miscarried despite anticoagulant therapy. The prevalences of abnormal chromosomes in either partner and uterine anomaly were higher than the prevalences in previous study. This finding suggests that the prognosis of abnormal chromosomes and uterine anomaly is poor.
The reported live birth rate in women with APS treated with low-dose aspirin plus heparin is 70–80% (Cowchock et al., 1992; Rai et al., 1997). The cumulative live birth rate in this group in the present study was relatively low, because women with APS gave up after the first treatment and failure. The reported prevalence range, in review articles, of APS is 5–15% (Branch et al., 2010). Several reports have indicated that about 10–15% of women with recurrent miscarriage are diagnosed with APS (Clifford et al., 1994; Rai et al., 1995; Yetman and Kutteh, 1996). However, it is unclear whether the aPLs persisted according to International Criteria (Miyakis et al., 2006). The prevalence of APS according to International Criteria was found to be 2.5% in the present study, presumably because the figure represents the prevalence after one treatment failure. The single positive rate of aPLs was 10.7% and the recurrent positive rate was 4.5% in our previous study (Sugiura-Ogasawara et al., 2008). There are many methods used for the detection of aPLs. However, there are limited reports on which method might be most suitable for prediction of recurrent miscarriage or intrauterine fetal death. The positive rate might be large if methods with a large false-positive rate were used. The prevalence of 'true' APS might be <5%, although it depends on the age of the women comprising the study population and the method used for the detection of APS.
The frequency of major congenital uterine anomalies has been reported to be between 3.2 and 6.9% in women with a history of recurrent miscarriage, the variation largely depending on the method of selection and the criteria used for the diagnosis (Sugiura-Ogasawara et al., 2011). Uterine anomalies are encountered in miscarriages associated with euploidy (Sugiura-Ogasawara et al., 2010). However, the prognosis in these cases is better than that in patients presenting with recurrent miscarriage of unexplained cause.
Recently, the mean age of the population has been increasing year by year, and the proportion of women in their 40s has been increasing in Japan. APS and uterine anomalies were found to be rare in subjects with secondary recurrent miscarriage and women over 40 years old in the present study. This should be borne in mind before evaluation of the screening tests.
The prevalence of recurrent miscarriage of truly unexplained cause, in whom the embryonic karyotype was ascertained to be normal, was found to be 24.5% in this study. Kaandorp et al. (2010) demonstrated, based on the results of an RCT, that aspirin plus heparin therapy has no beneficial effect in patients with two or more miscarriages. Further studies are needed in women with recurrent miscarriage of truly unexplained cause, after excluding cases with an abnormal embryonic karyotype, to confirm the conclusion.
Limitation
The prevalence of each cause differs among centers, depending on the background of the patients, such as the mean age and previous number of miscarriages of the women in the studied population and the selected method and criteria for the diagnosis.
Subjects with thrombophilia, infection, fibroid or deficiency of progesterone were excluded from the analysis, because the contribution of these factors to recurrent miscarriage has not yet been established (Branch et al., 2010).
Although the standard G-band technique is the gold standard for evaluating chromosomal abnormalities, it has several limitations, including the need for tissue culture and the possibility of maternal cell contamination. Additional analysis, such as by comparative genomic hybridization (CGH), could not be performed in the present study. Further abnormalities could be detectable and also contamination with maternal tissue could be distinguishable by CGH. A recent microarray CGH indicated that about 80% of sporadic spontaneous abortions were caused by an abnormal embryonic karyotype (Shimokawa et al., 2006). Thus, if the prevalence is similar in women with recurrent miscarring and they have no predisposing factors, the incidence of the abnormal embryonic karyotype as a cause of recurrent miscarriage can be estimated to be (0.8) in patients with n consecutive miscarriages.About 51% of patients caused by an abnormal embryonic karyotype can be expected to exist in patients with three miscarriages occasionally. The incidence might increase if the candidate gene such as SYCP3 or MLH affect.
Among subjects with abnormal chromosomes in the present study, 26.8% (19/71) carried embryos that were normal or balanced (Table I). It is possible that CGH would have revealed lack of balance, because 40% of 42 balanced translocation carriers as assessed by cytogenetic analysis were found to show a loss of balance as assessed by CGH (De Gregori et al., 2007).
