Embryo loss pattern is predominant in miscarriages with normal chromosome karyotype among women with repeated miscarriage

Mamoru Morikawa1, Hideto Yamada1,2, Emi H. Kato1, Shigeki Shimada1, Takashi Yamada1 and Hisanori Minakami1

Department of Obstetrics and Gynecology, Hokkaido University Graduate School of Medicine, Kita-ku N15 W7, Sapporo 060-8638, Japan

2 To whom correspondence should be addressed. Email: yhideto{at}med.hokudai.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
OBJECTIVE: The aim of this study was to assess pregnancy loss patterns in women with repeated miscarriage (RM), according to fetal chromosome karyotypes and aetiologies of RM. METHODS: In this cohort study, 168 fetal chromosome karyotypes of miscarriages were investigated. The pregnancy loss patterns were compared between 75 miscarriages from RM women who had a history of two or more consecutive miscarriages and 93 miscarriages from control women whose previous pregnancies ended in live births without a history of RM. By serial ultrasonography, embryo loss (EL) was defined as miscarriage before fetal heat movement was identified and fetal loss (FL) as miscarriage after fetal heat movement was identified. The EL rate was calculated as EL/(EL+FL). RESULTS: The EL rate (66.7%) in miscarriages with normal karyotypes among RM women (n=42) was higher (P<0.05) than that (45.7%) in controls (n=46), while the EL rate (30.3%) in miscarriages with abnormal karyotypes among RM women (n=33) did not differ from that (25.5%) in the controls (n=47). The EL rate (71.4%) in miscarriages with normal karyotypes among unexplained RM women (n=21) was much higher (P<0.05) than that in the controls. CONCLUSIONS: By evaluating fetal karyotypes, we demonstrated for the first time that EL was predominant in miscarriages with normal karyotype among RM women.

Key words: chromosome karyotype/embryo loss/recurrent spontaneous abortion/repeated miscarriage/ultrasonography


    Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
By ultrasonography, a variety of fetal growth patterns are routinely assessed early in pregnancy. It is well acknowledged that the gestational sac can be detected by 5 weeks of gestation (GW) and fetal heart movement (FHM) by 7 GW in all pregnancies in which the gestational ages are correctly determined and fetuses grow normally (Rempen, 1990Go). The FHM is usually detected by vaginal ultrasonography when the crown-rump length (CRL) of a fetus is >2 mm (Wisser et al., 1994Go).

In miscarriages, pregnancy loss patterns can be classified into two categories, embryo loss (EL) and fetal loss (FL), according to findings of ultrasonography. EL is defined as miscarriage before a FHM is identified and FL as miscarriage after a FHM is identified (Bricker and Farquharson, 2002Go; Li et al., 2002Go). It has been demonstrated recently that EL is more common than FL in miscarriages from women with repeated miscarriage (RM); the pregnancy loss patterns do not differ between aetiologic subgroups of RM (Bricker and Farquharson, 2002Go). However, this investigation included miscarriages with abnormal chromosome karyotypes as subjects and no data of fertile women were compared in study analyses. Generally, ~50% of miscarriages are caused by fetal chromosome abnormalities (Eiben et al., 1990Go). Among RM women, 53.8% of miscarriages carry abnormal fetal chromosome karyotypes (Morikawa et al., 2003Go). Additionally, >50% of RM has unexplained aetiology after excluding plausible causes (Stray-Pedersen and Stray-Pedersen, 1984Go; Yamada et al., 2001aGo). However, there have not yet been any reports concerning the pregnancy loss patterns in miscarriages with normal chromosome karyotype among women with explained and unexplained RM. We previously reported that live birth rates/miscarriage rates in women with unexplained RM did not differ from those in women with explained RM, by excluding pregnancies that ended in miscarriages with abnormal chromosome karyotype (Morikawa et al., 2003Go).

In this study, by prospectively evaluating the chromosome karyotypes of abortuses, we aimed to assess whether frequencies of miscarriages with normal chromosome karyotype increased in RM women and whether any of pregnany loss patterns were related to miscarriages with normal chromosome karyotype in women with unexplained RM.


    Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patient characteristics
Between January 1992 and June 2003, in this cohort study, 353 miscarriages at <12 GW were analysed karyotypically with written informed consent in the Hokkaido University Hospital. The G-banding technique was used for the chromosome karyotype, and all specimens were obtained from the curettage. During this study period, 17 women whose pregnancies ended in miscarriages refused karyotypic analyses of abortuses. Ten of the 353 karyotypic analyses failed due to insufficient specimen or contamination with the decidual cells and bacteria. Both biochemical pregnancies (i.e. miscarriage with a positive test for serum and/or urinary HCG and no gestational sac in the uterus) and complete miscarriages were excluded from this study because karyotypic analyses of these miscarriages were technically impossible.

In this study, 75 of 343 miscarriages were defined as an RM group. The 75 RM women consisted of 24 women with two primary consecutive RM without experiencing live birth, 39 women with three or more primary consecutive RM without experiencing live birth, and 12 women with three or more secondary consecutive RM after experiencing live births. In the RM group, the number of previous miscarriages was mean±SD (range) 3.5±1.6 (2–8). The age of RM women at conception was mean±SD (range) 33.5±5.4 (19–44) years. A control group consisted of 93 miscarriages in women whose previous pregnancies ended in live births without a history of consecutive miscarriages. In the controls, the number of previous live births was mean±SD (range) 1.5±0.8 (1–4) and the number of previous miscarriages was 0.4±0.3 (0–1). The age of control women at conception was mean±SD (range) 33.2±5.2 (20–46) years.

Prior to conception, all RM women had been subjected to examination by ultrasonography and hysterosalpingography to detect anatomical abnormalities of the genital tract or cervical incompetency. Measurements were made of serum testosterone, estradiol, early follicular phase FSH, LH and mid-luteal phase progesterone measurements; and endometrial biopsy was performed. Blood analyses were carried out for syphilis, anti-nuclear antibody (ANA), anti-DNA antibody, lupus anticoagulant, anti-cardiolipin antibody (aCL), {beta}2-glycoprotein I-dependent aCL and haemostatic molecular markers that included activated partial thromboplastin time, protein C activity, d-dimer and antithrombin III. If ANA or anti-DNA antibody was present, further serological tests, i.e. LE test, rheumatoid factor, anti-SSA(B) antibody, anti-RNP antibody and anti-Sm antibody, were performed and complements were measured. Karyotyping and screening of infectious agents in all couples were also performed.

Diagnosis of pregnant loss patterns
From a positive pregnancy test or the first visit to the hospital, all pregnancies were examined at least once a week by transvaginal ultrasonography with a 3.5 MHz transducer (Sonovista-EXTM, Sonovista-MSCTM, Mochida, Tokyo Japan) until 9 GW, and thereafter biweekly until 12 GW. When a pregnancy ended in miscarriage, pregnancy loss patterns were classified into two categories according to findings of ultrasonography, i.e. EL and FL. EL was diagnosed when miscarriage was confirmed before FHM was identified and included blighted ova (presence of a gestational sac without fetal echo or with a CRL of <3 mm). FL was diagnosed when FHM disappeared, or when miscarriage was confirmed with a CRL of ≥3 mm. The EL rates (%) were calculated as follows; number of embryo losses/(number of embryo losses+number of fetal losses)x100.

Statistical analysis
The t-test and the Fisher's exact test were used to analyse the results. P<0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Of the 75 women in the RM group, 18 (24.0%) had endocrine diseases such as hypothyroidism, luteal insufficiency or hyperprolactinaemia; eight (10.7%) had balanced-type chromosomal translocation; five (6.7%) had autoimmune disease, anti-phospholipid antibody or factor XII deficiency; and five (6.7%) had a uterine conformational abnormality as a plausible cause of RM. The other 39 (52.0%) were classified as having unexplained aetiology after exclusion of all other causes. Of the 75 miscarriages in the RM group, 63 pregnancies underwent therapies such as low dose aspirin (n=23), luteal support (n=53) and/or massive i.v. immunoglobulin therapy (n=10).

Between the RM group and the control group, there were no significant differences in the maternal ages or the numbers of previous pregnancies. Seventy-five miscarriages in the RM group consisted of 33 miscarriages with abnormal chromosome karyotype (MsAK) and 42 miscarriages with normal chromosome karyotype (MsNK) (Table I).


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Table I. Pregnancy loss patterns according to fetal chromosome karyotypes in the two groups

 
The 33 MsAK in the RM group included 23 miscarriages with trisomy (seven with trisomy-16, four with trisomy-21, two each with trisomy-14, -15 and -22, and one each with trisomy-3, -4, -5, -7, -9 and -20), three with triploidy, two with double trisomy, one with 45X, one with tetraploidy and three with structural aberration. On the other hand, the 93 miscarriages in the control group consisted of 47 MsAK and 46 MsNK. The 47 MsAK in the control group included 35 miscarriages with trisomy (six with trisomy-21, five with trisomy-16, five with trisomy-7, four with trisomy-22, three each with trisomy-9 and -15, two each with trisomy-14 and -17, and one each with trisomy-2, -6, -12, -13 and -18), six with triploidy, four with 45X and two with structural aberration. The male (46XY):female (46XX) ratios among MsNK were 19:23 in the RM group and 21:25 in the controls.

Of 42 MsNK in the RM group, 21 were derived from RM women with unexplained aetiology and the other 21 MsNK were derived from RM women with explained aetiology; nine had endocrine diseases, five had a uterine conformational abnormality, four had balanced-type chromosomal translocation, and three had autoimmune disease, anti-phospholipid antibody or factor XII deficiency.

The frequency of MsAK (33 out of 75, 44.0%) and MsNK (42 out of 75, 56.0%) in the RM group did not differ significantly from that of MsAK (47 out of 93, 50.5%) and MsNK (46 out of 93, 49.5%) in the control group. However, the EL rate (38 out of 75, 50.7%) in the RM group was significantly (P<0.05) higher than that in the controls (33 out of 93, 35.5%). The EL rate in MsNK (28 out of 42, 66.7%) was significantly (P<0.01) higher compared with that in MsAK in the RM group (10 out of 33, 30.3%). Similarly, the EL rate in MsNK (21 out of 46, 45.7%) was significantly (P<0.05) higher compared with that in MsAK in the controls (12 out of 47, 25.5%) (Table I).

In addition, the EL rate (66.7%) in MsNK of the RM group was significantly (P<0.05) higher than that in the control group (45.7%), while the EL rate (30.3%) in MsAK of the RM group did not differ from that in the controls (25.5%) (Figure 1). The EL rate (15 out of 21, 71.4%) in MsNK of unexplained RM women, but not of explained RM women (13 out of 21, 61.9%), was significantly (P<0.05) higher than that in the controls (45.7%) (Figure 1).



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Figure 1. Embryo loss rates according to fetal chromosome karyotypes in two groups. RM = repeated miscarriage. The embryo loss rates (%) are calculated as follows; number of embryo losses/(number of embryo losses + number of fetal losses)x100.

 
The EL rates in MsNK of explained RM women with each aetiology were as follows; 77.8% (seven of nine) in endocrine disease, 80.0% (four of five) in a uterine conformational abnormality, 0% (none of four) in balanced-type chromosomal translocation, and 66.7% (two of three) in autoimmune disease, anti-phospholipid antibody or factor XII deficiency. The EL rates in MsNK of endocrine disease (P<0.05) and of a uterine conformational abnormality (P<0.05) were significantly higher than that in balanced-type chromosomal translocation.

If we used different criteria for FL (i.e. when FHM disappeared or when miscarriage was confirmed with a CRL of ≥5 mm), the study results were similar. The EL rate (69.0%) in MsNK of the RM group was significantly (P<0.05) higher than that in the control group (47.8%), while the EL rate (33.3%) in MsAK of the RM group did not differ from that in the controls (27.7%). The EL rate (76.2%) in MsNK of unexplained RM women was much higher (P<0.05) than that in the control group.


    Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the current study, we demonstrated that the ratio of MsAK and MsNK in miscarriages did not differ between RM women and age-matched controls. In both groups, the EL rate in MsNK was found to be higher than that in MsAK. However, the EL rate increased in RM as compared with the controls. This increase in the EL rate was due mainly to a high frequency of EL in MsNK of RM; no increase in the EL rate was found in MsAK of RM. The EL rate in MsNK of unexplained RM was much higher than that in the controls. Thus, we demonstrated for the first time that EL was predominant in MsNK of RM women. It is suggested that the aetiology existing in very early gestation may underlie the pathophysiology of RM, especially of unexplained RM, causing the EL with normal chromosome karyotype. These aetiologies in very early gestation may include subclinical hormonal imbalance and immunological abnormalities. As regards risk factors of MsNK, it has been found that a pre-conception high natural killer (NK) cell activity (Yamada et al., 2003aGo), a high NK cell activity in early gestation (Yamada et al., 2001bGo) and a low level of macrophage migration inhibition factor in early gestation (Yamada et al., 2003bGo) are causally related to subsequent MsNK in women with unexplained RM.

It has been demonstrated recently that EL is more common than FL in miscarriages from RM women; the pregnancy loss patterns do not differ between aetiological subgroups of RM (Bricker and Farquharson, 2002Go). Another study has found that the frequency of MsAK in 105 karyotyped miscarriages out of a total of 191 miscarriages from RM women is 32.4% (Li et al., 2002Go); in the current study, the frequencies of MsAK in miscarriages of the RM group was 44.0%. Other investigators have shown that 16 (66.7%) of 24 first trimester FL from non-RM women were MsAK (Bessho et al., 1995Go). These percentages were compatible with results in the current study; frequencies of MsAK in FL were found to be 62.2% (23 out of 37) in the RM group and 58.3% (35 out of 25) in the controls. Therefore, it was unlikely that our subjects were significantly biased in the study design.

Transvaginal ultrasonography has enabled observers to document living embryos. The cardiac activity begins at approximately the 22nd day post-conception. This equates to ~36 menstrual days or a CRL of 1.5–3 mm (Filly, 1994Go). The mean heart rate at a CRL of 2 mm has been reported to be 106.8 b.p.m. (Yapar et al., 1995Go). Therefore, in the current study, we defined FL as if FHM disappeared or when miscarriage was confirmed with a CRL of ≥3 mm. However, earlier studies have recommended the FL as a CRL of ≥5 mm and negative cardiac activity (Levi et al., 1990aGo), because they have found that the cardiac activity was not detected in all normal embryos at a CRL of <4.0 mm (Levi et al., 1990bGo). We reanalysed the current study data and found the results were similar even when we used criteria of FL with a CRL of ≥5 mm.

There have been no reports as yet concerning the EL rate in MsNK among women with explained and unexplained RM. In the current study, by prospectively evaluating fetal karyotypes, we demonstrated for the first time that the EL was predominant in MsNK of RM women, especially of unexplained RM women. The information provided here could constitute a beneficial reference for clinical practice in the field of infertility. First, any therapeutic option could not alter subsequent miscarriages in conceptions with abnormal chromosome karyotype either in RM women or in non-RM women; frequencies of such MsAKs in RM women did not differ from those of non-RM women. Secondly, because the unknown aetiology causing the EL with normal chromosome karyotype may exist in unexplained RM, therapies for RM should be commenced very early in gestation or prior to conception although the maximum therapeutic option for unexplained RM has not yet been determined unequivocally. For example, it has been found that a high dose of immunoglobulin therapy that is commenced and completed during very early gestation is effective for severe cases of unexplained RM possibly through downregulation of NK cell activity and T-helper cell modification (Morikawa et al., 2001Go; Yamada et al., 2003cGo).

However, the current study had several limitations. The ratio of MsAK and MsNK in miscarriages did not differ statistically between RM women and age-matched controls. This result was controversial and probably depended on mean values of previous miscarriage number in the study population because it was reported that the frequency of MsNK increased with the number of previous miscarriages (Ogasawara et al., 2000Go). In the current study, an ~20% excess of female embryos in MsNK was found among RM and control women. An excess of females in MsNK might originate from maternal cell contamination. Therefore, a further confirmatory study is needed.


    Acknowledgements
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by a Grant-in-Aid (No. 14370521) from the Ministry of Education, Science, Sports, and Culture of Japan and by a Grant-in-Aid from the Ministry of Health, Labor and Welfare of Japan.


    References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on April 22, 2004; resubmitted on June 2, 2004; accepted on July 13, 2004.