- Email: [email protected]
Embryonic karyotype in recurrent miscarriage with parental karyotypic aberrations
Embryonic karyotype in recurrent miscarriage with parental karyotypic aberrations Howard Carp, M.B., B.S.,a Esther Guetta, Ph.D.,b Haya Dorf, M.Sc.,b David Soriano, M.D.,a Gad Barkai, M.D.,a,b and Eyal Schiff, M.D.a a
Department of Obstetrics and Gynecology and b Institute of Genetics, Sheba Medical Center, Tel Hashomer, Israel
Objective: To assesses chromosomal aberrations in the abortus in recurrent miscarriage, in the presence of parental chromosomal aberrations. Design: Retrospective comparative cohort study. Setting: Tertiary referral unit in university hospital. Patient(s): One thousand one hundred eight patients with 3–16 miscarriages before 20 weeks gestation; 113 patients with and 995 without chromosomal aberrations. Intervention(s): Karyotyping by standard G-banding techniques of both parents, and of 205 abortuses collected at curettage. Main Outcome Measure(s): The incidence of the euploidic and aneuploidic abortuses according to the parental karyotype. Result(s): Two hundred three abortuses were successfully karyotyped. In 164 embryos of patients with no parental chromosomal aberrations, 23.2% (38/164) had chromosome aberrations. Of the 39 abortuses karyotyped in patients with chromosomal aberrations, 17 had normal karyotypes, 8 had balanced translocations, 2 had inversions identical to the parents, and 12 (30.8%) had abnormal karyotypes. This difference is not statistically significant (odd ratio 1.47, 95% confidence interval 0.63–3.39). Only 4 of the 39 karyotyped abortuses had an unbalanced translocation. Conclusion(s): Parental karyotyping was not particularly predictive of a subsequent miscarriage as a result of chromosomal aberrations as 43.5% of abortuses were euploidic, and the parental aberration was only passed on to the abortus in 10% of cases. (Fertil Steril威 2006;85:446 –50. ©2006 by American Society for Reproductive Medicine.) Key Words: Abortion, karyotype, recurrent miscarriage, pregnancy loss, parental chromosomal aberrations
Recurrent miscarriage is usually defined as the loss of three or more consecutive pregnancies before 20 or even 28 weeks of gestation (1, 2). It has been recommended that the standard investigation of such patients should include karyotyping of both parents for chromosomal aberrations (3, 4). Karyotyping allows for the diagnosis of balanced reciprocal or Robertsonian translocations and mosaicism for numerical aneuploidy. When these aberrations are present it is often assumed that they are responsible for the miscarriages as the aberration may be inherited by the embryo in an unbalanced form. Embryonic chromosomal aberrations have been found in 29%– 60% of recurrent miscarriages (5– 8); however, most of these are numeric aberrations that probably arise de novo, rather than being inherited (e.g., 34 of 36 in the series by Carp et al. [7]). Three studies have examined the prognosis for a subsequent live birth in couples who have parental karyotypic aberrations (9 –11). Because there is disagreement about the subsequent prognosis, the present study examines the karyotype of the abortus in couples with recurrent miscarriage and Received December 5, 2004; revised and accepted July 19, 2005. Reprint requests: Howard Carp, M.B., B.S., Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer, 52621 Israel (FAX: 972-9-9574779; E-mail: [email protected]).
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parental chromosomal rearrangements in those couples who had a subsequent miscarriage. The incidence of embryonic karyotypic aberrations is compared in patients with and without parental chromosomal aberrations. MATERIALS AND METHODS The Sheba Medical Center is a tertiary referral center for patients with recurrent pregnancy losses. A karyotype was performed on both parents in 1,108 patients who presented to our service with three or more consecutive miscarriages before 20 weeks gestation between 1984 and 2003. This was performed as part of the primary investigation in addition to other presumptive causes of abortion, according to our previously described criteria (12). Before inclusion in the study, objective evidence of past pregnancies was required. This might have included original laboratory reports of a positive hCG test, histological confirmation after curettage, and ultrasonic reports of a pregnancy sac. Of the patients who miscarried again and had a curettage in our center, chromosomal analysis of the abortuses was offered and performed after obtaining informed consent. Institutional review board approval was not required as parental karyotyping is part of the routine investigation of recurrent miscarriage. Karyotyping of the abortus has been
Fertility and Sterility姞 Vol. 85, No. 2, February 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.07.1305
recommended as an essential part of the investigation of recurrent miscarriage by the Royal College of Obstetricians and Gynaecologists in the U.K. in a 1998 guideline, and revised in 2003 (4), and has been recommended by the American College in a practice guideline in 2001 (3). Peripheral Blood Karyotyping Chromosome studies were performed on routinely cultured peripheral blood lymphocytes. Slides were processed for G-banding using trypsin– giemsa by standard techniques. Colcimide was added 30 minutes before the cytological preparation. A minimum of 15 cells were microscopically analyzed at metaphase from two independent cultures prepared for each individual. Tissue Culture Tissue samples were obtained by curettage. Curettings of the abortus were collected into sterile saline or culture medium and transferred immediately to the laboratory where the tissue was identified visually. Two types of tissue were identified: placental villi comprised of trophoblast or fetal parts. In selected cases, the curettings were examined microscopically to exclude maternal decidua contaminating the culture and skewing the results. Culture initiation and propogation were carried out as previously described (7), with the following modification. Tissue that was identified as fetal was cut into small pieces mechanically, with a scalpel and divided into culture vessels. A portion of the sample was seeded directly onto coverslips in petri dishes and the remainder was seeded into 25-cm2 culture flasks containing RPMI medium (Biological Industries, Bet Ha’emek, Israel) supplemented with 20% fetal calf serum (FCS), 1% streptomycin/penicillin, and 1% glutamine or BIO-AMF-2 (samples obtained from 1999 onward). All cultures were incubated at 37°C in 5% CO2. Metaphase arrest was induced by incubating the cultures in colcimide (1g/ml) for 3 hours. The samples were processed using standard techniques for chromosome analysis. All specimens were G-banded using trypsin– giemsa to analyze and identify abnormalities in the cells. A minimum of 10 cells were scored, and at least 5 cells were analyzed, while the others were counted. All culture reagents
were manufactured by Biological Industries, Kibbutz Beit Haemek, Israel. Follow-Up All patients with parental aberrations were offered genetic counseling. The details of each subject were entered into a computerized database, using the SPSS program (SPSS Inc., Chicago, IL). The clinical features of each patient and her miscarriages were recorded, including whether the previous miscarriages occurred in the first or second trimesters, or whether the patients were primary aborters (all pregnancies terminating in miscarriage), or secondary aborters (one or more live births followed by at least three consecutive miscarriages). Patients were instructed to attend the clinic as soon as a subsequent pregnancy was diagnosed and the details of the pregnancy were followed up longitudinally until either delivery or a subsequent pregnancy loss occurred. Statistical Analysis Two-by-two tables were constructed to calculate the odds ratio (OR) for a live birth with 95% confidence intervals (CI). A difference was considered to be significant if the 95% CI did not exceed 1. Analysis was performed by Fisher’s exact test when the numbers were small. RESULTS Karyotyping was attempted on 248 abortuses. This procedure was successful in 203 abortuses, but in 45 abortuses there was either no growth in culture or infection of the specimen. Table 1 shows the details of the patients in the study with regard to age and gravidity at the time of fetal karyotyping, and success in fetal karyotyping. As can be seen, there were no significant differences between patients with parental karyotypic aberrations and those with no aberrations. Table 2 shows the parental karyotype in 37 patients who subsequently aborted either once or more than once and had fetal karyotyping attempted. Fetal karyotyping was successful in 81.8% of patients. Table 3 shows that
TABLE 1 Details of patients in the study.
No. patients Mean age ⫾ SD (Range) No. of abortions ⫾ SD Range
Parental karyotypic aberations
No parental karyotypic aberations
All patients
113 30.96 ⫾ 5.01 (20–44) 4.27 ⫾ 1.60 (3–10)
995 31.77 ⫾ 6.1 (20–43) 4.22 ⫾ 1.7 (3–16)
1,108 31.08 ⫾ 5.19 (20–44) 4.22 ⫾ 1.69 (3–16)
Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
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TABLE 2 Details of parental chromosomal aberrations in patients in whom subsequent embryonic karyotyping was performed. Maternal balanced translocation Paternal balanced translocation Maternal mosaic for numerical aberration Paternal mosaic for numerical aberration Maternal reciprocal inversion Paternal reciprocal inversion Total
16 6 8 1 6 0 37
Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
30.8% of fetal karyotypes were abnormal in parents with parental karyotypic aberrations (12 of 39) compared to 23.2% in patients with no parental chromosomal aberrations. However, this figure is not statistically significant (OR ⫽ 1.47, 95% CI 0.63–3.39). An additional eight embryos had balanced translocations and two had reciprocal inversions identical to the parents. Because the parents were normal in every way, these embryos were considered to be normal. They would be replaced in pregestational diagnosis cycles, and couples would not be advised to abort if these identical karyotypes to the parents were found at amniocentesis. The higher incidence of fetal karyotypic aberrations in patients with karyotypic aberrations can be explained by the four embryos with unbalanced translocations (Table 4). However, these only account for 8.5% of abortuses that could be karyotyped. Table 4 shows the details of the aberrations found in the abortus. As in our previous study (7), the most frequent chromosomal anomaly was trisomy, which was found in 33 of the 60 chromosomally abnormal embryos. Ten patients (of the 37 with parental karyotypic aberrations) had a (second) subsequent abortion karyotyped. Five
patients had had a normal initial karyotype, two with balanced translocations, two with trisomies, and one patient whose first karyotyped abortus did not grow in culture. Of the five patients with a normal embryonic karyotype, three subsequently lost embryos with balanced translocations, one miscarried a euploidic embryo in the (second) subsequent pregnancy, and one lost a trisomic embryo. Of the two patients who initially miscarried an embryo with a balanced translocation, one subsequently miscarried an embryo with the same balanced translocation, and one lost a euploidic embryo subsequently. Two patients originally lost trisomic embryos, one lost a subsequent euploidic embryo and the other lost a subsequent trisomic embryo. The patient with culture failure on the first attempted karyotype, subsequently lost an embryo with uniparental disomy for chromosome 14 (this patient had a maternal translocation from chromosome 13 to 14). DISCUSSION In this series of patients there were 113 patients with chromosomal aberrations in one of the parents. In these patients, 39 subsequent pregnancies which terminated in miscarriage, were subsequently karyotyped. The incidence of penetrance to the embryo was low as only 30.7% were karyotypically abnormal. This incidence was not statistically different to that seen in couples with no parental aberrations (where 23.2% had karyotypic aberrations). In our previous study (7), only 125 of 167 (74.9%) of the embryos grew in culture, and the incidence of embryonic chromosomal aberrations was low (29%). It has been suggested that this low incidence may have been due to the karyotypically normal embryos growing in cutlture, whereas the karyotypically abnormal embryos could not grow in culture (8). In the present study there was the same low incidence of embryonic chromosomal aberrations, although 89% of abortuses grew in culture, similar to the 88% in the series by Stephenson et al. (8). The low incidence of embry-
TABLE 3 Normal and abnormal fetal karyotypes.
No. patients Attempted fetal karyotypes Successful fetal karyotypes Normal karyotype Abnormal karyotype Identical karyotype to parent
Parental karyotypic aberations
No parental karyotypic aberations
37 47 39 (82.9%) 17 (43.6%) 12 (30.8%) 10 (25.6%)a
168 201 164 (81.6%) 126 (76.8%) 38 (23.2%)
All patients 205 248 203 (81.8%) 143 (70.4%) 50 (24.6%) 10 (4.9%)
Note: Figures in parentheses refer to the number of abnormal, normal karyotypes found as a proportion of those tested. a In patients with parental karyotypic aberrations, there were also eight abortuses with a balanced translocation identical to that found in the parents, and two with reciprocal inversions identical to those in the parents. Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
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Parental and embryonic karyotype in recurrent miscarriage
Vol. 85, No. 2, February 2006
TABLE 4 Details of anomalies found in abortus.
Anomaly Monosomy (45XO) Trisomya Triploidy Unbalanced translocations UPD Total
Parental karyotypic aberations
No parental karyotypic aberations
2 5 0 4
6 28 4 0
1 12
0 38
There were also eight balanced translocations and two reciprocal inversions, which were identical to those seen in the parents. a The following trisomies were found: Trisomies 13 and 22, each in five abortuses; trisomies 16 in four abortuses; trisomies 4, 15, 18, and 21, each in three abortuses; trisomy 7 in two abortuses; trisomies 8, 14, 20, and XXY were each found in one specimen. Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
onic chromosomal aberrations is more likely due to the relatively large number of previous miscarriages in the present series, 4.22, compared to 4.0 in the series by Stephenson et al. (8), 3.5 in the series by Stern et al. (5), and 3.8 in the one by Ogasawara et al. (6). Although disputed in the literature, Ogasawara et al. (6) have reported that the incidence of embryonic chromosomal aberrations decreases as the number of previous miscarriages increases. With recurrent miscarriage the patients usually ask about the cause, prognosis, and treatment. In this series the presence of parental aberrations did not diagnose the cause of miscarriage as only 12 of 39 miscarriages (30.7%) were chromosomally abnormal, indicating an alternative cause for the recurrent miscarriages. The chromosomal aberration was only passed on to the fetus in an unbalanced form in 4 of 39 karyotyped embryos (10.26%). In 10 of 39 abortuses a balanced translocation or inversion was found, which was similar to that of the parents. Because the parents were phenotypically normal in every way, these abortions must also have an alternative cause. Hence, it is unjustified to advise patients with parental karyotypic aberrations that their recurrent miscarriages are due to the aberration being inherited by the fetus in an unbalanced form. It has been suggested by Sugiura-Ogasawara et al. (9) that the presence of parental karyotypic aberrations defines a high risk group for subsequent miscarriages, as between 61% and 72% of their patients had subsequent miscarriages. However, in the study by Goddijn et al. (11) more than 70% of subsequent pregnancies resulted in live births, and in their previous study 55.3% of subsequent pregnancies resulted in Fertility and Sterility姞
live births, which was not statistically different from the 45% found in the control group. It seems more likely that the prognosis for a subsequent live birth is more dependant on factors known to affect the subsequent prognosis such as the number of previous miscarriages, maternal age, karyotype of the previous miscarriage, and primary or secondary aborter status (13) rather than parental karyotype. It is also questionable whether the presence of parental aberrations defines the need for treatment. The only treatment available for karyotypic aberrations is embryo biopsy after IVF and pregestational diagnosis. Most reports on pregestational diagnosis in the presence of parental chromosomal rearrangements have not been restricted to patients with recurrent miscarriage, but include patients with infertility. Simopoulou et al. (14) reported 11 pregestational diagnosis cycles in 8 recurrently miscarrying patients with various chromosomal rearrangements. Euploid embryos were replaced in all 11 cycles. Four pregnancies ensued, leading to three live births and one “biochemical” pregnancy. It is still too early to say whether this live birth rate is different to the 70% live birth rate in the series by Goddijn et al. (11) or the current result of 55.3% live births. The report by Rubio et al. (15) showed that most embryos with chromosomal aberrations failed to grow in culture. Hence, patients forming embryos with severe chromosomal aberrations may present with infertility rather than recurrent miscarriage. Accordingly, the formation of chromosomally abnormal embryos may explain the 32% infertility rate (16) seen in recurrently miscarrying patients and also explain why most karyotyped embryos are euploid in recurrent miscarriage. Pregestational diagnosis is only worthwhile if the patient with parental aberrations loses embryos with a karyotypic abnormality. In patients losing karyotypically normal embryos, pregestational diagnosis will not lead to a subsequent live birth, as the cause of pregnancy loss is more likely to be maternal in origin. In these cases treatment of maternal causes is appropriate. It still remains to be determined whether an IVF procedure with embryo biopsy to select karyotypically normal embryos for transfer can improve on the success rate of natural reproduction. Even if one anomalous embryo is lost, it is doubtful whether pregestational diagnosis is warranted. Although Coulam et al. (17) reported a theoretical risk of 35% of a repeated chromosomal anomaly after three losses increasing to 70% after six losses, Warburton et al. (18) reported that there was no increased risk for trisomy in a second spontaneous abortion after either a previous trisomic abortion or an abortion with another abnormal karyotype. In this series, 10 patients with a parental karyotypic aberration had two subsequent miscarriages karyotyped. Five had an initally euploidic miscarriage, one miscarried an embryo that failed to grow in culture. Of two patients with an initially trisomic abortus unrelated to the parental karyotypes, one miscarried a subsequently euploid abortus, and one miscarried a subsequent trisomic abortus. Of the two patients who initially lost an embryo with a balanced translocation, one subsequently 449
miscarried an embryo with the same balanced translocation, and one lost a euploidic embryo subsequently. Therefore it may not be enough to show aneuploidy in the abortus, it may be necessary to show repeat aneuploidy to determine the need for interventions such as pregestational diagnosis. If the subsequent pregnancy continues developing, in the presence of parental chromosomal aberations, the question arises as to whether chorionic villus sampling (CVS) or amniocentesis are indicated. Esrig and Leonardi (19) have reported an increased incidence of pregnancy loss after amniocentesis in patients with previous miscarriages compared to patients without previous miscarriages. However, Esrig and Leonardie (19) did not define when the previous miscarriages occurred in the first or second trimesters in their series, nor did they examine recurrent miscarriage specifically. Carp et al. (20) reported on 70 amniocenteses in recurrent miscarriage. There was not one subsequent abortion. Goddijn et al. (11) performed 26 amniocenteses in patients with recurrent miscarriage and parental karyotypic rearrangements. Fourteen pregnancies were euploid, 12 had balanced rearrangements similar to the parents. There were no unbalanced translocations or inversions. In this series 10 abortuses had karyotypic aberrations identical to the parents. If these chromosomal rearrangements were found at amniocentesis, it would be assumed that the transmission of the chromosome anomaly would have no phenotypic effect as the carrier is nornal. In these cases, abortion would not be advised. Although we offer amniocentesis to all patients with a parental karyotypic aberration, even if the woman is below the age where prenatal diagnosis would be advised, in our opinion, all these factors should be taken into account before advising genetic testing. Although parental karyotyping is part of the standard management of recurrent miscarriage, these results concur with those of Clark et al. (21), indicating that parental karyotyping is of limited value in diagnosing the cause, prognosis, or mode of treatment for recurrent miscarriage. Parental karyotyping is, at best, an indirect measure of the fetal karyotype. In our opinion, treatment and prognosis depend on the fetal karyotype. Hence, the fetal karyotype should be determined directly at abortion. Newer techniques such as embryoscopy (22) and comparative genomic hybridization (23) should improve the accuracy of fetal karyotyping. Parental karyotyping is a poor substitute.
1. Salat-Baroux J. Recurrent spontaneous miscarriages. Reprod Nutr Dev 1988;28:1555– 68. 2. Crosignani PC, Rubin BL. Recurrent spontaneous miscarriage. The recommendations of the ESHRE workshop on recurrent spontaneous miscarriage held in Anacapri on September 9 –11, 1990. Hum Reprod 1991;6:609 –10. 3. American College of Obstetricians and Gynaecologists. Management of recurrent early pregnancy loss. ACOG Practice Bulletin 2001;24:1–12. 4. Royal College of Obstetricians and Gynaecologists, Guideline No. 17. The investigation and treatment of couples with recurrent
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miscarriage. London: Royal College of Obstetricians and Gynaecologists, 2003. Stern JJ, Dorfman AD, Gutierez-Najar AJ, Cerrillo M, Coulam CB. Frequency of abnormal karyotype among abortuses from women with and without a history of recurrent spontaneous abortion. Fertil Steril 1996;65:250 –3. Ogasawara M, Aoki K, Okada S, Suzumori K. Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertil Steril 2000;73:300 – 4. Carp HJA, Toder V, Orgad S, Aviram A, Daniely M, Mashiach S, et al. Karyotype of the abortus in recurrent miscarriage. Fertil Steril 2001;5: 678 – 82. Stephenson MD, Awartani KA, Robinson WP. Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Hum Reprod 2002;17:446 –51. Sugiura-Ogasawara M, Ozaki Y, Sato T, Suzumori N, Suzumori K. Poor prognosis of recurrent aborters with either maternal or paternal reciprocal translocations. Fertil Steril 2004;81:367–73. Carp HJA, Feldman B, Oelsner G, Schiff E. Parental karyotype and subsequent live births in recurrent miscarriage. Fertil Steril 2004;81: 1296 –301. Goddijn M, Joosten JHK, Knegt AC, Van der Veen F, Franssen MTM, Bonsel GJ, et al. Clinical relevance of diagnosing structural chromosome abnormalities in couples with repeated miscarriage. Hum Reprod 2004;19:1013–7. Orgad S, Gazit E, Lowenthal R, Carp HJA. Influence of antipaternal antibodies on the incidence of live births in couples with consecutive recurrent abortions. Hum Reprod 1999;14:2974 –9. Recurrent Miscarriage Immunotherapy Trialists Group. Worldwide collaborative observational study and meta-analysis on allogenic leukocyte immunotherapy for recurrent spontaneous abortion. Am J Reprod. Immunol 1994;32:55–72. Simopoulou M, Harper JC, Fragouli E, Mantzouratou A, Speyer BE, Serhal P, et al. Preimplantation genetic diagnosis of chromosome abnormalities: implications from the outcome for couples with chromosomal rearrangements. Prenat Diagn 2003;23:652– 62. Rubio C, Simon C, Vidal F. Rodrigo L, Pehlivan T, Remohi J, et al. Chromosomal abnormalities and embryo development in recurrent miscarriage couples. Hum Reprod 2003;18:182– 8. Clifford K, Rai R, Watson H, Regan L. An informative protocol for the investigation of recurrent miscarriage: preliminary experience of 500 consecutive cases. Hum Reprod 1994;9:1328 –32. Coulam CB, Stephenson M, Stern JJ, Clark DA. Immunotherapy for recurrent pregnancy loss: analysis of results from clinical trials. Am J Reprod Immunol 1996;35:352–9. Warburton D, Kline J, Stein Z, Hutzler M, Chin A, Hassold T. Does the karyotype of a spontaneous abortion predict the karyotype of a subsequent abortion? Evidence from 273 women with two karyotyped spontaneous abortions. Am J Hum Genet 1987;41:465– 83. Esrig SM, Leonardi DE. Spontaneous abortion after amniocentesis in women with a history of spontaneous abortion. Prenat Diagn 1985;5: 321– 8. Carp HJA, Ben-Shlomo I, Toder V, Nebel L, Mashiach S. Congenital malformations after immunotherapy for habitual abortion: is there an increase? J Gynaecol Obstet Invest 1993;36:198 –201. Clark DA, Daya S, Coulam CB, Gunby J. Implication of abnormal human trophoblast karyotype for the evidence-based approach to the understanding, investigation, and treatment of recurrent spontaneous abortion. The Recurrent Miscarriage Immunotherapy Trialists Group. Am J Reprod Immunol 1996;35:495– 8. Ferro J, Martinez MC, Lara C, Pellicer A, Remohi J, Serra V. Improved accuracy of hysteroembryoscopic biopsies for karyotyping early missed abortions. Fertil Steril 2003;80:1260 – 4. Daniely M, Aviram-Goldring A, Barkai G, Goldman B. Detection of chromosomal abberation in fetuses arising from recurrent spontaneous abortion by comparative genomic hybridization. Hum Reprod 1998;13:805–9.
Parental and embryonic karyotype in recurrent miscarriage
Vol. 85, No. 2, February 2006
Department of Obstetrics and Gynecology and b Institute of Genetics, Sheba Medical Center, Tel Hashomer, Israel
Objective: To assesses chromosomal aberrations in the abortus in recurrent miscarriage, in the presence of parental chromosomal aberrations. Design: Retrospective comparative cohort study. Setting: Tertiary referral unit in university hospital. Patient(s): One thousand one hundred eight patients with 3–16 miscarriages before 20 weeks gestation; 113 patients with and 995 without chromosomal aberrations. Intervention(s): Karyotyping by standard G-banding techniques of both parents, and of 205 abortuses collected at curettage. Main Outcome Measure(s): The incidence of the euploidic and aneuploidic abortuses according to the parental karyotype. Result(s): Two hundred three abortuses were successfully karyotyped. In 164 embryos of patients with no parental chromosomal aberrations, 23.2% (38/164) had chromosome aberrations. Of the 39 abortuses karyotyped in patients with chromosomal aberrations, 17 had normal karyotypes, 8 had balanced translocations, 2 had inversions identical to the parents, and 12 (30.8%) had abnormal karyotypes. This difference is not statistically significant (odd ratio 1.47, 95% confidence interval 0.63–3.39). Only 4 of the 39 karyotyped abortuses had an unbalanced translocation. Conclusion(s): Parental karyotyping was not particularly predictive of a subsequent miscarriage as a result of chromosomal aberrations as 43.5% of abortuses were euploidic, and the parental aberration was only passed on to the abortus in 10% of cases. (Fertil Steril威 2006;85:446 –50. ©2006 by American Society for Reproductive Medicine.) Key Words: Abortion, karyotype, recurrent miscarriage, pregnancy loss, parental chromosomal aberrations
Recurrent miscarriage is usually defined as the loss of three or more consecutive pregnancies before 20 or even 28 weeks of gestation (1, 2). It has been recommended that the standard investigation of such patients should include karyotyping of both parents for chromosomal aberrations (3, 4). Karyotyping allows for the diagnosis of balanced reciprocal or Robertsonian translocations and mosaicism for numerical aneuploidy. When these aberrations are present it is often assumed that they are responsible for the miscarriages as the aberration may be inherited by the embryo in an unbalanced form. Embryonic chromosomal aberrations have been found in 29%– 60% of recurrent miscarriages (5– 8); however, most of these are numeric aberrations that probably arise de novo, rather than being inherited (e.g., 34 of 36 in the series by Carp et al. [7]). Three studies have examined the prognosis for a subsequent live birth in couples who have parental karyotypic aberrations (9 –11). Because there is disagreement about the subsequent prognosis, the present study examines the karyotype of the abortus in couples with recurrent miscarriage and Received December 5, 2004; revised and accepted July 19, 2005. Reprint requests: Howard Carp, M.B., B.S., Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer, 52621 Israel (FAX: 972-9-9574779; E-mail: [email protected]).
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parental chromosomal rearrangements in those couples who had a subsequent miscarriage. The incidence of embryonic karyotypic aberrations is compared in patients with and without parental chromosomal aberrations. MATERIALS AND METHODS The Sheba Medical Center is a tertiary referral center for patients with recurrent pregnancy losses. A karyotype was performed on both parents in 1,108 patients who presented to our service with three or more consecutive miscarriages before 20 weeks gestation between 1984 and 2003. This was performed as part of the primary investigation in addition to other presumptive causes of abortion, according to our previously described criteria (12). Before inclusion in the study, objective evidence of past pregnancies was required. This might have included original laboratory reports of a positive hCG test, histological confirmation after curettage, and ultrasonic reports of a pregnancy sac. Of the patients who miscarried again and had a curettage in our center, chromosomal analysis of the abortuses was offered and performed after obtaining informed consent. Institutional review board approval was not required as parental karyotyping is part of the routine investigation of recurrent miscarriage. Karyotyping of the abortus has been
Fertility and Sterility姞 Vol. 85, No. 2, February 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.07.1305
recommended as an essential part of the investigation of recurrent miscarriage by the Royal College of Obstetricians and Gynaecologists in the U.K. in a 1998 guideline, and revised in 2003 (4), and has been recommended by the American College in a practice guideline in 2001 (3). Peripheral Blood Karyotyping Chromosome studies were performed on routinely cultured peripheral blood lymphocytes. Slides were processed for G-banding using trypsin– giemsa by standard techniques. Colcimide was added 30 minutes before the cytological preparation. A minimum of 15 cells were microscopically analyzed at metaphase from two independent cultures prepared for each individual. Tissue Culture Tissue samples were obtained by curettage. Curettings of the abortus were collected into sterile saline or culture medium and transferred immediately to the laboratory where the tissue was identified visually. Two types of tissue were identified: placental villi comprised of trophoblast or fetal parts. In selected cases, the curettings were examined microscopically to exclude maternal decidua contaminating the culture and skewing the results. Culture initiation and propogation were carried out as previously described (7), with the following modification. Tissue that was identified as fetal was cut into small pieces mechanically, with a scalpel and divided into culture vessels. A portion of the sample was seeded directly onto coverslips in petri dishes and the remainder was seeded into 25-cm2 culture flasks containing RPMI medium (Biological Industries, Bet Ha’emek, Israel) supplemented with 20% fetal calf serum (FCS), 1% streptomycin/penicillin, and 1% glutamine or BIO-AMF-2 (samples obtained from 1999 onward). All cultures were incubated at 37°C in 5% CO2. Metaphase arrest was induced by incubating the cultures in colcimide (1g/ml) for 3 hours. The samples were processed using standard techniques for chromosome analysis. All specimens were G-banded using trypsin– giemsa to analyze and identify abnormalities in the cells. A minimum of 10 cells were scored, and at least 5 cells were analyzed, while the others were counted. All culture reagents
were manufactured by Biological Industries, Kibbutz Beit Haemek, Israel. Follow-Up All patients with parental aberrations were offered genetic counseling. The details of each subject were entered into a computerized database, using the SPSS program (SPSS Inc., Chicago, IL). The clinical features of each patient and her miscarriages were recorded, including whether the previous miscarriages occurred in the first or second trimesters, or whether the patients were primary aborters (all pregnancies terminating in miscarriage), or secondary aborters (one or more live births followed by at least three consecutive miscarriages). Patients were instructed to attend the clinic as soon as a subsequent pregnancy was diagnosed and the details of the pregnancy were followed up longitudinally until either delivery or a subsequent pregnancy loss occurred. Statistical Analysis Two-by-two tables were constructed to calculate the odds ratio (OR) for a live birth with 95% confidence intervals (CI). A difference was considered to be significant if the 95% CI did not exceed 1. Analysis was performed by Fisher’s exact test when the numbers were small. RESULTS Karyotyping was attempted on 248 abortuses. This procedure was successful in 203 abortuses, but in 45 abortuses there was either no growth in culture or infection of the specimen. Table 1 shows the details of the patients in the study with regard to age and gravidity at the time of fetal karyotyping, and success in fetal karyotyping. As can be seen, there were no significant differences between patients with parental karyotypic aberrations and those with no aberrations. Table 2 shows the parental karyotype in 37 patients who subsequently aborted either once or more than once and had fetal karyotyping attempted. Fetal karyotyping was successful in 81.8% of patients. Table 3 shows that
TABLE 1 Details of patients in the study.
No. patients Mean age ⫾ SD (Range) No. of abortions ⫾ SD Range
Parental karyotypic aberations
No parental karyotypic aberations
All patients
113 30.96 ⫾ 5.01 (20–44) 4.27 ⫾ 1.60 (3–10)
995 31.77 ⫾ 6.1 (20–43) 4.22 ⫾ 1.7 (3–16)
1,108 31.08 ⫾ 5.19 (20–44) 4.22 ⫾ 1.69 (3–16)
Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
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TABLE 2 Details of parental chromosomal aberrations in patients in whom subsequent embryonic karyotyping was performed. Maternal balanced translocation Paternal balanced translocation Maternal mosaic for numerical aberration Paternal mosaic for numerical aberration Maternal reciprocal inversion Paternal reciprocal inversion Total
16 6 8 1 6 0 37
Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
30.8% of fetal karyotypes were abnormal in parents with parental karyotypic aberrations (12 of 39) compared to 23.2% in patients with no parental chromosomal aberrations. However, this figure is not statistically significant (OR ⫽ 1.47, 95% CI 0.63–3.39). An additional eight embryos had balanced translocations and two had reciprocal inversions identical to the parents. Because the parents were normal in every way, these embryos were considered to be normal. They would be replaced in pregestational diagnosis cycles, and couples would not be advised to abort if these identical karyotypes to the parents were found at amniocentesis. The higher incidence of fetal karyotypic aberrations in patients with karyotypic aberrations can be explained by the four embryos with unbalanced translocations (Table 4). However, these only account for 8.5% of abortuses that could be karyotyped. Table 4 shows the details of the aberrations found in the abortus. As in our previous study (7), the most frequent chromosomal anomaly was trisomy, which was found in 33 of the 60 chromosomally abnormal embryos. Ten patients (of the 37 with parental karyotypic aberrations) had a (second) subsequent abortion karyotyped. Five
patients had had a normal initial karyotype, two with balanced translocations, two with trisomies, and one patient whose first karyotyped abortus did not grow in culture. Of the five patients with a normal embryonic karyotype, three subsequently lost embryos with balanced translocations, one miscarried a euploidic embryo in the (second) subsequent pregnancy, and one lost a trisomic embryo. Of the two patients who initially miscarried an embryo with a balanced translocation, one subsequently miscarried an embryo with the same balanced translocation, and one lost a euploidic embryo subsequently. Two patients originally lost trisomic embryos, one lost a subsequent euploidic embryo and the other lost a subsequent trisomic embryo. The patient with culture failure on the first attempted karyotype, subsequently lost an embryo with uniparental disomy for chromosome 14 (this patient had a maternal translocation from chromosome 13 to 14). DISCUSSION In this series of patients there were 113 patients with chromosomal aberrations in one of the parents. In these patients, 39 subsequent pregnancies which terminated in miscarriage, were subsequently karyotyped. The incidence of penetrance to the embryo was low as only 30.7% were karyotypically abnormal. This incidence was not statistically different to that seen in couples with no parental aberrations (where 23.2% had karyotypic aberrations). In our previous study (7), only 125 of 167 (74.9%) of the embryos grew in culture, and the incidence of embryonic chromosomal aberrations was low (29%). It has been suggested that this low incidence may have been due to the karyotypically normal embryos growing in cutlture, whereas the karyotypically abnormal embryos could not grow in culture (8). In the present study there was the same low incidence of embryonic chromosomal aberrations, although 89% of abortuses grew in culture, similar to the 88% in the series by Stephenson et al. (8). The low incidence of embry-
TABLE 3 Normal and abnormal fetal karyotypes.
No. patients Attempted fetal karyotypes Successful fetal karyotypes Normal karyotype Abnormal karyotype Identical karyotype to parent
Parental karyotypic aberations
No parental karyotypic aberations
37 47 39 (82.9%) 17 (43.6%) 12 (30.8%) 10 (25.6%)a
168 201 164 (81.6%) 126 (76.8%) 38 (23.2%)
All patients 205 248 203 (81.8%) 143 (70.4%) 50 (24.6%) 10 (4.9%)
Note: Figures in parentheses refer to the number of abnormal, normal karyotypes found as a proportion of those tested. a In patients with parental karyotypic aberrations, there were also eight abortuses with a balanced translocation identical to that found in the parents, and two with reciprocal inversions identical to those in the parents. Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
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TABLE 4 Details of anomalies found in abortus.
Anomaly Monosomy (45XO) Trisomya Triploidy Unbalanced translocations UPD Total
Parental karyotypic aberations
No parental karyotypic aberations
2 5 0 4
6 28 4 0
1 12
0 38
There were also eight balanced translocations and two reciprocal inversions, which were identical to those seen in the parents. a The following trisomies were found: Trisomies 13 and 22, each in five abortuses; trisomies 16 in four abortuses; trisomies 4, 15, 18, and 21, each in three abortuses; trisomy 7 in two abortuses; trisomies 8, 14, 20, and XXY were each found in one specimen. Carp. Parental and embryonic karyotype in recurrent miscarriage. Fertil Steril 2006.
onic chromosomal aberrations is more likely due to the relatively large number of previous miscarriages in the present series, 4.22, compared to 4.0 in the series by Stephenson et al. (8), 3.5 in the series by Stern et al. (5), and 3.8 in the one by Ogasawara et al. (6). Although disputed in the literature, Ogasawara et al. (6) have reported that the incidence of embryonic chromosomal aberrations decreases as the number of previous miscarriages increases. With recurrent miscarriage the patients usually ask about the cause, prognosis, and treatment. In this series the presence of parental aberrations did not diagnose the cause of miscarriage as only 12 of 39 miscarriages (30.7%) were chromosomally abnormal, indicating an alternative cause for the recurrent miscarriages. The chromosomal aberration was only passed on to the fetus in an unbalanced form in 4 of 39 karyotyped embryos (10.26%). In 10 of 39 abortuses a balanced translocation or inversion was found, which was similar to that of the parents. Because the parents were phenotypically normal in every way, these abortions must also have an alternative cause. Hence, it is unjustified to advise patients with parental karyotypic aberrations that their recurrent miscarriages are due to the aberration being inherited by the fetus in an unbalanced form. It has been suggested by Sugiura-Ogasawara et al. (9) that the presence of parental karyotypic aberrations defines a high risk group for subsequent miscarriages, as between 61% and 72% of their patients had subsequent miscarriages. However, in the study by Goddijn et al. (11) more than 70% of subsequent pregnancies resulted in live births, and in their previous study 55.3% of subsequent pregnancies resulted in Fertility and Sterility姞
live births, which was not statistically different from the 45% found in the control group. It seems more likely that the prognosis for a subsequent live birth is more dependant on factors known to affect the subsequent prognosis such as the number of previous miscarriages, maternal age, karyotype of the previous miscarriage, and primary or secondary aborter status (13) rather than parental karyotype. It is also questionable whether the presence of parental aberrations defines the need for treatment. The only treatment available for karyotypic aberrations is embryo biopsy after IVF and pregestational diagnosis. Most reports on pregestational diagnosis in the presence of parental chromosomal rearrangements have not been restricted to patients with recurrent miscarriage, but include patients with infertility. Simopoulou et al. (14) reported 11 pregestational diagnosis cycles in 8 recurrently miscarrying patients with various chromosomal rearrangements. Euploid embryos were replaced in all 11 cycles. Four pregnancies ensued, leading to three live births and one “biochemical” pregnancy. It is still too early to say whether this live birth rate is different to the 70% live birth rate in the series by Goddijn et al. (11) or the current result of 55.3% live births. The report by Rubio et al. (15) showed that most embryos with chromosomal aberrations failed to grow in culture. Hence, patients forming embryos with severe chromosomal aberrations may present with infertility rather than recurrent miscarriage. Accordingly, the formation of chromosomally abnormal embryos may explain the 32% infertility rate (16) seen in recurrently miscarrying patients and also explain why most karyotyped embryos are euploid in recurrent miscarriage. Pregestational diagnosis is only worthwhile if the patient with parental aberrations loses embryos with a karyotypic abnormality. In patients losing karyotypically normal embryos, pregestational diagnosis will not lead to a subsequent live birth, as the cause of pregnancy loss is more likely to be maternal in origin. In these cases treatment of maternal causes is appropriate. It still remains to be determined whether an IVF procedure with embryo biopsy to select karyotypically normal embryos for transfer can improve on the success rate of natural reproduction. Even if one anomalous embryo is lost, it is doubtful whether pregestational diagnosis is warranted. Although Coulam et al. (17) reported a theoretical risk of 35% of a repeated chromosomal anomaly after three losses increasing to 70% after six losses, Warburton et al. (18) reported that there was no increased risk for trisomy in a second spontaneous abortion after either a previous trisomic abortion or an abortion with another abnormal karyotype. In this series, 10 patients with a parental karyotypic aberration had two subsequent miscarriages karyotyped. Five had an initally euploidic miscarriage, one miscarried an embryo that failed to grow in culture. Of two patients with an initially trisomic abortus unrelated to the parental karyotypes, one miscarried a subsequently euploid abortus, and one miscarried a subsequent trisomic abortus. Of the two patients who initially lost an embryo with a balanced translocation, one subsequently 449
miscarried an embryo with the same balanced translocation, and one lost a euploidic embryo subsequently. Therefore it may not be enough to show aneuploidy in the abortus, it may be necessary to show repeat aneuploidy to determine the need for interventions such as pregestational diagnosis. If the subsequent pregnancy continues developing, in the presence of parental chromosomal aberations, the question arises as to whether chorionic villus sampling (CVS) or amniocentesis are indicated. Esrig and Leonardi (19) have reported an increased incidence of pregnancy loss after amniocentesis in patients with previous miscarriages compared to patients without previous miscarriages. However, Esrig and Leonardie (19) did not define when the previous miscarriages occurred in the first or second trimesters in their series, nor did they examine recurrent miscarriage specifically. Carp et al. (20) reported on 70 amniocenteses in recurrent miscarriage. There was not one subsequent abortion. Goddijn et al. (11) performed 26 amniocenteses in patients with recurrent miscarriage and parental karyotypic rearrangements. Fourteen pregnancies were euploid, 12 had balanced rearrangements similar to the parents. There were no unbalanced translocations or inversions. In this series 10 abortuses had karyotypic aberrations identical to the parents. If these chromosomal rearrangements were found at amniocentesis, it would be assumed that the transmission of the chromosome anomaly would have no phenotypic effect as the carrier is nornal. In these cases, abortion would not be advised. Although we offer amniocentesis to all patients with a parental karyotypic aberration, even if the woman is below the age where prenatal diagnosis would be advised, in our opinion, all these factors should be taken into account before advising genetic testing. Although parental karyotyping is part of the standard management of recurrent miscarriage, these results concur with those of Clark et al. (21), indicating that parental karyotyping is of limited value in diagnosing the cause, prognosis, or mode of treatment for recurrent miscarriage. Parental karyotyping is, at best, an indirect measure of the fetal karyotype. In our opinion, treatment and prognosis depend on the fetal karyotype. Hence, the fetal karyotype should be determined directly at abortion. Newer techniques such as embryoscopy (22) and comparative genomic hybridization (23) should improve the accuracy of fetal karyotyping. Parental karyotyping is a poor substitute.
1. Salat-Baroux J. Recurrent spontaneous miscarriages. Reprod Nutr Dev 1988;28:1555– 68. 2. Crosignani PC, Rubin BL. Recurrent spontaneous miscarriage. The recommendations of the ESHRE workshop on recurrent spontaneous miscarriage held in Anacapri on September 9 –11, 1990. Hum Reprod 1991;6:609 –10. 3. American College of Obstetricians and Gynaecologists. Management of recurrent early pregnancy loss. ACOG Practice Bulletin 2001;24:1–12. 4. Royal College of Obstetricians and Gynaecologists, Guideline No. 17. The investigation and treatment of couples with recurrent
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