RECURRENT PREGNANCY LOSS

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1 RECURRENT PREGNANCY LOSS FERTILITY AND STERILITY VOL. 81, NO. 5, MAY 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Parental karyotype and subsequent live births in recurrent miscarriage Howard Carp, M.B., B.S., a,c Baruch Feldman, M.D., Ph.D., a,b,c Gabriel Oelsner, M.D., a,c and Eyal Schiff, M.D. a,c Sheba Medical Center, Tel Hashomer, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Objective: To compare the subsequent live birth rate in recurrently miscarrying women with and without parental balanced chromosomal aberrations. Design: Retrospective comparative cohort study. Setting: Tertiary referral unit in a university hospital. Patient(s): Nine hundred sixteen patients with 3 16 miscarriages before 20 weeks: 99 patients with and 817 patients without chromosomal aberrations. Intervention(s): None. Main Outcome Measure(s): Outcome of the subsequent pregnancy in terms of live births or repeat miscarriage. Result(s): Of the 916 patients, 661 subsequently conceived, 73 (73.7%) with parental chromosomal aberrations and 588 (71.9%) without aberrations. In patients with and without chromosomal aberrations, 33 of 73 pregnancies (45.2%) and 325 of 588 pregnancies (55.3%), respectively, resulted in live births. The difference is not statistically significant. There was a similar prevalence of aberrations in primary, secondary, and tertiary aborters. The prevalence of aberrations was not related to the number of previous miscarriages. Translocations, inversions, and mosaicism were followed by a similar live birth rate. Conclusion(s): Patients with parental chromosomal rearrangements do not have a significantly lower live birth rate than patients without aberrations. Parental karyotyping might not be a good predictor of the outcome of subsequent pregnancies. (Fertil Steril 2004;81: by American Society for Reproductive Medicine.) Key Words: Abortion, karyotype, recurrent miscarriage, pregnancy loss Received November 13, 2002; revised and accepted September 11, Reprint requests: Howard Carp, M.B., B.S., FRCOG, Department of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer 52621, Israel (FAX: ; E- mail: carp@netvision.net.il). a Department of Obstetrics and Gynecology, Sheba Medical Center. b Institute of Genetics, Sheba Medical Center. c Sackler Faculty of Medicine, Tel Aviv University /04/$30.00 doi: /j.fertnstert Up to 60% of sporadic miscarriages (1 3) and between 29% and 60% of recurrent miscarriages (defined as the loss of three or more consecutive pregnancies before 20 weeks of pregnancy) might be caused by chromosomal aberrations in the embryo (4 6). In recurrent miscarriage, it is common clinical practice to karyotype both parents for chromosome aberrations, as officially recommended by both the American (7) and Royal (8) Colleges of Obstetricians and Gynecologists. It is generally assumed that recurrent miscarriage might be due to recurrent chromosomal anomalies in the fetus due to a balanced aberration in one of the parents being inherited by the offspring in an unbalanced form. The parental chromosomal aberration might be either a structural anomaly, such as reciprocal or Robertsonian translocations, or mosaicism for numeric aberrations. However, little information is available as to prognosis, compared with patients with unexplained pregnancy losses. The Sheba Medical Center is a tertiary referral center for patients with recurrent miscarriage. For the last 20 years, it has been our practice to karyotype both parents. This article summarizes the incidence and type of chromosomal anomalies in both partners with recurrent miscarriage and the prognosis for a subsequent live birth. MATERIALS AND METHODS A total of 916 patients presented to our service with three or more consecutive miscarriages before 20 weeks gestation between 1984 and Both partners were karyotyped as part of the primary investigation. Other presumptive causes of abortion were also investi- 1296

2 TABLE 1 Details of patients in the study. Chromosomal aberrations No aberrations All patients No. of patients Age (y) (23 43) (20 44) (20 44) No. of abortions (3 10) (3 16) (3 16) Time to conceive (mo) (1 90) (1 60) (1 90) Note: Data are presented as mean SD (range). gated, according to our previously described criteria (9), with [1] glucose tolerance test, [2] toxoplasmosis serology, [3] hysterosalpingogram or hysteroscopy to diagnose anatomic abnormalities, intrauterine adhesions, and cervical incompetence, [4] thyroid function assessment, [5] serum PRL measurement, [6] luteal phase assessment according to length of luteal phase and midluteal plasma P, [7] antinuclear factor assessment, by use of rat liver as substrate and fluoresceinated rabbit anti-human immunoglobulin G or by immunofluorescence (Hep 2000 kit; Immunoconcepts, Sacramento, CA), and [8] assessment of anticardiolipin antibody by enzyme-linked immunosorbent assay and lupus anticoagulant, according to the kaolin clotting time or Russell s viper venom time. Before inclusion in the study, objective evidence of past pregnancies was required, such as a positive hcg test, histology of fetal or placental tissue, or ultrasound-based reports of a pregnancy sac. Peripheral Blood Karyotyping Chromosome studies were performed on routinely cultured peripheral blood lymphocytes. Slides were processed for G-banding with Trypsin-Giemsa by standard techniques. Colchicine was added 4 hours before the cytologic preparation. A minimum of 15 cells were microscopically analyzed at metaphase from two independent cultures prepared for each individual. Follow-Up All patients with parental aberrations were offered genetic counseling. The details of each individual were entered into a computerized database with statistical software (SPSS, Chicago, IL). The clinical features of each patient and her miscarriages were recorded, with particular attention paid to whether the previous miscarriages occurred in the first or second trimester, or whether the patients were primary, secondary, or tertiary aborters. Patients were classified as primary aborters if they had no previous live births, secondary if there was a live birth followed by miscarriages. Tertiary aborters had abortions followed by a live birth and at least three subsequent abortions (10). 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 Analysis was performed by Pearson s 2 test and by Fisher s exact test when the numbers were small. A difference was considered to be significant at P.05. The relative risk ratio for a live birth was calculated with 95% confidence intervals where appropriate. RESULTS A total of 916 couples were karyotyped. A karyotypic aberration was found in 99 couples (10.8%). Table 1 shows the details of the patients in the study with regard to age and total number of miscarriages. As can be seen, there were no significant age differences between patients with chromosomal aberrations and those with no aberrations; the mean number of miscarriages was also similar in both groups of patients. Table 2 shows the prevalence of parental chromosomal aberrations according to the number of previous miscarriages. The prevalence of parental chromosomal aberrations was similar irrespective of the number of previous miscarriages. The prevalence of parental chromosomal aberrations was also similar among primary, secondary, and tertiary aborters (Table 3). The slightly higher prevalence in tertiary aborters was not statistically significant. TABLE 2 Prevalence of chromosomal aberrations according to number of previous miscarriages. No. of previous miscarriages Chromosomal aberrations No aberrations Total (10.6) 571 (89.4) (10.8) 246 (89.2) 277 Total 99 (10.8) 817 (89.2) 916 Note: Data are presented as n (%). P not significant ( 2 ) for the difference between 3 or 4, or 5 or more miscarriages. FERTILITY & STERILITY 1297

3 TABLE 3 Parental karyotype according to primary, secondary, or tertiary aborter status. TABLE 4 Details of anomalies found in parents. Anomaly Chromosomal aberrations No aberrations Total Primary 45 (10.6) 477 (89.4) 522 Secondary 42 (13.2) 276 (86.8) 318 Tertiary 12 (15.8) 64 (84.2) 76 Total 99 (10.8) 817 (89.2) 916 Note: Data are presented as n (%). P not significant ( 2 ) for the difference between primary and secondary or tertiary aborters. Maternal balanced translocation 31 Paternal balanced translocation 21 Maternal mosaic for numeric aberration 18 Paternal mosaic for numeric aberration 3 Maternal inversion 6 Paternal inversion 19 Chromosomal inversion in both parents 1 Total 99 No. Seventy-three of the 99 patients with chromosomal aberrations conceived (73.7%), and 588 of the 817 patients with no aberrations conceived (71.9%). However, the time to conception was longer in patients with chromosomal aberrations (mean SD, months, range 1 90) than in patients with no chromosomal aberrations ( months, range 1 60). Table 4 shows the details of the aberrations found. The most frequent chromosomal rearrangement was balanced translocations, which were found in 52 of the 99 patients tested. Translocations were found more often in the female partner than in the male, but this difference was not statistically significant. The second most prevalent anomaly was chromosomal inversions, which were found in 26 of the patients, with a significantly greater prevalence in the male partner (P.005). Mosaics were found for numeric aberrations in 21 patients; there was a significantly greater prevalence in the female partner (P.004). Table 5 shows the outcome of the subsequent pregnancy, according to the chromosomal status of the parents. It can be seen that the presence of a chromosomal aberration increased the relative risk of a subsequent pregnancy loss by 43%, but this risk was not statistically significant. The relative risk ratio of a live birth was also not statistically different in the presence of chromosomal aberrations when compared with the original number of patients enrolled in the study. Figure 1 shows the subsequent live birth rate according to the number of previous miscarriages. The live birth rate fell slightly as the number of previous miscarriages increased. The figures were not statistically significant for any number of previous miscarriages. The live birth rate was not different in subsequent pregnancies, whether the aberrations were translocations, inversions, or numeric mosaics (Table 6). In addition, the subsequent live birth rate was identical whether the chromosomal aberration was maternally or paternally derived. The live birth rate was 52.6% (10/19) for maternally derived, balanced translocations and 47.4% (9/19) (P nonsignificant [NS]) for paternally derived translocations. With regard to inversions, the live birth rate was 40% (3/8) and 60% (5/8) (P NS) for maternally and paternally derived inversions, respectively. In the case of numeric mosaics, all 6 live births occurred when the mother carried the mosaicism. DISCUSSION The prevalence of parental chromosomal aberrations was greater in this series (10.8%) than in most series in the literature, which quote a 3% 5% prevalence (11 13). We originally thought that the high prevalence might have been due to the relatively high number of previous miscarriages in this cohort of patients ( ). However, as Table 2 shows, the prevalence of chromosomal aberrations was independent of the number of previous abortions. The high prevalence might be because Sheba Medical Center is a tertiary referral unit that receives patients for whom alternative causes of abortion, such as antiphospholipid syndrome and uterine anomalies, have been excluded. Patients with alternative causes for miscarriage might not have been referred, thus leading to overrepresentation of parental chromosome aberrations. In this series, the presence of a parental chromosomal aberration decreased the chance of a live birth from 55.3% to 45.2%. However, the increased risk of subsequent miscarriage was not statistically significant. Power analysis (14) has shown that the present trend needs to continue for 2,265 patients to reach statistical significance. Analysis of the live birth rate according to the number of previous miscarriages (Fig. 1) showed that the incidence of live births decreased with the number of previous miscarriages, as has been described by other investigators (15, 16), but there was no significant difference in birth rates for any particular number of abortions. The time to conception was greater in couples with chromosomal aberrations ( months, compared with months in couples with no chromosomal aberrations). It is possible that a parental chromosome abnormality leads to preimplantation or occult losses, which might present as infertility. However, the fact that 73.7% of pa Carp et al. Parental karyotype, births, and recurrent miscarriage Vol. 81, No. 5, May 2004

4 FIGURE 1 Outcome of subsequent pregnancy according to number of previous miscarriages. The relative risk for a further abortion was not statistically significant for any particular number of previous miscarriages for the group as a whole (relative risk 1.43, 95% confidence interval ). tients with chromosomal aberrations conceived, compared with 71.9% in the group with no aberrations, indicates that the cause of the higher (but not significantly so) miscarriage rate (relative risk, 1.43) is recurrent miscarriage rather than prolonged infertility. The only form of therapy that can overcome the presence of chromosomal aberrations in TABLE 5 Outcome of subsequent pregnancy, according to chromosomal status of parents. Chromosomal aberrations No aberrations All patients No. of patients No. of pregnancies (%) 73 (73.7) a 588 (71.9) a 661 (72.2) a No. of subsequent live births (%) 33 (45.2) b 325 (55.3) b 358 (54.2) b Relative risk ratio for miscarriage (95% confidence interval) 1.43 ( ) c 1.00 Relative risk ratio for live birth (95% confidence interval) ( ) d a Proportion of pregnancies per patient in trial. Proportion of live births per pregnancy. Relative risk ratio of miscarriage per pregnancy. Relative risk ratio of live birth per patient enrolled in study. FERTILITY & STERILITY 1299

5 TABLE 6 Outcome of subsequent pregnancy, according to type of parental chromosomal aberration. Translocations Inversions Numeric mosaic Total Live births, n (%) 19/44 (43.2) 8/15 (53.3) 6/14 (42.9) 33/73 (45.2) Note: Live births are quoted as a proportion of the total number of pregnancies. P not significant (Fisher s exact test) for the difference between translocations, inversions, and numeric mosaics. the embryo is pregestational diagnosis (PGD) at IVF. This technique has been reported to result in an approximately 25% 30% pregnancy rate per transfer (17, 18), which is not very different from the pregnancy rate in IVF without PGD. It is generally assumed that in the presence of a parental chromosomal aberration, the previous and subsequent miscarriages are due to the balanced aberration being passed on to the offspring in an unbalanced manner. However, this requires confirmatory evidence from karyotyping of the abortus. In our previous karyotyping series of 125 embryos from recurrent miscarriages (6), only 36 had chromosomal aberrations. Of these, 2 were structural anomalies that could have been inherited, and the other 34 (94%) were trisomies that might have occurred by chance. Boue and Gallano (19) have reported that abnormal chromosomes might be passed on to the embryo in 40% of cases when chorionic villus sampling is used for diagnosis. Hence, 60% of subsequent embryos are euploidic. Most series (19, 20) investigating recurrent miscarriage claim that the untreated subsequent live birth rate is 60% with a 40% abortion rate. In addition, Hassold et al. (21) reported that after karyotyping the fetus, it is unlikely that aneuploidy detectable in peripheral blood preparations is an important indicator of fetal aneuploidy. The only indicator of fetal aneuploidy is karyotyping of the fetus itself. If fetal aneuploidy recurs, PGD might improve the subsequent live birth rate (22, 23). However, there is as yet no report on the subsequent live birth rate per pregnancy in women with three or more miscarriages after PGD. If the patient with a chromosomal aberration aborts a euploidic embryo, PGD will not improve the chance of a live birth. Clark et al. (24) have shown that failure to take fetal aneuploidy into account prevents proper interpretation of the results of treating recurrent pregnancy loss. It is interesting that the type of aberration had no appreciable effect on the subsequent live birth rate, whether the aberration was a translocation, inversion, or numeric mosaicism. Additionally, there was no effect if the aberration was maternally or paternally derived. Although the live birth rate was slightly higher if the chromosomal aberration was maternally derived (57.6% [19/33]), the numbers are too small to draw any conclusions. Fetal karyotyping is necessary to determine whether the incidence of fetal chromosomal aberrations is similar if the aberration is maternally or paternally derived. In the case of the translocation form of Down s syndrome, the recurrence risk is 10% 15% if the mother is the carrier but only 2% if the father is the carrier (19). If parental chromosomal aberrations are present, it is our view that amniocentesis should be offered to the patient in a subsequent pregnancy. Although there is concern that abortion subsequent to the procedure might be more frequent in cases of recurrent miscarriage than in women with no previous pregnancy losses (25), inversions and translocations might undergo crossing over, thus giving rise to new aberrations in which genetic material might be duplicated or deleted from the genome (26). These de novo aberrations might conceivably be insufficient to cause abortion but sufficient to give rise to anomalies. In addition, 89% of recurrent miscarriages occur in the first trimester (27). Hence, amniocentesis performed at 17 weeks occurs long after the stage of the previous miscarriages. Chorionic villus sampling is not recommended because the abortion rate is 3% (28, 29) and if performed at 10 weeks it often occurs close to the time of the previous miscarriages. In conclusion, it seems that further studies are necessary with a large cohort of patients to determine which patients lose genetically aberrant embryos, so that treatment to prevent further pregnancy losses can be specific for these particular patients. Parental karyotyping is a very indirect predictor of fetal aneuploidy, and its predictive value is limited. References 1. Boue J, Boue A, Lazar P. The epidemiology of human spontaneous abortions with chromosomal anomalies. In: Blandau RJ, ed. Aging gametes. Basel: Karger, 1975: Stein Z. Early fetal loss. Birth Defects 1981;17: Sanchez JM, Franzi L, Collia F, De Diaz SL, Panal M, Dubner M. Cytogenetic study of spontaneous abortions by transabdominal villus sampling and direct analysis of villi. Prenat Diagn 1999;19: Stern JJ, Dorfman AD, Gutierez-Najar MD. Frequency of abnormal karyotype among abortuses from women with and without a history of recurrent spontaneous abortion. Fertil Steril 1996;65: Ogasawara M, Aoki K, Okada S, Suzumori K. Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertil Steril 2000;73: Carp HJA, Toder V, Orgad S, Aviram A, Danieli M, Mashiach S, et al. Karyotype of the abortus in recurrent miscarriage. Fertil Steril 2001;5: American College of Obstetricians and Gynecologists. Management of recurrent early pregnancy loss. ACOG practice bulletin. Washington, DC: American College of Obstetricians and Gynecologists, Royal College of Obstetricians and Gynaecologists. Guideline no. 17. The management of recurrent miscarriage. London: Royal College of Obstetricians and Gynaecologists, 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: Carp HJA. Abstracts of contributors individual data submitted to the worldwide prospective observational study on immunotherapy for treatment of recurrent spontaneous abortion. Am J Reprod Immunol 1994; 32: De Braekeleer M, Dao TN. Cytogenetic studies in couples experiencing repeated pregnancy losses. Hum Reprod 1990;5: Carp et al. Parental karyotype, births, and recurrent miscarriage Vol. 81, No. 5, May 2004

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