April 2005 ⅐ Vol. 7 ⅐ No. 4 article Incidence and spectrum of chromosome abnormalities in spontaneous abortions: New insights from a 12-year study *Joshua Menasha, MD1, *Brynn Levy, MSc (Med), PhD1–3, Kurt Hirschhorn, MD1,2, and Nataline B. Kardon, MD1–3 Purpose: Despite advances in harvesting and culturing techniques, analysis of the impact of these improvements on the observed frequency of chromosomal abnormalities in spontaneous abortions (SAB) has not been deter- mined. We sought to evaluate the effect of these refinements on the success rate of our cultures and on the resulting frequency of detected chromosomal abnormalities. Methods: Between 1990 and 2002, 2301 specimens obtained from the products of conception (POC) of SABs were submitted to our laboratory for cytogenetic analysis. Due to refinements in specimen processing and culture techniques introduced at the end of 1997, our data were analyzed for two periods: Period A from 1990 through 1997 with 907 eligible specimens and Period B from 1998 through 2002 with 1273 eligible specimens. Results: Modifications in physician communication and sample processing contributed to significant improvements in the culture success rate and in the ratio of male-to-female cases with normal karyotypes. Additionally, increased detection of trisomic, triploid, and multiple aneuploid cases in Period B resulted in a significant increase in the percentage of cases with abnormal karyotypes (42.8% in Period A vs. 65.8% in Period B). Monosomy X accounted for Ͻ 10% of all abnormalities in Period B. Eighty five multiple aneuploid karyotypes, including 57 double trisomies, comprised 7.7% of our 1099 abnormal cases. These karyotypes were detected predominantly in POCs from the older women in our study. This collection of multiple aneuploidies is the largest published to date and includes abnormalities not reported in prior studies. We also present a table empirically derived from the data in Period B that indicates the likelihood of a specific abnormal karyotype based on maternal age. The table can be utilized by health care providers, who counsel patients after a spontaneous miscarriage. Conclusion: Improvements in laboratory technique have led to reduced contamination and growth failure of POCs, irrespective of maternal age. This in turn has led to a more balanced male-to-female ratio and to the detection of an increased number of abnormal cases. Genet Med 2005:7(4):251–263. Key Words: cytogenetics, spontaneous abortion, aneuploidy, karyotype, chromosome abnormality A correlation between chromosomal abnormalities and thus could not be evaluated for abnormalities. The data culled spontaneous abortions (SABs) has been observed since the from these studies have served as the basis for the estimated 1960s.1 This correlation was strengthened in the 1970s when abnormality rates in the general population even to this day. Boue et al.2 published one of the earliest large cytogenetic stud- In the mid-1980s, a shift in cytogenetic techniques emerged, ies. In the study, almost 1500 samples of fetal tissue were karyo- as studies utilizing chorionic villi from POCs began to appear typed and an abnormality rate of over 60% was found. Subse- in the literature.10–14 These studies had a much lower rate of quent large studies using harvested products of conception culture failure and a higher abnormality rate. Unfortunately, (POC) failed to match this abnormality rate, with rates of most of these studies were of small sample size and some in- 32%–54% reported.3–9 Furthermore, a large number of speci- volved women of more advanced age. This improvement in mens in these studies failed to grow successfully in culture and methodology, coupled with advances in the detection of early pregnancy, supported the belief that the incidence of chromo- some abnormalities was greater than previously estimated.15,16 From the 1Departments of Human Genetics, 2Pediatrics, 3Obstetrics, Gynecology, and Repro- Certain trends, however, have remained unchanged. Aneu- ductive Sciences, Mount Sinai School of Medicine, New York, New York. ploidy continues to account for the largest proportion of ab- Dr. Brynn Levy, Departments of Human Genetics, Mount Sinai School of Medicine, normalities observed in POCs.17 Although many theories have Box 1497, One Gustave L. Levy Place, New York, NY 10029. been proposed, the exact causes for this high rate of aneuploid *Both authors contributed equally to this study. conceptions in humans have remained unclear.18–20 Further- Received: August 31, 2004. more, multiple aneuploidies continue to be reported infre- Accepted: January 21, 2005. quently in the literature, predominantly as sporadic case re- DOI: 10.1097/01.GIM.0000160075.96707.04 ports, some involving liveborn conceptuses.21–23 Genetics IN Medicine 251 knowledge of cytogenetic abnormalities in SABs. update and to addabnormalities in an POCs. With additional this study, perspective weous hope methodological to to provide changes an the have had current ona the 12-year detection period rate in of an attempt to assess the impact theserent vari- reproductive trends, is needed torepresentation serve of current as abnormality a rates, comparison. which reflectto cur- determine the impact of these new techniques, an accurate to evaluate POC samples that havesitu failed hybridization to (iFISH) grow. has allowed cytogenetic laboratories genomic hybridization (CGH) and interphase fluorescenceprove in our diagnostic abilities. The utilization of comparative logbooks. After April 1997,and results April 1997, were results and entered demographic data into wereJanuary recorded a 1990 in through pass- December 2002. Betweenthe January specimens 1990 processed by theicine. Data cytogenetics were laboratory collected retrospectively from from recordsInstitutional of Review all Board of of the Mount Sinai School of Med- cytogenetic analysis of the POC wasobstetrician cancelled. was notified and,or in fetal parts most were recovered cases, fromstudies, the the is specimen, request referred the to attending for in thisflasks. article This as method, CV and harvesting. similar If methods no reportedCVS villi in protocol) previous in additionwere to centrifuged and being plated setDNAase on up coverslips was in (in used situ tissue toenzyme diagnostic culture digest mixture the consisting POC of collagenase, villi.villi hyauronidase, from The and placental digested tissue cells ascess. well as A tissue diligent from effort fetal was parts. made An to carefully dissectnoted chorionic a request for cytogenetic studies. netics laboratory before placing specimens intion, formalin, the if Pathology they Department agreedtion to contact of the the cytoge- specimenthe to cytogenetics laboratory the then forwarded Pathology the remaining Department. por- the In cytogenetics laboratory. addi- After culture initiation, thespecimens staff directly of from the operating roomring or obstetricians their were offices contacted to andchange instructed the to specimen send handling their and culturing processes. Refer- POC harvesting. in previous studies, isattached. This referred method, to along with throughoutaddition similar of this appropriate techniques tissue utilized article culture media as after themanually cells were and placed insolid a tissue T-25 techniques. tissueDepartment. Small culture The pieces flask samples of withcytogenetics were the tissue laboratory then after were being set processed minced infrom up the patients Pathology using in our standard institution were usually forwarded to the MATERIALS AND METHODS Menasha et al. 252 In this report, we present a large collection of data spanning Advances in technology and techniques continue to im- The study protocol was submitted to and approved by the In 1997, modifications were also made in the culturing pro- In the latter part of 1997, a concerted effort was made to Between 1990 and 1997, spontaneously aborted samples 24,25 In order Table 1 Specimen and karyotype breakdown per period Specimens Contaminated (% of No growth (% of Total Normal (% of 46,XY (% of 46,XX (% of Abnormal (% of set up specimens set up) submitted cases) analyzed analyzed cases) normal cases) normal cases) analyzed cases) Aneuploid Polyploid Structural 82.1%a 13%a 4.9%a Period A 907 6.2% 14.8% 717 57.2% 24.6% 75.4% 42.8% 35.2%b 5.6%b 2.1%b (n ϭ 56) (n ϭ 134) (n ϭ 410) (n ϭ 101) (n ϭ 309) (n ϭ 307) (n ϭ 252) (n ϭ 40) (n ϭ 15) 81.9%a 14.1%a 3.9%a Period B 1273 0.7% 4.8% 1203 34.2% 41.6% 58.4% 65.8% 54.0%b 9.3%b 2.6%b (n ϭ 9) (n ϭ 61) (n ϭ 411) (n ϭ 171) (n ϭ 240) (n ϭ 792) (n ϭ 649) (n ϭ 112) (n ϭ 31) Genetics Ͻ 0.05 Ͻ 0.05 Ͻ 0.05 Ͻ 0.05 Ͻ 0.05 Ͻ 0.05 0.96a 0.63a 0.47a P value Ͻ 0.05b Ͻ 0.05b 0.50b IN Total 2180 65 195 1920 821 272 549 1099 901 152 46 Medicine a% of abnormal cases. b% of analyzed cases. Chromosome abnormalities in spontaneous abortions word-protected computerized database. Only those patients Aneuploidy with documented spontaneous abortions were used in the Cases within the aneuploid group were divided into trisomy, study. POC specimens from induced abortions sent to the lab- monosomy, multisomy, and mosaic categories (Table 2). oratory for confirmation of abnormal results obtained by CVS or amniocentesis were not included in the study. Trisomy Of the initial 2301 specimens eligible for study, 121 were The proportion of aneuploid cases and the proportion of excluded, thus bringing the total to 2180. Reasons for exclu- abnormal cases that were trisomies did not significantly differ sion included cancellation by the ordering physician, submis- between the two periods. Trisomies, however, composed a sig- sion for research purposes, or inappropriate collection of the nificantly larger proportion of all of the cases analyzed in Pe- POC. riod B compared to Period A. Results from each year were analyzed and divided into two Figure 1 shows the chromosome distribution of the 723 specific blocks of time, with 1990 to 1997 forming Period A and cases with an extra chromosome.