Frequency of Chromosomal Abnormalities in Products of Conception: A Retrospective, Large-scale, Single-center study

INTRODUCTION

The major cause of pregnancy loss is due to fetal chromosomal aberrations. In view of this, it becomes essential to undertake laboratory investigations on the products of conception (POC). Results of such investigations will unravel the potential reasons for recurrent miscarriage.¹ Karyotyping has been the gold standard method to analyze the POC samples.^2,3 Karyotyping helps in identification of both the structural (translocations, deletions, and inversions) and the numerical chromosomal abnormalities. One of the major drawbacks of karyotyping is that it consumes substantial diagnosis time. In comparison to other tissues, the rate of culture failure in POC is relatively higher. This is because POC tissue demands persistent maceration and fixation in formalin, which may render it to relatively higher chances of contamination. Overall, this may hamper the success of diagnosis rate of chromosomal abnormalities in POC samples.

Under this background, molecular analyzes of POC samples to identify chromosomal aberrations have been proven useful. Molecular diagnostic tests exclusively study fetal DNA, without the need for culturing and fixing tissues. Advanced molecular diagnostic tests involve probe-based identification of chromosomal aberrations. The probe-based molecular diagnostic tests include fluorescence in situ hybridization (FISH), quantitative fluorescent polymerase chain reaction (QF-PCR), microarray and multiplex ligation-dependent probe amplification (MLPA).⁴

The current study focused on identifying the frequency of chromosomal aberrations in POC samples. The study protocol was approved by an independent ethics committee. The study obeyed the principles of good clinical practice and the Declaration of Helsinki.

MATERIALS AND METHODS

RESULTS

Our study included cytogenetic reports of 1,386 POC samples. Table 1 illustrates the incidences of normal and abnormal results analyzed by the karyotyping technique. Of the total 1,386 cases, 1150 (82.97%) showed apparently normal karyotype pattern or normal diploid status for 13/16/18/21/22/X and Y in FISH, while 236 (17.03%) cases showed chromosomal abnormalities. Of the 1,386 cases, 1058 (76.33%) showed female pattern and 328 (23.67%) showed male pattern. Among the aneuploidy cases, cytogenetic results revealed 36.02% trisomy, 0.85% double trisomy, 38.14% monosomy X, 15.25% triploidy, 8.90% tetraploidy, and 0.85% derivative. The most common chromosome aberration reported was monosomy X (38.14%). Trisomy 21 was detected in 14.41% cases, followed by trisomy 22 in 9.32% cases. Trisomy 18 and trisomy 16 were identified in 5.51% cases. This was followed by trisomy 13 (4.24%) in 10 cases. And, a solitary case of trisomy 20 was also identified. Of the total anomalies, triploidy and tetraploidy accounted for 15.25 and 8.90%, respectively. Out of 36 triploidy cases, XXY detected in 21 (58.33%) cases and 2019 detected in 15 (41.67%) cases. Also, among the 21 tetraploidy cases, 2019X was detected in 9 (42.86%) cases and XXYY was detected in 12 (57.14%) cases. Of the 236 abnormal cases, 209 (88.56%) showed female pattern and 27 (11.44%) showed male pattern. Of the 236 cases, 2 cases (0.85%) of structural abnormalities in the form of derivative were observed. Only 1 case (0.42%) with trisomy for 13 and 21 and 1 case (0.42%) with trisomy for 15 and 21 were detected. Total number of 71 cases (5.12%) with mosaic pattern XX/XY was also observed, but this is not taken into consideration because of the possibility of maternal contamination (Fig. 1).

DISCUSSION

The POC remain an ideal tissue for elucidating recurrent pregnancy loss (RPL) by cytogenetic testing. Investigation results of chromosomal abnormalities in POC samples assist in determining the exact causes of RPL, which, in turn, help genetic counselors and physicians to support the couple about future pregnancy.^5–7

In several instances of RPLs, cytogenetic reports of both mother and father reveal normal karyotype. Recurrent pregnancy loss in such circumstances can be due to fetal chromosomal abnormality during cell division errors or gonadal mosaicism.

Under such challenging circumstances, comprehensive assessment of POC samples becomes critical to ascertain accurate genetic reason for recurrent loss in pregnancy.

Our study reported aneuploidy in 17.03% of the POC samples. Fetal chromosomal abnormalities are the most common cause in early pregnancy loss, and they contribute the maximum for all the spontaneous abortions. Majority of the pregnancy losses with genetic as etiology show numerical abnormalities, and the most commonly seen is monosomy X, followed by triploidy, tetraploidy, and various autosomal numerical abnormalities. Chromosomal karyotype is the gold standard test for detecting numerical as well as structural chromosomal abnormalities, while FISH detects only numerical abnormalities. The POC specimen mostly shows high rates of culture failure since it suffers from maternal cell contamination. In such cases, FISH testing can be very important since FISH can be done directly on the available cells in interphase stage; while for karyotyping, we have to set up a long-term culture to make the cell divide and redivide to obtain a satisfactory numbers of cells and then to arrest the cell division to get the metaphases to study.^8,9

In view of these technical challenges and to improve the success rate along with making it cost-effective, our laboratory implemented a reflex testing strategy. In reflex testing strategy, if our laboratory does not obtain a karyotype/culture, then the case will be taken for FISH testing, which involves common aneuploidy detection, and reported accordingly. The reason for devising this strategy is that the repeat sampling of POC specimen cannot be achieved. Wherever applicable, with the consent of respective clinician and the concerned patient, the clinical specimen is taken for an additional FISH testing especially for microdeletion studies or the test is converted to microarray analysis.

Overall, this laboratory research study has successfully achieved the objective of finding the incidences of chromosomal defects from a large number of POC samples received from tertiary care laboratory.

CONCLUSION

Fluoroscent in situ hybridization offers rapid diagnosis of chromosomal alterations from POC specimens in lower costs over karyotyping methods. In spite of advantages, FISH fails to accurately diagnose chromosomal duplications, small deletions, chromosomal translocations, and mosaicism. Therefore, our study recommended that FISH complemented with karyotyping will cover all chromosomal alterations in POC samples.

REFERENCES

1. Teles TMA, Marques de Paula CM, Ramos MG, et al. Frequency of chromosomal abnormalities in products of conception. Rev Bras Ginecol Obstet 2017;39(3):14.

2. Wan TSK. Cancer cytogenetics: Methodology revisited. Ann Lab Med 2014;34(6):413–425. DOI: 10.3343/alm.2014.34.6.413.

3. Shah MS, Cinnioglu C, Maisenbacher M, et al. Comparison of cytogenetics and molecular karyotyping for chromosome testing of miscarriage specimens. Fertil Steril 2017;107(4):1028–1033. DOI: 10.1016/j.fertnstert.2017.01.022.

4. Moraes AC, Moron AF, Hashimoto EM, et al. Cytogenetic and molecular evaluation of spontaneous abortion samples. Rev Bras Ginecol Obstet 2005;27(9):554–560. DOI: 10.1590/S0100-72032005000900009.

5. López AGA, Huerta SB, Galván RH, et al. Diagnóstico citogenético en aborto espontáneo del primer trimestre. Ginecol Obstet Méx 2003;79(12):779–784.

6. Liu S, Song L, Cram DS, Xiong L, et al. Traditional karyotyping vs copy number variation sequencing for detection of chromosomal abnormalities associated with spontaneous miscarriage. Ultrasound Obstet Gynecol 2015;46(4):472–477. DOI: 10.1002/uog.14849.

7. Lathi G, Gray Hazard RB, Heerema-McKenney FK, et al. First trimester miscarriage evaluation. Semin Reprod Med 2011;29(6):463–469. DOI: 10.1055/s-0031-1293200.

8. Wan TS, Ma ES. Molecular cytogenetics: an indispensable tool for cancer diagnosis. Chang Gung Med J 2012;35(2):96–110. DOI: 10.4103/2319-4170.106161.

9. Wan TS, Ma ES. The role of FISH in hematologic cancer. Int J Hematol Oncol 2012;1(1):71–86. DOI: 10.2217/ijh.12.9.