Placenta
Volume 29, Supplement 2 , Pages 126-128, October 2008

Chromosomal Abnormalities in Miscarriages after Different Assisted Reproduction Procedures

  • D. Bettio

      Affiliations

    • Cytogenetic and Medical Genetic Laboratory, Operative Unit of Clinical Investigations, IRCCS Humanitas Clinical Institute, Via Manzoni, 56, 20089 Rozzano, Milan, Italy
    • Corresponding Author InformationCorresponding author. Tel.: +39 02 82244720; fax: +39 02 82244790.
    • ,
  • A. Venci

      Affiliations

    • Cytogenetic and Medical Genetic Laboratory, Operative Unit of Clinical Investigations, IRCCS Humanitas Clinical Institute, Via Manzoni, 56, 20089 Rozzano, Milan, Italy
    • ,
  • P.E. Levi Setti

      Affiliations

    • Operative Unit of Reproductive Medicine, IRCCS Humanitas Clinical Institute, Rozzano, Milan, Italy

Accepted 14 August 2008.

Article Outline

Abstract 

About 10–15% of all recognised pregnancies end in spontaneous abortion and around 60% of these show a chromosomal abnormality. The finding of an abnormal karyotype allows one to avoid unnecessary and controversial testing and treatment, providing accurate reproductive and genetic counselling to the couple. Consequently, chromosome study of products of conception (POC) is routinely performed in our Institute, starting from 2002. Cytogenetic analysis on spontaneous metaphases, obtained using direct method, was successfully performed on a total of 277 cases: 133 from patients who underwent assisted reproductive technology (ART) and 144 samples from infertile couples that had natural conceptions (NC). An abnormal karyotype was observed in 84 (63.2%) cases after ART and in 103 (71.5%) after NC demonstrating that the ART group doesn't seem subjected to a higher cytogenetic risk due to the application of technical procedures and higher than the expected risk in the fertile population. Moreover we did not observe a significant difference in the incidence of chromosome anomalies between intracytoplasmic sperm injection (ICSI) (61.5%) and in vitro fertilisation (IVF) (54.5%). However, in the ICSI with testicular sperm extraction (ICSI-TESE) group, 80% of the cases were abnormal with 50% showing triploid/tetraploid karyotypes. Chromosomal abnormalities were present in 54.5% of miscarriages which occurred after ICSI with cryo-preserved oocytes (cryo-ICSI) and in 85.7% after intra-uterine insemination (IUI). To our knowledge this study analysed the largest number of POCs after ART and NC in an infertile population. Moreover, for the first time the cytogenetic results of POC from different ART procedures were included.

Keywords: Miscarriages, Cytogenetic analysis, Direct method, Assisted reproductive techniques

 

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1. Introduction 

Foetal wastage has many causes, but genetic factors are by far the most common. The majority of spontaneous abortions occur during the first trimester and a chromosomal abnormality is observed in about 60% of the cases [1]. The cytogenetic anomalies include autosomal trisomies (27%), polyploidies (10%), chromosome X monosomy (9%) and structural rearrangements (2%) [2]. Double trisomies, as well as multiple trisomies, are infrequent, with an incidence of about 0.7% [3] and are considered the result of non-disjunction of two or more pairs of chromosomes in the successive cell lineage of the same zygote [4].

Cytogenetic analysis of products of conception (POC) is essential to determine the cause of the pregnancy loss and brings valuable information in the setting of infertility and assisted reproduction. We report the cytogenetic data obtained from miscarriages occurring in couples after ART, and compare the rate of chromosomal anomalies among different ART procedures. Moreover, we compare the rate of chromosome abnormalities between ART and NC group.

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2. Materials and methods 

Starting from 2002, a total of 362 POC samples of infertile couples were collected for cytogenetic analysis. All the miscarriages occurred in the first trimester (6–12 weeks). The mean maternal age was 36.5 years and 36.6 years for women who underwent ART or had NC, respectively. Samples were collected in sterile containers with 10ml RPMI-1640 w/o l-glutamine medium (Euroclone # ECB9006D) supplemented with 10% Foetal Bovine Serum (Euroclone # ECS0170D), 1% l-glutamine 200mM (Euroclone # ECB3000D). In the Cytogenetic Laboratory a trained technologist dissected and selected placental chorionic villi. In order to avoid maternal contamination and minimize external microbial contamination, chromosome study was performed on spontaneous metaphases obtained using direct preparation [5], [6] after 24–48h incubation at 37°C. At least 10 QFQ-banded metaphases were analysed for each case.

χ2-test and Fisher's exact test were applied for statistical evaluation in order to compare the abnormal karyotype rates between ART and NC group, IVF and ICSI group, ICSI and ICSI-TESE group. P<0.05 was considered statistically significant.

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3. Results 

Chromosome analysis was successfully performed on 277 out of 362 (76.5%) cases: 133 miscarriages occurred after ART and 144 after NC. In 70 of 362 cases (19.3%) we did not get any spontaneous metaphases because of absent or insufficient villi, while 15 samples (4.2%) presented bacterial contamination after 24h incubation. In the ART group, POCs derived from different procedures: 65 ICSI, 10 ICSI-TESE, 11 cryo-ICSI, 33 IVF and 14 IUI. The overall chromosome abnormalities' rate was 67.5%. An abnormal karyotype was observed in 84 of 133 (63.2%) cases of abortions after ART, in particular: 85.7% of IUI, 80% ICSI-TESE, 61.5% ICSI, 54.5% IVF and 54.5% cryo-ICSI (Table 1). In the ICSI-TESE group only autosomal trisomies (50%) or polyploidy (50%) was found (Table 2). An abnormal karyotype was observed in 103 of 144 (71.5%) samples in the NC group. Autosomal trisomy was the most frequent finding in the ART population as well as in the NC one (Table 3). In the ART group, trisomy 16 and 22 (21.2% each) were the most frequent anomalies, followed by trisomy 15 (13.5%), 13 (7.7%) and 7 (5.7%); trisomy 21 was found in one case only (1.9%). In the NC group, trisomy 22 (27.5%) was the most common, followed by trisomy 16 (18.8%), trisomy 15 (13.8%) and trisomy 21 (8.8%). Polyploidy was observed in almost twice as much cases that underwent ART (16.6%), compared to NC (9.7%) (Table 3).

Table 1. Normal vs. abnormal karyotypes of miscarriages after different ART procedures
NormalAbnormalTotal
IVF1518 (54.5%)33
ICSI2540 (61.5%)65
ICSI-TESE28 (80.0%)10
cryo-ICSI56 (54.5%)11
IUI212 (85.7%)14

Total4984 (63.2%)133
Table 2. Comparison of chromosome abnormalities in miscarriages after different ART procedures
Autosomal trisomy n (%)Autosomal double trisomy n (%)Triploidy n (%)Tetraploidy n (%)Monosomy X n (%)Structural anomaliesan (%)
IVF13 (72.2)1 (5.6)1 (5.6)1 (5.6)2 (11.0)–
ICSI23 (57.5)4 (10.0)4 (10.0)3 (7.5)4 (10.0)2 (5.0)
ICSI-TESE4 (50.0)–2 (25.0)b2 (25.0)c––
cryo-ICSI4 (66.7)––––2 (33.3)
IUI8 (66.7)3 (25.0)–1 (8.3)––

Total52 (61.9%)8 (9.5%)7 (8.3%)7 (8.3%)6 (7.2%)4 (4.8%)

ade novo: ICSI: 45,XX,t(13;14)(q10;q10); 46,XY,del(2)(q24q34); cryo-ICSI: mos 46,XY,add(14)(p12)/46,XY; mos 46,XX,add(22p)/46,XX.

b69,XXY; 68,XXX,der(14;21)(q10;q10).

c94,XXXX,+16,+16; mos 94,XXXX,+14,+14[5]/47,XX,+14[5].

Table 3. Comparison of chromosome abnormalities in miscarriages after ART and NC
ART 84/133 (63.2%) (%)NC 103/144 (71.6%) (%)
Autosomal trisomy61.977.7
Autosomal double trisomy9.52.9
Triploidy8.37.8
Tetraploidy8.31.9
Monosomy X7.27.8
Structural anomaliesa4.81.9

ade novo: ART – ICSI: 45,XX,t(13;14)(q10;q10); 46,XY,del(2)(q24q34); cryo-ICSI: mos 46,XY,add(14)(p12)/46,XY; mos 46,XX,add(22p)/46,XX. NC: 46,XY,add(5)(p15.3); 46,XX,del(18)(q21).

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4. Discussion 

Cytogenetic analysis of spontaneous abortions is essential to determine the cause of pregnancy loss and for counselling, especially in the setting of infertility and assisted reproduction. The incidence of chromosomal abnormalities in miscarriages in the general population ranges between 40 and 80%, depending on the culture methods adopted [6], [7], [8], [9]. It is well known that conventional tissue culturing of POC can give a bias in the rate of abnormal karyotypes detected, since the presence of decidua and overgrowth of maternal cells can lead to a normal female karyotype [10]. We applied the direct method with a success rate of 76.5%, and a chromosome abnormality was found in 67.5% of the cases. Our study showed an abnormal karyotype in 63.2% of the POC couples who underwent ART and in 71.5% of the NC cases. The difference was not statistically significant (P=0.13), demonstrating that the ART group didn't show a higher cytogenetic risk compared to the NC one, also in comparison with data from the literature in the normal population [6], [7], [8], [9]. These results cannot be explained by age since the mean maternal age in the ART group was 36.6 years and 36.5 years in the NC group. Although the slight increase in the incidence of karyotypically normal POC observed in our ART group compared to NC turned to be not statistically significant, this evidence could be correlated to gamete selection that possibly reduced the probability of chromosomally abnormal conceptions.

Several investigators have reported an increased rate of chromosomal aberrations in ICSI conceptions, however, the few studies reporting on the incidence of aneuploidy in POCs after IVF or ICSI showed conflicting results [11], [12], [13], [14] (Table 4). In our study we did not observe a significant difference for the total aneuploidy rate in the POC between the two groups, that were 61.5% for ICSI and 54.5% for IVF (P=0.50), but differences in the distribution of chromosome abnormalities in agreement with a previous study [14]. However, those authors reported an increased rate of acrocentric trisomies in the IVF group (32.4%) compared to ICSI group (11%), while we observed a comparable incidence being the acrocentric trisomies 15.5% of the abnormal IVF cases and 19.7% of the ICSI cases. Our data do not support the hypothesis raised by the previous authors that the mode of fertilisation can play a role in the kind of trisomy observed. Although only 10 ICSI-TESE cases were analysed, a great discrepancy between chromosome abnormalities detected in this group (80%) compared to ICSI (61.5%) was observed. Despite the low number of cases and the statistical analysis not reaching-significance (P=0.25), we think that it is important to underline that 50% showed triploid/tetraploid karyotypes, when in the other groups polyploidy did not exceed 17.5% (Table 2). We can't make a comparison with the literature since to our knowledge, this is the first report about the POC chromosome anomalies in this ICSI group. In order to explain the phenomenon we hypothesize that the use of immature testicular spermatozoa confers a higher risk of cytogenetically abnormal conceptions. No correlation was observed between ploidy and the etiology of male infertility and, since the ICSI procedure involves a single spermatozoon microinjected into the oocyte, the hypothesis of dispermy can be excluded [11]. For the triploid karyotype we can speculate that when azoospermia is due to the impairment of the spermatogenesis process, the extraction of immature testicular spermatozoa leads to a higher rate of unreduced diploid sperms. Moreover an incorrect activation of the oocyte by an immature speratozoon could prevent the second polar body expulsion, or during sperm injection, the needle itself could cause cytoskeleton and meiotic spindle perturbations in the oocyte, leading to an arrest in its maturation process [15]. But triploidy and tetraploidy can also be explained by a post-zygotic abnormality, in particular a failure of cytokinesis during the first divisions of a diploid zygote [12]. This last hypothesis well explains the 94,XXXX,+16,+16, and the 94,XXXX,+14,+14[5]/47,XX,+14[5] karyotypes we observed in two case of ICSI-TESE (Table 2). The cryo-ICSI group is the only one in which we did not find polyploidy supporting the idea that the major cause of polyploidy in the ICSI-TESE could be the use of immature sperm that can also cause a post-zygotic error rather than a teratogenic effect by ICSI itself [16]. In the IUI group the very high rate of chromosome abnormalities found is not statistically significant, compared to the ART group presenting an abnormal karyotype (P=0.09), due to the low number of cases investigated; since the mean maternal age was 36.5 this could not be the explanation of our results. Finally the comparison between the ART and NC groups did not show a statistically significant difference in the rate of POC chromosome abnormalities. Autosomal trisomy represented the most frequent anomaly with a lower incidence in ART (61.9%) compared to NC (77.7%) (Table 3). In conclusion our data showed that couples that underwent ART were not subjected to a higher cytogenetic risk compared to the NC group in our study and the fertile population investigated in other studies. On the contrary, infertile couples that had NC had a slightly higher risk of chromosomally abnormal conceptions, suggesting the importance of gametes selection in infertile patients. Moreover a statistically significant difference was not observed between the IVF and ICSI groups. Finally we found a very high percentage of anomalies in the ICSI-TESE group, especially triploid/tetraploid karyotypes that require a more extensive investigation. Further studies are needed in order to confirm our results.

Table 4. Incidence of chromosome abnormalities in miscarriages after ICSI and IVF
AuthorCytogenetic methodIVF n (%)ICSI n (%)
Present studyDirect18/33 (54.5)40/65 (61.5)
Causio et al. [12]Direct/culture15/35 (42.9)14/29 (48.3)
Lathi and Milki [13]Culture16/38 (41)16/21 (76)
Ma et al. [14]Culture24/34 (71)27/46 (59)

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5. Conflict of interest 

All authors declare that there are no financial and personal relationships with other people or organisations that can inappropriately influence (bias) their work.

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Acknowledgements 

The Authors thank the clinical and laboratory staff of the Operative Unit of Reproductive Medicine and Dr. Emanuela Morenghi of the Biometrical Unit for statistical analysis. We also thank Dr. Silvia Stolfi for the initial contribution to the manuscript.

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References 

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PII: S0143-4004(08)00261-0

doi:10.1016/j.placenta.2008.08.015

Placenta
Volume 29, Supplement 2 , Pages 126-128, October 2008

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