IRecurrent miscarriage (RM), one of the most frus.
trating problems faced by both patients and clinicians,
is recently defined by the American Society for Reproductive Medicine as the miscarriage of two or moreconsecutive pregnancies in the first or early secondtrimester of gestation (
When conventional cytogenetic techniques are used,
balanced chromosomal anomalies are detected inabout 5% of RM cases (
In the past decade, subtelomeric rearrangements
have been shown to be implicated in congenital malformations and intellectual disabilities (ID) (
Nevertheless, the effectiveness of such a tool in RM cases is still unclear and the exact incidence of such rearrangements remains uncertain. In this study, we screened subtelomeric regions in 21 couples having experienced two or more spontaneous abortions with or without stillbirth and/or children with CA to examine the rate of cryptic subtelomeric translocations in RM.
This cross-sectional study was undertaken in the Department of Cytogenetic and Reproductive Biology at Farhat Hached University Teaching Hospital (Sousse, Tunisia). We selected 21 clinically normal couples, from 01/07/2012 to 31/07/2013, based on the inclusion criteria of having at least two abortions and exhibiting “normal” karyotypes. These couples had normal endocrine function and had no medically assisted procreation attempt in the study period. The women had normal ovarian function, normal genital organs and had no anterior toxic exposure, trauma, radiotherapy, chemotherapy, chronic diseases or medications. The local Ethics Board approved the present study (IRB00008931) and all patients gave informed consent for this study.
R banding karyotyping on peripheral blood lymphocyte cultures was carried out systematically. FISH based on Vysis ToTelVysion Multi-Color FISH Probe Kit (Abbott ® Molecular Inc., Des Plaines, USA) was performed for the 21 couples according to the standard protocol. This kit contained 41 TelVysion probes which were specific to subtelomeric regions of chromosomes 1-12 and 16-20, subtelomeres of the q arm of acrocentric chromosomes and pseudo-autosomal region subtelomeres (Xp/Yp and Xq/Yq). For each chromosome, we analyzed at least ten cells and in case of translocations, more metaphases were considered.
Among the 21 selected couples, only one cryptic subtelomeric translocation was found in the female partner of the 21st couple who were referred to the Obstetrics and Gynecology Department and had a history of two abortions. The pedigree of this couple is illustrated in Figure 1. Around 62% had RM with history of stillbirth or CA/ ID and the other 38% had only RM. Using i. TelVysion 3p Spectrum Green (D3S4559) and TelVysion 3q Spectrum Orange (D3S4560) for chromosome 3, and ii. TelVysion 4p Spectrum Green (D4S3359) and TelVysion 4q Spectrum Orange (D4S2930) for chromosome 4, subtelomeric FISH analysis of this patient (III:4) showed a subtelomeric translocation between the long and short arms of chromosomes 3 and 4 respectively. Her chromosomal formula was 46,XX,ish t (3;4) (q28-p16+;p16,q28+) (D3S4559+, D3S4560-D4S3359+; D3S4560+, D4S3359, D4S2930+).
Family pedigree. The black arrow points to the couple number 21 (III: 4 and III: 5) in this study. *; Not available for analysis.
The partial karyotype of this patient (III:4) showed apparently
normal banding pattern of chromosomes 3 and 4
With this molecular information ignored by the family,
two years later, patient III:4 requested consultation after
giving birth to a daughter (IV:5) with congenital malformations
and ID. The classical cytogenetic analysis of IV:5
showed that she inherited the same translocation in its unbalanced
form (46, XX, ish der (
Karyotype of the mother (III: 4).
A review of the literature of screening for subtelomeric rearrangements is summarized in Table 1.
Review of studies screening subtelomeric regions in patients with recurrent miscarriage
|Study||Number of studied cases||Number of subtelomeric rearrangements||Number of miscarriages||Malformed/stillborn child||Translocation||Technique|
|Wakui et al. (12)||10||5||2 or more||+||46, XY, t (7;16)(q36; q22)||Dual-colour subtelomere FISH|
|+||46, XX, t (4; 7)(q35; p15.3)|
|+||46, XX, t(5; 10)(p15.1; p13|
|Giardino et al. (13)||1 family with 2 female carriers||1||2||+||46, XX, t(2;16)(q37.3;q24.3)||Multi-subtelomere FISH using the CytocellMultiprobe-T system|
|Benzacken et al. (14)||114||0||2 or more||NM||-||Multi-subtelomere FISH using the CytocellMultiprobe-T system|
|Fan and Zhang (15)||80||0||4 or more||NM||-||Multi-subtelomere FISH using the CytocellMultiprobe-T system|
|Joyce et al. (16)||2||2||2||+||46,XX,t(11;17)(p15.5;p13.3)||Dual-colour subtelomere FISH|
|4||+||46,XX, t(11;17)(p15.5;p13.3)||FISH analysis with telomere-specific probes|
|Yakut et al. (17)||10||2||5 or more||-||46,XY,ish t(3q; 10p)||Subtelomere specific FISH probe|
|5 or more||-||46,XX,ish t(20p;?Dp)||Multi-subtelomere FISH using the CytocellMultiprobe-T system|
|Jalal et al. (18)||53||0||Multiple||NM||-|
|Bruyere et al. (19)||1||1||3||+||46,XX, t(2;17) (q37.2; q25)||Multi-subtelomere FISH using the CytocellMultiprobe-T system|
|Cockwell et al. (20)||100||1||7||-||46,XX t(3:10)(q29;p15.3)||ToTelVysion Multi-Color FISH|
|Monfort et al. (21)||36||1||7||-||46,XX,t(2;3)||ToTelVysion Multi-Color + Miller-Dieker probe|
|Primerano et al. (22)||1||1||2||+||46,XX. t(5;17)||ToTelVysion Multi-Color FISH|
|Present study||42||1||2||+||46, XX, t(3;4)(q28;p16)|
+; Exist, -; Does not exist, and NM; Not Mentioned.
FISH on metaphase spread prepared from the women’s (III:4) blood with Mix 3 and Mix 4 from Vysis ToTelVysion Kit. A. Mixture 3: chromosome 3 (p spectrum green and q spectrum orange) and chromosome 22 (q spectrum green + spectrum orange), LCI bcr (22q11) spectrum aqua. The red signal of the 3q telomere probe is observed on the normal 3q chromosome (white arrow) and on the short arm of the derivative chromosome 4 (red arrow) and B. Mixture 4: chromosome 4 (p spectrum green and q spectrum orange) and chromosome 21 (q spectrum green+spectrum orange), LCI bcr (21q22) spectrum aqua. Similarly, as expected, the green signal of the probe specific for telomere 4p is observed on normal chromosome 4 (white arrow) and derivative 3 (red arrow).
Humans are characterized by a high rate of embryonic
failure at the early stages of development. RM, stillbirths
and the birth of children with multiple CA remain the
most spectacular varieties of this reproductive failure.
The cause of RM remains elusive in approximately 50%
of cases, although many studies have attempted to identify
the underlying mechanism (
The purpose of the present study was to examine whether
RM is associated with subtelomeric rearrangements.
Among the 42 individuals tested, one female showed a
cryptic translocation between the 3q and 4p arms with
distal breakpoints near the telomeres. Consistently, her
affected daughter (IV:5) showed inheritance of the same
translocation in its unbalanced form (46, XX, der 4,t(3;4)
(q28;p16)) based on classical cytogenetic analysis. In this
family, it was important to consider that not only a higher
risk of RM was observed, but also congenital anomalies
were present in subsequent pregnancies of carriers
of cryptic rearrangements. Depending on the type of the
reciprocal translocation, it has been estimated that the recurrent
risk varies from1 to 50% (
We reviewed the literature and identified eleven studies which had screened for subtelomeric regions in patients with RM. By combining these studies and our present report, 15 out of 448 patients showed subtelomeric translocations based on different sets of telomeric probes. This give a total rate of 3.34% for carriers of cryptic translocations while this rate was 4.76% in this study. Interestingly, these carriers not only had a history of RM, but also had a history of giving birth to children with ID and/or CA, or a clinically recognizable syndrome. This shows the importance of detailing the family history in improving diagnosis and suggesting the appropriate tool of exploration for precision in genetic counseling.
As previously mentioned, both conventional karyotyping
and more advanced techniques such as aCGH and
MLPA have limitations in detecting subtle genomic aberrations
including balanced rearrangements (
Identification of cryptic subtelomeric translocations is an active area of investigation. This study emphasizes the importance of screening these types of balanced rearrangements with subtelomeric FISH particularly in couples with RM. We conclude that subtelomeric FISH analysis should become mandatory for couples with RM and familial family history of stillbirth or children with CA or ID. This has a great impact when DNA or abortion products are no longer available. In fact, these products are not always accessible due to incompatibility with life of the congenital anomalies besides, in some cases, patient refusal to be tested. In addition, miscarriages and ID remain a somewhat offensive and delicate subject for parents, which makes the recruitment of patients more difficult in familial cases of RM. Furthermore, subtelomeric FISH is required to exclude any cytogenetic cause before searching for other spermatic factors such as sperm aneuploidy, sperm DNA integrity, chromatin packaging and semen parameters as we previously reported. Through this study, we highlight the importance of early clinical identification of such cases toward a more efficient diagnosis of subtelomeric translocations in RM cases.