Abstract
BACKGROUND:
Chromosomal abnormalities are a significant cause of miscarriages. Carriers of balanced chromosome rearrangement are often at risk of recurrent pregnancy loss (RPL), as they are more likely to produce gametes with unbalanced chromosome rearrangements.
OBJECTIVE:
This study evaluated the chromosomal abnormalities detected in couples with history of primary recurrent pregnancy loss.
DESIGN:
Retrospective, cross-sectional study
SETTING:
Single center, tertiary healthcare center in Turkey
PATIENTS AND METHODS:
This study reviewed conventional cytogenetic/molecular cytogenetic analysis data of 4030 patients (2015 couples) who visited the clinic from 2008–2024.
MAIN OUTCOME MEASURES:
Chromosomal abnormalities in patients diagnosed with primary recurrent pregnancy loss and genetic testing results of spontaneously achieved pregnancies in 16 patients with a balanced chromosomal rearrangement.
SAMPLE SIZE:
4030 individuals (2015 couples)
RESULTS:
Majority of couples had a history of two spontaneous miscarriages (59.4%), followed by couples with 3 miscarriages (28.1%), 4 miscarriages (7.5%), and 5 or more miscarriages (4.91%). Chromosomal abnormality was detected in 133 (3.3%) cases. Among the revealed abnormalities, 130 (97.7%) were structural chromosome anomalies, while only 3 (2.3%) numerical chromosome anomalies were observed, including sex chromosome aneuploidy in 2 cases and mosaic karyotype in one case. Among the detected 130 structural chromosome abnormalities, reciprocal translocations (86 cases, 66.2%) were most frequently observed, followed by Robertsonian translocations in 26 cases (20.0%), inversion in 11 cases (8.5%), marker chromosome in 5 cases (3.8%), and derivative chromosomes in 2 cases (1.5%). Products of conception (conceptus materials) were analyzed from 16 spontaneously conceived pregnancies in individuals identified as carriers of chromosomal rearrangements. Although reciprocal translocations involving acrocentric chromosomes are typically expected to result in 3:1 meiotic segregation, adjacent-1 segregation was observed in two female individuals carrying the translocation t(9;15)(p22;q23). This finding is likely due to the limited genetic content of the translocated segments. Additionally, a novel complex three-way translocation, t(5;7;13)(p12;p12;p11), was identified for the first time.
CONCLUSION:
Cytogenetic and molecular analyses are crucial components in the etiological investigations of couples with RPL.
LIMITATIONS:
Retrospective design
INTRODUCTION
Recurrent pregnancy loss (RPL), as defined by European Society of Human Reproduction and Embryology (ESHRE), is the loss of two or more pregnancies before the 24th week of gestation.1 It is an important reproductive health problem affecting 2% to 5% of couples.2–5 Prevalence rates might vary across regions and the diagnostic criteria used. In Turkey, the prevalence of RPL was observed to be Previous studies indicated that RPL is seen in 5%–5.9% of the women in Turkey.6,7
In many societies, having children is important for establishing and maintaining family unity. Consequently, couples with a history of RPL often face increased levels of depression, grief, psychosocial stress, and hopelessness.8 A study assessing depression, hopelessness, and adjustment in couples with women diagnosed with recurrent pregnancy loss (RPL) found that 18.2% of women experienced moderate hopelessness, while 13.6% experienced severe hopelessness. Additionally, 24.3% were mildly depressed, 30.3% moderately depressed, and 1.5% severely depressed, with a significant correlation observed between levels of hopelessness and depression.9 Considering these emotional burdens, genetic counseling plays a crucial role in supporting couples by providing clear explanations of possible genetic factors, recurrence risks, and available testing options.
RPL is categorized into primary and secondary types. While women who have never given birth to a live baby are grouped as primary RPL, those with multiple losses (including stillbirth and neonatal death) in their reproductive history with live birth are categorized as secondary RPL.10
Despite recent technological advancements, around 50% of cases still lack a definite cause. Risk factors for RPL include uterine anatomical issues (10–15%), endocrine disturbances (17–20%), immunological factors (20%), and parental chromosomal abnormalities.11 Chromosome abnormalities are the most common cause of clinical miscarriages, which mostly happen in the first trimester. A considerable percentage of pregnancy losses are attributed to fetal chromosomal abnormalities, according to studies, which highlights the significance of cytogenetic research in situations of recurrent miscarriage.12
Parental balanced chromosome rearrangement carriers are associated with an increased risk of RPL due to their predisposition to produce gametes with unbalanced chromosome rearrangements that are often non-viable.13 Additionally, carriers of balanced chromosome anomalies are also at an increased risk of having a child with congenital disabilities. These risks emphasize the importance of cytogenetic analyses in cases with recurrent miscarriage history. Identifying these genetic risks in balanced abnormality carriers is essential in providing accurate risk assessments, effective genetic counseling, and informed reproductive planning.12
Approximately 3–5% of the couples with a history of RPL have balanced translocations (reciprocal translocations (~60%) and Robertsonian translocations (~40%).14 In addition, chromosomal inversion, sex chromosome aneuploidy, and smaller percentages of supernumerary marker chromosomes are also observed in parental karyotypes.15
Therefore, the parental karyotype analysis is considered an essential examination for determining the cause of RPL as recommended by the American Colleges of Obstetricians and Gynecologists, American Society for Reproductive Medicine (ASRM), and The German, Austrian and Swiss Societies of Gynecology and Obstetrics.1
In this retrospective study, we aimed to determine the prevalence and types of chromosomal anomalies detected in Turkish couples referred to our clinic for RPL over a 16-year period. Additionally, we evaluated the clinical characteristics and pregnancy outcomes of the carriers.
PATIENTS AND METHODS
This retrospective, single-center study reviewed conventional cytogenetic/molecular cytogenetic analysis data of 2015 couples (4030 individuals) with a history of primary RPL who either visited our clinic or referred from other clinics between 2008 and 2024. This study was approved by the Institutional Ethics Review Board of the Eskişehir Osmangazi University Faculty of Medicine (Ethical committee number: 2023-170/21).
The study group consisted of individual adults aged 18 and above, evaluated by relevant clinics and deemed normal concerning known RPL risk factors. Our clinic's routine RPL evaluation algorithm accepts normal couples for genetic analysis without any known reasons regarding all risk factors associated with RPL. In line with this algorithm, the study group included women who showed no uterine anomalies in the ultrasonography and hysterosalpingography evaluations. Additionally, all immunological and biochemical risk factors, including antiphospholipid antibodies, antinuclear antibodies, and antithyroid antibodies were within normal limits. The women did not have endocrine disorders or autoimmune diseases, and the men did not have any sperm abnormalities. We excluded cases with a family history of monogenic diseases, anatomical abnormalities, endocrine disorders, thrombophilic gene mutations, and those diagnosed with immunological and biochemical risk factors.
After obtaining family histories and providing genetic counseling, we performed conventional cytogenetic analyses on Giemsa-Trypsin Giemsa-banded metaphases according to the standardized peripheral blood protocols.16 The chromosome constitutions of the couples were reported following the International System for Human Cytogenetic or Cytogenomic Nomenclature 2020 (ISCN 2020) standards.17
For cases with structural anomalies, fluorescence in situ hybridization (FISH) analysis was performed using specific probes to identify chromosome breakpoints and mosaicism rates.16 In the FISH Protocol, denaturation after dehydration of the slides, hybridization, post-hybridization washings, and visualization stages were performed according to previously reported protocols. Briefly, after the dehydration of the slides, denaturation processes were performed by applying probes in the hybridization buffer solution for the purpose, and overnight hybridization was performed using the Thermobrite Elite (TBE) system (Leica) at 37ºC. After hybridization, post-hybridization washing was performed according to the protocols specified by the manufacturers' directions, and the preparations were allowed to air dry. Then, the slides were counterstained with 4.6 diamidino-2-phenylindole and stored at −20ºC until analysis. The slides were analyzed using an automated CytoVision image analysis and capture system (Leica Biosystems Richmond Inc., Richmond, IL, United States). Chromosome-specific telomere, chromosome centromere, and locus-specific FISH probes were used depending on the suspected abnormality. For marker chromosome origin assessment, origin determination was performed using the Chromoprobe Multiprobe® OctoChrome™ (Cytocell) kit (OGT, Oxfordshire, UK), whereas analyses of the telomeric regions of the p and q arms were performed using the chromosome-specific TelVysion Subtelomere Probes (Vysis) (Abbott, Abbott Park, Illinois, USA), structural anomaly (translocations and inversions) breakpoints specific locus-specific probes (Vysis, Cytocell or Kreatech) (OGT, Oxfordshire, UK), and in mosaic cases, LSI SHOX (Xp22.33)/(Yp11.32)/CEPX (Cytocell) (OGT, Oxfordshire, UK) probes were used.
Families with a history of abortion were advised to send conceptus material for genetic analysis in case of future pregnancy losses to enable detailed cytogenetic/molecular studies. The study included analysis of conception products, amniocentesis and chorionic villus sampling (CVS) samples from 16 families. Part of the fetal tissue was used for quantitative fluorescent polymerase chain reaction (QF-PCR) analysis to check for maternal contamination and aneuploidies of chromosomes 13, 18, 21, X, and Y. The rest was cultured for cyogenetics and molecular cytogenetics analysis.
For QF-PCR analyses, DNA isolation from product of conceptions (POCs)/CVS and maternal peripheral blood samples was performed using the high pure PCR Template Preparation Kit (Roche, USA) following the manufacturer's protocol. Multiplex PCR was conducted with fluorescently labeled primers targeting short tandem repeat (STR) markers on chromosomes 13, 18, 21, X, and Y with the Devyser Complete Aneuploidy Detection kit according to the manufacturer's protocol. The PCR products were then analyzed by capillary electrophoresis on the Applied Biosystems™ 3130 DNA analyzer and fragment analysis was performed using GeneMapper software. Chromosomal aneuploidies were identified through abnormal STR peak ratios (e.g., 1:2, 2:1, or 1:1:1 for trisomies). Maternal cell contamination was evaluated for each sample by comparing the sizes of 33 STR (Short Tandem Repeat) markers of maternal and fetal DNA during QF-PCR.
Conventional cytogenetic analyses were performed from cultured samples, and single nucleotide polymorphism (SNP) array analyses were carried out by using Agilent (180K) (Agilent, Sta.Clara CA, USA) and Illumina (300K) (Illumina Inc, San Diego, CA, USA) platforms over time to reveal genomic copy changes in the samples.
Statistical analyses including descriptive statistics, Wilson Score test, Pearson correlation test, logistic regression analysis and Mann-Whitney tests were done using Graphpad Prism Version 10.4.1 program. A P value <.05 was considered statistically significant with 95% confidence intervals (CIs).
RESULTS
Patient characteristics
The median age of females was 27.6 (median: 19–46), and the median age of males was 31.5 (median: 20–51). The distribution of 2015 couples according to the number of pregnancy losses is given in Table 1. Due to the limited number of couples with a history of five or more RPLs, this group was grouped as 5 or more abortions. In the study cohort, 59.45% of couples had a history of two spontaneous miscarriages, followed by those with three miscarriages (28.14%), four miscarriages (7.5%), and five or more miscarriages with (4.91%). No significant association was found between frequency of miscarriage and either maternal age (P=.747) or paternal age (P=.953). In our study, the frequency of chromosomal anomalies in couples with a history of RPL was 6.6%.
Table 1.
Distribution of pregnancy losses numbers in couples.
| No. of pregnancy losses | No. of couples n (%) | Abnormality rate (n=133) n (%) |
|---|---|---|
| 2 | 1198 (59.4) | 85 (63.9) |
| 3 | 567 (28.1) | 39 (29.3) |
| 4 | 151 (7.5) | 6 (4.5) |
| 5 or more | 99 (4.9) | 3 (2.2) |
Chromosomal abnormalities
Normal chromosome constitutions were detected in 3897 cases, accounting for 96.7% (95% CI: [96.3–97.2%]), while chromosomal abnormalities were detected in 133 cases, representing 3.3% (95% CI: [3–3.6%]). Based on the number of previous miscarriages, the prevalence of total cytogenetic abnormalities (in both women and their partners) were found to be 63.9% (85/133), 29.3% (39/133), 4.5% (6/133) and 2.2% (3/133) in cases with a history of two, three, four, and five or more recurrent miscarriages, respectively. An analysis between the number of miscarriages and the rate of chromosomal abnormalities in patients revealed no significant difference (P=.1699). Among the revealed 133 abnormalities, 130 (97.7%) were structural chromosome anomalies, while 3 (2.3%) were numerical chromosome anomalies, which included sex chromosome aneuploidy (2 cases) and a mosaic karyotype (one case). No significant difference was observed in the number of miscarriages with respect to the presence of either numerical or structural chromosomal abnormalities in patients (P=.8881).
As seen in Table 2, among the 130 identified structural chromosome abnormalities, reciprocal translocations were the most frequently observed, accounting for 86 cases [66.2% (95% CI: 57.7%–73.7%)]. This was followed by Robertsonian translocations in 26 cases [20.0% (95% CI: 14.0%–27.7%)], inversions in 11 cases [8.5% (95% CI: 4.8%–14.5%)], marker chromosomes in 5 cases [3.8% (95% CI: 1.6%–8.7%)], and derivative chromosomes in 2 cases [1.5% (95% CI: 0.4%–5.4%)]. Table 3 shows the comprehensive list of all detected anomalies along with the parental genders. The female to male ratio was 1.50. Reciprocal translocations were evenly distributed across both genders, showing no clear gender-specific predominance. However, Robertsonian translocations were slightly more common in females, except rob(13;14) which was detected in seven males versus six in females. Inversions were found in both genders, as well. Overall, there was no significant difference in the distribution of chromosomal abnormalities between males and females (P=.658).
Table 2.
Distribution of structural chromosome abnormalities.
| Structural abnormalities | Female n (%) | Male n (%) | Total n (%) |
|---|---|---|---|
| Reciprocal translocation | 54 (41.5) | 32 (24.6) | 86 (66.1) |
| Robertsonian translocation | 14 (10.8) | 12 (9.2) | 26 (20.0) |
| Inversion | 5 (3.8) | 6 (4.6) | 11 (8.5) |
| Marker chromosome | 4 (3.1) | 1 (0.8) | 5 (3.8) |
| Derivative chromosome | 2 (1.5) | 0 (0) | 2 (1.5) |
| Total | 79 (60.8) | 51 (39.2) | 130 (100) |
Table 3.
Chromosomal abnormalities in patients with RPL.
| Chromosomal abnormalities Reciprocal translocations | |
|---|---|
| Female | Male |
| 46,XX,t(1;4)(q41;q34) | 46,XY,t(1;3)(q31;q22) |
| 46,XX,t(1;5)(p36;q23) | 46,XY,t(1;18)(q42;q21.3) |
| 46,XX,t(1;14)(q25;q24) | 46,XY,t(1;10)(q43;q25) |
| 46,XX,t(1;12)(q43;p12) | 46,XY,t(1;12)(p36.2;q24.2) |
| 46,XX,t(1;15)(p22;q25) | 46,XY,t(1;3)(q31.1;q25.3) |
| 46,XX,t(2;14)(q23;q24) | 46,XY,t(2;14)(q23;q24) |
| 46,XX,t(2;7)(q32;p23) | 46,XY,t(2;3)(p13;q26) |
| 46,XX,t(3;13)(p22;q31) | 46,XY,t(2;7)(q32;p21) |
| 46,XX,t(3;5)(q31;q24) | 46,XY,t(3;10)(p26;p12) |
| 46,XX,t(3;7)(q24;q33) | 46,XY,t(3;11)(q24;q25) |
| 46,XX,t(3;19)(p21;p13.3) | 46,XY,t(3;4)(p24;q34) |
| 46,XX,t(4;11)(p16;q23) | 46,XY,t(4;11)(p16.3;p15.4) |
| 46,XX,t(3;6)(q21;q25) | 46,XY,t(6;10)(q24;q23) |
| 46,XX,t(3;6)(q25;q21) | 46,XY,t(6;13)(q23;q32) |
| 46,XX,t(3;12)(p21.3;q24.3) | 46,XY,t(6;19)(q27;p11) |
| 46,XX,t(3;12)(p21.3;q24.1) | 46,XY,t(7;9)(p15;q22) |
| 46,XX,t(3;21)(p13;q11) | 46,XYt(7;21)(q21;q22) |
| 46,XX,t(4;6)(q28;p23) | 46,XY,t(8;22)(q23;q11) |
| 46,XX,t(4;9)(q31;p24) | 46,XY,t(9;11)(q21;p15) |
| 46,XX,t(4;14)(p15;q24) | a46,XY,t(9;15)(p22,1;q23) |
| 46,XX,t(4;15)(q26;q13) | 46,XY,t(9;21)(q12;q11) |
| 46,XX,t(4;15)(q35;q22) | 46,XY,t(10;17)(q26;q21) |
| 46,XX,t(4;12)(p11;p11) | 46,XY,t(10;18)(q25;21) |
| 46,XX,t(5;6)(q31;q25) | 46,XY,t(12;13)(p12;q12) |
| 46,XX,t(5;7)(q24;p12) | 46,XY,t(12;16)(q21;q24) |
| 46,XX,t(5;14)(p15;q32) | 46,XY,t(13;15)(q22;p13) |
| 46,XX,t(5;7;13)(p12;p12;p11) | 46,XY,t(14;18)(q22;q11) |
| 46,XX,t(5;14)(p15;q31) | 46,XY,t(15;22)(q21;q13) |
| 46,XX,t(5;18)(p13;p11.2) | 46,XY,t(16;17)(q13;q23) |
| 46,XX,t(6;9)(p22;q33) | 46,XY,t(16;20)(q11;q12) |
| 46,XX,t(6;9)(p12;q13) | 46,XY,t(17;19)(q12;p13.1) |
| 46,,XX,t(7;13)(q34;q33) | 46,XY,t(18;20)(q12;q12) |
| 46,XX,t(7;20)(q22.1;q11.1) | |
| 46,XX,t(5;18)(p13;p11.2) | |
| 46,XX,t(8;21)(q24;p11) | |
| 46,XX,t(8;13)(q23;q21) | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(p22;q23)a | |
| 46,XX,t(9;15)(q11;q11), inv(9)(p11.1q12) | |
| 46,XX,t(9;21)(p24;q21) | |
| 46,XX,t(10;11)(p11.2;q25) | |
| 46,XX,t(10;11)(q26;p11.2) | |
| 46,XX,t(10;14)(p12.3;q13) | |
| 46,XX,t(10;17)(q22;q22) | |
| 46,XX,t(11;17)(p14;p13) | |
| 46,XX,t(11;22)(q23;q11) | |
| 46,XX,t(11;22)(q22.2;q21.3) | |
| 46,XX,t(18;22)(q11.2;q11.1) | |
| Robertsonian translocations | |
| Female | Male |
| 45,XX,rob(13;13)(q10;q10) (3 cases) | 45,XY,rob(13;13)(q10;q10) (3 cases) |
| 45,XX,rob(13;14)(q10;q10) (6 cases) | 45,XY,rob(13;14 (q10;q10) (7 cases) |
| 45,XX,rob(14;21)(q10;q10) (4 cases) | 45,XY,rob(13;15)(q10;q10) (2 cases) |
| 45,XX,rob(15;22)(q10q10) | |
| Inversions | |
| Female | Male |
| 46,XX,inv2(q35q37) | 46,XY,inv(2)(p11.1q21) |
| 46,XX, inv4(q13q32) | 46,XY,inv(6)(p22q25) |
| 46,XX,inv11(p12q14) | 46,XY,inv(10)(p13q22) |
| 46,XX,inv12(p11q13) | 46,XY,inv(12)(p12;q15) |
| 46,X,inv(X)(q11q27) | 46,XY,inv(18)(p11q22) |
| 46,X,inv(Y)(p11.2q11.2) | |
| Marker chromosomes | |
| Female | Male |
| 47,XX+mar (4 cases) | 47,XY+mar(15)(p11.1-q11.1) |
| Derivative chromosomes | |
| Female | Male |
| 46,XX(der15) | |
| 46,XX,der(X)(:p11.4→qter) | |
| Numerical abnormalities | |
| Female | Male |
| mos45,X[23]/46,X,del(X)(p22.1→pter)[27] | 47,XYY (2 cases) |
Indicates the patients from the same village
Pregnancy outcomes of chromosomal abnormality carriers
As seen in Table 3, eight women and one man with a balanced reciprocal translocation t(9;15)(p22;q23) were all from the same village, and are likely distant relatives. They were all referred to our outpatient clinics for the investigation of RPLs at different times. Each woman had at least two miscarriages. Overall, it was noted that nine patients/spouses had a total of 44 clinically recognizable pregnancy losses. One of the females with t(9;15) (p22;q23), who had previously suffered two miscarriages, experienced a missed abortion in her 3rd spontaneous pregnancy. Karyotype and microarray analyses done from POC sample revealed the following chromosome constitution: der(9)(15qter→15q23::9p24→9qter) arr[GRCh37] 9p24.2p24.3(204193–4556061)x1, 15q24.2q26.3 (76124331–102403355)x3 resulting from adjacent-1 gamet segregation from the maternal reciprocal translocation.
Another female t(9;15)(p22;q23) carrier underwent amniocentesis during her 3rd pregnancy at 18th weeks of gestation. The fetal karyotype and microrray analysis revealed der(9)(15qter→15q23::9p24→9qter) arr[GRCh37] 9p24.2p24.3(202180-4553861)x1, 15q24.2q26.3(76126326-102403369)x3. Accordingly, the option of pregnancy termination was offered to the couple, along with clinical information related to the anomaly. Additionally, another female patient, also a t(9;15)(p22;q23) carrier had gone CVS sampling at 12th week of gestation. The fetal karyotype and microarray analysis resulted as 46,XX, arr[GRCh37] (X,1-22)x2. All couples received genetic counseling, and the prenatal/preimplantation genetic diagnosis options were recommended. Karyotype analysis was also offered to asymptomatic family members of the translocation carriers.
When a couple with a history of three first-trimester recurrent miscarriages presented to our clinic, chromosomal analysis was performed after excluding all maternal and paternal risk factors associated with miscarriage. The analysis revealed a balanced complex 46,XX,t(5;7;13)(p12;p12;p11) karyotype in the woman. In the evaluation of the proband's parents, normal chromosome constitutions were detected. Consequently, it was thought that this represented a de novo anomaly, and the couple received genetic counseling that included information about the risks in subsequent pregnancies and recommendations for prenatal diagnosis.
Prenatal diagnosis and genetic analysis of POC samples from spontaneous abortions were suggested to all patients referred to our clinics. To our knowledge, 54 patients with balanced chromosomal rearrangements experienced 68 spontaneous pregnancies after the diagnosis. Among those who did not undergo further evaluation at our center, it was learned that 32 out of 52 pregnancies (61.5%) resulted in live births, while 20 out of 52 pregnancies (38.5%) ended either in miscarriage or termination. In our center, 11 patients had invasive prenatal testing, and 5 patients had genetic testing of POC samples after pregnancy terminations because of fetal abnormalities and/or missed abortus. Pregnancy outcomes of these patients are summarized in Table 4. Finally, polymorphic chromosomal variants (heterochromatin, satellite increments, and chromosome 9 inversion in 159 (3.9%) cases were also detected. The most frequent polymorphic variant was inv9(p12;q13), revealed in 22 cases. Moreover, chromosomes 1, 9, and 16 heterochromatin increments (1qh+, 9qh+, and 16qh+) were detected in 36 cases.
Table 4.
Outcomes of 16 pregnancies achieved spontaneously during the follow-up of patients diagnosed with chromosomal rearrangements.
| Parental karyotype | Sample tested/week of gestation | QF-PCR | Fetal karyotype | Microarray | Pregnancy outcome |
|---|---|---|---|---|---|
| 45,XY,rob(13;14)(q10;q10) | CVS/12th week of gestation | Trisomy 13 | 46,XY,rob(13;14)(q10;q10)+13 | – | Termination |
| 45,XY,rob(13;14)(q10;q10) | CVS/12th week of gestation | Trisomy 13 | 46,XY,rob(13;14)(q10;q10)+13 | – | Termination |
| 45,XY,rob(13;14)(q10;q10) | POC/11th week of gestation | Trisomy 13 | 46,XY,rob(13;14)(q10;q10)+13 | – | Missed abortus/termination |
| 45,XY,rob(13;13)(q10;q10) | POC/13th week of gestation | Trisomy 13 | 46,XY,rob(13;13)(q10;q10)+13 | – | Missed abortus/termination |
| 45,XX,rob(13;14)(q10;q10) | POC/11th week of gestation | Trisomy 13 | 46,XY,rob(13; 14)(q10;q10)+13 | – | Missed abortus/termination |
| 45,XX,rob(13;13)(q10;q10) | CVS/12th week of gestation | Trisomy 13 | 46,XX,rob(13;13)(q10;q10)+13 | – | Termination |
| 45,XX,rob(13;13)(q10;q10) | POC/10th week of gestation | Trisomy 13 | 46,XX,rob(13;13)(q10;q10)+13 | – | Missed abortus/termination |
| 45,XX,rob(13;13)(q10;q10) | Amniocentesis/16th week of gestation | Normal | 45,XY,rob(13;13)(q10;q10) | arr[GRCh37] (X,Y)x1(1-22)x2 | Delivery |
| 45,XY,rob(13;14)(q10;q10) | CVS/12th week of gestation | Normal | 45,XX,rob(13;14)(q10;q10) | arr[GRCh37] (Xz1-22)x2 | Delivery |
| 46,X,inv(Y)(p11.2q11.2) | Amniocentesis/17th week of gestation | Normal | 46,X, inv(Y)(p11.2q11.2) | arr[GRCh37](X,Y)x1,(1-22)x2 | Delivery |
| 46,XX,t(9;15)(p22;q23) | Amniocentesis/18th week of gestation | Normal | 46,XY,der(9) (15qter→15q23::9p24→9qter) | arr[GRCh37] 9p24.2p24.3(202180-4555861)x1, 15q24.2q26.3(76126326-102405355)x3 | Termination |
| 46,XX,t(9;15)(p22;q23) | POC/13th week of gestation | Normal | 46,XY,der(9) (15qter→15q23::9p24→9qter) | arr[GRCh37] 9p24.2p24.3(204193-4556061)x1, 15q24.2q26.3(76126326-102405355)x3 | Missed abortus/termination |
| 46,XX,t(9;15)(p22;q23) | CVS/12th week of gestation | Normal | 46,XX | arr[GRCh37] (Xz1-22)x2 | Delivery |
| 46,XX,t(7;13)(q34;q33) | CVS/12th week of gestation | Normal | 46,XX, der(13)t(7;13)(q36;q31) | arr[GRCh37]13q31. 3q34(92270484-114342258)x1 z7q36. 1 q36.3(151708732-159327017)x3 | Termination |
| 46,XX,t(4;11)(p16;p15) | CVS/12nd week of gestation | Normal | 46,XY | arr[GRCh37] 4p 16.3(67746_3980138) x1, 11p15.5p15.4(259229_3414328)x3 | Termination |
| 46,XY,inv(2)(p11.1q21) | CVS/12th week of gestation | Normal | 46,XX,inv(2)(p11.1q21) | arr[GRCh37] (X,Y)x1(1-22)x2 | Delivery |
DISCUSSION
Parental chromosomal abnormalities are one of the leading genetic causes involved in the pathogenesis of RPL.18 Studies indicate that approximately 2.25–5% of couples experiencing RPL have one parent with a balanced chromosome rearrangement.19 Phenotypically normal spouses with balanced chromosomal anomaly carriers are at risk of producing gametes with unbalanced chromosome formations, which can result from meiotic abnormal segregations.20
The prevalence of chromosomal anomaly carriers in couples with a history of RPL varies across different populations, but it is generally higher than in the general population, where the frequency is about 0.3% to 0.4%.21,22 The incidence of chromosomal abnormalities among the couples with RPL has been reported to range from 1.2% to 12%.15,21,23 Compared to previous studies conducted in different regions of Turkey, our cohort size is larger. While the frequencies reported previously in these studies ranged from 3.9% to 8.7%, the frequency detected in this study (6.6%) falls within the established ranges.24–26
Previous studies have indicated that balanced chromosome rearrangements were observed more frequently than numerical anomalies, and this finding was confirmed (97.7%) in the current study as well. Numerical abnormalities were identified in three cases; two males who had 47,XYY sex chromosome aneuploidies. The other case was a phenotypically normal woman with a history of two abortions, who exhibited a mosaic 45,X/46,X,del(X)(p22.1→pter) karyotype. Mosaicism is a genetic phenomenon in which an individual has two or more distinct cell populations with different genetic compositions, originating from a single fertilized egg.27 This condition arises due to postzygotic errors or chromosomal nondisjunction during gametogenesis.28 The proportion and distribution of 45,X cells across different tissues significantly influence phenotypic severity, impacting growth, ovarian function, and systemic complications.29 Nonmosaic 47,XYY is a sex chromosome aneuploidy commonly associated with male infertility. Men with a 47, XYY chromosomal constitution exhibit highly variable sperm counts, ranging from normal to azoospermia.28 Although fertility varies in XYY men with normal semen parameters, previous studies have reported an increased incidence of chromosomally abnormal spermatozoa in their semen samples.30 Studies have revealed that XYY males often have impaired chromosome synapsis and are missing a meiotic recombination site, which increases the risk of aneuploidy within spermatozoa because of their susceptibility to meiotic errors.28,30
Reciprocal translocations occur in approximately 1 in 600 individuals in the general population but this rate rises to around 5% in couples with a RPL history. These chromosomal rearrangements occurs by the breakage of non-homologous chromosomes, followed by the mutual exchange of their segments.3 During the oogenesis/spermatogenesis process of the carrier spouse, there is always a risk of partial aneuploidy of translocated chromosomes due to adjacent or 3:1 segregations during meiosis.31 Consequently, reciprocal translocations are more common in couples with a history of RPL than other chromosomal abnormalities such as inversions, sex chromosome mosaicism, or Robertsonian translocations.20 The most common chromosomal abnormality in our study was reciprocal translocations, with a prevalence of 2.1%. Previous studies that reported the rate of reciprocal translocations in couples with a RPL history ranges between 1.3% and 5.9%.10,20,25,32–34 The variances in these prevalence rates can be attributed to differences in the selection criteria of the study cohorts. All autosomal chromosomes were involved in the balanced translocations detected in our study. Among the chromosomes involved in translocations, chromosomes 1, 3, 9, 10, and 15 were most frequently observed. As stated in previous publications, the frequency of balanced translocations tends to be higher in larger chromosomes compared to smaller ones. This may be related to the larger target size of the bigger chromosomes during double-strand break and repair processes.35
In our study, polymorphic chromosomal variants were detected in 4% of patients, a rate comparable to that observed in the general population.36 This finding further supports the notion that there is no significant association between chromosome heteromorphisms and reproductive problems.37
The frequency of consanguineous marriages in Turkey is around 21%, and in some regions, people do not prefer to marry outside the borders of their ethnic region. Although consanguineous marriages pose a risk in terms of recessive diseases and congenital malformations, we believe that our study is a good example that emphasizes the importance of comprehensive pedigree evaluation in couples with RPL history.
As one of the highlights of this study, we report a large family of balanced t(9;15)(p22;q23) carriers. Eight females and one male with a history of RPL were all from the same village and identified as distant relatives. The chromosomal constitutions of two fetal samples were found to be derived from maternal adjacent-1 gamete segregation. Although acrocentric chromosomes involved in a reciprocal translocation are typically expected to generate a 3:1 segregation due to the small lengths of their short arms, which creates significant asymmetry during quadrivalent formation, adjacent-1 segregation was detected in all analyzed conceptus material of the family because the translocated segments contain limited genetic content.
Another important finding of this study is the result of the CVS analysis conducted on the partner of a 46,XY,t(4;11)(p16;p15) translocation carrier, as mentioned in Table 4. Both QF-PCR and karyotype analyses revealed a normal chromosome constitution. However, subsequent microarray analysis from the cell culture detected a loss of 3 912 392 bp at chromosome 4 and a gain of 3 155 099 bp at chromosome 11, reported as arr[GRCh37] 4p16.3(67746_3980138)x1, 11p15.5p15.4(259229_3414328)x3. Microarray analysis plays a critical role for diagnosing submicroscopic chromosomal aberrations due to its high resolution and sensitivity.38 Even if the fetus/offspring appears to have a normal chromosome constitution or a balanced chromosomal rearrangement similar to the parent at conventional cytogenetic analyses, there remains a possibility of submicroscopic anomalies arising during meiotic segregation that could be overlooked. Therefore, we strongly recommend performing microarray analysis for fetuses with a parent who has balanced chromosomal rearrangements.
Additionally, we emphasize that rapid aneuploidy screening tests must be used cautiously. These tests are custom-designed panels and may be insufficient when used alone in individuals with balanced chromosomal rearrangements. In a female patient with t(7;13) (q34;q33) (Table 4), QF-PCR analysis of CVS material initially showed normal results concerning short tandem repeats markers related to chromosome 13. However, further cytogenetic and microarray analyses revealed an unbalanced chromosomal constitution in the fetus. The STR markers of chromosome 13 used in the QFPCR analysis did not overlap with the chromosomal segment included in the derivative chromosome, which is why the initial results were deemed normal. This finding highlights the importance of conducting more comprehensive cytogenetic and microarray analyses in individuals with balanced chromosomal rearrangements. It is concluded that genetic analyses should be supported by multiple methods to detect unbalanced rearrangements.
Genetic counseling, which includes discussing the risks in subsequent pregnancies and providing recommendations for prenatal diagnosis and preimplantation genetic diagnosis was offered to all patients with detected chromosomal abnormalities. We stress the importance of thorough genetic counselling and advocate for the support of genetic analyses by multiple methods to effectively detect unbalanced rearrangements in subsequent pregnancies. The limitations of our study include the absence of a control group for comparison and the lack of analysis using NGS panels or single-gene variants that could potentially elucidate the etiology of recurrent pregnancy loss.
CONCLUSION
Recently identified genetic factors, including epigenetic mechanisms and rare monogenic diseases, are increasingly important for explaining the causes of RP). Efforts are underway to address the unresolved 50% of RPL cases using new techniques. Once all known risk factors have been eliminated, different structural anomalies may be detected in the partners through cytogenetic and molecular cytogenetic analyses. More importantly, these relatively cost-effective approaches should be used in the management of couples with RPL and their conceptus samples in a combination manner to identify cytogenetic-based causes.
Funding Statement
None.
Footnotes
CONFLICT OF INTEREST: None.
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