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ORIGINAL ARTICLE
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Cytogenetics evaluation of 261 couples with first-trimester recurrent pregnancy loss: A prevalent case–control study


1 Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India
2 Department of Community Medicine, Adichunchanagiri Institute of Medical Sciences, Nagara, Karnataka, India

Date of Submission29-Jun-2022
Date of Decision06-Aug-2022
Date of Acceptance20-Aug-2022
Date of Web Publication10-Jan-2023

Correspondence Address:
Gurpreet Kaur Sagoo,
Department of Pathology, Armed Forces Medical College, Wanowrie, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmms.jmms_105_22

  Abstract 


Introduction: Recurrent pregnancy loss (RPL) is a common occurrence which affects approximately 15-20% of couples. Chromosomal abnormality is an important cause of recurrent abortions especially if either of the partner is a carrier of balanced translocation. The current study aimed to determine the prevalence of chromosomal abnormalities in couples suffering from first trimester RPL and compare with normal control. Materials and Methods: A prospective case-control study, in which 261 couples with history of two or more abortions were evaluated for various chromosomal abnormalities; and compared with 190 healthy couples with no history of abortion and at least one normal biological child. Peripheral blood T-lymphocytes were cultured using RPMI-1640 medium for obtaining metaphases and chromosomal analysis. SPSS software and Student's t test were used. p value < 0.05 was considered statistically significant. Results: Among 261 couples in RPL group, 240(91.95%) had normal karyotype, 17(6.51%) had major chromosomal abnormalities and 04(1.53%) had polymorphic variants. Most of the couples had two abortions (39.8%). Females were more commonly affected with M:F=0.214. Structural abnormalities (n=12,70.59%) were more frequent than numerical abnormalities (n=5,29.41%). There was no statistical correlation between age, number of abortions and major chromosomal abnormalities (p=0.06). Conclusion: Chromosomal aberrations in carrier parents, predispose them to RPL and can also be transmitted to the offspring which may results in imbalance in their genetic constitution, thus justifying the requirement of cytogenetic testing in these patients.

Keywords: Abortions, chromosomal abnormalities, karyotyping, recurrent pregnancy loss



How to cite this URL:
Singhal P, Raghavendra S K, Chakrabarty BK, Pendkur G, Pendharkar CS, Sagoo GK. Cytogenetics evaluation of 261 couples with first-trimester recurrent pregnancy loss: A prevalent case–control study. J Mar Med Soc [Epub ahead of print] [cited 2023 Feb 1]. Available from: https://www.marinemedicalsociety.in/preprintarticle.asp?id=367485




  Introduction Top


"Recurrent" pregnancy loss (RPL), "multiple" or "habitual" abortion is a distinct clinical condition which has been recently defined as two or more consecutive, involuntarily spontaneous pregnancy losses before 20 weeks of gestation.[1],[2] It is a frustrating problem for the couple and even more challenging for the treating obstetrician. The challenges for the obstetrician are to focus on a particular cause from various etiological factors enumerated in the literature and to differentiate sporadic miscarriage from RPL, as self-reported losses by the patients may not be accurate.[3] Despite all the resources currently available, the etiology of RPL is still idiopathic in approximately 50% of cases and a putative diagnosis with treatment can be made in the remaining cases.[4] The known causes of RPL include autoimmune and endocrine disorders, genetic factors, maternal age, uterine anomalies, male factors, infectious diseases, drugs or chemicals, and psychological causes.

Approximately 50% of products of the conception of first-trimester abortions are associated with sporadic chromosomal anomalies (CAs).[5] At present, it is hypothesized that these CA may be due to nondisjunction or loss of chromosomes during the process of germ cell maturation or in the early cleavage of fertilized eggs. Most of these CAs occur randomly and are of de novo in origin. However, advanced maternal age and the carrier partners with balanced structural CA also poses risk for embryonic chromosomal imbalance.[6]

Although the carrier partners are phenotypically normal, the transmission of chromosomal abnormalities through their gametes predisposes to the formation of abnormal zygotes. Numerical or structural rearrangements in the chromosomes of gamete result in meiotic errors, leading to an imbalance in the developing embryo/fetus. These imbalances may result in live births with congenital abnormalities or may even be lethal to the fetus/embryo culminating in pregnancy loss. The prevalence of CA among couples with RPL varies from 2% to 8% in various studies.[7],[8]

Cytogenetic workup of RPL is still an uncommon practice in India, due to a lack of awareness and the limited infrastructure of cytogenetic services. Cytogenetic analysis may help the treating physician to decide whether further investigations are warranted, in addition, abnormal genetic test results would also help in understanding the etiology of miscarriages. Such testing would also help to counsel the patients about the likelihood of a normal pregnancy so that informed choices may be made about future pregnancies and preimplantation genetic diagnosis. The primary aim of this prospective study was to determine the prevalence of chromosomal aberrations in RPL couples with idiopathic causes, referred to the infertility center of Western Maharashtra. The secondary aim was to compare the cytogenetics findings of RPL couples with healthy control couples.


  Materials and Methods Top


This prospective case–control study included 451 couples from January 2019 to March 2022. The couples were further subcategorized into two groups. Two hundred and sixty-one couples in Group A had a history of two or more idiopathic spontaneous abortions including stillbirths or malformed children as per the following inclusion and exclusion criteria. Group B was comprised 190 volunteered couples matched for age and ethnicity with at least one normal biological child and no history of miscarriage in the past.

Written informed consent from all the participants and approval from the local ethical committee and institutional review board to conduct the study (IEC No. 18-230) were obtained.

Inclusion criteria

All the participants (n = 261) were interviewed for relevant medical history, physical examination, and relevant investigations.

  • Two or more consecutive first-trimester miscarriages
  • Normal USG findings of reproductive tracts
  • Normal semen analysis of husbands
  • No history of iatrogenic/gonadotoxic drug intake.


Exclusion criteria

  • Positive serology for the TORCH panel or anticardiolipin antigen or lupus anticoagulant antibodies or antinuclear antigen
  • Presence of endocrine abnormalities, namely, thyroid dysfunction and diabetes mellitus.


Cytogenetics analysis

About 5 ml of peripheral blood was withdrawn from cases and control couples in sodium heparin. Chromosomal analysis was performed on peripheral blood T-lymphocytes cultured for 69–72 h, using RPMI-1640 medium with 15% fetal bovine serum and stimulated by 2% phytohemagglutinin. After 72 h, cells were harvested using hypotonic potassium chloride followed by GTG banding with trypsin. Twenty metaphases were analyzed. When mosaicism was suspected, 30 additional metaphases were analyzed. Chromosome's study was done using image processor and software (Cytovision) version 7.2 build 147 and abnormalities were reported according to An International System for Human Cytogenomic Nomenclature (ISCN, version: 2016) at band level 500–550.[9]

Special assays such as C-banding and florescence in situ hybridisation (FISH) were performed as and when required. FISH analysis was done for confirmation of low-level mosaicism on 500 viable interphase cells using commercially available in vitro diagnostics (IVD)-approved probes. Postcentromere probe validation in our laboratory, more than 4% of abnormal signals were considered as a mosaic.

The major chromosomal abnormalities (MCA) were further divided into two major anomalies which include structural abnormalities (SAs) and numerical abnormalities (NAs). Structural aberrations included balanced translocation, insertion, and Robertsonian translocation (RT). NAs included mosaic (mos) form, nonmosaic form, and marker chromosome (+mar). Another category of chromosomal aberrations having heteromorphic forms was classified under polymorphic variants (PVs), which included pericentric inversion (inv), variations in the size of the stalk of acrocentric chromosomes, and length of heterochromatin regions of nonacrocentric chromosomes.

Statistical analysis

The sample size was calculated by considering the prevalence of 15% chromosomal aberrations (based on the study by Ghazaey et al.)[10] with a 95% level of significance (α error of 0.05), ±5% absolute precision, and 10% attrition. The final sample was 216 per group (using EPI info software).

Statistical analysis was done using the SPSS software version 20. The difference between the two groups was determined using Student's t-test and Chi-square/Fisher's exact test, and P < 0.05 was considered statistically significant.


  Results Top


A total of 451 couples were investigated for CA by peripheral blood karyotype in this prevalent case–control study, out of which 261 couples had recurrent miscarriages. The age of the females and males with RPL ranged from 19 to 37 years (26.37 ± 4.1) and 23 to 42 years (29.78 ± 4.27), respectively. The number of abortions ranged from 2 to 7 (2.3 ± 1.07). There was no history of consanguinity in the affected group.

In the control group, the age range of females was 20–36 years (26.75 ± 4.16) and males was 22–45 years (30.6 ± 4.3). Against the initial calculation of 216 couples to be included in this study, 190 control couples turned out due to restricted movement during the COVID-19 pandemic.

Among 261 couples in the RPL group, 240 couples (91.95%) had a normal karyotype, 17 couples (6.51%) had MCA, and 4 couples (1.53%) had shown PV in their cytogenetic study. Most of the affected couples had only two abortions in their first trimester (39.8%) and four couples (1.5%) in this study even had up to seven abortions also [Table 1].
Table 1: Cases with number of first-trimester abortions

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Females with RPL were further grouped: <30 years and ≥30 years and number of abortions, i.e., 2, 3, and ≥4. In the present study, we did not find any statistical correlation (P = 0.06) between the age of females, number of abortions, and MCA [Table 2].
Table 2: Age of female with number of abortions and karyotype abnormalities

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Somatic chromosomal study: Couples with recurrent pregnancy loss

Among 261 couples, the overall prevalence of MCA in the present study was 6.5% (17/261) which included female counterparts (n = 14) who were more commonly involved as compared to their male counterparts (n = 3) [Table 3]. The male: female ratio was 0.214. Cytogenetic study of partners of these 17 cases with MCA had a normal chromosomal constitution. Of the 17 cases with MCA, 70.59% of cases (12/17) were found to have SAs, which was more frequent than NAs (n = 5, 29.41%). About 8 (47.05%) cases of balanced translocation were there in which the majority were females (n = 7). RT was observed only in females, affecting 11.67% (2/17) of cases of RPL. Two cases of insertion were seen involving both male and female partners equally.
Table 3: Gender- and age-wise distribution of cytogenetic aberrations in patients with recurrent miscarriages

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NAs were observed in five cases (29.41%) in which four were females and one single male partner with 47, XYY syndrome [Table 3]. Mosaic Turner syndrome (mos 45, X/46, XX), which was further confirmed on FISH, was the most common subtype of NA seen in two female patients. An isolated case of de novo marker chromosome was also seen in a female patient.

PV was observed in 2.68% (n = 7) RPL cases, of which three were female and four were male [Table 4]. Pericentric inversion of chromosome 9 and increased length of heterochromatin region in "q" arm of chromosome 1 was the most common PV. No statistical significance was observed between normal karyotype and PV of RM cases for age and number of abortions (P > 0.05) [Table 5].
Table 4: Distribution of polymorphic variants in cases and control group

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Table 5: Age and abortion in males and females with normal karyotype and polymorphic variants of RM group

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Somatic chromosomal study: Control couples

All the healthy control men and women had a normal chromosomal constitution. PV was the only cytogenetic abnormality which was observed in this category, affecting 4.2% of couples [Table 4]. In our cohort, both the sexes had an equal frequency of PV.


  Discussion Top


The loss of a wanted pregnancy among a couple raises multiple questions, including why it happened and will it be repeated? Answering these concerns requires careful consideration and evaluation of multiple underlying etiological factors which can help these couples to some extent. Although the prevalence of major cytogenetic abnormalities in carrier parents varies from 2% to 9%, it still remains a cost-effective screening modality for cases with recurrent abortions, as it would spare at least those couples from undergoing further invasive and expensive evaluations.[11]

The frequency of overall major chromosomal rearrangements seen in our study group was 6.51% among the couples with male:female ratio of 0.214. As per the previous studies and review of literature, the prevalence of cytogenetic abnormalities in RPL varied from 2.9% to 9.0%.[12],[13],[14],[15] These differences may be related to various sample sizes, maternal age, and inclusion criteria (e.g., number of abortions per couple, exclusion of already defined etiological factors, gestation age, consideration of PV as a chromosomal abnormality, and inclusion of couples or individuals as a denominator for prevalence calculation).

In our study group, SAs were the most common cytogenetic aberration affecting 4.59% of all couples with RPL and constituted 70.6% (12/17) of all the MCA. None of the control group individuals had any major SAs. These abnormalities were in the form of balanced reciprocal translocations (n = 8), RT (n = 2), and insertions (n = 2), involving autosomes only. As per the previous studies also with a larger sample size, structural chromosomal abnormalities were the most common MCA, with a slightly lower prevalence (1.75%) as compared to this study.[13]

In this study, we could not establish a definitive association between the chromosome involved or any specific breakpoint in it, which probably is due to the smaller sample size. We found chromosomes 2, 6, 7, and 8 were most frequently involved in balanced reciprocal translocations. Chromosome 14 was the most frequently involved chromosome in carrier patients with RT. Other studies also mentioned the frequency of commonly involved chromosomes in their cases but could not establish any definitive pattern of break points in the chromosomes involved and the number of abortions.[13],[16]

Carrier partners with these balanced translocations are phenotypically normal because there is no net loss or gain of genetic content in any of their somatic cells. However, due to various malsegregation patterns during gametogenesis (adjacent 2:2 or 3:1 or rarely 4:0), there is the formation of bivalents which may alter the gene expression pattern and eventually lead to the genesis of gametes with unbalanced genetic content.[17] This genetic imbalance may lead to variable phenotypic effects in the form of primary infertility, azoospermia, multiple abortions, or even offspring with congenital anomalies, dysmorphism, and intellectual disabilities. In the present study, a carrier mother having balanced genetic abnormality [Figure 1] also had a child with an interstitial deletion in the short "p" arm of chromosome 8 [Figure 2].
Figure 1: Mother with subtle balanced translocation: 46, XX, t(6;8)(p25;p23)

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Figure 2: Child with unbalanced translocation: 46, XX, del(8)(p12p23), deletion in short 'p' arm of chromosome 8 (Arrow)

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In the present study, sex chromosome aneuploidy, primarily mosaic Turner syndrome, represented the second largest category of chromosomal abnormalities in couples experiencing RPL. This abnormality constituted 1.53% of the couples and 23.5% of overall chromosomal rearrangements, which is higher than the study performed on a cytogenetic database of 16,692 couples (0.07%) by Braekeleer and Dao[18] but lesser in comparison to another study group.[19] It has been postulated that females with X chromosome aneuploidy are at increased risk for meiotic nondisjunction due to hypodiploidy or hyperdiploidy gametes.[20]

Female partners were approximately five times more common carriers of MCA as compared to their male counterparts, which was also found in other similar reports.[21],[22] Lower incidence of a balanced translocation in the male population can be explained due to meiotic blocking of spermatogenesis which eventually leads to azoospermia; however, oogenesis is usually conserved, leading to the production of defective gametes with unbalanced chromosomal abnormalities.[23]

The mean maternal age in our RPL group was 26.37 years. We could not establish any significant correlation between maternal age and MCA (P = 0.06), indicating that carriers of chromosomal aberrations do not have any high-risk behavior like advanced maternal age for screening of congenital abnormalities. There was also no increase in the number of abortions relative to the rate of chromosomal abnormalities in our cohort. This is in concordance with earlier reports.[24]

PV was seen in 2.68% and 4.2% of cases of RPL and control group, respectively, which was lesser in frequency as compared to other studies.[25] The most common variant in the present study was a pericentric inversion of chromosome 9. Other variants were prominent constitutive heterochromatin in chromosomes 1 and 16 and satellite stalk region prominence in acrocentric chromosomes 15 and 22. Other studies have also reported similar variants in RPL couples.[13] These variants were not included in MCA in the present study because the chromosomes with these variations, especiallyinv(9), behave normally during meiosis with a 1;1 segregation pattern consequently having a normal reproductive fitness,[26],[27] and it has also been found that PV eventually does not influence the outcome in IVF-embryo transfer treatment.[28]

Limitations of the study

There are certain limitations in the present study which should be highlighted. First, being a single-center study, it comprised a small number of patients that might be insufficient to represent the problem from different ethnicities. Second, cytogenetic analysis of products of conception could not be performed in all the cases as transportation of abortus samples was not feasible from distant sites, hence relevant pertaining data could not be complied. Third, follow-up of pregnancy outcome was not done, especially in cases with MCA, as most of the carrier partners were counseled and had opted for gamete donation as an institutional practice.


  Conclusion Top


Chromosomal rearrangements in carrier parents, predispose them to RPL. The present study demonstrated that major chromosomal aberrations were present in 6.51% of patients in the population studied which is consistent with other series of studies. These chromosomal aberrations are transmitted to the offspring from the parents resulting in an imbalance in their genetic constitution which may be lethal for survival. Therefore, our findings reemphasize that cytogenetic analysis should be part of the investigation work up for couples with two or more pregnancy losses of unknown origin. An individual with chromosomal abnormalities should be counseled and their subsequent pregnancies are a strong indication for prenatal diagnosis (amniocentesis, chorionic villus biopsy, or preimplantation genetic diagnosis) or even gamete donation.

Ethical approval

The Institutional Ethical Committee approval and informed consent were obtained from the patients for publication of this article.

Acknowledgments

We are thankful to all couples who participated in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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