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ISSN: 1476-7058 (Print) 1476-4954 (Online) Journal homepage: http://www.tandfonline.com/loi/ijmf20

Cytogenetic abnormalities in couples with ahistory of primary and secondary recurrentmiscarriage: a Brazilian Multicentric Study

Marcelo Borges Cavalcante, Manoel Sarno, Gabriela Gayer, Joanna Meira,Marla Niag, Kleber Pimentel, Ivana Luz, Bianca Figueiredo, Tatiana Michelon,Jorge Neumann, Simone Lima, Isabela Nelly Machado, Edward Araujo Júnior& Ricardo Barini

To cite this article: Marcelo Borges Cavalcante, Manoel Sarno, Gabriela Gayer, JoannaMeira, Marla Niag, Kleber Pimentel, Ivana Luz, Bianca Figueiredo, Tatiana Michelon, JorgeNeumann, Simone Lima, Isabela Nelly Machado, Edward Araujo Júnior & Ricardo Barini(2018): Cytogenetic abnormalities in couples with a history of primary and secondary recurrentmiscarriage: a Brazilian Multicentric Study, The Journal of Maternal-Fetal & Neonatal Medicine,DOI: 10.1080/14767058.2018.1494714

To link to this article: https://doi.org/10.1080/14767058.2018.1494714

Accepted author version posted online: 27Jun 2018.

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CYTOGENETIC ABNORMALITIES IN COUPLES WITH AHISTORY OF PRIMARY AND SECONDARY RECURRENT

MISCARRIAGE: A BRAZILIAN MULTICENTRIC STUDY

Running title: Cytogenetic and recurrent miscarriages

Type of article: Original article

Authors: Marcelo Borges Cavalcante1, Manoel Sarno2,3, Gabriela Gayer3, Joanna Meira4,Marla Niag3, Kleber Pimentel2, Ivana Luz3, Bianca Figueiredo5, Tatiana Michelon6, JorgeNeumann6, Simone Lima7, Isabela Nelly Machado7, Edward Araujo Júnior8, RicardoBarini7,9

Institutions:1Department of Obstetrics and Gynecology, Fortaleza University (UNIFOR), Fortaleza,Brazil2Department of Obstetrics and Gynecology, Federal University of Bahia (UFBA),Salvador, Brazil3Aloimune - Reproductive Immunology Centre, Salvador, Brazil4Department of Genetics, Federal University of Bahia (UFBA), Salvador, Brazil5Reproductive Immunology Centre, Rio de Janeiro, Brazil6Reproductive Immunology Centre, Porto Alegre, Brazil7Allovita Reproductive Immunology Centre, Campinas, Brazil8Department of Obstetrics, Paulista School of Medicine - Federal University of São Paulo(EPM-UNIFESP), São Paulo, Brazil9Department of Obstetrics and Gynecology, State University of Campinas (UNICAMP),Campinas, Brazil

Address for correspondence:Prof. Edward Araujo Júnior, PhDRua Belchior de Azevedo, 156 apto. 111 Torre VitoriaSão Paulo-SP, BrazilCEP 05089-030Tel/Fax: +55-11-37965944; E-mail: araujojred@terra.com.br

ABSTRACT

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Objective: evaluate the difference between chromosomal abnormalities between the

gender of couples affected by RM and if there is an association between previous obstetric

history and chromosomal abnormalities of the parents

Methods: multicenter, retrospective, observational study from seven different RM clinics

between 2006 and 2016. We enrolled 707 couples (1,014 participants) with a history

of RM. We compared the frequency of chromosomal abnormalities between groups of

couples with primary and secondary RM and separated between women and their partners.

Furthermore, we compared the prevalence of chromosomal abnormalities between groups

based on the number of previous spontaneous abortions.

Results: The overall prevalence of all cytogenetic abnormalities was 5.59% (n =

1,414, women and their partners). Excluding cases of polymorphism and inversion

of chromosome 9, which are considered variants of normality, the prevalence in all

individuals was 2.26% (n = 32/1,414). The comparative analysis of cases of chromosomal

abnormalities among couples with primary and secondary RM based on the number of

PM revealed a similar frequency between groups. The statistical analysis of the total

cases (primary PM + secondary PM) in these three groups were as follows: (a) couple,

2 PM vs. 3 PM vs. ≥4 PM, p = 0.514; (b) women, 2 PM vs. 3 PM vs. ≥4 PM, p =

0.347; and (3) partner, 2 PM vs. 3 PM vs. ≥4 PM, p = 0.959. Chromosomal abnormalities

were significantly more prevalent among women than among their partners (6.9% vs.

4.2%; p = 0.027). Moreover, the distribution of leading chromosomal abnormalities

among women was different compared with their partners. Among women, we observed

these abnormalities in the following frequency order: mosaicism (38.8%), polymorphism

(32.6%), translocation (16.3%), and inversion (12.3%). Among their partners, these

abnormalities were polymorphism (73.3%), inversion (13.3%), mosaicism (6.7%), and

translocation (6.7%).

Conclusion:the number of PM and the history of full term pregnancy does not correlate

with an increase or decrease in the prevalence of cytogenetic abnormalities in couples

with RM.

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Keywords: recurrent miscarriage, recurrent pregnancy loss, cytogenetic

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INTRODUCTION

Recurrent miscarriage (RM) has been historically defined by the World Health

Organization (WHO) as the occurrence of three or more spontaneous and consecutive

gestational losses, each occurring until 20 weeks of gestational age (1). However, this

concept has evolved over the years. The American Society for Reproductive Medicine

defined RM as two or more spontaneous abortions, consecutive or non-consecutive, but

with gestation proven ultrasonographically or through histopathological examination (2).

In contrast, the European Society for Human Reproduction and Embryology and the

Royal College of Obstetricians and Gynaecologists defined RM as the occurrence of three

consecutive (not necessarily intrauterine) gestational losses (3,4). Thus, the International

Committee for Monitoring Assisted Reproductive Technology and the WHO revised

Glossary on ART Terminology to standardize concepts and proposed that RM should be

defined as the occurrence of two or more spontaneous abortions before the 20th week of

clinical pregnancy, not necessarily consecutive (5).

Typically, RM is categorized into primary RM, when there are no previous

pregnancies of >20 weeks, or secondary RM, when multiple losses occur in a woman

who has already had a pregnancy beyond 20 gestational weeks, resulting in live birth

(more frequent), stillbirth, or neonatal death (6). Some studies reported primary and

secondary RM as a similar pathological entity; however, others suggested that distinct

groups comprised different etiologies, proposing different investigation and therapy (6,7).

RM affects about 2%–5% of couples in reproductive age, with a current increasing

trend in the incidence (6). Despite all technological advancements and novel concepts,

approximately 50% of the cases remain without definite cause (6). Some of the

factors associated with RM are parental chromosomal abnormalities, uterine anatomical

abnormalities, hormonal disorders, maternal obesity, and antiphospholipid syndrome.

However, other factors still lack further evidence, including hereditary thrombophilia,

chronic endometritis, and alloimmune factors (6).

Parental chromosomal abnormalities represent a well-established cause of RM,

with the incidence ranging between 2% and 13% (8,9). These abnormalities can be

structural or numerical; although structural rearrangements are the leading chromosomal

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anomaly among couples with RM (8,10), the screening with parental karyotypes in RM

couples remains debatable in the literature (11-13).

To date, the best therapeutic approach for RM couples with karyotype

abnormalities awaits establishment. The use of embryonic preimplantation genetic

diagnosis (PGD) did not elevate the rates of live births in these couples (14,15). Thus,

a better understanding of the correlation between genetic factors and RM is of high

relevance and must answer some questions (8,10,16). The objective of this study is

to evaluate the difference between chromosomal abnormalities between the gender of

couples affected by RM and if there is an association between previous obstetric history

and chromosomal abnormalities of the parents.

METHODS

In this multicentre, retrospective, observational study conducted in seven Brazilian

reproductive immunology centers (cities of São Paulo, Campinas, Rio de Janeiro,

Salvador, Porto Alegre, Recife, and Fortaleza) between January 2006 and December

2016, we reviewed the medical records of 707 couples with two or more consecutive

spontaneous abortions, with or without previous gestation(s) of >20 weeks, and who

undertook genetic testing through a peripheral blood G-banding karyotype.

In the karyotyping, the first stepcomprised the preparation of lymphocyte cultures

from the peripheral blood. The chromosome isolation technique involved the disruption

of the spindle fibers to inhibit the cells from proceeding to the subsequent anaphase

stage. Then, we treated the cells with a hypotonic solution and preserved them in their

swollen state with Carnoy’s fixative. Next, the cells were dropped on the slides. G-banding

involved trypsin treatment followed by staining with Giemsa to create characteristic light

and dark bands (17).Of note, 20 metaphases were analyzed per case. However, in cases

with suspected mosaicism, this number was expanded to 100.

We classified the karyotypes in accordance with the International System for

Human Cytogenetic Nomenclature. The images were acquired and analyzed using

BandView Software (Applied Spectral Imaging). We re-verified the results following

international criteria of assertiveness, tracking, and quality control (18). In addition,

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the frequency of chromosomal abnormalities was compared between groups of couples

with primary and secondary RM and separated between women and their partners.

Furthermore, the prevalence of chromosomal abnormalities was compared between

groups according to the number of previous spontaneous abortions.

The characteristics of the studied population were described as mean standard

deviation for continuous variables, these were described as numbers and percentages for

categorical variables. In addition, comparisons between groups were performed using

the Mann-Whitney test, Kruskal–Wallis test, the Fisher’s exact test, and the chi-squared

testwhen appropriate. We transferred data to an Excel 2007 worksheet (Microsoft Corp.,

Redmond, WA, USA) and used SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) for the

statistical analysis. We considered P < 0.05 as statistically significant. This study protocol

was approved by a local ethics committee (reference number 1.542.767).

RESULTS

We analyzed the results of 1,014 karyotypes (707 women and their 707 partners)

from 707 couples with a history of RM. The women’s age was 33.79 ± 4.8 years, with an

obstetric history of 2.85 1.1 previous pregnancies, 0.16 0.4 pregnancies that evolved

at term, and 2.68 0.9 spontaneous abortions. Most women (53.5%) had a history of two

spontaneous abortions, while 31.5% had three spontaneous abortions, 15% had a history

of four or more spontaneous abortions (Table 1).

Of all cases, we classified 602 (85.1%) couples as primary RM. The maternal

age, number of pregnancies, and previous gestational losses were considerably higher

among couples with secondary RM. The number of previous abortions (2.66 0.9 vs.

2.80 1.1; p = 0.162) and the percentage of couples with abnormal karyotypes (10.6%

vs. 10.5%; p = 0.936) were similar between the primary and secondary RM groups,

respectively (Table 1). Based on the number of previous abortions, the prevalence of total

cytogenetic aberrations (women and their partners) between groups was 5.7% (43/756),

6.1% (27/446), and 4.2% (09/212) for groups with two, three, and four or more previous

miscarriages (PM), respectively. According to the number of PM, the comparative analysis

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of cases of chromosomal abnormalities among couples with primary and secondary RM

revealed a similar frequency between groups (Table 2). In addition, the findings of the

statistical analysis of the total cases (primary + secondary) in the three groups were as

follows: (a) couple, 2 PM vs. 3 PM vs. ≥4 PM, p = 0.514; (b) women, 2 PM vs. 3 PM vs.

≥4 PM, p = 0.347; and (c) partner, 2 PM vs. 3 PM vs. >4 PM, p = 0.959 (Table 2).

We considered the karyotypes abnormal (including all anomalies) in 79 cases,

with a total prevalence of 5.59% (n = 1,414, women and their partners). Excluding

cases of polymorphism and inversion of chromosome 9, which are considered variants

of normality, the prevalence in all individuals was 2.26% (n = 32/1,414). In addition, we

observed concurrent abnormalities in both woman and their partner in four cases, two

couples, in the group of 2 PM (one primary RM and one secondary RM); one couple in the

group of 3 PM (one primary RM) and one couple in the group with >4 PM (one primary

RM). The prevalence of altered karyotype pairs (only the woman, only the partner, or both)

was 10.6% (n = 75/707) among the 707 couples. Furthermore, chromosomal abnormalities

were significantly more frequent among women than men (49/707 cases [6.9%] vs. 30/707

cases [4.2%], respectively; p = 0.027; Table 2).

The leading chromosomal abnormalities among women were mosaicism (19/49,

38.8%), polymorphism (16/49, 32.6%), translocation (8/49, 16.3%), and inversion (6/49,

12.3%). Among men, the leading karyotype alteration was polymorphism (22/30, 73.3%),

inversion (4/30, 13.3%), mosaicism (2/30, 6.7%), and translocation (2/30, 6.7%; Table

3). The chromosome that presented a higher frequency of balanced translocation was

chromosome 8 (three cases), followed by 1, 9, 11, 13, and 19 (all with two cases each).

Furthermore, cases of mosaicism mostly involved sex chromosomes, except for two

cases (one case among women and another among their partners) in which the karyotype

identified a marker chromosome (mar) (Table 4).

DISCUSSION

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The correlation between chromosomal abnormalities and high prevalence of RM is

well-established; hereby, couples’ cytogenetic analysis is part of almost all the protocols of

clinical investigation of RM worldwide (19,20,11). Of note, the prevalence of cytogenetic

abnormalities in couples with RM is variable in the literature, ranging from 1%–7% (8).

Perhaps, this variation in the prevalence depends on the population studied, inclusion

criteria, and cytogenetic findings considered altered (8,21,22).

Structural chromosome abnormalities, especially balanced translocations, are

more frequently described in couples facing RM. Moreover, Robertsonian translocations,

mosaicisms, and inversions are frequent. While the prevalence of structural abnormalities

varies from 1%–6%, numerical aberrations range from 0.4%–2% (8,23-25). Although

polymorphic variations are described more frequently in this group of women, no

consensus exists about their actual participation in mechanisms of gestational loss (25,26).

The number of PM does not correlate with an increase or decrease in the prevalence

of cytogenetic abnormalities in our analysis; 5.7%, 6.1%, and 4.2% for groups with two,

three, and four or more PM, respectively. Likewise, Elghezal et al. did not establish a

correlation between the number of previous abortions and the prevalence of cytogenetic

abnormalities in a group of 1,400 couples with three or more recurrent abortions; 6.3%

(45/1,432) and 3.8% (52/1,368) for couples with three PM and four or more PM,

respectively (16). Furthermore, Tunç et al. did not establish a correlation between the

number of PM and the incidence of cytogenetic alterations. A study of 1,510 RM couples

reported the incidence of 3%, 3%, 7%, and 7% of chromosomal abnormalities in couples

with a history two, three, four, or five or more PM, respectively (23).

According to the history of term gestation before the evaluation (primary or

secondary RM), cytogenetic abnormalities were similar between the groups. To date, a

few authors have compared the frequency of cytogenetic abnormalities in primary and

secondary RM couples. Similarly, Shapira et al. observed a similar number of abnormal

karyotypes in couples with a history of primary and secondary RM (3.1% and 6.8%; P =

0.196), respectively (7).

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Of all analyses performed by our group, the totality of cases of structural and

numerical anomalies was similar; however, the numerical abnormalities were always

found in mosaic. The numerical abnormalities (aneuploidies) corresponded to 21 cases

(26.6%), 19 involving sex chromosomes and two marker chromosomes. A marker

chromosome (mar) is a small fragment of a chromosome which generally cannot be

identified without specialized genomic analysis due to the size of the fragment. Therefore,

the clinical consequence of this marker will vary depending on which material is contained

in the marker. In order to perform this evaluation, the most appropriate exam would be

the arrayCGH (comparative genomic hybridization), however this exam was not included

in the methodology of this study. In addition, structural arrangements were noted in 20

cases (25.3%); 10 balanced translocations and 10 chromosomal inversions. One case of

a Robertsonian translocation involving chromosomes 13 and 15 was also observed. The

remaining 38 cases (48.1%) were classified as polymorphic variations. The literature data

presents a higher proportion of cases of structural abnormalities when compared with the

numerical ones, reaching ratios from 2:1 to 8:1 (9).

In our sample, chromosomal abnormalities were more prevalent among women

than men (1.6:1). However, excluding cases of polymorphism and inversion of

chromosome 9, this ratio increased to 7:1 (women:partners), demonstrating a higher

incidence rate when compared with other published studies [Tharapel et al. (1985), which

was 2.2:1; Elghezal et al. (2007), which was 3:1; and Tunç et al. (2016), which was 1.7:1]

(8,16,23).

The chromosome that presented a higher frequency of balanced translocation was

chromosome 8 (three cases), followed by 1, 9, 11, 13 and 19 (two cases each). Tharapel

et al. (1985), investigating a sample of 16,042 recurrent abortion couples (8,208 women

and 7,834 partners), reported a balanced translocation in all chromosomes, including

sex chromosomes, being chromosomes 1, 7, 6, 4, 3, and 11 most often involved (8).

Cytogenetic heteromorphisms and polymorphisms are described as variations in specific

chromosomal regions with no impact on phenotype. However, higher frequencies of these

variants have recently been reported in infertile and subfertile individuals, and so their

clinical significance has been frequently debated.

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Common chromosomal polymorphisms comprise heterochromatin regions on

short arms of acrocentric chromosomes and regions of chromosomes 1, 9, 16, and Y

(8). Reportedly, the prevalence of polymorphisms is marginally higher in men, with

heterochromias and Y polymorphisms corresponding to most variants detected (27),

which is corroborated by our study. A study reported that the prevalence of polymorphism

is considerably higher in men with oligozoospermia or azoospermia than in those with

normozoospermia (28).

Some studies have suggested an elevated frequency of heteromorphisms in

parents with multiple pregnancy losses, suggesting a correlation between these findings

with RM (22,29-32). Inversion of chromosome 9 has been reported to play a vital

role in chromosomal non-disjunction and exerts variable effects on spermatogenesis,

from azoospermia to the rigorously altered sperm morphology, motility, and meiotic

segregation (33,34). In addition, several studies suggest that heterochromatin plays an

essential crucial role in the spindle attachment and chromosome movement, meiotic

pairing, and sister chromatid cohesion (35). However, evidence from some controlled

surveys on healthy adults and parents with multiple pregnancy losses did not entirely

support this perspective (36,37).

Historically, the gold-standard genetic evaluation for couples with RM is the

peripheral blood karyotype. With the emergence of cytogenomics techniques, researches

seek new genetic markers present in these couples. Currently, chromossomal microarray

analysis (CMA) is proposed for investigation of chromosomal abnormalities in products

of conception, with encouraging results (38). However, the use of CMA in counseling

couples with recurrent miscarriage still requires further evidences.

Elucidating cytogenetic abnormalities in RM cases is imperative for

preconception counseling and devising a therapeutic plan for these couples. Seemingly,

submitting these cases to assisted reproduction cycles with PGD is a logical course but still

lacks scientific evidence compared with a natural pregnancy (15,25). Hence, we assume

that the evaluation of cytogenetics factors should be part of the investigation protocols of

couples with RM, irrespective of the obstetric history.

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CONFLICT OF INTEREST

None declared.

REFERENCES

1. WHO. Recommended Definitions, Terminology and Format for Statistical Tables

Related To the Perinatal Period and Use of a New Certificate for Cause of Perinatal

Deaths. Acta Obs Gynecoi Scand 1977;56:247–253.

2. Practice Committee of American Society for Reproductive Medicine. Definitions of

infertility and recurrent pregnancy loss: a committee opinion. Fertil Steril 2013;99:63.

3. Zegers-Hochschild F, Adamson GD, Mouzon J de, Ishihara O, Mansour R, Nygren

K, et al. International Committee for Monitoring Assisted Reproductive Technology

(ICMART) and the World Health Organization (WHO) revised glossary of ART

terminology, 2009. Fertil Steril 2009;92:1520–1524.

4. Jauniaux E, Farquharson RG, Christiansen OB, Exalto N. Evidence-based guidelines

for the investigation and medical treatment of recurrent miscarriage. Hum Reprod

2006;21:2216–2222.

5. Zegers-Hochschild F, Adamson GD, Dyer S, Racowsky C, Mouzon J de, Sokol R,

et al. The International Glossary on Infertility and Fertility Care, 2017. Fertil Steril

2017;108:393–406.

6. El Hachem H, Crepaux V, May-Panloup P, Descamps P, Legendre G, Bouet PE.

Recurrent pregnancy loss: current perspectives. Int J Womens Health 2017; 9:331–345.

7. Shapira E, Ratzon R, Shoham-Vardi I, Serjienko R, Mazor M, Bashiri A. Primary vs.

secondary recurrent pregnancy loss--epidemiological characteristics, etiology, and next

pregnancy outcome. J Perinat Med 2012;40:389–396.

8. Tharapel AT, Tharapel SA, Bannerman RM. Recurrent pregnancy losses and parental

chromosome abnormalities: a review. Br J Obstet Gynaecol 1985;92:899–914.

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9. Dutta UR, Rajitha P, Pidugu VK, Dalal AB. Cytogenetic abnormalities in 1162 couples

with recurrent miscarriages in southern region of India: report and review. J Assist

Reprod Genet 2011;28:145–149.

10. Stephenson MD, Sierra S. Reproductive outcomes in recurrent pregnancy loss

associated with a parental carrier of a structural chromosome rearrangement. Hum

Reprod 2006;21:1076–1082.

11. Practice Committee of American Society for Reproductive Medicine. Evaluation

and treatment of recurrent pregnancy loss: a committee opinion. Fertil Steril

2012;98:1103-1111.

12. Vlaanderen W. Recurrent miscarriage: guidelines could be improved. Hum Reprod

2014;29:1344–1345.

13. Bender Atik R, Christiansen OB, Elson J, Kolte AM, Lewis S, Middeldorp S, et al.

ESHRE guideline: recurrent pregnancy loss. Hum Reprod Open 2018;2018:.

14. De Krom G, Arens YH, Coonen E, Van Ravenswaaij-Arts CM, Meijer-Hoogeveen

M, Evers JL, et al. Recurrent miscarriage in translocation carriers: no differences in

clinical characteristics between couples who accept and couples who decline PGD.

Hum Reprod 2015;30:484–489.

15. Iews M, Tan J, Taskin O, Alfaraj S, AbdelHafez FF, Abdellah AH, et al.

Does preimplantation genetic diagnosis improve reproductive outcome in couples

with recurrent pregnancy loss owing to structural chromosomal rearrangement? A

systematic review. Reprod Biomed Online 2018; S1472-6483(18)30101-9 [Epub ahead

of print].

16. Elghezal H, Hidar S, Mougou S, Khairi H, Saâd A. Prevalence of chromosomal

abnormalities in couples with recurrent miscarriage. Fertil Steril 2007;88:721–723.

17. Howe B, Umrigar A, Tsien F. Chromosome preparation from cultured cells. J Vis Exp

2014;83:e50203.

18. McGowan-Jordan, Jean; Simons, Annet; Schmid, M (Michael); International Standing

Committee on Human Cytogenomic Nomenclature. ISCN: an international system for

human cytogenomic nomenclature (2016). Karger: Basel; 2016.

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19. Toth B, Würfel W, Bohlmann M, Gillessen-Kaesbach G, Nawroth F, Rogenhofer

N, et al. Recurrent Miscarriage: Diagnostic and Therapeutic Procedures. Guideline of

the DGGG (S1-Level, AWMF Registry No. 015/050, December 2013). Geburtshilfe

Frauenheilkd 2015;75:1117–1129.

20. Royal College of Obstetricians and Gynaecologists. The Investigations and Treatments

of Couples with Recurrent First-Trimester and SecondTrimester Miscarriage. London,

UK: Royal College of Obstetricians and Gynaecologists; 2011. Green-top Guidel; 17.

21. Mozdarani H, Meybodi AM, Zari-Moradi S. A cytogenetic study of couples with

recurrent spontaneous abortions and infertile patients with recurrent IVF/ICSI failure.

Indian J Hum Genet 2008;14:1–6.

22. Campanho Cde L, Heinrich JK, Couto E, Barini R. [Subfertility phenotype,

chromosome polymorphism and conception failures]. Rev Bras Ginecol Obstet

2011;33:246–251.

23. Tunç E, Tanrıverdi N, Demirhan O, Süleymanova D, Çetinel N. Chromosomal

analyses of 1510 couples who have experienced recurrent spontaneous abortions.

Reprod Biomed Online 2016;32:414–419.

24. Kiss A, Rosa RF, Dibi RP, Zen PR, Pfeil JN, Graziadio C, et al. [Chromosomal

abnormalities in couples with history of recurrent abortion]. Rev Bras Ginecol Obstet

2009;31:68–74.

25. Maithripala S, Durland U, Havelock J, Kashyap S, Hitkari J, Tan J, et al. Prevalence

and Treatment Choices for Couples with Recurrent Pregnancy Loss Due to Structural

Chromosomal Anomalies. J Obstet Gynaecol Can 2017; S1701-2163(17)30605-9

[Epub ahead of print].

26. Caglayan AO, Ozyazgan I, Demiryilmaz F, Ozgun MT. Are heterochromatin

polymorphisms associated with recurrent miscarriage? J Obstet Gynaecol Res

2010;36:774–776.

27. Liang J, Zhang Y, Yu Y, Sun W, Jing J, Liu R. Effect of chromosomal polymorphisms of

different genders on fertilization rate of fresh IVF-ICSI embryo transfer cycles. Reprod

Biomed Online 2014;29:436–444.

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28. Pylyp LY, Spinenko LO, Verhoglyad NV, Zukin VD. Chromosomal abnormalities in

patients with oligozoospermia and non-obstructive azoospermia. J Assist Reprod Genet

2013;30:729–732.

29. Sahin FI, Yilmaz Z, Yuregir OO, Bulakbasi T, Ozer O, Zeyneloglu HB. Chromosome

heteromorphisms: an impact on infertility. J Assist Reprod Genet 2008;25:191–195.

30. Purandare H, Fernandez NV, Deshmukh SV, Chavan S. Heterochromatic variations

and Pregnancy losses in Humans. Int J Hum Genet 2011;11: 167-175.

31. Akbaş H, Isi H, Oral D, Türkyılmaz A, Kalkanlı-Taş S, Simşek S, et al. Chromosome

heteromorphisms are more frequent in couples with recurrent abortions. Genet Mol Res

2012;11:3847–3851.

32. Baghbani F, Mirzaee S, Hassanzadeh-Nazarabadi M. Association of heteromorphism

of chromosome 9 and recurrent abortion (ultrasound diagnosed blighted ovum): A case

report. Iran J Reprod Med 2014;12:357–360.

33. Saran N, Kumar B, Kumar A. Chromosomal Heteromorphisms and Karyotype

Abnormalities in Humans. Int J Curr Microbiol App Sci 2017;6:2940–2953.

34. Morales R, Lledó B, Ortiz JA, Ten J, Llácer J, Bernabeu R. Chromosomal polymorphic

variants increase aneuploidies in male gametes and embryos. Syst Biol Reprod Med

2016;62:317–324.

35. Karpen G, Endow S. Meiosis: chromosome behaviour and spindle dynamics. In:

Endow SA, Glover DM, editors. Dynamics of Cell Division. Oxford University Press:

Oxford; 1998.

36. Verma RS. Heterochromatin: Molecular and Structural Aspects. Cambridge

University Press: New York; 1998.

37. Kosyakova N, Grigorian A, Liehr T, Manvelyan M, Simonyan I, Mkrtchyan H, et al.

Heteromorphic variants of chromosome 9. Mol Cytogenet 2013;6:14

38. Sahoo T, Dzidic N, Strecker MN, Commander S, Travis MK, Doherty C, et al.

Comprehensive genetic analysis of pregnancy loss by chromosomal microarrays:

outcomes, benefits, and challenges. Genet Med 2017;19:83-89

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Table 1. Demographic characteristics, obstetric history, and cytogenetic studies in thegroup of patients based on their gestational outcomes.

Variable All Patients(n = 707)

Primary RM (n = 602)

Secondary RM (n = 105)

P

Female age, mean SD 33.79 4.8 33.49 4.8 35.52 4.8 <0.001

Number of pregnancies,mean SD

2.85 1.1 2.67 0.9 3.90 1.1 <0.001

Number of pregnanciescarried to term, mean SD

0.16 0.4 0 1.10 0.3 <0.001

Number of miscarriages,mean SD

2.68 0.9 2.66 0.9 2.80 1.1 0.162

2 PM, n (%) 378 (53.5) 327 (54.3) 51 (48.6) 0.275

3 PM, n (%) 223 (31.5) 188 (31.2) 35 (33.3) 0.668

>4 PM, n (%) 106 (15.0) 87 (14.5) 19 (18.1) 0.334

Women abnormal karyotype, n(%)

49 (6.9) 40 (6.6) 09 (8.6) 0.473

Partners abnormal karyotype, n(%)

30 (4.2) 27 (4.5) 03 (2.9) 0.603

Couple abnormal karyotype,n (%)

75 (10.6) 64 (10.6) 11 (10.5) 0.936

RM, recurrent miscarriage; PM, previous miscarriage; SD, standard deviation.

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Table 2. Cytogenetic abnormalities according to the number of previous miscarriages andobstetric history.

Variables 2 PM (n = 378) 3 PM (n = 223) >4 PM (n = 106)

Primary(n = 327)

Secondary(n = 51)

P Primary(n = 188)

Secondary(n = 35)

P Primary(n = 87)

Secondary(n = 19)

P

Women, n (%) 24 (7.3) 03 (5.9) 0.70 13 (6.9) 05 (14.3) 0.14 03 (3.4) 01 (5.3) 0.70

Partners, n (%) 13 (4,0) 03 (5.9) 0.46 09 (4.8) 00 0.36 05 (5.7) 00 0.58

Couple, n (%) 36 (11.0) 05 (9.8) 0.79 21 (9.4) 05 (14.2) 0.59 07 (8.0) 01 (5.3) 0.67

Cytogenetics abnormalities were observed in four couples: two couples in the group of 2 PM (one primaryRM and one secondary RM); one couple in the group of 3 PM (primary RM); one couple in the group with>4 PM (primary RM). Statistical analysis of total cases (primary + secondary) in the three groups were asfollows: (a) couple, 2 PM vs. 3 PM vs. ≥4 PM, P = 0.514; (b) women, 2 PM vs. 3 PM vs. ≥4 PM, P = 0.347;and (c) partner, 2 PM vs. 3 PM vs. ≥4 PM; P = 0.959.

JUST A

CCEPTED

Table 3. Cytogenetic abnormalities according to the gender, number of previousmiscarriage, and obstetric history.

Women karyotypes Partners karyotypes

Results All (n = 49) Primary(n = 40)

Secondary(n = 09)

All (n = 30) Primary(n = 27)

Secondary(n = 3)

P

Mosaicism, n (%) 19 (38.8) 16 (40.0) 03 (33.3) 02 (6.7) 02 (7.4) 00 (0) 0.001

Translocation, n (%) 08 (16.3) 06 (15) 02 (22.3) 02 (6.7) 02 (7.4) 00 (0) 0.210

Inversion, n (%) 06 (12.3) 05 (12.5) 01 (11.1) 04 (13.3) 04 (14.8) 00 (0) 0.887

Polymorphism, n (%) 16 (32.6) 13 (32.5) 03 (33.3) 22 (73.3) 19 (70.4) 03 (100) 0.001

Translocation, includes all types of translocation; Inversion, includes all types of inversion; Primary, primaryrecurrent miscarriage couples; Secondary, secondary recurrent miscarriages couples.

JUST A

CCEPTED

Table 4. The frequency of cytogenetic abnormalities.

Women karyotypes (n = 49) Partners karyotypes (n = 30)

Mosaicism (n = 19) Mosaicism (n = 2)

46,XX (90) / 45,X0 (10) (n = 07)

46,XX (92) / 45,X0 (04) / 47,XXX (04) (n = 06)

46,XX (96) / 47,XXX (04) (n = 04)

46,XX (97) / 45,X0 (01) / 47,XXX (01) / 49,XXXXX(01) (n = 01)

46,XX (50) / 47,XX +mar (50) (n = 01)

46,XY (90) / 45,X0 (10) (n = 01)

46,XY (64) / 47,XY +mar (10) (n = 01)

Translocation (n = 8) Translocation (n = 2)

46,XX t(1;9)(p12q13) (n = 01)

46,XX t(13;15)(q10;q10) (n = 01)*

46,XX t(8;19)(p21q13) (n = 01)

46,XX t(1;3)(q42q11) (n = 01)

46,XX t(8;19)(q13q11) (n = 01)

46,XX t(11;18)(q11;q11) (n = 01)

46,XX t(11;22)(q25;q13) (n = 01)

46,XX t(6;9)(q23;p24) (n = 01)

46,XY t(5;13)(q11q12) (n = 01)

46,XY t(8;10)(q24.3p2) (n = 01)

Inversion (n = 6) Inversion (n = 4)

46,XX inv(9)(p12q13) (n = 05)

46,XX inv(6)(p23q23) (n = 01)

46,XY inv(9)(p12q13) (n = 04)

Polymorphism (n = 16) Polymorphism (n = 22)

46,XX,9qh+ (n = 04)

46,XX,1qh+ (n = 02)

46,XX,15ptsk+ (n = 02)

46,XX,15qh+ (n = 02)

46,XX,21ptsk+ (n = 02)

46,XX,22ps+ (n = 02)

46,XX,15ps+ (n = 02)

46,XY qh+ (n = 14)

46,XY 9qh+ (n = 02)

46,XY 7qh+ (n = 01)

46,XY 21pstk+ (n = 03)

46,XY 22pstk+ (n = 01)

46,XY 15ps+ (n = 01)

*Robertsonian translocation.

1

JUST A

CCEPTED