The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69
DOI 10.1186/s12881-016-0331-1

RESEARCH

Open Access

Associations of recurrent miscarriages withchromosomal abnormalities, thrombophilia
allelic polymorphisms and/or consanguinity
in Saudi Arabia
Rola F. Turki1,2†, Mourad Assidi2,3†, Huda A. Banni2,3, Hanan A. Zahed1, Sajjad Karim3, Hans-Juergen Schulten3,
Muhammad Abu-Elmagd2,3, Abdulrahim A. Rouzi1, Osama Bajouh1,2, Hassan S. Jamal1,
Mohammed H. Al-Qahtani3 and Adel M. Abuzenadah2,3*
From 3rd International Genomic Medicine Conference
Jeddah, Saudi Arabia. 30 November - 3 December 2015

Abstract
Background: Recurrent pregnancy loss (RPL) or recurrent spontaneous abortion is an obstetric complication that affects
couples at reproductive age. Previous reports documented a clear relationship between parents with chromosomal
abnormalities and both recurrent miscarriages and infertility. However, limited data is available from the Arabian
Peninsula which is known by higher rates of consanguineous marriages. The main goal of this study was to determine
the prevalence of chromosomal abnormalities and thrombophilic polymorphisms, and to correlate them with RPL andconsanguinity in Saudi Arabia.
Methods: Cytogenetic analysis of 171 consent patients with RPL was performed by the standard method of 72-h
lymphocyte culture and GTG banding. Allelic polymorphisms of three thrombophilic genes (Factor V Leiden,
Prothrombin A20210G, MTHFR C677T) were performed using PCR-RFLP (restriction fragment length polymorphism)and gel electrophoresis.
Results: Data analysis revealed that 7.6 % of patients were carrier of numerical or structural chromosomal
abnormalities. A high rate of translocations (46 %) was associated to increased incidence of RPL. A significant
correlation between consanguineous RPL patients and chromosomal abnormalities (P < 0.05) was found. Both
Factor V Leiden and Prothrombin A20210G allelic polymorphisms were significantly associated with a higher
prevalence of RPL.
(Continued on next page)* Correspondence: aabuzenadah@kau.edu.sa†
Equal contributors
2
Center of Innovation in Personalized Medicine, King AbdulAziz University,
P.O. Box: 80216, Jeddah 21589, Kingdom of Saudi Arabia
3
Center of Excellence in Genomic Medicine Research, King Abdulaziz
University, Jeddah, Saudi Arabia
Full list of author information is available at the end of the article© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

Page 16 of 23(Continued from previous page)

Conclusions: This study demonstrated a strong association between RPL and the prevalence of chromosomal
abnormalities and inherited thrombophilia. Given the high rate of consanguineous marriages in the Saudi
population, these results underline the importance of systematic cytogenetic investigation and genetic counselingpreferably at the premarital stage or at least during early pregnancy phase through preimplantation genetic diagnosis(PGD).
Keywords: Recurrent pregnancy loss, Chromosomal aberrations, Thrombophilia, Consanguinity, Cytogenetic analysis

Background
Recurrent miscarriages (RM) are clinically detectablepregnancies that fail to progress. They are common
pregnancy complications that affects 15–20 % of couples[1]. It is a common obstetric health concern that affects
around 5 % of women at the reproductive age [2, 3]. Regarding the RM etiology, it is due to several causes including chromosomal, genetic, anatomic, immune andinfective factors [1]. Although it is still a controversy
whether RM is considered after 2 pregnancy losses versus 3 or more, most of clinicians recommended initiating evaluations from the onset of the second miscarriage
since there was no significant difference in RM susceptibility between several patients with 2 versus 3 and morepregnancy losses [4].
Despite worthy studies in Obstetrics/Gynecology clinics
and IVF (in vitro fertilization) centers worldwide of thissporadic complication of early pregnancy, RM etiology remains poorly understood [5, 6]. Therefore, effective diagnosis and prevention/treatment approaches are stilllacking [7, 8]. Several causes underlying this failure to deliver a normal and viable embryo were reported including
aged mothers, uterine abnormalities, placental abruption,
incompetent cervix, parents' chromosomal and genetic
background, immune disorders and/or endocrine imbalances. Among these causes, three in particular were considered as the major factors of RM including: (i) structuraland numerical chromosomal abnormalities, (ii) inflammatory and autoimmune disorders, and (iii) allelic polymorphisms of some pro-thrombophilic genes [6, 9–12]. In
fact, positive correlations were reported between chromosomal abnormalities in the embryos and higher RM [13].
This fetal genomic incompatibility to life was associated tochromosomal aneuploidies and mosaicism within theembryo/abortus [14–17]. Moreover, couples who carry
chromosomal abnormalities were found to be at risk for
repeated miscarriages and therefore have lower chances todeliver a viable offspring [7, 18–21]. Spontaneous miscarriages caused by the chromosomal abnormalities may
arise from one of the parents producing defective gametesthat will lead to fetal abnormalities and mental disorders.
In fact, 3–6 % of RM were due to chromosomal abnormalities of one of the two partners [22–24]. Trisomies inchromosomes 13–16, 21 and 22 were the most common

chromosomal aneuploidies followed by monosomy X(45, X). Thus, parental karyotyping is a recommended
procedure to assess the cause of recurrent pregnancy
losses [23, 25–27].
Pro-thrombophilic factors have also been suggested as
one of the major causes of RM. In fact, some genetic
polymorphisms of prothrombin (FII G 20210A), Factor
V (Factor V Leiden, FVL) and methylene tetrahydrofolate
reductase (MTHFR, C677T gene variant) genes werestrongly associated with recurrent miscarriages [3, 28, 29].
These factors of inherited thrombophilia disturb normal
placental vascularization and formation leading to fetal
growth restrictions, pregnancy failure, placental abruption
and therefore miscarriages or stillbirth [30].
In addition to the RM incidence worldwide, Saudi
Arabia is well known by a high level of consanguineous
marriages driven by ethnic or tribal considerations [31].
These consanguineous marriages significantly increase theincidence of inherited recessive disorders and affect some
reproductive and developmental health parameters such asinfertility rates, recurrent miscarriages, and congenital disorders (e.g. thrombophilia) [32–35]. The objective of this study
is to assess possible correlations between chromosomal abnormalities and couples with history of RM. Additional investigations were also carried out to assess the presence ofsome thrombophilia genetic risk factors including factor V
Leiden, Prothrombin A20210G, MTHFR C677T mutations
that may further explain the RM outcomes.

Methods
Study approach

A cohort of patients with a history of two or more miscarriages up to 20 weeks was conducted at King Abdulaziz
University Hospital in the Western region of Saudi Arabia
between 2008 and 2013. This study included couples withrepeated pregnancy losses who had presented themselves
during this period for further investigations. Pregnancy
was confirmed by a positive human chorionic gonadotropin (HCG) test using serum or urine in combination
with ultrasounds.
Patients

Following King Abdulaziz University board approval andinformed consent of the patients, a cohort of 171 RM

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

patients (73 couples in addition to 25 women only because their husbands were not available for cytogenetic
analysis) were selected for peripheral blood collection
and a detailed counseling questionnaire covering theirpersonal details, family history and any laboratory results
or past investigations that had been conducted. Only
patients who experienced two or more miscarriages up
to 20 weeks of gestational age were included in thisstudy. Patients’ anonymity and data confidentiality were
preserved.
Cytogenetic analysis

The peripheral blood of each patient (5–10 ml) was subjected to standard 72-h lymphocyte culture to produce
Metaphases for cytogenetic analysis (karyotyping) using
standard harvesting protocols. GTG banding (G banding)was performed by a pretreatment of chromosomes withtrypsin followed by Giemsa staining. Chromosomes’ analysis was done using Cytovision software, a semi-automatic
Applied Imaging Karyotyper, and karyotypes were designed
according to International System for Human Cytogenetic
Nomenclature [36]. Karyotype analysis was performed
using at least 20 cells for each patient. In case of suspectedmosaicism, this number was expanded to 100 metaphases.

Page 17 of 23

Statistical analysis

Association between the patients’ clinical features and thecytogenetic analysis in the cohort of patients wereassessed using χ2 analysis and Fisher’s exact test. The statistical analysis was carried out using MATLAB R2012a(Version 7.14; The MathWorks, Natick, MA, USA).

Results
Patients’ cohort and RM

This study reported the chromosomal analysis of the
171 RM patients (73 couples + 25 women). There were
492 documented pregnancies in this cohort (mean pregnancy per couple = 5.02, SD = 2.79). The age of the subjects
ranged from 18 to 48 (mean 32.17, SD = 6.39) (Table 2)
and number of miscarriages were between 2 to 14 (meanmiscarriage per couple = 4.18; SD = 2.578). Out of 98
women, 32 were able to achieve successful pregnancies
which led to viable baby, whereas the remaining 67.35 %were unable to achieve any successful full term pregnancy
despite several attempts. Overall, most of clinical pregnancies (79.59 %) were terminated in their first trimester while
only 12.24 % and 8.16 % ended up respectively at the second and third trimesters.
Chromosomal analysis

Genetic risk factors causing thrombophilia

Since associations between thromphobilia and RM werepreviously reported [37, 38], we proposed in this study
to further explore the thrombophilic genetic polymorphisms in our patients’ cohort. The main genes investigatedusing PCR- RFLP (restriction fragment length polymorphism) were Factor V Leiden (FVL), Prothrombin A20210G
and MTHFR C677T. Briefly and following DNA extraction(QIAamp DNA Blood Maxi kit, Qiagen), a Taq-polymerase
based PCR using specific primers for each gene was performed (Table 1). The PCR product for each gene is thenfragmented using a specific restriction enzyme in order totarget potential Single Nucleotide Polymorphisms (SNPs)
and separated according to their base pairs’ size by gel electrophoresis as summarized in Table 1.

Following cytogenetic analysis, 13 patients were foundto be carrier of chromosomal abnormalities and/or polymorphisms (7.6 %), 10 of them were women (77 %) and3 (23 %) were men. The prevalence of mosaicism, balanced translocations, duplications, Robertson translocation, triple X syndrome, and allelic polymorphism were2.34 %, 1.17 %, 1.17 %, 0.58 %, 0.58 % and 1.17 % respectively. Very surprisingly in this study, the majority of young
women (≤35 years) (78.6 %) were carriers of chromosomal
aberrations and have a high average of pregnancy failures(Table 3).
Around 30 % of couples in this patients’ cohort had afamily history of consanguineous marriages. Interestingly,
a significant positive correlation (P = 0.046) between consanguineous marriages and chromosomal abnormalities

Table 1 Specific primers sequences, restriction enzymes and restriction digestion products’ sizes for FVL, Prothrombin A20210G and
MTHFR C677T. The PCR-RFLP products sizes are given according to the genotype polymorphisms
Gene

Length(bp)

Primer’s sequence

Restriction
enzymeRestriction digestion product size (bp)NormalHeterozygous

Homozygous

FVL

143

Forward: CATGAGAGACATCGCCTCTG
Reverse: GACCTAACATGTTCTAGCCAGAAG

MnII

25
37
81

25
37
81
118

25
118

Prothrombin

345

Forward: TCTAGAAACAGTTGCCTGGC
Reverse: ATAGCACTGGGAGCATTGAAGC

HindIII

345

23
322
345

23
322

MTHFR

198

Forward: TGAAGGAGAAGGTGTCTGCGGGA
Reverse: AGGACGGTGCGGTGAGAGTG

HinfI

198

23
175
198

23
175

References[77–80]

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

Page 18 of 23

Table 2 Summary of RM patients’ cohort age range, gender and chromosomal abnormalities incidence
Total RM patients

Women

Men

Chromosomal abnormalities

No. of cases

171

98

73

13 (7.6 %)Age range (years)

18 – 48

18 -47

24-48

23-34

Mean

32.17

29.98

35.45

30.50

Std Deviation

6.39

5.98

5.57

4.17

Missing data (%)

16 (9.36 %)

5 (2.92 %)

11 (6.43 %)1 (0.58 %)was recorded. However, correlation between the numberof miscarriages and consanguinity was not statistically significant (Table 4). A relatively higher number of spontaneous miscarriages in couples with abnormal karyotype(mean 4.78, SD = 3.11) was observed when compared tothose with normal karyotype (mean 4.12, SD = 2.57). Outof 52 pregnancies attempts performed by just 9 couples
with chromosomal abnormalities, only 9 (17 %) ended up
with live birth.

miscarriages and pregnancy demise [23, 39, 40]. Amongst
these genetic factors, thrombophilia was shown to be amain cause leading to recurrent miscarriages [3, 29].
Moreover, Saudi population is marked by a high rate ofconsanguineous marriages (about 58 %), the majority ofthem were between first-degree cousins [33, 34]. In thisparticular context, the current study was designed toassess two major parameters know to be causative of
RM: (i) the chromosomal abnormalities; and (ii) inherited thrombophilia.

Thrombophilia allelic polymorphisms and RM

Results of SNPs analysis of 3 thrombophilic genes
showed that the frequencies of FV Leiden, Prothrombin
and MTHFR mutations among RM patients comparedto general incidence reported in Saudi population. Allelic polymorphisms of mainly for FVL and Prothrombin
genes were relatively high supporting thus our hypothesis of considering these as RM genetic factors (Table 5).

Chromosomal abnormalities analysis

Following cytogenetic analysis of our patients' cohort,
77 % of carriers of numerical or chromosomal abnormalities were women. It means that a high female to male ratioof 4: 1 in couples carriers of chromosomal abnormalities
was recorded, which is higher than previously reported ratios (around 2:1) in Saudi Arabia and elsewhere[18, 41, 42]. This higher ratio in women facing recurrent
miscarriages could be explained by the heavy involvementof their oocytes in the fertilization process and early embryo development by providing all the molecular machinery of the new embryo start-up and early development[43–45]. Therefore, these molecular or cytogenetic imbalances look to affect the onset and/or the stability of theDiscussions
Recurrent miscarriages are clinically detectable pregnancies that fail to progress due to several causes including
chromosomal, genetic, anatomic, immune or infective
factors [1]. Chromosomal and genetic abnormalities areamong the most common factors leading to recurrent

Table 3 Cytogenetic results, number of miscarriages and age of RM patients with numerical and/or structural chromosomal
abnormalities
No.

Sex

Age

Graviditya

Parityb

Abortusc

Karyotype

1

F

30

8

3

5

45,XX,rob(14:21)(q11.1;q11.1)

2

F

23

2

0

2

46,XX,dup(1)(q11q21)

3

F

34

3

0

3

46,XX,dup(1)(q11q21)

4

F

33

9

2

7

46, XX,t(3;7) (p23;p22)

5

F

28

6

2

4

46,XX[97]/47,XX,+mar[3]////46,XY[76]/47,XY,+mar[4]

6

M

33

//

7

F

34

4

2

2

46,XX[96]/45,XO[4]////46,XY, add(Y)(p11.3)

8

M

32

//

9

F

34

4

0

4

46,XX[96]/45,X[2]/37-42,XX,-X,t(7;14) (q34;p10) + mer(cp2)

10

F

24

4

0

4

47,XXX

11

M

-

12

0

12

46,XY,t(3;4;13;6)(q25;q32;q31;q22)

12

F

35

12

0

11

46,XX,13 ps + (polymorphism)

13

F

34

8

2

6

46,XX,16qh + (polymorphism)aGravidity = No. of pregnancies; bParity = No. of full term pregnancies; cAbortus = No. of terminated pregnancies

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

Page 19 of 23

Table 4 Clinical features of RM patients with chromosomal
abnormalities
Normal karyotypes

Chromosomal
abnormalities

P value

Male

70

3

0.356

Female908Patient gender

although the number of patients carrying this syndrome
is not as expected in our cohort, since patients with TS
are known to face RM and are not routinely referred forthat. Concomitant with our results, other chromosomal
disorders, including duplications and deletions havebeen also reported in RM couples [17, 59].
Age, consanguinity and RM

Miscarriage stage
Trimester 1

68

10

Trimester 2 or 3

19

01

≤3

49

4

>3

40

7

Consanguineous

51

7

Non-consanguineous

109

4

Saudi

117

6

Non Saudi

43

5

0.452

Miscarriage frequency
0.339

Type of marriage
0.046*

Citizenship0.295*Significant Fisher exact test (α = 0.05)

pregnancy with higher incidence in women compared tomen. These findings are in line with previous reports describing associations between the maternal chromosomal
status and RM [46, 47].
Among the carriers’ cohort, more than 46 % of patients showed various types of translocations. This isconsistent with previous reports where chromosomal aberrations, mainly translocations in the parents or theabortus (embryo), were shown to be strongly associatedwith higher incidence of miscarriages [15, 46, 48–52].
The presence of chromosomal polymorphisms wereassociated with abnormalities of the heterochromatinconstitutively located in different loci of the chromosomes and might be associated with some diseases as infertility and RM [53–55].
Turner Syndrome (TS) is another chromosomal disorders reported in our patients’ cohort (Fig. 1) which usually is marked by a total or partial loss of one of the two
X chromosomes. In fact, recurrent miscarriages, fetal
perinatal death or malformed newborns are known to be
frequent in TS patients [56–58]. Our data also confirm
this strong association between women with TS and RM

The age range of the patients’ group involved in thisstudy was wide (from 18 to 48) (Table 2). One striking
finding is that the ages of all of the 13 patients (100 %)identified as carriers of chromosomal abnormalities were
below 35 years (Table 3). Despite their relative young
age of marriage, these couples were struggling to conceive
since they (both or one of them) are carrier of chromosomal abnormalities leading to higher risk of repeatedmiscarriages and lower chances to deliver a viable andhealthy offspring [18, 19]. These patients are also facing
an important social pressure to procreate [60] especiallyin conservative societies as Saudi Arabia. Such particular
context may explain the high average (≈5) of pregnancy
attempts per couple even at relatively young age. Such social pressure to procreate is very interesting to highlightcompared to other societies where the childfree is one of
the new reproductive lifestyle trends [60, 61]. However,
the high incidence of population inbreeding and consanguineous marriages could explain the high rate of RM at
this early age [31, 62]. In fact, around one third of the couples involved in this study were consanguineous (Table 4).
Although it is somehow decreasing, this consanguinity
driven by an old Arabic tradition is known to rise thefrequency of recessive genetic diseases as well as reproductive and developmental disorders such as infertility
rates, recurrent miscarriages, and congenital disorders(e.g. thrombophilia) [32–35]. A significant positive correlation between consanguinity and reported chromosomal aberrations and polymorphic variants (P = 0.046)
documented in this study supports that the RM ismainly due to genomic instability manifested in several
chromosomal abnormalities in this group (age ≤ 35) rather than reproductive aging or other known factors.
On the other hand, no significant correlation between
the number of RM and consanguinity was reported. This
result might be explained by the fact that the consanguinity negative effect is observed mainly in the presence ofTable 5 Prevalence of FV Leiden, Prothrombin and MTHFR mutations among RM patients
Gene
FVL

Prevalence in RM patients

Prevalence reported in others
studies in Saudi Arabia (%)c

Heterozygous (%)

Homozygous (%)

Total (%)

14.92

0.58

15.5a

1.3

a

Prothrombin A20210G

6

-

6

0.7

MTHFR C677T

23.75

1.75

25.5b

2.5

a

Significant (P < 0.05); bnot significant (P > 0.05); c[68]The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

Fig. 1 Incidence of numerical and structural chromosomal
rearrangements in patients with RM (%). (D): duplications; (A):
Additions; (RT): Robertsonian Translocations; (BT): Balanced
Translocations; (CT): Complex Translocations; (TS): Turner Syndrome;(CP) Chromosomal Polymorphisms; (SMC): Supernumerary
Marker Chromosomes

carried chromosomal and/or genetic abnormalities in one
or both partners, and only very large cohorts of patientsmight detect such impact.
However, the aging process seems to be the main causeof RM in couples beyond 35 years since no chromosomal
disorders have been detected. In fact, the miscarriage
frequency and subsequent reproductive failure werepositively correlated to the increase of paternal and/or
maternal age [63–65]. Such aging process (beyond 35
and 40 years respectively for women and men) is known
to cause genetic and chromosomal disorders during
gametogenesis, fertilization and early embryonic development [15, 46, 65]. Such fetal genetic alterations induce a genomic instability and therefore RM.
Out of 52 pregnancies among couples carrying chromosomal abnormalities, only 17 ended up with live birth.
This rate is lower than that reported in other studies
which showed up to 45 % live birth among couples withstructural chromosomal abnormalities. This could be explained by predisposition of the Saudi society to other RM
risk factors as thrombophilia [35, 66].
Thrombophilia genetic polymorphisms and RM

Coagulation anomalies are reported to induce important pregnancy complications. In this context, prothrombophilic factors were reported to be involved in
RM including FVL, Prothrombin A20210G and MTHFR
C677T. These factors are known to disrupt key events associated to placentation, fetal development and pregnancy
progression till the delivery [3, 28–30, 67]. In this study,
PCR- RFLP was used for molecular analysis of potential

Page 20 of 23

SNPs in three (3) thrombophilia-associated genes: FVL,
Prothrombin A20210G and MTHFR C677T as detailed inTable 1. The screening for potential SNPs in these 3 genes
showed that the frequencies of FVL, Prothrombin and
MTHFR mutations (including both homozygous and carriers) were respectively 15.5 %, 6 % and 25.5 (Table 5).
These results confirm the atypical and relatively high incidence of thrombophilic gene polymorphisms among Saudi
population reported in previous studies [68]. Beside national awareness campaigns, these findings support morepreventive measurements to be considered at the premarital and/or before the IVF procedures in ART clinics.
The prevalence of FVL and Prothrombin A20210G
mutations reported in patients’ cohort support a strong
relationship between these traits and RM. Following
analysis of the most important studies about inherited
thrombophilia, our data are in line with previous findings
where both mutations have been known as common
genetic disorders that predispose to early and late RM[3, 29, 69–71]. A recent study in Saudi Arabia also confirmed the involvement of mutations in these two particular genes (FVL and Prothrombin A20210G) in
increasing RM incidence [72]. Additionally, these twothrombophilic genetic traits were associated with obstetric complications including miscarriages, placental
abruption, intrauterine growth retardation (IUGR) or
death [69, 73–75]. These findings explain the significant
correlations with RM reported in this study and support
the assumption that both factor V Leiden and Prothrombin mutations are major risk factors for RM. Suitable
treatment of inherited and acquired thrombophilia will
improve the pregnancy outcomes as discussed elsewhere[69]. Therefore, a national medical program for routine
screening of these two genes in patients with repeatedpregnancy failure in Saudi Arabia is highly recommended.
For MTHFR C677T mutation in RM patients and inagreement with other studies, no significant (P > 0.05) association with RM were found [29]. The general practiceof folic acid supplementation during pregnancy seems tobe the reason of masking the effect of the MTHFR mutation in RM patients as reported elsewhere [76].
Since RM is a challenging obstetric complication withvarious psychological, societal and economic burdens on
both couples and the health care system in general, ourstudy contributed to report an effect of both structuraland numerical effects of chromosomal abnormalities on
RM, which were amplified by consanguinity mainly forcouples under 35 years. Moreover, thrombophilic polymorphisms of FVL and Prothrombin A20210G were significantly associated to higher prevalence of RM.

Conclusions
This study demonstrated a strong association between
RPL and the prevalence of chromosomal abnormalities

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

and inherited thrombophilia and confirms the high incidence of RM in the Western region of Saudi Arabia suggesting thus some main but non-exclusive causes of thisdisease. Our findings lay also foundation for larger
cohort-based studies to further validate and confirm theimpact of thalassemia gene polymorphisms and hemoglobinopathies in general, chromosomal aberrations andconsanguinity, but also to predict the involvement ofother anatomic, endocrine or auto-immune factors.
Our study highlights the importance of including cytogenetic and thrombophilia testing as part of the routine
clinical investigation of RM and during Preimplantation
Genetic Screening (PGS) in IVF clinics in order to suggest suitable management and/or treatment approaches.
Such genetic testing along with standard karyotyping arehighly recommended to be included in premarital test
especially for consanguineous partners. In the genomic
era, further studies focusing on the molecular mechanismof thrombophlic polymorphisms on placental pathologies
and pregnancy loss using high-throughput technologies
as array Comparative Genomic Hybridization (aCGH)
and Next Generation Sequencing (NGS) are highly recommended. We believe that a multidisciplinary and collaborative approach between obstetricians, geneticists,
hematologists, scientists and bioethicists combined witheffective awareness program will contribute to relieve theburden of RM.
Abbreviations
FVL: Factor V Leiden; HCG: Human chorionic gonadotropin; IVF: In vitro
fertilization; PGD: Preimplantation genetic diagnosis; RFLP: Restriction
fragment length polymorphism; RM: Recurrent miscarriages; RPL: Recurrent
pregnancy loss; SNPs: Single nucleotide polymorphisms; TS: Turner syndrome
Acknowledgments
Authors would like to thank the King Abdulaziz City for Science and Technology(KACST) for financial support of this study (Grant no. KACST APR-34-210).
Declaration
This article has been published as part of BMC Medical Genetics Volume 17
Supplement 1, 2016: Proceedings of the 3rd International Genomic Medicine
Conference: medical genetics. The full contents of the supplement areavailable online at http://bmcmedgenet.biomedcentral.com/articles/
supplements/volume-17-supplement-1.
Availability of data and material
Not applicable.
Authors’ contributions
RFT: Samples collection and data acquisition, drafting the manuscript;
MA: Study design, obtaining funds, contributed in manuscript drafting;
HAB, SJ and HJS: technical support, data acquisition and statistical analysis;
MAE: Contributed in study design and manuscript drafting; HAZ, AAR, OB and
HSJ: Contributed in samples collection, data analysis and patients’ follow-up;
MHA and AMA: Study design and supervision, technical and administrative
support; All authors critically reviewed and agreed on the final version of
the manuscript.
Competing interests
Authors declare no financial or other conflicts of interests that might bias
the results of this study.

Page 21 of 23

Consent for publication
Not applicable.
Ethics approval and consent to participate
This study was approved by the Research Committee of the Biomedical Ethics
Unit, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia.
Publication fees declaration
Publication fees for this article are paid by the Center of Excellence inGenomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah,
Kingdom of Saudi Arabia.
Author details
1
Obstetrics and Gynecology Department, King Abdulaziz University Hospital,
Jeddah, Saudi Arabia. 2Center of Innovation in Personalized Medicine, King
AbdulAziz University, P.O. Box: 80216, Jeddah 21589, Kingdom of Saudi
Arabia. 3Center of Excellence in Genomic Medicine Research, King Abdulaziz
University, Jeddah, Saudi Arabia.
Published: 10 October 2016
References
1. ASRM. Evaluation and treatment of recurrent pregnancy loss:
a committee opinion. Fertil Steril. 2012;98(5):1103–11.
2. Horne AW, Alexander CI. Recurrent miscarriage. The Journal of Family
Planning and Reproductive Health Care/Faculty of Family Planning &Reproductive Health Care, Royal College of Obstetricians & Gynaecologists.
2005;31(2):103–7.
3. Bennett SA, Bagot CN, Arya R. Pregnancy loss and thrombophilia: theelusive link. Br J Haematol. 2012;157(5):529–42.
4. Jaslow CR, Carney JL, Kutteh WH. Diagnostic factors identified in 1020
women with two versus three or more recurrent pregnancy losses. Fertil
Steril. 2010;93(4):1234–43.
5. McNamee K, Dawood F, Farquharson RG. Evaluation of array comparative
genomic hybridization in recurrent miscarriage. Br J Hosp Med (Lond).
2013;74(1):36–40.
6. Larsen EC, Christiansen OB, Kolte AM, Macklon N. New insights intomechanisms behind miscarriage. BMC Med. 2013;11:154.
7. Mathur N, Triplett L, Stephenson MD. Miscarriage chromosome testing:utility of comparative genomic hybridization with reflex microsatellite
analysis in preserved miscarriage tissue. Fertil Steril. 2014;101(5):1349–52.
8. Rubio C, Pehlivan T, Rodrigo L, Simon C, Remohi J, Pellicer A. Embryo
aneuploidy screening for unexplained recurrent miscarriage: a minireview.
Am J Reprod Immunol. 2005;53(4):159–65.
9. Franssen MTM, Korevaar JC, van der Veen F, Leschot NJ, Bossuyt PMM, Goddijn
M. Reproductive outcome after chromosome analysis in couples with two or
more miscarriages: an index-control study. BMJ. 2006;332(7548):1012.
10. Christiansen OB, Steffensen R, Nielsen HS, Varming K. Multifactorial etiologyof recurrent miscarriage and its scientific and clinical implications. Gynecol
Obstet Invest. 2008;66(4):257–67.
11. Rubio C. Update on preimplantation genetic diagnosis for chromosomal
abnormalities. Expert Rev Mol Diagn. 2010;10(8):973–6.
12. Brown S. Miscarriage and its associations. Semin Reprod Med. 2008;26(5):391–400.
13. Kiss A, Rosa RFM, Dibi RP, Zen PRG, Pfeil JN, Graziadio C, Paskulin GA.
Chromosomal abnormalities in couples with history of recurrent abortion.
Rev Bras Ginecol Obstet. 2009;31(2):68–74.
14. Kochhar PK, Ghosh P. Reproductive outcome of couples with recurrent
miscarriage and balanced chromosomal abnormalities. J Obstet Gynaecol
Res. 2013;39(1):113–20.
15. Hodes-Wertz B, Grifo J, Ghadir S, Kaplan B, Laskin CA, Glassner M, Munne S.
Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos.
Fertil Steril. 2012;98(3):675–80.
16. Chang LJ, Chen SU, Tsai YY, Hung CC, Fang MY, Su YN, Yang YS. An
update of preimplantation genetic diagnosis in gene diseases, chromosomaltranslocation, and aneuploidy screening. Clin Exp Reprod Med. 2011;38(3):126–34.
17. Elghezal H, Hidar S, Mougou S, Khairi H, Saad A. Prevalence of chromosomal
abnormalities in couples with recurrent miscarriage. Fertil Steril.
2007;88(3):721–3.
18. Franssen MTM, Korevaar JC, Tjoa WM, Leschot NJ, Bossuyt PMM, Knegt AC,
Suykerbuyk RF, Hochstenbach R, van der Veen F, Goddijn M. Inheritedunbalanced structural chromosome abnormalities at prenatal chromosome

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

19.

20.

21.

22.
23.

24.

25.

26.

27.

28.

29.

30.
31.

32.
33.

34.

35.

36.
37.

38.

39.

40.
41.

analysis are rarely ascertained through recurrent miscarriage. Prenat Diagn.
2008;28(5):408–11.
Hume Jr RF, Kilmer-Ernst P, Wolfe HM, Ebrahim SA, Treadwell MC, Johnson MP,
Evans MI. Prenatal cytogenetic abnormalities: correlations of structural
rearrangements and ultrasonographically detected fetal anomalies. Am J
Obstet Gynecol. 1995;173(4):1334–6.
Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, Debrock S,
Amyere M, Vikkula M, Schuit F, et al. Chromosome instability is common inhuman cleavage-stage embryos. Nat Med. 2009;15(5):577–83.
Rubio C, Simon C, Vidal F, Rodrigo L, Pehlivan T, Remohi J, Pellicer A.
Chromosomal abnormalities and embryo development in recurrent
miscarriage couples. Hum Reprod. 2003;18(1):182–8.
Branch DW, Gibson M, Silver RM. Clinical practice. Recurrent miscarriage.
N Engl J Med. 2010;363(18):1740–7.
Al-Hassan S, Hellani A, Al-Shahrani A, Al-Deery M, Jaroudi K, Coskun S.
Sperm chromosomal abnormalities in patients with unexplained recurrent
abortions. Arch Androl. 2005;51(1):69–76.
Zastavna D, Sosnina K, Terpylyak O, Huleyuk N, Bezkorovayna H, Mikula M,
Helner N. Cytogenetic and immunogenetic analysis of recurrent pregnancy
loss in women. Tsitol Genet. 2014;48(4):44–50.
Weier H, Munne S, Lersch RA, Hsieh H, Smida J, Chen X, Korenberg JR,Pedersen RA, Fung J. Towards a full karyotype screening of interphase cells:'FISH and chip' technology. Mol Cell Endocrinol. 2001;183 Suppl 1:S41–5.
Magli MC, Gianaroli L, Crippa A, Munne S, Robles F, Ferraretti AP.
Aneuploidies of chromosomes 1, 4, and 6 are not compatible with humanembryos' implantation. Fertil Steril. 2010;94(6):2012–6.
van den Boogaard E, Hermens RP, Verhoeve HR, Kremer JA, van der Veen F,
Knegt AC, Goddijn M. Selective karyotyping in recurrent miscarriage: arerecommended guidelines adopted in daily clinical practice? Hum Reprod.
2011;26(8):1965–70.
Parand A, Zolghadri J, Nezam M, Afrasiabi A, Haghpanah S, Karimi M.
Inherited thrombophilia and recurrent pregnancy loss. Iranian Red Crescent
medical journal. 2013;15(12):e13708.
Isaoglu U, Ulug P, Delibas IB, Yilmaz M, Kumtepe Y, Dogan H, Tasdemir S.
The association between inherited thrombophilia and recurrent pregnancy
loss in Turkish women. Clin Exp Obstet Gynecol. 2014;41(2):177–81.
Brenner B. Thrombophilia and adverse pregnancy outcome. Obstet Gynecol
Clin North Am. 2006;33(3):443–56. ix.
Abu-Elmagd M, Assidi M, Schulten H-J, Dallol A, Pushparaj P, Ahmed F,
Scherer S, Al-Qahtani M. Individualized medicine enabled by genomics in
Saudi Arabia. BMC Med Genomics. 2015;8 Suppl 1:S3.
Tadmouri GO, Nair P, Obeid T, Al Ali MT, Al Khaja N, Hamamy HA. Consanguinity
and reproductive health among Arabs. Reprod Health. 2009;6:17.
El Mouzan MI, Al Salloum AA, Al Herbish AS, Qurachi MM, Al Omar AA.
Consanguinity and major genetic disorders in Saudi children: a communitybased cross-sectional study. Ann Saudi Med. 2008;28(3):169–73.
El-Hazmi MA, Al-Swailem AR, Warsy AS, Al-Swailem AM, Sulaimani R,
Al-Meshari AA. Consanguinity among the Saudi Arabian population.
J Med Genet. 1995;32(8):623–6.
Settin AA, Alghasham A, Ali A, Dowaidar M, Ismail H. Frequency ofthrombophilic genetic polymorphisms among Saudi subjects comparedwith other populations. Hematology. 2012;17(3):176–82.
Simons A, Shaffer LG, Hastings RJ. Cytogenetic Nomenclature: Changes in the
ISCN 2013 Compared to the 2009 Edition. Cytogenet Genome Res. 2013;141:1–6.
Mazzucconi MG, De Sanctis V, Alfo M, Amendolea MA, Conti L, Santoro C,
Baldacci E, Peraino M, Masala C. Maternal Thrombophilia and Adverse
Pregnancy Outcome: A Case–control Study. Acta Haematol. 2015;133(2):242–8.
Lissalde-Lavigne G, Fabbro-Peray P, Cochery-Nouvellon E, Mercier E,
Ripart-Neveu S, Balducchi JP, DaurÈS JP, Perneger T, QuÉRÉ I, Dauzat M,
et al. IN FOCUS: Factor V Leiden and prothrombin G20210A
polymorphisms as risk factors for miscarriage during a first intended
pregnancy: the matched case–control ‘NOHA first’ study. J Thromb
Haemost. 2005;3(10):2178–84.
Munne S, Magli C, Bahce M, Fung J, Legator M, Morrison L, Cohert J,
Gianaroli L. Preimplantation diagnosis of the aneuploidies most commonly
found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22.
Prenat Diagn. 1998;18(13):1459–66.
Hassold TJ. A cytogenetic study of repeated spontaneous abortions. Am J
Hum Genet. 1980;32(5):723–30.
Al-Hussain M, Al-Nuaim L, Abu Talib Z, Zaki OK. Cytogenetic study in caseswith recurrent abortion in Saudi Arabia. Ann Saudi Med. 2000;20(3–4):233–6.

Page 22 of 23

42. Goddijn M, Joosten JH, Knegt AC, van der Veen F, Franssen MT, Bonsel GJ,
Leschot NJ. Clinical relevance of diagnosing structural chromosome
abnormalities in couples with repeated miscarriage. Hum Reprod.
2004;19(4):1013–7.
43. Krisher RL. The effect of oocyte quality on development. J Anim Sci.
2004;82(E-Supp):E14–23.
44. Assidi M, Montag M, Van Der Ven K, Sirard MA. Biomarkers of human oocyte
developmental competence expressed in cumulus cells before ICSI: a preliminary
study. J Assist Reprod Genet. 2011
45. Sirard M-A, Assidi M. Screening for Oocyte Competence. In: Krisher RL,
editor. Oocyte Physiology and Development in Domestic Animals.
Wiley-Blackwell; 2013. p. 191–206.
46. Choi TY, Lee HM, Park WK, Jeong SY, Moon HS. Spontaneous abortion andrecurrent miscarriage: A comparison of cytogenetic diagnosis in 250 cases.
Obstet Gynecol Sci. 2014;57(6):518–25.
47. Munne S, Chen S, Fischer J, Colls P, Zheng X, Stevens J, Escudero T, Oter M,
Schoolcraft B, Simpson JL, et al. Preimplantation genetic diagnosis reduces
pregnancy loss in women aged 35 years and older with a history ofrecurrent miscarriages. Fertil Steril. 2005;84(2):331–5.
48. Munne S, Morrison L, Fung J, Marquez C, Weier U, Bahce M, Sable D,
Grundfeld L, Schoolcraft B, Scott R, et al. Spontaneous abortions are
reduced after preconception diagnosis of translocations. J Assist Reprod
Genet. 1998;15(5):290–6.
49. Otani T, Roche M, Mizuike M, Colls P, Escudero T, Munne S. Preimplantation
genetic diagnosis significantly improves the pregnancy outcome oftranslocation carriers with a history of recurrent miscarriage andunsuccessful pregnancies. Reprod Biomed Online. 2006;13(6):869–74.
50. De Krom G, Arens YH, Coonen E, Van Ravenswaaij-Arts CM, MeijerHoogeveen M, Evers JL, Van Golde RJ, De Die-Smulders CE. Recurrent
miscarriage in translocation carriers: no differences in clinical characteristics
between couples who accept and couples who decline PGD. Hum Reprod.
2015;30(2):484–9.
51. Dutta UR, Rajitha P, Pidugu VK, Dalal AB. Cytogenetic abnormalities in 1162
couples with recurrent miscarriages in southern region of India: report andreview. J Assist Reprod Genet. 2011;28(2):145–9.
52. Ocak Z, Ozlu T, Ozyurt O. Association of recurrent pregnancy loss withchromosomal abnormalities and hereditary thrombophilias. Afr Health Sci.
2013;13(2):447–52.
53. Willatt L, Morgan S, Shaffer LG, Slovak ML, Campbell LJ. ISCN 2009 an
international system for human cytogenetic nomenclature. Hum Genet.
2009;126(4):603–4.
54. Madon PF, Athalye AS, Parikh FR. Polymorphic variants on
chromosomes probably play a significant role in infertility. Reprod Biomed
Online. 2005;11(6):726–32.
55. Baghbani F, Mirzaee S, Hassanzadeh-Nazarabadi M. Association ofheteromorphism of chromosome 9 and recurrent abortion (ultrasounddiagnosed blighted ovum): A case report. Iran J Reprod Med. 2014;12(5):357–60.
56. Tarani L, Lampariello S, Raguso G, Colloridi F, Pucarelli I, Pasquino AM, Bruni LA.
Pregnancy in patients with Turner's syndrome: Six new cases and review ofliterature. Gynecol Endocrinol. 1998;12(2):83–7.
57. Hewitt JK, Jayasinghe Y, Amor DJ, Gillam LH, Warne GL, Grover S, Zacharin MR.
Fertility in Turner syndrome. Clin Endocrinol. 2013;79(5):606–14.
58. Aboussair N, Jaouad IC, Dequaqui SC, Sbiti A, Elkerch F, Yahya B, Natiq A,
Sefiani A. Cytogenetic analysis of 5572 patients referred for suspected
chromosomal abnormalities in Morocco. Genet Test Mol Biomarkers.
2012;16(6):569–73.
59. De la Fuente-Cortés BE, Cerda-Flores RM, Dávila-Rodríguez MI, García-Vielma C,
De la Rosa Alvarado RM, Cortés-Gutiérrez EI. Chromosomal abnormalities andpolymorphic variants in couples with repeated miscarriage in Mexico. Reprod
Biomed Online. 2009;18(4):543–8.
60. Martins MV, Peterson BD, Almeida VM, Costa ME. Direct and indirect effectsof perceived social support on women's infertility-related stress. Hum Reprod.
2011;26(8):2113–21.
61. Doyle J, Pooley JA, Breen L. A phenomenological exploration of the childfree
choice in a sample of Australian women. J Health Psychol. 2013;18(3):397–407.
62. Warsy AS, Al-Jaser MH, Albdass A, Al-Daihan S, Alanazi M. Is consanguinity
prevalence decreasing in Saudis?: A study in two generations. Afr Health Sci.
2014;14(2):314–21.
63. de la Rochebrochard E, Thonneau P. Paternal age and maternal age are risk
factors for miscarriage; results of a multicentre European study. Hum Reprod.
2002;17(6):1649–56.

The Author(s) BMC Medical Genetics 2016, 17(Suppl 1):69

Page 23 of 23

64. Belloc S, Cohen-Bacrie P, Benkhalifa M, Cohen-Bacrie M, De Mouzon J,
Hazout A, Menezo Y. Effect of maternal and paternal age on pregnancy andmiscarriage rates after intrauterine insemination. Reprod Biomed Online.
2008;17(3):392–7.
65. Kleinhaus K, Perrin M, Friedlander Y, Paltiel O, Malaspina D, Harlap S. Paternal
age and spontaneous abortion. Obstet Gynecol. 2006;108(2):369–77.
66. Carp H, Feldman B, Oelsner G, Schiff E. Parental karyotype and subsequent
live births in recurrent miscarriage. Fertil Steril. 2004;81(5):1296–301.
67. Carp HJ. Thrombophilia and recurrent pregnancy loss. Obstet Gynecol Clin
North Am. 2006;33(3):429–42.
68. Saour JN, Shoukri MM, Mammo LA. The Saudi Thrombosis and Familial
Thrombophilia Registry. Design, rational, and preliminary results. Saudi Med
J. 2009;30(10):1286–90.
69. De Santis M, Cavaliere AF, Straface G, Di Gianantonio E, Caruso A. Inherited
and acquired thrombophilia: pregnancy outcome and treatment. Reprod
Toxicol. 2006;22(2):227–33.
70. Ali N, Bhatti FA, Khan SA. Frequency of hereditary thrombophilia in womenwith recurrent pregnancy loss in Northern Pakistan. J Obstet Gynaecol Res.
2014;40(6):1561–6.
71. Motha MB, Palihawadana TS, Perry DJ. Recurrent pregnancy loss andthrombophilia. Ceylon Med J. 2014;59(1):1–3.
72. Gawish G. The Prevalence of Inherited Thrombophilic Polymorphisms in
Saudi Females with Recurrent Pregnancy Loss Confirmed using Different
Screening Protocols of PCR. J Mol Genet Med. 2015;9(156):1747. 0862.1000156.
73. Robertson L, Wu O, Langhorne P, Twaddle S, Clark P, Lowe GD, Walker ID,
Greaves M, Brenkel I, Regan L, et al. Thrombophilia in pregnancy: a systematic
review. Br J Haematol. 2006;132(2):171–96.
74. Vormittag R, Pabinger I. Thrombophilia and pregnancy complications.
Hamostaseologie. 2006;26(1):59–62.
75. Kovalevsky G, Gracia CR, Berlin JA, Sammel MD, Barnhart KT. Evaluation of
the association between hereditary thrombophilias and recurrent pregnancy
loss: a meta-analysis. Arch Intern Med. 2004;164(5):558–63.
76. Li X, Jiang J, Xu M, Yang Y, Lu W, Yu X, Ma J, Pan J. Individualized
Supplementation of Folic Acid According to Polymorphisms of
Methylenetetrahydrofolate Reductase (MTHFR), Methionine Synthase
Reductase (MTRR) Reduced Pregnant Complications. Gynecol Obstet Invest.
2015;79(2):107–12.
77. Gray E, Hawkins JR, Morrison M, Hawkins M, Byrne E, Kitchen S, Jennings I,
Makris M, Preston FE, Metcalfe P. Establishment of the 1st International
Genetic Reference Panel for Factor V Leiden, human gDNA. Thromb
Haemost. 2006;96(2):215–9.
78. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic
variation in the 3'-untranslated region of the prothrombin gene isassociated with elevated plasma prothrombin levels and an increase invenous thrombosis. Blood. 1996;88(10):3698–703.
79. Simioni P, Tormene D, Manfrin D, Gavasso S, Luni S, Stocco D, Girolami A.
Prothrombin antigen levels in symptomatic and asymptomatic carriers ofthe 20210A prothrombin variant. Br J Haematol. 1998;103(4):1045–50.
80. Speletas M, Papadopoulos N, Daiou C, Katodritou E, Pavlitou-Tsiontsi A,
Galanopoulou V. Relationship between 5,10-methylenetetrahydrofolate
reductase C677T gene polymorphism and methotrexate related toxicity inpatients with autoimmune diseases receiving folic acid supplementation.
Ann Rheum Dis. 2005;64(12):1791–2.

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