International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 9, Issue 4, 2017
Original Article
KARYOTYPIC ANALYSIS OF CHROMOSOMAL POLYMORPHISM IN RELATION TO
REPRODUCTIVE FAILURE
KETAN K. VAGHASIA1, NIDHI D. SHAH1, PARTH S. SHAH1, VIDHI M. BHATT1, SANDIP C. SHAH1*,
MANDAVA V. RAO1,2
1Supratech
Micropath Laboratory and Research Institute, 2Ex. Director, School of Sciences, Gujarat University, Ahmedabad, Gujarat India
Email: [email protected]
Received: 20 Oct 2016 Revised and Accepted: 27 Feb 2017
ABSTRACT
Objective: This study was undertaken to elucidate the role of heteromorphism in causation of reproductive anomalies like infertility.
Methods: In our study, cytogenetic analysis of 830 suspected referral cases of both sexes were assessed using standard karyotypic technique with
Giemsa staining from their blood samples. We identified heteromorphism of D/G groups and non-acrocentric chromosomes following WHO
nomenclature.
Results: Our data revealed that most of our heteromorphic cases (38;4.58%) were related to p arm satellites (ps+) of the chromosomes and are
related to infertility and abortion. No significant gender variation was noticed in this study.
Conclusion: We hence, suggest that heteromorphism is associated with a loss of reproductive function, as heterochromatin may contain genes that
regulate cellular roles in reproduction. Further, it becomes important that such cases are considered for molecular studies, genetic counseling and
prenatal/pre-implantation screening.
Keywords: Chromosomal Polymorphism, Reproductive Failure, Cytogenetic Analysis. Acrocentric and non-acrocentric groups, Heterochromatin
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4. 0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i4.15787
INTRODUCTION
Karyotypic analysis and heteromorphism
Repeated DNA sequences in the genome are subject to be
heteromorphic and their regions are also highly polymorphic [1].
Chromosomal heteromorphisms include various sizes of
heterochromatin blocks, satellites, repeat sequence region and
inversion [2]. These heteromorphic variants are most commonly
present on non-acrocentric chromosome in the pericentric
heterochromatin on long arms (q) of 1, 9 and 16, short arms (p) of
acrocentric chromosomes and distal heterochromatin of Y
chromosome [2]. Increase/decrease in length of heterochromatin
regions of 1, 9, 16 chromosomes (1qh+, 9qh+and 16qh+as well as qh-)
and short arms of D and G groups, eg., 13pstk+, 13ps+, 14ps+, 15ps+,
21ps+, Yp+etc. are responsible for reproductive anomalies [3, 4].
Survey showed variants in higher frequency individuals [5].
Reproductive failure is defined as abnormal outcome such as
infertility, recurrent spontaneous abortions (RSAs), still birth and bad
obstetric history (BOH). Numerous studies revealed the relationship
between chromosome heteromorphism and reproductive failure. But
controversies also exist [1, 6]. Moreover, studies are rare in population
of Gujarat. The role of these heteromorphisms in relation to
reproductive failure hence needs to be investigated [4, 7]. Infertility
and hyperactivity disorder are also associated with the chromosomal
rearrangement [8, 9]. Hence, this study was undertaken to identify
heterochromatic regions of acrocentric and paracentric chromosomes
in Indian population and their relation to reproductive anomalies at
our Research Institute in Ahmedabad, India.
The peripheral blood samples of all patients were cultured for 72 h
following the method of Moorhead et al. [10]. For each case, 20
metaphase plates containing chromosome plates deemed
appropriate quality were analyzed on an automated Meta Systems of
Carl Ziess for karyotypic study and heteromorphism was reported
according to ISCN of WHO [11]. Satellite stalks (stk) and satellites (s)
of D and G groups and non-acrocentric chromosomes including Y
chromosome length variations were included in this report.
Statistical analysis was done as appropriate and frequencies and
percentage of each variant was calculated.
MATERIALS AND METHODS
Patients
We studied 830 referral cases ranging in age between 20-40 y collected
from different regions of India, over two years (2014-2016) at Supratech
Micropath Research Institute, Ahmedabad referred with suspected or
established reproductive issues. They were asked to collect blood
samples as per our instructions after filling patients’ consent form. This
work is approved by Gujarat University (GU) human ethical committee
(GU/HEC/001/15) for investigation.
RESULTS
Out of the total number of 830 referral cases analyzed, Only 38 cases
(4.58%) were detected to have heteromorphisms in their karyotypes.
A: D and G group satellites on short arm (P) (ps+)
13ps+Variant: The frequency was more in males (3) than females
(2) and the percent was found to be 14.2 in both male and females
having infertility and recurrent miscarriages (RMCs) (table 1, fig. 3).
14ps+Variant: This frequency was more in females (4)
comparatively. Only one male case was affected. The total percent
was 13.2 exhibiting above mentioned anomalies (table 1, fig. 3).
15ps+Variant: The frequency was found to be more in females (3)
comparatively. The percent was like that of 13 chromosome (14.2%)
and exhibited infertility (table 1, fig. 3).
21ps+Variant: The frequency was higher in males (4) than the
opposite sex (2) and the percent was also more than all above
variants (15.9%) with infertility and recurrent spontaneous
abortions (RSAs) (table 1, fig. 3).
22ps+Variant: It had the highest incidence amongst all
heteromorphisms detected in our study. Males (6) are affected
comparatively with higher frequency with (21.2%) which was
significant causing sterility and RSAs (table 1, fig. 3).
Shah et al.
13ps+, 14ps+, 15ps+, 21ps+, 22ps+variants: All variants were
present in karyotype of one female. Percentage and frequency were
Int J Pharm Pharm Sci, Vol 9, Issue 4, 140-143
same. This case had (BOH) with infertility and recurrent
spontaneous abortions (RSAs) (table 1, fig. 1, 3)
Table 1: Gender wise heteromorphic frequencies of D and G Groups
Karyotypes
46,XY,13ps+
46,XX,13ps+
46,XY,14ps+
46,XX,14ps+
46,XY,15ps+
46,XX,15ps+
46,XY,21ps+
46,XX,21ps+
46,XY,22ps+
46,XX,22ps+
46XX,13ps+,14ps+,
15ps+,21ps+,22ps+
Gender
3
2
1
4
2
3
4
2
6
2
1
Frequency M-16
0.089
0.027
0.053
0.106
0.159
-
Frequency F-14
0.053
0.106
0.089
0.053
0.053
0.027
Total frequency
0.142
Percentage (%)
14.2
0.133
13.3
0.142
14.2
0.159
15.9
0.080
08.0
0.027
02.7
Anomalies
Infertility and
RMCs
Infertility and
RMCs
Infertility and
RMCs
Infertility and
RSAs
Infertility and
RSAs
BOH and Abortion
Bad obstetric history= BOH., RSAs= Recurrent spontaneous abortions., RMCs= Recurrent miscarriages., M=Male., F=Female.
Fig. 1: Female karyotype showing multiple heteromorphisms of D and G groups, (46, XX, 13ps+, 14ps+, 15ps+, 21ps+and 22ps+)
9qh+Variant
Yp+Variant
inv(Y)
Fig. 2: Sample patient karyotype showing heterochromatin variants of 9 and Y chromosomes and inversion (Y)
B: Heteromorphic variation in 9 and Y Chromosomes
We did not detect any heteromorphisms in 1 and 16 chromosomes.
Its calculated percent was (14.2%) possessing reproductive
malformation included infertility and RSAs (table 2, fig. 4).
9qh+Variants: It was found on chromosome 9 and frequency
detected was 0.089 in females (3) and 0.053 in males (2).
Inv (Y) and Yp+: These were present in the male. Frequency of
inversion Y is more (2) than that of Yp+(1). Similarly percent was
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Shah et al.
also higher in respect to inversions (5.3%) and elucidated with
Int J Pharm Pharm Sci, Vol 9, Issue 4, 140-143
infertility including abortions (table 2, fig. 2, 4).
Table 2: Gender wise status of heteromorphic frequencies of 9 and ‘Y’ chromosomes with percent
Karyotype
Male (2) frequency
46,XY,9qh+
0.053
46,XX,9qh+
Y-Chromosome length
Variation (Male)
46,XY,inv(Y)
0.053 (2)
46,XY,Yp+
0.027 (1)
Female (3) frequency
0.089
Total frequency
-
0.05
0.02
0.142
Percent (%)
14.2
Anomalies
Infertility and
Abortion
5.3
2.7
Infertility
Infertility
Figure in parenthesis indicate case numbers of males/females
In our data high frequency of D and G group variants (ps+) were
noticed in both sexes. The percent of 22ps+was higher (21.2%)
followed by 21ps+(15.9%) and others (14.2%; 13ps+, 14ps+and
15ps+;13.3%) respectively. Madon et al. [4] documented these
variants in their study were correlated with reproductive effects
followed by chromosomal rearrangements of inv(9) and inv(Y) in
both cases in support of our data. Moreover, we observed in our
study that heteromorphism of ps+in all D and G groups was
observed leading to multiple clinical effects in the female patient
with bad obstetric history (BOH) too. Sofia et al. [14] and Pokale [16]
detected satellite karyotypes of acrocentric chromosome in both
cases as mentioned earlier that D/G groups with heterochromatin
variants led to infertility, spontaneous recurrent abortions (RSAs)
and recurrent miscarriages (RMCs) with BOH [1, 2, 7, 15, 17].
Fig. 3: Percent (%) distribution of variants in D and G groups
Fig. 4: Percent (%) distribution of chromosomal
heteromorphism of 9 and Y
DISCUSSION
In our study 38 karyotypes with heteromorphic variants were
detected. These included acrocentric chromosomal satellites, (ps+)
and non-acrocentric chromosomes 9 with qh+ and Y–chromosome
length variation p+(Yp+) and inv(Y) in our study.
There are various reports which are controversial on clinical effects
of chromosome variants with respect to reproductive failure [1, 4, 7,
12]. Earlier heterochromatin is considered to be ‘Junk’ inert DNA.
But now it is known to be essential for cell viability and fertility as it
contains genes for their vital activities [4]. It also plays an important
role in spindle attachment and chromosome movement, meiotic
paring and sister chromatid exchange (SCE) cohesion [12, 13]. Sofia
et al. [14] noticed the secondary construction variation regions and
satellite alterations among individuals with reproductive failure.
Mierla and Stolan [15], Christofolini et al. [2] and Pokale [16]
stressed the role of satellite variants of acrocentric and nonacrocentric chromosomes in induction of recurrent spontaneous
abortions (RSAs), recurrent miscarriages (RMCs), still birth and
male infertility strongly supporting our data.
Christofolin et al., [2] and Ogata et al., [18] reported Y length
variation and 22ps+variants in the male affecting its development
and fertility in support of our data, where inv(Y) and Yp+are related
to male reproductive failure (8%) as it is known to be more
polymorphic in Asians [19,20]. Recently other workers [7, 16, 21]
studied the inversion 9 and Y chromosomes causing multiple effects
in female and male partners, supporting our observation where
9qh+contributed 14.2% in causation of clinical effects. Boronova et
al., [22] and Purandare et al. [7] provided an exhaustive review in
regard to heteromorphic variations in acrocentric and nonacrocentric chromosomes to which our data corroborates.
Though the role of heteromorphic variants in etiology of RSA/RMC
and infertility is not well understood earlier, it is now considered
[23] that heterochromatien properties and molecular composition
are similar to that chromosomal regions involved in gene silencing,
position effect variegation and X-chromosome inactivation [23]. The
appearance of a specific kind of chromatin packaging initiated by
epigenetic signals in DNA like gene promoter hyper methylation and
histone acetylation is necessary for activation and silencing of genes.
A high incidence of chromosomal variants in infertile men/women is
suggestive of large heterochromatin blocks responsible for
weakening of chromosome pairing, spindle fiber attachment or
down regulation of active genes for meiotic arrest in gametogenesis
to cause sterility. Further nuclear organizer region (NOR), stalks of
satellites and satellites having heterochomatin are present in
acrocentric and paracentric chromosomes (1, 13, 19). Their
variations in heterochromatin under influence of heat stimuli such
as heat shock and heat shock protein (HSP) assembly and epigenetic
signals play a role in gene regulation and control affecting
reproductive function in both cases [24-26]. Previously, Boronova et
al., [22], Pokale, [16] and Mau et al., [27] have documented that
heterochromatin regions contain high amount of DNA and is
heterogenous. Chromosomal variants are an expression of
morphologic variability chromosome related modifications in
amount of heterochromatin. It is hence believed that an increase in
heterochromatin might develop the risk of nondisjunction of
chromosome segregation leading to chromosomally unbalanced
gamete formation in both sexes. Further variation in
heterochromatin in centromere causes defects in its function and
kinetochore assembly causing abnormal homologous chromosome
pairing and impacts on cell division affecting gamete formation[7,
22] leading to reproductive ill effects. However other investigators
[21, 28, 29] suggested an incidence of chromosomal variants need to
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Shah et al.
be debatable and further search is necessary to identify genes
responsible for normal reproduction in heterochromatic regions of
acrocentric and non-acrocentric groups and their activation and
inactivation mechanisms.
CONCLUSION
We hence conclude that karyotypic analysis and heteromorphism of
the D/G groups and 9 chromosomes with chromosomal rearrangements found in our study are related to infertility, and other
anomalies. It is therefore suggestive that couples undergo cytogenetic
detection followed by genetic analysis. The recent molecular methods
probably may unveil the relation between heteromorphism and
reproductive failure as we know that heterochromatin has been
regarded now playing crucial cellular roles.
ACKNOWLEDGEMENT
Authors are thankful to all staff of Supratech Micropath Research
Institute, Ahmedabad for their assistance.
CONFLICT OF INTERESTS
No conflicts of interest noticed amongst authors
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How to cite this article

Ketan K Vaghasia, Nidhi D Shah, Parth S Shah, Vidhi M Bhatt,
Sandip C Shah, Mandava V Rao. Karyotypic analysis of
chromosomal polymorphism in relation to reproductive failure.
Int J Pharm Pharm Sci 2017;9(4):140-143.
143