International Journal of Scientific & Engineering Research, Volume 5, Issue 7, July-2014 1107
ISSN 2229-5518
Cytogenetic Analysis of Couples with Recurrent
Pregnancy Loss
Jisha P, Sanuj C Breezevilla and Dinesh Roy D
50 years. The cytogenetic analysis results were correlated with various demographic, lifestyle and clinical aspects of couples with RPL. Increase in number of gestations, spontaneous abortions and MTPs showed increased incidence of abnormal karyotypes among the study subjects with RPL. The study demonstrated that there is a positive correlation between duration of married life, history of infection, chronic illness and drug intake with chances for recurrent pregnancy loss. A significant abnormal karyotype among the study subjects along with the above parameters is suggestive of increased risk of recurrent pregnancy loss. Modification of life style along with proper medication for teratogenic infection and awareness of the role of Genetics in the etiology of RPL will help in reducing the risk for recurrent pregnancy loss.
—————————— ——————————
nutritional and environmental factors [4].
Recurrent pregnancy loss (RPL), defined as two or more consecutive pregnancy losses before 20th week of pregnancy is a frequent obstetric complication [1]. Couples with recurrent miscarriage (RM) are facing an increased risk of being carriers of a structural balanced chromosome abnormality. The incidence of carrier status is ∼0.7% in the
general population worldwide and increases to 2.2% after
one miscarriage, 4.8% after two miscarriages and 5.2% after
three miscarriages [2].
Factors associated with RPL include parental chromosome translocations, uterine malformations, endocrine, hematological and autoimmune factors. In spite of exhaustive investigation, approximately 40% of RPL cases remain unexplained and are classified as idiopathic RPL (iRPL) [3]. Other risk factor includes loss of euploid pregnancy, loss after the first trimester, difficulty in conceiving, and delivery of a very low birth weight baby. The most prevalent abnormality of spontaneous abortion is chromosomal aneuploidy. Other etiologic factors of spontaneous abortions are anatomic anomalies, endocrine or hormonal problems, coagulation protein defects, and
————————————————
• Jisha P, Research scholar, Department of Medical Microbiology, School of Health Sciences, Thalassery campus, Palayad.P.O, Kannur- 670 661
• Sanuj C Breezevilla , Head, Dept. of Zoology, Sree Narayana
College, Cherthala – 688 582
• Dinesh Roy D, Genetika, Centre for Advanced Genetic Studies, Pettah.P.O, Thiruvananthapuram- 695 024
Several studies have implicated the role of female
factor in recurrent miscarriages, but the role of male factor has only recently been realized in couples experiencing sporadic assisted reproductive technique failure. Till date few studies have evaluated the role of sperm integrity in iRPL cases following spontaneous conception. Analysis of DNA integrity in understanding the role of sperm factor in iRPL may reduce the number of cases diagnosed as idiopathic and aid in providing most adapted therapeutics to the couple [3].
The clinical association of RPL in polycystic ovarian syndrome (PCOS) is more than common. However, the incidence rate between PCOS and recurrent miscarriage remains uncertain due to its wide variation in different studies. The high prevalence of hyper-secretion of LH and obesity in the syndrome contributes to the conclusion which has been reported as a risk factor for spontaneous abortion. Hyperinsulinemia has been proposed as the pathway for the effect of obesity on some reproductive abnormalities, probably through its effect on androgen production. There are many other reasons for recurrent miscarriages including chromosomal abnormalities, maternal age, medical illness, intrauterine illness, infections and immunological, endocrine and psychological factors [4].
Cytogenetic Analysis of recurrent miscarriages provides valuable insights into the cause of miscarriage which can eliminate further costly testing. In addition, recurrent risk estimates for subsequent pregnancies can also be determined. Genetic testing of prospective parents can identify those who carry a disease that can be passed
IJSER © 2014 http://www.ijser.org
International Journal of Scientific & Engineering Research, Volume 5, Issue 7, July-2014 1108
ISSN 2229-5518
onto their children [5]. Genetic testing of fetuses provides information about potential birth defects and gives couples high level of probability information upon which to base decisions. These new technologies have given sophisticated information and advanced technical solutions to this problems. Hence the present study was undertaken to evaluate various risk factors and chromosome abnormalities, if any, in couples with recurrent pregnancy loss and to evaluate the role of various demographic and clinical aspects of couples with RPL.
Fifty couples suffering with varying degrees of pregnancy loss were selected for this study. All these couples have at least one year duration of married life. These couples were referred from various infertility clinics and maternity centers of Kerala for genetic testing to Genetika, Centre for Advanced Genetic Studies, Trivandrum, Kerala. Detailed demographic and lifestyle characteristics were recorded using proforma. Chromosome preparation was done by peripheral blood lymphocyte culture method [6] and banded with GTG banding technique [7].
The fresh blood collected by venepuncture was transferred in to vacuutainer containing sodium heparin as anticoagulant. Added 5 to 6 drops of whole blood samples to a vial containing 10ml of RPMI 1640 medium supplemented with 20% foetal bovine serum. Then phytohaemagglutinin (PHA, 10µg/ml) was added to proliferate the lymphocyte cells and incubated at 37ºC for
72 hrs. At the 70th hour to the culture added a drop of
colchicine (0.04µg/ml) to arrest the cell division at
metaphase, then mixed gently and kept in incubator at 37ºC for 2 hours. Then transferred the whole content into a sterile centrifuge tube and centrifuged at 1000 rpm for 10 minutes. Discarded the supernatant, mixed the contents using cyclomixer, and re-suspend the pellet in pre-warmed hypotonic KCl (0.075M) and incubated for 20 minutes at 37
ºC. 2-3 drops of Methanol: Acetic acid fixative (3:1 ratio)
which was freshly prepared was added to the culture and centrifuged at 1000 rpm. Supernatant was discarded and the cell contents were mixed in a cyclomixer. 10ml of fresh fixative was added to the pellets present at the bottom of the centrifuge tube and kept for at least 30 minutes in
refrigerator. Again the preparation was centrifuged at 1000 rpm for 10 minutes and the supernatant was discarded. Added fresh fixative centrifuged and repeated the process 3 or 4 times until the supernatant appeared clears. Clear cell suspension was prepared and dropped the cell suspension, drop by drop on to pre cleaned labelled and chilled slides from a particular height so as to get good quality metaphases. The slides were air dried and the slides were stained with 10% Giemsa staining solution for 10 minutes. Washed the slides in distilled water and observed under a research microscope through 100x objective. For karyotyping and detecting the structural anomalies, GTG banding technique was performed. To detect numerical and structural abnormalities 20-25 metaphases were analyzed and 5-6 metaphases were karyotyped [8].
The demographic and clinical characteristic findings are given in the table1. The age of husbands and wives ranged from 20 to 50 years. The clinical characteristic findings are given in the table 2. The number of gestation of the couples was observed and among them the abnormal karyotype was found in 33.33% on those with number of gestation >6. Moreover subjects with more than six times spontaneous abortions showed increased (66.66%) incidence of abnormal karyotypes. Simultaneously couples with more medical termination of pregnancies (MTP) showed increased (40%) incidence of abnormal karyotypes. In short, increase in number of gestations, spontaneous abortions and MTPs showed increased incidence of abnormal karyotypes among the study subjects with RPL.
The life style characteristic findings are given in the table 3. The duration of married life of the couples were observed and among them those which had a duration >10 years showed 66.66% of abnormal karyotypes. Subjects with history of infections viz. fever, urinary tract infection, allergy due to dust, etc reported increased incidence of abortions. 27.2% of the couples with history of chronic illness had abnormal karyotype. The illnesses observed were PCOD, APLA Syndrome, asthma and diabetes. These findings demonstrated that there is a positive correlation between duration of married life, history of infection, chronic illness and drug intake with chances for recurrent pregnancy loss.
TABLE: 1
Distribution of Karyotype according to Demographic characteristics
IJSER © 2014 http://www.ijser.org
International Journal of Scientific & Engineering Research, Volume 5, Issue 7, July-2014 1109
ISSN 2229-5518
Age of Husband | Variable | Number (Percentage) | Abnormal Karyotype (Percentage) | Normal Karyotype (Percentage) |
Age of Husband | 21 – 30 | 10 (20%) | 1(10%) | 9 (90%) |
Age of Husband | 31– 40 | 32 (62%) | 1(3.12%) | 31(96.87%) |
Age of Husband | 41 – 50 | 8 (16%) | 2 (25%) | 6 (75%) |
Age of Wife | 21-30 | 30(60%) | 1(3.3%) | 29 (96.6%) |
Age of Wife | 31-40 | 15(30%) | 1(6.66%) | 14(93.33%) |
Age of Wife | 41-50 | 5(6%) | 3(60%) | 2(40%) |
IJSER © 2014 http://www.ijser.org
International Journal of Scientific & Engineering Research, Volume 5, Issue 7, July-2014 1110
ISSN 2229-5518
TABLE: 2
Distribution of Karyotype according to Clinical characteristics
Number of Gestations | Variable | Number (Percentage) | Abnormal Karyotype (Percentage) | Normal Karyotype (Percentage) |
Number of Gestations | <6 | 47(94%) | 2(4.25%) | 45(95.74%) |
Number of Gestations | >6 | 3(6%) | 1 (33.33%) | 2(66.66%) |
Number of Spontaneous Abortions | <6 | 47(94%) | 1(2.12%) | 46(97.8%) |
Number of Spontaneous Abortions | >6 | 3(6%) | 2(66.66%) | 1(33.33%) |
Number of MTPs | 0-1 | 45(90%) | 1(2.22%) | 43(95.5%) |
Number of MTPs | 2-3 | 5(10%) | 2 (40%) | 3(60%) |
IJSER © 2014 http://www.ijser.org
International Journal of Scientific & Engineering Research, Volume 5, Issue 7, July-2014 1111
ISSN 2229-5518
TABLE: 3
Distribution of Karyotype according to Lifestyle characteristics
Duration of Married Life | Variable | Number (Percentage) | Number of Abnormal Karyotype (Percentage) | Number of Normal Karyotype (Percentage) |
Duration of Married Life | <10 | 44 (88%) | 5 (11.36%) | 39 (88.6%) |
Duration of Married Life | >10 | 6 (12%) | 1 (16.6%) | 5 (83.3%) |
H/o Infection | YES | 3 (6%) | 2 (66.66%) | 1 (33.33%) |
H/o Infection | NO | 47 (94%) | 1 (2.12%) | 46 (97.8%) |
H/o Illness | YES | 11 (22%) | 3 (27.2%) | 8 (72.7%) |
H/o Illness | NO | 39 (78%) | 2 (5.12%) | 37 (94.87%) |
H/o Drug Intake | YES | 9 (18%) | 3 (33.33%) | 6 (66.66%) |
H/o Drug Intake | NO | 41 (82%) | 2 (4.87%) | 39 (95.12%) |
Recurrent Pregnancy Loss (RPL) also referred to as Recurrent Miscarriage or Habitual Abortion is a distinct disorder defined by two or more failed clinical pregnancies,
and up to 50% of cases of RPL will not have a clearly defined etiology. Approximately 15-20% of clinically recognizable pregnancies end in spontaneous abortion. The incidence of chromosomal abnormalities in those abortions
IJSER © 2014 http://www.ijser.org
International Journal of Scientific & Engineering Research, Volume 5, Issue 7, July-2014 1112
ISSN 2229-5518
is as high as 50%. A modest but clinically important proportion of spontaneous abortions are caused by a balanced chromosomal aberration in one of the parents. This results from the production of gametes and embryos with unbalanced chromosome sets. The clinical consequences of such abnormal gametes include sterility, repeated abortions, and giving birth to malformed children [9].
In most cases more than 99% of chromosomally abnormal pregnancies result in miscarriage which occurs prior to 10 weeks of gestation. Such miscarriages are thought to occur on a random basis, with increasing frequency of trisomy with advancing maternal age [10]. In this study the cytogenetic analysis of 50 couples with recurrent pregnancy loss was conducted. Regarding the parental chromosome analysis 10% out of 100 subjects were found abnormal.
Some couples with habitual abortion, have an increased proportion of aneuploid cells in their peripheral blood lymphocytes. Some studies have described increased sex chromosome aneuploidy in couples with recurrent abortions [11]. In the present study, the karyotype analysis was found 10% abnormal for the male subjects ranging from 21-30, 3.12% abnormal for the male subjects ranging from 31-40 years of age group and 25% abnormal karyotype for the male subjects of age group 41-50 years. The karyotype analysis was observed as 3.33% and 6.66% abnormal for the women ranging from 21 - 30 and 31 - 40 years respectively and 60% abnormal for the female subjects from age group of 41-50 years. Epidemiological evidence shows a steep increase in sporadic miscarriage rate in women aged more than 36 years [12]. This age related risk is due to a higher number of aneuploides, mainly trisomies [10]. The age dependent increase of trisomies, with repeated miscarriages as its manifestation, may be due to a recurrence of sporadic chromosome abnormalities [13].
Numerous studies have shown that abortion is closely associated with other confounding factors, such as smoking, alcohol, drug use, promiscuity and venereal disease. These behaviors may help women to cope with unresolved anxiety and depression after abortion. Women with a history of abortion are especially likely to persist in using tobacco, alcohol, and illegal drugs during subsequent pregnancies [14]. This maybe because the subsequent wanted pregnancy is a connector to unresolved issues after abortion. In the present study, the karyotype analysis on duration of married life among couples showed 11.36%
abnormal karyotype from the group of less than 10 years and 16.6% abnormal karyotype from the group of more than 10 years.
The major cause of Spontaneous abortion is fetal
chromosomal abnormalities, contributing to about 50∼60%
of the cases. The most prevalent abnormality of
Spontaneous abortion is chromosomal aneuploidy. Other
etiologic factors of Spontaneous abortion are anatomic
anomalies, endocrine or hormonal problems, coagulation protein defects, and nutritional and environmental factors [4]. In the present study 94% of the subjects had spontaneous abortions less than 6 times with 2.12% abnormal karyotype and 6% subjects had spontaneous abortions more than 6 times with the abnormal karyotype of 66.66%.
Recurrent pregnancy loss (RPL) in polycystic ovary
syndrome (PCOS), which occurs in ∼50% of total
pregnancies, is a frequent obstetric complication. Polycystic
ovary syndrome (PCOS) is a common condition estimated to affect 4 – 18% of women of reproductive age [15]. Several studies have implicated the role of female factor in recurrent miscarriages. In the present study 22% of subjects were found with history of illness having 27.2% abnormal karyotype and 18% of subjects were found with history of drug intake and the abnormal karyotype analysis was found to be 33.33%. Thus the present study suggests the evaluation of genetic instabilities in couples with recurrent pregnancy loss especially in idiopathic cases.
The couples who had reported for the recurrent pregnancy loss showed a higher percentage of abnormal karyotype. The abnormal karyotype was found higher in those couples with an increased age, increased duration of married life, increased number of gestations, increased number of spontaneous abortions and increased number of MTPs. The incidence of RPL was found also prevalent in those who had history of infection, illness and drug intake. The recurrent risk is more in couples with structural anomalies and so such couples are suggested to undergo genetic counseling before they are planning to get conceived again to avoid further miscarriages. This study has shown that the incidence and distribution of chromosomal abnormalities among couples with repeated fetal loss is comparable to that reported worldwide. Thus cytogenetic studies should be offered to all couples with more than 2 spontaneous abortions especially in idiopathic cases. Increasing awareness of the role of Genetics in the
IJSER © 2014 http://www.ijser.org
International Journal of Scientific & Engineering Research, Volume 5, Issue 7, July-2014 1113
ISSN 2229-5518
etiology of RPL and its overall impact on the burden imposed on individuals, families and society has led to the emergence on modern Clinical Cytogenetics, through this individuals can be better informed about extent of DNA damages, Genetic risks and reproductive options.
1. Pratip Chakraborty (2013); Recurrent Pregnancy Loss in Polycystic Ovary Syndrome: Role of Hyperhomocysteinemia and Insulin Resistance; 8(5).
2. E.Van den Boogaard (2011); Selective karyotyping in
recurrent miscarriage: are recommended guidelines adopted in daily clinical practice? ; Human Reproduction; 26 (8); 1965-1970.
3. Kishlay Kumarl (2012); Predictive value of DNA
integrity analysis in idiopathic Recurrent Pregnancy
Loss following spontaneous conception; 29(9); pp:
861-867.
4. Ji Hyae Lim (2013); Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy; 8(2): e56787.
5. Andersen N, Wohlfahrt J, Christens P, Olsen J, Melbye M (2000); Maternal age and fetal loss; Population based register linkage study. Br Med J; 320: 1708-1712.
6. Moorhead P S, Nowell P C, Wellmann W J (1960);
Chromosome preparation of Leukocyte culture from
Human Peripheral Blood. Exp. Cell.Res., vol. 20, pp. 613-
617.
7. Seabright M. A (1971); Rapid banding technique for human chromosome. Lancet. 11, 971-972.
8. Mittelman F, Ed, Karger S, Basal 1995; International
System for Human Cytogenetic Nomenclature (ISCN).
9. Philipp T (2003); Embryoscopic and cytogenetic analysis of 233 missed abortions: factors involved in the pathogenesis of developmental defects of early failed pregnancies, Human reproduction; 18 (8); 1724 -
1732.
10. Hassold, T and Chiu (1985) Maternal age specific rates of numerical chromosome abnormalities with special reference to trisomy; Human Genetics; 70; 11-17.
11. Hecht, S.M. (2000) Bleomycin: new perspective on the mechanism of action.J.Nat.Prod. 63, 158-168.
12. Cowchock FS, Gibas Z, Jackson LG (1993); Chromosome errors as a cause of spontaneous abortion: The relative importance of maternal age and obstetric history; Fertil Steri; 59: 1101-4.
13. Lee RM, Silver RM (2000); Recurrent pregnancy loss:
Summary and Clinical recommendations; Sem Report
Med; 18:433-40.
14. Lipmann-Hand A (1980): Genetic counselling and
human reproductive loss, in porter IH, Hook EH (eds): Human embryonic and foetal death. New York, Academic Press; 1980; pp: 303.
15. Manikumar R, Dinesh Roy D, Viji Krishnan and T
Vijayakumar (2013). Association of DNA damage and
dyslipidemia with Polycystic ovarian syndrome,
Journal of Medical and Allied Sciences; 3 (1): 15-21.
IJSER © 2014 http://www.ijser.org