|Year : 2020 | Volume
| Issue : 3 | Page : 121-126
Primary amenorrhea in North Kerala: A cytogenetic study
KS Lekha1, N Seena2, Lola Das3, V Bhagyam4
1 Associate Professor, Department of Anatomy, Government Medical College, Thrissur, Kerala, India
2 Assistant Professor, Department of Anatomy, Government Medical College, Thrissur, Kerala, India
3 Professor, Department of Anatomy, Government Medical College, Thrissur, Kerala, India
4 (Rtd) Professor, Department of Anatomy, Government Medical College, Kozhikode, Kerala, India
|Date of Submission||01-May-2020|
|Date of Decision||19-May-2020|
|Date of Acceptance||05-Oct-2020|
|Date of Web Publication||15-Oct-2020|
K S Lekha
Mammavalappil House, Pazhanji, Thrissur - 680 542, Kerala
Source of Support: None, Conflict of Interest: None
Background and Objectives: Primary amenorrhea (PA) is a major cause of female infertility. PA with the absence of secondary sexual characters is usually due to endocrine or chromosome abnormalities. The purpose of the present study is to estimate the frequency and type of chromosomal anomalies (CAs) in patients with PA, referred for karyotyping, mainly from the gynecology and endocrinology departments. Materials and Methods: In this cross-sectional study, the cytogenetic analysis was carried out in 53 patients with PA from North Kerala. Based on the standard protocol, peripheral blood lymphocyte culture was done. Chromosomal analysis was done with the help of an automated karyotyping system, after G-banding of chromosomes. In each case, 30 metaphase spreads were analyzed to detect CAs. When mosaicism was suspected, at least 50 metaphase spreads were examined. Results: Out of the 53 patients with PA studied, 67.92% (n = 36) showed normal female karyotype (46, XX). Abnormal karyotype was detected in 32.08% of cases (n = 17). CAs can be grouped into the following four types: (1) The most frequent anomaly was aneuploidy of X chromosome 64.7% (n = 11). (2) Structural anomalies of the X chromosome were detected in 11.76% (n = 2). (3) Mosaicism of X chromosome aneuploidy with a structural anomaly of the X chromosome was found in 11.76% (n = 2). (4) Male karyotype 46, XY was present in 11.76% (n = 2). Conclusion: This study emphasizes the importance of karyotyping in the diagnosis and management of patients with PA as it reveals the incidence of a significantly high number of cases of CAs.
Keywords: Chromosomal anomalies, cytogenetic study, karyotyping, primary amenorrhea
|How to cite this article:|
Lekha K S, Seena N, Das L, Bhagyam V. Primary amenorrhea in North Kerala: A cytogenetic study. Natl J Clin Anat 2020;9:121-6
|How to cite this URL:|
Lekha K S, Seena N, Das L, Bhagyam V. Primary amenorrhea in North Kerala: A cytogenetic study. Natl J Clin Anat [serial online] 2020 [cited 2021 Apr 22];9:121-6. Available from: http://www.njca.info/text.asp?2020/9/3/121/298166
| Introduction|| |
Primary amenorrhea (PA) is defined as the failure to attain menarche by 16 years of age, in the presence of secondary sexual characteristics and normal growth or by 14 years of age, in the absence of normal growth and secondary sexual characteristics. Amenorrhea affects 2%–5% of women of childbearing age , and accounts for 20% of patients with infertility. Etiological basis of PA is formed mainly by hormonal, structural, genetic, or environmental factors. Hormonal factors include mainly disorders affecting hypothalamic–pituitary function, caused by congenital, acquired, or functional conditions. This leads to dysfunction of the hypothalamic–pituitary ovarian pathway. Hormonal factors also include conditions such as congenital adrenal hyperplasia, juvenile hypothyroidism, and androgen insensitivity syndrome (AIS). Endocrine factors account for about 40% of the causes of PA, and the remaining 60% have developmental origins (structural or genetic). Structural anomalies are the result of defective development of the genital system.,
Following improvements in hygiene and health care, there is a marked decline in the number of diseases caused by environmental factors, but the contribution of genetic factors to morbidity and mortality is on an increase. Failure to attain menarche can cause a severe psychological impact on the affected person and her family members. The karyotyping is one standard procedure to find the incidence of chromosomal anomalies (CAs) in PA. Determination of genetic factors is very important for diagnosis, genetic counseling, and further management. Cytogenetic studies show that the incidence of CAs in PA is 20%–40%. In India, reported studies are few on the genetic basis of amenorrhea.
Only very few studies have been conducted in patients with PA in Kerala. PA with the absence of secondary sexual characters is usually due to endocrine or chromosome abnormalities. Because of the above, the present study was undertaken. The purpose of this study is to reveal the correlation of genetic factors in cases of PA.
| Materials and Methods|| |
It is a cross-sectional study.
Study subjects included patients with PA referred to the cytogenetic laboratory of the Department of Anatomy, Government Medical College, Kozhikode, Kerala. Patients were referred from various departments of the medical college, mainly obstetrics and gynecology, general medicine, endocrinology, and plastic surgery for chromosomal analysis. The age of the participants ranged from 14 to 36 years. This study was conducted as per the norms of the Institutional Ethics Committee over a period of 6 years.
Patients with failure to attain menarche by 14 years of age, in the absence of normal growth and secondary sexual characters, or by 16 years of age in the presence of normal growth and secondary sexual characters were included.
Patients below this age limit and above 40 years of age were excluded. Patients on treatment with hormones or drugs like antiepileptics and patients under extreme physical or psychological stress were excluded. Patients with anatomical defects like menstrual outflow obstruction and those with secondary amenorrhea were also excluded.
The sample size was calculated using the formula , the observed prevalence of CA in patients with PA being 14%–78%. Informed consent was obtained for each patient along with detailed pro forma.
Furthermore, from each patient/guardian, consent was obtained that their images and the information of the study may be discussed, presented, or published after concealing the identity strictly maintaining the confidentiality of the patient.
The procedure of human blood lymphocyte culture
Blood cell karyotyping is the foundation of modern cytogenetics, and it has excellent growth potential after mitogen stimulation. One milliliter of peripheral blood was collected for each patient in a heparinized syringe. Five hundred micro litres (0.5ml) of heparinized whole blood was inoculated into labeled culture tubes containing 10 ml of peripheral blood medium (Sigma/Gibco). The main components of the medium are as follows: RPMI-1640 with L-gluten, fetal bovine serum, phytohemagglutinin which acts as a mitogen, and antibiotic gentamicin. The culture tubes were incubated for about 72 h in a CO2 incubator.,
Harvesting of peripheral blood culture
To each culture tube, 100 μl of colchicine (Colcemid, Gibco) was added to arrest cell division at metaphase by preventing mitotic spindle formation. The solution was mixed by inverting the tube and incubated again at 37°C for 20 min. Then, the tubes were gently shaken, contents transferred to 15 ml centrifuge tubes, and centrifuged at 1000 rpm for 10 minutes The supernatant was aspirated leaving 0.5 ml of the solution above pellets. Ten milliliters of prewarmed (37°C) hypotonic solution (0.075 M potassium chloride) was added to each tube and incubated for 20 min at 37°C. The tubes were again centrifuged, and the supernatant was pipetted out. Then, 10 ml of Carnoy's fixative (75% methanol and 25% glacial acetic acid) was added and allowed to sit at room temperature for 5 min. The tubes were spun at 1000 rpm for 10 min. The supernatant was removed, again 10 ml of Carnoy's fixative was added, and culture was allowed to stand in fixative for 30 min. These steps were repeated until the sediment is clear, and the fixed cell pellets were used to prepare chromosome spreads.
Preparation of slides
Pre cleaned slides were refrigerated (4°C) in triple-distilled water for 2–3 h. Then, the slide was kept at an angle of 45°, and 4–5 drops of cell suspension were dropped to slide surface from a height of two feet. 5–6 slides were prepared for each case and kept at room temperature for 2–3 days before staining. The slides were immersed in trypsin solution for 20–40 s and rinsed with cold phosphate-buffered saline, then stained in Giemsa for 5 min, rinsed in distilled water, and air-dried. Karyotyping was done with the help of an automated karyotyping system (Cytovision software version 7). Thirty metaphase spreads were analyzed in each case for CAs. In suspected mosaicism, 50–100 metaphase spreads were analyzed.
| Observation and Results|| |
A total of 53 cases of PA in the age group of 14–36 years were subjected to peripheral blood lymphocyte culture and chromosome analysis.
Among the patients, 67.92% showed a normal female karyotype (46, XX) and 32.08% showed chromosome anomalies. CA can be classified into the following four main groups:
- Most frequent anomaly was X chromosome aneuploidy (n = 11, 64.7%) which included Turner's syndrome (TS) 45, X (n = 10, 58.88%) and mosaic TS 45, X/46, XX (n = 1, 5.88%)
- SAs were detected in 11.76% of cases (n = 2). One was found to have isochromosome of the long arm of X – 46, X, i(X)(q10) – and one with deletion of the long arm of X chromosome 46, X, del(X)(q21-2)
- Mosaicism of X chromosome aneuploidy with structural anomaly of X was found in 2 cases, 45, X (77%)/46, X, i(X)(q10) (23%) and 45, X (87%)/46, X, der(X) (13%)
- Male karyotype (46, XY) was present in 2 cases (11.76%). One of the cases of 46, XX showed heterochromatin in q arm, a coincidental finding. Karyotype distribution is tabulated in [Table 1].
|Table 1: Incidence of various chromosomal anomalies in primary amenorrhea|
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| Discussion|| |
In the present study of 53 cases of PA, 36 patients showed a normal karyotype. CAs were detected in 17 cases (32.08%) revealing the significance of cytogenetic analysis in patients with PA [Figure 1].
|Figure 1: Percentage of abnormal karyotype and normal karyotype in patients with primary amenorrhea|
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Etiological factors contributing to PA are mainly hormonal, anatomical, genetic, and environmental. Endocrine factors account for about 40% of cases of PA, and the remaining 60% have genetic or developmental anomalies. Sex chromosome anomalies play a major role in PA. The high psychological impact of PA prevents many from seeking genetic evaluation. In rural parts of India, patients with sex-related anomalies do not visit a clinic for investigations. Hence, actual frequencies of CA in PA remain undetected.
Numerical anomalies of the X chromosome, which included pure and mosaic TS, were more in our study. The next frequent was the structural anomaly of X. SA of X was present in two cases, and out of three cases of mosaic TS, two showed SAs of the X chromosome. The SAs observed in our study were isochromosome (2 cases), one in pure form and one in mosaic form, deletion (1 case), and derivative chromosome X (1 case). Two cases of sex reversal (46, XY karyotype) were also obtained [Figure 2].
|Figure 2: Frequency and distribution of various chromosomal anomalies found in participants with primary amenorrhea|
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The frequency of various CAs in our study is compared with that of previous studies in [Table 2]. CAs play an important role in PA, and this includes both numerical and SAs and sex reversal. These findings indicate the importance of cytogenetic study in patients with PA to detect the underlying cause and to plan further management accordingly.
|Table 2: Frequency of various chromosomal anomalies in the present study compared with earlier studies|
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Nearly 32.08% of CAs in our study are comparable to the reported frequency in literature. CAs reported in 1997 based on 29 studies from 1961 to 1992 was 32.32%. The overall frequency of ten independent studies conducted in 1961 to 1984 is 33%. The percentage of chromosome anomalies in our study corresponds to this and is comparable to that of the studies given in [Table 2].
Reported frequencies of CA show a wide range. According to literature, the frequency of chromosome anomalies ranges from 14%–78%.,,,,,, Reason for this wide variation is the difference in sample size and selection criteria of the study population.
In our study, numerical anomalies of the chromosome were more and included mainly TS [Figure 3] and also mosaic TS. The incidence of pure TS in our study was high, 10 cases (58.88%), and is comparable to the high incidence of TS reported from North Kerala by Ashalatha et al. The reported percentage of pure TS in previous studies [Table 2] is 23%–47%. According to published reports, the main anomaly in PA is TS., Our study also supports the same.
As clinical data were lacking in many cases, we could not correlate karyotype with phenotype in all cases, but the absence of secondary sexual characters and short stature were noticed in all cases of pure TS, and most of them presented with skeletal anomalies [Figure 4]a and [Figure 4]b. Short stature is said to be triggered by the lack of a second sex chromosome. Genes whose absence leads to somatic abnormalities of TS may be located along the length of Xp or middle of Xq (Xq21–Xq26), whereas genes involved in gonadal function are located on the proximal part of Xp and distal part of Xq. The most frequent cause of short stature in TS is a short-stature homeobox gene in the pseudoautosomal region 1, other possible cause being inadequate production of estrogen leading to osteoporosis which can cause a decrease in height and exacerbation of spinal curvature. The distribution of CA, age, height, and secondary sexual characters in patients with PA is shown in [Table 3].
|Figure 4: (a) Patient with Turner's syndrome showing bilateral cubitus valgus. (b) Feet of the same patient showing bilateral fourth metatarsal shortening|
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|Table 3: Distribution of cytogenetic abnormality, age, height, and secondary sexual characters in patients with primary amenorrhea|
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The structural anomaly was the next frequent anomaly detected in our study. Isochromosome of the long arm of X is a frequent SA reported by many authors.,,,,, Two cases of isochromosome of the long arm of X were found in our study, one in pure form and one in the mosaic form [Figure 5]. Other SAs detected in our study were the deletion of Xq and the derivative of the X chromosome. These were reported by several authors.,,, However, our study failed to detect reported SAs such as isochromosome Xp, ring chromosome,, marker chromosome,,, and X-autosome translocation.,,, One patient showed the presence of heterochromatin(46, XX,16qh+). A similar finding had reported by Rajangam and Nanjappa. and mentioned it as a coincidental finding as it is known to have no significant effect in the phenotype of the patient. Genes located on the q arm of X are very important for normal gonadal function, and hence, structural changes can lead to gonadal dysfunction.,, If the inactivated X chromosome is the structurally abnormal one, then the cellular function disturbances will be minimal.
|Figure 5: Karyotype showing the presence of isochromosome X – 46,X,i(Xq)|
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Mosaicism of X chromosome aneuploidy with a structural anomaly of X was reported by many authors.,,, Isochromosome of Xq is the most frequent structural anomaly reported in mosaicism also. Our study showed two mosaic TS with a structural anomaly of X, one case with isochromosome Xq and the other with a derivative of X.
46, XY karyotype was seen in 11.76% of cases. This is a little less than the reported incidence in all studies mentioned in [Table 2] which ranges from 15% to 35%. Anupam Kaur et al. in 2004 reported a 20% incidence of 46, XY cases.
We collected the clinical data of the two cases of sex reversal in our study and found that the first one was a case of complete AIS caused by mutations of androgen receptor gene. This is an X-linked gene that stops its activity following mutations. This condition was formerly called testicular feminization syndrome. The second was a rare type due to a deficiency of 17 alpha-hydroxylase enzymes [Figure 6]a, [Figure 6]b and [Figure 7]a, [Figure 7]b.
|Figure 6: (a) Patient with 46,XY karyotype (androgen insensitivity syndrome) showing female external genitalia and scar of bilateral inguinal surgery done. (b) Patient with 46,XY karyotype showing female external genitalia and absence of secondary sexual characters|
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|Figure 7: (a) Karyotype of case 1 showing 46,XY. (b) Karyotype of case 2 showing 46,XY|
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The first case was a 14-year-old girl with a history of surgery for bilateral inguinal hernia at the age of 6 years. Her histopathology report confirmed removed testicular tissue, indicative of a case of testicular feminization syndrome caused by the complete insensitivity of androgen receptors. In this case, androgen insensitivity has affected the development of male internal and external genitalia.
Let us review a few steps in the development of gonads and genital ducts which give insight into the phenotype of this case. Until the 6th week of the embryo, bipotential gonads are undifferentiated and both Mullerian and Wolffian ducts coexist. The testis-determining factor is the sex-determining region of Y gene on the short arm of Y chromosome. This gene triggers the differentiation of medulla of undifferentiated gonad into the testis. Leydig cells of the testis secrete testosterone which stimulates the Wolffian duct to form male internal genitalia. Dihydrotestosterone formed from testosterone by 5-alpha reductase leads to the masculinization of external genitalia. Mullerian duct system regression is caused by Mullerian inhibitory factor (MIF) produced by Sertoli cells of the testis. Although the testes had secreted testosterone in the first case mentioned, its effects were blocked due to the insensitivity of androgen receptors. The individual had no Mullerian development because of the action of MIF produced by Sertoli cells. This girl presented with an absent uterus, fallopian tubes, and a short vagina which was a blind-ending.
The second case was a 21-year-old girl who presented with hypertension, hypokalemia, and the absence of secondary sexual characters. This patient had a rare type of congenital adrenal hyperplasia due to the deficiency of 17 alpha-hydroxylase enzymes, resulting in a block in pathway of synthesis of glucocorticoids and sex steroids, without affecting the mineralocorticoid synthesis. Increased production of mineralocorticoids due to adrenocorticotropic hormone stimulation resulted in clinical features such as hypertension, hypokalemia. Male internal and external genitalia were not formed in this case due to a lack of testosterone.
The main cause of sex reversal is AIS or pure XY gonadal dysgenesis. Detection of the Y chromosome is very important as it is an indication for immediate removal of gonads because of increased risk of gonadal tumor., Roy and Banerjee  detected two cases of complete testicular feminization syndrome in the study of 60 cases of PA.
Limitations of the study
Correlation between phenotype and karyotype could not be done due to the unavailability of complete clinical data.
The authors' suggestion is to conduct investigations for nongenetic causes before undertaking genetic evaluation.
| Conclusion|| |
This cytogenetic study reveals the incidence of a significantly high number of cases of CAs in patients with PA and emphasizes the importance of early karyotyping to distinguish the genetic causes of PA. The cytogenetic analysis helps precise diagnosis, appropriate management, and counseling of patients with PA. Genetic counseling should include the importance of surgical excision of gonads in women with the Y chromosome because of its increased chance of malignant transformation, reconstructive surgery of the genital tract, and early starting of hormone replacement therapy to prevent osteoporosis.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, patients/guardians have given their consent for images and other clinical information to be reported in the journal. Patients/guardians understand that names and initials will not be published and due efforts will be made to conceal patient identity, but anonymity cannot be guaranteed.
The authors thank the clinical team for directing the patients for karyotyping, also the technical staff of cytogenetic lab, and Mrs. Manju M., Senior Scientific Assistant.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]