Original Study

Frequency and Types of Chromosomal Abnormalities in TurkishWomen with Amenorrhea

Osman Demirhan PhD *, Nilg€un Tanrıverdi PhD, Erdal Tunc PhD, Nihal _Inandıklıo�glu PhD,Dilara S€uleymanova PhD, MDDepartment of Medical Biology and Genetics, Faculty of Medicine, Cukurova University, Balcali-Adana, Turkey

a b s t r a c t

Study Objective: To estimate the frequency and the type of chromos

omal abnormalities (CA) in patients with primary (PA) and secondaryamenorrhea (SA).Design: This retrospective study was comprised of patients had been referred to our laboratory between 1990 to 2008 and designed asoriginal article.Setting: Medical Faculty of Cukurova University in Turkey.Participants: Chromosomal analysis was carried out on 393 patients with PA and SA that were referred to Cytogenetic laboratory of MedicalBiology and Genetic Department, Faculty of Medicine, Cukurova University.Interventions: Lymphocyte culturing depended karyotyping.Main Outcome Measures: Standard lymphocyte culturing procedure and karyotyping was performed to all samples.Results: PA and SAwere identified in 393 patients. The karyotype was normal in 337 cases (85.8%) and abnormal in 56 (14.2%) patients. CAswere found in 54 (13.7%) and 2 (0.5%) of women with PA and SA, respectively. Females carrying rearrangements between autosomal andsex chromosomes were detected in 2% (8/393). The numerical abnormalities of the X chromosome were detected in 39.3% (22/56)(monosomy and mosaic). Structural abnormalities of the X and the other chromosomes were detected in 25.5% (13 of 56). Structuralmosaicism of X chromosome was found in 5.4% (3 of 56). Male karyotype (46, XY) was found in 33.9% (19/56). The most frequentlydetected abnormality were X chromosome monosomies or mosaics.Conclusions: Our study revealed that some causes of amenorrhea could be due to CAs. Therefore, cytogenetic study should be importanttest in the evaluation of patients with PA or SA. The most common abnormality seen is 45,X karyotype (monosomy X/Turner Syndrome)and its variants.Key Words: Primary and secondary amenorrhea, Karyotyping, Chromosomal abnormalities

Introduction

Menstrual disorders are among the most commonlyseen gynecologic problems in adolescent girls, composing75% of such problems worldwide.1 Amenorrhea meansabsence of menstruation during puberty or later in life.There are 2 types of amenorrhea: primary (PA) e thefailure of menses to occur by the age of 16, and secondary(SA) e in which the menses appear at puberty but subse-quently cease.2 Amenorrhea is a symptom with severaldifferent causes, including pregnancy, absence of uterusand vagina, hormonal imbalance, excess of male testos-terone, endometritis, improper functioning of ovaries,3,4

and M€ullerian agenesis. Cytogenetic investigations haveshown the importance of chromosomal abnormalities(CAs) as a major cause of amenorrhea.5,6 The percentage ofCAs reported varies greatly, from 15.9% to 63.3% for pri-mary amenorrhea7e11 and from 3.8% to 44.4% for second-ary amenorrhea.4,8,11,12 This wide variation is due todifferent sample size of selection group or to the different

The authors indicate no conflicts of interest.* Address correspondence to: Prof. Dr. Osman Demirhan, PhD, Department of

Medical Biology and Genetics, Faculty of Medicine, Cukurova University, 01330Balcalı-Adana, Turkey; Phone: 90-322-3387140; fax: 90-322-3386572

E-mail address: osdemir@cu.edu.tr (O. Demirhan).

1083-3188/$ - see front matter � 2014 North American Society for Pediatric and Adolehttp://dx.doi.org/10.1016/j.jpag.2013.11.013

selection criteria of different studies. A number of surveysin various parts of the world have endeavored to ascertainthe contribution of sex CAs to the problem of amenorrhea.Many amenorrhea patients show a variety of CAs such as45,X and sex reversals, that is, female phenotype withmale chromosomal complements and also other auto-somal translocations. This study was undertaken todetermine the frequency and type of CAs that result in PAin the southern region of Turkey.

Materials and Methods

All women with PA and SA were recruited who werereferred to our genetics laboratory from May 1, 1992, toApril 28, 2009. Karyotypes of patients referred withamenorrhea were retrospectively analysed. The initialdiagnosis of PA or SA as made by the referring gynecologistwas followed up in the Division, based on the availablemedical history, which included clinical details, hormo-nal profile, and ultrasonography. PA was defined as theabsence of menstruation and secondary sexual character-istics in phenotypic women aged 14 years or older, or aged16 years or older. In SA cases, secondary sexual charac-teristics were present. Patients with SA had at least 1

scent Gynecology. Published by Elsevier Inc.

O. Demirhan et al. / J Pediatr Adolesc Gynecol 27 (2014) 274e277 275

spontaneous bleeding episode, followed by no menstrua-tion for a minimum of 12 months at or before the age of42 years. Patients were referred to us from gynecology andother services for genetic analysis after exclusion of non-genetic causes. The age of the analyzed population ran-ged between 15 and 40 years and the average age was19.3 years. The cytogenetic analyses were performed in theCytogenetics Laboratory, at the Department of MedicalBiology and Genetics, Faculty of Medicine, Cukurova Uni-versity. Metaphase chromosome preparations from pe-ripheral blood were made according to the standardcytogenetic protocols. Twenty metaphases were analyzedin all the patients, but in cases of abnormalities andmosaicism the study was extended up to 50 metaphases.All CAs were reported according to the current interna-tional standard nomenclature (ISCN, 2009).

Results

A total of 393 cases were evaluated for both PA and SA.The details of the karyotypes and percentage of abnormal-ities of all cases are given in Table 1. We had classified CAsinto 3 main types with or without mosaicism. They arenumerical and structural abnormalities of X chromosome,abnormalities of Y chromosome and females carryingrearrangements between autosomal and sex chromosomes.

The karyotype results were normal in 337 cases (85.8% ofall cases). However, CAs was present in 56 (14.2%) of thecases with PA and SA (Table 1). These abnormalities wasfound in 54 (13.7%) and 2 (0.5%) of women with PA and SA,respectively (Table 1). The most frequent karyotype was Xchromosome aneuploidy. X CAs (either structural or nu-merical) were found in 29 (7.4% of all cases and in 51.8% ofall aberrations) patients. The most frequent abnormalitydetected was X chromosome monosomies or mosaics. Thenumerical abnormalities of the X chromosome weredetected in 4.6% of all cases and in 32.1% of all aberrations.

Table 1Frequencies and Distributions of the Karyotypes in Patients with Amenorrhea

Cytogenetic Grade Karyot

The total number of cases with PA and SANormal karyotypes 46,XXAbnormal karyotypes Numerical and structAbnormalities of X chromosomePure Turner 45,XMosaic Turner with numerical aberrations of X 46,XX/45,XMosaic Turner with structural abnormalities of X 45,X/46,X,i(Xq)Structural abnormalities of X 46,X,i(Xq)Mosaic normal with structural abnormalities of X 46,XX/46,X,i(Xq)

TotalAbnormalities of Y chromosomePure XY females 46,XYPure XY females with autosomal structural abnormalities 46,XY,inv(9)(p11-q13

46,XY,18qþTotalFemales with autosomal and sex chromosome structural

abnormalities46,XX,fra(3q32)46,XX,fra(1q32)45,XX,robt(13; 14)46,XX,inv(9)(p11-q1246,XX,15pþ46,XX,t(13; X)(q22; q46,XX,t(8,X)

Total

Structural abnormalities of the X chromosome weredetected in 1.8% of all cases and in 12.5% of all aberrations.Mosaicism of X chromosome was found in 1% of all casesand in 7.1% of all aberrations. In those with PA, the secondmost common abnormalities was male karyotype (46,XY),which accounted for 19 (in 4.8% of all cases and in 33.9% ofall aberrations) patients. Females carrying rearrangementsbetween autosomal and sex chromosomes were detected in2% of all cases and in 14.3 % of all aberrations. Two caseswere found to have autosomal-sex chromosome trans-locations (Table 1).

Discussion

Amenorrhea is one of the important reasons for patientreferral to an endocrine or gynecologic clinic. Its etiology isheterogeneous. Occurrence of CAs have a significant impacton both human fitness and sexual development. A largenumber of surveys have been undertaken worldwide in abid to ascertain the frequency of CAs in patients who pre-sent with PA or SA. Majority of studies have had small pa-tient numbers. In this study, we included a large number ofpatients. A review of the literature shows that CAs appear tobe one of the main causes of PA. The estimated frequency inour study was compared with the CAs described in theliterature and is presented in Tables 1 and 2.

In the present study, the karyotype was normal in 337cases. The frequency of CAs was 14.3% which is comparableto other studies (Table 2). CAs were numerical and struc-tural. The frequency of abnormal karyotypes has been re-ported to vary between 16% and 63.3% among women withPA in different parts of theworld.4,7 Our results (14.3%) werein accordance with other reports in similar studies: Josephand Thomas,9 16%, and Rajangam et al,5 16.3%, and lowerthan in other studies10,13e22 (Table 2). These findings showthat CAs appear to be a cause of amenorrhea. When welooked at the wide scale of results from other studies, the

ype No. of Cases Frequency inall Cases (%)

Frequency inAberrations (%)

393 (371PAþ22SA)337 85.8

ural abnormalities 56 (54 PA þ 2 SA) 14.2

18 4.6 32.14 1.0 7.12 0.5 3.64 1.0 7.11 0.5 1.8

29 7.4 51.8

16 4.1 28.6) 1

20.8 5.4

19 4.8 33.9

)

28)

11121118 2.0 14.3

Table 2Review of the Literature in Comparison with the Present Study

Author TotalCases

X ChromosomalAneuploidy (%)

StructuralAbnormalities (%)

MarkerChromosome (%)

MaleKaryotype (%)

AbnormalChromosome (%)

Joseph & Thomas (1982)9 63 8 3 2 3 16Opitz et al (1983)4 88 17 7 . 4 28Anglani et al (1984)13 145 25 10 . 11 46Ten et al (1990)7 117 8 7 2 14 31Roy & Banerjee (1995)10 60 60 . . 3.3 63.3Temocin et al (1997)8 9 ... .... .... ..... 11.1Lakshmi and Satyanarayana (1997)14 70 25.7 . . 2.8 28.5Ramirez et al (2000)15 96 89.6 4.2 ... 7.9 36.7Wong & Lam (2005)16 237 12.6 2.9 0.4 8.4 24.5Rajangam &Nanjappa (2007)5 865 45.5 23.3 . 31.2 23.4Kong et al (2007)17 10 20 50 . 30 58.9Safaei et al (2010)20 220 10.9 3.6 . 6.4 20Kalavathi et al (2010)19 979 10.4 6.9 . 6.2 23.4Vijayalaksmi et al (2010)18 140 14.3 7.1 . 6.4 27.8Dutta et al (2013)21 600 8.7 9.2 . 3 21Butnariu et al (2011)22 269 82.2 8.17 .. 5.2 54.6Our cases (2013) 390 7.2 4.6 .. 5.1 14.4

O. Demirhan et al. / J Pediatr Adolesc Gynecol 27 (2014) 274e277276

differences between the present results and that of theprevious studies may be due to different selection criteria aswell as different population genetics throughout the world.

The karyotype was normal in 337 cases (85.8%), and thefrequency of CAs was 14.3% (54 cases) in patients with PA.The frequency of normal karyotypes was 91% (20 cases) andthe frequency of CAs was 9.1% (2 cases) in patients with SA.The percentage of CAs reported for SA varies greatly, from3.9% to 44.4%. Thismay be due to thewide variation inpatientselection criteria of different studies. Our result was inaccordance with some reports in similar studies: 7.08%19 and9.09%.16 In other studies the frequency of CAs was variable:Rajangam et al,5 16.32%, Devi and Benn,11 13.3%. In our study,CA frequencies for SA (in 0.5% of all cases) were much lessthan for PA (in 13.7% of all cases, in 96.4% of all aberrations). Ina previous study, a comparison of PA and SA in the southwestof Iran showed a higher prevalence of CAs in PA (20%) than inSA (5.3%).20 The high percentage of CAs detected in our pa-tients with PA suggested the major role of CAs in theabnormal gonadal development and function, as well as theimportance of genetic investigation of patients with PA.

In our study, the most frequent abnormalities detectedwere numerical and structural abnormalities of X chromo-some (7.4% of all cases and 51.8% of all aberrations). Themostfrequent X CAs were monosomy (45,X) and mosaic (45,X/46XX) (5.6% of all cases and 39.2% of all aberrations, TurnerSyndrome-TS). Our results (5.6%) were in accordance withother reports in similar studies (Joseph and Thomas9 andTen et al7) and lower than in other studies (Opitz et al,4

Rajangam and Nanjappa,5 Anglani et al,13 Roy and Bane-rjee,10 Lakshmi and Satyanarayana,14 Ramirez et al,15 Wongand Lam,16 Kong et al,17 Vijayalaksmi et al,18 Kalavathi et al,19

Safaei et al,20 Dutta et al,21 Butnariu et al22). See Table 2. TheTurner phenotype is quite variable, even among womenwith the same karyotype. The correlation between genotypeand phenotype is not yet well understood, but generallypatients with a 45,X karyotype tend to have a more severephenotype than those who are mosaic with a normal cellline.23 Absent pubertal development and PA occurs in mostindividuals with TS, due to accelerated loss of oocytes in the45,X ovary, leaving few follicles in a fibrous streak by birth.

Approximately one third of girls with TS undergo sponta-neous puberty, but in only half of them is puberty complete.Chromosome studies should be carried out in women withabsence of menstruation, particularly when they show anyof the following features: sexual infantilism, unexplainedshort stature, signs suggestive of TS, ambiguous genitalia,developmental abnormalities of the uterus and vagina,inguinal hernia and gonads, evidence of gonadal dysgenesis,raised follicle stimulating and luteinizing hormones, familyhistory of primary amenorrhea, and family history oftesticular feminization syndrome.MonosomyX results fromnondisjunction as a result of failure of the sex chromatids toseparate during meiosis in the parental gametes or in theearly embryonic divisions.11We noticed the lower frequencyof chromosomal mosaics (45; X/46,XX), with 2 or more celllines, in 4 of 393 cases (1%). The percentage of mosaicismranged from 10% to 70% in different studies.18,20,24

Premature ovarian failure may be secondary to X chro-mosome deletions. The patients with chromosome Xdeletions involving Xq13-q26 bands, present primaryamenorrhea determined by an ovarian dysgenesis.25,26 Re-ports of patients with premature ovarian failure and Xqdeletions suggest that there is a gene localized to Xq21.3-q27 or within Xq26.1-q2718-20 and a gene localized toXq13.3-q21.1.19. The second line with an unbalancedstructural abnormality of X chromosome: themost frequentabnormality detected was isochromosomes Xq [i(Xq)]. In 7cases (1.8% of all cases) we identified i(Xq) (Table 1). Therole of sex chromosome in the initiation and maintenanceof normal menstruation needs no emphasis. The integrity ofa critical area in the X chromosome (q13q26) is essentialfor normal ovarian function. In TS cases, the genes involvedin gonadal function are located on the proximal part of Xpand also on the distal part of the Xq, whereas the geneswhose absence is responsible for somatic features of thesyndromemay be distributed along the length of Xp and themiddle section of Xq.27 Based on these hypotheses thephenotype of patients with i ( Xq) can be correlated withshort stature and some Turner stigmata (sexual infantilism,gonadal dysgenesis) and association with other congenitalmalformations.

O. Demirhan et al. / J Pediatr Adolesc Gynecol 27 (2014) 274e277 277

In the present study, the second frequent group of CAsdetected in patients was represented by the karyotypeshaving a cell line with Y chromosome (46; XY females, 20cases: 5.1%). In almost all the studies reviewed, the mostfrequent chromosomal anomaly in amenorrhea patientsis 45,X followed by a male karyotype.16 Male karyotypeand mosaics karyotypes having a cell line with Y chro-mosome were present in a significant percentage of thepatients (ranging from 3.3% to 13.7%) with PA in previousstudies.20 Our results were comparable with these studiesand were similar to Vijayalaksmi et al,18 who detected6.4% cases with Y chromosome. In these cases prophy-lactic gonadectomy is recommended, because of the highrisk (7%-30%) of gonadoblastoma in the dysgeneticgonads.9,19,28,29

Autosomal abnormalities are rare in patients withamenorrhea. In our study, cases carrying rearrangementsbetween autosomal and sex chromosomes were detected in2% (8 of 393, Table 1). Jyothy reported 1 case of 46,XY,13pþin secondary amenorrhea patients.30 We also detected 1case of 46,XX,15pþ and 3 cases of the translocations[45,XX,robt(13; 14); 46,XX,t(13; X)(q22; q28); 46,XX,t(8,X)]in PA. In studies of Russell and Bangham31,32 and Russell33 aportion of an autosome had been translocated to an X. Thevariegated expression of the autosomal genes in thesetranslocations is one of the main lines of evidence for thehypothesis of X-chromosome inactivation.33,34

Conclusion

A significant number of patients had sex chromosomalabnormalities; thus early cytogenetic investigation is pru-dent to guide further management. Patients with amenor-rhea should be initially screened by primary physicians andgynecologists for non-genetic causes. After exclusion ofnon-genetic causes, patients with amenorrhea, in particularprimary amenorrhea, should receive prompt referral forgenetic study. The reason for referral should be explained tothe patient. If cytogenetic abnormalities are detected, a fullexplanation should be given to the patient by a geneticist orgynecologist with experience in genetics. Counselingshould include the risk of premature menopause for pa-tients with Turner syndrome and the use of hormonalreplacement therapy, the possibility of infertility in thefuture children of patients with mosaic Turner, and the riskof gonadal malignancy for patients with XY gonadaldysgenesis. Counseling should be performed tactfully,bearing in mind that sensitive issues related to femininityare involved. An experienced counselor and clinical psy-chologist would be helpful. The incidence of CAs in womenwith amenorrhea is similar to that reported in the literature.CAs are identified often enough to warrant karyotyping ofall women with amenorrhea. The present study showedthat karyotyping is necessary to detect the causes of pri-mary amenorrhea. This study also revealed the incidence ofCAs in women with primary amenorrhea in Turkey issimilar to that reported in previous literatures. This studyalso illustrates the frequency and distribution of karyotypes

causing amenorrhea, and we think will contribute to liter-ature about this condition.

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