Cytogenetic Abnormalities Identified in Cases of Amenorrhea

INTRODUCTION

Amenorrhea has been described as the cessation or absence of the menses.¹ The two types described in literature are primary amenorrhea (PA) and secondary amenorrhea (SA).¹ Secondary amenorrhea differs from the former as being described as the occurrence of amenorrhea after menarche.¹ Although there are known similarities in the causation for the two types of amenorrhea, the conditions warrant timed evaluation.¹

Guidelines have stated prompt evaluation of the patient if there is a failure to menstruate by the age of 15 in the presence of normal secondary sexual characteristics, or within 5 years of breast development if it has occurred before 10 years of age.¹ Major causes of amenorrhea are polycystic ovarian syndrome (PCOS), hyperprolactinemia and ovarian failure.¹ There are also genetic causes for the same that either underline these problems or may occur independently.¹ Such genetic causes are due to abnormal karyotype like Turner’s syndrome and mosaicism.¹ Neuroendocrinological problems like functional hypothalamic amenorrhea and hyperprolactinemia are other documented causes of amenorrhea.²

Associations between idiopathic hypogonadotropic hypogonadism and rare variants in genes have been established.³ These are found in women with hypothalamic amenorrhea.³ It is anticipated that such mutations may be the contributing factor to the variable susceptibility of women to the functional changes in GnRH secretion (Fig. 1).³

Another presentation of SA is primary ovarian failure (POF).⁴ Chromosomal abnormalities have been confirmed as causes for POF, while other causes for the same are autoimmune, infectious, metabolic, and iatrogenic causes.^4,5 Abnormal karyotypes have been reported in women with PA to vary between 20 and 31%.⁶

Hence, the absolute importance of cytogenetic analysis in the evaluation of women with amenorrhea is warranted for better management of the patient.⁶ This will aid in genetic counseling and clinical treatment.⁶ As there are numerous causes for PA and SA, we were determined to find out the cytogenetical causes for the cause of amenorrhea.

AIMS AND OBJECTIVE(S) OF THE STUDY

Primary: To determine the cytogenetic abnormalities in women with amenorrhea who presented in a tertiary health care hospital, South India.

MATERIALS AND METHODS

This was a retrospective descriptive record study. Patients with amenorrhea who had presented themselves for karyotyping at St. John’s Medical College and Hospital were included in the study. The study included all patients who presented for amenorrhea during the past 1.5 years (January 1, 2020 to June 30, 2021). Records of the genetic analysis report and their presentations were reviewed retrospectively by looking into the past folders of the patients. The cytogenetic testing was done from the Division of Human Genetics and the report was analyzed. All records that identified any cytogenetic abnormality that was the suggested cause of amenorrhea was documented. However, exclusion of other genetic abnormalities was done for those cases which did not present with amenorrhea. Patient records were coded to prevent the identification of any individual subject and only the research team handled the data, keeping full confidentiality. Ethical approval was sought (IEC No 169/2022). Patients were given genetic counseling when they had received their report. The physicians had no part in the decision-making of the outcome of the patient and absolute independence of the decision of the patient and family to decide on the continuation of the treatment as per their doctor’s advice was ensured at the time of dispatching the report (Fig. 2).

The chromosomes were arranged according to size, length, and position of the centromeres. They were divided into seven groups A–G. Normal male has 22 pairs of autosomes, one X chromosome and one Y chromosome. Normal female has 22 pairs of autosomes and two X chromosomes.⁷ Culturing and harvesting process required 2–4 mL of blood. After attaining appropriate growth of cells, at metaphase stage, a chemical called colchicine is added to arrest the cell cycle. The sample was then cultured, harvested, prepared on slides and banded with GTG refers to G bands by trypsin using Giemsa. Chromosomes were analyzed under a trinocular microscope using Applied Bioimaging Automated Karyotyping software version 7.2 under 100× oil immersion objective (Fig. 3).

The patients were given a unique hospital number and personal information including name, age and relevant history was noted. A detailed pedigree showing three generations to know the presence of consanguinity in the family was seen. Additional medical history including scanning reports, menstrual history, obstetric history, and hormone analysis was noted.

RESULTS

A total of 54 patients presented to the Division of Human Genetics for counseling and cytogenetic analysis over a period of 2.5 years with complaints of amenorrhea. Out of the 54 patients, 11 patients had cytogenetical abnormalities and the others had a normal karyotype. About 9 (82%) patients presented with PA and 2 (185) presented with SA out of the 11 who presented with amenorrhea. The mean age of the study subjects was 22 ± 6.7 years. Some of the patients were on medications for withdrawal bleeding. Peripheral blood was the specimen that was analyzed in the 11 patients. And 36% of the patients had a history of being born of a consanguineous marriage out of the 11 patients as seen in Table 1.

In Table 2, it is seen that 3 patients had normal secondary sexual characteristics but had an abnormal karyotype. On patient with mos 45,X[12]/46, XY female [13] karyotype, had presented with PA, short neck, bowed legs, and right cubitus valgus, with a normal echo report. One patients with a 46, XY female karyotype had an enlarged clitoris and the differential diagnosis of MRKH/Mullerian agenesis was considered. Another patient with 46, XY female karyotype, presented with PA had an ultrasound study that detected an absent uterus and B/L ovaries? Testicular tissue in ectopic location.

Table 3 shows the clinical findings of the patients on examination. The mean BMI was 23 ± 3.7 kg/m². One patient had an enlarged clitoris with a normal vaginal orifice, blind vagina, normal labia majora, and a labia minora. Another 16-year-old girl with PA with a karyotype of 46,X,derX,t(X;3)(q22;q13.3)[20] translocation of chromosome X and 3 had a blind vagina, with normal external genitalia. Out of the documented reports, it was seen that 6 patients were reported to have a gynecoid pelvis.⁸

Table 4 shows the basic biochemical testing done in most cases of amenorrhea. The mean FSH level was high for the subjects studied when compared with LH. AMH was low suggesting absence of follicles to produce the same. A 19-year-old patient with mos45,X[16]/46,X,i(X)(q10)[24] Turners variant karyotype had PA, who showed deranged thyroid function (Anti-TPO: 9.08 IU/mL and TSH: µIU/mL) and increased cholesterol levels (Total cholesterol: 215 mg/dL).

DISCUSSION

It is a normal clinical presentation to manifest as amenorrhea prepubertal girls.⁹ Pregnancy and postmenopausal females are also known to present like this.⁹ About 20% of women present with infertility, due to disruption of the physiology of menstruation and reproduction, which is linked to the expression of the X chromosome.⁹ Vijayalakshmi et al. showed that out of 140 women with amenorrhea 71.2% women had a normal karyotype and 27.8% showed an abnormal karyotype.⁹ Similar findings were seen in our study.

It was known that 46, XY female karyotype was seen in 5.5% of women in an Iranian study with X aneuploidies being the most frequent abnormalities seen in women with PA.¹⁰ In our study, out of the 9 women with PA and with abnormal karyotype, 22.2% of the women had a 46, XY female karyotype. The XY karyotype was detected in 29.8% women with primary amenorrhea as revealed by Korgaonkar et al.¹¹ Hence, initial referral and cytogenetic evaluation is needed for the identification chromosomal aberrations in such women.^9,10,12 Similar results were reported by Ghosh et al. and Soltani et al.^10–13

Kalpana and Satyanarayana studied 70 cases of PA and found normal chromosomal Karyotype in 71.43% and abnormal karyotype in 28.57%. Four cases were with 45,X (5.71%), 11 (15.71) with 45,X/46,XX; 1 each with mosaicism of 45,X/47,XXX and 45,XX/47,XXX and 47,XXX (1.43%) and 2 cases were with 46,XY constitution.¹⁴ Our study showed that six patients had an abnormal X karyotype and mosaic pattern. One patient had translocation of chromosome X and 3. A study concluded that the precise molecular characterization and unique breakpoint regions could help identify new genes in PA.^13,15

Mosaics X/XY and their variants were seen in 3.34% of women with PA. One patient in our study had mos 45,X/46,XY female. Cytogenetic abnormalities have resulted in the detection of abnormal presence of Y chromosomes.^11,13

Balwan et al. showed that out of 37 women with PA, 6 cases had an abnormal karyotype.¹⁶ Four of the patients had a typical Turners syndrome and two patients had mosaic pattern in the Kashmir study.¹⁶ Our study had a no patients with a typical Turner syndrome although mosaic variants were seen more commonly. Turners was attributed as the most common cause for PA.¹⁷ Female patients can be offered advanced therapies like hormone therapy and marriage counseling.¹⁶ Psychological support being the utmost important and central to genetic counseling.¹⁶

Korgaonkar et al. showed 81.9% of high frequency of abnormal uterus and ovaries (86.7%).¹¹ Korgaonkar et al. detected a total of 121 (24.7%) cases with abnormal karyotype.¹¹ The study showed such individuals (PA) with anatomical abnormalities (84.3%) had chromosomal aberrations (24.6%).¹¹ In our study, out of the two women with SA, the ultrasound study has shown that one case was found to have a hypoplastic uterus, B/L atrophic ovaries, and the other case had a normal uterus and B/L ovaries. Out of the 9 cases of PA with abnormal karyotype 2, women had a normal uterus while the others had either a hypoplastic uterus or an absent uterus.

Safai et al. showed chromosomal abnormalities in women with SA in 5.3% women.¹⁸ Out of the 11 cases with chromosomal abnormalities 2 patients had presented with SA in our study. The causes of secondary amenorrhea was attributed to abnormal karyotype, namely, 46,XX?del(9q)[50] possible interstitial deletion of light chain chromosome 9 in one patient and mos45,X[3]/46,XX[37] low mosaicism for X in the other patient in our study. It is revealed that the causes of SA could be due to chromosomal abnormalities in some patients and hence needs knowledge of the same for investigation.¹⁸

One patient with karyotype of mos45,X[3]/46,XX[37] low mosaicism for X and normal uterus and B/L ovaries as seen on ultrasound presented with SA. Hence, the cytogenetic data forms a first line diagnostic tool in women with amenorrhea, especially in patients with gonadal dysgenesis.¹⁹ Surico et al. stated that in cases a cytogenetic alteration may be present in the cellular level of the ovary and may be accompanied without an abnormal chromosome finding.¹⁹ The study showed that a patient had to undergo an ovarian wedge biopsy and cytogenetic analysis of the ovarian tissue to reveal a 45,X/46,XX-type mosaicism,¹⁹ thus indicating the need for a gonadal biopsy in patients with a normal karyotype if no other causes for amenorrhea is determined.¹⁹

CONCLUSION

This study shows the possible cytogenetic abnormalities that present in women in a case of amenorrhea. Although most women presenting with amenorrhea have a normal karyotype, its testing remains vital for genetic counseling. Clinical examination of the patient and ultrasound reporting of the internal genital system hold its unique place in the management of a case of amenorrhea.

ACKNOWLEDGMENTS

The study team remains grateful to the Department of Genetics St. John’s Medical College Hospital, for their immense support. The study team also like to thank Ms Smita Bernadet Kujur (DHG/SJMC) and Ms Vaishnavi S (JSSAHER, Mysuru) for their unconditional support.

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