Chromosomal abnormalities & oxidative stress in women with premature ovarian failure (POF).

A total of 75 cases (19.27±1.72) yr and 50 female controls (18.98±1.54) yr were analysed. Eighteen chromosome variations were identified in POF patients (24%) by conventional cytogenetic analysis of 50 G-banded well spread metaphases. X-chromosome variations were found in 21 (28%) cases. 45,XO/46,XX mosaicisms or other complex mosaicisms involving X chromosome were also observed in patients. 45,XO chromosome complement was found in 11 (14.66%) cases whereas 8 (10.66%) were found to be Turner mosaic. Three cases (4%) were found to harbour autosomal chromosome abnormality. Chromosomal complement of patients with their clinical phenotype is shown in Table I and Fig . Clinical phenotype of POF patients with their chromosome complement Karyotype distribution of POF patients. ROS levels were assessed in all cytogenetically normal cases and controls. Overall median ROS range was found to be significantly higher ( P <0.05) in POF patients 50480 (120, 132966) RLU/min when compared to controls [340 (120, 5094) RLU/min]. However, 50 per cent of the POF patients had very high ROS levels, 20 per cent had medium elevation and 30 per cent were found to have normal values comparable to controls. The mean values of the FSH, age and height of the patients and controls are shown in Table II . The Table describes the values according to the karyotype distribution of the patients. A significant difference ( P <0.01) in mean FSH level was found between Turner and other POF patients. Whereas patients (other than 45,XO) harbouring X-chromosome abnormalities had higher ( P <0.001) FSH levels than patients having normal karyotype. The mean height was found to be significantly lower ( P <0.001) in POF patients as compared to controls. 45,XO patients showed significantly ( P <0.001) low mean height as compared to other POF patients. No family showing blepharophimosis or with conditions related to cancer cluster nor families presenting cases having fragile X syndrome or other forms of mental retardation were found. Mean values of FSH, age and height of the POF patients

Seventy five consecutive women with idiopathic POF were selected over a 2 years period from January 2008 to July 2010, and 50 women who had two children (enrolled in family planning OPD and had regular menses with no history of infertility or autoimmune disease) were enrolled as controls. The criterion considered for inclusion as a POF patient was absence of menstruation along with a FSH concentration of >40mIU/ml. Physical and clinical examination was done to identify secondary sexual characteristics or any syndromic features. All the patients having primary amenorrhea were included in the study. A complete gynaecological, occupational and medical history was taken from each patient. Patients having secondary amenorrhea including pregnancy were excluded. The patients were referred from Department of Obstetrics and Gynaecology to Laboratory for Molecular Reproduction and Genetics Department of Anatomy, All India Institute of Medical Sciences, New Delhi, for genetic analysis. The study was approved by institutional review board (IRB#IRB00006862; All India Institute of Medical Sciences, Delhi, India). The study protocol was approved by institutional review board, and all participants gave their written informed consent. All cases which were found be cytogenetically normal were assessed for ROS levels. Chromosome preparation 19 : In POF patients, chromosome analysis was done to identify any numerical or structural chromosomal aberrations. Lymphocyte cultures were set up and chromosomes were analyzed by G banding. Heparinized blood (5 ml) was drawn and kept in an upright position at 37°C for 30 min. This helps in the separation of plasma from red blood cells. The plasma and the settled lymphocyte (PLS, plasma lymphocyte suspension) in buffy coat was tapped gently and mixed together. The needle was bent and 0.5 ml of PLS was transferred into a sterile culture vial containing 5 ml of media RPMI-1640 and 0.2 ml phytohaemagglutinin (PHA; Gibco). The cultures were incubated for 72 h at 37°C. After 70 h of incubation, 0.1 ml (0.2%) of colcemid (Gibco) was added to the cultures. At 72 h the samples were washed for removing colcemid and centrifuged at 67 g for 10 min. The supernatant was discarded and freshly prepared pre-warmed hypotonic solution (0.56% KCl) was added and incubated for 20-25 min at 37°C. The cell suspension was centrifuged again and after discarding the supernatant, freshly prepared chilled carnoy's fixative (methanol:acetic acid/3:1) was added to the cell pellet slowly. At least three changes of fixative were given till the pellet became pale. Two drops of cell suspension were dropped from a height of 30-100 cm on a clean wet slide. G-banding 20 : Giemsa staining of chromosome preparation after proteolytic enzyme treatment revealed G-banding. The 3 days old matured unstained chromosome preparations were flooded with 0.25 per cent Trypsin for 10-15 sec, the slides were rinsed in phosphate buffer saline p H 7.4. The slides were stained in 2 per cent Gimesa stain for 5-7 min and were washed in distilled water. Metaphases were analyzed using cytovision software (Applied Imaging, USA) (Zeiss Microscope, Olympus, Japan) and were classified according to ISCN 2005 21 . At least 20 metaphases in each patient were analyzed and karyotyped. In case of abnormal karyotype more metaphases (~100) were examined. ROS measurement : One ml of fresh blood was centrifuged at 300 g for 7 min and the plasma was removed and transferred into a microcentrifuge tube. To 400 μl of the plasma 10 μl of 5M luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione; Sigma, USA), was added to the mixture and served as a probe. Levels of ROS were assessed by measuring the luminol-dependant chemiluminescence with the luminometer (Sirius, Berthold) in the integrated mode for 15 min. The results were expressed as median (minimum, maximum) relative light unit/minute (RLU/min) per 400 μl of blood plasma. Statistical analysis : The significance difference in ROS levels between patients and controls was found by applying Mann-Whitney test. The values were expressed as median (minimum, maximum) range. FSH levels, age and height were compared by one-way ANOVA. P <0.005 was considered as significant. The statistical analyses were performed using Stata 9.0 version (StataCorp LP, TX, USA).

In this study chromosomal abnormalities were found in 24 per cent of POF patients. The frequency of sex chromosome abnormalities was found to be 28 per cent, similar to that reported 3 8 9 . The involvement of the X chromosome may not be limited to POF but may influence the broader spectrum of menopausal age 22 . In this study, X chromosome structural anomalies were found in 8 per cent of the POF patients (three cases of Xq deletions, one case of ring X chromosome and two cases of iso X-chromosome). One of three patient showed deletion in Xq13.3-q21.1 which involved the critical region. Deletions involving Xq region are known to cause amenorrhoea between the age of 16 and 21 yr because of haplo-insufficiency of genes resulting in accelerated apoptosis of germ cells 8 11 . In the presence of only one X chromosome as in Turner's syndrome, ovarian follicles degenerate by birth due to accelerated atresia of germ cells which may be the result of a lack of diploid dosage of one or more vital genes, both alleles of which are active in oogenesis 12 . Gonadal dysfunction due to chromosomal abnormalities can occur for several reasons commonly linked to gene expression being disrupted by the rearrangement 14 . Our findings related to breakpoints falling within a region of repetitive elements in Xq are in favour of alterations in chromosome meiotic pairing or position effect. Some microdeletions may be escaped by routine cytogenetic analysis. For detecting these microdeltions comparative genome hybridization (CGH) would be ideal method. Two cases of dicentric isochromosome X, dic (Xp-), with premature ovarian failure have been reported 23 . Turner's syndrome associated with X ring chromosome, r(X) is rare and there has been no report of pregnancy in Turner's syndrome with 45,X/46,Xr(X)/46,XX mosaicism except in one study 24 . We identified a patient with 46,Xr(X)/46,XX,i(2q)/46,X mosaicism. The physical and clinical characteristics of the patient were similar to Turner's syndrome. Very few cases with r(X) chromosome have been reported in the literature 25 and as it is present with Turner cell line the correlation of r(X) and ovarian phenotype in patient could be suggestive only. Chromosome 2 contains the gene for FSH and leutinizing hormone (LH) receptors and mutation in FSH receptor gene in a Finnish family presented several cases of primary amenorrhoea 26 . Chromosome abnormalities involving chromosomes 9 and 2 have not been reported in POF, though inv(9) has been previously reported in female infertility and other congenital anomalies 26 . The mechanisms of origin of inversions 9 are highly complex. It was not possible to study the karyotype of family members of these patients’ pedigrees in order to exclude a polymorphic inheritance pattern of these chromosome variants. In this study ROS levels were found to be significantly higher in cytogenetically normal POF cases as compared to controls. Supraphysiological free radicals damage both nuclear and mtDNA and induce mutations, and result in decreased ATP production 18 28 29 . The pathological role of ROS has been demonstrated in various female reproductive disorders such as endometriosis, polycystic ovarian disease, spontaneous abortions, hydatidiform mole and pre-eclampsia 30 . Oxidative stress induces accumulation of lipid peroxide products (MDA, acetaldehyde, etc .) and may also alter protein structure which may change its function and cause chemotaxis of inflammatory cytokines. Though physiological free radicals promote oogenesis and follicle formation, excess ROS levels adversely affects female reproductive events 30 . In a study from our laboratory we found increased number of nucleotide alterations in mtDNA and raised ROS levels in POF cases compared to age matched controls 18 . Mitochondrial mutations adversely affect ATP production but produce high ROS levels 18 28 which further induce nucleotide alterations. Low ATP levels may impair oogenesis and result in accelerated apoptosis of germ cells. The mammalian oocyte and embryo are very sensitive to oxidative stress and though physiological levels subserve several important functions, high levels impair oocyte maturation 31 . It has been reported that oocytes maintain a delicate balance of pro and anti-oxidants to minimize oxidative stress 28 . High ROS levels may thus induce mtDNA damage and results in nuclear DNA fragmentation and increased apoptosis 32 . Accelerated apoptosis may result in premature cessation of ovarian function. Women with POF have a 5 per cent chance of spontaneous conception after diagnosis, as in some cases hormone levels fluctuate and return to biochemical normality 5 . The diagnosis of POF may have a deleterious psychological impact and the emotional importance of the condition is often underestimated. In case where POF can be diagnosed at an early age the patient can be counselled for ova cryopreservation to be used later for ART. Cytogenetic analysis should be considered for women presenting with unexplained POF, even when there are no other clinical features suggestive of chromosomal abnormality. In clinical cytogenetics, the precise identification of a chromosomal abnormality is a key factor when considering genotype-phenotype correlation and with advancement of new high resolution techniques like CGH microdeletions can also be detected. In conclusion, we identified 28 per cent cases with X chromosome abnormalities in a small population of idiopathic POF patients and 50 per cent cytogenetically normal cases had raised ROS levels. Women having POF before the age of 30 should undergo cytogenetic and ROS level analysis. Since a large number of idiopathic cases have raised ROS levels, such cases if diagnosed at an early stage, can be administered antioxidants which may prevent mt and nuclear DNA damage and can also delay or slow down germ cell apoptosis. Thus, cytogenetic and oxidative stress analyses are important tools in the evaluation of idiopathic cases of POF.