Chromosomal abnormalities and hormonal disorders of primary amenorrhea patients in Egypt

BACKGROUND:

Primary amenorrhea is defined as the absence of menstruation and secondary sexual characteristics in phenotypic women aged 14 years or older. Hormonal disorders are main causes of primary amenorrhea. Common hormonal cause of primary amenorrhea includes pituitary dysfunction and absent ovarian function. The aim of this study was to estimate the incidence and types of chromosomal abnormalities in patients with primary amenorrhea in Egypt.

MATERIALS AND METHODS:

Chromosomal analysis and hormonal assay were carried out on 223 patients with primary amenorrhea that were referred from different parts of Egypt to Cytogenetic laboratory of Genetic Unit, Children Hospital Mansoura University, from July 2008 to December 2010. FISH technique was carried out in some of cases to more evaluation.

RESULTS:

The frequency of chromosomal abnormalities was 46 (20.63%) in primary amenorrhea patients. The chromosomal abnormalities can be classified into four main types. (1) The numerical abnormalities of the X chromosome were detected in 23 (50 %). (2) Structural abnormalities of the X chromosome were detected in 11 (23.91%). (3) Mosaicism of X chromosome was found in 10 (21.74%). (4) Male karyotype 46, XY was presented in 2 (4.35%).

CONCLUSION:

The present study showed that karyotype and FISH are necessary to detect the causes of primary amenorrhea. This study also revealed the incidence of chromosomal abnormalities in women with primary amenorrhea in Egypt is similar to that reported in previous literatures.     

Chromatid association in acrocentric chromosomes of abnormal sexual development (ASD) cases

Concordant/discordant associations at chromatid level were compared and found significant (P less than 0.05) in females with primary amenorrhea. This probably suggested that the acrocentric association pattern in this group of ASD and infertility did not follow a random segregation in subsequent cell divisions and that the concordant acrocentric chromosomes have regularly established physical connections with one another, held together for several cell cycles. It could only be speculated that the association of acrocentric chromosome anomalies in some females with abnormal sex chromosomes are due to this reason. In the event that chromosome association has a bearing on chromosome aberrations, the non-random pattern of acrocentric association probably would increase the choice for translocation and non disjunction in the somatic cells in females with primary amenorrhea during ontogenesis.     

Idiopathic premature ovarian failure in 63 young women

BACKGROUND: Premature ovarian failure (POF) in adolescents is defined as primary or secondary amenorrhea associated with high follicle-stimulating hormone (FSH) levels. In normal 46,XX patients, its etiology is most often unknown. We have evaluated the clinical, hormonal and ovarian phenotypes in patients with a normal karyotype who were diagnosed with POF before the age of 18. METHODS: Sixty-three patients were included in this retrospective study. RESULTS: The mean patient age was 20.4 years. The patients presented with three clinical patterns: lack of pubertal development (n = 23), primary amenorrhea with interrupted puberty (n = 18), and secondary amenorrhea with normal puberty (n = 22). Ten patients had a familial history of POF and 6 presented with hypothyroidism. The FSH, estradiol and inhibin B levels were not statistically different in the three clinical groups. Fifty percent of the patients presented small ovaries (length <2 cm) at ultrasonography. The presence of follicles was found at histology in only 7 of the 27 patients who underwent an ovarian biopsy. CONCLUSION: 46,XX patients presenting with early POF rarely presented a specific, identifiable disorder. We discuss the clinical management and different diagnosis strategies to improve our current knowledge of this syndrome.     

The buccal smear: Reassessment of its usefulness.

Over a 7-year period 43 patients who underwent sex-chromatin and cytogenetic studies in the investigation of a disorder related to reproductive function were found to have abnormalities of the sex or autosomal chromosomes that could not have been detected by routine buccal smear. Therefore, testing for sex chromatin is of no value to the clinician, because full chromosome analysis must be performed irrespective of the findings from the buccal smear. However, the sex-chromatin test is an aid to the cytogeneticist in interpreting the chromosome analysis. In addition to those with amenorrhea and oligospermia or aspermia, persons with hypospadius and those to be treated with fertility drugs should undergo cytogenetic studies.     

Giemsa-11 technique elucidating three structurally altered nonfluorescent Y chromosomes: r (Y), idic (Yp), dir tan dup (Yp)

Three patients are presented in whom a structurally altered Y chromosome was finally diagnosed using Giemsa-11 technique. The first patient, a 19-year-old woman with primary amenorrhea and some features of Turner syndrome had ring (Y). The second patient, a 2-year-old boy with small stature and incomplete masculinization was found to have an isodicentric (Yp). In the third patient who was examined because of ambigous genitalia the chromosome abnormality, a nonfluorescent pseudodicentric (Y) was interpreted as a direct tandem duplication of the short arm, centromere, and a piece of the long arm, a rearrangement not described before. In each of the patients Q-, G-, and C-bandings failed to elucidate the kind of chromosome abnormality. Since clarification of a given Y structural rearrangement by cytogenetic methods cannot be avoided even in the era of molecular genetics, Giemsa-11 technique should be applied in the analysis of every dubious small sex chromosome.     

Identification of Y-chromosomal DNA in a Turner syndrome mosaic by polymerase chain reaction.

A 15.5-year-old female was referred for primary amenorrhea and slow development of secondary sex characteristics. The karyotype revealed 45,X/46,X,+mar (75%/25%). The small marker chromosome was C-band and Q-band negative. It appeared to be primarily centromeric with some light G-band staining material on either side. Females with Y-chromosomal material are at an increased risk for gonadal neoplasia and this patient was studied further to investigate the possibility that the marker was a deleted Y chromosome. Polymerase chain reaction (PCR) analysis of this patient's DNA revealed the presence of Y-chromosomal material presumably derived from the marker chromosome. These results indicate that the PCR technique, in conjunction with cytogenetic analysis, can identify possible Y-chromosomal material. This testing provides critical information necessary for correct medical followup of Turner syndrome mosaic patients.     

Late-replicating ring X-chromosomes identified by r-banding after BrdU pulse

Summary A detailed chromosome analysis was carried out in 3 patients presenting the mosaicism, 45,XO/46,Xr(X). In 2 patients, the r(X) chromosome was found to be latereplicating using R-banding techniques after a BrdU pulse administered 6 h before harvesting the leukocyte culture. The clinical symptomatology presents the great variability already observed in association with this karyotype. Only a short stature was common to the 3 patients. Case 1 is a 6-year-old girl with moderate mental retardation; case 2 is a 16-year-old girl with primary amenorrhea and absence of secondary sexual characteristics; case 3 is a 24-year-old woman whose puberty and normal sexual development occurred at age 17.     

Chromosome abnormalities in secondary pig oocytes matured in vitro

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30-48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8 %). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0 %) than in the groups matured for 30 and 36 h (9.0 %). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy (n=19-1) was identified in 22 cells (3.59 %) and a hyperhaploid (n=19+1) set of chromosomes was identified in 15 cells (2.45 %). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9 %. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.     

131例原发性闭经女性的细胞遗传学分析(含世界首报异常核型3例)

原发性闭经是一种原因复杂的疾病, 染色体异常则是发病的主要原因。通过对131例原发性闭经患者的外周血淋巴细胞染色体的G带核型分析, 发现其中83例为正常女性核型, 占63.36%; 各种异常核型48例,占36.64%, 其中包括3例世界首次报道的异常核型[46,X,t(X;1)(q22;p34); 46,X,t(X;5;6)(p11.2;q35;q16); 46,XX,t(4; 9)(q21;p22),t(6;10)(p25;q25),t(11;14)(q23;q32)]。另外, 将33例Turner’s综合征患者的主要异常体征及核型分布分别与Elsheikh等的报道进行比较, 发现矮身材、蹼颈、后发迹低和肘外翻的发生率与文献资料存在显著差异, 说明东西方Turner’s综合征患者临床体征的表现可能存在差异。通过对2例X-常染色体易位携带者的分析, 认为Xp11.2和Xq22区域可能与原发性闭经有关。     

Lipid and lipoprotein profile in menopausal transition. Effects of hormones, age and fat distribution.

The behavior of lipoproteins during the menopausal transition and their relationship with sex hormones and body fat distribution is still unclear. Our aim was to evaluate atherogenic IDL, LDL, Lp(a) and antiatherogenic HDL lipoproteins in four groups of women: premenopausal (n = 20), menopausal transition women with menstrual bleeding (n = 31), menopausal transition women with 3 to 6 months amenorrhea (n = 36), and postmenopausal women (n = 30). We also measured their FSH, LH and estradiol levels along with BMI and waist circumference. Menopausal transition and postmenopausal women showed higher values of waist circumference (p < 0.0032), LDL-cholesterol (p < 0.002), IDL-cholesterol (p < 0.002) and apoprotein B (p < 0.0001) than premenopausal women. Total-cholesterol (p < 0.0001), triglycerides (p < 0.004), IDL-cholesterol and Lp(a) were higher in menopausal transition women with amenorrhea and in postmenopausal women in comparison with premenopausal women. After adjustment according to age and waist circumference, multiple regression analysis showed the increase in total-cholesterol and LDL-cholesterol to be linearly associated to menopausal status and estradiol concentration, whereas Lp(a) was only related to menopausal status. Age was found to be an independent variable in relation to apoprotein B concentration changes. The effect of menopausal status on TG levels did not remain in the model when age, waist and BMI were included (beta = 0.05, p = 0.356). HDL-cholesterol levels were the same in all the groups. Menopause, age and the increase in abdominal fat distribution were three independent and significant factors impairing lipoprotein profiles from the beginning of the menopausal transition.     

A karyological study of the spirurid nematode Mastophorus muris (Nematoda: Spirocercidae)

The karyotype of Mastophorus muris (Gmelin, 1790) comprises four pairs of small autosomal chromosomes and two larger sex X chromosomes in females or one X chromosome in males (2n = 8 + XX/XO). All pairs of chromosomes possess rather uniform morphology without distinct primary or secondary constrictions. No heterochromatin bands were found by C-banding analysis. The absolute chromosome length ranges from 4.02 to 2.24 microns. The mean total length of the haploid complement is 14.34 microns. The course of gametogenesis represents a typical pattern common in the order Spirurida. The recently available karyotypes of spirurid nematodes have been reviewed.     

Chromosome elimination and sex determination in springtails (Insecta, Collembola).

A post-zygotic mechanism of sex determination is described in the two symphypleonans Dicyrtomina ornata (Nicolet) and Ptenothrix italica Dallai. The process consists of the loss of two sex chromosomes from the male embryo. At the end of the first meiotic division of spermatogenesis, a second chromosome elimination occurs, allowing half the secondary spermatocytes, later transformed into spermatids, to receive a complete haploid set of chromosomes. The secondary spermatocytes, which receive an incomplete set of chromosomes, degenerate. Males of the two collembolan species, therefore, produce a reduced number (50%) of spermatozoa. Females of D. ornata have 2n = 12 and males 2n = 10 chromosomes; females of P. italica have 2n = 14 and males 2n = 12 chromosomes. In both species, oogenesis proceeds normally and chromosomes pair and form chiasmata in meiotic prophase. The adaptive significance of this post-zygotic mechanism of sex determination is discussed. The mechanism seems to be a characteristic feature of the suborder Symphypleona. The neanurid Arthropleona Anurida maritima (Guérin), which was studied for comparative analysis, has 2n = 8 chromosomes and normal spermatogenesis producing haploid nuclei with four chromosomes. J. Exp. Zool. (Mol. Dev. Evol.) 285:215-225, 1999.     

Chromosomal studies of male infertility

Prometaphase and metaphase chromosome analyses performed on 70 consecutive men with primary infertility (for a period of at least 2 years) revealed 8 (11.42%) men with some kind of chromosomal abnormality. The highest frequency of abnormal karyotypes (10%) was found among patients with azpospennia and the most frequent anomaly was 47, XXY chromosomal constitution, found in 6 (8.57%) patients. All the chromosomal aberrations found in this study was sex chromosomal type and we did not find any autosomal aberration. All patients with numerical chromosomal anomalies had azoospermia. The incidence of structural aberration in our study was 1.42%. Fifteen patients had different chromosomal variants (21.38%). We suggest that men with azoospermia should be considered for cytogenetic investigation and we report that "variants of the Y chromosome" have no influence on the sperm count (million/ml) and fertility of men.     

Impact of electro-acupuncture and physical exercise on hyperandrogenism and oligo/amenorrhea in women with polycystic ovary syndrome: a randomized controlled trial

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism, oligo/amenorrhea, and polycystic ovaries. We aimed to determine whether low-frequency electro-acupuncture (EA) would decrease hyperandrogenism and improve oligo/amenorrhea more effectively than physical exercise or no intervention. We randomized 84 women with PCOS, aged 18-37 yr, to 16 wk of low-frequency EA, physical exercise, or no intervention. The primary outcome measure changes in the concentration of total testosterone (T) at week 16 determined by gas and liquid chromatography-mass spectrometry was analyzed by intention to treat. Secondary outcome measures were changes in menstrual frequency; concentrations of androgens, estrogens, androgen precursors, and glucuronidated androgen metabolites; and acne and hirsutism. Outcomes were assessed at baseline, after 16 wk of intervention, and after a 16-wk follow-up. After 16 wk of intervention, circulating T decreased by -25%, androsterone glucuronide by -30%, and androstane-3α,17β-diol-3-glucuronide by -28% in the EA group (P = 0.038, 0.030, and 0.047, respectively vs. exercise); menstrual frequency increased to 0.69/month from 0.28 at baseline in the EA group (P = 0.018 vs. exercise). After the 16-wk follow-up, the acne score decreased by -32% in the EA group (P = 0.006 vs. exercise). Both EA and exercise improved menstrual frequency and decreased the levels of several sex steroids at week 16 and at the 16-wk follow-up compared with no intervention. Low-frequency EA and physical exercise improved hyperandrogenism and menstrual frequency more effectively than no intervention in women with PCOS. Low-frequency EA was superior to physical exercise and may be useful for treating hyperandrogenism and oligo/amenorrhea.     

Synergism between psychosocial and metabolic stressors: impact on reproductive function in cynomolgus monkeys

The role of energy imbalance versus psychosocial stress in the pathogenesis of female reproductive dysfunction characterized by anovulation and amenorrhea remains controversial. In women, functional hypothalamic amenorrhea can develop in the absence of significant weight loss, excessive exercise, or profound psychosocial disruption. We posited, therefore, that commonplace, seemingly minor stressors that alone would have minimal impact upon reproductive function might interact synergistically such that combinations of stressors would cause a greater impairment of the reproductive axis than any single stressor alone. We then developed a monkey model to test this hypothesis. Adult female cynomolgus monkeys with normal menstrual cycles were randomized into three experimental groups and studied over four menstrual cycles. The groups were: low-level psychosocial stress (i.e., moving to a new housing environment; Move, n = 8), moderate energy imbalance (Exercise + Diet, n = 9); and all stressors in combination (Move + Exercise + Diet, n = 10). Food intake, body weight, menstrual cyclicity, and reproductive hormones were assessed for two control menstrual cycles followed by two experimental cycles during which the monkeys experienced the stressors. Abnormal cycles were considered to be abnormally long or anovulatory cycles. Few abnormal cycles occurred in the Move group (1 of 8 monkeys) and in the Exercise + Diet group (1 of 9 monkeys). In contrast, 7 of 10 monkeys in the Move + Exercise + Diet group displayed at least one abnormal cycle (chi(2) = 9.61, P = 0.008). These findings suggest that infertility due to hypothalamic hypogonadism can result from the combination of commonplace, seemingly minor stressors that often escape clinical attention.     

A comparative study of the estrogenic potential of two synthetic estrogens (mestranol and ethinylestradiol)

The purpose of this study was to establish the relative estrogenicity of ethinylestradiol and mestranol and to establish .05 and .1 mg of mestranol as standard reference preparations for the comparative study of other compounds. Patients were selected from a large group of primary amenorrhea, secondary amenorrhea, and postmenopausal women. All required hormonal replacement therapy. Patients with chromosomal abnormalities or those with diminished or abnormal response were excluded. Vaginal cytology was used as the main method for the assessment of the response. Before treatment all had either an atrophic pattern (Maturation Index = 50-50-0) or a hypertrophic pattern (MI=5-95-0) with a mean value of 10-87-3 for patients with primary amenorrhea and 18-79-3 in the other 2 groups. Patients with a proliferative intermediate cell pattern and postmenopausal women thought to have continuing endogenous estrogen activity were omitted. Ethinylestradiol or mestranol were given orally in doses of .025, .05, and .1 mg for a period of 10 days during which frequent vaginal smears were made. When cell patterns showed extremely marked cytolysis of the glycogen-containing intermediate cells and a low percentage of karyopyknotic superficial cells these patients were excluded. 40 women were finally included. The effects of estrogen on the vaginal epithelium in all 40 women after 10 days therapy were quite uniform. In those cases showing delayed regression of estrogenic effects 4 mg of chlormadinone acetate was given daily for 10 days. These patients then showed a reduction in the superficial cell percentage accompanied by a shift to the left in the maturation index such as 0-40-60 before to 0-95-5 after treatment. A few women had an insignificant reduction in the superficial cell percentages. Changes in the percentages of the intermediate type of cells were not significant. The regression was considered to be complete when the smear contained mainly small intermediate cells and parabasal cells with numerous leukocytes and some necrotic debris. Both estrogen preparations produced the same vaginal response at .05 mg doses. Tablets of .05 and .1 mg mestranol were chosen for further study. The increase in the superficial cell index to 30 (SD plus or minus 18.5) following treatment with .05 mg and the increase to 54.1 (SD plus or minus 26.7) following .1 mg have been adopted as standard vaginal responses. The term estrogenic effect used in vaginal cytology is taken to mean the local vaginal effect induced by estrogens.     

Cytogenetic investigations in 150 cases with complaints of sterility or primary amenorrhea

Summary Cytogenetic investigations were carried out on 150 individuals. Out of these 107 were females and 43 males. Eighty seven of the above (43 males and 44 females) had been referred for sterility. Sixty three patients had primary amenorrhea and had been referred directly to this laboratory by clinicians, having been suspected of genetic abnormalities. Twenty-two cases (14.7%) involved in this study showed chromosomal abnormalities and seven cases (4.7%) showed chromosomal polymorphism. Of the 107 females (44 sterile and 63 with primary amenorrhea), 11 (10.2%) showed numerical or structural sex chromosomal abnormalities. Five patients (4.67%) showed chromosomal polymorphism involving the paracentromeric and centromeric regions of chromosomes 1 and 9, double satellites, and giant satellites. Of the 43 males, 11 (25.59%) showed numerical and structural abnormalities. Ten cases were anomalies involving the sex chromosomes. One case of a triple autosomal translocation in an otherwise phenotypically normal azoospermic male was of particular interest. Two cases (4.65%) showed double satellites of the acrocentrics.     

Chromosomal studies in infertile men

Prometaphase and metaphase chromosome analyses performed on 70 consecutive men with primary infertility (for a period of at least 2 years) revealed 8 (11.42%) men with some kind of chromosomal abnormality. The highest frequency of abnormal karyotypes (10%) was found among patients with azoospermia and the most frequent anomaly was 47, XXY chromosomal constitution, found in 6 (8.57%) patients. All the chromosomal aberrations found in this study, was sex chromosomal type and we did not find any autosomal aberration. All patients with numerical chromosomal anomalies had azoospermia. The incidence of structural aberration in our study was 1.42%. 15 patients had different chromosomal variants (21.38%). We suggest that men with azoospermia should be considered for cytogenetic investigation and we report that "variants of the Y chromosome" have no influence on the sperm count (Million/ml) and fertility of men.     

Chromosomal Studies in Infertile Men

Prometaphase and metaphase chromosome analyses performed on 70 consecutive men with primary infertility (for a period of at least 2 years) revealed 8 (11.42%) men with some kind of chromosomal abnormality. The highest frequency of abnormal karyotypes (10%) was found among patients with azoospermia and the most frequent anomaly was 47, XXY chromosomal constitution, found in 6 (8.57%) patients. All the chromosomal aberrations found in this study was sex chromosomal type and we did not find any autosomal aberration. All patients with numerical chromosomal anomalies had azoospermia. The incidence of structural aberration in our study was 1.42%. Fifteen patients had different chromosomal variants (21.38%). We suggest that men with azoospermia should be considered for cytogenetic investigation and we report that variants of the Y chromosome have no influence on the sperm count (million/ml) and fertility of men.