Molecular and cytogenetic evaluation of Y chromosome in
Transkript
Molecular and cytogenetic evaluation of Y chromosome in
Journal of Cell and Molecular Biology 7(2) & 8(1): 45-52, 2010 Haliç University, Printed in Turkey. http://jcmb.halic.edu.tr Research Article Molecular and cytogenetic evaluation of Y chromosome in spontaneous abortion cases Gülşah KOÇ1, Korkut ULUCAN*,1, Deniz KIRAÇ2, Deniz ERGEÇ1, Tufan TARCAN3, A. İlter GÜNEY1. 1 Marmara University, Faculty of Medicine, Department of Medical Genetics, Istanbul, Turkey. Yeditepe University, Faculty of Medicine, Department of Biochemistry, Istanbul Turkey. 3 Marmara University, Faculty of Medicine, Department of Urology, Istanbul, Turkey. (* author for correspondence; korkutulucan@hotmail.com) 2 Received: 21 April 2010; Accepted: 05 May 2010 Abstract Infertility is defined as not being able to get pregnant despite having frequent, unprotected sex for at least a year. Several conditions contribute to infertility and 50% is considered to be caused by a male-related factor. Spontaneous abortion (SAB) is noninduced embryonic or fetal death or passage of products of conception before the 20th week of pregnancy and is the most common complication of early pregnancy. SAB can occur by teratogenic factors, advanced maternal age, genetic factors such as Y chromosome microdeletions and chromosomal anomalies. In order to investigate the etiology of recurrent pregnancy loss (RPL) and to develop an appropriate therapeutic strategy, it is necessary to ascertain the molecular and cytogenetic basis of these defects. In this study, we aimed to reveal the relations between male infertility, Y chromosome microdeletions and SAB. Thirty couples with a spontaneous abortion history and thirty fertile men were recruited to the study. All the women were 46, XX and men were 46, XY. We couldn’t detect any Y chromosome microdeletion that could be the reason for SAB. In order to evaluate effect of chromosome anomalies and Y chromosome microdeletions on SAB, further studies with increased number of cases and controls need to be carried on. Keywords: Infertility, spontaneous abortion, Y chromosome microdeletions. Spontan düşük vakalarında Y kromozomunun moleküler ve sitogenetik incelemesi Özet Çiftlerin çocuk sahibi olma isteklerine ve düzenli cinsel ilişkiye rağmen, bir yıl içerisinde gebelik elde edilmemesine infertilite (kısırlık) adı verilmektedir. İnfertiliteye etki eden birçok faktör bulunmaktadır ve bunların %50’sinde etken erkek infertilitesidir. Gebeliğin ilk 20 haftası içinde, dışarıdan herhangi bir müdahale olmadan, doğal nedenlerle, embriyo veya fetus ve eklerinin tamamının veya bir kısmının uterus kavitesi dışına atılması olayına spontan düşük (abortus) denilmektedir ve gebeliğin erken döneminde en çok gözlenen komplikasyondur. Spontan düşükler, teratojenik faktörler, ileri anne yaşı gibi nedenlerin yanında, Y kromozomu mikrodelesyonları ve kromozomal anomaliler gibi genetik faktörlere bağlı olarak da oluşabilmektedir. Tekrarlayan gebelik kayıplarının etyolojisini belirlemek ve uygun bir tedavi yöntemi geliştirmek için bu defektlerin moleküler ve sitogenetik temellerinin incelenmesi gerekmektedir. Bu çalışmada, erkek infertilitesi, Y kromozom mikrodelesyonları ve spontan düşükler arasındaki ilişkinin ortaya çıkarılması amaçlanmıştır. Spontan düşük hikayesi bulunan 30 çift ve fertil 30 erkek çalışmaya dahil edilmiştir. Çalışmaya dahil olan bireylerin kromozom analizi sonuçlarına göre, tüm kadınlar 46,XX ve erkekler ise 46,XY‘dir. Çalışmamızda spontan düşüklere neden olabilecek herhangi bir Y kromozom mikrodelesyonu belirlenememiştir. Kromozom anomalilerinin ve Y kromozomu mikrodelesyonlarının spontan düşükler üzerindeki etkisinin değerlendirilebilmesi için vaka ve kontrol sayılarının arttırılarak başka çalışmalar yapılması gerekmektedir. Anahtar Sözcükler: İnfertilite, spontan düşük, Y kromozom mikrodelesyonu. 46 Gülşah Koç et. al. Introduction Infertility is the inability of being pregnant after one year of unprotected sexual intercourse. Infertility comprises up to 15% of couples of reproductive age in which 50% is caused by a male factor (Noordam and Repping, 2006). Several factors contribute to male infertility, such as gene defects, hormonal milieu, chromosomal aberrations and genital infections (Stipoljev et al., 2006). Genetic factors are considered to affect almost 30% of severe male infertility cases (Noordam and Repping, 2006). The diagnosis of male infertility include anamnesis, physical examination, semen analysis, hormonal screening and genetic factors of somatic cells (Stipoljev et al., 2006). Spontaneous abortion (SAB) is the expulsion of an embryo or fetus due to accidental trauma or natural causes before approximately 22nd week of gestation. It effects up to 15% clinically recognized pregnancies and considered to be the most common adverse outcome of pregnancy. Although several studies tried to explain the etiology of SAB, the results are still controversial. Beside the teratogenic factors and advanced maternal age, genetic factors such as Y chromosome microdeletions and chromosomal anomalies are considered to be the main reason of SAB (Dewan et al., 2006; Pryor et al., 1997). Y chromosome is essential not only for human sex determination but also for maintenance of sex cells and sex cell development. Y chromosome (Yq) microdeletions represent the most frequent molecular genetic cause of severe infertility, observed with a prevalence of 10-15% in nonobstructive azoospermia and severe oligozoospermia (Sinclair et al.,1990). The regions responsible for male infertility of Y chromosome are located on the long arm of chromosome and are termed as AZFa, AZFb, AZFc (AZF: Azoospermia Factor) ( Burgoyne, 1998) (Stouffs et al., 2009). The AZFa locus is located on proximal Yq11 (Yq11.21), while AZFb and AZFc are located on distal Yq11 (Yq11.23). These AZF genes code RNA binding proteins and may be involved in the regulation of gene expression, RNA metabolism, RNA packaging and RNA transportation from nucleus to cytoplasm (Li et al., 2008). Deletions of these regions result in spermatogenic arrest and are associated with oligozoospermia, azoospermia and also with a extended testis histological profile range from Sertoli cell only (SCO), maturation arrest and hypospermatogenesis (Vollrath, 1992) (Vogt et al., 1996) (Briton-Jones and Haines, 2000). The prevalence of the Y chromosome microdeletions in the proximal AZFc region was found higher in men from recurrent pregnancy loss (RPL) couples than from fertile or infertile couples. Although these patients are from a tertiary referral center that may not reflect the population informations, one may consider proximal AZFc region detecting in the evaluation of RPL couples when all other tests fail to reveal the etiology (Dewan et al., 2006). Before performing a molecular test, cytogenetic analysis is necessary for an accurate approach to elucidate the causes of spontaneous abortion. Chromosomal anomalies which may cause male infertility can be determined by cytogenetic techniques. It is also known that approximately 50% of recurrent spontaneous abortions in the first trimester is caused by chromosomal anomalies. Besides these, recent data show that Y chromosome microdeletions can also be a major factor in these cases. These findings suggest a potential relation between RPL and microdeletions in AZF regions. In order to investigate the etiology of RPL and to develop an appropriate therapeutic strategy, it is necessary to ascertain the molecular and cytogenetic basis of these defects. So in this study, we aimed to reveal the relations between male infertility, Y chromosome microdeletions and recurrent spontaneous abortions. Material and methods Patient and Control Groups Thirty couples that applied to Marmara University, Department of Urology and Kartal Education and Research Hospital with a spontaneous abortion history were recruited to the study. Thirty fertile men, at least having one child, were examined as the control group. Written informed consent was taken from all cases. Chromosome Analyses from Peripheral Blood Cell Culture Lymphocytes from 400 µl peripheral blood were cultured for 72 hours at 37ºC culture medium containing 8.5 ml RPMI, 1.5 ml fetal bovine serum, Y chromosome microdeletions in spontaneous abortions 47 200 µl L-Glutamin, 20 µl penicillin- streptomycin and 200 µl phytohaemagglutinin. After incubation at 37ºC for 72 hours, 200 µl Colchicine was added to arrest the cells at metaphase. Following an additional incubation at 37ºC for 30 minutes and centrifugation at 20ºC for 8 min. at 1500 rpm the supernatant was removed. The pellet was resuspended with up to 10 ml hypotonic solution (0.4% KCl solution) vortexed immediately. All the samples were kept at 37ºC for 20 minutes and again centrifuged at the same condition. After removing supernatant from the samples, the pellet which contains cells at metaphase, was homogenised. Fixative solution (methanol and acetic acid mixed with 3:1 ratio) was added and the tubes were vortexed for the fixation of chromosomes. Then samples were centrifuged after adding up to 5 ml of fixative solution. Supernatant was discarded from the samples and fresh fixative solution was added to the tubes. This procedure was repeated until the samples were clarified. According to the cell density, up to 0.5 ml fixative solution was added to the samples. Then samples were homogenized and cells were lied onto slide glasses, which were kept at 4ºC in distilled water till they are used. After spreading the cells on the slides, the samples were dried at room temperature and kept overnight at 60ºC. Karyotyping GTG (Giemsa-Trypsin) banding technique was performed. When the banding of the chromosomes was not successful, the protocol was repeated. After staining, at least 20 metaphase plaques were analysed for each sample (Figure 1). Detection of Y chromosome microdeletions DNA isolation from blood DNA was extracted from 200 µl peripheral blood by using High Pure PCR Template Preparation Kit (Roche-Germany) according to the manufacturer’s protocol. Multiplex polymerase (multiplex PCR) chain reaction For detection of Y chromosome microdeletions, isolated DNA was amplified by multiplex PCR. AB ANALITICA–The AZF Extension Kit, which is recommended by European Andrology Association was used in multiplex PCR. By using this kit, 13 different regions could be investigated at the same time by performing 3 multiplex PCRs for each sample. Three primer sets, each containing primers that is unique to ZFX/Y locus which also exist in X chromosome are shown in Table 1. Table 1. Primers used for multiplex PCR and the length of amplicons. MIX1 Amlicon length (bp) MIX2 Amlicon length (bp) MIX3 Amlicon length (bp) ZFX/Y 495 ZFX/Y 495 DBY 689 SRY 472 SRY 472 ZFX/Y 495 sY 254 380 sY 95 303 SRY 472 sY 86 320 sY 117 262 sY 84 326 sY 127 274 sY 125 200 sY 134 301 sY 255 120 DFFRY 155 48 Gülşah Koç et. al. In addition to the mixtures which are found in the AZF Extension Kit, 0.3µl Taq DNA polymerase and 8µl DNA sample were added to each tube during multiplex PCR. The conditions of PCR amplification were as follows: a denaturation step at 94˚C for 5 min followed by 35 cycles at 94˚C for 1 min, 60˚C for 1 min, 72˚C for 1 min and a final extension at 72˚C for 7 min and stop at 4˚C. After multiplex PCR, products were electrophoresed on 2% agarose gel. After performing lymphocyte cell culture, metaphase plaques were analyzed for the detection of karyotypes of patient and control groups. According to karyotype analyses, all the males and females were found as 46, XY and 46, XX respectively in the patient group, whereas all the males were found as 46, XY in the control group (Figure 1). Results After multiplex PCR, PCR products were examined by electrophoresis on 2% agarose gel. Y chromosome microdeletions were not found in patient and control groups. Karyotyping Figure 1. Karyotype analyses of a male (46, XY) patient. Detection of Y chromosome microdeletions Y chromosome microdeletions in spontaneous abortions 49 Figure 2. Multiplex PCR analyses of Y chromosome microdeletions (M: 50 bp ladder (Fermentas, Germany); Mix1a, Mix1b and Mix1c: 3 sets of PCR reactions that amplify different loci on Y chromosome for sample a; Mix1b, Mix2b, Mix3b for sample b; boxes indicate the region and the length of the amplicons. Discussion Chromosomal abnormalities, including translocations and deletions, are higher in infertile men and are recognized as one of the main causes of spontaneous abortions with an estimated frequency of 50–70% (Svetlana et al., 2005) In couples experiencing RPL, the incidence of chromosomal translocations is higher than the incidence present in newborn series (De Braekeleer and Dao 1991). There is also evidence which indicates that the presence of translocations changes the spermatogenic process. It has been found that the incidence of reciprocal translocation carriers is seven times more than in newborn series. As a general rule reciprocal translocation carriers produce more unbalanced sperm than normal or balanced sperm. The proportion of unbalanced forms depends on the characteristics of the reorganization. Also deletions which remove Y chromosomal genes required for spermatogenesis may effect infertility and susceptibility of RPL (Byrne and Ward, 1994) (Simpson, 1981). As the severity of the spermatogenic defect increases, the frequency of the microdeletions also increases. In this study, primarily, cytogenetic evaluation was performed from peripheral blood samples of the couples in spontaneous abortion cases. 30 couples who had a spontaneous abortion history were karyotyped to detect the chromosome anomalies. According to karyotype analyses, all the women and men were found to be 46, XX and 46, XY, respectively. In our study we couldn’t detect any numerical and structural chromosome anomalies that can be detected by karyotype analyses. Other genetic abnormalities such as Y chromosome microdeletions may effect spermatogenesis, fertilization and post-zygotic metabolism and may influence male infertility and RPL. 50 Gülşah Koç et. al. So we used multiplex PCR for the detection of microdeletions on the long arm of the Y chromosome. In this study, “AB ANALITICA–The AZF Extension Kit” used for the analysis of microdeletions rather than AZF-MX Extension kit. Diagnostic sensitivity is considered to be the capacity of the device to correctly identify the deleted samples with reference to AZF locus under investigation. The results obtained from an experimental investigation show that the diagnostic sensitivity of the system is 100%. The kit is in premix format as all the reagents for the amplification are pre-mixed and aliquoted in single dose tubes in which only additional Taq polymerase and the extracted DNA should be added. This premix format allows the reduction of the manipulation in preamplification steps, with considerable time saving for the operator, the repeated freezing/thawing of reagents (that could alter the products’ performances) is avoided and, above all, this form minimizes the risk of sample contamination and the risk of false positive results. The amplified regions of the Y chromosome are not polymorphic and are well known to be deleted specifically in men affected by oligo/azoospermia according to the known, clinically relevant microdeletion pattern (Viswambharan, 2007). Based on the experience of many laboratories and the results of external quality control and considering the multiplex PCR format, the first choice of STS primers recommended in the first version of the guidelines remains basically valid. These primers include the regions: For AZFa: sY84, sY86 For AZFb: sY127, sY134 For AZFc: sY254, sY255 The usage of this primer set will enable the detection of almost all the clinically relevant deletions and of over 95% of the deletions reported in the literature in the three AZF regions and is sufficient for routine analysis (Simoni, 2004). In this study, the set of PCR primers as best choice for the diagnosis of microdeletion of the AZFa, AZFb and AZFc region (sY14 (SRY), ZFX/ZFY, sY84, sY86, sY127, sY134, sY254, sY255) used in multiplex PCR reactions. We couldn’t detect any Y chromosome microdeletions in AZFa, AZFb and AZFc regions. Genes that are located on Y chromosome and responsible from spermatogenesis have a mosaic structure at somatic and/or germ cells. When leukocytes from blood were used, usually the results can not be suitable for Y chromosome microdeletion analysis because there may have been deletions in germ cells (Martin, 2008). There may be a mosaicism between seminiferous tubules in terms of the expression of genetic material. Some seminiferous tubules have aplasia whereas some tubules can be normal or mutant arrest at testes. In the identification of deletions this situation may show different outcomes when cells from blood or semen were used. When fibroblasts or leukocytes are used in genetic analysis, the proportion of a detection of Y chromosome microdeletion is slightly low because the deletions occuring in germ line cells have an independent nature from other tissues. In this study, we used peripherial blood leukocytes for the detection of Y chromosome microdeletions, however we couldn’t find any deletions. But the possibility of having deletions in germline cells shouldn’t be omitted. We are looking forward to extend our study by adding spermial Y chromosome microdeletion analysis from the same individuals. Dewan et al. (2006) reported the relation between RPL and proximal AZFc deletions and found a significant correlation. Although, they detected proximal Y chromosome AZFc microdeletions in 14 of 17 patients (82%), they couldn’t find any deletion in control group. Karaer et al. (2008) reported 43 infertile men among which 7 of them have sY 220 (AZFb) deletions (16%) of the 4 examined region, stating the importance of AZF deletions in the aetiology of RPL. In the previous studies, sequenced tagged site (STS) numbers which were selected for detection of Y chromosome microdeletions are different from each other. After physical mapping of Y chromosome, more than 300 STS were produced. It was stated that, analysing of low number of STS can be insufficient for detection of deletion regions also high number of STS can give false-positive results as polymorphic regions may identified as deletions (Simoni, 2001). One of the most important criteria for the detection of Y chromosome microdeletions is the selected STS. For this reason, European Andrology Association and European Molecular Genetics Y chromosome microdeletions in spontaneous abortions 51 Quality Network improved a standardization to distinguish the differences of deletion proportions between different laboratories. So they proposed 6 STS for detecting of AZFa, AZFb ve AZFc regions. In the present study, although 13 STS including 6 STS which were suggested by European Molecular Genetics Quality Network were analyzed, we couldn’t detect any microdeletions on Y chromosome. We propose the evolution of the results by increasing the analysed STS. Due to limited knowledge of the metabolism and the progress of the genes on Y chromosome, we can not predict the answers of the questions including Y chromosome microdeletion’s effect on RPL. For this reason researches should be focused on the relationship of Y chromosome microdeletions, male infertility and RPL. References Briton-Jones C, Haines CJ. 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