AbstractObjective: To determine the frequency, types of chromosomal ab-normalities and Y chromosome microdeletions in patients with severe male factor infertility, and the association between clini-cal background and genetic abnormality. Study design: A total of 322 infertile men; 136 men with severe oligozoospermia (sperm count <5 million/ml) and 196 with nonobstructive azoospermia were studied between April 2004 and November 2006 at the Dr. Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. Blood, semen sam-ples, and testicular biopsies of patients were obtained. Hor-monal analysis (follicle-stimulating hormone (FSH), lutein-izing hormone (LH), and testosterone levels), semen analysis, karyotype analysis, and PCR screening for Y chromosome microdeletions were performed. Result(s): Forty-eight out of 332 (14%) infertile men had a genetic abnormality. Twenty-four (7.2%) cases with karyotype abnormality were detected. The frequencies of karyotype abnormalities were Klinefelter’s syndrome 17/24 (71%), translocation 3/24 (12%), mix go-nadal dysgenesis 2/24 (8%), XX male 1/24 (4%), and 46XYY 1/24 (4%). Twenty cases (6%) infertile men had only Y chro-mosome microdeletions. The frequencies of the deleted areas were azoospermia factor (AZF)c 42%, AZFb 25%, AZFa 21%, AZFb, c 8%, and AZFa, c 4%. Four of the cases with Y chromosome microdeletions also had a concurrent karyotype abnormality. Conclusion(s): All patients with nonobstructive azo-ospermia and severe oligozoospermia (sperm count <5 million/ml) should undergo genetic screening.
Keywords: Azoospermia, chromosomal abnormality, infertility, oligozoospermia, Y chromosome microdeletion
Introduction
Infertility affects ~10–15% of couples and is an impor-tant part of clinical practice for many clinicians (Mosher & Pratt, 1991). Male factor has been identified in 50%
of cases and has increased in recent years (Bashin et al., 1994). Many cases of male infertility, previously classified as unknown etiology, can be the result of genetic factors, including cytogenetic abnormalities and microdeletions of the Y chromosome (van der Ven et al., 1997).
The prevalence of chromosome abnormalities is higher in infertile men; the overall incidence of a chromosomal factor in infertile males ranges between 2 and 8%, with a mean value of 5% (Foresta et al., 2002; 2005). In 1976, Tiepolo and Zuffardi (Tiepolo & Zuffardi, 1976) discovered that the long arm of the Y chromosome carried genetic information essential for spermatogenesisand research over the last 20 years has indicated that deletions of the long arm of the Y chromosome can disrupt spermatogenesis, leading to male infertility (Maeda et al., 1976). This spermatogenesis locus, lying in Yq11.23, has been termed the “azoospermia factor” or “AZF” and is located within intervals five and six. The reported incidence of Y deletions varies between 1 and 55% in infertile men and this could be due to selection criteria, methodology, and ethnic differences (Maeda et al., 1976).
The aim of this study was to discover the frequency and types of chromosomal abnormalities and Y chro-mosome microdeletions in patients with severe male factor and the association between clinical features and types of genetic abnormality.
Materials and methods
Between April 2004 and November 2006, 332 consec-utive men with severe oligozoospermia (sperm count <5 million/ml) or azoospermia attending the In-vitro
Genetic evaluation of severe male factor infertility in Turkey: A cross-sectional study
SABRI CAVKAYTAR1, SERTAç BATIOgLu2, MuFIT guNEL3, SERDAR CEYLANER4 & ABDuLLAH KARAER5
1Kecioren Education and Research Hospital, Department of Obstetrics and Gynecology, Ankara, Turkey, 2Ondokuz Mayıs University Faculty of Medicine, Department of Obstetrics and Gynecology, Samsun, Turkey, 3Dr. Zekai Tahir Burak Woman Health Education and Research Hospital, Department of Urology, Ankara, Turkey, 4Intergen Genetics Center, Ankara, Turkey, and 5Inonu University, Faculty of Medicine, Department of Obstetrics and Gynecology, Malatya, Turkey
Fertilization (IVF) unit of the Dr. Zekai Tahir Burak Woman Health Research and Education Hospital, Ankara, Turkey, were enrolled prospectively into the study. All patients gave informed written consent which was approved by the Dr. Zekai Tahir Burak Woman Health Research and Education Hospital Ethics Committee.
Patients with a past or present history of testicular malignancy, radiotherapy or chemotherapy, vasectomy or vasectomy reversal, congenital absence of the vas def-erens, orchidectomy, and endocrinological abnormality were excluded.
Infertile men completed comprehensive question-naires related to their medical, surgical, sexual and family histories, lifestyle habits (such as smoking, alco-hol use, and drug use), and exposure to gonadotoxins (such as drugs used in cancer chemotherapy). All the patients underwent a urological examination for the anatomical integrity of the genital system. Blood sam-ples were obtained for karyotype and Y chromosome microdeletion analysis and determination of serum follicle-stimulating hormone (FSH), luteinizing hor-mone (LH), and testosterone levels. In all cases, semen analysis was carried out according to WHO guidelines (World Health Organization, 1999), on two occasions separated by a 3 week interval, following 3 days of sexu-al abstinence. Scrotal ultrasound scanning of the testes was performed to determine subclinical varicocele and parenchymal lesions compatible with neoplasms. If available, testicular biopsy results were recorded. The microscopic examination of testicular tissue classified the patients into the following histological categories (Foresta et al., 1995).
1. Hypospermatogenesis, characterized by a quanti-tative reduction in the absolute number of germ cells with respect to Sertoli cells.
2. Sertoli cell-only (SCO) syndrome, also known as germ cell aplasia, diagnosed when extensive analy-sis of all the testicular tissue demonstrated the presence of SCO and the complete absence of any spermatogenetic cell.
3. Maturation arrest, an interruption of spermato-genesis preventing the formation of mature sper-matozoa.
4. Atrophy.
Cytogenetic analysis
Chromosomal analysis was performed on phytohemag-glutinin-stimulated peripheral lymphocyte cultures using standard cytogenetic methods. Twenty to 30 metaphases were analyzed per individual and, in cases of suspected mosaicism, the number of metaphases was increased to a total of 100 for analysis. A resolu-tion of 400-band stage was considered as a minimum; for a more detailed structural analysis, a 550–700-band stage was preferred. The routine analysis was based on gTg-banded staining.
Y chromosome microdeletion analysis
genomic DNA was extracted from peripheral ve-nous blood lymphocytes using a standard phenol-choroform protocol. Diagnostic testing of deletions was performed by PCR amplification of selected re-gions of the Y chromosome using MSY-specific STS primers to amplify both anonymous sequences of the chromosome or genes. For the diagnosis of microde-letions it is important to use a panel of STS primers derived from regions of the Y chromosome, which are not polymorphic and are well-known to be deleted specifically in men affected by oligo/azoospermia ac-cording to known, clinically relevant microdeletion patterns (Skaletsky et al., 2003). In principle, the analysis of only one nonpolymorphic STS locus in each AZF region is sufficient to determine whether a STS deletion is present in AZFa, AZFb or AZFc. 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 prim-ers recommended in the first version of the guidelines remains valid (Simoni et al., 2004). The STSs used for the detection of microdeletions included sY84, sY86 (AZFa) sY127, sY134 (AZFb) sY254, sY255 (AZFc) sY14 (SRY), ZFX/ZFY. SRY gene was used as a control for the testis-determining factor on the short arm of the Y chromosome. ZFX/ZFY was used as an internal PCR control since the primers amplify a unique fragment in both male and female DNA, respectively. These are the first choice of STS primers recommended by the International Symposium on genetics of male infertility in Florence, Italy, in 2003 (Simoni et al., 2004).
Briefly, 100 ng of genomic DNA was used as a template in a 25 mL reaction mix, comprising 13 mL amplification buffer, 1 mmoL dNTPs, 10–25 pmol of each primer, and 1.25 Iu of Taq DNA polymerase. After an initial denaturation step of 5 min, each PCR reaction was carried out, the annealing temperature specific for each primer pair. PCR products were seperated on 2% agarose gel by electrophoresis, stained with ethidium bromide and visualized using ultraviolet transillumination. Deletion of a specific locus was indicated by the failure of amplification in two independent multiplex PCRs. Whenever failure of amplification was detected, two additional PCRs were performed to confirm the absence of the unamplified STSs.
Statistical analysis
Statistical analysis was carried out by SPSS for Win-dows, version 13 (SPSS, Chicago, IL). χ2 test, Fisher test, Student’s t-test, and Mann–Whitney u test were used. P < 0.05 was accepted as significant.
Results
Three hundred and thirty-two patients were en-rolled into this study from different regions of Turkey
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and forty eight out of 332 (14%) infertile men had a genetic abnormality. Table I shows the characteristics of patients with genetic abnormalities and indicates that 32% (106/332) of patients had a past or present his-tory of varicocele or varicocelectomy. The incidence of past or present varicocele or varicocelectomy was 21% (10/48) in patients with a genetic problem and 34% (96/284) in patients with no genetic problem.
Serum FSH, LH, and testosterone levels of patients were available in 33 out of 48 patients with a genetic problem and in 139 out of 284 patients with no genetic problem (Table II). Serum FSH and LH levels of patients with a genetic problem was significantly higher than in patients with no genetic problem (P < 0.001). Serum testosterone levels of patients with a genetic problem were significantly lower than in patients with no genetic problem (P = 0.004).
Testicular biopsy results were available in 19 out of 48 patients with a genetic problem and in 36 out of 284 patients with no genetic problem (Table III). SCO syndrome was the most common result in both groups with an incidence of 47% in the genetic group and 39% in the nongenetic group. In the patients with no genetic problem, 6% (2/36) of testis biopsy results were normal, but no normal histology was detected in the patients with a genetic problem. Testicular biopsy results showed no statistically sig-nificant differences between the patients with and without genetic problems.
In total, 24 cases of karyotype abnormality were detected in 332 infertile men, with a frequency of 7.2%. Table IV shows the clinical and laboratory characteristics of patients with karyotype abnormality. Klinefelter’s syndrome was detected in 8.7% (17/196) of azoospermic males but there was none in the oli-gozoospermic group. The frequencies of karyotype ab-normalities were Klinefelter’s syndrome 17/24 (71%), translocation 3/24 (12%), mix gonadal dysgenesis 2/24 (8%), XX male 1/24 (4%), and 46XYY 1/24 (4%). Testicular biopsy results were available in five patients with karyotype abnormalities with a frequency of 60% SCO and 40% maturation arrest.
Table V shows the clinical and labarotory charac-teristics of patients with Y chromosome microdele-tions. Twenty out of 332 (6%) infertile men had only
Y chromosome microdeletions. The frequencies of the deleted areas were 42% AZFc, 25% AZFb, 21% AZFa, 8% AZFb, c, and 4% AZFa, c. However, four of the cases with Y chromosome microdeletions also had a concurrent karyotype abnormality (Table V). Testicular biopsy results were available in 12 patients with Y chromosome microdeletions and showed a frequency of 50% SCO, 25% maturation arrest, 17% hypospermatogenesis, and 8% atrophy. Twelve of 24 patients with Y chromosome microdeletions under-went TESE, and sperm were retrieved in 3 (25%) cases.
Discussion
In this study, forty-eight out of 332 (14%) infertile men had a genetic abnormality of which 24 cases were due to a karyotype abnormality; a frequency of 7.2%. Of these karyotype abnormalities 71% due to Klinefelter’s syndrome, 12% were translocations, 8% mixed gonadal dysgenesis, 4% XX male, and 4% 46XYY. Klinefelter’s syndrome was detected in 8.7% (17/196) of azoospermic males but there were none in the oligozoospermic group. Klinefelter’s syndrome have been reported as the most common chromosomal abnormality (Foresta et al., 2002). In our study, testicular biopsy revealed three out of 17 Klinefelter’s syndrome which were all SCO. These three patients underwent TESE and sperm was retrieved in 2 (33%) patients but no pregnancy could be achieved. In the literature, testicular biopsy for Klinefelter’s syndrome has also been generally reported as SCO (Aksglaede et al., 2006).
The effects of chromosomal translocations on spermatogenesis are obvious (De Braekeleer & Dao,
Table I. Clinical features of patients.Patients with
Table IV. Clinical and laboratory features of patients with karyotype abnormality.Patient number Age Sperm analysis FSH Testis biopsy Karyotype Treatment Sperm Pregnancy
1991). In the present study, there were three patients (46XX t (1;10), 46X t (Y;16) (q12:q13), 46XY t (2;3) (q21:q25)) with chromosomal translocations. Y chromosome and autosomal translocations are very rare and our patient with 46X t (Y;16) (q12:q13) is the sixth case in the literature (gunel et al., 2008).
The other karyotype abnormality, 46XX, has been re-ported with an incidence of 0.9% in azoospermic males In the current study, three patients had a SRY+XX karyotype and one of them was mosaic (45X/46XX). SRY was detected in the short arm of X chromosome in all three patients. AZFa+b microdeletion was detected in a patient with 45X/46XX and AZFb in the other patient with 46XX. In 2002, Patsalis et al. (Patsalis et al., 2002) screened 12 patients who had a 45X/46XY karyotype and presented with Turner stigmata or sexual ambiguities for Y chromosome microdeletions and re-ported distal Yq microdeletions in four patients. In 2000, Siffroi et al. (Siffroi et al., 2000) reported that Yq microdeletions may lead to 45, XO cell lines associated with Y chromosomal instability. This may explain the high incidence of spontaneous abortion in the female partners of men with Y chromosome microdeletion. In our study, there were three patients with 45X/46XY karyotype and two of them with Y chromosome micro-deletions (AZFb and AZFb+c). The two patients with chromosome microdeletions underwent TESE but no sperm were retrieved. There were four mosaic patients with 45XO cell lines and 75% (3/4) had Y chromosome microdeletions. This may have been due to a de novo or paternal transmitted Y chromosome instability.
Since (Tiepolo & Zuffardi, 1976) reported that the long arm of the Y chromosome carried genetic informa-tion essential for spermatogenesis, there have been many reports concerning Y chromosome microdeletions and male infertility (Pryor et al., 1997; Mallidis et al., 1998). The incidence of Y chromosome microdeletions chang-es according to patient selection criteria, methodology, and ethnicity (Maeda et al., 1976; Mallidis et al., 1998). There is no correlation between the frequency of mi-crodeletions and the number of STS analyzed. (Krausz et al., 1999; Peterlin et al., 2002). In the present study, STS primers were used as recommended (Simoni et al., 2004). The incidence of Y chromosome microdeletions was 6% (20/332) in our study, 8.7% (17/196) in the azoospermic group, and 2.2% (3/136) in the severe oli-gozoospermic group. These findings are consistent with similar studies (Krausz et al., 1999; Rao et al., 2004). The frequencies of the deleted areas were 42% AZFc, 25% AZFb, 21% AZFa, 8% AZFb, c, and 4% AZFa, c but four of the cases with Y chromosome microdeletions also had a concurrent karyotype abnormality (Table V). In 2001, Foresta et al. (Foresta et al., 2001) reviewed the extent of Y chromosome microdeletions and reported 60% AZFc, 16% AZFb, 5% AZFa, and 14% with two or three microdeletion regions together and 5% outside the AZF region (Foresta et al., 2001).
In some studies, the deleted area was only in the AZFc region (Rao et al., 2004; Vicdan et al., 2004). In
our study, although the AZFc was the most frequently deleted region, AZFa and AZFb microdeletions had higher incidences than in other reports. These differ-ences probably depend on selection criteria and ethnic-ity. Histologically, these deletions are associated with various spermatogenetic alterations, (van golde et al., 2001; Luetjens et al., 2002). Complete deletion of the AZFa region results in SCO syndrome and azoosper-mia (Vogt et al., 1996; Krausz et al., 2000; Kamp et al., 2001; Hopps et al., 2003). However, isolated gene dele-tions on AZF a region have been associated with differ-ent testicular phenotypes (Ferlin et al., 1999; Foresta et al., 2000). In complete deletions of AZFa and AZFb, it is impossible to retrieve spermatozoa (Krausz et al., 2000; Hopps et al., 2003), but deletions of the AZFc region have been associated with residual spermatogen-esis and sperm can be retrieved via TESE (Page et al., 1999; Peterlin et al., 2002). However, transmission of Y chromosome microdeletion to the male offspring is possible (Nap et al., 1999; Oates et al., 2002).
Testicular biopsy results were available in 19 out of 48 patients with a genetic problem and in 36 out of 284 patients with no genetic problem. SCO syndrome was the most common result in both groups. In the patients with no genetic problem, 6% (2/36) of testis biopsy results were normal, but normal histology was not de-tected in the patients with a genetic problem. Twelve of 24 patients with Y chromosome microdeletions under-went TESE and sperm were retrieved in 3 (25%) cases and the deleted areas were AZFa, AZFb, and AZFc. A pregnancy via the sperm from the man with AZFa de-letion resulted in spontaneous abortion. In the patient with severe oligozoospermia with AZFc deletion, ICSI resulted in pregnancy with a healthy birth.
Thirty-two percent (106/332) of patients had a past or present history of varicocele or varicocelectomy. The incidence of varicocele was 21% (10/48) in patients with a genetic problem and 34% (96/284) in patients with no genetic problem; a difference which was not statistically significant. Varicocele affects approximately 40% of men attending infertility clinics and its role in male infertility is still unclear (Marmar, 2001). Some recent studies report that genetic defects and varicocele may coexist (Moro et al., 2000; Rao et al., 2004).
The frequency of azoospermia was 90% (43 out of 48) in the group with genetic problems and 54% (153 out of 284) in the group with no genetic problem (P < 0.001).
As a result, azoospermia has a wider genetic basis than oligozoospermia. Serum FSH and LH levels of pa-tients with a genetic problem were significantly higher than in patients with no genetic problem (P < 0.001). Serum testosterone levels of patients with a genetic problem was significantly lower than in patients with no genetic problem (P = 0.004). This may be due to the high number of Klinefelter’s syndrome patients (11/33) in the group with serum testosterone levels. Hormone profiles of our patients were consistent with testicular insufficiency.
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Genetic evaluation of severe male factor infertility in Turkey 105
In conclusion, all patients with nonobstructive azo-ospermia and severe oligozoospermia (sperm count <5 million/ml) with a history of varicocele or not, should undergo genetic screening. Preimplantation diagnosis should be offered to men with a genetic problem if an embryo is produced by ICSI in order to prevent the transmission of a genetic abnormality to the offspring.
Acknowledgment
This work was done by Dr. Zekai Tahir Burak Women’s Health Education and Research Hospital, Department of Obstetrics and gynecology, Division of Reproduc-tive Endocrinology and Infertility, Ankara, Turkey. No financial support received.
Declaration of interest: The authors report no con-flicts of interest. The authors alone are responsible for the content and writing of the paper.
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