Abstract
It has been indicated that approximately 20% of azoospermic patients have chromosomal anomalies, 90% of which are sex-chromosome abnormalities. Even azoospermic patients with sex-chromosomal anomalies might be able to father children using an advanced assisted reproductive technique such as microdissection testicular sperm extraction (micro-TESE) with intracytoplasmic sperm injection (ICSI). To evaluate the effect of micro-TESE in azoospermic patients with various sex-chromosomal anomalies, we reviewed their clinical results. A chromosomal survey using the G-banding technique was performed on males whose semen analysis demonstrated azoospermia at the Division of Male Infertilities at our institution between January 2004 and December 2009. Forty-two of these subjects demonstrated sex-chromosomal anomalies. The mean patient age was 34.4 ± 4.3 years. We classified them into two groups: Klinefelter syndrome (47,XXY) and other sex-chromosome abnormalities. Thirty-five patients showed Klinefelter syndrome and seven patients showed other sex-chromosome abnormalities. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels were 36.3 ± 14.0 IU/L, 15.8 ± 6.7 IU/L, and 3.2 ± 2.0 ng/ml in Klinefelter syndrome, and 20.8 ± 10.4 IU/L, 8.2 ± 5.2 IU/L and 4.1 ± 1.5 ng/ml in other sex-chromosome abnormalities, respectively. The mean testicular volume was 4.0 ± 2.1 ml in Klinefelter syndrome and 9.9 ± 4.6 ml in other sex-chromosome abnormalities. Serum FSH and LH in Klinefelter syndrome were significantly higher than those in other sex-chromosome abnormalities, and the mean testicular volume in Klinefelter syndrome was significantly smaller than that in other sex-chromosome abnormalities. The sperm retrieval rate (SRR) for micro-TESE showed no significant difference between the two groups (42.4% vs. 42.9%). In this study, the outcome of micro-TESE appeared not to differ between Klinefelter syndrome and other sex-chromosome abnormalities.
Keywords:
Introduction
Infertility is defined as a failure to conceive in a couple trying to reproduce for a period of one year. Approximately 15 percent of couples are infertile, and among these couples, male factor infertility accounts for approximately 50 percent [Poongothai et al. Citation2009]. Male infertility is a multifactorial syndrome encompassing a wide variety of disorders; the cause of infertility can be idiopathic and can be congenital or acquired.
The presence of an association between human male infertility and chromosomal anomalies has been known for a long time. The incidence of karyotype abnormalities among infertile men has been reported to range between 2.2 and 19.6% [Chandley et al. Citation1972; De Krester et al. Citation1972; Nakamura et al. Citation2001; Yatsenko et al. Citation2010]. Thus, it is well known that infertile men have a higher than normal incidence of chromosomal abnormalities. Karyotyping of white blood cells of every male attending an infertility clinic would be necessary to identify those with genetic defects. However, this process is time-consuming and expensive and the relationship between many of the variants and impaired spermatogenesis is not clear, even after cloning of the many genes involved in spermatogenesis.
Specific genetic defects have been identified in fewer than 20% of infertile males and most causes of infertility remain to be elucidated. The most common cytogenetic defects associated with azoospermia are numerical and structural chromosome abnormalities, including Klinefelter syndrome and Y chromosome microdeletions. Even azoospermic patients with sex-chromosomal anomalies might be able to father children using an advanced assisted reproductive technique such as micro-TESE with ICSI. The objective of this study was to evaluate the effect of micro-TESE in azoospermic patients with sex-chromosomal anomalies.
Results
Of the group of 209 patients, 42 (20.1%) showed sex-chromosomal abnormalities. The mean patient age was 34.4 ± 4.3 years (25 − 44) overall. The overall serum FSH, LH, and T levels were 33.7 ± 14.6 IU/L (7.2 − 78.3), 14.5 ± 7.0 IU/L (3.8 − 41.2), and 3.4 ± 1.9 ng/ml (0.7 − 9.0), respectively ().
Table 1. Comparison of several parameters in 42 patients including patients with sex chromosomal anomalies.
There were 35 individuals with Klinefelter syndrome, constituting 16.7% of the total group studied; 83.3% of the patients with an abnormal blood karyotype were found to have Klinefelter syndrome. Of these 35 men, 32 had a chromosomal complement, while three exhibited mosaicism. The mean age of patients with Klinefelter syndrome was 34.4 ± 4.6 years (25 − 44). Their serum FSH, LH, and T levels were 35.6 ± 13.2 IU/L (7.2 − 78.3), 15.7 ± 6.8 IU/L (3.8 − 41.2), and 3.3 ± 2.0 ng/ml (0.7 – 9.0), respectively.
There were 7 individuals with other sex-chromosomal abnormalities, who constituted 3.3% of the total group studied; 15.6% of these patients were found to have an abnormal blood karyotype. The mean age of patients with other sex-chromosomal abnormalities was 35.6 ± 3.7 years (30 - 40). Their serum FSH, LH, and T levels were 20.8 ± 10.4 IU/L (10.5 – 42.3), 8.2 ± 5.2 IU/L (4.7 – 19.5), and 4.1 ± 1.5 ng/ml (1.6 – 6.4), respectively. Most of this group had numerical or structural changes of the Y chromosome; for example, there were a deletion of Y chromosome, a supernumerary Y chromosome, a ring Y chromosome, and an isodicentric Y chromosome. Of these 7 individuals, one patient showed a 47,XYY configuration; the incidence of this anomaly was only 0.5% among all patients and 2.4% of all men with chromosomal abnormalities. Four men had structural abnormalities of the Y chromosome; the incidence of this type of anomaly was 1.9% among all patients and 9.5% of all men with chromosomal abnormalities.
The serum FSH and LH levels in cases of Klinefelter syndrome were significantly higher than those in other sex-chromosome abnormalities, and the mean testicular volume in Klinefelter syndrome was significantly smaller than that in other sex-chromosome abnormalities. There were no significant differences in age and serum levels of T between these two groups ().
In this series, 40 of 42 patients with sex-chromosomal abnormalities underwent therapeutic sperm retrieval by micro-TESE at our institution. Sperm was successfully retrieved from 17 of these 40 patients (42.5%). There were no significant differences in several clinical parameters between successful and unsuccessful sperm retrieval groups (). Sperm from 14 of the 33 with Klinefelter syndrome (42.4%; non-mosaic, 40%; mosaic, 66.7%) was retrieved. Meanwhile, sperm from 3 of the 7 with other sex-chromosomal abnormalities (42.9%) was retrieved. Results for each sex-chromosomal aberration type are shown in . There was no significant difference in the SRR between these two groups (). Moreover, SRR was not associated with the deletion of Yq in this study ().
Table 2. Comparison of several parameters in 40 patients with successful and unsuccessful sperm retrieval.
Table 3. Comparison of SRR between KFS and other sex chromosome anomalies.
Table 4. Comparison of deletion of Yq between successful and unsuccessful sperm retrieva l.
Discussion
Chromosomal abnormalities are common in infertile and subfertile males. However, the exact mechanism by which chromosomal abnormalities induce infertility is not clear. It is likely that the presence of abnormally distributed chromatin may interfere with meiotic division and reduce sperm production. Spermatozoa harboring abnormal chromosomes may cause abnormal embryonic development, which can cause early pregnancy loss.
In this series, the incidence of sex-chromosomal abnormalities was 20.1% (42/209). This was higher than in previous studies on the Japanese population [Yoshida et al. Citation1997]. However, the reported incidence of chromosomal abnormalities among infertile men depends on several factors. The most important of these is the definition of the population. Thus, clinics that carry out chromosomal analyses on all men from infertile couples may have a lower incidence of such abnormalities than those clinics that perform such analysis only on patients who have a defined seminal abnormality. However, determination of a white blood cell karyotype is a time-consuming and costly procedure. Therefore, it would be advantageous for clinicians to be able to predict the presence of such abnormalities because this would help reduce the overall cost of fertility evaluation.
Klinefelter syndrome is the most frequent cause of sex-chromosomal abnormality and leads to male infertility. Approximately 14% of azoospermic males have Klinefelter syndrome [Oates Citation2008]. Approximately 95% of men with Klinefelter syndrome have a 47,XXY chromosomal complement [Walsh et al. Citation2009]. Men with Klinefelter syndrome have reduced testicular size, elevated FSH and LH levels, as well as low T levels. However, these men can father their own children by ICSI with sperm extracted from their testes. The SRR of men with Klinefelter syndrome by micro-TESE has been described to range from approximately 21% to 72% [Levron et al. Citation2000; Westlander et al. Citation2001; Madgar et al. Citation2002; Vernaeve et al. Citation2004; Schiff et al. 2005; Koga et al. Citation2007]. The series previously reported SRRs of Klinefelter syndrome men by micro-TESE of 40-50% with the exclusion of the highest and lowest samples. It seems that the SRR in this series is comparable to those in previous reports.
In this series, a patient presented with a 45,X/46,XY mosaic karyotype. The mitotic loss of 1 Y chromosome during early embryonic development produces a 45,X/46,XY mosaic karyotype [Maduro and Lamb Citation2002]. Although 80% of 45,X/46,XY individuals have a normal sex-determining region on the Y chromosome (SRY), approximately 60% of these individuals present with mixed gonadal dysgenesis with a streak gonad and a testis, a uterus, and a vagina [Walsh et al. Citation2009]. This karyotype is usually seen in cases harboring azoospermic factor region (AZF) deletions, in which, because of chromosome instability, the Y chromosome is lost, resulting in mosaicism. In this case, sperm retrieval in micro-TESE was successful.
Most of the other sex-chromosomal anomalies in our study involved partial deletion of the Y chromosome long arm (Yq). Four patients showed partial deletion of the Yq and three did not. The SRR for micro-TESE were 25% and 66.7%, respectively. However, there were no significant differences between the two groups. Deletions of Yq are one of the most frequently occurring chromosomal abnormalities in men and are believed to arise from recombination events between long stretches of highly repetitive DNA sequences during meiosis or early pre-implantation development [Poongothai et al. Citation2009]. It was established that Yq is required for spermatogenesis. The region that is important for germ cell development and differentiation on Yq is the AZF region. The AZF is divided into four recurrently deleted non-overlapping subregions designated as AZFa, AZFb, AZFc, and AZFd. These regions likely contain multiple genes required for different stages of spermatogenesis; for example, USP9Y (ubiquitin-specific protease 9 on Y) and DBY (dead box on Y) are in the AZFa locus, RBMY (RNA-binding motif on Y) is in the AZFb locus, and DAZ (deleted in azoospermia) is in the AZFc locus. In this series, testicular sperm could not be found in patients with deletions of Yq.
Within the past decade, there has been an enormous proliferation of tests available for men with infertility. Initially, hormonal tests, semen analysis, and karyotype were all that were available to test men with infertility. These tests, although important, could only begin to elucidate the potential causes of male infertility. Routine cytogenetic studies performed on peripheral blood can detect Y chromosome structural abnormalities; however, cytogenetic analysis alone cannot detect microdeletions of the Y chromosome, or whether a cytogenetically visible deletion extends into AZF regions and small interstitial deletions in AZF regions. In this series, there were no significant differences in SRR between the patients with Klinefelter syndrome and those with other sex-chromosomal anomalies despite the significant differences of FSH, LH, and testicular volume among the two groups. These results clearly reveal that conventional physical examinations or pituitary-testicular hormones and chromosomal assay are deficient for preoperative examinations in azoospermia patients. Although we could not examine AZF genes in our institution in 2004-2009, the presence of these genes seems to be a strong predictor of sperm recovery in micro-TESE, so we now examine these genes in almost all patients with azoospermia or severe oligozoospermia. With the advent of sophisticated genetic testing and more specific diagnostic tests, specific causes of male infertility are more likely to be determined.
The introduction of assisted reproduction treatment (ART) and more particularly micro-TESE and ICSI has revolutionized the management of patients with chromosomal abnormalities. However, increasing concern is now being expressed about the risk of aneuploidy in the offspring upon the use of ART such as ICSI. In particular, some reports have revealed that spermatozoa of patients with Klinefelter syndrome tend to have high rates of sex and autosomal chromosome aneuploidy [Staessen et al. Citation2003; Morel et al. Citation2003]. These data indicate that these individuals should be informed of their risks and presented with options including prenatal diagnosis or preimplantation genetic screening (PGS) [Goossens et al. Citation2009; Kahraman et al. Citation2003].
Materials and Methods
In a 5-year period from January 2004 to December 2009, 323 patients were evaluated for male factor infertility at the Division of Male Infertilities at Kobe University Hospital. Blood samples from 209 patients aged 25 to 44 years old were submitted for cytogenetic analysis after informed consent was provided. Each patient provided their history and the obstetric history of their wife, and underwent physical examination and semen sampling at least 2-3 times. Semen analyses were performed on at least two separate occasions for each patient according to the methods described in the World Health Organization manuals [WHO 1999]. The endocrinologic evaluation included assays of serum FSH, LH, and T. The blood sample was drawn between 9:00 and 10:00 a.m.
Cytogenetic studies were carried out as part of routine evaluation in males with severe male factor infertility, and then cases of sperm density less than 5 × 106/ml and those suspected of involving genetic causes of infertility were routinely screened for karyotype abnormalities. Chromosomal analyses were performed using cultures of peripheral blood lymphocytes by standard methods of Giemsa banding and/or fluorescence in situ hybridization (FISH). At least 20 cells were analyzed.
We classified patients with sex-chromosomal abnormalities into two groups: Klinefelter syndrome and other sex-chromosome abnormalities. Statistical comparison of clinical parameters was performed using Student's t-test and chi-square test. P values < 0.05 were considered to be statistically significant. Differences were examined using an unpaired t-test. This study was approved by the Institutional Review Board of the hospital and all participants provided written informed consent, permitting the use of their tissue samples in this study.
Declaration of interest: The authors have no conflict of interest.
Author contributions: Conception and design, acquisition of data, analysis and interpretation of data, and drafting the manuscript: MA; Conception and design, acquisition of data, analysis and interpretation of data, and revising critically for important intellectual content: KY; Acquisition of data: KC; Conception and design, and drafting the manuscript: HM; Final approval of the version to be published: MF.
Abbreviations
| micro-TESE: | = | microdissection testicular sperm extraction |
| ICSI: | = | intracytoplasmic sperm injection |
| FSH: | = | follicle-stimulating hormone |
| LH: | = | luteinizing hormone |
| T: | = | testosterone |
| SRR: | = | sperm retrieval rate |
| AZF: | = | azoospermic factor region |
| Yq: | = | Y chromosome long arm |
References
- Chandley, A.C., Christie, S., Flestcher, J., Frackiewicz, A. and Jacobs, P.A. (1972) Translocation heterozygosity and associated subfertility. Cytogenet Cell Genet 11: 516–533.
- De Krester, D.M., Burger, H.G., Fortune, D., Hudson, B., Long, A.R., Paulsen, C.A.. (1972) Hormonal, histological and chromosomal studies. J. Clin.Endocrinol. Metabol 35: 392–440.
- Goossens, V., Harton, G., Moutou, C., Traeger-Synodinos, J., Van Rij, M. and Harper, J.C. (2009) ESHRE PGD Cousortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 24(8): 1786–1810.
- Kahraman, S., Findikli, N., Berkil, H., Bakircioglu, E., Donmez, E., Sertyel, S.. (2003) Results of preimplantation genetic diagnosis in patients with Klinefelter's syndrome. Reprod Biomed Online 7: 346–352.
- Koga, M., Tsujimura, A., Takeyama, M., Kikuchi, H., Takao, T., Miyagawa, Y. (2007) Clinical comparison of successful and failed microdissection testicular sperm extraction in patients with nonmosaic Klinefelter syndrome. Urology 70: 341–345.
- Levron, J., Aviram-Goldring, A., Madgar, I., Raviv, G., Barkai, G. and Dor, J. (2000) Sperm chromosome analysis and outocome of IVF in patients with non-mosaic Klinefelter's syndrome. Fertil Steril 74: 925–929.
- Madgar, I., Dor, J., Weissenberg, R., Raviv, G., Menashe, Y. and Levron, J. (2002) Prognostic value of the clinical and laboratory evaluation in patients with nonmosaic Klinefelter syndrome who are receiving assisted reproductive therapy. Fertil Steril 77: 1167–1169.
- Maduro, MR. and Lamb, D.J. (2002) Understanding new genetics of male infertility. J Urol 168(5):p2197-2205.
- Morel, F., Bernicot, I., Herry, A., Le Bris, M.J., Amice, V. and De Braekeleer, M. (2003) An increased incidence of autosomal aneuploidies in spermatozoa from a patient with Klinefelter's syndrome. Fertil Steril 79 Suppl3: 1644–1646.
- Nakamura, Y., Kitamura, M., Nishimura, K., Koga, M., Kondoh, N., Takeyama, M. (2001) Chromosal variants among 1790 infertile men. Int J Urol 8: 49–52.
- Oates, R.D. (2008) The genetic basis of male reproductive failure. Urol Clin North Am 35: 257-270.
- Poongothai, J., Gopenatch, T.S. and Manonayaki, S. (2009) Genetics of human male infertility. Singapore Med J 50: 336–347.
- Schiff, JD., Palermo, GD., Veeck, LL., Goldstein, M., Rosenwaks, Z., Schlegel, PN. (2005) J Clin Endocrinol Metab 90(11):6263–6267
- Staessen, C., Tournaye, H., Van Assche, E., Michiels, A., Van Landuyt, L., Devroey, P.. (2003) PGD in 47,XXY Klinefelter's syndrome patients. Hum Reprod Update 9: 319–330.
- Vernaeve, V., Staessen, C., Verheyen, G., Van Steirteghem, A., Devroey, P. and Tournaye, H. (2004) Can biological or clinical parameters predict testicular sperm recovery in 47,XXY Klinefelter's syndrome patients? Hum Reprod 19: 1135–1139.
- Walsh, T.J., Pera, R.R. and Turek, P.J. (2009) The genetics of male infertility. Semin Reprod Med 27: 124–136.
- Westlander, G., Ekerhovd, E., Granberg, S., Hanson, L., Hanson, C. and Bergh, C. (2001) Testicular ultrasonography and extended chromosome analysis in men with nonmosaic Klinefelter syndrome: a prospective study of possible predictive factors for successful sperm recovery. Fertil Steril 75: 1102–1105.
- World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 4th ed. Cambridge University press.
- Yatsenko, A.N., Yatsenko, S.A., Weedin, J.W., Lawrence, A.E., Patel, A., Peacock, S.. (2010) Comprehensive 5-year study of cytogenetic aberrations in 668 infertile men. J Urol 183: 1636–1642.
- Yoshida, A., Miura, K. and Shirai, M. (1997) Cytogenetic survey of 1,007 infertile males. Urol Int 58: 166–176.