1 Departments of Obstetrics and Gynaecology, 2 Human Genetics and 3 Epidemiology and Biostatistics, University Medical Centre Nijmegen, Nijmegen, The Netherlands
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Abstract |
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Key words: assisted reproduction outcome/ICSI/male infertility/microdeletions
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Introduction |
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Microdeletions of the Y chromosome occur in between 1% and 29% of subfertile men (Foresta et al., 1997; Kremer et al., 1997
; Van der Ven et al., 1997
). The frequency is dependent on the definition of male subfertility and on the choice of sequence tagged sites used for screening (Simoni et al., 1999
). The AZFc locus containing the DAZ gene cluster is the most frequently deleted region of the Y chromosome in men with non-obstructive infertility (Kostiner et al., 1998
; Simoni et al., 1998
). Histologically, these deletions are associated with various spermatogenetic alterations, including Sertoli cell-only syndrome, maturation arrest and hypospermatogenesis.
Following the introduction of intracytoplasmic sperm injection (ICSI), it was possible for men with a microdeletion of the Y chromosome to father children, despite their severe oligozoospermia or azoospermia, using ejaculated or surgically retrieved spermatozoa.
When a microdeletion of the Y chromosome is diagnosed, it is important that a couple is informed about their reproductive options. These options are ICSI, artificial insemination with donor semen, or no treatment. Most infertile couples (79%) with a microdeletion of the Y chromosome choose ICSI as treatment for their fertility problem (Nap et al., 1999). The couple should also take into account the success rates of these options, besides the fact that when ICSI is used, the male offspring inherit the same deletion and presumably the related fertility problem (Mulhall et al., 1997
).
Although many reports have detailed the success rate of ICSI treatment, virtually no information is available (other than a few case reports) regarding the success rates of ICSI in couples with severe oligozoospermia due to microdeletions of the Y chromosome. In the present study, the outcome of ICSI treatment using spermatozoa from men with a microdeletion of the Y chromosome was compared with that of ICSI treatment using spermatozoa from oligozoospermic men without this deletion.
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Materials and methods |
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The fertilization rate, embryo quality, pregnancy rate, implantation rate and take-home baby rate after ICSI in couples in which the man had microdeletions of the Y chromosome and couples without this genetic disorder were compared retrospectively. Observations were commenced when oocytes were able to be punctured at the time of oocyte retrieval.
The success of ICSI treatment is mainly influenced by maternal age, and whether the subfertility is primary or secondary (Sherins et al., 1995; Abdelmassih et al., 1996
; Stolwijk et al., 2000
). For this reason, controls with a primary subfertility which had been treated within the same year and at the same age as one of the patients, were considered. Among this group, only couples who had started their first ICSI cycle in 1996 or later were included. Thus, matching was carried out without using a fixed matching ratio.
Because of possible interference with the outcome of ICSI treatment, the following couples were excluded from the control group: (i) chromosomal abnormalities in the male ICSI candidate; (ii) cryopreserved spermatozoa used for ICSI; (iii) using ejaculated spermatozoa from a man with a vaso-vasostomy; and (iv) ICSI treatment performed because of total failure of previous IVF treatment, despite normal sperm parameters.
ICSI treatment
Ovarian stimulation was performed by means of a long protocol of gonadotrophin-releasing hormone (GnRH) agonist, that was started on day 21 of the previous cycle, followed by human menopausal gonadotrophin (HMG). Oocyte retrieval occurred 36 h after HCG injection, and ICSI was performed according to a published technique (Palermo et al., 1992). Fertilization rates were scored 1822 h after the ICSI procedure, with respect to injected oocytes. On day 3 after oocyte retrieval the embryos were scored for cell number and percentage of fragmentation using a modification of a previously published scoring system (Bolton et al., 1989
): no fragmentation = excellent; <10% fragmentation = good; >10% fragmentation = fair. The best embryos were selected for embryo transfer. Before 1997, a maximum of three embryos was transferred; however, since January 1997 the maximum number of transferred embryos has been two. Clinical pregnancy was defined as a positive (>50 IU/l) urinary ß-HCG test on day 18 after oocyte retrieval. An ongoing pregnancy was defined as one or more gestational sacs in utero, 5 weeks after embryo transfer. The implantation rate per embryo was defined as the fraction of transferred embryos developing to gestational sac. The take-home baby rate was defined as a dichotomous variable: one (or more) child per pregnancy was considered to be a positive result, and the ratios were calculated in relation to an embryo transfer and in relation to an oocyte retrieval.
Statistical analysis
Two sample tests according to Student and Wilcoxon have been used with respect to number of oocytes per oocyte retrieval, percentage of injected oocytes per retrieval (injection rate), and the fertilization rate.
Fisher's exact tests were carried out with respect to the categorical variable for embryo quality of the transferred embryos and the dichotomous variables for the occurring of pregnancy, ongoing pregnancy, take-home baby rate per embryo transfer and take-home baby rate per oocyte retrieval. Testing according to Wilcoxon's two sample test was also performed with respect to the implantation rate of embryos transferred. Only SAS procedures were used.
In order to justify the above statistical analyses, a more sophisticated approach was applied by taking into account the patient dependence of cycle results. First, an alternative analysis was performed with respect to fertilization rate on the basis of a general linear mixed model with random patient effects (SAS procedure mixed). In addition, an alternative analysis was carried out with respect to the pregnancy and take-home baby rates on the basis of a logistic regression analysis with random patient effects (SAS macro GLIMMIX).
A P-value 0.05 was considered to be significant.
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Results |
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The outcome after ICSI treatment until fertilization of couples with an AZFc deletion (n = 8) and control couples (n = 107) is shown in Table I. In the former group, 19 retrieved oocytes resulted in a significantly lower fertilization rate of 55% (95% CI: 4169%) compared with 71% (95% CI: 6774%) in the control group (P = 0.01). In addition, the embryo quality was significantly poorer (P < 0.001) among the group of Y-deleted men (Table II
).
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Five healthy female babies were born as a result of the three pregnancies.
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Discussion |
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Although in IVF the fertilization rate may be affected by sperm and/or oocyte factors, success rates after ICSI are most likely not related to sperm parameters (Mansour et al., 1995; Nagy et al., 1995
). The injection rate and number of oocytes were not significantly different between the two groups in this study. Furthermore, it was apparent that the same staff, using the same protocols during the same time period, performed the ICSI procedures.
It remains unknown how spermatozoa with an AZFc deletion influence the fertilization rate and embryo quality. Until now, it has been thought that the main function of the AZFc region of the Y chromosome is its involvement in spermatogenesis. It may be postulated that either the quality of the spermatozoa or the sperm function in fertilization and embryo development is impaired due to the AZFc deletion. The current study might indicate that Y-bearing spermatozoa of these men have a lower chance of fertilization and normal embryo development. If this were the situation, it would only occur in male embryos, and result in a lower male:female ratio. Therefore, it would be interesting to investigate the sex of the supernumerary embryos in those couples with an AZFc deletion.
This hypothesis is supported in the current Y-deleted group, because only daughters were born to these couples. However, male offspring have been born after ICSI using spermatozoa from men with an AZFc deletion (Page et al., 1999).
As argued above, it may be important to maximize the available number of good quality embryos in couples with an AZFc deletion, perhaps by attempting to increase the number of oocytes collected. In this way the number of embryos available for transfer can be increased.
No significant difference was found in the take-home baby rate after ICSI treatment in couples with microdeletions in the AZFc region of the Y chromosome, although due to the low frequency of microdeletion of the Y chromosome in oligozoospermic men the number of patients in this study was small. Research on a larger group of men may show an influence on both pregnancy and take-home baby rates. If the pregnancy and take-home baby rates in the current study are considered to be true rates, then increasing the number of ICSI treatments with oocyte retrieval in couples with Y deletions to at least 150 and 375 respectively would be required to reach statistical significance at the 5% level, assuming a power of 80% for the statistical test.
In the present group of patients we were confronted with azoospermia in one patient at the time of ICSI, though pre-treatment semen analysis from this patient revealed oligozoospermia. Men with an AZFc deletion sometimes have azoospermia. In The Netherlands it is not permissible at present to use spermatozoa from epididymal or testicular origin (TESE), and such men can only be treated at a fertility centre in another country. A progressive reduction in sperm number over several months has been described in some men with AZFc deletions (Girardi et al., 1997; Simoni et al., 1997
), and it is these men who may benefit from early detection combined with cryopreservation of their semen.
Although in some centres Y chromosome deletion analysis is still not offered, it is feasible that a couple can benefit from testing. First, little is known about the origin of male subfertility and about the consequences and risks of ICSI. Furthermore, it has been shown that a reasonable percentage (21%) of couples with a microdeletion of the Y chromosome decide to refrain from ICSI treatment (Nap et al., 1999). Therefore, we consider that we have an obligation to inform the couple with regard to their situation, and only then can they make a well-informed choice about their reproductive status. Moreover, as argued above, the finding of a Y chromosome microdeletion might be an adverse prognostic finding for future treatment, and patients must be informed of the situation and possible prognostic aspects (Krausz et al., 2000
). Deletions including and extending beyond the AZFc region (AZFb+c and AZFa+b+c) are associated with a total absence of testicular spermatozoa (Silber et al., 1998
), and the presence of AZFb deletion is a significantly adverse prognostic finding for TESE (Brandell et al., 1998
). Nonetheless, mature spermatozoa have been found in ~50% of azoospermic patients with AZFc deletions. The present study adds to the ongoing discussion that couples with an AZFc deletion show a poorer outcome in ICSI treatment. However, further investigation using larger numbers of couples with AZFc microdeletions of the Y chromosome is required to study the possible prognosis of such deletions on ICSI treatment.
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Acknowledgments |
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Notes |
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References |
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Bolton, V.N., Hawes, S.M., Taylor C.T. et al. (1989) Development of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates, and development to blastocyst. J. In Vitro Fertil. Embryo Transfer, 6, 3035.[ISI][Medline]
Brandell, R.A., Mielnik, A., Liotta, D. et al. (1998) AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum. Reprod., 13, 28122815.
Foresta, C., Ferlin, A., Garolla, A. et al. (1997) Y-chromosome deletions in idiopathic severe testiculopathies. J. Clin. Endocrinol. Metab., 82, 10751080.
Girardi, S.K., Mielnik, A. and Schlegel, P.N. (1997) Submicroscopic deletions in the Y chromosome of infertile men. Hum. Reprod., 12, 16351641.[Abstract]
Hoefsloot, L.H., Tuerlings, J.H., Kremer, J.A. et al. (1997) PCR analysis of Y-chromosome deletions in subfertile men [letter; comment]. Lancet, 349, 1400.
Kostiner, D.R., Turek, P.J. and Reijo, R.A. (1998) Male infertility: analysis of the markers and genes on the human Y chromosome. Hum. Reprod., 13, 30323038.[Abstract]
Krausz, C., Quintana-Murci, L. and McElreavy, K. (2000) Prognostic value of Y deletion analysis, what is the clinical prognostic value of Y chromosome microdeletion analysis. Hum. Reprod., 15, 14311434.
Kremer, J.A., Tuerlings, J.H., Meuleman, E.J. et al. (1997) Microdeletions of the Y chromosome and intracytoplasmic sperm injection: from gene to clinic. Hum. Reprod., 12, 687691.[Abstract]
Lahn, B.T. and Page, D.C. (1997) Functional coherence of the human Y chromosome. Science, 278, 675680.
Ma, K., Inglis, J.D., Sharkey, A. et al. (1993) A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermia factor AZF controlling human spermatogenesis. Cell, 75, 12871295.[ISI][Medline]
Mansour, R.T., Aboulghar, M.A., Serour, G.I. et al. (1995) The effect of sperm parameters on the outcome of intracytoplasmic sperm injection. Fertil. Steril., 64, 982986.[ISI][Medline]
Mulhall, J.P., Reijo, R., Alagappan, R. et al. (1997) Azoospermic men with deletion of the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic sperm injection. Hum. Reprod., 12, 503508.[ISI][Medline]
Nagy, Z.P., Liu, J., Joris, H. et al. (1995) The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum. Reprod., 10, 11231129.[Abstract]
Nap, A.W., Van Golde, R.J., Tuerlings, J.H. et al. (1999) Reproductive decisions of men with microdeletions of the Y chromosome: the role of genetic counselling. Hum. Reprod., 14, 21662169.
Page, D.C., Silber, S. and Brown, L.G. (1999) Men with infertility caused by AZFc deletion can produce sons by intracytoplasmic sperm injection, but are likely to transmit the deletion and infertility. Hum. Reprod., 14, 17221726.
Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Reijo, R., Lee, T.Y., Salo, P. et al. (1995) Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genet., 10, 383393.[ISI][Medline]
Sherins, R.J., Thorsell, L.P., Dorfmann, A. et al. (1995) Intracytoplasmic sperm injection facilitates fertilization even in the most severe forms of male infertility: pregnancy outcome correlates with maternal age and number of eggs available. Fertil. Steril., 64, 369375.[ISI][Medline]
Silber, S.J., Alagappan, R., Brown, L.G. et al. (1998) Y chromosome deletions in azoospermic and severely oligozoospermic men undergoing intracytoplasmic sperm injection after testicular sperm extraction. Hum. Reprod., 13, 33323337.[Abstract]
Simoni, M., Gromoll, J., Dworniczak, B. et al. (1997) Screening for deletions of the Y chromosome involving the DAZ (Deleted in AZoospermia) gene in azoospermia and severe oligozoospermia. Fertil. Steril., 67, 542547.[ISI][Medline]
Simoni, M., Kamischke, A. and Nieschlag, E. (1998) Current status of the molecular diagnosis of Y-chromosomal microdeletions in the work-up of male infertility. Initiative for international quality control [see comments]. Hum. Reprod., 13, 17641768.
Simoni, M., Bakker, E., Eurlings, M.C. et al. (1999) Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int. J. Androl., 22, 292299.[ISI][Medline]
Stolwijk, A.M., Wetzels, A.M. and Braat, D.D. (2000) Cumulative probability of achieving an ongoing pregnancy after in-vitro fertilization and intracytoplasmic sperm injection according to a woman's age, subfertility diagnosis and primary or secondary subfertility. Hum. Reprod., 15, 203209.
Tiepolo, L. and Zuffardi, O. (1976) Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum. Genet., 34, 119124.[ISI][Medline]
van der Ven, K., Montag, M., Peschka, B. et al. (1997) Combined cytogenetic and Y chromosome microdeletion screening in males undergoing intracytoplasmic sperm injection. Mol. Hum. Reprod., 3, 699704.[Abstract]
Vogt, P.H., Edelmann, A., Kirsch, S. et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped different subregions in Yq11. Hum. Mol. Genet., 5, 933943
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge, UK.
Submitted on July 31, 2000; accepted on October 20, 2000.