Decreased fertilization rate and embryo quality after ICSI in oligozoospermic men with microdeletions in the azoospermia factor c region of the Y chromosome

Ron J.T. van Golde1, Alex M.M. Wetzels1, Ruurd de Graaf3, Joep H.A.M. Tuerlings2, Didi D.M. Braat1 and Jan A.M. Kremer1,4

1 Departments of Obstetrics and Gynaecology, 2 Human Genetics and 3 Epidemiology and Biostatistics, University Medical Centre Nijmegen, Nijmegen, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microdeletions of the azoospermia factor (AZF) region of the Y chromosome occur in between 1 and 29% of oligozoospermic and azoospermic men, and most deletions are found in the AZFc region. These men can father children when intracytoplasmic sperm injection (ICSI) is used, but the success rate is unclear. Thus, the success rate of 19 ICSI treatments in eight couples with a microdeletion in the AZFc region of the Y chromosome was analysed retrospectively. These were compared with a control group of 239 ICSI treatments in 107 couples undergoing ICSI treatment with ejaculated spermatozoa. The fertilization rate was significantly lower in the group of Y-deleted men (55%; 95% CI: 41–69%) compared with controls (71%; 95% CI: 67–74%; P < 0.01). The embryo quality was also significantly poorer among Y-deleted men (P < 0.001). Pregnancy, implantation and take-home baby rates were not significantly lower in the Y-deleted group. This study shows that ICSI in oligozoospermic men with microdeletions in the AZFc region of the Y chromosome leads to a lower fertilization rate and poorer embryo quality.

Key words: assisted reproduction outcome/ICSI/male infertility/microdeletions


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The azoospermia factor (AZF) region of the Y chromosome has been described previously (Tiepolo and Zuffardi, 1976Go), based on the observation of cytogenetic deletions in infertile men. Many studies have investigated the role of this region in spermatogenesis. Until now, it has been suggested that some of the genes in the Yq11 region control spermatogenesis (Ma et al., 1993Go; Reijo et al., 1995Go; Lahn and Page, 1997Go). This region can be divided into three non-overlapping regions of the Y chromosome: AZFa, b and c (Vogt et al., 1996Go).

Microdeletions of the Y chromosome occur in between 1% and 29% of subfertile men (Foresta et al., 1997Go; Kremer et al., 1997Go; Van der Ven et al., 1997Go). The frequency is dependent on the definition of male subfertility and on the choice of sequence tagged sites used for screening (Simoni et al., 1999Go). 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., 1998Go; Simoni et al., 1998Go). 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., 1999Go). 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., 1997Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
In our clinic, subfertile men are candidates for ICSI if their ejaculate contains <106 spermatozoa with propulsive motility (WHO a and b; World Health Organization, 1992Go). Since 1996, these men have been screened for microdeletions of the AZFa, b and c regions of the Y chromosome (Hoefsloot et al., 1997Go).

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., 1995Go; Abdelmassih et al., 1996Go; Stolwijk et al., 2000Go). 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., 1992Go). Fertilization rates were scored 18–22 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., 1989Go): 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Between 1996 and 1998, a total of 300 men with <106 spermatozoa with propulsive motility in their ejaculate (azoospermia) was screened for microdeletions in the AZF region of the Y chromosome. Among these men, eight (2.7%) appeared to have a microdeletion in the AZFc region of the Y chromosome. No microdeletions were detected in the AZFa or b regions. The mean sperm count among these men was 2.3x106 per ml, with 19% propulsive spermatozoa. All eight couples suffered from primary subfertility; the mean (± SD) age was 34 ± 5 years for females, and 36 ± 5 years for males.

The outcome after ICSI treatment until fertilization of couples with an AZFc deletion (n = 8) and control couples (n = 107) is shown in Table IGo. In the former group, 19 retrieved oocytes resulted in a significantly lower fertilization rate of 55% (95% CI: 41–69%) compared with 71% (95% CI: 67–74%) 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 IIGo).


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Table I. Results of ICSI treatment and fertilization rates of couples with Y-deletion, and in controls
 

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Table II. Embryo quality score of the transferred embryos and their number in each group of the scoring system in couples with Y-deletion, and in controls
 
In 17 of the 19 cycles, embryos could be transferred in the Y-deletion group. No fertilization occurred in two cases: in one case this was because the man had azoospermia at the time of the ICSI procedure; in the other case there was no fertilization of the single oocyte that had been injected. The pregnancy rate, implantation rate and take-home baby rate per embryo transfer were not significantly lower in the Y-deleted group (Table IIIGo).


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Table III. Pregnancy, implantation and take-home baby rate in couples with Y-deletion, and in controls
 
The take-home baby rate was also calculated per oocyte retrieval procedure, but was not significantly different for the Y-deleted and control groups (16% versus 25% respectively).

Five healthy female babies were born as a result of the three pregnancies.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study it was found that ICSI with ejaculated spermatozoa from men with a microdeletion in the AZFc region of the Y chromosome resulted in a reduced fertilization rate and embryo score.

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., 1995Go; Nagy et al., 1995Go). 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., 1999Go).

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., 1997Go; Simoni et al., 1997Go), 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., 1999Go). 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., 2000Go). 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., 1998Go), and the presence of AZFb deletion is a significantly adverse prognostic finding for TESE (Brandell et al., 1998Go). 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.


    Acknowledgments
 
The authors wish to thank A.Nap and L.Hoefsloot for their contributions to this study.


    Notes
 
To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: j.kremer{at}obgyn.azn.nl Back


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Submitted on July 31, 2000; accepted on October 20, 2000.