Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia

J.M. Vendrell1,3, F. García1, A. Veiga1, G. Calderón1, S. Egozcue2, J. Egozcue2 and P.N. Barri1

1 Reproductive Medicine Service, Department of Obstetrics and Gynaecology, Institut Universitari Dexeus, Universitat Autònoma de Barcelona, Passeig Bonanova 67, E-08017 Barcelona and 2 Department of Cellular Biology and Physiology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of meiotic abnormalities and their relationship with different spermatogenic parameters was assessed in 103 male patients with presumably idiopathic severe oligoasthenozoospermia (motile sperm concentration <=1.5x106/ml). Meiosis on testicular biopsies was independently evaluated by two observers. Meiotic patterns included normal meiosis and two meiotic abnormalities, i.e. severe arrest and synaptic anomalies. A normal pattern was found in 64 (62.1%), severe arrest in 21 (20.4%) and synaptic anomalies in 18 (17.5%). The overall rate of meiotic abnormalities was 37.9%. Most (66.7%) meiotic abnormalities occurred in patients with a sperm concentration <=1x106/ml. In this group, total meiotic abnormalities were found in 57.8% of the patients; of these, 26.7% had synaptic anomalies. When the sperm concentration was <=0.5x106/ml, synaptic anomalies were detected in 40% of the patients. In patients with increased follicle stimulating hormone (FSH) concentrations, total meiotic abnormalities occurred in 54.8% (synaptic anomalies in 22.6%). There were statistically significant differences among the three meiotic patterns in relation to sperm concentration (P < 0.001) and serum FSH concentration (P < 0.05). In the multivariate analysis, sperm concentration <=1x106/ml and/or FSH concentration >10 IU/l were the only predictors of meiotic abnormalities.

Key words: intracytoplasmic sperm injection /meiosis/meiotic chromosome abnormalities/oligoasthenozoospermia/sperm parameters


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In couples suffering from severe idiopathic oligoasthenozoospermia, results of assisted fertilization methods prior to the introduction of intracytoplasmic sperm injection (ICSI) were highly discouraging. In 1992, Palermo et al. reported the first ongoing pregnancies and deliveries following ICSI. Presently, ICSI is the most efficient assisted fertilization technique in cases of severe male factor infertility (Van Steirteghem et al., 1993aGo,bGo; Calderón et al., 1995aGo; Nagy et al., 1995Go). Furthermore, it has been shown that testicular sperm extraction plus ICSI allows the development of viable embryos and the establishment of viable pregnancies in apparently azoospermic (obstructive or non-obstructive) men (Schoysman et al., 1993Go; Devroey et al., 1994Go, 1995Go; Silber et al., 1994Go, 1995Go; Tournaye et al., 1994Go; Calderón et al., 1995bGo, 1997Go). Thus, genetically normal epididymal, testicular or ejaculated spermatozoa can be used for ICSI.

On the other hand, chromosome abnormalities may be responsible for male infertility. Indeed, the prevalence of somatic chromosome abnormalities detectable in the karyotype is 10 times higher in infertile men (5.3%) than in the general population (0.6%) (Egozcue, 1989Go). Numerical and structural anomalies of the sex chromosomes occur with a high frequency, mainly in azoospermic and severely oligoasthenozoospermic men (Retief, 1986Go). In addition, the incidence of synaptic chromosome anomalies restricted to the germ cell line and only detectable by meiotic studies is 4–7.7% in cases of male infertility (Egozcue et al., 1983Go; De Braekeleer and Dao, 1991Go).

The aim of this study was to evaluate the relationship between different spermatogenic parameters [testicular size, sperm concentration and motility, baseline serum follicle stimulating hormone (FSH) and count of mature spermatids per tubule] and the presence of meiotic abnormalities (severe arrest and synaptic anomalies) in patients with severe oligoasthenozoospermia and therefore likely to undergo ICSI.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The population studied consisted of 103 male patients who were consecutively referred to the unit of andrology of our institution because of the detection of extreme oligoasthenozoospermia (motile sperm concentration <=1.5x106/ml), presumably of idiopathic origin, in at least two previous sperm samples. The ICSI protocol was approved by the Ethical Committee of Institut Universitari Dexeus, and written informed consent was obtained from all participants.

In all patients, salient features of clinical history and physical examination were recorded as well as results of the following investigations: testicular volume, semen evaluation, baseline serum FSH concentration, and a testicular biopsy.

Testicular dimensions were measured with calipers. Testicular volume was estimated by the following formula (Ley and Leonard, 1985Go): V = 4/3 p (a/2)2 (b/2), where `a' equals the short testicular axis, and `b' equals the long testicular axis (in cm). Volumes are expressed in ml as the average of the two testicles.

Seminal fluid was collected by masturbation after 3–5 days of abstinence and in the absence of fever for 3 months before the study. All samples were analysed for volume (ml). Sperm concentration (x106/ml), motility (%) and motile sperm concentration (x106/ml) were assessed by means of the Makler chamber (Makler, 1978Go; Makler et al., 1980Go). Baseline serum FSH was measured by radioimmunoassay with a commercial kit (Abbott Laboratories, S.A.). A single blood sample for each patient was analysed.

A testicular biopsy was taken under local anaesthesia. The biopsy was performed unilaterally for the study of meiosis according to the method described by Egozcue et al. (1983) when a histological diagnosis was already available, and bilaterally for meiotic studies, for histological evaluation (Levin, 1979Go) and for counting of the number of mature spermatids per 20 tubules (Silber and Rodriguez-Rigau, 1981) when a previous histological diagnosis was not available. Meiosis on testicular biopsy material was independently evaluated by two observers. Three meiotic patterns were defined: normal meiosis and two meiotic abnormalities, i.e. severe arrest (presence of pachytenes and occasional spermatozoa, but no metaphase I figures found) and synaptic anomalies (chromosome pairing anomalies). Peripheral blood karyotypes were also evaluated.

Statistical analysis
The four quantitative parameters were dichotomized as follows: sperm concentration, <=1x106/ml versus >1x106/ml; motile sperm concentration, <=0.5x106/ml versus >0.5x106/ml; testicular volume, <15 ml versus >=15 ml (normal range); and serum FSH concentration, >10 IU/l versus <=10 IU/l (normal range). Meiotic abnormalities (arrest and synaptic anomalies) were analysed together and separately. The Student's t-test and the analysis of variance (ANOVA) were used for the comparison of quantitative variables and the chi-square test ({chi}2) for categorical variables. The independent predictive value of significant variables in the univariate analysis was assessed by means of a logistical regression model. All statistical tests were performed at the 5% level of significance. The Statistical Package for the Social Sciences (SPSS) for Windows was used for the analysis of data.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General characteristics of the population studied are shown in Table IGo. Ages of the patients ranged between 26 and 59 years and time of infertility between 1 and 22 years. The seminal volume varied from 0.8 to 9 ml and sperm concentration between 0.01 and 10.6x106/ml. Motile sperm concentration ranged between 0 and 1.5x106/ml being less than 1x106/ml in 88 (85.4%) of the 103 patients. Testicular volume ranged between 2.75 and 25.8 ml. It was <15 ml in 73 (70.9%) patients. Baseline serum FSH concentrations were determined in 89 patients ranging between 1 and 37.49 IU/l. Increased FSH concentrations (>10 IU/l) were found in 31 (34.8%) patients. Quantitative analysis of testicular biopsy was performed in 71 (68.9%) patients in whom the mean number of mature spermatids per tubule ranged between 0.15 and 21.5. Histological diagnosis was incomplete arrest of spermatogenesis in 91.3% of cases and hypo-spermatogenesis in the remaining cases.


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Table I. General characteristics of 103 male patients with severe oligoasthenozoospermia
 
A total of 70 men, randomly chosen, was karyotyped and a single case of 46,XY/47,XXY mosaicism was found. The distribution of meiotic abnormalities in the karyotyped group (normal 62.8%, arrest 18.6%, synaptic anomalies 18.6%) was not significantly different (P = 0.77) from that in patients not karyotyped (normal 60.6%, arrest 24.2%, synaptic anomalies 15.2%).

Meiotic studies in testicular samples were performed in all 103 patients. A normal pattern was found in 64 (62.1%), severe arrest in 21 (20.4%), and synaptic anomalies in 18 (17.5%) (Figure 1Go). The overall rate of meiotic abnormalities was 37.9%. There were no statistically significant differences among patients with normal or abnormal meiotic patterns in relation to mean age, length of time of infertility, seminal volume, percentage of motile spermatozoa, and number of mature spermatids per tubule (Table IIGo). However, in patients with sperm concentration <=1x106/ml, motile sperm concentration <=0.5x106/ml and serum FSH concentrations >10 IU/l, meiotic abnormalities were significantly more frequent than normal meiotic patterns (Table IIIGo). Total meiotic abnormalities and synaptic anomalies accounted respectively for 57.8 and 26.7% of patients with sperm counts <=1x106/ml and for 54.8 and 22.6% of patients with FSH concentrations >10 UI/l. After multivariate analysis, sperm concentration and serum FSH concentration appeared to be the only independent predictive factors of normal or abnormal meiotic patterns (Table IVGo). Motile sperm concentration was not a predictive factor because there was a statistically significant correlation with sperm concentration (r = 0.653; P < 0.0001).



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Figure 1. Metaphase I with a severe reduction in the number of chiasmata in several bivalents (arrows). The XY bivalent is indicated (original magnification x3000).

 

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Table II. Relationship between severe oligoasthenozoospermia and meiotic patterns
 

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Table III. Frequency distribution of meiotic patterns in different subgroups of patients
 

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Table IV. Results of logistic regression analysis. Risk factors for normal or abnormal meiosis
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although the incidence of somatic chromosome anomalies in infertile male patients with severe oligoasthenozoospermia or azoospermia has been well documented (Retief, 1986Go; Egozcue, 1989Go; Reijo et al., 1995Go), there are few studies on the incidence of meiotic chromosome abnormalities (Egozcue et al., 1983Go; De Braekeleer and Dao, 1991Go; Lange et al., 1997Go), especially in male ICSI candidates (Pieters et al., 1998Go).

The population studied suffered from severe non-obstructive oligoasthenozoospermia, i.e. low motile sperm concentration (mean 0.49x106/ml), moderately hypoplastic testicular volume (mean 11.74 ml); slightly elevated baseline serum FSH level (mean 10.7 IU/l), and low count of mature spermatids per tubule (mean 5.33) and met the criteria for inclusion in an ICSI programme. In this population, we found a high incidence of meiotic abnormalities (37.9%), 17.5% of which corresponded to synaptic anomalies. This incidence is ~2.5 times higher than in heterogeneous male infertility (Egozcue et al., 1983Go), suggesting that the higher frequency of meiotic chromosome abnormalities may often be responsible for infertility in patients with severe oligoasthenozoospermia.

The present results also demonstrate that most total meiotic abnormalities (66.7%) and synaptic anomalies (66.7%) were found in patients with sperm concentrations <=1x106/ml, affecting 57.8 and 26.7% of the patients respectively. When the sperm concentration was <=0.5x106/ml, 32% of patients had a normal meiotic pattern, 28% had a severe arrest and 40% had synaptic anomalies (P < 0.001). The incidence of synaptic anomalies is about five to six times higher than for the general infertile male population. These data suggest a relationship between the incidence of meiotic chromosome abnormalities and the degree of impairment of spermatogenesis. In our opinion, this high incidence of meiotic abnormalities in severe oligoasthenozoospermia makes meiotic studies in male infertility advisable before ICSI if they are available. Meiotic studies can be performed on testicular tissue samples (Egozcue et al., 1983Go; Lange et al., 1997Go) or in semen (Sperling and Kaden, 1971Go; Templado et al., 1980Go), although in the ejaculate sufficient material for a consistent diagnosis is only obtained in 25–30% of cases.

Meiotic abnormalities include severe meiotic arrest and synaptic anomalies. Severe meiotic arrest due to synaptic anomalies or to unknown causes results in an arrest of spermatogenesis, usually at the stage of primary spermatocyte, resulting in oligozoospermia or azoospermia. Synaptic anomalies limited to the germ cell line in patients with normal karyotype and, therefore, only detectable by meiotic studies, are usually associated with an incomplete meiotic arrest and oligoasthenozoospermia (or azoospermia in case of complete meiotic arrest) and/or subsequent production of chromosomically abnormal spermatozoa (sperm aneuploidies or diploidies), which may be responsible for male infertility, spontaneous abortions during the first trimester of pregnancy or fetal chromosome abnormalities (Egozcue et al., 1983Go). In the present series, a former history of spontaneous abortions was recorded in three cases (2.9%). All three patients had a normal karyotype but synaptic anomalies were found in two of them. The single patient with an abnormal karyotype (46,XY/47,XXY) had a normal meiotic pattern.

In our group of 31 patients with increased FSH levels (>10 IU/l), total meiotic abnormalities occurred in 54.8% and synaptic anomalies in 22.6%. In contrast with others (Novero et al., 1997Go), this high incidence of meiotic abnormalities leads us to recommend the inclusion of FSH measurement in the routine evaluation of infertile male patients with oligoasthenozoospermia for ICSI.

We conclude that in male infertility due to oligoasthenozoospermia, sperm concentrations <=1x106/ml and/or baseline serum FSH levels >10 IU/l are significant predictors of meiotic abnormalities. In these cases, meiotic chromosome studies can be performed to identify and characterize cytogenetic errors. This would allow the characterization of a particular `high-risk' group that needs genetic counselling and prenatal diagnosis in case of establishment of a pregnancy in ICSI cycles.

Meiotic studies are predictive, but they not provide information on the final status of the gametes. On the other hand, fluorescent in-situ hybridization (FISH) on decondensed sperm heads only provides information on the status of the chromosome pairs analysed with the probes used, may be difficult to perform on a significant number of spermatozoa in cases with very low sperm counts. If possible, we would recommend sperm chromosome studies, although they take a long time and are expensive, which makes them unpractical in clinical work.


    Acknowledgments
 
We are indebted to J.C.Surís, MD, for statistical analysis of data, to Ms M.J.Lafuente for secretarial assistance, to Marta Pulido, MD, for editing the manuscript and translating it into English and to the Càtedra d'Investigació en Obstetricia i Ginecologia (Universitat Autónoma de Barcelona) for its support.


    Notes
 
3 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 28, 1998; accepted on November 17, 1998.