Meiotic chromosome abnormalities in fertile men: are they increasing? Laia Uroz, Ph.D.,a Osvaldo Rajmil, Ph.D.,b and Cristina Templado, Ph.D.a a
Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Medicina, Universitat Autonoma de Barcelona, Bellaterra; and b Servei d’Andrologia, Fundacio Puigvert, Barcelona, Spain
Objective: To determine the basal frequencies of meiotic chromosome abnormalities in fertile men. Design: Descriptive design. Setting: Research university laboratory and clinical andrology service. Patient(s): Seventeen fertile donors undergoing vasectomy. Intervention(s): Analysis of testicular biopsies. Main Outcome Measure(s): Meiotic chromosome abnormalities in metaphase I spermatocytes. Result(s): A total of 1,407 spermatocytes I was analyzed. The main meiotic abnormality was absence or low chiasma number of individual bivalents (23.4%), followed by structural (3.3%) and numerical (0.7%) abnormalities. Sex chromosomes and G-group chromosomes were the most commonly found as univalents at metaphase I. Statistically significant heterogeneity was found for meiotic abnormalities among fertile men, caused by interindividual variation in the level of dissociated sex chromosomes (ranging from 3.2% to 43.7%). The mean total percentage of meiotic abnormalities in spermatocytes I from fertile men was 27.4%, 1.7 times higher than those described a few decades ago in fertile and even in infertile men. Conclusion(s): Fertile men are a heterogeneous group for meiotic errors, with individuals showing percentages of meiotic abnormalities as high as 50%. From these findings, caution is recommended when using meiotic studies to diagnose and provide genetic counselling to patients consulting for infertility. (Fertil Steril 2011;95:141–6. 2011 by American Society for Reproductive Medicine.) Key Words: Meiotic abnormalities, fertile men, spermatocytes, achiasmate bivalents
Chromosome abnormalities are responsible for approximately 20% of male infertility (1), including somatic chromosome abnormalities and meiotic chromosome disorders present in germ cells of men with a normal karyotype. Meiotic chromosome disorders may cause partial or total meiotic arrest, resulting in oligozoospermia and azoospermia, respectively. Moreover, these abnormalities could lead to chromosomally abnormal spermatozoa, causing pregnancy loss and affected offspring. Meiotic studies allow the identification of meiotic abnormalities, and their use is especially relevant when diagnosing patients with idiopathic infertility. As a consequence of the increased frequency of de novo chromosome abnormalities found in pregnancies obtained by intracytoplasmic sperm injection (2), meiotic studies have been recommended in infertile men undergoing this technique (3,4). Despite their significance, direct meiotic studies are scarce, because they are carried out in spermatocytes obtained from a testicular biopsy. Most meiotic studies have applied conventional cytogenetic techniques to metaphase I (MI) spermatocytes from heterogeneous infertile series to determine the causes of their infertility (5–9). Received March 9, 2010; revised June 11, 2010; accepted June 16, 2010; published online August 2, 2010. L.U. has nothing to disclose. O.R. has nothing to disclose. C.T. has nothing to disclose. This research was supported by the Ministerio de Ciencia y Tecnologıa, Spain (BFI2002-01193) and the Generalitat de Catalunya, Spain (2005FI00399, 2009SGR-01107). Reprint requests: Cristina Templado, Ph.D., Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Medicina, Universitat noma de Barcelona, Bellaterra, Barcelona 08193, Spain (FAX: Auto 34-935811025; E-mail:
[email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.06.042
These studies revealed that meiotic arrest involved 18.5% of infertile men, and meiotic chromosome abnormalities affected 6% of cases (reviewed by Egozcue et al. [8]). In selected patients with severe oligoasthenozoospermia the incidence of meiotic chromosome disorders may increase to 17.5% (3). The most frequent meiotic chromosome disorder described in infertile men is abnormal chiasma frequency, observed for the first time as a decrease in the number of chiasmata affecting all bivalents in some or all cells (6, 10–17). Templado et al. (18) reported that this reduction in the number of chiasmata can affect only individual bivalents. A low number of chiasmata in MI results in monochiasmate medium-sized bivalents and in dissociated small univalents. The presence of two dissociated sex chromosomes has been described in all series of fertile and infertile men, and it has been suggested that percentages higher than 30% may lead to infertility (19). To our knowledge, cytogenetic meiotic analyses in MI spermatocytes from fertile men are scarce, and little is known about the baseline chromosomal abnormalities in these individuals. Several of these studies have been carried out in infertility clinic patients with ‘‘normal meiosis’’ (5, 9), and all of them reported the dissociation of sex chromosomes (5, 7, 9). One single series of eight fertile men (6) described dissociation of both sex chromosomes and small autosomes. We carried out meiotic chromosome studies, using the airdrying technique, in MI spermatocytes from 17 fertile male donors undergoing surgical vasectomy. The main purposes of this work were to investigate [1] the basal frequencies of meiotic chromosome abnormalities in fertile men, and [2] the differences between our results and those previously reported by other authors in similar studies.
Fertility and Sterility Vol. 95, No. 1, January 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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MATERIALS AND METHODS Subjects Testicular samples were collected from 17 healthy fertile men, selected from individuals attending the Andrology Service of Puigvert Foundation for vasectomy. The ages of the donors ranged from 28 to 47 years (mean, 38.3 years). All of them had fathered at least one naturally conceived child. Among these 17 donors, 7 were smokers (cases 1, 3, 4, 10, 14, 15, and 16), and none had been exposed to known mutagens or radiation. The frequencies of meiotic chromosome abnormalities in spermatocytes I and II from cases 1 and 2 have been described elsewhere (20) using the centromere-specific multicolor fluorescence in situ hybridization (FISH) technique, and these data have not been included in this work. The study was approved by our University and institutional ethics committees, and all donors signed an informed consent form before surgery.
System for Human Cytogenetic Nomenclature (23). To avoid technical artefacts, a conservative estimate of aneuploidy was calculated as being twice the hyperploidy frequency. Hypoploid metaphases were not included in this study, except those showing other chromosome abnormalities. Chiasma frequency was counted in each individual in 15 well-spread spermatocytes I with a normal karyotype (23,XY).
Statistical Analyses To evaluate the interdonor variation in the incidence of meiotic chromosome abnormalities, the two-tailed Fisher exact test was performed. To analyze correlation between age and abnormality frequencies in the 17 fertile donors, the Spearman correlation test was applied. To investigate whether there were statistically significant differences among individuals in terms of chiasma frequency, we used the Kruskal-Wallis nonparametric test.
MI Spermatocyte Preparation
RESULTS
Testicular tissue was collected under local anaesthesia during surgical vasectomy, through the same scrotal incision used for deferens resection. Meiotic cells were fixed using the air-drying technique (21) with minor modifications (22). Meiotic chromosome preparations were preserved at 20 C until 40 ,6-diamidino-2-phenylindole (DAPI) stain.
A total of 1,407 spermatocyte metaphases I was analyzed using the DAPI stain technique (Fig. 1). The DAPI-stain images are similarly informative to those reported in classic meiotic studies using uniform stain. In our laboratory, DAPI stain is used because the same spreads may be sequentially subjected to FISH techniques for further studies. The percentages of meiotic chromosome abnormalities are shown in Table 1, and abnormal karyotypes are listed in Table 2. Meiotic chromosome abnormalities (27.4%) affected principally the number of chiasmata of individual bivalents (85%), followed by structural (12%) and numerical (3%) abnormalities. Low chiasma count was observed both as [1] two separated univalents for sex chromosomes (22.1%) (Fig. 1A) or for small autosomes (0.4%) (achiasmate chromosomes) (Fig. 1B), and [2] medium-sized lineal bivalents with a single distal chiasma (0.9%) (monochiasmate bivalents). Structural abnormalities (3.3%) were principally acentric fragments (Fig. 1C), followed by chromosome breaks (Table 2). Trisomy (0.4%), found in most cases as a bivalent and an extra univalent, was significantly correlated with age (P¼.005). Significant interdonor heterogeneity was observed for the total meiotic chromosome abnormalities (range, 8.1%–46.5%) (P
