Analysis of sperm chromosomes in six carriers of rare and common Robertsonian translocations^*

Approximately, 15% of couples in the reproductive age are infertile. About half of infertility cases results from the female side, followed by male reasons of the problem (20–30%), while the combined causes from both partners constitute 20–30% [2]. Beside the known infertility reasons, such as: disturbances in genetic background, changes in hormonal levels, infections, environmental factors or epigenetics, the etiology of many infertility cases remains unclear (idiopathic). Amongst the genetic background of reproductive failure, chromosomal aberrations (both structural and numerical) are observed quite frequently (6/1000 of live births). The most common structural aberrations observed in general population are chromosomal translocations with the frequencies 0.143% for reciprocal (RCT), and 0.123% for Robertsonian (RobT) translocations [24, 51, 52, 54]. In infertile males, those frequencies rise from 5 to 9 times [16, 47]. More than half of the translocation carriers have decreased semen parameters [48, 75]. At the same time, among infertile men with oligozoospermia, RobT is the most frequently detected aberration of autosomal chromosomes [47]. However, the details of its influence on fertility remain unclear – an infertile carrier can be a son or a brother of a fertile carrier of the same aberration within one family.

Robertsonian translocations can be inherited (mostly maternally) or appear de novo [60]. RobT arise as a consequence of centric fusion between two long (q) arms of acrocentric chromosomes, leading to formation of one derivative chromosome, followed by the loss of short (p) arms. Thus, centric fusion leads to reduced number of chromosomes in a karyotype of the carrier – 45 instead of 46 in human, but without any phenotypic effects (except infertility/reproductive problems). It was suggested that similar DNA sequences (repetitive or satellite III type) in short (p)arms of acrocentrics may facilitate the formation of RobT. However, detailed molecular mechanisms of their formation still remain unclear [36, 38]. In human, RobTs concern five acrocentric chromosomes: 13, 14, 15, 21 and 22, with short (p) arms built from repetitive satellite DNA and nucleolar organizing regions (NORs; sequences for 18S and 28S rRNA). A derivative chromosome may be dicentric (~90% of all RobT) with two centromeres from both acrocentrics (but only one active, resulting from a centric fusion after breakpoints in (p) arms of both involved chromosomes), or monocentric (observed in only ~10%) with one centromere from one of the involved acrocentrics (centric fusion after breakpoints: one in (p) arm of the first chromosome and the second in (q) arm of the other chromosome involved) [29, 38]. There is 15 different possible RobTs, including 10 heterologous (built from two different chromosomes; 95% of all cases) and 5 homologous (only 5% of all centric fusions, including isochromosomes) rearrangements. The most common heterologous rearrangements are rob(13;14)(q10;q10), and rob(14;21)(q10;q10) observed in approximately 91% of all Robertsonian translocations (Fig. 1B) [49]. It was suggested that a high frequency of those two common RobTs may result from a specific mechanism that involves homological recombination between opposite-directed DNA sequences in chromosomes 13 and 21 vs. 14 [18, 57, 61, 82]. Other types of RobTs are known as rare cases [3, 82, 90].

Fig. 1

Presence of Robertsonian karyotype determines the production of genetically unbalanced gametes. In RobT carriers, a trivalent pachytene figure is being formed during meiosis from chromosomes involved in a rearrangement and determines 8 different types of segregants, produced in three distinct segregation patterns: alternate, adjacent and 3:0 (Fig. 2). Only the alternate segregation leads to genetically normal (46 chromosomes) and balanced gametes (45 chromosomes including one derivative chromosome from two acrocentrics). All other types of segregants are genetically unbalanced and may lead to miscarriages or an offspring with abnormal karyotype [21]. Results of meiotic segregation pattern in male carriers of RobT, were published in at least 212 cases, so far. Majority of data concerned the two common Robertsonian translocations: der(13;14) (116 cases) and der(14;21) (38 cases) with mean value of about 20% of genetically unbalanced gametes. Other data are related to rare RobT cases, also with a mean value of about 20% of genetically unbalanced gametes [3, 4, 5, 10, 12, 13, 14, 16, 22, 23, 25, 26, 31, 32, 37, 40, 41, 43, 44, 45, 46, 50, 52, 53, 55, 56, 57, 62, 65, 66, 67, 68, 70, 71, 72, 73, 79, 80, 85, 86, 87, 89]. However, a range of results concerning the frequency of genetically unbalanced gametes in all analyzed RobT cases is much wider: from 0.2 to 49.1% [41, 89].

Fig. 2

Closely associated with the carrying of RobT is the problem of the increased frequency of spermatozoa with aneuploidy of chromosomes that are not involved in the rearrangement. The so-called “interchromosomal effect” (ICE) was analyzed inabout 30 cases of different rare and 90 cases of common RobTs [1, 4, 5, 8, 9, 11, 17, 19, 23, 26, 28, 31, 37, 39, 43, 44, 53, 57, 63, 64, 69, 72, 74, 77, 78, 81, 85, 86]. An increased frequency of aneuploid gametes can arise from a disrupted disjunction of chromosomes during meiosis and is one of the documented causes of reproductive failures [81]. The elevated level of aneuploidy of at least one of the chromosomes analyzed was observed in more than half of RobT carriers [1, 31, 80].

The published data regarding both the aneuploidy levels and the meiotic segregation patterns in carriers of various heterological RobTs are still insufficient and they do not meet the criteria for meta-analyzes [27]. Considering the frequency of RobT occurence and its influence on reproductive failure, research on new RobTs cases, especially of rare ones, is vital and important for genetic counseling. In this work, we examined the level of hyperhaploidy of chromosomes 7, 9, 18, 21, 22, X and Y in sperm cells of 6 RobT carriers: two unrelated carriers with rare rob(13;15), one with rare rob(13;22), and three carriers of the most common rob(13;14). The results were compared with the control data obtained froma group of 7 fertile men with normal karyotype. Additionally, for one of the carriers of rare rob(13;15) we offered an analysis of meiotic segregation pattern. For the other carriers, the meiotic segregation results were presented earlier [87].

In the present study we performed an aneuploidy screening in spermatozoa of three new cases of rare RobT: two rob(13;15) and one rob(13;22), and three cases of common rob(13;14) (Table 2). Also, theanalysis of meiotic segregation pattern in one case of rare rob(13;15) was performed (Table 3).

According to the literature, aneuploidy levels have been recognized in at least 126 RobT carriers, and meiotic segregation pattern in 212 RobT carriers, including rare and common cases [1, 3, 4, 5 8, 9, 10, 11, 12, 13, 14, 17, 19, 22, 23, 25, 26, 28, 31, 32, 37, 39, 40, 41, 43, 44, 45, 46, 50, 53, 55, 56, 57, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 77, 78, 79, 80, 83, 84, 85, 86, 87, 89]. However, these data – although seemingly numerous, are still insufficient and they do not meet criteria for meta-analyzes [27]. This is due to the fact that the data are very heterogenous, e.g., they concern carriers with different type of spermiogram abnormalities, most often the number of patients in the studied group is small, and the number of analyzed spermatozoa in each case differ significantly. Due to the relatively high frequency of RobTs carriers among infertile men, it is necessary to obtain more results with a considerably larger group of patients. This is especially important for carriers of rare RobTs because data for individual types of rare RobT are very little [41].

Published aneuploidy results for individual RobT carriers concern the most of chromosomes, but predominant analyzes were concentrated on hyperhaploidy of chromosomes 13, 15, 16, 18, 21, 22, X and Y (and also diploidy). These chromosomes have a special tendency to undergo non-disjunction, and/or these aneuploidies can generate potentially viable aneuploid embryos. So far, it has been known the results of aneuploidy analysis of 18, 20, 21, and sex chromosomes (plus diploidy) in only 6 cases of rare rob(13;15) [9, 23, 43, 57, 63, 83], and of chromosomes 6, 9, 15, 18 and 21 (plus diploidy) in only 4 cases of rare rob(13;22) [5, 11, 26, 86]. All these carriers had abnormal spermiograms, and there were only two (2/9) carriers who showed normal aneuploidy levels. In 79 cases of analyzed common rob(13;14) there were presented the aneuploidy results of 1, 2, 3, 7, 8, 12, 15, 17, 18, 21, 22 and sex chromosomes (plus disomy) (for chromosomes 1 and 15 available data are only from 3 carriers, for chromosomes 2, 3, 12 and 17 only from 2 carriers) [4, 8, 9, 17, 19, 23, 26, 28, 37, 43, 44, 53, 57, 74, 78, 85, 86]. Most of the analyzed cases (> 70%) were characterized by both elevated level of the hyperhaploidy of at least one of the tested chromosomes and abonormal spermiogram. From the data presented in the Table 1 it is clear that all our carriers R1-R6 demonstrated spermatogenic impairment. At the same time, we found the hyperhaploidy of at least one of the tested chromosomes in each of the carriers. Thus, our patients’ aneuploidy results were typical for infertile RobTs carriers.

The fact that most of infertile RobT carriers have an altered spermatogenesis, i.e. they have mostly oligoasthenoteratozoospermia (OAT) with varying degree of intensity, has been known since the beginning of the RobTs study [15, 41, 48, 56, 84]. Moreover, unusual ultrastructural sperm anomalies related to immaturity of the cells were observed [8]. Additionally, in approximately half of described RobT carriers, so-called: interchromosomal effect (ICE; increased frequency of spermatozoa with aneuploidy of chromosomes that are not involved in rearrangement) was observed [4, 6, 8, 17, 19, 26, 53, 57, 72, 77]. Increased frequency of aneuploid gametes arises from a disrupted disjunction of chromosomes during meiosis leading to an association of asynaptic chromosomal regions preferentially with the sex chromosomes or with the acrocentric ones [30, 34, 35]. It is known that the presence of aneuploidyis one of the documented causes of decreased semen concentration and reproductive failures [6, 11, 39, 43]. It can be postulated that in RobT carriers the ICE frequency is higher because of the increased probability of association of additional chromosome to the asynaptic regions during formation of the trivalent in prophase I. However, there are also literature data concerning RobT carriers without ICE phenomenon [1, 31, 80]. Also, an increased frequency of spermatozoa with aneuploidy can be observed in males with normal karyotype but those usually demonstrate reproductive failure [30, 34, 35]. Thus, each RobT carrier with ICE presence should be evaluated individually.

It is estimated that in most carriers of different nonhomologous RobTs (a total of 212 analyzed cases), the frequency of genetically normal/balanced segregants is close to 80%, although the results range from 51% to 99% [5, 41]. The highest frequency of spermatozoa after alternate segregation (>50%) and also the proportion of other segregantsis characteristic for all carriers of RobT regardless of the type of RobT [41]. Luciani f [42] suggested that the dominant number of normal and genetically balanced segregants can be the consequence of the fact that in the pachytene the trivalent of pairing chromosomes occurs in the cis configuration. It is also known that in the most carriers of nonhomologous RobT, the number of offspring with normal and balanced karyotypes are similar, which is compliant with theoretical expectations [20]. According to many authors, the above data can indicate a “homogenous segregation behaviour” of Robertsonian translocations independent of the chromosome pairs involved [5, 57, 67, 73].

So far, results of meiotic segregation pattern of rare rob(13;15) have been published only for 16 carriers [12, 13, 23, 41, 51, 52, 57, 62, 66, 68, 70, 87]. About 70% of the rob(13;15) carriers had normal/balanced sperm cells frequency above 75%, and our R1 carrier also belongs to this group (75.96% of alternate segregants in Table 3).

It is generally assumed that the reduced fertility in RobT carriers is mainly the consequence of the formation of chromosomally unbalanced sperm cells as a result of the segregation of the chromosomes involved in the meiotic prophase trivalent. The second critical impact on carriers’ reproductive ability seems to be closely related with the multifactorial nature of aneuploidy of chromosomes unrelated to those involved in the particular translocation. In some RobT carriers the relatively low frequency of aneuploidy of some chromosomes can be caused by a meiotic checkpoints triggering apoptosis. At the same time however, the elimination rate of these abnormal cells is not totally efficient, leading to both: a reduction in gamete production and not preventing aneuploidy as such. However, it should be noted that it is hard to clearly assess the influence of RobT on infertility, since there are also fertile carriers among the carriers of the same RobT. This could suggest that sperm production impairment is due to global interactions between the translocated chromosomes and the whole genome, perhaps influencing not shown epigenetic changes. Also, the genetic counselling (including prenatal genetic diagnostics, when necessary) for the RobT carriers, who have reproductive problems has to be implemented to evaluate the individual risk level of the miscarriages and/or a probability of having a healthy offspring.