1 Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Applied Biology, University of Siena, 2 C.N.R. Center for the Study of Germinal Cells, and 3 Regional Referential Center for Male Infertility, Azienda Ospedaliera Senese, Via T. Pendola, 62, 53100 Siena, Italy
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Abstract |
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Key words: consanguinity/genetic defects/male infertility/spermatozoa/ultrastructure
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Introduction |
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In the years 19902000, we examined, by transmission electron microscopy (TEM), the ejaculate of a large population of sterile patients. The ultrastructural observations of spermatozoa have been evaluated by the mathematical-statistical formula of Baccetti et al. (Baccetti et al., 1995). The family history of the patients has been considered. Many epidemiological and statistical studies of consanguineous marriages have been carried out in the past, particularly because this kind of marriage is traditional and very common in Middle Eastern culture. Inbreeding is known to have adverse effects on morbidity and mortality, particularly in relation to autosomal recessive disorders, which are more than twice as common in inbreeding (Hoodfar and Teebi, 1996
). Inbreeding increases the power to detect the role of a recessive or a quasi-recessive disease susceptibility factor (Genin and Clerget-Darpouxn, 1996
). Effects of inbreeding on the fertility and primary sterility of couples were investigated in marriages among uncles and nieces or among first or second degree cousins. On the contrary, the level of primary sterility was found to be similar both in consanguineous and in non-consanguineous couples (Edmond and De Braekeleer, 1993
).
In this paper, we present the results of the study performed on our large group of infertile patients, some of them characterized by consanguinity of various degrees. The sperm submicroscopical characteristics were analysed by TEM, in order to evaluate the relationship between consanguinity and sperm alterations.
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Materials and methods |
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We identified a group of male patients presenting different degrees of consanguinity in their history. Among them were siblings (1/2 consanguinity), sons of 1st degree cousins (therefore their chance of homozygosity was 1/16), sons of 2nd degree cousins (their chance of homozygosity was 1/64) and sons of 3rd degree cousins (their chance of homozygosity was 1/256).
Light and transmission electron microscopy
Ejaculates obtained by masturbation after at least 3 days of sexual abstinence were examined after liquefaction: for each sample, pH, sperm concentration and progressive motility at 30 min were evaluated. For electron microscopy, spermatozoa were fixed in cold Karnovsky fixative and maintained at 4°C for 2 h. The fixed semen was centrifuged at 3000 g for 15 min. The fixed pellet was removed from the centrifuge tubes, washed in 0.1 mol/l cacodylate buffer (pH 7.2) for 12 h, postfixed for 1 h at 4°C in 1% buffered osmium tetraoxide, dehydrated and embedded in Epon Araldite. Ultrathin sections were cut with an ultramicrotome (Supernova; Reickert Jung, Vienna, Austria) and collected on copper grids, stained with uranyl acetate and lead citrate, observed and photographed with a Philips CM 10 electron microscope (Philips Scientifics, Eindhoven, The Netherlands). Submicroscopical characteristics of sperm organelles were evaluated using the mathematical statistical formula of Baccetti et al. which calculates the number of spermatozoa free of structural defects (`healthy') (Baccetti et al., 1995). On the basis of this ultrastructural evaluation, the presence or absence of genotypic sperm defects was identified.
Statistical analysis
The frequencies of the values of the supposed genetic sperm defects were calculated and compared between the consanguineous patients and the non-consanguineous patient groups, using the 2-test.
In order to test the possibility that consanguinity may influence only the phenotypic characteristics, a randomized trial was designed. Non-consanguineous patients, ranging from 16 to 46 years, were grouped into seven classes of age, excluding the carriers of genotypic sperm defects. Subsequently, the consanguineous patients, excluding the carriers of genotypic sperm defects, were grouped in the same seven classes of age. The class widths were equal. According to the number of consanguineous individuals (cases) distributed in each age class, the same number of non-consanguineous ones (controls) was randomly assigned in the same correspondent class.
The ShapiroWilk W-test was used to determine whether values conformed to a normal distribution. Where the data were normally distributed, the Student t-test between independent groups was performed, where they were not, the MannWhitney U-test was used to compare the values of the percentage motility, the `healthy' sperm numbers and percentages in the carriers of only non-genetic sperm defects (phenotypic) between the two groups (cases and controls). Statistical significance was set as P < 0.05.
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Results |
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Various degrees of consanguinity
The 1/2 degree consanguinity (sib-ship) group included a pair of sterile siblings, carriers of `miniacrosome' (acrosome hypoplasia) (Table I), a defect clearly affecting the total sperm population and candidate for a genetic nature. A further very interesting case is a triplet of sterile siblings, affected by the genetic defect `stunted' tail (`stump`) in the total sperm population (Table I
). A third, more peculiar case, is that of a pair of siblings (1/2 consanguinity) with consanguineous parents (1/64 degree cousins). They represented a sib-ship with the characteristic of `detached tail' spermatozoa (Table I
). In our case record, we have also observed three pairs of siblings devoid of any genotypic sperm defect. In total (Table II
), the 13 observed 1/2 consanguineous males presented seven carriers of a genotypic sperm defect (55%).
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In total, the 62 consanguineous individuals included 17 cases of genotypic sperm defects (27.5%) (Figure 10). It is interesting to distribute the percentages of genotypic sperm defects in the four major classes of consanguinity explored in this study (Table II
). In siblings (1/2 of consanguinity), seven out of 13 patients were affected (54%), while the smaller degrees of consanguinity, due to consanguineous ancestors, gradually reduce the percentage of patients affected, passing from 30%, to 15%, to 0% (Figure 11
).
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Sperm ultrastructure and motility percentage in 1536 sterile men, free from genotypic defects: consanguineous versus non-consanguineous
Ultrastructural sperm examination of 45 of the 62 consanguineous patients confirmed the absence of genotypic defects and assessed in each patient only the presence of well-known defects of a phenotypic nature concerning the various sperm organelles, without affecting the whole sperm population. It is interesting to observe that among these 45 patients, one, a Mediterranean anaemia carrier, had two sisters affected by `Kartagener syndrome'. In the same group, three patients had sterile brothers and a fertile brother of one of these patients had two daughters affected by neuro-psychomotor disorders. Therefore it can be assumed that even if genetic sperm defects are not evident, the genome of some consanguineous sterile men contains some recessive factor which may become evident in homozygosity. In this case, the percentage of consanguineous carriers of genotypic sperm defects could be still larger. Also in this event, more than half of the consanguineous individuals would be affected by only phenotypic sperm defects, as are most of the non-consanguineous patients.
In order to substantiate the assumption that the two groups contain spermatozoa with only phenotypic defects, the ultrastructural and kinetic sperm characteristics of the 45 randomly allocated consanguineous individuals (cases) were compared with 45 non-consanguineous patients (controls) also not affected by genotypic defects. The mean percentage progressive motility for the spermatozoa in the consanguineous patient group was 20.35%, whereas in the other group it was 27.18%. When the percentage motility values of these two groups were compared, a significant difference was found using the MannWhitney U-test (P = 0.02), as well as the t-test (P = 0.03). However, as regards ultrastructure, the percentages of `healthy' spermatozoa, and the total numbers of `healthy' spermatozoa in the two groups (cases and controls), calculated by the formula of Baccetti et al. (Baccetti et al., 1995), did not differ (MannWhitney U-test).
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Discussion |
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In conclusion, the presence of genetic sperm defects is clearly related to consanguinity. Most of these defects, inducing male sterility, seem to be due to recessive autosomal mutations. This possibility has been envisaged both in `dynein arms deficiency syndrome' (Rott et al., 1983; Edwards et al., 1989; Walter et al., 1990
) and in `stunted tail syndrome' (Bisson et al., 1979
; Chemes et al., 1998
), in `round headed,' in `miniacrosome' and in `detached tails'. These alterations affect particularly the tail organelles, namely fibrous sheath, axonemal tubules and accessory fibres, all of them favoured targets of mutations. As a consequence, sperm motility in most of the genetic sperm defects is completely absent or abnormal. A further interesting observation is that the portion of genes shared and the chance of homozygosity by descent are significantly related to the amount of genotypic sperm defects.
The remaining group of 1506 non-consanguineous individuals presented genotypic sperm defects in only 15 cases. The cause of these genotypic sperm defects in individuals devoid of consanguineous relationships is intriguing. Evidently, the presence of a not-declared or distant relationship cannot be excluded in the carriers of these alterations. However, in addition, it could be assumed that a proportion, usually very small, of the lethal genetic defects leading to infertility in the natural population arises from novel mutations. Moreover, we cannot exclude, for at least two (`round head' and `fibrous sheath dysplasia') of these defects, both relatively frequent in the non-consanguineous category, the possibility of a recessive mutation carried by the X chromosome and transmitted by the mother.
All the 1536 sterile patients whose spermatozoa were devoid of genotypic defects and were therefore affected only by phenotypic defects showed the same structural sperm quality both in the consanguineous (45 individuals) and in the non-consanguineous (1491 individuals) groups. This observation substantiates the role of consanguinity in inducing the conditions of homozygosity necessary to reveal genetic structural sperm affections.
In contrast, the percentage sperm motility was significantly lower in the 45 consanguineous patients with spermatozoa affected only by phenotypic defects. This discordant characteristic, evident in consanguinity but apparently devoid of any ultrastructural basis, could be dependent on a genetic defect not (or not yet) revealed by electron microscopy.
A very important aspect of this matter is the chromosomal localization of the mutations involved in sperm sterility. Sometimes attempts have been made to relate these defects to cytogenetic disorders. However, convincing indications of a chromosome region involved in the presence or in the absence of a characteristic with a mutation-deletion system have not yet been produced. Only Moss et al. (1997) in the mouse, reported the localization in the X chromosome of the AKAP82 gene that encodes for a major fibrous sheath protein (Moss et al., 1997). At present, there are no reports of gene anomalies in human patients with fibrous sheath dysplasia (Chemes et al., 1998
). Baccetti et al. (1997) reported many sperm tail alterations (including dysplasia in the fibrous sheath) in the total sperm population of two unrelated carriers of the same autosomal (chromosome 9) pericentric inversion (Baccetti et al., 1997
). However, the large number of sperm characteristics involved suggests that this chromosome region could also have a general role in spermatogenesis. Ramesh and Verma (1996) observed that the same chromosome alterations have no phenotypic consequences in spermatozoa (Ramesh and Verma, 1996
).
Therefore, the current data on consanguinity substantiate the demonstration that several very peculiar, monomorphic and rare sperm defects, affecting the total sperm population of sterile men, have a genetic basis. Most of them are probably determined by a recessive autosomal mutation.
A consequence of this demonstration is that these mutations present in spermatozoa can be transmitted to the offspring by the ICSI technique (Anderson and Makinen, 2000) overcoming the usual impossibility of oocyte penetration by the affected spermatozoa. At least in the case of patients with high degrees of consanguinity, an accurate sperm ultrastructural examination of the ejaculate is recommended before starting an ICSI procedure.
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Acknowledgements |
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Notes |
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References |
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Submitted on December 29, 2000; accepted on March 22, 2001.