Cytogenetic determinants of male fertility BACKGROUND Cytogenetic abnormalities have been known to be important causes of male infertility for decades . Introduction Infertility is a significant problem , affecting up to 15 % of couples of reproductive age -LRB- de Kretser , 1997 -RRB- . For many years , it was assumed that most reproductive problems could be attributed to the female partner but research in recent years has demonstrated that ∼ 30 -- 50 % of infertility is caused by a male factor -LRB- Lipshultz and Howards , 1997 -RRB- . Treatment of male-factor infertility by in vitro fertilization -LRB- IVF -RRB- was largely unsuccessful . However , the development of intracytoplasmic sperm injection -LRB- ICSI -RRB- -LRB- Palermo et al. , 1992 -RRB- revolutionized the treatment of male infertility , providing many men the chance to father their own children . ICSI also greatly stimulated research into the causes of male infertility , and our knowledge has increased tremendously in the past decade . The development of new techniques has provided a wealth of information on the cytogenetics of human sperm . The human sperm -- hamster oocyte fusion system allowed the first analysis of human sperm chromosomes -LRB- Rudak et al. , 1978 ; Martin et al. , 1983 ; Brandriff et al. , 1985 ; Mikamo et al. , 1990 ; Templado et al. , 1996 -RRB- . This technique provides precise karyotypes in which numerical and structural abnormalities can be assessed for each chromosome -LRB- Fig. 1 -RRB- . Unfortunately , the technique is very difficult with only a handful of laboratories achieving success ; also the data yield , though of great significance , is low . Figure 1 : Q-banded karyotype of a normal 23 , X sperm . Fluorescence in situ hybridization -LRB- FISH -RRB- analysis with chromosome-specific DNA probes was developed in the 1990s , providing a faster , cheaper , easier alternative for detecting aneuploidy in human sperm -LRB- Martin et al. , 1993 ; Robbins et al. , 1993 ; Wyrobek et al. , 1994 ; Martin et al. , 1996 -RRB- . Also , sperm hampered by abnormalities in motility or other aspects of fertilization can be assessed using FISH analysis -LRB- Downie et al. , 1997 ; Egozcue et al. , 1997 ; Robbins et al. , 1997 ; Martin et al. , 1999a ; Hristova et al. , 2002 -RRB- . This is a simple technique that has been embraced by many laboratories , but it must be remembered that it is indirect : fluorescent signals , rather than chromosomes , are counted -LRB- Fig. 2 -RRB- . Figure 2 : Disomic XY sperm , with green X signal , red Y signal and a single blue chromosome 1 signal -LRB- an internal control to distinguish disomy from diploidy -RRB- . Single sperm polymerase chain reaction -LRB- PCR -RRB- or single sperm typing is a difficult , time-consuming technique but it can be used in a very powerful manner for specific studies on recombination in delimited areas of the genome -LRB- Shi et al. , 2001 , 2002 ; Tiemann-Boege et al. , 2006 -RRB- . The latest development to shed light on the causes of chromosomal abnormalities and infertility is meiotic analysis of the synaptonemal complex -LRB- SC -RRB- . Various important meiotic structures can be identified by the use of immunofluorescence . Antibodies against SCP1 -LRB- transverse elements -RRB- or SCP3 -LRB- lateral elements -RRB- can be used to visualize the SCs -LRB- the proteinaceous structure linking homologous chromosomes in prophase of meiosis I -RRB- . The centromere can be localized with CREST antisera . Most importantly , recent studies have demonstrated that antibodies against the DNA mismatch repair protein MLH1 identify the sites of meiotic exchange in SCs in both mouse -LRB- Baker et al. , 1996 ; Anderson et al. , 1999 -RRB- and human spermatocytes and oocytes -LRB- Barlow and Hultén , 1998 ; Lynn et al. , 2002 ; Tease et al. , 2002 -RRB- . The use of a multicentromeric FISH technique in conjunction with SC meiotic analysis is a powerful tool to study recombination and the fidelity of pairing in individual identified chromosomes -LRB- Sun et al. , 2004b , 2006b , 2007a ; Oliver-Bonet et al. , 2006 -RRB- -LRB- Fig. 3 -RRB- . These studies have provided some important information on the meiotic abnormalities in infertile men and the association between meiotic recombination and aneuploidy -LRB- Gonsalves et al. , 2004 ; Sun et al. , 2004a , 2006a , 2007b ; Codina-Pascual et al. , 2005 ; Martin , 2005 -RRB- . Figure 3 : -LRB- upper -RRB- Human pachytene spermatocyte with SCs shown in red , centromeres in blue and MLH1 foci in yellow . -LRB- lower -RRB- Subsequent cenM-FISH analysis permits identification of individual chromosomes so that recombination -LRB- MLH1 -RRB- foci can be analyzed for each SC . It is clear that cytogenetic abnormalities -LRB- both somatic and meiotic -RRB- are a major cause of male infertility . This review will summarize our knowledge of somatic chromosome abnormalities and their effects on sperm , susceptibility to sperm chromosome anomalies in chromosomally normal infertile men , and meiotic errors in translocation carriers and infertile men with a normal chromosome constitution . Materials and Methods Research publications from 1978 to 2008 , from PubMed and the author 's personal library , were reviewed . Methods for selecting and synthesizing the data were based on personal experience . Somatic chromosomal abnormalities Somatic chromosomal abnormalities are relatively common in humans . These can be numerical chromosomal abnormalities , such as an extra chromosome , or structural abnormalities such as a translocation . It has long been recognized that somatic chromosomal abnormalities are associated with infertility , an increased probability of pregnancy loss and the birth of handicapped children . The frequency of somatic chromosomal abnormalities in infertile men varies from 3 % to 19 % : 3 % in the cases of mild infertility and 19 % in men with non-obstructive azoospermia -LRB- NOA -RRB- -LRB- Yoshida et al. , 1997 -RRB- . Thus , it is imperative that chromosome karyotyping be performed in all infertile men so that they can be apprised of their risks . Somatic chromosomal abnormalities have definite consequences on the cytogenetic abnormalities observed in sperm and consequently those observed in newborns . Sex chromosomal abnormalities 47 , XYY Men with a 47 , XYY karyotype are generally fertile , but they are seen more frequently in infertile populations . Many 47 , XYY men produce normal children , but there has been no systematic analysis of children born to these men . Theoretically , 50 % of the sperm cells should be abnormal . In a study of 75 sperm karyotypes from a 47 , XYY male , we found no sperm disomic for a sex chromosome -LRB- Benet and Martin , 1988 -RRB- . Our results supported the hypothesis that the extra sex chromosome is eliminated during spermatogenesis . FISH analysis on the same male with 10 000 sperm studied demonstrated a small but significant increase for XY disomy to 0.6 % -LRB- Martin et al. , 1999b -RRB- . Other laboratories have demonstrated increased frequencies of sperm aneuploidy for the sex chromosomes ranging from 0.3 % to 15 % -LRB- Mercier et al. , 1996 ; Chevret et al. , 1997 ; Shi and Martin , 2000b -RRB- . The majority of the more-stringent three-color FISH studies have demonstrated a low risk of 24 , YY or 24 , XY sperm of ∼ 1 % . However , a recent study of two 47 , XYY men with severe oligozoospermia demonstrated a higher frequency of aneuploid sperm of 37 -- 38 % , with approximately one-half of the abnormalities caused by sex chromosomal aneuploidy -LRB- Gonzalez-Merino et al. , 2007 -RRB- . This group found that the frequency of sperm aneuploidy was concordant with the frequency of aneuploidy in preimplantation embryos -LRB- 32 % -RRB- . Since many 47 , XYY men have normal semen parameters , the severe oligozoospermia observed in these men may indicate more perturbations during meiotic pairing , subsequent loss of germ cells and the production of aneuploid sperm . 47 , XXY Patients with Klinefelter syndrome -LRB- 47 , XXY -RRB- or mosaic variants of Klinefelter syndrome have greatly impaired spermatogenesis , with severe oligozoospermia or azoospermia . Nevertheless , these men are candidates for ICSI , particularly with the new methods used for recovering testicular spermatozoa . Studies on sperm chromosomes from men with Klinefelter syndrome have also demonstrated that the extra sex chromosome appears to be eliminated during spermatogenesis . FISH analysis has demonstrated that the frequency of aneuploidy for the sex chromosomes varies from 1.5 % -LRB- Lim et al. , 1999 -RRB- to 7 % -LRB- Kruse et al. , 1998 -RRB- in sperm from Klinefelter mosaics , and 2 % -LRB- Rives et al. , 2000 -RRB- to 45 % -LRB- Estop et al. , 1998 -RRB- in the sperm of men who appear to have a non-mosaic 47 , XXY karyotype . The majority of babies born to 47 , XXY men have been normal although chromosomally abnormal fetuses have been reported -LRB- Ron-el et al. , 2000 ; Friedler et al. , 2001 -RRB- . Staessen et al. -LRB- 2003 -RRB- studied 113 embryos by preimplantation genetic diagnosis -LRB- PGD -RRB- and found a significantly increased frequency of autosomal and sex chromosomal abnormalities . Thus , there appears to be a small increased risk for these men . Structural aberrations Translocations Robertsonian translocations : Robertsonian translocation carriers have a fusion of the long arms of two acrocentric chromosomes . The fused short arms -LRB- containing redundant DNA -RRB- are generally lost so that the carrier has a balanced chromosomal constitution with 45 chromosomes . When the chromosomes pair during meiosis , they do so as a trivalent , and the resulting gametes can be chromosomally normal or aneuploid with an extra or missing chromosome q arm . This can cause translocation Down 's syndrome or Patau 's syndrome , e.g. sperm karyotyping studies in our laboratory , and others have demonstrated that the actual frequency of unbalanced sperm in seven men is lower than theoretically expected , with 3 -- 27 % of sperm being unbalanced because of the translocation -LRB- Martin , 1995 ; Ogawa et al. , 2000 -RRB- . Similarly , FISH studies in 71 Robertsonian translocation heterozygotes have demonstrated that 7 % to 40 % are unbalanced , with a mean of 15 % unbalanced -LRB- Rousseaux et al. , 1995 ; Blanco et al. , 2000 ; Escudero et al. , 2000 ; Honda et al. , 2000 ; Vegetti et al. , 2000 ; Frydman et al. , 2001 ; Morel et al. , 2001 ; Pellestor et al. , 2001 ; Acar et al. , 2002 ; Baccetti et al. , 2002 ; Anton et al. , 2004 ; Anahory et al. , 2005 ; Douet-Guilbert et al. , 2005 ; Machev et al. , 2005 ; Brugnon et al. , 2006 ; Hatakeyama et al. , 2006 ; Moradkhani et al. , 2006a , b ; Ogur et al. , 2006 ; Chen et al. , 2007 ; Nishikawa et al. , 2008 -RRB- . Thus , all Robertsonian translocations have relatively similar segregation behaviors despite the participation of different acrocentric chromosomes . The risks of chromosomal imbalance at prenatal diagnosis are even lower , with generally only 1 -- 2 % of paternally derived Robertsonian translocations being unbalanced -LRB- Boué and Gallano , 1984 -RRB- . Even though the risks are low , prospective parents deserve to be informed of them as the abnormalities can be devastating , and trisomy 13 fetuses have been detected after ICSI using sperm from a father with a Robertsonian translocation -LRB- I n't Veld et al. , 1997a -RRB- . Also , a special category of Robertsonian translocation , if found , would nullify the usefulness of ICSI : a Robertsonian translocation between the same two chromosomes , e.g. two chromosomes 13 , would produce only abnormal embryos , trisomy 13 or monosomy 13 , with no hope of long-term survival . A case with this particular problem was discovered in the Netherlands after three unsuccessful ICSI attempts -LRB- I n't Veld et al. , 1997b -RRB- . Reciprocal translocations : reciprocal translocations occur when there are exchanges of chromosome material between any chromosomes . During meiosis , four chromosomes must pair in reciprocal translocation heterozygotes and the resulting segregations have a higher frequency of unbalanced chromosomes than Robertsonian translocations . Sperm karyotyping studies of 37 reciprocal translocation heterozygotes have shown that 19 -- 77 % of spermatozoa are unbalanced -LRB- Estop et al. , 1995 ; Martin and Spriggs , 1995 ; Cifuentes et al. , 1999 -RRB- . FISH analyses of chromosome segregations in 99 reciprocal translocation heterozygotes have also shown a large range in the frequency of unbalanced sperm , from 37 % to 91 % -LRB- Goldman and Hultén , 1993 ; Spriggs and Martin , 1994 ; Rousseaux and Chevret , 1995 ; Estop et al. , 1997 , 1998 , 1999 , 2000 ; Van Hummelen et al. , 1997 ; Blanco et al. , 1998a , 2000 ; Martini et al. , 1998 ; Mercier et al. , 1998 ; Cifuentes et al. , 1999 ; Giltay et al. , 1999 ; Honda et al. , 1999 ; Vegetti et al. , 2000 ; Oliver-Bonet et al. , 2001 , 2002 , 2004 ; Pellestor et al. , 2001 ; Cora et al. , 2002 ; Geneix et al. , 2002 ; Trappe et al. , 2002 ; Baccetti et al. , 2003 ; Escudero et al. , 2003 ; Lim et al. , 2003 ; Anton et al. , 2004 ; Morel et al. , 2004 ; Douet-Guilbert et al. , 2005 ; Machev et al. , 2005 ; Brugnon et al. , 2006 ; Midro et al. , 2006 ; Yakut et al. , 2006 ; Perrin et al. , 2007 ; Wiland et al. , 2007 ; Nishikawa et al. , 2008 ; Vozdova et al. , 2008 -RRB- . In one study , four male family members of a kindred segregating a chromosome 15 ; 17 translocation were studied by FISH analysis -LRB- Cora et al. , 2002 ; Vozdova et al. , 2008 -RRB- . The segregation patterns were very similar in all four men , with ∼ 50 % of sperm chromosomally unbalanced . Also , Morel et al. -LRB- 2004 -RRB- found similar frequencies of imbalance of 37 % and 43 % in two brothers heterozygous for a chromosome 7 ; 8 translocation . These studies demonstrate that the risk of meiotic imbalance is primarily determined by the characteristics of the chromosomes involved , and the break-point positions . They also demonstrate the reproducibility of the method . In both karyotyping and FISH studies , the mean frequency of sperm with unbalanced chromosomes is ∼ 50 % in reciprocal translocation carriers . Many of these imbalances are not compatible with survival , and the average frequency of paternally derived translocation imbalances at prenatal diagnosis is 12 % -LRB- Boué and Gallano , 1984 -RRB- . However , some translocations have higher risks of imbalance and survival , and all have serious consequences of mental and physical handicaps . A number of fetuses with unbalanced segregations of reciprocal translocations have been reported after ICSI -LRB- Baschat et al. , 1996 ; Meschede et al. , 1997 -RRB- . Because the frequency of chromosome abnormality is very high , some men carrying reciprocal translocations have undergone PGD to implant only chromosomally normal or balanced embryos . Studies comparing the frequency of chromosome abnormalities in sperm and embryos from reciprocal translocation carriers show a close agreement in the abnormality frequencies -LRB- Escudero et al. , 2003 -RRB- . Inversions Inversions occur when two chromosome breaks occur in the same chromosome and the breaks heal in an inverted order . All the genes are present in the correct number but difficulties arise during pairing of homologous chromosomes during meiosis . If a single crossover occurs in the inverted region of the paired chromosomes , offspring with chromosomal duplications and deficiencies can result from recombinant chromosomes . Paracentric inversions : paracentric inversions occur when both break points are in one chromosome arm . If a single crossover occurs within the inversion pairing loop , one-half of the gametes are normal , one-quarter are acentric -LRB- and would be lost -RRB- and one-quarter are dicentric -LRB- which could lead to a break between the centromeres and chromosome imbalance -RRB- . Paracentric inversions are rarely reported , since they can only be detected by the use of banding procedures -LRB- Pettenati et al. , 1995 -RRB- . Some investigators have suggested that paracentric inversions in man are generally harmless -LRB- Madan , 1995 -RRB- ; however , recombinant chromosomes have been observed in newborns , and the risk of viable recombinants has been estimated to be 3.8 % -LRB- Pettenati et al. , 1995 -RRB- . Only two men with paracentric inversions have been studied by sperm karyotyping -LRB- Martin , 1986 , 1999 -RRB- . Neither showed any recombinant chromosomes in sperm , suggesting that either an inversion loop was not formed or that crossing over was suppressed within the loop . Similarly , one case has been studied by FISH analysis and 1 % of sperm were recombinant , with both dicentric and acentric chromosomes observed -LRB- Devine et al. , 2000 -RRB- . Thus , the risk for paracentric inversions appears to be small.Pericentric inversions : pericentric inversions occur when the chromosome breaks occur in both chromosome arms and include the centromere in the inversion . Sperm karyotyping studies have been performed in seven men heterozygous for pericentric inversions -LRB- Martin , 1999 -RRB- . Four men had no recombinant chromosomes and three men had frequencies of imbalance varying from 11 % to 31 % . FISH studies have been performed in 24 pericentric inversion carriers with the frequency of recombinant chromosomes varying from 0 % to 54 % -LRB- Anton et al. , 2007 ; Chantot-Bastaraud et al. , 2007 ; Morel et al. , 2007 -RRB- . The inversions that produce recombinant chromosomes are , in general , large inversions encompassing more than half of the chromosome length . An overall risk at prenatal diagnosis has been estimated to be 10 -- 15 % -LRB- Daniel et al. , 1989 -RRB- , but it is clear that the risks are dependent on the individual inversion . Interchromosomal effects A number of researchers have suggested that there is an increased frequency of chromosomal abnormalities unrelated to the structural abnormality and have termed this an ` interchromosomal effect ' -LRB- Aurias et al. , 1978 -RRB- , e.g. an increased frequency of trisomy 21 children born to Robertsonian translocation carriers . Sperm karyotype studies have not shown any support for an interchromosomal effect in translocation -LRB- Martin and Spriggs , 1995 -RRB- or inversion carriers -LRB- Martin , 1999 -RRB- , despite the fact that all chromosomes can be analyzed . However , the number of sperm karyotypes is limited with a maximum of 548 sperm cells analyzed in one translocation carrier -LRB- Spriggs et al. , 1992 -RRB- . Thus , a small interchromosomal effect would be missed by these studies . Studies employing FISH analysis have the advantage of much larger sample sizes , with a few hundred to several thousand sperm assessed . There is some suggestion for an interchromosomal effect in 58 % -LRB- 21/36 studied for an interchromosomal effect -RRB- of Robertsonian translocations and 64 % -LRB- 35/55 -RRB- of reciprocal translocations studied for the segregation of other chromosomes by FISH analysis , since an increased frequency of numerical abnormalities was observed in at least one of the chromosomes evaluated . For inversions , only one of seven cases demonstrated a possible interchromosomal effect -LRB- Amiel et al. , 2001 -RRB- . However , it is doubtful whether this is a true interchromosomal effect , since carriers of translocations and inversions are often infertile , with altered sperm profiles . In fact , almost all the Robertsonian translocation carriers studied for an interchromosomal effect had abnormal semen profiles , with only three normospermic men -LRB- none of whom demonstrated an interchromosomal effect -RRB- . In reciprocal translocation heterozygotes , abnormal semen profiles were much more common among the cases demonstrating an interchromosomal effect -LRB- 67 % -RRB- compared with those that did not -LRB- 11 % -RRB- . Infertile men with oligozoospermia , asthenozoospermia or teratozoospermia are known to have an increased frequency of chromosome abnormalities in their sperm -LRB- Moosani et al. , 1995 ; Martin , 1996 ; Aran et al. , 1999 ; Pang et al. , 1999 ; Nishikawa et al. , 2000 -RRB- . Therefore , it is possible that the increased frequency of numerical chromosomal abnormalities in some carriers of structural rearrangements may be related to infertility factors rather than the rearrangement . On the other hand , clear examples of translocations with asynaptic segments pairing with sex chromosomes or autosomes suggest that this type of interchromosomal effect may be causing the infertility -LRB- Oliver-Bonet et al. , 2005a -RRB- . PGD studies have demonstrated that embryos of translocation carriers have a high frequency of genetic imbalance . Some studies have suggested an interchromosomal effect for Robertsonian translocation carriers , since there appears to be a higher frequency of aneuploidy -LRB- Conn et al. , 1998 , 1999 ; Gianaroli et al. , 2002 -RRB- , whereas others have not found a significant increase -LRB- Scriven et al. , 2001 ; Munné et al. , 2005 -RRB- . In summary , an interchromosomal effect may be a reality for some translocations , especially in infertile patients , and sperm chromosome studies may be useful to determine the level of risk . Y chromosome microdeletions Tiepolo and Zuffardi -LRB- 1976 -RRB- first recognized that six azoospermic men had deletions of the long arm of the Y chromosome , large enough to be recognized by light microscopy . Since that time , it has been determined that the majority are Yq microdeletions and therefore require analysis by molecular means . Most studies demonstrate that 4 -- 14 % of azoospermic or severely oligozoospermic men have an Yq microdeletion , making this a major contribution to male infertility -LRB- Foresta et al. , 2001 -RRB- . Of significance is the fact that all sons are expected to be infertile . Three regions have been delineated as azoospermic factor -LRB- AZF -RRB- a , b and c with AZFc holding the best prognosis for viable testicular sperm retrieval -LRB- Foresta et al. , 2001 ; Vogt , 2004 -RRB- . An interesting aspect of Y chromosome microdeletions is the instability of the chromosome suggested by recent studies . Patsalis et al. -LRB- 2002 -RRB- studied 12 mosaic 46 , X/46 , XY patients with Turner syndrome trails or sexual ambiguities and 4/12 had Y chromosome microdeletions . Ferlin et al. -LRB- 2007 -RRB- performed sperm FISH studies in 11 men with AZFc deletions and found only 33 % Y-bearing sperm compared with 49 % in controls , and a significant increase in disomic XY sperm and sperm nullisomic for the sex chromosomes . More research is clearly indicated , but this suggests that men with a Y chromosome microdeletion may have an increased risk of 45 , X and 47 , XXY offspring as well as mosaic offspring because of loss of the Y chromosome . Germinal mosaics Men with a normal somatic karyotype may still have an abnormal cell line in their testes . These men are called ` germinal mosaics ' , and it is difficult to discover them without a testicular biopsy . Studies have discovered that 1 -- 17 % of infertile men are germinal mosaics -LRB- Chandley et al. , 1976 ; Hendry et al. , 1976 -RRB- , so this is still a risk after a normal karyotype result , but the risks for abnormal offspring would be lower than those for non-mosaic individuals . Infertile men with a normal karyotype With the advent of ICSI , it has become clear that infertile men with a normal somatic karyotype also have an increased risk of chromosomally abnormal sperm . Moosani et al. -LRB- 1995 -RRB- were the first to report that infertile men with a normal 46 , XY karyotype have an increased risk for autosomal and sex chromosomal abnormalities in their sperm . More than 30 FISH studies have confirmed this association of increased sperm aneuploidy frequencies in 46 , XY infertile men -LRB- Aran et al. , 1999 ; Pang et al. , 1999 ; Nishikawa et al. , 2000 -RRB- . Most studies have reported the increase of sperm chromosome abnormalities in infertile men to be about three times higher than in control donors -LRB- Moosani et al. , 1995 ; Lahdetie et al. , 1999 ; Acar et al. , 2000 -RRB- . Reports based on prenatal diagnosis of ICSI pregnancies -LRB- Van Steirteghem et al. , 2002 -RRB- and newborns -LRB- Aboulghar et al. , 2001 -RRB- have indicated the risk of de novo chromosome abnormalities to be ∼ 2 % to 3 % , which is 3-fold higher than that in normal pregnancies . Thus , the increased frequency of chromosome abnormalities in ICSI pregnancies and newborns mirrors the increased frequency observed in the sperm of infertile ICSI patients . Furthermore , studies have indicated that these chromosome abnormalities are of paternal origin -LRB- Van Opstal et al. , 1997 -RRB- , underscoring the fact that chromosomally abnormal sperm in ICSI patients become chromosomally abnormal fetuses and children . The first studies of sperm chromosomal abnormalities in infertile men tended to lump all types in infertility together . However , it is possible that some subsets of infertility have an elevated risk of sperm chromosomal abnormalities whereas others do not . We have studied men with asthenozoospermia -LRB- motility defects ; Hristova et al. , 2002 -RRB- , teratozoospermia -LRB- abnormalities of form ; Templado et al. , 2002 -RRB- , various degrees of oligozoospermia -LRB- low concentration ; Martin et al. , 2003b -RRB- and azoospermia -LRB- no sperm in the ejaculate ; Martin et al. , 2003a -RRB- . To our surprise , we found that men with any type of infertility had an increased frequency of sperm chromosomal abnormalities , varying from 2 to 10 times higher than that of control donors . Thus , it seems that any perturbation of spermatogenesis confers an increased risk of aneuploid sperm . One rare type of teratozoospermia appears to confer a very high risk is men with a high percentage of macrocephalic , multinucleated , multiflagellate sperm . A number of studies have reported very high frequencies of aneuploidy and polyploidy in these men -LRB- 50 -- 100 % -RRB- -LRB- Benzacken et al. , 2001 ; Devillard et al. , 2002 ; Lewis-Jones et al. , 2003 -RRB- . It has also been suggested that abnormalities of the centrosome may exist in surgically retrieved sperm , a problem that may lead to increased mitotic non-disjunction and mosaicism in resulting embryos -LRB- Silber et al. , 2003 -RRB- . Utility of aneuploidy assessment in sperm There have been a number of studies which have found a high frequency of sperm aneuploidy in men who subsequently fathered chromosomally abnormal children -LRB- Blanco et al. , 1998b ; Martinez-Pasarell et al. , 1999a , b ; Moosani et al. , 1999 ; Soares et al. , 2001 ; Nagvenkar et al. , 2005 -RRB- . For example , in our original study of infertile men , one male had a frequency of 24 , XY sperm that was 9-fold higher than controls -LRB- Martin , 1986 -RRB- . This man subsequently had ICSI and fathered a pregnancy that resulted in a 47 , XXY fetus -LRB- Moosani et al. , 1999 -RRB- . Gianaroli et al. -LRB- 2005 -RRB- studied sperm aneuploidy and correlated it to results in blastomeres after PGD in couples . They found a higher incidence of monosomies and trisomies in embryos from microepididymal sperm aspiration and testicular sperm extraction sperm , and aneuploidy for the sex chromosomes increased proportionally to the severity of the male-factor condition . These authors suggested that it is important to include sperm FISH analysis in preliminary tests given to infertile couples , especially in the case of repeated IVF failures . These preliminary studies suggest a correlation between sperm aneuploidy frequencies and ICSI outcome . However , unpublished results from our research laboratory demonstrated that only 10 % of infertile men with a normal karyotype have a 24 , XY sperm frequency that is ≥ 5 times that of controls , and only 3 % have a frequency ≥ 10 times that of controls . Thus , it appears that a high frequency of sperm aneuploidy is relatively rare in 46 , XY infertile men . Recombination analysis by single sperm PCR Our studies , and those of others , have shown that the frequency of aneuploidy in sperm is elevated for the sex chromosomes compared with the autosomes , for both fertile -LRB- Martin et al. , 1991 ; Spriggs et al. , 1996 ; Scarpato et al. , 1998 -RRB- and infertile men -LRB- Shi and Martin , 2000a ; Hristova et al. , 2002 -RRB- . The XY bivalent normally has only one crossover in the pseudoautosomal region during meiosis . If recombination is reduced or absent for these chromosomes , they may be particularly susceptible to non-disjunction . Indeed , it has been shown that 47 , XXY of paternal origin is associated with a decreased recombination frequency -LRB- Hassold et al. , 1991 ; Lorda-Sanchez et al. , 1992 -RRB- . For a direct test of whether recombination is associated with non-disjunction in human sperm , we performed single sperm PCR analysis for a sex specific locus -LRB- STS/STS pseudogene -RRB- and a pseudoautosomal locus -LRB- DXYS15 -RRB- -LRB- Shi and Martin , 2001 -RRB- . Individual unisomic sperm -LRB- 23 , X or Y -RRB- were isolated using a FACStarPlus flow cytometer into PCR wells . To identify disomic 24 , XY sperm , 3-color FISH analysis was performed with probes for chromosomes X , Y and 1 . The 24 , XY cells were identified using fluorescence microscopy , each disomic sperm was scraped off the slide using a glass needle attached to a micromanipulator and then put into a PCR well . Hemi-nested PCR analysis of the two markers was performed to determine the frequency of recombination . The frequency of recombination between the two DNA markers was 38 % for the normal unisomic sperm compared with 25 % for the 24 , XY disomic sperm that had undergone non-disjunction . This difference was highly significant , and demonstrates that lack of recombination in the pseudoautosomal region is associated with XY non-disjunction and the production of aneuploid sperm . SC analysis The discovery that lack of recombination is associated with non-disjunction is significant because it provides a definite molecular correlate with aneuploidy . The next logical step would be to assess recombination in other chromosomes . However , this is extremely difficult and time-consuming by single sperm PCR analysis . Luckily , new immunocytogenetic techniques allow assessment of recombination and chromosome pairing by visualization of the SC in early meiosis -LRB- Barlow and Hultén , 1998 ; Lynn et al. , 2002 ; Sun et al. , 2004a , 2006b , 2007b -RRB- . The SC can be analyzed throughout the stages of prophase -LRB- leptotene , zygotene , pachytene , diplotene -RRB- to assess the progress of meiosis and the fidelity of chromosome pairing and synapsis . The frequency and location of recombination sites on individual chromosomes can also be analyzed by use of antibodies to the mismatch repair protein MLH1 -LRB- Baker et al. , 1996 ; Marcon and Moens , 2003 ; Sun et al. , 2004b -RRB- combined with cenM-FISH to identify individual chromosomes -LRB- Fig. 3 -RRB- . This technique provided the first recombination maps for every autosome -LRB- Sun et al. , 2004b , 2006b -RRB- . Studies in a number of laboratories have demonstrated that normal healthy men have ∼ 50 recombination foci per pachytene cell , with a wide range in the mean number of recombination sites per cell and in the number of sites in individual pachytene cells -LRB- Barlow and Hultén , 1998 ; Lynn et al. , 2002 ; Sun et al. , 2004b , 2006b -RRB- . This number of recombination sites is very similar to chiasma counts at diakinesis -LRB- Hultén , 1974 ; Laurie , 1985 ; Laurie and Hultén , 1985 -RRB- and also to the corresponding genetic length obtained from linkage data -LRB- Kong et al. , 2002 -RRB- . SC analysis in translocation carriers Reciprocal translocations are known to generate meiotic disturbances that affect both quantitative and qualitative sperm production . The chromosomes involved in a reciprocal translocation must pair as a quadrivalent , which is clearly visible at the SC level during pachytene -LRB- Oliver-Bonet et al. , 2005a ; Sun et al. , 2005b -RRB- . Meiotic studies of infertile men carrying chromosomal rearrangements have shown that quadrivalent configurations have different degrees of asynapsis around the break points . It has also been observed that these regions sometimes interact with the sex body . Associations between an autosome and the sex body can be visualized as continuous proteinaceous filaments that connect the two together . This association has been suggested to be the cause of infertility in carriers -LRB- Gabriel-Robez et al. , 1986 -RRB- . It has been hypothesized that this may be due to the activation of X-linked genes , or a spreading of inactivation to the autosomes . Study of the SC permits analysis of these two hypotheses . The X and Y chromosomes pair in only a small region , with the rest of the chromosomes remaining unpaired . Meiotic sex chromosomes are inactivated with many proteins locating to the sex body -LRB- Oliver-Bonet et al. , 2005b -RRB- . It has been hypothesized that the association of an autosome with the sex body causes reversal of the sex body inactivation and allows expression of some genes , with lethal results for the cell -LRB- Lifschytz and Lindsley , 1972 -RRB- . Others have suggested a spreading of sex body inactivation toward the autosomes connected to the sex body -LRB- Jaafar et al. , 1993 -RRB- . Recent studies have shown normal timing and progression of condensation through the pachytene stage for the sex body in a carrier of a t -LRB- Y ; 1 -RRB- translocation -LRB- Sun et al. , 2005b -RRB- and for the sex body associated with the quadrivalent in a t -LRB- 13 ; 20 -RRB- carrier -LRB- Oliver-Bonet et al. , 2005b -RRB- . These results , together with the fact that there is a strong relationship between sex body inactivation and XY condensation -LRB- Fernandez-Capetillo et al. , 2003 -RRB- , do not support the model of gene activation on the X chromosomes . In addition , the observation that autosome arms invading the sex body show gradual heterochromatinization , mimicking the behavior of the sex chromosomes , suggests that spreading of inactivation to the autosome is taking place . It has been suggested that transcriptional repression might not be an exclusive mechanism of the sex chromosomes , but rather a general mechanism that acts to silence any asynapsed region in the cell -LRB- Baarends and Grootegoed , 2003 -RRB- . Such a general mechanism has been described in Neurospora crassa -LRB- Shiu et al. , 2001 -RRB- , and in the mouse -LRB- Turner et al. , 2005 -RRB- . It is possible that a similar mechanism is operative in humans and that the spreading of sex body inactivation toward the translocated chromosome is a consequence of asynapsed regions within the quadrivalent attaching to the sex body . In this case , genes important in meiosis might be repressed with resultant destruction of the cell . The meiotic process in two translocation carriers with different fertility outcomes , one normozoospermic and the other azoospermic , have been compared with interesting results -LRB- Oliver-Bonet et al. , 2005a -RRB- . A significant number of quadrivalents were attached to the sex body in the azoospermic carrier , whereas such an association was never detected in the normozoospermic carrier . In addition , this normozoospermic patient displayed heterologous synapsis within the quadrivalent . Thus , it appears that unpaired regions within the quadrivalent are likely to be detected by the pachytene checkpoint , so asynapsed regions seek each other out and try to pair , in order to escape the checkpoint and avoid apoptosis of the cell . The other option to shelter unsynapsed regions from the checkpoint is association with the sex body , but this situation may lead to anomalies disrupting the proper segregation of the chromosomes . SC analysis in infertile men Studies of infertile men have demonstrated a number of meiotic errors . Most of these meiotic studies have been performed on men with NOA , but some men with obstructive azoospermia and oligoasthenoteratozoospermia have also been studied . Approximately one-half of the men with NOA have no meiotic cells -LRB- Gonsalves et al. , 2004 ; Sun et al. , 2005a ; Topping et al. , 2006 , 2007b -RRB- . In those with meiotic cells , the progression of meiosis is altered , with significantly more cells observed in the early stages of prophase -LRB- leptotene and zygotene ; Gonsalves et al. , 2004 ; leptotene and zygotene ; Ferguson et al. , 2007 ; Sun et al. , 2007b -RRB- . This suggests problems in the development of the lateral and transverse elements of the SC , leading to difficulties in pairing and synapsis of homologous chromosomes . Sun et al. -LRB- 2005a , c , 2007b -RRB- determined that NOA men have a significant increase in the proportion of pachytene cells with unsynapsed regions . Also , a number of studies have demonstrated that NOA males have a significant reduction in the frequency of MLH1 foci compared with controls -LRB- Gonsalves et al. , 2004 ; Sun et al. , 2005a , 2007b -RRB- , although two studies have not observed this reduction -LRB- Codina-Pascual et al. , 2005 ; Topping et al. , 2006 -RRB- . Achiasmate bivalents -LRB- with no crossovers -RRB- have been observed with a significantly elevated frequency in NOA males compared with controls -LRB- leptotene and zygotene ; Gonsalves et al. , 2004 ; Sun et al. , 2005a , 2007b ; Ferguson et al. , 2007 -RRB- . For example , Sun et al. -LRB- 2007b -RRB- observed that 29 % of pachytene cells had at least one bivalent with no recombination foci , compared with 5 % in controls . This is a dangerous situation , since without a crossover there is no mechanism to ensure orderly chromosome segregation at metaphase I . This could lead to engagement of the pachytene checkpoint and meiotic arrest or sperm aneuploidy . A minimum number of recombination sites -LRB- crossovers -RRB- for correct alignment and segregation of the chromosomes is one per chromosome arm -LRB- except for the short arms of the acrocentric chromosomes , which rarely recombine -RRB- . Achiasmate chromosomes -LRB- those without a recombination site -RRB- are rare in humans , except for the sex chromosome pair , which has a visible recombination site only 56 -- 93 % of the time -LRB- Codina-Pascual et al. , 2005 ; Sun et al. , 2006a ; Ferguson et al. , 2007 -RRB- . However , when achiasmate chromosomes are observed in autosomes , they occur most frequently for chromosomes 21 and 22 -LRB- Codina-Pascual et al. , 2006 ; Sun et al. , 2006a -RRB- . This is interesting , since it parallels data on aneuploidy in human sperm . Both studies on human sperm karyotypes -LRB- Martin and Rademaker , 1990 -RRB- and FISH analysis -LRB- Williams et al. , 1993 ; Spriggs et al. , 1996 ; Blanco et al. , 1998b ; Scarpato et al. , 1998 -RRB- have demonstrated that chromosomes 21 and 22 , and the sex chromosomes have the highest frequency of aneuploidy . This strengthens the association between lack of recombination and aneuploidy . However , these studies on meiotic recombination and sperm aneuploidy have not been performed on the same men . A more robust test of the association between meiotic recombination and aneuploidy would be to correlate both analyses in the same men . When we performed just such a study in vasectomy reversal patients , we found no correlation between meiotic recombination in individual chromosomes and sperm aneuploidy for the same chromosome -LRB- Sun et al. , 2008 -RRB- . We hypothesized that our population of fertile men may not have reached the threshold of meiotic abnormalities necessary to demonstrate the relationship between recombination and aneuploidy , since achiasmate bivalents were rare in this group of men . Ferguson et al. -LRB- 2007 -RRB- found a significant inverse correlation between meiotic recombination for the sex chromosomes and XY disomy in sperm . They also found a significant relationship between the frequency of achiasmate chromosome 21 and sperm disomy 21 . This analysis was performed in a mixed group of control donors -LRB- vasectomy reversals -RRB- and infertile men . When we studied men with NOA , we also demonstrated that a low frequency of meiotic recombination in the sex bivalent was significantly correlated with a high frequency of aneuploidy for the sex chromosomes -LRB- unpublished results -RRB- . Thus , it is possible that men with more dramatic meiotic abnormalities are infertile because of loss of meiotic cells at the pachytene checkpoint , and also face an increased risk of aneuploid sperm because of non-recombinant cells that escape the checkpoint but are still susceptible to errors of chromosome segregation . Egozcue 's group in Barcelona has performed elegant research in this area . They suggest that the best candidates for a meiotic study would be : infertile males with a normal karyotype and unexplained infertility , and among them , infertile males with normozoospermia and long-term sterility , or IVF failures -LRB- embryonic factor , no fertilization , repeated IVF failure -RRB- , or infertile males with a severe oligozoospermia -LRB- < 5 × 106 sperm/ml -RRB- or a severe oligoasthenozoospermia -LRB- < 1.5 × 106 motile sperm/ml -RRB- -LRB- Egozcue et al. , 2005 -RRB- . Funding R.H.M. holds the Canada Research Chair in Genetics and her research is funded by the Canadian Institutes of Health Research . Funding to pay the Open Access publication charges for this article was provided by the Canadian Institutes of Health Research .

O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

male infertility
meiosis
sperm aneuploidy
sperm chromosome abnormalities


The UNESCO Universal Declaration on Bioethics and Human Rights : Perspectives from Kenya and South Africa In October 2005 , UNESCO -LRB- the United Nations Educational , Scientific and Cultural Organization -RRB- adopted the Universal Declaration on Bioethics and Human Rights . This was the culmination of nearly 2 years of deliberations and negotiations . As a non-binding instrument , the declaration must be incorporated by UNESCO 's member states into their national laws , regulations or policies in order to take effect . Based on documentary evidence and data from interviews , this paper compares the declaration 's universal principles with national bioethics guidelines and practice in Kenya and South Africa . It concentrates on areas of particular relevance to developing countries , such as protection of vulnerable persons and social responsibility . The comparison demonstrates the need for universal principles to be contextualised before they can be applied in a meaningful sense at national level . The paper also assesses the ` added value ' of the declaration in terms of biomedical research ethics , given that there are already well-established international instruments on bioethics , namely the World Medical Association Declaration of Helsinki and the CIOMS -LRB- Council for International Organizations of Medical Sciences -RRB- guidelines on biomedical research . It may be that the added value lies as much in the follow-up capacity building activities being initiated by UNESCO as in the document itself . Introduction In October 2005,1 the United Nations Educational , Scientific and Cultural Organization -LRB- UNESCO -RRB- adopted the Universal Declaration on Bioethics and Human Rights . The preamble states : `` It is necessary and timely for the international community to state universal principles that will provide a foundation for humanity 's response to the ever-increasing dilemmas and controversies that science and technology present for humankind and the environment '' -LSB- 20 , p. 3 -RSB- . As the declaration is non-binding , this universal foundation will be implemented at the level of the nation-state . The onus is on UNESCO 's member states to incorporate the declaration 's provisions into their national laws , regulations or policies . This paper examines the synergies between the declaration and bioethics regulation in two countries , Kenya and South Africa . Before the UNESCO declaration was adopted , international instruments such as the World Medical Association -LRB- WMA -RRB- Declaration of Helsinki and the International Ethical Guidelines for Biomedical Research Involving Human Subjects of the Council for International Organizations of Medical Sciences -LRB- CIOMS -RRB- were already well-established in bioethics -LSB- 5 -RSB- . Indeed , the Helsinki declaration and the CIOMS guidelines are noted in the preamble of the UNESCO declaration -LSB- 20 -RSB- . These instruments have had a significant influence on the national bioethics policies of developing countries -LSB- 1 -RSB- , including Kenya and South Africa . The objectives of this paper are twofold . The first is to assess the translation of universal principles into national practice . The comparison between the UNESCO declaration and Kenyan and South African bioethics forms the basis for this analysis . The second is to locate the ` added value ' of the declaration , particularly to developing countries , given the pre-existence of international level bioethics documents . Empirically based , the paper draws on documentary evidence and interviews conducted in Kenya and South Africa in 2005 and 2006 . Background The empirical research for this paper was carried out as part of a larger doctoral project on the global governance of human genomic and biomedical research and in particular the part developing countries play in this . The theoretical framework for this project is provided by international relations , a sub-discipline of political science . While international relations theory is not referred to overtly in this paper 's analytical sections , it provides the context for the understanding of universality contained therein . That is , universality is explored pragmatically , with regard to the relationship between broad principles negotiated at international level and their subsequent adaptation to national level policy and practice , rather than philosophically , in terms of universal versus pluralist moral reasoning . ` Bioethics ' What exactly is meant by ` bioethics ' is notoriously difficult to determine . This is illustrated by the fact that the UNESCO declaration contains no clarification of the term . While a definition appeared in earlier drafts , the impossibility of reaching a consensus on wording necessitated its being left out of the final version -LSB- 18 -RSB- . For the purposes of this paper , bioethics is understood specifically in terms of the regulation of biomedical research . The UNESCO declaration itself has a wider remit , covering medical practice and protection of biodiversity and the environment as well as research ethics -LSB- 20 -RSB- . Since the overall doctoral project is concerned chiefly with human subjects research , however , these broader considerations will not be discussed here . Methodology and Limitations Much of contemporary international relations theory concerns the roles of both state and non-state actors in global governance mechanisms . Reflecting this , the doctoral fieldwork consisted of 70 semi-structured interviews with a range of persons considered to be stakeholders in genomics and bioethics in France , the United Kingdom , Kenya and South Africa .2 The sample was chosen to reflect different societal perspectives on genomics and bioethics and thus consisted of those who formulate policy at international and national levels , those who must implement these policies in laboratories and ethics committees , those who claim to represent public concerns and those with commercial interests . The breakdown of sectors was as follows : policy-makers -LRB- 20 -RRB- , scientists -LRB- 17 -RRB- , ethicists3 -LRB- 18 -RRB- , civil society representatives -LRB- 12 -RRB- and businesspersons -LRB- 3 -RRB- .4 Of these 70 interviews , the data used in this paper draw on only 22 and come mostly from those with members of research ethics committees . Thus they are illustrative rather than representative . Studying a declaration as it evolves5 makes for exciting and contemporary research . It also carries limitations , however ; in this instance , fieldwork may have been conducted too early to enable an assessment of the social and political impact of the UNESCO declaration in Kenya and South Africa . In the wider context of the doctoral research project , this limitation will be addressed by the inclusion of UNESCO 's previous declarations on genomics and genetics , which have had longer to become established within national policy frameworks .6 It is worth noting that the research process became by default an awareness-raising exercise , in that many of the people interviewed were previously unaware of the bioethics declaration , or indeed its predecessors . Previous Studies The UNESCO declaration features in several publications . Developing World Bioethics devoted a whole issue to the draft text in September 2005 . The articles were largely critical , questioning the content of the draft declaration , how it had been put together and whether UNESCO was the right body to be taking on such an endeavour -LSB- 8 -RSB- . Professor Henk ten Have , Director of the Division of Ethics of Science and Technology at UNESCO , responded that the journal 's contributors were perhaps not au fait with how UN agencies work -LSB- 14 -RSB- . After the declaration was adopted , Herman Nys wrote an editorial for the European Journal of Health Law outlining its basic tenets and comparing it to the European Convention on Human Rights and Biomedicine . He emphasised the obligations of states to take on the declaration , despite its being legally non-binding -LSB- 10 -RSB- . Professor ten Have has himself written about the declaration , in the wider context of UNESCO 's activities in ethics . In his article in the Kennedy Institute of Ethics Journal he described how these activities fall into three areas , namely the adoption of normative instruments such as the declaration and their subsequent implementation through national level capacity building and awareness raising -LSB- 6 -RSB- . There has been a growing literature in recent years on research ethics in developing countries . A few examples , taken from the British Medical Journal , the Bulletin of the World Health Organization and PloS Medicine , will serve to illustrate that contextualisation of international instruments at national level and capacity for ethical review have been among the major concerns raised . Sylvester Chima has suggested that international guidelines need to be interpreted legislatively at local and regional levels and has thus called on the African Union to pass binding directives that are nevertheless adaptable to the laws of each state -LSB- 3 -RSB- . Kass et al. , in a case study published in January 2007 reviewing the practices of research ethics committees in several African countries , including Kenya and South Africa , highlighted insufficient funding and training as the biggest challenges facing these committees and proposed that workshops be set up on how to apply ethical principles at local levels . They also called for more empirical investigation of ethics in African research -LSB- 7 -RSB- . Peter Singer and Solomon Benatar , in a 2001 article on the Helsinki declaration , contended that building capacity in research ethics will have far more impact on ethical standards than `` revisions of this or any other research ethics code , '' implying that declarations themselves are of limited use unless the capacity exists to implement them -LSB- 15 , p. 747 -RRB- . Zulfiqar Bhutta has also argued that strengthening local capacity in bioethics is key to promoting ethical health research in developing countries -LSB- 1 -RSB- . The UNESCO Declaration The Universal Declaration on Bioethics and Human Rights was adopted `` by acclamation '' at the UNESCO General Conference on 19 October 2005 -LSB- 20 , p. 5 -RRB- . This was the end of a process that began with an invitation by the 2001 General Conference to the UNESCO International Bioethics Committee to report on the possibility of elaborating a universal instrument on bioethics . The Committee recommended that this instrument be declaratory in nature -LRB- that is , non-binding -RRB- and the drafting process was launched in January 2004 -LSB- 17 , 19 -RSB- . Thus the actual negotiation period lasted under 2 years . The declaration is aimed primarily at states , but can also be implemented by `` individuals , groups , communities , institutions and corporations , public and private '' where appropriate -LSB- 20 , p. 6 -RSB- . It covers a wide range of bioethical principles , several of which had already become customary in bioethics and feature in documents such as the Helsinki declaration and the CIOMS guidelines -LRB- informed consent , for example -RRB- . Some of the principles of particular relevance to developing countries will be elaborated further below . Bioethics in Kenya and South Africa Kenya and South Africa were chosen as fieldwork destinations because of their significant involvement in genomics and bioethics at local , national , regional and international levels . Both countries have recently adopted national guidelines on bioethics : in Kenya the 20047 National Council for Science and Technology -LRB- NCST -RRB- Guidelines for Ethical Conduct of Biomedical Research Involving Human Subjects in Kenya -LRB- ` human subjects guidelines ' -RRB- and the 2005 Ministry of Health -LRB- MoH -RRB- Kenya National Guidelines for Research and Development of HIV/AIDS Vaccines -LRB- ` vaccines guidelines ' -RRB- and in South Africa the Department of Health -LRB- DoH -RRB- Ethics in Health Research : Principles , Structures and Processes , which were drawn up by members of both the Department and the Interim National Health Research Ethics Committee , appointed under the National Health Act of 2003 -LSB- 13 -RSB- . Both Kenya and South Africa decided that national bioethics guidelines were necessary partly in order to protect poor and marginalised people from being exploited by unscrupulous researchers -LSB- 9 , 13 -RSB- . Among other texts , the guidelines draw on the Helsinki declaration , the CIOMS guidelines8 and several documents from the World Health Organization and the United States , but are tailored to their national contexts , with specific provisions addressing the vulnerabilities that may have enabled past abuses -LSB- 9 , 11 , 13 -RSB- . Participants from ethics committees cited a similar assortment of guidelines and regulations -- international , regional and national -- as influential , including some from Europe , the United Kingdom and Australia . Synergies between the UNESCO Declaration and Bioethics in Kenya and South Africa As the UNESCO declaration is non-binding , if its principles are to be applied universally they will necessarily have to be reflected in national level documents and systems . This section compares the main tenets of the declaration that are of special relevance for developing countries with Kenyan and South African bioethics policy and practice . The comparison has two purposes . The first is to illustrate how internationally determined , universal principles might be implemented at national levels in developing countries . The second is to show to what extent these principles were already reflected in national systems , before the adoption of the declaration . Community Consent -- Article 6 The UNESCO declaration states that for a research project on a group or community , agreement from representatives may be sought , in addition to that of the individual participants -LSB- 20 -RSB- . Community Advisory Boards facilitate this in some areas of Kenya -LRB- interviews , K_06:2005 and K_25:2005 -RRB- and dialogue with community members through such boards is a requirement for HIV/AIDS vaccines research -LSB- 11 -RSB- . The South African guidelines stipulate community involvement and consultation for research involving ` collectivities ' , on issues such as ownership of data and distribution of benefits and harms -LSB- 13 -RSB- .9 Vulnerability -- Article 8 This article holds that `` individuals and groups of special vulnerability should be protected and the personal integrity of such individuals respected '' -LSB- 20 , p. 8 -RSB- . The Kenyan human subjects guidelines give special instructions concerning research with underdeveloped communities , prisoners , married women in rural areas and pregnant or lactating women -LSB- 9 -RSB- , while the vaccines guidelines state that the vulnerable and poor must be protected from exploitation -LSB- 11 -RSB- . The South African guidelines invite ethics committees to be `` especially vigilant when considering research proposals involving vulnerable populations '' -LSB- 13 , Preamble -RSB- and contain detailed provisions for research involving pregnant women , foetuses , prisoners and vulnerable communities -LSB- 13 -RSB- . Cultural Diversity and Pluralism -- Article 9 Under the declaration these should be given `` due regard '' -LSB- 20 , p. 8 -RSB- . Again , the national guidelines contain specific examples of what this might entail . The Kenyan human subjects guidelines , in the context of gaining informed consent from married women in rural communities , remind researchers that each of Kenya 's 42 tribes will have `` unique sociocultural backgrounds '' -LSB- 9 , p. 11 -RSB- , while the South African guidelines , in a section on indigenous medical systems , call on researchers to respect the cultures and traditional values of all communities -LSB- 13 -RSB- . The Kenyan vaccines guidelines are less detailed and , in a similar vein to the UNESCO declaration , simply require that research teams be sensitive to `` sociocultural issues , '' without specifying what these issues might be -LSB- 11 , p. 30 -RSB- . Social Responsibility -- Article 14 This article of the UNESCO declaration is considered particularly innovative -LSB- 6 -RSB- . It is perhaps not surprising , then , that it should prove more specific than any of the national guidelines as to how social responsibility could be implemented . Where the Kenyan and South African guidelines require research to be relevant to study populations and each country in general , by addressing either `` health needs '' -LRB- Kenya -RRB- -LSB- 9 , pp. 13 , 16 -RSB- or `` broad health and development needs '' -LRB- South Africa -RRB- -LSB- 13 , p. 3 -RSB- , the declaration states that scientific and technological progress should advance : access to healthcare and medicines ; adequate nutrition and water ; improved living conditions and environment ; elimination of marginalisation and exclusion ; and reductions in poverty and illiteracy -LSB- 20 -RSB- . Benefit Sharing -- Article 15 The declaration is similarly specific with regard to the sharing of benefits , giving seven examples of what form this could take , including sustainable assistance to research participants and provision of new health products stemming from research -LSB- 20 -RSB- . It is the latter of these examples which features most prominently in the national guidelines . In Kenya , if research produces positive results , treatments should be available to participants -LSB- 9 -RSB- . Also , before HIV/AIDS vaccine research can take place , the `` availability , affordability and accessibility '' of its potential products must be considered -LSB- 11 , p. 30 -RSB- . Research proposals in South Africa must indicate whether there is a reasonable likelihood that participants will benefit from the research and whether they will receive long-term therapy after the study -LSB- 13 -RSB- . More widely , research findings must `` be translatable into mechanisms for improving the health status of South Africans '' -LSB- 13 , p. 3 -RSB- . In practice , ethics committees in both countries try to assess the extent to which participants will benefit from a research project -LRB- interviews , K_25:2005 and SA_19:2006 -RRB- , although they do not always find this a straightforward endeavour -LRB- interviews , SA_10:2006 and SA_35:2006 -RRB- . Transnational Practices -- Article 21 This article calls on states to combat illicit trafficking of `` organs , tissues , samples , genetic resources and genetic-related materials '' -LSB- 20 , p. 10 -RSB- . Transfer of tissues overseas has been a particular concern in both Kenya and South Africa , as reflected in their guidelines . The Kenyan vaccines guidelines aim to eliminate the unauthorised transfer of research materials and to this end contain a sample Biological Material Transfer Agreement -LSB- 11 -RSB- . Under chapter 810 of the South African National Health Act , an appendix to the DoH guidelines , the Health Minister may regulate `` the importation and exportation of tissue , human cells , blood , blood products or gametes '' -LSB- 12 , pp. 70 , 62 -RSB- . Ethics committees in both countries take tissue transfer equally seriously , examining closely any research protocol that involves movement of samples across borders . Generally they prefer a project to train people to analyse data in-country -LRB- interviews , K_17:2005 , K_21:2005 , K_25:2005 , SA_19:2006 , SA_21:2006 and SA_30:2006 -RRB- . Bioethics Education , Training and Information -- Article 23 Under this article states are invited to foster bioethics training and education `` at all levels '' and to encourage information dissemination on bioethics -LSB- 20 , p. 11 -RSB- . Such training and education is perhaps more widespread in South Africa than Kenya , although the UNESCO National Commission was in the process of setting up a bioethics centre to serve the East Africa region at the time of interview -LRB- interviews , K_01:2005 and K_16:2005 -RRB- . Several South African universities run courses in ethics or bioethics and two training initiatives , IRENSA and SARETI , serve sub-Saharan Africa as a whole -LRB- including Kenya -RRB- .11 All three set of guidelines advocate bioethics training -LSB- 9 , 11 , 13 -RSB- . Universal Principles in the National Context The South African guidelines read : The challenge to international research ethics is the development of universal rules for research at a time when health care is being delivered within very different health care systems and in a multicultural world in which people live under radically different economic conditions . -LSB- 13 , p. 7 -RSB- This paper asks whether the UNESCO declaration has met this challenge . Although promulgated with the expressed purpose of stating universal principles , the declaration acknowledges that such cultural and economic differences exist , through its articles on community consent , cultural diversity , vulnerability , social responsibility and benefit sharing . The Kenyan and South African guidelines demonstrate how the declaration might be applied at state level , with their prescriptions concerning , for example , how researchers should engage with communities and which particular members of society should receive special attention as vulnerable persons . That states may need to adopt particular interpretations of the declaration 's principles in order to realise them in national and local contexts is highlighted by ten Have in his paper on UNESCO 's ethical activities . He describes the adoption of the declaration as only the `` start of a long process of detailed elaboration and consequent application '' -LSB- 6 , p. 342 -RSB- and in the context of the articles on consent and social responsibility states : As principles they are universally adopted , but in practice their application must be tailored in multiple ways to accommodate different types of research and health care , categories of patients and problems , and cultural settings and traditions . -LSB- 6 , pp. 342 -- 343 -RSB- The need for any national implementation of the UNESCO declaration to be adequately contextualised is brought out in the interview data from Kenya and South Africa , as are differences in opinion as to the usefulness of international guidelines in general . In Kenya , an academic scientist and advisor to both the government and UNESCO stated that it would be necessary to work with those government officers responsible for effecting international documents in law , in order to `` translate it -LSB- the declaration -RSB- into what is happening locally '' -LRB- interview , K_13:2005 -RRB- . Similarly , the head of a research institution attested : Something which became more and more legalistic in the detail and binding but which ignored local realities would be unhelpful . Something which tried to establish clear , agreed principles , which had been widely consulted , not just between member states , but with the kind of communities that are affected , would be very useful . -LRB- Interview , K_07:2005 -RRB- In South Africa , one ethics committee member thought that , given different cultural contexts and sensitivities , declarations such as the UNESCO one would have to be formulated as generally as possible to enable universal applicability -LRB- interview , SA_08:2006 -RRB- . Another was of the view that national and local ethics guidelines are `` the things to follow , '' because what is applicable in one country may not be applicable in another -LRB- interview , SA_19:2006 -RRB- . Several saw at least some value in the UNESCO endeavour , but cautioned that universal principles must not be applied unthinkingly . Perhaps mirroring ten Have 's prediction that elaboration of the declaration will be a long process , they described how working out how to apply such principles in different contexts is often the most challenging aspect of implementing international instruments -LRB- interviews , SA_10:2006 , SA_17:2006 and SA_24:2006 -RRB- . Added Value of the UNESCO Declaration The UNESCO declaration is considered to be of added value because it is the first intergovernmental instrument on bioethics . Ten Have describes the commitment by governments to an agreed set of principles on bioethics as the `` innovative dimension '' of the declaration -LSB- 6 , p. 342 -RSB- . The Helsinki declaration and the CIOMS guidelines , by contrast , have been adopted by professional organisations -LRB- although CIOMS is in official relations with the World Health Organization -RRB- . Where the former is officially directed at physicians or researchers , however , the latter , like the UNESCO declaration , are to be used in designing national policy on biomedical research ethics , particularly in developing countries -LSB- 4 , 21 -RSB- . It may take time for the significance of the UNESCO declaration 's governmental backing to filter through to those who practise research ethics . An anonymous reviewer of this paper described the declaration as the `` common denominator of global ethical thought , '' but for many stakeholders the Declaration of Helsinki and the CIOMS guidelines fulfil this role , their professional origins notwithstanding . The Helsinki declaration is generally considered the foremost document globally on medical research ethics -LSB- 2 , 5 -RSB- and , with the CIOMS guidelines , forms the bedrock of research ethics in many developing countries -LSB- 1 -RSB- . In Kenya and South Africa , where policy-makers and ethics committees have looked to these two documents for guidance at the global level , several of those interviewed were unconvinced that the UNESCO declaration , which they saw as simply another international instrument on bioethics , was necessary . As the declaration was adopted only relatively recently , such perceptions may change as both its contents and its intergovernmental status become more widely established -LRB- particularly as it construes bioethics in broader terms than only medical research ethics -RRB- . If the UNESCO declaration is not to `` remain paperwork , '' as a non-binding instrument it must be effected by states -LSB- 6 , p. 343 -RSB- . Kenya and South Africa already have national bioethics guidelines that complement the declaration to a large degree . They also have research ethics committees at institutional and national levels , or plans for constituting them . For those countries which have not already established bioethics systems , however , the UNESCO declaration could act as a catalyst to galvanise them into doing so . To this end , UNESCO 's ethics programme supports states in building bioethics capacity , through various activities . The first is to construct a database with information on ethics experts , institutions , teaching programmes and policies around the world . The second is to promote bioethics education , through teacher training and curriculum development . The third is to support the setting up of ethics committees and their subsequent operations -LSB- 6 -RSB- . These activities were broadly welcomed among those interviewed in both countries , with the caveat that they should not overlap too far with the initiatives of other organisations . UNESCO is in fact piloting an ethics teacher training course in Kenya in July 2007 -LSB- 16 -RSB- , where there are fewer bioethics courses available than in South Africa . One area where ethics committees in Kenya and South Africa are in need of support is in the monitoring of research projects once they have been approved , as required by all three sets of national guidelines -LSB- 9 , 11 , 13 -RSB- . In practice , as found by Kass et al. , funding can be an issue . One prominent Kenyan ethics committee had only recently carried out its first on-the-spot inspection at the time when interview data were obtained in November 2005 . Prevented from conducting these inspections more often by financial constraints , the committee generally relies on reports from investigators and word of mouth -LRB- interviews , K_17:2005 and K_25:2005 -RRB- . The South African DoH guidelines require at a minimum that ethics committees request annual reports from principal investigators and establish a complaints procedure -LSB- 13 -RSB- ; members of two different ethics committees described such measures as `` passive monitoring '' . As in Kenya , committees do not have the resources to carry out site visits -LRB- interviews , SA_10:2006 and SA_17:2006 -RRB- . The UNESCO declaration itself does not offer much by way of assistance , the only article that could possibly be taken to relate to post-approval monitoring stating , `` Appropriate assessment and adequate management of risk related to medicine , life sciences and associated technologies should be promoted '' -LSB- 20 , p. 10 -RSB- .12 Perhaps UNESCO 's ethics programme could help with training in these areas or encourage better funding , however . As highlighted by Singer and Benatar , capacity building is likely to advance research ethics further than are reformulations of bioethical principles . It may be , then , that the added value of the declaration will prove to lie more in UNESCO 's follow-up activities than in the document itself , at least in the medium term , the innovative sections on social responsibility and benefit sharing notwithstanding . Conclusion This paper has revealed something of a loose consensus , at least between UNESCO and those involved in bioethics in Kenya and South Africa , on two counts . With regard to universal principles , any attempt to implement them at national levels must be contextualised . Working out how to apply such principles in particular social and economic contexts is arguably as challenging as reaching agreement on how they should be constituted in the first place . With regard to the usefulness of the UNESCO declaration , the significance of its adoption as the first intergovernmental instrument on bioethics must be matched by action in the form of capacity building for it to be of added value in the realm of biomedical research ethics . The scope of this paper has been limited to a primarily pragmatic analysis of how universal principles can be applied at national level . The paper has not commented on whether the value of the universality that UNESCO has aimed towards in terms of a foundation for humanity is compromised if these applications are very different . Deeper reflection on the nature of universality in this context would require the input of trained bioethicists and philosophers . This paper highlights , then , the need for a cross-disciplinary approach to the analysis of international bioethics instruments . The scope of the study that engendered this paper was necessarily limited by the time and resource constraints of doctoral research . Further research could explore whether the experiences of other African countries have been similar to those of Kenya and South Africa . Francophone and lusophone states , in particular , may have quite different bioethical traditions and thus have had very different experiences in implementing bioethics policies .

O B O O O O O O O O O O B O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O B I O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O 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kenya
national bioethics
south africa
unesco
universal principles