Giandomenico Palka

Male infertility: role of genetic background.

Male infertility represents one of the clearest examples of a complex disease with a substantial genetic basis. Numerous male mouse models, mutation screening and association studies reported over the last few years reveal the high prevalence of genetic causes of spermatogenic impairment, accounting for 10-15% of severe male infertility, including chromosomal aberrations and single gene mutations. Natural selection prevents the transmission of mutations causing infertility, but this protective mechanism may be overcome by assisted reproduction techniques. Consequently, the identification of genetic factors is important for appropriate management of the infertile couple. However, a large proportion of infertile males are diagnosed as idiopathic, reflecting poor understanding of the basic mechanisms regulating spermatogenesis and sperm function. Furthermore, the molecular mechanisms underlying spermatogenic damage in cases of genetic infertility (for example Yq microdeletions) are not known. These problems can be addressed only by large scale association studies and testicular or spermatozoal expression studies in well-defined alterations of spermatogenesis. It is conceivable that these studies will have important diagnostic and therapeutic implications in the future. This review discusses the genetic causes of male infertility known to date, the genetic polymorphisms possibly associated with male infertility, and reports novel results of global gene expression profiling of normal human testis by microarray technology.

Microdeletions in interval 6 of the Y chromosome detected by STS-PCR in 6 of 33 patients with idiopathic oligo- or azoospermia

It has been proposed that interval 6 of the human Y chromosome contains the gene or genes that control spermatogenesis (AZF, azoospermia factor). We have studied this region in 33 patients with oligo- or azoospermia, using PCR amplification of the YRRM1 (RBM1) gene and of 13 sequence-tagged sites (STSs), all mapping within interval 6. Six of the 33 patients showed no amplification of specific STSs, whereas there was no failure of amplification in normal male controls. We deduce that these six patients had microdeletions in interval 6 of the Y chromosome that correlated with the oligo- or azoospermia of these individuals. On biopsy of the testis, two of these patients showed a low number of germ cells, and four showed arrest with spermatides. We conclude that PCR amplification of Y-specific regions is a powerful and very sensitive tool for screening infertile men.

Folate gene polymorphisms and the risk of Down syndrome pregnancies in young Italian women.

Maternal impairments in folate metabolism and elevated homocysteinemia are known risk factors for having a child with Down syndrome (DS) at a young age. The 80G>A polymorphism of the reduced folate carrier gene (RFC‐1) has been recently demonstrated to affect plasma folate and homocysteine levels, alone or in combination with the 677C>T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene. We performed the present study on 80 Italian mothers of DS individuals, aged less than 35 at conception, and 111 Italian control mothers, to study the role of the RFC‐1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C genotypes to the risk of a DS offspring at a young maternal age. When polymorphisms were considered alone, both allele and genotype frequencies did not significantly differ between DS mothers and control mothers. However, the combined MTHFR677TT/RFC‐1 80GG genotype was borderline associated with an increased risk (OR 6 (CI 95%: 1.0–35.9), P = 0.05), and to be MTHF1298AA/RFC‐1 80(GA or AA) was inversely associated with the risk (OR 0.36 (CI 95%: 0.14–0.96), P = 0.04). Present results seem to indicate that none of the RFC‐1 80G>A, MTHFR 677C>T, and MTHFR 1298A>C polymorphisms is an independent risk factor for a DS offspring at a young maternal age; however, a role for the combined MTHFR/RFC‐1 genotypes in the risk of DS pregnancies among young Italian women cannot be excluded.

A quarter of men with idiopathic oligo-azoospermia display chromosomal abnormalities and microdeletions of different types in interval 6 of Yq11.

Cytogenetic investigations and molecular analysis of the Y chromosome by the polymerase chain reaction amplification of sequence-tagged sites (STS-PCR) technique were performed in 126 patients affected by idiopathic oligo-azoospermia following accurate selection of cases. Seventeen patients evidenced an abnormal karyotype. Fourteen patients with a normal karyotype had microdeletions of the Y chromosome within interval 6. In azo-ospermic patients microdeletions were scattered along different subintervals, while in oligozoospermic patients they were clustered in subinterval 6E. The size of the deletion was not apparently related to the severity of the disease. These results suggest that cytogenetic analysis and the STS-PCR technique can detect a genetic cause of infertility in about one-quarter of patients with idiopathic oligo-azoospermia.

The methylenetethrahydrofolate reductase (MTHFR) C677T polymorphism and male infertility in Italy.

The 677T allele of the MTHFR gene has been suggested to represent a factor of risk for male infertility. In order to confirm this association, we investigated the presence of the 677T allele in 93 Italian infertile patients, selected after the exclusion of other possible genetic causes of infertility, and in 105 Italian fertile controls. The homozygous 677TT genotype was present in 20.4% of patients and 27.6% of controls. These results do not support an association between the MTHFR 677T allele and male infertility in Italy.

Association of maternal polymorphisms in folate metabolizing genes with chromosome damage and risk of Down syndrome offspring

We analyzed the role of six common polymorphisms in folate metabolizing genes as possible risk factors for having a child with Down syndrome (DS) in 94 Italian mothers of a DS child (MDS) and 113 matched control mothers, both aged less than 35 years at conception. Investigated polymorphisms include methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, and thymidylate synthase (TYMS) 28bp repeat and 1494del6. We also measured the amount of chromosome damage in peripheral blood lymphocytes of 42 MDS and 41 matched controls, by means of the micronucleus assay, and searched for association between this cytogenetic endpoint and any of the studied polymorphisms. Micronuclei in peripheral blood lymphocytes have been analyzed several years after conception: the mean age at sampling was 45.6+/-11.4 years for MDS and 47.95+/-6.9 years for controls. The combined MTHFR 677TT/MTR 2756AA genotype was associated with increased DS risk (P=0.034), and the combined MTHFR 1298AC/TYMS 2R/2R genotype with reduced risk (P=0.003). Moreover, we observed a significant increased frequency of micronucleated lymphocytes in MDS as compared to controls (P<0.0001) and, in the total population, a significant correlation between micronucleated cells and both MTHFR 677C>T (P=0.031) and 1298A>C (P=0.047) polymorphisms.

C677T mutation in the 5,10-MTHFR gene and risk of Down syndrome in Italy.

The C677T polymorphism of the MTHFR gene has been associated to maternal risk of Down syndrome, due to the detection of an higher prevalence of the T allele among mothers of children with trisomy 21, compared to control mothers. In order to confirm this association, we studied the presence of the C677T in 64 mothers of Down syndrome children and 112 controls from central Italy. An higher incidence of the mutant T allele in controls (48.2%) than in Down syndrome children mothers (44%) was detected. These results do not support the presence of an increased risk of Down syndrome in mothers carriers of the T allele in the Italian population.

Genetics of syndromic and nonsyndromic cleft lip and palate.

Cleft of the lip with or without cleft palate (CL/P) represents one of the commonest congenital malformations in Western countries. Based on their association with specific malformative patterns or their presence as isolated defects, CL/P can be classified as syndromic and nonsyndromic, respectively. Both forms of CL/P are characterized by a strong genetic component. Syndromic forms are in many cases due to chromosomal aberrations or monogenic diseases. Among these, the Van der Woude syndrome, caused by mutation of the IRF6 gene, represents the commonest form of syndromic CL/P, accounting for about 2% of all cases. On the other hand, nonsyndromic CL/P is a multifactorial disease derived by the interaction between genetic and environmental factors. In recent years, great efforts have been made to identify the genes involved in the susceptibility to nonsyndromic CL/P and to disclose their relationship with specific environmental risk factors, to get information about the pathogenic mechanism leading to the malformation. In this article, we will review the most recent findings about the genes involved in the pathogenesis of syndromic and nonsyndromic CL/P, to provide information about the opportunity in the future to use specific genetic testing for the identification of at-risk mothers and the prevention of the disease based on a personalized approach.

Zinc Sulfate Supplementation Improves Thyroid Function in Hypozincemic Down Children

In subjects affected by trisomy 21 (Down syndrome), hypothyroidism is the most common endocrinological deficit. Plasma zinc levels, which are commonly detected below the normal range in Down patients, are related to some endocrinological and immunological functions; in fact, zinc deficiency has been shown to impair immune response and growth rate. Aims of this study were to evaluate (1) the role of zinc deficiency in subclinical hypothyroidism and (2) thyroid function changes in Down children cyclically supplemented with zinc sulfate. Inverse correlations have been observed between age and triiodotironine (T3) and between zinc and thyroid-stimulating hormone (TSH); higher TSH levels have been found in hypozincemic patients at the beginning of the study. After 6 mo of supplementation, an improvement of thyroid function (TSH levels: 3.96 ± 1.84 vs 2.64 ± 1.33 mUI/mL basally and after 6 mo, respectively) was observed in hypozincemic patients. In the second cycle of supplementation, a similar trend of TSH was observed. At the end of the study, TSH significantly decreased in treated hypozincemic subjects (4.48 ± 1.93 vs 2.96 ± 1.20 mUI/mL) and it was no longer different in comparison to normozincemic patients. We suggest zinc supplementation to the diet in hypozincemic Down children as a simple and useful therapeutic tool.

Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

BackgroundStem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.ResultsThe described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.ConclusionThe described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.