Male idiopathic infertility and the TP53 polymorphism in codon 72.

Many environmental agents affect the development of male germ cells at different stages. Apoptosis is common during normal spermatogenesis; it plays an important role in controlling the number of germ cells and the disposal of defective stem cells to produce functional sperm. The presence of p53 in primary spermatocytes suggests that it plays a role in the prophase of meiosis. p53 is expressed in the testis in both spermatocytes and spermatogonia. This suggests that the p53 gene (TP53) is important for apoptosis regulation during spermatogenesis, and may be associated with male infertility. The main causes of male infertility are genetic, physical, and pathological abnormalities, intense and prolonged exercise, aging, drug use, and long periods of sexual abstinence. Approximately 20% of male infertility is idiopathic. The Trp53 gene is involved in meiosis in male rats and mice suggesting that the p53 plays a critical role in spermatogenesis. We investigated the association between the TP53 polymorphism in codon 72 and idiopathic male infertility in 208 semen samples: 106 showed abnormal semen analysis results and were from infertile men, and 102 were from fertile individuals (the control group). Changes in Trp53 expression are associated with the main phase regulating meiotic progression with a peak in the pachytene stage, and Trp53-deficient mice exhibit degenerative syndrome (giant cells). The genotypic and allelic frequencies were not significantly different among the groups in this study; the results suggest that the TP53 polymorphism in codon 72 is not associated with the pathogenesis of idiopathic male infertility or failure of spermatogenesis.

Efeito do extrato aquoso de camomila (Chamomilla recutita L. ) na prenhez de ratas e no desenvolvimento dos filhotes / Effect of aqueous extract of chamomile (Chamomilla recutita L. ) on rat pregnancy and offspring development

As plantas medicinais possuem substâncias ativas e, muitas vezes, o efeito tóxico sobre o organismo é desconhecido ou ignorado pelos usuários. A camomila é muito utilizada pela população, porém, contra-indicada para gestantes por possuir indícios de atividade emenagoga e relaxante da musculatura lisa. Por esses motivos, o objetivo do presente estudo foi avaliar os efeitos do extrato aquoso de camomila na gestação e nos filhotes gerados. Foram utilizadas 9 ratas da linhagem Wistar, divididas em 3 grupos, D1 e D2 que receberam infusão de camomila a 5% e 10% respectivamente, e o grupo controle que recebeu soro fisiológico. Os tratamentos foram administrados por via oral, desde o 1º ao 7º dia após o cruzamento. Os parâmetros estudados foram: prevalência de abortos, ganho de peso materno durante a prenhez, morte fetal e materna, malformações fetais grosseiras, número de recém-nascidos, peso dos filhotes, e análise de reflexos neurológicos dos filhotes (postural, preensão e orientação) no 1º, 3º, 5º e 10º dias de vida. Houve gestação em 70% do grupo controle, 40% do D1 e 80% do D2. Não houve diferença no ganho de peso materno no 7º e 21º dia, porém, os grupos tratados obtiveram ganho de peso menor em relação ao controle no 14º dia de gestação (p=0,04). As diferenças entre o número de recém-nascidos não foram significantes. Quanto ao peso dos recém-nascidos, os animais tratados apresentaram menor ganho de peso aos dias 1, 3, 5, e 10 após o nascimento (p=0,005; p=0,001; p<0,001; p<0,001; respectivamente). Ocorreram diferenças no reflexo postural no 1º dia, ocorrendo aceleração (p=0,005); já no reflexo de preensão (p=0,006), e no reflexo de orientação (p=0,01) houve retardo no desaparecimento, sem alteração nos demais dias sobre os outros parâmetros avaliados. A camomila pode influenciar, tanto no ganho de peso materno durante a gestação, como no dos filhotes após o nascimento, e pode provocar alterações nos reflexos neurológicos. Não se observou alteração nos demais parâmetros estudados.

Medicinal plants have active substances and their toxic effect on the organism is often unknown or ignored by users. Chamomile has been widely used by the population; however, it is contraindicated for pregnant women due to evidence of its emmenagogue and relaxing activity on smooth muscles. For these reasons, the aim of this study was to evaluate the effects of aqueous extract of chamomile on pregnancy and generated offspring. Nine Wistar rats were divided into three groups, D1 and D2, which received chamomile infusion at 5% and 10%, respectively, and the control group which received saline solution. Treatments were administered from the 1st to the 7th day after breeding. The studied parameters were abortion prevalence, maternal weight gain during pregnancy, fetal and maternal death, gross fetal malformation, newborn number, weight and neurological reflexes of the offspring on the 1st, 3rd, 5th and 10th days of life. There was pregnancy in 70% of control group, 40% of D1 and 80% of D2. There was no difference in maternal weight gain on the 7th and 21st days; however, treated groups had smaller weight gain, compared to control, on the 14th day of pregnancy (p=0.04). The differences among newborn numbers were not significant. As to weight of newborns, treated animals had smaller weight gain on days 1, 3, 5 and 10 after birth (p=0.005; p=0.001; p<0.001; p<0.001; respectively). For neurological effects, there were differences in postural reflex on the 1st day, resulting in acceleration; on the other hand, for grasp (p=0.006) and orienting reflex (p=0.01), there was a delay in disappearing, and no changes occurred on the remaining days for the other assessed parameters. Chamomile may influence maternal weight gain during pregnancy, as well as offspring weight gain after birth, and may cause changes in neurological reflexes. There was no alteration in the remaining studied parameters.

Analysis of p53 codon 72 gene polymorphism in Brazilian patients with endometriosis.

We examined the frequency of p53 codon 72 polymorphism in 38 patients with endometriosis whose diagnosis was confirmed using videolaparoscopy. Half of the women were infertile. There were no significant differences in the genotype (P = 0.0927) or allele frequencies (P = 0.1430) for p53 Arg72Pro polymorphism between the two groups. We found a significant association between the heterozygous and homozygous proline genotypes and intense pain in the patients. Sixty-four percent of the patients were homozygous or heterozygous for proline in patients with degree III or IV endometriosis, but there was no significant difference compared to homozygous arginine genotype (P = 0.6115). We found that the proline allele is associated with substantial complaints (infertility associated with pain), when compared to the homozygous arginine genotype; we also found that the proline allele was more frequent in endometriosis patients.

Primary open angle glaucoma was not found to be associated with p53 codon 72 polymorphism in a Brazilian cohort.

Primary open angle glaucoma (POAG) is the most common type of glaucoma. The p53 codon 72 Arg-Pro (CGC to CCC) polymorphism of exon 4 affects various biological properties; recently, it was reported that this polymorphism affects the ability to induce apoptosis in vitro. Various genotypes have been found to be significantly associated with POAG. We examined the distribution of this polymorphism in 104 unrelated POAG patients and in 58 normal healthy individuals without history of POAG at the Pronto Clínica de Olhos in Goiânia, Brazil. The controls were recruited among individuals undergoing ophthalmological examination. Their genomic DNA was analyzed for p53 gene codon 72 polymorphism by polymerase chain reaction. The Arg72 allele was more common than the Pro72 allele in both groups. There was no significant difference in the distribution of the codon 72 polymorphism between groups (p= 0.3311). The genotype distribution in the POAG group was 23.07 Arg homozygote, 75 heterozygote, and 1.93% Pro homozygote, while in the control group it was 31.04 Arg homozygote, 68.96 heterozygote, and 0% Pro homozygote. We concluded that the p53 codon 72 Arg/Pro polymorphism is not associated with glaucoma in Brazilian patients.

TP53 gene expression, codon 72 polymorphism and human papillomavirus DNA associated with pterygium.

Pterygium is a disease of unknown origin and pathogenesis that can be vision threatening. Several researchers believe that pterygium is UV-related and that abnormal expression of p53 protein and infection with human papillomavirus (HPV) are risk factors for pterygium, but their experiments have been inconclusive. We investigated its relation with p53 protein expression, p53 gene codon 72 polymorphism and infection with HPV DNA. Pterygial samples were obtained from 36 patients; 21 normal conjunctival samples were used as controls. Expression of p53 protein was studied by immunohistochemistry, using the antibody DO-7. Analysis for the p53 genotype was made by polymerase chain reaction, using specific primers for the arginine and proline alleles, and an analysis for HPV was made of the pterygium patients and control group. Fourteen of the 36 pterygial specimens were positive for abnormal p53 expression. Thirty-one of the patients were heterozygotic and three were homozygotic for the proline allele; two were homozygotic for the arginine allele; in the control group 12 of 21 were heterozygotic and seven of these 21 were homozygotic for the proline allele; two were homozygotic for the arginine allele. Twenty-one of the pterygium patients were positive for HPV; HPV type 1 was found in nine of these, type 2 in seven and both types in five. Only two of the 21 controls had HPV; both had type 16. We suggest that abnormal expression of p53, p53 codon 72 polymorphisms and HPV DNA are required co-factors for the development of pterygium.

Homologous recombination between HERVs causes duplications in the AZFa region of men accidentally exposed to cesium-137 in Goiânia.

In September 1987, in Goiânia, Brazil, one of the most serious radiological accidents occurred at a radiation therapy unit involving a source of cesium-137. The current study examined the occurrence of possible germline mutations at the AZF region of the exposed men and in their male offspring. Genomic DNA samples of 16 individuals were analyzed for microdeletions. All exposed individuals amplified sequence tagged sites; however, sY84 and sY86 showed a duplication in 75% (12/16) of the exposed group. Exposed families designated as B and E showed a duplication of sY84 and sY86, both in the fathers and their sons. Fathers of families A, C, D, and F did not show a duplication in the AZF region, but their sons did. The children in A and D had duplications of sY84 and sY86, while children in families C and F had a duplication exclusively of sY84. Family G showed a duplication of sY84 in all three generations from grandfather to grandson. Two human endogenous retroviral sequences (HERV) exist in the AZFa region, and non-allelic recombination between these sequences could cause chromosomal rearrangements, such as deletions or duplications, and a mutational mechanism intrinsic to non-allelic recombination could be increased by individual exposure to ionizing radiations from cesium-137. Consequently, the hotspots inside HERV mediated recombination in AZFa, and the duplication diversity was compatible with male fertility, since to date, none of the exposed individuals have demonstrated fertility disorders.

Tracking microdeletions of the AZF region in a patrilineal line of infertile men.

Male infertility is considered to be a difficult-to-treat condition because it is not a single entity, but rather reflects a variety of different pathologic conditions, thus making it difficult to use a single treatment strategy. Structural alterations in the Y chromosome have been the principal factor responsible for male infertility. We examined 26 family members of 13 patients with male infertility who showed deletions in the AZF region. In family 1, the father and a brother did not show microdeletions. However, a son showed a microdeletion in AZFa (sY84) and an azoospermic sperm analysis, but another son had a microdeletion in AZFa (sY84) and AZFb (sY127) and a normal sperm analysis. The father of family 2, with severe oligozoospermia, had a microdeletion in the AZFa region (sY84) and his son, conceived by intracytoplasmic sperm injection, also showed the same microdeletion. In the other families, only the men with an altered sperm analysis had a microdeletion. It is possible that in family 1, the father and brother who did not show microdeletions in this study, could have microdeletions in regions upstream or downstream of the one analyzed. The treatment with intracytoplasmic sperm injection can result in vertical transmission of microdeletions of the AZF region and can also cause the expansion of a de novo mutation. This finding reinforces the necessity of an investigation of microdeletions of the Y chromosome in individuals who are candidates for assisted reproduction, as well as genetic counciling and follow-up.

Y chromosome microdeletions in Brazilian fertility clinic patients.

Microdeletions in Yq are associated with defects in spermatogenesis, while those in the AZF region are considered critical for germ cell development. We examined microdeletions in the Y chromosomes of patients attended at the Laboratory of Human Reproduction of the Clinical Hospital of the Federal University of Goiás as part of a screening of patients who plan to undergo assisted reproduction. Analysis was made of the AZF region of the Y chromosome in men who had altered spermograms to detect possible microdeletions in Yq. Twenty-three patients with azoospermia and 40 with severe oligozoospermia were analyzed by PCR for the detection of six sequence-tagged sites: sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, and sY254 and sY255 for AZFc. Microdeletions were detected in 28 patients, including 10 azoospermics and 18 severe oligozoospermics. The patients with azoospermia had 43.4% of their microdeletions in the AZFa region, 8.6% in the AZFb region and 17.4% in the AZFc region. In the severe oligozoospermics, 40% were in the AZFa region, 5% in the AZFb region and 5% in the AZFc region. We conclude that microdeletions can be the cause of idiopathic male infertility, supporting conclusions from previous studies.

Y chromosome microdeletions in Brazilian fertility clinic patients

Microdeletions in Yq are associated with defects in spermatogenesis, while those in the AZF region are considered critical for germ cell development. We examined microdeletions in the Y chromosomes of patients attended at the Laboratory of Human Reproduction of the Clinical Hospital of the Federal University of Goiás as part of a screening of patients who plan to undergo assisted reproduction. Analysis was made of the AZF region of the Y chromosome in men who had altered spermograms to detect possible microdeletions in Yq. Twenty-three patients with azoospermia and 40 with severe oligozoospermia were analyzed by PCR for the detection of six sequence-tagged sites: sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, and sY254 and sY255 for AZFc. Microdeletions were detected in 28 patients, including 10 azoospermics and 18 severe oligozoospermics. The patients with azoospermia had 43.4% of their microdeletions in the AZFa region, 8.6% in the AZFb region and 17.4% in the AZFc region. In the severe oligozoospermics, 40% were in the AZFa region, 5% in the AZFb region and 5% in the AZFc region. We conclude that microdeletions can be the cause of idiopathic male infertility, supporting conclusions from previous studies.