RESUMO
Quantitative and qualitative spermatogenic impairments are major causes of men's infertility. Although in vitro fertilization (IVF) is effective, some couples persistently fail to conceive. To identify causal variants in patients with severe male infertility factor and repeated IVF failures, we sequenced the exome of two consanguineous family members who underwent several failed IVF cycles and were diagnosed with low sperm count and motility. We identified a rare homozygous nonsense mutation in a previously uncharacterized gene, RNF212B, as the causative variant. Recurrence was identified in another unrelated, infertile patient who also faced repeated failed IVF treatments. scRNA-seq demonstrated meiosis-specific expression of RNF212B. Sequence analysis located a protein domain known to be associated with aneuploidy, which can explain multiple IVF failures. Accordingly, FISH analysis revealed a high aneuploidy rate in the patients' sperm cells and their IVF embryos. Finally, inactivation of the Drosophila orthologs significantly reduced male fertility. Given that members of the evolutionary conserved RNF212 gene family are involved in meiotic recombination and crossover maturation, our findings indicate a critical role of RNF212B in meiosis, genome stability, and in human fertility. Since recombination is completely absent in Drosophila males, our findings may indicate an additional unrelated role for the RNF212-like paralogs in spermatogenesis.
Assuntos
Infertilidade Masculina , Ligases , Sêmen , Humanos , Masculino , Aneuploidia , Fertilização in vitro , Infertilidade Masculina/genética , Ligases/genética , Espermatozoides , Domínios RING FingerRESUMO
BACKGROUND: MicroRNAs are involved in the regulation of spermatogenesis, are detected in semen and may be useful as molecular markers for predicting residual complete spermatogenesis in azoospermic men. OBJECTIVES: To study the biomarker potential of microRNAs that are detected in semen and testicular tissue. MATERIALS AND METHODS: MicroRNA profiles were analyzed in semen fractions of normozoospermic (n = 3) and azoospermic (n = 6) men by small RNA deep sequencing. Specific microRNAs were further analyzed by reverse transcription and quantitative polymerase chain reaction in eight testicular samples and 46 semen supernatants. The semen supernatant samples included 18 normozoospermic and 28 azoospermic men with various pathologies. RESULTS: The sequenced microRNA profiles of semen supernatant fraction samples were distinct from the other fractions. Significant expression differences were observed between the semen supernatant of normozoospermic and azoospermic men. Further analysis by reverse transcription and quantitative polymerase chain reaction revealed that expression of miR-202-3p was considerably reduced (undetectable in most samples) in the azoospermic semen supernatants. The expression of miR-202-3p was significantly lower in the azoospermic specimens than in the normozoospermic specimens and a trend was observed for miR-629-5p (p = 0.03 and 0.06, respectively). Differences in expression levels in the semen supernatant were observed among the various pathologies but not to a level of significance, possibly because of the small subgroups. miRNA-370-3p was significantly higher in semen supernatant samples from azoospermic men without sperm cells in testis (p = 0.05). In testes, the three microRNAs were expressed at higher levels in the obstructive and spermatocyte maturation arrest pathologies than in mixed atrophy and Sertoli cell only. miR-202-3p was detected in all testicular samples. CONCLUSIONS: MicroRNA expression profiles in semen were distinguishable between azoospermic and normozoospermic men. The microRNA profile also diverged among azoospermic men subdivided according to their testicular pathologies. The levels of specific microRNAs in testis and in the semen supernatant were not directly correlated.
Assuntos
Azoospermia , MicroRNAs , Humanos , Masculino , Testículo/metabolismo , Sêmen/metabolismo , Espermatogênese/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Biomarcadores/metabolismoRESUMO
Klinefelter syndrome (KS) is the most prevalent genetic disorder of infertile males. This study aimed to determine in Klinefelter patients (KS) the expression levels of spermatogenic markers and testicular growth factors that might predict spermatogenesis based on conventional testicular sperm extraction (TESE). The expression levels of the pre-meiotic (OCT4, CD9, GFR-α1, α-6-INTEGRIN, SALL4, C-KIT), meiotic (CREM-1), and post-meiotic (protamine) markers, as well as the colony stimulating factor-1 (CSF-1) were examined in testicular biopsies with and without mature sperm of KS and normal karyotype of azoospermic patients (AZO) with complete spermatogenesis. In the biopsies of AZO, the expression levels (fold of expression compared to the PPI of the same sample) of OCT4 were 9.68± 7.93, CREM 42.78± 28.22, CSF-1 3.07 ± 3.19, and protamine 78498.12 ± 73214.40. Biopsies from KS included 7 with sperm and 17 without sperm. Among the biopsies with sperm, the expression levels of OCT4 were 7.27± 9.29, CREM 3.13± 7.89, CSF-1 35.5 ± 48.01, and protamine 902.97 ± 2365.92. In 14 biopsies without sperm, we found low expression levels of OCT4, CREM and CSF-1, and no expression of protamine. However, in three of the biopsies without sperm that highly expressed OCT4 and CSF-1, the expression levels of CREM-1 and protamine were high. These results may be used for further consulting with patients considering repeating conventional TESE or micro TESE and cryopreservation for possible future in-vitro spermatogenesis.
Assuntos
Azoospermia , Modulador de Elemento de Resposta do AMP Cíclico , Síndrome de Klinefelter , Fator Estimulador de Colônias de Macrófagos , Fator 3 de Transcrição de Octâmero , Adulto , Azoospermia/patologia , Biópsia , Modulador de Elemento de Resposta do AMP Cíclico/genética , Humanos , Integrinas , Síndrome de Klinefelter/genética , Fator Estimulador de Colônias de Macrófagos/genética , Masculino , Fator 3 de Transcrição de Octâmero/genética , Protaminas , Sêmen , Recuperação Espermática , Espermatogênese/genética , Espermatozoides/patologia , Testículo/patologiaRESUMO
OBJECTIVE: To examine the effect of the BNT162b, mRNA, SARS-CoV-2 virus vaccine on sperm quality. METHODS: This was a prospective cohort study conducted on sperm donors at the sperm bank of a tertiary, university affiliated medical center. All sperm donors donated sperm repeatedly and the average sperm parameters of all available samples were compared before and after receiving the SARS-CoV-2 vaccine. Each donor served as his own control. For all participants, at-least one sperm sample was received 72 days after completing the second vaccine. Main outcome measures included total sperm count, total motile count and percent of motile sperm. RESULTS: A total of 898 sperm samples from 33 sperm donors that were vaccinated with the Pfizer BNT162b, mRNA, SARS-CoV-2 virus vaccine were analyzed, 425 samples were received before the vaccine, while 473 samples were received after vaccination. Total sperm count and total motile count increased after the second vaccine compared to samples before vaccination. Percent of motile sperm did not change after vaccine. CONCLUSION: The Pfizer BNT162b, SARS-CoV-2 vaccine has no deleterious effect on sperm quality. Patients and physicians should be counseled accordingly.
Assuntos
COVID-19 , Motilidade dos Espermatozoides , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Masculino , Estudos Prospectivos , RNA Mensageiro , SARS-CoV-2 , EspermatozoidesRESUMO
OBJECTIVE: To investigate the prevalence of testicular microlithiasis and its association with sperm retrieval rates and histopathology in men with non-obstructive azoospermia. METHODS: A total of 120 men underwent scrotal ultrasonography prior to microsurgical testicular sperm extraction. Sperm retrieval rate, testicular histopathology, testicular size, reproductive hormones, karyotyping, Y chromosome microdeletion analyses, and presence of varicoceles and hydroceles were compared between men with and without testicular microlithiasis. RESULTS: The total sperm retrieval rate was 40%. Ten men with normal spermatogenesis were excluded. The remaining 110 men with non-obstructive azoospermia were analyzed and testicular microlithiasis was detected in 16 of them (14.5%). The sperm retrieval rate in that subgroup was only 6.2% (1/16) as opposed to 39.4% (37/94) in men with non-obstructive azoospermia and no evidence of microlithiasis (P = 0.009). The mean right and left testicular diameters were significantly lower in the microlithiasis group (P = 0.04). On multivariate logistic regression analysis, the presence of mictolithiasis (odds ratio 7.4, 95% confidence interval 2.3, 12.2; P = 0.01) was the only independent predictor of unsuccessful sperm retrieval. The 15 patients with microlithiasis and without successful sperm extraction were diagnosed by histopathology as having Sertoli cells only. The 16th patient with successful sperm retrieval had a histopathology of mixed atrophy and was diagnosed with Klinefelter syndrome. CONCLUSION: The presence of testicular microlithiasis is associated with low sperm retrieval rates among our cohort of men with non-obstructive azoospermia undergoing scrotal ultrasonography prior to microsurgical testicular sperm extraction. Larger, prospective studies should be conducted to confirm these findings.
Assuntos
Azoospermia , Doenças Testiculares , Azoospermia/diagnóstico por imagem , Azoospermia/epidemiologia , Cálculos , Humanos , Masculino , Estudos Prospectivos , Estudos Retrospectivos , Recuperação Espermática , Doenças Testiculares/diagnóstico por imagem , Doenças Testiculares/epidemiologia , Testículo/diagnóstico por imagemRESUMO
Infertility affects one in six couples, half of which are caused by a male factor. Male infertility can be caused by both, qualitative and quantitative defects, leading to Oligo- astheno-terato-zoospermia (OAT; impairment in ejaculate sperm cell concentration, motility and morphology). Azoospermia defined as complete absence of sperm cells in the ejaculation. While hundreds of genes are involved in spermatogenesis the genetic etiology of men's infertility remains incomplete.We identified a hemizygous stop gain pathogenic variation (PV) in the X-linked Germ Cell Nuclear Acidic Peptidase (GCNA), in an Azoospermic patient by exome sequencing. Assessment of the prevalence of pathogenic variations in this gene in infertile males by exome sequence data of 11 additional unrelated patients identified a probable hemizygous causative missense PV in GCNA in a severe OAT patient. Expression of GCNA in the patients' testes biopsies and the stage of spermatogonial developmental arrest were determined by immunofluorescence and immunohistochemistry. The Azoospermic patient presented spermatogenic maturation arrest with an almost complete absence of early and late primary spermatocytes and thus the complete absence of sperm. GCNA is critical for genome integrity and its loss results in genomic instability and infertility in Drosophila, C. elegans, zebrafish, and mouse. PVs in GCNA appear to be incompatible with male fertility in humans as well: A stop-gain PV caused Azoospermia and a missense PV caused severe OAT with very low fertilization rates and no pregnancy in numerous IVF treatments.
Assuntos
Infertilidade Masculina/genética , Mutação , Proteínas Nucleares/genética , Adulto , Humanos , Infertilidade Masculina/patologia , Masculino , Proteínas Nucleares/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patologiaRESUMO
BACKGROUND: Data on who among the infertile male population may benefit from round spermatid injections (ROSI) are lacking. OBJECTIVE: To determine the probability of finding round spermatids suitable for ROSI in men with non-obstructive azoospermia (NOA) in whom no spermatozoa were retrieved at testicular sperm extraction. MATERIALS AND METHODS: Four-hundred fifty-seven consecutive men with azoospermia underwent testicular sperm extraction. Clinical examination included age, secondary sexual characteristics, testicular size, reproductive hormone estimation, karyotyping, and Y chromosome microdeletion analyses. Histologic examination was performed, and histologic classification was determined by the most advanced spermatogenetic cell identified in the combined histologic and cytologic examination. RESULTS: Of the 457 azoospermic men, 342 were diagnosed with NOA, and 148 (148/342, 43%) had mixed atrophy on histopathology and retrievable spermatozoa. No spermatozoa were found in 194/342 men with NOA (57%). Histopathology diagnosed 145/194 (75%) of them with Sertoli cell only, 45/194 (23%) with spermatocyte maturation arrest, and 4/194 (2%) with spermatid maturation arrest. CONCLUSIONS: Histopathologically identified round spermatids without spermatozoa were rare in men with NOA. Only very few of them are likely to reap the benefits of ROSI, thus presenting the need to reconsider its actual clinical value.
Assuntos
Azoospermia , Recuperação Espermática , Espermátides , Adulto , Humanos , Masculino , Estudos RetrospectivosRESUMO
Refreezing of sperm samples would provide the possibility of performing more cycles of fertility treatments. Although the effect of repeated cycles of freezing on sperm quality was studied, the effect of the length of the time interval between each freeze-thaw cycle has not been reported. Hence, we assessed the effect of incubation time on the sperm quality of thawed sperm after repeated freezing. One-hundred samples of potential sperm donations with normal sperm quality were evaluated. The fresh semen samples were analyzed and cryopreserved in liquid nitrogen until use. After thawing, the samples were divided randomly to two groups and reanalyzed for motility, vitality, and DNA fragmentation. They were incubated at room temperature and reanalyzed after either 90 min (group A) or 180 min (group B) of incubation, and once again after a repeated cycle of freezing and thawing. Our results showed that the sperm parameters of fresh samples of both groups were similar. After one freeze-thaw cycle, both groups still had comparable values. At the end of their respective incubation time periods, however, there was a significant difference in the mean values of the assessed parameters between the two groups (p < 0.01). An additional freeze-thaw cycle further exacerbated those differences, with group B undergoing an even more substantial decline (p < 0.001). Our data suggest that thawed human spermatozoa sustain a significant decline in sperm parameters in association with longer incubation time, which is further exacerbated by an additional freeze-thaw cycle.
Assuntos
Criopreservação , Congelamento , Espermatozoides , Fragmentação do DNA , Humanos , Masculino , Motilidade dos EspermatozoidesRESUMO
STUDY QUESTION: Are there genetic variants that can be used for the clinical evaluation of azoospermic men? SUMMARY ANSWER: A novel homozygous frame-shift mutation in the MEIOB gene was identified in three azoospermic patients from two different families. WHAT IS KNOWN ALREADY: Up to 1% of all men have complete absence of sperm in the semen, a condition known as azoospermia. There are very few tools for determining the etiology of azoospermia and the likelihood of sperm cells in the testis. The MEIOB gene codes for a single-strand DNA binding protein required for DNA double-strand breaks repair during meiosis. MEIOB appears to be exclusively expressed in human and mouse testis, and MeioB knockout mice are azoospermic due to meiotic arrest. STUDY DESIGN, SIZE, DURATION: Two brothers with non-obstructive azoospermia (NOA) underwent whole-exome sequencing followed by comprehensive bioinformatics analyses. Candidate variations were further screened in infertile and fertile men, as well as in public and local reference databases. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 159 infertile and 77 fertile men. The exomes of two Arab men were completely sequenced. In addition, 213 other men of the same Arab ethnicity (136 infertile and 77 fertile men) underwent restriction fragment length polymorphism (RFLP) screening, as did 21 NOA men, of other ethnicities, with testicular impairment of spermatocyte arrest. All of the infertile men underwent Y-chromosome microdeletion and CFTR gene mutation assessments. Comprehensive bioinformatics analyses were designed to uncover candidate mutations associated with azoospermia. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous frame-shift mutation in the MEIOB gene was identified in two brothers of Arab ethnicity. This frame-shift is predicted to result in a truncated MEIOB protein, which lacks the conserved C-terminal DNA binding domain. RFLP screening of the mutation in 157 infertile men, including 112 NOA patients of Arab ethnicity, identified an additional unrelated NOA patient with the same homozygous mutation and a similar testicular impairment. This mutation was not found in available public databases (n > 160 000), nor in the 77 proven fertile men, nor in our database of local Israeli population variations derived from exome and genome sequencing data (n = 500). LIMITATIONS, REASONS FOR CAUTION: We have thus far screened for only two specific MEIOB probable pathogenic mutations in a relatively small local cohort. Therefore, the relative incidence of MEIOB mutations in azoospermia should be further assessed in larger and diverse cohorts in order to determine the efficiency of MEIOB sequence screening for clinical evaluations. WIDER IMPLICATIONS OF THE FINDINGS: The relatively high incidence of likely NOA-causing mutations in MEIOB that was found in our cohort supports the idea that a complete screening of this gene might be beneficial for clinical evaluation of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This research was supported in part by a grant to EA from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC grant agreement (616088). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Azoospermia/genética , Proteínas de Ligação a DNA/genética , Meiose/genética , Mutação , Testículo/metabolismo , Adulto , Árabes/genética , Azoospermia/diagnóstico , Azoospermia/etnologia , Azoospermia/patologia , Estudos de Coortes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Irmãos , Sequenciamento do ExomaRESUMO
BACKGROUND: The study is aimed to describe a novel strategy that increases the accuracy and reliability of PGD in patients using sperm donation by pre-selecting the donor whose haplotype does not overlap the carrier's one. METHODS: A panel of 4-9 informative polymorphic markers, flanking the mutation in carriers of autosomal dominant/X-linked disorders, was tested in DNA of sperm donors before PGD. Whenever the lengths of donors' repeats overlapped those of the women, additional donors' DNA samples were analyzed. The donor that demonstrated the minimal overlapping with the patient was selected for IVF. RESULTS: In 8 out of 17 carriers the markers of the initially chosen donors overlapped the patients' alleles and 2-8 additional sperm donors for each patient were haplotyped. The selection of additional sperm donors increased the number of informative markers and reduced misdiagnosis risk from 6.00% ± 7.48 to 0.48% ±0.68. The PGD results were confirmed and no misdiagnosis was detected. CONCLUSIONS: Our study demonstrates that pre-selecting a sperm donor whose haplotype has minimal overlapping with the female's haplotype, is critical for reducing the misdiagnosis risk and ensuring a reliable PGD. This strategy may contribute to prevent the transmission of affected IVF-PGD embryos using a simple and economical procedure. TRIAL REGISTRATION: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. DNA testing of donors was approved by the institutional Helsinki committee (registration number 319-08TLV, 2008). The present study was approved by the institutional Helsinki committee (registration number 0385-13TLV, 2013).
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Haplótipos/genética , Diagnóstico Pré-Implantação/normas , Espermatozoides/fisiologia , Espermatozoides/transplante , Doadores de Tecidos , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Humanos , Masculino , Diagnóstico Pré-Implantação/métodosRESUMO
PURPOSE: Up to 1% of all men experience azoospermia, a condition of complete absence of sperm in the semen. The mechanisms and genes involved in spermatogenesis are mainly studied in model organisms, and their relevance to humans is unclear because human genetic studies are very scarce. Our objective was to uncover novel human mutations and genes causing azoospermia due to testicular meiotic maturation arrest. METHODS: Affected and unaffected siblings from three families were subjected to whole-exome or whole-genome sequencing, followed by comprehensive bioinformatics analyses to identify mutations suspected to cause azoospermia. These likely mutations were further screened in azoospermic and normozoospermic men and in men proven to be fertile, as well as in a reference database of local populations. RESULTS: We identified three novel likely causative mutations of azoospermia in three genes: MEIOB, TEX14, and DNAH6. These genes are associated with different meiotic processes: meiotic crossovers, daughter cell abscission, and possibly rapid prophase movements. CONCLUSION: The genes and pathways we identified are fundamental for delineating common causes of azoospermia originating in mutations affecting diverse meiotic processes and have great potential for accelerating approaches to diagnose, treat, and prevent infertility.Genet Med advance online publication 16 February 2017.
Assuntos
Azoospermia/diagnóstico , Azoospermia/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Sequência de Aminoácidos , Biomarcadores , Biópsia , Estudos de Casos e Controles , Consanguinidade , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Dineínas/genética , Família , Testes Genéticos , Genótipo , Humanos , Hibridização in Situ Fluorescente , Masculino , Linhagem , Espermatozoides/metabolismoRESUMO
The bromodomain testis-specific (BRDT) protein belongs to the bromodomain extra-terminal (BET) family of proteins. It serves as a transcriptional regulator of gene expression during spermatogenesis, and is an essential factor for the normal spermatogenesis process. In this study, we characterized mice of several age groups who lacked the Brdt gene. The testes of Brdt mutant mice aged 8 weeks exhibited complete spermatocyte maturation arrest with a significantly increased number of apoptotic cells. The weights of the testes and accessory glands as well as the testosterone levels of the mutant mice were significantly lower compared to the normal mice. The mutant mice had delayed puberty, with normal levels of testosterone and accessory gland weights at the age of 14 and 28 weeks. The testes of the mutant mice at older ages also exhibited round spermatids. The presence of the BRDT protein was identified in the mice pituitary gland. Microarray analysis of mice pituitaries showed that 28 genes were down-regulated while 26 genes were up-regulated in the absence of the Brdt gene. Our results suggest that in addition to its critical role in the spermatogenesis process, the BRDT protein is also responsible for scheduling male puberty by regulation of the pituitary-gonad axis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Nucleares/genética , Espermatogênese/genética , Animais , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/biossíntese , Sistema Hipófise-Suprarrenal/crescimento & desenvolvimento , Sistema Hipófise-Suprarrenal/metabolismo , Espermátides/crescimento & desenvolvimento , Espermátides/metabolismo , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/patologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismoRESUMO
PURPOSE: Mature sperm cells can be found in testicular specimens extracted from azoospermic men with non-mosaic Klinefelter syndrome (KS). The present study evaluates the expression of various known molecular markers of spermatogenesis in a population of men with KS and assesses the ability of those markers to predict spermatogenesis. METHODS: Two groups of men with non-obstructive azoospermia who underwent testicular sperm-retrieval procedures were included in the study: 31 had non-mosaic KS (KS group) and 91 had normal karyotype (NK group). Each group was subdivided into mixed atrophy (containing some mature sperm cells) or Sertoli cell only syndrome according to testicular histology and cytology observations. Semi-quantitative histological morphometric analysis (interstitial hyperplasia and hyalinization, tubules with cells and abnormal thickness of the basement membrane) and expression of spermatogenetic markers (DAZ, RBM, BOLL, and CDY1) were evaluated and compared among those subgroups. RESULTS: Clear differences in the histological morphometry and spermatogenetic marker expression were noted between the KS and NK groups. There was a significant difference in the expression of spermatogenetic markers between the subgroups of the NK group (as expected), while no difference could be discerned between the two subgroups in the KS group. CONCLUSION: We conclude that molecular spermatogenetic markers have a pattern of expression in men with KS that is distinctively different from that of men with NK, and that it precludes and limits their use for predicting spermatogenesis in the former. It is suggested that this difference might be due to the specific highly abnormal histological morphometric parameters in KS specimens.
Assuntos
Azoospermia/metabolismo , Síndrome de Klinefelter/metabolismo , Espermatogênese/genética , Testículo/metabolismo , Adulto , Azoospermia/complicações , Azoospermia/patologia , Biomarcadores/metabolismo , Humanos , Síndrome de Klinefelter/complicações , Síndrome de Klinefelter/patologia , Masculino , Recuperação Espermática , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/metabolismo , Espermatozoides/patologiaRESUMO
OBJECTIVE: To test the effect of sperm specimen volume in the freezing-thawing process on specimen quality. DESIGN: Experimental prospective study. SETTING: Tertiary academic medical center. PATIENT(S): Fifty high-quality sperm donors donated â¼3 times each. Sperm samples were split into two aliquots and frozen in volumes of 0.25 mL and 0.5 mL. INTERVENTION(S): Semen analyses. MAIN OUTCOME MEASURE(S): Eight sperm quality parameters of thawed specimens. RESULT(S): Thawed 0.5-mL specimens had a higher percentage of motility and viability, progressive motility concentration, percentage of cells with high mitochondrial membrane potential, and intact chromatin compared with 0.25-mL specimens. Although there were fewer cells with intact acrosomes in the 0.5-mL thawed samples, they had a similar ability to respond to ionophore by acrosome reaction as the 0.25-mL specimens. Both groups had similar percentages of cells with oxidative stress and numbers of cells that bound to the zona pellucida. The remaining air volume in the straw and freezing medium composition had a minimal effect on tested parameters. CONCLUSION(S): Better quality thawed human sperm was achieved after cryopreservation of high volumes compared with low volumes of specimens. Air volume in the straw had no influence on specimen quality.
Assuntos
Tamanho Celular , Criopreservação/métodos , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Acrossomo/fisiologia , Humanos , Masculino , Estudos Prospectivos , Distribuição Aleatória , Contagem de Espermatozoides/métodosRESUMO
Sperm cryopreservation is the best modality to ensure future fertility for males diagnosed with cancer. The extent to which cryopreserved sperm is actually used for impregnation, the fertility treatment options that are available and the success rates of these treatments have not been investigated in depth. The medical records of 682 patients who cryopreserved sperm cells due to cancer treatment were analyzed. Seventy of these patients withdrew their frozen sperm for fertility treatments over a 20-year period (most within the first 4 years after cryopreservation). Sperm quality of different malignancies and outcomes of assisted reproduction treatment (ART) for pregnancy achievement in relation to the type of treatment and the type of malignancy were evaluated. The results showed that the rate of using cryo-thawed sperm from cancer patients for fertility treatments in our unit was 10.3%. Sperm quality indices differed between different types of malignancies, with the poorest quality measured in testicular cancer. Conception was achieved in 46 of the 184 ART cycles (25%), and resulted in 36 deliveries. The use of intracytoplasmic sperm injection (ICSI) methodology yielded a significantly higher pregnancy rate (37.4%) than intrauterine insemination (IUI; 11.5%) and was similar to other groups of infertile couples using these modalities. In vitro fertilization (IVF) failed to produce pregnancies. In conclusion, the rate of use of cryopresseved sperm in cancer patients is relatively low (10.3%). Achievement of pregnancies by ICSI presents the best option but when there are enough stored sperm samples and adequate quality, IUI can be employed. Cryopreservation is nevertheless the best option to preserve future fertility potential and hope for cancer patients.
Assuntos
Criopreservação , Neoplasias/complicações , Resultado da Gravidez , Técnicas de Reprodução Assistida/estatística & dados numéricos , Análise do Sêmen , Espermatozoides , Feminino , Preservação da Fertilidade , Humanos , Infertilidade Masculina/etiologia , Linfoma/complicações , Linfoma/terapia , Masculino , Neoplasias/terapia , Gravidez , Taxa de Gravidez , Centros de Atenção Terciária , Neoplasias Testiculares/complicações , Neoplasias Testiculares/terapiaRESUMO
OBJECTIVE: To investigate genetic, molecular and functional aspects of human zona pellucida (ZP) in oocytes with an abnormal appearance. STUDY DESIGN: The study included three women with unexplained infertility whose oocytes had an abnormal ZP appearance and the mother and fertile sister of one of them. The coding exons and their flanking intron regions of the four ZP genes and the regulatory element for the ZP3 gene were sequenced. Immunofluorescence staining of discarded oocytes using monoclonal antibodies against recombinant human ZP glycoproteins and a hemizona assay were performed. RESULTS: No new mutations were observed in the ZP1 (12 exons), ZP2 (19 exons), ZP3 (9 exons), ZP4 (12 exons) genes or in the ZP3 regulatory element of the three studied women. Sequencing of the genes revealed eight synonymous and non-synonymous reported polymorphisms only in ZP1, ZP2 and ZP3. Immunofluorescence staining of the discarded oocytes of two women showed clear and strong staining of the ZP1, ZP2 and ZP4 proteins, but weak staining of the ZP3 protein, although their ZP displayed normal sperm binding ability in the hemizona assay. Intracytoplasmic sperm injection yielded good pregnancy outcomes, even though few injected oocytes developed normally up to day 3. CONCLUSIONS: The abnormal oocyte ZP appearance in the three study women may not have been due to the genetic changes in the ZP genes. Moreover, sperm binding was normal despite low ZP3 staining observed, suggesting that ZP3 profile may play a subordinate role in the reported cases. Our findings support previous studies which claim that abnormal oocyte morphology is not associated with a decrease in fertilization rates or birth outcomes in couples undergoing intracytoplasmic sperm injection.
Assuntos
Proteínas do Ovo/genética , Infertilidade Feminina/terapia , Glicoproteínas de Membrana/genética , Doenças Ovarianas/genética , Polimorfismo Genético , Receptores de Superfície Celular/genética , Elementos Reguladores de Transcrição , Zona Pelúcida/patologia , Adulto , Análise Mutacional de DNA , Ectogênese , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Éxons , Saúde da Família , Feminino , Humanos , Infertilidade Feminina/etiologia , Nascido Vivo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Mutação , Oócitos/metabolismo , Oócitos/patologia , Doenças Ovarianas/metabolismo , Doenças Ovarianas/patologia , Doenças Ovarianas/fisiopatologia , Gravidez , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Injeções de Esperma Intracitoplásmicas , Interações Espermatozoide-Óvulo , Zona Pelúcida/metabolismo , Glicoproteínas da Zona PelúcidaRESUMO
OBJECTIVE: To evaluate the frequency of complete and partial AZFa Y-chromosome microdeletions among infertile Israeli men. To review the published frequencies and histologic findings of AZFa deletions. DESIGN: Retrospective study. SETTING: Academic medical center. PATIENT(S): A total of 1,260 infertile Israeli men. Literature review (2000-2010) of reports on men with AZFa deletions and their testicular findings. INTERVENTION(S): The DNA of 1,260 infertile men was evaluated for AZF microdeletions. The DNA of 657 of them with undetected microdeletions was analyzed for partial AZFa deletion in the USP9Y and DDX3Y genes using sequence-tagged sites beyond EAA/EMQN recommendations. MAIN OUTCOME MEASURE(S): The frequency of complete and partial AZFa microdeletions. Availability of sperm cells for intracytoplasmic sperm injection in men with complete/partial microdeletions. RESULT(S): Two men had complete AZFa deletion (a frequency of 0.28% among nonobstructive azoospermic men). None had partial AZFa deletions. CONCLUSION(S): The likelihood of finding sperm cells in men with complete AZFa deletions is negligible. Complete AZFa deletion is rare and usually associated with azoospermia and absence of sperm cells in testicular tissue. The low frequency of partial AZFa deletions and the inconsistent prospects for spermatogenesis reported in the literature question the need for routine assessment of microdeletions in genes, such as USP9Y or DDX3Y.
Assuntos
Azoospermia/diagnóstico , Azoospermia/genética , Cromossomos Humanos Y/genética , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/genética , Adulto , Azoospermia/complicações , Deleção Cromossômica , Frequência do Gene , Humanos , Infertilidade Masculina/etiologia , Masculino , Programas de Rastreamento/métodos , Repetições de Microssatélites/genética , Valor Preditivo dos Testes , Estudos Retrospectivos , Deleção de Sequência/genética , Aberrações dos Cromossomos Sexuais , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/diagnóstico , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/genética , Adulto JovemRESUMO
There has been considerable concern worldwide about possible semen quality deterioration over the last 2 decades. The aim of this study was to evaluate freezability and semen quality of healthy young males during the years 1992-2010. A total of 1211 young (20-32 years old) candidates for sperm bank donation were recruited into the study with no exclusion criteria. They were instructed to observe 2 to 3 days of abstinence from sexual activity, and most of them supplied 2 specimens each. Average values of the various semen parameters, including freezing survival, were calculated for each participant. The change in different semen parameters over years, according to yearly and monthly average temperatures, was evaluated by SAS PROC SURVEYREG analysis. During that period, there were significant increases in motility and vitality percentages, as well as in the percentage of thawed sperm motility. The parameters of volume, concentration, normal morphology, total count, and total motile count showed a significant decrease with years (P < .01). The significant increase in average yearly temperature (P < .004) had limited, nonsignificant association with any of the semen variables. However, average monthly temperature contributed significantly to the trend of semen quality parameters (ie, specimen volume, concentration, percentage of normal morphology, and thawed motility). To the best of our knowledge, this is the first demonstration of the occurrence of an improvement in percent thawed motility over the years, and its significance lies in enabling a higher proportion of sperm bank candidates to be suitable for donation. It is suggested that the global warming phenomenon might have only partial contribution to semen variable changes over the years.
Assuntos
Criopreservação , Análise do Sêmen , Sêmen , Bancos de Esperma , Espermatozoides/patologia , Doadores de Tecidos , Adulto , Forma Celular , Sobrevivência Celular , Seleção do Doador , Aquecimento Global , Humanos , Israel , Masculino , Estações do Ano , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Fatores de Tempo , Adulto JovemRESUMO
OBJECTIVE: To characterize the BET gene expression in human testis with spermatogenetic impairments; to examine BRDT protein expression in testis and semen. DESIGN: Prospective study. SETTING: Fertility clinic. PATIENT(S): Azoospermic men (n = 120) who underwent testicular sperm extraction and who were classified as either normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest, or Sertoli cells only according to their combined histologic and cytologic testicular findings and three normozoospermic men who donated sperm. INTERVENTION(S): Evaluation of testicular biopsies by qualitative and quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical staining, and analysis of spermatozoa by immunofluorescence. MAIN OUTCOME MEASURE(S): Expression of the four BET genes in testis and localization of BRDT protein in testicular tissue and ejaculated spermatozoa. RESULT(S): The BRDT gene was not expressed in testicular tissue from patients with Sertoli cells only, whereas the other three genes of the BET family retained expression in all the pathologies. The BRDT protein was localized in the nuclei of spermatocytes, spermatids, and ejaculated spermatozoa. Expression of BRDT protein was almost nil in testicular tissue specimens with spermatocyte maturation arrest despite normal transcript levels. CONCLUSION(S): Human BRDT expression pattern differs from mouse BRDT expression. In human, BRDT is the only BET gene expressed exclusively in testicular germ cells. Its expression in elongated spermatids and ejaculated spermatozoa raises the possibility that it is involved in unidentified additional functions.
Assuntos
Azoospermia/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/genética , Síndrome de Células de Sertoli/genética , Fatores de Transcrição/genética , Azoospermia/patologia , Biópsia , Proteínas de Ciclo Celular , Epigênese Genética/fisiologia , Expressão Gênica/fisiologia , Humanos , Masculino , Proteínas Nucleares/metabolismo , Estudos Prospectivos , Síndrome de Células de Sertoli/patologia , Espermátides/patologia , Espermátides/fisiologia , Espermatogênese/genética , Espermatozoides/patologia , Espermatozoides/fisiologiaRESUMO
OBJECTIVE: To evaluate the molecular markers CDY1 and BOULE in the same testicular biopsy for predicting success of sperm retrieval in azoospermic men. DESIGN: Prospective study. SETTING: University-affiliated medical center. PATIENT(S): Azoospermic men (n = 92) who underwent testicular sperm extraction (TESE) and who were classified as normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest, or Sertoli cell only according to their combined histological and cytological testicular findings. INTERVENTION(S): Quantitative and qualitative evaluation of testicular biopsies by histological and qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) expression methodologies. MAIN OUTCOME MEASURE(S): CDY1 and BOULE expression and the presence of sperm cells in testicular tissue. RESULT(S): Both transcripts significantly predicted the presence of sperm cells by qualitative and quantitative methodologies. Although CDY1 had the best sensitivity by qualitative RT-PCR (98.3%), assessing both transcripts simultaneously had an additive efficacy compared with assessing CDY1 alone, improving the specificity from 84.4% to 96.3%. CONCLUSION(S): Assessing the expression of both CDY1 and BOULE by qualitative RT-PCR is a sensitive and feasible test for predicting the presence of sperm cells in testicular tissue and may serve as a predictive tool if repeated TESE is required.