Upregulation of the RNF8 gene can predict the presence of sperm in azoospermic individuals / 대한생식의학회지

Objective@#In this study, specimens from testicular biopsies of men with nonobstructive azoospermia (NOA) were used to investigate whether RNF8 gene could serve as a biomarker to predict the presence of sperm in these patients. @*Methods@#Testicular biopsy specimens from 47 patients were classified according to the presence of sperm (positive vs. negative groups) and investigated for the expression of RNF8. The level of RNF8 gene expression in the testes was compared between these groups using reverse-transcription polymerase chain reaction. @*Results@#The expression level of RNF8 was significantly higher in testicular samples from the positive group than in those from the negative group. Moreover, the area under the curve of RNF8 expression for the entire study population was 0.84, showing the discriminatory power of RNF8 expression in differentiating between the positive and negative groups of men with NOA. A receiver operating characteristic curve analysis showed that RNF8 expression had a sensitivity of 81% and a specificity of 84%, with a cutoff level of 1.76. @*Conclusion@#This study points out a significant association between the expression of RNF8 and the presence of sperm in NOA patients, which suggests that quantified RNF8 expression in testicular biopsy samples may be a valuable biomarker for predicting the presence of spermatozoa in biopsy samples.

Association between JMJD1A expression and sperm retrieval in non-obstructive azoospermic patients

Identification of molecular markers which can predict the outcome of sperm retrieval non-invasively in patients with non-obstructive azoospermia [NOA] are valuable in clinical andrology. Jumonji domain-containing 1a [JMJD1A] is a significant epigenetic regulator during spermatogenesis, which plays an important role in the differentiation of post-meiotic germ cells into mature spermatozoa. We therefore aimed to examine the potential association between JMJD1A expression and the outcome of sperm retrieval in patients with NOA. Testicular biopsy specimens from 50 NOA patients with either successful sperm retrieval [sperm+, n=22] or failed sperm retrieval [sperm-, n=28] were collected and then examined for JMJD1A expression by reverse transcription-quantitative polymerase chain reaction [RT-qPCR]. In addition, conventional clinical parameters including luteinizing hormone, follicle-stimulating hormone, testosterone, age, and testicular volume were compared between the two NOA groups. The expression of JMJD1A in the sperm+ group was significantly higher than in the sperm- group [P<0.001], however, no significant difference was observed between the two groups in clinical parameters. The receiver operating characteristic [ROC] curve of JMJD1A expression in predicting the sperm retrieval outcome showed a sensitivity of 90.91% and a specificity of 89.29% with significant discriminatory ability between the sperm+ and sperm- groups [area under the ROC curve [AUC]= 0.91]. This study demonstrates a significant association between the expression of JMJD1A and the success of sperm recovery in patients with NOA, and thus suggests that JMJD1A expression quantification in testicular biopsies may be a valuable biomarker along with conventional parameters in predicting the presence of spermatozoa

Sperm retrieval in patients with klinefelter syndrome: a skewed regression model analysis

Background: The most common chromosomal abnormality due to non-obstructive azoospermia [NOA] is Klinefelter syndrome [KS] which occurs in 1-1.72 out of 500-1000 male infants. The probability of retrieving sperm as the outcome could be asymmetrically different between patients with and without KS, therefore logistic regression analysis is not a well-qualified test for this type of data. This study has been designed to evaluate skewed regression model analysis for data collected from microsurgical testicular sperm extraction [micro-TESE] among azoospermic patients with and without non-mosaic KS syndrome

Materials and Methods: This cohort study compared the micro-TESE outcome between 134 men with classic KS and 537 men with NOA and normal karyotype who were referred to Royan Institute between 2009 and 2011. In addition to our main outcome, which was sperm retrieval, we also used logistic and skewed regression analyses to compare the following demographic and hormonal factors: age, level of follicle stimulating hormone [FSH], luteinizing hormone [LH], and testosterone between the two groups

Results: A comparison of the micro-TESE between the KS and control groups showed a success rate of 28.4% [38/134] for the KS group and 22.2% [119/537] for the control group. In the KS group, a significantly difference [P<0.001] existed between testosterone levels for the successful sperm retrieval group [3.4 +/- 0.48 mg/mL] compared to the unsuccessful sperm retrieval group [2.33 +/- 0.23 mg/mL]. The index for quasi Akaike information criterion [QAIC] had a goodness of fit of 74 for the skewed model which was lower than logistic regression [QAIC=85]

Conclusion: According to the results, skewed regression is more efficient in estimating sperm retrieval success when the data from patients with KS are analyzed. This finding should be investigated by conducting additional studies with different data structures

Isolation, culture and characterization of human sertoli cells by flow cytometry: development of procedure

Background: the sertoli cells in the testis create unique and safe environment to protect seminiferous tubules from auto antigens and invading pathogens. These cells produce the survival factor of the blood-testis barrier and produce special materials such as androgen binding proteins and contribute to the coordinated action of spermatogenesis. Given that the sertoli cells play an essential role in spermatogenesis and the lack of these cells leads to the disruption of spermatogenesis, it is necessary to investigate the behavior and performance of these cells. To achieve this goal, the cells must first be extracted. The aim of this study was to develop a procedure to isolate, culture, and characterize human sertoli cells

Methods: in order to isolate the sertoli cells of azoospermia patients who underwent [testicular sperm extraction] TESE surgery, washing up and multi_stage enzyme digestion of single cells, culture on petri dishes impregnated with datura stramonium lectin agglutinin [DSA] were done and then the cells were passaged for several times and isolated. For more purification, flow cytometry method with FSH receptor antibody was used. Immunocytochemistry assays and Elisa test for identification of these cells were employed

Results: the purification method resulted in more than 97% purity. The nature of sertoli cells was confirmed by morphology evaluation, detecting anti-mullerian hormone in sertoli cell culture media and the presence of FSH receptor on sertoli cells

Conclusion: this study introduced and applied a method to isolate, culture, and purify human sertoli cells with high purity which made possible further researches on these cells

expression of TLR2 and TLR3 in sertoli cells of azoospermic patients

Objective: Toll-like receptors [TLRs] on Sertoli cells are thought to have essential roles in sperm protection. This study was conducted to investigate the expression of TLR2 and TLR3 in Sertoli cells of men with azoospermia

Materials and Methods: In this experimental study, testicular biopsies were taken from ten azoospermic men. Following enzymatic dissociation, the samples were moved to lectin coated petri dishes. After a few passages, all cells were cultivated and Seroli cells were sorted by flow cytometry. To confirm Sertoli cell purification, alkaline phosphatase activity [ALP] and immunohistochemistry assays were employed. The expression of TLR2 and TLR3 at the transcript and protein levels was examined with real-time quantitative reverse transcription-polymerase chain reaction [RT-QPCR] and western blot, respectively

Results: Isolation, purification and cultivation of human Sertoli cells were performed successfully. Efficacy of purification of Sertoli cells by fluorescence-activated cell sorting [FACS] sorter was 97%. The type of cultured cells was confirmed by vimentin and follicle-stimulating hormone [FSH] receptor markers. Furthermore, the existence of anti- Mullerian hormone in culture was confirmed. RT-PCR showed that both genes were expressed in Sertoli cells. Consistently, proteins of both were also expressed in Sertoli cells. Moreover, QPCR showed that the relative expression of TLR3 transcripts was significantly higher than TLR2 in Sertoli cells. Although both genes are expressed in fibroblast cells, their level of expression was significantly lower than in Sertoli cells

Conclusion: This study confirmed expression of TLR2 and TLR3 in human Sertoli cells. This may be an indicator of their roles in developing immunity against pathogens as well as allo- and auto-antigens or viral antigens in seminiferous tubules

[Introduction of a new method for mono-specific antibody production by sequential use of recombinant proteins and synthetic peptides [PrIPeP model]]

Objective: This study attempted to generate monospecific antibodies through immunization with recombinant proteins and subsequent purification by synthetic peptides [the PrIPeP model]

Methods: The SRY gene was cloned on a pet-28a vector and the recombinant protein was expressed in the Escherichia coli [E.coli] BL21 strain. The purified antigen was emulsified in Freund's adjuvant and injected into rabbits according to a standard time table. Then, a specific peptide was designed, synthesized, and conjugated to sepharose 4B to generate an affinity purification column. As a control, the peptide was conjugated to KLH and used for immunization, as above. Antisera against the conjugated peptide [Pepantisera] and SRY recombinant protein [Pro-antisera] were evaluated by ELISA and subsequently subjected to the affinity purification column. Sensitivity and specificity of the purified antibodies against SRY recombinant protein as well as negative controls [recombinant HSFY, RBMY, and RPSFY] were assessed by Western blot analysis

Results: Titration by ELISA confirmed proper immunization and specificity of both antigens. Western blot analysis validated the specificity and sensitivity of the IgG class purified antibodies

Conclusion: By applying the PrIPeP model, it is possible to develop antibodies against the native structure of a protein whilst avoiding challenges of peptide-carrier protein conjugation

New single nucleotide polymorphism G5508A in the SEPT12 gene may be associated with idiopathic male infertility in Iranian men

Male infertility is a multifactorial disorder, which affects approximately 10% of couples at childbearing age with substantial clinical and social impact. Genetic factors are associated with the susceptibility to spermatogenic impairment in humans. Recently, SEPT12 is reported as a critical gene for spermatogenesis. This gene encodes a testis specific member of Septin proteins, a family of polymerizing GTP-binding proteins. SEPT12 in association with other Septins is an essential annulus component in mature sperm. So, it is hypothesized that genetic alterations of SEPT12 may be concerned in male infertility. The objective of this research is exploration of new single nucleotide polymorphism G5508A in the SEPT12 gene association with idiopathic male infertility in Iranian men. In this case control study, 67 infertile men and 100 normal controls were analyzed for genetic alterations in the active site coding region of SEPT12, using polymerase chain reaction sequencing technique. Fisher exact test was used for statistical analysis and p<0.05 was considered as statistically significant. Genotype analysis indicated that G5508A polymorphic SEPT12 alleles were distributed in three peaks of frequency in both control and diseases groups. Categorization of the alleles into [GG], [GA], [AA] types revealed a significant difference between infertile patients [azoospermic and asthenospermic] and normal controls [p=0.005]. According to our finding we suggest that G5508A polymorphism in SEPT12 gene can affect spermatogenesis in men, the opinion needs more investigation in different populations

Predictive factors of successful microdissection testicular sperm extraction in patients with presumed sertoli cell-only syndrome

To evaluate predictive factors of successful microdissection-testicular sperm extraction [MD-TESE] in patients with presumed Sertoli cell-only syndrome [SCOS]. In this retrospective analysis, 874 men with non-obstructive azoospermia [NOA], among whom 148 individuals with diagnosis of SCOS in prior biopsy, underwent MD-TESE at Department of Andrology, Royan Institute, Tehran, Iran. The predictive values of follicle stimulating hormone [FSH], luteinizing hormone [LH], and testosterone [T] levels, testicular volume, as well as male age for retrieving testicular sperm by MD-TESE were analyzed by multiple logistic regression analysis. Testicular sperm were successfully retrieved in 23.6% men with presumed SCOS. Using receiver operating characteristic [ROC] curve analysis, it was shown that sperm retrieval rate in the group of men with FSH values >15.25% was 28.9%. This was higher than the group of men with FSH

Comparative expression analysis of septin 14 in testes of infertile men with normal spermatogenesis and spermatogenic failure

Septins are an evolutionary conserved group of GTP-binding and filament-forming proteins that have diverse cellular roles. An increasing body of data implicates the septin family in the pathogenesis of diverse states including cancers, neurodegeneration, and male infertility. The objective of the study was to evaluate the expression pattern of Septin14 in testis tissue of men with and without spermatogenic failure. The samples retrieved accessible random between infertile men who underwent diagnostic testicular biopsy in Royan institute. 10 infertile men with obstructive azoospermia and normal spermatogenesis and 20 infertile men with non-obstructive azoospermia were recruited for real-time reverse transcription [RT]-PCR analysis of the testicular tissue. Total RNA was extracted with trizol reagent. Comparison of the mRNA level of septin14 revealed that in tissues with partial [n=10] or complete spermatogenesis [n=10], the expression of septin 14 was significantly higher than sertoli cell only tissues. The testicular tissues of men with hypospermatogenesis, maturation arrest and sertoli cell only had lower levels of septin 14 transcripts than normal men. These data indicates that Septin 14 expression level is critical for human spermatogenesis

rare de novo complex chromosomal rearrangement [CCR] involving four chromosomes in an oligo-asthenosperm infertile man

Complex chromosomal rearrangements [CCRs] are rare events involving more than two chromosomes and over two breakpoints. They are usually associated with infertility or sub fertility in male carriers. Here we report a novel case of a CCR in a 30-year-old oligoasthenosperm man with a history of varicocelectomy, normal testes size and normal endocrinology profile referred for chromosome analysis to the Genetics unit of Royan Reproductive Biomedicine Research Center. Chromosomal analysis was performed using peripheral blood lymphocyte cultures and analyzed by GTG banding. Additional tests such as C-banding and multicolor fluorescence in situ hybridization [FISH] procedure for each of the involved chromosomes were performed to determine the patterns of the segregations. Y chromosome microdeletions in the azoospermia factor [AZF] region were analyzed with multiplex polymerase chain reaction. To identify the history and origin of this CCR, all the family members were analyzed. No micro deletion in Y chromosome was detected. The same de novo reciprocal exchange was also found in his monozygous twin brother. The other siblings and parents were normal. CCRs are associated with male infertility as a result of spermatogenic disruption due to complex meiotic configurations and the production of chromosomally abnormal sperms. These chromosomal rearrangements might have an influence on decreasing the number of sperms

Expression analysis of RNA-binding motif gene on Y chromosome [RBMY] protein isoforms in testis tissue and a testicular germ cell cancer-derived cell line [NT2]

Background: RNA-binding motif gene on Y chromosome [RBMY], a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages

Methods: full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms

Results: selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate

Conclusion: the results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility. Iran. Biomed. J. 17[2]: 54-61, 2013

[Predictive value of semen parameters and age of the couple in pregnancy outcome after Intrauterine insemination]

Intrauterine insemination [IUI] is one of the most common methods in infertility treatment, but its efficiency in infertile couples with male factor is controversial. This study is a retrospective study about correlation between semen parameters and male and female age with successful rate of IUI in patients attending to Royan Institute. A total of 998 consecutive couples in a period of 6 months undergoing IUI were included. They were classified into two groups: couples with successful and unsuccessful pregnancy. Main outcome was clinical pregnancy. Data about male and female ages and semen analysis including concentration, total sperm motility, class A motility, class B motility, class A+B motility and normal morphology was extracted from patients' records. Semen samples were collected by masturbation or coitus after 2 to 7 days of abstinence. Their female partners were reported to have no chronic medical conditions and have normal menstrual cycles. One hundred and fifty seven of total 998 cycles [15.7%] achieved pregnancy. The average of female age in successful and unsuccessful group was 28.95 +/- 4.19 and 30.00 +/- 4.56 years, respectively. Mean of male age was 33.97 +/- 4.85 years in successful group and 34.44 +/- 4.62 years in unsuccessful group. In successful and unsuccessful groups, average of sperm concentration was 53.62 +/- 38.45 and 46.26 +/- 26.59 [million sperm/ml], normal morphology of sperm was 8.98 +/- 4.31 [%] and 8.68 +/- 4.81 [%], sperm total motility was 47.24 +/- 18.92 [%] and 43.70 +/- 20.22 [%] and total motile sperm count was 80.10 +/- 63.61 million and 78.57 +/- 68.22 million, respectively. There was no significant difference in mean of females' age and males' age between successful and unsuccessful groups [P<0.05]. In addition, there was no significant difference in semen parameters including concentration, total sperm motility, class A motility, class B motility, class A+B motility and normal morphology between two groups. It was shown that common semen analysis and male and female ages cannot predict IUI outcome.

Enzymatic digestion improves testicular sperm retrieval in non-obstructive azoospermic patients

In patients with non-obstructive azoospermia [NOA], vital spermatozoa from the tissue is obtained from testes by enzymatic treatment besides the mechanical treatment. To increase the sperm recovery success of testicular sperm extraction [TESE], with enzymatic digestion if no sperm is obtained from testis tissue by mechanical method. Tissue samples were collected from 150 men who presented with clinical and laboratory data indicating NOA by means of TESE and micro dissection TESE methods. Initially, mature spermatozoa were examined for by mechanical extraction technique shredding the biopsy fractions. In cases whom no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was followed by enzymatic digestion using DNaseI and collagenase type IV. Surgery type, pathology, AZF, karyotype, hormones and testis size were compared in patients. Of 150 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery in 13 [about 9%] patients. Comparison of parameters revealed that level of FSH and LH were significantly different [p=0.04 and 0.08 respectively] between two groups that response negative and positive to enzymatic digestion. The combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa. The benefit of the mincing combined with enzyme to sperm retrieval for NOA firstly shorten the mechanical searching time, leading to minimizing further cellular damage as well as exposure to external conditions, and secondly reduce the number of cases with sperm recovery failures. Also, the serum level of FSH and LH are factors that influence the chance of sperm retrieval