[Evaluating the presence of testis specific transcripts in mature human spermatozoa]

Progress and completion of spermatogenesis is related to simultaneous expression of various genes. Recent studies show that many genes are expressed in the sperm and several RNA copies are present in the mature spermatozoa. Identification of these genes and evaluation of their functions would improve our understanding of the molecular basis of fertilization, early embryo cleavage and the causes of many types of unexplained male infertility. In this study, we investigated the expression of DAZ, PRM1, PRM2, TSGA10, SYCP3 and AKAP4 genes in ejaculated human spermatozoa. Semen samples were collected from men referring to Avicenna Infertility Clinic. Normal semen samples [According to WHO criteria] were subjected to density-gradient centrifugation to specifically recover the pure fraction of motile spermatozoa with normal morphology. Total RNA was extracted from sperm pellets and cDNA was synthesized using RT-PCR. The presence of DAZ, TSGA10, PRM1 and PRM2 cDNAs were evaluated using appropriate primers. Expression of SYCP3 [Testis specific gene] was evaluated by nested RT-PCR. The cDNA synthesized from normal testis tissues was used as positive control. Study on cDNAs showed that DAZ, TSGA10, PRM1 and PRM2 transcripts were present in normal human testis and all of the evaluated mature spermatozoa samples but not AKAP4 or SYCP3 transcripts. According to our previous study, the expression of SYCP3 and AKAP4 genes is started from spermatocyte level in human testis during spermatogenesis process. However, we did not found any transcripts of these genes in mature spermatozoa. It is estimated that mRNAs of TSGA10, PRM1, PRM2 and DAZ and other testis specific genes in spermatozoa may participate in later sperm functions such as fertilization and early embryo cleavage. Therefore, further studies are needed to understand the role of these transcripts in the process of fertilization and early embryo development

expression profile of indolamine 2,3-dioxygenase in murine endometrium during estrous cycle

Activation of Indolamine 2,3-dioxygenase [IDO], an enzyme responsible for tryptophan catabolism, has been reported to be a necessary requirement to achieve immunological tolerance against the fetus and protection against intracellular and extracellular pathogens. The objective of this study was to evaluate the expression of IDO gene in murine endometrium and its expression rate in different phases of estrous cycle. Noticing the role of this enzyme especially in the survival of a semi-antigenic embryo, the results of this study may be used as a basis for practical studies on the immunologic bases of recurrent abortions. In this experimental study, we studied the expression of IDO in the female BALB/c mice endometrium during four stages of estrous cycle. The phases of estrous cycle were determined by examining vaginal cytology .At each phase, endometrium was pealed away and the relative expression of IDO mRNA was detected by semi-quantitative RT-PCR using specific primers to IDO and mGAPDH as a housekeeping gene. The specificity of reaction was confirmed by enzymatic digestion of amplicon which yielded to 138bp and 259bp fragments. Our results showed, for the first time, that IDO is expressed in the endometrium of cycling mice during all stages of estrous cycle. The expression of IDO was highest at estrus and lowest at diestrus [p<.001]. Expression of IDO in endometrium during all phases of estrous cycle reveals that this enzyme as an effective arm of innate immune system may serve a role in protecting the female reproductive tract against ascending infections. Also regarding the fact that, mating only occurs at estrus phase, the high expression of IDO in this phase, may act as the main mechanism in inducing immunological tolerance to the fetus

Intrafollicular fluid antigamete antibodies in infertile patient candidates for ICSI

Immunologic disturbances must be considered as a major cause of infertility. Antigamete antibodies like antisperm antibodies [ASA] and to anti-zona antibodies [AZA] seem to be implicated in the etiology of infertility. These antibodies affect fertilization and embryo development. It is important to screen these antibodies in infertile women who are candidates for in-vitro fertilization [IVF], because the presence of these antibodies may switch the treatment from IVF to intra-cytoplasmic microinjection [ICSI]. The objective of this study was to determine the presence of ASA and AZA in the follicular fluids [FF] of women who sought candidacy for ICSI. In this prospective study, the follicular fluids of 96 infertile women [20 to 39 years old, mean 31.5 +/- 5.1], who were candidates for ICSI, were evaluated. According to the etiologies, 80 women had explained whereas 16 had unexplained infertility. All the follicular fluids were evaluated for the presence of ASA by ELISA and Sperm MAR test and also for the presence of AZA by ELISA. The data were analyzed by Chi-square test using SPSS soft-ware and the significance level was considered p<0.05. According to the results of ELISA and Sperm MAR test, none of the patients had ASA in their follicular fluids. However, twenty samples [20.8%] were positive for AZA. In patients with unexplained infertility, autoantibodies to zona pellucida were significantly higher in the follicular fluid than the group with proven etiologies for infertility [p=0.001]. The low incidence of ASA and the high incidence of AZA in the infertile women in this study, especially in women with unexplained infertility in Iran have to be considered seriously. Determination of AZA is highly recommended in the evaluation of infertile couples, especially those with unexplained infertility

Attitudes of infertile women towards gamete donation: a case study in Tehran

On average, around 10-15 percent of couples in Iran are infertile. There have been major achievements in the area of modern technologies on infertility treatment and infertile couples have no obstacles or legal barriers to use these technologies in Iran. However, not all infertile couples are using modern technologies in Iran, and their decision making to use these technologies is influenced by their perceptions as well as societal expectations and attitudes. A deeper understanding of the perceptions and attitudes of infertile couples would shed light on socio-cultural aspects of their decision making in using these modern technologies. The main objective of the present paper was to study the attitudes of infertile women on gamete and embryo donation for infertility treatment in Tehran. The data were collected from thirty women by using qualitative methods and in-depth interviews. Fifty percent of the respondents were selected from women visiting Avesina Infertility Clinic in Tehran and the rest were randomly selected from infertile women referring to five Health Centers of Shahid Beheshti Health Deputy in Tehran. The data for this study was collected during July and September 2005. The results of this study indicate that, there is a difference between the attitudes of respondents from Avesina Infertility Clinic [AIC] and their counterparts from Shahid Beheshti Health Centers. All respondents from AIC were familiar with gamete donation and believed that it was a medical step towards treatment of infertility, and their religious beliefs did not contradict their decision. However, the respondents from Shahid Beheshti Health Centers did not have any information regarding religious leaders' attitudes towards those methods, and the majority of them considered those methods as forbidden or illegal. In addition, the infertile women in the study were afraid of their treatment not kept confidential and that other people would not consider their child as their biological offspring. Providing appropriate information related to new treatment methods, laws, and religious edicts not only will increase public awareness but also will change individuals' stereotypical views on infertility, thus promote infertile couples' decision making on the use of gamete and embryo donation for infertility treatment

[Evaluation of Spermatogenesis in non-obstructive azoospermic patients with histopathological and cytological methods]

The ability to use only a few spermatozoa using testicular biopsy and by microinjection technique to achieve fertilization and pregnancy has revolutionized the potentials to treat patients suffering from azoospermia. However, spermatogenesis is defective in men with non-obstructive azoospermia [NOA] resulting in failure to detect spermatozoa. In order to achieve a more sensitive and reliable method for detecting sperm/spermatid in testes of NOA patients, we compared histopathological and cytological methods. Eighty six NOA patients were included in a prospective study. History taking, physical examination and hormonal profile [including FSH] were done initially. Thirty six patients had testis pathology report before enrolling in to our study. The patients underwent multiple bilateral testis biopsies until successful retrieval of sperm/spermatid. Half of each biopsy specimen underwent cytological evaluation [mechanical and enzymatic] and the other half was sent for pathological evaluation [TBX]. The male and female mean ages were 37 [25-59] and 32 [23-42] years, respectively. The mean infertility duration was 7 years. The mean volume of right and left testis were 16.4 and 16.2 ml, respectively. Mean FSH level was 18.1 +/- 14.2 ml U/ml. In cytology, sperms and spermatids were seen in 65 and 18 patients, respectively and in pathology slides in 51 and 16 patients, respectively. In our study, the relationship between visualizing testicular sperm/spermatid and TESE had sensitivity of 80% and negative predictive value of 15%. Sixty one patients had sufficient number of sperm/spermatid for ICSI and with this treatment fifty seven embryos were transferred and seven clinical pregnancies were observed. In conclusion, in men with non-obstructive azoospermia, TESE is more sensitive and reliable than histopathology evaluation. This means that TESE may help in deciding for treatment of severe male factor infertility, even when histopathologic examination is inconclusive

[Evaluation of rat sperm motility by CASA]

When mammalian spermatozoa first enter the epididymis from testis they display little or no progressive motility. On transit along the epididymis, spermatozoa gradually acquire the potential for full progressive motility. This process which begins after sperm leave the testis, is called sperm maturation and requires specific factors from the epididymal epithelium. The aim of this study was to investigate the use of computer-assisted sperm analysis for the objective determination of rat sperm motility. Having validated this system, it would be used to assess in vitro and in vivo sperm maturation. Sperm samples from various regions of the epididymis and ejaculates, were prepared. Primary cultures of the epithelium from the rat epididymis were established. A novel computer-aided sperm analysis was used to analyze and characterize the motility of the rat spermatozoa during in vitro and in vivo maturation. Sperm motility analysis also examined during co-culture. Image analyses of sperm movement from video recording tapes were also examined. This study used a novel CASA system and image analysis to monitor the changes in motility as spermatozoa undergo maturation in the rat epididymis, during ejaculation and during co-incubation with epididymal epithelial cultures and medium preparations. It is interesting to note that changes in epididymal sperm motility were occurred in vivo and in vitro. The co-incubation of immature rat spermatozoa from the caput epididymis with epithelial cultures led to significant changes in progressive sperm motility parameters associated with maturation. This study indicate the CASA and other image analysis techniques, were able to characterize in detail the objective determination and changes in motility parameters of rat spermatozoa. Validation of this system provides the best facility to assess rat sperm maturation in vitro

[Intracytoplasmic sperm injection [ICSI] of one and two day[s]-old oocytes after complete in vitro fertilization [IVF] failure]

The objective of this study was to evaluate the outcome of late [one and two days] Intracytoplasmic sperm injection [ICSI] after total fertilization failure in IVF. 35 IVF cycles that were part of our regular IVF program and showed no evidence of fertilization 16-46 hours after insemination [oocytes were observed at 16-18 hours and again 42-44 hours after the IVF procedures], were assigned to two treatment groups. Assisted fertilization with ICSI was carried out at 24 and 48 hours after oocyte retrieval. Group I [injected-day 1], consisted of 21 patients with 72 failed-fertilized metaphase II oocytes injected 1 day after ovum pick-up; and group II [injected-day 2], included 14 patients with 45 failed-fertilized metaphase II oocytes injected 2 days after ovum pickup. A single spermatozoon from the patient's husband [same as that used for insemination in IVF program] was injected into the cytoplasm of each of these oocytes. Resultant embryos were transferred 72 and 96 hours after oocyte retrieval in group I and II, respectively. Fertilization was achieved with ICSI in most patients with fertilization failure. In group I, [80.5%] oocytes fertilized, whereas in group II, 46.6% of oocytes fertilized. Cleavage rate was 79.3% of injected oocytes in group I, and 42.8% in group II. Finally, in group one 19 of 21 [94%] embryos well transferred. The transfer rate for group two was 11 of 14 [78%]. These results indicate significant differences between fertilization and cleavage rates in both groups. One of the singleton pregnancies resulted from transfer of the embryos in group II, and none in group I. This is the first known pregnancy achieved from late [two days] ICSI and late transferred embryos, after failed IVF. In conclusion, late [24 and 48 hours] ICSI after complete failed fertilization in IVF, can give good fertilization and good cleavage rates. This method can be used as an ideal protocol in IVF programs, to increase chance of pregnancy in infertile couples using the advantages of two main assisted reproductive treatments including IVF and ICSI

[Role of sperm surface proteins with epididymal origin on fertilization]

During transit of spermatozoa through the epididymis, where develop its capacity to fertilize oocytes, several new antigenic determinants appear on surface of spermatozoa. Many of these are proteins and glycoproteins with epididymal origin. The aim of this study was evaluation the role of epididymal secretory proteins on fertilizing ability of spermatozoa. In This study, epithelial cells from proximal portion of rat epididymis were cultured in RPMI medium supplemented with growth factors and androgens. To identify epididymal secretory proteins, pulse labeling with [[35]S]-[Met, Cys] was used. Labeled conditioned medium recovered and subjected to SDS-PAGE and flurography. Antiserum against 8 secretory proteins were produced in Rabbits. The immunogens were lyophilized narrow strip of polyacrylamide gel from preparative electrophoresis with ultrapure proteins. Incubation of intact rat spermatozoa with these antisera was revealed secretory proteins on surface of spermatozoa. The effects of these antisera on in vitro fertilization were evaluated. The results showed that epithelial cells secreted about 20-30 proteins in culture medium. High titer antisera for 8 secretory proteins were produced. Three antisera were reacted with surface of rat spermatozoa [20, 24 and 72 kD proteins], and only 20 kD protein antiserum decreased fertilization rate. [28% in treatment group against 89% for control group]. In Conclusion, the epididymal maturation process of spermatozoa was associated with the secretion of a number proteins by epididymal epithelial cells, many of these proteins bound to plasma membrane of spermatozoa. Only secreted 20 kD protein has a critical role in fertilizing ability of spermatozoa