Evaluation of mouse oocyte in vitro maturation developmental competency in dynamic culture systems by design and construction of a lab on a chip device and its comparison with conventional culture system

Objective: In conventional assisted reproductive technology [ART], oocytes are cultured in static microdrops within Petri dishes that contain vast amounts of media. However, the in vivo environment is dynamic. This study assesses in vitro oocyte maturation through the use of a new microfluidic device. We evaluate oocyte fertilization to the blastocyct stage and their glutathione [GSH] contents in each experimental group

Materials and Methods: In this experimental study, we established a dynamic culture condition. Immature oocytes were harvested from ovaries of Naval Medical Research Institute [NMRI] mice. Oocytes were randomly placed in static [passive] and dynamic [active] in vitro maturation [IVM] culture medium for 24 hours. In vitro matured oocytes underwent fertilization, after which we placed the pronucleus [PN] stage embryos in microdrops and followed their developmental stages to blastocyst formation after 3 days. GSH content of the in vitro matured oocytes was assessed by monochlorobimane [MCB] staining

Results: We observed significantly higher percentages of mature metaphase II oocytes [MII] in the passive and active dynamic culture systems [DCS] compared to the static group [P<0.01]. There were significantly less mean numbers of germinal vesicle [GV] and degenerated oocytes in the passive and active dynamic groups compared to the static group [P<0.01]. Fertilization and blastocyst formation rate in the dynamic systems were statistically significant compared to the static cultures [P<0.01]. There was significantly higher GSH content in dynamically matured oocytes compared to statically matured oocytes [P<0.01]

Conclusion: Dynamic culture for in vitro oocyte maturation improves their developmental competency in comparison with static culture conditions

Comparison of mitochondrial-related transcriptional levels of TFAM, NRF1 and MT-CO1 genes in single human oocytes at various stages of the oocyte maturation

The aim of the current study was to assess the mRNA levels of two mitochondria-related genes,including nuclear-encoded NRF1 [nuclear respiratory factor 1], mitochondrial transcription factor A [TFAM], and mitochondrial-encoded cytochrome c oxidase subunit 1 [MT-CO1] genes in various stages of the human oocyte maturation. Oocytes were obtained from nine infertile women with male factor undergoing in vitro fertilization [IVF]/intra-cytoplasmic sperm injection protocol. Mitochondrial-related mRNA levels were performed by single-cell TaqMan real-time PCR. the expression level of the target genes was low at the germinal vesicle stage [P>0.05]. Although the mRNA level of NRF1gene remained stable in metaphase I, the mRNA level of TFAM and MT-CO1 increased significantly [P<0.05].In metaphase II, the expression level of all genes increasedcompared to metaphase I [P<0.05]. The overexpression levels of NRF1, TFAM, and MT-CO1 genes are related to the oocyte maturation. Therefore, the current study could be used clinically to improve the successrate of IVF.

Effect of human ovarian tissue vitrification/warming on the expression of genes related to folliculogenesis

Ovarian tissue cryopreservation is an alternative strategy to preserve the fertility of women predicted to undergo premature ovarian failure. This study was designed to evaluate the expression of folliculogenesis-related genes, including factor in the germline alpha [FIGLA], growth differentiation factor-9 [GDF-9], follicle-stimulating hormone receptor [FSHR], and KIT LIGAND after vitrification/warming of human ovarian tissue. Human ovarian tissue samples were collected from five transsexual women. In the laboratory, the ovarian medullary part was removed by a surgical blade, and the cortical tissue was cut into small pieces. Some pieces were vitrified and warmed and the others were considered as non-vitrified group [control]. Follicular normality was assessed with morphological observation by a light microscope, and the expression of FIGLA, KIT LIGAND, GDF- 9, and FSHR genes was examined using real-time RT-PCR in both the vitrified and non-vitrified groups. Overall, 85% of the follicles preserved their normal morphologic feature after warming. The percentage of normal follicles and the expression of FIGLA, KIT LIGAND, GDF-9, and FSHR genes were similar in both vitrified and non-vitrified groups [P > 0.05]. Vitrification/warming of human ovarian tissue had no remarkable effect on the expression of folliculogenesis-related genes

effect of melatonin on maturation, glutathione level and expression of HMGB1 gene in brilliant cresyl blue [BCB] stained immature oocyte

Nutrients and antioxidants in the medium of immature oocyte have a profound effect on maturation, fertilization and development of resulting embryos. In this study the effects of melatonin as an antioxidant agent on maturation, glutathione level and expression of high mobility group box-1 [HMGB1] gene were evaluated in immature oocytes of mice stained with brilliant cresyl blue [BCB]. In this experimental study, immature oocytes were harvested from ovaries of Naval Medical Research Institute [NMRI] mice. Oocytes were stained with 26 Micro M BCB for 90 minutes and transferred to in vitro maturation medium containing varying doses of melatonin [10[-12], 10[-9], 10[-6], 10[-3] M] and without melatonin, for 22-24 hours. Maturation was monitored using an inverted microscope. Glutathione was assessed by monochlorobimane [MCB] staining and HMGB1 expression in mature oocyte was analyzed using real-time polymerase chain reaction [PCR]. Melatonin in the concentration of 10[-6] M had the most effect on maturation and HMGB1 expression of BCB+ oocytes [p<0.05]. Meanwhile melatonin had no effects on glutathione levels. Additionally in immature BCB- oocytes, compared to the control group, melatonin did not affect cytoplasm maturation [p>0.05]. In vitro treatment with melatonin increases the maturation and HMGB1 expression in BCB+ immature oocytes and has no significant effect on glutathione levels

New single nucleotide variation in the promotor region of androgen receptor [AR] gene in hypospadic patients

Hypospadias is one of the most common congenital abnormalities in the male which is characterized by altered development of urethra, foreskin and ventral surface of the penis. Androgen receptor gene plays a critical role in the development of the male genital system by mediating the androgens effects. In present study, we looked for new variations in androgen receptor promotor and screened its exon 1 for five single nucleotide polymorphisms [SNP] in healthy and hypospadias Iranian men. In our study, at first DNA was extracted from patients [n=100] and controls [n=100] blood samples. Desired fragments of promoter and exon 1 were amplified using polymerase chain reaction. The promoter region was sequenced for the new variation and exone 1 screened for five SNPs [rs139767835, rs78686797, rs62636528, rs62636529, rs145326748] using restriction fragment length polymorphism technique. The results showed a new single nucleotide variation [CT] at -480 of two patients' promoter region [2%]. None of the mentioned SNPs were detected in patients and controls groups [0%].This finding indicates that new single nucleotide polymorphism in androgen receptor promoter may have role in etiology of hypospadias and development of this anomaly

Expression of glycogen synthase kinase 3-beta [GSK3-beta] gene in azoospermic men

The Wnt/beta- The Wnt/beta-catenin signaling pathway is involved in many developmental processes in both fetal and adult life; its abnormalities can lead to disorders including several types of cancers and malfunction of specific cells and tissues in both animals and humans. Its role in reproductive processes has been proven. This study was designed to evaluate the expression of the key regulator of this signaling pathway GSK3-beta and its presumed role in azoospermia. WNT3[a] protein concentration and GSK3-beta gene expression levels were measured and compared between two groups of infertile men. The test groups consisted of 10 patients with obstructive and 10 non-obstructive azoospermia. The control group was selected among healthy men after vasectomies that were willing to conceive a child using a testicular biopsy technique. Samples were obtained by testicular biopsy and screened for the most common mutations [84, 86 and 255] in the SRY region before analyzing. GSK3-beta gene expression was assessed quantitatively by real time-PCR. The WNT3[a] protein concentration had no significant difference between the two test groups and controls. Expression of GSK3-beta was down-regulated in non-obstructive azoospermia [3.10 +/- 0.19] compared with normal [7.12 +/- 0.39] and obstructive azoospermia [6.32 +/- 0.42] groups [p=0.001]. Down-regulation of GSK-3beta may cause to non-obstructive azoospermia. Regulation and modification of GSK-3beta gene expression by drugs could be used as a therapeutic solution

Association between increased expression of endothelial isoform of nitric oxide synthase in the human fallopian tube and tubal ectopic pregnancy

Tubal ectopic pregnancy [tEP] is the most common type of extra-uterine pregnancy and the most common cause of maternal mortality. Nitric oxide [NO] is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies have demonstrated the possible role of endothelial isoform of nitric oxide synthase [eNOS] enzyme in the regulation of many reproductive events that occur in the fallopian tube [FT]. The aim of this study was to evaluate the expression of eNOS in the FTs of women with tEP. In this case-control study, a total number of 30FTs samples were obtained from three groups including: 10 FTs of women that bearing an EP, 10 FTs from the non-pregnant women at luteal phase of the menstrual cycle, and 10 FTs of healthy pregnant women [n=10]. Samples were fixed in 10% buffered formalin and then were evaluated by immunohistochemistry. Localization of eNOS was seen in secretory and ciliated luminal epithelium and vascular endothelium of all groups. However, we did not observed the expression of eNOS in smooth muscle cells of all groups. Expression of eNOS in luminal epithelium of women with EP compared to non-pregnant women at luteal phase of menstrual cycle and healthy pregnant group showed statistically significant increase [p=0.00]. Significant difference in expression of eNOS was not observed in luminal epithelium of FTs of women at luteal phase compared to healthy pregnant groups [p=0.78]. This study indicates that changes in expression of eNOS in luminal epithelium of FT may lead to development of EP.

Evaluation of an aqueous-ethanolic extract from Rosmarinusa officinalis [Rosemary] for its activity on the hormonal and cellular function of testes in adult male rat

Rosmarinus officinalis has been used in traditional medicine extensively. This study evaluated the hormonal and cellular effects of Rosmarinus officinalis extract on testes of adult rats. Thirty male Wistar rats [in three groups] received 50 or 100 mg/Kg b.w of Rosmarinus officinalis extract [made from the plant's leaves, flower and stem] [treatment groups] and 10 mL/Kg b.w normal saline [control group] respectively, on a daily bases by gavage route for 60 days. Then, spermatological properties, histometric parameters and sperm dynamics, testis and body weight, testicular cell population and serum testosterone level were analyzed by an acceptable method. Results showed that the mean serum testosterone level was decreased significantly in both treatment groups [50 and 100 mg/Kg b.w] during the experiment time, compared with control group [p < 0.05]. However, Rosmarinus officinalis did not change the total count, motility and viability of sperm. In addition, Rosmarinus officinalis at both doses did not change body and testes weight and their ratio. Furthermore, Rosmarinus officinalis increased the number of Spermatogonia at both doses, Spermatocyte at doses of 50 mg/Kg b.w, Leydig cell and Spermatid at dose of 100 mg/Kg b.w significantly [p < 0.05]. Rosmarinus officinalis did not significantly affect the number of Spermatozoid and Sertoli cells. In conclusion, it seems that Rosmarinus officinalis may have some hormonal and cellular effects on the testes which can contribute the spermatogenesis process in rat. Rosmarinus officinalis may have antiandrogenic effect potentially indicating the possibility of developing herbal male contraceptive

The expression of IL-6Ralpha and Gp130 in fallopian tubes bearing an ectopic pregnancy / 대한해부학회지

Women with tubal ectopic pregnancies have high levels of circulating interleukin 6 (IL-6). IL-6 treatment in vitro significantly reduces the ciliary activity of tubal epithelium. The effects of IL-6 on target cells occur via the formation of a high-affinity complex with its receptors IL-6Ralpha and glycoprotein 130 (Gp130). IL-6Ralpha is specifically expressed in the cilia of the epithelial cells. In this study, we performed a quantitative reverse transcriptase polymerase chain reaction to determine the mRNA expression of IL-6Ralpha and Gp130 in the fallopian tubes obtained from 12 women with ectopic pregnancies, 12 women with normal pregnancies, and 12 healthy nonpregnant women in the luteal phase of their menstrual cycle. Fallopian tubes were evaluated from specimens taken during tubal ligation in normal pregnancies and nonpregnant fertile women or during tubal surgery in ectopic pregnancies. We observed that IL-6Ralpha mRNA expression in fallopian tubes was increased in ectopic pregnancy compared with that in the midluteal phase. We also found that the Gp130 mRNA expression was significantly lower in fallopian tubes from ectopic pregnancies than in those from nonpregnant women during the midluteal phase of their menstrual cycle, although its expression was noticeably high in fallopian tubes in the midluteal phase, which suggests that high Gp130 levels may possibly contribute to embryo transport into the uterus.

Immunohistochemical localization of endothelial nitric oxide synthase in endometrial tissue of women with unexplained infertility

Nitric oxide [NO] is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies suggested the possible role of endothelial isoform of nitric oxide synthase [eNOS] enzyme in female infertility. The aim of this study is to evaluate the expression of endothelial nitric oxide synthase in endometrial tissue of women with unexplained infertility. In this case-control study a total of 18 endometrial tissues obtained from 10 women with unexplained infertility and 8 normal and fertile women by endometrial biopsy, 6 to 10 days after LH surge. Specimens were fixed in 4% paraformaldhyde fixative and frozen sectioned for semi-quantitative immunohistochemical evaluation using monoclonal anti-human eNOS antibody. Hematoxilin and Eosin was used for Histological dating. Localization of endothelial nitric oxide synthase was seen in glandular and luminal epithelium, vascular endothelium and stroma in both fertile women and women with unexplained infertility. Although there were differences in immunoreactivity of glandular epithelium [p=0.44], vascular endothelium [p=0.60] and stroma [p=0.63] but only over-expression of eNOS in luminal epithelium [p=0.045] of women with unexplained infertility compared to fertile women was statistically significant [p<0.05]. This study suggests that changes in luminal expression of eNOS may influence receptivity of endometrium

Immuno-histochemical localization of endothelial nitric oxide synthase in testicular cells of men with non-obstructive azoospermia

Non obstructive azoospermia [NOA] is one of the causes of male infertility in which spermatogenesis process is disturbed. Recent studies suggested the possible role of endothelial nitric oxide synthase [eNOS] in spermatogenesis process. The aim of the present study is to evaluate the expression of eNOS in human testicular tissue in men with NOA and men with normal spermatogenesis by using immunocytochemistry. In this case-control study, testicular biopsies were obtained from 10 men with NOA and 7 men with normospermia who were attended to infertility center for diagnosis or infertility treatment. Immunohistochemistry was used to localize the isoform of eNOS in these tissues and the intensity of staining was semi quantitively assessed. In addition, the histopathological evaluation was examined in both groups. The isoform of eNOS enzyme activity was detected in the cytoplasm of sertoli and leydig cells in both groups. There was, however, a considerable variability in the intensity of staining between two groups. The expression of eNOS in Leydig cells in control group was significantly [p<0.05] higher than those in the NOA group. In contrast, expression of eNOS in Sertoli cells in NOA was more than those in the control group. eNO Simmune staining was absent in the normal germ cells but was intense in the abnormal germ cells with piknotic neucleous. The most histopathological finding were hypospermatogenesis [27.2%], Sertoli cell only syndrome [18.1%] and tubular fibrotic [13.6%]. These results suggested that increase level of eNOS may play an important role in the apoptosis process in the abnormal germ cells and disturbance of spermatogenesis process

Increase in concentration of soluble HLA-G in high-quality embryos after intracytoplasmic sperm injection / 대한해부학회지

Non-invasive methods are normally preferred to conventional invasive methods when selecting suitable embryos to improve pregnancy rates after assisted reproduction techniques. One of the most recognized non-invasive methods is to examine the supernatants of embryo culture media. Soluble human leukocyte antigen, class I, G (sHLA-G) antigen is a non-classical class I molecule that has been widely considered as a marker of pregnancy failure or implantation success. In the current study of some Iranian patients, we examined the concentration of sHLA-G at different time points after intracytoplasmic sperm injection and compared the rates to the morphology and quality of the selected embryos. We showed that the concentration of sHLA-G increases over time in high-quality embryos. We conclude that there is a positive relationship between morphology, quality, and sHLA-G concentration. We suggest that this relationship can be used to increase the chance of a successful pregnancy.

Effect of matrigel on function and morphology of human endometrial epithelial cell in vitro

The importance of extra cellular matrix [ECM] in development and function of different cells has been reported but little is known about its role in human endometrial epithelial cells. The aim of the present study was to examine effects of artificial ECM [Matrigel] and progesterone on the function and morphology of human endometrial epithelial cells in vitro. Endometrial samples were removed, with informed patients consent and Ethics Committee approval, from 17 previously fertile women undergoing total abdominal hysterectomy. The tissue was dissociated and centrifuged to provide an epithelial rich suspension which was cultured either on plastic or seeded into Matrigel to produce polarized cells and then supplemented with or without progesterone [10-6 M]. The amount of nucleic acid content of the cells in both in vitro model systems was examined by DNA, RNA extraction methods. The DNA and RNA content were later measured by spectrophotometry. The amount of total RNA in cells grown on Matrigel [23 +/- 1.5 pg/cell] was more than double that in cells grown on pl1astic [9.1 +/- 1.4 pg/cell]. Cells cultured on both in vitro model systems had RNA induced by steroid hormones, but the extent of induction was greater in cells grown on Matrigel [30 +/- 2 pg/cell] than those on plastic [12 +/- 1.9 pg/cell]. Cells cultured on Matrigel were differentiated and became polarized but cells grown on plastic proliferated to full confluency. Cells grown on Matrigel with progesterone supplementation were highly polarized, euchromatic and had greater mitochondria and accumulation of glycogen, when compared to unsupplemented cultures. These results suggest that ECM plays an important role in gene expression, polarization and differentiation of human endometrial epithelial cells in vitro. Endometrial cells grown on ECM responded to steroid hormone in a manner to that reported in endometrial cells in vivo

Induction of endometriosis by implantation of endometrial fragments in female rats

Endometriosis is defined as the growth of endometrial tissues in ectopic places outside the uterus. This disease has an important effect on the health and fertility of affected women. It's etiology is not clearly known. For better understanding the pathophysiology of this disease, many researchers study on several aspects of the disease on animals. In this experimental study endometriosis was induced in female rats surgically and then its side effects were investigated with special focus on adhesion formation that is a major problem in women with this disease. In Protestrous phase, female rats were randomly divided into two groups. In both groups, under intra peritoneal anesthesia, laparotomy was done and left horn and associated fat were removed. In experimented group [A], the removed endometrium was cut to six square pieces [2mm each] and they were sutured to the peritoneum, near ovaries and subcutaneous. In sham group [B], the same procedure was done for the fat tissues around the removed horn and the pieces were sutured to the same places. After 8 weeks, in Protestrous phase, clinical adhesion and size of implants were evaluated. The total mean size of implants was calculated in each group, and this was significantly larger in experimented group [25.4 mm versus 2 mm p=0.000]. The mean diameter of implants that calculated for each site of implantation in experimented group were significantly larger in left peritoneum [p=0.002], followed by right [p=0.000] and left [p=0.000] ovaries. The endometrial tissues grew in 100% of implants in subcutaneous area. Clinical adhesions [Score. 2] were detected in 7 out of 10 in experimented group and in 2 out 10 in control group. The number of Esterous cycle were similar in both groups. Our study showed that after inducing endometriosis by surgical approach, only endometrial implants grew as a cystic structures and this is a unique aspect of endometrial cells. Our results showed that endometriosis had a direct effect on adhesion formation, not surgery alone and induction of this disease didn't have any adverse effect on ovarian function in female rats