In vitro effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the expression of genes related to sperm motility and energy metabolism and intracytoplasmic sperm injection outcomes in obstructive azoospermic patients.

BACKGROUND: The presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor in various testicular cells and spermatozoa suggests a potential role in enhancing spermatogonial and postmeiotic cell development. Moreover, GM-CSF activates the pivotal pathways implicated in sperm motility regulation and glucose metabolism. However, the impact of GM-CSF on testicular biopsies from patients with obstructive azoospermia (OA) remains unexplored. Therefore, this study aimed to investigate the in vitro effects of GM-CSF on the expression of genes related to glucose transporters and signaling pathways, sperm motility, and viability in testicular biopsies. METHODS AND RESULTS: Following testicular sperm extraction from 20 patients diagnosed with OA, each sample was divided into two parts: the experimental samples were incubated with medium containing 2 ng/ml GM-CSF at 37 °C for 60 min, and the control samples were incubated with medium without GM-CSF. Subsequently, the oocytes retrieved from the partner were injected with sperm from the treatment and control groups. The sperm parameters (motility and viability), the expression levels of sperm motility-related genes (PIK3R1, PIK3CA, and AKT1), and the expression levels of sperm energy metabolism-related genes (GLUT1, GLUT3, and GLUT14) were assessed. Furthermore, the fertilization and day 3 embryo development rate and embryo quality were evaluated. Compared with those in the nontreated group, the motility parameters and the mRNA expression levels of PIK3R1, AKT1, and GLUT3 in testicular sperm supplemented with GM-CSF were significantly greater (p < 0.05). However, no significant differences in the mRNA expression of PIK3CA, GLUT1, or GLUT14 were detected. According to the ICSI results, compared with the control group, the GM-CSF treatment group exhibited significantly greater fertilization rates (p = 0.027), Day 3 embryo development rate (p = 0.001), and proportions of good-quality embryos (p = 0.002). CONCLUSIONS: GM-CSF increased the expression of genes related to motility and the energy metabolism pathway and effectively promoted the motility of testis-extracted spermatozoa, consequently yielding positive clinical outcomes.

GM-CSF (granulocyte-macrophage colony-stimulating factor) treatment improves sperm parameters in men with oligoasthenoteratospermia via PI3K/AKT pathway.

Poor sperm quality in oligoasthenoteratospermia patients negatively affects assisted reproductive technology outcomes. Therefore, the development of sperm media is necessary to improve sperm parameters. This study investigated the effect of GM-CSF via PI3K/AKT pathway on sperm quality in OAT patients. Semen samples were collected from 20 OAT patients, and each sample was divided into two groups: Experiment and Control. In the experimental group, the samples were incubated with medium containing GM-CSF, and control samples were incubated without GM-CSF. Sperm parameters, mitochondrial membrane potential, acrosome reaction and DFI were studied; in addition, gene expression of PI3KR1, PI3KCA, GLUT1, GLUT3 and AKT1 was analysed, evaluation of PAKT/TAKT, and expression of GLUT 1, 3 was examined; subsequent fertilization rate and embryo quality were assessed. Our data showed that GM-CSF supplementation could significantly increase motility, mitochondrial activity, gene expression of PI3KCA, AKT1, the protein level of PAKT/TAKT and expression of GLUT 1, 3 while it decreases DNA fragmentation. The fertilization rate and embryo quality significantly improved in the treatment group. LY294002 had adverse effects on sperm motility and the PAKT/TAKT ratio. GM-CSF can improve in vitro sperm quality and could be a suitable supplement to sperm media for OAT patients.

Does prevalence of sexual dysfunction differ among the most common causes of infertility? A cross-sectional study.

BACKGROUND: Sexuality as a fundamental component of women's health, can be affected by infertility. The current study aimed at comparing the prevalence of sexual dysfunction among women with the most common causes of infertility. METHODS: The current cross-sectional study was conducted on 240 infertile females with infertility due to polycystic ovary syndrome (PCOS, n = 80), endometriosis (n = 80) and male factor (n = 80) at Royan Institute for Reproductive Biomedicine (Tehran, Iran) and 160 fertile women at health care centers, between May 2016 and June 2017. Sexual function was assessed by Female Sexual Function Index (FSFI). Data were analyzed using SPSS (version 25.00) and differences were regarded statistically significant at p < 0. 05. RESULTS: The prevalence of female sexual dysfunction was 98.8% in women with PCOS, 100.0% in those with endometriosis, and 80.0% in those with male factor infertility. Overall, 36.2% of the enrolled fertile women were suffering from sexual dysfunction. CONCLUSIONS: There was an association between the prevalence of female sexual dysfunction or individual domain scores of the FSFI, and infertility etiologies. Therefore, infertility care providers are required to take this into consideration and develop preventive strategies in this regard. Infertility as a major health care problem affects an estimated 8-12% of couples of reproductive age globally and sexuality as an important part of women's health, can be affected by infertility. In this study, the prevalence of sexual dysfunction among women with the most common causes of infertility has been evaluated. The present study was conducted on 240 infertile females with infertility due to polycystic ovary syndrome (PCOS, n = 80), endometriosis (n = 80) and male factor (n = 80) at Royan Institute (Tehran, Iran) and 160 fertile women at health care centers, between May 2016 and June 2017. Sexual function was assessed by Female Sexual Function Index (FSFI); a brief self-report measure of sexual functioning. Results highlight that the prevalence of sexual dysfunction in women with endometriosis and PCOS was higher than in other groups. As, the prevalence of female sexual dysfunction was 98.8% in women with PCOS, 100.0% in those with endometriosis, and 80.0% in those with male factor infertility. In total, 36.2% of the enrolled fertile women were suffering from sexual dysfunction. The results point to an association between the prevalence of female sexual dysfunction and causes of infertility. Therefore, infertility care providers are required to take this into consideration and develop preventive strategies in this regard.

The boosting effects of melatonin on the expression of related genes to oocyte maturation and antioxidant pathways: a polycystic ovary syndrome- mouse model.

BACKGROUND: Melatonin, as a free radical scavenger exhibiting genomic actions, regulates the antioxidant genes expression and apoptosis mechanisms. In polycystic ovary syndrome (PCOS) patients, an imbalance between free radicals and antioxidants in follicular fluid leads to oxidative stress, aberrant folliculogenesis, and intrinsic defects in PCOS oocytes. In this experimental mouse model study, oocytes of PCOS and the control groups were cultured in different melatonin concentrations (10- 5, 10- 6, and 10- 7 M) to investigate the expression of oocyte maturation-related genes (Gdf9/Bmp15), antioxidant-related genes (Gpx1/Sod1), apoptotic biomarkers (Bcl2/Bax) and total intracellular ROS levels. RESULTS: Gdf9 and Bmp15, Gpx1 and Sod1 were up-regulated in PCOS and control oocytes cultured in all melatonin concentrations compared to those cultured in IVM basal medium (P < 0.05). A significant decrease in the total ROS level was observed in all groups cultured in the supplemented cultures. Melatonin increased Bcl2 and decreased Bax gene expression in PCOS and control oocytes compared to non-treated oocytes. CONCLUSIONS: Melatonin increased antioxidant gene expression and regulated the apoptosis pathway, effectively reducing the adverse effects of culture conditions on PCOS oocytes. Furthermore, it influenced the expression of oocyte maturation-related genes in PCOS, providing valuable support during the IVM process.

Differential expression of innate/adaptive immunity genes induced by endometrial scratching as a hopeful approach for implantation boosting in unexplained, repeated implantation failure: An RCT.

BACKGROUND: Endometrial scratching (ES) has been proposed as a potential treatment for implantation improvement in unexplained repeated implantation failure (uRIF) patients, however, little is known about its exact molecular mechanisms. OBJECTIVE: This randomized controlled trial (RCT) was conducted on twenty uRIF patients to investigate the expression of innate and adaptive immune signaling genes after ES. METHODS: Ten uRIF patients in the intervention (twice endometrial sampling in follicular and luteal phases) and 10 uRIF patients in the control group (only luteal phase sampling) were randomly enrolled. Gene expression analysis with innate and adaptive immune response PCR-array kit between intervention and control groups were performed. RESULTS: Among innate immune-associated genes, a significant decrease was observed in the expression of APCS, CPR, CCL2, NLRP3, HLA-A, TLR3 and TLR4 in the intervention group. In adaptive immune-related genes, the expression level of CD80, CD86, CXCR3, IFNγ, IFNα1, IFNß, MBL2, CCR6, CCR8 and IL17A were decreased and CSF2, GATA3, and IL4 increased significantly in the intervention group (P < 0.05). Of 14 uRIF patients, five live birth (35.71 %) was achieved. CONCLUSION: ES in uRIF patients may exert positive effects on the endometrial preparation which increases its receptivity for embryo implantation by modulating the expression of an array of immune signaling pathway genes.

The role of sperm in inducing genomic changes in the implantation: An experimental study.

Endometrial receptivity and implantation are important topics in reproductive sciences. No evidence was found to support sperm involvement in endometrial receptivity and its associated factors. This study aimed to explore the effect of the normal human spermatozoa-endometrium cell interaction in regulating genes in the endometrial receptivity pathway. Semen samples were collected from a healthy and fertile man; then, they were incubated with endometrial cells for 24 hr and considered as the sperm group. A group was cultured without spermatozoa and considered as a control group. About 24 hr later, cells were collected from the bottom of the culture dish. The expressions of the VEGF, FGF2, HBEGF, LIFR, EGF, LIF, MUC1, HOXA10, CSF and PGR genes were evaluated in the two groups. Statistical analysis was performed using an independent sample test. Compared with the control group, in the sperm group, the mRNA levels of PGR (p = .0451), VEGF (p = .0101), HBEGF (p = .0163), EFG (p = .0339), FGF2 (p = .012), LIF (p = .0324), LIFR (p = .0321) and HOXA10 (p = .0098) were significantly upregulated. The results showed that there is a need for the interaction between spermatozoa and endometrium for implantation and can be used for preparing uterine in in vitro fertilisation cycles.

Proteome analysis of endometrial tissue from patients with PCOS reveals proteins predicted to impact the disease.

Polycystic ovary syndrome (PCOS) is a complex disease that causes an ovulatory infertility in approximately 10% of reproductive-age women. We searched for candidate proteins that might contribute to endometrial receptivity defects in PCOS patients, and result in adverse reproductive outcomes. Shotgun proteomics approach was used to investigate the proteome profile of the endometrium at the luteal phase in PCOS patients compared to healthy fertile individuals. Biological process and pathway analyses were conducted to categorize the proteins with differential expressions. Confirmation was performed for a number of proteins via immunoblotting in new samples. 150 proteins with higher abundance, and 46 proteins with lower abundance were identified in the endometrial tissue from PCOS patients compared to healthy fertile individuals. The proteins with higher abundance were enriched in protein degradation, cell cycle, and signaling cascades. Proteins with lower abundance in PCOS patients were enriched in extracellular matrix (ECM) composition and function, as well as the salvage pathway of purine biosynthesis. Metabolism was the most affected biological process with over 100 up-regulated, and approximately 30 down-regulated proteins. Our results indicate significant imbalances in metabolism, proteasome, cell cycle, ECM related proteins, and signaling cascades in endometrial tissue of PCOS, which may contribute to poor reproductive outcomes in these patients. We postulate that the endometria in PCOS patients may not be well-differentiated and synchronized for implantation. Possible roles of the above-mentioned pathways that underlie implantation failure in PCOS will be discussed. Our findings need to be confirmed in larger populations.

Restoration of estrous cycles by co-transplantation of mouse ovarian tissue with MSCs.

This study investigates the effect of bone marrow (BM-MSCs) and visceral peritoneum (VP-MSCs)-derived mesenchymal stem cells on the transplanted ovary. VP-MSCs and BM-MSCs were obtained from green fluorescent protein-expressing mice (GFP+). Six- to eight-week-old female NMRI mice were divided into four experimental groups, autograft ovarian tissue fragments (AO), autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel (AO-H), autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel containing BM-MSCs (AO-HB) and autograft ovarian tissue fragments encapsulated in fibrin-collagen hydrogel containing VP-MSCs (AO-HP). Intact ovary (IO) was the control group. The estrous cycles resumption time was monitored and at the third estrous cycle, the blood samples and grafted ovaries were evaluated using hormonal, histological and gene expression analysis. Onset of estrous cycles, especially at the second cycle, was earlier in AO-HB and AO-HP groups than in the AO-H group (P < 0.05). Moreover, E2 and FSH levels in AO-HB and AO-HP groups were returned to those of the intact group. However, folliculogenesis was still retarded as compared with the IO group. The gene expression of theca (Lhcgr, Cyp17a1, Gli2, Gli3 and Ptch1), granulosa (Amh and Fshr), oocyte (Zp3 and Gdf9), germ cells (Stella and Prdm1), angiogenesis (VEGF and bFGF) and apoptosis (Bax/Bcl2 and Caspase3) markers was similar in both AO-HB and AO-HP groups. Expression of Amh, Fshr, Gdf9 and VEGF increased only in the AO-HP group whereas expression of Ptch1 increased only in the AO-HB group, as compared with the AO group (P < 0.05). In conclusion, BM-MSCs or VP-MSCs can improve ovarian autotransplantation in mice with no superiority over each other.

Role of epigenetic modifications in the aberrant CYP19A1 gene expression in polycystic ovary syndrome.

INTRODUCTION: In this study, the global DNA methylation, histone acetylation and methylation levels of cumulus cells (CCs) in infertile polycystic ovary syndrome (PCOS) patients and the correlation of these epigenetic modifications with the expression of the ovarian aromatase gene (as an important marker in the etiology of PCOS) were investigated. MATERIAL AND METHODS: A cross-sectional study was conducted on 24 patients (12 PCOS patients and 12 healthy women), who underwent ovarian stimulation. Nucleosome ELISA was performed, in order to identify the global occupancy level of Mecp2 (as a marker of DNA methylation) and H3K9me2/H3K9ac as histone modification markers in chromatin fractions obtained from CCs. The CYP19A1 gene expression was measured by qRT-PCR. The level of DNA incorporation of MeCP2, histone modification markers and binding of estrogen receptor ß (ERß) to CYP19A1 regulatory sequences were examined by ChIP-QPCR assay. RESULTS: The data demonstrate a significant increase in global occupancy levels of MeCP2 and H3K9ac markers and a decrease of H3K9me2 to chromatin in CCs of PCOS patients vs. control group. Furthermore, CYP19A1 gene expression, and the incorporation of H3K9ac in PII, PI.3, and PI.4 promoters of CYP19A1 in PCOS, were higher than those of controls. Also, significant hypomethylation of H3K9 at PII and DNA hypomethylated at PII and PI.3 promoters and differential binding of ERß to three promoters were observed in PCOS patients (p < 0.05). CONCLUSIONS: Aromatase expression can be affected by epigenetic modifications and differential ERß binding to the proximal CYP19A1 promoters. These mechanisms may be involved in the enhanced aromatase transcription during ovarian stimulation in PCOS patients.

The myo-inositol effect on the oocyte quality and fertilization rate among women with polycystic ovary syndrome undergoing assisted reproductive technology cycles: a randomized clinical trial.

PURPOSE: The aim of the present study was to evaluate the effect of myo-Inositol administration on oocyte quality, fertilization rate and embryo quality in patients with PCOS during assisted reproductive technology (ART) cycles. METHODS: Fifty infertile PCOS patients were randomly designated in two groups. In the study group, patients received daily doses of 4 g myo-Inositol combined with 400 mg folic acid and in the control group patients received only 400 mg folic acid from 1 month before starting the antagonist cycle until the day of ovum pick up. Oocyte and embryo qualities were assessed according to European Society of Human Reproduction and Embryology (ESHRE) guidelines. The gene expression of PGK1, RGS2 and CDC42 as a factor of oocyte quality in granulosa cells was analyzed using real-time RT-PCR. Levels of total antioxidant capacity (TAC) and reactive oxygen species (ROS) were evaluated by chemiluminescence assay in follicular fluid. RESULTS: The percentage of metaphase II oocyte, fertilization rate and embryo quality significantly improved in the study group (p < 0.05), but the number of retrieved oocytes and follicle count were not statistically different between groups. Furthermore, the gene expression of PGK1, RGS2 and CDC42 was significantly higher in the study group (p < 0.05) but no differences were found between two groups in terms of TAC and ROS levels. CONCLUSIONS: The present study findings suggest that myo-Inositol alters the gene expression in granulosa cells and improves oocyte and embryo quality among PCOS patients undergoing ART.

The impact of the localisation of endometriosis lesions on ovarian reserve and assisted reproduction techniques outcomes.

This case-control study was designed to evaluate the impact of endometriosis and the presence of endometrioma (OMA) per se on the serum anti-Müllerian hormone (AMH) level and also to compare the in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) outcomes after therapeutic surgery in endometriosis patients, according to the localisation of endometriosis lesions. One hundred and fifty two infertile women ≤40 years with suspicious symptoms were surgically evaluated to detect the aetiology of infertility at the Royan Institute during this study and, in parallel, 131 patients with a male factor infertility diagnosis were considered as the control group. The serum AMH level and IVF/ICSI outcomes were compared according to the nature and extension degree of endometriosis lesions. The results demonstrated that the existence of a deep infiltrating endometriosis (DIE) with and without OMA was associated with a significant decrease in AMH level, antral follicle count and ovarian sensitivity index (OSI) (p < .001 and p = .007, respectively). The multivariable logistic regression analysis adjusted for confounding factors indicated that the OSI and the existence of DIE with and without OMA were a significant predictive variable for clinical pregnancy and for live birth. On the basis of our results, the severity of endometriosis and the location of its lesions could affect an ovarian reserve and the ovarian stimulation outcomes. Impact Statement What is already known on this subject? Previous studies have evaluated the impact of endometrioma (OMA) on ovarian reserve and the assisted reproduction technology (ART) outcomes and controversial results have been reported; therefore, it seems that this topic still needs further research. What the results of this study add? In the present study, the effect of endometriosis lesions' localisation on ovarian reserve and the success rate of the in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) cycle after therapeutic surgery were compared with that of the control group. It was found that the existence of a deep infiltrating endometriosis (DIE) with and without OMA was associated with a significant decrease in the anti-Müllerian hormone (AMH) level, antral follicle count, ovarian sensitivity index (OSI), clinical pregnancy and live birth rates. What the implications are of these findings for clinical practice and/or further research? The results of this study has a practical value in the decision making process for the ovarian stimulation protocol in patients with the different severity of endometriosis and the counselling regarding the success rate of IVF or ICSI/embryo transfer cycles.

Does the "delayed start" protocol with gonadotropin-releasing hormone antagonist improve the pregnancy outcome in Bologna poor responders? a randomized clinical trial.

BACKGROUND: Recently, a novel approach with delaying the start of controlled ovarian stimulation along with gonadotropin-releasing hormone (GnRH) antagonist pretreatment for 7 days after estrogen priming for further suppression of endogenous follicle stimulating hormone (FSH) during the early follicular phase, resulting in more FSH-responsive follicles and thus improving synchronous follicular development was introduced. Two clinical trials have examined this strategy and reported controversial results. This study aimed to compare the effect of delayed-start GnRH antagonist protocol and standard GnRH antagonist in patients with poor ovarian response (POR) undergoing in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI). METHODS: This randomized clinical trial was conducted at infertility department of Royan Institute from January 2017 to June 2018. Poor ovarian response was defined according to the Bologna criteria. The eligible women were randomly allocated into an experimental and control groups. In experimental group, patients received delayed-start GnRH antagonist protocol with estrogen priming followed by early follicular-phase GnRH antagonist treatment for 7 days before ovarian stimulation with gonadotropin and in control group, patients treated with estrogen priming antagonist protocol. IVF/ICSI outcomes were compared between groups. RESULTS: Among all the 250 patients examined 156 women were eligible for study and finally 120 patients were allocated to intervention (n = 60) and control (n = 60) groups. Demographic characteristics and hormonal profiles of the patients did not differ between groups. The statistical analysis showed that there were significant differences between groups regarding the total dose of used gonadotropins (P < 0.001), stimulation duration (P < 0.001), number of retrieved oocytes (P = 0.01) and top quality embryo (P < 0.001) and also cancellation (P = 0.002) and fertilization rates (P = 0.002). CONCLUSION: On the basis of present results the delayed-start protocol in poor responders can improve the fertilization rate and quality of embryos and reduce the cycle cancellation but have no significant effect on clinical pregnancy rate; however, larger randomized clinical trials are required to compare it with other protocols. TRIAL REGISTRATION: NCT, NCT03134690. Registered 1 May 2017 - Retrospectively registered, http://www.clinicaltrial.gov / NCT03134690.

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

RESEARCH QUESTION: What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? DESIGN: In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. RESULTS: Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. CONCLUSIONS: The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women.

Comparison of the symptoms and localisation of endometriosis involvement according to fertility status of endometriosis patients.

This cross-sectional study aimed to assess the prevalence of endometriosis in women who were referred for Diagnostic Laparoscopy Unit due to infertility or pelvic pain between January 2012 and January 2013 and compare the symptoms and laparoscopic signs among the three groups according to the fertility status. Four hundred and thirteen women were evaluated; of these, 383 patients for infertility and 30 patients for pelvic pain and/or cyst. Endometriosis symptoms were compared between fertile and infertile women with primary and secondary infertility. There was no statistically significant difference in the overall prevalence of endometriosis between the three study groups (52.9%, 45% and 40.7%, respectively, in primary, secondary infertile and fertile women). The endometriosis stage was categorised as early- (I and II) or late- (III and IV) stages and the extent of endometriosis was divided into peritoneal, ovarian and ovarian coexisting with peritoneal. There is no relationship between the frequency of dysmenorrhoea or non-cyclic pelvic pain and the disease stage; although these pain symptoms are significantly more prevalent in cases with both ovarian and peritoneal endometriotic implants. Infertility was more prevalent among the patients with peritoneal endometriosis in comparison to the ones with ovarian endometriosis. Further studies with a larger sample size are required to confirm these findings. Impact statement What is already known on this subject? Few studies have been done in this area and only one study compared the localisation of endometriosis lesions between fertile and infertile endometriosis cases; however, more study is needed to confirm their results. What the results of this study add? A possible relationship between localisation of endometriosis involvement and infertility was found in the present study in agreement to result of a previous study performance in this area. Although the present study includes a greater number of cases than that of the previous reported study, further studies with a larger sample size are required for the confirmation or refusal of this finding. What are the implications of these findings for clinical practice and/or further research? The results of this study could have clinical application in the consultation and decision-making in infertile women with an endometriosis diagnosis.

Study of telomerase reverse transcriptase and uterine-ovarian-specific genes expression in the endometrial tissue of ovariectomized female Sprague-Dawley rats.

BACKGROUND: An in vivo study was carried out to study of telomerase reverse transcriptase and Uterine-Ovarian-specific genes expression in the endometrial tissue of ovariectomized female Sprague-Dawley rats. MATERIAL AND METHODS: Twenty-four female Sprague-Dawley rats divided into 4 groups of six rats. The first and second groups were ovariectomized and given tamoxifen and tamoxifen-loaded SLN respectively for six days continuously. Group 3 served as the untreated ovariectomized control group and group 4 was made up of untreated normal healthy rats. At the end of the study, the rats were sacrificed and study of the genes expression and serum zinc and copper were carried out. RESULTS: The results showed that the expression of TERT in the group treated with tamoxifen, and tamoxifen-loaded solid lipid nanoparticles, significantly decreased (p<0.001) compared with ovariectomized control group. The results also revealed that the treatment with tamoxifen-loaded solid lipid nanoparticles increased expression of UO-44 gene compared to ovariectomized control group, while there was no difference between tamoxifen treated and control group. CONCLUSIONS: Encapsulation of tamoxifen in solid lipid nanoparticles increased its targeting efficiency and improved the impact of the drug on the serum levels of some trace elements.

Congenital Malformations in Singleton Infants Conceived by Assisted Reproductive Technologies and Singleton Infants by Natural Conception in Tehran, Iran.

BACKGROUND: Multiple pregnancies occur more frequently in assisted reproductive technology (ART) compared to normal conception (NC). It is known that the risk of congenital malformations in a multiple pregnancy are higher than single pregnancy. The aim of this study is to compare congenital malformations in singleton infants conceived by ART to singleton infants conceived naturally. MATERIALS AND METHODS: In this historical cohort study, we performed a historical cohort study of major congenital malformations (MCM) in 820 singleton births from January 2012 to December 2014. The data for this analysis were derived from Tehran's ART linked data file. The risk of congenital malformations was compared in 164 ART infants and 656 NC infants. We performed multiple logistic regression analyses for the independent association of ART on each outcome. RESULTS: We found 40 infants with MCM 29 (4.4%) NC infants and 14 (8.3%) ART infants. In comparison with NC infants, ART infants had a significant 2-fold increased risk of MCM (P=0.046). After adjusting individually for maternal age, infant gender, prior stillbirth, mother's history of spontaneous abortion, and type of delivery, we did not find any difference in risk. In this study the majority (95.1%) of all infants were normal but 4.9% of infants had at least one MCM. We found a difference in risk of MCMs between in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). We excluded the possible role of genotype and other unknown factors in causing more malformations in ART infants. CONCLUSION: This study reported a higher risk of MCMs in ART singleton infants than in NC singleton infants. Congenital heart disease, developmental dysplasia of the hip (DDH), and urogenital malformations were the most reported major malformations in singleton ART infants according to organ and system classification.

The effect of endometrial scratch injury on pregnancy outcome in women with previous intrauterine insemination failure: A randomized clinical trial.

AIM: Endometrial scratch injury (ESI) has been recently proposed to enhance the implantation rate in assisted reproductive technology cycles. The present study was conducted to determine the effect of ESI on pregnancy rate in women with intrauterine insemination (IUI) failure. METHODS: This prospective randomized controlled study was carried out in Imam-Khomeini Hospital and Royan Institute, Tehran, during a 12-month period from January 2013 to January 2014. After assessment, 169 patients who had IUI failure twice or more (no chemical or clinical pregnancy) with normal uterine anatomy and hysterosalpingography, were enrolled. They were randomly assigned into two groups. In the experimental group, all patients underwent ESI at day 8 or 9 of stimulation phase in the present IUI cycle, whereas no intervention was performed on the control group. IUI outcome was then compared between the two groups. RESULTS: A total of 150 patients completed the IUI cycle during the study. The chemical pregnancy rate was 10.7% and 2.7% in the experimental and control groups, respectively, without significant difference (P = 0.09). Also no significant differences were detected in terms of clinical pregnancy and miscarriage rates between the two groups (P > 0.05). CONCLUSIONS: No significant beneficial effect of ESI on fertility outcome in patients with repeated IUI failure was detected when it was carried out on day 8 or 9 of the same IUI stimulation cycle. Also, however, no negative impact secondary to ESI was observed. Therefore, confirmation or refutation of this hypothesis requires further studies with a larger sample size. IRCT201507271141N19.

The Effects of Calcium, Vitamins D and K co-Supplementation on Markers of Insulin Metabolism and Lipid Profiles in Vitamin D-Deficient Women with Polycystic Ovary Syndrome.

Data on the effects of calcium, vitamins D and K co-supplementation on markers of insulin metabolism and lipid profiles among vitamin D-deficient women with polycystic ovary syndrome (PCOS) are scarce. This study was done to determine the effects of calcium, vitamins D and K co-supplementation on markers of insulin metabolism and lipid profiles in vitamin D-deficient women with PCOS. This randomized double-blind, placebo-controlled trial was conducted among 55 vitamin D-deficient women diagnosed with PCOS aged 18-40 years old. Subjects were randomly assigned into 2 groups to intake either 500 mg calcium, 200 IU vitamin D and 90 µg vitamin K supplements (n=28) or placebo (n=27) twice a day for 8 weeks. After the 8-week intervention, compared with the placebo, joint calcium, vitamins D and K supplementation resulted in significant decreases in serum insulin concentrations (-1.9±3.5 vs. +1.8±6.6 µIU/mL, P=0.01), homeostasis model of assessment-estimated insulin resistance (-0.4±0.7 vs. +0.4±1.4, P=0.01), homeostasis model of assessment-estimated b cell function (-7.9±14.7 vs. +7.0±30.3, P=0.02) and a significant increase in quantitative insulin sensitivity check index (+0.01±0.01 vs. -0.008±0.03, P=0.01). In addition, significant decreases in serum triglycerides (-23.4±71.3 vs. +9.9±39.5 mg/dL, P=0.03) and VLDL-cholesterol levels (-4.7±14.3 vs. +2.0±7.9 mg/dL, P=0.03) was observed following supplementation with combined calcium, vitamins D and K compared with the placebo. Overall, calcium, vitamins D and K co-supplementation for 8 weeks among vitamin D-deficient women with PCOS had beneficial effects on markers of insulin metabolism, serum triglycerides and VLDL-cholesterol levels.

Effects of melatonin on oocyte maturation in PCOS mouse model.

The purpose of oocyte in vitro maturation is generation of mature oocytes that could support future development. Efforts have been made to enhance oocyte developmental competence by developing optimal culture conditions. The present study is conducted to determine melatonin effects on quality of polycystic ovarian syndrome (PCOS) oocytes when it has been added during in vitro maturation, and immature oocytes were cultured in defined conditioned medium with and without different melatonin concentrations. Melatonin could significantly improve nuclear maturation of PCOS oocytes (81.1% vs. 56.3%, P < 0.05 were achieved with 10-6 mol/L concentration). Cleavage rate was significantly higher in 10-5 mol/L concentration compared to untreated oocytes in PCOS (54% vs. 35%, respectively) and it was significantly higher with 10-6 mol/L concentration in the control group, 55% versus 38%, compared to untreated oocytes. This study showed that melatonin has the potential to induce oocyte nuclear maturation and guarantee fertilization potential. © 2016 Japanese Society of Animal Science.

Epigenetic alterations of CYP19A1 gene in Cumulus cells and its relevance to infertility in endometriosis.

PURPOSE: The purpose of the present study was to investigate the epigenetic mechanisms responsible for the aberrant aromatase expression (CYP19A1) in Cumulus Cells (CCs) of infertile endometriosis patients. METHOD: Cumulus cells were obtained from 24 infertile patients with and without endometriosis who underwent ovarian stimulation for intracytoplasmic sperm injection. Expression of CYP19A1 gene was quantified using Reverse Transcription Q-PCR. DNA methylation, histone modifications, and binding of Estrogen Receptor, ERß to regulatory DNA sequences of CYP19A1 gene were evaluated by Chromatin ImmunoPrecipitation (ChIP) assay. RESULTS: CYP19A1 gene expression in CCs of endometriosis patients was significantly lower than the control group (P = 0.04). Higher incorporation of MeCP2 (as a marker of DNA methylation) on PII and PI.4 promoters, and hypoacetylation at H3K9 in PII and hypermethylation at H3K9 in PI.4 were observed in CYP19A1 gene in endometriosis patients (P < 0.05). Moreover, a decreased level of ERß binding to PII and an increased level of its binding to PI.3 and PI.4 promoters of CYP19A1 were observed in endometriosis patients when compared to control. CONCLUSION: Significant reduction of CYP19A1 gene expression in CCs of endometriosis patients may be the result of epigenetic alterations in its regulatory regions, either by DNA methylation or histone modifications. These epigenetic changes along with differential binding of ERß (as a transcription factor) in CYP19A1 promoters may impair follicular steroidogenesis, leading to poor Oocyte and embryo condition in endometriosis patients.