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Background and purpose : Ënergy sources play an important role in preimplantation development. Ëffect of glucose, an importamt energy source, has been studied in different stages of embryo development. Ënergy sources play important role in embryos development in preimplantation peroid. Glucose is one of the main sources of energy supply, and it’s effect has been studied in different stages of embryo development. Since fructose and galactose are the important isomers of glucose, thus in addition to the effect of glucose, its isomers were compared on the development of mouse embryos.
Materials and methods : 4-6 weeks old swiss mice were supravulated by HMG and HÇG hormones. The mice were killed 48- 50h after last injection and 2- cell embryos were collected by flashing. The embryos were cultured randomly to hatching stage using T6 medium with glucose (T6+gl), galactose( T6+ gal), fructose( T6+fr) and without hexose(T6-h).
Results : The percentages of embryos reached to blastocyst were 91.25, 67. 09, 92.85 and 70. 92 respectivly. Âlso, 40.82, 25.21, 42.85 and 23.46 percent of embryos were hatched after 72h of culture respectively. The percentages of blastocyst and hatching embryos in T6+gl and T6+fr were significantly greater than those in T6+gal and T6-h (p<0.0001), but there were no obvious differences between embyos cultured in T6+gal and T6–h and those cultured in T6+gl and T6+fr in reaching to blastocyst and hatching stages.
Çonclusion : The results indicate that galactose can not be a proper replacement for glucose in primary embryos medium. But fructose can be an alternate for glucose in the medium, without causing any disorder in embryo development compared to glucose. But despite of this phenomenon, use of fructose as an alternate of glucose needs more studies.

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Background and purpose: The purpose of this study was to evaluate the effect of mouse embryonic fibroblast cells conditioned medium on resumption of meiosis, in vitro maturation of immature mouse oocytes, fertilization and derived embryos development. Material and Methods: Immature oocytes denuded at the germinal vesicle (GV) stage were obtained from female NMRI mice 46-48 hrs after injection of 7/5 IU PMSG. To preparate MEF, the fetuses were collected from the female mice at day 13 and cultured in DMEM. After the preparation of a monolayer, conditioned medium were collected and used for culture. Immature oocytes were randomly cultured in culture medium of MEM-α supplemented with different concentrations (0, 10, 30, 50 and 100%) of mouse embryonic fibroblast conditioned medium. After 14-16 hrs the matured oocytes were fertilized with spermatozoa in T6 medium and their development was assessed until blastocyst stage. Results: There was no significant difference in maturation rate between control and conditioned medium groups. But there were significant differences (P<0.05) in the percentage of fertilization and cleavage in conditioned medium with 30%-50% concentrations as compared to the control group (P<0.05). Conclusion: The conditioned medium of fibroblast cells increase in vitro fertilization (IVF) and embryo development. But there was no effect on the maturation of immature oocytes.

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Background and purpose: Chlorhexidine (CHX) is still considered the gold standard anti-plaque agent. The main disadvantage of chlorhexidine is its taste, and staining. Improvements of these disadvantages are time consume. This study was to determine if polyvinylpyrrolidone (PVP) could be added to chlorhexidine rinses to maintain efficacy and reduce staining. Materials and methods: This study divided to in vivo and in vitro.The in vivo was randomized, double blind study, and 40 patients with moderate to severe inflammation enrolled to study. The patients undergone dental polishing before the study. In case study and control used e Chlorhexidine + polyvinylpyrrolidone Chlorhexidine mouth wash for 2 weeks respectively. Plaque index (PI) and gingival index (GI), bleeding index (BI), and stain index were assessed before and after the intervention. The glass block and spectrophotometry was used to examine the staining intensity of each mouthwash in In vitro study. The data in SPSS and statistical methods were analyzed by Wilcoxon Signed Ranks Test. Results: PI, GI, BI, stain index difference between base line of Chlorhexidine + polyvinyl pyrrolidone and Chlorhexidine mouthwash group was not significant, But the amount of interference between the two groups was significant difference (P=0.006, 0.007, 0.042 respectively). Final severity of body and gingiva between base line of Chlorhexidine + polyvinylpyrrolidone and Chlorhexidine mouthwash group was significant (P<0.05). Final extent of body and gingiva between base line of two groups was significant (P<0.05). Conclusion: 0.2% concentration of Chlorhexidine and 5% PVP concentration in clinical practice to decrease side effect of Chlorhexidine but maintain Chlorhexidine effect

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Background and purpose: Oxidative stress induced by cyclophosphamide (CP), a chemotherapeutic agent, causes vulnerability of sperm and declining the fertility power. Then in this study the effect of crocin (saffron extract) on amelioration of these side effects was investigated. Materials and methods: In this study 24 adult male mice were divided in three groups (n=8). Control group received normal saline (0.2 ml/day, IP), CP group received cyclophosphamide (15 mg/kg/week, IP), and CP+Cr group received crocin (200 mg/kg/day, IP) along with CP for 35 days. Then the animals were euthanized and malondialdehyde (MDA) in testis was assayed. After collection of sperm from caudate of epididymis, the rate of DNA damage was calculated by acridine orange staining. After stimulation of ovulation in 72 female adult mice, oocytes were collected and transferred in HTF medium that contained BSA. Then, in-vitro fertilization (IVF) was done with capacitated sperms. The rate of fertilization and primitive embryonic growth were evaluated for 120 hours. Results: The results showed increase in fertilization, two cell embryos and blastocysts in CP+Cr group compared to those of the CP group (P<0.05). Also, the crocin in CP+Cr group prevented the increase of total number of arrested embryos and percentage of arrested embryos type I, II, and III (P<0.05). Decrease in MDA assay and sperms with damaged DNA were observed more in CP+Cr group (P<0.05). Conclusion: This study showed the efficiency of crocin in amelioration of fertility and growth of primitive embryo in animals that received CP.

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Background and Purpose: In vitro maturation of oocyte is a promising technology in treatment of infertility, however, its clinical application is still limited due to poor success rate. This study was devised to evaluate the effect of mouse embryo tail bud co-culture on maturation of immature mouse oocyte. Materials and Methods: Immature oocytes were recovered from NMRI female mice 48hr after injection of 7.5 IU PMSG (pregnant mare serum gonadotropin). Oocytes were divided into four groups in α-MEM medium containing 10% FBS. The maturation medium for positive control group contained hCG, while for negative control group it was without hCG. In experimental groups 1 and 2 the maturation medium contained no hCG, but 9.5-10.5 and 12-13 days mouse embryo tail bud. The oocytes in each group were cultured in CO2 incubator. The oocytes maturation was recorded under an invert microscope after 24 hr. Data analysis was performed applying ANNOVA in SPSS. Results: In vitro maturation rate in positive control group, negative control group, experimental groups 1 and 2 were 52, 48, 66 and 50%, respectively. These rates indicated a significant increase in matured MII oocytes (in the presence of 9.5-10.5 days mouse embryo tail bud) than those of the negative control group and the second experimental group (P<0.05). Conclusion: Co-culture of oocyte with 9.5-10.5 days mouse embryo tail bud, increased the maturation rate of immature oocyte

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Background and purpose: Stress is one of the decisive factors in infertility, which can interfere with spermatogenesis and reduce spermatozoa. Considering the immobilization stress-induced infertility disorders, this study aimed at examining the protective effect of selenium yeast cell wall (as an antioxidant) on in vitro fertilizing ability following immobilization stress in adult male rats.

Materials and methods: In an experimental study, 32 male adult rats were divided into 4 groups (n= 8 per group) including controls, stress, stress + cell wall of yeast (Saccharomyces cerevisiae) enriched with selenium (SECW) and a SECW group without inducing stress. To induce stress, the rats were immobilized in restraint device for 42 days 2hrs per day. The SECW (5×10⁸ CFU/ml) +268µg/gr SE were orally administrated for 42 days. Afterwards, the sperm samples were collected for in-vitro fertilization (IVF). Moreover, malondialdehyde (MDA) concentration in testicular tissue was assessed in all groups.

Results: Immobilization stress significantly reduced the percentage of zygote, two-cell embryos, blastocyst, hatched embryos and increased MDA levels compared to the control group (P <0.05). In the SECW+stress all parameters of fertilization and embryos development increased significantly (P <0.05) except the percentage of zygote compared to the stress group. Administration of SECW+ stress, significantly decreased the level of MDA tissue compared to the stress group (P <0.05).

Conclusion: Immobilization stress was proved to have harmful effects on fertility, therefore, SECW that has antioxidant compounds could inhibit oxygen free radicals thereby, increasing fertilization and fertility potential.

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Background and purpose: Mitomycin C (MMC) is used as an antiproliferative agent for feeder cells in cell culture. It may induce destructive effects on these cells and eventually on oocytes maturation. This study was conducted to investigate the effect of MMC and co-culturing with fibroblast and cumulus cells on in vitro maturation (IVM) of mouse immature oocytes.

Materials and methods: In this experimental study, germinal vesicle oocytes (GV) obtained from NMRI mature female mice were cultured in αMEM supplemented by FSH, hCG, fetal bovine serum (FBS), penicillin and streptomycin, at 37 °C and 5% CO2 in following groups: a control group and four experimental groups; 1– co-cultured with cumulus cells, 2 – co-cultured with mouse embryonic fibroblast (MEF) cells, 3 - co-cultured with cumulus cells treated with MMC, and 4 - co-cultured with MEF treated with MMC. After 24 hours, number of GV, GVBD, MII and degenerated oocytes were determined using invert microscope. Data were analyzed using ANOVA with Tukey test.

Results: The percentages of GV (24±2.75) and MII (51±2.28) oocytes in control group were significantly higher (8±2.25, 10±2.29, 10±2.28, 11±1.19) and lower (64±2.34, 63±2.62, 62±2.86, 62±2.47) than those in all experimental groups, respectively. Nevertheless, no significant difference was observed between experimental groups.

Conclusion: Co-culturing with fibroblast and cumulus cells promoted in vitro maturation of immature oocytes. There was no significant difference among experimental groups, therefore, removal of monolayer inactivation step by MMC from such protocols can be proposed.

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Background and purpose: Saponaria officinalis is a plant from the caryophyllaceae family and there are some contradictory reports about its antimicrobial effects. Metronidazole is commonly used in treatment of trichomoniasis but it has several side effects. On the other hand, finding an alternative drug from natural sources is very important. This study aimed at evaluating the hydroalcoholic extract of Saponaria officinalis leaf plant on the growth of Trichomonas vaginalis in vitro.

Materials and methods: The plant was approved in herbarium and hydro-alcoholic extracts were prepared. The experiment was done using 24 wells cell culture plate. In each well,  200 microliter of  TYM culture medium and 200 microliter of different concentrations of plant extract was added. Also metronidazole was added to positive control well. Then 100 µl TYM were added to all wells containing 500,000 parasites and the plates were incubated at 37 ° C. The experiment was performed as double blind and triplicate. The mean number of the parasites in different concentrations and times and effect of the extract were recorded.

Results: The number of parasites in 50, 100, 200, 400, 800 and 1600 micrograms per ml of the extract, during 24, 48, and 72 hr increased compared to the negative controls, while no growth of parasite was observed in positive controls (containing Metronidazole).

Conclusion: Saponaria officinalis leaf extract caused growth stimulation and increased the number of Trichomonas vaginalis, therefore, it could be used for mass cultivation of the parasite in vitro. However, further investigations are needed in future.

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Background and purpose: Embryo quality and endometrial receptivity are two parameters that determine the In Vitro Fertilization (IVF) results. This study aimed at investigating the relationship between endometrial and sub endometrial blood flow with prognosis of IVF treatment cycles.

Materials and methods: An analytical historical cohort study was conducted in which women undergoing IVF cycles were recruited. Doppler ultrasound for endometrial and sub endometrial arteries were performed three times in each treatment cycle. Then, the participants were divided into fertile and infertile groups according to treatment results.

Results: Significant relation was found in endometrial and sub endometrial blood flow between the two groups (P=0.012). In initiation of treatment and HCG administration there were significant differences in mean PI values between the fertile group and infertile group (P=0.001 and P=0.006, respectively). On the day of HCG administration, significant differences were observed in mean SD index between the two groups (P=0.032).

Conclusion: In this study absence of endometrial and sub endometrial blood flow was found to be correlated with infertility. Lower PI values in initiation of treatment and time of HCG administration and lower SD values on HCG administration are correlated with successful pregnancy.

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Background and purpose: Diabetes is one of the major factors that affects fertility and ovulation and can disrupt the formation of fetus. Omega-3 polyunsaturated fatty acids might be able to improve the quality of the embryo in maturation of oocytes. The aim of this study was to investigate the effects of omega-3 on ovarian tissue and the development of blastocysts in diabetic rats.

Materials and methods: In this study, 32 adult female rats were divided into 4 groups (n= 8 per group) including controls, diabetic, diabetic+ Omega-3 (300mg/kg /day), and diabetic+ Omega-3 (600mg/kg/day). Diabetes was induced by intraperitoneal injections of Streptozotocin (50 mg/kg). Omega-3 was administered by gavage for 45 days. Then PMSG (IU, IP25) was injected and 54 hours later HCG (IU, IP25) was injected. Afterwards, the ovarian samples were used in vitro fertilization.

Results: Compared with the control group, diabetes significantly reduced the percentage of healthy follicles, percentage of zygote, two cell embryos, blastocysts, and hatching embryos (P<0.05). Data analysis revealed that administration of omega-3 fatty acids in diabetic rats increased the percentage of healthy follicles and parameters associated with fertilization and embryo development in vitro compared with the diabetic group that did not receive omega-3 (P< 0.05).

Conclusion: Due to the harmful effects of diabetes on pregnancy, omega-3 fatty acids has the ability to inhibit the adverse effects of diabetes on the ovaries, thereby increasing the chances of fertility.

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Background and purpose: Trichomonas vaginalis, is one the most common sexually transmitted diseases (STD) in women. Although metronidazole is the drug of choice for trichomoniasis, but due to its side effects attempts have been made to explore an alternative drug particularly with herbal source. Therefore, this study aimed to determine in vitro activity of hydroalcoholic extracts of Chenopodium album (C. album) leaf on the growth of Trichomonas vaginalis.

Materials and methods: The plant (C.album) was approved in herbarium and hydro-alcoholic extracts were prepared. The experiment was done using 24 wells cell culture plate. In each well, 200 µl of modified axenic TYM culture medium and 200 µl of different concentrations (5, 37, 75, 150, 300, 600, 1200 μg/mL) of the plant extract was added. They were incubated for 24 and 48 hours and then the plates were incubated at 37 ° C. The experiment was performed as a double blind design and in triplicate. Then growth inhibition percent (GI%) of the parasites was evaluated in both times and in different concentrations of plant leaf extract.

Results: Compared with metronidazole, the 600 and 1200 μg/mL concentrations of hydro alcoholic leaf extracts of C. album showed 96% and 100% inhibitory effects on the growth of trophozoeites of T. vaginalis in 48 hours, respectively.

Conclusion: C. album as an herbal native plant with ant-trichomonas activity is favorable and could be a candidate for in vivo researches on trichomoniasis in future.

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Background and purpose: There is an increasing rate of drug resistance against trichomoniasis and current drugs used in treatment of this disease have many side effects, therefore, attempts have been made to explore alternative drugs favorably from natural sources. The present study aimed at evaluating the anti-Trichomonas effect of essential oil and that of nano-emulsion of Rosmarinus officinalis on the growth of Trichomonas vaginalis (T. vaginalis) in vitro.
Materials and methods: T. vaginalis reference strains were cultured in modified TYM medium. The components of rosemary essential oil were determined by gas chromatography. In addition, cytotoxicity effects of essential oils and nano-emulsions prepared from this plant were evaluated against macrophage cell line (J774.A.1). Serial concentrations of 10, 25, 50, and 100 (μg/mL) were prepared from rosemary essential oil and nano-emulsion. Then growth inhibition rate of the parasites in different concentrations was done in triplicate within 1, 2, and 3 hours.
Results: Rosemary essential oil and nano-emulsion had an inhibitory effect on the growth of Trichomonas and concentration of 100 μg/mL showed growth inhibition effect of 96.1% and 100% throughout 1,2 and 3 hours, respectively. Additionally, cytotoxicity assay against macrophage revealed no toxicity on the cells at 100 μg/mL.
Conclusion: According to the suitable anti-trichomonas activity of rosemary, this plant could be considered as a good candidate in treatment of Trichomonas infection, after complementary studies on animal model.
 

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Background and purpose: Glycyrrhiza glabra is a perennial plant with some major food and medicinal compounds that has received attention by food and pharmaceutical industries. The aim of this study was to investigate the effect of licorice on in vitro maturation of immature oocytes on NMRI mice.
Materials and methods: In this experimental study, the animals were randomly divided into five groups: a control, a sham and three experimental groups. The mice were injected intraperitoneally with 7.5 IU PMSG to stimulate ovulation. The control group, received only water and food. The sham animals were gavaged with 200 ml of distilled water for 7 days. The three experimental groups were given daily injection of 100, 200, and 500 μL licorice by oral gavage for 7 days. The mice were sacrificed on day 8, the ovaries were then removed and investigated for oocytes reaching metaphase II (MII) within 24 hours. The oocytes were fertilized by the sperm of male mice and investigated after 24 and 48 hours after in vitro fertilization (IVF).
Results: Compared with the control group, consumption of licorice extract in experimental groups (II and III) was found to have a positive effect on the number of follicles, oocytes, and number of 2 and 4 cell embryos.
Conclusion: This study showed the beneficial effects of licorice extract due to its Phyto-estrogenic properties on oocyte maturation and infertility.
 

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Background and purpose: Androgens play a key role in growth of ovarian follicles and female fertility in the process of in vitro fertilization. This study was performed to determine the relationship between androgen levels and success rate in Intracytoplasmic sperm injection (ICSI).
Materials and methods: A nested case-control study was carried out in 60 infertile women attending the Infertility Treatment Center in Sari Imam Khomeini Hospital. They underwent agonist protocol and their levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and dehydro epiandrosterone-sulfate (DHEA-S) were measured on days three of menstrual cycle and ICSI cycle, day six of gonadotropin injection, and the day of human chorionic gonadotropin (HCG) injection. Data were analyzed in SPSS V18 applying independent T-test.
Results: The mean age of participants was 30.1± 6.1 years. Among the subjects, 16 (26.7%) became pregnant and 44 (73.3%) did not become pregnant. On day three of ICSI cycle and before the onset of HCG injection, testosterone level was 0.88 ± 0.68 ng/ml in pregnant women and 0.53 ± 0.25 ng/ml in non-pregnant women (P= 0.012). The levels of DHEA-S were 2.79 ± 1.87 µg/l and 1.62 ± 1.45 µg/l in pregnant and non-pregnant women, respectively (P= 0.02).
Conclusion: Androgen levels can play a significant role in the success of ICSI cycle, so, androgen therapy can be an attractive hypothesis for improving ovarian response and used as a treatment option to increase the success of in vitro fertilization.
 

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Background and purpose: There are few treatment options available for treatment of toxoplasmosis and effective drugs have serious toxic effects. In this study, the in vivo and in vitro anti-toxoplasma activities of Heracleum persicum and Foeniculum vulgare fruits essential oils were investigated.
Materials and methods: In vitro, Vero cells were incubated with different concentrations of essential oils or pyrimethamine (positive control) and the cellular viability was determined. Next, Vero cells were infected with T. gondii (RH strain) and treated with agents. Then, the CC₅₀, IC₅₀, and selectivity index (SI) were calculated. Moreover, in vivo, the effect of oils on survival times of Balb/c mice infected with T. gondii were determined.
Results: In vitro results showed that the oils exhibited less cell toxicity than pyrimethamine.
The selective index was 2.94, 6.96, and 3.06 for Heracleum persicum, Foeniculum vulgare, and pyrimethamine, respectively. Also, the infected mice treated with F. vulgare-pyrimethamine showed a better survival rate than others (P<0.05).
Conclusion: The H. persicum and F. vulgare essential oils showed anti-toxoplasmic activity in vitro and in vivo, but, combination therapy with F. vulgare and pyrimethamine showed a better survival time in mice infected with T. gondii. Therefore, F. vulgare may be a useful candidate in treatment of Toxoplasmosis. However, further studies are needed to investigate the fractions of this plant against T. gondii.

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Background and purpose: One of the problems of in vitro fertilization (IVF) cycles is recurrent failure of implantation. The present study aimed to investigate the effect of intrauterine saline infusion in luteal phase of previous menstrual cycle on occurrence of pregnancy in infertile patients undergoing frozen-thawed embryo transfer.
Materials and methods: This clinical trial was conducted in 70 infertile women attending the infertility center in Sari Imam Khomeini Hospital who were undergoing frozen-thawed embryo transfer in 2020. The participants were randomly divided into an intervention group and a control group (n= 35 per group) using random number table. In intervention group, intrauterine injection of saline with ultrasound guidance was performed on day 21 of previous menstrual cycle, while the control group received no intervention. Pregnancy outcomes were compared between the two groups. Data analysis was performed using Chi-square and t-test.
Results: The intervention group and control group were found to be significantly different in rate of clinical pregnancy (6.5%, 28.1%) (P=0.003) and live birth (6.5%, 28.1%), respectively (P=0.02).
Conclusion: In this study, saline infusion sonohysterography did not have positive effect on clinical pregnancy and live birth, which could be due to small sample size, performing the procedure on day 21 of cycle, or ineffectiveness of this method in causing inflammation following scratches compared to conventional methods. Further molecular and cellular studies are needed to compare the effect of these methods on increasing the level of inflammatory factors.
 
(Clinical Trials Registry Number: IRCT20160815029374N8)

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 Background and purpose: Recurrent implantation failure is one of the problems associated with in vitro fertilization cycles. The aim of this study was to evaluate the results of pregnancy using intralipid infusion in infertile women with history of two implantation failures.
Materials and methods: A clinical trial was conducted in 80 infertile women in the infertility center in Sari Imam Khomeini Hospital, Iran 2020. In all samples, the level of peripheral blood natural killer (NK) cells was measured two weeks before the intervention using flow cytometry. The participants were divided into an intervention group (n=40) and a control group (n=40) by SAS statistical software. Two days before embryo transfer, patients in intervention group underwent intra lipid infusion (2 ml of 20% solution in 250 ml sterile saline) for two hours and the control group received normal saline (250 ml) infusion. Clinical pregnancy (detection of fetal heart rate by ultrasound) rate was compared between the two groups. Data were analyzed using Chi-square, Mann-whitney, and t-test.
Results: Clinical pregnancy rate was higher in intervention group after intralipid infusion (30%) compared with the control group (10%) (P=0.025). There were no significant differences between the two groups in the level of NK cells and peripheral blood lymphocytes (P<0.05).
Conclusion: Intralipid infusion improved clinical pregnancy rate in women with history of recurrent implantation failure. However, further studies are required on the possible mechanism of immunological effects of interlaipid in NK cells.

(Clinical Trials Registry Number: IRCT20140305016858N5)