Induction of sublethal oxidative stress on human sperm before cryopreservation: a time-dependent response in post-thawed sperm parameters

Objective: A recent innovative approach, based on induction of sublethal oxidative stress to enhance sperm cryosurvival, has been applied before sperm cryopreservation. The purpose of this study was to investigate the effects of different induction times of sublethal oxidative stress before cryopreservation on human post-thawed sperm quality

Materials and Methods: In this experimental study, we selected semen samples [n=20] from normozoospermic men according to 2010 World Health Organization [WHO] guidelines. After processing the samples by the density gradient method, we divided each sample into 5 experimental groups: fresh, control freezing, and 3 groups exposed to 0.01M sodium nitroprusside [SNP] [nitric oxide [NO] donor] for 30 [T30], 60 [T60], or 90 minutes [T90] at 37C and 5% CO2 before cryopreservation. Motion characteristics [computer-assisted sperm analyser], viability, apoptosis [annexin V/propidium iodide [PI] assay], DNA fragmentation [sperm chromatin structure assay [SCSA]], and caspase 3 activity [FLICA Caspase Detection Kit] were assessed after thawing. The results were analysed by using one-way ANOVA and Tukey's test. The means were significantly different at P<0.05

Results: Cryopreservation significantly decreased sperm viability and motility parameters, and increased the percentage of apoptosis, caspase 3 activity, and DNA fragmentation [P<0.01] compared to the fresh group. The T60 group had a higher significant percentage of total motility [TM] and progressive motility compared with other cryopreserved groups [P<0.05]. We observed a significantly lower percentage of apoptotic rate and caspase 3 activity in the T60 group compared to the other cryopreserved groups [P<0.05]. DNA integrity was not significantly affected by this time of sublethal stress induction [P>0.05]

Conclusion: Our results have demonstrated that the application of sublethal oxidative stress by using 0.01 MuM NO for 60 minutes before the freezing process can be a beneficial approach to improve post-thawed human sperm quality

Downregulation of extracellular matrix and cell adhesion molecules in cumulus cells of infertile polycystic ovary syndrome women with and without insulin resistance

Objective: The extracellular matrix [ECM] of the cumulus oocyte complex [COC] is composed of several molecules that have different roles during follicle development. This study aims to explore gene expression profiles for ECM and cell adhesion molecules in the cumulus cells of polycystic ovary syndrome [PCOS] patients based on their insulin sensitivity following controlled ovarian stimulation [COS]

Materials and Methods: In this prospective case-control study enrolled 23 women less than 36 years of age who participated in an intracytoplasmic sperm injection [ICSI] program. Patients were subdivided into 3 groups: control [n=8, fertile women with male infertility history], insulin resistant [IR] PCOS [n=7], and insulin sensitive [IS] PCOS [n=8]. We compared 84 ECM component and adhesion molecule gene expressions by quantitative real-time polymerase chain reaction array [qPCR-array] among the groups

Results: We noted that 21 of the 84 studied genes differentially expressed among the groups, from which 18 of these genes downregulated. Overall, comparison of PCOS cases with controls showed downregulation of extracellular matrix protein 1 [ECM1]; catenin [cadherin-associated protein], alpha 1 [CTNNA1]; integrin, alpha 5 [ITGA5]; laminin, alpha 3 [LAMA3]; laminin, beta 1 [LAMB1]; fibronectin 1 [FN1]; and integrin, alpha 7 [ITGA7]. In the IS group, there was upregulation of ADAM metallopeptidase with thrombospondin type 1 motif, 8 [ADAMTS8] and neural cell adhesion molecule 1 [NCAM1] compared with the controls [P<0.05]

Conclusion: Downregulation of ECM and cell adhesion molecules seem to be related to PCOS. Gene expression profile alterations in cumulus cells from both the IS and IR groups of PCOS patients seems to be involved in the composition and regulation of ECM during the ovulation process. This study highlights the association of ECM gene alteration as a viewpoint for additional understanding of the etiology of PCOS

impact of genetic variation and gene expression level of the follicle-stimulating hormone receptor on ovarian reserve

Objective: ovarian reserve is defined as the capacity of the ovary to provide fertile oocytes. Diminished ovarian reserve [DOR] is a disorder in which ovaries are prone to go through early menopause. Where this loss of function occurs before the age of 40, it results in the premature ovarian failure [POF] disease. Throughout folliculogenesis, the follicle-stimulating hormone receptor [FSHR] starts a signaling cascade in the granulosa cells where its inactivation leads to the arrest of follicle maturation and therefore adversely affects ovarian reserve. The aim of this study was to investigate the association of genetic variation [polymorphisms and inactivating mutations] of FSHR with POF and DOR

Materials and Methods: this case-control study comprised 84 POF, 52 DOR and 80 fertile Iranian women. To determine the presence of the 566C>T mutation and the -29G>A polymorphism in FSHR, PCR-RFLP method was used. SSCP-sequencing was used to identify any allelic variants in exon 10. The expression of human FSHR at the transcript level was also compared between DOR and fertile controls by real time-polymerase chain reaction [PCR]

Results: the 566C>T polymorphism was normal in all the cases. All genotypes of -29G>A and 919G>A [exon 10] polymorphisms were observed. Statistically significant differences were seen in the genotypic distribution of both polymorphisms when comparing the control group with the DOR patient group. A decrease was observed in FSHR expression of DOR patients compared with the control group but was not significant

Conclusion: we conclude that the -29G>A and 919G>A polymorphisms in FSHR may be associated with DOR. Although these polymorphisms had significant differences at the genic level, no significant variation was found at the transcript level

coasting valuable in all patients with any cause of infertility?

Objectives: This study aimed to assess the influence of coasting duration on the number and quality of oocytes and fertilization rate in male factor infertile women and those with polycystic ovary syndrome [PCOS]

Methods: In this prospective observational follow-up study, 114 patients undergoing coasting [53 women with male factor infertility and 61 women with PCOS] were evaluated at the Royan Institute Research Center, Iran, between 2010 and 2012

Results: The results were analyzed according to the coasting periods of 1-4 days. In normal females, the number of oocytes retrieved was significantly reduced after the second day [p = 0.004]. In addition, a statistically significant drop was observed in the number of metaphase II oocytes and fertilization rate after the third day [p = 0.006 and p = 0.006, respectively]. No significant differences were observed in the number and quality of oocytes retrieved and fertilization rate with regard to coasting days in PCOS patients

Conclusion: Coasting with duration of more than three days should be performed with caution in normal females who are at risk of developing ovarian hyperstimulation syndrome

Effect of prolonged culture of chromosomally abnormal human embryos on the rate of diploid cells

Background: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies

Materials and Methods: In this cohort study, we used fluorescence in situ hybridization [FISH] to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis [PGD] on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization

Results: Of the 30 analyzed blastocysts, mosaicism was observed in 26[86.6%], while 2[6.7%] were diploid, and 2[6.7%] were triploid. Of those with mosaicism, 23[88.5%] were determined to be diploid-aneuploid and 3[11.5%] were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 [P<0.05]; however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality

Conclusion: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells

Evaluating the effective factors in pregnancy after intrauterine insemination: a retrospective study

Controlled ovarian hyperstimulation [COM] in conjunction with intrau-terine inseminations [IUI] are commonly used to treat infertile couples. In this study we evaluated the relationship between IUI outcome and special causes of infertility. We also aimed to examine parameters that might predict success following IUI. In this cross-sectional study, we included 994 IUI cycles in 803 couples who referred to the infertility Institute. All statistical analyses were performed by using SPSS program, t tests and chi-square. Stepwise multiple linear regression analysis was performed to compare the association between dependent and independent variables. Logistic regression was conducted to build a prediction model of the IUI outcome. Overall pregnancy rate per completed cycle [16.5%] and live birth rate per cycle [14.5%]. The mean age in the pregnant group was significantly lower than that of the non-pregnant group [P=0.01].There was an association between cause of infertility and clinical pregnancies [P=0.001]. Logistic regression identified four significant factors in determining the success of the IUI [menstrual irregularites [OR:2.3, CI: 1.6-3.4, P<0.001], duration of infertility [OR:0.8, CLO.8-0.9, P<0.001], total dose of gonado-tropin [OR:1.02, CL1.003-1.04, P=0.02] and semen volume [ORrl.l, CL1.008-1.2, P=0.03]] which were the most predictive of IUI success. Our study defined prognostic factors for pregnancy in COH+IUI. These variables can be integrated into a mathematical model to predict the chance of pregnancy rate in subsequent COH+IUI cycles

Follicle development of xenotransplanted sheep ovarian tissue into male and female immunodeficient rats

This study aimed to assess follicle survival after xenotransplantation of sheep ovarian tissue into male and female immunodeficient rats. We evaluated the effects of gonadotropin treatment on follicular development in the transplanted tissue. In this experimental study, sheep ovarian cortical strips were transplanted into the neck back muscles of 8 male and 8 female immunodeficient, castrated rats. Fourteen days after surgery, each rat was treated with human menopausal gonadotropin [hMG] for 9 weeks. One day after the last injection, ovarian tissues were removed and fixed for histology assessment. Histology analyses were performed before and after grafting. Estradiol [E[2]] levels were measured before and after gonadectomy, and at the end of the experiment. The control group consisted of 7 male and 7 female non-castrated/non-grafted rats and the sham group comprised 7 male and 7 female castrated/ non-grafted rats for comparison of serum E2 concentrations. The percentage of primordial follicles decreased after transplantation in male [25.97%] and female [24.14%] rats compared to the control group [ovarian tissue non-grafted; 37.51%]. Preantral follicles increased in the male [19.5%] and female [19.49%] transplanted rats compared to the control group [11.4%]. Differences in antral follicles between male [0.06 +/- 0.0%] and female [0.06 +/- 0.0%] rats were not noticeable compared to control [1.25 +/- 0.0%] rats. We observed a significantly higher percent of mean E[2] secretion in grafted males compared to grafted females [P<0.05]. Despite significant differences in E[2] secretion between xenografted male and female rats, we observed no statistical differences in terms of follicular development

overview of the available methods for morphological scoring of pre-implantation embryos in in vitro fertilization

Assessment of embryo quality in order to choose the embryos that most likely result in pregnancy is the critical goal in assisted reproductive technologies [ART]. The current trend in human in vitro fertilization/embryo transfer [IVF/ET] protocols is to decrease the rate of multiple pregnancies after multiple embryo transfer with maintaining the pregnancy rate at admissible levels [according to laboratory standards]. Assessment of morphological feathers as a reliable non-invasive method that provides valuable information in prediction of IVF/intra cytoplasmic sperm injection [ICSI] outcome has been frequently proposed in recent years. This article describes the current status of morphological embryo evaluation at different pre-implantation stages

In vitro maturation, fertilization and embryo culture of oocytes obtained from vitrified auto-transplanted mouse ovary

The purpose of this study was to investigate the in vitro survival and developmental potential of oocytes obtained from vitrified mouse ovaries transplanted to a heterotopic site. In this experimental study, two-week-old mice were unilaterally ovariectomized after anesthesia. The ovaries were vitrified by cryotop. After two weeks, the ovaries were thawed and autotransplanted to the gluteus muscle tissue. Three weeks later the mice were killed, after which we removed and dissected the transplanted and opposite right ovaries. Cumulus oocyte complexes [COCs] and denuded oocytes were evaluated for in vitro maturation [IVM], in vitro fertilization [IVF] and in vitro development [IVD]. The control group consisted of sevenweek- old age-matched mice ovaries. All vitrified-transplanted [Vit-trans] ovaries contained some oocytes that survived. Following IVM, IVF and IVD, there were 41.7% out of 12 cultured zygotes that reached the 8-cell stage. Our experiment supports the progressive role of long-term graft survival after wholeovarian cryopreservation by vitrification and subsequent heterotopic transplantation. It is possible to recover viable follicles and oocytes that have the ability to develop in vitro

effects of ISM1 medium on embryo quality and outcomes of IVF/ICSI cycles

The aim of this study is to investigate the effect of ISM1 culture medium on embryo development, quality and outcomes of in vitro fertilization/intracytoplasmic sperm injection [IVF/ICSI] cycles. This study compares culture medium commonly used in the laboratory setting for oocyte recovery and embryo development with a medium from MediCult. We have assessed the effects of these media on embryo development and newborn characteristics. In this prospective randomized study, fertilized oocytes from patients were randomly assigned to culture in ISM1 [MediCult, cycles: n=293] or routine lab culture medium [G-1[TM]v5; Vitrolife, cycles: n=290] according to the daily media schedule for oocyte retrieval. IVF or ICSI and embryo transfer were performed with either MediCult media or routine lab media. Embryo quality on days 2/3, cleavage, pregnancy and implantation rates, baby take home rate [BTHR], in addition to the weight and length of newborns were compared between groups. There were similar cleavage rates for ISM1 [86%] vs. G-1[TM] v5 [88%]. We observed a significantly higher percentage of excellent embryos in ISM1 [42.7%] compared to G-1[TM] v5 [39%, p<0.05]. Babies born after culture in ISM1 had both higher birth weight [3.03 kg] and length [48.8 cm] compared to G-1[TM] v5 babies that had a birth weight of 2.66 kg and a length of 46.0 cm [p<0.001 for both]. This study suggests that ISM1 is a more effective culture medium in generating higher quality embryos, which may be reflected in the characteristics of babies at birth

Quantitative expression of developmental genes, Pou5f1 [Oct4] and Mest [Peg1], in vitrified mouse embryos

Embryo cryopreservation is the process that water is removed from the cell by cryoprotectant materials, and embryos are stored at temperature below zero. This process may affect the viability and developmental potential of embryos. In this study, the effect of the vitrification cryotop method on the expression level of Oct4 and Mest developmental genes in mouse blastocysts was examined. The collected 2-cell embryos of superovulated mouse by oviduct flushing were divided into non-vitrified and vitrified groups. These embryos were cultured to the blastocyst stage directly in the non-vitrified group and in the vitrified group, these embryos were cultured to 4-8 cell embryos, vitrified with cryotop in these stages and after 2-6 months, warmed and cultured to blastocyst embryos. Quantitative expression of two developmental genes, namely Oct4 and Mest, were performed in these groups, using RNA purification and Real-time RT-PCR. Quantitative PCR analysis showed that the expression level of both genes, Oct4 and Mest, was reduced significantly in the vitrified-warmed group relative to the control group [p=0.046 and p=0.001]. This study revealed that morphologically normal embryos show a reduced amount of Oct4 and Mest transcripts which indicate that the vitrification method negatively effects the expression level of these two developmental genes

Reproductive performance of mouse oocyte after in vivo exposure of the ovary to continuous wave ultrasound

There is a lack of studies regarding the effects of ultrasound [US] and replication of its exposure on pre-implantation events in mammals. Thus, this study assesses the reproductive performance of mouse oocytes that have been obtained from ovaries irradiated with US waves versus non-irradiated ovaries. Also comparision of their parthenogenesis, ovulation, fertilization, and pre-implantation development rates. In this experimental study, we divided extracted ovaries into three experimental groups that received the same dosage, but different replicates of radiation for each group. Results were compared with the control and sham groups. Continuous wave [CW] US, at a spatial average intensity of 355 mW/cm[2] and a frequency of 3.28 MHz, was administered for 5 minutes to the ovaries at an interval between pregnant mare serum gonadotropin [PMSG] and human chorionic gonadotropin [hCG] injections. Statistical analysis was performed using the ANOVA test and the level of significance was determined to be 0.05. Data collection was based on microscopic visualization. According to the obtained results, metaphase II [MII] oocyte numbers and the percentage of blastocysts significantly reduced in the US exposed groups versus the unexposed groups. Fertilization rate was comparable between groups while parthenogenesis was significantly higher in the US-exposed groups compared to the unexposed groups. Structural damage to cells, intracellular organelles and proteins, as well as changes in signaling pathways induced by US may be reasons for some of the observed adverse effects in groups that have received more US exposure.

Effects of saffron [crocus sativus L.] aqueous extract on in vitro maturation, fertilization and embryo development of mouse oocytes

Lower pregnancy rates of in vitro matured oocytes compared to those of in vivo stimulated cycles indicate that optimization of in vitro maturation [IVM] remains a challenge. Reduced developmental competence of in vitro matured oocytes shows that current culture systems for oocyte maturation do not adequately support nuclear and/or cytoplasmic maturation. Therefore this study evaluates the effects of different concentrations of saffron [Crocus sativus L.] aqueous extract [SAE], as an antioxidant agent on IVM of immature mouse oocytes. In this experimental study ,cumulus-oocyte complexes [COCs] were collected from 6-8 weeks old novel medical research institute [NMRI] female mice ovaries. COCs were cultured in IVM medium supplemented with 0 [control], 5, 10, 20 and 40 micro g/ml of SAE in 5% CO[2] at 37[degree sign] C. The rates of maturation, fertilization and development were recorded. ANOVA and Duncan's protected least significant test, using the SAS program was applied for all statistical analysis. The maturation rate was significantly higher in all groups treated with different concentrations of SAE compared with the control group [p<0.05]. However, the lower concentrations of SAE [10 and 5 micro g/ml] in maturation medium respectively increased the fertilization rate of oocytes and in vitro developmental competence when compared with the control group [p<0.05]. The results of this study indicate that lower concentrations of SAE are more appropriate to be added to maturation medium when compared with other experimental and control groups. Generally, we conclude that addition of appropriate amounts of natural extracts such as SAE to maturation medium improves oocyte maturation and embryo development

Nitric oxide-induced polycystic ovaries in the wistar rat

Nitric oxide [NO] involves in polycystic ovary syndrome [PCOS], a cause of infertility in women during the reproductive age. The PCOS is now categorized as an inflammatory phenomenon. The aim of this study was to evaluate the role of NO, a proinflammatory agent, in this syndrome at histological and biochemical levels. In this experimental study, animals were female Wistar rats [weighing 200-250 g] kept under standard conditions. L-Arginine [50-200 mg/kg], a precursor of NO, was injected intra-peritoneally [i.p.] through a period ranging from 9 to14 days/ once a day. The rats' estrous cycle was studied using Papanicolaou test; those showing phase of Diestrous were grouped into experimental and control groups. The control group solely received saline [1 ml/kg, i.p.] throughout all experiments. To evaluate the inflammatory effect of NO, the rats were treated an anti-inflammatory agent, naloxone hydrochloride [0.4 mg/kg, i.p.], prior to L-arginine. At the end of the treatment period all animals' ovaries were assessed for histopathological and histochemical investigations. Also, activation of NO synthase [NOS] in the experiments was studied using NADPH-diaphorase technique. The ovaries of rats treated with L-arginine showed polycystic characteristics in contrast to those collected from control or naloxone pretreated groups, based on image analysis. A difference in enzyme activation was also shown in the sections that belonged to the groups that received L-arginine when compared with the pre-naloxone and control groups. Based on these results, we believe that NO may play a major role in the pathophysiology of PCOS

[Effect of ultrasound on parthenogenic activation of mouse oocyte]

Artificial stimulation of mouse oocyte, in the absence of sperm contribution, can induce its parthenogenic activation of oocyte. Ultrasound is one of the newest methods for artificial activation of mammal oocytes, and its successful utilization in pig oocyte activation has been recently reported. Our objective was to assess the effect of ultrasound on mouse oocyte activation. Our groups included1 control group, 3 experimental groups consisting of 1, 2 and 3 repetitions of ultrasound exposure, and 3 sham groups handled similar to experimental groups but ultrasound system was off during treatments. In experimental groups, adult female NMRI mice at the interval between pregnant mare serum gonadotropin [PMSG] and human corionic gonadotropin [hCG] injections, were exposed to continuous ultrasound with 3.28 MHz frequency and peak intensity [I pk]=355 mW/cm2. Sixteen hours after injection of hCG, the mice were euthanized and their oocytes were collected; thereafter, parthenogenic oocytes were counted. Data analysis using the ANOVA test shows a significant increase in the number of parthenogenic oocytes in mice with 3 overall exposures to ovarian ultrasound [p<0.05]. A significant decrease in the number of metaphase II [MII] oocytes numbers was also seen in mice treated with ultrasound [p<0.05]. Ultrasound is thought to induce pores generation in oocyte membranes and provides an easier inward transport of Ca++ into oocytes. This phenomenon can induce meiosis resumption in immature oocytes. With increased exposure repetitions from 1 to 3 times and greater Ca++ arrival, oocytes can be parthenogenetically activated

[Quantitative expression of BAG1, BAX and BCL-2 genes in human embryos with different fragmentation grades derived from ART]

The purpose of this study was to evaluate the quantitative expressions of BAG1, BAX and BCL-2 in human embryos with different fragmentation grades as derived from assisted reproduction technology [ART]. Fragmented and normal human 8-cell embryos were scored according to the degree of fragmentation with an inverted microscope and divided into four grades [grade I: no or minimal fragmentation [<5%], grade II: embryos with <25% fragmentation, grade III: embryos with >25% fragmentation and grade IV: apoptotic induced embryos with actinomycin D]. In this study, TUNEL labeling was initially used to detect apoptosis, and then revers transcription polymerase chain reaction [RT-PCR] and quantitative PCR were used to define the quantitative expressions of experimental genes in human embryos with different fragmentation grades. The results of TUNEL labeling showed that embryos with higher fragmentation had a high number of apoptotic bodies. The results of RT-PCR and q-PCR analyses showed a significantly decreased amount of BAGI transcript expression from group I to group IV. The highest expression of BAX gene was observed in group II, however, the transcript of BCL-2 gene was not observed in any of the experimental groups. The effect of actinomycin D on transcript expression amounts of experimental genes in apoptotic induced embryos [group IV] compared to control embryos [group I] showed a significant decrease. mRNA expression of BAG1 gene can be used as a good marker to detect apoptosis in human embryos. However, the transcript of BCL-2 gene does not play a role in the detection of apoptosis in human embryos at the 8-cell stage

structure of human mesenchymal stem cells differentiated into cartilage in micro mass culture system

The aim of this study was to differentiate human mesenchymal stem cells [hMSCs] into cartilage in a micromass culture system and study of their structure by light and electron microscopy. Human bone marrow cells obtained from volunteer patients were plated in 75-cm2 flasks and their MSCs were expanded through several sub-cultures. The passage 4 cells were used to establish micromass culture system for chondrogenic differentiation. For this purpose, 200,000 fibroblastic cells were placed in centrifuge tubes and pelleted at 250 g for 5 minutes. About 0.5 ml chondrogenic induction medium was then added to the pellet and the culture incubated in 5% CO2 at 37°C for 21 days. Then, some pellets were utilized to evaluate chondrogenic differentiation by either RT-PCR analysis of some cartilage marker molecules or specific staining for detecting cartilage matrix, and other pellets were used for light and electron microscopic study of differentiated tissue. Primary culture of the bone marrow cells were initially composed of the spindle- and round shaped cells, from which the spindle cells remained and expanded through several passages. At the end of differentiation period, RT-PCR analysis showed high production of collagen II and X and aggrecan mRNA inside the differentiated cells, and toluidine blue staining indicated intermediate accumulation of the metachromatic matrix among the inddced cells. In general, light micrograph indicated a rather cellular state of the differentiated tissue in which the peripheral part had more metachromatic matrix than central zone. More detailed study of the sections revealed that induced aggregates of the cells were composed externally of very thin layer of elongated cells reminiscent of perichondrium and internally a mass of oval cells comprising the main part of the pellet. Ultra-thin sections showed that the cells in perichondrium-like layer were very similar to fibroblastic cells and those located centrally had a set of well-developed organelles, characteristic of highly active cells. Some fat cells were seen in central zone. Conclusion: Cartilage tissue differentiated from MSCs in micromass culture system seemed to be structurally very similar to developing cartilage not to adult mature cartilage