Effects of cryopreservation in the presence of Natural Deep Eutectic Solvents (NADESs) on sperm parameters.

Natural Deep Eutectic Solvents (NADESs) are being considered as a potential alternative to traditional cryoprotective agents (CPAs) in sperm freezing. The study aimed to assess the effects of NADESs as a CPA on human sperm parameters. A total of 32 normozoospermic semen samples were collected from the Alzahra infertility treatment center (Iran) between July 2021 and September 2022. The samples were categorized into eight different groups: 1) a control (nonfrozen), and groups frozen with 2) SpermFreeze Solution, 3) ChX (Choline chloride and Xylitol), 4) ChS (Choline chloride and D-sorbitol), 5) ChG (Choline chloride and Glucose), 6) ChU (Choline chloride and Urea), 7) EtP (Ethylene glycol and l-proline), and 8) GlyP (Glycerol and l-proline). The study also analyzed the quality of sperm parameters, such as chromatin condensation and integrity, acrosome integrity, and survival, along with the expression of some genes that affect sperm fertility (TRPV1, TRPV4, SPACA3, and OGG1). The study found there were notable variations in sperm parameters (such as viability, chromatin condensation and integrity, and acrosome integrity) among frozen groups with some NADESs compared to the SpermFreeze Solution and control groups (P < 0.05). Analysis of gene expression demonstrated that the levels of TRPV1, TRPV4, SPACA3, and OGG1 genes were superior in the GlyP group compared to the other groups (P < 0.05). Additionally, the ChS and ChU groups exhibited preserved expression of these genes compared with the SpermFreeze Solution group. The use of NADESs led to the discovery of a more appropriate CPA that has low toxicity and is highly effective in maintaining the fertility potential of sperm.

The Emerging Role of LncRNA FENDRR in Multiple Cancers: A Review.

Long noncoding RNAs (lncRNAs) are prominent as crucial regulators of tumor establishment and are repeatedly dysregulated in multiple cancers. Therefore, lncRNAs have been identified to play an essential function in carcinogenesis and progression of cancer at genetic and epigenetic levels. FENDRR (fetal-lethal noncoding developmental regulatory RNA) as a LncRNA is a hallmark of various malignancies. FENDRR is crucial for multiple organs' development, such as the lung and heart. The effects of FENDRR under signaling pathways in different cancers have been identified. In addition, it has been verified that FENDRR can affect the development and progression of various cancers. In addition, FENDRR expression has been associated with epigenetic regulation of target genes participating in tumor immunity. Furthermore, FENDRR downregulation was observed in various types of cancers, including colorectal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, liver cancer, gallbladder cancer, lung cancer, breast cancer, endometrial cancer, prostate cancer, chronic myeloid leukemia, osteosarcoma, and cutaneous malignant melanoma cells. Here, we review the biological functions and molecular mechanisms of FENDRR in several cancers, and we will discuss its potential as a cancer biomarker and as a probable option for cancer treatment.

Effect of in ovo feeding of folic acid on brain derived neurotrophic factor (BDNF) and gga-miR-190a-3p expression in the developing cerebral cortex of chickens.

Folate plays important role in biosynthesis of purine and pyrimidine nucleotides and is therefore crucial for DNA synthesis and neurogenesis in fetal brains. Many genes comprising brain derived neurotrophic factor (BDNF) and miRNAs have been shown to play important role in brain development. Gga-miR-190a-3p targets many genes including BDNF. The aim of this project was to study the effects of in ovo administration of folic acid (FA) on BDNF and gga-miR-190a-3p expression in the cerebral cortex of chick embryo. A total number of 120 hatching eggs with the correct shape and weight were used in this experiment. Forty eggs was injected by FA into the yolk sac at a dose of 150-µg per egg, 40 eggs by PBS (SHAM) on embryonic day 11 and 40 eggs were left without injection as controls. Then the cerebral cortex was collected on E19 and BDNF and gga-miR-190a-3p expression was studied using Real time PCR. The results showed that BDNF expression in the cortex of FA treated, SHAM and controls were 2.06 ± 0.29, 1.12 ± 0.12 and 1.02 ± 0.21 fold changes, respectively and for gga-miR-190a-3p were 0.72 ± 0.08, 0.95 ± 0.09 and 1.007 ± 0.12 fold change, respectively. Statistical analysis showed that there is significant increase in BDNF expression and decreased gga-miR-190a-3p expression in FA injected cerebral cortex as compared either with SHAM or controls. Although, no significant change in BDNF and gga-miR-190a-3p expression were observed between SHAM and controls. It is concluded that in ovo administration of FA increases BDNF and decreases gga-miR-190a-3p expression in the developing chick cerebral cortex.

Association between Glucose Consumption and Oocyte Maturation Competence in Mice with Polycystic Ovarian Syndrome.

BACKGROUND: This study evaluated association between glucose uptake by individually cultured oocyte and their maturation competence in mice with polycystic ovarian syndrome (PCOS). MATERIALS AND METHODS: In this experimental study, PCOS and non-PCOS cumulus-oocyte complexes (COCs), and cumulus-denuded oocytes (DOs) were cultured individually and categorized in four groups: i. PCOS DOs (n=83), ii. PCOS COCs (n=35), iii. Non-PCOS DOs (n=61) and iv. Non-PCOS COCs (n=62). After the culture period, 50 µl aliquots of the spent drops were used for glucose change analysis using high performance liquid chromatography. Polar NH2 column was used for the study of carbohydrates, acetonitrile with deionized water as the solvent phase and UV as detectors. Oocyte quality (growth differentiation factor 9: GDF-9), viability [bcl-2-like protein 4 (BAX) and B-cell lymphoma2 (BCL2)], in addition to fertilization and embryonic development rates were also evaluated in relation to glucose consumption rate of each oocyte. RESULTS: Maturation rate was significantly higher in non-PCOS COCs and DOs compared to PCOS COCs (IV: 70.9% vs. II: 45.71%) and DOs (III: 67.2% vs. 1: 53.01%), respectively. There was a significant negative correlation between high glucose intake (38.17 ppm) and BCL2 gene expression (P=0.03) in PCOS COCs compared to non-PCOS COCs. There was a significant difference in the GDF-9 gene expression from PCOS DOs (0.66 ± 0.02, P=0.003) and COCs (0.37 ± 0.02, P=0.0001) compared to non-PCOS DOs and COCs, respectively. A negative correlation was also observed between quality of PCOS-DOs and -COCs with glucose intake. Non-PCOS COCs significantly showed higher rate of successful IVF and development compared to PCOS COCs (P=0.01). CONCLUSION: Based on the importance of metabolic analysis, the glucose consumption by DOs and COCs in culture medium can be a suitable criterion for their quality assessment. So that, glucose consumption may reflect oocyte maturation competence.

TEffect of Single Embryo Blastomere Biopsy from Human Frozen Embryos on Assisted Reproductive Outcomes.

OBJECTIVE: Preimplantation genetic testing for aneuploidies (PGT-A) is used to determine chromosomal normality and achieve a successful live birth in infertile couples. There is a possible correlation between chromosomal aneuploidy, embryo development and pregnancy rate. This study evaluated the influence of single blastomere biopsy (SBB) on embryo development and pregnancy rates during frozen embryo transfer (FET) and fresh cycles. MATERIALS AND METHODS: This quasi-experimental study evaluated 115 intracytoplasmic sperm injection (ICSI) cycles, including 443 embryos (6-8 cells) with a grade A on day three, following PGT-A in the fresh or FET cycles from February 2018 to June 2020. In addition, the fresh cycles without PGT were included as a control group (n=166 embryos). SBB was done on day three and was grouped as FET-PGT (n=149) and the fresh-PGT (n=128). RESULTS: There is a more aneuploidy rate in the FET-PGT group compared to the fresh-PGT cycle (36.60% vs. 20.38%, P<0.001). There is a rate of higher development and blastocyst in the control group. While the embryos of PGT groups showed higher degrees of expansion (expansion 5) on day five. 8.6, 8.59, and 9.37% of expansion 3, 4, and 5 in the fresh-PGT embryos, 12.58, 2.78, and 14.84% of expansion 3, 4, and 5 in the FET-PGD embryos compared to 10.84and 33.73% of expansion 3 and 4 in the control group (without expansion 5; P<0.001). There was no significant relationship between 13, 18, and 21 chromosome aneuploidies with blastocyst development competence among the groups (P<0.1). Following embryo transfer (n=97), the spontaneous abortion rate was higher in the FET-PGT cycles compared to the fresh-PGT and control groups (50 vs. 22 and 11%, respectively; P<0.04). CONCLUSION: The process of SBB following vitrification significantly decreased embryo development and pregnancy outcomes. Therefore, a morphological analysis could not be reliable in selecting chromosomally normal embryos.

Morphometric evaluation of two-pronucleus zygote images using image-processing techniques.

Identifying embryos with a high potential for implementation remains a challenge in in vitro fertilization (IVF) cycles. Despite progress in IVF treatment, only a minority of generated embryos has the ability to implant. Another drawback of this practice is the high frequency of multiple pregnancies. This problem leads to economic and health problems. Therefore, the transfer of a single embryo with high implantation potential is the ideal strategy. Morphometric evaluation of two-pronucleus zygote images is a helpful technique when aiming to transfer a single embryo with a high implantation potential. In this study, an automated zygote morphometric evaluation algorithm, called the zygote morphology evaluation (ZME) algorithm, was created to analyze the zygote and provide morphological measurements. The first and most crucial step of the ZME algorithm is the noise reduction step, which was first applied to zygote images. After that, the proposed algorithm detects different parts of the zygote that are indicators of embryo viability and normality, that is the oolemma, perivitelline space, zona pellucida, and nucleolar precursor bodies (NPBs). In addition, a novel dataset was prepared for this task. This dataset consisted of 703 human zygote images, and called the human zygote morphometric evaluation dataset (HZME-DS). Our experimental results in the HZME-DS showed that the ZME algorithm was able to achieve 79.58% average accuracy in identifying the oolemma region, 79.40% average accuracy in determining the perivitelline space, and 79.72% accuracy in identifying the zona pellucida. To calculate the accuracy of identifying NPBs, the proposed algorithm uses Recall and Precision measures, and their harmonic average (F1 measure) reached values of 81.14% and 79.53%, respectively. These encouraging results for our proposed method, which is an automatic and very fast method, showed that the ZME algorithm could help embryologists to evaluate the best zygotes in real time and the best embryos subsequently.

Metformin, clomiphene citrate and flutamide effects on oocyte ultrastructure status and quality in PCOS mouse model.

RESEARCH QUESTION: Polycystic ovary syndrome (PCOS) alters oocyte developmental competence and so the treatment of PCOS patients with assisted reproductive techniques is a major challenge for an infertility specialist. Does ovulation induction therapy with clomiphene citrate, metformin and flutamide affect abnormal PCOS follicle gene expression, its quality, and nuclear and cytoplasmic maturation in the oocyte matured in vitro? DESIGN: Fifty NMRI mice (7-8 weeks old) were randomly divided into five groups, including non-PCOS, PCOS and PCOS groups treated with clomiphene citrate (18 mg/kg body weight for 2 days), metformin (50 mg/100 g body weight for 30 days) and flutamide (10 mg/kg body weight injection for 15 days). The oocytes were collected for quantitative real-time polymerase chain reaction analysis (the evaluation of genes associated with oocyte maturation), transmission electron microscopy (the evaluation of cytoplasmic maturation) and in-vitro maturation (IVM) and IVF outcomes. RESULTS: The dysregulated expression of some genes of insulin-like growth factor (IGF) families involved in oocyte competence and steroid metabolism (e.g. Cyp19a1 and Cyp11a1), transforming growth factor beta (TGF-ß) family (e.g. Tgfb1, Gdf9, Bmp15, Inhbb and Bmpr1a), and oestrogen receptor signalling (e.g. Ncoa3, Pgr) was, to some extent, restored in the PCOS follicles following their with clomiphene citrate, metformin and flutamide. The improved cytoplasmic maturation was observed particularly in the PCOS mice treated with clomiphene citrate. The better IVM-IVF outcomes were also observed in the clomiphene citrate and metformin groups compared with the PCOS group. CONCLUSIONS: This study opens up a new perspective on knowing the effect of ovulation induction therapy on not only nuclear maturation but also ultrastructural characteristics, IVM-IVF outcomes and PCOS oocyte developmental competence.

Using Deep Learning Algorithm: The Study of Sperm Head Vacuoles and Its Correlation with Protamine mRNA Ratio.

OBJECTIVE: It is necessary to evaluate fertility effective agents to predict assisted reproduction outcomes. This study was designed to examine sperm vacuole characteristics, and its association with sperm chromatin status and protamine-1 (PRM1) to protamine-2 (PRM2) ratio, to predict assisted pregnancy outcomes. MATERIALS AND METHODS: In this experimental study, ninety eight semen samples from infertile men were classified based on Vanderzwalmen's criteria as follows: grade I: no vacuoles; grade II: ≤2 small vacuoles; grade III: ≥1 large vacuole and grade IV: large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscopy (SEM). The chromatin integrity, condensation, viability and acrosome integrity, and protamination status were evaluated for vacuolated samples by toluidine blue (TB) staining, aniline blue, triple staining, and CMA3 staining, respectively. Also, Protamine-1 and protamine-2 genes expression was analysed by reverse transcription-quantitative polymerase chain reaction (PCR). The assisted reproduction outcomes were also followed for each cycle. RESULTS: The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (P=0.03 and P=0.02, respectively), and also, protamine-deficient (P=0.04 and P=0.03, respectively). The percentage of reacting acrosomes was significantly higher in the grades III and IV spermatozoa in comparison with normal group. The vacuolated spermatozoa with grade IV showed a high protamine mRNA ratio (PRM-2 was underexpressed, P=0.01). In the IVF cycles, we observed a negative association between sperm head vacuole and fertilization rate (P=0.01). This negative association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P=0.04 and P=0.03, respectively). CONCLUSION: The results of our study highlight the importance sperm parameters such as sperm head vacuole characteristics, particularly those parameters with the potency of reflecting protamine-deficiency and in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) outcomes predicting.

Staphylococcus saprophyticus and Escherichia coli: Tracking from sperm fertility potential to assisted reproductive outcomes.

OBJECTIVE: Bacteriospermia and urogenital infections are common problems in male infertility. This study aimed to evaluate the effects of bacteriospermia on sperm parameters and clinical outcomes in semen samples infected with two common bacteria (Staphylococcus saprophyticus and Escherichia coli) in northern Iran. METHODS: Microbiological tests were performed to isolate and identify organisms from 435 semen samples from infertile couples. Semen samples were assessed according to the World Health Organization criteria. The protamine status, chromatin structure, chromatin condensation, and acrosome reaction of sperm and assisted reproductive outcomes were determined in couples with different male infertility factors. RESULTS: Among the total cases, the two most prevalent pathogens were considered: S. saprophyticus (38.2%) and E. coli (52.9%). In the semen samples infected with E. coli, the spontaneous acrosome reaction and abnormal chromatin condensation were more common (p<0.05). Significant increases in abnormal chromatin condensation and deprotamination were seen in the presence of S. saprophyticus. In washed semen, tight adhesion between the sperm midpiece and S. saprophyticus was observed. There was also a significant decrease in the fertilization rate using semen samples infected with S. saprophyticus and E. coli during in vitro fertilization cycles (p<0.001). In addition, the presence of S. saprophyticus and E. coli in semen samples was associated with a lower likelihood of clinical pregnancy in couples with various factors of male infertility. CONCLUSION: Poor results of assisted reproductive techniques may be correlated with semen samples infected with two common bacteria in northern Iran.

A robust deep learning-based multiclass segmentation method for analyzing human metaphase II oocyte images.

BACKGROUND AND OBJECTIVE: The morphology of the human metaphase II (MII) oocyte is an essential indicator of the embryo's potential for developing into a healthy baby in the Intra-Cytoplasmic Sperm Injection (ICSI) process. In this case, characteristics such as oocyte and ooplasm area, zona pellucida (ZP) thickness, and perivitelline space (PVS) width are also linked to the embryo's implantation potential. Moreover, oocyte segmentation methods may be of particular interest in those countries' restrictive IVF legislation. METHODS: While the manual examination is impractically time-consuming and subjective, this paper concentrates efforts on designing an automated deep learning framework to take on the challenging task of segmentation in low-resolution microscopic images of MII oocytes. In particular, we have developed a deep learning network based on an improved U-Net model using our presented unique collection of human MII oocyte images (a new challenging dataset contains 1,009 images accompanied by manually labeled pixel-accurate ground truths). High-quality ground truth (GT) preparation is a labor-intensive task. However, we put considerable effort into assessing how different types of GT annotations (binary and multiclass) impact segmentation performance. RESULTS: Experimental results on 250 MII oocyte test images demonstrate that the proposed multiclass segmentation algorithm is able to segment complex and irregular ooplasm, ZP, and PVS structures more accurately than its two-class version. Furthermore, the proposed architecture outperforms two other state-of-the-art deep learning models, U-Net and ENet, for the MII oocyte segmentation task. CONCLUSIONS: The findings of this study provide a fascinating insight into the automatic and accurate segmentation of human MII oocytes.

The reaggregation of normal granulosa-cumulus cells and mouse oocytes with polycystic ovarian syndrome in vitro: An experimental study.

BACKGROUND: The dialogue between oocytes and their surrounding cells plays a major role in the progress of oocyte meiosis and their developmental potential. OBJECTIVE: This study aimed to evaluate the effect of co-culture of normal granulosa-cumulus cells (GCCs) with oocytes from polycystic ovarian syndrome (PCOS) mice. MATERIALS AND METHODS: Normal GCCs were collected from 10 virgin adult Naval Medical Research Institute female mice (30-35 gr, 7-8 wk old), and were cultured in an alpha-minimum essential medium supplemented with 5% fetal calf serum for 24-48 hr (1 × 10 6 cells/well). Then, germinal-vesicle oocytes from PCOS mice were cultured in the presence of cultured normal GCCs (experimental group) and without GCCs (control group). The maturation rate and quality of the PCOS oocytes were examined by evaluating TFAM and Cx43 gene expression (real-time PCR) and the connection among PCOS oocytes and normal GCCs after 24 hr of culture. RESULTS: The co-culture of normal GCCs and PCOS oocytes in the experimental group led to the formation of a complex called a PCOS oocyte-normal GCCs complex. The maturation rate of these complexes was significantly increased compared to that of the control group (p ≤ 0.001). A significant difference was also found in the expression of Cx43 (p ≤ 0.001) and TFAM (p < 0.05) genes in the experimental group compared with the control group. The connection between PCOS oocytes and normal GCCs was observed in the scanning electron microscope images. CONCLUSION: Co-culture with normal GCCs improves the capacity of PCOS oocytes to enter meiosis, which may result in the promotion of assisted reproduction techniques.

Is it necessary to focus on morphologically normal acrosome of sperm during intracytoplasmic sperm injection?

Background & objectives: The detailed assessment of sperm morphology is important in the semen of infertile men because there is a low proportion of normal spermatozoa. One of the parameters of such sperm morphology is the acrosome, and its effect on assisted reproductive outcomes is controversial. This study was undertaken to evaluate the association between different forms of acrosome on the chromatin status and the assisted reproductive outcomes. Methods: A total of 1587 unstained sperms from 514 infertile men were captured and analyzed for different acrosome forms (normal, large, small, skew, amorphous acrosome and without acrosome) in real time during intracytoplasmic sperm injection into oocytes. The association between the percentage of sperms with atypical acrosome and head shapes and the sperm chromatin status was studied. Fertilization, zygote and embryo quality and clinical pregnancy rates were calculated for different groups of sperms. Results: The highest frequency of irregular shapes of acrosomes, such as small, large and amorphous, was observed in abnormal ellipticity, anteroposterior symmetry and angularity parameters, respectively (P <0.05). The fertilization rate of injected sperms with large (P <0.01) and small (P=0.001) acrosomes and without acrosome (P=0.001) was significantly lower in comparison with normal acrosomes. The quality of zygotes (Z3, P=0.05), embryos (grade C, P <0.05) and the pregnancy rate (P=0.001) from injected sperms with large acrosomes were significantly lower compared with normal acrosomes. Interpretation & conclusions: Our findings showed that the different sperm acrosome morphologies (e.g., large, small, and without acrosome) might negatively relate with chromatin integrity and decrease the sperm's fertility potential and pregnancy rate during intracytoplasmic sperm injection (ICSI) cycles.

Applying data mining techniques for increasing implantation rate by selecting best sperms for intra-cytoplasmic sperm injection treatment.

BACKGROUND AND OBJECTIVE: Aspiration of a good-quality sperm during intracytoplasmic sperm injection (ICSI) is one of the main concerns. Understanding the influence of individual sperm morphology on fertilization, embryo quality, and pregnancy probability is one of the most important subjects in male factor infertility. Embryologists need to decide the best sperm for injection in real time during ICSI cycle. Our objective is to predict the quality of zygote, embryo, and implantation outcome before injection of each sperm in an ICSI cycle for male factor infertility with the aim of providing a decision support system on the sperm selection. METHODS: The information was collected from 219 patients with male factor infertility at the infertility therapy center of Alzahra hospital in Rasht from 2012 through 2014. The prepared dataset included the quality of zygote, embryo, and implantation outcome of 1544 injected sperms into the related oocytes. In our study, embryo transfer was performed at day 3. Each sperm was represented with thirteen clinical features. Data preprocessing was the first step in the proposed data mining algorithm. After applying more than 30 classifiers, 9 successful classifiers were selected and evaluated by 10-fold cross validation technique using precision, recall, F1, and AUC measures. Another important experiment was measuring the effect of each feature in prediction process. RESULTS: In zygote and embryo quality prediction, IBK and RandomCommittee models provided 79.2% and 83.8% F1, respectively. In implantation outcome prediction, KStar model achieved 95.9% F1, which is even better than prediction of human experts. All these predictions can be done in real time. CONCLUSIONS: A machine learning-based decision support system would be helpful in sperm selection phase of ICSI cycle to improve the success rate of ICSI treatment.

An efficient method for automatic morphological abnormality detection from human sperm images.

BACKGROUND AND OBJECTIVE: Sperm morphology analysis (SMA) is an important factor in the diagnosis of human male infertility. This study presents an automatic algorithm for sperm morphology analysis (to detect malformation) using images of human sperm cells. METHODS: The SMA method was used to detect and analyze different parts of the human sperm. First of all, SMA removes the image noises and enhances the contrast of the image to a great extent. Then it recognizes the different parts of sperm (e.g., head, tail) and analyzes the size and shape of each part. Finally, the algorithm classifies each sperm as normal or abnormal. Malformations in the head, midpiece, and tail of a sperm, can be detected by the SMA method. In contrast to other similar methods, the SMA method can work with low resolution and non-stained images. Furthermore, an image collection created for the SMA, has also been described in this study. This benchmark consists of 1457 sperm images from 235 patients, and is known as human sperm morphology analysis dataset (HSMA-DS). RESULTS: The proposed algorithm was tested on HSMA-DS. The experimental results show the high ability of SMA to detect morphological deformities from sperm images. In this study, the SMA algorithm produced above 90% accuracy in sperm abnormality detection task. Another advantage of the proposed method is its low computation time (that is, less than 9s), as such, the expert can quickly decide to choose the analyzed sperm or select another one. CONCLUSIONS: Automatic and fast analysis of human sperm morphology can be useful during intracytoplasmic sperm injection for helping embryologists to select the best sperm in real time.

Evaluation of attention-deficit hyperactivity disorder risk factors.

Background. Attention-deficit hyperactivity disorder (ADHD) is one of the most common psychiatric disorders among children. The aim of this study was to evaluate risk factors for ADHD in children. Method. In this case-control study, 404 children between 4 and 11 years old were selected by cluster sampling method from preschool children (208 patients as cases and 196 controls). All the participants were interviewed by a child and adolescent psychiatrist to survey risk factors of ADHD. Results. Among cases, 59.3% of children were boys and 38.4% were girls, which is different to that in control group with 40.7% boys and 61.6% girls. The chi-square showed statistically significance (P value < 0.0001). The other significant factors by chi-square were fathers' somatic or psychiatric disease (P value < 0.0001), history of trauma and accident during pregnancy (P value = 0.039), abortion proceeds (P value < 0.0001), unintended pregnancy (P value < 0.0001), and history of head trauma (P value < 0.0001). Conclusions. Findings of our study suggest that maternal and paternal adverse events were associated with ADHD symptoms, but breast feeding is a protective factor.

Melatonin effect during different maturation stages of oocyte and subsequent embryo development in mice.

BACKGROUND: It is important to protect oocytes and embryos from oxidative stress in the culture medium. Melatonin has been shown to be a direct free radical scavenger. OBJECTIVE: Effect of melatonin during in vitro oocyte maturation, fertilization and embryo development of mouse oocytes was evaluated. MATERIALS AND METHODS: Oocytes from supper-ovulated mouse were divided to two groups: cumulus oocyte complexes (COCs, group I) and denuded COC (d-COCs, group II). The oocytes were cultured in maturation medium with different doses of melatonin (1×10(1)-10(5) nM). The cumulus expansion and nuclear status were evaluated after 24 h of in-vitro maturation. The oocytes were used for in-vitro fertilization. The fertilized oocytes were cultured in medium supplemented with different doses of melatonin. RESULTS: The expansion (86.79%) and maturation (80.55%) rate of COCs increased in supplemented medium with 10 nM of melatonin vs. control group (73.33%), p=0.006 and p=0.026 respectively), but oocytes without cumulus cells indicated higher maturation rate at higher melatonin doses (10 and 100 M, 84.34% and 79.5% respectively( vs. 69.33% in control group (p=0.002). Fertilization rate was higher in treated medium with 1 µM of melatonin (93.75%, p=0.007). The rate of cleavage and blastocyst formation was promoted in medium supplemented with 10 and 100 nM of melatonin (92.37% and 89.36% vs. 81.25% in control group, p=0.002). We observed a dose dependent response to melatonin treatment in this experiment. CONCLUSION: Exogenous melatonin can promote cumulus cell expansion, in vitro oocyte maturation, and embryo development. However we investigated a dose-dependent response in different stages of maturation and development. It may reflect sensitive rate of oocytes and embryos to culture conditions.

The impact of different time intervals between hCG priming and oocyte retrieval on ART outcomes.

BACKGROUND: Abnormal oocyte morphology has been associated with the hormonal environment to which the gametes are exposed. OBJECTIVE: In this study, we evaluated the oocytes morphology, fertilization rate, embryos quality, and implantation rate resulted of retrieved oocytes in different times after human chorionic gonadotrophin (HCG) administration. MATERIALS AND METHODS: A total of 985 metaphase II oocytes were retrieved 35, 36, 37 and 38 h after the injection of HCG as groups 1, 2, 3, and 4 respectively. Oocyte morphology was divided into (I) normal morphology, (II) extracytoplasmic abnormalities, (III) cytoplasmic abnormalities and (IV) intracytoplasmic vacuoles and in each group, oocytes were evaluated according to this classification. RESULTS: Extracytoplasmic abnormalities were encountered in 17.76% and 31.1% of these oocytes (groups 3 and 4 respectively, p=0.007) in comparison with 12.23% group 2. Cytoplasmic abnormalities in group 4 were higher than other groups. 23.88% (p=0.039) and 43.25% (p=0.089) of resulted 2PN (two pronucleus) from groups 3 and 4 showed grade Z3 respectively in comparison to group 2 (16.44%). Normal and various categories of abnormal oocytes did not differ regarding fertilization and cleavage rates (p=0.061). However, group 4 showed significant difference in the rate of embryos fragmentation (grade III and IV embryo) in comparison with group 2 (40.96% vs. 24.93%, p=0.078). The pregnancy rate was higher in G2 and G3 groups (28.5 and 24.13% respectively). CONCLUSION: Oocyte retrieval time following HCG priming affected on oocyte morphology, 2PN pattern and embryos qualities subsequently. Both good quality embryo formation and pregnancy outcomes were noticeably higher when oocytes were retrieved 36 h after HCG priming in ART program.

The effect of melatonin on the developmental potential and implantation rate of mouse embryos.

OBJECTIVE: Melatonin is a scavenger agent that has been used to promote in vitro embryo development. This study was designed to show the effects of melatonin on the quality and quantity rate of preimplantation mouse embryo development and pregnancy. MATERIALS AND METHODS: In this experimental study, super ovulated, mated mice were killed by cervical dislocation to collect two-cell zygotes from the oviduct of pregnant 1 day NMRI mice. Zygotes were cultured to the hatching blastocyst stage and the numbers of embryos at different stages were recorded under an inverted microscope. The cleavage rates of two-cell zygotes were assayed until the blastocyst and hatching blastocyst stage in drops of T6 medium that contained either melatonin (1, 10, and 100×10(6), 10 and 100×10(9) M) or no melatonin. The cell numbers of blastocysts were determined by differential staining, implantation outcomes were studied, and development and pregnancy rate were compared by the Chi-square (development) and Fisher's exact (pregnancy rate) tests. RESULTS: The addition of 10 and 100 nM melatonin to the embryo culture media promoted the development of the two-cell stage embryos to blastocyst and hatching blastocysts (p<0.01) and caused a significant increase in total cell number (TCN), trophoectoderm (TE), and inner cell mass (ICM) of the blastocysts (p<0.01). A difference was observed in the percentage of transferred embryos that were successfully implanted between the control and treatment groups (p<0.05). CONCLUSION: The data indicate that 10 and 100 nM of melatonin positively impact mouse embryo cleavage rates, blastocyst TCN, and their implantation. Therefore, melatonin at low concentrations promotes an embryonic culture system in mice.

Evaluation of serial thawing-refreezing on human spermatozoa resistance using cryovials and straws.

BACKGROUND: We designed this study to detect the cryoinjury rate on human sperm after serial freezing and thawing, taking into consideration the effects of using cryovials and straws. MATERIALS AND METHODS: In this experimental study, semen specimens obtained from 15 subjects were divided into normozoospermic and oligozoospermic groups. Each of the normozoospermic and oligozoo spermic semen specimens were additionally divided into two groups: i. washed and ii. unwashed. Specimens were repeatedly freeze-thawed by using cryovials and straws with the fast liquid nitrogen vapor method, until no motile sperm remained. Sperm motility, recovery, and morphology rate were then determined after thawing, and compared between the groups while taking into consideration the effects of using cryovials and straws. RESULTS: Motile spermatozoa were observed in all normozoospermic samples up to thaw 6 with both cryovials and straws while in oligozoospermic specimens up to thaw 4 (straw) and thaw 3 (cryovial) in the freeze-thawing cycle. Normozoospermic sample analysis showed no significant difference in morphology rate. There was a significant increase in motility and recovery percentages for washed samples, which was observed with straws in compared to the unwashed groups. Oligozoospermic sample analysis indicated a significant increase in motility, recovery (p<0.01), and morphology (p<0.001) rates in washed specimens compared to unwashed specimens using straws. The importance of washing sperm was obvious for oligozoospermic specimens. CONCLUSION: Normozoospermic sperm resisted freezing longer than oligozoospermic sperm. Use of straws and cryovials made significant differences in motility, recovery, and morphology of sperm in each thaw. This difference was slightly higher for oligozoospermic specimens. Results indicated that the percentage of motility was higher for washed normozoospermic specimens in each thaw when straws were used, whereas the percentage of motility, recovery, and morphology were promoted after frozen oligozoospermic specimens were washed using straws.