The possible regulatory role of miR-514 and miR-642b in cumulus cells on the oocyte maturation in patients with polycystic ovary syndrome.

Polycystic ovary syndrome is a common endocrine disorder in reproductive-age women. Accordingly, abnormal microenvironment may negatively influence oocyte developmental competence as a result of the altered expression profile of cumulus cells (CCs), mainly the key players of oocyte maturation, such as epidermal growth factor receptor (EGFR) and prostaglandin E receptor-2 (PTGER2). This study aimed to examine the expression levels of miR-514, miR-642b, and their candidate target genes (EGFR and PTGER2, respectively) in CCs of immature and mature oocytes in patients with PCOS. A total of 40 oocytes at germinal vesicle (GV) and 40 oocytes at metaphase II (MII) stages were retrieved from 30 PCOS women. Quantitative real-time PCR was performed to analyze the expression level of miR-514, miR-642b, EGFR, and PTGER2 in cumulus cells (CCS) of each oocyte. The expression level of miRNAs and their candidate target genes were compared between CCs of GV and MII oocytes. Our study suggests an inverse relationship exists between the expression levels of miR-514 and EGFR, and miR-642b and PTGER2. Furthermore, we observed that CCs of GV oocytes had higher levels of EGFR and PTGER2 mRNA and lower levels of miR-514 and miR-642b expression compared to those of MII oocytes. The present study demonstrated that miR-514 and miR-642b can regulate oocyte development by targeting EGFR and PTGER2, respectively. Therefore, examination of these miRNAs in CCs could be promising parameters to predict oocyte competence in PCOS patients.

Detrimental effects of electromagnetic radiation emitted from cell phone on embryo morphokinetics and blastocyst viability in mice.

Electromagnetic radiation (EMR) has deleterious effects on sperm motility and viability, as well as oocyte membrane and organelle structure. The aim was to assess the effects of cell phone radiation on preimplantation embryo morphokinetics and blastocyst viability in mice. For superovulation, 20 female mice were treated with intraperitoneal (IP) injections of 10 IU pregnant mare's serum gonadotropin (Folligon® PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) after 48 h. The zygotes (n = 150) from the control group were incubated for 4 days. The experimental zygotes (n = 150) were exposed to a cell phone emitting EMR with a frequency range 900-1800 MHz for 30 min on day 1. Then, all embryos were cultured in the time-lapse system and annotated based on time points from the 2-cell stage (t2) to hatched blastocyst (tHDyz), as well as abnormal cleavage patterns. Blastocyst viability was assessed using Hoechst and propidium iodide staining. Significant increases (P < 0.05) were observed in the cleavage division time points of t2, t8, t10, and t12 of the experimental group compared with the controls. In terms of blastocyst formation parameters, a delay in embryo development was observed in the experimental group compared with the controls. Data analysis of the time intervals between the two groups showed a significant difference in the s3 time interval (P < 0.05). Also, the rates of fragmentation, reverse cleavage, vacuole formation, and embryo arrest were significantly higher in the experimental group (P < 0.05). Furthermore, the cell survival rate in the experimental group was lower than the control group (P < 0.05). Exposure to EMR has detrimental consequences for preimplantation embryo development in mice. These effects can manifest as defects in the cleavage stage and impaired blastocyst formation, leading to lower cell viability.

Detrimental impact of cell phone radiation on sperm DNA integrity.

Radiofrequency electromagnetic radiation (RF-EMR) from various sources may impact health due to the generation of frequency bands. Broad pulses emitted within frequency bands can be absorbed by cells, influencing their function. Numerous laboratory studies have demonstrated that mobile phones-generally the most widely used devices-can have harmful effects on sex cells, such as sperm and oocytes, by producing RF-EMR. Moreover, some research has indicated that RF-EMR generated by mobile phones can influence sperm parameters, including motility, morphology, viability, and (most critically) DNA structure. Consequently, RF-EMR can disrupt both sperm function and fertilization. However, other studies have reported that exposure of spermatozoa to RF-EMR does not affect the functional parameters or genetic structure of sperm. These conflicting results likely stem from differences among studies in the duration and exposure distance, as well as the species of animal used. This report was undertaken to review the existing research discussing the effects of RF-EMR on the DNA integrity of mammalian spermatozoa.

Sperm Selection Using Zona Pellucida-Binding Enhanced Embryo Morphokinetic and Clinical Outcomes in ICSI: A Sibling Oocytes Study.

The objective was to investigate the embryo morphokinitics using a time-lapse monitoring (TLM) system and assessment of clinical outcomes following intracytoplasmic sperm injection (ICSI) with zona pellucida (ZP)-bound sperm selection and conventional methods. A total of 371 metaphase II (MII) oocytes from 50 ICSI cycles were studied. Sibling oocytes were randomly divided into control (n = 199) and ZP-bound group (n = 172). All resulting zygotes were cultured and monitored in the TLM system up to Day 3 after ICSI. Fertilization rate, early embryo development, and clinical outcomes were evaluated. No significant differences were found in fertilization rate, time-lapse qualitative and quantitative measures, pronuclear fading time (PNF) t2, t3, t4, t5, t6, and t7 (times of cleavage to 2, 3, 4, 5, 6, and 7 cells), respectively. However, the t8 (time of cleavage to eight cells) and cc3 (duration of third cell cycle) revealed a significant difference between control and ZP-bound groups (p < .05). A significant difference between the two groups (p < .05) in the rates of Grade A embryos (according to Basile algorithm), chemical pregnancy, clinical pregnancy, and implantation was observed. Sperm selection using biological materials, such as ZP, improved both embryo quality and pregnancy outcomes, despite not affecting the early embryo development and morphokinetic parameters up to t8. This prospective randomized sibling oocyte trial was registered in October 2020 to January 2022 (IRCT20200705048021N1).

Quality of testicular spermatozoa improves with changes in composition of culture medium.

BACKGROUND: Spermatozoa retrieved from the testis and epididymis are deprived of the beneficial effects of seminal fluid. Thus applying an artificial medium with normal seminal fluid characteristics, known as artificial seminal fluid (ASF), may provide an appropriate condition for improving some sperm parameters in azoospermia. The objective was to investigate the impact of in vitro exposure of testicular and epididymal spermatozoa to ASF on sperm quality. The study was conducted on testicular (n = 20) and epididymal (n = 20) sperm specimens obtained from azoospermic men. Each sample was divided into two equal parts: Part I) for processing and incubation with Ham's F10 medium; Part II) for processing and incubation with ASF. RESULTS: After 2 h incubation, testicular sperm motility was significantly higher in ASF than in Ham's F10 medium. In comparison to 0 h, mitochondrial membrane potential levels of testicular spermatozoa were significantly higher after 2 h and 24 h in ASF and after 24 h in Ham's F10 medium. Furthermore, the data indicated significantly lower rates of epididymal spermatozoa with high MMP in both media after 24 h. There were no significant differences in the DNA fragmentation index of testicular and epididymal spermatozoa between ASF and Ham's F10 medium at different time points. CONCLUSION: The results demonstrated that in vitro incubation of testicular spermatozoa improved their motility more effectively than Ham's F10 medium in the short term (2 h), but had no effect on epididymal spermatozoa. Since the physiology of testicular spermatozoa is different from that of ejaculated spermatozoa, it seems that a special environment should be designed and used for each of them.

RéSUMé: CONTEXTE: Les spermatozoïdes prélevés dans les testicules et les épididymes sont privés des effets bénéfiques du liquide séminal. Ainsi, l'utilisation d'un milieu artificiel avec des caractéristiques normales du liquide séminal, connu sous le nom de liquide séminal artificiel (ASF), peut constituer une condition appropriée pour améliorer certains paramètres des spermatozoïdes obtenus dans l'azoospermie. L'objectif était d'étudier l'impact de l'exposition in vitro de spermatozoïdes testiculaires et épididymaires à l'ASF sur la qualité du sperme. L'étude a été menée sur des échantillons de spermatozoïdes testiculaires (n = 20) et épididymaires (n = 20) obtenus chez des hommes azoospermiques. Chaque échantillon a été divisé en deux parties égales: Partie I) pour le traitement et l'incubation avec le milieu F10 de Ham; Partie II) pour la transformation et l'incubation avec l'ASF. RéSULTATS: Après 2 h d'incubation, la mobilité des spermatozoïdes testiculaires était significativement plus élevée dans l'ASF que dans le milieu F10 de Ham. Par rapport à 0 h, les niveaux du potentiel de membrane mitochondriale (PMM) des spermatozoïdes testiculaires étaient significativement plus élevés après 2 h et 24 h dans l'ASF, et après 24 h dans le milieu F10 de Ham. En outre, les données ont indiqué des taux significativement plus faibles de spermatozoïdes épididymaires avec un PMM élevé dans les deux milieux après 24 heures. Il n'y avait pas de différences significatives dans l'indice de fragmentation de l'ADN des spermatozoïdes testiculaires et épididymaires entre l'ASF et le milieu F10 de Ham aux différents temps. CONCLUSION: Les résultats ont montré que l'incubation in vitro de spermatozoïdes testiculaires dans l'ASF améliorait leur mobilité plus efficacement que le milieu F10 de Ham à court terme (2 h), mais n'avait aucun effet sur les spermatozoïdes épididymaires. Étant donné que la physiologie des spermatozoïdes testiculaires est différente de celle des spermatozoïdes éjaculés, il semble qu'un environnement spécial devrait être conçu et utilisé pour chacun d'eux.

Human embryos derived from first polar body nuclear transfer exhibit comparatively abnormal morphokinetics during development.

OBJECTIVE: Reconstructed oocytes after polar body genome transfer constitute a potential therapeutic option for patients with a history of embryo fragmentation and advanced maternal age. However, the rescue of genetic material from the first polar body (PB1) through introduction into the donor cytoplasm is not yet ready for clinical application. METHODS: Eighty-five oocytes were obtained following in vitro maturation (IVM) and divided into two groups: PB1 nuclear transfer (PB1NT; n=54) and control (n=31). Following enucleation and PB1 genomic transfer, PB1 fusion was assessed. Subsequently, all fused oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured in an incubator under a time-lapse monitoring system to evaluate fertilization, embryonic morphokinetic parameters, and cleavage patterns. RESULTS: Following enucleation and fusion, 77.14% of oocytes survived, and 92.59% of polar bodies (PBs) fused. However, the normal fertilization rate was lower in the PB1NT group than in the control group (56.41% vs. 92%, p=0.002). No significant differences were observed in embryo kinetics between the groups, but a significant difference was detected in embryo developmental arrest after the four-cell stage, along with abnormal cleavage division in the PB1NT group. This was followed by significant between-group differences in the implantation potential rate and euploidy status. Most embryos in the PB1NT group had at least one abnormal cleavage division (93.3%, p=0.001). CONCLUSION: Fresh PB1NT oocytes successfully produced normal zygotes following PB fusion and ICSI in IVM oocytes. However, this was accompanied by low efficiency in developing into cleavage embryos, along with an increase in abnormal cleavage patterns.

Detrimental effects of radiofrequency electromagnetic waves emitted by mobile phones on morphokinetics, oxidative stress, and apoptosis in mouse preimplantation embryos.

Due to the increasing use of smart mobile phones, the impact of radiofrequency electromagnetic radiation (RF-EMR) on reproductive health has become a serious concern. This study investigated the effect of mobile phone RF-EMR with frequency 900-1800 MHZ on the mouse embryo morphokinetics and genotoxic effect in laboratory conditions. After ovarian stimulation in mice, the MII oocytes were collected and underwent by in vitro fertilization (IVF) method. The generated zygotes were divided into control and exposed groups. Then, the zygotes with 30 min of exposure to mobile phone RF-EMR, and the control zygotes without exposure, were incubated in the time-lapse for 5 days. The intracellular reactive oxygen species (ROS) level, morphokinetic, embryo viability rate, and Gene expression were evaluated. Exposure of zygotes to RF-EMR by inducing ROS caused a significant decrease in blastocyst viability (87.85 ± 2.86 versus 94.23 ± 2.44), delay in cleavage development (t3-t12) and also increased the time (in hours) to reach the blastocyst stage (97.44 ± 5.21 versus 92.56 ± 6.7) compared to the control group. A significant increase observed in mRNA levels of Hsp70 in exposed animals; while Sod gene expression showed a significant down-regulation in this group compared to the controls, respectively. However, there was no significant change in the transcript level of proapoptotic and antiapoptotic genes in embryos of the exposed group compared to the controls. RF-EMR emitted by mobile phone with a frequency of 900-1800 MHZ, through inducing the production of ROS and oxidative stress, could negatively affect the growth and development as well as the transcript levels of oxidative stress associated genes in the preimplantation embryos of mice.

Ultrastructural Evaluation of Mouse Oocytes Exposed In Vitro to Different Concentrations of the Fungicide Mancozeb.

Mancozeb is a widely used fungicide, considered to be an endocrine disruptor. In vivo and in vitro studies evidenced its reproductive toxicity on mouse oocytes by altering spindle morphology, impairing oocyte maturation, fertilization, and embryo implantation. Mancozeb also induces dose-dependent toxicity on the ultrastructure of mouse granulosa cells, including chromatin condensation, membrane blebbing, and vacuolization. We evaluated the effects on the ultrastructure of mouse oocytes isolated from cumulus-oocyte complexes (COCs), exposed in vitro to increasing concentrations of mancozeb. COCs were matured in vitro with or without (control) low fungicide concentrations (0.001-1 µg/mL). All mature oocytes were collected and prepared for light and transmission electron microscopy. Results showed a preserved ultrastructure at the lowest doses (0.001-0.01 µg/mL), with evident clusters of round-to-ovoid mitochondria, visible electron-dense round cortical granules, and thin microvilli. Mancozeb concentration of 1 µg/mL affected organelle density concerning controls, with a reduction of mitochondria, appearing moderately vacuolated, cortical granules, and microvilli, short and less abundant. In summary, ultrastructural data revealed changes mainly at the highest concentration of mancozeb on mouse oocytes. This could be responsible for the previously described impaired capability in oocyte maturation, fertilization, and embryo implantation, demonstrating its impact on the reproductive health and fertility.

A new cryotop vial device system provides an aseptic cryoprotectant-free and centrifuge-free cryopreservation of human spermatozoa (a closed system).

Single sperm cryopreservation is a new method to preserve small numbers of spermatozoa in small droplets. So far, several devices have been introduced for this technique, but more studies are needed for its optimization. The aim of this study was optimizing the previous device for low number of spermatozoa and low volume semen, which led to design of Cryotop Vial device. Normal semen samples from 25 patients were prepared by swim-up method and divided into four groups: Fresh (F), Rapid freezing (R) and ultra-rapid freezing with Cryotop Device (CD) and Cryotop Vial Device (CVD). In R group, the diluted sperm suspension with sperm freezing medium was cooled in the vapor phase and then immersed in liquid nitrogen. Ultra rapid freezing was performed with sucrose in small volume using the Cryotop Device (CD) or Cryotop Vial Device (CVD). Sperm viability, motility, fine morphology, mitochondrial activity and DNA fragmentation were assessed in all samples. All sperm parameters decreased significantly in all the cryo groups compared to the fresh group. The comparison between the cryo groups showed that progressive motility (69.28 ± 6.82 vs. 55.68 ± 9.04, and 54.76 ± 5.34, p < 0.001) and viability (77.36 ± 5.48 vs.68.84 ± 8.51, p < 0.001, and 70.04 ± 7.44, P = 0.002) were significantly higher in the CVD compared to the CD and R groups respectively. DNA fragmentation was also significantly lower in both ultra-rapid freezing groups (CD and CVD) compared to the R group. Fine morphology and mitochondrial activity were not different between the cryo groups. The CVD as a cryoprotectant and centrifuge-free technique preserved sperm motility, viability and DNA integrity after cryopreservation better than other groups.

Ischemia and vasculogenesis after transplantation of frozen and vitrified human ovarian tissue onto Chick Embryo Chorioallantoic Membrane (CAM).

OBJECTIVES: The first days of post-ovarian transplantation are critical periods, as the ischemic injury can diminish the success rate. In this study, the first day's events of ovarian transplantation in two dimensions of structure and ultrastructure following slow freezing and vitrification were assessed. STUDY DESIGN: Ovarian tissues (OTs) from 10 cancerous patients were frozen in two methods of slow freezing and vitrification. Tissues were transplanted onto the CAM and then retrieved at 5 and 10 days of culture. Nine groups were assigned as follows; I-III; fresh, 5 and 10 days culture, IV-VI; vitrification, 5 and 10 days culture, and VII-IX; slow freezing, 5 and 10 days culture. Structural and ultra-structural studies were done to assess the tissue viability and integrity following CAM transplantation. Image J software was used to measure the amounts of fibrosis and necrosis. RESULTS: The first sign of successful transplantation was found on day 3 post-transplantation. Vitrified tissues showed higher viability and transplantation rate compared to the slow frozen group (65% vs 57.5%) (p = 0.7). Tissue fibrosis and areas didn't increase significantly after cryopreservation using two methods (p > 0.05). The areas of fibrosis and necrosis and avian vessels increased significantly after 5 and 10 days of culture (p < 0.05). Large ultra-structural follicular deformities were noticed after 10 days of CAM transplantation. Better stromal ultrastructure features can be found after vitrified tissue culture. Also, the CAM transplantation technique had negative effects on the integrity of follicles, independent of the freezing procedure. CONCLUSION: Evaluation of early events of the ovarian post-transplantation is of amount importance, since the hypoxia during this period may accelerate follicular pool depletion, before the tissue stability. Vitrification can be considered a reliable alternative for slow freezing. CAM transplantation is a good technique for confirmation of tissue viability after warming but damaged the follicle ultrastructure in a short period.

Pentoxifylline treatment had no detrimental effect on sperm DNA integrity and clinical characteristics in cases with non-obstructive azoospermia.

The aim of this study was to assess the consequences of treatment with pentoxifylline (PTX), an inducer of sperm motility, on sperm DNA fragmentation (SDF) and clinical characteristics in non-obstructive azoospermia (NOA) patients. The pilot study included 15 NOA patients. Half of each sperm sample before and after rapid freezing, was treated with PTX (3.6 mM /l, 30 min) as the PTX group and the remaining samples were considered as the control. SDF and sperm motility were assessed in each group. The clinical study comprised 30 fresh testicular sperm extractions (TESE) and 22 post-thawed TESE intracytoplasmic sperm injection cycles. Half of the mature oocytes from each patient were injected with PTX-treated spermatozoa and the remaining oocytes were injected with non-treated spermatozoa. Fertilization was assessed at 16 h post injection. Embryo transfer was carried out on day 2 after fertilization. Chemical pregnancy was assessed 2 weeks after transfer. PTX was found to significantly increase (P < 0.05) sperm motility. There was an insignificant difference in SDF rates between the groups (P > 0.05). In patient ovaries given fresh TESE, there was not any significant difference in clinical characteristics (P > 0.05). In patient ovaries given post-thawed TESE, there was a significant difference in the number of 2PN and in embryo formation (P < 0.05). Differences in the results of chemical pregnancy were insignificant (P > 0.05) between the groups. In addition, there was not any correlation between DNA fragmentation index and sperm motility and laboratory outcomes. Therefore, obtaining viable spermatozoa using PTX was more effective in post-thawed TESE regime patients in terms of 2PN and in embryo formation, deprived of damaging effects on sperm DNA integrity.

Ultrastructure of human ovarian tissues and risk of cancer cells re-implantation after transplantation to chick embryo chorioallantois membrane (CAM) following vitrification or slow freezing.

Ovarian follicle depletion and premature ovarian failure are significant challenges in cancer patients subjected to radio- or chemotherapy. Ovarian tissue (OT) cryopreservation would be an option when other fertility preservation methods are not accessible. This study aimed to analyze the structure and ultrastructure of human OTs transplanted onto chick embryo chorioallantois membrane (CAM) after cryopreservation by vitrification or slow freezing. OTs from 10 cancer patients underwent cryopreservation. CAM transplantation was done on fresh and cryopreserved OTs, to assign samples to nine study groups as follows: 1) FI-FIII = fresh, 5- and 10-days post-CAM transplantation groups; 2) VI-VIII = vitrified, 5- and 10-days post-transplantation vitrified groups; 3) SFI-SFIII: slow frozen, 5- and 10-days post-transplantation slow freezing groups. Proliferation ability, folliculogenesis, and structural and ultrastructure were analyzed. The density of primordial follicles did not change after both freezing methods, but reduced after 5 (P ≥ 0.05) and 10 days (P ≤ 0.05) post-CAM transplantation. The follicular grade significantly decreased in all transplanted tissues (P ≤ 0.0). The proliferation marker increased after cryopreservation, but reduced after transplantation (P ≤ 0.05). TEM evaluation showed better follicular ultrastructure in the fresh group, after transplantation. Stromal ultrastructure appeared more preserved after vitrification compared with slow freezing. There was no sign of malignant cell contamination after transplantation. Some follicular TEM abnormalities were found in both methods of freezing, with a better transplantation rate after vitrification. Also, enhanced follicular activation resulted in faster follicular depletion in this method. The information regarding post grafting events would improve our knowledge for longer OTs' lifespans.

The effect of pentoxifylline and calcium ionophore treatment on sperm cell biology in oligoasthenoteratozoospermia samples.

The objective of this study was to assess the effects of pentoxifylline (PTX) and Ca2+ ionophore (CI) A12387 treatment on some biological characteristics of sperm cells in oligoasthenoteratozoospermia (OAT) patients. After processing, each sample was divided into four groups: 1, control; 2, exposed to 3.6 mM PTX; 3, exposed to 5 µm calcium ionophore (CI); and 4, exposed to both PTX and CI; 30 min at 37°C. Sperm motility was measured before and after preparation. Acrosome reaction (AR), status of sperm vacuoles, mitochondrial membrane potential (MMP) and DNA fragmentation were assessed using PSA-FITC staining, motile sperm organelle morphology examination (MSOME), JC-1 staining and sperm chromatin dispersion (CSD) test, respectively. Treatment with PTX and CI led to increased and decreased sperm motility, respectively (P < 0.05). Furthermore, vacuole status and rates of sperm DNA fragmentation were not significantly different among groups (P > 0.05). Moreover, the data showed that the rates of AR and disrupted MMP were significantly different between groups (P < 0.05). In conclusion, in vitro application of PTX not only did not have any adverse effects on sperm cell biology characteristics, but also can rectify the harmful effect of CI.

Improvement of human sperm properties with platelet-rich plasma as a cryoprotectant supplement.

PURPOSE: Platelet-rich plasma (PRP) is a remarkable substance, which involves the growth and proliferation of all cell types. As a source of growth factors, we evaluated whether sperm cryopreservation supplemented with PRP improves the rates of sperm motility, viability, and DNA integrity after vitrification compared with conventional cryo-medium. MATERIALS AND METHODS: 20 normal semen specimens were collected from healthy men. After swim-up preparation, each sample was divided into four aliquots. One, as control, received no treatment, and the other three experimental samples were treated with three different concentrations of PRP as cryoprotectant. Sperm parameters were examined before and after freezing procedure. RESULTS: PRP had no significant effect on sperm count. Meanwhile, the percentage of sperm progressive motility and viability in the PRP treated samples with 1×105 /µL concentration was significantly higher than control group. Besides, the rate of immotile sperms in these samples was significantly lower than the control. Sperm viability was significantly higher in the PRP samples at 1×105/µL concentration. In the case of DNA integrity, CMA3 staining showed that the lower PRP concentration was correlated with the higher rate of abnormal spermatozoa. SCD showed that the rate of abnormal sperms in the PRP samples with 1×105 /µL concentration was significantly lower than control group. CONCLUSIONS: This study showed a protective effect of PRP on human sperm quality at an optimized concentration after vitrification. Besides, the effects of PRP supplementation of sperms on successful fertility following sperm preservation will be of interest.

Prolonged semen incubation alters the biological characteristics of human spermatozoa.

OBJECTIVE: The present study assessed the biological characteristics of human spermatozoa at different time intervals (0, 1, 1.5, and 2 hours) after incubation at 37°C. METHODS: Twenty-five normozoospermic semen samples were incubated at 37°C. Incubation was performed at four time intervals of 0 (after liquefaction), 1, 1.5, and 2 hours. The samples were evaluated for sperm parameters at each time interval. RESULTS: The rate of sperm progressive motility decreased at 1.5 hours compared to 0 hours as well as 2 hours compared to 1 hour and 0 hours. The rate of non-motile spermatozoa also decreased after 2 hours compared to after 0 hours. No significant changes were observed in sperm viability (p=0.98) and non- progressive motility (p=0.48) at any time intervals. Abnormal sperm morphology increased at 1.5 hours of incubation time (p<0.001). No significant changes were observed in DNA fragmentation at 1 hour compared to 0 hours (median [interquartile range]: 19.5 [4] vs. 19 [4]), as well as at 1.5 hours compared to 1 hour (20 [5]). However, a significant increase in DNA fragmentation was observed at 1.5 hours compared to 0 hours. The mitochondrial membrane potential decreased remarkably after 1 hour of incubation time. No significant differences were observed in the acrosome reaction or malonaldehyde levels at any time point (p=0.34 and p=0.98, respectively). CONCLUSION: The incubation of normozoospermic samples before use in assisted reproductive technology should be less than 1.5 hours to minimize the destructive effects of prolonged incubation time on general and specific sperm parameters.

Comparison of the Efficiency of Magnetic-Activated Cell Sorting (MACS) and Physiological Intracytoplasmic Sperm Injection (PICSI) for Sperm Selection in Cases with Unexplained Infertility.

Background: The cases with unexplained infertility may have an abnormality in their sperm chromatin structure. Sperm selection methods can be used to separate sperm with low DNA fragmentation. The purpose of this study was to compare the efficacy of physiological intracytoplasmic sperm injection (PICSI) with magnetic-activated cell sorting (MACS) in assisted reproductive techniques in cases with unexplained infertility. Methods: The semen samples were collected from couples with unexplained infertility. After semen analysis and sperm DNA fragmentation (SDF) evaluations, samples were prepared with swim-up method. The rates of SDF in different fractions including raw semen (n=20), swim-up (n=20), only motile sperm after swim-up (swim-up selection) (n=20), MACS sperm selection (n=20), only motile sperm after MACS (MACS selection) (n=20), and PICSI sperm selection (n=16) were evaluated. Also, the main sperm characteristics and fine morphology of sperm suspension after MACS were assessed. Statistical analysis was performed using GraphPad Prism. The p<0.05 was considered statistically significant. Results: DNA fragmentation index (DFI) values in PICSI and MACS groups were significantly reduced as compared to the swim-up group. The rate of this reduction was more pronounced in MACS (58.20±13.02) than PICSI (36.57±15.52) group. Also, our results showed that MACS resulted in decreased sperm motility, with no alteration in their fine morphology. Conclusion: MACS was found to be more efficient in reduction of SDF rates than PICSI. However, none of the sperm selection techniques can not totally eliminated the spermatozoa with DNA fragmentation in the final sperm sample.

Free centrifuge sorting method for high-count sperm preparation improves biological characteristics of human spermatozoa and clinical outcome: A sibling oocytes study.

Sperm processing for assisted reproductive technologies (ART) aims to separate immotile and debris from the motile spermatozoa in the semen. The purpose of this study was to assess the effect of free centrifuge sorting (FCS) approach based on a combination of rheotaxis and swim-up on sperm biological characteristics and ICSI clinical outcomes. Each semen sample was splitted into two equal parts for 67 ICSI cycles with donation oocytes. Parts were processed with the Direct Swim Up (DSU) (control) and with the FCS method (experimental). Sperm quality was assessed in terms of motility, fine morphology, mitochondrial membrane potential, lipid peroxidation and sperm DNA fragmentation. Also Following ICSI, the clinical outcomes were compared between the groups. Sperm progressive motility (93.5 ± 4.1% vs. 78.6 ± 8.2%; p < 0.001), the fraction of Class I (good) morphology (30.2 ± 9.4% vs. 23.7 ± 8.5%; p < 0.0001) and the rate of mitochondrial membrane potential (77.4 ± 7.2% vs. 66.9 ± 5.7%; p < 0.0001) were significantly higher in the FCS compared to DSU groups. The level of lipid peroxidation (0.5 ± 0.05% vs. 0.6 ± 0.06%; p < 0.0001) and concentration of DNA fragmentation (DF) (7.4 ± 1.6% vs. 15.4 ± 2.6%; p < 0.0001) were lower in sperm from the FCS group compared to DSU group. There were higher rates of high-quality embryo formation (p < 0.001), implantation and clinical pregnancy rates (p = 0.03) in the FCS group compared to the control group. The processing of seminal samples using FCS collected spermatozoa with better biological quality and resulted in higher reproductive outcomes in ICSI cycles.

Target gene repression mediated by miR-144 and miR-224 in cumulus cells is related to the success of oocyte in vitro maturation and fertilisation in patients with polycystic ovary syndrome (PCOS).

CONTEXT: In vitro maturation (IVM) of oocytes is an alternative approach for patients with polycystic ovary syndrome (PCOS) predisposing to ovarian hyperstimulation syndrome (OHSS). Transcriptomic analysis of cumulus cells (CC) may help make IVM more efficient. The aim of this study was to examine the impact of miR-144 and miR-224 and their candidate target genes (COX-2 and PTX-3 , respectively) expression on oocyte development in PCOS patients. METHODS: Immature oocytes were retrieved from 20 PCOS patients. After IVM, samples were divided into two groups: matured (M) and immatured (I) oocytes. ICSI was performed and the embryo quality was evaluated. qPCR was used to analyse miR-144, miR-224, COX-2 and PTX-3 expression levels in CCs of each group. KEY RESULTS: We found that the expression levels of miR-144 and miR-224 were lower and the COX-2 and PTX-3 mRNA levels were higher in CCs of M group than in CCs of I group. The expression level of miR-144 and miR-224 in unfertilised oocytes were higher than fertilised oocytes. The contrary results were observed for COX-2 and PTX-3 . A reduction pattern in the expression level of miR-144 and miR-224 and increasing pattern in the level of COX-2 and PTX-3 expression were observed in high quality compared to low quality embryos. CONCLUSIONS: The selected miRNAs were related to oocyte maturation, fertilisation and embryo development. These results support their critical involvement in oocyte development. IMPLICATIONS: Our findings may help reveal the mechanisms of post-transcriptional regulation by miR-144 and miR-224 during IVM procedure.

Urtica dioica Agglutinin: A plant protein candidate for inhibition of SARS-COV-2 receptor-binding domain for control of Covid19 Infection.

Despite using effective drugs and vaccines for Covid 19, due to some limitations of current strategies and the high rate of coronavirus mutation, the development of medicines with effective inhibitory activity against this infection is essential. The SARS-CoV-2 enters the cell by attaching its receptor-binding domain (RBD) of Spike to angiotensin-converting enzyme-2 (ACE2). According to previous studies, the natural peptide Urtica dioica agglutinin (UDA) exhibited an antiviral effect on SARS-CoV, but its mechanism has not precisely been elucidated. Here, we studied the interaction between UDA and RBD of Spike protein of SARS-CoV-2. So, protein-protein docking of RBD-UDA was performed using Cluspro 2.0. To further confirm the stability of the complex, the RBD-UDA docked complex with higher binding affinity was studied using Molecular Dynamic simulation (via Gromacs 2020.2), and MM-PBSA calculated the binding free energy of the system. In addition, ELISA assay was used to examine the binding of UDA with RBD protein. Results were compared to ELISA of RBD-bound samples of convalescent serum IgG (from donors who recovered from Covid 19). Finally, the toxicity of UDA is assessed by using MTT assay. The docking results show UDA binds to the RBD binding site. MD simulation illustrates the UDA-RBD complex is stable during 100 ns of simulation, and the average binding energy was calculated to be -47.505 kJ/mol. ELISA and, MTT results show that UDA binds to RBD like IgG-RBD binding and may be safe in human cells. Data presented here indicate UDA interaction with S-protein inhibits the binding sites of RBD, it can prevent the virus from attaching to ACE2 and entering the host cell.

High efficiency of homemade culture medium supplemented with GDF9-ß in human oocytes for rescue in vitro maturation.

OBJECTIVE: Optimizing culture media for the incubation of immature oocytes is a vital strategy to increase the oocyte maturation rate during in vitro maturation (IVM) programs. This study evaluated the IVM and fertilization rates of human germinal vesicle (GV) and metaphase I (MI) oocytes using two different maturation media (commercial and homemade) with or without growth differentiation factor 9-ß (GDF9-ß). supplementation. METHODS: Immature oocytes from intracytoplasmic sperm injection (ICSI) cycles were collected and assigned to one of two IVM culture media (commercial or homemade; cleavage-stage base). After maturation, MII oocytes were examined under an inverted microscope for the presence of the polar body, zona pellucida (ZP) birefringence, and meiotic spindle (MS) visualization after maturation in four conditions (commercial or homemade medium, with or without GDF9-ß. ICSI was done for matured oocytes, and fertilization was confirmed by the visualization of two distinct pronuclei and two polar bodies. RESULTS: No significant differences were found between the two culture media in terms of the time and rate of oocyte maturation or the rate of fertilization (p>0.05). Growth factor supplementation increased the 24-hour maturation rate for both GV and MI oocytes only in homemade medium. The maturation rate after 24 hours was higher for MI oocytes (p<0.05). Similar results were observed for MS visualization and ZP structure in both types of media (p>0.05). CONCLUSION: Higher rates of oocyte maturation and fertilization were observed after application of homemade medium supplemented with GDF9-ß. Therefore, this combination may be recommended as an alternative for clinical IVM programs.