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AbstractOocyte cryopreservation is offered to women of various age groups for both health and social reasons. Oocytes derived from either controlled ovarian stimulation or in vitro maturation (IVM) are cryopreserved via vitrification. As maternal age is a significant determinant of oocyte quality, there is limited data on the age-related susceptibility of oocytes to the vitrification-warming procedure alone or in conjunction with IVM. In the present study, metaphase II oocytes obtained from 2, 6, 9, and 12 month old Swiss albino mice either by superovulation or IVM were used. To understand the association between maternal age and oocyte cryotolerance, oocytes were subjected to vitrification-warming and compared to non vitrified sibling oocytes. Survived oocytes were evaluated for mitochondrial potential, spindle integrity, relative expression of spindle checkpoint protein transcripts, and DNA double-strand breaks. Maturation potential and vitrification-warming survival were significantly affected (p < 0.001 and p < 0.05, respectively) in ovulated oocytes from the advanced age group but not in IVM oocytes. Although vitrification-warming significantly increased spindle abnormalities in ovulated oocytes from advanced maternal age (p < 0.01), no significant changes were observed in IVM oocytes. Furthermore, Bub1 and Mad2 transcript levels were significantly higher in vitrified-warmed IVM oocytes (p < 0.05). In conclusion, advanced maternal age can have a negative impact on the cryosusceptibility of ovulated oocytes but not IVM oocytes in mice.

AbstractPreimplantation-stage embryos are susceptible to various types of stress when cultured in vitro. Parthenogenetic embryos that lack spermatozoa contribution exhibit aberrant developmental dynamics due to their uniparental origin. Herein, we assessed whether the absence of paternal genome affects the susceptibility of the embryos to pH, osmotic and oxidative stress. Haploid parthenogenetic embryos (HPE) (activated oocytes with 1 pronucleus and 2 polar bodies) were generated by incubating cumulus oocyte complexes of Swiss albino mice with 10 mM strontium chloride for 3 h. Normally fertilized embryos (NFE) (fertilized oocytes with 2 pronuclei and 2 polar bodies) were derived using in vitro fertilization. At 2-cell stage, both HPE and NFE were exposed to various stressors including pH (6.8 to 8.2), osmotic (isotonic, hypotonic, and hypertonic), and peroxidatic oxidative (H2O2, 25 µM) stress. Endoplasmic reticulum stress response, mitochondrial membrane potential, and the rate of blastocyst development were assessed. HPE were susceptible to alteration in the pH that was well tolerated by NFE. Similarly, HPE displayed remarkable difference in sensitivity to hypertonic stress and oxidative stress compared to NFE. The results clearly indicate that the oocytes that develop into embryos in the absence of paternal contribution are more vulnerable to environmental stressors, further highlighting the importance of spermatozoa contribution and/or the ploidy status in mitigating these stressors and towards healthy early embryo development.

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AbstractSpermatozoon is a motile cell with a special ability to travel through the woman's reproductive tract and fertilize an oocyte. To reach and penetrate the oocyte, spermatozoa should possess progressive motility. Therefore, motility is an important parameter during both natural and assisted conception. The global trend of progressive reduction in the number and motility of healthy spermatozoa in the ejaculate is associated with increased risk of infertility. Therefore, developing approaches for maintaining or enhancing human sperm motility has been an important area of investigation. In this review we discuss the physiology of sperm, molecular pathways regulating sperm motility, risk factors affecting sperm motility, and the role of sperm motility in fertility outcomes. In addition, we discuss various pharmacological agents and biomolecules that can enhance sperm motility in vitro and in vivo conditions to improve assisted reproductive technology (ART) outcomes. This article opens dialogs to help toxicologists, clinicians, andrologists, and embryologists in understanding the mechanism of factors influencing sperm motility and various management strategies to improve treatment outcomes.

AbstractThis pilot study was conducted to explore the benefits of using a centrifugation-free device based on the migration–sedimentation (MS) technique over centrifugation-based techniques in selecting competent spermatozoa, as compared with using split human semen samples. Ejaculates from 35 men undergoing semen analysis were split into four parts where one part was retained as the neat (NE) and the other three parts were subjected to sperm selection by using migration–sedimentation (MS), density gradient (DG) separation, and swim-up (SU) techniques. Sperm functional characteristics along with mitochondrial integrity, tyrosine phosphorylation, acrosome reaction, and ultrastructure were measured. The ability of selection techniques in reducing spontaneous and radiation-induced sperm DNA lesions was assessed by the TUNEL assay. In results, MS-selected spermatozoa had higher viability (P < 0.001), longevity in terms of total motility at the end of 6 and 18 h post-extraction (P < 0.001), and mitochondrial integrity (P < 0.001) compared with those selected by DG. Furthermore, spontaneous DNA lesions were significantly reduced in MS and SU fractions compared with NE (P < 0.001). Similarly, radiation-induced sperm DNA lesions were significantly lower in MS and SU fractions (P < 0.001) compared with DG. Ultrastructural analysis using scanning electron microscopy suggested a moderate, non-significant increase in the number of spermatozoa with normal head and mid-piece in MS fraction compared with other methods. In conclusion, the MS-based device offers a centrifugation-free, efficient, and reliable sperm selection method, making it suitable for partially equipped intra-uterine insemination (IUI) laboratories or office IUI programmes. Further research should focus on the safety and clinical usefulness of the device in assisted conception programmes in general and IUI in specific.

AbstractThis study investigated whether combining metabolomic and embryologic data with machine learning (ML) models improve the prediction of embryo implantation potential. In this prospective cohort study, infertile couples (n=56) undergoing day-5 single blastocyst transfer between February 2019 and August 2021 were included. After day-5 single blastocyst transfer, spent culture medium (SCM) was subjected to metabolite analysis using nuclear magnetic resonance (NMR) spectroscopy. Derived metabolite levels and embryologic parameters between successfully implanted and failed groups were incorporated into ML models to explore their predictive potential regarding embryo implantation. The SCM of blastocysts that resulted in successful embryo implantation had significantly lower pyruvate (p<0.05) and threonine (p<0.05) levels compared to medium control but not compared to SCM related to embryos that failed to implant. Notably, the prediction accuracy increased when classical ML algorithms were combined with metabolomic and embryologic data. Specifically, the custom artificial neural network (ANN) model with regularized parameters for metabolomic data provided 100% accuracy, indicating the efficiency in predicting implantation potential. Hence, combining ML models (specifically, custom ANN) with metabolomic and embryologic data improves the prediction of embryo implantation potential. The approach could potentially be used to derive clinical benefits for patients in real-time.

AbstractCryopreservation of immature-testicular-tissue (ITT) prior to gonadotoxic treatment, while experimental, is the only recommended option for fertility preservation in prepubertal boys. The handling and manipulation of ITT before cryopreservation could influence the functionality of cells during fertility restoration, which this study explored by evaluating cellular niche and quality of mouse ITT subjected to various temperatures and time durations in vitro. ITT from 6-day-old mice were handled at ultraprofound-hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for varying time periods prior to 14-day organotypic culture. Viability, functionality, synaptonemal complex and chromatin remodeling markers were assessed. Results have shown that cell viability, testosterone level, and in vitro proliferation ability did not change when ITT were held at ultraprofound-hypothermic-temperature up to 24 h, whereas cell viability was significantly reduced (P < 0.01), when held at profound-hypothermic-temperature for 24 h before culture. Further, cell viability and testosterone levels in cultured cells from profound-hypothermic group were comparable to corresponding ultraprofound-hypothermic group but with moderate reduction in postmeiotic cells (P < 0.01). In conclusion, holding ITT at ultraprofound-hypothermic-temperature is most suitable for organotypic culture, whereas short-term exposure at profound-hypothermic-temperature may compromise postmeiotic germ cell yield post in vitro culture. This data, albeit in mouse model, will have immense value in human prepubertal fertility restoration research.

AbstractIn this study, we aimed to explore the beneficial properties of novel quinoline derivatives on human sperm motility and its functional competence. Nine novel quinoline derivatives were screened for their effect on motility in human spermatozoa from normozoospermic ejaculates. Compounds with impressive sperm motility enhancement properties were further assessed for their effect on functional competence of human spermatozoa. To determine the effect on the fertilizing ability of spermatozoa processed with quinoline derivatives and to assess developmental competence of embryos derived, in vitro fertilization (IVF) was performed using mouse model. Among the nine quinoline derivatives, 2 compounds (6MQT and 2,6DQT) exhibited significant enhancement in sperm progressive motility and survival at 24 h. Further, non-significant increase in curvilinear velocity (VCL), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) was observed. Capacitation, intracellular cAMP level and tyrosine phosphorylated sperm proteins were significantly higher in 6MQT (P < 0.05) and 2,6DQT (P < 0.001) compared to control. In vitro fertilization (IVF) experiments using Swiss albino mice revealed that spermatozoa processed with 6MQT had non-significantly higher blastocyst rate and a superior blastocyst quality, while, 2,6DQT resulted in significantly lower blastocyst rate (P < 0.05) compared to control. Quinoline derivative 6MQT has significant motility enhancement property under in vitro conditions.