Puberty Blocker and Aging Impact on Testicular Cell States and Function.

Spermatogonial stem cell (SSC) acquisition of meiotogenetic state during puberty to produce genetically diverse gametes is blocked by drugs collectively referred as 'puberty blocker' (PB). Investigating the impact of PB on juvenile SSC state and function is challenging due to limited tissue access and clinical data. Herein, we report largest clinically annotated juvenile testicular biorepository with all children with gender dysphoria on chronic PB treatment highlighting shift in pediatric patient demography in US. At the tissue level, we report mild-to-severe sex gland atrophy in PB treated children. We developed most extensive integrated single-cell RNA dataset to date (>100K single cells; 25 patients), merging both public and novel (52 month PB-treated) datasets, alongside innovative computational approach tailed for germ cells and evaluated the impact of PB and aging on SSC. We report novel constitutional ranges for each testicular cell type across the entire age spectrum, distinct effects of treatments on prepubertal vs adult SSC, presence of spermatogenic epithelial cells exhibiting post-meiotic-state, irrespective of age, puberty status, or PB treatment. Further, we defined distinct effects of PB and aging on testicular cell lineage composition, and SSC meiotogenetic state and function. Using single cell data from prepubertal and young adult, we were able to accurately predict sexual maturity based both on overall cell type proportions, as well as on gene expression patterns within each major cell type. Applying these models to a PB-treated patient that they appeared pre-pubertal across the entire tissue. This combined with the noted gland atrophy and abnormalities from the histology data raise a potential concern regarding the complete 'reversibility' and reproductive fitness of SSC. The biorepository, data, and research approach presented in this study provide unique opportunity to explore the impact of PB on testicular reproductive health.

Degradation meets development: Implications in ß-cell development and diabetes.

Pancreatic development is orchestrated by timely synthesis and degradation of stage-specific transcription factors (TFs). The transition from one stage to another stage is dependent on the precise expression of the developmentally relevant TFs. Persistent expression of particular TF would impede the exit from the progenitor stage to the matured cell type. Intracellular protein degradation-mediated protein turnover contributes to a major extent to the turnover of these TFs and thereby dictates the development of different tissues. Since even subtle changes in the crucial cellular pathways would dramatically impact pancreatic ß-cell performance, it is generally acknowledged that the biological activity of these pathways is tightly regulated by protein synthesis and degradation process. Intracellular protein degradation is executed majorly by the ubiquitin proteasome system (UPS) and Lysosomal degradation pathway. As more than 90% of the TFs are targeted to proteasomal degradation, this review aims to examine the crucial role of UPS in normal pancreatic ß-cell development and how dysfunction of these pathways manifests in metabolic syndromes such as diabetes. Such understanding would facilitate designing a faithful approach to obtain a therapeutic quality of ß-cells from stem cells.

Secretomes from Conventional Insemination and Intra-Cytoplasmic Sperm Injection Derived Embryos Differentially Modulate Endometrial Cells In Vitro.

Conventional Insemination (CI) and Intra-Cytoplasmic Sperm Injection (ICSI) are routinely used insemination methods in clinical Assisted Reproductive Technologies (ART) settings. However, the existing data on the developmental competence and implantation potential of CI and ICSI derived embryos are not unequivocal. This prospective study on 23 patients undergoing ART treatment explored whether the secretomes of CI- and ICSI-derived embryo differentially alter the expression of integrins (αv and ß3 integrin) and MUCIN-1 (MUC-1) in a human endometrial epithelial cell line (Ishikawa). Immunocytochemical data demonstrated that the secretome of CI-derived top quality (GI) embryos induced higher (p < 0.05) expression of ɑv ß3 compared to sibling ICSI derived G1 embryos in Ishikawa cells. Though, relative levels of the transcript for MUC-1, anti-adhesion molecule did not show a significant difference between the study groups, immunocytochemical analysis demonstrated significantly (p < 0.0001) higher expression of MUC-1 in cells treated with ICSI-derived embryo secretome, compared to that treated with CI -derived embryo secretome. These results suggest that secretomes from CI and ICSI embryos differentially modulate the endometrial cells in vitro. This hints at differences in the ability of CI- and ICSI- derived embryos to alter endometrial profile.

Advanced Maternal Age Affects the Cryosusceptibility of Ovulated but not In Vitro Matured Mouse Oocytes.

Oocyte 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.

Differential sperm histone retention in normozoospermic ejaculates of infertile men negatively affects sperm functional competence and embryo quality.

BACKGROUND: The unique epigenetic architecture that sperm cells acquire during spermiogenesis by retaining <15% of either canonical or variant histone proteins in their genome is essential for normal embryogenesis. Whilst heterogeneous levels of retained histones are found in morphologically normal spermatozoa, their effect on reproductive outcomes is not fully understood. METHODS: Processed spermatozoa (n = 62) were tested for DNA integrity by sperm chromatin dispersion assay, and retained histones were extracted and subjected to dot-blot analysis. The impact of retained histone modifications in normozoospermic patients on sperm functional characteristics, embryo quality, metabolic signature in embryo spent culture medium and pregnancy outcome was studied. RESULTS: Dot-blot analysis showed heterogeneous levels of retained histones in the genome of normozoospermic ejaculates. Post-wash sperm yield was affected by an increase in H3K27Me3 and H4K20Me3 levels in the sperm chromatin (p < 0.05). Also, spermatozoa with higher histone H3 retention had increased DNA damage (p < 0.05). Spermatozoa from these cohorts, when injected into donor oocytes, correlated to a significant decrease in the fertilisation rate with an increase in sperm histone H3 (p < 0.05) and H3K27Me3 (p < 0.01). An increase in histone H3 negatively affected embryo quality (p < 0.01) and clinical pregnancy outcome post-embryo transfer (p < 0.05). On the other hand, spent culture medium metabolites assessed by high-resolution (800 MHz) nuclear magnetic resonance showed an increased intensity of the amino acid methionine in the non-pregnant group than in the pregnant group (p < 0.05) and a negative correlation with sperm histone H3 in the pregnant group (p < 0.05). DISCUSSION AND CONCLUSION: Histone retention in spermatozoa can be one of the factors behind the development of idiopathic male infertility. Such spermatozoa may influence embryonic behaviour and thereby affect the success rate of assisted reproductive technology procedures. These results, although descriptive in nature, warrant further research to address the underlying mechanisms behind these clinically important observations.

Acute and sub-chronic toxicity of Liberin, an anti-diabetic polyherbal formulation in rats.

BACKGROUND: The polyherbal formulation (PHF) liberin, is known to exert anti-hyperglycemic effects in type 2 diabetes mellitus. Hence, it is important to study the safety profile of PHF in the current study through acute and chronic toxicity evaluation. OBJECTIVES: This research aims to assess the acute and sub-chronic toxicity of PHF in rats. MATERIALS AND METHODS: PHF was administered once orally (1000 mg/kg body weight), and the rats (male and female) were monitored for toxicity signs for a 14-day period. For a 28-day chronic toxicity study, rats were daily administered with PHF dose of 500 mg/kg and 1000 mg/kg body weight. Rats were followed up for mortality, weight changes, and other morbidities. Further haematological, biochemical, and histopathological changes were assessed. RESULTS: No death related to treatment or toxicity signs were recorded in the acute single-dose administration group. The results showed that the PHF was tolerated well up to a dose of 1000 mg/kg body weight. Even at the high dose of 1000 mg/kg body weight, sub-chronic tests did not show any significant difference between the dosed and normal groups. No significant changes were seen in the histopathological analysis of the liver, spleen, and kidney as well as haematological and biochemical parameters in acute, sub-chronic and satellite groups following the administration of PHF. CONCLUSION: The results confirmed that there was no adverse effect of this PHF at the maximum dose of 1000 mg/kg body weight in Wistar rats. Further, no adverse delayed effects related to PHF were observed in the satellite group. Therefore, this PHF appears safe for therapeutic purposes in the Ayurvedic medicinal system.

Oncofertility awareness among primary care physicians in India.

BACKGROUND: Primary care physicians not only coordinate referrals to oncology services but can play a crucial role in successful fertility preservation referrals in cancer-diagnosed patients. Hence, it is important to assess their knowledge and attitudes towards fertility preservation. METHODS: An eighteen-item oncofertility survey was administered to primary care physicians between May 2019 to September 2020.  Results: A total of forty-six responses were received and analysed. About 60% of primary care physicians did not have adequate knowledge about available fertility preservation options and only 26-32% were aware of international guidelines recommending fertility preservation in cancer patients.  Conclusions: Imparting awareness and knowledge of fertility preservation and its options to primary care physicians could enable an integrated cancer care model while also facilitating successful oncofertility referrals in countries like India.

Impact of prepubertal bovine ovarian tissue pre-freeze holding duration on follicle quality.

Ovarian tissue cryopreservation prior to gonadotoxic treatment is the only recommended option for fertility preservation in prepubertal girls. Due to the technical complexity of this technique, limited number of centres across the world are equipped to offer the facility. Hence, the retrieved ovarian tissue needs to be maintained at hypothermic temperature (4 °C) for long time during shipment. The time taken between tissue retrieval and cryopreservation could influence the functionality of cells during fertility restoration. This study explored the tissue integrity and follicle quality of ovarian cortical slices subjected to pre-freeze holding for various time durations in vitro. Prepubertal bovine ovarian tissue from < 12 months old animals were handled at hypothermic holding (4 °C) for 0, 24, 48 and 72 h. The tissues were assessed for follicle viability through confocal analysis of live-dead labelled samples, and follicle quality and tissue integrity through histology. Results have shown that follicle viability, and overall follicle quality were not significantly affected at the end of 72 h hypothermic holding. Though, the observation reassures extended hypothermic holding prior to freezing, findings need to be validated in human tissue prior to use in clinical fertility preservation programs.

Use of sensitivity-enhanced nuclear magnetic resonance spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe in identifying human sperm intracellular metabolites.

CONTEXT: The clinical value of human sperm metabolites has not been established due to the technical complexity in detecting these metabolites when sperm numbers are low. AIMS: To detect endogenous intracellular metabolites in fresh and post-thaw human spermatozoa using 800MHz nuclear magnetic resonance (NMR) spectroscopy equipped with a 1.7-mm cryo-probe. METHODS: Processed spermatozoa from 25 normozoospermic ejaculates were subjected to extraction of intracellular metabolites and then profiled by sensitivity-enhanced NMR spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe. In parallel, some of the processed sperm fractions were subjected to freeze-thawing and were then analysed for intracellular metabolites. KEY RESULTS: Twenty-three metabolites were profiled from only 1.25million sperm cells. Comparison of the metabolomic signature of pre-freeze and post-thaw sperm cells did not show significant changes in the levels of metabolites. CONCLUSIONS: Sensitivity-enhanced NMR spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe is a potential tool for identifying intracellular metabolites when sperm number is low. IMPLICATIONS: Use of sensitivity-enhanced NMR spectroscopy opens up the opportunity to test for endogenous metabolites in samples with a limited number of spermatozoa, to understand the patho-physiology of infertility.

High doses of clethodim-based herbicide GrassOut Max poses reproductive hazard by affecting male reproductive function and early embryogenesis in Swiss albino mice.

Clethodim is a widely used and approved class II herbicide, with little information about its impact on the reproductive system. Herein, we investigated the male reproductive toxicity of clethodim using a mouse model. GrassOut Max (26% clethodim-equivalent) or analytical grade clethodim (≥90%) were given orally to male mice for 10 d in varying doses. All parameters were assessed at 35 d post-treatment. Significant decrease in testicular weight, decreased germ cell population, elevated DNA damage in testicular cells and lower serum testosterone level was observed post clethodim based herbicide exposure. Epididymal spermatozoa were characterized with significant decrease in motility, elevated DNA damage, abnormal morphology, chromatin immaturity and, decreased acetylated-lysine of sperm proteins. In the testicular cells of clethodim-based herbicide treated mice, the expression of Erß and Gper was significantly higher. Proteomic analysis revealed lower metabolic activity, poor sperm-oocyte binding potential and defective mitochondrial electron transport in spermatozoa of clethodim-based herbicide treated mice. Further, fertilizing ability of spermatozoa was compromised and resulted in defective preimplantation embryo development. Together, our data suggest that clethodim exposure risks male reproductive function and early embryogenesis in Swiss albino mice via endocrine disrupting function.

Engineered Nano-carrier systems for the oral targeted delivery of follicle stimulating Hormone: Development, characterization, and, assessment of in vitro and in vivo performance and targetability.

Follicle stimulating hormone (FSH) is widely used for the treatment of female infertility, where the level of FSH is suboptimal due to which arrest in follicular development and anovulation takes place. Currently, only parenteral formulations are available for FSH in the market. Due to the drawbacks of parenteral administration and the high market shares of FSH, there is a need for easily accessible oral formulation. Therefore, enteric coated capsules filled with FSH loaded nanostructured lipid carriers (NLCs) or liposomes were prepared. Preliminary studies such as circular dichroism, SDS-PAGE, FTIR and ELISA were conducted to analyze FSH. Prepared formulations were optimized with respect to the size, polydispersity index, zeta potential, and entrapment efficiency using the design of experiments. Optimized formulations were subjected to particle counts and distribution analysis, TEM analysis, in vitro drug release, dissolution of enteric coated capsules, cell line studies, everted sac rat's intestinal uptake study, pharmacokinetics, pharmacodynamics, and stability studies. In the case of liposomes, RGD conjugation was done by carbodiimide chemistry and conjugation was confirmed by FTIR, 1HNMR and Raman spectroscopy. The prepared formulations were discrete and spherical. The release of FSH from enteric coated capsules was slow and sustained. The increased permeability of nano-formulations was observed in Caco-2 monoculture as well as in Caco-2 and Raji-B co-culture models. NLCs and liposomes showed an improvement in oral bioavailability and efficacy of FSH in rats. This may be due to mainly chylomicron-assisted lymphatic uptake of NLCs; whereas, in the case of liposomes, RGD-based targeting of ß1 integrins of M cells on Peyer's patches may be the main reason for the better effect by FSH. FSH was found to be stable chemically and conformationally. Overall, the study reveals the successful development and evaluation of FSH loaded NLCs and liposomes.

Haploid Parthenogenetic Embryos Exhibit Unique Stress Response to pH, Osmotic and Oxidative Stress.

Preimplantation-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.

Combining Machine Learning with Metabolomic and Embryologic Data Improves Embryo Implantation Prediction.

This 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.

Anti-tuberculosis drugs used in a directly observed treatment short course (DOTS) schedule alter endocrine patterns and reduce the ovarian reserve and oocyte quality in the mouse.

CONTEXT: Tuberculosis is one of the major infectious diseases, with people of reproductive age group having a high risk of infection. AIMS: The present study was designed to understand the consequences of anti-tuberculosis drugs (ATDs) used in DOTS (directly observed treatment short course) schedule on ovarian function. METHODS: Adult female Swiss albino mice were orally administered with combinations of ATDs used in the DOTS schedule every day for 4weeks. At 2weeks after the cessation of ATDs administration, the endocrine changes and ovarian function were assessed in mice. KEY RESULTS: Administration of ATDs to mice resulted in a prolonged estrous cycle, reduced ovarian follicle reserve, alteration in FSH, LH, and progesterone level, and decreased the number of ovulated oocytes. Further, the degree of fragmentation, degeneration, abnormal distribution of cytoplasmic organelles, abnormal spindle organisation, and chromosomal misalignment were higher in oocytes that were ovulated following superovulation. Blastocysts derived from ATDs treated mice had significantly lower total cell numbers and greater DNA damage. A marginal increase in the number of resorbed fetuses was observed in all the ATDs treated groups except in the multidrug resistance treatment group. Male progeny of ATDs treated mice had decreased sperm count and lower progressive motility, while female progeny exhibited a non-significant reduction in the number of oocytes ovulated. CONCLUSIONS: Theresults of this study suggest that ATDs can have significant adverse effects on the ovarian reserve, cytoplasmic organisation of oocytes, and can potentially cause transgenerational changes. IMPLICATIONS: The findings of the present study indicate ovarian toxicity of ATDs and warrant further research in the direction of identifying alternate drugs with minimal toxicity, and strategies to mitigate the ovarian toxicity induced by these drugs.

Semen characteristics of individuals before and after CovishieldTM vaccination.

Concern about fertility impairment after vaccination is one of the reasons for vaccine hesitancy in the population. This retrospective observational study aims to understand the impact of CovishieldTM (ChAdOx1 nCoV- 19 Corona Virus Vaccine, Recombinant) COVID-19 vaccination on ejaculate quality in fifty-three patients undergoing semen analysis between 2018 to 2021. A baseline semen profile was recorded from the subjects during their visit before the vaccination for fertility work-up. Follow-up ejaculates were provided approximately 82 (Q1:37, Q3:124) days after the second dose of vaccination. Semen characteristics such as volume, sperm concentration, sperm motility, and morphological abnormalities were recorded. Of the 53 subjects, 33 (62%) had semen characteristics above the WHO reference. In general, no significant variations in the semen parameters were observed except for a moderate decline in sperm morphology (p< 0.05). The baseline semen characteristics in 20 (38%) subjects were below the WHO reference range; however, no significant decline in the ejaculate quality was observed in their follow-up samples. Further, none of the ejaculates in both study groups were azoospermic during the follow-up evaluation. Our results affirm that CovishieldTM vaccine is not detrimental to male fertility.

ICSI in non-male factor infertility patients does not alter metabolomic signature in sibling embryos as evidenced by sensitivity enhanced nuclear magnetic resonance (NMR) spectroscopy.

Intracytoplasmic sperm injection (ICSI) was developed to overcome male factor infertility, however, there recently has been an increasing trend in ICSI usage irrespective of the etiology, demonstrating an overuse of this insemination technique. There is a limited knowledge on the behaviour of ICSI derived embryos in non-male factor infertility patients. Metabolomic assessment of preimplantation embryos in conjunction with morphological evaluation can provide better understanding of embryonic behaviour. Hence, this study was undertaken to explore if there are any metabolomic differences between IVF and ICSI derived sibling day-5 blastocysts from non-male factor infertility patients. This prospective study included nineteen couples with non-male factor infertility undergoing Assisted Reproductive Technology. The sibling oocytes retrieved from each patient were randomly assigned to two groups and inseminated either by IVF or ICSI. Spent culture media (SCM) in which embryos were cultured up to day 5 were collected and investigated using sensitivity enhanced NMR based metabolite profiling utilizing high resolution (800 MHz) NMR equipped with cryogenically cooled micro-coil (1.7 mm) probe. The metabolomic signature between IVF and ICSI derived sibling blastocysts was assessed. A significant reduction in the concentrations of pyruvate, citrate, glucose and lysine were observed in both IVF and ICSI sibling embryos compared to medium control (P< 0.05-0.001). Further, histidine and valine level was found lower in ICSI embryos compared to medium control (P<0.05) during 96 hours of in vitro culture. Notably, between IVF and ICSI SCM, no significant difference in the concentration of the metabolites was found. Our results suggest that ICSI in non-male factor does not alter the SCM metabolomic signature during 96 hours of embryonic development.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter medium composition.

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Experimentally Induced Hyperglycemia in Prepubertal Phase Impairs Oocyte Quality and Functionality in Adult Mice.

Reproductive abnormalities in women with a history of childhood diabetes are believed to be partially attributed to hyperglycemia. Prolonged hyperglycemia can negatively affect ovarian function and fertility during reproductive life. To address this in an experimental setting, the present study used streptozotocin-induced hyperglycemic prepubertal mouse model. The impact of prolonged hyperglycemic exposure during prepubertal life on ovarian function, oocyte quality, and functional competence was assessed in adult mice. The ovarian reserve was not significantly altered; however, the in vitro maturation potential (P < 0.001), mitochondrial integrity (P < 0.01), and meiotic spindle assembly (P < 0.05-0.001) in oocytes were significantly affected in hyperglycemic animals in comparison to control groups. The results from the study suggest that prepubertal hyperglycemia can have adverse effects on the oocyte functional competence and spindle integrity during the reproductive phase of life. Because these changes can have a significant impact on the genetic integrity and developmental potential of the embryos and fetus, the observation warrants further research both in experimental and clinical settings.

Distinctions in PCOS Induced by Letrozole Vs Dehydroepiandrosterone With High-fat Diet in Mouse Model.

Polycystic ovarian syndrome (PCOS) is a complex health condition associated with metabolic disturbances and infertility. Recent data suggest that the prevalence of PCOS is increasing among women globally, although the etiology of these trends is undefined. Consequently, preclinical models that better reflect the biology of PCOS are urgently needed to facilitate research that can lead to the discovery of prevention strategies or improved management. The existing animal models have several limitations as they do not reflect all the PCOS features metabolically and/or phenotypically. Therefore, there is no clear consensus on the use of appropriate animal model and selection of the most appropriate PCOS-inducing agent. To that end, we have established a Swiss albino mouse model of PCOS based on 3 weeks of daily treatment with letrozole (50 µg/day; intraperitoneal) and dehydroepiandrosterone (DHEA, 6 mg/100 g body weight; subcutaneous) in 5-week-old female mice fed on normal or high-fat diet (HFD). Mice were regularly assessed for body weight, blood glucose, and estrous cycle. Three weeks after drug administration, mice were sacrificed and assessed for blood-based metabolic parameters as well as ovarian function. Our results indicate that DHEA combined with HFD produces changes mimicking those of clinical PCOS, including elevated serum testosterone and luteinizing hormone, dyslipidemia, poor ovarian microenvironment, and development of multiple ovarian cysts, recapitulating cardinal features of PCOS. In comparison, normal diet and/or letrozole produced fewer features of PCOS. The data from the experimental models presented here can improve our understanding of PCOS, a growing concern in women's health.

Sperm characteristics in normal and abnormal ejaculates are differently influenced by the length of ejaculatory abstinence.

BACKGROUND: No association between the length of ejaculatory abstinence (LEA) and semen characteristics has been confirmed. A short LEA has been linked to improved sperm characteristics and a higher pregnancy rate, but its negative influence on sperm chromatin maturity and longevity may adversely affect reproductive outcomes. OBJECTIVES: We sought to determine the influence of LEA on (i) semen parameters in normozoospermic and abnormal ejaculates; and (ii) the outcomes of sperm-preparation methods in a large number of subfertile men undergoing infertility workups. MATERIALS AND METHODS: This retrospective registry-based cohort study analyzed the data of 10,674 ejaculates from 7972 subfertile men, who were then segregated into normozoospermic, oligozoospermic, asthenozoospermic, and oligo-asthenozoospermic cohorts. Variations in semen characteristics and post-wash outcomes were studied between four LEA intervals across 0-15 days. RESULTS: An age-adjusted analysis of covariance (ANCOVA) model linked significant increases in ejaculate volume, sperm concentration (except in the oligozoospermic cohort), and total sperm count to an increased LEA (p < 0.05). LEA was negatively associated with motility (except in the asthenozoospermic cohort) and vitality (p < 0.05). Large-headed spermatozoa were less common with an increased LEA only in the oligo-asthenozoospermic cohort (p < 0.05). In the normozoospermic cohort, a longer LEA led to fewer spermatozoa with amorphous heads but more spermatozoa with tapered heads and cytoplasmic droplets (p < 0.05). LEA extension resulted in greater sperm DNA fragmentation in the abnormal cohort (p < 0.01). The post-wash sperm concentration and total motile sperm count were significantly improved with a longer LEA in the normozoospermic cohort (p < 0.05). DISCUSSION AND CONCLUSION: Considering the findings in this study and existing literature, a generalized recommendation for long LEA has no clinical value. The LEA should be individualized based on the ejaculate profile and the need for specific clinical intervention.