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1.
Sci Rep ; 14(1): 23008, 2024 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-39362929

RESUMO

Preimplantation embryos can experience stress from laboratory interventions and a sub-optimal culture environment. Though research has demonstrated advanced maternal age impairs oocyte quality, the response of embryos derived from such oocytes to vitrification-thawing and culture in a high oxygen (O2) environment in the assisted reproductive technology laboratory is unknown. Therefore, in this study, embryos produced by in vitro fertilization (IVF) using oocytes from two- and eight-month-old Swiss albino mice were vitrified and thawed during their 6-8 cell stage. and cultured at low oxygen (5%) tension (LOT) and high oxygen (20%) tension (HOT). Embryo development, apoptosis, inner cell mass (ICM) outgrowth proliferation ability in vitro and pluripotency were assessed. Embryos from advanced maternal age cultured at HOT showed reduced fertilizing ability (p < 0.05), poor survival post-thawing (p < 0.05), and increased apoptosis (p < 0.01) in comparison to sibling embryos cultured at LOT. Importantly, the extended culture of vitrified-thawed embryos from advanced maternal age led to a significant (p < 0.001) reduction in complete ICM outgrowth formation at HOT in comparison to the LOT environment. The findings of this study suggest that HOT is detrimental to embryos from advanced maternal age, and importantly, vitrified-thawed embryos are more susceptible to stress, which could have negative implications, especially during the peri-implantation developmental period.


Assuntos
Criopreservação , Desenvolvimento Embrionário , Idade Materna , Oxigênio , Animais , Camundongos , Criopreservação/métodos , Oxigênio/metabolismo , Feminino , Blastocisto/citologia , Blastocisto/fisiologia , Fertilização in vitro/métodos , Vitrificação , Apoptose , Técnicas de Cultura Embrionária/métodos , Oócitos/citologia
2.
Reprod Fertil ; 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39401134

RESUMO

Historically, infertility has been stigmatized in the Indian society, primarily due to societal norms that equate marriage with procreation. In twentieth century, India focused primarily on over-fertility in its family planning programs, with little attention given to the complexities of infertility. The introduction of Assisted Reproductive Technology (ART) in the late 1970s made a global revolution, including in India, offering hope to infertile couples. Despite a significant rise in ART clinics offering a wide range of treatment options in the recent years, challenges remain, particularly regarding the affordability. In India, ART is typically dominated by the private sector as government support remains limited. Efforts to standardize ART practices, including the establishment of the National ART & Surrogacy Registry and ART act aim to regulate, improve outcomes and curb unethical practice. Despite these advancements, the high cost of treatment cycles and lack of insurance coverage limit many couples' ability to undergo fertility treatment. Addressing these issues requires a multifaceted approach, including policy reform, increased public awareness, and the development of affordable treatment options to ensure broader access to reproductive care across India.

3.
Reprod Sci ; 2024 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-39300034

RESUMO

Cryopreservation of testicular tissue holds an important role in the field of fertility preservation, particularly for prepubertal boys diagnosed with cancer. However, prepubertal testicular tissue cryopreservation is still considered to be in the experimental stage necessitating the refinement of cryopreservation protocol. Considering the fact that loss of membrane lipids is the primary cause of freeze-thaw-induced loss of testicular cell functions, in this study, we explored the beneficial properties of exogenous supplementation of membrane lipids in the form of liposomes in enhancing the cryosurvival of prepubertal testicular tissue. The freezing medium supplemented with liposomes (prepared from soy lecithin, phosphatidylethanolamine, phosphatidylserine, and cholesterol) was used for the experiments. Prepubertal testicular tissues from Swiss albino mice were cryopreserved in a liposome-containing freezing medium (LFM) composed of 0.25 mg/mL liposomes, 5% DMSO, and 30% FCS in the DMEM/F12 medium using a slow freezing protocol. The tissues were thawed and assessed for various testicular cell functions. Freezing in LFM mitigated the loss of viability, decreased malondialdehyde level (p < 0.05), and reduced apoptosis (p < 0.05) in the testicular cells compared to the testicular tissue cryopreserved in the control freezing medium (CFM). Further, DMSO (5%) appears to be the ideal penetrating cryoprotectant for prepubertal testicular tissue cryopreservation with liposome-based freezing medium. Similar enhancement in cryosurvival of cells was observed in adult human testicular tissue frozen with LFM. These findings highlight the translational value of liposome-based freezing medium in the cryopreservation of testicular tissue of prepubertal boys undergoing chemotherapy.

4.
Toxicol Appl Pharmacol ; 490: 117039, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39019093

RESUMO

The present study aimed to investigate the role of antidiabetic drug metformin on the cytoplasmic organization of oocytes. Germinal vesicle (GV) stage oocytes were collected from adult female Swiss albino mice and subjected to in vitro maturation (IVM) in various experimental groups- control, vehicle control (0.3% ethanol), metformin (50 µg/mL), high glucose and high lipid (HGHL, 10 mM glucose; 150 µM palmitic acid; 75 µM stearic acid and 200 µM oleic acid in ethanol), and HGHL supplemented with metformin. The metaphase II (MII) oocytes were analyzed for lipid accumulation, mitochondrial and endoplasmic reticulum (ER) distribution pattern, oxidative and ER stress, actin filament organization, cortical granule distribution pattern, spindle organization and chromosome alignment. An early polar body extrusion was observed in the HGHL group. However, the maturation rate at 24 h did not differ significantly among the experimental groups compared to the control. The HGHL conditions exhibited significantly higher levels of oxidative stress, ER stress, poor actin filament organization, increased lipid accumulation, altered mitochondrial distribution, spindle abnormalities, and chromosome misalignment compared to the control. Except for spindle organization, supplementation of metformin to the HGHL conditions improved all the parameters (non-significant for ER and actin distribution pattern). These results show that metformin exposure in the culture media helped to improve the hyperglycemia and hyperlipidemia-induced cytoplasmic anomalies except for spindle organization. Given the crucial role of spindle organization in proper chromosome segregation during oocyte maturation and meiotic resumption, the implications of metformin's limitations in this aspect warrant careful evaluation and further investigation.


Assuntos
Hiperglicemia , Hiperlipidemias , Metformina , Oócitos , Estresse Oxidativo , Fuso Acromático , Animais , Metformina/farmacologia , Feminino , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Camundongos , Fuso Acromático/efeitos dos fármacos , Hiperglicemia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Hiperlipidemias/tratamento farmacológico , Hipoglicemiantes/farmacologia , Citoplasma/metabolismo , Citoplasma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Cultivadas , Ácido Palmítico/toxicidade , Ácido Palmítico/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos
5.
Cells ; 13(11)2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38891086

RESUMO

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment.


Assuntos
Desenvolvimento Embrionário , Hiperglicemia , Oxigênio , Animais , Feminino , Oxigênio/metabolismo , Oxigênio/farmacologia , Camundongos , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Desenvolvimento Embrionário/genética , Apoptose/efeitos dos fármacos , Blastocisto/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Oócitos/metabolismo , Embrião de Mamíferos/metabolismo , Proliferação de Células
6.
Hum Fertil (Camb) ; 27(1): 2362980, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38842163

RESUMO

Fertility restoration potential of immature testicular tissue (ITT) depends on the number of spermatogonial cells in the retrieved tissue prior to cryopreservation in oncofertility programme. There are limited data on the association between type of malignancy and testicular germ cell population. Hence, this study is aimed to investigate the spermatogonial and Sertoli cell population in ITT retrieved from 14 pre-pubertal boys who opted for fertility preservation. Histopathological and immunochemical analysis of seminiferous tubules from haematological (N = 7) and non-haematological (N = 7) malignant patients revealed 3.43 ± 2.92 and 1.71 ± 1.81 spermatogonia per tubular cross section (S/T), respectively. The Sertoli cell number was comparable between haematological and non-haematological group (18.42 ± 3.78 and 22.03 ± 10.43). Spermatogonial quantity in ITT did not vary significantly between haematological and non-haematological cancers. This observation, though preliminary, would contribute to the limited literature on paediatric male oncofertility.


Assuntos
Preservação da Fertilidade , Neoplasias , Espermatogônias , Humanos , Masculino , Preservação da Fertilidade/métodos , Criança , Criopreservação , Testículo , Pré-Escolar , Neoplasias Hematológicas/terapia , Células de Sertoli , Infertilidade Masculina/etiologia , Infertilidade Masculina/terapia
7.
bioRxiv ; 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38585884

RESUMO

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.

8.
Reprod Sci ; 31(7): 2080-2091, 2024 07.
Artigo em Inglês | MEDLINE | ID: mdl-38472711

RESUMO

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.


Assuntos
Endométrio , Mucina-1 , Injeções de Esperma Intracitoplásmicas , Humanos , Feminino , Endométrio/metabolismo , Endométrio/citologia , Injeções de Esperma Intracitoplásmicas/métodos , Mucina-1/metabolismo , Adulto , Linhagem Celular , Estudos Prospectivos , Inseminação Artificial , Implantação do Embrião/fisiologia , Masculino
9.
Cell Biol Int ; 48(6): 759-776, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38499517

RESUMO

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.


Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Complexo de Endopeptidases do Proteassoma , Proteólise , Células Secretoras de Insulina/metabolismo , Humanos , Diabetes Mellitus/metabolismo , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Diferenciação Celular , Ubiquitina/metabolismo , Lisossomos/metabolismo
10.
Reprod Sci ; 31(5): 1420-1428, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38294668

RESUMO

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.


Assuntos
Criopreservação , Técnicas de Maturação in Vitro de Oócitos , Idade Materna , Oócitos , Vitrificação , Animais , Oócitos/fisiologia , Feminino , Camundongos , Criopreservação/métodos , Proteínas Mad2/metabolismo , Fuso Acromático/fisiologia , Fuso Acromático/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/fisiologia
11.
Andrology ; 12(4): 881-890, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-37801310

RESUMO

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.


Assuntos
Histonas , Infertilidade Masculina , Feminino , Humanos , Masculino , Gravidez , Histonas/metabolismo , Sêmen/metabolismo , Cromatina/metabolismo , Infertilidade Masculina/genética , Espermatozoides/metabolismo
12.
J Ayurveda Integr Med ; 14(6): 100804, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37847964

RESUMO

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.

13.
F1000Res ; 12: 153, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37767024

RESUMO

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.


Assuntos
Preservação da Fertilidade , Neoplasias , Médicos de Atenção Primária , Humanos , Neoplasias/terapia , Atitude , Índia
14.
Reprod Biol ; 23(3): 100794, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37531931

RESUMO

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.


Assuntos
Preservação da Fertilidade , Folículo Ovariano , Feminino , Animais , Bovinos , Humanos , Lactente , Congelamento , Ovário/patologia , Criopreservação/veterinária , Criopreservação/métodos , Preservação da Fertilidade/métodos
15.
Reprod Fertil Dev ; 35(14): 661-668, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37643634

RESUMO

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.


Assuntos
Preservação do Sêmen , Sêmen , Humanos , Masculino , Sêmen/fisiologia , Criopreservação/veterinária , Criopreservação/métodos , Espermatozoides/metabolismo , Congelamento , Preservação do Sêmen/métodos , Espectroscopia de Ressonância Magnética , Motilidade dos Espermatozoides/fisiologia
16.
Chemosphere ; 336: 139215, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37336444

RESUMO

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.


Assuntos
Herbicidas , Gravidez , Animais , Feminino , Camundongos , Masculino , Herbicidas/toxicidade , Herbicidas/metabolismo , Proteômica , Sêmen , Testículo/metabolismo , Espermatozoides/metabolismo , Desenvolvimento Embrionário
17.
Int J Pharm ; 637: 122868, 2023 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-36958606

RESUMO

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.


Assuntos
Portadores de Fármacos , Nanoestruturas , Humanos , Ratos , Feminino , Animais , Portadores de Fármacos/química , Lipossomos , Hormônio Foliculoestimulante , Células CACO-2 , Nanoestruturas/química , Administração Oral , Cápsulas , Oligopeptídeos , Tamanho da Partícula
18.
Reprod Sci ; 30(7): 2137-2151, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36690917

RESUMO

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.


Assuntos
Peróxido de Hidrogênio , Partenogênese , Animais , Masculino , Camundongos , Haploidia , Partenogênese/genética , Desenvolvimento Embrionário , Blastocisto/fisiologia , Oócitos/metabolismo , Estresse Oxidativo , Fertilização in vitro , Concentração de Íons de Hidrogênio
19.
Reprod Sci ; 30(3): 984-994, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36097248

RESUMO

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.


Assuntos
Implantação do Embrião , Transferência Embrionária , Humanos , Estudos Prospectivos , Transferência Embrionária/métodos , Embrião de Mamíferos , Blastocisto/metabolismo , Técnicas de Cultura Embrionária/métodos , Estudos Retrospectivos
20.
Reprod Fertil Dev ; 34(17): 1059-1077, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36219878

RESUMO

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.


Assuntos
Reserva Ovariana , Masculino , Camundongos , Feminino , Animais , Antituberculosos/farmacologia , Sêmen , Oócitos , Superovulação
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