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/fisiologiaRESUMO
Reopening fertility care services across the world in the midst of a pandemic brings with it numerous concerns that need immediate addressing, such as the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the male and female reproductive cells and the plausible risk of cross-contamination and transmission. Due to the novelty of the disease the literature contains few reports confirming an association of SARS-CoV-2 with reproductive tissues, gametes and embryos. Cryobanking, an essential service in fertility preservation, carries the risk of cross-contamination through cryogenic medium and thus calls for risk-mitigation strategies. This review aims to address the available literature on the presence of SARS-CoV-2 on tissues, gametes and embryos, with special reference to the possible sources of cross-contamination through liquid nitrogen. Strategies for risk mitigation have been extrapolated from reports dealing with other viruses to the current global crisis, for safety in fertility treatment services in general, and specifically for oncofertility.
Assuntos
COVID-19/epidemiologia , Criopreservação , Contaminação de Equipamentos/prevenção & controle , Preservação da Fertilidade , Células Germinativas , Pandemias , Criopreservação/normas , Feminino , Preservação da Fertilidade/métodos , Preservação da Fertilidade/normas , Humanos , Controle de Infecções/métodos , Controle de Infecções/organização & administração , Controle de Infecções/normas , Masculino , SARS-CoV-2/fisiologiaRESUMO
This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes' abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.
Assuntos
Criopreservação/métodos , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Vitrificação , Animais , Feminino , Preservação da Fertilidade , CamundongosRESUMO
Paternal genetic alterations may affect embryo viability and reproductive outcomes. Currently it is unknown whether embryo metabolism is affected by sperm-mediated abnormalities. Hence, using a mouse model, this study investigated the response to paternally transmitted DNA lesions on genetic integrity and metabolism in preimplantation embryos. Spent embryo culture media were analysed for metabolites by nuclear magnetic resonance spectroscopy and embryonic genetic integrity was determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay on embryonic Day 4.5 (E4.5). Metabolic signatures were compared between normally derived embryos (control) and embryos derived from spermatozoa carrying induced DNA lesions (SDL). SDL embryos showed a significant reduction in blastocyst formation on E3.5 and E4.5 (P<0.0001) and had an approximately 2-fold increase in TUNEL-positive cells (P<0.01). A cohort of SDL embryos showing delayed development on E4.5 had increased uptake of pyruvate (P<0.05) and released significantly less alanine (P<0.05) to the medium compared with the corresponding control embryos. On the other hand, normally developed SDL embryos had a reduced (P<0.001) pyruvate-to-alanine ratio compared with normally developed embryos from the control group. Hence, the difference in the metabolic behaviour of SDL embryos may be attributed to paternally transmitted DNA lesions in SDL embryos.
Assuntos
Apoptose/efeitos dos fármacos , Blastocisto/metabolismo , Dano ao DNA/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Blastocisto/efeitos dos fármacos , Cisplatino/farmacologia , Meios de Cultura , Fragmentação do DNA/efeitos dos fármacos , Técnicas de Cultura Embrionária , Masculino , CamundongosRESUMO
Female sperm storage is an intriguing adaptation exhibited by a wide array of both vertebrates and invertebrates. The mechanisms underlying female sperm storage have remained elusive. Using the Indian garden lizard Calotes versicolor as a model organism, we investigated the role of low and high molecular weight factors in this phenomenon. Previously, we demonstrated three distinct phases of the reproductive cycle in this animal with live, motile spermatozoa recovered from the uterovaginal region during the reproductive phase. In the present study, we analysed the uterovaginal contents using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified an abundant protein band corresponding to ~55 kDa regardless of the phase of the reproductive cycle. Analysis of the purified protein by liquid chromatography-tandem mass spectrometry suggested a unique protein without any homology to the National Center for Biotechnology Information database. Exogenous addition of this protein to washed spermatozoa derived from the epididymis reversibly inhibited sperm motility in a concentration- and time-dependent manner, suggesting it plays a key role in sperm storage. These studies are likely to offer new avenues to unravel the secrets of female sperm storage seen across the animal taxa and may have novel applications not only in reproductive biology, but also in general cell storage and preserving endangered animal species.
Assuntos
Proteínas Motores Moleculares/metabolismo , Espermatozoides/citologia , Útero/fisiologia , Vagina/fisiologia , Animais , Feminino , Lagartos , Masculino , Reprodução/fisiologia , Motilidade dos Espermatozoides/fisiologiaRESUMO
The advantage of using laser for assisted hatching in routine assisted reproductive technology (ART) practice is debatable. Recently, it has been shown that laser-manipulated mouse embryos had compromised genetic integrity. However, the impact of laser-assisted hatching (LAH) on the epigenetic integrity of the preimplantation embryos is not elucidated so far. Since continuous thermal stress on embryos was found to lower mRNA levels of de novo (bovine) methyl transferases in embryos, we hypothesize that thermal energy induced during LAH may alter the epigenetic signature through abnormal de novo methyl transferases (Dnmts) levels. Thus, using mouse model, we made an attempt to look into the expression of Dnmt3a and Dnmt3b in laser-manipulated embryos and their effects on global methylation. This experimental prospective study used mouse embryos from varying developmental stages (2-cell, 6-8-cell, and blastocyst) which were subjected to LAH using a 1480-nm diode laser. Two pulses of 350 µs frequency were applied to breach the zona pellucida, and then, embryos were assessed for the expression of two de novo methyl transferases (Dnmt3a and Dnmt3b) and LINE-1 (long interspersed element-1) methylation when LAH embryos developed to blastocyst stage. Results from this study have shown that blastocysts subjected to LAH at two-cell stage had significantly lower mRNA transcripts of Dnmt3a (P < 0.01) and Dnmt3b (P < 0.05) whereas LAH at six- to eight-cell and blastocyst stages did not affect the mRNA level significantly. On the other hand, LINE-1 methylation did not change significantly between LAH and control group in all the stages studied. These results suggest that two-cell-stage laser manipulation of embryos changes the mRNA level of Dnmts without affecting the global DNA methylation.
Assuntos
Blastocisto/metabolismo , Blastocisto/efeitos da radiação , Epigênese Genética , Lasers Semicondutores , Animais , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Feminino , Masculino , Camundongos , Estudos Prospectivos , DNA Metiltransferase 3BRESUMO
Earlier reports have suggested that exposure to radiation at workplace may induce cytogenetic abnormalities. However, the association between plasma antioxidants and the cytogenetic abnormalities in these patients has not been elucidated till now. Hence, the present study was undertaken to determine the relationship between the cytogenetic abnormalities, plasma antioxidant system, and the radiation exposure levels in men who were occupationally exposed to ionizing radiation. The study included 134 male volunteers, among whom 83 were occupationally exposed to ionizing radiation. Incidence of micronuclei and chromosomal aberration was assessed in lymphocytes. Total and reduced glutathione (GSH), total antioxidant capacity (TAC), superoxide dismutase (SOD), and lipid peroxidation were assessed in the plasma. The micronuclei frequency and chromosomal aberrations were significantly higher in the exposed group in comparison to the nonexposed group (P < 0.01-0.0001). Similarly, GSH, TAC, and SOD in the blood plasma were significantly higher in the exposed group than the nonexposed group (P < 0.01-0.0001). However, the level of malondialdehyde, which is an indicator of lipid peroxidation, did not differ significantly between both the groups. Importantly, radiation absorbed dose exhibited a positive correlation with the incidence of micronuclei in blood lymphocytes but not with chromosomal aberrations. This study shows that the susceptibility of peripheral blood lymphocytes to chromosomal damage is associated with plasma antioxidant levels. Furthermore, increased levels of blood plasma GSH, TAC, and SOD in occupationally exposed individuals could be an adaptive measure in response to oxidative stress to protect somatic cell genetic integrity.
Assuntos
Aberrações Cromossômicas/induzido quimicamente , Linfócitos/efeitos da radiação , Exposição Ocupacional/efeitos adversos , Lesões por Radiação/etiologia , Radiação Ionizante , Adulto , Glutationa/sangue , Pessoal de Saúde , Humanos , Peroxidação de Lipídeos/efeitos da radiação , Linfócitos/metabolismo , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Doses de Radiação , Superóxido Dismutase/sangue , Adulto JovemRESUMO
It has been shown that oocytes isolated from ovarian tissue cryopreservation acquire DNA damage during the process of freeze-thawing. Using a mouse model, here we have investigated the functional competence and phosphorylation of H2AX (γ-H2AX) in germinal vesicle (GV) and parthenogenetically activated oocytes derived from conventional ovarian tissue slow freezing and vitrification techniques. The number of GV-stage oocytes with γ-H2AX foci was not significantly different between the slow-freezing and vitrification groups. Although the in vitro maturation (IVM) potential of GV oocytes in the slow-freezing group showed a significant delay (P<0.0001) in the process of germinal vesicle breakdown, no difference in the maturation rate was observed between the two protocols. Nevertheless, parthenogenetic activation of IVM oocytes using strontium chloride showed a significantly lower activation rate in the slow-freezing group compared with the vitrification (P<0.05) and control (P<0.01) groups. Importantly, H2AX phosphorylation was significantly perturbed in the slow-freezing group in comparison to the control (P<0.05). Therefore, we conclude that impaired sensing of DNA strand breaks and repair processes are associated with the reduced functional competence of the oocytes recovered from the slow-freezing group, which may have a significant impact on the reproductive outcome.
Assuntos
Criopreservação/métodos , Histonas/metabolismo , Oócitos/metabolismo , Vitrificação , Animais , Feminino , Camundongos , FosforilaçãoRESUMO
This study aims to investigate the influence of two- (day 2) and six-to-eight-cell-stage (day 3) laser-assisted hatchings on the developmental potential and genetic integrity of the embryos. In this prospective experimental study, two- and six-to-eight-cell-stage mouse embryos were subjected to laser hatching using 1,480 nm diode laser, and then assessed for the developmental potential and DNA integrity in blastocysts. Similarly, four-cell-stage human embryos from 20 patients were also subjected to laser hatching, and then assessed for the developmental competence. Laser-assisted hatching in mouse embryos significantly enhanced the blastocyst hatching potential on day 4.5 (P < 0.0001). However, a significant decline in blastocyst total cell number (TCN) was observed in six-to-eight-cell-stage laser-hatched embryos (P < 0.001). Conversely, no significant difference in TCN was observed between laser-hatched and unhatched human four-cell-stage embryos after 24 h. Attempt to understand the genetic integrity in laser-hatched mouse blastocysts revealed significantly higher labeling index when hatching was done at two- (P < 0.01) and six-to-eight-cell stage (P < 0.05). DNA damage induced by the laser manipulation may affect implantation and postimplantation developmental potential of the embryos. However, further studies are required to elucidate the impact of laser-induced DNA damage on the reproductive outcome.
Assuntos
Blastocisto/efeitos da radiação , Técnicas de Cultura Embrionária , Idoso , Animais , Blastocisto/fisiologia , Contagem de Células , Dano ao DNA , Desenvolvimento Embrionário/efeitos da radiação , Humanos , Lasers Semicondutores , Lasers de Estado Sólido , CamundongosRESUMO
There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers.
Assuntos
Antioxidantes/metabolismo , Cromatina/efeitos da radiação , Sêmen/efeitos da radiação , Espermatozoides/efeitos da radiação , Adulto , Glutationa/metabolismo , Pessoal de Saúde , Humanos , Peroxidação de Lipídeos , Masculino , Radiação Ionizante , Estudos Retrospectivos , Sêmen/enzimologia , Superóxido Dismutase/metabolismoRESUMO
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 CélulasRESUMO
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/fisiologiaRESUMO
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/metabolismoRESUMO
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 Év 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 , MasculinoRESUMO
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/terapiaRESUMO
There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5) ) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential.
Assuntos
Alanina/metabolismo , Blastocisto/metabolismo , Transferência Embrionária/métodos , Espectroscopia de Ressonância Magnética/métodos , Diagnóstico Pré-Implantação/métodos , Ácido Pirúvico/metabolismo , Biomarcadores/metabolismo , Implantação do Embrião/fisiologia , Fertilização in vitro/métodos , HumanosRESUMO
PURPOSE: To investigate the influence of sperm DNA integrity on the zona binding ability of mouse spermatozoa in relation to their sex chromosomal constitution. METHOD(S): In this prospective experimental study, the sperm DNA fragmentation was induced by exposing testicular area of Swiss Albino mice (Mus musculus) to different doses of γ-radiation (0, 2.5, 5.0 and 10.0 Gy). Sperm DNA fragmentation was quantified by single cell gel electrophoresis (comet assay). In vitro sperm zona binding assay was performed and the numbers of zona bound X and Y bearing spermatozoa were determined using fluorescence in situ hybridization (FISH). RESULT(S): The assessment of zona pellucida bound X and Y-bearing spermatozoa using fluorescence in situ hybridization has revealed a unique binding pattern. The number of zona bound Y-spermatozoa declined significantly (P < 0.01 to 0.0001) with increase in the DNA damage. The skewed binding pattern of X and Y-bearing sperm was strongly correlated with the extent of sperm DNA damage. CONCLUSION(S): The zona pellucida may have a role in preventing DNA damaged mouse sperm binding especially towards Y-bearing sperm. However, the exact mechanism behind this observation needs to be elucidated further.
Assuntos
Fragmentação do DNA , Interações Espermatozoide-Óvulo/genética , Espermatozoides/fisiologia , Cromossomo Y/genética , Animais , Núcleo Celular , Dano ao DNA/genética , Hibridização in Situ Fluorescente , Masculino , Camundongos , Interações Espermatozoide-Óvulo/fisiologia , Cromossomo X/genética , Zona Pelúcida/fisiologiaRESUMO
Vitamin D is an essential micronutrient for bone health and the general cellular functions of the body. Its insufficiency/deficiency leads to the pathophysiology of disorders like diabetes, cancer, autoimmune, neurodegenerative, and cardiovascular diseases. Clinical interest in Vitamin D metabolites and their role in various medical disorders have contributed to an increase in laboratory demands for vitamin D measurements. For clinical and research laboratories worldwide, analysis of vitamin D and associated metabolites is a significant problem. The best way for determining vitamin D levels is constantly being debated. Various methods such as immunoassays and chromatographic techniques are available for determining vitamin D levels. Additionally, biosensors have recently been considered promising options for routine vitamin D analysis. The existing methods and other developments in the measurement of vitamin D metabolites and associated analytical challenges are discussed in this review.
Assuntos
Doenças Cardiovasculares , Deficiência de Vitamina D , Humanos , Vitamina D/análise , Vitamina D/metabolismo , Vitaminas , Deficiência de Vitamina D/diagnóstico , Doenças Cardiovasculares/diagnóstico , Osso e Ossos/química , Osso e Ossos/metabolismoRESUMO
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 , ÍndiaRESUMO
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.