-
Journal of Assisted Reproduction and... Dec 2016The goal of this study was to evaluate the ultrastructure of cytoplasmic fragments along with the effect of cytoplasmic fragment and perivitelline space coarse...
PURPOSE
The goal of this study was to evaluate the ultrastructure of cytoplasmic fragments along with the effect of cytoplasmic fragment and perivitelline space coarse granulation removal (cosmetic microsurgery) from embryos before embryo transfer on ART outcomes.
METHODS
One hundred and fifty intracytoplasmic sperm injection cycles with male factor infertility were included in this prospective study. Patients were divided into three groups of case (n = 50), sham (n = 50), and control (n = 50). Embryos with 10-50 % fragmentation were included in this study. Cosmetic microsurgery and zona assisted hatching were only performed in case and sham groups respectively. Extracted fragments were evaluated ultrastructurally by transmission electron microscopy (TEM). Rates of clinical pregnancy, live birth, miscarriage, multiple pregnancies, and congenital anomaly in the three groups were also compared.
RESULTS
Micrographs from TEM showed that mitochondria were the most abundant structures found in the fragments along with mitochondria-vesicle complexes, Golgi apparatus, primary lysosomes, and vacuoles. There were no significant differences in demographic characteristics, laboratory and clinical data, or embryo morphological features between the groups. The rate of clinical pregnancy in control, sham, and case groups had no significant differences (24, 18, and 18 %, respectively). The rates of live birth, miscarriage, multiple pregnancy, and congenital anomaly were also similar between the different groups.
CONCLUSIONS
Our data demonstrated that cosmetic microsurgery on preimplantation embryos had no beneficial effect on ART outcomes in unselected groups of patients. As mitochondria are the most abundant organelles found in cytoplasmic fragments, fragment removal should be performed with more caution in embryos with moderate fragmentation.
Topics: Abortion, Spontaneous; Adult; Blastocyst; Cleavage Stage, Ovum; Embryo Transfer; Female; Fertilization in Vitro; Humans; Lysosomes; Male; Microscopy, Electron, Transmission; Mitochondria; Pregnancy; Pregnancy Outcome; Reproductive Techniques, Assisted; Sperm Injections, Intracytoplasmic
PubMed: 27614632
DOI: 10.1007/s10815-016-0806-1 -
Clinical and Experimental Reproductive... Dec 2015To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection...
OBJECTIVE
To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection (ICSI).
METHODS
The fertilization rate (FR) and embryo quality were compared among 58 dysmorphic and 42 normal form oocytes (control 1) obtained from 35 consecutive ICSI cycles, each of which yielded at least one dysmorphic mature oocyte, performed over a period of 5 years. The FR and embryo quality of 441 normal form oocytes from another 119 ICSI cycles that did not involve dysmorphic oocytes served as control 2. Dysmorphic oocytes were classified as having a dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body (PB).
RESULTS
The overall FR was significantly lower in dysmorphic oocytes than in normal form oocytes in both the control 1 and control 2 groups. However, embryo quality in the dysmorphic oocyte group and the normal form oocyte groups at day 3 was similar. The FR and embryo quality were similar in the oocyte groups with a single abnormality and multiple abnormalities. Specific abnormalities related with a higher percentage of top-quality embryos were dark cytoplasm (66.7%), abnormal PB (50%), and cytoplasmic vacuoles (25%).
CONCLUSION
The fertilization potential of dysmorphic oocytes in our study was lower, but their subsequent embryonic development and embryo quality was relatively good. We were able to define several specific abnormalities related with good or poor embryo quality.
PubMed: 26815385
DOI: 10.5653/cerm.2015.42.4.156 -
Transgenic Research Aug 2017Swine are the only livestock species that produce both the second mammalian isoform of gonadotropin-releasing hormone (GNRH2) and its receptor (GNRHR2). Previously, we...
Swine are the only livestock species that produce both the second mammalian isoform of gonadotropin-releasing hormone (GNRH2) and its receptor (GNRHR2). Previously, we reported that GNRH2 and GNRHR2 mediate LH-independent testosterone secretion from porcine testes. To further explore this ligand-receptor complex, a pig model with reduced GNRHR2 expression was developed. Small hairpin RNA sequences targeting porcine GNRHR2 were subcloned into a lentiviral-based vector, lentiviral particles were generated and microinjected into the perivitelline space of zygotes, and embryos were transferred into a recipient. One GNRHR2 knockdown (KD) female was born that subsequently produced 80 piglets from 6 litters with 46 hemizygous progeny (57% transgenic). Hemizygous GNRHR2 KD (n = 10) and littermate control (n = 7) males were monitored at 40, 100, 150, 190, 225 and 300 days of age; body weight and testis size were measured and serum was isolated and assayed for testosterone and luteinizing hormone (LH) concentrations. Body weight of GNRHR2 KD boars was not different from littermate controls (P = 0.14), but testes were smaller (P < 0.05; 331.8 vs. 374.8 cm, respectively). Testosterone concentrations tended (P = 0.06) to be reduced in GNRHR2 KD (1.6 ng/ml) compared to littermate control (4.2 ng/ml) males, but LH levels were similar (P = 0.47). The abundance of GNRHR2 mRNA was reduced (P < 0.001) by 69% in testicular tissue from mature GNRHR2 KD (n = 5) versus littermate control (n = 4) animals. These swine represent the first genetically-engineered model to elucidate the function of GNRH2 and its receptor in mammals.
Topics: Animals; Animals, Genetically Modified; Female; Gene Expression Regulation; Gene Knockdown Techniques; Gonadotropin-Releasing Hormone; Hemizygote; Luteinizing Hormone; Male; RNA, Small Interfering; Receptors, LHRH; Swine; Testis; Testosterone
PubMed: 28534229
DOI: 10.1007/s11248-017-0023-4 -
Molecular Biomedicine Sep 2022Sperm-oocyte membrane fusion is necessary for mammalian fertilization. The factors that determine the fusion of sperm with oocytes are largely unknown. So far,...
Sperm-oocyte membrane fusion is necessary for mammalian fertilization. The factors that determine the fusion of sperm with oocytes are largely unknown. So far, spermatozoon factor IZUMO1 and the IZUMO1 counter-receptor JUNO on the oocyte membrane has been identified as a protein requiring fusion. Some sperm membrane proteins such as FIMP, SPACA6 and TEME95, have been proved not to directly regulate fusion, but their knockout will affect the fusion process of sperm and oocytes. Here, we identified a novel gene C11orf94 encoding a testicular-specific small transmembrane protein that emerges in vertebrates likely acquired via horizontal gene transfer from bacteria and plays an indispensable role in sperm-oocyte binding. We demonstrated that the deletion of C11orf94 dramatically decreased male fertility in mice. Sperm from C11orf94-deficient mice could pass through the zona pellucida, but failed to bind to the oocyte membrane, thus accumulating in the perivitelline space. In consistence, when the sperm of C11orf94-deficient mice were microinjected into the oocyte cytoplasm, fertilized oocytes were obtained and developed normally to blastocysts. Proteomics analysis revealed that C11orf94 influenced the expression of multiple gene products known to be indispensable for sperm-oocyte binding and fusion, including IZUMO1, EQTN and CRISP1. Thus, our study indicated that C11ORF94 is a vertebrate- and testis-specific small transmembrane protein that plays a critical role in sperm binding to the oolemma.
PubMed: 36050562
DOI: 10.1186/s43556-022-00092-1 -
International Journal of Reproductive... Dec 2020To increase the results of infertility treatment, many efforts have been made to improve the treatment methods. As assisted reproductive technology is mainly using cell...
BACKGROUND
To increase the results of infertility treatment, many efforts have been made to improve the treatment methods. As assisted reproductive technology is mainly using cell culture methods, one of the approaches to improve this technology is conditioned medium from different sources. It is desirable to apply maturation (IVM) and use oocytes from normal cycles instead of stimulating ovulation.
OBJECTIVE
To investigate the effect of human cumulus cell condition medium (hCCCM) on the IVM of immature mouse oocytes and morphology.
MATERIALS AND METHODS
In this experimental study, 240 germinal vesile oocytes were collected from four-six wk-old mice after 48 hr of 5IU pregnant mare serum gonadotropin (PMSG) injection and cultured in hCCCM (test group, n = 120) and DMEM + 20% FBS (control group, n = 120). The IVM rates and changes in perivitelline space (PVS) and shape were investigated at 8, 16, and 24 hr following the culture. The mature (MII) oocytes were subjected to in vitro fertilization (IVF) and the fertilization rate was assessed in three days.
RESULTS
A significant difference was observed between the maturation rates in the hCCCM and control groups (24.16% vs 0%; p = 0.001), as well as morphologic changes between the two groups (p = 0.04, p = 0.05). The development rate for MII oocytes attained from IVM in the hCCCM group was 27.58% (2-cell) and 6.89% (4-cell). Data displayed that hCCCM is an effective medium for oocytes maturation compared to the control medium.
CONCLUSION
hCCCM supports oocyte growth and maturation. Moreover, hCCCM changes the oocyte shape and size of perivitelline space.
PubMed: 33426412
DOI: 10.18502/ijrm.v18i12.8023 -
Brazilian Journal of Biology = Revista... Feb 2016The objective of this study was to describe the embryonic and larval development of Brycon amazonicus, featuring the main events up to 50 hours after fertilization (AF)....
The objective of this study was to describe the embryonic and larval development of Brycon amazonicus, featuring the main events up to 50 hours after fertilization (AF). The material was provided by the Aquaculture Training, Technology and Production Center, Presidente Figueiredo (AM). The characterization was based on stereomicroscopic examination of the morphology of eggs, embryos and larvae and comparison with the literature. Matrinxã eggs are free, transparent, and spherical, with a perivitelline space of 0.56 ± 0.3 mm. The successive divisions give rise to cells with 64 blastomeres during the first hour AF. The gastrula stage, beginning 02 h 40 min AF, was characterized by progressive regression cells and the formation of the embryonic axis, leading to differentiation of the head and tail 05 h 30 min AF. From 06 to 09 h AF the somites, notochord, otic and optic vesicles and otoliths were observed, in addition to heart rate and the release of the tail. The larvae hatched at 10 h 30 min AF (29.9 °C), with a total length of 3.56 ± 0.46 mm. Between 19 and 30 h AF, we observed 1) pigmentation and gut formation, 2) branchial arches, 3) pectoral fins, 4) a mouth opening and 5) teeth. Cannibalism was initiated earlier (34 h AF) which was associated with rapid yolk absorption (more than 90% until 50 h AF), signaling the need for an exogenous nutritional source. The environmental conditions (especially temperature) influenced the time course of some events throughout the embryonic and larval development, suggesting the need for further studies on this subject.
Topics: Animals; Characidae; Embryo, Nonmammalian; Embryonic Development
PubMed: 26909629
DOI: 10.1590/1519-6984.13914 -
Oncotarget Nov 2016Chitosan nanoparticles (CSNPs) are used as drug or gene delivery vehicles. However, a detailed understanding of the effects of CSNPs on embryonic development remains...
Chitosan nanoparticles (CSNPs) are used as drug or gene delivery vehicles. However, a detailed understanding of the effects of CSNPs on embryonic development remains obscure. Here, we show that CSNPs can be internalized into mouse blastocysts, such as the zona pellucida, the perivitelline space, and the cytoplasm. Consequently, CSNPs-induced endoplasmic reticulum (ER) stress increases both of Bip/Grp78, Chop, Atf4, Perk, and Ire1a mRNAs expression levels, and reactive oxygen species. Moreover, CSNPs show double- and multi-membraned autophagic vesicles, and lead to cell death of blastocoels. Conversely, treatment with rapamycin, which plays an important role as a central regulator of cellular proliferation and stress responses, decreased CSNPs-induced mitochondrial Ca+2 overloading, apoptosis, oxidative stress, ER stress, and autophagy. In vivo studies demonstrated that CSNPs injection has significant toxic effect on primordial and developing follicles. Notably, rapamycin rescued oxidative stress-induced embryonic defects via modulating gene expression of sirtuin and mammalian target of rapamycin. Interestingly, CSNPs treatment alters epigenetic reprogramming in mouse embryos. Overall, these observations suggest that rapamycin treatment could ameliorate CSNPs-induced developmental defects in preimplantation embryos. The data from this study would facilitate to understand the toxicity of these CSNPs, and enable the engineering of safer nanomaterials for therapeutic applications.
Topics: Animals; Autophagy; Blastocyst; Calcium; Chitosan; Embryonic Development; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Epigenesis, Genetic; Female; Humans; Male; Mice; Mitochondria; Nanoparticles; Ovarian Follicle; Reactive Oxygen Species; Signal Transduction; Sirolimus; Sirtuins; TOR Serine-Threonine Kinases; Unfolded Protein Response
PubMed: 27463007
DOI: 10.18632/oncotarget.10813 -
JBRA Assisted Reproduction Jul 2020Patients submitted to oncological fertility preservation with letrozole and gonadotropins seem to present a higher rate of immature oocytes and lower fertilization rates...
ABSTRACT
Patients submitted to oncological fertility preservation with letrozole and gonadotropins seem to present a higher rate of immature oocytes and lower fertilization rates in comparison to infertile patients submitted to IVF cycles with gonadotropins. The aim of this study was to evaluate the influence of letrozole on oocyte morphology in patients with breast cancer submitted to fertility preservation.
METHODS
Retrospective analysis performed at a public tertiary hospital in São Paulo, Brazil. The oocytes were retrieved from patients with breast cancer undergoing fertility preservation (n=69), and from infertile women undergoing in vitro fertilization (n=92). We evaluated 750 oocytes obtained from breast cancer patients submitted to ovarian stimulation with letrozole and gonadotropins, and 699 oocytes from patients without breast cancer submitted to ovarian stimulation for in vitro fertilization with gonadotropins only due to male factor infertility. The mature oocytes retrieved were analyzed for the presence of refractile bodies, ooplasm color and regularity, central granulation degree, cortical granules, zona pellucida staining and regularity, perivitelline space, presence of vacuoles or abnormal smooth-surfaced endoplasmic reticle and oocyte retraction.
RESULTS
There was a higher incidence of alterations in oocyte morphology in the letrozole group when compared to the control group: increased perivitelline space (p=0.007), irregular zona pellucida (p<0.001), refractile bodies (p<0.001), dark ooplasm (p<0.001), granular ooplasm (p<0.001), irregular ooplasm (p<0.001) and dense central granulation (p<0.001).
CONCLUSION
Letrozole is a risk factor for worse oocyte morphology. However, the clinical impact of ovarian stimulation protocol with combined use of gonadotropins and letrozole for fertility preservation remains unclear in this setting. These data underline the importance of establishing the predictive potential of morphological dimorphisms of human oocytes in IVF outcomes.
Topics: Adult; Antineoplastic Agents; Breast Neoplasms; Cell Shape; Cryopreservation; Female; Fertility Preservation; Humans; Infertility, Female; Letrozole; Oocyte Retrieval; Oocytes; Ovulation Induction; Retrospective Studies
PubMed: 32293820
DOI: 10.5935/1518-0557.20200002 -
Annals of Translational Medicine Nov 2022Long noncoding RNA (lncRNA) short nucleolar RNA host gene 15 () has been found to have an oncogenic function in numerous malignancies. Nevertheless, the biological...
BACKGROUND
Long noncoding RNA (lncRNA) short nucleolar RNA host gene 15 () has been found to have an oncogenic function in numerous malignancies. Nevertheless, the biological function and regulatory mechanisms of in breast cancer have not been fully elucidated.
METHODS
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of and in MDA-MB-231 breast cancer cells. The expression of was silenced using small interfering RNA (siRNA) technology. The proliferation and migration of the cells were examined by colony formation assays, cell counting kit 8 (CCK-8) assays, and transwell assays. For the zebrafish xenograft injection experiments, cultured cells labelled with the fluorescent dye CM-DiI were injected into the perivitelline space of the larvae.
RESULTS
This present study revealed that the expression of lncRNA () was significantly upregulated in breast cancer cells, and its overexpression was associated with the tumor. The relative expression of could be downregulated using siRNAs, and silencing inhibited the proliferation and the migration of MDA-MB-231 cells. experiments using the zebrafish xenograft model showed similar results. Mechanistically, the knockdown effect of could be restored by inhibiting the expression of the , confirming the negative regulation between and . Interestingly, cisplatin treatment combined with knockdown effectively inhibited MDA-MB-231 cell proliferation and migration in the zebrafish xenograft compared to negative controls.
CONCLUSIONS
In conclusion, knockdown increased expression and negated cisplatin resistance in breast cancer cells, and thus, may be a potential novel target for breast cancer therapy.
PubMed: 36467335
DOI: 10.21037/atm-22-5275 -
PloS One 2014To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage...
To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications.
Topics: Animals; Cumulus Cells; Female; Mice; Oocytes; Zona Pellucida
PubMed: 25144310
DOI: 10.1371/journal.pone.0105812