Science topic

Oocytes - Science topic

Female germ cells derived from OOGONIA and termed OOCYTES when they enter MEIOSIS. The primary oocytes begin meiosis but are arrested at the diplotene state until OVULATION at PUBERTY to give rise to haploid secondary oocytes or ova (OVUM).
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Hello everyone. I would like to discuss my ICSI procedure. I´ve been learning ICSI method for a year within my thesis, that focused on interspecific ICSI (specifically mouse oocytes and goat sperm). My problem is, that i can not reach satisfying numbers of viable injected oocytes. A lot of oocytes break and flow out during an injection. If they survive, some of them die later during cultivation in incubator. I hope you can understand, that I´m still a rookie, but I hope you can give me some good advices. The procedure is as follows:
  • PMSG stimulation
  • hCG stimulation (48 h after PMSG)
  • Isolation of oocytes (16-17h after hCG)
  • --- for isolation: M2 medium (10ml M2-HEPES + 10 mg BSA, stored in 4°C, filtered, heated to 36,8°C 1/2 h before use)
  • Removal of cumulus cells - in drops of hyaluronidase (2 mg HYA + 2 ml PBS, heated before use)
  • --- I have always been careful about this step for limited time (few seconds) that oocytes spend in drops. If cumulus cells have been removed, oocytes are washed in fresh drops of same M2 medium. Then oocytes are moved to drops of culture medium - usually MEM (40 mg BSA + 10 ml MEM + Na pyruvate gentamicin 100 μg).
  • Oocyte "rest" - in 4well in MEM medium in incubator with 5% CO2 . Recovering from isolation for 1/2 h.
  • ICSI - oocytes are moved from MEM to M2 medium. First in fresh drops outside, then M2 drops in manipulation dish (covered with mineral oil).
  • Sperm that is used for this procedure is frozen, so it is thawed and moved to M2 medium (centrifuged and washed from cyroprotectants)
  • Piezo system is used for this procedure. Injection pipette contains minimal amount of mercury. Pulses regulation depending on oocytes condition. Trying to use the bigger pulse for breaking through ZP and smaller pulse to get into cytoplasm. Than pull injection pipette slowly from oocyte.
Could you please tell me, if there is any critical thing in this procedure that could cause this problem. My master uses same protocol (except that he is a pro with many years of experience). I really understand that it is most likely my "hands" that make mistakes. So this is a little bit challenge for me, but I believe that you can understand that everyone had to start and learn.
Thank you all for your answers :)
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For your hyaluronidase step, do you use a supplemented PBS or just plain PBS? In human IVF we use mHTF (with HEPES) for that step on a warm (37degree surface) Osmolarity very important.
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Dear community,
I am interested in creating plasma membrane blebs from Xenopus oocyte by hypertonic treatment. Does any one have experience in doing so? The most important question would be to as do formed blebs harbour membrane expressed proteins?
Any help is greatly appreciated!
Thanks and kind regards
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Hello Yogesh,
Membrane blabbing is one of the morphological features characteristic of apoptosis commonly observed under in vitro culture conditions in various somatic cell types as well as in germ cells like oocytes under stressed or drug-induced conditions.
Hypertonic treatment to the Xenopus oocytes could induce a osmotic stress as the osmolarity of Xenopus oocytes is normally 240mOsmol. The hypertonic conditions will generate the hypertonic shock that may induce in the reduction of size due to oocyte shrinkage involving exocytosis and the other related to an ionic current. For detail see the link given below:
Release of ATP induced by hypertonic solutions in Xenopus oocytes. J Physiol. 2003 Feb 15; 547.
Membrane blabbing are seen in oocyte undergoing shrinkage during apoptosis in experimentally induced conditions and apoptosis under in vitro culture conditions.
Although we have not generated membrane blabbing in xenopus oocytes but we have observed membrane blabbing in rat oocytes undergoing apoptosis under in vitro cultured conditions.
The sequential development of membrane blabbing in rat oocytes cultured in vitro conditions can bee seen along with the detail protocol in our published paper link given below:
Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis 2005; Aug;10(4):863-74.doi: 10.1007/s10495-005-0367-8.
Hope this helps
Best
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I've been cryo-sectioning Xenopus oocytes and find these large internal branching structures in all of them (see attached image). I haven't been able to find a reference to it in the literature. I was tempted to say that it was ER, but I don't think it is. Does anyone have information on what this structure is?
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Hello Andrew Gipson,
In my opinion, it could be heterogeneous distribution of yolk platelets in the vegetal pole of Xenopus laevis oocytes. Two types of primordial yolk platelets (I and II) have been discriminated. After membrane fusion these precursors can be completely incorporated into the main body of existing platelets, numerous yolk crystals then merge and form one uniformly stratified core.
The yolk platelets ofXenopus laevis have been studied by thin-section and freeze-fracture electron microscopy to characterize the boundary membrane during yolk formation. Lipid droplets are tightly attached to the membrane at all developmental stages of yolk platelets. A direct connection of endoplasmic reticulum to the membranes of yolk platelets has not been observed. The proteinaceous yolk platelets tend to fracture along their periphery through the superficial layers.
For more detail see the following link:
Yolk organelles and their membranes during vitellogenesis ofXenopus oocytes. Roux's archives of developmental biology 198, 92–102 (1989).
Fluorescence lifetime imaging reveals heterogeneous functional distribution of eGFP expressed in Xenopus oocytes.
In this paper please see the figure 1B, similar to your attached image.
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I'm using JC-1 to measure the mitochondrial membrane potential by laser confocal microscopy. I used 100uM of FCCP as a positive control. Surprisingly, it caused lower green to red ratio and was significantly lower than the untreated cells. My cells are cryopreserved mice oocyte. On the other hand, FCCP did work and caused an elevation in calcium using Fluo 3 AM and caused plasmalemmal membranes depolarization.
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Dear Omar,
do you got the answer for this question? I've meet the same situation and tried to find the explanation.
Thanks.
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Studies related to ovarian tissue cryopreservation are relatively rare.
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As I have already replied that the ovarian tissue containing follicular oocytes are better tissue for cryopreservation. And clarified a difference between oocyte and ovum. Normally oocyte is a diploid and collected only from ovary. However after ovulation, ovum is a haploid female gamete and posses first polar body.
Few basic and important aspects of oocyte biology:
Oocyte is a large specialized germ cell that is arrested at diplotene stage of meiotic cell cycle within the follicular micro-environment. These oocytes are not yet matured in the ovary and subjecting them to cryopreservation process is risky. Due to its large size (about 100 micrometer in dia) there are several possibilities of its deterioration and oocytes may become fragile due to cryopreservation that could results in the loss of its quality.
Although ovum cryopreservation is done and IVF is conducted. In spite of having a significant advances in ART till date, the IVF rate is very poor due to several limiting factors. One of the major factor is the ovum factor. Normally in Fertility clinics, ovum is collected from stimulated ovary of infertile female. In the ovarian stimulation protocol, oocytes are pushed out due to supra physiological doses of gonadotropins (FSH & LH) or by ovulation inducing drugs like Clomiphene citrate and other similar drugs. These supra physiological doses of gonadotropins and ovulation inducing drugs like Clomiphene citrate and other similar drugs may have several adverse impact of the ovum quality as stated above by Dr. Elizabeth Rex citing ASRM quote and our animal studies published in ASRM Journal Fertility Sterility :
Chaube et al., Estradiol protects clomiphene citrate–induced apoptosis in ovarian follicular cells and ovulated cumulus–oocyte complexes. (Fertil Steril 2005;84(Suppl 2):1163–72. ©2005 by American Society for Reproductive Medicine.
Clomiphene citrate inhibits gonadotropin-induced ovulation by reducing cyclic adenosine 3=,5=-cyclic monophosphate and prostaglandin E2 levels in rat ovary. Fertil Steril 2006;86(Suppl 3):1106 –11. ©2006 by American Society for Reproductive Medicine
Melatonin protects against clomiphene citrate-induced generation of hydrogen
peroxide and morphological apoptotic changes in rat eggs. European Journal of Pharmacology 667 (2011) 419–424.
There is a big difference in naturally matured ovum collected after normal cycle of ovulation and ovum collected from stimulated ovary in terms of its competency. Hence to retain oocyte quality as good as naturally ovulated one, its important to take a piece of ovary containing follicular oocyte and cryopreserved. These tissues can be thawed and oocytes are collected and cultured in maturation medium so that the fist polar body is extruded (one of the most important oocyte function) in IVF Labs/Centers. After observing the first polar body, these ovum could be used for various ART programs (including IVF).
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As we know that the sperms function test is widely used for the management of male factor issues worldwide. However, similar parallel test is not available for the management of egg factor issue in female. Do the number of oocytes available for the purpose or the unavailability of the potential biomarkers for oocyte quality and other factors limit the development of oocyte function test? Kindly give your expert opinion.
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Thanks Prof. Omer Ucar for recognizing the quality issues of the oocytes. The scientific community need to generate the potential biomarkers that could be useful for the development of "oocyte function test". The experts in the field (you, me and others) should come forward to address this issue in order to develop oocyte function test (using non-human mammalian species) for the determination of oocyte quality prior to their use in ARTs.
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Hello, can someone help me? I am just random undergrad interested in oocytes and early embryos but I know almost nothing about their in vitro cultivation. What is the current state of the art of this topics? Where can I find what are the ingredients and what do they affect? Could you recommended me some reviews, articles, brochures, websites, anything about this topics?
Thank you very much!
Maria
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Hope you are interested in culture of mammalian oocytes/ovum in vitro. If yes, let me tell you that there are several culture medium available for oocyte in vitro for various purposes. For oocyte maturation, you can use M-199 (Sigma, HiMedia and several other companies), for fertilization (M-16, M--2 etc.) and embryo culture (several). All companies provides the ingredients of the culture medium and these media are provided both in powder form (to be reconstituted) as well as in liquide (ready to use) form with several options of buffering and alterations for in vitro culture such as Ca+2/Mg+ supplemented/free, etc. Actually you have to decide based on the source, stage and need of experiments and accordingly use the culture medium. Good luck
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Well, we read a lot about how in vitro manipulation of oocytes can induce stress and therefore reduce their potential or as people call it "quality". I've been looking into the studies mostly, they focus on the oxidative stress and reactive oxygen species relation with calcium regulation. Other studies investigated the heating as a stress factor. Well, how they established that this oocyte is healthy and this one is stressed? On what basis? What kind of methods they used? Are the polscope data useful?
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In vitro culture conditions never meets the body environment (in vivo conditions). Therefore, once the oocytes are retrieved for IVF purpose, they are always exposed to stress conditions (due to minor changes in physical, temperature and culture medium). Oocyte is more susceptible to the stress conditions due to larger surface area (100-120 microM in dia) and even minor stress conditions may initiate downstream signaling pathways to induce oocyte death under in vitro culture conditions. We have identified some morphological features that could be used to identify the stressed oocytes in vitro. The series of morphological changes that are identified in oocytes cutlured in vitro are:
1. The smoothness (clear) of oocyte cytoplam is reduced
2. Initiation of cytoplasmic granulation
3. Changes in Zona pelucida histoarchitecture
4. Polar body roughness (fragmentation)
5. Cytoplasmic dia shrinkage (more gap between zona and corona)
6. Membrane blabbings
7. Cytoplasmic fragmentation
and many more........
For more details, kindly see few of our published papers:
1. Apoptosis, 10: 863-875. IF=4.677
2. Fertility Sterility; 84; 1163-1172. IF= 7.329
3. Free Radical Research, 42:212-220 IF=4.148
4. Free Radical Research, 43, 287-294. IF=4.148
5. Eur J Pharmacol. 667; 419-424. IF =4.432
6. J Biomed Sci. 23;36,1-5. IF = 8.410
7. J Biomed Sci 25(1);36 (1-7). IF= 8.410
8. J Biomedical Science, 26;11 (1-6). IF=8.410
9. Stem Cell Reviews and Reports, 17(3): 777-784. IF=5.739
Good luck
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Our long standing understanding for mammalian ovary is that it has fixed number of germ cells and duty of the ovary is to ovulate high quality ovum for successful fertilization and early embryonic development. However, growing studies suggest that the oocytes can be generated from Oocyte like stem cell/neo-oogenesis/adult oogenesis and fertilized in vitro. Therefore, it is important tom know whether oocytes generated from these sources are as good as the follicular oocytes collected from mammals under natural conditions. Kindly give your opinion on the genomics, proteomics and metabolomics aspects of ocytes generated from Oocyte like stem cell/neo-oogenesis/adult oogenesis.
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Thank you Dr. Ke-Hui Cui M.D., Ph.D. for your personal opinion on introducing a new concepts of Hereditics, Cytohetics and Epicytohetics and considering both egg yolk and egg white as hereditary material. We know that the cytoplasmic inheritance (specially through mitochondrial genome) play important role in determining the fate of the individual (in mammals). I would not comment on your new concept and feel that it is premature to say anything on these new thoughts.
Whatever the research experience I have got on oocyte biology in last 30 years, I believe that the mammalian oocyte is a specialized cells and takes very long time (few oocytes takes very long time from puberty to menopause almost 30 years) to complete (oocyte maturation process; both nuclear maturation as well as cytoplasmic maturation in order to ensure the fully competent oocytes required for successful fertilization, early embryonic development and fate of the individuals).
As you know the mammalian oocytes are microlecithal with little or no yolk. However, mature human sperm are highly compact with no cytoplasm in the head region. As you know that the animal cloning for the first time Prof. Sir Ian Wilmut cloned Dolly and now several mammalian species have successfully been cloned using somatic cell nuclear transfer (SCNT) method. Keeping all these into consideration, I wonder how much these new concepts fit into specially in mammalian oocytes and helpful in oocyte biology of mammals.
Good luck for your new journal "Hereditics" in future.
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Hello - I need to retrieve GV stage oocytes from mice ovaries. I do not know where to get the small tool that is used to mush the ovaries into small pieces. I do not know how to fabricate it either. Does anyone know if it is commercially available? If not, do you have any spares you are willing to part with? Thank you very much in advance.
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Are you trying to isolated the GV stage oocytes for in vitro cultre and analysis? or trying to isolate them and use for biochemical/histological analysis. There is different protocol for different targets. Kindly clarify your purpose.
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Recent evidences suggest that mitochondria play important roles in determining the quality and fate of the oocytes in several mammalian species. Changes in shape, number as well as the distribution pattern of mitochondria alter oocyte physiology. More scientific discussion and suggestions are needed in order to develop a new biomarker for for the improvement of assisted reproductive technology outcome.
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Please read MRT syndrome in Https://www.hereditics.net. MRT syndrome is a new kind of artificial cytoplasmic hereditary diseases, shortened as Cytohetic diseases.
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The maturation promoting factor (MPF) is a key regulator of cell cycle that has regulatory subunit (Cyclins) and catalytic subunit (Cdks). Dissociation and degradation of specific cyclin destabilizes MPF, while its association with specific Cdk activate MPF.
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Thanks Prof. Yavuz Öztürkler for your reply. Our findings also supports your views. But the role of Cdk1 activation due to phosphorylation/dephosphorylation on specific amino acids sequences are seems to be important (as reported in other mammalian species).
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I want to culture oocytes in vitro and analyze gene expression from primordial follicles and primary follicles in mice, but am unsure how to collect isolated oocytes and granulosa cells. So far I've only seen articles with protocols involving hyaluronidase and mechanical methods for denuding the oocyte. I want to isolate the  oocytes from granulosa cells in mice. Does anyone have any particular methods that they could recommend to me please
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Your question is bit confusing. From your question, it seems that you want to isolate and denude diplotene arrested immature oocytes from primordial/primary follicles of mouse ovary and to study the gene expression in denuded oocytes cultured in vitro.
If it is so, then remember that due to larger in size as compare to other cell types in ovary, immature oocytes are more susceptible for any minor changes under in vitro culture conditions. These minor changes such as temperature, culture medium, addition of hyaluronidase or trypsin will have negative impact on the oocyte in vitro. Addition of hormones like hCG etc. may also have influence on the gene expression analysis of oocytes due to presence of encircling somatic cells.
You have to be very careful, while isolating and denudation of immature oocytes in vitro in terms of addition of either enzyme or hormones (both exposure time and concentration).
I would suggest you to reduce the diameter of micro-glass pipette, and keeping all physical and biochemical factors into consideration, make repeated pipetting in order to denude the immature oocytes. Yes, it is difficult task but not impossible for a technically skilled scientist.
Hope, you would be able to do it and best of luck
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I am doing a mammalian oocytes maturation, followed by nuclear meiotic evaluation using confocal microscopy, I mount the oocyte over 90% glycerol in a double sided adhesive tape, and I am curious because I don't want to smash the oocytes under the cover slip. is there any recommendation for a good commercial wax cushion to be used in this case. (oocyte diameter is 100 micron).
Thank you,
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Yes. It is matter of concern and although alternative methods are available in order to prevent oocyte damage during handling in vitro. The alternative methods have some or other type of negative impact on the oocyte. The development of an improved method/materials that can replace glycerol and prevent damage under experimental condition is required. To the best of my knowledge no such appropriate wax cushion is in the market which can be better in handling oocytes in vitro or for the assessment of maturation process in mammalian oocytes for confocal microscope.
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I would like to ask you what is the best model to mimic human corpus luteum functions in vitro. I saw in literature that Granulosa Cells can be isolated from follicular liquid during oocyte withdraw for IVF. What would be the best protocol to differentiate these cells in granulosa-lutein cells? would it be reliable and scientifically accepted?
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Good morning, I understand your interest for the best knowledge for high technology to FIV. It should be necessary to teach at the woman (before her hormonal stimulation and intervention) the knowledge of the natural signs of fertility, as cervical mucus for maximum days of fertility and basal temperature records to diagnose corpus luteum (less than 10 days of high level is insufficient CL, 10 days are sufficient CL, 16 days are maximum natural CL, 20 days diagnose a pregnancy.) If you are interested in more information about sympto-thermal method you can consult CaMen in Milan. All teachers of STM have cases of conception after unsuccessful FIV, but not all people conceive. It should be better to learn those signs before high technology. Regards
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Hey,
I recently started my masters thesis and for that matter I will patch oocytes. I need to prepare some solutions for the procedure and I need to get Cs-glutamate. I cannot find anything on the internet about how to make it. It is also not possible to order it anywhere.
Does anyone know how to make or get Cs-Glutamate?
Best,
Mary
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Glutamic acid + Cs-hydroxide
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Xenopus laevis oocytes are used in electrophysiology to make transient expression of injected mRNA fragments. Through the technique of 2-electrodes voltage-clamp we can record the physiological phenomena that originate from the activity of the generated protein that has been embedded in the oocyte membrane. Depending on its activity we will observe hyperpolarization or repolarization of the membrane, in the presence of different external stimuli (different concentrations of anions, amino acids, organic acids, etc.).
In our case, electrophysiology recordings in Xenopus laevis were assayed. A plant's membrane transporter working in an animal cell membrane.
But, in young and old, well-fed, and apparently healthy frogs, sometimes the experiments could not be as brilliant as on other occasions. The oocytes were of poor quality, without showing that polarized two-color differentiation, and after injecting the RNA they exploded most of them. Surviving oocytes showed strange electrical behaviors, mostly related to passive endogenous chloride input channels and ended up lysing soon.
Have you had any similar experiences? If you have an explanation I would be very grateful. We are trying to "dust off" old jobs that were abandoned in a drawer.
I want to thank Dr. Omar Homero Pantoja from IBT-UNAM (Cuernavaca, Mexico) for teaching me during my stay in his electrophysiology laboratory.
#xenopus #oocytes #2EVC #electrophysiology #recordings
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Hi Franco,
I hope things have already worked on your side.
I went through the same issue from March to May. I tried to adjust all the parameters I could think of (bought new frogs, tried a couple of collagenase and concentrations, remove follicular membrane manually, different types of solution, glass vials/falcon tubes, temperature, time, autoclaved everything, etc) but nothing worked. But after we entered June, the problem just disappeared by itself by using our original protocol. I can only say that the "bad period/good period" thing is real. We sometimes just have to be patient.
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Hi guys! I would like to know if someone could help me to solve a problem the following problem: After the parthenogenetic activation of oocytes, zygotes are degenerating after 72h of activation. I made some changes in the SOF environment, but there was no positive result. Who can help me, I appreciate it!!
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Leonardo, you can verify basic things like osmolarity and pH, if the gas mixture was changed recently, any expired reagent. If you use oil on your culture, this might also be a problem (we had problems with different lots of oil). During the years I experienced all kinds of problems that resulted in the embryos dying, from CO2 problems, expired reagents, external contamination with cleaning substances... If you can provide more information about any possible changes that occurred in your system, that might help to identify the problem.
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Must all groups in chi-square have the same number of for example cells (or embryos etc.)? We analyzed 30, 28, 37 and 36 oocytes in experimental groups.
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The chi² test of independence and the null hypothesis is that there is no difference in the distribution of responses to the outcome across comparison groups. This is often stated as follows: The outcome variable and the grouping variable (e.g., the comparison treatments or comparison groups) are independent (hence the name of the test).
Independence here implies homogeneity in the distribution of the outcome among comparison groups. One of the limitations of chi² test is that all participants measured must be independent, meaning that an individual cannot fit in more than one category. If a participant can fit into two categories a chi-square analysis is not appropriate.
Another limitation with using chi² test is that the data must be frequency data. For example if you are just counting how many embryos show (or not) protein expression in two separate conditions, than a chi² test is appropriate. Also when calculating the number of expected individuals for each class, there should be greater than 5 for each class (and not null) for the most appropriate use of chi² test.
One last consideration is that the chi² test statistic is sensitive to sample size. Most recommend that chi² test not be used if the sample size is less than 50 (so your 30, 28, 37 and 36 oocytes in experimental groups seems too low). If you have a 2x2 table with fewer than 50 cases many recommend using Fisher’s exact test.
Hoping that these statistical informations will help you in your data analyses.
Ludovic Dumont, Ph.D.
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I would like to avoid transfering oocytes from one solution to another by micropipeting and I am wondering if I can attach them somehow on a slide and employ fixation and staining procedure on them for immunocytochemisty.
Thank you in advance.
I am looking forward for your suggestions if possible.
Regards,
Nikos
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I think you can do
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Hello everyone! I am relatively new to the zebrafish world, and I am hoping to ask some questions about their reproductive biology. After breeding my fish, I often find what appear to be "smushed" or hollow-looking "eggs". I am not sure what they are, but suspect they are perhaps some sort of underdeveloped oocyte or follicle-related tissue? I am pretty certain they are not scales as they have some thickness to them; the shapes/sizes are also inconsistent.
I've attached some photos. In the larger photo, I have circled the "smushed" egg (Red) as well as an unfertilized oocyte (Yellow) for comparison, next to fertilized oocytes (uncircled). There is a close up image also attached. If anyone can direct me to some resources to learn more that would be great as well.
Thanks!
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Hi Melanie,
That picture or the shed part you see in your image is called as Chorion. If you follow the time span of how the eggs develop over 3-4 days you will see a very thin membrane that covers your egg. These are basically protective membranes that shed off by themselves as they grow. However, due to technical handling or shaking or any such issues the eggs may loose these membrane and you will have tiny transprent embryos in the medium (watch for them closely if dont want to loose it). Simply, you can assume it to be basically a shell protecting the main embryo. Refer Zebrafish book and you will learn more about it. If you are a beginner I would suggest to read through the questionnaires provided at the end of the book too. It will be way too helpful for you for maintaining and harvesting your egg cultures from zebrafish.
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There are several studies that have shown that different types of stem cells (mesenchymal stem cells, embryonic, endometrial, and ovarian stem cells) can be differentiated into both spermatozoa and oocytes in vitro, proving their potential clinical use in management and cure of infertility issues. Can these trials be applicable?
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Open pulled straws which are made by manually pulling heated French mini straws usually get contaminated during processing and after that how they can be sterilized. I mean the method recommended without any deleterious effect on the quality of oocytes and embryos.
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It is very difficult to avoid the contamination in open pulled straws, however, can be minimised by low pressure long time autoclaving i.e. 5-7 psi for 20-25 minutes.
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Thank you so much! 
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How to estimate oocyte numbers in the mouse fetal (1dpp) ovary?
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In this case-control study, 60 patients with in vitro fertilization (IVF)/intracytoplas- mic sperm injection (ICSI) indication were subdivided into 3 groups as follow: 20 subjects were assigned to control (fertile women with male infertility history) group, 20 subjects with PCOS were insulin resistant (IR) and 20 subjects with PCOS were insulin sensitive (IS). After puncture, retrieved oocytes were classified into metaphase II (MII) as mature and in metaphase I (MI) or germinal vesicle stage (GV) as immature. Regres- sion analyses were used to explore the association between MII oocyte number and demographic and clinical variables. LH/FSH ratio was significantly higher in PCOS-IR women compared to controls but not significantly dif- ferent from that of PCOS-IS group. PCOS-IR women had lower MII oocyte number compared with that of controls. According to multiple regression analysis, the number of previous assisted reproductive technology (ART) cycles was negatively associated with the number of MII oocy
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From this possibility you can influence the amount of M2
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Has anyone been able to successfully cryopreserve coral oocytes? I've only found one case for a Gorgonian (Junceella juncea) through vitrification (Tsai et al. 2015, attached below). I know that in some cases some researchers (personal communications) have been able to thaw and retrieve from liquid nitrogen, but though they are still 'alive', they loose their ability to be fertilized (infertile).
I appreciate your help providing me with your answers and/or experience in this matter.
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Everything is very simple. They are prepared in the same way as mammalian eggs. Deep freezing takes place in liquid nitrogen. There is also an article, unfortunately in Russian.
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Does anyone have a protocol for simultaneous collection of VG and MII oocytes in the mouse (from the same animal)?
Thanks
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Its the best option. However, I'm looking for an alternative to collect both GV and MII oocytes at once because there are few animals available now. I'll give it a try and comment here later.
If it doesn't work, I'll collect GV and MII separately. Thanks Mukesh Kumar Gupta !
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I bought a stereo microscope with led lighting and it makes very difficult to see oocytes /embryos. What microscope may you suggest for this kind of research? Thanks.
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You need stereo microscope with diascopic light stand.
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I want to know that can we use the cryotop device for vitrification in oocytes once we have thawed the already vitrified oocytes using this device...Is it recommended or not.....
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Agree with both, we reuse them for research/training purposes only, with no problem. We wash with sterile MilliQ water followed by ethanol and then UV it for 30 min before use.
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Hi,
I would like to know the Xenopus Oocyte approximate membrane thickness? Suggest me please.
Best,
Rajesh
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After a long time, I got only one answer. But, I want membrane thickness, not a vitelline layer.
Thanks for your reply.
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Not sure about breeding strategy,
To speed up offspring production with the required genotype (I only need hetero and hetero share the same phenotype with the WT), is it ok to set up breeding pairs:
homo mutant (male) cross with WT (female)
and homo mutant (female) cross with WT (male),
which can give me the desired genotype 100 % than hetero crossing at a Modelian ratio, let us forget the controls from one littermate for the time being that must be used for the experiment.
I guess my question is how to know whether there is individual variance in the offspring because of oocyte/sperm coming from different genotype or not.
Thanks !
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Yes there will be variance sir
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I want to inject equine oocytes using the femtojet but in the manual there is no explanation for the volume of injection. Only for the pressure and time. Does anyone know how to use both to determine the volume?
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Xiujuan Tan Hi there, I asked this question during my master project, so I am not doing this experiment anymore. If you wish to get into contact with that lab let me know.
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COC retrieved from same patient. Age: 28 Year and AMH: 2.5 ng/ml
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The reason I said gdf9, because gdf9 and bmp15 comes from oocyte and control the coc complex morphology and function
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During the process of oocyte maturation, asymmetric divisions result in segregation of the maternal genome and highly asymmetric partition of the cytoplasm, which generates the small polar body and the large oocyte. Why does the polar body stop cell division even after parthenogenetic activation?
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Thanks Wasim Al Shebli for your feedback. You are right, ribosomes might play a role in this.
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Can I use fluconazole? If yes what is the concentration of it?
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I TRIED ONCE USING FLUCONAZOLE, BUT I COUNT GET RID OF YEAST ...
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Hi. I want to do patch clamp on mouse oocyte. I have used poly L-lysine and either matrigel but no attachment was observed. Anybody knows another solution?
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We never attach oocytes and I doubt that it may be useful. For patch-clamp you will anyway use excised (most probably, outside-out) patch.
We are using chambers with a bath for oocyte where seal is formed and patch excised, and near it a flow bath for measurement. So, after excision the pipette with the patch is transferred (slowly!) into this flow bath, and oocyte is not interesting for you anymore.
In 2-electrode clamp experiments oocytes are also not attached, just help by electrodes themselves.
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What is the value of in vitro maturation and embryonic development of oocytes?What is the end result we want?
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We want to extract RNA from small number of oocytes for RNA-seq. I am afraid that I lose RNA with column extractio. I am thinking about using Tirzol. Any idea?
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Hi!
I used Trizol and similar reagents many times to extract RNA from small samples (about 2 mg of tissue). Always cleaning at least twice with 75% ethanol before elution. After total RNA extraction, I usually purify the RNA with the RNEasy MinElute CleanUp (Qiagen) kit. I had some sample loss, but nothing to be afraid of and enough to make RNAseq. If you use this approach, clean the columns twice with the RPE buffer.
The other alternative would be to use the appropriate kit suggested by the colleagues above, or RNeasy Plus Micro Kit (Qiagen).
Best Regards!
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Dear Colleagues!
IS it possible to observe oocytes in the adult clam male gonad histological section? The adult clam males were treated for 28 days with high temperature or/and endocrine-disrupting compounds.
Thanks a lot in advance;
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I would like to purchase an antibody that works for ICC on oocytes. However, I have found the antibodies for WB only. So if they are possible to use for ICC, I want to perform with them. Those who know well on this, please provide me any references or reasons that I can use them. Thank you! Also, if the antibodies for WB are monoclonal, can that be possible for ICC?
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@Schmitt
thank you for your specific answer. I agree with your comment that it is neccassary to check whether the Ab works in ICC by KO cells or overexpressed cells, when it has never been tested. However, I think it would be better to find references as I have some limitations for gene modification.
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Can anybody kindly give details about pGEMHE vector map? It's origin of replication, MCS etc. It is supposedly a oocyte expression vector, I want to know whether it can be expressed in animal/mammalian cell lines?
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The original reference for the construct is: Liman ER, Tygat J, Hess P (1992) Subunit Stoichiometry of a Mammalian K+ Channel Determined by Construction of Multimeric cDNAs. Neuron. 9:861-871. For more detailed information on the sequence you can check maps of constructs derived from this vector, for example: http://www.euroscarf.de/plasmid_details.php?accno=P30652.
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Hi,
I am struggling to find a way to measure oocytes on histological sections with the nucleus while excluding those without the nucleus. Since the measures would involve the whole histological section (i.e. a few hundreds of oocytes to manually be measured) I am trying segmenting and/or thresholding the pictures on imageJ, but without success. The idea underlying this approach is to find a way to automatically detect the "objects" I am specifically interested in.
I've already tried ImageJ Trainable Weka Segmentation and MorphoLibJ.
Here a couple of pictures of ovaries DAPi-stained and what I'd roughly like to obtain. Red and yellow circles for oocytes with and without nucleus, respectively.
Thank you in advance.
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I can advise you to read "Correlation between germinal vesicle and oocyte development in the adult Japanese quail ( Coturnix coturnix japonica)
J. Embryol. exp. Morph. vol 29, 1, 145-157, 1973.
With kind regards, Marc Callebaut.
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We would like to compare the aneuploidy rates of oocytes and embryos.
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Many thanks!
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I am working with embryos and oocytes, i am confused about how many an which internal controls to use for qPCR?
in literature, i have seen only one internal control has been used and study is published in reputed journal. but i am not getting clear idea.
Please suggest.
Thank you
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Hello Riddhi Kirit Pandya,
First of all, what do you call "controls" for RT-qPCR? Housekeeping genes ?
Indeed, results (the relative expression level of each gene) need to be normalized by two housekeeping genes (and not usually only on one); as recommended by the MIQE guidelines (Bustin et al., 2009) with the use of geometric mean to generate an accurate normalization; available at http://www.rdml.org/miqe.php.
Then, be careful to evaluate RT-qPCR results visely (fold-change, relative expression, the 2^(-ΔΔCt), etc...). After computing your RT-qPCR slopes & curves data you obtain your 2^(-ΔΔCt) data. During this procedure, you have to normalise all your data on housekeeping genes (a couple) and on a "control experiment" (associated as the "1:1" value) to compare them as ratio-values: data are presented as relative expression of your genes. --Be careful to use geometric means at this step!--
If you want more more precise and detailed informations, please see Livak and Schmittgen (2001).
Dear community of RG, be free to add modifications & precisions to my Answer, especially on commonly used internal controls to study mice oocytes and embryos!
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Hello,
I'm looking for an effective way to clean some COOK® Flexipet capillaries (100 um of inner diameter).
We tried by flushing ethanol and dH2O using a insulin syringe, with little results. Is it possible to clean them better (maybe with stronger mixes such as RCA1 or Piranha) given that they are made of polycarbonate?
Thanks
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Try 0.5mg/ml pronase in PBS and leave your cappilaries for 20 mins, then rinse with distilled water for flushing a couple of times before using.
Works in bovine oocyte manipulation. Hope it works
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We have been trying to collect metaphase II oocytes from mice for some time. We tried a variety of ways for super ovulation. In each experiment we wash oviducts but we can hardly find any oocytes. I need your advice. Thank you in advance
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We published all techniques of mouse reproductive technology on our website (CARD, Kumamoto University). Please check the superovulation protocol via following link:
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My questions are about meiosis chromosome spreads.
My experiment is about a gene which can result in primary ovarian insufficiency perhaps through meiotic prophase I abnormality.
I have read some papers and protocols about this experimental technique. I can replicate the protocol but cannot recognize the 5 substages of meiosis prophase I (so as the first figure below).
And I also have to identify the wildtype and mutant oocyte chromosome patterns ,however, I’ve no idea about the abnormality or normal chromosome pattern, which is as indicated below (the second figure below). So I want to get help from you about the specific instructions on the identification of substages of meiosis prophase I and the normal patterns of the chromosome. Literature, books and personal experience will be deeply appreciated.
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The first figure in your question is from my article
As stated, the slides were stained with CREST serum (red), which stains centromeres, and anti-SYCP3 (green), which stains the axial/lateral elements of the synaptonemal complex. Staining the centromeres will help you identify the differences between the 5 substages of Prophase I.
In Leptonema, there are many centromeres and the chromosomes are very thin. In lack of better terms, it's a mess. In a zygotene cell, the chromosomes are more distinct. By the pachytene stage, there should be 20 chromosomes (for murines) and the centromeres are at the ends of each chromsome. In diplonema, you see the ends of the chromosomes appear like zippers or split ends. In diakinesis, the chromosomes are no longer present, hence little specks.
Hope this helps.
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Hi everyone,
Im in Australia and we haven’t been able to import Folligon in for a few months now and all the alternative don’t seem to work on our mice. Does anyone have any other drug suggestions?
We use C57.B6/CBA females at 3 weeks of age. We inject 48 hours apart with 5IU and collect 13-15 hours later to collect MII oocytes.
We havw tried pregnecol as an alternative but no luck.
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Thanks so much! I’ll give it a try. How much do you administer your mice?
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I am trying to culture mouse follicles in a 2D system but the oocytes extrude earlier before adding hormones (after 6 days culture). Can anybody give any advice? what should I do ?
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thank you very much for your advice
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We will be collecting ovaries and surrounding tissues from slaughtered animals for oocyte collection. One of the goals is also to measure the diameter of arteries to ovary by ultrasound in a water bath. Do we need to ligate the vessels and fill them with fluid in order to be able to see them? Does anyone have suggestions/ a protocol for this?
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Jiří Šichtař thank you for your helpful insight!
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I am going to study the response of cumulus cells after using a kind of media. Considering the diffusion may associated with the volume of the cell group, I need to measure the volume of human cumulus cells collected from immature COCs, after removing the oocyte. The cumulus cells are still connected together. Does anyone have any ideas?
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You have cumulus clump and oocyte has been removed. I will suggest rupture the cumulus in smaller clumps with fine needles (27Gz attached with a syringe), Place smaller clumps on hemacytometer, cover with slide and use microscope to measure diameter of randomly picked 50 cells from 10-15 different clumps. Use formula to calculate volume once you have radius length in hand. Volume of cells near the oocyte may be different from cells in periphery of follicle. Take an average. Good luck.
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I have nucleolus samples isolated from oocytes. If someone has an experience with total RNA isolation from such samples for NGS, please share or advise.
Thanks
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Hi Niloufar,
Hi Ahmed,
Thanks for your answers. Actually, miRNeasy-mini-kit could works good with tissues or high number of cells but it will not be enough for fine samples as 25 nucleolus/sample.
I found this protocol which could be useful :
Thanks again for your help.
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Is JC-1 reliable probe to assess mitochondrial membrane potential in oocyte?
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Dear Marayam ,
It is good to guide me and explain me how is it work
Regards,
Usama
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Could the transfer of embryos (or oocytes and zygotes) between different culture media induce alterations in their cytoskeletal structure leading to cell division failure?
Thanks in advance.
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Dear Gabor, the following paper may help you:
Mechanical Stresses in Embryonic Tissues: Patterns, Morphogenetic Role, and Involvement in Regulatory Feedback
L.V.Beloussov, S.V.SavelievI, .NaumidiV. V.Novoselov
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We all struggle to define and reach the best criteria to define and standardize sperm morphology, these can be seen from variations in quality assessments worlwide. Surprisingly, we don't worry to much about the quality of morphoogy of TESE sperm cells.
I would like to have expert opinions about this issue.
Thanks to all of you for joining the discussion and providing insights.
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Dear Hemraj,
The morphology assessment of sperm cells obtained from the testicular tissue by means of microTESE procedure is limited to motility, contour of sperm head, tail length and mid piece in relation to presence/absence of cytoplasmic droplet only! Most of the Embryologists including me follow the similar criteria for selection of testicular sperms prior to inject oocytes to anticipate healthy fertilisation rate.
This is so because of practicality of the TESE-ICSI treatment option in relation to availability of the motile and viable sperms as many a time we are having a very few number of sperms for performing ICSI which restrict us to apply additional laboratory test for evaluation of the testicular Sperm morphology. Overall Immaturity (chemica/ cytoplasmic/ Nuclear) of the Sperm is one of the important aspect for the normal growth of quality embryos and subsequent success of such TESE-ICSI-ET cycles. I hope, you might be convinced by the answer. However, latest publication also suggest no significant benefit of ICSI in non-male factor ART cycle given as follows:
📷📷Article Navigation
ICSI does not increase the cumulative live birth rate in non-male factor infertility
Z Li A Y Wang M Bowman K HammarbergC Farquhar L Johnson N Safi E A SullivanHuman Reproduction, dey118, https://doi.org/10.1093/humrep/dey118Published: 12 June 2018 Article history
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Abstract
STUDY QUESTION
What is the cumulative live birth rate following ICSI cycles compared with IVF cycles for couples with non-male factor infertility?
SUMMARY ANSWER
ICSI resulted in a similar cumulative live birth rate compared with IVF for couples with non-male factor infertility.
WHAT IS KNOWN ALREADY
The ICSI procedure was developed for couples with male factor infertility. There has been an increased use of ICSI regardless of the cause of infertility. Cycle-based statistics show that there is no difference in pregnancy rates between ICSI and IVF in couples with non-male factor infertility. However, evidence indicates that ICSI is associated with an increased risk of adverse perinatal outcomes.
STUDY DESIGN, SIZE, DURATION
A population-based cohort of 14 693 women, who had their first ever stimulated cycle with fertilization performed for at least one oocyte by either IVF or ICSI between July 2009 and June 2014 in Victoria, Australia was evaluated retrospectively. The pregnancy and birth outcomes following IVF or ICSI were recorded for the first oocyte retrieval (fresh stimulated cycle and associated thaw cycles) until 30 June 2016, or until a live birth was achieved, or until all embryos from the first oocyte retrieval had been used.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Demographic, treatment characteristics and resulting outcome data were obtained from the Victorian Assisted Reproductive Treatment Authority. Data items in the VARTA dataset were collected from all fertility clinics in Victoria. Women were grouped by whether they had undergone IVF or ICSI. The primary outcome was the cumulative live birth rate, which was defined as live deliveries (at least one live birth) per woman after the first oocyte retrieval. A discrete-time survival model was used to evaluate the cumulative live birth rate following IVF and ICSI. The adjustment was made for year of treatment in which fertilization occurred, the woman’s and male partner’s age at first stimulated cycle, parity and the number of oocytes retrieved in the first stimulated cycle.
MAIN RESULTS AND THE ROLE OF CHANCE
A total of 4993 women undergoing IVF and 8470 women undergoing ICSI had 7980 and 13 092 embryo transfers, resulting in 1848 and 3046 live deliveries, respectively. About one-fifth of the women (19.0% of the IVF group versus 17.9% of the ICSI group) had three or more cycles during the study period. For couples who achieved a live delivery, the median time from oocyte retrieval to live delivery was 8.9 months in both IVF (range: 4.2–66.5) and ICSI group (range: 4.5–71.3) (P = 0.474). Fertilization rate per oocyte retrieval was higher in the IVF than in the ICSI group (59.8 versus 56.2%, P < 0.001). The overall cumulative live birth rate was 37.0% for IVF and 36.0% for ICSI. The overall likelihood of a live birth for women undergoing ICSI was not significantly different to that for women undergoing IVF (adjusted hazard ratio (AHR): 0.99, 95% CI: 0.92–1.06). For couples with a known cause of infertility, non-male factor infertility (female factor only or unexplained infertility) was reported for 64.0% in the IVF group and 36.8% in the ICSI group (P < 0.001). Among couples with non-male factor infertility, ICSI resulted in a similar cumulative live birth rate compared with IVF (AHR: 0.96, 95% CI: 0.85–1.10).
LIMITATIONS, REASONS FOR CAUTION
Data were not available on clinic-specific protocols and processes for IVF and ICSI and the potential impact of these technique aspects on clinical outcomes. The reported causes of infertility were based on the treating clinician’s classification which may vary between clinicians.
WIDER IMPLICATIONS OF THE FINDINGS
This population-based study found ICSI resulted in a lower fertilization rate per oocyte retrieved and a similar cumulative live birth rate compared to conventional IVF. These data suggest that ICSI offers no advantage over conventional IVF in terms of live birth rate for couples with non-male factor infertility.
STUDY FUNDING/COMPETING INTEREST(S)
No specific funding was received to undertake this study. There is no conflict of interest, except that M.B. is a shareholder in Genea Ltd.
TRIAL REGISTRATION NUMBER
N/A.
assisted reproduction, ICSI, IVF, IVF/ICSI outcome, infertility, non-male factor infertility, male factor infertility, cumulative rates, live birthIssue Section: Reproductive epidemiology
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Our stock is 5nmol.
Or is it different for each miRNA?
Thank you for answers.
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If I want to perform the RRBS technique on buffalo oocytes, how many oocytes I have to collect to make a consistent pool to be analysed?
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You need to have enough DNA for comfortable PCRing. It will be hit and trial. You may try with mouse oocytes after superovulation which are easy to get and if you are interested as a trial run. Buffalo oocytes will be much more difficult and expensive to pool for trial run. DNA of mouse and buffalo oocyte should be similar in quantity.
My other worry is contamination with granulosa cells, need several washings of oocytes to get rid of other cells,
More opinion from other experts can not be ruled out,
Best,
Subhash
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I am doing oocyte expression study in an ion channel. Can anyone please provide me the vector map of pMJB08 and pGEMHE?
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You can get the map from the company U purchased the vector. Or may be ask the fellow who has given you.
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Is anyone using IMSI for oocyte fertlization on a routine basis and what is your experience as compared to conventional ICSI method?
I am interested in hearing feedbacks .
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Dear Hemraz,
Please have a look at this useful ResearchGate link and request the authors for feedback.
Good luck!
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In vitro maturation of human oocytes could be an attractive strategy for in vitro fertilization treatment. However, current in vitro maturation protocols unfortunately produce oocytes with a poor clinical outcome. Over the last 30 years, advances in culture conditions have continuously improved IVM's efficiency but even now in-vitro maturation does not support all nuclear/cytoplasmic changes that occur physiologically as a result of ovulatory stimulus in vivo. Adding EGF family molecules (e.g. amphiregulin, epiregulin) to the culture media increases the IVM rate of immature human oocytes. Additionally, brain-derived neurotrophic factor (BDNF) and glial-cell-derived neurotrophic factor (GDNF) also could improve the IVM outcome. Delaying the maturation process by adding cAMP or kinase inhibitors is another option to combine with cytoplasmic maturation. Do you have experience with these supplements? What other options do you apply in your practice?
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Interesting question.
check this link
Best wishes
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Hi,I have recently started working on mitochondria patch clamp in xenopus oocytes. I am trying to find the best osmolarity for my solutions.However, since I cannot find the exact value for cytoplasmic osmolarity within the oocytes i am having a hard time.
Thank you for your help.
Liliya
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Follow this link maybe helpful
regards
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I want to classify compounds as inhibitors or non-inhibitors for some transport protein.
The experimental results were xx fmol/oocyte/min or fmol/oocyte/hr. I have never done such experiment and don't know what it means.
1. How can I classify them with the unit of fmol/oocyte.
Normally, if a compound has a IC50 < 100 microM or under 100 microM it has 50% inhibition, I'll classify as inhibitor.
And I don't know how to classify them according to their uptake . Whether less than 80% or 50%?
2. Is uptake means 1-inhibition? If not, how to classify according to the uptake.
Sometimes, the results are not absolute value. Maybe the inhibition is 50% of control.
3. Can I regard them as the same and check whether the inhibition is higher than 50% (of control)?
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P-value is a statistical parameter to decide whether a difference between 2 measurements may have occurred by chance at some threshold level of certainty. It may be used to decide whether the effect observed is likely to be real, or just random variation. If the experiment was designed to find out whether something was an inhibitor, the P-value allows you to state the likelihood that the effect observed was not due to chance, i.e. that there really was inhibition. The P-value, however, is not a measurement of the potency of inhibition.
If the experiment being performed was an inhibition measurement (i.e. there were 2 substances involved: a transport substrate and a potential inhibitor of the transporter), then the statement "uptake less than 50%" (referring to the transport substrate's uptake) indicates that inhibition was >50%. Therefore, ignoring any scatter in the data, the IC50 of the inhibitor would be less than the concentration of the inhibitor used in the experiment. However, an IC50 should be calculated based on the effect of several concentrations of inhibitor, not just one.
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An explanation of how it works in oocytes would be more helpful. Thanks.
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It's definitely possible to knock-down lncRNA or miRNAs in the nucleus with antisense.
You may find this information helpful:
and this webinar:
I have no personal experience in doing this in mouse oocytes, but would suggest you look at microinjection of the antisense oligo.
Below is a publication from Ionis scientists, the experts in antisense technology - maybe you could try and contact the first author and see if they can help?
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My undergrad thesis is all about contamination of water buffalo oocytes in the in vitro culture. Now, i've been researching for a possible antimycotic which I can use as a treatment. However, I've found Amphotericin B but it's too expensive, now I'm considering Ketoconazole as an alternative.
Does anyone tried using this? And may I know the concentration of it? Or could you suggest any other antimycotic which are not expensive?
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Unless you find multiple studies that made use of ketoconazole as antifungal for cell culture treatments, I advise you not to use it. Ketoconazole has been documented to antagonize GRs (glucocorticoid receptors) and if your aim is to maintain the integrity of the cells after an antifungal treatment, your drug of choice might not be appropriate.
Check if this supplement might be of lower price that Amphotericin: http://www.invivogen.com/fungin
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This is my undergraduate thesis as it will tackle the contaminants in the in vitro culture of oocytes. Furthermore, my professors recommended me to use Molecular Biology to identify these fungal contaminants and prevent them when I have already identified them. As for the prevention, what is the best measure you can advice to me? Can I use fungicide or just stick with the sterility of the laboratory?
Thank you!
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You have to work under completely asceptic conditions and in Laminar Flow Cabinet containing filtered air. Sterilize your hands and sterilize the cabinet you work in .Also lab. atmosphere should be sterilized overnight with UV and switch off in the morning befor you enter to your Lab. Also you can use fungicides
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Dear Colleagues,
During in vitro maturation of "camel" cumulus-oocyte complexes, I frequently noticed that cumulus cells start to attach to IVF non-treated dishes within few hours of culture.
I have not observed this attachment during my IVM work in bovine and porcine oocytes.
I think this attachment affects the quality of oocytes.
Do you have any explanation for that?
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Dear Islam,
I would recommend you to check the CO2 concentration in your incubator and the pH of your media. In general, somatic cells (like cumulus cells and fibroblasts) like to grow in slightly alkaline media (pH around 7.5 - 7.6). Maybe by just adjusting your culture conditions you can prevent them from growing so much and also your oocytes will like it better.
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I wanted to investigate about apoptosis in mouse oocytes, but it's a new field to me (both apoptosis and mouse oocytes- I only worked with human cell lines before), so I hesitated to choose the good options to check apoptosis in mouse oocytes. I saw people often use COMET assay, TUNEL assay, Anexin V (flow cytometry or immnufluorescence), Western blot with PARP or casp 3 cleavage in different studies. Are there any advantages or disadvantages of those to use in mouse oocytes? (From what I see Anexin V is used on non-fixed cells, while COMET/TUNEL can be on fixed cellsbutI'm not sure for oocytes if it's better to go with fixed or non-fixed methos. And Western blot and Flow cytometry might be hard since I need to have a high number of oocytes). 
Thank you.
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Tunel, although not 100% specific, is a good first step approach.
Best regards,
Daniela Rodler
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I am going to simulate mechanical aspects of patch-clamp process in Finite Element Software, but I do not know the approximate size of the micro-pipette and oocytes which were used in such studies. Information like approximate or typical diameter of the Xenopus oocyte and inner/outer diameter of pipette tip would help.
Attached is an example of papers I am dealing with
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To avoid what Anjul is mentioning, it is preferable to only do macropatches in Oocytes.
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So recently I injected a batch of xenopus laevis oocytes with some cRNA for some transport experiments. I noticed that there were quite a bit of internal content coming out right after the injection, but nothing too abnormal. After 12 hours or so, I checked the oocytes again and noticed that around 80% of them continued having intracellular content leaking out of the injection site and they soon turned pale and became necrotic. I've been doing oocyte experiments using the same technique for years and this is the first time I encounter such situation. I am wondering what is going on. If you can contribute some thoughts, it will be greatly appreciated. 
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Hi Haonan, 
I am not a Xenopus- guy but rather a fly/zebrafish one. I hope it is still useful.
There are a plethora of potential problems:
- age of the parents (egg quality drops with age)
-toxicity of the injected agent or the buffer it is dissolved in
- check the pH and osmolarity of all your buffers 
 - filter sterilize buffers if appropriate
-we keep the larvae for 2 days in 1/100 PenStrep in E3 with methylene blue and check 3 times the first day for dead larvae. They start to decompose very fast and infect the remaining. The puncture wound is an entry point-for bacteria.
- use fewer larvae per dish to prevent infection
- did you use some RNAse-inhibitor in the injection mix? That could have deleterious effects. 
- make a control mock- injection without the RNA  check the parameters of the injecting apparatus. 
-check the parameters of the injecting apparatus
-monitor uninjected larvae in your buffer(s)
-do a literature search for the RNA you're injecting, is it plausible that it causes lethality? 
- check the lab's protocols if any change in water-quality occurred adults might tolerate some change better than larvae
-check your needles /puller- parameters are the needles of good quality in terms of length 
-did you use the right glass for them
-check if the MilliQ filter is OK
I hope this gives you a few ideas where to start.
Cheers 
David 
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Which protocol is best for preparation of porcine oocyte and iPS extracts for treating donor cell before SCNT?
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A.Retrovirus based iPSC reprogramming protocol
Retroviral reprogramming using standard Oct4, Sox2, c-Myc, and Klf4 Factors
iPSC colonies are selected based on morphology
AP Staining and immunostaining of ESC markers (Nanog or Oct4, SSEA3 and TRA-1-81 included)
5 vials iPSC colonies will be provided (>2 x 10^5 cells/vial)
Timelines for human iPSCs: 3-4 months and mouse iPSCs: 2-3 months
B. Episomal vector based iPSC reprogramming protocol
Episomal DNA reprogramming using Oct4, Sox2, c-Myc, and Klf4 factors with small molecules
iPSC colonies are selected based on morphology
AP Staining and immunostaining of ESC markers (Nanog or Oct4, SSEA3 and TRA-1-81 included)
5 vials iPSC colonies will be provided (>2 x 10^5 cells/vial)
Timelines for human iPSCs: 4-5 months
C. Protein based iPSC reprogramming protocol
Protein reprogramming using standard Oct4, Sox2, c-Myc, and Klf4 factors with a cell-penetrating peptide (CPP)
iPSC colonies are selected based on morphology
AP Staining and immunostaining of ESC markers (Nanog or Oct4, SSEA3 and TRA-1-81 included)
iPSC colonies will be provided (>5 x 10^5 cells/vial)
Timelines for human iPSCs: 3-4 months
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Hey there. I need to isolate stem cells from mouse fetal membrane, for a project I'm working on. but I know nearly nothing about it, only a little about mating a male and female mice and pregnancy duration! but exactly how and when can I extract fetal membrane from pregnant mice and could it be after breeding the babies? I hope it would.
waiting.thanks a lot.
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tnx a lot!
sorry for  answer back this late, there was a problem with my connection
regards, Sara Vahdat
  • asked a question related to Oocytes
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Dear All,
I am looking for a protocol optimized for the DNA (RNA-free) isolation of mammalian (preferentially mouse) MII oocytes.
I have different pools of 20-80 oocytes and would like to proceed with DNA-specific analysis.
I performed some bibliographic review and I see every different paper with a different protocol, so I thought it would be useful to ear someone with experience in this particular protocol.
Thanks,
Dario 
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Dear De Jesus. I think the following article is so useful for your request. Kind regards. https://www.ncbi.nlm.nih.gov/pubmed/22907499