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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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I have values for both variables over time, I did a simple correlation between them for each time point, and the results are very variable, and If I do a global correlation (data from all times together), the correlation is not true. I have been looking for what is the best procedure, and I have found information about autoregression but this method is for correlating any variable along the time, but not the correlation between two variables along the time. Anybody could help me?
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I'm interested in the answer to this question too. I have two continuous variables measured at 7 timepoints. My advisor suggested doing spearman's rho to get a correlation coefficient for each participant (I only have 7 time points so I guess that's why it's spearman??). Then enter all of the coefficients in a single sample t-test, testing it against the value of zero. I'm having trouble finding an example of this and why though...because most resources suggest that pearsons is useful in most cases.
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Treatment modalities of sub-fertility
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Different stimulation protocols have been offered for PCOS patients undergoing ARTs : GnRH agonist and GnRH antagonist protocols. There is no clear evidence for the advantage of one over the other. The GnRH agonist was initially considered to be the protocol of choice in PCOS women. Due to increased risk of mid-cycle gonadotrophin flares, multiple pregnancies and OHSS its usage has been restricted to certain situation. GnRH antagonist has been shown to have an advantage over the agonist in form of quicker onset of action, short duration of treatment, lesser total dose of gonadotropins required, lower cost, better to be tolerated, lower incidence of LH surge, lower incidence of OHSS and being more patient friendly. Recent data have been obtained from systematic review and meta analysis studies were showed no significant difference in fertilization rate and pregnancy outcome when comparing the two GnRH analogues; agonist versus antagonist in PCOS and normal women. However another study showed higher clinical pregnancy when ultra-long agonist was used instead of long agonist protocol.
The aim of every IVF/ICSI program is to achieve multi-follicular development and collection of several appropriately matured oocytes without causing OHSS and with a comparable pregnancy rate. This is especially important in women with PCOS as they usually exhibit greater sensitivity to exogenous gonadotropin stimulation than women with normal ovaries.
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We are trying to create a transgenic mice in our lab for the first time. We currently have a microinjector that we use for ICSI or biopsies, which is fitted with mineral oil injectors, to control the pipettes. In all the papers I´ve read about this, people use air pressure-controlled injectors to inject a small volume into the embryo. Has any of you attempted something like this using the oil injector, or is it necessary to have the air injector? Thank you!
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Dear Rex,
It does not really matter, whether you are using and oil based injector or air based injector. Only thing you have to make sure is
1) Use of IVF/Cell Culture Grade Oil(mineral or paraffin).
2) Make sure oil does not enter your embryos.
And please do not use this for CRISPR technology, instead use it for other purposes.
Regards,
Sanketh
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more often we found degeneration in oocytes after ICSI ? was just curious to know what can be the reasons apart from technical error?
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one more: prolonged duration of ICSI in low or very high temperature (check the heating stage of the inverted miscroscope)
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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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I would like to model the human oocyte flowing into a micro-channel. I would like to find information about the density (weight) of the human oocyte. I would like to know if the density of the human oocyte is bigger than the density of the solution used in intra cytoplasmic sperm injection procedure. When the human oocyte is immersed into the solution, does it go down to the bottom of the micro-channel because of gravitation, or it is floating freely into the solution?
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Tihomir,
Kindly try to see if you can get in touch with scientists at Northwestern Univeristy, Dept of Reproductive Medicine as they might help you. As a far as I know they have cultured oocytes 3-D. May be the biophysicists involved might be of some help.
Thanks
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We previously did ICSI with this patients sperm and got 6 blastocysts which were tested normal using array CGH, yet no pregnancy outcome
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Are you determining DNA.chromatin fragmentation based on SCSA? If you did, you can assume that the germline of this patient has germline genomic instability which we determine by small pool PCR.We have found that a subset of these patients are at a high risk of developing testicular cancer as they age. Most of this genomic instability is due to
oxidative stress.
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Hi, I'm looking at embryonic quality score data for IVF/ICSI (multiple embryos per mother), which are categorical and ordinal. I want to compare 2 groups: 1 has had a certain type of surgery, the other hasn't.
The only way I know how to analyze categorical data is chi-square or fisher's exact. However, that doesn't take into account that the dependent variable is ordinal, and also leaves the problem that not all data are independent (multiple embryos from the same parents).
How could I best analyze this? (also preferably in SPSS)
Any help would be greatly appreciated.
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You can use proc catmod to run a regression on your data in SAS.
In SPSS a regression may help you.
Regards
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There seems to be variability in dosing of lupron triggers.
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1 mg leuprolide trigger SC
1 mg leuprolide trigger + 1500 mg hCG for dual trigger
 This is followed at our center. By we rarely use it. we just reduce the dose of hCG trigger and go fo segmented IVF OHSS prone patients
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32y old patient, candidate for frozen-thawed embryo transfer. Endometrium seemed resistant to estradiol validate, from 4 to 8 mg/day, and to estradiol patch, from 50 to 100 mug each 2 days.
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We have good results with the vaginal sildenafil application. Best regards. Pavel Travnik
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What dosing?
For the fresh or FET cycles.
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Dear Tarun,
we do not apply the Prometrium, but we have very extensive experience with the using of the Utrogestan, which has the same composition as Prometrium 100mg. We use them mainly in the cycles without corpus luteum (FET, oocyte donation), but also for the cycles with corpus luteum (fresh IVF cycles, FET in natural cycles) support. The usual dosing is from 2 capsules to 4 capsules daily per os, the vaginal application is also possible.
Best regards
Pavel Travnik
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pt' with necrozoospermia ( 100% immotile spermatozoa) , how we can get fertilization by ICSI?
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Agree with Prof. Tulay Irez:
Use HOS Test to identify sperm with intact normal DNA.
Those sperm exhibiting tail curling can be used for ICSI, we believe.
However, such a study should be reported.
Ref: Stanger et al, 2010. RBM Online 21, 474-484.
Best wishes and do report your experience.
John Yovich
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Does anyone have experience with this issue as all available data suggested no role of estrogen?
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I am developing a robotic system for cell injection that could be applied for ICSI. I would like to choose an IT platform and a language for programming. What kind of software is suitable for real time programming and control of different hardware devices as piezo-actuators, digital camera, microscope, micro-pumps. There are robotic toolbox, modules for image processing and many other modules for automation and control of robotic systems in real time in Labview and Matlab. I am not sure which language is better. In Matlab I can buy a specific hardware for control in opposite of Matlab, par example. The other possibility is to use linux OS, Qt4 programming language and OpenCV vision library as open source technology. The last option is to use Microsoft Visual Studio as a programming environment and selecting any programming language as C++ or C#. Could you please give me advice on what kind of a programming technology is best suited for development of software applications in real time? Thanks.
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Salamatin, Joint Institute for Nuclear Research. Now the new technology for On-line systems construction is in progress, and some of papers are avaliable, but in russian. Below is abstrfct of one of them. If it is what you need, please write to del@xaker.ru or salam@nf.jinr.ru.
Paper title:"Automation of neutron spectrometry experiments using network technologies". Abstract contents:
A new structure of a distributed software system for experiments automation (SEA) is proposed. The DSEA includes the following interacting parts: 1) a subsystem describing the experimental procedure (SDEP); 2) the experiment control program (ECP); 3) distributed components messaging environment (DICME); 4) sample environment at the instrument; 5) Data Acquisition (DAQ) system; 6) components for auxiliary operations.
SDEP contains data base (DB) and two dialog software: 1) a program producing passports of components which govern conditions of data registration; 2) program of Preparation of a Single Job (PSJ). The first one creates and writes to the database documentation for components, which contains: the name and description of controllers and devices connected to them, globally unique identifiers (GUID), used for addressing components, and parameters descriptions. The second one, PSJ, uses only terminology understandable to the experimenter, and represents the job as a list of ECP states descriptions in JSON format.
ECP program receives from SDEP the list of SEA states, selects the description of the next state of the system (list of the registration data conditions) and sends a description of each condition to the intermediary DiCME. The description of the condition carries information about the component (GUID), sufficient for its search and link to ECP, and it also contains a list of parameters. Communicating through DICME with all necessary components, the ECP serves as a components manager. The choice and procedures procession that implements the conditions of registration data is performed in the components that drive the hardware, based on the interpretation of the referred to them descriptions of the conditions. Each component, to which ECP sent a message, should return to ECP the end of job signal (DONE / ERROR). After receiving signals from all the components listed in the description of the required state, ECP activates the data acquisition subsystem DAQ. The signal of data exposition finishing allows the ECP to switch to processing of the next state description in the job file.
Mediator DiCME, developed using network technology, automatically searches and dynamically binds components. It provides an asynchronous mechanism for remote execution of procedures and access interface to component’s procedures independent on the computer network address. Information transfer and processing of all interactions of components in the SEA is performed by similar means. For dynamic components linking two algorithms are used in the SEA: 1) the "hard" one for the basic operations (conditions formation, registration and archiving of experimental data) , implying a mandatory delivery of the message from a "customer" and of the answer from the "implementer"; 2)the "soft" one: a "subscription" for auxiliary operations (visualization , preprocessing , ...), in which a component consumer declares interest in a particular type of information, and the event manager serves all the "Subscribed" consumers when the information appears. As a result, the components implemented in different experiments and in different SEA can be used without modification.
The presented approach was applied in the development of SEA software for several spectrometers at neutron sources IBR-2 and IRENE in JINR. It can be also applied in other areas, for example, to process automation.
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Could a frozen tissue saved from adjacent testis tumor jeopardize future offspring in those indiviiduals who need to undego to bilateral orquiectomy due to a TU?
Any guess and or suggestion?
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Dear Alireza.
Thanks for your suggestion.
We´ve done radical orchidectomy, saving tissue adjacent to the tumor (Seminoma)
We don´t do partial orchi.
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It's generally thought that mtDNA is only maternally transmitted. But is there any evidence at all for paternal mtDNA transmission? What about during ICSI? And if so, what are the implications? Does it change what we can say about mitochondrial "Eve."?
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There is an ongoing debate on this topic. However, it turns out that virtually all reported cases of paternal inheritance of mtDNA in mammals (and specifically in humans) were in fact artifacts of laboratory procedures. I don't have the PDFs with me right now but I'll give you some links later.
Take care
Marcin