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Assisted Reproductive Technology - Science topic

Assisted Reproductive Technology is an all about ART
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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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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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Hello there
As we know researches are being rapidly evolved in most majors. I would like to know where animal reproduction physiology researches will go to in future. where do animal reproduction physiologist see themselves in 5 to 10 years later?
In my opinion, it is vital for researchers in this field to find out which aspects of their major define future studies and accordingly researchers should empower their knowledge in growing areas.
It is my pleasure to know your idea about the future of animal reproduction physiology researches and related areas.
I would appreciate if any related papers, sites and other stuffs be suggested.
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Increased research pertaning to infertility, it physiological and pathological causes as prevelence of infertility in animals is presenting increased trend with time. Similarly, retention of placenta and increase in dystocia, it is all related to chane in physiology.
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Does anyone know a reliable methodology to estimate the initial values of theta1 (or A) and theta2 (or B) in a non linear model? For example, in a logistic model describing scrotal circumference growth:
SC = A/[1 + B exp(-kt)]
How to reliably estimate the initial values of "A", "B" and "k"?
I'm using NLIN Procedure on SAS.
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In your example, you have 3 parameters in your model, so you can pick 3 data points in your data pool and plug these 3 data points into your model to solve the parameter values. Although your model is nonlinear, the equations you need to solve may be linear. In the case that these equations are nonlinear equations, root-finding problem is still easier than your original problem. The solution for these 3 parameters can be used as a reasonable initial guess for nonlinear least squares fitting. More details of this approach (e.g., how to choose 3 data points from the data pool) can be found in the following paper:
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Hello there
I wanna know what challenges exist in canine semen preservation whether in cooled or freezed storage conditions.
please provide complementary references if it is necessary.
thanks in advance
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dear Omer Ucar
I really appreciate your comprehensive and beneficial explanation.
wish u the best
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We use this gonadotropin for superovulating mice. 
Thanks in advance!
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Yes, We can supply you,
PMSG,pregnant mare serum gonadotropin, Natural glycoprotein, pure natural animal hormone, extracted from serum of pregnant mares , Follicle stimulating and Luteinizing activites,improving fertility rate, inducing superovalutions and estrous synchoronization , used in livestock farming,Pig,prepuberal gilts,Cattle,Dairy,Sheep,Goat and other mammal animals
Full series of PMSG, from Raw mare serum, intermediate, lyophilized powder ,and injection (FDF),
now Scientific research and animal farm are using this products
For more and discussion, please contact us .tommy59@qq.com
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Hello everyone! I'm now a biomedical senior and get stuck selecting a research idea for my Bachelor thesis, especially, I'm very enthused in the field of Assisted Reproductive Technology. Actually, I don't know how to start, I mean the first steps of getting an idea. I would be grateful for all of your supports, thank you!!
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Thank you for sharing with me, it means a lots and I really appreciate that! :)
Best wishes!!
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India is becoming a hub of suurogacy outsourcing capitaL OF THE wORLD.Commercial surrogacy,egg donor programmes are becoming popular services provided by the fertility industry.Through lights on deeply regarding the issues.Well articulTED Suugestions are highly accepted.
Regards!
Md Osim Aquatar
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Dear Stefan S Du Plessis ,
Thank you so much for your awell articulated suggestions.Now,I got the bsic idea pertaining to the issue.
Regards!!
Osim
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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 are performing a superovulation and embryo collection programme in goats. We use commercial porcine FSH (Folltropin) and after dilution we store it for a maximum of 4 days at 4C. Could we freeze this FSH dilution remaining its stability?
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Our kharkiv`s scientifics stored diluted FSH in liquid nitrogen without losses of activity.
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Should we be using 5000 or 10000 IU hCG to trigger ovulation ?
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recently I have been in University of Brussel , the Belgian friend’s they depend on BWt , if > 80kg will trigger by 10,000 and of < 80 kg they trigger by 5000 , I think it dose make sense
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Does anybody know what I can use to measure lipid peroxidation by green fluorescence?
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Any advice on the following would be greatly appreciated
(1) cryopreservation solution, which is best for long term storage?
(2) removal of contaminating somatic cells, before or after freezing?
(3) cryo tubes or straws, which is best?
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Hi Maraia
if we spoke about old method coconut fresh was used as preservative for sperms before  And still used it for animals
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Dear Meng,
I believe this work will help : Eur Rev Med Pharmacol Sci. 2010 Oct ; 14 ( 10 ) : 891-6 .
Inositol activity in oligoasthenoteratospermia - an in vitro study .
Colone M1 , Marelli G , Unfer V , Bozzuto G , Molinari A, Stringaro A.
If you want I can send you some vials of Myoinositol for your experimentation.
Vittorio
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The last update of haploidization and its future perspectives was written by Drs Jan Tesarik and Carmen Mendoza in 2002 (Somatic cell haploidization: an update, Reproductive BioMedicine Online, 2002, 6(1):60–65) . Is there any new update or report of live births from this approach?
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We (my husband, Professor Neal First) and I had a student that studied haploidization in the bovine model.  She did manage to get development to the morula stage but not beyond that.  I will add that a few embryos did look as though they were "trying" to develop into blastocysts but they were far from normal.  With a little work I can dig out her thesis if there is interest.
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I want to use of 120uM Torlox  ( water soluble analogue of vitamin E) and I want to know can I make solution and store it for two month?
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Well, in my opinion I wouldn't make the solution and store it for so long. According to our experience in laboratory, if you want to use it to obtain a calibration line, for example, I recommend that you make your solution almost immediately, not waiting for more than a week. 
Greetings!
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How can you predict and prevent infertile women who undergo IVF of having ovarian hyperstimulation syndrome after IVF?
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Regarding prevent ovarian hyperstimulation syndrome, think about ORPI (ovarian response prediction index)
Reprod Biol Endocrinol. 2012 Nov 21;10:94. doi: 10.1186/1477-7827-10-94
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I know INRA96, but are there other good extenders for fresh and cooled use? 
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Hi,
INRA96 is the best extender for cooled stallion semen and INRA freeze is the the best for cryopreservation at -196 C
Magdi
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Dear All, 
I'm planning to perform epigenetic study on offspring born following Assisted Reproductive Technologies. I do not have an extensive experience on mouse model and I know that some strains are not so good for this kind of study. thus, which one should I use?
Thank you for your suggestion. 
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You can try BL6 mice.
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This is a cross match test to determine the presence of anti- lymphocyte antibody in female partners suffering from immune mediated infertility, Like that developed by Dr Alan Beer and currently offered in his Lab and Rosalind Franklin Lab, Chicago.  Would also be used in transplantation studies  
Thank you
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I believe there are kits for this.  The American Red Cross  (ARC)Specialty lab does this, as well as NIH. These are micro well plates.  These are serological tests.  I would contact ARC in S. California (formerly Dr. Garratty's lab). Dr. Garraty passed last year. There they do flow cytometry crossmatches.  They would be the first I would talk to. 
Mixed lymphocyte studies is a pretty old method to do this, but it actually works well.  This would be for cellular immunity.
Hope this helps 
Sergio
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Please, advise me any publications in this topic would also be helpful. 
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just because they are close does not make them the same!  The endometrium is a completely different environment and I would say not influenced by what is going on in the cervical region 
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How Acridine Orange stain is used in semen evaluation?
I want to know procedure and name of stain (Source of procurement-company name) used to detect DNA integrity in Bovine spermatozoa.
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Acridine Orange intercalates into nicked or damaged DNA and will foluresce "Red" on 488 blue laser.  Undamaged DNA or sperm cells with correctly packaged DNA will resist intercalation and the compound flouresces "green".  In this way the ratio of red to green can be calculated as the DNA fragmentation index (DFI).  I use AO from sigma aldrich (SIAL) or bioscience also have it. Important to use very high purity and concentration of stock solution should be 1mg/ml.  maintain AO powder at -20.
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I am injecting rRNA transcribed by mMESSAGE mMACHINE T3 Transcription Kit into Xenopus oocyte for 2 electrode recording.  I am injecting 1ul per egg.  What concentration is can be a start for this type of injection?
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It depends on what you are injecting & where you anticipate its expression. Check out Xenopus methods here...
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During artificial Insemination in Honeybee queens, someone uses chloroform to help endophallus eversion process in drones. Does anybody know if there is any paper concerning chloroform affecting sperm vitality (in any species.. not honeybee only)?
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sorry I dont know
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I am interested on legal and ethical aspects of human assisted reproduction in different cultures. Your opinion about the most questionable aspects is also welcome!
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My co-authored (with Maureen McBrien) book titled Assisted Reproductive Technolgy publishled by the American Bar Association (2011) covers the US. 
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I  want to dissolve the coconut oil in semen extender, can anyone guide me how to so this?   
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Be careful. If you mix the methanol solution with the extender, the oil will probably drop out of the solution.
Pavel
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I want to develop fluorescent assays to evaluate the membrane integrity, capacitation status and acrosome reaction in bull and stallion semen. So, what are the possible results of this assay, and what sperm populations can I obtain after this analysis?
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With regards to the acrosome reaction you can either use FITC-PSA or FITC-PNA. Please refer to the WHO Laboratory manual for the Examination and processing of human semen (2010) pages 146-152
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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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I would like culture media samples which contain none of the overlying oil in them.
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Just take a  sterile disposable pipette tip and pipette the medium from under the oil. I collected in this way medium after human embryo culture,to analyse the glycolitic activity between day 4 and day 5 of in vitro culture.  I worked in drops of 5 micro-liters.  Just keep in mind that if you are overlying cultures with oil that the oil will extract all lipo-soluble substances contained in the medium.  Good luck
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Due to assisted reproductive technologies and hormonal therapy there is higher risk of thromboembolism especially in cases with complicated course (for example with ovarian hyperstimulation syndrome)
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thank you very much. I'll read it with joy. I have recently published a book on the subject but there are still topic to include or expand.
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Blastocysts
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If you have blastocysts, they all have survived to that that stage of development. It is understandable that there is a strong desire to be able to assess them and then predict their chance for success. To meet that desire, some have developed protocols.
Yet, it is difficult to assess embryos with confidence.
In cattle, sheep, deer and mice, I have transferred blastocysts and found that. One thing I discovered that was useful was to examine the shape of the zona pellucida. If it was round and the blastocyst looked competent to hatch, we would get better pregnancy rates. However, even when the blastocysts had flattened zona pellucidae, if I cut them out of their zona with a micro-scapula, their pregnancy rates would go way up.
The idea of transferring the most advanced stage ones is interesting . It has been learned that, all other things being equal, the most advance stage mammalian embryos within a female, tend to be males. Other things can influence the relative stages of embryos such as the fact that all follicles are not always ovulated at the same time.
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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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I treated the mature cumulus-free oocytes with pronase 0.3% (w/v in PBS), the zona pellucida was digested within 60-90 seconds however when the oocytes were first electrically activated (parthenogenesis) the digestion time became more longer (120-180 seconds or even more). Is there any explanation?
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You should be seeing the effect of zona hardening, which is a byproduct of fertilization but might be brought about my the Ca++ transient elicited by activation, depending on the species you are working with.
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Primary cell culture of buffaloes
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I have done lot of work on primary cell culture in buffaloes, I have work both on buffaloes and cattle, Their is nothing change between two species, except for buffaloes you need to maintain CO2 incubator temp to 38.5C.
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I am developing microfluidic devices for immobilization of biological cells. It is necessary to create hydrophilic surfaces. I saw in the literature that there are many plasma cleaning equipment that is very expensive. I would like to ask do you know any technology to create hydrophilic surfaces with home made cheaper equipment?
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I am using PDMS and lexan.
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I have a percoll solution from sigma, and I want to prepare 80% 60% 40% and 20% percoll solutions to obtain pure sperm. How to prepare it and what is the buffer or other solution to be used for its preparation?
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I would like to know which genes are considered the best progesterone marker in endometrial cell line. If I treat the cells with progesterone, an expression in which gene or protein is the best to proof the treatment.
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Thanks a lot Rohan, I am reading the literature, and I saw there are a lot of genes, which is difficult to decide which one is better. I will read papers that you mentioned and thanks for points.
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Implantation of bone marrow stem cells in the testes is a hope for the treatment of males with primary testicular failure in humans. If this technique proves successful it will transform the treatment of male infertility. Basic research have shown the success of this technique in mouses.
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I`m quite curious & waiting for contributions in this so crucial issue. :
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I have a young patient, 31 years with amenorrhoea and very high LH and FSH. She wants to conceive. She bled when I challenged her with progesterone.
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Transvaginal sonogram is helpful before progesterone challenge test , because in atrophic endometrium , no bleeding is occurred with progesterone, if there is bleeding , so there is a thick endometrium.
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Does anybody know how can we get the SCSA software developed for sperm chromatin analysis on flow cytometer?
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Hi Loris,
Here is the website of Dr. Evenson's company. You can directly order from them.
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Do you get acceptable post-thaw sperm motility and what results have you had for clinical use?
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Thanks Miriam, please say hello to Shlomi for me. We used glycerol,egg-yolk,citrate (GEYC) cryoprotectant from 1976 until early 2013 with very good results. We have had to switch to a commercially available cryoprotectant because of lab accreditation requirements. Cryosperm has the advantage of not containing any biological products such as egg-yolk or albumin but still giving equivalent post-thaw motility recovery. Cryosperm does contain Raffinose which a previous student of mine (Puti Adla Runisa) found to give good motility recovery in the absence of egg-yolk or HSA (manuscript in preparation). Cryopreservation is a very complex subject and I am convinced that further significant advances in sperm cryopreservation can be made, for example, by developing cryoprotectants which are tailored to be optimal for different categories of semen.
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The principal idea of this technique is to have more GM-CSF and other growth factors, but the critical thing is how to know the concentrations precisely to avoid any contamination, alteration or apoptosis? How can we evaluate that? Any suggestions?
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See Ouhibi et al.
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What are the requirements of an IVF laboratory?
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I mean does someone think that this cut off is the same for poor responders, normal responders and high responders?
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At our center, the P4 result is just one factor used to determine day of hCG trigger. The ovulation induction protocol matters, as GnRH antagonist IVF cases will evoke a different P4 pattern compared to those receiving standard GnRH agonist downregulation. The value you mention (1.5ng/ml) probably would be a reasonable level, although P4 levels are best interpreted against corresponding E2 levels taken on the same day. Lower E2 responses could be triggered at a lower absolute P4..
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I´m starting to work on IVF and I have some doubts about the IVF room. How does the room need to be cleaned? How should the room be mopped? How does one take care of the air quality?
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There are those days product specially developped for embryology labs. I do not sell those products but could provide you advise visit www.quartec.co.
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Does somebody know how many times can I reused the tips from piettors?
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For basic rules of safety and possible cross contamination pipette tips should not be re-used.
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What's the best way to get a higher fusion rate when doing bovine cloning?
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Thanks! For the fusion medium, I use PVA instead of BSA.
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1. What are the advantage and disadvantages of GnRH agonist protocol for poor response to stimulation?
2. What are the advantage and disadvantages of antagonist protocols for poor response to stimulation?
3. Can using DHEA really improve the quality of life?
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We are 100% agree with that ideas the antagonist is not a golden protocol over the agonist but in our (>10 yrs in infertility treatment) experience the outcome of antagonist is better than antagonist protocols. In case of bad responders .. it is the most ,unfortunately, unwanted results for our kind persons. We found that OCP for at lest 3 months improved those bad responders to short antagonist protocols.
Prof. Dr Ahmed K. Allow
Infertility consultant
PhD and M Sc
Director of Allow IVF center
Sana'a Yemen