Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
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Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

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Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



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Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
Knowledge is treasure of a wise man. The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
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I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




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Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




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Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help ones reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journalsNo manuscriptsNo authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Experimental Research
Year : 2008 | Month : December | Volume : 2 | Issue : 6 | Page : 1234 - 1239

In Vitro Maturation And Fertilization Capacity Of Mouse GV-Stage Oocyte Following Stepwise Vitrification

Mahmoudi R * ,Ragardi Kashani I ** ,Abbasi M ** ,Amidi F** ,Sobhani A** ,Abolhasani F** Saadipour Kh***,Amiri I ****

*Department of Anatomy and Embryology, Medical school,Yasuj University of MedicalScience,Yasuj,(Iran)*Department of Anatomy and Embryology,Medical school,Tehran University of Medical Science,Tehran,(Iran).***Department of Physiology, Medical school,Yasuj University of Medical Science,Yasuj, (Iran).****Department of Anatomy and Embryology,Medical school,Hamadan University of MedicalScience,Hamadan, (Iran).

Correspondence Address :
Iraj Amiri,Ph.D in Anatomy,Institute Department of Anatomy and Embryology, Medical school,Hamadan University of Medical Science,Hamadan,(Iran)Email: amiri44@yahoo.com,TEL:+98-912-336-2561 Fax:+98-811-8276299

Abstract

Background: The storage of oocytes is important for advances in reproductive biology and infertility treatment. Although successful procedures for cryopreservation of human metaphase II oocytes have been reported, their results have proven unsatisfactory, and appear to require further progress. The microtubular spindle of MII oocytes is sensitive to temperature changes. Germinal vesicle (GV) stage oocytes do not have microtubular spindle, so its cryopreservation may be an alternative approach to the storage of gametes.
Aims: examining the viability and subsequent developmental ability of murine GV oocytes after vitrification.
Setting: Embryology research center at an academic institution.
Design: Original Article
Methods and Materials: Germinal vesicle oocytes with cumulus cells were transferred to vitrification solution, which was composed of 30% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose either by single step or in a step-wise way. After vitrification and storage in liquid nitrogen, the oocytes were thawed and washed two times in medium TCM199 and then subjected to in vitro maturation, fertilization and culture.
Results: The oocyte survival, maturation to MII, fertilization and development rates in step-wise exposure were found to be significantly higher (P<0.05) when compared with corresponding rates in the single step procedure. Collected data was analyzed by one way ANOVA test.
Conclusion: The results of present study indicate that GV stage oocytes have better morphology and viability after vitrification in stepwise method; and their rates of maturation and development to 2-cell stage are also higher as compared to oocytes cryopreserved via single step procedure.

Keywords

Oocytes Cryopreservation, Germinal Vesicle Oocyte, Vitrification, Microtubule.

Introduction
Cryopreservation of human gametes and embryos has become an integral part of assisted reproduction. The major developments in cryopreservation technology have mirrored the rapid expansion of reproductive technology over the past 2 decades. It is now possible to cryopreserve sperm, oocytes and embryos at their various stages of development (1). There are two methods of cell cryopreservation, slow cryopreservation and vitrification. Many cell types can be successfully cryopreserved by slow cryopreservation but some cell types can not be cryopreserved by slow equilibrium methods, or yield poor survivals.

The common reasons for failure are high sensitivity to extreme temperatures, inability to maintain an intracellular supercooled state, and disruption of complex cell-cell interactions by extracellular ice. One important cell type that has been found difficult to cryopreserve by slow freezing, is the mammalian oocyte. Generalized high sensitivity to extremes of temperature is one reason. Another, important reason is that mature oocytes are locked in the metaphase of Meiosis II; i.e., the chromosomes are arrayed on the meiotic spindle. The spindle is composed of microtubulin material, and like microtubules in general, it becomes disaggregated by being cooled to temperatures around 0°C(2),(3),(4),(5).

In case of cells and tissue types where equilibrium slow freezing yields inferior results, increasing efforts are being made to achieve cryopreservation by vitrification procedures. Vitrification has been used successfully for storage of mature (metaphase II stage) oocytes (6),(7),(8),(9),(10). But data about vitrification of germinal vesicle (GV)-stage oocytes are limited, although this mode of cryopreservation can be promising (11), (12).Up till the stage of Germinal vesicle (GV) oocytes have not formed spindles, which poses significant problems in successful cryopreservation of oocytes. At this stage, the chromosomes are decondensed and enclosed within nuclear membrane, potentially making them more resilient to cooling.
Regimes for collection of fully-grown GV-stage human oocytes have been devised that involve modification of mature oocyte collection techniques (13) and reduction in hormonal stimulation. Studies have shown that GV-stage oocytes which are stripped of cumulus cells have a reduced developmental capacity compared with that of cumulus-enclosed GV-stage oocytes (14). Cumulus cells play an important role on oocyte maturation, since they provide and transfer several known and unknown factors that are essential in regulation of meiotic progression, normal nuclear and cytoplasmic maturation of oocytes and subsequent embryonic development after fertilization (15),(16),(17),(18). The aim of this study was to compare the effects of stepwise and single step exposure to cryoprotectant on the developmental ability of vitrified mouse oocyte in ethylene glycol-sucrose in presence of cumulus cells.

Material and Methods

All chemicals were purchased from Sigma Chemical Co., St. Louis, MO, except for the ones specifically described.

Collection Of GV Oocytes
Oocytes were obtained from 3-4 week old ICR strain female mice, the animal were kept under controlled condition (14h light: 10h dark) and fed water and pellets ab libitum. Mice were stimulated by an i.p. injection of 7.5 IU pregnant mare serum gonadotropin (PMSG). 46h later the animals were killed by cervical dislocation and the ovaries were removed in Hepes-buffered human tubal fluid medium (HTF) (sigma) supplemented with 5mg/ml BSA. The GV-stage oocytes of ovarian antral follicles were released by puncturing with a 28G micro-injection needle under a stereomicroscope. The “GV cumulus oocyte complexes” were randomly allocated to three groups:control without treatment, stepwise preservation and single-step vitrification group.

Preparation Of Vitrification And Thawing Solutions
The solutions for vitrification and dilution were prepared using PB1 plus 20% fetal bovine serum. The stepwise vitrification solution consisted of solution A, which was composed of 10%(v/v) ethylene glycol, 4.5%(W/V) Ficoll-70, and 0.075M sucrose, and solution B which was composed of 20% (V/V) ethylene glycol, 9.0% (W/V) Ficoll-70, and 0.15 M sucrose, and solution C, which was composed of 30% (V/V) ethylene glycol, 18% (W/V) Ficoll-70, and 0.3 M sucrose. Single-step vitrification solution consisted only of 30% (V/V) ethylene glycol, 18% (W/V) Ficoll-70, and 0.3 M sucrose. The solutions for thawing were made of 0.5, 0.25 and 0.125 M sucrose.

Vitrification And Thawing
The GV-COC were randomly divided into either stepwise group or single-step group. In stepwise group, the GV-stage oocytes were exposed first for 5 min to 200-ml droplets of solution A, then for 2 min to 200- μl drop of solution B and finally for 1 min to 200- μl drop of solution C in 4-well plates. In single step group, the GV-stage oocytes were exposed for 1 min to 200- μl drop of solution C.

The procedures were performed at room temperature of 22-24ºC. After obtaining equilibrium, 10-15 GV oocytes were loaded into a 0.25 ml plastic straw (IVM, I Aigle, France). The straw was filled with 1 cm of vitrification medium, 0.5 cm of air, 2 cm of Vitrification medium containing oocytes, 0.5 cm of air and 3.5 cm of vitrification medium. The straw was heat sealed and plunged into liquid nitrogen. After storage for 1-5 days, straw was taken out of liquid nitrogen, held in air for 10 s, and then plunged into water of 37ºC for 10 s. Straw was then removed from water and wiped dry. It was cut with scissors and the contents containing oocytes were expelled into 400- μl drop with a sequential series of 0.5, 0.25, and 0.125 M sucrose by keeping for 90 s in each solution, and washed for 6 min in α-MEM medium supplemented with 20% FBS.

Maturation Of GV Oocytes
The vitrified-thawed GV oocytes or fresh GV oocytes (control group) were cultured in 100 μl drop of IVM medium composed of α-MEM supplemented with 0.23 mM sodium pyruvate, 1 mg/ml of fetuin, antibiotic and antimycotic solution (comprising 100 U of penicillin, 100 μg of streptomycin, and 0.25 μg of amphotericin B), 10 ng/ml of mouse epithelial growth factor of culture-grade (EGF, Upstate Biotechnology Inc, Lake placid, NY), 75 mU/ml of follicle-stimulating hormone (fertinome, serono, Geneva, Switzerland), and 3 mg/ml of bovine serum albumin (BSA, Fraction V, Sigma) under mineral oil of embryo-tested grade (sigma), and incubated at 37ºC in an atmosphere of 5% CO2 in humidified air. 16-18 h after culture, the GV-COC oocytes with first polar body were defined as mature MII oocytes.

In Vitro Fertilization And Development
Spermatozoa from male ICR mice of 12 weeks age were released by cauda Epididymis puncture in to IVF medium TYH medium supplemented with 4 mg/ml BSA. The sperms were suspended in 200 μl droplet of the IVF medium, were covered with mineral oil and incubated at 37ºC for 1-2 hrs in humidified atmosphere of 5% CO2 for capacitation. Mature oocytes (n=15-20) were placed in separate 200 μl droplet of IVF Medium under mineral oil. Sperm mixture (10-20 μl) was added to each droplet to obtain a concentration of 1-2 × 106 motile sperm/ml. after co-incubation for 5 h at 37ºC, the oocytes were then removed and washed in fresh TYH medium and placed in a 5% CO2 incubator at 37ºC. At 6-8 h post-insemination, embryos with two distinct pronuclei and a second polar body were classified as PN stage, observed under a phase-contrast inverted microscope. The pronuclei stages were then transferred to 100 μl of KSOM (potassium Simplex optimized medium) for 2-cell stage development.

Statistical Analysis
Collected data was analyzed by one way ANOVA test. The difference in values of Survival, Maturation, Fertilization and Developmental rate, were considered significant when p value was<0.05.

Results

A total of 570 GV oocytes with cumulus cells were obtained from 26 ovaries that were used for vitrified and non-vitrified (control) group.
Morphological figures of nonvitrified (controls) and vitrified GV oocytes using either single step or stepwise method are shown in (Table/Fig 3). Partly dispersed cumulus cells and large subzonal space are noticeable. In stepwise method COC were similar to those in the control group.

Survival and in vitro maturation of vitrified GV oocytes
The survival and maturation rates of GV oocytes after different treatments including exposure to cryoprotectant, vitrification and for non-vitrified oocytes are shown in (Table/Fig 1). The survival and maturation rate in the stepwise group was significantly higher than single step group (p<0.05). But maturation rate in control group was significantly higher than both single step and stepwise vitrified groups(P<0.05).

In Vitro Fertilization And Development Of Vitrified GV
In vitro fertilization and development after in vitro maturation of vitrified GV oocytes is shown in (Table/Fig 2). The rates of fertilization and development to 2-cell stage in the control group were significantly higher than the entire vitrified group. Among the vitrified groups, the fertilization and developmental rates in the stepwise group treatment with EFS10, EFS 20 and EFS 30% were significantly higher than those of the single step treatment only with EFS30%. Furthermore, in stepwise vitrified group the fertilization and developmental rates were significantly higher than those in single step group (p <0.05).


Discussion

In the present study mouse oocytes have been used as an easily available source. We have investigated effects of stepwise and single step exposure to cryoprotectant on the developmental ability of vitrified oocyte in ethylene glycol-sucrose in presence of cumulus cells. The vitrified oocytes were evaluated by post thawing survival, in vitro maturation, in vitro fertilization and developmental capacity to 2-cell stage. We provide evidence that vitrification of immature mouse oocytes in a stepwise manner results in satisfactory maturation and fertilization rates.

Also, this study emphasizes that the connection between cumulus cells and the oocyte in the GV-COCs is important for completion of normal oocyte maturation in vitro. Partly dispersed cumulus cells in the oocytes after single step vitrification, shows that the connection between cumulus cells and oocytes may be disturbed during vitrification and result in lower in vitro maturation rates in this group. The role of cumulus cells on oocyte maturation, ovulation, and fertilization has been identified before (18).Our results show that the presence of cumulus cells is important for oocyte maturation and fertilization after vitrification. In agreement to our study, Abe et al (19) reported survival, fertilization, maturation and developmental rate of bovine GV-COCs with using Nylon-Mesh and exposure with stepwise cryoprotectant to be significantly higher (p < 0.05) compared with the single-step vitrification. Also, Anon et al (20),(21) reported higher survival, maturation and development rates when using ultrarapid vitrification accompanied with step-wise equilibration in mouse GV oocytes than single step vitrified group.

In conclusion, the introduction of vitrification and rapid freezing techniques is gaining widespread recognition and might become the procedure of choice over traditional slow freezing methods. This study shows that GV oocytes can be vitrified successfully with both single step and stepwise methods. But, better survival and maturation and developement rates can be obtained when GV oocytes are vitrified by stepwise procedure with conventional straws.

References

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. Smith G, Silva E and Silva C. Developmental consequences of cryopreservation of mammalian oocytes and embryos. Reprod Biomed Online 2004; 9: 171–78.
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. Yoon TK, Chung HM, Lim JM, Han SY, Ko JJ and Cha KY. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2000; 74:180–81.
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