The primary focus of this division is to undertake research, postgraduate and doctoral level training in the area of Clinical Embryology. In India, the last two decades have seen an exponential growth of infertility clinics that use techniques requiring handling of spermatozoa or the oocyte outside the body, or the use of a surrogate mother. However, there is a tremendous shortage of trained clinical embryologists and researchers serving assisted reproductive centers.
Post Graduate program
Manipal University was first to introduce Master of Clinical Embryology (M.Sc.) in 2005 aiming to provide thorough knowledge, research experience, ethics and skills related to human embryology.
The main goal of the Masters in Clinical Embryology is to offer graduates in science the opportunity to gain the necessary experience to undertake employment in diverse areas of reproduction. This is the first and only postgraduate program of its kind being offered in whole of Asia. This program consists of didactic lectures, discussions, research projects, hands-on training and exposure to various practical procedures used in the Assisted Reproductive Technology Laboratory.
Major objectives of M.Sc. Clinical Embryology program:
The objectives of this course are to provide
• up-to-date knowledge in the areas of Assisted Reproductive Technology
• the skills required for working in IVF and micromanipulation clinics
• an appreciation of the ethics and attitude towards human assisted reproduction
• integration of the clinical and scientific aspects of embryology
• advanced knowledge and research exposure in specific areas in clinical embryology through a thesis dissertation
• adequate training to take up Ph.D. course in the area of Clinical Embryology
Ph.D program:
The goal of the Ph.D. program in Clinical Embryology is to train students to become leaders in the field and preparation for careers in research and academic medicine. The program focuses on the role of Clinical Embryology in the diverse fields of clinical and basic sciences. Specific training areas emphasize gamete biology, preimplantation embryogenesis, gamete and embryo manipulation, sperm, oocyte and embryo cryopreservation and fertility preservation.
The PhD program also combines interdisciplinary collaboration, developmental and molecular biological technology in basic and applied areas to investigate the pathophysiology of mammalian reproduction. More details about this may be obtained from
clinical.embryology@manipal.edu
Major areas of Research:
The general area of our research is sperm and oocyte disorders, chromatin abnormalities and improving functional competence of gametes and embryos to improve overall reproductive outcome. Following projects have been initiated in these areas:
Evaluation of sperm characteristics, sperm methylation errors and reproductive toxicity in radiation health workers
Recently, concern has been expressed on the modification of sperm DNA due to various endogenous and exogenous genotoxic factors including ionizing radiation. There is convincing evidence from animal studies that exposure of males to radiation can have detrimental effects on sperm chromatin which affects the fertilization, embryo viability and health of the offspring. Furthermore, in the recent observations made in our laboratory in collaboration with Kyoto University, Japan has shown p53 and p21 dependent checkpoint response during mouse early embryogenesis in the embryos derived from the sperm DNA damage induced by ionizing radiation. This was followed by the excessive apoptosis in the inner cell mass of the blastocyst resulting in defective implantation and postimplantation embryonic demise suggesting the influence of radiation-induced sperm DNA damage on the adverse reproductive outcome. Although earlier studies have demonstrated decreased conception rate, increased incidence of still birth, anomalies in children of parents who are occupationally exposed to radiation and altered reproductive health among radiographers, till date there have been no reports on the incidence of sperm functional, chromatin abnormalities and methylation errors among radiation health professionals. Individuals carrying DNA damaged sperm may father children, however, fertilization with such spermatozoa may increase the risk of miscarriage or genetic abnormalities in the offspring. In the clinical set up, the assessment of fertility still relies on the traditional method of standard semen analysis, which does not address integrity of the male genome. Therefore, this study has been undertaken to evaluate sperm abnormalities in the radiation health workers and then find out its association with the incidence of infertility and miscarriages in their partners. The outcome of the proposed research is expected to provide information on the influence of occupational radiation exposure on the sperm characteristics, methylation pattern and sperm functional competence.
Sperm DNA damage in male infertility and its influence on reproductive outcome
The integrity of sperm DNA can be affected by various endogenous and exogenous genotoxic factors. Recently, concern has been expressed in developed countries about the influence of sperm DNA integrity on the abnormal reproductive outcome. Although infertile men may father children with assisted conception, fertilization with DNA-damaged spermatozoa may increase the risk of genetic abnormalities offspring. In India, male factor infertility remains a significant problem contributing 50% of cases attending infertility clinics and its assessment still relies on the traditional method of standard semen analysis, which does not address integrity of the male genome. Since there have been no scientific studies carried out on the level of sperm DNA damage and its effect among Indian population, the result from the proposed study is expected to contribute information on the level of sperm DNA integrity in the infertile men seeking assisted conception with the hope of identifying some clues on possible association between sperm DNA damage with failed conception, repeated miscarriage and fetal abnormalities.
The impact of chromosomal and sub-chromosomal sperm abnormalities on the embryonic development and Assisted Reproductive Technology (ART) outcome
A male factor is implicated in more than 50% of couples treated with In vitro fertilization (IVF). However, neither the routine testing of male fertility potential nor its treatment addresses the specific mechanisms by which spermatozoal factors may impact upon reproductive outcome. Recently, concern has been expressed on the implications of various acquired spermatozoal factors like morphological disorders, numerical chromosomal abnormalities and DNA damage on the adverse reproductive outcome. The recent observations made by our group and others have clearly shown that the damage in mouse spermatozoa can lead to delayed chromosomal instability in blastocysts and subsequent developmental abnormalities. Based on these observations, now we would like to test whether similar trend exists in patients undergoing IVF treatment for male factor or idiopathic infertility as these groups of patients are known to have high incidence of chromosomal and sub-chromosomal abnormalities in their sperm. The specific objectives of this study are 1) to test the impact of chromosomal and sub-chromosomal sperm abnormalities on the embryogenesis and overall reproductive outcome 2) to develop a non invasive tool for the assessment of the embryos derived from the abnormal spermatozoa. There are many fundamental aspects in human embryos in response to fertilization with abnormal sperm are not elucidated till now due to ethical restrictions in performing invasive techniques. Hence here we combine mouse model with human patients to address the issues on the impact of sperm quality on embryogenesis and also to develop a non invasive quality assessment tool for the better selection of healthy embryos so that complications associated with implantation of abnormal embryos would be minimized.
Evaluation of laser manipulated embryos for their hatching potential, metabolic changes and laser induced effects
Repeated implantation failure in the in vitro fertilization (IVF) treatment is a growing concern and fertility clinics worldwide perform laser manipulation of the embryos prior to the embryo transfer so that chances of successful hatching, implantation and consequent pregnancy are expected to increase. However, the observations from the recent randomized controlled studies are contradictory and do not demonstrate any significant benefits of hatching on the pregnancy rate possibly due to technical differences in the protocols followed by the clinic or due to detrimental effects of laser manipulation on the embryos. Furthermore due to technical and ethical limitations, there is no report on how hatching takes place in human embryos after laser manipulation. Since human and mouse embryos have similarities in their size, behavior, and zonapellucida architecture, we use mouse as the study model to evaluate various laser manipulation techniques employed in IVF practice to evaluate their consequences on early embryo development The results of this investigation are expected to help fertility specialists to improve the embryo implantation potential and pregnancy rate by applying appropriate laser manipulation techniques. Although non contact diode lasers are being used in the embryo manipulation, recently, concern has been expressed on the safety of these techniques especially laser manipulation of cryopreserved embryos as these embryos experience extensive stress during the process of freeze-thawing. We hypothesize that laser energy delivered on the embryos during manipulation even at lowest level may have impact on the metabolic process and subsequent embryonic/fetal development. Hence we use non-invasive Nuclear magnetic resonance spectroscopy (NMR) based fingerprinting of spent embryo culture medium to assess the influence of laser hatching on the metabolism of human and mouse embryos. Besides, laser manipulated embryos are analyzed for the expression pattern of damage response genes in the embryos. The results from this study are expected to help fertility experts by acquiring valuable information on the appropriate technique for the manipulation with minimum or no adverse effects.
Intermediates of calcium signal process as oocyte activating agents and development of an optimum culture medium for mouse parthenotes
Embryonic stem cells (ESCs) hold great potential in cell and tissue replacement therapy while treating various pathological conditions. However, the ethical and technical hurdles in obtaining the human embryos for deriving the stem cell lines have urged the scientists to look for alternate sources to derive the ESCs. Therefore, parthenogenically activated embryos have a tremendous potential in replacing the normally fertilized embryos and overcome all the ethical hurdles associated with it. In addition to this, these embryos are good model to understanding the role of paternal and maternal factors on early embryo development.
Several agents have been used in the past to induce parthenogenesis in oocytes. Alcohol, strontium, electrical shock, calcium channel blockers and protein kinase inhibitors have been used successfully. However, these parthenotes are known to have very poor developmental potential. High degree of fragmentation and low blastocyst rate has been observed in mouse parthenotes. It is interesting to know whether this is due to 1) toxicity of the inducing agent, 2) absence of paternal factors in the activated oocyte, 3) altered metabolic requirement of activated oocytes.
Inositol trisphosphate (IP3) is a second messenger which is involved in calcium mediated signal transduction.Activation of membrane bound phospholipase C (PLC) leads to the formation of IP3 and di-acyl glycerol (DAG). During oocyte maturation, the number of IP3 receptors on the endoplasmic reticulum increases (Lisa et al, 1996) which regulates the calcium oscillations during oocyte activation. The entry of sperm activates phospholipase C enzyme which will produce IP3 and DAG. Therefore using these intermediates directly may induce calcium oscillation in oocyte and activate them. In addition, using IP3 and DAG may have minimum toxicity since they are the obligatory intermediates of calcium metabolism and therefore may act as an ideal parthenogenesis inducing agent. Our main focus is on 1) to study the efficacy of IP3 and DAG alone or in combination in oocyte activation, 2) to design an optimum culture medium to enhance to blastocyst rate in parthenotes, 3) to study the effect of sperm derived factors in culture media on development of parthenotes.