Mulyoto Pangestu

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

Cryopreservation of ovarian tissue is an important option for preserving the fertility of cancer patients undergoing chemotherapy and radiotherapy. In this study, we examined the viability and function of oocytes derived in vitro from pre-antral follicles as an alternative method for restoring fertility. Pre-antral follicles (specified as secondary follicle with a diameter around 100-130 μm) were mechanically isolated from vitrified-warmed and fresh adult mouse ovarian tissues and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. Oocytes were then released from these follicles, fertilized in vitro, and cultured to the blastocyst stage and vitrified. After storage in liquid nitrogen for 2 weeks, groups of vitrified blastocysts were warmed and transferred into pseudo-pregnant recipient females. Although most of the isolated mouse pre-antral follicles from fresh (79.4%) and vitrified (75.0%) ovarian tissues survived the 12-day in vitro culture period, significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than from the fresh controls (62.2 vs 86.4%, P<0.05). No difference was observed in embryo cleavage rates between these two groups, but the proportion of embryos that developed into blastocysts in the vitrification group was only half that of the controls (24.2 vs 47.2%, P<0.05). Nevertheless, live births of healthy normal pups were achieved after transfer of vitrified blastocysts derived from both experimental groups. This study shows that successful production of healthy offspring using an in vitro follicle culture system is feasible, and suggests that this procedure could be used in cancer patients who wish to preserve their fertility using ovarian tissue cryopreservation.

Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice

Ovarian tissue cryopreservation and transplantation can be used to preserve fertility for cancer patients. In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by solid surface vitrification or traditional slow-cooling using various in vitro and in vivo techniques, including allotransplantation, in vitro oocyte maturation, embryo culture in vitro, blastocyst cryopreservation, embryo transfer, and development. The importance of cumulus cells for oocyte maturation, fertilization, and embryo development was investigated. Graft recovery, follicle survival, and oocyte retrieval was similar in control, vitrified, and slow-cooled groups. High rates of oocyte maturation, cleavage, and blastocyst formation were achieved, with no significant differences between the control, vitrified or slow-cooled ovarian tissue grafts. The presence of cumulus cells was important for oocyte maturation, fertilization, and subsequent development. Cumulus-oocyte complexes with no surrounding cumulus cells (N-COCs) or with an incomplete layer (P-COCs) had significantly lower rates of oocyte maturation and blastocyst formation than cumulus-oocyte complexes with at least one complete layer of cumulus cells (F-COCs; maturation rate: 63, 78 vs 94%; blastocyst rate: 29, 49 vs 80%). Live births were achieved using vitrified blastocysts derived from oocytes taken from vitrified and slow-cooled ovarian tissue heterotypic allografts. Successful production of healthy offspring from these vitrified blastocysts suggests that this technique should be considered as a useful stage to pause in the assisted reproduction pathway. This provides an alternative protocol for restoring fertility and offering cancer patients a better indication of their chances of pregnancy and live birth.

Indonesian infertility patients’ health seeking behaviour and patterns of access to biomedical infertility care: an interviewer administered survey conducted in three clinics

BackgroundIndonesia has high levels of biological need for infertility treatment, great sociological and psychological demand for children, and yet existing infertility services are underutilized. Access to adequate comprehensive reproductive health services, including infertility care, is a basic reproductive right regardless of the economic circumstances in which individuals are born into. Thus, identifying and implementing strategies to improve access to assisted reproductive technology (ART) in Indonesia is imperative. The principle objectives of this article are to improve our understanding of infertility patients’ patterns of health seeking behaviour and their patterns of access to infertility treatment in Indonesia, in order to highlight the possibilities for improving access.MethodsAn interviewer-administered survey was conducted with 212 female infertility patients recruited through three Indonesian infertility clinics between July and September 2011. Participants were self-selected and data was subject to descriptive statistical analysis.ResultsPatients identified a number of barriers to access, including: low confidence in infertility treatment and high rates of switching between providers due to perceived treatment failure; the number and location of clinics; the lack of a well established referral system; the cost of treatment; and patients also experienced fear of receiving a diagnosis of sterility, of vaginal examinations and of embarrassment. Women’s age of marriage and the timing of their initial presentation to gynaecologists were not found to be barriers to timely access to infertility care.ConclusionsThe findings based on the responses of 212 female infertility patients indicated four key areas of opportunity for improving access to infertility care. Firstly, greater patient education about the nature and progression of infertility care was required among this group of women. Secondly, increased resources in terms of the number and distribution of infertility clinics would reduce the substantial travel required to access infertility care. Thirdly, improvements in the financial accessibility of infertility care would have promoted ease of access to care in this sample. Finally, the expansion of poorly developed referral systems would also have enhanced the efficiency with which this group of patients were able to access appropriate care.

Haptic Virtual Reality Training Environment for Micro-robotic Cell Injection

Micro-robotic cell injection is typically performed manually by a trained bio-operator, and success rates are often low. To enhance bio-operator performance during real-time cell injection, our earlier work introduced a haptically-enabled micro-robotic cell injection system. The system employed haptic virtual fixtures to provide haptic guidance according to particular performance metrics. This paper extends the work by replicating the system within a virtual reality (VR) environment for bio-operator training. Using the virtual environment, the bio-operator is able to control the virtual injection process in the same way they would with the physical haptic micro-robotic cell injection system, while benefiting from the enhanced visualisation capabilities offered by the 3D VR environment. The system is achieved using cost-effective components offering training at much lower cost than using the physical system.

Confirmed dioestrus in pseudopregnant mice using vaginal exfoliative cytology improves embryo transfer implantation rate.

Embryo transfer is a commonly performed surgical technique. In mice, protocols typically specify pairing recipient females with vasectomized males to induce a receptive uterine environment for embryo implantation. However, this induced receptive state is not always maintained until implantation occurs. The use of a well-characterized correlation between oestrous state and exfoliative vaginal cytology was therefore evaluated to assess uterine receptivity immediately before embryo transfer. Eight- to 12-week-old virgin female CD1 mice (n = 22) were paired overnight with vasectomized males and successfully mated, indicated by the presence of a vaginal plug. These dams underwent embryo transfer 3 days later with embryos obtained from superovulated 4-week-old F1 (C57BL/6 × CBA) females. Non-invasive vaginal lavage was conducted immediately before transfer. Dams were killed 6 days after transfer and the uterus collected for histological analysis. Embryo implantation rate in mice was 96% when cytological analysis of the lavage samples signified dioestrus (n = 6), whereas the implantation rate was <15% (n = 16) when cytology signified other stages of oestrous. This simple, quick, non-invasive measure of receptivity was accurate and easily adopted and, when applied prospectively, will avoid unnecessary surgery and subsequent culling of non-suitable recipients, while maximizing the implantation potential of each recipient female.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.

Haptic Technology for Micro-robotic Cell Injection Training Systems—A Review

AbstractCurrently, the micro-robotic cell injection procedure is performed manually by expert human bio-operators. In order to be proficient at the task, lengthy and expensive dedicated training is required. As such, effective specialized training systems for this procedure can prove highly beneficial. This paper presents a comprehensive review of haptic technology relevant to cell injection training and discusses the feasibility of developing such training systems, providing researchers with an inclusive resource enabling the application of the presented approaches, or extension and advancement of the work. A brief explanation of cell injection and the challenges associated with the procedure are first presented. Important skills, such as accuracy, trajectory, speed and applied force, which need to be mastered by the bio-operator in order to achieve successful injection, are then discussed. Then an overview of various types of haptic feedback, devices and approaches is presented. This is followed by disc...

Micro-robotic Cell Injection Training in a CAVE

This paper focuses on the design of an evaluation made to a large-scale virtual reality micro-robotic cell injection training system. The aim of the evaluation is to empirically investigate the usability and effectiveness of three distinct display configurations and the input controller employed in the system. The data was gathered through a set of experiments with human participants. Participants’ performance against metrics such as success rate and magnitude of error was considered in the evaluation. For the experiments, participants were randomly divided into six equal sized groups where each group was provided with a specific combination of display configuration and haptic guidance mode. The participants performed ten injections and the time and position of the virtual micropipette tip were recorded. Data was analysed using descriptive statistics and performance comparison between groups was conducted. Additionally three groups also underwent two subsequent sessions, training and post-training, as a basis to evaluate the effectiveness of the training with haptic guidance by comparing participants’ performance before and after the training session. The implementation of the designed evaluation has contributed to the conclusions drawn which suggest the proposed large-scale virtual reality system as a feasible training tool for micro-robotic cell injection procedure, and recommendations for future work are proposed.

Evaluation of a new virtual reality micro-robotic cell injection training system

Abstract This study considers a virtual reality (VR) micro-robotic cell injection training system developed to reduce the time and cost required for a trainee to become proficient in cell injection. The VR environment replicates a micro-robotic cell injection setup to be interacted with and controlled using either a keyboard or haptic device. Using these two input control methods, user training evaluation experiments were designed and conducted to evaluate trainee performance. The performance improvement of 13 participants after undergoing training was analyzed. Results demonstrate that the participants attained higher accuracy and success rates when utilizing the haptic device control method than when applying the keyboard control method. All participants successfully performed the required task when employing the haptic device control method with haptic guidance enabled.

Large-scale Virtual Reality micro-robotic cell injection training

Currently the micro-robotic cell injection procedure is performed manually by professional bio-operators. It is a challenging task requiring advanced skills including the ability to precisely control the movement of a micropipette. Developing these skills requires both lengthy and intensive training, and significant practical experience. This paper extends upon our previous work in desktop Virtual Reality (VR) cell injection training to introduce a large-scale VR micro-robotic cell injection system. Through utilization of large visual displays and the large workspace INCA 6D haptic device, the proprioception related to large arm movements (and corresponding visual representation) and the resulting movement of the micropipette in relation to the cell aims to provide the user with a better understanding of the spatial relationship between the micropipette and cell. The haptic device can be operated either with or without haptic guidance. When enabled, haptic guidance is provided in the form of virtual fixtures (VFs) and force feedback to assist the user in following the ideal trajectory towards the penetration point, applying appropriate force for penetration and stopping the micropipettes tip at the suitable deposition point. A user evaluation was conducted to study the usability of the system. Eighteen participants took part in the experiments and were randomly divided into six groups based on the display and haptic guidance modes assigned. The results demonstrated that the large-scale VR micro-robotic cell injection system is a feasible and effective method for bio-operator training where it is suggested that the skills and knowledge acquired can be transferred to the real-world task.