Soon-Chye Ng

Fertilizing ability of DNA-damaged spermatozoa

In order to investigate the fertilizing ability of DNA-damaged sperm, they were exposed to gamma radiation prior to insemination. The presence of DNA-strand breaks were detected by the TUNEL test. Fertilization rates of 64.3, 59.9, 58.5, and 61.1% were achieved when sperm were subjected to 5, 10, 50, and 100 GY, respectively. This rate was 53.2% in the control group with no significant difference (P > 0.01). The blastocyst development was decreased from 49.8% in the control group to 20.3, 7.8, 3.4, and 2.3% with sperm exposed to doses of 5, 10, 50, and 100 GY, respectively. Of the transferred blastocyst in the control group, 69.8% were implanted and 33.9% developed into live fetuses. These rates were 57.1 and 21. 4%, 20 and 0% when sperm were exposed to doses of 5 and 10 GY with a significant difference (P < 0.01). The present study clearly shows that DNA-damaged sperm (regardless of degree of damage) have the ability to fertilize the oocyte, but that embryonic development is very much related to the degree of DNA damage. However, the oocyte has the capacity to repair DNA damage of sperm when it is damaged less than 8%. Damage beyond this level will result in low rate of embryonic development and high early pregnancy loss. J. Exp. Zool. 284:696-704, 1999.

The sperm centriole: its inheritance, replication and perpetuation in early human embryos

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage. The sperm proximal centriole is introduced into the oocyte at fertilization and remains attached to the expanding spermhead during sperm nuclear decondensation, as it forms the male pronucleus. A sperm aster is initially formed after the centriole duplicates at the pronuclear stage. At syngamy, centrioles occupy a pivotal position on opposite spindle poles, when the first mitotic figure is formed. Bipolar spindles were found in the majority of embryos, while tripolar spindles were seen in four dispermic embryos at syngamy. Two single centrioles were detected at two poles of two tripolar spindles, while two additional centrioles were located on the sides of a bipolar spindle of a dispermic embryo. Sperm tails were detected near spindle poles at syngamy and in later embryos. Typical centrioles showing the characteristic pin-wheel organization of nine triplets of microtubules were evident. During centriolar replication, the daughter centriole grows laterally from the parent and gradually acquires pericentriolar material (PCM). The two centrioles are surrounded by a halo of electron-dense PCM, which nucleates microtubules, thus making it a typical centrosome. The usual alignment of diplosomes at right angles to each other was maintained. Centrioles were detected at all stages of embryonic cleavage from the 1-cell through 8-cell stages, right up to the hatching blastocyst stage. They were closely associated with nuclei at interphase, when they were often replicating, and were prominently located at spindle poles during the first four cell cycles. In blastocysts, they were detected in trophoblast, embryoblast and endoderm cells respectively. It is evident that the sperm centrosome is the functional active centrosome in human, while the female is inactive but may contribute some centrosomal material to the zygote centrosome. It is very likely that the paternal centriole is the ancestor of the centrioles in fetal and adult somatic cells.

Cocultures : a new lead in embryo quality improvement for assisted reproduction

One of the contributory causes to poor PRs in assisted reproduction has been the decreased viability of transferred embryos and the transfer of four-cell embryos into an environment that naturally would be receptive only to 5-day-old blastocysts. In this paper, we have reviewed our own work and that of others on the role of tubal ampullary cells (cocultures) to mimic the in vivo environment to bring about improved embryo quality and an increased number of blastocysts for replacement in IVF patients. The establishment, maintenance, and behavior of human tubal cell lines is first presented, followed by their use as cocultures for fertilization and cleavage of embryos. The mode of action, specificity, and cryopreservation of ampullary cells are also discussed. The currently available results of pregnancies after cocultures are presented together with future aspects of research that are necessary to refine the coculture system. The ultimate aim is to mimic in vivo conditions in vitro, so that at least the PRs of assisted conception can be parallel to normal fecundity in the human. Therefore, a very attractive future includes the freezing of blastocysts generated from coculture, thawing, and replacing them in natural cycles.

Improved pregnancy rate after transfer of embryos grown in human fallopian tubal cell coculture

OBJECTIVE To evaluate the embryonic behavior in vitro and the pregnancy and implantation rates of embryos grown in a human ampullary cell coculture system. DESIGN In a prospective study, two pronuclei embryos were cultured on human ampullary feeder layers up to the two to six-cell and blastocyst stages and replaced either as tubal, uterine, or sequential transfers. SETTING Assisted reproductive technology program in a university-based hospital. PATIENTS Fifty women with a mean age of 35.6 years who went through a single coculture cycle. Thirty of the patients were admitted for in vitro fertilization (IVF) and 20 for tubal embryo transfer (TET). RESULTS The overall clinical pregnancy rate (PR) for all 50 patients was 44% per cycle (IVF, 37%; TET, 55%) and the implantation rate was 31.8% (IVF, 31.0%; TET, 32.6%). Sixty-eight percent of pregnant patients were over 35 years, and 68% had two previously failed assisted reproduction cycles. Five of 9 patients who received sequential transfers became pregnant. Three of the 22 pregnancies aborted (2 after sequential transfer), and there was one ectopic. Overall, 88% of two to six-cell stage embryos were of good quality. CONCLUSIONS The human ampullary coculture system produces better quality embryos, increased numbers of blastocysts with improved PRs and implantation rates. The beneficial effects of the feeder layer may be through the release of embryotrophic factors and detoxification of the medium by the cells. Coculture is a new concept in assisted reproduction and has tremendous potential in boosting conception rates by mimicking the in vivo environment.

AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo

A major problem in gene therapy and tissue replacement is accessibility of tissue‐specific stem cells. One solution is to isolate tissue‐specific stem cells from differentiating embryonic stem (ES) cells. Here, we show that liver progenitor cells can be purified from differentiated ES cells using alpha‐fetoprotein (AFP) as a marker. By knocking the green fluorescent protein (GFP) gene into the AFP locus of ES cells and differentiating the modified ES cells in vitro, a subpopulation of GFP+ and AFP‐expressing cells was generated. When transplanted into partially hepatectomized lacZ‐positive ROSA26 mice, GFP+ cells engrafted and differentiated into lacZ‐negative and albumin‐positive hepatocytes. Differentiation into hepatocytes also occurred after transplantation of GFP+ cells in apolipoprotein‐E‐ (ApoE) or haptoglobin‐deficient mice as demonstrated by the presence of ApoE‐positive hepatocytes and ApoE mRNA in the liver of ApoE‐deficient mice or by haptoglobin in the serum and haptoglobin mRNA in the liver of haptoglobin‐deficient mice. This study describes the first isolation of ES‐cell‐derived liver progenitor cells that are viable mediators of liver‐specific functions in vivo.

Effect of Sperm Motility on Human In Vitro Fertilization

Several sperm motility parameters in semen prepared by the swim-up technique were compared with IVF rates in 84 patients. The patients were either on clomiphene + human menopausal gonadotrophin or follicle stimulating hormone + human menopausal gonadotrophin stimulation regimens. Motility ratings were assessed both manually according to World Health Organization guidelines as well as computer-automated semen analysis (Cellsoft, Cryoresources, USA). Motility ratings of greater than or equal to 2 yielded significantly higher fertilization rates (78-82%) than ratings below 2 (20-23%) (p less than 0.001) for patients on both regimens. Velocity (41, 55, 78 microns/sec) and mean amplitude of lateral head displacement (1.96, 3.29, 4.91 microns) correlated significantly with and between manual ratings of 1, 2, and 3, respectively (r = 0.83; p less than 0.01). No significant differences were observed in linearity and beat/cross frequency between the manual ratings, although beat/cross frequencies tended to reduce linearly with increases in intensity of motility. The velocity of sperm motility has a significant effect on fertilization rates, and cut-off points of greater than or equal to 2 or greater than or equal to 50 microns/sec predict the actual potential and likely success of in vitro fertilization. These criteria on the swim-up semen should be used in the selection of patients admitted to IVF programs, and they justify the necessity of research investigations to improve motility in those patients with sluggish motility.

Microinjection of human oocytes: a technique for severe oligoasthenoteratozoospermia *

Objective To determine outcome after microinjection with very poor quality sperm and after failed fertilization. Design Group 1, fresh oocytes from patients with very low sperm density and motility on the day of oocyte recovery; Group 2, 1-day-old oocytes that failed to fertilize. Setting All material was obtained from the National University Hospital. Patients One hundred and thirty-one from group 1; 35 from group 2. Interventions Sperm was injected subzonally or directly into the ooplasm. Main Outcome Measure Normal and abnormal fertilization and pregnancy. Results Subzonal transfer was done on 771 oocytes in group 1 and 188 oocytes in group 2. Multiple sperm were transferred [mean of 6.6 for group 1 and 7.3 for group 2]. Monospermic fertilization occurred in 16.6% and 14.9%, respectively. Polyspermy and parthenogenetic activation were low at 2.3% and 2.8%, respectively. Five pregnancies were obtained, but only one delivered. Ooplasmic injection (single sperm heads) was done in 38 oocytes from three patients with extremely severe oligozoospermia; only four two-pronuclear zygotes were obtained and replaced into two patients, without any resulting pregnancy. Conclusions Subzonal transfer may be a viable technique for patients with severe sperm problems.

Micromanipulation: its relevance to human in vitro fertilization

Micromanipulation has been practiced for almost a century in invertebrates and lower animals. But in the past 2 decades, many micromanipulation procedures were initiated with the mouse embryo, including removal of the zona pellucida, dissociation of blastomeres, aggregation of denuded embryos and blastomeres, injection of single or aggregated cells into the cavity of early blastocysts, and insertion of isolated blastomeres into foreign zona pellucidae. Micromanipulation in the human will probably be limited to the following: (1) diagnosis and correction of genetic diseases and chromosomal disorders, and (2) assisting fertilization in severe oligozoospermia or motility problems of the spermatozoa

An overview and synopsis of techniques for directing stem cell differentiation in vitro

The majority of studies on stem cell differentiation have so far been based in vivo, on live animal models. The usefulness of such models is limited, since it is much more technically challenging to conduct molecular studies and genetic manipulation on live animal models compared to in vitro cell culture. Hence, it is imperative that efficient protocols for directing stem cell differentiation into well-defined lineages in vitro are developed. The development of such protocols would also be useful for clinical therapy, since it is likely that the transplantation of differentiated stem cells would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated stem cells. The in vitro differentiation of stem cells, prior to transplantation in vivo, would also avoid spontaneous differentiation into undesired lineages at the transplantation site, as well as reduce the risk of teratoma formation, in the case of embryonic stem cells. Hence, this review critically examines the various strategies that could be employed to direct and control stem cell differentiation in vitro.

Intracytoplasmic injection of frozen-thawed epididymal spermatozoa in a nonhuman primate model, the cynomolgus monkey (Macaca fascicularis)

Intracytoplasmic sperm injection (ICSI) with frozen-thawed epididymal spermatozoa was performed in the cynomolgus monkey (Macacafascicularis) to produce embryos in vitro. Eleven sexually mature females were hyperstimulated with an GnRH agonist (1.8 mg active triptorelin per 2 kg body weight), followed (2 weeks later) by rFSH (37.5 IU per 2 kg daily) for 12 days, and finally 1000 IU of hCG. Epididymal spermatozoa were collected from a single adult male monkey. The first stimulation cycle resulted in 90 oocytes; 70% of which were metaphase II (MII). Sixty-four percent of these MII oocytes were fertilized. Comparing ovarian response of five monkeys that underwent a second stimulation cycle there was an increase in oocyte quantity (13.2 versus 9.2 oocytes per monkey) but the percentage of MII oocytes remained the same at 58%. Fertilization and cleavage rates were also reduced but there was an increase in the number of embryos available for transfer. Overall, four monkeys became pregnant resulting in the birth of two healthy infants and two abortions. These findings show that ovarian stimulation by GnRH-rFSH did not compromise the developmental competence of the oocytes; effective combination of cryopreservation of epididymal spermatozoa and ICSI is possible in nonhuman primate reproduction, and thus has potential application in the conservation of highly endangered nonhuman primate species, and the cynomolgus monkey is a reliable biomedical research model to study the potential risks and benefits associated with assisted reproductive techniques prior to approval for clinical trials on humans.