Human micro-insemination by injection of single or multiple sperm: ultrastructure

The process of micro-insemination by single or muhiple spermtransfer into the perivitelline space (PVS) or by direct sperminjection into oocytes was examined by transmission electronmicroscopy. Spermatozoa from normal and oligozoospermic menwere injected into oocytes, obtained from consenting IVF patients,mostly by zona-puncture using micromanipulators. Spermatozoawere washed by the Percoll or Ficoll methods and capacitatedusing Whittingham's T₆ or modified Tyrode's medium or incubatedin strontium medium before injection. The women were stimulatedby three IVF methods and oocytes were recovered by laparoscopyor ultrasonography. Sixty-one oocytes were cultured in T₆ orHam's F-10 media (3–24 h) and were subjected to micromanipulation.Four oocytes were also studied after zona-drilling. Normal 2-pronuclearova were developed after single-sperm transfer satisfying allmorphological criteria of fertilization. Both monospermic andpolyspermic fertilization resulted after multiple sperm transfer,indicating that a vitelline block to polyspermy may exist inhumans. The majority of oocytes examined were unfertilized.Spermatozoa with intact or reacted acrosomes and those undergoingthe acrosome reaction were found in the PVS and in the ooplasm.Abnormal spermatozoa were also seen in these locations. Quantitatlonof acrosomal status in 16 oocytes after multiple-sperm transfer,revealed that 24% of spermatozoa were acrosome reacted or reactingin the PVS following Ficoll entrapment, while 76% of spermatozoawere intact (33% of these abnormal). Sperm transfer seemed tobe the least invasive, while direct sperm injection was comparativelydestructive to oocytes. Drilling with acid made larger breachesin the zona when compared with mechanical perforation and spermatozoaoccasionally escaped through breaches. Three 2-pronuclear ovaobtained after multiple sperm transfer have resulted in twopregnancies, in cases of severe oligozoo spermia, during thecourse of this study.     

Micro-centrifugation of human spermatozoa: its effect on fertilization of hamster oocytes after micro-insemination spermatozoal transfer

Micro-centrifugation (MC) at 6500 r.p.m. (3352 g) has not beenused previously for spermatozoal concentration and subsequentfertilization. We investigated MC for micro-insemination spermatozoaltransfer (MIST) of human spermatozoa from normal donors intohamster oocytes. MC resulted in reduced penetration of hamsteroocytes, both after MIST [77.4% (41/53) versus 87.8% (43/49)for control; NS] and after exposure to zona-free hamster oocytes[60.8% (79/130) versus 72.7% (88/121) for control; P < 0.05].However, MIST under the zona resulted in better incorporationof sperm nuclei when compared with zona-free hamster oocytes,for spermatozoa exposed to micro-centrifugation (77.4 versus60.8%, P < 0.05) and controls (87.8 versus 72.7%, P <0.05). Polyspermy was higher after MIST [22.0% (9/41) versus13.9% (11/79); NS] for MC + , and [25.6% (11/43) versus 13.6%(12/88); NS] for MC-. We conclude that MC does have a negative,but minimal effect on the fertility of human spermatozoa withrespect to hamster oocytes.     

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.     

The Therapeutic Potential, Challenges and Future Clinical Directions of Stem Cells from the Wharton’s Jelly of the Human Umbilical Cord

Mesenchymal stem cells (MSCs) from bone marrow, adult organs and fetuses face the disadvantages of invasive isolation, limited cell numbers and ethical constraints while embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) face the clinical hurdles of potential immunorejection and tumorigenesis respectively. These challenges have prompted interest in the study and evaluation of stem cells from birth-associated tissues. The umbilical cord (UC) has been the most popular. Hematopoietic stem cells (HSCs) harvested from cord blood have been successfully used for the treatment of hematopoietic diseases. Stem cell populations have also been reported in other compartments of the UC viz., amnion, subamnion, perivascular region, Wharton’s jelly, umbilical blood vessel adventia and endothelium. Differences in stemness characteristics between compartments have been reported and hence derivation protocols using whole UC pieces containing all compartments yield mixed stem cell populations with varied characteristics. Stem cells derived directly from the uncontaminated Wharton’s jelly (hWJSCs) appear to offer the best clinical utility because of their unique beneficial properties. They are non-controversial, can be harvested painlessly in abundance, proliferative, possess stemness properties that last several passages in vitro, multipotent, hypoimmunogenic and do not induce tumorigenesis even though they have some ESC markers. hWJSCs and its extracts (conditioned medium and lysate) also possess anti-cancer properties and support HSC expansion ex vivo. They are thus attractive autologous or allogeneic agents for the treatment of malignant and non-malignant hematopoietic and non-hematopoietic diseases. This review critically evaluates their therapeutic value, the challenges and future directions for their clinical application.     

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.     

Evaluation of human sperm function after repeated freezing and thawing

Sperm storage via freezing has been useful for men who have difficulty masturbating during assisted reproductive technology (ART) programs and before impotency caused by chemotherapy, vasectomy, and other procedures. Studies were undertaken to evaluate the extent of cryoinjury to sperm after repeated freezing and thawing. The results showed that normozoospermic and oligozoospermic sperm survived after 3 repeated freeze-thaw cycles. The inclusion of seminal plasma did not seem to protect human sperm during freezing and thawing. There were no significant differences in recovery percentages for motile, vital, and morphologically normal sperm between slow and rapid freezing methods in thaws 1, 2, and 3 of normozoospermic and oligozoospermic unwashed (u), washed (w), and washed + seminal plasma (ws) samples. However, there were significant percentage drops in the recovery of motile and vital sperm between each thaw (ie, first to second thaw, and second to third thaw) using both slow and rapid freezing for u, w, and ws samples (P < .01). There were also no significant differences in percentage recovery of motile, vital, and morphologically normal sperm between u, w, and ws samples during thaws 1 to 3 in the normozoospermic and oligozoospermic groups. Sperm were capable of fertilizing hamster oocytes microinjected with single sperms after 3 freeze-thaw cycles as evidenced by the formation of 2 distinct pronuclei and 2 polar bodies in 22.2% and 17.2% of normozoospermic and oligozoospermic samples, respectively. The numbers of normal vital motile sperm after 3 serial freeze-thaw cycles are adequate for bringing about fertilization via intracytoplasmic sperm injection in ART programs. Thus, leftover washed sperm in laboratories that perform in vitro fertilization can be frozen, thawed, and refrozen several times without loss of the sperms' ability to fertilize. This approach has tremendous benefits for men who have difficulty producing sperm and for those with low and declining sperm counts.     

Fetal Blood Sampling and Its Complications Related to the Indications for Fetal Blood Sampling

Summary: A prospective study on fetal blood sampling (FBS) was conducted in the Fetomaternal Medicine Division of the Department of Obstetrics and Gynaecology at the National University Hospital, Singapore. FBS was performed on 159 occasions in 156 women between January, 1988 and December, 1991. The aim of this study was to identify the factors that were associated with an adverse outcome following the procedure.
Twenty four abnormal pregnancies were terminated; of the remaining 132 desired pregnancies the overall pregnancy loss was 44 (33.3%), which included those within 2 weeks and those after 2 weeks of the procedure and neonatal deaths. Fetal loss occurring within 2 weeks of the procedure is considered a procedure-related loss which occurred in 19 (14.3%) of the 132 pregnancies. When the fetal loss occurred within 2 weeks of the procedure 89% had a major abnormality on ultrasonographic scanning.
The conclusion from our study is that the risks of FBS were increased in abnormal pregnancies, most likely due to the underlying pathology.     

Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes

BACKGROUND: Myocardial infarction results in the death of cardiomyocytes, which are replaced by scar tissue. Cardiomyocytes cannot regenerate because they are terminally differentiated. Mesenchymal cells are pluripotent cells, which have the potential to differentiate to specialized tissues under appropriate stimuli. The aim of this study was to direct differentiation of the adult mesenchymal stem cells isolated from fatty tissue into cardiomyocytes using 5-azacytidine. METHODS: Adult mesenchymal stem cells were isolated from the fatty tissue of New Zealand White rabbits and cultured in RPMI medium. Second-passaged mesenchymal cells were treated with various concentrations of 5-azacytidine and incubated for different intervals of time. The cells were plated in six-well dishes at 500, 5,000, and 50,000 cells/well. These cells were treated with 1-, 3-, 6-, 9-, and 12-micromol/L concentrations of 5-azacytidine and incubated for 12, 24, 48, and 72 hours. Later, the medium was replaced with fresh medium and incubated in a CO2 incubator. The medium was changed once at 3 to 4 days. At 2 months, the cells were fixed with 0.4% glutaraldehyde for 2 hours and later washed with phosphate-buffered saline. The transformed cells were subjected to immunostaining for the myosin heavy chain, alpha actinin, and troponin-I. RESULTS: After treatment with 5-azacytidine, the adult mesenchymal stem cells were transformed into cardiomyocytes. At 1 week, some cells showed binucleation and extended cytoplasmic processes with adjacent cells. At 2 weeks, 20% to 30% of the cells increased in size and formed a ball-like appearance. At 3 weeks, these cells began to beat spontaneously in culture when observed under phase contrast microscope. Immunostaining of the transformed cells for myosin heavy chain, alpha actinin, and troponin-I was positive. The differentiated cells maintained the phenotype and did not dedifferentiate up to 2 months after treatment with 5-azacytidine. CONCLUSIONS: These observations confirm that adult mesenchymal stem cells isolated from fatty tissue can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.