Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo.

Bone marrow mesenchymal stromal cells (BMMSCs) have been used as feeder support for the ex vivo expansion of hematopoietic stem cells (HSCs) but have the limitations of painful harvest, morbidity, and risk of infection to the patient. This prompted us to explore the use of human umbilical cord Wharton's jelly MSCs (hWJSCs) and its conditioned medium (hWJSC-CM) for ex vivo expansion of HSCs in allogeneic and autologous settings because hWJSCs can be harvested in abundance painlessly, are proliferative, hypoimmunogenic, and secrete a variety of unique proteins. In the presence of hWJSCs and hWJSC-CM, HSCs put out pseudopodia-like outgrowths and became highly motile. Time lapse imaging showed that the outgrowths helped them to migrate towards and attach to the upper surfaces of hWJSCs and undergo proliferation. After 9 days of culture in the presence of hWJSCs and hWJSC-CM, MTT, and Trypan blue assays showed significant increases in HSC numbers, and FACS analysis generated significantly greater numbers of CD34(+) cells compared to controls. hWJSC-CM produced the highest number of colonies (CFU assay) and all six classifications of colony morphology typical of hematopoiesis were observed. Proteomic analysis of hWJSC-CM showed significantly greater levels of interleukins (IL-1a, IL-6, IL-7, and IL-8), SCF, HGF, and ICAM-1 compared to controls suggesting that they may be involved in the HSC multiplication. We propose that cord blood banks freeze autologous hWJSCs and umbilical cord blood (UCB) from the same umbilical cord at the same time for the patient for future ex vivo HSC expansion and cell-based therapies.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Differential regulation of cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzyme mRNA expression by gonadotrophins and cyclic AMP in human granulosa cells.

The co-ordinated biosynthesis of progesterone and oestradiol in the human ovary is critical for reproductive cyclicity and eventual pregnancy. The crucial regulatory enzymes for progesterone and oestradiol biosynthesis in granulosa cells are the cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzymes respectively. We utilized the cDNA sequences encoding P450arom and P450scc to examine the roles of FSH and LH, and their intracellular second messenger, cyclic AMP (cAMP), in regulating steroidogenic gene expression. Mature granulosa cells (aspirated before the onset of the endogenous LH surge) and granulosa lutein cells (obtained after an ovulatory dose of human chorionic gonadotrophin) were cultured for 4 days with FSH, LH or dibutyryl cAMP (dbcAMP). After the period of culture, total RNA was extracted from granulosa cells and Northern analyses were performed utilizing 32P-labelled cDNAs encoding P450arom and P450scc. Spent culture media were analysed for steroid and cAMP content. Both FSH and LH strongly stimulated P450arom mRNA expression and oestradiol production in mature granulosa cells. On the other hand, P450scc mRNA expression and progesterone biosynthesis were weakly induced by FSH; maximal synthesis occurred only in the presence of LH. With both gonadotrophins at equivalent concentrations, LH generated a 30-fold higher level of cAMP than FSH. Furthermore, the differential effects of FSH and LH on P450 mRNA expression were reproduced by the presence of low and high concentrations of dbcAMP respectively. LH (and high levels of dbcAMP) increased P450arom mRNA expression in mature granulosa cells but inhibited its accumulation in granulosa lutein cells. In contrast, it stimulated P450scc mRNA expression and progesterone synthesis in both mature granulosa and granulosa lutein cells. Therefore, FSH/low cAMP levels stimulated P450arom gene expression and oestradiol production, while LH/high cAMP levels maximally induced P450scc gene expression and function, in a development-related manner consistent with steroid production in vivo. These findings support the hypothesis that one set of genes (like P450arom) in human granulosa cells is regulated by FSH/low cAMP levels and another (like P450scc) by LH/high cAMP levels.

Effect of growth hormone therapy in men with severe idiopathic oligozoospermia.

Some studies have suggested that growth hormone (GH) may enhance folliculogenesis in women, and similarly may enhance spermatogenesis in men with hypogonadotrophic hypogonadism. In this prospective open-controlled pilot study, we investigated the effect of daily subcutaneous GH for 5 months in 12 endocrinologically normal men with severe idiopathic oligozoospermia (< 10 million/ml). All the men had normal karyotype and endocrine tests, including a GH response of > 20,000 mU/l to insulin hypoglycaemia. Nine men with similar sperm counts acted as controls. During treatment, each patient was examined monthly, asked for side effects and had glycosylated haemoglobin, glucose and blood counts monitored. Five semen samples were obtained in the 4 months before treatment, two samples per month during treatment and three samples after stopping treatment. The mean insulin-like growth factor I (IGF-I) was normal before treatment and 1 month after ending treatment, at 206 and 182 micrograms/l, respectively, but increased significantly during treatment to 444 micrograms/l (p < 0.0001, ANOVA). The mean (SD) sperm counts were 2.6 (2.5), 2.5 (3.7) and 2.3 (2.1) million/ml before, during and after GH treatment, respectively, and did not show any statistically significant differences (ANOVA). We conclude that GH does not increase or decrease sperm counts in men with severe idiopathic oligozoospermia.

Cocultures in human assisted reproduction. Support of embryos in vitro and their specificity.

Assisted reproductive techniques (ART) have contributed tremendously to alleviating infertility in childless couples. However, the "take-home baby" rate for in vitro fertilization (IVF) has been much below the rate for normal fertile couples and for the other tubal procedures such as gamete intrafallopian transfer (GIFT) and tubal embryo transfer (TET). It was therefore speculated that the tubal environment might be playing an important role towards the viability of the embryo and that, in IVF, 4-6 cell stage embryos that were usually replaced directly into the uterus might not be receiving the beneficial effects of the tubal environment. Pronuclear stage human and mouse embryos were thus cocultured with reproductive and other somatic cell types to evaluate the quality of the various cleaving stages and the percentage yield of blastocysts. Human ampullary cell cultures were established in 6 to 7 days and kept alive through one menstrual cycle. Cleavage to the compacted and cavitating stages was achieved in 78% and 69%, respectively, of human embryos cocultured in 24-48 hour human ampullary subcultures as compared to 50% and 33%, respectively, for embryos grown in culture medium alone. The percentages of expanded blastocysts and hatching stages in such cocultures were not significantly different from those of controls. However, human ampullary and cumulus and mouse ampullary and muscle fibroblast cultures supported cleavage and development of mouse embryos up to the hatching stages as well as or better than controls. It thus appears that there is no absolute specificity for a reproductive tract source of cocultured cells and that the beneficial effects of ampullary and cumulus cells are not species-specific. The coculture system also appears to overcome the embryonic blocks of the human and mouse, which may be mediated via growth factors that activate the embryonic genome.

Myocyte transplantation for myocardial repair: a few good cells can mend a broken heart.

Cell transplantation is a potential therapeutic approach for patients with chronic myocardial failure. Experimental transplantation of neonatal and fetal cardiac myocytes showed that the grafted cells can functionally integrate with and augment the function of the recipient heart. Clinical application of this approach will be limited by shortage of donors, chronic rejection, and because it is ethically contentious. By contrast skeletal myoblasts (satellite cells) are abundant and can be grafted successfully into the animal's own heart even after genetic manipulation in vitro. Functional integration of myoblasts, however, is hampered by the lack of intercellular gap junction communication and the difference in excitation-contraction coupling between skeletal and cardiac myocytes. In experimental studies several other cell types have been used to augment cardiac function. In this review we discuss the published results of myocyte transplantation with emphasis on potential sources of cells, the ethics of using donor embryonic and fetal cardiomyocytes, genetic transformation of skeletal myoblasts for myocardial repair, and the functional benefits of cell transplantation to the failing heart.

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.

Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm.

OBJECTIVE: To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. DESIGN: Prospective. SETTING: Assisted reproductive technology (ART) program. PATIENTS: Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. RESULTS: Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P less than 0.01). Zona-free oocytes had higher fertilization rates than zona-intact oocytes (48.1% to 60.9% versus 20.0% to 32.2%). Multiple pronuclei were high in zona-free oocytes (33.1% to 41.3%). Forty-eight to 54% of embryos generated after donor insemination had chromosome anomalies (mosaicism, aneuploidy, pulverization). CONCLUSIONS: One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients.

Human embryonic behavior in a sequential human oviduct-endometrial coculture system.

Embryonic behavior to blastocyst, hatching, and hatched stages were evaluated in 77, four-cell human embryos that were first grown in oviductal cell coculture and then equally allotted at the eight-cell stage to two coculture systems in a serum-free medium (34 continued on oviductal monolayers, 32 on endometrium monolayers). Sixty-three percent and 40% of embryos expanded and hatched in the sequential oviductal-endometrial coculture system when compared with 41% and 9% in the oviductal system alone, respectively. The sequential coculture system appears to be an improved system over the single human oviductal coculture system.

The evaluation of various culture media in combination with dimethylsulfoxide for ultrarapid freezing of murine embryos.

Bicarbonate-buffered HTF medium, Medicult, and T6 are as effective as PB1 medium when used in combination with DMSO in ultrarapid freezing of two-cell mouse embryos. However, the use of phosphate-buffered T6 results in reduced in vitro development and inner cell mass size as compared with bicarbonate- and Hepes-buffered T6 when used with 3.5 M of DMSO. Hence, the use of this media for ultrarapid freezing should be avoided when this concentration of DMSO is used.

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.

Preimplantation genetics: chromosomes of fragmented human embryos.

STUDY OBJECTIVE: To examine the chromosome makeup of fragmented human embryos. DESIGN: Prospective. SETTING: Assisted reproductive technology (ART) program. PATIENTS: One hundred twenty-one poor-quality embryos from 58 patients 31 to 40 years of age admitted for an ART program were examined for chromosome makeup. RESULTS: Chromosome anomalies were observed in 31.9% (29/91) of poor-quality embryos, 19.8% (18/91) displayed mosaicism (diploid/haploid, diploid/triploid, diploid/aneuploid), 5.5% (5/91) showed polyploidy, 2.2% (2/91) had pulverized chromosomes, 2.2% (2/91) revealed aneuploidy, 1.1% (1/91) had prematurely condensed chromosomes, and 1.1% (1/91) had structural rearrangements involving chromosome number 2. The mean age of patients showing anomalies (36.5 years) was not significantly higher than the mean for the entire group (35.5 years). CONCLUSIONS: The incidence of chromosome anomalies in fragmented human embryos is high. These anomalies originate either in the gametes or through mitotic nondisjunction within the embryos. It is not advisable to replace such embryos into patients going through in vitro fertilization.

Transfer of human sperm into the perivitelline space of human oocytes after zona-drilling or zona-puncture.

To evaluate the transfer of sperm from severely oligozoospermic men into the perivitelline space of mature oocytes, zona-drilling with acid phosphate-buffered saline (PBS) and direct zona-puncture in the presence of cytochalasin D were studied. Zona-drilling also was done for eggs from patients with previous failed in vitro fertilization (IVF). Forty-seven eggs from seven patients with oligozoospermia and three patients with failed IVF had a mean of between 2.6 and 3.6 sperm transferred into the perivitelline space. In the group whose eggs had zona-drilling with acid PBS, 1 of 13 eggs fertilized from the oligozoospermic category, while there was no fertilization from the failed IVF category. Karyotyping of the unfertilized eggs after zona-drilling revealed a high incidence (2/5 and 3/7, respectively) of possible arrest at anaphase II after reinitiation of meiosis. In the group whose eggs were directly punctured through the zona in the presence of cytochalasin D, there was no fertilization in 23 undamaged eggs. Two of the 15 interpretable karyotypes were aneuploid, but this incidence is within that observed for our unfertilized eggs after IVF. Hence, the use of acid PBS for zona-drilling is not advised. Moreover, transfer of sperm from men with previous failed fertilization resulted in poor fertilization rates.

The use of amniotic fluid and serum with propanediol in freezing of murine 2-cell embryos.

Human and mouse embryos have been cultured in amniotic fluid (AF). Human AF and human serum (HS) are used in the freeze-thaw of 2-cell mouse embryos. Two hundred seventy-five 2-cell embryos were collected into phosphate-buffered saline with 20% HS and 20% AF and into 100% HS and AF. The embryos were cooled with propanediol as cryoprotectant at a controlled rate. After thaw, they were cultured in T6 with 3 mg/ml bovine serum albumin. Blastocyst formation post-thaw was 56/79, 44/70, 51/61, and 56/79 of intact embryos from 20% HS, 20% AF, pure HS, and pure AF (NS). But blastocyst hatching was better from embryos frozen in pure HS (22/61, compared with 16/79 for 20% HS; P less than 0.05). Hence there is no advantage in using AF in freeze-thaw, but pure HS may be of use.

Chromosome analysis of two-cell mouse embryos frozen by slow and ultrarapid methods using two different cryoprotectants.

Three freezing methods (slow-1,2 propanediol; ultrarapid-dimethyl sulphoxide; ultrarapid-1,2 propanediol) were used to assess the effects of low temperature storage on morphologic features and chromosome make-up of 2-cell mouse embryos. Weekly batches (15 weeks) of 2-cell mouse embryos were frozen for a period of 7 days using these three freezing methods, then thawed and subjected to chromosome analysis. After thawing, all three freezing regimens yielded 70.2% to 75.8% of healthy intact 2-cell embryos, and 5.5% to 8.1% of embryos with one healthy blastomere intact, respectively, out of 817 embryos examined. Chromosome analysis was possible in all 1- and 2-cell embryos. The incidence of aneuploidy and polyploidy in frozen-thawed embryos of all three methods (2.8% to 3.4%; 4.0% to 6.5%) was not significantly different from that of control unfrozen embryos (3.0%; 6.0%) (P greater than 0.01). Mitotic crossing over was observed in 3.5% of embryos frozen-thawed by the ultrarapid-dimethyl sulphoxide method only. It is not clear how the mitotic crossing over was induced by ultrarapid freezing in the presence of dimethyl sulphoxide.

Improved sperm concentration, motility, and fertilization rates following Ficoll treatment of sperm in a human in vitro fertilization program.

Duplicate sperm samples from the spouses of 54 patients admitted to an in vitro fertilization program were prepared by the swim-up and a simplified procedure using Ficoll. Cellsoft (CRYO Resources, New York, NY) sperm curvilinear velocities (microns/sec) and mean amplitude of lateral head displacement values (microns) equivalent to grade 1 and 2 visual sperm motility were significantly higher for Ficoll as compared with swim-up samples (P less than 0.01). Fertilization rates were significantly higher in the Ficoll as compared with the swim-up group for poor semen samples (grade less than 2: 58% versus 24%; P less than 0.01) and normal semen samples (grade greater than or equal to 2: 85% versus 78%; P less than 0.05). Ficoll sperm separation appears to be an excellent method of yielding increased fertilization rates in in vitro fertilization programs.

Effects of peritoneal macrophages from women with endometriosis on endometrial cellular proliferation in an in vitro coculture model.

OBJECTIVE: To study the effects of peritoneal macrophages on endometrial cellular proliferation in an in vitro coculture model and to compare the magnitude of these effects between macrophages from women with endometriosis and normal women. DESIGN: Controlled study of peritoneal macrophage function. SETTING: University hospital. PATIENT(S): Patients with a normal peritoneal cavity (n = 15) and with pelvic endometriosis (n = 20) undergoing laparoscopy. INTERVENTION(S): Peritoneal macrophages were cocultured with endometrial epithelial and stromal cells; endometrial cell cultures without macrophage coculture acted as controls. MAIN OUTCOME MEASURE(S): Endometrial cellular proliferation measured by 3H-thymidine incorporation. RESULT(S): Endometrial epithelial cells cocultured with peritoneal macrophages from women with endometriosis showed significantly increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.56 versus 1.03 times, respectively, over control) and at 72 hours (1.55 versus 1.10 times over control). Endometrial stromal cells cocultured with peritoneal macrophages from women with endometriosis similarly exhibited increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.65 versus 1.17 times over control) and at 72 hours (1.65 versus 1.21 times over control). CONCLUSION(S): Peritoneal macrophages of patients with endometriosis stimulate cellular proliferation of endometrial epithelial and stromal cells in vitro.

Comparative evaluation of fresh and washed human sperm cryopreserved in vapor and liquid phases of liquid nitrogen.

Infectious organisms such as hepatitis B were recently shown to survive in liquid nitrogen. To prevent cross-contamination of semen samples via liquid nitrogen, studies were undertaken to evaluate human sperm survival in the vapor phase of liquid nitrogen at -189 degrees C. The study was conducted in 2 separate experiments. In the first experiment, a total of 30 unwashed, fresh semen samples (15 normozoospermic and 15 oligozoospermic) were evaluated for motility, vitality, and morphology after freeze-thaw survival in vaporous (-189 degrees C) and liquid nitrogen (-196 degrees C; control) phases. Similar evaluations were carried out in a second experiment on 27 samples (15 normozoospermic and 12 oligozoospermic) that were previously washed by the swim-up method. Motile sperm recovery rates were significantly different between liquid and vapor phases (unwashed, normozoospermic: 42.76% +/- 3.23% vs 45.52% +/- 4.44%, P < .05; washed, normozoospermic: 34.44% +/- 4.41% vs 37.58% +/- 3.90%, P < .05; unwashed, oligozoospermic: 16.53% +/- 3.34% vs 18.25% +/- 4.36%, P < .05; washed, oligozoospermic: 10.32% +/- 2.54% vs 12.25% +/- 2.81%, P < .05). Recovery rates for motility were much higher for unwashed samples compared with washed semen samples. In all experiments the recovery of normal and live forms showed no significant differences between the vapor and liquid nitrogen storage phases (P > .05). The results demonstrate that both washed and unwashed human sperm survive satisfactorily with good recovery in the vapor of liquid nitrogen and can be recommended for future storage in medically assisted conception programs.

Does sperm morphology play a significant role in increased sex chromosomal disomy? A comparison between patients with teratozoospermia and OAT by FISH.

Eight semen samples from men with teratozoospermia (T), along with samples from 3 men with normal fertility and 5 samples from men with oligoasthenoteratozoospermia (OAT) were analyzed for X and Y chromosomal anomalies with the use of fluorescently labeled centromeric probes. This study was undertaken to determine whether patients with abnormal sperm morphology (teratozoospermia) have increased or decreased incidence of a sex chromosomal anomaly. The sex chromosome disomy for the T samples was 0.36% and for the OAT group it was 0.61%, compared with baseline value for the normozoospermia group (0.09%).