Shee-Uan Chen

Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article.

Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.

Age is a better predictor of pregnancy potential than basal follicle-stimulating hormone levels in women undergoing in vitro fertilization

OBJECTIVE To analyze to what extent the parameters of ovarian functional reserve including female age and basal FSH levels will affect the results of ovarian hyperstimulation and IVF outcome. DESIGN Retrospective cohort study. University hospital infertility center. PATIENT(S) One thousand forty-five women undergoing their first cycle of IVF with ovarian stimulation after pituitary desensitization. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Cycle parameters, cancellation rate, implantation rate, and pregnancy rate. RESULT(S) Both increasing age and basal FSH were associated significantly with reduced numbers of oocytes collected, oocytes fertilized, and embryos transferred. The combined use of age and basal FSH significantly improves the predictive power for these parameters. Increasing age, but not basal FSH, was associated significantly with reduced implantation rate and pregnancy rate. Logistic regression analysis revealed that age, but not basal FSH, was an independent predictor of pregnancy rate. Neither age nor basal FSH had significant association with fertilization rate, miscarriage rate, or ectopic pregnancy rate. CONCLUSION(S) Both basal FSH and age contributed to the prediction of the quantitative ovarian reserve as reflected by the number of oocytes collected. However, age is a better predictor of pregnancy potential for women undergoing IVF.

Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws

OBJECTIVE To examine the effect of vitrification with ethylene glycol (EG) for mature human oocytes in straws. DESIGN Prospective, randomized, in vitro experiments. SETTING Reproductive unit of a university hospital. PATIENT(S) Immature oocytes from 110 patients undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S) The immature oocytes were incubated to reach metaphase II (MII). The MII oocytes were treated with EG-based cryoprotectants and vitrified in straws. They were diluted in sucrose solutions, inseminated by ICSI, and cultured in vitro. MAIN OUTCOME MEASURE(S) Survival, fertilization, and embryo cleavage. RESULT(S) The survival rates were greater for oocytes pretreated with 1.5 M of EG (65% for 0 minute, 93% for 5 minutes, and 96% for 10 minutes). The oocytes vitrified in 60 and 90 seconds had a greater rate of fertilization than those vitrified in 120 seconds. There were no differences in survival and fertilization for vitrified oocytes diluted by three or four steps. The cleavage rates to the six- to eight-cell stage were comparable with controls. However, no blastocyst formation was observed in vitrified oocytes. CONCLUSION(S) Vitrification of human oocytes with EG in straws achieves a high rate of survival, fertilization, and early cleavage of embryos. Further studies should be conducted for the improvement of blastocyst formation.

Decidual Natural Killer Cytotoxicity Decreased in Normal Pregnancy but Not in Anembryonic Pregnancy and Recurrent Spontaneous Abortion

PROBLEM: The natural killer (NK) cell activity is depressed in the decidua of early normal pregnancy. Recently Morii et al. (Am J Reprod Immunol 1993;29:1–4) found that all early intradecidual CD3+ T cells expressed either T cell receptor (TCR) α/β or γ/δ but that the expression of the CD3+/TCR complex was down‐regulated.

Decrease in interferon gamma production and impairment of T-lymphocyte proliferation in peritoneal fluid of women with endometriosis.

OBJECTIVE Our purpose was to verify regional immune modulations and to test the effect of gonadotropin-releasing hormone agonist in women with endometriosis. STUDY DESIGN Concentrations of peritoneal cytokines, including interleukin-1 beta, interleukin-2, soluble interleukin-2 receptor, interleukin-6, granulocyte-monocyte colony-stimulating factor, interferon gamma, and tumor necrosis factor-alpha were compared in women with and without endometriosis. Peritoneal cytokine and interleukin-2 production were examined by adding various mitogens to peritoneal fluid mononuclear cells of women with advanced endometriosis before and after gonadotropin-releasing hormone agonist treatment. RESULTS A significant increase in peritoneal interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha and a decrease in interferon gamma were noted in women with endometriosis. After gonadotropin-releasing hormone agonist treatment interleukin-6 decreased and interferon gamma increased. A significant impairment of interleukin-2 production of peritoneal fluid mononuclear cells by phytohemagglutinin and pokeweed mitogen stimulation was demonstrated in endometriosis, and production could be restored after gonadotropin-releasing hormone agonist treatment. CONCLUSION These results indicate that regional immunologic dysfunction might be invoked in the disease process of endometriosis.

Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)-embryo transfer (ET), but not that of IVF-tubal ET in patients with repeated IVF failures

OBJECTIVE To assess the effect of augmenting IVF with assisted hatching in the treatment of patients with repeated IVF failures. DESIGN Prospective randomized study. SETTING Division of Reproductive Endocrinology and Infertility of National Taiwan University Hospital. PATIENT(S) From July 1993 to February 1996, 49 patients with repeatedly failed IVF were treated with assisted hatching and were compared with 51 control subjects without assisted hatching. INTERVENTION(S) Assisted hatching. MAIN OUTCOME MEASURE(S) Pregnancy rate and implantation rate per embryo after IVF-ET or IVF-tubal ET (TET) were measured. RESULT(S) The pregnancy rate (PR) in the assisted hatching group was found to be 36.7% compared with 19.6% in the control group, but the difference was not significant. When only patients receiving IVF-ET were considered, it was observed that the PR was significantly higher in the assisted hatching group than the control group (42.4% versus 16.1%). With IVF-TET however, the PR was found to be similar in both assisted hatching and control groups (25.0% and 25.0%, respectively). The rate of embryonic implantation in the IVF-ET patients was 11.0%, which was significantly higher than that of control embryos (3.7%). CONCLUSION(S) These results implied that IVF-ET, combined with assisted hatching, may improve the PR and implantation rate in patients with repeated IVF failures, but the same was not true in the case of IVF-TET.

Slow Freezing or Vitrification of Oocytes: Their Effects on Survival and Meiotic Spindles, and the Time Schedule for Clinical Practice

Both the slow-freezing method with increased sucrose concentration and new vitrification techniques significantly improve the results of cryopreservation of human oocytes. The recent perfection for vitrification includes the concepts of increase of cooling and warming rates using minimum volume methods, and decrease of toxicity by reducing the concentration of cryoprotectants. In the recent literature, the survival of cryopreserved oocytes ranged from 74% to 90% using the slow-freezing method and from 84% to 99% by vitrification. Overall, the survival rate of oocytes from vitrification (95%, 899/948) appeared higher than that of the slow-freezing method (75%, 1,275/1,683). The microtubules of meiotic spindles are vulnerable to the thermal changes and will depolymerize. After incubation, the microtubules repolymerize. Spindle recovery is faster after vitrification than slow freezing. Even so, after 3 hours of incubation, spindle recuperation is similar between vitrification and slow freezing. Considering both aspects of spindle recovery and oocyte aging, the time schedule for oocyte cryopreservation program makes fertilization in the optimal time. Intracytoplasmic sperm injection is performed for oocytes at 3 hours of post-thaw incubation from the slow-freezing method and 2 hours from vitrification, with restoration of meiotic spindles. The pregnancy potential of cryopreserved oocytes is comparable to that of fresh oocytes or frozen embryos. Cryopreservation of oocytes would importantly contribute to oocyte donation and preservation of fertility for cancer patients.

Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos

Oocyte and embryo selection governs the success of assisted reproductive technologies. The imaging tools applied for selecting embryos may need to contain several key properties: noninvasiveness, high 3D resolution, and the contrast capability to provide as much information about the embryos as possible, such as spindle fibers, zona pellucida, and organelles. Currently adopted imaging techniques can only provide one or two of these desired properties and are with limited contrast of the embryos. Some image techniques can even damage the embryos. Previous studies have shown that harmonic generation microscopy (HGM), a virtual-transition based technology, can provide noninvasive imaging in zebrafish embryos with a sub-cellular 3D resolution and a millimeter penetration depth, and thus could be a suitable tool for future oocyte and embryo selection of assisted reproductive technologies. However to evaluate HGM in clinical use, the intrinsic contrast origin of the second harmonic generation (SHG) and third harmonic generation (THG) inside the mammal embryos has to be studied. In this work we performed HGM studies on the in vitro cultured mouse oocytes and embryos by combining the SHG and THG modalities, with a focus on the contrast origin evaluation. Through the noninvasive HGM imaging, we can clearly identify various structures in the whole oocytes and embryos, including spindle fibers, zona pellucida, polar bodies, cell membranes, and the laminated organelles in the cells. The origin of the THG contrast was further confirmed through the standard staining studies. Through SHG signals, we could not only observe the spindle fibers when the oocytes were arrested at metaphase II or during the cleavage of the embryos, but can also distinguish and analyze the thickness of the three layers of the zona pellucida. Combining two different higher-harmonic generation modalities, SHG and THG, HGM successfully revealed the sub-cellular structures of the whole mouse embryos with a high 3D spatial resolution.

Signal mechanisms of vascular endothelial growth factor and interleukin-8 in ovarian hyperstimulation syndrome: dopamine targets their common pathways

BACKGROUND Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovarian stimulation with massive ascites, pleural effusion and hemoconcentration. The pathophysiological signal mechanisms of OHSS are still unclear and merit further investigation. METHODS Various angiogenic cytokines of follicular fluid and ascites of patients with risk of OHSS were measured, and examined for inducing endothelial permeability. These include vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, basic fibroblast growth factor, tumor necrosis factor-alpha, IL-1alpha, IL-1beta and platelet-derived growth factor. We explore the molecular signal pathways of major contributing cytokines in granulosa-lutein cells and endothelial cells possibly involved in OHSS. RESULTS Neutralizing antibodies of VEGF or IL-8 significantly decreased follicular fluid- and ascites-induced endothelial permeability. Human chorionic gonadotrophin induced VEGF secretion of granulosa-lutein cells through the Sp1 and CREB dependent pathways. IL-8 activated CXCR1/2 of endothelial cells leading to VEGF receptor (VEGFR)-2 transactivation. Both VEGF and IL-8 of follicular fluid enhanced endothelial permeability via VEGFR-2-mediated Rho/Rock activation, actin polymerization and phosphorylations of VE-cadherin and occludin, resulting in opening of adherens junctions and tight junctions. Dopamine (2 microM) inhibited follicular fluid-induced VEGFR-2 signals and endothelial permeability, without diminishing migration and tube formation. CONCLUSIONS Our results suggest that VEGF and IL-8 secreted from corpora luteae may play major roles in OHSS. Delineation of signal pathways would be helpful for treatment. Dopamine may block VEGF- and IL-8-induced endothelial permeability by inhibiting common VEGFR-2 dependent signals.

Increase in the Production of Interleukin-10 Early After Implantation is Related to the Success of Pregnancy

PROBLEM: To study the correlation of interleukin (IL)‐10, IL‐11 leukemia inhibitory factor (LIF), placental growth factor (PlGF), and transforming growth factor (TGF)‐β and outcome of human pregnancy.
 METHOD OF STUDY: We prospectively measured the serum levels of these cytokines in patients undergoing in vitro fertilization (IVF) programs. A total of 60 women (non‐pregnant, n=27; early abortions, n=12; normal pregnancies, n=21) were enrolled.
 RESULTS: There was no difference in the cytokines studied on D0 and D14 among the three groups of women. The increase in PlGF from D0 to D14 after human chorionic gonadotropin (hCG) injection was greater in pregnant women than in non‐pregnant women; however, the difference did not reach significance (P=0.068). The increase in IL‐10 production from D14 to D21 was significant in women with successful pregnancies compared to women in the abortion group.
 CONCLUSIONS: This increase in IL‐10 may be important in sustaining a normal pregnancy early after implantation.