Kuang-Han Chao

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.

Activation status of T and NK cells in the endometrium throughout menstrual cycle and normal and abnormal early pregnancy

Endometrial lymphocytes were studied at all stages throughout the menstrual cycle and early pregnancy by flow cytometry to examine different lymphocyte subpopulations and the expression of the T- and NK-cell activation markers. After pregnancy, CD8+CD3+ lymphocytes were decreased in the decidua. In both endometrium and decidua, more T cells expressed CD69, CD71, HLA-DR, and CD38 antigens than in peripheral blood. After pregnancy, CD71+CD3+ lymphocytes were further increased. CD25+CD3+ lymphocytes decreased significantly in the endometrium and decidua of ectopic pregnancies, but not in the decidua of normal pregnancies. These findings indicate that T cells are regionally activated in the first trimester, and it may be the result of the stimulation by fetal antigens. NK cells were the most abundant cell type in the decidua, which expressed the phenotype CD16- CD56+, and CD57-CD56+. The proportion of activated decidual NK cells was increased in anembryonic pregnancies more than in normal pregnancies, although the total NK subpopulation was similar in both groups. This might result in increased NK cytotoxicity in anembryonic pregnancies. In conclusion, T cells are activated, but NK cytotoxicity is decreased in the decidua of early normal pregnancies. This might be important in the control of trophoblast growth and placental development.

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.

Increase in the expression of killer cell inhibitory receptors on peritoneal natural killer cells in women with endometriosis

OBJECTIVE Malfunction of peritoneal natural killer cells (NK) may result in endometriosis. The present study was designed to determine whether the decrease in NK cytotoxicity occurs at early and advanced stages of endometriosis and is due to the increase in the NK inhibition receptors. DESIGN A case control study. SETTING A tertiary-care infertility center . PATIENT(S) A total of 44 women (controls, n = 11; women with early-stage endometriosis, n = 11; and women with advanced-stage endometriosis, n = 22) were included in this study. INTERVENTION(S) Laparoscopic examination. MAIN OUTCOME MEASURE(S) NK cytotoxicity was determined by assay of (51)Cr release against K562 cells, and the expression of killer cell inhibitory receptors (KIR, including NKB1, GL183, and EB6) in NK cells was examined by flow cytometry. RESULT(S) Women with endometriosis showed a decrease in peritoneal NK cytotoxicities against K562 at early and advanced stages of endometriosis. The expression of KIR (NKB1 and EB6) was significantly elevated in the peritoneal NK cells of women with advanced-stage endometriosis compared with controls. KIR (NKB1) was also significantly increased in peritoneal NK cells of women with advanced-stage endometriosis, compared with those of women with early-stage endometriosis. CONCLUSION(S) The results of this study suggest that the decrease in peritoneal NK cytotoxicities against K562 is observed and that this disease may be partially due to the increased expression of KIR on these NK cells.

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.

Hormone replacement therapy reverses the decrease in natural killer cytotoxicity but does not reverse the decreases in the T-cell subpopulation or interferon-gamma production in postmenopausal women

OBJECTIVE To investigate the immunologic deviations of postmenopausal women before and after hormone replacement therapy (HRT). DESIGN Prospective study. SETTING University teaching hospital. PATIENT(S) Seventeen postmenopausal women (study group) and 17 women of reproductive age (control group). INTERVENTION(S) Continuous usage of E(2) valerate 2 mg/d and medroxyprogesterone acetate 5 mg/d in postmenopausal women in the study group. MAIN OUTCOME MEASURE(S) Immunophenotyping with flow cytometry, cytokine production with and without mitogen stimulation of the peripheral mononuclear cells, and a natural killer (NK) cell cytotoxicity test against K562 target cells by the (51)Cr-release assay were performed in the control group and in the study group before, 1 month after, and 6 months after HRT. RESULT(S) NK cytotoxicity, interferon-gamma production, and the T-cell subpopulation were significantly decreased, and the subpopulations of CD3(+)CD25(+) and CD3(+)HLA-DR(+) were increased in the study group before HRT when compared with those in the control group. After HRT was given for 6 months, however, the NK cytotoxicity increased significantly in the postmenopausal women to a value similar to that of the control group. CONCLUSION(S) Women after menopause are prone to impaired immune responses. Nevertheless, some of the impairment can be restored after HRT.