Chung-Hsien Liu

Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility

BACKGROUND Couples with unexplained infertility (UI) tend to have low fertilization rates with current IVF procedures. Here, we attempted to identify spermatozoa with apoptotic markers in couples with UI and unsuccessful intrauterine insemination (IUI) and we investigated the efficiency and benefit of magnetic-activated cell sorting (MACS) for sperm preparation in such patients. METHODS Sixty couples with UI and two IUI failures were recruited. The sperm were prepared by conventional density gradient centrifugation (DGC) and divided into two aliquots. One aliquot was used as a control and the other was further processed by MACS (D + M). Apoptotic markers were identified using fluorescence-labeled dye and flow cytometry, including externalization of phosphatidylserine (EPS), disrupted mitochondrial membrane potential (MMP) and DNA fragmentation. The fertilization potential of prepared spermatozoa was analyzed by basic semen analysis, computer-aided sperm analysis and the induced acrosome reaction test (IART). RESULTS After DGC, spermatozoa showed 18.6% EPS, 28.3% disrupted MMP and 13.5% DNA fragmentation. Numbers of spermatozoa with apoptotic markers were significantly reduced by D + M, versus DGC alone (P < 0.001). Although the motility of spermatozoa was slightly decreased after MACS, most sperm motion characteristics were not impaired. Interestingly, the IART significantly improved after D + M, versus DGC alone, especially for the couples with a normal hemizona assay (P < 0.001). CONCLUSIONS The spermatozoa prepared by D + M showed a reduced level of apoptotic markers. Improvement in the IART suggests a high fertilization potential of the processed spermatozoa. The identification of apoptotic markers and use of MACS may be helpful in directing the management plan for patients with UI and multiple IUI failures.

Impact of female age and male infertility on ovarian reserve markers to predict outcome of assisted reproduction technology cycles

BackgroundThis study was designed to assess the capability of ovarian reserve markers, including baseline FSH levels, baseline anti-Müllerian hormone (AMH) levels, and antral follicle count (AFC), as predictors of live births during IVF cycles, especially for infertile couples with advanced maternal age and/or male factors.MethodsA prospective cohort of 336 first IVF/ICSI cycles undergoing a long protocol with GnRH agonist was investigated. Patients with endocrine disorders or unilateral ovaries were excluded.ResultsAmong the ovarian reserve tests, AMH and age had a greater area under the receiving operating characteristic curve than FSH in predicting live births. Furthermore, AMH and age were the sole predictive factors of live births for women greater than or equal to 35 years of age; while AMH was the major determinant of live births for infertile couples with absence of male factors by multivariate logistic regression analysis. However, all the studied ovarain reserve tests were not preditive of live births for women < 35 years of age or infertile couples with male factors.ConclusionThe serum AMH levels were prognostic for pregnancy outcome for infertile couples with advanced female age or absence of male factors. The predictive capability of ovarian reserve tests is clearly influenced by the etiology of infertility.

DNA Fragmentation, Mitochondrial Dysfunction and Chromosomal Aneuploidy in the Spermatozoa of Oligoasthenoteratozoospermic Males

AbstractPurpose: This study determined the incidence of sperm nuclear DNA fragmentation, mitochondrial dysfunction, and chromosomal aneuploidy. The results were correlated with the semen analysis parameters and fertilization rates. Methods: Semen samples from 10 men showing oligoasthenoteratozoospermia (OAT) and undergoing ICSI treatment were analyzed. Another semen samples from 10 men showing normozoospermia and undergoing IVF treatment were analyzed for comparison. The samples were prepared using a two-step discontinuous Percoll gradient (80%–50%) and analyzed using a Hamilton-Thorne Integrated Visual Optical System (IVOS) Sperm Analyzer. DNA fragmentation was detected with a terminal deoxynucleotidyl transferase-mediated dUTP nick end label (TUNEL) assay. Functional integrity of mitochondria was detected using an ApoalertTM Mitochondrial Membrane Sensor Kit. Chromosomal aneuploidy was assayed by fluorescence in situ hybridization. Results: Higher sperm DNA fragmentation rate (18.8% vs. 2.8%), mitochondrial dysfunction rate (24.9% vs. 5.7%), and chromosomal aneuploidy rate (0.12% vs. 0.06%) were found in the oligoasthenoteratozoospermic patients in comparison with the normozoospermic patients. Conclusions: The result indicates that spermatozoa from oligoasthenoteratozoospermic patients contain greater DNA fragmentation, mitochondrial dysfunction, and chromosomal aneuploidy. Because extremely poor semen samples are the indication for ICSI treatment, the result indicates the importance of selecting good quality sperm for oocyte injection.

Blastocoel volume is related to successful establishment of human embryonic stem cell lines

Human embryonic stem cell (hESC) banks strive to establish hESC lines from discarded or surplus human embryos. The effect of embryo quality on establishing hESC lines was investigated by observing cultures derived from 28 Taiwanese fresh surplus and donated embryos that were cultured using the whole embryo method. Cultures of hESC lines were followed for 15 months. At the blastocyst stage, 14 of the 28 embryos were graded as good quality, defined as featuring a blastocoel volume of at least half of the embryo volume. Fourteen embryos did not meet these standards on day 5. Five successful hESC lines were derived from the good quality embryos (5/14; 35.7%); these hESC cells grew for 27-60 passages. In contrast, cells from poor quality embryos all stopped growing at the second or third passage. The successful hESC exhibited typical stem cell characteristics, including the capacity for pluripotent differentiation. Embryo quality on day 5, as defined by blastocoel volume, is thus a strong predictor for successful establishment of hESC lines.

The association between microenvironmental reactive oxygen species and embryo development in assisted reproduction technology cycles.

This study was designed to determine the relevance between the levels of reactive oxygen species (ROS) in microenvironment (follicular fluid or culture media) and the embryo development in IVF/ICSI cycles. A total of 466 follicles from 174 IVF/ICSI cycles were collected for this study. The ROS levels in monofollicular fluid and spent culture media were evaluated by chemiluminescence assay with luminol as a probe. The results demonstrated that it is in ICSI cycles that elevated ROS levels in follicular fluid were associated with day 3 poor embryo quality. The ROS levels in spent culture media were correlated with advanced degree of fragmentation. In addition, ROS levels in culture media, instead of in follicular fluid, were negatively correlated with implantation potential of embryos. The ROS levels in culture media may be viewed as an embryo metabolic marker and function as an adjuvant criterion for embryo selection.

Recipient age and pulsatility index affect uterine receptivity in oocyte donation programmes

Successful embryo implantation depends on interactions between the embryo and the uterus. Investigating factors related to the recipients in an oocyte donation programme could therefore improve the overall pregnancy outcome. In this study, the factors that affect outcomes following IVF after oocyte donation were evaluated in a retrospective cohort study. A total of 143 cycles were evaluated. All oocyte donors were younger than 30 years of age. In the youngest recipients (< 34 years old) the implantation rate was higher (P = 0.042) and the abortion rate was apparently lower than in the recipients of more advanced age (> or =40 years old). The implantation and pregnancy rates were higher when the pulsatility index was > or = 3.0. The pulsatility index on the day of embryo transfer in oocyte donation IVF cycles was an important determinant of successful pregnancy outcomes.

The association between polypronucleate zygote formation with certain motion characteristics of sperm and IVF outcome

PurposeTo determine the efficiency of sperm motion characteristics as predictors for normal (2PN) and polypronulceate (PPN) zygotes in IVF.MethodsA retrospective cohort analysis for a total of 230 couples undergoing IVF treatment in a single infertility center.Result(s)Subsequent to semen analysis and hemizona assay, unexpected fertilization failure would appear to have occurred only extremely rarely (1/236, 0.4%). The rate of PPN, however, did arise and appeared to be related to certain sperm motion characteristics, such as lateral head displacement and concentration of progressive motile sperm. Interestingly, the patients featuring a high PPN rate (>20%) was associated with a greater pregnancy rate than those featuring a low PPN rate (<20%).ConclusionThe sperm motion characteristics examined herein could be utilized to predict the rate of PPN in IVF. In order to enhance the rate of 2PN and maintain the relative high rate of clinical pregnancy, an efficient method needs further investigation and development.

Nitric oxide regualte mitochondrial activity and apoptosis through protein s-nitrosylation for preimplantation embryo development

Purpose Previous studies reported that patients with endometriosis had excess nitric oxide (NO) in the reproductive tract and poor embryo development in IVF cycles. This study aims to elucidate the effects of NO on early embryo development.