Chun-Chia Huang

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

BACKGROUNDnCouples 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.nnnMETHODSnSixty 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).nnnRESULTSnAfter 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).nnnCONCLUSIONSnThe 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.nMethods: 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.nResults: 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.nConclusions: 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.

Association of creatin kinase B and peroxiredoxin 2 expression with age and embryo quality in cumulus cells

PurposeThe purpose of this study was to identify age-related oocyte or embryo markers suitable for non-invasive analysis, as women over 38xa0years of age experience diminished pregnancy and ovulation rates.MethodsWe used real-time quantitative PCR to examine the gene expression profiles in cumulus cells acquired from older and younger age groups. We selected 11 genes involved in three functions that directly affect cellular aging: cell cycle control, apoptosis, and metabolism.ResultsCKB and PRDX2 were up-regulated in women older than 38xa0years, and the expression of these genes in cumulus cells was associated with embryo quality. In good-quality embryos, CKB expression was higher in the cumulus cells acquired from both older and younger age groups than in poor-quality embryos.ConclusionsThese potential relationships among cumulus cell gene expression, oocyte quality, and age may expand our understanding of oogenesis and embryo development. CKB and PRDX2 may serve as biomarkers or therapeutic targets for the developmental potential of oocytes.

Evaluation of telomere length in cumulus cells as a potential biomarker of oocyte and embryo quality

STUDY QUESTIONnIs the relative telomere length in cumulus cells associated with embryo quality and the subjects age?nnnSUMMARY ANSWERnThe relative telomere length in cumulus cells at the time of oocyte collection may be a new potential biomarker for selecting highly competent oocytes and good quality embryos.nnnWHAT IS KNOWN ALREADYnTelomeres play central roles in aging and in determining cell fate. In mammalian ovarian follicles, maturing oocytes are nurtured and supported by surrounding somatic cells, the mural granulosa and cumulus cells.nnnSTUDY DESIGN, SIZE, DURATIONnA total of 350 oocyte-cumulus complex samples were collected from 80 IVF cycles prospectively recruited for this study at the Lee Womens Hospital, Taichung, Taiwan.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnCumulus cells were manually separated from the oocyte-cumulus complex under a microscope. DNA was extracted from cumulus cells and assessed for telomere length by real-time quantitative PCR. We analyzed telomere length relative to a single copy marker gene (36B4) to evaluate the effect of the real reproductive age of cumulus cells on oocyte and embryo development.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThe relative telomere length was longer in cumulus cells from mature oocytes compared with cumulus cells from immature oocytes, and in cumulus cells from good-quality embryos compared with cumulus cells from poor-quality embryos. The cut-off value of the T/S ratio between good and poor-quality embryos on embryonic Day 3 was 4.235.nnnLIMITATIONS, REASONS FOR CAUTIONnOnly a limited number of cumulus cells were measured for each oocyte and the corresponding embryo.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe relative telomere length in cumulus cells at the time of oocyte collection is predictive of highly competent oocytes and good-quality embryos but may not be sufficiently discriminating to be clinically useful.nnnSTUDY FUNDING/COMPETING INTEREST(S)nNational Science Council, Taiwan (NSC 97-2314-B-040-018). The authors have no conflicts of interest to declare.

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.

The influence of female age on the cumulative live-birth rate of fresh cycles and subsequent frozen cycles using vitrified blastocysts in hyper-responders.

OBJECTIVEnThe aim of this research was to study the influence of female age on the cumulative live-birth rate of fresh and subsequent frozen cycles using vitrified blastocysts of the same cohort in hyper-responders.nnnMATERIALS AND METHODSnThis was a retrospective study of 1137 infertile women undergoing their first in vitro fertilization treatment between 2006 and 2013. The main outcome measure was cumulative live births among the fresh and all vitrified blastocyst transfers combined after the same stimulation cycle. The results were also analyzed according to age (i.e., <35 years, 35-39 years, and ≥ 40 years).nnnRESULTSnThe mean number of retrieved oocytes was 19.9 ± 8.5 oocytes. The cumulative pregnancy rate was 89.2% and the cumulative live-birth rate was 73.3%. The cumulative live-birth rate declined from 73.9% for women younger than 35 years old to 67.3% for women 35-39 years old to 57.9% for women 40 years or older.nnnCONCLUSIONnCombined fresh and vitrified blastocyst transfer cycles can result in a high cumulative live-birth rate. The cumulative live-birth rates among older women are lower than the rates among younger women when autologous oocytes are used.

Effects of reactive oxygen species levels in prepared culture media on embryo development: A comparison of two media

OBJECTIVEnThis study determined the correlation between the levels of reactive oxygen species (ROS) in prepared culture media and the early development of human embryos.nnnMATERIALS AND METHODSnThis was an autocontrolled comparison study. A total of 159 patients undergoing inxa0vitro fertilization/intracytoplasmic sperm injection treatment were recruited in this study. The pH values, osmolarity pressures, and ROS levels of 15 batches of two culture media were measured. Sibling oocytes or embryos from individual patients were randomly assigned to two culture groups with Quinns Advantage Cleavage and Blastocyst media (QAC/QAB) or GIII series cleavage and blastocyst media (G1.3/G2.3). The difference between the two culture groups was analyzed using one-sample t test.nnnRESULTSnThe QAC/QAB and G1.3/G2.3 media exhibited similar pH values and osmolarity pressures. However, the prepared QAC/QAB media were characterized to contain lower amounts of ROS than the G1.3/G2.3 media. Furthermore, the blastocysts that developed under the QAC/QAB media were morphologically superior to those that developed under the G1.3/G2.3 media.nnnCONCLUSIONnThe elevated ROS levels in culture media were associated with poor development of blastocyst-stage embryos. Measurement of ROS levels may be a valuable process for medium selection or modification.

Nitric oxide modulates mitochondrial activity and apoptosis through protein S-nitrosylation for preimplantation embryo development

PurposePrevious 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.MethodsZygotes from superovulated B6CBF1 mice were cultured to blastocysts in a variety of media. Sodium nitroprusside (SNP) and NG-nitro-L-arginine (LNA) were added to the culture medium as a NO donor and a NO synthase inhibitor, respectively. The localization and fluorescence intensity of S-nitrosylated (SNO) proteins within 2-cell stage embryos were analyzed with confocal microscopy. Apoptosis and ATP production in the blastocysts were measured.Result(s)Subsequent to NO exposure, the SNO proteins mainly colocalized with the mitochondria and endoplasmic reticulum and the intensity of SNO proteins increased. The addition of a quanylate cyclase inhibitor and a cyclic GMP mimic agent induced nonsignificant changes in SNO proteins, whereas addition of a superoxide scavenger or a reduced form of glutathione rescued the embryos from the effects of NO. However, superoxide scavenger supplementation resulted in decreased blastocyst ATP production.Conclusion(s)Elevated NO exerts deleterious effects on embryo development, possibly through protein S-nitrosylation in the mitochondria and endoplasmic reticulum. Including glutathione as a component in the culture medium might counteract this effect.