Maw-Sheng Lee

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.

Laser-assisted hatching of embryos is better than the chemical method for enhancing the pregnancy rate in women with advanced age

OBJECTIVE Assisted hatching may enhance embryo implantation. This study was conducted to examine the efficacy of the laser- and chemical-assisted hatching for promotion of implantation (IR), pregnancy (PR), and delivery rate (DR) in older women undergoing IVF cycles. DESIGN Prospective study. SETTING An IVF unit of a medical center. PATIENT(S) A total of 601 embryos from 141 women aged > or =38 years underwent controlled ovarian hyperstimulation (COH) and assisted hatching. INTERVENTION(S) The study population was divided into two groups: group 1 had laser-assisted hatching (n = 85) and group 2 had chemical-assisted hatching (n = 56). Before the transfer, the day 3 embryos were hatched by using a 1.48-microm noncontact diode laser or acid Tyrodes solution. MAIN OUTCOME MEASUREMENT(S) The IR, PR, and DR between the groups were compared. RESULT(S) There were no statistical differences between groups in age, E2 concentrations during hCG administration, gonadotrophin dosage, embryo grade, the numbers of oocytes retrieved, oocytes fertilized, and embryos transferred. Higher IR, PR, and DR were noted in the laser-assisted hatching group. The IR, PR, and DR were: group 1, 8.2%/31.8%/24.7% and group 2, 3.8%/16.1%/10.7%, respectively. CONCLUSION(S) Laser-assisted hatching of embryos is more effective than the chemical method in enhancing the IR and PR of women with advanced age. The laser system allows an easier, faster, and safer micromanipulation of the zona pellucida, which provided a better method in zona drilling.

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

STUDY QUESTION Is the relative telomere length in cumulus cells associated with embryo quality and the subjects age? SUMMARY ANSWER The 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. WHAT IS KNOWN ALREADY Telomeres 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. STUDY DESIGN, SIZE, DURATION A 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. PARTICIPANTS/MATERIALS, SETTING, METHODS Cumulus 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. MAIN RESULTS AND THE ROLE OF CHANCE The 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. LIMITATIONS, REASONS FOR CAUTION Only a limited number of cumulus cells were measured for each oocyte and the corresponding embryo. WIDER IMPLICATIONS OF THE FINDINGS The 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. STUDY FUNDING/COMPETING INTEREST(S) National Science Council, Taiwan (NSC 97-2314-B-040-018). The authors have no conflicts of interest to declare.

A single-nucleotide polymorphism of the DAZL gene promoter confers susceptibility to spermatogenic failure in the Taiwanese Han

BACKGROUND Deleted in AZoospermia-like (DAZL) is an autosomal homologue of Y chromosome-linked DAZ gene located on chromosome 3p24. DAZL is only expressed in the gonads and is critical to germ cell development in different species. However, the regulation of DAZL has not been explored. METHODS Reporter assays, electrophoretic mobility shift assays, supershift assays and bisulfate sequencing were used to identify the core promoter region of DAZL. Sequence analysis was used to identify single-nucleotide polymorphisms (SNPs) in the promoter region. A total of 337 infertile men with abnormal semen parameters and 203 fertile men with normal semen parameters were subjected to sequence analysis of the DAZL promoter region. RESULTS The DAZL gene core promoter is located 1 kb upstream of the transcription start site. Three SNPs (-792G>A, -669A>C and -309T>C) were identified in our population. Of these three SNPs, -792G>A was more prevalent in the infertile men (P= 0.0005). Quantitative analysis revealed that genotypes of -792G>A had effects on sperm concentration (P= 0.0025) and motility (P= 1.5 × 10(-7)). The G to A substitution was associated with decreased binding of the nuclear respiratory factor-1 (NRF-1) to the promoter region and decreased reporter gene activity. CONCLUSION We have identified the core promoter of the human DAZL gene. We also provide preliminary evidence for the role of a novel SNP of the DAZL gene promoter in human spermatogenic failure.

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.

OBJECTIVE The 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. MATERIALS AND METHODS This 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). RESULTS The 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. CONCLUSION Combined 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.

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.

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.