Telomere lengths in human oocytes, cleavage stage embryos and blastocysts

Telomeres are repeated sequences that protect the ends of chromosomes and harbour DNA-repair proteins. Telomeres shorten during each cell division in the absence of telomerase. When telomere length becomes critically short, cell senescence occurs. Telomere length therefore reflects both cellular ageing and capacity for division. We have measured telomere length in human germinal vesicle (GV) oocytes and pre-implantation embryos, by quantitative fluorescence in-situ hybridisation (Q-FISH), providing baseline data towards our hypothesis that telomere length is a marker of embryo quality. The numbers of fluorescent foci suggest that extensive clustering of telomeres occurs in mature GV stage oocytes, and in pre-implantation embryos. When calculating average telomere length by assuming that each signal presents one telomere, the calculated telomere length decreased from the oocyte to the cleavage stages, and increased between the cleavage stages and the blastocyst (11.12 vs 8.43 vs 12.22kb respectively, p<0.001). Other methods of calculation, based upon expected maximum and minimum numbers of telomeres, confirm that telomere length in blastocysts is significantly longer than cleavage stages. Individual blastomeres within an embryo showed substantial variation in calculated average telomere length. This study implies that telomere length changes according to the stage of pre-implantation embryo development

Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity

STUDY QUESTION: Can we identify compound(s) with reported phosphodiesterase inhibitor (PDEI) activity that could be added to human spermatozoa in vitro to enhance their motility without compromising other sperm functions? SUMMARY ANSWER: We have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples. WHAT IS KNOWN ALREADY: For &gt;20 years, the use of non-selective PDEIs, such as pentoxifylline, has been known to influence the motility of human spermatozoa; however, conflicting results have been obtained. It is now clear that human sperm express several different phosphodiesterases and these are compartmentalized at different regions of the cells. By using type-specific PDEIs, differential modulation of sperm motility may be achieved without adversely affecting other functions such as the acrosome reaction (AR). STUDY DESIGN, SIZE, DURATION: This was a basic medical research study examining sperm samples from normozoospermic donors and subfertile patients attending the Assisted Conception Unit (ACU), Ninewells Hospital Dundee for diagnostic semen analysis, IVF and ICSI. Phase 1 screened 43 commercially available compounds with reported PDEI activity to identify lead compounds that stimulate sperm motility. Samples were exposed (20 min) to three concentrations (1, 10 and 100 µM) of compound, and selected candidates (n = 6) progressed to Phase 2, which provided a more comprehensive assessment using a battery of in vitro sperm function tests.  PARTICIPANTS/MATERIALS, SETTING, METHODS: All healthy donors and subfertile patients were recruited at the Medical Research Institute, University of Dundee and ACU, Ninewells Hospital Dundee (ethical approval 08/S1402/6). In Phase 1, poor motility cells recovered from the 40% interface of the discontinuous density gradient were used as surrogates for patient samples. Pooled samples from three to four different donors were utilized in order to reduce variability and increase the number of cells available for simultaneous examination of multiple compounds. During Phase 2 testing, semen samples from 23 patients attending for either routine diagnostic andrology assessment or IVF/ICSI were prepared and exposed to selected compounds. Additionally, 48 aliquots of prepared samples, surplus to clinical use, were examined from IVF (n = 32) and ICSI (n = 16) patients to further determine the effects of selected compounds under clinical conditions of treatment. Effects of compounds on sperm motility were assessed by computer-assisted sperm analysis. A modified Kremer test using methyl cellulose was used to assess sperm functional ability to penetrate into viscous media. Sperm acrosome integrity and induction of apoptosis were assessed using the acrosomal content marker PSA-FITC and annexin V kit, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: In Phase 1, six compounds were found to have a strong effect on poor motility samples with a magnitude of response of ≥60% increase in percentage total motility. Under capacitating and non-capacitating conditions, these compounds significantly (P ≤ 0.05) increased the percentage of total and progressive motility. Furthermore, these compounds enhanced penetration into a cervical mucus substitute (P ≤ 0.05). Finally, the AR was not significantly induced and these compounds did not significantly increase the externalization of phosphatidylserine (P = 0.6, respectively). In general, the six compounds maintained the stimulation of motility over long periods of time (180 min) and their effects were still observed after their removal. In examinations of clinical samples, there was a general observation of a more significant stimulation of sperm motility in samples with lower baseline motility. In ICSI samples, compounds #26, #37 and #38 were the most effective at significantly increasing total motility (88, 81 and 79% of samples, respectively) and progressive motility (94, 93 and 81% of samples, respectively). In conclusion, using a two-phased drug discovery screening approach including the examination of clinical samples, 3/43 compounds were identified as promising candidates for further study. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and caution must be taken when extrapolating the results. Data for patients were from one assessment and thus the robustness of responses needs to be established. The n values for ICSI samples were relatively small. WIDER IMPLICATIONS OF THE FINDINGS: We have systematically screened and identified several compounds that have robust and effective stimulation (i.e. functional significance with longevity and no toxicity) of total and progressive motility under clinical conditions of treatment. These compounds could be clinical candidates with possibilities in terms of assisted reproductive technology options for current or future patients affected by asthenozoospermia or oligoasthenozoospermia

Impact of GnRH analogues on oocyte/embryo quality and embryo development in in vitro fertilization/intracytoplasmic sperm injection cycles: a case control study

<p>Abstract</p> <p>Background</p> <p>Despite the clinical outcomes of ovarian stimulation with either GnRH-agonist or GnRH-antagonist analogues for in vitro fertilization (IVF) being well analysed, the effect of analogues on oocyte/embryo quality and embryo development is still not known in detail. The aim of this case-control study was to compare the efficacy of a multiple-dose GnRH antagonist protocol with that of the GnRH agonist long protocol with a view to oocyte and embryo quality, embryo development and IVF treatment outcome.</p> <p>Methods</p> <p>Between October 2001 and December 2008, 100 patients were stimulated with human menopausal gonadotrophin (HMG) and GnRH antagonist in their first treatment cycle for IVF or intracytoplasmic sperm injection (ICSI). One hundred combined GnRH agonist + HMG (long protocol) cycles were matched to the GnRH antagonist + HMG cycles by age, BMI, baseline FSH levels and by cause of infertility. We determined the number and quality of retrieved oocytes, the rate of early-cleavage embryos, the morphology and development of embryos, as well as clinical pregnancy rates. Statistical analysis was performed using Wilcoxon's matched pairs rank sum test and McNemar's chi-square test. P < 0.05 was considered statistically significant.</p> <p>Results</p> <p>The rate of cytoplasmic abnormalities in retrieved oocytes was significantly higher with the use of GnRH antagonist than in GnRH agonist cycles (62.1% vs. 49.9%; P < 0.01). We observed lower rate of zygotes showing normal pronuclear morphology (49.3% vs. 58.0%; P < 0.01), and higher cell-number of preembryos on day 2 after fertilization (4.28 vs. 4.03; P < 0.01) with the use of GnRH antagonist analogues. The rate of mature oocytes, rate of presence of multinucleated blastomers, amount of fragmentation in embryos and rate of early-cleaved embryos was similar in the two groups. Clinical pregnancy rate per embryo transfer was lower in the antagonist group than in the agonist group (30.8% vs. 40.4%) although this difference did not reach statistical significance (P = 0.17).</p> <p>Conclusion</p> <p>Antagonist seemed to influence favourably some parameters of early embryo development dynamics, while other morphological parameters seemed not to be altered according to GnRH analogue used for ovarian stimulation in IVF cycles.</p

Discontinuation of rLH two days before hCG may increase the number of oocytes retrieved in IVF

<p>Abstract</p> <p>Background</p> <p>Administration of recombinant luteinizing hormone (rLH) in controlled ovarian hyperstimulation may benefit a subpopulation of patients. However, late follicular phase administration of high doses of rLH may also reduce the size of the follicular cohort and promote monofollicular development.</p> <p>Methods</p> <p>To determine if rLH in late follicular development had a negative impact on follicular growth and oocyte yield, IVF patients in our practice who received rFSH and rLH for the entire stimulation were retrospectively compared with those that had the rLH discontinued at least two days prior to hCG trigger.</p> <p>Results</p> <p>The two groups had similar baseline characteristics before stimulation with respect to age, FSH level and antral follicle count. However, the group which had the rLH discontinued at least two days prior to their hCG shot, had a significantly higher number of oocytes retrieved, including a higher number of MII oocytes and number of 2PN embryos.</p> <p>Conclusions</p> <p>When using rLH for controlled ovarian hyperstimulation, administering it from the start of stimulation and stopping it in the late follicular phase, at least two days prior to hCG trigger, may increase oocyte and embryo yield.</p

Administration of single-dose GnRH agonist in the luteal phase in ICSI cycles: a meta-analysis

<p>Abstract</p> <p>Background</p> <p>The effects of gonadotrophin-releasing hormone agonist (GnRH-a) administered in the luteal phase remains controversial. This meta-analysis aimed to evaluate the effect of the administration of a single-dose of GnRH-a in the luteal phase on ICSI clinical outcomes.</p> <p>Methods</p> <p>The research strategy included the online search of databases. Only randomized studies were included. The outcomes analyzed were implantation rate, clinical pregnancy rate (CPR) per transfer and ongoing pregnancy rate. The fixed effects model was used for odds ratio. In all trials, a single dose of GnRH-a was administered at day 5/6 after ICSI procedures.</p> <p>Results</p> <p>All cycles presented statistically significantly higher rates of implantation (P < 0.0001), CPR per transfer (P = 0.006) and ongoing pregnancy (P = 0.02) in the group that received luteal-phase GnRH-a administration than in the control group (without luteal-phase-GnRH-a administration). When meta-analysis was carried out only in trials that had used long GnRH-a ovarian stimulation protocol, CPR per transfer (P = 0.06) and ongoing pregnancy (P = 0.23) rates were not significantly different between the groups, but implantation rate was significant higher (P = 0.02) in the group that received luteal-phase-GnRH-a administration. On the other hand, the results from trials that had used GnRH antagonist multi-dose ovarian stimulation protocol showed statistically significantly higher implantation (P = 0.0002), CPR per transfer (P = 0.04) and ongoing pregnancy rate (P = 0.04) in the luteal-phase-GnRH-a administration group. The majority of the results presented heterogeneity.</p> <p>Conclusions</p> <p>These findings demonstrate that the luteal-phase single-dose GnRH-a administration can increase implantation rate in all cycles and CPR per transfer and ongoing pregnancy rate in cycles with GnRH antagonist ovarian stimulation protocol. Nevertheless, by considering the heterogeneity between the trials, it seems premature to recommend the use of GnRH-a in the luteal phase. Additional randomized controlled trials are necessary before evidence-based recommendations can be provided.</p

Epididymis Response Partly Compensates for Spermatozoa Oxidative Defects in snGPx4 and GPx5 Double Mutant Mice

We report here that spermatozoa of mice lacking both the sperm nucleaus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H2O2-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice

Effects of recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-agonist protocol: a matched case-control study

<p>Abstract</p> <p>Background</p> <p>Some studies have suggested that the suppression of endogenous LH secretion does not seem to affect the majority of patients who are undergoing assisted reproduction and stimulation with recombinant FSH (r-FSH). Other studies have indicated that a group of normogonadotrophic women down-regulated and stimulated with pure FSH preparations may experience low LH concentrations that compromise the IVF parameters. The present study aimed to compare the efficacy of recombinant LH (r-LH) supplementation for controlled ovarian stimulation in r-FSH and GnRH-agonist (GnRH-a) protocol in ICSI cycles.</p> <p>Methods</p> <p>A total of 244 patients without ovulatory dysfunction, aged <40 years and at the first ICSI cycle were divided into two groups matched by age according to an ovarian stimulation scheme: Group I (n = 122): Down-regulation with GnRH-a + r-FSH and Group II (n = 122): Down-regulation with GnRH-a + r-FSH and r-LH (beginning simultaneously).</p> <p>Result(s)</p> <p>The number of oocytes collected, the number of oocytes in metaphase II and fertilization rate were significantly lower in the Group I than in Group II (<it>P </it>= 0.036, <it>P </it>= 0.0014 and <it>P </it>= 0.017, respectively). In addition, the mean number of embryos produced per cycle and the mean number of frozen embryos per cycle were statistically lower (<it>P </it>= 0.0092 and <it>P </it>= 0.0008, respectively) in Group I than in Group II. Finally the cumulative implantation rate (fresh+thaw ed embryos) was significantly lower (<it>P </it>= 0.04) in Group I than in Group II. The other clinical and laboratory results analyzed did not show difference between groups.</p> <p>Conclusion</p> <p>These data support r-LH supplementation in ovarian stimulation protocols with r-FSH and GnRH-a for assisted reproduction treatment.</p

Paternal effect on genomic activation, clinical pregnancy and live birth rate after ICSI with cryopreserved epididymal versus testicular spermatozoa

<p>Abstract</p> <p>Background</p> <p>This study takes an in depth look at embryonic development, implantation, pregnancy and live birth rates with frozen epididymal and testicular sperm from obstructed (OA) and non-obstructed (NOA) patients.</p> <p>Methods</p> <p>Paternal effect of sperm source on zygote formation, embryonic cleavage, and genomic activation were examined. Additional outcome parameters monitored were clinical pregnancy rate (CPR), implantation rate (IR) and live birth rate.</p> <p>Results</p> <p>In this report, we retrospectively analyzed 156 ICSI cycles using cryopreserved epididymal sperm (ES; n = 77) or testicular sperm (TESE; n = 79). The developmental potential of embryos did not appear to be influenced by the type of surgically retrieved sperm. The average number of blastomeres observed on Day 3 was not different among different groups; 7.5 +/- 1.7 (ES), 7.6 +/- 2.1 (TESE-OA) and 6.5 +/- 2.3 (TESE-NOA). Compaction and blastulation rates, both indicators of paternal genomic activation, were similar in embryos derived from ICSI with ES or TESE from OA as well as NOA men. The only parameter significantly affected in NOA-TESE cases was the fertilization rate. CPR and IR with cryopreserved TESE (TESE-OA 59%, 34%, and TESE-NOA 37%, 20%) were also not statistically different, from that achieved with cryopreserved ES (61% and 39%). Live birth rates also appeared to be independent of sperm type. The 87 clinical pregnancies established using cryopreserved TESE and ES, resulted in the birth of 115 healthy infants. No congenital anomalies were noted.</p> <p>Conclusion</p> <p>Zygotic activation seems to be independent of sperm origin and type of azoospermia.</p

Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis..Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer