Human Tubal Fluid Medium Research Articles

TallyHO obese female mice experience poor reproductive outcomes and abnormal blastocyst metabolism that is reversed by metformin.

Obese women experience worse reproductive outcomes than normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay of offspring. The aim of the present study was to use a genetic mouse model of obesity to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. New inbred, polygenic Type 2 diabetic TallyHO mice and age-matched control C57BL/6 mice were superovulated to obtain morula or blastocyst stage embryos that were cultured in human tubal fluid (HTF) medium. Deoxyglucose uptake was determined for individual insulin-stimulated blastocysts. Apoptosis was detected by confocal microscopy using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and Topro-3 nuclear dye. Embryos were scored for TUNEL-positive as a percentage of total nuclei. AMP-activated protein kinase (AMPK) activation, tumour necrosis factor (TNF)-α expression and adiponectin expression were analysed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by BODIPY. Comparisons were made between TallyHO morulae cultured to blastocyst embryos in either HTF medium or HTF medium with 25 μg mL(-1) metformin. TallyHO mice developed whole body abnormal insulin tolerance, had decreased litter sizes and increased non-esterified fatty acid levels. Blastocysts from TallyHO mice exhibited increased apoptosis, decreased insulin sensitivity and decreased AMPK. A possible cause for the insulin resistance and abnormal AMPK phosphorylation was the increased TNF-α expression and lipid accumulation, as detected by BODIPY, in TallyHO blastocysts and decreased adiponectin. Culturing TallyHO morulae with the AMPK activator metformin led to a reversal of all the abnormal findings, including increased AMPK phosphorylation, improved insulin-stimulated glucose uptake and normalisation of lipid accumulation. Women with obesity and insulin resistance experience poor pregnancy outcomes. Previously we have shown in mouse models of insulin resistance that AMPK activity is decreased and that activators of AMPK reverse poor embryo outcomes. Here, we show for the first time using a genetically altered obese model, not a diet-induced model, that metformin reverses many of the adverse effects of obesity at the level of the blastocyst. Expanding on this we determine that activation of AMPK via metformin reduces lipid droplet accumulation, presumably by eliminating the inhibitory effects of TNF-α, resulting in normalisation of fatty acid oxidation and HADH2 (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit) activity. Metformin exposure in vitro was able to partially reverse these effects, at the level of the blastocyst, and may thus be effective in preventing the adverse effects of obesity on pregnancy and reproductive outcomes.

136 INVESTIGATION OF LEPTIN ON DEVELOPMENT OF MOUSE EMBRYOS

Leptin is a hormone-like protein of 167 amino acids. As an adipocyte-related hormone it has an important role in weight regulation and physical fitness but also has effects on reproductive and other physiological mechanisms. The aim of the present study was to investigate the effects of different concentrations of leptin added to the culture media, the quality, in vitro development rate, and in vivo rate of mouse embryos. Superovulated CB6F1 (C57BL/6XBalb/c) hybrid female mice (5–6 weeks of age) were killed ~18 to 20 h after hCG administration. Single-cell embryos were flushed from the oviducts of the dead mice with human tubal fluid medium supplemented with HEPES and 3 mg mL–1 of BSA. They were cultured in Quinn's cleavage medium supplemented with 4 mg mL–1 of BSA in 5% CO2, 37°C until reaching 2-cell stage. The 2-cell embryos were randomly divided into 4 groups and cultured in Quinn's blastocyst medium supplemented with 4 mg mL–1 BSA + 0, 10, 50, and 100 ng mL–1 leptin (L0, L10, L50, and L100) in 5% CO2, 37°C until the blastocyst stage. Some of the developing blastocysts were used for differential staining for the inner cell mass and trophectoderm (TE) cells [Mallol et al. 2013 Syst. Biol. Reprod. Med. 59,117–122]. Some of them were transferred into pseudopregnant females (CD1) on the 2.5 to 3.5th days and kept until the 13.5th day of pregnancy for the in vivo development rate. The results were evaluated using one-way ANOVA with Bonferroni post-hoc test in SPSS 22.0. The P-values <0.05 were considered statistically significant. Each experiment was repeated at least 4 times. The blastocyst development rates of L0, L10, L50, and L100 were 92.57% (162/175), 97.16% (205/211), 97.80% (178/182), and 97.85% (182/186), respectively. The in vitro development rates were significantly higher in the L10, L50, and L100 compared with L0 (P < 0.05). The inner cell mass cells of L0, L10, L50, and L100 were 13.17, 14, 16, and 15.43. There was no significant difference between the groups in terms of inner cell mass cells (P > 0.05). The TE cells of L0, L10, L50, and L100 were 47, 56.4, 53.7, and 58.57, respectively. The TE numbers were significantly increased in the presence of L10 and L100 compared with L0 (P < 0.05). The in vivo development rates of L0, L10, L50, and L100 were 13.51% (5/37), 48.72% (19/39), 15.38% (6/39), and 41.03% (16/39), respectively. The in vivo development rates of L10 and L100 were significantly higher than for L0 and L50 (P < 0.05). The resorption rates of L0, L10, L50, and L100 were 10.8% (4/37), 30.8% (12/39), 12.8% (5/39), and 20.5% (8/39), respectively. There was no significant difference between the groups in terms of the resorption rates (P > 0.05). This study found that L10, L50, and L100 were supporting the development of the embryos in the in vitro culture. The L10, L50, and L100 significantly increased the total cell numbers. The L10 and L100 were particularly effective on the number of the TE cells. In conclusion, 10 and 100 ng mL–1 leptin have a positive effect on the in vitro, quality and in vivo development of the mouse embryo. Therefore, leptin seems to play an important role on the embryo development and in vivo development. Research supported by TUBITAK-113O223.

MP66-15 ADVANCES IN THE ULTRA-RAPID VITRIFICATION OF HUMAN SPERMATAZOA WITH A MICROCAPILLARY SYSTEM

You have accessJournal of UrologyInfertility: Basic Research, Physiology & Pathophysiology1 Apr 2014MP66-15 ADVANCES IN THE ULTRA-RAPID VITRIFICATION OF HUMAN SPERMATAZOA WITH A MICROCAPILLARY SYSTEM Joseph W. McQuaid, Yahir Santiago-Lastra, Diane Wright, Mehmet Toner, Thomas L. Toth, and Cigdem Tanrikut Joseph W. McQuaidJoseph W. McQuaid More articles by this author , Yahir Santiago-LastraYahir Santiago-Lastra More articles by this author , Diane WrightDiane Wright More articles by this author , Mehmet TonerMehmet Toner More articles by this author , Thomas L. TothThomas L. Toth More articles by this author , and Cigdem TanrikutCigdem Tanrikut More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2062AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Freezing low concentrations of sperm from severely oligospermic patients while maintaining adequate post-thaw motility remains a challenge. We previously reported the feasibility of ultra-rapid vitrification using a microcapillary system. In an attempt to improve these results, several modifications were introduced including a larger capillary system and lower concentrations of non-toxic cryoprotectants: trehalose and propanediol. METHODS Using an IRB-approved protocol, 27 fresh ejaculate samples from healthy men were obtained and prepared via a swim-up protocol in human tubal fluid (HTF). 15 samples with an average concentration of 77 million/mL and pre-thaw total motility of 86% were included in our modified protocol. These samples were diluted in a drop-wise, 1:1 fashion with HTF media containing trehalose (0.05M) and propanediol (PrOH, 0.75M). After loading the samples into a series of 200 μm capillaries with heat-sealed ends, they were divided into two equal groups: 1) Control, from which the samples were immediately expelled, washed, then assessed via computer-assisted semen analysis, and 2) Experimental, which was vitrified in a super-cooled, liquid nitrogen bath, stored for up to 24 hours, then thawed, washed, and analyzed. A subset of specimens from the experimental group was used for viability staining. RESULTS Consistent post-thaw motility was achieved using this protocol. While a total motility of 48.6% was recorded in the control, the experimental group demonstrated an overall post-thaw motility of 10.5%. Subset analysis allowed stratification of samples based upon their ability to withstand this process. Of the highest-performing specimens, defined as those with a post-thaw motility >15%, we were able to conserve 22% of the sham control motility in our vitrified samples. CONCLUSIONS Building upon our prior experience, we sought to enhance sperm survival and improve post-thaw motility. Modifications included use of a larger capillary to reduce shear stress, conversion to a less viscous and less toxic media, and incorporation of a wash prior to final analysis. Gains were achieved in post-thaw motility and confirmed the feasibility of this system. However, it is clear from viability stains and subset analysis that there are yet undiscovered intrinsic and extrinsic variables affecting optimal recovery of specimens with this process. We will continue efforts to optimize this system for use in severely oligoospermic patients. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e746 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Joseph W. McQuaid More articles by this author Yahir Santiago-Lastra More articles by this author Diane Wright More articles by this author Mehmet Toner More articles by this author Thomas L. Toth More articles by this author Cigdem Tanrikut More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

2045 SUSPENDED ANIMATION OF HUMAN SPERM: ULTRA-RAPID VITRIFICATION USING A NOVEL MICROCAPILLARY SYSTEM

You have accessJournal of UrologyInfertility: Basic Research, Physiology, Pathophysiology1 Apr 20132045 SUSPENDED ANIMATION OF HUMAN SPERM: ULTRA-RAPID VITRIFICATION USING A NOVEL MICROCAPILLARY SYSTEM Yahir Santiago-Lastra, Joseph W. McQuaid, Diane Wright, Mehmet Toner, Cigdem Tanrikut, and Thomas L. Toth Yahir Santiago-LastraYahir Santiago-Lastra Boston, MA More articles by this author , Joseph W. McQuaidJoseph W. McQuaid Boston, MA More articles by this author , Diane WrightDiane Wright Boston, MA More articles by this author , Mehmet TonerMehmet Toner Boston, MA More articles by this author , Cigdem TanrikutCigdem Tanrikut Boston, MA More articles by this author , and Thomas L. TothThomas L. Toth Boston, MA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2464AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Freezing very low concentrations of sperm remains a challenge. Conventional slow-freeze methods require dilution with relatively large volumes and high concentrations of traditional cryoprotectants with potentially lethal osmotic and cytotoxic effects. Development of ultra-rapid vitrification techniques allows for freezing of very low volumes using low concentrations of nontoxic cryoprotectants, such as sugars, in addition to very low doses of permeable cryoprotectant. Our investigation was undertaken to determine the feasibility of ultra-rapid freezing via vitrification of human sperm samples with 0.5M trehalose and 3.0M propanediol (PROH) using a novel microcapillary system. METHODS 84 fresh ejaculated samples were obtained from healthy men and prepared using the swim-up method in human tubal fluid (HTF) media supplemented with 5% human serum albumin (HSA). All samples had at least 20 million spermatozoa/ml and at least 50% motility prior to swim-up preparation. These samples were then diluted to varying concentrations and divided into equal volumes for vitrification into the following groups: (1) 0.25M trehalose (a natural sugar) to act as an impermeable cryoprotectant, (2) vitrification with 0.25M trehalose and propanediol (PROH), (3) vitrification without cryoprotectant, or (4) no freezing/vitrification (control). Each sample was loaded into a 100μ microcapillary and the ends were heat-sealed to create a closed system. The samples undergoing vitrification were plunged into a liquid nitrogen bath for 20 seconds and then warmed in a 37°C solution of phosphate-buffered saline. Each sample was expelled from the capillaries and motility was qualitatively assessed via microscopic visualization. All samples were further assessed by computer-assisted semen analysis. RESULTS Preliminary results demonstrate consistent and statistically significant (p<0.05) recovery of motile sperm in samples that were vitrified in 100μ microcapillaries with HTF media plus 5% HSA supplemented by 0.25M trehalose and PROH. CONCLUSIONS Ultra-rapid vitrification of human sperm samples in microcapillaries is feasible using low volumes of a nontoxic sugar, such as trehalose, as an alternative to traditional toxic cryoprotectants. This is a particularly relevant issue with men who produce very little sperm in the ejaculate and in men who undergo any variety of surgical sperm extraction efforts for either obstructive or nonobstructive azoospermia. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e839 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yahir Santiago-Lastra Boston, MA More articles by this author Joseph W. McQuaid Boston, MA More articles by this author Diane Wright Boston, MA More articles by this author Mehmet Toner Boston, MA More articles by this author Cigdem Tanrikut Boston, MA More articles by this author Thomas L. Toth Boston, MA More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Cryoprotectants up-regulate expression of mouse oocyte AQP7, which facilitates water diffusion during cryopreservation

To investigate the effects of cryoprotectants on the expression of AQP7 in oocytes. Experimental animal study. University-based research laboratory. Adult female C57BL/6J mice. In metaphase II (MII) oocytes obtained from adult female C57BL/6J mice and from donations by fertile women, the mouse oocytes were treated with human tubal fluid medium containing 8% ethylene glycol (EG), 9.5% dimethylsulfoxide (DMSO), and 0.5 M sucrose, respectively; 293T cells transfected with GFP-hAQP7 expression vector were treated with the same solutions. AQP7 expression in oocytes examined by reverse-transcriptase-nested polymerase chain reaction and immunofluorescence, changes in the volume of mouse oocytes treated with different solutions calculated to determine their permeability to water, and survival rates of vitrified oocytes. AQP7 is expressed in human and mouse oocytes. Cryoprotectants, including EG, DMSO, and sucrose, up-regulated AQP7 expression in mouse oocytes and 293T cells transfected with GFP-hAQP7 expression vector. Compared with other cryoprotectants, DMSO stimulated higher expression of AQP7, and this was associated with faster cell volume recovery and lower survival rates of vitrified oocytes. DMSO up-regulates AQP7 expression in mouse oocytes more than EG. This may facilitate water diffusion and reduce the time for oocytes to reach osmotic balance with the cryoprotectant solution during cryopreservation.

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

Pregnancy outcome after intracytoplasmic sperm injection with strontium oocyte activation in a globozoospermic patient

202 SEX PRE-SELECTION IN RABBITS: AN ATTEMPT TO SKEW OFFSPRING SEX THROUGH PERCOLL AND SWIM-UP SPERM PREPARATION TECHNIQUES

Sperm preparation techniques could have an effect on the birth ratio (male vs female offspring) through enrichment of either X- or Y-bearing sperm populations, although studies have been poorly controlled and results have been inconclusive. In the rabbit, producers may be interested in producing a majority of males (meat producing systems) or females (hybrid producing systems). The objective of this study was to evaluate the effect of modified Percoll and Swim-up protocols on the enrichment of the male and female birth ratios in rabbits. Four hybrid mature bucks of adequate body condition score and proven fertility were used. The same four bucks were used throughout the study (8 replicates). Ejaculates were collected using a warmed, solid, artificial vagina. Progressive motility was assessed at 200× magnification under brightfield microscopy on a heated (37°C) stage. Sperm concentration for each buck was determined using a Neubaur chamber and adjusted (350 × 106 total spermatozoa mL–1); they were then combined (1 mL/buck) to form a heterospermic sample (final heteropsermic sample volume = 4 mL). The heteropsermic sample was then divided into 3 sperm preparation treatments: (1) diluted in commercial tri-buffered extender followed by immediate AI (control); (2) modified Percoll centrifugation in a 90 to 45% density gradient (diluted in human tubal fluid medium); and (3) Swim-up (in human tubal fluid medium). In total, 125 females were inseminated. Does were treated with PMSG (20 UI/doe, 48 h before AI) and inseminated with 30 to 40 × 106 spermatozoa; ovulation was induced with 20 μg of gonadotropin-releasing hormone (GnRH)/doe immediately after AI. Sex of newborn kits was determined 35 days after birth by an experienced technician through external visualization of genitalia. Results were statistically analyzed using ANOVA and Tukey test (Infostat). The insemination of does with heterospermic ejaculates (control) resulted in a progeny distribution of 49 ± 2% males and 51 ± 2% females, which is in good agreement with the expected theoretical 50:50 ratio. Sex ratios from modified Percoll and Swim-up treatments deviated significantly from the control (P &lt; 0.01). Percoll gradient resulted in a progeny distribution of 32 ± 2% males and 68 ± 2% females; Swim-up resulted in a 64 ± 2% male and a 36 ± 2% female distribution (P &lt; 0.01), whereas prolificacy, perinatal mortality and birth weight were not affected by treatment (Table 1). Our results indicate sperm preparation techniques could be used to skew birth ratios towards male or female offspring in rabbits; Percoll preparation could be used to increase proportion of female kits whereas Swim-up could be used to increase the proportion of males. Further studies are needed to determine the underlying mechanisms of action. Table 1.Progeny distribution after Percoll or Swim-up sperm preparation in rabbits

38 VITRIFICATION OF BOS TAURUS INDICUS AND BOS TAURUS INDICUS×BOS TAURUS TAURUS EMBRYOS PRODUCED IN THE PRESENCE OR ABSENCE OF FETAL CALF SERUM

In vitro-produced Bos taurus indicus (zebu) and Bos taurus indicus × Bos taurus taurus (cross-bred) embryos behave differently when vitrified. The present experiment aimed to examine the effect of vitrification on embryos produced in the presence or absence of FCS. Cumulus-oocyte complexes (COC) were matured in TCM-199 and fertilized in human tubal fluid medium with frozen Nelore bull semen. On Day 1 (Day 0 = IVF), presumptive zygotes were cultured with SOFaa + BSA in the presence of FCS (Group 2.5%) or in the absence of FCS (Group 0%) until Day 7. The cleavage was analysed on Day 3 and the blastocyst rate on Day 7. Blastocysts were vitrified and, after warming (Campos-Chillòn et al. 2006) the viability was evaluated. Data were analysed with ANOVA, using the general linear model (GLM) of SAS (SAS Inst Inc., Cary, NC, USA). Sources of variation in the model included FCS concentration and first-order interactions; all factors were considered fixed effects. The arcsine transformation (√y/100) was applied to percentage data. If the ANOVA was significant, means were separated using the Tukey test. There was no difference in cleavage (for zebu embryos: Group 0%: 87.2 ± 6.8; Group 2.5%: 87.4 ± 9.5; for cross-bred embryos: Group 0%: 79.6 ± 11.9; Group 2.5%: 73.1 ± 13.7; P &gt; 0.05). On the other hand, zebu embryos cultured in the presence of FCS reached blastocysts at a higher rate than cross-bred embryos in the absence of FCS (for zebu embryos: Group 0%: 33.3 ± 12.4ab; Group 2.5%: 46.8 ± 13.2a; for cross-bred embryos: Group 0%: 21.8 ± 8.3b; Group 2.5%: 33.6 ± 10.1ab; P &lt; 0.05). After vitrification and warming, no significant differences in re-expansion rate (zebu embryos: Group 0%: 82.7 ± 13.1; Group 2.5%: 75.0 ± 9.8; cross-bred embryos: Group 0%: 93.7 ± 8.8; Group 2.5%: 84.1 ± 11.3; P &gt; 0.05) and cell number per embryo (zebu embryos: Group 0%: 65.1 ± 34.7; Group 2.5%: 42.6 ± 17.2; cross-bred embryos: Group 0%: 64.3 ± 44.2; Group 2.5%: 52.0 ± 31.5; P &gt; 0.05) between species groups and within species were seen. However for zebu embryos, Group 0% showed a lower damaged cell rate than Group 2.5%. The same effect was not observed in the cross-bred embryos (zebu embryos: Group 0%: 20.3 ± 22.7c; Group 2.5%: 63.3 ± 27.0d; cross-bred embryos: Group 0%: 25.4 ± 24.3cd; Group 2.5%: 45.8 ± 34.6cd; P &lt; 0.05). The addition of 2.5% FCS had a higher deleterious effect on zebu embryos than cross-bred (zebu × taurine) embryos after vitrification. These results also reinforce the species differences observed between zebu and cross-bred, as they behaved differently in relation to the addition of FCS in the culture medium and in relation to their cryopreservation sensitivity. Supported by FAPESP 10/50410-2.

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94–99%) and blastocyst (90–96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94–98%) and blastocysts (91–93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.

Quinn’s advantage fertilization medium enhances zona pellucida-induced acrosome reaction compared with human tubal fluid medium

The aim of this study was to compare sperm velocity, hyperactivation, zona pellucida (ZP) binding and ZP-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF), human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from normozoospermic men and motile spermatozoa were prepared by gradient centrifugation (PureSperm). Unfertilized oocytes from clinical IVF were used for spermatozoa–oocyte interaction tests. Sperm velocity and hyperactivation were assessed using a Hamilton-Thorn motility analyser. When media were supplemented with human albumin, sperm motility and velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF (42 ± 22 versus 21 ± 18, P < 0.001). Sperm velocity, hyperactivation and sperm binding were also significantly higher in QAF than Ham’s F10 media. Supplementation of media with either human serum or human albumin showed no difference in effect on all sperm test results. In conclusion, QAF medium significantly enhances ZP-induced AR which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF. The aim of this study was to compare sperm velocity, hyperactivated motility, sperm binding and zona pellucida (ZP)-induced acrosome reaction (AR) between Quinn’s advantage fertilization (QAF) and human tubal fluid (HTF) and Ham’s F10 media. Semen samples were obtained from men with normal semen analysis results and all test results were compared in paired control samples between different media. When media were supplemented with human albumin, sperm motility, velocity and sperm binding were not significantly different between QAF and HTF. However, ZP-induced AR was significantly higher with QAF than HTF. Sperm velocity, hyperactivated motility and sperm binding were all significantly higher in QAF than Ham’s F10 media. Supplementation with either human serum or human albumin in media had no different effects on all sperm test results. In conclusion, Quinn’s advantage fertilization medium significantly enhances ZP-induced AR, which is essential for sperm penetration. Thus QAF appears to be a better medium than HTF for sperm fertilizing ability in conventional IVF.

Short-Term Storage of Human Spermatozoa in Electrolyte-Free Medium Without Freezing Maintains Sperm Chromatin Integrity Better Than Cryopreservation

Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage.

A protocol for rat in vitro fertilization during conventional laboratory working hours

In vitro fertilization (IVF) is a valuable technique for the propagation of experimental animals. IVF has typically been used in mice to rapidly expand breeding colonies and create large numbers of embryos. However, applications of IVF in rat breeding experiments have stalled due to the inconvenient laboratory work schedules imposed by current IVF protocols for this species. Here, we developed a new rat IVF protocol that consists of experimental steps performed during common laboratory working hours. Our protocol can be completed within 12h by shortening the period of sperm capacitation from 5 to 1h and the fertilization time from 10 to 8h in human tubal fluid (HTF) medium. This new protocol generated an excellent birth rate and was applicable not only to closed colony rat strains, such as Wistar, Long-Evans, and Sprague-Dawley (SD), but also to the inbred Lewis strain. Moreover, Wistar and Long-Evans embryos prepared by this protocol were successfully frozen by vitrification and later successfully thawed and resuscitated. This protocol is practical and can be easily adopted by laboratory workers.

238 ASSOCIATION BETWEEN SPERM MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION IN MICE

Although sperm morphology affects male fertility in several species, normally, it is not considered for in vitro fertilization (IVF) programs in mice. In order to correlate sperm morphology with IVF rates in murines, a total of 3336 oocytes were used in 11 identical IVF replicates using 8-wk-old B6D2 F1 (C57BL/6 × DBA/2J) males and 3- to 4-wk-old C57BL/6J donors females. For each replicate, 10 females were injected intraperitoneally with 5 IU of eCG (Novormon, Syntex, Buenos Aires, Argentina), and 5 IU of hCG (Ovusyn, Syntex) 48 h apart. Superovulated females were killed 14 to 16 h after hCG injection. The oviducts were isolated and cumulus–oocyte complexes were recovered and introduced into each sperm suspension drop. Previously, sperm was obtained from males killed by cervical dislocation, the cauda epididymis were recovered, minced with fine scissors in equilibrated human tubal fluid medium (HTF, EmbryoMax, Chemicon International, Phillipsburg, NJ, USA), and resuspended in a total volume of 200 μL of HTF. The sperm suspension was incubated at 0.05 or 3 × 106 spermatozoa/mL concentration in equilibrated 100-μL drops of HTF under embryo tested mineral oil (Sigma, St. Louis, MO, USA), at 37°C in 5% CO2, 95% air for 1 h before insemination. To evaluate sperm morphology, a sample of each replicate was fixed with 10% formalin and observed under phase-contrast microscopy (Olympus IX81) at 100× magnification. Sperm abnormalities regarding head and neck, tail, and cytoplasmic droplet were recorded from 200 cells. Five hours after insemination, the fertilized eggs were washed and transferred in groups of 50 to 100 into 100-μL drops of equilibrated culture medium (KSOM, EmbryoMax, Chemicon International) under the same culture conditions. The number of 2- (cleavage) and 8- (development) cell embryos was scored after 24 and 72 h in culture, respectively. Statistical analysis was performed by logistic regression taking into account the effect of the sperm abnormalities, the replicate, and the sperm dose. The effect of the sperm abnormalities is shown in Table 1. Results were not significantly influenced by the replicate or the sperm dose (P &gt; 0.05). In conclusion, sperm morphology affects cleavage and embryo development rates in mice, and it should be taken into account as a source of variation in the success of IVF. Table 1.Effect of total sperm abnormalities on cleavage rate and embryo development in mice

In vitro maturation (IVM) of murine and human germinal vesicle (GV)–stage oocytes by coculture with immortalized human fallopian tube epithelial cells

To improve the maturation rate of murine and human germinal vesicle (GV) oocytes using human tubal epithelial cells (hTECs). Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or cocultured with mouse embryonic fibroblasts (MEFs) or primary hTECs or immortalized hTECS (ihTECs) for various times. Rates of maturation to meiosis II (MII) were compared between groups. TECs were isolated from discarded salpingectomy specimens. One batch was immortalized with TERT and SV40 large T-antigen. GV oocytes (n = 710) were isolated from 8-week-old-mice at 40 hours after pregnant mare's serum gonadotropin stimulation. Discarded human GV oocytes (n = 62) were obtained from intracytoplasmic sperm injection cycle IVF center patients. Oocytes were cultured in HTF media alone or with MEFs, hTECs, or ihTECs. Maturation rates were assessed by standard morphological criteria and compared. The maturation rate of murine GV oocytes to MII at 12 and 24 hours increased significantly in coculture with hTECS and ihTECS compared with MEF and HTF media alone. In addition, the development rate after IVF was significantly higher with hTECS and ihTECS than in MEF and HTF media alone. Maturation of human GV oocytes to MII at 24 and 48 hours was significantly higher in hTECS and ihTECS compared with HTF media alone. Coculture with either primary or immortalized TECs might improve oocyte quality and significantly raise in vitro maturation rates for GV oocytes.

Attenuated oocyte fertilization and embryo development associated with altered growth factor/signal transduction induced by endometriotic peritoneal fluid

To investigate whether the embryotoxic effect of peritoneal fluid (PF) from infertile women with mild endometriosis on mouse oocytes and embryos is associated with changes in embryonic epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and their receptors. Experimental animal study. University-based research laboratory. Adult ICR mice. Peritoneal fluid was obtained from fertile women with no endometriosis (PF-NE) and infertile women with mild endometriosis (PF-E). In vitro fertilization was performed, and mouse two-cell stage embryos were cultured in human tubal fluid medium with or without PF. Rates of fertilization, cleavage, and blastulation. The embryonic EGF and IGF-I levels in culture medium were analyzed by enzyme-linked immunosorbent assay, and EGF receptor, IGF-I receptor, and phosphorylated extracellular signal-regulated protein kinases (p-ERK) expression was determined by immunofluorescence and confocal microscopy. When oocytes and embryos were cultured in media with PF-E, the fertilization capability of oocytes and the development potential of embryos were decreased. The levels of embryonic EGF, IGF-I, and their receptors were increased. However, p-ERK of the postreceptor signal transduction pathway was down-regulated. Endometriotic PF may attenuate oocyte and embryo development by impairing embryonic growth factor/receptor/signal transduction, resulting in endometriotic infertility.

In vitro maturation (IVM) of murine and human germinal vesicle (GV) stage oocytes using variations of a human fallopian tube epithelial feeder cell system

OBJECTIVE: To assess the maturation rate of murine and human GV oocytes using variations of a human tubal feeder cell system. DESIGN: Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or co-culture with primary (CP58) or immortalized (CP13IM) human tubal cells. Rates of maturation were compared between groups. MATERIALS AND METHODS: Tubal epithelial cells were isolated from salpingectomy specimens by digestion with DNase/Collagenase. One batch was immortalized (CP13IM). Mouse GV oocytes were isolated from 8 week-old mice 40 h after PMSG stimulation. Human GV oocytes (n=21) were obtained from the MGH IVF lab. Murine and human GV oocytes were cultured in HTF media alone or on feeder layers of CP58 and CP13IM cells. Maturation rates were assessed by standard morphological criteria and compared between groups. RESULTS: Maturation rates of GV oocytes under the various culture conditions are shown in table 1. Tabled 1Maturation stage (% of total) of GV oocytes with different culture conditionsMurine (n=210, 24 h)Human (n=21)Culture systemGVGVBDMIIMII (24 h)MII (48 h)HTF27.5021.2551.2512.537.5CP588.7513.7577.5062.575.0CP13 IM12.5017.5070.0060.060.0 Open table in a new tab For murine GV oocytes (n=210 in 3 independent experiments), the maturation rate to the MII stage was approximately 50% higher with CP58 cells and nearly 40% higher with CP13IM cells compared to media alone. For human GV oocytes, the rate of maturation to the MII stage was approximately 5-fold higher with both the CP58 and CP13IM feeder systems at 24 h and approximately 2-fold higher at 48 h. CONCLUSIONS: This study shows that co-culture with either primary or immortalized tubal cells improved IVM rates for murine and human GV oocytes, suggesting a promising new technique with future applications in clinical reproductive medicine or in basic science research, such as in the generation of new stem cell lines.

The impact of peritoneal fluid from healthy women and from women with endometriosis on sperm DNA and its relationship to the sperm deformity index

To study the effect of peritoneal fluid (PF) from healthy women and from women with endometriosis on sperm DNA and its relationship to sperm morphology as assessed by the sperm deformity index (SDI). Experimental study. Research laboratory at an academic hospital. Healthy women undergoing laparoscopic tubal ligation and women with endometriosis. Aliquots of prepared sperm from 10 healthy donors were incubated with PF from healthy women undergoing laparoscopic tubal ligation (treatment 1, n = 10), with PF from patients with endometriosis (treatment 2, n = 10), and with human tubal fluid media with 10% bovine serum albumin (control, n = 10). Sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and SDI. There was a significant increase in sperm DNA damage at 24 hours compared with at 1.5 and 3 hours. The SDI scores significantly correlated with sperm DNA damage after 1.5 and 24 hours of incubation in all aliquots. Percentage normal morphology did not correlate with DNA damage. In vitro exposure of sperm to PF from patients with endometriosis is associated with significantly increased DNA damage. There is evidence of interdependence between the sperm morphology as assessed by SDI scores and DNA damage. The significant increase in sperm DNA damage observed after 24 hours of incubation may be clinically relevant.

PTP1B Dephosphorylates N-Ethylmaleimide-sensitive Factor and Elicits SNARE Complex Disassembly during Human Sperm Exocytosis

The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.

Nitric oxide, antioxidants, and mitochondria activity for preimplantation development

OBJECTIVE: Nitric oxide (NO) is an important regulator of apoptosis for early embryo development and a documented modulator of mitochondrial activity for several types of somatic cells. This study attempts to elucidate the effect of NO on mitochondrial activity for early cleavage stage embryos. Furthermore, the capability of antioxidants to against the apoptotic effect of NO for preimplantation embryos is also investigated. DESIGN: Prospective, controlled study. MATERIALS AND METHODS: Zygotes from superovulated B6CBF1 female mice were retrieved from excised oviducts and cultured within modified human tubal fluid (mHTF) medium. Sodium nitroprusside (SNP) and NG-nitro-L-arginine (L-NA) were supplemented into the culture medium and functioned as NO donor and NOS inhibitor, respectively. Mitochondrial membrane potential and amount of ATP were measured for monitoring mitochondrial activity. Mn(III) tetrakis(4-benzoic acid) porphyrin choloride (TBAP), ebselen (EBS), and glutathione methyl ester (GSH) were applied into the culture medium to reduce oxidative stress. Apoptosis assay for blastomeres was performed with terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay. The blastocyst formation rate and number of apoptotic cells were recorded as an index of embryo development. RESULTS: Supplementation of SNP impaired embryo development and induced disruption of mitochondrial membrane potential in a dose-dependent manner. Although L-NA also arrested embryo development, L-NA did not break the integrity of mitochondrial membrane potential. The degenerated embryos after NO exposure exhibited apoptosis demonstrated by TUNEL stain. Subsequent to NO exposure, the addition of TBAP (superoxide scavenger) or GSH was able to rescue the embryos. However, the supplementation of TBAP was associated with a decline of blastocyst formation rate. Furthermore, EBS (peroxynitrite scavenger) was not able to rescue the embryos from NO toxicity. CONCLUSIONS: The NO donor exerted deleterious effect on embryo development, which might through disruption of mitochondrial membrane potential and induced mitochondria-dependent apoptosis. In order to counteract this adverse effect, GSH might be a required addictive of culture medium.

Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mul suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 degrees C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1-1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1+/-3.2%, P<0.05) when compared with controls (19.4+/-1.9%). The combination of HSA and sucrose (65.2+/-2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6+/-4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.