Human Tubal Fluid Medium Research Articles

Single Step, Two-Steps, and Microfluidics in Embryo Culture. How Do They Measure up in Improving Embryo Quality?

The issues surrounding the culture of the preimplantation human embryo are distinct from those associated with the propagation of somatic cells, given that the starting point of culture, the pronucleate oocyte, and the final stage of development, the blastocyst, are as distinct as any two cell types can be with regards to their nutrient requirements and metabolism. Furthermore, the cleavage stage period of embryo development is exquisitely sensitive to its environment and has limited capacity to regulate against disruptions in its surroundings, especially changes in pH. Historically the media used in human IVF, such as Human Tubal Fluid medium, were based on simple salt solutions but lacked key regulators of blastomere function, specifically amino acids. A resurgence of research in embryo metabolism and physiology in the 1980s paved the way for the development of Sequential media, designed not only to accommodate the changing requirements of the embryo itself (both in terms of carbohydrates and amino acids), but which were also based on available data on the dynamic/differing composition of the Fallopian tube and uterus. Sequential media, especially when used in combination with isolettes, were shown to be highly effective in supporting blastocyst development and significantly increased implantation rates, thereby facilitating the worldwide movement to single embryo transfer. However, while this approach partially satisfied the changing nutrient requirements of the embryo, and further facilitated the removal of toxins built up in the culture drop over 48h, such as ammonium, it necessitated moving embryos. Unless undertaken using warmed gassed isolettes, moving embryos inevitably resulted in shifts in temperature and pH. With the advent of time-lapse, there began a growing movement towards the use of one-step media in order to keep the embryo within an imaging incubator. To facilitate this, hybrid type media were formulated, and while they do not accommodate the dynamic requirements of the preimplantation period, their use meant embryos were not exposed to changes in either temperature or pH. To date, clinical data have reported similar outcomes using both media approaches, plausibly due to a trade-off of the inherent limitations of either approach. An alternative approach proposed decades ago, is to provide the embryo with a gradient of nutrients within a perfusion system, thereby facilitating a dynamic culture environment without the need to move embryos. Unfortunately, until recently, the technology to support this concept has not been available. However, the recent development of 3D printing using 2-photo lithography, has made it possible to create embryo-specific perfusion systems. Hence, we are now able to evaluate the potential of such dynamic systems and could find ourselves on the verge of a new era in embryo culture.

Understanding the impact of varicocele on sperm capacitation

To study the relationship between the seminal sample quality of men with varicocele and sperm capacitation. Cross-sectional observational study. Academic hospital. Seventy-six men (19 control and 57 with varicocele) were analyzed. Semen samples were submitted to a discontinuous density gradient for sperm selection. Sperm capacitation was induced using a human tubal fluid medium supplemented with bovine serum albumin. After capacitation induction, the sperm were assessed by capacitation state, computer-assisted sperm motility, mitochondrial activity, membrane integrity, acrosome reaction, and intracellular oxidative stress. The capacitation period increased sperm motility, showing an increase in the average path velocity and a decrease in the straightness compared with sperm before capacitation (paired analysis). After capacitation, the rate of capacitated sperm, motility, and mitochondrial activity showed differences between groups (control and varicocele). The varicocele group showed lower mitochondrial activity and capacitation than the control group. On the other hand, no significant differences were observed in the other variables evaluated. Varicocele men showed less viable sperm and mitochondrial activity than control men after capacitation sperm. The induction of capacitation altered motility by increasing path velocity and decreasing straightness in all of the studied groups, evidencing the occurrence of hyperactivation.

Acquisition of fertilization competence in guinea pig spermatozoa under different capacitation protocols

Guinea pig in vitro fertilization (IVF) are poorly developed due to the limited accessibility to oocytes and the lack of an efficient method of sperm capacitation. Thus, we aimed to evaluate different capacitation protocols that we validated through sperm analysis and using heterologous (He) IVF with zona-intact bovine oocytes. Spermatozoa of guinea pigs were collected and processed separately by 4 different protocols: A) Spermatozoa were obtained by flushing the lumen of one cauda epididymis and incubated in a minimal culture medium (MCM); B) One epididymis was placed in a prewarmed of M2 medium and gently minced with fine scissors. Spermatozoa were incubated in a modified human tubal fluid medium (HTF). In both protocols, the spermatozoa were capacitated at 37 °C under an atmosphere of 5% CO2 for 2 h. In the protocols C and D, the spermatozoa were collected by flushing the lumen of the cauda epididymis and selected by commercial density gradient Bovipure® (Nidacon Laboratories AB, Göthenborg, Sweden), according to the manufacturer's instructions. Then for Protocol C) spermatozoa were incubated in MCM medium supplemented with 10 mg/mL heparin (MCM-Hep); while for Protocol D) spermatozoa were incubated in FERT medium supplemented 10 mg/mL heparin (FERT-Hep). Incubation of C and D protocols were performed at 38.5 °C under an atmosphere of 5% CO2 for 2 h. Capacitation protocols C and D showed a higher percentage of viability, total and hyperactive-like motility, and acrosome reaction compared to protocols A and B. For this reason, protocols C and D were used for further He-IVF analysis. Guinea pig sperm and matured zona-intact bovine oocytes were co-incubated at 5% CO2 and 38.5 °C. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) and pronuclear formation (PrF) were evaluated at 18, 20, 22, 24 and 26 hpi, while the cleavage rate was evaluated at 48 hpi. In protocol D, PrF was significantly higher than in protocol C (P ≤ 0.05) at every time point evaluated. Also, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in He-IVF protocol D (69.8 ± 1.7%) compared to He-IVF protocol C (49.1 ± 1.1%). In conclusion, we determined the most adequate sperm capacitation conditions for guinea pig that allow zona-intact bovine oocyte penetration and lead to hybrid embryo formation, suggesting that these conditions could be optimal to develop IVF in guinea pigs.

SERPINA5 Protein in Cumulus-Oocyte Complexes Increases the Fertilisation Ability of Mouse Sperm.

Obtaining high-quality sperm is key to improving the success rate of assisted reproductive technology (ART). Although cytokines secreted by cumulus-oocyte complexes (COCs) bind to sperm surface receptors to improve sperm quality, the effects of adding mouse COCs to human tubal fluid (HTF) medium on sperm capacitation have not yet been explored. Eight-week-old ICR mouse COCs were added to HTF medium and crushed to obtain the post-modified HTF medium. Compared with using HTF medium, the fertilisation rate and number of sperm combined with the zona pellucida significantly increased after in vitro capacitation using the post-modified HTF medium (P < 0.01). Proteomic and Western blotting analyses showed that the level of SERPINA5 in sperm increased significantly following in vitro capacitation with the post-modified HTF medium (P < 0.05). Immunohistochemical staining analysis demonstrated that SERPINA5 protein was expressed in mouse cumulus cells. A SERPINA5 antibody was added in the post-modified HTF medium to block the effects of SERPINA5 after in vitro capacitation, which significantly decreased the fertilisation rate and the number of sperm combined with the zona pellucida (P < 0.05). Recombinant mouse SERPINA5 protein (1 ~ 2μg/ml) was added to HTF medium and the fertilisation rate and the number of sperm combined with the zona pellucida significantly increased (P < 0.01). Moreover, recombinant human SERPINA5 protein (5μg/ml) was added before human semen freezing. Compared with adding no SERPINA5 protein, the percentage of normal sperm morphology and the intact acrosome significantly increased (P < 0.05). Our study provides a reference method for optimising sperm quality in the process of in vitro capacitation.

Sperm motility in asthenozoospermic semen samples can be improved by incubation in a continuous single culture medium (CSCM®)

ABSTRACT Standard protocols for clinical in vitro fertilization (IVF) laboratories recommend incubating semen at 37°C in 5% CO2 without strictly specifying which medium should be used or for how long. This study aimed to test the most common different incubation media used in Latin American andrology and micromanipulation laboratories and verify which, if any, is the most appropriate medium to improve asthenozoospermic semen samples’ motility in the infertile male population. Ejaculates (136) collected from asthenozoospermic men were divided into two cohorts with similar characteristics (cohort 1; n = 28 and cohort 2; n = 108). Cohort 1 was used to evaluate the optimal incubation time with regard to unprepared asthenozoospermic sample sperm motility. After defining an optimal incubation period of 2 h, cohort 2 was used to evaluate which of the four media commonly used in IVF clinics (continuous single culture medium = CSCM®; SpermRinse medium = SR®; in vitro fertilization medium = G-IVF® and human tubal fluid medium = HTF®) was preferred for semen samples from asthenozoospermic patients. Overall, it was determined that a 2-h incubation in CSCM® medium led to the highest asthenozoospermic sperm motility. Thus, this simple, cost-effective, easily reproducible protocol could prove extremely useful for andrology laboratories working with IVF clinics dealing with asthenozoospermic semen specimens. This is particularly relevant since the incidence of the latter is on the rise as semen quality decreases around the globe. Abbreviations: ANOVA: Analysis of variance; ARTs: Assisted reproductive techniques; BWW: Biggers, Whitten, and Whittingham; CO2: Carbon dioxide; CPM: counted per minute; CSCM: Continuous Single Culture Medium; DAB: 3.3′- diaminobenzidine; DFI: DNA Fragmentation Index; DMSO: Dimethyl sulfoxide; G-IVF: In Vitro Fertilization Medium; GSH: Glutathione; GPx: glutathione peroxidase; HDS: High DNA Stainability; HSA: Human Serum Albumin; HTF: Human Tubal Fluid; HYP: Hyperactivity; ICSI: Intracytoplasmic sperm injection; IUI: Intrauterine insemination; IVF: in vitro fertilization; LIN: Linearity; ROS: Reactive Oxygen Species-level; SC: Sperm concentration; SCA: Sperm Computer Analysis; SCSA: Sperm Chromatin Structural Assay; SR: SpermRinse medium; SSS: Synthetic Serum Substitute; STR: Straightness; SOD: superoxide dismutase; TNE: Tris-Borate-EDTA; TSC: Total sperm count; VAP: Mean velocity; VCL: Curvilinear velocity; VSL: Linear velocity; WHO: World Health Organization; WOB: Wobble; spz: spermatozoa; AO: antioxidant

Tricostatin A-treated round spermatid enhances preimplantation embryo developmental competency following round spermatid injection in mice.

It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.

Investigation of Stem Cell Applications on In Vitro Fertilization in Rats

We aimed to search the effects of bone marrow-derived mesenchymal stem cell-conditioned media on in vitro fertilization by investigation of lifetime of germ cells cleavage, degeneration rates and embryo quality. For this purpose, firstly MSCs were isolated from femurs and tibias of the rat, and cells were cultured until the fourth passage. Sperm and oocytes were collected from male and female rats. Oocytes were added in Human Tubal Fluid Media (HTFM), Single Step Media (SSM), Alpha-MEM Media (AMM) and Bone Marrow-Derived Mesenchymal Stem Cell-Conditioned Media (CM). Thousand sperm were added into the media which including oocytes. Embryos were allowed to produce by IVF. The development of the embryos was followed until the 11th day, and the arrest, degeneration rates and alive embryos were established. The embryos reached 2, 4, 8, 16 cells stages and morula stage in the CM. While AMM had a negative effect on fertilization and embryo development, the most favourable effect was shown to be caused by CM in comparison with the other medias. These results have shown that the beneficial effects of CM in IVF would be a significant increase in the rate of fertility and development of embryos.

Factors of the human embryo culture system that may affect media evaporation and osmolality.

Which lab-related factors impact the culture system's capacity to maintain a stable osmolality during human embryo culture? Incubator humidity, the volume of mineral oil, the type of culture media and the design of time-lapse dishes have been identified as important parameters that can cause an impact on media evaporation and consequently osmolality during culture. Culture medium is a critical component in human embryo culture. Minimizing its evaporation during culture is an adequate strategy to stabilize osmolality and, as a result, improving culture conditions and clinical outcomes. The studied variables included media composition and supplementation; volume of mineral oil; incubator humidification; and the type of dish and incubator used. Additionally, six time-lapse dish models were compared in their ability to prevent evaporation. Dishes were incubated in parallel to analyze osmolality during culture between groups: synthetic oviductal medium enriched with potassium versus human tubal fluid medium; protein versus no protein supplementation; dry versus humid atmosphere; high versus low volume of mineral oil. Additionally, media evaporation was compared between six models of time-lapse dishes with distinct designs, cultured in a joint incubator. Two of them were retested in their corresponding incubator to analyze the dish-incubator fit. Daily osmolality measurements were compared between groups. Linear regression was performed to analyze evaporation rates. Protein supplementation did not significantly affect evaporation. Contrarily, humidity levels inside the incubators, the volume of mineral oil and the type of culture media, played an important role in osmolality stabilization. The design of time-lapse dishes and their recommended preparation protocol heavily influenced their evaporation rates, which were further altered by each incubator's characteristics. Media with initially high osmolalities had a bigger risk of reaching hypertonic levels during culture. While numerous, the studied variables are limited and therefore other factors could play a role in osmolality dynamics, as well. Incontrollable atmospheric factors could also result in some variation in the observed results between different centers and laboratories. Published literature has extensively described how hypertonic media may impair embryo development and negatively affect clinical outcomes; therefore, maintaining a stable osmolality during culture should be considered essential. This work is of interest both for embryologists when analyzing their culture system and methodologies, as well as manufacturers in charge of designing IVF consumables. This study was privately funded. N/A.

The duration of embryo culture after mouse IVF differentially affects cardiovascular and metabolic health in male offspring.

STUDY QUESTIONDo the long-term health outcomes following IVF differ depending upon the duration of embryo culture before transfer?SUMMARY ANSWERUsing a mouse model, we demonstrate that in male but not female offspring, adverse cardiovascular (CV) health was more likely with prolonged culture to the blastocyst stage, but metabolic dysfunction was more likely if embryo transfer (ET) occurred at the early cleavage stage.WHAT IS KNOWN ALREADYART associate with increased risk of adverse CV and metabolic health in offspring, and these findings have been confirmed in animal models in the absence of parental infertility issues. It is unclear which specific ART treatments may cause these risks. There is increasing use of blastocyst, versus cleavage-stage, transfer in clinical ART which does not appear to impair perinatal health of children born, but the longer-term health implications are unknown.STUDY DESIGN, SIZE, DURATIONFive mouse groups were generated comprising: (i) natural mating (NM)—naturally mated, non-superovulated and undisturbed gestation; (ii) IV-ET-2Cell—in-vivo derived two-cell embryos collected from superovulated mothers, with immediate ET to recipients; (iii) IVF-ET-2Cell—IVF generated embryos, from oocytes from superovulated mothers, cultured to the two-cell stage before ET to recipients; (iv) IV-ET-BL—in-vivo derived blastocysts collected from superovulated mothers, with immediate ET to recipients; (v) IVF-ET-BL—IVF generated embryos, from oocytes from superovulated mothers, cultured to the blastocyst stage before ET to recipients. Both male and female offspring were analysed for growth, CV and metabolic markers of health. There were 8–13 litters generated for each group for analyses; postnatal data were analysed by multilevel random effects regression to take account of between-mother and within-mother variation and litter size.PARTICIPANTS/MATERIALS, SETTINGS, METHODSC57/BL6 female mice (3–4 weeks old) were used for oocyte production; CBA males for sperm with human tubal fluid medium were used for IVF. Embryos were transferred (ET) to MF1 pseudo-pregnant recipients at the two-cell stage or cultured in synthetic oviductal medium enriched with potassium medium to the blastocyst stage before ET. Control in-vivo embryos from C57BL6 × CBA matings were collected and immediately transferred at the two-cell or blastocyst stage. Postnatal assays included growth rate up to 27 weeks; systolic blood pressure (SBP) at 9, 15 and 21 weeks; lung and serum angiotensin-converting enzyme (ACE) activity at time of cull (27 weeks); glucose tolerance test (GTT; 27 weeks); basal glucose and insulin levels (27 weeks); and lipid accumulation in liver cryosections using Oil Red O imaging (27 weeks).MAIN RESULTS AND THE ROLE OF CHANCEBlastocysts formed by IVF developed at a slower rate and comprised fewer cells that in-vivo generated blastocysts without culture (P < 0.05). Postnatal growth rate was increased in all four experimental treatments compared with NM group (P < 0.05). SBP, serum and lung ACE and heart/body weight were higher in IVF-ET-BL versus IVF-ET-2Cell males (P < 0.05) and higher than in other treatment groups, with SBP and lung ACE positively correlated (P < 0.05). Glucose handling (GTT AUC) was poorer and basal insulin levels were higher in IVF-ET-2Cell males than in IVF-ET-BL (P < 0.05) with the glucose:insulin ratio more negatively correlated with body weight in IVF-ET-2Cell males than in other groups. Liver/body weight and liver lipid droplet diameter and density in IVF-ET-2Cell males were higher than in IVF-ET-BL males (P < 0.05). IVF groups had poorer health characteristics than their in-vivo control groups, indicating that outcomes were not caused specifically by background techniques (superovulation, ET). No consistent health effects from duration of culture were identified in female offspring.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONResults from experimental animal models cannot be extrapolated to humans. Nevertheless, they are valuable to develop conceptual models, in this case, in the absence of confounding parental infertility, in assessing the safety of ART manipulations.WIDER IMPLICATIONS OF THE FINDINGSThe study indicates that longer duration of embryo culture after IVF up to blastocyst before ET leads to increased dysfunction of CV health in males compared with IVF and shorter cleavage-stage ET. However, the metabolic health of male offspring was poorer after shorter versus longer culture duration. This distinction indicates that the origin of CV and metabolic health phenotypes after ART may be different. The poorer metabolic health of males after cleavage-stage ET coincides with embryonic genome activation occurring at the time of ET.STUDY FUNDING/COMPETING INTEREST(S)This work was supported through the European Union FP7-CP-FP Epihealth programme (278418) and FP7-PEOPLE-2012-ITN EpiHealthNet programme (317146) to T.P.F., the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/F007450/1) to T.P.F., and the Saudi government, University of Jeddah and King Abdulaziz University to A.A. The authors have no conflicts of interest to declare.

A simple sperm-sexing method that activates TLR7/8 on X sperm for the efficient production of sexed mouse or cattle embryos.

The preferred sex of livestock differs among breeders; for example, dairy farmers prefer female calves for the production of milk, whereas cattle meat producers often prefer males. Sexing of laboratory animals is also beneficial in some research fields, including reproductive biology and metabolic studies. Most sexing methods separate X sperm and Y sperm with a cell sorter. Here, we describe a system in which treatment with the TLR7/8 ligand (R848) separates X sperm from Y sperm. Because this protocol does not require any special equipment or professional skills, it can be easily applied in laboratories where in vitro fertilization (IVF) is performed. The sperm are treated with 0.03 µM R848 in 1 mL of modified human tubal fluid (mHTF) medium (mouse sperm) or 3 mL of mHTF medium (bull sperm) for 60 min, and then the upper layer (400 µL in mouse sperm or 1 mL in bull sperm) and the precipitate are separately collected. After each sample is washed by centrifugation, the sperm are suspended in ligand-free IVF medium and can then be used for IVF. More than 90% of the embryos made with upper-layer sperm are XY in both mice and cattle, and >80% of the embryos made with precipitated sperm are XX in both species. Separation of X sperm and Y sperm for IVF can be completed within 2 h.

Comparison of DNA methylation patterns of parentally imprinted genes in placenta derived from IVF conceptions in two different culture media.

Study questionIs there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium?Summary answerWe found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium.What is known alreadyAnimal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta.Study design, size, durationTo identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008–2013 in the Netherlands as reference material.Participants/materials, setting, methodsTo identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects.Main results and the role of chanceWe found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture mediumgroups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon.Limitations, reasons for cautionDue to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues.Wider implications of the findingsIt has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed.Study funding/competing interest(s)This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest.Trial registration numberPlacental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.

The Effects of Calligonum Extract and Low-Intensity Ultrasound on Motility, Viability, and DNA Fragmentation of Human Frozen-Thawed Semen Samples.

BackgroundThe study aimed to evaluate the impact of Calligonum extract and US radiation on sperm parameters of cryopreserved human semen samples.Materials and MethodsIn this experimental study, twenty-five semen specimens were obtained from healthy semen donors and incubated in human tubal fluid (HTF) medium supplemented with 10% human serum albumin (HSA) for 45 minutes. Samples were treated with Calligonum extract (10 μg/ml) alone (CGM group) and US radiation (LIPUS- exposed group) alone or a combination of both treatments (CGM+LIPUS). The US group received US stimulation (in both continuous and pulsed wave modes) at a frequency of 1 MHZ and intensity of 200 mW/cm2 for 200 seconds. Sperm morphology was assessed by Diff-Quik staining. The DNA fragmentation was evaluated the Halo sperm kit. Sperm parameters was analyzed by a computer-assisted semen analysis system. Reactive oxygen species (ROS) was assessed by flow cytometry.ResultsThe results showed that the treatment with Calligonum extract significantly (P<0.05) increased the progressive motility of spermatozoa in the CGM group as compared with the control group. The application of low-intensity US significantly (P<0.05) decreased the motility and viability of spermatozoa in the US group when compared with the control group. Our findings also indicated that the use of both low-intensity US in continuous mode and Calligonum extract slightly increased progressive motility; however, such an increase was not statistically significant. The rate of DNA fragmentation was considerably higher (P<0.05) in control and LIPUS-exposed groups than the other groups.ConclusionTreatment of spermatozoa with Calligonum extract slightly improved the sperm parameters due to its antioxidant activity, on the other hand, according to our results, US radiation did not improve sperm parameters which may be due to interference with the motility of sperm, as well as its physical effects on spermatozoa.

156 Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional IVF has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. Thus, the first part of this study aimed to compare HTF (Summers and Biggers, 2003 Hum. Reprod. Update9, 557-582, DOI: 10.1093/humupd/dmg039) and Whitten’s (McPartlin et al. 2009 Biol. Reprod.81, 199-206, DOI: 10.1095/biolreprod.108.074880) media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine on sperm motility parameters was evaluated in both media at different incubation times. Fresh semen from 3 Chilote stallions was collected, diluted to 10×106 sperm mL−1 in capacitating (7mg mL−1 BSA and 25mM NaHCO3) and non-capacitating (without BSA and NaHCO3) HTF and Whitten’s media and incubated for 30 and 120min at 38°C in air atmosphere. Integrity and destabilisation of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester perchlorate, acrosome membrane integrity by peanut agglutinin/fluorescein isothiocyanate and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® in a FACSCanto II flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, USA). A total of 10,000 sperm events were acquired from each measurement (n=3 replicates for each stallion). Motility parameters were evaluated using the integrated semen analysis system (ISAS®, Selinion Medical, Brussels, Belgium). Percentage data were arcsine transformed and subjected to a 2-way ANOVA with Bonferroni’s post hoc test using Prism 7 software (GraphPad Software, La Jolla, CA, USA). We found no differences between Whitten’s and HTF media in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30 and 120min of incubation. Membrane fluidity (MC540) increased in both media at 30 and 120min of incubation compared with non-capacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2 and 4h of incubation compared with non-capacitating conditions, without differences between media. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm. Funding support was received from FONDECYT 1160467 CONICYT, Chile.

Effect of Lepidium meyenii on in vitro fertilization via improvement in acrosome reaction and motility of mouse and human sperm.

PurposeThe direct effects of Lepidium meyenii (Maca) on sperm remain unclear. Herein, we examined the direct effect of Maca on in vitro fertilization.MethodsWe examined the fertilization rate in a mouse model and the rate of acrosome reaction in sperm from transgenic mice expressing enhanced green fluorescent protein (EGFP) in a Maca extract‐containing human tubal fluid (HTF) medium. Using human sperm, we assessed acrosome status via fluorescein isothiocyanate‐conjugated peanut agglutinin (FITC‐PNA) staining and performed detailed analysis using a sperm motility analysis system (SMAS).ResultsIn the mouse model, the fertilization rate in the Maca extract‐containing HTF was significantly higher than that in the control medium. The acrosome reaction rate in sperm from transgenic mice expressing EGFP was also significantly higher in the Maca extract‐containing HTF than that in the control medium. Similarly, a high acrosome reaction rate, identified via FITC‐PNA staining of human sperm samples, was found in the Maca extract‐containing HTF compared with that in the control medium. Human sperm motility in the Maca extract‐containing HTF was also increased compared with that in the control medium as measured using an SMAS.ConclusionsMaca improved in vitro fertilization rates by inducing an acrosome reaction and increasing sperm motility.

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation.

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.

Quality of ram spermatozoa separated with modified swim up method

Semen was collected from four rams, using artificial vagina and viability%, motility% and plasma membrane integrity% were measured. Fresh ejaculates (n = 32) were separated by modified swim-up separation using modified human tubal fluid medium. Four fractions of supernatant were collected at 15-minute intervals. The mean volume, mass activity, concentration, motility%, viability%, normal morphology and membrane integrity% (HOST +ve) of fresh semen were 1.0 ± 0.14, 4.1 ± 0.1 × 109 spermatozoa/ml, 85.0 ± 1.3, 89.4 ± 1.0, 85.5 ± 0.7, 84.7 ± 0.5 respectively. There was no significant (P&gt;0.05) difference in fresh semen quality parameters between rams. The motility%, viability% and HOST +ve % of first, second, third and fourth fractions were 53.4 ± 0.5, 68.2 ± 0.3, 74.8 ± 0.3 and 65.5 ± 0.4; 55.5 ± 0.4, 66.2 ± 0.4, 74.5 ± 0.3 and 73.6 ± 0.3 and 66.7 ± 0.5, 66.8 ± 0.5, 65.2 ± 0.4 and 74.7 ± 0.5 respectively. The motility%, viability% and membrane integrity% of separated semen samples differed significantly (P&lt;0.05) between four fractions. The mean motility% and viability% were significantly higher (P&lt;0.05) in third fraction (74.8 ± 0.3%), whereas the mean HOST +ve% was significantly higher (P&lt;0.05) in fourth fraction (74.7 ± 0.5). All quality parameters of separated spermatozoa were significantly (P&lt;0.05) lower than that of fresh semen. The pregnancy rates were higher with fresh semen (71%) in comparison to that of separated sample (57%).Bangl. vet. 2016. Vol. 33, No. 2, 62-70

Cryoprotectant‐free vitrification of human spermatozoa in new artificial seminal fluid

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5mg/mL human serum albumin and 0.25mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09±10.33 vs. 36.80±13.75), grade A motility (36.59±11.40 vs. 16.41±11.24), and normal morphology (18.74±8.35 vs. 11.85±5.84) and viability (68.22±10.83 vs. 60.86±11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p=0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p<0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p<0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF.

Human Preimplantation Embryo Culture Media: Past, Present, and Future

Abstract: Systematic studies of mouse embryo culture beginning in 1949 led to an understanding of essential medium components for early mammalian embryos, and embryo culture from the zygote to the blastocyst stage was achieved in 1968. Since then, medium components that are either beneficial or detrimental for embryo culture have been identified. A variety of culture media that mimic the female reproductive tract, such as human tubal fluid medium and sequential media, were developed from the 1970s to the 1990s, and a single medium in which the concentrations of components were determined by a simplex optimization method was introduced for clinical use in 2002. While either sequential media or a single medium is currently used in most cases, no conclusion has yet been reached as to which of the two approaches is the best. That we are now easily able to culture embryos is the result of the work of pioneers. This review presents a chronological overview of media development from initial attempts at mouse emb...

Effect of repeated vitrification-thawing on sperm chromatin and morphology

Cryoinjury in sperm after repeated cycles of thaw-refreezing is of interest in patients with few sperm, cancer patients and for couples needing same donor sperm. Presently, there is little information on sperm chromatin integrity after repeated vitrification. The null hypothesis was that this process would not disrupt sperm chromatin and alter morphology. The objectives were: (1) to analyze sperm chromatin and morphology after repeated vitrification and thawing and (2) to compare vitrification with a standard cryopreservation method for preservation of chromatin integrity. In vitro study in an IVF center. Sperm cells (normozoospermic, WHO criteria) were washed by 2-gradient centrifugation and resuspended in either: (1) modified human tubal fluid (HTF) medium and Irvine Scientific Freezing Medium 90128 (standard freezing) or, (2) HTF medium with 0.25M sucrose with 1% albumin (Isachenko vitrification method). Aliquots were analyzed (pre-freeze parameters). For comparison, sperm from an oligozoospermic case was processed in a similar manner. Sperm (standard freezing) were placed in Nunc cryovials (0.25 mL each) and frozen in liquid nitrogen (LN2). In the vitrification group, sperm were aspirated into 0.25mL cryostraws, sealed in 0.5mL cryostraws and frozen in LN2. After 1-week, sperm were thawed and one vial/straw was analyzed. The remaining vials/straws were re-frozen. Three cycles of repeated freeze-thaw were carried out. Differences were tested using ANOVA and Student's t-test. In the vitrification group, sperm chromatin integrity decreased by 9.6, 29.1 and 47.1% with each subsequent freeze-thaw cycle. Decreased chromatin integrity in the standard freeze group were 0.4, 12.9, 27.0% respectively. In the oligozoospermic specimen, chromatin integrity (vitrification) decreased by 45.3, 48.4 and 68.5% compared with standard freeze reduction of 25.8, 37.1 and 57.2%. Normal morphology in the vitrification group were 17.7+/-0.4, 7.0+/-1.4, 5.3+/-0.4, 5.6+/-0.4% (mean+/-SEM) while the standard freeze group were 18.3+/-0.7, 12.0+/-0.7, 9.3+/-0.4, 7.7+/-1.1% for pre-freeze and 3 cycles of freeze-thaw respectively. Similar decreases were seen in the oligozoospermic case. Sperm viability and motility also decreased in a similar manner. The results showed that vitrification was less optimal than standard freeze method when repeated freeze-thaw cycles were involved. Furthermore, normal morphology was greatly decreased with repeat vitrification suggesting extensive structural disruption. Interestingly, after the third freeze-thaw cycle, 46% (vitrification) or 62% (standard freeze) of sperm had normal chromatin suggesting adequacy for conserving sperm for patients.

Sperm Selection Using Three Semen Processing Techniques.

This study aimed to assess the efficiency, in terms of recovered motile spermatozoa with normal morphology, of three sperm selection techniques: migration- sedimentation (SS), swim-up from fresh semen (SF), and swim-up from washed (SL) sperm. Samples from 20 normozoospermic men were divided into three equal aliquots and processed in parallel. SS was performed in a Jondet tube, using 1 ml of semen and 2.5 ml of Human Tubal Fluid medium (HTF+10% Synthetic Serum Supplement, Irvine, USA). For SF, 1 ml of HTF was layered over 1 ml of fresh semen (SF). For SL, 1 ml of sperm was first centrifuged (300 g, 10 min) and the pellet resuspended in 1 ml of HTF; a second layer of HTF was placed on top. Migration time was 1h (SF and SL) and 1h30' for SS at 37°C. After migration, 200 μl were removed from the top layer (SF, SL) and from the central cone (SS). Concentration, morphology and motility were determined. Recovery rates were 25% for SS, 10.1% for SF and 4.5% for SL. SS recovery rate was significantly higher (P<0.01) than the two swim-up techniques. Total motility was statistically different (P<0.001), with 93.6% for SS, 91.2% for SF, and 77% for SL. Sperm morphology was similar between the three techniques (P= 0.12). SS is an efficient technique for the recovery of motile spermatozoa from native semen preparations and yielded better results than SF and SL. Routine use for assisted reproduction remains to be evaluated.