Structure-based redesigning of pentoxifylline analogs against selective phosphodiesterases to modulate sperm functional competence for assisted reproductive technologies

AbstractPhosphodiesterase (PDE) inhibitors, such as pentoxifylline (PTX), are used as pharmacological agents to enhance sperm motility in assisted reproductive technology (ART), mainly to aid the selection of viable sperm in asthenozoospermic ejaculates and testicular spermatozoa, prior to intracytoplasmic sperm injection (ICSI). However, PTX is reported to induce premature acrosome reaction (AR) and, exert toxic effects on oocyte function and early embryo development. Additionally, in vitro binding studies as well as computational binding free energy (ΔGbind) suggest that PTX exhibits weak binding to sperm PDEs, indicating room for improvement. Aiming to reduce the adverse effects and to enhance the sperm motility, we designed and studied PTX analogues. Using structure-guided in silico approach and by considering the physico-chemical properties of the binding pocket of the PDEs, designed analogues of PTX. In silico assessments indicated that PTX analogues bind more tightly to PDEs and form stable complexes. Particularly, ex vivo evaluation of sperm treated with one of the PTX analogues (PTXm-1), showed comparable beneficial effect at much lower concentration—slower AR, higher DNA integrity and extended longevity of spermatozoa and superior embryo quality. PTXm-1 is proposed to be a better pharmacological agent for ART than PTX for sperm function enhancement.

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Ex vivo early embryo development and effects on gene expression and imprinting

Reproduction Fertility and Development ◽

10.1071/rd04103 ◽

2005 ◽

Vol 17 (3) ◽

pp. 361 ◽

Cited By ~ 103

Author(s):

David K. Gardner ◽

Michelle Lane

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Assisted Reproductive Technologies ◽

Ex Vivo ◽

Reproductive Technologies ◽

Developmental Competence ◽

Laboratory Animals ◽

Cell Physiology ◽

Mammalian Embryo

The environment to which the mammalian embryo is exposed during the preimplantation period of development has a profound effect on the physiology and viability of the conceptus. It has been demonstrated that conditions that alter gene expression, and in some instances the imprinting status of specific genes, have all previously been shown to adversely affect cell physiology. Thus, questions are raised regarding the aetiology of abnormal gene expression and altered imprinting patterns, and whether problems can be averted by using more physiological culture conditions. It is also of note that the sensitivity of the embryo to its surroundings decreases as development proceeds. Post compaction, environmental conditions have a lesser effect on gene function. This, therefore, has implications regarding the conditions used for IVF and the culture of the cleavage stage embryo. The developmental competence of the oocyte also impacts gene expression in the embryo, and therefore superovulation has been implicated in abnormal methylation and imprinting in the resultant embryo. Furthermore, the genetics and dietary status of the mother have a profound impact on embryo development and gene expression. The significance of specific animal models for human assisted reproductive technologies (ART) is questioned, given that most cattle data have been obtained from in vitro-matured oocytes and that genes imprinted in domestic and laboratory animals are not necessarily imprinted in the human. Patients treated with ART have fertility problems, which in turn may predispose their gametes or embryos to greater sensitivities to the process of ART. Whether this is from the drugs involved in the ovulation induction or from the IVF, intracytoplasmic sperm injection or culture procedures themselves remains to be determined. Alternatively, it may be that epigenetic alterations are associated with infertility and symptoms are subsequently revealed through ART. Whatever the aetiology, continued long-term monitoring of the children conceived through ART is warranted.

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Long-Term Effects Following Fresh/Vitrified Embryo Transfer Are Transmitted by Paternal Germline in a Large Size Rabbit Cohort

Animals ◽

10.3390/ani10081272 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1272 ◽

Cited By ~ 1

Author(s):

Ximo Garcia-Dominguez ◽

José Salvador Vicente ◽

María P. Viudes-de-Castro ◽

Francisco Marco-Jiménez

Keyword(s):

Embryo Transfer ◽

Assisted Reproductive Technologies ◽

Later Life ◽

Reproductive Technologies ◽

Early Embryo ◽

Embryo Cryopreservation ◽

Long Term Effects ◽

Adult Body Weight ◽

Female Germline

The concept of developmental programming suggests that the early life environment influences offspring phenotype in later life, whose effects may also be manifested in further generations. Valuable pieces of evidence come from the fields applying assisted reproductive technologies (ARTs), which deprive embryos of their optimal maternal environment and were thus associated with subsequent developmental deviations. Recently, we demonstrated that the in vitro manipulations during a vitrified embryo transfer procedure incurs a cumulative and transgenerational decline in the growth performance of the resulting offspring. Here, we provide a longitudinal study to investigate whether previous developmental deviations could be indistinctly paternally or maternally transmitted using crossbred mattings. Our findings revealed that early embryo manipulations through fresh and vitrified embryo transfer incurred paternally transmissible effects over the growth pattern and adult body weight, which seemed not inheritable via the female germline. Similar inheritable effects were observed after fresh and vitrified embryo transfer, suggesting that disturbing optimal embryo development through in vitro manipulations was the principal trigger of transmissible effects, rather than embryo cryopreservation per se.

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Investigating media that support red wolf (Canis rufus) sperm viability and capacitation in vitro

Reproduction and Fertility ◽

10.1530/raf-20-0042 ◽

2020 ◽

Vol 1 (1) ◽

pp. 83-92

Author(s):

Jennifer B Nagashima ◽

Marcia de Almeida Monteiro Melo Ferraz ◽

Sarah H Kamen ◽

Nucharin Songsasen

Keyword(s):

Tyrosine Phosphorylation ◽

Sperm Motility ◽

Assisted Reproductive Technologies ◽

Culture Media ◽

Reproductive Technologies ◽

Critically Endangered ◽

Sperm Viability ◽

Canis Rufus ◽

Red Wolf

The red wolf is a critically endangered canid, with ~250 and ~20 individuals in the ex situ and reintroduced wild populations, respectively. Assisted reproductive technologies such as sperm cryopreservation and in vitro fertilization therefore represent critically-needed tools to manage these populations. However, the motility of post-thaw red wolf sperm rapidly declines during in vitro incubation, hindering the ability to develop these technologies. In this study, we evaluated the influence of several culture media (a modified canine capacitation medium (mCCM), a modified North Carolina State University-23 medium (mNCSU-23), a synthetic oviductal fluid (SOF), a fertilization Tyrode’s medium base or Fert-TALP (FERT), and a TRIS-based buffer (TRIS)) on the survival and capacitation of red wolf sperm during extended (18 h) incubation at 38.5°C and 5% CO2. Red wolf sperm motility averaged (±s.e.m.) 73.8 ± 7.1% at the time of collection, and was better maintained over 4 h incubation in mCCM (55.0 ± 9.8%) and mNCSU-23 (54.7 ± 10.4), compared to mSOF (43.8 ± 8.3%), FERT (30 ± 10.5), and TRIS (16.4 ± 4.1%) solutions. Patterns of tyrosine phosphorylation signal, as assessed via immunocytochemistry, indicated induction of capacitation between 2 and 4 h in vitro culture. Tyrosine phosphorylation signal was particularly robust in mCCM and mNCSU-23 incubated sperm, although significant acrosome exocytosis was not observed in response to progesterone supplementation after 3 h incubation in any of the media. In sum, results indicate mCCM and mNCSU-23 are promising base media for the in vitro incubation and capacitation of red wolf sperm, for assisted reproduction applications. Lay summary Development of assisted reproductive technologies such as in vitro fertilization and artificial insemination is of high importance to the genetic management of critically endangered species such as the red wolf (Canis rufus). However, these technologies require the ability to maintain sperm viability and function during extended incubation, which has not been successful for the red wolf thus far. In this study, various culture media developed for sperm/egg/embryo culture in large mammalian species were evaluated for their ability to maintain red wolf sperm motility under physiological incubation conditions. Media and conditions previously utilized for domestic dog sperm were found to best support sperm incubation and capacitation (process of becoming competent to fertilize an egg) in the red wolf, representing a key step for future development of assisted reproductive technologies for the species.

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Comparison of Histone H3K4me3 between IVF and ICSI Technologies and between Boy and Girl Offspring

International Journal of Molecular Sciences ◽

10.3390/ijms22168574 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8574

Author(s):

Huixia Yang ◽

Zhi Ma ◽

Lin Peng ◽

Christina Kuhn ◽

Martina Rahmeh ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Histone Modifications ◽

Assisted Reproductive Technologies ◽

Histone H3 ◽

Reproductive Technologies ◽

Early Embryo ◽

Vital Role ◽

Vitro Fertilization ◽

Cord Blood Mononuclear Cell

Epigenetics play a vital role in early embryo development. Offspring conceived via assisted reproductive technologies (ARTs) have a three times higher risk of epigenetic diseases than naturally conceived children. However, investigations into ART-associated placental histone modifications or sex-stratified analyses of ART-associated histone modifications remain limited. In the current study, we carried out immunohistochemistry, chip-sequence analysis, and a series of in vitro experiments. Our results demonstrated that placentas from intra-cytoplasmic sperm injection (ICSI), but not in vitro fertilization (IVF), showed global tri-methylated-histone-H3-lysine-4 (H3K4me3) alteration compared to those from natural conception. However, for acetylated-histone-H3-lysine-9 (H3K9ac) and acetylated-histone-H3-lysine-27 (H3K27ac), no significant differences between groups could be found. Further, sex -stratified analysis found that, compared with the same-gender newborn cord blood mononuclear cell (CBMC) from natural conceptions, CBMC from ICSI-boys presented more genes with differentially enriched H3K4me3 (n = 198) than those from ICSI-girls (n = 79), IVF-girls (n = 5), and IVF-boys (n = 2). We also found that varying oxygen conditions, RNA polymerase II subunit A (Polr2A), and lysine demethylase 5A (KDM5A) regulated H3K4me3. These findings revealed a difference between IVF and ICSI and a difference between boys and girls in H3K4me3 modification, providing greater insight into ART-associated epigenetic alteration.

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Impacts of and interactions between environmental stress and epigenetic programming during early embryo development

Reproduction Fertility and Development ◽

10.1071/rd14049 ◽

2015 ◽

Vol 27 (8) ◽

pp. 1125 ◽

Cited By ~ 5

Author(s):

Michael J. Bertoldo ◽

Yann Locatelli ◽

Christopher O'Neill ◽

Pascal Mermillod

Keyword(s):

Embryo Development ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Culture Conditions ◽

Early Embryo ◽

Signalling Pathways ◽

Epigenetic Programming ◽

Altered Gene ◽

Vitro Development

The processes of assisted reproductive technologies (ART) involve a variety of interventions that impact on the oocyte and embryo. Critically, these interventions cause considerable stress and coincide with important imprinting events throughout gametogenesis, fertilisation and early embryonic development. It is now accepted that the IVM and in vitro development of gametes and embryos can perturb the natural course of development to varying degrees of severity. Altered gene expression and, more recently, imprinting disorders relating to ART have become a focused area of research. Although various hypotheses have been put forward, most research has been observational, with little attempt to discover the mechanisms and periods of sensitivity during embryo development that are influenced by the culture conditions following fertilisation. The embryo possesses innate survival factor signalling pathways, yet when an embryo is placed in culture, this signalling in response to in vitro stress becomes critically important in mitigating the effects of stresses caused by the in vitro environment. It is apparent that not all embryos possess this ability to adequately adapt to the stresses experienced in vitro, most probably due to an inadequate oocyte. It is speculated that it is important that embryos use their survival signalling mechanisms to maintain normal epigenetic programming. The seeming redundancy in the function of various survival signalling pathways would support this notion. Any invasion into the natural, highly orchestrated and dynamic process of sexual reproduction could perturb the normal progression of epigenetic programming. Therefore the source of gametes and the subsequent culture conditions of gametes and embryos are critically important and require careful attention. It is the aim of this review to highlight avenues of research to elucidate the effects of stress and the relationship with epigenetic programming. The short- and long-term health and viability of human and animal embryos derived in vitro will also be discussed.

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A Comparison Between the Effects of Global Sperm Washing® and FertiCult Flushing TM Media on Certain Sperm Function Parameters of Asthenozoospermic Men

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2017.11.2.519 ◽

2017 ◽

Vol 11 (2) ◽

pp. 37-41

Author(s):

Saad S. Al-Dujaily ◽

Khalid Al-Azzawi ◽

Zena Hussein ◽

Ban Al-Anii

Keyword(s):

Sperm Motility ◽

Assisted Reproductive Technologies ◽

Semen Analysis ◽

Reproductive Technologies ◽

World Health ◽

Sperm Function ◽

Sperm Activation ◽

Optimum Result ◽

Sperm Washing

The World Health Organization (WHO) and many studies considered the infertility as a disease and so many couples complaining from unsuccessful assisted reproductive technologies procedures to overcome their problem. One of the reasons of this dilemma is the sperm preparation method when no optimum result obtained even by using any of media found globally. However Global sperm washing®, and FertiCult flushingTM media were proved their capability to obtain good results of certain sperm function parameters. Nevertheless, the studies that compare between these media were rare. Therefore, this study aimed to compare between Global sperm washing medium®, FertiCult flushing TM media that used for sperm washing before using the partner sperm for ART procedure. After detecting asthenozoospermia in sixty semen samples, they were divided into two groups according to medium used for sperm activation in vitro Global sperm washing medium ® (n=31) and FertiCult flushing mediumTM (n=29) groups.The semen analysis was done after 3-5 days of abstinence as recommended by the manual of WHO (1999). Certain sperm function parameters were recorded. Semen fluid samples were treated with sperm activation media (Global sperm washing medium and FertiCult flushing medium TM) by using direct swim-up technique for in vitro sperm activation test. A significant (P<0.05) improvement was noticed between the two media regarding active sperm motility grades A and B when using FertiCult flushing mediumTM compared to Global sperm washing medium®. Whereas no significant (P>0.05) differences were detected between the two media regarding sperm motility grades C and D. There was no significant (P>0.05) differences in morphologically normal sperm following in vitro activation by using the two media. It is concluded that FertiCult flushing mediumTM was better than Global sperm washing medium® in improving active sperm motility of asthenozoospermic men which can be utilized in future for successful of assisted reproduction.

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Future developments in assisted reproduction in humans

Reproduction ◽

10.1530/rep.0.1230171 ◽

2002 ◽

pp. 171-183 ◽

Cited By ~ 27

Author(s):

K Hardy ◽

C Wright ◽

S Rice ◽

M Tachataki ◽

R Roberts ◽

...

Keyword(s):

Assisted Reproduction ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Early Embryo ◽

Embryo Biopsy ◽

Screening Methods ◽

Success Rates ◽

Aneuploidy Screening ◽

Vitro Fertilization

The advent of human in vitro fertilization (IVF) over 30 years ago has made the oocyte and preimplantation embryo uniquely accessible. This accessibility has given rise to new micromanipulation techniques, such as intracytoplasmic sperm injection for treatment of male infertility, as well as embryo biopsy for preimplantation diagnosis of both genetic disease and aneuploidy, a major cause of early embryo demise and miscarriage. In the UK, average pregnancy rates after IVF and embryo transfer are < 25%, even after transfer of several embryos. Unfortunately, a third of these pregnancies involve multiple gestations. Research is currently focusing on methods to improve IVF success rates while reducing twin and triplet pregnancies and their associated increased morbidity and mortality. One approach is to develop screening methods to identify the most viable embryos, so that transfer of fewer healthy embryos will result in a higher proportion of singleton pregnancies. Screening methods include optimizing culture conditions for prolonged culture and selection of viable blastocysts for transfer, or embryo biopsy and aneuploidy screening. Assisted reproduction is also increasingly important in other branches of medicine: survival rates for cancer sufferers are improving continually and there is now a significant need for approaches to preserve fertility after sterilizing chemo-and radiotherapy treatment. Techniques for cryopreserving male and female gametes or gonadal tissue are being developed, although systems to grow and mature these gametes are in their infancy. Finally, there are also concerns regarding the safety of these new assisted reproductive technologies.

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Papain and its inhibitor E-64 reduce camelid semen viscosity without impairing sperm function and improve post-thaw motility rates

Reproduction Fertility and Development ◽

10.1071/rd15261 ◽

2017 ◽

Vol 29 (6) ◽

pp. 1107 ◽

Cited By ~ 12

Author(s):

C. M. Kershaw ◽

G. Evans ◽

R. Rodney ◽

W. M. C. Maxwell

Keyword(s):

Sperm Motility ◽

Exposure Time ◽

Seminal Plasma ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Plasma Viscosity ◽

Dna Integrity ◽

Sperm Function ◽

Viscous Component ◽

Acrosome Integrity

In camelids, the development of assisted reproductive technologies is impaired by the viscous nature of the semen. The protease papain has shown promise in reducing viscosity, although its effect on sperm integrity is unknown. The present study determined the optimal papain concentration and exposure time to reduce seminal plasma viscosity and investigated the effect of papain and its inhibitor E-64 on sperm function and cryopreservation in alpacas. Papain (0.1 mg mL–1, 20 min, 37°C) eliminated alpaca semen viscosity while maintaining sperm motility, viability, acrosome integrity and DNA integrity. Furthermore E-64 (10 µM at 37°C for 5 min after 20 min papain) inhibited the papain without impairing sperm function. Cryopreserved, papain-treated alpaca spermatozoa exhibited higher total motility rates after chilling and 0 and 1 h after thawing compared with control (untreated) samples. Papain treatment, followed by inhibition of papain with E-64, is effective in reducing alpaca seminal plasma viscosity without impairing sperm integrity and improves post-thaw motility rates of cryopreserved alpaca spermatozoa. The use of the combination of papain and E-64 to eliminate the viscous component of camelid semen may aid the development of assisted reproductive technologies in camelids.

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Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies

Reproduction ◽

10.1530/rep-12-0237 ◽

2012 ◽

Vol 144 (4) ◽

pp. 393-409 ◽

Cited By ~ 81

Author(s):

Michelle M Denomme ◽

Mellissa R W Mann

Keyword(s):

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Early Embryo ◽

Cellular Mechanisms ◽

Apparent Lack ◽

Epigenetic Instability ◽

Genomic Imprints ◽

Epigenetic Disruption ◽

Genome Scale

Gamete and early embryo development are important stages when genome-scale epigenetic transitions are orchestrated. The apparent lack of remodeling of differential imprinted DNA methylation during preimplantation development has lead to the argument that epigenetic disruption by assisted reproductive technologies (ARTs) is restricted to imprinted genes. We contend that aberrant imprinted methylation arising from assisted reproduction or infertility may be an indicator of more global epigenetic instability. Here, we review the current literature on the effects of ARTs, including ovarian stimulation,in vitrooocyte maturation, oocyte cryopreservation, IVF, ICSI, embryo culture, and infertility on genomic imprinting as a model for evaluating epigenetic stability. Undoubtedly, the relationship between impaired fertility, ARTs, and epigenetic stability is unquestionably complex. What is clear is that future studies need to be directed at determining the molecular and cellular mechanisms giving rise to epigenetic errors.

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207 Female Reproductive Fluids and Epigenetics

Journal of Animal Science ◽

10.1093/jas/skab054.188 ◽

2021 ◽

Vol 99 (Supplement_1) ◽

pp. 114-114

Author(s):

Sebastian Canovas ◽

Raquel Romar ◽

Pilar Coy

Keyword(s):

Assisted Reproductive Technologies ◽

Culture Media ◽

Morphological Changes ◽

Adult Life ◽

Specific Effect ◽

Reproductive Technologies ◽

Early Embryo ◽

Vitro Fertilization

Abstract Physiological fertilization, and early embryo development, involves dramatic transcriptomic, epigenetic and morphological changes in a short temporal window. During this period gametes and early embryos are surrounded by reproductive fluids (oviductal and uterine), which contain nutrients, growth factors, hormones and extracellular vesicles acting as carriers of DNA, RNA, proteins and other factors with putative roles in intercellular communication. Under in vitro conditions, and in the absence of these fluids, embryos derived from Assisted Reproductive Technologies (ART) reveal transcriptional and epigenetic differences compared with in vivo embryos, which could result in long-term phenotypic consequences in adult life. Therefore, reproductive fluids supplementation in the culture medium offers an alternative to imitate physiological conditions and decrease these consequences. In vitro, oviductal fluid (OF) can modulate capacitation-associated events and sperm-zona pellucida interactions and contribute to the control of polyspermy in pigs. The use of in vitro fertilization media supplemented with reproductive fluids (Natur-IVF) improves embryo quality and blastocysts hatching ability. Moreover, Natur-IVF embryos show expression and methylation patterns closer to in vivo blastocysts. In cows, supplementation of culture media with reproductive fluids, or some isolated factors, improves blastocyst rate and survival after embryo transfer, and reverses the expression of some altered genes. However, considering the complexity of the oviductal and uterine fluids, it seems difficult that the use of just a few factors in isolation can reverse all undesired consequences of the IVP. On the other hand, sex-specific embryonic plasticity, as a consequence of the oviductal regulatory signals, have been proposed. Thus, we have analysed the sex-specific effect of supplementation with reproductive fluids in bovine embryos and data reveal sex-dependent impact in DNA methylation. All these results confirm that developmental programme can be modulated by reproductive fluids and it shows sex-specific effects. This strategy allows the possibility of minimizing undesired in vitro derived consequences.

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