Capacitation-associated Protein Tyrosine Phosphorylation As a Possible Biomarker of Sperm Selection
NCT ID: NCT04962100
Last Updated: 2024-10-08
Study Results
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Basic Information
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COMPLETED
NA
335 participants
INTERVENTIONAL
2021-06-29
2023-09-30
Brief Summary
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Detailed Description
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The sperm cells from the ejaculate of all mammals are unable to fertilize the oocyte. In order to acquire the fertilization competence, sperm must undergo a process called capacitation. Capacitation has been defined as a complex phenomenon that involves biochemical and physiological changes in the sperm.
These changes are such as:
* Loss of proteins or replacement by others of lower molecular weight
* Transformation of phospholipids, decreased cholesterol/phospholipid ratio
* Changes in the carbohydrate fraction of glycoproteins
* Lipid and protein mobility
Not all the events involved in the complex capacitation process are known.
The following stages can be differentiated:
* Cell membrane fluidification
* Increased intracellular Ca2+
* Generation of controlled amounts of reactive oxygen species
* Increased intracellular pH and hyperpolarization of sperm membrane potential
* Protein phosphorylation (serine, threonine and tyrosine residues)
Under in vivo conditions, motile sperm actively migrate through the cervical mucus and are separated from the rest of the ejaculate. In vitro, sperm capacitation can be achieved if they are subjected to particular culture conditions during the necessary period of time.
After capacitation, sperm acquires three characteristics:
1. Hyperactivation.
2. Acrosomic reaction.
3. Sperm-oocyte interaction.
The capacitation process is necessary for the sperm to be able to cross the layer of cells that surround the oocyte and to undergo the acrosome reaction. Only a fraction of sperm are known to reach the capacitation state at some point.
The capacitation process depends mainly on post-translational modifications of proteins. One of the most important modifications that these proteins undergo is phosphorylation. During sperm capacitation, serine, threonine and tyrosine residues are phosphorylated. However, phosphorylation of tyrosine residues is the best indicator of sperm capacitation.
The evaluation of the phosphorylation of the tyrosine residues of the sperm proteins by flow cytometry makes it possible to assess the degree of capacitation of the sperm in vitro and to determine how many of them have carried out this process adequately . As previously mentioned, not all sperm cells of a sample undergo the capacitation process and not all of them present the same percentage of phosphorylated proteins in tyrosine residues.
Certain events, such as the cryopreservation process and the incubation times of the samples until their use in fertility treatments (IA or IVF / ICSI), can affect the capacitation process. Therefore, the measurement of the levels of tyrosine phosphorylation of the sperm by flow cytometry can allow the investigators to know the level of capacitation of the sample. There are studies that report changes in success rates when samples are incubated different period of times. The incubation period affects the rate of acrosomal reaction of sperm and may even affect DNA fragmentation. Furthermore, the temperature at which such incubation is carried out also seems to affect sperm capacitation.
Based on the above, this project aims to analyze the possible correlation between the levels of tyrosine phosphorylation in sperm and the functional quality of the seminal sample, as well as the reproductive success of the cycle. In parallel, the investigators will analyze whether the sperm capacitation process is affected by the incubation times of the sample once it has been capacitated or whether it depends exclusively on the quality of the seminal sample and is intrinsic to the male.
Conditions
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Study Design
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NA
SINGLE_GROUP
BASIC_SCIENCE
NONE
Study Groups
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Study population
Male patients and donors who provide a sample of fresh ejaculated semen will be the population of this study, as well as female patients undergoing artificial insemination with their partner's semen or frozen donor semen.
Analysis of capacitation-associated tyrosine phosphorylation in human sperm by flow cytometry
After the assessment of the macroscopic and microscopic characteristics of the sample, we will wash the sample and process it using three layers of density gradients.
In the case of samples for diagnostic analysis, the remaining volume will be incubated in at 37ºC, and 0.1-0.2 ml aliquots will be taken from the end of the processing at t0 (just before introducing the sample in the incubator), at t1 (after one hour of incubation) and at t3 (after 3 hours of incubation). Additionally, a sample will be taken after washing, prior to processing the sample with the density gradients. In the case of samples processed for insemination, an additional aliquot will be taken at the exact moment in which the insemination is carried out and the elapsed incubation time will be noted.
The evaluation of the capacitation will be carried out by analyzing the state of phosphorylation of tyrosines by flow cytometry.
Interventions
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Analysis of capacitation-associated tyrosine phosphorylation in human sperm by flow cytometry
After the assessment of the macroscopic and microscopic characteristics of the sample, we will wash the sample and process it using three layers of density gradients.
In the case of samples for diagnostic analysis, the remaining volume will be incubated in at 37ºC, and 0.1-0.2 ml aliquots will be taken from the end of the processing at t0 (just before introducing the sample in the incubator), at t1 (after one hour of incubation) and at t3 (after 3 hours of incubation). Additionally, a sample will be taken after washing, prior to processing the sample with the density gradients. In the case of samples processed for insemination, an additional aliquot will be taken at the exact moment in which the insemination is carried out and the elapsed incubation time will be noted.
The evaluation of the capacitation will be carried out by analyzing the state of phosphorylation of tyrosines by flow cytometry.
Eligibility Criteria
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Inclusion Criteria
* Sexual abstinence (2 to 5 days).
* To be a male patient who delivers a fresh seminal sample for diagnosis or artificial insemination treatment.
* To be a male donor who delivers a fresh seminal sample for donation or for previous tests to include him in the donation programme.
* To be a female patient undergoing artificial insemination.
Exclusion Criteria
* Cryptozoospermia or oligozoospermia with sperm concentration \<1 mill/ml.
* Ejaculates obtained with more than 5 days of sexual abstinence.
* Frozen semen samples for artificial insemination cycles with partner's semen.
* Female patients who present uterine malformations that compromise the viability of the pregnancy (intramural or submucosal fibroids \> 3 cm, polyps, adenomyosis or congenital or acquired malformations). Female patients with hydrosalpinx.
* Patients who have suffered two or more previous abortions (repeated abortions).
* Patients with a body mass index greater than 30 Kg / m2.
18 Years
MALE
Yes
Sponsors
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Vida Recoletas Sevilla
OTHER
Responsible Party
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Principal Investigators
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Cristina González Ravina, PhD
Role: PRINCIPAL_INVESTIGATOR
IVI RMA Sevilla
Locations
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IVI RMA Sevilla
Seville, Sevilla, Spain
Countries
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References
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Said TM, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum Reprod Update. 2011 Nov-Dec;17(6):719-33. doi: 10.1093/humupd/dmr032. Epub 2011 Aug 25.
Aitken RJ, Nixon B. Sperm capacitation: a distant landscape glimpsed but unexplored. Mol Hum Reprod. 2013 Dec;19(12):785-93. doi: 10.1093/molehr/gat067. Epub 2013 Sep 26.
Bianchi E, Doe B, Goulding D, Wright GJ. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature. 2014 Apr 24;508(7497):483-7. doi: 10.1038/nature13203. Epub 2014 Apr 16.
Henkel R. Sperm preparation: state-of-the-art--physiological aspects and application of advanced sperm preparation methods. Asian J Androl. 2012 Mar;14(2):260-9. doi: 10.1038/aja.2011.133. Epub 2011 Dec 5.
Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P, Kopf GS. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development. 1995 Apr;121(4):1129-37. doi: 10.1242/dev.121.4.1129.
Visconti PE. Understanding the molecular basis of sperm capacitation through kinase design. Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):667-8. doi: 10.1073/pnas.0811895106. Epub 2009 Jan 14. No abstract available.
Liu DY, Clarke GN, Baker HW. Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm-zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction. Hum Reprod. 2006 Apr;21(4):1002-8. doi: 10.1093/humrep/dei435. Epub 2006 Jan 20.
Kumaresan A, Siqueira AP, Hossain MS, Johannisson A, Eriksson I, Wallgren M, Bergqvist AS. Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca(2)(+) concentration in boar spermatozoa during cryopreservation. Reprod Fertil Dev. 2012;24(4):531-42. doi: 10.1071/RD11074.
Escoffier J, Navarrete F, Haddad D, Santi CM, Darszon A, Visconti PE. Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation. Biol Reprod. 2015 May;92(5):121. doi: 10.1095/biolreprod.114.127266. Epub 2015 Apr 8.
Naresh S, Atreja SK. The protein tyrosine phosphorylation during in vitro capacitation and cryopreservation of mammalian spermatozoa. Cryobiology. 2015 Jun;70(3):211-6. doi: 10.1016/j.cryobiol.2015.03.008. Epub 2015 Mar 28.
Mansour RT, Serour MG, Abbas AM, Kamal A, Tawab NA, Aboulghar MA, Serour GI. The impact of spermatozoa preincubation time and spontaneous acrosome reaction in intracytoplasmic sperm injection: a controlled randomized study. Fertil Steril. 2008 Sep;90(3):584-91. doi: 10.1016/j.fertnstert.2006.11.176. Epub 2008 Mar 4.
Zhang XD, Chen MY, Gao Y, Han W, Liu DY, Huang GN. The effects of different sperm preparation methods and incubation time on the sperm DNA fragmentation. Hum Fertil (Camb). 2011 Sep;14(3):187-91. doi: 10.3109/14647273.2011.604817. Epub 2011 Aug 23.
Marin-Briggiler CI, Tezon JG, Miranda PV, Vazquez-Levin MH. Effect of incubating human sperm at room temperature on capacitation-related events. Fertil Steril. 2002 Feb;77(2):252-9. doi: 10.1016/s0015-0282(01)02982-x.
Other Identifiers
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1608-SEV-065-CG
Identifier Type: -
Identifier Source: org_study_id
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