Relationship Between Nitric Oxide (NO) in Follicular Fluid and Sperm Fertilization Ability

NCT03307655 | Unknown

• 1 other identifier: 1602-MUR-014JL
• Study Type: interventional
• Target: 82
• Locations: 1 country
• Sites: 1

Brief Summary

Several studies indicate that Nitric Oxide (NO) plays an important role in the physiology of the reproductive system in mammals. It has been shown that NO affects sperm motility, it regulates the tyrosine phosphorylation of different sperm proteins, it enhances the sperm binding ability to the zona pellucida and it modulates the acrosome reaction. The enzyme responsible for NO synthesis, the Nitric Oxide Synthase (NOS), has also been identified in the oocytes, cumulus and corona cells, as well as in the oviduct. For these reasons, the NOS presence at the fertilization site could be a key element to determine the success of this process. Therefore, carrying out in vitro studies to better understand NO's role in the fertilization process, especially in human sperm capacitation, could improve the outcome of the Assisted Reproductive Techniques (ARTs) due to an improvement both in the diagnosis of infertility and in the prognosis of treatment success. This study is carried out in collaboration with the Animal Physiology Department from the Veterinary Faculty (University of Murcia, Spain) and it is funded by the European Commission under the Horizon 2020 Programme.

Conditions

Male Infertility

Keywords

nitric oxide; follicular fluid; sperm; capacitation; fertilization; phosphorylation; nitrosylation

Outcome Measures

Primary Outcomes (1)

• Nitric Oxide levels in follicular fluid

  Upon collection and after oocyte removal, the follicular fluid samples will be centrifuged to remove cellular debris. An aliquot will be further centrifuged in tubes with 10 kDa filters to remove proteins. This alliquot will then be used to determine NO concentration with the help of a commercially available measurement system. NO is rapidly oxidized into two stable ions, namely nitrite and nitrate, which can be assayed by using ions selective electrodes. A software will then be used to calculate the initial concentration of Nitric Oxide (micromolar).

  Nitrit Oxide levels will be measured within 30 min after the follicular fluid sample is available.

Study Arms (2)

Control (fertile semen donors)

EXPERIMENTAL

Sperm from fertile men (donors who attend the IVI Murcia clinic) will be included in this arm.

Other: Control (fertile semen donors)

Patients (subfertile men)

EXPERIMENTAL

Sperm from patients (subfertile men, i.e. men who possess one altered spermiogram parameter, according to the WHO 2010 guidelines) will be included in this arm.

Other: Patients (subfertile men)

Interventions

Control (fertile semen donors) OTHER

The sperm will be capacitated in the presence and absence of follicular fluid (FF). In a first set of experiments, the FF will be supplemented with L-Arginine and the NOS inhibitor L-NAME. In another set of experiments the same supplementations to the capacitation medium will be examined, but without using FF. Changes in NOS expression, protein nitrosylation, PKA activity, tyrosine phosphorylation and tyrosine nitration in sperm will be determined.

Control (fertile semen donors)

Patients (subfertile men) OTHER

The sperm will be capacitated in the presence and absence of follicular fluid (FF). In a first set of experiments, the FF will be supplemented with L-Arginine and the NOS inhibitor L-NAME. In another set of experiments the same supplementations to the capacitation medium will be examined, but without using FF. Changes in NOS expression, protein nitrosylation, PKA activity, tyrosine phosphorylation and tyrosine nitration in sperm will be determined.

Patients (subfertile men)

Eligibility Criteria

• Age: 18 Years+
• Gender Eligibility Details: This study will include: fertile and subfertile men (who will provide the semen samples) and women donors (who will provide the follicular fluid samples).
• Healthy Volunteers: Yes
• Age Groups: Adult (18-64), Older Adult (65+)

You may qualify if:

• This study will include fertile males such as the semen donors attending the clinic IVI Murcia and a group of subfertile males composed of patients, who attend the clinic to undergo an in vitro fertilization treatment and who possess one altered spermiogram parameter (according to the WHO 2010 guidelines). These participants will be recruited to examine the modifications in the NOS expression as well as the protein nitrosylation in fertile and subfertile men.
• This study will also include couples who undergo an IVF or ICSI treatment, in order to investigate if the effects of human follicular fluid on sperm capacitation are associated to NO levels in the fluid and to identify the correlation between NO levels in the follicular fluid and the clinical outcomes from ARTs.

You may not qualify if:

• From this study will be excluded couples who undergo preimplantation genetic diagnosis, were diagnosed with repetitive abortion (three or more consecutive abortions before week 20) or implantation failure (lack of pregnancy after three embryo transfers with good quality embryos in women under 36 years of age or after two embryo transfers in women over 36 years of age), as well as women over 40 years of age.

Sponsors & Collaborators

• IVI Murcia: lead
• Universidad de Murcia: collaborator
• Harvard School of Public Health (HSPH): collaborator
• European Commission: collaborator

Study Sites (1)

IVI Murcia

Murcia, 30007, Spain

RECRUITING

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MeSH Terms

Conditions

Infertility, Male

Condition Hierarchy (Ancestors)

Genital Diseases, Male; Genital Diseases; Urogenital Diseases; Infertility; Male Urogenital Diseases

Study Officials

• Carmen Matas Parra, Professor

  University of Murcia (Spain)

  STUDY DIRECTOR

• Juan Carlos Martinez Soto, MD, PhD

  IVI Murcia (Spain)

  STUDY DIRECTOR

• Jorge Chavarro, MD, PhD

  Harvard University

  STUDY DIRECTOR

• Florentin-Daniel Staicu, MSc

  University of Murcia (Spain)

  PRINCIPAL INVESTIGATOR

Central Study Contacts

Juan Carlos Martinez Soto, MD, PhD

CONTACT

+34666676183; JuanCarlos.Martinez@ivi.es

Carmen Matas Parra, Professor

CONTACT

+34669031210; cmatas@um.es

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: NON RANDOMIZED
• Masking: NONE
• Purpose: DIAGNOSTIC
• Intervention Model: PARALLEL
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Model Details: Semen and FF samples will be obtained from donors and patients by masturbation after 2-7 days of sexual abstinence and they will be analyzed according to WHO guidelines (2010). FF samples will be obtained during the oocyte pick up procedure. After oocyte removal, the FF will be centrifuged for 10 min at 2000g and stored at -20°C until evaluation. NO concentration will be determined in the FF samples with the help of ion selective electrodes. Sperm will be capacitated in the presence and absence of FF. In a first set of experiments, the FF will be supplemented with L-Arginine and the NOS inhibitor L-NAME. In another set of experiments the same supplementations to the capacitation medium will be examined, but without using FF. Changes in NOS expression, protein nitrosylation, PKA activity, tyrosine phosphorylation and tyrosine nitration in sperm will be determined. The same treatment will be performed on all semen samples regardless of whether they come from patients or donors.

Study Record Dates

• First Submitted: October 6, 2017
• First Posted: October 12, 2017
• Study Start: September 21, 2017
• Primary Completion: December 1, 2018
• Study Completion: October 1, 2019
• Last Updated: October 19, 2017

Record last verified: 2017-10

Data Sharing

• IPD Sharing: Will not share

Locations

ES (1)