Sperm Phenotype and Differentially Methylated Regions (Epigenetics)

Association Between Anogenital Distance, Sperm Phenotype and Epigenetics in Infertile Men.

Testicular dysgenesis syndrome (TDS) is known to cause epigenetic abnormalities in spermatozoa. Anogenital distance (AGD) is considered to be a suitable clinical marker of TDS, but the direct link between AGD and epigenetic abnormalities is still missing.

Infertile men (n=10) presenting with shortened AGD and a control group of normal semen donors (n=10) with normal AGD will then be asked to provide one semen sample each. Using a flow cytometer and sorter (FACS) their spermatozoa will be sorted into populations of spermatozoa with/without DNA fragmentation or with/without chromatin decondensation. These sorted populations of spermatozoa will then be examined for differences in epigenetic imprinting differences using whole genome expression analysis. Whereas the sorting of spermatozoa will be carried out in Basel, the epigenetic analysis will be carried at the University of Geneva.

Study Overview

• Status: Completed
• Conditions: Infertility, Male
• Intervention / Treatment: Diagnostic test: obtention of up to three semen samples

Detailed Description

A subset of 10 men with shortened AGD (together with a control group of 10 fertile donors with normal AGD) will be asked to provide up to three semen samples, each of which then will be sorted with FACS into subpopulations with/without DNA fragmentation and into subpopulations with/without chromatin decondensation.

Spermatozoa with fragmented DNA will be separated through FACS-sorting of spermatozoa with intact DNA using the YoPro 1-dye, which has been shown to correlate significantly with the degree of DNA fragmentation in the nuclei of sperm.

In addition, spermatozoa with abnormal chromatin remodelling will be separated through sorting from spermatozoa with condensed chromatin using the fluorochrome chromomycin A3 (CMA3), which competes for protamin for binding to the minor groove of DNA thereby correlating with the persistence of histones in the sperm nuclei. Pilot experiments have demonstrated the highly significant and close correlation of CMA3 with anilin blue staining. Anilin blue staining is not suitable for the sorting experiment, because it requires fixation of the spermatozoa. Sorting based on CMA3 can be carried out with living spermatozoa.

The sorted and anonymized samples will then be sent frozen in dry ice to a laboratory at the University of Geneva for the assessment of differences in the epigenetic imprinting of the DNA using whole genome expression studies.

• Study Type: Observational
• Enrollment (Actual): 60

Contacts and Locations

Study Locations

• Switzerland
  • Basel, Switzerland, 4031
    • Christian De Geyter

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 20 years to 55 years (Adult)
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Both the 10 infertile men with shortened AGD and the fertile control men (semen donors) will be recruited and examined in the infertility consultation of the Clinic of Gyn. Endocrinology and Reproductive Medicine.

Description

Inclusion Criteria:

• infertile men with known anogenital distance

Exclusion Criteria:

• sperm concentration must be more than 15 million/ml to allow appropriate sorting with flow cytometry...

Study Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Prospective

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
subfertile men; 10 infertile men with shortened AGD (< 40 mm)	Diagnostic test: obtention of up to three semen samples; sorting of spermatozoa with flow cytometry. In the presence of insufficient numbers of spermatozoa after sorting (<15 mill), up to three semen samples will be collected.; Other Names: conventional semen analysis followed by sorting
fertile semen donors; 10 fertile semen donors with normal AGD (>40 mm)	Diagnostic test: obtention of up to three semen samples; sorting of spermatozoa with flow cytometry. In the presence of insufficient numbers of spermatozoa after sorting (<15 mill), up to three semen samples will be collected.; Other Names: conventional semen analysis followed by sorting

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
anogenital distance and epigenetics	number of differentially methylated transposable regulatory sequences in the genome of sorted spermatozoa.	6 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
sperm phenotype 1	based on conventional semen analysis: concentration of spermatozoa (in mill/ml).	6 months
sperm phenotype 2	based on conventional semen analysis: progressive motility (in %).	6 months
sperm phenotype 3	based on conventional semen analysis: normal morphology (in %), staining).	6 months
sperm phenotype 4	chromatin decondensation (as given by % of CMA3 staining).	6 months
sperm phenotype 5	DNA fragmentation (% of YoPro 1-staining).	6 months

Collaborators and Investigators

• Sponsor: University of Basel
• Collaborators: University of Geneva, Switzerland

Study record dates

Study Major Dates

• Study Start (Actual): November 1, 2017
• Primary Completion (Actual): January 30, 2021
• Study Completion (Actual): December 31, 2023

Study Registration Dates

• First Submitted: December 9, 2017
• First Submitted That Met QC Criteria: July 12, 2022
• First Posted (Actual): July 15, 2022

Study Record Updates

• Last Update Posted (Actual): March 20, 2024
• Last Update Submitted That Met QC Criteria: March 19, 2024
• Last Verified: March 1, 2024

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Urogenital Diseases; Male Urogenital Diseases; Genital Diseases, Male; Genital Diseases; Infertility; Infertility, Male
• Other Study ID Numbers: RME12072017

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No