## Protocol Section
### Identification Module
**NCT ID:** NCT05461079

**Acronym:** Epigenetics

**Brief Title:** Sperm Phenotype and Differentially Methylated Regions

**Official Title:** Association Between Anogenital Distance, Sperm Phenotype and Epigenetics in Infertile Men.

#### Organization Study ID Info
**ID:** RME12072017
#### Organization
**Class:** OTHER
**Full Name:** University of Basel
### Status Module
#### Completion Date
**Date:** 2023-12-31

**Type:** ACTUAL

#### Expanded Access Info
#### Last Update Post Date
**Date:** 2024-03-20

**Type:** ACTUAL

**Last Update Submit Date:** 2024-03-19

**Overall Status:** COMPLETED

#### Primary Completion Date
**Date:** 2021-01-30

**Type:** ACTUAL

#### Start Date
**Date:** 2017-11-01

**Type:** ACTUAL

**Status Verified Date:** 2024-03

#### Study First Post Date
**Date:** 2022-07-15
**Type:** ACTUAL
**Study First Submit Date:** 2017-12-09
**Study First Submit QC Date:** 2022-07-12
### Sponsor Collaborators Module

#### Collaborators

**Class:** OTHER

**Name:** University of Geneva, Switzerland

#### Lead Sponsor

**Class:** OTHER

**Name:** University of Basel

#### Responsible Party

**Investigator Affiliation:** University of Basel

**Investigator Full Name:** Christian De Geyter

**Investigator Title:** Prof. Dr.med.

**Type:** PRINCIPAL_INVESTIGATOR

### Oversight Module

**Is FDA Regulated Device:** False

**Is FDA Regulated Drug:** False

**Oversight Has DMC:** False

### Description Module

**Brief Summary:** 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.

**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.

### Conditions Module

**Conditions:**

- Infertility, Male

### Design Module

#### Bio Spec

**Description:** Spermatozoa sorted with FACS according to two different staining methods: YoPro1 (apoptosis) and CMA3 (histone content). After sorting, we obtained four different groups: 1. YoPro1 positive and 2. YoPro1 negative, CMA3 positive and CMA3 negative. All sorted samples were stored frozen and sent for analysis of whole genome methylation status.

**Retention:** SAMPLES_WITH_DNA

#### Design Info

**Observational Model:** CASE_CONTROL

**Time Perspective:** PROSPECTIVE

#### Enrollment Info

**Count:** 60

**Type:** ACTUAL

**Study Type:** OBSERVATIONAL

### Arms Interventions Module

#### Arm Group 1

**Description:** 10 infertile men with shortened AGD (\< 40 mm)

**Intervention Names:**

- Diagnostic Test: obtention of up to three semen samples

**Label:** subfertile men

#### Arm Group 2

**Description:** 10 fertile semen donors with normal AGD (\>40 mm)

**Intervention Names:**

- Diagnostic Test: obtention of up to three semen samples

**Label:** fertile semen donors

### Interventions

#### Intervention 1

**Arm Group Labels:**

- fertile semen donors

- subfertile men

**Description:** 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.

**Name:** obtention of up to three semen samples

**Other Names:**

- conventional semen analysis followed by sorting

**Type:** DIAGNOSTIC_TEST

### Outcomes Module

#### Primary Outcomes

**Description:** number of differentially methylated transposable regulatory sequences in the genome of sorted spermatozoa.

**Measure:** anogenital distance and epigenetics

**Time Frame:** 6 months

#### Secondary Outcomes

**Description:** based on conventional semen analysis: concentration of spermatozoa (in mill/ml).

**Measure:** sperm phenotype 1

**Time Frame:** 6 months

**Description:** based on conventional semen analysis: progressive motility (in %).

**Measure:** sperm phenotype 2

**Time Frame:** 6 months

**Description:** based on conventional semen analysis: normal morphology (in %), staining).

**Measure:** sperm phenotype 3

**Time Frame:** 6 months

**Description:** chromatin decondensation (as given by % of CMA3 staining).

**Measure:** sperm phenotype 4

**Time Frame:** 6 months

**Description:** DNA fragmentation (% of YoPro 1-staining).

**Measure:** sperm phenotype 5

**Time Frame:** 6 months

### Eligibility Module

**Eligibility Criteria:** 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...

**Maximum Age:** 55 Years

**Minimum Age:** 20 Years

**Sampling Method:** NON_PROBABILITY_SAMPLE

**Sex:** MALE

**Standard Ages:**

- ADULT

**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.

### Contacts Locations Module

#### Locations

**Location 1:**

**City:** Basel

**Country:** Switzerland

**Facility:** Christian De Geyter

**Zip:** 4031

### IPD Sharing Statement Module

**IPD Sharing:** NO

## Derived Section

### Condition Browse Module - Ancestors

- ID: D000091662

- Term: Genital Diseases

- ID: D000091642

- Term: Urogenital Diseases

- ID: D000005832

- Term: Genital Diseases, Male

- ID: D000052801

- Term: Male Urogenital Diseases

### Condition Browse Module - Browse Branches

- Abbrev: BXS

- Name: Urinary Tract, Sexual Organs, and Pregnancy Conditions

- Abbrev: All

- Name: All Conditions

### Condition Browse Module - Browse Leaves

- ID: M10290

- Name: Infertility

- Relevance: HIGH

- As Found: Infertility

- ID: M10292

- Name: Infertility, Male

- Relevance: HIGH

- As Found: Infertility, Male

- ID: M2876

- Name: Genital Diseases

- Relevance: LOW

- As Found: Unknown

- ID: M2875

- Name: Urogenital Diseases

- Relevance: LOW

- As Found: Unknown

- ID: M8944

- Name: Genital Diseases, Male

- Relevance: LOW

- As Found: Unknown

- ID: M27095

- Name: Male Urogenital Diseases

- Relevance: LOW

- As Found: Unknown

### Condition Browse Module - Meshes

- ID: D000007246

- Term: Infertility

- ID: D000007248

- Term: Infertility, Male

### Misc Info Module

- Version Holder: 2024-05-24
