Female Fertility, Environmental Agents and Stress Oxidant

Study Overview
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Brief Summary
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Synthetic products used in industrial, pharmaceutical, agro-alimentary or agricultural fields are found in our environment. Thus, humans could be simultaneously exposed to several of these pollutants. Furthermore, these environmental agents exert or could exert adverse actions on fertility, by altering gamete and embryo quality through endocrine disruptor effects or through increase in oxidative stress in gonads (cellular pathway known to be involved in several human reproductive pathologies). In this context, the objectives of the present project are to obtain descriptive and analytical data on woman and oocyte exposure to several environmental agents (bisphenols, ethynylestradiol and glyphosate). The relation between these pollutant measures in follicular fluid and urine (from women receiving follow-up of in vitro fertilization (IVF) protocol in the University hospital of Tours, France) and the oocyte quality, the IVF and pregnancy successes will be studied. Several oxidative stress biomarkers in blood and follicular fluid will be also measured for these women, who will complete a questionnaire on their lifestyles. Finally, thanks to in vitro approaches, the effects and the mechanisms of action (including oxidative stress) of these pollutants (alone or in cocktails) will be studied on granulosa cells from these patients.

Detailed Description
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After being informed about the project Fertenox during a meeting describing about the Fecondation In Vitro protocol, all women patients not opposing to participate in the study and to use individual information will be included in the research during the usual bacteriological exam performed 1-2 months before oocyte pick up in the University hospital. They will receive the questionnaire on their lifestyle during this exam. On the eve of the oocyte pick up (at the end of ovarian stimulation) during the usual blood collection, three additional tubes of blood will be collected for each patient in the University hospital. Women will also receive the screw-top container for morning-after urine sample and the completion of the lifestyle questionnaire will be checked (some advices will be done if necessary to help patients to fill out the questionnaire). Samples will be treated, aliquoted and stored according to requirements the same day. The day of the oocyte pick up, the first morning urine will be collected by patients at home (or otherwise in the hospital). The completed questionnaire will be recovered and follicular fluid and granulosa cells will be collected for each woman. Samples will be aliquoted and stored according to requirements the same day. Granulosa cells will be cultured for in vitro approaches in research labs the same day. Tubes of blood, urine and follicular fluid will be coded and kept at -80°C in the University hospital until their shipment to laboratories, where analyses of pollutants (bisphenols, ethynylestradiol and glyphosate) and analyses of oxidative stress biomarkers (antioxidant vitamins, FRAP), activity of several enzymes (catalase, superoxide dismutase, glutathione peroxidase) and oxidized lipids) will be performed.

Official Title
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FERTilité féminine, Agents ENvironnementaux et Stress OXydant

Conditions
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Female Infertility

Intervention / Treatment
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* Other: Blood sample
* Other: Urine sample
* Other: Follicular fluid and granulosa cells sample


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Woman aged 18 to 43 years old First oocyte puncture (IVF rank = 1) Exclusion Criteria: Opposition to data processing IVF rank equal or greater than 2 Egg donation Intracytoplasmic Sperm Injection with testicular biopsy Intracytoplasmic Sperm Injection with self-preservation straw Sperm donation

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 43 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| In Vitro Fertilisation protocol<br>Women followed in the department for an in vitro fertilisation protocol | Other: Blood sample<br>* Additional blood sampling at the end of ovarian stimulation monitoring (the eve of the oocyte pick-up)<br>Other: Urine sample<br>* Collection of the first urine in the morning on the day of the oocyte pick-up<br>Other: Follicular fluid and granulosa cells sample<br>* Collection of follicular fluid and granulosa cells during oocyte pick-up.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Presence or absence of ethinylestradiol in follicular fluid | Assessed by measure of ethinylestradiol in follicular fluid by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | Baseline | 
| Presence or absence of ethinylestradiol in urine | Assessed by measure of ethinylestradiol in urine by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | Baseline | 
| Presence or absence of bisphenols in follicular fluid | Assessed by measure of bisphenols in follicular fluid by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | Baseline | 
| Presence or absence of bisphenols in urine | Assessed by measure of bisphenols in urine by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | Baseline | 
| Presence or absence of glyphosate in follicular fluid | Assessed by measure of glyphosate in follicular fluid by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 
| Presence or absence of glyphosate in urine | Assessed by measure of glyphosate in urine by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 
| Concentration of ethinylestradiol in follicular fluid | Assessed by concentration of ethinylestradiol in follicular fluid by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 
| Concentration of ethinylestradiol in urine | Assessed by concentration of ethinylestradiol in urine by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 
| Concentration of bisphenols in follicular fluid | Assessed by concentration of bisphenols in follicular fluid by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 
| Concentration of bisphenols in urine | Assessed by concentration of bisphenols in urine by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 
| Concentration of glyphosate in follicular fluid | Assessed by concentration of glyphosate in follicular fluid by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 
| Concentration of glyphosate in urine | Assessed by concentration of glyphosate in urine by Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) | baseline | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Oocyte quality | Assessed by nuclear maturity of each collected punctured oocyte | baseline | 
| Embryo quality | Assessed by in vitro early embryo developmental competence of each punctured oocyte (Day 2 embryo morphology, ability to reach the blastocyst stage, Day 5/6 blastocyst morphology, blastocyst outcome (transfer, freezing, discarding) | Day 2 and day 5/6 after oocyte pick up | 
| Embryo implantation success (pregnancy success) | Assessed by confirmation of a clinical pregnancy (blood beta human chorionic gonadotropin assay: > 1000 Unity Intenational /L), by confirmation of on-going pregnancy (ultrasound measure of foetus cardiac activity) and by confirmation delivery of live and healthy birth | From day 7 after embryo transfer for beta hCG assay, at 8 weeks of amenorrhea for foetus cardiac activity and after birth | 
| Oxidative stress biomarkers in follicular fluid | Assessed by measure of antioxidant vitamins (carotenoids, vitamin E, retinol) by High Performance Liquid Chromatography | baseline | 
| Oxidative stress biomarkers in follicular fluid | Assessed by measure of oxidized lipids (isoprostanoids) by LC-MS/MS | Baseline | 
| Oxidative stress biomarkers in follicular fluid | Assessed by measure of antioxidant power (FRAP: Ferric ion Reducing Antioxidant Power) by spectrophotometry | Baseline | 
| Oxidative stress biomarkers in follicular fluid | Assessed by measure of glutathione peroxidase activity by spectrophometry | Baseline | 
| Oxidative stress biomarkers in blood | Assessed by measure of plasma antioxidant vitamins (carotenoids, vitamin E, retinol) by HPLC | On the eve of baseline | 
| Oxidative stress biomarkers in blood | Assessed by measure of plasma oxidized lipids (isoprostanoids) by LC-MS/M | On the eve of baseline | 
| Oxidative stress biomarkers in blood | Assessed by measure of plasma antioxidant power (FRAP) by spectrophotometry | On the eve of baseline | 
| Oxidative stress biomarkers in blood | Assessed by measure of activities of glutathione peroxidase, catalase and superoxide dismutase in red blood cells by UV and visible spectrophometry | On the eve of baseline | 
| Oxidative stress biomarkers in urine | Assessed by measure of oxidized lipids (isoprostanoids) by LC-MS/MS | baseline | 
| Information on woman lifestyles | Assessed by a questionnaire on sociodemographic characteristics, smoking, physical activity, professional and environmental expositions, consumption of dietary supplements (antioxidants), consumption of drinks (tap water, from plastic bottle, cans...), food reheating habit, consumption of food from tin box, use of hygiene and cosmetic products... | The 3 last months of lifestyles before baseline | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on cellular viability (CCK8, live-dead assay) | after 24 or 48h of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on proliferation (BrdU) | after 24 or 48h of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on steroid production (ELISA, LC-MS) | after 48 or 72h of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on energetic metabolism (mitochondrial and glycolytic activities by Seahorse and Omnilog assays) | after 1 or 24h of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on oxidative stress biomarkers (reactive oxygen production, Nrf2 nuclear translocation by confocal microscopy, cytometry and fluorimetry) | after 5, 6 or 24h of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on protein expression (Western Blotting) of several markers of the studied functions (including oxidative stress) | after 24, 48 or 72h of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on gene expression (RNAseq and Quantitative Reverse Transcription-PCR) of several markers of the studied functions (including oxidative stress) | after 6 or 24h of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on protein expression (Western Blotting ) of signaling pathways | after 1, 5, 10, 30 and 60 min of exposure | 
| Pollutant effects on patient granulosa cells in vitro | Assessed on gene expression (RNAseq and qRT-PCR) of signaling pathways | after 6h of exposure | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Environmental pollutants, Bisphenols, Ethynylestradiol, Glyphosate, Oxidative stress, Oocyte and embryo quality, In vitro fertilization, Pregnancy, Follicular fluid and urine biomarkers