Descriptive Analysis of Serum Immunological Markers During an Euploid Frozen Embryo Transfer in a Natural Cycle.

Study Overview
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Brief Summary
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To describe the peripheral serum levels of the anti-inflammatory cytokines IL-4 (Interleukin-4), IL-10 (Interleukin-10), TGF-ß1 (Transforming Growth Factor beta1), the pro-inflammatory cytokines IL-17 (Interleukin-17), IFγ (Interferon Gamma) and the immune mediator PIBF (Progesterone-Induced Blocking Factor) along a single frozen euploid blastocyst transfer in a natural cycle (NC) or Hormone Replacement Therapy (HRT).

Detailed Description
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This a prospective, observational study including 40 infertile patients undergoing a single embryo transfer (SET). Blood samples will be drawn for Progesterone-Induced Blocking Factor (PIBF), interleukin 17 (IL-17), Interferon Gamma (IFγ), Interleukin 10 (IL-10), Interleukin 4 (IL-4) and Transforming Growth Factor beta 1 (TGF-ß1) measurement on the day of ovulation in the natural cycle (NC) or first day of progesterone in the Hormone Replacement Therapy (HRT) protocol, on the day of embryo transfer (ET) and 3 and 10 days later, coinciding with the early and late embryo invasion stages respectively. In case of pregnancy, an additional blood test for PIBF, IL-17, IFγ, IL-10, IL-4, TGF-ß1 and ßHCG (Beta-Human Chorionic Gonadotropin) will be performed at 5 weeks +/- 3 days. The study will be performed at ART Fertility Clinics and the estimated completion time will be 12 months.

Official Title
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Descriptive Analysis of Serum Immunological Markers During an Euploid Frozen Embryo Transfer in a Natural Cycle (NC).

Conditions
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Infertility, Female

Intervention / Treatment
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* Drug: Endometrin 100Mg Vaginal Insert
* Drug: Estradiol Valerate


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Women aged 18 years to 40 years Having at least one good quality (grade A or B for inner mass cell and trophectoderm) day 5 or 6 chromosomally normal cryopreserved blastocyst available for transfer Single embryo transfer in a natural cycle (NC) or an Hormone Replacement Therapy (HRT) protocol Fresh ejaculate used for fertilization Exclusion Criteria: Body mass index lower than 18.5 or equal or higher than 29 kg/m2 Endometriosis or adenomyosis suspected by medical history (dysmenorrhea, dyspareunia, heavy or prolonged menstrual bleeding (> 8 days), chronic pelvic pain, catamenial rectal or bladder symptoms) or diagnosed by imaging (magnetic resonance imaging or ultrasonography) Uterine abnormalities Hydrosalpinx Insulin resistance or diabetes mellitus diagnosed by HbA1c 5.7 % Antiphospholipid syndrome Polycystic ovarian syndrome according to Rotterdam criteria: presence of at least two of the following: irregular cycles (< 21 or > 35 days or < 8 cycles a year), biochemical or clinical hyperandrogenism, ovarian ultrasound morphology (> 20 follicles per ovary on transvaginal scan) or Anti-mullerian hormone > 5,98 ng/ml History of recurrent miscarriage, defined as the loss of 2 or more pregnancies according to ESHRE guidelines History of implantation failure, considered as the lack of pregnancy after the transfer of 2 good-quality (grade A or B for inner mass cell and trophectoderm (29)) euploid embryos History or suspicion of Asherman syndrome Autoimmune disease Couple first degree consanguineous

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 40 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Natural Cycle Protocol<br>Ultrasound monitoring to monitor follicular and endometrial growth. Serial measurements of serum Lutenizing Hormone (LH), estradiol (E2), and progesterone (P4) to identify ovulation. Minimal endometrial thickness of 7 mm is required. No progesterone supplementation. Embryo transfer (ET) will be scheduled 5 days after the day of ovulation (120 hours of P4 exposure). Blood samples will be drawn to measure PIBF, IL-17, IFγ, IL-10, IL-4 and TGF-ß1 on day of ovulation (P4 rise), on the day before ET, on the day of ET and 3 and 10 days later, between 9 and 12 am. A blood test to measure beta human chorionic gonadotropin (ß-hCG) will be performed 3 and 10 days after ET and at 5 weeks +/- 3 days in case of pregnancy, between 9 and 12 am. In case of pregnancy, a blood test to measure PIBF, IL-17, IFγ, IL-10, IL-4 and TGF-ß1 will be performed at 5 weeks +/- 3 days between 9 and 12 am.. | |
| Hormone Replacement Therapy Protocol<br>Ultrasound monitoring of endometrial growth and exclude growing follicle Oral Estradiol Valerate (Progyluton) 4 mg on cycle day 2 at 7 pm for 2 days. On 3rd day, increase dose to 6 mg. The dose may be increased based on endometrial thickness. Once endometrial thickness is at least 7 mm and trilaminar, after 10-15 days of Estradiol Valerate administration, start Endometrin 100 mg at 1 pm and 9 pm. From 2nd day onwards increase to 3 times daily. Continue Progyluton & Endometrin until 12 weeks of pregnancy ET on the 5th full day of Endometrin administration Blood test to measure PIBF, IL-17, IFγ, IL-4, IL-10 and TGF-ß1 on the 1st day of Endometrin supplementation (before 1st administration), on the 4th day of Endometrin administration, on the day of ET and 3 and10 days after, 4 to 7 hours after the morning administration. Blood test to measure beta human chorionic gonadotropin (ß-hCG) will be performed 3 and 10 days after ET and at 5 weeks +/- 3 days if pregnancy. | Drug: Endometrin 100Mg Vaginal Insert<br>* Endometrin will start once endometrial thickness is equal or higher to 7 mm with a trilaminar appearance, after at least 10 days and always less than 16 days of estradiol administration. First day of supplementation a dose of 100mg will be administered at of at 1.pm and 9 pm. From the second day and onwards administration will be increased to three times daily, at 6 am, 2 pm and 10 pm<br>Drug: Estradiol Valerate<br>* Estradiol valerate dose will be reduced to 4mg daily at 7 pm during the first two days of vaginal natural micronized progesterone supplementation and increased afterwards to 6 mg daily (2 mg at 10 am and 4 mg at 7 pm).<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Blood levels of IL-4 (Interleukin 4) on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of IL-4 on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of IL-10 (Interleukin 10) on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of IL-10 on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of IL-17 (Interleukin 17) on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of IL-17 on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of TGF-ß1 (Transforming Growth Factor beta1) on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of TGF-ß1 on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of IFγ (Interferon gamma) on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of IFγ on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of PIBF on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of PIBF on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of IL-4 (Interleukin 4) on the day before embryo transfer | Blood levels of IL-4 on the day before embryo transfer | 1 day | 
| Blood levels of IL-10 (Interleukin 10) on the day before embryo transfer | Blood levels of IL-10 on the day before embryo transfer | 1 day | 
| Blood levels of IL-17 (Interleukin 17) on the day before embryo transfer | Blood levels of IL-17 on the day before embryo transfer | 1 day | 
| Blood levels of TGF-ß1 (Transforming Growth Factor beta1) on the day before embryo transfer | Blood levels of TGF-ß1 on the day before embryo transfer | 1 day | 
| Blood levels of IFγ (Interferon gamma) on the day before embryo transfer | Blood levels of IFγ on the day before embryo transfer | 1 day | 
| Blood levels of PIBF (Progesterone induced blocking factor) on the day before embryo transfer | Blood levels of PIBF on the day before embryo transfer | 1 day | 
| Blood levels of IL-4 (Interleukin 4) on the day of embryo transfer | Blood levels of IL-4 on the day of embryo transfer | 1 day | 
| Blood levels of IL-10 (Interleukin 10) on the day of embryo transfer | Blood levels of IL-10 on the day of embryo transfer | 1 day | 
| Blood levels of IL-17 (Interleukin 17) on the day of embryo transfer | Blood levels of IL-17 on the day of embryo transfer | 1 day | 
| Blood levels of TGF-ß1 (Transforming Growth Factor beta1) on the day of embryo transfer | Blood levels of TGF-ß1 on the day of embryo transfer | 1 day | 
| Blood levels of IFγ (Interferon gamma) on the day of embryo transfer | Blood levels of IFγ on the day of embryo transfer | 1 day | 
| Blood levels of PIBF (Progesterone induced blocking factor) on the day of embryo transfer | Blood levels of PIBF on the day of embryo transfer | 1 day | 
| Blood levels of IL-4 (Interleukin 4) three days after embryo transfer | Blood levels of IL-4 three days after embryo transfer | 3 days | 
| Blood levels of IL-10 (Interleukin 10) three days after embryo transfer | Blood levels of IL-10 three days after embryo transfer | 3 days | 
| Blood levels of IL-17 (Interleukin 17) three days after embryo transfer | Blood levels of IL-17 three days after embryo transfer | 3 days | 
| Blood levels of TGF-ß1 (Transforming Growth Factor beta1) three days after embryo transfer | Blood levels of TGF-ß1 three days after embryo transfer | 3 days | 
| Blood levels of IFγ (Interferon gamma) three days after embryo transfer | Blood levels of IFγ three days after embryo transfer | 3 days | 
| Blood levels of PIBF (Progesterone induced blocking factor) three days after embryo transfer | Blood levels of PIBF three days after embryo transfer | 3 days | 
| Blood levels of IL-4 (Interleukin 4) ten days after embryo transfer | Blood levels of IL-4 ten days after embryo transfer | 10 days | 
| Blood levels of IL-10 (Interleukin 10) ten days after embryo transfer | Blood levels of IL-10 ten days after embryo transfer | 10 days | 
| Blood levels of IL-17 (Interleukin 17) ten days after embryo transfer | Blood levels of IL-17 ten days after embryo transfer | 10 days | 
| Blood levels of TGF-ß1 (Transforming Growth Factor beta1) ten days after embryo transfer | Blood levels of TGF-ß1 ten days after embryo transfer | 10 days | 
| Blood levels of IFγ (Interferon gamma) ten days after embryo transfer | Blood levels of IFγ ten days after embryo transfer | 10 days | 
| Blood levels of PIBF (Progesterone induced blocking factor) ten days after embryo transfer | Blood levels of PIBF ten days after embryo transfer | 10 days | 
| Blood levels of IL-4 (Interleukin 4) in case of pregnancy at 5 weeks +/- 3 days | Blood levels of IL-4 in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of IL-10 (Interleukin 10) in case of pregnancy at 5 weeks +/- 3 days | Blood levels of IL-10 in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of IL-17 (Interleukin 17) in case of pregnancy at 5 weeks +/- 3 days | Blood levels of IL-17 in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of TGF-ß1 (Transforming Growth Factor beta1) in case of pregnancy at 5 weeks +/- 3 days | Blood levels of TGF-ß1 in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of IFγ (Interferon gamma) in case of pregnancy at 5 weeks +/- 3 days | Blood levels of IFγ in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of PIBF (Progesterone induced blocking factor) in case of pregnancy at 5 weeks +/- 3 days | Blood levels of PIBF in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of estradiol on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of estradiol on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of progesterone on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | Blood levels of progesterone on the on the day of progesterone rise (NC) or first day of progesterone administration (HRT) | 1 day | 
| Blood levels of estradiol on the day before embryo transfer | Blood levels of estradiol on the day before embryo transfer | 1 day | 
| Blood levels of progesterone on the day before embryo transfer | Blood levels of progesterone on the day before embryo transfer | 1 day | 
| Blood levels of estradiol on the day of embryo transfer | Blood levels of estradiol on the day of embryo transfer | 1 day | 
| Blood levels of progesterone on the day of embryo transfer | Blood levels of progesterone on the day of embryo transfer | 1 day | 
| Blood levels of estradiol three days after embryo transfer | Blood levels of estradiol three days after embryo transfer | 3 days | 
| Blood levels of progesterone three days after embryo transfer | Blood levels of progesterone three days after embryo transfer | 3 days | 
| Blood levels of estradiol ten days after embryo transfer | Blood levels of estradiol ten days after embryo transfer | 10 days | 
| Blood levels of progesterone ten days after embryo transfer | Blood levels of progesterone ten days after embryo transfer | 10 days | 
| Blood levels of estradiol in case of pregnancy at 5 weeks +/- 3 days | Blood levels of estradiol in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of progesterone in case of pregnancy at 5 weeks +/- 3 days | Blood levels of progesterone in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 
| Blood levels of ß-hCG (Beta-Human Chorionic Gonadotropin) three days after embryo transfer | Blood levels of ß-hCG three days after embryo transfer | 3 days | 
| Blood levels of ß-hCG (Beta-Human Chorionic Gonadotropin) ten days after embryo transfer | Blood levels of ß-hCG ten days after embryo transfer | 10 days | 
| Blood levels of ß-hCG (Beta-Human Chorionic Gonadotropin) in case of pregnancy at 5 weeks +/- 3 days | Blood levels of ß-hCG in case of pregnancy at 5 weeks +/- 3 days | 5 weeks | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Pregnancy rate | Number of patients with a ß-hCG > 5 IU out of the number of patients who underwent an embryo transfer. | 10 days | 
| Clinical pregnancy rate | Number of patients with an ultrasonographic visible sac out of the number of patients who underwent an embryo transfer | 5 weeks and 3 days | 
| Miscarriage rate | Number of patients with a pregnancy loss out of the number of patients who had a ß-hCG > 5 IU after embryo transfer | 12 weeks | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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infertilty, ivf, progesterone, estradiol, cytokines, immunology, Pharmacological effect of drugs, hormones