Scanning the Meiotic Spindle in Assisted Reproductive Techniques to Assess Oocyte Quality

Not Applicable

Active, not recruiting

• Conditions: Infertility, Male; Infertility, Female; Infertility
• Interventions: Procedure: ICSI; Procedure: Meiotic spindle status observation in polarized light

• Registration Number: NCT06539585
• Lead Sponsor: Charles University, Czech Republic

Brief Summary

The assisted reproduction success rate is affected by several factors including the age of the women, oocyte quality and maturation state, as well as sperm quality. Imaging of the meiotic spindle may be crucial for determining the oocyte maturation and the optimal time of oocyte fertilization by intracytoplasmic sperm injection (ICSI). A new accurate and non-invasive method for selecting quality maturated oocytes based on meiotic spindle imaging is for women over 35 years of age will be introduced. The evaluation of efficiency using meiotic spindle visibility in polarized light and its relative position to the polar body as indicator of oocyte maturity will be monitored and the optimal time for ICSI will be defined.

Detailed Description

One of the main strategies of infertility treatment is in vitro fertilization (IVF) . The IVF success rate is affected by several key factors including the age of the women, oocyte quality and maturation state, as well as sperm quality. Several authors have suggested that the presence, position and retardance of the optically birefringent meiotic spindle (MS) are related to oocyte developmental competence, affecting the quality of fertilization and embryo development. Oocytes with normal spindle morphology are significantly more likely to produce euploid embryos compared with oocytes with meiotic spindles that are not visible or abnormal. Adjusting the timing of intracytoplasmic sperm injection (ICSI) based on MS morphology has also been proposed. It was shown that adjusting the time of ICSI based on the position of the MS with respect to the polar body (PB) increases the probability of successful fertilization for women over 35 years of age. For the oocytes of patients over 35 years of age, the longer incubation time enabled a greater number of oocytes to become fully mature (MII phase) and prepared for ICSI. In this situation, the MS status was an important indicator of oocyte immaturity.

Patient will be randomized into three groups using Research Electronic Data Capture (REDCap): group 1, standard ICSI without MS imaging; group 2, MS imaging followed by standard ICSI, group 3, MS imaging and ICSI fertilization according MS status. Oocytes from group 3 patients with MS evaluation will be fertilized according to MS status either 5-6 hours after ovum pick-up (OPU) or 7-8 hours after OPU. Oocytes without MS evaluation will all fertilized 5-6 hours after OPU. MS evaluation will take place in pre-prepared glass-bottomed dishes with about 5μl medium with the HEPES buffer covered with paraffin oil, put to heat one hour before use. The oocyte will be rotated using a needle so that Polar Body (PB) and MS are both well visible, and photographs will be taken using optical microscope. The MS status and the angle (α) between MS and PB will be obtained 3-4 hours after oocyte pick-up (OPU). At the same time, the polarized-light microscopy image will be acquired (polarized light microscopy at ×100 magnification. Specially, for oocytes from group 3 patients with PB/MS in close proximity (angle between PB and MS \< 5◦) or MS not visible will be predicted as immature oocytes. For these oocytes, ICSI will be performed 4-5 h after the polarization microscopy evaluation, i.e., 7-8 h after OPU. For oocytes with MS clearly visible and PB/MS not in close proximity (angle between PB and MS \> 5◦) ICSI will be performed typically 2-3 h after the polarization microscopy evaluation, i.e., 5-6 h after OPU, as for patients from 1 and 2 groups. ICSI will be performed according to standard protocol using ICSI/holding micropipettes (Microtech IVF, Czech Republic), polyvinylpyrrolidone (ICSI™, Vitrolife, Sweden), and Eppendorf (Hamburg, Germany) micromanipulation system equipped with thermoplate (Tokaohit, Japan). The oocytes will be cultivated individually, and their order preserved, so that the other outcomes (data from timelapse, clinical results) can be associated with individual oocytes. We will also monitor the time from the human chorionic gonadotropin (hCG) administration to the completion of the actual fertilization. The oocytes will be denuded (HYASE-10X™, Vitrolife, Sweden) after OPU, and the maturation stage will be examined. Germinal Vesicle (GV) stage oocyte will not be included. Oocytes and embryos in time-lapse incubator from the Japanese manufacturer Astec will be cultivated in a timelapse dish Origio, Denmark Sage 1-Step™, Origio, Denmark under paraffin oil (OVOIL™, VITROLIFE, Sweden) at 37.0 °C, 6% CO2 and 5%O2. Fertilization after ICSI will be defined as the presence of two pronuclei and 2 polar body 16-20 h post ICSI. Embryos will be cultivated for 122-144 h.

Pilot findings will be confirmed in a randomized control trial, i.e. to the efficiency of using MS visibility and relative position to the polar body as indicators of oocyte maturation in order to optimize ICSI timing will be evaluated.

Basic Information
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Recruitment & Eligibility
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Study & Design
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Trial Locations

Study Timeline

• Study Start: 2024-07-18
• Study First Submitted: 2024-07-23
• Status Verified: 2024-08
• Study First Submitted QC: 2024-08-01
• Last Update Submitted: 2024-08-01
• Study First Posted: 2024-08-06
• Last Update Posted: 2024-08-06
• Primary Completion: 2027-12-31
• Study Completion: 2028-03-31

Recruitment & Eligibility

• Status: ACTIVE_NOT_RECRUITING
• Sex: Female
• Target Recruitment: 164

Inclusion Criteria

• Women who underwent ICSI;
• Spermiogram containing at least 0.5 mil/ml sperms;
• Morphlogy: normal morphology >1%;
• Stimulated cycles

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Exclusion Criteria

• Age under 35 years or over 40;
• Native cycles;
• Severe uterine abnormalities (submucosal fibroid, fibroid ≥5cm, uterine septum, endometrial polyps, uterus duplex)

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Study & Design

• Study Type: INTERVENTIONAL
• Study Design: PARALLEL

Arm && Interventions

Group	Intervention	Description
Standard ICSI without meiotic spindle imaging.	ICSI	A control group of patients who underwent a standard IVF cycle without microscopy of the meiotic spindle. In this control group of patients, ICSI fertilization will be performed 5-6 hours after oocyte collection. ICSI will be performed according to standard protocol \[6\]. The oocytes will be cultivated individually, and their order preserved, so that the other outcomes (data from timelapse, clinical results) can be associated with individual oocytes. Fertilization after ICSI will be defined as the presence of two pronuclei and 2 polar body. Embryos will be cultivated for 122-144 h.
Meiotic spindle imaging followed by standard ICSI.	Meiotic spindle status observation in polarized light	MS evaluation will take place in pre-prepared glass-bottomed dishes with about 5μl medium with the HEPES buffer covered with paraffin oil, put to heat one hour before use. The oocyte will be rotated using a needle so that PB and MS are both well visible, and photographs will be taken using optical microscope. The MS status and the angle (α) between MS and PB will be obtained 3-4 hours after oocyte pick-up (OPU). At the same time, the polarized-light microscopy image will be acquired (polarized light microscopy at ×100 magnification. ICSI will be performed typically 2-3 h after the polarization microscopy evaluation, i.e., 5-6 h after OPU. ICSI will be performed according to standard protocol \[6\]. Fertilization after ICSI will be defined as the presence of two pronuclei and 2 polar body. Embryos will be cultivated for 122-144 h.
MS imaging and ICSI fertilization according MS status.	Meiotic spindle status observation in polarized light	Oocytes with MS evaluation will be fertilized according to MS status either 5-6 hours after ovum pick-up (OPU) or 7-8 hours after OPU. MS evaluation will take place in pre-prepared glass-bottomed dishes. The MS status and the angle (α) between MS and PB will be obtained 3-4 hours after oocyte pick-up (OPU). At the same time, the polarized-light microscopy image will be acquired (polarized light microscopy at ×100 magnification. For oocytes with PB/MS in close proximity (angle between PB and MS \< 5◦) or MS not visible ICSI will be performed 4-5 h after the polarization microscopy evaluation, i.e., 7-8 h after OPU (these oocytes are supposed to be not fully mature). For oocytes with MS clearly visible and PB/MS not in close proximity (angle between PB and MS \> 5◦) ICSI will be performed typically 2-3 h after the polarization microscopy evaluation, i.e., 5-6 h after OPU. ICSI will be performed according to standard protocol \[6\].
Meiotic spindle imaging followed by standard ICSI.	ICSI	MS evaluation will take place in pre-prepared glass-bottomed dishes with about 5μl medium with the HEPES buffer covered with paraffin oil, put to heat one hour before use. The oocyte will be rotated using a needle so that PB and MS are both well visible, and photographs will be taken using optical microscope. The MS status and the angle (α) between MS and PB will be obtained 3-4 hours after oocyte pick-up (OPU). At the same time, the polarized-light microscopy image will be acquired (polarized light microscopy at ×100 magnification. ICSI will be performed typically 2-3 h after the polarization microscopy evaluation, i.e., 5-6 h after OPU. ICSI will be performed according to standard protocol \[6\]. Fertilization after ICSI will be defined as the presence of two pronuclei and 2 polar body. Embryos will be cultivated for 122-144 h.
MS imaging and ICSI fertilization according MS status.	ICSI	Oocytes with MS evaluation will be fertilized according to MS status either 5-6 hours after ovum pick-up (OPU) or 7-8 hours after OPU. MS evaluation will take place in pre-prepared glass-bottomed dishes. The MS status and the angle (α) between MS and PB will be obtained 3-4 hours after oocyte pick-up (OPU). At the same time, the polarized-light microscopy image will be acquired (polarized light microscopy at ×100 magnification. For oocytes with PB/MS in close proximity (angle between PB and MS \< 5◦) or MS not visible ICSI will be performed 4-5 h after the polarization microscopy evaluation, i.e., 7-8 h after OPU (these oocytes are supposed to be not fully mature). For oocytes with MS clearly visible and PB/MS not in close proximity (angle between PB and MS \> 5◦) ICSI will be performed typically 2-3 h after the polarization microscopy evaluation, i.e., 5-6 h after OPU. ICSI will be performed according to standard protocol \[6\].

Primary Outcome Measures

Name	Time	Method
Clinical pregnancy rate of patients whose ICSI time was established according to MS state	6-8 weeks	The percentage of all attempts that leads to pregnancy of patients whose ICSI fertilization was done according MS status. Meiotic spindle visibility in polarized light and its relative position to the polar body as indicator of oocyte maturity will be monitored and the optimal time for ICSI will be defined.

Secondary Outcome Measures

Name	Time	Method
clinical pregnancy rate of patients whose ICSI time was not established according to measured MS state	6-8 weeks	The percentage of all attempts that leads to pregnancy of patients whose ICSI fertilization was not done according MS status. But the meiotic spindle visibility in polarized light and its relative position to the polar body was measured.

Trial Locations

Locations (1)

General University Hospital in Prague; 🇨🇿 Prague, Czechia