We would like to thank the embryology laboratory of Reprofit International. We also acknowledge the core facility CELLIM of CEITEC supported by MEYS CR (LM2015062 Czech-Bioimaging) for their support with obtaining immunofluorescence imaging data presented here.

This protocol describes the clinical procedure which is an &#8216;add-on&#8217; to standard IVF treatment. It should be performed by experienced personnel in compliance with good laboratory practice and clinical guidelines24,25. Obtaining written informed consent of from the eligible patients is recommended. This protocol was approved by the institutional Ethics Committee.
1. Egg retrieval and denudation
<ol>
	<li>Induce ovarian stimulation using conventional stimulation protocols3. Adjust the dose to individual response. When two or more follicles, visualized by ultrasound scan, reach a diameter of 18 mm, induce oocyte maturation with application of 250 &#956;g human chorionic gonadotropin (hCG). Schedule oocyte pick-up (OPU) at 35&#8722;36 h post hCG injection.</li>
	<li>Collect retrieved cumulus-oocyte complexes (COCs) in CO2-independent handling medium (Table of Materials). Following a short incubation period (10&#8722;15 min) in a CO2-independent incubator, briefly (up&#160;to&#160;30&#160;s) expose collected COCs to hyaluronidase solution (Table of Materials). Under a stereomicroscope, mechanically remove cumulus-corona cells by gently pipetting COCs with a 200 &#181;L filter tip.</li>
	<li>Using denudation micropipettes with a gradually decreasing diameter (200 &#181;m, 180 &#181;m and 150 &#181;m), gently strip the oocytes from the remaining follicular cells and wash the oocytes 3x in handling medium.</li>
	<li>Assess the number and developmental status of the denuded oocytes according to the presence or absence of the nucleus and first PB (Figure 1).</li>
	<li>Check the inclusion criteria for PLM examination. Carry out an egg maturity assessment if there is (1) an unexpected poor response to conventional stimulation with fewer than 6 MII oocytes collected at OPU and (2) a history of previous fertilization failure or oocyte immaturity.</li>
	<li>Place GV, MI and MII oocytes into separate wells in an IVF dish, each containing 500 &#181;L of preequilibrated CO2-dependent culture medium (Table of Materials) covered with mineral oil (Table of Materials).</li>
	<li>Incubate for an additional 3&#8722;4 h at 37 &#176;C in a humidified atmosphere of 5% O2 and 6% CO2.</li>
</ol>
2. Preparation for PLM examination and subsequent ICSI
NOTE: The protocol provided here describes the PLM assessment performed using the OCTAX Polar AIDE system (Table of Materials). Alternatively, other commercially available spindle view systems can be used.
<ol>
	<li>Prepare plates for embryo cultivation.
	<ol style="margin-left: 40px;">
		<li>Depending on the number of oocytes to be examined (total of MII oocytes, and MI oocytes extruding a PB during preincubation period), prepare either a 4-well plate or a 12-well plate, fill each well with 500 &#181;L or 30 &#181;L of culture medium, respectively, and cover with previously equilibrated mineral oil.</li>
		<li>Ensure that both culture medium and mineral oil have been equilibrated in the CO2 incubator overnight. Keep the prepared dish in the CO2-dependent incubator for at least 2 h. Number the wells if necessary to track the developmental fate of individual oocytes.</li>
	</ol>
	</li>
	<li>Prepare the ICSI dish.
	<ol>
		<li>Use assigned plastic dish and make 5 &#181;L droplet of prewarmed handling medium for each PB-displaying oocyte and one extra droplet for needle washing. Make an additional droplet of polyvinylpyrrolidone (PVP) solution (Table of Materials) for sperm immobilization prior to ICSI (add sperm just before ICSI) and overlay with prewarmed mineral oil.</li>
		<li>Keep the prepared ICSI dish in the CO2-independent incubator for at least 20 min. Number the wells if necessary to track the oocytes after ICSI.</li>
	</ol>
	</li>
	<li>Prepare the PLM examination dish.
	<ol>
		<li>Use assigned glass bottom dish and make 5 &#181;L droplets of prewarmed handling medium for each PB-displaying oocyte. Overlay with prewarmed mineral oil.</li>
		<li>Keep the dish in the CO2-independent incubator for at least 20 min. Number the droplets if necessary to track the oocytes after PLM examination.</li>
	</ol>
	</li>
	<li>Set the microscope ready for PLM examination.
	<ol>
		<li>Switch the heated stage on the inverted microscope on well in advance to achieve correct warming temperature. Ensure that the settings are accurately adjusted to maintain 37 &#176;C in the handling medium droplets in the PLM/ICSI dish during micromanipulation procedures.</li>
		<li>Fit the sterile holding and ICSI needle into microinjection holders and bring them into focus. Alternatively, use a hatching needle.</li>
		<li>Select the appropriate objective (20x and 25x are the most suitable) and ensure that the condenser is in bright field position.</li>
		<li>Insert green interference filter (the light turns green) and set the liquid crystal analysis slider into working position.</li>
		<li>Set the shutter to ~50% and adjust circular polarizer to decrease background noise.</li>
	</ol>
	</li>
	<li>Set the computer ready for PLM examination.
	<ol>
		<li>Launch the imaging software.</li>
		<li>Select Video | Video Source | polarAIDE in the video menu in the top menu bar.</li>
		<li>Switch to live video by going to the Video page and activate spindle and zona analysis by hitting on the icon (Supplemental Figure 1) in the video toolbar.</li>
		<li>Select display mode for dynamic scaling during spindle imaging: (1) red (birefringence)/green (background) combined view, or (2) white (birefringence)/and black (background) view. The dynamic scoring mode is used for autoscoring of zona pellucida.</li>
	</ol>
	</li>
</ol>
3. Examination of egg maturity
<ol>
	<li>After the preincubation period (3&#8722;4 h), perform a PLM examination revealing the oocyte maturity status at the standard time of ICSI (39&#8722;40 h after hCG trigger).</li>
	<li>Transfer all oocytes into individual droplets on the PLM dish and place it under the inverted microscope prepared for spindle imaging. Remember to remove the plastic lid from the glass bottom dish before starting the examination.</li>
	<li>Bring the first oocyte into focus. If it is difficult to search for the cell under green light, pull out the green filter temporarily. Make sure the green filter is inserted before analysis.</li>
	<li>Observe the detected oocyte birefringence image (red/orange on green background) as it is computer-processed and displayed in real time on the computer screen. Remember that the signal is not visible in the eyepiece.</li>
	<li>If the message announcing light exposure is too low/high pops up, adjust the brightness to the suitable intensity using the microscope&#8217;s light intensity knob.</li>
	<li>Use a holding and ICSI needle to turn the oocyte so the PB is in the 12 o&#8217;clock position and focus to the PB.</li>
	<li>If the spindle birefringence is not visible at first sight in the vicinity of PB, gently turn the oocyte around each axis by slightly touching the zona pellucida to ensure the alignment of the polarized light with the array spindle fibers (Supplemental Video). Declare the absence of the MII spindle as long as the oocyte fails to show the spindle signal despite rigorous rotation.</li>
	<li>Based on the observed birefringence pattern, classify the oocytes into the following categories (Figure 2): (A) oocytes with bright signal of bipolar barrel-shaped MII spindle with clearly delineated boundaries and even distribution of birefringence; (B) oocytes with dysmorphic, apolar and translucent MII spindle with irregular boundaries and uneven distribution of the signal; (C) oocytes with no detectable MII spindle birefringence in ooplasm; &#160;&#160;(D) anaphase I/telophase I oocytes, showing a microtubular bridge (a connective strand between first PB and the oocyte) instead of MII spindle. 
	NOTE: Oocyte with detectable MII spindle signal (grade A or B) are suitable for immediate ICSI.</li>
	<li>Take a snapshot (F9) or record video for a report and/or subsequent image analysis. Keep documentation of image pattern to reduce subjectivity of the operator.</li>
	<li>Move to the position of a next oocyte and repeat steps 3.6&#8722;3.9.</li>
</ol>
4. Optimizing ICSI timing
<ol>
	<li>Transfer all spindle-positive oocytes (grade A or B, step 3.8) into the ICSI dish and subject them to ICSI according to standard protocols24,25.</li>
	<li>If the oocytes show no detectable MII spindle signal, place the PLM dish into the CO2- independent incubator and shift the ICSI into a later time.</li>
	<li>Perform PLM re-examination ~2&#8722;3 h later following steps 3.3&#8722;3.9. If some oocyte(s) still lack a MII spindle, further delay ICSI for additional 1&#8722;2 h.</li>
	<li>Transfer all oocytes into the ICSI dish and inject them according to standard protocols24. If the PLM dish is compatible with the bending angle of the microinjection needle, perform ICSI immediately in the PLM dish after switching to a bright field mode.</li>
	<li>Upon ICSI, transfer oocytes to prepared plates for embryo cultivation and culture until the blastocyst stage.</li>
</ol>

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Y., Schatten, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+aging:+cellular+and+molecular+changes,+developmental+potential+and+reversal+possibility.">Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility.</a> Human Reproduction Update. 15 (5), 573-585 (2009).</li><li>Pujol, A., Garcia, D., Obradors, A., Rodriguez, A., Vassena, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+there+a+relation+between+the+time+to+ICSI+and+the+reproductive+outcomes?.">Is there a relation between the time to ICSI and the reproductive outcomes?.</a> Human Reproduction. 33 (5), 797-806 (2018).</li><li>Yanagida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+oocyte+preincubation+time+on+fertilization+after+intracytoplasmic+sperm+injection.">Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection.</a> Human Reproduction. 13 (8), 2223-2226 (1998).</li></ol>

The authors have nothing to disclose.

The acentrosomal meiotic spindle in human oocytes is highly dynamic and a delicate structure1. Under suboptimal conditions, the microtubule fibers quickly depolymerize and meiotic spindle disassembles30,31,32,33. Hence, it is critically important to ensure that the oocyte culture and micromanipulation conditions, namely temperature and pH are in the optimal range. To minimize the risk of spindle disruption, the oocytes should be kept in a temperature-controlled environment maintaining 37 &#177; 0.5 &#176;C in the oocyte media. Use of microscopes and microinjection setup equipped with a heated stage is strictly required. All manipulation with oocytes outside of the incubator must be carried out in HEPES/MOPS buffered media to avoid pH fluctulations. To avoid potential adverse effects of excessive oocyte handing in ambient condition, the total time of PLM examination should not exceed 10 minutes. The oocytes have to be analyzed on the glass bottom dish with the plastic lid removed because positioning of a standard plastic in the beam path impairs image analysis. Because plastic and glass bottom dishes have different thermal characteristics, the temperature should be checked directly in the droplets of handling medium in the examination dish.
In addition to laboratory conditions, procedural differences and operator&#39;s micromanipulation skills could have an impact on PLM examination accuracy. Only highly assembled bipolar spindles can be noninvasively visualized. Observed signal is proportional to the degree of structural organization of the spindle. Nascent, apolar, loosened or disarrayed spindles display only blurred birefringence or none at all (Figure 3 and Figure 4). Besides, imperfect alignment of polarized light with microtubule arrays produces only a translucent and ill-defined birefringence despite the actual presence of welldeveloped bipolar spindle (Figure 4). Unless properly oriented, the spindle signal might be easily missed and oocyte maturity misdiagnosed (Supplemental Video). For optimal spindle imaging, the oocyte has to be properly turned around each axis. Since birefringence is not visible in eyepieces, the operator has to watch the computer screen while performing rotation of the oocyte. Previous experience with micromanipulation and training of spindle imaging in surplus in vitro matured oocytes is desirable before switching to clinical application.
Besides the spindle, the cumulus cells surrounding the oocyte, oolema, the inner layer of the zona pellucida and some cytoplasmic structures (e.g., refractile bodies, vacuoles) exhibit birefringence (Figure 5A-D). Unless thoroughly removed from the oocyte before imaging, tightly attached follicular cells produce background noise and compromise meiotic spindle detection. Be careful not to mistake a large refractile body for the spindle. Many cytoplasmic inclusions residing in the cytoplasm display high brightness which sharply contrasts with the dark background. Meiotic spindle, on the other hand, exhibit only moderate signal, blurred boundaries and typically attaches to the oolema beneath or adjacent to the first PB. Occasionally, PLM examination reveals a gross spindle deviation from its standard position (Figure 5E,F). If there is a major misalignment between the division apparatus and the PB, PLM helps to orient the oocyte to avoid the risk of spindle injury during microinjection. The relative position of the spindle within the oocyte does not appear to influence the developmental potential of the resulting embryos17,34. However, when the spindle competely detaches from the oolema, fertilization abnormalities occur. Interestingly, some poor-quality oocytes undergo chromatin decondensation immediately after PB extrusion instead of starting microtubule nucleation (Figure 4F). Using conventional light microscopy and the PB as the only marker of oocyte maturity, such sub-competent oocytes would be considered as fertilizable. Thus, on top of the routine morphological assessment, spindle imaging adds important information about the egg&#39;s maturity and can serve as an indirect marker of its quality.
The incidence of spindled MII oocytes is likely to be affected by characteristics of the study population (e.g., genetic background, medical condition, maternal age). In addition, the proportion of developmentally delayed oocytes within the cohort will influence the actual number of detected spindle-negative oocytes9,27. Meiotic spindle visualization makes it possible to clearly identify fertilizable oocytes arrested in the MII stage. Furthermore, the second inspection at a later time may reveal whether the spindle-negative oocyte is abnormal or has only recently progressed through MI/MII transition9,10,11. When allowed to develop a spindle prior to ICSI, even immature oocytes, which are routinely discarded, can produce viable embryos9. In cycles with very few oocytes available for fertilization, finely-timed ICSI of oocytes extruding PB can serve as a rescue strategy and alternative to cycle cancellation.
Nevertheless, extending the preincubation time should not be generalized to all oocytes. In vivo matured oocytes from normal responders typically exhibit an MII spindle9,20,21,27. Here, the chance of successful spindle imaging decreases with time as a consequence of post-ovulatory in vitro aging35. If possible, ICSI should be performed on the day of retrieval and should not exceed 9 hours (45 hours post hCG), the period associated with a decline in resulting embryo quality36,37. Oocytes displaying distinct bipolar spindles ought to be subjected to ICSI without further delay. In summary, individualized optimization of ICSI timing is worthwhile in poor-prognosis patients to exclude any risk of premature sperm injection. However, it is unnecessary, too time-consuming and laborious to be performed in all IVF cycles.
PLM analysis reveals whether the oocyte reached the MII stage. However, noninvasive visualization of the meiotic spindle provides no information about chromosome organization. There might be severe chromosome misalignment and/or maternal age-related chromatid splitting in oocytes featuring a bipolar spindle (Figure 5). Various other factors have significant impact on reproduction success (e.g., sperm factor, mitochondria, embryonic genome activation, irregular cleavage, epigenetics, endometrium, maternal immunity). Therefore, detection of the MII spindle per se, does not guarantee a positive clinical outcome of the IVF procedure.

To become a fertilizable haploid egg, the diploid oocyte has to extrude half of its genetic information into an adjacent cell, called the first polar body (PB), and align chromosomes in the equator of the bipolar metaphase II (MII) spindle. While PB can be clearly observed by conventional light microscopy, detection of genetic material and cytoskeletal structures typically requires invasive preparatory procedures that are incompatible with the oocyte&#39;s further use for fertility treatment. Hence, in clinical practice, the presence of the PB is regarded as a hallmark of oocyte maturity. However, live imaging of microtubule and chromosome dynamics during human oocyte maturation revealed that PB becomes visible a couple of hours before bipolar MII spindle is assembled and chromosomes are aligned1. Nevertheless, in the transmitted light, the MII arrested eggs are indistinguishable from the oocytes which just entered the process of chromosome segregation. Thus, a cohort of oocytes, classified as MII oocytes based only on the PB presence, might contain latematuring oocytes that have not yet completed their development and thus are not ready for fertilization.
The delay of oocyte maturation is likely to affect the population of poor responders with a low number of MII oocytes and a high proportion of immature oocytes collected unexpectedly in stimulated cycles2. In vivo, only a single, best-quality egg achieves maturity and becomes ovulated. In in-vitro fertilization (IVF) cycles, controlled ovarian hyperstimulation is used to recruit multiple oocytes for maturation. Gonadotropin surge triggers a resumption of the meiotic program and egg progenitors are supposed to reach the MII arrest stage within 36 hours3. However, oocytes retrieved from preovulatory follicles often constitute an assortment of PBdisplaying MII oocytes and immature oocytes, either at metaphase I (MI) or germinal vesicle stage (GV) (Figure 1). Only MII oocytes are subjected to intracytoplasmic sperm injection (ICSI) while immature oocytes are typically discarded. Yet, when cultivated in vitro, MI oocytes are commonly observed to extrude PB in vitro. Despite their general inferiority, late-maturing oocytes which spontaneously completed the first meiotic division during overnight culture have been successfully used as last resource oocytes, and live births have been reported4,5,6,7,8. Hence, the untimely injection of the sperm might be a primary reason underlying poor developmental outcomes of late-maturing oocytes reported in previous studies9,10,11.
Polarized light microscopy (PLM) combined with an image processing software allows for non-invasive visualization of the meiotic spindle in the live oocyte. The double reflection is generated by the interaction of polarized light beam with the highly ordered assembly of microtubules building the bipolar spindle. Since the polarized light is of normal intensity, the technique could be safely used in clinical settings to view the dynamics of the division apparatus11,12,13,14. The presence of the MII spindle birefringence within the oocyte has been identified as a marker of an egg&#39;s developmental competence9,15,16,17,18,19,20,21,22,23. Thus, it has been suggested that non-invasive meiotic spindle imaging could be used for egg quality control in clinical practice11,14,20.
Since the timeline for the microtubular dynamics during oocyte meiosis has been resolved1, the observed PLM pattern can be better related to the time course of MI to MII transition. Shortly after PB emission, the nascent MII spindle becomes undetectable by PLM. However, if the oocytes are kept in the culture, the birefringence signal may emerge later when the bipolar MII spindle reassembles9,10,11. Thus, in oocytes extruding a PB in vitro, the absence of the spindle might be only temporary corresponding to the physiological transition of the late-maturing oocyte from MI to MII stage. If the MII spindle signal is undetectable, sperm injection can be deferred to a later time point providing extra time for the MII spindle formation. PLM-aided optimization of ICSI time maximizes the chance of late-maturing oocytes to be clinically utilized and make a difference for poor prognosis patients9.
Below, we provide a step-by-step protocol of how to perform non-invasive spindle imaging in human oocytes. We also demonstrate how PLM can be employed to avoid the risk of premature fertilization of late-maturing oocytes.

<table><tbody><tr><td>Continuous Single Culture Complete with Human Serum Albumin</td><td>Irvine Scientific</td><td>90164</td><td>bicarbonate based single-step &lt;strong&gt;culture medium&lt;/strong&gt; for embryo culture</td></tr><tr><td>Denuding micropipette 150 &amp;micro;m</td><td>Microtech IVF</td><td>005-150C</td><td></td></tr><tr><td>Denuding micropipette 180 &amp;micro;m</td><td>Microtech IVF</td><td>005-180B</td><td>suitable for oocyte transfer between dishes</td></tr><tr><td>Denuding micropipette 200 &amp;micro;m</td><td>Microtech IVF</td><td>005-250-A</td><td>suitable for oocyte transfer between dishes</td></tr><tr><td>FluoroDish</td><td>World Precision Instruments</td><td>FD 5040-100</td><td>glass-bottom dish</td></tr><tr><td>(alternative: WillCo-dish GWST-5040 WillCo Wells)</td><td></td><td></td><td></td></tr><tr><td>Holding micropipette</td><td>Microtech</td><td>001-120-30</td><td>sterile glass microneedles</td></tr><tr><td>Hyaluronidase solution</td><td>Irvine Scientific</td><td>90101</td><td>for oocyte denudation</td></tr><tr><td>ICSI micropipette</td><td>Microtech</td><td>002-5-30</td><td>sterile glass microneedles</td></tr><tr><td>Micro Droplet Culture Dish</td><td>Vitrolife</td><td>16003</td><td>12-well plate for embryo culture</td></tr><tr><td>Multipurpose &lt;strong&gt;Handling Medium &lt;/strong&gt;(MHM) with Gentamicin</td><td>Irvine Scientific</td><td>90163</td><td>handling medium, MOPS/HEPES buffered</td></tr><tr><td>Nikon Eclipse TE 2000-U</td><td>Nikon</td><td></td><td>inverted microscope with heated stage</td></tr><tr><td>Nunc IVF Petri Dish, 60 mm</td><td>Thermo Fisher Scientific</td><td>150270</td><td>plastic ICSI dish</td></tr><tr><td>Nunc non-treated 4-well IVF dish</td><td>Thermo Fisher Scientific</td><td>179830</td><td>4-well plate for embryo culture</td></tr><tr><td>OCTAX polarAide</td><td>MTG</td><td></td><td>integrated PLM system</td></tr><tr><td>Oil for embryo culture</td><td>Irvine Scientific</td><td>9305</td><td>oil for overlay</td></tr><tr><td>Polyvinylpyrrolidone</td><td>Irvine Scientific</td><td>90123</td><td>for sperm immobilization prior to ICSI</td></tr></tbody></table>

human egg maturity assessment and its clinical application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

Polarized light microscopy makes it possible to instantly inspect whether the oocyte completed nuclear maturation and assembled MII spindle prior to ICSI. Due to its non-invasive character, it can be used to safely assess the human egg&#39;s readiness for fertilization in clinical settings11,12,13,14. Spindled oocytes are more likely to give rise to viable embryos than oocytes without a spindle9,15,16,17,18,19,20,21,22,23. Also, oocytes featuring a distinct bipolar spindle appear to have a higher developmental competence than oocytes with dysmorphic spindles9,21,22. Taken together, spindle imaging can serve as a tool to identify the oocytes with the greatest potential to be successfully fertilized, undergo preimplantation development and support full-term pregnancy9,15,16,17,18,19,20,21,22,23.
The reported incidence of the spindle varies (44&#8722;97%) reflecting the diversity of the studied population9,15,16,17,18,19,20,21,22,23,26,27,28. In vivo matured oocytes from normal responders typically show bipolar MII spindle at 40 h after hCG trigger9,18,27,29. However, in slow responders, the pool of oocytes retrieved from preovulatory follicles is affected by maturation delay9,27. Here, spindle imaging should be carried out to discriminate the oocytes arrested in the MII stage from those undergoing important maturational transition (Figure 3). We recommend the removal of follicular cells immediately upon retrieval, and the incubation of MI and MII oocytes in separate wells to be able to distinguish the In vivo matured oocytes and late-maturing oocytes extruding PB in vitro, shortly before ICSI. In case the denudation is performed just before ICSI, these two populations of oocytes are indistinguishable based on the PB appearance.
The phase-specific changes of the PLM pattern should be interpreted in the context of knowledge of the spindle dynamics during meiotic maturation. During the interkinesis, division apparatus undergoes extensive structural reorganization and PLM signal transiently disappears1,9,10,11. Thus, in developmentally delayed oocytes extruding PB in vitro, the absence of the MII spindle signal may not necessarily reflect cellular disturbance but it could indicate the oocyte&#39;s progression from MI to MII stage (Figure 3). If the sperm is injected at an unphysiological time point, the egg&#39;s developmental potential is compromised. Postponing ICSI provides late-maturing oocytes with more time to assemble an MII spindle whose presence is associated with better clinical results9,10,11. In the clinical study involving slow/poor responders, nearly 60% of initially spindle-negative oocytes developed spindle signal before PLM re-examination approximately 2 h later9. Depending on the developmental stage and overall fitness of late-maturing oocytes, the MII spindle appearance after PB extrusion typically takes 2&#8722;6 h (unpublished observation). The oocytes exhibiting a microtubule bridge (anaphase/telophase, grade D) during the first PLM examination require a longer time to develop MII spindle signal than oocytes with no visible spindle (emerging spindles, grade C). Individual adjustment of the time of ICSI to the egg&#39;s developmental stage prevents premature activation of the oocyte and results in an improved fertilization rate and successful embryo development9.
Although late-maturing oocytes extruding PB in vitro generally have a lower developmental competence than In vivo matured oocytes, the rate of embryo development is acceptable if they manage to assemble a detectable spindle prior to delayed ICSI (blastulation rate of 41.32%)9. Using this approach, even immature oocytes, which are normally rejected for fertility treatment, can be clinically utilized, produce transferable embryos, and give rise to full-term pregnancies. Evaluation of the maturational stage of each individual oocyte is especially beneficial in poor prognosis cycles yielding a small number of MII oocytes and/or after performing a rescue in vitro maturation of immature oocytes9.
Apart from egg maturity assessment, spindle imaging is also recommended prior to PB biopsy in order to avoid spindle destruction in anaphase I/telophase oocytes. In experimental embryology, visualization of the meiotic spindle is used during egg enucleation and/or spindle transfer procedure13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60058/60058fig1.jpg" /> 
Figure 1: Human oocyte maturation. In vivo matured oocytes (MII) exhibit PB at the time of retrieval. Immature oocytes (GV, MI) can spontaneously complete maturation in vitro. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60058/60058fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60058/60058fig2.jpg" /> 
Figure 2: Oocyte classification based on PLM-detected pattern. Representative examples of oocyte grades (A-D) based on the appearance of the spindle signal. PLM-detected pattern: (A) prominent signal of bipolar spindle, (B) translucent apolar MII spindle spindle, (C) no detectable spindle, and (D) microtubule bridge. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60058/60058fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60058/60058fig3.jpg" /> 
Figure 3: Stages of MI to MII transition in oocyte maturation. The appearance of oocytes in bright field (top row), combined with a fluorescence signal of chromosomes (cyan) and microtubules (magenta) (middle row), and in polarized light (bottom row) is shown. Each oocyte was first PLM-examined and immediately fixed. Fixed oocytes were (immuno)labeled with Hoechst (DNA) and anti-&#945;-tubulin antibody (microtubules). The yellow arrow indicates the presence of PB and the white arrow highlights the position of birefringent microtubules. Scale bar = 20 &#181;m. This figure has been modified from Holubcov&#225; et al., JARG 20199. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60058/60058fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60058/60058fig4.jpg" /> 
Figure 4: Correlation of chromosome-microtubule organization with birefringence pattern detected by polarized light microscopy. The appearance of oocytes in the bright field combined with a fluorescent signal for chromosomes (cyan) and microtubules (magenta) (top row), and in polarized light (bottom row) is shown. Each oocyte was fixed immediately after PLM-examination. DNA (Hoechst) and microtubule (&#945;-tubulin) were stained. (A-C) oocytes with PLM-detectable spindle yet misaligned chromosomes (A, B) or loosely focused spindle poles (C); (D-F) abnormal oocytes without PLM-detectable MII spindle showing underdeveloped (D), apolar (E) or no spindle (F). The white arrow highlights the position of birefringent microtubules, and asterisk (*) indicates the position of decondensed chromatin. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60058/60058fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60058/60058fig5v2.jpg" /> 
Figure 5: Birefringence structures in human oocytes. Representative examples of (A-D) birefringent structures detected by PLM in human oocytes. ZP = zona pellucida; PM = plasma membrane (oolema); RB = refractile bodies, vacuoles. White arrow, meiotic spindle in different stages of maturation. (E) MII spindle misalignment with polar body (PB). (F) Spindle detachment from plasma membrane. Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60058/60058fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Desktop layout of the image analysis software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60058/desktop_layout.jpg">Please click here to download this file.</a>
Supplemental Video: PLM examination procedure. Rotation of the oocyte is required to exclude the presence of the spindle (arrow). Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60058/Supplementary_video.avi">Please click here to download this file.</a>

Watch this Scientific Journal Video about Human Egg Maturity Assessment and Its Clinical Application at JoVE.com

Human Egg Maturity Assessment and Its Clinical Application

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

human-egg-maturity-assessment-and-its-clinical-application

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

19.0K Views.  Clinic of Reproductive Medicine. We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

Video: Human Egg Maturity Assessment and Its Clinical Application

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Biology

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Cancer Research

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

Bioengineering

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Neuroscience

Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage

Source: Hostelley T.L., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/56187/" target="_blank">Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish</a>. J. Vis. Exp. (2017)
The article describes a protocol to generate zebrafish embryos through natural mating and sorting their developmental stages. The protocol is used further for analysis of pancreatic &#946;-cells by using transgenic fishes.

Source: Hostelley T.L., et al. Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish. J. Vis. Exp. (2017)
The article ...

Human Egg Maturity Assessment and Its Clinical Application

Summary

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Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

An In Ovo Model for Testing Insulin-mimetic Compounds

Mating and Egg Staging: A Method to Generate Embryos and Sort Them by Developmental Stage