Zona Pellucida Birefringence Research Articles

Comparing the IVM laboratory outcomes between stimulated IVF with unstimulated natural cycles.

Recently, more attention has been raised towards fertility preservation in women with cancer. One option is in vitro maturation (IVM) of the immature oocytes as there is not enough time for induction of an ovarian stimulation protocol. The aim was to compare the IVM laboratory outcomes between stimulated and unstimulated (natural) in vitro fertilization (IVF) cycles. In total, 234 immature oocytes collected from 15 cancer patients who underwent an IVM programme (natural IVM) and 23 IVF cycles with a controlled ovarian hyperstimulation protocol (stimulated IVM) were analyzed. The oocyte morphology, zona pellucida (ZP), and meiotic spindle presence were measured using PolScope technology. Also, the rates of oocyte maturation and fertilization were assessed in both groups. The IVM rate was higher in the stimulated cycle (P < 0.05), but the fertilization rate was insignificant in comparison with unstimulated cycles. There were no significant differences in the spindle visualization and ZP birefringence scoring between the groups (P > 0.05). The oocyte normal morphology was better in the stimulated cycle compared with the natural cycle (P < 0.05). In conclusion, IVM can be recommended for cancer patients as an alternative treatment when there is insufficient time for conventional IVF before chemotherapy initiation.

High efficiency of homemade culture medium supplemented with GDF9-β in human oocytes for rescue in vitro maturation

ObjectiveOptimizing culture media for the incubation of immature oocytes is a vital strategy to increase the oocyte maturation rate during in vitro maturation (IVM) programs. This study evaluated the IVM and fertilization rates of human germinal vesicle (GV) and metaphase I (MI) oocytes using two different maturation media (commercial and homemade) with or without growth differentiation factor 9-β (GDF9-β). supplementation.MethodsImmature oocytes from intracytoplasmic sperm injection (ICSI) cycles were collected and assigned to one of two IVM culture media (commercial or homemade; cleavage-stage base). After maturation, MII oocytes were examined under an inverted microscope for the presence of the polar body, zona pellucida (ZP) birefringence, and meiotic spindle (MS) visualization after maturation in four conditions (commercial or homemade medium, with or without GDF9-β. ICSI was done for matured oocytes, and fertilization was confirmed by the visualization of two distinct pronuclei and two polar bodies.ResultsNo significant differences were found between the two culture media in terms of the time and rate of oocyte maturation or the rate of fertilization (p>0.05). Growth factor supplementation increased the 24-hour maturation rate for both GV and MI oocytes only in homemade medium. The maturation rate after 24 hours was higher for MI oocytes (p<0.05). Similar results were observed for MS visualization and ZP structure in both types of media (p>0.05).ConclusionHigher rates of oocyte maturation and fertilization were observed after application of homemade medium supplemented with GDF9-β. Therefore, this combination may be recommended as an alternative for clinical IVM programs.

Stratifying Cumulus Cell Samples Based on Molecular Profiling to Help Resolve Biomarker Discrepancies and to Predict Oocyte Developmental Competence.

To increase the efficiency of assisted reproductive techniques (ART), molecular studies have been performed to identify the best predictive biomarkers for selecting the most suitable germ cells for fertilization and the best embryo for intra-uterine transfer. However, across different studies, no universal markers have been found. In this study, we addressed this issue by generating gene expression and CpG methylation profiles of outer cumulus cells obtained during intra-cytoplasmic sperm injection (ICSI). We also studied the association of the generated genomic data with the clinical parameters (spindle presence, zona pellucida birefringence, pronuclear pattern, estrogen level, endometrium size and lead follicle size) and the pregnancy result. Our data highlighted the presence of several parameters that affect analysis, such as inter-individual differences, inter-treatment differences, and, above all, specific treatment protocol differences. When comparing the pregnancy outcome following the long protocol (GnRH agonist) of ovarian stimulation, we identified the single gene markers (NME6 and ASAP1, FDR < 5%) which were also correlated with endometrium size, upstream regulators (e.g., EIF2AK3, FSH, ATF4, MKNK1, and TP53) and several bio-functions related to cell death (apoptosis) and cellular growth and proliferation. In conclusion, our study highlighted the need to stratify samples that are very heterogeneous and to use pathway analysis as a more reliable and universal method for identifying markers that can predict oocyte development potential.

Vitrification has detrimental effects on maturation, viability, and subcellular quality of oocytes post IVM in cancerous women: An experimental study.

Background In vitro maturation (IVM) of immature oocytes retrieved from ovarian tissue has been considered as a valuable approach for fertility preservation in cancerous patients.ObjectiveTo evaluate the efficacy of vitrification on oocyte maturation, survival rates, as well as the subcellular oocyte quality post IVM.Materials and MethodsThe ovarian cortexes from 19 women with cervix and uterine malignancy aged 21–39 yr were collected. Cumulus-oocyte complexes were aspirated from all visible antral follicles. 102 immature oocytes were collected, and 43 oocytes were detected appropriately for IVM (control group). Also, 59 immature oocytes were vitrified, then matured in vitro (IVM) in two groups: with Growth/differentiation factor 9 (GDF9) (group 1) and without GDF9 (group 2) supplementation. Rates of oocytes viability, maturation, and survival along with meiotic spindle visualization and zona pellucida birefringence were assessed with Polyscope.ResultsThe rate of maturation was significantly higher in controls (55.8%) compared to the other groups. Maturation rate was 23.3% in oocytes cultured in IVM medium enriched with GDF9, and 27.6% in those cultured in IVM medium lacking GDF9 (p = 0.86). Also, the meiotic spindle was present in 74.4% of control oocytes which was significantly higher than the other groups. The proportion of high zona pellucida birefringence was higher in the controls when compared with group 1 (51.2% vs. 23.3%, respectively, p = 0.04).ConclusionVitrification had a detrimental effect on oocyte maturation, viability as well as the subcellular quality of the oocytes after IVM in cancerous women.

Zona pellucida birefringence and meiotic spindle visualization are not related to the time-lapse detected embryo morphokinetics in women with polycystic ovarian syndrome

ObjectivesOocyte parameters as well as embryo developmental kinetics can be applied as non-invasive predictors for selection of viable embryos and clinical outcomes. Time-lapse morphokinetic events in conjunction with zona pellucida birefringence (ZPB) and meiotic spindle visualization (MSV) have been evaluated for predicting pregnancy outcome. Study designWe assessed a total of 547 embryos from 100 intra cytoplasmic sperm injection cycles in women with polycystic ovary syndrome (PCOS) and tubal factor infertility. ZPB and MSV were evaluated in 100 oocytes using polarized microscope. Time-lapse archives were noted for time to pronuclear fading (tPNf), time to 2–8 cells (t2–t8), direct cleavage, reverse cleavage and also for the presence of multinucleation. ResultsThe mean timings of embryo morphokinetics were not significantly different between high and low ZPB and visible and not-visible meiotic spindle oocytes in the PCOS group (P > 0.05). Furthermore implantation rate was 4.6 fold higher in embryos derived from oocytes with visible meiotic spindles compared to those without visible meiotic spindle in PCOS patients (P < 0.05). We observed that chemical and clinical pregnancy rates as well as live birth were higher in transferred embryos which formed from oocytes with visible meiotic spindle (27.6% vs.16.7%),(24.1% vs. 8.3%) and (24.1% vs. 8.3%) respectively. According to logistic regression analysis, only tPNf significantly influenced clinical pregnancy and live birth (P < 0.05). ConclusionTime-lapse embryo parameters are not related to oocyte ZPB and MSV status. However, clinical pregnancy and live birth have been affected by the presence of meiotic spindle.

Is there any correlation between oocyte polarization microscopy findings with embryo time lapse monitoring in ICSI program?

The aim was to investigate the relationship between the presence of the meiotic spindle (MS) and zona pellucida (ZP) birefringence of MII oocytes with morphokinetics variables of derived embryos in ICSI setting. Using a polarization imaging system, the ZP birefringence and presence of MS were evaluated pre ICSI. Also, morphokinetics variables including time of second PB extrusion (tPB2), time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf), time of two to eight discrete cells (t2-t8) ECC1 (t2-tPB2), cc2a (t3-t2), S2 (t4-t3) and S3 (t8-t5) as well as irregular cleavage events of 368 embryos were analyzed with time lapse monitoring (TLM). t5 occurred earlier in high birefringent ZP (HB-ZP) compared with low birefringent oocytes (LB-ZP; p=0.001). In addition, t2 happened later in invisible MS compared to visible MS oocytes (p=0.013). There were significantly lower rates of cell fusion (Fu) in oocytes with HB-ZP and also the Fu and trichotomous mitoses (TM) together in visible MS oocytes (p=0.005, p=0.001 and p=0.001, respectively). Both t2 and t5 timings and irregular cleavage events of embryos were correlated with ZP birefringence and MS status, respectively. So, combining the information from both oocyte polarization microscopy imaging and embryo TLM can be a useful tool for single embryo transfer (SET) program.

Analysis of meiotic spindle and zona pellucida birefringenceof IVM oocytes in PCOS patients.

Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility. One of the best therapeutic options for PCOS patients is intracytoplasmic sperm injection (ICSI). In vitro maturation (IVM) can also be a useful technique for these women. The goal of this study was to evaluate both the zona pellucida (ZP) birefringence and meiotic spindle (MS) of in vivo- and in vitro-matured oocytes from PCOS patients using the PolScope system. Immature oocytes undergoing IVM and MII oocytes were obtained from PCOS patients in an ICSI program. Using PolScope, the presence of MS and ZP birefringence was assessed in both in vivo-matured oocytes (n = 32) and IVM oocytes (n = 24). Oocytes were classified as having highly birefringent (HB) ZP and lowly birefringent (LB) ZP. Furthermore, the rates of fertilization after ICSI were evaluated. The maturation rate was 68.5% after IVM. The percentage of a HB-ZP was significantly higher in the IVM oocytes than in vivo-matured ones (58.3% vs. 31.2%, respectively; P = 0.04). There were similar outcomes for the fertilization rates and MS detection between the two groups (P = 0.80 and P = 0.53, respectively). Clinical IVM is a safe technology for the maturation and maintenance of oocyte integrity in PCOS patients. The use of the noninvasive PolScope is recommended for detection of healthy oocytes in ICSI.

Methods for assessing the quality of mammalian embryos: How far we are from the gold standard?

Morphological embryo classification is of great importance for many laboratory techniques, from basic research to the ones applied to assisted reproductive technology. However, the standard classification method for both human and cattle embryos, is based on quality parameters that reflect the overall morphological quality of the embryo in cattle, or the quality of the individual embryonic structures, more relevant in human embryo classification. This assessment method is biased by the subjectivity of the evaluator and even though several guidelines exist to standardize the classification, it is not a method capable of giving reliable and trustworthy results. Latest approaches for the improvement of quality assessment include the use of data from cellular metabolism, a new morphological grading system, development kinetics and cleavage symmetry, embryo cell biopsy followed by pre-implantation genetic diagnosis, zona pellucida birefringence, ion release by the embryo cells and so forth. Nowadays there exists a great need for evaluation methods that are practical and non-invasive while being accurate and objective. A method along these lines would be of great importance to embryo evaluation by embryologists, clinicians and other professionals who work with assisted reproductive technology. Several techniques shows promising results in this sense, one being the use of digital images of the embryo as basis for features extraction and classification by means of artificial intelligence techniques (as genetic algorithms and artificial neural networks). This process has the potential to become an accurate and objective standard for embryo quality assessment.

Noninvasive assays of in vitro matured human oocytes showed insignificant correlation with fertilization and embryo development.

Recently, the upgrading of in vitro maturation (IVM) of human oocytes as a promising strategy has emerged in assisted reproductive technology (ART). The goal was to evaluate the correlation of the in vitro matured oocytes selected on the basis of the zona pellucida (ZP) birefringence and meiotic spindles (MS) detection with fertilization and subsequent embryo development in ICSI program. A total of 168 immature oocytes [germinal vesicle (n = 140) and metaphase I (n = 28)] obtained from patients undergoing oocytes retrieval for ICSI. After in vitro culture for 24-40 h, 112 (67 %) oocytes reached to MII stage. Using a polarized microscopy, the presence of MS and ZP birefringence were assessed in matured oocytes, followed by ICSI performance. The rates of fertilization in oocytes with spindles (51.3 %) were similar to that of the oocytes without spindles (50.7 %; P = 1.00). Moreover, the fertilization rates in high birefringence (HB) oocytes was not statistically different than oocytes with low birefringence (LB) (P = 0.44). The findings also showed that 64.9 % of the fertilized oocytes developed to embryos, in which 33.3 % were derived from spindle-detected oocytes. Regarding the ZP birefringence, 35.5 % of the embryos were derived from HB oocytes. There were insignificant relationships between the MS detection and ZP birefringence score with the rates of fertilization and embryo development in IVM oocytes.

Use of both cumulus cells' transcriptomic markers and zona pellucida birefringence to select developmentally competent oocytes in human assisted reproductive technologies.

BackgroundSelection of the best oocyte for subsequent steps of fertilization and embryo transfer was shown to be the crucial step in human infertility treatment procedure. Oocyte selection using morphological criteria mainly Zona pellucida (ZP) has been the gold standard method in assisted reproductive technologies (ART) clinics, but this selection approach has limitations in terms of accuracy, objectivity and constancy. Recent studies using OMICs-based approaches have allowed the identification of key molecular markers that quantitatively and non-invasively predict the oocyte quality for higher pregnancy rates and efficient infertility treatment. These biomarkers are a valuable reinforcement of the morphological selection criteria widely used in in vitro fertilization (IVF) clinics. In this context, this study was designed to investigate the relationship between transcriptomic predictors of oocyte quality found by our group and the conventional morphological parameters of oocyte quality mainly the ZP birefringence.ResultsMicroarray data revealed that 48 and 27 differentially expressed candidate genes in cumulus cells (CCs) were respectively overexpressed and underexpressed in the ZGP (Zona Good Pregnant) versus ZBNP (Zona Bad Non Pregnant) groups. More than 70% of previously reported transcriptomic biomarkers of oocyte developmental competence were confirmed in this study. The analysis of possible association between ZP birefringence versus molecular markers approach showed an absence of correlation between them using the current set of markers.ConclusionsThis study suggested a new integrative approach that matches morphological and molecular approaches used to select developmentally competent oocytes able to lead to successful pregnancy and the delivery of healthy baby. For each ZP birefringence score, oocytes displayed a particular CCs' gene expression pattern. However, no correlations were found between the 7 gene biomarkers of oocyte developmental potential and the ZP birefringence score. Further studies using larger lists of candidate markers are required to identify suitable genes that are highly correlated with the morphological criteria, and therefore able to reinforce the accuracy of oocyte selection and the effectiveness of infertility treatment.

Analysis of correlations between zona pellucida birefringence and molecular markers of oocyte developmental competence

Background Human infertility is the incapacity of a couple to conceive after one year of unprotected sexual intercourse. Selection of the best gametes for subsequent steps of fertilization and embryo transfer was shown to be the crucial step in infertility treatment procedure [1,2]. Oocyte selection using morphological criteria has been the gold standard method in assisted reproductive technologies (ART) clinics. Zona Pellucida (ZP) , a filamentous matrix of glycosylated glycoproteins surrounding the oocyte, is one of these morphological criteria of oocyte selection. In fact, ZP thickness and birefringence was reported to be positively correlated with higher ability of the oocyte to achieve successful pregnancy, but this selection approach has limitations in terms of accuracy, objectivity and constancy. Recent studies using OMICs approaches have identified key molecular markers in somatic cells (cumulus and/or granulosa cells) and follicular fluid that quantitatively and non-invasively predict the oocyte quality for better selection, higher pregnancy rates and efficient infertility treatment. These biomarkers could be a valuable reinforcement of the morphological selection criteria widely used in IVF clinics. In this context, this study was designed to study the relationship between some molecular predictors of oocyte quality found by our group and the conventional morphological parameters of oocyte quality. We expect to find a positive correlation between the ZP birefringence and molecular markers of oocyte competence. Such integrative strategy should lead to a powerful combined approach that will precisely predict the oocyte developmental potential, allowing therefore efficient infertility treatment and elective single embryo transfer (eSET).

Oocyte zona pellucida and meiotic spindle birefringence as a biomarker of pregnancy rate outcome in IVF-ICSI treatment.

IVF-ICSI procedures are accompanied by a continuous search for predictors of ART outcome. The properties of zona pellucida (ZP) have been believed to reflect the history of oocyte cytoplasmic maturation. The meiotic spindle (MS) is crucial for chromosomal alignment and proper separation of the maternal chromosomes. There is data suggesting that birefringent ZP and MS can clinically predict the oocyte quality and developmental potential of an embryo. The aim of the study was to examine the possible effect of ZP birefringent properties and MS visualization and localization as valuable predictors of IVF-ICSI effectiveness. The prospective study was performed during a 16-month period. A total of 51 patients undergoing in vitro fertilization--embryo transfer (IVF-ET) treatment procedure with intracytoplasmic sperm injection (ICSI) were included. Controlled ovarian hyperstimulation (COH) was done using either a long n = 32 (62.75%) or an antagonist protocol n = 19. In the group of the 48 examined patients (aged 25-40), 46 ET were performed, resulting in 24 positive pregnancy tests and 19 (39.59%) clinical pregnancies. Oocytes were examined as follows: ZP birefringence autoscoring (OCTAX PolarAIDE), numeral autoscoring, thickness and clinical evaluation; MS visualization, if MS was visualized, localization of MS in relation to the polar body (PB). On day 3, 64.3% of the embryos were of good and 40.3% were of top quality. Visible differences, not statistically significant, were observed in the numeral score of ZP between oocytes selected and non-selected for ET. In cases when embryos were not of good or top quality, ZP score was higher (p = 0.005 p = 0.001). ZP manual evaluation indicated significantly stronger birefringence when pregnancy was not achieved (p = 0.022). The rate of MS positive oocytes was the highest in the group with pregnancy but it did not reach statistical significance (p = 0.471). The MS localization in relation to the PB was in most oocytes very close (< 45 degrees) in 70.9% and not different in the studied groups. Unexpected polarization microscopy imaging and rating of ZP and MS cannot be a direct predictor of the IVF outcome.

Effects of M II stage oocytes zona pellucida birefringence on pregnancy outcome

ObjectiveTo explore the effects of different M II stage oocytes zona pellucida birefringence on pregnancy outcome. MethodsA total of 46 couples with infertile which induced by single cause received in-vitro fertilization treatment were analyzed retrospectively, and randomly divided into the high zona birefringence (HZB)/HZB group, HZB/low zona birefringence (LZB) group and LZB/LZB group according to different oocytes zona pellucida birefringence. Intracytoplasmic sperm injection outcome was analyzed and compared. ResultsThe proportion of HZB oocytes, implantation rate and the pregnancy rate were decreased in three groups (HZB/HZB group>HZB/LZB group>LZB/LZB group) (P<0.05). But there was no significantly different between the number of oocytes and fertilization rate of these groups (P>0.05). Logistic regression analysis showed that factors affect M II stage oocytes zona pellucida birefringence were age, basal FSH level and the LH level on the day of HCG injection. Age and FSH levels were negatively correlated with the single oocyte zona pellucida birefringence; While the LH level on the day of hCG injection was positively correlated with the single oocyte zona pellucida birefringence. ConclusionsThe primary influence factors on M II stage oocytes zona pellucida are age, basal FSH level and the LH level on the day of hCG injection. The birefringence value of zona pellucida can affect the pregnancy outcome.

Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression

In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3µm) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1µm) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence.

Zona pellucida birefringence and meiotic spindle visualisation of human oocytes are not influenced by IVM technology

The aim of the present study was to investigate the relationship between the presence of the meiotic spindle and zona pellucida (ZP) birefringence with morphology of in vivo- and in vitro-matured human oocytes. Germinal vesicles (n=47) and MI (n=38) oocytes obtained from stimulated ovaries of patients undergoing intracytoplasmic sperm injection (ICSI) underwent IVM. Using a PolScope (OCTAX PolarAID; Octax, Herbon, Germany), the presence of spindles and ZP birefringence was assessed in both in vivo-matured (n=56) and IVM (n=56) oocytes. In addition, the morphology of each matured oocyte was evaluated microscopically. There were insignificant differences for ZP birefringence and meiotic spindle between the in vivo-matured and IVM MII oocytes. Subanalysis revealed that the rates of morphologically abnormal oocytes did not differ significantly between the two groups, except in the case of irregular shape (P=0.001), refractile body (P=0.001) and fragmented polar body (P=0.03), which were higher in IVM oocytes. In the case of in vivo-matured oocytes, a significantly higher percentage of oocytes with intracytoplasmic and both intra- and extracytoplasmic abnormalities have a low birefringent ZP (P=0.007 and P=0.02, respectively). There was no relationship between morphological abnormalities and spindle detection. The findings suggest that clinical IVM is a safe technology that maintains the high maturation rate and integrity of oocytes. In addition, the use of the non-invasive PolScope is recommended for the detection of oocytes most suitable for ICSI.

160 ZONA PELLUCIDA BIREFRINGENCE CORRELATES WITH CUMULUS MORPHOLOGY AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY OF EQUINE OOCYTES

The present study was conducted to determine the predictive value of zona pellucida birefringence (ZPB) measurement for developmental capacity of equine oocytes. In vitro developmental capacity of equine oocytes is related to cumulus morphology as well as glucose-6-phosphate dehydrogenase (G6PD) activity. Therefore, we classified equine oocytes according to cumulus morphology and G6PDH activity into groups of different developmental capacities and analysed the correlation between ZPB measured by polarized light microscopy and developmental competence. Ovaries were obtained from an abattoir and were transported to the laboratory within 2 to 4 h at 25 to 30°C in 0.9% saline. Cumulus–oocyte complexes (COC) were recovered by scraping the granulosa layer from opened follicles using a bone curette and were classified as having an expanded (Ex, n = 86) or compact (Cp, n = 93) cumulus. Other COC were subjected to 26 μM brilliant cresyl blue (BCB) in modified PBS for 90 min at 38.5°C and were categorised into BCB+ (blue cytoplasm, low G6PD activity, n = 89) and BCB– (colourless cytoplasm, high G6PD activity, n = 41) according to their ability to convert the BCB stain from blue to colourless. Following maturation for 28 to 30 h at 38.5°C in 5% CO2 in DMEM-F12 supplemented with 10% FCS, 5 mU mL–1 of FSH and 25 μg mL–1 of gentamicin sulfate, oocytes were imaged individually to assess zona thickness and ZPB by polarized light microscopy using OCTAX polarAIDE software (OCTAX, Bruckberg, Germany). Subsequently, oocytes were subjected to intracytoplasmic sperm injection using frozen-thawed stallion spermatozoa followed by culture in DMEM/F-12 medium (10% FCS and 25 μg mL–1 of gentamicin sulfate) under mineral oil (38.2°C, 5% CO2, 5% O2) for 8 days. Development rates were compared by chi-square test; variations in thickness and birefringence were analysed by Tukey's test and ANOVA. Higher maturation (58.4 vs 40.2%) and blastocyst rates (11.9 vs 3.8%) of Ex oocytes compared with Cp oocytes as well as higher proportions of BCB+ oocytes that matured (57.7% vs 28.1%), cleaved (45.9 vs 29.0%) and developed to the blastocyst stage (9.2 vs 1.4%) compared with BCB– oocytes (all P &lt; 0.05) confirmed cumulus morphology and G6PDH activity as useful predictors for viability. Moreover, Ex oocytes had a thicker zona (18.2 ± 2.2 μm vs 17.3 ± 2.1 μm) and a higher birefringence (64.6 ± 5.2 vs 62.1 ± 4.2) compared with Cp oocytes (both P &lt; 0.05). Concurrently, oocytes classified as BCB+ had a thicker zona (18.8 ± 2.4 μm vs 16.1 ± 2.0 μm) and higher values for birefringence (63.1 ± 4.5 vs 61.3 ± 3.3) compared with BCB– oocytes (both P &lt; 0.05). Taken together, our results revealed that equine oocytes with higher developmental capacity have a thicker zona and greater birefringence scores compared with oocytes of lower developmental competence. Therefore, ZPB measurement provides a new, noninvasive method to assess the developmental competence of equine oocytes.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles

BackgroundThis study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles.ResultsThe ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11).ConclusionsThe current study demonstrated an absence of relationship between ZP-BF high/positive or low/negative score and nuclear and cytoplasmic in vitro maturation of oocytes from stimulation cycles.

Correlation of oocyte morphometry parameters with woman’s age

Aim of this study was to evaluate morphometric parameters of metaphase II oocytes, including cytoplasm diameter (CD), zona pellucida thickness (ZPT) and width of the perivitelline space (PS), in relation with zona pellucida birefringence, spindle presence and age of the woman. Oocytes were classified into groups according to zona birefringence (low or high zona birefringence, LZB and HZB, respectively) and presence or absence of a visible spindle (SP and aSP, respectively). HZB oocytes showed a thicker zona (17.7 ± 0.3 μm) than LZB oocytes (16.7 ± 0.3 μm, p < 0.01). Moreover, PS was narrower in HZB and SP oocytes than in LZB (p < 0,001) and aSP (p < 0,05) oocytes. Finally, we found that CD and ZPT linearly decrease with age of the woman (CD r = 0.028: p < 0.01; ZPT r = 0.050: p < 0.0001). Our results evidence an association in human oocytes between zona pellucida and spindle birefringence and defined morphometric parameters and a decrease of oocyte size and ZPT as a function of women's age.

Biomarkers of human oocyte developmental competence expressed in cumulus cells before ICSI: a preliminary study

To identify reliable genomic biomarkers expressed in cumulus cells that accurately and non-invasively predict the oocyte developmental competence and reinforce the already used morphological criteria. Eight consenting patients were selected for ovarian stimulation and ICSI procedures. Cumulus-oocyte complexes were transvaginally punctured and individually selected based on both good morphological criteria and high zona pellucida birefringence. Following ICSI, two 3-day embryos per patient were transferred. Pregnancy outcome was recorded and proven implantation was thereafter confirmed. Differential gene expression was assessed using two microarray platforms. Further real-time PCR validation, Ingenuity pathways analysis and intra-patient analysis were performed on 17 selected candidates. Seven genes were differentially (p ≤ 0.05) associated to successful pregnancy and implantation. These biomarkers could be used to predict the oocyte developmental competence. These genomic markers are a powerful reinforcement of morphological approaches of oocyte selection. Their large-scale validation could increase pregnancy outcome and single embryo transfer efficiency.