Triggering method in assisted reproduction alters the cumulus cell transcriptome.

RESEARCH QUESTION: How does the choice of triggering final oocyte maturation affect the cumulus cell transcriptome? DESIGN: Sixty patients undergoing gonadotrophin-releasing hormone antagonist (GnRH-ant) IVF cycles were recruited for this nested case-control study. Patients were stratified into three subgroups based on their ovarian reserve (high, normal and low). Triggering final oocyte maturation was accomplished by either single trigger (with human chorionic gonadotrophin [HCG] only or gonadotrophin-releasing hormone agonist [GnRH-ag] only) or dual trigger combining HCG and GnRH-ag. The choice of trigger was at the discretion of the treating physician. Within each group patients receiving a dual trigger were matched by demographic and pre-stimulation parameters with patients receiving a single trigger. The matching was performed to minimize the biological variability within each subgroup. Thirty patients were included in the final analysis. Cumulus cells were stripped away from the retrieved oocytes. Cumulus cells from three sibling oocytes were pooled, the RNA extracted and libraries prepared. Next-generation sequencing was performed on all samples. RESULTS: Dual triggering supports key ovarian pathways of oocyte maturation and extracellular matrix remodelling, while attenuating vasculo-endothelial growth and providing antioxidant protection to the growing follicles. CONCLUSIONS: This is the first study to delineate key transcriptomic changes under dual triggering of final oocyte maturation, across different patient populations. The findings underline the need for larger-scale studies validating transcriptomic effects of methods for triggering final oocyte maturation. Furthermore, there is a need for large-scale clinical randomized controlled studies to relate the findings of this study with clinical outcomes.

The use of coenzyme Q10 and DHEA during IUI and IVF cycles in patients with decreased ovarian reserve.

OBJECTIVE: The objective of this study is to compare the combination of dehydroepiandrosterone (DHEA) and coenzyme Q10 (CoQ10) (D + C) with DHEA alone (D) in intrauterine insemination (IUI) and in vitro fertilization (IVF) cycles among patients with decreased ovarian reserve. METHODS: We retrospectively extracted data from patients charts treated by DHEA with/without CoQ10 during IUI or IVF between February 2006 and June 2014. Prestimulation parameters included age, BMI, day 3 FSH and antral follicular count (AFC). Ovarian response parameters included total gonadotropins dosage, peak serum estradiol, number of follicles > 16 mm and fertilization rate. Clinical outcomes included clinical and ongoing pregnancy rates. RESULTS: Three hundred and thirty IUI cycles involved D + C compared with 467 cycles of D; 78 IVF cycles involved D + C and 175 D. In both IUI and IVF, AFC was higher with D + C compared with D (7.4 ± 5.7 versus 5.9 ± 4.7, 8.2 ± 6.3 versus 5.2 ± 5, respectively, p < 0.05). D + C resulted in a more follicles > 16 mm during IUI cycles (3.3 ± 2.3 versus 2.9 ± 2.2, respectively, p = 0.01), while lower mean total gonadotropin dosage was administered after D + C supplementation compared with D (3414 ± 1141 IUs versus 3877 ± 1143 IUs respectively, p = 0.032) in IVF cycles. Pregnancy and delivery rates were similar for both IUI and IVF. CONCLUSION: D + C significantly increases AFC and improves ovarian responsiveness during IUI and IVF without a difference in clinical outcome.

Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.-

The developmental potential of mature oocytes derived from rescue in vitro maturation.

OBJECTIVE: To examine the developmental competence of immature oocytes in stimulated cycles, that matured after rescue in vitro maturation (IVM) compared with their sibling in vivo matured oocytes. DESIGN: Retrospective cohort study. SETTING: IVF clinic. PATIENTS: A total of 182 patients underwent 200 controlled ovarian stimulation cycles with intracytoplasmic sperm injection cycles in which immature oocytes were retrieved and at least one mature oocyte was obtained through rescue IVM. INTERVENTION: In vitro culture of immature germinal vesicle (GV) and metaphase I (MI) oocytes, retrieved in stimulated cycles. MAIN OUTCOME MEASURES: Fertilization rate, cleavage rate, blastulation rate, ploidy of embryos evaluated using preimplantation genetic testing for aneuploidy, morphokinetic parameters and pregnancy outcomes. RESULTS: In total, 2,288 oocytes were retrieved from 200 cycles. After denudation, 1,056 of the oocytes (46% ± 16%) were classified as metaphase II (MII). A total of 333/375 (89%) of MI oocytes and 292/540 (54%) of GV oocytes matured overnight and underwent intracytoplasmic sperm injection. The fertilization rates of matured oocytes from MI rescue IVM (R-MI) and from GV rescue IVM (R-GV) were comparable with those of their sibling MII oocytes (71% vs. 66%; 66% vs. 63%, respectively). Early cleavage rates (80% ± 35% vs. 92% ± 20%; 80% ± 42% vs. 95% ± 28%, respectively) and blastulation rates (32 ± 40% vs. 62 ± 33%; 24 ± 37% vs. 60 ± 35%, respectively) were significantly decreased in rescue IVM matured oocytes (R-oocytes)-derived zygotes, but the blastocyst (BL) euploidy rate and "good quality" BL rate were comparable with those of MII sibling-derived embryos. In addition, rescue IVM embryos showed significantly higher levels of multinucleation at the 2- and 4-cell stages, as well as higher rates of zygote direct cleavage from one to 3 to 4 cells. Overall, 21 transfers of rescue IVM embryos resulted in 3 healthy live births. CONCLUSIONS: For patients with a low maturation rate and/or low numbers of mature oocytes at retrieval, rescue IVM may contribute more competent oocytes and additional viable BLs for transfer from the same stimulation cycle, maximizing the chances for pregnancy and live birth.

The impact of hyaluronan-enriched culture medium and intrauterine infusion of human chorionic gonadotropin on clinical outcomes in blastocyst transfer cycles.

Over the last few decades, advances in ovarian hormonal stimulation, embryology laboratory technologies and embryo genetic testing, have significantly enhanced clinical outcomes in human assisted reproduction technologies (ART). However, embryo implantation remains a major bottleneck in achieving better pregnancy and live birth rates. Thus, there is growing interest in establishing new approaches to enhance implantation efficiency after embryo transfer. With advanced molecular techniques, many promising biomarkers associated with embryonic and endometrial changes occurring prior to and during embryo implantation have been identified. However, despite the progress in applying novel procedures into IVF practice, clinical evaluation of those biomarkers has so far reached modest predictive value for enhancing blastocyst developmental potential and endometrial receptivity. Therefore, other simpler strategies have also been introduced to increase the rates of successful clinical pregnancies and live births. One of these approaches is to investigate the impact of using embryo transfer medium containing high concentrations of an adherence compound, such as hyaluronic acid (HA), on IVF outcomes. Additionally, intrauterine infusion of a small volume of human chorionic gonadotropin (hCG) at the time of embryo transfer (ET) has also been proposed as a technique that might be advantageous for increasing the clinical outcomes, considering the fact that hCG plays a critical role in synchronizing endometrial and fetal development. However, the current findings from both interventions remain controversial, demonstrating a mixture of positive and indifferent results of these treatments in ART cycles. Further research will be crucial for a better understanding of the molecular mechanism of cross-talk between the blastocyst and the maternal endometrium during the optimal implantation period when using either hyaluronan-enriched medium or hCG infusion before embryo transfers. Therefore, this review aims to present existing literature related to both treatments, emphasizing their effects on blastocyst implantation.Abbreviations: ART: assisted reproduction technologies; HA: hyaluronic acid; hCG: human chorionic gonadotrophin; IVF: in vitro Fertilization; ET: embryo transfer; pH: hydrogen ions; CO2: Carbone dioxide; O2: Oxygen; PGT: pre-implantation genetic testing; FET: frozen embryo transfer; PCOS: Polycystic ovarian syndrome; DNA: deoxyribonucleic acid; miRNA: micro-ribonucleic acid; EVs: extracellular vesicles; ERA: endometrial receptivity array; CD44 and RHAMM: primary hyaluronan surface receptors; RCT: randomized clinical trials; LBR: life birth rate; CPR: clinical pregnancy rate; IR: implantation rate.

Comprehensive profiling of Small RNAs in human embryo-conditioned culture media by improved sequencing and quantitative PCR methods.

Embryo implantation depends on two primary factors: the quality of the embryo and endometrial receptivity. Small RNAs have been shown to be potent epigenetic regulators influencing cell proliferation, differentiation, and communication even in the context of early embryonic development. However, previous reports are limited to miRNAs and lack sensitivity. Here, we describe a platform for non-invasive small RNA biomarker discovery and validation from embryo-conditioned culture media (ECCM). We hypothesize that small non-coding RNAs (sncRNAs) are secreted by the embryo into the ECCM and test the limit of detection for profiling sncRNA by deep sequencing and quantitative PCR. In the first set of experiments, we evaluated sequencing sensitivity by comparing sncRNA profiles from pools of 10, 5, 3, and single ECCM drops. Next, we performed a similar test for TaqMan qPCR sensitivity by measuring select sncRNAs in 5, 3 and single drop ECCM pools. Finally, we compared the expression of an sncRNA panel by qPCR in single ECCM vs no-embryo control media . We report the first comprehensive sequencing of sncRNAs in ECCM with a sequencing sensitivity of 3 single embryo drops, capturing ~150 miRNAs and an abundance of tRNA-derived small RNAs (tsRNAs). We then profiled 15 sncRNAs by qPCR and determined that the assay maintains sensitivity in single ECCM drops. Finally, we found significant differences in these sncRNA expression between control and ECCM drops. Improving embryo selection is crucial for reducing time to pregnancy. Here we describe a sensitive technique for biomarker discovery by sequencing and qPCR validation in ECCM, demonstrating that the majority of sncRNAs are embryo derived. We also report an abundance of tsRNAs which suggests these sncRNAs may have functions in endometrial-maternal communication beyond the microRNAs which have been described previously.Abbreviations: PGT-A: Preimplantation genetic testing for aneuploidies; ECCM: Embryo-conditioned culture media; sncRNAs: Small non-coding RNAs; miRNAs: microRNAs; EVs: Extracellular vesicles; PCA: Principal component analysis.

Transcriptomics of cumulus cells - a window into oocyte maturation in humans.

BACKGROUND: Cumulus cells (CC) encapsulate growing oocytes and support their growth and development. Transcriptomic signatures of CC have the potential to serve as valuable non-invasive biomarkers for oocyte competency and potential. The present sibling cumulus-oocyte-complex (COC) cohort study aimed at defining functional variations between oocytes of different maturity exposed to the same stimulation conditions, by assessing the transcriptomic signatures of their corresponding CC. CC were collected from 18 patients with both germinal vesicle and metaphase II oocytes from the same cycle to keep the biological variability between samples to a minimum. RNA sequencing, differential expression, pathway analysis, and leading-edge were performed to highlight functional differences between CC encapsulating oocytes of different maturity. RESULTS: Transcriptomic signatures representing CC encapsulating oocytes of different maturity clustered separately on principal component analysis with 1818 genes differentially expressed. CCs encapsulating mature oocytes were more transcriptionally synchronized when compared with CCs encapsulating immature oocytes. Moreover, the transcriptional activity was lower, albeit not absent, in CC encapsulating mature oocytes, with 2407 fewer transcripts detected than in CC encapsulating immature (germinal vesicle - GV) oocytes. Hallmark pathways and ovarian processes that were affected by oocyte maturity included cell cycle regulation, steroid metabolism, apoptosis, extracellular matrix remodeling, and inflammation. CONCLUSIONS: Herein we review our findings and discuss how they align with previous literature addressing transcriptomic signatures of oocyte maturation. Our findings support the available literature and enhance it with several genes and pathways, which have not been previously implicated in promoting human oocyte maturation. This study lays the ground for future functional studies that can enhance our understanding of human oocyte maturation.

Ultrastructural identification of CD9 positive extracellular vesicles released from human embryos and transported through the zona pellucida.

Extracellular vesicles (EVs) are highly specific and multi-purpose vesicular structures that are released by various cell and tissue types in the body. However, the secretion of EVs from mammalian embryos, especially human, has not been well characterized. Thus, the aim of this study was to 1) identify EVs in human preimplantation embryos at different stages of their development using scanning and electron microscopy, and 2) investigate whether EVs can cross the zona pellucida (ZP) and be released from human embryos cultured in vitro. Human oocytes, zygotes, cleavage embryos and blastocysts donated for research were labeled with the tetraspanin EV marker CD9 and analyzed by scanning and transmission electron microscopy. Embryo culture conditioned media collected 3- and 5-days post fertilization were examined for the presence of EVs using electron microscopy. We detected numerous CD9 positive vesicles released from all embryos examined. They were observed on the surface of the plasma membrane, within the perivitelline space as well as throughout the zona pellucida. Interestingly, EVs were not seen in the ZP of all mature metaphase II oocytes, however, were detected just after fertilization in the ZP of zygotes and embryos. Electron microscopy using negative staining, and nanoparticle tracking analysis (NTA) of embryo conditioned culture media also showed the presence of vesicles of various sizes, which were round shaped, and had a lipid bilayer. Their size ranged from 30 to 500 nm, consistent with the sizes of exosomes and microvesicles. In conclusion, the results of the study provide evidence that human preimplantation embryos at all developmental stages secrete EVs into the perivitelline space, which then traverse through the ZP, and are then released into the surrounding culture medium. Abbreviations: EVs: extracellular vesicles; ZP: zona pellucida; CD9, CD63, and CD81: tetraspanin EV markers; NTA: nanoparticle tracking analysis; ESCRT: endosomal sorting complexes required for transport; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TE: trophectoderm; ICM: inner cell mass; PVS: perivitelline space; MI: metaphase I; MII: metaphase II; GV: germinal vesicle; MVs/EXs: microvesicles/exosomes; hCG: human chorionic gonadotrophin; GnRH: gonadogrophin releasing hormone; ICSI: intracytoplasmic sperm injection; SPS: serum protein substitute; 1PN: one pronuclear zygote; 3PN: tri-pronuclear zygote; IgG: immunoglobulin G; PBS: phosphate buffer saline; ETHO: ethanol; ESED: Environmental Secondary Electron Detector; BSA: bovine serum albumin.

Impact of multinucleated blastomeres on embryo developmental competence, morphokinetics, and aneuploidy.

OBJECTIVE: To study the effect of human embryo multinucleation on the rate of aneuploidy, in vitro developmental morphokinetics, and pregnancy outcome. DESIGN: Retrospective study. SETTING: University-affiliated fertility center. PATIENT(S): A total of 296 patients undergoing IVF cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rate of multinucleation at the 2- and 4-cell stage, time-lapse morphokinetic parameters from zygote to blastocyst stage, ploidy of embryos analyzed by means of trophectoderm biopsy and array comparative genomic hybridization (PGS), and pregnancy outcome. RESULT(S): A total of 1,055 out of 2,441 (43.2%) embryos evaluated with the use of the Embryoscope time-lapse system showed blastomere multinucleation at the 2-cell stage (MN2). The frequency of this abnormality was substantially reduced in 4-cell-embryos (15.0%). Among all clinical factors analyzed, only maternal age had a positive correlation with multinucleation rate. The timing of cleavage divisions from the pronuclear fading to 5-cell embryo was significantly longer (1.0-2.5 h) in MN2 embryos than in non-MN2 control samples. Of the total embryos tested with the use of PGS (n = 607), the rates of multinucleation were similar in euploid versus aneuploid blastocysts (40.8% and 46.7%, respectively). All 24 chromosomes contributed to aneuploidy of MN2 embryos. There were 61 transfers of MN2 embryos that resulted in 45.9% clinical pregnancies and a 31.6% implantation rate. CONCLUSION(S): The frequency of multinucleation is high in human embryos cultured in vitro and equally affects euploid and aneuploid human embryos. It appears that most MN embryos have the capacity for self-correction during early cleavage divisions and can develop into euploid blastocysts resulting in healthy babies.

Secretion of human leukocyte antigen-G by human embryos is associated with a higher in vitro fertilization pregnancy rate.

OBJECTIVE: To investigate whether secretion of soluble human leukocyte antigen-G (HLA-G) by human embryos is associated with embryo development and IVF pregnancy outcome. DESIGN: Retrospective study. SETTING: In vitro fertilization program affiliated with a university research center. PATIENT(S): Infertile couples attending an IVF program were selected. INTERVENTION(S): Embryo culture conditioned medium (72 hours) from cases in which intracytoplasmic sperm injection was used for fertilization. MAIN OUTCOME MEASURE(S): Soluble HLA-G in embryo culture medium samples from IVF patients was assayed and associations between soluble HLA-G secretion and outcome measures were analyzed. RESULT(S): Two hundred seventy of 386 samples had detectable soluble HLA-G. Soluble HLA-G secretion was independent of embryo grade or patients' age. The cleavage rate of embryos secreting soluble HLA-G was significantly higher than that of those lacking it (blastomere number 6.71 +/- 0.09 vs. 5.86 +/- 0.22). The live birth rate from embryos with soluble HLA-G was significantly higher than that of those without (48.4% vs. 17.1%, chi(2) = 9.09). Combining soluble HLA-G detection and cleavage rate was most predictive of pregnancy. CONCLUSION(S): Our five conclusions are as follows: [1] embryonic secretion of soluble HLA-G protein is variable, [2] secretion of HLA-G is correlated with a higher cleavage rate, [3] secretion of HLA-G is associated with a higher pregnancy rate, [4] HLA-G secretion is a better independent predictor than cleavage rate alone, and [5] the combination of soluble HLA-G detection and high cleavage rate was the best predictor of outcome.

Sperm content of postacrosomal WW binding protein is related to fertilization outcomes in patients undergoing assisted reproductive technology.

OBJECTIVE: To determine the levels of postacrosomal WW binding protein (PAWP) in the spermatozoa of men that were used clinically for intracytoplasmic sperm injection (ICSI) and to correlate them with infertility treatment outcomes. DESIGN: Prospective clinical and laboratory study. SETTING: University-based laboratory and infertility clinic. PATIENT(S): Men undergoing ICSI for the treatment of couples' infertility (n=110). INTERVENTION(S): Quantitative analysis of sperm PAWP levels by flow cytometry and developmental analysis of PAWP expression by immunoblotting, immunofluorescence, and immunohistochemistry. MAIN OUTCOME MEASURE(S): PAWP flow-cytometric levels and immunolocalization in spermatozoa. RESULT(S): A strong positive correlation was found between PAWP expression levels and fertilization rates after ICSI, with high levels of PAWP being associated with higher fertilization rates; the positive correlation was independent of age, DNA fragmentation index, and other sperm parameters. PAWP expression levels were correlated with embryonic development, with high levels of PAWP being associated with a lower number of arrested embryos within 3-5 days post-ICSI. PAWP expression was detected during the late stages of human spermiogenesis in elongating spermatids, confirming previous findings in various animal models. CONCLUSION(S): Our clinical data from infertile couples demonstrate significant correlations between sperm PAWP levels and both fertilization rates and normal embryonic development after ICSI. Considering its proposed role in the initiation of oocyte activation, we suggest that PAWP could have potential applications in the diagnosis and treatment of infertility.

Expression of survivin in human oocytes and preimplantation embryos.

OBJECTIVE: To determine whether [1] survivin is expressed in human oocytes and embryos; [2] embryos grown in vitro secrete survivin protein; and [3] survivin levels are correlated with embryo cleavage rates. DESIGN: Experimental. SETTING: University-affiliated IVF clinic. PATIENT(S): Couples undergoing IVF-ET cycles. INTERVENTION(S): Conventional reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, immunohistochemistry, Western blot on oocytes, embryos and control choriocarcinoma JEG-3 cells, and ELISA analysis of conditioned culture media. MAIN OUTCOME MEASURE(S): Detection of survivin mRNA and protein in oocytes and preimplantation embryos and in JEG-3 cancer cells. Detection of survivin concentrations in embryo culture media. RESULT(S): Survivin mRNA and protein were expressed during human oocyte maturation, from germinal vesicle to metaphase II stage, and throughout embryo development, from pronuclear stage to blastocyst stage. Survivin was localized predominantly in the cytoplasm of all cells examined and in the oocytes on the chromatin of metaphase chromosomes and midbodies. Western blot analysis of human oocyte and cancer cell extracts detected a full-length (primary) survivin band of 16.5 kDa. Survivin was also detected in conditioned media samples from embryo cultures and showed a positive correlation with embryo cleavage rates. CONCLUSION(S): Our data have demonstrated for the first time that human oocytes/embryos not only express but also secret survivin, suggesting that survivin may play an important role in human oogenesis and embryogenesis.

Is the zona pellucida thickness of human embryos influenced by women's age and hormonal levels?

OBJECTIVE: To evaluate whether zona pellucida thickness (ZPT) of human embryos is correlated with maternal age, patient's hormonal status, embryo quality, and IVF outcomes. DESIGN: Prospective study. SETTING: University-affiliated IVF clinic. PATIENT(S): Couples undergoing IVF-ET cycles. INTERVENTION(S): Zona measurements, clinical data collection. MAIN OUTCOME MEASURE(S): Correlation between the ZPT and maternal age, basal FSH and E(2) levels, stimulation protocols, cause of infertility, embryo quality, and implantation/pregnancy rates. RESULT(S): The measurements of ZPT were collected from 5,184 day 3 human embryos originated from 744 IVF patients. The overall mean ZPT was 16.18 ± 2.00 µm. No significant correlation was observed between the ZPT and the patient's age, E(2) values on the day of hCG administration, basal concentration of serum FSH, stimulation protocol, infertility diagnosis, and implantation/pregnancy rates. The ZPT was strongly influenced only by the embryo quality: Embryos with good morphology exhibited considerably thinner ZP compared with those of less favorable morphology (mean 15.87 ± 2.48 µm vs. 16.36 ± 2.57 µm, respectively). The ZPT had no significant impact on the implantation and pregnancy rates. CONCLUSION(S): The thickness of the human ZP of day 3 embryos is not influenced by women's age and hormonal levels. The strong correlation between ZPT and embryo quality suggests that thickness of ZP depends on inherent embryo properties. The overall ZPT is not a good predictive indicator for IVF clinical outcomes.

Laser zona thinning in women aged

The objective of this prospective randomized double-blind clinical trial was to evaluate whether laser zona pellucida thinning of human embryos improves clinical outcomes in women

Time-dependent capability of human oocytes for activation and pronuclear formation during metaphase II arrest.

BACKGROUND: The purpose of this study was to investigate the fertilization rate and developmental potential of human oocytes in relation to the duration of their metaphase II (MII) arrest stage following the extrusion of the first polar body (1PB). METHODS: Immature metaphase I oocytes (MI; study oocytes, n = 468) that underwent meiotic maturation during brief in vitro culture and their matured in vivo, MII siblings (control oocytes, n = 3293) were subjected to ICSI. Fertilization and early cleavage were evaluated in both study and control groups. RESULTS: The overall fertilization rate was significantly lower in the oocytes matured in vitro than in those matured in vivo (42 versus 77%, P < 0.0001). A significant relationship was observed between oocyte activation potential and the length of MII arrest. The majority of study oocytes injected soon after PB extrusion remained unfertilized (64%; 98/154 oocytes). The proportion of normally activated oocytes that contained two pronuclei and two PBs gradually increased with prolonged time of MII arrest (43 and 61% at 2 and 3-6 h after 1PB extrusion). Significantly more embryos originating from the study than control oocytes were arrested soon after the first two cleavage divisions (39 and 17%; P < 0.0001) and exhibited multinucleated blastomeres (23 and 13%; P < 0.0001), which suggests the existence of chromosomal abnormalities. CONCLUSIONS: Human oocytes progressively develop the ability for full activation and normal development during the MII arrest stage.

Calcium-binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos.

BACKGROUND: The study aim was to investigate the presence and localization of Ca2+-binding proteins and Ca2+-release receptor channels in human maturing oocytes, pronuclear zygotes and preimplantation embryos. METHODS: Immunocytochemical analysis, using specific antibodies against the proteins being studied, followed with confocal laser microscopy, was performed on human oocytes and embryos. RESULTS: Calreticulin and calsequestrin (the two major calcium storage proteins of somatic cells), two types of calcium release receptors, the inositol trisphosphate and ryanodine receptors (InsP(3)R-2, RyRs-1,2,3), and the molecular chaperone, calnexin, were identified in all investigated cell types. Calreticulin was predominant in the cell cortex and in the nuclear envelope, while calsequestrin was distributed throughout the entire cytoplasm. Generally, localization of the InsP(3)R-2 and RyRs was similar to that of calreticulin and calsequestrin respectively. Both types of receptor were enriched in the subplasmalemmal region of meiotic oocytes. In addition, the InsP(3)R was detected in the nuclear structures of oocytes and blastomeres. Calnexin distribution overlapped with that of calreticulin but appeared to be present in distinct subcompartments. CONCLUSIONS: Human oocytes and embryos express the calcium sequestration and release proteins in highly organized and developmentally regulated patterns. Fine-tuning of these proteins may play a crucial role in regulation of Ca2+ transience during oocyte maturation, fertilization and early embryo development.

Morphological and cytogenetic analysis of human giant oocytes and giant embryos.

BACKGROUND: Giant binuclear oocytes occur with considerable frequency in human ovaries, but their ultimate fate remains unknown. We report the morphology, cytogenetics and developmental potential of human giant oocytes from patients undergoing assisted reproductive technologies. METHODS AND RESULTS: A total of 44 giant oocytes was collected from patients aged 22-44 years old, with an overall frequency of 0.3% (44/14 272 oocytes). Giant oocytes were approximately 30% larger in diameter than normal oocytes (mean 200.4 versus 154.7 micro m, P = 0.0001). Two different morphological patterns were observed among giant unfertilized and fertilized oocytes. All unfertilized oocytes appeared to be diploid and contained either one or two metaphase plates (46 or 2 x 23 chromosomes), and one or two polar bodies respectively. Consequently, fertilized giant oocytes exhibited either two or three pronuclei, or two or four polar bodies. Both types of giant zygotes were capable of normal cleavage and development to blastocyst stage. Four giant embryos were analysed by interphase fluorescence in-situ hybridization using probes for chromosomes 9, 22, X and Y, and all appeared chromosomally abnormal with numerical alterations indicative of ploidy change. CONCLUSIONS: Giant oocytes might be a possible source of human digynic triploidy. To avoid undesired miscarriages, giant embryos originated from either two- or three-pronuclear giant zygotes should be excluded from uterine transfers.