Mature MII Oocytes Research Articles

Comparing the effect of sitagliptin and metformin on the oocyte and embryo quality in classic PCOS patients undergoing ICSI.

Insulin resistance plays a major role in the pathogenesis of polycystic ovary syndrome (PCOS). Therefore, there is a growing interest in the use of insulin sensitizer drugs in the treatment of PCOS. Research in recent years has shown that sitagliptin has been reported to improve ovarian cycles and ovulation in PCOS patients. We aimed to compare the effects of metformin and sitagliptin on PCOS individuals undergoing ICSI. Sixty PCOS patients were divided into 3 groups: metformin, sitagliptin, and placebo group. Treatment was carried out 2months before the start of the ovulation cycle and continued until the day of oocyte aspiration. The serum levels of total testosterone, estradiol, and fasting insulin along with the total number of retrieved, normal and abnormal MII, and fertilized oocytes, the number of transferred embryos (grades I, II and III), and biochemical and clinical pregnancy rates as well as the ovarian hyperstimulation syndrome (OHSS) were evaluated. There was a significant reduction in the serum levels of Insulin and total testosterone in the treated groups compared with the placebo. The number of mature and normal MII oocytes increased significantly in the treated groups compared with the placebo. Moreover, the number of immature oocytes decreased significantly and the number of grade I embryos increases significantly in the sitagliptin group compared with the placebo group. We conclude that sitagliptin can improve the maturation of oocytes and embryos' quality more effectively than metformin, in PCOS patients undergoing ICSI. Trial registration is NCT04268563 ( https://clinicaltrials.gov ).

Long noncoding RNA 2193 regulates meiosis through global epigenetic modification and cytoskeleton organization in pig oocytes.

Long noncoding RNAs (lncRNAs) regulate a variety of physiological and pathological processes. However, the biological function of lncRNAs in mammalian germ cells remains largely unexplored. Here we identified one novel lncRNA (lncRNA2193) from single-cell RNA sequencing performed on porcine oocytes and investigated its function in oocyte meiosis. During in vitro maturation (IVM), from germinal vesicle (GV, 0 hr), GV breakdown (GVBD, 24 hr), to metaphase II stage (MII, 44 hr), the transcriptional abundance of lncRNA2193 remained stable and high. LncRNA2193 interference by small interfering RNA microinjection into porcine GV oocytes could significantly inhibit rates of GVBD and the first polar body extrusion, but enhance the rates of oocytes with a nuclear abnormality. Moreover, lncRNA2193 knockdown disturbed cytoskeletal organization (F-actin and spindle), and decreased DNA 5-methylcytosine (5mC) and histone trimethylation (H3K4me3, H3K9me3, H3K27me3, and H3K36me3) levels. The lncRNA2193 downregulation induced a decrease of 5mC level could be partially due to the reduction of DNA methyltransferase 3A and 3B, and the elevation of 5mC-hydroxylase ten-11 translocation 2 (TET2). After parthenogenetic activation of MII oocytes, parthenotes exhibited higher fragmentation but lower cleavage rates in the lncRNA2193 downregulated group. However, lncRNA2193 interference performed on mature MII oocytes and parthenotes at 1-cell stage did not affect the cleavage and blasctocyst rates of pathenotes. Taken together, lncRNA2193 plays an important role in porcine oocyte maturation, providing more insights for relevant investigations on mammalian germ cells.

Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes

This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes’ abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.

Combined Use of Autologous Bone Marrow-derived Stem Cells and Platelet-rich Plasma for Ovarian Rejuvenation in Poor Responders.

Background:The management of poor responders is still a challenge in modern-assisted reproductive technology. Several researches are showing encouraging results with autologous bone marrow-derived stem cells (ABMDSCs) and platelet-rich plasma (PRP) individually. Hence, we decided to study the synergistic effect of ABMDSCs with PRP.Aims and Objective:The aim of the study was to assess the safety and efficacy of intraovarian instillation of ABMDSCs combined with PRP in poor responders.Design:This was an interventional pilot study. Study Period: January 2017 to January 2019.Materials and Methods:We designed a pilot study using Patient-oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON) Group 3 and 4 poor responder patients (n = 20). The study group underwent laparoscopic/transvaginal intraovarian instillation of ABMDSCs combined with PRP and the outcome was analyzed – primary outcome – antral follicular count (AFC) and mature MII oocytes and secondary outcome – Anti-Mullerian hormone (AMH) levels and number of Grade A and B embryos frozen on day 3. The Wilcoxon signed-rank test and Pearson correlation were used for the statistical analysis and P < 0.05 was considered statistically significant.Results:After 6 weeks of intraovarian instillation ABMDSCs mixed with PRP, patients were reassessed for AFC and AMH and their response to subsequent controlled ovarian stimulation (COS) cycle was observed. Statistically significant improvement was seen in AFC, MII oocytes, and Grade A and Grade B embryos. AMH was also increased in some patients, but the result was not statistically significant.Conclusion:Our results suggest that intraovarian instillation of ABMDSCs combined with PRP is safe and it optimized the recruitment of existing dormant primordial follicles to improve oocyte yield and hence the number and quality of embryos after COS in POSEIDON Group 3 and 4 poor responders.

What is the true place of a double stimulation and double oocyte retrieval in the same cycle for patients diagnosed with poor ovarian reserve? A systematic review including a meta-analytical approach.

This systematic review including a meta-analytical approach aims to investigate the safety and efficacy of employing a double ovarian stimulation (DuoStim) and a subsequent double oocyte retrieval in the same menstrual cycle, in poor ovarian reserve (POR) patients. A systematic search of literature was performed in the databases of PubMed/MEDLINE, Embase, and Cochrane Central Library up until March 2019. Both prospective and retrospective cohort studies considered suitable for inclusion reported on women with POR undergoing a DuoStim in the follicular (FPS) and luteal phase (LPS) of the same menstrual cycle. Following the systematic review of the literature, a meta-analytical approach was attempted. This study indicates that DuoStim is correlated with a higher number of retrieved oocytes, mature MII oocytes, and good-quality embryos in comparison to conventional stimulation. Additionally, LPS seems to be correlated with an equal or an even higher overall performance in comparison to FPS. DuoStim favors an enhanced clinical outcome in regard to the total number of yielded oocytes, mature oocytes, and available embryos, along with the quality of obtained embryos. Sourced data indicate that LPS is not correlated with a higher aneuploidy rate. This option may present as promising for the time-sensitive nature of POR patients' management, by enabling a higher oocyte yield during a single menstrual cycle.

A novel zona-free culture system for severe cytoplasmic fragmentation cases: a pilot study using 3PN embryos and time-lapse cinematography

A study in 2017 observed perivitelline threads in more than 50% of cleavage-stage human embryos using time-lapse imaging, and the rate of cytoplasmic fragmentation (at the first cleavage) was significantly decreased in embryos without perivitelline threads (P < 0.001). While it is proposed that perivitelline threads play an important role in crosslinking the cumulus cells and oocyte during maturation, the mechanism underlying such a role remains unclear. It is also unknown whether the threads still function in mature MII oocytes. Therefore, in this study, zona pellucida of abnormally-fertilized oocytes which were donated by patients was removed at pronuclear stage. Those zona-free oocytes were observed in time-lapse culturing system in order to examine developmental morphology. Prospective study. This study used 57 abnormally fertilized (3PN) embryos (cIVF: n = 51, ICSI: n = 6) donated by assisted reproduction technology patients in our clinic with informed consent since 2017. After confirming the three pronuclei, we removed the ZP from each 3PN embryo using a laser, and the resultant zona-free embryos were cultured and observed in an incubator equipped with a time-lapse imaging system. For ZP removal, 3PN embryos were placed in drops of 0.125M sucrose-containing HEPES media that had been covered with mineral oil and warmed to 37°C. Despite a small reduction in ooplasm size, half of the ZP was removed by laser （Saturn 5; Origio、Lykos; Hamilton Thorne). Subsequently, the ooplasms were completely separated from their ZPs by pipetting, and these zona-free 3PN embryos were cultured continuously for 5 days with time-lapse imaging. Of 58 zona-free embryos in total, 54 (94.7%) were cleaved, and there was no significant decrease in cleavage rate compared to 2PN embryos (98%) used routinely in our clinic. Furthermore, 28 of the 54 embryos (51.9%) developed to the morula stage after third cleavage, and 18 embryos (33.3%) formed a blastocoel and became blastocysts. Thus, removing the ZP before cleavage did not adversely affect the embryo development. In terms of the amount of fragmentation, based on the modified Veeck’s criteria, 36 of 54 zona-free 3PN embryos (66.7%) showed less than 20% of the volume in fragments compared to the total volume of cytoplasm at the first cleavage (Grade 1 and 2), 14 (25.9%) showed 20-40% fragments (Grade 3), and only 4 (7.4%) showed > 40% fragments (Grade 4). These results suggested that the rate of fragmentation was decreased by ZP removal before the first cleavage. This study revealed that the ZP is not always necessary for normal development after the pronuclear stage because the zona-free embryos studied herein developed normally, maintained their cell adhesion well, and showed a decreased rate of fragmentation. This innovative culture system might provide the major breakthrough needed for patients who have difficulty obtaining good-quality embryos.

Altered cytoplasmic maturation in rescued in vitro matured oocytes.

Do culture conditions affect cytoplasmic maturation in denuded immature non-GV oocytes? The maturation rate of denuded non-GV oocytes is not affected by culture media, but in vitro maturation seems to alter the mitochondrial membrane potential, endoplasmic reticulum (ER) and actin cytoskeleton compared with in vivo maturation. In vitro maturation of denuded immature non-GV oocytes benefits cycles with poor in vivo MII oocyte collection, but maturation levels of non-GV oocytes are only scored by polar body extrusion. Since oocyte maturation involves nuclear as well as cytoplasmic maturation for full meiotic competence, further knowledge is needed about cytoplasmic maturation in in vitro culture. This basic research study was carried out between January 2017 and September 2018. A total of 339 denuded immature non-GV oocytes were cultured in SAGE 1-Step (177) or G-2 PLUS (162) for 6-8h after retrieval, and 72 in vivo matured MII oocytes were used as controls. Cultured immature non-GV oocytes were scored for polar body extrusion and analysed for mitochondrial membrane potential (ΔΨm), ER clusters, cortical granules number and distribution, spindle morphology and actin cytoskeleton organization. The obtained parameter values were compared to in vivo matured MII oocyte parameter values. The maturation rates of oocytes cultured in G-2 PLUS and SAGE 1-Step were similar (65% vs 64.2%; P= 0.91). The differences observed in cortical granule density were not statistically significant. Also spindle morphometric parameters were mostly similar between in vitro and in vivo matured MII oocytes. However, the number of ER clusters, the ΔΨm and the cortical actin thickness showed significant differences between in vivo MII oocytes and denuded immature non-GV oocytes cultured in vitro until meiosis completion. Frozen-thawed oocytes together with fresh oocytes were used as controls. Due to technical limitations (fixation method and fluorochrome overlap), only one or two parameters could be studied per oocyte. Thus, a global view of the maturation status for each individual oocyte could not be obtained. Characterization of in vitro matured oocytes at the cellular level will help us to understand the differences observed in the clinical outcomes reported with rescue IVM compared to in vivo MII oocytes and to improve the culture methods applied. This work was supported by intramural funding of Clinica Eugin and by the Torres Quevedo Program to A.F.-V. from the Spanish Ministry of Economy and Competitiveness. No competing interests are declared.

PO-01-080 Intracytoplasmic Sperm Injection (ICSI) Outcome Using Cryopreserved Testicular Sperm from Non-Obstructive Azoospermic Infertile Men with Varicocele

To study the outcome of ICSI using cryopreserved testicular sperm from infertile men with varicocele-associated non-obstructive azoospermia (NOA). The study included 40 infertile men with NOA who underwent ICSI with cryopreserved testicular sperm. Twenty two patients had clinically palpable varicocele (group 1) and 18 had no detectable abnormality in their genital examination (group2). Diagnosis of varicocele was confirmed by scrotal color Doppler ultrasound. Diagnosis of azoospermia was confirmed by repeated absence of sperms in centrifuged semen pellets. Men with abnormal hormonal profiles, genetic abnormalities or genitourinary anomalies suggestive of obstructive pathology were excluded. Couples with known female factor infertility were excluded. ICSI was done using mature MII oocytes with good morphology. The pronuclear formation was assessed at 17 ± 1 hours following injection. Embryo quality was assessed on day 3 (67 ± 2 hours) and day 5. Positive clinical pregnancy was considered upon finding of gestational sac(s) and fetal pulsation by trans-vaginal US, 15 days after BHCG assessment. Results were presented as mean ± standard deviation (SD) for continuous variables and frequency and percentage (%) for categorical variables. P-value <0.05 was significant.

Pregnancy following exclusion testing for SCA3 on polar bodies in a fresh cycle: a comparison of STR-based targeted haplotyping and genome-wide SNP analysis and meiomapping

Introduction Preimplantation genetic testing of monogenic disease (PGT-M) is allowed in Germany only with approval from the federal ethics committee and autosomal dominant disorders are often rejected. Polar body analysis, however, does not require ethical approval. A 34-year-old asymptomatic patient with a family history of spinocerebellar ataxia type 3 (SCA3) applied for PGT-M on trophectoderm samples but was rejected. The patient's father has a repeat expansion in the ATXN3 gene and is affected with SCA3, a progressive autosomal dominantly inherited neurological disease characterized by ataxia, spasticity and ocular movement abnormalities. As the patient does not want to know her status, we offered exclusion testing based on polar body analysis in a fresh cycle and have compared targeted haplotyping by STR analysis with genome-wide SNP genotyping and meiomap analysis. Material and methods The patient and her father were genotyped for 12 STR markers within 0.44 Mb of the ATXN3 gene on 14q32. 11 STRs were heterozygous (9 fully informative) and used for phasing high and low risk haplotypes. Eggs were collected (Day 0) and the first polar body (PB1) biopsied from mature MII oocytes. ICSI was then performed and PB2 biopsied from three fertilised zygotes on Day 1. After whole genome amplification (WGA) (Repli-g Single Cell Kit; Qiagen, Venlo, Netherlands) multiplex PCR was performed and the products run on a capillary sequencer. Haplotypes were constructed based on the pre-case workup. In addition, genome-wide SNP genotyping of the DNAs from the patient, her father and the WGA products from the polar bodies was carried out (HumanKaryomap-12 SNP array; Illumina, San Diego, USA) for maternal haplotyping and meiomap analysis. Results WGA and multiplex PCR was successful for all of the polar bodies except for the PB2 of Oocyte 1. As all 11 closely linked informative STRs were homozygous for the high-risk haplotype in PB1 from this oocyte, the corresponding PB2 and oocyte was deduced to have the low risk haplotype assuming normal segregation during meiosis. For Oocytes 2 and 3, the PB1 was heterozygous for most STRs and the PB2 of these oocytes were homozygous for the high and low risk haplotypes, respectively. Therefore, the embryos derived from Oocytes 1 and 2 were deduced to have the low risk maternal haplotype while the embryo from Oocyte 3 should have the high-risk haplotype. On Day 5, the blastocysts arising from Oocytes 1 and 2 were transferred, resulting in an ongoing pregnancy (currently week 10). Genome-wide SNP genotyping and meiomap analysis was concordant with STR analysis in all cases. Conclusions We report a pregnancy following targeted STR-based haplotyping and exclusion testing of an autosomal dominant disease by polar body analysis in a fresh cycle. Genome-wide SNP genotyping and meiomap analysis was fully concordant with the STR results but had the advantage that it also demonstrated that segregation of the maternal chromosome 14 was normal and predicted that the embryos from the two oocytes in which both polar bodies were analysed were euploid for all maternal chromosomes.

Impact of sperm DNA fragmentation on ICSI outcome and incidence of apoptosis of human pre-implantation embryos obtained from in vitro matured MII oocytes

BackgroundSperm DNA integrity and oocyte quality significantly affect embryo development and survival. The current study evaluated embryo development and quality, as well as the expression level of apoptosis-related genes and microRNAs in embryo derived from in vitro matured MII oocytes according to sperm DNA fragmentation (SDF) level. MethodsThe semen and immature oocytes were collected from 50 ICSI cycles with any recognizable female factor infertility. After ovarian stimulation, germinal vesicle stage (GV) oocytes were collected and incubated in in vitro maturation (IVM) medium for 24 h. Next, reactive oxygen species (ROS) level of media culture was determined. Using by sperm chromatin dispersion (SCD) test, the SDF levels of processed semen were assessed and categorized into SDF ≤ 30% and SDF>30%. Seventy two hours after intracytoplasmic injection, the embryo development and quality score were recorded in the groups I (GV-MII + SDF≤ 30%) and II (GV-MII + SDF> 30%). Also, the apoptosis incidence of embryos at morula stage was evaluated at molecular and cellular levels by quantitative real time PCR and TUNEL staining, respectively. ResultsCleavage rate did not differ between two groups. The quality score of embryos obtained from IVM matured oocytes and high level of SDF was significantly lower than that of low level of SDF (P < 0.05). The embryos from group II had a significant reduction of the expression of BCL-2 compared to group I (P < 0.05). Also, they showed an increase in relative transcription of pro-apoptotic microRNAs; miR 15a and miR 16-1 versus group I (P < 0.05). A rise of TUNEL positive blastomers of embryo was observed at group II versus group I, but it did not reach to significantly level. ConclusionThe IVM oocytes, probably, did not suffice to recover the high level of paternal genomic damage and inhibition of apoptosis pathway beginning.

Compensating human subjects providing oocytes for stem cell research: 9-year experience and outcomes.

Human oocytes are arguably one of the most important cell types in humans, yet they are one of the least investigated cells. Because oocytes are limited in number, the use of high-quality oocytes is almost entirely in reproduction. Furthermore, regulatory hurdles for research on gametes and regulations on funding related to research on gametes present significant obstacles to research and the advancement of reproductive treatments. Here we report the outcomes of the largest compensated oocyte donation program for research in the USA to date, and probably worldwide. Women who participated in oocyte donation for research between 2008 and 2017 were contacted in a phone interview and completed a standardized questionnaire. Of 114 participants, 98 oocyte donors completed donation, donating 1787 mature MII oocytes and a total of 86 skin biopsies. Complication rate, including minor complications, of oocyte donation was 8/98, or 8.1%, for which two involved follow-up. Fifty-seven donors answered questions about their experience. Participants were incentivized primarily by money and a desire to help others and reported an overall favorable experience. Most, but not all, human subjects recalled that they had donated for research, and approximately half recalled that their oocytes were being used specifically for stem cell research. Compensated oocyte donation provides a reliable path to obtaining high-quality oocytes for research and is reviewed favorably by oocyte donors. The continuation of programs that offer compensation for oocyte donation is invaluable to continued progress and advancements in stem cell research and human embryology, and for the advancement of novel reproductive treatments.

A retrospective analysis of adverse effects of an in vivo fluoroquinolone antibiotic enrofloxacin treatment on oocyte quality in the common marmoset

Here we report a retrospective analysis of negative effects of routine enrofloxacin treatment of recurrent diarrhea on the ovary and the developing oocytes of the common marmoset, a small New World primate. The most deleterious effect on oocytes was observed about two months post treatment suggesting that the enrofloxacin effect is on early growing follicles. Manifestations of toxicity included decreased numbers of growing follicles and recovered culturable oocytes, as well as signs of early atresia of granulosa cells. In addition, increased amounts of holed stroma after treatment strongly suggested increased death of the early growing follicles. Of the oocytes judged to be of adequate quality for culture, maturation rates were not affected but fertilization of in vitro matured MII oocytes and subsequent cleavage rates were severely reduced in the enrofloxacin treated animals. Further, the arrested oocytes, which failed to mature or fertilize, showed obvious meiotic spindle abnormalities.

Stage-Specific Profiling of Transforming Growth Factor-β, Fibroblast Growth Factor and Wingless-int Signaling Pathways during Early Embryo Development in The Goat.

This research intends to unravel the temporal expression profiles of genes in- volved in three developmentally important signaling pathways [transforming growth factor-β (TGF-β), fibroblast growth factor (FGF) and wingless/int (WNT)] during preand peri-implan- tation goat embryo development. In this experimental study, we examined the transcripts that encoded the ligand, receptor, intracellular signal transducer and modifier, and the down- stream effector, for each signaling pathway. In vitro mature MII oocytes and embryos at three distinctive stages [8-16 cell stage, day-7 (D7) blastocysts and day-14 (D14) blas- tocysts] were separately prepared in triplicate for comparative real-time reverse tran- scriptase polymerase chain reaction (RT-PCR) using the selected gene sets. Most components of the three signaling pathways were present at more or less stable levels throughout the assessed oocyte and embryo developmental stages. The transcripts for TGF-β, FGF and WNT signaling pathways were all induced in unfertilized MII-oocytes. However, developing embryos showed gradual patterns of decrease in the activities of TGF-β, FGF and WNT components with renewal thereafter. The results suggested that TGF-β, FGF and WNT are maternally active signaling pathways required during earlier, rather than later, stages of preand peri- implantation goat embryo development.

Autologous oocyte cryopreservation in women aged 40 and older using minimal stimulation IVF.

BackgroundThe value of oocyte cryopreservation in older women remains controversial. The aim of this study was to report the oocyte freezing experience in women aged 40 and older at a single fertility center.FindingsOne hundred fifty eight women (mean age 43.9 ± 0.2) who underwent minimal ovarian stimulation IVF were enrolled. IVF protocol included the use of clomiphene citrate (50 mg/day) or letrozole (2.5 mg/day) with or without low dose gonadotropins (started at 75 IU/day and increased as needed to 150 IU/day). 584 retrieved oocytes (2.1 ± 0.15 per patient) yielded 532 mature MII oocytes that were frozen. After thawing and fertilization by ICSI, a total of 344 embryos (1.9 ± 0.1 per patient) were formed. A total of 57 relatively good embryos were transferred and yielded three live births (5.3 % per embryo transfer), three spontaneous abortions, and one chemical pregnancy.ConclusionsThese data are important in counseling older women who desire autologous oocyte freezing.

Developmental Regulated Expression of Anti- and Pro-Apoptotic BCL-2 Family Genes During Human Early Embryonic Development

Apoptotic cell death has been reported in human oocytes and preimplantation embryos under in vivo and in vitro conditions. BCL-2 family proteins comprise both anti- and pro-apoptotic members, which are likely to play a key role in controlling oocyte and early embryo survival. However, very limited data are available on their expression kinetics during human early embryonic development. Using our DNA microarray data, we analyzed the expression pattern of 21 BCL-2 family genes in human mature MII oocytes, day 3 embryos and day 5/6 blastocysts from patients who underwent in vitro fertilization (IVF). Selected genes were further validated by qRT-PCR and their subcellular localization analyzed by immunofluorescence confocal microscopy. Our results suggest a switch from oocyte-inherited BCL-2 family transcripts, such as BCL2L10, to embryo-produced transcripts after embryonic genome activation, including BIK, BCL2L11 and NOXA. Moreover, the pro-apoptotic gene BCL2L13 was constitutively expressed throughout human early embryonic development. Remarkably, day 3 embryos expressed more BCL-2 pro-apoptotic genes than mature MII oocytes and day 5/6 blastocysts, suggesting that embryos at this stage are more prone to apoptosis. This is further supported by an absence of cleaved Caspase-3 in the oocyte and its presence in the embryo. Using a drug that induces apoptosis (gambogic acid), we were able to show activated Caspase-3 in the oocyte in addition to an alteration of BCL2L13 protein localization. Similarly BCL2L13 localization was altered in degenerated oocytes. This study opens new perspectives for understanding the molecular regulation of human oocyte and pre-implantation embryo survival and death.

Small RNAs: new candidates for the regulation of the human cumulus-oocyte complex crosstalk

To identify and quantify small RNAs, including microRNAs, in human cumulus cells (CCs) and mature MII oocytes and to characterize the biological relationships between miRNAs and the messenger RNA expression profiles of MII oocytes and CCs. Mature MII oocytes and CCs were collected from women who underwent IVF/ICSI under COS. Using Illumina/deep-sequencing technology, we dissected the small RNAome of pooled mature MII oocytes and CCs (n=24 and n=20 respectively). The correlation between these microRNAs and their corresponding validated mRNA targets was investigated using in silico prediction algorithms. Using oligonucleotide microarrays, genome-wide gene expression was studied in mature MII oocytes or CCs. TaqMan miRNA assays were used to confirm the sequencing results. The functional roles of microRNAs, were validated in an in vitro system of primary cultures of human CCs. Deep sequencing of small RNAs yielded more than one million raw reads. We identified known microRNAs that were abundant in MII oocytes (MIR100 and MIR10A) or CCs (MIR29a, MIR30d, and the LET7 family). Predicted target genes of the oocyte miRNAs were associated with regulation of transcription and cell cycle, whereas, genes targeted by CC miRNAs were involved in extracellular matrix and apoptosis. Comparison of the predicted miRNA target genes and mRNA microarray data resulted in a list of 224 target genes that were differentially expressed in MII oocytes and CCs, including CTGF (fold: 38, p<0.0001) and BMPR1B (fold: 15.4, p<0.0001) that are important for cumulus-oocyte communication. This study provides the first characterization of the microRNA profile in human CCs and mature MII oocytes.. These results might help improving our understanding of the roles of miRNAs in oocyte maturation. Moreover, many of the identified miRNAs might be used as a tools to monitor the oocyte health, viability and competence and consequently to improve IVF/ICSI outcome.

Reproductive potential of mature MII oocytes following natural cycle IVF / minimal ovarian stimulation: analysis of 2876 treatment cycles from a single Japanese center

In previous studies oocyte utilization following conventional ovarian stimulation was found to be constantly low (<5%) and has declined even further (<1%) in >37 years-old women. Although it was suggested that mild ovarian stimulation could yield better quality eggs to date there are no large-scale analyses on the reproductive potential of MII oocytes following mild IVF.

High FSH decreases the developmental potential of mouse oocytes and resulting fertilized embryos, but does not influence offspring physiology and behavior in vitro or in vivo

Do different concentrations of FSH in the assisted reproductive technology (ART) procedure in vitro or in vivo affect the developmental competence of oocytes, the embryos and the offspring conceived from these embryos? Improper FSH treatment (200 IU/l in vitro, 10 IU/ml in vivo and 200 IU/ml in vivo) impairs the development competence of oocyte and embryo, but does not influence offspring physiology and behavior. Exogenous FSH has been widely used in the field of ART. However, the effects of different concentrations of FSH on the developmental competence of oocytes, embryos and the offspring conceived from these embryos, are still unknown. In a prospective study, a total of 45 mice at 8-10 weeks of age were primed in vivo with different dosages of FSH (9 mice in the 10 IU/ml, 10 mice in the 50 IU/ml, 10 mice in the 100 IU/ml and 16 mice in the 200 IU/ml groups). Fresh MII oocytes were retrieved from ovaries: this was designated as in vivo group. Thirty six mice at 8-10 weeks of age were sacrificed by cervical dislocation to obtain ovaries without FSH treatment (9 mice in the 0 IU/l, 9 mice in the 50 IU/l, 8 mice in the 100 IU/l and 10 mice in the 200 IU/l groups), and then the immature oocytes were collected from these ovaries and cultured in vitro matured medium supplemented with 0, 50, 100 and 200 IU/l FSH: this was designated as in vitro group. Spindle assembly of matured MII oocytes was stained via an immunofluorescence method and the oocytes ratio of normal spindle was analyzed. The developmental competence of the resulting fertilized embryos in the pre- and post-implantation stages was examined in in vitro and in vivo groups. Furthermore, physiological index, including reproductive potential and body weight, of the offspring was investigated by mating experiments and behavior index, including learning, memory, probing and intelligence, was tested by Morris water maze in in vitro and in vivo groups. In the in vitro groups, the oocyte maturation competence, normal spindle assembly, blastocyst formation and implantation, as well as viable pup production were all impaired in the group treated with 200 IU/l FSH (P < 0.05). No differences were observed among the other three groups (P > 0.05). In the in vivo groups, 10 IU/ml FSH but not 200 IU/ml treatment influenced blastocyst formation and viable pup production (P < 0.05), although the high proportion of spindle assembly abnormality was only observed in the 200 IU/ml FSH treatment group (P < 0.05). Furthermore, there were no significant differences in terms of physiological index (reproductive potential and body weight) and behavior index (learning, memory, probing and intelligence) in offspring from in vitro and in vivo groups (P > 0.05). The mouse model was used in this study. The results of the mouse follicle growth and oocyte development in responding to different concentrations of FSH are not 100% transferable to human, because of the physiological differences between mouse and human. The findings indicated that FSH application in the field of ART is safe to the resulted offspring, but it should be more carefully used for each women in ART cycles because the inappropriate FSH concentration would decrease the oocyte developmental competence. This work was partially supported by the Ministry of Science and Technology of China Grants (973 program; 2011CB944504), the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (30825038), the National Natural Science Funds for Young Scholar (31000661) and by the Joint Research Fund for Overseas, Hong Kong and Marco Scholars (31128013/C120205). None of the authors has any conflicts of interest.

Altered gene expression profile in cumulus cells of mature MII oocytes from patients with polycystic ovary syndrome

Oocyte developmental competence is altered in patients with polycystic ovary syndrome (PCOS); is gene expression in cumulus cells (CCs) from mature metaphase II oocytes of patients with PCOS altered as well? Compared with CCs from non-PCOS patients, the gene expression profile of CCs isolated from mature oocytes of patients with PCOS present alterations that could explain the abnormal folliculogenesis and reduced oocyte competence in such patients. Abnormal mRNA expression of several members of the insulin-like growth factor (IGF) family in CCs from PCOS patients was previously reported. Moreover, the whole transcriptome has been investigated in cultured CCs from PCOS patients. This retrospective study included six PCOS patients diagnosed following the Rotterdam Criteria and six non-PCOS patients who all underwent ICSI for male infertility in the assisted reproduction technique (ART) Department of Montpellier University Hospital, between 2009 and 2011. CCs from PCOS and non-PCOS patients who underwent controlled ovarian stimulation (COS) were isolated mechanically before ICSI. Gene expression profiles were analysed using the microarray technology and the Significance Analysis of Microarray was applied to compare the expression profiles of CCs from PCOS and non-PCOS patients. The gene expression profile of CCs from patients with PCOS was significantly different from that of CCs from non-PCOS patients. Specifically, CCs from women with PCOS were characterized by abnormal expression of many growth factors, including members of the epidermal growth factor-like (EGFR, EREG and AREG) and IGF-like families (IGF1R, IGF2R, IGF2BP2 and IGFBP2), that are known to play a role in oocyte competence. In addition, mRNA transcripts of factors involved in steroid metabolism, such as CYP11A1, CYP1B1, CYP19A1 and CYP2B7P1, were deregulated in PCOS CCs, and this could explain the abnormal steroidogenesis observed in these women. Functional annotation of the differentially expressed genes suggests that defects in the transforming growth factor β and estrogen receptors signalling cascades may contribute to the reduced oocyte developmental competence in patients with PCOS. Owing to the strict selection criteria (similar age, weight and reasons for ART), this study included a small sample size (six cases and six controls), and thus, further investigations using a large cohort of patients are needed to confirm these results. This study opens a new perspective for understanding the pathogenesis of PCOS. This work was partially supported by a grant from the Ferring Pharmaceutical. The authors of the study have no competing interests to report. Not applicable.

Be careful what you inject, as late maturing ooocytes only abate your rates: developmentally young oocytes are not ready for the needle!

Intracytoplasmic sperm injection (ICSI) of normally mature MII oocytes (NMO) at ∼4hr post retrieval is the standard of care, however some centers also ICSI late maturing oocytes (LMO) that were not mature at the time of hyaluronidase (∼2.5hr post retrieval) but have subsequently matured to MII at the time of ICSI. Development delay of oocyte maturation post retrieval may be due to many factors that could influence the fertilization and developmental potential of oocytes. Our study investigated 6713 cycles and their individual oocytes maturation history, comparing laboratory and clinical data.