Transcriptome Signature of Immature and In Vitro-Matured Equine Cumulus-Oocytes Complex.

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).

IVM Advances for Early Antral Follicle-Enclosed Oocytes Coupling Reproductive Tissue Engineering to Inductive Influences of Human Chorionic Gonadotropin and Ovarian Surface Epithelium Coculture.

In vitro maturation (IVM) is not a routine assisted reproductive technology (ART) for oocytes collected from early antral (EA) follicles, a large source of potentially available gametes. Despite substantial improvements in IVM in the past decade, the outcomes remain low for EA-derived oocytes due to their reduced developmental competences. To optimize IVM for ovine EA-derived oocytes, a three-dimensional (3D) scaffold-mediated follicle-enclosed oocytes (FEO) system was compared with a validated cumulus-oocyte complex (COC) protocol. Gonadotropin stimulation (eCG and/or hCG) and/or somatic cell coculture (ovarian vs. extraovarian-cell source) were supplied to both systems. The maturation rate and parthenogenetic activation were significantly improved by combining hCG stimulation with ovarian surface epithelium (OSE) cells coculture exclusively on the FEO system. Based on the data, the paracrine factors released specifically from OSE enhanced the hCG-triggering of oocyte maturation mechanisms by acting through the mural compartment (positive effect on FEO and not on COC) by stimulating the EGFR signaling. Overall, the FEO system performed on a developed reproductive scaffold proved feasible and reliable in promoting a synergic cytoplasmatic and nuclear maturation, offering a novel cultural strategy to widen the availability of mature gametes for ART.

In-vitro maturation and transplantation of cryopreserved ovary tissue: understanding ovarian longevity.

RESEARCH QUESTION: Is it possible to use experience gained from 24 years of frozen ovarian transplantation, and from recent experience with in-vitro gametogenesis to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? DESIGN: A total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24-year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back. RESULTS: Every woman had a return of ovarian function 5 months after transplant, similar to previous observations. As observed before, anti-Müllerian hormone (AMH) concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had had leukaemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes. CONCLUSIONS: The authors concluded the following. First, ovary tissue cryopreservation is a robust method for preserving fertility even for women with leukaemia, without a need to delay cancer treatment. Second, many mature oocytes can often be obtained from ovary tissue with simple media and no need for ovarian stimulation. Third, ovarian stimulation only be necessary for removing the oocyte from the ovary, which can also be accomplished by simple dissection at the time of ovary freezing. Finally, pressure and just eight 'core genes' control primordial follicle recruitment and development.

Chromosome aneuploidy analysis in embryos derived from in vivo and in vitro matured human oocytes.

BACKGROUND: In vitro oocyte maturation (IVM) is being increasingly approached in assisted reproductive technology (ART). This study aimed to evaluate the quality of embryos generated by in-vitro matured immature follicles, as a guideline for further clinical decision-making. METHODS: A total of 52 couples with normal karyotypes underwent in vitro fertilization, and 162 embryos were donated for genetic screening. Embryos in IVF group were generated by mature follicles retrieved during gonadotrophin-stimulated in vitro fertilization (IVF) cycles. And embryos in IVM group were fertilized from IVM immature oocytes. RESULTS: The average age of the women was 30.50 ± 4.55 years (range 21-42 years) with 87 embryos from IVF group and 75 embryos from IVM group. The rate of aneuploid with 28 of the 87 (32.2%) embryos from IVF group and 21 of the 75 (28%) embryos from IVM group, with no significant difference. The frequency of aneuploid embryos was lowest in the youngest age and increased gradually with women's age, whether in IVF group or IVM group and risen significantly over 35 years old. The embryos with morphological grade 1 have the lowest aneuploidy frequency (16.6%), and increase by the grade, especially in IVF group. In grade 3, embryos in IVM group were more likely to be euploid than IVF group (60% vs 40%, respectively). CONCLUSIONS: IVM does not affect the quality of embryos and does not increase the aneuploidy rate of embryos. It is clinically recommended that women more than 35 years have a high aneuploidy rate and recommended to test by PGS (strongly recommended to screened by PGS for women more than 40 years). Women aged less than 35 years old for PGS according to their physical and economic conditions. Embryo with poor quality is also recommended to test by PGS, especially for grade III embryos.

Maturation conditions, post-ovulatory age, medium pH, and ER stress affect [Ca2+]i oscillation patterns in mouse oocytes.

Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca2+ concentration ([Ca2+]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca2+]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca2+]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca2+]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca2+]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca2+]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca2+]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca2+]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.

In vitro Maturation of Oocytes with Consecutive Clinical Pregnancy after Accidental Premature Ovulation Induction-A Rescue Strategy in Art.

BACKGROUND: The implementation of assisted reproductive techniques (ART) is a complex treatment requiring both a good cooperation between various professional groups in the fertility centre, and the patient's and her partner's cooperation. Accordingly, there are many sources of failure, such as using the wrong medication or not considering optimal times. If there is an artificial application of the ovulation induction injection, the success of the treatment is endangered and in some cases the cycle is discontinued, if the patient failed to administer the drug correctly. An alternative to cycle cancellation might be the maturation of the oocytes in vitro. WE REPORT: on a 31-year-old patient in whom we performed an oocyte retrieval procedure 24 hours after triggering ovulation followed by in vitro maturation of the immature oocytes over a period of more than 12 h. The treatment resulted in a healthy, ongoing pregnancy.

Induction of CIRBP expression by cold shock on bovine cumulus-oocyte complexes.

The aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5°C) and cold-stressed oocytes (33.5°C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.

Current state and future possibilities of ovarian tissue transplantation.

BACKGROUND: As a result of recent developments in cancer treatment, cancer survivorship and survivors' quality of life have been emphasized. Although ovarian tissue cryopreservation (OTC) is an experimental technique, it would be the sole technique for fertility preservation treatment for girls with malignant disease. Indeed, OTC requires ovarian tissue transplantation (OTT) for conception. As for OTC, there is room to investigate OTT. The present review focused on the current state and progress of OTT. METHOD: The literature regarding OTT, which is currently under development, was reviewed. MAIN FINDINGS: To improve the outcome of OTT, both efficacy and safety are important. Good surgical technique and the optimal site are important surgical factors, with orthotopic transplantation increasing. Treatment of growth factors, gonadotropins, antioxidants, apoptosis suppression factors, and cell therapy may improve the efficacy of OTT by inducing neo-angiogenesis and preventing damage. Artificial ovaries, complete in vitro primordial follicle culture technique, and non-invasive ovarian imaging techniques, such as optical coherence tomography, to select the best ovarian tissue are future possibilities. CONCLUSION: Improving neo-angiogenesis and preventing damage with optimization, as well as investigation of future techniques, may bring us to the next stage of a fertility preservation strategy.

Outcome of immature oocytes collection of 119 cancer patients during ovarian tissue harvesting for fertility preservation.

PURPOSE: Few clinical options for fertility preservation are available to females with cancer, and data about clinical outcomes is limited. Potential supplementary approaches to fertility preservation include retrieval of immature oocytes followed by in vitro maturation (IVM) and storage. The aim of this study was to evaluate post-thawing outcomes of immature oocytes collected both by transvaginal aspiration and from excised ovarian tissue. METHODS: We conducted a retrospective cohort study of patients treated in a single tertiary center. We reviewed the records of 119 cancer patients who underwent ovarian tissue cryopreservation and immature oocyte harvesting for fertility preservation. All embryos and oocytes that were frozen and thawed were included in the study. Post-thawing outcomes were evaluated. RESULTS: Thirty-five stored embryos from eight patients were thawed. Twenty-nine embryos survived (82% survival rate) and were transferred. Six oocytes were thawed, two oocytes survived, and no oocytes were fertilized. Only one PCOS patient became pregnant, resulting in the normal delivery of a healthy baby. CONCLUSIONS: Although a relatively high number of mature oocytes and embryos can be stored with the combined procedure, the limited rate of pregnancies represents a poor reproductive outcome. Therefore, this approach should be reserved for special groups with limited options.

Differing molecular response of young and advanced maternal age human oocytes to IVM.

STUDY QUESTION: What effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation? SUMMARY ANSWER: Although polyadenylated transcript abundance is similar between young and advanced maternal age (AMA) germinal vesicle (GV) oocytes, metaphase II (MII) oocytes exhibit a divergent transcriptome resulting from a differential response to in vitro oocyte maturation. WHAT IS KNOWN ALREADY: Microarray studies considering maternal age or maturation stage have shown that either of these factors will affect oocyte polyadenylated transcript abundance in human oocytes. However, studies considering both human oocyte age and multiple stages simultaneously are limited to a single study that examined transcript levels for two genes by qPCR. Thus, polyadenylated RNA sequencing (RNA-Seq) could provide novel insight into age-associated aberrations in gene expression in GV and MII oocytes. STUDY DESIGN, SIZE, DURATION: The effect of maternal age (longitudinal analysis) on polyadenylated transcript abundance at different stages was analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (YNG; < 30 years; average age 26.8; range 20-29) and five advanced maternal age (AMA; ≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 YNG (5 GV and 5 MII) and 10 AMA (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided oocyte retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 h incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads on HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, weighted gene correlation network analysis (WGCNA), Ingenuity Pathway Analysis) were performed. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 12 770 genes were determined to be expressed in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least three of five replicates for a minimum of one sample type). Differential gene expression analysis between YNG and AMA oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted P < 0.1 and log2 fold change >1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5 and PRDX1, which have been reported to affect oocyte developmental potential. Despite the similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were correlated (P < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation. LARGE SCALE DATA: Raw data from this study can be accessed through GSE95477. LIMITATIONS, REASONS FOR CAUTION: The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by IVM of patient oocytes. Although the approach has the benefit of identifying intrinsic differences between samples, it may not be completely representative of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Transcriptome profiles of YNG and AMA oocytes, particularly at the MII stage, suggest that aberrant transcript abundance may contribute to the age-associated decline in fertility. STUDY FUNDING/COMPETING INTEREST(S): J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.

Supplementation of SkQ1 Increases Mouse In Vitro Oocyte Maturation and Subsequent Embryonic Development by Reducing Oxidative Stress.

In vitro oocyte maturation (IVM) technology is important for assisted animal and human reproduction. However, the maturation rates and developmental potential of in vitro-matured oocytes are usually lower than those of in vivo-matured oocytes. Oxidative stress is a main factor that causes the lower maturation rates and quality of in vitro-matured oocytes. The purpose of this study was to investigate the effects of treatment with SkQ1, a mitochondria-targeted antioxidant, on mouse IVM and subsequent embryonic development. The results demonstrated that the supplementation of SkQ1 during IVM improves the maturation rates of mouse oocytes and the subsequent developmental competence of in vitro-fertilized embryos. The addition of SkQ1 to the IVM medium also decreased oxidative stress and apoptosis, and increased mitochondrial membrane potential in matured mouse oocytes. This study provides a new method through which to enhance the maturation rates and the quality of in vitro-matured mouse oocytes, thus promoting the application and development of assisted animal and human reproductive technology.

Effects of combination of melatonin and L-carnitine on in vitro maturation in mouse oocytes: An experimental study.

Background: Melatonin and L-carnitine are free radical scavengers with antiapoptotic and antioxidant properties that improve oocyte development. Objective: This study aimed to find the possible effect of combining 2 antioxidant agents of melatonin and L-carnitine on oocyte morphology, maturation, apoptosis, and expression of bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) genes in a mice model. Materials and Methods: To overstimulation, 60 female NMRI mice were injected intraperitoneally using mare serum gonadotropin. On day 2 post-injection, 70 cumulus-oocyte complexes were collected from each mouse. The collected oocytes randomly were then divided into 4 groups including, the control, melatonin, L-carnitine, and melatonin + L-carnitine groups. The morphology and maturation rate of the oocytes was evaluated using a light microscope. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and the expression of BMP-15 and growth and differentiation factor GDF-9 genes was also evaluated by real-time polymerase chain reaction. Results: Oocyte diameter significantly was increased in combination treatment of L-carnitine and melatonin compared to other groups (p < 0.05). L-carnitine group showed the highest mean percentage of oocyte cytoplasmic pattern. Results of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling indicated that the lowest apoptosis rate belonged to the melatonin + L-carnitine group. Moreover, the combination groups showed the highest number of oocytes and maturation rate. The BMP-15 and GDF-9 genes were significantly upregulated in all treatment groups compared to the control group. Conclusion: Our results suggested a combination of melatonin + L-carnitine administration as a more effective choice for in vitro promotion of oocyte maturation.

All-trans retinoic acid and fibroblast growth factor-2 enhance the fertility rate and embryo development in polycystic ovary syndrome mouse model.

Objectives: Polycystic ovary syndrome (PCOS) causes a developmental arrest of antral follicles and disrupts oocyte maturation. Retinoic acid (RA) and Fibroblast Growth Factor-2 (FGF2) are effective in follicle growth, thus their effects on histopathology and in vitro fertility of oocytes were investigated in PCOS-induced mice. Materials and Methods: Eighty female NMRI mice were randomly divided into 8 groups including 1-Normal mice, 2-PCOS mice without any treatment, 3-Normal mice treated with RA, 4-Normal mice treated with FGF2, 5-PCOS mice treated with RA, 6- PCOS mice treated with FGF2, 7- PCOS mice treated with RA and FGF2, and 8- Normal mice treated with RA and FGF2. Following PCOS induction, the mice were treated with intraperitoneal RA and FGF2 as a treatment. Then ovarian stimulation, for preparing the oocyte and embryo microscopic examinations was performed. After oocyte morphometry, through in vitro fertilization, the embryo formation was assessed. Data was analyzed by one-way ANOVA and Tukey tests. Results: The results showed simultaneous injection of RA and FGF2 into PCOS-induced mice increases antral follicles and corpus luteum, but decreases cystic follicles. Simultaneous injection of these two substances into healthy mice increases the pre-antral follicles and corpus luteum. Simultaneous injection of RA and FGF2 increases the number of embryos in both control and intervention groups. Conclusion: It can be concluded that RA and FGF2 increase the maturity of ovarian follicles, the number of two-celled embryos, and the number of grade-A embryos in mice with PCOS, which is more effective when these two substances are injected simultaneously.

In Vitro Maturation, In Vitro Oogenesis, and Ovarian Longevity.

This paper will review a remarkable new approach to in vitro maturation "IVM" of oocytes from ovarian tissue, based on our results with in vitro oogenesis from somatic cells. As an aside benefit we also have derived a better understanding of ovarian longevity from ovary transplant. We have found that primordial follicle recruitment is triggered by tissue pressure gradients. Increased pressure holds the follicle in meiotic arrest and prevents recruitment. Therefore recruitment occurs first in the least dense inner tissue of the cortico-medullary junction. Many oocytes can be obtained from human ovarian tissue and mature to metaphase 2 in vitro with no need for ovarian stimulation. Ovarian stimulation may only be necessary for removing the oocyte from the ovary, but this can also be accomplished by simple dissection at the time of ovary tissue cryopreservation. By using surgical dissection of the removed ovary, rather than a needle stick, we can obtain many oocytes from very small follicles not visible with ultrasound. A clearer understanding of ovarian function has come from in vitro oogenesis experiments, and that explains why IVM has now become so simple and robust. Tissue pressure (and just a few "core genes" in the mouse) direct primordial follicle recruitment and development to mature oocyte, and therefore also control ovarian longevity. There are three distinct phases to oocyte development both in vitro and in vivo: in vitro differentiation "IVD" which is not gonadotropin sensitive (the longest phase), in vitro gonadotropin sensitivity "IVG" which is the phase of gonadotropin stimulation to prepare for meiotic competence, and IVM to metaphase II. On any given day 35% of GVs in ovarian tissue have already undergone "IVD" and "IVG" in vivo, and therefore are ready for IVM.

Effect of three-dimensional collagen membrane culture and presence of cumulus cells on maturation of bovine oocytes.

OBJECTIVE: The use of animals as experimental models has been proposed to improve the techniques applied in human reproduction clinics. This prospective and observational study evaluates the effects of the use of cumulus cells and collagen membrane on the maturation process of bovine oocytes. METHODS: Design and Setting: Bovine oocytes with or without cumulus cells were cultured in maturation medium for 24 hours in the conventional system (2D), central well plates and in the three-dimensional (3D) system. Intervention: The oocytes were positioned in the collagen membrane and matured for the same period. The morphological evaluation was carried out with the parameters of maturation. Main Outcome Measure: Presence or absence of the first polar corpuscle, which were observed and classified as germinal vesicle (GV), meiosis I (MI) and meiosis II (MII). RESULTS: The percentage of oocytes in GV was higher (p<0.05) in treatments without cumulus cells than those with cells. The rates of MII were higher (p<0.05) in the treatments with cumulus cells, independent of the culture system. In general, oocytes with presence of cumulus cells have approximately 1.7 times more chances (p<0.001) of reaching MII after MIV than those matured without cells. CONCLUSIONS: The presence of the cells in the cumulus is essential for the maturation process of bovine oocytes; the three-dimensional collagen membrane culture system is favorable for the maturation process of bovine oocytes.

Use of platelet lysate for in-vitro embryo production and treatment of repeat breeding in cows.

Platelet-rich plasma (PRP) is a biological hemocomponent derived from blood after the complete removal of red blood cells and the partial or complete removal of white blood cells to concentrate platelets in an appropriate volume of plasma. Platelets have important growth factors, cytokines, and active metabolites that improve the endometrial environment and positively affect implantation. This study evaluated the effect of the addition of activated PRP (platelets lysate; PL) on in vitro bovine oocyte maturation and embryonic development and the effect of intrauterine (IU) infusion of autologous PL in repeat breeder (RB) cows. Experiment 1 examined the effects of allogeneic PL, fetal calf serum (FCS), mixed PL + FCS, or platelet-poor plasma (PPP) supplementations to in vitro maturation and development media on in vitro oocyte maturation and embryo development in good- and poor-quality oocytes of Japanese Black cows. Experiment 2 examined the IU infusion of autologous PL, 24 h post-insemination, in 21 RB Holstein-Friesian dairy cows. The cleavage rate of good-quality oocytes was higher in the PL group (85.93 ± 2.50%) than in the PPP group (67.16 ± 3.41%) (P < 0.05), while the cleavage rate of the poor-quality oocytes was higher in the PL alone (76.13 ± 4.04%) and mixed PL + FCS treated (73.59 ± 4.22%) groups than in the PPP group (54.64 ± 2.93%) (P < 0.05). The blastocyst rate of the good-quality oocytes was higher in the PL group (40.97 ± 3.03%) than in the FCS (27.97 ± 3.31%) and PPP (25.33 ± 2.15%) groups (P < 0.05). The blastocyst rate of poor-quality oocytes and the hatching rates of both good and poor-quality oocytes showed no significant differences among all groups. The conception rate in the autologous PL-treated group was 41.67% (5/12), while it was 11.11% (1/9) in the control group. The platelets' count in the pregnant PL-treated cows (n = 5; mean ± SEM, 1.07 ± 0.10 × 109/mL) was higher than in the non-pregnant ones (n = 7; 0.67 ± 0.10 × 109/mL) (P < 0.05). In conclusion, allogeneic PL was effective in stimulating the in vitro oocyte maturation and embryonic development in both good and poor-quality bovine oocytes, and post-insemination IU infusion of autologous PL derived from high platelets' count-PRP would be recommended for the treatment of RB cows.

Clinical and laboratory factors associated with the presence of dysmorphic oocytes in intracytoplasmic sperm injection cycles.

OBJECTIVE: This study investigated the clinical and laboratory factors associated with the presence of dysmorphic oocytes in intracytoplasmic sperm injection (ICSI) cycles. METHODS: The study involved 200 ICSI cycles, performed from 2020 to 2021, that yielded at least one mature oocyte. Clinical characteristics and ovarian stimulation methods were compared between 68 cycles with at least one dysmorphic oocyte (the dysmorphic group) and 132 cycles with normal-form oocytes only (the non-dysmorphic group). Dysmorphic oocytes were characterized by dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body. RESULTS: The ages of the women, indications for in vitro fertilization, serum anti-Müllerian hormone levels, and rates of current ovarian endometrioma were similar between the dysmorphic and non-dysmorphic groups. In both groups, the three ovarian stimulation regimens, two types of pituitary suppression, and total gonadotropin dose were employed similarly. However, the dual-trigger method was used more frequently in the dysmorphic group (67.6% vs. 50%, p=0.024). The dysmorphic group contained significantly more immature oocytes and exhibited significantly lower oocyte maturity (50% vs. 66.7%, p=0.001) than the non-dysmorphic cycles. Within the dysmorphic group, significantly lower oocyte maturity was found in the cycles using a dual-trigger, but not in those with a human chorionic gonadotropin trigger. CONCLUSION: ICSI cycles with dysmorphic oocytes are closely associated with reduced oocyte maturity. This association was observed exclusively in dual-trigger cycles.

Gonadotropin releasing hormone agonist triggering for in vitro maturation cycles.

The objective was to evaluate the outcomes of in vitro maturation (IVM) cycles using gonadotropin releasing hormone agonist (GnRH-ag) triggering. A retrospective cohort of IVM cycles from January 2015 to December 2019 in a single university-affiliated centre was examined. Main outcome measures were: (i) IVM maturation rate; and (ii) IVM maturation result. Secondary outcome measures were: (i) metaphase II (MII) rate on the day of egg retrieval; (ii) final MII maturation rate; and (iii) pregnancy rates. A total of 98 IVM cycles were performed during the study period: 50 (51%) were triggered with GnRH-ag (17 received FSH priming and 33 did not) and 48 cycles (49%) were triggered by hCG (37 with FSH priming and 11 without). A significant (p = 0.01) difference was noticed in maturation rate on egg retrieval day, in favour of the GnRH-ag group, although not in the final maturation rate achieved. Pregnancy rates were comparable between treatment sub-groups. GnRH-ag triggering in IVM cycles is an optional triggering mode and can be considered an acceptable option, especially when fertility preservation is a concern. GnRH agonists resulted in higher maturation rate on day of oocyte retrieval, but no difference in the total maturation rate.

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

OBJECTIVE: Optimizing 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-ß (GDF9-ß). supplementation. METHODS: Immature 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-ß. ICSI was done for matured oocytes, and fertilization was confirmed by the visualization of two distinct pronuclei and two polar bodies. RESULTS: No 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). CONCLUSION: Higher rates of oocyte maturation and fertilization were observed after application of homemade medium supplemented with GDF9-ß. Therefore, this combination may be recommended as an alternative for clinical IVM programs.

In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm.

BACKGROUND: The outcomes of the use of commercial in vitro maturation (IVM) medium to culture immature oocytes obtained from conventional ovulation induction, followed by rescue intracytoplasmic sperm injection (RICSI), are not ideal. It is thus difficult to widely adopt this approach in clinical practice. Therefore, it is necessary to explore methods for improving the clinical outcome of IVM. AIM: To study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture. METHODS: This was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020. RICSI was performed using sperm collected on the day of oocyte harvest (old) and sperm collected on the day of RICSI (fresh) and oocytes matured in vitro after 24 h of culture in conventional medium. The rates of in vitro oocyte maturation, normal fertilization, normal cleavage, day-3 top-quality embryos, and useful blastocyst formation were compared between the two groups. RESULTS: In total, 102 germinal vesicle (GV)-stage immature oocytes were cultured in the old sperm group. In the fresh sperm group, 122 GV-stage immature oocytes were collected and cultured in vitro for 24 h. There were no significant differences in the general conditions of males and females between the two groups (P > 0.05). The oocyte maturation, normal fertilization, and normal cleavage rates of the old and fresh groups were 51.0% vs 55.7%, 61.5% vs 64.7%, and 93.8% vs 93.2%, respectively. None of the rates differed significantly (P > 0.05) between the two groups. However, the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6% vs 63.4%; 6.67% vs 34.6%, respectively. The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group (P < 0.05). CONCLUSION: In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.