Oocyte Health and Quality: Implication of Mitochondria-related Organelle Interactions.

Among factors like hormonal imbalance and uterine condition, oocyte quality is regarded as one of the key factors involved in age-related decline in the reproductive capacity. Here, are discussions about the functions played by organelles within the oocyte in forming the next generation that is more suitable for survival. Many insights on the adaptation to aging and maintenance of quality can be obtained from: interactions between mitochondria and other organelles that enable the long life of primordial oocytes; characteristics of organelle interactions after breaking dormancy from primary oocytes to mature oocytes; and characteristics of interactions between mitochondria and other organelles of aged oocytes collected during the ovulatory cycle from elderly individuals and animals. This information would potentially be beneficial to the development of future therapeutic methods or agents.

Rutin enhances mitochondrial function and improves the developmental potential of vitrified ovine GV-stage oocyte.

Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca2+, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca2+, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca2+ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca2+ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.

Equine ART and antral follicle count: Can we deepen our understanding to improve outcomes?

Assisted reproductive technologies (ARTs) are performed worldwide in the equine industry to produce genetically valuable foals. Among them, ovum pick up (OPU) combined with intra-cytoplasmic sperm injection (ICSI) can now be more efficient than embryo transfer (ET) under optimal conditions. However, OPU is not a benign procedure for the mare and the process is costly. Improved efficiency is therefore in the interest of everyone, maximizing mare welfare and optimizing economics for the client. One of the key factors of success is the antral follicle count (AFC) at the time of OPU and subsequently the number of oocytes obtained. Variations in AFC are reported between individuals and between geographical areas. This leads to a significant increase in numbers of embryos produced per session in some countries compared to others, independent of the laboratory efficiency. This article revisits the basics of folliculogenesis involved in establishment of the antral follicle population and explores work in other species given the paucity of equine research in this area. The aim of the review is to elucidate interesting areas of further research that could generate essential information for clinicians and clients about the management and selection of the donor mare for OPU and potentially identify pharmacological targets for manipulation.

An integrated analysis of multiple datasets reveals novel gene signatures in human granulosa cells.

Granulosa cells (GCs) play crucial roles in oocyte maturation. Through gap junctions and extracellular vesicles, they mediate the exchange of molecules such as microRNAs and messenger RNAs. Different ovarian cell types exhibit unique gene expression profiles, reflecting their specialized functions and stages. By combining RNA-seq data from various cell types forming the follicle, we aimed at capturing a wide range of expression patterns, offering insights into the functional diversity and complexity of the transcriptome regulation across GCs. Herein, we performed an integrated bioinformatics analysis of RNA sequencing datasets present in public databases, with a unique and standardized workflow., By combining the data from different studies, we successfully increased the robustness and reliability of our findings and discovered novel genes, miRNAs, and signaling pathways associated with GCs function and oocyte maturation. Moreover, our results provide a valuable resource for further wet-lab research on GCs biology and their impact on oocyte development and competence.

The stony coral Fimbriaphyllia (Euphyllia) ancora's reproductive strategy involves a sex change every year.

A sex change phenomenon was reported in some free-living, non-sessile coral species of the Family Fungiidae. However, there are no reports describing sex change in sessile colonial species. Timing and cellular processes of sex change are also unclear in corals. Here, we report sex change of the colonial coral, Fimbriaphyllia ancora, and its cellular process. Of 26 colonies monitored at Nanwan Bay, southern Taiwan, about 70% changed their sex every year after annual spawning for least 3-4 consecutive years, i.e., colonies that were male two years ago became female last year, and male again this year. The remaining 30% were permanently male or female. Sex-change and non-sex-change colonies grew in close proximity or even side-by-side. No significant differences were found in colony size between sex-change and non-sex-change colonies. Histological analysis showed that, in female-to-male sex change, small oocytes were present up to 3 months in some gonads after spawning and disappeared by 5 months. This suggests that sex change occurred 4-5 months after spawning. In contrast, in male-to-female sex change, oocytes appeared weeks after sperm release and in most gonads by 3 months, suggesting that male-to-female sex change occurred 0-3 months after sperm release.

Calcium signaling in oocyte quality and functionality and its application.

Calcium (Ca2+) is a second messenger for many signal pathways, and changes in intracellular Ca2+ concentration ([Ca2+]i) are an important signaling mechanism in the oocyte maturation, activation, fertilization, function regulation of granulosa and cumulus cells and offspring development. Ca2+ oscillations occur during oocyte maturation and fertilization, which are maintained by Ca2+ stores and extracellular Ca2+ ([Ca2+]e). Abnormalities in Ca2+ signaling can affect the release of the first polar body, the first meiotic division, and chromosome and spindle morphology. Well-studied aspects of Ca2+ signaling in the oocyte are oocyte activation and fertilization. Oocyte activation, driven by sperm-specific phospholipase PLCζ, is initiated by concerted intracellular patterns of Ca2+ release, termed Ca2+ oscillations. Ca2+ oscillations persist for a long time during fertilization and are coordinately engaged by a variety of Ca2+ channels, pumps, regulatory proteins and their partners. Calcium signaling also regulates granulosa and cumulus cells' function, which further affects oocyte maturation and fertilization outcome. Clinically, there are several physical and chemical options for treating fertilization failure through oocyte activation. Additionally, various exogenous compounds or drugs can cause ovarian dysfunction and female infertility by inducing abnormal Ca2+ signaling or Ca2+ dyshomeostasis in oocytes and granulosa cells. Therefore, the reproductive health risks caused by adverse stresses should arouse our attention. This review will systematically summarize the latest research progress on the aforementioned aspects and propose further research directions on calcium signaling in female reproduction.

The Ovary Structure in Terrestrial Parasitengona Mites: The Case of Trombidiidae (Acariformes: Parasitengona).

Species of mites (Chelicerata: Arachnida) show a great variety of structures of the female gonads. In both evolutionary lines, Acariformes and Parasitiformes, the panoistic ovary, in which all germline cysts differentiate into oocytes, and the meroistic ovary, in which the oocytes grow supported by the nurse cells, have been documented. A less pronounced variation in the gonad structure could be expected at lower systematic levels, hence, we ask about the degree of differences within the family that is subordinate to Acariformes and represents the cohort Parasitengona. Based on the members of Trombidiidae (Acariformes: Trombidiformes, Parasitengona, Trombidioidea), we test the hypothesis that the general ovary type is constant at the family level. Our previous research on the female gonad in Allothrombium fuliginosum revealed that the meroistic ovary occurs in these mites. Here, we proceed with a detailed insight into the ovary structure in A. fuliginosum and examine the structure of the female gonad in other members of Trombidiidae, focusing on the following representatives of its nominotypical genus Trombidium: Trombidium brevimanum, Trombidium holosericeum, Trombidium heterotrichum, and Trombidium latum. For all species, studied with light, fluorescence, and transmission electron microscopy, we could confirm the presence of the meroistic ovary that is highly similar with respect to general architecture. The germline cysts show similarities in general morphology and the mode of germline cell differentiation; they consist of a few nurse cells and one oocyte. The connection between the nurse cells and oocytes is maintained by trophic cords that serve for the transport of organelles and macromolecules. Our results confirm the constancy of the structure of the female gonad at the intrageneric level and provide further support for the hypothesis on the lack of differences at the intrafamily level.

A novel heterozygous missense variant of PANX1 causes human oocyte death and female infertility.

Pannexin1 (PANX1) is a highly glycosylated membrane channel-forming protein, which has been found to implicate in multiple physiological and pathophysiological functions. Variants in the PANX1 gene have been reported to be associated with oocyte death and recurrent in vitro fertilization failure. In this study, we identified a novel heterozygous PANX1 variant (NM_015368.4 c.410 C > T (p.Ser137Leu)) associated with the phenotype of oocyte death in a non-consanguineous family, followed by an autosomal dominant (AD) mode. We explored the molecular mechanism of the novel variant and the variant c.976_978del (p.Asn326del) that we reported previously. Both of the variants altered the PANX1 glycosylation pattern in cultured cells, led to aberrant PANX1 channel activation, affected ATP release and membrane electrophysiological properties, which resulted in mouse and human oocyte death in vitro. For the first time, we presented the direct evidence of the effect of the PANX1 variants on human oocyte development. Our findings expand the variant spectrum of PANX1 genes associated with oocyte death and provide new support for the genetic diagnosis of female infertility.

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera.

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

G-Protein-Coupled Receptor Kinase 2 Inhibition Induces Meiotic Arrest by Disturbing Ca2+ Release in Porcine Oocytes.

G-protein-coupled receptor kinase 2 (GRK2) interacts with Gßγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (ßi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; ßi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; ßi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of ßi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.

Effect of histidine and L-Tyrosine supplementation in maturation medium on in-vitro developmental outcomes of buffalo oocytes.

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.

The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

Efficiency of the zinc chelator 1,10-phenanthroline for assisted oocyte activation following ICSI in pigs.

Context In vitro embryo production in pigs is an important tool for advancing biomedical research. Intracytoplasmic sperm injection (ICSI) circumvents the polyspermy problems associated with conventional IVF in porcine. However, the suboptimal efficiency for ICSI in pigs requires new strategies to increase blastocyst formation rates. Aim To investigate novel methods for assisted activation using the zinc chelator 1,10-phenanthroline (PHEN), and to improve embryo developmental competence and quality of ICSI porcine blastocyst. Methods ICSI embryos were treated with PHEN after or before sperm injection, recording pronuclear formation, blastocyst rate and the expression of SMARCA4, OCT4, SOX2 and CDX2. Key results Neither electrical nor PHEN significantly improves pronuclear formation rates before or after ICSI. Following in vitro culture to the blastocyst stage, no significant differences were observed in developmental rates among the groups. Moreover, the use of PHEN did not alter the total cell number or the expression of OCT4, SOX2 and CDX2 in pig ICSI blastocysts. Conclusions Assisted oocyte activation with PHEN does not affect the preimplantation development of ICSI-derived pig embryos. Implications These results hold significance in refining and advancing the application of assisted oocyte activation techniques. They offer insights into addressing fertility issues and propelling advancements in human and animal reproductive medicine.

foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Functional complementation of two splicing variants of Gustavus in Neocaridina denticulata sinensis during ovarian maturation.

Gustavus, a positive regulator in arthropod reproduction, features a conserved SPRY and a C-terminal SOCS box domain and belongs to the SPSB protein family. The SPSB family, encompassing SPSB1 to SPSB4, plays pivotal roles in higher animals, including immune response, apoptosis, growth, and stress responses. In Neocaridina denticulata sinensis, alternative splicing yielded two NdGustavus isoforms, NdGusX1 and NdGusX2, with distinct expression patterns-high in ovaries and muscles, respectively, and across all ovarian germ cells. These isoforms showed similar expression dynamics during embryogenesis and significant upregulation post-copper ion exposure (P < 0.05). The in situ hybridization result elucidated that NdGusX1 and NdGusX2 were expressed across the germ cell spectrum in the ovary, with NdGusX1 showing enhanced expression in oogonia and primary oocytes. In addition, RNA interference revealed functional complementation in ovaries and potential functional differentiation in muscles. Knockdown of NdGusX1 and NdGusX2 potentially disrupted endogenous vitellogenin synthesis, regulating vitellogenesis and reducing mature oocyte volume, affecting follicular cavity occupation. This study provides a theoretical framework for understanding the biological functions of the SPSB family in crustacean ovarian maturation.

The importance of social oocyte cryopreservation in supporting local municipalities: A prospective study.

BACKGROUND: With the trend toward late marriages and late childbearing, cryopreservation of oocytes for fertility preservation is attracting attention as a method to counteract the declining birthrate. OBJECTIVES: To examine the impact of social oocyte cryopreservation on local communities by assessing the significance of government assistance for cryofreezing and capturing the participants' subsequent feelings regarding this assistance. DESIGN: Descriptive study. METHODS: A prospective study was conducted on city-dwelling women <35 years old attending monthly seminars on oocyte retrieval/cryopreservation to whom the study concept was explained. Egg collection and storage management costs were free for 3 years after the project completed, and subsequent actual storage costs were borne by the individuals. After oocyte retrieval, we conducted a questionnaire on oocyte cryopreservation and administrative assistance. RESULTS: Of the 62 seminar participants, 2 became pregnant naturally without oocyte retrieval. Oocytes were retrieved in 34 women (average age: 32.8 years, number of oocytes obtained: 8.3), among whom 4 subsequently became pregnant and gave birth through natural pregnancy or artificial insemination, and 1 became pregnant and gave birth using frozen oocytes. In a follow-up questionnaire given to these 34 subjects, all responded that they were glad to have oocyte cryopreservation, but 23 subjects (67.6%) answered that they could not perform cryopreservation without financial assistance. Twenty-five participants (73.5%) wanted to try to conceive without using frozen oocytes as a post-cryopreservation plan. CONCLUSIONS: As a countermeasure against the declining birthrate, oocyte cryopreservation and associated workshops that can provide the information and education needed to conduct this task in a "planned" manner may be useful in providing women with additional reproductive options. Financial assistance will also be required to offer this service to the women who need it.

Women benefit when egg freezing is subsidized by local municipalitiesWhy was the study done? To prospectively examine the significance of egg freezing in a society in which the declining birthrate is an issue, particularly with regard to those who wish to undergo egg freezing and their trends when it is supported by the government. What did the researchers do? This project was conducted as a three-year endowed course by a local city government. Participants were women aged 20 to 34 who lived in the city and were recruited through the city's newsletter and website. They then attended a fertility workshop that was held once a month. Participants who wished to freeze their eggs were offered one free egg retrieval and three years of frozen storage. Participants were also asked to complete a questionnaire about their progress three years after the project ended. What did the researchers find? Sixty-two women participated in the three-year project, of whom 34 chose to freeze their eggs. Those who did not plan to conceive early, and two conceived naturally. Of those who froze their eggs, only one gave birth using the frozen eggs, and seven conceived naturally or through fertility treatments without using frozen eggs, two of whom had two pregnancies, resulting in 10 children being born. What do the findings mean? Three years after the project ended, the findings suggested that egg freezing itself may not have had a significant effect on pregnancy and childbirth but that holding workshops on fertility may have acted as an incentive for women to become pregnant and give birth.

Protein phosphatase 4 maintains the survival of primordial follicles by regulating autophagy in oocytes.

In mammalian ovary, the primordial follicle pool serves as the source of developing follicles and fertilizable ova. To maintain the normal length of female reproductive life, the primordial follicles must have adequate number and be kept in a quiescent state before menopause. However, the molecular mechanisms underlying primordial follicle survival are poorly understood. Here, we provide genetic evidence showing that lacking protein phosphatase 4 (PPP4) in oocytes, a member of PP2A-like subfamily, results in infertility in female mice. A large quantity of primordial follicles has been depleted around the primordial follicle pool formation phase and the ovarian reserve is exhausted at about 7 months old. Further investigation demonstrates that depletion of PPP4 causes the abnormal activation of mTOR, which suppresses autophagy in primordial follicle oocytes. The abnormal primordial follicle oocytes are eventually erased by pregranulosa cells in the manner of lysosome invading. These results show that autophagy prevents primordial follicles over loss and PPP4-mTOR pathway governs autophagy during the primordial follicle formation and dormant period.

D4Z4 Hypomethylation in Human Germ Cells.

Expression of the double homeobox 4 (DUX4) transcription factor is highly regulated in early embryogenesis and is subsequently epigenetically silenced. Ectopic expression of DUX4 due to hypomethylation of the D4Z4 repeat array on permissive chromosome 4q35 alleles is associated with facioscapulohumeral muscular dystrophy (FSHD). In peripheral blood samples from 188 healthy individuals, D4Z4 methylation was highly variable, ranging from 19% to 76%, and was not affected by age. In 48 FSHD2 patients, D4Z4 methylation varied from 3% to 30%. Given that DUX4 is one of the earliest transcribed genes after fertilization, the D4Z4 array is expected to be unmethylated in mature germ cells. Deep bisulfite sequencing of 188 mainly normozoospermic sperm samples revealed an average methylation of 2.5% (range 0.3-22%). Overall, the vast majority (78%) of individual sperm cells displayed no methylation at all. In contrast, only 19 (17.5%) of 109 individual germinal vesicle (GV) oocytes displayed D4Z4 methylation <2.5%. However, it is not unexpected that immature GV oocytes which are not usable for assisted reproduction are endowed with D4Z4 (up to 74%) hypermethylation and/or abnormal (PEG3 and GTL2) imprints. Although not significant, it is interesting to note that the pregnancy rate after assisted reproduction was higher for donors of sperm samples and oocytes with <2.5% methylation.

New Immunological Indexes for the Effect of Systemic Inflammation on Oocyte and Embryo Development in Women With Unexplained Infertility: Systemic Immune Response Index and Pan-Immune-Inflammation Value.

PROBLEM: Predicting the impact of systemic inflammation on oocyte and embryonic development in unexplained infertile women using the new immunological indexes. METHOD OF STUDY: This retrospective cohort study was conducted using the records of the In Vitro Fertilization Department of Ankara Gülhane Training and Research Hospital. After reviewing the records of patients who had undergone in vitro fertilization (IVF) for unexplained infertility (UI) and excluding all known factors that could cause systemic immune inflammation, the systemic immune response index (SIRI), and pan-immune score were calculated from the pre-treatment hemogram parameters between the embryo arrest (EA) group and the embryo transfer group. It was investigated whether there was a statistical difference between the two groups and whether an SIRI value affecting embryo quality was found. A receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off values for inflammatory markers to predict EA. RESULTS: The 108 EA group (embryos that were arrested during their development and could not be transferred) and the 140 embryo transfer group showed statistically significant differences in the parameters of systemic inflammatory index (SII), SIRI, pan-immune inflammation value (PIV), and neutrophil/lymphocyte ratio (NLR) (p < 0.05). These inflammatory parameters, which were examined before ovulation induction, also correlated positively with the required total dose of gonadotropin and negatively with the ovarian sensitivity index (OSI). SII, SIRI, PIV, and NLR have specific cut-off values with ROC analysis and determine the effect of the inflammatory status of the environment in which the oocyte develops on EA (p < 0.005). CONCLUSION: In women with UI, high levels of systemic immune inflammation have a negative impact on oocyte and embryo development, and treatments to suppress inflammation may improve IVF success.

Culture of the Intact Postnatal Naked Mole-Rat Ovary: From Meiotic Prophase to Single-Cell RNASeq.

Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the primordial germ cells (PGCs), and the initiation of the meiotic program occurs postnatally. This is opposite to almost all other mammals, including humans and mice, whose reproductive cycle begins very early in development. In both mouse and human, the ovaries become populated with PGCs in utero; these PGCs will later generate the oogonia. After mitotic proliferation, these cells will trigger the meiotic program and initiate meiotic prophase I. Given that all these processes happen in utero, their analysis has been very challenging; so the ability to study them postnatally and to manipulate them with inhibitors or other substances, in the naked mole-rat, opens new possibilities in the field. In this chapter, we present a comprehensive collection of protocols that permit the culture of whole naked mole-rat ovaries, followed by analysis of germ cells, from PGCs to oocytes, in meiotic prophase I, as well the obtention of single-cell suspension or single-nuclei suspension for RNASeq.