RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

Ultrastructure of mitochondria of human oocytes in different clinical conditions during assisted reproduction.

Infertility affects around 8% of couples with a slight change in percentage in the last years. Despite the significant efforts made in Assisted Reproductive Technologies (ARTs) in handling this disorder, oocyte quality remains a crucial factor for a positive outcome. A better understanding of the dynamics underlying oocyte maturation, fertilization, and embryo development remains one of the main areas for progress in the ARTs field. Mitochondria are believed to play an essential role in these processes. Mitochondria have a crucial part in producing energy for oocyte maturation and embryo development throughout precise cellular functions comprising Ca2+ homeostasis regulation, glycolysis, amino acid and fatty acid metabolism, and regulation of apoptosis. Recent studies suggest that mitochondrial structure, content, and function may be related to oocyte competence, embryo viability, and implantation success during ARTs. Their defects could lead to low fertilization rates and embryonic development failure. This review aimed to provide an overview of the available literature data surrounding the correlation between changes at ultrastructural level of mitochondria or correlated-mitochondrial aggregates and oocyte quality and ARTs treatments. Our reported data demonstrated that oocyte mitochondrial ultrastructural alterations could be partial or complete recovery during the early embryo stages. However, these changes could persist as quiescent during the pre-implantation embryo development, causing abnormalities that become evident only during fetal and postnatal life. These factors led to consider the mitochondria as a crucial marker of oocyte and embryo quality, as well as a strategic target for further prospective therapeutical approaches.

Does in vitro application of pentoxifylline have beneficial effects in assisted male reproduction?

Application of nonspecific phosphodiesterases inhibitors, such as pentoxifylline (PTX), is a strategy utilised to aid sperm selection from immotile sperm samples prior to ICSI. No extensive studies have yet been performed to verify the safety of the clinical outcomes of ICSI after PTX administration. In this article, we summarise the data reported in the literature that assess the implication of in vitro usage of PTX on sperm parameters, as well as clinical outcomes during assisted male reproduction programme.

Nickel-Titanium Rotary Instruments: An In Vitro Comparison (Torsional Resistance of Two Heat-treated Reciprocating Files).

AIM AND OBJECTIVE: The present study aims to evaluate the difference in torsional resistance of two reciprocating nickel-titanium (Ni-Ti) rotary files: WaveOne Gold and EdgeOne Fire. MATERIALS AND METHODS: A total of 40 nickel-titanium rotary instruments (n = 40): 20 WaveOne Gold Small (WOGS) and 20 EdgeOne Fire Small (EOFS) were divided into two groups. Each instrument was tested using a torsional resistance device already validated in previous studies to evaluate and compare torsional resistance. The static torsional test was implemented by blocking each instrument at 3 mm from the tip and rotating it until fracture with a reciprocating motion. Torque to fracture (TtF) and fragment length (FL) were measured and statistically analyzed. RESULTS: Statistical analysis of TtF found significant differences between the two groups (p<0.05). The EOFS showed higher TtF if compared to WOGS, with a mean value and a standard deviation of 3.05 ± 0.07 (N cm) against 2.97 ± 0.08 (N cm). Data for FL showed no significant differences (p>0.05) between the two groups. CONCLUSION: According to the results of this study, it is reasonable to assert that EOFS instruments showed a higher torsional resistance if compared to the WOGS. CLINICAL SIGNIFICANCE: As evidenced by this study, EOFS should be considered as a safer solution, in terms of torsional resistance, if compared to WOGS, reducing the risk of intracanal separation due to excessive torsional load.

Efficacy of the in vitro splitting of human preimplantation embryos from ART programs

Background/aim: The aim of this study was to evaluate the efficiency of in vitro embryo splitting (IES) procedures. We also assessed the quality of the blastocysts developed from embryos obtained from different sources. Materials and methods: Good quality embryos at 6­8-cell stages were categorized according to their fertilization sources: 1) frozenwarmed donated embryos, 2) chromosomally abnormal embryos, 3) parthenogenetic embryos, and 4) embryos derived from fertilization of in vitro matured oocytes (rescue IVM). After IES, splitting and developmental efficiency was assessed. Furthermre, the quality of the developed blastocysts was evaluated by Hoechst and propidium iodide (PI) staining. Results: The data showed a high rate of both splitting and developmental efficiency in the frozen-warmed embryos after IES (140% and 71.7%, respectively), followed by chromosomally abnormal embryos (96.8% and 52.5%, respectively). Results of the Hoechst and PI staining showed that the mean ± SD cell numbers of the control group were higher (113.11 ± 16.01) than that of twins A (donor blastomeres embryos, 58 ± 12.2) and B (recipient blastomeres embryos, 50.4 ± 8.5), respectively. Conclusion: Chromosomally normal embryos enrolled in IES are more potent to develop into viable blastocysts. For research purposes, 1PN and 3PN embryos are the best options for splitting procedures, regardless of the poor quality of developed blastocysts.

Germ cell cysts, a fetal feature in mammals, are constitutively present in the adult armadillo.

Formation and subsequent break down of ovarian germ cell (GC) cysts is a key and an evolutionary-conserved developmental event, described in phylogenetically diverse species of invertebrates and vertebrates. In mammals, cyst break down (CBD) ends at the time of, or soon after, birth with the formation of primordial follicles enclosing single oocytes, which constitute the sole reservoir of gametes available through the whole female's reproductive life. In this study, we challenge this paradigm demonstrating the constitutive presence of a large number of cysts, enclosing two-thirty GCs, in the ovary of the adult armadillo Chaetophractus villosus, belonging to the superorder Xenarthra, one of the earliest offshoots among placentals. We also describe that (a) GCs enclosed within cysts are connected by intercellular bridges-intercellular bridges-markers of their clonal origin; (b) CBD occurs through four main phases, ending with primordial follicles containing single oocytes; (c) GCs encompass meiotic prophase I stages, from leptotene to diplotene; (d) seasonal variations in the number of GCs enclosed within cysts, suggesting the presence of a GC multiplying activity. The armadillo C. villosus''s ovary emerges as an extraordinary resource to investigate folliculogenesis and to explore the evolutionary past of the mammalian ovary.

Type of protein supplement in cryopreservation solutions impacts on the degree of ultrastructural damage in frozen-thawed human oocytes.

Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media.

Repeated hyperstimulation affects the ultrastructure of mouse fallopian tube epithelium.

Controlled ovarian hyperstimulation (COH) is routinary used in assisted reproductive technologies (ARTs) to increase the yields of mature oocytes. The possibility that patients with a history of failures or poor-responders may develop side-effects following these treatments is still debated. Epidemiological studies reported controversial results about pregnancy outcome and the risk of developing gynecological cancers. By using a mouse model, here we compared the ultrastructural features of fallopian tubes (FTs) obtained from mice undergoing or not (control, CTR) four (4R) and eight (8R) rounds of gonadotropin stimulation. Although the morphological characteristics of oviductal layers seemed unaffected by repeated treatments, dose-response ultrastructural alterations in the ampulla appeared in the 4R group and even more in the 8R group. The targets were oviductal ciliated (CCs) and non-ciliated (NCCs) cells, which showed damaged mitochondria and glycogen accumulations in the cytoplasm. The drastic reduction of CCs, evident after 4R, was supported by the absence of cilia. After 8R, glycogen granules were significantly reduced and massive degeneration of mitochondria, which appeared swollen and/or vacuolated, occurred in NCCs. Moreover, disintegrated mitochondria were found at the periphery of mitophagic vacuoles with evident signs of cristolysis. The morphometric analysis evidenced a significant increase in the density and frequency of damaged mitochondria after 4R and 8R. The absence of cilia, necessary to sustain oviductal transport of oocytes, spermatozoa and embryos, may originate from either mitochondrial dysfunction or glycogen consumption. These results suggest that repeated COH treatments could induce alterations impairing fertilization and embryo transport toward the uterus.

Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos.

STUDY QUESTION: Does the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF? SUMMARY ANSWER: Embryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen. WHAT IS KNOWN ALREADY: ART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations. STUDY DESIGN, SIZE, DURATION: Zygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM). MAIN RESULTS AND THE ROLE OF CHANCE: Embryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Findings in mice embryos might be different from human embryos. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.

The impact of sperm DNA fragmentation on ICSI outcome in cases of donated oocytes.

PURPOSE: The aim of this study is to evaluate the sperm DNA fragmentation index (DFI) in oocyte donation cycles and correlate it with the sperm parameters, the male characteristics, the embryo quality and the outcome of intracytoplasmic sperm injection (ICSI). METHODS: A total of 150 couples participating in an oocyte donation program were included in the study. Sperm samples were assessed by conventional sperm analysis. DFI was evaluated using the Halosperm kit, a sperm chromatin dispersion test (SCD). RESULTS: The relations between DNA damage and epidemiological male factors (age, height, weight), standard semen parameters (concentration, total and forward motility, and morphology), and embryological and clinical parameters (fertilization rate, total blastocyst number, number of good quality blastocyst, clinical pregnancy) were analyzed. DFI was positively correlated with advanced male age (r = 0.23, p < 0.05) and negatively correlated with total sperm and forward motility (r = - 0.29, r = - 0.27, respectively; p < 0.05). DFI was not significantly correlated with pregnancy outcome in oocyte donation cycles (r = - 0.05, p > 0.05). When good quality blastocysts were chosen, a trend toward the development of good quality embryos was detected in the presence of a low DFI (r = - 0.20, p = 0.08). CONCLUSIONS: DFI does not significantly affect the outcome of ICSI in oocyte donation cycles. Even in cases of advanced paternal age that a high DFI resulted sperm DNA fragmentation seems not to adversely affect the final outcome.

Mancozeb impairs the ultrastructure of mouse granulosa cells in a dose-dependent manner.

Mancozeb, an ethylene bis-dithiocarbamate, is widely used as a fungicide and exerts reproductive toxicity in vivo and in vitro in mouse oocytes by altering spindle morphology and impairing the ability to fertilize. Mancozeb also induces a premalignant status in mouse granulosa cells (GCs) cultured in vitro, as indicated by decreased p53 expression and tenuous oxidative stress. However, the presence and extent of ultrastructural alterations induced by mancozeb on GCs in vitro have not yet been reported. Using an in vitro model of reproductive toxicity, comprising parietal GCs from mouse antral follicles cultured with increasing concentrations of mancozeb (0.001-1 µg/ml), we sought to ascertain the in vitro ultrastructural cell toxicity by means of transmission (TEM) and scanning (SEM) electron microscopy. The results showed a dose-dependent toxicity of mancozeb on mouse GCs. Ultrastructural data showed intercellular contact alterations, nuclear membrane irregularities, and chromatin marginalization at lower concentrations, and showed chromatin condensation, membrane blebbing, and cytoplasmic vacuolization at higher concentrations. Morphometric analysis evidenced a reduction of mitochondrial length in GCs exposed to mancozeb 0.01-1 µg/ml and a dose-dependent increase of vacuole dimension. In conclusion, mancozeb induced dose-dependent toxicity against GCs in vitro, including ultrastructural signs of cell degeneration compatible with apoptosis, likely due to the toxic breakdown product ethylenethiourea. These alterations may represent a major cause of reduced/delayed/missed oocyte maturation in cases of infertility associated with exposure to pesticides.

Human cumulus cell sensitivity to vitrification, an ultrastructural study.

SummaryCumulus cells (CCs) play an important role in the regulation of female gamete development, meiotic maturation, oocyte-sperm interaction, capacitation and acrosome reaction. However, their role in maintaining oocyte competence after vitrification is unclear as controversial data on their protecting action against oocyte cryoinjuries are available. Here we described the effects of vitrification on the ultrastructure of human CCs collected from women undergoing assisted reproductive technologies (ARTs). In total, 50 patches of CCs, sampled from high-quality human cumulus-oocyte complexes, were randomly allocated into two groups after patient informed consent: 1, fresh CCs (controls, n = 25); 2, vitrified CCs (n = 25). Samples were then prepared and observed by transmission electron microscopy. In fresh CCs, in which small cell clusters were visible, cell membranes were joined by focal gap junctions. Microvilli were rare and short. Nuclei, mitochondria, smooth endoplasmic reticulum (SER), Golgi apparatus and lipid droplets appeared well preserved; vacuoles were scarce. After vitrification, we observed two populations of CCs: light CCs, with a smooth appearance and few short microvilli; and dark CCs, with numerous and long microvilli. In both, most of the organelles appeared similar to those of fresh CCs. Lipid droplets were denser and more numerous, with respect to fresh CCs. They were mainly located in the peri-nuclear and sub-plasmalemmal regions. Numerous packed electron-negative vacuoles were visible. The vitrification procedure did not cause alterations in the fine structure of major organelles, except for an increased amount of lipid droplets and vacuoles. This specific sensitivity of human CCs to vitrification should be considered during ARTs.

Vitrification of human immature oocytes before and after in vitro maturation: a review.

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

EGF-FSH supplementation reduces apoptosis of pig granulosa cells in co-culture with cumulus-oocyte complexes.

In cattle breeding, co-culture with granulosa cells (GCs) is one of the strategies to improve oocyte maturation and fertilization potential, but yields are still suboptimal due to GC apoptosis. We previously set up an in vitro co-culture system of cumulus-oocyte-complexes (COCs) anchored to GC multilayers adhering to the basal lamina (COCGs), in which GC apoptosis was inhibited by FSH supplementation. Here, we assessed the antiapoptotic effect of EGF (5 ng/ml-EGF5) alone or in synergism to FSH (50mU/ml-FSH50) on pig COCGs. COCG morphology, apoptotic rate, procaspase-8 and-9 expression levels and surface ultrastructure were determined. Results showed an increased % of apoptotic GCs in control and EGF5 (≈80%) respect to sampling (≈3%) and caspase-8 and -9 activation. In contrast, apoptotic cells were significantly reduced by FSH50 (≈35%) supplementation, with inactive Procaspase-8 and -9 highly expressed. The pro-survival effect of FSH was strengthened by EGF (EGF5+FSH50), as evidenced by a significant reduction of apoptosis (≈15%) and high expression levels of Procaspase-8 and -9. Ultrastructural analysis revealed that GC multilayers were characterized by round-to-ovoid cells connected each other and to the basal lamina by cytoplasmic projections. Microvilli shortening/thickening/reduction, cytoplasmic projection rarefaction, blebbing of apoptotic bodies and degenerating/atresic GCs were observed in control and EGF5 groups. FSH50 induced the formation of an abundant mucinous matrix, due to granulosa expansion. Blebs and atresic areas were rarely observed. In EGF5+FSH50 group, GCs were well-preserved, richly covered by microvilli and connected by numerous cytoplasmic projections. Degenerative phenomena were rarely observed. In conclusion, EGF in synergism with FSH seems to better counteract GC apoptosis in a co-culture of pig GC multilayers.

Ultrastructure of human oocytes after in vitro maturation.

STUDY HYPOTHESIS: How does the ultrastructure of human oocytes matured in vitro compare with oocytes collected from women after full hormonal stimulation? STUDY FINDING: The ultrastructure of human oocytes matured in vitro is largely, but not entirely, similar to those matured in vivo. WHAT IS KNOWN ALREADY: Embryos derived from in vitro-matured oocytes often have limited developmental potential, possibly as an effect of inappropriate in vitro maturation (IVM) conditions. Transmission electron microscopy (TEM) is a valuable research tool to compare in vivo and in vitro matured oocytes. However, previous studies on the ultrastructure of human IVM oocytes were done with inadequate material or inappropriate IVM conditions, and have limited significance. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Immature cumulus cell-enclosed oocytes, retrieved from mid-sized antral follicles of women requiring IVM treatment, were matured in vitro for 30 h. No leftover germinal vesicle-stage oocytes collected from fully stimulated cycles were used. Control in vivo matured oocytes were obtained from age-matched women undergoing full ovarian stimulation. In vitro and in vivo matured oocytes were analysed by TEM and compared according to previously established morphometric criteria of oocyte quality. MAIN RESULTS AND THE ROLE OF CHANCE: All oocytes had normal ooplasm showing uniform distribution of organelles. Mitochondrial morphology appeared similar between the maturation conditions. Cortical granules were found typically stratified in a single, mostly continuous row just beneath the ooplasm in all oocytes. Microvilli were well preserved after IVM. Vacuoles were only occasionally found in all oocytes and, if present, they were frequently associated with lysosomes. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and mitochondria-vesicles (MV) complexes were commonly found in in vivo matured oocytes. However, large MV complexes partially replaced M-SER aggregates in IVM oocytes. LIMITATIONS, REASONS FOR CAUTION: As a note of caution it should be noticed that, being laborious and technically demanding, TEM cannot be applied to a large number of samples in a single investigation. Therefore, our data require further independent confirmation. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggests the notion that TEM remains a valuable research tool that can also offer quantitative data if associated with morphometric criteria of evaluation. Therefore, it can be adopted to test pre-clinically the performance of novel in vitro systems that are demanded to make oocytes IVM more successful in the human. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was independently funded by Biogenesi Reproductive Medicine Centre, Monza, Italy. All authors declare that their participation in the study did not involve factual or potential conflicts of interests.

Ultrastructure of cytoplasmic fragments in human cleavage stage embryos.

PURPOSE: The goal of this study was to evaluate the ultrastructure of cytoplasmic fragments along with the effect of cytoplasmic fragment and perivitelline space coarse granulation removal (cosmetic microsurgery) from embryos before embryo transfer on ART outcomes. METHODS: One hundred and fifty intracytoplasmic sperm injection cycles with male factor infertility were included in this prospective study. Patients were divided into three groups of case (n = 50), sham (n = 50), and control (n = 50). Embryos with 10-50 % fragmentation were included in this study. Cosmetic microsurgery and zona assisted hatching were only performed in case and sham groups respectively. Extracted fragments were evaluated ultrastructurally by transmission electron microscopy (TEM). Rates of clinical pregnancy, live birth, miscarriage, multiple pregnancies, and congenital anomaly in the three groups were also compared. RESULTS: Micrographs from TEM showed that mitochondria were the most abundant structures found in the fragments along with mitochondria-vesicle complexes, Golgi apparatus, primary lysosomes, and vacuoles. There were no significant differences in demographic characteristics, laboratory and clinical data, or embryo morphological features between the groups. The rate of clinical pregnancy in control, sham, and case groups had no significant differences (24, 18, and 18 %, respectively). The rates of live birth, miscarriage, multiple pregnancy, and congenital anomaly were also similar between the different groups. CONCLUSIONS: Our data demonstrated that cosmetic microsurgery on preimplantation embryos had no beneficial effect on ART outcomes in unselected groups of patients. As mitochondria are the most abundant organelles found in cytoplasmic fragments, fragment removal should be performed with more caution in embryos with moderate fragmentation.

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes.

PURPOSE: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. METHODS: Samples were studied by light and transmission electron microscopy. RESULTS: We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. CONCLUSIONS: This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism.

Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis.

BACKGROUND: Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification. METHODS: We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation. RESULTS: Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90-100 µm in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3-4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8-9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied. CONCLUSIONS: Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane "recycling" that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardening.