Effects of Simulated Microgravity In Vitro on Human Metaphase II Oocytes: An Electron Microscopy-Based Study.

The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria-vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocytes.

Cleavage-stage embryo micromanipulation in the clinical setting.

Embryo micromanipulation was developed after introduction of microinjection to overcome infertility. Embryo micromanipulation may be performed at any embryo stage from pronuclear to blastocyst. The technique started out as basic and turned out to be increasingly more complex. Embryo micromanipulation at the cleavage-stage includes a wide range of techniques, from opening the zona pellucida in order to improve the chance of implantation, to removing detrimental components from the embryo to enhance embryo development or blastomeres for preimplantation genetic diagnosis and embryo splitting. Evaluating the impact(s) of different micromanipulation techniques on epigenetics of the embryo and considering quality control during these techniques are important issues in this regard. This review aims to discuss the micromanipulation of cleavage-stage embryos in clinical assisted reproductive technology (ART). ABBREVIATIONS: ART: assisted reproductive technology; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; PGD: preimplantation genetic diagnosis; PZD: partial zona dissection; ZP: zona pellucida; SUZI: subzonal insemination; PVS: perivitelline space; AH: assisted hatching; LAH: laserassisted hatching; ZT: zona thinning; UV: ultraviolet; IR: infrared; PCR: polymerase chain reaction; FISH: fluorescent in situ hybridization; NGS: next generation sequencing; QC: quality control; QA: quality assurance.

Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments.

The aim was to study the ultrastructure of cytoplasmic fragments along with the effect of cosmetic micromanipulation (CM) on the morphology and development of vitrified-warmed embryos as well as assisted reproductive technology (ART) outcomes. A total of 96 frozen embryo transfer (FET) cycles were included in this prospective randomized study. They were divided into three groups of CM (n=32), sham (n=32) and control (n=32). In the CM group, the vitrified- warmed embryos were subjected to fragments and coarse granules removal (cosmetic micromanipulation) after laser assisted zona hatching (LAH); sham group subjected only to LAH and no intervention was taken for the control group. Fragmented embryo was evaluated by transmission electron microscopy (TEM). Significant improvement was observed in the morphological parameters, such as fragmentation degrees, evenness of the blastomeres and embryo grade during the subsequent development, after applying cosmetic micromanipulation, when compared to sham or control groups (P=0.00001). However, there were no differences in the clinical outcomes amongst the three studied groups e.g. the rates of clinical, ongoing and multiple pregnancies, implantation, delivery and live birth. In fine structure view, fragments exhibited uniform cytoplasmic texture containing majority of organelles that were observed in normal blastomeres including mitochondria. In conclusion, application of cosmetic micromanipulation in low-grade vitrified-warmed embryos showed significant improvement on embryo morphology parameters; however, did not result in noticeable improvements in clinical outcomes of the patients undergoing ART program. In addition, embryo vitrification had no adverse effects on fine structure of the fragments.

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.

A novel method for transmission electron microscopy study of cytoplasmic fragments from preimplantation human embryos.

Transmission electron microscopy (TEM) is the main tool for exploring the intracellular damage and organelle distribution. The cause of producing embryo cytoplsamic fragmentation is not completely understood. Since the fragments have detrimental effects on embryo development, the ultrastructural analysis of fragments may play an important role in fragmentation etiology and in embryo development as well. There are no studies regarding the ultrastructure of fragments in transferable embryos, because the preparation for TEM is not vital and embryos are discarded inevitably. This study aims to introduce a new method for ultrastructural evaluation of fragments without damaging the human cleaving embryos. Microsc. Res. Tech. 79:459-462, 2016. © 2016 Wiley Periodicals, Inc.

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.

In vitro maturation is slowed in prepubertal lamb oocytes: ultrastructural evidences.

BACKGROUND: In vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries may avoid side effects connected to hyperstimulation during IVF procedures, including the risk of cancer recurrence. In humans, the scarce availability of immature oocytes limits morphological studies. The monovular ovine may represent an experimental model for IVM studies. METHODS: To assess if the scarce developmental competence of prepubertal oocytes (PO) is related to morphological changes we analyzed, by light and transmission electron microscopy, cumulus-oocyte-complexes (COCs) from lambs (30-40 days old) and sheep (4-6 years old) at sampling and after 7 h, 19 h, 24 h of IVM. Meiotic progression was determined at the same time points. RESULTS: At sampling, the germinal vesicle (GV) of PO was round and centrally or slightly eccentrically located, whereas in adult oocytes (AO) it was irregularly shaped and flattened against the oolemma. PO, differently from AO, showed numerous trans-zonal projections. Organelles, including cortical granules (CGs), were more abundant in AO. After 7 h, the percentage of AO that underwent GVBD-MI transition increased significantly. In PO, the oolemma was juxtaposed to the ZP; in AO, it showed several spikes in correspondence of cumulus cells (CC) endings. In PO, organelles and isolated CGs were scattered in the ooplasm. In AO, groups of CGs were also present under the oolemma. After 19 h, PO underwent GVBD-MI transition; their oolemma showed several spikes, with CC projections retracted and detached from the ZP. AO underwent MI-MII transition; their oolemma regained a round shape. CGs were located beneath the plasmalemma, arranged in multiple, continuous layers, sometime discontinuous in PO. After 24 h, both groups reached the MII-stage, characterized by a regular oolemma and by expanded CCs. PO showed CGs distributed discontinuously beneath the oolemma, while AO showed a continuous monolayer of CGs. CONCLUSIONS: Even if PO were able of reaching morphological maturation after 24 h of IVM, our ultrastructural analysis allowed detecting the presumptive sequence of cytoplasmic alterations connected with the delay of nuclear maturation, that might explain the reduced developmental competence of such oocytes. Data from the sheep model are of interest for zootechny, and provide an experimental basis for improving human IVM technology.

Impact of different embryo loading techniques on pregnancy rates in in vitro fertlization/embryo transfer cycles.

BACKGROUND: Embryo transfer (ET) technique is one of the important factors of in vitro fertlization success. Among the different steps in ET technique, less attention has been given to embryo loading (EL). The aim was to compare the impact of two different techniques of EL on pregnancy rate in IVF/ET cycles. MATERIALS AND METHODS: In this retrospective study, 144 and 170 patients were placed in groups A and B, respectively. In Group A, the embryos were drawn directly into the ET catheter from culture microdrop under the oil. In Group B, the embryos were transferred from culture microdrop into G2 medium in center-well dish. Then, the embryos were drawn into the catheter and finally transferred into the uterus. Both groups were adjusted for other parameters based on the EL technique. The main outcome measure was pregnancy rate. RESULTS: There were insignificant differences for etiology of infertility, source of sperm, type of stimulation protocol, percent of IVF or intracytoplasmic sperm injection type of ET catheter, cycles with good quality embryos and transferred embryos between two groups. The rate of both chemical and clinical pregnancy was higher in Group B compared to A, but the difference was insignificant (P = 0.09 and P = 0.1, respectively). CONCLUSION: It seems that there is no difference in the outcome by loading the embryo from microdrop or center-well dish.

The effect of immature oocytes quantity on the rates of oocytes maturity and morphology, fertilization, and embryo development in ICSI cycles.

PURPOSE: The goal was to evaluate the role of the number of retrieved immature oocytes on mature oocyte counts and morphology, and also the rates of fertilization and embryo development in ICSI cycles. METHODS: 101 ICSI cycles were included in this prospective evaluation. Patients were divided into 2 groups of A (≤ 2 immature oocytes) and B (> 2 immature oocytes). In sub-analysis, the impacts of the number of GV and MI oocytes were assessed on the rates of fertilization and embryo development. Also, correlations between the numbers of immature and mature oocytes, as well as maternal age between two groups were analyzed. Assessments of oocyte morphology, fertilization, embryo quality and development were done accordingly. RESULTS: There was no correlation between the immature oocytes quantity with the number of mature ones. There were insignificant differences for embryo development between two groups, but fertilization rate was higher in group A (P = 0.03). In sub-analysis, insignificant differences were observed between two groups of ≤ and >2 GV and MI oocytes for rates of fertilization and embryo development. Also, the rates of clinical pregnancy and delivery were insignificant between groups. The rate of morphologically abnormal oocytes had no significant difference between two groups, except for wide perivitelline space (PVS) which was higher in group A (P = 0.03). There was no significant difference for maternal age between two groups. CONCLUSIONS: In cases with few retrieved immature oocytes, rates of fertilization and incidence of wide PVS may increase, although immature oocytes may not have any negative impacts on early embryo development, or the rates on number of mature oocytes.

Ultrastructure of isolated mouse ovarian follicles cultured in vitro.

BACKGROUND: In vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles. METHODS: The present study was designed to evaluate, by transmission and scanning electron microscopy, the ultrastructural features of isolated mouse preantral follicles cultured in vitro for 6 days in a standard medium containing fetal calf serum (FCS). The culture was supplemented or not with FSH. RESULTS: The follicles cultured in FCS alone, without FSH supplementation (FCS follicles), did not form the antral cavity. They displayed low differentiation (juxta-nuclear aggregates of organelles in the ooplasm, a variable amount of microvilli on the oolemma, numerous granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). Eighty (80)% of FSH-treated follicles formed the antral cavity (FSH antral follicles). These follicles showed various ultrastructural markers of maturity (spreading of organelles in ooplasm, abundant microvilli on the oolemma, scarce granulosa cell-oolemma contacts, granulosa cell proliferation). Areas of detachment of the innermost granulosa cell layer from the oocyte were also found, along with a diffuse granulosa cell loosening compatible with the antral formation. Theca cells showed an immature morphology for the stage reached. Twenty (20)% of FSH-treated follicles did not develop the antral cavity (FSH non-antral follicles) and displayed morphological differentiation features intermediate between those shown by FCS and FSH antral follicles (spreading of organelles in the ooplasm, variable amount of microvilli, scattered granulosa cell-oolemma contacts, signs of degeneration in granulosa cell compartment). CONCLUSIONS: It is concluded that FSH supports the in vitro growth of follicles, but the presence of a diffuse structural granulosa cell-oocyte uncoupling and the absence of theca development unveil the incomplete efficiency of the system. The present study contributes to explain, from a morphological point of view, the effects of culture conditions on the development of mouse in vitro grown follicles and to highlight the necessity of maintaining efficient intercellular communications to obtain large numbers of fully-grown mature germ cells.

Morphological expression of angiogenesis in the mammalian ovary as seen by SEM of corrosion casts.

In the mammalian ovary, follicular and corpus luteum cycle is associated with intensive microvascular remodelling. The complex angiogenic dynamics are finely tuned by numerous regulatory factors acting as activators (up-regulators) or inhibitors (down-regulators) of angiogenesis. Alterations of such a tight modulation are involved in several pathologies, including infertility, polycystic ovarian syndrome, ovarian hyperstimulation syndrome and ovarian cancer. We have demonstrated in several experimental models that ovarian function is critically and specifically dependent on angiogenesis for follicular development, ovulation, and corpus luteum growth. The aim of this review is to summarize the results we have obtained on the morphodynamic remodelling of ovarian microvascularization, in polyovulatory (rat, rabbit and pig) and monovulatory species (cow), using scanning electron microscopy of vascular corrosion casts. The knowledge of the morphological expression of the up- and down-regulation of angiogenesis occurring in mono and polyovulatory animals might provide useful information to preserve fertility and to increase of the effectiveness of reproductive management in species of domestic interest.

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols.

PURPOSE: To ascertain possible cell damage from cryopreservation, the ultrastructure of human oocytes cryopreserved by slow cooling was assessed. MATERIALS AND METHODS: Cryopreservation was performed through two protocols with one-step or two-step propanediol. Fresh control oocytes were examined for comparison. Samples were processed for transmission electron microscopy analysis. RESULTS: By light microscopy, both fresh and frozen-thawed oocytes appeared regularly rounded, with intact zona pellucida, and homogeneous cytoplasm. By electron microscopy observation, organelles were abundant and uniformly dispersed. Mitochondria-smooth endoplasmic reticulum associations appeared regular. However, both the amount and density of cortical granules appeared abnormally reduced in frozen-thawed samples. Slight to moderate vacuolization was also found in the ooplasm of oocytes of both frozen groups. CONCLUSIONS: Slow cooling ensures a good overall preservation of human oocytes. However, cytoplasmic vacuolization and cortical granule loss appears associated with cryopreservation, irrespective of the protocol used.

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats.

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.

Preservation of fertility in young cancer patients: contribution of transmission electron microscopy.

During the last decade, new technologies in reproductive medicine have emerged to preserve the fertility of women whose gonadal function is threatened by premature menopause or gonadotoxic treatments. To offer an individualized approach to these patients, different experimental procedures are under investigation, including oocyte cryopreservation and cryopreservation and transplantation of ovarian tissue in the form of cortical fragments, whole ovary or isolated follicles. This review shows that transmission electron microscopy (TEM), combined with other in-vivo and in-vitro analysis techniques, is a valuable tool in the establishment of new experimental protocols to preserve female fertility. Ultrastructural studies allow in-depth evaluation of the oocyte's unique morpho-functional characteristics, which explain its low cryotolerance, and provide essential information on follicular, stromal and endothelial cell integrity, as well as cellular interactions crucial for normal folliculogenesis. In order to be able to offer appropriate and efficient options in every clinical situation, oocyte in-vitro maturation and ovarian tissue transplantation need to be optimized. Further development of new approaches, such as follicular isolation and whole ovary transplantation, should be encouraged. Fine ultrastructural details highlighted by TEM studies will be useful for the further optimization of these emerging technologies.

Cryopreservation and xenotransplantation of human ovarian tissue: an ultrastructural study.

OBJECTIVE: To analyze the ultrastructure of human ovarian follicles after cryopreservation and short-term xenografting. DESIGN: Prospective experimental study. SETTING: Academic gynecology and anatomy research units. PATIENT(S): Ovarian cortical biopsy specimens were obtained from 13 patients. INTERVENTION(S): Each ovarian biopsy specimen was dissected into pieces of 1 mm(3) and divided into three groups: [1] fresh tissue, [2] frozen-thawed tissue, and [3] frozen-thawed tissue xenografted onto the peritoneum of nude mice for 3 weeks. MAIN OUTCOME MEASURE(S): Follicular ultrastructure was assessed by light and transmission electron microscopy in [1] fresh, [2] frozen, and [3] frozen-grafted tissue. RESULT(S): Thirty-five ovarian follicles were analyzed by light and transmission electron microscopy. Twenty-five primordial and primary ovarian follicles were found. Most of them exhibited ultrastructurally well preserved features (fresh [N = 8/10], frozen [N = 7/10], and frozen-grafted [N = 4/5] tissue). Ten secondary follicles were present in xenografts. By transmission electron microscopy, all the healthy-looking secondary follicles (70%) were shown to contain intact oocytes, with features typical of earlier developmental stages, surrounded by several layers of follicular cells. CONCLUSION(S): The present study demonstrates, for the first time, that cryopreservation and xenotransplantation do not appear to greatly affect human primordial/primary follicle ultrastructure. Interestingly, in frozen-thawed xenografts, secondary human ovarian follicles presented a well preserved ultrastructure, but asynchrony between oocyte and granulosa cell development was detected. The possible causes for this asynchrony are discussed.

Digital imaging of stem cells by electron microscopy.

This chapter deals with basic techniques of scanning and transmission electron microscopy applicable to stem cell imaging. It is sometimes desirable to characterize the fine structure of embryonic and adult stem cells to supplement the images obtained by phase-contrast and confocal immunofluorescent microscopy to compare with the microstructure of cells and tissues reported in the literature. This would help confirm their true identity whilst defining their surface and internal morphology. The intention is to put a face on stem cells during their differentiation.

Ultrastructural dynamics of human reproduction, from ovulation to fertilization and early embryo development.

This study describes the updated, fine structure of human gametes, the human fertilization process, and human embryos, mainly derived from assisted reproductive technology (ART). As clearly shown, the ultrastructure of human reproduction is a peculiar multistep process, which differs in part from that of other mammalian models, having some unique features. Particular attention has been devoted to the (1) sperm ultrastructure, likely "Tygerberg (Kruger) strict morphology criteria"; (2) mature oocyte, in which the MII spindle is barrel shaped, anastral, and lacking centrioles; (3) three-dimensional microarchitecture of the zona pellucida with its unique supramolecular filamentous organization; (4) sperm-egg interactions with the peculiarity of the sperm centrosome that activates the egg and organizes the sperm aster and mitotic spindles of the embryo; and (5) presence of viable cumulus cells whose metabolic activity is closely related to egg and embryo behavior in in vitro as well as in vivo conditions, in a sort of extraovarian "microfollicular unit." Even if the ultrastructural morphodynamic features of human fertilization are well understood, our knowledge about in vivo fertilization is still very limited and the complex sequence of in vivo biological steps involved in human reproduction is only partially reproduced in current ART procedures.

Morphological patterns of angiogenesis in ovarian follicle capillary networks. A scanning electron microscopy study of corrosion cast.

To describe the morphological characteristics of the ovarian follicle (F) capillary neoformation and regression, the angiogenic figures were studied by means of scanning electron microscopy of corrosion casts in developing and mature F of rabbit, pig, and cow. Developing F showed gradual neoformation of thecal capillaries characterized by budding and then sprouting, likely from preexisting interstitial vessels. Postcapillary venules frequently showed vasoconstriction rings (sphincters). Vasodilation followed capillary elongation. Mature F, in addition to vessel elongation and dilation, also presented infolding of dilated capillary walls, followed by capillary duplication and sinusoidalization. Periovulatory F mainly showed functional changes, such as capillary dilation, signs of iperpermeabilization, and ischemia, the latter being limited to the apical follicular area. Vessel regression was characterized by thinning of capillaries and presence of avascular areas within the atretic F wall at any stage. This study showed two main types of angiogenic patterns. (a) longitudinal elongation (in series, sprouting angiogenesis) characterizing the initial phase of F development and (b) parallel duplication (in parallel, infolding or intussusceptive angiogenesis), ending in capillary lateral replication or splitting, secondary to functional microvascular changes. Indirect evidence of the establishment of postcapillary resistances contributing to capillary remodeling, was also shown. It is concluded that the sequence of capillary neoformation in mammalian ovarian F occurs in six steps: (1) budding, (2) sprouting (and elongation), (3) dilation, (4) infolding (intussusception), (5) duplication (splitting and elongation), and (6) sinusoidalization. Capillary regression hits F at any stage and characterizes F atresia.

Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization.

The use of somatic cells for cocultures during in vitro fertilization (IVF) is currently finalized to obtain a higher number of healthy and viable embryos with a high potential of implantation. Among the different cell lines that can be used as feeder cells for cocultures, granulosa cells (GCs) are autologous cells, safe and easy to recover. The aim of the present study was to analyze the fine structure of human GCs used in a coculture system to evaluate, from a morphodynamic point of view, their role in supporting embryo development. GCs were collected during oocyte pick-up, 36 h after human chorionic gonadotropin administration, from patients undergoing IVF procedures, who had given their informed consent to be included in this protocol. After coculture, GCs were fixed and processed for light microscopy (LM) and transmission electron microscopy (TEM). By LM, GCs appeared as clusters of loosely packed cells, irregularly rounded or polyhedral in shape, varying in diameter from 18 to 25 microm. Mitotic cells, as well as regressing elements (with pyknotic nuclei or dense cytoplasm) and cell fragments were occasionally observed. By TEM, the plasma membrane was irregular due to the presence of cytoplasmic evaginations. Linear and annular gap junctions between neighboring GCs were found. GC nuclei, rounded and eccentrically located, contained finely dispersed chromatin, one (often two) prominent nucleoli and, infrequently, peripheral patches of heterochromatin. Numerous organelles populated the GC cytoplasm, among them, mitochondria were rod-shaped or elongated, usually provided with tubular-vesicular cristae but occasionally showing atypical, longitudinally oriented cristae. Membranes of smooth endoplasmic reticulum, Golgi stacks and vesicles, secretory-like granules, cisternae of rough endoplasmic reticulum (RER), free ribosomes and polysomes, lysosomal-like bodies, microfilaments, and lipid droplets were also seen in the GC cytoplasm. In most cells, RER was scarcely represented and numerous lipid droplets filled the perinuclear space. On the contrary, some GCs contained an abundant RER and rare lipid droplets scattered in the cytoplasm. In conclusion, our data demonstrated the presence, in a coculture system, of GCs provided with ultrastructural characteristics typical of healthy, metabolically active, mostly steroidogenic cells. Protein-synthetic cells have also been detected. These data, evaluated at the light of biochemical and clinical studies, sustain the capability of human GCs cocultures to positively affect early embryo development in vitro by the secretion of steroids and proteins, putative "embryotrophic" factors.

Surface morphology of the zona pellucida surrounding human blastocysts obtained after in vitro fertilization.

Human zona pellucida (ZP) is maintained up to the blastocyst stage prior to hatching. In in vitro fertilized (IVF) embryos, it eventually acts as a morphodynamic interface between the cultured embryo and its microenvironment. Ultrastructural data on the ZP of IVF blastocysts are scarce in humans. We employed correlated phase contrast microscopy (PCM) and scanning electron microscopy (SEM) to study retrospectively the ultrastructural morphology of the ZP outer surface of 20 IVF human blastocysts from 16 Japanese patients (28-44 years of age, average 36.7+/-4.2) with a history of infertility. Blastocysts were derived from conventional in vitro fertilization (cIVF) (n = 10) and from intracytoplasmic sperm injection (ICSI) (n = 10). Both cIVF and ICSI groups included "clear blastocysts" (n = 5) and "dark blastocysts" (n = 5). By PCM, the clear blastocysts exhibited a regular, round-shaped contour and consisted of clear and voluminous cells. By SEM, they displayed a spongy ZP with numerous fenestrations formed by networked filaments. By PCM, dark blastocysts appeared irregularly shaped and often collapsed, and comprised dark cells and debris. By SEM, their ZP were smooth with remnants of compact fenestrations. In conclusion, viable blastocysts presented a normal ZP outer surface ultrastructure, whereas unhealthy blastocysts showed an altered ZP outer surface, comparable to that of immature/atretic oocytes. Such alterations could reflect sub-optimal culture conditions and/or could be related to blastocyst degenerative processes. The blastocyst ZP surface ultrastructure was unaffected by the fertilization technique (cIVF or ICSI). These data suggest that blastocyst survival in vitro is related to ZP ultrastructure maintenance.