Oncofertility case report: live birth 10 years after oocyte in vitro maturation and zygote cryopreservation.

PURPOSE: This study aims to report a case of urgent fertility preservation in an oncological patient with collection of immature oocytes in the absence of ovarian stimulation that, through in vitro maturation (IVM), followed by ICSI and cryopreservation of zygotes resulted, 10 years later, in the live birth of a healthy baby. METHODS: In September 2008, our clinic performed IVM in a 32-year-old woman diagnosed with a ductal invasive carcinoma with positive estradiol receptors, negative progesterone receptors and positive human epidermal growth factor receptor 2. The retrieval of immature oocytes was performed in the absence of ovarian stimulation after a simple mastectomy and prior to any chemotherapy treatment. The compact cumulus-oocyte complexes (COCs) collected were placed in Lag medium for 2 h, followed by incubation in IVM medium, supplemented with heat inactivated patient serum, recombinant FSH, and recombinant LH. After 30 h in culture, cumulus cells were removed, the metaphase II oocytes were microinjected, and the zygotes obtained were cryopreserved. In 2017, the zygotes were thawed and cultured until day 3. One embryo was transferred and the other cryopreserved. RESULTS: Four compact COCs were collected and subjected to IVM. Two oocytes reached metaphase II and were microinjected. Two zygotes were obtained and were cryopreserved at the two pronuclear stage. Approximately 9 years later, the two zygotes were thawed and cultured until day 3. An embryo with 10 cells was transferred and implanted, resulting in the birth of a healthy baby. CONCLUSIONS: In cases where urgency to start adjuvant therapy requires immediate oocyte collection, IVM may be the only option to obtain fully competent mature oocytes allowing for effective preservation of the reproductive potential.

The educational and professional status of clinical embryology and clinical embryologists in Europe.

STUDY QUESTION: What is the recognition of clinical embryology and the current status of clinical embryologists in European countries, regarding educational levels, responsibilities and workload, and need for a formal education in assisted reproductive technology (ART)? SUMMARY ANSWER: It is striking that the profession of clinical embryology, almost 40 years after the introduction of IVF, is still not officially recognized in most European countries. WHAT IS KNOWN ALREADY: Reproductive medicine has developed into a sophisticated multidisciplinary medical branch since the birth of Louise Brown 37 years ago. The European Board & College of Obstetrics and Gynaecology (EBCOG) has recognized reproductive medicine as a subspeciality and has developed a subspeciality training for gynaecologists in collaboration with the European Society for Human Reproduction and Embryology (ESHRE). However, nothing similar exists for the field of clinical embryology or for clinical embryologists. STUDY DESIGN, SIZE, DURATION: A questionnaire about the situation in clinical embryology in the period of 2012-2013 in the respective European country was sent to ESHRE National representatives (basic scientists only) in December 2013. At this time, 28 European countries had at least one basic scientist in the ESHRE Committee of National Representatives. PARTICIPANTS/MATERIALS, SETTING, METHODS: The survey consisted of 46 numeric, dichotomous (yes/no) or descriptive questions. Answers were obtained from 27 out of 28 countries and the data were tabulated. Data about the numbers of 'ESHRE Certified Embryologists' were taken from the ESHRE Steering Committee for Embryologist Certification. MAIN RESULTS AND THE ROLE OF CHANCE: In 2012, more than 7000 laboratory staff from 1349 IVF clinics in 27 European countries performed over 700 000 fresh and frozen ART cycles. Despite this, clinical embryology is only recognized as an official profession in 3 out of 27 national health systems. In most countries clinical embryologists need to be registered under another profession, and have limited possibilities for organized education in clinical embryology. Mostly they are trained for practical work by senior colleagues. ESHRE embryologist certification so far constitutes the only internationally recognized qualification; however this cannot be considered a subspecialization. LIMITATIONS, REASONS FOR CAUTION: Data were obtained through different methods, by involving national embryologist societies and cycle registers, collecting information from centre to centre, and in some cases by individual assessment of the situation. For these reasons, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: This paper presents the current status of clinical embryology and clinical embryologists in Europe and is an important step towards implementation of clinical embryology as an officially recognized profession. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: No.

Oogenesis: Prospects and challenges for the future.

Oogenesis serves a singular role in the reproductive success of plants and animals. Of their remarkable differentiation pathway what stands out is the ability of oocytes to transform from a single cell into the totipotent lineages that seed the early embryo. As our understanding that commonalities between diverse organisms at the genetic, cellular and molecular levels are conserved to achieve successful reproduction, the notion that embryogenesis presupposes oogenesis has entered the day-to-day parlance of regenerative medicine and stem cell biology. With emphasis on the mammalian oocyte, this review will cover (1) current concepts regarding the birth, survival and growth of oocytes that depends on complex patterns of cell communication between germ line and soma, (2) the notion of "maternal inheritance" from a genetic and epigenetic perspective, and (3) the relative value of model systems with reference to current clinical and biotechnology applications.

Age and gonadotropins control Ca2+-spike acquisition in mouse oocytes isolated from early preantral follicles.

The action of gonadotropins upon the oocyte is known to be crucial at later stages of follicular development in mammals. However, its influence on oocytes at early preantral stages is still a matter of debate. In the present study we evaluated the onset of mouse oocyte's capacity to exhibit calcium spikes during preantral stages of follicular development, prior to meiotic competence acquisition. In particular, through the use of confocal microscopy, we probed for the specific effects of age and gonadotropin stimulation upon the calcium dynamics of preantral follicle oocytes. We found that important developmental changes on the Ca2+ signalling mechanisms take place early during follicular development. Specifically we demonstrate that both age and gonadotropin stimulation increase the capacity of oocytes recovered from preantral follicles to exhibit calcium spikes. We propose that a strictly morphological staging of follicular development is insufficient to predict oocyte behaviour and must take in consideration animal age and gonadotropin environment.

Electre Tri-C, a multiple criteria decision aiding sorting model applied to assisted reproduction.

OBJECTIVE: The aim of this paper is to apply an informatics tool for dealing with a medical decision aiding problem to help infertile couples to become parents, when using assisted reproduction. METHODS: A multiple criteria decision aiding method for sorting or ordinal classification problems, called Electre Tri-C, was chosen in order to assign each couple to an embryo-transfer category. The set of categories puts in evidence a way for increasing the single pregnancy rate, while minimizing multiple pregnancies. The decision aiding sorting model was co-constructed through an interaction process between the decision aiding analysts and the medical experts. RESULTS: According to the sample used in this study, the Electre Tri-C method provides a unique category in 86% of the cases and it achieves a sorting accuracy of 61%. Retrospectively, the medical experts do agree that some of their judgments concerning the number of embryos to transfer back to the uterus of the woman could be different according to these results. The current ART methodology achieves a single pregnancy rate of 47% and a twin pregnancy rate of 14%. Thus, this informatics tools may play an important role for supporting ART medical decisions, aiming to increase the single pregnancy rate, while minimizing multiple pregnancies. LIMITATIONS: Building the set of criteria comprises a part of arbitrariness and imperfect knowledge, which require time and expertise to be refined. Among them, three criteria are modeled by means of a holistic classification procedure by the medical experts.

Effects of mouse ovarian tissue cryopreservation on granulosa cell-oocyte interaction.

BACKGROUND: Current ovarian tissue cryopreservation protocols have yet to be assessed in terms of somatic-germ cell interaction. Accordingly, post-thaw analysis of antral follicles can yield relevant data on the disruption of the granulosa-oocyte interface. METHODS: We compared fresh mouse ovarian tissue with tissues that had been either cryopreserved using dimethylsulphoxide (DMSO) or glycerol as cryoprotectants, or exposed to such cryoprotectants without freezing. The assessed parameters were: number of immature oocytes retrieved per ovary, allocation of the oocytes to different classes regarding antral follicle size and oocyte-granulosa cell adhesion, and the relative density of transzonal processes containing filamentous actin (TZPs-Act). RESULTS: Although cryopreservation reduces the average number of oocytes retrieved per ovary, it increases the relative distribution of granulosa-free oocytes while decreasing that of granulosa-enclosed ones. Additionally, a post-thaw decrease in TZPs-Act density was recorded. This decrease was also observed after cryoprotectant exposure without freezing, although at a lower level. For the assessed parameters, DMSO was more effective than glycerol as a cryoprotectant. CONCLUSIONS: In situ cryopreservation of granulosa-oocyte complexes with current protocols disrupts the granulosa-oocyte interface. The different patterns of granulosa cell adhesion and interaction in oocytes derived from different-sized antral follicles further suggests that the granulosa-oocyte interface may be developmentally regulated.

Cytokeratin dynamics during oocyte maturation in the hamster requires reaching of metaphase I.

Cytoskeletal components like microfilaments and microtubules are known to play important roles during the processes of oocyte maturation, fertilization and early embryonic development in mammals. However, the roles of other components such as cytoplasmic intermediate filaments, during these critical events remain largely unknown. Oocyte maturation is the final step of oogenesis, immediately before ovulation. Several cytological changes involving the cytoskeleton take place during the maturation process, including meiotic spindle formation, redistribution of cell organelles, membrane polarization and first polar body emission. In this study we determined the organization and rearrangements of cytokeratins during hamster oocyte maturation. Fully grown oocytes were cultured and then visualised using microscopic immunolabelling techniques to monitor the cytokeratin dynamics at specific meiotic stages of the maturation process. In prophase-I-arrested fully grown hamster oocytes, cytokeratins are confined to 4-10 large cortical aggregates, corresponding to extensive meshworks of intermediate filaments. These large aggregates disperse into multiple small spots starting at metaphase I until the end of the maturation period at metaphase II, where cytokeratin exhibits a homogeneously distributed spotted pattern. However, meiotic progression to metaphase II is not necessary for cytokeratin redistribution to occur, since precociously arrested metaphase I oocytes also exhibit dispersed cytoplasmic foci at the end of the culture period. The redistribution of cytokeratins is insensitive to nocodazole and cytochalasin D suggesting it occurs independent of microtubules and microfilaments. In contrast, both cumulus cells and protein synthesis are required for cytokeratin modifications to take place during oocyte maturation. These results show that cytokeratin intermediate filaments are present in the fully grown hamster oocyte, and that a striking reorganization of cytokeratins, triggered by attainment of the metaphase I stage, occurs during maturation.

Hormonal regulation of meiotic maturation in the hamster oocyte involves a cytoskeleton-mediated process.

Maturation of the mammalian oocyte involves hormone-induced meiotic cell cycle progression from prophase I to metaphase II and extrusion of the first polar body (PB). In this study, the effects of gonadotrophins on meiotic cell cycle progression in cultured hamster oocytes were analyzed with respect to changes in cumulus cell-oocyte interactions and the oocyte cytoskeleton. Cumulus-oocyte complexes were obtained from large antral follicles (> or = 700 microns in diameter) of eCG-primed animals and were cultured in the presence or absence of gonadotropins alone (FSH, hCG, LH) or in combination (FSH + hCG or FSH + LH). Oocytes were analyzed using conventional, digital, and confocal fluorescence microscopy to monitor chromatin, actin, and tubulin organization under different culture conditions. Most oocytes (83%) cultured with FSH alone progressed to and arrested at metaphase II and extruded the first PB; in contrast, meiotic progression and PB extrusion were impaired (45-85%) in all other groups. The presence of cumulus cells associated with the oocyte was found necessary for progression to metaphase I and for first PB emission. Completion of meiotic maturation in the presence of FSH alone was correlated with enhanced cortical actin polymerization in the oocyte and the retraction of actin-containing transzonal cumulus processes. The results demonstrate that gonadotropins exert specific effects on meiotic progression, PB emission, cumulus-oocyte interactions, and oocyte cytoskeletal organization during in vitro maturation of hamster oocytes, indicating that the hormonal control of meiosis involves cytoskeletal changes in both the somatic and germ cells.

Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations.

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.

Cytokeratin in early hamster embryogenesis and parthenogenesis: reorganization during mitosis and association with clusters of interchromatinlike granules.

In vivo obtained golden hamster embryos were used to study, by immunofluorescence and immunoelectron microscopy, the main cytokeratin pattern rearrangements during completion of meiosis and the first cleavage division. Our results point to three major re-organization steps: (1) diffuse immunofluorescent cytokeratin spots characteristic of recently ovulated oocytes rearrange into large cortical patches interconnected by fibrils in one-cell embryos; (2) during mitosis a homogeneous cytokeratin spotty pattern reappears; (3) in two-cell embryos cortical and perinuclear cytokeratin fibrillar networks become prominent. Parthenogenotic oocytes were able to mimic the major cytokeratin patterns observed until the first embryonic mitosis, supporting the concept of a maternally established common response to activation. Despite the lack of fibrillar immunofluorescent reactivity during mitosis, electron microscopy demonstrates persistence of 10 nm filament meshworks. These cytokeratin meshworks often associate with clusters of interchromatinlike granules, which persist in the cytoplasm for a short period after nuclear envelope reassembly.

Cytokeratin filaments are present in golden hamster oocytes and early embryos.

Light and electron microscope methods were used to study cytokeratin expression in the recently ovulated oocytes, fertilized eggs and early embryos from the golden hamster. Two cytokeratin polypeptides (Mr 51,000 and 58,000) were detected in oocyte lysates by immunoblotting using a polyclonal antiserum to prekeratin. In the oocyte, cytokeratin occurred as patchy aggregates consisting of short anastomosing 10-nm filaments that formed tight meshworks distributed throughout the cytoplasm. After fertilization the aggregates appeared to merge, becoming larger and concentrated at the cortical region. Prominent immunofluorescent fibrils were seen interconnecting the aggregates. In the 2-, 4- and 8-cell embryos, networks of cytokeratin filaments extended throughout the cortical and perinuclear regions, while aggregates progressively disappeared.

Three-dimensional environments preserve extracellular matrix compartments of ovarian follicles and increase FSH-dependent growth.

In this study we performed a systematic comparative analysis of two culture environments-flat/adhesive liquid and three-dimensional collagen gel-upon in vitro ovarian follicle development. We paid particular attention to the effects of in vitro environments upon the preservation of follicular structure and of peri- and intra-follicular extracellular matrix. We show that flat/adhesive environment leads to an obvious distortion of follicle morphology, marked extracellular matrix modifications and high rates of spontaneous, i.e., FSH-independent, follicle disruption. In contrast, three-dimensional collagen gel environments are able to maintain follicular structure with an in vivo-like basal lamina architecture, minimizing spontaneous disruption. Follicle distortions found in flat/adhesive culture systems include a pronounced flattening, causing the follicle horizontal diameters not to adequately reflect follicle volume. Our volume data, based on three-axis follicle diameter measurements, indicate that three-dimensional collagen gel environments increase follicle growth, particularly in response to FSH. This study demonstrates that preservation of both peri- and intra-follicular extracellular matrix compartments during the in vitro growth and differentiation of ovarian follicles is highly desirable, and is now possible through the use of appropriate three-dimensional collagen gel culture environments. This system allows a better understanding of the specific roles played by each of the follicle compartments during development.