Oocyte cytoplasmic diameter of ≥130 µm can be used to determine human giant oocytes.

OBJECTIVE: To determine if a cytoplasmic diameter of ≥130 µm can help identify human giant oocytes (GOs) in clinical practice and confirm the presence of genetic abnormalities in GOs by assessing the spindle length and centromere numbers. DESIGN: Case-control study. SETTING: Private in vitro fertilization clinic. PATIENT(S): The subjects were women aged 20-49 years who underwent oocyte retrieval after ovarian stimulation from January 2014 to December 2020. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The oocyte diameter was measured; immunofluorescent staining was performed to assess the spindle diameter and centromere numbers. RESULT(S): Among the 254,337 oocytes examined, 561 (0.22%) had a diameter of ≥130 µm. The mean diameter ranges in the normal-sized metaphase II (MII) oocytes (MII group) and GO group were 103.0-119.0 and 132.3-175.9 µm. Spindle size could be measured in 6 GOs with 1 spindle (GO1), 10 GOs with 2 spindles (GO2), and 16 MII groups. The equatorial plane and pole-to-pole distance in the GO1 were significantly longer than in the GO2 and MII groups. The median numbers of centromeres were 86 in GOs with 1 spindle and 42 in each spindle for GOs with 2 spindles among 11 GO1s and 5 GO2s. CONCLUSION(S): This study is the first to define GOs as oocytes with a diameter of ≥130 µm and is a large-scale study surveying the incidence of GO. It is also the first study to analyze and elucidate the relationship between spindle numbers within the cytoplasm of GOs and spindle size and centromeres.

Gonadotropin levels at the start of ovarian stimulation predict normal fertilization after hCG re-trigger in GnRH antagonist cycles.

PURPOSE: To assess the appropriateness of human chorionic gonadotropin (hCG) re-trigger in poor responders to gonadotropin-releasing hormone agonist (GnRHa) trigger in controlled ovarian stimulation (COS) cycles. METHODS: The 2251 cycles in 2251 patients triggered with GnRHa for oocyte stimulation, with or without requiring hCG re-trigger between 2013 and 2018, were retrospectively analyzed to compare gonadotropin levels at the start of COS and the rate of normal fertilization between the re-trigger and non-re-trigger group. Furthermore, patients in the re-trigger group were stratified by the rate of normal fertilization (good: ≥60% or poor: <60%) to compare patient demographics, hormone profiles, and clinical outcome between the subgroups. RESULTS: In the re-trigger group, FSH and LH levels at the start of COS were significantly lower in the good fertilization group than in the poor fertilization group (P < .01). Receiver operating characteristic curves identified cutoff values of the FSH and LH levels of 1.30 and 0.35 mIU/mL, respectively, for predicting ≥60% normal fertilization. CONCLUSION: Gonadotropin levels at the start of COS are predictors of response to GnRHa trigger and hCG re-trigger necessity, and may serve as indicators to help clinicians appropriately choose hCG re-trigger rather than abandoning the cycles or continuing the first oocyte aspiration attempt.

Development of an automated two pronuclei detection system on time-lapse embryo images using deep learning techniques.

PURPOSE: To establish an automated pronuclei determination system by analysis using deep learning technology which is able to effectively learn with limited amount of supervised data. METHODS: An algorithm was developed by explicitly incorporating human observation where the outline around pronuclei is being observed in determining the number of pronuclei. Supervised data were selected from the time-lapse images of 300 pronuclear stage embryos per class (total 900 embryos) clearly classified by embryologists as 0PN, 1PN, and 2PN. One-hundred embryos per class (a total of 300 embryos) were used for verification data. The verification data were evaluated for the performance of detection in the number of pronuclei by regarding the results consistent with the judgment of the embryologists as correct answers. RESULTS: The sensitivity rates of 0PN, 1PN, and 2PN were 99%, 82%, and 99%, respectively, and the overlapping 2PN being difficult to determine by microscopic observation alone could also be appropriately assessed. CONCLUSIONS: This study enabled the establishment of the automated pronuclei determination system with the precision almost equivalent to highly skilled embryologists.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice.

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.

The acceleration of reproductive aging in Nrg1flox/flox ;Cyp19-Cre female mice.

Irregular menstrual cycles, reduced responses to exogenous hormonal treatments, and altered endocrine profiles (high FSH/high LH/low AMH) are observed in women with increasing age before menopause. In this study, because the granulosa cell-specific Nrg1 knockout mice (gcNrg1KO) presented ovarian and endocrine phenotypes similar to older women, we sought to understand the mechanisms of ovarian aging and to develop a new strategy for improving fertility in older women prior to menopause. In the ovary of 6-month-old gcNrg1KO mice, follicular development was blocked in bilayer secondary follicles and heterogeneous cells accumulated in ovarian stroma. The heterogeneous cells in ovarian stroma were distinguished as two different types: (i) the LH receptor-positive endocrine cells and (ii) actin-rich fibrotic cells expressing collagen. Both the endocrine and fibrotic cells disappeared following long-term treatment with a GnRH antagonist, indicating that the high levels of serum LH induced the survival of both cell types and the abnormal endocrine profile to reduce fertility. Moreover, follicular development to the antral stages was observed with reduced LH and the disappearance of the abnormal stromal cells. Mice treated with the GnRH antagonist regained normal, recurrent estrous cycles and continuously delivered pups for at least for 3 months. We conclude that endocrine and matrix alternations occur within the ovarian stroma with increasing age and that abolishing these alternations resets the cyclical release of LH. Thus, GnRH antagonist treatments might provide a new, noninvasive strategy for improving fertility in a subset of aging women before menopause.