ABSTRACT
Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.
Subject(s)
Biomarkers , Cumulus Cells/metabolism , Gene Expression Regulation , Oocytes/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Female , Gene Expression Profiling , Humans , RNA Stability , TranscriptomeABSTRACT
Oocyte morphology assessment is easy to implement in any laboratory with possible quality grading prior to fertilization. At present, comprehensive oocyte morphology scoring is not performed as a routine procedure. However, it may augment chances for successful treatment outcomes if a correlation with certain dysmorphisms can be proven. In order to determine a correlation between oocyte morphology and treatment outcome, we performed a systematic search in PubMed and Cochrane Controlled Trials Register following PRISMA guidelines. A total of 52 articles out of 6,755 search results met the inclusion criteria. Dark colour of the cytoplasm (observed with an incidence rate of 7%), homogeneous granularity of the cytoplasm (19%) and ovoid shape of oocytes (7%) appeared to have no influence on treatment outcome. Abnormalities such as refractile bodies (10%), fragmented first polar body (37%), dark zona pellucida (9%), enlarged perivitelline space (18%) and debris in it (21%) are likely to affect the treatment outcome to some extent. Finally, cytoplasmic vacuoles (4%), centrally located cytoplasmic granularity (12%) and clusters of smooth endoplasmic reticulum (4%) negatively impact infertility treatment outcomes. Nonetheless, morphological assessment is informative rather than predictive. Adding oocyte morphology to the artificial intelligence (AI)-driven selection process may improve the precision of the algorithms. Oocyte morphology assessment can be especially useful in oocyte donation cycles, during oocyte freezing for fertility preservation and finally, objective oocyte scoring can be important in cases of very poor treatment outcome as a tool for explanation of results to the patient.
Subject(s)
Artificial Intelligence , Infertility , Humans , Oocyte Donation , Oocytes , Zona PellucidaABSTRACT
The aim of this study was to investigate whether pH is stable when transporting ovarian tissue in media buffered with either HEPES or histidine. Furthermore, if the choice of transport media impacts the in vitro maturation rate of oocytes collected in connection with ovarian tissue cryopreservation. Human ovaries (n = 34) collected for ovarian tissue cryopreservation were transported immersed in either 30 ml of HEPES buffered (follicle flushing media (Origio; Denmark)) or histidine buffered media (Custodiol®-HTK, Koehler-Chemie, Germany). Tissue was transported on ice for 4-5 h. At arrival, the ovary was weighed, and the pH of the media was measured at 0 °C. From 15 patients, immature oocytes were collected for in vitro maturation, oocytes that matured to metaphase II were evaluated. The pH measured in the HEPES buffered media (pH = 7.5 ± 0.13, n = 18) was significantly higher (p < 0.001) than the pH measured in the histidine buffered media (pH = 7.2 ± 0.05, n = 16). The standard deviation of pH measurements for the histidine buffered media was significantly lower than for the HEPES buffered media measurements (p < 0.0001). A total of 170 and 247 immature oocytes were collected and in vitro matured from ovaries transported in HEPES and histidine buffered media, respectively. The maturation rate of immature oocytes after IVM was similar in the two groups. The results show that pH in the histidine buffered media is closer to the physiological level and more stable than in HEPES buffered medium and support the use of histidine buffered media for cooled transportation of human ovaries.
Subject(s)
Cryopreservation/methods , Histidine/metabolism , Ovary/drug effects , Adolescent , Adult , Female , Humans , Hydrogen-Ion Concentration , Young AdultABSTRACT
OBJECTIVE: To evaluate the use of cryopreserved ovarian tissue in the Danish fertility preservation cohort. DESIGN: Retrospective cohort study. SETTING: University hospitals and fertility clinics. PATIENT(S): Ovarian tissue cryopreservation (OTC) was performed for 1,186 Danish girls and women from 1999-2020, of whom 117 subsequently underwent ovarian tissue transplantation (OTT). Subgroup 1 included 759 patients with a follow-up period of >5 years. Out of these, OTT rates were further analyzed for those patients who were alive and aged >24 years in July 2020 (subgroup 2; n = 554). INTERVENTION(S): OTC and OTT. MAIN OUTCOME MEASURE(S): OTT, death, donation of tissue. RESULT(S): In subgroup 1, 14% of the patients had undergone OTT, 18% had died, 9% had donated their tissue for research, and 59% still had their tissue stored. In subgroup 2, 19% had undergone OTT and for most diagnoses the OTT rates ranged from 15% to 22% with benign hematologic diseases having the highest OTT rate (35%). On the basis of the entire cohort, stratified age analysis indicated that women aged ≥30 years at OTC were more likely to return for OTT than women aged 18-29 years at OTC; mean storage times were 3.7 and 3.6 years, respectively. Only 4% of the girls aged <18 years at OTC had undergone OTT. CONCLUSION(S): The OTT rates depended on the diagnosis, age at OTC, and follow-up time. Specific criteria are needed for reporting and comparing OTT rates. Six out of 10 patients still had their cryopreserved tissue stored and longer follow-up is needed, especially for younger girls.
Subject(s)
Cryopreservation/trends , Fertility Preservation , Fertility , Infertility, Female/therapy , Organ Transplantation/trends , Ovary/transplantation , Primary Ovarian Insufficiency/physiopathology , Adolescent , Adult , Denmark , Female , Humans , Infertility, Female/diagnosis , Infertility, Female/etiology , Infertility, Female/physiopathology , Pregnancy , Primary Ovarian Insufficiency/etiology , Retrospective Studies , Risk Factors , Time Factors , Young AdultABSTRACT
PURPOSE: To investigate the effect of different FSH concentrations on human oocyte maturation in vitro and its impact on gene expression of key factors in the surrounding cumulus cells. METHODS: The study included 32 patients who underwent unilateral oophorectomy for ovarian tissue cryopreservation (OTC) (aged 28 years on average). Immature oocytes were collected from surplus medulla tissue. A total of 587 immature oocytes were divided into three categories according to the size of the cumulus mass: large (L-COCs), small (S-COCs), and naked oocytes (NOs), and submitted to 44-h IVM with one of the following concentrations of recombinant FSH: 0 IU/L, 20 IU/L, 40 IU/L, 70 IU/L, or 250 IU/L. After IVM, oocyte nuclear maturation stage and diameter were recorded. The relative gene expression of FSHR, LHCGR, and CYP19A1 in cumulus cells before (day 0; D0) and after IVM were evaluated. RESULTS: Addition of 70 or 250 IU/L FSH to the IVM medium improved oocyte nuclear maturation compared to 0, 20, and 40 IU/L FSH by upregulating LHCGR and downregulating FSHR in the cumulus cells. CONCLUSION: FSH improved oocyte nuclear maturation at concentrations above 70 IU/L suggesting a threshold for FSH during IVM of ex vivo collected human oocytes from small antral follicles. Moreover, current results for the first time highlight that FSH function in vitro is mediated via cumulus cells by downregulating FSHR and upregulating LHCGR, which was also observed when the immature oocytes progressed in meiosis from the GV to the MII stage.
Subject(s)
Aromatase/genetics , Follicle Stimulating Hormone/genetics , In Vitro Oocyte Maturation Techniques , Receptors, FSH/genetics , Receptors, LH/genetics , Adult , Blastocyst/metabolism , Cumulus Cells/metabolism , Female , Fertilization in Vitro , Gene Expression Regulation, Developmental/genetics , Humans , Meiosis/genetics , Oocytes/growth & development , Oocytes/metabolism , Oogenesis/genetics , Ovarian Follicle/growth & development , Ovarian Follicle/metabolismABSTRACT
BACKGROUND: A dimeric dipeptide mimetic of the BDNF loop 4, bis(N-monosuccinyl- L-seryl-L-lysine) hexamethylenediamide (GSB-106) activates TrkB, PI3K/AKT, MAPK/ ERK, and PLC-γ1, and was created at the V.V. Zakusov Research Institute of Pharmacology. GSB-106 showed neuroprotective activity in vitro and in vivo at systemic administration. OBJECTIVE: In this work, we studied the GSB-106 effect on the cerebral infarct volume, as well as on neurogenesis and synaptogenesis under the experimental ischemic stroke induced by intravascular occlusion of the middle cerebral artery in rats. METHODS: GSB-106 was administered i.p. in a dose of 0.1 mg/kg, 24 h after the surgery and then once a day, with the end of administration on day 6 after surgery. On day 7, brain samples were collected for morphometric and biochemical (Western-blot) analysis. RESULTS: It was established that GSB-106 reduced the brain damage volume by 24%, restored impaired neurogenesis and/or gliogenesis (by Ki-67) in the hippocampus and the striatum, and completely restored the reduced immunoreactivity to synaptic markers synaptophysin and PSD-95 in the striatum. CONCLUSION: Thus, the dimer dipeptide BDNF mimetic GSB-106 exhibits neuroregenerative properties at a clinically relevant time window (24 h) in a model of ischemic stroke presumably due to the stimulation of neurogenesis (and/or gliogenesis) and synaptogenesis.
Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Ischemic Stroke/drug therapy , Neurogenesis/drug effects , Animals , Antidepressive Agents , Dipeptides/pharmacology , Hippocampus/metabolism , Male , Membrane Glycoproteins , Neuroprotective Agents/pharmacology , Phosphatidylinositol 3-Kinases , Rats , Receptor, trkBABSTRACT
RESEARCH QUESTION: What is the frequency of morphological dysmorphisms in immature human oocytes collected ex vivo from small antral follicles and matured in vitro? DESIGN: Human ovaries (nâ¯=â¯56) were excised for ovarian tissue cryopreservation (OTC). None of the patients had received exogenous gonadotrophins prior to the procedure. Immature oocytes released from small antral follicles were collected in connection with isolation of the cortex for OTC. The oocytes' maturation stage and the morphological characteristics of the cytoplasm, zona pellucida, perivitelline space and first polar body were assessed after in-vitro maturation (IVM). RESULTS: A total of 1649 immature oocytes were collected: 30% of oocytes matured to the metaphase II (MII) stage after IVM, while metaphase I (MI), germinal vesicle and degenerated oocytes accounted for 20%, 24% and 26%, respectively. The percentages of oocytes without any dysmorphisms were 53%, 92%, and 97% for the MII, MI and germinal vesicle stage oocytes, respectively. The most frequently observed dysmorphisms among the MII oocytes were first polar body fragmentation (22%), homogeneously distributed cytoplasmic granularity (16%) and an enlarged perivitelline space (14%). Interestingly, none of the oocytes at any stage had clusters of smooth endoplasmic reticulum (SER). CONCLUSIONS: Morphological dysmorphisms are present among in-vitro-matured oocytes at all maturation stages. The incidence of dysmorphisms increases as maturation progresses. The most frequent dysmorphism among MII oocytes after IVM was fragmentation of the first polar body. Clusters of SER were not observed in oocytes from unstimulated patients.
Subject(s)
In Vitro Oocyte Maturation Techniques , Oocytes/pathology , Adolescent , Adult , Endoplasmic Reticulum, Smooth , Female , Humans , Young AdultABSTRACT
PURPOSE: The aim of the present study was to improve the in vitro maturation (IVM) procedure using oocytes from surplus ovarian tissue after fertility preservation. METHODS: Twenty-five patients aged 17-37 years were included in the study. Maturation was compared between oocytes collected in HEPES-buffered medium or saline, and we determined whether transport on ice prior to oocyte collection affected maturation. Two different IVM media were used that were supplemented with and without recombinant human midkine. Mature oocytes were assessed for aneuploidy using next-generation sequencing (NGS). RESULTS: On average, 36 immature oocytes were collected from each patient (range 7-90, N = 895). Oocytes recovered from HEPES-buffered medium matured at a higher rate than oocytes recovered from saline (36% vs 26%, p < 0.01). Ovarian transportation on ice prior to the procedure negatively affected maturation compared with non-transported samples (42% vs 27%, p < 0.01). The addition of midkine improved maturation rate (34% vs 27%, p < 0.05). On average, 11 MII oocytes were obtained per patient (range 1-30). NGS of 53 MII oocytes and their first polar bodies indicated that 64% were euploid. CONCLUSIONS: The study demonstrated unexpectedly high number of immature oocytes collected from surplus ovarian tissue without any stimulation. The overall MII rate was one in three, resulting in a total number of MII oocytes that was similar to the number obtained after ovarian stimulation. If these MII oocytes prove suitable for IVF, they will provide a substantial improvement in fertility preservation for patients and advance IVM as an interesting platform for further improvements in assisted reproduction.
Subject(s)
Cryopreservation , Fertility Preservation/methods , Oocytes/growth & development , Ovary/growth & development , Adolescent , Adult , Female , Humans , In Vitro Oocyte Maturation Techniques , Oocyte Retrieval/methods , Oocytes/transplantation , Ovary/metabolism , Ovulation Induction/methods , Young AdultABSTRACT
Chromosome errors, or aneuploidy, affect an exceptionally high number of human conceptions, causing pregnancy loss and congenital disorders. Here, we have followed chromosome segregation in human oocytes from females aged 9 to 43 years and report that aneuploidy follows a U-curve. Specific segregation error types show different age dependencies, providing a quantitative explanation for the U-curve. Whole-chromosome nondisjunction events are preferentially associated with increased aneuploidy in young girls, whereas centromeric and more extensive cohesion loss limit fertility as women age. Our findings suggest that chromosomal errors originating in oocytes determine the curve of natural fertility in humans.
Subject(s)
Aging , Aneuploidy , Chromosome Segregation , Fertility , Oocytes/cytology , Adolescent , Adult , Child , Female , Humans , Meiosis , Nondisjunction, Genetic , Young AdultABSTRACT
BACKGROUND: In recent years, autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility. The slow freezing is the most effective technique which resulted in greater live birth incidence so far. Despite that, interest to vitrification of the ovarian tissue is swiftly growing, thereby undermining the necessity for further improvements in the technique. In present study, we evaluated possibilities to increase follicle survival rates adopting innovative multi-protectoral vitrification protocols, applied to the slivers of ovarian cortex or isolated early-antral follicles, frozen individually. These experimental protocols have been compared with with validated vitrification and slow freezing ones, clinically used for female fertility preservation. RESULTS: The results showed that third tested variation of experimental vitrification protocol, with four cryoprotectants in relatively low concentrations and applied to pieces of ovarian tissue at 0 °C during equilibration, increased survival rate of ovine ovarian tissue and improved results in comparison with conventional vitrification method. This variation of experimental protocol showed significant increase in percentage of follicles with good morphology (69,3%) in comparison with only commercially available vitrification protocol for ovarian tissue (62,1%). Morphology results were confirmed by TUNEL assay. Analysis of estradiol and progesterone production by cultured individual follicles after freezing/thawing revealed that steroids secretion remained significantly higher after multi-protectoral vitrification and slow freezing protocol, when follicles after standard vitrification protocol demonstrated decline in steroidogenic activity. CONCLUSIONS: The multi-protectoral approach represents a workable solution to improve vitrification outcome on ovarian tissue and isolated follicles. The reduction of individual cryoprotectants concentrations, while maintaining their sufficient cumulative level in the final freezing solution, helps to increase efficiency of the procedure. Moreover, equilibration with lower temperatures helped to decrease even further the toxic effects of cryoprotectants and preserve original quality of ovarian tissue. Therefore, multi-protectoral vitrification can be suggested as an improved method for the clinical cryopreservation of ovarian tissue.