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Purpose: Investigating whether pre-ovulatory follicular fluid (FF) levels of selected proteins differ between women who do or do not develop severe ovarian hyperstimulation syndrome (OHSS) and evaluate whether they potentially could guide a "freeze-all" strategy., Methods: FF was collected during a randomized controlled trial comparing OHSS in antagonist versus agonist protocol including 1050 women in their first assisted reproductive technology (ART) cycle during year 2009-2013. The present sub-study is a matched case-control study comparing FF levels of soluble urokinase plasminogen activator receptor (suPAR), C-reactive protein, placental growth factor, vascular endothelial growth factor, and angiopoietins 1 and 2 in OHSS cases (n = 25, severe OHSS, and ≥ 15 oocytes), high-risk controls (n = 25, no OHSS, and ≥ 15 oocytes), and low-risk controls (n = 25, no OHSS, and 5-8 oocytes)., Results: FF level of suPAR differed significantly between the three groups (p = 0.018) with mean (SD) levels of 2.3 (0.4) μg/L, 2.6 (0.8) μg/L, and 2.8 (0.6) μg/L in OHSS cases, high-risk controls, and low-risk controls, respectively. Receiver operating characteristic curve analysis demonstrated that suPAR levels could predict severe OHSS (AUC 0.678; 95% CI 0.553-0.803) with a sensitivity of 64% and a specificity of 66%. None of the other investigated proteins differed between the three groups or between OHSS cases and combined controls., Conclusion: The pre-ovulatory FF level of suPAR was significantly lower in women developing severe OHSS, indicating that the plasminogen activator system could be involved in the pathophysiology of OHSS. However, suPAR did not provide a satisfying predictive value for the prediction of OHSS.

Study Question: Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation?, Summary Answer: The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation., What Is Known Already: Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-β and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development., Study Design, Size, Duration: We performed a class comparison study on human oocytes from primordial (n = 436) and primary (n = 182) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy., Participants/materials, Setting, Methods: RNA was extracted from oocytes from primordial and primary follicles isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue., Main Results and the Role of Chance: We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human primordial-to-primary follicle transition (P < 0.05 and/or FPKM fold-change >2). IPA® enrichment analysis revealed known pathways ('mTOR Signaling', 'PI3K/AKT Signaling' and 'PTEN Signaling') as well as enriched canonical pathways not previously associated with human ovarian follicle development such as 'ErB Signaling' and 'NGF Signaling' in the down-regulated category and 'Regulation of eIF4 and P70S6K Signaling' and 'HER-2 Signaling in Breast Cancer' in the up-regulated group. Additionally, immunohistochemistry on human ovarian tissue explored the intraovarian localization of VASA, FOXO1 and eIF4E., Large Scale Data: http://users-birc.au.dk/biopv/published&#95;data/ernst&#95;2017/., Limitations, Reasons for Caution: This is a descriptive analysis and no functional studies were performed. The study was based on a limited number of patients and the experimental design could not take into account the natural biological variance in human samples. Therefore, qPCR was used to confirm selected genes alongside immunohistochemical stainings., Wider Implications of the Findings: This study shows, for the first time, a detailed molecular description of global gene transcription activities in oocytes from primordial and primary follicles, respectively. Knowing the global transcription profiles of human oocyte dormancy and activation are important in developing new clinical applications., Study Funding/competing Interest(s): E.H.E. was supported by Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.H. and S.F. were supported by an MRC (UK) project grant MR/M012638/1. K.L.H. was supported by grants from Fonden til Lægevidenskabens Fremme, Kong Christian Den Tiendes Fond. K.L.H. and L.S. were supported by the IDEAS grant from Aarhus University Research Foundation (AUFF). There are no conflicts of interest., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Objective: Evaluating the developmental competence of immature oocytes collected from surplus medulla tissue in connection with ovarian tissue cryopreservation for fertility preservation., Design: Cohort comparative study., Setting: University laboratory in Denmark from 2011-2012., Population: 69 girls and women (0-38 years of age) who each had one ovary cryopreserved for fertility preservation., Methods: Ovaries were obtained directly from the local hospital or from collaborating hospitals (two to five hours' transport on ice). Immature oocytes were aspirated from large antral follicles visible on the ovaries, and collected from the saline solution, containing surplus medulla tissue, following dissection of the ovarian cortical tissue for cryopreservation. The immature oocytes were cultured for 48 h in an Embryoscope™ Time-lapse System or in culture dishes overlaid with liquid paraffin using commercial and in-house supplemented culture media., Main Outcome Measures: Maturation rate for immature oocytes reaching metaphase II., Results: With a maturation rate of 3.1%, only 21 of 682 immature oocytes reached metaphase II. Immature oocytes from ovaries that had been transported on ice for two to five hours performed significantly poorer than those recovered immediately after surgery. Addition of epidermal growth factor and follicle fluid from human small antral follicles to the culture medium did not augment the maturation rate. Immature oocytes cultured in the Embryoscope performed significantly better than those in conventional culture dishes., Conclusions: In vitro maturation of immature oocytes should only be attempted clinically from visible antral follicles and where the ovary is not subjected to a cooling period prior to recovery of immature oocytes., (© 2013 Nordic Federation of Societies of Obstetrics and Gynecology.)

The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

Background: The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes., Methods: mRNA was isolated for whole genome gene expression microarray analysis from metaphase II (MII) oocytes donated by IVF or ICSI patients [10 women aged <36 years (younger) and five women aged 37-39 years (both inclusive) (older)] undergoing controlled ovarian stimulation. The oocytes were donated and prepared immediately after recovery from the follicle. RT-PCR on additional four younger and two older oocytes confirmed the array analysis., Results: On the basis of 15 independent replicates of single MII oocytes, 7470 genes (10 428 transcripts) were identified as present in the MII oocytes. Of these, 342 genes showed a significantly different expression level between the two age groups; notably, genes annotated to be involved in cell cycle regulation, chromosome alignment (e.g. MAD2L1 binding protein), sister chromatid separation (e.g. separase), oxidative stress and ubiquitination. The top signaling network affected by age was 'cell cycle and organism development' (e.g. SMAD2 and activin B1 receptor)., Conclusion: There is a substantial difference between younger and older oocytes in the transcriptional level of genes involved in central biological functions of the oocytes, thus providing information on processes that may be associated with the ageing phenomenon and possibly contributing to decreased fertility.

Autotransplantation of frozen/thawed ovarian tissue in women undergoing cancer therapy has so far led to the birth of two healthy babies. In both cases, it can be discussed whether the fertilized oocyte originated from the transplant or from the native ovary. We now present a biochemical pregnancy achieved after heterotopical autotransplantation of cryopreserved ovarian cortical tissue and hence the unquestionable proof that pregnancy can occur after transplantation of cryopreserved ovarian tissue. A woman diagnosed with Hodgkin's lymphoma had ovarian tissue cryopreserved at the age of 28, before receiving chemotherapy and radiation therapy that rendered her amenorrhoeic. After complete remission, she had autotransplantation of ovarian tissue to the remaining ovary, to the right pelvic wall and to a midline subperitoneal pocket on the lower abdominal wall. The transplanted tissue resumed hormone secretion and follicles developed in all three locations. Three times during 8 months, when follicles could not be visualized in other locations, oocytes were aspirated from the subperitoneal autotransplanted tissue on the lower abdominal wall. Twice, an oocyte was retrieved, fertilized by intracytoplasmatic sperm injection (ICSI) and transferred to the woman's uterus. One of the treatments resulted in a positive pregnancy test 14 days after transfer. Clinical pregnancy, however, was not achieved. In conclusion, heterotopic autotransplantation of cryopreserved ovarian tissue can sustain follicle development. The oocytes of aspirated mature follicles are capable of fertilization after ICSI, and the resulting embryo is competent of producing hCG at detectable levels.

Cryopreservation of human ovarian tissue is now an option for cancer patients facing treatment with gonadotoxic regimes, as a means of preserving their fertility. So far, there have been only a few reports on autotransplantation of frozen-thawed tissue with regard to restoration of ovarian function. The present report describes a 32-year-old woman diagnosed with Hodgkin's lymphoma, who had cryopreserved ovarian tissue transplanted orthotopically after secondary ovarian failure due to chemotherapy. Only 8 weeks after transplantation, ultrasonography of the remaining ovary revealed two follicles with diameters of 10 and 15 mm. Concomitantly, circulating concentrations of oestradiol increased, while concentrations of gonadotrophins decreased. In the following months, the patient menstruated three times. Subsequent pituitary down-regulation with a gonadotrophin-releasing hormone (GnRH) agonist and ovarian stimulation resulted in development of one pre-ovulatory follicle from which a metaphase II oocyte was retrieved; however, this oocyte was unable to sustain further development after intracytoplasmic sperm injection (ICSI). Intrafollicular concentrations of oestradiol and progesterone suggested a normal luteinizing response of the follicle to human chorionic gonadotrophin stimulation. A 7-month follow-up revealed continued vivid follicular activity and normal oestradiol concentrations. In conclusion, cryopreserved human ovarian tissue restored ovarian function for several cycles and sustained development of mature oocytes in a woman cured of cancer.

Some intermediates in the cholesterol biosynthesis between lanosterol and cholesterol are capable of inducing resumption of meiosis in cultured mouse oocytes without the presence of gonadotropins. The mechanism by which these so-called Meiosis Activating Sterols (MAS) activate the meiotic process is unknown, and it is uncertain whether they participate in the physiological control of resumption of meiosis. Recently, it has been shown that accumulation of MAS occurs in a liver cell line and in rat testis tissue cultured in the presence of micromolar concentrations of progesterone and 17 alpha-OH-progesterone. Such high concentrations of progesterone and 17 alpha-OH-progesterone only occur in fluid of preovulatory follicles. In connection with the mid-cycle surge of gonadotropins, this may represent one mechanism whereby follicular accumulation of MAS takes place. In the present study, the effect of 10 micro M progesterone and 10 micro M 17 alpha-OH-progesterone on resumption of meiosis was evaluated using mouse cumulus enclosed oocytes (CEO) cultured in the presence of 4mM hypoxanthine. By the end of the 24-h culture period, the frequency by which oocytes had resumed meiosis was assessed by the determination of germinal vesicle breakdown (GVBD). Neither progesterone nor 17 alpha-OH-progesterone or a combination showed any effect on GVBD. In addition, progesterone and 17 alpha-OH-progesterone in combination with a sub-optimal dose of FSH (4 IU/l) did not affect GVBD. In conclusion, accumulation of MAS to an extent that allows resumption of meiosis to occur in CEO is unlikely to be induced by progesterone and 17 alpha-OH-progesterone or a combination.

FSH exists as a family of isohormones exhibiting distinct oligosaccharide structures, and the released FSH isoform mixtures change during the follicular phase of the menstrual cycle. In addition, the pulsatile release of gonadotrophins seems to expose follicles to bursts of more less-acidic FSH isoforms and the follicle is likely to be exposed to an almost ever changing composition of FSH isoforms. The different isoforms causes a number of different and divergent biological effects. FSH promotes oocyte maturation, and 5-10 IU/l of less-acidic FSH isoforms are sufficient to induce oocyte maturation in vitro. Exposure of cumulus-oocyte complexes to less-acidic FSH isoforms in a pulse-like fashion results in a rapid pattern of cAMP accumulation exceeding that seen with acidic isoforms, which appear to sustain lower but more constant cAMP production. The presence of particularly less-acidic isoforms for a period exceeding 30 min causes an attenuated cAMP response. In conclusion, it appears that pulsatile and intermittent release of less-acidic/short-living FSH isoforms is sufficient to induce biological responses, while allowing the granulosa cells to regain responsiveness to further FSH stimulation. Together with the interpulse release of more acidic isoforms, overall FSH secretion seems to ensure proper follicular maturation resulting in the release of developmentally competent oocytes.

In the preovulatory follicle, oocyte meiotic resumption occurs soon after the LH surge and is associated with a decrease in cAMP. Inhibition of cAMP degradation blocks germinal vesicle breakdown as well as activation of meiotic promoting factor, both hallmarks of reentry into the cell cycle. In situ and pharmacological analysis of rodent ovaries suggested the presence of a phosphodiesterase 3 (PDE3) in the germ cell but not the somatic cell compartment. Here we have investigated the structure and properties of the PDE form expressed in mouse oocytes. Polymerase chain reactions using a mouse oocyte cDNA library as a template, and primers based on the conserved sequence of rat and human PDE3As, yielded partial fragments corresponding to mouse PDE3A. Further screening of the mouse oocyte cDNA library and subsequent ligation of individual cDNA clones yielded PDE3A cDNA containing the entire coding region of mouse PDE3A. To determine the kinetic properties of this PDE, the cDNAs encoding the full-length PDE3A and NH(2)-truncation forms Delta 1 (Delta346aa) and Delta 2 (Delta608aa) were expressed in mouse Leydig tumor cells. Whereas the full-length recombinant protein was always found in the particulate fraction, the Delta 1 and Delta 2 truncated PDE3As were recovered mostly in the soluble fraction. The Michaelis constant values for hydrolysis of cAMP of PDE3A Delta 1 and PDE3A Delta 2 were similar to those of intact full-length PDE3A or oocyte PDE (0.2-0.5 microM). More importantly, there was good correlation between the rank of potency of selective and nonselective compounds in inhibiting recombinant PDE3A or PDE activity derived from cumulus-oocyte complexes and in blocking resumption of meiosis. These data provide evidence that the PDE expressed in the oocyte is a soluble form of PDE3A and that activity of this enzyme is involved in the control of resumption of meiosis.

The ability of different isoforms of follicle stimulating hormone (FSH) to induce accumulation of cAMP in cultured mouse cumulus-oocyte-complexes (COC) was evaluated in a time course study. Using isoform fractions representing less acidic (pI 6.43-5.69), mid-acidic (pI 5.62-4.96) and acidic (pI 4.69-3.75) isoforms, the accumulation of cAMP was monitored after an exposure time of 0, 5, 10, 15, 30, 60, 120 and 180 min. In addition, cAMP production was monitored for 0, 5, 10, 15 and 30 min following a 5 min exposure to FSH isoform fractions. Based on FSH measurements using radioimmunoassays, the less and mid-acidic isoforms caused almost twice as much cAMP to be accumulated than the acidic isoform fraction, thereby confirming an enhanced biological activity of FSH isoforms with a isoelectric point (pI) of >5.0. For all isoform fractions, maximal accumulation of cAMP was achieved after 30 min of exposure, after which the production declined to background levels. After a 5 min exposure to isoform fractions, levels of cAMP were significantly higher in the less acidic isoform fractions, but after isoform removal, the decline in cAMP production to background levels followed a similar time course. The results demonstrate that FSH isoforms with a pI of >5.0 induced significant biological responses within a period of 30 min and that prolonged exposure caused attenuated signal transduction. The present results, set in the context of the pulsatile characteristics of FSH release from the pituitary and the reported half-life of less acidic isoforms of approximately 35 min, make it conceivable that isoforms with a pI >5.0 actually possess important physiological functions during the periovulatory period.

The mitogen-activated protein kinase-dependent and the cAMP-protein kinase A-dependent signal transduction pathways were studied in cultured mouse oocytes during induced and spontaneous meiotic maturation. The role of the mitogen-activated protein kinase pathway was assessed using PD98059, which specifically inhibits mitogen-activated protein kinase 1 and 2 (that is, MEK1 and MEK2), which activates mitogen-activated protein kinase. The cAMP-dependent protein kinase was studied by treating oocytes with the protein kinase A inhibitor rp-cAMP. Inhibition of the mitogen-activated protein kinase pathway by PD98059 (25 micromol l(-1)) selectively inhibited the stimulatory effect on meiotic maturation by FSH and meiosis-activating sterol (that is, 4,4-dimethyl-5alpha-cholest-8,14, 24-triene-3beta-ol) in the presence of 4 mmol hypoxanthine l(-1), whereas spontaneous maturation in the absence of hypoxanthine was unaffected. This finding indicates that different signal transduction mechanisms are involved in induced and spontaneous maturation. The protein kinase A inhibitor rp-cAMP induced meiotic maturation in the presence of 4 mmol hypoxanthine l(-1), an effect that was additive to the maturation-promoting effect of FSH and meiosis-activating sterol, indicating that induced maturation also uses the cAMP-protein kinase A-dependent signal transduction pathway. In conclusion, induced and spontaneous maturation of mouse oocytes appear to use different signal transduction pathways.

Meiosis-activating sterol (MAS) has been shown to induce mouse oocytes cultured in the presence of hypoxanthine (HX) to resume meiosis. The present research was conducted to determine whether amphotericin B or ketoconazole (a promoter and an inhibitor of production of MAS), affected oocyte maturation. Mouse cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) were cultured for 24 h in the presence of 4 mM HX with FSH or amphotericin B or ketoconazole. At the end of the culture, the frequency of germinal vesicle break down (GVBD) and polar body formation (PB) were recorded. The results demonstrated: (i) FSH (10-200 IU/l) induced dose-dependent oocytes maturation in CEO, but was without effect on DO. A maximum increase in GVBD and PB was observed with 25-50 IU/l FSH. The presence of FSH (50 IU/l) for 1 h was sufficient to induce meiotic resumption, which after 2 h reached a plateau similar to that of a continuous presence of FSH. (ii) CEO exposed to amphotericin B (0.0025-2.5 microg/l) underwent GVBD dose-dependently, whereas no effect was observed on DO. The presence of amphotericin B (0.025 microg/l) for 1 h stimulated oocyte resumption in a way similar to that of FSH. (iii) Amphotericin B (0.025 microg/l) and FSH (50 IU/l) did not show any additive effect on resumption of meiosis. (iv) Ketoconazole (10(-7)-10(-3) M) inhibited the effect of FSH on resumption of meiosis, but had no effect on oocyte spontaneous maturation. These results show that FSH and amphotericin B induce resumption of meiosis and indicate that they are likely to cause an accumulation of meiosis activating sterols in the CEO, but ketoconazole blocks the production of MAS. The present study supports the notion that MAS plays a physiological relevant role in triggering resumption of meiosis in mouse oocytes.

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.

Purpose: Our purpose was to evaluate the requirements for human chorionic gonadotropin (hCG) and recombinant luteinizing hormone (rec-LH) for follicular development and maturation in mice., Methods: We carried out ovarian stimulation of immature mice. Output parameters were the preembryos created in vivo and frequency of blastocyst formation in vitro., Results: hCG at 0 to 1 IU resulted in a dose-dependent recovery of preembryos (0 to 39.7 +/- 4.3; mean +/- SE) per mouse. hCG at 1 and 10 hCG gave similar results, whereas higher doses significantly reduced the number of preembryos. Potential for blastocyst formation was independent of hCG dose. hCG and rec-LH together exerted a synergistic effect on the recovery of preembryos., Conclusions: Optimal follicular development required a combination of 20 IU follicle stimulating hormone and 1-10 IU hCG. The potency of hCG was higher than that of rec-LH, but a synergistic effect of rec-LH and hCG was observed. The results may be pertinent for the development of strategies for ovarian stimulation of women with low levels of endogenous LH.

The ability of different isoforms of follicle stimulating hormone (FSH) to induce the resumption of meiosis in cultured mouse oocytes was evaluated. Oocytes were cultured in the presence of hypoxanthine to prevent spontaneous resumption of meiosis. Using serial dilutions of the isoform fractions representing less acidic isoforms (pI 6. 43-5.69), mid-acidic (pI 5.62-4.96) and acidic (pI 4.69-3.75), the concentration which caused 50% of the oocytes to resume meiosis and undergo germinal vesicle breakdown (GVBD) after a culture period of 24 h (i.e. ED(50%) GVBD) was determined. The FSH concentration of the isoform fractions was determined by radioimmunoassay, radio-receptor assay or through cAMP release in a Chinese hamster ovary-cell line expressing the human FSH-receptor. Determined by radioimmunoassay, the (ED(50%) GVBD) values were: less acidic 6.4 +/- 0.3 IU/l (mean +/- SD), mid-acidic 6.1 +/- 0.7 IU/l and acidic 12.2 +/- 0.7 IU/l. The less and mid-acidic isoforms were significant lower than the acidic (P < 0.0005). Similar relationships between the isoform fractions were obtained by the two other FSH assays. The results demonstrate that FSH isoforms with a pI of >5.0 induced resumption of meiosis significantly more efficiently than acidic isoforms. Less and mid-acidic isoforms may exert an important physiological function by inducing the resumption of meiosis in oocytes from pre-ovulatory follicles during the mid-cycle gonadotrophin surge.

The aim of this study was to test whether ovulation from an ovary affects the health of oocytes from dominant follicles in that ovary two cycles later. A total of 80 women each with two intact ovaries underwent 270 treatment cycles (155 natural cycles and 115 clomiphene citrate cycles) all showing unilateral ovulation. The results from the in-vitro fertilization (IVF) treatment were grouped according to whether ovulation (O) or anovulation (A) (no ovulation) was observed in the ovary with dominant follicle during the treatment cycle in the previous two cycles: O-O, A-O, O-A and A-A (previous second cycle-previous first cycle). The rate of pre-embryo formation in A-A was significantly higher than that of O-A. The pregnancy rate in A-A (29%) was also higher than those of O-A (13%), A-O (9%) and O-O (5%). These rates increased from O-O to A-A as the number of previous ovulations in an ovary decreased. The presence of a corpus luteum and/or a dominant follicle is likely to exert local negative effects on the health of the oocyte contained in the follicle selected to ovulate up to two cycles later. Anovulations in an ovary for two menstrual cycles may therefore provide improved conditions for the development of a healthier oocyte with an increased pregnancy potential.

In the follicles of the mammalian and amphibian ovary, oocyte maturation is arrested at the prophase of the first meiotic division. Prior to ovulation, oocytes reenter the cell cycle, complete the meiotic division, and extrude the first polar body. Work from several laboratories including ours has provided evidence that the cAMP-mediated signal transduction pathway plays an important role in regulation of meiosis, the cyclic nucleotide acting as a negative regulator of maturation. Since cAMP can be regulated both at the level of synthesis and degradation, our laboratory is investigating the role of phosphodiesterases (PDE) in the control of cAMP levels of oocytes. Using pharmacological and molecular tools, we have determined that a PDE3 is the enzyme involved in the control of cAMP levels in the oocytes. In vitro and in vivo studies have established that inhibition of the oocyte PDE3 blocks resumption of a PDE is per se sufficient to cause resumption of meiosis in an amphibian oocyte model. The pathways regulating this PDE isoform expressed in the oocyte is under investigation, as they may uncover the physiological signals controlling meiosis.

The activation of protein kinase B/Akt is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. Because insulin-like growth factor 1 stimulates the resumption of meiosis in Xenopus laevis oocytes via phosphoinositide 3-kinase activation, we investigated the Akt involvement in this process. Injection of mRNA coding for a constitutively active Akt in Xenopus oocytes induced germinal vesicle breakdown (GVBD) to the same extent as progesterone or insulin treatment. Injection of mRNA coding for the wild type Akt kinase was less effective in stimulating GVBD, whereas Akt bearing a lysine mutation in the catalytic domain that abolishes the kinase activity had no effect. A mutant Akt lacking a membrane-targeting sequence did not induce GVBD, despite high levels of expression and activity. As previously reported for insulin, induction of GVBD by Akt was prevented by incubating the oocytes with cilostamide, an inhibitor specific for the type 3 phosphodiesterase (PDE3), suggesting that the activity of a PDE is required for Akt action. That an increase in PDE activity in the oocyte is sufficient to induce meiotic resumption was demonstrated by expression of an active PDE protein. In addition, the constitutively active Akt caused a 2-fold increase in the activity of the endogenous PDE. These data demonstrate that Akt is in the pathway controlling resumption of meiosis in the Xenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation.

Purpose: The possible effects of circulating FSH levels as used during IVF treatment on oocyte maturation and subsequent preembryo development were evaluated., Methods: Serum levels of FSH and LH on days 1 and 8 of ovarian stimulation and on the day of oocyte retrieval (OR) were correlated with subsequent preembryo development in vitro. After pituitary downregulation, 244 normogonadotropic women followed a fixed protocol for the first 7 days of stimulation., Results: The average FSH concentration on day 8 of stimulation was 11.5 IU/L and exceeded the expected midcycle surge of FSH by more than 25%. In contrast, levels of LH were below an average of 2 IU/L throughout the stimulation period. The concentration of FSH on day 8 and on the day of OR showed a significant inverse correlation with cleavage rate, whereas levels of LH, age, and body mass index showed no such correlation., Conclusions: Supraphysiologic levels of FSH seems to affect oocyte maturation negatively. Premature resumption of meiosis, leading to retrieval of postmature oocytes with a reduced developmental potential, is suggested as the underlying mechanism.

The cortex-medulla growth pattern of oocytes in the developing ovary is formed by a rim of small non-growing oocytes at the periphery and growing oocytes at the inner part of the cortex and in the medulla. In this study we aimed to: (i) develop an in-vitro model using fetal mouse ovaries to evaluate when the cortex-medulla growth pattern is organized during ovarian differentiation; and (ii) study the interaction between small, non-growing and growing oocytes. Fetal mouse ovaries of days 11, 13 and 16 post coitus (E11, E13 and E16) were cultured for 14, 12 and 9 days respectively, corresponding to post-partum day 7 (P7). A cortex-medulla growth pattern had developed by P7 in most ovaries from E16 and in half of those from E13. No ovaries from E11 developed a cortex-medulla pattern. The organization of this pattern within the ovary is concurrent with the initiation of meiosis. Ovaries with a cortex-medulla growth pattern had the same number of growing follicles (approximately 87), regardless of the number of small, non-growing oocytes present. In contrast, in most ovaries without a cortex-medulla pattern, almost all oocytes began to grow. Hence, the geographically determined growth pattern of oocytes in the developing mouse ovary may be organized at the onset of meiosis and is sustained by a balanced interaction between small and growing follicles.

The effect of the different follicular cell types on resumption of meiosis was studied during stimulation with FSH. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), and cumulus and mural granulosa cells were used. The resumption of meiosis and oocyte maturation were assessed by the determination of the germinal vesicle breakdown (GVBD) and polar body formation (PB) at the end of a 24 hr culture period in the presence of 4 mM hypoxanthine (HX). The effects of recombinant LH (r-LH) and hCG were also evaluated. Oocyte exposure to the gonadotrophins varied from 5 min to 24 hr (i.e., priming time). Oocytes were obtained from immature gonadotrophin-stimulated and -unstimulated mice. 1. FSH (1 IU/L-75 IU/L) provoked a dose-dependent increase in GVBD and PB in CEO, but not in DO, in stimulated and unstimulated mice. Eight IU/L was sufficient for inducing resumption of meiosis. In contrast, LH and hCG (both 1 IU/L-1500 IU/L) were without effect on GVBD and PB in CEO and DO of oocytes from stimulated and unstimulated mice. A combination of 8 IU/L FSH and 4-8 IU/L hCG produced an additive effect, whereas combinations with LH and higher concentrations of hCG had no such effect. 2. A 2 hr priming with FSH (8 IU/L-75 IU/L) induced a dose-dependent oocyte maturation in CEO. Thirty minutes of priming with FSH (75 IU/L) was sufficient for induction of meiotic resumption in CEO. 3. Priming CEO with FSH for 2 hr followed by the separation and repooling of oocytes and cumulus cells induced oocyte maturation. GVBD of new, unprimed DO added to cumulus cells of primed CEO increased slightly but was significant, whereas GVBD in DO isolated from the primed CEO only increased marginally. DO cocultured with FSH-primed cumulus masses seem to be prevented from resuming meiosis. 4. Priming a coculture of granulosa cells and DO with FSH for 2 hr caused a significant increase in GVBD compared to the control, evaluated after 24 hr. In contrast, a 24 hr FSH-priming of a coculture of granulosa cells and DO was without effect on GVBD. 5. A spent medium in which unstimulated cumulus cells or mural granulosa cells had grown was without effect on GVBD in DO. However, a small fraction of the DO resumed meiosis after culture in a spent medium derived from a 2 hr priming of CEO and spent media from 24 hr priming of CEO induced a 2-3 times higher GVBD frequency in the DO compared to the controls. Heat treatment of spent media (70 degrees C, 30 min) from a 24 hr FSH-priming of CEO still induced GVBD in naive DO. The results showed that FSH, in a concentration of as little as 8 IU/L, but not r-LH and hCG, induced within 30 minutes the cumulus cells to produce and after 2 hr to secrete a diffusible heat stable meiosis activating substance. This substance overcame, in a paracrine fashion, the inhibiting effect of HX and induced oocyte maturation directly in DO. The production of this substance, however, was dependent on the initial connection between the cumulus cells and the oocyte, indicating an important 2-way communication between these 2 cell types. The mural granulosa cells did not produce a meiosis inducing activity by stimulation with FSH, but significantly, more DO matured after coculture with the nonstimulated granulosa cells for 24 hr than for 2 hr. It is proposed that the heat stable meiosis activating component of the spent media from the FSH-stimulated CEO belongs to the meiosis activating sterols, MAS, previously isolated from human follicular fluid and from adult bull testes.

Gonadotrophins and various growth factors, but not sex steroids, can induce resumption of meiosis in vitro, but only in oocytes enclosed by cumulus-granulosa cells. Follicular purines prevent resumption of meiosis. This process can be overcome, in vitro, by a transient elevation of cyclic AMP resulting in the production of a diffusible meiosis-inducing substance secreted by the cumulus cells. A meiosis-inducing activity has been detected in gonads of different species, for example, in preovulatory follicular fluid of women and in mouse testes. We report here the isolation and characterization of meiosis-activating sterols from human follicular fluid and bull testes and the synthesis of two closely related C29 sterols. All these sterols induce a resumption of meiosis in cultured cumulus-enclosed and naked mouse oocytes indicating their nonspecificity across species and sex. This family of sterols is for the first time considered crucial to meiosis.

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed.(ABSTRACT TRUNCATED AT 250 WORDS)

The most common indication for oocyte donation is ovarian insufficiency due to premature menopause or resistant ovarian syndrome and ovarian dysgenesis with either normal or abnormal (e.g. Turner's syndrome) karyotype. In Denmark, oocyte donation must be anonymous, and the donors have to be other infertile patients undergoing in vitro fertilisation (IVF), treatment. It is suggested, that the National Health Service offers oocyte donation to hypergonadotropic women with ovarian insufficiency, as well as to a few other groups who fulfil the criteria for IVF treatment, but where this treatment cannot be completed. Oocyte donors must be less than 35 years old, physically and mentally healthy and without major genetic diseases in the family. The donor must be screened for HIV, hepatitis, syphilis, chlamydia and gonorrhoea. We propose that those patients who have more than six oocytes aspirated, allowing "surplus" oocytes to be donated. It is also proposed that the departmental order from the Ministry of Health be changed, so that normally fertile women are allowed to donate oocytes. Oocyte donation should be reported to a central authority.

In Denmark, legislation has made cryopreservation of human oocytes and embryos possible since October 1992. The legislation is reviewed. Cryopreservation of embryos constitutes a significant improvement of infertility treatment. The number of oocyte pick-ups and the number of embryos transferred can be reduced without compromising the total likelihood of success of in-vitro fertilization (IVF) treatment. According to Danish law, frozen embryos can only be stored for one year. This limit will interfere with patient expectations in numerous cases, and the time limit should be expanded. Moreover, freezing of embryos should be allowed in connection with oocyte donation programmes.

In the treatment of infertility employing in vitro fertilisation and embryo transfer (IVF-EF), oocytes, spermatozoa and pre-embryos are cultured for 48 hours outside the woman's body before they are introduced into the uterus. In addition to the necessary salts, the media in which this culture takes place, also consists of a source of protein. In order to eliminate the variability of patient sera, a prospective, randomized investigation was performed to elucidate whether a well-defined source of protein such as human serum albumin (hSA-hSA 200 mg/ml, Statens Seruminstitute) can replace patient serum as source of protein in the culture of oocytes, spermatozoa and pre-embryos in IVF-ET treatment. The pregnancy rate per transplantation was increased from 30% in the serum group (21 pregnant out of 69 transplantations) to 39% in the albumin group (26 pregnant out of 66 transplantations) but the difference is not significant. On the other hand, the quality of the pre-embryos as assessed by morphological criteria became significantly better and the implantation rate per transplanted pre-embryo was found to be significantly increased in the albumin group. On the basis of this investigation, hSA is recommended as the source of protein, rather than the patient's own serum in the culture of oocytes, spermatozoa and pre-embryos in IVF-ET treatment.

Aspirates from 31 ovarian follicles and 2 corpora lutea of 26 women at different stages of the menstrual cycle were investigated for activity of meiosis-inducing substance (MIS) and meiosis-preventing substance (MPS). The aspirated follicles were classified as dominant (i.e., preovulatory), healthy, or atretic according to their size, steroid hormone content, and stage of the menstrual cycle. To test for MIS and MPS activity, gonads of sexually undifferentiated fetal mice were cultured in media containing either 15% follicular fluid aspirate (test gonads) or 15% human blood serum (control gonads). MIS activity is present in follicular fluid if the test testes contain more meiotic germ cells than the control testes. MPS activity is present if the test ovaries have less meiotic germ cells than their controls. MIS activity was present only in healthy follicles of the late follicular phase (12 of 15 follicles). No MIS activity was seen in healthy or atretic follicles from other phases of the menstrual cycle. The MIS activity is apparently unrelated to the composition of steroids in the follicular fluid. MPS activity was not found in any of the follicles. It is proposed that the preovulatory resumption of meiosis may be triggered by a MIS in the follicular fluid.