Final Stages Of Oocyte Maturation Research Articles

Association between pituitary adenylate cyclase-activating polypeptide and reproduction in the blue gourami

In order to gain a better understanding of the roles of pituitary adenylate cyclase-activating polypeptide (PACAP) in reproduction and growth, the expression of the PACAP gene during the reproduction cycle and its potential role in regulating gonadotropin and growth hormone (GH) gene transcription in blue gourami were investigated. The cDNA sequences of the full-length blue gourami brain PACAP and that of its related peptide ( PRP) were acquired. PACAP cDNA had two variants, obtainable by alternative splicing: a long form encoding for both PRP and PACAP and a short form encoding only for PACAP. In females, mRNA levels of PACAP were very high only in individuals with oocytes in the maturation stage, as compared to levels in unpaired vitellogenic and non-vitellogenic fish. The PACAP mRNA levels in males were high only in nest builders, as opposed to in non-nest building males and juveniles. In pituitary culture cells from high vitellogenic females, PACAP38 (the 38 amino acid form) only brought about an increase in βFSH levels, without altering GH and βLH mRNA levels. On the other hand, in adult non-reproductive male pituitary cells, PACAP38 decreased the GH mRNA level. Based on these results, we propose that in the blue gourami, PACAP is involved in the final oocyte maturation stage in females, whereas in males, it is associated with sexual behavior. In addition, the effect of PACAP38 on pituitary hormone gene expression is different in females and males, indicating that PACAP38 is potentially a hypophysiotropic regulator of reproduction, which mediates pituitary hormone expression.

Relationship between the monoamine and gonadotropin content in follicular fluid of preovulatory graafian follicles after superovulation treatment.

Noradrenaline (NA), serotonin (5HT), dopamine (DA), FSH, LH and prolactin (PRL) content was determined in 104 preovulatory follicular fluids obtained from 44 patients undergoing in vitro fertilization. The patients were given human menopausal gonadotropin (HMG) for ovarium stimulation, ovulation was induced with 10000 IU human chorionic gonadotropin (HCG) 34-36 hours prior to the follicular aspiration by vaginal ultrasound. Classification of the oocytes was performed by direct microscopic evaluation differentiating three groups of oocytes: Group I.: prophase I; Group II.: metaphase I; Group III.: metaphase II. There was no significant difference in monoamine and FSH content of follicular fluid at different stage of the oocyte maturation. LH and PRL significantly increased parallel with oocyte maturation (38.9; 48.8; 56.7 IU/l and 1324; 2382; 3134 IU/l). significant negative correlation was observed in Group I. between 5HT-LH (r = -0.64); in Group II. between NA-LH (r = -0.62) and NA-PRL (r = -0.51). Significant positive correlation were found in Group I. between FSH-LH (r = 0.63), in Group II. between LH-PRL (r = 0.56), in Group III. between NA-5HT (r = 0.66), NA-DA (r = 0.80) and 5HT-DA (r = 0.66). These observations suggest that action of LH and PRL may be negatively modulated by 5HT and NA in the final stage of oocyte maturation.

The effect of follicular fluid hormones on oocyte recovery after ovarian stimulation: FSH level predicts oocyte recovery

BackgroundOvarian stimulation for assisted reproductive technology (ART) overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery.MethodsThis is a prospective study involving patients undergoing ART by standard ovarian stimulation protocols at an urban academic medical center. A total of 143 follicular fluid aspirates were collected from 80 patients. Concentrations of FSH, hCG, estradiol, progesterone, testosterone and prolactin were determined. A multivariable regression analysis was used to investigate the relationship between the follicular fluid hormones and oocyte recovery.ResultsIntrafollicular FSH was significantly associated with oocyte recovery after adjustment for hCG (Adjusted odds ratio (AOR) = 1.21, 95%CI 1.03–1.42). The hCG concentration alone, in the range tested, did not impact the odds of oocyte recovery (AOR = 0.99, 95%CI 0.93–1.07). Estradiol was significantly associated with oocyte recovery (AOR = 0.98, 95% CI 0.96–0.99). After adjustment for progesterone, the strength of association between FSH and oocyte recovery increased (AOR = 1.84, 95%CI 1.45–2.34).ConclusionThe relationship between FSH and oocyte recovery is significant and appears to work through mechanisms independent of the sex hormones. FSH may be important for the physiologic event of separation of the cumulus-oocyte complex from the follicle wall, thereby influencing oocyte recovery. Current methods for inducing the final stages of oocyte maturation, with hCG administration alone, may not be optimal. Modifications of treatment protocols utilizing additional FSH may enhance oocyte recovery.

Growth, reproduction, and feeding of large monkfish, Lophius americanus

Abstract Johnson, A. K., Richards, R. A., Cullen, D. W., and Sutherland, S. J. 2008. Growth, reproduction, and feeding of large monkfish, Lophius americanus. – ICES Journal of Marine Science, 65: 1306–1315. The American monkfish, Lophius americanus, supports important commercial fisheries in the Northwest Atlantic. Although life history information is available for smaller L. americanus, the biology of large monkfish (>70 cm) is poorly understood because relatively few large fish are caught in standard resource surveys. Between 2006 and 2008, 699 L. americanus of 71–118 cm total length were collected from commercial gillnet fishers operating in the mid-Atlantic Bight (n = 689) and in the Gulf of Maine (n = 10) to investigate growth rates, reproductive biology, and feeding habits of large monkfish. All those collected were mature females ranging in age from 7 to 13 years. Growth was linear at an average annual rate of 7.6 cm. Hepatosomatic indices peaked in February and gonadosomatic indices between February and April. Postovulatory follicles and vitellogenic oocytes were observed in the same ovaries, evidence that monkfish spawn over a protracted period and possibly more than once annually. Food habits were similar to those reported for smaller benthic phase monkfish, but cannibalism was more prevalent in large fish (5.6% frequency of occurrence). Frequencies of feeding and cannibalism were greatest in females in the final stage of oocyte maturation.

Expression of the two cytochrome P450 aromatase genes in the male and female blue gourami ( Trichogaster trichopterus) during the reproductive cycle

In this study, the involvement of the cytochrome P450 aromatase gene ( CYP19) in the gametogenesis of the teleost blue gourami ( Trichogaster trichopterus) is described. The blue gourami brain CYP19 ( bgCYP19b) and gonadal CYP19 ( bgCYP19a) aromatase genes were cloned and their expression analyzed during the different reproductive stages. The cloned cDNAs of the bgCYP19b and bgCYP19a were found to contain segments of 1518 bp (an open reading frame encoding a deduced protein of 506 residues) and 489 bp (encoding a peptide of 163 residues), respectively. Although the mRNA levels of bgCYP19b were very low in females until the vitellogenic phase, they were significantly higher in the final oocyte maturation stage. The aromatase gene mRNA levels in the gonads were significantly lower in females in the high vitellogenic stage, as compared to females during early vitellogenesis or maturation. In males, the mRNA levels of bgCYP19b were significantly lower in juveniles than in mature individuals. However, no significant differences were observed between mature non-reproductive and reproductive males. In addition, there was no significant difference between the expression of bgCYP19a in juvenile and non-nest building mature males, although a significant increase was detected in mature reproductive males. Although CYP19b expression was similar in both sexes, the expression of CYP19a was significantly different between males and females.

Comparison of serum and follicular fluid hormone levels with recombinant and urinary human chorionic gonadotropin during in vitro fertilization

To study serum and follicular fluid (FF) hormone levels after the administration of urinary or recombinant hCG to initiate the final stages of oocyte maturation during IVF. Prospective randomized study between 250 microg of recombinant hCG and 7,500 IU of urinary hCG as the final trigger of ovulation during IVF. Private IVF center. Infertile women undergoing IVF/intracytoplasmic sperm injection (ICSI) using the long protocol and recombinant FSH. IVF treatment. Serum and FF hormone measurements on the day of oocyte collection. Serum and FF E(2), P, hCG, and T levels. Stimulation parameters, serum and follicular E(2), P, T, and hCG levels were similar in the recombinant and urinary hCG groups. The number of oocytes retrieved from follicles >14 mm, the proportion of mature oocytes, fertilization rate, and pregnancy rate (PR) were also comparable. Recombinant and urinary hCG provided similar serum and follicular hormonal environments during the final stages of oocyte maturation. The IVF outcome parameters were also comparable. The two medications appear to be equally effective.

In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media

The aim of the present research was to verify the influence of oviductal cell co-culture previously supplemented with steroids (estrogen, progesterone, or both) on IVM rates for oocytes from anestrous bitches that were cultured in vitro for 48, 72 and 96 h. Oocytes harvested from anestrous bitches were selected and allocated into four groups: Group 1 (co-culture in oviductal epithelial cells without hormonal supplementation-control); Group 2 (estrogen supplementation); Group 3 (progesterone supplementation); Group 4 (estrogen + progesterone supplementation). The oviductal epithelial cell culture was established 72 h prior to oocyte co-culture. After periods of 48, 72 and 96 h, the degree of oocyte nuclear maturation was assessed. Co-culture in oviductal epithelial cells with estrogen was not as beneficial for canine IVM as supplementation with progesterone and estrogen, or progesterone supplementation alone. Therefore, it was feasible to use co-culture with oviductal epithelial cells obtained from anestrous bitches for IVM (monolayer culture with oviduct cells previously supplemented with progesterone). Final stages of oocyte maturation were achieved at 72 and 96 h of culture; therefore, the duration of maturation for oocytes obtained from bitches in different stages of the estrous cycle should be taken into account.

Steroid-Induced Oocyte Maturation in Atlantic Croaker (Micropogonias undulatus) Is Dependent on Activation of the Phosphatidylinositol 3-Kinase/Akt Signal Transduction Pathway

Exposure of fully grown fish and amphibian oocytes to a maturation-inducing steroid (MIS) activates numerous signal transduction pathways to initiate the final stage of oocyte maturation. These events culminate in the activation of maturation-promoting factor and germinal vesicle breakdown (GVBD). In most species, exposure to MIS causes a transient decrease in oocyte cAMP levels. Whether this reduction in oocyte cAMP concentration is sufficient to induce GVBD is unclear. The current study tested the hypothesis that activation of cAMP-independent signal transduction pathways by the naturally occurring MIS, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), is necessary for GVBD in Atlantic croaker (Micropogonias undulatus) oocytes. Results indicate that although 20beta-S treatment of oocyte membranes significantly reduced cAMP production, incubation of follicles with the cell-permeable cAMP-dependent protein kinase (Prka) inhibitors Rp-cAMP or KT5720 did not promote GVBD in the absence of 20beta-S. Additionally, treatment of follicles with the phosphodiesterase (Pde) inhibitors Cilostamide (Pde3) or Rolipram (Pde4) significantly reduced GVBD, but they were not able to completely block it. In contrast, pharmacologic inhibition of the cAMP-independent phosphatidylinositol 3-kinase (Pik3)/Akt signal transduction pathway using the Pik3 inhibitors Wortmannin or LY294002, or the Akt inhibitor ML-9, blocked 20beta-S-induced GVBD. Finally, mitogen-activated protein kinase (Mapk1/3) activity increased after treatment with 20beta-S; however, inhibition of Mapk1/3 activity using PD98059 or U0126 had no effect on GVBD. These results demonstrate that activation of cAMP-independent signaling pathways, especially the Pik3/Akt pathway, is necessary for 20beta-S-induced GVBD in Atlantic croaker oocytes.

The Use of GnRH Agonist to Trigger Final Stages of Oocyte Maturation in Patients with Polycystic Ovarian Syndrome (PCOS) and High Responders During IVF Treatment

Background: Previous studies have suggested that triggering ooocyte maturation with GnRH agonist may potentially prevent the development of OHSS. However, there are no studies evaluating this approach in patients with PCOS or previous high responders.

Developmental capability of denuded bovine oocyte in a Co‐culture system with intact cumulus‐oocyte complexes: Role of cumulus cells, cyclic adenosine 3′,5′‐monophosphate, and glutathione

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.

A prospective, randomized and blinded comparison between 10,000 IU urinary and 250 μg recombinant human chorionic gonadotropin for oocyte maturation in in vitro fertilization cycles

To compare the efficacy and safety of u-hCG with r-hCG in IVF cycles. A prospective, investigator-blind, randomized, comparative study. Patients (n = 100) < or =35 years with IVF indication were randomly assigned on the day of hCG administration for oocyte maturation to receive either u-hCG (10,000 IU) or r-hCG (250 microg). No statistical differences were found between groups in relation to total number of oocytes retrieved, percentage of mature oocytes, number of injected oocytes, fertilization rates and number of embryos transferred. The data indicate a possible trend toward a higher incidence of pregnancy in the r-hCG group. Adverse events, predominantly injection-site reactions, were significantly more common in the u-hCG group. r-hCG is at least as effective for inducing final stages of oocyte maturation as 10,000 IU u-hCG and is also associated with significantly better patient tolerance and thus higher patient acceptability.

Cathepsin C transcripts are differentially expressed in the final stages of oocyte maturation in kuruma prawn Marsupenaeus japonicus

To elucidate the molecular mechanism of oocyte maturation in the kuruma prawn ( Marsupenaeus japonicus), subtractive suppression hybridization (SSH) was initially used to identify novel up-regulated genes during the final stages of oocyte maturation, followed by evaluation of the differential expression profile by macroarray and quantitative real-time RT–PCR analyses. The cathepsin C (dipeptidyl peptidase I) gene was thus found to exhibit a significantly higher expression around the onset of cortical rod (CR) formation (early CR stage, appearance of round CRs), progress to a higher mRNA level until the middle CR stage (elongation of CRs), then rapidly revert to a low expression level at the late CR stage (occurrence of germinal vesicle breakdown, GVBD), as also observed at the non-CR stage (previtellogenesis and vitellogenesis). In situ hybridization analyses revealed that the sites of the expression of cathepsin C transcripts in the ovary were distributed in both oocyte and follicle cells, particularly at the early CR stage. A full-length cDNA sequence of this stage-specific gene was subsequently determined by rapid amplification of the cDNA 3′ and 5′ ends (3′ and 5′ RACE). The deduced amino acid sequence of the 230-residue mature peptide shared 67–70% identity to the known cathepsin C in mammals. Western blot analysis showed that expression of procathepsin C protein was exclusively at CR stages. The storage site of procathepsin C protein was localized in CRs as revealed by immunohistochemical analysis. This is the first report on the full-length cDNA sequence of cathepsin C and a demonstration of its involvement in the final stages of oocyte maturation in crustacean species.

Gonadal Development and Sexual Dimorphism of Gobiomorus dormitor from the Estuarine System of Tecolutla, Veracruz, Mexico

The bigmouth sleeper, Gobiomorus dormitor, is a benthic, euryhaline species, and is very abundant in river mouths, coastal lagoons, and sites away from marine influence from south Florida to Dutch Guyana. There are few studies of its life history, ecology, and abundance, particularly within Mexican waters. Nine trips to Tecolutla estuary, Veracruz, Mexico, were taken between October 1995 and May 1998 to estimate the gonadal development and sexual dimorphism of G. dormitor. A total of 94 individuals ranging from 15–260 mm SL and 0.05–181 g were captured. Seventy-two specimens were adults (60 females, 12 males) and 22 were juveniles that did not show external sexual dimorphism. Both juvenile and adult stages of G. dormitor were captured year-round in seagrass beds and adjacent shallow, muddy or sandy areas. This study has shown that G. dormitor are resident and undergo sexual maturation in the Tecolutla estuary. Histological evidence suggests both males and females undergo gonadal recrudescence in the estuary and have an extended reproductive season from May through November. However, it is unclear if the species actually spawns in the estuary, since females in the final stages of oocyte maturation were not captured. Additional research on the reproductive biology and ecology of this under-studied species is necessary to determine its role in tropical estuaries in the southern Gulf of Mexico. Information learned from areas in the center of its distribution may aid in conserving the species at the periphery of its range in Florida, where it is considered threatened.

Human oocytes and preimplantation embryos express mRNA for growth hormone receptor

Human genetic expression of growth hormone receptor (GHR) gene was qualitatively analysed using reverse transcription polymerase chain reaction (RT-PCR) in cumulus cells, immature germinal vesicle (GV) and mature metaphase II (MII) stage oocytes and preimplantation human embryos. The transcripts encoding GHR were detected in cumulus cells and also in naked oocytes, either mature or not. In this case, a nested PCR is needed, as for early embryo preimplantation stages, before genomic activation. The GHR gene is highly expressed from the 4-day morula onwards. This suggests that GHR transcription follows a classical scheme associated with genomic activation. It is probable that, in human, growth hormone plays a role in the final stages of oocyte maturation and early embryogenesis as it does for several other mammalian species.

A prospective randomized and blinded comparison between urinary and recombinant HCG for oocyte maturation in IVF cycles

Objective: It is well known that both hCG and LH can stimulate the same receptor. The hCG extracted from urine from pregnant women has been widely used in the induction of oocyte maturation in IVF cycles. Lack of purity and variation in activity have been cited as possible disadvantages of urinary hCG. Choriogonadotropin alfa (Ovidrel®, Serono Inc., Sao Paulo, SP) is a recombinant hCG (rhCG) preparation purified by repeated chromatographic steps in order to produce a drug with a high specific activity. The objective of this study was to compare the effects of conventional urinary hCG (Profasi HP, Serono Inc., Sao Paulo, SP) to those of recombinant hCG (Ovidrel®, Serono Inc., Sao Paulo, SP) in IVF cycles.Design: Eighty patients undergoing IVF cycles were randomically assigned, on the day of hCG administration for oocyte maturation, to receive either urinary hCG (Profasi HP® 10000 IU, Serono Inc, São Paulo, SP) or recombinant hCG (Ovidrel® 250mg, Serono Inc., Sao Paulo, SP).Materials and Methods: Patients ≤ 35 years old with IVF indication were submitted to a standard long GnRH protocol of ovarian stimulation performed with recombinant FSH (Gonal F®, Serono Inc, São Paulo, SP). The response was monitored by ultrasound and the hCG prescribed when two or more follicles ≥ 18mm were detected. Physicians responsible for oocyte retrieval and embryo transfer as well as the laboratory staff were not informed regarding the patient’s randomization status. Statistical evaluation was performed with unpaired t test, Fisher’s exact and Mann-Whitney test as appropriate.Results: The comparison of outcomes for the two groups showed no statistical difference in relation to total number of retrieved oocytes, percentage of mature oocytes, number of injected oocytes, fertilization rates, number of embryos transferred and positive beta hCG, although our data indicate a possible trend toward a higher incidence of pregnancy in Ovidrel group. Tabled 1Conclusion: The recombinant hCG is a safe and at least equally efficient drug for inducing the final stages of oocyte maturation as urinary hCG. Objective: It is well known that both hCG and LH can stimulate the same receptor. The hCG extracted from urine from pregnant women has been widely used in the induction of oocyte maturation in IVF cycles. Lack of purity and variation in activity have been cited as possible disadvantages of urinary hCG. Choriogonadotropin alfa (Ovidrel®, Serono Inc., Sao Paulo, SP) is a recombinant hCG (rhCG) preparation purified by repeated chromatographic steps in order to produce a drug with a high specific activity. The objective of this study was to compare the effects of conventional urinary hCG (Profasi HP, Serono Inc., Sao Paulo, SP) to those of recombinant hCG (Ovidrel®, Serono Inc., Sao Paulo, SP) in IVF cycles. Design: Eighty patients undergoing IVF cycles were randomically assigned, on the day of hCG administration for oocyte maturation, to receive either urinary hCG (Profasi HP® 10000 IU, Serono Inc, São Paulo, SP) or recombinant hCG (Ovidrel® 250mg, Serono Inc., Sao Paulo, SP). Materials and Methods: Patients ≤ 35 years old with IVF indication were submitted to a standard long GnRH protocol of ovarian stimulation performed with recombinant FSH (Gonal F®, Serono Inc, São Paulo, SP). The response was monitored by ultrasound and the hCG prescribed when two or more follicles ≥ 18mm were detected. Physicians responsible for oocyte retrieval and embryo transfer as well as the laboratory staff were not informed regarding the patient’s randomization status. Statistical evaluation was performed with unpaired t test, Fisher’s exact and Mann-Whitney test as appropriate. Results: The comparison of outcomes for the two groups showed no statistical difference in relation to total number of retrieved oocytes, percentage of mature oocytes, number of injected oocytes, fertilization rates, number of embryos transferred and positive beta hCG, although our data indicate a possible trend toward a higher incidence of pregnancy in Ovidrel group. Tabled 1 Conclusion: The recombinant hCG is a safe and at least equally efficient drug for inducing the final stages of oocyte maturation as urinary hCG.

Spawning induction and hormonal levels during final oocyte maturation in the silver perch ( Bidyanus bidyanus)

The silver perch ( Bidyanus bidyanus Mitchell) (Teraponidae), is a native Australian freshwater fish that, due to its high potential for aquaculture, was introduced to Israeli fish farming. The objective of this study was to find an optimal method for inducing spawning of silver perch. The agents tested for this purpose were: human chorionic gonadotropin (hCG; 150 or 200 IU/kg BW); salmon gonadotropin releasing hormone analogue (sGnRHa at 10, 20, 30, or 40 μg/kg BW); mammalian GnRH analogue (mGnRHa at 30 μg/kg) and the combination of sGnRHa at 20 μg/kg and domperidone at 5 mg/kg BW. Based on spawning success and relative fecundity, sGnRHa at the dose of 30 μg/kg was found to be more efficient than hCG, mGnRHa or sGnRHa with domperidone. Since domperidone does not improve the GnRHa effect on final oocyte maturation (FOM) and spawning, it is suggested that the dopaminergic inhibition during the stages of FOM in the silver perch is weak. Therefore, the use of GnRHa alone is sufficient to induce spawning in this fish. Immunoreactive gonadotropin (IR-GtH) and estradiol levels increased after a single injection of sGnRHa, and peaked after 24 h. Plasma levels of 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) also increased significantly 24 h after the injection of mGnRHa, 12 h before spawning, suggesting that 17,20-P is the maturation-inducing steroid in silver perch. In order to reveal whether the heterologous gonadotropin may elicit an immunological reaction, silver perch was subjected to prolonged treatment with hCG. This treatment resulted in no detectable titer of antibodies against the mammalian gonadotropin. In conclusion, although hCG has no deleterious effects in this fish, and is the more commonly used for spawning induction, sGnRHa at 30 μg/kg is the recommended treatment for spawning induction of female silver perch under the conditions prevailing in Israeli aquaculture.

Induced sexual maturation of the European eel Anguilla anguilla and fertilisation of the eggs

Abstract The aim of the present study was to induce sexual maturation in females of the European eel Anguilla anguilla using a protocol similar to that successful for Anguilla japonica . Inducing maturation in the female European eel has proven difficult in the extreme, as only one research group has so far reported fertilisation with subsequent embryonic and larval development in A. anguilla . The present study performed two experiments. In experiment A, the 10 surviving females received a weekly injection with salmon pituitary extract (SPE) at a dose of 18.2 mg pituitary powder per week and fish, and in experiment B, females were injected twice weekly and received a dose of 36.4 mg pituitary per fish and week. Only two experiment B fish had viable eggs. In both experiments, final oocyte maturation and ovulation was induced by injection of 17,20 β-dihydroxy-4-pregnen-3-one (DHP) at about 2 μg/g body weight. Maturation time was 16–25 weeks in experiment A and 7.5–11.5 weeks in experiment B. In the final stages of oocyte maturation, female A. anguilla showed highly individual rates of oocyte development, and as injection protocol depended on oocyte development, six protocols had to be used. The only protocol which resulted in fertilisation of eggs with subsequent development of larval bodies—and in one case hatching, although delayed, of larvae—was weekly SPE injection followed ca. 40 h later by an extra SPE injection (18.2 mg pituitary powder), 10 h after which DHP was injected.

Reducing the dose of hCG in high responders does not affect the outcomes of IVF

Objective: Human chorionic gonadotropin (hCG) is used to induce final stages of oocyte maturation as part of most ovarian hyperstimulation protocols; however, it is also believed to contribute to the occurrence of the ovarian hyperstimulation syndrome (OHSS). There have been many strategies described in attempts to reduce the risk of this potentially life-threatening complication. Many programs reduce the amount of hCG given to high responders in an effort to minimize the risk of OHSS. The lowest effective dose of hCG however, has not been established. This study was carried out to confirm that a dose as low as 3,300 IU of hCG is sufficient to provide adequate oocyte maturation and fertilization. Design: A retrospective review of IVF clinical data. Materials/Methods: 94 IVF cycles at a single IVF center were analyzed with two cohort groups with similar demographics. When peak estradiol (E2) levels on the day of hCG administration were ≥ 2,500 pg/ml, patients received 5,000 IU of hCG (Group A, n=47). If E2 levels were ≥ 4,000 pg/ml, patients were triggered with 3,300 IU of hCG (Group B, n=47). Demographics of the groups were compared including age, type of infertility, and stimulation protocols. All patients received hCG when ≥ 3 follicles showed an average size ≥ 17mm. The incidence of mild, moderate and severe OHSS was also analyzed. Appropriate statistical methods were used for analysis. Results: Mean ages for groups A and B were 35.4 ± 0.7 and 33.2 ± 0.7 respectively (p = 0.018). Outcome measures for the 5,000 IU vs. 3,300 IU groups were: peak E2 2,907 ± 76 vs. 4,260 ± 129 pg/ml (p <0.001), mean number of eggs retrieved 15.9 ± 0.9 vs. 20.3 ± 1.2 (p = 0.003), proportion of mature eggs 81.6% vs. 81.9% (NS), fertilization rate 70.5% vs. 68.7% (NS), clinical pregnancy rate 58.7% vs. 58.7% (NS), and ongoing pregnancy rate 50.0% vs. 43.5% (NS). There was no statistically significant difference in the incidence of mild (8.5% vs. 6.3%), moderate (2.1% vs.10.6%) or severe OHSS (0% vs. 4.2%) between groups A and B, respectively. Conclusions: To our knowledge, this is the first study demonstrating that a reduced hCG dose of 3,300 IU results in a similar proportion of mature eggs, similar fertilization rates and ongoing pregnancy rates compared to a more standard dose of 5,000 IU. Nevertheless, reducing the dose of hCG does not appear to eliminate the risk of OHSS in a high risk group. Perhaps new approaches such as triggering ovulation with a GnRH agonist in patients treated with a GnRH antagonist, will provide a safer treatment alternative for these challenging patients. Supported by: None.

Identification and characterisation of known and novel transcripts expressed during the final stages of human oocyte maturation.

The final stages of oocyte maturation, from the germinal vesicle (GV) stage to metaphase II (MII) oocytes, are characterised by a series of dynamic events. These include germinal vesicle break down (GVBD), resumption of meiosis, and nuclear and cytoplasmic maturation to produce MII oocytes ready for fertilisation. To investigate the specific genes transcribed during these stages of oogenesis, we have prepared and analysed amplified cDNA representing the transcribed genes in a series of GV and MII oocytes. Differential display analysis disclosed that the overall gene expression profiles between different samples of GV oocytes are very similar, regardless of their source, while those between the MII oocytes are markedly variable. A comparison of expression profiles in oocytes and somatic (cumulus) cells identified several known genes preferentially-expressed in oocytes (e.g., a zona pellucida gene), as well as five novel sequences. Two of the five novel sequences are homologous to retrotransposon sequences, long terminal repeat (LTR) and long interspersed nuclear element (LINE) 1, and two other sequences show partial homology to known ESTs and genomic sequences. The remaining sequence, which is identical to shorter ESTs isolated from germ cell tumor cDNA libraries, was extended towards its 5' end by PCR, using the original cDNA preparation from which it was isolated as a template. Expression of the resultant 1.1-kb transcript is restricted to the testis and ovary, and its expression correlates with cell pluripotency in that it is expressed in embryonal carcinoma cells, but not in their differentiated derivative cells.

Use of gonadotrophin-releasing hormone agonists to trigger ovulation

The introduction of gonadotrophin-releasing hormone (GnRH) agonists combined with gonadotrophins is considered to be one of the most significant advances in the development of in vitro fertilization (IVF) treatment. However, ovarian hyperstimulation syndrome (OHSS) remains a significant complication of controlled ovarian hyperstimulation. One possible strategy to reduce the risk of this complication would be the use of GnRH agonists instead of human chorionic gonadotrophin (hCG) to trigger the final stages of oocyte maturation. GnRH agonists are able to induce an endogenous surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and the effect may be more physiological than that of exogenous hCG. Several uncontrolled and controlled clinical studies have confirmed the efficacy of GnRH agonists for triggering ovulation, and pregnancy rates are comparable to those achieved with hCG. The incidence of OHSS appears to be decreased, but larger controlled studies are required to confirm this observation. The recent introduction of GnRH antagonists has led to renewed interest in the use of GnRH agonists to induce final oocyte maturation. An international multicentre randomized controlled trial has been completed recently comparing the efficacy of GnRH agonist with hCG for triggering ovulation in women undergoing controlled ovarian hyperstimulation using the GnRH antagonist ganirelix for pituitary suppression. The aim of the study was to determine the efficacy of the novel protocol for ovarian stimulation before IVF, in terms of pregnancy outcomes and the prevention of OHSS.