Injection Of Follicle-stimulating Hormone Research Articles

Long-acting follicle-stimulating hormone versus daily follicle-stimulating hormone for women undergoing assisted reproduction

Follicle-stimulating hormone (FSH) given as a weekly injection in a medium dose is as safe and effective as a daily FSH injection for women undergoing assisted reproduction.

Long-acting FSH versus daily FSH for women undergoing assisted reproduction.

Assisted reproduction techniques (ART), such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI), can help subfertile couples to create a family. It is necessary to induce multiple follicles, which is achieved by follicle stimulating hormone (FSH) injections. Current treatment regimens prescribe daily injections of FSH (urinary FSH either with or without luteinizing hormone (LH) injections or recombinant FSH (rFSH)).Recombinant DNA technologies have produced a new recombinant molecule which is a long-acting FSH, named corifollitropin alfa (Elonva) or FSH-CTP. A single dose of long-acting FSH is able to keep the circulating FSH level above the threshold necessary to support multi-follicular growth for an entire week. The optimal dose of long-acting FSH is still being determined. A single injection of long-acting FSH can replace seven daily FSH injections during the first week of controlled ovarian stimulation (COS) and can make assisted reproduction more patient friendly. To compare the effectiveness of long-acting FSH versus daily FSH in terms of pregnancy and safety outcomes in women undergoing IVF or ICSI treatment cycles. We searched the following electronic databases, trial registers and websites from inception to June 2015: the Cochrane Central Register of Controlled Trials (CENTRAL), the Cochrane Menstrual Disorders and Subfertility Group (MDSG) Specialized Register, MEDLINE, EMBASE, PsycINFO, CINAHL, electronic trial registers for ongoing and registered trials, citation indexes, conference abstracts in the ISI Web of Knowledge, LILACS, Clinical Study Results (for clinical trial results of marketed pharmaceuticals), PubMed and OpenSIGLE. We also carried out handsearches. We included all randomised controlled trials (RCTs) comparing long-acting FSH versus daily FSH in women who were part of a couple with subfertility and undertaking IVF or ICSI treatment cycles with a GnRH antagonist or agonist protocol. Two review authors independently performed study selection, data extraction and assessment of risk of bias. We contacted trial authors in cases of missing data. We calculated risk ratios for each outcome, and our primary outcomes were live birth rate and ovarian hyperstimulation syndrome (OHSS) rate. Our secondary outcomes were ongoing pregnancy rate, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, any other adverse event (including ectopic pregnancy, congenital malformations, drug side effects and infection) and patient satisfaction with the treatment. Trials reported all outcomes, except patient satisfaction with the treatment. We included six RCTs with a total of 3753 participants and we graded the quality of the included studies as moderate. All studies included women with an indication for COS as part of an IVF/ICSI cycle with age ranging from 18 to 41 years. A comparison of long-acting FSH versus daily FSH did not show evidence of difference in effect on overall live birth rate (Risk ratio (RR) 0.95, 95% confidence interval (CI) 0.84 to 1.07; 2363 participants, eight studies; I² statistic = 44%) or OHSS (RR 1.00, 95% CI 0.74 to 1.37; 3753 participants, nine studies; I² statistic = 0%). We compared subgroups by dose of long-acting FSH. There was evidence of reduced live birth rate in women who received lower doses (60 to 120 μg) of long-acting FSH compared to daily FSH (RR 0.70, 95% CI 0.52 to 0.93; 645 participants, three studies; I² statistic = 0%). There was no evidence a difference between the groups in live births in the medium dose (150 to 180 μg) subgroup (RR 1.03, 95% CI 0.90 to 1.18; 1685 participants, four studies; I² statistic = 6%). There was no evidence of a difference between the groups in the clinical pregnancy rate (any dose), ongoing pregnancy rate (any dose), multiple pregnancy rate (any dose), miscarriage rate (low or medium dose), ectopic pregnancy rate (any dose), congenital malformation rate, congenital malformation rate; major or minor (low or medium dose). The use of a medium dose (150 to 180 μg) of long-acting FSH is a safe treatment option and equally effective compared to daily FSH in women with unexplained subfertility. There was evidence of reduced live birth rate in women receiving a low dose (60 to 120 μg) of long-acting FSH compared to daily FSH. Further research is needed to determine whether long-acting FSH is safe and effective for use in hyper- or poor responders and in women with all causes of subfertility.

Anatomical Targeting Improves Delivery of Unconjugated Nanoparticles to the Testicle

Nanoparticles, which are submicroscopic particles typically ranging from 100 to 300 nm, are interesting as potential treatment of testicular disorders because they can be engineered to allow delivery to privileged tissues, such as across the blood-brain barrier or theoretically the blood-testis barrier. We compared the effects of anatomical and/or ligand targeting on testicular nanoparticle uptake in a rat model. A total of 48 rats were divided into 6 groups, including a control group and groups that received intra-arterial injection of unconjugated nanoparticles with and without saline flush, intravenous injection of unconjugated nanoparticles, intra-arterial injection of follicle stimulating hormone conjugated nanoparticles, intravenous injection of follicle stimulating hormone conjugated nanoparticles and intra-arterial injection of transactivating transcriptor conjugated nanoparticles. A dose response curve was assessed for intra-arterially injected unconjugated nanoparticles. Using high performance liquid chromatography and histological analysis we determined nanoparticle uptake by the testicle at 4 hours. Intra-arterial injection resulted in a 5.8-fold increase in uptake compared to intravenous injection at 35 mg/kg of unconjugated nanoparticles (3.7 vs 0.6 μg nanoparticles per gm testicle, p = 0.04). Anatomical targeting failed to improve testicular uptake in FSH conjugated nanoparticles (intra-arterial vs intravenous injection 0.33 vs 0.38 μg FSH nanoparticles per gm testicular tissue, p = 0.73). On fluorescence microscopy nanoparticles were noted in the testicular interstitium and seminiferous tubules, and absent from the testicular vasculature. Arterial injection for anatomical targeting of nanoparticles to the testis is feasible, improves unconjugated nanoparticle delivery to testicular tissue and enables nanoparticles to cross the gonadal vascular endothelium and the blood-testis barrier.

Comparison of FSH and hMG on ovarian stimulation outcome with a GnRH antagonist protocol in younger and advanced reproductive age women.

PurposeTo compare the embryo outcomes of in vitro fertilization/intra‐cytoplasmic sperm injection with a gonadotropin‐releasing hormone (GnRH) antagonist protocol with follicle stimulating hormone (FSH) and with human menopausal gonadotropin (hMG).MethodsWe performed a retrospective cohort study in 465 patients. Stimulation was started by daily FSH injection, and either FSH was continued (FSH alone group) or hMG was administrated (FSH‐hMG group) after administration of a GnRH antagonist. Primary outcomes were the embryo profile (number of retrieved, mature, and fertilized eggs, and morphologically good embryos on day 3) and endocrine profile. Secondary outcomes were the doses and durations of gonadotropin. Data were stratified by the patients’ age into two groups: <35 years and ≥35 years.ResultsIn patients aged <35 years, the number of retrieved oocytes in the FSH alone group was significantly increased than that in the FSH‐hMG group (13.7 vs 9.2, P = 0.04), while there was no difference at other age groups. The FSH‐hMG group required a significantly greater amount of gonadotropins at any age (all ages, P < 0.001; <35 years, P = 0.013; ≥35 years, P < 0.001).ConclusionsExogenous FSH alone is probably sufficient for follicular development and hMG may not improve the embryo profile in a GnRH antagonist protocol across all age.

Cost-effectiveness analysis on the use of rFSH + rLH for the treatment of anovulation in hypogonadotropic hypogonadal women.

BackgroundHypogonadotropic hypogonadal women are characterized by ovarian functionality deficiency, caused by low concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). To recover reproduction functionality, recommended therapies for ovarian induction involve injections of FSH and LH medications.ObjectiveSince important differences exist between recombinant and urinary gonadotropin therapies in terms of efficacy and cost, the objective of this study was to develop a cost-effectiveness model to compare recombinant FSH (rFSH) + recombinant LH (rLH) and highly purified human menopausal gonadotropin (HP-HMG).MethodsA Markov model was developed, considering three cycles of therapy; probability of pregnancy and miscarriage were considered, and the efficacy was evaluated in terms of pregnancy occurrence. The perspective of the model was that of the Italian Health Service, so only direct cost (drugs, specialist visits, patient examinations, and hospitalizations) were included.ResultsrFSH + rLH is associated with a higher total cost (€3,453.50) and higher efficacy (0.87) compared with HP-HMG (€2,719.70 and 0.50). rFSH + rLH generated an incremental cost effectiveness ratio equal to €2,007.30 compared to HP-HMG; the average cost per pregnancy is estimated to be €3,990.00 for recombinant strategy and €5,439.80 for urinary strategy. Results of probabilistic sensitivity analysis were consistent with the abovementioned findings.ConclusionDespite the higher acquisition cost in comparison to HP-HMG, rFSH + rLH resulted in a higher pregnancy rate, which makes it the recommended choice when considering cost-effectiveness of LH in supporting FSH-induced follicular gonadotropins in hypogonadotropic hypogonadal women.

Purification, Characterization and Pharmacokinetic Study of a Novel Long-acting Follicle-stimulating Hormone

Follicle-stimulating hormone( FSH) has been utilized to treat male and female infertility. However,frequent injection of FSH to patients is required due to its short serum half-life. Developing a longacting recombinant FSH is clinically desirable. A DNA construct containing FSH-CTP-vFc was made by fusing the coding sequence of carboxyl-terminal peptide( CTP) of the β-subunit of human chorionic gonadotropin and a variant of human Immunoglobulin G2( IgG2) Fc fragment( vFc) into FSH gene. The DNA construct was transfected into Chinese Hamster Ovary( CHO) cells,and cell clones with highly and stably expressed FSHCTP-vFc fusion protein were selected. The cultured medium of cell was collected for protein purification by protein A based chromatography. Biochemical and physical characterization were performed for the fusion proteins purified from the samples of different culture time. ELISA assay and animal study shows that the purified FSHCTP-vFc protein has significant activity in vitro and in vivo. Pharmacokinetic research demonstrates that the serum half-life of the fusion protein is almost ten fold of the recombinant FSH's. Obviously,this novel long-acting FSHCTP-vFc protein could greatly reduce the injection frequency and improve the patient compliance.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

BackgroundHematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and MethodsTo address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. ResultsWe found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. ConclusionsOur data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories.▪ Disclosures:No relevant conflicts of interest to declare.

Embryo production with sex-sorted semen in superovulated dairy heifers and cows

The aim of this study was to examine the effect of sex-sorted semen on the number and quality of embryos recovered from superovulated heifers and cows on commercial dairy farm conditions in Finland. The data consist of 1487 commercial embryo collections performed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively. Superovulation was induced by eight intramuscular injections of follicle-stimulating hormone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days after the onset of standing estrus. The donors were inseminated at 9 to 15–hour intervals beginning 12 hours after the onset of estrus with 2 + 2 (+1) doses of sex-sorted frozen-thawed semen (N = 218) into the uterine horns or with 1 + 1 (+1) doses of conventional frozen-thawed semen (N = 1269) into the uterine corpus. Most conventional semen (222 bulls) straws contained 15 million sperm (total number 30–45 million per donor). Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 8–14 million per donor). Mean number of transferable embryos in recoveries from cows bred with sex-sorted semen was 4.9, which is significantly lower than 9.1 transferable embryos recovered when using conventional semen (P ≤ 0.001). In heifers, no significant difference was detected between mean number of transferable embryos in recoveries using sex-sorted semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized ova was higher when using sex-sorted semen than when using conventional semen in heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows (P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted semen remains to be found.

Evaluation effects of allopurinol and FSH on reduction of ischemia-reperfusion injury and on preservation of follicle after heterotopic auto-transplantation of ovarian tissue in mouse.

Allopurinol and FSH injection are applied to reduce ischemia-reperfusion injury and to increase survival rate for ovarian follicles after ovarian heterotopic autotransplantation in mice. Ovarian tissues from 6-week-old mice were grafted into back muscle then collected after 3 weeks. A total of five groups were included in this experiment as follows: control group (n = 5), sham-operated group (n = 5), allopurinol treatment group (AP) (n = 5), follicle stimulating hormone (FSH) treatment group (n = 5), as well as, allopurinol and FSH treatment group (APF) (n = 5). We investigated survival, number and development of follicles, vaginal cytology along with plasma malondialdehyde (MDA) concentration in grafted ovary. Total follicles count significantly increased in APF group compared with other treatment groups (p < 0.05). MDA concentration significantly decreased in AP group and APF treatment group compared with sham-operated group. In AP group, vaginal smears showed presence of cornified epithelial cells three-five day after surgery. We demonstrated that allopurinol, as a XO inhibitor, plays an important role in order to decrease ischemia injury and to increase survival rate for follicles. Also, FSH, as a folliculogenesis and angiogenesis factor, increases development of follicles. It seems that allopurinol can cause re-establishing hypothalamus-pituitary axis and finally can restore estrous cycle earlier than for the sham operated group, so it explains the increasing survival rate for follicles.

Rescue from dominant follicle atresia by follicle-stimulating hormone in mice

We investigated the effects of follicle-stimulating hormone (FSH) on atresia of the dominant follicle and changes in relevant apoptosis genes in granulosa cells of dominant follicles regulated by FSH in vivo. Four-week-old mice were administered FSH by intraperitoneal injection to induce follicular maturation. Granulosa cells of dominant follicles were collected at 48, 72, and 96 h after the first FSH injection. Phosphate-buffered saline was injected as a control. The mRNA levels of relevant granulosa cell apoptosis genes were examined by real-time quantitative polymerase chain reaction, and apoptosis of granulosa cells in dominant ovarian follicles was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Apoptosis in granulosa cells of dominant follicles was almost TUNEL-negative at 48, 72-66, 72, and 96-90 h after the first FSH injection, but granulosa cell apoptosis in dominant follicles was clearly detected at 96, 102, and 102-96 h by TUNEL. The BIM, caspase-3, and caspase-9 mRNA expression levels were significantly lower after FSH treatment at 72-66 and 96-90 h, compared with that at 72 and 96 h (P < 0.05). Caspase-8 and FasL mRNA expressions did not respond to FSH. FSH rescued granulosa cells from apoptosis when the relevant apoptosis genes were upregulated in early atretic follicles. FSH did not rescue granulosa cells from apoptosis if the DNA was cut into fragments by endonucleases. Thus, the rescue by FSH of granulosa cells from apoptosis and dominant follicle atresia may be accomplished by inhibition of apoptosis in mitochondria.

Superovulation with Different Doses of Follicle Stimulating Hormone in Kamphaeng Saen Beef Cattle

An experiment was conducted to investigate the effect of different doses of follicle stimulating hormone (FSH) used in a superovulation program on numbers of corpora lutea, total ova/embryos and transferable embryos in Kamphaeng Saen beef cattle. Cyclic cows (n=3) and heifers (n=3) of Kamphaeng Saen beef breed were assigned to two levels of FSH (200 and 250 mg. NIH-FSH-P1) in Crossover Design by which two changes over treatments were studied over two periods in all animals. Cows and heifers were estrous synchronized by Cloprostenol (500 µg). Estrus detection was performed by teaser bull (Day 0 = day of the onset of standing estrus). On day 9 after the onset of standing estrus, all animals were treated with FSH twice daily in decreasing doses over 4 days. On day 3 of FSH injection, each animal was treated with Cloprostenol (500 µg). At the first standing estrus, all animals were artificially inseminated three times at 12 h intervals. Two straws of frozenthawed semen of Kamphaeng Saen bull were used per insemination. All animals were treated with gonadotropin releasing hormone (10 µg of Buserelin) at first insemination. Numbers of corpora lutea were determined by rectal palpation and embryos were flushed seven days after the onset of standing estrus and classified according to the development stage and quality. The results showed that numbers of corpora lutea, percentages of total ova/embryos and percentages of transferable embryos were not significantly different (P>0.05) between treatments (FSH: 200 versus 250 mg) in either cows or heifers.

Modeling Follicle Stimulating Hormone Levels in Serum for Controlled Ovarian Hyperstimulation I: Comparing Gonadotropin Products

Despite the dangers of gonadotropin administration, our understanding of serum levels of gonadotropins in our patients is poor. We created a mathematical model of gonadotropin administration in order to learn about the temporal profiles of Follicle Stimulating Hormone (FSH) levels in patients related to the doses of different gonadotropin products administered. Model parameters for each product were determined from published pharmacokinetic information available from the manufacturers. For each product, serum levels of FSH rose then fell following a single injection of FSH. When a gonadotropin dose was injected repeatedly (daily), each injection resulted in a peak level of FSH that was higher than the level attained from the previous injection. Daily FSH peaks rose asymptotically, requiring 3-10 days to approach the maximum. The maximum averaged 3.5 times the peak of the first injection. Decline of FSH following the final injection required 3-10 days to approach the baseline. Time courses and peak values of FSH using different gonadotropin preparations varied widely. Knowledge of the slow changes of FSH that occur during modeled FSH administration will provide a framework for understanding the gonadotropin profiles that occur in patients during controlled ovarian hyperstimulation.

In vitro maturation: a five‐year experience

Although some patients may benefit from reduced follicle-stimulating hormone (FSH) application, in-vitro maturation (IVM) belongs to the rare treatment options in assisted reproduction. We summarize our five-year IVM experience. Retrospective, observational study. Reproductive Medicine, University Hospital. 115 patients with polycystic ovary syndrome (PCOS) as well as patients after ovarian hyperstimulation syndrome (OHSS) from February 2005 to December 2009. Stimulation started between day 3-10 of the menstrual cycle and FSH dosage was 125IU/day over three days. Ovulation was induced on the third day of FSH injection or one day afterwards and oocyte retrieval was performed 33-38 hours later. Oocytes were cultivated for 24 hours in IVM medium. Fertilization was carried out one day after oocyte retrieval and embryo transfer two days afterwards. Pregnancy rates. 115 patients were included and 215 oocyte retrievals (intracytoplasmic sperm injection: n=125, 59%; in vitro fertilization: n=73, 34.4%) with 177 embryo transfers performed. The main reasons for IVM were: PCOS (71.7%) and OHSS (15.0%). Mean number of oocytes was 8.9/oocyte retrieval with 5.9 (64%) becoming mature, 2.8 (45.1%) being fertilized and 2.1 transferred. Pregnancy rate per transfer was 15.3% (n=27) with 13 live births (7.3%), one intrauterine death (0.6%), four miscarriages (2.3%) and nine biochemical pregnancies (5.1%). In 61 cases, fertilized oocytes were frozen and 32 cryotransfers were performed, resulting in three pregnancies. Although the pregnancy rate was low, IVM is very convenient for patients due to low FSH dosages and few appointments at low cost.

Follicular dominance on the fifth day of controlled ovarian stimulation reduces implantation in long down-regulated ICSI cycles

Objective To determine if follicular dominance on the fifth day of controlled ovarian stimulation (COS) predicts implantation rates in down-regulated intracytoplasmic sperm injection (ICSI) cycles. Study design One hundred and sixty-two consecutive women undergoing ICSI treatment with long down-regulation and recombinant follicle-stimulating hormone injections were included in a prospective cohort analysis. The clinical pregnancy and implantation rates per transfer were compared between two groups, one with and the other without follicular dominance detected by ultrasound on the fifth day of COS. Results There was no significant difference between the groups regarding the number of good quality embryos transferred, but clinical pregnancy and implantation rate per transfer were higher in group with follicular synchrony. Conclusions Follicular dominance on COS day 5 results in reduced implantation rates after ICSI.

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43‐based gap junctions

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5 IU/animal; T3 group-100 µg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.

Real-time qRT-PCR analysis of EGF receptor in cumulus-oocyte complexes recovered by laparoscopy in hormonally treated goats

Ovarian stimulation with exogenous follicle stimulating hormone (FSH) has been used to increase the number of viable oocytes for laparoscopic oocyte recovery (LOR) in goats. The aim of this study was to evaluate the effect of two FSH protocols for ovarian stimulation in goats on the expression pattern of epidermal growth factor (EGF) receptor (EGFR) in cumulus-oocyte complexes (COCs) recovered by LOR. After real-time qRT-PCR analysis, expression profiles of morphologically graded COCs were compared prior to and after in vitro maturation (IVM) on a FSH protocol basis. The use of a protocol with higher number of FSH injections at a shorter interval resulted in GI/GII COCs with a higher level of EGFR expression in cumulus cells, but not in the oocyte, which was correlated with an elevated meiotic competence following IVM. Based on the maturation profile and EGFR expression patterns observed between groups, the morphological selection of COCs prior to IVM was not a good predictor of oocyte meiotic competence. Therefore, EGFR may be a good candidate marker for indirect prediction of goat oocyte quality. The IVM process of goat COCs increased the EGFR expression in oocytes and cumulus cells, which seemed to be strongly associated with the resumption of meiosis. In summary, differential EGFR expression in goat cumulus cells was associated with the in vivo prematuration process, and in turn, the upregulation in the entire COC was associated with IVM. Cause-and-effect relationships between such increased expression levels, particularly in the oocyte, and oocyte competence itself still need to be further investigated.

406 EFFECT OF DIFFERENT FOLLICLE-STIMULATING HORMONE DOSES ON SUPEROVULATORY RESPONSE OF Red Sindhi COWS

The use of bovine embryo transfer to increase the reproductive potential of animals with high genetic merit is unquestionable. However, studies that evaluate the response to exogenous FSH stimulation in Red Sindhi cows are rare. Therefore, the objective of this study was to compare the superovulatory response of Red Sindhi cows treated with 3 different doses of FSH (Folltropin®-V, Bioniche Animal Health, Belleville, Ontario, Canada). The animals used had body condition scores ranging from 3.5 to 4.0 (1-5 scale), were between 6 and 10 years of age, and were randomly assigned to 1 of 3 treatment groups. Three doses of FSH were administered: 100 mg (n = 16); 133 mg (n = 13), and 200 mg (n = 14). The treatments were initiated on random days of the estrous cycle. On Day 0, animals received 2 mg of estradiol benzoate i.m. (Estrogin®, Farmavet, Jaboticabal, Brazil) and an intravaginal device (CIDR; InterAg, Hamilton, New Zealand) with 1.9 g of progesterone. On Day 4, donors received FSH in 8 i.m. injections that decreased in dose during 4 consecutive days. A dose of 265 μg of cloprostenol (Ciosin®, Schering-Plough, Cotia, Brazil) was given i.m. concomitantly with the fifth FSH injection. Twelve hours after the last FSH injection, donors received 25 μg of lecirelin i.m. (GnRH; Gestran Plus®, ARSA SRL, Buenos Aires, Argentina). TheAI were performed 12 and 24 h after GnRH. Seven days after the first AI, embryos were recovered and classified as described in the IETS manual. Data were evaluated using KruskalWallis test at a 5% level of significance. Ovarian ultrasonography was also performed to count the number of CL present at the time of ova/embryo collection. Results are summarized in Table 1. The number of CL and total ova/embryos was higher in donors receiving 200 mg of FSH than in those in the other groups. However, the number of transferable or cryopreserved embryos did not differ among groups. In conclusion, the dosage of 200 mg of Folltropin®-V seemed to be the more adequate dosage for this breed of cattle. Table 1.Effect of different follicle-stimulating hormone (FSH) doses on CL number, total structures, transferable embryos, and cryopreserved embryos in Red Sindhi cows (mean ± SEM) Financial support provided by FAPERJ (E26-170.529/2007).

Reduced Expression of an RNA-binding Protein by Prolactin Leads to Translational Silencing of Programmed Cell Death Protein 4 and Apoptosis in Newt Spermatogonia

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3'-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3'-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.

Inhibin A Production after Gonadotropin Stimulus: A New Method to Detect Ovarian Tissue in Ovotesticular Disorder of Sex Development

Background/Aims: While laboratory methods for the detection of testicular tissue are well standardized, currently there is no available test to demonstrate the presence of ovarian tissue. We evaluated the effectiveness of gonadal stimulation with luteinizing hormone (LH)/follicle-stimulating hormone (FSH) for the detection of ovarian tissue in patients with disorders of sex development (DSD). Methods: Ten patients with congenital adrenal hyperplasia (CAH) as ovarian-positive controls, 10 with cryptorchidism (ovarian-negative controls), 13 patients with DSD of no defined etiology and 7 patients with ovotesticular DSD (true hermaphroditism, TH) were included in the study. They underwent a daily injection of both LH and FSH on 3 consecutive days. LH, FSH, estradiol, testosterone and inhibin A were measured before treatment, 24 h after the 1st dose and 24 h after the 3rd dose. Results: Estradiol increased in all CAH and TH patients, with a median value of 155.1 and 92.6 pg/ml, respectively, after the 3rd injection. Inhibin A also increased in all CAH and TH patients, with a median value of 70.4 and 32.2 pg/ml, respectively, after the 3rd injection. There was no change in these hormones in the other groups. Conclusion: The LH/FSH stimulation test might be a useful method to detect the presence of ovarian tissue.

293 SUBCUTANEOUS ADMINISTRATION OF FOLLICLE STIMULATING HORMONE FOR SUPEROVULATION OF HOLSTEIN COWS

Follicle stimulating hormone (FSH) is usually administered in a series of intramuscular (IM) injections to induce multiple ovulations for embryo production in cattle and other species. The objective of this study was to determine the superovulatory response of dairy cows to subcutaneous (SC) administration of FSH using a reduced number of injections in combination with a progesterone-releasing device. Eighteen non-lactating Holstein cows initially received 25 mg Prostaglandin F2α IM (PGF; Lutalyse; Pfizer Animal Health, Groton, CT, USA) on Day –7. All cows then received an intravaginal progesterone-releasing device (CIDR-B, 1.38 mg progesterone; Pfizer Animal Health) on Day 0, and 100 μg GnRH IM (Cystorelin; Merial Ltd, USA) on Day 2. Cows were randomly assigned to receive a total of 400 mg (20 mL) of FSH (Folltropin-V; Bioniche Animal Health, USA) either by IM injection (IM Group, n = 9 cows) given at 12 h intervals on Days 4 (60 mg, 60 mg), 5 (55 mg, 55 mg), 6 (45 mg, 45 mg) and 7 (40 mg, 40 mg), or by SC injection (SC Group, n = 9 cows) given at 24 h intervals on Days 4 (140 mg), 5 (140 mg) and Day 6 (120 mg). On Day 7, CIDR-B inserts were removed and cows received two 25 mg PGF IM injections given 12 h apart. Cows were artificially inseminated at 12 and 24 h after standing estrus. Blood samples were obtained from all cows at 0, 2, 4, 8, 12, 24, 36, 48, 60, 72, and 84 h after the first FSH injection for determination of serum FSH concentrations. Ovarian follicles and CL were monitored using ultrasonography on Days 4, 7, and 16. Embryos were recovered non-surgically on Day 16 (7 days after estrus). The effects of treatment on follicular response and embryo yield were analyzed by Wilcoxon test, and the response of cows to treatment was analyzed by chi-square test. The effects of treatment on concentrations of serum FSH were analyzed using ANOVA for repeated measures. There was no effect (P &gt; 0.05) of route of FSH administration on the concentrations of serum FSH at any time point. The superovulatory response of cows to treatment, defined as greater than 2 CL per cow, did not differ (P &gt; 0.05) between the IM (77.8%, 7/9 cows) and SC (88.9%, 8/9 cows) Groups. There was also no difference (P &gt; 0.05) between the IM and SC Groups for the number of 5 to 10 mm follicles prior to FSH treatment (mean ± SEM; 0.6 ± 0.2 v. 0.9 ± 0.4), the total number of follicles after FSH treatment (12.4 ± 1.6 v. 12.7 ± 2.2) or the number of CL at embryo recovery (6.4 ± 1.5 v. 10.4 ± 2.1). Similarly, there were no differences (P &gt; 0.05) between the IM and SC Groups for total number of oocytes/embryos (5.6 ± 2.6 v. 13.0 ± 4.3), transferable embryos (Grade 1, 2, 3; 3.0 ± 1.4 v. 6.1 ± 2.9) or Grade 1 embryos (2.9 ± 1.4 v. 4.3 ± 2.5). In conclusion, administration of FSH using 3 SC injections in combination with a progesterone-releasing device was an effective method for superovulation of Holstein cows. Supported by USDA Animal Health Formula Funds and the State of North Carolina.